PRESYNAPTIC IONOTROPIC RECEPTORS AND CONTROL OF TRANSMITTER RELEASE

PRESYNAPTIC IONOTROPIC RECEPTORS AND CONTROL OF TRANSMITTER RELEASE Holly S. Engelman and Amy B. MacDermott Presynaptic nerve terminals are dynamic structures that release vesicular packages of neurotransmitter, affecting the activity of postsynaptic cells. This release of transmitter occurs both spontaneously and after the arrival of an action potential at presynaptic terminals. How is the release process modulated? Although ionotropic receptors are commonly regarded as postsynaptic elements that mediate the effect of the released chemical signals, a wide variety of ionotropic receptors have also been found on presynaptic membranes near release sites, where they powerfully influence vesicle fusion. Here, we provide an overview of the presynaptic ionotropic receptors that modulate transmitter release, focusing on their proposed mechanisms of action. Department of Physiology and Cellular Biophysics and the Center for Neurobiology and Behavior, Columbia University, New York, USA. Correspondence to A.B.M. e-mail: abm1@columbia.edu doi:10.1038/nrn1297 In the nervous system, information is transferred between neurons at chemical synapses. Action potential invasion of the presynaptic terminal elicits Ca 2+ entry and subsequent release of vesicular packages of transmitter that activate postsynaptic receptors. The strength of synaptic communication is dependent on the postsynaptic sensitivity to transmitter and on the probability of transmitter release in response to action potential invasion at each individual release site. Synapses with a high probability of vesicular release influence excitability of postsynaptic targets more reliably than synapses with a low probability of release. Moreover, terminals with high probability of release are more likely to undergo activitydependent depression of transmission, whereas terminals with low probability of release readily show activitydependent facilitation of release. Each type of nerve terminal has unique molecular and morphological elements that contribute to its release properties 1 (FIG. 1).For example, the arrangement of voltage-gated ion channels in the pre-terminal and presynaptic membrane determines the shape of presynaptic action potentials, which in turn regulates the time course and amplitude of presynaptic Ca 2+ influx. Similarly, the number and arrangement of voltage-gated Ca 2+ channels (VGCCs) with respect to presynaptic vesicles also affects the efficacy of transmitter release. Multiple types of VGCC can be expressed at different locations on the same terminal and influence transmitter release differently 2 4, and intracellular Ca 2+ stores in the presynaptic terminal can also contribute to the dynamic changes in Ca 2+ concentration that influence transmitter release 4 6. Ionotropic transmitter receptors in the presynaptic membrane impact on these molecular and cellular elements, dynamically modulating release properties and synaptic strength. Here, we will discuss the effect that activation of different receptor types has on transmitter release. We will see that their effects on action potential-evoked release are sometimes quite different from their effects on spontaneous release (BOX 1). We will contrast the impact of anionic and cationic receptor activation, and consider when receptor permeability to Ca 2+ becomes crucial for the modulation of release. On the basis of these observations, it will become clear that permeability of the different receptors is not predictive of their effects on transmitter release in a simple way. Rather, cellular and molecular elements that form part of the terminal are also crucial in determining the nature of the modulation. Finally, we will present evidence for the activation of receptors by endogenous agonists and discuss the consequent frequency dependence of synaptic transmission. NATURE REVIEWS NEUROSCIENCE VOLUME 5 FEBRUARY 2004 135

Na + channel Na + channel Ca 2+ -activated K + channel SHUNTING A phenomenon by which membrane depolarization that is induced by a given current is attenuated because of an enhanced membrane conductance. INPUT RESISTANCE The voltage change elicited by the injection of current into a cell, divided by the amount of current injected. EVOKED POSTSYNAPTIC CURRENTS Synaptic currents that are elicited by firing an action potential in a population of axons. They commonly reflect release from many presynaptic fibres, although it is possible to study release from one or a few fibres using so-called minimal stimulation protocols. Ca 2+ channel Ligand-gated ion channel Ca 2+ store Ca 2+ channel Ligand-gated ion channel Fast-inactivating K + channel Figure 1 Presynaptic factors that influence transmitter release. Various channels can be expressed near transmitter release sites including those that enhance N t release, R i such Nas voltagegated Na + channels and voltage-gated Ca 2+ channels, and those that suppress release such as i different types of K + channels (fast-inactivating K + channels, Ca 2+ -dependent K + channels, inward rectifier channels (I h )). Ca 2+ stores in the terminals also contribute to the regulation of transmitter release. Activation of ligand-gated ion channels or presynaptic ionotropic receptors can modulate these cellular elements, thereby influencing release. Receptor-mediated presynaptic modulation Modulation of transmitter release at central synapses was first hypothesized by Frank and Fuortes 7,who found that activation of flexor afferent fibres led to depression of monosynaptic transmission onto extensor muscle motoneurons, despite the lack of direct synaptic connections. This depression, subsequently termed presynaptic inhibition, was later found to depend on the activation of a Cl conductance in the presynaptic membrane, mediated by presynaptic GABA A (γ-aminobutyric acid, subtype A) receptors (FIG. 2;see REF. 8 for review). Owing to the temporal correspondence between presynaptic inhibition and the depolarization of the primary afferent terminals, Eccles et al. 9 suggested that depolarization of the afferents was responsible for the inhibition of transmitter release. This seemingly paradoxical inhibition of excitation could be due to