Rat Brain Nuclei Research Articles

Aspartoacylase is a regulated nuclear‐cytoplasmic enzyme

Mutations in the gene for aspartoacylase (ASPA), which catalyzes deacetylation of N-acetyl-L-aspartate in the central nervous system (CNS), result in Canavan Disease, a fatal dysmyelinating disease. Consistent with its role in supplying acetate for myelin lipid synthesis, ASPA is thought to be cytoplasmic. Here we describe the occurrence of ASPA within nuclei of rat brain and kidney, and in cultured rodent oligodendrocytes. Immunohistochemistry showed cytoplasmic and nuclear ASPA staining, the specificity of which was demonstrated by its absence from tissues of the Tremor rat, an ASPA-null mutant. Subcellular fractionation analysis revealed low enzyme activity against NAA in nuclear fractions from normal rats. Whereas two recent reports have indicated that ASPA exists as a dimer, size-exclusion chromatography of subcellular fractions showed ASPA is an active monomer in both subcellular fractions. Western blotting detected ASPA as a single 38 kD band. Because ASPA is small enough to passively diffuse into the nucleus, we constructed, expressed, and detected in COS-7 cells a green fluorescent protein-human ASPA (GFP-hASPA) fusion protein larger than the permissible size for the nuclear pore complex. GFP-hASPA was enzymatically active and showed mixed nuclear-cytoplasmic distribution. We conclude that ASPA is a regulated nuclear-cytoplasmic protein that may have distinct functional roles in the two cellular compartments.

Effect of melatonin on the relationship between lipid peroxidation and proteolytic activity in basal nuclei of rat brain during acute hypoxia

We studied the effect of melatonin on the relationship between LPO intensity and proteolytic activity in basal nuclei (caudate nucleus, globus pallidus, amygdaloid complex, and nucleus accumbens septi) of rat brain during acute hypobaric hypoxia. Acute hypoxia was accompanied by LPO activation and increase in proteolytic activity. It should be emphasized that the intensity of proteolysis was higher in structures responding by more pronounced LPO activation (nucleus accumbens septi and globus pallidus). Intraperitoneal injection of 1 mg/kg melatonin 30 min before acute hypoxia inhibited LPO and prevented the increase in proteolysis in basal nuclei of the brain. The effect of melatonin was most pronounced in basal nuclei highly sensitive to acute hypoxia.

5-Aminolevulinate and 4, 5-dioxovalerate ions decrease GABA A receptor density in neuronal cells, synaptosomes and rat brain

Porphyrias are heme-associated metabolic disorders such as intermittent acute porphyria (IAP) and lead poisoning, where 5-aminolevulinate (ALA) accumulates. Effects of ALA on the CNS have been explained by ALA binding to GABA A receptors, followed by receptor lesions from oxyradicals and 4, 5-dioxovalerate (DOVA) generated from metal-catalyzed ALA oxidation by oxygen. We have characterized the effects of ALA and DOVA on GABA A receptor density in synaptosomes and neurons in vitro and also in brains of rats treated with ALA or succinylacetone methyl ester (SAME), a tyrosine catabolite derivative able to induce ALA accumulation. Radiolabeling assays revealed that following exposure to DOVA the concentration of synaptosomal GABAergic sites decreased by approximately 50%. Pretreatment with DOVA resulted in less GABA A receptor density in P19 and WERI cells and altered cell morphology. Furthermore, exposure to DOVA also induced a 5-fold increase in WERI cell mortality rate. Treatment with ALA resulted in loss of neuronal morphology and decrease of GABA A density in P19 neuronal cells. ALA and SAME treatment diminished the density of GABAergic receptors in the habenular complex and the parabigeminal nucleus of rat brain as studied by immunohistochemical procedures. Our results strongly suggest that ALA- and DOVA-promoted damage to GABA A receptors may contribute to the neurological manifestations of AIP and plumbism.

