Expression of c-Fos protein in medial septum/diagonal band of Broca and CA3 region, associated with the temporary inactivation of the supramammillary area.

The supramammillary (SuM) area is part of the diencephalic nuclei comprising the mammillary bodies, and is a key structure in the memory and spatial learning processes. It is a critical region in the modulation/generation of hippocampal theta rhythm. In addition, many papers have recently shown a clear involvement of this structure in the processes of spatial learning and memory in animal models, although it is still not known how it modulates spatial navigation and response emotional. The aim of the present research was to study the effect of the temporary inactivation of the SuM area on synaptic plasticity of crucial structures in the formation of spatial memory and emotional response. Sprague-Dawley rats were asigned in three groups: a control group where the animals were not subjected to any treatment, and two groups where the rats received microinjections of tetrodotoxin (TTX) in the SuM area (5ng diluted in 0.5µl of saline) or saline (0.5µl). The microinjections were administered 90min before the perfusion. Later, cellular activity in medial septum/diagonal band of Broca (MS/DBB) and CA3 region of the dorsal hippocampus was assessed, by measuring the immediate early gene c-fos. The results show a clear hiperactivity cellular in medial septum/diagonal band of Broca and a clear hypoactivity cellular in the CA3 region of the hippocampus when there was a functional inactivation of the SuM area. It suggests that the SuM area seems to be part of the connection and information input pathways to CA3 region of the hippocampal formation, key for proper functioning in spatial memory and emotional response.

Immunohistochemical screening for antibodies in recent onset type 1 narcolepsy and after H1N1 vaccination.

Narcolepsy type 1 patients typically have undetectable hypocretin-1 levels in the cerebrospinal fluid (CSF), as a result of a selective loss of the hypocretin containing neurons in the hypothalamus. An autoimmune attack targeting hypothalamic hypocretin (orexin) neurons is hypothesised. So far, no direct evidence for an autoimmune attack was found. One of the major limitations of previous studies was that none included patients close to disease onset. We screened serum of 21 narcolepsy type 1 patients close to disease onset (median 11 months), including 8 H1N1 vaccinated patients, for antibodies against hypocretin neurons using immunohistochemistry. No autoantibodies against hypocretin neurons could be detected.

Colocalization pattern of calbindin and cocaine- and amphetamine-regulated transcript in the mammillary body-anterior thalamic nuclei axis of the guinea pig.

The study describes for the first time the colocalization pattern of calbindin (CB) and cocaine- and amphetamine-regulated transcript (CART) in the mammillary body (MB) and anterior thalamic nuclei (ATN) - structures connected in a topographically organized manner by the mammillothalamic tract (mtt). Immunohistochemical study was performed on fetal (E40, E50, E60), newborn (P0) and postnatal (P20, P80) brains of the guinea pig, but the coexistence pattern of the substances was invariable throughout the examined developmental stages. CB and CART colocalized in the perikarya of the lateral part of the medial mammillary nucleus (MMl), whereas in its medial part (MMm) only CB was detected. In the mtt, which originates from the MB, both the substances were present and colocalized in single fibers. Next, fibers from the mtt spread toward the ATN in a particular way: fibers containing CB ran to both the anteromedial thalamic nucleus (AM) and anteroventral thalamic nucleus (AV), while fibers containing CART ran mostly to the latter one. In the ventral part of AV, CB and CART colocalized vastly in the neuropil. The lateral mammillary nucleus and anterodorsal thalamic nucleus were virtually devoid of CB- and CART-positive structures. Based on the known connections between the MB and ATN, we conclude that the studied substances may cooperate in the MMl-AV part of the axis and CB plays a significant role in the MMm-AM part.

Relationships among the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure from a viewpoint of elements.

