>> GOOD AFTERNOON, OUR SPEAKER TODAY IS DR. LEONARDO BELLUSCIO. LEO CAME TO US IN 2002 AS AN INVESTIGATOR, HE'S NOW A SENIOR INVESTIGATOR IN THE DEPARTMENTMENT -- CHIEF OF DEVELOPMENTAL NEURAL MASS AT THISTY SECTION. HE GOT HIS TRAINING AT MANHATTAN COLLEGE AS UNDERGARAGE WIT THEN DID Ph.D. WITH RICHARD AXIL AT COLUMBIA UNIVERSITY, EVEN BEFORE RICHARD GOT HIS NOBEL PRIZE WHICH SHOWS GOOD SENSE. THEN HE DID A POST DOC AT DUKE BEFORE COMING TO THE NIH. HE'S BEEN INTERESTED IN OLFACTION AND THE OLFACTORY SYSTEM FOR MOST OF HIS CAREER. AND IN FACT HAS BEEN FOCUSING ON DEVELOPMENTAL ASPECTS OF THE OLFACTORY BOWL AND SYNAPTIC PLASTICITY. AS A MODEL FOR UNDERSTANDING SYNAPTIC PLASTICITY IN GENERAL I THINK ALSO UNDERSTANDING HOW DISEASE PROCESSES IN THE NERVOUS SYSTEM CAN AFFECT THOSE DISTINCT PROCESSES. SO I'M REALLY DELIGHTED THAT YOU'RE HERE TODAY AND THE TALK IS ENTITLED, UNDERSTANDING NEURAL CIRCUITPLASTITY IN THE MAMMALIAN OLFACTORY SYSTEM. ONE OTHER THING. I FORGOT TO SAY THAT LEO WAS PRESIDENTIAL EARLY CAREER AWARDEE IN SCIENCE AND ENGINEERING AND WE JUST WANTED PEOPLE WHO GET THOSE AWARDS TO BE APPRECIATED AND ALSO FOR YOU TO KNOW THAT THERE'S LIFE AFTER THE FIRST YEAR OF TENURE TRACK. >> THERE'S CERTAINLY LIFE AFTER THE FIRST YEAR, I'M LIVING PROOF. THANK YOU FOR THAT VERY NICE INTRODUCTION. AND FOR INVITING ME TO GIVE THIS PRESENTATION. AS MICHAEL SUGGESTED, FIRST OF ALL ACTUALLY THIS IS A PHOTOGRAPH THAT WILL GIVE A PLUG FOR THE NEW NEUROSCIENCE CENTER WE'RE ALL VERY EXCITED ABOUT. IT'S -- I WOULD SAY COMPLETE AT THIS POINT AND WE'RE VERY EXCITED TO SEE IT UP AND RUNNING AND MOVING IT FULL CAPACITY QUITE SOON. THIS IS WHERE MY LAB IS LOCATED. BY -- MY TALK TODAY IS ACTUALLY GOING TO FOCUS ON THE MAMMALIAN OLFACTORY SYSTEM, IN PARTICULAR I'M GOING TO TELL YOU A COUPLE OF STORIES. AND P HOW THEY SORT OF FIT TOGETHER TO DESCRIBE HOW WE'RE TRYING TO UNDERSTAND AND EXPLORE NEURAL CIRCUIT PLASTICITY WITHIN THE SYSTEM. SO THE MAIN UNDERLYING GOAL OF THE LAB IS TO UNDERSTAND THE MECHANISMS THAT CONTROL THE FORMATION OR REFINEMENT AND MAINTENANCE OF NEURAL CIRCUITS. WE'RE PARTICULARLY INTEREST MISDEMEANOR THE ROLE PLASTICITY REGENERATION PLAY IN THIS PROCESS AS WELL AS EXPERIENCE -- SENSORY EXPERIENCE CAN BE USED TO ORGANIZE OR REORGANIZE THAT CIRCUITRY. MORE RECENTLY WE HAVE BEEN INTERESTED IN TRYING TO UNDERSTAND USE THE SYSTEM TO EXPLORE MEG ANYMORES OF DISRUPTION AND REPAIR WITH THE OLFACTORY SYSTEM AS A DISEASE MODEL. I DON'T HAVE TIME TO TALK ABOUT THIS WORK BUT I WILL SAY THAT OLFACTORY LOSS IS INTIMATELY ASSOCIATED WITH A LOT OF NEUROLOGICAL DISORDERS, PARTICULARLY ALZHEIMER'S AND PARKINSONS AND VERY OFTEN ONE OF THE EARLIEST SYMPTOM. BY TRYING TO UNDERSTAND HOW THIS SYSTEM FUNCTIONS IN ITS NATIVE STATE ALSO IN THE DISEASE STATE IT CAN GIVE US IMPORTANT INSIGHT INTO THE EARLY STAGES OF SOME OF THESE DISORDERS. BUT FOR TODAY I'LL BE TALKING ABOUT THE NATIVE STATE OF THIS SYSTEM AND ALL THE WORK THEY I'M GOING TO TALK ABOUT IS FOCUSING ON THE MOUTH OLFACTORY SYSTEM. SO I'M GOING TO START BY INTRODUCING THE SYSTEM, BASIC ORGANIZATION, WHAT YOU'RE LOOKING AT HERE IS ACTUALLY A CROSS SECTION MOUSE HEAD SPLIT DOWN THE MIDLINE. HERE IS THE TIP OF NOSE, HERE IS THE PALATE. WHAT YOU SEE HIGHLIGHTED IN THESE FOUR COLORS IS THE MALE OLFACTORY EPITHELIUM RESPONSIBLE FOR DETECTING ODORS AND RELAYING THE INFORMATION TO THE BRAIN VIA THE STRUCTURE HERE, THE MAIN OLFACTORY BULB. SO WHEN AN ODOR. COST TO NASAL CAVITY INTO CONTACT WITH SENSORY NEURONS THAT LIVE IN THE EPITHELIUM AND EACH NEURON SENDS A SINGLE AXON ACROSS THE CRIBRIFORM PLATE THAT FORMS SYNAPSES ON THE SURFACE OF THE OLFACTORY BULB AND GIVES RISE TO STRUCTURES AS GLUMMER LIE. THESE ARE THE SENSORY NEURONS COMMUNICATE WITH OUTPUT NEURONS IN THE OLFACTORY BULB. SO IN THE EARLY 90s RICHARD AXEL AND THE BUCK CLONED AL FACTORY RECEPTORS, THIS IS A LARGE FAMILY OF GENES NUMBERING ABOUT A THOUSANDS AND WHAT THEY WERE ABLE TO SHOW IS THESE SEVEN TRANSMEMBRANE VEEPTORS ARE EXPRESSEDDED SUCH THAT ANY GIVEN RECEPTORS WILL EXPRESS SUBSET OF NEURONS SO I HIGHLIGHTED THE PATTERN EXPRESS ONE OF THESE RECEPTORS WOULD GIVE. WHAT THEY ALSO SHOW IS ALL NEURONS THAT SELECT THE SAME RECEPTOR TO EXPRESS SEND AXONS TO CONVERGE THE SAME GLUE MERLIS ON THE SURFACE AND IT'S TWO GLUMMER LIE, ONE MEDIAL, ONE LATERAL. THE LOCATION OF THESE GLUMERULI IS INVARIANT FROM ANIMAL TO ANIMAL. SO THIS ILLUSTRATED HERE, THIS PHOTOGRAPH THAT LATER PETER CREATED TRANSGENIC ANIMALS WHICH HE USED HOMOLOGOUS RECOMBINATION TO LABEL THE EXPRESSION PATTERN OF A GIVEN RECEPTOR, ERP 2. SO ALL THE NEURONS THAT EXPRESS THIS P-2 RECEPTOR LABELED HERE IN BLUE. YOU CAN SEE AXONS KNOWING ACROSS THE CRIBRIFORM PLATE AND CONVERGING BEAUTIFULLY ON THE MEDIAL GLUMERULUS. SO SINCE THE OLFACTORY BULB IS COVERED WITH GLUMERULI, ROUGHLY 2,000 OR SO PER BULB AND OTHER ODOR RECEPTORS EXPRESSED BY SUBSETS OF NEURONS AND THEY CONVERGE TO THEIR FAVORITE LOCATION, THIS IS REALLY THE BASIS OF A MAP ON THE SURFACE OF THE OLFACTORY BULB THAT REALLY ODOR RECEPTOR IDENTITY. SO IF WE TAKE A CROSS SECTION THROUGH THIS AND SHOW IT TO YOU IN CARTOON FORM, THIS IS WHAT YOU'RE LOOKING AT HERE. HERE IS THE MEDIAL SIDE OF THE BULB, THE LATERAL SIDE, YOU'RE SEEING PAIRS OF GLUMERULI. I COLOR CODED TO DEMONSTRATE THE ODOR RECEPTOR NEURONS THAT GENERATE TO THOSE LOCATIONS. - THE FIRST UNDERSTANDING OF A MAP ORGANIZATION ON THE SURFACE TO HAVE MOLECULAR BULB OPT SURFACE OF THE FINGERPRINT. WE SHOWED THE TWO HALVES OF THE BULB ARE CONNECTED TO ONE ANOTHER IN A PRECISE MANNER FOR A SET OF INTRABULBAR PROJECTIONS SO IT'S THIS STRUCTURE HERE THAT OUR WORK FOCUSES ON SO I'LL GO DEEPER TO DESCRIBING IT ORGANIZATION AT CIRCUIT LEVEL. IF WE SPLIT THIS IN HALF AND LAY IT OUT LIKE A P MAP, ONE THING THAT SHOULD STRIKE YOU RIGHT AWAY, IT'S BEAUTIFULLY ORGANIZED AS A LAMINAR STRUCTURE. I KNOW THIS IS A CARTOON, BUT