Chronic Benzodiazepine Treatment Research Articles

Chronic benzodiazepine treatment decreases spine density in cortical pyramidal neurons

The adult brain retains a substantial capacity for synaptic reorganization, which includes a wide range of modifications from molecular to structural plasticity. Previous reports have demonstrated that the structural remodeling of excitatory neurons seems to occur in parallel to changes in GABAergic neurotransmission. The function of neuronal inhibitory networks can be modified through GABAA receptors, which have a binding site for benzodiazepines (BZ). Although BZs are among the most prescribed drugs, is not known whether they modify the structure and connectivity of pyramidal neurons. In the present study we wish to elucidate the impact of a chronic treatment of 21 days with diazepam (2mg/kg, ip), a BZ that acts as an agonist of GABAA receptors, on the structural plasticity of pyramidal neurons in the prefrontal cortex of adult mice. We have examined the density of dendritic spines and the density of axonal en passant boutons in the cingulate cortex. Although no significant changes were observed in their anxiety levels, animals treated with diazepam showed a decrease in the density of spines in the apical dendrites of pyramidal neurons. Most GFP-expressing en passant boutons in the upper layers of the cingulate cortex had an extracortical origin and no changes in their density were detected after diazepam treatment. These results indicate that the chronic potentiation of GABAergic synapses can induce the structural remodeling of postsynaptic elements in pyramidal neurons.

Synaptic and extrasynaptic GABAA receptors in benzodiazepine withdrawal: effects of pregnanolone and gaboxadol in benzodiazepine‐dependent monkeys

Benzodiazepines and neuroactive steroids positively modulate GABAA receptors through different binding sites. These drugs share many behavioral effects including the ability to reverse benzodiazepine withdrawal; however during chronic benzodiazepine treatment, tolerance develops to benzodiazepines and not to neuroactive steroids. This difference might be related to actions of neuroactive steroids at benzodiazepine‐insensitive extrasynaptic GABAA receptors. This study examined the ability of gaboxadol, an orthosteric agonist at extrasynaptic GABAA receptors, to attenuate flumazenil‐lever responding, a sign of benzodiazepine withdrawal. Four rhesus monkeys received 5.6 mg/kg/day diazepam and discriminated 0.1 mg/kg flumazenil. Gaboxadol (1.78–5.6 mg/kg) did not produce flumazenil‐lever responding or shift flumazenil dose‐response curves whereas pregnanolone, a neuroactive steroid, shifted the curves rightward. Because pregnanolone attenuated withdrawal whereas agonist action only at extrasynaptic GABAA receptors (gaboxadol) did not, it is unlikely that those receptors account for the difference between benzodiazepines and neuroactive steroids in benzodiazepine‐treated monkeys. This difference is more likely due to changes in synaptic GABAA receptors, such as subunit composition or coupling of binding sites. Supported by USPHS grants R01DA09157 and K05DA017918 (CPF).

Prolonged-release melatonin versus placebo for benzodiazepine discontinuation in patients with schizophrenia: a randomized clinical trial - the SMART trial protocol.

