Flurazepam Research Articles

GABAA Receptor β2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in the adult brain. In spite of progress in describing (mainly) the static structures of this receptor, the molecular mechanisms underlying its activation remain unclear. It is known that in the α1β2γ2L receptors, the mutation of the β2E155 residue, at the orthosteric binding site, strongly impairs the receptor activation, but the molecular and kinetic mechanisms of this effect remain elusive. Herein, we investigated the impact of the β2E155C mutation on binding and gating of the α1β2γ2L receptor. To this end, we combined the macroscopic and single-channel analysis, the use of different agonists [GABA and muscimol (MSC)] and flurazepam (FLU) as a modulator. As expected, the β2E155C mutation caused a vast right shift of the dose–response (for GABA and MSC) and, additionally, dramatic changes in the time course of current responses, indicative of alterations in gating. Mutated receptors showed reduced maximum open probability and enhanced receptor spontaneous activity. Model simulations for macroscopic currents revealed that the primary effect of the mutation was the downregulation of the preactivation (flipping) rate. Experiments with MSC and FLU further confirmed a reduction in the preactivation rate. Our single-channel analysis revealed the mutation impact mainly on the second component in the shut times distributions. Based on model simulations, this finding further confirms that this mutation affects mostly the preactivation transition, supporting thus the macroscopic data. Altogether, we provide new evidence that the β2E155 residue is involved in both binding and gating (primarily preactivation).

Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity.

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1β2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity.

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II Signaling within Hippocampal Glutamatergic Postsynapses during Flurazepam Withdrawal

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, Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates GluA1 subunits at Ser831, 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 (Thr286) 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.

Effect of the triazolobenzodiazepine estazolam on hepatic drug-metabolizing enzyme activity in rats.

Oral doses of the sedative/hypnotic estazolam (500 mg kg-1 day-1) to rats for 21 days caused statistically significant increases in liver weight, ascorbate excretion, cytochrome P-450 concentrations, and in aniline hydroxylase, ethylmorphine N-demethylase and glutathione S-transferase activities, as did approximately equivalent doses of flurazepam hydrochloride. Histologically, the centrilobular hepatocytes were enlarged. Some of these parameters were also increased after doses of estazolam of 100 mg kg-1 day-1, but not after 5 mg kg-1 day-1, which is about 50-fold greater than a clinical dose. Estazolam was a much less potent enzyme inducer than phenobarbitone under the conditions of these studies.

Down-Regulation of Synaptic GluN2B Subunit-Containing N-methyl-d-aspartate Receptors: A Physiological Brake on CA1 Neuron α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Hyperexcitability during Benzodiazepine Withdrawal

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.

Immunogold electron microscopic evidence of differential regulation of GluN1, GluN2A, and GluN2B, NMDA‐type glutamate receptor subunits in rat hippocampal CA1 synapses during benzodiazepine withdrawal

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.

Calcium/Calmodulin-Dependent Protein Kinase II Mediates Hippocampal Glutamatergic Plasticity During Benzodiazepine Withdrawal

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.

Positive allosteric activation of GABAA receptors bi-directionally modulates hippocampal glutamate plasticity and behaviour

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.

REDUCED MICROSCOPIC GABAR SENSITIVITY IN RAT HIPPOCAMPAL CA1 NEURONS DURING BENZODIAZEPINE WITHDRAWAL IS REVERSED BY CAMKII INHIBITION

Two days after 1‐week oral treatment with the benzodiazepine (BZ), flurazepam (FZP), L‐type voltage‐gated channel (L‐VGCC) whole‐cell Ca2+ current density is doubled and γ‐amino butyric acid type A receptor (GABAR) single‐channel open probability (Po) is reduced in cell‐attached patches from dissociated hippocampal CA1 neurons. Blockade of Ca2+ influx by systemic injection of the L‐VGCC antagonist, nimodipine 1 day after FZP treatment, restored sensitivity of FZP neurons to 5 µM GABA, increasing Po from 0.04 to 0.54 after 2 days to near control levels (0.68). We hypothesized that BZ‐induced elevation of [Ca2+]i may suppress GABAR function by activating Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Single‐channel recordings were obtained after slice pre‐incubation with membrane‐permeant CaMKII inhibitors. At 1 µM GABA, Po in control patches was 0.19, while no openings were observed in FZP patches. After pre‐incubation with KN93, Po in FZP patches was 0.73. No openings were seen with the inactive analog KN92. Pre‐incubation with mAIP, activated 'silent' single channels in FZP patches (Po=0.69) and increased the basal response of control patches (Po=0.57). Membrane‐impermeant AIP and the PKC inhibitor, chelerythrine had no effect. Persistent activation of CaMKII is required to maintain basal GABAR function as well as for BZ‐induced, negative modulation of GABAR function associated with BZ tolerance.

