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Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

Tau is a microtubule-associated protein (MAP) that is localized to the axon. In Alzheimer's disease (AD), the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. While the abnormal aggregated tau has been extensively studied in human patient tissues and animal models of AD, how normal tau localizes to the axon, which would be the foundation to understand how the mis-localization occurs, has not been well studied due to the poor detectability of normal unaggregated tau in vivo. Therefore, we developed immunohistochemical techniques that can detect normal mouse and human tau in brain tissues with high sensitivity. Using these techniques, we demonstrate the global distribution of tau in the mouse brain and confirmed that normal tau is exclusively localized to the axonal compartment in vivo. Interestingly, tau antibodies strongly labeled nonmyelinated axons such as hippocampal mossy fibers, while white matters generally exhibited low levels of immunoreactivity. Furthermore, mouse tau is highly expressed not only in neurons but also in oligodendrocytes. With super resolution imaging using the stimulated-depletion microscopy, axonal tau appeared punctate rather than fibrous, indicating that tau decorates microtubules sparsely. Co-labeling with presynaptic and postsynaptic markers revealed that normal tau is not localized to synapses but sparsely distributes in the axon. Taken together, this study reports novel antibodies to investigate the localization and mis-localization of tau in vivo and novel findings of normal tau localization in the mouse brain., (© 2018 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Estrogen-related receptor (ERR) is a member of the nuclear receptor superfamily that has strong homology with estrogen receptor (ER) α. Despite the lack of endogenous ligands, ERR serves as transcription factors through their constitutively active structure with or without interaction with ERα. Among the three subtypes of ERR (α, β, and γ), ERRγ is highly expressed in brain, but the distribution of ERRγ is poorly characterized. Therefore, we investigated ERRγ immunoreactivity throughout the rostro-caudal axis in rat brain. Immunohistochemistry revealed localization of ERRγ protein in the cell nucleus, and a ubiquitous distribution of ERRγ in brain regions including the olfactory bulb, cerebrum, brain stem, and cerebellum. Selective intense immunoreactivity was observed in the reticular thalamic nucleus, zona incerta, circular nucleus, interpeduncular nucleus, pontine nucleus, and parasolitary nucleus. Most ERRγ-immunoreactive (ir) regions were also positive for ERα and/or ERβ, which suggests that ERRγ is involved in modulation of estrogen signaling in adult rat brain. Double immunofluorescence demonstrated colocalization of ERRγ with ERα within the anteroventral periventricular nucleus of the preoptic area (AVPV) and medial preoptic nucleus (MPO), which are major target sites for estrogen action. The results of this study suggest that ERRγ function in the brain is affected by estrogens through an interaction with ERα. The findings also provide basic information on brain region-specific ERRγ function., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Oestrogen receptor (ER)α plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ERα-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ERα promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ERα mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ERα immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ERα-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ERα gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ERα controls expression of the ERα gene itself. Thus, the ERα promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ERα-expressing neurons and oestrogen and its receptor-mediated neuronal responses., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Sexual differentiation of the brain can be considered as a process during which effects of sex steroid hormones secreted during early development is maintained into adulthood. Epigenetic regulation is emerging as a potentially important mechanism of conveyance of long-lasting effects of the hormonal and environmental milieu in the developing brain. Evidence has accumulated to show that epigenetic regulation is involved in the control of sexual differentiation of the brain. In the preoptic area (POA), which is important for male sexual behavior, histones associated with the estrogen receptor (ER) α and aromatase (Arom) gene promoters are differentially acetylated between the sexes, and two subtypes of histone deacetylase (HDAC2 and 4) are associated with the same promoters at higher frequencies in males in the early postnatal period. Since ERα and Arom are essential genes in masculinization of the brain, these findings suggest that histone deacetylation in the early postnatal period is involved in masculinization of the brain. Indeed, inhibition of HDAC activity in males during this period abrogates brain masculinization: structural sexual dimorphism of the bed nucleus of the stria terminalis is eliminated and expression of male sexual behavior is reduced in adulthood. Previous reports have demonstrated that ERα gene expression in the POA is higher in females during the developmental and pubertal periods and in adulthood, indicating that sexually dimorphic ERα expression that appears in early postnatal development is maintained until adulthood by epigenetic programming. The ERα promoter is also more sparsely methylated in females, with an inverse correlation with ERα expression. In addition to the hormonal effect, the amount of maternal care received during postnatal development has a lasting effect on ERα expression mediated by DNA methylation of its promoter. Taken together, these results suggest that epigenetic mechanisms play a central role in the transduction and maintenance of early hormonal and social cues to organize sexually differentiated brain functions.

