UNDERSTANDING THE TRANSCRIPTOR CELLS AND COULD BE SORT OF NEURONS TOGETHER BASED ON WHAT WE KNEW THEY WERE EXPRESSING AND HE'S REALLY SORT OF RAN WITH THAT IN THE LAST FIVE OR SIX YEARS. SO IT'S A GREAT PLEASURE TO HAVE HIM COME TALK TO US ABOUT THAT. HE'S ALSO BEEN AN OUTSTANDING CITIZEN AND CERTAINLY IN THE SOCIETY OF NEUROSCIENCES -- MEMBERSHIP COMMITTEE FOR THE SOCIETY. HE WAS IN THE PUBLICATIONS COMMITTEE GROUP WAS A MEMBER AND THEN THE CHAIR COMMITTEE AND MORE RECENTLY HE WAS COUNSELOR FOR THE LAST YEAR. SO IT'S A GREAT PLEASURE TO WELCOME SASHA THIS MORNING. HE'S GOING TO TALK TO US ABOUT THE PHYSIOLOGICAL GENOMES OF -- SO WELCOME. >> IS MY MIC ON? CAN YOU HEAR ME OKAY? SO THANKS VERY MUCH, CHRIS, FOR HAVING ME. IT'S A HONOR TO BE HERE AS PART OF THE SEMINAR SERIES AND IT'S GREAT TO CATCH UP WITH OLD FRIENDS AND TO MEET MANY OF YOU WHOSE WORK I FOLLOWED FOR MANY YEARS BUT SOMEHOW I HADN'T MANAGED TO MEET UP UNTIL NOW. SO AS CHRIS MENTIONED FOR THE LAST SEVERAL YEARS MY LAB HAS REALLY BEEN INTERESTED IN THE PROBLEM OF NEURONAL CELL TIMES. THIS IS A CRITICAL BOTTLE NECK BETWEEN ON THE ONE HAND THE GENOME, THE GENES THAT ARE EXPRESSED IN SPECIFIC NEURONS, AND AT THE UPPER END, THE NEURAL CIRCUITS WHICH PRESUMABLY PRODUCE BEHAVIORS THAT WE'RE INTERESTED IN AND WHICH ARISE IN DISEASE. WHAT I WANT TO SUGGEST IS THAT REALLY THIS UNDERSTANDING OF THE BIOLOGY OF THE NEURONAL CELL TYPES IS THE CRITICAL JUNCTURE IN ORDER TO PUT THE TREMENDOUS ADVANTAGE IN GENOMICS TO WORK TO START TO UNDERSTAND NEURAL CIRCUITS. THIS IS SOMETHING THAT OF COURSE HAS BEEN PLAIN TO PEOPLE THAT WORK ON SIMPLER CIRCUITS, SO WHETHER FOR EXAMPLE YOU WORK ON -- OR THE SOMATIC GANGLION OF -- SHOWN BELOW LIKE MY COLLEAGUE -- THE ADVANTAGES OF THESE CIRCUITS IS THAT INDIVIDUALS CAN GO ACROSS THE WORLD FROM PREPARATION TO PREPARATION TO IDENTIFY NEURONS, STUDY THE PROPERTIES OF THOSE NEURONS, THE CONNECTIONS OF THOSE NEURONS AND REALLY BEGIN TO BUILD UP AN UNDERSTANDING OF THE CIRCUIT OF THE DEVELOPMENT. AND TREMENDOUS ADVANCES IN UNDERSTANDING HOW THESE CIRCUITS WORK HAVE COME FROM THE SORT OF CONCEPT OF IDENTIFIED NEURONS THAT'S BEEN LARGELY LACKING IN MANY OF THE VERTEBRATE SYSTEMS THAT I AND OTHERS CARE DEEPLY ABOUT. SO WHILE THERE ARE PLACES IN THE VERTEBRA BRAIN LIKE THE RETINA WHERE IT'S -- CELL TYPES THERE ARE AND HOW TO PARSE THEM. IN THE NEOCORTEX FOR EXAMPLE OR THE HIPPO CAMPUS AND OTHER BRAIN REGIONS, THIS HAS BEEN NOT AT ALL SOLVED PROBLEM. AND THERE IS OF COURSE IN THE CORTEX AND HIPPOCAMPUS WIDE SPREAD AGREEMENT THAT THE TWO MAIN FLAVORS. THERE ARE GABA URGIC INTERNEURONS THAT ARE INHIBITORY AND DIVERSE IN THEIR PROPERTIES AND IT'S THE PROJECTION OF PYRAMIDAL NEURONS -- AND PROJECT BOTH LOCALLY AND TO OTHER LONG RANGE TARGETS WITHIN THE BRAIN. WITH YOU HOW YOU PARSE THESE SUBTYPES MORE FINELY REALLY DEPENDS ON WHAT YOU LIKE TO DO WITH YOUR TIME. SO IF YOU'RE AN ANATOMIST WHO STUDIES THE MORPHOLOGY OF AXONS AND DENDRITES -- YOU MIGHT PARSE THEM IN ANOTHER WAY. AND OVER THE LAST DECADE, THERE'S BEEN A LOT OF CONGRUENCE BUT NOT PERFECT AGREEMENT AS TO SORT OF HOW TO BRING THESE CLASSIFICATION SCHEMES INTO REGISTER. BUT IN GENERAL WHAT ONE IS TRYING TO DO IS REALLY UNDERSTAND HOW THE CELLULAR PHENOTYPES, THESE ARE JUST A FEW OF THEM ARISE FROM THE GENES THAT EACH OF THESE INDIVIDUALS CELL TYPES EXPRESS. SO A NUMBER OF YEARS AGO WE HIT ON AN ALTERNATIVE METHOD THAT ATTEMPTED TO LOOK IN AN UNBIASED WAY ALL OF THE GENES THAT ARE EXPRESSED BY A PARTICULAR CELL TYPE USING MICRO ARRAY ANALYSIS OF A GLOBAL MRNA EXPRESSION. AND BASICALLY THE APPROACH IS TO START WITH TRANSGENIC MOUSE IN WHICH A POPULATION OF NEURONS ARE LABELED TO USE ANATOMY AND PHYSIOLOGY TO CHARACTERIZE AND TO BE CONVINCE ONE SELF THAT THIS IS REALLY A CELL TYPE AND EXTRACT RNA FROM THESE CELLS AMPLIFY IT FOR MICRORAY -- WHEN WE STARTED THIS THERE WERE REALLY SMALLER NUMBER OF AVAILABLE TRANSGENIC MOUSE LINES TO INVESTIGATE THAN THERE ARE TODAY. AND FOR EXAMPLE, FOR ANALYZING -- NEURONS THIS IS ONE OF THE ONES THAT'S AVAILABLE, THIS IS THE SO-CALLED -- PH LINE PRODUCED BY -- LAB PERHAPS THE MOST STUDIED TRANSGENIC MOUSE LINE IN NEURON SCIENCE. WHEN WE RECORD FROM THESE NEURONS AND SLICES, WE FOUND THAT THEY WERE CELLS THAT WE HAD KNOWN AND LOVED FROM STUDYING THEIR SYNAPTIC PLASTICITY. THESE ARE -- NEURON THEY PRODUCE OUTSIDE OF THE HUMAN SELF LAWN AND THEY HAVE A CHARACTERISTIC FIRING PATTERN WITH AN INITIAL BURST AND THEN NON-ADAPTING PATTERN. IF YOU RECORDED FROM OTHER CELLS UNLABELED THEY HAVE AN ADAPTIVE PATTERN BETWEEN THE SUCCESSIVE ACTION POTENTIALS THAT ARE LONGER AND LONGER THROUGHOUT THE TRAINING OR THEY FIRED REPETITIVE BURSTS, ETCETERA. AND SO BASED ON THEIR MORPHOLOGY, THE POSITION OF THEIR CELL NODES, THEIR DENDRITIC PATTERNS, THEIR AXONAL PROJECTIONS AND THEIR FIRING PATTERN WE BRIEF THAT CONSTITUTE THE CELL TYPE. THERE ARE TWO LAYERS OF THE NAWRNZ. THERE ARE THE THICK TUFTED ONE SO-CALLED BECAUSE THEY HAVE A THICK APICAL DENDRITE -- THEY HAVE THIS NON-ADAPTING TYPE AND THERE ARE THIN DRIETS PROJECTING EITHER TO THE CORTEX OR TRY ACTUAL -- LONG RANGE TARGETS LIKE THE SPINAL CORD AND BRAIN STEM. AND CLAB CONTINUE WORK WITH -- LAB, SUE HAD IDENTIFIED A TRANSCRIPTION FACTORS FEZF2 IS WHICH IS CRITICAL FOR THESE THICK TUFTED CELLS. WHEN THIS TRANSCRIPTION FACTOR IS ABSENT THERE IS NO PROJECTION. FOR EXAMPLE THE PYRAMIDAL TRACT IS MISSING IN THESE MICE AND WE FOUND BY RECORDING FROM THE CELLS AND THE FACT THEY ARE INTRINSIC PHYSIOLOGY ALSO MATCHED THIS ABSENCE OF A BIFURCATION OF IDENTITY. IF ONE MEASURED THE DEGREE OF ADAPTATION OF THESE