IT'S MY PLEASURE TO INTRODUCE BILL SCHAFER, BACHELOR'S DEGREE AT HARVARD, HEADED TO CALIFORNIA WHERE HE SPENT QUITE A WHILE, DID HIS Ph.D. AT UC BERKELEY, USING YEAST TO UNDERSTAND RAS AND G-PROTEIN MODIFICATION AND SIGNALING. AFTER HIS Ph.D. DID A POSTDOC AT UCSF, WORK IN C. ELEGANS, STUDIED AND CHARACTERIZED MUTANTS BEHAVIORALLY ALTERED, DOPAMINE, SEROTONIN, IDENTIFIED GENES FOR CALCIUM CHANNEL FUNCTION, AND THEN AFTER DOING POSTDOC CONTINUED TO UCSD, STARTED HIS OWN LAB, AND CONTINUED USING C. ELEGANS AND BEGAN TO PIONEER NEW APPROACHES TO STUDY NEURONAL CONTROL OF BEHAVIOR. FOR EXAMPLE, HE ESTABLISHED ONE OF THE FIRST WORM TRACKING SYSTEMS TO ALLOW US TO GET A HOLD ON HOW GENES AND 302 NEURONS THAT MAKE UP THE NERVOUS SYSTEM GENERATE BEHAVIOR, IN 2002 RIGHT BEFORE I JOINED THE LAB AS GRADUATE STUDENT HE COLLABORATED WITH ROGER GENERAL USING GENETICALLY CODED CALCIUM, COMBINED GENETICS BEHAVIOR AND IN VIVO, AND MOVED HIS LAB TO CAME BRITAIN, ENGLAND, IN 2006. I LEARNED A LOT OF THINGS DOING MY Ph.D. WITH BILL. ONE, COFFEE DOES MAKE YOU SMARTER, FOR SURE. TWO, SOMEHOW BILL NEVER SEEMS TO IN SPIRIT. IT'S AMAZING. I'M TRYING TO FIGURE OUT HOW STILL. THREE, HE TAUGHT ME HOW TO BE A PATIENT MENTOR. I DIDN'T KNOW THE DIFFERENCE BETWEEN A SYNAPSE AND NEURO TRANSMITTER, HE WAS PATIENT AND SOMETHING I TRY EVERY DAY TO CHANNEL IN MY OWN LAB. THANK YOU, AND WELCOME, BILL. >> MY TITLE IS DIFFERENT THAN ADVERTISED. I'M GOING TO TALK ABOUT HOW WE'VE BEEN USING C. ELEGANS TO INVESTIGATE WAYS TO PROBE STRUCTURE AND FUNCTION OF CONNECTOMES. YOU KNOW ABOUT CONNECTOMICS, WHICH IS THE NEXT BIG THING IN NEUROSCIENCE, AND CONNECTOMICS CAN MEAN DIFFERENT THINGS, REFERRING TO MACROCONNECTOMES TO MAP CORTICAL REGIONS IN THE HUMAN BRAIN BUT INCREASINGLY REFERRED TO MICROCONNECTOMES, EM SERIAL RECONSTRUCTION HAS BEEN USED TO RECONSTRUCT CIRCUITRY AT THE LEVEL OF INDIVIDUAL NEURONS AND SYNAPSES. THIS IS A BIG JOB IN HUMANS WHERE THERE'S 10 TO 11 NEURONS TO MAP, AND SO INCREASINGLY THERE HAVE BEEN CONNECTOME PROJECTS IN SIMPLER MODEL ORGANISMS LIKE MOUSE AND FLY. BUT OF COURSE IN THE NEMATODE C. ELEGANS WHERE I WORK THERE'S ALREADY A CONNECTOME, OBTAINED OVER 30 YEARS AGO BY PEOPLE AT MY CURRENT INSTITUTION, OBTAINED BY HAND TRACING SERIAL EM SECTIONS JUST LIKE THIS. IT WAS POSSIBLE TO IDENTIFY EACH NEURON, THEY HAVE FOUR-LETTER NAMES EACH, THE GENERAL STRUCTURE IS IDENTIFIED, EXON, DENDRITIC PROJECTIONS AND FINALLY EACH INDIVIDUAL SYNAPSE MAPPED INCLUDING SYNAPTIC PARTNERS. SO FROM THIS WE NOW HAVE A COMPLETE MAP OF THE ENTIRE C. ELEGANS CONNECTOME WHERE WE KNOW ALL OF THE 302 NEURONS, ALL THEIR SYNAPTIC INPUTS AND OUTPUTS AND SO FORTH. SO AT SOME POINT THIS MIGHT SEEM A LITTLE BIT DISTURBING, RIGHT? BECAUSE THERE'S ALL THIS EFFORT TO OBTAIN THE HUMAN AND MOUSE CONNECTOME AND SO FORTH. AND YET IN C. ELEGANS WE'VE HAD A CONNECTOME FOR 31 YEARS, IT'S A SMALL CONNECTOME, YET WE REALLY DON'T UNDERSTAND A LOT OF THINGS ABOUT HOW THE C. ELEGANS NERVOUS SYSTEM WORKS. IN FACT THERE'S AN ARTICLE IN SCIENTIFIC AMERICAN A FEW YEARS AGO, IS MAPPING THE MIND OF THE WORM WORTH IT? SCIENTISTS MAPPED IT, DID IT TEACH ANYTHING? IT'S UNFAIR TO SAY IT HASN'T TAUGHT US ANYTHING ABOUT ITS BEHAVIOR BUT IT IS TRUE HAVING THE CONNECTOME HASN'T PROVIDED A TRANSFORMATIVE UNDERSTANDING OF THE SYSTEMS LEVEL WORKINGS OF THE WORM NERVOUS SYSTEM THE WAY, SAY, THE GENOME PROJECT HAS. THERE'S A NUMBER OF REASONS OR EXCUSES ONE COULD MAKE FOR WHY THIS MIGHT BE TRUE. FOR ONE, WE SAY THIS IS A COMPLETE CONNECTOME BUT IT'S AN INCOMPLETE STRUCTURE. THERE'S A LOT OF EXTRA SYNAPTIC SIGNALING CRITICAL TO HOW THE C. ELEGANS AND ALL NERVOUS SYSTEMS MENTION AND THIS NEUROMODULATION OCCURS OUTSIDE THE WIRED SECRETARY -- CONNECTOME. WE NEED GOOD TOOLS TO ASSESS FUNCTIONS OF INDIVIDUAL NEURONS. FOR A LONG TIME WE'VE LACKED THE REALLY PRECISE PHENOTYPING TOOLS TO ALLOW US TO ASSESS THE PHENOTYPES OF NEURONAL ABLATION. IMPORTANT THING IS THAT IF WE THINK OF THE GENOME, IT'S PRETTY EASY TO UNDERSTAND HOW A DNA SEQUENCE RELATES TO THE FUNCTION OF ENCODED MOLECULES BECAUSE WE KNOW GENETIC CODE, BUT THERE ISN'T A CORRESPONDING THEORETICAL STRUCTURE TO UNDERSTAND HOW THE STRUCTURE OF A CONNECTOME RELATES TO ITS FUNCTION. AND SO I'M GOING TO TALK ABOUT EACH OF THESE THINGS IN TURN AND HOW WE'VE BEEN TRYING TO ADDRESS THESE PARTICULAR DEFICIENCIES IN C. ELEGANS AND HOPEFULLY MIGHT HAVE APPLICATION FOR PEOPLE STUDYING BIGGER CONNECTOMES. A LOT OF THINGS CONCERN CIRCUITS WITH RESPONSE TO MECHANOSENSORY STIMULATION AS SHOWN IN THIS MOVIE. THIS IS A WORM CRAWLING AROUND. THIS IS A POSTDOC WITH AN EYEBROW HAIR PRODDING THE WORM. IF YOU TOUCH THE WORM CRAWLING BACKWARDS, ON THE POSTERIOR PART OF ITS BODY IT WILL CHANGE DIRECTION AND GO FORWARD. IF IT'S GOING FORWARD AND YOU TOUCH IT IN THE FRONT, IT WILL CHANGE DIRECTION AND GO BACKWARDS. SO THIS IS AN ESCAPE REFLEX CALLED REVERSAL. THE IF WE -- THIS POSTDOC WASN'T HARASSING THE WORM, OFTEN THEY ARE FOLLOWED BY DEEP TURNS CALLED OMEGA BENDS. AND WE KNOW THE MOST IMPORTANT NEURONS FOR THIS BEHAVIOR, WHICH COME FROM JUST INSPECTION OF THE CONNECTOME, AND THAT IS THESE SENSORY NEURONS, GENTLE BODY TOUCH NEURONS, ALM AND AVM AND PLM IN THE TAILS DIRECT ON CODE NETWORKS, PRE-MOTOR NEURONS, HALF OF THIS NETWORK SYNAPSES ON MOTOR NEURON TO PROMOTE FORWARD MOVEMENT, THE OTHER HALF PROMOTE BACKWARD MOVEMENT, STIMULATION OF ANTERIOR NEURONS ACTIVATES THE BACKWARD NEURONS, VICE VERSA. DOING AND IMMEDIATE REVERSAL IS NOT THE ONLY THING THAT HAPPENS WHEN YOU POKE WORMS. THIS IS SHOWING IF YOU POKE WORMS THEY NOT ONLY DO REVERSALS BUT THEY ALSO GO FROM HANGING AROUND LEISURELY TO DOING MUCH HIGHER LEVELS OF ACTIVITY, AND THIS IS LOCO MOTOR AROUSAL, ACTIVATING THE