IT REALLY IS ORGANIZED THIS VERY NICE STRUCTURE HERE. SO HERE IS THE MEDIAL HALF AND HERE IS THE LATERAL HALFMENT YOU CAN SEE AT THE SURFACE YOU HAVE THE SENSORY INPUTS COMING IN FROM THE EPITHELIUM AND PAIRS OF GLUMERULI. WITHIN THAT THEY ACTUALLY COMMUNICATE WITH THE OUTPUT NEURONS TO HAVE BULB WHICH ARE MITRAL CELLS WHO LIVE IN THIS LAYER AND THEY SEND AN APICAL TOP TO FORM SYNAPSES WITH THE INPUT NEURONS HERE AND SEND OUTPUT TO THE CORTEX, THE OLFACTORY CORTEX. THERE'S TWO POPULATIONS OF INTERNEURONS. THE PERIGLUMMER ALREADY CELLS, THEY COMMUNICATE BOTH WITH THE INPUT AND OUTPUT NEURONS OF THE BUBBLE AND ESSENTIALLY MODULATE THE COMMUNICATION THERE. THEN THERE'S THE GRANULAR CELLS THAT LIVE DOWN HERE AND THEY SEND PROJECTIONS UP TOWARDS THE SURFACE AN FORM SYNAPSES ON TO THE LATERAL DENDRITES OF THE CELLS ABOVE. ALL THESE NEURONS LABELED IN RED ARE INTERNEURONS AND THEY ALL REGENERATE CONTINUOUSLY THROUGHOUT LIFE. THAT'S A POINT I'LL COME BACK TO LATER. THE LAST CELL TYPE I'LL ADD IS THE TOUGHED CELLS IN PARTICULAR THE SUPERFICIAL TUFTED CELLS. THESE MEDIATE THIS CONNECTION BETWEEN THE TWO HALVES OF THE OLFACTORY BULBS SO THE MEDIAL PROJECTED LATERAL AND LATERAL TO MEDIAL, VERY PRECISE FASHION. COUPLE OF THINGS SHOULD JURY OUT AT YOU, IT'S MADE OF COLUMNS OF CELLS THAT ARE REPEATED UNITS OR REPEATED COLUMNS THAT WE HAVE COME TO TERM ODOR COLUMNS. THE SECOND AT THE TESTIMONY OF ONE OF THESE -- TOP OF THESE IS THE GLUMERULIS WHICH IS A HUB AROUND WHICH MANY CELL TYPES CENTER. SO IF EXTRACT ONE OF THESE, THIS IS REALLY OUR MODEL CIRCUIT WE HAVE BEEN TRYING TO UNDERSTAND. IT GETS INPUT FROM THE OUTSIDE WORLD, IT SENDS PROJECTION DIRECTLY TO THE OLFACTORY CORTEX. IT'S MODULATED BY REGENERATING POPULATION OF INTERNEURONS, AND THEN IT HAS THIS CONNECTION THAT LINKS IT TO THE HIGH EVERY ORDER SORT OF STRUCTURE. SO WE WANT TO UNDERSTAND HOW THIS DEVELOPS, HOW IT FORMS AND TAKES SHAPE, HOW THIS REGENERATION PLAYS A ROLE IN ITS FORMATION AND HOW ACTIVITY FLOWS THROUGH IT CAN MODIFY ITS ORGANIZATION AND AFFECT DEVELOPMENT. SO WHEY I'M GOING TO SHOW YOU TODAY IS THESE THREE FACTORS REP GENERATION PLASTICITY AND ACTIVITY, ACTUALLY PLAY AN INTERRELATED ROLE IN ORGANIZING THIS ORGANIZING AND MAINTAINING THIS CIRCUMSTANCE. SO TO DO THAT I'M GOING TO FOCUS ON A SPECIFIC PART OF THIS CIRCUIT. THAT IS THIS INTRABULBAR PROJECTING STRUCTURE HERE. SHE'S FIRST ABLE TO VISUALIZE THIS PROJECTION USING TRACER INJECTIONS. WHAT WE'RE ABLE TO DO IS TAKE A LYNN OF MICE WHICH A PARTICULAR GLUMERULUS WAS LABELED WITH GFP LOOKING DOWN DORSAL SURFACE OF THE OLFACTORY BULB, YOU CAN SEE A SINGLE ONE IS LABELED WITH GFP. WE CAN TARGET WITH A TRACER AND ASK ABILITY PROJECTIONS -- ABOUT PROJECTIONS WITHIN THE OLFACTORY BULB TO THE OTHER SIDE. THIS HAS A PARTNER IN HERE, IT'S ON THIS SIDE, BUT IT'S ACTUALLY DEEP SO HARD TO SEE IN VIVO. HERE IS AN INJECTION, YOU CAN SEE THE TUFTED CELLS TAKEN UP TO DIE. WE FOLLOW THE PROJECTION TO THE OTHER SIDE WE FOUND IT FORMED A TERMINAL TOUGH DIRECTLY BENEATH THE PARTNER GLUMERULIS ON THE OTHER SIDE. VICE VERSA, IF WE INJECT WE SEE A TOUGH UNDER THIS GLUMERULUS. SO THESE PAIRS OF GLUMERULI THAT RECEIVE THE SAME INPUT FROM OLFACTORY NEURONS COMMUNICATE WITH ONE ANOTHER THROUGH THE INTRABULBAR LINK AND THROUGH THE ODOR COLUMN CIRCUITS. NOW, IF WE JUST HIGHLIGHT JUST THE PROJECTION TERMINAL TUFT HERE, ASSOCIATED WITH THESE, AND WE APPRECIATE THE FACT THERE'S MULTIPLE PAIRS OF GLUMERULI ON THIS SURFACE AND THEY SEND PROJECTIONS AND HAVE TUFTS THAT LINK THESE PAIRS, WHAT WE HAVE DONE IS SORT OF HIGHLIGHTED AN ORGANIZATIONAL MAP THAT SITS BENEATH THE SURFACE AND MADE UP OF CENTRAL NEURONS AND IT MIMICS THE GLUMERULAR MAP, IN ORGANIZATION AND SPECIFICITY. SO WE WANT TO UNDERSTAND HOW THIS INTRABULBAR MAP DEVELOPS AND FORMS SO WE LOOKED AT THESE PROJECTIONS AT VARIOUS STAGES DURING DEVELOPMENT, WE REALIZE THESE WERE PRESENT RIGHT AFTER BIRTH THOUGH AT THIS STAGE ONE WEEK OF AGE THESE PROJECTIONS WERE NOT THESE REFINED TUFT BUT WERE QUITE BROAD AN ENCOMPASSED A BROAD AREA. WE ALSO FOUND AS WE DID A DEVELOPMENTAL ANALYSIS OF THIS, THEY GRADUALLY REFINE AND BY SEVEN WEEKS OF AGE THEY REFINE INTO THESE TERMINAL -- THESE VERY SPECIFIC TERMINAL TUFTS THAT WE HAVE COME TO DEFINE AS MATURE PROJECTIONS. WE ALSO FOUND THESE -- THIS PROCESS IS ACTIVITY-DEPENDENT. IF E WE BLOCK SENSORY INDUCED ACTIVITY AND OLFACTORY SYSTEM THAT'S STRAIGHT FORWARD, YOU SIMPLY PLUG THE NOSTRIL AND THAT ELIMINATES ACTIVITY PROJECTING TO THAT OLFACTORY BULB. IF YOU DO THAT WE FOUND THESE PROJECTIONS FAILED TO REFINE AND SO ACTIVITY BLOCK CAUSES THEM TO SPREAD. WHAT WAS INTERESTING IS IF YOU DO THIS DURING DEVELOPMENT THEY FAIL TO REFINE BUT EVEN IF YOU PLUG ACTIVITY AFTER REFINED, YOU CAN CAUSE THEM TO SPREAD. HERE IS AN EXAMPLE OF THAT, IF WE INJECT TRACER TEN WEEK OLD CONTROL ANIMAL, HERE IS A TRACER INJECTION, YOU SEE THE TERMINAL TUFT, YOU SEE THE DIAMETER AT THE TOP ROUGHLY EQUIVALENT TO THE DIAMETER OF THE AREA INJECTED SO THERE'S SPATIAL PRESERVATION HERE AND WE USE THE RATIO OF PROJECTION TO PROJECTION RATIO AS A MEASURE HOW SPECIFIC THEY ARE. IF WE BLOCK SENSORY ACTIVITY, FROM 7 TO 10 WEEKS, YOU CAN SEE THE PROJECTION IS WIDER, ABOUT FOUR TIMES THE SIZE OF THE INJECTION DIAMETER HERE. SO ONE THING WE WERE CURIOUS ABOUT WAS IF WE DISRUPTED THIS,S IS NOW STUCK IN THIS MANNER OR CAN IT ACTUALLY WITH BE PREPARED. WE FOUND IF YOU PUG THE PLUG AND LET SENSORY ACTIVITY BACK IN, IT WILL GRADUALLY REREFINE AND TAKE SOMEWHERE BETWEEN 6 TO 9 WEEKS TIME TO GRADUALLY REFINE. HERE IS A 19 WEEK OLD CONTROL TO SHOW YOU THAT IT BASICALLY COMES BACK DOWN TO ABOUT A ONE TO ONE RATIO. THIS ESSENTIALLY ILLUSTRATED THIS -- BY RESTORING ODOR TO INDUCE ACTIVITY YOU WOULD ALLOW THE PROJECTIONS TO REREFINE BUT IT ALSO SHOWED THIS PLASTICITY IS RETAINED THROUGH ADULTHOOD AND THERE WAS NO CRITICAL PERIOD ASSOCIATED WITH CHANGES. IF