We have developed a simple non-radioactive in situ hybridization procedure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of various transcripts present at a wide range of expression levels in the central nervous system. Cerebellar hybridization signals for transcripts estimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and low (inositol polyphosphate-5-phosphatase) levels of abundance are demonstrated as examples. The sensitivity and cellular resolution were significantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protocol can be combined with immunocytochemistry as demonstrated for glial fibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are described in detail.

We have identified and studied potential ionotropic glutamate receptor genes in pigeon brain. Three cDNA clones exhibit significant amino acid sequence identity to members of a rodent ligand‐gated ion channel family. One of them, GluP‐II, encodes a full‐length AMPA‐sensitive glutamate receptor GluR2 (GluR‐B) homologue, whereas the other two partial clones, designated as GluP‐III and ‐IV, are nearly identical to rodent GluR3 (GluR‐C) and GluR4 (GluR‐D) receptor subunits. Northern analysis demonstrated that the avian genes are widely expressed in the brain. Within the brain regions analyzed by in situ hybridization histochemistry, the three avian GluR subunits showed distinct and regionally specific mRNA expression patterns in the adult. Most of the differences in their expression were observed in cell types of the telencephalon, certain thalamic nuclei, the optic tectum, and the cerebellar cortex. A particularly striking finding was the expression of GluP‐II in Golgi epithelial/Bergmann glial cells. In contrast, Bergmann glial cells in rat cerebellum do not express GluR2 (GluR‐B) subunit genes. Immunoreactivity for a monoclonal sequence‐specific antipeptide antibody was widespread and most prominent in Purkinje cell perikarya and their dendrites, neuronal cell bodies of the ectostriatum, and the deep optic tectum. These results demonstrate the existence of multiple subunits of the ionotropic glutamate receptor channel family in avians. Excitatory amino acid receptor genes appear to be highly conserved during evolution.

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