IT'S A GREAT PLEASURE FOR ME TO INTRODUCE TODAY'S SPEAKER, OUR DISTINGUISHED COLLEAGUE MICHAEL O'DONOVAN, MICHAEL RECEIVED HIS DEGREE FROM THE UNIVERSITY OF BRISTOL IN UK AND HIS DR. OF NEUROSCIENCE FROM THE UNIVERSITY OF LONDON. HE POST DOC WIDE LYNN MASSER IN THE BIOLOGY DEPARTMENT AT YALE IS SUBSEQUENTLY HELD FACULTY POSITIONS INCLUDING AS A ASSOCIATE PROFESSOR IN THE DEPARTMENT OF PHYSIOLOGY AND BIOPHYSICS AT THE UNIVERSITY OF IOWA WHERE AMONG MANY OTHERS HIS WORK ON SPINAL MOTOR SYSTEMS WAS SUPPORTED BY A PRIEST EDGEIOUS NIH RESEARCH CAREER DEVELOPMENT AWARD. IN 1988, HE JOINED BOB BURKE AND COMPANY IN WHAT WAS THEN THE RENOWNED LABORATORY OF NEURAL CONTROL AT NINDS. WHICH I JOINED AFTER MICHAEL DUE IN LARGE PART TO HIS AND BOB'S INFLUENCE. SINCE 1991 HE HAS BEEN CHIEF OF THE SECTION ON DEVELOPMENTAL NEUROBIOLOGY AT NINDS WHERE HIS GROUP IS FOCUSED ON UNDERSTANDING THE ORGANIZATION AND FUNCTION OF LOCOMOTOR NETWORKS INCLUDING IN THE DEVELOPING EMBRYONIC CHICK AND THE NEONATAL MOUSE SPINAL CORD. MICHAEL'S GROUP HAS MADE MANY INFLUENTIAL DISCOVERIES IN THE DEVELOPING SPINAL CORD FOR IDENTIFYING NOVEL FUNCTIONS AND A GLUTAMATE TRANSMITTER PHENOTYPE OF MOTOR NEURONS AS WELL AS FOR THE WORK IDENTIFYING SENSORY MOTOR DEFICITS IN A MOUSE MODEL SPINAL MUSCULAR ATROPHY. ALL OF HIS GROUP'S WORK HAS BEEN CONCEPTUALLY ELEGANT AND TECHNICALLY INNOVATIVE INCLUDING MICHAEL WORK, SOME OF THE EARLIEST NEUROSCIENCE USING DYNAMIC CALCIUM TO INDIVIDUALIZE CIRCUIT ACTIVITY ASSOCIATE WIDE BEHAVIOR AND ESPECIALLY IN THIS SPINAL CORD WHERE HE DEVELOPED NOVEL METHODS FOR LOADING CALCIUM AND VOLTAGE SENSITIVE DIES INTO MOTOR CIRCUITS TO VISUALIZE ACTIVITY. HIS TECHNIQUES HAVE BEEN WIDELY USED BY OTHER SCIENTISTS IN THE FIELD AND WE'RE VERY INFLUENTIAL IN FACT, MOO MY LAB'S WORK IMAGING BRAIN STEM MOTOR CIRCUITS. SO IN THE TRADITION OF CHAMPIONING MOTOR AND MOTOR SYSTEMS AT NINDS, MICHAEL HAS BEEN WAGING A SUCCESSFUL CAMPAIGN TO MAKE MOTOR NEURONS GREAT AGAIN. [LAUGHTER] COMPLETE WITH OFFICIAL FANFARE AND FASHION HE INTRODUCED IN 2017 AT OUR ANNUAL FSN SATELLITE SYMPOSIUM ON MOTOR SYSTEMS AT NIH WHICH MICHAEL HAS ORGANIZED SINCE 2008. SO HATS OFF TO YOU MICHAEL AND I'M SURE WE WILL ALSO PLEDGE TO KEEP MOTOR NEURONS GREAT AFTER WE HEAR THE LATEST AND GREATEST IN HIS LAB AND TALK TODAY TITLED SPINAL MOTOR NEURONS, SHERRINGTON'S FINAL PATHWAY OR MOTOR NETWORK REGULATORS. THAT WAS A MAGNIFICENT INTRODUCTION. LET'S GET STARTED. I'M GOING TO START WITH A QUOTATION FROM THE PHYSIOLOGIST RAMANI KAHALL TO CHARLES SHERRINGTON AND HE WAS THE 1 WHO BY THE WAY, CALLED THE BRAIN AN ENCHANTED LOOM. AND HE POINTS OUT THAT TO MOVE THINGS IS ALL THAT MANKIND CAN DO FOR SUCH THE SOLO EXECUTE ANT IS MUSCLE WHETHER IN WHISPERING, A SYLLABLE OR IN FILLING A FOREST, IN FACT SHERRINGTON WAS SOMETHING OF A POET AS YOU CAN SEE FROM THAT. AND HIS ARGUMENT WAS THAT MOTOR ACTIVITY IS THE RAISED ON DETRA OF NERVOUS SYSTEMS AND [INDISCERNIBLE] LIKES TO POINT OUT THAT THE SEA SQUIRT IS A VERY UNUSUAL ANIMAL WITH AN UNUSUAL LIFE CYCLE. INITIALLY IT'S MODEL CITIZEN TILE AND SWIMS AROUND IN A TADPOLE FORM AND HAS A NERVOUS SYSTEM AND SPINAL CORD BUT IN THE LATER PART OF ITS LIFE CYCLE IT ATTACHES TO A ROCK AND IT BECOMES IMMOBILE LIKE A PLANT. AND AT THAT POINT IT DIGESTS ITS OWN NERVOUS SYSTEM. AGAIN HE MAKES THE ARGUMENT THAT EVOLUTION, THE FUNCTION OF NERVOUS SYSTEMS WAS DETERMINED BY THEIR ABILITY TO MOVE WHICH IS WHY PLANTS DON'T HAVE NERVOUS SYSTEMS. SO IT'S A RANDOMIZED TRIALER GRANDIOSE JUSTIFICATION FOR WHAT I'M ABOUT TO STUDY BUT NEVERTHELESS, SO WE STUDY LOCOMOTION. I'VE ALREADY FORGOTTEN HOW THIS WORKS. TOP BUTTON. OKAY. OKAY, HERE WE GO. OH YES. SO LOCOMOTION REALLY, THE STUDY OF LOCOMOTION STARTED A LONG TIME AGO IN THE 1860S AND 70S AND YOU MAY REMEMBER MOY BRIDGE, EDWARD MOYBRIDGE, HE WAS COMMISSIONED TO ADDRESS A QUESTION AS TO WHETHER HORSE'S LEGS WERE ALWAYS OFF THE GROUND WHILE THEY WERE GALLOPING, AS CAN YOU SEE, HE WAS ABLE TO ESTABLISH THAT FACT BUT HE WASN'T THE EARLIEST PERSON TO USE EARLY FILM TECHNIQUES TO STUDY LOCOMOTION. IN FACT, THAT WAS A FRENCH MAN, OH DEAR HIS NAME SEEMS TO HAVE DISAPPEARED, WHAT'S HIS NAME. >> [INDISCERNIBLE] >> YEAH MARIN, I REQUIRE A FRENCH POST DOC TO DO THAT, THERE THEY ARE. NOW, MELANIE WHO JUST MENTIONED HADIS NAME THERE WAS VERY DISAPPOINTED WITH THIS VIDEO THAT I GOT OFF THE WEB BECAUSE OBVIOUSLY MOYBRIDGE'S LOOKS FAR BETTER THAN [INDISCERNIBLE], HOWEVER SHE POINTED OUT TO ME THAT HIS WIFE'S LOVER HAVING FILMED THEM TOGETHER AND HE GOT OFF BECAUSE IT WAS CONSIDERED JUSTIFIABLE HOMICIDE. SO I THINK MURRAY WINS THE DAY HERE. ALL RIGHT, SO, WHY STUDY LOCOMOTION? WELL THIS IS ACTUALLY A PICTURE OF STILLS FROM MURRAY'S WORK, I DON'T KNOW WHY HE'S WEARING A HAT BUT OBVIOUSLY MAYBE THAT DEFINED--THIS WAS IN THE ABSENCE OF SPOTS, THAT DEFINED WHERE THE LIMBS WERE AND I THINK THERE ARE LOTS OF REASONS WHY LOCOMOTION IS AN INTERESTING SYSTEM TO STUDY. AND I THINK THAT LIKE ALMOST ALL THE SYSTEMS IN THE TIMING AND MATURITY OF TIMING OF MUSCLE ACTIVITY IN THE LIMBS. ANOTHER REAP WHY IT'S VERY ADVANTAGEOUS TO STUDY SCIENTIFICALLY IS THAT IT'S SICKLICAL AND TO REALLY UNDERSTAND THEN HOW LOCOMOTION IS ORGANIZED, YOU ONLY HAVE TO UNDERSTAND 1 CYCLE AND HOW IT REPEATS. AND ANOTHER FACTOR WHICH I WILL SHOW YOU IN A MOMENT, THE EVIDENCE FOR THE BASIC PATENT IS GENERATED BY CIRCUITS WITHIN THE SPINAL CORD SO IT'S A CIRCUMSCRIBED SET OF CIRCUITS THAT CAN BE ISOLATED AND STUDIED INVITRO. IN MY OPINION LOCOMOTION IS LIKELY TO BE THE VERY FIRST BEHAVIOR THAT IS GOING TO BE UNDERSTOOD ON THE NEURAL LEVEL COMPLETELY. PERHAPS NOT IN THE MOUSE, CERTAINLY NOT IN HUMANS BUT PERHAPS IN