>> GOOD AFTERNOON, EVERYBODY. I'M SO DELIGHTED TO SEE ALL OF YOU HERE. I'M JANINE CLAYTON, DIRECTOR OF THE OFFICE OF RESEARCH ON WOMEN'S HEALTH, IT'S MY PLEASURE TO INTRODUCE THE WALS LECTURER, DR. YANG DAN. FOLLOWING UNDERGRADUATE STUDIES IN PHYSICS AT BEIJING UNIVERSITY, SHE COMPLETED A Ph.D. IN NEUROBIOLOGY AT COLUMBIA UNIVERSITY. SHE WAS A FELLOW AND MOVED TO POSTDOC WORK AT ROCKEFELLER AND HARVARD MEDICAL SCHOOL, HER LAB USES ELECTROPHYSIOLOGY IMAGING, OPTOGENETICS TO STUDY THE MAMMALIAN BRAIN, MICROCIRCUITS AND MECHANISMS FOR CORTICAL PLASTICITY FROM SYNAPSE TO PERCEPTION, HER LABOR MAD CONTRIBUTIONS UNDERSTANDING SYNAPTIC BASES. HER RECENT WORK REVEALED THE MECHANISMS BY WHICH BRAIN STEM I HYPOTHALAMUS AND BASAL EXERT ON SLEEP. TRAINEES HAVE GONE TO TENURE TRACK POSITIONS. SHE WILL SPEAK REGARDING NEURAL CIRCUITS REGARDING SLEEP. JOIN ME IN WELCOMING DR. YANG DAN. [APPLAUSE] >> CAN YOU HEAR ME? I'M HONORED TO BE GIVEN THE OPPORTUNITY TO PRESENT IN THIS PRESTIGIOUS LECTURE SERIES. THANK YOU SO MUCH FOR COMING HERE ON THIS SNOWY DAY. I'D LIKE TO TELL YOU ABOUT OUR STUDIES OVER THE LAST FIVE YEARS NEUROCIRCUITS CONTROLLING SLEEP. MAMMALS AND BIRDS HAVE WAKEFULNESS, REM AND NON-RECOMMEND, DISTINGUISHED WITH EEG AND EMG. WE HAVE VIVID SENSATIONS OF EXTERNAL EVENTS. THIS SHOWS THE DESYNCHRONIZED PATTERN, EMG SHOWS HIGH MUSCLE TONE AND DURING NON-REM SLEEP, ALSO CALLED SLOW WAVE SLEEP, THE EEG IS HAS LARGE SLOW WAVES, EMG SHOWS REDUCED BASAL TONE, ABSENCE OF SENSATION. REM SLEEP, RAPID EYE MOVEMENT SLEEP, WAS DISCOVERED ONLY ABOUT 60 YEARS AGO IN THE 1950s. IT'S ALSO CALLED PARADOXICAL SLEEP, IN THE SENSE THAT THE EEG IS VERY SIMILAR TO THE WAKEFULNESS EEG, BUT THE EMG SHOWS A LACK OF MUSCLE TONE, THE SO-CALLED MUSCLE ATONIA OR REM PARALYSIS, WHEN WE HAVE VIVID DREAMS AND SO THE IDEA IS THAT DESYNCHRONIZED EEG, WAKE-LIKE, IS WHAT ALLOWS US TO HAVE THIS SORT OF VIVID MENTAL EXPERIENCE, BUT MUSCLE PARALYSIS CAN PREVENT US FROM ACTING OUT OUR DREAMS. THE QUESTION THAT WE HAVE BEEN FOCUSING ON IS WHAT MECHANISMS COMPOSE SLEEP AND WAKEFULNESS? NOW, OVER THE PAST MANY DECADES, STUDIES FROM MANY LABS AROUND THE WORLD HAVE IDENTIFIED MULTIPLE BRAIN REGIONS IMPORTANT INCLUDING BRAIN STEM, HYPOTHALAMUS AND BASAL FOREBRAIN. THESE INCLUDE MANY OF THE WELL KNOWN NEUROMODULATORY CIRCUITS. FOR EXAMPLE, NEUROADRENALINE, HISTAMINE, DOPAMINE ANDER SEROTONIN AND SO FORTH, AND SOME NEUROPEPTIDES INCLUDING MPH AND OREXIN. MOST WELL KNOWN NEUROMODULATORY SYSTEM AS FAR AS SLEEP-WAKE REGULATION, THEIR FUNCTION IS TO WAKE UP THE BRAIN WITH VERY FEW EXCEPTIONS. THERE ARE MANY CONVENTIONAL NEURONS IN THESE BRAIN AREAS, AND AS IT TURNS OUT, IF YOU JUST RUN THEM THE NEURONS ACTIVATE, NINE OUT OF TEN CASES YOU FIND THE NEURONS COME FROM A WAKEFULNESS RATHER THAN DURING SLEEP. I DIDN'T NOTICE FIVE YEARS AGO WHEN I GOT INTO SLEEP RESEARCH, BUT NOW I NOTICE VERY WELL. JUST TO GIVE YOU AN IDEA WHAT I'M TALKING ABOUT, HERE IS THE HYPOTHALAMUS, EVERY TIME YOU SEE THE BLUE TURNING ON, WE TURN ON THE LASER. WE CAN SEE THE MOUSE IS GOING CRAZY IN THIS TRIAL, AND EVEN AFTER WE TURN OFF THE LASER HE WAS STILL GOING CRAZY. THIS IS THE SECOND TRIAL. HE WAS IN NON-REM SLEEP BUT YOU MOMENT YOU TURN ON THE LASER THEY STARTS RUNNING AROUND IN AN AGITATED WAY. THIS IS WHAT YOU SEE PRETTY MUCH MOST OF THE TIME IF YOU JUST OPT GENETICALLY ACTIVATE SOME NEURONS IN THESE BRAIN REGIONS. IN OTHER WORDS, IT'S EASY TO FIND NEURONS THAT CAN WAKE UP THE BRAIN, HARDER TO FIND NEURONS THAT CAN TRIGGER SLEEP. THE OBSESSION OF MY LAB RECENTLY HAS BEEN TO TRY TO SEARCH FOR THE NEURONS THAT CAN PROMOTE SLEEP, WITH THE HOPE THAT ONCE WE UNDERSTAND THE CIRCUIT WE CAN PERHAPS COME UP WITH WAYS TO IMPROVE SLEEP BECAUSE INSOMNIA IS A VERY COMMON SORT OF PROBLEM IN THE MODERN SOCIETY. I'M GOING TO FIRST FOCUS ON THIS REGION, THE BRAIN STEM, AND THEN IN THE SECOND PART I'LL MOVE TO THE MORE ANTERIOR PART OF THIS CIRCUIT. THE BRAIN STEM HAS BEEN TRADITIONALLY CLOSELY LINKED TO REM SLEEP, AND THIS WAS FIGURED OUT USING THE TRANSSECTION STUDIES, PIONEERED IN THE 1960s AND FOLLOWED BY MANY INVESTIGATORS, BUT THIS IS A DIAGRAM FROM REVIEW PAPER BY SIEGEL, WHAT PEOPLE FOUND IS THAT IF YOU MAKE A CUT OR TRANSECTION LEVEL A OR B, THIS IS A SAGITTAL VIEW SHOWING THE BRAIN STEMF YOU MAKE A CUT OR A OR B, THEN IN THE FOREBRAIN REGION ANTERIOR TO THE CUT YOU SEE NO SIGN OF REM SLEEP BUT YOU WOULD STILL SEE ONCE IN A WHILE REM ATONIA, LOSS OF MUSCLE TONE. A CUT AT C YOU SEE NORMAL REM IN THE FOREBRAIN BUT NO SPINALLY MEDIATED ATONIA. THIS IS SUMMARIZING MANY LESION STUDIES FROM MANY LABS, BUT THESE STUDIES SUGGEST THAT THE REGION BETWEEN A, B AND C WHICH INCLUDES THE PONS AND MEDULLA IS IMPORTANT. THOSE BRAIN REGIONS ARE INVOLVED IN ANY OTHER STUDIES, TO IDENTIFY SPECIFIC NEURONS IMPORTANT FOR SLEEP, THE TRADITIONAL METHOD HAS BEEN TO -- ONE METHOD IS TO USE FOS STAINING. YOU DO A REM SLEEP DEPRIVATION, THERE ARE DIFFERENT WAYS BUT THE GENERAL WAY IS EVERY TIME THE ANIMAL GOES INTO REM SLEEP YOU WAKE THEM UP, POKE