THIS IS JUST A FANTASTIC OCCASION, I AM SO EXCITED. TODAY AND TOMORROW WE ARE CELEBRATING THE PORTER BUILDING AND THIS RESEARCHS SOME BUT NOT ALL OF THE NEUROSCIENCES WHO ARE IN PORTER AND THEN ITS FOLLOWED TODAY BY DEDICATION AT 3:00 OF THE BUILDING AND AS YOU CAN SEE THERE'S A WONDAREFUL STAGE AND LOTS OF DIGNITARIES AND CONGRESS PEOPLE WHO HOPEFULLY WILL RECOGNIZE HOW WONDERFUL NEUROSCIENCE IS AND NIAND INCREASE OUR BUDGETS. AND I'M ALWAYS OPTIMISTIC. THE VISION FOR THIS BUILDING, THE PORTER NEUROSCIENCE BUILDING CAME FROM DR. STEVE HYEMAN AND JERRY FISHBATCH, STEVE WAS THE NIH DIRECTOR FROM 2006, AND JERRY WAS THE NIH DIRECTOR FROM 1998 TO 2001. THIS WAS THERE VISION. THIS BUILDING WAS THE GLEAM IN THEIR EYES AND I THOUGHT IT WOULD BE REALLY WONDERFUL TO HAVE JERRY AND STEVE TELL US A LITTLE BIT ABOUT THE VISION THAT RESULTED IN IF BUILDING. SO, JERRY'S HERE, COME ON UP, JERRY AND STEVE, I THINK IS ON HIS WAY. HE GOTOT 6:00 A.M. U.S. AIR FLIGHT FROM BOSTON AND IF HE COMES BEFORE 9:30, WE WILL ALLOW HIM TO SAY A FEW WORDS AS WELL. SO JERRY FISHBATCH IS CURRENTLY THE CHIEF SCIENTIST OF THE FISH FOUNDATION. I DON'T WANT TO TAKE ANY TIME. >> I STEPPED OUT BECAUSE THERE'S A LOT MORE. >> I KNOW. >> BUT--[LAUGHTER] >> I WISH THEY WERE HERE IMU I CAN'T TELL YOU HOW THRILLING THIS IS TO COME AROUND THE SIDE OF THE BUILDING, WALK OUT THE FRONT AND SEE THE MAGNIFICENCE OF THE WHOLE BUILDING AND WHAT IT REPRESENTS. WE DID PLAN THIS AT ABOUT 1999, 2000 IT WAS A DREAM THEN AND A DREAM FOR A LONG TIME AND EVERYONE HERE, BOB, EVERYONE, WORKED VERY HARD TO MAKE THIS DREAM COME TRUE. AND THE DREAM WAS THAT NEUROSCIENCE IS ONE DISCIPLINE AND THAT PEOPLE FROM DIFFERENT INSTITUTES AND DIFFERENT WALKS OF LIFE WOULD COME TOGETHER IN ONE OPEN INTERACTIVE SPACE. I'VE ALWAYS THOUGHT--SOME OF MY HAPPIEST YEARS WERE AT THE NIH AS A SCIENTIST AND THEN AS A SCIENCE ADMEN--ADMINISTRATOR. AND I'VE ALWAYS FELT THAT AS THE NIH GOES, SO GOES THE REST OF THE COUNTRY AND THE REST OF THE WORLD AND I BELIEVE THIS BUILDING IS THE EPITOME OF THAT IDEA. WE WILL SUCCEED WITH SCIENCE IF THE NIH SUCCEEDS AND I HOPE FOR THE BEST IT'S A WONDERFUL PLACE TO BE AND WORK AND I THINK THE BEAUTY WILL INFLUENCE THE SCIENCE FOR A LONG, LONG TIME TO COME SO I'M VERY, VERY HAPPY TO BE HERE. THANKS. [ APPLAUSE ] >> SO JERRY DIDN'T REALLY EXPOUND ON THE VISION IN QUITE THE WAY I HAD ANTICIPATED, MAYBE THAT BECAUSE I DIDN'T TALK ABOUT ALL HIS HONORS. SO THE VISION THAT JERRY AND STEVE HAD WAS TO BRING TOGETHER INTRAMURAL SCIENTISTS, NEURAL SCIENTISTS FROM MULTIPLE INSTITUTES THAT WERE WORKING IN 10 DIFFERENT BUILDINGS ACROSS THE CAMPUS, BRING THEM TOGETHER IN ONE BUILDING AND THEN TO ARRANGE THEM NOT WITH A FLOOR FOR NINDS AND A FLOOR FOR NIMH AND A FLOOR FOR THE EYE INSTITUTE AND A FLOOR FOR THE OTHER INSTITUTES THAT ARE HERE BUT REALLY TO PUT THEM TOGETHER ACCORDING TO THE QUESTIONS THEY WERE ASKING AND THE TECHNIQUES AND TOOLS THEY WERE USING. IT WAS LIKE CREATING A NEUROSCIENCE COMMUNITY WALLET THEITANT ARD INSTITUTIONAL INSTITUTE BOUNDARIES OR IF YOU WERE IN AN ACADEMIC DEPARTMENT CENTER BOUNDARY. SO THE PORTER BUILDING, IT WAS PUTTING THE BRAIN BACK TOGETHER WAS ONE OF THE PHRASES I THINK THEY USED. SO THE PORTER BUILDING WHEN A RECRUITMENTS ARE COMPLETE WILL HOWES 85 INDEPENDENT LABORATORIES FROM 10 DIFFERENT INSTITUTES AND THEY ARE WORKING ON A VERY DIVERSE SET OF QUESTIONS. GENETIC UNDERPINNINGS OF BRAIN DISORDERS ON ION CHANNEL AND SYNAPSE STRUCTURE AND FUNCTION ON BRAIN CIRCUIT DYNAMICS AND SENSORY FUNCTION AND AUDITORY, CHEMICAL SENSES AND PAIN. AND OF COURSE, THE APPLICATION OF THE FINDINGS FROM THOSE STUDIES TO BRAIN DISORDERS. THE ADDITIONAL SPACE ONCE PORTER TWO, HAS BEEN COMPLETED WILL ALLOW, HAS ALLOWED THE DEAFNESS INSTITUTE PROGRAM TO RETURN TO CAMPUS AND WE'RE DELIGHTED TO HAVE YOU HERE AND I'M SURE YOU WILL CONTRIBUTE TO OUR SCIENCE, THE SCIENCE OF THE PEOPLE IN PORTER AND THEY WILL CONTRIBUTE TO YOURS. IT ALLOWED THE NCAM, NATIONAL CENTER FOR COMPLEMENTAR SCHEALTERNATIVE MEDICINE TO CREATE A PROGRAM ON PAIN AND A PROGRAM FROM NIDCR, THE CRANIOFACIAL INSTITUTE TO MOVE TO THE SENSORY AND SCIENCE FROM NINDS, NICDHD, AND NICHD, AND ALSO ALLOWED NICHD SCIENTISTS WHO HAD BEEN WORKING IN TRAILERS AT THE FAR END OF THE CAMPUS TO ACTUALLY MOVE INTO THE BUILDING. SO, SO IT'S ALLOWEDITOUS AGGREGATE SCIENTISTS FROM ACROSS THE CAMPUS. NOW, THE PORTER BUILDING SHOULD DO MORE THAN JUST HOUSE SCIENTISTS IT TURNS OUT WHILE THEY'RE 85 WITH STUDENTS AND FELLOWS IN IN BUILDING THAT'S LESS THAN HALF OF THE NEUROSCIE THAT WORK ON THIS CAMPUS, DEPENDING ON HOW YOU COUNT AND WHOSE SELF-IDENTIFIES AND WHO WE IDENTIFY, THEY'RE PROBABLY AT LEAST 200 INTRAMURAL SCIENTIST WHO IS WORK ON QUESTIONS RELATED TO BRAIN STRUCTURE AND FUNCTION AND THE PERIPHERAL NERVOUS SYSTEM. SO INVESTIGATORS USING PRIMATES AND DOG ANALYSIS OF COMPLEX CIRCUITS ACTUALLY ARE IN THIS BUILD THEY'RE IN BUILDING 49. THERE WAS A DIFFICULT DECISION THAT WAS MADE THAT THAT IF WE HOWESED PRIMATES IN THIS BUILDING WE WOULD LOSE THE INVESTMENT WE MADE IN 49 AND IT WOULD TAKE UP A LOT OF SPACE AND RESOURCES THOSE INCLUDE INVESTIGATORS FROM NIMH AND INVESTIGATORS AND NINDS AND INVESTIGATORS WORKING WITH PATIENTS AND USING IMAGING TOOLS AS THEIR PRIMARY RESOURCE ARE IN THE CLINICAL CENTER. THERE ARE ALSO SCIENTISTS WHO ARE AT OFFSITE, OFFCAMPUS SITES WHILE DEAFNESS WAS ABLE TO MOVE BACK, NIAAA ARE AT A TWIN BOOK AND NIDA AND NIA SCIENTISTS ARE IN BALTIMORE, WHAT WE HOPE IS THAT THIS BUILDING WILL ACTUALLY SERVE AS A FOCAL POINT FOR INTRAMURAL SCIENTISTS WHEREVER THEY ARE BECAUSE OF THIS CONFERENCE CENTER, BECAUSE OF THE WONDERFUL RESOURCES THAT ARE HERE, THOSE FLOATING CONFERENCE--CONFERENCE AREAS. AND THAT THE JOURNAL CLUBS AND INTEREST GROUPS WILL SERVE TO BRING EVERYB TOGETHER HERE. NOW THAT'S A REALLY BIG ASK. IT'S MUCH TOO EASY TO GO INTO YOUR LABORATORY OR AT LEAST IT USED TO BE AT LEAST WHEN THE LABORATORY WAS IN BUILDING 36. HOW MANY OF YOU REMEMBER BUILDING 36; YEAH, YEAH, IT WAS A BUNCH OF 200 SQUARE FOOT CUBICLES. SOME OF THEM WERE 4 SQUARE FEET AND AND HERE THE LABS ARE OPEN, THERE'S OPPORTUNITIES FOR MINGLING, ALONG THE ALONG THE BALCONIES AND IN THE OPEN SPACES AND SO WE HOPE THAT THAT KIND OF OPPORTUNITY TO COMMUNICATE WILL ACTUALLY ENRICH EVERYONE'S SCIENCE AND THERE ARE MANY EXAMPLES FOR HOW THAT HAS HAPPENED IN PORTER ONE. NOW THERE ARE A THOUSAND PEOPLE PROPROBABLY NEED TO BE THANKED FOR THIS AND IN FACT IT WOULD HAVE TAKEN THE WHOLE 30 MINUTE FIST WE HAD DONE THAT. JERRY AND STEVE FOR THE VISION, BOB AND I AND THE SCIENTISTS WHO WERE HERE WHEN PORTER ONE WAS BUILT. THOSE OF YOU WILL REALIZE THAT WE HAD TO TEAR DOWN BUILDING 36 AND THERE WERE MORE PEOPLE AND SCIENTISTS IN 36 THAN THERE WAS SPACE IN PORTER ONE. VERY COMPLITED SET OF DISCUSSIONS ABOUT WHAT WERE THE THEMES FOR PORTER ONE WHO MET THE THEMES, WHO WAS COLLEGIAL BUT THEN WITH A BUILDING OF PORTER TWORKS WE RECOGNIZED THAT SOME OF THE PRINCIPLES OF PORTER ONE LIKE YOU HAD TO HAVE SIX INSTITUTES REPRESENTED EACH POT PROBABLILY NEEDED TO BE MODIFIED. RITA WARD WHO HAS WORKED ON THIS PROJECT WHO HAS WORKED ON THIS PROJECT, NOW, ONE OF THE INTERESTING PIECES, THOSE OF YOU LOOKING AT THIS BUILDING NOW MAY NOT RECOGNIZE IT WAS BUILT--YOU RECOGNIZE IT WAS STILL TWO PHASES BUT THERE WERE TWO DIFFERENT ORPHTECTS, RAFAEL VINOLI, AND PERKINS AND WILLS FOR THE SECOND HALF AND AS YOU WALK OUT INTO THE ATRIUM, YOU CAN SEE THAT PERKIN SON WELLS DID A SPECTACULAR JOB OF ACTUALLY MARRYING THE TWO BUILDINGS. WE WERE FORTUNATE IN THAT THERE WAS ONLY ONE CONTRACT MANAGER FOR THIS. AND BECAUSE OF THAT, A LOT OF INTEGRATION WAS MUCH SIMPLER THAN IT OTHERWISE MIGHT HAVE BEEN. NOW I HAVE TALKED LONGER THAN I PLANNED BECAUSE I WAS ASSUMING S WOULD GET HERE. I THINK WHAT WE'LL DO IS GO AHEAD AND START WITH THE SCIENTIFIC PROGRAM AND I SHOULD SAY WHEN STEVE GETS HERE WE'LL INTERRUPT THE SCIENTIFIC PROGRAM TO ALLOW HIM TO SAY A FEW WORDS. SO THE NEXT PART OF THIS IS THE FIRST SCIENTIST SYMPOSIUM AND AND ONE OF THE THEMES IN THE PORTER ONE AND WAS REFLECT INDEED PORTER TWO AS WELL AND THAT'S THE NOTION OF CONSTRUCTING NEURON CIRCUITS. SO OUR FIRST SPEAKER IS DR. JOSH SANES, SITTING IN THE FRONT ROW LOOKING ANXIOUS, HE IS THE JEFF C. TARR PROFESSOR OF MOLECULAR AND CELLULAR BIOLOGY AT THE PAUL J. FINNEGAN DIRECTOR CENTER FOR BRAIN SCIENCE AT HARVARD UNIVERSITY WHERE JOSH RECEIVED HIS DOCTORATE. HIS WORK FOCUSES ON THE COMPLEX NEUROCIRCUITS AND ASSEM BLELLED IN ANIMALS AND HOW THEY PROCESS INFORMATION IN ADULTS AND HE AND HIS COLLEAGUE VS PIONEERED WONDERFUL WAYS TO MARKET NEURONS IN THE SYNAPSE SIS THEY FORM AND IT HIS WILL BE SYNAPSE FORMATION IN THE RETINA. [ APPLAUSE ] >> LUCKY WE GOT 15 EXTRA MINUTES. SO THIS IS THIS IS A POINTER THAT SAYS RENTAL NUMBER TWO. >> PROBABLY BEST TO HAVE A PLUG PLUG--TALK IN THE MICROPHONE. YOU CAN NEVER TELL. >> OKAY, THANK YOU. THANK YOU FOR BEING HERE AND THANKS STORY AND EVERYBODY FOR INVITING ME. I WANT TO START BY JUST--I'VE NEVER BEEN IN THE PORTER BUILDING BEFORE, IT'S GLORIOUS BUT MY CAREER LAS BEEN SUPPORTED ALMOST IN ITS ENTIRETYY BY NIH. SO I'M REALLY GRATEFUL TO HAVE A CHANCE TO BE HERE, SAN SALUTE THE PROGRAM AND TALK A LITTLE BIT ABOUT SOME OF THE WORK THAT NIH HAS SUPPORTED. PARTICULARLY FOR TODAY NINDS, STORIES EXCELLENT LEADERSHIP HAS BEEN PRECEDED BY A NUMBER OF OTHERS, JERRY, WHO YOU'VE JUST SEEN, ZACH HALL MY POST DOCTORAL MENTOR OVER THE ENTIRETYY OF MY CAREER AND I'M INCREDIBLY GRATEFUL THAT NINDS, LIKE THE REST OF NIH HAS STOOD FIRM IN SUPPORTING BASIC RESEARCH AND DONE ITS BEST AS MONEY DECREASES AND AS THE PRESSURES TO MOVE TO TRANSLATIONAL AND CLINICAL RESEARCH GET EVER STRONGER AND I JUST HOPE YOU CAN STAY STRONG AT LEAST UNTIL THE END OF MY CAREER AND HOPEFULLY LONG AFTER THAT. FOR MYSELF AS I SAID, ALMOST ALL OF THE RESEARCH THAT MY COLLEAGUES AND I HAVE DONE FOR MORE THAN 30 YEARS HAS BEEN SUPPORTED BY NINDS, AND I'M VERY GRATEFUL FOR THAT. AND I'M ALSO GRATEFUL TO HAVE HAD THE OPPORTUNITY TO GIVE BACK A LITTLE BIT BY BEING PART OF A NUMBER OF COMMITTEES STARTING WITH THE NOTORIOUS NEUROSEEDS STUDY SECTION THAT SOME OF YOU MAY REMEMBER AND GOING ALL THE WAY THROUGH TO THE INCREDIBLE INSPIRATIONAL WORKING GROUP TO THINK UP WHAT THE NEW BRAIN INITIATIVE SOME BE DOING IN THE YEARS TO COME. SO WITH THAT LET'S GET ON TO THE SHOW. THERE'S STEVE. DO YOU WANT TO? [LAUGHTER] >> YEAH. >> FRESH FROM THE AIRPLANE. >> YEAH, EXACTLY. >> FRESH FROM THE ICE. >> LET'S SAY A FEW WORDS ABOUT PORTER? >> YEAH, SURE. >> I CAN WARM YOU UP. JERRY SAID THREE SENTENCES. >> OKAY. I WILL-- >> OKAY. >> MY TWO ASK HALF MINUTES. >> NO, NO. [INDISCERNIBLE] >> ALL RIGHT, WHOO. >> THAT 6:00 A.M. FLIGHT I'VE TAKEN IT SO MANY TIMES BUT THIS MORNING WE HAD TO BE DEICED. IT'S REALLY GREAT TO BE HERE AND THIS IS A VISION THAT YOU ALL KNOW AND HAVE HEARD, STARTED A LONG TIME AGO AND I REALLY CREDIT JERRY AS THE MORE IMPORTANT PARTNE BECAUSE WHILE WE AGREED ON THE VISION. JERRY, AS USUAL WENT AND TALKED TO HAROLD VA--VARMIS, A FEW TIMES AND BY THE TIME HE HAD HIS MEETING, HE WAS ALREADY WON OVER BECAUSE OF HIS IRRESISTIBLE COMBINATION OF SCHOLALY DEPTH, WARMTH AND JUST A SLIGHTEST HINT OF MENACE. B OUR SHARED VISION FOR THE BUILDING, JERRY HAVING DONE ALL THE HEAVY LIFTING, WAS THE--NOT VERY DIFFICULT OBSERVATION TO MAKE THAT THE ORGANIZATION OF NEUROSCIENCE AT THE NIH WAS JUST ABSOLUTELY BIZARRE. AS A PSYCHIATRIST TRYST, I KNOW WE DO A VERY BAD JOB OF DIVIDING UP THE WORLD. WE HAVE THE DSM FIVE, WHICH STOPS ALL OF THE WRONG THINGS AND INTO MADE UP CATEGORIES BUT THE WAY THE NIH CLASSIFIED NEUROSCIENCE WAS NOT ACCORDING TO SOMEBODY'S INTELLECTUAL INTEREST, ACCORDING TO THE COLLABORATORS, THEY NEEDED, ACCORDING TO SHARED EQUIPMENT BUT RATHER ACCORDING TO THE INSTITUTE THAT HAD HIRED THEM AND THAT HAD PUT A TATTOO ON THEIR BODY. AND IT SEEMS, IT ACTUALLY SEEMED THAT IT WOULD BE A RATHER EASY CASE TO MAKE THAT PEOPLE SHOULD BE CLUSTERED NOT BY THE INSTITUTE THAT SIGNED THEIR PAYCHECK BUT RATHER BY THEIR INTELLECTUAL INTEREST AND BY FORMING A CRITICAL MASS, BY THE RATHER ODD IDEA THAT MAYBE WE COULD BUY ONE SHARED SOMETHING INSTEAD OF EIGHT OR NINE OF THEM. YOU KNOW AND I KNOW EVERYBODY IN THOSE DAYS NEEDED THEIR OWN ULTRA CENTRIFUGE EVEN IF THEY USED IT AS MUCH AS ONCE A MONTH BUT IT SEEMED LIKE A GOOD IDEA TO SHARE ALL THESE THINGS AND BRING THINGS TOGETHER. AS EASY AS IT WAS TO CONVINCE HAROLD, AGAIN, EASY FOR ME BECAUSE JERRY HAD ALREADY DONE IT, TURNED OUT TO BE A PRETTY TOUGH CELL WITH OUR FELLOW INSTITUTE DIRECTORS BECAUSE THEY THEY WERE JUST SUSPICIOUS OF THIS IDEA, I DON'T MEAN THIS, YOU KNOW IN A PARTICULARLY NASTY WAY, IT'S THE WAY BUSINESS HAD ALWAYS BEEN DONE BUT THEY WERE SUSPICIOUS THAT THING SHOULD BE DRIVEN BY SHARED SCIENTIFIC INTEREST RATHER THAN BY LOYALTY TO YOUR COUNTRY OR SINCEITUTE BUT OVER TIME WE--WE--THROUGH A SERIES OF MEETINGS, ENDLESS MEETINGS CONVINCED PEOPLE TO JOIN AND THEN IT'S REALLY RATHER REMARKABLE TO THINK WE MET WITH OUR DELIGHTFUL COLLEAGUES AT OMB A NUMBER OF TIMES AND THEY VISITED WITH US AND WE HAD A TRANSLATOR BETWEEN NEUROSCIENCE AND WHATEVER LANGUAGE THEY WERE SPEAKING AND JUST WHEN THINGS LOOKED PARTICULARLY GRIM DON WHO WAS THEN SECRETARY OF EXPHELGT HUMAN SERVICES WHO HAD BEEN A UNIVERSIT CHANCELLOR UNIVERSITY OF WISCONSIN, NOW MIAMI, SHE GOES FOR BIG SPORTS TEAMS MADE THIS BUILDING HER NUMBER ONE PRIORITY FOR OMB FOR THE YEAR AND SHE REALLY IS AN UNSUNG HERO IN THIS BECAUSE SHE CONVINCED THE BUDGET EARS THAT THIS NOT ONLY MADE ENORMO SENSE SCIENTIFICALLY BUT IT MADE GREAT SENSE FOR GOOD GOVERNMENT, THAT IS ACTUALLY COLLECTING PEOPLE ACCORDING TO THEIR COLLABORATIVE DESIRES AND POTENTIAL USE OF SHARED EQUIPMENT AND SO, THE BUILDING WAS AWHEN YOU'VED AND IT TURNED OUT ONLY HALF OF IT GOT PAID FOR AND AS I WAS LEAVING IN--LEAVING NIH, AGAIN FOLLOWING JERRY WHO HAD LEFT, I THINK A YEAR OR TWO BEFORE THE CONSTRUCTION WAS UNDERWAY BUT IT ENDED UP OBVIOUS LOW--OBVIOUSLY WITH THE UGLY SCAR THAT HAS BEEN REPAIRED TO CREATE THIS MARVELOUS BUILDING. NOW WHEN WE LEFT, WE KNEW WE LEFT THE BUILDING IN GOOD HANDS BECAUSE BOB DESMON, BUT ABOVE ALL STORY LANDIS CARRIED THIS IDEA THAT SCIENCE AND COLLABORATIVE SCIENCE AND INTERDISCIPLINARY NEUROSCIENCE, AFTERALL NEUROSCIENCE IS IN ITS VERY BIRTH WAS INTERDISCIPLINARY, PEOPLE BROUGHT TOGETHER FROM ANATOMY, PHYSIOLOGY AND BIOCHEMISTRY, AND LATER GENETICS AND PSYCHOLOGY IN MANY OTHER FIELDS, STORY I THINK REALLY DESERVES ENORMOUS CREDIT FOR CARRYING THIS THROUGH BECAUSE AGAIN WHILE IT MAKES PERFECT SENSE WHILE WE'RE SITTING HERE IN IF WONDERFUL BUILDING AND EVERYTHING I'VE HEARD ABOUT THE WAY THE FIRST HALF HAS WORKED BUT PEOPLE BEING TOGETHER HAS BEEN VERY POSSIVE I JUST CAN'T STRESS ENOUGH HOW ABSOLUTELY COUNTER INTUITIVE THIS CONCEPT WAS AND SO HERE'S ME AND JERRY AND BOB, WE'RE NOW ALL LOOKING IN FROM THE OUTSIDE AND STORY CONGRATULATIONS TO YOU BECAUSE IN MANY WAYS YOU'RE THE PERSON WHO BROUGHT THIS TO FRUITION. SO WITH THAT, LET ME TURN IT OVER TO JOSH WHO MUST HAVE TAKEN THE FLIGHT LAST NIGHT. AND OVER TO SCIENCE. [ APPLAUSE ] >> ALL RIGHT, SEE IF WE CAN GET BACK INTO ACTION HERE. >> OKAY, A FEW GLITCHES HERE. ALL RIGHT, SO, WE START AGAIN THANKS STEVE FOR GIVING ME A FEW EXTRA MINUTES TO COLLECT MY THOUGHTS SO THE ISSUE THAT'S CONCERNED MY COLLEAGUES AND I OVER THE LAST DECADE OR SO HAS BEEN THE SELECTIVE SYNAPSE FORMATION. I DID THIS, I WON'T DO IT AGAIN. AND THE QUESTION THAT'S SKETCHED HERE IS HOW ITY IT'SAXONS ARE CHOSING SYNAPTIC PARTNERS TO FORM SYNAPSES ON THE CORRECT ONES AND SOMETIMES EVENOT CORRECT PART OF THE CORREC ONES EITHER A DENDRITIC CELL REORGANIZATION OF THE INSTITUTE OR A STUDIES OF MULTIPLE ENDOCRINIA OR ANYTHING TO MAKE UP THE CIRCUITS THAT UNDERLIE MENTAL ACTIVITIES AND WE'VE CHOSEN TO ADDRESS THAT ISSUE USING THE RETINA SO I WILL SPEND A COUPLE OF MINUTES TELLING YOU ABOUT HOW THE RETINA LOOKS AND HOW IT WORKS. BY WAY OF BACK GROUPED GO ON AND TELL BUT A SINGLE STORY OF A WAY IN WHICH WE'VE TRIED TO GET INSIGHT INTO THIS QUESTION. SO EAR'S A SKETCH OF THE RETINA. IT COMPRISES THREE LAYERS OUTER LAYER ON PHOTORECEPTORS RODS AND CONES AND IN THE FIRST APROMULGATEMATION, LIGHT DETECTORS THAT EMIT AN ELECTRICAL SIGNAL WHEN LIGHT SHINES UPON THEM AND YOU ARE ALL 40 MEGAPIXEL CAMERAS BECAUSE EACH OF YOUR EYE HAS ABOUT 40 MILLION RODS AND CONES. NOW THE INNER LAYER OF CELLS. GANGLION CELL LAYER CONTAINS RETINAL GANGLION CELLS AND IT'S THEIR AXONS THAT CONVEY ALL OF THE INFORMATION TO THE BRAIN THAT YOU EVER HAVE ABOUT THE VISUAL WORLD. AND THE IMPORTANT FEATURE OF THE RETINAL GANGLION CELLS IS THAT THEY'RE NOT LIGHT DETECTORS, THEY DON'T RESPOND WELL TO FIELD FUNCTIONS FUSE LIGHT BUT RATHER FEATURE DETECTORS TUNED TO RESPONSE FEATURES SUCH AS AN EDGE OR LIGHT AN OBJECT MOVING AT A PARTICULAR DIRECTION AND AND THAT TRANSFORMATION LIGHT DEFECTOR COMES ABOUT FROM THE INTERPOST LAYER OF NEURONS, THEY RECEIVE SYNAPSE SIS FROM THE PHOTORECEPTORS, PROCESS THAT SIGNAL AND THEN FEED DID ON TO THE RETINAL GINGLIAISON AN CELLS AND THE REASON THAT WE CAN DEFECT FEATURES AND HAVE SUBTLE VISUAL ABILITIES AND THERE ARE MANY TYPES OF RETINAL GANGLION CELLS, 30 EACH SENSIT TO A PARTICULAR FEATURE AND THEY ARE FEDOT ORDER OF SOMETHING LIKE 70 TYPES OF INTERNEURONS, EACH OF WHICH IS CONNECTED TO ONLY A FEW OF THE RETINAL GANGLION CELL TYPES AND THE GANGLION CELLS IN TURN EACH RECEIVE SYNAPSE SIS FROM ONLY A SMALL SUBSET OF THE INTERNEURONS AND ALL OF THIS SYNAPSE FORMATION HAPPENS IN IN INCREDIBLY COMPLICATED NEUROPILL CALLED THE INNER FORM LAYER WHERE THE DENDRITES ON DENDRITES, ELECTRICAL SYNAPSE SIS, ANY KIND OF SYNAPSE ANYBODY EVER STUDY SIDE REPRESENT INDEED THE INNER FLEXUS FORM LAYER. SO THE UPSHOT OF THE SYSTEM IS WHAT THE RETINA DOES IS DECOMPOSE AN IMAGE INTO 30 PARALLEL REPRESENTATIONS THAT ARE FED TO THE BRAIN. ONE SENSITIVE TO MOTION IN A PARTICULAR DIRECTION, ONE TO EDGES, ONE TO SHAPES, SEVERAL TO COLOR AND SO FORTH. AND IT'S THE BRAINS JOB, AS YOU MAY HEAR LATER IN THE SYMPOSIUM TO RECONSTRUCT THESE IMPOVERISHED IMAGES BACK INTO A SINGLE ONE, IT'S ONLY THE ILLUSION YOU'VE SEEN THE WORLD AS IT IS. YOU'VE NEVER SEEN THE WORLD AS IT IS YOU'VE ONLY SEEN PROVEERISHED REPRESENTATIONS WHICH THE VAST MAJORITY OF REPRESENTATION HAS BEEN DEPLETED. SO OUR AIM IS TO FIGURE OUT HOW THE CIRCUITS ASSEMBLE THAT LEAD TO THE SELECTIVE DETECTION OF T SHIRT OR SO FEATURES. AND AND ONE CAN GERONTOLOGYSTS BIN TO THINK ABOUT DOING THIS, IT WOULD HAVE BEEN SCIENCE FICTION 10 OR 20 YEARS AGO, BUT NOW IN THE MOUSE AT LEAST THERE ARE GENETIC TOOLS AND REAGENTS THAT MAKE IT POSSIBLE AND ALSO A HUGE FOUNDATION OF INFORMATION THANKS TO THE INCREASING NUMBER OF PEOPLE WHO ARE STUDYING RETINAL CIRCUITRY AND FUNCTION AND SO WE CAN BEGIN TO THINK ABOUT A PROGRAM IN WHICH WE CATEGORIZE THE CELLS, THE 30 TYPES OF FEATURE DETECTORS AND THE SEIVET TYPES OF INTERNEURONS GAIN GENETIC ACCESS TO THEM SO WE CAN SEE THEM AS A DEVELOP ASK CAN MANIPULATE THEM IN VARIOUS WAYS. MAP THEIR CONNECTIVITY ACCIDENT THE CONNECTOME, THE CIRCUITS AND THEN BEGIN TO FIND CANDIDATE MEDIATORS OF SPECIFIC CONNECTIVITY IN THIS CASE, IN THE INTERPLEXA FORM LAYER, WHAT ARE MOLECULES THAT MIGHT SPECIFY SOME OF THOSE SYNAPSES EARLY IN DEVELOPMENT WITH A HUNDRED SORTS OF NEURITES COME TOGETHER AND MIX IT UP IN A VERY SMALL SPACE WHERE UPON OVER A PERIOD OF OF A WEEK OR SO THEY'RE GOING TO SORT OUT AND MAKE SPECIFIC CONNECTIONS AND THEN USE THE GENETIC AND OTHER METHODS THAT ARE NOW AVAILABLE TO TEST THOSE CANDIDATES AND SEE IF THERE'S SOME THAT ARE REALLY INVOLVED. SO THE STORY TODAY BEGINS IN STEP FOUR IDENTIFYING MEDEIATORRORS OF CONNECTIVITY AND THE MEDIATORS ARE ONES THAT WE'LL THINK AND YOU'LL SEE THIS AT THE END PLAY SOME ROLE IN AN INTERESTING FEATURE OF THE INNER FLEXA FORMULATOR WHICH IS THAT IT'S DIVIDED INTO A NUMBER OF SUBLAMINAE, I'VE DRONE FIVE BUT THERE ARE 10 OR SO AND EACH DENDRITE OF A GANGLION CELL AND EACH AXON OF AN INTERNEURON HAS ITS ARBOR CONFINED TO JUST ONE OR A FEW OF THOSE SUBLAMINAE AND THEREFORE THE SUBLAMMENER SPECIFICITY OF CONNECTION SYSTEM A MAJOR DETERMINANT OF SFICITY ALTOGETHER. SO HERE ARE THE CANDIDATES THAT WE WERE THINKING ABOUT. THEY'RE MEMBERS OF THE SUPER FAMILY WHICH IS ONE OF THE TWO MAJOR FAMILIES THAT HAVE BEEN IMPLICATE INDEED CELL-CELL RECOGNITION WITHIN THE NERVOUS SYSTEM, THE OTHER BEING THE IMMUNOGLOBULIN SUPERFAMILY AND A FEW YEARS OKAY WE STUDIED, I'M NOT GOING TO TALK ABOUT IT TODAY, ROLES OF A SET OF CAD HEREONS, IN SETTING OUT DENDRITIC MORPHOLOGY AND IN PARALENS LENS LENS LENS WE WERE LOOKING AT THE EXPRESSION OF THE CLASSICAL CADHERONS CALLED TYPE ONE AND TWO WHICH ARE A FAMILY OF 20 OR SO CLOSELY RELATED GENES THAT ENCODE RECOGNITION PROTEINS THAT HAVE BEEN IMPLICATE INDEED MANY ASPECTS OF DEVELOPMENT, NOT JUST IN THE NERVOUS SYSTEM, BUT IN FACT, THROUGHOUT THE BODY. IN VERTEBRATES AND INVERTEBRATES AS L. FOR A SCREEN FOR INSITU HYBRIDIZATION, AND USING HID BRIDEIDESSATION TO FIND OUT WHERE THESE 20 GENES ARE EXPRESSED ARENA GRADUATE STUDENT OF THE TIME FOUND THAT TWO OF THEM CAD HERON EIGHT AND NINE ARE EXPRESSED IN SUBSITS OF BIPOLAR CELLS AND EXPRESSED QUITE SELECTIVELY IN THOSE CELL TYPE ASKS IT WAS A STARTING POINT OF THE STORY I WANT TO TELL YOU ABOUT DONE BY TWO POST DOCTORAL FELLOWS SHIN GWAN AND ARE--ADMINISTRATIVE GINT O SWARNY, AND THEY WERE EXPRESSED IN BI HOLEAR CELLS AND THE REASON IS THAT THE BIPOLARS ARE TYPES OF INTERNEURONS THAT ARE THE BEST CLASS JUSTIFIED IN THE RETINA AND WORKED BY BEES LA AND OTHERS HAVE SHOWN THAT THERE ARE 12 TYPES, 10 SHOWN IN THE DIAGRAM AND HE MADE THE CASE THAT HE'S CLOSE TO A HUNDRED%. HE CAN COUNT UP THE TYPES HE FOUND AND HE GETS 97% OF BIPOLAR CELLS WHERE THERE ISN'T A LOT LEFT OVER. SO THAT GAVE US A START IN PLAYING THE ROLE AND WE COULD USE THE MARKET THAT THEY HAD COME UP WITH TO SHOW THAT CAD HERON EIGHT AND NINE ARE EXPRESSED IN BIPOLAR CELLS TYPE TWO ASK FIVE SELECTIVELY. SO, BASED ON THAT, WE FOUND SOME ADDITIONAL MARKERS OF THESE CELLS, AND WE USE THEM TO GAIN GENETIC ACCESS TO THESE CELLS BY GENERATING KNOCK-IN LINES IN WHICH THESE GENES DROVE EXPRESSION OF CREE RECOMBIN ACE SO WE COULD IMPLAINT IN TYPE TWO OR FIVE MARKETERS OR PROTEINS THAT WOULD ALTER THE FUNCTION OF THESE BI-POLAR CELLS AND YOU CAN SEE THEY WORK PRETTY LNOT PERFECTLY, BUT WHEN MADE IT TO THE PROPER REPORTER, WE CAN SEE THE THAT THE ONE LINE DOES IN FACT MARK THE TYPE TWO CELLS, KC NG FOUR AND THE TYPE FIVE CELLS AND WHAT WE SAW IMMEDIATELY WAS THAT THESE CELLS OVERLAPPED AND I'M NOT SURE IF YOU CAN SEE IT AND I'VE BEEN TOLD THAT THE BUILDING IS NOT AT A STATE YET WHERE WE CAN TURN DOWN THE LIGHTS BUT YOU MAY BE ABLE--OR IF WE TURN THEM DOWN WE WON'T BE ABLE TO TURN THEM BACK UP SO TH PROBABLY MORE IMPORTANT BUT I HOPE CAN YOU SEE THESE TWO RED STRIPES HERE, THOSE ARE THE DENDRITES OF WHAT CALLED STAR BURST AMKRONE CELLS AND