Frequency Of Spontaneous Synaptic Currents Research Articles

Analyses of the Mode of Action of an Alpha-Adrenoceptor Blocker in Substantia Gelatinosa Neurons in Rats.

To elucidate why naftopidil increases the frequency of spontaneous synaptic currents in only some substantia gelatinosa (SG) neurons, post-hoc analyses were performed. Blind patch-clamp recording was performed using slice preparations of SG neurons from the spinal cords of adult rats. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were recorded. The ratios of the frequency and amplitude of the sIPSCs and sEPSCs following the introduction of naftopidil compared with baseline, and after the application of naftopidil, serotonin (5-HT), and prazosin, compared with noradrenaline (NA) were evaluated. First, the sIPSC analysis indicated that SG neurons reached their full response ratio for NA at 50 μM. Second, they responded to 5-HT (50 μM) with a response ratio similar to that for NA, but prazosin (10 μM) did not change the sEPSCs and sIPSCs. Third, the highest concentration of naftopidil (100 μM) led to two types of response in the SG neurons, which corresponded with the reactions to 5-HT and prazosin. These results indicate that not all neurons were necessarily activated by naftopidil, and that the micturition reflex may be regulated in a sophisticated manner by inhibitory mechanisms in these interneurons.

Functional expression of TrkB receptors on interneurones and pyramidal cells of area CA3 of the rat hippocampus

The dentate gyrus and hippocampal area CA3 region of the mammalian brain contains the highest levels of brain-derived neurotrophic factor (BDNF) and its canonical membrane receptor, tropomyosin-related kinase B (TrkB). Therefore, the present study examines the expression and physiological responses triggered by activation of TrkB on hippocampal area CA3 interneurones and pyramidal cells of the rat hippocampus. Triple immunolabelling for TrkB, glutamate decarboxylase 67, and the calcium-binding proteins parvalbumin, calbindin or calretinin confirms the somatic expression of TrkB in all CA3 sublayers. TrkB-positive interneurones with fast-spiking discharge are restricted to strata oriens and lucidum, whereas regular-spiking interneurones are found in the strata lucidum, radiatum and lacunosum-moleculare. Activation of TrkB receptors with 7,8-dihydroxyflavone (DHF) modulates amplitude and frequency of spontaneous synaptic currents recorded from CA3 interneurones. Furthermore, the isolated excitatory postsynaptic currents (EPSC) of CA3 interneurones evoked by the mossy fibres (MF) or commissural/associational (C/A) axons, show input-specific synaptic potentiation in response to TrkB stimulation. On CA3 pyramidal cells, stimulation with DHF potentiates the MF synaptic transmission and increases the MF-EPSP – spike coupling. The latter exhibits a dramatic increase when picrotoxin is bath perfused after DHF, indicating that local interneurones restrain the excitability mediated by activation of TrkB. Therefore, we propose that release of BDNF on area CA3 reshapes the output of this hippocampal region by simultaneous activation of TrkB on GABAergic interneurones and pyramidal cells.

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) modifies synaptic transmission in hippocampal CA3 neurons of rats

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an alternative to organotin antifoulants, such as tributyltin and triphenyltin. Since DCOIT is found in harbors, bays, and coastal areas worldwide, this chemical compound may have some impacts on ecosystems. To determine whether DCOIT possesses neurotoxic activity by modifying synaptic transmission, we examined the effects of DCOIT on synaptic transmission in a ‘synaptic bouton’ preparation of rat brain. DCOIT at concentrations of 0.03–1 μM increased the amplitudes of evoked synaptic currents mediated by GABA and glutamate, while it reduced the amplitudes of these currents at 3–10 μM. However, the currents elicited by exogenous applications of GABA and glutamate were not affected by DCOIT. DCOIT at 1–10 μM increased the frequency of spontaneous synaptic currents mediated by GABA. It also increased the frequency of glutamate-mediated spontaneous currents at0.3–10 μM. The frequencies of miniature synaptic currents mediated by GABA and glutamate, observed in the presence of tetrodotoxin under external Ca2+-free conditions, were increased by 10 μM DCOIT. With the repetitive applications of DCOIT, the frequency of miniature synaptic currents mediated by glutamate was not increased by the second and third applications of DCOIT. Voltage-dependent Ca2+ channels were not affected by DCOIT, but DCOIT slowed the inactivation of voltage-dependent Na+ channels. These results suggest that DCOIT increases Ca2+ release from intracellular Ca2+ stores, resulting in the facilitation of both action potential-dependent and spontaneous neurotransmission, possibly leading to neurotoxicity.

Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination.

Oligodendrocyte precursor cells (OPCs) are a major source of remyelinating oligodendrocytes in demyelinating diseases such as Multiple Sclerosis (MS). While OPCs are innervated by unmyelinated axons in the normal brain, the fate of such synaptic contacts after demyelination is still unclear. By combining electrophysiology and immunostainings in different transgenic mice expressing fluorescent reporters, we studied the synaptic innervation of OPCs in the model of lysolecithin (LPC)-induced demyelination of corpus callosum. Synaptic innervation of reactivated OPCs in the lesion was revealed by the presence of AMPA receptor-mediated synaptic currents, VGluT1+ axon-OPC contacts in 3D confocal reconstructions and synaptic junctions observed by electron microscopy. Moreover, 3D confocal reconstructions of VGluT1 and NG2 immunolabeling showed the existence of glutamatergic axon-OPC contacts in post-mortem MS lesions. Interestingly, patch-clamp recordings in LPC-induced lesions demonstrated a drastic decrease in spontaneous synaptic activity of OPCs early after demyelination that was not caused by an impaired conduction of compound action potentials. A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine). At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels. In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process. Hence, glutamatergic synaptic inputs may contribute to inhibit OPC proliferation and might have a physiopathological relevance in demyelinating disorders.

CAMP promotes the differentiation of neural progenitor cells in vitro via modulation of voltage-gated calcium channels

The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs) remain poorly understood. In this study we investigated the role of Ca2+ and cAMP (cyclic adenosine monophosphate) in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine) significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na+ and K+ channels, as well as neuronal firing frequency. Furthermore, we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca2+currents. Conversely, blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX, indicating that voltage-gated Ca2+ influx plays a major role in neuronal differentiation. Finally, we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein), indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca2+-signaling. Taken together, these data indicate that the up-regulation of voltage-gated L-type Ca2+-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge, this is the first demonstration of the acute effects of cAMP on voltage-gated Ca+2channels in NPC-derived developing neurons.

GABAergic synapse maturation: evidence of the instructive role of activity‐dependent BDNF release

