1. Chapter 7 Neurotransmitter release
From Cellular and Molecular Neurophysiology, Fourth Edition.
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2. From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure 7.1 Three examples of preparations in which synaptic transmission has been studied.
(a)The cerebellar cortex. Left: Schematic showing the connections between a granular cell axon (parallel fiber) and a Purkinje cell. These synapses are axo-spinous (right). (b)The calyx of Held. Left: Frontal section of the brainstem drawn at the level of the 8th nerve. The axon collaterals of the globular cells of the ventral cochlear nucleus (VCN) project to the neurons of the contralateral medial nucleus of the trapezoid body (MNTB). This synapse is axo-somatic and each MNTB neuron receives only one axon terminal that forms the calyx of Held (right). (c)Squid giant synapse. Secondary neurons, that receive sensory information from the primary ones (left), establish giant axo-axonic synapses with tertiary neurons (right). The tertiary neurons are responsible for contraction of the mantle muscles thus permitting expulsion of water and propelling the animal out of the danger zone. The dotted square indicates the region enlarged on the right of the figure. LSO, lateral superior olive.
Drawing (b) adapted from Forsythe ID, Barnes-Davies M, Brew HM (1995) The calyx of Held: a model for transmission at mammalian glutamatergic synapses. In: Excitatory Aminoacids and Synaptic Transmission, 2nd edn, New York: Academic Press, with permission. Drawing (c) from Llinas R (1982) Calcium in synaptic transmission Sci. Am.247, 56–65, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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3. Figure 7.2 Number n of active zones per presynaptic terminal.
Drawings of synaptic boutons of the central nervous system with presynaptic active zone(s) and postsynaptic membranes. The active zones are represented as a black bar with adjacent synaptic vesicles. The number of active zones per bouton is n = 1 in (a) and (b) but n = 3 in (c). The synaptic connections are such that a single presynaptic spike will activate a maximum of one active zone in (a), and a maximum of three in (b) and (c).
From Cellular and Molecular Neurophysiology, Fourth Edition.
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4. 4 Figure 7.3 Basic properties of synaptic transmission.
(a) In the neocortex, pyramidal neurons are Golgi type I neurons, whose main axons leave the cortex after giving off collaterals. Left: These collaterals establish excitatory synapses with dendrites of local interneurons (Golgi type II neurons). Three synapses are represented but their exact number in the experiment performed at right was not determined. Right: In response to each presynaptic action potential (AP) evoked in the pyramidal neuron (top trace), a single-spike EPSP is recorded in the postsynaptic interneuron: it fluctuates in amplitude from 0 mV (failure) to 5 mV (middle traces); EPSPs recorded in response to four successive presynaptic spikes are superimposed (bottom traces). Vm= −75 mV in middle and bottom traces. (b) The synaptic delay between a presynaptic action potential (AP) evoked by stimulation of the afferent axon and recorded in the calyx of Held, and the postsynaptic response (excitatory postsynaptic current, EPSC) recorded in the postsynaptic soma varies from 500 μs to 1 ms.
Drawing (a) from Thomson AM, personal communication. Drawing (b) from Borst JGG, Helmchen F, Sakmann B (1995) Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J. Physiol.489, 825–840, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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5. Figure 7.4 Ca2+ current flows into presynaptic terminal during the repolarizing phase of the presynaptic spike.