THE ZEBRA FISH OR A SIMPLER SYSTEM LIKE THAT. AND OBVIOUSLY STUDYING LOCOMOTION HAS CLEAR IMPLICATIONS FOR YOUR MUSCULAR DISEASE, SPINAL CORD INJURY, AND SO ON. SO LET'S DEFINE SOME OF THE PARAMETERS THAT WE'RE GOING TO DEAL WITH IN LOCOMOTION AGAIN, YOU SEE THIS PICTURE FROM THE TEXTBOOK WAS OBVIOUSLY TAKEN FROM MAREY'S IMAGES BECAUSE THE FELLOW HAS A HAT. IN ANY EVENT THIS IS A SINGLE LOCOMOTION AND THE ACTIVITY OF MOTOR NEURONS IN YOU LOOK AT THEM, VARIOUS JOINTS OF THE ANIMAL, THE EXTENSES ARE ACTIVE MORE OR LESS TOGETHER IN THE STANCE PHASE, THAT IS THE PHASE WHEN THE ANIMAL IS STANDING LIKE THIS AND DURING THE SWING PHASE THE FLEXORS ARE ACTIVE AND YOU CAN SEE THERE'S THIS NICE AFFAIRS TEAM LEADERERNATION OF ACTIVITY THAT CHARACTERIZES LOCOMOTION. NOW, THE FIRST DEMONSTRATION, THE SPINAL CORD CAN GENERATE THIS PATENT CAME IN 1911 WHICH IS INCREDIBLE WHEN YOU THINK ABOUT IT OVER A HUNDRED YEARS AGO AND THE WAY IT WAS DONE WAS A CAT--THE SPINAL CORD WAS DEAFFRONTED THAT IS ALL THE DORSAL ROOTS WAS CUT AND IT WAS TRANSECTED AT T13 AND T12. AND THEN THE TENDONS OF THE GASTRIC NEMUOUS WHICH IS AN ANKLE EXTENSOR AND THE TIBIALIS ANTERIOR WERE ATTACHED TO A CHIME O GRAPH WHICH IS ACTUALLY A SMOKED DRUM AND THEY--THEY THEN EITHER SPONTANEOUS ARELY, I'M NOT SURE HOW IT WAS INITIATED BUT THE ACTIVITY CAN BE SEEN TO BE ALTERNATING BETWEEN THE FLEXORS AND EXTENSORS AND THAT IS CHARACTERISTIC LOCOMOTION, THIS WAS THE FIRST TIME THE SPINAL CORD HAD BEEN IDENTIFIED AS THE SOURCE OF THESE BEHAVIORS. THESE FINDINGS INDICATE THAT THE CIRCUITRY WITHIN THE SPINAL CORD IS GENERATED BY WHAT'S CALLED A CENTRAL PATENT GENERATOR AND THAT IS THE TERM WAS FIRST USED TO DESCRIBE--LOCUS FLYING IN 1961 AND THE IDEA IS THAT IT'S ENTIRELY CONTAINED AND IT DOESN'T NEED INFORMATION FROM AFERENCE OR DESCENDING INPUT TO PRODUCE THE BEHAVIOR. SO LET'S BRIEFLY GO OVER THE SPINAL CORD, I PUT THIS PICTURE UP BECAUSE I INTEND TO DESTROY IT LATER ON IN THE TALK AND THIS A CLASSIC TEXTBOOK PATIENTS PICTURE OF THE ORGANIZATION OF THE SPINAL CORD. THE DORSAL ROOTOT DORSAL POSITION AT SIDE OF THE SPINAL CORD RECEIVED CENTURY INPUTS, RECEPTIVE INPUTS AND SO ON, THE VENTRAL ROOT IS WHERE MOTOR NEURONS COME AND IN SOME SIGNATURES MENTORSHIP SKILLS THE GANGLIONIC NEURON ALSO EXIT THE SPINAL CORD. MOTOR NEURONS ARE KNOWN TO HAVE 2 SYNAPTIC TARGETS WITHIN THE SPINAL CORD. THE FIRST CELLS WHICH WERE DISCOVERED IN 1941. ANOTHER CLASS OF INTERNEURON HAS BEEN DISCOVERED, AN EXCITATORY NEURON WHICH RECEIVES INPUT FROM NOTER NEURONS AS WELL. WE ALSO KNOW THAT THE CENTRAL PATENT GENERATOR FOR LOCOMOTION IS LOCALIZE INDEED VENTRAL PART OF THE SPINAL CORD AND THIS IS SPRNSED BY OTHERS WHERE THEY COULD REMOVE THE DORSAL POWER OF THE SPINAL CORD AND ALL THE BEHAVIOR AND LOCOMOTIVE BEHAVIOR COULD BE PRESERVED UNDER THESE CONDITIONS. SO THE CENTURY PART OF THE SPINAL CORD IS CONSIDERED THE DORSAL HOME AND IT SITS THERE'S AN INTERMEDIATE REGION AND THEN THE VENTRAL HORN LAMINA 8 AND 9 WHICH CONTAINS INTERNEURONS AND MOTOR NEURONS. NOW WE STUDY THE MOUSE. WHY DO WE STUDY THE MOUSE BECAUSE OBVIOUSLY IT'S A VERY CONVENIENT ANIMAL TO STUDY LOCOMOTION IN, ALL THE GENETIC TOOLS THAT ARE AVAILABLE MAKING IT WONDERFUL ANIMAL TO STUDY THAT AND WE'VE EXPLOITED THEM AS YOU WILL SEE IN THIS TALK AND ALTHOUGH THEY CAN'T LOCOMOTE AT BIRTH, THEY CAN ACTUALLY SWIM. SO YOU CAN SEE HERE A LITTLE MOUSE SWIMMING AROUND. MELANIE TOOK THIS NICE VIDEO. NOW WE WORK WITH THE ISOLATED SPINAL CORD AND WHAT I WILL SHOW YOU NOW IS A VIDEO A SPINAL CORD STILL ATTACHED TO THE HIND LIMBS AND TAIL, THE BRAIN STEM WILL BE STIMULATED AND THIS IS TYPICALLY HOW WE BELIEVE LOCOMOTION IS INITIATED FROM DESCENDING INPUT INTO THE BRAIN, NOT PATENT, TONIC PERHAPS AND WE CAN SEE THAT THIS PREPARATION CAN GENERATE WALKING IN THE DISH. SEEMS TO BE WALKING BETTER WITH 1 LEG THAN THE OTHER, BUT--SO THIS IS A VERY USEFUL PREPARATION. NOW YOU DON'T ACTUALLY NEED THE LIMBS ATTACHED, YOU CAN TAKE THEM OFF AND IT WORKED PERFECTLY WELL AS WELL. AND THAT'S KNOWN AS FICTIVE REWARDIVE LOCOMOTION. ONE OTHER THING ABOUT THE SPINAL CORD'S ORGANIZATION THAT PROVES USEFUL, THE LUMBAR CORD WHICH IS CONTROLLING THE HIND LIMB COMPRISES 6 SEGMENTS FROM L1-L6. AND RATHER CONVENIENTLY THESE SEGMENTS ARE SEGREGATED MOST IMPORTANTLY FUNCTIONALLY SO THE L1 TO L3 SEGMENTS ARE FLEXOR RELATED AND THE L5 AND L6 SEGMENTS ARE EXTENSOR RELATED. THAT'S USEFUL BECAUSE NOW YOU CAN RECORD THE ALTERNATING PATTERN NOT FROM MUSCLE NERVES BUT FROM INDIVIDUAL VENTRAL ROOTS. AND I WILL SHOW YOU THE PATENT, WHAT IT LOOKS LIKE WE TYPICALLY RECORD FROM AN EXTENSOR AND A BI LOTERAL FLEXORS AND HERE YOU SEE THE PATTERN OF ACTIVITY WITH BRAIN STEM STIMULATION IN WHICH YOU HAVE THIS BEAUTIFUL RHYTHMIC ACTIVITY WHICH IS ALTERNATING BETWEEN THE LEFT AND RIGHT SIDES AND THE IPSALATERAL FLECTORS AND EXTENSORS. OKAY, IT TURNS OUT THAT LOCOMOTION CAN BE PRODUCED BY SEVERAL OTHER METHODS, SO THAT'S BRAIN STEM STIMULATION, VERY EFFECTIVE BUT CAN YOU ALSO STIMULATE THE DORSAL ROOTS AND PRODUCE THIS PATENT, IT'S EPISODIC, INTERESTINGLY, YOU NEED A TRAIN OF STIMULI, YOU CAN'T TRIGGER THIS--YOU CAN'T TRIGGER IT BY JUST A SINGLE STIMULUS, YOU HAVE TO GIVER A TRAIN, TYPICALLY AROUND 2-4-HERTZ, WE DON'T UNDERSTAND WHY. YOU CAN ALSO GIVE DRUGS MDMN5 HT AND THAT PRODUCES LONG LASTER EPISODESOF LOCOMOTE OR LIKE ACTIVITY. NOBODY UNDERSTANDS EXACTLY HOW THESE VARIOUS FORMS OF STIMULATION AND DRUGS PRODUCE LOCOMOTION. IT APPEARS THAT THE LOCOMOTOR CIRCUITS, THE MOST LA BILE IN THE SPINAL CORD BECAUSE OBVIOUSLY SPINAL CORDS CAN DO OTHER THINGS APART FROM LOCOMOTE. THEY CAN SCRATCH, PAW SHAKE AND DO OTHER THINGS BUT THE THING WE SEE