CART expression in limbic regions of rat brain following forced swim stress: Sex differences

Our previous studies showed the modulation of cocaine and amphetamine regulated transcript (CART) positive neurons and CART mRNA by adrenalectomy and corticosterone replacement in hypothalamic nuclei of male rat brain. More recently, we have shown by CART immunohistochemistry that restraint and forced swim (FS) stress have sexually dimorphic and regionally specific effects on CART expression in the hypothalamic nuclei of male and female Sprague-Dawley rats. This study aimed to evaluate the effects of FS stress on CART peptide expression in hypothalamus, amygdala and hippocampus of male and female (in or near estrus) Sprague-Dawley rats. Initially basal CART levels in regions of interest were determined in male and female rats; no sex differences were observed. In FS test, rats were forced to swim on two consecutive days, in a Plexiglas cylinder for 15 and 6 min, respectively. Rats were decapitated on the second day, 10 min after the stress procedure. Hypothalami, amygdalae and hippocampi were dissected and homogenized. CART peptide expression in these regions was measured by Western blotting. In males, FS increased CART expression in hypothalamus and amygdala. On the other hand, in females, FS lowered CART expression in amygdala. CART expression in hippocampus was not affected by the stress procedure in either sex. Our results suggest sexually dimorphic modulation of CART expression in hypothalamus and amygdala by FS procedure. Although modulation of the CART peptide by glucocorticoids and gonadal hormones appears likely, future studies are needed to elucidate the underlying mechanisms in the involvement of CART peptide in stress response.

Altered catecholamine levels in brain nuclei of taste aversion resistant rats

Taste Aversion Prone (TAP) and Taste Aversion Resistant (TAR) rats were selected over 28 generations of selective breeding. This resulted in a strain of rats resistant to formation of conditioned taste aversion to a number of unconditioned stimuli (Alc. Clin. Exp. Res. 1992, 16: 928–34). It has long been recognized that food related cues and taste aversion learning involves catecholamine systems (Brain Res Brain Res Rev. 1998, 28:309–69). In these studies we have microdissected regions of the brains of TAP and TAR rats. These microdissected regions were analyzed for biogenic amine content by HPLC. We found that dopamine was elevated in the amygdala, ventral striatum and nucleus of the solitary tract of TAR rats compared to TAP rats. No differences were noted in other regions analyzed. Unlike dopamine, serotonin was not different in these nuclei, while norepinephrine tended to be elevated in hypothalamus. These observations suggest a role for dopamine in mediating the behaviors observed in TAR rats. Future studies will focus on modulating these behaviors by manipulating dopamine function. (Supported by NIH AA12911, VA Med. Res. Serv. & Dept of Psychiat & Health Behav.)

A pharmacological study on Berberis vulgaris fruit extract

Berberis vulgaris fruit (barberry) is known for its antiarrhythmic and sedative effects in Iranian traditional medicine. The effects of crude aqueous extract of barberry on rat arterial blood pressure and the contractile responses of isolated rat aortic rings and mesenteric bed to phenylephrine were investigated. We also examined effect of the extract on potassium currents recorded from cells in parabrachial nucleus and cerebellum rejoins of rat brain. Administration of the extract (0.05–1 mg/100 g body weight of rat) significantly reduced the mean arterial blood pressure and heart rate in anaesthetized normotensive and desoxycorticosteron acetate-induced hypertensive rats in a dose-dependent manner. Concentration–response curves for phenylephrine effects on isolated rat aortic rings and the isolated mesenteric beds in the presence of the extract were significantly shifted to the right. Application of the extract (1–50 μg/ml) shifted the activation threshold voltage to more negative potentials, leading to an enhancement in magnitude of the outward potassium current recorded from cells present in rat brain slices of parabrachial nucleus and cerebellum. This effect on potassium current may explain the sedative and neuroprotective effects of barberry. The present data support the hypothesis that the aqueous extract of barberry has beneficial effects on both cardiovascular and neural system suggesting a potential use for treatment of hypertension, tachycardia and some neuronal disorders, such as epilepsy and convulsion.