THEM OR USE OTHER HIGHER TECH METHODS FOR SEVERAL HOURS, THEN YOU LEAVE THE ANIMAL ALONE, THAT CAUSES REM SLEEP REBOUND BECAUSE BOTH REM AND NON-REM SLEEP ARE HEAVILY REGULATED HOMEOSTATICALLY. AFTER DEPRIVATION, YOU LEAVE THEM ALONE, THEY SPEND MORE TIME IN REM SLEEP. LEAVE THEM FOR TWO HOURS, ENOUGH TIME FOR THE IMMEDIATE FOS TO BE EXPRESSED. THIS IS THE STUDY, A CROSS SECTION CUT AT THIS LEVEL, YOU CAN SEE THE POSTERIOR IN THE BRAIN STEM, ACTUALLY THE MEDULLA, THE C-FOS+ NEURONS, GABAERGIC. IT'S A CASE THESE ARE REM SLEEP ACTIVE. THIS IS USUALLY THE FIRST STEP TO SAY PERHAPS THESE NEURONS ARE IMPORTANT FOR REM SLEEP BECAUSE IF THEY ARE NOT ACTIVE DURING REM IT'S HARD TO IMAGINE THEY ARE IMPORTANT FOR REM SLEEP. SO BUT THE QUESTIONS THAT WE ASK AFTER BEING INSPIRED BY THESE RESULTS IS FIRST DOES THE MEDULA DOES THE GABAERGIC CAUSE REM SLEEP, THERE'S A CORRELATION BUT IT DOESN'T MEAN CAUSALITY. SECOND, ARE THE NEURONS SPECIFICALLY ACTIVATED DURING REM? THIS IS IMPORTANT BECAUSE EVEN THOUGH THESE NEURONS ARE C-POS POSITIVE, WE SPEND 10% OF THE TOTAL TIME IN REM SLEEP, SO REM REBOUND CAUSES 10% TO INCREASE TO 40%, IT'S A FOUR-FOLD INCREASE, VERY DRAMATIC, EXCEPT THAT DURING THE TWO HOURS THE ANIMAL SPENDS 60% OF TIME IN OTHER BRAIN STATES SO THE FACT THAT THESE NEURONS ARE C-POS POSITIVE, WE'RE NOT SURE THESE ARE SPECIFICALLY ACTIVE DURING REM. THE ONLY WAY TO TELL IS RECORD FROM THE REM. AND THEN IF ACTIVITY CAUSES REM SLEEP, WHAT ARE THE DOWNSTREAM TARGETS, IF THE NEURONS ARE SPECIFICALLY ACTIVE DURING REM WHAT ARE THE INPUTS? THESE ARE THE STANDARD QUESTIONS WE ASK WHEN WE TRY TO SEE GABA NEURON CIRCUITS UNDERLINE BEHAVIOR. SO TO ANSWER THE FIRST QUESTION, WHAT WE DID IS STRAIGHTFORWARD AND SIMPLE. NOW WE HAVE A MAGIC TOOL, OPTOGENETICS, WE INJECT THE AMB VIRUS, WHERE THEY FOUND THE C-POS, LPGI, THE AED CARRIES THE GENE IN A PRE-DEPENDENT MANNER. IF WE INJECT INTO CRE, THEY WILL BE EXPRESSED IN THE GABANERGIC. THIS IS A PICTURE OF THE REGION. THEN WE TURN ON THE LIGHT AND SEE WHAT HAPPENS. SO HERE IS A MEDIAL, TO GIVE YOU AN IDEA WHAT THE EXPERIMENT IS LIKE. AGAIN ON THE LEFT IS THE VIDEO OF THE ANIMAL, SHE'S PROBABLY ASLEEP. WHEN YOU SEE A BLUE, WE TURN ON THE LASER, THIS UPPER PANEL SHOWS THE POWER SPECTRUM OF THE EEG. EEG IS A VERY IMPORTANT MEASURE FOR DETERMINING THE BRAIN STATE, SO IF YOU SEE A LOT OF WARM COLOR DOWN HERE AT THE LOW FREQUENCIES IT MEANS THAT THE SYNCHRONIZED STATE INDICATES REM SLEEP. IF YOU SEE A THIN BAND, 8 HERTZ, USUALLY THAT MEANS THE ANIMAL IS IN REM SLEEP, AND IF YOU SEE A REDUCTION OF THE LOW FREQUENCY, AND IF YOU SEE A HIGH CMG THAT MEANS THE ANIMAL IS AWAKE. HERE IN THE FIRST TRIAL YOU SEE A LOT OF LOW FREQUENCY ACTIVITY IN NON-REM SLEEP, YOU SEE AS SOON AS WE TURN ON THE LASER QUICKLY THERE'S A CHANGE OF THE EEG PATTERN. AND AT THIS POINT YOU START SEEING THIS VERY THIN BAND. SO THIS IS THE THETA BAND, IT STAYS LOW, THE ANIMAL WAS IN REM SLEEP. HERE IS ANOTHER TRIAL, AGAIN IN NON-REM SLEEP BECAUSE OF THE BROADBAND ACTIVITY IN THE EEG, TURN ON THE LASER PRETTY QUICKLY, YOU SEE A CHANGE, NOW YOU SEE A VERY THIN THETA BAND. YOU SEE THE INCREASE IN THE EMG AS IT WOKE UP. THIS IS HOW WE DO THE EXPERIMENT. AND THIS IS A SUMMARY OF THAT PARTICULAR MOUSE, BUT RECORDING OVER A LONGER PERIOD OF TIME. HERE WE APPLY LASER IN FOUR TRIALS. NOW, THE LASER WAS APPLIED FOR TWO MINUTES IN EACH TRIAL, AND INTERTRIAL INTERVAL IS 15 TO 25 MINUTES, SEMI RANDOM, CONTROLLED BY THE COMPUTER. NOW, SO IF YOU DON'T WANT TO ALWAYS PICK UP THE EEG AND EMG YOU CAN LOOK AT THE COLOR CODED BRAIN STATE DETERMINED BASED ON EEG AND EMG AUTOMATICALLY BY THE COMPUTER PROGRAM. SO YOU CAN SEE IN THREE OF THE FOUR TRIALS, RIGHT, THE LASER WAS TURNED ON WHEN THE ANIMAL WAS IN NON-REM SLEEP. THIS GRAY REGION. SOON AFTER THE LASER WAS TURNED ON, IT TURNED BLUE, THAT MEANS THE BRAIN TRANSITIONS TO REM SLEEP. THAT HAPPENED HERE, HERE AND HERE. IN ONE OF THE TRIALS WE TURN ON THE LASER WHEN THE ANIMAL WAS AWAKE, AND NOTHING HAPPENED. THIS ANIMAL STAYED AWAKE. SO THIS IS A SUMMARY OF THE DATA FROM SIX MICE, ALL THE TRIALS THAT WE APPLIED. WE ALIGNED THESE TRIALS BY THE KIND OF LASER STIMULATION, THIS BAR HERE, TWO MINUTES PER TRIAL. EYEBALLING IT DURING LASER PERIOD THERE'S A LOT OF LIGHT BLUE INDICATING HE WAS MOST OF THE TIME IN REM SLEEP. NOW, THESE LINES, SUM SHOW THE PROBABILITY OF THE SLEEP REM STATE, SO NON-REM LIGHT GRAY, SO BEFORE LASER IT SPENDS 60% OF TIME IN NON-REM SLEEP, AND THIS IS NORMAL FOR MICE BECAUSE THEY ARE NOCTURNAL, SO WE DO EXPERIMENTS DURING THE DAY, SO THIS IS THEIR AVERAGE PROBABILITY NON-REM SLEEP. AS SOON AS YOU TURN ON THE LASER, THIS GRAY LINE, YOU SEE THIS DRAMATIC DROP, ALMOST TO ZERO. AND ACCOMPANYING THAT THERE'S A DRAMATIC INCREASE OF THE LIGHT BLUE LINE, DRAMATIC INCREASE OF REM SLEEP. NOW, THERE'S ALSO SLOWER INCREASE OF THE PURPLE LINE WHICH IS THE PROBABILITY WAKEFULNESS. I JUST WANT TO SAY HERE ACTUALLY WE DON'T THINK THAT THIS IS BECAUSE THE LASER WAS TRIGGERING WAKEFULNESS. WE THINK THE INCREASE IS