MOST READILY WHEN WE DO THIS IS LOCOMOTOR LIKE BEHAVIOR. NOW I THOUGHT I WOULD SHOW YOU TO START WITH, A VIDEO THAT MELANIE MADE. THIS IS USING A CHAT GCAM 6 F SO THE GCAM 6 F CALCIUM SENSITIVE PROTEIN IS EXPRESSED EXCLUSIVELY IN COLLINERGIC NEURONS AND WHEN WE'RE VIEWING FROM THE VENTRAL PART OF THE SPINAL CORD THESE ARE ALMOST EXCLUSIVELY MOTOR NEURONS AND YOU CAN SEE THE PATTERN OF ACTIVITY IS GENERATED AND IN THIS CASE, TRIGGERED BY DORSAL ROOT STIMULATOR, DIFFERENCE IMAGE AND THAT IS AN IMAGE IN WHICH THE ONLY ACTIVITY, CHANGES IN ACTIVITY ARE SHOWN. OH DEAR, THEY TURNED THE SOUND OFF. WHERE'S THAT LITTLE MAN WHO RUNS THIS THING. THAT'S VERY UNSATISFIRY IT HELPS YOU APPRECIATE THE TIMING OF THIS. I DON'T KNOW WHAT HAPPENED BUT ANYWAY, I'LL CONTINUE ON TILL HE COMES BACK. >> SO WHAT ABOUT OTHER GLUTAMATE AGANISTS? >> [INDISCERNIBLE] DOES IT, OTHER 1S DO IT AS WELL. SO IT IS--IT IS PRETTY NONSPECIFIC. AND IN FACT, VARIOUS DRUG COMBINATIONS DO IT, DOPAMINE, SO AGAIN, WHAT IT'S DOING TO THE NETWORK NOBODY KNOWS. YOU CAN IMAGINE WHAT A OPPORTUNITY OR CONDUCT ABTS WERE IN THOSE NETWORKS SO IT TURNED IN DOPAMINE 5 HT. WHAT'S THAT? >> [INDISCERNIBLE] >> OH IT SHOULD WORK NOW, ALL RIGHT? LET'S GO BACK. THAT'S BETTER. [UNINTELLIGIBLE AUDIO ] THE SOUND ADDS TO IT. YOU SAW THE SILENT VIDEO BUT IT BRING ITS ALIVE. ALL RIGHT, SO,--AND WHAT WE CAN DO THEN IS MEASURE THESE CALCIUM TRANSIENTS AND HERE YOU CAN SEE ON THE IPSA LATERAL SIDE, THE RIGHT L2 ASK THE RIGHT L5, BEAUTIFUL ALTERNATING PATTERN OF ACTIVITY AND ON THE OTHER SIDE, IF YOU LOOK AT THE CONTRAT, THEN- LATERAL SIDES, THEY'RE ALSO ALTERNATING AS WELL. SO THIS IS A VERY NICE WAY OF LOOKING AT THE TOTALITY OF THE LOCOMOTOR NETWORK. NOW I'M GOING TO TACK A DIVERSION HERE AS JEFF MENTIONED, IN MY ORIGINAL WORK WAS DONE IN THE CHICK, AND I WORKED UNTIL ABOUT 2000 EXCLUSIVELY ON SPONTANEOUS ACTIVITY IN THE CHICK EMBRYO, BUT I WANT TO SHOW THIS RESULT BECAUSE IT INFORMED WHAT YOU'RE NOW GOING TO SEE AND WHERE THE DIRECTION IT TOOK MY WORK BECAUSE AT THE TIME IT WAS AN EXTREME STRIKING RESULT. SO WHAT YOU CAN SEE HERE OKAY, THIS IS A LITTLE TRICKY. I HAD THAT THING DISAPPEAR. SO THESE ARE SPONTANEOUS MOVEMENTS IN THE EGG. I HAD PUT THAT OUTLINE SO YOU CAN SEE WHAT IT LOOKS LIKE. IT'S A LITTLE BIT DIFFICULT TO SEE, BUT THIS GOES ON THROUGHOUT THE WHOLE OF DEVELOPMENT. IT STARTS AT ABOUT E4 IN THE CHICK EMBRYO AND IT'S TRUE IN VIRTUALLY EVERY ANIMAL THAT'S BEEN STUDIED INCLUDING THE NEMATODE THAT THEY GO THROUGH A PERIOD WHERE THEY HAVE SPONTANEOUS EPISODIC ACTIVITY. AND THAT'S THOUGHT TO BE INVOLVED ACTUALLY IN CIRCUIT DEVELOPMENT AND REGULATING THE STRENGTH OF SYNAPTIC CONNECTIONS. NOW IT'S POSSIBLE TO RECORD THE ELECTRICAL ACTIVITY FROM MUSCLES DURING THESE BEHAVIORS. YOU PUT ON A FLEXIBLE AND IT MOVES AROUND WITH THE LIMB AND YOU CAN SEE, THAT'S WHAT THE ELECTRICAL ACTIVITY LOOKS LIKE. OBVIOUSLY IN THE CHICK IN THE EGG IT'S TRICKY, BUT NOT IMPOSSIBLE, PEOPLE HAVE DONE INTRA CELLULAR RECORDING FROM MOTOR NEURONS IN THE EGG BUT WE DEVELOPED WHEN IS WAS IN THE [INDISCERNIBLE] LAB, AN ISOLATED SPINAL CORD PREPARATION THAT ALSO EXHIBITS SPONTANEOUS ACTIVITY AND IT'S MUCH SLOWER THAN THE ACTIVITY THAT OCCURS IN THE EGG, BUT NEVERTHELESS IT'S A NICE MODEL TO STUDY IT AND WHOIL WE WERE STUDYING THAT, AND I BEGAN MY IMAGING WORK, 1 OF THE THINGS I WAS INTERESTED IN KNOWING WAS WHAT ARE THE FIRST NEURONS THAT BECOME ACTIVE. THIS IS AN EPISODE, TYPICAL EPISODE OF SPONTANEOUS ACTIVITY THAT I SHOWED YOU, NOTICE THAT YOU GOT NICE FLEXAL EXTENSAL LOCATION, SUGGESTING THAT THE MOTOR CIRCUITRY IS EDEVELOPPED EARLY ON, THIS IS E-10 IN THE CHICKEN EMBRYO AND YOU ALREADY HAVE BEAUTIFUL ALTERNATING ACTIVITY BETWEEN FLEXORS AND EXTENSORS, SO THE QUESTION WAS WHAT ARE THE FIRST CELLS AND FIND THEM AND GO AFTER THEM AND STUDY THEM. BUT THE VIDEO, THIS IS A BID OF A CRUDE VIDEO, I'M AFRAID. IT WAS MADE IN 1989. HERE YOU CAN SEE THE ELECTRICAL ACTIVITY FROM THE NERVE, NO SOUND ON THIS 1 AND AT THE BEGINNING OF THAT EPISODE, THAT'S WHAT WE SAW. LET'S JUST DO THAT AGAIN. THESE ARE THE MOTOR NEURONS AND WHAT YOU SEE IS THIS PROPAGATING WAVE THAT STARTS WITHIN THE MOTOR NUCLEUS AND THEN PROPAGATES THROUGHOUT THE REST OF THE SPINAL CORD. SO THAT WAS SHOCKING TO--IT SUGGESTED THE MOTOR NEURONS WERE IN FACT CONTROLLING THE SPONTANEOUS ACTIVITY. THIS HAS NOW BEEN VALIDATED IN THE ZEBRA FISH WHERE IT'S BEEN SHOWING THE MOTOR NEURONS OF THE FIRST CELLS TO BE ACTIVE IN THAT NETWORK AND THAT INFORMED BY WORK IN THE-AND WE DID A LOT OF OTHER WORK INTRA CELLULAR RECORDING, VOLTAGE CENTER DISCIPLINARY-RECORDING TO VERIFY THIS WAS TRUE THAT MOTOR NEURONS DO INDEED DRIVE THE NETWORK EARLY IN DEVELOPMENT SO THE QUESTION IS: DO THEY DO THAT DURING LOCOMOTION? AMAZINGLY, THEY DO. NOW I HAD TOLD YOU PREVIOUSLY, THE METHODS ARE ACTIVATING THE GENERATOR, LOCOMOTOR GENERATOR AND THE INYONATAL MOUSE, DORSAL ROOT STIMULATION, DRUGS AND BRAIN STEM STIMULATION BECAUSE OF THAT RESULT, YOU WERE VERY INTERESTED TO KNOW IF STIMULATING MOTOR NEURONS CAN ALSO PRODUCE THIS LOCOMOTOR BEHAVIOR. AND IT TURNS OUT THAT A LITTLE BIT OF SERENDIPITY HELPED US HERE BECAUSE YES, [INDISCERNIBLE] WHO WAS DOING THESE EXPERIMENTS NOTICED WHEN SHE PULLED ON THE VENTRAL ROOT ITS TRIGGERED LOCOMOTION AND SO WE THEN STIMULATE INDEED A MORE SYSTEMATIC WAY, IN FACT HERE WE'RE STIMULATING THE SKITTOMA ATTIC NERVE AND WHEN WE WERE DOING THAT WE WANT TO MAKE CERTAIN THERE WAS NO CURRENT SPREAD INTO THE SPINAL CORD SO WE TOOK A LONG LENGTH OF SKITTOMAATIC NERVE, COVERING THE DORSAL ROOTS AND STIMULATED IT AND WE GOT LOCOMOTOR BEHAVIOR. NOW, WHAT WAS MORE SHOCKING IS WHEN WE BLOCKED ALL COLLINERGIC