[Receptors for oreksins: structure, localisation and activation mechanisms].

The review concerns morpho-functional characteristics of orexin receptors. The data on their structure, signal transduction pathways, biological effects of orexin receptors 1 or 2 depending on association with different G-proteins are presented. Localisation of orexin receptors in various CNS structures as well as in peripheral organs mediates regulation of different physiological functions by orexins. Low concentration of orexins in peripheral blood and orexin-containing cells in ganglions and internal organs suggests a possibility to activate orexin-sensitive cells distantly, paracrinely or autocrinely. The data on effects of selective or non-selective orexin receptor antagonists is analysed.

[Immunoreactivity of hypothalamic orexin neurons and expression level of preproorexin gene in them after lipopolysaccharide injection].

Hypothalamic orexin neurons are involved in the regulation of many physiological functions. The immunoreactivity of these neurons is shown to be altered after LPS injection. This phenomenon is characterized by definite time-space pattern and depends on dose of antigen applied. The expression level ofpreproorexin gene in rat hypothalamus was investigated in 2, 4 and 6 hours after injection of 25 and 500 mkg/kg b. w. LPS. Both injections of higher and lower doses resulted in the increase of expression level of preproorexin gene after 2 hours that could suggest an enhancement of orexin synthesis in neurons. There were no significant changes in 4 and 6 hours after injection. The comparative analysis of the data obtained earlier with immunohistochemistry, and the data shown in the present study suggest the mechanisms of orexin neurons reaction to injection of LPS in different doses, i. e. the more considerable prevalence of orexin utilization over its synthesis in hypothalamic cells after injection of subseptic (500 mkg/kg) dose of LPS.

[Response of hypothalamic orexin-containing neurons to stimuli of antigen and non-antigen nature].

Orexin is a hypothalamic peptide, a neurotransmitter described in 1998. Orexinergic neurons are localized in hypothalamic structures and play a significant role in regulation of various physiological functions. The localization oforexin-containing neurons and their projections in hypothalamus of Wistar rats and other structures of CNS are presented. The participation of orexinergic neurons in the regulation of feeding behavior and in the sleep/wake cycle as well as their involvement in the regulation of immune functions is discussed. There are experimental data, containing comparative analysis oforexin-containing neurons responses to stimuli of antigenic and non-antigenic nature, which suggest functional heterogeneity of orexin-containing neurons of hypothalamus that leads, particularly, to involvement of different neurons in the realization of brain reaction to antigen and non-antigen stimuli. Both analyses ofpreproorexin gene expression level and morphofunctional characteristics of orecxin-containing neurons of hypothalamus after antigen challenge suggest the possibility of their participation in the mechanisms of realization of brain reaction to antigen challenge.

[Comparative analysis of orexin-containing neurons reactions in the rat hypothalamus after lipopolysaccharide injection in different doses].

Nowadays many investigations are focused on the participation of orexin-containing neurons in CNS reactions caused by antigen injection, for instance lipopolysaccharide (LPS). The analysis of orexin-containing neuron: quantity in 2, 4 and 6 hours after injection of low non-septic dose of LPS in comparison with a higher subseptic dose, was carried on. The increase of the orexin-immunoractive neurons quantity was observed in 2 and 4 hours after administration of 25 meg/kg of LPS, whereas in 6 hours the decrease of it was demonstrated. Injection of higher dose of LPS (500 meg/kg) was shown to cause the more significant decrease of the quantity of orexin-positive neurons only in 6 hours after antigen administration. The obtained data evidence the alteration of orexin content detected in neurons somas and the shift of the balance of its synthesis and utilization processes in response to the LPS injection.

Morphometric analysis of hypothalamic cells showing c-Fos proteins after movement restriction and EHF-irradiation.

A morphometric analysis of hypothalamic cells containing c-Fos-like protein detected by an indirect immunoperoxidase method was carried out to clarify the responses to movement restriction and skin electromagnetic high frequency (EHF) irradiation (7.1mm, 42.2GHz, 20mW output power) simultaneously to three acupuncture projection areas. The morphometry of c-Fos-protein positive neurons by their number and type was analyzed. Movement restriction (40min) induced c-Fos protein expression primarily in cells with 10-50mum(2) sizes (associative type neurons) only in anterior hypothalamic nucleus and lateral hypothalamic area; while additional EHF-irradiation of acupuncture projection areas (under movement restriction) induced c-Fos expression in all hypothalamic structures and mostly in cells with 70-150mum(2) sizes (relay type neurons), i.e. changed the pattern of activated cell type distribution. In conclusion, the findings show that modest stress under which experimental animals often are exposed increase c-Fos protein expression in hypothalamic centers and skin EHF-irradiation of acupuncture projection areas seem to increase that.

Stress-induced changes in cellular responses in hypothalamic structures to administration of an antigen (lipopolysaccharide) (in terms of c-Fos protein expression).

Stress is known to affect the intensity of the immune response. The involvement of central regulatory structures in mediating these changes was addressed by analyzing the extent of activation of neurons in the hypothalamus (in terms of the number of c-Fos-positive cells) in rats 2 h after i.v. administration of lipopolysaccharide alone and on the background of electrical pain stimulation. Studies were performed using 52 male Wistar rats weighing 200-250 g. c-Fos protein expression was studied by immunohistochemical analysis. Increases in the quantity of c-Fos-positive cells 2 h after administration of lipopolysaccharide were seen in the following hypothalamic structures: AHN, PVH, LHA, VMH, DMH, and PH. After electrical pain stimulation, the number of c-Fos-positive cells increased in these same hypothalamic structures (AHN, PVH, LHA, VMH, DMH, and PH). The combination of electrical pain stimulation and lipopolysaccharide administration led to a decrease in the extent of activation in hypothalamic structures AHN, PVH, LHA, and VMH as compared with the characteristic reaction to lipopolysaccharide without electrical pain stimulation. Electrical pain stimulation suppressed the intensity of the immune response induced by lipopolysaccharide (as assessed by local hemolysis and counts of the numbers of spleen antibody-forming cells). Thus, changes in the extent of activation of hypothalamic structures (AHN, PVH, LHA, VMH) correlated with the development of stress-induced immunosuppression, i.e., morphofunctional mapping of the extent of activation of hypothalamic structures allowed identification of which changes in hypothalamic cell activity occurred with stress-induced changes in immune system responses to antigen administration.

