Autoreceptors do not regulate routinely neurotransmitter release: focus on adrenergic systems

The theory that neurotransmitter release is regulated locally at the individual terminals of neurons has achieved a rapid and seemingly secure status in our understanding of neuronal function both in the periphery and in the central nervous system. This concept of negative feedback control through the monitoring of the perineuronal concentration of previously released transmitter has been extended to a multiplicity of transmitters and utilized to explain the mechanisms of action of diverse classes of drugs, ranging from antihypertensives to antidepressants. It is my view that negative feedback by terminal and by somadendritic receptors cannot account for the existing body of experimental work. Analyses of the profiles of action of agonists and antagonists, and of the per pulse release of transmitter in the absence of drugs in a variety if peripheral organ systems, as well as in superfused brain slices, demonstrates the need for alternate interpretations of the available data. Evidence is provided that the actions of agonists to inhibit transmitter release and that of antagonists to enhance release occur at different cellular loci and that the purported unitary action of these two classes that is so central to the validity of presynaptic theory is unsupportable.     

Presynaptic k-Opioid and Muscarinic Receptors Inhibit the Calcium-Dependent Component of Evoked Glutamate Release from Striatal Synaptosomes

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.     

Presynaptic alpha-receptor control of adrenergic transmitter release in blood vessels

The presynaptic alpha-adrenergic receptor control of transmitter release in vascular tissues is discussed. A model of adrenergic innervation of the vascular bed is proposed based on ultrastructural and histochemical evidence. Evidence is presented to support the concept of intermittent or periodic release of norepinephrine (NE) from the varicosity. Intermittency combined with a mechanism such as presynaptic control to ensure spatial distribution of release sites, along with a slow effector response and recovery, results in a smooth, generalized change in tone and an overall economy of transmitter. The effective concentration of NE around the presynaptic membrane is maintained for considerably less than 0.1 s. It is argued that the transient presence of transmitter in the synapse combined with intermittency of release does not favor accumulation of transmitter at the cleft at physiological frequencies or desensitization of presynaptic receptors. In addition, intermittency provides an explanation for why exogenous NE is more effective presynaptically in influencing release than endogenous NE. The importance of cleft width in presynaptic control of transmitter release, the possible complications caused by facilitation, and resolution of some apparent problems with the presynaptic hypothesis are also discussed.     

Evidence that noradrenergic transmitter release is regulated by presynaptic receptors

A review is provided of the evidence in support of the existence of prejunctional alpha adrenoceptors on noradrenergic nerve terminals as well as the evidence for their physiological importance. The use of alpha-adrenoceptor agonists and antagonists has provided convincing data in support of the presynaptic receptor hypothesis. Moreover, there is ample evidence for the location of alpha adrenoceptors on nerve terminals. This evidence has often been forgotten in arguments opposing the presynaptic alpha-adrenoceptor hypothesis. The precise physiological role of presynaptic alpha adrenoceptors is still an open question, but there is support from a wide range of experiments in favor of a physiological role. Although it is not known which of these functions is most important, presynaptic alpha adrenoceptors may: regulate the pulse-to-pulse regulation of norepinephrine release during nerve stimulation, prevent noise, and protect the neuroeffector cell from excessive activation by transmitter during periods of rest or as physiological antagonists to the facilitation of transmitter release. In summary, evidence reviewed here strongly supports the existence of presynaptic alpha adrenoceptors. These receptors are clearly important pharmacologically and may play a physiological role in noradrenergic transmission. The exact physiological function must await further experimentation.     

Intrasynaptic ephaptic feedback in central synapses]

