Intermingled modulatory and neurotoxic effects of thimerosal and mercuric ions on electrophysiological responses to GABA and NMDA in hippocampal neurons.

The organomercurial, thimerosal, is at the center of medical controversy as a suspected factor contributing to neurodevelopmental disorders in children. Many neurotoxic effects of thimerosal have been described, but its interaction with principal excitatory and inhibitory neurotransmiter systems is not known. We examined, using electrophysiological recordings, thimerosal effects on GABA and NMDA-evoked currents in cultured hippocampal neurons. After brief (3 to 10 min) exposure to thimerosal at concentrations up to 100 µM, there was no significant effect on GABA or NMDA-evoked currents. However, following exposure for 60-90 min to 1 or 10 µM thimerosal, there was a significant decrease in NMDA-induced currents (p<0.05) and GABAergic currents (p<0.05). Thimerosal was also neurotoxic, damaging a significant proportion of neurons after 60-90 min exposure; recordings were always conducted in the healthiest looking neurons. Mercuric chloride, at concentrations 1 µM and above, was even more toxic, killing a large proportion of cells after just a few minutes of exposure. Recordings from a few sturdy cells revealed that micromolar mercuric chloride markedly potentiated the GABAergic currents (p<0.05), but reduced NMDA-evoked currents (p<0.05). The results reveal complex interactions of thimerosal and mercuric ions with the GABA(A) and NMDA receptors. Mercuric chloride act rapidly, decreasing electrophysiological responses to NMDA but enhancing responses to GABA, while thimerosal works slowly, reducing both NMDA and GABA responses. The neurotoxic effects of both mercurials are interwoven with their modulatory actions on GABA(A) and NMDA receptors, which most likely involve binding to these macromolecules.

DHEAS and POMS measures identify cocaine dependence treatment outcome.

UNLABELLED: Early attrition is a significant problem in the treatment of cocaine dependence, but it is unclear why some patients succeed in treatment while others relapse or drop out of treatment without a demonstrated relapse. The goal of this study was to determine whether baseline levels of select hormones, including the adrenal hormone and excitatory neurosteroid dehydroepiandrosterone sulfate (DHEAS), would distinguish between treatment outcome groups. Based on the literature, completion of 90 days of treatment was established as a key outcome variable. METHODS: Quantitative urine levels of the cocaine metabolite benzoylecgonine (BE) and other substance of abuse analytes, plasma levels of DHEAS, DHEA, cortisol, and prolactin, and the profile of mood states (POMS) were serially measured in 38 male cocaine-dependent (DSM-IV) patients and in 28 controls of similar gender and age over a six month study. Exclusion criteria for the patients and controls included Axis I mood, anxiety or psychotic disorders. The patients could not manifest substance dependence except to cocaine. The patients and controls received remuneration for urine and blood collection. Blood samples for hormone levels were obtained between 8 and 10 a.m. on days 1, 14 and 21 of a 21-day inpatient treatment program and throughout 6 months of outpatient study visits at 45-day intervals. RESULTS: Attrition from treatment and study appointments occurred predominately at the junction between inpatient and outpatient programs. Forty percent of patients made the transition to outpatient treatment and remained abstinent and in treatment for a median of 103 days (ABST). Forty-two percent of patients dropped out of treatment during the inpatient stay or never returned after completing the inpatient program (DO) and 18% had a documented relapse either during, or within the first week after, the inpatient stay (REL). POMS total scores were elevated at treatment entry for both the ABST and DO groups. Plasma DHEAS levels in the DO patients were decreased compared to controls and increased in the ABST patients. POMS total scores for the REL patients at baseline were at control levels. Baseline cortisol levels were not statistically different between the outcome groups, though they were elevated for all cocaine patient groups. When treatment outcome was collapsed into whether patients completed (ABST) or did not complete 90 days of treatment (90N), ABST plasma DHEAS and cortisol were significantly elevated compared to the 90N patients and controls across the first 3 weeks of cocaine withdrawal. CONCLUSIONS: At treatment entry, each of the three patient outcome groups was identified by levels of circulating DHEAS and distressed mood. In the ABST patients, distressed mood during withdrawal may have been mitigated through antidepressant-like actions of enhanced endogenous DHEAS activity, thus contributing to improved abstinence and treatment retention. Patients, such as the DO group, with high levels of distressed mood at treatment entry and low DHEAS levels may benefit from adjunctive pharmacotherapy that targets DHEAS and POMS measures. Patients, such as the REL group, who lack distressed mood at treatment entry, may require intense application of motivational approaches plus residential treatment.

