c-Fos IR Research Articles

Chronic Intermittent Sucrose Consumption Facilitates the Ability to Discriminate Opioid Receptor Blockade with Naltrexone in Rats.

The opioid antagonist naltrexone (NTX) decreases intake of preferred diets in rats at very low doses relative to doses needed to decrease intake of “bland” laboratory chow. In the absence of an opioid agonist, NTX is not discriminable using operant techniques. In the current study, we found that rats given intermittent access to a 25% sucrose solution learned to discriminate between various naltrexone doses and saline. None of the rats given only water learned to discriminate between naltrexone and saline. When access to the sucrose solution was discontinued for 14 days, the rats lost the ability to discriminate between NTX and saline. We also studied the changes of c-Fos IR in selected brain regions in rats treated with saline versus NTX that were drinking water or 25% sucrose. An injection of NTX or saline resulted in a significant drug, diet, and interaction effect in various brain regions associated with feeding behavior, particularly the amygdala, accumbens, and hypothalamic sites. Thus, we found that ingestion of a sucrose solution results in the ability of rats to reliably discriminate naltrexone administration. In addition, sucrose and naltrexone altered c-Fos IR in an interactive fashion in brain regions known to be involved in ingestion behavior.

Mild Hypophagia and Associated Changes in Feeding-Related Gene Expression and c-Fos Immunoreactivity in Adult Male Rats with Sodium Valproate-Induced Autism.

A core yet understudied symptom of autism is aberrant eating behaviour, including extremely narrow food preferences. Autistic individuals often refuse to eat despite hunger unless preferred food is given. We hypothesised that, apart from aberrant preference, underfeeding stems from abnormal hunger processing. Utilising an adult male VPA rat, a model of autism, we examined intake of ‘bland’ chow in animals maintained on this diet continuously, eating this food after fasting and after both food and water deprivation. We assessed body weight in adulthood to determine whether lower feeding led to slower growth. Since food intake is highly regulated by brain processes, we looked into the activation (c-Fos immunoreactivity) of central sites controlling appetite in animals subjected to food deprivation vs. fed ad libitum. Expression of genes involved in food intake in the hypothalamus and brain stem, regions responsible for energy balance, was measured in deprived vs. sated animals. We performed our analyses on VPAs and age-matched healthy controls. We found that VPAs ate less of the ‘bland’ chow when fed ad libitum and after deprivation than controls did. Their body weight increased more slowly than that of controls when maintained on the ‘bland’ food. While hungry controls had lower c-Fos IR in key feeding-related areas than their ad libitum-fed counterparts, in hungry VPAs c-Fos was unchanged or elevated compared to the fed ones. The lack of changes in expression of feeding-related genes upon deprivation in VPAs was in contrast to several transcripts affected by fasting in healthy controls. We conclude that hunger processing is dysregulated in the VPA rat.

Galanin(1-15) Potentiates the Antidepressant-like Effects Induced by Escitalopram in a Rat Model of Depression.

Selective 5-HT reuptake inhibitor antidepressants (SSRIs) are the first choice in major depressive disorder (MDD), but 50% of affected patients do not show improvement. Galanin(1-15) [GAL(1-15)] enhanced Fluoxetine antidepressant-like effects in an animal model of depression, the olfactory bulbectomy (OBX); however, further detailed analysis of GAL(1-15) effects as augmentation treatment in OBX rats are needed. In OBX rats, we analysed the effect of GAL(1–15) on Escitalopram (ESC)-mediated responses in behavioural tests related to despair. We studied whether GAL(1–15) effects involved 5-HT1AR using an in vivo model siRNA 5-HT1A knockdown rats. Moreover, we analysed by immunohistochemistry the expression of the immediate-early gene c-Fos (c-Fos IR) after the administration of GAL(1-15)+ESC in OBX rats in several nuclei involved in MDD. GAL(1-15) enhances the antidepressant-like effects of ESC, and the GALR2 antagonist M871 blocked GAL(1-15) mediated actions. The downregulation of 5-HT1AR by siRNA was sufficient to block GAL(1-15) effects. Our immunohistochemistry and principal component analysis (PCA) analysis suggest that two functional networks are involved in these effects; one includes the lateral (LHb) and medial (mHb) habenula, dorsal raphe (DR) and ventral tegmental area (VTA), and the other consists of the dentate gyrus (DG), and prefrontal cortex (PFC). The results open up the possibility of using GAL(1-15) in combination with SSRIs as a novel strategy for treating MDD.