A review is given of experiments performed in the author's laboratory on slices from the rat visual cortex and hippocampus. The aim was to test the existence of the positive feedback in central synapses according to a mechanism of electrical (ephatic) linking proposed by A. L. Byzow. The hypothesis predicts that, in a subset of central synapses, artificial postsynaptic membrane potential (MP) hyperpolarization should increase the amplitude of the excitatory postsynaptic current (EPSC) and potential (EPSP) not only due to a deviation from the equilibrium potential but also due to increased presynaptic transmitter release. In a part of the experiments, we found changes in several traditional parameters of transmitter release during hyperpolarization: number of response failures, coefficient of variation of response amplitude and quantal content of minimal EPSC/EPSP. The effects were especially prominent for the giant mossy fibre-CA3 synapses. For them, "supralinear" amplitude-voltage relations at hyperpolarized membrane potentials and voltage--dependent paired--pulse facilitation ratios were found. All these "non-classical" effects disappeared when composite, rather than minimal, EPSCs were evoked. These data were consistent with simulation experiments performed on the Byzov's synaptic model with the ephaptic feedback and therefore they strengthen the hypothesis. Independent of their interpretation, the data reveal a novel feedback mechanism. The mechanism provides a possibility for the central postsynaptic neurone to control the efficacy of a subset of synapses via postsynaptic MP modifications. The mechanism can essentially increase the efficacy of large ("perforated") synapses. It explains the significance of the increased number of such synapses following experimental challenges such as leading to induction of the long-term potentiation or to behavioural conditioning.     

Muscarinic receptors modulating acetylcholine release from insect synaptosomes

1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.     

Fast homeostatic plasticity of inhibition via activity-dependent vesicular filling

Synaptic activity in the central nervous system undergoes rapid state-dependent changes, requiring constant adaptation of the homeostasis between excitation and inhibition. The underlying mechanisms are, however, largely unclear. Chronic changes in network activity result in enhanced production of the inhibitory transmitter GABA, indicating that presynaptic GABA content is a variable parameter for homeostatic plasticity. Here we tested whether such changes in inhibitory transmitter content do also occur at the fast time scale required to ensure inhibition-excitation-homeostasis in dynamic cortical networks. We found that intense stimulation of afferent fibers in the CA1 region of mouse hippocampal slices yielded a rapid and lasting increase in quantal size of miniature inhibitory postsynaptic currents. This potentiation was mediated by the uptake of GABA and glutamate into presynaptic endings of inhibitory interneurons (the latter serving as precursor for the synthesis of GABA). Thus, enhanced release of inhibitory and excitatory transmitters from active networks leads to enhanced presynaptic GABA content. Thereby, inhibitory efficacy follows local neuronal activity, constituting a negative feedback loop and providing a mechanism for rapid homeostatic scaling in cortical circuits.     

The action of some analogues of the excitatory amino acids in the dentate gyrus of the rat

We have confirmed that gamma-D-glutamylglycine and the L-isomer of 2-amino-4-phosphonobutyric acid, and have shown also that L-2-amino-5-phosphonovaleric (L-APV) acid, are antagonists of synaptic excitations of dentate granule cells induced from both lateral and medial perforant paths. The N-methyl-D-aspartic acid (NMDA) antagonist D-APV is without effect. The synaptic antagonists reduce the presynaptic fibre volley particularly in the lateral path, suggesting that a reduced transmitter output contributes to their action. NMDA receptors exist upon the granule cells, but they are not involved with these synaptic process.     

Neurotransmitters as ion shuttles in regulation of neural activity.

A molecular framework is described within which a single neural cell can modulate its excitability, or the quantity of transmitter released upon stimulation, in relationship to past stimulation. The key elements in this regulating system are complexation in the synaptic area by the transmitter molecule with extracellular ions, interaction of the complexed transmitter with the presynaptic receptor, possibly followed by reuptake of complexed transmitter.A number of mechanisms are suggested by which the transmitter/metal ion complex can regulate cellular function. Calculations are made to estimate the possible degree of change in the interior calcium concentration of a catecholaminergic cell by a calcium ion complex formed in the synapse. Experimental evidence is cited, which(a) documents the existence in the catecholaminergic cell of the necessary machinery for a calcium-ion-regulated cell-use registry device.(b) supports the hypothesis that catecholamines transport metal ions in neural systems, and(c) indicates that the ionic shuttle function of neurotransmitters plays a significant, but not exclusive, role in the transport of calcium. Calcium transported in this manner may be uniquely distinguishable from that derived from other sources of intracellular calcium in its temporal or spatial distribution. The existing evidence is discussed and rationalized with respect to the hypothesis that one of the chief presynaptic functions of many neurotransmitters is to feedback regulate cell function by performance as an ion shuttle.     

