Hippocampal CA1 lacunosum-moleculare interneurons: modulation of monosynaptic GABAergic IPSCs by presynaptic GABAB receptors

1. Whole cell patch-clamp recordings were employed to characterize monosynaptic inhibitory postsynaptic currents (IPSCs) in morphologically and electrophysiologically identified interneurons located in the stratum lacunosum moleculare, or near the border of the stratum radiatum (LM interneurons), in the CA1 region of hippocampal slices taken from 3- to 4-wk-old rats. Monosynaptic IPSCs, evoked in the presence of glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM) and D-2-amino-5-phosphopentanoate (APV; 50 microM) were biphasic. The gamma-aminobutyric acid-A (GABAA) receptor antagonist, bicuculline (20 microM), blocked the fast IPSC, and the slow IPSC was blocked by the GABAB receptor antagonist CGP35348 (500 microM). 2. Monosynaptic IPSCs were evoked by electrical stimulation in several distant regions including the stratum radiatum, the stratum oriens, the stratum lacunosum-moleculare, and the molecular layer of dentate gyrus, suggesting an extensive network of inhibitory interneurons in the hippocampus. In paired recordings of CA1 interneurons and pyramidal cells, IPSCs were evoked by electrical stimulation of most of these distal regions with the exception of the molecular layer of dentate gyrus, which evoked an IPSC only in LM interneurons. 3. Frequent (> 0.1 Hz) stimulation depressed the evoked IPSCs. With a paired-pulse protocol, the second IPSC was depressed and the maximal depression (40-50%) was observed with an interstimulus interval of 100-200 ms. 4. The GABAB receptor agonist baclofen (1 microM) reduced the amplitude of evoked IPSCs and the paired-pulse depression of the second IPSC. The GABAB receptor antagonist CGP35348 (0.5-1 mM) had no significant effect on the amplitude of isolated IPSCs. However, CGP35348 reduced but did not fully block paired-pulse depression, suggesting that this depression is partly due to the activation of presynaptic GABAB receptors. 5. The paired-pulse depression depended on the level of transmitter release. Potentiation of synaptic release of GABA, by increasing the extracellular Ca2+ concentration to 4 mM and reducing the extracellular Mg2+ concentration to 0.1 mM, enhanced the depression. Reduction of transmitter release by increasing extracellular Mg2+ concentration to 7 mM diminished the paired-pulse depression of IPSCs. After potentiation of transmitter release, CGP35348 was less efficient in reducing the paired-pulse depression, suggesting that enhancement of depression by high-calcium/low-magnesium medium was preferentially due to the potentiation of a GABAB-independent component. 6. In summary, monosynaptic IPSCs recorded in LM interneurons show similar features to those recorded in pyramidal cells. The strong correlation between the level of transmitter release and the degree of paired-pulse depression may have important physiological consequences, because in synapses with a high level of activity and a high level of GABA release, inhibition is powerful, but depression can develop more readily.     

Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors

The effects of external pH (pHout) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pHout 6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 microM) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Na(i)] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Ca(i)] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pHout 6. On the contrary, the external alkalinization (pHout 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pHout was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pHout changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pHout variations on [3H]GABA release were independent on the presence of HCO3-. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels.     

Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis

In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.     

Modulation of pre- and postsynaptic calcium dynamics by ionotropic glutamate receptors at a plastic synapse

