Influence of benzodiazepine binding site ligands on fear-conditioned contextual memory

Abstract

Eight compounds that bind to the benzodiazepine binding site on the γ-amino butyric acid_A (GABA_A) receptor were assessed for their influence on contextual memory, an aspect of memory affected in various cognitive disorders including Alzheimer's disease. Using a Pavlovian fear-conditioning paradigm, each ligand was evaluated in C57Bl/6 mice in regards to its direct affect on contextual memory and whether the ligand could attenuate scopolamine-induced contextual memory impairment. Of the eight ligands tested, one impaired contextual memory (agonist), six attenuated scopolamine-induced contextual memory impairment (inverse agonists), and one antagonized the ability of an inverse agonist to attenuate scopolamine-induced contextual memory impairment. Hence, further demonstrating the bi-directional influence benzodiazepine binding site ligands are able to exert on memory modulation. This study serves as an initial starting point in the development of pharmacological tools to be used in deciphering how GABA_A receptors influence contextual memory.

Introduction

The striking failure of memory observed in senile dementia of the Alzheimer's type has been attributed to the degeneration of presynaptic neuronal function of the basal forebrain cholinergic system (for review, see Whitehouse, 1998). Pharmacological treatment strategies for the cognitive decline associated with Alzheimer's disease have primarily focused on cholinomimetic mechanisms to address the cholinergic hypofunction. To date, this approach has achieved limited utility (Sarter and Bruno, 1997). These strategies tend to result in direct postsynaptic stimulation causing constant, tonic neuronal activity, which is unfavorable to normal cognitive processing (Sarter and Bruno, 1997) An effective pharmacological treatment for memory dysfunction associated with cholinergic hypofunction is one that would augment performance-associated increases in the cortical acetylcholine efflux of the surviving cholinergic neuronal processes, while at the same time preserving the highly complex transmission patterns inherent to cortical cholinergic pathways (Sarter and Bruno, 1997). A potential, and under-appreciated, therapeutic approach to achieving this goal would be to disinhibit γ-amino butyric acid (GABA) mediated regulatory control over the surviving cholinergic neurons, but only when cognitive variables simultaneously activate cortical cholinergic activity, thereby selectively augmenting cognitive functions that are due to cortical acetylcholine efflux (Sarter and Bruno, 1997). Such an approach would likely take advantage of the unique bi-directional properties of benzodiazepine binding site ligands to allosterically affect GABA mediated activation of the γ-amino butyric acid_A (GABA_A) receptor chloride ionophore complex, and would consequently have no direct activational effect in the absence of GABA release Abe et al., 1998, Sarter and Bruno, 1997. Moreover, several benzodiazepine binding site ligands have been clinically evaluated in the treatment of cognitive deficits associated with senile dementia Robbins et al., 1997, Takada et al., 1996.

Benzodiazepine binding site ligands are structurally diverse compounds that bind to a specific binding site on the GABA_A receptor chloride channel complex. A benzodiazepine binding site ligand can augment (agonism), attenuate (inverse agonism), or block the effect of an agonist or inverse agonist (antagonism) on GABAergic mediated chloride flux. Consequently, benzodiazepine binding site ligands exhibit bi-directional influences that either diminishes or potentiates multiple behaviors including anxiety, sleep regulation, eating behavior, convulsive states, body temperature regulation, muscle tension, vigilance, and cognitive processing. The anterograde amnesic properties of several of the benzodiazepine binding site ligands and conversely, the enhancement of performance in learning and memory tasks observed with other benzodiazepine binding site ligands has been recognized for some time Lister, 1985, Venault et al., 1986.

GABA_A receptors are composed of a combination of transmembrane protein subunits, most of which exist in multiple polypeptide isoforms (α_1–6, β_1–4, γ_1–4, δ, ρ_1–3, ε, π, θ) Bonnert et al., 1999, Olsen and DeLorey, 1999. These subunits assemble to form heteropentameric ion channels with chloride ion selectivity and are found ubiquitously throughout the central nervous system Bonnert et al., 1999, Wisden et al., 1992. The pharmacology of a particular GABA_A receptor pentamer is influenced by the subunit isoforms that comprise it. Interestingly, studies on postmortem brains of Alzheimer's patients reveal GABA_A receptors to be relatively spared throughout the brain, even at terminal stages of the disease, while many other neurotransmitter receptors are clearly reduced (Mizukami et al., 1997). Additionally, positron emission tomography (PET) scans of the brains of living Alzheimer's patients, likewise, provide evidence for the preservation of cortical benzodiazepine binding sites (Meyer et al., 1995). Moreover, inverse agonists acting through the benzodiazepine binding site on GABA_A receptors have been reported to elicit marked cognitive improvement in rodent models of senile dementia of the Alzheimer's type, produced by chemically or electrolytically lesioning the basal forebrain (Sarter and Bruno, 1997). However, the efficacy of benzodiazepine binding site inverse agonists to attenuate lesion induced cognitive impairment is dependent on the extent of the lesion. Animals with almost complete (80–90%) loss of cortical cholinergic fibers do not exhibit much benefit from treatment, while the performance of animals with less pronounced lesions improve with treatment (McGaughy et al., 1996). The loss of cholinergic neurons in aging and dementia is far from complete, commonly in the 40–70% range Geula and Mesulam, 1996, Lehericy et al., 1993. Hence, the effects of inverse agonists acting through the benzodiazepine site on GABA_A receptors to restore neural transmission in animals with partial loss of cortical cholinergic inputs suggests potential benefit in all but late stage Alzheimer's patients. The cognitive enhancing properties of some benzodiazepine binding site ligands, as outlined above, coupled with the preservation of GABA_A receptors in Alzheimer's patients, warrants further consideration of these ligands in developing alternative therapeutic strategies in Alzheimer's disease.

In the present study, the cholinergic antagonist scopolamine was used to disrupt contextual memory, which was generated by Pavlovian fear conditioning, a rapidly acquired form of learning thought to model human explicit memory (Kim and Fanselow, 1992). More specifically, scopolamine suppresses cognitive function with respect to attention and visuo-spatial memory in both normal humans and rats by inducing cholinergic hypofunction Anagnostaras et al., 1995, Ebert and Kirch, 1998. Using a fear-conditioning paradigm, a reliable method by which to assess contextual memory in mice (Chen et al., 1996a), we have assessed a set of structurally diverse benzodiazepine binding site ligands for their ability to either directly impair contextual memory or attenuate scopolamine-induced impairment of contextual memory.