Shank3 Deficiency Results in a Reduction in GABAergic Postsynaptic Puncta in the Olfactory Brain Areas.

Dysfunctional sensory systems, including altered olfactory function, have recently been reported in patients with autism spectrum disorder (ASD). Disturbances in olfactory processing can potentially result from gamma-aminobutyric acid (GABA)ergic synaptic abnormalities. The specific molecular mechanism by which GABAergic transmission affects the olfactory system in ASD remains unclear. Therefore, the present study aimed to evaluate selected components of the GABAergic system in olfactory brain regions and primary olfactory neurons isolated from Shank3-deficient (-/-) mice, which are known for their autism-like behavioral phenotype. Shank3 deficiency led to a significant reduction in GEPHYRIN/GABAAR colocalization in the piriform cortex and in primary neurons isolated from the olfactory bulb, while no change of cell morphology was observed. Gene expression analysis revealed a significant reduction in the mRNA levels of GABA transporter 1 in the olfactory bulb and Collybistin in the frontal cortex of the Shank3-/- mice compared to WT mice. A similar trend of reduction was observed in the expression of Somatostatin in the frontal cortex of Shank3-/- mice. The analysis of the expression of other GABAergic neurotransmission markers did not yield statistically significant results. Overall, it appears that Shank3 deficiency leads to changes in GABAergic synapses in the brain regions that are important for olfactory information processing, which may represent basis for understanding functional impairments in autism.

Naja ashei venom induces mitochondria-mediated apoptosis in human colorectal cancer cells.

In the present study, we investigated the antiproliferative activity of Naja ashei full venom (NAV) on human colorectal cancer cells. The NAV-induced antiproliferative effect was associated with cell cycle arrest in S phase and increased number of cells with sub G0/G1 DNA content, which is considered a marker of apoptosis. Apoptosis has also been confirmed with annexin V/PI staining. Furthermore, flow cytometric analysis revealed loss of mitochondrial membrane potential with concomitant increase in cytochrome c and Smac/DIABLO protein content. These effects were associated with the activation of caspase-9 and caspase-3, as well as with PARP cleavage. Moreover, phosphorylation of antiapoptotic Bcl-2 protein in NAV-treated HCT116 was observed. In conclusion, our study for the first time documented antiproliferative/pro-apoptotic effect of NAV in colorectal cancer cells. Our results strongly suggest the involvement of mitochondria in NAV induced apoptosis of cancer cells. Future studies are needed to further examine the potential of NAV in the treatment of colon cancer.

Clozapine impact on c-Fos expression in mild stress preconditioned male rats exposed to a novelty stressor.

Clozapine (CLZ) stimulates several brain areas some of them being sensitive to stress. Aim of the present study was to reveal whether 7-day CLZ administration may: (1) activate the selected forebrain areas; (2) modulate response of these structures to a single forced swimming episode (FSW); (3) modulate response of these structures to FSW after 13-day preconditioning with mild unpredictable stress complex (CMS). Used groups of male Wistar rats: (a) vehicle or CLZ treated for 7 days; (b) vehicle or CLZ treated for 7 days and on the 7th day exposed to FSW; (c) CMS exposed for 13 days, from the 8th day injected with vehicle or CLZ and on the 14th day exposed to FSW. Vehicle or CLZ (10 mg kg-1  day-1 in 0.1% acetic acid) were administered intraperitoneally. c-Fos quantification was performed 90 min after FSW in the medial prefrontal cortex (mPFC), dorsolateral (dLS) and ventrolateral (vLS) septum, dorsolateral (DLStr) and dorsomedial (DMStr) striatum, nucleus accumbens shell (NAc shell) and core (NAc core), and hypothalamic paraventricular nucleus (PVN). In unstressed animals CLZ increased c-Fos expression in the mPFC, vLS, and PVN. After a single FSW, CLZ decreased the number of c-Fos immunoreactive cells in the vLS, DMStr, NAc shell, and NAc core. In CMS rats, CLZ suppressed c-Fos immunoreactivity in response to FSW in the PVN. Our data indicate that CLZ elicits different impact on neuronal activities in the brain areas studied and modifies the response of these structures to stress. CLZ effect seems to be affected by stress duration.