This study was conducted to assess the role of ionotropic glutamate receptors in the modulation of calcium dynamics on both sides of a vertebrate plastic synapse. Retrograde labeling of neuronal elements with high-affinity calcium-sensitive dyes was used in conjunction with confocal imaging techniques in an in vitro lamprey brain stem preparation. A prolonged calcium transient was measured both pre- and postsynaptically in response to a period of high-frequency ("tetanic") stimulation to the vestibulospinal-reticulospinal synapse. The ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and D,L-2-amino-5-phosphonopentanoate (D,L-AP5; 100 microM) reduced the calcium signal in both compartments of the synapse. The presynaptic D,L-AP5-sensitive component was enhanced markedly by the removal of Mg2+ from the superfusate. Increasing the extracellular stimulus intensity progressively augmented the presynaptic calcium signal, suggesting the recruitment of excitatory axo-axonic inputs onto these fibers. Further, the presence of an excitatory amino acid-mediated presynaptic potential underlying a component of the Ca2+ signal was demonstrated by electrophysiological recordings from vestibulospinal axons. Bath application of agonist, in the presence of tetrodotoxin (1 microM), confirmed the existence of N-methyl-D-aspartate receptors at the presynaptic element capable of modulating calcium levels. The postsynaptic Ca2+ response, which is known to be necessary for long-term potentiation (LTP) induction at this synapse, was localized to areas of the dendritic tree that correlated with the location of known synaptic inputs; thus the synaptically activated rise in postsynaptic calcium may confer the synapse specificity of LTP induction previously demonstrated. In summary, we have demonstrated the existence of physiologically activated presynaptic ionotropic glutamate receptors that are capable of modulating levels of intracellular calcium and have highlighted the importance of receptor-mediated increases in postsynaptic calcium for neuronal plasticity in the lamprey.     

An essential role for a small synaptic vesicle-associated phosphatidylinositol 4-kinase in neurotransmitter release

Glutamate release from nerve terminals is the consequence of Ca2+-triggered fusion of small synaptic vesicles with the presynaptic plasma membrane. ATP dependence of neurotransmitter release has been suggested to be founded, in part, on phosphorylation steps preceding membrane fusion. Here we present evidence for an essential role of phosphatidylinositol phosphorylation in stimulated release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes). Specifically, we show that a phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity resides on nerve terminal-derived small synaptic vesicles (SSVs) and that inhibition of the PtdIns 4-kinase activity in intact synaptosomes leads to attenuation of the evoked release of glutamate. The attenuation of transmitter release is reversible and correlates with respective changes in intrasynaptosomal PtdIns 4-kinase activity. Because only the Ca2+-dependent release of glutamate is affected, regulation appears to be at the level of exocytosis. Taken together, our data imply a mandatory role for PtdIns 4-kinase and phosphoinositide products in the regulated exocytosis of SSV in mammalian nerve terminals.     

Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process

Doc2alpha and Munc13-1 proteins are highly concentrated on synaptic vesicles and the presynaptic plasma membrane, respectively, and have been implicated in Ca2+-dependent neurotransmitter release. Doc2alpha interacts with Munc13-1 through the N-terminal region of Doc2alpha (the Mid domain; amino acid residues 13-37). Here we examine whether the interaction between Doc2alpha and Munc13-1 is required for Ca2+-dependent neurotransmitter release from intact neuron. A synthetic Mid peptide (the Mid peptide), but not a control mutated Mid peptide or a scrambled Mid peptide, inhibited the interaction between Doc2alpha and Munc13-1 in vitro. Introduction of the Mid peptide into presynaptic neurons of cholinergic synapses, formed between rat superior cervical ganglion neurons, reversibly inhibited synaptic transmission evoked by action potentials. In contrast, the control peptides did not inhibit synaptic transmission. This inhibitory effect depended on the presynaptic activity and was affected by extracellular Ca2+ concentrations. The onset of the Mid peptide effect was shortened when the neuron was stimulated at a higher frequency, and the inhibition was more potent at 1 mM Ca2+ than at 5.1 mM Ca2+. These results suggest that the Doc2alpha-Munc13-1 interaction plays a role in a step before the final fusion step of synaptic vesicles with the presynaptic plasma membrane in the evoked neurotransmitter release process.     

