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Hypoxia regulates neuronal ion channels, sometimes resulting in seizures. We evaluated the effects of brief sustained hypoxia (1% O(2), 4h) on voltage-gated calcium channels (VGCCs) in cultured rat primary cortical neurons. High-voltage activated (HVA) Ca(2+) currents were acquired immediately after hypoxic exposure or after 48h recovery in 95% air/5% CO(2). Maximal Ca(2+) current density increased 1.5-fold immediately after hypoxia, but reverted to baseline after 48h normoxia. This enhancement was primarily due to an increase in L-type VGCC activity, since nimodipine-insensitive residual Ca(2+) currents were unchanged. The half-maximal potentials of activation and steady-state inactivation were unchanged. The calcineurin inhibitors FK-506 (in the recording pipette) or cyclosporine A (during hypoxia) prevented the post-hypoxic increase in HVA Ca(2+) currents, while rapamycin and okadaic acid did not. L-type VGCCs were the source of Ca(2+) for calcineurin activation, as nimodipine during hypoxia prevented post-hypoxic enhancement. Hypoxia transiently potentiated L-type VGCC currents via calcineurin, suggesting a positive feedback loop to amplify neuronal calcium signaling that may contribute to seizure generation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cessation of one-week oral administration of the benzodiazepine flurazepam (FZP) to rats results in withdrawal anxiety after 1 day of withdrawal. FZP withdrawal is correlated with synaptic incorporation of homomeric GluA1-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) in the proximal stratum radiatum of CA1 neurons. After 2 days of withdrawal, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylates GluA1 subunits at Ser(831), increasing channel conductance. Secondary to AMPAR potentiation, GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), known binding partners of CaMKII, are selectively removed from the postsynaptic density (PSD). While activation of synaptic CaMKII is known to involve translocation to the PSD, CaMKII bound to NMDARs may be removed from the PSD. To distinguish these possibilities, the current studies used postembedding immunogold electron microscopy to investigate alterations in CaMKII signaling at CA1 stratum radiatum synapses after 2 days of FZP withdrawal. These studies revealed decreased total, but not autophosphorylated (Thr(286)) CaMKIIα expression in CA1 PSDs. The removal of CaMKII-GluN2B complexes from the PSD during drug withdrawal may serve as a homeostatic mechanism to limit AMPAR-mediated CA1 neuron hyperexcitability and benzodiazepine withdrawal anxiety.

Benzodiazepines (BDZs) depress neuronal excitability via positive allosteric modulation of inhibitory GABA(A) receptors (GABA(A)R). BDZs and other positive GABA(A)R modulators, including barbiturates, ethanol, and neurosteroids, can also inhibit L-type voltage-gated calcium channels (L-VGCCs), which could contribute to reduced neuronal excitability. Because neuronal L-VGCC function is up-regulated after long-term GABA(A)R modulator exposure, an interaction with L-VGCCs may also play a role in physical dependence. The current studies assessed the effects of BDZs (diazepam, flurazepam, and desalkylflurazepam), allopregnanolone, pentobarbital, and ethanol on whole-cell Ba(2+) currents through recombinant neuronal Ca(v)1.2 and Ca(v)1.3 L-VGCCs expressed with β(3) and α(2)δ-1 in HEK293T cells. Allopregnanolone was the most potent inhibitor (IC(50), ∼10 μM), followed by BDZs (IC(50), ∼50 μM), pentobarbital (IC(50), 0.3-1 mM), and ethanol (IC(50), ∼300 mM). Ca(v)1.3 channels were less sensitive to pentobarbital inhibition than Ca(v)1.2 channels, similar to dihydropyridine (DHP) L-VGCC antagonists. All GABA(A)R modulators induced a negative shift in the steady-state inactivation curve of Ca(v)1.3 channels, but only BDZs and pentobarbital induced a negative shift in Ca(v)1.2 channel inactivation. Mutation of the high-affinity DHP binding site (T1039Y and Q1043M) in Ca(v)1.2 channels reduced pentobarbital potency. Despite the structural similarity between benzothiazepines and BDZs, mutation of an amino acid important for diltiazem potency (I1150A) did not affect diazepam potency. Although L-VGCC inhibition by BDZs occurred at concentrations that are possibly too high to be clinically relevant and is not likely to play a role in the up-regulation of L-VGCCs during long-term treatment, pentobarbital and ethanol inhibited L-VGCCs at clinically relevant concentrations.