YOU BLOCK THE ACTIVITY YOU GET PROJECTIONS TO SPREAD, ALLOW IT BACK IN AND REREFINE TO SPECIFIC MATURE STATE. WHAT WE DIDN'T QUITE UNDERSTAND IS WHAT ROLE ACTIVITY REALLY PLAYING IN THIS PROCESS. AND ANY BROAD LEVEL FIND OUT IF IT WAS PLAYING A PERMISSIVE OR INSTRUCTIVE ROLE IN THIS PROCESS. SO TO GET AT THIS QUESTION WE TRANSFORM THIS TO A QUESTION WE COULD ADDRESS WHICH IS TO ASK WHETHER NON-SPECIFIC ACTIVITY SUFFICIENT TO ALLOW THIS REFINEMENT. SO WE USE LINE OF MICE WE AWIRED FROM RANDY REED'S LAB A FEW YEARS AGO. UBI-7 WHICH RANDY WAS TRYING TO EXPRESS A SINGLE ODOR RECEPTOR BROADLY THROUGHOUT THE EPITHELIUM. WHAT HE SUCCEEDED IN DOING WAS ACTUALLY INTRODUCING THIS RECEPTOR AS A SECONDARY RECEPTOR TO ALL SENSORY NEURONS. WHAT HAPPENED IS EVERY SENSORY NEURON EXPRESSES ENDOGENOUS RECEPTOR SO YOU GET A NORMAL ORGANIZATION OF GLUMERULAR MAP BUT ALSO A LITTLE BIT OF IL-7 HERE SO WHEN WE CROSSED WITH THE REPORTER LINE YOU CAN SEE THE M- 71 OLFACTORY SENSORY NEURONS CONVERGE NICELY TO THE GLUMERULI AND DO THE SAME WITH IBI-7 ANIMALS SO THIS MAP WE HAVE DONE WITH REPORTER LINES IS NICELY PRESERVED. BUT BECAUSE THERE'S A LITTLE BIT OF I-7 EXPRESSED THERE WE'RE ABLE TO ACTIVATE WITH THE ODOR THAT CAN STIMULATE I-7. SO ANY SENSORY NEURON THAT WE BASE -- THAT WE PATCH ON TO WILL RESPOND TO THE DOOR (INAUDIBLE). THIS IS AN EXAMPLE OF A REPORTING, A CELL ATTACHED RECORDING YOU CAN SEE THIS VERY NICE DEFOR WILLIZING RESPONSE. BY CONTRAST IF WE PUFF ON BUTANOU, A STRUCTURALLY RELATED MOLECULE BUT DOESN'T ACTIVATE THE IL-7 RECEPTOR, WE DON'T SEE A RESPONSE, IT'S VERY DIFFICULT TO FIND A RESPONDING NEURON AMONG THE MANY MILLIONS OF NEURONS SO IT'S QUITE DIFFICULT TO BE ABLE TO SELECT ONE OF THOSE. SIMILARLY, IF YOU LOOK AT THE OLFACTORY BULB YOU CAN USE INTRINSIC SIGNAL IMAGE FOR THE SAME FACT. YOU'RE LOOKING AT THE SURFACE AND SIGNAL IMAGING IS ACTUALLY TECHNIQUE THAT ALLOWS YOU TO USE CHANGES IN LIGHT REFLECTTANTS TO HIGHLIGHT AREAS OF HIGH METABOLIC ACTIVITY. SO IF YOU INTRODUCE AN ODOR LIKE BUTANOU WITH RESTRICTED RANGE OF SENSORY -- ACTIVATE YOU CAN ACTIVATE DISCRETE SPOTS WHICH CORRELATE TO GLUMERULAR ACTIVATION. IF WE PUT (INAUDIBLE) WHICH IS EXPRESSED IN ALL SENSORY NEURONS BY VIRTUE OF THIS TRANSGENE, WE CAN ACTIVATE THE ENTIRE OLFACTORY BULB SIMULTANEOUSLY. SO THIS THEN GAVE US A TOOLS TO BE ABLE TO DO SOME EXPERIMENTS WITH THE INTRABULBAR PROJECTIONS, WE FOUND IN THE UBI-7, THE INTRABULBAR PROJECTIONS ARE FORMED NORMALLY, NICELY REFINED IF WE INJECT SMALL AREA ONE SIDE WE SEE REFINED TUFT ON THE OTHER. HOWEVER IF WE RAISE THE ANIMALS IN THE PRESENCE OF OCTENOU WITH BROAD ACTIVATION FROM BIRTH WE FIND THAT THAT THE PROJECTIONS FAIL TO REFINE. WHAT MORE, WHAT'S MORE INTERESTING IS THAT EVEN IF WE ALLOW THIS REFINEMENT TO OCCUR AND WE EXPOSE ANIMALS TO OCTENOU IN ADULTHOOD ONE WEEK OF THE EXPOSURE PUT INTO THE CAGE ON A REGULAR BASIS CAUSES THE PROJECTIONS TO EXPAND, AND THIS IS IN ADULT HOOD. IF WE REMOVE IT FROM THE ENVIRONMENT AGAIN, PULL IT OUT OF THE CAGE, WE GET THIS REFINEMENT AND ALL THIS CAN OCCUR WITHIN A WEEK OF TIME, THIS QUANTIFIES THE INCREASE IN THE SIZE OF THE PROJECTIONS AND AGAIN, RECOVERY. SO THIS WAS ESSENTIALLY ILLUSTRATING THAT THIS INTRABULBAR MAP CAN REMODEL RAPIDLY, IT DOES SO AND CONTINUES TO DO SO IN ACTIVITY DEPENDENT MANNER. AND THAT ACTIVITY IS NOT SIMPLY PERMISSIVE BECAUSE IF IT -- ALL THESE THINGS NEEDED WAS JUST ODOR INDUCED ACTIVITY THEY HAVE GOT PLENTY OF IT. BUT IT'S RATHER PLAYING AN INSTRUCTIVE ROLE. IT WAS UNCLEAR IN WHAT WAY SO THESE INTRABULBAR PROJECTIONS SEENING OUT DISTINCT ACTIVITY PATTERNS AND BROAD ACTIVATION IS SUFFICIENT? SO WE GOT SOME INSIGHT INTO THIS, FROM EXPERIMENTS ASSOCIATED WITH A DIFFERENT PROJECT. AND THIS PROJECT WAS ACTUALLY SEEKING TO DISRUPT AXON GUIDANCE IN THE OLFACTORY SENSORY NEURONS AND WHAT WE DID WAS WE EXPRESS THIS PROTEIN FF-1 UNDER CONTROL OF A OLFACTORY SPECIFIC PROMOTER G GAMMA 8 AND YOU CAN SEE USING THESE REPORTER MICE THAT YOU SAW EARLIER IN THE TALK, YOU GET THIS VERY NICE KORVER CONSENSUS AT BIRTH -- CONVERGENCE AT BIRTH, IF YOU EXPRESS THE PROTEIN YOU SEE AXON WANDERING AROUND THE OLFACTORY BULB. IF WE WAIT ANIMALS REACH THREE WEEKS OF AGE YOU CAN SEE THE CONSEQUENCES OF THIS DISRUPTION, YOU GET THIS SCATTERED MOSAIC PATTERN OF GLUMERULI ACROSS THE BUBBLE. YOU'RE ONLY LOOKING AT ONE RECEPTOR BECAUSE IT TEASE ONE MARKED BUT THIS IS HAPPENING ALL OVER THE OLFACTORY BULB. WHAT'S INTERESTING ABOUT THIS IS THAT IF WE THEN TURN OFF THE EXPRESSION OF THIS PROTEIN, YOU CAN ACTUALLY GET THE MAP TO RECOVER. SO THIS IS QUITE USEFUL FOR US IN OUR EXPERIMENTS THAT WE'RE DOING IN TERMS OF CERTAIN DISRUPTION AND REPAIR, BUT FOR THE PURPOSES OF GOALS HERE, THIS WAS REALLY THE STATE THAT WE WERE CURIOUS ABOUT. ONCE WE SCRAMBLED THIS OLFACTORY MAP WHAT HAPPENS TO INTRABULBAR PROJECTIONS? SO WE INJECTED A TRACERREN ONE SIDE OF THESE -- ONE SIDE OF THE BULB IN THESE ANIMALS AND WHAT WE FOUND WAS THAT THE PROJECTIONS TO THE OPPOSITE SIDE WERE QUITE BROAD. AND IN FACT AS WE CUT THROUGH THE SECTIONS YOU CAN SEE THAT THE PROJECTIONS CONTINUED FOR MANY, MANY SECTIONS. BUT AS WE LOOK MORE CAREFULLY AT THESE PROJECTIONS, THOUGH SPREAD THROUGHOUT THE OTHER HALF OF THE OLFACTORY BULB, IT WAS A UNIFORM PROJECTION, IN FACT WE CAN SEE TUFTS APPEARING AS WE CUT DEEPER AND DEEPER ALMOST AS IF THEY WERE TRYING TO MIMIC THIS MOSAIC PATTERN ASSOCIATED WITH A GIVEN RECEPTOR. SO THIS WAS SUGGESTING HOW IS -- THE QUESTION WAS HOW IS ODOR INDUCED ACTIVITY USED TO ACHIEVE THIS TARGETING? WHAT WE WERE HYPOTHESIZING BUT THAT THESE INTRABULBAR PROJECTIONS TARGET BY MATCHING ACTIVITY PATTERNS BETWEEN ISOFUNCTIONAL GLUMERULAR PAIRS. SO THIS WAS SOMETHING WE CAN TEST USING SOME OF THESE TRANSGENIC LINES. AS I TOLD YOU AT THE BEGINNING THERE'S USUALLY A PAIR OF GLUMERULI FOR EACH ODERANT RECEPTOR TAGGED BUT AT SOME REASONABLE FREQUENCY YOU