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Cell and Systems Biology
Francis Bambico

Chapter 13 Release of Neurotransmitters  Release of neurotransmitter stimulus: depolarization of the nerve terminal  Release occurs as a result of Ca entry into the terminal through voltage-activated Ca channels  Invariably, a delay of about 0.5 milliseconds intervenes between presynaptic depolarization and transmitter release because : o The time taken for Ca channels to open o Time required for Ca to cause transmitter release  Transmitter is secreted in multimolecular packets (quanta), each containing several thousand transmitter molecules  1-300 quanta are released almost synchronously, depending on the type of synapse, in response to an AP  At rest, nerve terminals release quanta spontaneously at a slow rate, giving rise to spontaneous miniature synaptic potentials (minis)  At rest, there is also a continuous, nonquantal leak of transmitter from nerve terminals  One quantum of transmitter corresponds to the contents of one synaptic vesicle and comprises several thousand molecules of a low-MW transmitter  Release occurs by exocytosis, during which the synaptic vesicle membrane fuses with the presynaptic membrane and the contents of the vesicle are released into the synaptic cleft  The components of the vesicle membrane are then retrieved by endocytosis, sorted in endosomes, and recycled into new synaptic vesicles  The presynaptic terminals at vertebrate skeletal NMJ are typically too small for electrophysiological recording; however, this can be done at a number of synapses, such as the giant fiber synapse in the stellate ganglion of a squid Characteristics of Transmitter Release Axon Terminal Depolarization and Release  Stellate ganglion of the squid was used by Katz and Miledi to determine the precise relation between presynaptic membrane depolarization and the amount of transmitter released  Simultaneous records were made of the AP in the presynaptic terminal and the response of the postsynaptic fiber  Sketch of the stellate ganglion , illustrating the 2 large axons that form a chemical synapse o Pre and post synaptic axons are impaled with microelectrodes to record membrane potential o An additional microelectrode is used to pass depolarizing current into the presynaptic terminal  To further explore the relation between the potential amplitude and transmitter release, they placed a second electrode in the presynaptic terminal, through which they applied a brief (1-2ms) depolarizing current pulses, thereby mimicking a presynaptic AP  The relationship between the amp of the artificial AP and that of the synaptic potential was the same as the relation obtained with the failing AP during TTX poisoning  This result indicates that the normal fluxes of Na and K ions responsible for the AP are not necessary for transmitter release; only depolarization is req Synaptic Delay  There is a lag time between the onset of the presynaptic AP and the beginning of the synaptic potential known as the synaptic delay  Detailed measurements at the frog NMJ show a synaptic delay of .5ms – too long to be accounted for by diffusion of Ach across the cleft (usually 50 microseconds)  A: the motor nerve is stimulated while recording with an extracellular microelectrode at the frog NMJ. With this recording arrangement, current flowing into the nerve terminal or the muscle fiber is recorded as a negative potential  B: (S) stimulus artifact; (AP) the axon terminal AP; (EPC) end plate current; the synaptic delay is the time between the AP in the terminal and the beginning of the EPC  When ACh is applied to the junction ionophoretically from a micropipette, delays as little as 150 microseconds can be achieved, even though the pipette is much farther from the postsynaptic receptors than are the nerve terminals  Furthermore, synaptic delay is much more sensitive to temperature than would be expected if it were due to diffusion  Cooling the frog to 2.5 degrees increases the delay to as long as 7 ms, whereas the delay in the response to ionophoretically applied Ach is not perceptibly altered -> the delay is largely in the transmitter release mechanism Evidence that Ca is Required for Release  Ca is the essential link in the process of synaptic transmission  When its conc in the ECF is decreased, release of Ach at the NMJ is reduced and eventually abolished  Evoked transmitter release is preceded by Ca entry into the terminal and is antagonized by ions that block Ca entry  Transmitter release can be reduced either by removing Ca from the bathing soln or by adding a blocking ion  For transmitter release to occur, Ca must be present in the bathing soln at the time of a depolarization of the presynaptic terminal Measurement of Ca Entry into Presynaptic Nerve Terminals  Entry of Ca into the nerve terminal is through voltage-sensitive Ca channels that are activated upon depolarization by the presynaptic AP  the presynaptic terminal is voltage clamped and treated with TTX and TEA to abolish voltage-activated Na and K currents  A: records show potentials applied to the presynaptic fiber (upper trace), presynaptic Ca current (middle), and EPSP in the postsynaptic fiber (lower) o A voltage pulse from -70—18 mV results in a slow inward Ca current and after a delay of about 1 ms, an EPSP o A larger depolarization, to +60mV suppresses Ca entry o At the end of the pulse, a surge of Ca current is followed within about .2ms by an EPSP  B: if a voltage change identical in shape to a normal AP is produced by the voltage clamp, then the EPSP is indistinguishable from that seen normally (postsynaptic) o The black curve gives the magnitude and time course of Ca current o The synaptic delay between the beginning of the post synaptic response is due in part to the time for Ca entry to trigger transmitter release  The time required for the presynaptic terminal to depolarize and the Ca channels to open accounts for the first half of the synaptic delay  The time required for the Ca conc to rise within the terminal and evoke transmitter release accounts for the remainder  An experimental technique important for characterizing the role of Ca transmitter release is the use of Ca indicator dyes to estimate intracellular Ca conc  The first dye was aequorin, a Ca-sensitive luminescent protein extracted from the jellyfish Aequorea victoria.  EGTA – Ca chelator  Aequorin injected into the resting presynaptic terminal of the squid giant synapse (A) revealed discrete microdomains of free Ca, some with relatively high conc (red and yellow)  After a brief train of presynaptic AP (B), the intracellular Ca conc reached 100 to 200 µM Localization of Ca Entry Sites  Distribution of free Ca in the cytoplasm is not at all uniform  Ca entering the terminal through a single channel collects briefly in a small nanodomain with the conc falling rapidly over a radius of a few tens of nm from the channel as the ions diffuse into the bulk soln or are bound by intrinsic Ca chelators  Ca entering through a group of closely apposed channels occupies a microdomain that can spread over a distance of a few hundred nm from the channel cluster  Because of the restricted spread of incoming ions, the spatial relation between Ca channels and their associated transmitter release sites is of critical importance  Injection of BAPTA, a potent Ca buffer, into the presynaptic terminal resulted in a severe attenuation of transmitter release, without affecting the presynaptic AP  EGTA, a Ca buffer of equal potency, had little effect on release  This disparity is due to the fact that Ca is bound hundreds of times faster by BAPTA than by EGTA  Thus Ca ions have little opportunity to diffuse from their site of entry before being bound by BAPTA, but can traverse some distance before being captured by EGTA Quantal Release  Presynaptic depolarization -> Ca entry -> transmitter release  Fatt and Katz showed that ACh can be released from terminals in multimolecular packets, which they called quanta  Kuffler and Yoshikami showed that each quantum corresponds to approx. 7k molecules of ACh  Quantal release means that any response to a stimulation will consist of roughly 7000 molecules or 14k  At any given synapse, the number of quanta released from the nerve terminal in response to an AP
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