Restricted Expression of LGR8 in Intralaminar Thalamic Nuclei of Rat Brain Suggests a Role in Sensorimotor Systems

LGR8 was recently identified as a cognate receptor for insulin-like peptide-3 (INSL3), and INSL3-LGR8 signaling is best known for its role in testis descent during development. LGR8 mRNA has been detected in various human tissues including brain, but the regional and cellular distribution of LGR8 expression in the mammalian central nervous system is unknown. Therefore, in this study we investigated the presence and localization of LGR8 mRNA in rat brain using reverse transcription-polymerase chain reaction and in situ hybridization histochemistry. Results revealed a distinct distribution of LGR8 in forebrain, with transcripts principally restricted to the posterior thalamus and highest densities detected in the parafascicular nucleus of both adult and developing rats. Unexpectedly, INSL3 mRNA was not detected in brain by similar methods, but preliminary electrophysiologic studies of parafascicular neurons revealed that INSL3 altered their activity. These findings suggest that LGR8 signaling may be involved in sensorimotor control in the rat and perhaps other species, particularly via actions on parafascicular neurons that project to basal ganglia and are depleted in Parkinson's disease.

Expression of ORL 1 mRNA in some brain nuclei in neuropathic pain rats

The present study was designed to investigate changes of opioid receptor like 1 receptor (ORL 1, NOP) mRNA expression in some pain-related brain nuclei of neuropathic pain rats using in situ hybridization technique. Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of ORL 1, plays an important role in neuropathic pain through its receptor. There are ORL 1 mRNA expression in the nucleus of raphe magnus (NRM), ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) of rat mesencephalon. In the sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain model, a significant increase of ORL 1 mRNA expression was observed in these three regions on the 7th day after operation, and the changes lasted for 2 weeks. The result indicated that ORL 1 synthesis was increased in NRM, vlPAG and DRN of neuropathic pain rats, suggesting that ORL 1 was involved in nociceptive transmission of neuropathic pain.

Induction of Brain Region-Specific Forms of Obesity by Agouti

Disruption of melanocortin (MC) signaling, such as by ectopic Agouti overexpression, leads to an obesity syndrome with hyperphagia, obesity, and accelerated body weight gain during high-fat diet. To investigate where in the brain disruption of MC signaling results in obesity, long-term Agouti expression was induced after local injections of recombinant adeno-associated viral particles in selected brain nuclei of adult rats. Agouti expression in the paraventricular nucleus, a hypothalamic region with a high density of MC receptors, induced acute onset hyperphagia and rapid weight gain that persisted for at least 6 weeks. In contrast, obesity and hyperphagia developed with a 3 week delay when Agouti was expressed in the dorsal medial hypothalamus. Agouti expression in the lateral hypothalamus (LH) did not affect food intake and body weight during regular diet, despite the presence of MC receptors in this region. However, during exposure to a high-fat diet, animals with Agouti expression in the LH exhibited a marked increase in body weight. Here we show that the LH is important for the protection against diet-induced obesity by controlling caloric intake during consumption of a high-fat diet. Together, this study provides evidence that different aspects of the Agouti-induced obesity syndrome, such as hyperphagia and diet responsiveness, are mediated by distinct brain regions and opens challenging opportunities for further understanding of pathophysiological processes in the development of the obesity syndrome.

Repeated swim stress leads to down-regulation of vesicular monoamine transporter 2 in rat brain nucleus accumbens and striatum

We assessed the impact of chronic swim stress in rats (daily for 3 weeks) on vesicular monoamine transporter 2 (VMAT2) in the nucleus accumbens and striatum. Exposure to repeated swim stress resulted in significant reduction in VMAT2 density in nucleus accumbens (20%, p<0.01) and striatum subregions (21–38%, p<0.001). The down-regulation of VMAT2 in this dopaminergic regions may serve as an adaptatory mechanism in the response to prolonged stress, and may be relevant to chronic stress-induced depression.