THEY'RE THE COLONERGIC CELLS OF THE RETINA AND YOU CAN SEE THEY'RE HIGHLY LAMINATED AND THE REASON THAT WE FOUND THAT INCREEING TREEINGS--ENTREESING IS YOU CAN FIND THOSE OVERLAPPED WITH THE STAR BUST AMKRONE DENDRITES AND YOU SEE WHY WE'RE INTERESTED IN THESE DENDRITES THEY'RE WELL KNOWN TO BE PART OF A NEURAL CIRCUIT IN THE RETINAL LOCATION THAT WE KNOW SOMETHING ABOUT AND THAT IS A CIRCUIT THAT COMPUTES SELECTIVITY SO IN WAYS I DON'T HAVE TIME TO GO INTO, THE STAR BURST AMCRIP CELLS ARE THEMSELVES DIRECTION SELECTIVE AND THEY FEED THORS THESE CELLS, THEY'RE BI STRATIFIED AND THE TOP DENDRITIC CELL RIDE GETS INPUTS FROM THE TOP TAR BURST, BOTTOM FROM THE BOTTOM STAR BURST. THEY COMPUTE THE SIGNAL AND THEN THERE ARE FOUR TYPES OF DIRECTION SELECTIVE GANGLION CELLS EACH ONE TUNE TO MOTION IN A PARTICULAR DIRECTION, ONE NORTH, ONE SOUTH, ONE EAST AND ONE WEST. SO THE IDEA WAS THAT ANYONE WATCHES THESE CELLS, EXCITATORY TO THE CELLS BECAUSE THE BI POLES THEMSELVES ARE THE INTERNEURONS THAT RECEIVE THIS THAT RECEIVE INPUT FROM THE POTENT STATE O RECEPTORS, SO WE WANTED TO ASK WHETHER THEY REALLY DO MAKE THOSE SYNAPSE SIS AND WE CAN DO THAT AGAIN BECAUSE OF THE GENETIC TOOLS THAT ARE AVAILABLE IN MICE. WE HAVE DEVELOPED IN THE LAB, LINES THAT ALLOW US TO MARK THE STAR BURST AMKRONE CELLS TWO DIFFERENT CELLS OF THE ON OFF SELECTIVE CELLS AND NOW I'VE JUST TOLD YOU THAT WE HAVE LINES THAT ALLOW US TO MARK THESE TWO TYPES OF BIPOLAR CELLS THAT ARGUABLY PROVIDE THE INPUT TO THE CIRCUIT THAT PROCESS TO GIVE YOU SELECTION ACTIVITY. SO THEN WE SET UP AN OPTICAL IMAGES O GENETIC SYSTEM TO ALLOW HIM TO ASK WHETHER THESE PROVIDE THE INPUT OR IF THEY DO, WHETHER ALTERING CAD HERON EXPRESSION AFFECTS CONNECTIVITY. HERE'S HOW HE DID IT. HE TOOK A LINE THAT WE'VE MADE, ACTUALLY IN THIS CASE, ONE WE GOT FROM A COLLEAGUE THAT MARKS ONE SET OF DIRECTION SELECTIVE GANGLION CELLS WITH THE YELLOW FLUORESCENT PROTEIN AND HE MATED THAT TO A LINE IN WHICH THE TYPE TWO BIPOLARS EXPRESS CREE AND THEN HE INTRODUCED CHANNEL REDOBSIN BIPOLAR CHANNELS AND NEW USING IT TO STIMULATE A LARGE NUMBER OF BIPOLARS WHILE RECORDING FROM A SINGLE DIRECTION SELECTIVE RETINAL GANGLION CELL AND ASK WHETHER THERE'S CONNECTIVITY. AND THIS GIVES YOU AN EXAMPLE OF ONE OF HIS EXPERIMENTS WHERE HE CAN NOW TRAIN HIS LASER ON ONE BIPOLAR AFTER ANOTHER AROUND 200 IN THIS CASE ALL THE WHALE RECORD FREE RADICALS GENERATED A SINGLE DIRECTION SELECTIVE GANGLION CELL AND EVERY TIME HE STIMULATES HE GETS THE RESPONSE AND EACH LINE HERE INDICATES THE RESPONSE HE GOT WHEN STIMULATING THIS PARTICULAR BIPOLAR AND RECORDING FROM THIS GANGLION CELL AND YOU CAN SEE THERE'S CONNECTIVITY THAT'S QUITE SUBSTANTIAL NEARBY. AND IF YOU BLOW IT UP, YOU CAN SEE IN THIS CASE, IT'S INTERESTING, NO TIME TO TALK ABOUT IT, THAT THE BIPOLARS THAT ARE CONNECTED ALL END UP BEING OR MOSTLY END UP BEING ON ONE SIDE OF THIS PARTICULAR GANGLION CELL. WHAT THEY WERE ABLE TO DO IS SHOW THAT IN FACT, TYPE TWO AND FIVE PROVIDE EXCITEAT O ARE INPUT TO THE CELLS BUT THEY DO THEMSELVES MAKE INHIBITORY SYNAPSE SIS ON TO THE CELLS. SO NOW WITH THE CIRCUIT INHAND, W CAN ASK WHETHER THE ADHEREONS EIGHT AND NINE PLAY A ROLL IN ESTABLISHING IT. USING AN EIGHT AND NINE MOUSE THAT WE OBTAINED AND THE CAT NINE MUTANT THAT SHIN MADE. SO THE IDEA IS TO MARKET THESE CELLS USING THE LINES I DESCRIBED AND THEN ASK WHAT HAPPEN WHEN IS YOU GET RID OF CAD HEREON EIGHT, AND WHAT HAPPENS WHEN YOU GET RID OF CAD HEREON EIGHT TO THE CELLS THAT EXPRESS IT HALF OF THEM HAVE ARBOR THAT MOVE DOWN FROM THE NORMAL POSITION TO THE POSITION OVER LAPPING THE OTHER STEP OF STAR BURST AMCRIP CELLS. IF YOU GET RID OF THEM NINE THERE'S NO OBVIOUS EFFECT AND IF YOU QUANTIFY WHAT YOU CAN SEE THAT HALF OF THE CELLS ARE THE ARBORS DISPLAYED AND OTHER HALF DON'T AND I'M GOING TO COME BACK TO THAT PARTIAL PHENOTYPE LATE OR. AND THE SAME IS TRUE FOR CAD HEREON NINE AND THE BIPOLAR TYPE FIVE IF YOU GET RID OF CAD HEREON NINE, ABOUT HALF OF THEM HAVE THE ARBORS MOVE UP AND THE OTHER HALF STAY PUT AND NO EFFECT OF GETTING RID OF CAD HEREON EIGHT. SO RIGHT AWAY WE HAD PRETTY GOOD EVIDENCE THAT THE CAD HEREONS ARE AFFECTING THE LAMINAR POSITION OF THE BIPOLARAXONS OR THE BIPOLAR AXONS WITH THE EXPRESSED TWO CAD HEREONS SO AND AND IN THE NEXT EXPERIMENT I'LL TELL YOU ABOUT IS MAKE USE OF THESE LINES THAT GIVE US GENETIC ACCESS TO THE TYPE TWO AND FIVE BIPOLAR CELLS TO INTRODUCE CAD HERON EIGHT AND NINE TO ASK ARE THEY NOT ONLY NECESSARY FOR PROPER LAMINATION BUT COULD THEY BE SUFFICIENT TO SPECIFY LAMINATION AND WHAT HE FOUND IS IF THEY PUT THESE INTO THE BIPOLAR CELLS, THERE'S NO EFFECT OF FLOODING THEM BUT IF HE PUT IN CAD HERON NINE HE GOT THEIR RETURN OF RESULTS BORES TO MOVE--ARBORS TO MOVE DOWN AND THE CELLS THAT NORMALLY EXPRESS CAD HEREON NINE ARE AND TAKE NOTE I'LL COME BACK TO IT, IT'S A PRETTY COMPLETE EFFECT AND THE EFFECT WAS THE SAME BUT IN THE OPPOSITE DIRECTION FOR PUTTING CAD HEREON EIGHT INTO THE CELLS THAT NORMALLY EXPRESSED CAD HEREON NINE. SO HERE WE HAVE ALMOST A COMPLETE TRANSFORMATION AND THAT TELLS US THE CAD HEREONS ARE ACTING DIFFERENTIALLY. IT'S NOT JUST THAT EVERY CELL NEEDS A CAD HEREON AND ONE HAPPENS TO HAVE EIGHT AND ONE HAPPENS TO HAVE NINE BUT THEY'RE DOING SOMETHING DIFFERENT AND POSSIBLY EVEN ACTING TO INSTRUCT THE POSITION WHERE THE ARBOR SHOULD BE. THERE'S A WAY THAT THEY WORK AND THAT IS THAT THEY WORK HOME FILIBLY. AND THEY CAD HEREON BINDS TO ONE AND CAD HEREON TWO TO TWO AND SO FORTH AND THE AREA GOT COMPLICATED BUT BY AND LARGE IS THAT CAD HEREONS BIND TO OTHER CELLS OR MEDIATE ADHESION BE CELLS THAT EXPRESS THE SAME CAD HEREON SO WE TESTED THAT OBVIOUS IDEA AND THE WAY WE DID IT WAS TO PUT CAD HEREON EIGHT OR NINE INTO A FEW CELLS IN A BACKGROUND THAT WAS LACKING CAD HEREON EIGHT OR NINE. SO THE IDEA NOW IS THAT IF WE INSTALL CAD HEREON EIGHT INTO A CELL AND IT'S SURROUNDED BY CAD HEREON EIGHT NEGATIVE NEIGHBORS THEN BY THIS HOMOPHILIC IDEA IT SHOULD HAVE NO EFFECT AND THAT TURNS OUT TO NOT BE THE CASE. IT WAS A BIG SURPRISE. SO IF WE PUT CAD HEREON EIGHT OR NINE INTO A FEW CELLS THAT EXPRESSION WAS JUST AS EFFECTIVE WHEN ALL THE OTHER NEIGHBORS WERE CAD HEREON FREE AS WHEN THEY WERE WILD-TYPE SO THIS REMAINS A MYSTERY I SHOULD SAY ONE OF THE MYSTERIES WE FIND HOW THEY'RE WORKING WE IMAGINE THEY MUST BE RECOGNIZING SOMETHING ON THESE PARTNERS AND QUITE EXCITED TO WHAT IT COULD BE. SO WE ALSO WANT TO COME BACK AND WE AGAIN BEING SHIN HERE TO THIS ISSUE OF WHETHER THESE CAD HEREONS ARE REALLY ACTING INSTRUCTIVELY. I TRY TO SAY BEFORE, ON--ONE EXTREME YOU COULD IMAGINE THAT EVERY CELL JUST HAPPENS TO NEED A CAD HEREON AND ONE OF THEM HAPPENS TO HAVE CAT HEREON EIGHT AND ONE HAPPENS TO HAVE CAD HEREON NINE OR THE OTHER EXTREME YOU COULD IMAGINE THAT THEY REALLY ARE PROVIDING SOME SORT O POSITIONAL INFORMATION TO THESE CELLS WITH CAD HEREON EIGHT AND NINE PROVIDING DIFFERENT INFORMATION AND THE EXPERIMENT I TOLD YOU ABOUT A MINUTE AGO SUGGEST THAD THE LATERAL ALTERNATIVE IS THE CASE. BUT TO NAIL THAT WHAT SHIN DID WAS TO MAKE USE OF ANOTHER CELL WHICH WE GAINED GENETIC ACCESS IN THE LAB AND THIS WAS WORK LED BY JEREMY KAY, A FORMER POST DOCTORAL FELLOW WHO HAS HIS OWN LAB AT DUKE UNIVERSITY. IN STUDIES I WON'T GO INTO, HE HAD BEEN STUDYING A SET OF CELLS THAT CAN BE ACCESSED WITH A LINE CALLED NEXT CREE AND THE IMPORTANT THING FOR THIS PURPOSE IS THAT THOSE CELLS HAPPEN TO HAVE THE BULK OF THEIR ORBORE IN THE IN BETWEEN SPACE, IN BETWEEN THE OUTER STAR BURST AND INNER STAR BURST. SO WHAT THAT ALLOWED SHIN TO DO WAS TO PUT CAD HEREON EIGHT AND NINE INTO THESE CELLS WHICH NORMALLY EXPRESS NEITHER CAD HEREON EIGHT NOR CAD HEREON NINE AND THE RESULT WAS THAT IN THIS COMPLETELY ECTOPIC SITUATION EXPRESSING CAD HEREON EIGHT COULD MOVE THE ARBORS UP TO THE CAD HEREON EIGHT PLACE AND EXPRESSING CAD HEREON NINE COULD MOVE THE ARBORS DOWN TO THE NINE PLACE SO WE BELIEVE THAT I DO INARE INSTRUCTIVE AND THIS HAPPENS BY SOME MYSTERIOUS HETEROGENEOUS ROW FEELIC MECHANISM. SOY WHAT DOES THIS MEAN FOR THE SURGENT? SO WE COME BACK TO ASK WHAT HAPPENS TO THE SYNAPSE SIS THAT ARE MADE BY THE BY POLARS THAT ARE MOVED TO A DIFFERENT PLACE BY EITHER REMOVING THE PROPER CAD HEREON OR ADDING AN IMPROPER CAD HEREON AND YOU CAN DO THAT USING THE OPTICAL IMAGES O GENETIC SYSTEM THAT I ALREADY DESCRIBED HE CAN STIMULATE AS YOU RECALL THE TYPE TWO OR FIVE BIPOLAR CELLS, RECORD FROM ON OFF SELECTION SELECTIVE GANGLION CELL AND IF YOU RECALL WHEN WE GET RID OF IT, HALF OF THE THE ARBORS MOVE TO ANOTHER PLACE, SOME STAY IN THE SAME PLACE. THIS IS DATA FROM THE EXPERIMENT I SHOWED YOU BEFORE BUT NOW PLOTTED SO YOU LOOK AT EACH ONE HALF OF THE TIME AND HALF OF THEM ARE FINE AND HALF MOVE TO THE OTHER PLACE AND YOU MIGHT E THAT THEY WOULD BE PERFECTLY HAPPY TO MAKE SYNAPSES ON THE DENDROID OF THE VERY SAME CELL THAT THEY'RE MAKING SYNAPSES ON ON THE FIRST PLACE AND SURPRISINGLY THAT'S NOT THE CASE SO HERE'S ANOTHER SURPRISE WHEN WE GET RID OF THE SYNAPSES IS REDUCED BY 80% AND THE SAME IS TRUE IN THE OTHER DIRECTION SO THIS LEADS US TO THE COMPLICATED IDEA THAT THE CAD HEREONS MAY BE DOING TWO DIFFERENT THINGS FOR THIS CIRCUIT SO HAY MAY BE DIRECTING TO THE PROPER LAMINA AND THAT'S INSTRUCTIVE MECHANISM BUT THEY MAY BE BUT THEY MAY BE ALSO NECESSARY TO MATURE TO THE POINT WHERE THEY CAN TRANSMIT INFORMATION AND BE FULLY FUNCTIONAL AND AGAIN THAT'S SOMETHING WE'RE TRYING TO WORK OUT. SO AT THIS POINT, THEN WHAT WE HAVE IS A VERY HYPOTHETICAL MODEL. I WOULDN PUT A LOT OF MONOTHIS AND IT'S THE WAY WE'RE THINK BEING IT AND IT'S THE MODEL THAT'S GIDDING THE EXPERIMENTS THAT SHIN AND ARE--ADMINISTRATIVE GENERA ARE NOW DOG AND THE IDEA IS AS FOLLOWS AND WHAT WE SINGLE THAT THE INNER FLEXA FORM LAYER AT SOME EARLY STAGE GETS TO BE DIVIDEDDED INTO DOMAINS. SO WHAT THEY DO IS LEAD THE BIPOLARS TO CHOOSE FROM TWO POSSIBLE PERMISSIVE SITES OR TWO POSSIBLE ATTRACTIVE SITES. A THE REASON FOR THINKING THAT IS THAT WHEN WE GET RID OF CAD HEREON EIGHT OR NINE, WHAT WE SEE IS THAT THE ARBORS DON'T GO EVERYWHERE, THEY DON'T FEEL THE INNER FLEXA FORM LAYER, THEY DIVIDE EVENLY BETWEEN THESE TWO SEATS. SO, SO CAD HEREON NINE IS NOT GIVING THEM HIGHLY PATTERNED POSITIONAL INFORMATION BUT RATHER MAKING THEM EXECUTE A CHOICE BETWEEN THOSE TWO SITES? THE REASON WE THINK IT'S MAKING THEM MAKE THE CHOICE IS BECAUSE OF THE ECTOPIC EXPRESSION. THAT IS WHEN WE FLOOD A CELL WITH CAD HEREON EIGHT OR NINE, IT THEN CHOOSES WITH GREAT FIDELITY OF THESE TWO PARTICULAR SITES. THEN OF COURSE THESE AND SYNAPSING ON A NUMBER OF THE DENDRITES IN THE LAMINA WHERE THERE ARBORIZING AND WE DON'T REALLY KNOW WHAT'S GOING ON THERE BUT IN OTHER WORK I DON'T HAVE TIME TO TELL YOU ABOUT, WE FOUND THAT MEMBERS OF THE OTHER FAMILY OF RECOGNITION MOLECULES IMMUNOGLOBULIN SUPER FAMILY, GENES LIKE THE SIDE KICKS, DS-CAMS AND CONTACT ANTS SEEM TO BE CAPABLE OF MEDIATING INTRA LAMINAR CHOICES. SO LAST OF ALL WHAT DOES THIS DO FOR FEATURE DETECTION, WHICH IS WHERE WE STARTED AND WE'VE ONLY BEGUN HERE AND IN FACT WHEN ARE--ADMINISTRATIVE GENERATED NOW RECORDS FROM THE DIRECTION SELECTIVE CELLS AND MAPS SELECTIVITY, HE'S ABLE TO REPLICATE RESULTS THAT OTHERS FOUND, THAT IS THESE CELLS RESPOND BOTH WITH A DARK EDGE ENTERS THE FIELD, OFFRESPONSE AND DARK EDGE LEAVES THE FIELD ON RESPONSE AND THEY RESPOND IN THIS PARTICULAR CASE AND THIS LINE TO OBJECTS MOVING IN A VENTRAL DIRECTION. WE ALREADY KNOW THAT THESE TWO TYPES OF BIPOLAR CELLS ARE FEEDING THE CIRCUIT AND IN FACT WHEN WE GET RID OF CAD HEREON EIGHT OR NINE, WE WE CAN OBLIGATIONS BLAT THE MUTE ANT AND WHAT WE SEE WHETHER SURPRISING OR NOT IS THAT THERE'S VERY LITTLE URNT ACTION B THE TWO, IT LOOKS AS IF CAD HEREON EIGHT IS SPECIFYING OFFPATHWAY, CAD HEREON NINE IS SPECIFYING ONPATHWAY AND THIS GANGLION CELL IS PERFORMING AN EXTRA LEVEL OF GENERALIZATION THAT IS, IT'S ALREADY RESPONSIVE TO OBJECTS MOVING VENTRALLY AND NOW IT COMES TO BE ABLE TO GENERALIZE THAT TO DO IT FOR DARK OR LIGHT OBJECTS MOVING DOWN THANKS TO THESE PATHWAYS THATAD LINEARLY MORE OR LESS TO GENERATE THE FINAL RESPONSE. NOW WHAT WE WANT TO DO AS YOU MIGHT WELL IMAGINE IS TO GO ON TO THE NEXT STAGE AND ASK WHAT THE MOUSE THINKALL THIS TO SEE IF WE CAN DO SOME BEHAVIORIAL STUDIES IN MICE CONTRARY TO POPULAR OPINION ARE NOT BLIND. THEY HAVE A LOT OF VISUALLY GUIDED BEHAVIOR AND SO THE DIRECTION WE'RE GOING NOW IS TO ASK WHETHER--WHEN WEB CONNECTED GET RID OF SOME OF THESE POTENTIAL RECOGNITION MOLECULES CAN WE TEASE OUT ANY PARTICULAR BEHAVIORIAL DEFECTS THAT WOULD GIVE US A SENSE THAT WHAT WE THINK IS A FEATURE DETECTOR IN THE MOUSES RETINA IS SOMETHING THE MOUSE ALSO THINKS IS A FEATURE DETECTOR. SO WITH THAT I LINING TO THANK EVERYONE ON THIS PROJECT. THANK YOU VERY MUCH [ APPLAUSE ] SO IT WAS A GOOD THEN YOU SAW THE RESULTS THAT GOES AGAINST THE [INDISCERNIBLE] AND SUGGESTS A HETEROFELLIC INTERACTION, I WAS WONDERING IF THERE'S ALTERNATIVE HYPOTHESIS THAT THE FACT THAT A ISOLATED CELL EXPRESSING THE APPROPRIATE CAD HEREON CAN FIX THE PROBLEM, SUGGESTED THAT THERE MAY BE A C AUTONOMOUS OR CAD HEREONS SOMEHOW MEDIATING AN INTERACTION THAT HAPPENS WITHIN THE SAME CELL? AH, WE--WE DON'T KNOW. AND WE WILL SAY FORMALLY WE HAVE NO WAY OF RULING THAT OUT. SINCE THEY'RE GOING TO A PARTICULAR PLACE, WE IMAGINE THERE MUST BE RECOGNIZING SOMETHING IN THAT PLACE, THEY CAN DIRECT A MOLECULE RECOGNIZING SOMETHING THERE AND I COULD FIGURE OUT A WAY IT CAN HAPPEN BUT I CAN'T RULE IT OUT. >> ANY OTHER QUESTIONS? SO OUR NEXT SPEAKER AND DR. CHRISTOPHER Mc BANE, HE IS ININVESTIGATING DEVELOPMENT OF HIPPOCAMPAL NEURON CIRCUITS IN WHICH THEY ARE EMBEDDED. HE AND HIS COLLEAGUE VS PIONEERED NEW WAYS TO MARKET AND NEURONS AND HIS TALK, I'M SORRY. HE WILL DISCUS FIND, AND OF THE DEVELOPMENT AND MATURATION OF CORTICALE FEEDS, FORWARD INHIBITORY PATHWAYS. THANK YOU. >> WELL FIGHTER OF ALL LET ME THANK EVERYBODY FOR BEING HERE AND ALL THE ORGANIZERS FOR GIVING ME THE OPPORTUNITY TO SPEAK, IT'S A REAL PLEASURE TO AND AN HONOR TO REPRESENT THE CHILD HEALTH INSTITUTE IN PORTER AND CHILD EXPHELGT A GOOD PRESENCE HERE AND WE REALLY, REALLY ENJOY THE MANY INTERACTIONS WE HAVE WITH MANY SCIENTISTS FROM DIFFERENT INSTITUTES I CAN'T EXPRESS THAT ENOUGH. SO WHAT I'M GOING TO TALK ABOUT GIVEN THAT I'VE HAD ABOUT FOUR E-MAILS IN THE LAST 24 HOURS THAT I HAVE TO KEEP IT TO 18 MINUTES SO I WILL TALK A LITTLE BIT ABOUT THE MORE RECENT WORK UNPUBLISHED ON THE LAB AND SORT OF EXPLORING HOW THE HIPPOCAMPAL FORWARD INHIBITORY CIRCUIT IS MATURING AND WHEN I HAVE A TOLD, I FEND TO SPEAK WITH A SCOTTISH ACCENT SO IF YOU CAN'T UNDERSTAND ME I APOLOGIZE FOR THAT. SO AS EVERYBODY KNOWS HERE, IT'S ABOUT 80 BILLION NEURONS ARE GENERATED DURING HUMAN DEVELOPMENT AND NOT ONLY ARE THESE GENERATED BUT THEY'RE BEING INSTRUCTED, MIGRATING AND FORMING INTO THE PATHWAYS, NASCENT PATHWAYS THAT WILL SERVE THE HUMAN BRAIN FOR ITS ENTIRE LIFE AND WE'RE ALL AWARE AND CERTAINLY FROM JOSH'S A BEAUTIFUL TALK, THAT THE RULES THAT INSTRUCT THESE NEURONS AND CONNECTING TO EACH OTHER AND ELIMINATING CO NECKSS FROM EACH OTHER IS REALLY A CENTRAL GOAL OF THE NEUROSCIENCES. SO SO SO MUCH TO SAY THAT 90% OF A CHILD A CRITICAL BRAIN DEVELOPMENT HAPPENS BY AGE FIVE AND THERE ARE MANY PROTURBAATIONS THAT CAN HAPPEN ALONG THE WAY THAT CAN DEVELOP NEURONAL DEVELOPMENT BUT MATURATION THAT UNDERLIE A VARIETY OF MENTAL HEALTH DISORDERS THAT WE'RE CURRENTLY INTERESTED IN AND SOME OF THOSE ARE CATASTROPHIC AND ANOTHER PROTOBATION WILL HAVE RAMIFICATIONS DURING LIFE DEVELOPMENT BUT LATE OR IN LIFE AND PART OF THE GOAL OF MY LAB IS TO TRY AND UNDERSTAND THE CERTAIN RHEUM RULES THAT WOULD UNDERPIN CORTICALE AND HIPPOCAMPAL INFORMATION AND JUST TO PILOT THAT INTEREST IN THE CORTEX AND HIPPOCAMPUS IS A SLIDE I'VE TAKEN FROM THE PAPERS IT SEEMS TO UNDERSCORE BEAUTIFUL SIDEWALK IT TAKES FROM THE HYPOCAMPUS, IT'S BEEN THE WORK HORSE PREPARATION FOR PHYSIOLOGY FOR THE LAST 50 YEARS OR MORE AND OF COURSE MY LAB HAS SPENT THE LAST 20 YEARS LOOKING AT CIRCUITRY WITHIN THE CAMPUS AND WE'RE INTERESTED NOT IN GLUTEA MINERGIC AND LONG RANGING INTERNEURON THAT ARE SUBSERVED AND GABAMINERGICKIC AND ALTHOUGH THEY REPRESENT ONLY ABOUT APPROXIMATELY OF THE 10% OF ENTIRE CELL POPULATION THEY'RE DIVERSE POPULATION AND AT THIS TIME IN THE HIPPOCAMPUS ALONE, THERE ARE AT LEAST 25 DISTINCT POPULATIONS ALL OF WHICH CAN BE ASSIGNED A CERTAIN TASK, WE'RE ALL AWARE OF THIS BASKET SALE WHICH IS CRITICAL FOR PACING CIRC CUTRY OSCILLATIONS AND THEY DO SOMETHING DIFFERENT FOR ALL IT INHIBITS THE CALCIUM DEPENDENT FIRST THING AND TARGET NEURONS. SOY ALTHOUGH A LOT IS UNDERSTOOD AND APPRECIATE OF THE DEVELOPMENT OF GLUTEA MINERGIC IN THE CORTEX, STLE KNOWN ABOUT HOW INNER NEURONSONS AND DOVETAIL INTO THIS AND INTERNEURONS DEVELOP IN ITSELF AND THAT'S BECAUSE OF MARKERS WE USE AND OTHER CALCIUM NEUROPEPTIDE MARKERS ARE NOT MATURE UNTIL SEVERAL WEEKS POST NATALL AND IT'S BEEN HARD TO FOLLOW WHAT THESE CELLS ARE DOING EARLY ON IN EMBRYONIC AS WELL AS EARLY POST NATAL DEVELOPMENT AND THAT AS RECENTLY BEEN--WE'VE BEEN HELPED BY MANY, MANY LABS ENCLUEDING STEW ANDERSON AND THE LIKE WHO GENERATE A LARGE NUMBER OF MOUSE LINES AND EXPLOITED TRANSCRIPTION FACTORS AND EARLY DEVELOPMENTAL MARKERS THAT HAVE REALLY, REALLY DRIVEN THIS FIELD RAPIDLY FORD IN THE LAST TWO OR E YEARS AND WE TOOK ADVANTAGE OF A NUMBER OF THESE LINES AND THEY TAKE ADVANTAGE OF THIS IDEA THAT THE INNER NEURONS ARE GENERATE INDEED LARGE NUMBERS FROM THE VENTRAL TAIL INSEVERE ENCEPHALON IN THE GANGLIONIC AND UNLIKE PATHWAY GIVES RAMTAL CELL WHICH IS ARE GENERATED LOCALLY THEY HAVE TO MIGRATE OVER EXTRAORDINARY LARGE AND DISTANCES BEFORE THEY REACH THE CORTEX AND THE HIPPO HIPPOCAMPUS SO A LOT OF WORK HAS BEEN SPENT TRYING TO WORK OUT WHO'S COMING FROM WHERE IN TERMS OF MAPPING ON TO THE CORTEX BUT SURPRISINGLY ABSOLUTELY NOTHING WAS KNOWN ABOUT HOW THESE INNER NEURONS GET INTO THE HIPPOCAMPUS. SO I'M GOING TO SHOW YOU A COUPLE STORIES WE WORKED ON RECENTLY. AND THESE HAVE TAKEN ADVANTAGE OF THESE TWO MOUSE LINES, ONE IS A MOUSE LINE WHICH LABELED CELLS DERIVED FROM THE EXPEFDZ A TRANSCRIPTION FACTOR NTX 2.1 PART OF THE LECHECK SIX LINEAGE AND FOR TWO OF THIS MOUSE WHICH IS LABELED CG, IT'S A KNOCK-IN MOUSE AS WELL AS FIVE HT MOUSE THAT WE SURPLANT WIDE THE GAT 65 AND THIS SHOWS THAT CELLS ARE BORN AS EARLY AS E13 AND 14 THEY'RE GENERATED HUGE NUMBERS OF HIPPOCAMPUSS BARELY IN EXISTENCE HERE IF YOU FOLLOW THESE CELLS THEY MIGRATE, EXTRAORDINARILY NUMBERS ACROSS CONSIDERABLY DISTANCES OF THE NG AND THEN THE POPULATE INTO THE HIPPOCAMPUS AND RADIATE INTO THE FINAL POSITION SO THAT BY POST NATAL LIFE THEY TAKE UP POSITIONS THAT WE KNOW AND HAVE STUDIED FOR A FEW DECK'SADES. --DECADES. NOW THESE ARE GENERATED EARLY ON WITH LIFE AND PUTTING BACK OF THE NUMBER OF THESE CELLS SO WE'RE VERY INTERESTED IN MY LAB TO WORK AT ONE OF THE INSTRUCTIVE RULES THAT, LOW FOR INCORPORATION OF THESE CELLS BUT BEFORE WE COULD DO THAT WE NEED TO SORT OF CLASSIFY WHAT CELL TYPES WERE BORN FROM THE NG AND WHICH TYPES ARE BORN FROM THE CG. MY LAB IS PRIMARILY AN EIOLOGICAL LAB AND WE TOOK ADVANTAGE OF THOSE SKILLS AND COMBINED THEM WITH A WHOLE ARRAY OF TECHNIES AND WE CAN DO WHOLESALE PATCH CLAMP FREE RADICALS GENERATED THEM AND WE CAN PUT AGENTS INTO THERE THAT ALLOWS THEM TO INITIATE THE RTPC R REACTION AND WE CAN RECOVER THE CELL AFTER WE FINISHED OUR RECORDING AND DO ALL SORTS OF ANATOMICAL RECONSTRUCTIONS COMBINEDDED WITH THE IMMUNOHISTOCHEMISTRY SO THIS SORT OF MULTIPARAMETER APPROACH AS SERVED US VERY WELL IN IDENTIFY WAG SELL IS COMING FROM WHICH OF THESE OF THE GINGLIONIC, AND I WILL SHOW YOU AN EXAMPLE THAT'S A MOLECULARITY CELL AND A CELL BODY AND LIES IN THE PARALLEL TO THE PRINCIPLE CELL LAYER AND IT SENDSAXON ACROSS A CONSIDERABLE DIFFERENCE PROBABLY TO BASE OR INTERIVATE THE PATHWAY GIVES RAM TELECELLS AND THE FROM THE INTER ROANAL CORTEX SO THIS CELLS FIRES WITH A REGULAR SPIKING PATTERN IF YOU DREW A FAITHFUL ANALYSIS YOU SEE THERE'S LITTLE DECKRIMENT IN THE CELL AND THIS IS VERY TYPICAL ELECTROPHYSIOLOGICAL HALLMARK OF THE CELL WITH THE SACK THAT IS INDICATIVE OF THE [INDISCERNIBLE] IN MY LAB MANY YEARS AGO. TOGETHER WITH THE MULTIPLAYED RTPC R, YOU CAN SEE THE EXPRESS OF GAS FIVE AND 67, CAN YOU SEE THE INTERNEURON AND IT EXPRESSES THE STAT INAND A VARIETY OF OTHER TRANSCRIPTION FACTOR ASKS BY TAKES THIS APPROACH, ALMOST EVERYBODY IN MY LAB WHO WAS INVOLVED IN IN PROJECT ALLOWED US TO BUILD UP A REPRESENTATIVE LIBRARY OF ALL THE CELLS COMES FROM THE NG AND SO BY DOING THE--IN SEVERAL HUNDRED RECORDINGS, YOU CAN SEE THAT WE CAN ALMOST CAPTURE ALL THE CELLS WE KNOW AND LOVE ON A VARIETY OF ANATOMICAL VARIETIES IN THE LAST FEW DAYS. THE BASKET CELL, STRATACELL, SO ON AND SO FORTH. IT'S EXTRAORDINARILY A RICH WAY OF COLLECTING DATA AIAN REALLY--I WILL COLLAPSE A THREE YEAR PROJECT INTO THIS ONE SLIDE THAT ALLOWED US TO COLLECT THESE CELLS BEGIN TO UNDERSTAND THE DEVELOPMENTAL MECHANISMS THAT GET THEM OUT OF THE GANGLIONIC INSTANCES INTO THE HIPPOCAMPUS AND HOW THEY HOOK UP INTO THE NASCENT CIRCUITS INSIDE THE HIPPOCAMPUS. SO AS I SAID TO CUT A VERY LONG STORY SHORT IT TURNS OUT THAT THE MEDIAL GANGLIONIC EVIDENCE IS GIVING RISE TO SOPHISTICATED MATEOSTATIN AND THE CELL POPULATION WHICH IS MW WHITE OR POPULATION AS WELL AS ONE FORM OF NEUROGLIAL FORM CELL AND THE CG GIVES RISE TO ALL THE OTHER CELLS, PRIMARILY CCCAIN AND THE VIP CELLS WHICH RECEIVE AN EXTRAORDINARY AMOUNT OF ATTENTION IN THE LAST YEAR OR SO. SO MUCH OF THESE RULES ARE SHARED BY INNER NEURONS THAT MIGRATE INTO THE CORTEX BUT MUCH TO OUR SURPRISE THERE ARE MANY DEVIATIONS FOR THE RULES THAT ARE SHOWN BY MANY OTHER LABS AND IT SORT OF SERVES TO UNDERSCORE THAT, THE DEVIL REALLY IS IN THE DETAILS AND TO GET DOWN TO OUR UNDERSTANDING OF THESE CIRCUITS YOU NEED TO STUDY EACH OF THESE INDIVIDUAL INTERNEURON TYPES ONE IS AT A TIME. SO ONE STUDY WE'VE DONE RECENTLY, AND THAT ISOT BASKET CELL, THE BARKET CELL IS PROBABLY AND FOR REASONS THAT ARE REALLY NOT CLEAR, PROBABLY THE MOST WIDELY STUDIED INTERNEURON SITE THAT HAS A CONSISTENT AND OBVIOUS ARCHITECTURE AND HAS A DENDRITE THAT IS AT 90-DEGREES AND IT WRAPS ON THE SOPHISTICATED MAT O OR PATHWAY GIVES RAMTAL CELLS, HENS THE TERM THE BASKET CELL, AND ANYWHERE BETWEEN HALF A DOZEN TO A DOZEN INPUTS ON A SINGLE PATHWAY GIVES RAMTAL CELL AND PROVIDES EXWIZZIT INHIBITORY CONTROL OVER THE TIMES OF INTERNEURAL AND AXONAL FIRING AND PATHWAY GIVES RAMTAL CELLS IF YOU LOOK AT THE SAME PLOT ANALYSIS ON THE PREVIOUS SLIDE THIS, IS A CELL THAT'S EXTRAORDINARILY HIGH FREQUENCY FIRING AND HAS ALL THE MARKERS YOU WOULD TYPICALLY ASSOCIATE WITH THE BASKET CELLS. AND DEVELOPMENTALLY A LOT IS GOING ON IN THE LIFE OF A CELL EARLY ON IN THE FIRST WEEK OR TWO AND THERE ARE KEY PROTEINS THAT REALLY NEED TO BE UPREGULATED AND THOSE INCLUDE THE SELF-AND IT'S ABSENT IN