Multivesicular release and saturation of glutamatergic signalling at retinal ribbon synapses

Pronounced multivesicular release (MVR) occurs at the ribbon synapses of sensory neurones that signal via graded potential changes. As MVR increases the likelihood of postsynaptic receptor saturation, it is of interest to consider how sensory synapses overcome this problem and use MVR to encode signals of widely varying intensities. Here, I discuss three postsynaptic mechanisms that permit three different retinal synapses to utilize MVR.     

Selective muscarinic regulation of functional glutamatergic Schaffer collateral synapses in rat CA1 pyramidal neurons

Analysis of the cholinergic regulation of glutamatergic neurotransmission is an essential step in understanding the hippocampus because it can influence forms of synaptic plasticity that are thought to underlie learning and memory. We studied in vitro the cholinergic regulation of excitatory postsynaptic currents (EPSCs) evoked in rat CA1 pyramidal neurons by Schaffer collateral (SC) stimulation. Using 'minimal' stimulation, which activates one or very few synapses, the cholinergic agonist carbamylcholine (CCh) increased the failure rate of functional more (36 %) than of silent synapses (7 %), without changes in the EPSC amplitude. These effects of CCh were insensitive to manipulations that increased the probability of release, such as paired pulse facilitation, increases in temperature and increases in the extracellular Ca²⁺ : Mg²⁺ ratio. Using 'conventional' stimulation, which activates a large number of synapses, CCh inhibited more the pharmacologically isolated non-NMDA (86 %) than the NMDA (47 %) EPSC. The changes in failure rate, EPSC variance and the increased paired pulse facilitation that paralleled the inhibition imply that CCh decreased release probability. Muscarine had similar effects. The inhibition by both CCh and by muscarine was prevented by atropine. We conclude that CCh reduces the non-NMDA component of SC EPSCs by selectively inhibiting transmitter release at functional synapses via activation of muscarinic receptors. The results suggest that SCs have two types of terminals, one in functional synapses, selectively sensitive to regulation through activation of muscarinic receptors, and the other in silent synapses less sensitive to that regulation. The specific inhibition of functional synapses would favour activity-dependent plastic phenomena through NMDA receptors at silent synapses without the activation of non-NMDA receptors and functional synapses.     

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain.     

GABA(B) receptors at glutamatergic synapses in the rat striatum

Although multiple effects of GABA(B) receptor activation on synaptic transmission in the striatum have been described, the precise locations of the receptors mediating these effects have not been determined. To address this issue, we carried out pre-embedding immunogold electron microscopy in the rat using antibodies against the GABA(B) receptor subunits, GABA(B1) and GABA(B2). In addition, to investigate the relationship between GABA(B) receptors and glutamatergic striatal afferents, we used antibodies against the vesicular glutamate transporters, vesicular glutamate transporter 1 and vesicular glutamate transporter 2, as markers for glutamatergic terminals. Immunolabeling for GABA(B1) and GABA(B2) was widely and similarly distributed in the striatum, with immunogold particles localized at both presynaptic and postsynaptic sites. The most commonly labeled structures were dendritic shafts and spines, as well as terminals forming asymmetric and symmetric synapses. In postsynaptic structures, the majority of labeling associated with the plasma membrane was localized at extrasynaptic sites, although immunogold particles were also found at the postsynaptic specialization of some symmetric, putative GABAergic synapses. Labeling in axon terminals was located within, or at the edge of, the presynaptic active zone, as well as at extrasynaptic sites. Double labeling for GABA(B) receptor subunits and vesicular glutamate transporters revealed that labeling for both GABA(B1) and GABA(B2) was localized on glutamatergic axon terminals that expressed either vesicular glutamate transporter 1 or vesicular glutamate transporter 2. The patterns of innervation of striatal neurons by the vesicular glutamate transporter 1- and vesicular glutamate transporter 2-positive terminals suggest that they are selective markers of corticostriatal and thalamostriatal afferents, respectively. These results thus provide evidence that presynaptic GABA(B) heteroreceptors are in a position to modulate the two major excitatory inputs to striatal spiny projection neurons arising in the cortex and thalamus. In addition, presynaptic GABA(B) autoreceptors are present on the terminals of spiny projection neurons and/or striatal GABAergic interneurons. Furthermore, the data indicate that GABA may also affect the excitability of striatal neurons via postsynaptic GABA(B) receptors.     

Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex

Thalamocortical synapses provide a strong glutamatergic excitation to cortical neurons that is critical for processing sensory information. Unit recordings in vivo indicate that metabotropic glutamate receptors (mGluRs) reduce the effect of thalamocortical input on cortical circuits. However, it is not known whether this reduction is due to a reduction in glutamate release from thalamocortical terminals or from a decrease in cortical neuron excitability. To directly determine whether mGluRs act as autoreceptors on thalamocortical terminals, we examined the effect of mGluR agonists on thalamocortical synapses in slices. Thalamocortical excitatory postsynaptic currents (EPSCs) were recorded in layer IV cortical neurons in developing mouse brain slices. The activation of group II mGluRs with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) reduced thalamocortical EPSCs in both excitatory and inhibitory neurons, while the stimulation of group I or group III mGluRs had no effect on thalamocortical EPSCs. Consistent with a reduction in glutamate release, DCG IV increased the paired pulse ratio and the coefficient of variation of the EPSCs. The reduction induced by DCG IV was reversed by the group II mGluR antagonist, LY341495, and mimicked by another selective group II agonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC). The mGluR2 subtype appears to mediate the reduction of thalamocortical EPSCs, since the selective mGluR3 agonist, N-acetylaspartylglutamate (NAAG), had no effect on the EPSCs. Consistent with this, we showed that mGluR2 is expressed in the barrels. Furthermore, blocking group II mGluRs with LY341495 reduced the synaptic depression induced by a short stimulus train, indicating that synaptically released glutamate activates these receptors. These results indicate that group II mGluRs modulate thalamocortical processing by inhibiting glutamate release from thalamocortical synapses. This inhibition provides a feedback mechanism for preventing excessive excitation of cortical neurons that could play a role in the plasticity and refinement of thalamocortical connections during this early developmental period.     

Cannabinoids modulate synaptic strength and plasticity at glutamatergic synapses of rat prefrontal cortex pyramidal neurons

Cannabinoids receptors have been reported to modulate synaptic transmission in many structures of the CNS, but yet little is known about their role in the prefrontal cortex where type I cannabinoid receptor (CB-1) are expressed. In this study, we tested first the acute effects of selective agonists and antagonist of CB-1 on glutamatergic excitatory postsynaptic currents (EPSCs) in slices of rat prefrontal cortex (PFC). EPSCs were evoked in patch-clamped layer V pyramidal cells by stimulation of layer V afferents. Monosynaptic EPSCs were strongly depressed by bath application (1 microM) of the cannabinoid receptors agonists WIN55212-2 (-50.4 +/- 8.8%) and CP55940 (-42.4 +/- 10.9%). The CB-1 antagonist SR141716A reversed these effects. Unexpectedly, SR141716A alone produced a significant increase of glutamatergic synaptic transmission (+46.9 +/- 11.2%), which could be partly reversed by WIN55212-2. In the presence of strontium in the bath, the frequency but not the amplitude of asynchronous synaptic events evoked in layer V pyramidal cells by stimulating layer V afferents, was markedly decreased (-54.2 +/- 8%), indicating a presynaptic site of action of cannabinoids at these synapses. Tetanic stimulation (100 pulses at 100 Hz, 4 trains) induced in control condition, no changes (n = 7/18), long-term depression (LTD; n = 6/18), or long-term potentiation (LTP; n = 5/18) of monosynaptic EPSCs evoked by stimulation of layer V afferents. When tetanus was applied in the presence of WIN 55,212-2 or SR141716-A (1 microM) in the bath, the proportion of "nonplastic" cells were not significantly changed (n = 7/15 in both cases). For the plastic ones (n = 8 in both cases), WIN 55,212-2 strongly favored LTD (n = 7/8) at the apparent expense of LTP (n = 1/8), whereas the opposite effect was observed with SR141716-A (7/8 LTP; 1/8 LTD). These results demonstrate that cannabinoids influence glutamatergic synaptic transmission and plasticity in the PFC of rodent.     

Somatostatin presynaptically inhibits both GABA and glutamate release onto rat basal forebrain cholinergic neurons

