Synaptically Released Research Articles

Blockade of Ca2+-Permeable AMPA/Kainate Channels Decreases Oxygen–Glucose Deprivation-Induced Zn2+Accumulation and Neuronal Loss in Hippocampal Pyramidal Neurons

Synaptic release of Zn2+ and its translocation into postsynaptic neurons probably contribute to neuronal injury after ischemia or epilepsy. Studies in cultured neurons have revealed that of the three major routes of divalent cation entry, NMDA channels, voltage-sensitive Ca2+ channels (VSCCs), and Ca2+-permeable AMPA/kainate (Ca-A/K) channels, Ca-A/K channels exhibit the highest permeability to exogenously applied Zn2+. However, routes through which synaptically released Zn2+ gains entry to postsynaptic neurons have not been characterized in vivo. To model ischemia-induced Zn2+ movement in a system approximating the in vivo situation, we subjected mouse hippocampal slice preparations to controlled periods of oxygen and glucose deprivation (OGD). Timm's staining revealed little reactive Zn2+ in CA1 and CA3 pyramidal neurons of slices exposed in the presence of O2 and glucose. However, 15 min of OGD resulted in marked labeling in both regions. Whereas strong Zn2+ labeling persisted if both the NMDA antagonist MK-801 and the VSCC blocker Gd3+ were present during OGD, the presence of either the Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) or the extracellular Zn2+ chelator Ca2+ EDTA substantially decreased Zn2+ accumulation in pyramidal neurons of both subregions. In parallel experiments, slices were subjected to 5 min OGD exposures as described above, followed 4 hr later by staining with the cell-death marker propidium iodide. As in the Timm's staining experiments, substantial CA1 or CA3 pyramidal neuronal damage occurred despite the presence of MK-801 and Gd3+, whereas injury was decreased by NAS or by Ca2+ EDTA (in CA1).

Synaptically released 5-HT modulates the activity of tonically discharging neuronal populations in the rostral ventral medulla (RVM).

There is substantial evidence for an important modulating role of monoamines (catecholamines and serotonin, 5-HT) in the rostral ventral medulla (RVM), a region which plays an important role in cardiovascular and nociceptive functions. We investigated in slices the role of endogenous monoamines in the synaptic control of the activity of rat RVM neuronal populations using intracellular recordings in the lateral RVM plus lateral aspect of nucleus paragigantocellularis lateralis. A triple-labelling protocol allowed us to identify the location of impaled neurons and their eventual monoaminergic phenotype within the serotonergic and catecholaminergic populations of the RVM. Focal electrical stimulation revealed the existence of a functional monoaminergic input onto RVM neurons which was mediated by endogenous 5-HT acting at inhibitory 5-HT1A receptors but did not involve noradrenergic neurotransmission. The slow 5-HT-mediated inhibitory postsynaptic potential (IPSP) was only observed in the regularly discharging neurons, which were found to be neither catecholaminergic nor serotonergic. The synaptic release of 5-HT was, itself, under an inhibitory control involving GABAA (gamma-aminobutyric acid) receptors. Moreover, we characterized the effect of the 5-HT-releasing agent fenfluramine on this functional 5-HT-mediated synaptic transmission. Our results show that the effect of fenfluramine is biphasic consisting of an initial prolongation of the serotonergic IPSP followed by a decrease in amplitude. Our data provide a basis for the previously reported inhibitory effects of exogenously applied serotonin agonists/antagonists on the autonomic functions controlled by the RVM. This 5-HT pathway, which functionally links the serotonergic and catecholaminergic regions, might play an important role in cardiovascular and nociceptive functions.

Synaptically released glutamate reduces gamma-aminobutyric acid (GABA)ergic inhibition in the hippocampus via kainate receptors.