THE BASKET CELL AND THERE'S A HOST OF SODIUM AND POTASSIUM CONDUCTANCES THAT WILL ENTHE CELL WITH THE FIRING FREQUENCY AND FROM OUR POINT OF VIEW, THE MOST IMPORTANT IS THE ACQUISITION AND IN PARTICULAR GLUTEA MATE RECEPTOR SUBTYPES AND IN PARTICULAR I WILL SHOW YOU THE NEXT FEW SLIDES IS THE ACQUISITION OF THE FOUR SUBUNIT THAT IMPARTS A RAPID FORM OF SYNAPTIC TRANSMISSION ON THESE CELLS, IT'S KEY IN ESTABLISHING THE CELL IN ITS CIRCUITRY AND AT THE SAME TIME THERE'S SYNAPTIC RELEASE OF THE MACHINERY WHICH IS MATURING WHICH IS INVOLVED IN DRIVING THESE CELLS AS WELL AS THE AFFERENT OUTPUT OF THESE CELLS WHICH ARE ESSENTIAL AND KIND OF UNDERSCORING THE MATURE PHENOTYPE. AND JUST TO KIND OF GIVE YOU A HINT OF WHAT THE CELL DOES ONCE IT'S REACHED MATURITY, YOU SAW A B FLASH OF LIGHT THERE AND THAT'S' APPLIED FOR THE INNER NEURON AND YOU SEE THE LOOK OF THE POTENTIAL OF THE PATHWAY GIVES RAMTAL SALE THERE AND YOU SEE THE FIRING AT THE FREQUENCY AND IT SHOWS THE FEES OF THE OSCILLATION AND THESE NEURONS ARE VERY, VERY, IMPORTANT IN PACING THE GAMMA OSCILLATION AS WELL AS OTHER TYPES INSIDE THE CORTEX AND SO IT'S NOT UNTIL T CELL ACQUIRES ALL OF THESE MATURE PHENOTYPES THAT THIS HAS THIS ADEQUATE MACHINERY THAT ALLOWS TOXIC EFFECTS PACE ADEQUATELY SO CAN YOU IMAGINE THAT ANYTHING THAT PERTURB THANKSGIVING FINAL MATURATION WILL HAVE AN IMPACT ON HOW THE CELL BEHAVES AND THE CIRCUIT. SO AS I SAID THEY'RE EXPRESSED ON MANY, MANY DIFFERENT TYPES OF NEURONS AND AROUND THIS TYPE THAT WE GOT INTO COLLABORATION WITH PAUL WHORLY WHO WAS INTERESTED IN MANYIARYS ON ROUGH ATOM--MANY, MANY YEARS ON THE NEURONNAAL PEN TRACT ANTS IT WAS INVOLVED IN CLUSTERING GLUTEA MATE RECEPTORS AND OF COURSE AS SOON AS THEY REALIZE TODAY WAS CLUSTERING THE NEWONS IT SHRUGGED AND WENT OFF SOMEWHERE ELSE AND SAID WE HAVE TO GET BACK TO PATHWAY GIVES RAMTAL CELLS AND WAY MORE INTERESTING IN THE NEURONS SO THIS IS WHERE MY LAB CAME IN AND PAUL APPROACHED US TO--TO TRY AND GET US TO WORK OUT WHAT THE ROLE OF THESE NEURONAL PEN TRACTIONS WERE IN THE NEURAL SITES AND IT TURNS OUT THERE ARE KEY FEATURES AND IF WE LOOK AT JUST A SERIES OF PICTURES SHOWING THE DEVELOPMENT PROFILE OF INHIBITORY NEURON AND THEIR EXPRESSION OF OF THE NKs 2.1 AND YOU CAN SEE IT'S VERY LITTLE EXPRESSED EARLY ON AND OVER TWO OR THREE WEEKS GLUE R FOUR IS IBT--INTEGRATE HICKIT AND IT TURNS OW THIS IS THE BASK THE CELL AND THE ACQUISITION OF GLUE R FOUR CONVERTS THE TRANSMISSION INTO EXTRAORDINARY TIGHT AND RAPID GLUTEA MINERGIC TRANSMISSION AND A KEY PROPERTY IN THIS RAPID SYNAPTIC TRANSMISSION IS THAT THE EXPRESSION OF THE PENETRANT ACTS ARE BEING ELEVATED TO THE TIME THAT GLUE R FOUR IS BEING EXPRESSED ON THESE MASKET CELLS SO CAN YOU SEE THAT P11 AND P20 THERE'S AN UPREGULATION OF NARP AND AN UPREGULATION OF THE NPR AND ATTRACT ANT WHICH IS ARE CRITICAL FOR CLUSTERING GLUE R FOUR IN THESE INNER NEURONS. SO PAUL MADE A SERIES OF COMBINATIONS AND WE GOT--ORIGINALLY WOO HAD A PAPER HIM DESCRIBING THE KNOCK OUT OF THE SINGLE PIN TRACT ANT AND I WILL SHOW YOU DATA FROM THE DOUBLE AND IN THE DOUBLE KNOCK OUT WHAT WE CAN SEE IS AN ALMOST COMPLETE LOSS OF THE GLUE R FOUR EXPRESSION PROFILE AND THIS IS AN INVITRO SLICE PREPARATION, YOU CAN SEE IT'S INVOLVED IN HEURONS ABSENCE OF GLUE R FOUR IF YOU TAKE THE CULTURE, CAN YOU SEE THAT IT'S EXPRESSED ALL OVER THE DENDRITIC DENDRITES OF THESE CELLS, ALMOST COMPLETELY ABSENT IN THE DOUBLE KNOCK OUT IF YOU DO WESTERN BLOT OF TOTAL MEMORY, CAN YOU SEE THAT GLUE R FOUR IS ELIMINATE INDEED THE DOUBLE KNOCK OUT. SO, IT TURNS OUT THAT THIS--THIS COMBINATION OF PEN TRACT ANT SYSTEM CRITICAL FOR A LOSS OR FOR ACQUISITION OF GLUE R FOUR CLUSTERING AT THESE AND GLUTEA MINERGIC BASKET CELLS AND AS ONE WOULD EXPECT, A LOSS OF GLUE R FOUR HAS AN IMPACT ON SYNAPTIC TRANSMISSION SO WE THEN MADE RECORDINGS FROM THESE BASKET CELLS AND UNDERWILD-TYPE WE CAN FIND THE CURRENTS WE CAN FIND AMPPLICATED CURRENTS IN THE DOUBLE KNOCK OUT WE CAN SEE THAT THE CURRENTS ARE ALMOST IDENTICAL TO THOSE OBSERVED IN WILD-TYPE BUT THERE'S A HUGE COMPROMISE OF THE CURRENT THAT'S CARRIED TO THESE AMPORECEPTORS AND SIMARLY THESE ARE MONITORS THEM IN THE SPONTANEOUS ACTIVITY BETWEEN WILD-TYPE AND THE DOUBLE KNOCK OUT. CAN YOU SEE THEREYA A HUGE DEFICIT IN THE AMPLITUDE OF THESE AMP O AND GATED SYNAPTIC CURRENTS. SO THE LOSS OF GLUE R FOUR IS REALLY ALLOWING UNDERREPRESENTATION OF THESE CELLS SO WHAT IMPACT DOES THE LOSS OF DRIVE ON THESE INTERNEURONS HAVE ON THE SYNAPTIC TARGETS DOWN STREAM. IF WE MOVE ONE SYNAPSE DOWN IN THE CHAIN OF THE NETWORK AND WE MEDE RECORDINGS FROM CATHREE PATHWAY GIVES RAMTAL CELL WHICH IS ARE DRIVEN BY THE THESE BARKET CELLS AND WE USE A CLIFF TRICK WHERE WE CAN VOLTAGE CLAMP THE CELL AT TWO DIFFERENT VOLTAGES TO LOOK AT THE COMOPPOSITE BEHAVIORIAL PHENOTYPE SPENT THE GABBA COMOPPOSITE BEHAVIORIAL PHENOTYPENT, YOU CAN SEE THAT UNDER WILD-TYPE CONDITIONS WE GET A VERY NICE EXCITATORY PATHWAY GIVES RAMTAL CELLS AND WE SEE THE INHIBITORY DRIVEOT PATHWAY GIVES RAMTAL CELL ALSO. BUT IN THE DOUBLE KNOCK OUT WE CAN SEE A SIMILAR TYPE OF GLUTEA MATE DRIVE ON TO THE PATHWAY GIVES RAMTAL CELL, WE SEE A VERY LARGE ATTENUATION OF THE AMOUNT OF GABBA THAT IS AVAILABLE FOR INHIBITORY DRIVE AND ONE CAN IMAGINE THAT A LOSS OF FEET FORWARD AND INHIBITORY DRIVE ON THE CELL WILL COMPROMISE THE EXCITATION AND INHIBITION RATIO THAT ENGAGES THE CELL IN THE OSCILLATING CIRCUIT. AND SURE ENOUGH, IF WE TAKE THIS ONE STEP FORWARD AND LOOK AT THE NETWORK IN THE TOTAL ACTIVITY DURING NETWORK OSCILLATIONS, CAN YOU SEE THAT UNDERWILE TYPE CONDITIONS IF WE TICK A LITTLE AND PUT IT ON THERE TO ACTIVATE THE COLONERGIC RECEPTORS WE CAN TRIGGER THE ROBUST ACTIVITY ASK IN THE ABSENCE OF THESE PENETRANT ANT WHICH IS RESULTS IN THE GLUE R FOUR AND THE CONSTREAM CONSEQUENCES OF THAT, WE ALMOST HAVE A COMPLETEULOSIS OF FUNCTION OR COMPLETE LOSS OF THIS GAMMA OSCILLATION INSIDE THE CIRCUIT. SO THE LOSS OF ONE SUBUNIT NOT ONLY HAS A HUGE IMPACT ON THE DRY ON TO THE BASKET CELL BUT IT ALSO HAS KNOCK-ON CONSEQUENCES FOR EVERYTHING THE SALE WILL COME IN CONTACT DOWN STREAM A BIT. SO I'M GOING TO WRAP UP THERE IN THE INTEREST OF TIME AND I WANT TO SUMMARIZE WHAT I'VE TOLD YOU. WE'VE TAKEN A GENETIC APPROACH TO STUDY THE--THE EMBRYO GENESIS IF YOU LIKE THAT THEY'RE MIGRATING OUT OF THE MEDIAL AND THE GANGLIONIC EVIDENCE FEDS AND THEY'VE ALLOWEDITOUS CATALOG AND WORK AT WHAT INHIBITORY INTERNEURONS ARE DERIVE FRIDAY WHICH EVIDENCE AND MANY OF THE RULES THAT ARE SHARED IN THE CORTEX THAT ARE A LARGE NUMBER OF DEVIATIONS AND THIS IS REALLY BEEN THE MOST REWARDING ASPECT OF THIS IS THAT IT'S THE GIFT THAT KEEPS ON GIVING, THE MORE YOU STUDY INNER NEURONS, THE MORE YOU LEARN THAT ARE HUGE DEVELOPMENTAL DEVIATIONS FROM WHAT WE EXPECT IN THE CORTEX AND I TOLD BUT THE PENETRANT ACTS OF GLUE R FOUR AND THE COMBINED LOSS OF THESE CONTRACT ANTS CAUSES A REDUCTION IN THE ACQUISITION ON THE SALES AND THAT HAS EXTRAORDINARILY DOWN STREAM CONSEQUENCES FOR EVERY ASPECT OF THE CIRCUIT THEY DON'T HAVE TIME TO GO INTO ALL THE FEATURES OF THAT, SO THE LOSS OF THE GAMMA OSCILLATION IN THE BEHAVING ANIMAL WILL BE CATASTROPHIC FOR ITS FUNCTIONS SO OF COURSE, I WOULD BE A MESS IF I DIDN'T TELL WHO YOU DID ALL THIS WORK. I'M JUST THE GUY THAT BARKS OUT THE ORDERS. MOST OF THIS WORK WAS ACCORDED BY AN EXTRAORDINARY STAFF IN MY LAB, KEN AND COLLEAGUES, EMBRYON AND I CAN GENETIC WORK WAS STARTED BY LUKE WHO'S NOW IN FRANCE AND ALL THE REST OF THE PEOPLE CONTRIBUTED AND MAJORLY TO ALL THE WORK I'VE SHOWN YOU TODAY. SO THANK YOU VERY: [ APPLAUSE ] >> I THINK WE HAVE THYME FOR ONE QUESTION. DID THESE MICE SURVIVE THE SEIZURES? >> YES. >> DID YES THAT'S WHERE WE'RE HEADING RIGHT NOW IS WHAT WERE THE CONSEQUENCES. >> THANKS, GREAT JOB. SO THE NEXT SPEAKER IS DR. MATT KELLIE WHO'S HEAD OF THE LABORATORY OF COCHLEAR DEVELOPMENT HIS LABORATORY SEEKS TO IDENTIFY THE IN LECULAR AND CELLULAR FACTORS THAT PLAY DEVELOPMENT IN THE MALE CO LEA, WIRE THE BRAIN'S MICRO PHONE WILL ELUCIDATE FACTORS THAT REGULATE THE INNERVATION OF CELLS IN THE COCHLEA. THANK YOU VERY MUCH. >> OKAY, THANK YOU VERY MUCH. IT'S A REAL HONOR TO BE HERE TO REPRESENT THE DEAFNESS INSTITUTE AND I WANT TO THANK THE ORGANIZERS AND THE LABORATORY FOR MAKING THAT HAPPEN. SO MY SPECIALTY AND UNDERSTANDING HOW THE COCHLEA DEVELOPS OVER TIME AND WE HAVE LOOKED AT THE HAIR CELLS WHICH ARE THE PRIMARY TRANCE DUCERS OF CHANGING AUDITORY SIGNALS INTO THE NERVOUS IMPULSES BUT A FEW YEARS AGO WE DEVELOPED A NEW INTEREST LOOKING AT THE GANGLI NEURON WHICH IS IS THE PRIMARY NEURONS THAT CARRY THE SIGNALS FROM THE HAIR CELLS INTO THE BRAIN AND THAT INTEREST CO INSIDED WITH THE ARRIVAL OF TOM COAT IN MY LAB, AND MUCH OF THE WORK IS WHAT TOM DO. CONTRARY TO HIS ARRIVAL, WE WERE WORKING ON THE WRONG TYPE, AND WE SHOULD BE MOVING ON AND LOOKING AT THE OTHER NEUR- OWNS. I'M NOT POSITIVE BUT I WILL TALK ABOUT IT. THIS IS A CLASSIC SORT OF HUMAN ANATOMICAL CROSS SECTION THROUGH THE EAR HERE. THIS IS THE OUTER HERE AND THEN THE MIDDLE EAR WITH THE THREE MIDDLE EAR BONES AND THEN HERE'S THE COCHLEA HERE AND THIS IS A BRANCH HERE, THE AUDITORY BRANCH AND IF WE CUT A CROSS SECTION THROUGH HERE AND ZOOM IN A LITTLE BIT WE CAN SEE THAT WHAT LOOKS LIKE A SINGLE COIL IN THE CO LEA IS IN FACT THREE TUBES THAT GO AROUND IN A COIL STARTING HERE AND MOVING UP THIS WAY AND THE MOST RELEVANT AND THE TUBE IN THE MIDDLE AND SITTING ON THE FLOOR OF THAT TUBE ARE THE MECHANO SENSORY HAIR CELLS AND INTERNAL AUDITERVATING THOSE HAIR CELLS ARE A CLUSTER OF NEURONS THAT HAVE THE CELL BODIES LOCATED RIGHT HERE IN EACH TURN. THAT PROJECT PERIPHERALAXONS OUT TO THE HAIR CELLS AND CENTRAL AXONS THAT COALESCE THAT LEAVE THEINER HERE AS THE NERVE. SO IF WE ZOOM IN MORE WE CAN SEE THAT RELATIONSHIP MORE CLEARLY HERE. HERE'S THE CELL BODIES FOR THE SPIRAL GANGLION NEURONS AND THESE ARE THE PERIPHERALAXONS HERE AND THEN THEY MAKE CONTACTS WITH THE HAIR CELLS HERE WHICH CAN YOU SEE. AND ONE OF THE FIRST THINGS YOU'LL NOTICE IS THAT THESE STOPOT FIRST HAIR CELL, AND PROJECT OUT BECAUSE THESE THREE HAIR CELLS REFER TO AS THE OUTER AND THEY COME BACK TO THAT IN A MINUTE. SO THE NAME DERIVED FROM THE SORT OF THREE DIMENSIONAL STRUCTURE OF THESE NEURONS WHICH YOU CAN SEE IN THIS PICTURE HERE SO THIS IS THE ENTIRE COCHLEAR FROM A MOUSE TO ABOUT TWO TURNS THESE ARE THE CELL TELES AGAIN ILLUSTRATED AND YOU CAN SEE THE SINGLE CELL SEPARATED FROM THESE OTHER THREE CELLS BY THIS GAP HERE AND IF WE NOW OVERLAY THE NEURONS CAN YOU SEE THEM HERE. THEY SPIRAL AROUND THE MEDILUS TO GIVE NAME FOR THE STRUCTURE AND YOU SEE THE PERIPHERAL AXONS THAT EXTEND HERE, RAD YALE FIBER BUNDLES THAT COME OUT AND MAKE CONTACTS WITH THE HAIR CELLS. WE ZOOM IN ON THE HAIR CELL ON THIS REGION HERE AND YOU WE CAN SEE THE INTERESTING ANATOMY THAT EXIST HERE IN TERMS OF INNERVATION. SO HERE'S THE FIRST ROW OF HAIR CELLS THE INNER HAIR CELLS AND YOU NOTICE THAT MOST FORM THEIR SYNAPSE SIS HERE AND THESE ARE SINGLE BUT TON ENDINGS SO THIS I A SINGLE FIB THEY'RE SENDS ONE, ONE PROJECTION THAT MAKES JUST ONE--WHOOPS, MAKES JUST ONE SINGLE HERE ON AN INNER HAIR CELL AND THERE ARE ON AVERAGE 20 TO 30 INAPSE SIS OF GINGLIAISON AN NEURONS FOR EACH HAIR CELL AND A BIG RATIO DIFFERENCE HERE BETWEEN THESE HAIR CELLS AND THESE FIBERS COMPRISE BETWEEN 90 AND 95% OF ALL OF THE TYPE ONE FIBERS. THE REMAINING FIVE TO 10% ARE WHAT ARE CALLED TYPE TWO FIBERS AND THESE ARE ONES THAT DON'T SYNAPSEOT--SYNAPSE ON THE INNER HAIR CELLS AND THEY MAKE A SHARP TURN TOWARDS THE BASE OF THE CO LEA AND THEY'LL TURN ALONG THE OUTER HAIR CELL ROW AND MAKE BETWEEN FIVE AND 10 SYNAPSES WITH FIVE OR 10 DIFFERENT OUTER HAIR CELLS SO TWO VERY DIFFERENT P FOR THESE TWO CELL TYPES. NOW THERE'S ALSO CONSIDERABLY DIFFERENCES HERE I'LL SHOW YOU THIS. THIS IS A SLIGHTY DIFFERENT HAIR CELL, CAN YOU SEE THE DIFFERENT FIBERS EXIST HERE REFIRED TO AS--REFERRED TO AS HIGH SPONTANEOUS AND LOW SPONTANEOUS RATE FIBERS. THEY'RE CHARACTERIZED BY TWO CHARACTERISTICS BEYOND THE PHYSIOLOGICAL ONES WHICH ARE IMPLIED IN THE NAME. THEY TEND TO HAVE A LARGER CALIBER AND THEY FORM SYNAPSE SISOT LARGER FORM HERE, THEY HAVE A SMALLER CALIBER AND THEY TEND TO FORM SYNAPSE SISOT OTHER SIDE RIGHT HERE AND SO ONE OF THE THINGS THAT TOM WAS INDIVIDUAL IN INVESTIGATING WHEN HE STARTED IN MY LAB WAS HOW DOES THIS PHENOTYPIC DIVERSITY IN EMERGING ITS OF INNERVATION BOTH SYNAPTIC LOCATION AND PATTERNING DEVELOPMENT WITHIN THE EAR AND ONE OF THE PROBLEMS THAT TOM FACED EARLY ON WAS THAT THERE ARE A LOT OF THESE NEURONS AND IT'S HARD TO SEE THESE SYNAPSES IN MOST PREPS AND I'LL SHOW YOU AN EXAMPLE HERE AND WE'RE LOOKING AT A SURFACE VIEW OF THE HAIR CELLS AND YOU CAN SEE THE INNER ROW HERE AND THREE ROWS OF OUTER HAIR CELLS AND IF YOU INNER LAY THESE ON TOP OF THAT, YOU SEE THAT THERE'S A LOT OF FIBERS IN HERE AND IT'S REALLY HARD TO SEE ANY SORT OF SPECIFICITY IN TERMS OF THE TYPES OF FIBER--INNERVATION PATTERNS WE SEE HERE SO THE PROBLEM THAT TOM HAD HERE WAS HE COULDN'T SEE THE TREES FOR THE FOREST. SO HE DECIDED TO DO WAS TO USE A SERIES OF GENETIC LABELING TECHNIQUES AND MOUSE LINES TO TRY AND MAKE THINGS EASIER FOR HIMSELF. SO HE TOOK A MOUSE THAT WAS GENERATED BY LISA GOODRICH AT HARVARD THAT USED THE PROMOTER FOR NEUROGENERATED ONE THAT WAS EXPRESSED IN THE CELLS TO EXPRESS AN INDUCIBLE FORM OF CREE, CALL CREE RTTWO AND THIS IS RTWORKS SIX ERROR & REPORTER AND TOOK ADVANTAGE OF AND LIMIT THE NUMBER OF CELLS THIS WEEK IN WHICH THE CREE RECOMBINED AND THEN ORDER TO MAKE THE TARGETS HE COMBINED THE THIRD LINE ON HERE WHICH AND WHICH IS EIGHT TO ONE GFP LINE SO THIS USES A DIFFERENT TRANSCRIPTION FACTOR EIGHT TO ONE THAT HAPPENS TO BE EXPRESS INDEED ALL HAIR CELLS THAT LABEL ALL THE TARGETS. SO WHEN TOM COMBINED THESE LINES HE'S ABLE TO CHANGE THIS VERY DIFFICULT PATTERN WITH THE INNERVATION. TO THIS ONE HERE. WHERE YOU CAN SEE THE SUBSET OF THESE FIBERS AND VERY CLEARLY PICK OUT INDIVIDUAL INNERVATION PATTERNS AND WE ZOOM IN WE CAN IDENTIFY FIBERS THESE ONES THAT CROSS AND TRAVEL ALONG THE ROADS OUTER HAIR CELLS AND YOU CAN ALSO IDENTIFY, ONE OF THESE HIGH SPOT FIBERS, AND A LOW FIBER LIKE THIS ONE THAT HAS A MORE NARROW CALIBER AND FORMS A SYNAPSE ON THIS SIDE OF THE HAIR CELL HERE. SO USING THIS TECHNIQUE, TOM WAS ABLE TO SET UP A SYSTEM WHERE WE COULD LOOK AT INDIVIDUAL NEURONAL BEHAVIOR AND ASK WHAT WAS GOING ON DURING SYNAPSE SELECTION AND NEURONAL PATTERNING. I WANT TO SHOW YOU ONE OTHER PICTURE HERE BEFORE MOVING ON TO SOME OF THESE QUESTIONS AND THAT'S A PROJECT UNDERTAKEN BY HANNAH SHARE ILLEGALS A GRADUATE IN THE--HANNAH SHARE ILLEGALS ILLEGALS--HANNAH CHERYL AND SHE'S GENERATING CLONES OF CELLS THAT ARE SPARSELY LABELED EACH ONE EXPRESSES A DIFFERENT CHLOR O FOR AND THEN TWO CELLS THAT ARE EXPRESSING CFP A BLUE VERSION AS WELL. SO USING HANNAH'S GOING TO BE ABLE TO ANSWER QUESTIONS ABOUT WHAT THE RELATIONSHIPS ARE BETWEEN THESE CELLS IN TERMS OF MAKING CHOICES ABOUT TYPE ONE VERSES TYPE TWO PHENOTYPES. OKAY, SO MOVING BACK IS ONE OF THE FIRST THING HE DID WAS TO REVISIT A QUESTION THAT'S EXISTED FOR QUIEM SOMETIME IN THE FIELD AND NOT WELL ANSWERED. AND THATTA THE QUESTION OF HOW CELLS, HOW INDIVIDUAL CELLS NEURONS DETERMINE WHETHER THEY'RE GOING TO DEVELOP TYPE ONE OR TWO FIBERS. VERY OLD STUDIES THAT USED GOLGI STANDARDS THAT USE FIBERS THAT SEEM TO BE MAKING SYNAPSES OR AT LEAST CONTACTING BOTHINNER AND OUTER HAIR CELLS SUGGESTING THEY REACH THEIR TARGETS AND RELY OFFICE OF DIVERSITY ENVIRONMENTAL INTERACTIONS AND DECIDE WHETHER THEY WERE GOING TO FORM A SYNAPSE ONINNER HAIR CELLS OR ON TO FORM INNER HAIR CELLS. MORE RECENT DATA US -ING GENETIC LABELING HAD REPORTED THE OPPOSITE FINDINGS THAT CELLS EITHER STOPPED OR MOVED IN TO THE HAIR CELL REASON, THEY WERE PREDETERMINED TO CHOOSE ONE FATE OVER OTHER. AND TOM WANTED TO REVISIT THIS QUESTION LIEUSING THE LOW, LOW LABELING TECHNIQUE HE DEVELOPED SO TO GIVE YOU AN IDEA ABOUT THE T PERIOD, THESE ARE CROSS SECTIONS THAT WERE DONE BY KEVIN AS THE SENIOR THESIS IN THE LAB WHEN HE WAS AN UNDERGRAT YOD AT THE UNIVERSITY OF MARYLAND AND IT SHOWS CROSS SECTIONS IN THE COCHLEAR DUCT IN BLUE HERE USING EIGHT TO ONE AGAIN THE TRANSCRIPTION FACTOR SPECIFIC FOR HAIR CELLS ISSUE. SHOW YOU WHERE THE HAIR CELLS FORM HERE AND HERE AND HERE AND THESE BEGIN TO PENETRATE THE EPITHELIUM EMBRYONIC DAY 14 AND FORMING THE INNER HAIR CELL AND SPREAD THIS WAY TO MAKE CONTEXT WITH THE OUTER HAIR CELL AS WELL THIS CONTINUES FOR A COUPLE DAYS DWHRAWND AND SO THIS IS TIME PERIOD THAT I'LL MOSTLY BE TALKING ABOUT BETWEEN E15.5 AND E16.5 HERE. OKAY SO ONE OF THE FIRST THINGS TOM WANTED TO DO WAS ASK IF HE COULD OBSERVE PATH FINDING ACTIVITY IN KNEES NEURONS. SO HE TOOKED THESE TRIPLE CHAINS GENIC MICE WHICH HE LIKE THIS IS PICTURE, AND HE GENOTYPE THESE GUYS UNDER THE FLUORESCENT MICRO SCOPE AND THEY'RE POSITIVE IN NEUROGENERATED ONE WHICH IS SHOWN IN MAGENTA AND THEN THE GFP, THE EIGHT TO ONE IS SHOWN HERE IN GREEN SO WHEN TOM SEES A MOUSE LIKE THIS, HE KNOWS HE HAS THE RIGHT GENO TYPE AND HE WOULD DISSECT OUT THE DEVELOPING COCHLEA FROM THOSE MICE BASICALLY CUT THAT INTO A HALF COCHLEAR OR A THIRD OF A CO LEA THAT WILL LAY FLAT AND PUT THAT UNDER OR ON A SPINNING DISCONFOCAL, FLATERN IT OUT SO HE CAN GET IN GOOD VISION, GOOD VIEW OF IT AND GENERATE TIME LAPSE MICROSCOPY OF THESE CELLS AS THEY'RE MAKING DECISIONS SO IT'S IMPORTANT TO REMEMBER THAT UNLIKE A LOT OF NEURONAL PATH FINDINGS EXPERIMENTS DONE WITH NEURONS IN A DISH, THESE ARE STILL WITHIN THEIR INTACT EPITHELIUM AND THEY'RE EXACTLY WHERE THEY'RE SUPPOSED TO BE, THE BASE METROPOLITAN MEMBRANE INTACT AND ALL OTHER CELL TYPES THEY'RE INTERACTING WITH SO WE'RE GETTING AN OPPORTUNITY TO SEE THE REAL BEHAVIOR OF THESE CELLS AS THEY'RE MAKING DECISIONS. THIS IS WHAT ONE OF THESE LOOKS LIKE. SO CAN YOU SEE HERE, THESE ARE INDIVIDUAL NEURONS HERE CAN YOU SEE THEM WITH T TARGET THAT THEY'RE IN. O AND--ONE OF THE FIRST THINGS WE NOTICED WAS A LOT OF COMPLEXITY IN THESE GANGLI NEURONS WHICH YOU SEE RIGHT HERE AND RIGHT HERE. THIS IS ONE THAT MIGHT BE--GOING TO DEVELOP AS A TYPE ONE FIBER, SEEMS TO HAVE A TERMINATION HERE BUT HAS VARIOUS BRANCH THIS IS WAY, THIS SEEMS MORE LIKELY TO DEVELOP AS A TYPE TWO, ALTHOUGH AT THIS STAGE WE CAN'T BE SURE. OKAY WHAT I ASK YOU TO DO NOTICE IS YOU LOOK ANYWHERE YOU WANT BUT I ASK THAT YOU LOOK AT THIS NEURIDE HERE WHERE YOU RUN THIS MOVIE AND YOU SEE FIVE HOURS OF TIME LAPSE AND YOU'LL NOTICE A LOT OF ACTIVITY WITHIN THESE FIBERS AND TOM SOW A OF PROBING SO CAN YOU SEE THIS PARTICULAR FIBER HERE IS EXTENDING INTO THE OUTER HAIR CELLS. IN FACT, IF YOU LOOK AT THIS ONE FIBER OVERTIME YOU CAN SEE THESE THREE PRIMARY BRANCHES AND THEY'RE FAIRLY DYNAMIC IN TERMS OF BEHAVIOR INTO THE OUTER HAIR CELL REGION AND ALONG THE INNER HAIR CELL REGION AS WELL. AND SO THESE RESULTS SUGGESTED TWO THINGS, OR PROVIDED TWO USEFUL PIECES OF INFORMATION. FIRST WE'RE SEEING WHAT LOOKS LIKE A LOT OF PROBING OF THE ENVIRONMENT BY THESE NEURIDES MAKING US THINK THEY HAVEN'T MAIDS A DECISION ABOUT WHERE THEY'LL FORM THE SYNAPSE SIS AND ASKING FOR CUES FROM THE ENVIRONMENT TO HELP US MAKE THOSE DECISIONS. THE SECTIONAL ANALYSIS THING TOM NOTICE WIDE THIS IS A LOT OF COMPLEXITY IN TERMS OF THE NUMBER OF BRANCHES IN THE NEURIDES AND THESE HAVE BEEN MISS INDEED THE LITERATURE AND APPARENTLY BECAUSE WHEN YOU FIX THESE, THEY COLLAPSE AS A RESULT OF FIXATION SO WE LEARNED--LEARNING THE POWER OF LIVE IMAGES HERE LEARNING WHAT THE FIBERS ARE DOING. SO THERE'S THAT. SO WHAT TOM WANTED TO DO NEXT WAS TO TRY AND IDENTIFY THE MOLECULAR PLAYERS THAT MIGHT BE MEDIATING THESE RESPONSES AND ONE OF THE FIRST THING HE DID WHEN HE BECAME INTERESTED IN THIS WAS TO TAKE ADVANTAGE OF THE SERIES OF MRNI EXPRESSION PROFILES THAT WE GENERATE FRIDAY THE LAB SEVERAL YEARS BEFORE HE GOT THERE. AND WHAT WE'VE DONE IS DISSECT OFF THE COCHLEAR EPITHELIUM HERE AND ASK WHAT MRNAs ARE EXPRESSED THERE USING THE MICROARRAYS WHICH WERE THE COOL TOPIC, COOLED OPTION AT THE TIME. AND WHAT TOM FOUND WAS EXPRESSED BY NUMBER OF MOLECULES CALLED SEMAPHORIN THREE AND THESE ARE REALLY INTRIGUING BECAUSE THEY'RE SECRETED MOLECULES THAT BIND TO A COMPLEX RECEPTORS ON NEURONS AND PLAY ROLES IN MEDIATING PATTERNING DECISIONS IN THOSE CELLS SO VERY BRIEFLY IN THE DIAGRAM HERE YOU CAN SEE THE PLAYERS WE'RE TALKING ABOUT, SEMAPHORIN'S BIND TO ONE OF TWO RECEPTORS CALLED NEUROPILLINS, NEUROPILLIN ONE OR TWO AND YOU NOTICE THERE'S NEUROPILLINS HAVE NO--TRANSMEMBRANE PROTEINS BUT NONAPOPTOTIC INTRACELLULAR DOMAIN. THEY ACCOMPLISH SIGNALING WITH ANOTHER SET OF MOLECULES CALLED FLEXIN A'S AND THEY HAVE A CELLULAR DOMAIN AND THEY ACTIVATE GTPA SIGNALING AND MAP KINASE SIGNALING PATHWAYS HERE SO WHEN A SEMAPHORIN IS PSSENT IT WILL GO TO A MEDIATED DECISION WITHIN THOSE CELLS. A LOT OF WORK, MUCH OF WHICH WAS DONE BY DAVID GINTY UP AT JOHNS HOPKINS, DEMONSTRATE THAD FOR THE MOST PART, SEMAPHORIN NEUROPILLIN INTERACTIONS TEND TO BE THERE IN NATURE SO THEY ARE MOVING BEYOND THE NORMAL POSITIONS AND THEY PLAY A ROLE IN SYNAPSE PAIRING OR ELIMINATION AND SO TOM WAS REALLY INTERESTED IN WHETHER THIS PATHWAY MIGHT PLAY A RELL IN LIMITING WHERE SPARROW GANGLION NEURONS WOULD GO. SO AS A FIRST STEP HE DID A LOT OF INSITU HYBRIDIZATIONS THAT ME FOUND IN THE MICROARRAY AND WHAT I'M GOING TO SHOW YOU ABOUT IS THE SEMAPHORIN THREE F AND IT'S--THIS SHOWS A LITTLE SCHEMEATIC AND THREE F BINDS TO NEUROPILLIN TWO AND THEN THERE HERE'S FLEX AND I KNOW HERE THE THREE TURNS OF THE COICALLY BASE AND YOU CAN SEE THE EXPRESSION HERE IN PURPLE AND IF WE ZOOM INOT MIDDLE, MIDDLE CROSS SECTION HERE YOU SEE THE INTERESTING EXPRESSION PATTERN WHICH IS EXPRESSION HERE WITH THE EPITHELIUM BUT WITH A STARK BOUNDARY HERE THAT YOU CAN SEE AND WHEN TOM OVERLAID IMMUNOLOGICAL DETECTION FOR HAIR CELLS AND GANGLION NEURONS IN THIS SECTION, WE SEE AN INTERESTING PATTERN OF EXPRESSION. SO HERE'S THE NEURONS IN GREEN. INNER HAIR CELL HERE IN READY AND OUTER HAIR CELLS ARE HERE IN RED AS WELL AND THEN HERE IN WHITE IS THE EXACT SAME EXPRESSION, AND YOU WILL NOTICE THAT THE BOUNDARY, CORRELATES EXACTLY WITH THE DIVIDING LINE BETWEEN TYPE ONE FIBERS AND TYPE TWO FIBERS, TYPE TWO FIBERS CROSSING BEYOND THAT REGION. SO THAT SUGGESTED A REALLY PROVOCATIVE POSSIBLE INTERACTION HERE BEFORE I MOVE ON TO SHOW YOU DATA TOM WOPTED TO BE SURE THAT THE GANGLION NEURONS COULD RESPOND TO THAT SO HE DID IMMUNO LABELING FOR SEMAPHORIN FOR NEUROPILLIN TWO AND HE FOUND NEUROPILLIN TWO IN THE CELL BODY SO THIS IS TGACONE AND THIS IS NEUROPILLIN TWO AND MORE IMPORTANTLY IN THE PERIPHERALAXONS THEMSELVES THIS, IS A DIFFERENT MARKER WITH THE TAG ONE SHOWIN THE GANGLION NEURONS AND CAN YOU SEE THIS EXPRESSED OUT HERE ON THE TIPS OF THOSE FIBERS SO BASED ON EXPRESSION PATTERN ONE CAN MAKE A FAIRLY STRAIGHT FORWARD PREDICTION, AND SIGNALING INVOLVE PREVEND BEING