>> IT'S A REAL PLEASURE FOR ME TO BE ABLE TO WELCOME DR. NATHANIEL HEINTZ FOR THE SEMINAR SERIES THIS MONDAY. HE'S THE HEAD OF THE LAB OF BIOLOGY AT ROCKEFELLER UNIVERSITY IN NEW YORK AND INVESTIGATOR OF THE HOWARD HUGHES. WENT TO WILLIAMS COLLEGE AND DID HIS PH.D. AT THE UNIVERSITY OF ALBANY AT THE UNIVERSITY OF NEW YORK. FROM THERE HE DID A POST DOC FELLOWSHIP WITH ROB RUTTER AND CONTINUED TO MOVE ON WITH ROB RUTTER TO ROCKEFELLER WHERE HE ESTABLISHED HIS OWN LABORATORY IN MOLECULAR BIOLOGY. OVER THE LAST THREE DECADES HE'S MOVED UP AT ROCKEFELLER WHERE HE'S NOW THE JAMES AND MARYLAND SIMMONS PROFESSOR IN THE LAB OF MICROBIOLOGY. OVER THE YEARS HE'S REALLY EXCELLED IN SEVERAL DISTINCT AREAS. HE MOVED ON FROM SORT OF FUNDAMENTAL MOLECULAR BIOLOGY IN THE LATE 80'S AND THEN BEGAN A VERY DISTINGUISHED STUDY IN NEURONAL DEVELOPMENT AND HOW IT'S AFFECTED VARIES DEVELOPMENTAL DISORDERS. IT'S A PLEASURE TO WELCOME NAT HEINTZ AND COMPLEXITY TOWARDS A CNS PHARMACOLOGY. >> THANKS FOR INVITING ME. I'LL TELL YOU BASICALLY THE BROAD SPAN OF WHAT WE DO, A COUPLE NICE NEW STORIES THAT ARE OVEN PUBLISHED AT THE END. I'VE BEEN INTERESTED SINCE I STUDY THE NEUROSCIENCE TRYING TO UNDERSTAND WHAT THE PROPERTIES OF INDIVIDUAL CELL TYPES ARE. YOU KNOW, IT SHOULD HAVE BEEN, AND IT WAS EVIDENT FROM THESE BEAUTIFUL DRAWINGS A HUNDRED YEARS AGO, THAT NEUROCELL TYPES ARE VERY DIFFERENT. THERE ARE SEVERAL HUNDRED OF THEM AND EVEN THEN THEY KNEW THAT THEY WERE CONNECTED IN THE CIRCUITRY. IT SEEMED TO ME FROM THE BEGINNING OF MY STUDIES IN NEUROSCIENCE THAT WE COULD BE APPROACH THE MOLECULAR EVENT THAT ARE IMPORTANT FOR CNS FUNCTION WITHOUT KNOWING MORE ABOUT THESE CELL TYPES. SO TO DO THAT, WE CLONED THE GENE QUITE A LONG TIME AGO THAT HAD A BEHAVIORAL PHENOTYPE, IT WAS A CHANNEL THAT'S EXPRESSED IN THE PERKINKY CELLS AND THAT GAVE US THE INSIGHT THAT THESE CELLS ARE VERY DIFFERENT FROM ONE ANOTHER. AT THE END OF THE TALK TODAY I'M GOING TO TALK ABOUT A FUNDAMENTAL ASPECT OF CELL BIOLOGY WHICH IS WHY THEIR NUCLEI ARE ABOUT 10 TIMES BIGGER THAN OTHER NEURONAL NUCLEI AND HAVE NO HEADER CHROMATIN. AT ANY CASE IF YOU LOOK AT ANY CNS DISORDER, NOT ANY, ACTUALLY TODAY I GOT A LOT OF INFORMATION ABOUT ONE THAT'S NOT LIKE THIS. BUT LET'S SAY YOU LOOK AT SOMETHING LIKE DEPRESSION AND YOU ASK WHAT BRAIN AREAS ARE INVOLVED. THERE ARE MANY BRAIN AREAS, THEY INTERACT. THE OUTCOME OF DYSFUNCTION IN THIS CIRCUIT IS DECRETION. IN EACH ONE OF THESE STRUCTURES, THERE ARE AT LEAST MANY, SOMETIMES DOZENS OF CELL TYPES THAT ARE IMPACTING LOCALLY. IT SEEMS TO ME WE NEED TO KNOW THE PROPERTY OF ALL THESE DIFFERENT CELL TYPES TO UNDERSTAND HOW THESE DISORDERS WORK. THE FIRST THING YOU NEED FOR THAT IS ACCESS TO THEM GENETICALLY. IN AN ANIMAL YOU CAN STUDY GENETICALLY LIKE MICE. A LONG TIME AGO A STUDENT IN MY LAB INVENTED THIS THING THAT'S NOW CALLED RECOMBINEERING WHICH IS ALL THE PLANKING INFORMATION FOR CORRECT EXPRESSION. SO UNLIKE WHEN YOU TAKE A SMALL PROMOTER AND PUT IT INTO A MOUSE AND IT GETS POSITION EFFECTS AND DOESN'T EXPRESS PROPERLY, IF YOU MANIPULATE THESE CONSTRUCTIONS, PUT IT IN THE MOUSE AND THE GENE AND ALL THE REGULATORY INFORMATION. WHEN YOU DO THAT YOU CAN REPRODUCE GENE EXPRESSION. SO HERE'S A GENE, IT'S CALLED -- IT'S EXPRESSED IN THE HIP CAMPO NEURON, ESSENTIALLY ONLY IN THE CNS. YOU MAKE A MOUSE EXPRESSING GSP IT WILL EXPRESS USING THAT GENE IN CA1 PYRAMIDAL CELLS. YOU NOW HAVE ACCESS TO STUDY THEM IN THE CONTEXT OF THE LIVING ANIMAL. HERE'S ANOTHER CASE WHERE THERE'S A CELL, WE HAVE NO IDEA WHAT ITS PROPERTIES ARE FROM HYBRIDIZATION, IT WAS DONE BY ALAN. ALL WE KNOW IT'S AT THE BASE OF THE CEREBRAL CORTEX. YOU SEE IN THE MOUSE ARE EXCITATORY NEURONS AND IT'S AXONS PROJECT NOT OUT OF THE CEREBRAL CORTEX BUT BACK INTO THE CORTICO. THIS HAS NEVER BEEN STUDIED NOW THERE ARE SEVERAL GROUPS ACTUALLY STUDY YING IT, INCLUDING OURS. SO WHAT YOU GET FROM THIS IS THE ABILITY NOT ONLY TO VISUALIZE ITSELF BUT THEN GO IN AND ANALYZE IT. NOW PEOPLE ASK DOES IT EXPRESS ACCURATELY. IF YOU DO THIS PROPERLY IN ABOUT 90% OF THE CASES YOU CAN COMPLETELY REPRODUCE THE ENDOGENOUS EXPRESSION USING BACTERIAL ARTIFICIAL CHROMOSOME. IF THE GENE IS LESS THAN ABOUT 200 KILO BASIS. IT'S GAINING GENETIC ACCESS TO INDIVIDUAL CELL TYPES. BECAUSE OF THAT, ABOUT A DECADE 12 OR 13 YEARS AGO IT WAS APPLIED VERY BROADLY AND TRIED TO GET EXPRESSION INFORMATION BUT ALSO FOR ME, THE FACTORS THAT WOULD ALLOW ME TO STUDY ANY CELL TYPE I'M INTERESTED IN. SO LARGE PROJECT WOULD SET UP. IT'S BEEN A REALLY ENJOYING COLLABORATION IN THE LAST, I DON'T KNOW, EIGHT OR SEVEN OR EIGHT YEARS WITH DR. LAURA HERE WHO IS THE PROJECT OFFICER. -- JOINED THE TEAM IN THE LAST FIVE YEARS TO HELP US WITH THE MICE. IT'S A LARGE PROJECT THAT WAS SET UP TO TRY TO MAKE MICE FOR ALL CELL TYPES IN THE CNS. SO IT'S DIFFERENT