Increased AMPA receptor GluR1 subunit incorporation in rat hippocampal CA1 synapses during benzodiazepine withdrawal

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.

Chronic Benzodiazepine Administration Potentiates High Voltage-Activated Calcium Currents in Hippocampal CA1 Neurons

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.

Chronic benzodiazepine-induced reduction in GABA A receptor-mediated synaptic currents in hippocampal CA1 pyramidal neurons prevented by prior nimodipine injection

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 μM), significantly greater than attained in vivo (1 μM) 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.

Benzodiazepine-induced hippocampal CA1 neuron α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor plasticity linked to severity of withdrawal anxiety: differential role of voltage-gated calcium channels and N-methyl-D-aspartic acid receptors

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.

Benzodiazepine Withdrawal-Induced Glutamatergic Plasticity Involves Up-Regulation of GluR1-Containing α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptors in Hippocampal CA1 Neurons

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.

Metabolism and toxicity of two new benzodiazepine-type antileishmanial agents in hepatocytes and macrophages

With increasing reports of resistance of Leishmania to antimonials (Thakur et al., 2004) and other traditional antileishmanial drugs, the need for the discovery of new antileishmanial agents is rising. In an attempt to find new antileishmanial agents, two new benzodiazepine (BNZ) analogues (7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benodiazepine-2,5-dione (BNZ-1) and 4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2)) were synthesised, and found to be effective against leishmaniasis in mice. This study investigates the metabolism of these two drugs together with the prototype BNZ, flurazepam (FZP), using rat hepatocytes, and investigates their interaction with glutathione in macrophages. Hepatocytes (>80% viability by Trypan Blue exclusion isolated by liver perfusion with collagenase) were prepared from male Sprague-Dawley rats (200-250 g). Drugs (100 μM) were incubated with 2 × 106 viable cells/ml in Krebs-Hepes buffer, pH 7.4, in rotating round bottomed flasks under an atmosphere of 95% O2/5% CO2, at 37 °C for 3 h, and timed samples taken for metabolite measurement. Samples were extracted twice with ethyl acetate at pH 10, the combined organic phases evaporated to dryness and stored at −20 °C until analysis. Metabolites were separated by HPLC using an ACE C18 column (50 mm × 3.0 mm i.d., 5 μm packing), and a solvent gradient consisting of 0.1% formic acid: acetonitrile (starting composition 95:5%, and composition after 25 min 65:35% for FZP and 70:30% for both BNZ 1 and 2). Flow rate was 0.5 ml/min, and detection was at 230 nm. Identification of the metabolites was by mass spectrometry with both positive and negative ion electronspray ionization. The effects of 24 h exposure to the compounds (100 μM) was investigated in the macrophage cell line J774.1 in terms of reduced glutathione content (GSH) and the activity of glutathione reductase (GR). There was no evidence of significant cytotoxicity with any of the compounds at the concentration used. FZP (m/z 388) was metabolised by dealkylation of the two N-1 ethyl substituents (m/z 360 and m/z 332), followed by hydroxylation on the BNZ ring. There was no evidence for either N- or O-glucuronidation of the resulting metabolites. BNZ-1 (m/z 321) was metabolised by N-demethylation (m/z 307) followed by hydroxylation (m/z 323), mono- and di-hydroxylation of the parent (m/z 337 and m/z 353) and by glucuronidation of the mono-hydroxylated metabolite (m/z 513). BNZ-2 (m/z 287) was transformed by N-demethylation (m/z 273) and hydroxylation of the parent (m/z 303), with the latter further metabolised by O-glucuronidation (m/z 479). In addition, the hydroxylated N-demethylated product (m/z 289) was also formed. Macrophages did not produce detectable metabolism of any of the drugs. All the drugs depleted macrophage GSH significantly (p < 0.05 by ANOVA followed by Dunnett's test) with BNZ-1 and BNZ-2 causing greater depletion than FZP (40.6 ± 4.0 and 45.8 ± 8.4, respectively, compared with 55.5 ± 4.9 nmol/mg protein with FZP, n = 3). Control macrophage GSH was 74.1 ± 6.6 nmol/mg protein. The depletion in GSH was not due to inhibition of GR: only FZP caused a significant decrease in macrophage GR activity (28.0 ± 5.9 compared with 42.1 ± 8.0 nmol/ml/min in control cells, p < 0.05 by ANOVA followed by Dunnett's test, n = 3). The marked depletion of macrophage GSH indicates a potential toxic interaction in mammalian cells. The new BNZ analogues were rapidly metabolised by hepatocytes, producing N-dealkylated and multiple hydroxylated phase I metabolites, followed by glucuronidation. This rapid metabolism may limit the therapeutic effect of BNZ 1 and 2 if their metabolites are inactive against leishmaniasis and suggest the need to optimise these lead structures further to obtain compounds with reduced rates and extent of metabolism.