Epigenetic histone modifications are emerging as important mechanisms for conveyance of and maintenance of effects of the hormonal milieu to the developing brain. We hypothesized that alteration of histone acetylation status early in development by sex steroid hormones is important for sexual differentiation of the brain. It was found that during the critical period for sexual differentiation, histones associated with promoters of essential genes in masculinization of the brain (estrogen receptor α and aromatase) in the medial preoptic area, an area necessary for male sexual behavior, were differentially acetylated between the sexes. Consistent with these findings, binding of histone deacetylase (HDAC) 2 and 4 to the promoters was higher in males than in females. To examine the involvement of histone deacetylation on masculinization of the brain at the behavioral level, we inhibited HDAC in vivo by intracerebroventricular infusion of the HDAC inhibitor trichostatin A or antisense oligodeoxynucleotide directed against the mRNA for HDAC2 and -4 in newborn male rats. Aspects of male sexual behavior in adulthood were significantly reduced by administration of either trichostatin A or antisense oligodeoxynucleotide. These results demonstrate that HDAC activity during the early postnatal period plays a crucial role in the masculinization of the brain via modifications of histone acetylation status.

Brain protection is important during aortic arch surgery, especially in patients with cerebral ischemia. We clinically applied the effectiveness of a novel protocol of retrograde cerebral perfusion with intermittent pressure augmentation for brain protection in a canine model, as described in a previous report. Although, in our patient the brachiocephalic artery and left subclavian artery were occluded as a result of aortitis, there was a history of right cerebral infarction, recovery of consciousness, and no neurologic sequelae. Near-infrared oximetry showed recovery of intracranial blood oxygen saturation every time the pressure was augmented., (Copyright © 2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Circulating levels of androgens determine the sexual differentiation of the brain and spinal cord at a critical period. Although estradiol, which is converted from testosterone by aromatase action, can explain the cytological basis for the sexual dimorphism, androgen has its own regulatory mechanism to promote male-specific behavior through receptors. The central nervous system (CNS) employs a sex-specific neuronal network involving peptides and steroids for the expression of the sexual phenotype. Elucidation of the molecular mechanism along with the neuroanatomical background should guide the development of novel pharmacotherapeutics for sexual behavior dysfunction.

Studies with green fluorescent protein (GFP) have revealed the subcellular distribution of many steroid hormone receptors to be much more dynamic than previously thought. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) are powerful techniques with which to examine protein-protein interaction and the mobility of tagged proteins, respectively. FRET analysis revealed that steroid treatment (with corticosterone or testosterone) induces direct interaction of the glucocorticoid receptor (GR) and importin alpha in the cytoplasm and that, shortly after nuclear entry, the GR detaches from importin alpha. The mineralocorticoid receptor (MR) and androgen receptor (AR) show the same trafficking. Upon oestradiol treatment, ERalpha and ERbeta in the same cell are relocalised to form a discrete pattern and are localised in the same discrete cluster (subnuclear foci). FRAP analysis showed that nuclear ERalpha and ERbeta are most dynamic and mobile in the absence of the ligand, and that mobility decreases slightly after ligand treatment. Genomic as well as non-genomic actions of steroid hormones influence the cellular function of target tissues spacio-temporally.