THE OUTER HAIR CELL REGION AND SHOULD RESULT IN MORE FIBERS CROSSING SO TOM OBTAINED THE NEUROPILLIN TWO KNOCK OUT MOUSE FROM THE LAB AND WAS LABEL TO ADDRESS THAT SPECIFIC QUESTION. THIS IS A FAIRLY EARLY TIME POINT 15.5 AND SO TOM HAS USED A DIFFERENT MARKER AND THE 271 AND LOOKING DOWN ON SURFACE HERE, THIS IS WHERE THE HOW THER AIR CELL PERFORMS AND INNER HAIR CELLS WERE FORM. IF WE SEE THE WILD-TYPE AT THE INNER HAIR CELL PRESIDENCY IN THE YOU HADITANT WE SEE A LOT MORE FIBERS CROSSING OUT AND THAT'S MORE EASILY SEEN HERE IN A THREE DIMENSIONAL RECONSTRUCTION, HERE'S OUR CONTROL, INNER HAIR CELL WOULD BE RIGHT HERE, AND MOST OF THOSE FIBERS ARE STOPPING HERE AND THERE'S A SMALLER NUMBER CROSSING BUT IN THE MUTANT WE SEE VERY FEW FIBERS AND MOST OF THEM CROSSES RIGHT HERE INTO THE OUTER HAIR CELL REGION, SUGGESTING THAT THE NEUROPILLIN TWO SIGNAL IS CERTAINTY IMPORTANT FOR STOPPING THE FIBERS AT THE HAIR CELL. WHEN I WAS A GRADUATE STUDENT, I WENT TO A SOCIETY FOR NEUROSCIENCE MEETING WHICH I ATTENDED A SESSION CALLED A PATIENTS IN THE CLINIC SURWORTH A THOUSAND WORDS BUT A NUMBER IS WORTH A THOUSAND PICTURES SO I'VE SEN ILLEGALSEN THEN TRYING TO SPEND A LOT OF TIME SAYING WE NEED TO QUANTIFY EVERYTHING WE CAN. SO WHAT TOM'S G BEEN DOING IS TO COMBINE NEUROPILLIN DEPLETION WITH THE HETEROGENEOUS O ZYGOTES TO--HETEROZYGOTES TO SEE IF WE CAN CHANGE NUMBERS HERE AND YOU CAN AN EXAMPLE, WHICH IS A PHENOTYPE SIMILAR TO THE HOMOZYGOUS AND WE SEE THE FIBERS FORMING TYPE TWO FIBERS CROSSING OUT INTO THE HAIR CELL REGION AND POINTED OUT BY QUANTIFICATION HERE SO IF YOU LOOK AT THE TOLLAL AND GET A SIGNIFICANT INCREASE IN TWO LOOKED LIKE TYPE TWO FIBERS IN THE ABSENCE OF THE PILLIN TWO AND TOM ASKED THE FOLLOW O UP WHICH IS IF THERE ARE MORE TYPE TWO FIBERS OUT HERE THEN DO WE SEE MORE SYNAPSES BETWEEN TYPE TWO FIBERS AND OUTER HAIR CELL. TO DO THESE, WE LOOK AT UNIQUE HAIR CELLS FOR GINGA LANGUAGE SYNAPSE, WHICH IS EXPRESSES A PARTICULAR MOLECULE CALLED RIB EYE WHICH FORMS HERE AND FORMS SMALL INAPTIC PUNTA ON THE CELLS. EACH OF THESE CIRCLES AND A SINGLE SYNAPSE IS A WILD-TYPE YOU'LL SEE THE NUMBERS ARE FAIRLY SMALL IN OUTER HAIR CELLS BUT IN THE NEUROPILLIN TWO MUTTITANT, CAN YOU SEE THERE'S AN INCREASE SUGGESTING WE ARE IN FACT GETTING MORE TYPE TWO FIBERS AND MORE SYNAPSES IN THE ABSENCE OF NEUROPILLIN TWO. NOW THE REVERSE POSSIBLE EXPERIMENT CAN BE DONE WHICH IS TO ASK WHAT HAPPENS IF WE INCREASE THE AMOUNT WITHIN THE EPITHELIUM. YOU RECALL THIS IS OUR SYMMETRY F EXPERIMENT HERE AND SO WHAT TOM DID HERE WAS TO SET UP COCHLEAR PLANTS AS HE HAD DONE FOR LIVE IMAGING AND CONTROL FUSION IMMUNO GLOBUE LYNN FUSION PROTEIN OR SEMITHREE F AS A FUSION PROTEIN AND WHAT HE SAW, WHAT HE SEES THISSEN IS A FAIRLY STRIKING DIFFERENCE IN THE MORPHOLOGY OF THE GROWTH CONES OF THE GANGLION NEURONS. SO HERE ARE THREE CONTROL SPIRAL GANGLION GROWTH CONES AND YOU SEE A HUGE AMOUNT OF COMPLEXITY AND YOU SUES THEM TO EXPLORE THE ENVIRONMENT IN CONTRACT AFTER 24 HOURS IN MEDIA CONTAINED SYMMETRY F, YOU SEE A REAL CLOSES OF COMPLEXITY WITHIN THOSE FIBERS. SO WITHOUT SEMESTERITRY F, THEY LOSE THE ABILITY TO PROBE THE ENVIRONMENT. THAT'S BORN OUT AGAIN QUANTITATIVELY, THE NUMBER OF NEURONS THAT MAKE IT INTO THE EPITHELIUM IS NOT CHANGED BUT WHEN HE LOOK AT NUMBER OF BRANCHES PER NEURON IT'S DECREASED SIGNIFICANTLY AND SUGGESTING THAT IT'S DECREASING THE ABILITY OF THESE CELLS NOT TONAL SCAN THIS WAY BUT ALSO WITHIN THE INNER HAIR CELL REGION HERE AS WELL. SO WITH THAT I'LL SUMMARIZE MORE GINGLIONS HERE WE KNOW THAT THE FIBERS FORM THE BUT TONS AND THEY CROSS AND MAKE MULTIPLE SYNAPSES IN THE OUTER HAIR CELL REGION THERE WAS QUESTION ABOUT WHETHER THIS WAS AN INTERACTION CONTROLLED BY ENVIRONMENTAL OR PREDETERMINED CUES, IT LOOKS LIKE FROM THE WORK WE'VE DONE SPECIALLY WORK AT TOM COPE THAT S TO BE THE CASE WHERE THESE CELLS ARE RELYING ON THE ENVIRONMENT TO HELP MAKE THOSE DECISIONS WITH TYPE ONES VERSES TYPE TWOS AND THE INTERACTION BETWEEN SYMMETRY THREE F IN THE EPITHELIUM ITSELF AND NEUROPILLIN ITSELF SEEMS TO BE MEDIATING SOME OF THAT DECISION. A COUPLE THINGS WE'RE DOING IN THURE TO LOOK AT THIS, FIRST IS WHEN TOM TREATED THESE WITH THE SEMESTERITRY THREE F, THEY DIDN'T HAVE ANY EFFECTOT TYPE TWO FIBERS SUGGESTING THEY WERE NOT BEING MEDIATED BY CEMETERY THREE F SINCE THERAPY AND M INTO THIS REGION. WHEN TOM LOOKED AT EXPRESSING PL ATHREE AND IS THE KORESEPTORSOR THAT'S REQUIRED FOR NEUROPILLIN SIGNALING WHAT HE OBSERVED WAS, HERE IT IS IN GREEN, TYPE TWO FIBERS HERE YOU SEE CROSSING OVER DON'T EXPRESS A-THREE WHERE IT'S EXPRESS INDEED THE TYPE ONE FIBERS. THE TYPE TWOS EXPRESS IN A-THREE UNTIL THEY DETAILS G TOTE THIS POINT AND TURN IT OFF. EITHER WAY IT SEEMS THAT THE FLEXIN AND TYPE TWO FIB E-PRESCRIBINGS MAY EXPLAIN WHY THEY'RE ABLE TO MIGRATE INTO THE OUTER CELL REGION. SECOND QUESTION, THAT WE'RE REALLY INTERESTED IN PURSUING IS HOW THORS BOUNDARY FORMS SO THIS BOUNDARY HERE SEEMS TO BE CRUCIAL FOR THIS DECISION WHICH WE TYPE ONE AND TYPE TWO FIBERS, IT ALSO IS A VERY IMPORTANT BOUNDARY FOR DEVELOPMENTAL ASPECTS OF THEINER SEER SO WE'RE INTERESTED IN UNDERSTANDING WHERE EMBRY O LOGICALLY THE BOUNDARY IS ESTABLISHED AND HOW IT'S MAINTAINED. OKAY, FINALLY I'LL ACKNOWLEDGE THE MEMBERS OF MY LAB AND TOM COAT RIGHT HERE WHO I CAN TELL YOU WITH BOTH HAPPINESS AND SADNESS IS LEAVING THE LAB THE AT END OF SUMMER TO START HIS OWN LAB. MY SADNESS AND OFFSET BY THE FACT HE'S NOT MOVING FOR FAR, HE'S MOVING TO GEORGETOWN UNIVERSITY SO WE CAN KEEP WORKING ON THE SPIRAL GANGLION 92 NEURONS AND I THE INNOVATION AND WITH THAT I'LL THANK YOU FOR YOUR ATTENTION. [ APPLAUSE ] >> WE HAVE TIME FOR ONE VERY QUICK QUESTION. >> SO WHILE ARE WORK TO THINK OF THIS AS ONE SIN CISION OF SCIENTISTS IT'S CLEAR IT'S NOT COMPLETELY TAKEN SINCE CHRIS SAID HE WAS REPRESENTING NICHD AND MATT SAID HE WAS REPRESENTING--NO CHRIS SAID NICHD AND NIDCD, AND YOU'LL NOTICE THAT MANY OF THE TOOLS THAT WERE USED IN THE TWO TALKS, THREE TALKS WE'VE HEARD ARE VERY SIMILAR AND IF WEREN'T FOR THE MOUSE GENOME, I DON'T KNOW WHERE DEVELOPMENTAL NEUROSCIENCE WOULD BE. SO THE NEXT SPEAKER IS DR. LEO BALUSIO, HE'S A SENIOR INVESTIGATOR IN THE NEURAL PLASTICITY SECTION AND I WON'T TELL YOU WHAT INSTITUTE. HE FACTORS ON THE OLFACTORY FUNCTION AND NATURALLY REGENERATING AT THE PERIPHERY AND SENSORY. HE WILL DISCUSS HIS LABS WORK AT HOW CIRCUITRY IS FORMED AND MAINTAINED THROUGH SENSORY EXPERIENCE. THIS SYMPOSIUM IS NOT BEING WEBCAST NOW BUT IT IS BEING RECORDED AND WILL BE UP ON THE WEB FOR PEOPLE WHO ARE HAVE MISSED TALKS OR WANT TO HEAR THE TALKS AGAIN IN ABOUT A WEEK. THANKS, LEO. >> THANK YOU. IT'S A PLEASURE TO BE PART OF THIS SYMPOSIUM. IT'S ALSO PLEASURE TO BE HERED. SORRY, IS THIS BETTER? ENTREE, THANK YOU FOR THAT INTRODUCTION, IT'S PLEASURE TO BE PART OF THIS. THIS IS MY VERSIO OF THE NEUROSCIENCE PICTURE, I TOOK IT FROM THE ROOF OF THE PARKING LOT ACROSS THE CRETE USING MY IPHONE IT'S NOT AS FANCY AS THE ONE YOU SAW EARLIER BUT I LIKE IT BETTER IT SHOWS THE DUMPSTER AND THE CONES AND IT'S A LITTLE MORE REALISTIC AND GHETTO AS MY DAUGHTER WOULD SAY. SO AS STORY MENTIONED I WORK ON THE OLFACTORY SYSTEM AND NOT FOR ANY PARTICULAR INSTITUTE, JUST HERE AT THE NIH: [LAUGHTER] AND I WILL TELL YOU ABOUT THE ORGANIZATION OF THIS SYSTEM AS WELL AS SOME OF THE WORK WE'VE BEEN DOING TO UNDERSTAND HOW SENSORY EXPERIENCE HELPS TO ESTABLISH THE CIRCUITRY AND MODIFY IT AS L. SO THE FOCUS OF MY LAB IS REALLY TOO UNDERSTAND THE MECHANISMS THAT CONTROL THE FORMATION, REFINEMENT AND NATIVE AMERICANS IN THE NORTHERNINENCE OF NEURAL CIRCUITS AND WE'RE PARTICULARLY INTERESTED IN THE ROLE THAT PLASTICITY REGENERATION PLAY IN THIS PROCESS AS WELL AS HOW SENSORY EXPERIENCE CAN BE USED TO EITHER ORGANIZE OR REORGANIZE THAT CIRCUITRY AND MORE RECENTLY WE'VE ALSO BECOME INTERESTED IN USING THIS SYSTEM TO STUDY CIRCUIT DISRUPTION AND REPAIR SOABTED WITH NEURODEGENERATIVE DISORDERS AND I DON'T HAVE TIME TO TALK ABOUT THIS TODAY, BUT I WILL STATE THAT OLYMPIC FADGERY LOSS IS ASSOCIATE WIDE A LOT OF NEUROLOGICAL DISEASE AND IT'S A VERY EARLY SYMPTOM OF DISORDERS LIKE PARKINSONS AND ALZHEIMERS AND SO WE REALLY BELIEVE THAT BY UNDERSTANDING THE ORGANIZATION AND FUNCTION OF THIS SYSTEM IN ITS NATIVE STATE AND REALLY ASSESSING WHAT GOES AWRY IN THESE CONDITIONS GIVE US INSIGHT INTO THE EARLY STAGES OF THESE DISORDERS. BUT FOR TODAY I WILL TALK ABOUT THE NATIVE STATE AND ALL THE WORK I'LL BE DESCRIBING WILL BE IN THE MOUSE OLFACTORY SYSTEM. AND JUST FOR A BRIEF INTRODUCTION, THIS IS A MOUSE HEAD THAT'S BEEN SPLIT DOWN THE MIDLINE AND WHAT YOU'RE LOOKING AT IS IT A MIDSAGEINAL VIEW, TIP OF THE NOSE, PALLET AND HIGHLIGHTED IN THESE COLOR SYSTEM THE MINE OLFACTORY EPITHELIUM WHICH IS RESPONSIBLE FOR DETECTING ODORS THAT ENTER THE NASAL CAVITY THROUGH LARGE POPULATION SENSORY NEURONS AND RELAYING THE INFORMATION TO THE BRAIN VIA THIS STRUCTURE HERE AND THE MAIN OLFACTORY BULB. THIS ORGANIZATION IS ACTUALLY VERY SIMILAR TO THE HUMAN SYSTEM, HERE'S THE TURBINATES IN HUMANS AND HIGHLIGHTED HERE IN YELLOW, A CLOSE UP IS THE OLFACTORY BULB. PORTIONS ARE DIFFERENT AS CAN YOU SEE AND HERE THE SENSORY NEURONS THAT POKE THROUGH THE PE AND DETECT ODORS AND SEND INFORMATION TO THE OLFACTORY BULB NOW THE LOGIC OF THE SYSTEM WAS EXPOSED DURING THE 90S WITH THE CLONING OF THE ODOR RECEPTORS, BY RICHARD AXEL AND DEBUCKET AND WHAT THEY BASICALLY SHOWED IS THAT A SENSE OF STELL IS MEDIATED BY A VERY LARGE FAMILY OF SEVEN TRANSMEMBRANE DOMAIN PROTEINS ENCODED BY ABOUT A THOUSAND DIFFERENT GENES. AND SO, TO GO INTO A LITTLE MORE DETAIL AND AND TO WHAT THIS DISCOVERY REALLY SHOWED US, I'M GOING TO GIVE YOU CROSS SECTION OF THESE TWO STRUCTURES AND SHOW IT TO YOU HERE AND SORT OF CARTOON FORM, THIS IS THE EPITHELIUM AND THE OLFACTORY BULBS, AND THESE LITTLE DOTS THAT YOU SEE HERE, REAP RESENT THE DIFFERENT OLFACTORY SENSORY NEURONS THAT ARE EXPRESSED IN THE EPITHELIUM AND SO FROM THE CLONING OF THESE RECEPTORS THERE WERE SORT OF FOUR BASIC PRINCIPLES THAT EMERGED ABOUT THIS ORGANIZATION. AND THOSE REALLY WERE THAT EACH OLFACTORY RECEPTOR IS REALLY ONLY EXPRESS INDEED JUST A SUBSET OF NEURONS AND I INDICATED THAT BY COLOR COATING SOME OF THESE DOTS AND COLORS SHOWING THEY EXPRESS DIFFERENT RECEPTORS, THE OTHER IS THAT EACH SENSORY NEURON EXPRESSES A SINGLE OR AND OLFACTORY SENSORY NEURON REGENERATE SO IT'S AN EXPRESSION EARLY ON IN THEIR GENERATION AND THEY CONTINUE TO EXPRESS THAT RECEPTOR THROUGHOUT A LIFETIME. THE THIRD IS THAT ALL THE OLFACTORY SENSORY NEURONS THAT EXPRESS THE SAME OR PROJECT AXONS TO THE SAME GLUE MAR LIE AND THESE ARE STRIPTURESOT SURFACE OF THE BULB AND THAT LOOKS SOMETHING LIKE THIS SO YOU CAN SEE THAT ALL THE AXONS AND THE NEURONS THAT EXPRESS THE SAME RECEPTOR USUALLY PROJECT TO MEDIAL AND LATERAL GLUE MAR LIAISONS AND THE GLUE MAR LIE TAKEN--THEY RECEIVE INFORMATION AND THAT I RESPOND TO THE SAME ARRAY OF ODORS AND THE THEN THE FOURTH PRINCIPLE IS THAT THE LOCATION OF ANY GIVEN LIST IS FAIRLY CONSTANT. IT'S A BIT OF VARIABILITY BUT BECAUSE OF THIS CONSTANT STABILITY OF THIS, THIS REALLY IS THE RATIONAL FOR A MAP ON THE SURFACE OF THE OLFACTORY BULB AND ODOR RECEPTOR MAP, I SHOULD SAY A GLUE MARRIAL MAP THAT REFLECTS IDENTITY AND IN REALITY THERE'S TWO MAPS, ONE ON THE MEDIAL AND LATERAL SIDE OF EACH OLFACTORY BULB AND WE HAVE TWO BULBS AND TWO MAPS ON THIS SIDE. NOW, OUR OWN WORK THEN LOOKED A BIT MORE DETAIL AT THE ORGANIZATION WITHIN THE BULB SO IF WE FOCUS ON THAT, WE DEMONSTRATED THERE'S A CONNECTION BETWEEN THE TWO BULB THAT'S SPECIFIC AND LINKED THE TWO HALF ANDS N A SPECIFIC AMERICAN. AND SO THIS IS REALLY THE STRUCTURE THAT DRAWS A LOT OF OUR ATTENTION, A LOT OF EXPERIMENTS AND SO, GOING TO TAKE JUST ANOTHER MINUTE TO DESCRIBE THE CIRCUITRY THAT UNDERLIE THIS IS STRUCTURE HERE. O I'M GOING TO SPLIT THIS IN HALF AND GIVE YOU A QUICK WIRING DIAGRAM AND THE SURFACE OF THE OLFACTORY BULB AND MEDIAL AND LATERAL SIDE AND THIS IS A BEAUTIFULLY LAMINAR STRUCTURE SIMILAR TO THE RETINA FACT, YOU SEE THE ACTION LIKE JOSHUA IS SAYING IS IN THE IPL. SENSORY NEURONS COME IN ON THE SURFACE AND THEY FORM GLUE MARIA, AND HERE'S THE PAIRS,OT SURFACE. THEY COMMUNICATE WITH THE PLAIN OUTPUT NEURONS OF THE BULB AND THE MIGHTERAL CELLS AND THE CELL BODIES LIVE DOWN HERE AND THEY SEND PROJECTIONS UP TOWARDS THE SURFACE AND FORM SYNAPSE SIS WITH THE INPUT AND THEIR OUTPUTS GO DIRECTLY TO THE CORTEX, THE PERIPHERAL CORTEX AND THERE ARE TWO POPULATIONS OF INTERNEURONS, THERE ARE THE PERIGLUE MARRIAL CELLS AND THESE SURROUND EACH AND COMMUNICATE WITH THE INPUT AND OUTPUT NEURONS AND MOTTULATE THAT COMMONICATION AND THEN THERE ARE THE GRANULAR CELLS AND THEY LOOK DOWN HERE AND THEY SEND UP TOWARDS THE SURFACE AND FORM SYNAPSIS ON THE DENDRITES ON THE CELLS ABOVE. NOW THE LAST JAP I WILL ADD, THESE ARE THE ONES THAT ACTUALLY MEDIATE THIS LINK BETWEEN THE TWO HALVES INVOLVED SO THAT THE MEDIAL PROJECTED LATERAL AND THE LATERAL TO THE MEDIAL. AND YOU LOOKA THIS DIAGRAM AND THERE SHOULD BE A FEW THAT JUMP OUT AT YOU AND ONE THAT IT'S REPEATED UNITS OR MODULES OR COLUMNS OF CELLS AND WE CALL THESE EACH WON OF THESE AN ODOR COLUMN AND THE SECOND IS THAT AT THE TOP OF ONE OF THESE COLUMNS IS THE LIST THAT ACTS AS AN ORGANIZING HUB AROUND WHICH MANY OF THE CELL TYPES ARE ARRANGED BUT ALSO DETERMINES THE RESPONSE PROPERTIES BELOW BECAUSE IT'S REALLY THE INPUT FROM THE SENSORY NEURONS THAT REALLY DETERMINES WHAT ACTIVATE THIS IS AMOUNT OF NEURONS. SO IF WE EXTRACT ONE OF THESE AND WE FOCUS ON THIS IT, THIS IS A MODEL CIRC NUT OUR LAB THAT WE'VE BEEN FRYING TO UNDERSTAND IT GETS INPUT DIRECT FROM THE ENVIRONMENT, THE OUTPUT GOES TO THE CORTEX AND IT HAS TWO POPULATIONS OF INTERNEURONS AND THEN MODULATE THE ACTIVITY AND THE ASSOCIATIONAL CONNECON THAT LINKS IT TO SORT OF THE HIGHER ORDER STRUCTURE. SO WE REALLY WANT TO UNDERSTAND HOW THIS FORMS AND HOW ACTIVITY THAT MAY FLOAT FLEW THE CIRCUITRY HELPS TO SHAPE AND MAINTAIN IT. AND WHAT I WILL TELL YOU IS THAT THERE ARE THREE FACTORS HERE WITH GENERATION PLASTICITY AND ACTIVITY THAT ARE ACTUALLY INTERACTING IN SORT OF AN INTERDEPENDENT WAY TO BOTH HELP ESTABLISH AND MAINTAIN THE CIRCUITRY. SO TO DO THAT I WILL FOCUS ON A CENTRAL COMPONENT OF THIS CIRCUIT AND THAT IS THIS BULGING NEURON. WE FIRST BECAME AWARE OF HOW SPECIFIC THIS PROJECTION REALLY WAS BY MAKING TRACER INJECTIONS IN TRANSGENIC ANIMALS IN WHICH A SINGLE GLUE MAR LIAISONS LABELED WITH GFP AND THAT IS WHAT THAT LOOKS LIKE HERE AND YOU'RE LOOKING AT THE DORSAL SERVICE IN WHICH THIS HERE IS LABELED WITH GFP AND THIS ALLOWED US TO TARGET IT HERE AND ASK ABOUT WHAT CONNECTIONS IT'S FORMING WITH ITS OUTPUT. HERE AIAN SECTION HERE AND THEY DETERMINEINATE DIRECTLY BENEATH PARTNER ON THE OPPOSITE SIDE OF THE BALL AND VICE VERSA IF YOU SEE ETERNAL TOUGH HERE BENEATH THIS ONE EMPLOY. AND WHAT THIS IS SAYING WAS THAT PAIRS OF IT ARE COMMUNICATING WITH ONE ANOTHER, THROUGH ODOR COLUMN CIRCUITRY IN A SPECIFIC MANNER, SO IF WE JUST HIGHLIGHT THE TERMINAL HERE AND COLON COAT THEM THE WAY OF FROM WHERE THEY EMERGE AND THEN WE REALIZE THAT THERE ARE MANY PAIRS OF THE THIS IS THEY FORM THE SAME--CONNECTIONS WHAT WE REALLY D SIDE A MAP THAT'S--LIES UNDERNEATH THE SURFACE, MADE UP OF CENTRAL NEURONS BUT SIMILAR IN BOTH AN ORGANIZATION AND SPECIFICITY TO THE MAP OF THE SRFACE. SO WE REALLY WANT TO UNDERSTAND THE NATURE OF THIS AND HOW IT'S DEVELOPED AND SEE WE STARTED LOOKING AT THE EARLY STAGES OF THESE NEURONS AND I WILL SUMMARIZE MANY YEARS OF WORK AND WE FOUND THAT THESE PROJECTIONS ARE ACTUALLY PRESENT AT BIRTH AND IN THEIR IMMATURE STATE THEY'RE QUITE BROAD AND THEY SPAN A VERY BROAD AREA OF INFLUENCE. THROUGH DEVELOPMENT, THEY THEN MATURE AND BY SEVEN WEEKS OF AGE THEY REACH THIS WHAT WE CALL ADULT SPECIFICITY. WE ALSO FOUND OUT THAT ACTIVITY IS NECESSARY FOR THIS MATURATION PROCESS SO IF YOU BLOCK ACTIVITY THE PROJECTIONS SPREAD. NOW THE INTERESTING PART OF THIS IS THAT THEY NOT ONLY SPREAD MEANING IF YOU BLOCK ACTIVITY AT BIRTH, THEY FAIL TO REFINE BUT EVEN AFTER REFINEMENT IF YOU BLOCK ACTIVITY THEY SPREAD OUT. HERE'S AN EXAMPLE OF THAT AND YOU CAN SEE ONE OF THESE INJECTIONS HERE, CONTROL, FORMS A SMALL TERMINAL TUST ON THE OTHER SIDE, THIS IS THE CONTROL OF MATURE PROJECTION. HERE WE BLOCKED ACTIVITY FROM SEVEN-10 WEEKS OF AGE AND THEN THE OLFACTORY SYSTEM IS EASY TO BLOCK ACTIVITY, YOU PLUG THE NOSTRIL AND YOU BLOCK ACTIVITY. SO WE BASICALLY SHOWED HERE THAT THE ROUGH ATOM GEEKSS ARE QUITE A BIT BROADER THAN THEY ARE IN THE CONTROL STATE. NOW WHAT WE WEREN'T AWARE WAS WHETHER WHEN WE'VE DISRUPTED THESE PROJECTIONS WHETHER TE SORT OF STUCK IN THIS PHASE AND A TALENTED POST DOC IN THE LAB ANNA CUMMINGS PIONEERED THE REVERSIBLE CONCLUSIONS SO HE CAN PUT IN A PLUG AND REMOVE IT AT A LATER STAGE AND WE FOUND IF YOU FULL THE PLUG AND LET NORMAL ODORS BACK IN, THESE PROJECTIONS RESILIENCE FINE AND IT TAKES SIX TO NINE WEEKS OF TIME BUT THEY REALLY COME BACK DOWN TO MATURE LEVELS. THIS IS JUST A NINE WEEK CONTROL. SO THIS REALLY IMSTRAIGHTED THAT WHEN WE RESTORE ODOR INDUCED ACTIVITY WITH THIS DISRUPTION, THESE PROJECTIONS ARE CAPABLE OF RESILIENCE FINING. THAL EFFECT WE SEE CONTINUES THROUGHOUT THE LIFE OF THE ANIMAL. SO THIS SAID TO THAT THESE WERE ATTAINED THROUGH ADULTHOOD AND DON'T FORM THROUGH ANY CHRISICAL PERIOD THAT WE'RE AWARE OF. SO THE ABERRANT QUESTION IS HOW DO THEY MAINTAIN THIS CONSTANT STATE OF PLASTICITY. THERE'S VERSE FORMS OF PLASTICITY THAT OCCUR IN THE SENSORY NEURONS BUT THE SENSORY NEURONS REGENERATE. THESE CELLS WERE NOT AWARE OF REGENERATING SO WE STARTED TO ASK, PERHAPS THERE ARE OTHER NEURONA POPULATIONS THAT INTERACT WITH THESE THAT MODULATE THE CIRCUITRY. AND INDEED, THERE ARE UP UNTIL NOW I'VE BASICALLY SHOWN YOU, THE INTERPROJECTION SORT OF AN ISOLATION, THIS IS JUST A TRANSGENIC LINE IN WHICH ALL OF THESE NEURONS HAVE BEEN LABELED THE INTERPROTECTING NEURONS HAVE B LABELED WITH THE TOMATO AND HERE YOU SEE THE IPL LAYER AND THAT JUST SHOWS AN INTENSE DENSITY OF THESE PROJECTIONS. I'LL LET YOU SEE THIS CLOSE UP AND CAN YOU SEE THE COMPLEXITY OF THESE OBJECTIONS WITH THE TRACER OF OBJECTIONS AND THESE PROJECTIONS DON'T EXIST IN ISOLATION. IN FACT THERE ARE MANY MANY CELL TYPES THAT ARE PRESENT NOT ONLY IN THE THIS LAYER BUT RUNNING THROUGH THE LAYER AND IN PARTICULAR THE INTERNEURONS GRANULAR CELLS THAT ARE SHOWN EARLY ON THAT THE CELLS LIKE TO FORM SYNAPTIC CONNECTIONS WITH. AND YOU CAN SEE THAT THESE GRANULE CELLS EVEN THOUGH I LUMP THEM INTO ONE CATEGORY COME IN QUITE A VARIETY IF WE JUST USE A COUPLE DIFFERENT MARKERS HERE WE SUBDIVIDE THEM INTO MANY CELL TYPES NOT ONLY IN SHAPES BUT WHERE THEY PROJECT AND THE AREAS THEY INFLUENCE AND THEY ALL--EITHER LIVE IN OR PASS THROUGH THE IPO LAYER. IF WE ASK ABOUT WHAT HAPPENS WITH THE INTERLABEL PROJECTIONS WE FIND THEW THESEAXONS LIKE TO WRAP AROUND THESE GLUE MARIOUSICOSITYS ON THE BODIES, THE CELL TYPES WHICH USUALLY INDICATE SYNAPTIC ACTIVITY SO WE KNOW THESE LIKE TO ASSOCIATE WITH THESE INTERNEURONS BUT THE IMPORTANT FACTOR HERE I NEED TO REEMPHASIZE IS THAT THESE OLFACTORY BULB ISHT ARE NEURONS REGENERATE AND WHAT DO WE KNOW ABOUT THESE. THESE ARE NEURONS THAT ORIGINATE IN THE SUBVENTRICULAR ZONE. THEY MIGRATE INTO THE BULB TO THE MIGRATORY STREAM AND ONCE A REACH THE CENTER OF THE BUTB THEY RADIATE TO THE SURFACE AND BECOME GRANULAR CELLS OR GLUE MARRIAL CELLS SO THIS IS A CONSTANTLY REGRENERATING--REGENERATING POPULATION. NOW WE USE MICE CALLED GAB 65 GFP IF WHICH THESE NEURONS LABELED WITH GFP AND I WANT TO GIVE YOU AN APPRECIATE FOR THE AMOUNT OF REGENERATION THAT'S REAMEE GOING ON HERE. THIS IS BASICALLY JUST A SHORT CLIP FROM A SLICE PREPARATION THAT SHOWS YOU THE MIGRATION OF THESE NEURONS THROUGH THE RMS. THIS IS ABOUT TWO HOURS OF TIME AND YOU CAN SEE THERE'S A FLOOD OF NEURONS, THOUSANDS OF NEURONS THAT INTEREST CONTINUOUSLY THROUGHOUT THE LIFETIME OF THIS ANIMAL. ONCE THEY REACH THE OLFACTORY BULB, THEY TAKE ON DIFFERENT CHARACTER AND SIMILAR TO WHAT MAT WAS SHOWING THEY HAVE THIS LEADING PROCESS THAT BASICALLY EXTENDS OUT AND ESSENTIALLY REACHING AROUND FOR CUES AS THEY CLIMB UP THROUGH THE LAYERS TO TRY AND FIGURE OUT HOW THEY'RE GOING TO INTEGRATE INTO INTO CIRCUITRY. SO THEN THE QUESTION WAS REALLY DO THESE REGENERATING INTERNEURONS PLAY A ROLE I THIS PLASTICITY. SO TO DO THIS WE COLLABORATE WIDE A COLLEAGUE AT NIH, AND IRRELEVANT WHAT INSTITUTE SHE'S IN BUT SHE'S WORKING ON THE HIPPOCAMPAL PLASTICITY AND SHE DEVELOPED A NEW LINES OF MICE THAT WE THOUGHT WOULD BE PERFECT FOR OUR EXPERIMENTS SO SHE'S DRIVING THE HSV, THE HERPES SIMPLEX VIRUS PROTEIN UNDER THE CONTROL OF TWO DIFFERENT PROMOTERS THAT ARE ACTIVE IN EARLY PROJECT GENITOR NEURONS AND IN PARTICULAR THE SVZ AREA. SO NEURONS THAT EXPRESS THIS IN THE PRESENCE OF GANNA PSYCHOVIRTHIS IS KILLING THE NEURONS SO THIS WAS AN ATTEMPT OF WIPING OUT THE FLOW OF NEW NEURONS COMING IN. SO WE CROSS THESE WITH GAB GFP MICE AND CAN YOU SEE IN THE MINUS THESE ARE ANIMALS THAT DO NOT HAVE GENES AND HERE'S THE SVZ REGION AND RMS AND OLFACTORY BULB WITH LARGE NUMBERS OF NEURONS THERE AND IN THE PRESENCE OF THE TK, YOU CAN SEE THAT WE'VE ESSENTIALLY ELIMINATED THE RMF. SO THE QUESTION IS, NOW WE'VE GOTTEN RID OF THIS, FLOW WITH NEURONS WHAT EFFECTS DOES THIS HAVE ON THE PLASTICITY WE'VE B STUDYING AND SO, TO WUT TO THE CHASE WITH BOTH LINES OF MICE WE COMPARED ANIMALS THAT HAVE AN RMSP AND THOSE THAT DON'T HAVE AN RMS. AND WE FOUND IF YOU ELIMITINATE THE RMS, THE INJECTIONS SPREAD. SO THE FLOW OF NEURON SYSTEM NECESSARY AND THIS QUANTIFYS THE CHANGES THAT WE SEE HERE THAT OUR EYES ARE SHOWING US BASICALLY. THIS BASICALLY EMPHASIZES THAT THESE RMS NEURONS ARE NECESSARY TO KEEP THE CENTRAL BULB OR MAP REFINED BUT IT'S SORT OF A BROADER LEVEL IT SHOWS THESE ARE CRITICAL IN MAINTAINING THIS ORGANIZATION AND THIS IS ACTUALLY AN IMPORTANT POINT. THIS IS A DEFINED CIRCUIT AND FIRST TIME THIS HAS BEEN SHOWN. SO, I'LL SUMMARIZE THIS WAY. THIS IS THE CIRCUIT WE'VE BEEN STUDYING AND TRIED TO DESCRIBE HOW WE SORT OF COME TO FOCUS ON THIS. I TOLD BUT THE GLUE MARRIAL LAB AND BULGAR MAP AND HOW THEY REGENERATING NEURONS PARTICIPATE IN THIS ORGANIZATION AND WHAT I HOPE I LABORATORIED THE SEED WITH THE FACTORS REPLASTITY ARE WORKING TOGETHER IN AN INTERDEPENDENT WAY TO CONTRIBUTE TO THE FUNCTIONAL STABILITY OF THE CIRCUIT AND PROBABLY MOST IMPORTANT ALL OF THIS IS HAPPENING DURING ADULTHOOD AND CONTINUES TO HAPPEN. SO, I WANT TO THANK THE FOLKS INVOLVED AND I DIDN'T HAVE TIME TO TALK ABOUT ALL THE VARIOUS PROJECTS THAT ARE GOING ON IN OUR LAB. AND IN FACT MOST OF THE WORK I TALKED ABOUT IS DONE BY TWO PEOPLE, DIANNA CUMMINGS AND CAROLYN MARKS, TWO FORMER PEOPLE IN THE LAB, IT'S A SMALL LAB, EVERYBODY PARTICIPATE INDEED THIS WORK AND OF COURSE MY TWO COLLABORATORS THAT I HAVE SORT OF ONGOING COLLABORATIONS WITH, AND NICK AND