BECAUSE OF THE FACT THAT IN RODENTS, AFTER REM SLEEP THEY ALWAYS WAKE UP. THEY DON'T GO BACK TO NON-REM SLEEP, FOR SOME REASON. I THINK HUMANS CAN DO THAT BUT RODENTS CAN DO THAT. BECAUSE THE LASER IS DRIVING HIGH PROBABILITY OF REM, AFTER REM YOU HAVE TO WAKE UP. I'LL SHOW YOU THE DATA TO SUPPORT THAT CLAIM. BUT BACK TO THIS DATA, RIGHT, IF WE STORE THE TRIALS AND SAY LET'S LOOK AT ALL THE TRIALS WHEN WE TURN ON THE LASER WHEN THE ANIMAL WAS IN NON-REM SLEEP, SO WE STORED THE TRIALS SHOWN HERE, RIGHT? YOU SEE AMONG THESE TRIALS THE PROBABILITY OF TRIGGERING REM SLEEP IS ABOUT 90%. SO IT'S HIGHLY, HIGHLY EFFICIENT. BUT IF YOU LOOK AT THE TRIALS WHERE WE TURN ON THE LASER WHEN THE ANIMAL WAS AWAKE, ACTUALLY WE DON'T SEE REALLY ANY REM. SO THE EFFECT OF LASER DEPENDS ON WHICH BRAIN STATE THE ANIMAL WAS IN. SO IN ADDITION TO LOOK AT THE PROBABILITY OF THE THREE BRAIN STATES, WE LOOK AT THE TRANSITION OF PROBABILITY. SO THIS IS A PLOT SHOWING THE PROBABILITY OF TRANSITION FROM NON-REM TO REM, AND SO THE WAY WE ANALYZE THAT IS SAY, OKAY, AT EACH TIME POINT, TIME POINT DEFINED RELATIVE TO THE TIME OF LASER STIMULATION, SO EACH TIME POINT WE LOOK IT'S A HOW MANY TRIALS IN WHICH THE ANIMAL WAS IN NON-REM SLEEP, AND THEN WE SEE 20 SECONDS IN HOW MANY CASES DID THE ANIMAL TRANSITION INTO REM, RIGHT? LET'S SAY YOU WITH 100 NON-REM TRIALS, WITHIN 20 SECONDS 30 GOT INTO REM, SO THE PROBABILITY WOULD BE 30%. SO THAT'S HOW WE CALCULATED THIS. YOU CAN SEE THAT LASER IS THIS SHADING PERIOD. BEFORE LASER, THE TRANSITION PROBABILITY IS INCREDIBLY LOW, MAYBE 5% OR SOMETHING. BUT DURING LASER STIMULATION, THERE'S THIS VERY DRAMATIC INCREASE, I THINK 20 TO 30-FOLD INCREASE OF THE TRANSITION PROBABILITY FROM NON-REM TO REM. WHICH IS CONSISTENT WITH WHAT WE SEE IN THE LAST SLIDE. NOW IF YOU LOOK AT THE REM TO WAKE TRANSITION, ACTUALLY IF ANYTHING THERE'S A DECREASE. THIS IS WHY I'M CLAIMING EVEN THOUGH IN THE LAST SLIDE WE DO SEE A SLOW INCREASE OF WAKEFULNESS, WE DON'T THINK THIS IS TRIGGERED BY LASER BECAUSE THE PROBABILITY FROM REM TO WAKE IS ACTUALLY DECREASED BY LASER. THIS IS THE PROBABILITY FROM NON-REM TO WAKE, THERE'S NO SIGNIFICANT CHANGE. SO BECAUSE THESE ARE THE ONLY TWO TRANSITIONS THAT CAN CAUSE WAKEFULNESS, AND ONE IS DECREASED, THE OTHER IS NO CHANGE, THAT'S WHY I'M SAYING LASER DOES NOT DIRECTLY TRIGGER WAKEFULNESS. IT'S ONLY PROMOTING REM. THE LAST PROBABILITY I WANTED TO SHOW IS THAT THE WAKE TO REM TRANSITION, IT TURNS OUT IF YOU'RE A NORMAL SUBJECT YOU DON'T HAVE THE DIRECT TRANSITION. YOU HAVE A DIRECT WAKE TO REM TRANSITION, ONLY IF YOU'RE NARCOLEPTIC. SOME OF YOU NOTICE THAT. THAT'S A PATHOLOGIC CONDITION. NOW, AS YOU SEE BEFORE LASER, THESE MICE DON'T HAVE THE TRANSITION, AND LASER DOES NOT CAUSE THAT TRANSITION EITHER. THE BOTTOM LINE IS THAT EVEN THOUGH OUR LASER STIMULATION OF THESE CAN REALLY PROMOTE REM SLEEP, IT'S ONLY THE NORMAL NON-REM TO REM TRANSITION, THE PATHOLOGICAL WAKE TO REM TRANSITION. OKAY. SO IF YOU DO OPTOGENETIC EXPERIMENTS, YOU HAVE TO DO CONTROLS. BECAUSE YOU'RE STICKING OPTIC FIBER INTO THE BRAIN, MAYBE THE MOUSE CAN SEE THE LIGHT, YOU EXPRESS YFP. EVERYTHING ELSE IS THE SAME. USING THE SAME PROTOCOL WE'RE NOT INCREASING REM SLEEP OR ANYTHING ELSE. THAT'S REALLY DUE TO THE ACTIVATION OF THE NEURONS, AND NOT JUST LIGHT, PER SE. IN ADDITION, IF WE ACTUALLY EXPRESS THE VIRUS NOT IN THE GABANERGIC, THEN ONLY 10 SECONDS OF LASER STIMULATION DRAMATICALLY INCREASES PROBABILITY OF WAKEFULNESS AND DECREASE OF BOTH NON-REM AND REM, RIGHT? THIS SUGGESTS THE EFFECT IS NOT ONLY SPECIFIC TO THE AREA BUT ALSO SPECIFIC TO THE CELL TYPE. IF YOU ACTIVATE AROUND CELL TYPE YOU INCREASE WAKEFULNESS. LIKE I SAID EARLIER, YOU KNOW, PROMOTING WAKEFULNESS, THOSE NEURONS ARE EVERYWHERE, IT'S EASY TO FIND THEM. SO IN THAT EXPERIMENT, WHAT'S WE CALL THE STIMULATION, THE COMPUTER IS BLIND TO WHAT THE MOUSE IS DOING, JUST TURNS ON THE LASER ONCE EVERY 15 TO 25 MINUTES. BUT HERE IS AN EXPERIMENT THAT WE USED FOR STIMULATION, WE KNOW WE CAN TRIGGER REM, RIGHT? BUT WE ALSO WANTED TO KNOW WHETHER WE CAN MAINTAIN REM. IF THE ANIMAL IS ALREADY INTO REM SLEEP, CAN WE PROLONG THE REM SLEEP? AND SO FOR THAT WE LIKE TO LEAVE THE TRANSITION INTO REM TO THE ANIMAL HERSELF OR HIMSELF, RIGHT? AND WE ONLY TURN ON THE LASER AFTER THE TRANSITION HAS HAPPENED. WHAT WE DID HERE WAS USE THE COMPUTER TO HAVE A REALTIME MONITORING THE EEG AND EMG, AND EVERY TIME WE DETECT A REM SLEEP, SO THIS IS WHAT THE ANIMAL DID ON THEIR OWN, AND THEN WE TELL THE LASER, THE COMPUTER TELLS THE LASER TO TURN OFF, AND THIS HAPPENS ABOUT 50% OF THE TIME. AND THE LASER WOULD STAY ON AS LONG AS THE REM IS THERE. SO ONLY WHEN THE REM -- AND WE TURN OFF THE LASER. IN OTHER WORDS, WE WANT TO KEEP THE LASER ON, RIGHT? WE DON'T WANT TO TURN OFF THE LASER TO TRIGGER THE END OF REM. WE JUST WANT TO STIMULATE AND SEE HOW LONG WE CAN PROMOTE REM. SO HERE IS AN EXAMPLE EXPERIMENT, AND YOU CAN SEE HERE IS A REM PERIOD. WHETHER YOU TURN ON THE LASER IS DETERMINED BY THE COMPUTER AT 50%, SO THIS