>> GOOD AFTERNOON, EVERYBODY. IT'S MY GREAT PLEASURE TO INTRODUCE MY OLD FRIEND RALF SCHNEGGENBURGER. RALF SCHNEGGENBURGER HAS A DEGREE IN 1994 -- OF BIOPHYSICAL CHEMISTRY. IN 1994-96 HE DID POST DOCTOR AT TRAINING IN PARIS, FRANCE. IN 1996 TO 2000 HE DID POST DOC -- 2001 TO FIVE HE IS GROUP LEADER IN THE SAME INSTITUTE. IN 2005 HE WAS PROMOTED TO AN ASSOCIATE PROFESSOR IN EPSL THE SWISS INSTITUTE FOR TECHNOLOGY. LAST YEAR HE WAS PROMOTED TO FULL PROFESSOR. PERSONALLY -- I'LL GIVE YOU SOME EXAMPLES THAT HE HAS MADE A LOT OF IMPORTANT FINDINGS IN HIS CAREER. FOR EXAMPLE, IN 2000 HE -- HE FIGURED OUT THE TRIGGERING MECHANISM AND CALCIUM TO RELEASE. HE DESCRIBED A MODEL WITH FIVE CALCIUM RELEASE WHICH IS NOW USED AS A STANDARD NOW FOR THE SYNAPSE. RECENTLY HE FOUND A FEW MOLECULES INVOLVED IN CONTROLLING THE CALCIUM CHANNEL NUMBERS IN THE SYNAPSE SIDE WHICH IS ALSO VERY CRITICAL FOR DETERMINING THE SYNAPTIC STRENGTH. I HAVE BEEN INFLUENCED VERY MUCH EVERY TIME I WRITE A PAPER I CHECK THE PAPER WHAT RALF SCHNEGGENBURGER IS DOING. IF YOU CAN SEE HIS NAME AS 15 CHARACTERS I CAN MEMORIZE ALL THE CHARACTERS. THE LONGEST NAME I CAN REMEMBER IN MY LIFE. >> THANK YOU VERY MUCH. FOR THE VERY KIND INTRODUCTION. CAN YOU HEAR ME ALL RIGHT? IS MY LOCAL MICROPHONE ON I'D LIKE TO THANK HIM FOR INVITE KNOWLEDGE ME TO GIVE THIS NICE NEUROSCIENCE CENTER TALK, IT'S A GREAT HONOR. A LOT OF OUR RESEARCH INTERESTS HAVE BEEN IN UNDERSTANDING THE NEUROTRANSMITTER RELEASE SYNAPSIS. ALSO TO GET MORE INTERESTED IN THE MOLECULAR FACTORS AND FACTORS IN THE DEVELOPMENT OF THE WRITTEN THAT WOULD SHAPE THE CONNECTIVITY BUT I WILL NOT SPEAK ABOUT THIS ASPECT OF MY RESEARCH TODAY. AS YOU ALL KNOW NEURONS HAVE ESSENTIALLY THREE VERY IMPORTANT COMPARTMENTS. THE AXON, DENDRITE. BUT THE ACTION POTENTIAL HIT THE NERVE TERMINAL AND THERE AN ACTION POTENTIAL HAS TO BE TRANSMITTED VIA TRANSMITTER RELEASE IN TO A POST SYNAPTIC POTENTIAL CHANGE IN THE POST SYNAPTIC MEMBRANE. FOR THIS NEURON TO EVOLVE WHAT I CALL THE FOURTH COMPARTMENT OF THE NEURON WHICH IS REALLY THE NERVE TERMINAL. SO IN THE NERVE TERMINAL YOU HAVE INCREDIBLY COMPLEX REGULATED TRANSMITTER RELEASE. YOU HAVE A CYCLE OF RECYCLING VESICLES THAT HAVE TO BE FILLED WITH TRANSMITTERS. SO THIS APPARATUS IN THE NERVE TERMINAL REQUIRES A VERY SPECIALIZED AND HIGHLY ADAPTIVE MOLECULAR MACHINERY THAT CONSISTS OF PROTEINS TO THE APPARATUS. TO ACTUALLY CALCIUM SENSOR AND TO -- JUST MAYBE TO NAME THE MOST IMPORTANT ONES. YOU ALL KNOW THAT THIS YEAR'S NOBEL PRIZE IN PHYSIOLOGY WAS AWARDED TO RANDY AND TOM FOR THEIR DISCOVERY AND DESCRIPTION OF THE MOLECULAR MACHINERY THAT ESSENTIAL LOW DRIVES VESICLE FUSION AND MEMBRANE TRAFFICKING INSIDE CELLS. SO I WILL SORT OF NOT SO MUCH GO IN TO MOLECULAR DETAILS. SYNAPSIS ACTUALLY HAVE HIGH DEGREE OF DIVERSITY AMONGST THEMSELVES, SOME ARE DEPRESSING, THERE ARE OTHER THAT ARE FACILITATING IT IS ONE OF OUR INTERESTS IN AN INTERMEDIATE RANGE TO ALSO FIND OUT WHAT DETERMINES WHETHER SINUOUS IS DEPRESSING OR FACILITATING. ANOTHER PROPERTY OF THE SYNAPSE IS THAT USUALLY THERE IS A VESICLE AND THE FUSION PROBABILITY AMONGST THOSE VESICLES IS ACTUALLY SURPRISINGLY LOW. ONLY ONE OUT OF TEN VESICLES FUSES WITH A MEMBRANE AS RESEARCH LAST ABOUT TEN YEARS HAS SHOWN. BUT RELEASES ACTUALLY TIME LOSS TO THE ACTION POTENTIAL THERE'S A VERY SHARP BASIC COMPONENT OF RELEASE AND THEN THERE IS A MORE ASYNCHRONOUS RELEASE OUR INTEREST RIGHT NOW IS ALSO IN FINDING OUT WHAT ARE BOTH THE INTRINSIC MECHANISMS, SO IT IS CLEAR THAT AT THE MEMBRANE PROBABLY FAST SENSOR FOR VESICLE WHICH WORKS FROM TOM'S LAB MAINLY HAS SHOWN MOST PROBABLY REPRESENTATIVE BY SYNAPTIC ISOFORMS ONE AND TWO I'LL TALK ABOUT THIS TODAY. THERE IS MAYBE A SLOW SENSOR THAT IS MOLECULARLY NOT SO WELL DEFINED. IN THE FIRST PART OF THE TALK I'D LIKE TO GO A LITTLE BIT IN TO THESE INTRINSIC MECHANISMS OF CALCIUM SENSING AND WHAT DISTINGUISHES FAST VERSUS SLOW FORMS OF RELEASE. IN THE SECOND AND SHORTER PART OF MY TALK I'D LIKE TO BRING THE QUESTION HOW CALCIUM CHANNELS MUST BE COUPLED TO THESE VESICLES BECAUSE SPECIAL COUPLING OF VESICLES AND CALCIUM CHANNELS ALSO VERY IMPORTANT TO DETERMINE VERSUS ASYNCHRONOUS. SO WE'VE BEEN DOING OUR STUDIES AT A VERY SPECIALIZED TIME. THE MECHANICS IS A GIANT SINUS THAT IS FOUND IN THE CENTRAL NERVOUS SYSTEM OF MAMMALS THIS ACTUALLY PART OF THE AUDITORY BRAINSTEM AND JUST -- AXON NOT SO BIG BUT THAT IT CONNECTS TO THE POST SYNAPTIC CELL WITH A VERY BIG NERVE TERMINAL, SORT OF THE BUY NEARS OF RESEARCH, ALSO IAN IN ENGLAND AND OTHERS. I LEARNED MY FIRST PREPARATION FROM LIN WHEN HE WAS POST DOC INHALEDBERG HE WORKED, ALSO STARTED WORKING AT THE GIANT SYNAPSE. CAN BE PUT EASILY ON THE NERVE TERMINAL AND GAIN DIRECT -- MOST IMPORTANTLY WE CAN VOLTAGE PLAN THE NERVE TERMINAL, WE CAN MEASURE CURRENT BUT WE CAN RELATE CALCIUM IN FLUX TO RELEASE AND WE CAN OPTICALLY CONTROL THE CALCIUM IN THE NERVE TERMINAL BY MEASURING OR IMAGING CALCIUM OR BY IMPOSING CALCIUM WHICH WE HAVE PIONEERED IN THE LAB. NOW MECHANICS HAVE BEEN BEEN A LITTLE BIT DIFFICULT BUT REASONS WORK FROM OUR LAB AND OTHER LABS HAVE SHOWN THAT ALSO QUITE NICE TO APPLY CONDITIONAL OR