HEATHER WHO YOU WILL HEAR FROM. [ APPLAUSE ] >> THANK YOU FOR A GREAT TALK. IT OCCURRED TO ME IN THE SPIRIT OF MICKING UP INSTITUTES WHILE MICE USE EACH YEAR TO DISTINGUISH DIRECTION CAN A MOUSE USE THE NOSTRILS TO ALSO DETECT DIRECTION? THE TIMING BETWEEN THE BULBS SEEM TO BE PLAYING A ROLE. WHETHER THEY'RE OVERLAPPING ROLES OVERLAPPING OR DISTINCT ISN'T REALLY CLEAR BUT I THINK SOME OF THE SAME PRINCIPLES ARE INVOLVED. >> THANK YOU. SO I WANT TO THANK ALL THE SPEAKERS FOR STAYING ONTIME. I THINK THEY'VE DONE A SPECTACULAR JOB NEXT SPEAKER IS DR. NICK RIFFA IN THE SENSORY BIOLOGY AT NIDCR WHERE HE'S STUDYING HOW TASTE PERCEPTOR CELLS STIMULATION TRANSMITTED TO THE BRAIN IS RAINS MITTED TO THE BRAIN AND ENCODED THERE. HE WILL DISCUSS LOGIC BY WHICH THEY DETECT THE PERRIFFRY REPRESENT INDEED THE BRAIN AND HOW THIS REPRESENTATION HELPS GUIDE BEHAVIOR. NICK, THANKS. ACCIDENT. >> THANK YOU VERY MUCH, STORY: I HAVE TWO CONFESSIONS, FIRST IS THAT WE'RE NOT DEVELOPMENTAL BIOLOGISTS BUT BECAUSE OF THE STRONG SENSORY FOCUS THIS SESSION, I PUSHED STORY TO PUT US-- >> VERY HARD, VERY HARD. >> TO PUT MY TALK INTO IT. AND THE SECOND CONFESSION IS THAT BECAUSE THIS IS SUCH A BROAD SYMPOSIUM I TRIED TO KEEP MY TALK VERY GENERAL. SO IT'S A GREAT PLEASURE TO BE TALKING HERE TODAY AND ALL THE MORE SO BECAUSE MIRROR IMAGE GROUP AND OTHER SENSORY BIOLOGISTS FOR THE ISSUES HAVE JUST MOVED INTO FANTASTIC NEW SPACE IN THIS BEAUTIFUL BUILDING WHERE WE'RE SURROUNDED BY NEUROSCIENTISTS WITH SIMILAR FOCUS AND USING TECHNIQUES, VERY OFTEN COMPLIMENTARY. AS I SAID, I WILL KEEP THE SENSORY FOCUS OF THIS MORNING'S SYMPOSIUM AND TALK A LITTLE BIT ABOUT OUR WORK ON TASTE. WHICH AS YOU KNOW IS A SENSE THAT PROVIDES US WITH ENJOYMENT THROUGHOUT OUR LIVES. BUT FOR ANIMALS EVOLVE FOOD PROTEIN INTAKE AND AS SUCHs THE SENSE OF TASTE IS AN EXTREMELY SIMPLE ONE. ANIMALS DISCRIMINATE BETWEEN JUST A FEW DIFFERENT TASTE QUALITIES. HUMANS DISTINGUISH PERHAPS JUST FIVE DIFFERENT TASTES ALL OF WHICH ARE VERY FAMILIAR THROUGH BITTER, SOUR, SWEET, SALTY AND THE FIFTH TASTE, WHICH IS EQUALLY FAMILIAR, SAVORY TASTE BUT GOES BY THE LESS FAMILIAR NAME [INDISCERNIBLE]. IMPORTANTLY. SIMPLE COMPOUNDS ELISSEITY VERY STRONG AND EASILY RECOGNIZABLE PERCEPTUAL QUALITIES SO FOR EXAMPLE, THIS MOLECULE, CYCLOHEX MID TASTES BETTER WHEREAS THIS MOLECULE WHICH I THINK IS SACCHARIN IS CLEARLY SWEET. AND MORE OVER THIS TASTE QUALITY HAVE INEIGHT MEANING--INNATE MEANING. NOT A LOOED THE WORK BUT A LOT OF HARD WIRING AND THE CIRCUITRY AND SPECIFIED BY IRG, SO WE THINK THAT TASTE PROVIDINGS AN EXTRAORDINARY POWERFUL SIMPLE PLATFORM TO START TO EXPLORE HOW SENSORY INFORMATION IS DETECTED AND THEN HOW ABOUT THAT SENSORY INFORMATION IS REPRESENTED IN THE BRAIN IN ORDER TO ELISSEITY DEFINED PERCEPTIONS. AND NOT ONLY TO TASTE PROVIDE US INFORMATION BUT OLES COMBINE OTHER SENSORY INPUT AND ACTUALITY MATILY WE WOULD LIKE TO KNOW HOW FOR EXAMPLE, OLFACTION AND TASTE COMBINE TO PRODUCE FLAVORS OF FOOD THAT WE COMMONLY REFER TO AS TASTE. AND--OKAY, SORRY. --AND MORE OVER, HOW HOW I EXPERIENCE AN INTERNAL STATE FOR INSTANCE OF HUNGER OR SATETY OR HOW WE USE TASTE TO GUIDE BEHAVIOR. SO IN WONDERFUL AND LONG-TERM WIDE RANGING COLLABION WITH CHILD ZUKA WHO'S NOW AT COLUMBIA UNIVERSITY, WE'VE DEMONSTRATED OVER THE PAST FEW YEARS A LIMITED NUMBER OF RECEPTORS AND CELL TYPES INVOLVED IN DETECTING TASTE IN MICE. AND I SHOULD STATE THAT BASED ON THE GENES WE WOULD SAY THAT HUMANS REALLY ARE ESSENTIALLY JUST VERY LARGE MICE. SO TWO FAMILIES OF PROTEIN LINKED RECEPTORS, TONE Rs, THE SMALL FAMILY OF THREE RECEPTORS AND A LARGER FAMILY OF 30 TTWO R INVOLVED IN DETECTING SWEET AND BITTER TASTE. AND IN FACT THE THREE T ONE ERROR COMBINED FOR TWO RECEPTORS, ONE MADE TONE ARE ONE AND TR-THREE ACTIVATED BY AMINO ACIDS AND MODULATED ALISTERICALLY BY PURE NEW CLE O TIDE AND RESPONSIBLE FOR THE TASTE. AND THE OTHER RECEPTOR MADE UP OF TONE RTWO, AGAIN COMBINED FOR TONE RTHREE BUT FORMS A SINGLE RECEPTOR THAT DETECTS ALL THREE TASTES INCLUDING NATURAL SWEETNESS LIKE SUGAR AND ARTIFICIAL SWEETENERS LIKE SACCHARIN OR HK. THE T-TWO RS LARGER FAMILY OF 30 RECEPTORS INDIVIDUALLY REDETECTED A SMALL RANGE OF BITTER COMPOUNDS AND MANY OF THESE BITTER COMPOUNDS IN FACT ARE HIGHLY TOXIC AND ARE COMPOUNDS THAT ANIMALS WERE TO ENCOUNTER IN THEIR NATURAL LIFE. SO HOUR AND SALTY TASTE ARE MEDIATED BY MECHANISMS DESPITE THE FACT THAT THEY'RE SUCH SIMPLE POLARIZED KIEWLS TRIGGER AND SOUR TASTE AND MEDIATED BY CELL WHICH IS EXPRESS THIS ION CHANNEL PKD 12-ONE AND THE TASTE OF SODIUM CHLORIDE AND MEDIATED BY ANOTHER ION CHANNEL ENABBING AND CLEARLY THE MOST DEFINITIVE DATA SHOWING THAT THESE--THESE ARE IN FACT B RECEPTORS MEDIATE TASTE COME FROM GENETICALLY ENGINEERED ANIMALS SO WHAT I'M SHOWING HERE ARE NEVER RESPONSES FROM WILD-TYPE MICE AND GENETICALLY ENGINEERED MICE IN RESPONSE TO TASTE STIMULATION OF THE TASK AND YOU CAN SEE THAT ALL FIVE DIFFERENT BASIC TASTES QUALITIES ELISSEITY STRONG RESPONSES IN WILD-TYPE MICE. BUT IN THE KNOCKOUT MICE SPECIFIC AND SELECTIVE RESPONSES ARE LOST THE RECEPTOR T-ONE R-ONE SELECTIVELY ELIMITINATES THE MAMMY RESPONSES WHILE PRESERVING ALL OTHER RESPONSES. KNOCKOUT MICE WITH LACKING TONE RTWO HAVE LOST SWEET TASTE RESPONSES. KNOCK OUT TO THE CORECEPTOR T-ONE R-THREE ELIMINATE THE RESPONSES KNOCK OUT OF TTWO R FIVE ELIMINATE RESPONSES FOR THE LIGAND WHILE PRESERVING OTHER TASTE RESPONSES. KILLING PKD 201 CELLS ELIMINATES SOUR TASTE. AND ENABBING IS CLEARLY NECESSARY FOR'S SPONSES TO SODIUM CHLORIDE. SO WE JUST HAVE TO MEDIATE TASTE BUT THEY ALSO TELL US A LOT MORE ABOUT HOW THE TASTE SYSTEM WORKS. I WANT TO CONCENTRATE ON JUST STREET POINTS. THE FIRST IS THAT THE RECEPTOR PREPPER 24 FOR THE ANIMAL IS RESPONSE FOR ITS PARTIC TASTE SENSITIVITY. THE SECOND IS THAT THE DIFFERENT TASTE QUALITIES ARE REPRESENTED BY COMPLETELY NONOVERLAPPING POPULATIONS IN CELLS AND THE THIRD IS THAT THE ACTIVATION OF THE CELL POPULATIONS RATHER THAN RECEPTORS PER SE OR EVEN CONTASTE WHICH IS IS WHAT IS DETECTED AND IN EACH CASE, I JUST WANT TO GIVE ONE EXAMPLE. I SAID EARLIER THAT MICE ADJUST VERY LITTLE TO HUMANS, BUT THIS FACT, THIS REAL ISN'T THE CASE BECAUSE THIS TASTE ANT ASPAR TAME OTHERWISE KNOWN AS NUTRISWEET IS TASTED TO HUMANS BUT MICE ARE UNINTERESTED IN IT IN BEHAVIORIAL ACIDS BUT IF WE TRANSGENICLY EXPRESS THE HUMAN SWEET TASTE RECEPTOR TONE RTWO I THE SWEET CELLS OF MICE WE HUMANIZE RESPONSES OF MICE AND THEY NOW SHOW MARKED BEHAVIOR PREFERENCE TO ASPARTAME. THE SECOND SPOINT THAT THE RECEPTORS ARE DIFFERENT IN NONOVERLAPPING SITUATIONS AND WHAT I'M SHOWING HERE IS HYBRIDIZATION THROUGH A REGION OF THE TIME WHERE THEIR TASTE BUTS AND WHAT YOU CAN SEE IS THAT THE THREE PERCEPTORS IN CREE ARE IN COMPLETELY SEPARATE CELLS FROM THE RECEPTORS SHOWN IN RED. AND AS I SAID, IT'S IT IS CELLS, NOT THE RECEPT THEMSELVES PER SE, AND THEY'RE IMPORTANT FOR DETECTION. AND TO ILLUSTRATE THIS, WHAT I WANT TO SAY IF WE ACTIVATE SWEET CELLS WE GET A SWEET RESPONSE NO MATTER HOW WE ACTIVATE THE CELLS. AND THE INCUT, SHOW YOU THAT WE--WE DID THIS WHICH WE ACTUALLY MISS EXPRESSED A BITTER TASTE RECEPTOR, HUMAN TTR-16 IN SWEET, SWEET CELLS AND THE BEHAVIOR OF MICE FROM RESPONDING AS IF THEY'RE DETECTING SOMETHING BETTER IN DETECTING SOMETHING SWEET. AND THIS EXPERIMENT IS POSSIBLE AGAIN BECAUSE MICE AND HUMANS HAVE A DIFFERENT REPERTOIRE OF BITTER TASTE RECEPTORS AND BITTER TASTE. SO, WHEREAS THIS COMPOUND IS BETTER TO HUMAN, AND IT ACTS THROUGH A SPECIFIC HUMAN, TTWO R, MICE HAVE NO BEHAVIOR RESPONDENT TO IT, IF THEY LACK THIS, THIS RECEPTOR. IF WE TAKE THIS RECEPTOR AND NOW EXPRESS IT IN THE BITTER CELL OF MICE, THE MICE SHOW HUMANIZED RESPONSES AND ADVERSION TO THIS BITTER TASTE, BUT IF INSTEAD WE PUT THE VERY SAME RECEPTOR NOW INTO THE SWEET CELL, THE MICE NOW SHOW STRONG ATTRACTIVE RESPONSES THEY'RE TASTE THANKSGIVING BITTER CHEMICAL THROUGH A BITTER RECEPTOR AS IF IT WERE SWEET. OER ALL OUR STUDIES ON THE PERFIVERY SUGGEST A SIMPLE PATTERN AND TASTE CODING AND REALLY THE BRAIN SIMPLY READS THE PATTERN OF TRC ACTIVATION TO DETERMINE THE QUALITY AND THE APPROPRIATE REACTION. I HAVE TO SAY THAT OUR RECENT TODAYS PARTICULARLY WITH HIGH CONCENTRATIONS OF SORTS SUGGEST THIS IS A BIT OF AN OVERLIMPNIFICATION BUT IT'S CLOSE. BUT WE ALL KNOW THAT TASTE ISN'T REALLY ABOUT SIMPLE SINGLE CHEMICALS. IT'S ABOUT MIXTURES. AND REALLY WE ALL KNOW AS WELL, THAT WHEN JUST MIX WIDE A BIT OF SUGAR, TASTES COMPLETELY DIFFERENT FROM EITHER THE LEMON JUICE OR THE SUGAR ALONE EVEN THOUGH THE Ph HASN'T BEEN ALTERED. SO IN ORDER TO UNDERSTAND THIS, WE HAVE TO UNDERSTAND HOW THE SIGNALS ARE TRANSMITTED TO THE ENTERTAIN AND THEN REPRESENT IT THERE BOTH TO GENERATE PARTICULAR PERSONS AND TO GUIDE BEHAVIOR. AND IN THE REST OF THIS TALK, I WILL CONSCION TRAITOT GANGLIA FOR TWO REASONS, FIRST OF ALL BECAUSE TIME'S PRESSING AND SECONDLY BECAUSE IT ACTUALLY PREVENTS AN INTERESTING DEVELOPMENTAL PROBLEM. THE IN EFFECTOUS SYSTEM--SORRY, THE NERVOUS SYSTEM AS I--AS I'M SURE THAT YOU'RE--YOU'RE ALL WELL AWARE IS REALLY VERY CONSTANT BUT THE CASE RECEPTOR CELLS HAVE A LIMITED LIFE. THEY LIVE FOR ONE TO THREE WEEKS AND ARE CONTINUE CONTINUOUSLY TURNING OVER SO THERE'S DEVELOPMENTAL PROBLEM ALL THE TIME OF HOW YOU WIRE THESE CELLS CORRECTLY TO PRESERVE THE CONSTANT SCENE OF TASTE WE ALL KNOW. SO THE BEST WAY TO DETERMINE THE SPECIFICITY OF GANGLION CELLS WOULD BE TO EMAN THE NEURAL RESPONSES, WE COULD MEASURE MANY CELLS SIMULTANEOUSLY, BUT WE HAD TO OVERCOME TWO PROBLEMS, FIRST OF ALL WE NEEDED A ROBUST FUNCTIONAL REPORTER ACTIVITY AND SECONDLY THE GANGLION ARE IN ACCESSIBLE. SO TO OVERCOME FIRST, WE WERE AIDED BY THE DEVELOPMENT OF NEW GENETICALLY ENCODED CALCIUM SENSORS WHICH PROVIDE FOURFUL TOOLS THE IMPAGGING NEURAL ACTIVITY AND WE DEVELOPED BOTH TRANSGENIC AND APPROACHES FOR THESE VIRAL SENSES IN GANGLION NEURONS. TH SECOND PROBLEM WAS A BIT MORE COMPLICATED. I THINK MOST PEOPLE ARE WELL AWARE THAT 2-PHOTON MICROSCOPY HAS BEEN USED TO GREAT EFFECT TO IMAGE DEEP IN THE INTO TEX AND IMAGE IMAGE THE RESPONSE INTO T CORTEX BUT IT HAS A LIMITED PENETRATION OF ABOUT .4 TO .5-MILLIMETERS AND THE TASTE GANGLIA ARE VARIED DEEP IN AEN BOY CAVITY UNDERNEATH THE BRAIN ABOUT 4-MILLIMETERS FROM THE SURFACE. SO IN ORDER TO IMAGE FROM THE GANGLION WE NEEDED TO ADD FIRST OF ALL AN APPROACH TO ACCESS THE GANGLIA IN THE MICE WITHOUT--WITHOUT DAMAGING EITHER THE NERVES OR THE GANGLIA AND SECONDLY WE MADE USE OF GRADED REFRACTED INDEX LENSES TO ACT AS MICRO ENDOSCOPES TO PROJECT AN IMAGE OF THE GANGLIA FROM THE DEEP WITHIN THE MOUSE TO THE SURFACE WHERE IT COULD LEND THE IMAGE IN TAKING THIS APPROACH WE COULD MANAGE THE G-COUNT IN THE GANGLIA NEURON. WE HAVE GOOD RESOLUTION AND WE COULD SEE RESPONSES FROM THESE NEURONS, WHAT I'M SHOWING HERE IS THE RESPONSE TO TASTE IN FOUR DIFFERENT EXPOSURES REPEATED TWICE FOR SOUR AND SWEET TASTE AND WHAT YOU CAN SEE IS THAT IT'S CERTAIN POPULATIONS TO CELLS THAT IN EACH OF THE FOUR PANELS RESPOND ROBUSTLY AND ONCE MORE IF YOU LOOK CAREFULLY YOU SEE THE ACTIVATION OF THE PANELS WAS VERY SIMILAR AND SWEET AND SOUR ELISSEITYED QUITE DIFFERENT RESPONSES. AND I WANT TO SHOW THAT AGAIN, THIS TYPE YOU CAN TRUENESS TRAIT ON WHAT YOU WANT ON SEE, THE SAME PATTERN OF YOU'REONS IS ACTIVATED IN PANELS AND THEY ARE ACTIVATED BY A DIFFERENT TASTE MPLET IN SUMMARY, SUMMARIZING THE RESPONSES OF A LARGE NUMBER OF GANGLION NEURONS WE GET STRIKING PICTURE, BASICALLY 70 PR OF THE NEURONS ARE ATTUNED JUST EXACTLY LIKE THE TASTE RECEPTOR. RESPONDING TASTING UPON THEM. AND MORE THAN HALF OF THE REST RESPOND TO JUST TWO OF THE POSSIBLE 26 COMBINATIONS THAT THAT COULD BE. A LARGE NUMBER RESPOND TO THE TWO PRINCIPLE ADVERSIVE TASTES PITTER AND SOUR, AND SMALLER BUT SIGNIFICANT NUMBER RESPOND TO THE TWO ATTRACTIVE TASTES. SWEETENED AND SOUR. I DON'T WANT TO DWELL ON THIS BUT THIS IMAGES APPROACH PROVIDE ACE PLATFORM FOR NOW IDENTIFYING MARKERS THAT WILL ALLOW US TO FUNCTIONALLY DISTINGUISH BETWEEN THE DIFFERENT TYPES OF GANGLION CELLS PERHAPS DEFINING MORE OF THIS HANDOUT SHAKE BETWEEN THE PRCs AND THE NERVOUS SYSTEM AND EXPLORING THE CONSTANCY OF TASTE IN THE LIGHT OF TURNOVER OF THE CONTINUOUS TURNOVER OF THE TASTE RECEPTORS. AND FINALLY, I WANT TO MENTION THAT STATE DOESN'T JUST GET QUALITY IT PROVIDES VALENT INFORMATION, SOME OF TASTE SEEMS TO INVOLVE CORTICALE PROCESSES SO THE CONSCIENCE DECEPTION OF TASTE AND SOME GOES BY UNCONSCIOUS OR SUBCONSCIENCE MEANING PRODUCING ALMOST REFLEX RESPONSES, IMMEDIATE TASTE RESPONSES PROBABLY SINCE THE BRAIN STEM. AND THIS PLATFORM PERHAPS PROVIDES A GREAT WAY OF ESTABLISHING BETWEEN THESE TWO. SO I WANT TO FINISH BY GOING BACK TO THE VIEW FROM THE PORTER--NEUROSCIENCE BUILDING AS A BACK DROP TO ACKNOWLEDGE A LOT OF PEOPLE WHO WERE INVOLVED IN THIS WORK. THIS PICTURE WAS TAKEN YESTERDAY MOR ACCOUNTING FOR THE DARK RESPONSE GRAYNESS AND LOOKIN AT THE DIRECTION OF THE PICTURE WAS TAKEN FROM AND IN PARTICULAR I WANT TO HIGHLIGHT THREE PEOPLE, CHARLES ZUKA WHO EASY BEEN A WONDERFUL COLLABORATOR. CHARLES AS MOST OF YOU DON'T KNOW IS A BRILLIANT SCIENTIST AND ALSO A CLOSE FRIEND. A JEREMY AND MARK WHO WAS INVOLVED WITH THIS WORK FROM THE START. AND GER AMY IS ON A FARM WHERE HE'S TRYING TO IMAGE ON VERY FINE DETAIL A LONG RANGE NEURAL CONNECTIONS IN THE WHOLE BRAIN AND MARK HAS A GROUP HERE IN THE BUILDING WHERE HE'S WORKING ON SOMATIC MUTATION PARTICULARLY STUDYING [INDISCERNIBLE] PAIN NICHE. SO IN CLOSING I WANT TO SHOW YOU THIS IMAGE. THIS IS--THIS IS CLEARLY-HAD OF AS PHILOSOPHERS AND ARTISTS HAVE POINTED OUT, IT'S NOT A BRAIN AT ALL, BUT MERELY AN IMAGE THAT OUR BRAINS RECOGNIZE. IT'S A GREAT DEAL OF OPTIMISM INDEED THE WHOLE NEUROSCIENCE COMMUNITY BUT OVER THE NEXT FEW YEARS, NEUROSCIENCE WILL START TO UNRAVEL WHAT MAKES BRAINS A MUST AND WE HOPE THAT IN PERHAPS THE SMALL WAY WE ARE WORK ON TASTE CAN HELP. THANK YOU VERY MUCH. KD--SALLY PLACE. [ APPLAUSE ] >> [INDISCERNIBLE]. SO THE LAST SPEAKENER IN SESSION IS DR. HEALTHEN CAMERON. CHIEF OF THE UNIT SIX. NEUROPLASTICITY IN ONE OF THE OTHER INSTIT IN THE PORTER BUILDING WHERE SHE'S FOCUSED ON ADULT NEUROGENESIS AND YOU HEARD OF ONE OF HER CONTRIBUTIONS. THIS IS THE FIELD SHE'S BEEN INVESTIGATING FOR MORE THAN 20 YEARS AND TODAY SHE WILL DISCUSS THE REGULATION OF ADULT NEUREE GENESIS IN THE ROLE OF NEWLY GENERATED DISEASES AND IN THE HIPPIE CAMPUS. THANK YOU VERY MUCH HEALTH HEATHER. >> THANK YOU FOR THE NICE INTRODUCTION AND FOR INCLUDING ME IN THIS GREAT SESSION THAT IS ALMOST OVER NOW. >> SO THE WORK IN MY LAB IS UNDERSTANDING ADULT NEUROGENESIS AND AS CHRIS ALLUDED TO EARLIER, MOST OF THE NEURONS IN THE BRAIN ARE PRODUCED BEFORE BIRTH AND CAN'T BE REPLACED EVEN IF THEY'RE LOST DUE TO ILLNESS OR INJURY, HOWEVER THERE ARE A FEW POPULATIONS OF NEURONS THAT CONTINUE TO BE GENERATED THROUGHOUT LIFE. THESE INCHUTE THE OLFACTORY ONES WHICHLY O TALKED ABOUT AND THE NEURONS IN THE SHI REGION OF THE HIPPOCAMPUS AND THESE ARE WORK THAT MY FOCUS ON. SO THE WORK IN THE HIPPOCAMPUS CONTINUES THROUGHOUT LIFE BUT NOT AIAN CONSTANT RATE. IT'S HIGHLY REGULATED BY EXPERIENCE AND OTHER EXTREENSIC FACTORS SO IT'S INHIBITED BY STRESS, AS WELL AS GIEWT O CORTICOIDS WHICH ARE INDOCKS NOWS ZONES ALSO WIDELY USED ANTIINFLAMMATORY MEDICATIONS, INHICK THE BIDE DRUGS OF ABUSE AND UNFORTUNATELY ALSO BY AGING. ON THE OTHER HAND NEUROGENESIS CAN BE INCREASED BY ENRICHMENT, RUNNING AND ESTRO JEAN AND MANY DIFFERENT FORMS OF ANTIDEPRESSANT TREATMENT. MOST OF THIS REGULATION, ALL OF THIS WORK IS ORIGINALLY FROM RODENTS SOME OF THESE FINDINGS HAVE BEEN REPLICATED IN NONHUMAN PRIMATES BUT WE DON'T YET KNOW FOR SMSM THAT RUNNING INCREASES NEUROGENESIS IN HUMANS SO WE DON'T HAVE TO GET ON THE TREAD MILES RIGHT AWAY. SO THIS PHOTOSHOWS A CROSS SECTION THROUGH THE GYRUS AND YOU SEE THE NEURONS SITTING HERE ON THE INNER EDGE OF THE GRANYULE CELL LAYER WITH THE MATURE COUNTERPARTS UP HERE IN BLUE. SO THE GRANULAR NEURONS ARE VERY SMALL, TIGHTLY PACKED NEURONS AND UNLIKE THE NEUROGENESIS THAT OCCURS IN THE OLFACTORY BULB THESE NEURONS ARE BORN FROM NEURAL STEM CELLS TO SIT RIGHT HERE IN SUBGRANULAR ZONE WITH THE NEURONS SO THEY MIGRATE VERY LITTLE IF AT ALL. THESE NEURONS FORM COMPLEX FOR DENDRITIC TREES WHICH YOU THINK I CAN SEE UP HERE IN GREEN, THEY SEND AXONS INTO THE REGION LIKE THE NORMAL--LIKE THE MATURE DEVELOPMENTALLY BORN GRANULAR NEURONS, SLIGHT PHYSIOLOGY EXPERIMENT SO THEY RECEIVE SYNAPTIC INPUT AND THEY MAKE ACTION POTENTIALS AND USING IMMEDIATE EARLY GENES WHICH ARE SHOWN HERE AS RED AS MARKERS OF NEURONAL ACTIVATION, CAN YOU SEE THESE BORN IN ADULTHOOD ARE ACTIVATED IN VIVO BY NORMAL PHYSIOLOGICAL EXPERIENCE. SO ALL SIGNS POINT TO THESE BEING FUNCTIONAL NEURONS BUT WHAT IS THEIR FUNCTION. SO THIS IS THE MAIN FOCUS OF OUR WORK RIGHT NOW. WE ARE TRYING TO--WE WOULD LIKE TO KNOW WHAT THE ROLE OF NEW NEURONS IS IN LIVING AND MEMORY SO THESE SIT IN THE HIPPOCAMPUS WHICH IS PRIMARILY A ROLE--THE PRIMARY ROLE OF THE HIPPOCAMPUS IS IN LEARNING AND MEMORY SO IT MAKES SENSE THIS THESE MUST DO SOMETHING THAT RELATES TO L AND MEMORY. BUT AS I MENTIONED ANTIDEPRESSANTS ALSO INCREASE NEUROGENESIS, SO DO THESE NEW NEURONS PLAY SOME ROLE IN ANXIOUS AND DEPRESSION. ONE WAY TO BEGIN TO LOOK AT BOTH OF THESE QUESTIONS IS TO ASK WHAT HAPPENS IF WE STOP ADULT NEUROGENESIS? AND TO DO THIS, WE MADE THE MICE THAT LEALLY O DISCUSSED BEFORE. THESE MICE EXPRESS HE WERIES VIRUS UNDER THE GFP PROMOTER SO ALL OF THE YAIDIAL STEM CELLS THAT THE NEURONAL PRECURSORS IN THE GRANULAR CELL LAYER EXPRESS THIS VIRAL GENE AS DO ASTRO SIGHTS. BUT THIS GENE HAS NO FUNCTION ON ITS OWN SO THESE ANIMALS CAN GROW AND DEVELOP NORMALLY AND THEN IN ADULTHOOD WHEN WE GIVE AN ANTIVIRAL DRUG VAL CYCLOVIRTHIS KILLS ANY CELL THAT TRIES TO DIVIDE. SO IT KILLS THESE PRECURSORLE CELLS WHILE LEAVING THE ASTRO SIGHTS UNDISTURBED. SO THIS IS A PREDICTION AND HAPPILY IN THIS CASE, THIS LOOKS TO BE TRUE. SO YOU CAN SEE THESE ARE TREATED TRANSGENIC ANIMALS AND TREATED WILD-TYPE AND YOU CAN SEE THE ASTRO SIGHTS HERE IN RED IN SIMILAR NUMBERS IN BOTH CASES AND WHEN WE COUNT THEM WE FIND THAT'S THE CASE, WE SEE NO LOSS OF ASTRO SITES THANKFULLY IN THESE MICE. ON THE NEURONSOT OTHER HAND ARE COMPLETELY ELIMINATED IN THE TRANSGENICS THAT HAVE BEEN GIVEN THIS VAL PSYCHE LOW VIRDRUG SO NOW WE HAVE A DRUG WHERE WE CAN INHIBIT NEUROGENESIS. SO WE THOUGHT THAT ONCE WE HAD THIS MODEL ALL WE WOULD HAVE TO DO IS RUN THE ANIMALS THROUGH NEURAL HIPPOCAMPUS BEHAVIOR DEPENDENT TASKS OF WHICH ARE THERE SEVERAL THAT ARE CHARACTERIZED AND WE WOULD FIGURE OUT WHICH ONES ARE AFFECTED. SO WE SPENT TWO YEARS LOOKING AT DIFFERENT VERSIONS OF THE WATER MAZE WHICH IS THE SPACIAL NAVIGATION TASK AND CONTEXTURAL FEAR CONDITIONING IN AVERSIVE MEMORY TASK WITHOUT MUCH SUCCESS. SO, THIS ACTUALLY ISN'T OUR DATA THIS IS FROM MICHAEL DREW'S LAB BECAUSE HE AS A MUCH NICER GRAFF OF THIS BUT CAN YOU SEE THESE ANIMAL VS NO NEUROGENESIS AND HIS EXPERIMENT, THEY USED IRRADIATION TO ELIMINATE THE NEUROGENESIS, THESE LEARN THE WATER MAZE TASK JUST FINE AND TWO WEEKS LATER THE ANIMAL CANS STILL FIND THE HIDDEN PLATFORM WITH NO PROBLEMS. WE LOOKED AT CONTEXTURAL FEAR AND ANIMALS DID THESE TASKS. WE DID MANY DIFFERENT VERSIONS OF THE WATER MAZE, MOVING THE PLATFORM AROUND, MOVING THE CUES, ROUND, CONTEXTURAL FEAR CONDITIONING WE MADE THE ANIMALS DISCRIMINATE BETWEEN DIFFERENT CONTEXT AND WE HAD VERY FEW EXPERIMENTS IN WHICH WE FOUND ANY DIFFERENCES. LUCKILY WE FOUND A FEW EXPERIMENT WHERE IS WE FOUND BEHAVIORIAL DIFFERENCES WE COULDN'T REPLICATE THEM RELIABLE BUT WHEN WE WENT BACK AND LOOKED AT BEHAVIOR IN THE EXPERIMENTS WHERE WE SAW THIS HINT OF A DIFFERENCE WHAT WE FOUND WAS IN THE MORRIS WATER MAZE THE ANIMALS THAT WERE BEHAVING DIFFERENTLY WERE CIRCLING THE EDGE OF THE POOL AND THIS HAS BEEN DESCRIBED BEFORE AS A STRESS RESPONSE, SO WE WONDERED MAYBE THESE ANIMALS DON'T HAVE LEARNING AND MEMORY DEFICIT BUT MAYBE THEY HAVE A DIFFERENCE IN THEIR STRESS RESPONSE SO TO LOOK AT THEW WE DID THE SIMPLEST TACT WE COULD THINK OF WHICH WAS TO TAKE THE ANIMALS RESTRAIN THEM IN THE TUBE A FOR 30 MINUTES AND THEN MEASURE THEIR STRESS RESPONSE BY MEASURING LEVELS OF CORTICOSTER OWN, THE ENDOGENOUS RODENT GLUCOCORTICOID. THE PRIMATE AN LOG WOULD BE CORTISOL. SO WHEN WE DO THIS RESTRAINT IMMEDIATELY AFTER--AT THE END OF THIS RESTRAINT PERIOD WE SEE NO DIFFERENCE. THE ANIMALS WITHIN AND WITHOUT NEUROGENESIS HAVE A ROBUST GLUCOCORTICOID RESPONSE BASELINE WOULD BE 50 HERE WHEN WE LACK AT LATER TIME POINTS YOU CAN SEE THAT THE ANIMALS WITHOUT NEUROGENESIS PROA LONGED RESPONSE. THEY DON'T RETURN TO BASELINE LEVELS OF KORT AS QUICKLY. SO THIS SURPRISED US BECAUSE THERE'S NO LEARNING OR MEMORY INVOLVED IN IN EXPERIMENT. SO WE REPEATED THE SAME TEST USING A DIFFERENT MODEL, USING ERADIATION TO ELIMINATE NEUROGENESIS AND WE FOUND THE SAME THING. NO DIFFERENCE IMMEDIATELY AT END OF RESTRA BUT THE ANIMALS TAKE LONG TORE RETURN TO BASELINE. AND USING IRRADIATION WE CAN ALSO SEPARATE OUT THE HIPPO HIPPOCAMPAL NEUROGENESIS FROM THE OLFACTORY NEUROGENESIS SO WE WEREN'T SUCCESSFUL IN ALL THE ANIMALS BUT IN THREE OF THESE ANIMALS WE HAD--WE DIDN'T TOUCH OLFACTORY NEUROGENESIS AT ALL THEY WE ONLY OBLIGATIONS BLATTED NEUROGENESIS IN THE HIPPOCAMPUS AND YOU CAN SEE THAT THESE CLEARLY SHOW THIS EFFECT. SO SO WE'RE CONFIDENT THAT THIS INCREASED STRESS RESPONSE IS PRODUCED BY THE LOSS OF THESE HIPPOCAMPAL NEURONS. SO WE WANTED TO KNOW IF THIS ENHANCED STRESS RESPONSE HAD A BEHAVIORIAL PHENOTYPE SO SINCE STRESS AND DEPRESSIVE BEHAVIOR ARE CLOSELY ASSOCIATED WE DECIDED TO LOOK AT DEPRESSION TEST NOVELTY SUPPRESSED FEEDING SO IN THIS TEST, YOU PUT HUNGRY ANIMALS INTO A LARGE ARENA WITH FOOD IN THE CENTER. AND THEN YOU ASK HOW LONG IT TAKES THE ANIMALS TO OVERCOME THEIR FEAR OF THIS WIDE OPEN SPACE IN THE CENTER OF THE ARENA TO BEGIN EATING THE FOOD AND AS YOU CAN SEE UNDER CONTROL CONDITIONS THE ANIMALS WITHOUT NEUROGENESIS IN BLUE, SHOW THE SAME BEHAVIOR AS THE NORMAL ANIMALS AND THIS--THIS FIT WITH WHAT RENE'S GROUP HAD ALSO SEEN. HOWEVER WHEN WE STRESS THE ANIMAL USING THE EXACT SAME PROTOCOL I SHOWED IN THE LAST SLIDE, 30 MINUTES OF RESTRAINT AND THEN PUT ANIMALS IN THE TEST, THIS STRESS HAD NO EFFECT ON THE NORMAL ANIMALS. BUT THE ANIMALS WITHOUT NEUROGENESIS SHOW THE INCREASED DEPRESSIVE LIKE BEHAVIOR. SO,--SO IT SEEMS THAT HAVING NEW NEURONS ALLOW BUFFERS AGAINST STRESS AFFECTING--STRESS EFFECTOT BEHAVIOR OF THESE ANIMALS. SO NOW OUR QUESTION IS HOW IS THIS WORKING HOW DOES THIS AFFECT THE BEHAVI OF ANIMALS IN IN TASK. SO ONE POSSIBILITY IS THAT GLUCOCORTICOIDS DIRECTLY AFFECT THE BEHAVIOR. Y KNOW THAT GLUCOCORTICOIDS INJECTED INTO AN ANIMAL CAN CHANGE BEHAVIOR BUT THIS ENHANCED GLUCOCORTICOID RESPONSE REWE REIS DELAYED NEGATIVE FEEDBACK CAUSES BEHAVIORIAL CHANGE AND SO THE NEW NEURONS WOULD ACT HERE ENHANCING KORT WHICH WOULD THEN AFFECT