Involvement of superoxide in malaoxon-induced toxicity in primary cultures of cortical neurons.

Organophosphorus compounds (OP) represent a class of insecticides that are used most globally. The neurotoxic effects are attributed mainly to acetylcholinesterase (AChE) enzyme inhibition, which is responsible for cholinergic manifestations in individuals acutely exposed to OP. However, AChE inhibition alone cannot account for the wide range of symptoms that were reported following OP exposures. In agreement with this, evidence shows that non-cholinergic events may be mechanistically linked to OP-induced neurotoxicity. The aim of this study was to investigate the potential occurrence of oxidative stress as a critical step in the toxicity induced by the OP malaoxon(MAL) using primary cultures of mouse cortical neurons, as well as to distinguish MAL-induced oxidative stress and cell toxicity from an action on AChE blockade. Primary cultures of mouse cortical neurons were treated with MAL (0.01; 0.1; 1; 10; or 100 µM) at varying time points (1, 3, 6, 24, 48, or 144 hr) and the following biochemical parameters determined including cell viability, AChE activity, and superoxide production. MAL significantly reduced cell viability in a concentration- and time-dependent manner. Of note, 1 µM MAL significantly inhibited (approximately 75%) AChE activity after 48 hr incubation. Pralidoxime (PRAL) (600 µM), a classical AChE reactivator, significantly protected against MAL-induced AChE blockade; however, PRAL did not affect MAL-mediated fall in cellular viability, indicating that AChE inhibition is not necessarily correlated with insecticide-induced decrease in cell survival. MAL-induced diminished cell viability was preceded by a significant increase in superoxide anion production. The antioxidant agent ascorbic acid (AA) (200 µM), which significantly protected against MAL-induced superoxide anion production, did not alter MAL-induced AChE inhibition and significantly prevented insecticide-mediated fall in cell survival. Data show that increased superoxide anion production is an event that precedes MAL-induced cell toxicity in primary cultures of mouse cortical neurons. Based on the preventative effects of AA against MAL-mediated superoxide anion production and reduced cell viability, evidence indicates that oxidative stress represents an important step mediating MAL-induced toxicity in neurons and that AChE inhibition is not necessarily correlated with lowered cell survival noted in insecticide-exposed cells.

Tryptophan depletion affects compulsive behaviour in rats: strain dependent effects and associated neuromechanisms.

RATIONALE: Compulsive behaviour, present in different psychiatric disorders, such as obsessive-compulsive disorder, schizophrenia and drug abuse, is associated with altered levels of monoamines, particularly serotonin (5-hydroxytryptamine) and its receptor system. OBJECTIVES: The present study investigated whether 5-HT manipulation, through a tryptophan (TRP) depletion by diet in Wistar and Lister Hooded rats, modulates compulsive drinking in schedule-induced polydipsia (SIP) and locomotor activity in the open-field test. The levels of dopamine, noradrenaline, serotonin and its metabolite were evaluated, as well as the 5-HT2A and 5-HT1A receptor binding, in different brain regions. METHODS: Wistar rats were selected as high (HD) or low (LD) drinkers according to their SIP behaviour, while Lister hooded rats did not show SIP acquisition. Both strains were fed for 14 days with either a TRP-free diet (T-) or a TRP-supplemented diet (T+) RESULTS: The TRP depletion diet effectively reduced 5-HT levels in the frontal cortex, amygdala and hippocampus in both strains of rats. The TRP-depleted HD Wistar rats were more sensitive to 5-HT manipulation, exhibiting more licks on SIP than did the non-depleted HD Wistar rats, while the LD Wistar and the Lister Hooded rats did not exhibit differences in SIP. In contrast, the TRP-depleted Lister Hooded rats increased locomotor activity compared to the non-depleted rats, while no differences were found in the Wistar rats. Serotonin 2A receptor binding in the striatum was significantly reduced in the TRP-depleted HD Wistar rats. CONCLUSIONS: These results suggest that alterations of the serotonergic system could be involved in compulsive behaviour in vulnerable populations.

Coatings of nanostructured pristine graphene-IrOx hybrids for neural electrodes: Layered stacking and the role of non-oxygenated graphene.