depolarization-dependent inactivation of the voltage-gated Na + channels that drive the action potential 10,11,or to the Cl -mediated SHUNTING of the Na + currents, as found at the crayfish neuromuscular junction. Indeed, at the crayfish synapse, shunting does not depend on depolarization, as this effect was accompanied by membrane hyperpolarization 12, and therefore could not be due to Na + channel inactivation. Although these early examples of modulation of transmitter release were shown to depend on the presynaptic action of an ionotropic receptor, subsequent experimental attention focused on the presynaptic actions of G-protein-coupled receptors (BOX 2). I h Only over the past ten years has new evidence been put forward for potent regulation of transmitter release through presynaptic ionotropic receptors. Presynaptic ionotropic receptors that enhance as well as inhibit transmitter release have been identified. For example, there is clear evidence for enhanced GABA and glutamate release that depends on activation of presynaptic nicotinic acetylcholine (nach) receptors cation channels that are permeable to Ca 2+ and monovalent cations. In this case, the observation that Ca 2+ entry through nach channels enhanced spontaneous release provided evidence for receptor localization near release sites 13,14.In other studies, activation of nach receptors enhanced spontaneous release, but the effect was blocked by tetrodotoxin (TTX) 15 17,a selective blocker of the voltage-gated Na + channels. The fact that the agonistenhanced release depended on action potential firing indicated that the nach receptors might have been remote from the release sites. In addition to receptors for ACh and GABA, other ionotropic neurotransmitter receptors are found on presynaptic terminals in many areas of the central nervous system (CNS). In the following sections, we will discuss the specific mechanisms by which these ionotropic receptors and their cognate ligands are thought to influence transmitter release. Modulation through membrane potential Ligand-gated receptors that are permeable to anions and to monovalent cations modulate transmitter release primarily by influencing presynaptic membrane potential and INPUT RESISTANCE. Anionic presynaptic receptors might hyperpolarize or depolarize nerve terminals, whereas non-selective cationic receptors depolarize the presynaptic membrane. Depolarization and the subsequent activation of VGCCs can enhance spontaneous neurotransmitter release, an effect that is sensitive to VGCC blockade. By contrast, effects of the activation of presynaptic anionic receptors on evoked release are more difficult to predict. Depolarization that activates VGCCs and enhances spontaneous release might directly enhance evoked release. Alternatively, inhibition of evoked release could occur, owing to blockade of action potential invasion of the presynaptic terminal or decreasing the Ca 2+ entry that is necessary for evoked release (FIG. 3). In some cases, the concentration of presynaptic agonists determines the sign of the effect, giving it a biphasic nature. At low agonist concentrations, activation of a few receptors might cause a small depolarization, enhancing evoked release by bringing the membrane potential closer to threshold. At higher agonist concentrations, activation of many receptors might shunt or depress action potential amplitude to decrease release. Alternatively, complex dependence on agonist concentration is sometimes an indication of recruitment of several different receptor types. A biphasic effect of presynaptic receptors might be quite common but has been investigated in few systems. We now turn our attention to some specific examples of presynaptic modulation that involve anionic and cationic receptors. 136 FEBRUARY 2004 VOLUME 5 www.nature.com/reviews/neuro

Box 1 Evoked and spontaneous release Evoked neurotransmitter release requires the invasion of presynaptic terminals by an action potential. The associated membrane depolarization activates voltage-gated Ca 2+ -channels (VGCCs) within the terminal, including channels that are strategically placed near vesicle docking sites. Ca 2+ entry stimulates release by promoting fusion of synaptic vesicles with the plasma membrane. Action potentials travelling down a single axon can synchronously evoke release of neurotransmitter from multiple terminals on the same postsynaptic neuron, thereby producing a large response called an EVOKED POSTSYNAPTIC CURRENT (epsc) that can be either excitatory or inhibitory. Many of the experiments that we review in this article have tried to distinguish whether a given change in epsc amplitude is due to presynaptic or postsynaptic effects. Making this distinction has generally entailed several experimental approaches including QUANTAL ANALYSIS, analysis of TRANSMISSION FAILURES and analysis of changes in paired-pulse facilitation or depression. It must be emphasized that none of these approaches are rigorously diagnostic of pre- versus postsynaptic changes. In the absence of action potentials, transmitter release occurs in a random, unsynchronized way by the spontaneous fusion of vesicles with the presynaptic membrane. These release events lead to MINIATURE POSTSYNAPTIC CURRENTS (mpscs). The frequency of mpscs is modulated by changes in intra- and extracellular Ca 2+, although they continue to occur in the absence of extracellular Ca 2+.Because mpscs do not require action potential-evoked Ca 2+ entry to initiate release, they are experimentally distinguishable from evoked release by recording them in the presence of the Na + channel blocker tetrodotoxin. Transmitter-induced changes in the frequency of mpscs with no change in their amplitudes has been commonly interpreted as an indication of the presence of presynaptic ionotropic receptors near the release site, although postsynaptic insertion of receptors has been shown to account for changes in mpsc frequency in at least one system 89.Changes in mpsc frequency are sensitive to relatively small changes (< 500 nm) in Ca 2+ concentration at the presynaptic terminal 90.So, changes in frequency are a sensitive measure of the presence of Ca 2+ -permeable ionotropic receptors, activation of VGCCs or release of Ca 2+ from intracellular stores. Flexor afferent Excitation Interneuron Anionic presynaptic receptors. As mentioned previously, GABA A receptors were the first presynaptic ionotropic receptors shown to inhibit evoked neurotransmitter release. Activation of GABA A and glycine receptors, which are permeable to anions, drives membrane potential towards the Cl reversal potential. Inhibition Extensor afferent Extensor motoneuron Excitation Figure 2 Presynaptic inhibition in the spinal cord. As demonstrated by Frank and Fuortes 7, flexor afferents activate inhibitory interneurons that terminate on extensor afferent central terminals. Mediated through GABA (γ-aminobutyric acid) release from the inhibitory interneuron, the flexor afferent inhibits transmitter release from the extensor afferent in a process known as presynaptic inhibition. This potential depends on the concentrations of Cl in the extracellular space and in the cytoplasm. Whereas the concentration of extracellular Cl tends to remain the same throughout development, cytoplasmic Cl levels often decrease in the CNS, especially during brain maturation 18.As a result, anionic presynaptic receptors often elicit depolarization early in development, but hyperpolarization in mature tissue. However, there are also examples of depolarizing responses to GABA and glycine at presynaptic sites in the postnatal 19 and adult 20 nervous systems. GABA A receptors are found on the presynaptic terminals of dorsal root ganglia (DRG) neurons, and their role in inhibiting presynaptic release has been extensively studied and reviewed 8.The relative importance of changes in membrane potential versus passive shunting as the mechanism for GABA A receptormediated inhibition of release has been investigated, and support has been found for each of these at different synapses 10,21,22. Interestingly, GABA A receptors have recently been shown to enhance spontaneous release of glycine in a manner that requires VGCC activation, while decreasing evoked release from early postnatal rat dorsal horn neurons 23.These observations indicate that GABA A receptor activation might depolarize the terminals, resulting in VGCC activation and enhanced spontaneous release, but simultaneously blocks or shunts the presynaptic action potential, thereby inhibiting evoked release. Surprisingly, anionic receptors have also been shown to enhance evoked release. Glycine receptors have an excitatory effect on synaptic transmission at the calyx of Held a giant synapse at the medial nucleus of the trapezoid body in the brainstem 19.Glycine enhances the frequency of miniature excitatory postsynaptic currents (mepscs) in a manner that is sensitive to the VGCC blocker Cd 2+,consistent with depolarization and subsequent activation of VGCCs as the underlying mechanism. Glycine also increases the amplitude of evoked EPSCs (eepscs) at this synapse, in contrast to the effect of GABA A receptor activation on evoked release of glycine in the dorsal horn. The large size of the presynaptic terminal at the calyx of Held has allowed the direct monitoring of the effect of receptor activation on presynaptic voltage. PERFORATED PATCH CLAMP recordings from the presynaptic terminal, leaving intracellular Cl concentration intact 24,revealed a depolarizing action of glycine. Furthermore, when whole-cell recordings were made and intraterminal Cl levels were deliberately decreased, the glycine-induced depolarization and eepsc enhancement were blocked. The ability of glycine to enhance the eepsc was therefore dependent on depolarization of the presynaptic terminal. Recently, activation of GABA A receptors has also been shown to enhance eepsc amplitude at the calyx of Held 25, although its effects on spontaneous release were not explored. GABA A receptors are present early during the development of the calyx, before glycine receptors are present. NATURE REVIEWS NEUROSCIENCE VOLUME 5 FEBRUARY 2004 137

a b Na + channel c Na + channel 100 Kainate Kainate receptor Na + Na + Kainate receptor Normalized amplitude (%) 50 0 50 Na + / Na + Na + / Na + / Ca 2+ Ca 2+ Ca 2+ Ca 2+ channel Ca 2+ Ca 2+ Ca 2+ channel Na + 100 5 20 50 200 500 5000 Kainate (10 9 M) Glutamate receptors Glutamate receptors Figure 3 Concentration-dependent effect of agonist on transmitter release. a Kainate receptors enhance or suppress transmitter release, depending on the concentration of agonist that they are exposed to. The dose response curve shows this effect of different concentrations of kainate on evoked excitatory postsynaptic current (eepsc) amplitude recorded from hippocampal CA3 neurons, normalized to control (modified from REF. 39). b, c The schematics show how activation of presynaptic ionotropic kainate receptors might enhance (b) or suppress (c) evoked EPSCs. Kainate receptors might be permeable to Ca 2+, providing additional Ca 2+ to the terminal to enhance release. The depolarization that accompanies kainate receptor activation might enhance activation of voltage-gated Ca 2+ channels at the terminal or inhibit hyperpolarization-activated K + channels at the terminal. On the other hand, more powerful depolarization owing to activation of more kainate receptors might cause voltage-gated Na + and Ca 2+ channel inactivation. Cationic, presynaptic receptors. At some synapses, the activation of presynaptic cationic receptors such as kainate receptors depresses evoked transmitter release 26,27.For example, excitatory transmission at synapses between DRG and dorsal horn neurons is Box 2 Presynaptic modulation by G-protein-coupled receptors G-protein-coupled receptors (GPCRs) are expressed on presynaptic terminals and can modulate synaptic transmission. Many presynaptic GPCRs, such the GABA B (γ-aminobutyric acid, subtype B), adenosine and cannabinoid receptors inhibit transmitter release. These receptors act by inhibiting voltage-gated Ca 2+ channels (VGCCs) near release sites or by enhancing K + -channel activation 91,92. They can also decrease release by acting downstream of Ca 2+ entry 93.Other GPCRs, such as serotonin receptors in Aplysia californica,enhance neurotransmitter release by decreasing K + currents and prolonging the presynaptic action potential 94. Many terminals, such as sympathetic axons 