THE BEHAVIOR SO THIS IS OUR ORIGINAL THOUGHT OF HOW THIS WORKED BUT WE NOW HAVE PILOT DATA SHOWING IF WE CLAMP CORD LEVELS AND DON'T ALLOW THEM TO INCREASE IN RESPONSE TO STRESS, WE SEE THIS SAME BEHAVIORIAL CHANGE SO WE NOW THINK THAT THE NEW NEURONS MUST BE ACTING UPSTREAM OF BOTH KORT, AFFECT AND BEHAVIORIAL EFFECT AND ENDOCRINE DEHAIRIAL EFFECT AND THESE TWO BEHAVIORIAL EFFECTS ARE SOMEWHAT INDEPENDENT OF EACH OTHER. E WHAT COULD THIS BE? OUR CURRENT THINK SUGGEST THAT WHAT THE NEW NEURONS MIGHT BE DOING HERE IS AFFECTING STRESS PERCEPTION. SO MEANING--AFFECTING THE MOUSE'S ABILITY, MOUSE'S DETERMINATION WHEN IT SEES SOMETHING LIKE THIS GOOFY CAT IT HAS TO DETERMINE WHETHER THIS IS A REAL THREAT, WHETHER THIS ANIMAL--HOW LIKELY IT IS THAT THIS STIMULUS IS GOING TO COME CHASE HIM AND WHETHER HE NEEDS TO CHANGE HIS BEHAVIOR IN RESPONSE TO THIS POTENTIAL THREAT SO NOW INSTEAD OF GIVING A SUMMARY, I WANT TO TOUCH A LITTLE BIT ON NEUROGENESIS AND OTHER REGIONS OF THE BRAIN. SO I MENTIONED EARLIER THAT NEUROGENESIS IS SEEN IN THE OLFACTORY BULB AND IN THE GYRUS, THIS IS WHERE IT'S ACCEPTED BY VIRTUALLY EVERYBODY AT THIS POINT, I THINK BUT ADULT NEUROGENESIS IS DESCRIBED IN SEVERAL OTHER REGIONS WHERE IT'S STILL VERY CONTROVERSIAL BECAUSE MANY PEOPLE LOOK IN KNEES BRAIN REGIONS AND THEY SEE NO NEUROGENESIS. BUT I THINK THIS MIGHT BE BECAUSE OF THE POPULATIONS THAT ARE ACTUALLY BEING PRODUCED IN THESE OTHER BRAIN REGIONS. SO SEVERAL YEARS AGO, IN MY GROUP WE LOOKED AT CORTICALE NEUROGENESIS IN THE RAT. THERE'S A LOT OF TIME IN THE ADULT NEUROGENESIS DESCRIBED IN THE PRIMATE BUT IT WAS VERY, VERY CONTROVERSIAL. SO WHEN WE LOOKED IN THE RAT WE DID FIND NEW NEURONS BEING BORN. THEY WERE LABELED WITH PAN NEURONAL MARKERS AND--BUT WHAT WE FOUND WAS THAT THEY WEREN'T THE--THE BIG PATHWAY GIVES RAMTAL NEURONS WE EXPECT TO FIND BECAUSE PATHWAY GIVES RAMTALS MAKE UP SEIVET% OF THEM IN THE CORTEX SO THAT'S WHERE WE STARTED OUR SEARCH BUT IT TURNED OUT THAT THE NEW NEURONS WERE GABAergic INTERNEURONS AND NOT ALL OF THE GABAergic INTERNEURONS BUT THEICAL BINNIAN AND POSITIVE NEURONS WHICH ARE SMALL IN NUMBER AND SMALL IN SIZE AND I THINK IT'S THESE DIFFERENCES THAT MAKE IT SO DIFFICULT TO FIND NEUROGENESIS NOT ONLY IN THE CORTEX BUT ALSO IN ALL OF THE OTHER BRAIN REGIONS WHERE ALL OF THE EVIDENCE TO DATE SUGGESTS THAT NSIS IN THIS REGION IS ALSO IN SUBPOPULATIONS OF INTERNEURONS. SO THIS CARTOON JUST SHOWS THAT ESSENTIALLY THEY THINK THE DENT A GYRUS AND OLFACTORY BULB ARE NOT BECAUSE OF ADULT NEUROGENESIS BUT THEY'RE SPECIAL BECAUSE OF THE ORGANIZATIONS AND THE LARGE NUMBER OF NEURONS IN THESE POPULATIONS MAKE NEUROGENESIS EASIER TO FIND. WISE IN OTHER REGIONS, THESE SMALL INTERNEURON POPULATIONS ARE SO SMALL THAT IF YOU REGENERATE THE SAME--SAME PROPORTION OF NEW NEURONS AS YOU DO IN THE DENTATE, YOU WILL END UP WITH A SMALL NUMBER OF CELLS AND THEY'RE WIDELY SCATTER INDEED THESE COMPLEX AND VERY COMPLEX REGIONS AND DIFFERENT ORGANIZATIONS MYSELFATIONS AND THEY THERAPIST MAKES THEM DIFFICULT TO FIND. BUT THEY ARE THERE, AND I THINK AS SOON AS WE HAVE THE TOOLS THAT ALLOW US TO FIND THESE NEURONS MORE EASILY, I THINK THIS WILL OPEN UP A WHOLE NEW FRONTIER FOR UNDERSTANDING ADULT NEUROGENESIS AND IT'S ROLES IN THE BRAIN. SO I GIST WANT TO END THERE BY THANKING THE PEOPLE IN MY LAB AND ESPECIALLY PEOPLE WHO HAVE NOW LEFT MY LAB INCLUDING JASON SNYDER OTHER DID THE WORK, AND ALEX DAYER WHO DID THENY O CORTEX WORK AND JIM MITCHELL WHO HELPED US WITH THE IRRADIATION EXPERIMENTS. [ APPLAUSE ] >> THANK YOU. >> [INDISCERNIBLE]. >> HI, VERY NICE TALK. DID YOU CHECK IN OLDER ANIMALS THE ROLE OF THE [INDISCERNIBLE] THAT YOU KILL THIS BECAUSE THAT'S ALREADY A STRESS CONDITION IN SOME SENSE AND I WAS WONDERING IF YOU FIND SOMETHING? >> SO IT'S KNOWN THAT ADULT MUREEE GENESIS RECREASES IN OLDER ANIMALS AND A LOT OF THE BEHAVIORIAL FINDINGS THAT YOU SEE IN OLD ANIMALS ARE CONSISTENT WITH A ROLE FOR THESE YOUNG NEURONS OR ARE CONSIST WENT A DROP IN NEURONS BEING THE CAUSE BUT IT HASN'T ACTUALLY BEEN DEMONSTRATED WHICH--BECAUSE WE STILL ARE KIND OF DISAGREEING ON WHAT THE FUNCTION OF THE NEW NEURONS IS, WE CAN'T TELL WHICH OF THE DEFINITES YOU SEE IN AGING ARE DUE TO THE LOST NEURONS. >> [INDISCERNIBLE]. >> IF YOU KILL THIS THEM IN OLD AGE. THAT'S A GOOD QUESTION, WE HAVEN'T D IT. >> GREAT. I HAVE A FEW MESSAGES. THANK YOU VERY MUCH. THE SPEAKERS AND THE SESSION ORGANIZERS IF THEY COULD GO TO THE YELLOW SKY BOX THAT WOULD BE GREAT. THE CAP--SECOND FLOOR. THE CAFETERIA IS SERVIN LUNCH. WE NEED TO BE BACK AT 1:00 O'CLOCK PROMPTLY. BECAUSE THE DEDICATION STARTS AT THREE AND WE NEED TO BE UP THERE AND ABLE TO GO. AND I WOULD LIKE TO ADD TO WHAT STEVE SAID, WHILE DONNA AND OMB WERE RESPONSIBLE FOR GETTING US THE MONEY FOR PHASE ONE, THE FIRST PART, PHASE TWO WAS FUNDED BY ARRA, THE STIMULUS MONEY AND IT WAS FUNDED BY ARRA BECAUSE WHEN THE ARRA MONEY WAS GRANTED OR PUT IN THE NIH BUDGET OF 10 MILLION DOLLARS, WE HAD PLANS, 95% PLANS THAT MEANT THAT THE BING WAS SHOVEL READY SO, YES, SHOVEL READY AND THAT MEANT IT WAS THE END OF THE GREEN PLASTIC WALL. THANK YOU VERY MUCH AND THANK THE SPEAKERS. THEY DID A GREAT JOB OF BEING ONTIME AND WONDERFUL SCIENCE. IT'S REALLY MY PLEASURE TO CHAIR THE SECOND SESSION OF THIS SYMPOSIUM IN CELEBRATION OF THE NEW PORTER BUILDING. FOR THOSE OF YOU WHO DON'T KNOW ME, I'M SUSAN OMARA, AND I'M THE SCIENTIFIC DIRECTOR, THE NIMH INTRAMURAL PROGRAM. IT'S MY PLEASURE TO BEGIN INTRODUCING OUR FIRST SPEAKER, CURRENTLY THE PROFESSOR OF FIZZ PHYSIOLOGY AND BIOFIPHYSICS AT THE UNIVERSITY OF CALIFORNIA IN SAN FRANCISCO AND ALSO A MEMBER OF THE HOWARD HUGHES MEDICAL IN INSTITUTE. SHE EARNED HER DOCTOR AL DEGREE IN FIPHYSICS AND BIOFIZPHYSICS FROM CALTECH, WORKING WITH SEYMOUR, WHO IS REALLY THE FATHER OF MANY OF APPROACHES TO USING GENETICS TO STUDY THE NERVOUS SYSTEM AND OTHER SYSTEMS AS WELL. SO TOGETHER WITH HER LONGTIME COLLABORATOR, JUNG, SHE CONDUCT CONDUCTED GENETIC STUDIES OF FRUITFLIES IN MICE THAT REALLY HAVE YIELDD MAJOR INSIGHTS INTO BRAIN FUNCTION AND DEVELOPMENT. LITTLY WAS ACTUALLY THE FIRST TO MOLECULAR LLY IDENTIFY NUMBERS OF THE POTASSIUM CHANNEL FAMILY, WHICH ARE CRITIC AL REGULATOR S HE OF CELLULAR CREDIBILITY AND KEY ELEMENTS IN THE SIGNALING SYNAPSE THAT OCCURS. AND IN THAT WAY, SHE REALLY DID A REMARKABLE FEAT, WHICH OPENED THE WAY FOR STRUCTURE FUNCTION STUDIES OF ION CHANNELS AND MANY OF THE ASPECTS THAT WE KNOW AND HAS FUNCTIOND AS THE KEY ELEMENTS OF BIOCHANNEL FUNCTION. SHE'S A RESCIPIENT OF NUMEROUS CARCINOGEN AWARDS, INCLUDING THE 2012 NON-SCIENCE PRIZE, THE JER GERARD PRIZE FROM THE OWE SEW SOCIETY FOR NEWUROSCIENCE AND ONE OTHER ELEMENT, WHICH I'D LIKE TO HIGHLIGHT ABOUT LITTLY IS THAT SHE'S BEEN A SPECTACULAR MENT OR TO MANY PEOPLE WHO HAVE GONE ON TO BE LEADING FIGURES IN THE FIELD. AND SO SHE'S RECEIVED THE OUTLINSTANDING MENT OORSHIP AWARD FROM THE POST DOCTOR AL SCHOLARS ASSOCIATION AT U COMMERCIALIC SF. AND TODAY WE JUST HAVE THE PLEASURE OF HAVING HER COME TO SPEAK TO US AND SHE'S GOING TO TELL US ABOUT THE ROLE OF CHANNELS IN HEALTH AND DISEASE. WELCOME, LITTLY. IT'S MY PLEASURE TO WELCOME YOU. APPLAU [APPLAUSE] >> THANK YOUCH, SUSAN. AND VERY HAPPY TO BE HERE FOR THIS CELEBRATION. SO IN MY LONG TERM COLLABORATION TO STUDY MODULATION OF NEWURO NEUROACTIVITY, WE RECKOND IT HAS SO MANY DIFFERENT TYPES OF ION CHANNELS SO WE THOUGHT TO STUDY THE CHANNEL FUNCTION AL REGULATION. AND WE START WITH MOLECULAR IDENTIFICATION SO THAT WE CAN STUDY THESE CHANNELS ONE TYPE AT A TIME. AND SO TODAY, I WILL FIRST GIVE A QUICK SUMMARY OF THE A APPROACHES WE'VE TAKEN TO IDENTIFY THE FAMILY MEN'S FOR THESE THREE FAMILIES OF ION CHANNELS AND THEN I WILL TELL YOU ABOUT TWO RECENT STUDIES AS EXAMPLES FOR HOW UR INITIAL STUDIES OF CHANNELS IN THE FRUIT FRUITFLIES AND THE FROGS HAVE TAKEN US TO LEARN ABOUT THE CHANNEL INVOLVED IN MANY MAM AL IAN FIPHYSIOLOGY AND HUMAN DISEASE DISEASE. SO THE COLLABORATION STARTED WHEN WE WERE BOTH POSTDOCS IN SEYMOUR'S LAB AND WE SCREENED FOR BEHAVIORAL MUTANTS IN THE FRUITFLIES TO IDENTIFY WITH IN INCREASED EXCITABILITY AT A NEWUROMUSK COLLAR JUNCTION. THIS HYPEREXCITABILITY COULD BE FEPHENOCOPIED IN THE WILD TYPE PREPARATION BY JUST TREATMENT WITH THE POTASSIUM CHANNEL BLOCK BLOCKERS. AND THAT LED TO THE REALIZATION THAT THESE CHANNELS, OR THIS MUTANTS REFUSED ANCONNECTIVITY. AND THE CHANNELS ARE NO TORIOUS LY RARE AND HEATTEROGENIUS. SO LEARNING THAT IT WAS INITIAL LY IDENTIFIED BASED ON THE FEPHENO PHENOTYPE HAS REDUCED CHANNEL ACTIVITY, RAISED THE POSSIBILITY OF POSITION AL CLONING OF POTAUS POTASSIUM CHANNEL WITHOUT HAVING TO PURIFY THE CHANNEL. AND AT THAT TIME, CLONING WAS ONLY CONCEIVABLE IN THE FRUIT FLY BECAUSE OF THE CHROMAT CHROMOSOME. FOR EXAMPLE, IT'S MAPPED TO THIS X CHROMOSOME. AND STARTING WITH THE GENOMIC DNA, THAT HOPEFULLY IS NOT TOO FAR AWAY. ONE CAN IN PRINCIPLE, WORK AROUND THE CHROMOSOMES BY ISOLATING OVERLAPPING DNAS UNTIL ONE GETS TO THAT GENE OF INTEREST. YOU KNOW IN THE STUDY IN OUR LAB AT U COMMERCIALIC SF SHORTLY AFTER WE STAR STARTED IN THE LAB, RECEIVING NIH SUPPORT IN THE FORM OF RO1 FOR THE CHANNEL STUDIES. IT TOOK 6 YEARS OF TEAMWORK, BY SCHWARZ AND BRUCE FOR THEIR CHROMOSOME WORK. EL, IT TOOK BRUCE TEMPLE BUT ONCE WE KNOW POTASSIUM JUST 3 MONTHS USING SHAKER AS THE PROBE TO ISOLATE THE MAM AL IAN HOM OLOG BECAUSE OF THE STRONG SEQUENCE SIMILAR ITY. AND IN THE NEXT DECADE, THAT HAPPENED TO BE THE DECADE OF THE BRAIN, IT WAS LINK D TO EPISODEIC ATACXIA TYPE 1 BY CLINICIANS AND BIOFIPHYSICIST S IS IN ORGANIZEGON, AND THAT HAPPENED TO BE THE FIRST CHANNELOPTHY REPORTED. AND BY NOW, WE KNOW THAT THE CHANNELS SERVED EVOLUTION AARY TO pFUNCTIO N OF ENSURE IING JUST IUNI UNIDIRECTIONAL PROPAGATION IN BOTH VERTEBRAE AND INVERT BRATES LIKE SQUIDS AND FLIES. SO WITHOUT CHANNEL FUNCTION, THERE WOULD BE HYPEREXCITABILITY HYPEREXCITABILITY, AS SEEN IN THE SHAKER MUTANTS AND ALSO IN HUMAN PATIENTS WITH 81. AND WITH THE CONCERT WITHD EFFORTS OF MANY LABS IN THE CHANNEL FIELD, OTHER RELATED CHANNELS HAVE BEEN ISOLATED AND SEVERAL MEMBERS OF THIS CHANNEL FAMILY HAVE BEEN LINK D TO TEHE DISEASE OF THE HEART, BRAIN, AND ALSO FOR HEARING IN THE COKE LAR LAR. IN -- COKE LEIA. IN CONTRAST TO DIFFERENT SPECIES SPECIES, THERE IS NOT ENOUGH SEQUENCE HOMOLOGY FOR SHAKER TO BE USED AS A PROBE TO ISOLATE POOH TASSIUM CHANNELS THAT CONTROL HEART RATE. AND SO FOR THAT, IT WAS NECESSARY TO USE A DIFFERENT STRATEGY THAT WAS PIONEERD BY DAVID JULIUS, CALLED EXPRESSION CLONING. SO WITH THIS, IT IDENTIFIES. AND JUST INJECT POOLS OF C RNA TO ASSESS GENERATE THE CHANNEL WITH THE TECHNICAL CURRENT. THEN WHEN IN PRINCIPLE, SUBDIV SUBDIVIDE THE POOL UNTIL JUST ONE CLONE IS ISOLATED. SO THAT WAS HOW WE ISOLATED ELELECTIN BIORNA 1 BY MY UC SF COLLEAGUE. THE BROAD DIFFERENCE EXPLAINS THE HYPERTROPHIC EFFECT OF SYMPTOMS WITH PATIENTS WITH ANDERSON SYNDROME WITH PROBLEMS IN THE HEART, IN THE MUSCLE, AND ALSO NEWURODERIVED TISSUE. AND THE CONCERTD EFFORTS AGAIN OF THE CHANNEL LED TO US OF IDENTIFYING OTHER FAMILY MEMBERS THAT ARE LINK D TO DISEASES OF THE KIDNEY AND THE PANCREAS. AND WHILE WE ARE STUDYING INDIVIDUAL MEMBERS OF THESE CHANNEL FAMILIES FOR THEIR CONTRIBUTION TO NEWUROSIGNALING, WE ARE AWARE THAT EVEN IN THIS DAY AND AGE, THERE ARE STILL OR ORPHAN CHANNELS WITH END RESULT MOLECULAR IDENTITY. AND THE EXAMPLE HERE IS CASTLCIUM CALCIUM-ACTIVATED COLORIDE CHANNELS, C COMMERCIALIC. AS YOU CAN TELL FROM THIS TITLE OF THE REVIEW NOT SO LONG AGO. THESE CHANNELS WERE IDENTIFIED SOME TIME AGO, FOR INSTANCE, FOR THE CHANNEL IN A NEWURON. SO IT'S KNOWN FOR A LONG TIME, AND PEOPLE TRIED VERY HARD TO IDENTIFY THE CHANNE MOLECULE, PARTLY BECAUSE OF THE POTENTIAL OF THAERNT VALUES OF CACC MOD MODULATEORS AND FOR THE INTRINES INTRINSIC FUNCTION OF ALL THESE DIFFERENT CELL TYPES, INCLUDING THE GENE ALGAE. THIS IS A VERY ANCIENT, PRESENT ON EARTH BEFORE THERE WERE ANY CREATURES ON LAND. THESE ALGAE CELLS ARE VERY LARGE LARGE. THEY ACTUALLY USE ACTION POTENTIALS TO CONTROL PROTOTYPES STEERING. BUT THEY DON'T HAVE SODIUM CHANNELS, SO THEY RELY ON CASTLE CALCIUM-ACTIVATED COLORIDE CHANNELS TO GENERATE ACTION POTENTIAL. AND THE PROBLEM WITH THE MOLL MOLECULAR IDENTIFICATION PROBABLY COMES FROM THE EXPRESSION IN DIFFERENT CELLS, INCLUDING THE OOCYTE. CACC IS RESPONSIBLE FOR PERMIA. PREVENTING COL H AND WE CANNOT USE THEOOOH CITES AS THE EXPRESSION SYSTEM. AND FOR THAT REASON, WE DON'T SHOW -- WE RESORT TO TESTING THE FIPHYSIOLODGGIC SPERMIC EXOL OTTO AND HAVING THE INDODENDOGENOUS UCC EXPRESSION SYSTEM. AND THE OSI AS THE SOURCE FOR R RNA. THIS AGAIN TOOK 6 YEARS FOR EXPRESSION CLONING OF THE TM AND 16 A. TMEM STANDS FOR TRANSMEMBRANE PROTEIN FUNCTION. IS T JUST SO HAPPENS A GROUP IN KOREA AND A GROUP IN ITALY USED COMPLETELY DIFFERENT APPROACHES AND ENDED UP WITH THE SAME CONCLUSION AT THE SAME TIME THAT TM 16 A GAVE RIBES TO CASTLE JANELLI ACTIVATED COLORIDE CHANNELS. IN MAMMALS, THIS FAMILY HAS 10 MEMBERS. IT SHOWS THAT 16 A AND 16B BOTH GIVE RISE TO CACC AND BY NOW, FROM THE STUDIES OF MANY GROUPS, ING WE KNOW THAT 16 A HAS FAIRLY RESTRICTED EXPRESSION IN THE NERVOUS SYSTEM. BUT IT IS PROBABLY EXPRESSED IN DIFFERENT PERIPHERAL CELL TYPES. IT CONTROLS SECRETION FROM EXO EXOCRIN GLANDS LIKE THE SLAVER ARY GLANDS AND ALSO FROM AIR WWAY EPTHIELIA. IT CONTROLS RITHYTHMIC CONTRACTION OF THE GLANDS AND IT CONTRIBUTES TO THE POSITIVE FEEDBACK TO SUST SUSTAIN CASTLE NUM RISE AND CON CONTRACTION OF SMOOTH MUSCLE. 16B HAS VERY BROAD EXPRESSION IN THE NERVOUS SYSTEM. IT ACCOUNTS FOR THE LOW NOISE, HIGH AMPLE PLIIFICATION IN THE BACTERIA NEWURONS. IT PROVIDES FEEDBACK REGULATION OF CASTLCIUM ENTRY FOR THE LE CEPT CEPTOR TERMINALS. AND IT CONTROLS NEWURON AAL SIGNALING IN THE CENTRAL NERVOUS SYSTEM. TO OUR SURPRISE, CLOSELY RELATED FAMILY MEMBER GMEM 16 F IS NOT A CHLORIDE CHANNEL. WE FOUND THAT IT FORMS A SMALL CONDUCTOR CASTLCIUM ACTIVATED NON- NON-SELECTIVE KET ITINE CHANNEL HAS HIGHLY PERMEABLE TO CASTLCIUM. HOW COULD IT BE THAT THESE THREE SIMILAR APROTEINS WITH SIMILAR PROTEIN SEQUENCE WILL FORM IN ONE CASE CAT IION CHANNEL AND ANOTHER CASE AN IION CHANNEL? WHEN WE LOOK AT THE SEQUENCE ALIGNMENT, WE SEE IN THE TRANS TRANSMEMBRANE SEGMENT IN THIS POSITION. IT'S OCCUPIED BY LICE ENE IN CHLORIDE CHANNELS BUT GLUT AM INE IN THE OTHER CHANNELS. REPLACING MUTEAM ININE WITH LICE ENE REPLACES THE SELECTIVITY OF , FORMED BY 16 THE CAT I F AND REPLACING LICINE WITH DMRUT AM IINE REDUCED SELECTIVITY OF THE CHLORIDE CHANNE AND WHILE WE ARE WORKING WITH THESE CHANNELS, WE WERE SURPRISED AGAIN BY THE REPORT THAT TMEM 16 F IS LINK D TO THE CASTLCIUM. IT'S ACTUALLY NAMED AFTER THE PATIENT. AND IT GENERATED 16 F KNOCKOUT MICE TO FIND THAT IT IS A MOUSE MODEL FOR THIS BLEEDING DISORDER DISORDER, INDICATING THAT THIS PRO TEEN IS SOMEHOW REQUIRED FOR THE CASTLCIUM-ACTIVATED ACTIVITY IN PLATELETS. AND SO THE KNOCKOUT MICE WILL HAVE PROBLEMS WITH BLOOD QUOING COAGULATION TO STOP BLEEDING AND ALSO WITH STRONG -- STROFSHUS FORMATION AND WHICH IS A CAUSE FOR STROKE AND OTHER ARTERIAL DISEASES. SO TO SUMMARIZE WHAT THEY HAVE FOUND IN COLLABORATION WITH JOHN COCHRAN'S LAB, TMEM 16 F FORMS A CASTLCIUM ACTIVATED CAT IOION CHANNEL THAT'S PRESENT IN THE MEGACKARYO CITES OF WILD TYPE MICE. AND THIS IS SOMEHOW REQUIRED FOR THE CASTLCIUM-ACT AVAILABILITYD SCRAM BLAISE ACTIVITY TO EXPOSE FOPHOSPHOTIRINE JEEFRMENTZ THIS SERVED AS A PAD FOR VARIOUS PRO -- PROT AASES THAT IS IMPORTANT FOR BLOOD QUOING COAGLATION. A GROUP FOUND TMEM 16 AND FOUND THAT IT BEHAVES RATHER LIKE ONE MIGHT EXPECT FOR A PROTEIN. THAT IF IT'S ACTIVATED BY CASTLE CALCIUM, IT PERMEATES BOTH AN ION AND CAT IION AND ITS LIPIDS. AND FINDING BOTH AN IION AND CAT CHANNEL IN THE FAMILY -- SAME FAMILY IS NOT A SURPRISE. WE RECENTLY CAME ACROSS TM ENOL 16C AS A COLLABORATOR WITH A N OPOTASSIUM CHANNEL, AND I WILL TELL YOU ABOUT THIS STORY IN A LITTLE WHILE. BUT FIRST, LET'S GET BACK TO THE POTASSIUM CHANNEL. THIS IS THE FAMILY PORTRAIT OF POTASSIUM CHANNEL BY THE BRAIN. FAIRLY EARLY ON, MORGAN AND I'LL EILEEN REALIZED POTASSIUM CHANNELS IN THE DENDRITES ACTUALLY HAVE DIFFERENT COMPOSITION. SO TO STUDY CHANNEL REGULATION OF NEWURON AAL ACTIVITY, WE MIGHT NEED TO KNOW WHERE THE CHANNEL IS, THE COMPARTMENTS, AS WELL AS THE NUMBER AND PROPERTIES SINCE THE CHANNEL ACTIVITY REALLY DEPENDS ON BOTH THE NUMBER AND THE PROBABILITY. AND CONVERSELY, TO STUDY NEWURONO REGULATION OF CHANNELS, WE NEED TO KNOW ABOUT THE PLACEMENT OF THESE CHANNELS, AS WELL AS THE CHANNEL DENSITY AND PROPERTY. TRO NEWURON, WITH MANY FOR A S THOUSANDS OF SYNAPSES DISTRIBUTE DISTRIBUTED ON THE DENDRITES, AND YET WITH THE CAPACITY TO RESPOND TO A PARTICULARLY ACTIVE SYNAPSE AND REMODEL JUST THAT ONE IN A PROTEIN SYNTHESIS DEPENDENT MANNER, IT IS IMPORTANT OR TEMPTING TO CONSIDER THE POSSIBILITY THAT FOR SOME OF THE SIGNALING MOLL MOLECULES, THE RNA MAY BE DISTRIBUTED IN THE DENDRITE. SO THE LOCAL PROTEIN SYNTHESIS COULD BE REGULATED BY SIGNIFICASYNAPTIC ACTIVITY NEARBY. WE HAVE SEEN, FOR BOTH KV 4.2, THE MESSENGER RNA IN THE DEN DENDRITE AND THE LOCAL CHANNEL SYNTHESIS IN THE DENDRITE CAN BE REGULATED BY N M. B. A. RECEPT RECEPTOR ACTIVITY THROUGH A SIGNALING MATH WAY DWAY -- PATH WAWAY THAT INVOLVES MOLECULES LINK DD TO DISEASES THAT CAUSE A GREATER RISK FOR AUTISM. AND THAT IS TUBERCULOSIS AND SYNDROME. AND WE ARE CONTINUING WITH THESE STUDIES TO SEE HOW THE POTASSIUM CHANNEL DISREGULATION MIGHT CONTRIBUTE TO THE BEHAVIORAL FEPHENOTYPES IN THE MOUSE MODEL FOR THE SYNDROME. AS I MENTIONED BEFORE, TB 1 HAS THE EVOLUTION AARY FUNCTION FOR CONTROLLING PROPAGATION IN THE OX ONS. AND THEY HAVE MICROTUBULES ORIENT IING WITH DIGIT AAL. FOUND AX ONOTARGETING OF CHANNELS REQUIRES TWO MICROTUBULE PRO PROTEINS THAT BIND TO THE BETA TUBULES OF KD 1 CHANNEL AND T 3 AND THE PLASMA-BINDING PROTEIN E EB 1. ALL THESE CHANNEL PROTEINS AND THE MICROTUBULE-ASSOCIATED PRO PROTEINS ARE EVOLUTION AARILY CONSERVED. SO THAT PROBABLY ACCOUNTS FOR THIS UNIVERSAL TARGETING OF KD 1 CHANNELS. IN THE CASE OF EA 1 AND SEVERAL EPLEAPSS, SUCH AS B MCHLMC, THE DISEASE IS CAUSED BY A MUTATION IN THE POTASSIUM CHANNEL. THERE ARE OTHER DISEASES THAT ARE ASSOCIATED WITH OTHER EXPRESSION LEVELS OF CHANNELS. AND THE EXAMPLE I WILL USE IS THE UPREGULATION OF EA GCHG 2 IN A SUBSET OF MEDULOBLAST OOMA. THIS IS THE MOST COMMON PED YEAH PEDIATRIC BRAIN CANCER. AND THE TREATMENT WITH SURGERY, CHEMOTHERAPY AND RADIATION, AS YOU EXPECT, HAVE SIDE EFFECTS. AND THESE EFFECTS ARE PARTICULARLY SEVERE FOR LUNG PATIENTS. AND THE PROBLEMS INCLUDE CONTINUING DECLINE IN WORKING MEMORY, NEWUROEND CRINE DISEASE, HEARING LOSS, AND ALSO SECOND ARY MALIANG NANG NANCEIES THAT COULD BE CAUSED BY EITHER RADIATION DAMAGE OR METAS SAYS OF THE BRAIN TUMOR. AND BLAST OOMA IS QUITE HEATER GENIUS. THERE ARE DIFFERENT SUBGROUPS, GROUP 3, GROUP 4, AND MICE WITH ACTIVATION OF THE PATH WWAY WOULD DEVELOP MEDULOBLAST OOMA. BUT HWANG INITIAL LY WITH MICROW A ARAY ANALYSIS, EG 2 IS THE MOST UPREGULATED CHANNEL IN MOUSE MED MEDULOBLAST OOMA. HE THEN WENT DOWN TO ASSESS THAT HUMAN MEDULO BLAST OOMA WITH DIFFERENT HISTOLOGY AND MOLL MOLECULAR SUBGROUPS ALSO HAVE UP UPREGULATIONS OF EAG 2. AND WHEN HE INTRODUCED HUMAN MED MEDULOBLAST OOMA CELLS INTO MICE AND HAVE THESE CELLS TREATED WITH EITHER SCRAMBLED SH RNA, HE FOUND THAT REDUCTION OF EA GCHG 2 CAUSED A REDUCTION IN THE TUMOR GROWTH AND PROLONGD THE SURVIVAL OF THESE MICE WITH FEPHENOGRAPHS. -- XENOGRAPHS. AND LIKE WIWISE, IN THE KNOCKOUT MICE WITHOUT EA GCHG 2, THERE IS RE REDUCED GROWTH OF THE MOUSE, MED MEDULOBLAST OOMA, AND PROLONGD SURVIVAL. SO HOW WOULD COULD IT BE THAT THE CHANNEL IS IMPORTANT FOR THE TUMOR GROWTH? WHEN SHE LOOKED FOR THE EA GCHG 2 PROTEIN, SHE FINDS IT IN THE IN INTRACELLULAR COMPARTMENTS OF CELLS IN INTERPHASE. BUT THEN IT MOVES TO THE CELL SURFACE, AND STAYS ON THE SURFACE THROUGH MIGHT OTOSIS. KNOCKDOWN OF EA GCHG 2 CAUSE THESE M MB CELLS TO HAVE MUCH LARGER VOL VOLUME IN THE G 2 PHASE AND THIS LED TO CELL CYCLE APPARATUS. WHILE THE CONTROL CELLS TREATED WITH SCRAMBLE RNA WILL GO ON AND DIV WHIIDE. SO TO SUMMARIZE THIS PART OF THE FINDING, EA GCHG 2 POTAUSSIUM CHANNELS MOVED TO THE CELL SURFACE DURING CELL MIGHT O!TOSIS TO CAUSE THIS VOLUME REDUCTION, KNOWN AS THE PREMIGTOTIC CONDENSE CONDENSATION. AND WITHOUT THIS CHANNEL ACTIVITY, THE CELLS ARE M 2 DUE TO THE ACTIVATION OF THE 238 PATHWAY. AND THIS IS MOST LIKELY FOR THE VOLUME TO GO DOWN, IT INVOLVES THE FLOW OF WATER AND IONS, WHICH REQUIRE THE ION CHANNEL ACTIVITY. AND BUT IN THESE EXPERIMENTS, SHE NOTE -- NOTICED THAT THE CONTROLLED HUMAN MB CELLS WILL MOVE TO THE SPINAL CORD, BUT THERE IS NO METAS -- MET ASTAS IIS OF THE MB CELLS TREATED WITH SH RNA. AND THAT IS THE CASE, EVEN WHEN SHE WAITED SO THAT THE PLAYBILL ARY TUMORS ARE OF COMPARABLE SIZE. AND IF THAT'S THE CASE WITH -- AND OUR COLLABORATOR, MIKCHAEL TAILOR TALERY, AT THE HOSPITAL WITH SICK CHILDREN, ALSO NOTICED THAT FROM THE SAME PATIENTS, THE MET ASTATIC TUMOR SAMPLES TEND TO HAVE HIGHER LEVELS OF EA GCHG 2, COMPARED TO THE PRIMARY TUMOR. AND IN THE MOUSE MODEL FOR MEDUL MEDULOBLAST OOMA, THEY SAW A MUCH HIGHER LEVEL OF EA GCHG 2 PROTEIN IN THE MET ASTATIC TUMOR IN THE BRAIN STEM AND THE SPINAL CORD. THEN THE QUESTION IS HOW WOULD THIS CHANNEL AFFECT MET ASTAS IIS? HERE AGAIN, LOOKING AT THE CHANNEL DISTRIBUTION, WHILE AT THE INTERPHASE IT'S INTERCELL INTERCELLULAR. WHEN THE CELLS START TO MOVE EITHER SPONTANEOUS LY OR IN RESPONSE TO, THE CHANNEL MOVES TO THE POSTERIOR END OF MIGRATE MIGRATING CELLS. THE CELL MIGRATION ACTUALLY INVOLVES LOCAL MOVOLUME REGULATION REGULATION. THE FRONT END HAS INCREASE IN VOLUME AND THE BACK END HAS TO DECREASE IN VOLUME. AND THAT INVOLVES THE OUTFLOW, AGAIN, OF WATER IN IONS. THEN THAT IS LIKELY THE FUNCTION OF EA GCHG 2 AT THE EDGE. KNOCKDOWN OF EA GCHG 2, OR BLOCKING THE CHANNEL ACTIVITY WITH A CHANNEL BLOCK ER CAUSE THESE CELLS TO HAVE DIFFICULTY LIFTING UP ITS REAR END TO MOVE FORWARD. AND SO THE CELLS CANNOT REALLY POLARIZE, AND THEY CANNOT REALLY EXTEND THE CODE IA AT THE LEADING EDGE. AND AS A RESULT, THE CELL MOBILITY IS DECREASED BY EITHER KNOCKDOWN OR THE CHANNEL BLOCK ER. THE CHANNEL BLOCK ER IS HERE, RIGHT IN THE EARLY EXPERIMENTS. IT'S ACTUALLY AN ANTI-HISTAM IINE, AND MY COLLEAGUE, WHO MOVED TO TORONTO, BRIAN SHORTAN, POINTED OUT TO US THAT ANTI-HISTAMINES BY DESIGN NOT TO CROSSBARIERS, SO IT WOULD NOT MAKE IT. SO WE MOVED TO ANTI-SCOPSYCHOTICS, DESIGNED TO CROSS THE BLOOD BAR BARRIER. SHE HAS FOUND FDA-APPROVED ANTI- ANTI-SCOPSYCHOTICS THAT WILL BLOCK EA GCHG 2 AT THE MICRO MOLAR CON CONCENTRATION THAT CAN BE ACHIEVED CLINICALLY. AT THIS CONCENTRATION, THE ANTI- OTIC WILL REDUCE MD ANTI-SCO CELL PROLIFERATION IN VITRO, AND ALSO CELL MIGRATION IN VITRO WITH SIMILAR EFFECTS ON POLAR POLARIZATION AND THE EXTENSION OF LAM ELIPOD IIA. AND AS A RESULT, THE