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.

Abstract-Prolonged flurazepam exposure regulates the expression of selected (a1, b2, b3) 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 (a1, a2, b1-3 and g2) 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 a1 and b3 subunit messenger RNA expression detected in a previous study, relative a1 and b3 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 b3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in b3 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 a2, b1 or b2 subunit antibody immunostaining were found in any brain region. g2 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 (a1 and b3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance. ᭧ 1999 IBRO. Published by Elsevier Science Ltd.Key words: tolerance, flurazepam, quantitative immunohistochemistry, dentate gyrus, limbic system, CA1 pyramidal cells.Although effective anxiolytics and hypnotics, the clinical use of benzodiazepines as anticonvulsants is limited by tolerance development during prolonged administration. A large body of evidence has indicated that benzodiazepine tolerance is related to a reduction in GABA A receptor-mediated fast inhibitory synaptic transmission (for reviews see Refs 2 and 27). The native GABA A receptor is a pentamer composed of combinations of homologous proteins derived from five subunit families with multiple variants (a1-6, b1-4, g1-3, d1 and e1). 8,11,34 The GABA A receptor macromolecule contains binding domains for GABA and several allosteric modulators, including a well-characterized benzodiazepine binding site. 59Benzodiazepines potentiate GABA A receptor-mediated inhibitory function by increasing the opening probability of the integral Cl Ϫ channel, 52,62 and may increase Cl Ϫ channel conductance. 13Although the synaptic mechanisms mediating benzodiazepine tolerance have not been...

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 RIIβ 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.

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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.