CAN GET MULTIPLE GLUMERULI ON ONE SIDE. SO FOR ALL PRACTICAL PURPOSES THE GLUMERULI ARE EQUIVALENT, THEY'RE RECEIVING INPUT FROM THE SAME OLFACTORY SENSORY NEURONS. SO THIS SET UP AN OPPORTUNITY FOR US TO SEE WHAT DO THESE PROJECTIONS DO WHEN GIVEN A CHOICE OF TWO GLUMERULI THAT ARE EQUIVALENT. SO AGAIN, WE INJECTED TRACER INTO THE ONE GLUMERULUS ON ONE SIDE. WHEN WE CUT THROUGH THE LAYERS THE PROJECTIONS FORM AD NICE TUFT UNDERNEATH THE PARTNER, THE FIRST GLUMERUUS. AS WE CONTINUE TO CUT WE SEE A SMALL TUFT APPEAR IN THE SECONDG GLUMERULUS AS WELL. IN BETWEEN IS 200 MYCONTROLS, THERE WAS NO TUFT. SO THESE PROJECTIONS ARE NOT CLEARLY JUST RANDOMLY TARGETING GLUMERULI, THEY ARE TARGETING DISTINCT GLUMERULI, IT'S USING THE INFORMATION BEING PRODUCED BY THIS GLUMERULUS AND PASSING THROUGH THE ODOR COLUMN CIRCUIT TO DISTINGUISH FROM ITS NEIGHBORS. SO I WILL TAKE YOU THROUGH THIS MODEL WE ARE USING AS OUR GUIDING MODEL. I SHOWED YOU THE MATURE INTRABULBAR MAP HERE IN THE IPL LAYER IS SIMILAR TO THE GLUMERULAR MAP AT THE SURFACE IN STRUCTURE AND ORGANIZATION BUT IT DOESN'T START THAT WAY. THE PROJECTIONS FROM THE OTHER SIDE ACTUALLY START OUT QUITE DIFFUSE. THEN THROUGH A GRADUAL DEVELOPMENTALLY FINEMENT, THEY MATURE AND BECOME A MAP WE CALL A MATURE INTRABULBAR MAP. IF THERE'S MULTIPLE BILL GLUMERULI ON ONE SIDE AS WE SAW, THE PROJECTIONS ON THE OPPOSITE SIDE TARGET BOTH. SO HOW ARE THESE PROJECTIONS THEN TRYING TO OR CAPABLE OF DETERMINING WHICH ARE THE GLUMERULI ON THIS SIDE? I MENTIONED BEFORE, THE READING SIGNALS COMING THROUGH THE GLUMERULAR LAYER, THE OLFACTORY BULB LAYERS, THAT'S HOW THEY'RE DISTINGUISHING THEM. SO IN THE CASE OF UBI-7 ANIMALS WHERE THESE PROJECTIONS START OUT BROAD AND THEN EXPOSE ANIMALS TO OCTENOU WHICH ACTIVATES THE ENTIRE OLFACTORY BULB THE SIGNALS COMING THROUGH THE LAYERS ARE ALL EQUIVALENT. SO PROJECTIONS COMING FROM THIS PARTICULAR GLUMERULUS CAN'T DISTINGUISH BETWEEN SIGNALS COMING THROUGH ANY OF THESE. THEY'RE ALL EXPRESSING UBI-7. SO THEY REMAIN UNREFINED AND THEY FAIL TO MATURE. WE KNOW AN ODOR IS CAPABLE OF ACTIVATING MULTIPLE GLUMERULI. BUT EVEN IF IT DOES TAKE AN ACTIVE GLUMERULI DIFFERENT ODOR RECEPTORS PROJECT TO DIFFERENT LOCATIONS SO THERE'S DIFFERENT PATTERNS OF ACTIVITY. SO HERE, SORT OF ILLUSTRATED THIS BY SHOWING THEY ACTUALLY RESPOND WITH DIFFERENT FREQUENCIES. SO PROJECTIONS COMING FROM A GIVEN GLUMERULUS DISTINGUISH BETWEEN THE GLUMERULI THOUGH THEY RESPOND TO THE SAME ODOR AND IN THIS RESPECT REFINE ACCURATELY AND GIVE RISE TO MATURE MAP. KEEP IN MIND THIS ISN'T SOMETHING THAT OCCURS ONCE DURING DEVELOPMENT, IT CONTINUES TO OCCUR THROUGH ADULT HOOD BECAUSE IF YOU BLOCK ACTIVITY THIS PROJECTION WILL SPREAD AND IF YOU ALLOW ACTIVITY BACK IN, THEY WILL REREFINE AND REGAIN ACCURACY. SO THIS RAISES THE QUESTION HOW DO THESE PROJECTIONS MAINTAIN THIS CONSTANT STATE OF PLASTICITY, HOW ARE THEY ABLE TO DO THAT? SO WE THOUGHT A CLUE MIGHT COME FROM ASKING THIS NEXT QUESTION. ARE THERE NEURONAL POPULATIONS MODULATING THE CIRCUITRY AND MAYBE THEY PLAY A ROLE IN THIS PROCESS. INDEED WE KNEW THAT FROM EARLY STUDIES IN MICHAEL SHIPLY'S LAB AT UNIVERSITY OF MARYLAND CONNECTS BETWEEN THE TWO BULBS ACTUALLY FORM BETWEEN INFORM -- AND P FORM ON TO REGENERATING GRAND JEWEL CELLS. SO THIS POPULATION OF REGENERATING NEURONS THESE ARE GENERATING GRANULE CELLS ARE THE TARGETS OF THESE INTRABULBAR PROJECTIONS. NOW, I HAVE DRAWN THESE AS UNIFORM CELLS JUST LABELING THEM IN RED BUT IN ACTUALITY THIS IS QUITE A DIVERSE POPULATION OF NEURONS. SO YOU CAN JUST SEE BY USING A COUPLE OF MOLECULAR MARKERS THAT EVEN THOUGH THESE ARE ALL REGENERATING NEURONS, REGENERATING INTERNEURONS THEY HAVE VERY DISTINCT CHARACTERS, NOT ONLY EXPRESSED IN DIFFERENT LOCATIONS BUT THEY HAVE DIFFERENT SHAPES AND THEY TARGET DIFFERENT AREAS SO BY TARGETING ANY ONE OF THESE POPULATIONS YOU CAN IMAGINE THAT THESE INTRABULBAR PROJECTIONS REALLY MODIFY A DISTINCT PORTION OF THE CIRCUITRY ON THE OTHER SIDE. WHEN WE LOOK AT THE EXPRESSION OF THESE WITH RESPECT TO SOME OF THESE TRACER INJECTIONS, YOU CAN SEE THAT THE TRACERS ACTUALLY LOVE THE WRAP AROUND THE CELL BODIES OF VARIOUS TYPES OF NEURONS. THEY FORM THESE LITTLE VERY COSTTIES WHICH ARE INDICATIVE OF SYNAPTIC CONNECTIVITY. SO THESE INTRABULBAR AXONS LIKE TO ASSOCIATE WITH NEURONS AND WE KNOW THE NEURONS REGENERATE, WE'RE JUST GOING TO TELL YOU A LITTLE BIT ABOUT THEM. THESE ARE NEURONS THAT ORIGINATE IN THE SUBVENTRICULAR ZONE, THEY MIGRATE TO THE OLFACTORY BULB THROUGH THE MIGRATORY STREAM AND ONCE IN THE BULB THEY MIGRATE RADIALLY TO THE SURFACE AND INTEGRATE INTO THE CIRCUITRY OF THE BULB. AND BECOME GRANULE CELLS AS A LARGE CATEGORY OR PERIGLUMERULAR CELLS AS WELL. NOW, USING A LINE OF MICE WHICH WE CAN LABEL THESE -- THIS LARGE POPULATION OF NEURONS USING CAD 65, THEY'RE ALL INHIBITORY NEURONS. WE CAN SEE THAT WHAT THE MIGRATION LOOKS LIKE HERE, I WANTED TO GIVE YOU A LITTLE BIT OF A FEEL FOR WHAT THESE NEURONS ARE DOING AS THEY'RE MIGRATING. HERE IS THESE NEURONS MIGRATING IN FROM THE MIGRATORY STREAM, THIS IS TWO HOURS OF TIME HERE CONDENSED DOWN TO 15 SECONDS. YOU CAN SEE THIS IS NOT JUST ONE OR TWO NEURONS MIGRATING IN, THIS IS A LARGE FLOW OF NEURONS THAT CONTINUES TO MIGRATE IN THROUGHOUT LIFE IN THESE ANIMALS. ONCE THEY REACH THE OLFACTORY BULB, THEY MIGRATE RADIALLY AND THIS FORM OF MIGRATION IS A LITTLE BIT DIFFERENT BECAUSE YOU DON'T HAVE A MASS FLOW. YOU CAN SEE NEURONS ARE TRYING TO CLIMB INTO THE CIRCUITRY AND FIGURE OUT WHERE IT IS THAT THEY'RE ACTUALLY BELONG AND HOW THEY MODIFY THE CIRCUIT THEY THEN WILL JOIN P. THE QUESTION BECAME DO THESE REGENERATING NEURONS PLAY A ROLE IN THIS INTRABULBAR PLASTICITY. TO ADDRESS THIS QUESTION WE ACTUALLY COLLABORATED WITH A COLLEAGUE AT NIMH, DR. HEATHER CAMERON, HEATHER IS INTERESTED IN THIS REPOPULATING GENERATION OF NEURONS AT THE HIPPOCAMPUS SO SHE HAD SEVERAL ANIMALS