Transient Expression of Acetylcholinesterase in the Posterior Ventral Cochlear Nucleus of Rat Brain

In this report we partially characterize a pathway projecting to the posterior ventral cochlear nucleus (PVCN) of the rat brain that transiently expresses a high level of acetylcholinesterase (AChE). The AChE-positive axons form a network that envelops a discrete region of the PVCN that includes the octopus cell region and some cells rostral to it. AChE is first detectable by postnatal day 3 (P3), peaks in expression at about P7-10, and is barely detectable in our preparations by P15. We previously reported that neurons in the octopus cell region express high levels of alpha7 nAChR mRNA and alpha-bungarotoxin binding during the same time period. In light microscopic immunocytochemical studies using antibodies to the vesicular acetylcholine transporter (VAChT), we could not identify immunopositive boutons in the developing regions of the PVCN that express high levels of AChE-positive fibers despite distinct punctate labeling in other brain regions. Systematic electron microscopic examination of AChE histochemical staining throughout the PVCN revealed intense labeling of axons, but synaptic sites were devoid of reaction product. The source of the AChE-positive fibers is not known, but the fibers are not auditory nerve axons and probably not collaterals of the olivocochlear bundle.

C-fos expression in rat brain nuclei following incisor tooth movement.

In the rat experimental model, molar tooth movement induced by Waldo's method is known to cause a temporally and spatially defined pattern of brain neuronal activation. Since orthodontic correction usually involves the entire dental arch, we used a spring-activated appliance to extend the investigation to incisors, and we included brain regions related to antinociception. Adjustment of the non-activated appliance on incisors resulted in c-fos expression in the dorsal raphe, peri-aqueductal gray matter, and the locus coeruleus, in addition to trigeminal sensory subnuclei and the parabrachial nucleus, where neuronal activation has already been detected in previous studies on molar tooth movement. Appliance activation with a 70-g force resulted in a further increase in Fos-immunoreactive neurons in the trigeminal sensory subnucleus caudalis and in the dorsal raphe. This result suggests that there is a recruitment of neurons related to nociception and to antinociception when tooth movement is increased.

Electroconvulsive shock increases serotonin transporter in the rat frontal cortex

The antidepressive action of electroconvulsive shock (ECS) is thought to involve the alteration in serotonin (5-HT) neurotransmission, including the increase in 5-HT release and uptake. In our previous study, 5-HT transporter (5-HTT) mRNA expression was decreased after single and repetitive ECS in rat raphe nucleus. In the present study, we investigated the effects of single and repetitive ECS on the protein levels of 5-HTT in the frontal cortex, hippocampus and raphe nucleus of rat brain using quantitative Western blot analysis. Single ECS did not alter 5-HTT protein expression in any brain regions examined. Repetitive ECS stably increased 5-HTT protein in the frontal cortex, but not in the hippocampus and raphe nucleus. Because ECS is known to facilitate the release of neurotransmitters, our results suggest that the increased 5-HTT protein expression in the frontal cortex might be a compensatory change against the enhanced 5-HT release by ECS in presynaptic terminals.

Suppressive effect of endogenous regucalcin on protein tyrosine phosphatase activity in the nucleus of rat brain: Attenuation with increasing age