Cocaine addiction as a neurological disorder: implications for treatment.

Clinical and preclinical studies provide convincing evidence for persistent neurological/psychiatric impairments and possible neuronal degeneration associated with chronic cocaine/stimulant abuse. These impairments include multifocal and global cerebral ischemia, cerebral hemorrhages, infarctions, optic neuropathy, cerebral atrophy, cognitive impairments, and mood and movement disorders. These findings may encourage the placement of stimulant addiction into the category of organic brain disorders. Functional and microanatomical anomalies in the frontal and temporal cortex as well as other brain regions may be responsible for certain aspects of phenomenology and neuropsychopathology that are characteristic of stimulant polydrug addictions. These may include broad spectrum of deficits in cognition, motivation, and insight; behavioral disinhibition; attention deficits; emotional instability; impulsiveness; aggressiveness; depression; anhedonia; and persistent movement disorders. Although it is still debated whether the hypofrontality and other brain anomalies observed in stimulant abusers are a consequence or an antecedent of drug abuse, this debate seems purely academic and irrelevant with respect to the importance of compensating for these deficits in the development of treatment strategies. The neuropsychiatric impairments accompanying stimulant abuse may contribute to the very high rate of relapse in addicts that can take place after long periods (years) of abstinence. It is possible that the neurological deficits present in stimulant addicts, whether they are primary or secondary to stimulant abuse, are responsible for perpetual drug abuse which may be a form of self-medication (Weiss et al. 1991, 1992). In this context, addiction to stimulants, once fully developed, may represent a true biological dependency on drugs that temporarily compensate for existing neurological deficits. The concept of self-medication by drug addicts is supported by major theories of biological psychiatry. While a majority of drug addicts are polydrug users, there seems to be a preference for a particular type of drug among different populations of addicts. Addicts who experience distress, anxious dysphoria, and turbulent anger prefer the calming actions of opiates, whereas addicts with preceding attention deficit disorder, depression, or bipolar disorder often prefer stimulants (Khantzian 1985). Figure 1 presents conceptual relationships between brain damage and cocaine/stimulant abuse. More clinical studies are needed to establish unequivocally the epidemiological relationships between preexisting neurological deficits-resulting either from genetic, developmental, traumatic, or neurotoxic factors- and vulnerability to drug addictions. Nonetheless, deducing from the results of preclinical studies, it is conceivable that individuals with neurological deficits associated with attention deficit disorder, developmental neuroanatomical abnormalities, lead poisoning, alcoholism, posttraumatic brain lesions, and PTSD may be more vulnerable to stimulant addiction. This notion has significant empirical support as preclinical studies have shown that animals with lesioned prefrontal cortex became supersensitive to cocaine (Schenk et al. 1991) and animals with lesions at the amygdala, VTA, or raphe nuclei manifest more rapid acquisition of amphetamine self-administration than control rats (Deminiere et al. 1989). The above arguments, postulating neuropathology as an intrinsic component of stimulant addiction, should be taken into consideration with the caveat that the clinical manifestations of the disease are heterogenous and addicts may express varying stages and degrees of the disease as determined by environmental and genetic factors. Therefore, it is likely that stimulant addicts who have less advanced neuropathology may recover spontaneously after detoxification with proper nutritional and psychotherapeutic support if they can sustain abstinence. (ABSTR

Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance.