Exogenous application of agonists at the kainate subtype of glutamate receptors has been shown to depress evoked monosynaptic inhibition by gamma-aminobutyric acid (GABA)ergic interneurons in the hippocampus. This observation has led to the hypothesis that synaptic release of endogenous glutamate might have a disinhibitory effect on neuronal circuits, in addition to depolarizing neurons via postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and N-methyl-D-aspartic acid (NMDA) receptors. It is not known, however, if glutamate released from excitatory neurons has the same kainate receptor-mediated effect on monosynaptic inhibitory transmission as exogenous agonist application. Indeed, the recent demonstration that excitatory synaptic signals elicited in interneurons are partly mediated by kainate receptors suggests that these receptors may have a pro- rather than disinhibitory role. Here, we examine the effect of synaptically released glutamate on monosynaptic inhibitory signaling. In the presence of antagonists to AMPA and NMDA receptors, brief bursts of activity in glutamatergic afferent fibers reduce GABAergic transmission. This depression of inhibition is reversibly abolished by blocking kainate receptors. It persists when GABA(B) receptors are blocked and is enhanced by blocking metabotropic glutamate receptors, possibly explained by presynaptic regulation of glutamate release from excitatory afferents by metabotropic autoreceptors. We conclude that the net kainate receptor-mediated effect of synaptically released glutamate is to reduce monosynaptic inhibition. Since this form of disinhibition may contribute to seizure initiation, kainate receptors may constitute an important target for anticonvulsant drug development.

The Concentration of Synaptically Released Glutamate Outside of the Climbing Fiber–Purkinje Cell Synaptic Cleft

AMPA receptors and glutamate transporters expressed by cerebellar Bergmann glial cells are activated by neurotransmitter released from climbing fibers (). Based on anatomical evidence, this is most likely the result of glutamate diffusing out of the climbing fiber-Purkinje cell synaptic clefts (). We used the change in the EC50 of the Bergmann glia AMPA receptors produced by cyclothiazide (CTZ) to estimate the concentration of glutamate reached at the glial membrane. The decrease of the EC50 gives rise to a concentration-dependent potentiation of the AMPA receptor-mediated responses (). By comparing the increase in amplitude of the AMPA receptor response in the Bergmann glia (840 +/- 240%; n = 8) with the shift in the glutamate dose-response curve measured in excised patches (EC50, 1810 microM in control vs 304 microM in CTZ), we estimate that the extrasynaptic transmitter concentration reaches 160-190 microM. This contrasts with the concentration in the synaptic cleft, thought to rapidly rise above 1 mM, but is still high enough to activate glutamate receptors. These results indicate that the sphere of influence of synaptically released glutamate can extend beyond the synaptic cleft.

GABA Activity Mediating Cytosolic Ca2+Rises in Developing Neurons Is Modulated by cAMP-Dependent Signal Transduction

In the majority of developing neurons, GABA can exert depolarizing actions, thereby raising neuronal Ca2+. Ca2+ elevations can have broad consequences during development, inducing gene expression, altering neurite outgrowth and growth cone turning, activating enzyme pathways, and influencing neuronal survival. We used fura-2 and fluo-3 Ca2+ digital imaging to assess the effects of inhibiting or activating the cAMP signal transduction pathway on GABA activity mediating Ca2+ rises during the early stages of in vitro hypothalamic neural development. Our experiments stemmed from the finding that stimulation of transmitter receptors shown to either activate or inhibit adenylyl cyclase activity caused a rapid decrease in Ca2+ rises mediated by synaptically released GABA. Both the adenylyl cyclase activator forskolin and the inhibitor SQ-22,536 reduced the Ca2+ rise elicited by the synaptic release of GABA. Bath application of the membrane-permeable cAMP analogs 8-bromo-cAMP (8-Br-cAMP) or 8-(4-chlorophenylthio)-cAMP (0.2-5 mM) produced a rapid, reversible, dose-dependent inhibition of Ca2+ rises triggered by synaptic GABA release. Potentiation of GABAergic activity mediating Ca2+ rises was observed in some neurons at relatively low concentrations of the membrane-permeable cAMP analogs (20-50 microM). In the presence of tetrodotoxin (TTX), postsynaptic Ca2+ rises triggered by the bath application of GABA were only moderately depressed (13%) by 8-Br-cAMP (1 mM), suggesting that the inhibitory effects of 8-Br-cAMP were largely the result of a presynaptic mechanism. The protein kinase A (PKA) inhibitors H89 and Rp-3', 5'-cyclic monophosphothioate triethylamine also caused a large reduction (>70%) in Ca2+ rises triggered by synaptic GABA release. Unlike the short-term depression elicited by activation of the cAMP signal transduction pathway, Ca2+ depression elicited by PKA inhibition persisted for an extended period (>30 min) after PKA inhibitor washout. Postsynaptic depression of GABA-evoked Ca2+ rises triggered by H89 (in the presence of TTX) recovered rapidly, suggesting that the extended depression observed during synaptic GABA release was largely through a presynaptic mechanism. Long-term Ca2+ modulation by cAMP-regulating hypothalamic peptides may be mediated through a parallel mechanism. Together, these results suggest that GABAergic activity mediating Ca2+ rises is dependent on ongoing PKA activity that is maintained within a narrow zone for GABA to elicit a maximal Ca2+ elevation. Thus, neuromodulator-mediated changes in the cAMP-dependent signal transduction pathway (activation or inhibition) could lead to a substantial decrease in GABA-mediated Ca2+ rises during early development.