FOR ME. I RAN A SEPARATE LAB 20 PEOPLE ALL DOING THE SAME THING ALL THE TIME WE MADE 14,000 LINES. ABOUT 1500 OF THEM WERE BEAUTIFUL AND VERY SPECIFIC. SO WE'RE MAKING SEVEN OR EIGHT IN MANY CASES FOR EACH GENE. PICKED THE BEST ONE, PUT IT ON-LINE. AND WHAT YOU GET IS THE ABILITY TO TRANSFORM THESE BEAUTIFUL DIAGRAMS INTO MICE THAT HAVE FACTORS ASSOCIATED WITH THEM. SO IF YOU WANT SOMETHING LIKE A -- CELL YOU CAN GO IN AND DO A GENETIC MANIPULATION IN THAT CELL TYPE. NOT ALL CELL TYPES ARE DESCRIBED IN THE CNS OF A MAMMAL SO YOU GET NOVEL INFORMATION ABOUT ACCESSING CELL TYPES IN LET'S SAY THE CEREBRAL CORTEX OR REGIONALLY EXPRESSED GENE CORTEX. IN SOME CASES, REALLY VERY BEAUTIFUL AND SPECIFIC EXPRESSION IN TINY DOMAINS OF MOUSE CORTEX. IN THIS CASE, I THINK IT WOULD BE VERY INTERESTING WHEN WE CHARACTERIZE THESE CELLS TO SEE IF THIS DOMAIN ACTUALLY EXPANDS AS YOU GO UP THE EVOLUTIONARY LADDERS. AND THIS IS THE ANTECEDENT TO SOME FUNCTIONAL DOMAIN THAT HAS MUCH MORE IMPORTANCE LET'S SAY IN PRIMATES. NOW, EVEN FOR WELL-KNOWN CELL TYPES, LIKE CORTICO SPINAL TRACK NEURONS THAT STARTED OUT IN CORTEX PROJECT ALL THE WAY DOWN TO THE SPINAL CORD. IF YOU DO THIS GENETIC TARGETING, INSTEAD OF TRACING SPINAL CORD BACK, WHAT YOU DO IS IMMEDIATELY SUBDIVIDE THESE CELLS INTO FIVE OR SIX OR SEVEN DIFFERENT CELL POPULATIONS THAT ARE PRESENT ALL IN LAYER FIVE BUT IN DIFFERENT REGIONS OF LIAR FIVE, THE CELL BODIES ARE DIFFERENT AND PROJECT DIFFERENT SIZE OF THE SPINAL CORD. WE DON'T KNOW WHAT THE DETAILED FUNCTIONS OF EACH OF THESE CELL TYPES ARE IS BUT WE CAN NOW STUDY THEM HAVING THESE FACTORS IN HAND. HERE'S ANOTHER CASE, YOU KNOW. THIS LAYER SIX CORTICO NEURONS. I THOUGHT WHEN I STARTED JUST OUT OF IGNORANCE THAT LAYER SIX CORTICO THALAMIC. IT PROJECTS ALL OF THE THALAMUS EXCEPT FOR THE -- NUCLEUS AND THIS ONE WHICH IS IN THE POSTERIOR ASPECT FILLS IN THAT SPOT. IT ACTUALLY TURNS OUT IF YOU LOOK IN DETAIL, THESE TWO TOGETHER DON'T LABEL THE WHOLE POPULATION. THERE'S ABOUT ANOTHER FOUR DRIVERS THAT PROJECT SPECIFICALLY TO THALAMUS. SO THIS OPENS UP INTERESTING EXPERIMENTAL OPPORTUNITIES FOR STUDYING THE CORTICO THALAMIC FEEDBACK GROUP AND WHY THESE SPECIFIC CELL TYPE POPULATIONS ARE DIFFERENT. OKAY. ONE FINAL THING BECAUSE THEY'RE SORT OF THE BACKBONE OF EVERYTHING THAT WE DO. WE HAVE A PROBLEM IN MAMMALS STUDYING INTERSECTIONAL INTERSECTIONAL INTERSECTIONAL-ALLELES. YOU NEED DIFFERENT ALLELES IN ORDER TO DO THAT. THAT'S VERY HARD IF YOU'RE TARGETING THE GENOME. BY THE TIME YOU GET TO THREE ALLELES, IT'S ONE IN 60, FOUR ALLELES IS ONE IN 32 ANIMALS, YOU USE THEM ONLY BASICALLY YOU WILL BREAK THE BUDGET OF ANY LAB. SO WHEN WE DO MULTIPLE STUFF, WE INJECT MULTIPLY MODIFIED ACTS IS HE SAME TIME AND ACT AS A SINGLE ALLELE. BECAUSE THEY CARRY THE REGULATION, IN THIS EXAMPLE, YOU CAN LABEL -- I THINK THIS IS IMPORTANT BECAUSE WE FACE THIS PROBLEM OF MULTIPLE ALLELES ALL THE TIME IN THE MILLION GENETIC RESEARCH. AND THIS IS A VERY USUL LITTLE DETAIL THAT PEOPLE DON'T USUALLY APPRECIATE. OKAY. LAST THING IS THEY ARE REPRODUCIBLY EXPRESSED. IF YOU MAKE FIVE LINES FOR TWO DIFFERENT ANIMALS, THAT'S SAY GFP AND CREE, ONE OR TWO OF EACH OF THOSE SETS OF ANIMALS WILL EXPRESS ACCURATELY. THIS IS NOT PERFECT. WHEN IT GOES INTO SOME REGIONS OF THE GENOME, IT WON'T BE EXPRESSED ACCURATELY. BUT IF YOU DO THIS RIGHT IN A SINGLE DAY OF INJECTION, YOU WILL GET ANIMALS THAT REPRODUCIBLY EXPRESS IN THE RIGHT CELL TYPE. AND BECAUSE OF THAT -- TURNED TOWARDS MAKING IS -- MICE IN IN THE LAST THREE YEARS THIS IS A COLLABORATION WITH CHIP'S LAB HERE. THE ADVISORY COMMITTEE HAD A BIG ROLE IN THIS AT NIH BECAUSE SELECTING WHICH LINES TO ACTUALLY KEEP AND ARCHIVE REQUIRES A LOT OF IN-DEPTH ANALYSIS. AND LAURA AT ROCKEFELLER WHERE THEY KEEP PEOPLE HERE. WE MADE THESE LINES, AND BASICALLY THEY TURNED REPORTER MICE INTO THESE BEAUTIFUL CRE LINES. IF YOU WANT TO DO CHANNEL -- OR SOME CELLS WITH SPECIFIC GENETIC MANIPULATION OR INTERSECTIONAL STUFF BY INJECTING INTO THESE CRE LINES. YOU CAN FIND THEM ON THE WEBSITE OR ON THE INTERNAL WEBSITE HERE. THERE'S A FEW HUNDRED UP NOW THAT I THINK ARE OF VERY WIDE INTEREST OF CNS COMMUNITY BECAUSE THEY ARE BEING USED BY HUNDREDS OF LABS ALREADY. WHAT DO WE GET. WE GET REAGENT, WE GET MICE AND INSIGHT INTO HOW MANY CELL TYPES THERE ARE IN CNS. IN CORTEX FOR EXAMPLE IN MOUSE CORTEX, HAVING DONE THIS, THERE ARE AT LEAST 70 DIFFERENT PYRAMIDAL CELL POPULATIONS. I THINK MOST PEOPLE WOULD ARGUE SOMEWHERE BETWEEN 20 AND 30 OR SORE INTERNEURON POPULATION, A FEW DIFFERENT KINDS OF GLIA AND AT LEAST TWO AL GORE DENDRITE POPULATION. THESE ARE GOING TO BE RELEVANT TO THAT QUESTION. PEOPLE SAY WHAT DO YOU MEAN BY A CELL TYPE. WHAT I MEAN IS OPERATIONALLY DENINED. SO WE TAKE THE FACTOR, WE CAN REPRODUCIBLY HIT THIS LAYER OF PYRAMIDAL POPULATION. THIS