>> GOOD AFTERNOON. WELCOME TO THE FIRST OF THE NIH INURE O SCIENCE SEMINAR SERIES PROGRAMS TO BE HELD IN THIS BEAUTIFUL RECENTLY COMPLETED PORTER NEUROSCIENCE CENTER. THE PNRC HAS A SPECIAL MEANING TO US AT NIDCD BECAUSE IT ENABLED US FOR THE FIRST TIME IN OUR 25 YEAR HISTORY TO HAVE ALL OF OUR INTRAMURAL SCIENTISTS LEICATEED ON THE NIH CAMPUS. TODAY WE FEATURE THE FOURTH ANNUAL WENDEL MEMORIAL LECTURE--HE SERVED AS SCIENTIFIC DIRECTOR FOR 10 YEARS FROM 1998-2008. TODAY WE ARE CELEBRATING BOB'S CONTRIBUTIONS TO SCIENCE AND NIH WITH A LECTURE TITLED UNCOVERING THE MICRO CIRCUITRY OF THE AUDITORY SYSTEM DELIVERED BY DR. LAURENCE TRUSSELL, WHO WAS A GOOD FRIEND OF BOB'S. SO PLEASE JOIN US FOR A LIGHT RECEPTION AT THE END. I WANT TO THANK THE FOUNDATION IN THE EDUCATION FOR SCIENCES FOR THEIR GENEROUS DONATION OF THE REFRESHMENTS WHICH YOU WILL ENJOY. >> THANK YOU TO EVERYBODY FOR COMING AND FOR THE FAMILY OF BOB, I AM VERONICA AND I AM A INVESTIGATOR AT THE NIAAA, AND I WANT TO START BY EXPRESSING MY GRATITUDE TO BOB WHO I THINK IS FAIR AND HONEST TO SAY I WOULDN'T BE AT NIH IF IT WASN'T FOR HIM. HE WAS VERY INSTRUMENTAL IN GETTING ME HERE AT THE NIH AND STUDIED IN THE TRUE SPIRIT OF COLLABORATION BETWEEN NIH INSTITUTES, HE PUT TOGETHER PEOPLE FROM THE AAA, AND NINDS TO FINALLY HIRE ME. I'VE BEEN VERY HAPPY HERE AND TODAY IS MY REALLY PLEASURE AND HONOR TO INTRODCE LARRY TRUSSELL, HE IS AT THE OHSU, AND SCIENTISTS AT THE BON HAM INSTITUTE AS WELL, HE RECEIVED HIS Ph.D. FROM UNIVERSITY OF CALIFORNIA, LOS ANGELES, AND WHERE HE DID HIS THESIS WORK WITH DR. ALAN GRUNNELL, STUDYING SIN APTATION OF SYNAPTIC STRENGTHS OF THE FROG NEUROMUSTULAR CONJUNCTION. HE CONTINUED HIS POST DOC TRAINING AT UNIVERSITY OF WASHINGTON UNIVERSITY SAN LUIS WHERE HE WORKED WITH GERALD FISHERBATCH WHERE HE STUDIED THE ROLL IN SYNAPTIC TRANSMISSION. IN THE 1990SULARLY JOINED THE FACULTY OF THE DEPARTMENT--1990S LARRY JOINED THE FACULTY DEPARTMENT WHERE HE STUDIES THE GLUTEA MATE RECEPTOR STATION AND FUNCTION BUT NOW WITHIN SYNAPSES OF THE AUDITORY SYSTEM. IN 1919 SO NINE YEARS AFTER THAT HE JOINED THE OHSU AS PROFESSOR OF THE OREGON RESEARCH CENTER AND ALSO SCIENTISTS AT THE BONHAM, SINCE THEN HIS LABORATORY HAS GONE ON BEFORE THEM THEN TO SIGNIFICANT CONTRIBUTIONS OF OUR UNDERSTANDINGS ON HOW SYNAPSES CAN WORK AND RELAY VERY PRECISE TIME AND INFORMATION. HIS LABORATORY STUDIES MAKEANISM NEUROTRANSMISSION RELEASE AND CLEARANCES WITHIN THE SYNAPSES WITHIN THE COCHLEAR NUCLAS AND THE--OTHER NEWICALLIAS WITHIN THE ODD--NUCLAS WITHIN THE SYSTEM AND OPTICAL APPROACHES TO UNDERSTAND THE MECHANISMS ALSO OF LONG-TERM PLASTICITY WITHIN THIS AUDITORY CIRCUIT. VERY BRIEFLY, I ALSO WANT TO MENTION SOME OF THE CONTRIBUTIONS THAT DON'T MAKE IT SO MUCH TO THE CV TO THE WHOLE HONOR OF THE SCIENCE COMMUNITY. LARRY SERS INVOLVED WITH--WAS INVOLVED WITH DR. JACKSON WITH THE FIRST SYNAPTIC TRANSMISSION CONFERENCE AND ALSO INVOLVED IN THE REVAMPING OF THE ION CHANNELS [INDISCERNIBLE] WHO HELPED TRAIN PROBABLY A LOT OF PEOPLE HERE AND MORE GREAT SCIENTISTS TO COME AND ELECTROPHYSIOLOGY SO THANK YOU FOR THAT AS WELL. AND TODAY HE IS GOING TO TELL US ABOUT UNCOVERING THE MICRO SYMMETRY OF THE AUDITORY SYSTEM. [ APPLAUSE ] >> THANK YOU FOR THAT VERY KIND INTRODUCTION. I WOULD LIKE TO THANK THE WENDTOHLD FAMILY AND THE NIH FOR HAVING THE LECTURE SERIES IN BOB'S HONOR. I FIRST BECAME AWARE OF BOB'S WORK WHEN I WAS AT THE UNIVERSITY OF WISCONSIN, BOB WAS THERE AS A FACULTY MEMBER AND HAD LEFT TO COME BACK TO THE NIH JUST BEFORE I ARRIVED. BUT WE BOTH HAD A COMMON INTEREST IN THE NEUROTRANSMITTER SYSTEMS OF THE AUDITORY PATHWAYS. SO, I BEFRIENDED BOB AND HE, AS EVERYBODY KNOWS HE'S VERY GUS WITH THE REAGENT SO HE SENT US ANTIBODIES WHICH WERE A BIG HELP FOR US. BUT MORE SO HE INVITED ME OUT TO THE NIH ON SEVERAL OCCASIONS AND I GOT TO SEE WHAT KIND OF SCIENTIST HE WAS AND WHAT WAS WAS--WHAT KIND OF PERSON HE WAS AND HOW HE INTERACTED WITH PEOPLE IN THE LAB AND HOW MUCH THE PEOPLE IN HIS LAB RESPECTED HIM. SO FROM THOSE KIND OF INTERACTIONS, I LEARNED THIS WAS THE KIND OF SCIENTISTS, I WANTED TO BE, AND THOSE ARE BIG SHOES TO FILL. BUT IN THINKING ABOUT IT WHAT I REALIZED WAS, WE REALLY JUST KEEP THE MEMORY OF SOMEBODY LIKE BOB ALIVE THEN THAT WILL ALWAYS HOLD THE STANDARD OF THE SCIENTIFIC STANDARD AND THE STANDARD FOR GOOD WILL AND INTEGRITY AND DEEP THAT GOING IN OUR FIELD. SO I'M VERY GRATEFUL FOR HIS CAREER. I WOULD LIKE TO BEGIN BY THANKING PEOPLE IN MY LAB. MY CURRENT LAB MEMBERS AND ALSO PAST LAB MEMBERS. AND IN PARTICULAR, I WANT TO GIVE A SHOUT OUT TO PIERRE APOSTOLIDES, WHOSE WORK WILL BE SHOWN TODAY AND I WANT TO THANK THESE THREE INDIVIDUALS WITH WHOM I DO NOT COLLABORATE, ENRICO, PAUL AND DONATA, THEY WORK HAS AFFECTED VIRTUAL EVERYTHING WE DO IN THE LAB AND I THINK ABOUT THEIR WORK EVERY DAY AND I'M GRATEFUL FOR THAT. SO CAN YOU HEAR ME IN THE BACK? LIKE MUSICAL CHAIRS BACK THERE. [LAUGHTER] SO I WANT TO START BY REMINDING YOU ALL OF THE FUNCTION OF