CART (cocaine- and amphetamine-regulated transcript) peptide specific binding sites in PC12 cells have characteristics of CART peptide receptors.

CART (cocaine- and amphetamine-regulated transcript) peptide is a neuropeptide with a powerful central anorexigenic effect. Specific CART peptide binding sites, most likely CART peptide receptors, have been found in PC12 cells. This study further characterizes the CART peptide binding sites in PC12 cells. After differentiation to a neuronal phenotype with nerve growth factor, the number of CART peptide binding sites in PC12 cells tripled. Following dexamethasone treatment, which transforms PC12 cells into chromaffin-like cells, the number of CART peptide binding sites substantially decreased. CART peptide did not affect the differentiation or acetylcholinesterase activity of PC12 cells, indicating that CART peptide does not participate in differentiation or neuronal activity. CART peptide increased the phosphorylation of SAPK/JNK (stress-activated protein kinase/c-Jun-amino-terminal kinase) and subsequent c-Jun protein expression. These effects were reversed by SP600125, a specific JNK-kinase inhibitor. CART peptide did not significantly affect ERK (extracellular signal-regulated kinase), CREB (cAMP responsive element binding protein), or p38 phosphorylation and c-Fos protein expression. Central administration of CART peptide into mice also resulted in increased c-Jun positive cells in dorsomedial hypothalamic nucleus and nucleus of the solitary tract, areas involved in food intake regulation. Activation of c-Jun by CART peptide might indicate a possible role of CART peptide in managing stress conditions rather than a role in cell proliferation or differentiation as well as the more complex and/or specific regulation ways by transcription factors in some nuclei involved in food intake regulation. The characteristics of stress that CART peptide potentially mediates should be further studied.

Effect of anorexinergic peptides, cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART) peptide, on the activity of neurons in hypothalamic structures of C57Bl/6 mice involved in the food intake regulation.

The hypothalamus plays an important role in food consumption, receiving information about energy balance via hormonal, metabolic, and neural inputs. Its neurons produce neuropeptides influencing energy balance. Especially important to regulation of food consumption are certain hypothalamic structures, including the arcuate (ARC) and ventromedial (VMN) nuclei and the lateral hypothalamic area (LHA). We determined the impact of cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART) peptide, on activity of ARC and VMN neurons and hypocretin (Hcrt) synthesizing neurons in LHA. ARC is an integrative nucleus regulating food consumption, VMN is considered to be a satiety centre, and LHA a hunger sensing centre. After overnight fasting, male C57Bl/6 mice received intraperitoneal injection of (i.p.) saline (SAL) or CCK (4microg/kg) or intracerebroventricular injection of (i.c.v.) CART peptide (0.1microg/mice) or CCK (i.p.) followed by CART peptide (i.c.v.) 5min later. Sixty minutes later, the presence of Fos or Fos/Hcrt immunostaining indicated activity of ARC and VMN neurons, as well as of Hcrt cells in LHA. CCK alone did not influence neuronal activity in any of the nuclei studied. CART peptide stimulated neurons in ARC and VMN (p<0.01) but decreased Hcrt neuronal activity in LHA (p<0.05). Co-administration of both peptides synergistically stimulated ARC neurons (p<0.01) and asynergistically inhibited LHA Hcrt neurons (p<0.01). Results indicate that CCK may modify the effect of CART peptide and thus substantially influence activity of neurons in hypothalamic structures involved in regulation of food intake.

Effect of liver ischemia-reperfusion injury on the activity of neurons in the rat brain.