TRIAL WE DIDN'T TURN ON THE LASER. THIS TRIAL WE DID. THESE TWO TRIALS WE DIDN'T TURN ON THE LASER. THIS TRIAL WE DID. NO LASER, LASER, RIGHT? EVEN JUST EYEBALLING IT YOU CAN SEE IN THESE THREE TRIALS WHERE WE TURN ON THE LASER IT SEEMED LIKE THE REM WAS LASTING LONGER, RIGHT? IF WE SUMMARIZE ALL THE MICE, ALL THE TRIALS, SO THESE ARE WITHIN THE SUBJECT CONTROL, EACH ANIMAL YOU HAVE SOME REM WITH NO LASER, SOME WITH LASER, IF YOU COMPARE THE REM DURATION NO LASER IS ABOUT 70 SECONDS ON AVERAGE, BUT WITH LASER IT DOUBLED DURATION. THIS DOESN'T HAPPEN IF WE USE THE EYG CONTROL MICE, THERE'S NO CHANGE. NOW WHEN WE USE THE STIMULATION, IF WE TURN ON -- IF WE ACTIVATE ARCH, OR HALO ADAPTER, WE CAN REDUCE REM DURATION, AND AGAIN THAT DOESN'T HAPPEN WITH YFP CONTROL. SO JUST TO SUMMARIZE THE ANSWER TO THE FIRST QUESTION, WE SHOWED ACTIVATING THE VM GABAERGIC NEURONS CAN CAN INDUCE NON-REM TO REM SLEEP AND DOUBLE THE DURATION OF REM SLEEP EPISODE. ARE THE NEURONS SPECIFICALLY ACTIVATED DURING REM? IMPORTANT BECAUSE OPTOGENETICS HAS TAKEN THE FIELD BY STORM. YOU TURN ON THE LIGHT, USUALLY THE ANIMAL, SOMETHING HAPPENS. ONE THING YOU ALWAYS HAVE TO ASK YOURSELF IS THAT WHETHER YOUR EFFECT IS PHYSIOLOGICAL, YOU DON'T KNOW WHAT THE NEURONS ARE NATURALLY DOING, SO WHAT IF YOU'RE ACTIVATING NEURONS IN A NON-PHYSIOLOGICAL CONDITION, CAUSING SOME BAGGAGE, IT DOESN'T TELL YOU HOW THE BRAIN WORKS. WE WANTED TO KNOW REALLY WHAT WHEN WE'RE NOT DRIVING THEM WITH LASER. THE PROBLEM HERE IS THAT IN THIS REGION, THERE ARE NOT ONLY GABANERGIC, THERE ARE ALSO SEROTONIN NEURONS CLOSE BY. HOW DO WE KNOW THEY WERE RECORDING HOW MUCH GABANERGIC? WITH CHANNEL DOPSIN, WE USE THIS SO-CALLED OPTIC FIBER SURROUNDED BY BUNDLES OF WIRES FOR RECORDING. WHEN WE INSERT THIS INTO THE BRAIN WE LOOK FOR NEURONS THAT ARE RELIABLY DRIVEN BY LASER. YOU CAN SEE THE SPIKES. THESE ARE THE SPONTANEOUS SPIKES. THIS IS WHERE WE'RE TURNING ON THE LASER. EACH LASER PULSE IS 5 MILLISECONDS, WE APPLY THE PULSE AT I THINK 20 HERTZ, PRETTY HIGH FREQUENCY, YOU CAN SEE EVERY LASER PULSE IS ASSOCIATED. WHEN WE SEE A NEURON THAT RESPONDS RELIABLY, WE'RE FAIRLY SURE THIS IS THE NEURON THAT COMPRESSES CHANNEL DOPLINS, THERE'S OTHER WAYS TO SHOW THE NEURONS RELIABLY. AND ONCE WE IDENTIFY NEURONS LIKE THAT WE RECORD ITS ACTIVITY OVER LONG PERIODS OF TIME. YOU CAN SEE EEG POWER SPECTRUM, EMG AMPLITUDE, THIS IS THE COMPUTER AUTOMATIC CLASSIFICATION, THE BRAIN STATE, THIS IS THE FINE RATE OF THE NEURON I SHOWED IN THE LAST SLIDE. YOU CAN SEE THIS NEURON SHOWS A HIGH FIRING RATE WHEN THERE'S A REM, RIGHT? SO HERE, HERE, HERE, HERE IS ALWAYS HIGH. DURING NON-REM SLEEP IT'S GENERALLY PRETTY LOW. DURING WAKEFULNESS IT'S ACTUALLY SORT OF VARIABLE, AND I'LL COME BACK TO THAT POINT. BUT THIS IS JUST THE AVERAGE FIRING RATE WITHIN EACH BRAIN STAGE, CAN SEE THIS UNIT HAS THE HIGHEST FIRING RATE DURING REM SLEEP, IT'S 26, 27 SPIKES PER SECOND, DURING NON-REM IT'S SLOW, WAKEFUL INTERMEDIATE. THE FIRST APPROXIMATATION, WE'RE ADDING 20 SPIKES PER SECOND, SO THE NATURAL FIRING RATE OF THIS NEURON DURING REM IS 27 SPIKES PER SECOND, SO THAT SUGGESTS OUR STIMULATION PORT IS NON-PHYSIOLOGICAL BECAUSE WE'RE ADDING NUMBERS BACK THAT MORE OR LESS SORT OF ARE NATURAL. THIS IS ONE NEURON, THIS IS THE SUMMARY OF 20 IDENTIFIED GABANERGIC NEURONS. THIS IS HARD TO SEE BUT THIS IS SUSTAINED 20 NEURONS, FOR EACH ONE OF THEM WE NORMALIZE FIRING RATE BY O MAXIMUM, ALL 20 NEURONS HAVE THE HIGHEST FIRING RATE DURING REM SLEEP. AS FAR AS MODULATION, THE NEURONS ARE FAIRLY HOMOGENOUS, ALL FIRING AT MAXIMUM RATE DURING REM SLEEP. WE ALSO LOOKED AT THE TRANSITION, CHANGE IN FIRING RATE DURING TRANSITION. THIS IS THE AVERAGE FIRING RATE AT THE NEURONS AT THE ONSET OF REM. NORMALLY THE ANIMALS WILL GO FROM NON-REM TO REM SLEEP, THIS IS THE POINT WHERE THE TRANSITION OCCURS, YOU CAN SEE THE NEURONS ACTUALLY ARE FINALLY CREEPING UP, 20 SECONDS BEFORE THE ACTUAL TRANSITION, AND THEY STAY AT HIGH RATE DURING REM SLEEP. THIS IS REM OFFSET, SO AFTER REM THEY WAKE UP, THERE'S ABRUPT DECREASE OF FIRING RATE. THE POINT IS THAT THERE'S THE INCREASE EVEN BEFORE THE TRANSITION, AND FIRING RATE STAYS HIGH DURING REM. WE THINK THIS KIND OF FIRING PATTERN IS IDEALLY SUITED FOR BOTH THE INDUCTION OF REM SLEEP AND ALSO MAINTENANCE, BECAUSE IF YOU WANT TO INDUCE REM YOU'VE GOT TO START HIGH RATE BEFORE THE TRANSITION HAPPENS. SO THIS IS THE ANSWER TO THE SECOND QUESTION. SO WE KNOW MOST ACTIVE DURING REM, FIRING RATE CHANGE FITS THE FUNCTION ROLE, THAT WE THINK THAT THEY SHOULD PERFORM. SO THE THIRD QUESTION, WHAT ARE THE DOWNSTREAM TARGETS, WE KNOW THE NEURONS CAN INDUCE REM SLEEP, HOW DO THEY DO IT? SO FIRST WE LOOK AT THE ANATOMY, AND IN FACT WE KNOW FROM EARLIER STUDIES, WE KNOW THE GABANERGIC NEURONS IN MEDULLA, SOME PROJECT TO THE PONS, SOME TO THE SPINAL CORD. WE KNOW THE PONS IS IMPORTANT FOR REM SLEEP. WE CAN SEE FOR EXAMPLE HERE THIS IS THE LOCUS JULIUS, RED IS HYDROXYLASE, A SPECIFIC MARKER FOR NEURONS, YOU CAN SEE GREEN FIBER. WHEN WE DID THE