The study of the actions of brain-derived neurotrophic factor (BDNF) on central neurons and their synapses has revealed a constellation of observations ranging from neuronal survival and differentiation during early development to rapid effects on membrane conductance and intracellular Ca2+ levels in mature neurons (Amaral et al. 2007). Unfortunately, most studies in this abundant ‘BDNF literature’ are phenomenological and lack mechanistic insights, and even more importantly, direct evidence that the endogenous – native – protein is able to trigger an effect similar to that produced by application of recombinant BDNF. In addition, a few studies of BDNF actions generated long-standing controversial argumentations. Luckily, other BDNF effects have been consistently replicated, such as its role in the developmental maturation of GABAergic synapses. The paper by Kuczewski et al. (2008) in this issue of The Journal of Physiology presents direct evidence that endogenously released BDNF plays an instructive role in a new form of GABAergic synaptic plasticity in area CA3 of the neonatal hippocampus. The fact that BDNF was necessary for the maturation of GABAergic synapses was known from studies in the cortex of Bdnf overexpressing mice (Huang et al. 1999), but its source and mechanism of activity-dependent release remained unexplored. Now, a series of rigorous experiments provide solid evidence that glutamatergic activity causes Ca2+-dependent release of BDNF from postsynaptic CA3 neuron dendrites, which leads to an enduring potentiation of GABAAR-mediated synaptic currents. Despite the fact that BDNF actions on GABAergic synapses were described early on (Marty et al. 1997), the majority of studies focused on its role in excitatory synaptic transmission and plasticity (Poo, 2001); those studies revealed both pre- and postsynaptic sites of action, as well as indirect effects, generating a highly controversial landscape. Presynaptic mechanisms included modulation of probability of transmitter release via presynaptic Ca2+ levels, and modulation of synaptic vesicle mobilization, docking, depletion and recycling (Tyler et al. 2002), whereas postsynaptic mechanisms included modulation of NMDA receptor subunits or K+ channels and surface delivery of AMPA receptor subunits. On the other hand, the effects of BDNF on inhibitory synaptic transmission seemed more consistent (Tanaka et al. 1997), although the sites of action also spanned the synaptic cleft, e.g. modulation of presynaptic Ca2+ channel clustering (Baldelli et al. 2005) and postsynaptic modulation of GABAA receptors (GABAAR) (Brunig et al. 2001) or Cl− transport (Wardle & Poo, 2003). In the current study presented in this issue, Kuczewski et al. (2008) continue their exploration of the mechanisms by which spontaneous glutamatergic neuronal activity participates in the maturation of the neonatal (P1–P6) rat hippocampal network and uncover an enduring potentiation of GABAAR responses. This long-lasting potentiation of GABAAR-mediated synaptic activity (LLPGABA-A) was induced in CA3 pyramidal neurons by a brief and transient recovery following blockade of glutamatergic synaptic transmission, and was mediated by Ca2+-dependent postsynaptic release of endogenous – native – BDNF. Activity-dependent release of endogenous BDNF is necessary for LLPGABA-A induction (i.e. permissive role), and was demonstrated by its blockade with the extracellular BDNF scavenger TrkB-IgG. The BDNF effect was instructive, demonstrated by the rescue of LLPGABA-A induction by exogenously applied recombinant human met-BDNF under conditions that prevented endogenous BDNF release (e.g. the use of glutamate receptor antagonists or L-type Ca2+ channel block). Despite a well-designed experimental strategy, there are important issues that remain unclear, some from a technical standpoint and others from a conceptual perspective. For example, loading postsynaptic CA3 neurons with the Ca2+ chelator BAPTA prevented the increase in the frequency of spontaneous GABAAR-mediated synaptic currents. This observation seems counterintuitive, since only the one cell loaded with BAPTA was unable to release BDNF, and the frequency of spontaneous synaptic currents is likely to reflect the excitability of the entire neuronal network connected to the cell under recording. More conceptual voids are the mechanism(s) of expression/maintenance of LLPGABA-A (e.g. which steps take place between postsynaptic Ca2+ elevations and potentiation of GABAAR-mediated synaptic currents?) and whether there is a physiological consequence at the CA3 or hippocampal network level. It is immediately obvious that an enduring enhancement of GABAAR-mediated synaptic currents represents an important feature of the maturation of GABAergic inhibitory synapses. Certainly, more work will be necessary to fully comprehend the mechanisms by which endogenous BDNF is able to drive the activity-dependent developmental refinement of inhibitory synaptic networks, especially in a brain region like the hippocampus that – due to its recurrent and reentrant circuits that evolved for learning and memory – is always at the border of epileptiform activity.