Fructus Viticis methanolic extract attenuates trigeminal hyperalgesia in migraine by regulating injury signal transmission.

Migraine, characterized by hyperalgesia of the trigeminovascular system, is a severe condition that leads to severe reductions in the quality of life. Upon external stimulation, the levels of various neurotransmitters, including aspartic acid (Asp), glutamic acid (Glu), γ-amino butyric acid (GABA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), are significantly altered; this directly or indirectly promotes trigeminal hypersensitivity. Fructus Viticis is a Traditional Chinese Medicine with analgesic properties to provide efficient relief of migraine. In the present study, the underlying mechanisms of the analgesic effect of Fructus Viticis methanolic extract were assessed in rats with nitroglycerin-induced migraine. The plasma levels of the neurotransmitters calcitonin gene-related peptide (CGRP) and substance P (SP), as well as the amount of c-fos immunoreactive cells (c-fos IR cells) in the brain, were detected. The analgesic effect was obvious, as Fructus Viticis methanolic extract ameliorated migraine-like behaviours in nitroglycerin-induced rats. The levels of 5-HT, GABA and NE in the brain of migraine model rats was lower compared with that of control rats, whereas opposite observations were made in the contents of excitatory amino acids. Pre-treatment with Fructus Viticis methanolic extract elevated the levels of 5-HT, GABA and NE, and also lowered the levels of excitatory amino acids, including Glu and Asp. In addition, treatment with Fructus Viticis methanolic extract lowered the plasma levels of CGRP and SP and decreased the c-fos IR cells in the brainstem. The present study provided a further scientific basis for the anti-migraine effects of Fructus Viticis.

Orchiectomy increases food intake and perifornical orexin A neuron's activity during glucoprivation

We examined the effect of orchiectomy on food intake and lateral hypothalamic neuron fs activity during glucoprivation induced by i.v. 2‐deoxy‐D‐glucose (2DG) administration in male rats. Rats (7W) were bilaterally orchiectomized (OrX) or sham‐operated (Sham). 2DG (400 mg/kg) was infused two weeks after the operation and food intake was measured for 3 hours after the injection. The same experiment was performed for immunohistochemical examination of c‐Fos and orexin A expression in the perifornical area (PFA) and arcuate nucleus (Arc). Glucoprivation induced food intake, and food intake in OrX rats was larger than Sham rats, and food intake was inversely correlated with plasma testosterone concentration. Glucoprivation stimulated c‐Fos expression in the orexin A neurons at the PFA, and c‐Fos expression in the Arc. The number and fraction of c‐Fos expressed orexin A neurons was significantly higher in OrX rats than Sham rats. The number of c‐Fos ir cells in the Arc was also higher in OrX rats than Sham rats. These data indicate that testosterone attenuates food intake induced by reduced glucose availability via attenuated perifornical orexin A neuron's activity and Arc neuron's activity.