CONVENTIONAL MOUSE KNOCK OUT APPROACHES COUPLED WITH VIRUS VECTOR TECHNIQUES TO MANIPULATE OR CONTROL PRESYNAPTIC PROTEINS. IF YOU COMBINE THESE MOLECULAR TECHNIQUES WITH THE BIOPHYSICAL TECHNIQUES YOU HAVE ADVANTAGE THAT YOU CAN STUDY -- THAT YOU CAN PERTURB THE SINUS RELATIVELY DIRECTLY LOOK AT THE CORRELATE AND MECHANISMS THAT UNDERLIE YOUR MOLECULAR PERTURBATION. AND SO I WOULD FIRST LIKE TO INTRODUCE THE IS IN NAP PARTICULARS A LITTLE BIT BETTER. THEY ARE ACTUALLY PRESENT IS IN NAP TOE TAG MEAN THIS IS VERY VERY IMPORTANT FOR STEP AS I WILL ARGUE IN A MINUTE. THERE ARE ACTUALLY FIVE RESIDUES THAT COMPLEX THESE TWO CALCIUM IONS AT THE TIP OF THE Ca2+ WHICH IS SHOWN IN THE LAB OF TOM AND BY OTHERS. MECHANICS HAVE HELD WE AND OTHERS HAVE FOUND EVIDENCE A FEW YEARS AGO, ABOUT TEN YEARS AGO THAT TAGMINA IN BOTH CASES WE USED THE MONO ANTIBODY THIS CLEARLY SHOWS THAT SYNAPOTO TAGMIN 1 AND 2 IS LOCALIZED AT OTHER SYNAPSIS ON THE NEURONS. TURNS OUT THAT THESE OTHER SINUSES ARE INHIBITORY AND EXCITATORY. THE KNOCK-OUT MOUSE WAS MADE BY ROBERTO THEN LATER USED BY TOM. THIS WAS ACTUALLY A KNOCK IN MICE I THINK LIN WHO USED THIS MODEL IN HIS RESEARCH. THIS EXPRESSION NICELY DEMONSTRATES A LITTLE BIT ALSO THE EXPRESSION PATTERN OF CYP 1 VERSUS CYP 2. ZIT AS IS NOW TRANSPIRING WHERE AS THE AREAS ARE LARGELY THOUGHT TO USE SYNAPOTO TAGMIN 1 AS THEIR CALCIUM TRANSFER THEY ONLY START DYING ABOUT THREE WEEKS OF AGE. BUT FORTUNATELY WE CAN USE THESE MICE AND WE CAN ALSO THEN SINCE THEY SURVIVE WE CAN MEASURE THEIR KNOCK OFF PHENOTYPE BUT ALSO REEXPRESS SYNAPOTOPROTEIN TO WITH SYT 2 TOGETHER WITH ALEXI AND LIKE JUST TO SUMMARIZE A FEW RESULTS OF THIS STUDY. SO THIS SLIDE JUST SHOWS THAT WE CAN USE A VIRUS METED EVACUATED TECHNIQUES THAT WE HAVE ACTUALLY SORT OF INHERITED FROM SAM YOUNG FROM THE INSTITUTE IN FLORIDA. THE SECOND GENERATION, TURNS OUT TO BE RELATIVELY NICE EXPRESSION VECTOR IF WE TRANSECT NEURONS IN THE NUCLEUS WHERE THE MECHANICS ARE, A FEW DAYS LATER YOU CAN SEE THESE AXONS THAT CROSS THE MID LINE THAT MAKE THE INNERVATION WITH THE CONTRA LATERAL SIDE OF THE BRAIN. YOU CAN SEE THE GAP IS EVERYWHERE IN THE NEURONS, BUT SYNAPOTOTAGMIN IS TRANSPORTED TO THE NERVE TERMINALS. THE TARGETING SEEMS TO BE QUITE NICE. SO YOU SEE THEY ARE YELLOW HERE WHERE AS THEY ALSO CONTAIN GFP BECAUSE WE'RE USING DIFFUSIBLE GFP. WE'VE SHOWN WITH THIS EXPRESSION METHOD WE CAN -- NOW WE ARE MEASURING VESICLE FUSION WITH THE TECHNIQUE OF CALCIUM UNCAGING THAT I HAVE ALREADY ALLUDED TO EARLIER. CALCIUM UNCAGING IS NOT JUST IN A SENSE A GYM MIC, WE LIKE IT A LOT BECAUSE WHAT IT ACTUALLY DOES IS BY INTRODUCING PHOTO LIESABLE. CHELATOR IN TO THE PRESYNAPTIC TERMINALS THEN PHOTOIZING. WE GET HOMOGENEOUS. WE CAN PRESSURE THIS CALCIUM JUMP BUT THE CAST YUM JUMP THAT WE ARE MEASURING IS IMMEDIATELY RELEVANT FOR THE VESICLE FUSION THAT HAPPENS IN THE ACTIVE ZONE. WE'RE PURPOSESFULLY AVOIDING ANY GRADIENT OF CALCIUM, ANY MICRO DOMAINS THAT HAVE EXTREME GRADIENT. WE LEAVE CALCIUM CHANNELS CLOSED, FLASH THE CALCIUM AND WE CAN READ OUT THE CAST YUM THAT IS IMMEDIATELY RELEVANT FOR VESICLE FUSION. IF WE DO THIS IN A KNOCK OUT ANIMAL YOU CAN SEE THAT THERE'S NO RELEASE RESPONSE. THERE IS VERY LITTLE RELEASE RESPONSE. BUT INTERESTINGLY IN THE KNOCK OUT IN THE SYT 2 KNOCK OUT THERE IS VESICLE FUSION. IF WE BRING IN AGAIN SYNAPOTOTAGMIN 2 WE HAVE TO PATCH PLAN ON TO EGFP POSITIVE NERVE CELLS WHICH INDICATE SUCCESSFULLY TRANSFUSED NERVE TERMINALS. IF WE DO THE SAME EXPERIMENT IN THE NERVE TERMINAL THAT EXPRESSES SYNAPOTOTAGMIN 2 WILDTYPE CONSTRUCT YOU CAN SEE THAT WE CAN NICELY RECONSTITUTE OR RESCUE VESICLE FUSION AND TO OUR BIG SURPRISE BEE ALSO RESCUED ACTUALLY THIS. THIS INITIALLY WAS A SURPRISE FOR US. THIS SHOWS IMMEDIATELY THAT SYNAPOTOTAGMIN 2 HAS TWO SEPARATE AFFECTS. ON THE ONE HAND IT DRIVES CALCIUM DEPENDENT FUSION TO A VERY STRONG DEGREE, THIS IS ABOUT 500 FOLD DIFFERENCE IN RELEASE RATE. ON THE OTHER HAND IT SUPPRESSES BY FACTOR OF ABOUT TEN THIS SPONTANEOUS RELEASE. AND SO IN THE NEXT EXPERIMENT JUST TO SUMMARIZE THIS A LITTLE BIT WE ASSOCIATED THE CLAMPING AFFECT FROM THE CALCIUM AFFECT. WE MUTATED ONE OF THESE RESIDUES THAT HELPS COMPLEXING THE CALCIUM IONS. WE NEUTRALIZED THIS. ON THE OTHER HAND WE LOOKED AT THE STRETCH IN THE C2B DOMAIN. PREVIOUS WORK HAS ACTUALLY ALREADY SUGGEST R SUGGESTED THAT THIS STRETCH MIGHT BE INVOLVED IN THE CLAMPING FUNCTION OF SYNAPOTOTAGMIN 2. VERY BRIEFLY, WHEN WE DID THESE EXPERIMENTS, JUST CONCENTRATE ON THESE TWO PANELS, HERE WE ACTUALLY RESCUED IN THE KNOCK OUT WITH THE CALCIUM BINDING DIMINISHED MUTATION, YOU CAN SEE THAT IN THIS RESCUE EXPERIMENT WE DO NOT RESCUE ANY OF THE CALCIUM RELEASED BUT WE NICELY OBTAINED CLAMPING. YOU CAN SEE THERE'S NO ENHANCED MINI FREQUENCY. BUT WHEN WE NOW TAKE IN THE CHARGED NEUTRALIZATION AT THE POLYLYSINE SITE. COMPLETELY UNAFFECTED OR NICELY RESCUED. BUT THE CLAMPING FUNCTION IS NOW NOT WORKING. SO CONCLUSION IS THAT THE SITE IN THE C2B DOMAIN ARE MORE IMPORTANT FOR CLAMPING ARE THE POLYLYSINE ON THE SIDE OF THE C2B DOMAIN. WHERE AS THE RESIDUES ON THE TIP OF THE C2B DOMAIN ARE MORE IMPORTANT FOR THE CALCIUM DRIVING FUNCTION. AND