Cytochrome P-450 activities in human and rat brain microsomes

The role of cytochrome P450 in the metabolism of dextromethorphan, amitriptyline, midazolam, S-mephenytoin, citalopram, fluoxetine and sertraline was investigated in rat and human brain microsomes. Depending on the parameters, the limit of quantification using gas chromatography-mass spectrometry methods was between 1.6 and 20 pmol per incubation, which generally contained 1500 microg protein. Amitriptyline was shown to be demethylated to nortriptyline by both rat and human microsomes. Inhibition studies using ketoconazole, furafylline, sulfaphenazole, omeprazole and quinidine suggested that CYP3A4 is the isoform responsible for this reaction whereas CYP1A2, CYP2C9, CYP2C19 and CYP2D6 do not seem to be involved. This result was confirmed by using a monoclonal antibody against CYP3A4. Dextromethorphan was metabolized to dextrorphan in rat brain microsomes and was inhibited by quinidine and by a polyclonal antibody against CYP2D6. Only the addition of exogenous reductase allowed the measurement of this activity in human brain microsomes. Metabolites of the other substrates could not be detected, possibly due to an insufficiently sensitive method. It is concluded that cytochrome P450 activity in the brain is very low, but that psychotropic drugs could undergo a local cerebral metabolism which could have pharmacological and/or toxicological consequences.     

Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain.

Brain macrophages and microglia play important roles in central nervous system (CNS) development, especially during regressive events in which particular neuronal and glial constituents are eliminated. The purpose of this study is to provide a complete map of brain macrophage and microglia distribution in all regions of the neuraxis from birth to sexual maturity. We have utilized morphology and immunostaining with the specific antibodies OX-42 and ED1 to distinguish between brain macrophages and microglia. Brain macrophages are large, round cells, 10-15 microns in diameter, with few or no cytoplasmic processes; these cells are ED1- and OX-42-immunopositive. Microglia have small cell bodies with numerous, ramified cytoplasmic processes. These cells are OX-42-positive, and ED1-negative. We found a specific pattern of distribution of brain macrophages, targeting specific cortical and subcortical areas transiently, including developing fiber tracts. These cells disappeared completely by the third postnatal week. In contrast, OX-42-positive microglia exhibited a gradual increase in number and were distributed uniformly throughout gray matter and within white matter tracts. These cells remain in the adult CNS, constituting the resident microglia population. We suggest that these two distinct phagocytic cell populations perform unique functions in the developing brain, including remodeling of restricted CNS areas by brain macrophages that is part of a normal morphological process.     

Expression of metabotropic glutamate receptors in rat meningeal and brain microvasculature and choroid plexus

This study investigated the distribution of metabotropic glutamate receptors (mGluRs) in meningeal and parenchymal microvasculature and in choroid plexus by means of Western blot analysis and immunohistochemistry. Western blot analysis demonstrated mGluR expression in both rat and human leptomeningeal tissues. In the rat, mGluR expression was developmentally regulated, with only mGluR2/3 showing expression at the embryonic day 19 developmental stage. In contrast, mGluR1 alpha, mGluR2/3, mGluR4a, and mGluR7 were expressed in leptomeninges from adult rats. Immunohistochemical analyses showed intense mGluR1 alpha immunoreactivity in the pia mater and blood vessels in the subarachnoid space and in the arachnoid layer of the meninges. mGluR2/3, mGluR4a, mGluR5, and mGluR7 were also expressed in meningeal microvasculature. In addition, the parenchymal microvasculature and choroid plexus were strongly immunoreactive for mGluR1 alpha, mGluR2/3, mGluR4a, mGluR5, and mGluR7. We used antibodies specific for phenotypic markers of microvascular and glial cells to characterize the cell type(s) immunopositive for mGluRs. Comparison of staining with anti-von Willebrand factor antibody and anti-mGluR antibodies revealed that mGluR immunoreactivity was present in cells that surrounded the luminal surface labeled by the endothelial cell marker. In these cells, smooth muscle actin and mGluR immunoreactivity overlapped, suggesting that, in addition to endothelial cells, pericytes within the microvasculature also express mGluRs. Furthermore, expression of mGluR1 alpha was also observed in pure pericyte cultures isolated from bovine retina. These data suggest that glutamate by means of activation of mGluRs may have a broad sphere of physiological influence in the brain which in addition to modulating synaptic transmission may also have a role in determining microvascular function and dysfunction.     