PROJECTS TO THE -- THALAMUS. IF YOU DO IT TWICE AND PICK GOOD ANIMALS, THEY LOOK THE SAME WAY, THEY EXPRESS THE SAME WAY, IN FACT THEY HAVE EXACTLY THE SAME GENE EXPRESSION PATTERNS IN THESE NEURONS. IF YOU DO THAT FOR DIFFERENT POPULATIONS, AND THEN GO IN AND STUDY THEM IN DETAIL, SO YOU DO THE ANATOMY ACROSS CORTEX. THIS WAS DONE WITH PATRICK CRAIGER AND BERT. WHAT THEY FOUND OUT IS THAT ALTHOUGH THERE WERE SLIGHT DIFFERENCES IN THESE TWO POPULATIONS IN A SINGLE POPULATION AS YOU WENT THROUGH DIFFERENT AREAS OF CORTEX. THEIR BASIC PROPERTIES WERE THE SAME. AND YET THEY WERE DIFFERENT BUT THE CORTICO STRIDAL CELLS WERE DIFFERENT. SO I THINK OF THIS AS A CELL TYPE BECAUSE IT IS RELATIVELY UNIFORM. OF COURSE IF IT'S GETTING DIFFERENT INFORMATION AND VISUAL CORTEX VERSUS SOME OTHER PIECES OF CORTEX IT MAY EXPRESS SLIGHTLY DIFFERENT GENES AT THAT TIME BUT THE GROUND STATE OF THIS CELL AND ITS FUNCTION IN THESE CIRCUITS I WOULD SAY IS SIMILAR ACROSS CORTEX. SO THAT'S WHAT I MEAN BY THALAMUS. NOW FOR ME, ALL THIS IS A PRELUDE TO TRY TO UNDERSTAND WHAT THE BIO CHEMISTRY OF INDIVIDUAL CELL TYPES IS AND HOW THAT'S ALTERED IN THE GENES. SO WHAT WEED TO DO IS THE ABILITY TO LOOK AT WHAT PROTEINS ARE BEING MADE IN THE CELL UNDER ANY CONDITION. WE TRY TO DO THIS BY FACT SORTING THE CELLS. GOT A LOT OF DATA. PROBABLY COULD HAVE PUBLISHED A LOT OF IT. I BASICALLY DIDN'T BELIEVE IT BECAUSE ONCE THE CELLS ARE TAKEN OUT OF THE TISSUE, THEIR AXONS AND DENDRITES PULL OFF FIVE OR SIX HOURS SINCE THEY'VE BEEN RESIDENT IN THE TISSUE. YOU DO GET THE MAJOR MARKERS. BUT IF I HAD TWO POST DOCKS DOING THE SAME EXPERIENCE THEIR PROFILES WOULD BE DIFFERENT. SO I SAID OKAY, THIS IS NOT SOMETHING THAT I'M GOING TO TO DO AND THOUGHT OF A METHOD WITHOUT ISOLATING CELLS. IT'S BASED ON A REALLY SIMPLE PRINCIPLE. SO ALL MESSENGER RNAs AND ALL PROTEINS ARE DECODED BY RIBOSOMES. SO IF YOU WERE ABLE TO ISOLATE A RIBOSOME FROM A SPECIFIC CELL YOU WOULD ISOLATE THE ASSOCIATED MRAs WITHOUT ISOLATING THE CELLS. THIS IS CALLED TRANSLATIONAL PROFILING. IT WAS DEVELOPED IN COLLABORATION WITH PAUL GREENGUARD'S LAB. MIRIAM REALLY WAS THE LEAD PERSON. SHE'S AT MIT NOW. HE'S A TERRIFIC YOUNG SCIENTIST. SO THE IDEA IS SIMPLE. YOU EXPRESS A FUSION PROTEIN TO ARRIVE AT THE CHROMOSOMAL PROTEIN. IT TOOK US A LONG TIME TO FIND IT. THEN EXPRESS IT WITH A SPECIFIC CELL TYPE. INSTEAD OF ISOLATING THE CELLS TO JUST GRIND UP THE BRAIN AND ISOLATE THE POLYSOMES. ALL OF THEM ARE GFP TAGGED. SO THE GFP TAG AND ALL THE OTHER CELLS IN CORTEX ARE NOT STAGGED. INSTEAD OF ISOLATING THE CELL YOU TAKE THE ANIMAL, SACRIFICE IT, GRIND UP THE CORTEX. TEN MINUTES LATER, YOU CAN IAP THE POLYSOMES THAT USED TO BE HERE FROM ALL THE OTHER ONES THAT ARE NOT PEGGED. THAT'S THE IDEA. ACTUALLY IT WORKS PHENOMENALLY WELL. THIS IS USED FOR IGPF FROM A REGULAR REPORTER LINE. THIS IS A BEAD FROM A TRAP LINE. YOU CAN SEE ON THE SURFACE OF THE BEAD THERE'S NOTHING BECAUSE GFP IS SMALL PROTEIN. THE SURFACE OF THIS BEAD IS ALL THIS STUFF AND IT'S A TEXTBOOK CASE OF A POLYSOME. SO THE REASON THIS WORKS IS THAT UNLIKE LET'S SAY A NORMAL RNA BINDING PROTEIN, THE RIBOSOME IS A MACHINE. YOU CAN FREEZE IT IN PLACE FOR HOURS AND HOURS AND HOURS WITH CYCLE HEXMEAN. THIS DOESN'T FALL APART AND REDISTRIBUTE BETWEEN YOUR CELL AND THE OTHER LET'S SAY THOUSAND MORE ABUNDANT CELL TYPES THAT ARE PRESENT IN THE MIXTURE. OKAY. SO THIS IS USEFUL. WE WANT TO KNOW WHAT THE PROPERTIES OF THEY CELLS ARE. WHAT WE'VE DONE IS MAKE A LOT OF THESE MICE. SO WE MADE ABOUT A HUNDRED REALLY WELL-CHARACTERIZED LINES. PROCESSED IT. YOU TAKE IT BACK, USUALLY EITHER FROM THE LITERATURE OR MOST OFTEN FROM LET'S SAY THE GEN SET DATA BASE. A FUSION PROTEIN TO MAKE MICE. DO THE ANATOMY SO THAT YOU KNOW THAT IT'S EXPRESSING LET'S SAY IN THE RIGHT STRUCTURE. VISUALIZE IT AND YOU CAN TELL IF THE FUSION IS WORKING BECAUSE RIBOSOMES ARE ASSEMBLED IN THE NUCLEOLUS AND KNOW IT'S BEING ASSEMBLED. YOU IT THE SPECIFIC RNAs AND PROFILE GENE EXPRESSION. NOW YOU CAN DO THIS OBVIOUSLY WITH ANY DRIVER YOU WANT SO IF YOU WANT TO HIT THE RETICULAR NUCLEUS OF THE THALAMUS, YOU PICK THIS DRIVER AND HIT THE RETICULAR NUCLEUS OF THE THALAMUS. BECAUSE IT'S GFP YOU CAN'T RECORD FROM THE CELLS BUT YOU CAN DOUBLE STAIN, FOR EXAMPLE, IF YOU WANT TO KNOW IF THESE ARE -- CELLS, YOU CAN LABEL THEM WITH SEROTONIN TRANSPORTER. THE ANATOMY'S CRITICAL. OBVIOUSLY IF YOU DON'T TARGET THE RIGHT CELL TYPE YOU WON'T GET ROBUST INFORMATION AND YOUR CONCLUSIONS WILL BE WRONG. BUT IT'S VERY EASY TO DO THE ANATOMY WITH THE GFP TAG. SO WHAT HAVE YOU GOT FROM THIS? WELL WE'VE DONE A LOT OF IT AND I'M GOING TO SHOW YOU ONE OF THE EARLY SLIDES FROM ONE OF THE ORIGINAL PAPERS THAT DONE BY -- IN THE LAB. THEY PROFILED A LARGE NUMBER OF DIFFERENT CELL TYPES. AND THEN THEY PROFILED PIECES OF THE BRAIN. STRIETAL, COURT