WE FELT WOULD BE USEFUL ALSO IN THE OLFACTORY SYSTEM. USING THESE TWO PROMOTERS GFAP OR NESTIN WE WERE ABLE TO DRIVE EXPRESSION OF HERPES SIMPLEX VIRUS TK GENE, IMMATURE POPULATIONS OF NEURONS. THESE ARE PROMOTERS THAT ARE ACTIVE IN THIS NEUROGENIC REGION AT THE SUBVENTRICULAR ZONE SO EXPRESSION OF THIS TK IF IN THE PRESENCE OF GANSCCLOVIR WILL INTERFERE WITH REPLICATION AND KILL THE NEURONS SO THIS IS A WAY TO ELIMINATE THE REGENERATING NEURONS IN THE OLFACTORY BULB AND ASK WHAT EFFECT IT WOULD HAVE ON THE ORGANIZATION OF THE CIRCUITRY. TO GET A GAUGE AS TO HOW A THICK -- HOW EFFECTIVE THIS WAS, WE CROSS WITH GAD 65 REPORTER MICE. YOU CAN SEE TK GENE IS PRESENT HERE, THERE'S NO DRUG, NO GAD CYCLOVICR SO WE HAVE A NICE MIGRATORY STREAM AND HERE IS THE OLFACTORY BULB. YOU SEE A LARGE MASS PRESENT IN THE OLFACTORY BULB. IN THE PRESENCE OF THE DRUG WE COMPLETELY ELIMINATED ROSS MIGRATORY STREAM THOUGH MANY MATURE NEURONS REMAIN. THIS IS QUITE EVIDENCE. WE WERE ABLE TO ASK THE QUESTION WHAT EFFECT DOES THIS HAVE ON THE INTRABULBAR PROJECTIONS. SO WE EXAMINED A GROUP OF MICE BOTH IN THE GFAP AND THE NESTIN CONDITIONS, BOTH WITH AND WITHOUT AN RMS, AND ESSENTIALLY WHAT WE FOUND WAS SURPRISING IN THE PRESENCE WHEN THE RMS WAS PRESENT WE HAD A VERY NORMAL REFINED SPECIFIC PROJECTION. HOWEVER IF THE RFMS IS ELIMINATED THE PROJECTIONS SPREAD. THIS HAPPENED IN BOTH CASES WITH THE NESTIN AND THE GFAP. THIS QUANTIFIED THE CHANGE IN THE SIZE OF THE PROJECTIONS. WHAT THIS REALLY SHOWED WAS THAT THESE ANIMALS, THERE WAS NOTHING ELSE THAT WE HAD DONE TO THEM, ALL WE DID WAS ELIMINATE THE RMS. THIS REGENERATING POPULATION. IT REALLY SAYS THESE NEW NEURONS ARE NECESSARY TO KEEP THE INTRABULBAR MAP REFINED. MORE IMPORTANTLY REALLY ALSO DEMONSTRATED FOR THE FIRST TIME THESE ADULT BORNE NEURONS PLAY A CRITICAL ROLE MAINTAINING A CIRCUIT ORGANIZATION. THAT'S A VERY DISTINCT DIFFERENCE THAN INTEGRATING A NEURON TO MODIFY CIRCUIT ASSOCIATED WITH LEARNING AND MEMORY. THIS IS BASICALLY SAYING THIS CONSTANT NEURONS ARE ESSENTIAL TO MAINTAIN A CIRCUIT IN ITS STABLE ORGANIZATION. THIS IS QUITE EXCITING. IF WE GO BACK TO THIS, I WANT TO REMIND YOU THAT THERE'S PLENTY OF MATURE GRANULOCELLS PRESENT, WHAT'S MISSING ARE THE IMMATURE NEURONS, SO IF WE JUST USE A MARKER FOR IMMATURE NEURONS, YOU CAN SEE THAT THE GFAP TK ANIMALS OUTGAINCYCLOVIR HAVE NEURONS COMING IN BUT GONE IN THE PRESENCE OF GANCYCLOVIR. SO THE NEW NEURONS ARE MISSING. SO ONE THING I HAVEN'T TOUCHED ON AND I WANT TO GO INTO A LITTLE BIT, IS WHAT THE PURPOSE OF THESE INTRABULBAR PROJECTION? WE KNOW THEY LINK THE CONNECTIONS BETWEEN THE TWO GLUMERULAR MAPS BUT FIRST WHY DO WE HAVE MULTIPLE MAPS INVOLVED? AND ONE HYPOTHESIS WE WERE WORKING WITH IS THE MAPS FUNCTION RELATIVE TO ONE ANOTHER RA THEY WERE INDEPENDENTLY SO THERE MIGHT BE A COORDINATION BETWEEN THE TWO SIDES. SO WE'RE ABLE TO GET INSIGHT ON THIS THROUGH SERIES OF EXPERIMENTS THAT POST DOC IN MY LAB DID SHESHANG JOU WHO PERFORMED A SERIES OF MULTI-ELECTRICAL RECORDINGS WHICH HE WAS ABLE TO MEASURE ACTIVITY BETWEEN THE TWO HALVES OF THE OLFACTORY BULB AND ASK IF THERE'S ANYTHING DIFFERENT GOING ON BETWEEN ONE-HALF AND THE OTHER HALF THAT WOULD REQUIRE THE TWO TO BE THERE. SO USING THIS MULTI-ELECTRODE ARRAY WITH 16 ELECTRODES HE CAN VAMP BILL ACTIVITY ACROSS THE OLFACTORY BULB IN ANECESSARY THESE TIDESSED MICE WHILE DELIVERING ODORS TO THE NOSE. HERE IS AN EXAMPLE OF THE RECORDING, 16 TRACES, HERE IS THE LATERAL SIDE, HERE IS THE MEDIAL SIDE AND THESE ARE THE ELECTRODES NUMBER 1 TO 16, RESPIRATION, BREATHING RATE OF THE ANIMAL. AND WHEN HE PUFFS ODE RON AFTER A FEW BREATHS YOU CAN SEE THE RESPONSE APPEAR AND FROM THIS RAW SUGGESTIONNAL WE CAN EXTRACT THE SPIKES, THE FIRING OF THE NEURONS IN THE VARIOUS LAYERS. SO WHAT YOU'RE ACTUALLY SEEING HERE IS THE SENSORY INFORMATION ODOR INFORMATION AS IT FLOWS THROUGH THE LAYERS OF THE BULB FROM BOTH SIDES. SO YOU CAN SEE HERE WHEN WE LINE THIS UP, WE SUDDENLY REALIZE THAT EVEN AT THIS LOWER RESOLUTION, THERE'S ACTUALLY A SLIGHT DIFFERENCE IN THE RESPONSE BETWEEN MEDIAL AND LATERAL SIDE. THAT DIFFERENCE IS A TIME DREAM THROUGH ANALYSIS HERE IN THE SPIKE HISTOGRAM BECOMES MORE EVIDENT YOU CAN SEE THE MEDIAL SIDE RESPONDS SLIGHTLY BEFORE THE LATERAL SIDE. AND AGAIN, ILLUSTRATED IN THE CROSS CORRELATION MATCH THE RESPONSE IN THE VARIOUS LAYERING YOU CAN SEE THAT THERE'S A TEMPORAL SHIFT BETWEEN MEDIAL AND LATERAL SIDE, MEASURE WITH THE THE OFFSET LATENCY AS WELL. THE MEDIAL SIDE HAS A SHORTER LATENCY THAN THE LATERAL SIDE. NOW, WHAT WAS MORE INTERESTING IS AS YOU INCREASE THE ODOR CONCENTRATION, THAT THIS LATENCY OR THIS DECREASE, THIS DELAY ACTUALLY GETS SHORTER AND SHORTER. AND THIS HAPPENS BOTH IN THE MEDIAL SIDE AND LATERAL SIDE. THE DELAY IS THE SHADED AREA HERE. YOU CAN ACTUALLY SEE THAT AS YOU INCREASE THE CONCENTRATION IT GETS SMALLER, BUT IT ACTUALLY DECREASES A LITTLE BIT FASTER IN THE LATERAL THAN IN THE MEDIAL SIDE. EFFECTIVELY WHAT YOU'RE DOING IS AS YOU'RE INCREASING THE CONCENTRATION YOU'RE ACTUALLY BRINGING THE TWO SIDES INTO ALIGNMENT. THAT'S ILLUSTRATED HERE WHEN WE MEASURE THE -- WHEN WE PLOT THE CONCENTRATION AGAINST THE ONSET LATENCY, YOU SEE A DOWNWARD SLOPE ON BOTH SIDES BUT THE SLOPE ISN'T THE SAME SO AT SOME POINT THEY WILL MEET. SO THE HIGH CONCENTRATION THE MEDIAL LATERAL SIDE ARE BROUGHT INTO SYNCHRONY. SO HOW DO MEDIAL AND LATERAL -- THESE MEDIAL LATERAL TIMING DIFFERENCES RELATE TO THE INTRABULBAR PROJECTIONS? WHAT WE BELIEVE IS THAT THESE INTRABULBAR PROJECTIONS REGULATE TIME OF THE MITRAL CELL ACTIVITY TO COORDINATE THIS OUTPUT. DO WE HAVE ANY PRECEDENT FOR THIS? WE HAVE INFORMATION FROM A STUDY JESHANG DID A COUPLE OF MONTHS AGO RECORDING THE ACTIVITY OF MITRAL CELLS AND ASK WHAT EFFECT DOES THE FIRING OF THE TUFTED CELLS HERE THAT MEDIATE THE BULBAR PROJECTION HAVE ON THE TIMING OF MITRAL CELL, WHAT WE WERE