Modulation of transmitter release by action potential duration at the hippocampal CA3-CA1 synapse

Presynaptic Ca2+ influx through voltage-dependent Ca2+ channels triggers neurotransmitter release. Action potential duration plays a determinant role in the dynamics of presynaptic Ca2+ influx. In this study, the presynaptic Ca2+ influx was optically measured with a low-affinity Ca2+ indicator (Furaptra). The effect of action potential duration on Ca2+ influx and transmitter release was investigated. The K+ channel blocker 4-aminopyridine (4-AP) was applied to broaden the action potential and thereby increase presynaptic Ca2+ influx. This increase of Ca2+ influx appeared to be much less effective in enhancing transmitter release than raising the extracellular Ca2+ concentration. 4-AP did not change the Ca2+ dependence of transmitter release but instead shifted the synaptic transmission curve toward larger total Ca2+ influx. These results suggest that changing the duration of Ca2+ influx is not equivalent to changing its amplitude in locally building up an effective Ca2+ concentration near the Ca2+ sensor of the release machinery. Furthermore, in the presence of 4-AP, the N-type Ca2+ channel blocker omegaCgTx GVIA was much less effective in blocking transmitter release. This phenomenon was not simply due to a saturation of the release machinery by the increased overall Ca2+ influx because a similar reduction of Ca2+ influx by application of the nonspecific Ca2+ channel blocker Cd2+ resulted in much more inhibition of transmitter release. Rather, the different potencies of omega-CgTx GVIA and Cd2+ in inhibiting transmitter release suggest that the Ca2+ sensor is possibly located at a distance from a cluster of Ca2+ channels such that it is sensitive to the location of Ca2+ channels within the cluster.     

Ontogenesis of presynaptic GABAB receptor-mediated inhibition in the CA3 region of the rat hippocampus

gamma-Aminobutyric acid-B(GABAB) receptor-dependent and -independent components of paired-pulse depression (PPD) were investigated in the rat CA3 hippocampal region. Intracellular and whole cell recordings of CA3 pyramidal neurons were performed on hippocampal slices obtained from neonatal (5-7 day old) and adult (27-34 day old) rats. Electrical stimulation in the hilus evoked monosynaptic GABAA postsynaptic currents (eIPSCs) isolated in the presence of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)2-amino-5-phosphovaleric acid (-AP5, 50 microM) with 2(triethylamino)-N-(2,6-dimethylphenyl) acetamine (QX314) filled electrodes. In adult CA3 pyramidal neurons, when a pair of identical stimuli was applied at interstimulus intervals (ISIs) ranging from 50 to 1,500 ms the amplitude of the second eIPSC was depressed when compared with the first eIPSC. This paired-pulse depression (PPD) was partially blocked by P-3-aminoprophyl -P-diethoxymethylphosphoric acid (CGP35348, 0.5 mM), a selective GABAB receptor antagonist. In neonates, PPD was restricted to ISIs shorter than 200 ms and was not affected by CGP35348. The GABAB receptor agonist baclofen reduced the amplitude of eIPSCs in a dose-dependent manner with the same efficiency in both adults and neonates. Increasing the probability of transmitter release with high Ca2+ (4 mM)/low Mg2+ (0.3 mM) external solution revealed PPD in neonatal CA3 pyramidal neurons that was 1) partially prevented by CGP35348, 2) independent of the membrane holding potential of the recorded cell, and 3) not resulting from a change in the reversal potential of GABAA eIPSCs. In adults the GABA uptake blocker tiagabine (20 microM) increased the duration of eIPSCs and the magnitude of GABAB receptor-dependent PPD. In neonates, tiagabine also increased duration of eIPSCs but to a lesser extent than in adult and did not reveal a GABAB receptor-dependent PPD. These results demonstrate that although GABAB receptor-dependent and -independent mechanisms of presynaptic inhibition are present onGABAergic terminals and functional, they do not operate at the level of monosynaptic GABAergic synaptic transmission at early stages of development. Absence of presynaptic autoinhibition of GABA release seems to be due to the small amount of transmitter that can access presynaptic regulatory sites.     

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.     

