RESUMEN

One week oral flurazepam (FZP) administration in rats results in reduced GABA(A) receptor-mediated synaptic transmission in CA1 pyramidal neurons associated with benzodiazepine tolerance in vivo and in vitro. Since voltage-gated calcium channel (VGCC) current density is enhanced twofold during chronic FZP treatment, the role of L-type VGCCs in regulating benzodiazepine-induced changes in CA1 neuron GABA(A) receptor-mediated function was evaluated. Nimodipine (10 mg/kg, i.p.) or vehicle (0.5% Tween 80, 2 ml/kg) was injected 1 day after ending FZP treatment and 24 h prior to hippocampal slice preparation for measurement of mIPSC characteristics and in vitro tolerance to zolpidem. The reduction in GABA(A) receptor-mediated mIPSC amplitude and estimated unitary channel conductance measured 2 days after drug removal was no longer observed following prior nimodipine injection. However, the single nimodipine injection failed to prevent in vitro tolerance to zolpidem's ability to prolong mIPSC decay in FZP-treated neurons, suggesting multiple mechanisms may be involved in regulating GABA(A) receptor-mediated synaptic transmission following chronic FZP administration. As reported previously in recombinant receptors, nimodipine inhibited synaptic GABA(A) receptor currents only at high concentrations (>30 muM), significantly greater than attained in vivo (1 muM) 45 min after a single antagonist injection. Thus, the effects of nimodipine were unlikely to be related to direct effects on GABA(A) receptors. As with nimodipine injection, buffering intracellular free [Ca(2+)] with BAPTA similarly prevented the effects on GABA(A) receptor-mediated synaptic transmission, suggesting intracellular Ca(2+) homeostasis is important to maintain GABA(A) receptor function. The findings further support a role for activation of L-type VGCCs, and perhaps other Ca(2+)-mediated signaling pathways, in the modulation of GABA(A) receptor synaptic function following chronic benzodiazepine administration, independent of modulation of the allosteric interactions between benzodiazepine and GABA binding sites.

Asunto(s)

Benzodiazepinas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Nimodipina/farmacología , Terminales Presinápticos/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Flurazepam/farmacología , Antagonistas del GABA/farmacología , Moduladores del GABA/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Masculino , Nimodipina/metabolismo , Técnicas de Placa-Clamp , Piridinas/farmacología , Ratas , Zolpidem

RESUMEN

Excitatory and inhibitory ionotropic receptors are regulated by protein kinases and phosphatases, which are localized to specific subcellular locations by one of several anchoring proteins. One of these is the A-kinase anchoring protein (AKAP150), which confers spatial specificity to protein kinase A and protein phosphatase 2B in the rat brain. The distribution of AKAP150 was examined at rat hippocampal CA1 pyramidal cell asymmetric and symmetric post-synaptic densities and with respect to the distribution of markers of excitatory (vesicular glutamate transporter 1, glutamate receptor subunit 1) and inhibitory receptors (vesicular GABA transporter, GABA receptor type A beta2/3 subunits, gephyrin) and the Golgi marker, trans-Golgi network glycoprotein 38. AKAP150 was close to asymmetric synapses, consistent with numerous molecular and biochemical studies suggesting its interaction with components of the excitatory postsynaptic density. In contrast, we did not find AKAP150-immunoreactivity associated with inhibitory synapses in rat CA1 neurons, despite reports demonstrating an in vitro interaction between AKAP150 and GABA receptor type A receptor beta subunits, and the reported co-localization of these proteins in rat hippocampal cultures. There was some overlap between AKAP150 and GABA receptor type A receptor beta2/3-immunoreactivity intracellularly in perinuclear clusters. These findings support previous work indicating the integration of kinase and phosphatase activity at excitatory synapses by AKAP150, but do not support a role for selective targeting of AKAP150 and its accompanying proteins to inhibitory synapses.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hipocampo/citología , Células Piramidales/metabolismo , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Anclaje a la Quinasa A , Animales , Proteínas Portadoras/metabolismo , Inmunohistoquímica/métodos , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica/métodos , Células Piramidales/citología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de GABA-A/metabolismo , Fracciones Subcelulares/metabolismo , Sinapsis/ultraestructura , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo

RESUMEN

One-week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAA receptor (GABAR) subunit protein and miniature inhibitory post-synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1-week FZP treatment, there were significant decreases in basal (- 30%) and total (- 25%) PKA activity, and a 40% reduction in PKA RIIbeta protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole-cell recording in vitro showed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP-treated rats responded to 8-bromo-cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP-treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.