BackgroundTreatment of schizophrenia frequently includes prolonged benzodiazepine administration despite a lack of evidence of its use. It is often difficult to discontinue benzodiazepines because of the development of dependence. We aim to assess if melatonin can facilitate the withdrawal of prolonged benzodiazepine administration in patients with schizophrenia. Furthermore, we aim to investigate the association of benzodiazepine dose reduction with the following clinically important variables: sleep, psychophysiology, cognition, social function, and quality of life.Methods/DesignRandomized, blinded, two-armed, parallel superiority trial. We plan to include 80 consenting outpatients diagnosed with schizophrenia or schizoaffective disorder, 18-55 years of age, treated with antipsychotic drug(s) and at least one benzodiazepine derivative for the last three months before inclusion. Exclusion criteria: currently under treatment for alcohol or drug abuse, aggressive or violent behavior, known mental retardation, pervasive developmental disorder, dementia, epilepsy, terminal illness, severe co morbidity, inability to understand Danish, allergy to melatonin, lactose, starch, gelatin, or talc, hepatic impairment, pregnancy or nursing, or lack of informed consent. After being randomized to prolonged-release melatonin (Circadin®) 2 mg daily or matching placebo, participants are required to slowly taper off their benzodiazepine dose. The primary outcome measure is benzodiazepine dose at 6 months follow-up. Secondary outcome measures include sleep, psychophysiological, and neurocognitive measures. Data are collected at baseline and at 6 months follow-up regarding medical treatment, cognition, psychophysiology, sleep, laboratory tests, adverse events, psychopathology, social function, and quality of life. Data on medical treatment, cognition, psychophysiology, adverse events, social function, and quality of life are also collected at 2 and 4 months follow-up.DiscussionThe results from this trial will examine whether melatonin has a role in withdrawing long-term benzodiazepine administration in schizophrenia patients. This group of patients is difficult to treat and therefore often subject to polypharmacy which may play a role in the reduced life expectancy of patients compared to the background population. The results will also provide new information on the association of chronic benzodiazepine treatment with sleep, psychophysiology, cognition, social function, and quality of life. Knowledge of these important clinical aspects is lacking in this group of patients.Trial RegistrationClinicalTrials NCT01431092

Chronic benzodiazepine treatment does not alter interactions between positive GABAA modulators and flumazenil or pentylenetetrazole in monkeys

Benzodiazepines and neuroactive steroids are positive c-aminobutyric acid(A) (GABA(A)) modulators acting at distinct binding sites; during benzodiazepine treatment, tolerance develops to many behavioral effects of benzodiazepines, although cross tolerance typically does not develop to neuroactive steroids. To determine whether differential changes in binding sites contribute to these behavioral differences, interactions between GABA(A) modulators were studied in two groups of four monkeys: one otherwise untreated group discriminated 0.178 mg/kg of the benzodiazepine midazolam; the other received 5.6 mg/kg/day of diazepam and discriminated 0.1 mg/kg of flumazenil, which binds to benzodiazepine sites without modulating GABA(A) receptors. In untreated monkeys, flumazenil antagonized midazolam but not the neuroactive steroid pregnanolone, whereas pentylenetetrazole (a negative modulator acting at a third site) antagonized both positive modulators. In diazepam-treated monkeys, 0.1 mg/kg of flumazenil or 32 mg/kg of pentylenetetrazole produced flumazenil-lever responding, which was reversed by midazolam and pregnanolone. As the flumazenil dose increased, larger doses of midazolam, but not pregnanolone, were needed to reverse flumazenil-lever responding. When the pentylenetetrazole dose increased, larger doses of both positive modulators were needed. Thus, interactions between GABA(A) modulators were not different between diazepam-treated and untreated monkeys and do not reveal changes in binding sites that could account for reported differences between benzodiazepines and neuroactive steroids.

Selective changes in sensitivity to benzodiazepines, and not other positive GABAA modulators, in rats receiving flunitrazepam chronically

Tolerance and dependence can develop during chronic benzodiazepine treatment; however, cross tolerance and cross dependence to positive modulators acting at other sites on GABA(A) receptors might not occur. The current study evaluated changes in sensitivity to positive GABA(A) modulators during chronic treatment with the benzodiazepine flunitrazepam to determine whether cross tolerance and cross dependence varied as a function of site of action. Eight rats responded under a fixed ratio 20 schedule of food presentation. Dose-effect curves were determined before, during and after chronic treatment with one or two daily injections of 1 mg/kg of flunitrazepam. Prior to chronic treatment, benzodiazepines (flunitrazepam, midazolam), a barbiturate (pentobarbital), a neuroactive steroid (pregnanolone), and drugs with primary mechanisms of action at receptors other than GABA(A) receptors (morphine, ketamine) dose-dependently decreased responding. Twice daily treatment with flunitrazepam produced 9.5- and 23-fold shifts to the right in the flunitrazepam and midazolam dose-effect curves, respectively. In contrast, dose-effect curves for other drugs either were not changed or were shifted less than or equal to fourfold to the right. Decreased sensitivity to benzodiazepines and not to a barbiturate or a neuroactive steroid during chronic flunitrazepam treatment indicates that tolerance and cross tolerance developed only to benzodiazepines. Despite similar acute behavioral effects among positive GABA(A) modulators, the differential development of cross tolerance suggests that adaptations at GABA(A) receptors produced by chronic benzodiazepine treatment differentially affect positive modulators depending on their site of action; such differences might be exploited to benefit patients treated daily with positive GABA(A) modulators.

