Elsevier

Brain Research

Volume 738, Issue 1, 28 October 1996, Pages 103-108
Brain Research

Research report
Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex

https://doi.org/10.1016/0006-8993(96)00764-0Get rights and content

Abstract

In the present communication we have investigated the pharmacological properties of the GABAA receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABAA receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABAA receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [3H]flunitrazepam revealed that neither the Kd values or the Bmax were modified during aging. Moreover, Cl 218 872 displacement of [3H]flunitrazepam showed no age-related modifications on either the Kis or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [3H]muscimol demonstrated a decrease in the Bmax of the low affinity [3H]muscimol binding sites in aged rats (4.3 ± 0.8 pmol/mg protein vs. 2.3 ± 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABAA receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.

Introduction

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter at the vertebrate central nervous system (CNS). GABA binds to two different receptor types expressed in CNS: the ionotropic GABAA receptors and the metabotropic GABAB receptors. The GABAA receptor complex is the target of some clinically relevant drugs, such as the benzodiazepines and barbiturates, which allosterically modulate the GABA transmission 41, 42. On the other hand, 13 different subunits of the GABAA receptor have been cloned. Based on the sequence similarity, four different families could be discriminated. Each family comprised several isoforms: α1–6, β1–3, γ1–3 and δ (see 7, 26, 38for reviews). In recombinant receptors, the expression of α, β and γ subunits is the minimal conformation to resemble all the pharmacological properties of native GABAA receptor complex 28, 30, 38.

In isolated cortical membranes, two different types of benzodiazepine binding sites could be discriminated by, among others, the triazolopyridazine Cl 218 872 and the imidazopyridine zolpidem. The Type I displays high affinity whereas the Type II has low affinity for these compounds 16, 37, 39. On the other hand, it has been postulated that the α subunits determine the pharmacological properties of the benzodiazepine site 27, 28.

Concerning to the age-dependent modifications of the GABAA receptor complex controversial results have been reported, depending on the experimental approach, the rat strain and the parameters tested. We have previously analyzed the age-related modifications on the pharmacological properties of GABAA receptors in membranes from rat hippocampus and cortex 33, 34, 36. Our results demonstrated a strong regional specificity. In this sense, at the hippocampal formation the proportion and the allosteric interactions of the Type I benzodiazepine binding sites increased with aging 33, 34. On the other hand, in membranes obtained from whole cerebral cortex, no age-related modifications were observed [36]. However, the cerebral cortex (as many other brain regions) is heterogeneous (see [17]). Thus, local alterations in the pharmacological properties of the GABAA receptors could exist in absence of extensive cortical changes. In this sense, it is known that the prefrontal cortex participates in cognitive and mnemotecnic functions and in the mediation or arousal and stress 17, 20and, on the other hand, it is also known the existence of age-dependent modifications in both processes, i.e. the memory and/or learning capacity decreases and the sensitivity to stress increases 2, 8, 10, 12, 15, 24. The GABAA receptor could be implicated in both processes. Therefore, in this communication we have analyzed the pharmacological properties of the GABAA receptors from prefrontal cortical membranes of 3-month-old and 24-month-old Wistar rats.

Section snippets

Materials

[3H]Flunitrazepam (86.6 Ci/mmol), [3H]zolpidem (58.3 Ci/mmol) and [3H]muscimol (30.3 Ci/mmol) were purchased from New England Nuclear. The triazolopyridazine Cl 218 872 was a gift from Cyanamid. All other benzodiazepines were kindly provided by Hoffman-LaRoche. GABA was from Sigma.

Membrane preparation

Three-month-old and 24-month-old male Wistar rats were killed by decapitation and the prefrontal cortex was dissected as described elsewhere [43]. Briefly, the olfactory bulbs were cutting off and the prefrontal

Absence of age-dependent modifications on the benzodiazepine binding sites

The benzodiazepine binding properties of adult (3 months old) and aged (24 months old) prefrontal cortical membranes were analyzed by [3H]flunitrazepam saturation experiments (Fig. 1A and Table 1) and by Cl 218 872 displacement experiments.

The Scatchard analysis of the saturation curves are shown in Table 1. As expected, [3H]flunitrazepam binds to a single high affinity binding site in both ages (Hill factor close to unity, not shown). No age-dependent modifications were observed in either the

Discussion

We have previously investigated the age-dependent modifications on the pharmacological properties and sub-unit composition of the GABAA receptor in several rat brain regions (33, 34, 36; Gutierrez et al., in press). In the present communication we have analyzed the pharmacological properties of the GABAA receptors expressed in prefrontal cortical membranes from adult and aged Wistar rats. The prefrontal cortex plays an important role in cognitive and/or memory processes [17]and in the mediation

Acknowledgements

This work was supported by Grant PB93-0739 from DGICYT.

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