Complete purification of two identical Na(+)-pump inhibitors isolated from bovine hypothalamus and hypophysis.

We have completely purified, in parallel, a low molecular weight, non-specific, non-lipidic, Na+,K(+)-ATPase inhibitory factor from bovine hypothalamic and pituitary tissues. In the final purification step we obtain, from both tissues, a single, homogeneous peak, with a maximal absorbance at 247 nm. This factor, at physiological concentrations of potassium (5-25 mM), inhibits in a dose-response manner Na+,K(+)-ATPase and displaces ouabain from its receptor at the enzyme structure. The factor isolated from both tissues is identical, being the specific activity per weight of tissue higher in hypophysis. No factor was found in cerebral cortex, used as tissue control.

Regional distribution of progesterone and 5 alpha-pregnane-3,20-dione in rat brain during progesterone-induced "anesthesia".

Progesterone and 5 alpha-pregnane-3,20-dione (5 alpha-DHP) concentrations were measured in seven brain areas and in plasma during "anesthesia" induced by progesterone (1-2 mg IV) in female rats. The highest levels of progesterone were detected in the striatum and hypothalamus (23.3 +/- 5.27 and 22.7 +/- 4.30 micrograms/g +/- SEM, respectively); these concentrations were approximately 1000 times higher than those during the post-ovulatory phase. Highest levels of 5 alpha-DHP were detected in the striatum and hippocampus (11.5 +/- 1.74 and 10.4 +/- 3.15 micrograms/g +/- SEM, respectively). The ratio of 5 alpha-DHP to progesterone was approximately 100 times higher in brain tissue than in plasma. We conclude that a conversion of progesterone to 5 alpha-DHP occurs in the brain during the course of progesterone-induced "anesthesia". This metabolic step may be an important contributory factor to the anesthetic potency of progesterone.

Effect of dexamethasone on neurotransmitter amines in a rat glioma model.

Vasogenic brain edema was produced by transplantation of rat glioma C6 cells into rat brain. Using this model, we measured neurotransmitter concentrations and water content of regional brain tissue. In the tumor-implanted controls, monoamine neurotransmitters in the hypothalamus, cortex, and striatum significantly decreased. When treated with DEX, these monoamines tended to return to the levels of the sham-operated controls. Additionally, DEX markedly lowered tumoral monoamines. In the nonsurgical animals, DEX significantly increased norepinephrine but not DA. Regardless of treating with DEX or not, water content showed no changes in the hypothalamus and striatum in the tumor-implanted animals. However, the increase in water content in the cortex was significant, and this increase could be reduced by DEX to the levels of controls. Water content of tumor tissue could also be markedly reduced by DEX. In the nonsurgical animals, there were no changes in water content between DEX-treated and nontreated animals. In conclusion, brain edema produced by the brain tumor may reduce noradrenergic and dopaminergic activities. This is more likely due to compression anoxia caused by the tumor mass, glial hydrops, and edema fluid. It is presumed that the effect of DEX is due to reduction of water content of the tumor and peritumoral white matter as well as by increasing noradrenergic activity.

The effect of repeated administration of antidepressant drugs on the thyrotropin-releasing hormone (TRH) content of rat brain structures.

The present study investigated the effect of repeated treatment with the antidepressant drugs imipramine, amitryptyline, citalopram and mianserin (10 mg/kg PO, twice daily for 14 days) on levels of thyrotropin-releasing hormone (TRH) in several brain structures (cerebral cortex, amygdala + pyriform cortex, hippocampus, nucleus accumbens, striatum and hypothalamus) of the rat. Amitriptyline caused a marked increase in the TRH content in the striatum and nucleus accumbens. Citalopram and mianserin produced a smaller but significant increase in the TRH content in the striatum only, while imipramine did not significantly affect the TRH concentrations in any of the brain structures. None of the antidepressant drugs administered acutely significantly affected the TRH concentrations in the nucleus accumbens or the striatum. These results indicate that changes in brain TRH induced by antidepressant drugs are not related to their therapeutic activity.