ABLE TO DEMONSTRATE IN THIS STUDY, I'LL SUMMARIZE IT FOR YOU, IF SIGNAL IS COMING IN FROM THE OUTSIDE, SO AN ODOR SIGNAL IS COMING IN, AND THE TUFTED CELL ACTUALLY FIRES AFTER THE SIGNAL STARTS TO COME IN, IT WILL POTENTIATE THE RESPONSE OF THE MITRAL CELL. ON THE OTHER HAND IF THE SIGNAL IS TOM COMING IN AND THE MITRAL CELL IS CONTINUOUSLY FIRINGING, THE TUFTED CELL GETS THE BUST OF ACTIVITY, IT CAN FIRE THE MITRAL CELL AN SHIFT TIMING SO THERE'S A MECHANISM IN PLACE FOR ACTIVATION OF THESE TUFTED CELLS TO BE ABLE TO SHIFT FIRING OF THE MITRAL CELLS. AND CARL, WE'RE TRYING STOKES EMPLOYER WHETHER THAT'S SOMETHING FUNCTIONALLY USED IN VIVO TO ADJUST THE TIMING BETWEEN THE TWO SIDES SOME BACK TO THIS MODEL TO ADD THIS INFORMATION. INITIALLY I SHOWED YOU THAT WHEN AN ODOR COMES IN IT WILL ACTIVATE MULTIPLE P G PLUMERULI. I ILLUSTRATED IT THIS WAY. WHAT YOU CAN SEE IS THAT EACH PAIR IS RESPONDING AT THE SAME TIME, THOUGH THEY'RE RESPONDING THE DIFFERENT FREQUENCIES. THIS IS NOT EXACTLY TRUE. BECAUSE WHAT WE ESSENTIALLY JUST FOUND IS THAT THE TWO HALVES ARE NOT RESPONDING AT PRECISELY THE SAME TIME. SO IF WE JUST FOCUS ON THIS -- THESE TWO GLUMERULI THIS IS THE MORE ACCURATE. THEY'RE FIRING AT THE SAME FREQUENCY OR WITH SIMILAR RESPONSE BUT TIMING BETWEEN THE TWO IS NOT SYNCHRONOUS. SO THE OUTPUT OF THESE GLUMERULI TO THE CORTEX WOULD FOLLOW SUIT. WE KNOW LOW MOTOR CONCENTRATION WE'RE GOING TO ACTIVATE THE TWO SIDES BUT THE ACTIVITY BETWEEN THE TWO IS NOT SYNCHRONIZED. I'M EXAGGERATING HERE BECAUSE THE TIMING DIFFERENCE IS ONLY ABOUT 50 MILLISECONDS TO 100 MILLISECONDS DEPENDING UPON THE REGION OF THE B YOU'RE MEASURING AT HIGH CONCENTRATION WHAT YOU DO IS YOU TAKE THE TWO ACTIVATED SIDES AND BRING THEM INTO ALIGNMENT SO THEY'RE NOW RESPONDING SYNCHRONOUSLY SO THE INFORMATION THAT REACHES THE CORTEX IS GOING TO HAVE DIFFERENT TEMPORAL STRUCTURE. WE BELIEVE THAT INFORMATION IS THEN USED FOR SPRYGRATION OF THIS -- THESE SIGNALS IN THE CORTICAL AREAS POSSIBLY RECRUITING NEW PATTERNS OF ACTIVITY. THE INTRABULBAR PROJECTIONS ARE CAPABLE OF MODULATING THE FIRING OF MITRAL CELLS AND THAT'S WHAT THEY'RE DOING IN TERMS OF MODULATING ACTIVITY BETWEEN THE TWO SIDES. WE KNOW REGENERATING NEURONS AND POPULATION NEURONS THAT CONTINUES TO FLOW IN, CAN AFFECT THE DEGREE OR SIZE OF THE INTRABULBAR PROJECTIONS AND THEREFORE INFLUENCE THE SPAN OF THESE PROJECTIONS AND WHAT AREA THEY'RE ABLE TO SORT OF -- WHAT AREA THEY'RE ABLE TO MODULATE. SO HOW ARE WITH FOLLOWING UP ON THIS? WE'RE TRYING TO DO THIS WITH TWO DIFFERENT APPROACHES. ONE IS TO ESSENTIALLY DO IN VIVO IMAGING, AND P FOLLOW CHANGES IN REAL TIME. AND I JUST SHOW THIS ILLUSTRATION HERE. YOU CAN SEE EVEN DURING DEVELOPMENT FROM THREE DAYS TO THREE WEEKS YOU CAN SEE HOW MUCH CHANGE IS OCCURRING IN THE LAYERS OF THE OLFACTORY BULB. HERE I'M SHOWING THE SENSORY INPUTS COMING IN AND THE MITRAL CELLS. BUT EVEN FIRST WEEK YOU CAN SEE HOW COLUMN TAKE SHAPE AND THERE'S A LOT THAT CAN BE LEARNED BY MONITORING AND IMAGING THIS PROCESS AS IT'S OCCURRING. ESPECIALLY IF YOU CAN LOOK AT PAIRS OF GROUPS OF NEURONS THAT I HAVE SEEN SIMULTANEOUSLY AND INTERACTIONS CHANGING OVER TIME. THE SECOND IS TO ACTUALLY CONTROL THIS MEDIAL LATERAL ACTIVATION DIRECTLY IN THE OLFACTORY BULB AND RECORD THE OUTPUT IN THESE AWAKE BEHAVING ANIMALS. WE MADE A LINE OF MICE THAT WE CAN EXPRESS THIS MODIFIED CHANNEL RHODOPSIN IN A CELL TYPE WE LIKE BY CROSSING IT WITH A SPECIFIC -- BY SPECIFIC PROMOTER, IN THIS CASE OMP WHICH DRIVES EXPRESSION IN OLFACTORY SENSORY NEURONS. AND YOU CAN SEE THAT THE SENSORY NEURONS HERE ARE NICELY LABELED. WE CAN TAKE A CROSS SECTION THROUGH THE EPITHELIUM AND SEE HOW MANY OF THESE NEURONS ARE LABELED AND WE ALSO SEE THAT BEGET NICE COVERAGE ACROSS THE OLFACTORY BULB. SO BY THEN RECORDING ACROSS THE OLFACTORY BULB, WE CAN USE LIGHT THEN TO STIMULATE VARIOUS REGIONS OF THE OLFACTORY BULB AND THIS IS JUST AN EXAMPLE HERE OF RED LINE ACTUALLY DOESN'T ACTIVATE THIS CHANNEL. AND THIS IS ESSENTIALLY BACKGROUND ACTIVITY BUT IF YOU ACTIVATE WITH BLUE LIGHT WE CAN THEN ACTIVATE ONE-HALF OF THE BULB VERY SPECIFICALLY. VERY NICELY IN RESPOND TO THIS SLIDE SIGNAL. IF WE RETUNE THE LIGHT REARRANGE IT WE CAN RECRUIT ACTIVATION TWO THE TWO SIDES SO THIS GIVES A TOOL TO BE ABLE TO INTRODUCE PATTERNS OF ACTIVITY BETWEEN THE TWO SIDES AND SEE IF THIS TIMING ACTUALLY CHANGES THE RESPONSE OF THE ANIMALS AND THEIR ABLE TO SMELL. SO THEN WE CAN PUT IT TOGETHER IN AWAKE BEHAVING STUDIES IN ANIMAL LIKE THIS WHERE WE HAVE IMPLANTED ELECTRODE INTO THE OLFACTORY BULB AND YOU CAN SEE THIS ANIMAL SITTING IN A BOX, NOTHING INTERESTING TO SMELL, THIS IS RECORDING ACTIVITY IN THE OLFACTORY BULB ON THIS ANIMAL. AND JESHANG WILL INTRODUCE A Q TIP, YOU SEE THE ANIMAL EXPLORE THIS AND YOU SEE BURST OF ACTIVITY OCCURRING HERE. SO BY THEN COMBINING THIS KIND OF RECORDING WITH INTRODUCING OUR OWN PATTERNS OF ACTIVITY, WE CAN MEASURE THROUGH BEHAVIORAL STUDIES WHAT EFFECT THAT HAS AND ANIMAL ABILITY TO DECRIMINATE ODORS OR DETECT ODORS OF DIFFERENT QUALITY. I THINK A LITTLE BIT -- HOW AM I DOING ON TIME HERE? SO THE CONCLUSIONS FROM THIS, THE INTRABULBAR MAP UNDERGOES THIS DEVELOPMENTAL REFINEMENT WITH THESE DIFFUSE PROJECTIONS AT BIRTH. REFINES TO PRECISE MAP POSTNATALLY. THE FORMATION OF THIS INTRABULBAR PROJECTION IS INDEPENDENT OF ODOR INDUCED ACTIVITY. HOWEVER, THIS ACTIVITY IS NECESSARY BOTH REFINEMENT AND MAINTENANCE. WHAT I DIDN'T SHOW YOU HERE WAS THAT WHEN YOU ELIMINATE THE ACTIVITY OF OLFACTORY SENSORY NEURONS AT BIRTH YOU GET PROJECTIONS CAPABLE OF REACHING THE OTHER SIDE, THEY JUST FAIL TO REFINE. SO THE ACTIVITY IS REALLY NECESSARY FOR THE REFINEMENT PROCESS. I ALSO SHOW YOU BROAD ODOR INDUCED ACTIVITY CAN BLOCK POSTNATAL REFINEMENT OF INTRABULBAR PROJECTIONS, IT CAN ALSO DISRUPT ORGANIZATION OF THIS MATURE MAP AND SUGGEST ODOR INDUCED ACTIVITY SEEMS TO PLAY AN INSTRUCTIVE