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Notes: The presence of metabotropic receptors for GABA, GABAB, on primary afferent terminals in mammalian spinal cord has been previously reported. In this study we provide further evidence to support this in the rat and show that the GABAB receptor subunits GABAB1 and GABAB2 mRNA and the corresponding subunit proteins are present in the spinal cord and dorsal root ganglion. We also show that the predominant GABAB1 receptor subunit mRNA present in the afferent fibre cell body appears to be the 1a form. In frozen sections of lumbar spinal cord and dorsal root ganglia (DRG) GABAB receptors were labelled with [3H]CGP 62349 or the sections postfixed with paraformaldehyde and subjected to in situ hybridization using oligonucleotides designed to selectively hybridize with the mRNA for GABAB(1a), GABAB(1b) or GABAB2. For immunocytochemistry (ICC), sections were obtained from rats anaesthetized and perfused-fixed with paraformaldehyde. The distribution of binding sites for [3H]CGP 62349 mirrored that previously observed with [3H]GABA at GABAB sites. The density of binding sites was high in the dorsal horn but much lower in the ventral regions. By contrast, the density of mRNA (pan) was more evenly distributed across the laminae of the spinal cord. The density of mRNA detected with the pan probe was high in the DRG and distributed over the neuron cell bodies. This would accord with GABAB receptor protein being formed in the sensory neurons and transported to the primary afferent terminals. Of the GABAB1 mRNA in the DRG, ≈ 90% was of the GABAB(1a) form and ≈ 10% in the GABAB(1b) form. This would suggest that GABAB(1a) mRNA may be responsible for encoding presynaptic GABAB receptors on primary afferent terminals in a manner similar to that we have previously observed in the cerebellar cortex. GABAB2 mRNA was also evenly distributed across the spinal cord laminae at densities equivalent to those of GABAB1 in the dorsal horn. GABAB2 mRNA was also detected to the same degree within the DRG. Immunocytochemical analysis revealed that GABAB(1a), GABAB(1b) and GABAB2 were all present in the spinal cord. GABAB(1a) labelling appeared to be more dense than GABAB(1b) and within the superficial dorsal horn GABAB(1a) was present in the neuropil whereas GABAB(1b) was associated with cell bodies in this region. Both 1a and 1b immunoreactivity was expressed in motor neurons in lamina IX. GABAB2 immunoreactivity was expressed throughout the spinal cord and was evident within the neuropil of the superficial laminae.

Notes: The involvement of protein kinase C (PKC)-dependent processes in adaptive and plastic changes underlying neuronal plasticity was tested in an in vivo animal model characterized by targeted cellular ablation of cortical and hippocampal neurons, cognitive impairment and lack of induction of long-term potentiation. [3H]Phorbol ester binding performed on brain slices revealed a 67.4 and 35.0% increase in membrane-bound protein kinase C in the cortex and hippocampus respectively of rats treated with methylazoxy-methanol acetate compared with saline-treated control rats, and there was no modification in the expression of mRNAs of different protein kinase C isozymes. In situ phosphorylation experiments performed with 32Pi-labelled synaptosomes from the affected areas demonstrated that the phosphorylation of the nervous tissue-specific presynaptic membrane-associated protein kinase C substrate B-50lGAP-43 was increased by 51.4 and 44.8% in cortex and hippocampus respectively. Western blot analysis of protein kinase C in synaptosomal cytosol and membrane fractions prepared from cortex and hippocampus showed an increased proportion of protein kinase C in the membrane compartment in treated animals, but no change in the total synaptosomal protein kinase C activity. Our data are consistent with increased activity of presynaptic protein kinase C and predict a sustained increase in glutamate release in methylazoxy-methanol-treated rats.