Age-dependent changes in the inactivation of thyrotrophin-releasing hormone by different areas of rat brain.

Age-dependent changes in the inactivation of thyrotrophin-releasing hormone (TRH) were investigated in two subcellular fractions prepared from hypothalamus, thalamus, cortex and cerebellum of male rats. It was found that, in both supernatant and particulate fractions from the four brain areas, enzyme activity increased to a peak at 10-25 days of age and then decreased gradually until adult levels were reached. The changes in TRH degradation observed may be related to alterations in hypothalamic TRH content with age and to maturation of the tripeptide's putative neurotransmitter/neuromodulator function.     

Local degradation of growth hormone-release-inhibiting hormone (somatostatin) in the central nervous system

Peptidases inactivating growth hormone-release-inhibiting hormone (somatostatin) were found to be present in two fractions prepared from the thalamus, hypothalamus, cortex and cerebellum of male and female rats. Somatostatin was also detected in significant quantities in fractions from the thalamus, hypothalamus, and cortex, after peptidase activity had been removed by heat denaturation. These results support the suggestion that somatostatin may function as a neurotransmitter or modulator of neuronal activity in the central nervous system.     

Thyrotropin releasing hormone (TRH): its widespread distribution in discrete hypothalamic nuclei and areas in rat brain

The precise distribution of thyrotropin releasing hormone (TRH) in 23 discrete brain nuclei and areas of Wistar strain male rats was determined by specific radioimmunoassay. TRH was detected in most of these areas. The highest concentration was found in the median eminence (27.52 +/- 2.84 ng/mg protein). The arcuate nucleus (4.92 +/- 0.58 ng/mg protein), dorsomedial nucleus (4.77 +/- 0.59 ng/mg protein) and medial preoptic area (3.94 +/- 0.15 ng/mg protein) also contained a considerable concentration of TRH. However, no TRH was detected in cerebral cortex, cerebellar hemisphere, anterior pituitary or pineal body. The data indicated that TRH was widely distributed throughout the hypothalamus; in particular, high concentrations occure in relatively restricted areas: in the median eminence, arcuate nucleus, dorsomedial nucleus and medial preoptic area. These areas coincide well with the so-called "thyrotropic" area of the hypothalamus.     

Major localization of aminopeptidase M in rat brain microvessels

The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 3.4.11.2) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.     

Local degradation of luteinizing hormone-releasing hormone (LH-RH) in the rat central nervous system

Peptidases inactivating luteinizing hormone-releasing hormone (LH-RH) were found to be present in two subcellular fractions prepared from the following areas of the central nervous system of male and female rats: thalamus, hypothalamus, cortex and cerebellum. LH-RH immunoreactivity was also detected in these fractions after peptidase activity had been removed by heat denaturation. These findings support the concept that LH-RH may have a role in modulating neuronal activity within the central nervous system (CNS).     

Evidence of dopamine D1 receptor mRNA and binding sites in cultured human amniotic epithelial cells

The noradrenergic innervation of the frontal cortex and the hypothalamus arises from different brainstem nuclei and neurones projecting to these two brain regions differ topographically and morphologically. In the present study, we investigated whether there is a difference in noradrenaline clearance in these two brain areas using dual-probe microdialysis in freely-moving rats. Noradrenaline clearance was determined simultaneously in each brain area by measuring the noradrenaline extraction fraction using the ‘No Net Flux’ method, with both brain regions perfused with the same solutions. The noradrenaline extraction fraction was greater in the frontal cortex than in the hypothalamus (72 and 54%, respectively). This finding suggests that the processes involved in noradrenaline clearance, especially uptake, are more efficient in the former brain area, which could explain the differences in the increase in noradrenaline efflux when these brain areas are challenged with drugs that alter the function of the central nervous system.     

Regional distribution of phosphoinositide-specific phospholipase C activity in rat brain

The phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolytic activities of phosphoinositide-specific phospholipase C (PLC) were measured in membrane and cytosol fractions from 7 discrete areas of the rat brain. Both the PI-PLC and PIP₂-PLC specific activities were found to differ significantly among the 7 discrete brain areas. In the membrane fraction, the PIP₂-PLC activity was higher than that of PI-PLC in each region, suggesting that the PLC in membranes prefers PIP₂ to PI as substrate. The PIP₂-PLC activities in the membrane were high in prefrontal cortex and cerebellum, but rather low in medulla oblongata and hypothalamus. The PI-PLC specific activity in the cytosol was significantly higher than that in the membrane of all brain areas examined. The PI-PLC specific activity in membranes is inversely proportional to its activity in the cytosol. In the cytosol fraction, the distribution pattern of PI-PLC specific activity resembled that of PIP₂-PLC. These results indicate that PLCs are differently distributed in various regions of rat brain, and suggest the regional differences in neuronal transduction.     