Asunto(s)

Ansiolíticos/administración & dosificación , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Flurazepam/administración & dosificación , Células Piramidales/efectos de los fármacos , Receptores de GABA-A/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Fraccionamiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Esquema de Medicación , Activación Enzimática/efectos de los fármacos , Hipocampo/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Isoenzimas/análisis , Isoenzimas/genética , Isoenzimas/fisiología , Masculino , Técnicas de Placa-Clamp , Células Piramidales/química , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Péptido Intestinal Vasoactivo/farmacología

RESUMEN

Rats are tolerant to benzodiazepine (BZ) anticonvulsant actions two days after ending one-week administration of the BZ, flurazepam (FZP). Concurrently, GABA(A) receptor-mediated inhibition is reduced and AMPA receptor-mediated excitation is selectively enhanced in CA1 pyramidal neurons in hippocampal slices. In the present study, the effects of chronic FZP exposure on NMDA receptor (NMDAR) currents were examined in CA1 pyramidal neurons in hippocampal slices and following acute dissociation. In CA1 neurons from chronic FZP-treated rats, evoked NMDAR EPSC amplitude was significantly decreased (52%) in slices, and the maximal current amplitude of NMDA-induced currents in dissociated neurons was also significantly reduced (58%). Evoked NMDAR EPSCs were not altered following acute desalkyl-FZP treatment. Using in situ hybridization and immunohistochemical techniques, a selective reduction in NR2B subunit mRNA and protein expression was detected in the CA1 and CA2 regions following FZP treatment. However, total hippocampal NMDAR number, as assessed by autoradiography with the NMDAR antagonist, [(3)H]MK-801, was unchanged by FZP treatment. These findings suggest that reduced NMDAR-mediated currents associated with chronic BZ treatment may be related to reduced NR2B subunit-containing NMDARs in the CA1 and CA2 regions. Altered NMDAR function and expression after chronic BZ exposure may contribute to BZ anticonvulsant tolerance or dependence.

Asunto(s)

Benzodiazepinas/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Autorradiografía , Maleato de Dizocilpina/farmacología , Electrofisiología , Potenciales Evocados/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Flunitrazepam/farmacología , Moduladores del GABA/farmacología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Técnicas In Vitro , Indicadores y Reactivos , Masculino , N-Metilaspartato/farmacología , Células Piramidales/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biosíntesis , Transmisión Sináptica/efectos de los fármacos

RESUMEN

Benzodiazepines are used to treat the anxiety associated with cocaine withdrawal, as well as cocaine-induced seizures. Since cocaine exposure was shown to affect BZ binding density, abuse liability, subjective hypnotic actions and seizure susceptibility, we assessed whether chronic cocaine alters diazepam's anxiolytic and anticonvulsant actions. Changes in GABA(A) receptor subunit protein expression were also assessed as they may relate to BZ activity at the receptor. Male Sprague-Dawley rats were injected with cocaine-HCl (15 mg/kg, i.p.) or saline once daily for 14 days. One day after the last injection, DZP (1 mg/kg i.p.) significantly increased time spent on and entries into open arms of an elevated plus maze in both saline- and cocaine-treated groups, yet the effect was greater in cocaine-treated rats. Eight days after cessation of treatment DZP did not have a significant anxiolytic effect in either group. To assess the effect of cocaine on DZP's anticonvulsant actions, PTZ was infused at a constant rate via the lateral tail vein and clonus onset was recorded in the presence and absence of DZP (5 mg/kg, i.p). DZP significantly elevated seizure threshold in both groups of rats. Chronic cocaine also had no effect on the beta-CCM seizure threshold. Quantitative immunohistochemistry of GABA(A) receptor subunit protein demonstrated significant regulation of alpha2 (-10%) and beta3 (+9%) subunits in the hippocampal dentate gyrus and CA1 regions, respectively. Small changes in GABAR subunit expression in specific brain areas may relate to DZP's enhanced anxiolytic effectiveness whereas it's anticonvulsant actions likely remain intact following cocaine administration.

Asunto(s)

Ansiolíticos/uso terapéutico , Anticonvulsivantes/uso terapéutico , Ansiedad/tratamiento farmacológico , Cocaína/farmacología , Diazepam/uso terapéutico , Inhibidores de Captación de Dopamina/farmacología , Receptores de GABA-A/efectos de los fármacos , Convulsiones/tratamiento farmacológico , Animales , Convulsivantes , Interacciones Farmacológicas , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Pentilenotetrazol , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Convulsiones/inducido químicamente