THESE DATA IS ALSO SUMMARIZED HERE SO WE MAY DETAIL THOSE RESPONSE CURVES THAT WERE INTRODUCED TO YOU LATER WHEN WE RESCUE ACTUALLY WITH THE WILDTYPE CONSTRUCT WE GET VERY DYNAMIC RELEASE RESPONSE. SO WITH CALCIUM TO TEN OR 20 MICRO MOLAR WE SEE HIGH RELEASE RATE. THE REST DATA POINTS SHOW KNOCK OUT DATA THAT HAVE RESPONSE CURVES CONSISTENT WITH PREVIOUS DATA. BUT WHEN WE OVER EXPRESSED ESSENTIAL LOW OUR CALCIUM BINDING DECISION WHERE THE CLAMPING IS STILL EFFECTIVE AS I SHOWED YOU YOU CAN SEE THAT THE CLAMPING ESSENTIALLY PULLS DOWN THE REMAINING RELEASE IN THE KNOCK OUT BY A FACTOR OF ABOUT TEN. PLEASE NOTE THAT THOSE RESPONSE CURVES IS PLOTTED IN A COORDINATE SO THIS IS ABOUT TENFOLD CHANGE. THIS IS IMMEDIATELY SHOW YOU THE RANGE OF CALCIUM DEPENDENT BINDING AND CLAMPING RAGE. IN THE ABSENCE OF CLAMPING IN THE SYT 2 KNOCK OUT. P WE BRING BACK THE MUTANT WHERE THE CALCIUM BINDING IS FIRST CONCLUSION THEN TO CONCLUDE A LITTLE ONE-THIRD OF MY TALK IS WHILE I JUST SHOWED YOU WITH THOSE RESPONSE DURING ACTION POTENTIAL THE RELEASE RATE INCREASES ABOUT ONE MILLION OR EVEN TEN MILLION FOLD OVER. THIS IS CONSISTENT WITH OUR PREVIOUS DATA THAT WE OBTAINED. NOW THIS LARGE DYNAMIC RANGE OF CALCIUM EVOKE RELEASE IS LARGELY ACTUALLY IMPARTED BY CALCIUM BINDING REACTION IN THE C2B DOMAIN. THIS ACCOUNTS FOR ABOUT A THOUSAND FOLD DYNAMIC RANGE. AND SUPPRESSION AND CLAMPING ACCOUNTS FOR TENFOLD SUPPRESSION OF THE ASYNCHRONOUS. THERE ARE DEFINITELY INTRINSIC MECHANISMS WHICH IS REPRESENTED BY SYNAPOTOTAGMIN 2 AS I JUST SHOWED YOU AND THAT SOMEHOW SUPPRESSES THE ACTION OF A SLOW SENSOR. UNFORTUNATELY WE DON'T KNOW YET THE MOLECULAR IDENTITY OF THE SLOW SENSOR AND I WILL NOT BE ABLE TO TELL YOU THIS TODAY EITHER. THEY ARE INTRINSIC MECHANISMS THAT SUPPRESS SLOW RELEASE IN FAVOR FAST RELEASE. BUT IN THE SECOND PART OF MY TALK I WOULD NOW LIKE TO ADDRESS THE QUESTION WHETHER ALSO MECHANISMS OF CALCIUM BUFFERING AND CALCIUM DIFFUSION MIGHT BE IMPORTANT IN SEPARATING THE FAST FROM THE SLOW RELEASE COMPONENT. FOR THIS IT IS IT IS IMPORTANT TO REALIZE I THINK WHAT MOST OF YOU KNOW THAT THE SIGNAL FOR TRANSMITTER RELEASE IS IMMEDIATE MICRO DOMAIN SIGNAL. WHEN THE VOLTAGE DATA CHANNELS OPEN, CALCIUM THERE'S IN THERE'S VERY SHARP RISE OF CALCIUM DOMAIN CLOSED. I WILL TALK ABOUT THIS MICRO DOMAIN SIGNAL MORE IN THE THIRD PART OF MY TALK. AFTER ABOUT FIVE MILLISECOND WHEN ACTION POTENTIAL IS OVER WHEN CALCIUM CHANNELS HAVE CLOSED. AND SECOND CALCIUM WILL HAVE BOUND TO ENDOGENOUS BUFFER SUBSTANCE WHOSE IDENTITY WE ALSO DON'T KNOW VERY WELL. AND ENDOGENOUS BUFFER USUALLY BINDS 40 CALCIUM IONS ONLY LEAVES ONE FREE CALCIUM ION. THE BUFFER HAS VERY STRONG INFLUENCE ON THE AMOUNT OF RESIDUAL CALCIUM WHICH REMAINS AFTER THE ACTION POTENTIAL. I WILL TRY TO CONVINCE YOU IN THE SECOND PART OF MY TALK THAT THE ENDOGENOUS CALCIUM BUFFERING ACTUALLY CONTRIBUTES STRONGLY TO LIMIT THE AMOUNT OF ASYNCHRONOUS RELEASE IN THE NERVE TERMINAL. AND THE WAY THAT WE FOUND THIS OUT WAS WE WANTED TO SORT OF CLEAN UP THIS COMPLICATED INTER-ACTION BETWEEN CALCIUM SENSORY MECHANISMS AND CALCIUM BUFFERING MECHANISMS. WE THOUGHT IT MIGHT BE INTERESTING TO PROBE THIS INTRICATE BALANCE BETWEEN CALCIUM BUFFERING AND CALCIUM BAYOU'SING AN ALIEN INTRACELLULAR ION WHICH IS A STRONG SEAL. IT'S A VERY SIMILAR ION, IT HAS RADIUS JUST A LITTLE BIT LARGER THAN CALCIUM. BUT IT ACTUALLY ACTIVATES CALCIUM USUALLY WITH THE LOWER AFFINITY THAN CALCIUM. HOWEVER IT WAS KNOWN BEFORE THAT IN THE PRESENCE THERE IS ACTUALLY AN INCREASED ASYNCHRONOUS RELEASE. THIS WAS A BIT COUNTER INTUITIVE AND MOST RESEARCHERS IN THE FIELD BELIEVE THAT IT WOULD ACTIVATE THE SLOW SENSOR WITH THE HIGHER AFFINITY RELATIVELY SPEAKING THAN CALCIUM WOULD THERE BY ESSENTIALLY FAVOR ASYNCHRONOUS RELEASE. WE THOUGHT IT IS NICE TO USE THIS TO PROBE THE FAST AN SLOW SENSOR AND KNOW THE CALCIUM THEN WANT TO KNOW CALCIUM AND BUFFERING CAPACITY IN THE NERVE TERMINAL. SO THAT'S WHAT I PLAN TO TELL YOU IN THE NEXT TEN OR 15 MINUTES. SO THIS EXPERIMENT JUST RECATCHES WHAT HAS BEEN DONE IN THE -- IF YOU REPLACE CALCIUM THAT WE USUALLY HAVE IN THE BACK WITH STRONG SEAL BOTH AT TWO MILLIE MOLAR YOU CAN CHARACTERISTIC CHANGES IN THE DEPRESSION BUT MOST IMPORTANT PART SHOWN HERE. AFTER A HUNDRED HERTZ IN THE WILDTYPE VERY LITTLE ASYNCHRONOUS RELEASE. WE CAN ACTUALLY NICELY SEE A FEW MINI BUT THERE IS NOT MUCH MORE COMING OUT. THERE'S THIS HUGE DYNAMIC RANGE BETWEENPHASIC RELEASE RESPONSE AND THIS LATE ASYNCHRONOUS RELEASE. THIS IS THE QUANTIFICATION YOU SEE IN THE WILDTYPE, ASYNCHRONOUS RELEASES TO TEN HERTZ BUT -- SORRY IN THE PRESENCE OF CALCIUM. BUT THEN IN THE PRESENCE OF -- THE IMMEDIATE RELEASE GOES UP BY FACTOR OF ABOUT FIVE TO TEN. THIS EFFECT PERSISTS IT WILL EVENTUALLY SETTLE BECAUSE RELEASE RATE RELATIVELY SIMILAR BETWEEN CALCIUM. WE WANTED TO FIND OUT ABOUT THE MECHANISMS. THE FIRST THING WE DID THEN, WE BEEN PRE AND POST SYNAPTIC RECORDING. HERE WE FOUND THAT CHANGING CALCIUM MADE THE CALCIUM CURRENT MUCH BIGGER. IT PERMEATES MORE THIS WAS KNOWN BEFORE IT WAS NOT A BIG SURPRISE. BUT PHASIC RELEASE IN RESPONSE TO THESE STIMULI, THEY ARE SMALLER. THIS IS A BIT OF A