High fat diet impact on Fos expression in ovariectomized female C57BL/6 mice: effect of colchicine and response of different neuronal phenotypes.

OBJECTIVES: Activity of neuropeptide Y (NPY), tyrosine hydroxylase (TH), corticoliberine (CRH), and oxytocin (OXY) producing cells was investigated in the ovariectomized (OVX) female C57BL/6 mice kept on the high fat diet for 16 weeks and their response to colchicine stress in selected brain areas, including the hypothalamic paraventricular (PVN), dorsomedial (DMN) and arcuate (ARC) nuclei, A1/C1 (in the ventrolateral medulla), and A2/C2 (in the nucleus of the solitarii tract, NTS) catecholaminergic cell groups. METHODS: The OVX female C57BL/6 mice kept on high fat diet were sacrificed by transcardial perfusion with fixative 48 h after intracerebroventricular injection of colchicine (18 µg mice). Dual Fos/neuropeptide immunohistochemistry was employed to investigate Fos/neuropeptide colocalizations. RESULTS: In the OVX saline-treated mice (sham control) with standard diet (St diet), no immunopositive CRH and NPY neurons were identified in the PVN and weak Fos immunostainig was visible in TH neurons in the DMN and ARC nuclei. Colchicine treatment in the OVX mice with St diet increased the number of CRH and OXY immunopositive neurons in the PVN as well as the number of NPY and TH neurons in DMN and ARC nuclei and NPY neurons in the middle NTS (mNTS) and A1/C1 cell group. Prolonged HF diet in OVX sham control mice moderately increased the number of Fos/TH neurons in the mNTS and commissural NTS (cNTS) in comparison with St diet mice. However, prolonged HF diet in OVX colchicines-treated mice reduced the number of Fos/NPY neurons in the anterior NTS (aNTS) and A1/C1 cell group in comparison with colchicines-treated animals with St diet as well as Fos-TH neurons in the mNTS and cNTS in comparison with saline-treated animals with HF diet. CONCLUSION: The data of this pilot study indicate that prolonged high fat diet might: 1) represent itself a light/moderate stimulus for activation of TH neurons in the NTS and A1/C1 cell group as well as NPY neurons in the A1/C1 cell group and 2) interfere with colchicines-induced and time-delayed Fos activation in the NPY and TH neurons in both the above mentioned brain nuclei.

Ghrelin agonists impact on Fos protein expression in brain areas related to food intake regulation in male C57BL/6 mice.

Many peripheral substances, including ghrelin, induce neuronal activation in the brain. In the present study, we compared the effect of subcutaneously administered ghrelin and its three stable agonists: Dpr(3)ghr ([Dpr(N-octanoyl)(3)] ghrelin) (Dpr - diaminopropionic acid), YA GHRP-6 (H-Tyr-Ala-His-DTrp-Ala-Trp-DPhe-Lys-NH(2)), and JMV1843 (H-Aib-DTrp-D-gTrp-CHO) on the Fos expression in food intake-responsive brain areas such as the hypothalamic paraventricular (PVN) and arcuate (ARC) nuclei, the nucleus of the solitary tract (NTS), and area postrema (AP) in male C57BL/6 mice. Immunohistochemical analysis showed that acute subcutaneous dose of each substance (5mg/kg b.w.), which induced a significant food intake increase, elevated Fos protein expression in all brain areas studied. Likewise ghrelin, each agonist tested induced distinct Fos expression overall the PVN. In the ARC, ghrelin and its agonists specifically activated similarly distributed neurons. Fos occurrence extended from the anterior (aARC) to middle (mARC) ARC region. In the latter part of the ARC, the Fos profiles were localized bilaterally, especially in the ventromedial portions of the nucleus. In the NTS, all substances tested also significantly increased the number of Fos profiles in neurons, which also revealed specific location, i.e., in the NTS dorsomedial subnucleus (dmNTS) and the area subpostrema (AsP). In addition, cells located nearby the NTS, in the AP, also revealed a significant increase in number of Fos-activated cells. These results demonstrate for the first time that ghrelin agonists, regardless of their different chemical nature, have a significant and similar activating impact on specific groups of neurons that can be a part of the circuits involved in the food intake regulation. Therefore there is a real potency for ghrelin agonists to treat cachexia and food intake disorders. Thus, likewise JMV1843, the other ghrelin agonists represent substances that might be involved in trials for clinical purposes.