Objective: For cerebral protection during aortic surgery, we introduced a novel retrograde cerebral perfusion method with intermittent pressure augmentation. We then assessed whether this novel method provides benefits similar to those provided by antegrade selective cerebral perfusion., Methods: Eighteen dogs were randomly divided into 3 groups: the RCP-INT group, intermittent-retrograde cerebral perfusion at 15 mm Hg with intermittent pressure augmentation to 45 mm Hg (n = 6); the ASCP group, antegrade selective cerebral perfusion at a flow rate of 10 mL x kg(-1) x min(-1) (n = 6); and the sham group, no circulatory arrest (n = 6). Cooling (26 degrees C) with cardiopulmonary bypass and 60 minutes of circulatory arrest were performed in the RCP-INT and ASCP groups. The levels of tau protein in the cerebrospinal fluid and the diameters of the retinal vessels were measured. The neurologic deficit scores and the histopathologic damage scores of the brains were determined., Results: The total postoperative tau protein levels (calculated as the area under the curve) did not differ significantly between the RCP-INT and ASCP groups (203 +/- 87 pg x mL(-1) x h vs 154 +/- 69 pg x mL(-1) x h, P = .95). The retinal vessels were effectively dilated at an augmented pressure of 45 mm Hg in the RCP-INT group. The total neurologic deficit score (0 = normal, 500 = brain death) and histopathologic damage score (0 = normal, 40 = worst) were not significantly different between the RCP-INT and ASCP groups (neurologic deficit score: 75 +/- 21 vs 70 +/- 21, P = .98; histopathologic damage score: 13.5 +/- 1.5 vs 14.2 +/- 1.3, P = .84)., Conclusions: Intermittent augmented pressure dilated the cerebral vessels, allowing adequate blood supply without injuring the brain. This retrograde cerebral perfusion method provides adequate neuroprotection during moderate hypothermia.

Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects in the central nervous system that regulate the stress response, mood, learning and memory, and various neuroendocrine functions. Corticosterone actions in the brain are mediated by two corticosteroid receptors, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and they show a high degree of colocalization in the hippocampal region. These receptors predominantly reside in the cytoplasm without ligand and are translocated into the nucleus upon ligand binding to act as transcriptional factors. Thus, their subcellular localizations are an important component of their biological activity. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between the cytoplasm and the nucleus and their interactions are regulated by ligand or other molecules to exert transcriptional activity. In this review, the authors focus on the nucleocytoplasmic and subnuclear trafficking of GR and MR in neural cells and nonneural cells and discuss various factors affecting the dynamics of these receptors.

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

We examined changes in 5-hydroxytriptamine (5-HT, serotonin) neurons in pyrithiamine-induced thiamine deficiency in mice immunohistochemically. Extensive decreases in the densities of 5-HT-immunoreactive fibers were detected in the lateral septal nucleus, the thalamus, the medial mammillary nucleus, the dorsal and the median raphe nuclei, the raphe obscurus nucleus, the tegmental area, the cerebellum and the vestibular nucleus, though only a small decrease was detected in the inferior colliculus. Most remarkably, degenerative winding fibers were detected between the deep mesencephalic nucleus and the ventral tegmental area. Increases in intensity of 5-HT immunoreactivity in the dorsal raphe nucleus and decreases in the number of 5-HT-immunoreactive cell bodies in the dorsal and the median raphe nuclei were detected. These results demonstrated the differential vulnerability of 5-HT neurons in thiamine-deficient mice. This is the first report to demonstrate changes in 5-HT neurons immunohistochemically throughout the brain of pyrithiamine-induced thiamine deficient mouse.

