Developmental switch in the expression of GABA_A receptor subunits α₁ and α₂ in the hypothalamus and limbic system of the rat

Abstract

The GABA_A receptor is a pentameric ligand gated ion channel complex assembled from a family of at least 17 different subunits encoded by distinct genes. Two subunits, α₁ and α₂, exhibit age dependent expression throughout several areas of the brain. In general, the density of immunoreactive product for α₁ is greatest in the adult brain, while α₂ is highest in younger tissue. Since the developmental switch in α₁ and α₂ coincides with the end of the sensitive period for steroid-mediated sexual differentiation of the brain, we hypothesized that GABA_A receptor subunit expression may be involved in this process. We have examined the age-dependent expression of α₁ and α₂ in discrete regions of the hypothalamus and limbic system of males and females. While we did not detect any dramatic sex differences in α₁ or α₂ immunoreactive density, each region exhibited a unique developmental profile. In the ventromedial nucleus of neonatal animals immunoreactivity is highest for α₁, while in the adult the signal for α₂ is greater; the opposite of that observed in the ventrolateral thalamus. There is no age dependent change for α₁ in the preoptic area, while α₂ shows a small, but significant increase. Immunoreactive densities for both subunits increase in the arcuate nucleus and the hippocampus, but decrease in the lateral amygdala. We conclude that these regional differences in subunit expression across development determine individual characteristics of brain areas and may play a role in establishing unique physiological responses to GABA.

Introduction

More appropriately described as a receptor complex, the GABA_A receptor forms a ligand gated chloride channel that contains many modulatory binding sites. In the adult brain, activation of the GABA_A receptor usually results in hyperpolarization of the cell membrane and reduces neuronal firing. A channel consists of five subunits [55]and a variety of subunits have been cloned: α_(1–6), β_(1–4), γ_(1–4), δ and ε (see Ref. [54]). A functional channel is comprised of an α and β subunit and may include a γ subunit, but the exact stoichiometry of the complex has not been established 2, 14. Some specific subunits have been associated with distinct pharmacological properties of the receptor complex. For example, the γ₂ subunit is known to confer sensitivity for classical benzodiazepines 7, 67. However, not much is known about the specific influence of receptor subtypes on the overall function of the channel and this area remains one of active investigation.

Expression patterns of this multitude of subunits have been heavily investigated in many areas of the brain. Some subunits (such as α₁) are widely expressed in several brain areas 22, 82, while others (such as α₆) exhibit a highly restricted distribution 3, 32, 39. Subunit expression also changes over development 23, 34, 52. One brain region that has escaped in-depth examination of subunit expression is the hypothalamus. Several hypothalamic nuclei are known to contain mRNA for the rate limiting enzyme in GABA synthesis, glutamic acid decarboxylase (GAD; 16, 49, 60), and the hypothalamus binds the GABA_A receptor agonist, muscimol 47, 74. In addition, the GABAergic system in the hypothalamus has been shown to play a role in the regulation of several physiological responses, including the LH surge required for ovulation and reproductive behaviors (for review, see Ref. [46]). Both of these responses are sexually dimorphic and determined by neonatal exposure to gonadal steroids.

Sexual differentiation of the brain is a steroid-mediated process that occurs during the perinatal period. The developing brain is sensitive to circulating gonadal steroids during a restricted window of time, which for the rat is from embryonic day 18 to approximately postnatal day 7–10 (PN7-10, see Ref. [26]). During this period exposure to either testosterone or its aromatized product, estrogen, results in masculinization of the brain, a phenomenon characterized by specific anatomical and physiological features present in the adult animal.

We have been examining sex differences in amino acid neurotransmission as a possible mechanism of sexual differentiation of the rat brain [48]. We have found sex differences in both GAD mRNA content [16]and GABA concentrations [18]on PN1, with males having significantly higher levels than females. These sex differences are no longer apparent outside the sensitive period of steroid-mediated brain differentiation or prior to puberty. Several investigations have demonstrated that GABA acts as an excitatory neurotransmitter during early development and induces depolarizing potentials [15]as well as rises in internal calcium [58]in neonatal rat hypothalamic tissue. The excitatory action of GABA gradually converts to inhibitory around PN4-10 6, 58. Excitatory GABA acting at the GABA_A receptor acts as a trophic factor both in vivo [41]and in vitro [5]during early development. These observations have led us to hypothesize that an increase in GABA induced excitation in the male brain during the perinatal period may be a mechanism involved in masculinization of the neonatal brain. Since we have found sex differences in both GAD mRNA and GABA levels, we have now turned our focus to the receptor.

Though there are an abundant number of GABA_A receptor subunits, immunocytochemical evidence by Fritschy et al. [23]illustrated a developmental change in the GABA_A receptor subunits α₁ and α₂, but no change in β_2,3. Immunoreactivity for α₁ is barely detectable in several brain regions at PN0, but increases over development and is very high at PN20. In contrast, α₂ immunoreactivity is higher on PN0 and is diminished at PN20. The “switch” from α₂ to α₁ occurs at approximately PN4, which correlates with the termination of GABA acting as an excitatory neurotransmitter; however, there is currently no evidence supporting the hypothesis that the change in these two subunits plays a role in changing the nature of GABAergic neurotransmission. Rather, the change in GABA from excitatory to inhibitory is due to a change in the internal chloride concentration, which is regulated by specific chloride pumps and transporters 40, 70, 71. The timing of the α₁ and α₂ subunit switch also coincides with the beginning of the end of the sensitive period for sexual differentiation of the rat brain. These data suggest that α₁ and α₂ subunit expression may play a role in sexual differentiation. In order to further understand the function of the developmental switch in α₁ and α₂ we proposed to (1) characterize the developmental expression of α₁ and α₂ in the hypothalamus and limbic system and (2) test the hypothesis that there would be differences in the timing of the switch from α₁ to α₂ between male and female brains given that this switch correlates with the termination of the sensitive period for sexual differentiation of the brain.

We have examined several areas of the hypothalamus and limbic system for sex and age dependent differences in α₁ and α₂ immunoreactivity in the neonatal brain. Several region specific variations in the developmental patterns of immunoreactive product were observed in these brain regions, but there were no major sex differences in these products. Therefore, we conclude that the switch in GABA_A receptor subunits is not causally involved with the termination of the sensitive period; however, it may be associated with other physiological characteristics of the receptor complex which then confer regionally specific sensitivity to GABA and other modulators of the GABA_A receptor.