Expression of Connexin36 in the adult and developing rat brain¹

Abstract

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.

Introduction

Gap junctions are specialized membrane regions composed of aggregates of transmembrane channels that directly connect the cytoplasm of adjacent cells. The passage of ions and small molecules (MW<900 Da in vertebrates) through gap junction channels results in metabolic and electrical coupling of cells. Each intercellular channel is formed by the conjunction of two hemichannels, or connexons, formed by the hexameric assembly of subunit proteins, called connexins. Connexins are encoded by a large multigene family and, today, we know 16 different members of this family in mammals. Each connexon can contain either a single type of connexin (homomeric) or multiple connexins (heteromeric) and each intercellular channel can be defined as homotypic or heterotypic depending on whether the two apposed connexons have the same or a different molecular composition (for reviews see [6], [48]).

Gap junctions are present in almost all organs of vertebrate organisms, including the nervous system, where the existence of gap junction-mediated communication has been shown both in glial cells and neurons by both functional (dye- or electrotonic coupling) and morphological assays (electron microscopy). Interneuronal gap junctions, the anatomical substrate of ‘electrical synapses’ [3], [4], may play important roles in both adult and developing mammalian brain. It is well-established that gap junction-mediated communication between neurons is widespread and intense in critical perinatal periods [7], [8], [11], [33], [36], [37], [45], [46], [47], while it persists only in selected brain regions in the adult (for a review see [9]). In the latter regions, interneuronal communication is thought to be essential for the synchronization of neural activity

Better understanding of the roles played by gap junction-mediated communication between neurons requires the identification of the connexins expressed in neuronal cells. Recently, we have reported that Cx36 is a major connexin of neuronal cells [2], [10]. Thus, analysis of Cx36 mRNA distribution revealed an intense expression of this connexin in various brain regions and in retina [2], [10], [41], [44]. Moreover, in situ hybridization analysis after the induction of neurotoxic lesions suggested that Cx36 mRNA is predominantly expressed in neuronal cells of specific brain regions [10].

We have now extended our screening for Cx36 in the adult rat brain by analysing the expression of this protein using novel antibodies [39] and relating it to that of Cx36 mRNA, as assessed by in situ hybridization. Moreover, the neuronal localization of Cx36 in several adult brain areas was determined by combining the in situ hybridization for Cx36 mRNA with immunohistochemistry for the general neuronal marker NeuN [31]. Since previous studies have indicated the presence of gap junctions in striatal and hippocampal GABAergic interneurons [18], [19], which contain the calcium-binding protein parvalbumin, we have also performed double labeling experiments for Cx36 mRNA and parvalbumin.

The extensive interneuronal communication mediated by gap junction in the developing brain (for a review see [16]) and the up-regulation of Cx36 mRNA in whole brain during early stages of postnatal development [41] prompted us to also analyse Cx36 expression during postnatal development of selected brain regions.