Schwann Cell-Derived Factors Modulate Synaptic Activities at Developing Neuromuscular Synapses

Glial cells are active participants in the function, formation, and maintenance of the chemical synapse. To investigate the molecular basis of neuron-glia interactions at the peripheral synapse, we examined whether and how Schwann cell-derived factors modulate synaptic function at developing neuromuscular junctions (NMJs). Schwann cell-conditioned medium (SC-CM) from Xenopus Schwann cell cultures was collected and applied to Xenopus nerve-muscle cocultures. We found that SC-CM increased the frequency of spontaneous synaptic currents (SSCs) within 3-15 min by an average of approximately 150-fold at developing neuromuscular synapses. The increase in SSC frequency by SC-CM is a presynaptic effect independent of neuronal excitability and requires the influx of Ca2+. In contrast to its potentiating effect on spontaneous transmitter release, SC-CM suppressed the evoked transmitter release. The SC-CM effect required the presence of motoneuron soma but not protein synthesis. Using molecular weight cutoff filters and dialysis membranes, we found that the molecular weight of functional factor(s) in SC-CM was within 500 and 5000 Da. The SC-CM effect was not attributable to currently known factors that modulate synaptic efficacy, including neurotrophins, glutamate, and ATP. SC-CM also enhanced spontaneous synaptic release at developing NMJs in Xenopus tadpoles in situ. Our results suggest that Schwann cells release small molecules that enhance spontaneous synaptic activities acutely and potently at developing neuromuscular synapses, and the glial cell-enhanced spontaneous neurotransmission may contribute to synaptogenesis.

Spontaneous Neurotransmitter Release Depends on Intracellular Rather than ER Calcium Stores in Cultured Xenopus NMJ

Calcium ions are important in many vital neuron processes, including spontaneous neurotransmitter release. Extracellular calcium has long been known to be related to spontaneous neurotransmitter release, but the detailed mechanism for the effect of intracellular Ca 2+ on synaptic release has not yet been understood. In this research, 1,2-bis-( o-aminophenoxy)-ethane-N, N, N’, N’-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM) was used to combine with cytosolic free Ca 2+ in a calcium free medium of cultured Xenopus neuromuscular junctions (NMJ). The spontaneous synaptic current (SSC) frequency was obviously reduced. Then, drugs were applied to interrupt and activate the Ca 2+ release channels in the endoplasmic reticulum (ER) membrane, but the SSC frequency was not affected. The results show that spontaneous neurotransmitter release depends on intracellular rather than ER calcium in cultured Xenopus NMJ without extracellular calcium.

Mechanism of β-bungarotoxin in facilitating spontaneous transmitter release at neuromuscular synapse

The mechanism of the action of β-bungarotoxin (β-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to β-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca 2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by β-BuTx. The β-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca 2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca 2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP 3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by β-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the β-BuTx-induced facilitation of synaptic transmission. The β-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDP βS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that β-BuTx elicits Ca 2+ release from the IP 3 sensitive intracellular Ca 2+ stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

Mechanism of β‐bungarotoxin in the facilitation of spontaneous transmitter release at developing neuromuscular synapse

The mechanism of action of β-bungarotoxin (β-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to β-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by β-BuTx. The β-BuTx-enhanced SSC frequency was abolished when pharmacological Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by β-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished β-BuTx-induced facilitation of synaptic transmission. The β-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPβS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that β-BuTx elicits Ca2+ release from IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3Kγ/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

A rapid, nongenomic pathway facilitates the synaptic transmission induced by retinoic acid at the developing synapse

We have previously shown that retinoic acid (RA), a factor highly expressed in spinal cord, rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture, using whole-cell patch-clamp recording. We have now further investigated the underlying mechanisms that are involved in RA-induced facilitation on the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by RA. The prompt RA-enhanced SSC frequency was not abolished when Ca2+ was eliminated from the culture medium or there was bath application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influx through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or ryanodine receptors effectively blocked the increase of SSC frequency elicited by RA. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) and with the phospholipase Cgamma (PLCgamma) inhibitor U73122, abolished RA-induced facilitation of synaptic transmission. Preincubation of the cultures with pharmacological inhibitors, either genistein, a broad-spectrum tyrosine kinase inhibitor, or PP2, which predominantly inhibits the Src family of nonreceptor tyrosine kinase, completely abolished RA-induced synaptic facilitation. Taken collectively, these results suggest that RA elicits Ca2+ release from Ins1,4,5P3 and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PLCgamma/PI 3-kinase signaling cascades and Src tyrosine kinase activation, leading to an enhancement of spontaneous transmitter release.