Nanodomain coupling between Ca2+ channels and Ca2+ sensors promotes fast and efficient transmitter release at a cortical GABAergic synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.     

Adrenergic presynaptic receptors: examination of a hypothesis in guinea pig vas deferens.

The hypothesis was examined that phenoxybenzamine enhances both the overflow of noradrenaline and the mechanical response in guinea pig vas deferens by blockade of presynaptic inhibitory receptors located on adrenergic nerve terminals which serve a negative-feedback function. Preparations were stimulated with a constant small number of pulses but at three different frequencies (1, 5, and 15 Hz) and the relative effectiveness of phenoxybenzamine in enhancing overflow assessed. According to the presynaptic receptor hypothesis inhibition of transmitter output should increase with increasing frequency due to increased activation of receptor sites by endogenously released noradrenaline. The antagonist enhanced the overflow of tritium but did so to a similar extent at all three frequencies, regardless of the length of the interval between pulses. Similarly, no evidence for a greater sensitization of the mechanical response by phenoxybenzamine at the higher frequencies was obtained. The conditions of the present experiment were considered optimal for the operation of the negative-feedback system and the results indicate that the physiological relevance of such a system is questionable.     

Long-term potentiation in the piriform cortex is blocked by lead

Summary 1. Long-term potentiation (LTP) is a prolonged increase in synaptic efficacy that is triggered by a brief tetanic stimulation at certain central synapses. LTP is one of the best available model systems available to the neurophysiologist of neuronal plasticity such as that underlying learning and memory.2. We have studied the susceptibility of LTP to blockade by lead as a test of the hypothesis that the negative effect of lead on intelligence in children may result from interference with this process. LTP was studied in slices of rat piriform cortex. At this site, as in many other central synapses, LTP requires activation of postsynapticN-methyl-d-aspartate (NMDA) receptors, and we investigated whether lead actions, if any, were mediated via effects on NMDA-activation ion channels or, alternatively, at voltage-activated calcium channels.3. We find that lead blocks LTP at low micromolar concentrations. However, concentrations of lead that totally block LTP had no apparent effect on either NMDA-activated responses or presynaptic calcium channels, as monitored by transmitter release from presynaptic terminals.4. While the mechanism of lead blockade of LTP remains to be determined, these observations are consistent with the hypothesis that the cognitive effects of lead neurotoxicity may result from effects on LTP.     

Glutamate inhibitors in the crayfish neuromuscular junction

1. The effects of chlorisondamine and TI-233 on the crayfish neuromuscular junction were investigated in order to compare the action of glutamate with that of the excitatory transmitter. 2. The glutamate-induced synaptic current was inhibited by both of these two drugs. Excitatory junctional potentials were significantly reduced by chlorisondamine, whereas they were increased by TI-233. 3. It is suggested that chlorisondamine and TI-233 are powerful non-competitive antagonists for glutamate. 4. A quantum analysis of extracellular EJPs demonstrated that chlorisondamine did not possess presynaptic action in the crayfish neuromuscular junction. Chlorisondamine shortened the decay phase of extracellular EJPs, and the decay was frequently fitted by a double exponential in relatively low concentrations. 5. Semilogarithmic plots of the decay phase of the glutamate current evoked by a short glutamate pulse were nearly linear, but they shifted from linearity to some extent in the presence of chlorisondamine, showing prolongation of the glutamate current tails. 6. When TI-233 was added to the bathing solution at a concentration of 0.1 mM, the quantum content of extracellular EJPs was increased by about two times, but the average unit size was not changed. 7. There was no change in the rise time and the decay phase of the glutamate potential in the presence of TI-233. 8. Pharmacological difference between glutamate responses and EJPs was revealed in the presence of chlorisondamine and TI-233. Unless this difference can be explicated with a reasonable explanation on the glutamate transmitter hypothesis, it is difficult to confirm that glutamic acid is an excitatory transmitter at the crayfish neuromuscular junction.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

Diversification of synaptic strength: presynaptic elements

Synapses are not static; their performance is modified adaptively in response to activity. Presynaptic mechanisms that affect the probability of transmitter release or the amount of transmitter that is released are important in synaptic diversification. Here, we address the diversity of presynaptic performance and its underlying mechanisms: how much of the variation can be accounted for by variation in synaptic morphology and how much by molecular differences? Significant progress has been made in defining presynaptic structural contributions to synaptic strength; by contrast, we know little about how presynaptic proteins produce normally observed functional differentiation, despite abundant information on presynaptic proteins and on the effects of their individual manipulation. Closing the gap between molecular and physiological synaptic diversification still represents a considerable challenge.     

