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