Analysis of the chronic intake of and withdrawal from diazepam on emotional reactivity and sensory information processing in rats

It has been demonstrated that, on abrupt withdrawal, patients with chronic exposure can experience a number of symptoms indicative of a dependent state. In clinical patients, the earliest to arise and most persistent signal of withdrawal from chronic benzodiazepine (Bzp) treatment is anxiety. In laboratory animals, anxiety-like effects following abrupt interruption of chronic Bzp treatment can also be reproduced. In fact, signs that oscillate from irritability to extreme fear behaviours and seizures have been described already. As anxiety remains one of the most important symptoms of Bzp withdrawal, in this study we evaluated the anxiety levels of rats withdrawn from diazepam. Also studied were the effects on the motor performance and preattentive sensory gating process of rats under diazepam chronic treatment and upon 48-h withdrawal on three animal models of anxiety, the elevated plus-maze (EPM), ultrasonic vocalizations (USV) and startle + prepulse inhibition tests. Data obtained showed an anxiolytic- and anxiogenic-like profile of the chronic intake of and withdrawal from diazepam regimen in the EPM test, 22-KHz USV and startle reflex. Diazepam chronic effects or its withdrawal were ineffective in promoting any alteration in the prepulse inhibition (PPI). However, an increase of PPI was achieved in both sucrose and diazepam pretreated rats on 48-h withdrawal, suggesting a procedural rather than a specific effect of withdrawal on sensory gating processes. It is also possible that the prepulse can function as a conditioned stimulus to informing the delivery of an aversive event, as the auditory startling-eliciting stimulus. All these findings are indicative of a sensitization of the neural substrates of aversion in diazepam-withdrawn animals without concomitant changes on the processing of sensory information.

Changes in relative potency among positive GABAA receptor modulators upon discontinuation of chronic benzodiazepine treatment in rhesus monkeys

Benzodiazepine treatment can result in dependence as evidenced by signs of withdrawal upon discontinuation of use. Positive GABA(A) receptor modulators were examined for their capacity to attenuate flumazenil-like discriminative stimulus effects (i.e., withdrawal) that emerge upon discontinuation of chronic benzodiazepine treatment. Rhesus monkeys receiving chronic diazepam (5.6 mg(-1) kg(-1) 24 h(-1) p.o.) discriminated flumazenil (0.1 mg/kg s.c.) from vehicle. Upon discontinuation of diazepam treatment, responding switched from the vehicle to the flumazenil lever, although at different times among monkeys. The shorter-acting benzodiazepine lorazepam (3.2 mg(-1) kg(-1) 8 h(-1)) was substituted for diazepam and, 11 h after lorazepam, monkeys consistently responded on the flumazenil lever. Flumazenil-lever responding after acute lorazepam deprivation was attenuated not only by benzodiazepines (lorazepam and midazolam) but also by positive GABA(A) receptor modulators acting at neuroactive steroid (pregnanolone and alfaxalone) and barbiturate sites (pentobarbital). Deprivation-induced responding on the flumazenil lever was not attenuated by low efficacy positive GABA(A) modulators (bretazenil and L-838,417) or non-GABA(A) receptor ligands (ketamine and cocaine). Neuroactive steroids were relatively more potent than other positive GABA(A) receptor modulators in attenuating deprivation-induced flumazenil-lever responding, as compared to their relative potency in monkeys discriminating midazolam and otherwise not receiving benzodiazepine treatment. These results suggest that positive GABA(A) receptor modulators acting at different sites attenuate withdrawal induced by discontinuation of benzodiazepine treatment, consistent with previous studies suggesting that the same compounds attenuate flumazenil-precipitated withdrawal. Differences in the relative potency of positive modulators as a function of acute versus chronic benzodiazepine treatment suggest that neuroactive steroids, in particular, are especially potent in benzodiazepine-dependent animals.