CELL MOTE MOTIVEITY IS DECREASED. AND THE AMEMBERO WITH THIS XENO XENOGRAPH OF HUMAN IN B CELLS, BY JUST TREATING THE MICE WITH IT INGENERATION DAILY FOR 2 WEEKS OF THIS ANTI-SCOPSYCHOTIC EAG 2 BLOCKER HAS NORMALLY USED FOR TREAT HIGHWAY SKITS FLANICS, THAT WAS SUFFICIENT TO REDUCE THE TUMOR GROWTH AND PROLONG SURVIVAL. AND IN SOME CASES IT SEEMED TO BE REMISSION. WE COULDN'T SEE ANY SIGNS OF THESE TUMOR CELLS BY LUMIN ES SCENCE. SO THESE STUDIES ARE STILL GOING ONGOING. SO LET ME TELLING YOU ABOUT THE TM ENOL 16B AND THAT'S A CLOSE RELATIVE OF THE CASTLCIUM-ACT CALCIUM-ACTIVATED CHLORIDE CHANNEL IN THIS NOVEL FAMILY OF ION CHANNELS. HE HAS FOUND THAT 26 C IS PRESENT -- 16 C IS PRESENT IN THE IB-4 POSITIVE NEWURONS IN THE DORSAL GANG LLIA. THESE NEWURONS ALSO EXPRESS SLACK SLACK, WHICH IS A POTASSIUM CHANNEL 6 TRANSMEMBRANE SEGMENT. IT IS ACTIVATED BY SODIUM ON THE CIYTOPLASMIC SIDE OF THE MEMBRANE MEMBRANE. IN COLLABORATION WITH A COMPANY THAT GENERATED RADS WITH TRANS TRANSPOSI INSERTION, HE FOUND THAT AT THIS TRANSPOSING IN INSERTION CAUSED A TOTAL EL ELIMINATION OF THE 16C PROTEIN, AS DETECT ABIBLE BY WESTIN. SO IT'S A KNOCKOUT RAT. AND IN THIS KNOCKOUT RAT, THERE IS ALSO REDUCED PROTEIN LEVEL OF SLACK, POTASSIUM CHANNEL. BY COL OIMMUNOPRECIPITATION, THEN FOUND THAT SLACK AND TMEM 16C FORMED A CONTRAST IN VIVO AND ALSO IN H GCHGK CELLS EXPRESS INING BOTH PROTEINS. AND TOGETHER WITH OUR COLLEAGUE JUAN, USING THE STORED METHOD HE DEVELOPED FOR A SUPERSOLUTION MI MICRO SCOPSCOPY. IT'S ADDED THAT LAB PROTEINS ARE RIGHT NEXT TO EACH OTHER IN THE COMPLEX. AND WHEN HE COMPARED CELLS EXPRESS IING JUST SLACK OR SLACK PLUS 16C, SHE FOUND THAT 16C PROMOTES THE SODIUM ACTIVATED PO POTASSIUM CHANNEL ACTIVITY BY IN INCREASING THE SODIUM SENSE SENSITIVITY. SO, HAVING SEEN THAT 16C AND SLACK FORM A CONTRAST, AND 16C PROMOTES THE SODIUM ACTIVATED PO POTASSIUM CHANNEL ACTIVITY, AS WELL AS THE PROTEIN STABILITY, AS INDICATED IN THE KNOCKOUT RATS, IT'S PERHAPS NOT SURPRISING THAT JUAN ALSO FOUND IN THE KNOCKOUT RAT THERE IS RE REDUCED SODIUM ACTIVATED POTAUS POTASSIUM CURREN, WHICH CAN BE ISOLATED EITHER BY BLOCKING THE SODIUM TDX RESIST ANANT CURREN WITH CAD MYIUM OR BY REPLACING SODIUM WITH NICEIUM, WHICH CANNOT ACT ACTIVATE THE SODIUM ACTIVATED POTASSIUM CHANNEL. AND A REDUCTION OF THE POTASSIUM CHANNEL IN THESE TYPE POSITIVE NEWURONS WOULD INCREASE THE NEWER NEURONOEXCITABILITY, AS EVIDENT FROM THE INCREASE IN EXO EXOPOTENTIAL DURING DURATION AND ALSO THE LOWERING OF EXO EXOPOTENTIAL THRESHOLD. AND THESE IB 4 POSITIVE NEWURONS NOT ONLY EXPRESSED 16C BUT B 1. THEY ARE NEWURONS AND ONE WOULD EXPECT AN INCREASE IN EXCITE EXCITABILITY WOULD AFFECT THE SENSITIVITY. SHE FOUND A POST DOC WITH ELLEN TO SHOW THAT INDEED THESE KNOCK KNOCKOUT RATS HAVE HEIGHTEND BOTH IN SENSITIVITY TO BOTH HEAT AND MECHANICAL STIMULI. AND SINCE THESE NEWURONS EXPRESS BOTH 16C AND SLACK AND GIVEN THE INTEROAACTION BETWEEN THESE, WE EXPECTED AND WE WERE HAPPY TO SEE THAT THAT IS THE CASE BY KNOCKDOWN OF SLACK IN THE RATS HAVE A SIMILAR EFFECT, AS THE KNOCKOUT OF 16C. AND IT WAS KIND OF CURIOUS THAT RECENTLY WE SEE THE LINKAGE OF BOTH SLACK AND 16C TO GIVE THE HUMAN DISEASES. SLACK IS LINK D TO TWO DIFFERENT FORMS OF CHILDHOOD ONSET EPLEAPS EPILEPSIES. AND TMEM 16C IS LINK D IN SEVERAL FAMILY STUDIES TO A SURVEY CALLED DIST ONIA. TMEM 16C HAS A FAIRLY HIGH LEVEL OF EXPRESSION IN THE STRATIUM, THE CORTEX AND THE HIP OCAMPUS. SO IT'S THE NEXT QUESTION -- IT'S A COMPLETELY OPEN QUESTION -- WHAT IS DMOEFBLT CHANNEL DPAEM IS DOING IN THE BRAIN? THOSE THIS NOVEL CHANNEL. AND TO SUMMARIZE WE'VE BEEN STUDYING THE CHANNEL CONTRIBUTION TO NEWURONOSIGNALING IN SEVERAL WAYS THAT WE COULD ANTICIPATE BASED ON THE ELECHTRO ELECTROFIPHYSIOLODGGICAL STUDIES OF THESE DECADES SCOMBRUVENLT THEN, IT ALSO LED US TO COMPLETELY UN UNEXPECTED FINDINGS, SUCH AS THE EA GCHG 2 POTAUSSIUM CHANNEL CONTRIBUTION TO TUMOR GROWTH AND MET ASTAS IIS. AND THIS NOVEL PROTEIN ACTUALLY WORKING TOGETHER WITH POTASSIUM CHANNEL TO MODULATE SENSITIVITY. THE STUDIES HAVE BEEN SUPPORTED ALL THESE YEARS BY NIMH, NIM DS AND HMMI, PLUS A MEMBER -- NUMBER OF DIFFERENT FELLOWSHIPS, PREDOCTOR AL AND POST DOCTOR AL FELLOWS, TO OUR CURRENT LAB MEMBERS AND THE FORMER LAB MEMBERS NOW WITH THEIR OWN LABORATORIES. AND THIS IS A STUDY WITH SEVERAL OF OUR COLLEAGUES AT UC SF AND ALSO VAND EERBILT AND CHILDREN'S HOSPITAL FOR SICK CHILDREN IN TORONTO. THANK YOU. APPLAU [APPLAUSE] >> TIME FOR ONE OR TWO QUESTIONS. >> SO OUR NEXT SPEAKER IS ANTON ANTONINA. SHE JOINED THE NIH UNIT AT NIN D SLEDS. SHE RECEIVED MULTIPLE AWARDS AND A 2010 SCHOLAR AWARD FOR INVESTIGATIONS INTO THE COMPLEX INTERPLAY BETWEEN MICRO TUBE SXULZ THEIR REGULATORS. HER TALK TODAY ABOUT WILL FOCUS ON THE SIGNS OF THIS INTRACELL PATHWAY.LULAR MICRO TUBULE PAT- >> I WANT TO THANK DR. LAND IS AND GORSEY FOR GIVING MY THE OPPORTUNITY TO SPEAK TO YOU TODAY HERE ON THIS VERY HAPPY OCCASION. AS YOU CAN SEE HERE, MY LAB HAS GROWN UP AT THE SAME TIME AS THE PORTER AND NEWUROSCIENCE CENTER. AND WE ARE HERE TODAY FOR THE DEDICATION AS A RESULT OF THE WORK OF MANY PENAL. BUT WE HAVE NOT BEEN IDLE IN THE LAST FOUR YEARS. AND THE PEOPLE IN MY LAB, AS YOU CAN SEE HERE, HAVE ALSO BEEN HARD AT WORK, AS THE CONSTRUCTION IN PORTER. AND FINALLY, WE ARE HERE IN THIS BEAUTIFUL BUILDING, AND AS YOU CAN SEE HERE, MY GROUP ENJOYS BOTH THE SCIENTIFIC, AS WELL AS THE ARK TECH YOU'CHITECTURAL SPACE OF PORT PORTER. INTRAMURAL PROGRAM HAS ALLOWED ME TO START AND COMPLETE -- IN A COMPLETELY NEW AREA AND UNDER UNDEREXPLORED AREA OF BIOLOGY AND THAT IS THE INVESTIGATION OF THE ROLES OF MODIFICATION IN CELL FIPHYSIOLOGY. BY 25% OF THE PROTEIN MATTER IN OUR BRAIN IS A BUILDING BLOCK OF MICRO TUBULES AND POLYMERS THAT ARE RESPONSIBLE FOR THE E ELABORATE SHAPES THAT NEWURONS TAKE. AS YOU CAN SEE HERE THIS ART ISTIC REPRESENTATION OF NEWURONS BASED ON MORE THAN 100-YEAR-OLD DRAWING BY MONICA HUB. MICRO TUBULES GIVE RISE TO VERY VARIOUS CELLULAR STRUCTURES THAT VARY IN THEIR BEHAVIOR. MICRO TUBULES CAN TAKE THE FORM OF RADIAL INTERFACE ARRAY, THE SUMMERTIYMMETRIC CRYSTALLINE IN NATURE ARRAY OF FLAGELLA. AND ALL THESE STRUCTURES THE CELLS CONSTANTLY REGULATES THE RELATIVE LEVELS OF STABLE, MI MICRMICRO TUBULES AND DINYNAMIC SHORT-LIVED MICRO TUBULES. AND AN EXAMPLE OF AMITOTE IS, WE -- A LARGE PART OF THE MICRO TUBULES, WHILE IN STRUCTURES LIKE OXOM IMINE, MICRO TUBULE STRUCTURES ARE STABLE FOR A DAYY SO THE FUNDAMENTAL QUESTION THAT MY LAB ULTIMATELY WANTS TO ANSWER IS HOW DOES A SINGLE BUILDING BLOCK THAT GIVE RISE TO THIS GREAT VARIETY IN ARK ARCHITECTURE AS WELL AS DIYNAMIC BEHAVIOR? AND WE CAN SEE THE NETWORK IS A FUNCTION OF DIFFERENT OPERATIONS ON THIS COMMON SUBSTRAIT, AND THE REGULATORS THAT ACT ON THE COMMON SUBSTRATE CAN BE UKETTEORS, FACTORS THAT MICRO TEORS TUBULES WITH RESPECT TO EACH OTHER. AND THE MICRO TUBULE NETWORK IS ALSO REGULATED BY A LESS WELL- WELL-UNDERSTOOD MECHANISM AND THAT IS THE MODIFICATION OF TUB TUBULIN. AND IN THIS MODIFICATION -- THESE MODIFICATIONS HAVE THE POTENTIAL TO CHANGE BOTH THE BEHAVIOR OF THE CELLS AS WELL AS CHANGING THE ACTIVITY OF THESE VARIOUS FACTORS. SUBJECT TO A VAST ARRAY OF MODIFICATIONS THAT ARE I WILL ILLUSTRATED HERE ON THE STRUCTURE OF THE ALPHA BETA AND IN ADTION TO MODIFICATIONS THAT MANY OF YOU ARE MAYBE FAMILIAR WITH, LIKE FOSS PHOSPHORYLATION, TUB UULIN IS SUBJECTED TO MORE SPECIALIZED MODIFICATION, LIKE TIYROSIN ATIOATION AND ALPHA OR BETA TUB UHULIN, THAT IS THE GLYCOLATION, THE MULTIPLE OF GLYCEINE RATES. AND THE MAJORITY OF THESE MODIFICATIONS RESIDES ON THE TUB TUBULIN, WHICH PROJECM THE MICRO TUBULE SHAFT. THEY ARE DISORDERD, HIGHLY NEGATIVELY CHARGED, ALMOST CREATING A CLOUD OF NEGATIVE CHARGE AROUND THE MICRO TUBULE SHAFT. AND THERE IS ONLY ONE EXCEPTION TO ALL THESE MODIFICATIONS, AND THAT IS ASELF ATIATION OF MICRO MODIFICATION. THESE MODIFICATIONS IN FACT MARK DIFFERENT SPECIALIZED POPULATION POPULATIONS OF MICRO TUBULES AND THEIR CELLS. MICRO TUBULES AND INTERPHASE CELLS ARE MOSTLY TIYROSIN ATATED. WHILE THOSE IN COMPLEX CELLS LIKE THE NEWURONS DISPLAY A LARGE COMBINATION OF DIFFERENT MODIFICATIONS WITH COMPARTMENTS, FOR EXAMPLE, WHILE THE AXON IS ENRICHED. BUT AS RE WE REACH THE HIGHLY DIYNAMIC GROWTH. FOR EXAMPLE, SELLIUM, IT'S HIGHLY ENRICHED IN DMRUTAMUL ATION AND GLYCOEL ATIATION. AND THIS IS CHEMICAL LY HIGHLY ECULAR MOTORS, WHICH WILL SEE DIVERSE AND AFFECTS ASSOCIATED A DIFFERENT TYPE OF MICRO TUBE TUBULES SUBSTRATES, DEFENDING ON WHAT LOCATION OF CELLS AND ALSO TRACK THEIR JOURNEY AS THEY ARE GOING ON THE MICROW TTUBULE TRACK. AND BECAUSE THE MICROW TTUBULE BIND BINDING SITES ARE VERY CLOSE TO WHERE THESE MODIFICATIONS ARE, THESE MODIFICATIONS TUNE THE BEHAVIOR OF THESE REGULATORS. AND IN FACT, THIS IS THE BASIS FOR WHAT HAS BEEN PROPOSED TO BE A TUB UULIN CODE AKIN TO A BETTER UNDERSTOOD HISTIN CODE. THIS MODIFICATION AS WELL AS THE ENZYMES THAT INTRODUCE THEM ARE HIGHLY CONSERVED. IN FACT, THE MORE COMPLEX THE ORGANISM THE MORE MODIFICATION ENZYME IT HAS AND MORE COMPLEX MICROW TTUBULE ARRAYS. AND THEY ARE IMPORTANT IN BASIC CELLULAR PRACTICES SUCH AS NEWURO NEUROGENESIS AND REGULATION LEADS TO HUMAN PATHOLOGY, SUCH AS DEGENERATION AS WELL AS RE RESIST ANANCE TO CHEMOTHERAPY. SO WHAT TDO YOU NEED TO UNDERSTAND THE CODE -- HOW THE CODE, YOU NEED TO UNDERSTAND THE SUBSTRATE ORN WHICH THE CODE IS WRITTEN SO THAT SOMEBODY CAN READ IT. SO OUR REGULATORS NEED TO UNDERSTAND HOW THE REGULAORS RESPOND TO THE CHEMICAL CODE ITSELF. ONE IS EVEN THOUGH MODIFICATIONS HAVE BEEN KNOWN FOR MORE THAN 13 YEARS, WE HAVE A VERY POOR ABIDES IING FOR THE MOLECULAR MECH MECHANISMS OF TUB UULIN CHEMICAL DIVERSITY. THERE ARE TWO MAIN REASONS. ONE IS THAT THE ENZYMES RESPONSIBLE FOR THE MODIFICATION MODIFICATIONS WERE NOT KNOWN SECOND AND THE MAIN REASON FOR THIS IS THE WAY WE WENT ABOUT THE STUDY. TUB UULIN FOR THE LAST 40 YEARS, DEFA FACTO MESSAGE WAS FROM BRAIN TISSUE BECAUSE IT IS ENRICHED IN TUBUBLEIN AND IT IS OBTAINED THROUGH REPEATED CYCLES OF POLL POLYMERIZE ATIATION AND IS HIGHLY HETEROJENNOUS WITH ICSOFORMS AND MULTIPLE TEXTURE MODIFICATIONS, AS SHOWN. BUT I THINK THE MOST IMPORTANT OUTCOME, OR RELEVANT OUTCOME OF THIS METHOD IS THAT ALL TOPO TOPOGRAPHIC AL INFORMATION IT STILL ORIGINALLY HAD IN THIS MODIFICATION HAVE NOW BEEN SCRAMBLED AND WE HAVE A MICRO TUBUL SUBSTRATE HAS MORE VAGUE AND HAS MORE THAN 30 TYPES OF DIFFERENT TUB UULIN ICSOFORMS. SO MY LAB DECIDED TO START SIMPLY. WE SOLVED THE TUB UULIN PROBLEM AND THAT IS BY OBTAINING TUB UULIN THAT IS UNMODIFIED AND HAS A SIMPLIFIED GROUP OF TUB UHULIN ISO ISOFORMS AND THIS UB TUB UULIN IS ISOLATED FROM CELLS THAT HAVE VERY LOW LEVELS OF TUB UULIN MODIFICATION. AND THIS IS COMPETENT FOR POLL POLYMERIZE ATIATION. WITH SOME WORK OF MULTIPLE YEARS IN WHICH WE'VE ASSEMBLED A STOCK STOCKPILE OF VARIATION MODIFICATION ENZYMES THAT WE NOW HAVE CHARACTERIZED VERY WELL IN THE LAB, WE CAN NOW CHEMICAL LY MANIPULATE THIS TUB UULIN AND MAKE DIFFERENT FLAVORS OF TUB UULIN. AND HERE YOU CAN SEE DIFFERENT FLAVORS WITH . TIYROSIN ATATED TUB UHULIN, WHICH IS EN ENRICHED IN DIYNAMIC CELLS THAT HAVE -- ASSCETYL ATATED TUB UHULIN, WHICH IS A TYPE OF TUB UULIN RICH IN HYPERSTABILIZED CELLS WITH LIFETIMES OF MORE THAN TWO, IN AS LONG AS 16 HOURS. AND THEN MET AB OOL ATATED TUB UHULIN THAT IS RICH IN TUB UUL STRUCTURES STRUCTURES. ONE THING I WOULD LIKE TO POINT OUT IS THAT THESE TWO MODIFICATIONS ARE MON AH MODIFICATIONS, SO RESULT, -- AS A RESULT, THEY CAN FUNCTION WITH SIMPLE ON A ON OFF SWITCHES. BUT DUE TO THE TYPE OF CONTROLS THAT THE GLUTAM AMATE CHANGE CAN HAVE, THIS TYPE OF MODIFICATION CAN WORK AS A REAL STATICDSTATIN CELLS. WITH THESE TYPES OF MICRO TUBULES THAT WE CAN MAKE, WE THEN DIRECTED OUR ATTENTION TOWARDS THE READER OF THE CODE. AND IN PARTICULAR, I DECIDED TO FOCUS ON AN ENZYME THAT'S VERY DEAR TO MY HEART, AND THAT IS STATSIN. THIS IS A PROTEIN THAT I DISCOVERED WHEN I WAS A POST DOC DOC. IT IS MUTATED IN A MAJORITY OF HEREDITARY CASES, AND THIS IS A GROUP OF DISORDERS THAT ARE CHARACTERIZED BY THE EXXON OPAT THE Y OF AX ONS. SO IF YOU LOOK, THIS ENZYME IS ABLE TO SEVER ROBUST MICROW T B TUBULES THAT ARE ASSEMBLED FROM BRAIN TUB UULIN. AND YOU SEE HERE MICROW T TUB UHLS THAT ARE BEING SEVERED IN THE PRESENCE OF ATP. BUT AS I TOLD YOU EARLIER, THEY ARE HIGHLY MODELED AND HETERO HETEROGEJENNOUS. THESE ARE IMPORTANT FOR THE SEVERING ACTIVITY OF STROSSIN BECAUSE IN THEIR ABSENCE, TUBUL TUBULIN ARE RESIST AANT TO SEVERING. AND SO WE DECIDE TOAD LOOK THEN ON UNMODIFIED MICROW T TUBULES. AND HERE YOU CAN SEE THAT IN FACT THE ACTIVITY OF UNMODIFIED MICROW T TUBULES, WHICH ARE IN FACT YOU FIND IN NEWURON AAL CELLS ARE RESIST AANT -- ARE VERY POOR SUB SUBSTRATES, BUT AS THE SUBSTRATE IS FULLY DETAMULATED, I RECOVER SEVERING ACTIVITY. AND IT'S PROPORTION AL WITH THE LEVEL OF POLY GLUTAMIN ATIOATION OF THE SUBSTRATE, IN A WAY DEMONSTRATINGLE THE ABILITY FOR BIODMRUTAMIN ATIATION. I WILL BES WANT TO POINT OUT TO YOU THAT IN FACT, MICROW T TUBULES IN THE AX ON WHERE THIS ENZYME ACT ARE PREDOMINANT LY FUEL LLY MUTILATED. WHILE MICROW T TUBULES IN THE DEN DENDRITE ARE TIYROSIN ATATED OR HAS ONLY ONE GROUP. WE ARE NOW TRYING TO UNDERSTAND THE MECHANISTIC BASICS FOR PO PTIAL AND AT THE SAME TIME EXTENDING OUR INVESTIGATION TOWARDS OTHER MICROW TTUBULE REGULATORS. I WILL SHOW YOU AN EXAMPLE OF HOW THE ACTIVITY OF A REGULATOR IS ACTIVITYD BY TUB LAR MODIFICATION AND I WANT TED TO TELL YOU A VERY SHORT STORY ABOUT HOW A WRITER OR AN ENZYME CAN GIVE RISE TO PATHS OF MODIFICATION THAT ARE COMPLEX THEMSELVES. I AM GOING TO TELL YOU ABOUT AN ENZYME THAT IS QUITE UNIQUE. AND THAT IS TUB UULOTRANSLATED AND ASSCETYL ATATES THAT IS LOCATED INSIDE THE MICROW TTUBULE UNITS. AS OPPOSED TO ALL THE MODIFICATIONS THAT ARE KNOWN TO ACT ON THE --. IT HAS BEEN SHOWN THROUGH IDENTIFICATION THAT THIS MODIFICATION ASSCETYL ATIOATION IS VERY SLOW. AND IN VITRO AS WELL AS IN CELLS CELLS. AND IN FACT, IS A MARKER FOR A LONG LIST AND HAVE BEEN USED BY CELL BIOLOGISTS EXACTLY FOR THAT FOR MANY YEARS. AND SO IS THIS SLOW PROCESS DUE TO THE FACT THAT THE AXIS OF THIS ENZYME IS LIMITING ITS ACTIVITY OF THE SUBSDMRAET IS THAT WHAT'S GIVING RISE FOR THE SPEC FIIFICITY OF ALL THE MICROW TUB TUBULES IN THE CELLS? A DIFFUSION-LIMITED PROCESS THAT WOULD OCCUR THROUGH THE MICROW TUBUL WITH RESPECT -- WOULD EXPECT A MANIPULATION WAY BIAS TOWARD THE END VITEOF THE MICRO TUB TUBULES. BUT IF YOU VISUALIZE ASSCETYL ATION IN VITRO, HERE IS RAND AOMLY DISTRIBUTED VERY SIMILAR TO THE TYPE OF DISTRIBUTION OF ASSCETYL ATION THAT YOU SEE IN CELLS, WITHOUT SHOWING A BIAS FOR ANY IST ENDS. MOREOVER, IF THE ACTIVITY IN, IN FACT, NOT ENHANCED WHEN IT CO COPOLARIZED OR WHEN USED IN A SUBSTRATES, DIFFERENT TYPES OF OLYMER THAT HAVE AN OPEN ARK ARCHITECTURE THAT WE CAN MANIPULATE IN THE TEST TUBE. IN FACT, THE HIGHEST ACTIVITY AF SETTIL ATIATION IS ENCLOSED. SO HOW DOES TRANSFERATES ENTER AND ACCESS? SO WE DECIDED TO JUST PUT A GFP ON IT AND LOOK AT A SINGLE-MOLL SINGLE-MOLECULE TASK GFT USING REFLECTION MICRO SCOPSCOPY. AND HERE YOU CAN SEE THE MODULES THAT THEY ARE DIFFUSE IION THE MICROW TTUBULE BIODIRECTIONAL LLY. FROM THAT, WE CAN CALCULATE AND S ENZYME DUTIESS WITH AN MEASURE A DIFFUSION FOR -- AND AVERAGE INTEROAACTION TIME OF ABOUT ONE AND A HALF SECONDS. IT TURNS OUT THAT THIS DIFFUSION COEFFICIENT IS ORDERS OF MAG MAGNITUDE FASTER THAN THE CAT L-- CAT LALYTIC RATE OF THE ENZYME, WHICH IS VERY VERY SLOW. AND IN AGREEMENT WITH THE STRUCTURE OF A BOUND TO THE AN ANALOGUE, IT SHOWS AN ACTIVE THAT IS SLOW FOR CATHIC WEIGHT. AND BECAUSE THE CATALYTIC WEIGHT IS CLOSE, THE STABLE MICROW T B TUBULES IN THE CELLS AND BYPASS THOSE TUBULES THAT HAVE A VERY FAST TURNOVER. THE THIS STORY, I THINK, I WILL ILLUSTRATES QUITE NICELY HOW UNDERSTANDING THE KIN EIGHETICS OF AN ENZYME IS ABLE TO GIVE YOU AN UNDERSTANDING OF A CRUCIAL BIOLOGICAL -- IN THE CELL. BECAUSE MICROW T TUBULES ARE DINE DYNAMIC POLYMERS, THIS TYPE OF COMPLEX PATTERN OF MODIFICATION CAN THUS BE FORMED, NOT ONLY BY CONTROL BUT ALSO BY KIN EIGH!ETIC CONTROL OF THE MODIFICATION EN ENZYME. AND SO IT IS USED TO MAKE A COMPLEX PATTERN IN WHICH ALL MI MICROW T TUBULES HAVE ONE TYPE OF MARKER AND THOSE THAT SLAY FAVOR TURNOVER HAVE A CHEMICAL MARK THAT CAN BE RECOGNIZED BY MICRO TUBUL REGULATORS. SO WHAT DOES THE FUTURE HOLD? WE ARE INTERESTED IN HOW THIS COMPLEX PATTERNS ARE GENERATED IN CELLS. AND SO WITH OUR DIFFERENT TYPES OF MICROW T TUBULES FLAVORS WE WANT TO REPRODUCE COMPLEX PATTERNS, WE ARE INTERESTED TO KNOW HOW THESE PATTERNS ARE DECODED. AND THEN ALSO THESE MARKS MIGHT NOT BE JUST MARKERS OF MICRO TUB TUBUL POLYMER BUT THEY CAN ALSO MODIFY THE PROPERTIES OF THE POLYMER ITSELF. AND ALSO WOULD LIKE TO GAIN A MORE WHOHOLISTIC UNDERSTANDING OF HOW THE PROTEOME IS COLLECTED THROUGH DIFFERENT TYPES OF MODIFIED MICROW T TUBULES. SCIENCE IS A SPORT AND THIS WORK IS MADE FUN BY A GREAT TEAM OF PEOPLE. ANNIE DEAN AND CHRIS HAVE WORKED TOGETHER ON GENERATING DIFFERENT TYPES OF MICRO TUBULES AND TAX WRITING. JEFF SPECT ER AND AG YEAA HAVE WORDING ON TRANSFER AATE STRUCTURE AND SINGLE MOLECULE WORK. MAX VALEN IINE IS WOKS FOCUSING ON THE REGULAION AND JANE SPARK IN THE LAB IN THE ROLE OF MODIFICATION AND CELL RE REGENERATION. AND IAN VIEW IS PRIMARILY WORKING ON A NEW MODIFICATION AND THAT IS I'D LIGLYCOLATION OF MI MICRMICRO TUBEUOLES. AND THANK YOU SO MUCH FOR YOUR ATTENTION. APPLAU [APPLAUSE] >> I THINK WE HAVE TIME FOR ONE QUESTION. ONE SHORT QUESTION. >> THE MET ABUL ATIATION. IS IT INDUCED BY LOCAL ENZYMES? >> INAUDIB[INAUDIBLE] >> ENZYMES HAVE -- THEY'RE VERY FEW BINDING PATTERNS IDENTIFIED SO FAR. IT LOOKS LIKE THESE BINDING PARTNERS CAN AFFECT THEIR LOCAL LOCALIZATION. BUB I WOULD ARGUE THAT THIS TYPE OF PATTERN BUT YOU PROBABLY OB OBTAIN BY SPATIAL CONTROL, AS WELL AS KIN EIGHETIC CONTROL, MEANING THAT THESE ENZYMES ARE TUNED TO CONNECT FOR DIFFERENT TYPES OF MICRO TUBULES WITH DIFFERENT TYPES OF DIYNAMICS. >> AND THEIR ADVANTAGE? >> THAT YOU ARE MARKING A PARTICULAR POPULATION WITH A CHEMICAL MARK THAT THEY CAN BE RECOGNIZE AND ENFORCE A SIGNAL. SO FOR EXAMPLE, WHEN YOU SENT A NEWER IITE, YOU HAVE HIGHLY DINE DYNAMIC MICRO TUBULE, BUT THEN YOU ACTUALLY NEED THE INFRA INFRASTRUCTURE TO STABILIZE THE CELL ELONG ATIATION AND THOSE STABLE MICRO TUBULES ARE MODIFIED WITH VARIOUS MODIFICATIONS AND THE HYPOTHESIS IS THAT THOSE MODIFICATIONS THEN ENFORCE THE INITIAL DECISION OF MAKING THAT ASSERTIC EXTENSION AND RECRUIT NEW FACTORS IN THE MODIFICATION- MODIFICATION-SPECIFIC MANNER. >> OUR NEXT SPEAKER KEN SCHWARTZ IS A SENIOR INVESTIGATE INVESTIGATOR IN THE MOLECULAR FIPHYSIOLOGY AND BIOFIZPHYSICS SECTION AT NIS DF. HE HAS JOINED THE INSTITUTE IN 1997 AND INVESTIGATED THE STRUCTURE OF ACTIVATED ION CHANNELS AND EXCLUSIVE MOLECULAR MECHANICS -- MECHANICS BY WHICH THESE CHANNELS OPEN AND CLOSE. LL SCUTES HIS WORK USING SPIDER VENOM TO CLOBLOCK THE ION CHANNELS AS A MEANS TO UNDERSTANDING HOW THESE ION CHANNELS FUNCTION. DISCUSS. >> THANK YOU, SUSAN AND THANKS FOR DOING A WONDERFUL JOB ORGANIZING TODAY'S MEETING. JERRY WAS MY CHAIRMAN WHEN I WAS A GRADUATE STUDENTS AT HARVARD. I CAME HERE SOON AFTER I MOVED HERE IN 1997 AND BE IT'S REALLY WONDERFUL TO CATCH UP. I WANT TO TELL YOU TODAY ABOUT WORK MY LABORATORY HAS BEEN DOING ON CLEAVING A LICHT OF OVERVIEW AND USING TOXINS TO EXPLORE ION CHANNEL. AND JUST TO KIND OF MAKE SHOURE WE'RE STARTING OFF ON THE SAME PAGE, OBVIOUSLY IN THIS KIND OF AUDIENCE, -- NEWEUROSCIENCE AUDIENCE, VERY FAMILIAR WITH THE NEWURONS AND MEASURING INTERESTING AND COMPLEX PARTNERS OF ELECTRO ACTIVITY. THE GOAL OF COURSE ARISES FROM TWO FUNDAMENTAL. FIRST OF ALL, THE CONCENTRATION OF PERMEATING IONS. IN THIS EXAMPLE SHOWN PEER FOR POTASSUP, CAN VARY ACROSS THE NEWUROCELL MEMBRANE. THIS IS HIGH INSIDE, LOW OUTSIDE OUTSIDE. AND IF YOU COMBINE THAT WITH A SELECTIVE ION PERM IAABILITY OF THE MEMBRANE TO THAT ION, WHAT HAPPENS OF COURSE WHEN THAT CHANNEL OPENS, POTASSIUM FLOWS OUT, AND GENERATES ELECTRICAL POTENTIAL. THIS IS BASIC THE FUNDAMENTAL MECHANISM BY WHICH NORMAL NEWER NEURONS GENERATE ELECTRICAL SIGNALS AND SO IT'S REALLY NOT SURPRISING THAT A REALLY HUGE NUMBER OF GENES ENCODE FOR PRO PROTEINS. AND LITTLY DID A FANTASTIC JOB OF GIVING US AN OVERVIEW OF THAT AND SHE PLAYED A VERY IMPORTANT ROLE IN IDENTIFYING AND CHARACTERIZING. THIS IS MY ATTEMPT TO TAKE THE COMPLEXITY THAT CURRENTLY ARE TRYING TO UNDERSTAND AND JUST CONCEPTUALIZE IT BASED ON THE DATA ALL THE WAY DO SOMETHING SIMPLER. SO MY LABORATORY HAS WORKED BY CHANNELS AND I WILL TELL YOU A LITTLE BIT ABOUT THAT AND THEN I WANT TO QUICKLY TRY TO FOCUS ON SOME MORE RECENT WORK ON PROTEIN RECEPTOR CHALLENGES. MUCH OF OUR WORK WAS TAKING ADVANTAGE OF REALLY THE AMAZING REPRESEERTOIRE THAT EXISTS IN THE NATURAL WORLD, OF US VENOMOUS CREATURES. AND DO SO BY TARGETING ION UCE IN THEIR VENOM. CHANNELS THROUGH THE TOXINS THEY AND BY AND LARGE, YOU CAN THINK OF THE VENOMOUS KINGDOM OUT THERE OF BEING A VERY GOOD AT TARGETING ION CELLS THAT GIVE RISE TO NERVE IMPULSE.