GABAergic synaptogenesis marks the onset of differentiation of basket and stellate cells in mouse cerebellum

Abstract

Type 2 glycine transporter (GlyT2) mediates intracellular glycine transport and is expressed selectively in glycinergic neurons. Expression of GlyT2 gene promoter-driven enhanced green fluorescent protein (eGFP) in BAC transgenic mice allows selective visualization of glycinergic neurons by fluorescence microscopy. Here, we show that cerebellar interneuron precursors identified by the transcription factor Pax2, including gamma-aminobutyric acid (GABA)ergic interneurons of the molecular layer (ML; basket and stellate cells), transiently express GlyT2-eGFP during development. In contrast, expression of endogenous GlyT2 is restricted to glycinergic Golgi cells. Comparison with knock-in mice expressing eGFP in GABAergic neurons [glutamic acid decarboxylase (GAD)67-eGFP] revealed that GlyT2-eGFP expression often precedes GAD67-eGFP and is therefore a marker of immature GABAergic interneurons. In the internal granule cell layer, GABAergic Golgi cells differentiated shortly after birth, prior to glycinergic Golgi cells. In the ML, GlyT2-eGFP-positive precursor cells migrated until the boundary with the external granule cell layer, forming an inside-out maturation gradient that determined the final position of interneurons in the ML. After migration, GlyT2-eGFP gradually disappeared, while interneurons differentiated morphologically and became immunoreactive for parvalbumin, the GABA(A) receptor alpha1 subunit, and the K(+)Cl(-) exchanger KCC2 (K(+)Cl(-) cotransporter type 2). Numerous presumptive GABAergic synaptic terminals were seen on immature ML interneurons as early as P4, preceding the expression of these neurochemical markers. These results suggest that GABAergic synaptogenesis marks the onset of differentiation of basket and stellate cells in the mouse cerebellum, and that GABAergic synaptic function might contribute to the differentiation of interneurons in the cerebellar cortex.

Laminar Fate and Phenotype Specification of Cerebellar GABAergic Interneurons

“…GFP expression intensity correlates with anti-Pax-2 immunolabeling but persists after the downregulation of Pax-2 protein that occurs in mature basket and stellate cells (Maricich and Herrup, 1999). In Gad67-GFP mice, GFP is consistently expressed in all GABAergic neurons of the cerebellum, including Purkinje cells and inhibitory interneurons (Tamamaki et al, 2003;Yamanaka et al, 2004;Simat et al, 2007a).…”

“…To investigate the early phases of interneuron development, we examined Pax-2 expression in wildtype mice or GFP fluorescence in Pax-2-GFP mice. According to previous studies, in the cerebellum anti-Pax-2 immunostaining identifies a population of neuronally restricted GABAergic cells (Maricich and Herrup, 1999), corresponding to the entire repertoire of cerebellar inhibitory interneurons (Maricich and Herrup, 1999;Simat et al, 2007a). In addition, in Pax-2-GFP mice, the Pax-2 protein and GFP are faithfully colocalized .…”

“…jneurosci.org as supplemental material). In all instances, Pax-2-expressing cells displayed morphological and neurochemical features of cerebellar interneurons (see below) Simat et al, 2007a). On the contrary, immunolabeling of the same cerebella with antibodies that recognize glial lineages never colocalized with GFP (supplemental Fig.…”

“…3e-h), where they acquired mature phenotypes following typical morphogenic phases (Fig. 3g-h) (Altman and Bayer, 1997;Simat et al, 2007a). Therefore, donor interneurons do not engraft in ectopic positions or follow unusual migratory pathways to reach their final destination, but faithfully replicate transit through the PWM and cortical dispersal of their endogenous counterparts.…”

Immunolocalization of Ephexin-1 in the Developing Canine Cerebellum

“…The immunostaining patterns of several markers appear and peak around the second postnatal week as described in a previous report [19]. Parvalbumin, another calcium-binding protein, labeling was observed initially in the soma and extending gradually to the dendritic arborization later than CB, which was in parallel with an increase in neuronal activity including synaptogenesis [4] [5] [21]. Proper dendritic arborization of Purkinje cells is thought to be a prerequisite for cerebellar motor coordination.…”