Distributional changes of serotonergic fibers associated with aging were demonstrated immunohistochemically. Old rat brains were morphologically characterized by the presence of peculiar features of serotonergic fibers not found in the young adult brain. In 24-month-old rats, these aberrant serotonergic fibers were subdivided into two groups according to morphological alterations: type 1 fibers consisting of thin fibers with moderately enlarged varicosities, and type 2 fibers consisting of much thicker fibers that have even larger varicosities and a tortuous course. These two types of fibers were distributed differentially in the forebrain. Type 1 fibers were found mainly in the striatum and frontoparietal cortex, whereas type 2 fibers were found in the posterior cingulate cortex and dentate gyrus of the hippocampal formation. Both types of aberrant fibers were seen in amygdala, frontoparietal cortex, hypothalamus, and thalamus. In 36-month-old rats, more highly degenerating arborizations were detected, and these aberrant ramifications were classified as follows based on shape as: type 3 fibers consisting of highly arborized thin fibers with a larger number of larger varicosities, and type 4 fibers consisting of thick fibers with abundant larger varicosities. Distributional difference indicated that type 1 fibers progress into type 3 fibers, whereas type 2 develop into type 4 fibers. These findings suggest the possibility that one set of pathological fibers emanate from the dorsal raphe nucleus and the other from the median raphe. Moreover, both two sets of serotonergic fibers show age-related aberrations in their morphology over same time course.

Steroid hormones regulate several important functions of the brain by altering the expression of particular genes through their receptors. First in this paper the localization of glucocorticoid receptor immunoreactivity and mRNA in the brain was examined. Second biphasic effects of glucocorticoid on the hippocampus was described and particular emphasis was given on the apoptosis. Third the significance of estrogen receptor in the sexually dimorphic areas was discussed. These results suggest that steroids modulate the gene expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.

The hippocampus and caudo-dorsal cortex of the homozygote of polydactyly mutant mouse (Polydactyly Nagoya, Pdn/Pdn) were markedly reduced in S-100 beta positive astrocytes and serotonergic fibers as compared to the heterozygote (Pdn/+) and wild type (+/+) [39]. The Pdn/Pdn mice die within 2 days after birth, so it is impossible to examine postnatal changes. To demonstrate the developmental change of Pdn/Pdn hippocampal tissue, we transplanted hippocampal pieces of neonatal Pdn/Pdn and +/+ mice into the right and left hippocampus of the same adult +/+ mice, respectively, and immunocytochemically examined them. Two weeks after transplantation, +/+ hippocampal tissue contained a large number of glial fibrillary acidic protein (GFAP) and S-100 beta positive astrocytes and a number of serotonergic fibers. While Pdn/Pdn hippocampal tissue contained numerous GFAP positive astrocytes, S-100 beta positive astrocytes and serotonergic fibers were not observed. Two months after transplantation, GFAP and S-100 beta were expressed in the Pdn/Pdn hippocampal tissue similar to the +/+ tissue. Serotonergic fibers were distributed in the +/+ tissue, while no serotonergic fibers were observed in the Pdn/Pdn transplant tissue. In contrast, no difference was observed in the tyrosine hydroxylase positive fibers between Pdn/Pdn and +/+ grafts. The expression of 5-HT1A receptor-like immunoreactivity was higher in the +/+ tissue than that of Pdn/Pdn tissue. The present results suggest that the expression of S-100 beta in the astrocytes of early stage of transplantation is a critical for fiber ingrowth of serotonergic neurons and expressions of 5-HT1A receptor.

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of cholesteryl esters, phospholipids and triglycerides between the lipoproteins, and regulates plasma high-density lipoprotein levels. We examined CETP-like immunolabeling in non-neurological and Alzheimer's disease (AD) liver and brain tissues. The anti-CETP antibodies showed positive staining in round cells in the liver sinusoid and in brain astrocytes. In the brains of non-neurological cases, positively stained astrocytes were preferentially distributed in the white matter. In AD tissue, many reactive astrocytes in the gray matter as well as the white matter astrocytes had CETP-like immunoreactivity. CETP-positive astrocytes may play a role for AD pathology such as tissue repair.