Regional distribution of neuropeptide Y and its receptor in the porcine central nervous system.

The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200-1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50-200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained less than 50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0-5.87 pmol/mg of protein. Intermediate binding (0.5-1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but less than 0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.

Concentrations of tryptoline and methtryptoline in rat brain.

Combined gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry have been used to identify and quantify tryptoline, methtryptoline, 5-hydroxytryptoline, and 5-hydroxymethtryptoline as their heptafluorobutyryl derivatives in extracts of rat brain. Tryptoline and methtryptoline were identified on the basis of their retention times and mass spectral characteristics: they were reliably detected in brain tissue extracts without interference from artifactual formation; their whole brain concentrations ranged between 0.2 and 3 ng/g; and they had a similar neuroanatomical distribution, with the highest concentrations in the cerebellum and the cortex. Smaller quantities of 5-hydroxytryptoline and 5-hydroxymethtryptoline were also identified on the basis of their retention times and mass spectral characteristics. However, the significance of this finding is unclear, because these two compounds were accompanied by larger quantities of their tetradeuterated analogues formed from tetradeuterated-5-hydroxytryptamine added at the time of tissue homogenization; this result suggests that formation of 5-hydroxytryptoline and 5-hydroxymethtryptoline occurred during tissue homogenization, sample preparation, or both.

Iodinated-NPY binding sites: autoradiographic study in the rat brain.

Several authors have described the presence of iodinated neuropeptide-Y binding sites on membranes of the mammalian CNS. In the present study we show a mapping of iodinated-NPY binding sites in the rat brain using receptor autoradiography. The sections were incubated with 125I-Bolton-Hunter coupled NPY (0.5-03 nM), in the absence or presence of 1 microM cold NPY. Some autoradiograms are studied by means of an image analyzer (VDC 501 Tesak) equipped with the host computer PDP 11 Digital, in order to enhance the contrast of the labeling. A very high density of NPY receptors is present in the limbic regions (hippocampus, amygdaloid complex, septal nuclei), in the cortex, and in some thalamic nuclei, while in some hypothalamic regions (paraventricular nucleus and median eminence) we detected a lower amount of NPY receptors. At the mesencephalic level, the substantia nigra presents a very high density of NPY receptors.

Characterization and localization of a novel neuroreceptor for the peptide sarafotoxin.

We have recently shown that the rat atrium and brain contain specific high affinity receptors for the novel snake vasoconstrictor peptide sarafotoxin-b (SRTXb), and demonstrated toxin-induced phosphoinositide hydrolysis. Here we report on the characteristics of 125I-SRTXb receptors and their regional distribution in rat brain. 125I-SRTX receptors in the rat brain bind the toxin rapidly and with high affinity. The binding was not inhibited by ligands of known neurotransmitter receptor and ion channels. 125I-SRTX receptors have a distinctive regional distribution. The highest densities were observed in the cerebellum, thalamus and hypothalamus (850, 550 and 450 fmol/mg protein, respectively) and the lowest densities in the caudate and cerebral cortex (82 and 62 fmol/mg protein, respectively). Taken together our results suggest that mammalian brains contain a hitherto undetected neuroreceptor that may operate in neurotransmission with a "SRTX-like" brain peptide, similar to the SRTX homologous vasoconstrictor peptide of the mammalian endothelium endothelin.

Glycosaminoglycans in cortical autopsy samples from Alzheimer brain.

Each of the known classes of mammalian glycosaminoglycans, with the exception of keratan sulphate, was found in cerebral cortex samples from patients with Alzheimer-type dementia and age-matched controls. These molecules were quantitated, after electrophoresis and staining with Alcian Blue dye, by scanning densitometry. No significant differences were found between the mean levels of each of the above glycosaminoglycans in frontal cortex from patients with dementia compared with controls. An increase (26%; p less than 0.05) in the mean level of hyaluronate, but not of other glycosaminoglycans, was found in temporal cortex samples. On the other hand, the uronic acid content of hyaluronate degradation products following Streptomyces hyaluronidase treatment of brain glycosaminoglycans did not reveal any statistically significant changes in Alzheimer's disease. HPLC of disaccharide products from Arthrobacter chondroitinase AC digests did not reveal any significant changes in sulphate substitution of chondroitin sulphate in Alzheimer brain.