TRANSMISSION WE STILL GOT THAT BEHAVIOR. YOU CAN SEE IT'S WEAKER THAN IT IS, BUT YOU CAN SEE THE PHASING OF ACTIVITY IS PRESERVED AND WHAT DID KNOCK IT OUT WAS GLUTAMATE ANTAGONISTS. WELL THAT WAS SHOCKING BECAUSE WHY WOULD GLUTAMATE ANTAGONISM BLOCK, THEY MIGHT BLOCK THE NETWORK BUT THEY SHOULDN'T BE BLOCKING MOTOR NEURAN ACTIVATION WHICH WAS COLLINERGIC, WELL ON THE SAME EXPERIMENTS DONE LATE ARE BY GEORGE [INDISCERNIBLE] IN THE LAB, WE LOOKED AT THE MOTOR NEURON TRANSMITTERS THAT WERE RELEASED ON TO THE CELLS, THESE WERE THE ONLY KNOWN SYNAPTIC TARGETS OF MOTOR NEURONS AND WHEN YOU STIMULATE A [INDISCERNIBLE] CELL, YOU GET PROLONGED EPSP. AND WHAT GEORGE FOUND IS IF YOU GAVE GLUTAMATE ANTAGONIST, THE GLUTAMATE WAS REDUCED AND IF YOU GET COLONERGIC, ITLESS ALSO REDUCED BUT NOT COMPLETE, IT WAS ABOUT 70% REDUCED. NOW THIS WAS PART OF WHAT WAS FOUND ABOUT 70 YEARS AGO BECAUSE WHEN YOU DID THESE EXPERIMENTS IN THE CAT, THEY COULD NEVER COMPLETELY ABOLISH [INDISCERNIBLE] EXCITATION BY MOTOR NEURONS BY GIVING COLON IRNLGIC ANTAGONISTS AND THE REASON THEY ASSUMED WAS BECAUSE THEY COULDN'T, YOU KNOW IT WAS IN THE INTACT, TACT, GIVEN SYSTEMICALLY AND PRESUMABLY AND IT SIMPLY WASN'T AFFECTED SO THEY MISSED THE POSSIBILITY THAT DPLIEWT MATE WAS ACTUALLY RELEASED AS WELL AS ACETA COALIN. NOW, THIS CONNECTION ACTUALLY IS STRONG ENOUGH TO FIRE THE CELLS, THAT MEANS THE CIRCUIT CAN BE ENGAGED, IN THE ABSENCE OF ANY COLLINERGIC TRANSMISSION. NOW MELANIE AND KD--SALLY QUAIN THE LAB ALSO DEMONSTRATED THAT SOME OF THESE MOTOR NEURONS APPEAR TO IMRESES THE GLUTAMATE TRANSPORTER, THE B-GLIEWT 2, WE ALSO VERIFIED THAT BY LOOKING AT THE MRNA AND IN SOME COLON IRNLGIC NEURONS YOU CAN SEE THE IN, RNA, IT'S AT A FAIRLY LOW LEVEL COMPARED TO OTHER NEURONS AND IN FACT, WE CHEMICALLY WE COULD NOT DETECT ANY OF THE STANDARD GLUTAMATE TRANSPORTERS IN MOTOR NEURON TERMINALS. BUT WE THINK IT PROBABLY IS THAT OR BE AT THE LOW LEVEL. NOW WHAT I'VE SHOWN YOU UP TO NOW IS THE SYNCHRONOUS MOTIVATION BY ROOT STIMULATION CAN TRIGGER LOCOMOTION BUT THAT'S COMPLETELY UNPHYSIOLOGICAL. TO THEFT THE POSSIBILITY THAT ONGOG MOTOR NEURON FIRING COULD CHANGE THE LOCOMOTOR RHYTHM, MELANIE DID OPTOGENETIC EXPERIMENTS SO IN THIS CASE, WE'RE EXPRESSING [INDISCERNIBLE] REDOBSIN, AN INHIBITORY [INDISCERNIBLE] IN THE NEURON AND WHEN YOU SHINE GREEN LIGHTED ON THEM, CAN YOU SEE IT SLOWS THE RHYTHM AND BLOCKS MOTOR NEURON FIRING AND WHAT'S MORE REMARKABLE IF YOU LOOK HERE, THIS INTRA CELLULAR RECORDING, YOU CAN SEE THESE BEAUTIFUL DRIVE POTENTIALS HERE AND THESE ARE ASSUMED TO BE PRESYNAPTIC COMING INTO THE NETWORK FROM THE OUTSIDE. THEY'RE COMPLETELY GONE IN THE PRESENCE OF LIGHT. SUGGESTING PERHAPS MOTOR NEURONS MIGHT EVEN BE PART OF THE CPG, THAT REMAINS CONTROVERSIAL. AND MELANIE DEVELOPED A VERY NICE METHOD TO QUANTIFY THESE CHANGES USING A BOOT STRAP TECHNIQUE WHERE IT COMPARES THE EFFECT OF WILD TYPE ON ANIMALS SHOWN IN GRAY WITH THE CHANGES THAT WE SAW IN THE ANIMALS EXPRESSING OPPOSITE. SO CAN YOU SEE THESE ARE SIGNIFICANT, VERY SIGNIFICANT CHANGES. ALL RIGHT, SO, 1 QUESTION THAT AROSE IS OBVIOUSLY THERE ARE OTHER CELLS IN THE SPINAL CORD THAT ARE COLLINERGIC APART FROM MOTOR NEURONS, MAYBE THESE EFFECTS ARE DUE TO AN ACTION ON THESE CELLS. SO WHAT WE DID THEN WAS USE ANOTHER MOUSE IN WHICH THE [INDISCERNIBLE] WAS DRIVEN BY EYELET 1, IT'S A TRANSCRIPTION FACTOR EXPRESSED IN MOTOR NEURONS, PRIMARY AFER ENSEL AS CAN YOU SEE, THESE ARE THE PRIMARY AFER ENSEL IN THE DORSAL HORN COME NOTHING, INNERIVATING THE MOTOR NEURONS AND PREGINGLIONIC NEURONS AND WHEN WE DID THAT EXPERIMENT, THE RESULT WAS THE SAME. SO NOW, IT SEEMED PRETTY CLEAR THAT WHAT WAS COMMON BETWEEN THESE 2 PREPARATIONS OR MOTOR NEURONS, NOT ONLY CAN THEY ACTIVATE THE GENERATOR WHEN YOU STIMULATE THEM BUT THEY CAN ALSO INHIBIT IT. AND THEN FINALLY MELANIE ALSO DID THE EXPERIMENT WITH CHANNEL ROUGH ATOM DOBSIN NOW TO EXCITE COLONERGIC NEURONS AND YOU TRIGGER LOCOMOTOR ACTIVITY BY PULSES OF PLURIBU LIGHT APPLIED TO THE SPINAL CORD. NOW 1 OF THE WAY TAC MOTOR NEURONS COULD IN PRINCIPLE MODULATE THE RHYTHM IS THROUGH THE CONNECTIONS WITH THE [INDISCERNIBLE] CELLS IN FACT WHEN WE DID THOSE EXPERIMENTS THOSE WERE THE ONLY KNOWN CONNECTIONS. SO WHAT MELANIE DID IN THESE EXPERIMENTS TO EXPRESS ARCHEROROUGH ATOM DOBSIN AND YOU CAN HYPER POLARIZE THESE CELLS AND ASK CAN VENTRAL ROOT STIMULATION TRIGGER LOCOMOTION? THE ANSWER IS YES. SO THIS STRONGLY SUGGESTS THAT THE RENTERAL CELL POGHT WAY WHICH IS NOT SURPRISING THE PATHWAY'S INHIBITORY PROJECTS INHIBITORY NEURONS AND INHIB THORS IDENTITIORY NEURONS AND A NUMBER OF OTHER CELLS SO TO SUMMARIZE THE ACTIVATION OF LOCOMOTOR NETWORKS BY VENTRAL ROOT STIMULATION IS DUE TO STIMULATION OF MOTOR NEURON AXONS AND NOT COLONERGIC AND NOT MEDIATE BIDE PROJECTION CELLS AND CAN BE SEEN IN ONGOING MODULATION OF LOCOMOTOR LIKE ACTIVITY. NOW MORE RECENTLY IT'S BEEN SHOWN THAT MOTOR NEURONS ARE IN FACT REGULATING CPGs AND A NUMBER OF OTHER SYSTEMS APART FROM THE MOUSE RECENTLY WAS SHOWN IN ZEBRA FISH, ADULT ZEBRA FISH LOCOMOTION AND THE MOTORING WAS THERE RETROGRADE COULD MODIFY THE SWIMMING RHYTHM, AND IN ADDITION THEY CAN ALSO REGULATE FROG LOCALIZATIONS THAT'S ALSO A RHYTHMIC CPG. SO NOW THE QUESTION ARISES WHAT NEURONS ARE ACTIVATED BY ROOT STIMULATION AND THIS IS WHERE THE THEORETICAL PART OF THE TALK COMES IN, SO THOSE WHO WORK ON THE DORSAL HORN, I HOPE TO AMAZING YOU. --AMAZE YOU. IF I DON'T LET ME KNOW. [LAUGHTER] SO THE WAY MELANIE DID THESE EXPERIMENTS WAS TO EXPRESS GCAMP 6 IN [INDISCERNIBLE] GAB 67 COLONERGIC NEURONS AND THEN FICIAL VISE THE SPINAL CORD FROM ALL THE ANGLES SHE COULD, NOW UNLIKE THE CORE