CELLS, THERE WERE TWO POPULATIONS, ONE THAT WERE HIGHLY ADAPTING AND ONE THAT WERE NOT ADAPTING IN THESE KNOCKOUT MICE THEY WERE ONLY THE ADAPTING CELLS AND THE NON-ADAPTING CRITICAL -- WERE MISSING. WHAT ABOUT THE -- INTERNEURONS. HERE'S AN EXAMPLE OF A LINE THAT WAS PRODUCED BY MY COLLEAGUE JOSH -- THIS IS A BACK TRANSGENIC IN WHICH A HUGE CHUNK OF THE GAD ONE GENE THAT EMPHASIZES THE GABA WAS PUT INTO A BACTERIA ARTIFICIAL CHROMOSOME AND THIS WAS INSERTED INTO THE GENOME RANDOMLY. BECAUSE OF POSITIONAL EFFECTS AND I'LL DISCUSS THIS MORE IN A MOMENT, IT IS NOT ALL GABA ALLERGIC NEURONS THAT ARE LABELED IN THIS LINE BUT ACTUALLY TURN OUT A SPECIFIC SUBSET IN THE NEO CORTEX AND THIS IS CALLED -- CELLS. U SEE THAT THEY'RE CAPABLE OF FIRING ACTION POTENTIALS OF VERY VERY HIGH RATES. THEY HAVE THE SPECIFIC POTASSIUM CHANNELS THAT REPOLARIZE THE MEMBRANE VERY QUICKLY AND ALTHOUGH OTHER NEURONS ARE LABELED WITHIN THE BRAINS OF THESE ANIMALS THAT ARE ALSO GABA ALLERGIC BUT DIFFERENT PROPERTIES WITHIN THE CORTEX THESE ARE ALSO CALLED POSITIVE BASKET CELLS. WE CALL THEM BASKET CELLS BECAUSE THEIR AXONS FORM BASKETS AROUND PYRAMIDAL NEURONS AND THEY ARE A SOURCE. IT'S IMPORTANT TO REALIZE IN THOSE THESE CASES THE PH STRAIN IS THE DYE ONE PROMOTER IN THE G42 LINE I JUST SCUSD IT'S THE GABA PROMOTER THAT'S DRIVING. THESE PROMOTERS ARE NOT RECAPITULATING THE EXPRESSION BUT ONLY A TINY SUBSET OF IT. AND THE PRESUMED REASON IS THAT THE INSERTION OF THE TRANSGENE CAUSES INTERACTION BETWEEN ELEMENTS THAT ARE IN THE PROMOTER AND OTHER ELEMENTS THAT ARE NEARBY ENHANCERS OR REPRESSERS TOGETHER COLLABORATE FOR EXPRESSION. THERE ARE NUMEROUS EXAMPLES OF THIS AS ONE LOOKS AT CROSS TRANSGENIC LINES AND TALKED TOWARDS THE END ABOUT TRYING TO EXPLOIT THE STRATEGY TRYING TO GENERATE NEW LINES TO BE USED TO ANALYZE ADDITIONAL CELL TYPES. SO THE BASIC STRATEGY THEN IS TO CUT SLICES FROM A TRANSGENIC MOUSE THAT HE -- EXPRESSIONS FLUORESCENT PROTEIN -- A PARTICULAR REGION IN THIS CASE PRIMARY SCHEMATIC SENSORY CORTEX TO ASSOCIATE THE NEURONS AND THEN MANUALLY SORT THEM. WE STARTED THIS USING A FACT SORTER AND INITIALLY REALLY JUST GOT A GREEN GOO AT THE OTHER END. SUBSEQUENTLY WE'VE BEEN ABLE TO ADJUST FACT SORTING TO WORK. BUT IN THIS CASE IT TURNS OUT THAT MANUALLY SORTING THIS WORKS EXTREMELY WELL ALSO. AND WE CAN THEN EXTRACT RNA AMPHI AND SCREEN MICRO RAYS. PART OF THE REASON WHY THIS WORKS IS THAT THE TECHNIQUES FOR AMPLIFICATION WORKED OUT FOR COLLEAGUES ARE EXTREMELY EFFICIENT SO THAT STARTING WITH ONLY 30 OR 40 NEURONS WHICH YOU CAN EASILY SORT, YOU CAN SEE SATURATED NUMBERS OF TRANSCRIPTS, DIFFERENT TRANSCRIPTS PRESENT ON THESE MICRO RAYS. SO IF YOU START WITH ONE OR TWO KNIFE OR TEN CELLS THEN YOU SEE A SMALLER NUMBER OF TRANSCRIPTS PRESENT ON THE CHIP PRESUME ME BECAUSE THE LESS ABUNDANT TRANSCRIPTS FAILED TO AMPHI. BUT IF YOU START WITH 30 OR 40, THEN YOU SEE SATURATING OUT TO 500 CELLS AND MOWERS YOU DON'T SEE MORE TRANSCRIPTS SUGGESTING WE'RE ACTUALLY SEEING EVERYTHING THAT'S THERE -- THE SECOND THING IS THAT THIS MANUAL SORTING TECHNIQUE TURNS OUT TO YIELD VERY HIGH PURITY WITH VERY LOW BACKGROUND. SO YOU CAN ASSESS THIS BY LOOKING FOR TRANSCRIPTS THAT SHOULDN'T BE THERE. FOR EXAMPLE IF YOU'RE SORTING EXCITATORY NEURONS, YOU CAN LOOK FOR TRANSCRIPTS THAT ARE PRESENT IN -- WHEN YOU DO THIS YOU SEE IF YOU DON'T SORT CELLS AT ALL YOU HE SEE THE TRANSCRIPTS PRESENT AT HIGH LEVELS. IF YOU DON'T WASH THEM OFF -- YOU SEE SOME BACKGROUND OR LESS. IF YOU CARRY OUT THE PROCEDURE AS WE HAVE, THEN YOU ACTUALLY SEE VERY LOW LEVELS OF THESE TRANSCRIPTS THAT ARE NOT THERE. NOW IT TURN OUT THAT A NUMBER OF OTHER PEOPLE HAVE NOW DEVELOPED METHODS FOR LOOKING AT CELL TYPE SPECIFIC TRANSCRIPTION. AND WE RECENTLY HAVE LOOKED MORE CLOSELY AT WHETHER THE WAY THAT YOU ISOLATE THE CELLS MATTER. SO YOU MIGHT WORRY FOR EXAMPLE THAT WHEN YOU DISASSOCIATE CELLS YOU ACTUALLY CHANGE TRANSCRIPTION. THE ACT OF DISASSOCIATING THEMSELVES AFTER THE EARLY GENES AND CELL DEATH GENES, ETCETERA. AND SO WE HAVE COMPARED DATA FROM METHODS LIKE THESE CAPTURE MICRO DISSECTION THAT DOESN'T DISASSOCIATE CELLS OR THE TRAP METHOD INVENTED -- THAT USES AN AFFINITY PURIFICATION OF A TAGGED RED SOMAL PROTEIN TO PULL DOWN WITHOUT NEEDING TO DISASSOCIATE NEURONS FROM METHODS LIKE FACT SORTING OR LIKE THE MANUAL SORTING THAT WE'VE USED OR PANNING AND IMMUNOPULL DOWN METHOD THAT -- HAVE USED. AND ALL OF THESE METHODS IT TURNS OUT LEAD TO HIGHLY REPRODUCIBLE RESULTS. THESE ARE CORRELATION CO-COEFFICIENTS ACROSS BIOLOGICAL RENTAL KITS. YOU CAN SEE WE'RE TALKING ABOUT DIFFERENCES BETWEEN .98 AND .99 THERE. HOWEVER, THE DEGREE OF BACKGROUND CONTAMINATION DIFFERS SIGNIFICANTLY. SO WITH LCM AS YOU MIGHT EXPECT, IT'S VERY HARD NOT TO BE PULLING ALONG WITH THE SAMPLE THAT YOU'RE INTERESTED IN. SOME AMOUNT OF BACKGROUND MATERIAL THAT CONTAINS TRANSCRIPTSZ THAT ARE NOT IN THE CELLS THAT YOU'RE INTERESTED IN. THE SAME IS TRUE WITH THIS IP METHODS OF TRAPS AND IT'S LESS TRUE WITH THESE METHODS THAT DISASSOCIATE AND SORT THEM. WHAT ABOUT THE ACTIVATION OF OTHER GENETIC PROGRAMS. HERE WHAT WE'VE DONE IS JUST LOOK AT A WHOLE BUNCH OF STRESS GENES, APOPTOSIS GENES AND ON THE NEXT SIDE IS EARLY GENES. AND YOU CAN SEE THAT ALTHOUGH THESE GENES -- THERE'S NO SYSTEMATIC DIFFERENCE BY METHOD WHICH IS LISTED DOWN AT THE BOTTOM. THERE'S A SOLICIT DIFFERENCE IN THE CASE OF IMMEDIATE EARLY GENES THIS IMMUNOPANNING TECHNIQUE. THIS MAY REFLECT A SLIGHTLY LONGER TIME COURSE OVER WHICH THIS PANNING BY THE FACT THAT SOME OF THESE ARE GLIAL CELLS THAT ARE MORE SENSITIVE TO THIS. BUT THE