>> THIS IS THE LAST IN THE SEMINAR SERIES IN YEAR AND LAST BUT NOT LEAST, WE HAVE CLIFFORD WOOLF, SO I'M HONORED TO INTRODUCE THIM. CLIFFARD IS THE DIRECTOR OF THE NEUROBIOLOGY CENTER AND THE PROFESSOR OF NEUROLOGY AND NEUROBIOLOGY AT HARVARD MEDICAL SCHOOL. CLIFFORD EARNED HIS MD Ph.D. IN SOUTH AFRICA AND THEN MOVED ON TO LONDON FOR ALMOST TWEBT YEARS HE WORK AT THE UNIVERSITY OF COLLEGE LONDON AND BECAME A PROFESSOR THERE AND IN 1997 HE CAME OVER TO THE STATES AND WAS RECRUITED TO HARVARD MEDICAL SCHOOL WHERE HE WAS THE FIRST RICHARD KILTZ, PROFESSOR OF ANESTHESIOLOGY. AND IN 2007 HE WAS APPOINTED THE PRINCIPAL FACULTY MEMBER OF HARVARD STEM CELL EYEWITNESS UT IN 1998 AND NAMED DIRECTOR OF THE BIOLOGY CENTER IN BOSTON CHILDREN'S HOSPITAL. SO DR. WOOLF HAS SINCE BECOME THE DEPUTY DIRECTOR OF THE INTELLECTUAL DEVELOPMENT DISABILITY DISORDER CENTER AT BESTON CHILDREN'S HOSPITAL AND THE CO DIRECTOR OF NEUROSCIENCE PROGRAM AT HARVARD STEM CELL INSTITUTE. SO, CLIFFORD'S WON MANY HONORS AND PRIZES OVER THE YEARS AND SOME OF THEM THAT HE WAS JUST AWARDED THE GAIL DISTINGUISHED SCIENTIST AWARD AND WAS GIVEN THE CARE AWARD FROM THE AMERICAN PAIN SOCIETY, FOUNDER AWARD OF THE AMERICAN ACADEMY OF THE PAIN, MEDICINE, HONOREE FELLOW OF THE IRISH COLLEGE OF ANESTHES THESEIOLOGYSTS AND [INDISCERNIBLE] IN ISRAEL AND A LARGE NUMBER OF OTHER 1S WHICH I WON'T GO INTO THEM BECAUSE I WANT TO LEAVE SOME TIME FOR CLIFFORD TO TALK. HE'S A FOUNDER OF 3 COMPANIES AND [INDISCERNIBLE] PATTERNS AND SOMEHOW IN ALL OF THIS, HE'S ABLE TO FIND TIME TO BE ON THE BSC FOR [INDISCERNIBLE] AND YOU WILL PROBABLY SEE HIM AS A FAMILIAR FACE. HE'S BEEN ON THE NIMS BCS SINCE 2015. SO CLIFFORD WORKS ON PAIN AND ON THE REGENERATION, DEGENERATION OF THE NERVOUS SYSTEM WITH PARTICULAR FOCUS ON THE NEUROBIOLOGY DISEASE MODELS AND DRUG SCREENING AND DRUG PATIENT IPS CELL, DERIVED CELLS AND TODAY HE'S GOING TO TALK TO US ABOUT SOMETHING THAT'S VERY IMPORTANT AND IN THE NEWS AND THAT IS CONFRONTING THE OPIOID CRISIS BY DEVELOPING NEW MEASURES AND BIOMARKERS FOR PAIN. THANK YOU. [ APPLAUSE ] >> THANKS VERY MUCH MARK, IT'S A REAL PLEASURE TO BE HERE, I FEEL THAT I SPEND MOST OF MY LIFE AT NIH, EITHER WRITING GRANTS FOR THE NIH OR REVIEWING YOUR GROUP. WHAT I WOULD LIKE TO DO IS SHARE WITH YOU 3 STORIES FROM--WHICH ARE UNPUBLISHED WORK IN MY LAB THAT HAVE BEEN EXPLORING DIFFERENT ASPECTS OF PAIN AS WE'VE BEEN STRUGGLING TO DEAL WITH THIS COMPLEX SENSORY PHENOMENON THAT HAS CONTRIBUTED TO THE OPIOID CRISIS AND I HOPE THAT'S AT LEAST SOME OF THESE MAY BE RELEVANT IN SOME OF THE INITIATIVES, THE NEW INITIATIVES THAT HAS RECENTLY BEEN ANNOUNCED BY WALTER AND NORA FOLKOFF. THIS IS AN EXCITING TIME FOR PAIN ALTHOUGH THE FACT THAT IT IS ASSOCIATED WITH THIS SOCIETAL CATASTROPHE OF THE OPIOID CRISIS REALLY FOCUSES OUR ATTENTION ON HOW IMPORTANT IT IS FOR US TO UNDERSTAND PAIN AND USE THAT UNDERSTANDING AS A BASIS FOR THE DEVELOPMENT OF NOVEL NONABUSE LIABLE ANALGESICS AND THAT CERTAINLY IS THE KEY OBJECTIVE OF MOST OF THE WORK IN MY LAB. ONE OF THE REAL PROBLEMS WE ALL CONFRONT IS: ARE THEY OBJECTIVE MEASURES OF PAIN AND HOW CAN WE POSSIBLY DO THAT. PAIN IN THE END IS OBVIOUSLY A SENSORY EXPERIENCE. IT'S SOMETHING WE FEEL WE CAN EMPATHIZE WITH OTHER PEOPLE WHO REPORT PAIN BUT ITS SUBJECTIVITY MAKES IT EXTREMELY DIFFICULT TO MEASURE IN HUMANS, NEVER MIND IN PRECLINICAL MODELS. AND YET IF WE ARE TO MAKE PROGRESS IN OUR UNDERSTANDING OF PAIN, WE NEED NOVEL APPROACHES TO MEASURE PAIN RELATED PHENOMENA THAT WILL ALLOW US TO TEST THE CONTRIBUTION OF PARTICULAR TARGETS IN THE GENERATED RAITIONZ OF PAIN--GENERATION OF PAIN OR VALIDATE NOVEL THERAPEUTIC STRATEGIES. SO THE QUESTION THEN IS HOW TO DO THAT. SO A GRADUATE STUDENT IN MY LAB, DAVID ROBISON SENT HIMSELF THIS CHALLENGE, HE SAID: CAN RODENT BEHAVIOR REPRESENT PAIN? CAN WE ACTUALLY GET SOME MEASURE FROM RODENT BEHAVIOR THAT REFLECTS THE PRESENCE IN THAT ANIMAL, OF SENSATION OF PAIN, IN ADDITION HE ALSO WANTS TO TACKLE ANOTHER PROBLEM WHICH IS THAT MANY ATTEMPTS AT DEVELOPMENT OF NEW THERAPIES FAILED NOT ONLY BECAUSE OF ABSENCE OF EFFICAY WHICH IS OBVIOUSLY A MAJOR PROBLEM BUT ALSO BECAUSE THERE ARE UNEXPECTED CNS SIDE EFFECTS AND CNS SIDE EFFECTS ARE EXTREMELY COMMON, PATIENTS MAY REPORT THEM AS JUST FEELING DIZZY OR FEELING SOME CHANGE IN THEIR MOOD OR COGNITIVE FUNCTION AND TOITORIOUSLY, THESE ARE AS DIFFICULT TO MEASURE PRECLINICALLY AS PAIN IS HE SET HIMSELF THE GOAL OF TRYING TO POTENTIALLY ADDRESS BOTH OF THESE. HOW TO DO THAT? AND HIS IDEA WAS INSTEAD OF THE STANDARD REFLEX OF MEASURES OF PAIN THAT MOST OF US IN THE FIELD HAVE USED OVER THE LAST SEVERAL DECADES WHERE YOU