Steroid hormones modify several functions of the nervous system by altering expression of particular genes that are relevant for cell-to-cell communication, neuronal structure, and differentiation. There are many regions of the brain showing structural differences between male and female. These sexual dimorphisms are primarily based on cytological features of the nervous system brought about by the organizational action of sex steroid hormones. Glucocorticoids also have a great influence on neuronal survival and differentiation. First, in this paper the estrogen effect on the phenotypic changes of neurons in the preoptic area in vivo was described, particularly in the neuronal system of methionine-enkephalin and calcitonin gene-related peptide. Second, the tissue culture system for the preoptic area was developed and the estrogen effect on process extension associated with cytoskeletal changes was examined. Third, glucocorticoid suppression of the differentiation of adrenal chromaffin cells and transmitter/neuropeptide plasticity was studied in the dissociated culture system. The results suggest that steroids modulate neurotransmitters and neuropeptides expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.

After the intraventricular injection of 5,6-dihydroxytryptamine (5,6-DHT), the course of degeneration and regeneration of the serotonergic fibers in the rat brain was studied immunohistochemically by using serotonin antiserum. Three days after 5,6-DHT treatment, an extensive disappearance of serotonin immunoreactive fibers was observed throughout the brain. Degenerative serotonergic fibers characterized by droplet-like swelling and intense staining by serotonin antiserum were detected in the following discrete areas, e.g., medial forebrain bundle (MFB), cingulate cortex, septal nucleus, diagonal band of Broca (DBB), lateral preoptic area, bed nucleus of stria terminalis, perifornical area, stria-terminals, raphe nuclei, ventral tegmental area, periaqueductal gray lateral reticular nucleus and nucleus of the solitary tract. The swelling were regarded as the proximal stumps of the chemically injured serotonin fibers. Sprouting serotonin fibers emanating from these swellings were observed 5 days after injection. The swollen thick fibers were seen up to 3 months thereafter throughout the brain, although they gradually decreased in number until 1 month after injection. On the contrary, the sprouting fibers extended their terminal fields throughout the brain. Three types of reorganization pattern of regenerating serotonergic fibers were distinguished: hyperinnervation, normoinnervation (similar to the normal innervation) and hypoinnervation patterns. In the areas with hyperinnervation, the density of the serotonergic fibers gradually increased for 3 months. These hyperinnervation patterns of regenerative serotonergic fibers were observed in the MFB, cingulate cortex, diagonal band of Broca, perifornical area, raphe nuclei, ventral tegmental area, motor trigeminal nucleus, facial nucleus, lateral reticular nucleus, inferior olivary complex and hypoglossal nucleus. On the other hand, the hypoinnervation displayed by a few regenerating serotonergic fibers was observed in the periventricular part of the prosencephalon, the ventromedial part of the hypothalamus, the dorsal hippocampus, the neocortex, the superior and inferior colliculi, the cerebellum, the dorsal tegmental nucleus of Gudden, the vestibular nuclei, the gracile nucleus and the cuneate nucleus. These reorganization patterns were formed 3 months after injection, and continued for a long time (2 years). The mechanisms of serotonergic fiber reorganization are discussed.

c-Fos expression in the rat central nervous system following sciatic nerve section was compared using an immunohistochemical procedure with control skin-incisions. c-Fos-immunoreactive (IR) materials were localized in the nuclei of nerve cells, and these positive cells were most abundantly observed 120 min after the operation. In the spinal cord, c-Fos-IR cells were seen in the posterior horn on the operated, but not in the contralateral side. The anterior horn and the dorsal root ganglia did not contain c-Fos-IR cells. The experimental animals had the largest number of c-Fos-IR cells in the lumbosacral segments, especially in laminae I and II of L4 and L5 segments. The difference in the number of c-Fos-IR cells and IR intensity between the experimental and control animals was conspicuous in the lumbosacral segments, but there was no significant difference between them in the cervical and thoracic segments. In the brain, an apparent difference in positive cell distribution was not recognized between the experimental and control animals. c-Fos-IR cells were observed in various regions of the brain. A high concentration of positive cells was seen in the temporal cortex, the clastrum, the lateral septal nucleus (ventral part), the hypothalamic nuclei such as periventricular, suprachiasmatic, paraventricular, arcuate, dorsomedial and supramammillary nuclei, the paraventricular thalamic nucleus, the ventral lateral geniculate nucleus (magnocellular part), the pontine nuclei, the dorsal and dorsolateral tegmental nuclei, the parabrachial nucleus (Kölliker-Fuse nucleus), the locus coeruleus, and the nucleus of the solitary tract. Among them, the paraventricular and suprachiasmatic hypothalamic nuclei exhibited the most dense concentration of c-Fos-IR cells and intensity. These results indicate that the c-Fos expression in the brain is induced by transsynaptic stimulation. The findings that there was a clear difference in the distribution of c-Fos-IR cells and in intensity between the experimental and control animals in the lumbosacral segment of the spinal cord but not in the upper portion and the brain, suggested that there is poor discrimination of nociceptive information in the brain between stimulation by nerve section and skin-incision.

