Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.     

Neuromuscular synaptic transmission in aged ganglioside-deficient mice

Gangliosides are sialylated glycosphingolipids that are present in high density on neuronal membranes, especially at synapses, where they are assumed to play functional or modulating roles. Mice lacking GM2/GD2-synthase express only the simple gangliosides GD3 and GM3 and develop progressive motor behaviour deficits upon ageing, apparently due to failing complex ganglioside-dependent maintenance and/or repair processes or, alternatively, toxic GM3/GD3 accumulation. We investigated the function of neuromuscular junctions (NMJs) of aged (>9 month-old) GM2/GD2-synthase null-mutant mice, because synaptic dysfunction might develop with age and could potentially contribute to the late-onset motor phenotype. In addition, we studied NMJs of old mice lacking GD3-synthase (expressing only O- and a-series gangliosides), which do not show an overt neurological phenotype but may develop subclinical synaptic deficits. Detailed electrophysiological analyses showed subtle changes in presynaptic neurotransmitter release. Acetylcholine release at 40 Hz nerve stimulation at aged GM2/GD2-synthase null-mutant NMJs ran down slightly more pronounced than at wild-type NMJs, and spontaneous acetylcholine release rate at GD3-synthase null-mutant NMJs was somewhat higher than at wild-type, selectively at 25 °C bath temperature. Interestingly, we observed faster kinetics of postsynaptic electrophysiological responses at aged GD3-synthase null-mutant NMJs, not previously seen by us at NMJs of young GD3-synthase null-mutants or other types of (aged or young) ganglioside-deficient mice. These kinetic changes might reflect a change in postsynaptic acetylcholine receptor behaviour. Our data indicate that it is highly unlikely that transmission failure at NMJs contributes to the progressive motor defects of aged GM2/GD2-synthase null-mutants and that, despite some kinetic changes of synaptic signals, neuromuscular transmission remains successful in aged GD3-synthase null-mutant mice. Apparently, mutual redundancy of the different gangliosides in supporting presynaptic function, as observed previously by us in young mice, remains adequate upon ageing or, alternatively, gangliosides have only relatively little direct impact on neuromuscular synaptic function, even in aged mice.     

Characterization of acetylcholine release and the compensatory contribution of non-Ca(v)2.1 channels at motor nerve terminals of leaner Ca(v)2.1-mutant mice

The severely ataxic and epileptic mouse leaner (Ln) carries a natural splice site mutation in Cacna1a, leading to a C-terminal truncation of the encoded Ca(v)2.1 alpha(1) protein. Ca(v)2.1 is a neuronal Ca(2+) channel, mediating neurotransmitter release at many central synapses and the peripheral neuromuscular junction (NMJ). With electrophysiological analyses we demonstrate severely reduced ( approximately 50%) neurotransmitter release at Ln NMJs. This equals the reduction at NMJs of Cacna1a null-mutant (Ca(v)2.1-KO) mice, which display a neurological phenotype remarkably similar to that of Ln mice. However, using selective Ca(v) channel blocking compounds we revealed a compensatory contribution profile of non-Ca(v)2.1 type channels at Ln NMJs that differs completely from that at Ca(v)2.1-KO NMJs. Our data indicate that the residual function and presence of Ln-mutated Ca(v)2.1 channels precludes presynaptic compensatory recruitment of Ca(v)1 and Ca(v)2.2 channels, and hampers that of Ca(v)2.3 channels. This is the first report directly showing at single synapses the deficits and plasticity in transmitter release resulting from the Ln mutation of Cacna1a.     