HAVE A MOUSE OR A RAT AND YOU EXPOSE IT TO A STIMULUS AND THEN YOU LOOK FOR INSTANTANEOUS CHANGE IN BEHAVIOR, THAT IS THE STANDARD WAY, A REFLEXIVE RESPONSE. WHAT HIS DEAL WAS, WE NEED SOMETHING COMPLETELY DIFFERENT AND WE ALSO NEED TO AVOID THE PRESENCE OF HUMANS BECAUSE MICE AND RATS ARE PREY ANIMALS, THEY ARE INTENSELY AWARE OF THE PRESENCE OF PREDATORIES AND THEY CONSIDER US AS POTENTIAL PREDATORS, SO IF WE ARE PRESENT WITH THE ANIMALS THEY HAVE HIGH DEGREE OF STRESS AND THEIR BEHAVIOR CHANGES. IN ADDITION IF WE ARE THERE, WE TEND TO MEASURE THEIR BEHAVIOR IN THE LIGHT. BUT MICE ARE NOCT URNAL ANIMALS SO WE'RE NOT SWITCHING AROUND THE BEHAVIOR BOTH BY EXPOSING THEM BY OBSERVERS AND DOING THIS IN THE LIGHT. SO HIS VIEW WAS CAN WE MEASURE BEHAVIOR IN A PLACE OF REFUGE, A SMALL DARK CHAMBER SOMETHING A MOUSE WOULD NATURALLY CHOOSE. SO WHAT ARE THE FEATURES HE SET HIMSELF UP TO TRY AND--NO VISIBLE LIGHT, THE OTHER THING IS TO MOVE AWAY FROM THE SNAPSHOTS OF BEHAVIOR, IN OTHER WORD FIST YOU APPLY A HEAT STIMULUS IN THE ANIMAL WITHDRAWS FROM THIS, THAT'S GREAT BUT MAYBE 102ndS--10-SECONDS LATER THE ANIMAL WOULD HAVE SOME BEHAVIOR SO YOU WANT TO CAPACITY OF LONG DURATION BEHAVIORS, LONG DURATION RECORDINGS THAT WOULD ENABLE A MUCH MORE INTEGRATED VIEW OF WHAT THE ANIMALS WERE DOING OVERTIME RATHER THAN WHAT HAPPENED THEA A PARTICULAR FRACTION OF TIME. YOU'LL OBSERVE PREDATORS ARE MENTIONED AND IN ADDITION SOME WAY OF COLLECTING HIGH TEMPORAL AND HIGH SPATIAL RESOLUTION INFORMATION. AND TO DO THIS, HE--HE FELT IT WAS IMPORTANT AS WE HAD A FULLY AUTOMATED DATA ACQUISITION AND ANALYSIS PACKAGE, AND HE SET UP--HE WAS AN MBA WHO CAME TO MY LAB AS A Ph.D. IN THE PROGRAM AT NEUROSCIENCE AT HARVARD MEDICAL SCHOOL AND THEN DEVELOPED ENGINEERING SKILLS TO TRY AND MEET THESE GOALS. AND THEN 1 OF THE QUESTIONS HE ALSO WANTED TO ADDRESS WAS THE STANDARD MEASURES OF ANNAL GEEZIA IN ANALGESIA, REQUIRE DOSES OF ANALGESICS THAT ARE ORDERS OF MAGNITUDE HIGHER THAN THOSE REQUIRED IN PATIENTS AND THERE'S BEEN WORK AROUNDS. WHY IS THAT TRUE? WHY WOULD A MOUSE REQUIRE 5-MILLIGRAMS PER KILOGRAM OF MORPHINE TO SEE AN ANALGESIC SIGNAL WHERE IN A HUMAN 5-MILLIGRAMS TOTAL IS ENOUGH TO SEE ANALGESIC AND 1 POTENTIAL IS THAT THE STANDARD OUTCOMES WE USE ARE SO INSSENSITIVE THAT YOU REQUIRE MASSIVE OVERDOSE TO SEE THE EFFECT AND SO WE WANTED TO SEE POTENTIALLY COULD WE ILLICIT SOME KIND OF READ OUT THAT WOULD BE SUFFICIENTLY SENSITIVE THAT WE COULD FIND WHAT ARE THE CLINICALLY RELEVANT DOSES. THE OTHER THING IS WHEN WE USE REFLEX OF MODELS, WE ARE FORCED TO MOVE AWAY FROM CLINICAL PAIN STATES. A PATIENT THAT COMES IN DOES NOT SAY, I HAVE HEAT HYPER ALGEEZIA, THEY COMPLAIN OF PAIN, USUALLY SPONTANEOUS PAIN AND SOME KIND OF DISCOMFORT THAT MAY CHANGE AT 1 MOMENT. AND IT MAY BE ASSOCIATED WITH PAIN ON A RESPONSIVE NOCCUE LOWS STIMULI, IT MAY BE BURNING, IT IT IS A COMPLEX EVOLVING THING. AND WE NEED AS WE EXPLORE THIS, SURROGATES OF HUMAN PAIN CONDITIONSA BEST WE CAN BECAUSE THAT WILL REVEAL MORE ABOUT WHAT ARE THE ACTUAL PATHOPHYSIOLOGICAL PROCESSES THAT ARE OCCURRING IN OUR PATIENTS AND IF WE CAN MODEL THAT, WE CAN THEN POTENTIALLY SEE WHICH--WHAT ARE THE CIRCUIT BASES FOR THOSE CHANGES THAT OCCUR IN OUR PATIENTS AND WHAT ARE THE MOLECULAR DRIVERS OF THAT WHICH MAY REVEAL NEW TARGETS FOR TREATMENT. SO AS YOU CAN SEE THIS IS A PRETTY AMBITIOUS, THIS WAS HIS QUALIFYING EXAM. THIS IS WHAT HE PRESENTED TO HIS EXAMINERS AND THEY ALL ROLL THEIR EYES AND SAID CRAZY. BUT SOMETIMES IF YOU TRY HARD, YOU CAN AT LEAST POTENTIALLY MAKERS AND A BREAK THROUGH. SO HE--HIS APPROACH, HIS IS THE LITTLE BLACK BOX THAT HE DEVISED AND HIS VIEW WAS RATHER THAN LOOKING AT THE MOUSE FROM THE SIDE WHICH TYPICALLY AN OBSERVER DOES OR FROM A CAMERA ON THE TOP, HOW ABOUT LOOKING AT THE MOUSE FROM UNDERNEATH. INSTEAD OF TOP DOWN FROM A BOTTOM UP APPEARANCE AND THE REASON WE DID THAT, THE REASONING FOR THAT WAS THAT AS PREY ANIMALS, EVOLUTION WOULD HAVE POTENTIALLY CHANGED BEHAVIORS SUCH THAT THE ANIMAL WOULD NOT DISCLOSE TO AN OBSERVER FROM THE SIDE OR THE TOP THAT THE ANIMAL HAD ANY DISEASE STATE. BUT FROM THE BOTTOM DOWN WHICH AN OBSERVER COULD COULD NOT NORMALLY SEE, YOU MAY SEE SUBTLE CHANGES THAT WOULD OTHERWISE NOT BE DETECTABLE. SO HOW TO DO THAT? WELL, HE SAID HIMSELF, HE BUILT THIS BOX AND HE PUT UNDERNEATH IT, AN INFRARED MICROSCOPE AND WORK THAT HAD HIGH SENSITIVITY, HIGH SPATIAL RESOLUTION AND HIGH TEMPORAL RESOLUTION AND THEN HE--HIS IDEA WAS TO USE A WELL KNOWN PHYSICAL PHENOMENON WHICH IS CALLED FRUSTRATED TOALTAL REFLECTION AND THAT INVOLVES IF YOU HAVE A LIGHT ON THE SIDE OF A GLASS SURFACE, THE LIGHT BOUNCES AROUND