Role of protein kinase A in GABAA receptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment.

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.

19F NMR spectrometric determination of the partition coefficients of some fluorinated psychotropic drugs between phosphatidylcholine bilayer vesicles and water

A simple 19F NMR spectrometric method was proposed for the determination of the partition coefficients of fluorinated psychotropic drugs, trifluoperazine (TFPZ), flunitrazepam (FNZ) and flurazepam (FZ) between phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUVs) and water (buffer). Each 19F NMR spectrum of these drugs in the presence of PC SUV showed a single signal accompanying a PC concentration-depending shift change and broadening, which indicated a fast exchange of these drugs between the water phase and the PC bilayer of SUV. From the relationship between the 19F chemical shift change (Δ δ) of each drug and the PC concentration, the molar partition coefficients ( K p's) were calculated and obtained with a good precision of RSD below 6%. The fractions of the partitioned drugs calculated by using the obtained K p-values were in a good agreement with the experimental values. The results demonstrate that the 19F NMR method can be usefully applied to the determination of partition coefficients of many drugs having fluorine atom(s) without any separation procedure, especially for drugs which do not have absorption in the ultraviolet or visible region, or those having absorption but show insignificant spectral changes according to their incorporation to PC bilayers (e.g. FNZ).

Chronic benzodiazepine administration alters hippocampal CA1 neuron excitability: NMDA receptor function and expression

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, [ 3H]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.

Selective enhancement of AMPA receptor-mediated function in hippocampal CA1 neurons from chronic benzodiazepine-treated rats

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, [ 3H]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.

Effect of phosphatidylserine content on the partition coefficients of diazepam and flurazepam between phosphatidylcholine-phosphatidylserine bilayer of small unilamellar vesicles and water studied by second derivative spectrophotometry.

The affinity of the psychotropic benzodiazepine drugs diazepam (DZ) and flurazepam (FZ) to phosphatidylserine (PS) was examined since PS is abundantly contained in brain membranes. The effect of PS content on the partition coefficients (K(p)s) of these drugs between phosphatidylcholine (PC)-PS bilayer membranes of small unilamellar vesicles (SUV) and water was measured using second derivative spectrophotometry. The second derivative spectra of DZ and FZ measured in the solutions containing various amounts of PC-PS SUV clearly showed derivative isosbestic points and a distinct derivative intensity change depending on the amount of the SUV added. The derivative intensity differences (AD) of the drugs before and after addition of the SUV suspension were measured at a specific wavelength. Using the AD values, the Kp values were calculated and obtained with relative standard deviation of below 10%. The Kp values of both drugs increased according to the PS content in the PS-PC bilayer membranes of the SUV proving that both have higher affinity to the PC-PS bilayer membranes than to PC membranes. The effect was much larger for FZ, i.e., the Kp value of FZ at 30 mol% PS content increased to about five times the value for the PC SUV. This can be explained by the fact that at the experimental pH of 7.4, 80% of FZ molecules are in a cationic form (pKa=8.1), so that these molecules are highly accessible to the negatively charged PS molecules. The results support the rapid and high distribution of DZ and FZ in the central nervous system after their administration.