RESUMEN

Chronic flurazepam treatment substantially impairs the function of GABAergic synapses on hippocampal CA1 pyramidal cells. Previous findings included a significant decrease in the synaptic and unitary conductance of CA1 pyramidal neuron GABA(A) receptor channels and the appearance of a GABA(A)-receptor mediated depolarizing potential. To investigate the ionic basis of the decreased conductance, whole-cell voltage-clamp techniques were used to record evoked, GABA(A) receptor-mediated IPSCs carried by HCO(3)(-)-Cl(-) or Cl(-) alone. Hippocampal slices were prepared from rats administered flurazepam orally for 1 week, 2 days after ending drug treatment. Slices were superfused with HCO(3)(-)-aCSF or with HEPES-aCSF (without HCO(3)(-)) plus 50 microM APV and 10 microM DNQX. The micropipette contained 130 mM CsCl and 1 microM QX-314. GABA(A) receptors located on pyramidal cell somata or dendrites were activated monosynaptically by maximal stimulation of GABAergic terminals at the stratum oriens-pyramidale (SO-SP) or stratum lacunosum-molecular (S-L-M) border, respectively. In HCO(3)(-)-aCSF, there was a significant reduction in synaptic-conductance in flurazepam-treated neurons following both SO-SP (control: 1058 pS, flurazepam: 226 pS, P<0.01) and S-L-M (control 998 pS, flurazepam: 179 pS, P<0.01) stimulation, as well as the total charge transfer, indicating a decreased HCO(3)(-)-Cl(-) flux. In HEPES-aCSF, the synaptic conductance and total charge transfer, and thus Cl(-) flux, was unchanged in flurazepam-treated neurons (SO-SP: control 588 pS, flurazepam: 580 pS, P>0.05; S-L-M: control 595 pS, flurazepam: 527 pS, P>0.05). Taken together, these findings suggest that a reduction in HCO(3)(-) flux may play a prominent role in mediating the action of GABA and that a loss of HCO(3)(-) conductance may significantly contribute to impaired GABA(A) receptor function after chronic benzodiazepine treatment.

Asunto(s)

Benzodiazepinas/farmacología , Bicarbonatos/metabolismo , Tolerancia a Medicamentos/fisiología , Hipocampo/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Bicarbonatos/farmacología , Líquido Cefalorraquídeo/metabolismo , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Estimulación Eléctrica , HEPES/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Células Piramidales/citología , Células Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/fisiología

RESUMEN

One week oral flurazepam (FZP) administration in rats results in anticonvulsant tolerance in vivo, tolerance measured in vitro in hippocampal CA1 pyramidal cells, and regulation of hippocampal gamma-aminobutyric acid(A)-receptor subunit protein expression. A single injection (4 or 20 mg/kg i.p) of the benzodiazepine antagonist flumazenil (FLM) was given 1 day after FZP treatment, and tolerance and subunit protein expression were evaluated 1 day later. In vivo tolerance was measured by a reduced ability of the alpha(1)-subunit-selective agonist zolpidem to suppress pentylenetetrazole-induced seizures. This tolerance was reversed by 20 but not 4 mg/kg FLM. In in vitro hippocampal slices, there was tolerance to the effect of zolpidem to prolong the decay of pyramidal cell miniature inhibitory postsynaptic currents, which was reversed by FLM (4 mg/kg) pretreatment. A reduction in miniature inhibitory postsynaptic current amplitude ( approximately 50%) was also restored by FLM injection. [(3)H]Zolpidem binding measured 0, 2, and 7 days after FZP treatment was significantly decreased in the hippocampus and cortex at 0 days but not thereafter. Changes in alpha(1)- and beta(3)-subunit protein expression were examined via quantitative immunohistochemical techniques. alpha(1)-Subunit protein levels were down-regulated in the CA1 stratum oriens and beta subunit levels were up-regulated in the stratum oriens and stratum radiatum of the CA3 region. Chronic FZP effects on alpha(1)- and beta(3)-subunit protein levels were also reversed by prior FLM injection. FLM's effect on both functional and structural correlates of benzodiazepine tolerance suggests that each of these measures plays an interdependent role in mediating benzodiazepine tolerance.

Asunto(s)

Anticonvulsivantes/farmacología , Benzodiazepinas/farmacología , Antagonistas de Receptores de GABA-A , Hipocampo/fisiología , Animales , Ansiolíticos/farmacología , Autorradiografía , Conducta Animal/efectos de los fármacos , Convulsivantes/farmacología , Tolerancia a Medicamentos , Electrofisiología , Flumazenil/farmacología , Flurazepam/farmacología , Moduladores del GABA/farmacología , Hipocampo/efectos de los fármacos , Hipnóticos y Sedantes/metabolismo , Hipnóticos y Sedantes/farmacología , Inmunohistoquímica , Masculino , Técnicas de Placa-Clamp , Pentilenotetrazol/antagonistas & inhibidores , Pentilenotetrazol/farmacología , Piridinas/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Convulsiones/fisiopatología , Zolpidem

RESUMEN

Prolonged flurazepam exposure regulates the expression of selected (alpha1, beta2, beta3) GABA(A) receptor subunit messenger RNAs in specific regions of the hippocampus and cortex with a time-course consistent with benzodiazepine tolerance both in vivo and in vitro. In this report, the immunostaining density of six specific GABA(A) receptor subunit (alpha1, beta2, beta1-3 and gamma2) antibodies was measured in the hippocampus and cortex, among other brain areas, in slide-mounted brain sections from flurazepam-treated and control rats using quantitative computer-assisted image analysis techniques. In parallel with the localized reduction in alpha1 and beta3 subunit messenger RNA expression detected in a previous study, relative alpha1 and beta3 subunit antibody immunostaining density was significantly decreased in flurazepam-treated rat hippocampal CA1, CA3 and dentate dendritic regions, and in specific cortical layers. Quantitative western blot analysis showed that beta3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in beta3 subunit immunostaining (16-21%), were reduced to a similar degree (18%). The latter findings provide independent support that relative immunostaining density may provide an accurate estimate of protein levels. Consistent with the absence of the regulation of their respective messenger RNAs immediately after ending flurazepam administration, no changes in the density of alpha2, beta1 or beta2 subunit antibody immunostaining were found in any brain region. gamma2 subunit antibody staining was changed only in the dentate molecular layer. The selective changes in GABA(A) receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABA(A) receptors involving specific subunits (alpha1 and beta3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance.