To elucidate the relationships among the brain regions belonging to the limbic system, the authors investigated the relationships among the hippocampus, dentate gyrus, mammillary body, and fornix, using the anterior commissure as a control, from a viewpoint of elements. After ordinary dissections at Nara Medical University were finished, the hippocampi, dentate gyri, mammillary bodies, fornices, and anterior commissures were resected from identical cerebra of the subjects. The subjects consisted of 23 men and 23 women, ranging in age from 70 to 101 years (average age = 83.5 ± 7.5 years). After ashing with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. With regard to seven elements of Ca, P, S, Mg, Zn, Fe, and Na, it was examined whether there were significant correlations among the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure. It was found that there were extremely or very significant direct correlations among all of the five brain regions of the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure in the P content. Likewise, with regard to the Fe content, there were significant direct correlations among the four brain regions belonging to the limbic system, except for the anterior commissure. In both the Ca and Zn contents, there were extremely or very significant direct correlations among the hippocampus, dentate gyrus, and mammillary body of the gray matter.

Gender differences in elements of human anterior commissure and olfactory bulb and tract.

To examine whether there were gender differences in the various brain regions, the authors investigated the gender differences in seven element contents of the anterior commissure, mammillary body, and olfactory bulb and tract by direct chemical analysis. After ordinary dissection at Nara Medical University was finished, the anterior commissures, mammillary bodies, and olfactory bulbs and tracts were resected from the cerebra cut at median line. The brain samples were treated with 99.5% ethanol three times to remove lipids. After ashing with nitric acid and perchloric acid, the seven element contents Ca, P, S, Mg, Zn, Fe, and Na were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the Zn content was significantly higher in the anterior commissures of men than in those of women. In the olfactory bulbs and tracts, it was found that the Ca, P, and Zn contents were significantly higher in men than in women. In contrast, no significant difference was found between the mammillary bodies of men and women regarding the seven element contents.

Polysynaptic pathways from the vestibular nuclei to the lateral mammillary nucleus of the rat: substrates for vestibular input to head direction cells.

The activity of some neurons in the lateral mammillary nucleus (LMN) of the rat corresponds with the animal's current head direction (HD). HD cells have been studied extensively but the circuitry responsible for the generation and maintenance of the HD signal has not been established. The present study tested the hypothesis that a polysynaptic pathway connects the vestibular nuclei with the LMN via one or more relay nuclei. This circuitry could provide a substrate for the integration of sensory input necessary for HD cell activity. This hypothesis is based upon the prior demonstration that labyrinthectomy abolishes HD selectivity in thalamic neurons. Viral transneuronal tracing with pseudorabies virus (PRV) was used to test this hypothesis. We injected recombinants of PRV into the LMN and surrounding nuclei of adult male rats and defined the patterns of retrograde transneuronal infection at survival intervals of 60 and 72 h. Infected medial vestibular neurons (MVN) were only observed at the longest postinoculation interval in animals in which the injection site was localized largely to the LMN. Robust infection of the dorsal tegmental nucleus (DTN) and nucleus prepositus hypoglossi (PH) in these cases, but not in controls, at both survival intervals identified these nuclei as potential relays of vestibular input to the LMN. These data are consistent with the conclusion that vestibular information that contributes to the LMN HD cell activity is relayed to this caudal hypothalamic cell group via a polysynaptic brainstem circuit.

Immunohistochemical analyses of thyroid-specific enhancer-binding protein in the fetal and adult rat hypothalami and pituitary glands.

Thyroid-specific enhancer-binding protein (T/EBP), also known as NKX2.1 or TTF-1, regulates the expression of thyroid- and lung-specific genes. The t/ebp/Nkx2.1-null mutant mouse was stillborn but lacked the thyroid gland, pituitary gland, ventral region of the forebrain and normal lungs. These data demonstrated that T/EBP/NKX2.1 plays an important role not only in tissue-specific gene expressions in adults but also in genesis of these organs during development. Although the expression of t/ebp/Nkx2.1 in the brain has been reported, its function in the brain remains unknown. The present study was designed to determine the localization of T/EBP/NKX2.1 in the hypothalamus and pituitary gland of fetal and adult rats by immunohistochemistry as the first step toward understanding the function of T/EBP/NKX2.1 in the rat brain. In the fetal rat hypothalamus, T/EBP/NKX2.1 was localized widely in the ventral hypothalamic areas. In the adult rat brain, T/EBP/NKX2.1 was localized in the ventromedial hypothalamic nucleus, medial tuberal nucleus, arcuate nucleus and mammillary body. No T/EBP/NKX2.1 immunoreactivity was observed in the anterior or intermediate lobe of the pituitary gland in either fetal or adult rats. On the other hand, immunoreactive T/EBP/NKX2.1 was found in the posterior lobe of the pituitary gland. This paper presents results of detailed analyses of the distributions of T/EBP/NKX2.1 protein in the fetal and adult rat hypothalami and pituitary glands, and these results should provide important information for understanding the function of T/EBP/NKX2.1 in the brain.