95, auditory giant synapses 96 and hippocampal mossy fibres 42,are regulated by both GPCRs and ionotropic receptors. The actions of GPCRs and ionotropic receptors on presynaptic terminals might differ greatly; differences in receptor affinity, and activation and desensitization properties between the two types of receptor will determine their net effect at a given presynaptic terminal. So, GPCRs might act in the order of seconds or minutes, whereas ionotropic receptors activate within a few milliseconds. These differences in activation can lead to different temporal regulation of neurotransmitter release 87. GPCRs and ionotropic receptors can also differ in their desensitization characteristics. GPCRs desensitize within seconds of agonist activation 97,whereas ionotropic receptors desensitize over widely variable time courses. In addition, GPCRs and ionotropic receptors that respond to the same agonist can have markedly different affinities. Simultaneous activation of GPCRs and ionotropic receptors can have effects that are profoundly different from the effects of activating either type alone. For example, at terminals where GPCRs inhibit release by suppressing Ca 2+ entry through VGCCs, activation of a Ca 2+ -permeable ionotropic receptor might offset this effect. Equally, inhibitory GPCR activation might counteract the action of ionotropic receptors that affect release by altering Ca 2+ entry through VGCCs. A recent study showed a requirement for coincident activation of presynaptic NMDA (N-methyl-D-aspartate) receptors, ionotropic glutamate receptors and cannabinoid receptors to induce a longlasting form of synaptic depression 59.This interaction between GPCRs and ionotropic receptors increases the possibilities for signal integration at presynaptic release sites. depressed by activation of kainate receptors 27.This presynaptic action is consistent with the observation that kainate receptors are present on dorsal roots and near the primary afferent terminals, where they mediate the depolarization of the primary afferent fibres 28,29.As with GABA A receptors, the simplest interpretation is that depolarization of the primary afferent fibre drives presynaptic inhibition of transmitter release. However, an alternative interpretation has recently been put forward: that this depression of evoked release is due to a metabotropic function of kainate receptors that results in inhibition of VGCCs (see later in text). The relative impact of receptor-mediated depolarization or metabotropic function at the terminals remains to be resolved. Kainate receptors also decrease eipsc amplitude at synapses between CA1 inhibitory interneurons and pyramidal cells 30 32.Interestingly, a bell-shaped, concentration-dependent effect of kainate on the inhibition of the evoked inhibitory postsynaptic currents (eipscs) was observed. This effect was postulated to be related to the STEADY-STATE CURRENT in response to kainate 30.Activation of kainate receptors also increased the frequency of SPONTANEOUS IPSCS (sipscs) in the absence of TTX, consistent with a depolarizationinduced firing of action potentials in the inhibitory neuron as the underlying mechanism. Kainate action on miniature IPSC (mipsc) frequency was not uniform among these studies. Whereas two groups 30,32 have seen a decrease in the frequency of mipsc with kainate application, another group has found no modulation of mipscs at this synapse 31. AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor activation, both by exogenous agonist and endogenous stimulation, decrease the amplitude of eipscs between cerebellar basket cells and Purkinje cells in 11 18-day-old rats 33.High-frequency 138 FEBRUARY 2004 VOLUME 5 www.nature.com/reviews/neuro

QUANTAL ANALYSIS This type of analysis aims to describe release as a function of three basic parameters: the number of release sites (n), the probability of release at each site (p), and the postsynaptic response elicited by a single transmitter vesicle (q). The amplitude of a synaptic event can be described by the product npq. TRANSMISSION FAILURES Cases in which a presynaptic action potential fails to produce a postsynaptic response. MINIATURE POSTSYNAPTIC CURRENTS Currents that are observed in the absence of presynaptic action potentials, thought to correspond to the response that are elicited by a single vesicle of transmitter. Changes in their frequency are indicative of presynaptic modifications, whereas changes in their amplitude are often interpreted as alterations of postsynaptic responsiveness to the transmitter. PERFORATED PATCH CLAMP Variation on the patch-clamp technique in which it is not necessary to break the cell membrane to gain access to the cytoplasm of the cell to control its voltage. Instead, the recording pipette contains a molecule that can perforate the membrane (often an antibiotic), generating pores through which cations can flow. STEADY-STATE CURRENT The residual current that is observed after a given receptor has become desensitized or fully activated. SPONTANEOUS POSTSYNAPTIC CURRENTS Currents that are observed in the absence of evoked action potentials. However, spontaneous currents are distinct from miniature currents in that they are sensitive to Na + channel blockers such as tetrodotoxin. CLIMBING FIBRES Cerebellar afferents that arise from the inferior olivary nucleus, each of which forms multiple synapses with a single Purkinje cell. conditioning stimulation of excitatory CLIMBING FIBRES decreased eipsc amplitude and increased PAIRED-PULSE FACILITATION at basket cell Purkinje cell synapses. The decrease of eipsc amplitude was antagonized by selective AMPA receptor antagonists and was enhanced by cyclothiazide, a drug that decreases AMPA receptor desensitization. This effect occurred without any change in sipsc frequency. It is possible that presynaptic AMPA receptors on basket cell terminals were activated by glutamate spillover from the climbing fibres, accounting for the depression of eipscs. Recently, functional AMPA receptors have been shown to inhibit release from central terminals of some sensory neurons 29 (FIG. 4).In a spinal cord slice preparation with the dorsal root attached, activation of AMPA receptors reversibly decreased the amplitude