Asunto(s)

Benzodiazepinas/farmacología , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Receptores de GABA-A/biosíntesis , Animales , Ansiolíticos/farmacología , Corteza Cerebral/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Flurazepam/farmacología , Hipocampo/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley

RESUMEN

The effect of prolonged benzodiazepine administration on GABA(A) receptor subunit (alpha1-6, beta1-3, gamma2) messenger RNAs was investigated in the rat hippocampus and cortex, among other brain areas. Rats were orally administered flurazepam for one week, a protocol which results in benzodiazepine anticonvulsant tolerance in vivo, and in the hippocampus in vitro, in the absence of behavioral signs of withdrawal. Autoradiographs of brain sections, hybridized with [35S]oligoprobes in situ, were examined immediately (day 0) or two days after drug treatment, when rats were tolerant, or seven days after treatment, when tolerance had reversed, and were compared to sections from pair-handled, vehicle-treated controls. Alpha1 subunit messenger RNA level was significantly decreased in CA1 pyramidal cells and dentate granule cells at day 0, an effect which persisted only in CA1 neurons. Decreased "alpha1-specific" silver grain density over a subclass of interneurons at the pyramidal cell border suggested concomitant regulation of interneuron GABA(A) receptors. A reduction in beta3 subunit messenger RNA levels was more widespread among hippocampal cell groups (CA1, CA2, CA3 and dentate gyrus), immediately and two days after treatment, and was also detected in the frontal and parieto-occipital cortices. Changes in beta2 subunit messenger RNA levels in CA1, CA3 and dentate gyrus cells two days after ending flurazepam treatment suggested a concomitant up-regulation of beta2 messenger RNA. There was a trend toward an increased level of alpha5, beta3 and gamma2 subunit messenger RNAs in CA1, CA3 and dentate gyrus cells, which was significant for the beta3 and gamma2 subunit messenger RNAs in the frontal cortex seven days after ending flurazepam treatment. There were no flurazepam treatment-induced changes in any other GABA(A) receptor subunit messenger RNAs. The messenger RNA levels of three (alpha1, beta2 and beta3) of nine GABA(A) receptor subunits were discretely regulated as a function of time after ending one-week flurazepam treatment related to the presence of anticonvulsant tolerance, but not dependence. The findings suggested that a localized switch in the subunit composition of GABA(A) receptor subtypes involving these specific subunits may represent a minimal requirement for the changes in GABA(A) receptor-mediated function recorded previously at hippocampal CA1 GABAergic synapses, associated with benzodiazepine anticonvulsant tolerance.

Asunto(s)

Química Encefálica/efectos de los fármacos , Flurazepam/farmacología , Moduladores del GABA/farmacología , ARN Mensajero/biosíntesis , Receptores de GABA-A/biosíntesis , Animales , Autorradiografía , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Sondas de Oligonucleótidos , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Factores de Tiempo

RESUMEN

CA1 pyramidal cells were voltage clamped, and GABA was applied to individual cells with a modified U-tube, rapid drug application system. With Vh = -50 mV, inward currents elicited by 10 microM GABA were inhibited by GABAA receptor (GABAR) antagonists and were baclofen insensitive, suggesting that GABA actions on isolated CA1 pyramidal cells were GABAR mediated. GABA concentration-response curves averaged from all cells were fitted best with a two-site equation, indicating the presence of at least two GABA binding sites, a higher-affinity site (EC50-1 = 11.0 microM) and a lower-affinity site (EC50-2 = 334.2 microM), on two or more populations of cells. The effects of GABAR allosteric modulators on peak concentration-dependent GABAR currents were complex and included monophasic (loreclezole) or multiphasic (diazepam) enhancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inhibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhancement of GABAR currents by diazepam suggested three different sites on GABARs (EC50-1 =1.8 nM; EC50-2 = 75.8 nM; EC50-3 = 275.9 nM) revealing GABAR heterogeneity. The imidazopyridine zolpidem enhanced GABAR currents in 70% of cells with an EC50 = 222.5 nM, suggesting a predominance of moderate affinity alpha2 (or alpha3-) subtype-containing BZ Type IIA receptors. A small fraction of cells (10%) had a high affinity for zolpidem, something that is suggestive of alpha1 subtype-containing BZ Type I receptors. The remaining 30% of cells were insensitive to or inhibited by zolpidem, suggesting the presence of alpha5 subtype-containing BZ Type IIB receptors. Whether BZ Type I and Type II receptors coexist could not be determined. The beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibited GABAR currents in all cells at midnanomolar concentrations, but in addition, potentiated GABAR currents in some cells at low nanomolar concentrations, characterizing two groups of cells, the latter likely due to functional assembly of alpha5betaxgamma2GABARs. In all cells, GABAR currents were moderately sensitive (EC50 = 9 microM) to loreclezole, consistent with a relatively greater beta3 subtype, than beta1 subtype, subunit mRNA expression. Two populations of cells were identified based on their sensitivities to zinc(IC50 = 28 and 182 microM), suggesting the presence of at least two GABAR isoforms including alpha5beta3gamma2 GABARs. Consistent with the heterogeneity of expression of GABAR subunit mRNA and protein in the hippocampus and based on their differential responses to GABA and to allosteric modulators, distinct populations of CA1 pyramidal cells likely express multiple, functional GABAR isoforms.