A significant link was previously established between benzodiazepine withdrawal anxiety and a progressive increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) potentiation in hippocampal CA1 neurons from rats withdrawn up to 2 days from 1-week oral administration of the benzodiazepine flurazepam (FZP). Despite AMPAR current potentiation, withdrawal anxiety was masked by a 2-fold reduction in CA1 neuron N-methyl-D-aspartate receptor (NMDAR) currents since preinjection of an NMDA antagonist restored NMDAR currents and unmasked anxiety in 2-day FZP-withdrawn rats. In the current study, GluN subunit levels in postsynaptic density (PSD)-enriched subfractions of CA1 minislices were compared with GluN2B-mediated whole-cell currents evoked in CA1 neurons in hippocampal slices from 1- and 2-day FZP-withdrawn rats. GluN1 and GluN2B, although not the phosphoSer1303-GluN2B ratio or GluN2A subunit levels, were decreased in PSD subfractions from 2-day, but not 1-day, FZP-withdrawn rats. Consistent with immunoblot analyses, GluN2B-mediated NMDAR currents evoked in slices from 2-day FZP-withdrawn rats were decreased in the absence, but not the presence, of the GluN2B subunit-selective antagonist ifenprodil. In contrast, ifenprodil-sensitive NMDAR currents were unchanged in slices from 1-day withdrawn rats. Because AMPA (1 μM) preincubation of slices from 1-day FZP-withdrawn rats induced depression of GluN2B subunit-mediated currents, depression of NMDAR currents was probably secondary to AMPAR potentiation. CA1 neuron NMDAR currents were depressed ∼50% after 2-day withdrawal and offset potentiation of AMPAR-mediated currents, leaving total charge transfer unchanged between groups. Collectively, these findings suggest that a reduction of GluN2B-containing NMDAR may serve as a homeostatic feedback mechanism to modulate glutamatergic synaptic strength during FZP withdrawal to alleviate benzodiazepine withdrawal symptoms.

Benzodiazepine withdrawal-anxiety is associated with enhanced α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawal-anxiety is associated with a reduction in N-methyl-D-aspartate receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A, and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2 days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZP-withdrawn rats. Similar decreases were observed for GluN2B subunits; however, the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles associated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal., (© 2010 Wiley-Liss, Inc.)

Benzodiazepine withdrawal anxiety is associated with potentiation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) currents in hippocampal CA1 pyramidal neurons attributable to increased synaptic incorporation of GluA1-containing AMPARs. The contribution of calcium/calmodulin-dependent protein kinase II (CaMKII) to enhanced glutamatergic synaptic strength during withdrawal from 1-week oral flurazepam (FZP) administration was further examined in hippocampal slices. As earlier reported, AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, along with increased single-channel conductance in neurons from 2-day rats, estimated by non-stationary noise analysis. Input-output curve slope was increased without a change in paired-pulse facilitation, suggesting increased AMPAR postsynaptic efficacy rather than altered glutamate release. The increased mEPSC amplitude and AMPAR conductance were related to CaMKII activity, as intracellular inclusion of CaMKIINtide or autocamtide-2-related inhibitory peptide, but not scrambled peptide, prevented both AMPAR amplitude and conductance changes. mEPSC inhibition by 1-naphthyl acetyl spermine and the negative shift in rectification index at both withdrawal time points were consistent with functional incorporation of GluA2-lacking AMPARs. GluA1 but not GluA2 or GluA3 levels were increased in immunoblots of postsynaptic density (PSD)-enriched subcellular fractions of CA1 minislices from 1-day FZP-withdrawn rats, when mEPSC amplitude, but not conductance, was increased. Both GluA1 expression levels and CaMKII alpha-mediated GluA1 Ser(831) phosphorylation were increased in PSD-subfractions from 2-day FZP-withdrawn rats. As phospho-Thr(286)CaMKII alpha was unchanged, CaMKII alpha may be activated through an alternative signaling pathway. Synaptic insertion and subsequent CaMKII alpha-mediated Ser(831) phosphorylation of GluA1 homomers contribute to benzodiazepine withdrawal-induced AMPAR potentiation and may represent an important hippocampal pathway mediating both drug-induced and activity-dependent plasticity.