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the limbic system of the rat.

We used immunocytochemistry to obtain a complete cellular and subcellular mapping of the 1,25-dihydroxyvitamin D3 receptor protein (VDR) in the rat limbic system. We observed specific VDR immunostaining in the nucleus as well as in the perinuclear cytoplasm of neuronal cells. The limbic system consists of a variety of neuronal structures, and is known to have influence on memory, behavior, emotions and reproduction. In the hippocampal formation, we found strong nuclear staining as well as less distinguished cytoplasmic VDR staining in CA1, CA3 and CA4. The CA2 area showed a unique cytoplasmic predominance of VDR. The amygdala was found to exhibit specific patterns of VDR distribution in the various regions of the nucleus. We observed distinct differences of VDR localization within the limbic preoptic areas of the hypothalamus. Further parts of the brain we analyzed included the mammillary bodies, the indusium griseum and the cingulate cortex. The subcellular distribution of VDR in regions of the limbic system suggests a specific functional role of the receptor protein and indicates a role for calcitriol as a neuroactive steroid.

Group II selective metabotropic glutamate receptor agonists and local cerebral glucose use in the rat.

The novel mGluR agonist LY354740 and a related analogue LY379268 are selective for mGluR2/3 receptors and are centrally active after systemic administration. In this study, rates of local cerebral glucose use were measured using the [14C]2-deoxyglucose autoradiographic technique to examine the functional consequences of their systemic administration in the conscious rat. Both LY354740 (0.3, 3.0, 30 mg/kg) and LY379268 (0.1, 1.0, 10 mg/kg) produced dose-dependent changes in glucose use. After LY354740 (3.0mg/kg), 4 of the 42 regions measured showed statistically significant changes from vehicle-treated controls: red nuclei (-16%), mammillary body (-25%), anterior thalamus (-29%), and the superficial layer of the superior colliculus (+50%). An additional 15 regions displayed significant reductions in function-related glucose use (P < .05) in animals treated with LY354740 (30 mg/ kg). LY379268 (0.1, 1.0, 10 mg/kg) produced changes in glucose metabolism in 20% of the brain regions analyzed. Significant increases (P < .05) in glucose use were evident in the following: the superficial layer of the superior colliculus (+81%), locus coeruleus (+57%), genu of the corpus callosum (+31%), cochlear nucleus (+26%), inferior colliculus (+20%), and the molecular layer of the hippocampus (+14%). Three regions displayed significant decreases: mammillary body (-34%), anteroventral thalamic nucleus (-28%), and the lateral habenular nucleus (-24%). These results show the important functional involvement of the limbic system together with the participation of components of different sensory systems in response to the activation of mGluR2 and mGluR3 with LY354740 and LY379268.

Localization of amino acids, neuropeptides and cholinergic neurotransmitter markers in identified projections from the mesencephalic tegmentum to the mammillary nuclei of the rat.