TEST, ETCETERA. IT WAS REVEALING. ALL OF PIECES OF THE BRAIN CLUSTERED TOGETHER. THAT'S BECAUSE IN MOST PLACES IN THE BRAIN ABOUT 50% OF THE CELLS ARE GLIAL CELLS AND DEPENDING ON WHAT PIECE OF TISSUE YOU'VE GOT, YOU'VE EITHER GOT HUNDREDS OF DIFFERENT CELL TYPES OR AT LEAST A DOZEN OR SO. SO ALL OF THAT INFORMATION THAT'S BEEN COLLECTED FOR YEARS AND YEARS FROM PIECES OF THE BRAIN, IT IS USEFUL BUT IT IS INFORMATION. WHEN YOU DO INDIVIDUAL CELL TYPES, WHAT YOU FIND OUT IS THEY CLUSTER NEXT TO EACH OTHER BUT THEY'RE QUITE DISTINCT FROM ONE ANOTHER. THE ONLY PIECE OF BRAIN THAT CLUSTERS WITH A CELL TYPE IN IT IS CEREBELLUM BECAUSE CEREBELLUM IS ABOUT 70% CRANIAL CELLS. THE SECOND THING WE LEARNED IS IN A COUPLE CASES IN THE STRIATUM -- AND THEN WE COMPARED THEM WITH DATA FROM THE STRIATUM AND CEREBELLUM. IT'S ASTOUNDING. WHEN YOU PROFILE WHOLE PIECES OF TISSUE YOU'RE MISSING ABOUT ON 40% OF THE TRANSCRIPTS IN THAT ISSUE BECAUSE THEY ARE CELL TYPES THAT ARE RELATIVELY RARE. NOT ONLY ARE YOU MISSING THOSE TRAN TRANSCRIPTS BUT THOSE ARE THE ONCE MOST INTERESTING, THEY GIVE TO ME ANY THE CELL ITS BASIC CHARACTER. SO THIS IS A BIG ADVANCE IN TRYING TO UNDERSTAND WHAT THE PROPERTIES OF INDIVIDUAL CELL TYPES ARE. IF YOU COMPARE THEM YOU GET MODULES THAT ARE SELF SPECIFIC, ETCETERA, ETCETERA. THIS IS WHAT WE WOULD EXPECT IS THAT EACH NEURONAL CLASS HAS DIFFERENT POPULATIONS OF MOLECULES IN IT AND THEY DEFINE ITS DIFFERENTIATED STATE. PEOPLE ARE INTERESTED IN HOW SENSITIVE THIS IS. IF YOU'RE WONDERING ABOUT THAT, WE FOUND A LOT OF EXPERIMENTS ON DISBURSE NUCLEI. FOR EXAMPLE THIS CONTROLS SLEEP. THEY HAVE ONLY A COUPLE THOUSAND CELLS IN THEM. I'M GOING TO SHOW YOU THE EXAMPLE FROM HCRT OR THE -- CELLS. THEY MADE A TRAP LINE ONLY EXPRESSED IN THE HYPO THALAMUS IN THE CORRECT CELL POPULATION. THAT'S ABOUT 2,000 OF THESE CELLS IN MOUSE. WE'RE TRYING TO LOOK AT A CELL POPULATION THAT'S A FEW THOUSAND CELLS OUT OF HUNDREDS OFIANS NATURAL ACTUALLY DISSECTING FROM THE ANIMAL. SO THE QUESTION IS CAN YOU SEE THESE GUYS AND WHAT ARE THEIR PROPERTIES. SO WE DID THE ENRICHED TRANSCRIPT AND THE IP, THESE ARE THE ONES NOT ENRICHED SO THE REST OF THE TISSUE. SO HYPO CREEDEN IS ENRICHED 36 FOLD IN THE IP. THIS FOR ME WAS REALLY AMAZING FOR TWO REASONS. ONE IS HYPO CRETIN WAS THE ONLY KNOWN MARKER AT THE TIME WE DID THIS SO WE GOT ANOTHER FEW HUNDRED GENE PRODUCTS THAT GIVE THESE CELLS PARTICULAR CHARACTERISTICS. WE'RE NOT DOING WITH THREE FOLD DIFFERENCES IN EXPRESSION. WE'RE SEEING ENRICHMENT OF 10,20 FOLD. SO THE STATISTICAL ANALYSIS IS MUCH EASIER FOR THIS ROBUST DATA. NOW THIS CAN BE USED TO IDENTIFY NEW MOLECULES AS TARGETS FOR DRUG DEVELOPMENT. WE STARTED A COMPANY WHICH I'M A CONSULTANT FOR, CALLED ON-WAY THERAPEUTICS -- WHO WAS ON -- CEO. WHAT THEY'VE DONE IN CONTRAST TO EXISTING DRUG TARGETS. SO HERE'S AN EXISTING DRUG TARGET. I'M NOT GOING TO MA LINE IT BY TELLING YOU WHICH ONE IT IS. BUT YOU LOOK AT RELATIVELY LOW LEVELS IN MANY CELL TYPES. THE ONES WE'RE INTERESTED IN ARE LIKE THIS. THE ONES THAT ARE EXPRESSED AT EXTREMELY HIGH LEVELS IN CELL TYPES THAT ARE VERY PARTICULAR FOR THIS CIRCUITRY THAT IS IMPORTANT IN THE DISORDER, AND SO WHAT WE'VE DONE IS PICKED THOSE AND ACTUALLY GO FORWARD IN DRUG DEVELOPMENT. IF YOU START WITH NON-SPECIFIC TARGETS, YOU'RE GOING TO HAVE A LOT OF OFF LINE NON-SPECIFIC EFFECTS. THE LAST TWO THINGS I THINK YOU NEED, YOU'VE PROBABLY HEARD A LOT OF ALREADY SO I'M NOT GOING TO TALK ABOUT THEM TODAY. METHODS FOR MODULATING NEURONAL ACTIVITY TO MAP CIRCUITS. AND I THINK WE NEED SOMETHING THAT WE DO WORK A LOT ON BUT HAVE HAD NO SUCCESS WITH SO FAR IS CELL TYPE SPECIFIC VIRAL FACTORS SO THAT WE CAN CROSS SPECIES BARRIERS BECAUSE RIGHT NOW WE'RE STUCK IN MICE WITH ALL THIS FANCY METHODOLOGY. AND I WOULD LIKE TO BE ABLE FOR EXAMPLE TO ASK EVEN A VERY SIMPLE QUESTION LIKE IS THE PERKIN -- HOW DOES THAT LOOK FOR DIFFERENT CELL TYPES WHEN YOU GO ACROSS SPECIES. I SUSPECT THEY WILL BE VERY VERY SIMILAR BU NO ONE'S ACTUALLY EVER PROVEN THAT. SO I WOULD LIKE TO DO THAT. FLOWER -- I'M GOING TO TURN NOW TO WHAT I THINK THIS IS THE CORE OF WHAT MY LAB DOES. TRY TO UNDERSTAND ON A MOLECULAR LEVEL WHAT OCCURS EITHER IN DEVELOP OR IN NEUROLOGIC OR PSYCHIATRIC DISORDERS. I WOULD FRAME THREE QUESTIONS I THINK ARE POWER PRESENTLY. THE FIRST ONE IS A GENETIC PARADOX. WHY DO MUTATIONS IN VERY BROADLY EXPRESSED GENES CAUSE SPECIFIC PHENOTYPES. IN SOME CASES AND I GOT CRAIG GAVE ME A REAL IN-DEPTH LESSON ABOUT THIS. IN SOME CASES THEY ACTUALLY ACT IN SPECIFIC CELL TYPES. IN OTHER CASES, THEY ACT EVERYWHERE BUT THE LIMITING FOR A PARTICULAR CELL TYPE AND THAT'S WHAT GENERATES THE PHENOTYPE. WHY DO DRUGS THAT RAISE NEUROTRANSMITTERS EVERYWHERE HAVE SUCH A SPECIFIC THERAPEUTIC EFFECT. THE THIRD IS WHY THERE'S SOMETHING LIKE FEVER. WHY IS IT