Chemical sympathectomy suppresses fibrosarcoma development and improves survival of tumor-bearing rats.

Both experimental and clinical data indicate that the sympathetic nervous system may affect the development of certain tumors. To test this, in the present study we combined in vivo and in vitro approaches to study the effect of the sympathetic nervous system on proliferation of BP6-TU2 fibrosarcoma cells. First, we investigated the effect of 6-hydroxydopamine-induced sympathectomy on tumor development and survival of tumor-bearing rats. One week after chemical sympathectomy, we injected the BP6-TU2 fibrosarcoma cells intraperitoneally into male Wistar rats. The sympathectomy significantly reduced the incidence of intraperitoneal tumors and resulted in significantly improved survival of tumor-bearing rats compared to those with intact sympathetic innervation. Using immunohistochemical methods, we found neuron-specific enolase immunopositive structures within fibrosarcoma tissue, indicating innervation of tumors. Finally, an in vitro study showed elevated proliferation of BP6-TU2 fibrosarcoma cells in response to adding norepinephrine to the culture medium. Our findings indicate that sympathetic nerves directly potentiate the proliferation of BP6-TU2 fibrosarcoma cells in rats.

Brain-liver interactions during liver ischemia reperfusion injury: a minireview.

KEYWORDS: The liver is a vital organ, with a wide range of functions. This organ plays an important role in the metabolism, including the glycogen storage, decomposition of red blood cells, plasma protein synthesis, hormone production, and detoxification. The liver is innervated by sympathetic and parasympathetic nerves which are involved in the regulation of the hepatic metabolism. Tissue injury connected with ischemia and reperfusion has been implicated in several clinical settings, including myocardial infarction, brain ischemia, and organ transplantation. Consequences of the liver ischemia reperfusion injury (LIRI) induce first of all an organ failure and afterwards multiorgan system damages that may eventually lead to a death. Many models with an attempt to reduce harmful consequences of the LIRI, directing to develop a variety of prophylactic strategies, has been introduced including models of warm, cold or normothermic ischemia, ischemic pre- and post-conditionings, pharmacological interventions, etc. In spite of the improvements in the medical care and accumulation of a large amount of experimental data concerning the prevention of ischemia and reperfusion related injuries, many destructive processes explanation still remains problematic.

Activation of different neuronal phenotypes in the rat brain induced by liver ischemia­reperfusion injury: dual Fos/neuropeptide immunohistochemistry.

The aim of the present study was to reveal the effect of liver ischemia­reperfusion injury (LIRI) on the activity of selected neuronal phenotypes in rat brain by applying dual Fos-oxytocin (OXY), vasopressin (AVP), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), corticoliberine (CRH), and neuropeptide Y (NPY) immunohistochemistry. Two liver ischemia­reperfusion models were investigated: (i) single ligation of the hepatic artery (LIRIa) for 30 min and (ii) combined ligation of the portal triad (the common hepatic artery, portal vein, and common bile duct) (LIRIb) for 15 min. The animals were killed 90 min, 5 h, and 24 h after reperfusion. Intact and sham operated rats served as controls. As indicated by semiquantitative estimation, increases in the number of Fos-positive cells mainly occurred 90 min after both liver reperfusion injuries, including activation of AVP and OXY perikarya in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and TH, NPY, and PNMT perikarya in the catecholaminergic ventrolateral medullar A1/C1 area. Moreover, only PNMT perikarya located in the A1/C1 cell group exhibited increased Fos expression 5 h after LIRIb reperfusion. No or very low Fos expression was found 24 h after reperfusion in neuronal phenotypes studied. Our results show that both models of the LIRI activate, almost by the same effectiveness, a number of different neuronal phenotypes which stimulation may be associated with a complex of physiological responses induced by (1) surgery (NPY, TH, PNMT), (2) hemodynamic changes (AVP, OXY, TH, PNMT), (3) inflammation evoked by ischemia and subsequent reperfusion (TH), and (4) glucoprivation induced by fasting (NPY, PNMT, TH). All these events may contribute by different strength to the development of pathological alterations occurring during the liver ischemia­reperfusion injury.