The abundant CNS cholesterol and its sulfate derivative serve as precursors of different neurosteroids, which bidirectionally modulate neuronal excitability, by potentiating or inhibiting function of the GABAA receptors. The regulation of GABAA receptors in the CNS by the steroids of central or peripheral origin may constitute a vital means of brain-body communication, essential for integrated whole organism responses to external stimuli or internal signals. Modulation of the brain GABA receptors by neurosteroids may form the basis of a myriad of psychophysiological phenomena, such as memory, stress, anxiety, sleep, depression, seizures and others. Therefore, the aberrant synthesis of centrally-active steroids may contribute to defects in neurotransmission, resulting in a variety of neural and affective disorders. The biosynthesis of neurosteroids may also be altered by diet and certain psychotropic drugs, thereby affecting excitation of neurons. Hereditary differences in the level of synthesis and catabolism of different neurosteroids may underlie individual variations in CNS excitability, contributing to differences in personality traits, including the inherited susceptibility to drug addition.

Steroid control of uterine motility via gamma-aminobutyric acidA receptors in the rabbit: a novel mechanism?

gamma-Aminobutyric acid (GABA) is a regulator of uterine motility. Stimulation of GABAA receptors tonically inhibited contractions of rabbit uterine strips, while stimulation of GABAB receptors enhanced contractions. Steroids appeared to interact with GABAA receptors to modulate uterine contractility: tetrahydroprogesterone (THP) inhibited while pregnenolone sulphate (PS) increased contractions. THP rapidly antagonized the stimulatory effect of PS, but progesterone inhibited the contractions after a delay, suggesting that the known 'silencing' actions of progesterone on the uterus could be mediated via the metabolite THP, which potentiates the inhibitory function of GABAA receptors. This novel mechanisms may play a role in uterine function during pregnancy and parturition.

Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABAA receptor.

Recently we demonstrated that [3H]dehydroepiandrosterone sulfate binds specifically to two populations of sites in rat brain membranes [Majewska et al. (1990) Eur. J. Pharmac. 189, 307-315]. As an extension of this work, we studied the biochemical and pharmacological properties of [3H]dehydroepiandrosterone sulfate binding to brain membranes and the effects of dehydroepiandrosterone sulfate on GABA-induced currents in cultured neurons. [3H]Dehydroepiandrosterone sulfate binding depended upon incubation time, pH, protein concentration, and incubation temperature. Thermal denaturation or pretreatment of the membranes with protease or phospholipase A2 reduced the binding by 54-85%. The higher affinity [3H]dehydroepiandrosterone sulfate binding sites appeared to be associated with protein and with the GABAA receptor complex. Among substances known to interact with the GABAA receptor complex, pregnenolone sulfate, pentobarbital, and phenobarbital inhibited the binding of [3H]dehydroepiandrosterone sulfate. High micromolar concentrations of dehydroepiandrosterone sulfate inhibited [3H]muscimol and [3H]flunitrazepam binding to rat brain membranes, primarily by reducing the binding affinities. Dehydroepiandrosterone sulfate also produced a concentration-dependent block of GABA-induced currents in cultured neurons from ventral mesencephalon (IC50 = 13 +/- 3 microM). The results of this study are consistent with an action of dehydroepiandrosterone sulfate as a negative noncompetitive modulator of the GABAA receptor. Because concentrations of dehydroepiandrosterone sulfate in the brain undergo physiological variations, this neurosteroid may play a vital role in regulation of neuronal excitability in the central nervous system.

Regulation of the NMDA receptor by redox phenomena: inhibitory role of ascorbate.

Redox phenomena seem to modulate activity of the N-methyl-D-aspartate receptor. Some reductants (ascorbate, hydroquinone) inhibit, while others (dithiothreitol, mercaptoethanol, penicillamine) potentiate NMDA receptor function. Ascorbate inhibits binding of [3H]glutamate and [3H]thienylcycohexylpiperidine to the NMDA receptor complex, and impedes NMDA-gated currents in isolated neurons; dithiothreitol-like reductants enhance NMDA-induced currents. The ability of reductants to alter function of the NMDA receptor is abolished by oxidation.

Ascorbic acid protects neurons from injury induced by glutamate and NMDA.

We have previously shown that redox phenomena regulate the function of the NMDA receptor in the brain and that ascorbic acid (AA) behaves as a receptor antagonist. Here we examined the potential neuroprotective effects of AA against toxicity induced by NMDA and glutamate in rat cerebral cortical neurones in cultures. AA completely protected against injury induced by 100 microM NMDA and markedly reduced cell death induced by 500 microM NMDA or 50 microM glutamate. Dehydroascorbic acid (DHAA) did not provide significant neuroprotection. Hence, we propose that AA may serve in the CNS as a neuroprotectant.

