Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin.

Extensive research has failed to clarify the mechanism of action of nitrous oxide (N2O, laughing gas), a widely used inhalational anesthetic and drug of abuse. Other general anesthetics are thought to act by one of two mechanisms-blockade of NMDA glutamate receptors or enhancement of GABAergic inhibition. Here we show that N2O, at anesthetically-relevant concentrations, inhibits both ionic currents and excitotoxic neurodegeneration mediated through NMDA receptors and, like other NMDA antagonists, produces neurotoxic side effects which can be prevented by drugs that enhance GABAergic inhibition. The favorable safety record of N2O may be explained by the low concentrations typically used and by the fact that it is usually used in combination with GABAergic anesthetics that counteract its neurotoxic potential.

Action potential initiation and propagation: upstream influences on neurotransmission.

Axonal action potentials initiate the cycle of synaptic communication that is key to our understanding of nervous system functioning. The field has accumulated vast knowledge of the signature action potential waveform, firing patterns, and underlying channel properties of many cell types, but in most cases this information comes from somatic intracellular/whole-cell recordings, which necessarily measure a mixture of the currents compartmentalized in the soma, dendrites, and axon. Because the axon in many neuron types appears to be the site of lowest threshold for action potential initiation, the channel constellation in the axon is of particular interest. However, the axon is more experimentally inaccessible than the soma or dendrites. Recent studies have developed and applied single-fiber extracellular recording, direct intracellular recording, and optical recording techniques from axons toward understanding the behavior of the axonal action potential. We are starting to understand better how specific channels and other cellular properties shape action potential threshold, waveform, and timing: key elements contributing to downstream transmitter release. From this increased scrutiny emerges a theme of axons with more computational power than in traditional conceptualizations.

Selective depression of low-release probability excitatory synapses by sodium channel blockers.

Sodium channels (NaChs) play a central role in action potential generation and are uniquely poised to influence the efficacy of transmitter release. We evaluated the effect of partial NaCh blockade on two aspects of synaptic efficacy First, we evaluated whether NaCh blockade accounts for the ability of certain drugs to selectively depress glutamate release. Second, we evaluated the contribution of NaChs to intraneuronal variability in glutamate release probability (p(r)). The antiglutamate drug riluzole nearly completely depresses glutamate excitatory postsynaptic currents (EPSCs) at concentrations that barely affect GABAergic inhibitory postsynaptic currents (IPSCs). NaCh inhibition explains the selective depression. Unlike other presynaptic depressants, partial NaCh blockade increases paired-pulse EPSC depression. This result is explained by selective depression of low-p(r) synapses. We conclude that local variations in the action potential contribute to p(r) variability among excitatory synapses.

Ultrafast exocytosis elicited by calcium current in synaptic terminals of retinal bipolar neurons.

Using high resolution capacitance measurements, we have characterized an ultrafast component of transmitter release in ribbon-type synaptic terminals of retinal bipolar neurons. During depolarization, capacitance increases to a plateau of approximately 30 fF with a time constant of approximately 1.5 ms. When not limited by activation kinetics of calcium current, the small pool is depleted even faster, with a time constant of 0.5 ms. After the ultrafast pool is depleted, capacitance rises with a slower time constant of approximately 300 ms. EGTA (5 mM) depresses the slower capacitance rise but leaves the ultrafast phase intact. BAPTA (5 mM) depresses both components of exocytosis. With paired-pulse stimulation, the ultrafast pool recovers from depletion with a time constant of approximately 4 s. The ultrafast component may represent fusion of docked vesicles at the base of the synaptic ribbon, while the slower component represents more distal vesicles on the ribbon.

Redox modulation of T-type calcium channels in rat peripheral nociceptors.

Although T-type calcium channels were first described in sensory neurons, their function in sensory processing remains unclear. In isolated rat sensory neurons, we show that redox agents modulate T currents but not other voltage- and ligand-gated channels thought to mediate pain sensitivity. Similarly, redox agents modulate currents through Ca(v)3.2 recombinant channels. When injected into peripheral receptive fields, reducing agents, including the endogenous amino acid L-cysteine, induce thermal hyperalgesia. This hyperalgesia is blocked by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the T channel antagonist mibefradil. DTNB alone and in combination with mibefradil induces thermal analgesia. Likewise, L-cysteine induces mechanical DTNB-sensitive hyperalgesia in peripheral receptive fields. These data strongly suggest a role for T channels in peripheral nociception. Redox sites on T channels in peripheral nociceptors could be important targets for agents that modify pain perception.

