Acute hydrogen sulfide poisoning. Demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels.

The possibility of measuring sulfide levels in the central nervous system (CNS) opens up many avenues for exploration. In acute hydrogen sulfide (H2S) poisoning, death results from loss of central respiratory drive. To date, however, measurement of brain sulfide has not been possible. By employing gas dialysis and ion chromatography coupled to electrochemical detection, rat brain sulfide levels could be measured either following inhalation of H2S or after injection of sodium hydrosulfide (median lethal dose, [LD50] = 14.6 +/- 1.00 mg/kg). Accumulation of brain sulfide was linearly proportional to the dose over the range 0.50 LD50 to 3.33 LD50 units, and was strongly correlated with mortality data (R = 0.947). Furthermore, analysis of untreated (control) brain showed an endogenous sulfide level of 1.57 +/- 0.04 micrograms/g (mean +/- SE; N = 16). Studies on various rat brain regions (brainstem, cerebellum, hippocampus, striatum and cortex) showed that the endogenous sulfide level of brainstem, 1.23 +/- 0.06 micrograms/g, was significantly lower than that of the other brain regions. Net uptake of sulfide was greatest in the brainstem (3.02 micrograms/g) compared to the other regions as was the selective accumulation of sulfide as calculated from normalized blood flow rates. The results of subcellular fractionation demonstrated that sulfide was detectable in fractions enriched in myelin, synaptosomes and mitochondria. Approximately one-quarter of the endogenous sulfide content of whole rat brain was found in the mitochondrial fraction. The sulfide content of these fractions increased 2- to 3-fold after 50 mg/kg NaHS, the greatest increases occurring in myelin- and mitochondrial-enriched fractions.

Sulfide-induced perturbations of the neuronal mechanisms controlling breathing in rats.

The effects of sulfide on neonatal rat respiration were studied. Two in vitro experimental models were utilized: the isolated brain stem-spinal cord preparation and the medullary slice preparation containing respiratory rhythm-generating regions from neonatal rats. Plethysmographic measurements of the effects of sulfide on the breathing patterns of unanesthetized neonatal rats were also made to compare the sensitivities of neonatal and adult rats to sulfide toxicity. In vitro, sulfide acted at sites within the ventrolateral medulla to depress the frequency of respiratory rhythmic discharge by approximately 50-60%. However, the neuronal network underlying respiratory rhythmogenesis continued to function in the presence of concentrations of sulfide far beyond those deemed to be lethal in vivo. Intraperitoneal administration of sulfide caused a dose-dependent decrease in the frequency and amplitude of breathing of neonatal rats of all ages (0-19 days postnatal), although the sensitivity to sulfide increased with age. We hypothesize that the rapid suppression of breathing caused by sulfide is due to changes in neuronal excitability within respiratory rhythm-generating centers rather than, as previously hypothesized, to perturbations of cellular oxidative metabolism.

Direct comparison of hallucinogenic phenethylamines and D-amphetamine on dorsal raphe neurons.

We compared the effects of the hallucinogens 2,5-dimethoxy-4-methylamphetamine (DOM), mescaline and the simulant D-amphetamine, applied by microiontophoresis to rat dorsal raphe (DR) units. DR neuron firing rate was relatively insensitive to DOM and unaffected by mescaline, but was clearly inhibited by D-amphetamine. Intravenous DOM usually inhibited, but this effect was correlated with blood pressure changes; i.v. D-amphetamine produced inconsistent responses. These results suggest that most of the effects seen on i.v. administration of phenethylamines are not mediated directly on the serotonergic cell.

Comparison of the effects of amphetamine and a fluorinated analogue on locomotion and exploratory activity in the mouse.

(+)-Amphetamine (AM) and its fluorinated analogue (+)-2-amino-3-fluoro-1-phenylpropane (fluoroamphetamine, FAM) were compared with regard to their effects on locomotor and exploratory activity in mice. Both drugs caused a reduction in spontaneous exploration, but this effect was more marked with FAM than with AM at 1 h after injection. Both compounds increased locomotor activity 10 min after injection, but FAM had sedative effects after 1 h, while AM continued to be stimulatory.