AND WILL NOT EXIT THE GLASS UNLESS THERE'S CONTACT ON THE SURFACE. SO IF YOU HAVE A MOUSE OR A RAT WALKING ON A GLASS SURFACE WHERE YOU HAVE INFRARED LIGHT THAT'S BOUNCING AT THE SITE OF THE CONTACT, THE LIGHT WILL GO THROUGH THE GLASS AND YOUR CAMERA UNDERNEATH CAN THEN PICK IT UP AND THEN PROVIDES YOU THEN WITH A WAY OF PICKING UP THE CONTACT OF PAWS WITH THE SURFACE AND AS CAN YOU SEE HERE, READILY SEE, THIS IS--NOT THE PICTURES OF THE PAWS, THIS IS--THOSE ELEMENTS OF THE PAWS THAT ARE IN CONTACT WITH THE SURFACE AND THE LUMINESCENT INTENSITY IS A REFLECTION OF THE DEGREE OF CONTACT WITH THAT SURFACE. CAN YOU THEN USE PSEUDOCOLLOR AND OTHER ANALYTIC METHODS TO ACTUALLY PICK UP BOTH THE SPATIAL, THE INTENSITY AND THE CHANGES. ONE OF THE PROBLEMS YOU QUICKLY SAW WAS THAT IF YOU TOOK A SINGLE FRAME, SOMETIMES YOU SAW SOMETHING THAT YOU DIDN'T KNOW WHAT IT WAS AND SO, HERE IS THE ACTUAL MOUSE. HERE AT THAT PARTICULAR MOMENT, THIS WAS THE ONLY CONTABLGHT IN THE SURFACE, SO HOW TO DEAL WITH THAT. AND SO THE IDEA HE CAME UP WITH WAS TO HAVE ALTERNATING FRAMES, 1 FRAME RED COMING UNDERNEATH SO YOU COULD SEE THE WHOLE ANIMAL AND THE WHOLE FRAME DOING THE TOTAL REFLECTION SO YOU CAN SEE THE CONTACT WITH THE SURFACE. THEN THE NEXT PROBLEM WAS HOW TO IDENTIFY WHICH PAW IS WHICH, WHICH MAY SEEM TRIVIAL BUT ACTUALLY TURNED OUT TO BE VERY COMPLICATED. ONE WAY WE'RE ATTEMPTED TO DO WAS TO DEVISE AN ALGORITHM THAT WOULD LOOK AT THE TAIL AND LOOK AT THE NOSE AND FROM THAT WOULD PREDICT WHICH IS THE 2 PAWS AND THEN 1 COULD ASSESS THE BEHAVIOR. SO HERE WE HAVE THE 2 ALTERNATING FRAMES. THIS IS A MOUSE AFTER THE INJECTION OF A FORMULA, WHERE THE DOZE APPLIED IS A AAA AGANIST AND YOU CAN CAN SEE THE ANIMAL IS BITING AND LICKING ITS PAW AND YOU CAN SEE IF YOU WATCH CLEARLY THERE'S A MAJOR DIFFERENCE BETWEEN THE EXPOSURE OF THE UNINJECTED PAW WHICH IS--THERE'S AN INTENSE SIGNAL AND THE INJECTED 1 WHICH IS LIFTED ALMOST NOT PRESENT. SO HOW DO YOU NORMALLY MEASURE THIS? WELL THE STANDARDWAY AND YOU TAKE VIDEO THE SIDE AND THEN HUMANS SIT THERE WITH A STOP WATCH COUNTING HOW MANY BITES OR ELEVATIONS OF THE PAW TAKE PLACE. AND AMGEN FOR EXAMPLE IN THEIR ANALGESIC PROGRAMS, THE WAY THEY DEAL WITH IT IS THE ENTIRE NEUROSCIENCE DIVISION HAS 5 HOURS A WEEK WHERE THEY HAVE TO LOOK THROUGH--FROM THE HEAD OF THE PROGRAM THROUGH TO THE JUNIOR RESEARCH ASSISTANT HAVE TO MANUELLY DO THIS. BUT HERE WE HAVE THE POTENTIAL WE, WE CAN NOW DO THIS AUTOMATICALLY. AND USING THIS PALM READER AND USING THE LUMINESCE ENSEL, THIS IS THE LUMINESCE ENSEL SIGNAL YOU GET FROM A HEALTHY CONTROL MOUSE. EACH PAW IDENTIFIED AS INDICATED THERE AND HERE IS THE LUMINESCE ENSEL SIGNAL FROM A FORMA LYNN TREATED MOUSE AND CONTRARY TO THE WAI WE THOUGHT ABOUT IT, THE BIG SIGNAL COMES FROM THE HEALTHY PAW BECAUSE THE ANIMAL IS NOW LIFTING THE PAINFUL PAW AND PUTTING MORE PRESSURE ON THE HEALTHY PAW, SO THE SIGNAL GOES DOWN ON THE EFFECTED PAW BUT IT GOES UP AND BUT IT BECOMES A VERY GOOD SURROGATE OF THIS AUTOMATIC CHANGE IN BEHAVIOR. AND NOW SUDDENLY INSTEAD OF NEEDING MANUEL OBSERVERS TO MEASURE THIS CHANGE IN BEHAVIOR, WE CAN VERY QUICKLY DEAL WITH THE WHOLE OF THE AMGEN NEUROSCIENCE DIVISION AND LITERALLY 10 MINUTES. WE ARE PLANNING TO SELL THIS INSTRUMENT TO AMGEN. SO HERE IS A MOUSE AFTER A MODEL OF INFLAMMATORY PAIN, THIS PAW OVER HERE WAS INJECTED WITH COMPLETE ADJUVANT 24 HOURS BEFORE THIS RECORDING AND YOU CAN READILY NOTICE THAT AGAIN THE SIGNALOT EFFECTED SIDE IS MUCH LOWER THANOT HEALTHY SIDE. --THAN ON THE HEALTHY SIDE. THIS IS THE SAME MOUSE, GIVEN A NONSTEROIDAL ANTIINFLAMMATORY DRUG, AND YOU CAN NOW SEE THAT THE PAW EXPOSURE IS PRETTY EQUAL BETWEEN THE 2 PAWS. WHAT IS IMPORTANT HERE IS THAT THE DOSE OF [INDISCERNIBLE] WE'VE GIVEN HER IS 10 TIMES LOWER THAN THAT IS REQUIRED TO EFFECT ANY REFLECTIVE BEHAVIOR, LIKE A THERMAL EVOKED RESPONSE OR MECHANICAL RESPONSE. SO WE HAVE AS HIGHER SENSITIVITY OF SEEING A CHANGE IN BEHAVIOR, A NORMALIZATION OF BEHAVIOR THAN THAT USING STANDARD MODELS AND AGAIN THIS TURNS OUT THAT THIS DOSE, THE DRUG EXPOSURE IS NOW IDENTICAL TO THAT SEEN IN PATIENTS. SO WOE DON'T NEED TO OVERDOSE OUR ANIMALS TO SEE A CHANGE IN BEHAVIOR. WHAT WE DO NEED ARE SENSITIVE OUTCOME MEASURES THAT REFLECT THE ACTUAL PROCESS THAT THE DRUG IS SUPPOSED TO BE ACTING ON. SO NOW FOR THE FIRST TIME IN NIGH CAREER, I HIRED A SOFTWARE ENGINEER TO HELP US TAKE THE OUTPUT FROM THIS INSTRUMENT, THE PALM READER AND WE NEED TO MEASURE THE LUMINESCE ENSEL OF EACH OF THE PAWS AS INDICATED HERE, WE CAN CAN GET THESE MEASURES. WE CAN ALSO START TO MEASURE OTHER FEATURES FOR EXAMPLE, LOOK AT THE INDIVIDUAL TOES, LOOK AT THE