EFFECT IS ACTUALLY QUITE SMALL. OKAY. SO OF COURSE ONE WOULD ALSO LIKE TO KNOW THERE'S A RECENT TECHNOLOGY OF RNA SEQUENCING HIGH SEQUENCING AND WE STARTED DOING THIS IN COLLABORATION WITH GROUPS AT -- AND THIS FOR EXAMPLE IS DATA FROM -- NEURON SHOWING THE AX ALWAYS -- THAT'S NOT EXPRESSED WHAT WE HAVEN'T DONE IS THE KIND OF SYSTEMATIC GENOME-WIDE COMPARISON ACROSS A NUMBER OF CELL TYPES. THAT'S SOMETHING THAT'S INTERESTING TO KNOW IN THE FUTURE. ENOUGH OF THIS SORT OF TECHNICAL CAVEATS TO THIS. THE FIRST STUDY THAT WE DID WITH THIS LOOKED AT A DOZEN DIFFERENT CELL TYPES ISOLATED FROM THESE FIVE DIFFERENT TRANSGENIC LINES. WE LOOKED AT THESE PYRAMIDAL NEURONS IN SINGULAR CORTEX AND HIPPOCAMPUS IN ANOTHER AREA OF NEOCORTEX PRIMARY SOMATOSENSORY CORTEX AND THE MEDULLA AND WE LOOKED AT SEVERAL SUBTYPES OF NEURONS BOTH IN SINGULAR CORTEX AND OTHER REGIONS OF THE FOREBRAIN. IN ONE CASE IN THE DIENCEPHALON. AND FOR EACH OF THESE CELL TYPES, WE COULD PICK OUT LARGE NUMBERS OF TRANSCRIPTS SHOWN IN YELLOW HERE THAT WERE SELECTIVELY EXPRESSED IN THAT CELL TYPE AND OTHERS. IN SOME CASES, WE COULD PICK OUT SETS OF TRANSCRIPTS THAT WERE EXPRESSED IN LARGER GROUPINGS OF NEURONS, FOR EXAMPLE ALL OF THE GABA URGIC NEURONS FOR ALL OF THE GABA URGICS NEURONS. THIS IS PARTICULARLY GOOD TO LOOK AT BECAUSE A LOT OF THESE WERE KNOWN. SO HERE'S FOR EXAMPLE COMPARISONS OF THREE DIFFERENT INTERNEURON SUBTYPES IN SINGULAR CORTEX WITH TWO SUBTYPES OF PYRAMIDAL NEURONS LAYER FIVE AND LAYER SIX NEURONS WERE UNSORTED CELLS. AND YOU CAN SEE THAT GENES LIKE THE -- INHIBITTITY AMINO ACID -- RECEPTOR, THE SYNTHESIS GAB ONE AND TWO. THESE NEURONS ARE THERE. OTHER TRANSCRIPTION FACTORS LIKE -- DLX6, THIS IS ANOTHER TRANSPORTER. AS WELL AS LOTS OF THING THAT WERE NOT KNOWN. AND IN LOOKING OVER THIS DATA FOR BOTH THE INHIBITORY AND EXCITATORY EVERYTHING THAT'S SUPPOSED TO BE THERE AND YOU DON'T SEE ANYTHING THAT'S NOT SUPPOSED TO BE THERE WITH A COUPLE OF EXCEPTIONAL THAT RELATE TO -- ON THE CHIP. SO WE THINK THAT THIS IS ACTUALLY A FAIRLY ACCURATE WAY TO DETERMINE THE COMPLETE TRANSCRIPTTON OF A PARTICULAR CELL TYPE. WE'VE EXTENDED THIS TO OTHER CELL TYPES. THIS IS JUST ONE EXAMPLE OF THE LOCUS -- NEURONS THAT ARE LABELED IN THE TYROSINE HYDROX LACE PROMOTER GFL LINE. OTHER POPULATIONS OF DOPAMINE URGIC -- NEURONS ARE LABELED AS WELL BUT YOU CAN SEE THE LOCUS -- HERE AND AGAIN WE CAN PICK OUT A LARGE NUMBER OF TRANSCRIPTS SELECTIVELY EXPRESSED IN THESE LC NEURONS BUT NOT IN OTHER CELL TYPES THAT WERE PROFILED. OF THESE ARE THINGS SHOWN IN GREEN THAT WERE KNOWN BEFORE LIKE TYROSINE HYDROX LACE ITSELF. LIKE A NUMBER OF TRANSCRIPTION FACTORS HYDROXLACE ETCETERA AND MANY THING THAT WERE NOT KNOWN. ONES THAT WERE STARTED WE CAN CONFIRM LOOKING AT THE ALAN BRAIN ATLAS THAT THESE WERE EXTRESSED LARGELY CONFINED TO LOCUS -- OR OTHER NEARBY STRUCTURES. CAN WE ACTUALLY USE THIS TO CLASSIFY NEURONS? WELL THIS IS UNBIASE YOU'RE PRIMARY DETERMINANTS WHICH ARE MORE IMPORTANT THAN THE OTHER. THOSE ARE MAJOR DETERMINANTS. THIS IS A MORE RECENT DATA SET THAT INCLUDES FOR EXAMPLE THE -- NEURONS AND SOME DEVELOPMENTAL DATA THAT SHOWS THIS SORT OF UNBIASED CLUSTERING IS LIKELY TO SCALE TO A MUCH LARGER DATA SETS AND WE'RE NOW TRYING TO GET TO A HUNDRED OR MORE CELL TYPES IN COLLABORATION WITH PEOPLE THAT ARE USING -- SO THERE ARE SOME IMPORTANT CAVEATS TO THE IDEA OF ACTUALLY TRYING TO USE EXPRESSION PROFILES TO CLASSIFY CELL TYPES. THEY SHOULD BE FAIRLY OBVIOUS TO ANYONE WHO KIND OF THINKS ABOUT THIS FOR MORE THAN FIVE MINUTES. THOSE ARE FIRST OF ALL DEVELOPMENT, DEPENDS ON WHEN YOU DO THIS. SO OF COURSE IF YOU DO THIS VERY EARLY IN DEVELOPMENT OR YOU LOOK AT JUVENILES, YOU SEE DIFFERENT PATTERNS OF EXPRESSION. EVEN BETWEEN JUVENILES AND ADULTS YOU SEE CHANGES IN EXPRESSION. OF COURSE THAT SHOULDN'T SURPRISE ANY PARENT OR TEENAGERS, BUT I WAS SURPRISED AT THE SIZE OF THIS. BUT WE'VE USED THESE KIND OF DEVELOPMENTAL CHANGES TO LOOK SPECIFICALLY AT THE EMERGENCE OF PHYSIOLOGICAL PROPERTIES AND UNDERLYING -- GENE EXPRESSION IN THESE BASKET CELLS THAT I MENTIONED. YOU CAN APPRECIATE HERE BY LOOKING AT THESE CURVES THAT RELATE FIRE TO INJECTION CURRENTS -- HOW DIFFERENT THESE ARE FROM OTHER NEURONS. HERE ARE SOME OTHER SUBTYPES OF NEURONS CLUSTERED HERE TOWARDS THE ORIGIN. AND HERE ARE THE, IS THE -- OF THESE FAST SPIKING NEURONS THAT CAN FIRE IN HUNDRED OF HERTZ. IT HOWEVER TAKES A LOT OF CURRENT TO GET THEM TO START FIRING. THEY HAVE VERY LOW INPUT RESISTANCE AND THEY HAVE TO RECEIVE A LOT OF SYNAPTIC INPUT NEURONS TO START PRODUCERRING THIS PROFOUND SOURCE OF INHIBITION WHICH AS I MENTION IT'S PROFOUND NOT ONLY BECAUSE OF HOW FAST THEY CAN FIRE BUT BECAUSE OF THEIR AXONS ARE PROXIMAL AND SURROUND THE PYRAMIDS THEY IN NERVATE. THESE PHYSIOLOGICAL PROPERTIES DON'T HAPPEN ALL AT ONCE. THESE CELLS MIGRATE DURING THE FIRST WEEK OR SO, ARE EVEN WITH THAT INTO THE CORTEX. AND AS SOON AS WE CAN IDENTIFY THEM IN THE CORTEX A FEW DAYS POSTNATALLY, JUST BARELY ABLE TO FIRE REPETITIVELY. BY THE END OF A WEEK THEY CAN FIRE REPETITIVELY BUT THEY CAN'T FIRE AT THE PHENOMENAL RATES THEY'RE CAPABLE OF AS ADULTS. THESE VARIOUS OTHER PROPERTIES LIKE THEIR INPUT RESISTANCE, THE DEGREE TO WHICH THEY ADAPT AND SO ON, THE CURRENT IT TAKES TO DRIVE THEM TO FIRE ALL CHANGE IN A SYSTEMATIC WAY WITH DEVELOPMENT. AT THE SAME TIME THERE ARE MASSIVE CHANGES IN THEIR UNDERLYING GENE EXPRESSION. AND IF ONE JUST SORT OF PLOTS, IF ONE JUST LOOKS AT WHICH GENES ARE CHANGING, IT'S ON THE ORDER OF THOUSANDS OF GENES AT CRITERION LIKE TWO-FOLD OR GREATER AND A HIGH SIGNIFICANCE LEVEL. IF