THE AUDITORY SYSTEM WHICH IS TO IDENTIFY SOUNDS AND LOCALIZE SOUNDS AND WE TYPICALLY SEE THE AUDITORY PATHWAYS AS REPRESENTED BY THIS ASCENSION FROM--WHOOPS--SORRY. THIS ASCENSION FROM THE COCHLEA WHERE SOUNDS ARE FIRST TRANSDUCED AND THEN THROUGH THE BRAIN STEM, MIDBRAIN AND THALAMUS AND INTO THE CORTEX. AND IN LOOKING AT THIS KIND OF DIAGRAM IT GIVES THE IMPRESSION THAT WE'RE DEV--DEALING WITH A SERIES OF RELAYS, BUT WE KNOW THAT IS NOT TRUE BECAUSE INFORMATION IS ALSO PASSED FROM HIGHER REGIONS BACK DOWN TO LOWER REGIONS SO THESE AUDITORY PATHWAYS ARE HIGHLY INTERACTIVE. AND WHAT'S MORE IN EACH REGION, EACH SUBSTATION OF AUDITORY PROCESSING THERE ARE MICROCIRCUITS AND THE WORK IN MY LAB IS DIRECTLY NOW TO TRYING TO UNDERSTAND THE COMPUTATION, THE COMPUTATIONS THAT TAKE PLACE WITHIN THESE MICROCIRCUITS. IN PARTICULAR, THE COCHLEAR NUCLEUS, THIS FIRST STATION FOR AUDITORY PROCESSING IN THE BRAIN. SO HOW CAN WE UNRALPH THESE MICROCIRCUITS--UNRAVEL THESE MICROCIRCUITS? WELL THIS IS A DIFFICULT PROBLEM FOR A NUMBER OF REASONS. ONE IS WE NOT ONLY HAVE TO KNOW WHAT THE CELLS ARE WITHIN THE CIRCUIT, HOW THEY'RE INTERCONNECTED, BUT ALSO HOW DO THEY TALK TO EACH OTHERS, WHAT ARELET DYNAMIC INTERACTIONS THAT TAKE PLACE BETWEEN DIFFERENT TYPES OF NEURONS. AND PEOPLE HAVE APPROACHED THESE DIFFICULT PROBLEMS IN DIFFER WAYS OVER THE YEARS. THE FIRST APPROACH THAT PEOPLE TOOK WAS THROUGH NEUROANATOMY. SO THIS SPECTACULAR DRAWING IS FROM LORENTA DENO THIS IS HIS SIGNATURE FROM WHICH HE DREW THIS IN 1931 IT LOOKS LIKE ASK IT SHOWS THE COCHLEA AND NUCLEUS IN THIS TWO PAGE DRAWING, YOU CAN SEE THE CELL TYPES ARE QUITE DIVERSE AND SO THIS APPROACH TELLS US WHO'S THERE BUT IT DOESN'T SAY ANYTHING ABOUT HOW THE CELLS ARE CONNECTED OR HOW THEY TALK TO EACH OTHER OR HOW THEY RESPOND TO AUDITORY ACTIVITY. AND IN LATER YEARS PEOPLE APPROACH THIS PROBLEM BY DOING RECORDINGS FROM ANIMALS DURING EXPOSURE TO SOUND. SO IN THESE KINDS OF STUDIES AND ELECTRODE WOULD BE PLACED IN THE BRAIN AND THE SPIKE ACTIVITY, THE ACTION POTENTIAL ACTIVITY OF INDIVIDUAL NEURONS IN RESPONSE TO A SOUND STIMULUS WOULD BE RECORDED AND THEN THAT NEURON WOULD BE LABELED AND RECONSTRUCTED AND SO, WE COULD LEARN HOW A PARTICULAR KIND OF CELL RESPONDS. BUT THESE TWO APPROACHES HAVE LIMITATIONS AND THAT LIMITATION IS FIRST OF ALL IT DOESN'T TELL YOU MUCH ABOUT HOW THE DIFFERENT TYPES OF CELLS, WITHIN THIS SUBREGION ARE INTERCONNECTED AND THEN HOW THOSE DIFFERENT TYPES OF CELLS TALK TO EACH OTHER. SO WE'VE APPROACHED THIS, WE AND OTHER VS APPROACHED THIS PROBLEM USING BRAIN SLICE STUDIES. SO WE AND DONATA ORTEL AND OTHER LABS FIND THAT IF YOU MAKE A THIN SLICE OF THE BRAIN AND PUT IT IN A DISH, YOU CAN IDENTIFY FROM CELL TYPES, YOU AND CAN YOU SEE HOW CELLS ARE COMMUNICATING AND IN MY OWN LAB WE'VE TAKEN THE PARTICULAR APPROACH OF DOING PAIRED RECORDINGS, SO RECARDING FROM TWO CELLS THAT ARE INAPTICALLY COUPLE--SYNAPTICALLY COUPLED AND THIS MICRO GRAPH SHOWS AN EXPERIMENT DURING AN EXPERIMENT WHERE WE HAVE TWO ELECTRODES, TWO CELL BODIES. AND WE ADD TO THIS, A FEW ADDITIONAL TECHNIQUES, ONE IS WE GENERALLY RECORD FROM ANIMALS IN WHICH A GENETICALLY ENCODES FLORA FLORS LIKE GTD OR TOMATO ARE IDENTIFIED IN THE CELL SO WE CAN KNOW BEFORE WE PATCH IT SO WE KNOW WHAT KIND OF CELL WE'RE RECORDING FROM SO I CHARACTERIZED THE CELLS BY HAVING THEM DIFFERENT COLORS. NOW LEST, YOU THINK THAT IT'S A HOPELESS TASK TO TRY TO UNDERSTAND BRAIN CIRCUITRY, YOU KNOW THE MILLIONS OF CONNECTIONS IN THE BRAIN, TWO CELLSA AT A TIME, I'D SAY A COUPLE THINGS. ONE IS: WELCOME TO MY LIFE. [LAUGHTER] THE OTHER IS, I HOPE BY THE END OF THE SEMINAR YOU'LL SEE THAT IT'S ACTUALLY POSSIBLE TO LEARN SOME FUNDAMENTAL FACTNEURONAL COMMUNICATION JUST STUDYING CELLS AT THIS LEVEL. SO, AN ADDITIONAL TECHNIQUE WE'RE BRINGING TO THE TABLE IN THIS IS THE TECHNIQUE OF OPTICAL IMAGES O GENICS, AND THE--OPTICALOPTOGENETICS AND WE CAN STUDY TWO TABLES AT THE SAME TIME. IN FACT, INDIVIDUAL NEURONS RECEIVE POPULATIONS OF NEURONS VERY OFTEN AND THOSE POPULATIONS OF INPUT CELLS ALSO TARGET OTHER TYPES OF NEURONS WHICH THEN SYNAPSE ON A PARTICULAR NEURON OF INTEREST, SO BY PUTTING CHANNEL REDOBSIN WITHIN A SPECIFIC POPULATION OF CELLS AND THEN SHINING BLUE LIGHT ON THAT POPULATION, WE CAN RECORD THE ACTIVITY OF A GIVEN NEURON TO THAT POPULATION OF INPUT FIBERS AND TO THE OTHER CELLS THAT ARE CONTACTED BY THOSE INPUT FIBERS SO IT EXPANDS OUR RANGE OF INFORMATION WE CAN OBTAIN WITH THE ELECTROPHYSIOLOGICAL APPROACH. SO THE WORK WE'RE DOING IS CENTERED ON THE MICRO CIRCUITRY OF THE COCHLEA NUCLEUS AS I MENTIONED AND IN PARTICULAR THE DORSAL CO-NUCLEUS, WHICH IS ONE OF TWO DIVISIONS OF THE COICALLIA NUCLEOTIDES CLEAR COMPLEX AND THE COCHLEA NUCLEUS OR DCN HAS AT ONE LEVEL A FAIRLY SIMPLE LAY OUT. I HAVE A PRIMARY PRINCIPAL CELLED CALLED THE FUSI FORM CELL, WHICH RECEIVES INPUT FROM TWO DIFFERENT STREAMS OF INFORMATION, ONE IS AUDITORY INPUT I HAVE AT AUDITORY NERVE AND THIS INPUT IS THEN PROCESSED BY POPULATION OF