ESCAPE CIRCUIT CAUSING INCREASED IN FORWARD LOCOMOTOR SPEEDS, LASTING FOR A MINUTE OR TWO. THIS IS SORT OF A STANDARD TYPE OF LOCOMOTOR AROUSAL. ONE THING THAT'S OFTEN ASSOCIATE WITH AROUSAL IS NOT ONLY IS IT AN INCREASE IN VOLUNTARY MOTOR ACTIVITY, WE'VE ALSO SEEN ENHANCED RESPONSIVENESS TO SENSORY STIMULI WHICH IS OFTEN CROSS-MODAL. YOU MIGHT EXPECT FOR EXAMPLE THAT MECHANOSENSORY EVOKED AROUSAL MIGHT SENSITIZE OTHER SENSORY PATHWAYS THAT ARE INVOLVED WITH BEHAVIOR. WE WANTED TO SEE WHETHER THIS WAS A GENERALIZABLE FORM OF ALLOWSAL. WE -- FORM OF AROUSAL, TEST TO SEE WHETHERRER SENSORY INPUTS THAT CAUSE BEHAVIOR ARE SENSITIZED BY AROUSAL. [ MUSIC ] I APOLOGIZE. IT DOES THE SAME REVERSAL BEHAVIOR. AND SO THIS AGAIN INVOLVES THE SAME SORT OF CIRCUITRY THAT WE SEE FOR THE LIGHT TOUCH AVOIDANCE. THIS NEURON CALLED ASH, THE ONE THAT SENSES THIS REPELLANT, DIRECTS SYNAPTIC OUTPUT ON PRE-MOTOR INTERNEURONS AND SWITCHES THE FORWARD HALF OF THE MOTOR CIRCUIT TO GOING BACKWARDS. SO WE WANTED TO TEST TO SEE WHETHER IF WE AROUSED THE WORMS BY STIMULATING THIS MECHANOSENSORY PATHWAY WHETHER IT WOULD SENSITIZE THIS CHEMOSENSORY AVOIDANCE RESPONSE, AND SO TO TEST THIS IN THIS PARTICULAR EXPERIMENT WE MADE A WORM THAT CELLS EXPRESS CHANNEL REDOPSIN, WE AROUSE WORMS BY TOUCHING THE DISHENED WHETHER IT'S BY ASH ACTIVATION ENHANCED BY THIS AROUSAL. YOU CAN SEE IT IS. THIS IS JUST MEASURING THE MAGNITUDE OF THIS BEHAVIOR AND YOU CAN SEE IT'S ABOUT TWICE AS -- HAS TWICE AS STRONG OF AVOIDANCE RESPONSES AFTER THIS MECHANOSENSORY AROUSAL THAN BEFORE. NOW, ONE THING YOU MIGHT WANT TO KNOW, WHERE IN THE NERVOUS SYSTEM DOES THIS AROUSAL TAKE PLACE. SO IS IT SENSITIZING THE SENSORY NEURON ITSELF OR IS IT SENSITIZING OTHER PARTS OF THE CIRCUITRY, SO TO TEST THIS WE WANT SOME WAY TO DO CALCIUM IMAGING IN DIFFERENT PARTS OF THE CIRCUITRY, TO SEE WHERE THE RESPONSE IS ENHANCED. SO TO DO THIS WE COLLABORATED WITH A LAB OF HAN LU, MICROFLUIDIC EXPERTS FROM GEORGIA TECH, THEY DESIGNED SOME MECHANOSENSORY CHIPS THAT ALLOW US TO FEED WORMS AT HIGH THROUGHPUT INTO THIS DEVICE, AND SO YOU'LL SEE THE WORMS WILL EVENTUALLY GO INTO THIS CHANNEL AND THEY ARE TRAPPED BY THIS TRAPPING VALVE HERE, AND THEN THIS WORM WILL JUST FLUSH OUT. THIS WORM WILL APPLY MECHANOSTIMULUS BY USING PRESSURE VALVES, AND YOU CAN SEE WE CAN APPLY PRESSURE TO THE ANTERIOR OF THE BODY, OR TO THE POSTERIOR OF THE BODY. AND THE RESPONSES WE SEE, FOR EXAMPLE, IF WE IMAGE FROM THE SENSORY NEURONS ARE SPECIFIC TO THE RECEPTOR FIELDS OF THE NEURONS. SO THIS IS APPLYING ANTERIOR STIMULUS TO THE ANTERIOR TOUCH RECEPTOR, WE SEE A RESPONSE BUT NOT IN THE POSTERIOR TOUCH RECEPTORS. AND VICE VERSA. SO AGAIN THIS CORRELATES WITH WHAT WE'D EXPECT AND SO THEN BY MODIFYING THIS SLIGHTLY, POSSIBLE TO APPLY MECHANOSENSORY AND TEST TO CHEM REPELLANT. TRAP THE WORM IN THIS CHAMBER, YOU HAVE ANOTHER VALVE HERE, NORMALLY BUFFER WOULD FLOW ACROSS ITS NOSE, YOU COULD SWITCH TO HAVING CHEMICAL REPELLANT ACROSS ITS NOSE. SO THIS IS JUST SHOWING IF YOU DON'T AROUSE THE WORMS THIS REPELLANT OF GLYCEROL PRODUCES A MODEST RESPONSE. THIS SHOWS MECHANOSENSORY AROUSAL IS ENHANCING THE EXCITABILITY OF THESE CHEMICAL NOCICEPTORS IN C. ELEGANS. ONE THING WE THOUGHT MIGHT BE INVOLVED WITH BE NEUROPEPTIDES. WORMS HAVE A HUGE NUMBER CONSIDERING THERE'S 302 NEURONS, 125 PUTATIVE PEPTIDE RECEPTORS, MANY ARE HOMOLOGS OF WELL-KNOWN VERTEBRATES. THERE'S EXPANSION OF THE FAMILY SPECIFIC TO NEMATODES, AND SIGNALING APPEARS TO BE MOSTLY EXTRASYNAPTIC. WE TESTED LOTS OF THESE MANY NEUROPEPTIDE MUTANTS, THERE ARE A LOT OF THEM TO TEST. WE DID FIND ONE MUTATION IN A GENE CALLED FLP-20, AND MUTANTS IF WE LOOK IN OUR AROUSAL ASSAY SEE THIS SHOW THIS LITTLE SPIKE HERE WHICH IS THE REVERSAL BUT THEIR AROUSAL IS SUBSTANTIALLY REDUCED COMPARED TO WILD TYPE. AND FURTHERMORE, IF WE PUT BACK WILDTYPE FLP-20 IN THE TOUCH NEURONS WE BETTER THAN RESCUE, IF WE OVEREXPRESS THE PEPTIDE WE SEE MORE AROUSAL IN WILD TYPE. FLP-20 ARE BEING RELEASED FROM THE TOUCH NEURONS AND THAT'S SOMEHOW LEADING TO BOTH LOCOMOTOR AROUSAL AND AS I'LL SHOW YOU HERE ALSO TO SENSORY AROUSAL. SO THIS IS JUST USING THAT ASSAY, YOU SEE WILDTYPE WORMS SHOW MUCH INCREASED CHEMOSENSORY AVOIDANCE AFTER MECHANOSENSORY AROUSAL. FLP-20 MUTANTS ARE DEFECTIVE IN THIS SENSITIZATION. WE CAN RESCUE THIS PHENOTYPE BY PUTTING BACK FLP-20 UNDER'S OWN PROMOTER OR UNDER TOUCH NEURON-SPECIFIC PROMOTER. AND AGAIN WE CAN SEE THE SAME SORT OF RESCUE IF WE USE OUR DUAL CHEMO MECHANOCHIP EXPERIMENT, THIS IS WILDTYPE WHERE WE SEE RESPONSE TO SEE A BIGGER RESPONSE TO CHEMICAL STIMULUS IN NAIVE ANIMALS, NO ENHANCEMENT BY MECHANOSENSORY AROUSAL. LOOKS LIKE FLP-20 PEPTIDES ARE RELEASED FROM THE TOUCH NEURONS, CAUSING SENSITIZATION AND LOCOMOTOR AROUSAL. HOW DOES THIS WORK? WE NEED TO KNOW WHAT THE RECEPTOR IS FOR THE PEPTIDE. THIS WAS NOT NONE WHEN WE STARTED EXPERIMENTS. SO THE LAB IN BELGIUM HAS A SETUP WHERE YOU CAN EXPRESS C. ELEGANS NEURAL PEPTIDES IN MAMMALIAN CULTURE, YOU CAN USE CALCIUM IMAGING. WE SCREENED A LIBRARY OF 80 RECEPTORS TO FIND ONE THAT RESPONDS TO FLP-20 PEPTIDE. AND WE FOUND ONE, AND ONLY ONE. AND THIS IS THIS RECEPTOR FRPR3, ALL OF THE FLP-20 LIGAND ACTIVATE THE FRPR3. WE SEE THE SAME LOCOMOTOR PHENOTYPE IN FLP-20 AND SEE THE FRPR3 MUTANT IS THE (INDISCERNIBLE). NOW WHAT ABOUT SENSORY SENSITIZATION? WHAT DID SENSITIZATION OF CHEMO SENSORY NEURON? IT HAS THE SAME DEFECT IN THE FLP-20 MUTANTS. HERE IS WILD TYPE, SIGNIFICANT AROUSAL, FRPR3 NO AROUSAL. WE ALSO SEE INVOLVEMENT OF THIS PATHWAY NOT JUST IN AROUSAL BUT ALSO IN ANTI-AROUSAL, OR SLEEP-LIKE BEHAVIOR. THERE'S A LOT OF ASSAYS PEOPLE DEVELOPED FOR SLEEP-LIKE BEHAVIOR IN C. ELEGANS, THIS IS ONE OF THE MORE, I DON'T KNOW, THIS