TYPES THIS IS A VERY BAD SYSTEM TO IMAGE BECAUSE YOU HAVE ALL THIS WHITE MATTER ON THE OUTSIDE AND WE FOUND THE 2 PHOTON IMAGING DOESN'T DO WELL TRYING TO LOOK THROUGH THAT INTO THE CELLS AND FURTHER MORE 2 PHOTON IMAGE SUGGEST LIMITED IN THE [INDISCERNIBLE] WHICH IS VIRTUALLY THE WHOLE LENGTH OF THE SPINAL CORD. AND THIS JUST SHOWS YOU THE CELLS, UNFORTUNATELY YOU CAN'T SEE--CAN WE DIM THE LIGHTS A BIT? MAYBE NOT? SHOULD WE GET THE LITTLE MAN AGAIN? NO? [LAUGHTER] , [LAUGHTER] , YOU CAN SEE THE CELLS ARE GREEN AND--HERE'S WHAT'S NICE ABOUT THIS IS YOU CAN SEE THAT SOME OF THESE MOTOR NEURONS, COLLINERGIC MOTOR NEURONS ARE THE GLUE 2 POSITIVE. ALL RIGHT, SO THAT'S THE STRATEGY. NOW, I'M GOING TO SHOW YOU WHAT HAPPENS WITH GRADED STIMULATION OF A VENTRAL ROOT. THIS IS THE CONFIGURURATION, DORSAL IS TAUGHT, VENTRAL IS BELOW, HERE'S THE VENTRAL ROOT ELECTRODE HERE AND IT'S [INDISCERNIBLE]. SO WE'RE GOING TO STIMULATE AT A LOW INTENSITY FIRST AND THIS WILL JUST SHOW YOU MOTOR NEURONS BEING ACTIVATED ANTIDERMICALLY. MELANIE IS GIVING 2 TRAINS SEPARATED BY 250 MILLI SECONDS. NOW IF WE TURN OFF THE INTENSITY, THIS IS WHAT WE SEE. THIS IS WHERE THE HEARSAY BEGINS WE SEE THIS STIMULATING THE VENTRAL ROOT. VERY SURPRISING. AND THIS JUST SHOWS YOU IN STATIC IMAGE IT IS THAT AVERAGES THE VARIOUS THINGS SO 20, 40, 50 AND MICROGRAMS YOU CAN SEE THE DORSAL HORN NETWORK IS ACTIVATED, IT'S VERY SUPERFICIAL NETWORK YOU CAN SEE THE INDIVIDUAL CELLS ON THE SUPERFICIAL ASPECT OF THE DORSAL HORN VERY, VERY CLEARLY. AND WE CALL THIS THE SUPERFICIENCY DORSAL NETWORK, ALSO A DEEP DORSAL NETWORK, WHICH TENDS TO PROPAGATE ITS ACTIVITY ALONG THE SPINAL CORD AND WE WILL SHOW YOU A LITTLE BIT MORE OF THAT IN A MINUTE. INTERESTINGLY, IT'S NOT JUST [INDISCERNIBLE] NEURONS THAT ARE ACTIVATED BY THIS STIMULUS, IT'S ALSO INHIBITORY NEURONS IN A SIMILAR LOCATION TO THE [INDISCERNIBLE] NEURONS SO YOU'RE ACTIVATING VERY COMPLICATED NETWORKS BY THIS VENTRAL ROOT STIMULATION. NOW, I TOLD YOU PREVIOUSLY, THAT ACTIVATING LOCOMOTOR LIKE ACTIVITY PERSISTED IN THE PRESENCE OF COLIGEERGIC ANTAGONISTS SO WE WERE VERY INTERESTED TO KNOW, IS THE SAME TRUE FOR ACTIVATION OF THESE DORSAL NETWORKS. ARE THEY ALSO DO THEY ALSO PERSIST IN COLLINERGIC ANTAGONISTS AND THE ANSWER IS YES. SO THESE ANTAGONIST WE PUT ON THE ANTAGONIST AND A [INDISCERNIBLE] AS WELL, MAKE SURE THAT WE'RE BLOCKING ALL COLLINERGIC TRANSMISSION AND YOU CAN SEE MOTENT ACTIVATION OF THOSE DORSAL NETWORKS. NOW THE NEXT THING WE WANT TO HAVE A LOOK AT WAS WHAT WAS THE TIMING OF THE ACTIVITY IN THESE VARIOUS NETWORKS. SO HERE WE LOOKING AT TRANSFERS, CUT FACE OF THE SPINAL CORD, THE L5 AND THIS IS CALLED L5 AND 6. AND THE REST OF THE CORD AND WHAT WE SEE WHEN WE DO THAT STIMULATE THIS VENTRAL ROOT HERE WE SEE THIS. NOW IF WE SLOW DOWN IT'S EASIER TO SEE THE TIMING. SEE THE VERY DORSAL POSITION AT NETWORK GOES FIRST AND IT SEEMS TO PROPAGATE INTO THIS REAMINGION HERE. THIS REGION IS VERY INTERESTING REGION BECAUSE THIS REGION IS WHERE THE LATERAL SPINAL NUCLEUS IS. AND THOSE DORSAL HORN OFFICKIAN ADOS MAY KNOW WHAT WE'RE TALKING ABOUT OR NOT. AND SO THIS JUST SHOWS YOU THE QUANTIFICATION OF THAT ACTIVITY, SO HERE'S THE TRANSVERSE PHASE, LATERAL VIEW, AND YOU CAN SEE THAT THIS REGION APPEARS TO GO FIRST, SOMETIMES BY A VERY SMALL AMOUNT BUT HERE IT'S MORE OBVIOUS AND THEN THIS REGION IS ACTIVATED SO IT'S POSSIBLE THAT THAT VERY DORSAL REGION IS TRIGGERING THE ACTIVITY OF THAT MORE--THAT DEEP DORSAL NETWORK. NOW, CALCIUM IMAGING DOESN'T ALLOW YOU TO REALLY INFER MUCH TIMING INFORMATION. SO WHAT MELANIE DID IN THIS CASE, SHE'S LOOKING AT THE SUPERFICIAL SURFACE OF THE DORSAL HORN AND VENTRAL ROOT STIMULATION ACTIVATES A PATCH AND BY THE WAY, THIS IS VERY STRANGE. SO WHERE IS THE DEEP DORSAL NETWORK, IS THIS VERY BROAD NETWORK, PROPAGATES QUARTERLY, THE VERY SUPERFICIAL DORSAL HORN ACTIVATION IS ALWAYS IN PATCHES. AND THESE PATCHES--THIS IS A CIRCUMSCRIBED AREA, ALSO LIKE A RECEPTIVE FIELD. AND WHEN SHE DID THAT SHE'S RECORDING WITH THE EXTRA CELLULAR LECTURE TO RECORD AN EXTRA CELLULAR FIELD, SHE FOUND YOU GET A NICE FIELD POTENTIAL AND RELATIVELY LATENCY, AND 10-152ndS AND THAT'S NOT MONOSYNAPTIC BUT IT'S STILL REMARKABLY SHORT WHEN YOU CONSIDER THIS IS THE VERY SUPERFICIAL DORSAL HORN. NOW TO SEE IF THAT VERY SUPERFICIAL DORSAL NETWORK WAS IN FACT CAUSEALLY RESPONSIBLE FOR TRIGGERING LOCOMOTOR LIKE ACTIVITY. CAN YOU PUT AN ELECTRODE INTO 1 OF THOSE PATCHES, A MICROELECTRODE AND YOU CAN STIMULATE LOW INTENSITY CURRENT AND THE AUDIO HERE IS A MORE REMOTE. THIS IS THE L5 SEGMENT YOU'RE LOOKING AT AND YOU'RE HEARING THE L2 VENTRAL ROOT. SO YOU SEE THE DEEP DORSAL NETWORK COMING UP AND ACTUALLY THE MOTOR NEURONS DOWN THERE ARE RHYTHMICALLY ACTIVE. ARE REMARKABLE AND THIS JUST SHOWS YOU THE QUANTIFICATION OF THE SIGNALS FROM THE SUPERFICIAL DORSAL NETWORK AND DEEP DORSAL NETWORK AND MOTOR NEURONS AND THIS THE L2 VENTRAL ROOT. MOTOR NEURONS SEEM TO BE EXPRESSING THE MOST OBVIOUS BEHAVIOR AND WELL IS EVIDENCE IN THE DEEP DORSAL NETWORK OF THE CANNED WHAT BUT NOTHING IN THE SUPERFICIAL DORSAL NETWORK. IS IT CAUSEALLY REQUIRED FOR THE EXPRESSION OF RHYTHMIC ACTIVITY. SO TO ADDRESS THAT QUESTION, WHAT MELANIE DID WAS TO REMOVE THE SUPERFICIAL LAYERS OF DORSAL HORN AND THEN ASK CAN THE VENTRAL ROOT TRIGGER LOCOMOTION. AND SO THAT IS SHOWN IN THE NEXT IMAGE SO THIS IS WHAT SHE DID. SHE TOOK A VIBE RATONE AND REMOVED THE DORSAL HORN OVER L2, TO L5 OR L4 IN THIS CASE, STIMULATED THE VENTRAL ROOT, THE TOP TRACE IS WHAT YOU HAD BEFORE, BECOME TRACE IS WHAT YOU HAVE AFTERWARDS, YOU CAN'T GET ANYTHING. NOTHING. HOWEVER, IF YOU STIMULATE A SAN FRANCISCO KRAL ROOT IN