The need to enhance charge capacity in neural stimulation-electrodes is promoting the formation of new materials and coatings. Among all the possible types of graphene, pristine graphene prepared by graphite electrochemical exfoliation, is used in this work to form a new nanostructured IrOx-graphene hybrid (IrOx-eG). Graphene is stabilized in suspension by IrOx nanoparticles without surfactants. Anodic electrodeposition results in coatings with much smaller roughness than IrOx-graphene oxide. Exfoliated pristine graphene (eG), does not electrodeposit in absence of iridium, but IrOx-nanoparticle adhesion on graphene flakes drives the process. IrOx-eG has a significantly different electronic state than graphene oxide, and different coordination for carbon. Electron diffraction shows the reflection features expected for graphene. IrOx 1-2 nm cluster/nanoparticles are oxohydroxo-species and adhere to 10nm graphene platelets. eG induces charge storage capacity values five times larger than in pure IrOx, and if calculated per carbon atom, this enhancement is one order magnitude larger than the induced by graphene oxide. IrOx-eG coatings show optimal in vitro neural cell viability and function as cell culture substrates. The fully straightforward electrochemical exfoliation and electrodeposition constitutes a step towards the application of graphene in biomedical systems, expanding the knowledge of pristine graphene vs. graphene oxide, in bioelectrodes.

Nanocomposites of iridium oxide and conducting polymers as electroactive phases in biological media.

Much effort is currently devoted to implementing new materials in electrodes that will be used in the central nervous system, either for functional electrostimulation or for tests on nerve regeneration. Their main aim is to improve the charge capacity of the electrodes, while preventing damaging secondary reactions, such as peroxide formation, occurring while applying the electric field. Thus, hybrids may represent a new generation of materials. Two novel hybrid materials are synthesized using three known biocompatible materials tested in the neural system: polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT) and iridium oxide (IrO2). In particular, PPy-IrO2 and PEDOT-IrO2 hybrid nanocomposite materials are prepared by chemical polymerization in hydrothermal conditions, using IrO2 as oxidizing agent. The reaction yields a significant ordered new hybrid where the conducting polymer is formed around the IrO2 nanoparticles, encapsulating them. Scanning electron microscopy and backscattering techniques show the extent of the encapsulation. Both X-ray photoelectron and Fourier transform infrared spectroscopies identify the components of the phases, as well as the absence of impurities. Electrochemical properties of the final phases in powder and pellet form are evaluated by cyclic voltammetry. Biocompatibility is tested with MTT toxicity tests using primary cultures of cortical neurons grown in vitro for 6 and 9days.

GABAergic pharmacological activity of propofol related compounds as possible enhancers of general anesthetics and interaction with membranes.

Phenol compounds, such as propofol and thymol, have been shown to act on the GABAA receptor through interaction with specific sites of this receptor. In addition, considering the high lipophilicity of phenols, it is possible that their pharmacological activity may also be the result of the interaction of phenol molecules with the surrounding lipid molecules, modulating the supramolecular organization of the receptor environment. Thus, in the present study, we study the pharmacological activity of some propofol- and thymol-related phenols on the native GABAA receptor using primary cultures of cortical neurons and investigate the effects of these compounds on the micro viscosity of artificial membranes by means of fluorescence anisotropy. The phenol compounds analyzed in this article are carvacrol, chlorothymol, and eugenol. All compounds were able to enhance the binding of [(3)H]flunitrazepam with EC50 values in the micromolar range and to increase the GABA-evoked Cl(-) influx in a concentration-dependent manner, both effects being inhibited by the competitive GABAA antagonist bicuculline. These results strongly suggest that the phenols studied are positive allosteric modulators of this receptor. Chlorothymol showed a bell-type effect, reducing its positive effect at concentrations >100 µM. The concentrations necessary to induce positive allosteric modulation of GABAA receptor were not cytotoxic. Although all compounds were able to decrease the micro viscosity of artificial membranes, chlorothymol displayed a larger effect which could explain its effects on [(3)H]flunitrazepam binding and on cell viability at high concentrations. Finally, it is suggested that these compounds may exert depressant activity on the central nervous system and potentiate the effects of general anesthetics.

Mechanisms of manganese-induced neurotoxicity in primary neuronal cultures: the role of manganese speciation and cell type.