YOU THEN DO A PRINCIPAL COMPONENT ANALYSIS TO SEE WHAT PATTERNS OF CHANGE THERE ARE, MOST OF THESE GENES ARE CHANGING-INCREASING MONOTOPICALLY. THERE ARE VERY POINTS IN THE MIDDLE HERE WHERE THIS SORT OF PEAKY SECOND PRINCIPAL COMPONENT IS HIGH AND THE FIRST PRINCIPAL COMPONENT IS CLOSE TO ZERO. PERHAPS -- WE WOULD HAVE SEEN A LARGER NUMBER OF TRAN TRANSCRIPTORS TURNING ON AND OFF THE TIME POINTS. DURING THIS POSTNATAL MATURATION IT APPEARS TO BE SORT OF A SMOOTH TRANSITION BETWEEN DEVELOPMENTALLY REGULATED GENES AND THINGS THAT ARE PRESENT IN THE ADULT. NOT SURPRISING, A LARGE NUMBER OF THE GENES THAT ARE DEVELOPMENTALLY REGULATED BOTH UP AND DOWN ARE ION CHANNEL GENES. AND SO WE SEE BOTH THING THAT ARE BEING TURNED OFF. THESE REPRESENT A SUBUNIT SWITCHES, FOR EXAMPLE, OR DOWN REGULATION OF SPECIFIC SUBUNITS OF SODIUM POTASSIUM AND CALCIUM CHANNELS. OR CHANNELS THAT ARE BEING UPREGULATED. SOME OF THESE WERE KNOWN BEFORE SO FOR EXAMPLE KCNC1 AND T OR KB3.1 AND 3.2 ARE VERY SPECIALIZED RAPIDLY ACTIVATING POTASSIUM CHANNELS SUBUNITS THAT ARE CRITICAL FOR THE FAST SPIKING PHENOTYPE. THEY HAVE THOSE VERY FAST SPIKE, VERY FAST REPOLARIZATION IN ORDER TO PROTECT THEIR SODIUM CURRENT FOR BEING INACTIVATED WHEN THEY FIRE AT SUCH HIGH RATES. IF THESE ARE KNOCKED OUT, THE PHENOTYPE IS IMPAIRED. WE ALSO IDENTIFIED SOME POTASSIUM LEAK CHANNELS, TAX ONE AND TWEAK ONE, K3 AND WE HYPOTHESIZE THESE MIGHT BE IMPORTANT, THE UPREGULATION OF THESE MIGHT BE IMPORTANT FOR THE TRULY DROPPING INPUT RESISTANCE OF THESE CELL AS THEY DEVELOP. SO WE CAN CONFIRM BOTH AT THE PROTEIN LEVEL USING ANTIBODIES THAT THERE WAS ALSO AN INCREASE DEVELOPMENTALLY AN EXPRESSION OF AT LEAST ONE OF THESE FOR GOOD AND BOTH TASK ONE. AND BY BLOCKING OTHER CURRENTS IN THESE CELLS, WE CAN ISOLATE A WEAK CURRENT AND ALTHOUGH THE PHARMACOLOGY FOR THESE CHANNELS IS NOT AS GOOD AS MANY OTHER POTASSIUM CHANNELS. THERE ARE A BLOCK OF -- THAT BLOCKS TASK ONE AND WE CAN SHOW THE FRACTION OF THE CURRENT THAT WAS BLOCKED BY THE -- WAS GROWING AS WELL. AND THEN SUBSEQUENTLY THE -- COLLEAGUES ACTUALLY KNOCKED OUT THIS WEAK CHANNEL AND SHOWED THAT IN FACT INHERENTLY MATURATION OF THE INPUT RESISTANCE OF THESE CELLS. SO CLEARLY, IT'S CRITICAL IN CLASSIFYING CELLS TO KNOW SOMETHING ABOUT, TO NORMALIZE IN A SENSE WHEN ONE IS LOOKING AT THESE. AND THESE REALLY REPRESENT SNAP SHOTS OF DEVELOPMENTAL PROGRAM OF EXPRESSION. BUT ONE WHICH YOU COULD USE THIS APPROACH ACTUALLY AND LEARN SOMETHING ABOUT. THE SECOND ISSUE REALLY GETS AT THE HEART OF THE QUESTION OF WHAT IS A CELL TYPE. THE WAY THAT I THINK ABOUT THIS IS SORT OF TREE. YOU CAN SORT OF PARSE OUT THE MAJOR BRANCHES OF THIS TREE, EXCITATORY, INHIBITORY THREE MAJOR BRANCHES, ETCETERA. BUT HOW FINELY DO YOU HAVE TO GET OUT TO THE BRANCHES? SO ULTIMATELY WE'D LIKE TO BE ABLE TO RECOGNIZE THE SAME CELL TYPES ACROSS ORGANISMS. MOST OF US WORK ON RODENTS BUT WE'D LIKE TO BE ABLE TO RECOGNIZE THIS SAME CELL TYPES IN PRIMATES AND OURSELVES. WE WOULD LIKE TO ALSO RECOGNIZE ALL OF THE CELL TYPES ACROSS DIFFERENT REGIONS OF THE NEOCORTEX -- HUGE AND IT'S KIND OF AN OPEN QUESTION TO WHAT DEGREE THE CELL TYPES IN ONE REGION ARE IDENTICAL TO THOSE IN ANOTHER. OR TO WHAT DEGREE THERE ARE REGIONAL SPECIALIZATIONS. OBVIOUSLY THERE MUST BE SOME DEGREE OF SPECIALIZATION BUT THE MAGNITUDE OF THESE SPECIALIZATIONS ARE NOT KNOWN. SO ALREADY WE CAN BEGIN TO LOOK AT THIS IN THE INITIAL DATA BY COMPARING FOR EXAMPLE HOMOLOGOUS PYRAMIDAL NEURONS OR INTERNEURONS ISOLATED FROM PRIMARY SOMATOSENSORY CORTEX. YOU CAN SEE FROM BOTH OF THESE WE COULD MAINLY FIND MARKERS THAT WERE PRESENT IN BOTH REGIONS INDICATING THAT THESE WERE REALLY HIGHLY SIMILAR. FOR THE INTERNEURONS IN FACT, WE COULDN'T FIND ANY DIFFERENCE BETWEEN THESE. FOR THE PYRAMIDAL NEURONS WE COULD FIND SOME SUBTLE DIFFERENCES THAT DIDN'T MAKE IT INTO THIS SORT OF HIGH UP THE LIST. BUT MORE RECENTLY, WE FOUND EXAMPLES OF PHYSIOLOGICAL PROPERTIES. THEY ARE RARE ADMITTEDLY, THAT ARE DRAMATICALLY DIFFERENT DEPENDING ON WHERE IN THE NEOCORTEX YOU'RE RECORDING. AND SO THIS IS THIS THICK TUFTED PYRAMIDAL NEURON I MENTIONED IS LABEL IN THIS YPH NEURON. THEY CONTRIBUTE TO THE PYRAMIDAL TRACT. AND SURE ENOUGH AS A -- IN THE PRIMARY M OTOR CORTEX THEY HAVE A DIFFERENT FIRING TYPE. THEIR FIRING ACTUALLY ACCELERATES OVER YOU SPIKE THE INTEGRALLAL A BUT IN FACT WE WERE ABLE TO SHOW THAT IT'S DUE TO SLOW, A SLOWLY CLOSING POTASSIUM CURRENT, SO-CALLED D CURRENT. THESE CURRENTS ARE SHAKER TYPE CURRENTS THAT ARE SENSITIVE TO A VARIETY OF VERY SPECIFIC TOXINS. THIS IS ONE OF THE LESS SELECTIVE ONES WE SELECTED FOR THESE SHAKERS BUT FOR SEVERAL OF THEM DENDRO TOXINS WHICH CHANGES THE -- FROM ACCELERATING TO NON-ADAPTING. WE CAN SHOW ALSO THE VOLTAGE PLANT THAT IT BLOCKS THE UNDERLYING SO THE INACTIVATING POTASSIUM CURRENT. BUT INTRIGUINGLY WHEN WE LOOKED FOR THE TRANSCRIPTS THAT MEDIATE THIS, WE COULD SEE NO DIFFERENCE BETWEEN THESE KCNA1, 2 AND 3 FOR EXAMPLE BETWEEN SOMATOSENSORY CORTEX -- EVEN AT THE PROTEIN LEVEL WE COULD SHOW BY ANTIBODY STANDING THAT THESE SAME SUBUNITS WERE HIGHLY DIFFERENTIALLY EXPRESSED IN THE CELL AND PROXIMAL DENDRITES IN THESE -- SO IT SUGGESTS OF COURSE AS AGAIN MIGHT BE OBVIOUS IF YOU THINK ABOUT THIS AT ALL THAT THERE'S A LOT MORE BIOLOGY THAT HAPPENS AFTER TRANSCRIPTION AND TRANSLATION MAY BE SPECIFICALLY REGULATING THE CELL TYPES IN A SPECIFIC WAY AND OF COURSE TRAFFICKING AND TRANSLATIONAL MUD -- MODIFICATIONS AS WELL AND THE DIFFERENCES BETWEEN CELL TYPES OR SPECIALIZATIONS OF CELL TYPES YOU HAVE TO HAVE MORE HIGH -- TO EXAMINE THESE AS WELL. OKAY. WELL