Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels.

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.

Synaptic transmission: exciting times for presynaptic receptors.

Presynaptic receptors play an important role - typically inhibitory--in modulating the strength of synaptic transmission in the brain. Recent studies now reveal that neurotransmitters can enhance synaptic strength by activating ligand-gated ion channels in presynaptic nerve endings.

Mechanisms governing dendritic gamma-aminobutyric acid (GABA) release in the rat olfactory bulb.

In the olfactory bulb, synaptic transmission between dendrites plays an important role in the processing of olfactory information. Glutamate released from the dendrites of principal mitral cells excites the dendritic spines of granule cells, which in turn release gamma-aminobutyric acid (GABA) back onto mitral cell dendrites. Slow N-methyl-d-aspartate (NMDA) receptors on granule dendrites are particularly effective in driving this reciprocal dendrodendritic inhibition (DDI), raising the possibility that calcium influx through NMDA receptors may trigger GABA exocytosis directly. In this study, I show that NMDA receptor activation is not an absolute requirement and that DDI can be evoked solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors when granule cell excitability is increased or under conditions that slow AMPA receptor kinetics. In physiological extracellular Mg(2+), DDI elicited by photolysis of caged calcium in mitral dendrites is blocked by cadmium and toxins to N- and P/Q-type voltage-gated calcium channels. DDI is largely unaffected after granule dendrites have been loaded with the slow calcium chelator EGTA, suggesting a tight coupling between the site of calcium influx and the release machinery governing GABA exocytosis. These results indicate that voltage-gated calcium channels play an essential role in dendritic GABA release during reciprocal feedback inhibition in the olfactory bulb.

Spillover in the spotlight.

Fast neurotransmission in the brain is typically mediated by local actions of transmitters at ionotropic receptors within synaptic contacts. Recent studies now reveal that, in addition to point-to-point signaling, amino-acid transmitters mediate diffuse signaling at extrasynaptic metabotropic receptors.

Glutamate spillover mediates excitatory transmission in the rat olfactory bulb.

In the CNS, glutamate typically mediates excitatory transmission via local actions at synaptic contacts. In the olfactory bulb, mitral cell dendrites release glutamate at synapses formed only onto the dendrites of inhibitory granule cells. Here, I show excitatory transmission mediated solely by transmitter spillover between mitral cells in olfactory bulb slices. Dendritic glutamate release from individual mitral cells causes self-excitation via local activation of N-methyl-D-aspartate (NMDA) receptors. Paired recordings reveal that glutamate release from one cell generates NMDA receptor-mediated responses in neighboring mitral cells that are enhanced by blockade of glutamate uptake. Furthermore, spillover generates spontaneous NMDA receptor-mediated population responses. This simultaneous activation of neighboring mitral cells by a diffuse action of glutamate provides a mechanism for synchronizing olfactory principal cells.

Modulation of motoneuron N-methyl-D-aspartate receptors by the inhibitory neurotransmitter glycine.

Previous studies in the central nervous system have shown that glycine is a co-agonist with glutamate at central N-methyl-D-aspartate receptors (NMDA-Rs). However, there is considerable controversy as to whether the glycine site on NMDA-Rs is saturated. If this site were not saturated then glycine released from glycinergic synaptic terminals might 'spill-over' and activate NMDA-Rs. Since motoneurons have both NMDA and glycine synapses these neurons present an optimal substrate for testing whether the glycine binding site of NMDA-Rs is activated by transmitter released from glycine synaptic terminals. Using an in vitro brainstem slice preparation we report on initial experiments to investigate whether the glycine binding site of NMDA-Rs is saturated in motoneurons. Specifically, we investigated the question of whether the response of neonatal rat hypoglossal motoneurons (HMs) to a brief application of NMDA is enhanced by the presence of exogenous glycine. We found that exogenously applied glycine (1 mM) enhanced the NMDA activated membrane current. We conclude that in brainstem slices the glycine site at motoneuronal NMDA-Rs is not saturated, and that synaptically-released glycine may modulate NMDA-Rs mediated responses.

GABAB receptor-mediated modulation of presynaptic currents and excitatory transmission at a fast central synapse.