THE SAME PREPARATION WHICH IS REMOTE NOW FROM THAT LESION, IN THE SACRALL, CORD, CAN YOU TRIGGER THE ROOT. IN OTHER WORDS THE CIRCUITRY FOR LOCOMOTION IS INTACT, IT SIMPLY CANNOT BE ACCESSED BY THE VENTRAL ROOT. AGAIN, AMAZING. THINK ABOUT IT, I WAS STRUGGLING THROUGH SEVERAL BSCs TO WORK THIS OUT AND THEY CHASTISED ME FOR NOT DOING IT FAST ENOUGH, WELL THE PROBLEM IS NOBODY EXPECTED THIS CIRCUITRY TO BE IN THE DORSAL HORN. WE EXPECT ALL THE LOCAL CIRCUITRY WITHIN THE VENTRAL CORD TO BE RESPONSIBLE BUT THAT'S NOT WHAT WE SEE. SO TAKE THAT BSC. [LAUGHTER] SO ... [LAUGHTER] I SEE WALTER OVER THERE. I'M JOKING. SO HERE WHAT WE'RE DOING NOW IS ASKING WELL WHAT ABOUT THAT DEEP DORSAL NETWORK. WHAT DOES THAT DO IN AND SO WHAT MELANIE DID HERE IS INSTEAD OF STIMULATING THE DORSAL PATCHES SHE PUT THE ELECTRODE, MICROELECTRODE IN THAT BAND THAT'S ACTIVATED LIKE THIS. OKAY? THE ELECTRODES VANISHED BUT NEVERTHELESS, OH NO SHE DIDN'T DO THAT THEM. SORRY. I'M GET BEING AHEAD OF MYSELF HERE, WHAT HE DID THERE--SO PREVIOUSLY I SHOWED THAT MOTOR ACTIVITY WAS BLOCKED WHEN YOU RENIEWF THE DORSAL POSITIONA HORN, WHAT ABOUT ACTIVATION OF THESE DORSAL NETWORKS IS THAT ALSO BLOCKED. SO, WHAT SHE SHOWED HERE WAS IN THE TOP TRACES, THE CONTROL, YOU CAN SEE THE MOTOR NEURONS AND THE DORSAL NETWORKS ARE ACTIVATED AND THENOT RIGHT WE USE A DORSAL POSITION AT ROOT STIMULATED DORSAL ROOT AND YOU CAN SEE THE D DWE'RE CALLING IT IS BY DORSAL ROOT STIMULATION AND ALSO BY ROOT STIMULATION BUT WHEN SHE REMOVES THE SDN AND SIMULATES THE SDN ROOT YOU CANNOT SEE THE DDN, IT'S PRESENT BECAUSE WHEN YOU SIMULATE THE SAN FRANCISCO KRAL DORSAL ROOT, CAN YOU ACTIVATE IT SUGGESTING THAT THAT SUPERFICIAL DORSAL NETWORK IS ACTIVATING THE DEEP DORSAL NETWORK. ALL RIGHT. SO WHAT HAPPENS WHEN YOU HAVE DEEP STIULATION, HERE IT IS, DEEP DORSAL NETWORK, MELANIE NOW PUTS AN ELECTRODE IN THIS AND STIMULATINGS IT AND WHAT YOU CAN SEE IS, SO HERE'S VENTRAL ROOT SUBMISSION, THE EXPECTATIONS ELEBT RODE ACTIVATES THAT WHOLE NETWORK AND PROPAGATES OUT AND WHEN YOU MERGE THE 2, CAN YOU SEE THEY HAVE MANY CELLS IN COMMON NA. IS LOCAL MICROSTIMMULATION OF THIS NETWORK CAN AUTORECRUIT IT AND IT PROPAGATES ROSTROCORDALLY, AND IT SHOWS YOU THIS PHENOMENON. SO HERE'S REALTIME STIMULATING VENTRAL ROOT AND YOU CAN--SORRY STIMULATING THE ELECTRODE, MICROSTIMMULATING AND YOU CAN SEE IT PROPAGATES ROSTROCORDALLY, YOU CAN SEE THE PROPAGATION IF YOU SLOW IT DOWN. THIS AN AUTORECRUITING NETWORK ONCE TRIGGERED IT CAN RECRUIT ITSELF FROM PROPAGATING BOTH DIRECTIONS. AND THAT OF COURSE MAY BE NECESSARY TO COORDINATE ACTIVITY IN THE DIFFERENT SEGMENTS OF THE SPINAL CORD. NOW, 1 OF THE VERY DRAMATIC WAYS TO SEE THIS IF YOU DISINHIBIT THE SPINAL CORD, THE REMOVAL INHIBITION BY STRYCHNINE, YOU NOW GET BURSTS LIKE THE 1S THAT ARE SHOWN BELOW THAT CAN BE ENTRAINED BY VENTRAL ROOT STIMULATION AND THIS IS WHAT YOU SEE WHEN YOU VISUALIZE THE CORD. WHAT YOU'RE HEARING NOW IS THIS ROOT HERE, YOU'LL HEAR THE KRRRRRR, OF THE STIMULUS FOLLOWED BY THE BURST. [AUDIO PLAYS ] SO NOW THE PROIGATION IS SPED UP, ENORMOUSLY POTENT AND IF WE LOOK NOW THAT SLOWED DOWN, YOU CAN SEE IT STARTS AS A LOCUS AND THEN GRADUALLY PROPAGATES OUT UNTIL IT BEGINS TO ACTIVATE VENTRAL MOTOR NEURONS AS WELL. AGAIN, CONSISTENT WITH THE IDEA THAT ONCE YOU GET A SMALL STIMULATION INTO THIS NETWORK IT RECRUITS ITSELF AND THIS BY THE WAY IS CHARACTERISTIC OF THE BEHAVIOR OF EMBRYONIC SPINAL CORDS. THEY ARE FUNCTIONALLY EXCITATORY THESE CORDS AND A SINGLE STIMULUS CAN TRIGGER NETWORK ACTIVITY AND IN FACT, WE COME UP--WE HAVE COME UP WITH THE IDEA OF A NETWORK ACTION POTENTIAL WHICH IS COMPLETELY ANALOGOUS TO A SODIUM BASED ACTION POTENTIAL AND THAT'S WHAT YOU SEE HERE. STIMULATION ACTIVATES A SMALL PORTION OF THE NETWORK BUT WHEN A SUFFICIENTLY LARGE PORTION OF THE NETWORK IS ACTIVATED, IT CAN ACTIVATE THE WHOLE NETWORK. NOW, THOSE OLD ENOUGH TO KNOW THE LITERATURE FROM THE 1980S WILL PERHAPS REMEMBER THAT [INDISCERNIBLE] ARGUED THAT THERE WERE SENSORY NEURONS IN THE VENTRAL ROOTS. NOW SUBSEQUENT WORK DONE BY HILDEBRAND AND HIS GROUP SHOWED THAT YES THERE ARE SENSORY NEURONS IN THE DORSAL ROOTS BUT THEY DON'T GO INTO THE GRAY MATTER. THEY INNERIVATE THE PEER, MAKE LOOPS AND COME OUT. SO THE CONVENTIONAL WISDOM IS THAT NOW ALTHOUGH THERE ARE AFERRANDIS ENSEL IN THE VENTRAL ROOTS THEY DON'T ACTUALLY ENTER THE GREAT MATTER BUT THEY COULD DO EARLY IN DEVELOPMENT AND SO, EVERYTHING THAT I'VE SHOWN YOU UP UNTIL NOW COULD BE SIMPLY DUE TO ME STIMULATING A41S GOING INTO THE SPINAL CORD. THIS WOULD BE DISAPPOINTING OF COURSE BECAUSE IT WOULD NOT BE INTERESTING OR HERETHICALL, NOW LET'S REVIEW, THE ANATOMY OF THE SPINAL CORD OVER HERE CAN YOU SEE THE TRANSSECTION THROUGH THE SPINAL CORD, YOU SEE THE DORSAL ROOT COMES OUT, THE DORSAL GANGBIAN AND IT SITS RIGHT UNDERNEATH THE DRG. THEN IT COMES TOGETHER INTO THE SPINAL NERVE. SO IT'S QUITE POSSIBLE THAT SOME OF THOSE AFFRONTS COULD LEAP INTO THE VENTRAL ROOT AND INTO THE GRAY MATTER. SO HOW DID SHE ADDRESS THAT? SO WHAT SHE DID FIRST WAS TO CUT THE DORSAL ROOT, AND THEN STIMULATE [INDISCERNIBLE] ASK WHEN YOU DO THAT, YOU GET WHAT I'VE SHOWN YOU BEFORE, YOU GET THE DEEP DORSAL NETWORK AND THE SUPERFICIAL DORSAL NETWORK AND THEN SHE STIMULATED THE DORSAL ROOT GANGLION. STILL ATTACHED, SO THAT IF THERE WERE ANY FIBERS ENTERING THE VENTRAL ROOT, YOU SHOULD BE ABLE TO MIMIC THE EFFECT, CAN YOU SEE DOWN HERE, I WILL SHOW YOU A VIDEO IN A MINUTE BUT HERE, ALL THE 2 NEURONS IN THIS, ALL THE [INDISCERNIBLE] NEURONS LIGHT UP IN THE DRG BUT YOU DON'T SEE ANYTHING IN THE SPINAL CORD. SO LET'S LOOK AT THAT AND SEE WHAT IT LOOKS LIKE AS A VIDEO. SO FIRST OF