Liver ischemia-reperfusion injury (LIRI) influences different body cells. Little is known about the effect of LIRI on the activity of neurons. Response of neurons to: (1) single ligation of hepatic artery (LIRIa) for 30 min and (2) combined ligation of portal triade (common hepatic artery, portal vein, common bile duct, LIRIb) for 15 min was investigated in Wistar rats. Ninety minutes, 5 h, and 24 h after liver reperfusion, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), interleukin 1alpha (IL-1alpha), and tumor necrosis factor alpha (TNFalpha) serum levels were analyzed and Fos-immunolabeled cells counted in subfornical organ (SFO), suprachiasmatic (SCH), paraventricular (PVN), supraoptic (SON), arcuate (ARC), and ventromedial (VMN) hypothalamic nuclei, locus coeruleus (LC), nucleus of the solitary tract (NTS), and A1/C1 catecholaminergic cell groups. LIRIb increased ALT serum level after 90 min and 24 h while AST activity only after 24 h in all experimental groups. IL-1alpha serum level was increased only after 90 min of LIRIb while TNFalpha level did not change. Ninety minutes after surgeries more Fos-immunostained cells occurred in both LIRIs than sham-operated animals in all structures studied. More distinct Fos expression occurred after LIRIb than LIRIa in SON, PVN, VMN, and NTS. Five hours after both LIRIs, Fos increased in the parabrachial nucleus (PBN) and NTS. Twenty-four hours after both LIRIs Fos incidence decreased in all groups. Although the present data indicate that increased neuronal activity after both LIRIs is mainly a consequence of the liver damage itself partial impact of non-specific factors can not be excluded. However, the anatomical distribution of Fos occurrence detected after LIRIs gives great opportunity to perform a targeted phenotypic identification of the activated neurons by LIRIs in the subsequent experiments.

Alpha2-adrenergic impact on hypothalamic magnocellular oxytocinergic neurons in long evans and brattleboro rats: effects of agonist and antagonists.

We have previously demonstrated that alpha2-adrenoceptors regulate hypothalamic magnocellular oxytocinergic (OXY) neurons in Sprague Dawley rats. Here we investigated whether activation/inhibition of alpha2-adrenoceptors may similarly trigger/downregulate the activity of OXY neurons in control Long Evans (+/+) and permanently osmotically stressed Brattleboro (di/di) rats. The effect of alpha2-adrenoceptor agonist, xylazine (XYL) and alpha2-adrenoceptor antagonists, atipamezole (ATIP), and idazoxan (IDX) were evaluated in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Saline (SAL, 0.1 ml/100 g), XYL (10 mg/kg), ATIP, (1 mg/kg), and IDX (10 mg/kg) and IDX or ATIP followed by XYL were applied intraperitoneally. Rats were sacrificed 90 min later and Fos/OXY co-labelings analyzed in microscope. In control +/+ rats no or few Fos/OXY co-labelings occurred in SON and PVN. XYL significantly increased Fos incidence in OXY neurons in both nuclei. ATIP significantly suppressed the effect of XYL in both nuclei and IDX only in SON. In di/di controls 81% of OXY neurons in SON and 44% in PVN revealed Fos presence and XYL did not further elevate Fos number in SON OXY neurons and slightly increased Fos number in PVN. ATIP or IDX only partially reduced Fos in SAL or XYL treated di/di rats. Our data indicate that: (1) XYL stimulation is not effective in di/di rats because of sustained upregulation of OXY neurons activity and (2) neither ATIP nor IDX reduced significantly the OXY activity in control di/di rats. These findings suggest that alpha2-adrenoceptors have only a limited impact in maintaining OXY cells activity upregulation in PVN and SON of di/di rats.

Fos expression in hypocretinergic neurons in C57B1/6 male and female mice after long-term consumption of high fat diet.

