Action potentials must admit calcium to evoke transmitter release.

There are two hypotheses to explain how neurons release transmitter. The calcium hypothesis proposes that membrane depolarization is necessary only for opening calcium channels and increasing internal calcium concentration ([Ca2+]i) near membrane transmitter-release sites. These calcium ions trigger a transient release of neurotransmitter. The calcium-voltage hypothesis postulates that voltage induces a conformational change in a membrane protein rendering it sensitive to calcium such that, in the presence of high [Ca2+]i, depolarization directly triggers transmitter release. Here we report that when calcium influx is blocked by cobalt or manganese ions in a calcium-free Ringer, as measured with Fura-2, and [Ca2+]i is elevated by liberation from a caged calcium compound, transmitter release at the crayfish neuromuscular junction is unaffected by presynaptic action potentials. These results support the calcium hypothesis.     

Presynaptic spike broadening reduces junctional potential amplitude

Presynaptic modulation of action potential duration may regulate synaptic transmission in both vertebrates and invertebrates. Such synaptic plasticity is brought about by modifications to membrane currents at presynaptic release sites, which, in turn, lead to changes in the concentration of cytosolic calcium available for mediating transmitter release. The 'primitive' neuromuscular junction of the jellyfish Polyorchis penicillatus is a useful model of presynaptic modulation. In this study, we show that the durations of action potentials in the motor neurons of this jellyfish are negatively correlated with the amplitude of excitatory junctional potentials. We present data from in vitro voltage-clamp experiments showing that short duration voltage spikes, which elicit large excitatory junctional potentials in vivo, produce larger and briefer calcium currents than do long duration action potentials, which elicit small excitatory junctional potentials.     

Activation of protein kinase C augments evoked transmitter release

In view of the emerging role of the phosphoinositide system in cellular communication we examined its involvement in quantal-transmitter release, which is a key element in synaptic transmission. Transmitter release is normally activated by an increase in intracellular calcium, achieved either by entry of calcium ions through the presynaptic membrane or by intracellular calcium liberation. One of the targets of the phosphoinositide signalling system is the enzyme protein kinase C (PKC), which can be activated experimentally by tumour promoting phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). Such activation of PKC may be implicated in transmitter release in two ways. First, phorbol esters were found to increase secretion and enhance calcium currents; it might therefore be expected that they would increase synaptic transmitter release. But phorbol esters also inhibit the calcium current in dorsal root ganglion neurones. We report that the phorbol ester TPA augments synaptic transmission at the neuromuscular junction by increasing transmitter liberation. Activation of PKC also depends synaptic depression.     

Is hyperosmotic neurosecretion from motor nerve endings a calcium-dependent process?

Spontaneous liberation of neurotransmitter quanta is strongly affected by the osmotic pressure of the extracellular fluid. Elevation of the osmolarity by 20-30% increases the rate of release from motor nerve endings by more than one order of magnitude. In this respect the neuromuscular junction resembles some other secretory systems. The mechanism of this hyperosmotic neurosecretion is not yet understood; extracellular calcium ions are not directly responsible, since this effect can be produced in their absence. Recently, it has been suggested that the liberation of neurotransmitter is regulated by the intracellular concentration of free calcium ions. We have therefore examined the hypothesis that hyperosmotic neurosecretion originates from an increase in internal calcium concentration ([Ca]in). At the frog neuromuscular synapse however, it is impossible at present to estimate directly free [Ca]in; hence we used an indirect technique, which is based on two assumptions; first, the frequency of the miniature endplate potentials (m.e.p.p.s.) reflects free [Ca]in. Second, the movement of calcium ions across the presynaptic membrane is governed by the electrochemical gradient, and by the calcium conductance (g(Ca)). If hyperosmotic neurosecretion is caused by an increase in [Ca]in, then increasing g(Ca), under reversed electrochemical gradient for the calcium should cause a reduction in the effect of hyperosmotic stress on transmitter release. We report that hyperosmotic neurosecretion is dependent on [Ca]in.     

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.     

Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse

Glycine and GABAA (gamma-aminobutyric acid A) receptors are inhibitory neurotransmitter-gated Cl- channels localized in postsynaptic membranes. In some cases, GABAA receptors are also found presynaptically, but they retain their inhibitory effect as their activation reduces excitatory transmitter release. Here we report evidence for presynaptic ionotropic glycine receptors, using pre- and postsynaptic recordings of a calyceal synapse in the medial nucleus of the trapezoid body (MNTB). Unlike the classical action of glycine, presynaptic glycine receptors triggered a weakly depolarizing Cl- current in the nerve terminal. The depolarization enhanced transmitter release by activating Ca2+ channels and increasing resting intraterminal Ca2+ concentrations. Repetitive activation of glycinergic synapses on MNTB neurons also enhanced glutamatergic synaptic currents, indicating that presynaptic glycine receptors are activated by glycine spillover. These results reveal a novel site of action of the transmitter glycine, and indicate that under certain conditions presynaptic Cl- channels may increase transmitter release.     