Regional distribution of the GABAA/benzodiazepine receptor (alpha subunit) mRNA in rat brain.

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.

[The corticofugal pathways of the visual system]. / Les voies corticofuges du système visuel.

Cortical neurons belonging to the same topological ensemble send axons to thalamic and mesencephalic structures and also to contra and ipsilateral cortical areas. The projections are called the corticofugal system. This review addresses the organization and the functions of the efferent cortical fibers within the visual network. For example, the cortico-geniculate fibers participate in shaping the structure of the concentric receptive fields of geniculate cells. Namely, the size of the surround area depends on descending impulses from the cortex. By contrast, cortico-mesencephalic fibers have a more global influence on visual responses. Following the interruption of cortical activity all responses to visual stimuli decline; although in rodents and lagomorphs cortical inactivation does not eliminate those visual responses that are sent to the superior colliculus or pretectum directly from the retina. In each hemisphere it has been demonstrated that contra-lateral cortico-cortical fibers participate in the continuity of the two visual hemi-fields, as the interruption of the callosal impulses results in a truncated field in which the contralateral part of the receptive field is missing., overlaps the vertical meridian is missing. Finally, ipsilateral cortico-cortical fibers allow a consolidation of visual properties of cortical cells. It must be added that there are considerable differences among species in the organization of cortico-cortical relationships. However, this survey seems to indicate that all corticofugal axons are excitatory.

The supramammillary region of the cat sends substance P-like immunoreactive axons to the hippocampal formation and the entorhinal cortex.

A combined method of the tracing of WGA-HRP (wheat germ agglutinin-conjugated horseradish peroxidase) and the immunohistochemistry of substance P (SP) showed that many SP-like immunoreactive neurons in the supramammillary nucleus of cat hypothalamus sent their axons to the hippocampal formation. SP-like immunoreactive axons in the hippocampal formation and entorhinal cortex were markedly reduced in number ipsilaterally after placing an electrothermic lesion in the supramammillary region of the hypothalamus.

Altered phosphoinositide fatty acid composition, mass and metabolism in brain essential fatty acid deficiency.

This study describes the specific alterations in phosphoinositide mass and fatty acid composition observed in brain essential fatty acid deficiency (EFAD). These investigations were motivated by the observation that alterations in volatile anesthetic potency were associated with changes in brain arachidonyl-phosphatidylinositol (PI) content, and were aimed at defining whether EFAD might alter the generation of chemical second messengers via the PI cycle. Analyses of cerebral cortical phosphoinositide mass and fatty acid composition showed that EFAD results in specific and preferential depletion of arachidonate (20:4(n - 6); 5,8,11,14-eicosatetraenoic acid) from cerebral cortical polyphosphoinositides, and that this depletion is reversed by parenteral supplementation with linoleic acid (18:2(n - 6); 9,12-octadecadienoic acid). These analyses also showed that, while phosphoinositides containing 20:3(n - 9) (5,8,11-eicosatrienoic acid) accumulated in EFAD, linoleate supplementation decreased 20:3(n - 9)-PI and 20:3(n - 9)-phosphatidylinositol 4-phosphate (PIP), but resulted in accumulation of 20:3(n - 9)-phosphatidylinositol 4,5-bisphosphate (PIP2). Comparison of the fatty acid composition of brain polyphosphoinositides and 1,2-diacylglycerols between treatment groups showed that diacylglycerols contain a lower molar percentage of 20:3(n - 9) and a higher percentage of arachidonate than the corresponding polyphosphoinositides. The combined results of these studies suggest the existence of fatty acid substrate specificity for the hydrolysis of PIP2 by phospholipase C. The biological relevance of these findings is suggested by a strong correlation between the mass of cerebral cortical arachidonyl-PIP2 and the potency of the anesthetic halothane.