ROLE RATHER THAN A PERMISSIVE ROLE IN THIS PROCESS. I SHOW YOU REEXPOSURE TO ENVIRONMENT TO ODERANTS CAN RESULT IN THE REREFINEMENT OF INTRABULBAR PROJECTIONS, WITH DISRUPTING OF THEIR SECLUSION, REQUIRE MORE TIME TO RECOVER THAN DISRUPTION FROM BROAD ACTIVATION THAT'S POINTING OUT THE FACT WHEN YOU BLOCK IT TAKES SOMEWHERE BETWEEN 6 TO 9 WEEKS FROM REREFINEMENT TO OCCUR, WHEREAS WITH BROAD ACTIVATION IT RECOVERS RAPIDLY. WHAT WE THINK IS THE REASON FOR THAT, WHEN YOU BLOCK ACTIVITY THROUGH A BLOCK YOU LOSE REGENERATING NEURONS SO THIS BRINGS INTO PLAY THE FACT THAT YOUR AFFECTING TWO FACTORS, YOU'RE AFFECTING NOT ONLY LOSS OF ACTIVITY BUT YOU'RE LOSING THIS POPULATION OF NEURONS THAT WE LATER FOUND OUT IS IMPORTANT FOR MODULATING THESE CONNECTIONS. REGENERATING NEURONS AS I JUST MENTIONED ARE NECESSARY FOR THIS INTRABULBAR MAP RESTORATION BUT ALSO MAINTENANCE. AND THE INTRABULBAR PROJECTIONS SPECIFICALLY PROJECT TO MULTIPLE GLUMERULI EXPRESSING THE SAME ODERANT PROCORRECTOR AN ACTIVITY PATTERNS THROUGH THE GLUMERULI THROUGH ODOR CIRCUITRY ARE USED TO LINK. I ILLUSTRATEDDED THIS BY SHOWING WHEN YOU HAVE GLUMERULI PROJECTIONS ARE CAPABLE OF TARGETING BOTH OF THEM. SO OUR IDEA IS WE KNOW THERE EXISTS A TIMING DIFFERENCE BETWEEN -- IN THE ODERANT INDUCED RESPONSE MEDIAL AND LATERAL OLFACTORY BULB AND THIS IS CONCENTRATION DEPENDENT AND WE BELIEVE THIS IS THEN USED FOR SENSORY CODING AND INTEGRATION OLFACTORY BULB SIGNALS IN THE CORTEX AND WHAT WE'RE TRYING TO UNDERSTAND IS WHAT THE NATURE OF THAT CODE IS AND WHAT IT'S USED FOR. SORT OF AS A FINAL WRAP UP, THIS IS THE CIRCUIT THAT WE STARTED OUT WITH. AND I TOLD YOU A LITTLE BIT ABOUT HOW THESE PROJECTIONS FORM MAPS NOT ONLY ON THE SURFACE BUT ALSO THE INTRABULBAR MAP AND WHAT THE RELATIONSHIP IS BETWEEN THESE TWO CONNECTIONS. ALSO HOW REGENERATING NEURONS PLAY A ROLE IN ORGANIZING AND MAINTAINING THIS CIRCUITRY AS WELL AS HOW INFORMATION COMING THROUGH THIS IS UTILIZED. SO WHAT I HOPE I HAVE CONVINCED YOU OF IS THESE THREE FACTORS, REGENERATION, PLASTICITY AND ACTIVITY, ARE REALLY ALL WORKING TOGETHER IN A VERY BALANCED WAY TO BE ABLE TO BRING FUNCTIONAL STABILITY TO THIS CIRCUIT. AND PROBABLY MOST IMPORTANTLY, KEEP IN MIND THAT THIS IS DURING ADULT HOOD AND CONTINUES TO HAPPEN THROUGHOUT LIFE. FROM Z SO WITH THAT I WANT TO THANK THE FOLKS THAT DID ALL THE WORK. THIS IS MY CURRENT LAB, I DIDN'T HAVE TIME TO TALK ABOUT ALL OF THE WORK THEY'RE DOING BUT THE WORK THAT I DESCRIBE WAS PRIMARILY DONE BY DAN CUMMINGS, WHO DID ALL THE TASER INJECTIONS -- TRACER INJECTIONS AN ANATOMICAL STUDIES, (INDISCERNIBLE) WITH THE UBI-7 AS WELL AS OPTO GENETIC STUFF. SHSHANG JOU WHO DID ALL THE RECORDINGS AND MULTI-ELECTRODE STUFF AND COMBINATION WITH THE OPTO GENETIC WORK. AND THEN FORMER STUDENT CAROLYN MARKS AND FORMER POST DOC WHO NOW MOVEDDED ON TO BIGGER AND P BETTER THINGS. OF COURSE MY COLLABORATORS WHOM WITHOUT THEIR SUPPORT AND HELP, I WOULD NOT HAVE BEEN ABLE TO DO THIS. SO THANKS VERY MUCH AND I'M HAPPY TO TAKE QUESTIONS. [APPLAUSE] >> QUESTIONS? COULD YOU GO TO THE MICROPHONE? WE ARE INSTRUCTED TO SEND YOU THE MICROPHONE. >> I'M WONDERING ABOUT THE ROLE OF REGENERATING NEURONS THAT ARE COMING IN. MORE PARTICULARLY ABOUT CELLS THEY'RE REPLACING SO PRESUMABLY YOU'RE REGENERATING NEURON BECAUSE GRANULE CELLS THERE ARE TURNING OVER. THAT'S A GUESS ON MY PART. HOW FAST ARE THE CELLS THAT ARE ALREADY THERE GOING AWAY? IS SOME OF THE SPREAD YOU GET IN ABSENCE OF REFINEMENT BECAUSE GUYS THAT ARE LEFT HAVE TO REACH OUT AND COVER MORE TERRITORY? >> ALL GOOD QUESTIONS THAT I DON'T THE SPECIFIC ANSWER TO. THERE'S A LOT OF DIFFERENT ANSWERS TO THAT, DIFFERENT THEORIES. PART OF IT IS THAT THE REGENERATING POPULATION OF NEURONS MIGHT ACTUALLY BE A UNIQUE AND DISTINCT GROUP WHEREAS THE STABLE ONES MIGHT JUST BE THERE. THERE'S SOME PRECEDENCE FOR THAT. THE FACT THAT THESE INTRABULBAR PROJECTIONS ARE THE FIRST CLEAR ANATOMICAL SIGN OF WHAT'S CHANGING, NOT JUST LOSS OF CELLS IN THE BULB BUT WHAT CHANGES WHEN NEW NEURONS ARE ELIMINATED, I THINK GIVES US NOW A FOOTHOLD INTO SAYING WHAT'S CHANGING IN THIS PARTICULAR CONNECTION THAT MIGHT BE ABLE TO ANSWER THIS QUESTION BUT I DON'T HAVE A CLEAR ANSWER FOR YOU. >> YOU MENTION ABOUT THE MIGRATION DEGENERATION AND CONNECTIONS. DO YOU HAVE ANY EXTERNAL MATRIX WHAT IS HAPPENING IN TERMS OF THIS PARTNER THERE FORMING A LINK BREAKING AND MOVING FORWARD? -- CELLS TO MIGRATE AND MAKE ALL THE CONNECTIONS SO ANY IDEA ABOUT THIS EXTRA CELLULAR MATRIX THAT MIGHT BE DYNAMIC ENVIRONMENT? >> RIGHT. WE HAVEN'T LOOKED AT EXTRA CELLULAR MATRIX PROTEINS AT ALL. OTHER THAN THE FACT THAT THE BULB ITSELF ACTUALLY CHANGES IN SIZE WITH SOME OF THE LOSS AND RECOVERY OF THE NEURONS. SO CERTAINLY VOLUME WISE THERE ARE NOTABLE CHANGES. WHICH SPECIFIC FOCUS WITH EXTRA CELLULAR MATRIX PROTEIN WE HAVEN'T LOOKED. >> YOU GET OLDER LIKE ME IN TERMS OF THE RECEPTION TO THESE DIFFERENT SMELLS. >> YOUNG MICE, ANY AFFECT OF THE AGING ON SYSTEM? >> AGING OF THE SYSTEM. OUR FOCUS HAS BEEN ON THESE PROJECTIONS AND HOW THOSE ARE AFFECTED BY ACTIVITY. THERE'S CERTAINLY PLENTY OF INFORMATION OUT THERE TO DEMONSTRATE THAT OLFACTORY LOSS OCCURS GRADUALLY WITH AGING. HOW THIS CORRELATES WITH INTRABULBAR PROJECTIONS WE HAVEN'T LOOKED AT SO I DON'T REALLY KNOW. >> THAT WAS A GREAT TALK. >> THANK YOU. >> I WAS PARTICULARLY INTERESTED IN THE FIRST PART WHEN YOU'RE TALKING ABOUT THE UBI-7 MICE. YOU LOST THE FUNCTIONAL SPECIFICITY OF THE MAP, YOU CREATE AD MAP THAT A SINGLE (INAUDIBLE) ACTIVATE IT IS WHOLE -- AND THE FUNCTION SPECIFICITY IS THERE TO GUIDE BEHAVIOR INTERACTION TO FOOD, PREDATORS, ET CETERA. SO DID YOU SEE ANY OBVIOUS BEHAVIORAL CHANGES, MODIFICATIONS IN THE UBI-7 MICE? AND ALSO HOW THIS CHANGING SPECIFICITY FUNCTION SPECIFICITY IN THE OLFACTORY BULB MAP IS REFLECTED IN THE CORTEX, ANY OBVIOUS CHANGES IN OLFACTORY CORTEX? >> SO THE FIRST