Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons

Localization of nitric oxide synthase indicating a neural role for nitric oxide

Excitatory amino acid transmitters

Two metabolites of the steroid hormones progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-dihydroprogesterone and 3 alpha, 5 alpha-tetrahydrodeoxycorticosterone, are potent barbiturate-like ligands of the gamma-aminobutyric acid (GABA) receptor-chloride ion channel complex. At concentrations between 10(-7) and 10(-5)M both steroids inhibited binding of the convulsant t-butylbicyclophosphorothionate to the GABA-receptor complex and increased the binding of the benzodiazepine flunitrazepam; they also stimulated chloride uptake (as measured by uptake of 36Cl-) into isolated brain vesicles, and potentiated the inhibitory actions of GABA in cultured rat hippocampal and spinal cord neurons. These data may explain the ability of certain steroid hormones to rapidly alter neuronal excitability and may provide a mechanism for the anesthetic and hypnotic actions of naturally occurring and synthetic anesthetic steroids.

https://science.sciencemag.org/content/sci/232/4753/1004.full.pdf

Steroid Hormone Metabolites are Barbiturate-Like Modulators of the GABA Receptor

The molecular and cellular actions of three classes of abused drugs--opiates, psychostimulants, and ethanol--are reviewed in the context of behavioral studies of drug dependence. The immediate effects of drugs are compared to those observed after long-term exposure. A neurobiological basis for drug dependence is proposed from the linkage between the cellular and behavioral effects of these drugs.

https://science.sciencemag.org/content/sci/242/4879/715.full.pdf

Cellular and molecular mechanisms of drug dependence.

Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system.

New concepts on the mechanism of action of benzodiazepines.

A dopamine-sensitive adenylate cyclase with characteristics similar to those measured in the striatum is present in the rat substantia nigra. Destruction of dopamine cell bodies by intranigral 6-hydroxydopamine application failed to abolish the response of nigral adenylate cyclase to dopamine. In contrast, brain hemitransection between the striatum and substantia nigra, or a more circumscribed lesion of striatonigral pathways, abolished the dopamine stimulation of adenylate cyclase in the substantia nigra. These results suggest that dopamine receptors within the substantia nigra are not located on dopamine cell bodies but are associated with a pathway, containing gamma-aminobutyric acid or substance P, which projects from forebrain structures to the substantia nigra.

https://science.sciencemag.org/content/sci/195/4277/503.full.pdf

Dopamine-sensitive adenylate cyclase: location in substantia nigra.

Antisera against L-glutamate decarboxylase (GAD), the synthesizing enzyme of γ-aminobutyric acid (GABA) were used to locate GABAergic neurons and nerve terminals in the septal complex of the rat by using the peroxidase-antiperoxidase method. Varying densities of immunoreactive terminals were observed in saline-treated rats but nerve cell bodies were only demonstrated after intraventricular or intraseptal injections of colchicine. Small and medium-sized GAD-positive neurons were found in lateral septal nuclei, the largest number of these cells being in the pars dorsalis, and in the bed nucleus of the stria terminalis. Several GAD-immunoreactive neurons were located in the medial septal nucleus and the nucleus of the diagonal band of Broca (DB), where the cells were larger in the ventral than dorsal parts of the region. In the medial septal nucleus and in DB the GAD-positive cell bodies were distributed similarly to cholinergic neurons. Large GAD-positive neurons were also found in the septofimbrial nucleus. Intense immunoreactivity in nerve terminals was observed in the lateral septal nucleus, around the island of Calleja magna, between the DB and nucleus accumbens, and in the septofimbrial and triangular septal nuclei. In contrast, the medial septal nucleus, the DB, and the bed nucleus of the stria terminalis only showed weak to moderate immunoreactivity.



These results provide direct morphological evidence for the presence of neurons capable of synthesizing GABA in septal nuclei. We suggest that there are two different GABAergic neuronal systems operating in the septum: a population of small cells in the lateral septal nucleus and a group of large cells in the medial septum and DB.

An immunohistochemical study on the location of GABAergic neurons in rat septum

Coupling high performance liquid chromatography with gas chromatography-mass fragmentography has made it possible to simultaneously measure subpicomolar concentrations of allopregnanolone and its precursors, pregnenolone, progesterone and 5 alpha-dihydroprogesterone (5 alpha-DHP) in various brain areas. Allopregnanolone was measured in the brain of adrenalectomized/castrated (ADX/CX) rats in nanomolar concentrations long after peripheral sources of allopregnanolone were removed. A partial decrease (approximately 30%) in the content of allopregnanolone was found in the brains of ADX/CX rats compared to sham-operated rats. Moreover, the content of allopregnanolone in brains of sham-operated as well as ADX/CX rats was nonuniformly distributed (olfactory bulb > striatum > cortex > hippocampus) and was one to two orders of magnitude higher than in plasma or liver. Infusion of pregnenolone sulfate in ADX/CX rats elicited a fourfold increase in 5 alpha-DHP and progesterone content and a seven- to eightfold increase in the content of allopregnanolone in brain but not in liver or plasma. Furthermore, the content of allopregnanolone in brain increased to the same extent in both sham-operated and ADX/CX rats following pregnenolone sulfate infusion. The 5 alpha-reductase inhibitor, (17 beta)17[[bis(1-methylethyl)amino]carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF 105111), reduced the brain content of allopregnanolone and blocked the increased formation of allopregnanolone in brain following pregnenolone sulfate infusion. The results clearly demonstrate that the synthesis of allopregnanolone from 5 alpha-DHP and progesterone occurs in the brain and that a significant amount of allopregnanolone is synthesized locally in brain from its precursors. These experiments suggest that the brain, like adrenals and gonads, is a steroidogenic organ which produces allopregnanolone as one of its own most important physiologically relevant steroids.

https://www.jneurosci.org/content/jneuro/15/6/4641.full.pdf

Gas chromatographic-mass fragmentographic quantitation of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats.

A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites.

E. Costa

Papers

New concepts on the mechanism of action of benzodiazepines.

Dopamine-sensitive adenylate cyclase: location in substantia nigra.

An immunohistochemical study on the location of GABAergic neurons in rat septum

Gas chromatographic-mass fragmentographic quantitation of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats.

A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites.