Frequency Of Spontaneous Synaptic Currents Research Articles

GABAA-mediated local synaptic pathways connect neurons in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) in mammals functions as the biological clock controlling circadian rhythms, but the synaptic circuitry of the SCN is largely unexplored. Most SCN neurons use the neurotransmitter gamma-aminobutyric acid (GABA), and anatomic studies indicate many GABAergic synapses and local axon collaterals; however, physiological evidence for synaptic communication among SCN neurons is indirect. We have used three approaches to investigate local circuitry in the SCN in acute hypothalamic slices from rat. First, tetrodotoxin was used to block action-potential-dependent synaptic release, which resulted in a decrease in the frequency of spontaneous synaptic currents in SCN neurons, suggesting that spontaneously active neurons in the slice connect synaptically to SCN neurons. Postsynaptic currents in SCN neurons were also evoked by the selective stimulation of other SCN neurons with glutamate, which avoids direct activation of axons that might originate outside the SCN. Two different methods of glutamate microapplication (i.e., pressure ejection and ultraviolet photolysis of caged glutamate) indicated that SCN neurons receive GABAA-receptor-mediated synaptic input from other SCN neurons. In contrast, glutamate-receptor-mediated synaptic connections between SCN neurons were not detected. The GABAergic synapses that comprise the network described here could conceivably be a substrate for the synchronization and amplification of the circadian rhythm of SCN firing. Alternatively, this circuitry might mediate other aspects of clock function such as the integration of environmental and physiological information.

Activity-Dependent Expression of NT-3 in Muscle Cells in Culture: Implications in the Development of Neuromuscular Junctions

Although activity-dependent expression of neurotrophins has been studied extensively in the CNS, its physiological role during synapse development is not well established. At the developing neuromuscular junction in culture, exogenous application of the neurotrophin BDNF or NT-3 has been shown to acutely potentiate synaptic transmission and chronically promote synapse maturation. Using the same cell culture model, we have investigated activity-dependent neurotrophin expression in muscle cells and its role in developing neuromuscular synapses. Membrane depolarization, elicited by either depolarizing agents or repetitive electric stimulation, rapidly and specifically increased the levels of NT-3 mRNA in developing Xenopus laevis muscle cells in culture. NT-3 gene expression also was enhanced by acetylcholine (ACh), the neurotransmitter that causes muscle membrane depolarization. The effects of depolarization were mediated by increasing intracellular calcium concentration. Moreover, factor(s) induced by membrane depolarization appeared to enhance synaptic transmission at the developing neuromuscular junction. The frequency of spontaneous synaptic currents (SSCs) recorded from neuromuscular synapses was increased significantly after treatment with conditioned medium from depolarized muscle cultures. The amplitude, rise time, and decay time of SSCs were not affected, indicating a presynaptic action of the conditioned medium. The effects of the conditioned medium were blocked, partially, by the NT-3 scavenger TrkC-IgG, suggesting that the potentiation of synaptic efficacy was attributable, at least in part, to elevated NT-3 as a consequence of muscle depolarization. Thus, activity-dependent expression of muscle NT-3 may contribute to the development of the neuromuscular synapse.

Role of chloride-homeostasis in the inhibitory control of neuronal network oscillators.

1. Spontaneous synaptic activity in networks formed by dissociated neurons from embryonic rat midbrain was analyzed in tight seal whole cell recordings. 2. Application of furosemide (0.5 mM) to the cell and its surrounding area increased the frequency of spontaneous synaptic currents. Incubation of the culture with furosemide resulted in "rhythmic" burst activity. 3. Furosemide (0.1-0.5 mM) changed equilibrium potentials of inhibitory postsynaptic currents, gamma-aminobutyric acid-A (GABAA) or glycine receptor-mediated Cl- currents by a blockade of Cl(-)-outward transport. Furosemide did not alter the slope conductance of GABAA receptor-mediated currents. Membrane conductance and cell excitability were also unaffected. 4. We conclude that furosemide locked the activity of the network in "burst activity" mode through impairment of inhibition resulting from the disturbance of Cl- homeostasis.

Synaptic Modulation by Neurotrophic Factors: Differential and Synergistic Effects of Brain-Derived Neurotrophic Factor and Ciliary Neurotrophic Factor