Two whole-cell electrodes are positioned in the calyx of Held; one measures the membrane potential (V) and the other one injects current (I). It is a two-electrode voltage clamp configuration. The preparation is bathed in 2 mM external Ca2+ with TTX and TEA to block the voltage-gated Na+ and K+ currents. (a) The voltage clamp command (VCmd) is an action potential waveform (V) from a holding potential of −80 mV. A reduced and inverted action potential waveform is also applied, scaled and re-inverted to measure the passive current. Top, recorded voltages. The two action potential waveforms are superimposed. Due to series resistance, the repolarization is somewhat slower during the full action potential than during the scaled action potential. Middle, currents. The current flowing during the full-sized action potential has a larger inward component (labeled ICa). The two passive transients overlay well. Bottom, calcium current. The calcium current is obtained by subtracting the passive current from the current measured during the full action potential. All traces are the average of 11. Vertical dotted lines denote peak of the action potential waveform and of the calcium current. (b) This Ca2+ current is reduced in 1 or 0.5 mM external Ca2+ ([Ca2+]o)
Part (a) from Borst JG, Sakmann B (1998) Calcium current during a single action potential in a large presynaptic terminal of the ra t brainstem. J. Physiol. 506, 143–157, with permission. Part (b) adapted from Borst JGG, Sakmann B (1996) Calcium influx and transmitter release in a fast CNS synapse. Nature383, 431–434, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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6. 6 From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure 7.5 N- and P/Q-type Ca2+ channel blockers reduce presynaptic Ca2+ influx and synaptic transmission at a cerebellar synapse.
(a) Transverse cerebellar slice with the relative locations of labeling with the dye furaptra (fill site), stimulus electrode and recording sites (whole-cell recording of EPSC from a Purkinje soma and recording of presynaptic fluorescence transients in the molecular layer). (b) Amplitude of furaptra fluorescence transients (ΔF/F) in the presence of increasing concentrations of ω-conotoxin GVIA (CgTx) to block the N-type Ca2+ current (top traces). Concomitant recording of the postsynaptic current is shown in the bottom trace. Each furaptra transient is elicited by a single stimulus of the parallel fiber tract and is a measure of the presynaptic Ca2+ influx. The inset shows superimposed fluorescence transients in control conditions and after addition of 0.5 and 1 μM CgTx. (c) The same experiment as in (b) but in the presence of increasing concentrations of ω-agatoxin IVA (ω-Aga IVA) to block the P/Q-type Ca2+ current. The inset shows superimposed fluorescence transients in control conditions and after addition of 200 and 400 nM ω-Aga IVA. Concomitant recording of the postsynaptic current is shown in the bottom trace. (d) Additive effects of the sequential application of saturating concentrations of the toxins on the furaptra transients.
Adapted from Mintz I, Sabatini BL, Regehr WG (1995) Calcium control of transmitter release at a cerebellar synapse. Neuron15, 675–688, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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7. Figure 7.6 Microdomains of Ca2+ increase in the presynaptic terminal of the squid giant synapse.
(a) Fluorescence image of a presynaptic terminal injected with n-aequorin-J. When the presynaptic fiber is fully loaded, it is continuously stimulated at 10 Hz for 10 s. (b) The acquisition during these 10 s of tetanic stimulation reveals stable quantum emission domains that appear as white spots. The background fluorescence shown in (a) disappears in (b) due to subtraction. (c) Superposition of the fluorescent images in (a) and (b) reveals that the distribution of the microdomains of high calcium coincides with the presynaptic terminal. Emission domains in an unstimulated terminal (d) and in the same terminal during tetanic stimulation (e) are shown at high magnification.
Adapted from Llinas R, Sugimori M, Silver RB (1992) Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677–679, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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8. 8 From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure 7.7 Presynaptic N-type Ca2+ channels are clustered at active zones.
(a) In the squid giant synapse, presynaptic zones of [Ca2+]i increase in response to a train of brief presynaptic stimuli (0.5 s at 80 Hz) are visualized with the FURA-2 technique. They are localized at active zones. The diagram illustrates the synapse and the box indicates the region studied. (b) In the frog neuromuscular junction, N-type Ca2+ channels and nicotinic acetylcholine receptors (nAChR) are labeled with two different selective toxins coupled to different fluorescent dyes. The preparation is viewed with a confocal laser microscope. The diagram illustrates the structure of the neuromuscular junction and the box indicates the region scanned by the microscope. The images showing the distribution of presynaptic Ca2+ channels (top) and postsynaptic nAChRs (bottom) are separated for clarity but they are in fact superimposed. (c) Predicted topological structure of the α1-subunit of N-type Ca2+ channel. The rectangle indicates the synprint site. (d) Theoretical model of interaction of presynaptic Cav2.2 and 2.1 channels (N- and P/Q-type Ca2+ channels) with SNARE proteins (syntaxin, SNAP-25 and synaptotagmin) at the presynaptic plasma membrane.