Asunto(s)

Hipocampo/citología , Células Piramidales/química , Células Piramidales/fisiología , Receptores de GABA-A/fisiología , Regulación Alostérica , Animales , Anticonvulsivantes/farmacología , Carbolinas/farmacología , Convulsivantes/farmacología , Diazepam/farmacología , Relación Dosis-Respuesta a Droga , Moduladores del GABA/farmacología , Hipnóticos y Sedantes/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/química , Triazoles/farmacología , Zinc/farmacología , Zolpidem , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Modulation of GABA function following 1 week oral administration of flurazepam (FZP) was investigated in chloride-loaded, rat hippocampal CA1 pyramidal neurons. Rats were sacrificed 2 or 7 days after ending drug treatment, when anticonvulsant tolerance was present or absent in vivo, respectively. Spontaneous (s)IPSCs and miniature (m)IPSCs were recorded using whole-cell voltage-clamp techniques. s/mIPSCs were bicuculline-sensitive, voltage-dependent, and reversed their polarity at 0 mV, the predicted E(Cl-). Comparisons of s/mIPSCs between FZP-treated and control groups were made at Vh = -90, -70, and -50 mV. The frequency of sIPSCs, but not mIPSCs, was significantly decreased in FZP-treated neurons 2 days, but not 7 days, after FZP treatment, suggesting a decrease in interneuron activity. These conclusions were supported by the negative findings of additional studies of [3H]GABA release from hippocampal slices and [3H]GABA uptake from hippocampal synaptosomes. The lack of change in the paired-pulse depression of GABA(B)-mediated IPSPs suggested that autoreceptor function was also not impaired following chronic FZP treatment. A large reduction in both sIPSC and mIPSC amplitude (60%) in FZP-treated neurons, the absence of mIPSCs in one-third of FZP-treated cells, and a measurable reduction in synaptic and unitary conductance confirmed that postsynaptic GABA(A) receptor function was profoundly impaired in FZP-treated CA1 neurons. Zolpidem, an alpha1-selective benzodiazepine receptor ligand, enhanced mIPSC amplitude and decay, but its ability to prolong mIPSC decay was reduced in FZP-treated neurons. Several pre- and postsynaptic changes at GABAergic synapses on CA1 pyramidal cells might be related to the decreased tonic GABA inhibition in FZP-treated CA1 neurons associated with the expression of benzodiazepine anticonvulsant tolerance.

Asunto(s)

Ansiolíticos/farmacología , Flurazepam/farmacología , Moduladores del GABA/farmacología , Hipocampo/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Sinapsis/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Animales , Tolerancia a Medicamentos , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Hipocampo/metabolismo , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Conducción Nerviosa/efectos de los fármacos , Técnicas de Placa-Clamp , Células Piramidales/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismo , Zolpidem , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

GABAergic synaptic responses were studied by direct, monosynaptic activation of GABAergic interneurons in the CA1 region of in vitro hippocampal slices from rats made tolerant to the benzodiazepine, flurazepam. Monosynaptic IPSPs were elicited in CA1 pyramidal neurons, following 1 week oral flurazepam administration, by electrical stimulation at the stratum oriens/stratum pyramidale or stratum radiatum/ stratum-lacanosum border < or = 0.5 mm from the recording electrode plane. Excitatory input to pyramidal cells and interneurons was eliminated by prior superfusion of the glutamate receptor antagonists, APV (50 microM) and DNQX (10 microM). GABAA receptor-mediated early IPSPs were further isolated by perfusion of the GABAB antagonist, CGP 35348 (25 microM) or by diffusion of Cs- from the recording electrode. GABAB receptor-mediated late IPSPs were pharmacologically isolated by perfusion of the GABAA antagonist, picrotoxin (50 microM). There was a significant decrease in the amplitude of pharmacologically isolated early and late IPSPs in FZP-treated neurons without a change in passive membrane properties. A shift of the early IPSP, but not the late IPSP, reversal potential in FZP-treated neurons suggested that a change in the driving force for anions, presumably Cl, in CA1 neurons was one important factor related to the decreased early IPSP amplitude after prolonged activation of GABAA receptors by flurazepam. A decreased early IPSP amplitude accompanied by a decreased late IPSP amplitude suggested that presynaptic GABA release onto FZP-treated pyramidal cells may also be reduced. We conclude from these data that an impairment of GABAergic transmission in CA1 pyramidal neurons associated with the development of tolerance during chronic benzodiazepine treatment may be related to the regulation of both pre- and postsynaptic mechanisms at the GABA synapse.

