Ammonia inhibits long-term potentiation via neurosteroid synthesis in hippocampal pyramidal neurons.

Neurosteroids are a class of endogenous steroids synthesized in the brain that are believed to be involved in the pathogenesis of neuropsychiatric disorders and memory impairment. Ammonia impairs long-term potentiation (LTP), a synaptic model of learning, in the hippocampus, a brain region involved in memory acquisition. Although mechanisms underlying ammonia-mediated LTP inhibition are not fully understood, we previously found that the activation of N-methyl-d-aspartate receptors (NMDARs) is important. Based on this, we hypothesize that metabolic stressors, including hyperammonemia, promote untimely NMDAR activation and result in neural adaptations that include the synthesis of allopregnanolone (alloP) and other GABA-potentiating neurosteroids that dampen neuronal activity and impair LTP and memory formation. Using an antibody against 5α-reduced neurosteroids, we found that 100 µM ammonia acutely enhanced neurosteroid immunostaining in pyramidal neurons in the CA1 region of rat hippocampal slices. The enhanced staining was blocked by finasteride, a selective inhibitor of 5α-reductase, a key enzyme required for alloP synthesis. Finasteride also overcame LTP inhibition by 100 µM ammonia, as did picrotoxin, an inhibitor of GABA-A receptors. These results indicate that GABA-enhancing neurosteroids, synthesized locally within pyramidal neurons, contribute significantly to ammonia-mediated synaptic dysfunction. These results suggest that the manipulation of neurosteroid synthesis could provide a strategy to improve cognitive function in individuals with hyperammonemia.

Functional network dysfunction in anxiety and anxiety disorders.

A recent paradigm shift in systems neuroscience is the division of the human brain into functional networks. Functional networks are collections of brain regions with strongly correlated activity both at rest and during cognitive tasks, and each network is believed to implement a different aspect of cognition. We propose here that anxiety disorders and high trait anxiety are associated with a particular pattern of functional network dysfunction: increased functioning of the cingulo-opercular and ventral attention networks as well as decreased functioning of the fronto-parietal and default mode networks. This functional network model can be used to differentiate the pathology of anxiety disorders from other psychiatric illnesses such as major depression and provides targets for novel treatment strategies.

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.

Structurally diverse amphiphiles exhibit biphasic modulation of GABAA receptors: similarities and differences with neurosteroid actions.

BACKGROUND AND PURPOSE: Some neurosteroids, notably 3alpha-hydroxysteroids, positively modulate GABA(A) receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions. EXPERIMENTAL APPROACH: Xenopus laevis oocytes were challenged with Triton X-100, octyl-beta-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations. KEY RESULTS: These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 microM) promoted potentiation and a high concentration (20 microM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (alpha1V256S) and positive (alpha1Q241L or alpha1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in alpha1V256S-mutated receptors, but potentiation was unaffected. In alpha1Q241L- or alpha1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact. CONCLUSIONS AND IMPLICATIONS: Structurally diverse amphiphiles have antagonist actions at GABA(A) receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroid-positive modulators. Thus, such antagonism at GABA(A) receptors does not have a clear pharmacophore requirement.

Glial-neuronal interactions underlying fructose utilization in rat hippocampal slices.

Although fructose is commonly used as a sweetener, its effects on brain function are unclear. Using rat hippocampal slices, we found that fructose and mannose, like pyruvate, preserve ATP levels during 3-h of glucose deprivation. Similarly, fructose and mannose restored synaptic potentials (excitatory postsynaptic potential, EPSPs) depressed during glucose deprivation. However, restoration of synaptic responses was slow and only partial with fructose. EPSPs supported by mannose were inhibited by cytochalasin B (CCB), a glucose transport inhibitor, but were not inhibited by alpha-cyano-4-hydroxycinnamate (4-CIN), a monocarboxylate transport inhibitor, indicating that neurons use mannose via glucose transporters. In contrast, both CCB and 4-CIN depressed EPSPs supported by fructose, suggesting that fructose may be taken up by non-neuronal cells through CCB sensitive hexose transporters and metabolized to a monocarboxylate for subsequent use during neuronal respiration. Supporting this possibility, 20 minutes of oxygen deprivation in the presence of fructose resulted in functional and morphological deterioration whereas oxygen deprivation in the presence of glucose or mannose had minimal toxic effects. These results indicate that neuronal fructose utilization differs from glucose and mannose and likely involves release of monocarboxylates from glia.