Regional distribution of the membrane-bound pyroglutamate amino peptidase-degrading thyrotropin-releasing hormone in rat brain

The brain regional distribution of membrane-bound pyroglutamate aminopeptidase-degrading thyrotropin-releasing hormone (TRH) in rat was studied using a specific radiometric assay. The distribution was not homogeneous: a 10-fold difference was observed between regions. The highest activity was detected in olfactory bulb while the lowest was in the cervical part of spinal cord. There was no correlation with the regional distribution of enzyme activity vs TRH levels, previously reported TRH receptors or in vitro TRH release. The differential distribution of this enzyme is consistent with the hypothesis that it is responsible for extracellular degradation of neuroactive peptides.     

Neuropeptides and their receptors in aged-rat brain

Age-associated changes in methionine-enkephalin (ENK) and thyrotropin releasing hormone (TRH) concentrations, and their receptors were examined in discrete regions of the rat brain. The ENK and TRH concentrations in aged rats were nearly identical to those in young adult rats, except for a slightly lower TRH value in the hypothalamus of the aged rats. On the other hand, the ENK and TRH receptor levels in the cerebral cortex of aged rats was markedly lower than that of young adults rats. The results suggest that determinations of both neuropeptide and receptor levels are indispensable for evaluation of peptide-mediated neural systems in the central nervous system.     

Regional glucose metabolism in the rabbit brain in control and TRH-treated animals

The local cerebral metabolism in urethane anaesthetized control and TRH-treated rabbits was studied with the [14C]2-deoxyglucose method. In the controls, the glucose use was found to be highest in regions known to have a high blood flow and low in regions with low flow. The glucose consumption was, calculated using the constants found by Kennedy et al. in monkeys, 23.5 +/- 6.0 mumol 100 g-1 min-1 in parietal cortex. The TRH was infused at a dose of 0.06 mg kg-1 min-1 which is known to cause vasodilation in the brain. No marked influence of the peptide on the glucose use was detected. It was concluded that the previously reported cerebral vasodilation caused by TRH is not due to an increase in cerebral metabolism.     

Changes in the content of GFAP in the rat brain during pregnancy and the beginning of lactation

Several changes in brain function, including learning and memory, have been reported during pregnancy but the molecular mechanisms involved in these changes are unknown. Due to the fundamental role of glial cells in brain activity, we analyzed the content of glial fibrillary acidic protein (GFAP) in the hippocampus, frontal cortex, preoptic area, hypothalamus and cerebellum of the rat on days 2, 14, 18, and 21 of pregnancy and on day 2 of lactation by Western blot. A differential expression pattern of GFAP was found in the brain during pregnancy and the beginning of lactation. GFAP content was increased in the hippocampus throughout pregnancy, whereas a decrease was observed in cerebellum. GFAP content was increased in the frontal cortex and hypothalamus on days 14 and 18, respectively, with a decrease in the following days of pregnancy in both regions. In preoptic area a decrease in GFAP content was observed on day 14 with an increase on days 18 and 21. In the frontal cortex and cerebellum, GFAP content was increased on day 2 of lactation, while it was maintained as on day 21 of pregnancy in the other regions. Our data suggest a differential expression pattern of GFAP in the rat brain during pregnancy and the beginning of lactation that should be associated with changes in brain function during these reproductive stages.     