A whole cell patch-clamp study was carried out in slices obtained from young rat brain to elucidate the roles of somatostatin in the modulation of synaptic transmission onto cholinergic neurons in the basal forebrain (BF), a region that contains cholinergic and GABAergic corticopetal neurons and somatostatin (SS)-containing local circuit neurons. Cholinergic neurons within the BF were identified by in vivo prelabeling with Cy3 IgG. Because in many cases SS is contained in GABAergic neurons in the CNS, we investigated whether exogenously applied SS can influence GABAergic transmission onto cholinergic neurons. Bath application of somatostatin (1 muM) reduced the amplitude of the evoked GABAergic inhibitory presynaptic currents (IPSCs) in cholinergic neurons. SS also reduced the frequency of miniature IPSCs (mIPSCs) without affecting their amplitude distribution. SS-induced effect on the mIPSC frequency was significantly larger in the solution containing 7.2 mM Ca(2+) than in the standard (2.4 mM Ca(2+)) external solution. Similar effects were observed in the case of non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). SS inhibited the amplitude of evoked EPSCs and reduced the frequency of miniature EPSCs dependent on the external Ca(2+) concentration with no effect on their amplitude distribution. Pharmacological analyses using SS-receptor subtype-specific drugs suggest that SS-induced action of the IPSCs is mediated mostly by the sst(2) subtype, whereas sst subtypes mediating SS-induced inhibition of EPSCs are mainly sst(1) or sst(4). These findings suggest that SS presynaptically inhibits both GABA and glutamate release onto BF cholinergic neurons in a Ca(2+)-dependent way, and that SS-induced effect on IPSCs and EPSCs are mediated by different sst subtypes.     

Postsynaptic specializations at excitatory and inhibitory cholinergic synapses

Summary In serial sections of neurons in the paravertebral ganglia of the frog (Limnodynastes dumerili), the postsynaptic structures termed postsynaptic bar (PSB) and junctional subsurface organ (JSO) were never observed in the same ganglion cell. Further, PSBs were found mostly in small ganglion cells (less than 22 m), while JSOs were found mostly in large ganglion cells (up to 45 m). Between 10 and 22 PSBs were located at both spine and non-spinous somatic synapses of the smaller ganglion cells; while 8 to 16 JSOs were located largely in the axon hillock region of the larger ganglion cells.Based on these observations, it is suggested that the two ganglion cell populations represent the B and C cell types defined according to electrophysiological data. Further, since the nerve terminals adjacent to both these postsynaptic structures appear to be cholinergic according to their vesicular content, this provides some basis for suggesting that JSOs are associated with slow excitatory synapses, while PSBs are present at slow inhibitory synapses.     

Mitochondria and release at hippocampal synapses

Mitochondria are present in some, but not all presynaptic terminals in the hippocampus. Mitochondria are capable of sequestering and storing large amounts of calcium, but it is unclear whether they influence release probability at these synapses. Using FM dye imaging techniques and confocal microscopy, we have examined the relationship between mitochondrial presence/absence and presynaptic vesicle release from rat hippocampal neurones in primary dissociated culture at room temperature. Following staining with the mitochondrial dye mitotracker green, we were able to resolve putative individual mitochondria associated with neuronal processes. The majority of mitochondria were positionally stable, although some exhibited periods of rapid motility (up to 0.4 m/s) interspersed with periods of immobility. Co-staining with mitotracker green and the synaptic vesicle dye FM 4–64 indicated that 180 of 506 (36%) synapses were devoid of mitochondria. A comparison of vesicular release in response to stimulation at 1 Hz and at 10 Hz revealed no differences in release properties between synapses with and without mitochondria.     

Chronic ethanol and withdrawal differentially modulate pre- and postsynaptic function at glutamatergic synapses in rat basolateral amygdala

Withdrawal anxiety is a significant factor contributing to continued alcohol abuse in alcoholics. This anxiety is long-lasting, can manifest well after the overt physical symptoms of withdrawal, and is frequently associated with relapse in recovering alcoholics. The neurobiological mechanisms governing these withdrawal-associated increases in anxiety are currently unknown. The basolateral amygdala (BLA) is a major emotional center in the brain and regulates the expression of both learned fear and anxiety. Neurotransmitter system alterations within this brain region may therefore contribute to withdrawal-associated anxiety. Because evidence suggests that glutamate-gated neurotransmitter receptors are sensitive to acute ethanol exposure, we examined the effect of chronic intermittent ethanol (CIE) and withdrawal (WD) on glutamatergic synaptic transmission in the BLA. We found that slices prepared from CIE and WD animals had significantly increased contributions by synaptic NMDA receptors. In addition, CIE increased the amplitude of AMPA-receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs), whereas only WD altered the amplitude and kinetics of tetrodotoxin-resistant spontaneous events (mEPSCs). Similarly, the frequency of sEPSCs was increased in both CIE and WD neurons, although only WD increased the frequency of mEPSCs. These data suggest that CIE and WD differentially alter both pre- and postsynaptic properties of BLA glutamatergic synapses. Finally, we show that microinjection of the AMPA-receptor antagonist, DNQX, can attenuate withdrawal-related anxiety-like behavior. Together, our results suggest that increased glutamatergic function may contribute to anxiety expressed during withdrawal from chronic ethanol.     