The immediate early gene early growth response gene 3 mediates adaptation to stress and novelty.

Stress and exploration of novel environments induce neural expression of immediate early gene transcription factors (IEG-TFs). However, as yet no IEG-TF has been shown to be required for the normal biological or behavioral responses to these stimuli. Here we show that mice deficient for the IEG-TF early growth response gene (Egr) 3, display accentuated behavioral responses to the mild stress of handling paralleled by increased release of the stress hormone corticosterone. Egr3-/- mice also display abnormal responses to novelty, including heightened reactivity to novel environments and failure to habituate to social cues or startling acoustic stimuli. In a Y-maze spontaneous alternation task, they perform fewer sequential arm entries than controls, suggesting defects in immediate memory. Because stress and novelty stimulate hippocampal long-term depression (LTD), and because abnormalities in habituation to novelty and Y-maze performance have been associated with LTD deficits, we examined this form of synaptic plasticity in Egr3-/- mice. We found that Egr3-/- mice fail to establish hippocampal LTD in response to low frequency stimulation and exhibit dysfunction of an ifenprodil-sensitive (NR1/NR2B) N-methyl-d-aspartate receptor subclass. Long term potentiation induction was not altered. The NR2B-dependent dysfunction does not result from transcriptional regulation of this subunit by Egr3, because NR2B mRNA levels did not differ in the hippocampi of Egr3-/- and control mice. These findings are the first demonstration of the requirement for an IEG-TF in mediating the response to stress and novelty, and in the establishment of LTD.

Modulation of hippocampal long-term potentiation by slow increases in ethanol concentration.

To determine how acute ethanol intoxication may alter memory processing, we examined the effects of stepwise increases in ethanol on long-term potentiation (LTP) in rat hippocampal slices. LTP was inhibited by acute administration of 60 mM ethanol, but was readily induced if ethanol was increased gradually to 60 mM over 75 min. Administration of 2-amino-5 phosphonovalerate (APV), an N-methyl-D-aspartate receptor (NMDAR) antagonist, during the stepwise increase in ethanol inhibited LTP, suggesting involvement of NMDARs in the development of tolerance. However, APV and nifedipine, an inhibitor of L-type calcium channels, failed to inhibit LTP when administered following the slow increase in ethanol. Ethanol-tolerant LTP was inhibited by thapsigargin, suggesting a major role for intracellular calcium release in this form of plasticity. The unique properties of ethanol-tolerant LTP suggest that memories formed during binge drinking are not acquired by standard synaptic mechanisms and that acute tolerance may involve the induction of novel mechanisms to maintain function.

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.

Expression of fructose sensitive glucose transporter in the brains of fructose-fed rats.

Glucose transporters play a critical role in mammalian brain energy metabolism because glucose is the principal brain energy source and these transporters promote glucose movement into neural cells. When glucose is unavailable, fructose can serve as an alternative energy source. Using real-time polymerase chain reaction and actin as a reference mRNA, we investigated the impact of fructose feeding on rat brain and other tissue mRNA expression of glucose transporter 5 which has high affinity for fructose. Brain mRNA levels of glucose transporter 5 increased 1.5-fold in 35-day old rats after 7 days of fructose feeding compared with controls, whereas it increased 2.5-fold in jejunum. Semi-quantitative analysis of protein expression by immunofluorescence of glucose transporter 5 in rat hippocampi indicated a 2.4-fold increase. We demonstrated the specificity of fructose feeding on glucose transporter 5 expression by showing that the expression of the neuronal glucose transporter 3 and insulin-regulated glucose transporter 4 were unaffected. In addition, the expression of glucose transporter 5 increased in fructose fed older adult rats (8-months and 12-months old) when compared with controls. These results suggest that short-term fructose feeding increases the expression of glucose transporter 5 in both young and aging adult rats. Increased brain expression of glucose transporter 5 is likely to be important in the role of fructose as an alternative energy source.