WHAT IS ALL OF THIS GOOD FOR? I SPENT A LOT OF TIME SORT OF TELLING YOU ABOUT THE METHOD AND SOME CAVEATS. WE GOT INTO THIS TO IDENTIFY COMPONENT CELL TYPES AND BEGIN TO CLASSIFY CELLS AND UNDERSTAND THEIR CIRCUITRY. BUT I THINK I'VE ALREADY I HOPE CONVINCED YOU THAT THIS IS ALSO A POTENTIALLY POWERFUL WAY TO UNDERSTAND THE CELLULAR BASIS OF THE MOLECULAR BASIS OF SPECIFIC PHENOTYPES. FOR EXAMPLE HOW FIRING TYPES ARISE FROM THE PATTERN OF ION CHANNELS THAT ARE EXPRESSED. OR WHAT WE HOPE TO LOOK AT IS HOW FOR EXAMPLE CONNECTIVITY PHENOTYPES ARISE FROM THE SETS OF CELL ADHESION MOLECULES THAT THESE CELLS EXPRESS. BUT IT'S POTENTIALLY A MUCH MORE SENSITIVE WAY TO LOOK AT HOW EXPRESSION CHANGES IN PLASTICITY OR DURING DISEASE STATES. I'LL TAKE A FEW MINUTES TO TELL YOU WITH ONE SUCH APPROACH WE'VE TAKEN TO LOOK AGAIN AT EXPRESSION IN A SPECIFIC DISEASE STATE. AND THE DISEASE THAT WE'VE CHOSEN TO LOOK AT IS RET SYNDROME. SO RET SYNDROME HAS BEEN CLASSIFIED IN THE PAST AS ONE OF THE AUTISM SPECTRUM DISORDERS. IT'S CLEARLY DISTINCT FROM SYNDROMIC AUTISM ALTHOUGH IT SHARES SOME FEATURES. IT'S THE SECOND MOST COMMON GENETIC CAUSE SINGLE GENE CAUSE AFTER -- OF MENTAL RETARDATION. AND IT'S A DEVASTATING DISEASE FOR THE FAMILIES THAT OF THESE GIRLS, AND I'LL MENTION WHY GIRLS IN A MOMENT. BECAUSE THE GIRLS INITIALLY DEVELOPMENT NORMALLY SO THE FIRST SIX TO 18 MONTHS OF LIVER, THEY APPEAR NORMAL. THEY START TO ACQUIRE LANGUAGE AND NORMAL MOTOR ABILITIES, COGNITIVE ABILITIES. AND THEN THERE'S A CHARACTERISTIC REGRESSION, ALSO A CHARACTERISTIC OF SOME OTHER FORMS OF AUTISM. AND THEY MAY BE CONFINED TO A WHEELCHAIR, LOSE ALL PURPOSE OF HAPPENED MOVEMENT. LOSE WHAT SPEECH THEY'VE HAD. BUT REMARKABLY UNLIKE OTHER FORMS OF AUTISM AND MANY OTHER FORMS OF MENTAL RETARDATION. THIS IS DUE TO DEFECTS IN A SINGLE GENE MCB2. THIS IS A METHYL DNA BINDING PROTEIN WHICH WAS ORIGINALLY ISOLATED BY -- TO UNDERLIE RET SYNDROME. THEY WENT ON TO ACTUALLY KNOCK OUT NACP2 WHICH IS -- AND FOUND THAT IT RECAPITULATES MANY FEATURES OF THE DETAIL. I MENTIONED EXCELLENT SO THAT THE GIRLS THAT ARE SUBJECT TO THE DISEASE HAVE ONE NORMAL COPY OF -- MUTANT COPY. NOW IT'S TYPICALLY NOT RECOGNIZED BECAUSE THEY HAVE A MUCH MORE RAPID COURSE HAVING NO COPY OF NECP2 IF THE SINGLE ONE IS KNOCKED OUT. AND THEY DIE WITHIN A FEW WEEKS OF AGE AS IS ALSO TRUE IN THE MICE. SO IN TERMS OF THE UNDERLYING NEUROPATHOLOGY THERE ARE SUBTLE DIFFERENCES, THE BRAIN ARE SOLICITLY SMALLER. PYRAMIDAL NEURONS IN THE FOREBRAIN ARE SLIGHTLY SMALLER -- MUCH MORE SUBTLY THAN IS INDICATED IN THIS CARTOON FROM A REVIEW BY -- BUT MEASURABLE DIFFERENCES EXISTS. IT'S CLEARLY NOT FOR EXAMPLE A GENERATION OF THE SPECIFIC CELL TYPE. AND SO WE'VE WORKED ON SORT OAF A TWO ENDS OF THIS PROBLEM -- OF TWO ENDS OF THIS PROBLEM. ONE IS UNDERSTANDING THE CORTICO CIRCUITS OF THESE MP2 KNOCKOUT MICE AND WHAT DIFFERENCES IN GENE EXPRESSION MIGHT EXPLAIN THIS. SO WE FOUND QUITE EARLY ON THAT IF YOU RECORD UNDER CONDITIONS IN WHICH THE SPONTANEOUS ACTIVE TEE, YOU CAN DO THIS BY SLIGHTLY LOWERING CALCIUM AND MAGNESIUM AND SLIGHTLY ELEVATED POTASSIUM IN THE SPINAL FLUID AND YOU FIND THERE'S ACTUALLY GREATLY REDUCED ACTIVITY IN THE PYRAMIDAL NEURONS RECORDED IN PRIMARY SOMATOSENSORY CORTEX. THIS IS APPARENT EVEN BEFORE THE MICE BECOME CLEARLY SYMPTOMATIC. NOW, THIS SORT OF THREE MAIN WAYS IN WHICH ACTIVITY COULD BE REDUCED IN THIS RECURRENT CIRCUIT. EITHER THE EXCITABILITY OF THE NEURONS THEMSELVES COULD BE ALTERED AND THAT TURN OUT NOT TO BE TRUE IF YOU BLOCK OFF TRANSMISSION AND JUST LOOK AT THE RELATIONSHIP BETWEEN FIRING AND INJECTED CURRENT. IT'S THE SAME FOR THE WILD TYPE AND -- NEURONS OR IT COULD BE REDUCED RECURRENT EXCISATION. BOTH ARE TRUE. IT'S A PROM DUCT REDUCTION IN THE SPONTANEOUS EXCITATORY INPUT TO THESE NEURONS AND A MORE MODEST INCREASE IN INHIBITORY INPUTS TO THESE NEURONS. AND SO -- WHO BEGAN THESE EXPERIMENTS IN MY LAB WANTED TO LOOK IN GREATER DETAIL AT THE SORT OF UNDERLYING BIOPHYSICAL MECHANISM. THERE WERE SORT OF TWO IDEAS ABOUT THIS. ONE, IT COULD BE A BLOCKADE OF THE PLASTICITY MECHANISMS LIKE LONG TERM POTENTIALATION THAT ENHANCE RECURRENT EXCITATION IN THESE CIRCUITS AND THAT THEREFORE AS A RESULT OF SELECTIVELY BLOCKING INHIBITION WHICH HAD BEEN NOTED BLOCKING LONG TERM POTENTIALATION WHICH HAD BEEN MOTED BEFORE, YOU LOSE THIS EXCITATORY SYNAPTIC TRANSMISSION OR THE REVERSE COULD BE TRUE. THAT IS BECAUSE THESE RECURRENT EXCITATORY SYNAPSES ARE WEAKER, IT'S HARDER TO ACTUALLY INDUCE LONG TERM POTENTIALATION. THE WAY TO GET AT THIS QUESTION IS TO ICE LACE THE SYNAPTIC CONNECTION BETWEEN A PAIR OF THESE PYRAMIDAL NEURONS AND SEE WHETHER IN THE ABSENCE OF ACTIVATION OF THE ENTIRE CIRCUIT IN WHICH THE BALANCE BETWEEN EXCITATION AND INHIBITION WILL GOVERN -- PAIRING PROTOCOL WITH PAIRED FIRING OF THE PRESYNAPTIC NEURON ABOUT ACTIVATION OF PO -- SYNAPTIC INPUT OR WITH THE SPIKED TIMING PROTOCOL WHERE THE PRE AND POST SYNAPTIC NEURONS FIRED WITH A SHORT INTERVAL BETWEEN THE TWO. BASICALLY THERE'S NORMAL LONG TERM POTENTIATION IN THESE. THESE ARE QUITE NOISY BECAUSE THEY REPRESENT INDIVIDUAL MONO SYNAPTIC CONNECTIONS -- WE AVERAGE OVER POPULATIONS OF CELLS. YOU CAN SEE THAT IF YOU JUST CONTINUALLY RECORD WITHOUT INDUCING -- THE STRENGTH IS CONSTANT. BUT THAT EITHER IN WILD TYPE OR NEURAL, WILD TYPE OR KNOCK OUTCELLS YOU SEE THIS LASTING POTENTIATION. IN CONTRAST THE BASELINE AMPLITUDE OR PROBABILITY OF CONNECTIVITY IS GREATLY IMPAIRED IN THESE ANIMALS. AND WHEN SO IF YOU LOOK FOR EXAMPLE AFTER FOUR WEEKS OF AGE, THE AMPLITUDE OF THESE UNITARY CONNECTION IS