Retrograde labelling has been combined with immunohistochemistry to localize neurons containing GABA, glutamate, choline acetyltransferase, leu-enkephalin, neurotensin and substance P-like immunoreactivity in the projection pathways from the midbrain tegmental nuclei to the mammillary nuclei in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the medial mammillary nucleus resulted in retrogradely labelled neurons in the ventral tegmental nucleus of Gudden, whereas injections into the lateral mammillary nucleus resulted in large numbers of retrogradely labelled neurons in the ipsilateral dorsal tegmental nucleus of Gudden and in the laterodorsal tegmental nucleus. In the ventral tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also immunolabelled for GABA and approximately ten to 18 WGA-HRP-labelled neurons per section were immunoreactive for leu-enkephalin. In addition, small numbers of WGA-HRP-labelled neurons in the principal subnucleus of the ventral tegmental nucleus were immunoreactive for Glu whereas small numbers of retrogradely labelled neurons in the compact subnucleus of the central superior nucleus displayed neurotensin-like immunoreactivity. In the ventral subnucleus of the dorsal tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also GABA-immunoreactive and approximately ten to 14 WGA-HRP labelled neurons per section were immunoreactive for leu-enkephalin. The ventral subnucleus of the dorsal tegmental nucleus also contained small numbers of retrogradely labelled neurons that displayed either glutamate or substance P-like immunoreactivity. In addition, moderate to small numbers of WGA-HRP-labelled neurons (five to 20 per section) in the laterodorsal tegmental nucleus were immunoreactive for choline acetyltransferase. These results are compatible with the possibility that tegmentomammillary projection neurons use several different neurochemicals as neurotransmitter(s) and/or neuromodulator(s).

Glutamate/aspartate and leu-enkephalin immunoreactivity in mammillothalamic projection neurons of the rat.

We have used retrograde transport and immunohistochemistry to study glutamate, aspartate, and enkephalin-like immunoreactive pathways from the mammillary nuclei to the anterior nuclei of the thalamus. Injections of wheat germ agglutinin conjugated to horseradish peroxidase into the anterodorsal thalamic nucleus resulted in retrogradely labelled cell bodies in the lateral mammillary nucleus, bilaterally, whereas injections into the anteroventral thalamic nucleus resulted in retrogradely labelled neurons in the ipsilateral medial mammillary nucleus. In three parallel series of sections immunoreacted for glutamate, aspartate, and enkephalin, respectively, 50-60% of the retrogradely labelled cell bodies were also immunolabelled for glutamate, 50-60% for aspartate, and 40-50% for enkephalin. The enkephalin-immunoreactive neurons may coincide with or constitute a separate population from the glutamate/aspartate-containing neurons. These results are compatible with the possibility that mammillothalamic projection neurons may use glutamate and/or aspartate and enkephalin as neurotransmitters.

Severe reduction of rat defensive behavior to a predator by discrete hypothalamic chemical lesions.

Nonspecific lesion and stimulation methods have suggested that the hypothalamus is critical for the expression of defensive behavior, although the organization of neural circuits mediating such behavior is unclear. In the rat hypothalamus, we found that increased Fos levels were restricted to specific cell groups following presentation of a stimulus (predator) known to elicit partly innate defensive responses. The dorsal premammillary nucleus showed the most striking increase in Fos levels, and cell body-specific chemical lesions therein virtually eliminated two major components of defensive behavior but increased exploratory behavior, suggesting that this caudal hypothalamic nucleus plays a critical role in the expression of behavioral responses sometimes critical for survival of the individual. We have previously shown that the Fos-responsive cell groups in the medial hypothalamus are interconnected in a neural system distinct from those mediating reproductive and ingestive behaviors.

Immunohistochemical studies of localization and co-localization of glutamate, aspartate and GABA in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat.

Localization and possible co-localization of glutamate, aspartate and GABA immunoreactivities was examined in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat by double antigen immunohistochemistry using diaminobenzidine and benzidine dihydrochloride in one series and double immunofluorescence labelling with rhodamine and fluorescein in a second series of animals. In three of these regions, retrosplenial granular cortex, anterior thalamic nuclei, and mammillary nuclei, glutamate immunoreactivity was co-localized with aspartate immunoreactivity in a majority of the projection neurons (pyramidal neurons, predominantly in layers V and VI in retrosplenial granular cortex; rounded polygonal multipolar neurons throughout the rostrocaudal extent of the anterior thalamic and mammillary nuclei). None of the cells showing glutamate and/or aspartate immunoreactivity in these regions also displayed GABA immunoreactivity, which was present in non-pyramidal cells in the retrosplenial granular cortex (chiefly in layers I-III) and in small numbers of cells within the anterior thalamic nuclei. In the thalamic reticular nucleus, in contrast, most neurons were immunoreactive for GABA and in the majority of these neurons glutamate (and/or aspartate) immunoreactivity was co-localized with GABA.