The α2-adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons ion the Long Evans and Brattleboro rats.

The hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei are activated by body salt-fluid variations. Stimulation of alpha(2)-adrenoceptors by an agonist-xylazine (XYL) activates oxytocinergic but not vasopressinergic magnocellular neurons. In this study, tyrosine hydroxylase (TH), corticoliberine (CRH), and neuropeptide Y(NPY) magnocellular phenotypes, were analysed in response to alpha(2)-adrenoceptor manipulations and sustained hyperosmolality in vasopressin deficient homozygous Brattleboro (di/di) rats. Saline (0.9% NaCl, 0.1 ml/100g/bw), XYL (10 mg/kg/bw), atipamezole (ATIP, alpha(2)-adrenoceptors antagonist, 1 mg/kg/bw), and ATIP 5 min later followed by XYL, were applied intraperitoneally. Presence of immunolabeled Fos peptide signalized the neuronal activity. Ninety minutes after injections, the rats were anesthesized and sacrificed by transcardial perfusion with fixative. Coronal sections of 30 mum thickness double immunolabeled with Fos/neuropeptide were evaluated under light microscope. Under basal conditions, di/di in comparison with control Long Evans rats, displayed significantly higher number of TH, CRH, and NPY immunoreactive neurons in the SON and PVN (except NPY cells in PVN) and more than 90%, 75%, and 86% of TH, NPY, and CRH neurons, respectively, displayed also Fos signal in the SON. XYL did not further increase the number of Fos in the PVN and SON and ATIP failed to reduce the stimulatory effect of hypertonic saline in all neuronal phenotypes studied. Our data indicate that hyperosmotic conditions significantly influence the activity of TH, CRH, and NPY magnocellular neuronal phenotypes, but alpha(2)-adrenoceptors do not play substantial role in their regulation during osmotic challenge induced by AVP deficiency.

Fos expression in tyrosine hydroxylase containing hypothalamic neurons in CRH-KO mice: effect of immobilization stress.

OBJECTIVE: Little is known about the response of tyrosine hydroxylase (TH) containing hypothalamic neurons to stress in corticoliberine deficient (CRH-KO) mice. This study was aimed to extend this issue and reveal the data leading to a better understanding of physiological/anatomical plasticity of hypothalamic TH cells in response to acute immobilization stress (IMO) as well as of possible of CRH body deficiency contribution in the regulation of TH cells during stress. We examined the topographic distribution of TH protein immunolabeled perikarya in selected hypothalamic structures including the paraventricular (PVN), supraoptic (SON), periventricular (PeVN), arcuate (ArcN), dorsomedial (DMN), and ventromedial (VMN) nuclei and extrahypothalamic zona incerta (ZI) in CRH-KO and wild type (WT) mice. METHODS: The animals were perfused with fixative 120 min after a single IMO stress. The brains were removed, cryo-sectioned throughout the hypothalamus and Fos-TH co-localizations were processed immunohistochemically. Fos protein was visualized by diaminobenzidine (DAB) intensified with nickel ammonium sulphate, while TH cells were labeled only with DAB chromogen. The evaluation of Fos-TH co-labeled perikarya was performed with the use of computerized Leica light microscope and expressed as the percentage of total amount of TH labeled cells. RESULTS: From the qualitative point of view, the present data indicate similar anatomical distribution of TH immunoreactive perikarya in all brain structures investigated in both WT and CRH-KO mice, while from the quantitative point of view only TH cells in the DMN of CRH-KO mice showed a trend for increased activation by IMO. CONCLUSIONS: In several hypothalamic structures the basic population of TH neurons was not affected by the absence of endogenous CRH. Based on the data of this study it can also be assumed that despite of the presence of direct reciprocal connections between PVN and DMN neurons, PVN CRH neurons possibly are not participating in the regulation of TH neurons in the DMN during IMO stress. KEYWORDS: Hypothalamic nuclei - Fos-immunohistochemistry - Tyrosine hydroxylase - Immobilization stress - CRH knockout mice.