Asunto(s)

Benzodiazepinas/farmacología , Hipocampo/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Benzodiazepinas/administración & dosificación , Masculino , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

We used several approaches to assess the reliability and sensitivity of computer-assisted densitometry to detect regional changes in tissue antigen content as a function of immunohistochemical staining density. We designed a model system to mimic variations in antigen concentration in postfixed, slide-mounted rat brain sections by varying the ratios of conjugated (biotinylated) to unconjugated secondary antibody. Antigen concentration was also varied in tissue discs made from mixing rat brain homogenate with increasing amounts of tissue embedding compound. The monoclonal antibody bd-17 to the beta2/3 subunit of the GABAA receptor was used as the primary antibody. Immunostaining density was visualized with diaminobenzidine (DAB). There was a significant, positive linear relationship (r = 0.97-0.99) between immunostaining intensity and antigen concentration. With this approach, changes in antigen content of less than 10%, as reflected in immunostaining intensity, were detectable in brain sections. The low degree of variability in measures of regional variation in immunostaining in sections from naive rats (n = 7) suggested that the method was suitable for quantitative analysis and indicated the reliability of the method. This systematic study of the utility of computer-assisted image analysis for semiquantitative immunohistochemical analysis found the method to be both reliable and sensitive.

Asunto(s)

Encéfalo/metabolismo , Densitometría/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Proteínas/metabolismo , Animales , Antígenos/metabolismo , Biotina/metabolismo , Encéfalo/inmunología , Masculino , Proteínas/inmunología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/inmunología , Sensibilidad y Especificidad

RESUMEN

The cDNAs encoding alpha 5 and gamma 2L subunit subtypes of the gamma-aminobutyric acid (GABA) type A receptor (GABAR) were transfected into L929 cells together with cDNAs encoding either the beta 1, beta 2, or beta 3 subunit subtype. Properties of expressed recombinant alpha 5 beta X gamma 2L (where X = 1,2, or 3) GABARs were studied with the use of whole-cell, patch-clamp techniques. In cells voltage-clamped at -70 mV with equlvalent bath and pipette chloride concentrations, the application of GABA produced a concentration-dependent inward chloride current with all three alpha 5 beta X gamma 2L isoforms. Minimal or no responses were recorded from cells transfected with only two subunit cDNAs, demonstrating that all three subunits were required for functional receptor assembly in these cells. The GABA concentration producing a half-maximal current was similar for beta 2 and beta 3 subtype-containing receptors (6 microM) but higher for beta 1 subtype-containing receptors (26 microM). alpha 5 beta 3 gamma 2L receptors were zinc and diazepam sensitive but zolpidem insensitive. In response to low GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed outward rectification of the current-voltage relationship, whereas current-voltage responses of beta 2 subtype-containing receptors were relatively linear. Likewise, at high GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed less desensitization at positive than at negative membrane potentials. Beta 2 subtype-containing receptors displayed faster desensitization at depolarized potentials. These voltage-dependent properties were characteristic of alpha 5 but not alpha 1 or alpha 6 subtype-containing receptors and were similar to responses recorded from hippocampal CA1 pyramidal neurons. Based on the pharmacological and biophysical similarities to hippocampal GABAR responses, the alpha 5 beta 3 gamma 2L isoform could represent a native GABAR subtype.

Asunto(s)

Neuronas/fisiología , Células Piramidales/fisiología , Receptores de GABA-A/química , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/farmacología , Animales , Benzodiazepinas/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Clonación Molecular , ADN Complementario , Diazepam/farmacología , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Células L , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/efectos de los fármacos , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transfección , Zinc/farmacología , Zolpidem

RESUMEN

The relative ability of derivatives of 2-piperidinecarboxylic acid (2-PC; pipecolic acid) and 3-piperidinecarboxylic acid (3-PC; nipecotic acid) to block maximal electroshock (MES)-induced seizures, elevate the threshold for electroshock-induced seizures and be neurotoxic in mice was investigated. Protective index (PI) values, based on the MES test and rotorod performance, ranged from 1.3 to 4.5 for 2-PC benzylamides and from < 1 to > 7.2 for 3-PC derivatives. PI values based on elevation of threshold for electroshock-induced seizures and rotorod performance ranged from > 1.6 to > 20 for both types of derivatives. Since preliminary data indicated that benzylamide derivatives of 2-PC displace [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine (TCP) binding to the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor in the micromolar range and such low affinity uncompetitive antagonists of the NMDA receptor-associated ionophore have been shown to be effective anticonvulsants with low neurological toxicity, the 2-PC derivatives were evaluated in rat brain homogenates for binding affinity to the PCP site. Although all compounds inhibited [3H]TCP binding, a clear correlation between pharmacological activity and binding affinity was not apparent. Select compounds demonstrated minimal ability to protect against pentylenetetrazol-, 4-aminopyridine- and NMDA-induced seizures in mice. Corneal and amygdala kindled rats exhibited different sensitivities to both valproic acid and the nonsubstituted 2-PC benzylamide, suggesting a difference in these two models. Enantiomers of the alpha-methyl substituted benzylamide of 2-PC showed some ability to reduce seizure severity in amygdala kindled rats.