Adenosine A1 receptors in the human brain: a quantitative autoradiographic study

The distribution of adenosine A1 receptors in the human brain was studied by autoradiography in post mortem brain tissues from 26 subjects without reported neurological disease. N6-[3H]Cyclohexyl-adenosine was used as the ligand. For comparison, adjacent sections of some regions were examined histochemically for 5'-nucleotidase activity. The receptor sites were heterogeneously distributed throughout the CNS. The highest receptor densities were found in the stratum oriens, pyramidale and radiatum of the hippocampus. High densities were also found in the cerebral cortex and the striatum. In the thalamus there was a heterogeneous distribution of binding sites with a high density in structures such as the medial and anterior nucleus. Intermediate receptor densities were found in the accumbens, the olfactory tubercle and most parts of the amygdala among others. The hypothalamus had low receptor densities. In the brainstem and the spinal cord very low receptor concentrations were found. However, in some structures such as the substantia nigra, the colliculus superior and the substantia gelatinosa of the spinal cord a low level of binding could be measured. The cerebellar cortex showed low densities of receptors. Structures showing high levels of 5'-nucleotidase activity were the hippocampus, the striatum and parts of the cerebral cortex among other regions. In general there was a poor correlation between the localization of A1 receptors and the 5'-nucleotidase activity. Some regions, however, showed a similar distribution of these two markers. In general, the distribution of adenosine A1 receptors found in the human brain is comparable to that found in previous autoradiographic studies in the rat brain. However, some regional differences were observed in, for example, the cerebral cortex, the striatum and the cerebellar cortex. These differences may prove to be functionally relevant.     

Acute starvation decreases acetylcholinesterase activity in different regions of rat brain.

The activity of acetylcholinesterase (AChE) was assayed spectrophotometrically in four brain regions of rats that had been deprived of food for 96 h. A significant decrease in the total AChE activity (by 4-45%) as well as in its specific activity (by 14-28%) was observed in the supernatant and total particulate fractions from cerebral hemispheres, cerebellum, brainstem and diencephalon + basal ganglia. Similarly, blood glucose, body weight and protein content of subcellular fractions from most brain regions showed decreases after starvation.     

Effect of nembutal anesthesia,electric shock,and shock avoidance conditioning on acetylcholinesterase activity and protein content in various regions of the rat brain

Nembutal [pentobarbital sodium] was found to increase the protein content in some regions of the rat brain (posterior cortex, caudate nucleus) and to decrease the protein content in the ventral cortex. Acetylcholinesterase (AChE) activity, expressed in terms of wet tissue weight, increased in the cerebellum and medulla, but decreased in the medial cortex, hippocampus, thalamus, and the caudate nucleus. These changes in AChE activity were not explicable in terms of a direct effect of the anesthetic on the enzyme. A decrease in the protein content was observed in the frontal cortex, ventral cortex, hippocampus, and the caudate nucleus after electric shock is applied. Following shock avoidance conditioning, a decrease in protein content was observed in the medial cortex, posterior cortex, cerebellum, and the ventral cortex; the protein content increased in the thalamus. An increase in AChE activity was observed in the frontal cortex and in the medulla after shock, but its content decreased in the caudate nucleus, hypothalamus, thalamus, and the olfactory bulb. Following shock avoidance conditioning AChE activity increased in the posterior cortex, hippocampus, thalamus, and hypothalamus, but decreased in the ventral cortex.Translated from Zhurnal Evclyutsionnoi Biokhimii i Fiziologii, Vol. 15, No. 3, pp. 254–262, May–June, 1979.     

Efflux of 5-HIAA from 5-HT neurons: a membrane potential-dependent process.

The effect of the membrane potential on the efflux of 5-HIAA from 5-HT neurons was studied in anesthetized (halothane: 1% in gas mixture of N2O: 70% and O2: 30%) cats. The endogenous 5-HT and its metabolite 5-HIAA were measured continuously from the cortex, the thalamus, the hypothalamus and the raphe nuclei using brain microdialysis technique combined with HPLC-ED monoamine measurements. Membrane potential variations were induced by changing the extracellular concentration of potassium through the microdialysis membrane. The levels of the extracellular 5-HIAA varied according to the different regions of the brain, being highest in the hypothalamus and lowest in the cerebral cortex. Increases in the extracellular potassium from 4 to 120 mM invariably produced a decrease of the extracellular 5-HIAA in all the tested brain regions. This decrease was inversely proportional to the logarithm of extracellular potassium concentration. Thus, it is postulated that the 5-HIAA is moved from inside the cell to extracellular space by an active mechanism of transport electrically coupled to the membrane potential.     

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.     

Stimulation of mono- and diacylglycerol lipase activities in ibotenate-induced lesions of nucleus basalis magnocellularis.

Ibotenic acid was injected into the nucleus basalis magnocellularis region of rat brain in order to study whether an elevation of lipase activities was associated with the degeneration of cholinergic neurons in this potential animal model of Alzheimer's disease. Two plasma membrane fractions were prepared from different regions of ibotenate injected (right hemisphere) and non-injected (left hemisphere) rat brain. One plasma membrane fraction was from synaptosomes (SPM) and the other from glial and neuronal cell bodies (PM). Activities of mono- and diacylglycerol lipases in these plasma membrane fractions were markedly increased (3- to 5-fold) in hippocampus, midbrain and frontal cortical regions of rat brain at 10 days after the injection of ibotenate. The activity of choline acetyltransferase was decreased in frontal cortex but unchanged in hippocampus and midbrain. Our results suggest that the increase in lipase activity is much more widespread and non-specific than is the decrease in cholinergic function.     