ALL, WE WILL STIMULATE THE VENTRAL ROOT EN P A SSANT. --PASS ANT. HERE'S AGAIN LOOKING AT DIFFERENCE IMAGES NOW, WE WILL STIMULATE THE DORSAL ROOT. SEE THE WHOLE DORSAL ROOT IS ACTIVATED UNDER THOSE CONDITIONS. WE SEE NOTHING IN THE SPINAL CORD AND WE REPEATED THIS MULTIPLE TIMES SO WE--IN AGREEMENT WITH THE HILDEBRAND WORK WE DON'T THINK THESE RESULTS CAN BE EXPLAINED BY MOTOR NEURONS ENTERING THE GRAY MATTER. SO ALL OF THE EXPERIENCE I DESCRIBED SO FAR HAVE BEEN NEONATAL, IS THERE ANY EVIDENCE THAT ANY OF THIS OCCURS IN THE ADULT. WELL IT TURNS OUT THAT THE RECURRENT EXCITATION, DPLIEWTA MITTURGIC MOTOR NEURONS ARE PRESENT IN THE ADULT MOUSE. THIS WAS WORK DONE BY [INDISCERNIBLE] LAB AND CAN YOU SEE HE'S RECORDING FROM MOTOR NEURON TO MOTOR NEUROON AND YOU CAN SEE A MONOSYNAPTIC CURRENT HERE WHICH IS ABOLISHED BY DPLIEWTA MATE ANTAGONISTS SO THE DPLIEWTA MITTERGIC PROJECTIONS AND MOTOR NEURONS ARE PRESENT IN THE ADULT. NOW WE'RE GOING TO GO TO A PAPER BY [INDISCERNIBLE] IN THE 1980S SHOWING IN THE ADULT CAT, THE VENTRAL ROOT STIMULATION CAN ACTIVATE NEURONS IN THE DORSAL HORN. SO HERE IS AN EXAMPLE. THESE ARE TRAIN--BRIEF TRAIN OF STIMULI, THE POST STIMULUS TIME INSTAGRAM OF THIS NEURON, AND IT ALSO WERE ABLE TO CHARACTERIZE ITS RECEPTIVE FIELD AND IF YOU LOOK AT THE NEURONS THAT WERE ACTIVATED BY VENTRAL ROOT STIMULATION, IN THE ADULT CAT, THEY'RE VERY SIMILAR TO THOSE THAT WE'VE SEEN HERE IN THE DEEP DORSAL NETWORK. AT THE TIME, HE INTERPRETED THIS AS BEING DUE TO THE STIMULATION OF AFFRONTS IN THE VENTRAL ROOT. ALSO THE TIMING WAS SLOW WHICH WAS CONSISTENT WITH THE IDEA THAT A VERY SMALL AFFRONTS IN THE VENTRAL ROOTS WITH SLOW CONDUCTION VELOCITIES COULD ACTIVATE THE CELLS. AND THS MAY INDICATE THAT ACTIVATION OF THESE DORSAL POSITION AT NETWORKS IS IN FACT DUE TO STIMULATION OF THE MOTOR NEURONS. IT'S AN INTERESTING BIT OF SOCIOLOGY AND SCIENCE. I MEAN NOT ONLY DID ECLES MISS DPLIEWTA MITTURGIC RELEASE FROM MOTOR NEURONS, EVEN THOUGH IN RETROSPECT WE CAN SEE HOW WE MISINTERPRETED THE EXPERIMENTS BUT HERE IN THE 80S THIS WAS MISINTERPRETED, TOO. AND INTERESTINGLY ENOUGH, NOBODY'S EVER FOLLOWED THAT UP. YOU THINK THIS WOULD BE REVOLUTIONARY, BUT IT WAS SIMPLY IGNORED. UNTIL NOW. ALL RIGHT. SO HERE'S THE SUMMARY. SO VENTRAL ROOT STIMULATION ACTIVATES DORSAL HOME NETWORKS, PRESENCE OF SUPERFICIAL DORSAL NETWORK IS NECESSARY FOR GENERATING LOCOMOTOR ACTIVITY, DEEP DORSAL NETWORK RUNS ROSTERCORDIALLILY AND RUN RSY CREWMENT SO THE QUESTION IS HOW DO THEY ACTIVATE THESE DORSAL HORN NETWORKS? NOW WE THINK NEITHER OF THESE CELLS, I VBT SHOWN YOU THIS BUT B3 WHEN THEY'RE STIMULATED ACTUALLY SLOW THE NOTER RHYTHM. THEY DO NOT INITIATE IT, THEREFORE THOSE ARE PROBABLY NOT INVOLVED, GAP JUNCTIONS ARE NOT INVOLVED, I'M SHOWING YOU THE PROLONGED APPLICATION OF BLOCKERS DOES NOT PREVENT MOTOR LIKE ACTIVITY OR THE MODULATION BY ONGOING ASYNCHRONOUS ACTIVITY. SO EITHER THERE'S ANOTHER TARGET, THE MOTOR NEURONS AND THIS IS SOMETHING WE THINKING WE FAVOR, IT'S DENDRITIC EXCITATION, MOTOR NEURON DENDRITES SPREAD VERY FAR INTO THE DORSAL HORN. AND IT'S QUITE POSSIBLE, EITHER BY RELEASE OF TRANSMITTER OR PERHAPS BY DENDRITIC CELL ROW DENDRITIC SIGN APSES THEY CAN ACTIVATE THOSE DORSAL HORN NEURONS, AGAIN THE NOTION THAT MOTOR NEURONS HAVE THIS SINGLE OUTPUT TO MUSCLES IN THIS LOCAL INPUT, BUT NOW IF MOTOR NEURONS ARE ACTIVATED IN THE CIRCUITS IN THE DORSAL HORN THROUGH DENDRITES, THE WHOLE NOTION OF THE MOTOR NEURONS IS THE FINAL COMMON PATHWAY HAS TO BE PROVIDED. WHAT'S THE SIGNIFICANCE OF THIS? WELL MOTOR NEURONS REGULATE SPINAL AND I'VE--SPINAL NETWORK AND MOTOR FUNCTION AND SENSORY FUNCTION. I SAY THE REDISCOVERY OF VENTRAL ROOT PATHWAY TO THE DORSAL HORN BECAUSE [INDISCERNIBLE] WORK THEORETICALLY PROCEEDED THIS, THIS A DEEPER UNDERSTANDING OF HOW THE NERVOUS SYSTEM GENERATES LOCOMOTION AND FINALLY THE DORSAL HORN CAN NO LONGER BE CONSIDERED EXCLUSIVELY SENSORY. WE COVERY OF FUNCTION OF SPINAL CORD INJURY, SO HERE'S THE LAB, MELANIE WHO IS SITTING HERE IN THE FRONT HERE DID ALMOST ALL OF THE EXPERIMENTS. BUT A NUMBER OF OTHER PEOPLE CONTRIBUTED AS WELL. THANK YOU VERY MUCH. [ APPLAUSE ] >> WHAT COMPETITION COULD DESCENDING PATHWAYS HAVE PRESIN APTAME INCREASE IN BODY INHIBITION FOR THE SYNAPSE, MIEN IT'S A VERY INTERESTING QUESTION, BUT ALSO EARLY DEVELOPMENT [INDISCERNIBLE]. >> SURE THESE--MOST OF THE EXPERIMENTS ARE DONE IN P0-P4, AND THESE WERE DONE IN ISOLATED SPINAL CORD WHERE WE ARE TYPICALLY CUTTING THE CORD AT THE THORACIC LEVEL NOTED TO SAY, I MEAN THERE COULD BE PATHWAYS THAT ARE PRESENT AND MAYBE MOTOR NEURONS INTERACT WITH INTH OF THOSE AS WELL AS YOU SAY, WE DON'T KNOW WHAT THE MECHANISM IS. YEAH? >> YOUR FRAME OF REFERENCE SIDE REFERENCES THE FRAME [INDISCERNIBLE]-- >> SORRY I THOUGHT IT WAS THE PERSON BEHIND YOU ASKING THE QUESTION. HE PUT HIS HAND UP AND I SAID YES AND THEN THE VOICE CAME FROM A COMPLETELY DIFFERENT LOCATION, SORRY, MICHAEL. [LAUGHTER] NO, NO, GO AHEAD. >> THE FRAME OF REFERENCE COMES FROM THE [INDISCERNIBLE] AND I'M NOT SURE CHICKEN AND THE EGG QUESTION, RIGHT SO THE MOTOR NEURONS GET STIMULATED BUT JUST [INDISCERNIBLE]-- >> CORRECT. >> [INDISCERNIBLE]-- >> OKAY, YEAH,-- >> [INDISCERNIBLE] >> ALL RIGHT, SO THE WAY I LIKE TO THINK ABOUT IT WHERE WE'VE LOOKED AT IT IN OTHER SYSTEMS IT IS FEEDBACK CONTROL OF THE GENERATOR BUT OBVIOUSLY IT'S DOING OTHER THINGS APART FROM REGULATING LOCOMOTION. SO WHY WOULD THAT BE. IF YOU THINK ABOUT IT, MOTOR NEURONS ARE THE FINAL COMMON PATHWAY OF SHERRINGTON AND IT'S THE LAST OPPORTUNITY TO MODIFY OUTPUT OF THE NERVOUS SYSTEM THAT'S CONVERTED INTO BEHAVIOR. SO YOU MIGHT WANT FROM AN EVOLUTIONARY POINT OF