Asunto(s)

Anticonvulsivantes/farmacología , Encéfalo/metabolismo , Ácidos Nipecóticos/farmacología , Ácidos Pipecólicos/farmacología , Prolina/análogos & derivados , Convulsiones/fisiopatología , 4-Aminopiridina , Amígdala del Cerebelo/fisiología , Animales , Convulsivantes , Excitación Neurológica , Masculino , Ratones , Ratones Endogámicos , Estructura Molecular , N-Metilaspartato , Neurotoxinas/farmacología , Pentilenotetrazol , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de Fenciclidina/efectos de los fármacos , Receptores de Fenciclidina/metabolismo , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Relación Estructura-Actividad , Ácido Valproico/farmacología

RESUMEN

Oral administration of the benzodiazepine, flurazepam, for one week results in tolerance in vivo and in vitro and in a reduction in recurrent and feedforward inhibition in vitro in the CA1 pyramidal cell region of hippocampus. In the present study CA1 pyramidal cells were examined intracellularly in vitro in rat hippocampal slices (500 microns) from rats sacrificed two or seven days after cessation of oral flurazepam treatment. Following drug treatment, the membrane characteristics of CA1 pyramidal cells were not significantly different from control neurons. GABAA-mediated, early inhibitory postsynaptic potentials were significantly reduced in amplitude (60%) in pyramidal neurons from rats killed two days, but not in those killed seven days, after the end of drug administration. The decrease in early inhibitory postsynaptic potential amplitude was observed using just-subthreshold, threshold and supramaximal orthodromic stimulation as well as following antidromic activation. The magnitude of the decrease in the early inhibitory postsynaptic potential amplitude was similar in the presence of the GABAB antagonist, CGP 35348, and could not be attributed to differences in the strength of afferent stimulation between flurazepam-treated and control groups. The size of the GABAB-mediated, late inhibitory postsynaptic potentials was also significantly decreased (45%) in comparison to control cells. Reversal potentials for both the early (-72 mV) and late (-92 mV) hyperpolarizations were not significantly different between groups. Following high intensity orthodromic stimulation, in the presence of an intracellular sodium channel blocker (QX-314) which also blocks the GABAB-mediated late hyperpolarization, a bicuculline-sensitive late depolarizing potential was unmasked in neurons from FZP-treated rats, but never from control cells. Excitatory postsynaptic potential amplitude was significantly increased in flurazepam-treated neurons and the threshold for the synaptically-evoked action potential was significantly increased. Following depolarizing current injection, the duration and frequency of pyramidal cell discharges and the action potential threshold were not altered by oral flurazepam treatment. The amplitude of the fast afterhyperpolarization was also not changed. Overall, the findings indicate an impairment of transmission at GABAergic synapses onto hippocampal CA1 pyramidal cell neurons after chronic benzodiazepine treatment at a time when rats are tolerant to the anticonvulsant effects of the benzodiazepines in vivo.

Asunto(s)

Flurazepam/farmacología , Hipocampo/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Sinapsis/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Acetatos/farmacología , Ácido Acético , Anestésicos Locales/farmacología , Animales , Depresión Química , Estimulación Eléctrica , Electrofisiología , Potenciales Evocados/efectos de los fármacos , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-B , Hipocampo/citología , Técnicas In Vitro , Lidocaína/análogos & derivados , Lidocaína/farmacología , Masculino , Compuestos Organofosforados/farmacología , Ratas , Ratas Sprague-Dawley , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo

RESUMEN

The strength of feedforward inhibition was assessed in the CA1 region of in vitro hippocampus of benzodiazepine tolerant rats 2 and 7 days after 1 week oral flurazepam (FZP) administration. Feedforward inhibition was activated with a Schaffer collateral stimulation just-subthreshold for a population spike followed at intervals of 10-100 ms by test population spikes elicited from the subicular side of the recording electrode. The amplitude of test spike was reduced to a lesser degree 2 days, but not 7 days, after FZP treatment, suggesting a decrease feedforward inhibition in treated rats coinciding with the time course for reversal of anticonvulsant tolerance in vivo.