Long-term BZ (benzodiazepine) anxiolytic therapy increases the risk of physical dependence manifested as withdrawal anxiety. BZ-induced potentiation of GABA(A)R (gamma-aminobutyric acid type-A receptor) function by 1-week oral administration of FZP (flurazepam) bi-directionally modulates excitatory glutamatergic synaptic transmission in hippocampal CA1 neurons during drug withdrawal. Previous electrophysiological studies on acutely isolated and intact CA1 neurons, as well as immunofluorescence and post-embedding immunogold electron microscopy studies, suggest increased synaptic insertion of GluR (glutamate receptor) 2-lacking AMPARs (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors) in 2-day FZP-withdrawn rats. Preliminary studies indicated a similar increase in GluR1, then phospho-Ser(831)-GluR1, as well as CaMKIIalpha (Ca(2+)/calmodulin-dependent protein kinase IIalpha), but not phospho-Thr(286)-CaMKII levels at the same time point. In our studies, whole-cell recordings in hippocampal slices revealed that AMPAR mEPSC [miniature EPSC (excitatory postsynaptic current)] amplitude was increased in 1-day FZP-withdrawn rats followed by an increase in estimated single-channel conductance in 2-day-FZP-withdrawn rats. Enhanced conductance was no longer observed in slices pre-incubated for 2 h in the CaMKII inhibitor KN-93, but not the inactive analogue KN-92. To evaluate whether CaMKII-mediated AMPA potentiation could occlude LTP (long-term potentiation), LTP was induced by TBS (theta burst stimulation) and recorded using whole-cell and extracellular techniques. LTP was induced in both groups, but only maintained for <15 min in 2-day FZP-withdrawn rats. LTP was fully restored after 7-day withdrawal. Despite the lack of LTP maintenance, impairment of object recognition, place and context was not observed in 2-day-FZP-withdrawn rats. Since L-VGCC (L-type voltage-gated calcium channel) current density was doubled on drug withdrawal and up to 2 days, Ca(2+) entry through L-VGCCs and perhaps subsequently through Ca(2+)-permeable AMPARs are proposed to be responsible for enhanced CaMKIIalpha levels and AMPAR potentiation. Mechanisms associated with several different models of activity-dependent plasticity may underlie BZ physical dependence.

Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR)-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2, subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined by postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2% +/- 2.2%) compared with controls (74.4% +/- 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared with control rats (FZP: 14.1 +/- 0.3 gold particles/mum; CON: 10.8 +/- 0.4 gold particles/mum). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity.

Signs of physical dependence as a consequence of long-term drug use and a moderate abuse liability limit benzodiazepine clinical usefulness. Growing evidence suggests a role for voltage-gated calcium channel (VGCC) regulation in mediating a range of chronic drug effects from drug withdrawal phenomena to dependence on a variety of drugs of abuse. High voltage-activated (HVA) calcium currents were measured in whole-cell recordings from acutely isolated hippocampal CA1 neurons after a 1-week flurazepam (FZP) treatment that results in withdrawal-anxiety. An approximately 1.8-fold increase in Ca(2+) current density was detected immediately after and up to 2 days but not 3 or 4 days after drug withdrawal. Current density was unchanged after acute desalkyl-FZP treatment. A significant negative shift of the half-maximal potential of activation of HVA currents was also observed but steady-state inactivation remained unchanged. FZP and diazepam showed use- and concentration-dependent inhibition of Ca(2+) currents in hippocampal cultured cells following depolarizing trains (FZP, IC(50) = 1.8 microM; diazepam, IC(50) = 36 microM), pointing to an additional mechanism by which benzodiazepines modulate HVA Ca(2+) channels. Systemic preinjection of nimodipine (10 mg/kg), an L-type (L)-VGCC antagonist, prevented the benzodiazepine-induced increase in alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid receptor (AMPAR)-mediated miniature excitatory postsynaptic current in CA1 neurons 2 days after FZP withdrawal, suggesting that AMPAR potentiation, previously linked to withdrawal-anxiety may require enhanced L-VGCC-mediated Ca(2+) influx. Taken together with prior work, these findings suggest that enhanced Ca(2+) entry through HVA Ca(2+) channels may contribute to hippocampal AMPAR plasticity and serve as a potential mechanism underlying benzodiazepine physical dependence.

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.

Withdrawal from 1-week oral administration of the benzodiazepine, flurazepam (FZP) is associated with increased alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor (AMPAR) miniature excitatory postsynaptic currents (mEPSCs) but reduction of N-methyl-D-aspartic acid (NMDA) receptor (NMDAR)-evoked (e)EPSCs in hippocampal CA1 neurons. A positive correlation was observed between increased AMPAR-mediated mEPSC amplitude and anxiety-like behavior in 1-day FZP-withdrawn rats. These effects were disrupted by systemic AMPAR antagonist administration (GYKI-52466, 0.5 mg/kg, intraperitoneal) at withdrawal onset, strengthening the hypothesis that CA1 neuron AMPAR-mediated hyperexcitability is a central component of a functional anatomic circuit associated with the expression of withdrawal anxiety. Abolition of AMPAR current upregulation in 2-day FZP withdrawn rats by GYKI-52466 injection also reversed the reduction in NMDAR-mediated eEPSC amplitude in CA1 neurons from the same rats, suggesting that downregulation of NMDAR function may serve a protective, negative-feedback role to prevent AMPAR-mediated neuronal overexcitation. NMDAR antagonist administration (MK-801, 0.25 mg/kg intraperitoneally) had no effect on modifying increased glutamatergic strength or on withdrawal anxiety, whereas injection of an L-type voltage-gated calcium channel antagonist, nimodipine (10 mg/kg, intraperitoneally) averted AMPAR current enhancement and anxiety-like behavior, suggesting that these manifestations may be initiated by a voltage-gated calcium channel-dependent signal transduction pathway. An evidence-based model of likely cellular mechanisms in the hippocampus contributing to benzodiazepine withdrawal anxiety was proposed implicating regulation of multiple CA1 neuron ion channels.