TRAJECTORY, THE MOVEMENTS, ET CETERA SX AND WE'RE DOING ALL OF THOSE. AND THAT ENABLES US NOW FOR EXAMPLE LOOKING AT INDIVIDUAL--THE LUMINESCE ENSEL FROM EACH OF THE PAWS THAT CAN GET THIS KIND OF SIGNAL OVER THERE. BUT THE BIG PROBLEM THAT WE CONFRONTED WAS THE 1 I MENTIONED BEFORE. HOW TO KNOW WHICH PAW IS WHICH. HERE IS THE ALGORITHM THAT TOOK US 18 MONTHS TO DEVELOP WORKING WITH SOME APPLIED MATHEMATICIANS AT HARVARD, IDENTIFYING THE TAIL AND THE NOSE THROUGH THE SHAPE. YOU CAN CAN SEE THE SHAPE CHANGES QUITE DRAMATICALLY, THE CENTER OF THE IMAGE THERE WHICH TURNS OUT TO BE QUITE INFORMATIVE BUT IF THE ANIMAL STANDS UP, IF IT GROOMS, YOU LOSE THE ALGORITHM CANNOT COPE WITH THAT. SO HOW TO DEAL WITH THAT, AT THE TIME WE ARE STRUGGLING WITH THIS, THERE'S BEEN AN ENORMOUS EXPLOSION, AS SOME OF YOU WILL NOTE IN THE DEVELOPMENT OF THE APPLICATION OF NEURAL NETWORKS, DEEP LEARNING, USING NEURAL NETWORKS AND WHAT THIS INVOLVES IS TRAINING A MACHINE TO RECOGNIZE DIFFERENT IMAGES. SO WHAT WE'VE DONE HERE IS TAKE EVERY POSSIBLE IMAGE WE CAN OF THE HIND PAWS AND TRAIN NEURAL NETWORK TO RECOGNIZE THEM. AND THIS IS TRULY TRANSFORMATIVE. SO NOW WE HAVE A PROGRAM WHEREBY WE HAVE DIFFERENT COLORS. WE HAVE A COLOR FOR THE TAILS, FOR EACH OF THE PAWS. THIS IS A HEALTHY ANIMAL WALKING AROUND, DOING ITSELF STUFF AND INSTEAD OF WITH OUR ALGORITHM, OUR ORIGINAL ALGORITHM WHICH WAS ONLY ABOUT 85% CORRECT IN TERMS OF IDENTIFYING WHICH PAW IS WHICH AND TO PUT THE APPROPRIATE LUMINESCE ENSEL TO THAT PAW, THIS--THIS OUTCOME NOW DRIVEN BY THIS NEURAL NETWORK LEARNING IS NOW ABOUT 99% CORRECT. SO WE KNOW EXACTLY WHAT THE ANIMAL IS DOING, WHERE IT IS AND WHICH PART THIS BODY IS ENABLED. HERE WE HAVE ANOTHER--THIS IS THE ANIMAL WITH THE FORMALIN AGAIN, WE CAN IDENTIFY THE PAW EVEN THOUGH IT'S NOT MAKING CONTACT WITH THE SURFACE, EVEN WHEN THE ANIMAL BITES IT, YOU CAN DIFFERENTIATE THE FRONT PAW AND THE BACK PAW. NOW WE CAN GET ENORMOUS INFORMATION FROM THIS INSTRUMENT. SO NOW WE ARE--THIS IS VERY RECENT, IT'S LITERALLY ABOUT 3 WEEKS AGO THAT WE FINALLY SOLVED THAT MAJOR PROBLEM AND WE ARE NOW ABOUT TO ERM BARK ON A--'EM BARK ON A OF USING IT TO EXPLORE READ OUTS THAT MAY REFLECT THE PRESENCE OF PAIN IN CONDITIONS OF NERVE INJURY, AFTER TREATMENT WITH CHEMO THERAPEUTIC AGENTS AND ALSO TO EXPLORE WHETHER WE CAN DETECT CHANGES THAT REFLECT CNS SIDE EFFECTS. AS INDICATED EARLIER 1 OF THE COMPLAINTS IS PATIENTS FEEL DIZZY. WELL HO HOW DO YOU MEASURE DIZZINESS IN THE AMOUNT? WE CAN PRODUCE DRUGS THAT PRODUCE DIZZINESS AND SEE IF WE CAN SEE A DIFFERENCE IN BEHAVIOR. ONE OF THE A. I. FEATURES OF THIS WHICH IS THE OPPOSITE OF THE NEURONET WORK WHERE YOU TRAIN THE MACHINE TO RECOGNIZE CERTAIN FEATURES IS THAT USING ARTIFICIAL INTELLIGENCE, THE DATA THAT WILL BE GENERATED BY AN ANIMAL RUNNING AROUND DOING ITSELF STUFF WILL PRESENT PATTERNS AND OUR ALLEGOR IPGHT IMPEDIMENTSS WILL BE ABLE TO SAY THIS ANIMAL IS DIFFERENT FROM THIS ANIMAL AND THIS ANIMAL CHANGED IN RESPONSE TO THIS TREATMENT AND WE'RE HOPEFUL THIS WILL REFLECT A NOVEL APPROACH TO THE EXPLOITATION OF PRECLINICAL MODELS FOR THE STUDY OF MAIN AND THIS WILL HELP US AS WE TEASE OUT WHAT ARE THE TARGETS OF DIFFERENT PAIN REGIONS AND WHAT ARE THE EFFORTS OF STRATEGIES IN MANAGING THAT PAIN AND HOPEFULLY WE CAN MOVE TO A STAGE WHERE WE DON'T HAVE GREAT SUCCESS IN CLINICAL MODELS THAT THEN FAILS TO TRANSLATE IF PHASE 2 B PROOF OF HUMAN CONCEPT STUDIES. SO THAT HAS BEEN THE MOTIVATION BEHIND THIS. THE SECOND PART OF THIS IS WHAT ABOUT BEING ABLE TO MEASURE PAIN OBJECTIVELY, ARE THERE OTHER BIOMARKERS OF PAIN AND THIS IS WORK THAT--COLLABORATION WITH TOM SCAMMEL, WHO IS A NEUROLOGYST IN BOSTON AND A POST DOC WHO WAS A POST DOC AT THE TIME IN MY LAB [INDISCERNIBLE] AND HIS WIFE CLOE, [INDISCERNIBLE] WHO IS IN TOM'S LAB AND THEY EXPLORED THE POSSIBILITY THAT SLEEP COULD BE A POTENTIAL BIOMARKER OF THE PRESENCE OF PAIN. AND THE MODEL THERE IS USING MICE AND THE MICE HAVE ELECTRODES IMPLANTED TO HAVE EG RECORDINGS AS WELL AS EMG RECORDINGS FROM THE NECK AND THE MICE ARE FREELY BEHAVING AGAIN AND ABLE TO SLEEP AND WAKE AND IT IS POSSIBLE THEN TO IDENTIFY WITH FROM THE EGs FROM THE STATE OF THE ANIMAL WHETHER IT'S AWAKE, ASLEEP OR REMETABOLISM OR REMETABOLISM SLEEP AND THEN THEY LOOKED AT 2 DIFFERENT NEUROPATHIC PAIN MODELS, 1 IS AN INJURY MODEL WHICH PRODUCES PRO PER CYSTENT DAMAGE AND PERSISTENT HYPER SENSITIVITY AND ANOTHER 1 IS SCIATIC NERVE CRUSH WHERE THERE'S TEMPORARY DAMAGED REINITIATION OF THE PERIPHERY AND THE HYPER SENSITIVITY IS TRANSIENT. SO THEY FIRST LOOKED WHETHER THERE WAS A CHANGE IN THE DEGREE TO WHICH AFTER ANY OF THESE MODELS THERE WAS