Intracellular expression of c-Fos protein in various structures of the hypothalamus in electrical pain stimulation and administration of antigens.

Application of different stimuli activated different hypothalamic structures. Immunohistochemical methods demonstrated changes in the numbers of c-Fos-positive cells in various hypothalamic structures after electrical pain stimulation and i.v. administration of antigens (bovine serum albumin (BSA) and lipopolysaccharide (LPS)). Increases in the numbers of c-Fos-positive cells in all the hypothalamic structures studied occurred after electrical pain stimulation and i.v. administration of antigens (BSA and LPS). The highest level of activation in hypothalamic structures was seen in the anterior hypothalamic nucleus (AHN) and posterior hypothalamic area (PH) after electrical pain stimulation and in the paraventricular nucleus (PVN) and lateral hypothalamic area level 28 (LHA-28) after i.v. administration of LPS. Comparative analysis showed that the level of activation of hypothalamic structures after electrical pain stimulation was significantly greater in the AHN, PVN, LHA, and PH than after i.v. administration of antigens (LPS and BSA). Administration of LPS led to more marked activation of cells in hypothalamic structures APH, PVN, LHA-28, dorsomedial hypothalamic nucleus (DMH), and PH (in terms of the numbers of c-Fos-positive cells) than administration of BSA.

Responses of hypothalamic orexin-containing neurons to cyclophosphamide, EHF-irradiation of the skin, and their combination in rats.

Orexins are neuromediators that participate in the regulation of feeding behavior, energy metabolism, circadian rhythms and perception of pain. The aim of the present study was to clarify the responses of the hypothalamic orexin-containing neurons to an intraperitoneal injection of cyclophosphamide (CPA), extremely high frequency (EHF)-electromagnetic stimulation of skin, which is used to modulate side effects of cytostatics and their combination. The activation of orexin-containing neurons was determined by recording of the intensity of c-Fos protein expression. Injection of cyclophosphamide (40mg/kg) or EHF-irradiation of the skin decreased the staining of orexin-containing neurons, which was most pronounced in the subfornical region of the lateral hypothalamic area (LHAs). A redistribution of orexin from the perinuclear space to the processes of these cells took place, which occurs after the activation and the expression of the c-fos-gene. c-Fos protein was expressed in most neurons with minimum content of orexin, i.e. activation of these neurons correlated with the redistribution of orexins caused by skin EHF-irradiation and injection of cyclophosphamide (CPA). EHF-irradiation of the skin before and after injection of CPA increased the staining of orexin-containing neurons, i.e. it prevented the redistribution of orexin.

[Stress-induced changes of hypothalamic structure cell responses to antigen injection (LPS) (revealed by c-Fos protein expression)].

Stress stimuli are known to influence the intensity if immune response. To elucidate the role of central regulating structures in this changes, analysis of activation level of hypothalamic neurons (revealed by quantity of c-Fos-positive cells) was carried out in rats within 2 hours after intravenous LPS injection and after this--impact associated with electric pain stimulation (EPS). The investigation was carried out in 52 male Wistar rats, 200-250 g. The c-Fos protein expression was analyzed with immunohistochemical method. The increase of c-Fos-positive cells number in 2 hours after LPS injection was observed in AFTN, PVH, LHA, VMH, DMH and PH. After electrical pain stimulation, the quantity of c-Fos-positive cells increased in the same structures. Combined application of electric pain stimulation and LPS injection results in diminished activation level in AHN, PVH, LHA and VMH as compared with typical response to single LPS injection without EPS. The EPS suppresses intensity of the immune response induced by injection of LPS (revealed by local hemolysis method with calculation of antibody-forming cells quantity (%) in the rat spleen). Thus the activation level changes of hypothalamic structures (AHN, PVH, LHA, PH) correlate with development of stress-induced immunosuppression, i. e. morphofunctional description of hypothalamic structures activation as revealed by pattern of activated cell alterations in hypothalamic structures during realization of stress-induced changes of immune system responses to antigen injection.

[c-FOS protein expression in cells of the variuos hypothalamic structures after electric pain stimulation and injections of antigens].

Hypothalamic structures become activated after stimul. Alteration of c-Fos-positive cells quantity in different hypothalamic structures after electric pain stimulation (EPS), intravenous (iv) injection of antigens (lioppolysaccharide (LPS) and bovine serum albumir (BSA)) was detected with immunohistochemical method. EPS and iv injection of antigens (LPS and BSA) result in c-Fos-positive cells quantity increase in all observed hypothalamic structures. The highest activation level was in AHN and PH after EPS and in AHN, PVH, LHA-28, and PH after iv LPS injection. Comparative analysis of results showed, that c-Fos-positive cells quantity increase after EPS in AHN, PVH, LHA and PH was more significant than after iv injection of antigens (LPS and BSA). LPS injection results in more pronounced cell activation in AHN, PVH, LHA-28 and DMH (according to quantity of c-Fos-positive cells), than BSA injection.