Abstract 201: Direct but not Indirect Activation of Sympathetic Ganglia Leads to Higher Neuronal Activation in Normotensive Than Deoxycorticosterone-salt Hypertensive Rats

Sympathetic activity is chronically elevated in hypertensive human patients and in many animal models of hypertension, including the deoxycorticosterone (DOCA)-salt rat model. Sympathetic ganglia receive signals from the central nervous system and relay them to blood vessels and peripheral organs. How sympathetic ganglia respond to activation signals differently in hypertension is not fully understood. We examined how direct and indirect activators of sympathetic ganglia would influence neuronal activation. 2-deoxy-D-glucose (2-DG) is a glucose analog that causes hypoglycemia leading to centrally-mediated, indirect activation of celiac ganglia, a prevertebral sympathetic ganglion. Nicotine directly activates sympathetic ganglia by acting on cholinergic receptors found on most sympathetic neurons. Acute neuronal activation was examined by measuring c-fos immunoreactivity (ir). We hypothesized that since there is higher chronic sympathetic activity in DOCA-salt hypertensive than normotensive rats, both direct and indirect acute activators will induce higher c-fos expression in sympathetic ganglia of normotensive than hypertensive rats. Maximal c-fos expression for 2-DG (800mg/kg i.p.) and nicotine (2mg/kg s.c.) treatments was observed 2hrs post-treatment and for all following experiments, celiac ganglia were dissected and fixed 2hrs post-treatment. Following nicotine treatment, c-fos expression was significantly higher in celiac ganglia of normotensive (25.5 ± 9.7 c-fos ir neurons/m^2) than hypertensive (12.0 ± 4.8 c-fos ir neurons/m^2) rats. However, following 2-DG treatment, c-fos expression was similar in celiac ganglia of normotensive (49.1 ± 17.6 c-fos ir neurons/m^2) and hypertensive (49.4 ± 16.0 c-fos ir neurons/m^2). In contrast with our hypothesis, only the direct but not the indirect activation of sympathetic ganglia induced higher sympathetic neuronal activation in normotensive than hypertensive rats. Thus, a centrally-mediated metabolic stimulus does not activate pathways to sympathetic ganglia differentially in normotension versus hypertension. However, direct ganglionic stimulation leads to greater neuronal activation in normotensive than hypertensive ganglia.

Neuroanatomical evidence demonstrating the existence of the vagal anti‐inflammatory reflex in the intestine

The cholinergic anti-inflammatory pathway is proposed to be part of the so-called vago-vagal 'inflammatory reflex'. The aim of this study is to provide neuro-anatomical evidence to support the existence of a functional neuronal circuit and its activation in response to intestinal inflammation. The expression of c-fos was evaluated at different levels of the neurocircuitry in the course of postoperative ileus (POI) in a mouse model. Specific activation of the motor neurons innervating the inflamed intestine and the spleen was monitored by retrograde tracing using cholera toxin-b. The role of the vagal afferent pathway nerve was evaluated by selective vagal denervation of the intestine. Abdominal surgery resulted in subtle inflammation of the manipulated intestine at 24 h (late phase), but not after 2 and 6 h (early) after surgery. This local inflammation was associated with activation of neurons in the nucleus of the solitary tract and in the dorsal nucleus of the vagus. The vagal output mainly targeted the inflamed zone: 42% of motor neurons innervating the intestine expressed c-fos IR in contrast to 7% of those innervating the spleen. Vagal denervation of the intestine abolished c-fos expression in the brain nuclei involved in the neuronal network activated by intestinal inflammation. Our data demonstrate that intestinal inflammation triggers a vagally mediated circuit leading mainly to activation of vagal motor neurons connected to the inflamed intestine. These findings for the first time provide neuro-anatomical evidence for the existence of the endogenous 'inflammatory reflex' and its activation during inflammation.

Distribution of c-Fos immunoreactivity in the rat brain following abuse-like toluene vapor inhalation