Non-genomic regulation of transmitter release by retinoic acid at developing motoneurons in Xenopus cell culture.

Although the long-term effects of all-trans retinoic acid (RA) on neuronal growth and differentiation have been intensively studied, nothing is known about its effect on synaptic transmission. Here we show that RA rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch-clamp recording. Acute addition of RA dose-dependently and reversibly enhances the frequency of spontaneous synaptic currents (SSCs). Application of the lipophilic RA analogue all-trans retinol or RA metabolites produced by light-induced decomposition failed to provoke similar changes in SSC frequency, indicating the specificity of RA-induced facilitation of spontaneous transmitter release. Protein synthesis inhibitors anisomycin or cycloheximide had no effect on RA-induced SSC frequency facilitation. Treating cells with pan RA receptor (RAR) selective agonist or RARbeta-selective agonist, but not RARalpha-, RARgamma- or retinoid X receptor (RXR)-selective agonists, mimicked the action of RA. These results suggest that RA acts through the activation of RARbeta, to induce a rapid, non-genomic increase in the frequency of spontaneous transmitter release at developing neuromuscular synapses.

Both A chain and B chain of β-bungarotoxin are functionally involved in the facilitation of spontaneous transmitter release in Xenopus nerve–muscle cultures

In the present study, Xenopus nerve–muscle cultures were used to explore the functional roles of A chain (a phospholipase A 2 subunit) and B chain (a non-phospholipase A 2 subunit) of Bungarus multicinctus β-bungarotoxin. It was found that β-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve–muscle cultures. Modification of β-bungarotoxin with pyridoxal-5′-phosphate or substitution of Ca 2+ with Ba 2+ in buffer abolished the phospholipase A 2 activity of β-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A 2 activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of β-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve–muscle cultures.

Potentiation of quantal secretion by insulin-like growth factor-1 at developing motoneurons in Xenopus cell culture.

Although evidence suggests that insulin-like growth factor (IGF) plays an important role in the development and growth of the nervous system, the effect of IGF-1 in the regulation of neurotransmitter release in the peripheral nervous system remains unknown. Here we show that acute application of IGF-1, a factor widely expressed in developing myocytes, dose-dependently enhances the spontaneous acetylcholine (ACh) secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording. We studied the role of endogenously released IGF-1 by examining the effect of IGF-1 antibody on the frequency of spontaneous synaptic currents (SSCs) at high-activity synapses, and found SSC frequency was markedly reduced at these high-activity synapses. The IGF-1-induced synaptic potentiation was not abolished when Ca2+ was eliminated from the culture medium or there was bath-application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influxes through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) or ryanodine receptors effectively occluded the increase of SSC frequency elicited by IGF-I. Treating cells with the phosphoinositide-3 kinase (PI3-K) inhibitors wortmannin or LY294002, and with phospholipase Cgamma (PLCgamma) inhibitor U73122, but not the inhibitor of mitogen-activated protein (MAP) kinase PD98059, abolished IGF-1-induced synaptic potentiation. Taken collectively, these results suggest that endogenously released IGF-1 from myocytes elicits Ca2+ release from IP3- and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3-K and PLCgamma signalling cascades, leading to an enhancement of spontaneous transmitter release.

Modulation of protein kinase A activation by fibronectin matrix proteins at developing neuromuscular synapses in Xenopus laevis cell cultures.

Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.

Regulation of acetylcholine release by extracellular matrix proteins at developing motoneurons in Xenopus cell cultures.

