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G-protein-gated K+ (KG) channels generate slow inhibitory postsynaptic potentials in the brain. Current opinion suggests that neuronal KG channels are heterotetramers of Kir3.1 and Kir3.2. In substantia nigra (SN), however, mRNA of Kir3.1 does not express, whereas that of Kir3.2 clearly does. Therefore, we have characterized the KG channels containing Kir3.2 subunits in SN using biochemical and immunological techniques. We found that they were composed of only Kir3.2 subunits and did not contain significant amounts of either Kir3.1 or Kir3.3. Furthermore, at least some of the KG channels in SN were assemblies of the splicing variants Kir3. 2a and Kir3.2c. The channels were localized specifically at the postsynaptic membrane on the dendrites of dopaminergic neurons. Kir3. 2c, but not Kir3.2a, could bind a PDZ domain-containing protein, PSD-95. The heterologously expressed KG channels composed of Kir3.2a plus Kir3.2c or Kir3.2a alone were activated by G-protein stimulation, but expression of Kir3.2c alone was not. This study reveals that the Kir3.2 splicing variants play distinct roles in the control of function and localization of some of the KG channels in dopaminergic neurons of SN.

A sensitive method for acetylcholinesterase (AChE) histochemistry has been developed which permits simultaneous observation of fme fiber processes and neuron cell bodies. In rat brain, distinctive configurations can be observed which have been difficult to see by other techniques. Ihe staining procedure involves two steps. Tissue sections are incubated first in Karnovsky and Roots medium diluted one-hundredfold; and then with a mixture containing diaminobenzidine (DAB) and H202. The reaction product of the first step induces deavage of hydrogen peroxide in the second step, with a resulting oxidation of DAB to yield a

Glial fibrillary acidic protein (GFA) has been visualized by direct peroxidase antiperoxidase (APA) immunohistochemistry in various vertebrates (cyclostomes, teleosts, amphibians, reptiles, birds, and several placental mammals). In this study GFA-immunoreactivity (GFA-I) was observed in all species examined except in cyclostomes and amphibians. Two types of immunoreactive elements were observed: astrocytes and long processes without visible somata. Astrocytic cells with GFA-I were first found in the snake, and more cells were in birds where the pattern of distribution was similar to that of mammals. Within mammals, few differences in the manner of localization were observed among different species, except in the corpus callosum and the ependymal and subependymal layers. Long straight processes were observed in the lower submammalians--the lamprey, carp, and turtle. They radiated through the neuropil from the ventricular wall and followed nerve fiber bundles in the white matter. An uncommon feature was observed in the turtle brain, which possessed very intense GFA-I within the ependymal layer. The presence of GFA-containing profiles in the ependyma of adult animals is discussed in relation to GFA-positive structures seen in the human brain during ontogeny or under certain pathological conditions.

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The present study has demonstrated that the sleep-promoting potency of 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine (CGS21680), a selective agonist for the adenosine A2A receptor, varies depending on the location of the administration. CGS21680 was continuously administered to rats through a chronically implanted cannula for 6 h during their active phase. The tip of the cannula was located in the subarachnoid space or the brain ventricle neighbouring the established brain areas implicated in the regulation of sleep-wake phenomena, i.e. rostral basal forebrain, medial preoptic area, lateral preoptic area, posterior hypothalamus, and dorsal tegmentum of the pons and medulla. At an infusion rate of 2.0 pmol/min, the magnitude of increase in non-rapid eye movement sleep varied from 14 min (a 15% increase) to 96 min (a 103% increase), and those of rapid eye movement sleep varied from 6 min (a 40% increase) to 28 min (a 264% increase) from the respective baseline values. The largest increases in both types of sleep occurred when CGS21680 was administered to the subarachnoid space underlying the rostral basal forebrain. These findings were interpreted to mean that the major, if not the only, site responsible for the CGS21680-inducing sleep was located in or near the rostral basal forebrain. This interpretation was supported by the findings that the administration of CGS21680 to the rostral basal forebrain produced predominant expression of Fos within the shell of the nucleus accumbens and the medial portion of the olfactory tubercle, and that the microdialysis perfusion of CGS21680 into the shell of the nucleus accumbens also exhibited a sleep-promoting effect.

The presence of nerve fibers in bone marrow has been noted by various investigators, and recent developments in immunohistochemistry have enabled differential localization of the intramedullary nerve fibers. Much interest has been devoted to the efferent activities of the afferent fibers, which probably act on the target tissues by secreting a variety of neurotransmitters. The present study aimed to further characterize intramedullary substance P, calcitonin gene-related peptide, and tyrosine hydroxylase-immunoreactive nerve fibers of the rat lower limb by comparing those of the knee, ankle, and tarsal joints. The ultrastructural details of intramedullary calcitonin gene-related peptide-immunoreactive axons were also investigated to provide a morphological basis for their possible efferent actions. Intramedullary calcitonin gene-related peptide and substance P-immunoreactive fibers in the proximal tibia and the knee joint were found to be as reported earlier, but the marrow of the distal metaphysis was also noted to be richly innervated, and the tarsal joints displayed dense innervation at the subchondral regions that underlie the joint cartilage. The articular and intramedullary innervations that function for joint protection might participate in characteristic clinical features of joint damage secondary to the neuropathies. Ultrastructurally, the intramedullary calcitonin gene-related peptide-immunoreactive axons were minimally engulfed by the Schwann cell, and naked intramedullary calcitonin gene-related peptide-immunoreactive axons were noted along an extraordinarily long extension, suggesting much efferent activity.