of eepscs elicited by dorsal root stimulation. Whereas activation of AMPA receptors did not alter action potentials that propagated down primary afferent axons, AMPA applied selectively to the spinal cord in the presence of a low Ca 2+ concentration (to block secondary release of transmitters in the dorsal horn) elicited depolarization of primary afferents, indicating the presence of AMPA receptors near DRG central terminals. So, similar to kainate and GABA A receptors, the AMPA receptormediated depolarization is believed to mediate presynaptic inhibition by directly or indirectly suppressing the action potential-mediated activation of release. Presynaptic cationic receptors that are permeable to Ca 2+ might be expected to enhance transmitter release owing to Ca 2+ entry through the receptor itself. However, if the receptor is not located close enough to the terminal, the depolarizing action might predominate in causing any change in transmitter release. For example, activation of nachrs in the ventrobasal thalamus elicits increases in mipsc frequency in a Cd 2+ - sensitive manner, indicating that the effect is not dependent on Ca 2+ entry through the nachrs but through VGCCs. Activation of nachrs enhances evoked release by decreasing the failure rate of the eipsc 34.It is possible that these receptors act by eliciting just enough depolarization to enhance, but not inhibit, the excitability of presynaptic terminals. Modulation that affects Ca 2+ near release sites Ca 2+ -permeable presynaptic receptors modify evoked synaptic transmission by depolarization and by altering input resistance, similar to the anionic and cationic receptors that we discussed earlier. In addition, Ca 2+ entry through these receptors could lead to enhancement or inhibition of evoked release by eliciting Ca 2+ -dependent changes in action potential shape, or by summating with Ca 2+ entering through VGCCs or released from intracellular stores. In the case of spontaneous transmitter release, activation of Ca 2+ -permeable ionotropic receptors located on the presynaptic terminal is expected to increase release independently of VGCC activation. Many families of cation permeable presynaptic receptors are permeable to Ca 2+.We will consider the complex presynaptic actions that are associated with activation of these different receptor types. SYM2081 Baseline SYM2081 + GYKI53655 Baseline SYM2081 Kainate SYM2081 + GYKI53655 Kainate 200 ms SYM2081 Wash Vm = +50mV Vm = 50mV 20 ms 40 pa Figure 4 Presynaptic inhibition and AMPA receptors. Presynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors expressed on primary afferent neurons depress release of glutamate onto dorsal horn neurons 29. In these experiments, the NMDA (N-methyl-D-aspartate) receptordependent evoked excitatory postsynaptic current (eepsc) was used to monitor glutamate release so that the impact of blocking AMPA and kainate receptors (with GYKI53655 and SYM2081, respectively) on eepsc amplitude could be tested. Presynaptic kainate receptors at the synapse between MOSSY FIBRES and CA3 pyramidal cells have been extensively studied. Interestingly, facilitation and LONG-TERM POTENTIATION (LTP) at this synapse are sensitive to kainate receptor antagonists 35, as well as to philanthotoxin a blocker of Ca 2+ -permeable glutamate receptors. Therefore, at least some kainate receptors at this synapse are believed to be Ca 2+ -permeable 4.It is possible that Ca 2+ entering through these kainate receptors has a role in presynaptic modulation by recruiting intracellular Ca 2+ stores (see below). At the same time, receptor activation depolarizes the membrane potential. Activation of kainate receptors has a concentration-dependent biphasic effect on eepsc amplitudes elicited in CA3 neurons by stimulation of mossy fibres 36 38 (FIG. 3). The concentration-dependent effects of kainate receptor agonists on eepsc amplitude might be mediated by depolarization, as they are mimicked by application of different K + concentrations 39.In fact, a biphasic effect of somatic depolarization on action potentialevoked Ca 2+ transients in mossy fibre terminals has been reported recently 40.However, the interaction between presynaptic kainate receptor activation, mossy fibre action potentials and transmitter release is complex and cannot be solely explained by the excitability of presynaptic fibres. At the synapse between mossy fibres and CA3 neurons, effects on presynaptic action potentials can be distinguished from effects on release by examining the properties of the PRESYNAPTIC VOLLEY.After application of 50 500 nm kainate, the mossy fibre presynaptic volley is enhanced, possibly because the kainate-induced depolarization brings additional fibres closer to threshold 41,42. The increase in the number of activated fibres must contribute to the reported enhancement of the eepsc by 50 nm kainate 36.However, 500 nm kainate first increases NATURE REVIEWS NEUROSCIENCE VOLUME 5 FEBRUARY 2004 139

PAIRED-PULSE FACILITATION If two stimuli are delivered to an axon in close succession, the postsynaptic response to the second stimulus is often larger than to the first one. This phenomenon is referred to as paired-pulse facilitation, and is thought to depend on the accumulation of Ca 2+ that ensues after successive stimuli. MOSSY FIBRES Axons of hippocampal granule cells, which form synapses with CA3 pyramidal neurons. Mossy fibre terminals are among the largest in the central nervous system. LONG-TERM POTENTIATION (LTP). An enduring increase in the amplitude of excitatory postsynaptic potentials as a result of high-frequency (tetanic) stimulation of afferent pathways. It is measured both as the amplitude of excitatory postsynaptic potentials and as the magnitude of the postsynaptic-cell population spike. LTP is most often studied in the hippocampus, and is often considered to be the cellular basis of learning and memory in vertebrates. PRESYNAPTIC VOLLEY The wave of a synaptic potential with the shortest latency. It is proportional to the number of active presynaptic fibres, and its amplitude serves to estimate the strength of an afferent input. UNITARY POSTSYNAPTIC CURRENTS Currents that result from the activation of a single presynaptic fibre. They often