CONTRADICTION, THERE'S MORE ENTERING BUT LESS FAST RELEASE AS YOU CAN SEE HERE. THIS ACTUALLY THEN STIMULATED US TO DO DIRECT UNCAGING EXPERIMENTS IN ORDER TO MEASURE THE SENSITIVITY OF THE FAST SENSOR HERE. THEN IN ORDER TO DO THIS WE HAD TO GO THROUGH SERIES OF CALIBRATION EXPERIMENTS OF OUR -- WE DO RATIO METRIC. WE DO FLORESCENCE INTENSITY AT DIFFERENT CONCENTRATIONS. WE CAREFULLY USE DIFFERENT BUFFERS LIKE EGTA, CTDA IT TURNS OUT THAT MOST OF THESE BUFFERS HAVE LOWER AFFINITY THAN FOR CALCIUM. FACT THAT I ALREADY ALLUDED ON BEFORE. THE INDICATORS ALSO HAVE TEN TO 20 TO 0 FOLD LOWER AFFINITY FOR -- YOU HAVE TO RECALIBRATE. WE WERE THEN ABLE TO DO UNCAGING EXPERIMENTS. SAME LOGIC, JUST USING NOW -- CHELATOR. WE LOAD THIS VERY SIMILAR TO THE CALCIUM EXPERIMENT WHEN WE DO THEN UNCAGING WE FOUND OUT RIGHT AWAY THAT WE NEED MUCH HIGHER STEPS TO ACTUALLY EVOKE SIMILAR RELEASE RATE FOR TRANSMITTER RELEASE. WHEN WE THEN FOR THESE CALCIUM UNAGING DATA CONSTRUCT WHOLE DOSE. IT WOULD ACTIVATE WHICH UNDERLIES -- WITHIN ABOUT SIX FOLD LOWER AFFINITY. IT WAS VERY INTERESTING TO SEE THAT THE SLOPE OF THESE CURVES IS ESSENTIALLY THE SAME. YOU CAN SHIFT THIS CURVE ON TO THIS CURVE. THIS MEANS THAT THE SAME NUMBER OF CALCIUM IONS THAT BINDS TO TO THE CENTER, IS ACTUALLY ALSO THE SAME NUMBER OF IONS THAN ALSO BINDS TO SYNAPOTOTAGMIN 2 WITH A LOWER AFFINITY. CONCLUSION IS ABOUT SIX FOLD LESS EFFICIENTLY ACTIVATED AND WHEN WE USED THIS DATA WITH -- IT TURNED OUT THAT LOWER AFFINITY WAS BOTH DUE TO A DECREASE RATE OF BINDING AND TO INCREASED OFF RATE OF BINDING. WE NEXT WANTED TO DO THESE EXPERIMENTS WITH CALCIUM SENSOR THAT IS IN THE ABSENCE IN ORDER TO SEE WHERE NOW ACTIVATES THE SLOW SENSOR RELATIVELY SPEAKING BETTER THAN CALCIUM. SO THIS JUST SHOWS YOU A RECORDING IN SYNAPOTOTAGMIN 2 WITH CALCIUM UNCAGING, RELEASE STRONGLY DESYNCHRONIZED RELATIVE TO THE WILDTYPE. RESPONSE OF ABOUT 200 MILLISECONDS. THERE IS TENFOLD IF NOT HUNDRED FOLD SLOWING IN THE KINETICS OF RELEASE WE THEN REPEATED THE EXPERIMENT IN THE KNOCK OUT ANIMAL USING NOW UNCAGING AND TO OUR BIG SURPRISE IT WAS ALSO MUCH LESS EFFICIENT AT THE SLOW SENSOR REMAINING IN THE SYT 2 KNOCK OUT THAN CALCIUM. THIS IS SHOWN HERE. WE AGAIN NEEDED MUCH HIGHER FLASHES TO EVEN ONLY EVOKE A VERY SMALL RELEASE RESPONSE HERE. WHEN WE NOW PLOT THE DATA FOR THE ENTIRE DATA SET YOU CAN SEE THAT THERE IS -- CALCIUM DATA IN BLACK ACTUALLY LEFT TO THE DATA IN RED. THERE IS AGAIN ABOUT FIVE TO SIX FOLD SHIFT IN THOSE RESPONSES. OTHER MESSAGE THAT YOU CAN SEE HERE IS THAT THE SLOW SENSOR THE ABSENCE OF SYNAPOTOTAGMIN TWO HAS MUCH MORE SHALLOW COOPERATE TIFT THAN FAST. THIS FITS WITH LINE IN DOUBLE SCALES. SLOPE WOULD BE ONE. WE DEVELOPED OR EXPANDED ON PREVIOUS MODEL BY SOME DOCTORS SO THERE WOULD BE ONE SENSOR THE SLOW SENSOR THAT BINDS CALCIUM QUITE SLOWLY CAN THEN INTRODUCE VESICLE FUSION AND THERE WOULD BE ANOTHER SENSOR, PREVIOUS SENSOR THAT BINDS CALCIUM FASTER BUT NEEDS TO BIND FIVE CALCIUM. SINCE WE'RE NOW LOOKING AT THE KNOCK OUT DATA WE CAN SIMPLIFY THIS MODEL WE CAN JUST LOOK AT THE SLOW SENSOR IN ISOLATION. THE MODEL WAS THEN FITTED WITH THE SLOW SENSOR PART OF THE MODEL THAT ACTUALLY PROVIDES JUST ONE ION BINDING SITE THAT CAN MORE OR LESS SATISFACTORILY DESCRIBE THIS DATA WHEN WE GET OUT TWO SETS OF PARAMETERS, ONE FOR CALCIUM THE OTHER FOR SR YOU CAN SEE IN THESE EXPERIMENTS THAT SR IS ALSO LESS EFFICIENT AS THE SLOW SENSOR. THERE IS POTENTIALLY THERE IS SOMETHING WRONG WITH OUR OLD EX PLAINTIFF NATION WHY THE SR PRODUCES MORE ASYNCHRONOUS RELEASE BECAUSE SR IS NOT ACTING MORE EFFICIENTLY THAN CALCIUM AT THE SLOW SENSOR FOR RELEASE. WE WENT ON AND CONTINUED THESE EXPERIMENTS BY MEASURING THE SR ACTIVITY ALSO IN THE LOW PART OF THE DOSE RESPONSE CURVE THAT I HAVEN'T SHOWN YOU BEFORE. IN THE WILDTYPE ANIMALS. THIS IS THE WILDTYPE DOSE RESPONSE CURVE FOR CALCIUM AND AS WE AND ALSO THE GROUP HAS SHOWN BEFORE IN THE PART ABOUT TWO MICRO MOLAR THERE IS A STEEP RELATIONSHIP. IT BECOMES MORE AND MORE SHALLOW AND THIS IS DUE TO THE FACT THAT THE SLOW SENSOR THAT JUST BINDS ONE CALCIUM ION THAT IS RESPONSIBLE TO THAT ARE PART OF THE RELEASE. EVEN IN THE WILDTYPE ANIMAL SR IN THE LOW -- IN THE LOW PART OF THE DOSE RESPONSE CURVE IN RED IS LESS EFFICIENT THAN CALCIUM. SO THIS MEANS THAT CONTRARY TO OUR INITIAL IDEA THAT SR SHOULD PREFERENTIALLY ACTIVATE A SLOW SENSOR IF THIS HAD BEEN THE CASE THE TWO DOSE RESPONSE CURVES WOULD HAVE TO CROSS OVER SO THAT SR AT LOW CONCENTRATIONS WOULD ACTIVATE THE SLOW SENSORS WITH STRONGER EFFICIENCY. SINCE THIS WAS NOT THE CASE THE REASON FOR THE DIFFERENT EFFICIENCY OF SR INVOKING SLOW RELEASE MUST BE SUGGESTION THAT VALUES MIGHT RICE TO MUCH HIGHER VALUES THAN CALCIUM. AND WE WENT ON TO TEST THIS SIMPLY BY USING A LOW KD CALCIUM INDICATOR IN THE PRESYNAPTIC PATCH PIPE. WHICH HAS EIGHT MICRO MOLAR. ACCORDING TO OUR CALIBRATION THE KD IS MUCH LOWER THIS IS NICE BECAUSE IN BOTH CASES WERE NOT STRONGLY INFLUENCED CALCIUM ENDOGENOUS CALCIUM. AND WHAT WE COULD SHOW THEN IS THAT IF WE FIRST MEASURE THE CALCIUM THE THE BUILD UP OF RESIDUAL WE SEE RESPONSE TO HUNDRED HERTZ STIMULATION A BUILD UP TO THREE MICRO MOLAR