Part (b) from Robitaille R, Adler EM, Charlton MP (1990) Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron5, 773–779, with permission. Part (c) from Sheng ZH, Rettig J, Takahashi M, Catterall WA (1994) Identification of a syntaxin-binding site on N-type calcium channels. Neuron13, 1303–1313, with permission. Part (d) from Catterall WA (2000) Structure and regulation of voltage-gated Ca2+channels. Annu. Rev. Cell Dev. Biol16, 521–555, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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9. Figure 7.8 Ca2+ clearance mechanisms in a presynaptic terminal.
While Ca2+ ions enter at the level of the presynaptic active zone through high-voltage-activated Ca2+ channels, they are rapidly buffered by cytoplasmic Ca2+-binding proteins (Ca-B) (1). Ca2+ ions are also actively cleared from the intracellular medium towards the extracellular medium via Ca-ATPases of the plasma membrane (PMCA pumps) (2) and the Na–Ca exchangers (3). They are also cleared by active transport toward endoplasmic reticulum via another type of Ca-ATPase (SERCA pumps) (2′). This clearing has a time constant of the order of tens of milliseconds to seconds.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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10. Figure 7.9 Relative contribution of the different mechanisms of Ca2+ clearance in Purkinje somata.
[Ca2+]i transients are evoked in FURA-2-loaded Purkinje cells by a depolarizing current pulse of varying duration (60–250 ms). Effects on [Ca2+]i transients of (a) cyclopiazonic acid (CPA), a blocker of SERCA pumps, (b) 5,6-succinimidyl carboxyeosin (CE), a blocker of PMCA pumps, and (c) Li2+ saline, a blocker of Na–Ca exchanger. (d) Total Ca2+ clearance rate is presented in comparison with the rate of the different components characterized in the above experiments. Clearance rate is plotted as a function of the [Ca2+]i in the range between 50 nM and 2 μM. The clearance rate is calculated as follows: (i) The decay phase of each transient is fitted by a single or double exponential function and the derivative function (d[Ca2+]i/dt) is calculated from the fit. (ii) 2d[Ca2+]i/dt is then plotted as a function of the [Ca2+]i values obtained from the experimental fit. (iii) The plots from transients with equal peak [Ca2+]i in each condition (control versus inhibitor) are pooled and fitted with a polynomial function of fifth to seventh order.
Adapted from Fierro L, DiPolo R, Llano I (1998) Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices. J. Physiol.510, 499–512, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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11. Figure 7.10 Diagram of the hypothetical synaptic vesicle cycle in a presynaptic terminal.
The same synaptic vesicle is shown at different stages. Sites of docking, priming and fusion have been separated for clarity. NT, neurotransmitter.
Adapted from Südhof TC (1995) The synaptic vesicle cycle: a cascade of protein–protein interactions. Nature375, 645–653, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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12. 12 Figure 7.11 The SNARE discovery.
Scheme of the experiment that identified the integral membrane proteins (SNAREs) of the vesicle and presynaptic plasma membranes of brain synapses. A stable complex between the NSF protein, the SNAPs and their membrane receptors can be isolated in the presence of a non- hydrolyzable analog of ATP (ATPγS). Inversely, the membrane-bound form of the NSF protein is released from the membranes to the cytoplasm by ATP hydrolysis (i.e. in the presence of ATP and Mg2+). Solubilized brain membranes and NSF–SNAPs were immobilized on beads via a specific anti-myc antibody (NSF is tagged with the marker myc), in the presence of the non- hydrolyzable analog of ATP, ATPγS, and in the absence of Mg2+. The stable complex [NSF–SNAP–membrane proteins] was thus captured. It was then dissociated in the presence of ATP and Mg2+ in NSF on the one hand and the complex SNAPs-membrane proteins on the other hand. Eluted proteins were collected and SNAREs characterized. For clarity, solubilized membrane proteins are represented by a black rectangle inserted in a lipid bilayer.
