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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.

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.

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.

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 substantia nigra pars reticulata (SNpr), a brain area rich in GABA and benzodiazepine receptors, is thought to be involved in the regulation of seizure activity. It has been shown to be a site of anticonvulsant action of substances that affect GABA transmission. The anti-pentylenetetrazol (PTZ) activities of intranigral of muscimol, a GABAA receptor agonist; two benzodiazepines, midazolam and flurazepam; and GABA were examined. Microinjection of a wide dose range of both GABA and muscimol into the SNpr failed to show anti-PTZ seizure activity. Intranigral injections of midazolam and flurazepam showed clear, dose-dependent anti-PTZ effects. Ro15-1788, a benzodiazepine receptor antagonist, reversed the anticonvulsant effects of midazolam when both were infused intranigrally. Intranigral infusion of muscimol or flurazepam protected rats from bicuculline-induced tonic seizures. The results suggest that the anti-PTZ effects of benzodiazepines in SNpr might not be mediated through GABAA receptors. Another possibility is that nigral neurons bearing GABAA receptors functionally linked to benzodiazepine sites may not be representative of the whole population of nigral neurons inhibited by GABA agonists. This could result in different patterns of inhibition of nigral efferent activity by GABAA agonists and benzodiazepines.

The effect of a benzodiazepine, flurazepam, on the spontaneous activity of neurons in the pars reticulata of the substantia nigra was studied in chloral hydrate anesthetized rats. Flurazepam produced a dose-related suppression of neuronal activity. In rats that were chronically treated with flurazepam, tolerance to flurazepam was present after 7 and 28 days, but not after only 3 days of treatment. Tolerance persisted at least 2, but not 7 days after 4 weeks of chronic treatment.