Quantitative estimation of potentiation and antagonism by dose ratios corrected for slopes of dose-response curves deviating from one.

A shift of dose-response curves of a receptor agonist A by a receptor antagonist B to the right is frequently expressed or quantitated by calculating the dose ratio (DR) from the ED50 values obtained in the absence and presence of B. A comparison of ED50 values or a DR is also used in a more general way to express the effects of other antagonists or of potentiators. For this situation, where B is not competing with A for a binding site, slope-values may often deviate from one. Because the slope of shifted dose-response curves (deviating from one) affects the magnitude of enhancement or diminution at a given DR, we have to take it into account. For example, the same changes in effects are associated with DR = 10 at curves with slope = 1, but with DR = 2.15 in case of slope = 3. Enhancement and diminution expressed by dose ratios is more or less underestimated in case of curves with slope > 1. We therefore propose to quantitate potentiation and antagonism by a corrected DR (DRcorr), which can simply be calculated from the uncorrected DR at a given slope. Consequently, a DRcorr reflects a true measure of enhancement or diminution for curves with slope = 1, equivalent to that which would have been observed for curves with slope = 1. The practical value of this modification is exemplified and illustrated by analysis of experimental data.

Increased levels of amino acid neurotransmitters in the inferior colliculus of the genetically epilepsy-prone rat.

Previous studies have shown an increase in the number of GABAergic and total neurons in the inferior colliculus (IC) of the genetically epilepsy-prone rat (GEPR). Amino acid analysis of the central nucleus of the IC, as well as cerebellum, sensorimotor, temporal, and occipital cerebral cortices of GEPRs with high pressure liquid chromatography showed significant increases in the levels of GABA, taurine and glutamate. The IC of GEPR displayed a 2.3-fold increase in GABA as compared to that of non-epileptic rats, a 2.4-fold increase of taurine, and a 1.9-fold increase of glutamate. In addition, taurine and glutamate were increased in the sensorimotor and temporal cortex, respectively. These results are consistent with previous anatomical data on the GABAergic system in the IC and provide additional information. The increase in taurine and glutamate in the IC indicates that other neurotransmitters could be involved in the mechanism of seizure activity.

Cerebrovascular responses to subarachnoid blood and serotonin in the monkey.

Preliminary in vitro experiments were performed to determine the serum concentration of serotonin in the monkey, and the ability of cyproheptadine to block serotonin and serum-induced contractions in monkey cerebral arteries. Thirty-four cynomolgus monkeys were subsequently used to study changes in regional cerebral blood flow (CBF) obtained by the intracartoid 133Xe technique, and in the angiographic cerebral arterial caliber resulting from subarachnoid injection of artificial cerebrospinal fluid (CSF), blood, and serotonin. Five animals in each injection group were given 1.0 mg/kg intravenous cyproheptadine (a serotonin-blocking agent) during the post-injection period. Subarachnoid injection of artificial CSF produced no change in CBF or arterial caliber. Post-injection administration of cyproheptadine also had no effect on these parameters. A subarachnoid injection of fresh autogenous blood produced a significant but transient (less than 1 hour) decrease in CBF and moderate vasospasm, which lasted at least 3 hours. This vasospasm was essentially unaffected by intravenous cyproheptadine. The CBF and arterial caliber were unchanged following a subarachnoid injection of serotonin at concentrations (5 x 10(-6)M) present in normal monkey serum. In contrast, 5 x 10(-6) M serotonin invariably produced near maximal contractions in the in vitro cerebral artery preparations. Higher (x10) serotonin concentrations caused a transient CBF response similar to that obtained with blood. However, the cerebral vasospasm induced was of shorter duration than that obtained with blood. These results do not support a major role for serotonin in the production of post-subarachnoid hemorrhage vasospasm. Moreover, our data indicate that in vitro experiments do not reflect the ability of serotonin to constrict cerebral arteries in the intact animal.