Inhalation of vapors from toluene-containing products results in euphoria accompanied by a variety of cognitive impairments and motor dysfunctions. The profound behavioral changes observed during and following toluene inhalation suggest changes in the activity of cells in potentially many brain regions; however, a comprehensive assessment of the neuroanatomical structures activated by toluene vapor has not been completed. Thus in the present study we systematically mapped in over 140 brain structures the distribution of c-Fos immunoreactivity (c-Fos IR), a proxy for neural activation, following exposure to an abuse-like concentration (~5000ppm) of toluene vapor for 0, 5, 10 or 30min. Quantitative analyses revealed increases in c-Fos IR in about one-third of the brain structures examined, with most of these structures significantly activated only after prolonged toluene exposure. The majority of brain structures activated by toluene were found in the forebrain and midbrain, with particularly pronounced activation in nuclei implicated in the processing of rewarding, emotional, and olfactory stimuli, and those controlling motor output. These structures included the ventral tegmental area, nucleus accumbens, select regions of the amygdala and hypothalamus, cingulate cortex, olfactory nuclei, piriform cortex, secondary motor cortex and caudate-putamen. In contrast, all subregions of the hippocampus and most thalamic nuclei were not significantly activated by toluene vapor. In the brainstem, effects of toluene vapor were restricted to select nuclei in the pons. The pattern of c-Fos IR evoked by inhalation of toluene vapor appears distinct from other psychoactive substances, consistent with the unique and complex behavioral outcomes associated with acute toluene inhalation.

Estrogen attenuates food intake and perifornical orexinergic neuron′s activity during glucoprivation in ovariectomized rats

We examined the effect of estrogen (E2) replacement in ovariectomized rats on food intake and lateral hypothalamic neuron′s activity during glucoprivation induced by i.v. 2‐deoxy‐D‐glucose (2DG) administration. Rats were ovariectomized and implanted E2 or vehicle (cholesterol) subcutaneously. Two weeks after the replacement, rats were infused with 2DG (400 mg/kg) and food intake was measured for 3 hours. The same experiment was performed for immunohistochemical examination of c‐Fos, melanin concentrating hormone (MCH), and orexin A expression in the lateral hypothalamic area (LH) and arcuate nucleus (Arc). Glucoprivation induced c‐Fos expression in the orexin A neurons locating at the perifornical region of LH, but did not activate MCH neurons. Glucoprivation significantly induced food intake, and food intake in E2 replaced group was significantly lower than E2 deficit group. The number and fraction of c‐Fos expressed orexinergic neurons was significantly lower in E2 replaced group than E2 deficit group. The number of c‐Fos ir cells was less in E2 replaced group than E2 deficit rats. These data indicate that E2‐induced reduction of food intake induced by 2DG is possibly due to the reduced perifornical orexinergic neuron′s activity and to the reduced Arc neuron′s activity.

Comparison of nociceptive behavior in prostaglandin E, F, D, prostacyclin and thromboxane receptor knockout mice

Antagonist at specific prostaglandin receptors might provide analgesia with a more favourable toxicity profile compared with cyclooxygenase inhibitors. We analyzed nociceptive responses in prostaglandin D, E, F, prostacyclin and thromboxane receptor knockout mice and mice deficient of cyclooxygenase 1 or 2 to evaluate the contribution of individual prostaglandin receptors for heat, mechanical and formalin-evoked pain. None of the knockouts was uniformly protected from all of these pain stimuli but COX-1 and EP4 receptor knockouts presented with reduced heat pain and EP3 receptor and COX-2 knockout mice had reduced licking responses in the 2nd phase of the formalin assay. This was accompanied with reduced c-Fos immunoreactivity in the spinal cord dorsal horn in EP3 knockouts. Oppositely, heat pain sensitivity was increased in FP, EP1 and EP1 + 3 double mutant mice possibly due to a loss of FP or EP1 receptor mediated central control of thermal pain sensitivity. Deficiency of either EP2 or DP1 was associated with increased formalin-evoked flinching responses and c-Fos IR in dorsal horn neurons suggesting facilitated spinal cord pain reflex circuity. Thromboxane and prostacyclin receptor knockout mice showed normal pain behavior in all tests. The results suggest a differential, pain-stimulus and site-specific contribution of specific PG-receptors for the processing of the nociceptive stimuli, a differential modulation of nociceptive responses by COX-1 and COX-2 derived prostaglandins and compensatory and/or developmental adaptations in mice lacking specific PG receptors.