INTERNEURONS AND THEN THERE'S A NONAUDITTORY INPUT THAT I WILL CALL THE MULTISENSORY INPUT WHICH COMES IN, AGAIN SEES ITS OWN COLLECTION OF INTERNEURONS AND THEN SYNAPSES ON THESE--ON OTHER SET OF DENDRITES IN THE FUSOFORM CELLS AND BOB AND RUBY DID SEVERAL REALLY NICE STUDIES DESCRIBING THE GLUTEA MATE RECEPTORS THAT ARE SELECTIVELY TARGETED TO THESE TWO DENDRITIC DOMAINS OF THE FUSI FORMS. NOW, IF WE LOOK BEYOND JUST THE ONE CELL AT THE TYPES OF CELLS WITHIN THE COCHLEA NUCLEUS, WE SEE THE DORSAL COCHLEA NUCLEUS, WE SEE SOMETHING INTERESTING WHICH IS THAT THERE'S AN ARRAY OF EXCITATORY GLUTAMINERGIC NEURONS WITH INTERESTING NAMES LIKE FUSI FORM AND GIANT BRUSH AND GRANULE. THERE'S ALSO AN EQUALLY SIZED ARRAY OF OF THE NEURONS, THE SUPERFICIAL CELL, GOLGI, CART WHEEL AND TUBER CULOVENTRAL CELL. AND LOOKS AT THIS CARTOON IS QUITE DOMINANT AND SELECTED FOR WITHIN THE DCN AND THAT'S APPARENT USING OTHER TECHNIQUES BESIDES CARTOONING SO HERE IS A STUDY OF DONATO WHERE SHE MADE A STUDY OF THE DORSAL NUCLEUS AND SHE LABELED IT USING AN ANTIBODY OF THE NEURODEVELOPPED AND YOU CAN SEE THAT WITHIN THE VENTRAL DIVISION THERE'S SOME LABELING, IT'S NOT PARTICLARLY DARK BUT ONCE YOU GET INTO THE DCN, IT'S ALMOST BLACK, SUGGESTING THAT INHIBITION IS VERY IMPORTANT IN THIS BRAIN REGION. AND WE'VE APPROACHED THIS MORE RECENTLY USING A SOMEWHAT DIFFERENT TECHNIQUE. SO HERE WE'RE USING A MOUSE IN WHICH IDENTIFIES GLUE MARIOUS SEENERGIC NEURONSOT BASIC EXPRESSION OF THE NEURONAL GLYTTWO-GFP, SO THE GFP IS EXPRESSED WITH THE GLUE MARIOUS SYNERGIC NEURONS SO IF YOU LOOK AT THIS SECTION, HERE'S THE STEM, THE DCN, AND THE CEREBELLUM. AND YOU CAN SEE THAT THE DCN LOOKS LIKE A CHOCOLATE CHIP COOK O ST. PATRICK'S DAY, IT'S LIT UP WITH NEURONS. AND THIS AGAIN SUGGESTS THAT GLYSIGN AND AN ININCREASE IN BODYITION IN GENERAL IS IMPORTANT IN THE DCN. SO WE TAKE ADVANTAGE OF THIS LOOKING CLOSER AND LOOK AT THESE CELL TYPES FOR OUR RECORDINGS. SO WE CAN IDENTIFY WITHIN THIS DEEP LAYER THE TUBERCULAR CELL AND IT IS CART WHEEL CELLS AND THEN FINALLY IN THE OUTER EDGE A REGION CALLED THE MOLECULAR LAYER, WE CAN SEE THE SUPERFICIAL CELL CHRS THESE LITTLE GUYS KIND OF PLASTERED UP AGAINST THE EDGE OF THE BRAIN ALMOST FALLING OUT OF THE BRAIN. SO WE'VE USED THESE APPROACHES THAT I MENTIONED TO COME UP WITH CIRCUIT DIAGRAMS OF THE DORSAL COCHLEA NEW CLEUS. AND THIS IS WHAT WE COME UP WITH AND THIS IS BASED ON THE WORK OF OTHER LABORATORIES AND ALSO ON OUR STUDIES USING PAIRED RECORDINGS AND IDENTIFIED CELLS, SO THE RED NEURONS AND ORANGE NEURONS ARE THE INHIBITORY CELLS A THE BLUE ONES ARE THE EXCITATORY CELLS AND IT SHOWS THE QUITE COMPLICATED PATTERNS OF CONNECTIONS. WE HAVE THE AUDITORY PATHWAY COME NOTHING INNERIVATING THE FUSI FORM CELL, AND THE NONAUDITTORY PATHWAY AS MOSI FIBERS INHIBITORRING CELLS AND THEN THE PARALENS LENS LENS LENS FIBERS FROM THE--PARALLEL CELLS WHICH FORM THE OWN DOMAIN OF CIRCUITRY HERE AND IN THE MOLECULAR DOMAIN. AND I'M SHOW THANKSGIVING DIAGRAM MAINLY TO EMPHASIZE THE IMPORTANCE OF THE INTERNEURONS AND THE SEEMINGLY COMPLETE PICTURE WE HAVE OF HOW GLUTEA MITERGIC NEURONS INTERACT WITHIN THIS REGION. BUT WHAT'S LEFT OUT HERE IS ANOTHER TYPE OF SIN APINGS WHICH IS ACTUALLY RECEIVED VERY LITTLE ATTENTION WITHIN THE AUDITORY SYSTEM AND THAT'S THE ELECTRICAL SYNAPSE. SO THE ELECTRICAL SYNAPSES ARE FORMED WHEN TWO CELLS OPPOSE EACH OTHER AND ARE CONNECTED BY A PROTEIN COMPLEX CALLED A CONNECKSON AND FORMED FROM SUBUNITS. AND THIS GAP JUNCTION CONNECTION ALLOWS IONS AND SMALL MOLECULES TO PASS BETWEEN TWO COUPLED CELLS AND IT PERMITS BOTH CHEMICAL COUPLING AND ELECTRICAL COUPLING BETWEEN THE CELLS. SO WHAT'S THE ROLE OF THE ELECTRICAL SIGNALING PLAY A ROLE IN THE AUDITORY SYSTEM? WELL THERE'S INDICATION THAT IT MIGHT IF YOU LOOK AT THIS MICROGRAPH, THIS IS AN OLD FROM THE LAB OF THOSE TINY INTERNEURONS I MEKSED, THE SUPERFICIAL SATELLITE CELLS AND HE DESCRIBED GAP JUNCTIONS SCRIBED WITH EM AS BEING QUITE COMMON IN THE SUPERFICIAL STELLATE CELLS AND HE SUGGESTED THAT MIGHT FORM A NOTE WORK OF INTERNEURONS. SO WE EXAMINE THAT USING OUR BRAIN SLICE PREPARATION, RECORD FREE RADICALS GENERATED TWO STELLATE CELLS AT A TIME AND FOUND THAT PREDICTED BY THE GROUP, THAT THEY ARE INDEED ELECTRICALLY COUPLED SO IF WE INJECT A CURRENT WITHIN ONE CELL IT HYPER POLARIES THAT CELL BUT RECORDING IN THE NEIGHBORING CELL, CAN YOU SEE THAT HYPER POLARIZATION TRANSMITTED THROUGH AN ELECTRICAL CONNECTION AND THEN IF WE SWITCH IT AROUND AND INJECT CURRENT IN THE OTHER SELL, THEN THAT CURRENT IS TRANSMITTED INTO THE FIRST CELL, SO THIS IS THE HALLMARK OF ELECTRICAL COUPLING AND WE CAN MEASURE THE STRENGTH OF THAT COUPLING AS THE RATIO OF THE POST JUNCTIONAL RESPONSE HERE TO THE FREE RADICALS JUNCTIONAL RESPONSE AND IT GIVES US A COUPLE AND CO-EFFICIENT OF A FEW% CAN COUPLING MEASUREMENTS THAT ARE INTERESTED WITHIN THE NETWORKS OF THE NERVOUS SYSTEM. BUT WHAT WAS SURPRISING TO US WAS THAT NOT ONLY ARE THESE STEALATE CELL WHICH IS ARE PREDOMINANTLY