Presynaptic Mechanisms Evoked vs. spontaneous exocytosis Quantal release—What is the evidence? Alternative techniques for monitoring exocytosis Calcium & exocytosis Calcium channels Molecular mechanisms underlying exocytosis SNARE proteins Synaptotagmin

Nerve-evoked and spontaneous synaptic transmission Note multiple release sites Synaptic transmission Spontaneous miniature EPSPs Muscle is bathed in calcium-free solution with magnesium added. “A focal electrode filled with 0.5M CaCl2 used for controlled application of calcium ions. By adjusting the electrical bias on the pipette, efflux of calcium could be prevented or accelerated in a graded manner. In some experiments, twin pipettes were used with separate barrels containing, respectively, 0.5M Nacl for recording and 0.5M CaCl2 for controlled calcium application. The same results, however, could be obtained by using a single CaCl2 pipette which was connected both to cathode follower and condenser-coupled amplifier, and, through a 50 to 100MOhm resistance, to the adjustable d.c. bias. Synaptic spots along the non-myelinated terminations of the nerve were located by looking for spontaneous external miniature potentials (which persist, unlike the evoked response, in the Ca-deficient medium). Pre- and post-synaptic responses to nerve stimuli, and the effects upon them of ionophoretic calcium release, were then recorded.” Nerve-evoked EPSP + AP Fatt & Katz, 1952

Quantal Hypothesis: Each evoked response is made up of a number of unitary events (quanta) that are the size of spontaneous events. Probability determines just how many: m = np m = mean quantal content n = number of quanta (or release sites) available p = probability of release where * “Suppose we have, at each nmj, a population of n units capable of responding to a nerve impulse. [GO TO NEXT SLIDE] Suppose, further, that the average probability of responding is p…then the mean number of units responding to one impulse is m = np. Under normal conditions, p may be assumed to be relatively large…However, as we reduce the Ca and increase the Mg concentration, the chances of responding are diminished and we observe mostly complete failures with an occasional response of one or two units. Under these conditions, when p is very small, the number of units x which make up the epp in a large series of observations should be distributed in the characteristic manner described by Poisson’s Law.”

“Suppose we have, at each nmj, a population of n units capable of responding to a nerve impulse.” Del Castillo & Katz 1954 Some people consider “n” to be the number of release sites Others consider “n” to be the total number of vesicles available for release If only one vesicle can be released from any one release site in response to a single action potential (which seems to be the case for many synapses, most of the time), then these two ways of defining “n” will give the same value--so we won’t worry about resolving this now

n = number of quanta p = probability of release m = quantal content

“Suppose, further, that the average probability of responding is p…then the mean number of units responding to one impulse is m = np. Under normal conditions, p may be assumed to be relatively large…However, as we reduce the Ca and increase the Mg concentration, the chances of responding are diminished and we observe mostly complete failures with an occasional response of one or two units. Under these conditions, when p is very small, the number of units x which make up the epp in a large series of observations should be distributed in the characteristic manner described by Poisson’s Law.” Del Castillo & Katz 1954 Poisson’s Law describes the probability of observing a particular number of low probability events--e.g. Prussian cavalry members killed by horse kicks each year Assumptions all hold for the nmj (although NOT so good for CNS synapses) Poisson Distribution can describe the probability of releasing different numbers of vesicles from trial to trial assuming: The pool of vesicles (and/or individual release sites) is large Each vesicle (and/or individual release site) behaves identically The probability of release for each vesicle is independent of release of other vesicles The probability of release is small P(x) = probability that a particular # of quanta are released n = number of vesicles (or release sites) available p = probability of release m = mean quantal content Where must be large must be low (& each vesicle must behave identically and independently)

For the special case of failures (no release, x = 0) —which are easy to detect accurately— this simplifies to: e-m P(0) = m0 0! 1 Ln P(0) = - m Substituting for P(0)=(Number of Failures)/(Number of Stimuli) and rearranging: m = Ln (Total # of Stimuli/# of Failures) Mean EPP amplitude m also is equal to Mean mEPP amplitude* *spontaneous event- aka single quantum m calculated both ways can be compared for verification

For 1st, simply count failures For 2nd, compare mean amplitudes of spontaneous and evoked events spontaneous evoked Frog nmj ?mark failures? del Castillo & Katz, 1954 Then compare results from both methods in the same cell