ROUGHLY IN HALF. AND IT'S EVEN A GREATER THAN TWO FOLD REDUCTION IN THE PROBABILITY THAT ANY TWO CELLS WOULD BE CONNECTED. THIS IS SOMEWHAT HEROIC UNDERTAKING TO RECORD ALL THESE POTENTIAL PAIRS IN THESE OLDER ANIMALS. BUT PERSISTED AND FOUND THAT THIS WAS THE CASE. SO WHAT IS IT THAT CAUSES THIS? WELL WHAT DO WE KNOW ABOUT NACP2 FUNCTION. WELL THE IDEA IS NACP2 ACTUALLY BINDS TO THESE METHYLATED CPG'S. IT RECRUITS REPRESSER COMPLEXES THAT THEN REGULATE TRANSCRIPTION. THERE'S SOME HINTS THAT DEPENDING ON WHETHER THE METHYLATION IS IN THE CELL BODY OR IN THE PROMOTER, IT COULD ACTUALLY HELP TO SERVE AS AN ACTIVATOR RATHER THAN REPRESSER. BUT IT WAS SURPRISING IN LIGHT OF THIS THAT THE FIRST KNOCKOUTS WHEN THEY DID MICRO RAY STUDIES FOWNLTD REALLY REMARKABLY SUBTLE EFFECTS IN TRANSCRIPTION. SO TAKING OUT CHUNKS OF CORTEX OR CEREBELLUM ETCETERA, THE COLLEAGUES FIRST FOUND THAT OKAY WELL IF THERE WAS 7% DIFFERENCE IN EXPRESSION IN THIS GENE AND A 7% DIFFERENCE IN EXPRESSION ON THAT GENE, AND 9% DIFFERENCE IN EXPRESSION IN THAT GENE, THAT THEY COULD TELL THE DIFFERENCE BY LOOKING ACROSS THESE TEN GENES OR ANY NUMBER OF TEN GENE CLASSIFICATION WHAT THE DIFFERENCE BETWEEN THE WILD TYPE MUTANT WAS BUT ANY OF THESE DIFFERENCES WERE BARELY OR NOT SIGNIFICANT. AND WE REASONED OF COURSE THAT THIS COULD REFLECT WHAT WE'VE COME TO CALL THE DILUTION PROBLEM. THE IDEA THAT IF THERE ARE SPECIFIC GENES BEING REGULATED BY METHYLATION IN SPECIFIC CELL TYPES, THEN WHEN YOU GRIND TOGETHER A BUNCH OF DIFFERENT CELL TYPES, YOU MIGHT ACTUALLY SORT OF WASH OUT MUCH LARGER GENE EXPRESSION. SO YOU CAN JUST SEE THAT THE PRINCIPAL HERE, IMAGINE THAT THERE'S A TENFOLD CHANGE IN EXPRESSION OF SOME TRANSCRIPT THAT THIS CELL TYPE ONLY REPRESENTS 1% OF THE TOTAL TISSUE. WHEN YOU NOW GRIND THINGS UP, YOU'RE ONLY GOING TO SEE A TINY BARELY SIGNIFICANT 9% CHANGE IN EXPRESSION. SO IN ORDER TO LOOK AT THIS, WE MADE THE -- BETWEEN NO MICE TO REPORTER NICE THAT WOULD ALLOW US TO LOOK AT FOUR DIFFERENT CELL TYPES, INHIBITORY NEURONS IN THE CORTEX -- BASKET CELLS. THESE THICK TUFTED PYRAMIDAL NEURON THAT I MENTIONED LOCUS -- NEURONS IN THE PURKINJE -- WHAT WE FOUND IS MUCH LARGER CHANGES IN EXPRESSION THAT HAVE BEEN FOUND AT THE TISSUE LEVEL. SO TEN, 50 FOLD CHANGES IN EXPRESSION IN SOME TRANSCRIPTS. SOME OF THESE ARE TANTALIZINGLY BEEN RELATED TO ASPECTS OF THE DISEASE IN HUMAN STUDIES. IF WE LOOKED ACROSS A LARGE NUMBER OF GENES THAT WERE SIGNIFICANTLY AFFECTED A TOTAL OF 850 GENES, MOST OF THESE ARE AFFECTED IN ONE CELL TYPE BUT NOT IN ANOTHER. SO THING THAT ARE DOWN REGULATED ARE SHOWN IN BLUE. THINGS THAT ARE UPREGULATED ARE SHOWN IN RED. AND YOU CAN SEE THAT LET'S SOME GENES THAT ARE AFFECTED IN MORE THAN ONE CELL TYPE BUT MOST OF THESE GENES ARE NOT. SO IT MAKES SENSE IF YOU GRIND THINGS YOU YOU ACTUALLY WILL SEE A MUCH SMALLER CONTRIBUTION TO THE WHOLE TISSUE LEVEL. SO EVEN THOUGH WE SAW DIFFERENT GENES BEING AFFECTED IN DIFFERENT CELL TYPES, WE SAW REPEATEDLY THE SAME CATEGORY OF GENE AND THAT IS CELL ADHESION MOLECULES BEING AFFECTED ACROSS CELL TYPES. HERE IS THREE OF THE FOUR CELL TYPES. AND YOU CAN SEE THAT OF THESE MANY CATEGORIES OF GENES THAT ARE OVERREPRESENTED IT'S REALLY CELL ADHESION THAT IS OVERREPRESENTED. HERE ARE SOME OF THE ACTUAL BIOLOGICAL CATEGORIES OF GENES INCLUDING -- SECRETED MOLECULES LIKE COLLAGENS AND -- THIS IS A TEXT CULL ANALYSIS HOW LIKELY THIS IS TO OCCUR BY CHANCE. THE BIGGER THE CIRCLE THE LESS LIKELY THIS IS TO OCCUR BY CHANCE AND THE COLOR INDICATES IT'S SOMETHING THAT OCCURS ACROSS FOUR CELL TYPES OR A SMALLER NUMBER OF CELL TYPES. AND THIS IS A SORT OF HIERARCHICAL REPRESENTATION OF ALL OF THE DIFFERENT GENE TOLOGY DEFINED TO THE LOCALIZATION OF GENES. YOU CAN SEE IF YOU SORT OF SIMULATE THIS HAPPENING BY CHANCE THAT THE CHANCE OF THIS WILL HAPPEN IS, YOU KNOW, MUCH MUCH LAST THAN WHAT YOU ACTUALLY SEE. SO THIS IS WHERE WE ARE IN TERMS OF ANALYZING RET SYNDROME. WE'D LIKE TO OF COURSE BE ABLE TO DRAW A CAUSAL RELATIONSHIP BETWEEN THESE CHANGES IN EXPRESSION OF CELL ADHESION MOLECULES THAT WE SEE IN SPECIFIC CELL TYPES AND THE DISRUPTION OF SPECIFIC CIRCUITS WE THINK RISE TO DISEASE. WE'RE NOT AT THAT POINT. WHAT WE NEED IS THE ABILITY TO ACTUALLY MANIPULATE SOME OF THESE GENES IN A CELL TYPE SPECIFIC WAY. SO THAT IS ACTUALLY SOMETHING WE'VE BEEN WORKING ON TO A GREAT EXTENT OVER THE LAST FEW YEARS. SO I MEAN I THINK THIS IS A GENERAL RECOGNIZED ISSUE THAT IT'S NOT ENOUGH TO SIMPLY BE ABLE TO DESCRIBE THE PATTERNS OF EXPRESSION THAT ARE DIFFERENT BETWEEN GENES AND ACTUALLY LINK UP THOSE DIFFERENCES TO FUNCTIONAL DIFFERENCES. YOU GOT TO ACTUALLY BE ABLE TO DISRUPT GENES IN A CELL TYPE SPECIFIC WAY. AND SO YOU KNOW MANY PEOPLE HAVE TRIED TO DO THIS IN MANY SYSTEMS AND THERE'S A SORT OF GENERAL STRATEGY HERE WHICH IS TO USE A COMMONTORIAL SYSTEM -- DRIVER RESPONDER SYSTEM WHERE YOU HAVE ONE GENE LIKE THE RECOMBINATION -- DRIVER EXPRESSED IN A SPECIFIC SET OF CELL TYPES AND YOU CAN COMBINE THAT WITH ALLELES OR RESPONDER ALLELES OR -- RECOGNIZED BY TETRA ACTIVATOR AND IN THAT WAY YOU DON'T NEED TO REGENERATE. AND YOU CAN SIMPLY MAKE DRIVERS TO RESPONDERS IN ORDER TO LOOK ACROSS CELL TYPES. SO THE TRIED AND TRUE METHOD FOR DO THIS AND THE METHOD THAT'S FORMED THE BASIS OF THE NAH BLUEPRINT PREDRIVER PROJECT THAT WE'RE A PART OF THAT'S -- IS TO IDENTIFY SPECIFIC PROMOTERS THAT ARE ACTIVE IN SPECIFIC CELL TYPES. AND THEN USE THOSE TO TARGET THE CELL TYPES OF INTEREST. THIS HAS BEEN HIGHLY SUCCESSFUL. HERE'S A PAPER FOR EXAMPLE FROM THE DRIVER PROJECT THAT ICE LATES KNOCK-IN CRETE LINES THAT CAN BE