Asunto(s)

Retroalimentación/efectos de los fármacos , Flurazepam/farmacología , Hipocampo/fisiología , Animales , Dendritas/efectos de los fármacos , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Masculino , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/fisiología , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/efectos de los fármacos

RESUMEN

This report continues the in-depth evaluation of methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate , 1 (ADD 196022), and methyl 4-(benzylamino)-6-methyl-2-oxocyclohex-3-en-1-oate, 2, two potent anticonvulsant enaminones. These compounds were evaluated employing the amygdala kindling model. Neither 1 nor 2 was active against amygdala kindled seizures, further supporting the corneal kindled model as a definitive tool for antielectroshock seizure evaluation as previously reported. Additional intraperitoneal (ip) data on 1 revealed toxicity at 24 h at 100 mg/kg. Several active analogs have been prepared with the view to minimizing toxicity. In a special ip rat screen developed by the Antiepileptic Drug Development (ADD) Program, these newer analogs were evaluated for protection against maximal electroshock seizures (MES) at 10 mg/kg and neurotoxicity at 100 mg/kg. From this screen, several compounds were shown to be safer alternatives, the most notable was methyl 4-[(p-bromophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate, 13. Compound 13 had an ip ED50 of 4 mg/kg in the rat and a TD50 of 269 mg/kg, providing a protective index (TD50/ED50) of > 67. By variation in the ring size, additional aromatic substitutions and the synthesis of acyclic analogs, these newer compounds provide a more definitive insight into the structure-activity correlation. CLOGP evaluation and molecular modeling studies are also provided to further elaborate the molecular characteristics of potential anticonvulsant enaminones.

Asunto(s)

Anticonvulsivantes/síntesis química , Anticonvulsivantes/farmacología , Bencilaminas/síntesis química , Ácidos Ciclohexanocarboxílicos/síntesis química , Ciclohexilaminas , Convulsiones/tratamiento farmacológico , Animales , Anticonvulsivantes/química , Bencilaminas/química , Bencilaminas/farmacología , Ácidos Ciclohexanocarboxílicos/química , Ácidos Ciclohexanocarboxílicos/farmacología , Excitación Neurológica/efectos de los fármacos , Masculino , Ratones , Modelos Moleculares , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad

RESUMEN

Prolonged benzodiazepine treatment of rats results in anticonvulsant tolerance in vivo. Studies of in vitro hippocampal slices following 1 wk flurazepam administration show reduced GABA-mediated inhibition in the CA1 region, and a decrease in GABAA agonist and benzodiazepine potency to inhibit CA1 pyramidal cell-evoked responses. To investigate the molecular basis of benzodiazepine tolerance in the hippocampus, in situ hybridization techniques were used to evaluate the expression of the mRNAs for the alpha 1, alpha 5, and gamma 2 subunits of the GABAA receptor in the hippocampal formation and frontal cortex of chronic flurazepam-treated rats. A discretely localized decrease in alpha 1, but not alpha 5 or gamma 2 mRNA expression was found in the CA1 region (35-40%) and in layers II-III and IV of cortex (50-60%) 2 d after cessation of flurazepam treatment. The decrease in the expression of alpha 1 subunit mRNA in cortex is similar to that reported following other chronic benzodiazepine treatment regimens. This is the first report of a reduction in alpha 1 subunit mRNA expression in the hippocampal formation.

Asunto(s)

Corteza Cerebral/efectos de los fármacos , Flurazepam/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Receptores de GABA/biosíntesis , Animales , Corteza Cerebral/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Hipocampo/metabolismo , Hibridación in Situ , Masculino , Proteínas del Tejido Nervioso/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de GABA/química , Receptores de GABA/genética

RESUMEN

The potency and efficacy of selective gamma-aminobutyric acidA (GABAA) agonists (GABA, muscimol, isoguvacine and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol), the GABAB agonist, baclofen, and the benzodiazepine agonist, diazepam, were examined using extracellular recording techniques in in vitro hippocampal slices from rats sacrificed 2 days after 1 week of flurazepam treatment. Population spikes elicited by stimulation of Schaffer collaterals were recorded in the CA1 pyramidal cell region with NaCl-containing glass micropipettes. GABA agonists were superfused in increasing concentrations for 5 min. Drug responses, averaged over the last 2 min for each concentration, were compared to the predrug base line. GABAA agonists, but not baclofen, showed a significant, 2-fold, decrease in potency, but not efficacy, to reduce CA1-evoked responses in treated vs. control slices. The benzodiazepine effect was evaluated by the shift in the isoguvacine dose-response curve in the absence, then presence, of diazepam. A reduction in diazepam potency was demonstrated in vitro by a significantly reduced shift in the isoguvacine curve by 300 nM, but not 1 microM, diazepam after chronic but not acute in vivo pretreatment. The results indicated a selective GABAA agonist subsensitivity and diazepam tolerance in hippocampus after 1 week of flurazepam treatment and establish the hippocampal slice preparation as a valuable substrate for investigating synaptic mechanisms of benzodiazepine tolerance.

Asunto(s)

Flurazepam/farmacología , Hipocampo/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Diazepam/farmacología , Potenciales Evocados/efectos de los fármacos , Ácidos Isonicotínicos/farmacología , Masculino , Ratas , Ratas Endogámicas