Large nerve terminals (calyces of Held) in the medial nucleus of the trapezoid body (MNTB) offer a unique opportunity to explore the modulation of presynaptic channels at a mammalian central synapse. In this study I examined gamma-aminobutyric acid-B (GABAB)-mediated presynaptic inhibition at the calyx of Held in slices of the rat auditory brain stem. The selective GABAB agonist baclofen caused a potent inhibition of synaptic transmission and presynaptic Ca2+ current. The inhibition of presynaptic Ca2+ channels was associated with a slowing of the activation kinetics of the underlying current, and the inhibition was relieved by strong depolarization. The inhibition of both synaptic transmission and presynaptic Ca2+ current was abolished by N-ethylmaleimide, a sulfhydryl alkylating agent that uncouples the G(o)/Gi class of G proteins from receptors. Baclofen does not activate a potassium conductance in the presynaptic terminal. Taken together, these results suggest that GABAB receptors inhibit synaptic transmission via G protein-mediated modulation of presynaptic Ca2+ channels at this large central synapse. Furthermore, these findings demonstrate that basic mechanisms of G protein-mediated inhibition of Ca2+ channels, proposed from recordings of neuron cell bodies, are well conserved at nerve endings in the mammalian brain.

Olfactory reciprocal synapses: dendritic signaling in the CNS.

Synaptic transmission between dendrites in the olfactory bulb is thought to play a major role in the processing of olfactory information. Glutamate released from mitral cell dendrites excites the dendrites of granule cells, which in turn mediate GABAergic dendrodendritic inhibition back onto mitral dendrites. We examined the mechanisms governing reciprocal dendritic transmission in rat olfactory bulb slices. We find that NMDA receptors play a critical role in this dendrodendritic inhibition. As with axonic synapses, the dendritic release of fast neurotransmitters relies on N- and P/Q-type calcium channels. The magnitude of dendrodendritic transmission is directly proportional to dendritic calcium influx. Furthermore, recordings from pairs of mitral cells show that dendrodendritic synapses can mediate lateral inhibition independently of axonal action potentials.

NMDA and non-NMDA receptors are co-localized at excitatory synapses of rat hypoglossal motoneurons.

We used whole-cell patch clamp recordings in a rat brainstem slice preparation to characterize the properties of miniature excitatory postsynaptic currents (mEPSCs) in hypoglossal motoneurons. The distinct kinetic characteristics of N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated synaptic responses allowed us to study dual component mEPSCs mediated by the two receptor types. Using this approach, NMDA and non-NMDA receptors were found to be co-localized at the same synaptic locations. In addition, some sites contain only NMDA receptors since a large proportion of mEPSCs were apparently mediated by NMDA receptors only. Furthermore, the amplitudes of pharmacologically isolated NMDA receptor-mediated mEPSCs were highly variable in individual cells and their decay kinetics were modulated by membrane potential.

GABA(B)-mediated presynaptic inhibition of excitatory transmission and synaptic vesicle dynamics in cultured hippocampal neurons.

Local recycling of synaptic vesicle membrane at nerve terminals is necessary to maintain a readily releasable pool of transmitter. To what extent are the dynamics of vesicle recycling subject to modulation? We examined the influence of presynaptic GABA(B) receptors on vesicle dynamics at single synapses using optical imaging of FM1-43 in cultured rat hippocampal neurons. The kinetics of FM1-43 destaining indicate that synapses from a single neuron have a unimodal distribution of release probabilities, and GABA(B)-mediated inhibition occurs uniformly at all sites. Electrical and optical recordings from single cells show that the inhibition of excitatory transmission is entirely accounted for by a rapidly reversible reduction of exocytosis. In contrast, GABA(B) receptors do not alter the rate or extent of endocytosis.

Amplitude and time course of spontaneous and evoked excitatory postsynaptic currents in bushy cells of the anteroventral cochlear nucleus.

1. Spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded in slices of the rat anteroventral cochlear nucleus (AVCN) at the endbulb-bushy cell synaptic connection. 2. The amplitudes of alpha-amino-3-hydroxy-5-methy-4-isoxa-zolepropionic acid (AMPA)-receptor-mediated spontaneous EPSCs were large (54 +/- 6 pA, mean +/- SD; membrane potential = -70 mV, 22-25 degrees C) and, in the same cell, exhibited a very wide range of peak amplitudes (CM = 0.42 +/- 0.01, n - 15 cells). There was no significant correlation between rise times or decay time constants and the peak amplitudes of spontaneous EPSCs recorded in the same cell, demonstrating that electrotonic attenuation is not responsible for the large amplitude variability of spontaneous EPSCs. 3. Cyclothiazide, a potent blocker of AMPA-receptor desensitization, did not affect the amplitude of spontaneous EPSCs in AVCN bushy cells, suggesting that background desensitization of AMPA receptors is not significant in these cells. However, the decay time constant of spontaneous EPSCs was prolonged significantly (2.6-fold increase). In addition, cyclothiazide produced a marked increase (approximately 40%, n = 6 cells) in the frequency of spontaneous EPSCs, indicating a likely presynaptic site of action of this drug. 4. Cyclothiazide produced a small increase (approximately 10%, n = 7 cells) in the peak amplitude of the evoked endbulb EPSC, but this effect could be explained by the action of cyclothiazide to increase the decay time constant of the underlying quantal EPSCs in conjunction with the asynchrony of quantal transmitter release at the endbulb synapse. 5. These results indicate that neither electrotonic attenuation nor receptor desensitization are responsible for the wide range of peak amplitudes of spontaneous EPSCs in bushy cells. The large quantal variability therefore is likely to be due entirely to intrinsic fluctuations at each release site and site-to-site variability in the numbers of available receptors.

Counting quanta: direct measurements of transmitter release at a central synapse.

Contradictory hypotheses regarding the nature of synaptic transmission in the CNS have arisen from indirect methods of quantal analysis. In this study, we directly count the quanta released following nerve stimulation to examine synaptic transmission at a fast glutamatergic synapse in the mammalian auditory brainstem. Our results demonstrate the relationship between spontaneous and nerve-evoked synaptic events, indicate that asynchronous transmitter release governs the time course of evoked transmission, and show that the stochastic quantal release process, as originally proposed at the neuromuscular junction, is highly conserved at this central synapse.

Channels underlying the slow afterhyperpolarization in hippocampal pyramidal neurons: neurotransmitters modulate the open probability.

The slow afterhyperpolarization in hippocampal pyramidal neurons is mediated by a calcium-activated potassium current (IAHP) and is a target for variety of different neurotransmitters. The characteristics of the channels underlying IAHP and how they are modulated by neurotransmitters are, however, unknown. In this study, we have examined the properties of the channels underlying IAHP using fluctuation analysis of the macroscopic current. Our results indicate that this channel has a unitary conductance of 2-5 pS and a mean open time of about 2 ms. When the peak amplitude of IAHP was maximal, these channels have an open probability of 0.4. Noradrenaline and carbachol reduced IAHP amplitude by lowering open channel probability. These result indicate that a novel calcium-activated potassium channel underlies IAHP. This channel is modulated in a similar fashion by two different transmitter systems that utilize distinct protein kinases.

Receptors underlying excitatory synaptic transmission in slices of the rat anteroventral cochlear nucleus.

1. The anteroventral cochlear nucleus (AVCN) contains two principal cell types that receive input from the auditory nerve. Stellate cells receive conventional synapses on their dendrites, and bushy cells of the AVCN receive axosomatic input via large, calyceal terminals (the end bulbs of Held). We have used whole cell patch-clamp recording techniques to study excitatory postsynaptic currents (EPSCs) in these two principal cells of the rat AVCN. 2. EPSCs evoked in stellate cells by stimulation of the auditory nerve were graded with stimulus strength, indicating a high degree of convergence of input to these cells. At depolarized membrane potentials, EPSCs evoked in stellate neurons had a dual-component time course. The slow component was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), and the fast component was abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 3. EPSCs evoked in bushy cells by auditory nerve stimulation were large (50 nS average conductance) and all-or-none at the threshold stimulus level. At -70 mV, the time course of the EPSC was very brief (average time constant of decay 700 microseconds at room temperature). Membrane depolarization revealed a slow component to the EPSC. The fast and slow components were mediated by non-NMDA and NMDA receptors, respectively. The switch-off of end bulb NMDA EPSCs by voltage jumps to the EPSC reversal potential was very rapid, suggesting that the NMDA component arises from sites on or close to the soma. 4. Miniature EPSCs, recorded in the presence of tetrodotoxin (TTX) at depolarized potentials, also had a dual-component time course. The fast and slow components of the miniature EPSCs were blocked by CNQX and APV, respectively. This result indicates that NMDA and non-NMDA receptors can be co-localized at the same, presumably end bulb, release sites. 5. The relative contribution of the slow, NMDA component to the end bulb EPSC declined significantly with age (postnatal days 11-22). 6. These results indicate that both NMDA and non-NMDA receptors underlie excitatory synaptic transmission in the AVCN of young rats. The end bulb synapse onto bushy cells generates a non-NMDA receptor-mediated EPSC with very fast kinetics. NMDA receptors can also mediate synaptic transmission at the end bulb synapse, but their contribution becomes less as the auditory system matures. This finding suggests that NMDA receptors may play an important role in the development of this synapse.