Effect of glucocorticoids on galactosylceramide sulfotransferase activity in rat brain

Previous studies have shown that glucocorticoids can induce the sulfatide-forming enzyme galactosylceramide sulfotransferase (GalCer-ST) in cultured glioblastoma cells. To investigate whether a similar process occurs in vivo, we administered corticosterone to infant and adult adrenalectomized rats and then assayed brain GalCer-ST activity and sulfatide content. Both measures were unexpectedly decreased rather than increased by hormone treatment, indicating that glucocorticoids may not regulate brain sulfatide biosynthesis in the same manner as observed in clonal cell lines.     

BMP2 expression in the adult rat brain

Bone morphogenetic protein-2 (BMP2) is a member of the transforming growth factor-β (TGF-β) superfamily and plays important roles in multiple biological events. Although BMP2 expression has been well described in the early development of the central nervous system (CNS), little information is available on its expression in the adult CNS. We thus investigated BMP2 expression in the adult rat CNS by using immunohistochemistry. Here we show that BMP2 is widely expressed throughout the adult CNS. In addition, besides intense BMP2 expression in almost all neurons, we found BMP2 expression in astrocytes and ependymal cells. Interestingly, we found that the axons of olfactory sensory neurons express BMP2. In addition, in the glomerular layer, BMP2 was very strongly expressed in some glomeruli, whereas the other glomeruli were weakly stained, suggesting that the variations in BMP2 expression level in each glomerus might be cues for each axon to find its adequate target and to keep its identity. Furthermore, we compared the expression patterns of BMP2 and BMP4. Interestingly, BMP4 was preferentially expressed in the dendrites of several neurons, whereas BMP2 was basically not expressed in the dendrites; however, it was detected in the axons. This means that in a single neuron the localizations of BMP2 and BMP4 are differentially regulated. These data indicate that BMP2 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP2 also plays pivotal roles in the adult brain.     

Merlin在大鼠脑的分布

目的：对正常成年大鼠脑中NF2基因表达产物Merlin的分布进行了观察。方法：运用免疫组化ABC法。结果：（1）Merlin阳性反应较强的脑区和核团有大脑皮质，杏仁核，丘脑前核群，丘脑内侧核群，丘脑板内核群，丘脑中线核群，脑干前庭内侧核以及小脑皮质颗粒层等。（2）中等阳性免疫反应的结构有海马结构，隔外侧核，隔内侧核，下丘脑室周核，下丘脑室旁核，蜗神经背侧核，蜗神经腹侧核，前庭内侧核等。（3）阳性免疫反应较弱的脑区和核团有尾壳核，屏状核，苍白球，下丘脑外侧前核，下丘脑背侧核，三叉神经脊束核，巨细胞网状核等。结论：Merlin在大鼠脑中有广泛的表达，但在不同的脑区和核团其表达强度不同。     

Decreased serotonin turnover in the dorsal hippocampus of rat brain shortly after adrenalectomy: selective normalization after corticosterone substitution

Pargyline-induced accumulation of serotonin (5-HT) was used as an index of 5-HT turnover rate in the dorsal hippocampus. One hour after bilateral removal of the adrenals, 5-HT turnover was significantly reduced when compared to that of the sham-operated controls. A low dose of corticosterone given immediately after adrenalectomy restored the 5-HT response, while the same dose of dexamethasone was ineffective. Pretreatment with dexamethasone blocked the 5-HT response to corticosterone in the acutely adrenalectomized rat. The specificity of the 5-HT response in the hippocampus corresponds to the properties of the glucocorticoid receptor system in rat hippocampal neurons.     

Immunohistochemical studies of FMRF-amide-like immunoreactivity in rat brain

The anatomical distribution of neuronal perikarya and nerve fibres containing FMRF-amide-like immunoreactivity in the brain, spinal cord and pituitary of the rat has been studied by immunohistochemistry. In animals pretreated with colchicine, the highest concentration of nerve cell bodies occurred in hypothalamic nuclei. Cells were also present in the cortex, striatum, septum, thalamus and in the brainstem. Beaded nerve fibres were abundant in the septum, nucleus of the striae terminalis, hypothalamus, medial regions of the thalamus, the parabrachial nucleus, the ventrolateral medulla, the substantia gelatinosa of the spinal trigeminal nucleus and the dorsal horn of the spinal cord. Fibres were also present in the cortex, striatum, amygdala, pons, ventral spinal cord and the neural lobe of the pituitary. The localization was specific in that preabsorbtion of the antisera with FMRF-amide, but not structurally related molecules such as Met-Enk-Arg6Phe7, APP or BPP, completely abolished the localization. The mammalian counterparts of FMRF-amide may have a neurotransmitter or neuromodulatory role.     