GENERAL PHYSIOLOGIC RESPONSE LIKE THAT ABLE TO MODULATE A COMPLEX BEHAVIORAL PHENOTYPE LIKE AUTISM. SO I THINK THAT THIS IS DUE TO THE FACT THAT THESE CELLS ARE BIO CHEMICALLY DIFFERENT. EVERY CIRCUIT IS DIFFERENT WITH RESPECT TO THE DETAILS OF THE CELL TYPES IN IT. AND SO WHEN YOU MAKE -- THEY ACT ON SPECIFIC CELL TYPES DIFFERENTLY. THAT'S A HYPOTHESES AND WE'RE TRYING TO INVESTIGATE THAT. WE'RE DOING IT IN MANY DIFFERENT TYPES OF STUDIES, BOTH OF PHARMACOLOGY AND GENETIC MODELS OF DISEASE. I WOULD SAY THAT THE MAIN CONCLUSIONS ARE IF THE CELL TYPE IS IMPORTANT IN THE RESPONSE AT A MOLECULAR LEVEL IT WILL BE IMPACTED VERY STRONGLY. THIS IS TRUE FOR GENETIC DISORDERS. BUT IT'S ALSO TRUE FOR DRUG TREATMENTS. AND IT'S NOT INVOLVED IN THE CIRCUITRY UNDERLYING THAT DISORDER, IT WILL HAVE ALMOST NO EFFECT WHEN YOU LOOK AT THE MUTATION ON THE BACK. I'M GOING TO SUPPORT THAT WITH OUR STUDIES OF ANTI-DEPRESSANT TREATMENTS. SO FOR THOSE OF YOU WHO AREN'T FAMILIAR WITH THIS, A LOT OF NEURO IMAGING STUDIES HAVE BEEN DONE ON DEPRESSION. I WOULDN'T BE ABLE TO EVALUATE THESE ON MY OWN BUT FROM THE REVIEWS THAT HAVE BEEN WRITTEN FROM THE FRONTAL CORTEX IS ALTERED IN DEPRESSED INDIVIDUALS. AND THAT TREATMENTS THAT ARE EFFECTIVE NORMALIZE THIS ACTIVITY. SO WE THOUGHT OKAY, LET'S SEE WHETHER THERE ARE CELL TYPES IN CEREBRAL CORTEX THAT ARE GOING TO BE IMPORTANT AND THAT ARE MODULATED BY ANTI-DEPRESSANT TREATMENT. SO TO DO THAT, WE NEEDED A MOUSE MODEL OF DEPRESSION. PAUL'S LAB CALLED GREENGARD'S LABORATORY A CLOSE FRIEND AND COLLABORATOR OF MINE HAS MADE A NICE DISCOVERY. THERE'S A SMALL PROTEIN THAT CHAPERONES SEROTONIN RECEPTORS AND THEY HAVE DEPRESSIVE LIKE BEHAVIORS. THAT'S BASICALLY THE BOTTOM LINE FOR THESE MICE. WHAT WE NOTICED IS THAT THAT PROTEIN IS EXPRESSED IN CEREBRAL CORTEX IN A LAMINAR WAY IN LAYER FIVE. SO THE QUESTION IS WHAT ARE THESE CELLS AND DO THEY CARRY THE INFORMATION FROM CEREBRAL CORTEX TO SUBCORTICO SITES LIKE STRIATUM THAT HAVE BEEN IMPLICATED IN HUMAN DEPRESSIVE DISORDERS. SO ERIK SCHMIDT IS A TERRIFIC POST DOC IN THE LAB. HE MAY TRAP MICE WITH THIS P11 -- IT'S ACTUALLY THE GENE IS CALLED S100A10. IT'S ALMOST 100% CORRESPONDENCE WITH P11 ENDOGENOUS PROTEIN IN THE LABELED CELLS. WE CAN ASK WHAT THESE CELLS ARE. HE LOOKED ALL THROUGH THE ORGANISM AND BASICALLY EVERYWHERE WHERE P11 IS EXPRESSED THIS REPRODUCES THE EXPRESSION. WE HAVE A GOOD ANIMAL ON OUR HANDS. WHAT ERIK IS INCORRECT TOXINS INTO DIFFERENT STRUCTURES AND ASK WHERE DO THESE CELLS PROJECT. BASICALLY WHAT HE WAS ABLE TO SHOW IS THAT THEY PROJECT TO THE DORSAL STRIATUM BUT NOT VENTRAL STRUCTURES IN THE STRIATUM AND THEY PROJECT -- 50 TO 70% OF THE CELLS ARE LABELED. THEY ARE LABELED IN THE CORTEX. IF YOU DO CONTRA LATERAL CORTEX YOU GET A NICE LABEL ALSO. SO THESE ARE CORTICO STRIATAL NEURONS FROM THE CORTEX TO THE DORSAL STRIATUM. NOW HE CHOSE AS A CONTROL POPULATION TO STUDY THESE CELLS WHICH ARE ALSO PRESENT IN LINEAR FIVE. THEY ARE DEEPER IN LAYER FIVE AND THEY PROJECT TO RESPOND ON FROM THE THALAMUS. THE HYPOTHESIS WAS PERHAPS ANTI-DEPRESSANTS ARE AFFECTING THESE CELL TYPES AND NOT THESE. I FORGOT TO TELL YOU THE KNOCK OUT HAS BOTH DEPRESSIVE-LIKE BEHAVIORS AND CANNOT RESPOND TO ANTI- DEPRESSANTS. SO WE'RE INTERESTED IN THAT. OKAY. SO THE FIRST THING IS WHEN YOU PROFILE THESE TWO PYRAMIDAL CELLS THEY ARE DIFFERENT FROM EACH OTHER. THEY ARE DIFFERENTIALLY EXPRESSING ABOUT 250 DIFFERENT GENE PRODUCTS, SOME OF WHICH WERE KNOWN TO BE SPECIFIC TO THE CORTICO STRIATAL POPULATION AND SOME OF THEM WEREN'T. SO VERY DIFFERENT CELL TYPES AT A MOLECULAR LEVEL. IF YOU PROFILE THEM IN RESPONSE TO CHRONIC ANTIDEPRESSANTS. HERE ARE THE CORTICO STRIATAL ONES. WHAT YOU CAN SEE IS FIRST OF ALL YOU DO SEE THE CHANGE IN P11 THAT PAUL REPORTED FROM INSIDE THE HYBRIDIZATION STUDIES. BUT YOU ALSO SEE THERE ARE ABOUT 70 OR 80 GENE PRODUCTS THAT ARE ALTERED EITHER UP OR DOWN IN THIS CORTICO STRIATAL POPULATION THAT ARE NOT CHANGED AT ALL IN THE CORTICO -- CELLS. SO THE FIRST CONCLUSION IS THAT SOME CELL TYPES DO RESPOND TO ANTI DEPRESSANT TREATMENT AND SOME DON'T. ACTUALLY WE'VE DONE THIS NOW IN A FEW DIFFERENT CELL POPULATIONS. THIS IS THE ONLY ONE THAT RESPONDS ROBUSTLY TO CHRONIC ANTI DEPRESSANT TREATMENT. OKAY. AND IF YOU PROFILE SEROTONIN RECEPTORS YOU GET A REALLY INTERESTING RESULTS. THESE CELLS EXPRESS MULTIPLE RECEPTORS BUT WHEN YOU DO CHRONIC ANTI-DEPRESSANT TREATMENT ONLY HTR4 IS REGULATED. IT'S NOT ONLY REGULATED IN OTHER CELL TYPES OR IN WHOLE CORTEX. SO THIS IS A POTENTIALLY INTERESTING FINDING. BECAUSE YOU'RE TREATING WITH ANTI-DEPRESSANT AND THE INCREASE OF SEROTONIN. IT'S ACTING ALL THROUGHOUT THE NERVOUS SYSTEM. WHAT DOES THIS DO, IT INCREASES HGR4 MAKING IT MORE SENSITIVE TO THE SEROTONIN SSRIs. OKAY. WHAT HAPPENS HERE'S IF YOU KNOCK OUT P11. THE SMALL PROTEIN THAT'S CAUSING THE DEPRESSIVE-LIKE BEHAVIORS. AND THE RESPONSE TO ANTI- DEPRESSANTS. THESE ARE IN OTHER STRUCTURES. WE DID A CORTEX-SPECIFIC KNOCK OUT AT 911. YOU LOSE COMPLETELY THE RESPONSE TO CHRONIC SSRIs AND YOU ALSO LOSE THE BEHAVIORAL RESPONSES. SO FROM THESE STUDIES WE KNOW AT IS LEAST IN THIS MOUSE MODEL HOW FAR DEPRESSION OR DEPRESSION-LIKE STAGE, THAT WHEN YOU, THE ENTIRE RESPONSE TO ANTIDEPRESSANTS IS DUE TO THEIR ACTIONS ON THIS ONE CORTICO STRIATAL CELL POPULATION. THAT'S NOT, I THINK IT'S SURPRISING BUT IT'S A REASON WE DID THE STUDY. AND POTENTIALLY IT COULD EXPLAIN WHY ON NORMALIZATION OF THE CIRCUITRY BETWEEN CORTEX AND SUBCORTICO SITES OCCURS PARTICULARLY STRIATUM OCCURS ON ANTI-DEPRESSANT TREATMENT. THE SECOND THING THAT IS IMPORTANT WITH RESPONSE TO THIS, WITH RESPECT TO THIS IS PAUL'S LAB HAS DONE ANOTHER STUDY STUDYING THE NUCLEUS INCUMBENTS. AND WHAT JENNIFER WARNER FOUND IN HER STUDIES IS THAT IF YOU LOOK AT COLONURGIC CELLS AND MANIPULATE ONLY THEM, THE BEHAVIORS THAT ARE DEPRESSIVE ARE GENERATED THERE BUT NOT THE RESPONSE TO THE ANTIDEPRESSANTS. SO THIS IS VERY INTERESTING. THE ACTUAL PHENOTYPE OF THE MICE IS JEAN RATED BY MISFUNCTION. THAT'S WHAT PAUL'S LAB SHOWS. BUT THE TREATMENT RESPONSE AS THE CORTEX TO THE SPECIFIC POP LITTLE. THIS IS REMINISCENT OF SOMETHING LIKE DEEP BRAIN STIMULATION WHERE YOU CAN GO SOMEWHERE ELSE IN THE CIRCUIT TO COMPENSATE FOR A PROBLEM THAT'S OCCURRING IN A PARTICULAR CELL TYPE. I THINK THE SECOND THING IS IMPORTANT HERE IS THAT TRAP STUDIES GOES BEYOND SAYING THIS CELL TYPE IS IMPLICATED IN THIS DISORDER. IT GIVES YOU AN INSIGHT INTO A MECHANISM THAT MIGHT BE RELEVANT EITHER FOR TREATMENT OR FOR THE DISORDER ITSELF. AND BASED ON THAT, FOR EXAMPLE, I THINK HTR4 REGULATION MIGHT BE CRITICAL FOR ANTI DEPRESSANT RESPONSES. AND HDR4 AGONISTS ARE RAPID ACTING ANTI-DEPRESSANTS BUT ACR4 IS REPLACING MANY PATHOLOGIC. IF WE COULD SOMEHOW STIMULATE THESE CELLS THROUGH A RECEPTOR THAT IS PRESENT IN THEM, I THINK IT WOULD BE AN INTERESTING POSSIBILITY FOR DEVELOPMENT. SO THIS IS THE MODEL. YOU TREAT WITH SSRIs. YOU INCREASE SEROTONIN. THAT INCREASES EXPRESSION OF A NUMBER OF GENE PRODUCTS INCLUDING HTR4 BECAUSE YOU ENHANCE SENSITIVITY TO THE SSRI AND NORMALIZATION OF A CORTICO SUBCORTICO CIRCUIT. WE DON'T KNOW WHETHER THIS IS TRUE IN OTHER CASES, WHETHER IT'S A GENERAL FINDING. WHETHER IT'S RELEVANT TO HUMAN DEPRESSION. WE DON'T KNOW ANY OF THAT STUFF. BUT IT'S A PLACE TO START AND I THINK IT'S AN INTERESTING PLACE TO START. SO AS I SAID, THIS WAS DONE BY ERIK SCHMIDT AND JENNIFER WARNER SCHMIDT IN PAUL'S LAB DID THE BEHAVIORAL PHENOTYPE BEING FOR THE GENERATION OF THE PHENOTYPE. NOW IN THE LAST FEW MINUTES, THE LAST SECTION HERE, I WANT TO RETURN TO THE PROPERTIES OF INDIVIDUAL CELL TYPES. AND I'VE BEEN STUDYING PERKINKY CELLS FOR A LONG TIME. ONE OF THE INTERESTING THINGS ABOUT THEM, IS THIS. IF YOU LOOK AT THE EM HAS -- DID IN THE 50'S. WHAT YOU SEE IS NEURONAL NUCLEI DIFFERENCES. PURKINJE ARE HUGE AROUND THE NUCLEOLUS. WHEREAS IF YOU LOOK AT SOMETHING LIKE A GRANULAR CELL THEY'RE LIKE MOST CELLS IN CULTURE. THIS IS WHAT THE PURKINJE CELLS, WHAT A CELL NUCLEUS I WORKED ON FOR MANY YEARS LOOKS LIKE. THE QUESTION IS WHY ARE THESE BIG AND WHAT IS THE FUNCTION OF HAVING SUCH A LARGE CHROMATIC NUCLEUS. NOW WHEN I WAS TAUGHT ABOUT GENE EXPRESSION I WAS TAUGHT YOU NEEDED CHROMATIN TO KEEP GENES OFF. WELL IT'S CLEARLY NOT THE CASE BECAUSE THE PROFILES OF THESE CELLS ARE VERY VERY SPECIFIC AND THEY HAD NO STITCH OF CHROMATIN IN THEM. SO WE STARTED TO ASK, IS THIS DUE TO THE LEVEL OF MANIPULATION IN THESE CELLS AND WE DISCOVERED SOMETHING REALLY INTERESTING A FEW YEARS AGO. I'M JUST SHOWING THIS BECAUSE I HAVE NO IDEA WHY THEY DREW THIS PERSON ON THIS COVER. BUT ANYWAY, WE DISCOVERED THAT THERE'S AN ALTERED NEUCLO TYPE. IT'S CALLED HYDRO XY METHYL CYTOSINE. I WANT TO TELL YOU A LITTLE BIT ABOUT IT BECAUSE I THINK IT'S AN INTERESTING CASE OF JUST INVESTIGATING BIOLOGY AND FINDING SOMETHING NEW. SO I HAD BEEN INTERESTED IN THIS BUSINESS OF NUCLEAR STRUCTURE AND FUNCTION FOR A LONG TIME. I STARTED MY CAREER STUDYING HISTONE GENES, AND YOU KNOW, CLONING THEM AND FIGURE OUT THERE ARE LOTS OF DIFFERENT HISTONE GENES, ETCETERA ETCETERA. BUT WE HAD NO WAY TO ISOLATE RARE NUCLEI. AND AS SOON AS WE SAW THE PICTURES OF THE TRAPPED MICE, IT BECAME CLEAR, DO YOU KNOW WHAT, THESE GUYS HAVE FLUORESCENT NUCLEI AND WHERE WE'RE GETTING RID OF THE NUCLEUS AND ALL STRUCTURAL STUFF. WHAT WE DID IS TAKE THEY MICE AND ASK IF WE ISOLATE THESE NUCLEI AND CHARACTERIZE THEM. HETERO CHROMATIN IS SUPPOSED TO FORM ON METHYLATED DNA THAT PURKINJE CELLS WOULD HAVE LESS METHYL CYTOSINE IN THEM. SO WHAT WE DID IS HE FIGURED OUT A WAY TO DO THIS. IF HE SORTS OUT DAY HE CAN GET ENOUGH FROM THE PURKINJE CELLS TO LOOK AT T WE HAVE TO USE THE