Binding of pregnenolone sulfate to rat brain membranes suggests multiple sites of steroid action at the GABAA receptor.

The steroid pregnenolone sulfate (PS) interacts with the GABAA receptor complex in a mixed GABA-agonistic/antagonistic manner in binding experiments. However, in functional assays pregnenolone sulfate (at micromolar concentrations) behaves as an allosteric GABAA receptor antagonist, similar to the convulsant, picrotoxin. In the present work, we examined the binding of [3H] pregnenolone sulfate to membranes from rat brain. We report that this steroid binds to two or three populations of sites: (Kd1 300-500 nM, Kd2 about 20 microM and Kd3 about 200-300 microM. [3H]Pregnenolone sulfate binding is thermostable and resistant to protease digestion. Picrotoxin inhibits about 40% of 5 nM [3H]pregnenolone sulfate binding, but other GABA receptor ligands are inactive. The data suggest that [3H]pregnenolone sulfate binding sites are connected with or adjacent to the ionic channel of the GABAA receptor, but that they differ from picrotoxin recognition sites. The high and intermediate affinity pregnenolone sulfate binding sites may mediate GABA-agonistic and antagonistic actions of pregnenolone sulfate, respectively.

The neurosteroid dehydroepiandrosterone sulfate is an allosteric antagonist of the GABAA receptor.

Binding of the neurosteroid dehydroepiandrosterone sulfate (DHEAS) to rat brain synaptosomal membranes was studied in vitro, and the interaction of DHEAS with the GABAA receptor was tested using biochemical and electrophysiological assays. DHEAS bound to two populations of sites, and its binding was inhibited by barbiturates. DHEAS interfered with barbiturate-mediated enhancement of benzodiazepine binding. In cultured neurons from ventral mesencephalon, DHEAS reversibly blocked GABA-induced currents, behaving as an allosteric antagonist of the GABAA receptor.

Steroid regulation of the GABAA receptor: ligand binding, chloride transport and behaviour.

Certain endogenous steroids are modulators of GABAA receptors. Tetrahydroprogesterone (THP, 5 alpha-pregnan-3 alpha-ol-20-one) and tetrahydrodeoxy-corticosterone (THDOC, 5 alpha-pregnane-3 alpha, 21-diol-20-one) behave as allosteric agonists of GABAA receptors whereas pregnenolone sulphate acts as an antagonist. THP and THDOC modulate ligand binding to GABAA receptors like barbiturates; they potentiate binding of the GABAA receptor agonist muscimol and the benzodiazepine flunitrazepam and they allosterically inhibit binding of the convulsant t-butylbicyclophosphorothionate. THP and THDOC also stimulate chloride uptake and currents in synaptoneurosomes and neurons. Pregnenolone sulphate acts principally as an allosteric GABAA receptor antagonist; it competitively inhibits binding of [35S] TBPS and blocks GABA agonist-activated Cl- uptake and currents in synaptoneurosomes and neurons. In behavioural experiments the GABA-agonistic steroid THDOC shows anxiolytic actions whereas the GABA-antagonistic steroid pregnenolone sulphate antagonizes barbiturate-induced hypnosis. Changes in physiological levels of GABAergic steroids may alter GABAA receptor function, influencing neuronal excitability and CNS arousal. For example, pregnancy and the puerperium are associated with alterations in GABAA receptor binding which might be attributable to steroid actions.

Interactions of noncompetitive inhibitors with nicotinic receptors in the rat brain.

