NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affnity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPHdependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.The NADPH diaphorase histochemical technique is based on the presence in certain neurons of an enzyme that can catalyze the NADPH-dependent conversion of a soluble tetrazolium salt to an insoluble, visible formazan (1,2). This method has proven useful for the examination of select populations of neurons in both experimental studies and in human neuropathology. In particular, NADPH diaphorase has been shown to be a selective marker for forebrain neurons containing both somatostatin and neuropeptide Y (3) and for the ascending cholinergic reticular system in the mesopontine tegmentum (4). This method has, therefore, been used to examine these neurons in Huntington disease (5), Alzheimer disease (6, 7), progressive supranuclear palsy (8), and ischemia (9, 10). NADPH diaphorase-containing neurons appear relatively resistant to anoxia and excitotoxic damage (11-13), and those in the striatum are selectively spared in Huntington disease (5).Although the NADPH diaphorase activity has been well defined histochemically (14), the function of this enzyme has remained a mystery. Previous attempts to characterize the enzyme biochemically have been hampered by lack of a specific assay (15, 16) because several proteins can exhibit NADPH-dependent diaphorase activity in brain homogenate (17). Therefore, we have used both a biochemical assay and an antibody that specifically recognizes neuronal NADPH diaphorase (18,19) to monitor purification of this enzyme from rat brain. Because NADPH diaphorase is an NADPHdependent enzyme, a purification protocol similar to that recently used for the NADPH-dependent brain enzyme, nitric oxide synthase, was att...

Bluegills were stimulated electrically while swimming free in an aquarium containing other bluegills, a mirror, gravel and food. Stimulation sites have been plotted on representative frontal sections for which a nomenclature system was developed. Nestbuilding was evoked in both sexes by stimulation near the area dorsalis telencephali pars centralis, while strong after-responses were elicited from the ventral preoptic region. Stimulation in the preoptic region slightly rostral to the habenula simultaneously inhibited aggressive behavior and evoked courtship. Feeding (snapping up prey, gravel or debris) and aggressive behavior (chasing and biting another bluegill) resulted from stimulation in the region surrounding the lateral recess of the third ventricle of the inferior lob:: of the hypothalamus. In several cases feeding and aggressive responses alternated during stimulation; a similar pattern was observed as an after-response to stimulation in the nucleus rotundus. These results are discussed with regard to possible anatomical systems controlling reproductive, feeding and aggressive behavior in fishes.Electrical stimulation of the brain in free-ranging unanesthetized birds and mammals has been used to identify areas of the telencephalon and hypothalamus associated with reproductive, feeding and aggressive behavior (Akerman, '66a,b; Harwood and Vowles, '66; Kaada, '67; Roberts et al., '67; Robinson and Mishkin, '66, '68; see Demski, '69, for additional references). Similar investigations have located some comparable regions in fishes (Fiedler, '64, '67; Grimm, '60) and amphibians (Schmidt, '68). More detailed information on the areas related to the above responses in the lower vertebrates could lead to a better understanding of the functional evolution of the forebrain. For this reason, the present mapping experiments were carried out. The bluegill sunfish was chosen as a representative species because of its availability, adaptability to the laboratory and wellstudied social behavior (Breder, '36; Miller, '63 Nieuwenhuys, '59; Tuge et al., '68), none were found that covered the entire region in a form suitable for plotting stimulation sites. For this reason, a series of representative frontal sections and a nomenclature system were developed for these areas of the bluegill brain. The indifferent lead consisted of a bare wire placed in the water of the test aquarium, Stimulation was provided by a Grass S-8 stimulator with two stimulus isolation units feeding into transistorized constant current converters modified after Bignall ('63). The output current was measured on a Hewlett-Packard 130C oscilloscope as the voltage drop across a 1000 ohm resistor in series with the fish. The stimulus used consisted of a pair of square wave pulses of opposite polarity, with a half wave duration of 1-2 msec, a frequency in the range of 2 to 100 Hz and a maximum current strength of 400 microamp. The length of the stimulation ranged from 5 sec-3 min, depending on the response being studied. MATERIALS AND METHODS Anim...

expansion was studied in young harbor seals. Negative pressure breathing did not increase rate of urine flow. Expansion of plasma volume with seal plasma or with 1% gelatin in saline caused a solute diuresis with increase in glomerular filtration rate similar to the changes seen in the post-prandial diuresis of the seal. I t is suggested that the post-prandial diuresis of the seal is mediated via a volume receptor.A continually mounting volume of research, summarized regularly ( 1-3), has explored mechanisms of neural regulation af ACTH secretion. The most intense interest is centered about isolation and characterization of the ACTH-regulating neurohumor (4-7) and identification of the hypothalamic area(s) in the region of the median eminence which represent the terminal link (s) between the central nervous system and the adenohypophysis (8-1 1 ) . More recently attention has k e n directed also to other regions of the brain which may have direct connections with the final hypothalamic effector sites or which may influence indirectly the amount of ACTH neurohumor produced there. Among these areas are the cerebral cortex( 12,13), limbic system( 14,l 5), brain stem and reticular formation ( 12,16-18). The present report describes alterations in basal levels of plasma free corticoids (PFC) and in the pattern af PFC response to stress occurring in rats with lesions of hippocampus or amygdala.Materials and methods. Bilateral electrolytic lesions ( 3 ma, 3 0 sec) were placed by stereotaxic means in hippocampus, amygdala * Supported by U.S.P.H.S. and N.S.F. grants. and habenular complex of adult male rats. The insulated stainless steel needle used for lesioning was bared approximately 1 mm at its tip for placement af lesions in amygdala and habenula; for hippocampus, the needle tip was bared 1.5-2 mm. Using a stereotaxic instrument with reference planes adjusted to the atlas of de Groot( 19), the anterior vertical and lateral coordinates used for lesioning amygdala, hippocampus and habenula were (+5.4, -2.8, & 4.5), (+3.0, -2.5, t 5.0) and (+3.4, +0.8, 20.5) respectively. Two months after lesioning, 1131 uptake was measured by counting the external neck activity 18-24 hrs after intraperitoneal administration of 5 pc carrier-free N a P ; a region of the hind limb was counted as a measure of the non-thyroidal neck activity. After one additional month, peripheral PFC levels were determined. Basal PFC levels and PFC response to 30 min and 4 hrs of continuous physical immobilization (paws bound with adhesive tape) were examined in both control and lesioned animals. Animds were sacrificed by decapitation, blood collected in heparinized beakers and PFC determined fluorometrically according to Guillemin et al.

No abstract

The effect of the monosodium glutamate (MSG) induced lesion of the arcuate nucleus on catecholamines in the arcuate nucleus and median eminence of the mouse hypothalamus was determined using the Falck-Hillarp histofluorescence technique. The number of fluorescent perikarya in the arcuate nucleus of treated animals was decreased approximately 60%; the fluorescence intensity of surviving neurons was notably reduced. These changes were accompanied by a reduction in the intensity of fluorescence in the median eminence. Pretreatment of control and MSG-lesioned animals with a monoamine oxidase inhibitor (pargyline) greatly increased fluroescence in the median eminence and arcuate nucleus of both groups. However, the number of fluorescing perikarya of the arcuate nucleus of the normal pargyline treated group far exceeded that of the pargyline MSG animals. It is concluded that neonatally administered MSG caused destruction of a large number of dopaminergic arcuate perikarya.