OLD LAYER CHROMATOGRAPHY THAT I USED WHEN I WAS TRAINING AND ASK WHAT IS THE COMPOSITION. WHAT WE'RE HOPING TO CEASE THAT THERE WAS -- TO SEE IS THERE WAS A DECREASE IN THESE CELLS. INSTEAD WHAT HE SAW WAS METHYL C AND ALSO A NEW SPOT THAT WAS HIGHLY ENRICHED IN PURKINJE CELLS. SO THE QUESTION IS WHAT IS THIS. NOW I WORKED ON BACTERIA PHAGE SPO1 AND T4 WHEN I WAS IN GRADUATE SCHOOL. THEY HAVE HYDROXY -- SO I SUSPECTED THIS MIGHT BE REAL BUT SCARY AS A SKEPTIC. HE WENT THROUGH AND DID SO MANY CONTROLS YOU CAN'T IMAGINE HOW CAREFUL THIS GUY IS. HE'S A TREMENDOUS SCIENTIST NOW AT OXFORD. ANYWAY HE WENT THROUGH, RULED OUT ALL THE CONTROLS AND THEN ISOLATED THIS STUFF AND IDENTIFIED IT BY MASS SPECTROMETRY. WHAT ARE THESE CELLS DOING. IS IT PRESENT IN ALL CELLS? IT IS PRESENT AT LOW LEVELS IN MOST CELLS BUT VERY EXCEEDINGLY LOW LEVELS. THAT'S WHY IT WAS MISSED. IT'S NOT PRESENT IN CULTURED CELLS, MOST CULTURED CELLS EXCEPT FOR ES CELLS. IT'S PRESENT IN DIFFERENT NEURONS AND DIFFERENT LEVELS. BUT THE ONES THAT CONTAIN THE MOST ARE THESE ONES THAT HAVE VERY LARGE CHROMATIC NUCLEI. IT'S NOT A SPECIES-SPECIFIC THING. IF YOU TAKE JUST WHOLE CEREBELLUM FROM HUMAN POST MORTEM TISSUE THERE'S PLENTY THERE. SO THIS IS COOL. WE GO FROM THE NEW NEUCLO TYPE UNTIL TWO YEARS AGO, AT THE SAME TIME THAT WE DISCOVERED IT UNBEKNOWNST TO US -- LAB WAS WORKING ON THE GENOME IN EARLY DEVELOPMENT AND SHE DISCOVERED THE ENZYMES THAT CONVERT METAL -- TO HYDROXY METHYL C. SO THIS IS A NEW BIOLOGICAL EPIGENETIC MECHANISM. THERE'S A LOT OF UNANSWERED QUESTIONS. THERE'S ALREADY A HUNDRED PAPERS ON THIS IN THE LITERATURE. I DON'T THINK THEY'RE ALL AT THE HIGHEST QUALITY THAT YOU COULD ENVISION BUT THERE'S A LOT OF INFORMATION IN THERE. MOST OF THIS DATA COMES FROM EITHER WHOLE TISSUE WHICH IF YOU'VE BEEN LISTENING TO ME I DON'T LIKE TO DEAL WITH WHOLE TISSUE I WOULD MUCH RATHER DEAL WITH INDIVIDUAL CELL TYPES OR NEUCLO TYPES OR ES CELLS. WHAT WE'VE BEEN DOING LATELY IS TO TRY TO UNDERSTAND WHAT IS THE FUNCTION IN NEUROCELLS. THIS IS MARION MELON A POST DOC IN THE LAB. THEY PICKED THREE CELL TYPES. PURKINJE CELLS WHICH HAVE THIS HUGE NUCLEI, GRANULE CELLS WHICH HAVE VERY TINY CONDENSED NUCLEI AND BURGMAN GLIAL CELLS THAT HAVE SORT OF INTERMEDIATE NUCLEI. THEY DID TWO THINGS. MARION SEQUENCED THE DRAFT RNA FROM A THESE THREE CELL POPULATIONS. WE GOT BEAUTIFUL QUANTITATIVE DATA. THEN THEY USED A METHOD THAT WAS PUBLISHED BY -- WHERE THEY USED A MODIFIED GLUCOSE. SO IT TURNS OUT IF YOU HAVE HMC. PHAGE MODIFY IT BY PUTTING GLUCOSE ON IT. YOU CAN GET THE ENZYME THAT DOES THAT AND ATTACH A GLUCOSE TO HMC. IT CLEARLY WON'T GO ON TO METHYL C BECAUSE THERE'S NO HYDROXY GROUP. YOU TAKE GLUCOSE SO YOU CAN TAKE GENOMIC DNA, MODIFY IT WITH THIS AND ISOLATE IT. AND THEREFORE YOU CAN MAP VERY SPECIFICALLY WHERE THESE THINGS OCCUR. SO WHAT MARION DID IS COLLECT THE TRAP DATA, SHE GOT BEAUTIFUL DATA. IT'S ACTUALLY MUCH MORE BEAUTIFUL THAN ARRAY DATA BECAUSE WHEN YOU LOOK, IT'S LINEAR FOR BASICALLY THE ENTIRE RANGE. SO A LOT OF THIS GENE EXPRESSION STUFF UP HERE FOR ABUNDANT TRANSCRIPTS WILL BE LOST ON MICRO RAY DATA. SO SHE DID THIS, ALL THE CONTROLS ARE RIGHT. THE PRODUCTS ENRICHARD IN -- ENRICHED. THEY CLUSTER DIFFERENTLY. BEAUTIFUL QUANTITATIVE DATA. WE AREN'T GOING TO LEARN ANYTHING MORE THAN EXCEPT THE FIRST EXPERIMENT SHE DID WAS TO DISPLAY THE EXPRESSION VERSUS THE SPECIFICITY. WHAT SHE FOUND WAS REALLY INTERESTING. IN THIS QUADRANT, THESE ARE HIGHLY, VERY HIGHLY EXPRESSED AND SPECIFICALLY EXPRESSED GENE PRODUCTS. AS IT TURN OUT FOR KNEES REALLY HIGHLY DIFFERENTIATED CELLS LIKE BURGMAN GLIA AND PURKINJE CELLS ABOUT 25% OF THEIR CAPACITY IS DEDICATED TO CELL SPECIFIC GENE EXPRESSION. THESE GUYS ARE THE MOST HIGHLY EXPRESSED GENE PRODUCTS IN THAT CELL. AND IN GRANULE CELLS HOUSEKEEPING GENES ARE THE MOST ABUNDANT. THIS SURPRISED ME. MY CONCEPT OF CELLS, I THINK I REALLY NAIVE WAS THAT OKAY THERE'S A BASIC CELL AND YOU EXPRESS A FEW THINGS AND YOU DO THE BELLS AND WHISTLE AND THAT CELL WILL BE A DIFFERENT CELL. INSTEAD, FOR THESE HIGHLY DIFFERENTIATED CELLS, THEY'RE SPENDING MOST OF THEIR ENERGY MAKING GENE SPECIFIC PRODUCTS. THAT'S WHAT GIVES THEM THEIR CHARACTERISTIC APPEARANCE WHICH BIO CHEMICAL FUNCTION. SO WHAT ABOUT 5HMC. WELL IT TURNS OUT THAT HMC DOES CORRELATE WITH GENE EXPRESSION. IT'S NOT A VERY STRONG CORRELATION IN ALL CELL TYPES. IT'S SLIGHTLY DIFFERENT IN DIFFERENT CELLS. BUT BASICALLY IF YOU HAVE MORE HMC AND YOU'RE A DIFFERENTIATED NEURO CELL TYPE, YOU WILL EXPRESS BETTER. HMC IS PRESENT IN THE GENE BODIES BUT YOU CAN SEE HERE IF YOU MAP HMC AND THREE CELL TYPES ACROSS GENE BODIES, FOR PURKINJE CELL SPECIFIC GENES BASICALLY THE SAME AMOUNT OF HMC IN ALL THREE CELL TYPES. WHEREAS GRANULE CELLS ARE USED HMC AS A VERY SPECIFIC SIGNAL. SO THE UTILIZATION OF HMC IS