Modification of glutamatergic synaptic function, a mechanism central to neuronal plasticity, may also mediate long-term drug effects, including dependence and addiction. Benzodiazepine withdrawal results in increased glutamatergic strength, but whether alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs) are functionally and structurally remodeled during benzodiazepine withdrawal is uncertain. Whole-cell recordings of rat hippocampal CA1 neurons, either acutely dissociated or in hippocampal slices, revealed that AMPAR function was enhanced up to 50% during flurazepam (FZP) withdrawal, without changes in whole-cell channel kinetic properties. Agonist-elicited AMPA currents showed a negative shift in rectification in the presence of spermine, suggesting augmented membrane incorporation of glutamate receptor (GluR) 2-lacking AMPARs. As GluR1-containing AMPARs are critical for activity-dependent alterations in excitatory strength, we sought to determine whether changes in GluR1 subunit distribution in CA1 neurons occurred during benzodiazepine withdrawal. Confocal image analysis revealed that FZP withdrawal promoted GluR1 subunit incorporation into somatic and proximal dendritic membranes of CA1 neurons without GluR2 subunit alterations. Findings of immunoblot studies were consistent with immunofluorescent studies indicating increased GluR1, but not GluR2, subunit protein levels in cytosolic, crude membrane and postsynaptic density-enriched fractions from CA1 minislices. As with long-term potentiation (LTP), the FZP-withdrawal-induced GluR1 incorporation into CA1 neuron membranes may require the GluR1-trafficking protein, synapse-associated protein 97, which was also elevated in membrane-associated fractions. Together, our findings provide evidence that during FZP withdrawal, increased membrane incorporation of GluR1-containing AMPARs and associated up-regulation of AMPAR functions in hippocampal CA1 pyramidal neurons share fundamental similarities with the mechanisms underlying LTP. This implies that glutamatergic neuronal remodeling observed in LTP also subserves physiological adaptations to drug withdrawal.

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.

Withdrawal from 1-week oral administration of the benzodiazepine (BZ), flurazepam (FZP) is associated with enhanced AMPA receptor (AMPAR)-mediated and reduced NMDA receptor (NMDAR)-mediated excitation in CA1 pyramidal neurons 2-days after cessation of FZP administration. The present study examined temporal regulation of glutamate receptor-mediated whole-cell currents in CA1 neurons from hippocampal slices prepared from 0-, 1-, 2-, and 4-day FZP-withdrawn rats in relation to expression of anxiety-like behavior during BZ withdrawal. AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude was significantly increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, while evoked NMDAR EPSC amplitude was reduced only in neurons from 2-day FZP-withdrawn rats. Withdrawal-anxiety, measured in the elevated plus-maze, was observed 1 day, but not 0, 2, or 4 days, after FZP treatment with 1-day withdrawn rats spending significantly reduced time in open arms compared to controls. CA1 neuron hyperexcitability was evident from the significant increase in the frequency of extracellular, 4-AP-induced spike discharges in slices from 1-day FZP-withdrawn rats. Systemic injection of the NMDAR antagonist MK-801 (0.25 mg/kg) on day 1 of withdrawal prevented reduced NMDAR-mediated currents in CA1 neurons from 2-day FZP-withdrawn rats, whereas AMPAR-mediated currents remained upregulated. Furthermore, MK-801 'unmasked' withdrawal-anxiety in the same 2-day FZP-withdrawn rats. Systemic injection of the AMPAR antagonist GYKI-52466 (0.5 mg/kg) at the onset of withdrawal blocked increased AMPAR-mediated currents and withdrawal-anxiety in 1-day FZP-withdrawn rats. These findings suggest that increased CA1 neuron AMPAR-mediated excitation may contribute to hippocampal hyperexcitability and expression of withdrawal-anxiety after prolonged BZ exposure via NMDAR-mediated neural circuits.

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.

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.