TRACING WE'VE SHOWN INDEED THESE NEURONS MAKE DIRECT SYNAPTIC CONNECTIONS WITH NEURONS. THAT'S ONE INTERESTING TARGET. THE OTHER TARGET AT LEAST WE CAN SEE THE AXON FIBERS, YOU SEE A LOT OF THEM IN THE PATH, THE VL PATH, INTERESTING BECAUSE EARLIER STUDIES THAT WHICH PEOPLE SHOW IF YOU PHARMACOLOGICALLY INACTIVATE BY INJECTING INTO THE GABA AGONIST, THEN THE AMOUNT OF REM SLEEP INCREASES SO THAT SUGGESTS THE PATH, YOU HAVE TO SUPPRESS FOR REM TO HAPPEN. NOW WE SEE THE GABANERGIC NEURONS, MAYBE THIS IS HOW NATURALLY YOU INDUCE REM BECAUSE YOU CAN USE THE GABANERGIC NEURONS TO SUPPRESS THE VL PATH. SO WHAT WE DID HERE WAS WE LOOKED AT THE VL PATH NEURONS, SO VLPAG HAS GABANERGIC NEURONS, IF WE EXPRESS CHANNEL DOPSIN AND TURN ON THE LIGHT YOU SEE WHAT THEY DO TO THE BRAIN. INCREASE OF NON-REM SLEEP, DECREASE OF WAKEFULNESS AND SUPPRESSION OF REM SLEEP, CONSISTENT WITH WHAT PEOPLE BELIEVED BEFORE USING PHARMACOLOGICAL MANIPULATION, EXCEPT THAT IN ADDITION TO EXPRESSING REM, IT ALSO DECREASES WAKEFULNESS, IT REALLY INCREASES NON-REM SLEEP. SO BECAUSE THE OTHER PART OF MY LAB IS ALSO INTERESTED IN LOOKING FOR MECHANISMS THAT PROMOTE NON-REM SLEEP, SO THIS CAME AS A PLEASANT SURPRISE BECAUSE THIS IS WHAT WE'RE LOOKING FOR, BUT DECREASE OF WAKEFULNESS IS SORT OF INTERESTING FINDING. BUT JUST TO COME BACK TO REM, IF WE DO STIMULATION, WE SAW DECREASE OF REM DURATION, SO THAT SUGGESTS THE VLPAG GABANERGIC NEURONS SUPPRESS REM SLEEP. MANY OF YOU ARE FAMILIAR WITH THIS TECHNIQUE, TRACING. WE TARGETED THE VLPAG GABANERGIC NEURONS. I WON'T GO INTO DETAIL BECAUSE THESE ARE PUBLISHED BUT THIS IS THE STARTING REGION, AND WE ALSO SEE A LOT OF LABELING IN THE MEDULLA, THEY ARE TWO MILLIMETERS APART AND WE SEE ALL THE NEURONS REALLY SHOWS THAT A LOT OF THESE NEURONS DIRECTLY ENERVATE. WE SAID, OKAY, WE KNOW THAT THESE NEURONS RIGHT HERE, THEY SUPPRESS, AND THERE'S SYNAPTIC CONNECTION, THE PREDICTION IF YOU ACTIVATE THE AXON TERMINALS HERE THAT SHOULD BE SUFFICIENT TO INDUCE REM BECAUSE THE REM INDUCTION WAS TO ACTIVATE CELL BODIES HERE BUT GIVEN THE ANATOMY, THAT'S WHAT WE FOUND. IF WE DO THE OPEN LOOP STIMULATION, WE SAW THE DECREASE IN NON-REM, INCREASE OF REM, AND SLOW INCREASE OF WAKEFULNESS. PRETTY MUCH EXACTLY WHAT WE SAW WITH CELL BODY STIMULATION. AND ALSO IF WE DO THE CLOSAL STIMULATION, WE SAW THE DOUBLING OF REM DURATION, PRETTY MUCH WE THINK ACTIVATING THIS AXON TERMINAL IS NOT ACCOUNTING FOR ALL THE EFFECT OF ACTIVATING CELL BODIES, MORE LIKE 90% OF THAT EFFECT, SO WE THINK THE PATHWAY IS REALLY THE MAIN PATHWAY DOING THAT JOB. SO TO SUMMARIZE THIS PART OF THE THIRD QUESTION, WE KNOW THE VM GABANERGIC NEURONS ENERVE EIGHT, EXPRESSING NEURONS, IF WE ACTIVATE THE AXON TERMINALS THEY CAN INDUCE AND PROLONG REM SLEEP. IT MAY NOT BE THE ONLY ONE BUT WE THINK IT'S AN IMPORTANT ONE. THIS IS ONLY THE BEGINNING, THE OTHER POTENTIAL DOWNSTREAM TARGETS. NOW QUESTION NUMBER FOUR, WHAT ARE THEIR INPUTS? PREVIOUS STUDIES, THIS IS A STUDY DONE, THEY USE C-FOS THAT PROJECT TO THE MEDULLA, CDB IS RETROGRADE LABELING DYE, AND THEN THEY LOOK AT C-FOS. WE THINK THIS IS A VERY GOOD CANDIDATE FOR THE UPSTREAM REGION THAT MIGHT BE DRIVING THE VM NEURONS TO BE TOUGH DURING REM. THAT WAS C-FOS. WE WANTED TO TAKE A CLOSER LOOK. WE USED THIS RELATIVELY NEW TECHNIQUE, MICRO ENDOSCOPE, SLD IS DEEP IN THE BRAIN, YOU CAN'T SEE IT WITH A REGULAR TWO-PHOTON ON THE SURFACE. YOU INSERT THIS INTO THE REGION AND ATTACH THIS TINY CAMERA DEVELOPED BY MARK'S LAB, SOME PEOPLE CALL IT THE SHKNEESER SCOPE, IT'S SMALL, EVEN THE MOUSE WITH A SMALL BODY CAN CARRY IT. WE WANTED THE MOUSE TO BE MOVING IN A CAGE, THAT'S WHY THEY SLEEP WITH THAT, RIGHT? THE SMALL CAMERA IS VERY IMPORTANT. HERE IS A VIDEO SHOWING GLUTAMATERGIC NEURONS. WE'RE LOOKING AT CALCIUM. THIS MOUSE WAS BASICALLY MOVING AROUND THE CAGE AND HE CAN SLEEP OR WAKE WHEN HE WANTS. RIGHT NOW HE'S IN NON-REM SLEEP. YOU CAN SEE SOME KIND OF FLICKERING IF YOU LOOK, FLICKERING DURING NON-REM, THEN YOU SEE HE GOT INTO REM BECAUSE OF THIS DATA, YOU SEE THE WHOLE FIELD LIGHTS UP AND THEN THESE NEURONS GENERALLY HAVE MUCH HIGHER ACTIVITY. SO OKAY. JUST TO SUMMARIZE EVERYTHING I TOLD YOU SO FAR, I THINK THAT THESE ARE THE EARLIER PARTS, FOR THE INPUT PART IN THE SLB WHICH PROJECTS DD CONTAINS THE POPULATION OF REM NEURONS, THE EXAMPLE I SHOWED YOU IN THE LAST MOVIE. HERE I'M PUTTING TOGETHER THIS LITTLE CIRCUIT DIAGRAM OF SO FAR WHAT WE FIGURED OUT IN THIS PART OF THE BRAIN. WE KNOW THESE ARE COLOR-CODED, LIGHT BLUE ARE REM ACTIVE, HOPEFULLY REM PROMOTING NEURONS, A POPULATION IN THE MEDULLA. THEY PROJECT TO THE LOCUS JULIUS, INHIBITING THE NEURONS, THEY ALSO PROJECT TO THE VL PATH WHERE THE GABANERGIC NEURONS. IF YOU LOOK AT THE BLACK, NON-REM NEURONS, GABANERGIC, THEY PROJECT SLD, THEY ENERVATE THE CYAN AND PINK. THESE ARROWS ARE THE ONES WE FIGURED OUT, BUT OBVIOUSLY THERE ARE MORE CONNECTIONS. IT SEEMS LIKE SO FAR MAYBE PARTLY WISHFUL THINKING BUT WE THINK THAT THE CIRCUIT IS PROBABLY DESIGNED WITH SIMPLE LOGIC, WHICH IS THAT