From Rothman JE (1994) Intracellular membrane fusion. Adv. Second Messenger Phosphoprotein Res.29, 81–96, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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13. Figure 7.12 The structures of three SNARE subfamilies and model of a trans-SNARE complex.
(a) Qa-SNAREs have N-terminal antiparallel three-helix bundles. Qbc-SNAREs represent a small subfamily of SNAREs – the SNAP-25 (25-kDa synaptosome-associated protein) subfamily – that contain two SNARE motifs connected by a linker that is frequently palmitoylated (zig-zag lines), and most of the members of this subfamily function in constitutive or regulated exocytosis. The various N-terminal domains of R-SNAREs are represented by a basic oval shape. Dashed domain borders highlight domains that are missing in some subfamily members. (b) Model of a trans-SNARE complex and synaptotagmin. The regions that interact are indicated with brackets.
Part (a) adapted from Jahn R, Scheller RH (2006) SNAREs-engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643, with permission. Part (b) adapted from Littleton JT, Bai J, Vyas B et al. (2001) Synaptotagmin mutants revel essential functions for the C2B domain in Ca-triggered fusion and recycling of synaptic vesicles in vivo. J. Neurosci.21, 1421–1433, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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14. Figure 7.13 Hypothetical SNARE conformational cycle during vesicle docking and fusion.
As an example, we consider three Q-SNAREs (Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on an acceptor membrane (red and green) and an R-SNARE on a vesicle (blue). Q-SNAREs, which are organized in clusters (top left), assemble into acceptor complexes. Acceptor complexes interact with the vesicular R-SNAREs through the N-terminal end of the SNARE motifs, and this nucleates the formation of a four-helical trans-complex. Trans-complexes proceed from a loose state (in which only the N-terminal portion of the SNARE motifs are ‘zipped up’) to a tight state (in which the zippering process is mostly completed), and this is followed by the opening of the fusion pore. In regulated exocytosis, these transition states are controlled by late regulatory proteins that include complexins (small proteins that bind to the surface of SNARE complexes) and synaptotagmin (which is activated by an influx of calcium). During fusion, the strained trans-complex relaxes into a cis-configuration. Cis-complexes are disassembled by the AAA1 (ATPases associated with various cellular activities) protein NSF (N-ethylmaleimide-sensitive factor) together with SNAPs (soluble NSF attachment proteins) that function as co-factors. The R- and Q-SNAREs are then separated by sorting (e.g. by endocytosis).
Adapted from Jahn R, Scheller RH (2006) SNAREs – engines for membrane fusion. Nat. Rev. Mol. Cell Biol.7, 631–643, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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15. Figure 7.14 Diaminopyridine (DAP) increases the duration and amplitude of motor endplate current. The postsynaptic endplate current of a frog sartorius muscle cell is evoked by stimulation (2 μA intensity, 5 ms duration) of the motor nerve (VH = −90 mV). (a) The average amplitude of the evoked postsynaptic current is 0.5 μA in control Ringer solution. (b) The postsynaptic current evoked by the same stimulation in the presence of DAP (1 mM) is always greater than 1 μA and can reach 3.3 μA. These inward currents are represented upwardly, which is unusual.
Adapted from Katz B, Miledi R (1979) Estimates of quantal content during chemical potentialization of transmitter release. Proc. R. Soc. Lond. B 205, 369–378, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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16. Figure 7.15 Botulinum toxins strongly decrease synaptic transmission without affecting presynaptic Ca2+ current.
