Neurogranin expression by cerebellar neurons in rodents and non‐human primates

Abstract

Neurogranin (NG) is a brain-specific protein kinase C substrate involved in the regulation of calcium signaling and neuronal plasticity. A rostrocaudal expression profile, with large amounts in telencephalic brain regions and low expression levels in phylogenetically older brain structures, was reported previously. In the cerebellum, expression of NG has not been described. By using immunocytochemistry and in situ hybridization, we found that NG is expressed in the mouse (C57Bl/6), rat (Wistar), and monkey (Cercopithecus aetiops) cerebella. In the mouse cerebellum, Golgi cells were strongly immunoreactive for NG, whereas other cerebellar neurons were devoid of this protein. Cell counts showed 1.6-fold more immunopositive Golgi cells in the hemispheres (61.1 +/- 8.0 cells/mm(2)) than in the vermis (37.5 +/- 3.3 cells/mm(2)). Developmental studies showed detectable NG in the mouse cerebellum as early as on postnatal day 10 (P10). In contrast to the mouse, in the rat cerebellum we found only a few Golgi cells containing NG (hemispheres, 2.4 +/- 0.5 cells/mm(2); vermis, 1.5 +/- 0.3 cells/mm(2)). In the monkey cerebellum, unipolar brush cells, localized in the granular layer, were heavily labeled, whereas Golgi cells were devoid of NG. This study demonstrated that NG is strongly expressed in specific gamma-aminobutyric acidergic neurons in the rodent cerebellum. In addition, NG expression in the primate cerebellum by brush cells, which are excitatory, showed remarkable cell type-specific and species-specific expression patterns of a postsynaptic protein mediating calcium signaling mechanisms.

Golgi Cell Dendrites Are Restricted by Purkinje Cell Stripe Boundaries in the Adult Mouse Cerebellar Cortex

“…In addition, a few basolateral dendrites extend in the granular layer (for review, see De Schutter and Bjaalie, 2001). In the adult mouse cerebellar cortex, neurogranin immunoreactivity is restricted to the somata and dendritic arbors of Golgi cells (Singec et al, 2003) [plus weak expression in a small subset of Purkinje cells, restricted primarily to the nodular zone, which is easily distinguished from Golgi cell immunoreactivity (Larouche et al, 2006)]. We have therefore used double-label neurogranin/zebrin II fluorescence immunohistochemistry to explore the behavior of Golgi cell apical dendrites at the boundaries between Purkinje cell compartments (Fig.…”

“…Antizebrin II is a mouse monoclonal antibody produced by immunization with a crude cerebellar homogenate from the weakly electric fish Apteronotus (Brochu et al, 1990) and subsequently shown to bind the respiratory isoenzyme aldolase C (Ahn et al, 1994;Hawkes and Herrup, 1996); it was used directly from spent hybridoma culture medium. Rabbit anti-neurogranin was raised against full-length recombinant rat neurogranin protein (Millipore, Billerica, MA; catalog #AB5620; used diluted 1:1000); it recognizes Purkinje cells in the neonatal cerebellum and Golgi cells in the adult cerebellum, as previously reported (Singec et al, 2003;Larouche et al, 2006), and on Western blots of newborn or postnatal day 20 cerebellar homogenates reveals a single polypeptide band of apparent molecular weight 10 kDa (Larouche et al, 2006), consistent with that of neurogranin/RC3 (Watson et al, 1990). Mouse hybridoma cell line TIB200 secreting monoclonal anti-HNK-1 (Abo and Balch, 1981) was obtained from the American Type Culture Collection (Rockville, MD).…”

“…Each hypothesis has unique structural implications that can be distinguished experimentally. First, anti-neurogranin immunohistochemistry, to identify the dendritic arbors of Golgi cells (Singec et al, 2003), was combined with Purkinje cell stripe antigens, zebrin II/aldolase C (Brochu et al, 1990;Ahn et al, 1994), HNK-1 (Marzban et al, 2004), and phospholipase C␤4 (Sarna et al, 2006), to explore interneuron cytoarchitecture in the context of cerebellar topography. Next, Golgi-Cox impregnation was combined with anti-zebrin II immunohistochemistry to relate Golgi cell dendrites to stripe boundaries.…”

Neuronal Migration Defect of the Developing Cerebellar Vermis in Substrains of C57BL/6 Mice: Cytoarchitecture and Prevalence of Molecular Layer Heterotopia

“…A number of Golgi cells also synthesize and release glycine and express the type-2 glycine transporter ( Slc6a5 ; [41]). Additional molecular markers that distinguish Golgi cells include neuregulin ( Nrg1 ; [42]), neurogranin [43], acetylcholinesterase ( Ache ; [44,45]), and proenkephalin ( Penk1 ; [46,47]). As shown in figure 2e and f, MLH was observed in a case hybridized for Slc6a5 .…”

Neurogranin, a link between calcium/calmodulin and protein kinase C signaling in synaptic plasticity

“…In the neocortex and hippocampus, Ng is only expressed by principal excitatory neurons (13,14). In the cerebellum, originally believed to contain no Ng, this protein is present in a subset of GABAergic Golgi cells (15,16) and also transiently expressed in a subpopulation of Purkinje neurons during development (E15-P20) (17). More recently, Ng has been detected in the granule cell inhibitory interneurons of the mouse olfactory bulb, a continuously renewed cell type that also express CaM kinase IV (CaMKIV), but not the calcium binding protein calretinin (18).…”