Characterization of inducible cyclooxygenase in rat brain

Considerable debate exists regarding the cellular source of prostaglandins in the mammalian central nervous system (CNS). At least two forms of prostaglandin endoperoxide synthase, or cyclooxygenase (COX), the principal enzyme in the biosynthesis of these mediators, are known to exist. Both forms have been identified in the CNS, but only the distribution of COX 1 has been mapped in detail. In this study, we used Western blot analysis and immunohistochemistry to describe the biochemical characterization and anatomical distribution of the second, mitogen-inducible form of this enzyme, COX 2 in the rat brain. COX 2-like immunoreactive (COX 2-ir) staining occurred in dendrites and cell bodies of neurons, structures that are typically postsynaptic. It was noted in distinct portions of specific cortical laminae and subcortical nuclei. The distribution in the CNS was quite different from COX 1. COX 2-ir neurons were primarily observed in the cortex and allocortical structures, such as the hippocampal formation and amygdala. Within the amygdala, neurons were primarily observed in the caudal and posterior part of the deep and cortical nuclei. In the diencephalon, COX 2–ir cells were also observed in the paraventricular nucleus of the hypothalamus and in the nuclei of the anteroventral region surrounding the third ventricle, including the vascular organ of the lamina terminalis. COX 2–ir neurons were also observed in the subparafascicular nucleus, the medial zona incerta, and pretectal area. In the brainstem, COX 2-ir neurons were observed in the dorsal raphe nucleus, the nucleus of the brachium, of the inferior colliculus, and in the region of the subcoeruleus. The distribution of COX 2-ir neurons in the CNS suggests that COX 2 may be involved in processing and integration of visceral and special sensory input and in elaboration of the autonomic, endocrine, and behavioral responses.     

Postnatal loss of axons in normal rat sciatic nerve

Myelinated and unmyelinated axons were counted in sciatic nerves of newborn, 5-day-old, 14-day-old, and adult rats. Myelinated axons increase from essentially none at birth to approximately 8,000 in adulthood, but total axon numbers decrease steadily from 33,954 at birth to 22,872 in adulthood. Thus there is a significant postnatal loss of axons from rat sciatic nerve. This loss is, in our opinion, not associated with the death of the cells that give rise to these axons. This is thus an example of a regressive event that probably is of importance in normal neural development, namely the postnatal elimination of axons unaccompanied by death of the neurons that give rise to axons. These findings presumably imply a considerable amount of proximal peripheral axon branching, and the postnatal elimination of axons in the sciatic nerve presumably results from a reduction of this branching. Thus postnatal elimination of processes on, for example, somatic muscle cells may be at least partially the result of long axon elimination rather than local withdrawal of presynaptic processes, as is usually thought to be the case. In addition, an increased number of axons resulting from early postnatal manipulations may indicate cessation of axon loss rather than formation of new axons.     

Sexual dimorphism of glucocorticoid binding in rat brain

Glucocorticoids bind with high affinity to intracellular receptors located in high density within discrete regions of the rodent and primate brain. The binding of [3H]corticosterone was compared in the brains of male vs female rats. The number and affinity of cytosol receptors in the hippocampus and hypothalamus were examined in vitro. The cytosolic binding capacity of the hippocampus is greater in the female than in the male. This difference in binding capacity is not dependent on the presence of gonadal steroids: the effect of gonadectomy was not significant for either sex. The difference is not due to transcortin since the binding capacity of [3H]dexamethasone is also greater in the female hippocampus. Receptor affinity in the female hippocampus is half that of the male value. In the hypothalamus, the dimorphism is in the opposite direction: the number of [3H]corticosterone cytosolic binding sites was found to be greater in the male. The male hypothalamus also showed a greater affinity for [3H]corticosterone than did the female. Ovariectomy increased the number of binding sites in the female hypothalamus. In vivo nuclear uptake of a tracer dose of [3H]corticosterone was determined in animals having intact gonads. The percent of tissue [3H]corticosterone present in cell nuclei from 4 brain regions, including the hippocampus and hypothalamus, was calculated per unit DNA. The concentrations of [3H]corticosterone in nuclei relative to tissue homogenates were higher in females than males for the 4 brain regions, but not for the pituitary or liver. The data are interpreted as suggesting that glucocorticoid secretion under basal conditions and during stress may differentially effect specific brain structures in male vs female rats.     