A neuromodulator of synaptic transmission acts on the secretory apparatus as well as on ion channels

The mechanisms that underlie synaptic plasticity have been largely inferred from electrophysiological studies performed at sites remote from synaptic terminals. Thus the mechanisms involved in plasticity at the secretory sites have remained ill-defined. We have now used somatic synapses of cultured Helisoma neurones to directly assess presynaptic ion conductances and study the secretory apparatus. At these synapses we determined the actions of a modulatory neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFa), on the release of the neurotransmitter acetylcholine (ACh). Using voltage- and calcium-clamp techniques, we have demonstrated that FMRFa causes a presynaptic inhibition of ACh release by (1) reducing the magnitude of the voltage-dependent calcium current, and (2) regulating the secretory apparatus. The photolabile calcium cage, nitr-5 (refs 3-8), was dialysed into the presynaptic cell. In response to ultraviolet light, calcium was released from nitr-5 and ACh secretion was stimulated. Under conditions of constant internal calcium, FMRFa reduced the rate of ACh release. Thus we conclude that FMRFa reduces the influx of calcium during the action potential and decreases the sensitivity of the secretory apparatus to elevated internal calcium, thereby contributing to a presynaptic inhibition of transmitter release.     

Membrane depolarization evokes neurotransmitter release in the absence of calcium entry

The discovery that Ca2+ is necessary for the release of neurotransmitter, the primary means by which nerve cells communicate, led to the calcium hypothesis of neutransmitter release, in which release is initiated after an action potential only by an increase in intracellular Ca2+ concentration near the release sites and is terminated (1-2 ms) by the rapid removal of Ca2+. Since then, the calcium-voltage hypothesis has been proposed, in which the depolarization of the presynaptic terminals has two functions. First, in common with the calcium hypothesis, the Ca2+ conductance is increased, thereby permitting Ca2+ entry. Second, a conformational change is induced in a membrane molecule that renders it sensitive to Ca2+, and then binding of Ca2+ to this active form triggers release of neurotransmitter. When the membrane is repolarized, the molecule is inactivated and release is terminated, regardless of the local Ca2+ concentration at that moment. This hypothesis, in contrast to the calcium hypothesis, accounts for the insensitivity of the time course of release to experimental manipulations of intracellular Ca2+ concentration. Furthermore, it explains rapid termination of release after depolarization, even though Ca2+ concentration may still be high. Here we describe experiments that distinguish between these two hypotheses and find that our results support the calcium voltage hypothesis.     

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

Glutamate is taken up avidly by glial cells in the central nervous system. Glutamate uptake may terminate the transmitter action of glutamate released from neurons, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.     

Is extracellular calcium buffering involved in regulation of transmitter release at the neuromuscular junction?

During synaptic activity at the neuromuscular junction, sodium, potassium and calcium ions flow through both the postsynaptic and presynaptic membrane. These ionic fluxes can cause changes in the local extracellular concentration in the synaptic gap: a decrease in the concentration of the inwardly flowing ions (sodium and calcium) and an increase in the outwardly flowing potassium ions. To check whether depletion of calcium ions in the synaptic gap is involved in transmitter release, we have used calcium buffers to keep the extracellular calcium concentration almost constant. The expectation was that if depletion does occur, transmitter release will increase; if no depletion occurs, there will be no change in quantal release when the calcium concentration is the same in buffered and unbuffered bathing solutions. We report here that, surprisingly, perfusing the frog neuromuscular preparation with a calcium-buffered solution caused a decrease in transmitter release compared with that in an unbuffered solution with the same calcium concentration. This presumably indicates that the calcium level in the synaptic cleft is higher than that in the bulk extracellular medium. If such a mechanism operates physiologically, it may provide an energetically economical way to determine the level of evoked transmitter release and thus synaptic efficiency.     

Intracellular calcium dependence of transmitter release rates at a fast central synapse

Calcium-triggered fusion of synaptic vesicles and neurotransmitter release are fundamental signalling steps in the central nervous system. It is generally assumed that fast transmitter release is triggered by elevations in intracellular calcium concentration ([Ca2+]i) to at least 100 microM near the sites of vesicle fusion. For synapses in the central nervous system, however, there are no experimental estimates of this local [Ca2+]i signal. Here we show, by using calcium ion uncaging in the large synaptic terminals of the calyx of Held, that step-like elevations to only 10 microM [Ca2+]i induce fast transmitter release, which depletes around 80% of a pool of available vesicles in less than 3 ms. Kinetic analysis of transmitter release rates after [Ca2+]i steps revealed the rate constants for calcium binding and vesicle fusion. These show that transient (around 0.5 ms) local elevations of [Ca2+]i to peak values as low as 25 microM can account for transmitter release during single presynaptic action potentials. The calcium sensors for vesicle fusion are far from saturation at normal release probability. This non-saturation, and the high intracellular calcium cooperativity in triggering vesicle fusion, make fast synaptic transmission very sensitive to modulation by changes in local [Ca2+]i.     