THAN FAST CK AND RESIDUAL CALCIUM SEAL. TO OUR BIG SURPRISE THERE WAS ACTUALLY A HUGE BUILD UP OF THE SR CONCENTRATION. IF YOU JUST SUMMER IMPOSE THESE CURVES ON TO ONE YOU CAN SEE THE CALCIUM BUILD UP IS ABOUT THREE MICRO MOLAR BUT SR GOES UP TO 40. THERE'S A TENFOLD HIGHER INCREASE IN THE FREE SR CONCENTRATION THAT MEANS AFTER CESSATION THERE IS TENFOLD MORE SR BEING LEFT IN THIS LATE PHASE DURING WHICH TRANSMITTER RELEASE ACTUALLY TAKES PLACE. WHAT HAS BEEN THE REASON FOR THIS MUCH BIGGER BUILD UP IN THE NERVE TERMINAL COMPARED TO CALCIUM. I WANT TO ACTUALLY DRIVE THIS POINT HOME A LITTLE BIT FASTER THAN I PLANNED ORIGINALLY. AS I HELPED INITIALLY CALCIUM IS BOUND TO ENDOGENOUS BUFFER WHICH IS -- WHICH WE DON'T KNOW YET SO WELL THE BINDING CAPACITY OF THIS BUFFER IS NORMALLY 40 MEANING THAT AMONGST 40 INCOMING CALCIUM IONS AFTER THE BREAK DOWN THERE IS ONLY ONE CALCIUM ION LEFT OVER. WE NOW SUSPECT THAT THIS BINDING RATIO. INDEED THIS IS WITH WE COULD SHOW IN THESE EXPERIMENTS SO WITH CALCIUM WE TYPICALLY SEE A FREE CALCIUM TRANSIENT OF HALF A MICRO MOLAR. SO THAT IN THESE LINEAR CURVES YOU CAN SEE DATA IN THE CURVES OF INVERT AMPLITUDE VERSUS EXOGENOUS BUFFERING FALLS MUCH LOWER. EXTRAPOLATED BUFFERING CAPACITY OF THE NERVE TERMINAL FOR SR IS NOW MUCH LOWER THAN ONE FOR CALCIUM. SO IN CONCLUSION THEN THIS LOWERING BUFFERING EMITS HIGHER SIGNAL AS COMPARED TO CALCIUM. IN CONCLUSION FOR SECOND PART OF THE TALK I HOPE THAT I CONVINCED YOU THAT SR IS LESS EFFICIENT AT BOTH SENSORS WHICH IS REPRESENT BY SYT 2 BUT ALSO AT THE SLOW SENSOR. THIS WAS A SURPRISE. IT MEANS TAKING THE FIRST PART AND SECOND PART OF THE TALK TOGETHER THAT THE HIGH DYNAMIC RANGE BETWEEN FAST AND SLOW TRANSMITTER RELEASE IS ON THE ONE HAND DETERMINED BY AN INTRINSIC MECHANISMS. FAST ON AND OFF RATE THAT HE IS SPECIALIZED FOR CALCIUM AND I SHOWED YOU THAT THE SR ON RATES AND OFF RATES ARE DIFFERENT. BUT THE HIGH DYNAMIC RANGE BETWEEN FAST VERSUS SLOW RELEASE ALSO DETERMINED BY CALCIUM BUFFERING AND BY THE VERY EFFECTIVE REMOVAL OF RESIDUAL CALCIUM IF YOU WOULD IMAGINE IF CALCIUM WOULD BE ONLY AS EFFECTIVELY REMOVED FROM THE TERMINAL AS SR IT WOULD BE HUGE RESIDUAL CALCIUM WHICH WOULD NEED TO LEAK OUT -- ASYNCHRONOUS LEAK OUT OF THE VESICLES FROM THE NERVE TERMINAL. THE SURPRISING CONCLUSION IS THAT CALCIUM BUFFERING CONTRIBUTES TO THE CESSATION OF ASYNCHRONOUS RELEASE MUCH STRONGER TO WHAT WE HAVE THOUGHT BEFORE. SO IN THE THIRD AND SHORTER PART OF MY TALK I WOULD LIKE TO RAISE A LITTLE QUESTION WHERE CALCIUM COMES IN TO THE NERVE TERMINAL BUT NATURALLY IDEA CALCIUM CHANNEL COLOCALIZATION WITH VESICLES IS VERY IMPORTANT HERE. WHY IS THIS SO IMPORTANT WHEN CALCIUM CHANNEL OPENSS IT FLOWS IN FROM THE OUTSIDE CREATES A MICRO DOMAIN. AS LONG AS THE CHANNEL IS OPEN THERE IS LIKE STANDING WAVE OF CALCIUM THAT ENTERS THROUGH THE CHANNEL THAT GETS DILUTE BY INFUSION AT THE EDGE OF THE MICRO DOMAIN. IN THIS SITUATION AS WAS SHOWN NICELY, THIS IS ACCORDING TO HIS EQUATION THERE IS A SHARP DROP OFF OF THE MICRO DOMAIN CONCENTRATION WITH LATERAL DISTANCE TO THE VOLTAGE. IF YOU COMBINE THIS WITH THE HIGHLY NONLINEAR CALCIUM DEPENDENCE OF VESICLE FUSION THAT WE HAVE DISCUSSED THERE WOULD BE VERY STEEP DEPENDENCE OF THE RELEASE PROBABILITY ON THE DISTANCE AWAY FROM VOLTAGE DATED CALCIUM CHANNELS. HOW ARE VOLTAGE GATED CALCIUM CHANNELS ORGANIZED WITH RESPECT TO VESICLES. RESEARCH IN THE LAST 10 TO 15 YEARS HAVE SORT OF SUBDIVIDED TWO LIMITING CASES. IMAGINE THESE WERE THREE, THIS WOULD BE MANY SHOWN IN RED ARRANGED AROUND THE VESICLES AND THERE IS ANOTHER LIMITING CASE WHERE MAYBE THE CALCIUM CHANNELS ARE -- ONLY ONE OR TWO CHANNELS IMMEDIATELY AT THE -- IF YOU DO HE CAN PAYMENT, WHERE YOU CHANGED THE NUMBER OF OPEN CALCIUM CHANNELS, IN THE LIMIT OF THE SINGLE CHANNEL CONTROL RELEASE CASE THERE WOULD BE SLOPE OF ONE. IN THE OTHER PLOT OF RELEASE VERSUS ENTERING CALCIUM BECAUSE WITH EVERY NEW CHANNEL THAT YOU RECRUIT YOU'RE RECRUITING A NEW VESICLE THAT WILL BE RELEASED. BUT IN THE DOMAIN OVERLAP SITUATION THE CALCIUM FOR MANY CHANNELS MAY BE 10 TO 20 CHANNELS TO COMBINE TO GET MIXED CHANNEL AT THE CENTER. THIS SITUATION YOU CAN CONTROL, THIS WAS FIRST POINTED ABOUT 20 YEARS AGO YOU RECOVER THE HIGH INTRINSIC CALCIUM COOPERATION. YOU CAN USE AN INDIRECT EXPERIMENT, THIS RELEASE EXPERIMENT WHICH FOR EXAMPLE HAD BEEN USED TEN OR 15 YEARS AGO. AND HE HAS FOUND GOOD EVIDENCE FOR HIGHLY COOPERATIVE RELATIONSHIP WHEN HE INVESTIGATES PQ TYPE CHANNELS, ARGUING I THINK AT THE TIME THAT SEVERAL PQ TYPE CHANNELS CONTRIBUTE TO THE RELEASE OF ONE VESICLE. ON THE OTHER HAND SORT OF THE SECOND INDIRECT EXPERIMENT TO INVESTIGATE THE TYPE OF COUPLING BETWEEN THE CHANNEL AND VESICLE IF BY USING FAST AND SLOW ON RATE CALCIUM BUFFERS. THE IDEA FIRST PROVIDED IS THAT A SLOW BUFFER SHOULD ACTUALLY NOT BE ABLE TO INFLUENCE THE CALCIUM SIGNAL VERY SLOWS TO THE VOLTAGE. I THINK YOU WERE ALL AWARE OF THIS ARGUMENT. WE REINVESTIGATED THE QUESTION OF COUPLING AND MECHANICS AND IT HAS BEEN SUGGESTED PREVIOUSLY IN THE LAB THAT THERE IS DOMAIN OVERLAP SITUATION WHERE THIS ACTUALLY BEING CHANGED TO A ONE TO ONE OR SO-CALLED NANODOMAIN COUPLING