Bone morphogenetic protein-4 expression in the adult rat brain

Bone morphogenetic protein-4 (BMP4) is a member of the transforming growth factor-beta (TGF-beta) superfamily and plays important roles in multiple biological events. Although BMP4 expression has been well described in the early development of the central nervous system (CNS), little information is available on its expression in the adult CNS. Therefore, we investigated BMP4 expression in the adult rat CNS by using immunohistochemistry. BMP4 is intensely expressed in most neurons and their dendrites. In addition, intense BMP4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes also express BMP4 protein. These data indicate that BMP4 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP4 plays pivotal roles also in the adult brain.     

Serotonin turnover in rat brain during semistarvation with high-protein and high-carbohydrate diets

Summary Serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) were determined in seven brain regions of semistarved and control male rats. After semistarvation on a high carbohydrate diet serotonin turnover, as indicated by 5-HIAA/5-HT ratio, was increased in the total brain and several regions both three and 24 hours after the last meal. In contrast, after semistarvation on a high-protein diet serotonin turnover was decreased three hours after ingestion of the final meal, but increased 24 hours thereafter. Compulsary running wheel activity for one hour did not influence diet inducet changes in serotonin turnover. Alterations in plasma corticosterone during semistarvation were not related to changes in central serotonin turnover. Data suggest that the interaction of caloric restriction and diet composition determines serotonin turnover during semistarvation.     

Effects of corticosterone and 5 alpha-dihydrocorticosterone on brain excitability in the rat

The effects of corticosterone (B) and its reduced metabolite 5 alpha-dihydrocorticosterone (DHB) on CNS activity in the rat were examined. Two indices of brain excitability were evaluated: 1) amplitude of population responses (evoked potentials [EP] to sciatic nerve stimulation) and 2) changes in the rate of firing of tonically discharging neurons--both at pontine brainstem regions of the reticular formation. Experiments were carried out in adrenalectomized rats, and recordings were obtained from animals under urethane anesthesia. Steroids were dissolved in a 4:1 saline:Cremophor-El (Sigma) solution and doses of 750 micrograms/0.5 ml were injected (IV). The effects of B on EPs were bidirectional. Increases (8 animals) and decreases (6 animals) of the amplitude responses in different animals were observed. In 4 animals, no changes were detected. In contrast, injection of DHB produced a consistent and significant reduction of brainstem sciatic evoked potentials in 10 of 12 animals tested; 2 animals did not respond to the steroid. At the neuronal level, the effects of the steroids were evaluated by the changes they induced in the mean firing frequency (P less than 0.01) measured during 5-min intervals as determined by a one-way analysis of variance and analysis with a test of multiple comparisons. Only cells that fired in a stationary mode for 15 min before the steroid injection were studied. A more consistent pattern of responses to B was observed at the single-cell level. From 31 neurons that responded to the hormone, of 76 examined, 27 showed an increase in their firing rate and only 4 neurons showed a decrease. The increase in firing rate had an onset latency of 2-5 min (means = 3.5, SE 0.43) with a duration of 16-25 min (means = 17.5, SE 2.7). Of 69 neurons that were tested with DHB, 51 showed a significant decrease in their mean firing frequency. Onset latency of the effect was 2-8 min (means = 4.0, SE 1.21) and the duration of the induced changes was 16-40 min (means = 30.0, SE 3.47). Central interactions of DHB and B when sequentially administered were examined in 28 neurons. Of these, 21 responded to DHB administration with a significant decrease in their firing rates. In 11 of these neurons, injection of B, 5 min after DHB, was followed by a rapid (1-2 min) return of the neurons to baseline firing rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Priming with interleukin-1beta suppresses experimental allergic encephalomyelitis in the Lewis rat

Lewis rats exhibit multiple defects in their hypothalamus-pituitary-adrenal (HPA) system that are considered to play a causal role in the susceptibility of this strain to autoimmune diseases, i.e. experimental allergic encephalomyelitis (EAE). In the present study, we aimed to modulate the HPA response of the Lewis rat and establish its consequences for the susceptibility to EAE. Because in Wistar rats, single administration of interleukin (IL)-beta (priming) is known to induce long-lasting (weeks) sensitization of HPA responses to stressors and immune stimuli, Lewis rats were given a single dose of hIL-1beta or vehicle 1 week prior to induction of EAE by immunization with myelin basic protein (MBP). Subsequently, neurological deficits were monitored once daily. The results show that IL-1 priming markedly suppresses the neurological symptoms of EAE, without affecting the onset or duration of the disease. Measurement of vasopressin and corticotropin releasing hormone (CRH) in the external zone of the median eminence revealed that, as compared to Wistar rats, Lewis rats exhibit low vasopressin but identical CRH, and that IL-1 priming increases (0.001) vasopressin without affecting CRH stores, which is consistent with a shift to vasopressin-dominated control of adrenocorticotropic hormone (ACTH) secretion as described in Wistar rats under conditions of HPA hyper(re)activity. However, IL-1 priming did not affect a.m. corticosterone levels following immunization with MBP or during the clinical phase of EAE. IL-1 priming of Lewis rats attenuated the ACTH responses to an IL-1 challenge 11 days later, which may relate to an increase in resting corticosterone levels. Thus, the mechanisms underlying IL-1 induced suppression of EAE are not related to enhanced HPA responses. In addition, we did not find IL-1 priming-induced alterations in MBP-specific immunoglobulin (Ig)M, IgG1, IgGa and IgGb plasma titres, or gross alterations in T cell activation as reflected in spontaneous or concanavalin-induced T cell proliferation. We therefore speculate that IL-1-induced elevation of resting corticosterone levels may influence the development of EAE.     