GDNF and GFRalpha1 promote formation of neuronal synapses by ligand-induced cell adhesion

The establishment of synaptic connections requires precise alignment of pre- and postsynaptic terminals. The glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 is enriched at pre- and postsynaptic compartments in hippocampal neurons, suggesting that it has a function in synapse formation. GDNF triggered trans-homophilic binding between GFRalpha1 molecules and cell adhesion between GFRalpha1-expressing cells. This represents the first example of a cell-cell interaction being mediated by a ligand-induced cell adhesion molecule (LICAM). In the presence of GDNF, ectopic GFRalpha1 induced localized presynaptic differentiation in hippocampal neurons, as visualized by clustering of vesicular proteins and neurotransmitter transporters, and by activity-dependent vesicle recycling. Presynaptic differentiation induced by GDNF was markedly reduced in neurons lacking GFRalpha1. Gdnf mutant mice showed reduced synaptic localization of presynaptic proteins and a marked decrease in the density of presynaptic puncta, indicating a role for GDNF signaling in hippocampal synaptogenesis in vivo. We propose that GFRalpha1 functions as a LICAM to establish precise synaptic contacts and induce presynaptic differentiation.     

Glia cell line-derived neurotrophic factor regulates the distribution of acetylcholine receptors in mouse primary skeletal muscle cells

It was recently reported that glia cell line-derived neurotrophic factor (GDNF) facilitates presynaptic axonal growth and neurotransmitter release at neuromuscular synapses. Little is known, however, whether GDNF can also act on the postsynaptic apparatus and its underlying mechanisms. Using biochemical cold blocking of existing membrane acetylcholine receptors (AchRs) and biotinylation of newly inserted receptors we demonstrate that GDNF increases the insertion of AChRs into the surface membrane of mouse primary cultured muscle cells and that this does not require protein synthesis. Quantitative data from double-label imaging indicate that GDNF induces a quick and substantial increase in AchR insertion as well as lateral movement into AchR aggregates, relative to a weak effect on reducing the loss of receptors from pre-existing AchR aggregates, which in contrast to the effect of PMA. These effects occur in both innervated and un-innervated muscles, and GDNF affects nerve-muscle co-cultures more than it affects muscle-only cultures. Neurturin, another member of GDNF-family ligands has similar effects on AchRs as GDNF but the unrelated growth factor, EGF does not. Studies on protein phosphorylation and specific inhibitors of cell signal transduction indicate that GDNF function is mediated by receptor GFRalpha1 and involves MAPK, cAMP/cAMP responsive element-binding factor and Src kinase activities. GDNF may signal through c-Ret as well as NCAM-140 pathways since both the signaling receptors are expressed in the neuromuscular junction (NMJ). These data suggest that GDNF is an autocrine regulator of NMJ to promote the insertion and stabilization of postsynaptic AchRs. In vivo, GDNF may function as a synaptotrophic modulator for both pre- and postsynaptic differentiation to strengthen the functional and structural connections between nerve and muscle, and contribute to the synaptogenesis and plasticity of neuromuscular synapses.     

alpha-Neurexins are required for efficient transmitter release and synaptic homeostasis at the mouse neuromuscular junction

Neurotransmission at chemical synapses of the brain involves alpha-neurexins, neuron-specific cell-surface molecules that are encoded by three genes in mammals. Deletion of alpha-neurexins in mice previously demonstrated an essential function, leading to early postnatal death of many double-knockout mice and all triple mutants. Neurotransmitter release at central synapses of newborn knockouts was severely reduced, a function of alpha-neurexins that requires their extracellular sequences. Here, we investigated the role of alpha-neurexins at neuromuscular junctions, presynaptic terminals that lack a neuronal postsynaptic partner, addressing an important question because the function of neurexins was hypothesized to involve cell-adhesion complexes between neurons. Using systems physiology, morphological analyses and electrophysiological recordings, we show that quantal content, i.e. the number of acetylcholine quanta released per nerve impulse from motor nerve terminals, and frequency of spontaneous miniature endplate potentials at the slow-twitch soleus muscle are reduced in adult alpha-neurexin double-knockouts, consistent with earlier data on central synapses. However, the same parameters at diaphragm muscle neuromuscular junctions showed no difference in basal neurotransmission. To reconcile these observations, we tested the capability of control and alpha-neurexin-deficient diaphragm neuromuscular junctions to compensate for an experimental reduction of postsynaptic acetylcholine receptors by a compensatory increase of presynaptic release: Knockout neuromuscular junctions produced significantly less upregulation of quantal content than synapses from control mice. Our data suggest that alpha-neurexins are required for efficient neurotransmitter release at neuromuscular junctions, and that they may perform a role in the molecular mechanism of synaptic homeostasis at these peripheral synapses.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of non-obese diabetic mice