WHERE A FEW OR AT MOST MAYBE TWO OR THREE CHANNELS CONTROL RELEASE. HOWEVER WHEN WE REDO THESE EXPERIMENTS AND WE CAN DO DIRECT PATCH RECORDINGS UP TO P15 TO P16 WE ARE NOT AS BRAVE AS OTHERS, WE CANNOT GO AS I THINK HE WENT UP TO P18. WE STILL SEE VERY STEEP SLOPES. HOWEVER THE SLOPES, EFFICIENCY OF THE VOLTAGE GATED NOW HIGHER. THIS IS CONSISTENT WITH EARLIER RESULTS. WE THINK THERE IS A PARALLEL SHIFT WITH AGE TO THE LEFT WHICH IS SHOWN HERE. BUT THERE IS ALSO A SLIGHT SHALLOWING OF THE SLOPE BUT THE SLOPES ARE ACTUALLY STILL IN THE RANGE OF THREE AT THIS AGE OF P15 TO P16. WE THINK THAT THERE MIGHT ACTUALLY STILL BE DOMAIN OVERLAP SITUATION ALSO IN THESE YOUNG MICE. IN ORDER TO PUSH THIS A BIT FURTHER WE USE EGTAM COULD NOW GO TO REAL ADULT MOUSE TO P75 MOUSE. TO OUR INITIAL SURPRISE I SHOULD SAY IF WE USE 200 MICRO MOLAR AND WE ARE CURRENTLY REVIEWING LOWER CONCENTRATIONS YOU CAN SEE THAT WIPES OUT OR BLOCKS RELEASE WITH ALMOST THE SAME EFFICIENCY. THESE DATA ACTUALLY WOULD SUGGEST THAT AT LEAST THE VERY FAST RELEASE PHASE, COUNTER INTUITIVELY IS NOT ACTUALLY DETERMINED BY A SINGLE CALCIUM CHANNEL RELEASE COUPLING BUT STILL DETERMINED BY DOMAIN OVERLAP. HOW CAN THIS WORK? ISN'T THIS A CONTRADICTION? EVERYBODY IN THE FIELD SAYS PERHAPS YOU NEED TO HAVE NANODOMAIN CALCIUM SIGNALLING. THEN IN ORDER TO WORK THIS OUT WE ACTUALLY TURNED TO A MODEL OF THE ACTIVE ZONE. WE WANTED TO MODEL REALISTIC CNS, CENTRAL NERVOUS SYSTEM ACTIVE ZONES. WE'VE BEEN DOING THIS TOGETHER WITH OTHERS AND WE THOUGHT IT IS NOISE TO FIRST LOOK AT SAMPLES OF ACTIVE ZONE FROM ELECTRON MICROGRAPH MATERIAL AND THE LAB HAD PREVIOUSLY -- 15 ACTIVE ZONES THESE ARE THE TOP DOWN VIEWS YOU CAN SEE IN BLUE ANDD BLUE THE SURFACES OF THE ACTIVE ZONES THE LIGHT BLUE ARE DARK VESICLES THAT SHE FOUND. YOU CAN SEE WE HAVE ALL OF THEM HERE TO INCREASING SIZE YOU CAN SEE IN GENERAL THE NUMBER OF DARK VESICLES CORRELATES QUITE WELL WITH THE ACTIVE ZONE AREA. THIS WAS KNOWN BEFORE. SMALL ACTIVE ZONES HAVE FEW DARK VESICLES, BIGGER ONES HAVE BIG ONES. YOU CAN SEE THAT THESE VESICLES THEY ARE SORT OF LOCALIZED QUITE RANDOMLY. THERE HAVE BEEN PREVIOUS MODEL FOR DOMAIN OVERLAP THAT BY OTHERS WHO HAVE ARGUED THAT CALCIUM CHANNELS HAVE TO SIT IN A CLUSTER. IF YOU LOOK AT THESE ACTIVE ZONES WE THOUGHT THAT THERE IS NO VERY EVIDENT SITUATION TO ACTUAL LOW PLACE CALCIUM CHANNEL. WE STARTED WITH EASIER ASSUMPTION, IN THE NEXT MODELING I WILL SHOW YOU WE PICKED OUT THIS ACTIVE ZONE FOR MODELING REPLACED REALISTIC NUMBER OF CALCIUM CHANNELS IN TO THIS ACTIVE ZONE. THIS CALCIUM CHANNEL NUMBER HAPPENS TO BE 14. OPEN RANDOMLY IN RESPONSE TO ACTION POTENTIAL. THE OPENING PROBABILITY WAS ABOUT 0.6. AVERAGE SINGLE CHANNEL CURRENT WAS 0.12 WE NATURAL LOW HAD OUR MODEL. SO IF WE DO THIS MODEL AND IN THE FIRST ASSUMPTION WE JUST PUT 14 CHANNELS BE RANDOMLY DISTRIBUTED THEM OVER OUR ACTIVE ZONE AND THEN WE ACTUALLY SIMULATE CALCIUM CURRENT RELEASE COOPERATIVE EXPERIMENT SHOWN HERE. WE PUT ON SINGLE ACTION POTENTIAL BUT WE ALSO PUT ON ACTION POTENTIAL WITH LONGER DURATION AND WITH SHORTER DURATION IN ORDER TO RECRUIT WE FOUND WITH THE RANDOM DISTRIBUTION THIS PRODUCED VERY LOW SLOPE OF ABOUT 1.2 TO 1.4. SO THIS MEANS THAT IN THIS ARRANGEMENT ACTUALLY THE FUSION OF EACH VESICLE IS BY CHANCE. CLOSE TO VESICLES ANOTHER FEATURE THAT YOU CAN SEE RELEASE PROBABILITY HERE FOR EACH VESICLE WAS ABOUT 50 TO 60% THERE BY UNREALISTICALLY HIGH. IT MEANS IF WE PUT CALCIUM CHANNEL RANDOMLY IN TO ACTIVE ZONE WITH REALISTIC DISTRIBUTION OF VESICLES. SO THIS LED US TO A SECOND SORT OF ARRANGE W. WE SAID, LET'S USE SAME NUMBER OF CALCIUM CHANNEL. IN TO THE ACTIVE ZONE WE CALCIUM CHANNEL CAN BE PLACED ANYWHERE BUT NOT INSIDE THE EXCLUSION ZONE. YOU CAN SEE THE DISTRIBUTION OF CALCIUM IS HIGHEST CALCIUM IS BURNED AWAY A LITTLE BIT FROM THE VESICLE. SO THIS ARRANGEMENT ESSENTIALLY GUARANTEES THAT THE HIGH CALCIUM CONCENTRATION AT THE MOUTH OF THE OPEN CHANNEL IS NOT SEEN BY THE SENSOR. BECAUSE OUR WORK SUGGESTS THAT THE SENSOR HAS A SENSITIVITY IN THE RANGE OF 20 TO 40 MICRO MOLAR SO IF YOU WOULD ELEVATE CALCIUM TO HUNDRED MICRO MOLAR, THE VESICLE WOULD SIMPLY FUSE. YOU HAVE TO BRING CALCIUM CHANNEL SOME DISTANCE AWAY. TO OUR DELIGHT WHEN WE DO THIS AND CHECK FOR THE SLOPES IN THE CALCIUM CURRENT COOPERATIVE SLOPES THEY ARE QUITE HIGH IN THE RANGE OF 2.TO THREE. WE CAN EXPLAIN DOMAIN OVERLAP IN OUR EXCLUSION ZONE MODEL. THE PROBABILITY ON AVERAGE ARE NOW 10% SO WE ACTUALLY CALIBRATED EXCLUSION ZONE DISTANCE IN ORDER TO GET RELEASE PROBABILITY OF 10%, THIS IS SHOWN HERE. IF WE TAKE LONGER ZONE WE'LL GET LORI LEASE PROBABILITY F. WE TAKE SHORTER EXCLUSION ZONE ALLOW CALCIUM CHANNEL TO GO CLOSER TO THE VESICLES WE NATURALLY GET INCREASE. SO THIS WAS NICE BUT WE RECEIVE SOME CRITICISM BECAUSE IT WAS ACTUALLY ARGUED THAT MAYBE THIS RELEASE, ACTIVE ZONE MAYBE NOT SO REPRESENTATIVE AND SO WE NATURALLY -- DATA MISSING HERE. THE NICEST PICTURE I THINK MISSING, LET ME JUST SEE WHETHER IT'S HIDDEN. NO, IT'S NOT HIDDEN. I WILL CONCLUDE THEN, I WILL JUST TELL YOU THAT WE DID SEE SIMULATIONS AT ANOTHER ACTIVE ZONE WHICH IS ACTUALLY BIGGER SO I TOLD YOU INITIAL LOW WE