Effect of starvation on insulin receptors in rat brain

To determine the effect of starvation on brain insulin receptors, rats were fed 4 g of chow/day for 14 days and then P2 fraction membranes were prepared from different brain regions. Compared to the fed state, there was an 18% reduction of insulin binding in olfactory bulbs from starved animals, but no change in the cerebellum, frontal cortex, amygdala, medial hypothalamus or lateral hypothalamus. A 15% reduction of olfactory bulb insulin binding was obtained by totally starving animals for four days. When membrane content was measured using the plasma membrane marker Na/K ATPase, insulin binding decreased by 26% and 14% in olfactory bulb membranes from starved and totally starved animals, respectively. The starvation-induced change in olfactory bulb binding was due to a loss of binding sites and not a decrease in binding affinity. Non-specific catabolism of protein and a change in the composition of membranes following starvation were excluded as causes for this effect. As streptozotocin induced diabetes had no effect on brain insulin binding, it was concluded that hypoinsulinaemia associated with starvation had not caused the reduction in olfactory bulb binding. Under similar conditions of starvation and diabetes, insulin binding in liver plasma membranes increased 26% and 38%, respectively. At 8 and 14 days of starvation, the reductions in olfactory bulb insulin binding and body weight were similar. On refeeding for three days, there was no increase in insulin binding, although body weight increased 7%. On refeeding for eight days, olfactory bulb insulin and body weight had returned to near normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

补肾药延缓老年大鼠下丘脑—垂体—甲状腺轴的功能退化

用放射免疫分析和高效液相测定不同组织和血液的激素和神经递质含量，以观察补肾益精主药－－固真方对老年大鼠下丘脑－垂体－甲状腺轴作用的影响。结果表明：（１）与青年对照组比较，老年大鼠下丘脑Ｔ３Ｒ、ＴＲＨ明显降低，垂体ＴＳＨ呈低下趋势；（２）老年大鼠血清Ｔ２、Ｔ４含量明显下降，而ｒＴ３则显著增高；血清ＴＲＨ和ＴＳＨ呈代偿性增高；而血冰亮氨酸脑啡肽（Ｌ－ＥＫ）含量明显降低：（３）老年大鼠在脑皮层去甲肾上腺     

Recognition by a mouse monoclonal antibody of a glycoprotein antigen of rat brain which is expressed intracellularly by neurons

We describe a previously uncharacterised glycoprotein antigen of rat brain. The antigen was localised by immunofluorescence on 10 μm cryostat tissue sections, and was found to be present intracellularly in neurons. No other cell types or structures within the brain were stained. The antigen is recognised by a mouse monoclonal antibody called NGP41. The antibody was produced after immunising a mouse with glycoproteins purified by lentil lectin affinity chromatography of solubilised rat brain membranes. Spleen cells from the immunised mouse were fused with the myeloma P3X63Ag8. The antigen is expressed by neurons in all brain regions, and also in the dorsal root ganglion neurons of the peripheral nervous system. In all brain regions, the large projection neurons are the most intensely stained by immunofluorescence, but some small neurons also express the antigen. Although dendrites were not stained, sections of sciatic nerve were stained by NGP41, suggesting that the antigen is expressed by axonal processes. The cell bodies of neurons in the inferior olive were stained by NGP41, but their terminals on Purkinje cell dendrites in the cerebellar cortex were not stained, suggesting that the antigen is absent or expressed below the limit of detection in terminals. Both crude brain membranes and a lentil lectin affinity purified brain glycoprotein fraction absorbed the antibody, suggesting that the antigen is a membrane bound glycoprotein. In immunoblotting experiments, the antigen was detected in homogenates of brain and spinal cord membranes, where it appeared as a triplet of bands with molecular weights of 41K, 38K and 36K. Antigen was not detected by immunoblotting in homogenates of six different tissues of non-nervous origin. The antigen was enriched in glycoprotein fractions from adult and juvenile cerebellum as assessed by immunoblotting. Adult brain glycoprotein preparations had a triplet structure similar to that in the homogenates, although most of the antigenic activity of the juvenile preparation was found in a position corresponding to the upper two bands of the triplet.