Quantal Hypothesis predicts: Neurotransmitter is released in discrete packets—how could this be accomplished? ln C/D A = mean EPP B = mean mEPP C = # of stimuli D = # of failures A/B = loge C/D A/B del Castillo & Katz, 1954

“Can the miniature discharges be attributed to molecular leakage of transmitter from nerve endings?” + ACh ? or “If the miniature discharges were to be regarded as the local depolarizing effect of individual ACh molecules then… the application of ACh in solution should greatly increase the frequency of the miniature discharges; in fact, the steady depolarization which is produced by applied Ach would have to be regarded as a fusion of miniature e.p.p.'s…This conclusion, however, is contrary to our observations. A moderate concentration of ACh which depolarized the end-plates by a few mV, did not appreciably alter the frequency of the miniature discharges. The only noticeable change was a slight reduction of amplitudes...incompatible with the suggestion that the miniature potential might be attributed to the action of one (or very few) ACh molecules.” Why is there a slight reduction in mini amplitudes in the presence of exogenous agonist? If “discrete packets” aren’t individual molecules, what else might they be? Fatt & Katz, 1952

EM Synapse Are these vesicles the physical correlate of quanta? Sanford Palay high resolution EM of synapses, late 1950’s

Freeze-slam image of NMJ immediately after stimulation “omega” shapes -Note “omega” shapes believed to represent fusing/fused vesicles; junctional fold is where AChR accumulate (on “shoulders” of folds) 1970’s -INDEPENDENT AND DIRECT MORPHOLOGICAL EVIDENCE THAT SVs ARE NT STORAGE ORGANELLES EXOCYTOSIS IS AN NT RELEASE MECHANISM -Liquid helium -Heuser saw with stimulation a decrease in SV #, increase is membrane surface area, and increase in HRP uptake Are they fusing vesicles?

Freeze-fracture EM of active zone fused vesicles bumps are some kind of integral membrane protein— calcium channels? Detwiler, Oreo-esque 1970’s Freeze in vacuum, fracture occurs b/w lipid bilayers of membrane, exposed surface coated w/carbon and platinum

Recycling of synaptic vesicles Neurotransmitter release is quantal Vesicles are quantal packets of transmitter

What techniques can be used to monitor presynaptically exocytosis So far, all “real time” examples monitor exocytosis by measuring a postsynaptic response using electrophysiology. What techniques can be used to monitor presynaptically exocytosis directly? Postsynaptic response may be an imperfect reporter of neurotransmitter release

Capacitance measurements of vesicle cycling at a ribbon synapse

AMPEROMETRY uses electrochemistry to detect catecholamine release from chromaffin cells, while CAPACITANCE measurements report changes in membrane surface area capacitance Note: amperometry can only be used for monoaminetransmitters (dopamine, serotonin, norepinephrine, but not glutamate or GABA) Chromaffin cells = neuroendocrine cells, release peptides, hormones, and monoaminetransmitters from large dense core vesicles, a close cousin of the small SVs of neurons, used as a model system to study exocytosis Note that capacitance trace will eventually come “back down” as vesicles are retrieved (endocytosed) from the surface amperometry Ales et al., 1999

FM1-43 stains recycling vesicles Lipophilic fluorescent dyes can be used to monitor vesicle cycling at small CNS synapses FM1-43 stains recycling vesicles

FM1-43 loaded NMJ destains with stimulation Rate of destaining (dye loss) is proportional to rate at which vesicles are fusing/transmitter is being released Bill Betz video

FM1-43 fluorescent dye loaded vesicles Cultured hippocampal neurons Individual release sites, not individual vesicles, labeled here Absolute intensity of fluorescence is a measure of how many vesicles were released/endocytosed in presence of dye during stim

FM1-43 reveals quantal transmission in cultured hippocampal neurons when FM dye is loaded under low Prelease conditions where only a few vesicles are expected to be released & take up dye Murthy & Stevens, 1999

Are there different modes of exocytosis—i.e. “kiss & run”? Under some conditions, release of transmitter can be observed without release of FM1-43 dye—this could indicate that release is occurring through a fusion pore that is big enough to let transmitter but not dye to get out What is a fusion pore?

Are there different modes of exocytosis—i.e. “kiss & run”?

FM dyes suggest “full fusion” of vesicles Ryan et al 1996