The effect of chronic lorazepam administration in aging mice

To assess benzodiazepine tolerance in aged animals, lorazepam or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice. Pharmacodynamic and neurochemical endpoints were examined on days 1 and 14 of drug administration. There was no age-related significant difference in plasma lorazepam levels. Young and middle-aged animals demonstrated behavioral tolerance to lorazepam, while the aged animals showed a similar trend which failed to reach significance. In addition, aged animals also showed a trend toward tolerance to the anticonvulsant effects of lorazepam. There were no changes in α1 mRNA levels in cortex or hippocampus following administration of lorazepam when compared to vehicle-treated animals in any age group. Aged animals, however, had an initial increase in α1 mRNA expression in cortex and hippocampus on day 1 of vehicle treatment followed by decreased expression on day 14. These age-related changes were abolished by lorazepam administration. In summary, age-related sensitivity to the effects of lorazepam was not demonstrated in the present study. However, comparison of these data to other studies indicates that the effect of chronic benzodiazepine treatment may be specific to the benzodiazepine administered, the technique used to quantify mRNA expression changes, the subunits of the GABA A receptor investigated and the brain region analyzed. The phenomenon of benzodiazepine sensitivity in the elderly is an area of research which remains controversial and may well be compound specific. Determining benzodiazepines that do not produce pharmacodynamic sensitivity, such as lorazepam, may allow more careful prescribing and dosing of these drugs, and perhaps even the development of specific agents which could avoid this sensitivity.

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.

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.

Chronic benzodiazepine treatment of cells expressing recombinant GABA(A) receptors uncouples allosteric binding: studies on possible mechanisms.

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA(A) receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [3H]flunitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.

Role of bicarbonate ion in mediating decreased synaptic conductance in benzodiazepine tolerant hippocampal CA1 pyramidal neurons

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 μM APV and 10 μM DNQX. The micropipette contained 130 mM CsCl and 1 μM 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.

Silent GABAASynapses during Flurazepam Withdrawal Are Region-Specific in the Hippocampal Formation

Whole-cell patch-clamp recordings were made from CA1 pyramidal and dentate gyrus granule cells (GCs) in hippocampal slices to assess the effects of withdrawal from chronic flurazepam (FRZ) treatment on the function of synaptic GABAA receptors. In slices from control rats, acute perfusion of FRZ (30 microM) increased the monoexponential decay time constant of miniature IPSCs (mIPSCs) in CA1 and GCs (from 3.4 +/- 0.6 to 7.6 +/- 2.1 msec and from 4.2 +/- 0. 6 to 7.1 +/- 1.8 msec, respectively) but did not change their mean conductance, 10-90% rise time, or frequency of occurrence. Withdrawal (2-5 d) from chronic in vivo FRZ treatment (40-110 mg/kg per day, per os) resulted in a dramatic loss of mIPSCs in CA1 neurons. On day 5 of withdrawal, no mIPSCs could be recorded in 40% of CA1 pyramidal cells. In the remaining 60% of the neurons, mIPSCs had a reduced mean conductance (from 0.78 +/- 0.12 nS in vehicle-treated controls to 0.31 +/- 0.05 nS) and a diminished frequency of occurrence (from 20.7 +/- 7.9 to 4.1 +/- 0.6 Hz). We have estimated that >80% of GABAA synapses on CA1 pyramidal cells had become silent, whereas at still-active synapses the number of functional GABAA receptor channels decreased by 60%. This reduction rapidly reverted to control levels on day 6 of withdrawal. FRZ withdrawal did not alter mIPSC properties in GCs. Our results are consistent with the hypothesis that chronic benzodiazepine treatment leads to a reduced number of functional synaptic GABAA receptors in a region-specific manner that may stem from differences in the subunit composition of synaptic GABAA receptors.