Growth hormone-releasing factor immunoreactivity in the hypothalamus and cortex of the rat: in vivo and in vitro studies.

Utilizing a specific RIA for rat (r) GRF, hypothalamus and cerebral cortex from adult rat and long term dissociated fetal rat hypothalamic and cerebral cortical cell cultures were investigated for the presence of rGRF immunoreactivity (IR-GRF). After homogenization in an acidic medium, tissues and cultures were extracted on octadecylsilyl-silica columns, and IR-GRF and somatostatin (IR-SS) were measured by RIA. In extracts from the hypothalamus from the adult rat the content of IR-GRF was 3.02 +/- 0.16 ng ( +/- SE) per hypothalamus. IR-GRF was identical with synthetic rGRF on gel filtration chromatography and by parallel displacement of dilutions of extract in RIA. In extracts from cerebral cortex isolated from the adult rat, no IR-GRF was detected. In extracts from long term dissociated cell cultures from fetal hypothalami, 7.3 +/- 7 pg/10(6) cells of IR-GRF were present and were identical with synthetic rGRF by chromatographic and immunological criteria. In the extracts from cerebral cortical cell cultures cross-reacting material was present which on gel filtration chromatography revealed two peaks of immunoreactivity of higher mol wt than synthetic rGRF. There was nonparallelism on dilution of the extract. The ratio of IR-SS to IR-GRF by weight (IR-SS/IR-GRF) was calculated to compare the relative abundance of IR-GRF in cultured hypothalamic cells. In the hypothalamus isolated from the adult rat the ratio of IR-SS/IR-GRF by weight was 15.8 +/- 1.4 as compared to 48.9 +/- 10.3 in hypothalamic cultures. We conclude that IR-GRF indistinguishable from synthetic rGRF is present in long term dissociated hypothalamic cell cultures, but is relatively less abundant than in the hypothalamus of the adult rat when compared on the basis of IR-SS. No IR-GRF was detectable in cerebral cortex of the adult rat. At least one cross-reacting molecular species is detected in cerebral cortical cultures by the rGRF RIA, but exhibits nonparallelism and has a higher mol wt than synthetic rGRF. The increase of the ratio of IR-SS/IR-GRF in hypothalamic cell cultures in vitro compared to hypothalamus in vivo suggests that the culture conditions change differentially the expression of IR-SS and IR-GRF.

Immunocytochemical localization of vasoactive intestinal polypeptide (VIP) in the brain of the little brown bat (Myotis lucifugus).

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity has been examined in the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry and the indirect antibody enzyme method of Sternberger. Animals were sacrificed at three different and discrete levels of physiological activity: euthermic, hypothermic and hibernating. The density and distribution of immunoreactive neurons and fibres was compared in the three animal groups with the aid of a computerized image analysis system. Our results were compared with those of previous studies in laboratory species such as the rat and cat. Our study has demonstrated marked changes in the density of VIP-immunoreactive fibres and plexuses in the anterior hypothalamic area which correspond to the physiological state of the animal. In addition we have demonstrated the presence of VIP immunoreactive perikarya in a number of previously unreported locations. These include the paraventricular and periventricular hypothalamic nuclei, the linear raphe nucleus, nucleus interfascicularis, and in neurons embedded in the fibres of the dorsal tegmental decussation.

Assay of 2-hydroxyputrescine in various regions of rat brain by gas chromatography-mass spectrometry.

2-Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography-mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2-hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and midbrain. The level of 2-hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.

Effects of chronic diazepam and medazepam treatment on the level of biogenic monoamines in different rat brain areas.