(a) In the squid giant synapse, at the stellate ganglion, presynaptic and postsynaptic intracellular electrodes are implanted to allow simultaneous recording of the presynaptic Ca2+ current (voltage clamp mode) and the postsynaptic response (EPSP, current clamp mode). A presynaptic voltage step (ΔV) evokes a presynaptic Ca2+ current (ICa) and, after a delay, a postsynaptic response (EPSP, control). After injection of botulinum toxin (BoT) through the presynaptic electrode, the EPSP decreases with time. Note that in the same time the presynaptic Ca2+ current is unchanged. (b) Dose–response curve of the effect of botulinum neurotoxin E (BoNT/E) on SNAP-25 proteolysis (purple squares, continuous line) and glycine release (green circles, broken line) in cultured neurons. The right-hand panel shows a schema of SNAP-25 and the position of BoNT/E cleavage.
Part (a) adapted from Marsal J, Ruiz-Montasell B, Blasi J et al. (1997) Block of transmitter release by botulinum C1 action on syntaxin at the squid giant synapse. Proc. Natl. Acad. Sci. USA94, 14871–14876, with permission. Part (b) adapted from Keller JE et al. (2001) Uptake of botulinum neurotoxin into cultured neurons. Biochemistry43, 526–532, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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17. Figure 7.16 Cascade of events leading to neurotransmitter release and its clearance from the synaptic cleft.
Schematic of some steps of synaptic transmission (between the presynaptic action potential and the postsynaptic response, EPSP or IPSP). In the presynaptic and glial plasma membranes, only one example of each channel, pump or transporter, is represented owing to the lack of space. In the postsynaptic membrane, several examples of ionotropic glutamatergic (AMPA and NMDA) or GABAergic (GABAA) channels are represented. nT, neurotransmitter.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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18. Figure 7.17 Summary (example of the calyx of Held).
Time course of the signaling cascade, showing (top to bottom) the presynaptic action potential (AP) waveform and resulting Ca2+ current (ICa), (inferred) release rate and postsynaptic EPSC.
Adapted from Meinrenken CJ, Borst JGG, Sakmann B (2003) Local routes revisited: the space and time-dependence of the Ca2+ signal for phasic transmitter release at the rat calyx of Held. J. Physiol. 547, 665–689 with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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19. Figure A7.1 Miniature postsynaptic potentials.
Miniature endplate potentials (right mEPPS) recorded at the frog neuromuscular junction as shown (a). Recordings are obtained in the presence of a low external Ca2+ concentration (b).
Adapted from Fatt P, Katz B (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. (Lond.)117, 109–128, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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20. 20 From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure A7.2 Demonstration of the probabilistic nature of acetylcholine release at the neuromuscular junction.
(a) Recordings of spontaneous miniatures and evoked endplate potentials (mEPP and EPP). The nerve–muscle preparation is bathed in a low-Ca2+ high-Mg2+ concentration medium. In such conditions, spontaneous mEPPs have nearly the same unitary amplitude whereas evoked EPPs have an amplitude that is a multiple of the mEPP amplitude. (b) The distribution of mEPP amplitude is unimodal and their average amplitude is q = 0.4 mV. (c) Histogram of evoked EPPs recorded in response to a presynaptic action potential. The cases where no postsynaptic response is recorded (failures) are represented at 0 mV (18 cases). The theoretical distribution calculated from Poisson’s law (represented by the green line) fits the distribution of the amplitude of recorded EPPs (histogram). Numbers on top of the histogram peaks indicate equivalent mEPP charges.
Part (a) adapted from Liley AW (1956) The quantal component of the mammalian endplate potential. J. Physiol. (Lond.)133, 571–587, with permission. Parts (b) and (c) from Boyd IA, Martin AR (1956) The endplate potential in mammalian muscle. J. Physiol. (Lond.)132, 74–91, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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