USED TO TARGET A VARIETY OF DIFFERENT FOREBRAIN INTERNEURONS AFFECT GABA URGIC NEURONS THROUGHOUT THE BRAIN. THEY ARE BECOMING MORE AVAILABLE AS WE SPEAK. BUT IT'S IMPORTANT TO RECOGNIZE THAT THERE ARE SOME LIMITATIONS TO THIS APPROACH. AND THAT IS THAT OFTEN THERE'S ONLY MODEST SPECIFICITY. THAT IS IF YOU'RE STAYING WITHIN A PARTICULAR STRUCTURE LIKE THE RETINA OR THE CORTEX YOU MAY BAY TARGETING CELL TYPE BUT ACROSS THE WHOLE BRAIN IT'S LIKELY THAT ANY GIVEN GENE IS EXPRESSED IN MANY DIFFERENT CELL TYPES. ANY GIVEN CELL TYPE IS OF COURSE REALLY REPRESENTS THE CONJUNCTION OF A BUNCH OF DIFFERENT GENES. AND SO WE'VE BEEN TRYING TO DEVELOP AN ALTERNATIVE APPROACH TO THIS. AND AS A MOTIVATION FOR OR ILLUSTRATION OF HOW THIS SORT OF LACK OF SPECIFICITY CAN CAUSE PROBLEMS I JUST DESCRIBED SOME ONGOING EXPERIMENTS WHERE WE'RE TRYING TO LOOK AT THE QUESTION OF WHETHER THE ENZYME DICER WHICH IS CRITICAL IN THE BIOGENESIS OF MICRO ORGAN SUBPOENAS, SMALL RNAS THAT REGULATE TRANSLATION ARE IMPORTANT FOR THIS DEVELOPMENT OF FAST SPIKING CELLS THAT I MENTION. INITIALLY WHAT WE DID WAS MAKE -- MATE A PV CREE ANIMAL TO A DICER ALLELE THAT HAD ALREADY BEEN MADE. WE WERE VERY EXCITED WHEN WE FIRST LOOKED AT THESE ANIMALS. SO COMPARED TO NORMAL ANIMALS WHICH SEEMS SORT OF WANTED RING AROUND THE KITCHEN SINK -- PHENOTYPE AT FIRST WHEN WE SAW THESE, ARE THESE SEIZURES, WHAT IS THIS. FINANCIAL WE'RE NOT USED TO ANALYZING BEHAVIOR AS OPPOSED TO PHYSIOLOGY AND ANATOMY. BUT SUFFICE IT TO SAY IT TURNS OUT THAT THE PV NEURONS IN THE CORTEX ARE ALMOST ENTIRELY NORMAL AND THESE BASELINE THAT DO SHOW SOME INTEREST IN PLASTICITY DEFICITS. AND PROBABLY THIS PHENOTYPE HAS TO DO WITH POSITIVE NEURONS IN THE SPINAL CORD -- WE'RE STUDYING A LITTLE BIT OF WHAT HAPPENS TO THOSE NEURONS. BUT AGAIN, IT WOULD BE NICE IF WE HAD SOMETHING THAT WOULD TARGET JUST THE PV NEURONS AND THE CORTEX. AND SO THAT IS SORT OF SERVES AS MOTIVATION FOR THIS OTHER STRATEGY THAT WE'VE BEEN PURSUING RECENTLY THAT HAS THE POTENTIAL FOR REVEALING CELL TYPES WITH GREATER SPECIFICITY. ALTHOUGH IT HAS A MAJOR DISADVANTAGE THAT IT'S NOT TALKED IN THE WAY THAT THE -- STRATEGY IS. THIS IS AN ENHANCIVE TRAP STRATEGY. IT'S BEEN USED TO GREAT SUCCESS IN FLIES. WHERE P ELEMENTS CAN BE USED TO MOBILIZE GENETIC ELEMENTS. THE IDEA IS YOU HAVE A WEAK PROMOTER THAT IS DRIVING TRANSCRIPT OF INTEREST. AND BECAUSE IT LANDS NEXT TO AT VARIOUS PLACES IN THE GENOME DIFFERENT ENHANCERS AND REPRESSERS, IT GETS PRESSED IN DIFFERENT PATTERNS AND DIFFERENT LINES. IN MILES WITH A DON'T HAVE P ELEMENTS. WE'RE LOOKING AT USING -- TO DO THIS BUT MOST OF WHAT WE'VE DONE SO FAR IS USING -- VIRUS. THIS IS IN COLLABORATION WITH CARLOS -- AT UMASS. THE IDEAS THAT YOU INJECT THE BIOS THAT CONTAIN THIS WEAK PROMOTER AND INTO SINGLE CELL FERTILIZED EMBRYO. AND -- EVENTUALLY YOU CAN SCREEN FOUNDERS AND LOOK AT INTERESTING PATTERNS OF EXPRESSION. I WON'T GO THROUGH THE VARIOUS CONSTRUCTS THAT WE'VE USED EXCEPT TO POINT OUT THAT THE BASIC IDEA IS THAT THE DRIVER IN THIS CASE, THE TRANSACTIVATOR IS INSISTS -- DRIVEN BY A TRE -- IN ORDER TO SCREEN THEM. THE SAME TTA CAN THEN BE USED TO DRIVE EXPRESSION OF OTHER GENES AND CELLS OF INTEREST. SO THIS TURNS OUT TO THE LABEL CELLS REMARKABLY SPECTACULARLY SPECIFICALLY IN MANY CASES. SO HERE'S AN EXAMPLE OF A LINE THAT LABELS -- CELLS. THERE IS A -- LINE THAT CAN ALSO LABEL THESE CELLS AND THEY LOOK VERY SIMILAR IN THE HIPPOCAMPUS OR IN THE CORTEX. YOU CAN SEE THEM IF YOU LOOK EARLIER IN DEVELOPMENT. HERE ARE TWO LINES -- NEURONS IN THE VPM. ANOTHER THAT LABELS ONE OF TWO MAIN SUBTYPES PYRAMIDAL NEURONS IN -- CORTEX. THESE ARE SO-CALLED -- CELLS. HERE'S A -- THERE'S A SMATTERING OF CELLS LABELED ALSO IN THE PRIMARY SOMATOSENSORY NUCLEUS. THERE ARE MANY GENES THAT LABEL WELL THERE ARE SEVERAL GENES THAT LABEL ALL NEURONS. THERE ARE NO GENES THAT LABEL JUST -- NEURONS. YOU CAN SEE THAT PROJECTIONS OF THESE NEURONS TO -- DELINEATE HERE IN B1 AND B2 -- LAYER ONE AND LAYER SIX. HERE'S ANOTHER LINE THAT LABELS THAT ARE -- SELECTIVELY IN PRIMARY SENSORY AREAS. THIS IS A LINE THAT LABELS A SUBSET OF PROTOCO-FIVE NEURONS JUST IN PREFRONTAL CORTEX AND THESE ARE NEURONS THAT PROJECT TO MODULATORY NUCLEI LIKE SEROTONIN NEURONS. AND THIS IS FINALLY A LINE THAT LABELS DEEP LAYER SIX NEURONS. THESE ARE CORTICO -- NEURONS BUT IT TURNS OUT AS WE INVESTIGATED THESE FURTHER THIS IS ONE OF TWO DIFFERENT SUBTYPES OF NEURONS. SO THE CLASSIC CORTICO -- NEURONS PROJECT BACK TO THE PRIMARY SENSORY NUCLEI THAT PROVIDE NEURONIC INTO THE PUT THAT REGION. FOR EXAMPLE IN THE -- THESE ARE NOT THOSE NEURONS. THESE ARE NEURONS THAT PROJECT TO SECONDARY SENSORY. SO TO LATERAL -- NUCLEUS IN THE CASE OF THE USUAL CORTEX OR TO -- IN THE CASE OF SOMATOSENSORY CORTEX. THEY AVOID THE PRIMARY SENSORY NUCLEUS. YOU CAN PUT TRACER INTO THE NUCLEUS AND SHOW THEY ARE NON-OVERLAPPING WITH THE LABELED CELLS AND SO ON. IT'S IMPORTANT HERE IS NOT THIS PARTICULAR FINE POINT OF CORTICO ANATOMY BUT THAT THIS STRATEGY PROVIDES ACCESS POTENTIALLY TO MUCH MORE SELECTIVE POPULATIONS OF NEURONS THAN IS ACCESSIBLE IN THE STANDARD KNOCK OUTAPPROACH. THIS IS JUST A SUMMARY OF WHERE WE'RE AT IN THIS AS OF A COUPLE MONTHS AGO. WE HAD INTERESTING PATTERN WITHIN THE BRAIN -- NOT AS SPECIFIC AS THE ONES I'VE SHOWN YOU BUT THERE ARE MANY THERE ARE. AND THE RATES VARIOUS A LOT DEPENDING ON WHAT EXACT CONSTRUCT THAT YOU USE. SO I THINK I'LL STOP THERE AND JUST SAY THAT I THINK THAT THESE ARE REALLY VERY EXCITING TIMES TO BE WORKING IN THE NEUROSCIENCE BECAUSE A LOT OF THE GENETIC AND GENOMIC TRICKS HAVE BEEN