Stress-induced increases in brainstem amino acid levels are prevented by chronic sodium hydrosulfide treatment.

Neurotransmitter amino acid levels were measured in select brain regions of rats and mice after chronic treatment with sublethal doses of sodium hydrosulfide (NaHS). Brainstem aspartate, glutamate, glutamine, taurine and GABA levels increased in chronically but not acutely saline-treated rats. These increases may have been due to stress from frequent handling, and were prevented by chronic NaHS treatment (7.5 mg/kg ip every 8 hr for 3 consecutive days). In contrast, aspartate, glutamate and glutamine increased in female but not in male ICR mouse brainstems after once daily treatment with 7.0 mg/kg NaHS for 5 consecutive days. These effects of NaHS may indicate chronic low level H2S neurotoxicity. Differences between chronic and acute treatments, female and male responses, and treatment paradigms may complicate interpretations of such toxicity studies.

Hydrogen sulfide in combination with taurine or cysteic acid reversibly abolishes sodium currents in neuroblastoma cells.

Patch clamp studies of neuroblastoma cells have shown that in the presence of sodium hydrogen sulfide (NaHS; the in vitro precursor of H2S), addition of the sulfonated amino acids, taurine or cysteic acid resulted in reversible abolition of the inward sodium currents. This effect could also be demonstrated by preincubating cells for 3-20 min with 5-10 mM NaHS followed by replacement of the solution with taurine or cysteic acid in sulfide-free saline. Neither NaHS, taurine nor cysteic acid alone had any effect. The sulfhydryl reagents, beta-mercaptoethanol and dithiothreitol, were also found to abolish reversibly the sodium currents. As the effects of the above treatments were nearly identical, the synergistic action of NaHS with taurine or cysteic acid may result from reduction of the disulfide bonds between subunits comprising the sodium channel. The responses to NaHS and taurine, a putative neurotransmitter/neuromodulator, suggest that reductions in sodium channel function may be the mechanism(s) responsible for loss of central respiratory drive during H2S poisoning.

Effects of acute intoxication with hydrogen sulfide on central amino acid transmitter systems.

The acute effects of hydrogen sulfide (H2S) on brain amino acid levels were examined in five regions of the rat brain following administration of either saline (controls), or 10 or 30 mg/kg i.p. of sodium hydrosulfide (NaHS). These doses represented sublethal (0.66 x LD50) as well as lethal (2 x LD50) amounts. No significant changes in amino acid levels were found in the cerebral cortex, striatum or hippocampus. In the cerebellum, aspartate and glycine levels declined at 10 mg/kg NaHS. The region showing the greatest change was the brainstem where aspartate, glutamate, glutamine, GABA, glycine and taurine and alanine all increased. It would appear then, that acute intoxication results in substantial changes in brainstem amino acid levels. As some of these amino acids have been implicated in the neuronal control of breathing, one of the underlying causes of death following H2S may be the alteration of amino acid neurotransmitter levels and metabolism resulting in the arrest of central respiratory drive.

Effects of repeated exposures of hydrogen sulphide on rat hippocampal EEG.

Exposure to high levels of hydrogen sulphide (H2S) in humans has been associated with a number of respiratory and neurological symptoms. Acute toxicity following exposure to high concentrations is well-documented, however, there is little scientific information concerning the effects of exposure to low concentrations. The effects of low levels of H2S on electroencephalographic (EEG) activity in the hippocampus and neocortex were investigated on the freely moving rat (Sprague-Dawley). Hippocampal electrodes were implanted in the dentate gyrus (DG) and CA1 region. Activity was recorded for 10 min just prior to H2S exposure in the presence of air (pre-exposure). Rats were exposed to H2S (25, 50, 75, or 100 ppm) for 3 h/day; data was collected during the final 10 min of each exposure. The total power of hippocampal theta activity increased in a concentration-dependent manner in both DG and CA1; repeated exposures for 5 consecutive days resulted in a cumulative effect that required 2 weeks for complete recovery. The effects were found to be highly significant at all concentrations within subjects. Neocortical EEG and LIA (Large Amplitude Irregular Activity) were unaffected. The results demonstrate that repeated exposure to low levels of H2S can produce cumulative changes in hippocampal function and suggest selectivity of action of this toxicant.