Postsynaptic Ca2+ influx mediated by three different pathways during synaptic transmission at a calyx-type synapse

Whole-cell recordings and Ca2+ flux measurements were made at a giant calyx-type synapse in rat brainstem slices to determine the contribution of glutamate receptor (GluR) channels and voltage-dependent Ca2+ channels (VDCCs) to postsynaptic Ca2+ influx during synaptic transmission. A single presynaptic action potential (AP) evoked an EPSP, followed by a single AP. The EPSP-AP sequence caused a postsynaptic Ca2+ influx of approximately 3.0 pC, primarily through VDCCs ( approximately 70%) and NMDA-type (up to 30%) channels but also through AMPA-type (<5%) GluR channels. At -80 mV, the fractional Ca2+ current (Pf) mediated by AMPA receptor (AMPAR) and NMDA receptor (NMDAR) channels was 1.3 and 11-12%, respectively. Simulations of the time course of Ca2+ influx through GluR channels suggested that the small contribution of AMPAR channels occurred only during the first few milliseconds of an EPSP, whereas influx through NMDAR channels dominated later. The NMDAR-mediated Ca2+ influx was localized in regions covered by the presynaptic terminal, whereas the Ca2+ influx mediated by VDCCs was more homogeneously distributed. Because of the temporal and spatial differences, calcium ions entering through the three different pathways are likely to activate different intracellular targets in the postsynaptic cell.     

A postsynaptic interaction between dopamine D1 and NMDA receptors promotes presynaptic inhibition in the rat nucleus accumbens via adenosine release

The mechanism underlying dopamine D1 receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonist D/L-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1 receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.     

Membrane currents evoked by ionotropic glutamate receptor agonists in rod bipolar cells in the rat retinal slice preparation

1. With the use of the whole cell voltage-clamp technique, I have recorded the current responses to ionotropic glutamate receptor agonists of rod bipolar cells in vertical slices of rat retina. Rod bipolar cells constitute a single population of cells and were visualized by infrared differential interference contrast video microscopy. They were targeted by the position of their cell bodies in the inner nuclear layer and, after recording, were visualized in their entirety by labeling with the fluorescent dye Lucifer yellow, which was included in the recording pipette. To study current-voltage relationships of evoked currents, voltage-gated potassium currents were blocked by including Cs+ and tetraethylammonium+ in the recording pipette. 2. Pressure application of the non-N-methyl-D-aspartate (non-NMDA) receptor agonists kainate and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) from puffer pipettes evoked a long-latency conductance increase selective for chloride ions. When the intracellular chloride concentration was increased, the reversal potential changed, corresponding to the change in equilibrium potential for chloride. The response was evoked in the presence of 5 mM Co2+ and nominally O mM Ca2+ in the extracellular solution, presumably blocking all external Ca2(+)-dependent release of neurotransmitter. 3. The long latency of kainate-evoked currents in bipolar cells contrasted with the short-latency currents evoked by gamma-aminobutyric acid (GABA) and glycine in rod bipolar cells and by kainate in amacrine cells. 4. Application of NMDA evoked no response in rod bipolar cells. 5. Coapplication of AMPA with cyclothiazide, a blocker of agonist-evoked desensitization of AMPA receptors, enhanced the conductance increase compared with application of AMPA alone. Coapplication of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the response to kainate and AMPA, indicating that the response was mediated by conventional ionotropic glutamate receptors. 6. The conductance increase evoked by non-NMDA receptor agonists could not be blocked by a combination of 100 microM picrotoxin and 10 microM strychnine. Application of the GABAC receptor antagonist 3-aminopropyl (methyl)phosphinic acid (3-APMPA) strongly reduced the response, and coapplication of 500 microM 3-APMPA and 100 microM picrotoxin completely blocked the response. These results suggested that the conductance increase evoked by non-NMDA receptor agonists was mediated by release of GABA and activation of GABAC receptors, and most likely also GABAA receptors, on rod bipolar cells. 7. Kainate responses like those described above could not be evoked in bipolar cells in which the axon had been cut somewhere along its passage to the inner plexiform layer during the slicing procedure. This suggests that the response was dependent on the integrity of the axon terminal in the inner plexiform layer, known to receive GABAergic synaptic input from amacrine cells. 8. The results indicate that ionotropic glutamate receptors are not involved in mediating synaptic input from photoreceptors to rod bipolar cells and that an unconventional mechanism of GABA release from amacrine cells might operate in the inner plexiform layer.     