Immunohistochemical mapping of nitric oxide synthase in the rat hypothalamus and colocalization with neuropeptides.

The localization and distribution of nitric oxide synthase in the hypothalamus have been studied with an immunohistochemical technique using antibodies to neuronal rat nitric oxide synthase. Subsequent double-labeling experiments examined the colocalization patterns of nitric oxide synthase and several peptides. Our results demonstrate a widespread occurrence of nitric oxide synthase-immunoreactive nerve cell bodies and processes throughout the hypothalamus, especially in various parts of the preoptic region, in the supraoptic and paraventricular nuclei, the lateral hypothalamic area, the ventromedial and dorsomedial nuclei, the arcuate nucleus and various parts of the mammillary region. Double labeling experiments showed that nitric oxide synthase-like immunoreactivity coexists with substance P-like immunoreactivity in the medial preoptic area, with oxytocin-, cholecystokinin-and galanin message-associated peptide-like immunoreactivity in the supraoptic nucleus, with enkephalin, oxytocin- and corticotropin releasing factor-like immunoreactivity in the paraventricular nucleus and with enkephalin-like immunoreactivity in the arcuate nucleus. Furthermore, in the ventromedial nucleus, nitric oxide synthase-like immunoreactivity coexisted with enkephalin-, substance P-, and somatostatin-like immunoreactivity, and in the dorsomedial nucleus with enkephalin-, galanin message-associated peptide-and substance P-like immunoreactivity. In the mammillary region nitric oxide synthase-like immunoreactivity coexisted with enkephalin-, cholecystokinin-, and substance P-like immunoreactivity. Among these neuropeptides, enkephalin and substance P were most frequently found in nitric oxide synthase-immunoreactive neurons. We conclude that nitric oxide synthase-immunoreactive neurons contain neuropeptides in various parts of the hypothalamus, and that nitric oxide in the hypothalamus may be involved in a variety of neuroendocrine and autonomic functions.

Functional sexual differences in rat mammillary bodies: a quantitative Ag-NOR study.

The mammillary bodies of the posterior hypothalamus are one of the CNS structures in which structural sexual dimorphism has already been described. This study quantifies the argyrophilic nucleolar organizer regions (NORs) of the neurons from this region in the male and female rats, the latter during two major phases of the estrous cycle (estrus-diestrus). The number and relative area of these NORs stained with a silver nitrate technique are considered as an index of the global protein synthesis of the neurons in the different nuclei from the mammillary bodies. Our results show the existence of statistically significant differences between sexes and estrous cycle phases. These findings suggest a significant influence of gender and the hormonal state on the neural activity of the MB.

Reserpine- and tetrabenazine-sensitive transport of (3)H-histamine by the neuronal isoform of the vesicular monoamine transporter.

The transport of (3)H-histamine by the endocrine-specific (VMAT1) and neuronal (VMAT2) isoforms of the vesicular monoamine transporter has been evaluated in digitonin-permeabilized fibroblasts transfected with either VMAT1 or VMAT2. Transport of (3)H-histamine by both VMAT1 and VMAT2 was reserpine-sensitive but only transport by VMAT2 was inhibited by tetrabenazine. Maximal equilibrated levels of (3)H-histamine accumulation by VMAT2 (K(m) 300 mu M) were approximately three times greater than that mediated by VMAT1 when using a subsaturating concentration of exogenous (3)H-histamine (50 mu M). The expression of VMAT2 in histaminergic neurons in the rat brain was examined with polyclonal antipeptide antibodies specific for VMAT1 or VMAT2. VMAT2-positive and tyrosine hydroxylase-negative immunoreactive cell bodies were localized to the ventral part of the posterior hypothalamus in the region of the mamillary nuclei. The transport properties of VMAT2 and the distribution of VMAT2 in cell bodies in the tuberomammillary nucleus of the posterior hypothalamus reported here and the apparent absence of VMAT1 and VMAT2 in tissue mast cells support previous findings of reserpine-sensitive and reserpine-resistant pools of histamine in brain and peripheral tissues.