IS ONE THAT IS SEEN IN ADULT WORMS. NOW, WORMS DON'T HAVE A STRONG CIRCADIAN LINK WITH SLEEP SO WORMS SHOW SLEEP-LIKE BEHAVIOR STOCHASTICALLY. IF YOU TAKE WORMS AND TRACK THEM OVER LONG PERIODS OF TIME THEY SEEM TO. THEY ARE NARCOLEPTIC AND SLEEP MORE. THIS PATHWAY IS INVOLVED WITH SORT OF SENSITIZATION TYPE AROUSAL BUT SEEMS TO PLAY A ROLE IN SLEEP-LIKE BEHAVIOR. WHERE DOES THE FRPR3 RECEPTOR FUNCTION? WE LOOKED AT REPORTERS, IT'S EXPRESSED IN A SMALL NUMBER OF HEAD NEURONS AND TESTED RESCUE FOR BEHAVIORS AND ONLY NAILED THIS TO LOCOMOTOR AROUSAL, AND THIS ONE NEURON IS THE COMPLICATED THING HERE BUT IF YOU LOOK AT THIS HERE IS WILD TYPE IN BLACK, THIS IS THE MUTANT, AND YOU CAN SEE THAT THESE ARE THREE DIFFERENT RESCUE LINES WHERE WE PUT THE FRPR3 RECEPTOR BACK IN RID AND GET OVERRESCUE WHICH OFTEN HAPPENS WHEN WE OVERRESCUE PROTEINS. IF YOU EXPRESS? A DIFFERENT NEURON, AVK, YOU DON'T SEE ANY RESCUE OF LOCOMOTOR AROUSAL. WHAT IS THE LINK WHEN RID AND LOCOMOTORIAL AROUSAL? MECHANOSENSORY STIMULATION LEADS TO INCREASE IN ACTIVITY OF RID AND THIS INCREASE IS DEPENDENT ON FLP-20. IF WE KNOCK OUT 20 THERE'S NO ENHANCEMENT WITH MECHANOSENSORY ALLOWSAL AND ONLY A LITTLE WITH FLP-20. NOW, IT TURNS OUT RID IS AN INTERESTING NEURON BECAUSE RID HAS THIS LONG PROCESS THAT GOES THROUGHOUT THE DORSAL CORD OF THE WORM AND HAS LOTS OF DENSE CORE VESICLES. IN A PAPER LAST YEAR THEY SHOWED OPTOGENETIC PROVIDED MORE FORWARD MOVEMENT AND THIS IS PARTIALLY DEPENDENT ON ANOTHER NEUROPEPTIDE CALLED FLP-14. WHAT IT LOOKS LIKE IS GOING ON IS THAT FLP-20 IS RELEASED FROM TOUCH NEURONS, MECHANOSENSORY STIMULATION, AND THIS ENHANCES ACTIVITY OF RID, AND RID IS RELEASING THIS OTHER NEUROPEPTIDE, FLP-14, RECEPTORS IN THE NEURONS. RATHER THAN GOING THROUGH THE CONNECTOME THERE'S SORT OF ALTERNATIVE CONNECTOME WHERE THE EXTRASYNAPTIC EVENTS ARE BYPASSING WIRED CONNECTIONS AND GIVING YOU THIS CHANGE IN BEHAVIORAL STATE. IN OTHER NEURONS WE THINK THERE ARE MAYBE OTHER CHAINS OF NEUROPEPTIDE SIGNALING AFFECTING OTHER BEHAVIORS. AVK CONTAINS THIS FACTOR, CRF 1 CAN ENHANCE LOCOMOTOR ACTIVITY AND PROBABLY ACTIVATE OTHER STRESS PATHWAYS. THIS SORT OF MAKES YOU WONDER THEN HOW CRITICAL IS THE SYNAPTIC CONNECTOME FOR THESE BEHAVIORS, AT THEIR VERY LEAST IS THIS ONLY PART OF THE STORY THAT'S GOING TO ALLOW US TO UNDERSTAND BEHAVIORS. AND IT TURNS OUT THIS ISN'T JUST NEUROPEPTIDES FOR WHICH THIS IS AN ISSUE. WE CAN LOOK AT MONOAMINES AND ASK HOW MUCH OF THE SIGNALING IN C. ELEGANS IS SYNAPTIC VERSUS EXTRASYNAPTIC BECAUSE WE KNOW WHICH NEURONS MAKE EACH OF THE FOUR MONOAMINE NEURO TRANSMITTER IN C. ELEGANS, AND WE KNOW ALL THE NEURONS THAT EXPRESS ALL OF THE KNOWN RECEPTORS FOR THESE. SO THESE ARE ALL THE NEURONS THAT ARE POST-SYNAPTIC TO RIM LEFT AND RIGHT, AND YOU CAN SEE SOME EXPRESS TYRAMINE SOME DON'T. WE CAN SEE WITH CONFIDENCE THEY DO NOT RECEIVE SYNAPSIS FOR THE NEURONS. THESE ARE THE NEURONS THAT RECEIVE SYNAPSIS AND NONE OF THE NEURONS THAT EXPRESS OCTOPAMINE ARE PART OF THE GROUP. MOST MUST BE SYNAPTIC. THIS MEANS THE CONNECTOME IS NOT REALLY ONE CONNECTOME BUT REALLY A MULTIPLEX NETWORK WITH WHERE EACH LAYER HAS THE SAME NODES, THE SAME NEURONS, BUT THE DIFFERENT LAYERS WHICH DESCRIBE DIFFERENT TYPES OF INTERNEURONAL INTERACTION WITH DIFFERENT PROPERTIES HAVE DIFFERENT STRUCTURES. SO FOR EXAMPLE THESE SYNAPTIC CONNECTIONS ARE RELATIVELY FAST, UNIDIRECTIONAL, AND THEY ARE WIRED. THERE ARE GAP JUNCTION CONNECTIONS ALSO VERY FAST, BUT THEY ARE BIDIRECTION. AND THEN WE ALSO HAVE THESE SLOWER WIRELESS CONNECTIONS INVOLVING MONOAMINES AND NEUROPEPTIDES. THE QUESTION IS CAN WE MAP OTHER LAYERS OF THE CONNECTOME AND FIND OUT THINGS ABOUT HOW THEIR STRUCTURES INTERACT. I'M GOING TO GO THROUGH THIS BRIEFLY BECAUSE I'VE GOT OTHER THINGS TO TALK ABOUT. BUT I CAN JUST SAY THAT WE'VE COMPILED FROM THIS INFORMATION ABOUT THE EXPRESSION PATTERNS OF ALL OF THESE MONOAMINE RECEPTORS AND ALSO THE NEUROPEPTIDE RECEPTORS, AND WE CAN COMPARE MATRICES FOR MONOAMINE CONNECTOME WITH THAT OF THE SYNAPTIC CONNECTOME AND WE SEE THERE'S RELATIVELY LITTLE OVERLAP. WE CAN ALSO LOOK AT VARIOUS NETWORK MEASURES TO LOOK AT WHAT THE TOPOLOGY TO THE NETWORKS LOOK LIKE COMPARED TO SYNAPTIC CONNECTOME, THEY ARE QUITE DIFFERENT. SO ALL THESE DIFFERENT LAYERS SHOW MODULARITY WHICH IMPLIES THERE'S FUNCTIONAL SEGREGATION BUT MONOAMINE NETWORK HAS THIS SORT OF HIGHLY DISASSORTED STAR-LIKE STRUCTURE, WHICH IS DIFFERENT, MUCH MORE DISASSORTATIVE THAN THE SYNAPTIC OR MONO -- OR THE NEUROPEPTIDE CONNECTOME. IN CONTRAST NEUROPEPTIDE NETWOR SHOWS HIGHLY CLUSTERED STRUCTURE WHERE YOU SEE HIGHLY -- YOU SEE LARGE PROPORTION OF INTERCONNECTED TRIPLES, WHICH IMPLIES THESE ARE IMPORTANT FOR FUNCTIONAL INTEGRATION. I GUESS WHAT I WOULD SAY IS THAT ONE OF THE THINGS WE'RE INTERESTED IN IS GETTING MORE INFORMATION ABOUT THESE OTHER LAYERS OF THE C. ELEGANS ONE THING IS THAT FOR THE MONOAMINES WE KNOW WHERE RECEPTORS ARE, FOR NEUROPEPTIDES THERE'S A LOT INFORMATION WE DON'T HAVE ABOUT INTERACTION BETWEEN LIGAND AND RECEPTOR PAIRS AND WHERE THEY ARE EXPRESSED. THIS IS WORK WE'RE CARRYING OUT IN COLLABORATION WITH LILLY ANN AND OLIVER. WE'D LIKE TO GET SOME INFORMATION ABOUT THE FUNCTIONAL ROLES OF THESE NEUROPEPTIDE SYSTEMS. THIS REQUIRES US, WE CAN GET NEUROPEPTIDE MUTANTS BUT NEED BETTER WAYS TO PHENOTYPE THEM TO FIND ROLES OF BEHAVIOR. I'M GOING TO TALK ABOUT HIGH THROUGHPUT PHENOTYPING LEADING TO THE LAST PART OF THE TALK. WHY WOULD WE NEED MORE QUANTITATIVE PHENOTYPING? IF WE LOOK AT GENES IMPORTANT FOR NEUROMODULATION IN C. ELEGANS, FOR EXAMPLE, THESE THINGS INVOLVED WITH DEGRADATION, THESE PEPTIDES AND RECEPTORS, THERE'S LOTS OF THESE GENES IN THE GENOME BUT MOST OF THEM WHEN THEY HAVE BEEN KNOCKED