ATP stimulates sympathetic transmitter release via presynaptic P2X purinoceptors

ATP is a fast transmitter in sympathetic ganglia and at the sympathoeffector junction. In primary cultures of dissociated rat superior cervical ganglion neurons, ATP elicits noradrenaline release in an entirely Ca2+-dependent manner. Nevertheless, ATP-evoked noradrenaline release was only partially reduced (by approximately 50%) when either Na+ or Ca2+ channels were blocked, which indicates that ATP receptors themselves mediated transmembrane Ca2+ entry. An "axonal" preparation was obtained by removing ganglia from explant cultures, which left a network of neurites behind; immunostaining for axonal and dendritic markers revealed that all of these neurites were axons. In this preparation, ATP raised intraaxonal Ca2+ and triggered noradrenaline release, and these actions were not altered when Ca2+ channels were blocked by Cd2+. Hence, Ca2+-permeable ATP-gated ion channels, i.e., P2X purinoceptors, are located at presynaptic sites and directly mediate Ca2+-dependent transmitter release. These presynaptic P2X receptors displayed a rank order of agonist potency of ATP >/= 2-methylthio-ATP > ATPgammaS > alpha,beta-methylene-ATP approximately beta,gamma-methylene-L-ATP and were blocked by suramin or PPADS. ATP, 2-methylthio-ATP, and ATPgammaS also evoked inward currents measured at neuronal somata, but there these agonists were equipotent. Hence, presynaptic P2X receptors resemble the cloned P2X2 subtype, but they appear to differ from somatodendritic P2X receptors in terms of agonist sensitivity. Suramin reduced depolarization-evoked noradrenaline release by up to 20%, when autoinhibitory mechanisms were inactivated by pertussis toxin. These results indicate that presynaptic P2X purinoceptors mediate a positive, whereas G-protein-coupled P2Y purinoceptors mediate a negative, feedback modulation of sympathetic transmitter release.     

Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors

We have investigated the role of metabotropic glutamate receptors linked to phosphoinositide hydrolysis in the control of glutamate release in cerebrocortical nerve terminals. The activation of these receptors with the agonist 3,5-dihydroxyphenylglycine enhanced intra-synaptosomal diacylglycerol and facilitated both the depolarization-induced increase in the cytosolic free Ca2+ concentration and the release of glutamate. However, 5 min after receptor activation, a second stimulation of the pathway with the agonist failed to produce diacylglycerol and to facilitate glutamate release. Interestingly, during the period in which the diacylglycerol response was desensitized, a strong agonist-induced inhibition of Ca2+ entry and glutamate release was observed. This change in the presynaptic effects of 3,5-dihydroxyphenylglycine is reversible since 30 min after the first stimulation, the agonist-induced inhibition of release disappeared, whereas both the production of diacylglycerol and the facilitation of glutamate release were recovered. The tonic elevation of the extracellular glutamate concentration from basal levels (0.8 microM) up to 5 microM also produced the switch from facilitation to inhibition in the receptor response. The existence of this activity-dependent switch in the presynaptic control of glutamate release suggests that release facilitation is limited to conditions under which an appropriate clearance of synaptic glutamate exists, probably to prevent the neurotoxic accumulation of glutamate in the synapse.     

The effect of a reduction in temperature on the quantal release of transmitter at the mouse neuromuscular junction

1. The effects of a reduction in temperature were examined on evoked and spontaneous release of transmitter quanta and on presynaptic negative signals, blocked by Cd2+, measured externally at neuromuscular junctions in mouse diaphragm muscles in low-Ca2+, high-Mg2+ Krebs-Ringer solutions. 2. The evoked release was enhanced with lowering of the temperature, whereas the extent of spontaneous release was reduced. Cooperativity of Ca2+ in the evoked release was slightly reduced by lowering the temperature. 3. The presynaptic negative signals increased in duration with lowering of the temperature. 4. These results support the hypothesis that the effect of a reduction in temperature reflects the improved efficacy of the calcium-mediated mechanism of transmitter release, manifested as a prolongation of the inflow of Ca2+. The process involved in the evoked release is probably attributable to an almost passive mechanism.     

Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons

Astrocytes exhibit a form of excitability and communication on the basis of intracellular Ca2+ variations (Cornell-Bell et al., 1990; Charles et al., 1991) that can be initiated by neuronal activity (Dani et al., 1992; Porter and McCarthy, 1996). A Ca2+ elevation in astrocytes induces the release of glutamate (Parpura et al., 1994; Pasti et al., 1997; Araque et al., 1998;Bezzi et al., 1998), which evokes a slow inward current in neurons and modulates action potential-evoked synaptic transmission between cultured hippocampal cells (Araque et al., 1998), suggesting that astrocytes and neurons may function as a network with bidirectional communication. Here we show that a Ca2+ elevation in astrocytes increases the frequency of excitatory as well as inhibitory miniature postsynaptic currents (mPSCs), without modifying their amplitudes. Thapsigargin incubation, microinjection of the Ca2+ chelator BAPTA, and photolysis of the Ca2+ cage NP-EGTA demonstrate that a Ca2+ elevation in astrocytes is both necessary and sufficient to modulate spontaneous transmitter release. This Ca2+-dependent release of glutamate from astrocytes enhances mPSC frequency by acting on NMDA glutamate receptors, because it is antagonized by D-2-amino-5-phosphonopentanoic acid (AP5) or extracellular Mg2+. These NMDA receptors are located extrasynaptically, because blockage specifically of synaptic NMDA receptors by synaptic activation in the presence of the open channel blocker MK-801 did not impair the AP5-sensitive astrocyte-induced increase of mPSC frequency. Therefore, astrocytes modulate spontaneous excitatory and inhibitory synaptic transmission by increasing the probability of transmitter release via the activation of NMDA receptors.     

Pharmacological identification of two types of presynaptic voltage-dependent calcium channels at CA3-CA1 synapses of the hippocampus

The effects of voltage-dependent Ca channel (VDCC) antagonists on synaptic transmission were investigated at CA3-CA1 synapses of guinea pig hippocampal slices. After selectively loading presynaptic structures in area CA1 with the calcium indicator fura-2, we simultaneously recorded a presynaptic calcium transient ([Ca]t) and the corresponding field excitatory postsynaptic potential (fEPSP) evoked by a single stimulus given to the Schaffer collateral-commissural (SCC) pathway. Application of nifedipine did not reduce either the [Ca]t of the fEPSP, suggesting that nifedipine-sensitive Ca channels do not significantly contribute to evoked synaptic transmission at low stimulation frequency. Application of omega-conotoxin GVIA (omega-CgTX) or omega-agatoxin-IVA (omega-Aga-IVA) dose-dependently blocked both the [Ca]t and the fEPSP. The time course of the block of the [Ca]t was similar to that of the fEPSP. About 40% of the total [Ca]t was omega-CgTX sensitive, and more than 20% was omega-Aga-IVA sensitive. Combined application of these two blockers showed no overlap of the omega-CgTX-sensitive with the omega-Aga-IVA-sensitive [Ca]t. These results suggest that there are at least two types of presynaptic VDCCs at CA3-CA1 synapses of the hippocampus: omega-CgTX-sensitive and omega-Aga-IVA-sensitive Ca channels. Our results also suggest that these two types of Ca channels are colocalized at a single presynaptic terminal. During application of omega-CgTX or omega-Aga-IVA, the initial slope of the fEPSP varied approximately as the fourth power of the amplitude of the [Ca]t, suggesting that omega-CgTX-sensitive and omega-Aga-IVA-sensitive Ca channels have about equal efficacy in triggering transmitter release. These results in combination with similar findings at the squid giant synapse suggest that the nonlinear relationship between transmitter release and the Ca influx is well conserved from the molluscan to the mammalian nervous system.     