Skeletal muscle is one of the main targets of the metabolic alterations in diabetes, in which protein synthesis is markedly reduced followed by increased proteolysis. Ultrastructural and functional changes in the presynaptic compartment of the neuromuscular junction (NMJ) have been demonstrated, but little attention has been paid to the proteins in the postsynaptic muscle fiber membrane. In the present work, we studied the changes in acetylcholine receptors (AChRs) and nerve terminal distribution in the NMJ of non-obese diabetic (NOD) mice. The sternomastoid muscles of adult female NOD mice were double-labeled for AChR and nerve terminal observation by fluorescence and reflected light confocal microscopy. In 62.4% of the diabetic endplates, AChR branches broke apart into receptor islands that stained less than in the normal mice. These patches had regular junctional folds. At most of the endplates studied, the nerve terminals colocalized with AChRs, and sprouts were seen in 10% of the diabetic endplates. The intramuscular nerve branches and axons in the nerve to the sternomastoid muscle showed no degenerative disorders. These results suggest that metabolic alterations in the diabetic muscle fiber can affect the distribution and expression of molecules, such as AChRs, in the postsynaptic membrane of the neuromuscular junction.     

Assembly, plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at "non-plastic" NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.     

Changes in the Kinetics of Evoked Secretion of Transmitter Quanta – an Effective Mechanism Modulating the Synaptic Transmission of Excitation

The main presynaptic mechanisms of synaptic plasticity are generally believed to consist of changes in the numbers of neurotransmitter quanta released in response to a nerve spike (the quantum composition of postsynaptic responses) and quantum size. However, studies in recent years have demonstrated the existence of a further, previously unconsidered but effective mechanism modulating the synaptic transmission of excitation, which is associated with changes in the secretion time course (kinetics) of the release of the individual quanta forming the multiquantum postsynaptic response. This review discusses current data (including our own results) on the kinetics of the evoked release of neurotransmitter quanta from motor nerve endings in peripheral synapses, the mechanisms of its modulation, and quantitative methods for its analysis.     

Presynaptic CB1 receptors regulate synaptic plasticity at cerebellar parallel fiber synapses

Endocannabinoids are potent regulators of synaptic strength. They are generally thought to modify neurotransmitter release through retrograde activation of presynaptic type 1 cannabinoid receptors (CB1Rs). In the cerebellar cortex, CB1Rs regulate several forms of synaptic plasticity at synapses onto Purkinje cells, including presynaptically expressed short-term plasticity and, somewhat paradoxically, a postsynaptic form of long-term depression (LTD). Here we have generated mice in which CB1Rs were selectively eliminated from cerebellar granule cells, whose axons form parallel fibers. We find that in these mice, endocannabinoid-dependent short-term plasticity is eliminated at parallel fiber, but not inhibitory interneuron, synapses onto Purkinje cells. Further, parallel fiber LTD is not observed in these mice, indicating that presynaptic CB1Rs regulate long-term plasticity at this synapse.     

GABA transporter-1 (GAT1)-deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus

After its release from interneurons in the CNS, the major inhibitory neurotransmitter GABA is taken up by GABA transporters (GATs). The predominant neuronal GABA transporter GAT1 is localized in GABAergic axons and nerve terminals, where it is thought to influence GABAergic synaptic transmission, but the details of this regulation are unclear. To address this issue, we have generated a strain of GAT1-deficient mice. We observed a large increase in a tonic postsynaptic hippocampal GABAA receptor-mediated conductance. There was little or no change in the waveform or amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) or miniature IPSCs. In contrast, the frequency of quantal GABA release was one-third of wild type (WT), although the densities of GABAA receptors, GABAB receptors, glutamic acid decarboxylase 65 kDa, and vesicular GAT were unaltered. The GAT1-deficient mice lacked a presynaptic GABAB receptor tone, present in WT mice, which reduces the frequency of spontaneous IPSCs. We conclude that GAT1 deficiency leads to enhanced extracellular GABA levels resulting in an overactivation of GABAA receptors responsible for a postsynaptic tonic conductance. Chronically elevated GABA levels also downregulate phasic GABA release and reduce presynaptic signaling via GABAB receptors thus causing an enhanced tonic and a diminished phasic inhibition.     

Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal CA3 pyramidal neurons in an isolated neuron/bouton preparation

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABA_A receptors to the GABA_A receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55212-2 (mesylate(R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid 2-arachidonoylglycerol also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-βS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of non‐obese diabetic mice

Skeletal muscle is one of the main targets of the metabolic alterations in diabetes, in which protein synthesis is markedly reduced followed by increased proteolysis. Ultrastructural and functional changes in the presynaptic compartment of the neuromuscular junction (NMJ) have been demonstrated, but little attention has been paid to the proteins in the postsynaptic muscle fiber membrane. In the present work, we studied the changes in acetylcholine receptors (AChRs) and nerve terminal distribution in the NMJ of non‐obese diabetic (NOD) mice. The sternomastoid muscles of adult female NOD mice were double‐labeled for AChR and nerve terminal observation by fluorescence and reflected light confocal microscopy. In 62.4% of the diabetic endplates, AChR branches broke apart into receptor islands that stained less than in the normal mice. These patches had regular junctional folds. At most of the endplates studied, the nerve terminals colocalized with AChRs, and sprouts were seen in 10% of the diabetic endplates. The intramuscular nerve branches and axons in the nerve to the sternomastoid muscle showed no degenerative disorders. These results suggest that metabolic alterations in the diabetic muscle fiber can affect the distribution and expression of molecules, such as AChRs, in the postsynaptic membrane of the neuromuscular junction. Anat Rec 267:112–119, 2002. © 2002 Wiley‐Liss, Inc.     

Retrograde signaling in the regulation of synaptic transmission: focus on endocannabinoids

This review covers recent developments in the cellular neurophysiology of retrograde signaling in the mammalian central nervous system. Normally at a chemical synapse a neurotransmitter is released from the presynaptic element and diffuses to the postsynaptic element, where it binds to and activates receptors. In retrograde signaling a diffusible messenger is liberated from the postsynaptic element, and travels "backwards" across the synaptic cleft, where it activates receptors on the presynaptic cell. Receptors for retrograde messengers are usually located on or near the presynaptic nerve terminals, and their activation causes an alteration in synaptic transmitter release. Although often considered in the context of long-term synaptic plasticity, retrograde messengers have numerous roles on the short-term regulation of synaptic transmission. The focus of this review will be on a group of molecules from different chemical classes that appear to act as retrograde messengers. The evidence supporting their candidacy as retrograde messengers is considered and evaluated. Endocannabinoids have recently emerged as one of the most thoroughly investigated, and widely accepted, classes of retrograde messenger in the brain. The study of the endocannabinoids can therefore serve as a model for the investigation of other putative messengers, and most attention is devoted to a discussion of systems that use these new messenger molecules.     

Polymodal activation of the endocannabinoid system in the extended amygdala

The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(2+) channels and the subsequent release of 2-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, 2-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.     

Impaired GABAergic inhibition in the visual cortex of brain-derived neurotrophic factor heterozygous knockout mice