OBJECTIVE: It is generally known that hypocretin (Hcrt) neurons in lateral hypothalamus (LH) are involved in feeding behaviour. The aim of this study was to reveal the activity response of Hcrt neurons, as measured by Fos protein incidence, to prolonged high fat (HF) diet in the LH of both genders of C57B1/6 mice. METHODS: Standard (St) and high fat (HF) diets were available to mice for 16 weeks and thereafter the animals were perfused transcardially with fixative. Then the brains were removed, soaked with 15 % sucrose in 0.1 M phosphate buffer (PB), and cryo-sectioned throughout the hypothalamus into 35 microm thick coronal sections. Fos/Hcrt co-localizations were processed by employing avidin-biotin-peroxidase (ABC) complex and diaminobenzidine chromogen for Hcrt labeling. Fos immunoproduct was intensified by nickel chloride as a black color inducer. Evaluation of the incidence of Fos/Hcrt co-labeled perikarya was performed using a computerized Leica light microscopy. RESULTS: The effect of of mice gender, applied diet, and gender plus applied diet on the activation of Hcrt neurons was found. Turkey s test revealed significant (p<0.05) rise in Fos labeled Hcrt neurons in male vs. female mice after consumption of both types of diets: St (44.64 +/- 2.28 % vs. 1.47 +/- 0.195 %, resp.) and HF (44.15 +/- 3.77 % vs. 24.32 +/- 0.7 %, resp.). This showed that HF diet significantly elevated the number of activated Hcrt neurons only in female mice (24.32 +/- 0.7 % in HF fed vs. 1.47 +/- 0.195 % in St fed, p<0.05). The body weight and accumulation of body fat in animals (body fat weight expressed as % of body weight) were influenced by gender and applied diet, although the body fat weight was influenced by HF diet (more noticeably in females). CONCLUSION: The data indicated a positive correlation between body weight, fat gain, and Hcrt activities in females but not in males, thus accentuating the importance of the gender impact.

Synergistic effect of CART (cocaine- and amphetamine-regulated transcript) peptide and cholecystokinin on food intake regulation in lean mice.

BACKGROUND: CART (cocaine- and amphetamine-regulated transcript) peptide and cholecystokinin (CCK) are neuromodulators involved in feeding behavior. This study is based on previously found synergistic effect of leptin and CCK on food intake and our hypothesis on a co-operation of the CART peptide and CCK in food intake regulation and Fos activation in their common targets, the nucleus tractus solitarii of the brainstem (NTS), the paraventricular nucleus (PVN), and the dorsomedial nucleus (DMH) of the hypothalamus. RESULTS: In fasted C57BL/6 mice, the anorexigenic effect of CART(61-102) in the doses of 0.1 or 0.5 microg/mouse was significantly enhanced by low doses of CCK-8 of 0.4 or 4 microg/kg, while 1 mg/kg dose of CCK-A receptor antagonist devazepide blocked the effect of CART(61-102) on food intake. After simultaneous administration of 0.1 microg/mouse CART(61-102) and of 4 microg/kg of CCK-8, the number of Fos-positive neurons in NTS, PVN, and DMH was significantly higher than after administration of each particular peptide. Besides, CART(61-102) and CCK-8 showed an additive effect on inhibition of the locomotor activity of mice in an open field test. CONCLUSION: The synergistic and long-lasting effect of the CART peptide and CCK on food intake and their additive effect on Fos immunoreactivity in their common targets suggest a co-operative action of CART peptide and CCK which could be related to synergistic effect of leptin on CCK satiety.

Response of substances co-expressed in hypothalamic magnocellular neurons to osmotic challenges in normal and Brattleboro rats.

