Cerebellar fastigial nuclear glutamatergic neurons regulate immune function via hypothalamic and sympathetic pathways.

We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-ß-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.

Role of cerebellohypothalamic GABAergic projection in mediating cerebellar immunomodulation.

Our previous studies have shown that the cerebellar interposed nucleus (IN) modulates lymphocyte functions. As the cerebellum does not have a direct contact with the immune system, it is required to explore the pathway mediating the cerebellar immunomodulation. In this study, both lymphocyte percentage in peripheral leukocytes and lymphocyte proliferation induced by concanavalin A were reduced by the bilateral IN lesions with kainic acid. Anterograde tracing of nerve tracts with biotinylated dextran amine (BDA) from the cerebellum to the hypothalamus revealed that the BDA-labeled fibers from the cerebellar IN neurons traveled through superior cerebellar peduncle (SCP), crossed in SCP decussation, and primarily terminated in lateral hypothalamic area (LHA). Retrograde tracing with wheat germ agglutinin-horseradish peroxidase from the LHA to the cerebellar IN combined with immunohistochemistry for gamma-aminobutyric acid (GABA) or glutamate in the cerebellar sections displayed that the neuronal projections from the cerebellar IN to the LHA mostly were GABAergic. Blockage of GABA(A) receptors in the LHA with hydrastine led to a reduction in the lymphocyte percentage and proliferation, similar to the IN lesions. These results show a direct GABAergic projection from cerebellar IN to LHA and suggest that the projection mediates cerebellar immunomodulation.

Chronic electric stimulation of the midbrain ventral tegmental area increases spleen but not blood natural killer cell cytotoxicity in rats.

Previously we found that in conscious, freely behaving rats chronic electric stimulation of the lateral hypothalamus (LH) caused significant augmentation of natural killer cell cytotoxicity (NKCC) and a large granular lymphocyte (LGL) number more pronounced in the spleen than in the peripheral blood. The LH belongs to the so-called "brain reward system", a collection of the central structures whose activation produce positive emotions. The midbrain ventral tegmental area (VTA) is another prominent reward-relevant structure. In the present work, chronic electric stimulation of VTA (constant current 0.1 ms duration cathodal pulses delivered at frequency 50 Hz during 60 min daily session for 14 consecutive days) caused in rats an increase in the spleen but not in the peripheral blood NKCC (chromium release assay) without simultaneous effect on the number of large granular lymphocytes (LGL) (morphological method) and plasma level of prolactin (PRL), growth hormone (GH), corticosterone (COR), and testosterone (TST). This effect was anatomically specific as no influence of analogous thalamic stimulation on immune and endocrine response was found. The results obtained indicate that both reward-related areas VTA and LH enhance the cell-mediated immune response, represented by natural killer cytotoxicity, especially in the spleen. However, the effect pronounced by VTA is weaker than that of LH, possibly due to additional connections of LH with the hormonal and/or autonomic control systems.

The effects of lateral hypothalamic lesions on peripheral blood natural killer cell cytotoxicity in rats hyper- and hyporesponsive to novelty.

Individual variability in the central control of the cellular immune responses is the main subject of the study. Previously, it was found that destruction of the lateral hypothalamus (LH) produced long-term depression of the cytotoxicity of NK cells (NKCC) and their number (LGL). In the present experiment we compared changes in the peripheral blood NKCC, LGL number, as well as leukocyte and lymphocyte number, their mitogenic activity and plasma corticosterone level evoked by electrolytic LH lesions in rats which were categorized as either high (HR) and low (LR) responders according to their locomotor response to a new environment. It was found that: (1) before the lesion NKCC (measured by 51Cr release assay) was higher in the HRs than in LRs; (2) LH damage caused a drop in NKCC and LGL number (21st postlesion day) preceded by a transient enhancement (5th postlesion day) significant for HRs only. As a result of a greater decrease in the HRs than LRs the baseline differences between groups disappeared by 21st postlesion day; (3) NKCC and LGL depression was not accompanied by changes in lytic activity of a single NK cell (agarose assay) which indicates that NKCC decrease concerned the population level and was dependent on LGL redistribution and/or recycling rate; (4) on the 21st postlesion day there was a significant leuko- and lymphopenia in the lesioned groups both HRs and LRs; (5) proliferative lymphocyte response to PWM (colorimetric assay) and plasma corticosterone level were not affected either by the motility level or by the lesion. The results emphasize the importance of individual differences in behavioral reactivity for NKCC regulation and a possible involvement of LH in the mechanism which connects high locomotor activity with stimulation of NKCC.