Distinct roles of P2X receptors in modulating glutamate release at different primary sensory synapses in rat spinal cord

Using spinal cord slice preparations and patch-clamp recordings in lamina II and lamina V regions, we tested a hypothesis that P2X receptor subtypes differentially modulate glutamate release from primary afferent terminals innervating different sensory regions. We found that activation of P2X receptors by alpha,beta-methylene-ATP increased glutamate release onto >80% of DH neurons in both lamina regions. However, two distinct types of modulation, a transient and a long-lasting enhancement of glutamate release were observed. In lamina II recordings, >70% of the modulation was transient. In contrast, P2X receptor-mediated modulation was always long-lasting in lamina V. Pharmacologically, both transient and long-lasting types of modulation were blocked by 10 microM pyridxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium, a broad-spectrum P2X receptor antagonist. Transient modulation was not observed in the presence of 1 microM trinitrophenyl-ATP (TNP-ATP), a subtype-selective P2X receptor antagonist, suggesting that homomeric P2X3 receptors may be involved in the transient modulation in lamina II. The long-lasting modulation remained in the presence of 1 microM TNP-ATP. Selective removal of P2X3-expressing afferent terminals by the targeting toxin saporin-conjugated isolectin B4 or surgical removal of superficial DH did not affect P2X receptor-mediated long-lasting modulation in lamina V. Taken together, these results suggest that P2X receptor subtypes play distinct roles in sensory processing in functionally different sensory regions.     

Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission.     

Development of nociceptive synaptic inputs to the neonatal rat dorsal horn: glutamate release by capsaicin and menthol

To study the postnatal development of nociceptive synaptic inputs in the superficial dorsal horn of the neonatal rat spinal cord, we examined the effect of capsaicin and menthol on glutamatergic mEPSCs in postnatal day (P) 0–1, P5–6 and P9–11 slices of spinal cord. Capsaicin (100 n m to 2 μ m ) increased the mEPSC frequency in a concentration-dependent manner at all ages tested, with a significant enhancement of the effect between P5 and P10. This effect was sensitive to vanilloid receptor (VR) antagonists. The elevation in mEPSC frequency occurred at concentrations of capsaicin (100 n m ) that did not alter the distribution of mEPSC amplitudes and was abolished by a dorsal rhizotomy, demonstrating that capsaicin acts via presynaptic VR1 receptors localized on primary afferents. Menthol significantly increased the mEPSC frequency with a similar developmental pattern to capsaicin without consistently affecting mEPSC amplitude. The increase in mEPSC frequency following capsaicin did not depend on transmembrane calcium influx since it persisted in zero [Ca²⁺]_o. The facilitation of spontaneous glutamate release by capsaicin was sufficient to evoke action potentials in neonatal dorsal horn neurons but was accompanied by a block of EPSCs evoked by electrical stimulation of the dorsal root. These results indicate that VR1-expressing nociceptive primary afferents form functional synaptic connections in the superficial dorsal horn from birth and that activation of the VR1 receptor increases spontaneous glutamate release via an undetermined mechanism. In addition, the data suggest that immature primary afferents express functional menthol receptors that are capable of modulating transmitter release. These results have important functional implications for infant pain processing.     

Receptor targeting and heterogeneity at interneuronal nicotinic cholinergic synapses in vivo

Within a single neuron the correct targeting of the diverse neurotransmitter receptor types to discrete synaptic regions is crucial for proper function. However, the molecular mechanisms that underlie neuronal receptor clustering and targeting are still largely undefined. Here we report advances in defining the mechanisms that mediate nicotinic acetylcholine receptor (nAChR) targeting to interneuronal synapses. Recent in vivo studies have demonstrated that one subunit plays a critical role in the differentiation of nicotinic cholinergic synapses on vertebrate autonomic neurons. The major cytoplasmic loop of the α3 subunit targets specific nAChR subtypes to the synapse. In contrast, nAChR complexes that lack the α3 targeting domain are excluded and are perisynaptic. Additional studies have demonstrated a greater complexity to α3-nAChR targeting due to a unique postsynaptic receptor microheterogeneity – under one presynaptic terminal, α3-nAChR clusters are separate, but proximal to, glycine receptor (GlyR) clusters in discrete postsynaptic membrane microregions. The surprising coexistence under one nerve ending of separate clusters of receptors that respond to different fast-acting transmitters with opposing functions may represent a novel mechanism for modulating synaptic activity. Overall, the receptor targeting and clustering studies reviewed in this issue suggest that a common mechanism underlies the formation of the diverse types of interneuronal synapses but differs from that responsible for neuromuscular junction assembly in vertebrates.