Region specific galanin receptor/neuropeptide Y Y1 receptor interactions in the tel- and diencephalon of the rat. Relevance for food consumption

The aim of this work was to determine the interactions between NPY and GAL receptor (GALR) subtypes in the hypothalamus and the amygdala using quantitative receptor autoradiography to analyze the binding characteristics of NPY-Y1 and Y2 receptor subtypes in the presence and absence of GAL. Food intake in satiated animals was evaluated after intraventricular coinjections of GAL and NPY-Y1 or Y2 agonists. The expression of c-Fos IR in both regions was also investigated. GAL decreases NPY-Y1 agonist binding in the arcuate nucleus by about 15% ( p < 0.01), but increases NPY-Y1 agonist binding in amygdala (18%) ( p < 0.01). These effects were blocked with the GAL antagonist M35. Y2-agonist binding was not modified by GAL. GAL blocked the food intake induced by the Y1 agonist ( p < 0.01). Coinjections of Y1 agonist and GAL also reduced the c-Fos expression induced by the Y1 agonist in the arcuate nucleus and the dorsomedial hypothalamic nucleus but increased c-Fos expression in amygdala. These results indicate the existence of antagonistic interactions between GALR and NPY-Y1 receptors in the hypothalamus and their functional relevance for food intake. In contrast, a facilitatory interaction between GALR and Y1 receptors exists in the amygdala which may be of relevance for fear related behaviour.

Effect of opioid receptor ligands injected into the rostral lateral hypothalamus on c- fos and feeding behavior

The lateral hypothalamic area (LHa) is an important brain site for the regulation of food intake. Central injection of opioids increases food intake, and the LHa contains mu and kappa opioid receptors, both of which are involved in feeding behavior. It is unclear whether opioids impact feeding when injected directly into the rostral portion of the LHa (rLHa) in rats. We performed a series of studies in which free-feeding rLHa-cannulated rats were injected with opioid agonists (DAMGO, morphine, dynorphin, U-50488H) followed by the measurement of food intake at 1, 2, and 4 h postinjection. To determine whether opioid receptor ligands administered into the rLHa affect neuronal activation in this brain site, we studied cFos immunoreactivity (cFos IR) in response to rLHa stimulation with naltrexone. We found that the only compound that stimulated feeding behavior was morphine. The other agonists had no effect on food consumption. Naltrexone injection into the rLHa increased neural activation in the LHa, indicating the presence of functional opioid receptors in this region. These data suggest that although neuronal activity is affected by opioid agents acting in the rLHa, administration of selective mu and kappa opioid ligands in this subdivision of the LHa does not have a reliable effect on feeding behavior.

Essential role of the synaptic vesicle protein synapsin II in formalin-induced hyperalgesia and glutamate release in the spinal cord

The synaptic vesicle protein synapsin II plays an important role in the regulation of neurotransmitter release and synaptic plasticity. Here, we investigated its involvement in the synaptic transmission of nociceptive signals in the spinal cord and the development of pain hypersensitivity. We show that synapsin II is predominantly expressed in terminals and neuronal fibers in superficial laminae of the dorsal horn (laminae I–II). Formalin injection into a mouse hindpaw normally causes an immediate and strong release of glutamate in the dorsal horn. In synapsin II deficient mice this glutamate release is almost completely missing. This is associated with reduced nociceptive behavior in the formalin test and in the zymosan-induced paw inflammation model. In addition, the formalin evoked increase in the number of c-Fos IR neurons is significantly reduced in synapsin II knockout mice. Touch perception and motor coordination, however, are normal indicating that synapsin II deficiency does not generally disrupt sensory and/or motor functions. Antisense-mediated transient knockdown of synapsin II in the spinal cord of adult animals also reduced the nociceptive behavior. As the antisense effect is independent of a potential role of synapsin II during development we suggest that the hypoalgesia in synapsin II deficient mice does involve a direct ‘pain-facilitating’ effect of synapsin II and is not essentially dependent on potentially occurring developmental alterations. The distinctive role of synapsin II for pain signaling probably results from its specific localization and possibly from a specific control of glutamate release.