ACCESSIBLE TO COLLEAGUES FOR MANY YEARS ARE NOW REALLY COMING ON-LINE FOR US. AND I THINK HAVING A HANDLE ON THE GENETICS AND GENOMICS OF CELL TYPES IS GOING TO START TO ALLOW US TO UNRAVEL HOW THESE CELL TYPES INTO CIRCUITS AND DO WHAT THEY DO. LET ME THANK THE PEOPLE IN MY LAB WHO CONTRIBUTE TO DO THIS WORK. IT STARTED WITH A TALENTED GRADUATE STUDENT -- KEN HAS NOW MOVED ON TO -- FARMS AND IN COLLABORATION WITH -- THERE AND SEAN EDDY, WERE CONTINUING NOW USING RNAC TO PROFILE SPECIFIC CELL TYPES. THE PHYSIOLOGY PHYSIOLOGICAL CHARACTERIZATION OF THESE CELL TYPES IN THE CORTEX WAS DONE BY MARK MILLER WITH HELP FROM -- AND -- AND MOUSE MAKING IN THE LAB HAS REALLY LARGELY BEEN THE WORK OF -- IN COLLABORATION WITH CARLOS LOUIS AND JOSH -- I TALKED BRIEFLY ABOUT SOME OF THE WORK OF -- AND THE RET PROJECT WAS STARTED BY -- CONTINUED BY -- WITH COLLABORATIONS FROM -- LAB. AND THE DATA ON EXPRESSION PATTERNS IS AVAILABLE AT CREW DRIVER.ORG IF YOU'D LIKE TO FOLLOW UP ON ANYTHING IN PARTICULAR. THANKS VERY MUCH FOR YOUR ATTENTION. [APPLAUSE] >> [INDISCERNIBLE] THOSE KIND OF ANALYSIS OF WHAT'S BEEN CALLED GENE BATTERIES WOULD BECOME MORE FEASIBLE. THAT'S ONE OF THE THING THAT WE'RE REALLY HOPING TO GET OUT OF YOU KNOW A HUNDRED CELL TYPES AS OPPOSED TO A DOZEN CELL TYPES. NOW IT'S A, YOU KNOW, YES. >> [INDISCERNIBLE] >> YES. SO THERE WAS A PAPER NOT TOO LONG AGO SUGGESTING, THERE HAVE BEEN A COUPLE PAPERS SUGGESTING THAT COLONIALLY RELATED CELLS ARE MORE LIKELY TO BE CONNECTED AND COLONIAL CELLS ARE MORE LIKELY TO SHARE SOME PROPERTIES. WE ACTUALLY LOOKED AT IT SO ONE OF CARLOS' ORIGINAL LINES CLEARLY WAS LABELING CLONES OF NEURONS IN THE CORTEX. AND HE DID A BUNCH OF -- AND FOUND NO EVIDENCE OF THIS WHATSOEVER. IT TURNS OUT THAT YOU KNOW IF YOU, I THINK IN THAT PAPER WE'RE LOOKING AT A PARTICULAR CONNECTION ACROSS LAYERS AND THERE YOU CAN SEE SOME EVIDENCE OF IT. I THINK IT'S A WEAK EFFECT, YOU KNOW, IF AT ALL. WELL, I SHOULDN'T SAY IF AT ALL. I'M CONVINCED BY THE DATA IN THOSE PAPERS AND I THINK OVERALL IN A SENSE THE CORTEX IS CONSTRUCTED TO DISTRIBUTE CLONES AS MUCH AS POSSIBLE GIVEN THE UNDERLYING DEVELOPMENTAL MECHANISM RATHER THAN THE SORT OF CRYSTALLINE COLUMNAR STRUCTURE WHICH MANY OF US KIND OF GREW UP WITH. I REALLY THINK THAT CIRCUITS ARE DESIGNED TO MULTIPLEX THAT AND COMPLETE THAT RATHER THAN TO PRESERVE IT. >> [INDISCERNIBLE] >> RIGHT. THAT'S INTERESTING. YOU MUST HAVE BEEN ONE OF THE REVIEWERS OF OUR PAPER. I'M JUST KIDDING. IN FACT, NECB2 COMES ON AROUND BIRTH SO IT INCREASES AN EXPRESSION SO IT'S CLEARLY IMPORTANT IN LATER POSTNATAL DEVELOPMENT. BUT IN PARTICULAR, ONE OF THE REVIEWERS WANTED US TO REPEAT THIS EXPERIMENT EARLIER IN DEVELOPMENT. SO WE'VE JUST DONE THAT AND ARE TRYING TO GET THE POST DOCS MOVED ON TO REANALYZE THE DATA BUT IT'S VERY SLOW. BUT ANYWAY THE ANSWER IS THERE ARE SMALLER EFFECTS BUT OF THE SAME SORT EARLY ON. BUT NECB2 ITSELF IS MAINLY EXPRESSED DURING THIS SORT OF PERIOD OF LATER DEVELOPMENT. YES. >> [INDISCERNIBLE] >> YES. SO WE CAN AMPHI SUFFICIENTLY FOR RNA USING THE MANUAL SORTED METHOD. WE'RE ALSO TRYING TO LOOK AT EPI GENETIC MODIFICATION OF GENOMIC DNA AND AT CHIP SEAT FROM IDENTIFIED NEURONS AND TO GET ENOUGH GENOMIC DNA WE ABSOLUTELY NEED TO FACT SORT. FACT SORTING WORKS. IT JUST A MATTER OF BASICALLY HAND TUNING OR FINE TUNING THE PARAMETERS FOR EACH CELL TYPES. SO WHAT ENABLE THE CELL TO GET THROUGH THAT IN FACT AS IT TURNS OUT FROM CELL TO CELL. FROM CELL TYPE TO CELL TYPE. SO YES, IT'S NOT QUITE AS PURE. YOU GET BY NECESSITY SOME OTHER STUFF IN THERE. BUT IT'S A PERFECTLY GOOD WAY. IT TURNS OUT THAT IN ALL THIS, THE MANUAL SORTING IS NOT ACTUALLY A SUPER DIFFICULT PART, IT'S REALLY THE AM -- AMPLIFICATION IS MORE DIFFICULT THAN THE SORTING ITSELF. >> [INDISCERNIBLE] YES, DEFINITELY. WE HAVE A PAPER SUBMITTED FROM -- GROUP WHERE HE'S TRIED TO BASICALLY COMBINE THESE CELL TYPE SPECIFIC PROFILES WITH THE ALAN TO PARSE OUT THE DISTRIBUTION OF CELL TYPES ACROSS THE ATLAS. AND YOU KNOW I THINK THEY ARE COMPLEMENTARY SORTS OF DATA SO WE CAN SEE ALL THE GENES EXPRESSED IN A SPECIFIC CELL TYPE BUT DON'T KNOW ANYTHING ABOUT THE OVERALL DISTRIBUTION OF THAT GENE -- CAN SEE THE OVERALL DISTRIBUTION OF THAT BUT NOT SORT OF CELLULAR LEVEL FOR LOCALIZATION. WE WANT TO FOCUS ON CELL TYPES THAT ARE GENETICALLY ACCESSIBLE SO PEOPLE CAN GO BACK AND SAY THESE SUBUNITS ARE IN THE CELL SO THESE ARE THE ONE I NEED TO TEST IF I'M INTERESTED IN THESE PROPERTIES. IT'S POSSIBLE TO PROFILE OTHER SORTS OF CELLS LIKE YOU COULD JUST LABEL INJECTIONS AND SO ON. BUT I THINK THAT'S USABLE AND THEREFORE LESS USEFUL. I THINK A FAIR AMOUNT OF THIS WILL BE USEFUL. >> [INDISCERNIBLE] >> YOU GET IT ALL FOR FREE SO YOU DON'T NEED TO RESTRICT IT. CERTAINLY IN TERMS OF ANALYZING IT YOU'VE GOT TO CHOOSE WHAT TO LOOK AT OR YOU'VE GOT TO DO A STATISTICAL TEST WHAT'S DIFFERENT. I HAD NAIVELY THOUGHT WHEN I ENTERED THIS BUSINESS EVERYBODY'S BEEN EXPRESSING THE POTASSIUM CHANNEL FOR UMPTEEN YEARS, IT'S GOING TO BE TRIVIAL. I KNOW WHAT THEY WILL DO BY VIRTUE OF THE FACT THEY'RE EXPRESSED IN THE CELL TYPE. THAT TURNS OUT TO REALLY NOT BE TRUE. FOR A FEW THINGS YOU CAN SEE CLEAR FUNCTIONAL OBJECTS AND IT'S A LOT BUT IT DOESN'T REPLACE THE HARD BIOLOGY OF KNOCKING THEM OUT AND MODIFYING THEM AND SEEING WHETHER THEY DO OR DON'T. IT JUST SORT OF GREATLY RESTRICTS THE REGION WHICH YOU HAVE TO LOOK. >> [INDISCERNIBLE] >> ABSOLUTELY. I SHOWED YOU AN EXAMPLE WHERE AT THE PROTEIN LEVEL YOU TO DON'T SEE SOMETHING THAT AT THE TRANSCRIPT LEVEL YOU DON'T SEE SOMETHING THAT YOU DO SEE AT THE -- LEVEL. THANK YOU.