OUT DON'T HAVE DESCRIBED PHENOTYPES IN WORM BASE, THE REASON IS IF YOU LOOK AT THEM TO THE NAKED EYE THEY LOOK NORMAL. I MEAN, YOU MIGHT THINK THESE WOULD BE VERY IMPORTANT BUT IF YOU WERE LOOKING UNDER THE MICROSCOPE THEY DON'T LOOK THAT MUTANT. HOWEVER, IF YOU USE VIDEO ANALYSIS, YOU CAN DETECT -- OH, THIS IS FROM KATIE. I DIDN'T KNOW I HAD ONE OF KATIE'S PAPERS. THIS IS HOW YOU CAN USE QUANTITATIVE PHENOTYPE TO DETECT SUBTLE PHENOTYPE. KATIE FOUND FROM LOOKING AT ALL THESE VIDEOS THAT THEY HAVE THIS SUBTLE DEFECT IN HOW IT MOVES ITS NOSE BACK AND FORTH, WHICH IS A BEHAVIOR IN C. ELEGANS CALLED FORAGING, A QUANTITATIVE DEFECT THAT'S HIGHLY ROBUST BUT YOU WOULDN'T NOTICE FROM JUST LOOKING AT WORMS UNDER A MICROSCOPE. WE DECIDED TO DEVELOP TECHNIQUES TO COLLECT VIDEO DATA AT HIGH THROUGHPUT AND DEVELOP MEANS TO ANALYZE THIS DATA TO USE IN PHENOTYPING. AND SO AS A FORMER GRADUATE STUDENT IN THE LAB DEVELOPED INEXPENSIVE WORM TRACKERS THAT WE COULD SET UP LOTS OF THEM IN OUR TRACKER ROOM, AND WE CAN COLLECT THOUSANDS OF VIDEOS OF WILD TYPE AND MUTANT WORMS. ONE OF THE THING THAT'S NICE ABOUT C. ELEGANS IN THE LAB ENVIRONMENT THEIR MOVEMENTS ARE IN TWO DIMENSIONS AND THEY DON'T HAVE ANY APPENDAGES SO YOU CAN REDUCE BODY POSTURE OF THESE WORMS TO BASICALLY COORDINATES OF THIS SKELETON OR MIDLINE DOWN THE WORM, SO IF YOU DO ELEMENTARY SEGMENTATION OF IMAGES TO FIND CONTOUR OF THE WORM AND DETERMINE ITS MIDLINE, YOU FIND ITS HEAD AND TAIL, YOU CAN REDUCE INFORMATION IN THESE GRAY SCALE IMAGES CONSIDERABLY. AND THEN HOW DO WE ANALYZE THIS? ONE WAY YOU CAN DO IT IS BY USING FEATURES, THINGS WE KNOW ARE INTERESTING ABOUT BEHAVIORS, SO SPEED, WE TALKED ABOUT AROUSAL, WORMS MOVE FASTER SO WE CAN MEASURE SPEED OVER TIME. WE CAN MEASURE THINGS WILL SHAPE LIKE ECCENTRICICITY, AMPLITUDE OF BODY BENDS, REVERSAL, OMEGA TURNS AND SO FORTH. AND SO IF WE DO THIS, AND THEN WE CAN LOOK AT SUBFEATURES, AT THE SPEED OF WORM MOVING FORWARD OR BACKWARD, CURVATURE AT DIFFERENT POINTS IN THE BODY, SO FORTH. WE CAN BASICALLY PARAMETERIZE DATA AND COME UP WITH UNDERSTAND HAD OF WAYS TO DESCRIBE THE IMAGES THAT WE EXTRACT FROM MOVIES. SO ONE THING WE CAN DO IS WE CAN USE FEATURES TO TRY TO CLASSIFY MUTANTS. THIS IS MUTANTS FROM OUR DATABASE. MOST OF THESE, SAY ONC AFTER THEM, IDENTIFIED BY SIDNEY BRENNER AS BEING UNCOORDINATED. YOU CAN FIND FEATURES AND FIND CLUSTERING MAKES A FAIR AMOUNT OF SENSE. YELLOW IS ALLELES THAT CLUSTER CLOSELY TOGETHER, AUTOMATED CLUSTERING IS DOING A GOOD JOB FINDING THINGS WITH SIMILAR PHENOTYPES. WE CAN ALSO FIND THAT MUTATIONS IN GENES WHOSE MOLECULES FUNCTION IN A COMMON PROCESS ALSO CLUSTER TOGETHER, TWO SUBUNITS OF SODIUM CHANNEL, THESE ARE TWO SUBUNITS OF GAP JUNCTION PROTEIN, AND NICOTINIC RECEPTOR. IF WE DO THIS FOR UNANNOTATED MUTANTS, NEUROPEPTIDE MUTANTS THAT DON'T HAVE DESCRIED PHENOTYPES, WE FIND CLUSTERING STILL KIND OF WORKS BUT THE DIFFERENCES WE DETECT WITH WILD TYPE ARE MUCH LESS PRONOUNCED. THAT'S WHY THIS HEAT MAP LOOKS SO WHITE. AND ALSO, YOU KNOW, THESE ARE THREE ALLELES OF THE SAME GENE, YOU CAN SEE THEY DON'T CLUSTER TOGETHER. THAT'S BECAUSE IN THE CASE OF THE SUBTLE PHENOTYPES BACKGROUND EFFECTS OF THESE DIFFERENT ALLELES, A LOT OF DIFFERENCES TO WILDTYPE ARE PROBABLY NOT DUE TO THE TRIP 2 MUTATION BUT BACKGROUND. WE NEED A BETTER WAY TO DEAL WITH SUBTLE PHENOTYPES. ONE POSSIBILITY IS THAT MAYBE BY REDUCING THIS DATA TO A SUMMARY OF FEATURES, WE'VE THROWN OUT TOO MUCH OF THE DATA. AND ANOTHER THING IS THAT IF WE'RE BASING FEATURES ON BEHAVIORS WE ALREADY KNOW, IF THERE'S BEHAVIORS THE WORM IS DOING THAT WE DON'T KNOW ABOUT WE'RE JUST NOT USING ANY OF THAT INFORMATION. SO A POSTDOC NAMED ANDRE BROWN DECIDED TO EXPLORE THE POSSIBILITY OF USING UNSUPERVISED BEHAVIORAL ANALYSIS TO TRY TO ANALYZE DATA. SO APPROACH HE TOOK WAS BASED ON EARLIER WORK BY A GUY NAMED GREG STEVENS, AND HE TOOK THESE SKELETONS THAT I ALREADY MENTIONED, AND SO BASICALLY SKELETONS YOU COULD PUT I THINK 49 POINTS ALONG THEM AND YOU CAN BASICALLY REPRESENT THESE SKELETON SHAPES IN TERMS OF THE ANGLES BETWEEN THE POINTS. AND SO WHAT GREG DID WAS THEN TAKE THESE SKELETONS OF WILDTYPE WORMS AND RUN PCA ON THEM AND SEE IF THERE ARE ANY SORT OF PROTOTYPICAL SHAPES THAT ALL REAL SHAPES WERE A COMBINATION OF. HE CALLED THESE IgAN WORMS, THAT YOU FIND IF YOU LOOK AT WILD TYPE WORMS ON FOOD, SIMILAR TO GREG'S EIGENWORMS. YOU CAN SEE THE SHAPES AND IN PRINCIPLE REPRESENT BEHAVIOR OF ALL THOSE WORMS IN THE MOVIE, NOT AS SEQUENCE OF GRAY SCALE IMAGES BUT FOUR, THE FOUR EIGEN WORMS. THIS THING IN BLUE IS THE EIGENWORM RECONSTRUCTION, AND CONTOUR IS THE REAL CONTOUR. YOU CAN SEE THAT MORE OR LESS IT'S CAPTURING THE POSTURES OF THE WORM THROUGH TIME PRETTY WELL. NOW, OF COURSE, THIS ONLY MEANS THAT THIS WORKS FOR WILDTYPE WORMS, BUT WHAT WE'RE INTERESTED IN APPLYING YOU APPLYING THIS TO MUTANT WORMS. IF YOU LOOK AT THE SHAPES BY MUTANT WORMS THEY DON'T LOOK LIKE SHAPES BY WILD TYPE. THESE ARE WILDTYPE WORMS, THESE ARE MUTANT WORMS. THESE HAVE A DEVELOPMENTAL DEFECT IN THE NERVOUS SYSTEM. THESE ARE MISSING THE MAIN CAV2 CALCIUM CHANNEL, NERVOUS SYSTEM DOESN'T WORK WELL AT ALL. IT'S MAKING SHAPES THAT DON'T LOOK ANYTHING LIKE WILDTYPE SHAPES. YOU MAY ASK WHETHER THE EIGENWORMS DESCRIBES THE MUTANT SHAPES. IT DOES A VERY GOOD JOB. THESE ARE THE WORST WORMS, THE EIGENWORMS DESCRIBES TO 86% ACCURACY. MOREOVER, IF YOU LOOK AT THE EIGENWORMS FROM ALL THE MUTANTS IN OUR DATABASE, 300 WORMS IN THE DATABASE, THE RED ONES ARE WILD TYPE EIGENWORMS, MIGHTANT ARE THE SAME AS WILD TYPE EIGENWORMS. SO SURPRISINGLY, I THINK, THIS EIGENWORM IS A GOOD WAY TO DESCRIBE NOT JUST WILDTYPE WORMS BUT WORMS WITH SEVERE ABNORMALITIES IN THE NERVOUS SYSTEM, AND PRESUMABLY