Manganese (Mn) is an essential trace element required for the proper functioning of a variety of physiological processes. However, chronic exposures to Mn can cause neurotoxicity in humans, especially when it occurs during critical stages of the central nervous system development. The mechanisms mediating this phenomenon as well as the contribution of Mn speciation and the sensitivity of different types of neuronal cells in such toxicity are poorly understood. This study was aimed to investigate the mechanisms mediating the toxic effects of MnCl(2), Mn(II) citrate, Mn(III) citrate, and Mn(III) pyrophosphate in primary cultures of neocortical (CTX) and cerebellar granular (CGC) neurons. Cell viability, mitochondrial function, and glutathione levels were evaluated after Mn exposure. CGC were significantly more susceptible to Mn-induced toxicity when compared with CTX. Moreover, undifferentiated CGC were more vulnerable to Mn toxicity than mature neurons. Mitochondrial dysfunction was observed after the exposure to all the tested Mn species. Ascorbate protected CGC against Mn-induced neurotoxicity, and this event seemed to be related to the dual role of ascorbate in neurons, acting as antioxidant and metabolic energetic supplier. CTX were protected from Mn-induced toxicity by ascorbate only when coincubated with lactate. These findings reinforce and extend the notion of the hazardous effects of Mn toward neuronal cells. In addition, the present results indicate that Mn-induced neurotoxicity is influenced by brain cell types, their origins, and developmental stages as well as by the chemical speciation of Mn, thus providing important information about Mn-induced developmental neurotoxicity and its risk assessment.

A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Studies with neuronal cells: From basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity.

Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity.

The organochlorine pesticides gamma-hexachlorocyclohexane (lindane), alpha-endosulfan and dieldrin differentially interact with GABA(A) and glycine-gated chloride channels in primary cultures of cerebellar granule cells.

The neurotoxic organochlorine pesticides gamma-hexachlorocyclohexane, alpha-endosulfan and dieldrin induce in mammals a hyperexcitability syndrome accompanied by convulsions. They reduce the GABA-induced Cl(-) flux. The strychnine-sensitive glycine receptor also regulates Cl(-)-flux inhibitory responses. We studied the effects of these compounds on Cl(-) channels associated with glycine receptors in cultured cerebellar granule cells in comparison to the GABA(A) receptor. Both GABA (EC(50): 5 microM) and glycine (EC(50): 68 microM) increased (36)Cl(-) influx. This increase was antagonized by bicuculline and strychnine, respectively. Lindane inhibited with similar potency both GABA(A) (IC(50): 6.1 microM) and glycine (5.0 microM) receptors. alpha-Endosulfan and dieldrin inhibited the GABA(A) receptor (IC(50) values: 0.4 microM and 0.2 microM, respectively) more potently than the glycine receptor (IC(50) values: 3.5 microM and 3 microM, respectively). Picrotoxinin also inhibited the glycine receptor, although with low potency (IC(50)>100 microM). A 3D pharmacophore model, consisting of five hydrophobic regions and one hydrogen bond acceptor site in a specific three-dimensional arrangement, was developed for these compounds by computational modelling. We propose that the hydrogen bond acceptor moiety and the hydrophobic region were responsible for the affinity of these compounds at the GABA(A) receptor whereas only the hydrophobic region of the molecules was responsible for their interaction with the glycine receptors. In summary, these compounds could produce neuronal hyperexcitability by blocking glycine receptors besides the GABA(A) receptor. We propose that two zones of the polychlorocycloalkane pesticide molecules (a lipophilic zone and a polar zone) differentially contribute to their binding to GABA(A) and glycine receptors.

Translocation of apoptosis-inducing factor in cerebellar granule cells exposed to neurotoxic agents inducing oxidative stress.

We have previously shown that the neurotoxic compounds colchicine, methylmercury (MeHg) and hydrogen peroxide (H2O2) cause apoptosis in primary cultures of cerebellar granule cells (CGC), characterized by nuclear condensation and high-molecular weight DNA fragmentation. However, only colchicine triggers the activation of caspases, suggesting that factors other than caspase-activated DNase (CAD) are responsible for DNA cleavage in the other two models. Here we report that the two agents that cause oxidative stress, MeHg (1 micro m) and H2O2 (50 micro m), induce translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus in CGC. Our data suggest that, in absence of caspase activity, AIF translocation could be a key event leading to chromatin condensation and DNA degradation in CGC exposed to MeHg and H2O2.

Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.

Pharmacological characterization of the effects of methylmercury and mercuric chloride on spontaneous noradrenaline release from rat hippocampal slices.

The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.

Trimethyltin and triethyltin differentially induce spontaneous noradrenaline release from rat hippocampal slices.