Calcium channels in the GABAergic presynaptic nerve terminals projecting to meynert neurons of the rat

Effects of selective Ca2+ channel blockers on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in the acutely dissociated rat nucleus basalis of Meynert (nBM) neurons attached with nerve endings, namely, the "synaptic bouton" preparation, and in the thin slices of nBM, using nystatin perforated and conventional whole-cell patch recording modes, respectively. In the synaptic bouton preparation, nicardipine (3 x 10(-6) M) and omega-conotoxin-MVIIC (3 x 10(-6) M) reduced the frequency of spontaneous postsynaptic currents by 37 and 22%, respectively, whereas omega-conotoxin-GVIA had no effect. After blockade of L- and P/Q-type Ca2+ channels, successive removal of Ca2+ from external solution had no significant effect on the residual spontaneous activities, indicating that N-, R-, and T-type Ca2+ channels are not involved in the spontaneous GABA release. Thapsigargin, but not ryanodine, increased the frequency of spontaneous IPSCs in both the synaptic bouton and slice preparations, suggesting the partial contribution of the intracellular Ca2+ storage site to the spontaneous GABA release. In contrast, omega-conotoxin-GVIA (3 x 10(-6) M) and omega-conotoxin-MVIIC (3 x 10(-6) M) suppressed the evoked IPSCs by 31 and 37%, respectively, but nicardipine produced no significant effect. The residual evoked currents were abolished in Ca2+-free external solution but not in the external solution containing 10(-5) M Ni2+, suggesting the involvement of N-, P/Q-, and R-type Ca2+ channels but not L- and T-type ones in the evoked IPSCs. Neither thapsigargin nor ryanodine had any significant effects on the evoked IPSCs. It was concluded that Ca2+ channel subtypes responsible for spontaneous transmitter release are different from those mediating the transmitter release evoked by nerve stimulation.     

Dynamics of clot growth induced by thrombin diffusing into nonstirred citrate human plasma

We have tested whether action potential-evoked Ca2+ influx is required to initiate clathrin-mediated synaptic vesicle endocytosis in the lamprey reticulospinal synapse. Exo- and endocytosis were temporally separated by a procedure involving tonic action potential stimulation and subsequent removal of extracellular Ca2+ (Ca2+e). A low concentration of Ca2+ ([Ca2+]e of 11 microM) was found to be required for the induction of early stages of endocytosis. However, the entire endocytic process, from the formation of clathrin-coated membrane invaginations to the generation of synaptic vesicles, proceeded in the absence of action potential-mediated Ca2+ entry. Our results indicate that the membrane of synaptic vesicles newly incorporated in the plasma membrane is a sufficient trigger of clathrin-mediated synaptic vesicle endocytosis.     

Conductances evoked by light in the ON-beta ganglion cell of cat retina

We have addressed the role of Ca2+ channels in mossy fiber synaptic transmission and long-term potentiation (LTP). Whereas the induction of mossy fiber LTP is entirely normal when synaptic transmission is blocked by the glutamate receptor antagonist kynurenate, LTP is blocked in the absence of extracellular Ca2+. These findings suggest that presynaptic Ca2+ entry is essential for mossy fiber LTP. Therefore, the role of different types of presynaptic Ca2+ channels in synaptic transmission and LTP was investigated. Mossy fiber responses were little affected by the L-type Ca2+ channel blocker nifedipine. They were blocked partially by omega-conotoxin-GVIA (N-type) and almost entirely by omega-agatoxin-IVA (P-type). None of these antagonists blocked mossy fiber LTP, nor was its expression associated with a change in sensitivity of synaptic transmission to either of the two toxins. These results, together with previous findings, suggest that the induction of mossy fiber LTP is critically dependent on the entry of Ca2+ into the presynaptic terminal to trigger a series of steps resulting in the long lasting enhancement of evoked glutamate release. Whereas P-type Ca2+ channels are of primary importance in mossy fiber synaptic transmission, both the induction and expression of mossy fiber LTP can occur in the absence of P-type (or N-type) Ca2+ channels.