Actions of anesthetics on excitatory transmitter-gated channels.

Excitatory transmitter-gated receptors are found in three gene families: the glutamate ionotropic receptors, the Cys-loop receptor family (nicotinic and 5HT3), and the purinergic (P2X) receptors. Anesthetic drugs act on many members of these families, but in most cases the effects are unlikely to be related to clinically relevant anesthetic actions. However, the gaseous anesthetics (xenon and nitrous oxide) and the dissociative anesthetics (ketamine) have significant inhibitory activity at one type of glutamate receptor (the NMDA receptor) that is likely to contribute to anesthetic action. It is possible that some actions at neuronal nicotinic receptors may make a smaller contribution to effects of some anesthetics.

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions.

BACKGROUND AND PURPOSE: Neuroactive steroids are potent modulators of GABA(A) receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABA(A) receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABA(A) receptor, such as (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP, allopregnanolone). EXPERIMENTAL APPROACH: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between gamma-cyclodextrin and neuroactive steroids of different structural classes. KEY RESULTS: Both a bioassay based on electrophysiological assessment of GABA(A) receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that gamma-cyclodextrin sequesters steroids rather than directly influencing GABA(A) receptor function. Neither a 5beta-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and gamma-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and gamma-cyclodexrin ranged from 10-60 microM. Although gamma-cyclodextrin accommodates a range of natural and synthetic steroids, C(11) substitutions reduced inclusion complex formation. Using gamma-cyclodextrin to remove steroid not directly bound to GABA(A) receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3alpha- hydroxysteroids but not inhibition by sulphated steroids. CONCLUSIONS AND IMPLICATIONS: We conclude that gamma-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids.

Pregnenolone sulfate modulates inhibitory synaptic transmission by enhancing GABA(A) receptor desensitization.

We examined the effects of the neurosteroid pregnenolone sulfate (PS) on GABA(A) receptor-mediated synaptic currents and currents elicited by rapid applications of GABA onto nucleated outside-out patches in cultured postnatal rat hippocampal neurons. At 10 microm, PS significantly depressed peak responses and accelerated the decay of evoked inhibitory synaptic currents. In nucleated outside-out patches, PS depressed peak currents and speeded deactivation after 5 msec applications of a saturating concentration of GABA. PS also increased the rate and degree of macroscopic GABA receptor desensitization during prolonged GABA applications. In a paired GABA application paradigm, PS slowed the rate of recovery from desensitization. In contrast to its prominent effects on currents produced by saturating GABA concentrations, PS had only small effects on peak currents and failed to alter deactivation after brief applications of the weakly desensitizing GABA(A) receptor agonists taurine and beta-alanine. However, when beta-alanine was applied for a sufficient duration to promote receptor desensitization, PS augmented macroscopic desensitization and slowed deactivation. These results suggest that PS inhibits GABA-gated chloride currents by enhancing receptor desensitization and stabilizing desensitized states. This contention is supported by kinetic modeling studies in which increases in the rate of entry into doubly liganded desensitized states mimic most effects of PS.

Slow death of postnatal hippocampal neurons by GABA(A) receptor overactivation.

Neurotransmitters can have both toxic and trophic functions in addition to their role in neural signaling. Surprisingly, chronic blockade of GABA(A) receptor activity for 5-8 d in vitro enhanced survival of hippocampal neurons, suggesting that GABA(A) receptor overactivation may be neurotoxic. Potentiating GABA(A) receptor activity by chronic treatment with the endogenous neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one caused massive cell loss over 1 week in culture. Other potentiators of GABA(A) receptors, including benzodiazepines, mimicked the cell loss, suggesting that potentiating endogenous GABA activity is sufficient to produce neuronal death. Neurosteroid-treated neurons had lower resting intracellular calcium levels than control cells and produced smaller calcium rises in response to depolarizing challenges. Manipulating intracellular calcium levels with chronic elevated extracellular potassium or with the calcium channel agonist Bay K 8644 protected neurons. The results may have implications for the mechanisms of programmed cell death in the developing CNS as well as implications for the long-term consequences of chronic GABAmimetic drug use during development.

Substrate turnover by transporters curtails synaptic glutamate transients.