Extracellular application of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) to developing neuromuscular junctions in Xenopus nerve-muscle cultures resulted in an increase in the frequency of spontaneous synaptic currents (SSCs) and in the amplitude of nerve-evoked synaptic currents. Analyses of the amplitude and time course of the SSCs suggest that these effects are attributable to elevation of presynaptic transmitter release. The actions of these two factors on the transmitter secretion process, however, are distinctly different. Fura-2 Ca2+ imaging showed that an increase in presynaptic cytosolic Ca2+ ([Ca2+]i) accompanied the synaptic potentiation by BDNF, whereas no change in [Ca2+]i was observed during synaptic potentiation by CNTF. Removing external Ca2+ also abolished the potentiating effect of BDNF but did not influence the CNTF effect. Moreover, the two factors exerted different effects on the short-term synaptic plasticity. Paired-pulse facilitation normally found at these synapses was reduced by BDNF but unaffected by CNTF; CNTF, but not BDNF, reduced the extent of synaptic depression during high-frequency tetanic stimulation. Finally, the potentiation effect of BDNF and CNTF on spontaneous transmitter release was additive when both factors were applied together to the synapse at saturating concentrations (100 ng/ml) and was highly synergistic when low doses (1 and 10 ng/ml) of both factors were used. These results suggest that because of their differential effects on the secretory machinery, BDNF and CNTF may act cooperatively in modulating the development and functioning of synapses.

Potentiation of neurotransmitter release by activation of presynaptic glutamate receptors at developing neuromuscular synapses of Xenopus.

1. Glutamate receptors play important roles in synaptic plasticity and neural development. Here we report that, at the developing neuromuscular synapses in Xenopus cultures, the activation of presynaptic glutamate receptors at motor nerve terminals potentiates spontaneous acetylcholine (ACh) release. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes using whole-cell recording. Bath application of glutamate (10 microM) markedly increased the frequency of SSCs, and the action of glutamate was reversible. 3. Pretreatment with 0.3 microM tetrodotoxin, which blocks Na+ channels and the conduction of action potentials, only slightly inhibited the potentiating action of glutamate on SSCs. Furthermore, the enhancement of ACh secretion was much more prominent when glutamate was applied locally to the synaptic region. 4. Three types of glutamate receptor agonists, kainate, quisqualate, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and NMDA (N-methyl-D-aspartate), were effective in inducing the potentiating effect. The ranking order was: glutamate > kainate > NMDA > AMPA > quisqualate. Glycine potentiated the effects induced by NMDA. Metabotropic receptors were not involved in the potentiating action of glutamate. 5. The potentiating effect of glutamate depended on the influx of Ca2+ through both L-type Ca2+ channels and NMDA-gated channels. 6. Since glutamate is known to be co-released with ACh at some cholinergic nerve terminals, the released glutamate may serve as a positive feedback regulation of ACh secretion at developing neuromuscular junctions via its action on presynaptic glutamate receptors.

Regulation of postsynaptic responses by calcitonin gene related peptide and ATP at developing neuromuscular junctions.

Neuronal factors co-released with neurotransmitters may play an important role in synapse development and function. Calcitonin gene related peptide (CGRP) and adenosine 5'-triphosphate (ATP), two principal neuromodulators present in the motor nerve terminals, were studied for their roles and mechanisms during early development of neuromuscular synapses in Xenopus nerve--muscle co-cultures. CGRP treatment increased the decay time and amplitude of spontaneous synaptic currents (SSCs) recorded from innervated myocytes, without affecting SSC frequency, suggesting a postsynaptic mechanism. ATP also increased the SSC amplitude and decay time. In addition, ATP was shown to potentiate the responses of isolated myocytes to iontophoretically applied acetylcholine (ACh). Single-channel recording from isolate myocytes showed that both CGRP and ATP specifically increased the open time of embryonic-type, low-conductance ACh channels. Pharmacological experiments suggest that the CGRP actions were mediated by cAMP-dependent protein kinase (PKA), while ATP exerted its effects by binding to P2 purinoceptors and thereby activating protein kinase C (PKC). Moreover, the effects of CGRP and ATP on ACh channel activity were restricted to immature myocytes. Taken together, these results suggest that endogenous CGRP and ATP co-released with ACh from the nerve terminal may promote synaptic development by potentiating postsynaptic ACh channel activity during the early phase of synaptogenesis.

Depression of developing neuromuscular synapses induced by repetitive postsynaptic depolarizations

Effect of postsynaptic activity on the synaptic efficacy was studied in Xenopus nerve-muscle cultures. Repetitive postsynaptic depolarizations induced by injection of current pulses into singly innervated myocytes resulted in significant reduction in the frequency of spontaneous synaptic currents and the amplitude of nerve-evoked synaptic currents at the majority of synapses that showed immature synaptic properties. Repetitive hyperpolarizations and steady depolarizations of similar duration were without effect. The depolarization-induced synaptic depression appeared to result predominantly from a reduced ACh secretion from the presynaptic nerve terminal. Buffering the myocyte cytosolic Ca2+ at a low level with intracellular loading of a Ca2+ buffer, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), significantly reduced the effect of the depolarizations. Thus postsynaptic electrical activity can regulate the synaptic efficacy of the developing neuromuscular synapases and the regulation may be mediated by retrograde transsynaptic interactions.

Potentiation by ATP of the postsynaptic acetylcholine response at developing neuromuscular synapses in Xenopus cell cultures.