Different antipsychotics elicit different effects on magnocellular oxytocinergic and vasopressinergic neurons as revealed by Fos immunohistochemistry.

Acute administration of antipsychotics elicits regionally distinct patterns of Fos expression in the rat brain. Stimulation of oxytocin (OXY) and vasopressin (AVP) release in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei indicates that antipsychotics may play a role in autonomic, neuroendocrine, and behavioral processes. This study was focused to reveal the responsiveness of hypothalamic OXY- and AVP- producing magnocellular neurons, in terms of quantitative and topographical distinctions, to antipsychotics displaying different pharmacological profiles. Naive male Wistar rats were injected intraperitoneally with haloperidol (1 mg/kg), clozapine (30 mg/kg), olanzapine (30 mg/kg), risperidone (2mg/kg), and vehicle (5% chremophor) and were sacrificed 60 min later by a fixative. Fos, Fos/OXY, and Fos/AVP labelings were visualized by immunohistochemistry in the SON, 5 accessory (ACS) cell groups, and 4 distinct PVN subdivisions using a computerized light microscope. Most apparent activation of single Fos, Fos/OXY, and Fos/AVP cells was induced by clozapine and olanzapine; effects of risperidone and haloperidol were substantially lower; no colocalizations were revealed in naive or vehicle treated control rats. The data indicate the existence of a substantial diversity in the stimulatory effect of the selected antipsychotics on quantity of Fos, Fos/OXY, and Fos/AVP immunostainings with the preferential action of the atypicals clozapine over olanzapine and little effects of risperidone and haloperidol. Variabilities in Fos distribution in the PVN, SON, and ACS induced by antipsychotics may be helpful to understand more precisely the extent of their extra-forebrain actions with possible presumption of their functional impact and side effect consequences.

Response of hypothalamic oxytocinergic neurons to immobilization stress is not dependent on the presence of corticotrophin releasing hormone (CRH): a CRH knock-out mouse study.

This study explores the quantitative patterns of immunolabeled Fos protein incidence in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) oxytocinergic (OXY) neurons in response to immobilization (IMO) stress in corticotrophin releasing hormone deficient (CRH-KO) mice. Adult male mice, taken directly from cages or 120 min after a single IMO, were sacrificed by intracardial perfusion with fixative. Coronal brain sections of 30 mum thickness were processed for dual Fos/OXY immunohistochemistry. In control wild type (WT) and CRH-KO mice, scattered Fos immunoreactivity was observed in hypothalamus, including the PVN where scanty Fos signal occurred in both parvocellular and magnocellular PVN subdivisions. Dual Fos/OXY immunostainings revealed higher basal Fos expression in the PVN of control CRH-KO mice. IMO evoked a marked rise in Fos expression in OXY neurons of the PVN and SON in both WT and CRH-KO groups of mice. The present data demonstrate that 1/ CRH deficiency upregulates the basal activity of hypothalamic PVN OXY cells in CRH-KO mice and 2/ IMO stress in both WT and CRH-KO mice affects distinctly the activity of OXY cells in both SON and PVN. Our data indicate that CRH deficiency does not alter the responsiveness of PVN and SON OXY cells to IMO stress.