We have clarified that rhoA p21 purified from bovine aortic smooth muscle is geranylgeranylated at the cysteine residue in the C-terminal CAAX motif (A is an aliphatic amino acid and X is any amino acid). In this paper, a geranylgeranyltransferase for rhoA p21 (rhoA p21 GGT) was partially purified from bovine brain cytosol. This enzyme transferred a geranylgeranyl moiety from geranylgeranyl pyrophosphate to rhoA p21 having the CAAX motif (rhoA p21-CAAX) but not to rhoA p21 lacking the AAX portion. rhoA p21 GGT was separated from the previously reported farnesyltransferase for ras p21s (ras p21 FT) by column chromatographies and did not geranylgeranylate or farnesylate c-Ha-ras p21-CAAX. ras p21 FT did not geranylgeranylate or farnesylate rhoA p21-CAAX. These results indicate that rhoA p21 GGT distinct from ras p21 FT is present in bovine brain cytosol.

The widespread distribution of neurons containing alpha-atrial natriuretic polypeptide-like immunoreactivity in the rat brain was demonstrated using radioimmunoassay and immunohistochemistry in conjunction with specific antisera. The highest concentrations of alpha-atrial natriuretic polypeptide-like immunoreactivity were in the hypothalamus and septum, with low but still appreciable concentrations in the mesencephalon, cerebral cortex, olfactory bulb and thalamus by radioimmunoassay. Immunohistochemical studies clearly showed that the perikarya of immunoreactive neurons are most prevalent in the ventral part of the lateral septal nucleus, periventricular preoptic nucleus, bed nucleus of the stria terminalis, periventricular and dorsal parts of the paraventricular hypothalamic nucleus, ventromedial nucleus, dorsomedial nucleus, arcuate nucleus, median mamillary nucleus, supramamillary nucleus, zona incerta, medial habenular nucleus and the periaqueductal grey matter. Scattered neurons were seen in the cingulate cortex, endopiriform nucleus, lateral hypothalamic area, and pretectal and dorsal thalamic areas. In addition to the areas mentioned above, high concentrations of immunoreactive varicose fibers were seen in the glomerular layer of the olfactory bulb, external layer of the median eminence, central to paramedian parts of the interpeduncular nucleus and the paraventricular hypothalamic nucleus. The globus pallidus, medial and central amygdaloid nuclei, dorsal raphe, dorsal parabrachial nucleus, locus coeruleus, vagal dorsal motor nucleus, solitary nucleus and some circumventricular organs, including the subfornical organ and organum vasculosum laminae terminalis, contained considerable numbers of immunoreactive varicose fibers. In dehydrated rats and homozygous Brattleboro rats, the pattern of alpha-atrial natriuretic polypeptide-immunoreactive neurons and varicose fibers was qualitatively similar to that seen in normal conditioned rats. This study gives an atlas of the distribution of the alpha-atrial natriuretic polypeptide-containing neuronal system in the rat brain and provides the groundwork for studying the influence of this new peptide on various brain functions.