Acute effects of ethanol on hippocampal long-term potentiation and long-term depression are mediated by different mechanisms.

To determine potential mechanisms contributing to ethanol-induced cognitive impairment, we examined acute effects of ethanol on hippocampal N-methyl-d-aspartate receptors and forms of synaptic plasticity thought to underlie memory processing. In the CA1 region of rat hippocampal slices, ethanol partially inhibited N-methyl-d-aspartate receptor-mediated synaptic responses at concentrations up to 180 mM. The block of synaptic N-methyl-d-aspartate receptors by 60mM ethanol occluded the effects of 10 microM ifenprodil, an agent that has relative selectivity for N-methyl-D-aspartate receptors expressing NR1 and NR2B subunits. Ethanol did not occlude the effects of a low concentration of 2-amino-5-phosphonovalerate, an antagonist with less N-methyl-d-aspartate receptor subtype selectivity. Recent studies indicate that ifenprodil and other NR2B-selective antagonists inhibit N-methyl-D-aspartate receptor-dependent long-term depression but not long-term potentiation. We found that ethanol reversibly inhibited long-term depression in a manner consistent with its effects on synaptic N-methyl-D-aspartate receptors. Ethanol also inhibited the induction of N-methyl-D-aspartate receptor-dependent long-term potentiation, but the actions on long-term potentiation were complex and largely irreversible over the time course of our experiments. Furthermore, ethanol inhibited a form of long-term potentiation induced by very high frequency stimulation that does not depend on N-methyl-D-aspartate receptor activation. The effects of ethanol on both forms of long-term potentiation, but not on long-term depression, were at least partially reversed by block of GABA type A receptors with picrotoxin. These results indicate that pharmacologically relevant concentrations of ethanol exert preferential effects on a subtype of synaptic N-methyl-D-aspartate receptors in the CA1 hippocampal region. Inhibition of synaptic N-methyl-D-aspartate receptors appears to contribute strongly to ethanol-mediated long-term depression inhibition, but effects on long-term potentiation are complex, involving, at least partially, changes in GABAergic transmission.

A single day of ethanol exposure during development has persistent effects on bi-directional plasticity, N-methyl-D-aspartate receptor function and ethanol sensitivity.

To determine factors that contribute to the learning deficits observed in individuals with fetal alcohol syndrome, we examined the effects of early postnatal ethanol exposure on forms of synaptic plasticity thought to underlie memory. Treatment of rat pups with ethanol on postnatal day 7 impaired the induction of N-methyl-D-aspartate receptor-dependent long-term potentiation and abolished homosynaptic long-term depression in the CA1 region of hippocampal slices prepared at postnatal day 30. An N-methyl-D-aspartate receptor-independent form of long-term potentiation induced by very high frequency stimulation could be induced in slices from ethanol-treated rats. Defects in long-term depression correlated with a diminished contribution of ifenprodil-sensitive N-methyl-D-aspartate receptors to synaptic transmission and defects in a spontaneous alternation behavioral task. Rats exposed to ethanol on postnatal day 7 also exhibited diminished sensitivity of synaptic N-methyl-D-aspartate receptors to block by ethanol at postnatal day 30 and decreased behavioral sedation to systemic ethanol injections. These results indicate that changes in synaptic plasticity and N-methyl-D-aspartate receptor function are likely to provide a neural substrate for the cognitive and behavioral changes that follow developmental ethanol exposure.

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.

Effects of insulin on long-term potentiation in hippocampal slices from diabetic rats.