The environmental contaminants trimethyltin (TMT) and triethyltin (TET) stimulated the spontaneous release of [(3)H]noradrenaline ([(3)H]NA) from hippocampal slices in a time- and concentration-dependent manner. TMT was the most potent compound, exhibiting an EC50 value 10-fold lower (3.8 microM) than that of TET (39.5 microM). Metal-evoked [(3)H]NA release did not increase in the absence of desipramine and was completely blocked by reserpine preincubation, indicating a vesicular origin of [(3)H]NA release but not a mechanism involving reversal of the transmitter transporter. The voltage-gated Na(+) channel blocker tetrodotoxin (TTX) did not affect metal-evoked [(3)H]NA release. [(3)H]NA release elicited by TMT was partially extracellular Ca(2+)-dependent, since it was significantly decreased in a Ca(2+)-free EGTA-containing medium, whereas TET induced an extracellular Ca(2+)-independent release of [(3)H]NA. Neither inhibitors of Ca(2+)-entry through Na(+)/Ca(2+)exchanger and voltage-gated calcium channels, nor agents that interfere with Ca(2+)-mobilization from intracellular stores affected [(3)H]NA release induced by TMT. TET-evoked [(3)H]NA release was reduced by ruthenium red, which depletes mitochondrial Ca(2+)stores, but was not modified by caffeine and thapsigargin, which interfere with Ca(2+)mobilization from endoplasmic reticulum. The fact that TET effect was also attenuated by DIDS, an inhibitor of anion exchange, indicates that the effect of TET on spontaneous [(3)H]NA release may be mediated by intracellular mobilization of Ca(2+) from mitochondrial stores through a Cl(-) dependent mechanism.

Effects of the conformationally restricted GABA analogues, cis- and trans-4-aminocrotonic acid, on GABA neurotransmission in primary neuronal cultures.

The effects of the GABA analogues, cis- and trans-4-aminocrotonic acid (ACA) on GABA(A) receptor function and GABA uptake, together with the presence of p-1 subunit mRNA and putative GABAc receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl- influx, which was inhibited by bicuculline, t-butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and gamma-hexachlorocyclohexane (gamma-HCH or lindane). [3H]-flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the transisomer completely inhibited the [3H]GABA uptake, whereas the cis-isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABAc receptors was investigated only in neocortical cultures by using RT-PCR in order to detect the presence of the mRNA encoding the p-1 subunit which assembles to form homooligomeric Cl-channels. The results presented here show that p-1 subunits, and thus GABAc receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA(A) receptors, although with very different potencies.

Polychlorocycloalkane insecticide action on GABA-and glycine-dependent chloride flux.

The inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine directly cause an increase in conductance to Cl- by binding to ligand-operated ion channel receptors at the postsynaptic membranes, so that opening of Cl- channels usually leads to a net hyperpolarization. The GABA(A) receptor has separate but allosterically interacting binding sites for GABA, benzodiazepines, barbiturates, anesthetic steroids and the convulsant picrotoxinin. The GABA(C) receptor also forms a Cl- channel, however its pharmacology differs from that of the GABA(A) receptor. Neurotoxic organochlorine pesticides belonging to the group of polychlorocycloalkanes (cyclodienes and gamma-hexachlorocyclohexane or lindane) induce in mammals an hyperexcitability syndrome that can progress until the production of tonic-clonic convulsions. They act as non-competitive GABA antagonists interacting with the picrotoxinin site both in membranes and in intact cultured neurons, thereby inhibiting the GABA-induced Cl- flux following activation of either GABA(A) or GABA(C) receptors. We also report the effects of polychlorocycloalkanes on glycine-induced 36Cl- flux in primary neuronal cultures. The delta isomer of hexachlorocyclohexane is a depressant compound, that increases the GABA-induced Cl- flux and allosterically increases benzodiazepine binding at the GABA(A) receptor. We discuss the mechanism of action of these compounds in relation to the disruption of ligand-operated Cl- channel receptors and the relevance of their convulsant/depressant actions.

Behavioral and monoaminergic changes after lindane exposure in developing rats.