The effect of endogenous regucalcin, which is a regulatory protein of Ca2+ signaling, on protein tyrosine phosphatase activity in the nucleus of brain tissues of young and aged rats was investigated. Phosphotyrosine was used as the substrate for assay of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity in the brain nucleus of young (5 weeks old) rats was significantly increased in the presence of calcium chloride (5-50 micro M) in the enzyme reaction mixture. The increase was completely blocked by the addition of trifluoperazine (10-50 micro M), an antagonist of calmodulin, indicating that the enzyme is activated by endogenous Ca2+/ calmodulin. The addition of regucalcin (10(-4)-10(-8) M) in the enzyme reaction mixture caused a significant decrease in protein tyrosine phosphatase activity in the absence or presence of calcium chloride (20 micro M). Brain nuclear protein tyrosine phosphatase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The increase was completely prevented by the addition of regucalcin (10(-6) M). In the brain nucleus of aged (50 weeks old) rats, protein tyrosine phosphatase activity was elevated significantly as compared with that of 5 weeks old rats. The effect of anti-regucalcin monoclonal antibody in increasing the enzyme activity in the brain nucleus of aged rats was seen in the presence of 50 ng/ml of the antibody. Such an effect was not found by the antibody of 10 and 25 ng/ml. Regucalcin protein in brain nucleus was detected by Western blot analysis. This level was significantly decreased by increasing age. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein tyrosine phosphatase activity in the nucleus of rat brain, and that this regulation is attenuated with increasing age.

Nuclei and subnuclei gene expression profiling in mammalian brain

Information on the neuroanatomical expression of a given gene is critical to understanding its function in the central nervous system. The integration of laser capture microdissection (LCM), T7-based RNA amplification and cDNA microarrays allows for this information to be simultaneously generated for thousands of genes. To validate this integrative approach, we catalogued the gene expression profiles of seven rat brain nuclei or subnuclei. A hundred cells from the following seven brain nuclei were analyzed: locus coeruleus (LC), dorsal raphe nucleus (DR), parvocellular division (PA) and magnocellular division (MG) of the hypothalamic paraventricular nucleus (PVN) and CA1, CA3 and dentate gyrus (DG) divisions of the hippocampal formation. Of the 2145 genes investigated, 1402 genes (65%) gave a hybridization signal statistically different from the background level that was defined by non-specific hybridizations to 15 different plant genes. Validation of our microarray data on four arbitrarily selected genes was confirmed by Real-Time PCR. Previous research showing expression patterns of ‘signature’ genes ( n=17) for specific brain nuclei are consistent with our findings. For example, as previously shown, enriched mRNA expression encoding the serotonin transporter or tyrosine hydroxylase was found in DR and LC cells, respectively. Interestingly, expression of the serotonin 5-HT 2B receptor mRNA was also found in DR cells. We confirmed this new finding by in-situ hybridization. The hierarchical clustering analysis of gene expression shows that the two divisions of the PVN (PA and MG) are closely related to each other, as well as the three regions of the hippocampal formation (CA1, CA3 and DG), which also showed similar gene expression profiles. This study demonstrates the importance, feasibility and utility of cellular brain nuclei profiling.

Localization of pore-forming subunit of the ATP-sensitive K +-channel, Kir6.2, in rat brain neurons and glial cells

Kir6.2, a subunit of the ATP-sensitive K + channel (K ATP), was localized in adult rat brain by immunohistochemistry and in situ hybridization. The Kir6.2 mRNA was widely expressed in most rat brain neuronal populations and nuclei examined, intensely in the mitral cell layer and tufted cells of the olfactory bulb, pontine nucleus, pontine reticular nucleus, motor and spinal trigeminal nuclei and cuneate nuclei of the brain stem, moderately in the neocortex and cerebellar Purkinje cells, and weakly in the granular cell layer of the olfactory bulb and the granular layer of the cerebellum. In addition, glial cells also expressed the Kir6.2 gene weakly in the corpus callosum and cerebellar white matter. This wide localization of the gene was quite similar to that of Kir6.2 protein. Double stainings with anti-GFAP and anti-Kir6.2 antibodies were performed in this study. Glial cells showing immunoreactivity to both anti-Kir6.2 and anti-GFAP were confirmed to be astrocytes, and those showing only immunoreactivity to anti-Kir6.2 but not to anti-GFAP were presumed to be oligodendrocytes and confirmed by immunoelectron microscopy. Thus, it may be concluded that both oligodendrocytes and astrocytes contain Kir6.2. Under the electron microscope, we showed in vivo for the first time that the immunoreactive products were localized in the endoplasmic reticulum and Golgi apparatus as well as the plasma membranes of neurons and glial cells.