This study was performed to determine how drugs that inhibit the function of peripheral nicotinic receptors (noncompetitive inhibitors), interact with nicotinic receptors in the brain. By using [3H]MCC (methylcarbamylcholine ) as a ligand for nicotinic receptors, competition studies at a fixed concentration of radioligand and saturation studies were performed with various noncompetitive inhibitors. [3H]MCC labeled high affinity nicotinic receptor sites in the rat brain at equilibrium. The sites appeared to represent desensitized nicotinic receptors, comprising a fraction of the total pool of these receptors. At micromolar concentrations, noncompetitive inhibitors interacted distinctively with [3H]MCC binding sites. Mecamylamine behaved as an allosteric inhibitor, as it decreased the apparent density of [3H]MCC binding sites. Tetracaine had mixed allosteric/competitive properties, reducing both the density and the affinity of binding. Chlorpromazine manifested a biphasic effect, increasing receptor density at concentrations of approximately 50 to 500 microM and reducing the affinity at higher concentrations. The results suggest that noncompetitive inhibitors bind to different, but interacting sites associated with desensitized nicotinic receptors in the brain, as well as to recognition sites for acetylcholine.

Modulation of uterine GABAA receptors during gestation and by tetrahydroprogesterone.

Binding of a GABAA receptor agonist, [3H]muscimol, was studied in rat uterine membranes of non-pregnant rats and those at days 15 and 19 of pregnancy. Also, an interaction of the uterine GABAA receptors with tetrahydroprogesterone was examined. At day 15 of gestation [3H]muscimol binding was twice as high as in non-pregnant rats, but at day 19 it was reduced. These changes resulted from an increase of the receptor affinity and decrease of the receptor density, at days 15 and 19, respectively. Since tetrahydroprogesterone, in vitro, also increased [3H]muscimol binding, we propose that this steroid may participate in regulation of uterine function during pregnancy.

Pregnenolone sulfate antagonizes barbiturate-induced hypnosis.

The potential influence of the neurosteroid pregnenolone sulfate (PrS) on barbiturate-induced hypnosis was tested in rats. PrS, when injected intracerebroventricularly or intraperitoneally, significantly shortened the sleep-time produced by pentobarbital. The results suggest an important physiological and pharmacological role for PrS in the regulation of CNS excitability.

Divergent ontogeny of sigma and phencyclidine binding sites in the rat brain.

The postnatal developmental patterns of sigma (sigma) and phencyclidine (PCP) binding sites were compared in the rat brain. The results show diametrically different ontogenic patterns for the sites. While both the affinity and the density of sigma sites remain constant throughout the developmental period tested (postnatal day 1 to 1 year), the density of PCP binding sites increases from the time of birth, reaching the adult level by postnatal day 14. The differences in developmental patterns provide evidence for distinctive properties of cerebral sigma and PCP binding sites.

Pregnancy-induced alterations of GABAA receptor sensitivity in maternal brain: an antecedent of post-partum 'blues'?

Pregnancy increases affinity of [3H]muscimol binding to GABAA receptors in the rat forebrain. Post-partum, the receptor affinity is further increased concomitantly with a reduction of the receptor density. These changes may result from an action of endogenous placental and adrenal steroids, tetrahydroprogesterone and tetrahydrodeoxycorticosterone, which in vitro behave as allosteric agonists of GABAA receptors. The alterations may contribute to the psychological phenomena associated with pregnancy and the puerperium.

Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons.

Our earlier biochemical studies suggested that the neurosteroid pregnenolone sulfate (PS) may reduce gamma-aminobutyric acid (GABA) action at the Cl- channel associated with GABAA receptors. In the present electrophysiological study the interaction of PS with the GABAA receptor was tested, using whole-cell voltage-clamp recordings from isolated cerebral cortical neurons of neonatal rats. At micromolar concentrations PS reversibly inhibited GABA-induced current, behaving as an allosteric receptor antagonist.

Interaction of ethanol with the GABAA receptor in the rat brain: possible involvement of endogenous steroids.

The effect of ethanol on the binding of gamma-aminobutyric acid (GABA) agonist, [3H]muscimol, to crude synaptosomal membranes prepared from various rat brain regions was investigated, in vitro, at 37 degrees C. Ethanol altered specific muscimol binding in a biphasic manner--reducing it at concentrations less than 10 mM and subsequently increasing specific binding at concentrations greater than 10 mM. The former effect was due to a decrease of the receptor affinity for an agonist, and the latter, due to an increase of the receptor density. Ethanol interfered also with the effects of "GABAergic" modulatory steroids on muscimol binding. This suggests that steroid-ethanol interactions, occurring at the level of the plasma membrane, may be involved in the molecular mechanism of action of ethanol on the GABAA receptor.