The intention of this review is to emphasize the current knowledge about the extent and importance of the substances co-localized with magnocellular arginine vasopressin (AVP) and oxytocin (OXY) as potential candidates for the gradual clarification of their actual role in the regulation of hydromineral homeostasis. Maintenance of the body hydromineral balance depends on the coordinated action of principal biologically active compounds, AVP and OXY, synthesized in the hypothalamic supraoptic and paraventricular nuclei. However, on the regulation of water-salt balance, other substances, co-localized with the principal neuropetides, participate. These can be classified as (1) peptides co-localized with AVP or OXY with unambiguous osmotic function, including angiotensin II, apelin, corticotropin releasing hormone, and galanin and (2) peptides co-localized with AVP or OXY with an unknown role in osmotic regulation, including cholecystokinin, chromogranin/secretogranin, dynorphin, endothelin-1, enkephalin, ferritin protein, interleukin 6, kininogen, neurokinin B, neuropeptide Y, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, TAFA5 protein, thyrotropin releasing hormone, tyrosine hydroxylase, and urocortin. In this brief review, also the responses of these substances to different hyperosmotic and hypoosmotic challenges are pointed out. Based on the literature data published recently, the functional implication of the majority of co-localized substances is still better understood in non-osmotic than osmotic functional circuits. Brattleboro strain of rats that does not express functional vasopressin was also included in this review. These animals suffer from chronic hypernatremia and hyperosmolality, accompanied by sustained increase in OXY mRNA in PVN and SON and OXY levels in plasma. They represent an important model of animals with constantly sustained osmolality, which in the future, will be utilizable for revealing the physiological importance of biologically active substances co-expressed with AVP and OXY, involved in the regulation of plasma osmolality.

Activity of oxytocinergic neurons in the supraoptic nucleus under stimulation of alpha2-adrenoceptors in Brattleboro rats.

Vasopressin (AVP) deficient homozygous Brattleboro rats exhibit severe osmotic challenges due to waterless chronic hypernatremia and hyperosmolality. We investigated the effect of xylazine, an alpha2-adrenoceptor agonist, on the activity of oxytocinergic (OXY) neurons in the supraoptic nucleus (SON) of homozygous (di/di), heterozygous (di/+), and control (+/+) rats. Ninety minutes after saline (0.1 mL/100 g b.w., i.p.) or xylazine injection (10 mg/kg, i.p.) rats were anaesthetized with pentobarbital (50 mg/kg i.p.) and sacrificed by transcardial perfusion with fixative. Activity of OXY neurons was evidenced by nuclear Fos protein immunoreactivity. Fos/OXY colabelings were analyzed on 40-mum thick coronal sections using computerized light microscope. As expected, plasma osmolality and water intake revealed high heterogenity within the di/di group of rats. Fos expression in SON of di/di rats was correlated with osmolality of each rat. In saline-treated rats, maximum activation of Fos reached around 4% in +/+, 20% in di/+ rats, and as much as 60% in di/di rats. Xylazine activated in SON about 70% of OXY-ergic neurons in +/+, 60% in di/+ rats, and more than 80% in di/di rats. The present findings indicate that in spite of the high spontaneous activity of SON OXY-ergic neurons due to the AVP deficiency in di/di rats, many of the silent OXY-ergic neurons in the SON remained acceptable for alpha2-adrenoceptor stimulation.

Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat.

Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immunohistochemistry. Zolpidem was administered intraperitoneally in dose 10 mg/kg of BW and 60 min later the animals were sacrificed by transcardial perfusion with fixative. The Fos/OXY co-labelings were analyzed on 40 microm thick serial coronal sections using computerized light microscopy. Zolpidem elicited a concordant Fos/OXY staining in all four PVN sub-areas investigated, including the anterior (15.71+/-2.35%), middle (14.52+/-2.53%), dorsal (13.34+/-2.61%), and periventricular (18.21+/-4.75%) ones, however, had no significant stimulatory effect on OXY cells in the SON. In response to Zolpidem, statistically significant activations were also seen in certain groups of accessory structures including the circular nucleus (13.99+/-3.43%), small clusters of accessory neurons (10.55+/-1.94%), and the lateral hypothalamic perivascular nucleus (9.42+/-2.74%). Between the naive and vehicle controls, the dual Fos/OXY labelings did not elicit any significant differences. Our data provide insight into the topographic patterns of brain activity within the clusters of magnocellular OXY cells in the hypothalamus associated with stimulation of GABA(A) benzodiazepine receptors and for the first time illustrate the triggering contemporaneousness within the cells of the principal and accessory magnocellular nuclei in response to Zolpidem treatment. The present study provides a comparative background that may help in the further understanding of a possible extend of Zolpidem effect on the brain.