^ THERE ARE TWO KINDS OF FUNDAMENTAL MECHANISMS AND JUST A REVIEW. IT'S BY SIMPLIFIED CARTOON OF A POTASSIUM CHANNEL UNDERGOING A CHANGE IN RESPONSE TO CHANGE IN VOLTAGE. AND WHEN THESE CHANNELS ARE OPEN, THERE IS A SELECTIVE PATH WAY FOR THAT ION TO DIFFUSE OUT OF ITS CONCENTRATION RADIANCE. SHE'S -- THESE ARE CLOSED, IT PREVENTS THE FLOW OF ION AT THE CLOSED PHASE. AND THIS IS CONTROLLED EXCLUSIVE LY HIGH FIDELITY BY THESE VOLTAGE CELLS. THIS IS THE BASIC DESIGN OF A VOLTAGE ACTIVATED CHANNEL. YOU CAN IMAGINE TWO DIFFERENT MECHANISMS BY WHICH TOXINS WILL BE ABLE TO INTERFERE WITH ACTIVITY AND CAUSE PARALYSIS. AND THE FIRST IS ILLUSTRATED HERE AND A SIMPLE CORE-BLOCKING TOXIN THAT BINDS THE OUTER VES VESICLE. AND BEHAVES MUCH LIKE A CORK SO IT PREVENTS THE FLOW OF ION. A THIS IS KIND OF AN EXAMPLE OF ONE OF THESE SCORPIONS FOUND. AND THESE TOXINS WERE VERY VITAL IN TELLING US WHAT PARTS OF THE PROTEIN ACTUALLY FORMED THE PORE PORE. LITTLY FIRST CLONED HER CHANNEL, CHRIS MILLER'S LABORATORY BROUGHT MCKENIN AND USED THIS TOXIN TO ACTUALLY SHOW US WHAT PARTS OF THE PROTEIN FORM THE PATHWAY. IOON PRODUCTION PATHWAY. THE FIRST TYPE OF MECHANISM IS I WILL STRAIGHTD HERE. AND THE FOUND IING MEMBER OF TOX TOXINS THAT WORK THROUGH THIS MECHANISM IS A CANOTOXIN AND PART OF OUR STRUCTURE AND IT'S ISOLATED -- WHEN I WAS A GRADUATE STUDENT --. SO WE CURRENTLY DO HAVE NOT A STRUCTURE OF CANOTOXIN BOUND TO AN ION CHANNEL. BUT THE PROTEIN WE BELIEVE FORMS A RECEPTOR IS I WILLUSTRATED HERE IN THIS TRANSHELIX MONTTIF CONTAINED. A TOP VIEW LOOKING ON THE EXTRA CELLULAR SIDE OF A STRUCTURE. AND TOXIN DIVIDES THIS MONTIF AND BASICALLY INHIBITS THE CHANNEL IN A CLOSED CONFORMATION. AND THE OTHER INTERESTING FEATURE OF TOXINS IS I WILLUSTRATED HERE BY VIRTUE OF THE FACT THAT THEY SEEM TO BIND BY FIRST PAR PARTITION IING THE MEMBRANE. I DON'T HAVE TIME TO GO INTO ALL THE WORK THAT KIND OF LED TO THIS GENERAL IDEA. BUT I WANT TO KIND OF SHOW YOU THAT THE STRUCTURE OF IT, WHICH IDENTIFIES A REALLY INTERESTING THEME AND CLUSTER OF HIRYDROPHOBIC SHOWN HERE IN GREEN, SURROUND BY POLAR ATTRIBUTES AND IN BLUE AND RED. REALLY FROM A STRUCTURAL PERCENT PERSPECTIVE, THESE TOXINS ARE VERY APOPATHIC AND YOU WOULD EXPECT THE INTEROAACTION WITH A SUBSTRATE. AND SO THIS IS WORK FROM A VARIETY OF LABORATORIES, INCLUDING ROB MCKININ BUT ALL ARE CONSISTENT WITH THESE AN ANIMATIONS. SO A NUMBER OF YEARS AGO, WE STARTED TO ASK THE QUESTION OF WHETHER WE MIGHT BE ABLE TO FIND TOXINS AGAINST OTHER TYPES OF I ION CHALLENGES. AND THESE, OF COURSE, ACTUALLY TOUCHED IN FEW TALKS TODAY ALREADY, OF A VERY DIVERSE FAMILY IING OF IONS ACTIVATED BY SOME OF THE MOST INTERESTING LIG LIGANS IN THE PLANT FAMILIES. THE ACTIVE INGREDIENT, CHILI PEPPERS AND THERE IS A CHANNEL IS ONE THAT IS ACTIVATED BY CAPS CAPSASIN. WOULD THERE BE TOX CLANS OUT THERE AND FU THINK ABOUT IT, EVEN THOUGH THE FIRST TRANSIENT CHANNEL IS CLONED, IT'S ACTUALLY A PHOTO RE SERECEPTORS AND RESPONSIBLE FOR PHOTOTRANSHE WAS. THEY DON'T SHOW DIVERSE CHANNELS THAT DON'T SHOW UP IN INSECTS SOS NOT GOIN G TO BE A MECHANISM THAT VENOMOUS CREATURES USE FOR THEIR PREY. THESE ARE A DIVERSE FAMILY OF MAMMAL AND IN PARTICULAR THE CAP CAPSASIN RECEPTOR PLAYS A CRITIC AL ROLE. AND WE BEGAN LOOKING AT THESE CHANNELS. OF COURSE, THESE CHANNELS ARE THE BIOLOGICAL THERMOSENSORS. THERE ARE OTHER VARIETIES ACT ACTIVATED. TRIP M 8. AND SO WE WANTED TO SEE IF WE COULD FIND TOXINS AND WE SPENT A FEW YEARS COMING UP EMPTY. AND THEN IN DAVID JULIUS' LAB IN 2010 IDENTIFIED IT AS A VERY POTENT ACTIVATOR. AND CAN THIS IS A SEQUENCE OF TOXINS THAT THEY IDENTIFIED. AND WHAT THEY FOUND THAT IT LOOKS LIKE THE TOXIN IS ORGANIZED INTO 2 DOMAINS. EACH OF WHICH LOOKS LIKE, BASED ON THE PATTERNS DISULFIDE, AND THIS IS OUR ORIGINAL STRUCTURE OF THE TOXIN WITH THE BONDS. SO IN DAVID'S LABORATORY, PROPOSED A DOUBLE -- THIS IS TWO ACTIVE LOBES THAT WOULD BE MUCH LIKE VERY SIMILAR TO AN ANTIBODY ANTIGEN COMPLEX. SO WE BEGAN STUDYING THIS IN THE LABORATORY AND HAS BEEN LEADING OUR FUNCTION AL EFFORTS TO STUDY THE MECHANISM. AND THESE ARE EXAMPLES OF RECORDINGS FROM CELL EXPRESS IING THE CHANNEL, EITHER WHERE HE'S GIVING A WHOLE SERIES IN VOLTAGE STEPS FROM NEGATIVE TO POSITIVE VOLTAGES. AND YOU CAN SEE VERY LOW CURRENT IN CONTROL, ROBUST ACTIVATION AND THEN WHEN' PLIES IT TO THE EXTERNAL SIDE OF THE MEMBRANE, ALSO ROBUST ACTIVATION. AND YOU CAN SEE ALSO IN THESE PLOTS VOLTAGE RELATIONSHIPS, THEY CONTROL. NOW THE INTERESTING THING ABOUT THIS TOXIN IS IT ASSOCIATES VERY SLOWLY. YOU CAN SEE THE PLOTS THAT AFTER ACTIVATION OF THE CHANNEL BY TOX TOXIN, YOU GET VERY SLOW DIS DISORBIT TOXIN AFTER MOVING IT FROM THE EXTERNAL RECORDING SOLUTION. SO ONE THAT HE FOUND DISCOVERED USING AN INTERESTING CONSTRUCT THAT HAS BEEN MADE IT IN THE LABORATORY, WHERE HE HAD IT ON THE TOP. AND THE HIST OTOXIN IS ACTIVE AND IT ACTIVATES THE CELL. BUT IT PRODUCES A VERY ODD RELATIONSHIP WHERE YOU SEE ROBUST OUTWARD OCCURRENCE BUT REALLY NO INWARD TO SPEAK OF. USING A VARIETY OF APPROACHES, LOOTING AT PH. AND IT IS ACTUALLY ACTING LIKE A CHARGE BLOCK ER TO BLOCK THE CHANNEL. EVEN THOUGH THIS TOXIN IS BIND BINDING TO THE SURFACE OF THE PROTEIN AND TRAPPING IT IN AN OPEN SPACE. OKAY, SO JEN00 IN THE LABORATORY HAS BEEN WORKING ON THE STRUCTURAL BIOLOGY, KIND OF DOING THE STRUCTURE OF THE TX AND HE HAS USED A SOLUTION, WHICH IS MUCH MORE DIFFICULT TO LOOK AT THE TOXINS. AND HAS ACCEPTED AND VERY SIMILAR TO OTHER INHIBITOR TOXIN FOLDS. WHAT WAS KIND OF SURPRISING IS IT TURNS OUT THAT IF YOU COLOR- COLOR-CODE LIKE I DID FOR THE TOXIN, THESE TOXINS ARE VERY EMP EMPOPATHIC. THE LABORATORY HAS SHOWN THAT THESE TOXINS CAN INTERACT VERY WELL WITH THE MEMBRANES. AND THE WORKING MODEL WE HAVE IS THAT WE BORROWD THIS GENERAL DESIGN OF A POTASSIUM CHANNEL AND THINK ABOUT IT IN THE CON CONTEXT OF CHANNELS, IS THESE FINDINGS KIND OF IN THE PRIME MINISTER RY OF THE DOMAINS WHERE THEY INTERACT WITH THE MEMBRANES MEMBRANES. BUT WITH A TAG CAN MAKE ITS WAY AND PRODUCE VOLTAGE. SO THIS IS WHERE WE WERE ABOUT TWO MONTHS AGO. AND IN JULIUS' LAB IN COLLABORATION WITH CHEN, A BIOLOGIST, HE -- GIEBEL ING ING TO PLAY THIS MOVIE QUICKLY SO YOU CAN LOOK AT THE DIFFERENT PARTS TO PROTEIN. AND THE PENETRABLE DOMAINS, RI WHICH ARE CONSISTENT WITH MICHELLE'S LABORATORY. THE TRANSMEMBRANE DOMAINS OPEAR TO A STRUCTURE THAT'S VERY SIMILAR TO THE VOLTAGE. LOOK AT THE TOP. THESE GREEN OBJECTS ARE FOUR HAN HANOTOXIN MODULES THAT CORRESPONDS TO 2 DKTX MOLECULES BOUND TO THE CHANNELS. THESE ARE SHOWN HERE AT VARYING LEVELS. AND SO BECAUSE THE SOLUTION STRUCTURE OF TKS WAS NOT AVAILABLE, ONE OF THE FIRST THINGS WE WANTED TO DO WAS TO SEE IF WE COULD ACTUALLY BEGIN DOC IKING IT INTO THE STRUCTURE. AND THIS IS A DOC IKING DAYCARE MOVIE TO SHOW THE PROCESS OF DOCKET IING. AND WE'RE STARTING OFF LOOKING AT THE DENSITY FROM THE OUTSIDE LOOKING IN. WE'RE NOW STANDING AT THE CENTRAL AXIS LOOKING OUT. AND WHAT CHEN HAS DONE IS TO FIRST DOCK IN THE BACK SCAAND SHOW YOU ONE OF THE KNOTS THAT HE INVOLVEDSOLVED. NOW FU COMPARE IT, FU COMPARE IT IT, IT REALLY DOES A FANTASTIC JOB. AND SO ONE OF THE KEY THINGS THAT THIS SIMPLE EXERCISE DOES FOR US IS IT ANSWERS A VERY FUNDAMENTAL QUESTION ABOUT HOW THIS TOXIN IS ACTUALLY ORIENT ING THE PROTEIN. AND THIS IS THE ENTERMUS OF THIS DKTS. WE'RE LOOKING AT IT FROM THE OUTSIDE. SO WHAT THIS TELLS US IS THAT IT'S POSITIONED COUNTERCLOCK WISE RELATIVE. AND BECAUSE THE LINKAGE IS NOT AS RELATIVELY SHORT, IT ACTUALLY TELLS US THAT THIS TOXIN MUST BE ORIENTD IN KIND OF A CLOCK WIWISE CONFIGURATION ON THE OUTER PART. AND SO CHEN IS TRYING TO UNDERSTAND THIS PROTEIN-TO PRO PROTEIN INTERFACE IN DETAIL IN ART BECAUSE IT WILL TELL SOMETHING ABOUT HOW THE STRUCTURE OF ITS UNDERLYING RECEPTOR CHANGES AND HOW A MOD MODULATOR CAN ACTUALLY INFLUENCE THE DIYNAMICS TO THE PROTEIN. AND I WANT TO JUST END QUICKLY BY TELLING YOU ABOUT A FEW RESEARCH EXPERIMENTS HE HAS BEEN DOING TO LOOK AT THE QUESTION OF HOW THE DIFFERENT STIMULI THAT ACTIVATE IT ARE KIND OF COUPLED TOGETHER. I MENTION AID COUPLE OF THEM IN VARIOUS PARTS DURING THE TALK. ONE I HAVE. BUT THESE ARE VOLTAGE ACTIVATED CHANNELS AND YOU CAN SEE THAT THE PLOTS OF WHERE WE'RE LOOKING OF NEGATIVE 90 MILLIVOLTS. YOU SEE THESE MICRO SCOPIC AND THAT'S BECAUSE OF THE VOLTAGE IN PART DUE TO VOLTAGE DEPENDENCY. NOW HE'S WASHED OUT THE TOP. HE'S GOING BACK AND FORTH BETWEEN POSITIVE AND NEGATIVE MEMBRANE VOLTAGES. AND WHAT THIS SHOWS YOU IS THREATS TOXINS AND YOU CAN ACTUALLY CLOSE THIS WITHOUT ACTUALLY FORCING THE ASSOCIATION OF TOXINS, WHICH REMAIN STABLEY ATTACHED TO THE CHANNEL EVEN THOUGH IT'S NOT PRESENT IN THE EXTERNAL SOLUTION. SO THAT TELLS US THE VOLTAGE MECHANISM IS INDEPENDENT OF THE ACTIVATION METHOD. HE ALSO LOOKED AT THE SITES TOWARD THE INTERNAL SITE OF THE PROTEINS AND THIS IS AN EXPERIMENT WHERE SIMILAR KINDS. HERE IS ACTIVATION OF THE CHANNEL BY CAPSASIN. AND THEN BINDING SITES ANDS AND WHAT YOU CAN SEE IS IT PRODUCES THE ACTIVATED CHANNEL. AND AFTER WASHING OUT THE CAPA CAPASIN, IT REMAINS AT THE SAME LEVEL. IT'S NOT LIKE YOU LOST THAT LEVEL OF ACTIVATION, WHICH YOU MIGHT EXPECT. SO THAT SHOULD SUGGESTION -- SUGGEST THAT THIS ASSOCIATION AND YOU CAN PROVE THAT BY REAH REAPPLYING AND SATURATE IING CON CONSTRATHSS AND GET FULL ACT ACTIVATION OF THE CHANNEL BACK. SO WHAT THIS ACTUALLY DEMONSTRATES IS EXTRAORDINARILY TYPE COUPLE IING FROM THIS INTRAY INTRACELLULAR SIDE OF THE PRO PROTEIN AND THIS EXTERNAL SIDE. AND OLAST ONE I WANT TO SHOW YOU IS NOW LOOKING AT TEMPERATURE, WHICH PLAYS A KEY ROLE IN SENSE SENSORY FUNCTIONS. SO NOW WHAT HE HAS DONE IS COOLED THE CHAMBER AS RAPIDLY AS HE CAN AND THIS IS SOMEHOW WE'RE WORKING TIME PROVE IN TERMS OF SPEED. AND COOLING THE CHAMBER ACTUALLY CAUGHSES INHIBITION IN THE CHANNELS. AND FU LOOK AT THE TEMPERATURE DEPENDENCE FOR ACTIVATION EITHER IN CONTROL HERE OR AFTER DKTS, WHAT YOU ARE SIS -- THIS IS THE MULTI ATIATION DESCRIBED THAT INFLUENCE IN THES THIS PROCESS BUT NOTHING ALONG THESE LINES. THE MAGNITUDE REALLY MAKE THEY THINK THIS TOXIN IS HONING IN PRETTY INTIMATE LY TO THE CORE THERMOSENSORY MECHANISM. SO I JUST WANT TO FINISH AND BY HIGHLIGHT IING THIS IDEA IN THE CONTEXT OF THESE STRUCTURES. AND I SHOWED YOU THE DKTX OPEN SPACE FOU-- SOLVED HA -- THE STRUCTURAL CHANGES ARE NOT VERY LARGE, RIGHT? THE BACKBONE IS ONLY AROUND THESE STRUCTURES AND THEY JUST OPENED SNUFF ENOUGH. AND I THINK THE RESULTS ARE TELLING US THAT THIS WHOLE CORE PORE-FORMING DOMAIN IS THE GATE ING MODULE TO THESE CHANNELS. THAT CAPASIN IS BINDING TOWARDS THE OUTSIDE. DKTX HAS A VERY STRONG CAGE COUPLING WITH EACH OTHER. I WANT TO STOP THERE AND REMIND YOU OF THE PEOPLE THAT CONTRIBUTED. I MENTIONED SEVERAL OF THEM. ANDRE, DEMETRY, I HAVEN'T HAD TIME TO TALK ABOUT JANG'S WORK, ALL OF WHO ARE HELPING ON OUR STRUCTURAL EFFORTS. THANKS. [APPLAUSE] >> IN THE INTEREST OF TIME, I DR. SCHWARTZ UNTIL AFTER THE SESSION. I WANT TED TO MAKE ONE ANNOUNCEMENT, WHICH IS AT 3:00 THERE IS GOING TO BE THE DED DEDICATION CEREMONY, WHICH WILL BE LOCATED OUT HERE AND YOU ARE ALL INVITED. I MIGHT RECOMMEND, THOUGH, THAT IN THE WALKWAYS AROUND MIGHT BE A GREAT PLACE IS LIKE HAVING A -- DOWN BELOW. SO THERE WILL BE ROOM THROUGH FOR MORE PEOPLE. SO THE FINAL SPEAKER FOR THE AFTERNOON IS A SENIOR INVESTIGATOR IN THE SURGICAL NEUROLOGY BRANCH OF NIN DS. MIGTOCON DRAHONDRIAL FISSION, FUSION AND TOXICOLOGY TO CONTROLLING ORGANIELLE QUALITY. AND TODAY HE'S GOING TO EXPLAIN THE WORLD IN FAMILIARIAL PARK PARKINSON'S DISEASE. >> IT'S A JOY TO PARTICIPATE IN THIS DEDICATION. IT'S A WONDERFUL BUILDING TO WORK IN. BEEN HERE SINCE THE BEGINNING OF PORTER 1. I'LL TRY TO USE THIS CURSOR SO THE POSTDOCS DON'T SEE HOW NERVOUS I AM. SO PARKINSON'S DISEASE IS LARGE LARGELY LATE-ONSET, SPRAORADIC DISEASE WITH NO CLEAR GENETIC COMPONENT. AND THE TREATMENT IS PAL YELIATIVE. THERE IS NO WAY TO SLOW OR BLOCK THE PROGRESSIVE NEWURO NEURODEGENERATION IN THESE PATIENTS. SO ONE OF THE CHALLENGES FOR DEVELOPING THERAPEUTICS IS WE DON'T KNOW THE CAUSE OF THE SPLIT OF PARK SDOINZ DISEASE. WE DON'T KNOW WHY SOME PEOPLE GET IT AND SOME PEOPLE DON'T. PARKINSON'S DISEASE. THERE ARE A CERTAIN SUBSET OF PATIENTS THAT ARE EARLY ONSET AND MIGHT HAVE CAUSES OF PARK PARKINSON'S. THESE GENES, THESE MUTATIONS IN THESE GENES CAN GIVE ITS CLEAR HINTS ON WHAT CAUSES THEIR DISEASE AND MAY GIVE ADDITIONAL INSIGHT INTO THE CAUSE OR SPRATT SPORADIC PARKINSON'S. THE FIVE GENES I HAVE LISTED UP THERE -- IT'S A LARGER LIST NOW AND TO A LARGE EXTENT DISCOVERED BY ANDY SINGLETON, WHO WORKS IN THIS BUILDING WHO IS GOING TO TALK TOMORROW. THE TWO BOTTOM ONES, THOSE ARE AUTOSOMEONE'LL-DOMINANT DISEASE IT'S VERY HARD TO KNOW WHAT NEW FUNCTIONS THESE MUTATED PROTEINS CAN GENERATE. HOWEVER, THE TOP TWO I'VE LISTED THROUGH ARE AUTOSOMAL RECESSIVE, INDICATING THAT YOU HAVE TO USE BOTH COPIES OF THE GENE, INDICATING THAT THEY ARE LIKELY LOSS OF FUNCTION. IT'S A LITTLE BIT MORE RATIONAL TO FIGURE OUT WHAT'S GOING WRONG BECAUSE YOU NEED TO FIND OUT THE NORMAL FUNCTION OF THESE TWO GENE PRODUCTS. NOW, THE FIRST THING PEOPLE DID TO TACKLE THIS PROBLEM IS LOOK AT THE SEQUENCE OF THE PROTEINS AND YOU CAN LOOK AT PINK 1 AND SEE IT IS A PREDICTED KIN AASE. AND IT ALSO HAS A PREDICTED MIGMIT MITOCON DRHONDRIAL SCOMBRINES IT IS TO BE A KIN AASE AND LOCALIZED TO MIGTOCONDRE IIA. THAT PUTS IT ON THE MAP OF A POTENTIAL PARKINSON'S DISEASE. IF YOU LOOK AT THE SEQUENCE OF PARKINSON'S. AN E 3 UBTIN LIGAN LOCALIZED IN THE CIYTOSOL AND THAT HAS ALL BEEN CONFIRMED. AND I THINK THE NEXT MAJOR ADVANCE IN THESE TWO GENE PRODUCTS WAS MADE BY LEO PLANK AT THE UNIVERSITY OF WASHINGTON. HE DEVELOPED MUTATIONS IN THE PARKIN GENES AND FOUND OUT THOSE FLIES HAVE MUSCLE WASTING AND MIGTOKINE AAL DIYSFUNCTION SHOT.^ THERE IS ANOTHER HINT. THERE IS ANOTHER STUDY THAT SHOWS THAT PINK 1 IN PARKINS, MUTATIONS COPY ONE ANOTHER AND AFTER THE EXPERIMENTS, THEY FUNCTION IN THE SAME PATH WWAYS. THE QUESTION IS WHAT IS THAT PATH WWAY? THAT IS WHERE WE ENTERD THIS. AND I WILL JUST POINT OUT THIS GENERAL SUMMARY SLIDE AND GIVE YOU A SUMMARY OF IT. BUT I ALSO WANT TO NOTE THAT CHANGES NEW TOP IC BE FOR US, SO WE STARTED AFTER WE ENTERD THIS BUILDING. AND IT WOULDN'T HAVE HAPPENED WITHOUT THE GREAT LEAGUES COLLEAGUES IN THIS BUILDING AND PARTICULARLY MARK, WHO INTRODUCED US WITH THIS TOP IC AND COLLABORATED WITH US EARLIER ON AND MY COLLEAGUES CAKATHERINE AND CRAIG BLACKSTONE, WHO WERE PARTICIPANT OF THE ENTERPRISE. THE SUMMARY OF WORK SCAAND OTHERS WAS TO LOOK AT THESE FIVE MIGTO MITOCON DRHONDRIA AND IMAGINE THEY ARE THIS ONE CELL. ONE OF THEM IN YELLOW AC ACCUMULATES DAMAGE. AND WHEN THAT HAPPENS, IT'S QUITE INTERESTING. THIS KIN AASE I MENTIONED PINK 1, ACCUMULATES ON THE OUTER MEM MEMBRANE OF THE MIGTOCON DRAHONDRIA. AND KIM GOING TO GET INTO HOW IT KNOWS THAT MIGTOCON DRAHONDRIA IS DAMAGED, COMPARED TO THE GREEN ONES. AND WHEN IT ACCUMULATES ON THE MIGTOCON DRAHONDRIA, IT RECRUITS PARK PARKINS FROM THE CIYTOSOL TO THE MIGTOCON DRAHONDRIA AND ACTIVATES ITS ENZYME ACTIVITY AND THEN PARKIN PROCEEDS TO UBTIN ANATE THE MEM MEMBRANE PROTEINS. THAT'S WHAT THIS UV STANDS FOR. AND THAT INDUCES AN AUTOFAGO AUTOFAGOSOME, A BODY OF SELF-EN SELF-ENGULFMENT HERE, TO ENGULF IT AND DEGRADE IT. SO IF YOU PUT ALL THIS TOGETHER, IT SUGGESTS THAT THESE TWO PRO PROTEINS ARE PARTICIPATING IN A MIGTOCON DRAHONDRIA CONTROL PATH WAWAY. SO I WILL SHOW YOU SOME EXPERIMENTS. ZED ORN THE OUTER MIGTO ARE LOCAL MITOCONDRI IA MEM BRAIN AND MUTE MUTATED IN A TYPE OF DISEASE. AND WHEN YOU KNOCK OUT BOTH THOSE GENES, MIGTOCON DRAHONDRIA AC ACCUMULATE DAMAGE. AND AS YOU CAN SEE IN THE UPPER RIGHT, THE GENE PARKIN WILL AC ACCUMULATE TO A HIGH DEGREE AND 0% OF THE CELLS IT WILL ACCUMULATE ON THE MIGTOCON DRAHONDRIAL MEMBRANES. THIS IS A PURELY GENETIC DAMAGE AND PARKIN IS BEING RECRUITD. AND THE IT'S INTERESTING THAT FU LOOK AT A HIGH RESOLUTION, YOU CAN SEE THIS SQUARE. AND ALL THE MIGTOCON DRAHONDRIA IN THAT SQUARE ARE LABELED WITH SICYTOSOME C AND THESE IMAGES ARE MEASURED IN MEMBRANE POTENTIAL OR THEIR FUNCTION. YOU CAN SEE THE ONE ON THE LEFT HAS LOW FUNCTION AND THE ONE ON THE RIGHT HAS HIGH FUNCTION. THEN YOU CAN LOOK AT PARKIN AND LYIIGASE AND YOU CAN SEE AC ACCUMULATED ON THE LEFT ONE BUT NOT THE RIGHT ONE. AND THERE IS OTHER EXAMPLES IN THIS ONE CELL. BUT SOMEHOW WHEN ONE MIGTOCONDRI IA GETS DAMAGED IN THE CELL OR A HANDFUL, PARKINS ACCUMULATE AND ON THOSE DAMAGED MIGTOCON DRAHONDRIA, IT REDUCES ACCUMULATIONS. YOU DON'T HAVE TO GO THROUGH A FAIRLY LABORIOUS GENETIC PROCESS PROCESS. YOU CAN ACTUALLY JUST ADD A VERY SIMPLE NON-TOXIC COMPOUND CALLED AN ONC COUPLE R USED AS A DIET ARY SUPPLEMENT THROUGH WEIGHT LOSS. AND WHAT DEREK FOUND AS FU ADD AN ENCOUPLE R OF CELLS SGRUCHLT LOOK AT THESE FIVE CELLS IN THIS SLIDE, GREEN IS PARKIN AND MIGTO MITOCON DRHONDRIA LABELED IN RED. THEN IF YOU ADD THE ENCOUPLE R, YOU CAN SEE OVER A 30-MINUTE TIME POINT THAT THAT GREEN WILL MOVE FROM THE CIYTOSOL ON TO THE MIGTOCONDRADRI IIA AND THIS WAY YOU CAN MAGNIFY THE PROCESS AND MAN MANIPULATE IT VERY EASILY BY DOING THESE KINDS OF EXPERIMENTS IN CULTURE. WHAT I DIDN'T GET TO IS THIS PROCESS OF ACCUMULATION IS 100% DEPENDENT ON THE KIN AASE PINK 1. SO YOU KNOCK OUT PINK 1, IT DOESN'T GO AT ALL. THE QUESTION IS HOW DOES PINK 1 SENSE THE MIGTOCON DRAHONDRIAL DAMAGE ACCUMULATION AND HOW DOES THIS KIN AASE THEN ACTIVATE IT? SO HERE IS SOME REALLY NICE EXPERIMENTS HERE. THIS BY DEREK AND SCOTT JANET WILLOUGHBY JEN AND -- MICHAEL LASSRO. WITHOUT ANY TREATMENT WHATSOEVER WHATSOEVER, YOU CAN HARDLY SEE THE PINK 1. AND THIS HAS BAIN PROBLEM IN THE FIELD BECAUSE PEOPLE REALLY COULDN'T SEE PINK 1 AND SAID THE ANTIBODIES WEREN'T ANY GOOD. THE FIELD BY AND LARGE OVER OVEREXPRESSED PINK 1 BECAUSE THE ANTIBODIES WEREN'T ANY GOOD. BUT LOOK WHAT HAPPENS WHEN YOU ADD C COMMERCIALIC P. IT'S A 3 MOTHER AND YOU GET A AND THIS IS THE EXACT SITUATION THAT RECRUITS PARKIN FROM THE CIYTOSOL TO THE MEMBRANES. BUT LOOK WHAT HAPPENS FU WASH OUT THE C COMMERCIALIC P FOR 2 1/2 MINUTES OR 5 MINUTES. THE PINK 1 IS IMMEDIATELY EL ELIMINATED. THE EXPERIMENT SHOWS THAT IT TURNS OUT THAT IT'S PROTEOLIESD. IT'S BEING MADE AND IMPORTD INTO THE MIGTOCON DRAHONDRIA SO THE BASAL RATE IS ALMOST UNDETECTIBLE. WE FOUND AN INTERMEMBRANE PROT ASE FAMILY TO KNOCK IT SUIT AND LOOKS WHAT HAPPENS IN 0 TIME WITHOUT ANY TREATMENT WHATSOEVER WHATSOEVER. THAT IS THE POTE AASE THAT'S LINK ING PINK 1 AND ELIMINATING IT. AND I WILL GET TO A MODEL THAT SUMMARIZES THIS WHOLE THING, FOR THOSE OF YOU WHO SAY IT'S NOT MIGTOCON DRAHONDRIA. IT HAS TWO MEMBRANE. OUTER AND INNER. IT'S GOT IMPORT AND ION CHANNELS CHANNELS. IT'S GOT IMIMPORTANT CHANNELS. WHAT NORMALLY HAPPENS IN HEALTHY CELLS THEY ARE IMPORT IING PRO PROTEINS ALL UT TIME. THE IT IMPORTS PINK 1 BY THE INNER MEMBRANE. THAT RELEASE S S IT FROM THE CHALLENGES. IT FLOATS BACK OUT TO THE CIYTO CYTOSOL AND IS ELIMINATED BY THE ANDROOL. AND IF YOU DAMAGE THE MIGTOCONDRI IA, WHAT YOU SEE IS THAT PREVENTS -- ANY KIND OF DAMAGE THAT WILL PREVENT IMPORT HERE, IT COULD BE CHEMICAL OR SKWLENT SKWLENTIC DAMAGE -- THEN IT AC ACCUMULATES, BOUND TO THE COMPLEX THROUGH AN ACTIVITY OF TOM 1K37 FROM THERE IT RECRUITS PARKINS TO THE MIGTOCON DRAHONDRIA. AS I MENTIONED, YOU DON'T HAVE TO HAVE THE CHEMICALS LIKE CC P, ALTHOUGH THOSE WORK QUITE ERRONEOUS LY. GENETIC DAMAGE. AND IN THE KNOCKOUT CELLS I SHOW YOU. PEOPLE THAT HAVE MIGTOCON DRAHONDRIAL DNA MUTATIONS, THEY WILL AC ACCUMULATE PARKIN ON THEIR MIGTO MITOCON DRHONDRIA. BUT MOST OF THESE CAUSE A CLEAR DEPOLARIZATION TO MIGTOCON DRAHONDRIA. BUT WHAT WE'VE RECENTLY FOUND IS PROTEINS REDUCE CAPARKIN TRANS TRANSPOSITION. SO THE COMMON DEFOMENOMINATOR A APPEARS TO BE ANYTHING THAT IN INHIBITS PINK 1 IMPORT IN DESTRUCTION. LET ME SHOW YOU NEWS NEW RESULTS RESULTS. IF YOU DELETE, -- SO HE DEVELOPED THAT INDUCIBLE CELL LINES TO EXPRESS NO TREATMENT. AND AS IN NEW LITERATURE, YOU CAN SEE THIS IS EXPRESSED SPECIFICALLY WITH DOX INDUCTION AND SMALL COMPLOORL WEIGHT. SO IF YOU SEPARATE THESE PRO PROTEINS IN SOLUBLE AND NONSOL NONSOLUBLE. YOU CAN SEE ALMOST 100% OF THE SOLUBLE AND DELTA IS IN THE IN INSOLUBLE FACTION. AND IF YOU LOOK AT PINK 1, IT HAS NO EFFECTS. BUT IF YOU LOOK AT THE MEMBRANE POTENTIAL, OF COURSE, TOXICYCLE TOXICYCLEIN TREATMENT DOES NOT. THIS IS A NEW AREA TO VEG INVESTIGATE. AND YOU CAN SEE HERE IS ANOTHER EXAMPLE THAT C COMMERCIALIC P CAUGHTSES PI NK 1 TO GO UP. AND IT ALSO TRANSLOCATES SIMPLY FROM THIS GOING UP TO PROTEINS OUT.^ DON'T NEED TO ADD ANY CHEMICAL TREATMENT. SO I THINK WE'VE GOT A GOOD HANDLE ON HOW PINK 1, KIN AASE, THE SENSOR MIGHT KNOW DAMAGE. IT ACCUMULATES UPON DAMAGE. NOW THE BIG MYSTERY FOR US FOR MANY YEARS WE'VE BEEN TRYING TO FIGURE OUT IT RECRUITS. WHAT IS THE SUBSTRATE OF THIS KINASE THAT CAUGHTSES PARKINS TO BECOME ACTIVATED? I'M REALLY HAPPY THAT WE SOLVED THAT THIS YEAR. EARLIER THIS YEAR, A POST DOC DID A PH. D IN MASS SPECTROMETRY AND FOUND THAT THAT SUBSTRATE WILL ACTIVATE PARKIN ACTIVITY. I WILL BRIEFLY MENTION THAT. THE WAY WE DID IT IS WE KNOCKED OUT PINK 1 AND THAT ALLOWED US TO DIRECTLY COMPARE INDOCENDOGENOUS PINK 1 CELLS WITH PINK 1 KNOCK KNOCKOUT CELLS. AND YOU CAN SEE IN THE FIRST SLIDE PINK 1. PINK 1 NORMALLY WOULDN'T BE THERE. FIRST WE HAD TO ADD CC P. AND THEN WHAT LESLIE DID WAS ISOLATED MIGTOCON DRAHONDRIA FROM THESE TWO TYPES OF CELLS AND PROTEO PROTEOLIESD THE PROTEIN AND THIS IS THE TIME OF TRIPSIN. SHE PROTEOLIESD THE OUTER MEM MEMBRANE SO SHE DIDN'T DISTURB INNER MEMBRANE PROTEINS. AND SHE WAS LOOKING FOR FOPHOSPHO PHOSPHOPEPTIDES THAT WERE IN THE WILD TYPE BUT NOT IN THE PINK KNOCKOUT. AND THERE IS ONE ROBUST RESULT, WAY WAS A SURPRISE TO US BECAUSE THE ANSWER IS THE PEPTIDE IN INGREDIENT IN RED, AND SER IINE IS FOPHOSPHORYLATED AND IT'S UBTIN IN ITSELF, WHICH IS A SMALL PROTEIN NOTIFICATION. AND IT APPEARS THAT PINK 1 IS POST TRANSLATION ALLY MODIFYING THE POST TRANSLATIONAL MODIFICATION. NOW TO CONFIRM THIS, THE WHOLE TEAM RALLIED BEHIND LESLIE AND HELPED HER GET THIS OUT QUICKLY. YOU GOT PURE PINK 1 AND PURE UBT UBTIN AND ATP AND MIX IT IN A TEST TUBE AND DAY SCOMBLOT COMPARED WILD TYPE PINK 1 AND MUTANT PINK 1 AND FOSPHOSPHORYLATE AND YOU SEE PINK 1S LEFT AND RIGHT. AND THERE IS UBTIN LEFT AND RIGHT. BUT THIS IS A P 32 CRAYEDIO GRAM AND PINK 1 IS KNOWN FO -- PINK 3 31 AND IT'S KNOWN TO FOSS PHOSPHORYLATE ITSELF. AND AS FAR AS I CAN TELL, THIS PINK 1 IS THE FIRST UBIQUITOUS KINASE KNOWN. SO THE FIRST QUESTION IS PINK 1 IS KNOWN TO ACTIVATE THE LIG AASE ACTIVITY. THE QUESTION IS HOW? IS THIS THE SUBSTRATE THAT ACT ACTIVATES PINK 1? THE ANSWER IS YES. WE CAN SIMPLY MIX UBIQUITIN AND GET FOPHOSPHORYLATE SUDDEN AND LOOK AT THE WILD TYPE AND THE MUTANT UBIQUITIN AND THIS IS THE LAT -- LADDER OF THE CHAIN. REMOVING THE PINK 1 FROM THE SYSTEM AND THE BOOM.^ LINE IS MIXING FOPHOSPHOUBIQUITIN WITH PARKIN WILL ACTIVATE THE PARKIN TRANSMUTATION IN CELLS. CELLS SHOULD NOT BE FOSS PHOSPHORYLATED LARGELY TO PREVENT THIS PARKIN TRANS TRANSLOCATION THAT I SHOWED YOU IN THAT MOVIE EARLIER. SO IT'S VERY INTERESTING BECAUSE THE PRODUCT, WHAT PARKIN DOES, COULD BE THE SUBSTRATE FOR PINK 1 TO GENERATE FOPHOSPHOUBIQUITIN, WHICH IS A LIGAN. AND THIS MAY RESULT IN THE ROBUST UBIQUIT NOT A ATIATION OF MIGTOCON DRAHONDRIA AND THERE IS NO CE YOU START THE CYCLE, IT KEEPS GOING. I THINK -- HOW IS THE TIME DOING THERE? OKAY, JOPLII AM GOING TO SKIP THIS REALLY COOL STORY. IN VIVO, WHAT WE SHOW IS THAT PARKIN IS ACTIVE. AND JUST TO CONCLUDE, THIS PATH WAY SHOWS BIOCHEMICAL AND CELL BIOLOGICAL RELATIONSHIPS BETWEEN 3 GENE PRODUCTS LINK D TO PARK PARKINSON'S DISEASE. PINK 1 ACTIVATES PARKIN TO THIS NEW LIGAN AND THE FUNCTION OF THIS PATH WWAY MAY LEAD TO PARK SARNS. MOST IMPORTANT SLIDE. THESE ARE THE PEOPLE IN MY LAB. THIS IS DEREK WENDRA, WHO IS A GRADUATE STUDENT WHO DISCOVERED THIS ORIGINALLY. AND HE IS NOW A RESIDENT IN NEUROLOGY AT HARVARD. LESLIE CHAIN FROM HOPKINS WEB LOT OF HELP WITH MIKE LAZARO AND SCOTT, WHO COVERED AND SHER ERENE, VERY INSTRUMENTAL IN HELPING US. I MENTIONED THIS NEW STEP I DIDN'T GET TO TALK ABOUT, BECAUSE ONE THING THAT'S QUITE INTERESTING IS THERE IS LOWER AND MORE GENES ARE BEING IDENTIFIED AND MUTATED IN PARK PARKINSON'S DISEASE. THE QUESTION IS ARE ANY OTHERS INVOLVED IN THE PATH WWAY? WE PUBLISHED A PAPER EARLIER THIS YEAR AND THERE IS MUTATION IN ANOTHER LAB MI-- LINK ED TED TO PARK PARKINSON'S DISEASE. SO I THINK THERE IS MORE OF THIS PATHWAY IN THE FUTURE. THANK YOU. APPLAU [APPLAUSE] >> THANK YOU FOR THAT BEAUTIFUL STORY. THANK YOU FOR THIS AFTERNOON. THAT WRAPS US UP. AND WE HOPE TO SEE YOU OUT THERE AT THE OPENING CEREMONY.