A CHANGE IN HOW MUCH WAKE TIME, HOW MUCH TIME THE ANIMAL WAS IN NONREM SLEEP AND REM SLEEP AND THERE WAS NO DIFFERENCE AT ALL. LITERATURE IS FULL WITH THE DISCUSSION BUT VERY LITTLE HARD DATA THAT THE PRESENCE MUCH PAIN--PRESENCE OF PAIN INTERRUPTS SLEEP AND IF YOU LOOK IN THIS WAY, THERE WAS NO INTERRUPTION, NO CHANGE. IF YOU LOOK AT THE DISCIPLINARY URNAL VARIATION, THIS IS IN THE AMOUNT OF NONREM SLEEP OVER 24 HOURS, THIS IS IN THE SNI MODEL OF NEUROPATHIC PAIN, THIS IS THE SAME ANIMAL BEFORE INJURY, AND YOU CAN SEE THIS IS ABSOLUTELY NO DIFFERENCE, THIS WASN'T LOOKING VERY PROMISING. SO THE QUESTION THEN WAS WHAT POTENTIALLY MAY BE THE DRIVER OF NEURO--WHAT ARE THE DRIVERS OF SPONTANEOUS NEUROPATHIC PAIN AND WHAT ARE THE CONSEQUENCES OF THAT. WELL, SPONTANEOUS NEUROPATHIC PAIN IS ASSOCIATED WITH ONGOING SPONTANEOUS ACTIVITY IN INJURED AND NEIGHBORING NONINJURED SENSORY FIBERS. WE PERFORMED A STUDY LAST YEAR THAT SHOWED THAT IF YOU OPT OGENETICLY ACTIVATE SUSCEPTORS AS FEW AS 10 OF THEM TRANSDERMALLY YOU GET CHANGE IN BEHIEEVER. SO THE MIND SET IS IF YOU HAD SPONTANEOUS ACTIVITY IN AN INJURED NERVE, IN SLEEP WOULD THAT PRODUCE SOME CHANGE OR NOT. AND INDEED THAT TURNED OUT TO BE THE CASE. SO THAT ALTHOUGH THE TOTAL AMOUNT OF TIME THAT THE ANIMAL IS AWAKE OR IN NONREM SLEEP OR NONREM SLEEP DID NOT CHANGE AT ALL. IF WE LOOK CLOSELY AT THE ACTUAL PATTERNS OF SLEEP, HERE'S THE MOUSE IN NONREM SLEEP AND THEN IT SHOWS A BRIEF PARDONS OF AWAKENING, THE ANIMAL AWAKES IN AN EEG SENSE, THE MOUSE MAY NOT REALIZE IT'S AWAKE BECAUSE IT'S SHOW SHORT, THIS IS A MATTER OF SECONDS OVER HERE, 4-SECONDS AND AT THE TIME IT WAKES THERE IS A CLEAR EMG REFLECTION OF THIS AWAKENING. AND IF YOU THEN LOOK AT TRAINING OF EEGs, YOU CAN SEE THAT THESE LITTLE BURSTS OF BRIEF AWAKENING, OCCUR REPEATEDLY BUT ONLY IN THE CONTEXT OF INJURED--OF AN INJURED NERVE, SO HERE WE HAVE RECORDINGS OF THE NUMBER OF BRIEF AWAKENS OF PERCENTAGE OF THE BASE LINE AND YOU CAN SEE 14 DAYS AFTER SPREADING INJURY, AT THE TIME THERE'S MARKED HYPER SENSITIVITY, THERE'S A SIGNIFICANT INCREASE IN THE NUMBER OF BRIEF AWAKENINGS AND THIS PERSISTS FOR 49 DAYS AFTER THE SURGERY SO THAT WE HAVE A ROBUST MEASURE OF SOMETHING THAT IS GOING ON IN THE MOUSE WHEN IT HAS AN INJURED NERVE FIBER. ANOTHER WAY OF MEASURING THIS IS TO LOOK AT THE DURATION OF THE NONREM SLEEP AND THE LENGTH OF THE SLEEP AND IN THE MICE WITH THE [INDISCERNIBLE] INJURY, THERE IS REDUCTION IN THE LENGTH BECAUSE IT'S INTERRUPTED BY THESE BRIEF AWAKENINGS AND THE NUMBER OF EPISODES SIGNIFICANTLY INCREASES SO WE HAVE 3 DIFFERENT WAYS OF MEASURING THESE BRIEF AWAKENING THAT ARE OCCURRING IN THE SETTING OF AN INJURED NERVE THAT IS ASSOCIATED WITH PAIN HYPER SENSITIVITY. AND INDEED, IT TURNS OUT THERE IS A VERY GOOD CORRELATION BETWEEN THESE BRIEF AWAKENINGS, SO THIS IS 7 DAYS, UP TO 42 DAYS AFTER THE PRODUCTION OF THIS PARTIAL NERVE INJURY, WE HAVE THIS ONGOING SIGNIFICANT PRESENCE OF BRIEF AWAKENINGS THAT IS HIGHLY CORRELATED WITH THE PRESENCE OF MECHANICAL HYPER SENSITIVITY AND HYPER SENSITIVITY TO COLD. WHEN WE LOOK IN THE SCIATIC NERVE INJURY, WHERE THERE IS BROAF AWAKENING BUT THEN RETURN TO BASE LINE. THIS, TOO, CORRELATED WITH THE CHANGES IN SENSITIVITY TO A MECHANICAL AND COLD STIMULI. IN BOTH CASES HERE, THERE'S A TEMPORARY HYPER SENSITIVITY UNTIL THERE'S REINNOVATION AND THEN IT RETURNS AS THIS REINNOVATION, THE HYPER SENSITIVITY RETURNS FROM NORMAL AND THE BRIEF AWAKENINGS OCCUR, SO THIS SUGGESTS THE POSSIBILITY THAT THESE BRIEF AWAKENINGS ARE REA REFLECTION OF THE PRESENCE IN THESE MICE, WHILE THEY SLEEP OR THE BURST OF ACTIVITY THEY WOULD PRODUCE SPONTANEOUS PAIN IN A PATIENT BUT THEN PRODUCES THIS BRIEF AWAKENING AND IF SHOW, THIS COULD THEN BE A BIOMARKER OF THE PRESENCE OF SPONTANEOUS PAIN AND SPONTANEOUS NEUROPATHIC PAIN. HOW TO TEST THOSE, WELL, LIKE MANY PEOPLE WE ARE EXTREMELY--WE WANT TO TAKE ADVANTAGE OF THE EXTREMELY POWERFUL GENETICALLY TARGETED TOOLS, THE 1 THAT WE USED HERE WAS TO USE A TETANUS TOXIN UNDER A LOX-P SITE THAT WOULD ENABLE US WITH A CREE DRIVER TO EXPRESS TOXIN ONLY IN SMALL DIAMETER SENSORY NEURONS AND THAT WOULD PRESENT THE RELEASE OF TRANSMITTERS CENTRALLY AND THIS WOULD ENABLE US TO SILENCE THESE IN A TAMOXIFEN DRIVEN WAY. THE ACTIVITY OF THE SMALL SENSORY NEURONS. AND WHEN WE DID THIS, WE FOUND--THIS IS THE WILD TYPE LITTER MATE CONTROL, WE HAD SLEEP EPISODE LENGTH AND REDUCED BECAUSE OF THE BRIEF AWAKENINGS THAT INTERRUPT THE SLEEP AND YOU CAN SEE THAT IN AN ANIMAL WHERE THE DOSE RECEPTORS ARE SILENCED, THERE IS THE BRIEF AWAKENINGS NO LONGER OCCUR. OTHER BRIEF AWAKENINGS BEING DRIVEN BY ECTOPIC ACTIVITY IN THE