Region-dependent effects of acute and chronic tranylcypromine in vivo on [ 3H]2-BFI binding to brain imidazoline I 2 sites

An imidazoline I 2 site has been localised to monoamine oxidase. However, in vitro studies of the effect of monoamine oxidase inhibitors on imidazoline I 2-site radioligand binding have produced conflicting findings. Using the technique of autoradiography, we examined the effect of in vivo administration of the irreversible monoamine oxidase inhibitor tranylcypromine on binding of the imidazoline I 2 site-specific ligand [ 3H]2-(-2-benzofuranyl)-2-imidazoline ([ 3H]2-BFI) in four rat brain nuclei which are known to possess a high density of imidazoline I 2 sites, together with cerebral cortex and cerebellum which show weaker binding. A single acute pre-treatment with tranylcypromine significantly increased imidazoline I 2 site-specific binding in four regions: arcuate nucleus, interpeduncular nucleus, pineal gland and area postrema, but effects in cortical areas and cerebellum were not significant. The extent of the increase was proportional to the control binding in each region. In contrast, five daily treatments with the same dose of tranylcypromine significantly reduced [ 3H]2-BFI binding in these same areas. The potential role of monoamine oxidase isoforms in these changes is discussed.

Galanin-Like Peptide mRNA in Neural Lobe of Rat Pituitary

Galanin-like peptide (GALP) was recently identified in the porcine hypothalamus, pituitary gland and gut, and has reported selectivity for the GalR2, c.f. the GalR1 receptor. GALP cDNAs have been cloned from pig, rat and human, and GALP mRNA expression is restricted to the arcuate nucleus in normal rat brain. This study examined the regional and cellular distribution of GALP mRNA in the rat pituitary gland, and subsequently determined the effect of osmotic stimulation on GALP transcript levels. GALP mRNA was not detected in the anterior or intermediate lobes, but moderate levels of GALP mRNA were present in the neural (posterior) lobe, in presumed pituicytes, of normal male and female rats. Osmotic stimulation by dehydration or salt loading produced a time-dependent increase in GALP mRNA levels in the neural lobe. Thus, dehydration for 4 days increased GALP mRNA 40-fold, while salt loading for 4, 7 or 10 days increased GALP levels 14-, 21- and 25-fold, respectively (p ≤ 0.001). Levels of vasopressin (VP) mRNA in the neural lobe were also increased by these treatments, consistent with previous reports. Galanin (GAL) and GalR2 receptor mRNAs were not detected in the neural lobe, under normal or osmotic stimulation conditions. In addition, GALP mRNA levels in the arcuate nucleus were not altered in dehydrated or salt-loaded rats; and GALP mRNA was not detected in magnocellular neurons of the supraoptic or paraventricular nucleus, despite the characteristic up-regulation of VP and GAL mRNA in these cells. In view of the established anatomy and function of VP/oxytocin neurons in the hypothalamo-neurohypophysial system and the role played by pituicytes in their regulation, the likely synthesis/release of GALP by these specialized astrocytes strongly suggests a role for this novel peptide in regulation of pituicyte morphology/function and/or neurohormone release.

Effect of morphine on the number and branching of astrocytes in various regions of rat brain.

We studied the effects of morphine in high doses on astrocytes in the nucleus accumbens, lateral septum, and caudate nucleus of rat brain. Activation of astrocytes in the nucleus accumbens and lateral septal nucleus was manifested in hyperplasia and elongation of astrocyte processes. In the caudate nucleus, the total length of astrocyte processes and branching of individual astrocytes decreased.

Morphogenesis of asymmetry of rat brain nuclei under normal conditions and during exposure to microgravitation.

Histological and morphometrical analysis of the brain and peripheral analyzers revealed proliferative migration abnormalities of brain development and neuron differentiation in mammals during a space flight, which can limit space exploration.