Although inhibitors of glutamate transport prolong synaptic currents at many glutamate synapses, the cause of the current prolongation is unclear. Transport inhibitors may prolong synaptic currents by simply interfering with synaptic glutamate binding to transporters, by inhibiting substrate translocation, or by promoting accumulation of ambient glutamate, which may act cooperatively at receptors with synaptic glutamate. We show that reversal of the membrane potential of astrocytes surrounding the synapse prolongs synaptic currents but does not decrease the apparent affinity of transporters or significantly alter glutamate-dependent kinetics of macroscopic transporter currents in excised membrane patches. Positive membrane potentials do not affect binding of a nontransported glutamate analog, nor do positive membrane potentials alter the number of transporters available to bind analog. We also test the hypothesis that glutamate accumulation during uptake inhibition by transporter substrates is the direct cause of synaptic current prolongations. Transporter substrates elevate ambient glutamate near synapses by fostering reverse transport of endogenous glutamate. However, increases in ambient glutamate cannot account for the prolongations of synaptic currents, because a nonsubstrate transport inhibitor does not foster reverse uptake yet it prolongs synaptic currents. Moreover, exogenous glutamate does not mimic synaptic current prolongations induced by substrate inhibitors. These results provide strong support for a major role of substrate translocation in determining the time course of the glutamate concentration transient at excitatory synapses.

Astrocytes exert a pro-apoptotic effect on neurons in postnatal hippocampal cultures.

We investigated conditions that promote basal and activity-dependent neuronal apoptosis in postnatal rat hippocampal cultures. Low-density mixed cultures of astrocytes and neurons exhibited lower sensitivity than high-density cultures to basal neuronal death and activity-sensitive neuronal death, induced with glutamate receptor blockers, sodium channel blockers, or calcium channel blockers. Although elevations of [Ca(2+)](i) protect neurons from apoptosis, low-density microcultures and mass cultures exhibited only minor differences in resting [Ca(2+)](i) and Ca(2+) current density, suggesting that these variables are unlikely to explain differences in susceptibility. Astrocytes, rather than neurons, were implicated in the neuronal loss. Several candidate molecules implicated in other astrocyte-dependent neurotoxicity models were excluded, but heat inactivation experiments suggested that a heat-labile factor is critically involved. In sum, our results suggest the surprising result that astrocytes can be negative modulators of neuronal survival during development and when the immature nervous system is challenged with drugs that dampen electrical excitability.

Neural activity and survival in the developing nervous system.

Recent evidence suggests that blockade of normal excitation in the immature nervous system may have profound effects on neuronal survival during the period of natural cell death. Cell loss following depression of electrical activity in the central nervous system (CNS) may explain the neuropsychiatric deficits in humans exposed to alcohol or other CNS depressants during development. Thus, understanding the role of electrical activity in the survival of young neurons is an important goal of modern basic and clinical neuroscience. Here we review the evidence from in vivo and in vitro model systems that electrical activity participates in promoting neuronal survival. We discuss the potential role of moderate elevations of intracellular calcium in promoting survival, and we address the possible ways in which activity and conventional trophic factors may interact.

Neuroprotective agent riluzole potentiates postsynaptic GABA(A) receptor function.

The antiepileptic drug riluzole is a use-dependent blocker of voltage-gated Na(+) channels and selectively depresses action potential-driven glutamate over gamma-aminobutyric acid (GABA) release. Here we report that in addition to its presynaptic effect, riluzole at higher concentrations also strongly potentiates postsynaptic GABA(A) responses both in cultured hippocampal neurons and in Xenopus oocytes expressing recombinant receptors. Although peak inhibitory postsynaptic currents (IPSCs) of autaptic hippocampal neurons were inhibited, 20-100 microM riluzole significantly prolonged the decay of IPSCs, resulting in little change in total charge transfer. The effect was dose-dependent and reversible. Riluzole selectively increased miniature IPSC fast and slow decay time constants, without affecting their relative proportions. Miniature IPSC peak amplitude, rise time and frequency were unaffected, indicating a postsynaptic mechanism. In the Xenopus oocyte expression system, riluzole potentiated GABA responses by lowering the EC(50) for GABA activation. Riluzole directly gated a GABA(A) current that was partially blocked by bicuculline and gabazine. Pharmacological experiments suggest that the action of riluzole did not involve a benzodiazepine, barbiturate, or neurosteroid site. Instead, riluzole-induced potentiation was inhibited by the lactone antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolatone (alpha-IMGBL). While most anticonvulsants either block voltage-gated Na(+) channels or potentiate GABA(A) receptors, our results suggest that riluzole may define an advantageous class of anticonvulsants with both effects.