VIEW, YOU MAY WANT SOME REGULATION OF THAT LEVEL WHERE THE OUTPUT IS NO LONGER PREDETERMINED, BUT THERE ARE COMPLEX REGULATORY SYSTEMS THAT CAN CONTROL WHAT ULTIMATELY IS DELIVERED TO THE MUSCLES. SO I TEND TO LOOK AT IT IN THAT WAY, NOW WHAT THE FUNCTION OF THESE, YOU CAN SPECULATE ABOUT WHAT THE FUNCTION OF THESE SPOTS IS. ONE POSSIBILITY, EARLY IN DEVELOPMENT WHEN ANIMALS MOVE, THEY MOVE SPONTANEOUSLY AND THEY THEN HIT OBJECTS IN THEIR ENVIRONMENT AND THOSE SENSORY FIELDS ARE FACILITATED BY THAT MOTION. SO IT IS POSSIBLE THAT BY ACTIVATING THE RECEPTIVE FIELD OF A CERTAIN CLASS OF NEURONS THAT YOU WOULD ACTIVATE, SOMEHOW FACILITATING THAT CIRCUMSTANCE UTR. THAT'S 1 EXTREMELY SPECULATIVE IDEA. NO 1 IS EFERENCE COPY. IN CONTROL SYSTEMS EFER ENSEL COPY IS CONSIDERED A VERY POTENTIALLY VERY IMPORTANT WAY OF MONITORING THE OUTPUT OF A SYSTEM, CONSIDER MOTOR NEURONS, YOU DRIVE THEM, THEY DRIVE THE MUSCLE, BUT THE INPUT-OUTPUT RELATION OF THE MUSCLE MOTOR NEURONS ARE PROBABLY UNKNOWN TO THE NERVOUS SYSTEM, SO IT'S A NERVOUS SYSTEM CAN MONITOR HOW MOTOR NEURONS FIRE, THAT'S A SIGNAL BEING DELIVERED TO MUSCLES SO THAT AGAIN COULD BE AN IMPORTANT FUNCTION OF THIS RECURRENT PATHWAY. YOU KNOW AT THE MOMENT IT'S SPECULATIVE I'M AFRAID. >> [INDISCERNIBLE]. >> CORRECT. >> [INDISCERNIBLE]. >> SO COULD MOTOR NEURONS BE INVOLVED IN THAT? >> [INDISCERNIBLE]. >> CORRECT. >> [INDISCERNIBLE]. >> PROBABLY BOTH, WE THAT IF YOU YOU--NO IT'S NOT, THAT'S EVOLUTION MICHAEL SO THE THING IS, IF YOU STIMULATE WITHOUT LOCOMOTION, SO IT IS RAY RECIPROCAL PROCESS. >> [INDISCERNIBLE]. CORRECT THE DEEP DORSAL HORN AS I SAID MENTIONED THOSE LOCATIONS CORRESPOND TO THE LATERAL SPOONAL NUCLEUS WHICH CONTAINS PROP RIO SPINAL NEURONS WHICH RUN UP AND DOWN THE CORD WHICH MAY BE THE SUBSTRATE FOR THIS PROPAGATING ACTIVITY WE SEE, THEN MULTIMODAL, NO SUSCEPTIVE INPUT AND OTHER CLASS OF INPUT AS WELL INTO THOSE CELLS. >> [INDISCERNIBLE]. >> WHAT'S THAT? >> [INDISCERNIBLE] >> POTENTIALLY ABSOLUTELY. >> [INDISCERNIBLE] >> NOW OBVIOUSLY IF THE COPY IS TO BE TRULY USEFUL, IT'S GOING TO THE CORTEX, THE CEREBELLUM, OTHER LOCATIONS AND THE CENTRAL NERVOUS SYSTEM, YEAH. YEAH? THIS TIME I AM ANSWERING THAT FELLOW. >> IT SEEMS AS THOUGH THE LIGHT ACTIVATED SUPPRESSION WAS ONLY TRANSIENTLY-- >> CORRECT. >> [INDISCERNIBLE] >> YEAH. SO THERE WERE SEVERAL POSSIBILITIES, NUMBER 1 IS THE CHANNEL ROUGH ATOM DOBS KNOW WE'RE USING IT EITHER ADAPTS TO THE LIGHT STIMULUS AND IN FACT THE MEMBRANE POTENTIAL AND WHEN YOU LOOK AT IT, MEMBRANE POTENTIAL DECAYS BACK TO THE BASE LINE WITH A SIMILAR TIME COURSE TO THE CHANGES WE SAW. IT COULD BE AN ACTIVATION OF SOCIETYIUM CHANNELS UNDERLYING MECHANISMS, WE DON'T KNOW WHAT THEY ARE, SO WE FOCUS ONED FIRST 10-152nd WHICH IS IS SEVERAL STEP CYCLES AND UNDER THOSE CONDITIONS WHEN YOU DO THAT THESE ARE VERY--THEY CAN BE EVEN MORE MARKED IN SOME EXPERIMENTS. ONE OF THE THINGS I DIDN'T MENTION IS THAT THESE APPROXIMATE ROOT STIMULI ARE VARIABLE, SOMETIMES THEY PRODUCE LOCOMOTION AND SOMETIMES THEY DON'T, IT'S A DIRTY LITTLE SECRET OF ACEOLATED SPINAL CORD PREPARATION AND WE THINK IT'S BECAUSE THE CORD IS ISOLATED THE EXCITABILITY OF THE TISSUE IS VARYING FROM PREPARATION TO PREPARATION. BUT YES, AS FAR AS THE OPTOGENETIC EXPERIMENTS I THINK IT'S--FUNNY ENOUGH, IT WASN'T TRUE WHEN WE DID THE CHANNEL ROUGH ATOM DOBSIN NEURONS, IT TENDED TO STAY UP FOR THE DURATION--MELANIE, I DON'T LIKE THAT EXPRESSION, AM I SAYING SOMETHING WRONG? >> [INDISCERNIBLE]. >> OH YES. OKAY. YEAH? >> [INDISCERNIBLE] >> WHAT DO YOU MEAN THE IDENTITY? >> [INDISCERNIBLE] >> WHAT ARE THEY? >> YEAH, THATIA --THAT'S A VERY GOOD QUESTION. WE DON'T KNOW AT THE MOLENT. THERE ARE SEVERAL DIFFERENT TYPES, ALPHA MOTOR NEURON ARE AT LEAST 3 CATEGORIES THEY INNOVATE THE SPENDLE AND THE FIBERS THEN HAVE YOU SEVERAL TIMES OF GAMMA MOTOR NEURONS, SO WHEN I TALK ABOUT MOTOR NEURONS, IN FACT, IT'S A HETEROGENEOUS ROW GENIUS IT'S A VERY IMPORTANT QUESTION AS TO WHAT WHICH NEURONS ARE EXPRESSING AND AT THE MOMENT WE DON'T KNOW. YES? SORRY WALTER, I KNOW HAVE YOU A BAD LEG. >> [INDISCERNIBLE] >> ABSOLUTELY, HOW DO WE KNOW THE CIRCUITRY ISN'T BEING TRAINED BY THEY RETROGRADE ACTION OF MOTOR NEURONS ON THOSE CIRCUITS. WE DON'T. THE ASSUMPTION HAS ALWAYS BEEN ACTIVATION OF THE LOCOMOTION OF THE MOTOR CIRCUITS ARE, THEN OBVIOUSLY, MANIPULATING MOTOR NEURON ACTIVITY IN VARIOUS WAYS COULD FACILITATE TRAINING AND LOCOMOTOR RECOVERY AFTER INJURY. THE OTHER THING I THINK IS VERY IMPORTANT BE TO AWARE OF IS NEURODEGENERATIVE MOTOR NEURON DISEASE. IT IS ALSO BEEN ASSUME THAD THE MOTOR NEURONS ARE ISOLATED ENTITY BUT IF THEY HAVE THESE CONNECTION INTOS NEOF THE WORKS WITHIN THE SPINAL CORD, THEN THE CHANGES IN FUNCTION YOU SEE WHEN THE MOTOR NEURONS ARE GENERATING ARE GOING TO BE FAR MORE COMPLEX THAN SIMPLY THE LOSS OF MOTOR NEURON ACTIVITY OR INNOVATION OF MUSCLE. MICHAEL? ONE MORE? TWO MORE? YEAH. >> [INDISCERNIBLE] >> OF THE WHAT SYSTEM? >> LIFE CYCLE OF THE SQUID? >> YES, YES. >> ANTICIPATING YOU TO FOLLOW UP TO SAY MAYBE THERE WILL COME A DAY WHEN THE ENTIRE [INDISCERNIBLE] LOCAL CIRCUITRY WILL [INDISCERNIBLE] >> [LAUGHTER] , I'M NOT THAT HERETICAL YET. NO. [LAUGHTER] >> POSSIBLY A MISSING CHOLINNER GRAPHIC SYSTEM? MY WORLD IS [INDISCERNIBLE] >> BY THE WAY GLUE MARIOUSA ACTIVATION IS SOMETHING I DIDN'T TOUCH ON, THAT REMAINS--WE HAVE TRIED TO TEST WHETHER THESE EFFECTS ARE MEDIATED BY GLIAL CONNECTIONS TO THE DORSAL HORN. SO FAR THE EXPERIMENTS HAVE BEEN ACBIGUOUS, DIFFICULT TO INTERPRET BECAUSE IT'S DIFFICULT TO CANNED WHAT UNAMBIG USELY WITHOUT ANYTHING ELSE, SO, THAT REMAINS AN OPEN QUESTION. AN OPEN WOO WOO QUESTION. OKAY? [APPLAUSE ]