The uptake inhibitor L-trans-PDC enhances responses to glutamate but fails to alter the kinetics of excitatory synaptic currents in the hippocampus.

1. We have used patch-clamp recording techniques to study the physiological properties of a recently described glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), in the CA1 region of the guinea pig hippocampus. 2. L-trans-PDC markedly potentiated the action of exogenously applied glutamate and raised the ambient extracellular levels of glutamate in hippocampal slices. Despite these actions, L-trans-PDC did not affect the time course of either the N-methyl-D-aspartate (NMDA) or non-NMDA receptor-mediated synaptic currents evoked by the stimulation of a large number of neighboring synapses. 3. These findings are consistent with models of fast synaptic transmission in which transmitter is rapidly cleared from the synaptic cleft by diffusion. However, in marked contrast to fast gamma-aminobutyric acid A (GABAA) synapses in the hippocampus, uptake does not appear to play a role in regulating the "spill-over" of transmitter from neighboring, co-activated glutamatergic synapses. Therefore, either diffusion alone can effectively limit the temporal and spatial domain of synaptically released glutamate, or alternatively, L-trans-PDC like other currently available blockers is not sufficiently potent to reveal a role for transmitter uptake at glutamatergic synapses.

Local and diffuse synaptic actions of GABA in the hippocampus.

In the CNS, gamma-aminobutyric acid (GABA) acts as an inhibitory transmitter via ligand-gated GABAA receptor channels and G protein-coupled GABAB receptors. Both of these receptor types mediate inhibitory postsynaptic transmission in the hippocampus. In addition to these direct postsynaptic actions, GABAB receptor agonists inhibit excitatory transmission through presynaptic receptors on excitatory afferent terminals. However, a physiological role for the GABAB receptors on excitatory nerve endings has not been established. In this study, we have found a brief, heterosynaptic depression of excitatory synaptic transmission in the CA1 region of the hippocampal slice following short-lasting repetitive stimulation and determined that this inhibition is mediated by presynaptic GABAB receptors. The inhibition of GABA uptake greatly enhanced both the presynaptic action of GABA and the slow GABAB-mediated inhibitory postsynaptic current. Transmitter uptake was also found to regulate the "spill-over" of GABA at conventional GABAA synapses. These results suggest that uptake mechanisms restrict the spatial range of both point-to-point synaptic transmission mediated by GABA and its action at a distance.

Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus.

Aniracetam is a nootropic drug that has been shown to selectively enhance quisqualate receptor-mediated responses in Xenopus oocytes injected with brain mRNA and in hippocampal pyramidal cells [Ito, I., Tanabe, S., Kohda, A. & Sugiyama, H. (1990) J. Physiol. (London) 424, 533-544]. We have used patch clamp recording techniques in hippocampal slices to elucidate the mechanism for this selective action. We find that aniracetam enhances glutamate-evoked currents in whole-cell recordings and, in outside-out patches, strongly reduces glutamate receptor desensitization. In addition, aniracetam selectively prolongs the time course and increases the peak amplitude of fast synaptic currents. These findings indicate that aniracetam slows the kinetics of fast synaptic transmission and are consistent with the proposal [Trussell, L. O. & Fischbach, G. D. (1989) Neuron 3, 209-218; Tang, C.-M., Dichter, M. & Morad, M. (1989) Science 243, 1474-1477] that receptor desensitization governs the strength of fast excitatory synaptic transmission in the brain.

Importance of endogenous angiotensin II in the cardiovascular responses to sympathetic stimulation in conscious rabbits.