Local synaptic release of glutamate from neurons in the rat hypothalamic arcuate nucleus.

1. The hypothalamic arcuate nucleus (ARC) contains neuroendocrine neurons that regulate endocrine secretions by releasing substances which control anterior pituitary hormonal release into the portal blood stream. Many neuroactive substances have been identified in the ARC, but the existence of excitatory neurons in the ARC and the identity of an excitatory transmitter have not been investigated physiologically. 2. In the present experiments using whole-cell current- and voltage-clamp recording of neurons from cultures and slices of the ARC, we demonstrate for the first time that some of the neurons in the ARC secrete glutamate as their transmitter. 3. Using microdrop stimulation of presynaptic neurons in ARC slices, we found that local axons from these glutamatergic neurons make local synaptic contact with other neurons in the ARC and that all evoked excitatory postsynaptic potentials could be blocked by the selective ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and D,L-2-amino-5-phosphonovalerate (AP5; 100 microM). To determine the identity of ARC neurons postsynaptic to local glutamatergic neurons, we used antidromic stimulation to reveal that many of these cells were neuroendocrine neurons by virtue of their maintaining axon terminals in the median eminence. 4. In ARC cultures, postsynaptic potentials, both excitatory and inhibitory, were virtually eliminated by the glutamate receptor antagonists AP5 and CNQX, underlining the functional importance of glutamate within this part of the neuroendocrine brain. 5. GABA was secreted by a subset of ARC neurons from local axons. The GABAA receptor antagonist bicuculline released glutamatergic neurons from chronic inhibition mediated by synaptically released GABA, resulting in further depolarization and an increase in the amplitude and frequency of glutamate-mediated excitatory postsynaptic potentials.

Selection of postsynaptic serotonin receptors during reinnervation of an identified leech neuron in culture

Serotoninergic Retzius neurons reform an inhibitory synapse onto pressure-sensitive mechanosensory (P) neurons when the cells are removed from the nervous system of the leech and are juxtaposed in tissue culture. The somas of P cells in situ and single (uninnervated) P cells in culture have both a depolarizing and Cl-dependent hyperpolarizing response to application of the transmitter serotonin (5-HT). Synaptic release of 5-HT by a Retzius cell in situ and in culture evokes a Cl-dependent postsynaptic response but does not appear to activate the depolarizing receptors. We have characterized the ionic currents induced by synaptically released and applied 5-HT in voltage-clamped P cells in culture in order to determine the responses to transmitter and their modifications following innervation. When 5-HT was applied to single P cells, 2 types of channels were activated by 5-HT and could be distinguished by changing the ionic composition of the superfusion solution. In an impermeant cation (TrisCl) solution, a 5-HT-dependent Cl current was activated. When single P cells were superfused with a Cl-free solution (Cl replaced by impermeant SO4), 5-HT activated a monovalent cation current. Following innervation of a P cell by a cocultured Retzius cell, the reversal potential of the peak postsynaptic current depended on the Cl gradient and the synaptic response was blocked by the Cl channel blocker 9-anthracenecarboxylic acid. Thus, synaptic release of 5-HT activated solely the Cl channels and not the cationic channels. Pipette application of 5-HT onto innervated P cells activated a Cl conductance comparable in magnitude to the synaptic response. In contrast, the cationic conductance was reduced roughly 5-fold. It is concluded that innervation of a P cell by a Retzius cell resulted in clustering of the synaptic 5-HT receptors, which activate Cl channels and reduction of the nonsynaptic, cationic response. The result is a selection of receptors in the cultured P cell that mimics the pattern observed in vivo.