Pregnenolone sulfate and dehydroepiandrosterone sulfate inhibit GABA-gated chloride currents in Xenopus oocytes expressing picrotoxin-insensitive GABA(A) receptors.

We examined the effects of picrotoxinin, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) on gamma-aminobutyric acid (GABA) responses in Xenopus oocytes injected with wild type alpha1, beta2 and gamma2 GABA(A) receptor subunits and in oocytes injected with wild type alpha1 and beta2 subunits and a mutated gamma2 subunit that eliminates picrotoxin sensitivity. All three agents inhibited GABA currents in oocytes injected with wild type subunits. Oocytes injected with the mutated gamma2 subunit showed no inhibition of GABA responses by picrotoxinin at concentrations up to 100 microM. PS and DHEAS inhibited GABA currents at similar concentrations in both sets of oocytes. These results indicate that PS and DHEAS do not require a functional picrotoxin site for inhibition of GABA responses.

Potential clinical uses of neuroactive steroids.

Neuroactive steroids rapidly modulate gamma-aminobutyric acid (GABA) and glutamate receptors. GABA-enhancing steroids have potential clinical utility as anesthetics, hypnotics, anticonvulsants and anxiolytics. Furthermore, GABAergic neurosteroids may participate in regulating mood and the effects of alcohol on the nervous system, suggesting a potential role in major psychiatric disorders. Neuroactive steroids that alter the function of glutamate receptors could be useful for treating neurodegenerative disorders, and as cognitive enhancers. Recent progress in developing water-soluble steroids and steroids with enhanced oral efficacy foster optimism that certain neuroactive steroids will be developed for clinical use.

Pharmacological and physiological properties of a putative ganglionic nicotinic receptor, alpha 3 beta 4, expressed in transfected eucaryotic cells.

Neuronal nicotinic acetylcholine receptor subunits alpha 3 (PCA48E) and beta 4S (ZPC13) were expressed in human embryonic kidney (HEK)-293 cells by calcium phosphate transfection. In the presence of atropine, acetylcholine (ACh) induced fast activating currents which exhibited desensitization and inward rectification. The EC50 for ACh was 202 +/- 32 microM with a Hill coefficient of 1.9 +/- 0.4. The rank order of nicotinic agonist potency was 1,1-dimethyl-4-phenylpiperozinium (DMPP) > cytisine = nicotine approximately equal to ACh. The maximal response elicited by DMPP was substantially less than that elicited by other agonists, suggesting that DMPP is a partial agonist. ACh (500 microM) responses were very effectively blocked by equimolar concentrations (100 microM) of the ganglionic antagonists d-tubocurarine, mecamylamine and hexamethonium. Equal concentrations of the potent muscle receptor antagonist decamethonium and the competitive antagonist dihydro-beta-erythroidine were much less effective. alpha bungaro-toxin (1 microM) had little effect on ACh-induced responses. This physiological and pharmacological profile is consistent with a ganglionic nicotinic response.

Temporal ordering of pathogenic events following transient global ischemia.

Rats were subjected to transient global ischemia (four vessel occlusion) and time-related changes in the selectively vulnerable hippocampal field CA1 were characterized. The assessment included ex vivo field responses to afferent stimulation, silver staining, calpain-induced spectrin breakdown, chromatolysis, and cell death, beginning at 6 h post-ischemia and continuing until total disintegration of the pyramidal cells occurred several days later. The earliest change observed was a modest increase in the slope and amplitude of field CA1 potentials (at 6 h). The hyperresponsiveness was most apparent at higher stimulation currents and persisted unchanged at 16 h post-ischemia. Three effects became detectable within 24 h, post-ischemia: (a) an increase in concentrations of calpain-mediated, spectrin breakdown products; (b) enhanced silver staining in the deep pyramidal neurons of the field CA1 with lesser, though still apparent, staining of stratum radiatum, and (c) a decrease in amplitude and slope of field CA1 responses to afferent stimulation. Both the concentration of spectrin breakdown products and the intensity of silver staining progressively increased to a maximum at four days post ischemia, while the amplitude and slope of the field responses dropped to a very low level between 24 and 48 h. Disturbances of Nissl staining were finally evident at 48 h, with nearly complete disappearance of staining at five days post-ischemia. This study provides the first demonstration of a close and early temporal relationship between calpain proteolysis, subcellular damage to the pyramidal cells and their loss of function following global ischemia, prior to their eventual death.

Potential clinical uses of neuroactive steroids.