Effect of cholecystokinin on feeding is attenuated in monosodium glutamate obese mice.

Treatment of newborn mice with monosodium glutamate (MSG) is neurotoxic for hypothalamic arcuate nucleus (ARC) and causes obesity. In the MSG-treated 16-week-old NMRI mice, we detected specific ablation of ARC neuronal cells, 8 times higher fat to body mass ratio but unchanged body mass compared to controls, advanced hyperglycemia and hyperinsulinemia--both more pronounced in males, and hyperleptinemia--more severe in females. After fasting, the MSG-treated mice showed attenuated food intake compared to controls. Cholecystokinin octapeptide, which decreased food intake in a dose-dependent manner in 24 h fasted controls, did not significantly affect food intake in the MSG-treated animals. We propose that the obesity-related changes in the feeding behavior of the MSG-treated obese mice were the result of missing leptin and insulin receptors in ARC and consequent altered neuropeptide signaling. This makes the MSG model suitable for clarifying generally the central control of food intake.

Fos expression variances in mouse hypothalamus upon physical and osmotic stimuli: co-staining with vasopressin, oxytocin, and tyrosine hydroxylase.

Fos expression in the hypothalamus and its quantification in vasopressinergic (AVP), oxytocinergic (OXY) and tyrosine hydroxylase (TH) immunoreactive cells in the hypothalamic paraventricular (PVN), supraoptic (SON), suprachiasmatic (SCh), and arcuate (Arc) nuclei was performed in response to physiologically two different, i.e. osmotic (i.p. hypertonic saline, HS) and immobilization (IMO), stimuli in mouse using a dual Fos-neuropeptide immunohistochemistry. Both 60 min of HS and 120 min of IMO evoked Fos induction in many hypothalamic structures, whereas, HS evoked more extensive Fos labeling than IMO in the SON, ventromedial (VMN) and dorsomedial (NDM) hypothalamic nuclei and the retrochiasmatic area (RCh). Other hypothalamic structures including the anterior hypothalamic area (AHA), the latero-anterior hypothalamic nucleus (LA), the Arc, the perifornical nucleus (PeF), and the lateral hypothalamic area (LH) showed similar Fos incidence after both HS and IMO. However, after both stimuli explicitly most extensive Fos expression was observed in the PVN. In addition, in the PVN substantially more Fos-AVP (62-67% versus 10-15%) and Fos-OXY (38-45% versus 4-8%) perikarya were observed after HS than IMO, respectively. Incidence of TH-immunoreactive Fos labeled cells in the PVN was also more frequent after HS. In the SON, HS activated more than 50% of AVP and OXY neurons while IMO less than 4%. The number of TH activated neurons in Arc was also higher after HS (11%) than IMO (4%). Lowest number of colocalizations was revealed in the SCh where both HS and IMO activated around 2% of AVP neurons. The present data demonstrate that both HS and IMO are powerful stimuli for the majority of hypothalamic structures displaying considerable topographic similarity in Fos expression suggesting their multifunctional involvement. The quantity and phenotypic differences of activated hypothalamic neurons may speak out for functional dissimilarities in response to HS and IMO.

Prenatal immune challenge affects growth, behavior, and brain dopamine in offspring.

It is known that the development and plasticity of the neuroendocrine system can be affected by many factors, and that adverse events during the prenatal period can result in long-lasting changes in adulthood. This study was aimed at evaluating the possible consequences for offspring from chronic inflammation during pregnancy. Chronic inflammation was simulated by treatment with increasing doses of lipopolysaccharide (LPS) to dams on days 15 through 19 of pregnancy. Attempts were made to prevent possible negative alterations by keeping animals in an enriched environment (EE). Maternal exposure to LPS resulted in a significant reduction of body weight of male offspring during the weaning period. This difference remained until the age of 63 days in controls (C), but not in animals reared in EE. The content of dopamine in the nucleus accumbens was found to be lower in prenatally stressed (PS) adult males. Furthermore, prenatal exposure to maternal immune challenge was associated with lower locomotor activity in elevated plus maze and increased number of skips in the beam-walking test, as observed in female offspring. No differences in ACTH and corticosterone concentrations with regard to prenatal treatment were found; however, both groups kept in EE showed increased levels of corticosterone as well as enlarged adrenal glands. Thus, immune activation during pregnancy may induce long-term changes in brain catecholamines and behavior, but it is not harmful to basal hormone secretion in the offspring.