Brain sulfide levels in anaesthesia: a comparison with hydrogen sulfide intoxication.

Although sublethal concentrations of hydrogen sulfide produce a state not unlike anaesthesia, measurement of rat brain sulfide levels by gas dialysis and IC with electrochemical detection after either 1.5 g/kg urethane or 42 mg/kg pentobarbital failed to demonstrate any changes as compared with endogenous brain sulfide levels of saline-injected controls. This suggests that the mechanisms underlying anaesthesia are not directly linked to endogenous cerebral sulfide levels.

Determination of sulfide in brain tissue by gas dialysis/ion chromatography: postmortem studies and two case reports.

An analytical method for the determination of sulfide in human and rat brain is described. It utilizes a continuous flow gas dialysis pretreatment and quantitation by ion chromatography with electrochemical detection. Rat brain sulfide levels were reliably measured after fatal intoxication by intraperitoneal injection of NaHS. By expeditious analysis of samples it was possible to demonstrate the presence of endogenous levels of sulfide in both rat and human brain as well as to measure elevated brain levels of sulfide after intoxication. In postmortem rat brain tissue, elevated sulfide levels could still be reliably demonstrated 96 h after death if the bodies had been refrigerated at 4 degrees C. Two case studies of human hydrogen sulfide inhalation fatalities are presented. The described method was able to measure significantly elevated sulfide levels in both cases.

Release of exogenous gamma-[3H]aminobutyric acid during seizure activity in chronically denervated and normal cat cortex.

The hypothesis that the seizure susceptibility of chronically denervated cortex is due to interruption of recurrent inhibitory pathways was tested by examining the release of 3H-labeled gamma-aminobutyric acid ([3H]GABA) from chronic slabs and normal cortex of cats. Seizure activity was maintained throughout the test periods in both normal and chronically isolated cortex. When methacholine was used to evoke seizure activity, [3H]GABA release was depressed in both normal and epileptic cortex, suggesting that the mechanism of seizure genesis by cholinomimetics involves suppression of inhibitory neuron activity. Pentylenetetrazol-induced seizures evoked a small, equal increase in [3H]GABA efflux from epileptic and normal cortex. Continuous electrical stimulation evoked a large, and again equal increase in [3H]GABA release. Preseizure efflux of [3H]GABA was the same from chronic slabs and normal cortex in all experiments. Since the interruption of recurrent inhibitory pathways by chronic denervation would result in a decreased resting and seizure-evoked release of [3H]GABA, results obtained do not support the above-mentioned hypothesis.

Lack of effect of antagonists on serotonin-induced inhibition in rat hippocampus.

Four putative central nervous system 5-hydroxytryptamine antagonists, methysergide, cyproheptadine, metergoline, and ketanserin and also lysergic acid diethylamide were applied by iontophoresis to firing CA1 hippocampal pyramidal cells to test their action on the inhibition produced by 5-hydroxytryptamine. In contrast to a previous report, none of these peripherally active 5-hydroxytryptamine antagonists altered the inhibitory response to submaximal doses of 5-hydroxytryptamine, but they did block after-excitations that followed the inhibitions. All the antagonists and lysergic acid diethylamide produced a depression of firing. When picrotoxin was used to drive the cells, 5-hydroxytryptamine was still able to produce a normal inhibition. The results of this study suggest that CA1 hippocampus is another structure, innervated by serotonergic neurones, where all (peripherally active) serotonin antagonists tested to date are ineffective against 5-hydroxytryptamine induced inhibition.