AIMS/HYPOTHESIS: Cognitive deficits occur commonly in diabetic patients. It is unclear whether these impairments result from hypoglycaemia during intensive insulin therapy, or from the diabetes itself. The aim of this study was to examine if impaired energy utilization resulting from insulin deficiency contributes to impaired long-term potentiation (reflecting impaired synaptic plasticity). As long-term potentiation is considered a candidate cellular mechanism underlying learning and memory, understanding how diabetes alters long-term potentiation may provide insight into mechanisms producing cognitive deficits in diabetes. METHODS: Electrophysiologic recordings were used to study long-term potentiation in the CA1 region of hippocampal slices from healthy rats and rats with streptozotocin-induced diabetes. RESULTS: Long-term potentiation was difficult to induce in slices from diabetic rats in standard recording buffer (contains 10 mmol/l glucose). In slices from diabetic rats, increasing extracellular glucose failed to recover long-term potentiation induction, but 10 mmol/l pyruvate added to standard buffer enabled long-term potentiation induction. Moreover, incubation of slices from diabetic rats with insulin enabled long-term potentiation induction in standard buffer. Acute administration of streptozotocin alone did not impair long-term potentiation in slices from healthy animals, and changing extracellular glucose concentrations over the range of 5 mmol/l to 30 mmol/l did not alter long-term potentiation in slices from control rats. CONCLUSIONS/INTERPRETATION: These observations suggest that impaired energy utilization from insulin deficiency, rather than the accompanying hyperglycaemia, impair long-term potentiation in diabetes. Impaired hippocampal synaptic plasticity could contribute to learning and cognitive impairment in diabetic patients.

Effects of fructose-1,6-bisphosphate on morphological and functional neuronal integrity in rat hippocampal slices during energy deprivation.

D-fructose-1,6-bisphosphate, a high energy glycolytic intermediate, attenuates ischemic damage in a variety of tissues, including brain. To determine whether D-fructose-1,6-bisphosphate serves as an alternate energy substrate in the CNS, rat hippocampal slices were treated with D-fructose-1,6-bisphosphate during glucose deprivation. Unlike pyruvate, an endproduct of glycolysis, 10 mM D-fructose-1,6-bisphosphate did not preserve synaptic transmission or morphological integrity of CA1 pyramidal neurons during glucose deprivation. Moreover, during glucose deprivation, 10-mM D-fructose-1,6-bisphosphate failed to maintain adenosine triphosphate levels in slices. D-fructose-1,6-bisphosphate, however, attenuated acute neuronal degeneration produced by 200 microM iodoacetate, an inhibitor of glycolysis downstream of D-fructose-1,6-bisphosphate. Because (5S, 10R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, an antagonist of N-methyl-D-aspartate receptors, exhibited similar protection against iodoacetate damage, we examined whether (5S, 10R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine and D-fructose-1,6-bisphosphate share a common neuroprotective mechanism. Indeed, D-fructose-1,6-bisphosphate diminished N-methyl-D-aspartate receptor-mediated synaptic responses and partially attenuated neuronal degeneration induced by 100-microM N-methyl-D-aspartate. Taken together, these results indicate that D-fructose-1,6-bisphosphate is unlikely to serve as an energy substrate in the hippocampus, and that neuroprotective effects of D-fructose-1,6-bisphosphate are mediated by mechanisms other than anaerobic energy supply.

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.

Recent developments in structure-activity relationships for steroid modulators of GABA(A) receptors.

GABAergic neurotransmission can be both positively and negatively modulated by steroids. The steroid effects are thought to be mediated by binding of steroids to specific sites on GABA(A) receptors. It appears that the receptor sites for positive and negative modulatory steroids are different. Thus far, the location and number of binding sites for steroids on these receptors have not been established. In this brief review, we concentrate largely on results from our own structure-activity studies. Novel analogues have been studied to further delineate the structural features required for compounds to modulate receptor function via steroid binding sites. Non-naturally occurring enantiomers of both positive and negative modulators have been studied to provide further evidence for the existence of specific steroid binding sites on the receptors.

Effects on gamma-aminobutyric acid (GABA)(A) receptors of a neuroactive steroid that negatively modulates glutamate neurotransmission and augments GABA neurotransmission.