ENGINEER NERVE FIBERS, DAVID [INDISCERNIBLE] A POST DOC IN MY LAB WHO IS 1 OF THE PIONEERS OF USING G-CAMP RECORDINGS FROM SENSORY NEURONS IS WORKING WITH [INDISCERNIBLE] TO TRY AND SEE IF THERE'S A CORRELATION BETWEEN THE BRIEF AWAKENING AND SPONTANEOUS BURSTS OF ACTIVITY IN NEURONS. AND THAT'S A WORK IN PROGRESS. WHAT WE HAVE SHOWN THOUGH IS THAT 1 OF THE POSSIBILITIES WAS THAT THE ANIMAL, THE BRIEF AWAKENINGS OCCURRED FROM THE ANIMAL MOVING SLIGHTLY AND ACTIVATING PERIPH RAT INPUTS WHICH WOULD THEN PRODUCE THESE AWAKENINGS AND IF WE THEN ANESTHETIZED THAT PORTION OF THE PAW THAT WAS STILL INNOVATED AFTER THE [INDISCERNIBLE] OF INJURY, WE FOUND NO CHANGE IN THE REDUCTION IN SLEEP EPISODE LENGTH SUGGESTING THAT THE INPUT THAT'S DRIVING THESE BRIEF AWAKENINGS DOES NOT COME IN TACT FROM SENSORY FIBERS BUT FROM THE INJURED FIBERS THEMSELVES. CAN WE USE THIS BIOMARKER AS A MEASURE OF ANALGESIC EFFICACY? SO WE STARTED OFF AT LOOKING AT GABA PENTINE REGIMENIN WHICH IS A STANDARD TREATMENT FOR NEUROPATHIC PAIN GIVING 3 DOSES, 30-MILLIGRAMS PERKILOGRAM IS THE DOSE THAT IS REQUIRED IN STANDARD NEUROPATHIC PAIN MODELS FOR FOR FLEXIBLIVE MEASUREMENTS AND WE LOOKED AT SLEP AMOUNT AND EVEN THOUGH GABA PENTINE REGIMENIN IS THOUGHT TO BE A SEDENTARIAATIVE, WE SAW NO AMOUNT THERE IN TERPS OF TOTAL AMOUNTS OF SLEEP. HOWEVER WHEN WE LOOK AT THE SLEEP--THE DURATION OF THE SLEEP EPISODES WHICH WAS INTERRUPTED BY THE [INDISCERNIBLE] OF INJURY, WE CAN SEE A VERY NICE DOSE RESPONSE RETURNING THE SLEEP EPISODES TO THEIR NORMAL LENGTH. AND WHAT IS I THINK PARTICULARLY IMPORTANT HERE IS THAT WE SEE EFFECTS HERE AT 3-MILLIGRAMS PERKILOGRAM WHICH IS A 10th OF A DOSE THAT IS REQUIRED IN STANDARD MODELS SO AGAIN, IF YOU HAVE THE RIGHT OUTCOME MEASURE, YOU CAN GET SOME OF THE SENSITIVITY. SO WE'RE ENCOURAGED BY THIS TO CONSIDER THAT BRIEF AWAKENINGS MAY REPRESENT A BY O MARKER OF SPONTANEOUS NEUROPATHIC PAIN. THIS IS JUST THE TIME COURSE AFTER ADMINISTRATION OF THE GABA PENTINE REGIMENIN AND YOU CAN SEE THIS ACTUALLY MATCHES EXACTLY THE PK OF THE T-HOF MUCH THE KINETICS OF GABA PENTINE REGIMENIN WHERE YOU HAVE HIGH ACTIVITY, SOME ACTIVITY AT 2-4 HOURS AND THEN RETURN TO NORMAL WHEN THE LEVELS DROP VERY LOW. CLOE AND [INDISCERNIBLE] ARE NEW FACULTY NEIGHBORS OF YOURS AT JOHNS HOPKINS. AND WILL BE EXTENDING THIS WORK INTO HUMAN PATIENTS IN THE NEAR FUTURE. TO END OFF, FIRST 2 STORIES RELATE TO--ATTEMPT TO IMPROVE THE WAY IN WHICH WE CAN USE PRECLINICAL MODELS TO MEASURE PAIN AND TO GET A BIOMARKER OF PAIN THAT HAS HOPEFULLY TRANSLATABLE ELEMENT TO IT, THE LAST BIT I WOULD LIKE TO JUST DISCUSS WITH YOU IS ACTUALLY MODELING PAIN USING HUMAN NEURONS. AND THIS INVOLVES THE APPLICATION OF STEM CELL TECHNOLOGY WHEREBY WE CAN DIFFERENTIATE HUMAN INDUCED PLURIPOTENT CELLSY POTENTIAL CELLS INTO SPECIFIC DIFFERENT KINDS OF NEURONS AND IN THIS CASE, WE'RE MAKING NOSE SUSCEPTORS FROM IPSCs. THIS IS WHAT THEY LOOK LIKE. IF YOU THEY LOOK SURPRISINGLY SIMILAR IF YOU TAKE A MOUSE DRG AND PLATE THE CELLS, IT'S DIFFICULT TO TELL THEM APART FROM THE NEURONS WE MAKE HERE AND THEY EXPRESS FOR EXAMPLE NAV1.7 WHICH IS 1 OF THE VOLTAGE SETTING CHANNELS THAT IS FOUND SELECTIVELY IN THOSE SUSCEPTORS. WE--1 OF THE GREAT ADVANTAGES OF USING THESE KINDS OF DIFFERENTIATION PROTOCOLS FROM INDUCED PLURIPOTENT STEM CELLS IS THAT YOU CAN PRODUCE NEURONS OF THE SINGLE TYPE. A GRADUATE STUDENT OF MY LAB HAS USED 2 PROTOCOLS HERE, COMBINED THEM AND DONE SINGLE CELL SEQUENCING AND BASICALLY IN THE 1 PROTOCOL, WE MAKE A SET OF NEURONS THAT ARE NONPATHOGENIC, NO SUSCEPTORS AND THE OTHER 1AR PATHOGENIC SUSCEPTIBLES AND THE NEURONS IN THESE PROCOLS ARE NO SUSCEPTORS OF 1 TYPE WHICH MAKE ITS EASIER CONDUCT FORMAL SCREENS AND TO USE THEM TO DISSECT OUT THEIR PARTICULAR MECHANISMS. ONE WAY WE CAN DO THAT IS ELECTROPHYSIOLOGICALLY BY USING WHOLESALE PARCH RECORDINGS AND IF YOU DO THAT FROM OUR HUMAN SUSCEPTORS WE NOTICE THAT THE ACTION POTENTIALS FROM THESE NEURONS HAVE THE TYPICAL FEATURE OF NO SUSCEPTORS WHICH IS THEY HAVE BROAD ACTION PROTENTIALS DUE TO THE PRESENCE OF MARKED POTASSIUM CURRENTS AND CALCIUM CURRENTS. WE CAN ALSO DISSECT WHICH OF THE BIG DRIVERS OF EXCITABILITY IN THESE NEURONS AND IN THIS PARTICULAR PLACE WE'RE USING A TOXIN, PROTOXIN 2 WHICH ACTS ON NAV 1.7. SO THIS IS THE CURRENT, NORMALLY WE THEN GIVE THE PROTOXIN 2, IT'S REDUCED AND WE CAN PROTRACT IT, THIS IS THE 1.7 CONTRIBUTION TO THE TOTAL CURRENT IN THESE HUMAN NOSE SUSCEPTORS AND THIS ENABLES US TO EXPLORE CHANNEL OOPERATING GLOBALLYATHYS WHICH PLAY A PROMINENT FEATURE IN AT LEAST HUMAN CONDITIONS. WE CAN ALSO RECORD FROM NEEZ NEURONS USING MULTIELECTRODE ARRAY RECORDING