In situ hybridization histochemistry and immunocytochemistry, including double immunofluorescence, were used to study the populations of neurons expressing the alpha subunit of type II calcium/calmodulin-dependent protein kinase (CAM II kinase-alpha) or glutamic acid decarboxylase (GAD) in the somatic sensory and motor areas of the macaque monkey cerebral cortex. Sections were subjected to in situ hybridization using radioactive, complementary RNA probes specific for monkey CAM II kinase-alpha or 67 kDa GAD mRNAs. Others were stained immunocytochemically for CAM II kinase-alpha and/or GABA. CAM II kinase-alpha and GAD-67 are expressed in different populations of cells, with no colocalization. CAM II kinase-alpha is expressed in pyramidal cells of layers II-VI, especially layers II and III, as well as in certain small nonpyramidal cells of layer IV in areas 3a, 3b, 1, and 2 and of middle regions of area 4. Both cell types produce excitatory amino acid transmitters. Therefore, as in subcortical regions, CAM II kinase-alpha will be found on the presynaptic side of excitatory synapses but on the postsynaptic side only when these synapses occur on excitatory neurons in the sensory-motor cortex. Quantitative examination showed that CAM II kinase-alpha immunoreactive cells form, on average, approximately 50% of the total neuronal population in each area, while GABA immunoreactive or GAD cRNA hybridized cells form approximately 25-30%. Thus, CAM II kinase-alpha expressing cells cannot account for the total population of non-GABAergic cortical cells, and a certain proportion of the pyramidal cells probably do not express it. In other cortical areas, gene expression for the two molecules is regulated by afferent activity. Therefore, the present results form a necessary basis for studies aimed at determining the role of activity-dependent changes in the balance of excitation and inhibition as a mechanism underlying plasticity of representational maps in the primate sensory-motor cortex.

In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin- dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non- GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate. This suggests that the kinase may be differentially engaged in pre- and postsynaptic functions at certain synapses.

Increases in immunocytochemically detectable type II calcium-calmodulin-dependent protein kinase (CaM II kinase) and decreases in immunocytochemically detectable glutamic acid decarboxylase (GAD) are known to occur in the visual cortex of adult monkeys following brief periods of monocular visual deprivation. In the present study, GAD and CaM II kinase gene expression was investigated under these conditions. The polymerase chain reaction (PCR) was used to generate species-specific cDNA clones that were used to make antisense RNA probes. A second form of CaM II kinase alpha, CaM II kinase alpha-33, which contains an additional phosphorylation consensus sequence, was identified. In situ hybridization in normal visual cortex revealed a complex sublaminar organization of GAD-expressing cells within layers IVC and VI and a distribution of CaM II kinase alpha-expressing cells that was greatest in layers II, III, IVB, and VI. In situ hybridization in the cortex from animals that had been monocularly deprived revealed enhanced CaM II kinase mRNA levels in deprived-eye columns of layer IVC and, associated with the deprived eye, cytochrome oxidase-stained periodicities in other layers. In layer IV, the enhancement of labeling in deprived-eye stripes was, on average, 16% greater than in normal-eye stripes. By contrast, GAD, mRNA levels appeared unchanged in all layers, suggesting a posttranscriptional regulatory mechanism.

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