Cristina Suñol

Clozapine and haloperidol differently suppress the MK-801-increased glutamatergic and serotonergic transmission in the medial prefrontal cortex of the rat

The administration of noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine and ketamine has been shown to increase the extracellular concentration of glutamate and serotonin (5-HT) in the medial prefrontal cortex (mPFC). In the present work, we used in vivo microdialysis to examine the effects of the more potent noncompetitive NMDA receptor antagonist, MK-801, on the efflux of glutamate and 5-HT in the mPFC, and whether the MK-801-induced changes in the cortical efflux of both transmitters could be blocked by clozapine and haloperidol given systemically or intra-mPFC. The systemic, but not the local administration of MK-801, induced an increased efflux of 5-HT and glutamate, which suggests that the NMDA receptors responsible for these effects are located outside the mPFC, possibly in GABAergic neurons that tonically inhibit glutamatergic inputs to the mPFC. The MK-801-induced increases of extracellular glutamate and 5-HT were dependent on nerve impulse and the activation of mPFC AMPA/kainate receptors as they were blocked by tetrodotoxin and NBQX, respectively. Clozapine and haloperidol blocked the MK-801-induced increase in glutamate, whereas only clozapine was able to block the increased efflux of 5-HT. The local effects of clozapine and haloperidol paralleled those observed after systemic administration, which emphasizes the relevance of the mPFC as a site of action of these antipsychotic drugs in offsetting the neurochemical effects of MK-801. The ability of clozapine to block excessive cortical 5-HT efflux elicited by MK-801 might be related to the superior efficacy of this drug in treating negative/cognitive symptoms of schizophrenia.

Probucol Increases Glutathione Peroxidase-1 Activity and Displays Long-Lasting Protection against Methylmercury Toxicity in Cerebellar Granule Cells

Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here, we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase, isozyme 1 (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen peroxide or tert-butyl peroxide and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake, and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated with increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants, ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (1) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (2) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides.

The organochlorine pesticides γ-hexachlorocyclohexane (lindane), α-endosulfan and dieldrin differentially interact with GABAA 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.

Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons

Thymol is a naturally occurring phenolic monoterpene known for its anti-microbial and anti-oxidant properties. It is used in dental practice and in anaesthetic halothane preparations. Recent studies have reported enhanced GABA(A) receptor-operated chloride channel activity and increased binding affinity of [(3)H]flunitrazepam in the presence of thymol. In the present work, we more closely examined the pharmacological action of thymol on the native GABA(A) receptor by using primary cultures of cortical neurons. Thymol enhanced GABA-induced (5 microM) chloride influx at concentrations lower than those exhibiting direct activity in the absence of GABA (EC(50) = 12 microM and 135 microM, respectively). This direct effect was inhibited by competitive and non-competitive GABA(A) receptor antagonists. Thymol increased [(3)H]flunitrazepam binding (EC(50) = 131 microM) and showed a tendency to increase [(3)H]muscimol binding. These results confirm that thymol is a positive allosteric modulator of the GABA(A) receptor. The thymol structural analogues menthol and cymene, which lack an aromatic ring or a hydroxyl group, did not affect [(3)H]flunitrazepam binding. Using a pharmacophoric model that includes a hydrogen bond donor group as well as an aromatic ring with two aliphatic substituents, we propose to demonstrate the molecular essential features of these compounds to interact with GABA(A) receptors. Thymol (0-1 mM) did not affect cellular viability.

Impulsivity Characterization in the Roman High- and Low-Avoidance Rat Strains: Behavioral and Neurochemical Differences

The selective breeding of Roman high- (RHA) and low-avoidance (RLA) rats for rapid vs extremely poor acquisition of active avoidance behavior in a shuttle-box has generated two phenotypes with different emotional and motivational profiles. The phenotypic traits of the Roman rat lines/strains (outbred or inbred, respectively) include differences in sensation/novelty seeking, anxiety/fearfulness, stress responsivity, and susceptibility to addictive substances. We designed this study to characterize differences between the inbred RHA-I and RLA-I strains in the impulsivity trait by evaluating different aspects of the multifaceted nature of impulsive behaviors using two different models of impulsivity, the delay-discounting task and five-choice serial reaction time (5-CSRT) task. Previously, rats were evaluated on a schedule-induced polydipsia (SIP) task that has been suggested as a model of obsessive-compulsive disorder. RHA-I rats showed an increased acquisition of the SIP task, higher choice impulsivity in the delay-discounting task, and poor inhibitory control as shown by increased premature responses in the 5-CSRT task. Therefore, RHA-I rats manifested an increased impulsivity phenotype compared with RLA-I rats. Moreover, these differences in impulsivity were associated with basal neurochemical differences in striatum and nucleus accumbens monoamines found between the two strains. These findings characterize the Roman rat strains as a valid model for studying the different aspects of impulsive behavior and for analyzing the mechanisms involved in individual predisposition to impulsivity and its related psychopathologies.