1. Extracellular application of ATP to developing Xenopus neuromuscular synapses in culture resulted in a marked increase in the amplitude and frequency of spontaneous synaptic currents, using whole-cell recording. 2. The postsynaptic action of ATP was examined by studying the response of isolated muscle cells to iontophoretically applied acetylcholine (ACh). ATP enhanced the responses of the muscle membrane to ACh. The order of potency for various nucleotides (ATP = ADP >> AMP, adenosine, GTP) suggests that ATP acts through P2-purinoceptors. The effect of ATP on whole-cell currents was also abolished by the protein kinase inhibitor H-7. 3. Single-channel measurements indicate that ATP increased the mean open time of low-conductance ACh channels. No change in the conductance of ACh channels was observed. 4. Local application of ATP to one region of the elongated myocyte surface resulted in potentiated ACh responses only at the ATP-treated region, suggesting that the cytosolic second messengers were effectively confined within the muscle cytoplasm. 5. The results of the present study suggest that ATP released from the nerve terminals may potentiate the ACh response of developing muscle cells during the early phase of synaptogenesis, and that the action of ATP can be restricted to the subsynaptic region exposed to the secreted ATP.

Potentiation by endogenously released ATP of spontaneous transmitter secretion at developing neuromuscular synapses in Xenopus cell cultures.

1. Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh. In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP. 2. Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP. 3. We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses. In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin. 4. Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect. The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers. On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses. 5. These results suggest that endogenous release of ATP at developing neuromuscular synapses is responsible for the maintenance of high levels of spontaneous ACh release, which is known to play a crucial role in regulating postsynaptic differentiation.

L-type Ca 2+ channel is involved in the regulation of spontaneous transmitter release at developing neuromuscular synapses

Involvement of an L-type Ca 2+ channel in the regulation of spontaneous transmitter release was studied in Xenopus nerve-muscle cultures. The frequency of spontaneous synaptic currents, which reflects impulse-independent acetylcholine release from the nerve terminals, showed a marked increase in high-K + medium or after treatment with a phorbol ester, 12- O-tetradecanoyl-phorbol 13-acetate, a drug that activates protein kinase C and depolarizes the presynaptic neuron. The potentiation effect of high K + and 12- O-tetradecanoyl-phorbol 13-acetate requires Ca 2+ influx through the L-type Ca 2+ channel in the plasma membrane, since it was significantly reduced by the presence of nifedipine, verapamil or diltiazem and enhanced by Bay K 8644, an L-type Ca 2+ channel agonist. It was shown recently that adenosine 5'-triphosphate markedly potentiates the spontaneous acetylcholine release at these synapses through the binding of P 2-purinoceptors and the activation of protein kinase C. We found in the present study that potentiation effects of adenosine 5'-triphosphate are inhibited by L-type Ca 2+ channel blockers, suggesting that the L-type Ca 2+ channel is responsible for the positive regulation of spontaneous acetylcholine secretion by adenosine 5'-triphosphate at the developing neuromuscular synapses. Our data suggest that modulation of the L-type Ca 2+ channel in embryonic motor nerve terminals is important for the regulation of spontaneous transmitter release.

Retrograde modulation at developing neuromuscular synapses: Involvement of G protein and arachidonic acid cascade

Intracellular loading of nonhydrolyzable GTP analogs into innervated muscle cells in Xenopus cultures led to a marked increase in the frequency of spontaneous synaptic currents (SSCs), while extracellular application of the drugs at the same concentration was without effect. The increase in SSC frequency appeared to be unrelated to changes in the muscle membrane sensitivity toward acetylcholine (ACh), but resulted from an elevated spontaneous ACh secretion from the presynaptic nerve terminal. Postsynaptic loading of arachidonic acid (AA) produced a similar effect as the GTP analogs, and the potentiation effect of both GTP analogs and AA was reversed by an inhibitor of AA metabolism, AA861. Further studies indicate that a lipoxygenase metabolite, 5-HPETE, appears to be a likely candidate for the retrograde factor involved in modulating ACh secretion. These results suggest that G protein activation of the AA cascade in the postsynaptic cell could produce a retrograde signal to modulate transmitter secretion from the presynaptic nerve terminal at developing synapses.

ATP potentiates spontaneous transmitter release at developing neuromuscular synapses

Extracellular application of ATP, a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiates the spontaneous secretion of acetylcholine at developing neuromuscular synapses in Xenopus cell culture, as shown by a marked increase in the frequency of spontaneous synaptic currents recorded in the postsynaptic muscle cell. The effect of ATP is apparently mediated by the activation of cytosolic protein kinases and requires the influx of Ca 2+ through the plasma membrane. Since spontaneous acetylcholine release is known to regulate the development of contractile properties of the postsynaptic muscle cell, extracellular ATP may serve as a positive trophic factor at developing neuromuscular synapses.