PART OF THAT WHICH IS HOW THE -- I GUESS MAYBE I WASN'T CLEAR BUT THESE ANIMALS ARE EXPRESSING NORMAL REPERTOIRE OF ODERANT RECEPTORS, SO WE DON'T NOTICE ANY FUNCTIONAL DIFFERENCE BETWEEN THEIR ABILITY TO EAT AND FORGE FOR FOOD. IN FACT WE HAVE TESTD THEM IN ANY NUMBER OF ASSAYS AND THEY APPEAR QUITE NORMAL. WE'RE EXPLORING WHAT THE AFFECT IS OF INTRODUCING IT INTO THE ENVIRONMENT WHILE THEY'RE TRYING TO DO THESE THINGS, THAT'S SORT OF ONGOING WORK. BUT OTHERWISE THEY ACTUALLY FUNCTION QUITE NORMALLY. WITH RESPECT TO THE PROJECTIONS FROM THE OLFACTORY BULB TO THE CORTEX, WE'RE DOING RECORDINGS THERE BUT PROJECTIONS TO THE CORTEX ARE MORE COMPLICATED. THEY'RE VERY DIFFUSE. AND SO IT'S DIFFICULT TO SEE WHAT THE CONSEQUENCES ARE ANATOMICALLY. E WHICH WE MIGHT BE ABLE TO DETECT EFFECTS USING ELECTROPHYSIOLOGY. >> YOU SHOWED YOUR WORK SHOWS THAT CONSTANT INFLUX OF REGENERATIVE NEURONS LIKE CELLS IS REQUIRED FOR KEEPING THIS REFINED STATEMENT IN OTHER SENSORY MODALITIES WHERE THERE'S ALSO REFINED STATES AND THERE'S NOT A CONSTANT INFLUX. SO WHAT DO YOU SUPPOSE -- IT'S MAINTAINED, WHAT DO YOU SUPPOSE THE OLFACTORY SYSTEM IS A LITTLE BIT DIFFERENT THAT WAY? COMPARED TO OTHER MODALITIES? Q. I THINK THE OLFACT ROUGH SYSTEM IS SPECIAL. -- I THINK THE OLFACTORY SYSTEM IS SPECIAL. TO SAY THAT SORT OF GETTING INTO BIGGER QUESTIONS ABOUT WHAT'S CAPABLE OF FORMING A FUNCTIONALLY STABLE CIRCUIT AND IN SOME WAYS I THINK THIS IS ALSO GETTING INTO THE QUESTION OF WHY THE OLFACTORY SYSTEM HAS HIGH LEVEL PLASTICITY TO BEGIN WITH. IT'S KIND OF A CHICKEN AND EGG THING BECAUSE I THINK PART OF THE REASON WHY THIS CONSTANT TURN OVER AND REPOPULATING OF THE CIRCUITRY IS THERE, IS REALLY TO COMPENSATE FOR HIGH LEVEL PLASTICITY THAT'S THERE. SO IT IS A BALANCE BETWEEN THE TWO AS I WAS TRYING TO ILLUSTRATE. WHY THAT'S THE CASE IN THE OLFACTORY SYSTEM, I CAN SPECULATE BUT I DON'T REALLY KNOW FOR SURE. THERE'S IDEAS OUT THERE THAT IT'S ACTUALLY ONE OF THE FEW PLACES WHERE YOU HAVE SENSORY NEURONS EXPOSED TO THE ENVIRONMENT SO THEY'RE REALLY EXPOSED TO LOTS OF DAMAGE, SO THIS TURN OVER RATE FROM THE PERIPHERY, FOR INSTANCE, IS REALLY A WAY OF COMPENSATING FOR THE LOSS OF NEURONS THAT WOULD COME THROUGH DAMAGE SAY. THEN YOU CAN THEN EXTEND THAT ARGUMENT TO SAY THEN THE CHANGES THAT ARE OCCURRING DOWNSTREAM IN THE OLFACTORY BULB ARE REALLY COMPENSATING FOR THE CHANGES THAT ARE GOING ON IN THE PERIPHERY BUT THIS IS REALLY ALL JUST SORT OF HANDLING. >> THIS IS A BIT RELATED. WONDERING YOU HINTED AT THIS POSSIBILITY THAT THE NEW NEURONS IN THE OLFACTORY BULB SEEM TO BE PROMOTING STABILITY MIGHT BE DIFFERENT FOR NEW NEURONS OR WHAT NEW NEURONS ARE DISTRIBUTING IN OTHER REGIONS. AND IT MIGHT BE CONTRIBUTING TO LEARNS AND MEMORY. CAN YOU EXPAND ON THAT A LITTLE? THE ROLE OF KNEW NEURONS IN YOUR SYSTEM VERSUS OTHER? >> RIGHT. I THINK THIS IN SOME WAY TOUCHES ON THE PREVIOUS QUESTION WHICH IS IN THE ROLE OF KNEW NEURONS AND SYSTEM THAT MIGHT BE STABLE AND FIXED, THEY MIGHT BE PLAYING A SLIGHTLY DIFFERENCE ROLE THAN -- DIFFERENT ROLE THAN A SYSTEM IN THE OLFACTORY SYSTEM WHERE THERE'S INHERENT PLASTICITY BUILT IN AND PERHAPS THE KNEW NEURONS COMING IN AND COMPETING FOR SPACE IS PART OF THE MECHANISM THAT THE SYSTEM USES TO SAY TO FUNCTION SO TO SPEAK. AGAIN I DON'T KNOW, THAT'S NOT REALLY CLEAR, BOTTOM LINE I DON'T KNOW WHY IT'S HAPPENING, IT'S ONE OF THE PLACES WHERE YOU HAVE A CONSTANT FLOW OF NEURONS AN THIS WAS BIT OF A SURPRISE TO US THAT WE SAW THERE WERE NECESSARY TO MAINTAIN THIS ORGANIZATION. >> BEAUTIFUL IMAGES, BEAUTIFUL MODEL. I'M WONDERING IF YOU HAVE THOUGHTS ABOUT WHAT ELEMENTS IN YOUR MODEL AND SYSTEM, SEEM TO BE SENSITIVE IN PARKINSON'S DISEASE? GIVEN THAT LOSS OF SMELL IS EARLY MARKER AND THERE'S A LOT OF INTEREST IN THAT. ALSO WHETHER EITHER FROM HUMAN HUMAN MATERIALS OR MOUSE MODELS THAT EXPRESS GENES INONU SOCIALIATED WITH PARKINSON'S DISEASE WHETHER ANYONE HAS MADE A CONNECTION BETWEEN ANY OF THESE COMPONENTS OF THE SYSTEM AND POINT OF SENSITIVITY WEAKNESS IN THE PARKINSON ANIMAL. >> RIGHT. WE ACTUALLY NOT EXPLORED VERY DEEPLY TYPE THE PARKINSON CONNECTION THERE ARE TH NEURONS IN THE OLFACTORY BULB AND THAT'S A ENTICING PLACE TO SAY ARE THERE LOSS OF NEURONS ASSOCIATED WITH THE L OLFACTORY LOSS PARKINSON. MORTEMMED TO SAY IT'S THE SENSORY NEURONS THAT ARE LOST. WE HAVE BEEN TO FOCUSING MORE MORE HEAVILY ON CONNECTION WITH ALZHEIMERS AND WHERE THE EVIDENCE IS POINTING TO THE SENSORY NEURONS THE HIGHLY SENSITIVE PART OF THAT THAT'S LOST. WHERE THAT'S THE BEGINNING OF THE PROCESS THAT THEN EXTENDS FURTHER, THAT IS ONE OF THE THINGS THAT WE'RE LOOKING AT. >> WHEN YOU BLOCK THE RMS, THIS IS ACTUALLY A BEAUTIFUL EXPERIMENT AND YOU SEE THOSE ANATOMICAL CHANGES. DO THEY SEE CHANGES IN THE ABILITY TO DISCRIMINATE BETWEEN DIFFERENT ODOR? FROM ANIMALS THAT BLOCKED IN ANIMAL WHERE IS YOU DIDN'T BLOCK IT? >> RIGHT. STAND BY. WE'RE DOING THOSE EXPERIMENTS PART OF WHAT WE'RE TRYING TO DO WE HAVE VARIOUS MODEL CAPABLE OF DISRUPTING THESE PROJECTIONS THROUGH DIFFERENT MECHANISMS. SO IT'S BEEN VERY DIFFICULT TO TEASE APART WHAT THE EFFECT THE FUNCTIONAL EFFECT OF THESE CHANGES REALLY ARE BECAUSE MICE ARE VERY GOOD AT SMELLING. ANYTHING THAT WOULD COMPLETELY DISRUPT THE SYSTEM, WOULD BE EASY TO FIND BUT WHAT WE IMAGINE HAPPENING, THIS IS A TUNING OF THE SYSTEM AND NOT SO MUCH AN ELIMINATION OF ABILITY TO SMELL. SO ZEROING IN EXACTLY ON WHAT THE AFFECT OF THESE CHANGES REALLY ARE, WE HAVE GOTTEN SOME CLUES SO I THINK WE'RE NOW TESTING THE RIGHT THINGS BUT WE WANT TO TEST ALL OF THE VARIOUS MODELS TO SEE IF THIS IS A COMMON ELEMENT IN ALL OF THEM. THAT IS ONE OF THE ONES WE'RE QUITE INTERESTING IN TESTING. >> IF THERE ARE NO FURTHER QUESTIONS LET'S THANK LEO FOR A BEAUTIFUL ELEGANT TALK. [APPLAUSE] >> I'M SURE THERE ARE OTHER QUESTIONS AND LEO WILL BE HERE TO TALK TO YOU FOR AT LEAST A FEW MORE MINUTES. SO THANKS.