WE HAVE THREE BRAIN STATES, WAKEFUL INSIDE, REM AND NON-REM. IF YOUR NEURON WANTS TO PROMOTE A STATE, YOU HAVE TO INHIBIT THE OTHER TWO, SLOW NEURON INHIBITSES PINK AND BLACK, BLACK INHIBITS PINK AND LIGHT BLUE. THIS IS SO FAR WHAT WE HAVE SEEN. IT SEEMS LIKE THE SCHEME MAKES SENSE. LIKE I SAID THERE'S A LOT MORE TO BE MAPPED OUT IN THIS REGION. OH, THE OTHER THING, I SHOWED YOU IN THE SLD REGION THERE ARE REM ACTIVE NEURONS, UNFORTUNATELY THERE ARE WAKE IF I HAVE NEURONS, SPATIALLY PRETTY CLOSE TO EACH OTHER, SO IT'S ACTUALLY QUITE A CHALLENGE TOLL TRY TO SEPARATE THESE TWO POPULATIONS. THAT WAS THE BRAIN STEM. AND I'M ALSO RUNNING OUT OF TIME. I'LL TRY TO MOVE TO THE ANTERIOR PART OF THE BRAIN AND I'LL TRY TO SPEAK FASTER. I'M ALREADY TOO FAST BUT ANYWAY. I'LL QUICKLY TELL YOU ABOUT OUR STUDY IN THE BASAL FOREBRAIN, PREVIOUS STUDIOUS SITUATION LESION CAUSES INSOMNIA, INCREASE IN SLOW EEG IN OTHER CASES, THE SLEEPIER BRAIN STATE. THAT SOCIALS SUGGESTS IMPORTANCE FOR SLEEP AND WAKEFULNESS. CHOLINERGIC NEURONS ARE DAMAGED IN THE EARLY PART OF ALZHEIMER'S DISEASE, A LOT OF GLUTAMATERGIC AND GABAERGIC NEURONS. HERE IS A RECORDING WITH CELL TYPE IDENTIFICATION. THIS IS CHOLINERGIC. WE FOLLOW THIS OVER BRAIN STATES, THIS NEURON IS ACTIVE IN WAKEFULNESS AND ALSO REM, AND COMPLETELY SHUT DOWN DURING NON-REM SLEEP. THE COLOR SCHEME HERE IS TOTALLY DIFFERENT FROM EARLIER SLIDE BECAUSE THESE PROJECTS ARE DONE BY DIFFERENT POSTDOCS, THEY HAD FAVORITE COLORS AND I COULD NEVER CONVINCE THEM TO CHANGE SO YOU HAVE TO READ THE LEGEND. CHOLINERGIC NEURONS ARE LIKE THIS, RIGHT? THIS IS THE OTHER NOTATION. ON THE HORIZONTAL AXIS WE'RE PLOTTING FIRING RATE DIFFERENTLY BETWEEN WAKE AND NON-REM STATE, NORMALIZED, THE VERTICAL AXIS IS THE DIFFERENCE BETWEEN REM AND NON-REM. EACH COLOR IS A SINGLE, CIRCLES ARE REM AND WAKE ACTIVE. YOU CAN SEE ALL 12 CHOLINERGIC NEURONS ARE IN THE UP RIGHT QUADRANT. THIS WAS KNOWN BEFORE. BARBARA JONES IN CANADA, SHE ACTUALLY RECORDED CHOLINERGIC NEURONS BEFORE. BUT WE JUST WANTED TO SORT OF USE A NEW TECHNIQUE TO CONFIRM WHAT PEOPLE STATED BEFORE. NOW, IF WE ACTIVATE A LOT OF THESE CHOLINERGIC NEURONS WE SAW DECREASE OF NON-REM SLEEP, INCREASE OF WAKEFULNESS, NOT TOO MUCH CHANGE IN REM SO CHOLINERGIC NEURONS ARE WAKE PROMOTING. WE LOOK AT THE GLUTAMATERGIC. PREVIOUSLY NOT MUCH WAS KNOWN. I'M SHOWING 31 GLUTAMATERGIC, MOST IN THE UPPER RIGHT QUADRANT AGAIN, ONLY A COUPLE IN THE OTHER QUADRANTS, CLOSE TO THE ORIGIN SO THESE ARE THE CELLS WEAKLY MODULATED. MOST OF THEM ARE REALLY WAKE AND REM ACTIVE. AND IF WE ACTIVATE A LOT OF THESE GLUTAMATERGIC WE SEE DECREASE OF NON-REM, INCREASE OF WAKEFULNESS, NO CHANGE IN REM. WHAT ABOUT A GABAERGIC NEURONS? THEIR DIVERSE. WE KNEW WE HAD TO SEPARATE THEM AND THE WAY TO DO THAT SINCE I CAME FROM THE CORTEX IS TO USE THESE MOLECULAR MARKERS, SO IN THE CORTEX IT'S VERY WELL KNOWN THERE ARE PV NEURONS, AND SOM+, POST EXIST IN BASAL FOREBRAIN, LARGELY SEPARATE. FIRST THE PV NEURONS, MOST IN THE UPPER RIGHT QUADRANT, A COUPLE IN UPPER LEFT, THESE ARE REM ACTIVE BUT NOT WAKE ACTIVE. WHEN WE ACTIVATE THESE PV CELLS AGAIN DECREASE OF NON-REM, INCREASE OF WAKEFULNESS, CONFIRMING WHAT I TOLD YOU AT THE VERY BEGINNING, REALLY EASY TO FIND NEURONS THAT PROMOTE WAKEFULNESS, RIGHT? SO THREE OUT OF FOUR CELL TYPES. FORTUNATELY WHEN WE MOVE TO SOM WE WERE BORED TO DEATH. WAKE, WAKE, WAKE. WHEN WE MOVE TO THIS WE SAW SOME ARE SLEEP ACTIVE, RIGHT? HERE YOU SEE THIS NEURON IS COMPLETELY SHUT DOWN DURING GRAY, WAKE, BUT IT'S REALLY ACTIVE DURING NON-REM, DURING WHITE REGION, AND REM YELLOW IS INTERMEDIATE. WHEN WE LOOKED AT THE -- WHEN WE PLOTTED ALL THESE NEURONS YOU SEE IT'S ALL OVER THE PLACE. WE ALSO SEE SOME WAKE AND REM ACTIVE NEURONS, WE ALSO SAW REM ACTIVE, NON-REM ACTIVE NEURONS, ALSO SOME IN THE FOURTH QUADRANT, THEY ARE EVERYWHERE, RIGHT? IT'S A VERY DIVERSE POPULATION. AT LEAST THIS IS THE ONLY POPULATION WE SEE NEURONS WAY OUT HERE, REALLY THE NON-REM ACTIVE NEURONS. WHEN WE ACTIVATE THE ENTIRE POPULATION, WE ACTUALLY FINALLY SAW WE CAN PROMOTE NON-REM SLEEP A LITTLE BIT, DECREASE WAKEFULNESS A LITTLE BIT. BASICALLY WHAT'S GOING ON IS IT'S A DIVERSE POPULATION BUT THE OVERALL FACT IS TO PROMOTE NON-REM SLEEP. SO THESE ARE THE FUNCTIONAL CHARACTERIZATION OF THE FOUR CELL TYPES, WE'RE ALSO INTERESTED IN THE CIRCUIT. ONE THING WE DID WAS TO LOOK AT THE LOCAL CIRCUIT IN THE BASAL FOREBRAIN. THIS ILLUSTRATES OUR TECHNIQUE. WE WOULD EXPRESS CHANNEL DOPSIN IN ONE CELL TYPE, IN THIS CASE CHOLINERGIC, AND FLUORESCENT MARKER IN THIS CASE PD, PATCH ON THE RETINA, TURN ON THE RIGHT, LOOK AT THE SYNAPTIC CONNECTION. THIS IS OUR DATA, RIGHT? I DON'T EXPECT YOU TO LOOK THROUGH ALL OF THEM BUT BASICALLY WHAT IT IS IS THAT THERE ARE FOUR PANELS, RIGHT? EACH ONE SHOWS ONE PRESYNAPTIC CELL TYPE, WITHIN EACH PANEL THERE'S THREE BOXES, THAT SHOWS EACH OF THE THREE POST-SYNAPTIC CELL TYPES, MAPPING THE CONNECTIONS, A LOT OF WORK. AND HERE I'LL SUMMARIZE WHAT WE FOUND. THESE ARE THE THREE WAKE PROMOTING NEURONS, I'M