Role of vanilloid receptor 1 (VR1) in increased nociceptive signaling in experimental pancreatitis

Background/Aims: Vanilloid receptor 1 (VR1) plays a fundamental role in the transduction of pain due to peripheral tissue injury/inflammation. The aim of our study was to determine its role in visceral pain in a rat model of pancreetitis by measuring spinal cord c-fos as a functional marker. Methods: Pancreatitis was induced with two i.p. injections of L-arginine; control animals received saline and experiments were performed 5 days post-injection. Afferent responses were quantified by counting the number of c-fos immunoreactive neurons in spinal cord segments T8-TlO two hours after intraductal (i.d.) infusion of capsaicin (0.5 mg/kg), acid(pH 6) or vehicle with or without pre-treatment with the VR1 antagonist capsazepine (i.d.,O.5 mg/kg). To determine whether the number of pancreatic afferents expressing VR1 was increased in pancreatitis, the number of neurons in dorsal root ganglia T9-T11 positive for the retrograde label Dil and VR1 in pancreatitis and control rats was compared. Results: C-fos IR was significantly increased after acid or capsaicin stimulation compared to the vehicle injected group(40.9+-10.2/52.6+-11.8 vs 19.6+_6.6; p O.O5),capsazepine effectively blocked c-fos expression induced by acid and capsaicin(28.4+_8.6,p O.O5).Conciusions: The increased expression of c-los in response to capsaicin or acid stimulation in pancreatitis rats indicates that pancreatic afferents are sensitized in this model. Inhibition of the responses to acid and capsaicin by pre-treatment with a VR1 antagonist suggests that these responses require VR1 activation and that sensitization of afferent neurons in pancreatitis involves an increase in VR1 activity. These results imply that VR1 plays a critical role in pain transmission in pancreatitis. Our results also suggest that capsazepine may be an effective therapeutic agent in treating pain in pancreetitis.

Counteraction of NYP-induced c-Fos expression in the nucleus tractus solitarii by alpha 2 receptor agonists.

Neuronal c-Fos protein-like immunoreactivity (c-Fos IR) in parts of the nucleus tractus solitarii (NTS)-dorsal motor nucleus of the vagus nerve (dmnX) complex of the anaesthetized rat was studied 90 min after NTS microinjections of neuropeptide Y (NPY) and/or adrenergic receptor agonists in relevant cardiovascular doses. Microinjections of NPY and l-adrenaline alone into the NTS induced neuronal c-Fos IR in parts of the NTS-dmnX complex, while clonidine had no action. No double-labelled GFAP/c-Fos immunoreactive cells were found in the NTS-dmnX complex after the microinjection of NPY into the NTS. Coinjections of NPY and l-adrenaline or clonidine into the NTS reduced the NPY-induced increase in c-Fos IR. The results emphasize that during cardiovascular regulation NPY receptors can control neuronal gene expression in the NTS-dmnX complex via c-fos and that stimulation of alpha 2-adrenoceptors can attenuate NPY receptor transduction also in terms of induction of c-Fos IR in the NTS-dmnX complex via an antagonistic alpha 2/NPY receptor interaction.

C-fos Protein-like immunoreactivity in cultured human glial cells

The expression of the c-fos protein(s) in cultured glial cells of the human brain and sympathetic ganglia was studied immunocytochemically using an antibody to a synthetic fragment of the c-fos protein (M-peptide). GFAP (glial fibrillary acidic protein) and S-100 antibodies were used as glial cell markers. The c-fos immunoreactivity ( c-fos IR) was observed in both brain and peripheral ganglion derived cells maintained 7–22 days in culture. In general, c-fos IR was stronger in small spindle-shaped cells than in large cells. Deprivation of serum for 24 h decreased drastically c-fos IR in both types of cultures. The results demonstrate a serum-dependent expression of c-fos in the human glial cells derived from central and peripheral nervous systems.