ONES WITH SUBTLE AS WELL. THE OTHER THING THAT'S NICE IS THE FOR EIGENWORMS CORRESPOND TO REAL THINGS WORMS DO. IF WE LOOK AT WORMS WITH A HIGH CONTENT OF EIGENWORM 1 THEY ARE MAKING DEEP BENDS, OMEGA BENDS, UNIFORM CURVATURE ACROSSES THE WHOLE BODY. 2 AND 3 ARE DIFFERENT PHASES OF THE TRAVELING WAVE THAT YOU SEE WITH SINUSOID MOVEMENT. AND THIS IS THE HEAD AND SALE. NEXT THING ANDRE DID WAS SEE IF HE COULD USE EIGENWORMS AS A BASIS TO FIND NEW BEHAVIORS. HE DID WHAT IS CALLED MICRO BEHAVIORAL MOTIF DISCOVERY, WHERE IN EACH OF HIS MOVIES HE LOOKED FOR THE MOST CLOSELY REPEATED SEQUENCE OF A GIVEN LENGTH IN THAT MOVIE. IF YOU TOOK -- THIS WAS A 15-SECOND MOTIF, HE LOOKED AT THE 15 SECONDS MOST CLOSELY REPEATED WITHIN THE MOVIE. SO A LOT OF THESE MOTIFS ARE THINGS LIKE FOR WILD TYPE SINUSOIDAL MOVEMENT LIKE THIS BUT IN WILDTYPE OR EVEN MUTANTS YOU FIND THEM DOING FUNNY THINGS. THIS IS IN THE DOPAMINE DEFICIENT MUTANT, YOU CAN SEE IT'S DOING THIS SORT OF WEIRD SHUDDERING PAUSE-LIKE STATE, AND I DON'T KNOW IF YOU WANT TO LIKE ANTHROPOMORPHISE THIS AS BEING A PARKINSONIAN WORM BUT IT'S SOMETHING WILDTYPE WORMS DON'T DO. YOU CAN USE A DICTIONARY AND USE THIS TO CLASSIFY MUTANTS IN YOUR DATABASE. THIS IS WHAT ANDRE DID HERE. THIS IS A CLUSTERING HE DID BASED ON EIGENWORM BASED MICRO BEHAVIORS, THESE ARE WILD TYPE SO A LOT OF THINGS CLUSTER CLOSELY TO WILD TYPE, A LOT OF NEUROPEPTIDE MUTANTS AND MONOAMINE MUTANTS AND SO FORTH ARE QUITE CLEARLY DISTINGUISHABLE FROM WILD TYPE. THIS ISN'T GOING TO BE THAT IMPRESSIVE FOR PEOPLE THAT HAVE EVER LOOKED AT DOPAMINE DEFICIENT OR SEROTONIN DEFICIENT WORMS BUT YOU WOULD NOT KNOW THERE WAS ANYTHING WRONG WITH THESE WORMS AT ALL AND YET ONLY DO THEY CLUSTER AWAY FROM WILD TYPE BUT MONOAMINES CLUSTER DIFFERENT FROM EACH OTHER SO SEROTONIN DEFICIENT HERE, DOPAMINE DEFICIENT HERE AND SO FORTH, EXCEPT FOR THE ONLY SEROTONIN DEFICIENT MUTANTS THAT CLUSTER ARE LACKING. THEY DOES A GOOD JOB OF IDENTIFYING SUBTLE PHENOTYPES AND FINDING ONES THAT ARE ACTUALLY RELATED TO THE UNDERLYING MOLECULAR DEFECT. THIS IS JUST SHOWING HOW WE CAN APPLY THIS TO NEURAL PEPTIDES, THIS IS THE MUTANT WE FOUND, THIS IS JUST LOOKING AT THE DIFFERENT AVERAGE EIGEN PROJECTION FOR MUTANTS. SUB3 AND CRF MUTANTS SHOW REDUCED VALUES FOR 2 AND 3 AND RESCUE OVEREXPRESSION RESCUE CAUSES TEASE THESE TO BE HIGHER THAN WILD TYPE. IF YOU COMPARE YOU SEE THE MUTANT SHOWS A SUBTLY BUT SHALLOWING WAVE. CAN USE THESE TO IDENTIFY SUBTLE PHENOTYPES AND RELATE THEM TO MOLECULAR PHENOTYPES. OKAY. NOW I'M GOING TO GET TO THE LAST PART OF THE TALK HERE, THE TALK ABOUT HOW WE CAN TRY TO COME UP WITH A THEORETICAL BASIS FOR UNDERSTANDING THE CONNECTOME. THIS IS JUST ANOTHER REPRESENTATION OF THE C. ELEGANS CONNECTOME WITH ALL THESE DIFFERENT NEURONS HERE, ALL THE CONNECTIONS, AND YOU CAN SEE IT KIND OF LOOKS LIKE A MESS. SO THE QUESTION IS, IS THERE ANY WAY THAT WE CAN TAKE THE PATTERN OF CONNECTIVITY HERE AND MAKE SOME PREDICTIONS ABOUT WHERE THE INFORMATION FLOW IS IN THIS NETWORK? THIS IS SOMETHING THAT REALLY IS SOMETHING THAT HAS OCCUPIED PEOPLE IN THE C. ELEGANS FIELD FOR A LONG TIME. AND SO ONE OF THE PROPOSALS FOR HOW YOU COULD THEY -- A THEORETICAL APPROACH CAME OUT A FEW YEARS AGO, FROM A WELL-KNOWN NETWORK SCIENTIST, HIS PROPOSAL WAS THAT YOU COULD USE CONTROL THEORY TO UNDERSTAND COMPLEX BIOLOGICAL NETWORKS LIKE THE CONNECTOME. THE IDEA IS THAT THE FUNCTION OF THE BRAIN OF NEURAL NETWORKS IS TO CONTROL THE STATE OF EFFECTORS LIKE MUSCLES IN PARTICULAR, AND SO IF YOU WERE DESIGNING A BRAIN THEN YOU COULD USE CONTROL THEORY TO PREDICT HOW AN INPUT SIGNAL COULD PROPAGATE THROUGH THE NETWORK TO CONTROL YOUR ANDROID'S BODY MOVEMENTS. AND SO THE IDEA IS THAT -- AND ONE OF THE MAIN CONCLUSIONS OF CONTROL THEORY IS THAT THE STRUCTURAL CONTROLLABILITY OF A NETWORK DEPENDS ON ITS GEOMETRY. IF THE BIOLOGICAL NETWORKS ARE ALSO DESIGNED AT LEAST THROUGH EVOLUTION UNDER CONTROL PRINCIPLES, THEN THIS MIGHT -- THEN CONTROL THEORY THAT WAS DEVELOPED FOR ARTIFICIAL CONTROL OF MECHANISMS MIGHT ALLOW US TO REVERSE ENGINEER THESE NETWORKS TO UNDERSTAND THE STRUCTURE REQUIREMENTS FOR THEIR FUNCTION. OKAY. SO HOW DOES THIS ACTUALLY WORK? THIS IS JUST AN EXAMPLE FROM THEIR THEORY PAPER THAT CAME OUT A FEW YEARS AGO, IF YOU TAKE THIS SIMPLE NETWORK WHERE YOU HAVE THREE NODES IN THE NETWORK AND THESE COULD BE NEURONS, THEN IF YOU APPLY CONTROL SIGNAL TO THIS NEURON YOU WOULDN'T BE ABLE TO INDEPENDENTLY CONTROL THESE TWO DIFFERENT OUTPUTS BECAUSE SIGNAL THEY RECEIVE FROM NEURON ONE IS ALWAYS GOING TO BE EXACTLY CORRELATED. YOU NEED DO HAVE A SECOND INPUT TO ONE OF THESE OTHER NEURONS TO GIVE YOU FULL CONTROLLABILITY. YOU COULD ALSO CHANGE THE NETWORK AND ADD A CONNECTION BETWEEN NODES 2 AND 3, AND NOW THIS NETWORK IS FULLY CONTROLLABLE, YOU CAN SHOW THIS USING CLASSIC EQUATION OF CONTROL THEORY CALLED COLEMAN'S RANK CONDITION. IF THIS CONTROL MATRIX IS A FULL RANK, THEN YOU CAN SAY THAT THIS NETWORK IS FULLY CONTROLLABLE. NOW, UNFORTUNATELY FOR NETWORKS THAT ARE MUCH BIGGER THAN THIS, THIS IS A VERY DIFFICULT FORMULATION TO WORK WITH BECAUSE THIS MATRIX, THE NUMBER OF COMBINATIONS YOU HAVE TO SOLVE WILL INCREASE EXPONENTIALLY. EVEN FOR A NETWORK OF 100 NEURONS, THIS IS NOT GOING TO ALLOW YOU TO DETERMINE CONTROLLABILITY WITH NORMAL AMOUNTS OF COMPUTER POWER. HOWEVER, WHAT FARABOSI AND COLLEAGUES SHOWED, BECAUSE THIS CONTROLLABILITY IS DETERMINED GEOMETRICICALLY YOU CAN USE MAXIMUM MATCHING, YOU DETERMINE MAXIMUM NUMBER OF LINKS THAT HAVE UNIQUE STARTING AND ENDPOINTS AND THEN IF YOU DO THAT, ALL OF THE MATCH NODES WITH DIRECTED PATHS FROM AN INPUT ARE STRUCTURAL CONTROLLABLE. AND THIS IS MUCH MORE SOLVABLE WITH NORMAL COMPUTATIONAL POWER. SO YOU CAN