Depression of early and late monosynaptic inhibitory postsynaptic potentials in hippocampal CA1 neurons following prolonged benzodiazepine administration: role of a reduction in Cl- driving force.

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.

Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect is independent of GABA, and that the benzodiazepine and GABA sites may not be equally coupled to the down-regulation process, with the benzodiazepine site being the more dominant. The biochemical mechanism underlying the benzodiazepine-mediated down-regulation of the alpha 1 subunit protein seems to involve the activity of staurosporine-sensitive protein kinases.

THE BEHAVIOURAL AND NEURONAL EFFECTS OF THE CHRONIC ADMINISTRATION OF BENZODIAZEPINE ANXIOLYTIC AND HYPNOTIC DRUGS

Benzodiazepine anxiolytic and hypnotic drugs are some of the most widely prescribed drugs in the Western world. Despite this fact, the mechanisms that underlie the development of tolerance to, and dependence upon, benzodiazepines are poorly understood. The aim of this review is to summarize and critically evaluate the experimental evidence relating to the chronic behavioural and neuronal effects of benzodiazepines. Behavioural studies in animals generally indicate that tolerance gradually develops to the muscle relaxant, ataxic, locomotor and anticonvulsant effects of benzodiazepines. The evidence relating to the development of tolerance to the anxiolytic effects of benzodiazepines is less clear. The literature on the possible mechanisms of benzodiazepine tolerance and dependence is large, highly complex and difficult to interpret. The effect of chronic benzodiazepine treatment varies enormously as a function of the benzodiazepine used and the treatment schedule employed. Many studies have demonstrated a down-regulation of benzodiazepine binding sites, although affinity is usually unchanged. The evidence relating to the number and affinity of GABA A binding sites is unclear. Some studies suggest that chronic benzodiazepine administration results in a reduction in the number of C − channels associated with the GABA A receptor complex, although it is not clear that the efficacy of the GABA binding site in operating the Cl − channel necessarily changes. There is, however, substantial evidence to support the hypothesis that chronic benzodiazepine treatment results in a reduction in the coupling between the GABA A and benzodiazepine binding sites (the “functional uncoupling hypothesis”). Although some electrophysiological studies suggest that chronic benzodiazepine treatment results in a subsensitivity to GABA, this effect seems to be highly area-specific.

γ-Aminobutyric acid A receptor regulation: heterologous uncoupling of modulatory site interactions induced by chronic steroid, barbiturate, benzodiazepine, or GABA treatment in culture

Prolonged administration of anxiolytic, sedative, and anticonvulsant drugs that act through the GABA A receptor (GAB AR) can evoke tolerance and dependence, suggesting the existence of an endogenous mechanism(s) for altering the ability of such agents to interact with the GABA AR. Uncoupling appears to be one such mechanism. This is a decrease in the allosteric interactions between the benzodiazepine (BZD) recognition site and other agonist or modulator sites on the GABA AR, as measured by potentiation of [ 3H]flunitrazepam ([ 3H]FNZ) binding. To investigate the mechanism(s) of uncoupling, neuronal cultures were treated chronically with 3α-hydroxy-5β-pregnan-20-one (pregnanolone), pentobarbital, flurazepam, or GABA, then tested for enhancement of [ 3H]FNZ binding by these substances. The results indicate that BZDs, barbiturates, and steroids, as well as GABA itself, are capable of inducing both heterologous and homologous uncoupling. Surprisingly, different chronic drug treatments produce different patterns of homologous and heterologous uncoupling. Chronic exposure to pregnanolone, GABA, flurazepam or pentobarbital induces complete uncoupling of barbiturate-BZD site interactions, partial uncoupling of GABA-BZD site interactions, but different amounts of uncoupling of steroid-BZD site interactions. In addition, the EC 50 for pregnanolone-induced homologous uncoupling (1.7 μM) is over an order of magnitude greater than that for heterologous uncoupling of GABA and BZD sites (82 nM). Moreover, heterologous uncoupling by pregnanolone is inhibited by the GABA site antagonist SR-95531, whereas homologous uncoupling by pregnanolone is resistant to SR-95531. Therefore, there are at least two distinct ways in which GABA AR modulatory site interactions can be regulated by chronic drug treatment.