GABAergic, THEY'RE COUPLED TO ONE ANOTHER BUT THEY'RE ALSO COUPLED TO THE GLUTEA MA TERNALLIC CELLS, THE FUSI FORM NEURONS. SO HERE WE'RE INSTRUCTING A THIS INTO THE NEURON AND YOU CAN SEE THE HYPER POLARIZATION TRANSMITTED INTO THE STELLATE CELL AND THEN VICE VERSA. WHAT WAS INTERESTING HERE WAS THAT WHEN WE COMPARED THESE COUPLING CO EFFICIENTS GOING FROM THE STELLATE CELL TO THE FUSIFORM CELL, AND THEN IN THE REVERSE DIRECTION, THE COUPLING IS THE UNITEAM ION, THE COUPLING WAS STRONGER GOING FROM THE FUSIFORM INTO THE STEALATE CELL. SO IT SEEMED LIKE AN ELECTRICKIFYING CONNECTION. SO WE EXPLORED THAT IN THIS ELECTRICAL CONNECTIONS INDEED DUE TO GAP JUNCTION. SO THESE CONNECTIONS, THESE ELECTRICAL CONNECTIONS CAN BE BLOCKED BY CLASSIC BLOCKER OF GAP JUNCTIONS, AND THEY'RE ABSENT IN A MOUSE LINE IN WHICH THE 36 SYSTEM IS KNOCKED OUT. SO HERE WE'RE INFUSING THE HYPER STEALATE CELL AND IN THE OTHER STELLATE CELL WE SEE NOTHING. AND MORPH OVER THE GENERATED CITE REPORTS CONNECTION OF 36 WITHIN LARGE NEURONS WHICH ARE UNDOUBTEDLY FUSIFORM CELLS. SO THESE SUGGIEST THAT THE INTERNEURONS AND THE PRINCIPLE CELLS ARE ELECTRICALLY COUPLED VIA CONNECTION 36 CONTAINING GAP JUNCTIONS. SO WHAT IS TRANSMITTED TO THESE GAP JUNCTIONS. WELL, IF WE RECORD IN THE STELLATE CELL, WE CAN SEE SPONTANEOUS MINIATURE ACTION POTENTIALS OCCURRING SO THESE ACTION POTENTIALS INSTEAD OF BEING LIKE MOST SELF-RESPECTING ACTION POTENTIALS THERE ARE ONLY A FEW AND AMPLITUDE AND THEY'RE ABSENT IN CONNECTION WITH KNOCKOUT MICE SUGGESTING THAT THESE ARE--THESE SO CALL SPIKELETS ARE TRANSMITTED THROUGH THE GAP JUNCTIONS FROM FUSI FORM CELLS AND INDEED IF WE RECORD FROM A PAIR OF CELLS, A FUSI FORM AND A STELLATE CELL AND INJECT CURRENT IN THE FUSI FORM CELL TO TRIGGER ACTION POTENTIALS, WITHIN THE CELL WE SEE THE ONSET OF SPIKELETS THAT ARE SYNCHRONOUS WITH THOSE IN THE FUSI FORM NEURON. SO HOW DO THESE SPIKES GET FROM THE FUSI FORM CELL INTO THE STELLATE CELL? SO IN THIS MICROGRAPH WE'RE RING FROM AN ELECTRICALLY COUPLED PAIR OF CELLS, THE BIG GREEN GUY IS THE FUSI FORM CELL AND THE RED ONE IS THE STELLATE CELL AND THE--YOU CAN SEE THAT THE SITE OF INTERACTION BETWEEN THESE TWO CELLS IS RIGHT AT THE TIPS OF THE DENDRITES SO IF WE GO UP INTO HIGHER POWER, WE CAN SEE THAT THE POINTS OF CONTACT BETWEEN THE STELLATE CELL AND THE FUSI FORM CELLS ARE ON THE DENDRITES OUT ON THE OUTER EDGE OF THE MOLECULAR LAYER AND EAR'S A POINT OF CONTACT WHICH IS A SINGLE 2-PHOTON SECTION WHICH SUGGESTS THAT THESE POINTS OF APPOSITION BETWEEN THE TWO CELLS MIGHT BE WHERE THE GAP JUNCTIONS ARE. SO WHAT IT INDICATE SYSTEM THAT AN ACTION POTENTIAL GENERATED WITHIN THE FUSI FORM CELL HAS TO BACK PROPAGATE INTO THE DISTAL DENDRITES AND THEN INTO THE STELLATE CELL. NOW, THAT SUGGESTS THAT WHAT'S TRANSMITTED ARE THESE LITTLE SPIKELETS WHICH DON'T SEEM VERY IMPRESSIVE SO WE--WE LOOKED FOR ANOTHER, SEVERAL OTHER WAYS TO ASSAY THE IMPACT OF THIS CONNECTIVITY BETWEEN THESE TWO CELLS. SO IN THIS EXPERIMENT WHAT WE DID WAS STIMULATE--INSTEAD OF STIMULATING THE FUSI CELL, WE RECORD FRIDAY A STELLATE CELL SHOWN HERE AND THEN STIMULATED AUDITORY NERVE FIBERS THAT THEN SYNAPSE ON TO THE FUSI FORM CELL AND WHAT WAS RECORDED THEM WITHIN THE STELLATE CELL STLES A MUCH LARGER DEPOLARIZATION FOLLOWED BY A LATE AFTER HYPER POLARIZATION, I WILL SHOW YOU A LOT OF PICTURES OF THIS KIND OF WAY FORM AND EXPLAIN BY THE END OF THE TALK WHAT'S REALLY GENERATING IT. SO THIS IS AN INTERESTING RESULT IN ITSELF BECAUSE IT SUGGESTS THAT SIGNALINGS COMING INTO THIS AUDITORY DOMAIN OF THIS PROCESSING CAN FUNNEL THEIR WAY INTO THIS MULTISENSORY PROCESSING DOMAIN THROUGH THE FUSI FORM CELL AND THE GAP JUNCTIONS WITH THE STELLATE CELL, SO WE WONDERED WHETHER THIS IS A SIMPLY A CELL-CELL CONTACT PHENOMENON LIKE I'M SHOWING HERE OR WHETHER MULTIPLE FUSI FORM CELLS MIGHT CONVERGE ON A SINGLE STELLATE CELL SO THE WHY SIDE THAT--IDEA IS THAT STELLATE CELLS MIGHT BECAUSE OF THE ARRAY OF THE DENDRITES RECEIVE REFUSE THESE FORMS AND MIGHT INCREASE THE POWER OF THEIR RECEPTION OF AUDITORY SIGNALS OCCURRING DOWN HERE. SO WE TESTED THAT USING THE METHOD OF OPTICAL IMAGES O GENETICS SO WE FOUND SEVERAL MOUSE LINES IN WHICH CHANNEL ROUGH ATOM DOBSIN WAS EXPRESSED--REDOBSIN WAS EXPRESSED IN THE CHANNEL CELLS. SO ONE OF THESE WAS A VGLUT SIGNAL AND THAT IS A TRANSPORTER AND ONE OF OF THE THYONE CHANNEL RECOPINGSON CHAI ARE--REDOBSIN CELLS AND IF WE CUT AND FUSI, WE CAN SEE A GENERATE IN THE FUSI FORM CELL AND WE CAN RECORD ACTION POTENTIALS AND NOW RECORD FREE RADICALS GENERATED THE STELLATE CELL, WHAT WE SEE DURING SIMULATION IS IN VOLTAGE CLAMP IS AN INWARD CURWENT LOTS OF LITTLE SPIKELETS GENERATED AND YOU CAN SEE THAT THE SPIKELETS HAVE DIFFERENT AMP LITUDE WHICH IS WE THINK MEANS EACH SPIKELET IS COMING FROM A DIFFERENT FUSIFORM CELL WITH A DIFFERENT COUPLING CO EFFICIENCY FROM THE CELL WE'RE RECORDING FROM AND IN CURRENT CLAMP THAT GENERATE ACE STUDY DEPOLARIZATION AND AT THE END OF THE BLUE LIGHT FAULT