mGluR7 is a metaplastic switch controlling bidirectional plasticity of feedforward inhibition

Plasticity of feedforward inhibition in the hippocampal mossy fiber (MF) pathway can dramatically influence dentate gyrus-CA3 dialog. Interestingly, MF inputs to CA3 stratum lucidum interneurons (SLINs) undergo long-term depression (LTD) following high-frequency stimulation (HFS), in contrast to MF-pyramid (PYR) synapses, where long-term potentiation (LTP) occurs. Furthermore, activity-induced potentiation of MF-SLIN transmission has not previously been observed. Here we report that metabotropic glutamate receptor subtype 7 (mGluR7) is a metaplastic switch at MF-SLIN synapses, whose activation and surface expression governs the direction of plasticity. In naive slices, mGluR7 activation during HFS generates MF-SLIN LTD, depressing presynaptic release through a PKC-dependent mechanism. Following agonist exposure, mGluR7 undergoes internalization, unmasking the ability of MF-SLIN synapses to undergo presynaptic potentiation in response to the same HFS that induces LTD in naive slices. Thus, selective mGluR7 targeting to MF terminals contacting SLINs and not PYRs provides cell target-specific plasticity and bidirectional control of feedforward inhibition.     

Subtype-specific expression of group III metabotropic glutamate receptors and Ca2+ channels in single nerve terminals

The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca(2+) channel activity. By imaging Ca(2+) in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca(2+) channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca(2+) channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for l(+)-2-amino-4-phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca(2+) response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca(2+) channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca(2+) channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca(2+) channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity.     

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25^+/− mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.     

Inactivation kinetics of voltage-gated calcium channels in glutamatergic neurons are influenced by SNAP-25

SNAP-25 forms part of the SNARE core complex that mediates membrane fusion. Biochemical and electrophysiological evidence supports an accessory role for SNAP-25 in interacting with voltage-gated calcium channels (VGCCs) to modulate channel activity. We recently reported that endogenous SNAP-25 negatively regulates VGCC activity in glutamatergic neurons from rat hippocampal cultures by shifting the voltage-dependence of inactivation of the predominant P/Q-type channel current in these cells. In the present study, we extend these findings by investigating the effect that manipulating endogenous SNAP-25 expression has on the inactivation kinetics of VGCC current in both glutamatergic and GABAergic cells recorded from 9-13 DIV cultures. Silencing SNAP-25 in glutamatergic neurons significantly slowed the inactivation rate of P/Q-type VGCC current whereas alterations in SNAP-25 expression did not alter inactivation rates in GABAergic neurons. These results indicate that endogenous SNAP-25 plays an important role in P/Q-type channel regulation in glutamatergic neurons.     

Inactivation kinetics of voltage-gated calcium channels in glutamatergic neurons are influenced by SNAP-25

SNAP-25 forms part of the SNARE core complex that mediates membrane fusion. Biochemical and electrophysiological evidence supports an accessory role for SNAP-25 in interacting with voltage-gated calcium channels (VGCCs) to modulate channel activity. We recently reported that endogenous SNAP-25 negatively regulates VGCC activity in glutamatergic neurons from rat hippocampal cultures by shifting the voltage-dependence of inactivation of the predominant P/Q-type channel current in these cells. In the present study, we extend these findings by investigating the effect that manipulating endogenous SNAP-25 expression has on the inactivation kinetics of VGCC current in both glutamatergic and GABAergic cells recorded from 9-13 DIV cultures. Silencing SNAP-25 in glutamatergic neurons significantly slowed the inactivation rate of P/Q-type VGCC current whereas alterations in SNAP-25 expression did not alter inactivation rates in GABAergic neurons. These results indicate that endogenous SNAP-25 plays an important role in P/Q-type channel regulation in glutamatergic neurons.     

Modulating modulation: crosstalk between regulatory pathways of presynaptic calcium channels

The activity of some voltage-gated calcium channels (VGCCs) can be inhibited by specific G protein beta subunits. Conversely, in the case of N-type VGCCs, protein kinase C can relieve Gbeta-dependent inhibition by phosphorylating at least one specific site on the calcium channel. A recent publication describes a newly identified method of intracellular regulation of specific VGCCs. Wu et al. have uncovered that VGCC activity can be regulated by phosphatidylinositol-4',5'-bisphosphate (PIP2). Whereas PIP2 is important for maintaining the activity (open state) of Cav2.1 (N-type) and Cav2.2 (P/Q-type) channels, the enzymatic breakdown of PIP2 leads to the inactivation of these channels. Additionally, PIP2 can cause changes in voltage-dependent activation of Cav2.2 (P/Q-type) channels that make it more difficult for these channels to open (from the closed state). Furthermore, protein kinase A activity can circumvent PIP2-mediated inhibition. Thus, the PIP2-mediated regulation of VGCCs is tightly controlled by the functions of kinases (and phosphatases), as well as phospholipases. Wu et al. stress that because PIP2 can be found at synapses, PIP2-dependent control of VGCCs "could have profound consequences on synaptic transmission and plasticity."     