DEVICES WHICH ENABLE US TO MONITOR THE SPONTANEOUS FIRING OF THE CELLS. BUT WHY NAV1.7. IT TURNS OUT THERE ARE A NUMBER OF WELL CHARACTERIZED MUTATIONS IN THAT 1.7 WHICH PRODUCE VERY DISTINCT PAIN PHENOTYPES. TWO OF THEM PRODUCE PAIN DUE TO GAIN OF FUNCTION MUTATIONS INHERITED FROM [INDISCERNIBLE]. WHICH MANIFESTS IN CHILDHOOD HAS SEVERE BURNING PAIN AND ARETHEMIA, PARTICULARLY IN THE LIMBS, THE PAIN IS SO BAD THAT SOME OF THEM WALK AROUND WITH BUCKETS OF ICE AND WHEN THEY GET 1 OF THESE, THEY PUT THE LUMS IN THIS BUCKET OF ICE TO EXTENT THEY SOMETIMES GET ESCHEMIC GANG GREEN FROM THE INTENSE COLD. ANOTHER DEFINITE SET OF GAIN OF FUNCTION MUTATIONS PRODUCE A PHENOMENON KNOWN AS [INDISCERNIBLE] PAIN DISORDER AND IT'S MARKED IN INFANTS. THE PAIN IS TYPICALLY PERIRECTAL OR IN THE FACE WITH THIS VERY MARKED SKIN CLESHING, ON 1 DAY IT CAN BE HALF THE BODY, THE OTHER DAY IT CAN BE THE OTHER HALF WITH NO EXPLNATION WHY IT SHOULD BE LIKE THAT AND IT'S VERY--THE INFANTS EXPERIENCE THE MOST EXCRUCIATING PAIN IMAGINABLE. LOSS OF FUNCTION MUTATIONS IN N AV1.7 PRODUCE CONGENITAL INSENSITIVITY PAIN WHERE THEY ARE BORN WITHOUT A LACK OF THE CAPACITY TO FEEL PAIN AND RESPONSE TO NOXIOUS STIMULI AND CONSEQUENCE THEY DAMAGE THE TIPS OF THEIR FINGERS AND TOES, PARTICULARLY THEIR FINGERS BECAUSE WE EXPLER THE WORLD WITH OUR FINGERS. THEY ALSO DAMAGE THEIR LIPS AND TONGUES BECAUSE WHEN WE EAT, OFTEN WITHOUT BEING AWARE OF IT, WE BITE INTO OUR TONGUES OR OUR CHEEK AND WE CORRECT THAT IMMEDIATELY BUT THEY ALSO CANNOT DIFFERENTIATE BETWEEN WARMTH AND SCALDING HOT AND TYPICALLY THESE INDIVIDUALS HAVE NO PAIN, DIE IN THEIR 20S. THIS IS ALSO A WARNING FOR US AS WE ATTEMPT TO DEVELOP NEW ANALGESICS IS THAT OUR GOAL SHOULD NOT BE TO SWITCH OFF PAIN BECAUSE PAIN HAS EVOLVED FOR A REASON, IT IS A PROTECTIVE MECHANISM THAT INFORMS US OF THE PRESENCE OF POTENTIAL DANGER OR INJURY AND HELPS US AVOID THEM. AND WHAT WE THEED TO DO IS NORMALIZE THE PAIN THAT OUR PATIENTS EXPERIENCE BUT NOT SWITCH OFF THEIR CAPACITY TO FEEL PROTECTIVE PAIN AND THIS IS SOMETHING THAT SOMETIMES PHARMA HAS NOT FULLY APPRECIATED. SO THESE ARE THE MUTATIONS IN NAV1.7 THAT PRODUCE THESE DIFFERENT CLINICAL SYNDROMES AND THE BEAUTY NOW IS USING CRSPR, GENOME EDITING TECHNOLOGY, WE CAN CREATE NAV1.7 MUTANT MODELS AND WHAT WE'VE BEEN DO SUGGEST TO HAVE PARALLEL MODELS WHERE WE MAKE MUTANT MICE OR HUMAN IPSC LINES AND IN THAT WAY WE CAN COMPARE THE PRIMARY NEURONS WITH THE INDUCED NEURONS AND WE CAN GET--WE CAN CREATE MUTATIONS AS WELL AS TRY AND GET PATIENT LINES AND SEE WHETHER THEY ARE PHENOCOPIED AND IDLY WE CAN LOOK AT BEHAVIOR. SO SO FAR WE'VE MANAGED TO GENERATE 10 CRSPR LINES WITH THESE 3 MUTATIONS AND 3 MOUSE LINES, INCREASINGLY IT'S CLEAR TO US THAT THE MOUSE LINES DO NOT PHENOCOPY THE PATIENTS AND YET THEY ARE FEATURES OF THE--THE HUMAN IPSC DERIVED NEURONS WHICH SUGGEST THAT THEY MAY PHENOCOPY OF THE PATIENTS. SO HERE ARE MULTIELECTRODE ARRAY RECORDINGS, THESE ARE CONTROL--AT THE CONTROL IPSC LINES, THESE ARE 2 INDEPENDENT CLONES WITH THE PARTICULAR MUTATION. THIS IS RECORDED AT 33-DEGREES, THIS AT 37, YOU CAN SEE THAT QUITE CLEARLY IN BOTH TEMPERATURES THERE'S MORE SPONTANEOUS ACTIVITY IN THE--WHEN THE LINE HAS THESE MUTATION AND IN ADDITION THERE IS A TEMPERATURE DEPENDENCE AND THIS IS IMPORTANT BECAUSE THAT'S EXACTLY WHAT THE PATIENT'S COMPLAIN OF. I MENTIONED BEFORE THEY WALK AROUND WITH BUCKETS OF ICE AND THE REASON FOR THAT IS THAT IF THE SKIN IS WARM, IT PRODUCES 1 THE [INDISCERNIBLE] OF PAIN AND WE CAN REPLICATE THIS FEATURE OF THE PAIN PHENOTYPE IN THE DISH. THIS JUST SHOWS THE QUANTIFICATION OF THESE CHANGES. AND WE FEEL THIS IS GOING TO HELP US IN SOME WAYS TO BRIDGE THE GAP BETWEEN PRECLINICAL MODELS AND THEIR LIMITATIONS WHICH IS THAT WE CAN'T KNOW WHAT A MOUSE IS FEELING AND EVEN WHAT IT IS FEELING MAY BE DIFFERENT FROM WHAT OUR PATIENTS ARE FEELING BUT NEVERTHELESS, I HOPE I CONVINCED YOU THAT USING NEW IMAGE CAPTURE CAN ANALYSIS MORE INFLATION FROM MICE THAN WE COULD IN THE PAST AND THEN USING ARTIFICIAL INTELLIGENCE AND MACHINE LEARNING, YOU WILL BE ABLE TO INTERROGATE THE DIFFERENCES BEHAVIOR AND OBSERVE IN AN INDEPENDENT WAY AND THAT MAY HELP US DISSECT OUT THE MECHANISMS THAT ARE DRIVING PAIN AND THE CONSEQUENCES OF MANIPULATING THE TARGETS WITHIN THAT--IN THE PAIN NETWORK. IN ADDITION, THE PRECLINICAL MODELS HAVE HINTED TO US, THE POSSIBILITY THAT BRIEF AWAKENINGS MAY BE A SURROGATE OF THE SPONTANEOUS ECTOPIC ACTIVITY THAT OCCURS IN AN INJURED NERVE AND THEREFORE MAY ENABLE US TO USE IT AS AN OBJECTIVE MEASURE OF THE PRESENCE OF SPONTANEOUS PAIN IN PRECLINICAL MODELS BUT EVEN MORE SO IN PATIENTS. IF