THERE'S AFTER HYPER POLARIZATION. SO WHAT IS THIS AFTER HYPER POLARIZATION HERE ALL ABOUT? SO THE DEPOLARIZATION IS FROM THE ACTION POTENTIALS AND THE CHANNEL REDOBSIN CURRENT IN THE FUSI FORM CURRENT BUT WHAT'S THIS NEGATIVE GOING DIP, SO WE THINK THAT IS INHERITED FROM THE FUSIFORM CELL. SO HERE I'M SHOWING YOU ACTION POTENTIAL GENERATED IN A FUSI FORM CELL AND AFTER THE END OF THE ACTION POTENTIALS GENERATED EITHER WITH CURRENT INJECTION OR A BLUE LIGHT STIMULUS, THERE'S AFTER HYPER POLARIZATION AND THIS SHOWS THAT AFTER PIPER POLARIZATION AT HIGHER GAIN. NOW IF WE LOOK IN THE STELLATE CELL, WE CAN SEE AN AFTER HYPER POLARIZATION FOLLOWING A STIMULUS BUT IF WE JUST INJECT CURRENT INTO THE STELLATE CELL TO GIVE A DEPOLARIZATION ABOUT THE SAME SIZE AS WE GET WITH NOW BLUE LIGHT WE SEE NO AFTER HYPER POLARIZATION SO THAT TELLS US THAT THE NEGATIVE GOING AWAY FORM IS TRANSTRANSMITTED THROUGH THE FUSI FORM CELL AND THAT HAS INTERESTING CONSEQUENCES WE THINK FOR PROCESSING IN THIS REGION. SO, AND THAT WAS TESTED IN THIS EXPERIMENT, SO THIS IS A LITTLE COMPLICATED SO LET ME TAKE YOU THROUGH IT. SO HERE WE RECORD FREE RADICALS GENERATED A STELLATE CELL AND WITHIN THE STEALATE CELL WE'RE INJECTING ELECTRICAL CURRENT TO POLARIZE THE STELLATE CELLS AND TRIGGER ACTION POTENTIALS. SO WE'RE DOING THAT JUST REPEATEDLY SO EACH 1 OF THESE SPIKES IS DUE TO CURRENT INJECTION AND IN ADDITION TO THAT, WE ARE SHINING A PULSE OF BLUE LIGHT AND THE TIMING OF THE BLUE LIGHT PULSE WITH RESPECT TO THE CURRENT INJECTION IS SHIFTED IN DIFFERENTLY IN EACH 1 OF THESE PANELS. AND WHAT YOU CAN SEE IS THAT AS THAT BLUE LIGHT APPROACHES IN TIME, THE CURRENT INJECTION, THE ACTION POTENTIALS THAT ARE GENERATED BY THAT CURRENT INJECTION ARE INHIBITED. AND THEN WHEN THEY OCCUR AT THE SAME TIME, THE NUMBER OF ACTION POTENTIALS INCREASES OVER THE CONTROL AND THEN EVERYTHING RETURNS TO NORMAL AFTER THE BLUE--THE TIMES OF THE BLUE LIGHT PULSE PASSES, SO IF WE PLOT THE CHANGE IN SPIKE RATE AS A FUNCTION OF THE TIMING OF THAT PULSE, WE CAN SEE IT'S INTERESTING BI-PHASIC TIMING CURVE. SO WHAT IT WILLS US IS THAT THE XIEWNICATION VIA THE GAP JUNCTION BETWEEN THE FUSI FORM CELL AND THE STELLATE CELL, IS BOTH EXCITEATOR SCHEINHIBITTORY AND IT DEPENDS ON THE RELATIVITY OF THE TIMING ON THE 2 CELL TYPES WHAT ASPECT OF THAT EXCITATION OR INHIBITION WILL BE TRANSMITTED. ALL RIGHT, SO THESE ARE THE CONCLUSIONS FOR THIS PART OF THE TALK THAT FIRST OF ALL THAT GAP JUNCTIONS OCCUR BETWEEN INHIBITORY STELLATE CELLS AND FUSI FORM CELLS AND THESE ENABLE THE FUSI FORM CELL TO MODIFY THE ACTIVITY OF THE STELLATE CELLS AND INAPTIC TARGETS AND I DIDN'T--I DIDN'T PLAN ON HAVING TIME TO SHOW YOU THIS, BUT WE'VE ALSO LOOKED AT THE SYNAPTIC TARGETS OF THE STELLATE CELL, SO THE CELLS OWNAXON SYNAPSES ON TO OTHER INTERNEURONS AS WELL AS FUSI FORM CELLS AND THIS ACTIVITY THAT'S TRANSMITTED FROM THE FUSI FORM CELL IS THEN TRANSMITTED VIA ACTION POTENTIALS ON TO THE SYNAPTIC TARGETS OF THE STELLATE CELL. SO WHAT I WANT TO TURN TO NOW IS A SOMEWHAT DIFFERENT QUESTION. SO WHAT I TOLD YOU IS THAT IF WE EXCITE THE CELL, DRIVE ACTION POTENTIALS IN THE FUSI FORM CELL, THAT THOSE ACTION POTENTIALS CAN BE TRANSMITTED AND THEN EXCITE THE STELLATE CELL AND THEN AFTER THAT PERIOD OF EXCITATION THERE'S HYPER POLARIZATION AND NOW WHEY WANT TO TURN - IS WHETHER SUBCLERK HOLD, LITTLE SYNAPTIC RESPONSES ARE ALSO TRANSMITTED. AND THE WAY WE'RE GOING TO LOOK AT THAT IS BY RECORD FREE RADICALS GENERATED THE STEAL CELL OR THE FUSI FORM CELL AND THEN STIMULATING THE PARALLEL FIBERS, THEY'RE GLUTEA MITTERGIC AXONS THAT INNERIVATE BOTH THE STELLATE CELL AND THE DENDRITES OF THE FUSI FORM CELLS AND YOU CAN SEE WHETHER THESE SYNAPTIC POTENTIALS THAT ARE GENERATED WITHIN THE FUSI FORM CELL ARE TRANSMITTED TO THE CELL AND THE REASON THAT WE THOUGHT THIS IS AN OPEN QUESTION HAS TO DO WITH THIS IMAGE HERE, SO WHAT I'M SHOWING YOU HERE IS A FULL BLOWN ACTION POTENTIAL IN THE FUSI FORM CELL AND THE SPIKELET THAT'S THEN TRANSMITTED THROUGH THE GAP JUNCTION HONDURAS INTO THE STELLATE CELL AND IF YOU COMPARE THESE, CAN YOU SEE THAT THE SPIKELET IS--THAT'S TRANSMITTED BECOMES VERY SLOW, BUT IT ALSO BECOMES VERY SMALL. SO THIS SPIKELET IS ONLY 3 MILLI VOLTS IN AMPLITUDE COMPARED TO ALMOST A HUNDRED MILLI VOLTS FOR THE ACTION POTENTIAL. SO A FULL BLOWN ACTION POTENTIAL IS ATTENUATE SEVERELY GOING INTO THE STELLATE CELL THROUGH THE GAP JUNCTIONS. SO HOW MUCH MORE SO WOULD A SYNAPTIC RESPONSE BE ATTENUATE SINCE SYNAPTIC RESPONSES ARE MUCH SMALLER. SO WE INVESTIGATED THAT IN THE MANNER I JUST DESCRIBED AND WHAT WE SAW WAS SOMETHING VERY STRANGE. SO, AGAIN WE'RE RECORDING HERE FROM THE STELLATE CELL AND STIMULATING THE PARALLEL FIBERS USING A STIMULUS ELECTRODE. AND WHAT WE RECORD IN THE STELLATE CELL IS THIS VERY WEIRD LOOKING SYNAPTIC CURRENT. SO, WE GET AN INWARD CURRENT THAT RISES QUICKLY AND FOLDS QUICKLY AND THEN IT'S GOT A SECOND PHASE WHICH IS VERY SLOW AND THEN FINALLY AN OUTWARD DISPAIZ THESE ARE HUGELY