Chronic electrical stimulation of the lateral hypothalamus increases natural killer cell cytotoxicity in rats.

Previously, we found that in rats coagulation of the lateral hypothalamus (LH) caused depression of the peripheral blood natural killer cell cytotoxicity (NKCC) and the number of large granular lymphocytes (LGL). In the present work, we have tested the effects on both spleen and blood NKCC of acute (1 day) and chronic (21 days) electrical stimulation of LH, and LGL number in conscious, freely behaving animals. Five groups of male Wistar rats were used: LH stimulated (n=22), thalamic (Thal) stimulated control (n=4), operated but non-stimulated LH sham controls (n=7), non-operated normal control group (n=8) and spleen baseline group (n=10). Chronic stimulation of LH caused significant augmentation of NKCC (51Cr-release assay) and LGL number (a morphological method), more pronounced in the spleen than in the peripheral blood. Rats responding to LH stimulation with feeding showed a slightly greater effect than those responding with a locomotor reaction. The observed effects were anatomically specific as no influence of Thal stimulation or the sham procedure was found. The results are discussed in terms of the involvement of LH in reward phenomena and the hormonal control of the immune system.

Modulation of natural killer cell activity affected by electroacupuncture through lateral hypothalamic area in rats.

Electroacupuncture (EA) has been reported to modulate natural killer cell (NK cell) activities. Also it is well known that hypothalamus directly mediates the effects of EA on analgesia. Especially lateral hypothalamic area (LHA) is related to splenic NK cell activities. In order to investigate the relationship between hypothalamus and effects of EA on NK cell activity, lesions have been made bilaterally at LHA of Spraque-Dawley rats. Subsequently, NK cell cytotoxities of normal and lesioned rats were measured with (51)Cr release immunoassay after EA stimulation for 2 and 14 days. NK cell activity of EA group was significantly higher than sham group. In addition, lesions abolished effects of EA on NK cell activity. These results strongly suggest that LHA is closely related to increase of NK cell activity induced by EA.

Expression of immunoregulatory cytokines in neurons of the lateral hypothalamic area and amygdaloid nuclear complex of rats immunized against human IgG.

Present results showed that interleukin-1 (IL-1), IL-6 and transforming growth factor-beta (TGF-beta) were constitutively expressed in the supraoptic and paraventricular nuclei of the rat hypothalamus. Immunoreactive cells were also detected, but to a lesser extent, in other parts of hypothalamus as well as in the cerebral cortex. In rats immunized with IgG, there was moderate increase in immunoreactivities of the cytokines. A notable feature, however, was the induction of the cytokine expression in the lateral hypothalamic area and the amygdaloid nuclear complex, suggesting that the neurons in these two areas are involved in possible immune regulation.

[Areas of brain involved in immunoregulation].

OBJECTIVE: To study the location of brain areas involved in immunoregulation. METHODS: Immunohistochemistry and immunofluorescence were used to detect the different distribution of cytokines immunopositive cells in the brain of rats immunized via both intraperitoneal and subcutaneous injections. RESULTS: The cytokines immunopositive cells were distributed in the supraoptic (SO) and paraventricular (PV) nuclei of the hypothalamus, the anterior hypothalamic (AH) nucleus, arcuate and median eminence, the lateral hypothalamic nucleus (LH) and the amygdaloid nuclear complex while only the appearance of the cytokines immunopositive cells in LH and amygdaloid nuclear complex in hypothalamus was related with immunization status of the animals. Double-labelling results showed that the cytokines immunopositive cells were neurons. CONCLUSIONS: We have observed that neurons of the LH and amygdaloid nuclear complex in hypothalamus as a main source of the neuroimmunoregulation played a key role in neuroimmunoregulation and they participated in the neuroimmunoregulation at an early stage of the immune response.