USE THIS FOR LARGER NETWORKS, FOR EXAMPLE IN THIS CASE YOU CAN ALSO NOT ONLY SHOW HOW MANY INPUTS YOU NEED TO CONTROL, YOU CAN ALSO FIND SUBNETWORKS THAT COULD BE CONTROLLED BY PARTICULAR INPUT. SO THE IDEA IS COULD WE USE THIS TO LOOK -- SO ONE OF THE PROBLEMS OF COURSE IS THAT THERE'S NO EMPIRICAL EVIDENCE THAT BIOLOGICAL NETWORKS ARE DESIGNED UNDER CONTROL PRINCIPLES. IT'S DIFFICULT TO TEST THIS, RIGHT? BECAUSE MOST BIOLOGICAL NETWORKS ARE NOT MAPPED TO DEGREE OF COMPLETENESS THAT WOULD ALLOW EXPERIMENTAL TEST. IN C. ELEGANS WE HAVE A COMPLETE CONNECTOME, SO IN COLLABORATION WE DECIDED TO SEE WHETHER WE COULD SEE WHETHER CONTROLLABILITY HAD ANYTHING TO DO WITH C. ELEGANS. WE KNOW THE NEURONS IN THE C. ELEGANS NETWORK AND CAN ABLATE THEM BY USING A LASER TO KILL ANY PARTICULAR NEURON OR COMPUTATION NEURONS WE WANT. SO IF WE PREDICT THE CONTROLLABILITY OF THE INTACT NETWORK AND ALSO CONTROLLABILITY OF NETWORKS CONTAINING PARTICULAR ABLATIONS, WE COULD THEN ASK WHETHER ABLATIONS OF NEURONS THAT LEAD TO REDUCTION IN MUSCLE CONTROLLABILITY WHEN THEY ARE MOVED FROM THE NETWORK, SEE WHETHER THAT AFFECTS BEHAVIOR. TO DO THIS WE STARTED WITH OUR FAVORITE INPUT, WHICH IS THESE TOUCH NEURONS, WHICH AS YOU KNOW ACTIVATION OF THIS NEURON IS SUFFICIENT TO PROMOTE LOCOMOTOR ACTIVITY. AND THEN WE ASKED WHAT FIRST OF ALL IS THE CONTROLLABILITY OF THE BODY MUSCLES IN RESPONSE TO SIGNAL FROM PLM, AND WE FOUND THAT -- OR THE GROUP FOUND THAT NOT ALL OF THE MUSCLES ARE INDEPENDENTLY CONTROLLABLE BUT A SURPRISINGLY LARGE NUMBER WITH STRUCTURAL CONTROLLABLE. AND THEN THEY ASK WHICH NEURON CLASSES IF YOU REMOVE THEM FROM THE NETWORK WOULD REDUCE THIS CONTROLLABILITY OF THE BODY MUSCLES, AND SO THIS WAS DONE FIRST BY JUST LOOKING AT NEUROCLASSES, SOME CLASSES CONTAIN QUITE A FEW NEURONS. AND SO MANY OF THE NEURONS THAT WERE FOUND TO BE REQUIRED FOR CONTROLLABILITY WERE NOT A SURPRISE. SO, OH, I SHOULD SAY -- LET ME ANIMATE THIS FIRST. THESE ARE -- THESE NEURONS HERE ARE THE VENTRAL CORD, MOTOR NEURONS WE KNOW ARE IMPORTANT FOR PROMOTING PROMOTE LOCOMOTION, REDUCING THE WORM'S ABILITY TO MOVE. ALSO THESE ARE THESE PRE-MOTOR INTERNEURONS, THE DIRECT -- THEY ARE THE NEURONS THAT THESE SENSORY NEURONS SYNAPSE ONTO AND WE KNOW AGAIN THESE ARE IMPORTANT FOR TRIGGERING FORWARD OR BACKWARD LOCOMOTION. THESE VENTRAL CORD MOTOR NEURONS, THERE'S TEN OF EACH, A LOT OF THEM. THESE NEURONS ARE THE MOST HIGHLY CONNECTED NEURONS IN THE NETWORK. THESE WILL DISRUPT A LOT OF LINKS IF WE GET RID OF THEM. PDB IS ONLY ONE NEURON, NOT PARTICULARLY WELL CONNECTED. THE FACT THIS WOULD CONTROL CONNECT ABILITY BECAME A SURPRISE. IF WE LOOK AT A STARTING POINT ANY OF THESE INPUTS THAT EVOKE ESCAPE REFLEX, WE GET THE SAME SET OF PREDICTIONS. WHAT THESE PREDICTIONS ARE, ABOUT THE MOTOR CIRCUIT, NOT SENSORY INPUT PER SE. OKAY. SO WHAT ABOUT PDB? IF WE'RE PREDICTING IT SHOULD AFFECT BEHAVIOR, DOES IT? WELL, THIS IS SHOWING THE SPECIFIC PREDICTIONS WHICH IS PDB SHOULD AFFECT CONTROLLABILITY MUCH A SMALL SET OF VENTRAL MUSCLES NEAR THE WORM'S TAIL. SO THAT'S NOT A LARGE NUMBER OF MUSCLES. IF WE LOST CONTROLLABILITY OF THOSE MUSCLES, IT'S NOT GOING TO PARALYZE THE WORM OR ANYTHING LIKE THAT. SO THIS IS A PRIME EXAMPLE OF SOMETHING WHERE USING THESE SORT OF EIGENWORM BEHAVIOR ACKNOWLEDGE SIS WOULD COME INTO PLAY. WE FOUND WHEN WE LOOKED AT PDB-ABLATED ANIMALS WE FOUND SUBTLE BUT SIGNIFICANT ABNORMALITY IN EIGENWORM 1 FEATURES, BENDS THAT CORRELATE WITH OMEGA BIG TURNS IN THE WORM. IN PARTICULAR, WHAT WE SAW WAS THAT THESE -- WHEN THE EIGENWORM 1 ADOPTED NEGATIVE VALUES IT WAS MORE EXTREME IN ABLATED ANIMALS SUGGESTING SOMETHING, JUST AN EXAMPLE, THIS IS A WILD TYPE OR MOCK ABLATED, THIS IS ABLATED ANIMAL, WELL, ONE THING THAT OCCURRED TO US IS THAT WHEN NORMAL WORMS DO AN OMEGA TURN THEY ALMOST ALWAYS DO IT ON THE VENTRAL SIDE. IN FACT THEY DO IT 85% OF THE TIME ON THE VENTRAL SIDE. A NEGATIVE VALUE OF EIGENWORM 1 MEANS A BIG TURN ON THE DORSAL SIDE SO WE WONDERED WHETHER THE ABLATION MIGHT AFFECT THE POLARITY. WE WENT BACK TO USING OMEGA TURN DETECTOR ALGORITHM, THIS IS WILD TYPE OR MOCK ABLATED, 80%, WE LOSE MOST OF THE VENTRAL BIAS. THIS WAS COOL BECAUSE WE IDENTIFIED A VERY SUBTLE BUT SIGNIFICANT FUNCTION OF THIS ONE NEURON USING THIS PREDICTION. YOU MIGHT THINK IN TERMS OF TEST OF THE THEORY IT'S NOT SO GOOD, RIGHT? BECAUSE WE PREDICTED ONE NEURON AND SAW A PHENOTYPE. IDEALLY YOU'D LIKE SOMETHING MORE FALSIFIABLE THAN THAT. THEY MADE ANOTHER PREDICTION THAT WAS I THOUGHT MUCH MORE COUNTERINTUITIVE AND SOMEWHAT MORE FALSIFIABLE WHICH IS CONCERNING THIS DD CLASS OF VENTRAL CORD MOTOR NEURONS, 60 SCATTERED FROM HEAD TO TAIL, AND THESE ARE THE MAJOR INHIBITOR MOTOR NEURONS THAT SYNAPSE WITH DORSAL BODY MUSCLE. NOW, IT WAS SHOWN BY MARTY MANY YEARS AGO IF YOU ABLATE ALL THE DDs, YOU GET SEVERE ABNORMALITYS IN LOCOMOTION. CONTROL THEORY PREDICTSS IF YOU ABLADE INDIVIDUAL MEMBERS, DD 1, 2 OR 3, THIS SHOULD NOT AFFECT CONTROLLALITY, IF YOU ABLATE 4, 5 OR 6 IT SHOULD AFFECTS CONTROLLABILITY IN THE POSTERIOR HALF OF THE WORM. THIS SEEMED LIKE SOMETHING I DIDN'T REALLY BELIEVE WOULD BE TRUE BUT WE DECIDED TO TEST IT. WE ABLATED SOME OF THESE AND SOME OF THESE TO SEE WHAT HAPPENS. AND THE ANSWER IS THAT THE POSTERIOR DDS DO IN FACT HAVE SPECIFIC EFFECTS ON TAIL LOCOMOTION. THIS IS THE EIGENWORM 4, SMALL MOVEMENT AT HEAD AND SALE. THE EIGENWORM FOR 4 OR 5 ARE CLOSER TO ZERO THAN MOCK ABLATED OR DD 2 OR DD 3 ABLATED, YOU CAN SEE FOR MOCK ABLATED ANTERIOR DD ABLATED THEY FLUCTUATE OVER POSITIVE AND NEGATIVE, AROUND ZERO, MUCH MORE TIGHTER AROUND ZERO THAN THE DD5 ABLATED ANIMALS. IF YOU LOOK AT THE -- COMPARE LOOKS LIKE WILD TYPE. YOU CAN SEE DD 5 IS DRAGGING ITS TAIL. YEAH, SO AGAIN