Hypertonic saline and immobilization induce Fos expression in mouse brain catecholaminergic cell groups: colocalization with tyrosine hydroxylase and neuropeptide Y.

The aim of the present study was to reveal stress-type dependent differences in hindbrain catecholaminergic (CA) cells and parabrachial nuclei (PBN) in the wild-type mouse. Neuronal activities were evaluated based on the incidence of Fos-labeling analyzed 60 min after injection of hypertonic saline (HS; 400 microL, 1.5 M, i.p.) or 120 min of immobilization (IMO) stress. The phenotypic nature of neurons was identified by costaining of Fos with either tyrosine hydroxylase (TH) or the neuropeptide Y (NPY) antibody. Generally, HS elicited broader Fos-staining than IMO. In comparison with IMO, HS induced more extensive Fos activation in the nucleus tractus solitarii-area postrema complex, and in TH- and NPY-positive cells in the A1 and C1 areas. Locus coeruleus (LC) cells displayed similar Fos activation after HS and IMO, and both stimuli also evoked evident TH-Fos colocalizations. Both stimuli also induced TH-Fos costainings in the A5 area. In contrast, IMO failed to activate PBN cells. The data indicate that the activity of TH and NPY hindbrain neurons responds differently to HS and IMO stress, supporting the notion that different stressors have different effects on the activity of autonomic centers.

Fos induction in the rat deep cerebellar and vestibular nuclei following central administration of colchicine: a qualitative and quantitative time-course study.

The present study was conducted to demonstrate Fos expression at four levels (anterior, prefastigial, postfastigial, posterior) of the cerebellar-vestibular nuclear complex in rats exposed to 1, 6, 24, and 48h of colchicine treatment using a light microscopic avidin biotin peroxidase (ABC) immunohistochemistry. Intracerebroventricular administration of colchicine (60microg per 10microl saline) elicited a continuous increase in the number of Fos-positive cells in the main cerebellar (fastigial, interpositus, dentatus) and vestibular (superior, medial, lateral, spinal, Y) nuclei. One and six hours after colchicine treatment, intensive Fos labeling was observed only in the pyriform cortex and the hypothalamic paraventricular nucleus, respectively, and there was no Fos immunolabeling in any of the cerebellar or vestibular structures investigated. On the other hand, moderate number of Fos-positive cells was visible in each of the cerebellar and vestibular nuclei 24h after colchicine treatment. Exposure of the animals to 48h of colchicine treatment induced an additional, more than 50%, rise in the accumulation of Fos-positive profiles in almost all the cerebellar and vestibular nuclei. In addition, at this time-point, a characteristic pattern of Fos distribution appeared almost in all of the cerebellar and vestibular nuclei, however, the numerical incidence of Fos-positive profiles in paired structures along the neuroaxis was bilaterally symmetric. The present data demonstrate for the first time that the central administration of colchicine causes a persistent and, in comparison with other brain areas, time-delayed activation of certain population of neurons in both cerebellar and vestibular nuclei. We assume that the delayed Fos activation in these structures indicate that the cerebellar and vestibular nuclei are not the primary targets of the central effect of colchicine and their activation seems to be rather a result of a postponed functional consequences of the central action of colchicine probably related to the coordination of motor performance.

Detection of oxytocin mRNA in hypertonic saline Fos-activated PVN neurons: comparison of chromogens in dual immunocytochemical and in situ hybridization procedure.