DIFFERENT. AND THE REASON THAT THIS LOOKS LIKE THIS IS THAT WHAT CORRELATES WELL, THIS MAKES SENSE, IS NOT WHETHER YOU HAVE HMC OR DON'T HAVE HMC. IT'S THE RATIO OF HYDROXY METHYL CYTOSINE TO METHYL CYTOSINE THIS MAKES SENSE. IF YOU'RE A GENE AND GOING TO BE REGULATED AND ASSEMBLING ON HCC -- IT'S WHO WINS THE COMPETITION. THE MUSCLE C BINDING PROTEINS OR THE HMC BINDING PROTEANTZ. I WOULD SAY IN PURKINJE CELLS THE HMC HAS ALMOST NO EFFECT WHEREAS IN GRANULAR CELL IT HAS A HUGE EFFECT. SO THIS IS SOMETHING THAT'S FUNDAMENTAL AND I THINK IT REFLECTS THE FACT THAT THESE CELL TYPES WE KNOW EXPRESS DIFFERENT SETS OF THE METHYL BINDING PROTEINS. WE DON'T WHICH ONES BIND HMC WELL SO I CAN'T GIVE YOU THAT SIDE OF THE STORY. SO THIS IS EVIDENCE FOR A LOT OF REASONS, EVIDENTLY IMPORTANT. ONE OF THE THING THAT'S OBVIOUS IS IN NUCLEI, EVEN IF YOU JUST SUSTAIN THEM. SO THE ANTIBODY PHAGE MC ARE GOOD ENOUGH FOR SUSTAINING NUCLEI. THE DISTRIBUTION IS DIFFERENT. SO THINGS THAT ARE REALLY HIGHLY METHYLATED HAVE ALMOST NO HMC. AND THINGS THAT HAVE A TREMENDOUS AMOUNT OF HMC HAVE VERY LITTLE METHYL C. THIS MAKES SENSE. IT'S A PRECURSOR PRODUCT RELATIONSHIP. BUT SOME CELL TYPES HAVE MOSTLY HMC AND OTHERS HAVE METHYL C. SO WHAT WE THINK AND I DON'T HAVE TIME TO GO THROUGH THIS DATA, IS THAT HMC HAS A CRITICAL ROLE IN MAKING THIS NUCLEUS CHROMATIC BUT DEMETHYLATION OF THE GENOME IS EQUALLY IMPORTANT FOR GENE EXPRESSION. IN THESE SMALL HETERO CHROMATIC CELLS, HEAVY METHYLATION GIVES YOU HEAVIER CHROME CONTINUE BUT IN THE ANTITHESES OF THE CHROMATIC SITES IT'S THE RATIO OF HMC TO METHYL C THAT'S IMPORTANT. MARION IS WRITING A PAPER NOW TO DESCRIBE. OKAY. SO BASICALLY WHAT I'VE TOLD YOU IS I THINK THAT IF WE'RE GOING TO UNDERSTAND DISEASE, EVEN BASIC CELL BIOLOGY OF NEURONS, YOU NEED TO BE ABLE TO ACCESS THE INDIVIDUAL CELL TYPES, YOU NEED TO ACCESS ALL OF THEM. AND DEPENDING ON WHAT YOU'RE INTERESTED IN, THE COMPARATIVE ANALYSIS OF CELL TYPES IS CRITICAL. HAVING DONE THAT AND USING TRAP PROFILING WE FOUND OUT THAT NEURONS ARE VERY FINE TUNED. THEIR PROPERTIES ARE DISTINCT. FINDING THEIR OWN POPULATIONS, YOU CAN'T DETERMINE AS DIFFERENCE FROM LOOKING AT THEM OR LOOKING ARE QUITE DIFFERENT. I THINK TRAP PROFILING CAN GIVE US THE DETAILS OF BIO CHEMICAL FUNCTION AND THAT MOLECULAR PHENOTYPING IS A USEFUL METHODOLOGY. IN ANATOMIC OR DEGENERATIVE PHENOTYPE. IN DEGENERATIVE DISORDERS WHERE YOU MAKE A SINGLE MUTATIONS WHERE CELLS AREN'T DYING IN TWO YEARS THAT THE ANIMALS LIVE. THAT DOES NOT MEAN THAT THE MOLECULAR PHENOTYPES WON'T TELL YOU SOMETHING ABOUT EARLY STAGE PATHOGENESIS OF THAT DISORDER. 5HMC IS A CRITICAL FOR NEUROCELL TYPES IN REGULATING GENE EXPRESSION IN NUCLEAR STRUCTURE. AND ALTHOUGH I DIDN'T SHOW YOU THIS, THE MECHANISMS THAT DECODE 5HC VERSUS METHYL C AND DIFFERENT CELL TYPES ARE DIFFERENT AND THEY REFLECT THE PROTEINS EXPRESSED IN THOSE CELLS TO DECODE THIS INFORMATION. AND JUST TO FINISH HERE BY ACKNOWLEDGING ALL THESE PEOPLE. THIS IS ENDING BECAUSE IT'S RUN FOR TEN YEARS. IT'S A WONDERFUL COLLABORATION WITH NINDS. THE OTHER NIH INDIVIDUALS. I THINK THESE PEOPLE HERE ... ETCETERA, THEY DESERVE A LOT OF CREDIT FOR BASICALLY WORKING FOR TEN YEARS ON A PROJECT THAT'S VERY DIFFICULT IN PUBLISHING ONE PAPER. THIS IS NOT NORMALLY DONE IN SCIENCE BUT I THINK IT'S A VERY IMPORTANT THING. IT'S LED THE WAY TO A LOT OF STUDIES. THERE ARE LITERALLY HUNDREDS OF LABS WITH MICE NOW. THE TRANSLATIONAL PROFILING BASICALLY EVERYBODY IN MY LAB IS DOING IT NOW. BUT IT WAS DEVELOPED IN COLLABORATION WITH PAUL GREENGARD -- AND AN SHAFFER WHO IS NOW AT SINAI ALONG WITH JOE DOYLE AND DOCKERRITY. HE'S. NOW THE HMC STORY REALLY THE KEY PROTAGNESS THERE WAS -- HE'S A FANTASTIC SCIENTIST. MARION HAS DONE AN UNBELIEVABLY AGGRESSIVE JOB CHARACTERIZING ITS ROLE IN THE INDUSTRIAL CELL TYPE. THANK YOU. [APPLAUSE] >> [INDISCERNIBLE] >> IT'S ACQUIRED POST MITOTICALLY AND THAT MAKES SENSE BECAUSE HMC CAN'T BE TRANSFERRED BY DMA MUSCLE TRANSASES. HMC MAY BE USED FOR PASSIVE DEMETHYLIZATION. FOR NEURONS AND GLIAL CELLS, THEY ACQUIRE HMC LATE AFTER THEIR LAST DIVISION AND THEY MAINTAIN IT IN THE ADULT ANIMAL. AND IT DOES CORRELATE WITH GENE EXPRESSION BUT NOT PERFECTLY. DEPENDS ON WHAT CELL IT IS, HOW IMPORTANT IT IS. >> I HAVE ONE QUESTION. YOU MENTIONED AT THE VERY END THERE ONE OF THE THINGS, DID YOU MENTION WHY THE NUCLEUS IS SO MUCH LARGER IN THE PURKINJE. >> I THINK WE KNOW THAT. LET'S A PROTEIN THAT'S EXTREMELY ABUNDANT IN PURKINJE CELLS THAT BIND HMC. WE DON'T HAVE THE DEFINITIVE DATA THAT THAT'S THE CAUSATIVE PROTEIN BUT I THINK IT IS. I THINK WHAT HAPPENS IS THE HMC IS BOUND BY A VERY ABUNDANT PROTEIN AND THAT LOOSENS CHROMATIN. >> ANY MORE QUESTIONS FROM ANYONE? WELL, IF ANYONE CAN THINK OF ANY, WE'LL BE HERE FOR A FEW MORE MINUTES AFTER THE SESSION. AGAIN THANK YOU AGAIN