HAD SAMPLE OF 15 ACTIVE ZONES. WE WENT TO A BIGGER ACTIVE ZONE I THINK WE CAN SHOW THAT THE BIGGER ACTIVE ZONE CAN ALSO BE -- CAN ALSO SLAIN RELEASE QUITE NICELY WITH THE EXCLUSION ZONE MODEL HOWEVER IT BECOMES APPARENT ESPECIALLY AT THE BIG ACTIVE ZONE PUTTING INDIVIDUAL LARGE CALCIUM CHANNEL CLUSTER IS QUITE UNREALISTIC SITUATION BECAUSE CALCIUM CHANNELS ARE CONCENTRATED ALL ON ONE POINT WHERE AS VESICLES ARE SCATTERED OVER A LARGER AREA AND CALCIUM WOULD NEVER REALLY REACH THE FAR AWAY VESICLES. IN CONCLUSION WE THINK THAT THE EXCLUSION ZONE MODEL MAY BE PROVIDED AN INTERESTING IDEA OF HOW CALCIUM CHANNEL VESICLE COLOCALIZATION MIGHT BE REGULATED IN CNS ACTIVE ZONE. BECAUSE I THINK OUR MODELING HAS SHOWN THAT IN A DOMAIN OVERLAP SITUATION RANDOM PLACEMENT OF CALCIUM CHANNELS NOT AN OPTION BECAUSE THEN CALCIUM CHANNELS WILL GET TOO CLOSE TO VESICLES. SO THE CONCLUSIONS ARE MAYBE OPPOSITE TO WHAT WE THOUGHT BEFORE. DOMAIN OVERLAP SITUATION PROBABLY REPRESENTS A NONRANDOM ORGANIZATION OFF THE ACTIVE ZONE WITH RESPECT TO CALCIUM CHANNEL. THERE MIGHT BE SOME FORCES OR SOME TETHERS OR SOME, YOU KNOW, PROTEINS THAT MIGHT FORM ESSENTIALLY A LITTLE BIT BARRIERS FOR CALCIUM TO AVOID CALCIUM CHANNELS COMING TOO CLOSE TO VESICLES. IN THE FUTURE IT WILL BE INTERESTING TO FIND OUT WHICH ACTIVE ZONES, PROTEINS ENABLE DOMAIN OVERLAP CONTROL AND ALSO WHICH MOLECULAR PROGRAMS DETERMINE SINGLE CHANNEL CALCIUM VESICLE COUPLING WHICH OBVIOUS LEO CURES AT MANY CNS. AND DOES THIS IN SPECIFIC SETTING. WITH THIS, LET ME JUST ESSENTIALLY LEAD OUT A LITTLE BIT, LET ME GIVE YOU OUTLOOK OF WHAT I TOLD YOU SO I DO THINK THAT IT IS IMPORTANT TO STUDY THE BIOPHYSICAL MECHANISMS AND MOLECULAR MECHANISMS THAT DETERMINE CALCIUM OF THE VESICLE FUSION APPARATUS WITHIN THE NERVE TERMINAL AND CALCIUM CHANNEL VESICLE COLOCALLIZATIONS. HOWEVER I THINK THAT IN THE FUTURE IT WILL BE IMPORTANT TO ALSO ADDRESS QUESTIONS LIKE TO TRY TO EXPLAIN WHAT ACTUALLY DETERMINES THE IMMENSE COMPLEXITY OF SYNAPTIC CONNECTIVITY IN BRAIN SO I HAVE STARTED OUT MY TALK WITH SAYING THAT THE NERVE TERMINAL SORT OF THE MOST -- IS THE FOURTH COMPARTMENT OF THE NEURONS THAT MEDIATES. NORMALLY A NEURON HAS MANY DIFFERENT OUTPUT SINUSES, THEY CAN HAVE TARGETS SPECIFIC PROPERTIES. SO IT SEEMS THAT NEURONS SEEM TO HAVE SORT OF BIT INTRINSIC DESIRE TO CONNECT WITH OTHER CELLS. I THINK THE CELL BIOLOGICAL MECHANISMS OF THIS INTRINSIC DESIRE OF CONNECTING WITH OTHER NEURONS AND OF THEN ACTUALLY SETTING UP SPECIFIC CONNECTIONS DEPENDING ON THE POST SYNAPTIC NEURON POOL IS ACTUALLY ISSUE THAT NEEDS MORE ATTENTION IN THE FUTURE. THIS ACTUALLY RELATES TO THE IDENTITY AND SPECIFICITY OF SPECIFIC SYNAPSIS. WE HAVE RECENTLY BEEN ABLE TO SHOW THAT THE MECHANICS SEEM TO BE UNDER PROGRAM OF B&P THAT IS IMPORTANT FOR THE GROWTH OF THE LARGE HELIX I THINK IN THE TO YOU FUTURE IT WILL BE IMPORTANT TO CONNECT TO SIGNALING MECHANISMS THAT PLAY A ROLE FOR MATURATION AND SPECIFICATION ALSO OF OTHER SINUSES IN OTHER BRAIN AREAS AND I THINK THAT WITH STUDYING THIS IDENTITY AND SPECIFICITY OF SINUSES THERE MIGHT BE KEY TO UNDERSTAND SOME NEURODEVELOPMENTAL DISORDERS THAT MIGHT HAPPEN AT THE SYNAPSE LIKE AUTISM AND OTHERS. WITH THIS I'D LIKE TO THANK MY COLLABORATORS. THANK YOU FOR YOUR ATTENTION. [APPLAUSE] [ NOT AUDIBLE ] >> THAT'S A GOOD COMMENT. NATURALLY PEOPLE ALSO IN THE POST SYNAPTIC FIELD ARE MUCH MORE AWAY THAT RECEPTORS ARE HEAVILY MOBILE. AND IT SEEMS TO SUGGEST FROM SOME WORK THAT CHANNELS COULD ALSO BE MOBILE. BUT I THINK -- DASH I HAVE TO STATE DISCLAIMER IN OUR MODEL VESICLES AND CALCIUM CHANNELS ARE NOT MOBILE. WE ASSUME A STATIC SITUATION IN A SENSE BUT ONLY SIMULATE FOR A FEW MILLISECONDS ANYWAY. WE COULD MAYBE ARGUE, WE JUST LOOK FOR RELEASE EVENT TO GET INITIATED AND TO TERMINATE BECAUSE THESE MODEL SIMULATIONS ARE -- WHERE WE HAVE TO TRACK THOUSANDS OF INCOMING CALCIUM IONS THIS IS COMPUTATIONALLY INTENSE. I AGREE WITH YOUR POINT. OTHERS SUGGEST THAT THE VESICLES MIGHT BE MOW BIBLE MOBILE THAT THE VESICLES AFTER BEING INTRINSICALLY THEY MIGHT HAVE TO FIND WAY TO ACQUIRE HIGHER POSITIONAL RELEASE. LATERAL MOVEMENT OF VESICLES ALSO NOT BUILT IN TO OUR MODEL. WE FEEL THAT OUR MODEL IS MORE PRINCIPLE MODEL. WE COULD LATER BUILD IN THESE THINGS. I WOULD JUST LIKE TO COMMENT TO YOUR IDEA THERE IS NICE REPORT WHEN THEY KNOCKED OUT A PROTEIN WHICH IS STRUCTURAL PROTEIN WAS ALSO LOCALIZED AT THE ACTIVE ZONE THEY FOUND SURPRISINGLY DECREASE IN RELEASE PROBABILITY -- SORRY INCREASE IN POSITIONAL RELEASE PROBABILITY. MAYBE SR IS PART OF COMPLEX THAT NORMALLY KEEPS AWAY THE CALCIUM CHANNEL. WE THINK STILL THAT THE EXCLUSION ZONE IS REASONABLE IDEA BECAUSE INDEED IF YOU JUST PLACE CALCIUM CHANNELS AND VESICLE RADOMLY RELEASE PROBABILITIES ARE JUST TOO HIGH. WE MADE REASONABLE ASSUMPTIONS ABOUT BUFFERS WHICH I DIDN'T SINGLE OUT HERE WE KNOW ENDOGENOUS SIX BUFFER, PHYSICAL PROPERTY QUITE WELL. ALSO PUT IN ATP AS A BUFFER AND, WE KNOW THE SENSITIVITY OF THE CALCIUM SENSOR FOR MANY OF OUR PREVIOUS WORK. IF YOU WOULD PUT CALCIUM CHANNEL AT 10 OR 20 NANOMETER FROM THE VESICLE IT WOULD SIMPLY FUSE THAT VESICLE WITH HIGH FUSION PROBABILITY THIS WE DON'T