Lateral hypothalamus modulates the intrinsic splenic natural killer cell activity in rats.

Electrical stimulation of the lateral hypothalamus (LH) in rats increased splenic natural killer (NK) cell activity, whereas electrical ablation of the LH decreased it. However, the percentage of NK cells, as detected by the anti-NKR-P1 monoclonal antibody, in the spleen did not change significantly. These results suggest that the LH does not modulate the splenic NK cell activity by increasing the NK cell number but by increasing the intrinsic NK cell activity itself.

Acute electrical stimulation of lateral hypothalamus increases natural killer cell activity in rats.

Natural killer cell (NK) activity in WKA and SD rats was found to be significantly higher following electrical stimulation of the lateral hypothalamus (LH) compared to sham operated. There was no such difference between sham operated rats and those receiving electrical stimulation in the frontal cortex as a control. Operations were performed under sodium pentobarbital anesthetic, and NK activity against YAC-1 target cells was measured 20 h later using 51Cr release assay. The LH area stimulated is a potent reward center and that stimulation of this point increased NK activity opens the possibility that pleasure might play a role in cellular immunity.

Modulation of cellular immunity by a lesion of the lateral hypothalamic area (LHA) in rats.

The subject of our investigation was the effect of bilateral damage to the lateral hypothalamic area (LHA) on the cellular immune response (CIR), assessed by measuring the diameter of skin infiltration (DSI) 24 and 48 h after intradermal administration of tuberculin in adult male Wistar rats, previously immunized with BCG vaccine. It has been shown that: 1) in the hypothalamo-lesioned rats (HLR) DSI measured 24 and 48 h after tuberculinization did not differ, 2) in the sham-operated rats (SOR) DSI measured 48 h after tuberculin injection was significantly higher (p < 0.02) than that found 24 h after the antigen administration, 3) the damage-induced loss of body weight and the 24-48 h difference in DSI in the responders of the HLR group correlated negatively with each other (r = -0.806, p < 0.05). These results indicate that damage to the LHA enhances the rate of formation, but not the maximal size, of the tuberculin-induced skin infiltration. Thus, the LHA seems to be involved in the regulation/modulation of processes engaged in the expression of tuberculin reaction.

Electrolytic lesions of the lateral hypothalamus influence peripheral blood NK cytotoxicity in rats.

Bilateral electrolytic lesions of the lateral hypothalamic (LH) area in Wistar rats result in a time-dependent blood NK cytotoxicity changes as measured by the 51Cr-release (for entire cell population) and agarose (for a single-cell) assays. NK activity against YAC-1 and K-562 cells shifts from depression through enhancement to another depression on the 2nd, 5th and 21st post-lesion day, respectively, as compared to both LH sham-operated animals and the pre-lesion baselines. This effect is not attributable to malnutrition and dehydration resulting from ingestive impairments evoked by LH lesions. No significant change in NK cytotoxicity was found after destruction of the medial hypothalamus (MH). The results indicate that LH, under normal conditions, which may be considered as a dynamogenic and stressogenic hypothalamic area is essential for proper regulations of NK cytotoxicity at both population and single-cell level.

Hypothalamic melanin-concentrating hormone and alpha-neoendorphin-immunoreactive neurons project to the medial part of the rat parabrachial area.

Neurons in the middle and posterior parts of the lateral hypothalamus project to the parabrachial area, and in particular to the gustatory relay-station located in the medial part of this area. In the present study we have examined some of the neuropeptide immunoreactivities of the lateral hypothalamus neurons that project to the gustatory region of the parabrachial area. By coupling retrograde transport and immunohistochemistry, we found that 50-60% of medial parabrachial area-projecting cells located in the juxta-capsular region of the posterior lateral hypothalamus are labeled by rat melanin-concentrating hormone antiserum, while 28% of the retrogradely labeled neurons located in the perifornical lateral hypothalamus are visualized with alpha-neoendorphin antiserum. Moreover, a large number of terminals distributed throughout the parabrachial nucleus are immunoreactive to melanin-concentrating hormone or alpha-neoendorphin antisera. These immunoreactivities are not co-localized within the same lateral hypothalamic neurons. The potential role of these peptidergic projections in the reward mechanisms elicited in the medial parabrachial area and in the control of palatability is discussed.