The effects of lindane on behavior and central monoaminergic systems were studied in rat pups at 15 days of postnatal age. Pups were previously given nonconvulsant lindane PO doses, either a single 20 mg/kg or 7-day repeated 10 mg/kg doses. Both treatment schedules improved the passive avoidance acquisition but only the acute administration prolonged the step-through latency. Acute lindane decreased the motor activity, whereas the repeated dosing increased it. Increases of the ratio 5-HIAA/serotonin in several brain regions and of the ratio DOPAC/dopamine in the mesencephalon after a single dose of lindane suggest an enhanced monoaminergic turnover. In contrast, repeated lindane doses decreased monoamine/metabolite ratios excluding the striatum, where an increase of DOPAC/dopamine ratio correlates with the higher motor activity of these animals. It is postulated that both the imbalance of the central monoaminergic systems and the lindane-induced GABAergic blockade may be the basis of the behavioral alterations.

Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABA(A) receptors.

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (y-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 microM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA(A) receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 microM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA(A) antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA(A) receptor. When 100 microM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 microM lindane. The GABA(B) receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA(A) receptor.

Cytotoxic action of lindane in cerebellar granule neurons is mediated by interaction with inducible GABA(B) receptors.

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (gamma-HCH, lindane) was studied in cultured mouse cerebellar granule neurons maintained in the presence or absence of the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). The cells were exposed for 24 hr to lindane (30-300 microM) in the culture medium. Changes in mitochondrial function were investigated by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The results showed that lindane-induced cytotoxicity was concentration-dependent. In cerebellar granule cells not treated with THIP, lindane-induced cytotoxicity did not appear to be related to GABA(A) or GABA(B) receptors. However, in THIP-treated cultures, lindane-induced cytotoxicity was found to be mediated by an action of the insecticide on GABA receptors. In the latter case, GABA reduced the lindane-induced cytotoxicity, but the protective effect was not potentiated by flunitrazepam. The GABA(A) receptor agonist muscimol (50 microM) also protected the THIP-treated cultures against lindane-induced cytotoxicity. In addition, the GABA(B) receptor agonist R(+)baclofen protected the cells from lindane-induced cytotoxicity and the effect of baclofen was blocked by GABA(B) receptor antagonists. Pertussis toxin was found to reverse the protective effect of baclofen only at the highest lindane concentration (300 microM). The lindane-induced cytotoxicity could be partly explained as being secondary to excitotoxicity as a mixture of the excitatory amino acid receptor antagonists APV (D-(-)-2-amino-5-phosphonopentanoate) and CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) shifted the concentration-response curve for lindane-induced cytotoxicity to the right. It is suggested that the cytotoxic effects of lindane in THIP-treated cerebellar granule neurons are primarily related to an action of lindane on GABA(B) receptors and to a lesser extent on inducible low-affinity, benzodiazepine insensitive GABA(A) receptors.

Antidepressant drugs inhibit a glial 5-hydroxytryptamine transporter in rat brain.

We assessed the role of glial cells in the uptake of serotonin (5-hydroxytryptamine, 5-HT). Primary cultures of rat and mouse cortical astrocytes took up and deaminated 5-HT. The antidepressants citalopram, clomipramine, fluoxetine, fluvoxamine, paroxetine and sertraline inhibited this process. The presence of the mRNAs for the 5-HT transporter and monoamine oxidase-A (MOA-A) was established in cultured astrocytes and in adult rat brain areas with (midbrain and brainstem) and without (frontal cortex) serotonergic cell bodies after reverse transcription-polymerase chain reaction and hybridization with probes complementary to the cloned neuronal 5-HT transporter and MAO-A. To examine in vivo the role of astrocytes in the elimination of 5-HT from the extracellular brain space, 5-HT was perfused through dialysis probes implanted in the frontal cortex of conscious rats and its concentration was measured at the probe outlet. Tissue 5-HT recovery was dose-dependently inhibited by the concurrent perfusion of citalopram, fluoxetine and paroxetine, showing that it essentially measured uptake through the high-affinity 5-HT transporter. Rats lesioned with 5,7-dihydroxytryptamine (5,7-DHT; 88% reduction of tissue 5-HT) displayed tissue 5-HT recovery slightly higher than sham-operated rats (55 +/- 2 vs. 46 +/- 3%, P < 0.001), a finding perhaps attributable to the astrogliosis induced by 5,7-DHT denervation. Rats lesioned with 6-hydroxydopamine showed tissue 5-HT uptake similar to controls, suggesting negligible reuptake of 5-HT by catecholaminergic terminals. These results are consistent with the presence of a glial component of 5-HT uptake in the rodent brain, sensitive to antidepressants, which takes place through a 5-HT transporter very similar or identical to that present in neurons.