OBJECTIVE: The aim of the present experiments was to elucidate: 1. the stability and usefulness of a 3,3 -diaminobenzidine tetrahydrochloride (DAB) chromogen intensified with nickel and cobalt (DAB-Ni-Co) in the dual immunocytochemical and in situ hybridization procedure using Fos-protein antibody and oxytocin mRNA (OXY mRNA) radiolabeled probe; 2. the susceptibility of the free floating and mounted cryostat sections, freshly prepared or stored for 24 month at -20 degrees C. METHODS: The dual staining procedure was tested on neurons of the hypothalamic paraventricular nucleus (PVN) activated by an intraperitoneal injection of hypertonic saline (HS, 1.5 M, 5 ml, 60 min). Two dual labeling procedures were compared: 1/ Fos-immunostaining with DAB alone and combined with OXY mRNA in situ hybridization and 2/ Fos-immunostaining with DAB-Ni-Co and combined with OXY mRNA in situ hybridization. In both experiments free floating and mounted cryostat sections, freshly prepared or stored for 24 month at -20 degrees C, were tested. RESULTS: HS strongly stimulated both the parvicellular and magnocellular population of PVN neurons followed by an extensive Fos-immunolabeling in many cell nuclei. The first staining sequence with Fos-DAB labeling resulted in a good staining quality on both the fresh and for 24 month stored mounted sections. Although the free floating sections during the in situ procedure showed the same staining properties as the mounted ones, with respect to their increased fragility on the end of the hybridization procedure, they were difficult to mount and stretch on poly-L-lysine coated slides. On the other hand, Fos-immunolabeling with DAB-Ni-Co exhibited improved staining density of the single DAB chromogen in the first staining sequence of the dual staining procedure. However, DAB-Ni-Co mixture showed up as an unstable chromogen complex, which after completing the in situ hybridization process completely disappeared from each type of section. CONCLUSIONS: The results of the present dual immunocytochemical-in situ hybridization staining utilizing Fos-antibody and OXY mRNA oligoprobe indicate that this procedure is applicable on free floating as well as mounted cryostat sections, freshly prepared or stored for 24 month at -20 degrees C. However, the dual procedure is only successful when the immunoproduct in the first sequence is visualized with an unintensified DAB and not with combined DAB-Ni-Co chromogen and when the histological sections in the second sequence are not processed as free floating but are attached to a poly-L-lysine coated microscopic glasses.

Dexamethasone attenuates by colchicine induced Fos expression in the rat deep cerebellar and vestibular nuclei.

1. The intent of the present study was to find out whether dexamethasone pretreatment may affect the induction of Fos protein in cell nuclei of the cerebellar vestibular neuronal complex (CVNC) elicited by central administration of colchicine. Specifically, the rate of the dexamethasone-sensitive cell population was analyzed and compared at different levels of the CVNC using a light microscopic avidin-biotin peroxidase immunohistochemistry. 2. Male Wistar rats were pretreated with dexamethasone 3 days prior (2.5 mg/kg/day, s.c.) and 24 h after an intracerebroventricular delivery of colchicine (60 microg/10 microL). Animals were sacrificed 48 h after colchicine treatment by a transcardial perfusion with fixative. 3. Dexamethasone in itself had no effect on the activity of cells of the CVNC. However, in colchicine treated animals, which exhibited a large number of Fos-positive cells over the entire CVNC, the dexamethasone elicited a substantial reduction in the number of the Fos-immunoreactive cells over the CVNC. Distinct dexamethasone dependent reduction (50-90%) of Fos-immunoreactivity was observed in each of the deep cerebellar nuclei. On the other hand, less number of dexamethasone-sensitive cells were recognized in the vestibular structures. From these, maximal Fos-inhibition by dexamethasone was recognized in the medial vestibular nucleus, however, even in this case the number of suppressed cells did not exceed 50%. 4. The results provide for the first time evidence about the dexamethasone dependent reduction of Fos-immunoreactivity in the cells of the CVNC in response to stimulation elicited by colchicine. The data also indicate that the glucocorticoids might be involved in the regulation of some functions of the CVNC under stress conditions.