THIS EXACTLY MATCHES TO PREDICTIONS OF THE THEORY AND IT IMPLIES THERE'S SPECIFIC FUNCTION OF POSTERIOR DDs IN CONTROLLING MOVEMENTS OF THE TAIL. YOU MIGHT THINK ONE QUESTION YOU MIGHT ASK, DO YOU NEED TO HAVE AN EXACTLY ACCURATE CONNECTOME FOR THIS TO WORK? AND ACTUALLY C. ELEGANS CONNECTOME IS PROBABLY NOT 100% ACCURATE, BASICALLY ONLY DONE WITH TWO WORMS. SO IF THERE'S ANY VARIABILITY THEN PROBABLY SOME OF THE CONNECTIONS WE FOUND ARE PROBABLY GOING TO BE VARIABLE. AND SO IF YOU ACTUALLY TRY EITHER RANDOMLY DELETING WEAK LINKS, ADD, REWIRE, MOST PREDICTIONS ARE ROBUST UP TO REMOVAL OR REWIRING OF QUITE A SIGNIFICANT NUMBER OF LINKS. IT SEEMS LIKE THAT ACTUALLY THIS MIGHT ACTUALLY BE FAIRLY USEFUL, EVEN IN CONNECTOMES YOU DON'T KNOW ARE 100% ACCURATE. SO OBVIOUSLY THIS IS KIND OF COOL BECAUSE MAYBE THIS COULD ALLOW US TO INVESTIGATE OTHER CIRCUITS AND OTHER CONNECTOMES. SO I TOLD YOU ABOUT THESE EXPERIMENTS WE WERE PICKING INPUTS THAT GO TO THE PRE-MOTOR INTERNEURONS TO ADDRESS MOTOR CIRCUIT BUT THERE'S OTHER CIRCUITS OF THE WORM THAT GO THROUGH MANY MORE LAYERS OF PROCESSING, THESE ARE NAVIGATION CIRCUITS INVOLVED WITH ACHEMOTAXIS, THESE ARE GO THROUGH THREE OR FOUR LAYERS, SO WE'RE INTERESTED IN SEEING WHAT NEURONS HERE ARE REQUIRED FOR CONTROLLABILITY IN, SAY, THE HEAD MUSCLES DOWN HERE. ALSO OBVIOUSLY MAYBE THIS COULD BE A WAY TO LOOK AT THE CONNECTOMES OF OTHER ANIMALS AS OTHER CONNECTOMES COME ONLINE. JUST TO REMIND YOU WE FOUND THIS PEPTIDE SYSTEM INVOLVED WITH ALLOWSAL AND SENSITIZATION THAT ACTINGS EXTRASYNAPTICALLY WITH A COMPLEX MULTI LAYER CONNECTOME. WE'VE TALKED WITH UNSUPERVISED BEHAVIORAL ANALYSIS TO DETECT GENETIC AND ABLATION PHENOTYPES AND USING CONTROL THEORY AS A WAY TO PROBE THE STRUCTURES OF THESE CONNECTOMES AND HOPEFULLY THESE TOOLS MAY BE USEFUL FOR OTHER PEOPLE LOOKING AT MORE COMPLEX BRAINS. FINALLY I'D LIKE TO THANK THE PEOPLE THAT DID THE WORK. A LOT OF WORK WITH WAS DONE BY THIS POSTDOC, THE AROUSAL STUFF, A LOT OF WORK ON THE CONTROLLABILITY TEST, DENISE HELPED WITH CONTROLLABILITY TEST, AND ANDRE DEVELOPED UNSUPERVISED BEHAVIORAL ANALYSIS. I MENTIONED COLLABORATION WITH HANGLU AND THESE ARE THE THEORISTS INVOLVED WITH THE COLLABORATION ON CONTROL THEORY. THAT'S IT. THANK YOU. [APPLAUSE] YEAH? >> (INAUDIBLE). >> THAT'S RIGHT, YEAH. >> (INAUDIBLE). >> YEAH, THAT'S ACTUALLY A REALLY GOOD POINT. OBVIOUSLY WE WOULD LIKE -- I THINK THE REASON THAT IT WORKED IS THAT I THINK THE DIFFERENT TYPES OF NEURONAL INTERACTIONS ACT ON DIFFERENT TIME SCALES, SO THE TIME SCALE OF MOST OF THOSE THINGS INVOLVING NEUROMODULATORS IS SLOWER THAN THE SYNAPTIC CONNECTIONS, SO THE BEHAVIORS WE LOOKED AT, YOU KNOW -- YEAH, THE TYPES OF LOCOMOTION WE LOOKED AT ABOUT THE WIGGLING THE TAIL AND GOING THE LOCOMOTOR WAVE, THOSE DON'T DEPEND ON NEUROMODULATORS AND YOU CAN KNOCK OUT MAJOR NEUROPEPTIDE PROCESSS ENZYME AND GET PERFECTLY GOOD MOTOR WAVES. WE CAN GET AROUND THAT PROBLEM BUT IT WOULD BE NICE TO LOOK AT THE NEUROMODULATORY NETWORKS AS WELL, AND IF WE KNEW MORE OF THE STRUCTURE WE COULD APPLY THE SAME APPROACH. UNFORTUNATELY FOR THE NEUROPEPTIDE NETWORKS WHICH I THINK ARE THE MORE INTERESTING TOPOLOGICALLY WE JUST DON'T KNOW THE STRUCTURE OF THOSE NETWORKS WELL ENOUGH BUT THAT'S A DIRECTION WE WOULD LIKE TO GO. YEAH? >> (INAUDIBLE). >> WELL, YEAH. I DON'T THINK THERE'S A DIFFERENCE. BASICALLY WHEN YOU DO PCA THOSE ARE THE THINGS IT SPITS OUT. BASICALLY EIGEN 2 AND 3 ARE BASICALLY DIFFERENT PHASES OF THE SAME PROCESS. BY DOING EIGENWORMS, IT'S NOT PERFECT, BECAUSE OF THE WAY THE PCA GENERATES ITS SHAPES, EIGENWORMS 4 IS HEAD AND TAIL, PROBABLY CONTROLLED DIFFERENTLY, SO I THINK SOME OF THOSE ARE ARTIFACTS OF PCA AND IF WE HAD BETTER WAYS TO GENERATE SHAPES WE COULD WORK EIGENWORMS 2 AND 3 AND MAYBE SPLIT 4 AND SEE HOW IT PANS OUT. >> (INAUDIBLE). >> YEAH, THAT'S A VERY GOOD POINT. I MEAN I THINK PART OF THE REASON FOR STARTING WITH EIGENWORMS, IF YOU HAVE TOO MANY FEATURES, STATISTICS GETS DIFFICULT BECAUSE YOU'VE GOT LOTS OF MULTIPLE COMPARISON CORRECTIONS TO DO. THEN IT GETS VERY HARD TO GET SIGNIFICANCE TO ANYTHING. HOWEVER, IF WE WOULD HAVE -- YEAH, ACTUALLY THE OTHER THING IS THAT THE POLARITY OF OMEGA TURNS WAS NOT ACTUALLY ONE OF THE FEATURES WRITTEN INTO THE CODE. SO IT WAS WRITTEN INTO THE CODE TO DETECT OMEGA TURNS, AND IT WAS WRITTEN INTO THE CODE WHETHER THEY ARE DORSAL OR VENTRAL BUT HE DIDN'T HAVE A THING TO COMPILE. I WENT BACK AFTER I SAW THE EIGENWORMS FEATURE AND COMPILED IT MYSELF. I THINK IF YOU WERE -- IF YOU WERE SMART ENOUGH THAT YOU HAD A COMPLETE SET OF FEATURES, THEN THE FEATURES WOULD PROBABLY GIVE YOU MORE ROBUST THINGS. BUT THE DANGER THERE IS THAT MAYBE YOUR LIBRARY OF FEATURES ISN'T COMPLETE ENOUGH SO I THINK MAYBE DOING A COMBINATION OF MOST IS STILL USEFUL. >> (INAUDIBLE). >> WELL, THE PATH WAS ME LOOKING AT THE DATA, RIGHT? BECAUSE, YEAH, LOOKING AT IT, YOU KNOW, YEAH, BASICALLY THE THOUGHT PROCESS WAS WHAT I DESCRIBED IN THE SLIDE. I COULD SEE THE NEGATIVE VALUES OF EIGENWORMS 1 WAS MORE PRONOUNCED IN MUTANTS, NEGATIVE WAS A DORSAL BEND, SO IT HAD MORE EXTREME DORSAL BEND, WILD TYPE SHOULDN'T MAKE DORSAL OMEGA TURNS, A CERTAIN AMOUNT COMES FROM KNOWING THE BEHAVIOR. IN TERMS OF HAVING AUTOMATED PIPELINE TO DO THAT, I DON'T KNOW. I GUESS, YEAH, IDEALLY THAT WOULD BE GREAT BUT I'M NOT AGAIN SURE THAT WE'RE THERE YET. >> (INAUDIBLE). >> IN MAMMALS AROUSAL USED IN THREE SUBTLY DIFFERENT WAYS, DIFFERENCE BETWEEN SLEEP STATES AND WAKE STATES WHERE WE'RE AROUSED IN THE WAKE STATE AND THEN THERE'S THE MEANING OF AROUSAL WHERE WITHIN THE WAKE STATE YOU CAN BE MORE OR LESS AROUSED, AND THEN THAT'S PASSED INTO TWO TYPES OF AROUSAL, GENERALIZED WHERE YOU'RE MORE SENSITIVE TO ALL THE