Dynamic aspects of presynaptic calcium currents mediating synaptic transmission

Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers transmitter release. Direct recording of Ca2+ currents from the calyx of Held nerve terminal revealed that presynaptic VGCCs undergo various modulations via presynaptic G protein-coupled receptors (GPCRs), Ca2+-binding proteins and a developmental switch of their alpha1 subunits. Dynamic changes of presynaptic VGCCs alter synaptic efficacy, thereby contributing to a variety of modulations of the CNS function.     

Co-expression of metabotropic glutamate receptor 7 and N-type Ca(2+) channels in single cerebrocortical nerve terminals of adult rats

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in cerebrocortical nerve terminals of adult rats, the inhibition of glutamate release is mediated by mGluR7. In this preparation, the major component of glutamate release is supported by P/Q-type Ca2+ channels (72.7%). However, mGluR7 selectively reduced the release component that is associated with N-type Ca2+ channels (29.9%). Inhibition of P/Q channels by mGluR7 is not masked by the higher efficiency of these channels in driving glutamate release when compared with N-type channels. Thus, activation of mGluR7 failed to reduce the release associated with P/Q channels when the extracellular calcium concentration, ([Ca2+]o), was reduced from 1.3 to 0.5 mm. Through Ca2+ imaging, we show that Ca2+ channels are distributed in a heterogeneous manner in individual nerve terminals. Indeed, in this preparation, nerve terminals were observed that contain N-type (31.1%; conotoxin GVIA-sensitive) or P/Q-type (64.3%; agatoxin IVA-sensitive) channels or that were insensitive to these two toxins (4.6%). Interestingly, the great majority of the responses to l-AP4 (95.4%) were observed in nerve terminals containing N-type channels. This specific co-localization of mGluR7 and N-type Ca2+-channels could explain the failure of the receptor to inhibit the P/Q channel-associated release component and also reveal the existence of specific targeting mechanisms to localize the two proteins in the same nerve terminal subset.     

Presynaptic glycine receptors on hippocampal mossy fibers

Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of ∼600 pS in young to ∼130 pS in adult animals. Single-channel analysis revealed multiple conductance states between ∼20 and ∼120 pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8-17 per young, and 1-3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber - CA3 synapse.     

The CACNA1A Mutant Disrupts Lysosome Calcium Homeostasis in Cerebellar Neurons and the Resulting Endo-Lysosomal Fusion Defect Can be Improved by Calcium Modulation

Mutations in P/Q type voltage gated calcium channel (VGCC) lead severe human neurological diseases such as episodic ataxia 2, familial hemiplegic migraine 1, absence epilepsy, progressive ataxia and spinocerebellar ataxia 6. The pathogenesis of these diseases remains unclear. Mice with spontaneous mutation in the Cacna1a gene encoding the pore-forming subunit of P/Q type VGCC also exhibit ataxia, epilepsy and neurodegeneration. Based on the previous work showing that the P/Q type VGCC in neurons regulates lysosomal fusion through its calcium channel activity on lysosomes, we utilized CACNA1A mutant mice to further investigate the mechanism by which P/Q-type VGCCs regulate lysosomal function and neuronal homeostasis. We found CACNA1A mutant neurons have reduced lysosomal calcium storage without changing the resting calcium concentration in cytoplasm and the acidification of lysosomes. Immunohistochemistry and transmission electron microscopy reveal axonal degeneration due to lysosome dysfunction in the CACNA1A mutant cerebella. The calcium modulating drug thapsigargin, by depleting the ER calcium store, which locally increases the calcium concentration can alleviate the defective lysosomal fusion in mutant neurons. We propose a model that in cerebellar neurons, P/Q-type VGCC maintains the integrity of the nervous system by regulating lysosomal calcium homeostasis to affect lysosomal fusion, which in turn regulates multiple important cellular processes such as autophagy and endocytosis. This study helps us to better understand the pathogenesis of P/Q-type VGCC related neurodegenerative diseases and provides a feasible direction for future pharmacological treatment.

     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Presynaptic LTP and LTD of Excitatory and Inhibitory Synapses

Ubiquitous forms of long-term potentiation (LTP) and depression (LTD) are caused by enduring increases or decreases in neurotransmitter release. Such forms or presynaptic plasticity are equally observed at excitatory and inhibitory synapses and the list of locations expressing presynaptic LTP and LTD continues to grow. In addition to the mechanistically distinct forms of postsynaptic plasticity, presynaptic plasticity offers a powerful means to modify neural circuits. A wide range of induction mechanisms has been identified, some of which occur entirely in the presynaptic terminal, whereas others require retrograde signaling from the postsynaptic to presynaptic terminals. In spite of this diversity of induction mechanisms, some common induction rules can be identified across synapses. Although the precise molecular mechanism underlying long-term changes in transmitter release in most cases remains unclear, increasing evidence indicates that presynaptic LTP and LTD can occur in vivo and likely mediate some forms of learning.At several excitatory and inhibitory synapses, neuronal activity can trigger enduring increases or decreases in neurotransmitter release, thereby producing long-term potentiation (LTP) or long-term depression (LTD) of synaptic strength, respectively. In the last decade, many studies have revealed that these forms of plasticity are ubiquitously expressed in the mammalian brain, and accumulating evidence indicates that they may underlie behavioral adaptations occurring in vivo. These studies have also uncovered a wide range of induction mechanisms, which converge on the presynaptic terminal where an enduring modification in the neurotransmitter release process takes place. Interestingly, presynaptic forms of LTP/LTD can coexist with classical forms of postsynaptic plasticity. Such diversity expands the dynamic range and repertoire by which neurons modify their synaptic connections. This review discusses mechanistic aspects of presynaptic LTP and LTD at both excitatory and inhibitory synapses in the mammalian brain, with an emphasis on recent findings.     