Characterization of the Clonidine Receptor Site

At the cardiac sympathetic nerve terminal the α2-adrenoceptor is presynaptic and appears to be located at an extrasynaptic site. This is suggested by (1) absence of evidence of autoinhibitory feedback at physiologic stimulus levels up to about 50 percent of the maximum chronotropic response in the isolated guinea pig right atrium, and (2) absence of significant competition between clonidine and synaptically released noradrenaline (NA) for the presynaptic site. In the central nervous system (CNS) cardiovascular α2-receptors are probably located at a postsynaptic site in bulbospinal regions of the brain, since they produce effects identical to those of synaptic release of NA. Experiments with the clonidine analog alinidine (ST 567) suggest that there are differences in central receptor type subserving clonidine-mediated baroreflex heart rate and blood pressure changes. At the cardiac sympathetic nerve terminal the α2-adrenoceptor is presynaptic and appears to be located at an extrasynaptic site. This is suggested by (1) absence of evidence of autoinhibitory feedback at physiologic stimulus levels up to about 50 percent of the maximum chronotropic response in the isolated guinea pig right atrium, and (2) absence of significant competition between clonidine and synaptically released noradrenaline (NA) for the presynaptic site. In the central nervous system (CNS) cardiovascular α2-receptors are probably located at a postsynaptic site in bulbospinal regions of the brain, since they produce effects identical to those of synaptic release of NA. Experiments with the clonidine analog alinidine (ST 567) suggest that there are differences in central receptor type subserving clonidine-mediated baroreflex heart rate and blood pressure changes.

Characterization of the Clonidine Receptor Site

At the cardiac sympathetic nerve terminal the alpha 2-adrenoceptor is presynaptic and appears to be located at an extrasynaptic site. This is suggested by (1) absence of evidence of autoinhibitory feedback at physiologic stimulus levels up to about 50 percent of the maximum chronotropic response in the isolated guinea pig right atrium, and (2) absence of significant competition between clonidine and synaptically released noradrenaline (NA) for the presynaptic site. In the central nervous system (CNS) cardiovascular alpha 2-receptors are probably located at a postsynaptic site in bulbospinal regions of the brain, since they produce effects identical to those of synaptic release of NA. Experiments with the clonidine analog alinidine (ST 567) suggest that there are differences in central receptor type subserving clonidine-mediated baroreflex heart rate and blood pressure changes.

GABA-mediated biphasic inhibitory responses in hippocampus.

γ-Amlnobutyric acid (GABA) can have a biphasic effect on membrane potential when applied to certain central nervous system neurones1–3. Typically with iontophoresis, this effect is a negative–positive sequence of deflections and both phases are associated with an increase in conductance. However, iontophoretic application of neurotransmitter substances can produce effects even in cases where ordinary synaptic transmitter release is impossible; for example, on dorsal root ganglion cells. Thus, it is important in assessing the physiological relevance of the depolarising GABA response to determine if similar actions are produced synaptically. In addition, although the depolarising aspect of the GABA response has been attributed to activation of dendritic receptors, this identification has remained uncertain. Here, we have used the hippocampal slice preparation to demonstrate that a biphasic inhibitory response results from orthodromic, but not antidromic electrical stimulation in the presence of anaesthetic concentrations of pentobarbital. (For the sake of brevity, we use the term ‘biphasic inhibitory postynaptic potential (i.p.s.p.)’ to describe the response complex, although it seems likely that a combination of two distinct events, rather than two parts of a single event, occurs.) GABA antagonists and tetrodotoxin (TTX) were used to show that the depolarising phase is due to synaptically released GABA acting on dendritic sites in the CA1 region. Both negative and positive phases are associated with a large conductance increase, and both block cell firing, indicating their inhibitory nature. The ionic mechanism of the depolarising phase is not fully understood but seems to involve a chloride conductance increase. Although the functional significance of the depolarising sign of the i.p.s.p. is not clear, the dendritic location would provide very effective control over dendritic excitability.