Elizabeth I. Tietz - US grants

This research will use an integrated approach to examine the adaptations in CNS function which occur with the prolonged use of CNS depressants, and to understand the mechanisms by which their chronic use alters CNS inhibitory/excitatory tone. Converging lines of evidence, including recent findings from this laboratory, suggest that the substantia nigra pars reticulata (SNpr) is a major subcortical regulatory center of CNS excitability and of basal ganglia motor output and is an ideal location in which to attempt to correlate alterations in receptor function with their behavioral consequences. The SNpr is a major site of benzodiazepine receptor downregulation following chronic benzodiazepine treatment. Further, the inhibitory GABA syste, prominent in SNpr, is also altered by chronic benzodiazepine treatment. Preliminary studies indicate an alteration in behavioral sensitivity in SNpr to locally applied GABA and benzodiazepine agonists following chronic benzodiazepine treatment. The first major goal of this research is to use both standard and autoradiographic binding techniques to quantitate and localize changes in GABA receptor number after chronic benzodiazepine administration. The time course of changes in the GABA receptor system will be compared to those already known for the benzodiazepine system. The second major goal is to use SNpr function as a substrate to evaluate the extent of tolerance and dependence to the benzodiazepines and its relationship to altered GABA and benzodiazepine receptor number, and to altered function of the GABA-benzodiazepine-chloride ionophore complex. SNpr function will be measured by the motor response following local microinjection of benzodiazepine receptor agonists and antagonists, GABA receptor agonists and an antagonists and picrotoxin. The results of this proposal may have bearing on the differential tolerance which has been observed to occur to the benzodiazepines in man. This system may provide a behavioral assay for the benzodiazepine "antagonist" Ro15-1788 by evaluation of the effects of microinjection of Ro15-1788 into SNpr. Ro15-1788 can precipitate abstinence signs following chronic benzodiazepine treatment and so may prove a valuable tool for examining dependence. This SNpr system will be useful for exploring the molecular and functional correlates of tolerance and dependence to other CNS depressants e.g., barbiturates and ethanol, whose mechanisms have well established links with alterations in GABA systems and should also be useful for evaluating the role of other drug or neuro-transmitter systems in the mechanisms underlying tolerance and dependence.

DESCRIPTION: (Applicant's Abstract) The development of tolerance to benzodiazepine (BZ) anticonvulsant actions limits their clinical value and may relate to patterns of chronic abuse. BZs potentiate GABA inhibition at the GABAA receptor (GABAR) increasing Cl conductance. Regulation of the GABAR following chronic BZ treatment is well established as one mechanism underlying BZ tolerance, yet the sequence of events at brain GABA synapses which result in BZ tolerance are not well understood. Findings of electrophysiological studies in in vitro hippocampal slices, ongoing GABAR autoradiographic studies, and our initial in situ hybridization and immunohistochemical studies of GABAR subunit mRNA and protein have established that the BZ tolerant rat hippocampus provides a useful model for studying the synaptic mechanisms of BZ tolerance and have provided a basis for the proposed studies. Studies designed to evaluate the temporal relation between the functional changes associated with chronic BZ treatment and the regulation of GABARs will be carried out in the hippocampus at several time-points after discontinuing 1 week oral flurazepam (FZP) treatment. Molecular biological, immunohistochemical and electrophysiological methods will be used to address three hypotheses: 1) GABAR subunit composition is modified by chronic BZ treatment; 2) BZ and GABA actions are attenuated by chronic BZ treatment as a function of changes in presynaptic, as well as postsynaptic, GABA transmission; and 3) that changes in GABAR composition resulting from changes in mRNA expression, thus subunit protein expression, are localized to hippocampal layers associated with GABA-mediated inhibition. The magnitude and time-course of the development and reversal of chronic BZ-induced changes in the GABAergic inhibitory system and BZ and GABA agonist sensitivity are related to the degree and time-course of changes in GABAR subunits. A change in the expression of the genes encoding GABAR subunits, thus a change in subunit composition is proposed as one mechanism for GABAR regulation, therefore the expression of mRNAs for GABAR subunits (alpha (1-5), beta (1-3) and gamma (1-2)) will be systematically studied using quantitative in situ hybridization methods in hippocampal layers and temporally correlated with changes in subunit proteins using quantitative immunohistochemical methods developed in our lab. Presynaptic GABA release will be indirectly measured by a change in the frequency of mini IPSCs. The functional consequences of chronic FZP treatment on GABA and BZ pharmacology will be measured using whole-cell patch-clamp methods to measure GABA-induced currents in CAl pyramidal cells in hippocampal slices and in acutely dissociated CAl neurons. Changes in diazepam and zolpidem's effects to potentiate GABA currents will also be assessed in these two models. A functional and molecular reorganization of GABAR synapses may provide a basis for BZ tolerance.