THIS IS TRUE, AND THE SAME SENSITIVITY TO ANALGESICS OCCURS, 1 CAN ANTICIPATE THEN, CLINICAL TRIALS WHEREBY THE PATIENT SLEEPS. WE MEASURE THEIR EEG, LOOK AT BASAL LEVEL OF BRIEF AWAKENINGS AND THEN SEE IF NEW THERAPIES CAN INTRODUCE THAT. AND IF THAT TURNS OUT TO BE TRUE, WE WOULD THEN HAVE A QUITE TRANSFORMATIVE AND DIFFERENT APPROACH TO THE DEVELOPMENT OF NOVEL ANALGESICS AND THEN FINALLY, I'VE INDICATE THAD WE CAN USE--INDICATED THAT WE CAN USE HUMAN AND PATIENT NEURONS AS A WAY OF TEASING OUT SOME FEATURES OF THE MECHANISMS THAT DRIVE PAIN. THE HUMAN NEURON TEAM INCLUDED POST DOC [INDISCERNIBLE] A GRADUATE STUDENT, [INDISCERNIBLE] FORMER POSTDOC IN [INDISCERNIBLE]. AND THIS WORK HAS ALL BEEN FUNDED BY THE NIH AND I'M VERY GRATEFUL FOR THE SUPPORT. THANK YOU. [ APPLAUSE ] >> I HAVE A QUESTION, SO IF I FEEL PAIN I WILL REALLY HAVE DIFFICULT FALLING ASLEEP, SO DO MOUSE HAVE THE SAME PROBLEM AND USE THE SAME BIOMARKER INSTEAD OF THE FREQUENCY TO WAKE UP? >> SO THAT WAS OUR EXPECTATION AND WE LOOKED--THAT'S WHAT WE LOOKED FOR AND WE FOUND ABSOLUTELY NO DIFFERENCE WHATSOEVER IN THE TOTAL DURATION OF SLEEP IN ANIMALS WITH THESE PAIN MODELS. WITH WE USE INFLAMMATORY, I DIDN'T SHOW THE DATA BUT WHEN WE USE STANDARD INFLAMMATORY MODELS, WE FOUND A SURPRISE WHICH IS THE ANIMALS SLEPT MORE THAN WE WOULD EXPECT AND AGAIN WE NEED TO TEASE OUT WHAT THAT MEANS. SO OF COURSE WE'RE AWARE OF THE FACT THAT PATIENTS REPORT THAT THEY--THEY FIND THE SLEEP INTERRUPTED, ALBAN AND CLOE WERE PUBLISHERS IN A PAPER WHERE WE SHOWED THAT DISRUPTING SLEEP INCREASED PAIN SO HAVE YOU A VICIOUS CYCLE SO IF YOU SLEEP LESS YOUR PAIN GETS WORSE BUT THAT'S A DIFFERENT ISSUE FROM JUST USING THE FACT THAT IF YOU ARE ASLEEP, EACH IF IT'S DIFFICULT, THEN AND YOU HAVE A SPONTANEOUS BURST OF ACTIVITY IN THOSE SUSCEPTORS THIS IS SUFFICIENT TO JUST CAUSE A BRIEF PERIOD OF AWAKENING SUCH THAT YOU WOULDN'T BE AWARE OF IT, IT'S LITERALLY A MATTER OF SECONDS AND YOU GO BACK TO SLEEP BUT IT'S--IF IT HOLDS OUT, THAT MEANS WE CAN MEASURE FOR THE FIRST TIME THE PRESENCE OF THE ONGOING PRESENCE IN A PATIENT OF SPONTANEOUS PAIN RELATED ACTIVITIES. >> I REALLY LIKE THE PALM READER, DETAILED BEHAVIOR OVER PERIODS OF TIME, I WANT TO BUY 1. >> [LAUGHTER] >> BUT MAYBE YOU CAN MAKE 1 FOR A RAT AND-- >> YEAH, WE HAVE WOAN FOR A RAT AND IN FACT YOU COULD MAKE 1 FOR A HUMAN. [LAUGHTER] I MENTIONED WHO DAVID ROBISON WHO INITIATED THIS HAS AN MBA, WE ARE AT THE LAST STAGES OF FINALIZING THE SOFTWARE. WE WANT TO DEVELOP A PACKAGE SO IT'LL BE USER FRIENDLY THAT SOMEONE DOESN'T NEED TO KNOW ALL THE CODE IN ORDER TO DO THIS. AND WE HOPE TO RELEASE--WE GET SOME OF THIS FOR PUBLICATION THIS SUMMER AND DAVID IS THEN GOING TO EXPLORE THE OPTIONS ABOUT HOW TO MAKE IT AVAILABLE AND AT CHEAP PRICE TO ACADEMICS AND HIGH PRICE TO INDUSTRY. >> THAT WAS MY SECOND QUESTION. THANKS. >> CLIFFORD SO THIS IS A GREAT NEW APPROACH, HOW YOU MAKE THIS MORE APPROACHABLE TO STUDIES IN HUMANS. AT LEAST IN YOUR SLEEP EXPERIMENTS, YOU SHOWS THAT THE EXPERIMENTS WERE DONE IN MALE MICE. DID YOU REPEAT THEM IN FEMALE MICE. >> YES, WE--THIS WAS FUNDED BY THE NIH AND I KNOW THAT IF WE DIDN'T DO IT IN FEMALES I WOULD BE IN BIG TROUBLE. I WOULD BE IN BIG TROUBLE ANYWAY. YES, WE HAVE DONE THEM IN FEMALES AND WE SEE EXACTLY THE SAME THING. >> EXACTLY THE SAME THING. AND THANKS FOR POINTING THAT OUT. >> SO I HAVE A QUESTION. SINCE YOU MENTION THAT YOUR MICE WILL EXPERIENCE BRIEF PERIODS OF AWAKENESS DURING THEIR SLEEP, HAVE YOU TRIED PUTTING THEM THROUGH ANY COGNITIVE OR SOME SORT OF TESTING OF THEIR ABILITIES AFTERWARDS. >> WE VERMEN HAVEN'T. I THINK IT'S AN IMPORTANT ISSUE BECAUSE PATIENTS DO COMPLAIN WHETHER THIS DEGREE OF SLEEP DISRUPTION IS ENOUGH TO PRODUCE THE KINDS OF COGNITIVE CHANGES THAT ARE WELL KNOWN TO OCCUR IN OTHER FORMS OF SLEEP DISRUPTION IS SOMETHING THAT'S WORTH EXPLORING. WE WILL NEED TO SEE THE EXTEND OF WHICH THESE CHANGES IN MICE, THE PATTERNS OF SLEEP IN A MOUSE ARE VERY DIFFERENT FROM HUMANS, THEY HAVE--AS IT IS ANYWAY, THEY HAVE VERY BRIEF PERIODS OF NONREM AND REM SLEEP FOLLOWED BY PERIODS OF AWAKENING, NOT A CONTINUOUS SLEEP AS WE DO. SO WE'RE VERY KEEN TO MOVE FORWARD AS QUICKLY AS WE CAN TO WHICH OUR SIMILARITIES ORTENT- DIFFERENCES. >> COOL. >> SOCIAL INTERACTIONS, CAN YOU--DOWN THE LINE CAN YOU HAVE A BOX WHERE YOU HAVE 2 ANIMALS IN THERE AND-- >> IF YOU ASKED ME 3 WEEKS AGO BEFORE WE GOT THIS NEURAL NETWORK GOING, I WOULD HAVE SAID WE'RE STRUGGLING WITH 1, JUST TO NOW THERE'S ABSOLUTELY FOR SURE, IT'S GOING TO BE FINE. >> COOL. >> AND SO, YEAH. WE CAN NOW SEE, EXPLORE EXACTLY THAT. SSO IF THERE'S NO MORE QUESTIONS, ... [ APPLAUSE ]