Relationship between lindane concentration in blood and brain and convulsant response in rats after oral or intraperitoneal administration

The relationships between brain and blood concentrations of lindane and its convulsant effects have been studied in rats after different po and ip doses. A good correlation was observed between dose and the frequency and time to onset of tonic seizures, the intensity of the response and lethality. An oral ED50 (84 mg/kg) and an ip ED50 (131 mg/kg) for the half-maximal incidence rate of lindane-induced tonic seizures was determined. The incidence of response was directly proportional to the log of lindane concentration in brain and blood, with an EC50 of 5.3 μg/g and 1.5 μg/ml, respectively. The threshold concentration of lindane eliciting tonic seizures was estimated to be ∼ 5 μg/g in brain and ∼ 1.5 μg/ml in blood. The kinetics of lindane in brain and blood were examined after a single 60 mg/kg po or ip dose over a period from 5 min to 1 week. Concentrations of lindane in brain and blood were found to be highly correlated.

Induction of GABAergic phenotype in a neural stem cell line for transplantation in an excitotoxic model of Huntington's disease

The implementation of cell replacement therapies for Huntingtons disease using multipotent neural stem cells (NSCs) requires the specific differentiation into gamma-aminobutyric acid (GABA) neuronal subtype before transplantation. Here we present an efficient culture procedure that induces stable GABAergic neurons from the immortalized striatal neural stem cell line ST14A. This process requires sequential retinoic acid treatment and KCl depolarization. Initial addition of 10 microM retinoic acid increased cell survival and promoted neuronal differentiation. Subsequent stimulation with 40 mM KCl induced specific differentiation into GABAergic neurons, yielding 74% of total cultured cells. KCl-evoked Ca(2+) influx reduced cell proliferation and nestin expression, and induced neurite outgrowth and GABAergic markers as well as GABA contents, release, and uptake. Characterization of the integration, survival, and phenotype of these predifferentiated GABAergic neurons following transplantation into the adult brain in a model of Huntingtons disease revealed long-term survival in quinolinate-lesioned striata. Under these conditions, cells maintained their GABAergic phenotype and elaborated neurite processes with synaptic contacts with endogenous neurons. In conclusion, we have generated a homogeneous population of functional GABAergic neurons from a neural stem cell line, which survive and maintain their acquired fate in vivo. These data may lend support to the possibility of cell replacement therapies for Huntingtons disease using neural stem cells.

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 µm) and H2O2 (50 µm), 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.

Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels

Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl– channels in neuronal excitotoxicity using either N‐methyl‐d‐aspartic acid (NMDA) or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K+ concentrations ([K+]o) for 5 min. Under these conditions, a Ca2+‐dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 µm, cell viability was significantly reduced by 30–40%. The NMDA receptor antagonists (MK‐801 and d‐2‐amino‐5‐phosphonopentanoic acid) prevented cell death. Exposure to high [K+]o produced a 36Cl– influx which was significantly reduced by picrotoxinin. In addition, the GABAA receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K+]o‐triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl– channel blockers niflumic acid and 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K+]o‐induced 36Cl– influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K+]o. Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K+]o exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABAA receptors and niflumic acid‐sensitive Cl– channels.

Physical exercise improves synaptic dysfunction and recovers the loss of survival factors in 3xTg-AD mouse brain.

Physical exercise has become a potentially beneficial therapy for reducing neurodegeneration symptoms in Alzheimers disease. Previous studies have shown that cognitive deterioration, anxiety and the startle response observed in 7-month-old 3xTg-AD mice were ameliorated after 6 months of free access to a running wheel. Also, alterations in synaptic response to paired-pulse stimulation were improved. The present study further investigated some molecular mechanisms underlying the beneficial effects of 6 months of voluntary exercise on synaptic plasticity in 7-month-old 3xTg-AD mice. Changes in binding parameters of [(3)H]-flunitrazepam to GABAA receptor and of [(3)H]-MK-801 to NMDA receptor in cerebral cortex of 3xTgAD mice were restored by voluntary exercise. In addition, reduced expression levels of NMDA receptor NR2B subunit were reestablished. The synaptic proteins synaptophysin and PSD-95 and the neuroprotective proteins GDNF and SIRT1 were downregulated in 3xTgAD mice and were recovered by exercise treatment. Overall, in this paper we highlight the fact that different interrelated mechanisms are involved in the beneficial effects of exercise on synaptic plasticity alterations in the 3xTg-AD mouse model.