Two days following one-week administration of the benzodiazepine, flurazepam (FZP), rats exhibit anticonvulsant tolerance in vivo, while reduced GABA(A) receptor-mediated inhibition and enhanced EPSP amplitude are present in CA1 pyramidal neurons in vitro. AMPA receptor (AMPAR)-mediated synaptic transmission in FZP-treated rats was examined using electrophysiological techniques in in vitro hippocampal slices. In CA1 pyramidal neurons from FZP-treated rats, the miniature excitatory postsynaptic current (mEPSC) amplitude was significantly increased (33%) without change in frequency, rise time or decay time. Moreover, mEPSC amplitude was not elevated in dentate granule neurons following 1-week FZP treatment or in CA1 pyramidal neurons following acute desalkyl-FZP treatment. Regulation of AMPAR number was assessed by quantitative autoradiography with the AMPAR antagonist, [(3)H]Ro48-8587. Specific binding was significantly increased in stratum pyramidale of hippocampal areas CA1 and CA2 and in proximal dendritic fields of CA1 pyramidal neurons. Regulation of AMPAR subunit proteins was examined using immunological techniques. Neither abundance nor distribution of GluR1-3 subunit proteins was different in the CA1 region following FZP treatment. These findings suggest that enhanced AMPAR currents, mediated at least in part by increased AMPAR number, may contribute to BZ anticonvulsant tolerance. Furthermore, these studies suggest an interaction between GABAergic and glutamatergic systems in the CA1 region which may provide novel therapeutic strategies for restoring BZ effectiveness.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The acute anticonvulsant efficacy of diazepam (1.5 mg/kg, i.p.) was evaluated by repeated test injection in kindled rats subcutaneously implanted with diazepam-filled or empty silastic tubes for 3 weeks. Tolerance developed to acute test injections in both diazepam- and sham-implanted rats. Tolerance developed to a lesser extent in another group of diazepam-implanted rats which did not receive acute intermittent anticonvulsant tests. The hypothesis that contingent tolerance had developed to the anticonvulsant actions of benzodiazepines (diazepam, 1.5 mg/kg, i.p. and clobazam, 10 mg/kg, i.p.) in kindled rats given acute intermittent injections was investigated using a 'before-after' design. Significant contingent tolerance developed in rats which received intermittent benzodiazepine treatment before, but not after, amygdala stimulation. Tolerance developed to different extents depending on the seizure measure evaluated (forelimb clonus duration, amygdala afterdischarge duration, motor seizure latency and duration, and seizure stage). Contingent tolerance to both benzodiazepines developed at a similar rate. The findings suggest that contingent tolerance may contribute a sizeable component to the overall functional benzodiazepine tolerance measured in long-term anticonvulsant drug studies in kindled rats. Several questions regarding contingent tolerance phenomena are posed and the implications of these findings for studies using repeated anticonvulsant testing are discussed.

Paired-pulse inhibition was studied extracellularly in in vitro hippocampal slices from rats sacrificed 48 h or 7 days after 1 week flurazepam (FZP) treatment. Population spikes and field excitatory postsynaptic potentials (EPSPs) were recorded with NaCl-containing glass micropipettes in the stratum pyramidale and stratum radiatum, respectively, of the CA1 region. Conditioning pulses were delivered by stimulating Shaffer collaterals (orthodromic) or the alveus (antidromic). Orthodromic test pulses were delivered with interpulse intervals of 10-200 ms. There was a significant reduction in paired-pulse inhibition in slices from treated vs control rats in both the orthodromic-orthodromic and antidromic-orthodromic paradigms. Reduced inhibition was evident 48 h, but not 7 days, after the end of FZP treatment. Furthermore, there was a significant prolongation of the half decay time of the field EPSP, without a significant change in the initial slope or maximum amplitude. The results may suggest an impairment of endogenous gamma-aminobutyric acid function in the hippocampus after chronic benzodiazepine (BZ) treatment and may provide a basis for a mechanism of BZ tolerance.

Rats were treated for one week with flurazepam (FZP). After an additional two days with no treatment, each rat was injected with one of seven benzodiazepines (BZs). Several different doses of each BZ were evaluated. Ten min later, 100 mg/kg pentylenetetrazol (PTZ) was injected, IP, and convulsive activity was recorded. Rats treated for a week with FZP were tolerant to ataxia induced by each of the seven BZs tested. There was a dose-dependent anti-PTZ effect for each BZ. Whether or not tolerance to the anti-PTZ effect was found depended on the particular BZ used. Tolerance was found for four of the drugs: diazepam, clobazam, flurazepam and desalkylflurazepam. However, no tolerance was found to the anti-PTZ actions of midazolam, triazolam or clonazepam. Brain BZ levels were measured by the ability of brain extracts to displace specifically bound [3H]flunitrazepam in vitro. There was no significant effect of one week of flurazepam treatment. It was proposed that differences among BZs in their interactions with receptors allowed some to circumvent the mechanism responsible for tolerance to the anti-PTZ effect.