Possible association of alcohol tolerance with increased synaptic Ca2+ sensitivity

The inhibitory effect of ethanol on neurotransmitter release has been suggested to be due to either reduced Ca2+ entry or increased removal of free intracellular Ca2+ from the synapse. The use of the Ca2+ ionophore, A23187, to allow direct access of external Ca2+ to the presynaptic interior should help to determine which of these two factors is the more important, as ethanol should inhibit A23187-induced release of transmitter only if increased Ca2+ removal from the synapse is important. Here we show in rat striatal slices that, although 3H-dopamine release evoked by depolarization with 40 mM K+ is inhibited by 50 mM ethanol, the release evoked by A23187 is enhanced by the presence of ethanol in vitro. The results suggest that ethanol reduces depolarization-induced transmitter release by reducing Ca2+ entry to the presynaptic terminal. However, for brain slices taken from rats made tolerant to ethanol, 3H-dopamine release in the absence of ethanol showed altered characteristics; both K+ depolarization and A23187 released a significantly greater fraction of 3H-dopamine from these slices than from controls. Thus tolerance to the inhibitory effect of ethanol on release may develop by a mechanism involving increased sensitivity of the terminal to Ca2+ entry.     

Quantal transmitter secretion from myocytes loaded with acetylcholine.

It is well known that transmitter secretion requires specialized secretory organelles, the synaptic vesicles, for the packaging, storage and exocytotic release of the transmitter. Here we report that when acetylcholine (ACh) is loaded into an isolated Xenopus myocyte, there is spontaneous quantal release of ACh from the myocyte which results in activation of its own surface ACh channels and the appearance of membrane currents resembling miniature endplate currents. This myocyte secretion probably reflects Ca(2+)-regulated exocytosis of ACh-filled cytoplasmic compartments. Furthermore, step depolarization of the myocyte membrane triggers evoked ACh release from the myocyte with a weak excitation-secretion coupling. These findings suggest that quantal transmitter secretion does not require secretory pathways unique to neurons and that the essence of presynaptic differentiation may reside in the provision of transmitter supply and modification of the preexisting secretion pathway.     

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.     

Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurones

We have identified a serotonin-sensitive K+ channel with novel properties. The channel is active at the testing potential; its gating is moderately affected by membrane potential and is not dependent on the activity of intracellular calcium ions. Application of serotonin to the cell body or intracellular injection of cyclic AMP causes prolonged and complete closure of the channel, thereby reducing the effective number of active channels in the membrane. The closure of the channel can account for the increases in the duration of the action potential, Ca2+ influx, and transmitter release which underlie behavioural sensitization, a simple form of learning.     

GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels

The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones.     

Synaptic potentiation onto habenula neurons in the learned helplessness model of depression

The cellular basis of depressive disorders is poorly understood. Recent studies in monkeys indicate that neurons in the lateral habenula (LHb), a nucleus that mediates communication between forebrain and midbrain structures, can increase their activity when an animal fails to receive an expected positive reward or receives a stimulus that predicts aversive conditions (that is, disappointment or anticipation of a negative outcome). LHb neurons project to, and modulate, dopamine-rich regions, such as the ventral tegmental area (VTA), that control reward-seeking behaviour and participate in depressive disorders. Here we show that in two learned helplessness models of depression, excitatory synapses onto LHb neurons projecting to the VTA are potentiated. Synaptic potentiation correlates with an animal's helplessness behaviour and is due to an enhanced presynaptic release probability. Depleting transmitter release by repeated electrical stimulation of LHb afferents, using a protocol that can be effective for patients who are depressed, markedly suppresses synaptic drive onto VTA-projecting LHb neurons in brain slices and can significantly reduce learned helplessness behaviour in rats. Our results indicate that increased presynaptic action onto LHb neurons contributes to the rodent learned helplessness model of depression.     

Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.     

Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus

Long-term potentiation (LTP) is a widely studied model of the synaptic basis of information storage in the mammalian brain. The induction of LTP is triggered by the postsynaptic entry of calcium through the channel associated with the N-methyl-D-aspartate (NMDA) receptor, whereas its maintenance is mediated, at least in part, by presynaptic mechanisms. To explain how postsynaptic events can lead to an increase in transmitter release, we have postulated the existence of a retrograde messenger to carry information from the postsynaptic side of the synapse to recently active presynaptic terminals. Candidates for a retrograde messenger include arachidonic acid or one of its lipoxygenase metabolites. Here we report that weak activation of the perforant path, when given in the presence of arachidonic acid, leads to a slow-onset persistent increase in synaptic efficacy both in vivo and in vitro. The activity-dependent potentiation thus produced is accompanied by an increase in the release of glutamate, and is non-additive with tetanus-induced LTP. These observations indicate a role for arachidonic acid as a retrograde messenger in the later, but not the initial, stages of LTP.