Comparative analysis of the expression of c-Fos and interleukin-2 proteins in hypothalamus cells during various treatments.

The aim of the present work was to perform a combined analysis of the degree of activation of the anterior hypothalamus of the rat and expression of the interleukin-2 gene during treatments of different types: mild stress ("handling") and adaption to it, as well as intranasal administration of physiological saline and the peptides Vilon (Lys-Glu) and Epitalon (Ala-Glu-Asp-Gly). Changes in the numbers of c-Fos-and IL-2-positive cells in structures of the lateral area (LHA) and anterior (AHN), supraoptic (SON), and paraventricular (PVN) nuclei of the hypothalamus in Wistar rats. Ratios of the quantities of c-Fos-and IL-2-positive cells were determined in intact animals and after activation of brain cells initiated by different treatments; the influences of adaptation to handling on the nature of changes in the expression of these proteins was also studied. Combined analysis of the intensity of expression of these two proteins - c-Fos, a marker of neuron activation and a trans-factor for the IL-2 cytokine gene and other inducible genes, and IL-2 - in intact animals and after various treatments showed that the process of cell activation in most of the hypothalamic structures studied correlated with decreases in the quantity of IL-2-positive cells in these structures; different patterns of changes in the numbers of c-Fos-and IL-2-positive cells were seen in response to different treatments in conditions of stress and adaptation to it.

[Effects of antagonists of 1A and 2A/2C subtypes of serotonin receptors on the depressive behavior and expression of c-Fos protein in hypothalamus of ovariectomized rats].

We have studied the influence of a chronic administration of the 5-HT(2A/2C) receptor antagonist ketanserin (0.1 mg/kg, i.p.) and the 5-HT(1A) receptor antagonist NAN-190 (0.1 mg/kg, i.p.) alone or in combinations with 17beta-estradiol (0.5 mg per animal, i.m.) for 14 days on the depressive behavior and expression of c-Fos protein in the paraventricular nucleus of hypothalamus in adult ovariectomized (OVX) female rats. The depression in rats was modeled by the Porsolt test. The c-Fos protein expression in the paraventricular nucleus of hypothalamus was determined using immunohistochemical techniques. In the Porsolt test, 17beta-estradiol in OVX rats reduced the immobilization time to some extent. Ketanserin alone significantly decreased the immobilization time in OVX rats. The chronic administration of ketanserin in combination with 17beta-estradiol in OVX females potentiated the antidepressant effect of ketanserin. At the same time, ketanserin administration led to a significant decrease in the level of c-Fos protein in the hypothalamus in OVX rats as compared to the intact control. These results are indicative of a substantial interaction between the ovarian hormonal system and the serotoninergic brain system involved the mechanisms of depression.

[Parallel analysis of c-Fos protein and interleukin-2 expression in hypothalamic cells under different influence].

The objective of this work was to perform a parallel analysis of activation of the rat anterior hypothalamus cells as judged by c-Fos protein expression, and of the expression of interleukin-2 (IL-2) under different influences, i. e., mild stress (handling) and adaptation to it, and intranasal administration of saline and the peptides Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly). Changes in the counts of cells positive for c-Fos- and IL-2 proteins were studied in structures of the lateral (LHA) area, anterior (AHN), supraoptic (SO) and paraventricular (PVH) nuclei of Wistar rat hypothalamus. Quantity of the interleukin-2-positive and c-Fos-positive cells was calculated. The findings were: a negative correlation between the activation of cells and the amount of IL-2 in the cells in the hypothalamic structures under study, and the specific patterns of changes in the counts of cells positive for c-Fos and IL-2 under stress and adaptation to stress.

Interleukin-2 concentration in hypothalamic structures of rats receiving peptides during mild stress.

The number of hypothalamic IL-2-containing cells changed in rats receiving Vilon and Epithalon during mild stress (handling). The number of IL-2-positive cells in hypothalamic structures decreased 24 h after intramuscular injection of Epithalon and 2 h after intranasal administration of the test peptides. Adaptation of animals to experimental conditions prevented the decrease in the number of IL-2-positive cells in the supraoptic nucleus after intranasal administration of Epithalon.

Synthesis of IL-2 mRNA in cells of rat hypothalamic structures after injection of short peptides.

In situ hybridization on paraffin sections of the rat brain showed that synthetic peptides Vilon, Epithalon, and Cortagen modulated the expression of IL-2 gene in vivo in cells of some hypothalamic structures depending on the terms and routes of administration.

[In vivo expression of copper transporting proteins in rat brain sections].

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (CP), a copper-containing ferroxidase. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of CP gene in the brain is cell membrane-bound CP. Secretory CP, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the vascular plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. The Atp7a gene is active in all brain sections, whereas the Atp7b gene is active only in the hypothalamus. The membrane-bound CP is expressed in glial cells of all types and in ependyma cells. ATP7b and ATP7a are expressed predominantly in ependyomyocytes and neutrons, respectively. The organization of copper transport in mammalian brain is discussed.

In vitro effect of short peptides on expression of interleukin-2 gene in splenocytes.

Synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) in vitro activated interleukin-2 mRNA synthesis in splenocytes from CBA mice in the absence of specific inductors. The intensity of interleukin-2 mRNA synthesis in splenocytes depended on the type, concentration, and duration of treatment with the peptides. Vilon and Epithalon were most potent, while Cortagen produced a less pronounced effect on interleukin-2 mRNA synthesis.

Antigen-induced activation of hypothalamic cells (assessed by expression of the c-fos gene).

The aim of the present work was to study the activation of the expression of the c-fos gene (by in situ hybridization) in cells from rat (Sprague Dawley) hypothalamic structures 0.5, 2, 6, and 16 h after i.v. injections of tetanus toxoid (200 microg/kg). Tetanus toxoid was selected as the antigen because it does not induce any general non-specific body reactions. Control animals received i.v. doses of apyrogenic physiological saline. The number of c-fos mRNA-positive cells in all the hypothalamic structures studied was insignificant 30 min after injections of tetanus toxoid. c-fos mRNA-positive cells were seen in the posterior, lateral, and anterior hypothalamic fields and in the dorsomedial and ventromedial hypothalamic nuclei 2 h after injections of tetanus toxoid. The intensity of c-fos mRNA expression decreased in the posterior, lateral, and anterior hypothalamic fields 6 h after injections of tetanus toxoid. The maximum number of c-fos mRNA-positive cells in the anterior field and the paraventricular nucleus of the hypothalamic induced by tetanus toxoid, as compared with reactions to administration of physiological saline, were seen at 6 h. Administration of tetanus toxoid and physiological saline did not active the synthesis of c-fos mRNA in the arcuate or supraoptic nuclei at any time point. The number of c-fos mRNA-positive cells returned to baseline by 16 h after tetanus toxoid injections. Thus, this study revealed the temporospatial pattern of activation of hypothalamic structures in response to exposure to an antigen.

[Cell activation in the hypothalamus after exposure to an antigen (based on c-fos gene expression)]. / Aktivatsiia kletok gipotalamusa posle antigennogo vozdeistviia (po ékspressii gena c-fos).

Increase of the c-fos mRNA positive cells number was insignificant in 30 min. following activation of the rat hypothalamic structures with the tetanus toxoid (TT). Elevation of the c-fos mRNA positive cells number occurred in the hypothalamic' posterior (PHA), lateral (LHA), anterior (AHA), areas dorsomedial (DMH), and ventromedial (VMH) nuclei within 2 hours of the TT administration. In 6 hours the c-fos mRNA positive cells number decreased in PHA, LHA, DMH. The c-fos mRNA expression was stable in arquate and supraoptic hypothalamic nuclei following either the TT or saline administration.

C-fos and IL-2 gene expression in rat brain cells and splenic lymphocytes after nonantigenic and antigenic stimuli.

Immunostimulatory or immunosuppressive stress models were used: (1) rotation stress (RS) and (2) immobilization (restraint) stress (IS). Intravenous injection of tetanus toxoid (anatoxin) (TT) was chosen as the antigenic stimulus (500 micrograms/kg weight), and intravenous injection of saline solution was used as the control. Splenic lymphocytes (CBA mice) or different brain structures (Wistar and Sprague-Dawley rats) were analyzed. The c-fos and interleukin-2 (IL-2) mRNA expression was measured using a digoxigenin (Dig)-labeled cDNA probe by spot or in situ hybridization. Rotation stress stimulated IL-2 mRNA synthesis in lymphocytes in the presence of ConA and rIL-2 by 40%. IL-2 mRNA synthesis in lymphoid cells obtained from animals after IS and after IS in combination with the administration in vitro of the cytotoxic drug CsA to the splenic lymphocytes was inhibited (30% and 99%), accordingly, as compared with control rats. Induction of c-fos mRNA synthesis in rat brain cells was noted 30 minutes after RS in the hypothalamus (lateralis hypothalamic area, LHA), thalamus, corpus collosum, and sensorimotor zone of the brain cortex. IL-2 mRNA synthesis was shown two hours after RS in the same structures. The increased number of c-fos mRNA-positive cells two hours after TT injection was shown in the posterior hypothalamus area (PHA), LHA, dorsomedial nucleus (DMH), ventromedial nucleus (VMH), and anterior hypothalamus area (AHA) as compared to the effect of i.v. saline injection. Moreover, IL-2 mRNA-positive cell induction was noted in the PHA, DMH, and VMH. Six hours after TT injection, c-fos mRNA expression was decreased in the PHA, LHA, and AHA. Activation of c-fos and IL-2 mRNA was detected in the paraventricularis nucleus 6 hours after TT i.v. injection. Thus, inhibition or stimulation of IL-2 gene expression in lymphoid cells depends on the nature of the stressors. RS or antigenic stimuli induce c-fos and IL-2 gene expression in definite structures of the brain. The dynamics of this process are time dependent. The partial correlation between c-fos and IL-2 mRNA expression in localization in brain structures and time dependence was shown.