Enrichment of unipolar brush cell-like neurons in primary rat cerebellar cultures

Abstract

Unipolar brush cells are a distinct class of excitatory interneurons situated in the granular layer of the cerebellar cortex, where they form giant synapses with individual mossy fiber terminals. We have previously shown that primary cerebellar cell cultures from embryonic and postnatal rodents contain neurons displaying morphological and chemical phenotypes characteristic of unipolar brush cells in situ, including intense staining with calretinin antiserum. In cultures from both embryonic and postnatal rats, the large majority of calretinin-positive neurons are unipolar brush cells, while granule cells are usually calretinin-negative. A small percentage of putative Golgi/Lugaro cells also express calretinin. We demonstrate here that the developmental stage of the source tissue, the concentration of potassium in the medium, and treatment with glutamate after differentiation have substantial effects on the density of putative unipolar brush cells in the cultures. In dissociated cultures obtained from embryos at gestation day E18 and E20 and from pups at postnatal day P0, P2, P5, P8, and P10 grown in 25 mM KCl, the percentage calretinin-positive cells progressively decreases from 24% to 0.1% of total cells. In cultures from E20 embryos grown in physiological potassium (5 mM KCl), calretinin-positive cells are enriched to approximately 60% of total cells, while the majority of calretinin-negative cells die. In embryonic cultures exposed to high concentrations of glutamate after 12 days in vitro, calretinin-positive neurons have a survival advantage over calretinin-negative cells and represent up to 83% of total cells.

Phospholipase Cβ4 Expression Identifies a Novel Subset of Unipolar Brush Cells in the Adult Mouse Cerebellum

“…The CR+ phenotype is expressed in UBCs in dissociated primary cultures from which extracerebellar mossy fiber innervation is absent [43]. Therefore, to test if the PLCβ4+ UBC phenotype is induced by extracerebellar mossy fiber input, PLCβ4 expression was examined in primary mouse cerebellar cultures (E18, 21 days in vitro).…”

Besides Purkinje cells and granule neurons: an appraisal of the cell biology of the interneurons of the cerebellar cortex

“…In primary cerebellar cultures, which lack extracerebellar mossy Wbers, UBCs develop and may be recognized based on their expression of calretinin. While some cultured UBCs develop the name-giving morphology of these cells, that of most cells is suYciently variable to argue in favour of a strong inXuence of orderly local cues for the proper morphogenesis of these cells (Anelli et al 2000;Anelli and Mugnaini 2001). We do not know whether and how aVerent innervation impinges on the neurochemical diVerentiation and diversiWcation of UBCs mentioned above.…”

The unipolar brush cell: A remarkable neuron finally receiving deserved attention

“…Successively, CR-immunoreactivity was also used to distinguish UBCs from granule cells and Golgi-like neurons in dissociated cerebellar cell cultures (Marini et al, 1997; Anelli et al, 2000). UBCs were sparse in classical granule cell cultures prepared from P7-8 rodents, but their density increased substantially in cultures prepared from late embryonic stages (E17-18 mice and E17-20 rats; Anelli and Mugnaini, 2001). Although CR-antibodies raised in different species to this day remain an excellent tool to study the chemical phenotypes of UBC sublineages (Sekerkova et al, 2004; Englund et al, 2006), ultimately the CR + UBCs turned out to be a relatively small subset of UBCs (see sections below: The UBC Subclasses).…”