DIFFERENT SO THIS FAST 1 DECAYS IN A FEW MILLI SECONDS BUT THE SLOW 1 AS YOU CAN SEE FROM THE TIME BASE LASTS FOR HUNDREDS OF MILLI SECONDS AS DOES THIS LATE OUTWARD PHASE AND WE THOUGHT AT FIRST THAT THIS MUST BE DUE TO SOME SLOW MODLATTORY TRANSMITTER SYSTEM, A GPCR PERHAPS THAT'S BEING OR ACTIVATING A SLOW INWARD AND OUTWARD GATING ION CHANNEL BUT IN FACT THIS ENTIRE RESPONSE CAN BE BLOCKED BY THE AMPORECEPTORRAN TAGANIST MBQX. WHAT WAS ALSO STRANGE WAS THAT WHEN WE REPEATED THE STIMULI WITH STIMULUS STRENGTH THAT WAS CLOSE TO THRESHOLD, SOMETIME THIS IS VAST RESPONSE WOULD BE PRESENT AND THIS SHOWS THIS SAME SUITE THAT SPREAD APART SO CAN YOU SEE THAT HIGH SWEEP SPEED, SOMETIME THIS IS FAST RESPONSE IS PRES SPENT THEN SOMETIME ITS WOULD DISAPPEAR BUT THE SLOW RESPONSE WOULD BE PERFECTLY NORMAL. SO WHAT WE REASONED IS THAT THIS FAST RESPONSE MUSTINGING FROM THE SYNAPSES THAT FORM BETWEEN THE PARALLEL FIBERS AND THE STELLATE CELL AND THE SLOW RESPONSE IS COMING FROM THE SYNAPSES THAT FORM BETWEEN THE PARALLEL FIBERS AND THE FUSI FORM CELL AND IT'S THEN TRANSMITTED INTO THE STELLATE CELL THROUGH THE GAP JUNCTIONS AND THIS IS DEMONSTRATED HERE BECAUSE WHEN WE RECORDED IN THE CONNECTION 36 KNOCKOUT MICE WICH LACKED THOSE GAP JUNCTIONS, NOW, WE SEE ONLY A FAST RESPONSE. AND AGAIN WHEN WE LOOK AT THIS FLUCTUATION AND AMPLITUDE, IT'S A FLUCTUATION BETWEEN A FAST RESPONSE AND A 0 RESPONSE, NOT A SLOW RESPONSE. SO WHAT THE STELLATE CELL SEE SYSTEM SYNAPTICALLY IS 2 KINDS OF SYNAPTIC RESPONSE. ONE IS SOMETHING COMING FROM ITS OWN PARALLEL FIBER SYNAPSE SYSTEM AND SOMETH THAT'S COMING THROUGH THIS PARALYLE FIBER SYNAPSES ON THE NEIGHBORING FUSI FORM CELL AND IN FACT, CAN YOU SEE FROM THE SIZE OF THESE CURRENTS, THE AMOUNT OF ELECTRICAL CHARGE FLOWING THROUGH THIS ELECTRICALLY CONDUCTED PATHWAY IS MUCH GREATER THAN WHAT THE STELLATE CELL SEES FROM THE OWN PARALLEL FIBER SYNAPSE, SO THE MAJORITY OF TRANSMISSION OF THE STELLATE CELL IS ACTUALLY COMING THROUGH THE GAP JUNCTION. SO THE QUESTION NOW IS WHAT IS GOING ON. WHAT IS CAUSING THIS SUPERSLOW INAPTIC RESPONSE--SUPER SLOW SYNAPTIC RESONS--RESPONSE THAT IS FROM THE RECEPTOR? SO WE EXPLORE THAD HERE. WE'RE TESTING THE HYPOTHESIS WHICH I SINGLE CORRECT THAT THE SLOW IN-WARD CURRENT IS GENERATED NOT BY A SLOW AMPORECEPTOR BUT BY A SODIUM CNNEL. SO THE IDEA IS THAT WHEN THE FUSI FORM CELL IS DEPOLARIZED BY THE AMPORECEPTORS THAT THAT WILL THEN ACTIVATE SODIUM CHANNELS WHICH WILL THEN SLOW DOWN OR AMPLIFY THAT SYNAPTIC RESPONSE SO THAT'S TESTED HERE BY RECORDING FROM THE FUSI FORM CELL, INJECTING A CURRENT WAVE FORM WHICH LOOKS LIKE A SYNAPTIC CURRENT, AND THEN WE'VE DONE THAT AT 2 DIFFERENT AMPLITUDES OF CURRENT, SO SMALL CURRENT WHICH PRODUCES THIS SIZE OF DEPOLARIZATION, SHOWN IN BLACK AND THEN A LARGER CURRENT, WHICH PRODUCES A MUCH LARGER SYNAPTIC POTENTIAL AND CAN YOU SEE THAT BLACK SYNAPTIC POTENTIAL LASTS FOR OVER A HUNDRED MILLI SECONDS AND IT'S FOLLOWED BY THIS SLOW AFTERHYPER POLARIZATION. THEN WHEN THE SODIUM CHANNEL BLOCKER IS APPLIED, THAT SLOW WAVE FORM, BOTH THE SLOW DEPOLARIZATION AND AFTER HYPER POLARIZATION ARE ELIMINATED AND NOW WE HAVE A CONVENTIONAL LOOKING FAST EPSP. SO WE THINK THE SLOW SYNAPTIC RESPONSE IS MEDIATED BY AMPORECEPTORS WHICH THEN DEPOLARIZE THE FUSI FORM CELL AND ACTIVATE SODIUM CHANNEL WHICH IS THEN AMPLIFY THAT AMPORECEPTOR MEDIATED POTENTIAL. SO THEN WHAT HAPPENS IN THE STELLATE CELL? SO HERE WE'RE RECORD FREE RADICALS GENERATED BOTH A STELLATE CELL AND A FUSI FORM CELL THAT ARE ELECTRICALLY CONNECTED AND THEN INJECT THANKSGIVING CURRENT WAVE FORM THAT I JUST DESCRIBED WITHIN THE FUSI FORM CELL AND THAT PRODUCES THIS BLACK WAVE FORM, A SLOW EPSP FOLLOWED BY A LITTLE UNDERSHOOT HERE, AND THEN IN THE STELLATE CELL WE SEE THE SLOW INWARD CURRENT BUT IT'S INJECT WIDE A DRUG CALLED QX314 WHICH BLOCKS SODIUM CHANNELS BUT IT ONLY BLOCKS SODIUM CHANNELS IN THE CELL WE'RE RECORDING FROM SO THE SLOWNESS OF THIS RESPONSE MUST BE--WE WOULD CONJECTURE IS DUE TO THE SODIUM CHANNELS WITHIN THE FUSI FORM CELL AND WE CAN CONFIRM THAT BY ADDING TOXINS AND THAT SPEEDS UP THE CELL RESPONSE IN BOTH CELL TYPES. SO THAT CONFIRMS THAT SODIUM CHANNELS WITH THE F SI FORM CELL--FUSIFORM CELL AMPLIFY THE SYNAPTIC POTENTIAL AND THAT'S WHAT THE STELLATE CELL IS SEEING. SO NOW WHAT ABOUT THAT SLOW AFTER HYPER POLARIZATION THAT I'VE MENTIONED MANY TIMES NOW? WE THINK THAT IS GENERATED BY ATHER TYPE OF ION CHANNEL CALLED THE HCN CHANNEL, A HYPER POLARIZATION ACTIVATED ION CHANNEL. SO THIS CHANNEL HAS FOLLOWING PROPERTIES AND THESE ARE DATA THAT WERE COLLECTED IN FUSI FORM CELLS BY A POST DOC IN THE LAB R Z.TANG AND HE BOLTED THEM TO DIFFERENT POTENTIALS AND WHEN YOU GIVE A VOLTAGE STEP TO THESE, YOU CAN SEE AN ACTIVATION OF AN IONIC CURRENT THAT INCREASES WITH HYPER POLARIZATION SO THIS IS THE AMPLITUDE OF THE CONDUCT ANTS. IT INCREASING WITH HYPER POLARIZATION, IT'S CHARACTERISTIC OF THE H-CHANNEL. AND SO WE REASON THAT WHAT'S HAPPENING IN THE FUSIFORM CELL IS