Immunoreactivities for antisera to three putative neuropeptides of the rat melanin-concentrating hormone precursor are coexpressed in neurons of the rat lateral dorsal hypothalamus.

Antisera (AS) raised against rat melanin-concentrating hormone (rMCH) and against two additional peptides sequences derived from the rat MCH precursor (neuropeptide glutamic acid-isoleucineamide (NEI), and neuropeptide glycine-glutamic acid (NGE)) exclusively stained the hypothalamic neurons previously described using AS to salmon MCH, human somatocrinin 1-37 (GRF37) and alpha-MSH. Liquid phase and dot-blot controls for specificity indicated that rMCH-, NEI- and NGE-AS bound epitopes recognized by sMCH-, alpha-MSH- and GRF-37-AS, respectively. The distinct intracellular patterns of immunoreactivity obtained in control animals with rMCH-, NGE- and NEI-AS, as well as the changes observed after intracerebroventricular injection of colchicine matched previous findings using sMCH-, GRF37- and alpha-MSH-AS.

Distribution of substance P-immunoreactive elements in the preoptic area and the hypothalamus of the rat.

The localization and morphology of neurons, processes, and neuronal groups in the rat preoptic area and hypothalamus containing substance P-like immunoreactivity were studied with a highly selective antiserum raised against synthetic substance P. The antiserum was thoroughly characterized by immunoblotting; only substance P was recognized by the antiserum. Absorption of the antiserum with synthetic substance P abolished immunostaining while addition of other hypothalamic neuropeptides had no effect on the immunostaining. The specificity of the observed immunohistochemical staining pattern was further confirmed with a monoclonal substance P antiserum. The distribution of substance P immunoreactive perikarya was investigated in colchicine-treated animals, whereas the distribution of immunoreactive nerve fibers and terminals was described in brains from untreated animals. In colchicine-treated rats, immunoreactive cells were reliably detected throughout the preoptic area and the hypothalamus. In the preoptic region, labeled cells were found in the anteroventral periventricular and the anteroventral preoptic nuclei and the medial and lateral preoptic areas. Within the hypothalamus, immunoreactive cells were found in the suprachiasmatic, paraventricular, supraoptic, ventromedial, dorsomedial, supramammillary, and premammillary nuclei, the retrochiasmatic, medial hypothalamic, and lateral hypothalamic areas, and the tuber cinereum. The immunoreactive cell groups were usually continuous with adjacent cell groups. Because of the highly variable effect of the colchicine treatment, it was not possible to determine the actual number of immunoreactive cells. Mean soma size varied considerably from one cell group to another. Cells in the magnocellular subnuclei of the paraventricular and supraoptic nuclei were among the largest, with a diameter of about 25 microns, while cells in the supramammillary and suprachiasmatic nuclei were among the smallest, with a diameter of about 12 microns. Immunoreactive nerve fibers were found in all areas of the preoptic area and the hypothalamus. The morphology, size, density, and number of terminals varied considerably from region to region. Thus, some areas contained single immunoreactive fibers, while others were innervated with such a density that individual nerve fibers were hardly discernible. During the last decade, knowledge about neural organization of rodent hypothalamic areas and mammalian tachykinin biochemistry has increased substantially. In the light of these new insights, the present study gives comprehensive morphological evidence that substance P may be centrally involved in a wide variety of hypothalamic functions. Among these could be sexual behavior, pituitary hormone release, and water homeostasis.

[Steady potential dynamics in hypothalamic structures in response to a tolerogen and an immunogen]. / Dinamika postoiannogo potentsiala v strukturakh gipotalamusa pri vvedenii toleragena i immunogena.

DC potential shifts due to induction of immune tolerance and immune memory were studied in hypothalamic structures. The lost capability of immune cooperation after tolerogen administration was accompanied by a monophasic negative shift of DC potential. Immunogen administration induced a positive shift of DC potential within 1-3 days. An immunogen fraction induced a pyrogenic response. There seems to be a correlation between the pyrogenic and immunogenic actions of antigens and the reorganization of hypothalamic neurons function.