require that the pre- and postsynaptic cells be recorded simultaneously to ensure that the postsynaptic response occurs as a result of an action potential in the presynaptic cell. and then decreases the eepsc 36,41.Likewise, although 200 nm kainate increases the fibre volley, it decreases voltagedependent Ca 2+ influx and the amplitude of the eepsc 42. Therefore, concentrations of kainate that enhance mossy fibre action potentials can actually decrease release. This raises the possibility that, at moderate concentrations of kainate receptor agonist, release might be decreased by mechanisms that are independent of changes in fibre volley at this synapse. At high concentrations (5 10 µm), kainate decreases the presynaptic volley, presumably through depolarization, subsequent inactivation of Na + channels and blockade of action potentials 36,41. Synaptically released glutamate also acts bi-directionally through kainate receptors at mossy fibre terminals. Glutamate release elicited by brief trains of stimulation of either mossy fibres or CA3 collaterals leads to a facilitation of the EPSC, that is mediated by kainate receptors. By contrast, longer stimulation trains to CA3 collaterals decrease eepsc amplitude 36,39. Kainate receptors also modulate inhibitory neurotransmission in a biphasic manner in the basolateral amygdala 43,where high concentrations of kainate receptor agonists depressed eipsc amplitude and low agonist concentrations enhanced it 43.These effects were mirrored by an increase and a decrease in mipsc frequency, respectively, which were not prevented by blocking VGCCs with Cd 2+,possibly indicating Ca 2+ entry through kainate receptors. The authors raise the possibility that two different receptors using the GluR5 kainate receptor subunit might be responsible for each component of the biphasic effect, analogous to the two types of kainate receptors that are postulated to inhibit GABA release from hippocampal interneurons 44. In the hippocampus 44,depression of GABA release is mediated by a kainate receptor with metabotropic properties, and this effect was selectively activated by low concentrations of glutamate without depolarization of the interneurons. However, in the case of the amygdala, no direct evidence for two receptor populations has been presented. Glutamate respectively increased and decreased evoked and spontaneous GABA release at similar concentrations. So, the underlying mechanisms of the biphasic dose dependence of agonist action on transmitter release seem to be different for different synapses and will have to be established independently in each case. Activation of the capsaicin or vanilloid receptor TRPV1,a ligand- and heat-sensitive Ca 2+ -permeable ion channel, increases spontaneous EPSC (sepsc) and mepsc frequency at synapses on dorsal horn neurons 45, and routinely blocks the C-fibre-mediated eepsc 46. Capsaicin elicits a powerful depolarization of C-fibre cell bodies and a suppression of the C-fibre-mediated compound action potentials 28.Inhibition of eepsc amplitude is probably due to depolarization of the primary afferent fibres, preventing action potential invasion of the terminal, or to a shunting of the action potential by TRPV1 channels. In addition to deploarizing sensory neurons, activation of TRPV1 also causes a potent elevation of intracellular Ca 2+ concentration. Ca 2+ must then drive the enhancement of mepsc frequency, despite the depolarization-induced presynaptic inhibition. Activation of the NMDA (N-methyl-D-aspartate) receptor a Ca 2+ -permeable ionotropic glutamate receptor causes an increase in mipsc frequency and a decrease in eipsc amplitude, in inhibitory cerebellar interneurons. Such changes can be induced by direct depolarization of the interneurons 47.The NMDAinduced decrease in eipsc amplitude might therefore be due to depolarization of either the soma or the presynaptic terminal. In entorhinal cortex, blocking endogenous activation of NMDA receptors decreases both sepsc and mepsc frequency, indicating that glutamate significantly enhances spontaneous release of glutamate by acting through NMDA receptors 48. Recently, presynaptic NMDA receptors have been found at synapses between cortical layer V pyramidal neurons. Blockade of these NMDA receptors elicited a decrease in mepsc frequency and decreased amplitude of UNITARY EPSPS evoked by high frequency stimulation of pyramidal cells 49.These data show that endogenously released glutamate can enhance glutamate release through presynaptic NMDA receptor activation. ATP, acting on Ca 2+ -permeable P2X receptors, modulates transmitter release at many central synapses. Activation of P2X receptors enhances glutamate release at DRG dorsal horn synapses in culture 50 and in spinal cord slices 51,52,consistent with the ability of these receptors to depolarize the membrane potential and with their permeability to Ca 2+.Another agonist α,β-methylene ATP (α,β-meatp) stimulates both an increase in mepsc frequency and a decrease in failures of transmission at the synapse between Aδ fibres and the dorsal horn. Inhibition of ecto-atpases enzymes that break down extracellular ATP causes a decrease in failures of transmission and an increase in eepsc amplitude, indicating that endogenous ATP might modulate this synapse 52. P2X receptors have also been shown to modulate mipsc frequency in a subset of dorsal horn neurons in culture 53.At synapses between dorsal horn neurons, ATP, but not α,β-meatp, increases mipsc frequency and the amplitude of eipscs. This pharmacological profile indicates that the relevant P2X receptors might be different from those expressed on primary afferent nerve terminals. A similar pharmacological profile has been shown in acutely dissociated dorsal horn neurons that retained their presynaptic terminals 54.In this system, ATP increases mipsc frequency, even in the presence of Cd 2+,ruling out a requirement for depolarization-activated VGCCs. In all of these examples, Ca 2+ entry through P2X receptors increases the frequency of spontaneous transmitter release and enhances evoked transmitter release. Presynaptic nachrs with variable degrees of Ca 2+ permeability regulate release in various preparations from the CNS and peripheral nervous system 55.A nachr-mediated enhancement of glutamatergic transmission was first shown in the chick by McGehee et al. 14, studying the projection from the medial habenula to interpeduncular neurons. Both evoked and spontaneous EPSCs were enhanced by nicotine, and these effects were antagonized by α-bungarotoxin, pointing to 140 FEBRUARY 2004 VOLUME 5 www.nature.com/reviews/neuro