OBSERVE. THE CONQUINCE THAT YOU HAVE TO PLACE CALCIUM CHANNELS AT SOME DISTANCE EVEN MORE SO IF YOU HAVE LARGE NUMBER OF CALCIUM CHANNEL AT EACH EXIT ZONE. OUR ASSUMPTION THAT THERE SHOULD BE LARGE NUMBER OF CALCIUM CHANNELS AGREES WITH OTHER'S WORK WHO HAS SHOWN DIRECT PATCH ON SINGLE ACTIVE ZONES BUT WE MIGHT CONSIDER THAT HIS AVERAGE ACTIVE ZONE WAS MAYBE A LITTLE BIT BIGGER THAN THE ACTIVE ZONE WE SHOWED HERE. >> WHAT YOU'RE TALKING ABOUT HOW THINGS COULD BE MOVING AROUND, WHERE IS IT NECESSARILY NOT A TENABLE SITUATION TO HAVE CLUSTER OF CHANNELS TOO FAR AWAY FROM A DOCKING SITE AT ONE PARTICULAR POINT IN TIME IF EVERYTHING IS MOVING AROUND. >> THIS DATA IS MISSING. THERE IS A DATA SLIDE MISSING, ESPECIALLY FOR A LARGE ACTIVE ZONE, LARGE ACTIVE ZONE CAN HAVE A DIMENSION OF 300 BY 400 NANOMETER. THAT WOULD BE SIZE OF ABOUT 0.1 MICRO METER SQUARE WHICH IS ACTUALLY NOT YET HUGE. IMAGINE DIFFUSION TIME THAT YOU ALREADY HAVE ACTIVE ZONE OF 300 BY 400 NANOMETER. IF YOU NOW PUT SINGLE CLUSTER THERE, THE VESICLES THAT ARE FAR AWAY WILL HAVE SUCH LOW RELEASE PROBABILITY THAT THEY WILL ESSENTIALLY NEVER BE RELEASED. WHERE AS OTHER VESICLES THAT ARE CLOSE TO THE CLUSTER WILL JUST BE BURNED AWAY. >> THEY COULD BE RELEASED 50 MILLISECOND LATER IF EVERYTHING IS ALLOWED TO REARRANGE ON THAT TIME SCALE. THAT WOULD BE A WAY OF PROTECTING IT AGAINST DEPLETION. >> COULD EVEN BE RELEASED 50 MILLISECONDS LATER IF YOU DON'T ALLOW THINGS TO BE REARRANGED BECAUSE -- VESICLES ARE 200 NAN MOW METER AWAY WILL ALSO EXPERIENCE CERTAIN AMOUNT OF CALCIUM. IF YOU KEEP THE CALCIUM CHANNEL OPEN IN THE 50 MILLISECONDS USED BY OTHERS THEN EVENTUALLY FAR AWAY VESICLES WOULD ALSO FUSE. THIS MIGHT EXPLAIN THE LOW RELEASE COMPONENT THAT THEY OBSERVED. >> I'M SORRY, THE QUESTION I WAS GOING TO ASK BEFORE I GOT SIDETRACKED, IT'S INTERESTING YOUR RESTRICTION ZONE GIVES RISE WOULD PREDICT HETEROGENEOUS RELEASE PROBABILITY. I WAS WONDERING IF YOU HAD DONE ANY EXPERIMENTS THAT MIGHT ADDRESS WHETHER YOU THINK THAT THE RELEASE PROBABILITY OF THE VESICLES ARE REL TESTIFILY HOMOGENEOUS OR HETEROGENEOUS. >> IS IT POSSIBLE TO PUT DATA UP AGAIN. WHAT THE DATA SLIDE THAT IS MISSING ACTUALLY SHOWS OUR SIMULATION WITH THE SUPER CLUSTER MODEL. AND HETEROGENEITY. THERE ARE SOME VESICLES THAT SEE SOME CALCIUM. WE ARGUE THAT TWO LARGE RETRO GENTLEMAN ANYWAY TEE IS NOT REEL LESS PARTICULAR. WE HAVE TO CALIBRATE WITH KNOWN EXPERIMENTS IN THE FIELD BEFORE. SO NATURALLY WE TOGETHER WITH OTHERS ARGUED FOR LONG TIME THAT COMPONENT OF RELEASE IS REPRESENTED BY VESICLES THAT ARE CLOSER TO BOTH. SLOW ROW LEASE COMPONENT REPRESENTED BY VESICLES THAT ARE FARTHER AWAY. USING DESTAINING EXPERIMENTS ALSO CUMULATIVE DEPENDENT BLOCK RELEASE SORT OF HETEROGENEOUS. PROBLEM IS ALWAYS THAT WE ARE ALSO LOOKING OVER VARIOUS ACTIVE ZONES, THIS IS OFTEN DONE AT SEVERAL ACTIVE ZONES. I JUST WANT TO POINT OUT IN OUR SIMULATION SUPER CLUSTER MODEL PRODUCES HIGHER HETEROGENEITY. THIS WAS ONE REASON TO EXCLUDE THE SUPER CLUSTER MODEL. OTHER IF WE LOOK AT RECENT END DATA FROM OTHERS WHERE THEY USE FREEZE FRACTURE THEN EM STAINING, WHERE THE EPITOPES BECOME VISIBLE THEN THEY CAN LOCALIZE WITH GOLD PARCEL IT LOOKS LIKE A CLUSTER DISTRIBUTION, A LITTLE BIT CLUSTERED OVERALL RANDOM DISTRIBUTION. WE SORT OF THINK THAT THIS DOESN'T LOOK SO DIFFERENT FROM WHAT WE ASSUMED IN OUR MODEL WITH THE EXCLUSION ZONE. THE PROBLEM CURRENTLY THAT WE CANNOT GET DATA TO BOTH SEE LOCALIZATION OF THE VESICLES AND LOCALIZATION OF THE CALCIUM CHANNEL. IN OUR TRADITIONAL EM DATA WE SEE LOCALIZATION BUT WE CANNOT SEE CALCIUM CHANNEL. DID WITH FREEZE FRACTURE METHOD WE CAN SEE -- WELL OTHERS CAN SEE IT. BUT THEY CANNOT SEE THE LOCALIZATION OF THE VESICLES. IN THIS SITUATION I THINK THAT THE MODEL IS A GOOD IDEA TO EXPLORE WHAT IS GOING ON. [ NOT AUDIBLE ] >> YOU ARGUE FROM EM DATA IT LOOKS LIKE CALCIUM CHANNEL HAVE TO BE CLOSE TO THE VESICLE. [ NOT AUDIBLE ] NO. YOU DON'T KNOW. BECAUSE IN THIS DATA YOU DON'T KNOW WHERE THE VESICLES OR LOCALIZED BECAUSE YOU COULD ARGUE THAT THE PSEUDOCLUSTERS OF CHANNELS ARE ACTUALLY LOCALIZED AT THOSE POINTS THAT WOULD BE THE INTERPRETATION IN OUR MODEL WHERE THERE ARE NO VESICLES BECAUSE THERE CAST YUM CHANNELS WOULD CLUSTER BECAUSE THEY CAN NO THE ENTER OUR HYPOTHESIZED EXCLUSION ZONE. [ NOT AUDIBLE ] HE ASKED THAT OTHERS SHOWED THAT EGTA CANNOT BLOCK RELEASE BUT I THINK THIS REFERS TO LOU'S DATA. I HAVE TO THINK HOW THEY DID THIS. THEY DID THIS BY PATCH RECORDINGS. SURE ENOUGH WE DID OUR WORK BY EGTAM, PATCH RECORDINGS COULD GO UP TO P18 ANIMALS. WE COULD SORT OF CHOOSE OUR AGE. I WOULD ARGUE WE'D HAVE TO DO MORE EXPERIMENTS TO FIND OUT THE DIFFERENCE OF THE CONCLUSION. I AGREE WITH YOU I DIDN'T CONSIDER THE LOU'S EXPERIMENT. HE SHOWED THAT EGTA BECOMES LESS EFFICIENT. IN A SENSE I DON'T WANT TO APPEAR TOO BRUTAL ON THIS CONCLUSION. I JUST WANT TO SAY THAT I ACTUALLY DO THINK BECAUSE OF THE DEVELOPMENT THAT DIFFERENCE BETWEEN CALCIUM CHANNELS AND VESICLES BECOMES LESS. ALSO IN AGREEMENT WITH OUR OWN WORK IN RATS THAT WE DID BEFORE. SEVERAL CALCIUM CHANNEL, IS THAT CONTROL THE RELEASE OF ONE VESICLE THIS CONCLUSION WOULD BE IMPORTANT. [APPLAUSE]