Bilateral injection of 2-amino-7-phosphonoheptanoate (2-APH), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, into substantia nigra pars reticulata (SNpr) significantly and dose-dependently suppressed tonic pentylenetetrazol (PTZ) seizures (100 mg/kg, i.p.). The results confirm the anticonvulsant effectiveness of 2-APH and the capacity of SNpr to modulate PTZ seizure activity. The anti-PTZ effect of 2-APH was significantly attenuated by muscimol, co-infused into SNpr. This result supports the hypothesis, drawn from earlier studies, that intranigral muscimol can interfere with the anti-PTZ actions of other intranigral treatments.

Subsensitivity to gamma-aminobutyric acid (GABA) agonists was sought in rats treated 1 or 4 weeks with flurazepam (FZP). Sensitivity to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) was assessed by measuring contralateral rotation following unilateral microinjection of drug into the substantia nigra pars reticulata (SNpr). Immediately and 48 h after chronic treatment GABA, 200 micrograms or THIP, 60 ng was infused into SNpr. Immediately, but not 48 h after 1 week of FZP treatment, GABA subsensitivity was shown by a significantly reduced total number of contralateral turns and peak rotation rate. There was no change in the response to THIP after 1 week FZP treatment. Following 4 week FZP treatment, no subsensitivity to GABA or THIP was evident. Previous results showed subsensitivity to muscimol after 4, but not 1 week of FZP treatment. Since muscimol and THIP are not subject to uptake, there may be increased uptake of GABA after 1 week of FZP treatment, though it may not persist during continued treatment. Differential regulation of GABA agonist effects in SNpr may be related to their acting at differing GABAA receptor subpopulations, and variable responses of these subpopulations to chronic BZ treatment.

gamma-Aminobutyric acid (GABA)-stimulated 36chloride (Cl-) influx into membrane vesicles derived from cerebral cortex, hippocampus, cerebellum and midbrain/brainstem was evaluated in naive rats and amygdala or sham kindled rats. GABA-stimulated 36Cl- uptake was greatest in cortex and hippocampus of naive rats. One week after amygdala kindling, 10 microM GABA-stimulated 36Cl- influx was significantly reduced in each brain region, except midbrain/brainstem, indicating a regional variation in impaired GABAergic function. Midazolam potentiation of GABA-mediated Cl- flux showed a regional variation. The effect of midazolam in kindled rats indicated either an increase in efficiency of GABA/benzodiazepine coupling in areas of reduced GABA function or its ability to restore Cl- channel function to the pre-kindled state.

In a previous study, infusion of flurazepam and midazolam, but not GABA or muscimol into the pars reticulata of substantia nigra (SNpr), blocked the tonus induced by a high dose of pentylenetetrazol (PTZ). It was hypothesized that the divergence of GABAA agonist and benzodiazepine (BZ) actions might be due to a complex action of the GABAA agonists in SNpr that is not shared by the BZs, and which is specific for certain experimental seizures. This was tested in the present study in which GABA, muscimol, midazolam, and combinations of midazolam with GABA and with muscimol were tested against seizures induced by PTZ (40 or 100 mg/kg, i.p.), bicuculline (0.5 mg/kg, i.v.) and maximal electroshock (MES). Intranigral midazolam was effective against PTZ, bicuculline and MES seizures. Intranigral GABA or muscimol, infused together with midazolam, prevented midazolam from blocking tonus induced by the high dose of PTZ. For bicuculline seizures, midazolam alone had a better overall anticonvulsant effect than did the combination of midazolam plus muscimol. Intranigral muscimol, midazolam and a combination of two were equally effective against the low dose PTZ (40 mg/kg, i.p.) and MES seizures. Since the effect of intranigral drugs was model-specific, it was suggested that different populations of nigral output neurons were involved in regulating the generalization of these seizures.

The spontaneous activity of neurons in the pars reticulata of substantia nigra (SNpr) was studied in chloral hydrate anesthetized rats. As a function of dose, intravenous diazepam decreased, and methyl-beta-carboline-3-carboxylate (beta CCM) increased discharge frequency. Two days after terminating a one week treatment with flurazepam (FZP), both diazepam and beta CCM showed decreased ability to alter SNpr neuronal activity. Neither residual FZP nor down-regulation of benzodiazepine receptors can account for these results. In contrast, behavioral testing revealed no change in the ability of i.v. beta CCM to cause convulsions, suggesting that sites other than the SNpr are of prime importance in expressing the convulsant actions of systemically injected beta CCM.