Cell surface antigens on neoplastic lymphoid cells of T cell lineage were analyzed with a fluorescence-activated cell sorter (FACS-II) by utilizing rabbit antisera raised against human brain tissue (anti-Br) and fetal thymocytes (anti-Ty). Both before and after extensive absorption with normal peripheral blood lymphocytes (PBL), the anti-Br antiserum was capable of staining neoplastic lymphoid cells from patients with Sézary syndrome, mycosis fungoides, thymoma, and chronic lymphocytic leukemia. The absorption of anti-Ty with normal PBL, however, resulted in loss of the ability to stain normal bone marrow cells and preabsorbed with normal PBL was found to stain normal bone marrow cells and neoplastic myeloid cells but not fetal thymocytes. It is suggested that the absorbed anti-Br is capable of detecting differentiation antigens present on immature hematopoietic cells in the bone marrow, which would be anomalously expressed on neoplastic lymphoid cells to T cell lineage.

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.

Using the immunoperoxidase technique in conjunction with specific antisera to alpha-atrial natriuretic polypeptide (alpha-ANP), it was shown that immunoreactive cell bodies and varicose fibers are widely distributed throughout the rat brain. The highest concentrations of alpha-ANP-containing neuronal cell bodies and fibers were found in the hypothalamus and septum. This result confirms the radioimmunological determination of alpha-ANP immunoreactivity in the rat brain.

We have devised a method for the parallel determination of choline acetyltransferase (CAT) and muscarinic cholinergic receptor (mCh-R) in the same brain tissue. The method for CAT activity determination is more rapid, simplified, stable, and economical than the usual Fonnum's method. With our method, age-associated changes in CAT activity and mCh-R levels were examined. Although CAT activity hardly changed with age except in a few areas, mCh-R binding of aged-rats was markedly reduced in all areas. These results suggest that the change in mCh-R represents an age-associated biochemical change in the brain and that determination of CAT activity is not sufficient for the study of age-associated changes in the brain cholinergic system.

The occurrence of CRF (corticotropin-releasing factor)-immunoreactive nerve fibers in the circumventricular organs of adult male monkeys, Macaca fuscata, was studied on serially sectioned brains, by means of the peroxidase-antiperoxidase technique in combination with a highly specific and sensitive CRF antiserum. CRF-containing nerve fibers were found in high concentrations in the infundibulum and, in addition, in small numbers in the posterior lobe, organum vasculosum laminae terminalis, subfornical organ, and area postrema; they were missing in the pineal body and the subcommissural organ. The CRF-immunoreactive nerve fibers distributed in these organs were located in the proximity of the blood vessels.

Abstract: Vasopressin (AVP) plays an important role in anxiety-related and social behaviors. Single-prolonged stress (SPS) has been established as an animal acute severe stress model and has been shown to induce a lower adrenocorticotropic hormone (ACTH) response upon cortisol challenge. Here, we show results from immunoassays for AVP, ACTH, and corticosterone (CORT), and in situ hybridizations for AVP mRNA performed 7 days after SPS exposure. Immunofluorescence for AVP was also performed during the 7-day period following SPS exposure and after an additional forced swimming stress paradigm. We observed that the plasma concentrations of AVP, ACTH, and CORT were not altered by SPS; ACTH content in the pituitary and AVP mRNA expression in the supraoptic nucleus (SON) were significantly reduced by SPS. During the 7-day period following SPS, the intensity of immunoreactivity, the size of the soma, and the immunoreactive optical density of the dendrites of AVP neurons in the SON all increased. An apparent reduction in the intensity of AVP immunoreactivity was observed in the SON at 4 h after additional stress. Additional forced swimming led to a rapid increase in the dendritic AVP content only in the controls and not in the SPS-treated rats. These findings suggest that AVP is a potential biomarker for past exposure to severe stress and that alterations in AVP may affect the development of pathogenesis in stress-related disorders. [Copyright &y& Elsevier]