Pharmacological evidence indicates that angiotensin (Ang II) converting enzyme inhibitors attenuate cardiovascular responses to sympathetic stimulation. To investigate the physiological significance of this attenuation, the pressor and heart rate responses to bilateral carotid occlusion (BCO) were studied before and after administration of captopril and again during Ang II replacement in conscious, aortic nerve-sectioned rabbits with chronically implanted carotid occluders. In the control period, BCO produced increases (p less than 0.05) in mean arterial pressure (MAP) and heart rate (HR) of 37.3 +/- 3.0 mm Hg and 21.7 +/- 5.4 beats/min from baseline values of 79.1 +/- 2.5 mm Hg and 255.4 +/- 16.7 beats/min. Captopril (5 mg/kg i.v.) markedly reduced (p less than 0.05) both the pressor (10.2 +/- 2.6 mm Hg) and HR (5.0 +/- 4.0 beats/min) responses to BCO, in parallel with a decrease in plasma Ang II of 75%. Infusion of a subpressor dose of Ang II (5-25 ng/kg/min i.v.) increased plasma Ang II to precaptopril levels and fully restored (p less than 0.05) the pressor (33.0 +/- 5.7 mm Hg) and HR (19.8 +/- 7.7 beats/min) responses to BCO. In two additional series of experiments, the mechanism of the effects of captopril and Ang II were investigated. In the first series, cardiac baroreflex curves (pulse interval versus MAP) were generated by increasing or decreasing blood pressure with phenylephrine or nitroprusside (5-20 micrograms/kg/min i.v.). The slope of the linear region of the curve (2.9 msec/mm Hg) was not changed significantly by captopril treatment (3.1 msec/mm Hg) or Ang II replacement (3.2 msec/mm Hg), indicating that cardiac baroreflex sensitivity was not altered by blockade of the renin-angiotensin system. In the second series, the effect of captopril on the pressor response to exogenous norepinephrine (0.1-2.5 micrograms/kg/min i.v.) was tested. The response was reduced by less than 40%, indicating only a modest postsynaptic component to the action of captopril. These results provide physiological evidence for an important action of endogenous Ang II in facilitating the cardiovascular responses to sympathetic stimulation in conscious rabbits. This facilitation is not due to an action upon the baroreflex per se but results, at least in part, from a presynaptic action of Ang II.

Solubilization, partial purification, and properties of omega-conotoxin receptors associated with voltage-dependent calcium channels from rat brain synaptosomes.

These experiments provide a starting point for biochemical characterization of Ca channels from neuronal membranes, using omega-CgTX as a specific marker. The purification of the omega-CgTX receptors is far from complete. Each of the purification steps described results in only a two- to fivefold enrichment of the receptor proteins, and is accompanied by a loss of receptor concentration and stability, so the maximal specific activity achieved by a combination of these steps falls several orders of magnitude short of that of a large, homogeneous, active protein. Nevertheless, these studies have yielded important information about the omega-CgTX receptor. The Stokes' radius, determined from gel exclusion chromatography, is approximately 87 A, and the sedimentation coefficient, determined from sucrose gradient sedimentation, is approximately 19 S. These values are similar to those found for the DHP receptors solubilized in digitonin. We have also found that at least some of the omega-CgTX receptors have complex carbohydrate moieties that are recognized by WGA, together with evidence of heterogeneity of receptor glycosylation. Additionally, we have been able to use the solubilized, partially purified receptors in cross-linking experiments to tentatively identify the molecular weights of the omega-CgTX targets from rat brain. A large peptide of approximately 300 kDa, similar to that identified in photoaffinity studies, is very clearly labeled by the chemical incorporation of [125I]omega-CgTX into partially purified receptor preparations, but some ambiguity remains because of the faint labeling of peptides in the 120-170-kDa range. The approximately 300-kDa peptide is much larger than any single peptide component of DHP receptors from skeletal muscle, and it may be related to a molecular combination of the 170-kDa and 135-kDa subunits of the DHP receptor. Because [125I]omega-CgTX presumably labels both N- and L-type neuronal Ca channels, both channel types will probably be found in the purified preparations. Thus, at some time, it will be necessary to separate DHP-sensitive L-type channels from preparations of L- and N-type channels identified by omega-CgTX binding.