Neurosteroids positively and negatively modulate gamma-aminobutyric acid (GABA)(A) receptors and glutamate receptors, which underlie most fast inhibition and excitation in the central nervous system. We report the identification of a neuroactive steroid, (3 alpha,5 beta)-20-oxo-pregnane-3-carboxylic acid (3 alpha 5 beta PC), with unique cellular actions. 3 alpha 5 beta PC positively modulates GABA(A) receptor function and negatively modulates N-methyl-D-aspartate (NMDA) receptor function, a combination that may be of particular clinical benefit. 3 alpha 5 beta PC promotes net GABA(A) potentiation at low steroid concentrations (<10 microM) and at negative membrane potentials. At higher concentrations, the steroid also blocks GABA receptors. Because this block would presumably counteract the NMDA receptor blocking actions of 3 alpha 5 beta PC, we characterize the GABA receptor block in some detail. Agonist concentration, depolarization, and high extracellular pH increase the block. The apparent pK for both potentiation and block was 6.4 to 6.9, substantially higher than expected from carboxylated steroid in an aqueous environment. Block is not dependent on the stereochemistry of the carboxylic acid at carbon 3 and is relatively insensitive to placement of the carboxylic acid at the opposite end of the steroid (carbon 24). Potentiation is critically dependent on the stereochemistry of the carboxylic acid group at carbon 3. Consistent with the pH dependence of potentiation, effects of the amide derivative (3 alpha,5 beta)-20-oxo-pregnane-3-carboxamide, suggest that the un-ionized form of 3 alpha 5 beta PC is important for potentiation, whereas the ionized form is probably responsible for block. Further refinement of the neuroactive steroid to promote GABA potentiation and NMDA receptor block and diminish GABA receptor block may lead to a clinically useful neuroactive steroid.

Ca(v)3.2 channel is a molecular substrate for inhibition of T-type calcium currents in rat sensory neurons by nitrous oxide.

Although nitrous oxide (N(2)O; laughing gas) remains widely used as an anesthetic and analgesic in clinical practice, its cellular mechanisms of action remain inadequately understood. In this report, we examined the effects of N(2)O on voltage-gated Ca(2+) channels in acutely dissociated small sensory neurons of adult rat. At subanesthetic concentrations, N(2)O blocks low-voltage-activated, T-type Ca(2+) currents (T currents), but not high-voltage-activated (HVA) currents. This blockade of T currents was concentration dependent, with an IC(50) value of 45 +/- 13%, maximal block of 38 +/- 12%, and Hill coefficient of 2.6 +/- 1.0. No desensitization of the response or change in current kinetics was observed during N(2)O application. The magnitude of T current blockade by N(2)O does not seem to reflect any use- or voltage-dependent properties. In addition, T current blockade was not altered when intracellular GTP was replaced with guanosine 5'-(gamma-thio)triphosphate or guanosine 5'-0-(2-thiodiphosphate) suggesting a lack of involvement of G-proteins in the inhibition. N(2)O selectively blocked currents arising from the Ca(v)3.2 but not Ca(v)3.1 recombinant channels stably expressed in human embryonic kidney (HEK) cells in a concentration-dependent manner with an apparent affinity and potency similar to native dorsal root ganglion currents. Analogously, the block of Ca(v)3.2 T currents exhibited little voltage- or use-dependence. These data indicate that N(2)O selectively blocks T-type but not HVA Ca(2+) currents in small sensory neurons and Ca(v)3.2 currents in HEK cells at subanesthetic concentrations. Blockade of T currents may contribute to the anesthetic and/or analgesic effects of N(2)O.

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.

Lactate dehydrogenase release is facilitated by brief sonication of rat hippocampal slices and isolated retinas following acute neuronal damage.

Although useful for determining neuronal damage in cell cultures, the lactate dehydrogenase (LDH) assay is not suitable for acute brain preparations because LDH release is typically delayed relative to neuronal deterioration. The slow release suggests that LDH may remain trapped inside damaged cells until late in the degenerative process. To test this, we examined whether brief sonication facilitates LDH release from acutely damaged neurons. In rat isolated retinas and hippocampal slices, LDH release was minimal following acute administration of iodoacetate or kainate. However, these toxins promoted significant LDH release, when toxin exposure was followed by brief sonication. Increases in extracellular LDH correlated with changes in neuronal morphology. These findings suggest that sonication may facilitate the use of the LDH assay in acute brain preparations.