Integrins mediate cell-extracellular matrix connections and are particularly important during neuronal development. We here investigated the regulatory role of extracellular matrix (ECM) proteins on the synaptic transmission at developing motoneurons. Synaptic currents were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. Soluble fibronectin and laminin had no significant effect on the frequency of spontaneous synaptic currents (SSCs) by themselves and markedly increased SSC frequency in the presence of low concentration of protein kinase C (PKC) activators. Pretreatment with Gly-Arg-Gly-Asp-Ser peptide inhibited the SSC increasing action of 12-o-tetradecanoyl-phorbol-13-acetate (TPA, 0.5 microM) plus fibronectin, but not that of TPA plus laminin. Genistein but not cytochalasin D inhibited the SSC increasing action of TPA plus fibronectin or laminin. High concentration of TPA (5 microM) markedly increased the SSC frequency by itself and occluded the SSC increasing action of fibronectin. Very low concentration of TPA (0.05 microM) markedly enhanced the SSC frequency when the cells were plated onto fibronectin- or laminin-coated substratum for 1 day. The SSC frequency increased markedly right after a train stimulation, which was defined as post-train potentiation (PTrP), when the cultures were plated onto fibronectin substratum and chronically treated with brain-derived neurotrophic factor (BDNF). The PTrP phenomenon is not observed upon chronic treatment with neurotrophin-3, glial cell line-derived neurotrophic factor, or ciliary neurotrophic factor. Our results suggest that the activation of PKC and tyrosine kinase but not actin reorganization plays a role in the SSC potentiating action of fibronectin. BDNF exerts synergistic effects in increasing synaptic transmission in neurons grown on fibronectin substratum. ECMs in concert with neurotrophic factor may play a role in regulating synaptic function at developing motoneurons.

A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1.

ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.

Nerve Terminal Currents Induced by Autoreception of Acetylcholine Release

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.

Regulation of Presynaptic NMDA Responses by External and Intracellular pH Changes at Developing Neuromuscular Synapses

NMDA receptors play important roles in synaptic plasticity and neuronal development. The functions of NMDA receptors are modulated by many endogenous substances, such as external pH (pHe), as well as second messenger systems. In the present study, the nerve-muscle cocultures of Xenopus embryos were used to investigate the effects of both external and intracellular pH (pHi) changes on the functional responses of presynaptic NMDA receptors. Spontaneous synaptic currents (SSCs) were recorded from innervated myocyte using whole-cell recordings. Local perfusion of NMDA at synaptic regions increased the SSC frequency via the activation of presynaptic NMDA receptors. A decrease in pHe from 7.6 to 6.6 reduced NMDA responses to 23% of the control, and an increase in pHe from 7.6 to 8.6 potentiated the NMDA responses in increasing SSC frequency. The effect of NMDA on intracellular Ca2+ concentration ([Ca2+]i) was also affected by pHe changes: external acidification inhibited and alkalinization potentiated [Ca2+]i increases induced by NMDA. Intracellular pH changes of single soma were measured by ratio fluorometric method using 2,7-bis (carboxyethyl)-5, 6-carboxyfluorescein (BCECF). Cytosolic acidification was used in which NaCl in Ringer's solution was replaced with weak organic acids. Acetate and propionate but not methylsulfate substitution caused intracellular acidification and potentiated NMDA responses in increasing SSC frequency, intracellular free Ca2+ concentration, and NMDA-induced currents. On the other hand, cytosolic alkalinization with NH4Cl did not significantly affect these NMDA responses. These results suggest that the functions of NMDA receptors are modulated by both pHe and pHi changes, which may occur in some physiological or pathological conditions.

Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures.

1. The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve-muscle co-cultures. 2. Spontaneous and evoked ACh release of motoneurons was monitored by using whole-cell voltage-clamped myocytes. Intracellular alkalinization with 15 mM NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. 3. Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. 4. Impulse-evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. 5. Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. 6. We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.