Brain derived neurotrophic factor (BDNF) promotes the formation, maturation and stabilization of inhibitory synapses in the central nervous system. In addition, BDNF has been suggested to regulate the critical period for ocular dominance plasticity in the visual system. Here we further evaluated the role of BDNF in the visual cortex by studying the GABAergic synaptic transmission under conditions of chronically reduced levels of BDNF. Whole-cell patch-clamp recordings were performed from pyramidal neurons located in layers II/III of visual cortical slices in heterozygous BDNF knockout mice (BDNF (+/-)) and their wild-type littermates at the age of 21-25 days. The BDNF (+/-) mice showed a decreased frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) as well as a reduced amplitude and prolonged decay time constant of evoked IPSCs. Further analyses indicated an impaired presynaptic GABAergic function in BDNF (+/-) mice, as shown by the decreased release probability, steady-state release and synchronous release of GABA. However, the number of functional release sites remained unchanged. In line with these observations, an impaired glutamate-driven GABA release was observed in BDNF (+/-) mice. Furthermore, the overall balance in the strength of cortical excitation to inhibition shifted towards a decreased inhibition. Finally, the reversal potential for chloride-mediated evoked IPSCs was not affected. These findings suggested that chronically reduced levels of BDNF strongly impair the GABAergic inhibitory function in visual cortex by altering postsynaptic properties and by reducing presynaptic GABA release as well as the overall strength of inhibition onto pyramidal neurons within the cortical network. These impairments of inhibitory function are compatible with a rather immature status of the GABAergic system in BDNF (+/-) mice, which supports the hypothesis that the level of expression for BDNF critically affects maturation and function of the GABAergic inhibition.     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

Assessing the long-term role of L-type voltage dependent calcium channel blocker verapamil on short-term presynaptic plasticity at dentate gyrus of hippocampus

High-voltage-activated Ca(2+) channels on presynaptic nerve terminals are known to play an important role in neurotransmitter release at both excitatory and inhibitory synapses. Whereas there is currently debate over the contribution of L-type voltage dependent Ca(2+) channels (L-type VDCCs) on the short-term presynaptic plasticity which is a defining feature of neuronal activity, the underlying mechanisms are poorly understood. In the present study, the L-type VDCCs chronically was inhibited with different doses of verapamil (10, 20 and 50 mg/kg; orally) to evaluate hippocampal dentate gyrus (DG) inhibitory interneuron function and its involvement on short-term plasticity using paired pulse stimulation in perforant path-DG of hippocampus. Our data show that chronic oral treatment of verapamil at dose of 50 mg/kg but not at lower doses, facilitated the excitability of DG cells at inter-stimulus intervals 20, 30 and 50 ms (P<0.03, 0.01 and 0.001; respectively) in population spike amplitude ratio, which is indicative of paired pulse potentiation in perforant path-DG synapses. While there are no significant differences in field excitatory postsynaptic potential slope ratio at all doses. We suggest that DG neurons facilitation is caused by inhibition of inhibitory interneurons directly and/or indirectly via inhibition of glutamate release in hippocampal DG. Therefore, these experiments indicate that chronic use of verapamil has effect on short-term presynaptic plasticity.     

Presynaptic NMDA receptor-mediated modulation of excitatory neurotransmission in the mouse dorsal motor nucleus of the vagus

Activity of neurons in the dorsal motor nucleus of the vagus nerve (DMV) is closely regulated by synaptic input, and regulation of that input by glutamate receptors on presynaptic terminals has been proposed. Presynaptic N-methyl-d-aspartic acid (NMDA) receptors have been identified in a number of brain regions and act to modulate neurotransmitter release, but functional presynaptic NMDA receptors have not been adequately studied in the DMV. This study identified the presence and physiological function of presynaptic NMDA receptors on synaptic input to DMV neurons. Whole-cell patch-clamp recordings from DMV neurons in acute slices from mice revealed prevalent miniature excitatory postsynaptic currents, which were significantly increased in frequency, but not amplitude, by application of NMDA. Antagonism of NMDA receptors with dl-2-amino-5-phosphonopentanoic acid (100 μM) resulted in a decrease in miniature excitatory postsynaptic current frequency and an increase in the paired pulse ratio of responses following afferent stimulation. No consistent effects of presynaptic NMDA receptor modulation were observed on GABAergic inputs. These results suggest that presynaptic NMDA receptors are present in the dorsal vagal complex and function to facilitate the release of glutamate, preferentially onto DMV neurons tonically, with little effect on GABA release. This type of presynaptic modulation represents a potentially novel form of glutamate regulation in the DMV, which may function to regulate glutamate-induced activity of central parasympathetic circuits.