PENTRAXIN (or Pentaxin). Protein of discoid appearance under the electron microscope, consisting of five non-covalently bound subunits. PDZ DOMAIN A peptide-binding domain that is important for the organization of membrane proteins, particularly at cell cell junctions, including synapses. It can bind to the carboxyl termini of proteins or can form dimers with other PDZ domains. PDZ domains are named after the proteins in which these sequence motifs were originally identified (PSD95, Discs large, zona occludens 1). the involvement of nachrs that contain the α7 subunit. Gray et al. 56 also showed a nicotine-mediated enhancement of glutamate release in CA3 neurons, which was sensitive to the α7-subunit-containing nachrs antagonists methyllycaconitine and α-bungarotoxin. These authors also reported that Ca 2+ concentration in mossy fibre terminals changed in response to nicotine application. The localization of nachrs at mossy fibre terminals and the existence of the Ca 2+ response have been recently challenged 57, although variable detection of Ca 2+ transients after activation of nachrs might be owing to inconsistent triggering of Ca 2+ -induced Ca 2+ release under different experimental conditions 5. Activation of kainate receptors enhances neurotransmission between hippocampal CA1 inhibitory interneurons in a Cd 2+ -insensitive manner. Activation with 250 nm kainate increases mipsc frequency and decreases failures of the eipsc at synapses made on interneurons 58.Mulle et al. 59 observed an increased mipsc frequency in response to 10 µm kainate at this synapse in wild-type mice and in mice lacking the kainate receptor subunit GluR5. However, they found no modulation in mice lacking GluR6, another kainate receptor subunit. Therefore, the increase in mipsc frequency at the synapse between interneurons is probably due to activation of Ca 2+ -permeable, GluR6-containing kainate receptors. AMPA receptors enhance spontaneous inhibitory transmitter release. In stellate cells of the developing (11 13-days-old) mouse cerebellum, AMPA receptor agonists elicit increases in both sipsc and mipsc frequency 60.These effects are only partially blocked by Cd 2+,implicating the presynaptic involvement of Ca 2+ - permeable AMPA receptors. In the adult cerebellum, this presynaptic modulation is no longer present, pointing to the developmental loss of this presynaptic receptor. We have also obtained evidence for AMPA receptormediated increases in mipsc frequency in the dorsal horn (H.E., R. L. Anderson and A.B.M, unpublished data). AMPA application increases mipsc frequency, but not amplitude, in a subpopulation of dorsal horn neurons. This increase shows varying sensitivity to Cd 2+, indicating that Ca 2+ -permeable AMPA receptors might allow Ca 2+ entry into the terminal, which in turn supports the release of GABA/glycine. In contrast to the cerebellum, this modulation persists in the dorsal horn of the young adult. Activation of nachrs enhances mipsc frequency in the dorsolateral geniculate nucleus of the thalamus 34. Nicotinic agonists increase mipsc frequency in this nucleus in a Cd 2+ -resistant manner. Nicotine can also evoke an increase in the frequency of glycine-mediated mipscs in the dorsal horn of the spinal cord 61.This effect is also Cd 2+ -resistant, indicating that Ca 2+ entry directly through the receptor is involved in the effect. As discussed previously, however, other examples of nachr activation do not seem to indicate a role for Ca 2+ entry through the receptor, possibly owing to its location. Nicotine elicits a concentration-dependent increase in sipsc frequency, but has a biphasic effect on eipsc amplitude in the lateral spiriform nucleus of chick brain 62.Low concentrations of nicotine (50 100 nm) enhance eipsc amplitude, whereas higher concentrations (0.5 1 µm) decrease it. Because mipscs were not assessed in this study 62, the site on which nicotine is acting in this system in not known. Ca 2+ release from intracellular stores. Recently, two groups have reported presynaptic modulation of glutamate release onto CA3 hippocampal neurons by Ca 2+ - permeable ionotropic receptors; modulation that involves Ca 2+ -induced Ca 2+ release from intracellular stores 4,5.Activation of nachrs elicits an increase in frequency and amplitude of mepscs recorded from CA3 pyramidal neurons. These increases are sensitive to extracellular Ca 2+ and are blocked by drugs that empty or prevent refilling of intracellular Ca 2+ stores 5.These results indicate that Ca 2+ entering through nachrs elicits Ca 2+ release from intracellular stores, which then facilitates vesicle fusion. Recruitment of Ca 2+ stores results in the potent effect of nicotinic agonists in this preparation. Kainate receptors also modulate transmission at the mossy fibre CA3 synapse. As mentioned previously, mossy fibre LTP is antagonized by kainate receptor antagonists 35, and these receptors are thought to be Ca 2+ permeable. It has been shown that LTP at this synapse is reduced by emptying Ca 2+ stores, and that the effect of the antagonists is also reduced by this manipulation 4. So, Ca 2+ entry through kainate receptors is thought to trigger Ca 2+ -induced Ca 2+ release from intracellular stores, contributing to mossy fibre LTP 4.The role of Ca 2+ stores in the actions of presynaptic ionotropic receptors at other synapses remains to be investigated. Modulation independent of ion flow Ionotropic receptors are known to associate with many other proteins through their cytoplasmic and extracellular domains. AMPA receptors, for example, can be clustered extracellularly by a protein known as neuronal activity-regulated PENTRAXIN (NARP) 66.The cytoplasmic tails of the different AMPA receptor subunits can bind PDZ DOMAIN-containing proteins, such as synapseassociated protein 97 (SAP97) 67,glutamate-receptorinteracting protein (GRIP) 