Benzodiazepines (BZs) which act through the GABA/A receptor (GABAR), are potent anti-convulsant for a variety of epilepsies. Their clinical usefulness is limited by the appearance of functional tolerance, commonly-held to be mediated by changes at the post-synaptic on CA1 pyramidal cells in the in vitro hippocampus. In addition to changes in post-synaptic GABAR structure (alpha1 and beta3 subunit mRNA and protein down-regulation), measured in situ, and function (decreased mIPSC amplitude and C1-channel conductance), changes in GABAergic interneuron activity are proposed to contribute to BZ tolerance. Rapid BZ antagonist-induced restoration of GABAR subunit protein levels and mIPSC amplitude suggested that both translational and post-translational mechanisms may interdependently contribute to BZ tolerance. Preliminary studies of PKA-mediated modulation of GABAR currents and the ability of a cAMP analogue to partially restore mIPSC amplitude in BZ-treated rats suggests that a modulation of PKA-mediated events, in part, contribute to GABAR system dysfunction. Additional studies have identified changes in excitatory amino acid part, contribute to GABAR system dysfunction. Additional studies have identified changes in excitatory amino acid receptor (EAAR) subunit mRNAs and protein suggesting that excitatory systems are also regulated by chronic BZ treatment in response to reduced GABA inhibition. Three central hypotheses were generated from these findings in in vitro and in situ hippocampus which will be addressed by 3 SPECIFIC AIMS: SPECIFIC AIM 1: to examine the role of intrinsic and extrinsic interneuron function in reducing GABA tone by sampling subpopulations of visualized CA1 interneurons using intracellular and whole-cell patch techniques. Specific Aim 2: is to explore the role of both 2A) translational, i.e., GABAR alpha1 subunit protein redistribution following chronic agonist and acute antagonist administration, using fluorescent co-localization and quantitative EM immunogold techniques and 2B) post-translational mechanisms, i.e., the effect of exogenous and endogenous stimulators of PKA-mediated protein phosphorylation to modulate mIPSC amplitude in CA1 pyramidal cells. SPECIFIC AIM 3: is to examine the compensatory changes which occur in excitatory amino acid receptor (EAAR) structure and function in CA1 pyramidal cells: 3A) Structural measures include: quantitative immunohistochemical techniques and Western blot analysis of microdissected hippocampus and changes in EAAR receptor binding using autoradiographic techniques. 3B) Whole-cell slice patch techniques will e used to measure EAAR receptor-mediated, evoked and miniature EPSC amplitude and decay kinetics. EAAR function will also be assessed using microfluorometric measurements of Ca2+ uptake into acutely dissociated CA1 pyramidal cells. Understanding the nature of the changes at inhibitory and excitatory synapses may allow the design of drugs and approaches to circumvent tolerance and allow us to gain a better understanding of basic mechanisms involved in the dysfunction of these receptor systems during anticonvulsant drug treatment of epileptic patients.

DESCRIPTION (provided by applicant): The neurophysiological mechanisms underlying withdrawal syndromes reflecting physical dependence on a variety of drugs of abuse, including the benzodiazepines (BZs), are unknown. Using a well-established rat model of chronic BZ treatment, we have identified changes in hippocampal excitatory amino acid receptors temporally associated with anxiety-like behavior, a sign of withdrawal. CA1 neuron changes include increases in alpha-amino-3-hydroxy-5-methyl-4-isozaxolepropionic acid receptor (AMPAR) current amplitude and conductance, and increases in AMPAR binding and GluR1 subunit levels. When AMPAR currents increase, N-methyl-D-aspartate receptor (NMDAR)-evoked currents, NMDA efficacy and NR2B subunit levels are reduced. NMDA antagonist treatment during withdrawal reverses NMDAR down regulation, allowing more prolonged expression of anxiety-like behavior. AMPAR antagonist treatment prevents subsequent AMPAR upregulation. These findings suggest that enhanced AMPAR function contributes to BZ-induced withdrawal through NMDAR-dependent hippocampal pathways, and are reminiscent of well-described mechanisms underlying activity-dependent plasticity of hippocampal excitatory synapses. Similar mechanisms may be involved in behavioral plasticity during BZ withdrawal. The working hypothesis is that localized remodeling of hippocampal CA1 neuron excitatory synapses is a central feature underlying BZ physical dependence, expressed as withdrawal-induced anxiety-like behavior, and has essential characteristics analogous to those associated with activity-dependent synaptic plasticity. Three specific hypotheses focus on the subunit dependence of the functional and structural alterations that occur at CA1 neuron EAAR synapses after withdrawal from 1-week flurazepam treatment. AMPA and NMDAR function will be studied at selected time-points after drug removal, when anxiety-like behavior is expressed, using whole-cell and outside-out patch techniques in hippocampal slices and acutely dissociated CA1 neurons. The first aim will be to explore AMPAR channel properties using GluR1 subunit-selective neurophysiological and pharmacological tools. The second aim will use a similar approach to study the NR2B-subunitdependence of decreased NMDAR function at CA1 synapses. The third aim is to use light microscopic and electron microscopic immunohistochemical approaches to investigate the structural changes in AMPARs and NMDARs at CA1 synapses that contribute to changes in hippocampal excitatory function and to anxiety-like behavior. Rational approaches to the treatment of physical dependence on drugs of abuse can emerge from a better understanding of the neurophysiological mechanisms underlying withdrawal phenomena.