Studies were made of the effect of 19-day administration of diazepam (1 mg.kg-1, i.p.) and medazepam (5 mg.kg-1, i.p.) on the level of biogenic monoamines in different rat brain areas: cerebral cortex, striatum, hippocampus, and hypothalamus. The noradrenaline (NA), dopamine (DA) and serotonin (5-HT) levels were determined. After chronic treatment with diazepam no alterations in the NA content in the cortex, striatum and hypothalamus were found. Medazepam increased the NA level in the cortex, striatum, and hippocampus without changing it in the hypothalamus. Repeated administration of diazepam or medazepam led to an increase in the DA level in the striatum. Both benzodiazepines provoked a significant increase in the hippocampal 5-HT level, which could be attributed to the antianxiety effect of these drugs.

The regulation of TRH and serotonin receptors: chronic TRH and analog administration in the rat.

Thyrotropin releasing hormone (TRH) and serotonin (5-HT) are co-transmitters in spinal and medullary neurons. To study the functional significance of this relationship and the regulation of TRH receptors, we chronically administered TRH and analogs MK-771 and CG-3509 to rats at a dose which evoked behavioral abnormalities. TRH reduced specific binding at spinal (24%) and hypothalamic (31%) TRH receptors and decreased TRH stimulated motor behaviors, such as rearing and cage crossing (locomotion). 5-HT1 and 5-HT2 receptors were unaffected except for an 11% increase in specific binding at spinal 5-HT1 sites. 5-HT and NE concentrations measured by HPLC were not altered in brainstem or hippocampus. In contrast, spinal TRH receptor specific binding increased 11% in rats treated intracisternally with 5,7-dihydroxytryptamine, but the effect was not significant. In competition studies in vitro, MK-771, TRH, and CG-3509 had no activity at 5-HT2 receptors in neocortex, brainstem, or spinal cord, and little activity at 5-HT1 sites (IC 50s greater than 100 uM). A mixed competitive and non-competitive binding profile at 5-HT1 sites resulted in the presence of 100 uM TRH. Conversely, 5-HT agonists had minimal effect at TRH receptors in spinal cord. These data suggest reciprocal or independent regulation of 5-HT1 and TRH receptors in co-transmitter and non-cotransmitter regions, respectively, in response to chronic TRH administration.

Prostacyclin (PGI2) induces rapid depletion of cyclic AMP levels in brain nuclei of rats.

Intravenous injection of prostacyclin (100 micrograms/kg) in rats resulted in a decrease of systolic blood pressure within 2 minutes. Concentrations of cAMP in 15 brain regions and nuclei were determined by radioimmunoassay. In lower brain stem nuclei, such as the nucleus of the solitary tract and the lateral reticular nucleus (A1 and C1 catecholaminergic cell groups) cAMP levels were depleted significantly, while in others, including the locus coeruleus and the periaqueductal central gray, cAMP levels did not show any alterations. Levels of cAMP were also depleted in some of the hypothalamic nuclei (periventricular, anterior hypothalamic, ventromedial), and in cerebral cortical areas. Lowered cAMP levels in brain areas might indicate lower cellular activity in cells participating in baroreceptor control mechanisms.

Pentylenetetrazol seizure threshold and extracellular levels of cortical amino acids in taurine-deficient kittens.

Directly after weaning, kittens were raised on a semisynthetic diet supplemented with 0.4% taurine or devoid of this amino acid. Eight to twelve weeks later the blood plasma concentration of taurine was decreased by 98-99% in kittens fed a taurine-free regimen. Parietal cortex dialysis, performed in anaesthetized kittens, revealed a selective, but less marked, reduction of extracellular taurine. When kainic acid was included in the dialysis buffer, taurine was doubled in taurine-supplemented kittens, but it was only slightly affected in deficient animals. The animals were also used for determination of the threshold of pentylenetetrazol-induced epilepsy 3 days after the dialysis experiment. This was not significantly different between the groups. The present work shows that taurine deficiency in its own right does not elevate interstitial glutamate, an effect previously observed in the 2-guanidino-ethane sulphonic acid model for taurine deficiency. The results further suggest that taurine is better retained in neural cells in the taurine-deficient state. Moreover, the findings argue against a role for endogenous taurine in the control of epileptiform discharge initiation and/or spread.