Distribution of prosaposin-like immunoreactivity in rat brain

Prosaposin is the precursor for saposins A, B, C, and D, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. With a monospecific anti-saposin C antibody, which cross-reacts with prosaposin but not with saposin A, B, or D, the present immunoblot experiments showed that the rat brain expresses an unprocessed ～ 72 kDa protein (possibly prosaposin) and little saposin C. Regional analysis demonstrated that prosaposin is abundant in the brainstem, hypothalamus, cerebellum, striatum, and hippocampus, and less abundant in the cerebral cortex. Consistent with this finding, prosaposin-like immunoreactive neurons and fibers as revealed by immunohistochemistry were observed frequently in subcortical regions. The medial septum, diagonal bands, basal nucleus of Meynert, ventral striatum, medial habenular nucleus, and motor nuclei of cranial nerve had significant numbers of immunoreactive neurons. There were also nerve fibers with prosaposin-like immunoreactivity in several projection fields of the above nuclei. Other brain areas that contained prosaposin-like immunoreactive neurons and/or processes were: several brain nuclei (nucleus caudate putamen, globus pallidus, substantia nigra, red nucleus) constituting the so-called extrapyramidal system, reticular thalamic nucleus, entopeduncular nucleus, mammillary nuclei, auditory relay nuclei, cerebellum, sensory cranial nerve nuclei, and the reticular formation. The distribution pattern of prosaposin is apparently different from that of other neuroactive substances so far examined, and thus prosaposin may be involved in novel central events. © 1993 Wiley-Liss, Inc.     

Quantitative autoradiography of [H]corticosterone receptors in rat brain

We have quantified corticosterone receptors in rat brain by optical density measurements of tritium-film autoradiograms. Rats were injected i.v. with 500 μCi [³H]corticosterone to label brain receptors. Frozen sections of brain were cut with a cryostat and exposed for 2 months against tritium-sensitive sheet film (LKB Ultrofilm). Tritium standards were used to convert optical density readings into molar concentrations of receptor. High levels of corticosterone receptors were present throughout the pyramidal and granule cell layers of the hippocampus. Moderate levels of receptors were found in the neuropil of the hippocampus, the lateral septum, the cortical nucleus of the amygdala and the entorhinal cortex. All other brain regions had low levels of receptors. These results extend previous non-quantitative autoradiographic studies of corticosterone receptors and provide a general procedure for the quantitative autoradiography of steroid hormone receptors in brain tissue.     

Effect of adrenalectomy and corticosterone substitution on glucose and glycogen metabolism in rat brain

Summary In non-nervous tissues, glucocorticoids (GCs) counteract the effects of insulin and stimulate gluconeogenesis. The present study was designed to investigate whether or not adrenalectomy (ADX) and glucocorticoid substitution influence the pathway of both glucose and glycogen metabolism in cerebral parietotemporal cortex and hippocampus, and if so how. The activities of respective key enzymes, such as hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), glucose-6-phosphatase (G6Pase) and phosphorylase a (PLa), and the concentrations of the intermediates, such as glucose (Glu), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (F16PP), pyruvate (Pyr), lactate (Lac), glycogen (Glyc) and glucose-1-phosphate (G1F), were measured in the brains of 1-year-old male Wistar rats under controlled conditions 3 days after ADX or sham operation and in a pilot study after ADX and substitution with corticosterone (CST) suspended in sesame oil or after ADX and subcutaneous administration of the vehicle only. An increase in both glycolytic flux and glycogen breakdown and a decrease in gluconeogenesis in cerebral cortex but not in hippocampus were observed after ADX. After substitution with CST in adrenalectomized rats the effect of ADX on enzyme activities was reversed: significant differences from adrenalectomized rats that received vehicle only was shown for PK and G6Pase activities in both areas of the rat brain investigated.