GABA immunoreactivity in the developing rat thalamus and Otx2 homeoprotein expression in migrating neurons

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Abstract

We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.

Introduction

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult mammalian central nervous system (CNS), is also one of the most abundant neurotransmitters in embryonic life [7], [32]. It is widely accepted that GABA in these early stages, before the emergence of its role in neurotransmission, acts to influence CNS developmental events, such as cell proliferation, migration and differentiation [28]. Immunohistochemical and in situ hybridization studies have shown that GABAergic neurons become visible very early during embryogenesis in distinct brain areas of different species [9], [35], [47], [56].

During ontogenesis of the rat CNS, GABA has been detected by immunohistochemistry in fibers running in the developing brain at embryonic day (E) 12 and, between E14-18, in cell bodies situated in several discrete brain regions [3], [27], [46], [53]. A wealth of data pointed out the early appearance of cortical GABAergic neurons in the developing telencephalon and their tangential migration from the ganglionic eminence to the lateral part of the cortex in the primordial plexiform layer [25].

Despite the abundance of studies on GABAergic neurons in the mammalian developing telencephalon, a few investigations have been devoted so far to the appearance of GABA in the developing diencephalon. In particular, in situ hybridization studies in monkey and ferret showed that GABAergic neurons which compose the reticular (Rt) and ventral lateral geniculate (VLG) nuclei appear at early stages of development, while GABAergic intrinsic neurons appear much later in the dorsal thalamus, when proliferative activity has ceased and differentiation of dorsal thalamic nuclei is virtually complete [17], [20].

In the adult rat thalamus, Rt is entirely composed of GABAergic neurons [10], [18], [50] while, with the exception of the lateral and medial geniculate nuclei [39], GABAergic neurons are virtually absent in other dorsal thalamic nuclei [5], [37]. Data on GABA expression in the rat thalamus during early development are, however, still lacking. An in situ hybridization study reported the presence since E17 of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), in neurons which belong to Rt [6]. Such neurons are the earliest generated cells in the thalamus, since they appear between days E13 and E15, and then migrate along the reticular thalamic migration region (Rtm), located between the dorsal and ventral thalamic primordia [1], [2]. Within Rtm and dorsal thalamus, scattered GABA-immunopositive cells were detected at E15 [13], [38]. The narrow territory that separates the embryonic dorsal and ventral thalamus is also called zona limitans intrathalamica (Zli) in the prosomeric model [12], [43], [44] and is implicated as patterning centre in development of the thalamus through the expression of specific transcription factors. In embryos at midgestation, the Zli appears to be a migratory stream [24], [36] and is characterized by heterogeneity of expression of different genes encoding transcription factors such as brx1, dlx1, arx, ptx2, lhx1, nkx2.2 and otx2 [24], [31], [36], [44], [49], [51]. Recently, it has been reported that the co-expression of multiple transcription factors controls the neurotransmitter phenotype and the positional identity of excitatory and inhibitory progenitors [21], [31], [34], [42], [41]. In rat embryos, at midgestation a subset of neurons located in the Rtm/Zli can be identified by the expression of calretinin (CR), a calcium-binding protein which is a marker for some thalamic populations during early embryogenesis until adulthood [14]. Tracing experiments in living embryonic diencephalic slices [13], [38] have demonstrated that some neurons detach from the cluster of cells migrating in the Rtm/Zli and move towards the dorsal thalamus, providing evidence on the different routes of migration of early generated thalamic neurons.

The aim of the present study was to examine the spatio-temporal expression of GABA in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy, focusing our attention on dorsal thalamus and Rtm/Zli. In addition, we revealed a clear correspondence among GABA, CR and Otx2 immunoreactivities in midgestation stage of embryonic development (i.e., E15–E16) and we investigated the relationship between neurons located in Rtm/Zli and in the dorsal thalamus, identified by the neuronal markers CR and GABA, and the expression of the homeoprotein Otx2.

Section snippets

Animals

Experiments were performed on 29 Sprague–Dawley rats (Charles River, Calco, LC, Italy) ranging from E14 to postnatal day (P) 40. The day of detection of a vaginal plug was designated as E1. The experiments were undertaken in accordance with the guidelines defined by the European Communities Council Directive of 24 November 1986 (directive 86/609/EEC) and all the efforts were made to reduce the number of animals used.

Perfusion and histology

A total of 19 rat embryos (E14, n = 4; E15, n = 4; E16, n = 5; E18, n = 3; E19, n = 3)

Light microscopy

The peak of neurogenesis of Rt cells occurs at E14 [2]. At this stage, the neuroepithelial region from which Rt neurons derive (rt) was clearly distinguishable as a small protrusion into the third ventricle. Laterally to this region, a zone of differentiating cells, the Rt primordium (Rtp), was also evident (Fig. 1A). GABA immunohistochemical staining revealed the presence in Rtp of immunoreactive (ir) neurons showing an immature morphology and intensely labeled somata, as well as longitudinal

Discussion

The present study demonstrates that GABA immunoreactivity appears early during thalamic development (E14) in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. Confocal and electron microscopy revealed the axonal nature of GABA-immunopositive processes. GABA expression in fibers, as well as in radially and non-radially oriented thalamic neurons, characterized the embryonic stages. We here showed that GABA immunoreactivity in fibers gradually decreased

Acknowledgements

The authors are grateful to Dr. Giorgio Corte for providing Otx2 antibody. This work was supported by grants of the Italian Ministry of Health to C.F.

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