THAT DURING THIS SLOW DEPOLARIZATION, MEDIATED BY THE SODIUM CHANNELS THAT IT'S TURNING OFF HCN CHANNELS THAT ARE NORMALLY OPEN AT THE RESTING POTENTIAL. AND SO WHAT THAT'S GOING TO DO IS SINCE HCN CHANNELS PRODUCE INWARD CURRENT, THAT'S GOING TO REDUCE AN INWARD CURSPENT PROVIDE A HYPER POLARIZEITION DRIVE TO THE CELL. SO WOO CAN DEMONSTRATE THAT IN THIS EXPERIMENT IN WHICH WE ADD A BLOCKER OF THE HCN CHANNELS AND WHAT IT DOES IS ELIMINATE THAT SLOW OUTWARD CURRENT, THE 1 THAT PRODUC THE HYPER POLARIZATION AND IT ALSO ELIMINATES THE SLOW INWARD CURRENT AS WELL. SO, YOU MAY BE SCRATCHING YOUR HEADS ABOUT THAT SO LET ME EXPLAIN THAT HERE. SO HERE WE'RE--AGAIN RECORDING IN THE FUSI FORM CELL, WEB CONNECTED INJECT OUR CURRENT. WE GET A DEPOLARIZATION AND THAT PRODUCES CONTROL TRACE IN BLACK AND NOW WE WASH ON C-ZIUM, WHICH IS SELECTIVE FOR THE HCN CHAN AND HE WILL WE'VE DONE THIS WITH OTHER BLOCKERS OF THE HCN CHANNEL AND YOU CAN SEE THAT THE RESPONSE IS NOW TRANSFORMED INTO A FAST EPSP. BUT WHEN WE ADD C-ZIUM, IT NOT ONLY BLOCKS THE HCN CHANNELS IT HYPER POLARIZES THE CHANNEL BECAUSE THEY'RE HYPER POLARIZED AT THE RESTING CHANNEL SO THEN WE REBOOST THE CELL BACK TO ITS NORMAL RESTING POTENTIAL AND REPEATED THAT CURRENT INJECTION AND NOW WHAT WE SEE AND THAT'S IN THE GRAY TRACE HERE IS WE SEE A BROAD EPSP AGAIN BUT NOW IT LACKS THIS AFTER HYPER POLARIZATION PHASE SO I'M SHOWING THAT HERE AT HIGHER GAIN SO IN THE PRESENCE OF C-ZIUM, AND REPOLARIZING THE CELL, WE COMPLETELY LOSE THAT AHP. IF WE GO INTO THE STELLATE CELL AND START OFF WITH ORIGINAL EXPERIMENT, ACTIVATE THE PARALLEL FIBERS, AGAIN WE GET OUR FAST AND SLOW PHASES AND WE ADD C-ZIUM CHLORIDE IT ELIMINATES THE SLOW INWARD CURRENT AND THE SLOW OUTWARD CURRENT COMPLETELY. SO IT LOOKS LIKE A VERY REMARKABLE MECHANISM AS BEEN GENERATED WITHIN THESE FUSI FORM CELLS TO DEAL, TO TRANSFORM THE AMPORECEPTOR RESPONSE. SO FIRST AMPORECEPTORS ARE ACTIVATED BY GLUTEA MATE AND THAT PRODUCE ACE FAST DEPOLARIZATION. BUT THEN UPON DEPOLARIZATION, THAT ACTIVATES SODIUM CHANNELS IN THE CELL WHICH BROADENS THE EPSP AND LEADS TO A LONGER LASTING DEPOLARIZATION. AND DURING THAT STRONGER DEPOLARIZATION, THE VOLTAGE SENSITIVE HCN CHANNELS CLOSE DOWN AND THEN THAT LEADS TO THIS REBOUND, HYPER POLARIZATION OF THE MEMBRANE AND THEN AFTER THE CELL THEN REPOLARIZES, NOW, IT'SA AT A VOLTAGE WHERE SODIUM CHANNELS WILL CLOSE DOWN AND HDN CHANNELS REOPEN SO EVERYTHING RETURNS TO REST. SO WE HAVE THIS REMARKABLE SEQUENCE STARTING WITH A FAST EPSP, THEN A SLOW EPSP AND A SLOW AFTER HYPER POLARIZATION AND A WHOLE SEQUENCE OF SLOW EXITATION AND INHIBITION IS THEN TRANSMITTED THROUGH THE GAP JUNCTIONS TO THE STELLATE CELLS SO I THINK IT'S A VERY INTERESTING MECHANISM THAT IS GENERATED BECAUSE REMEMBER, THE GAP JUNCTIONS DON'T TRANSMIT ACTION POTENTIALS VERY FAST. THEY DON'T TRANSMIT FAST SIGNAL BECAUSE THEY ACT LIKE AN ELECTRICAL FILTER SO BY SLOWING EVERYTHING DOWN, NOW THE GAP JUNCTIONS BECOME VERY POTENT IN TRANSMITTING SYNAPTIC SIGNALS TO THE STELLATE CELL AND SO, TO SEE HOW POTENT THEY ARE, WE DID THE FOLLOWING EXPERIMENT. SO IN THIS--IN THIS EXPERIMENT, WE'RE RECORDING AGAIN FROM A PAIR OF CELLS AND THE FUSI FORM CELL IS BEING DEPOLARIZED WITH AN EPSP LIKE WAVE FORM, 1 THAT'S FAIRLY SUBSTANTIAL BUT IT'S STILL SUBTHRESHOLD, NOT GENERATING ACTION POTENTIALS WITHIN THE FUSI FORM CELL, BUT IF YOU RECORD FROM THE STELLATE CELL THAT DEPOLARIZATION WITHIN THE FUSI FORM CELL IS STILL THERE TO DERIVE THE SPIKES IN THE CELL. SO THIS IS ACTION POTENTIALS IN RESPONSE TO SUBTHRESHOLD SYNAPTIC POTENTIALS WITHIN THE FUSI FORM, AND THIS IS THE AVERAGE DATA SHOWING THAT THE SPIKE RATE INCREASES MANY 10S OF FOLD DURING SUBTHRESHOLD EPSPs WITHIN THE FUSI FORM CELL. SO THE CONCLUSION HERE, I THINK IS QUITE REMARKABLE, IT SAYS THAT THE SEQUENCE INITIATED BY THE SUBTHRESHOLD SIGNAL SYSTEM SUFFICIENT TO DRIVE SPIKES IN THE STELLATE CELL. SO WITH THAT, I'M--THAT'S A LOT OF ELECTROPHYSIOLOGY, BUT WITH THAT I'LL SUMMARIZE WHAT I TOLD YOU. FIRST THAT GAP JUNCTIONS MEDIATE COMMUNICATION BETWEEN DIVERSE TYPES OF NEURONS WITHIN THE AUDITORY SYSTEMS AND I'VE SHOWN YOU 2 TYPES OF NEURONS AND NOW WE'RE SEEING GAP JUNCTION COUPLING WITHIN OTHER CELL TYPES WITH THE COCHLEA AND NUCLEUS AS WELL, SO THIS IS OPENING THE DOOR TO NEW AREA OF RESEARCH WITHIN THE AUDITORY SYSTEM. AND THIS COMMUNICATION CAN BE BOTH EXCITATORY AND INHIBITORY. IT HAS A DEPOLARIZING PHASE AND A HYPER POLARIZING PHASE AND IT DOES THIS REGARDLESS OF THE PHENOTYPE OF THE COUPLED CELLS. WE ARE SEEING CELLS THAT ARE GLUTEA MITTERGIC AND GABAergic. THIS CONTROLS ACTION POTENTIALS IN NEIGHBORING NETWORKS WITHOUT THEMSELVES FIRING ANY SPIKES. SO A EPSP IN THE FUSIFORM BY MEANS OF THE SLOW EXCITATION AND INHIBITION SEQUENCE THAT'S GENERATED BY THE ION CHANNEL CANS DRIVE SPIKES IN NEIGHBORING CELLS. AND WE THINK THAT THIS MECHANISM MIGHT ALLOW SIGNALING BETWEEN DIFFERENT COMPONENTS OF THE NEURAL CIRCUIT, IN THIS CASE THE AUDITORY DOMAIN AND THE MULTISENSORY DOMAIN, IN RESPONSE TO RELATIVELY SPARSE SENSORY ACTIVITY. SO WITH THAT, I AM GOING TO FINISH AND I WOULD LIKE TO AGAIN THANK THIS VERY TALENTED GRADUATE STUDENT NOW A POST DOC AT GENILIA PH ARM, PIERRE APOSTOLIDES, FOR ALL THE WORK HE DID MY LAB SO I THANK YOU VERY MUCH FOR YOUR ATTENTION. >> THERE'S CERTAINLY TIME FOR QUESTIONS IF YOU HAVE ANY. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, I'LL REPEAT THE QUESTION. THE QUESTION IS: IS IT POSSIBLE TO EMIC THE ACTIVITY IN THE NERVE TO SEE IF THAT CAN BE TRANSMITTED? AND THAT IS POSSIBLE, THAT'S SOMETHING THAT'S ACTUALLY FAIRLY STRAIGHT FORWARD TO DO. WE KNOW THAT TIMING, THE STATISTICS OF FIRING IN THE EIGHTH NERVE AND WE SHOULD BE ABLE TO GENERATE PATTERNS OF SYNAPTIC POTENTIALS SIMULATED SYNAPTIC POTENTIALS WITHIN THE FUSI FORM CELLS SO THAT'S A GOOD IDEA. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, SO THE QUESTION IS: WHAT IS THE HEARING PROFILE IN A CONNECTION 36 KNOCK OUT MOUSE? AND WE TEST THAD USING ABRs AND THEY ARE PERFECTLY FINE. BUT IT'S IMPORTANT TO REMEMBER THAT THE ABR DOESN'T TEST, DOESN'T PROBABLY GET ANY SIGNAL FROM THE DCN. AVR IS ASYNCHRONOUSLY FIRING NEURONS SO TELL BE DOMINATED BY OTHER CELL TYPES. SO WHAT MIGHT BE THE SOUND LOCALIZATION PHENOTYPE IN THIS ANIMAL IS SOMETHING WE WOULD LIKE TO KNOW. WE CAN'T REALLY EASILY ASSAY THAT WITH OUR TECHNIQUES. >> WHAT'S REALLY BEAUTIFUL AND I THINK IT--YOU REALLY EXCEL--THE THING I LIKE THE MOST ABOUT YOUR SCIENCE IS THAT YOU SHOWED US A REALLY NICE MECHANISM BUT THEN YOU ALSO SHOW US WHAT IT MEANS FOR THE SET AND THE INPUTS AND REALLY BEAUTIFUL. I WANTED TO KNOW ARE THESE CONJUNCTIONS [INDISCERNIBLE] PERMEABLE AND ARE YOU GOING TO LOOK INTO THAT AS WELL? >> YEAH. THAT'S A GOOD QUESTION, WHAT GOES THROUGH THE GAP JUNCTIONS BESIDES ELECTRICAL CURRENT. AND THAT'S SOMETHING WE WOULD LIKE TO LOOK INTO FOR SURE. BUT WE DON'T KNOW. >> [INAUDIBLE RESPONSE FROM AUDIENCE ] >> RIGHT. SO WHERE ARE THE GLUE MARIOUS SEEN RECEPTOR EXPRESSING NEURONS FACTOR INTO THIS OR GLUE MARIOUS SEEN EXPRESSING NEURONS? BECAUSE ALL THESE--YEAH, THE GLUE MARIOUS SEENERGIC NEURONS ARE FIRST OF ALL THE STELLATE CELLS ARE ALSO IT TURNS OUT ALSO GLYCNERGIC SO THEY'RE CO-RELEASING CELL WHICH IS SEEMS TO BE HOW THINGS ARE AWIVE DONE IN THE AUDITORY SYSTEM. SOPHISTICATED THE MORE DOMINANT NEURONS ARE THE CART WHEEL CELLS AND THE VENTRICULAR CELLS AND THOSE DON'T SEEP TO BE GETTING GAP JUNCTION COUPLING TO THE BEST OF OUR KNOWLEDGE BETWEEN IT IS FUSI FORM CELL OR RAUGHT TORE EACH OTHER. RATHER THEY RECEIVE CONVENTIO AND INHIBITORY SYNAPSE SYSTEM AND IF WE LOOKS AT GLUTEA MITTERGIC INPUT INTO THE KNOCK OUT ANIMALS AND WE HAVEN'T SEEN ANY OBVIOUS DIFFERENCES WITHIN THESE BUT I'LL BET THERE IS, THERE MUST BE SOME DIFFERENCES. IF YOU LOOK AT THE HOST, THE CONNECTION KNOCK OUT ANIMALS ARE CLEARLY RUNTIER, THEY'RE SMALLER THAN WILD-TYPE ANIMALS, I WOULDN'T BE SURPRISED IF THERE ARE A NUMBER OF SUBTLE EFFECTS WITHIN THE CNS. SORRY? >> [INAUDIBLE QUESTION FROM AUDIENCE ] NO ALL WE'VE DONE IS LOOKED AT AVRs WHICH ARE NORMAL. YES? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, SO THE QUESTION IS: WHY IS THERE APPARENT PREFERENTIAL TRANSMISSION FROM THE FUSIFORM CELL INTO THE STELLATE CELL? THIS IS KIND OF LIKE A RUSSIA UKRAINE THING. IF YOU REMEMBER THE MICROGRAPH I SHOWED YOU THE FUSI FORM CELL IS HUGE AND THE STELLATE IS TINY SO ELECTRIC AT CURRENTS WITHIN THE FUSIFORM CELL WILL BE--IF THEY'RE BIG ENOUGH TO DEPOLARIZE, THEY WILL DEFINITELY DEPOLARIZE THE STELLATE CELL, BUT GOING IN THE OTHER DIRECTION, IT'S LIKE A DROP OF INK IN THE OCEAN, IT WILL GET DILUTED SPEW THE LARGE CAPACITANTS OF THE FUSI FORM CELLS, IT'S IN THE REPLICATION, NOT SOMETHING ABOUT THE GAP JUNCTION. YES? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, SO THE QUESTION IS INSTEAD OF ADDING CEZIUM, AND JUST CHANGE THE RESTING POTENTIAL CAN WE REPRODUCE THIS EFFECT, AND THE ANSWER IS YES, WE JUST HYPER POLARIZE A LITTLE. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> THE RESTING POTENTIAL CHANGES DURING SYNAPTIC ACTIVITY, DURING IPSPs SO IT'S DEFINITELY A MODULATED PARAMETER. SOMETHING WE'RE WORKING ON QUITE AVIDLY. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> BECAUSE THE CONNECTION 36 KNOCK OUT MOUSE WAS AVAILABLE FROM THE LAB 4 FLOORS DOWN AND CONNECTION 36 HAPPENS TO BE THE DOMINANT CONNECTION WITHIN NEURONS IN THE CNS SO IT WAS A LIKELY CANDIDATE. YOU KNOW? I LUCKY 1. OKAY? ONE MORE? >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> YEAH, SO THE QUESTION IS: ARE THESE GAP JUNCTIONS HOMOTYPIC OR HETEROGENEOUS ROW TYPIC AND I THINK THERE COULD BE OTHER SUBUNITS THERE HAS WELL? BUT THEY SOMEHOW CONNECTION 36 IS THE DOMINANT 1 BUT WE HAVE SEEN A FEW CASES WHERE IN THE KNOCK OUT ANIMAL WE CAN STILL SEE COUPLING AND YOU KNOW LIKE 1 OR 2 CELLS BUT IT'S RARE BUT THAT SUGGESTS RIGHT THERE THAT THERE'S EITHER UPREGULATION OF SOME OTHER SUBUNIT OR IF THERE'S AN ADDITIONAL SUBUNIT. >> OKAY, THANK YOU VERY MUCH. [ APPLAUSE ]