DIFFERENT SENSORY THINGS AND SPECIFIC OR LOCALIZED AROUSAL WHERE YOU MAY BE MORE SELECTIVELY ATTENDING TO CERTAIN THINGS. BECAUSE WORMS SEEM TO HAVE NO CIRCADIAN CONTROL OF SLEEP-WAKE STATES IS IT POSSIBLE WHAT YOU'RE REALLY LOOKING AT IS GENERALIZED AROUSAL AFFECT THE SAME AS WAKE STATE AND DO YOU HAVE EVIDENCE FOR SELECTIVE AROUSAL? >> GOOD QUESTION. I DON'T KNOW THE COMPLETE ANSWER TO IT. SO FAR THE -- WE HAVEN'T -- ONE THING I WOULD LIKE TO TEST IN TERMS OF SENSORY, INCREASE IN SENSORY ACUITY TO TEST SENSORY CUES THAT ARE ATTRACTIVE AS OPPOSED TO ADVERSIVE. WE HAVEN'T TESTED. THAT'S PARTLY BECAUSE THE TIME SCALE TO BEHAVIORALLY LOOK AT HEMOTAXIS IS LONGER THAN DURATION OF AROUSAL. WE COULD LOOK AT IT THROUGH CALCIUM IMAGING OF THE SENSORY NEURONS, WE HAVEN'T DONE THAT YET. IN TERMS OF WHETHER SLEEP IN WORMS IS JUST ANTI--- JUST ANTI-AROUSAL, I THINK PROBABLY WE DON'T KNOW ENOUGH ABOUT THAT TO KNOW. THERE WAS ALSO DIFFERENT TYPES OF BEHAVIOR CALLED SLEEP-LIKE BEHAVIOR IN C. ELEGANS THAT IT'S NOT CLEAR THEY ARE ALL THE SAME. SO THERE'S THIS ADULT-TYPE SLEEP WHERE WORMS GO INTO QUIESCENCE AS ADULTS BUT CERTAIN DIAPODS WILL SHOW CERTAIN SLEEP-LIKE BEHAVIOR, SOME MAY BE DIFFERENT, OBVIOUSLY PEOPLE WANT TO CONNECT ALL THESE TO SLEEP BECAUSE SLEEP IS SORT OF COOL. BUT I'M NOT SURE THEY AR ALL KNOWN TO BE THE SAME, YEAH. >> HI. >> CAN I JUST THROW IN ONE LITTLE RANDOM THING, WHICH IS THAT IT TURNS OUT INTEREST -- THERE IS IT ANOTHER WEIRD LINK. SOME RECEPTORS, NPR 22, A WEAK LIGAND FOR FLP-20 IS ONE OF THE THINGS IDENTIFIED AS AFFECTING DIAPODS, THERE MAY BE OTHER CONNECTIONS LIKE, YOU KNOW, CERTAIN PEPTIDES INVOLVED WITH CERTAIN TYPES OF AROUSAL, WEAKLY INTERACTING WITH THINGS THAT AFFECT SLEEP, THEY MIGHT BE DIFFERENT PROCESSES, SO, YEAH, IT COULD BE KIND OF COMPLICATED. YEAH? >> I WAS LOOKING FOR HELP CONCEPTUALIZING MULTI-LAYER NETWORK YOU WERE TALKING ABOUT EARLIER. IN TERMS OF NETWORKING I'M USED TO NOT THE SAME NODE, IN MOVIES YOU HAVE ACTORS, MOVIES, AND GENRES. THESE WOULD BE THREE LAYERS OF A NETWORK THAT WOULD BE CONNECTED BUT THERE'S IN-BETWEEN AS WELL. HERE YOU HAVE THE SAME NODES BUT YOU HAVE DIFFERENT CONNECTION TYPES BETWEEN THEM AND THEN SOME OF THEM SEEM TO BE BROADCASTING VERSUS OTHER, NORMAL SYNAPTIC QUOTE/UNQUOTE. IS THERE ANY WORK MESHING OR BRINGING THESE TOGETHER OR STILL POINTING OUT THERE ARE DIFFERENT LAYERS AND WE SHOULD BE TAKING THEM INTO CONSIDERATION? >> YEAH, SO I'M SORT OF NEW TO NETWORK SCIENCE MYSELF BUT I THINK THERE'S TWO DIFFERENT THINGS YOU'RE TALKING ABOUT. THE THING ABOUT MOVIES AND ACTORS AND SO FORTH, THOSE ARE WHAT PEOPLE OFTEN THINK OF AS LIKE TRIPART TYPE NETWORKS, DIFFERENT CATEGORIES CONNECTED, YOU CAN PROJECT INTO AN ACTOR, ACTOR NETWORK AND MOVIE NETWORK OF LIKE ACTORS THAT SHARE MOVIES AND MOVIES THAT SHARE ACTORS. IN THIS CASE THIS IS WHAT OFTEN PEOPLE REFER TO AS MULTIPLEX NETWORKS WHERE THE NODES ARE THE SAME BUT THE LINKS ARE DIFFERENT SO THERE IS -- SO PEOPLE SOMETIMES THINK ABOUT THIS IN COMMUNICATION NETWORKS WHERE LIKE SAY THERE'S PEOPLE YOU COMMUNICATE BY E-MAIL, PEOPLE YOU TALK TO ON THE PHONE. I THINK THERE'S A FAIR AMOUNT OF INTEREST THESE DAYS IN STUDYING THESE MULTIPLEX NETWORKS OF THAT SORT. ONE OF THE THINGS THAT I THINK IS KIND OF NICE ABOUT THIS NETWORK FROM THE PERSPECTIVE OF NETWORK SCIENTIST, A LOT OF THE MULTIPLEX NETWORKS THAT PEOPLE THINK ABOUT LIKE THE ONE I WAS JUST TALKING ABOUT ARE NOT REALLY AS MULTIPLEX AS YOU THINK, THE PEOPLE THAT YOU SEND E-MAILS TOO ARE OFTEN THE SAME PEOPLE THAT YOU COMMUNICATE WITH BY PHONE AND SO THEY ARE NOT REALLY TWO NETWORKS, JUST DIFFERENT MANIFESTATIONS OF THE SAME NETWORK. IN THIS CASE, AT LEAST IN THE CASE OF SYNAPTIC VERSUS NEUROMODULATOR THEY ARE DIFFERENT NETWORKS, VERY DIFFERENT TYPOLOGY, SO I THINK, YEAH, IN TERMS OF TRYING TO JUST LOOK REAL WORLD EXAMPLES OF MULTIPLEX NETWORKS THIS IS ONE OF THE BETTER EXAMPLES I'M AWARE OF. INTERESTINGLY, THE GAP JUNCTION IN SYNAPTIC NETWORK OVERLAP A LOT. SO I DIDN'T ACTUALLY SHOW THE SLIDE BUT IF YOU ACTUALLY LOOK AT THE LEVELS OF OVERLAP BETWEEN DIFFERENT LAYERS GAP JUNCTION AND SYNAPTIC NETWORK MAP REALLY CLOSE TOGETHER. AND CLEARLY THEY ARE VERY DIFFERENT STRUCTURES BUT MAYBE THE WAY THE NETWORK IS WIRED UP GAP JUNCTIONS ARE FORMED BETWEEN NEURONS THAT TEND TO MAKE SYNAPSES AND THEY ARE COMMUNICATING WITH THE SAME GROUPS OF NEURONS. YEAH. DID THAT ANSWER YOUR QUESTION. >> I GUESS WHAT'S THE NEXT STEP? WE KNOW THERE'S A MULTIPLEX NETWORK. IS THERE A WAY TO CONSIDER ALL AT ONCE? >> THERE ARE THINGS YOU CAN DO. ONE OF THE THINGS YOU CAN DO WITH A MULTIPLEX NETWORK, AGAIN I DIDN'T HAVE TIME TO GO INTO THIS, ONE THING YOU CAN DO IS LOOK AT MOTIFS, FOR EXAMPLE. IF YOU HAVE A MULTIPLEX NETWORKS LINKS CAN BE DESCRIBED NOT JUST IN TERMS OF LINKS BUT LAYER, YOU CAN HAVE A MULTI LINK, WHAT TYPES OF LINKS THERE ARE IN PARTICULAR BETWEEN TWO NEURONS AND SO, YOU KNOW, IF THERE'S LIKE, YEAH, IF THERE'S A RECIPROCAL SYNAPSE AND ALSO AN EXTRASYNAPTIC INTERACTION. WE HAVE DONE SOME OF THAT WITH THE MONOAMINE AND SYNAPTIC NETWORK AND ALSO WITH NEUROPEPTIDES AND LOOKED FOR MULTI-LINKS OVER OR UNDERREPRESENTED IN THE NETWORK AND THAT GIVES YOU SOME IDEA AS TO, YOU KNOW, WHAT TYPES OF COMBINATIONS OF SIGNALING EVENTS MIGHT HAVE FUNCTIONAL IMPORTANCE, THINGS OVERREPRESENTED MIGHT BE THINGS DOING SOMETHING. THERE'S ALSO WAYS YOU CAN DO MULTIPLEX ANALYSIS OF THE CENTRALITY OF NODES. YOU FIND THERE ARE DIFFERENT NODES THAT ARE IMPORTANT FOR COMMUNICATING IN THE SYNAPTIC NETWORK AND EXTRASYNAPTIC NETWORKS BUT SOME NODES ARE IMPORTANT IN BOTH LAYERS, CAN YOU STRESS THAT AND THAT CAN GIVE YOU INSIGHT INTO HOW DEFICIENCY RUNT COMMUNICATIONS IN THE SYSTEM ARE GENERATED AND SO FORTH, YEAH. [APPLAUSE]