Flumazenil before Electroconvulsive Therapy

Elana B. Doering, M.D., Ph.D., Research Fellow, Department of Anesthesia.William A. Ball, M.D., Ph.D., Inpatient Medical Director, Department of Psychiatry, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104.To the Editor:--We were very interested to read Hanania's report of flumazenil administration before electroconvulsive therapy (ECT). [1]In his two cases, flumazenil, given immediately before ECT, reversed the sedative effects of benzodiazepine premedication; seizures of acceptable duration followed. This report raises several issues that deserve further consideration, regarding the seizures and the therapeutic outcome of ECT.Regarding the seizures, the effects of flumazenil are unclear. In Hanania's cases, flumazenil was given before any ECT stimulus was delivered. We do not know what the outcome would have been without flumazenil in these two patients. Furthermore, in one case, extra flumazenil was given after an unsuccessful ECT attempt; however, Hanania does not note whether the stimulus current was increased at the same time, as is usually done when the first seizure is inadequate. So, we cannot surely attribute the subsequent success to the extra flumazenil. On the other hand, acute benzodiazepine administration has been shown to raise seizure threshold and shorten seizure duration. [2](In this context, it is interesting that flumazenil did not cause unacceptably long seizures.) We may guess that flumazenil led to better seizure outcomes than otherwise might have occurred after benzodiazepine administration, but this remains to be demonstrated.Clinically, of course, the most important issues concern the effects of flumazenil and benzodiazepines on the therapeutic outcome of ECT. Several questions arise here. First, do benzodiazepines affect clinical outcome, independent of seizure duration? Second, do acute versus chronic benzodiazepines have different effects? Third, does flumazenil reverse benzodiazepines' effects on ECT outcome? Finally, does flumazenil have relevant psychoactive effects of its own?Several authors have raised the question of benzodiazepine interference with therapeutic effects of ECT. In one study in humans, the authors determined that unilateral ECT was less effective in patients who were receiving chronic benzodiazepines, despite adequate seizures. [2]A study in mice investigated behavioral responses to serial electroshocks. Behavioral changes that usually followed the shocks did not occur when diazepam was given, despite apparently identical seizures. This was noted whether diazepam was given before or after the shocks, suggesting that diazepam's antitherapeutic effects are independent of its effects on the seizures. [3].The question of chronic versus acute benzodiazepine administration in this setting remains largely unaddressed. Hanania's patients received lorazepam only once, immediately before ECT. In contrast, the patients in the human study cited above received chronic benzodiazepine treatment. Because long-term psychoactive effects of medications often require chronic treatment, we might suppose that one-time dosing is relatively benign. However, the mouse study suggests that the presence of benzodiazepines after ECT, even from one dose of a long-acting agent, may alter the behavioral response.The effects of flumazenil on ECT outcome remain unexplored. Of note, some authors have implicated benzodiazepine receptors and enhancement of the effects of gamma-aminobutyric acid in recovery from depression. [4]Will flumazenil reverse benzodiazepine effects on ECT outcome? Will it turn out to have its own clinical effects in patients receiving ECT or in other depressed patients? Answering these questions may clarify the mechanism of ECT therapeutic effects and illuminate the biochemical features of depression as well.In the meantime, we may consider benzodiazepine sedation and flumazenil reversal in ECT patients who will not otherwise accept treatment. However, we should be prepared for the possibility of a reduced clinical antidepressant response.Elana B. Doering, M.D., Ph.D., Research Fellow, Department of Anesthesia.William A. Ball, M.D., Ph.D., Inpatient Medical Director, Department of Psychiatry, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104.(Accepted for publication May 31, 1995.)

Reduction of GABA-mediated inhibitory postsynaptic potentials in hippocampal CA1 pyramidal neurons following oral flurazepam administration.

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.

Impairment of feedforward inhibition in CA1 region of hippocampus after chronic benzodiazepine treatment

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.