Modulation/physiology of calcium channel sub-types in neurosecretory terminals

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, μ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.     

Lambert-Eaton myasthenic syndrome as an autoimmune calcium channelopathy

Lambert-Eaton myasthenic syndrome, often associated with small-cell lung carcinoma, is a disease of neuromuscular transmission in which antibodies directed against voltage-gated calcium channel (VGCC)(P/Q-type) in the motor nerve terminal play a crucial role in causing a deficient quantal release of acetylcholine. The motor nerve terminal and carcinoma cell may share a common antigen. The study using synthetic peptides and recombinant protein specified the extracellular S5-S6 linker regions in 3 of 4 domains as immunodominant sites in the molecular structure of P/Q-type VGCC alpha1 subunit. Also, the study by use of peptides and recombinant protein corresponding to synaptotagmin I suggested that in this functionally VGCC-associated presynaptic protein, the segment which exposes extracellularly during exocytosis can be immunogenic for the syndrome.     

Structural plasticity at crustacean neuromuscular synapses

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.     

Long-term depression of kainate receptor-mediated synaptic transmission

Kainate receptors (KARs) have been shown to be involved in hippocampal mossy fiber long-term potentiation (LTP); however, it is not known if KARs are involved in the induction or expression of long-term depression (LTD), the other major form of long-term synaptic plasticity. Here we describe LTD of KAR-mediated synaptic transmission (EPSC(KA) LTD) in perirhinal cortex layer II/III neurons that is distinct from LTD of AMPAR-mediated transmission, which also coexists at the same synapses. Induction of EPSC(KA) LTD requires a rise in postsynaptic Ca(2+) but is independent of NMDARs or T-type voltage-gated Ca(2+) channels; however, it requires synaptic activation of inwardly rectifying KARs and release of Ca(2+) from stores. The synaptic KARs are regulated by tonically activated mGluR5, and expression of EPSC(KA) LTD occurs via a mechanism involving mGluR5, PKC, and PICK1 PDZ domain interactions. Thus, we describe the induction and expression mechanism of a form of synaptic plasticity, EPSC(KA) LTD.     

Regulation of presynaptic Ca(V)2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity

Short-term synaptic plasticity shapes the postsynaptic response to bursts of impulses and is crucial for encoding information in neurons, but the molecular mechanisms are unknown. Here we show that activity-dependent modulation of presynaptic Ca(V)2.1 channels mediated by neuronal Ca(2+) sensor proteins (CaS) induces synaptic plasticity in cultured superior cervical ganglion (SCG) neurons. A mutation of the IQ-like motif in the C terminus that blocks Ca(2+)/CaS-dependent facilitation of the P/Q-type Ca(2+) current markedly reduces facilitation of synaptic transmission. Deletion of the nearby calmodulin-binding domain, which inhibits CaS-dependent inactivation, substantially reduces depression of synaptic transmission. These results demonstrate that residual Ca(2+) in presynaptic terminals can act through CaS-dependent regulation of Ca(V)2.1 channels to induce short-term synaptic facilitation and rapid synaptic depression. Activity-dependent regulation of presynaptic Ca(V)2.1 channels by CaS proteins may therefore be a primary determinant of short-term synaptic plasticity and information-processing in the nervous system.     

Long-term plasticity mediated by mGluR1 at a retinal reciprocal synapse

The flow of information across the retina is controlled by reciprocal synapses between bipolar cell terminals and amacrine cells. However, the synaptic delays and properties of plasticity at these synapses are not known. Here we report that glutamate release from goldfish Mb-type bipolar cell terminals can trigger fast (delay of 2-3 ms) and transient GABA(A) IPSCs and a much slower and more sustained GABA(C) feedback. Synaptically released glutamate activated mGluR1 receptors on amacrine cells and, depending on the strength of presynaptic activity, potentiated subsequent feedback. This poststimulus enhancement of GABAergic feedback lasted for up to 10 min. This form of mGluR1-mediated long-term synaptic plasticity may provide retinal reciprocal synapses with adaptive capabilities.     

Synthesis and biological evaluation of nonpeptide mimetics of omega-conotoxin GVIA

A benzothiazole-derived compound (4a) designed to mimic the C(alpha)-C(beta) bond vectors and terminal functionalities of Lys2, Tyr13 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low microM binding affinity to N-type VGCCs (IC(50)=1.9 microM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC(50)= 4.1 microM) showed a greater than 25-fold selectivity for the N-type channel.