Comparative regulation of potassium and amphetamine induced release of 3H-norepinephrine from rat brain via presynaptic mechanisms.

This study examined the ability of various drugs to modify the potassium (K) or d-amphetamine (d-A) induced release of ³H-norepinephrine ³HNE) from chopped rat cortical tissue. The K induced release of the transmitter, which occurs from reserpine sensitive sites of cortical tissue, was significantly reduced by the beta receptor antagonist propranolol, the alpha receptor agonist clonidine and also by PGE₂. Pretreatment with eicosatetrynoic acid, an inhibitor of prostaglandin synthesis, did not influence the effect of clonidine on ³HNE release; thus this latter effect appears to be independent of enhanced prostaglandin formation. The proposed alpha receptor mediated negative feedback exhibits stereospecificity since addition of exogenous 1-, but not d-, NE decreased release of the transmitter. Blockade of alpha receptors by phentolamine or stimulation of beta receptors by isoproterenol significantly enhanced the K induced release of ³HNE from cortical tissue. By contrast, the d-A induced release of ³HNE which occurs from reserpine-insensitive sites, was reduced by propranolol and clonidine; and was not altered by phentolamine, isoproterenol or PGE₂. These data indicate that the K, but no d-A, induced release of ³HNE from cortical tissue is modified in accordance with postulated presynaptic negative and positive feedback mechanisms.     

Presynaptic receptors regulating the time course of neurotransmitter release from vertebrate nerve endings

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β₁-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.     

Do they or don't they? Presynaptic Receptors and the Question of Autoregulation of Neurotransmitter Release sponsored by the New York Academy of Sciences, Philadelphia, PA, USA, December 4-6, 1989

It is felt that the objectives of this conference were met and a great deal of new information was presented on the characterization of presynaptic receptors and their mechanisms of action and pathophysiological roles. There is still a difference of opinion concerning the precise physiological role of presynaptic autoreceptors. It is possible that autoregulation may not exist at all junctions or synapses. It is also not clear if autoregulation takes place at each varicosity, upstream of terminal varicosities, or by lateral regulation (one varicosity to another). Possible physiological functions of autoregulation may include: pulse-to-pulse regulation of transmitter release during nerve stimulation; upstream or lateral regulation of release from various varicosities; modulation of release during periods of rest or during periods in which the frequency of nerve stimulation is low; and modulation of release during periods of very intense stimulation. Finally, autoregulation may serve as a physiological antagonist to facilitation of transmitter release that is known to take place in some neurons during nerve stimulation. The conference provided a clearer understanding of the objections to attributing a physiological role to autoregulation and an understanding of what information is lacking. Challenges for future research will be to unravel the precise physiological and pathophysiological roles of presynaptic receptor-modulation of neurotransmission, to explain better why some results are inconsistent with the autoregulation hypothesis, and to define further the mechanisms by which activation of autoreceptors and heteroreceptors are linked to inhibition or facilitation of transmitter release.     

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.     

Evidence for the Action of Endogenous Adenosine in the Rabbit Retina: Modulation of the Light-Evoked Release of Acetylcholine

Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent. The cholinergic amacrine cells of the rabbit retina were labeled with [3H]choline, and the effects of enzymatic adenosine degradation or adenosine antagonists on the light-evoked efflux of acetylcholine were evaluated. When endogenous adenosine was degraded by addition of adenosine deaminase, the light-evoked release of radioactivity derived from [3H]choline was significantly increased compared with control values. A similar response was observed when rabbit eyecups were superfused with a selective adenosine A1 receptor antagonist. The effect elicited by adenosine deaminase could be almost completely reversed by addition of cyclopentyladenosine, a highly selective A1 receptor agonist. These effects were observed in either the presence or the absence of picrotoxin. The results demonstrate a modulation of retinal physiology by adenosine.