Neuroactive steroids rapidly modulate gamma-aminobutyric acid (GABA) and glutamate receptors. GABA-enhancing steroids have potential clinical utility as anesthetics, hypnotics, anticonvulsants and anxiolytics. Furthermore, GABAergic neurosteroids may participate in regulating mood and the effects of alcohol on the nervous system, suggesting a potential role in major psychiatric disorders. Neuroactive steroids that alter the function of glutamate receptors could be useful for treating neurodegenerative disorders, and as cognitive enhancers. Recent progress in developing water-soluble steroids and steroids with enhanced oral efficacy foster optimism that certain neuroactive steroids will be developed for clinical use.

The neurosteroid 5ß-pregnan-3α-ol-20-one enhances actions of etomidate as a positive allosteric modulator of α1ß2γ2L GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids potentiate responses of the GABAA receptor to the endogenous agonist GABA. Here, we examined the ability of neurosteroids to potentiate responses to the allosteric activators etomidate, pentobarbital and propofol. EXPERIMENTAL APPROACH: Electrophysiological assays were conducted on rat α1ß2γ2L GABAA receptors expressed in HEK 293 cells. The sedative activity of etomidate was studied in Xenopus tadpoles and mice. Effects of neurosteroids on etomidate-elicited inhibition of cortisol synthesis were determined in human adrenocortical cells. KEY RESULTS: The neurosteroid 5ß-pregnan-3α-ol-20-one (3α5ßP) potentiated activation of GABAA receptors by GABA and allosteric activators. Co-application of 1 µM 3α5ßP induced a leftward shift (almost 100-fold) of the whole-cell macroscopic concentration-response relationship for gating by etomidate. Co-application of 100 nM 3α5ßP reduced the EC50 for potentiation by etomidate of currents elicited by 0.5 µM GABA by about three-fold. In vivo, 3α5ßP (1mg kg(-1) ) reduced the dose of etomidate required to produce loss of righting in mice (ED50 ) by almost 10-fold. In tadpoles, the presence of 50 or 100 nM 3α5ßP shifted the EC50 for loss of righting about three- or ten-fold respectively. Exposure to 3α5ßP did not influence inhibition of cortisol synthesis by etomidate. CONCLUSIONS AND IMPLICATIONS: Potentiating neurosteroids act similarly on orthosterically and allosterically activated GABAA receptors. Co-application of neurosteroids with etomidate can significantly reduce dosage requirements for the anaesthetic, and is a potentially beneficial combination to reduce undesired side effects.

Hydrophobic anions potently and uncompetitively antagonize GABA(A) receptor function in the absence of a conventional binding site.

BACKGROUND AND PURPOSE: A 'lock-and-key' binding site typically accounts for the effect of receptor antagonists. However, sulphated neurosteroids are potent non-competitive antagonists of GABA(A) receptors without a clear structure-activity relationship. To gain new insights, we tested two structurally unrelated hydrophobic anions with superficially similar properties to sulphated neurosteroids. EXPERIMENTAL APPROACH: We used voltage-clamp techniques in Xenopus oocytes and hippocampal neurons to characterize dipicrylamine (DPA) and tetraphenylborate (TPB), compounds previously used to probe membrane structure and voltage-gated ion channel function. KEY RESULTS: Both DPA and TPB potently antagonized GABA(A) receptors. DPA exhibited an IC50 near 60 nM at half-maximal GABA concentration and antagonism with features indistinguishable from pregnenolone sulphate antagonism, including sensitivity to a point mutation in transmembrane domain 2 of the α1 subunit. Bovine serum albumin, which scavenges free membrane-associated DPA, accelerated both capacitance offset and antagonism washout. Membrane interactions and antagonism were explored using the voltage-dependent movement of DPA between membrane leaflets. Washout of DPA antagonism was strongly voltage-dependent, paralleling DPA membrane loss, although steady-state antagonism lacked voltage dependence. At antagonist concentrations, DPA failed to affect inhibitory post-synaptic current (IPSC) amplitude or decay, but DPA accelerated pharmacologically prolonged IPSCs. CONCLUSIONS AND IMPLICATIONS: Neurosteroid-like GABA(A) receptor antagonism appears to lacks a conventional binding site. These features highlight key roles of membrane interactions in antagonism. Because its membrane mobility can be controlled, DPA may be a useful probe of GABA(A) receptors, but its effects on excitability via GABA(A) receptors raise caveats for its use in monitoring neuronal activity.