[3H]Flunitrazepam (FNP) and [3H]methyl beta-carboline-3-carboxylate (MCC) binding was examined in soluble and particulate fractions from membranes solubilized with Triton X-100 or in subfractions of synaptosomal membranes obtained by a physical separation technique. Results using both methods demonstrate that benzodiazepine and beta-carboline sites reside on both pre- and postsynaptic membranes. Further, subfractionation experiments indicate that the binding sites for both ligands are unequally distributed within the synapse and among brain regions. For example, in cerebral cortical presynaptic membranes there are twice as many FNP as MCC sites whereas in postsynaptic membranes this ratio is reversed. The number of FNP and MCC sites are equal in the presynaptic fraction from cerebellum. The postsynaptic membranes derived from cerebellum have three times the number of FNP compared to MCC sites. In hippocampus this ratio varies between 1.5 and 2.8 in each subfraction. These results support the idea that benzodiazepine and beta-carboline binding sites represent different recognition sites.

Tolerance to the anticonvulsant action of benzodiazepines was studied in rats that had been treated for 4 weeks with 100-150 mg/kg per day of flurazepam. Previous studies had shown that this treatment produced tolerance to motor impairment induced by benzodiazepines and also down-regulation of benzodiazepine receptors in brain, which was seen as a reduced number of binding sites with no change in binding affinity. In the present study, seizures were produced using pentylenetetrazol (PTZ). In rats that had been chronically treated with flurazepam, pretreatment with diazepam was significantly less effective in blocking pentylenetetrazol-induced seizures, thus indicating tolerance. This tolerance could not be explained by a change in sensitivity to pentylenetetrazol resulting from chronic treatment, nor by any differences in levels of active drug in the brain following doses of diazepam. Residual amounts of flurazepam and its active metabolites may have artifactually reduced the apparent degree of tolerance measured 12 hr after the end of chronic treatment, but not at later times. Tolerance to the antipentylenetetrazol action of diazepam was evident up to the fourth day following chronic treatment with flurazepam, but tolerance had largely disappeared a week after chronic treatment. The duration of tolerance was much longer than that reported for tolerance to motor impairment induced by benzodiazepines, and for down-regulation of receptors. These results suggest that different mechanisms or different neural systems must mediate tolerance to these different actions of benzodiazepines. Furthermore, an adaptive reduction in the number of benzodiazepine receptors does not seem to be a likely mechanism for tolerance to the anticonvulsant action of these drugs.

A daytime nap procedure was used to evaluate the daytime sleepiness associated with antihistamines, as well as to assess their hypnotic potential. Healthy, normal subjects received diphenhydramine (150 mg), terfenadine (120 mg), and placebo and went to bed at 900, 1100, 2000, and 2200 h with the instruction to try to fall asleep. The remained in bed for 60 min while standard sleep recordings were made. Across all conditions latency to stage 1 sleep increased significantly from nap 1 to nap 4 and the amount of sleep (all nonstage 1 sleep) decreased significantly. Over the four naps the mean latency to stage 1 sleep with diphenhydramine was significantly shorter than terfenadine and placebo, which did not differ. On the other hand, there were no differences among the drug conditions in the amount of nonstage 1 sleep. In sum, diphenhydramine at this dose produces sleepiness but shows little potential as a hypnotic, and accumulated sleep across the day makes people progressively more alert.

Studies suggest that the 1,5-benzodiazepine clobazam possesses a favorable anticonvulsant profile due to its minimal neurotoxicity. The anticonvulsant and motor impairment effects of clobazam and 2 1,4-benzodiazepine, diazepam and clonazepam, were compared by dose-response analysis in amygdala-kindled rats and on 3 tests of motor function: gross motor impairment, a vertical screen test, and muscle tone. All drugs produced a significant, dose-dependent decrease in the duration of both behavioral and electrographic kindled seizure measures. Forelimb clonus suppression was the most sensitive measure of anticonvulsant drug effect. The order of potency for all effects was clonazepam greater than diazepam greater than clobazam. ED50s for the benzodiazepines' effects on motor impairment were compared to their ability to protect rats from forelimb clonus. Different spectrums of action for the various benzodiazepines were found depending on the comparison measure. Clonazepam had the most favorable ratio of potency for anticonvulsant vs. motor impairment activity when ataxia rating was the comparison measure. Diazepam had the most advantageous profile when the more sensitive screen test was used for comparison. Clobazam was not found to have a superior spectrum of action when compared across these measures. The results emphasize the importance of dose-response analyses and the consideration of behavioral measures used to assess beneficial and adverse effects of anticonvulsants.