68, AMPA receptor-binding protein (ABP) 69,protein that interacts with C kinase 1 (PICK1) 70 and stargazin 71,all ofwhich might form part of larger signalling complexes. In addition, the cytoplasmic domain of the AMPA receptor subunit GluR2 associates with the N-ethylmaleimide-sensitive fusion protein (NSF) 72,73.AMPA receptors can also associate with the tyrosine kinase lyn 74 and with G-protein subunits 75.So, through their association with these proteins, AMPA receptors might trigger changes that are independent of ion flux, which might in turn promote changes in the readily releasable vesicle pool or in vesicle fusion. There are several examples of ionotropic receptors that modulate transmitter release for which the underlying mechanism seems to be independent of the permeating ions. Rodríguez-Moreno et al. 44,76 have proposed a metabotropic-like function for kainate receptors that are present near hippocampal GABA release sites on CA1 interneurons. In this case, the NATURE REVIEWS NEUROSCIENCE VOLUME 5 FEBRUARY 2004 141

Table 1 Modulation of spontaneous and evoked release mediated by presynaptic ionotropic receptors Neuro- Ionotropic Effect on Effect on evoked Brain area IPSC or EPSC References transmitter receptors spontaneous release release Glutamate NMDAR Increase Decrease Cerebellum IPSC 47 Kainate R Increase Biphasic Dorsal horn IPSC 26 Kainate R Decrease Decrease Hippocampus, IPSC 30,32 onto CA1 Kainate R No change Decrease Hippocampus, IPSC 31 onto CA1 Kainate R Increase Increase Hippocampus, IPSC 57 (decrease failures) between inhibitory neurons in CA1 Kainate R Biphasic Biphasic Amygdala IPSC 43 ACh nachr Increase Increase Medial habenula EPSC 14 Ca 2+ -perm. nachr Increase Increase Ventrobasal IPSC 34 Ca 2+ -perm. thalamus nachr Increase Increase Dorsolateral IPSC 34 Ca 2+ -imperm. geniculate thalamus ATP P2X Increase Increase Dorsal horn IPSC 52 P2X Increase Increase Dorsal horn EPSC 51 (decrease failure) GABA GABA A Increase Decrease Dorsal horn IPSC 23 Glycine Glycine Increase Increase Trapezoid body, EPSC 19 auditory brainstem? VR1 Increase Decrease Dorsal horn EPSC 45,46 ACh, acetylcholine ; EPSC, excitatory postsynptic current; GABA, γ-aminobutyric acid; imperm., impermeable; IPSC, inhibitory postsynaptic current ; NMDAR, N-methyl-D-aspartate; R, receptor; P2X, purinergic receptor 2X; perm., permeable. PERTUSSIS TOXIN The causative agent of whooping cough, pertussis toxin causes the persistent activation of G i proteins by catalysing the ADPribosylation of the α-subunit. PARALLEL FIBRES The axons of cerebellar granule cells. Parallel fibres emerge from the molecular layer of the cerebellar cortex towards the periphery, where they extend branches perpendicular to the main axis of Purkinje neurons and form so-called en passant synapses with this cell type. LONG-TERM DEPRESSION (LTD). An enduring weakening of synaptic strength that is thought to interact with long term potentiation (LTP) as cellular mechanisms of learning and memory in structures such as the hippocampus and cerebellum. G-protein inhibitor PERTUSSIS TOXIN and selective blockers of protein kinase C prevented the inhibition of eipscs by kainate, without influencing the ability of kainate to drive action potential firing in the inhibitory neurons 44. Rozas et al. 77 have subsequently shown that activation of kainate receptors in DRG neurons causes inhibition of VGCCs, and that this effect is sensitive to pertussis toxin 77.This pathway provides a possible mechanism for the inhibitory action of kainate both at synapses of sensory afferents in the dorsal horn and in hippocampal interneurons. Presynaptic NMDA receptors at the PARALLEL FIBRE Purkinje cell synapse in the cerebellum constitute another case in which the modulation of release does not depend directly on ion flux through the receptor 78. Instead, activation of NMDA receptors induces the synthesis of nitric oxide, which functions as a modulator at this synapse. Application of NMDA decreases the eepsc, but it does not change the presynaptic volley, nor does it alter paired-pulse facilitation, indicating that NMDA does not act by directly affecting the presynaptic action potential or altering release. However, presynaptic NMDA receptors are believed to be responsible for this effect for two reasons. First, NMDA receptors are not believed to be present on Purkinje cells (the postsynaptic neuron in this case) in mature animals. Second, the application of NMDA receptor agonists must be paired with parallel fibre stimulation to elicit eepsc depression. This NMDAinduced depression is sensitive to inhibitors of nitric oxide (NO) synthase and of guanylate cyclase. The authors hypothesize that NMDA triggers NO production, and that NO travels to the postsynaptic site to stimulate guanylate cyclase and decrease the response of the Purkinje cell. It is worthwhile noting that when postsynaptic Ca 2+ buffering is reduced, NMDA elicits a longer depression, which occludes the induction of LONG-TERM DEPRESSION (LTD) at this synapse. These presynaptic NMDA receptors might be involved in this form of cerebellar LTD, an idea that is further supported by the sensitivity of LTD to the NMDA receptor antagonist 2-amino-phosphonovalerate (APV) 79. TABLE 1 provides a summary of the action of the different receptors that we have discussed, trying to distinguish between effects on spontaneous and evoked release. Source of endogenous agonist The sources of endogenous agonists that activate ionotropic presynaptic receptors have generally been difficult to identify, with the exception of synapses between reciprocally connected cells or in the case of autoreceptors. As most ionotropic receptors have relatively low affinity, the source of the transmitter should be close by to ensure a transmitter concentration that is sufficiently high for receptor activation. This implies that the most effective means for activating presynaptic ionotropic receptors is through release from closely apposed terminals, although spillover from nearby release sites might also be effective. This release could be glial 80,dendritic (BOX 3),axonal or from the presynaptic terminal itself in the case of autoreceptors. 142 FEBRUARY 2004 VOLUME 5 www.nature.com/reviews/neuro