USING A LIGHT COLOR TO REPRESENT THEM, THIS IS THE CELL TYPE THAT PROMOTES NON-REM SLEEP. WE FOUND THE WAKE PROMOTING NEURONS SEEM TO BE ORGANIZED IN THE HIERARCHY. THE CHOLINERGIC NEURONS INTERESTINGLY EXCITE A PV NEURON BUT MOSTLY INHIBIT, THERE'S A HIERARCHY BECAUSE FEEDBACK IS EITHER INHIBITORY, WE COULD NEVER DETECT THE CONNECTION. THE MORE IMPORTANT THING HERE IS THAT THE ONE TYPE OF SLEEP PROMOTING NEURON PROVIDE BROAD INCUBATION TO ALL THREE TYPES OF WAKE-PROMOTING NEURONS. IN FACT THIS IS WHAT WE SEE IN THE OTHER BRAIN REGIONS AS WELL. IN THE SENSE IF YOU WANT TO PROMOTE SLEEP, YOU BETTER BROADLY INHIBIT A LOT OF THE WAKE PROMOTING NEURONS. OKAY. AT THIS POINT, RIGHT, WE KNOW IT'S A DIVERSE POPULATION, SO WHAT DO WE DO? WE CAN KEEP TRYING OTHER MARKERS. I HAVE A VERY LONG LIST OF CANDIDATE MARKERS, BUT TRYING EACH MARKER TAKES SEVERAL MONTHS. MY POSTDOCS WERE IN THE INTERESTED IN DOING THAT, RIGHT? SO BELIEVE ME, I TRIED COERCION AND BRIBING AND NOTHING WAS WORKING. WE DECIDED TO TAKE A DIFFERENT APPROACH, WHICH IS, YOU KNOW, INSPIRED BY THE FINDING THE SLEEP NEURONS INHIBITED WAKE NEURONS, WE THOUGHT COULD WE USE THAT FEATURE TO TRY TO HUNT DOWN A SLEEP NEURON BASED ON THE FACT THAT THEY MIGHT WANT TO INNERVATE. THIS IS DONE BY SHINJAE CHUNG IN MY LAB. WE INJECT THE THE RETROGRADE BEADS INTO POST-SYNAPTIC TARGETS, REGIONS THAT PROMOTE WAKEFULNESS, NEURONS ARE IMPORTANT FOR WAKEFULNESS, WE DID A CFOS, AFTER SLEEP REBOUND, WE FOUND THE NEURONS LABELED, IF WE LOOK AT THE SLEEP ACTIVE NEURONS BASE ON CFOS SCREENING WE SEE A LOT PROJECTING TMN SUGGESTING IT MIGHT BE AN INTERESTING TARGET TO GO FOR. WE COLLABORATED WITH ALI CETIN WHO DEVELOPED A COOL LENTIVIRUS TOOL, PSEUDOTYPED WITH A VIRUS MAKING IT TRAVEL RETROGRADE, NORMALLY IT DOESN'T TRAVEL BACKWARDS BUT THESE DO. WHAT WE DID WAS INJECT THE LENTIVIRUS IN THE REGION, SO THE VIRUS CAN TRAVEL BACKWARDS TO THE PRE-OPTIC AREA AND THIS VIRUS, IF WE INJECT THIS WE CAN LABEL LABEL GABANERGIC NEURONS, IT HAS TO BE CELL TYPE, ALSO PROJECTION SPECIFIC. SO THEN WE DID CFOS STAINING FOR SLEEP ACTIVE NEURONS, YOU CAN SEE A LOT OF OVERLAP SUGGESTING THIS STRATEGY MIGHT BE A USEFUL STRATEGY. AND SO WE ACTIVATED THOSE NEURONS IN THE PRE-OPTIC AREA, BASE TO THE BASAL FOREBRAIN. IN ONE TRIAL THE ANIMAL WAS AWAKE. WE TURNED ON THE LASER HERE, THE BLUE SHADING, QUICKLY THE MOUSE GOT INTO NON-REM SLEEP, HERE IS ANOTHER TRIAL, THE MOUSE WAS IN NON-REM SLEEP, AFTER WE TURNED ON THE LASER THE MOUSE GOT INTO REM SLEEP. THESE ARE TWO TRIALS. BUT HERE IS A SUMMARY OF THE PROBABILITY, RIGHT? SO YOU CAN SEE THAT WE TURNED ON THE LASER, AND THAT AS SOON AS YOU TURN ON THE LASER THERE'S SHARP INCREASE OF PROBABILITY OF NON-REM SLEEP, DECREASE OF WAKEFULNESS THAT STAYED LOW DURING THIS TWO-MINUTE PERIOD THAT WE TURNED ON THE LASER. SO THIS SEEMS TO PROMOTE REM AND NON-REM SLEEP, IN CONTRAST TO THE EFFECT OF ACTIVATING OTHER GABAERGIC NEURONS. WE SAW THIS DRAMATIC INCREASE OF WAKEFULNESS, WHAT I TOLD YOU BEFORE, IF YOU RANDOMLY DO IT YOU WAKE UP THE ANIMALS. THIS IS A SUBSET, THE OPPOSITE EFFECT, WE THINK IT WAS REALLY USING THIS METHOD TO SORT OUT MINORITY OF SLEEP PROMOTING NEURONS IN THE PRE-OPTIC REGION. IF WE DO THIS FOR THE GLUTEMANERGIC WE SAW PROMOTION OF WAKEFULNESS SUGGEST FIGURE YOU WANT TO GET SLEEP YOU HAVE TO TARGET -- IT HAS TO BE CELL-TYPE SPECIFIC AND PROJECT SPECIFIC, IF YOU MOVE EITHER ONE YOU GET WAKEFULNESS. AND THEN WE GET TO THE TAGGING AND COURTING, IDENTIFY THE NEURONS, USING THE OPTOGENETIC METHOD, HERE IS ONE EXAMPLE, THIS IS A SUMMARY OF 17 IDENTIFYING NEURONS, GABANERGIC PROMOTING, ACTIVE DURING REM, INTERMEDIATE DURING NON-REM, LEAST ACTIVE DURING WAKE, WHAT WE WERE HOPING FOR. THIS IS ANOTHER USING THE TWO DIMENSIONAL SCATTER PLOT, IN THIS CASE WE CHANGED AXES SO ON THE RIGHT IT MEANS IT'S MORE ACTIVE DURING NON-REM, UP IS MORE ACTIVE DURING REM, SO THE BLUE DOTS ARE IDENTIFIED GABANERGIC NEURONS, REM AND NON-REM ACTIVE. SO THIS IS DIFFERENT FROM THE RANDOMLY COLLECTED GABANERGIC NEURONS NOTICE REGION, THEY ARE ALL OVER THE PLACE, SOME ARE WAKE ACTIVE. AGAIN, USING THIS RETROGRADE WE'RE SORTING OUT A SUBSET THAT ARE GABANERGIC AND SLEEP PROMOTING. A QUICK SUMMARY, I WON'T READ IT. WE'RE RUNNING OUT OF TIME. LAST, SO FAR WE'RE USING THE LENTIVIRUS, IT'S GREAT BECAUSE WITHOUT THAT WE COULDN'T HAVE SORTED OUT THE POA NEURONS, BUT LENTIVIRUS IS ALSO NOT THAT COMMON SO EVERY TIME WE WANT TO DO SOMETHING DIFFERENT WE HAVE TO BEG ALI TO MAKE A DIFFERENT CONSTRUCT FOR US AND RELY (INDISCERNIBLE) I HATE TO BAD MOUTH BUT THE LAST FEW VIRUSES HAD A GREAT EXPERIENCE, WE COULD RATHER USE AED, EVERYBODY USES THEM AND THEY ARE RELIABLE BUT HOW DO WE DO THAT? AED IS NOT RETROGRADE. WE'VE GOT TO FIND MOLECULAR MARKER TO USE A CRE LINE, YOU CAN USE CRE DEPENDENT, DOUGH BACK TO THE EASIER METHODS. HOW DO WE FIND A MARKER? FOR THIS WE DID GENE PROFILING, SINGLE CELL RNA-SEQ IN COLLABORATION WITH ALI INSTITUTE, THEY DID ALL OF THIS BECAUSE IN MY LAB WE DIDN'T HAVE THE EXPERTISE. THIS IS WHAT THE