Rotational behavior was elicited by unilateral microinjection of the benzodiazepine flurazepam, and the gamma-aminobutyric acid (GABA) agonist, muscimol, into the substantia nigra pars reticulata (SNpr). This response was used to quantitate benzodiazepine tolerance and GABAergic subsensitivity after chronic benzodiazepine treatment. Studies in naive rats established the dose requirements for inducing contralateral circling and demonstrated the reproducibility of the behavioral response as a measure of SNpr function. There was a large difference in potency between the two drugs for causing dose-related rotation. The response to microinjected flurazepam could be blocked by 16 mg/kg of the benzodiazepine antagonist, Ro15-1788. Tolerance to intranigral flurazepam (50 micrograms) was measured by a reduction in the turning response after a 1- or 4-week chronic flurazepam treatment. The time course for the reversal of tolerance after a 4-week benzodiazepine treatment correlates with the time course of the reversal of benzodiazepine receptor down-regulation in the SNpr. Subsensitivity of the GABAergic system was demonstrated by the decreased rotational response to muscimol (10 ng), confirming the idea that the GABAergic system is also functionally altered by chronic benzodiazepine treatment. The time course of the decreased sensitivity to muscimol does not coincide with the development and reversal of tolerance to the turning produced by flurazepam or with benzodiazepine receptor down-regulation. These data suggest differential regulation of SNpr sensitivity to benzodiazepine and GABA agonists following chronic benzodiazepine treatment and may provide a basis for differential tolerance; the development of tolerance to some but not other benzodiazepine actions.

The present study evaluated the efficacy of 25 mg of quazepam, a new benzodiazepine hypnotic, in a population of chronic insomniacs. The results indicate that a single dose (25 mg) administered for one night was efficacious when measured both objectively by polysomnographic recording and subjectively by questionnaire with no reported side-effects. The change in the objective measures paralleled the direction of change in subjective measures. Sleep efficiency and sleep maintenance were improved without EEG changes in Stages 2, 3-4, and REM. Further study is needed to evaluate the effects of chronic administration of different doses of quazepam in chronic insomniacs.

Benzodiazepines are effective anticonvulsants, but long-term clinical usefulness is limited by development of tolerance. Tolerance to the actions of three prototype anticonvulsant benzodiazepines (BZDs)--diazepam (DZP), clonazepam (CZP), and clobazam (CLB)--was studied in amygdala-kindled rats. Fully kindled rats were dosed three times daily for 2 or 4 weeks. Amygdala stimulation was given 30 min after drug administration on days 1, 2, 3, 5, and 7 of chronic treatment and then three times weekly. During treatment, tolerance was observed as a loss of drug effect to suppress behavioral and EEG manifestations of seizure activity. Seizure activity remained stable in rats treated with vehicle. Tolerance to the anticonvulsant effects developed most rapidly during CLB treatment and most slowly during CZP treatment. Tolerance to the motor impairment caused by the drugs developed more rapidly. Assay of the amount of drug in brain extracts, using a BZD receptor assay, showed that tolerance was functional, not metabolic. Doubling the dose did not readily restore full anticonvulsant activity. The response to amygdala stimulation 24 h after treatment was stopped showed no residual BZD effect, but there was a rebound in duration of some seizure measures in rats that had been treated with CLB or DZP. Retesting 48 h after treatment was stopped showed that rats were still tolerant. The amygdala-kindled rat is a reliable and sensitive model for studying long-term actions of anticonvulsant BZDs.

The present study investigated the effects of the benzodiazepine, 7-chloro-5-(2-fluorophenyl)-1,3-dihydro-1-(2,2,2-trifluoroethyl)-2H-1,4-benzodiazeepine-2-thione (quazepam), on hypnotic efficacy and sleep staging in chronic insomniacs. We evaluated the acute withdrawal effects of several doses (0, 7.5, 30 and 45 mg) of quazepam. The effects of the different doses were assessed using both objective (polysomnographic) and subjective sleep parameters in a double blind repeated measures design (n = 15). The results indicated that daily administration of quazepam over three nights produced increased effects on sleep which suggested drug accumulation. Lower doses (7.5 and 15 mg) only had significant effects on sleep (sleep induction and maintenance and sleep architecture) with three days of administration, while 45 mg of quazepam produced significant effects on the first night of drug administration. Contrary to previous research investigating longer periods of administration, acute administration of low doses of quazepam did not produce carryover effects on withdrawal, while higher doses did. The accumulation of the parent compound or its active metabolites thus seemed to mediate these effects on sleep in a dose related fashion.