DATA LOOKS LIKE. EACH COLUMN REPRESENTS A -- EACH ROW REPRESENTS A SINGLE SCREENING, EACH COLUMN IS A SINGLE CELL, 70 CELLS IN THIS BATCH. RED ROW MEANS THAT PARTICULAR GENE IS HEAVILY EXPRESSED IN MAJORITY OF THE CELLS WE SORTED OUT SO WE FOUND ONE OF THEM IS PATH 1, GENE THAT ENCODES NEUROPEPTIDE, AND THERE'S ANOTHER ONE. THERE'S A PRE-MOUSE LINE, WE DECIDED TO TEST THAT. SO AGAIN BECAUSE I'M RUNNING OUT OF TIME I'LL QUICKLY GO THROUGH, THIS IS SHOWING TAC1 NEURONS DO OVERLAP, TAC1 OVERLAPS, AND THIS SHOWS IF WE EXPRESS IN THE TAC1 NEURONS AND LOOK AT FIBERS WE SEE THEM, THEY PROJECT THIS REGION, SO THE RNA-SEQ WAS GIVEN SERIOUS EFFECT. WHAT IF WE EXPRESS WITH CHANNEL DOPSIN. WE TURNED ON THE LASER, WE SAW NOT MUCH CHANGE IN REM, AT LEAST WE'RE PROMOTING SLEEP. RNA-SEQ IS ONE METHOD. THE OTHER IS THE SO-CALLED TRAP METHOD THE PURIFICATION METHOD, THE IDEA IS INJECT THIS VIRUS, ALSO RETROGRADE, WE'RE LABELING THE PARTICULAR TARGET WITH RIBOSOMAL TARGET, USE THE ANTIBODY AGAINST H-A, ATTACHED TO THE MAGNETIC BEAD, SO WE CAN PULL DOWN THE RIBOSOME, AND BECAUSE RIBOSOME IS DOING THE JOB ATTACHED TO RNA WE CAN PULL DOWN mRNA TOGETHER AND SEQUENCE THE mRNA. THIS IS WHAT THE DATA LOOKS LIKE. EACH DOT IS A SINGLE GENE, PLOTTING EXPRESSION LEVEL OF THE RETROGRADELY LABELED POPULATION AGAINST THE ENTIRE POPULATION, IF YOU SEE A DOT ABOVE THE DIAGONAL THE GENE IS MORE HEAVILY EXPRESSED IN THE RETROGRADE POPULATION. WE FOCUSED ON TWO, SOME LITERATURE SUGGESTS THEY MIGHT BE INVOLVED IN Seq REGULATION, THEY HAVE THE CRE LINES SO WE CAN TEST THEM. THESE ARE JUST ANATOMY TO SHOW THESE PROJECT TMN. WHEN WE ACTIVATE, YOU SEE THE INCREASE IN NON-REM, ALSO INCREASE OF REM, DRAMATIC DECREASE OF WAKEFULNESS, THIS IS THE EFFECT. WE THINK USING THE GENE PROFILING IS A MORE PRINCIPLED WAY TO SORT OF PULL OUT THE MARKERS THAT MIGHT LABEL A SUBSET THE GABANERGIC NEURONS. SO THAT'S WHERE WE ARE. THERE'S MORE WORK TO BE DONE BUT THIS IS JUST A PROGRESSIVE WORK. THESE ARE THE PEOPLE IN MY LAB TWO DID THE WORK, FRANZ WEBBER DID THE STUDY ON REM SLEEP, JULIA GOT ENDOSCOPE IMAGING TO WORK, MIN XU AND SHINJAE CHUNG DEVELOPED KEY TECHNIQUES, RESPONSIBLE FOR GENE PROFILING PROJECT I TALKED ABOUT IN THE LAST PART. THANK YOU VERY MUCH. [APPLAUSE] OKAY, SO ARE THERE ANY QUESTIONS? >> HI. I'LL ASK A QUESTION ABOUT THE FIRST PART, THE MEDULLA. SO YOU SHOWED THAT THE STIMULATION OF THE PROJECTION FROM THE MEDULLA TO THE VLPAG INDUCES REM, HAVE YOU DONE FUNCTIONAL OCCLUSION EXPERIMENT, IF THEY WERE BOTH DONE IN THE SAME MOUSE LINES, GAD LINES, COULD YOU PUT CHANNEL DOPSIN IN THE UPSTREAM IN THE MEDULLA AND VLPAG AND OCCLUDE THE EFFECT OF STIMULATING THAT PROJECTION? >> WE'VE NOT DONE THAT BUT THAT WOULD BE AN INTERESTING EXPERIMENT. INJECT THE VIRUS IN THE VM AND ALSO IN THE VLPAG AND STICK AN OPTIC FIBER. I GUESS SO YOU HOPE TO SEE TO COMPARE THAT WITH JUST THE VM OR JUST THE VLPAG. >> AND IF IT'S TRULY DOWNSTREAM. >> IT'S DOABLE EXCEPT IT'S A SUBJECT COMPARISON, THAT WOULD BE I THINK YOU NEED MORE DATA TO BE SURE ABOUT THAT. >> SURE. >> BUT I THINK IT'S A MEANINGFUL EXPERIMENT. >> I WAS WONDERING IF YOU WOULD EXPLAIN FOR THE SECOND PART OF YOUR TALK HOW DO YOU DECIDE WHAT FREQUENCY TO STIMULATE THE NEURONS THAT YOU'RE PROFILING TO SEE WHAT THEIR EFFECTS ARE FOR PROGRAM? >> SO, YOU KNOW, IDEALLY WHAT YOU WANT TO DO IS FIRST TO FIGURE OUT THEIR NATURAL FIRING RATE AND THEN LATER FREQUENCY ACCORDING TO THAT BUT IN REALITY REPORTING IS A LABOR INTENSEIVE TECHNIQUE, RIGHT? TO BE HONEST, IT DEPENDS, DIVERSE POPULATION KENT CELL TYPE, IT'S EVEN HARDER BUT I WILL SAY THE AVERAGE, ONCE EVERY THREE DAYS IF YOU'RE GOOD. INITIALLY IT TAKES A LONG TIME TO REALLY GET THAT INFORMATION, SO MY ATTITUDE IS THAT INITIALLY YOU WANT TO USE A RELATIVELY LOW FREQUENCY, IF YOU CAN SEE THE EFFECT, AND THEN YOU'RE SAFER, BECAUSE MORE LIKELY TO BE WITHIN A PHYSIOLOGICAL RANGE, IF YOU USE TOO HIGH, NORMALLY IT WOULD NEVER GO TO 30 HERTZ BECAUSE THEN YOU'RE PROBABILITY BETWEEN NON-PHYSIOLOGICAL IS MUCH HIGHER. >> REALLY NICE STUFF. >> THANK YOU. >> THE IT LOOKED LIKE IF I UNDERSTAND THIS CORRECTLY THE GABAERGIC WERE SUPPRESSING WAKEFUL NEURONS, IF REALLY GABA THAT'S DOING IT? THERE'S OFTEN A LOT OF PEPTIDES CO-EXPRESSED WITH THAT. SO DO YOU HAVE ANY EVIDENCE OF THAT YET? >> THAT'S A REALLY, REALLY GOOD QUESTION. AT THIS POINT, WE'RE REALLY SORT OF IN OUR FIRST ORDER OF BUSINESS, RIGHT? JUST TO TRY TO FIND THE MARKER AND SEE, YOU KNOW, WHO ARE THESE NEURONS, CAN WE USE THE MARKER TO RELIABLE GET REPRODUCIBLE EFFECTS? THE DOWNSTREAM MECHANISM, THERE'S MORE TO BE DONE. FIRST THE MARKERS THAT ARE THE PEPTIDE MARKERS, IMMEDIATELY OPENING THE QUESTION, IS IT GABA OR PEPTIDE, RIGHT? THOSE ARE IMPORTANT QUESTIONS. IN THE END MY GUESS IS THE ANSWER IS BOTH. >> JOIN ME IN THANKING DR. DAN. I WANT TO ACKNOWLEDGE DR. GOTTESMAN IN THE FRONT ENJOYING US AND INVITE YOU TO A RECEPTION IN THE NIH LIBRARY IMMEDIATELY FOLLOWING THE LECTURE. DR. DAN WILL BE AVAILABLE TO ANSWER MORE QUESTIONS THERE. THANK YOU FOR COMING.