Increased c-Fos Immunoreactivity Research Articles

The central anorexigenic mechanism of adrenocorticotropic hormone involves the caudal hypothalamus in chicks

Adrenocorticotropic hormone (ACTH), consisting of 39 amino acids, is most well-known for its involvement in an organism's response to stress. It also participates in satiety, as exogenous ACTH causes decreased food intake in rats. However, its anorexigenic mechanism is not well understood in any species and its effect on appetite is not reported in the avian class. Thus, the present study was designed to evaluate central ACTH's effect on food intake and to elucidate the mechanism mediating this response using broiler chicks. Chicks that received intracerebroventricular (ICV) injection of 1, 2, or 4nmol of ACTH reduced food intake, under both ad libitum and 180min fasted conditions. Water intake was also reduced in ACTH-injected chicks under both feeding conditions, but when measured without access to feed it was not affected. Blood glucose was not affected in either feeding condition. Following ACTH injection, c-Fos immunoreactivity was quantified in key appetite-associated hypothalamic nuclei including the ventromedial hypothalamus (VMH), dorsomedial hypothalamus, lateral hypothalamus (LH), arcuate nucleus (ARC) and the parvo- and magno-cellular portions of the paraventricular nucleus. ACTH-injected chicks had increased c-Fos immunoreactivity in the VMH, LH, and ARC. Hypothalamus was collected at 1h post-injection, and real-time PCR performed to measure mRNA abundance of some appetite-associated factors. Neuropeptide Y, pro-opiomelanocortin, glutamate decarboxylase 1, melanocortin receptors 2–5, and urocortin 3 mRNA abundance was not affected by ACTH treatment. However, expression of corticotropin releasing factor (CRF), urotensin 2 (UT), agouti-related peptide (AgRP), and orexin (ORX), and melanocortin receptor 1 (MC1R) mRNA decreased in the hypothalamus of ACTH-injected chicks. In conclusion, ICV ACTH causes decreased food intake in chicks, and is associated with VMH, LH, and ARC activation, and a decrease in hypothalamic mRNA abundance of CRF, UT, AgRP, ORX and MC1R.

Phosphodiesterase 10A inhibitor, MP-10 (PF-2545920), produces greater induction of c-Fos in dopamine D2 neurons than in D1 neurons in the neostriatum

Studies described here tested the hypothesis that phosphodiesterase 10A inhibition by a selective antagonist, MP-10, activates the dopamine D2 receptor expressing medium spiny neurons to a greater extent than the D1 receptor expressing neurons. We used regional pattern of c-Fos induction in the neostriatal subregions of rodents and direct assessment of D1-positive and -negative neurons in the DRd1a-tdTomato mice for the purpose. MP-10 (1, 3, 10 or 30 mg/kg, PO) dose-dependently increased c-Fos immunopositive nuclei in all regions of neostriatum. However, the effect was statistically greater in the dorsolateral striatum, a region known to be activated preferentially by the D2 antagonism, than the D1-activated dorsomedial striatum. The D2 antagonist, haloperidol (0.3, 1, or 3 mg/kg, PO) produced an identical, regional pattern of c-Fos induction favoring the dorsolateral striatum of the rat. In contrast, the D1 agonist, SKF82958 (0.5, 1, or 2 mg/kg, PO), induced greater expression of c-Fos in the dorsomedial striatum. The C57Bl/6 mouse also showed regionally preferential c-Fos activation by haloperidol (2 mg/kg, IP) and SKF82858 (3 mg/kg, IP). In the Drd1a-tdTomato mice, MP-10 (3 or 10 mg/kg, IP) increased c-Fos immunoreactivity in both types of neurons, the induction was greater in the D1-negative neurons. Taken together, both the regional pattern of c-Fos induction in the striatal sub-regions and the greater induction of c-Fos in the D1-negative neurons indicate that PDE10A inhibition produces a small but significantly greater activation of the D2-containing striatopallidal pathway.

A novel role for xenopsin: Stimulation of food intake

Xenopsin (XPN), an extract from frog skin, is comprised of 80 amino acids and exerts effects on the mammalian digestive tract. The purpose of the study presented here was to determine if XPN would affect food intake using chicks as models. Chicks which had been fasted for 180min did not change food or water intake after central injection of XPN. However, ab libitum fed chicks which received 1 and 3nmol central XPN increased food intake while water intake was not affected. When the dose was increased to 9nmol chicks did not increase food intake but their water intake was reduced suggesting malaise. Chicks injected with XPN had increased c-Fos immunoreactivity in the lateral hypothalamus, but other hypothalamic appetite-associated nuclei were not affected. When XPN was directly injected into the lateral hypothalamus food intake was increased, suggesting a primary site of action. When the expression of appetite-associated neuropeptide mRNA was quantified chicks injected with XPN had increased proopiomelanocortin mRNA. Lastly, a comprehensive behavior analysis was performed and while XPN injected chicks had an increase in the number of feeding pecks, jumping, preening, deep rest and sitting were all decreased. Thus, we conclude that exogenous XPN functions as an orexigenic factor in chicks and its effects are mediated by the lateral hypothalamus.

Neurotrophin selectivity in organizing topographic regeneration of nociceptive afferents

Neurotrophins represent some of the best candidates to enhance regeneration. In the current study, we investigated the effects of artemin, a member of the glial derived neurotrophic factor (GDNF) family, on sensory axon regeneration following a lumbar dorsal root injury and compared these effects with that observed after either NGF or GDNF expression in the rat spinal cord. Unlike previously published data, artemin failed to induce regeneration of large-diameter myelinated sensory afferents when expressed within either the spinal cord or DRG. However, artemin or NGF induced regeneration of calcitonin gene related peptide positive (CGRP+) axons only when expressed within the spinal cord. Accordingly, artemin or NGF enhanced recovery of only nociceptive behavior and showed a cFos distribution similar to the topography of regenerating axons. Artemin and GDNF signaling requires binding to different co-receptors (GFRα3 or GFRα1, respectively) prior to binding to the signaling receptor, cRet. Approximately 70% of DRG neurons express cRet, but only 35% express either co-receptor. To enhance artemin-induced regeneration, we co-expressed artemin with either GFRα3 or GDNF. Co-expression of artemin and GFRα3 only slightly enhanced regeneration of IB4+ non-peptidergic nociceptive axons, but not myelinated axons. Interestingly, this co-expression also disrupted the ability of artemin to produce topographic targeting and lead to significant increases in cFos immunoreactivity within the deep dorsal laminae. This study failed to demonstrate artemin-induced regeneration of myelinated axons, even with co-expression of GFRα3, which only promoted mistargeted regeneration.

Acute sacral nerve stimulation reduces visceral mechanosensitivity in Rat through spinal opioid pathway.

Sacral nerve stimulation (SNS) is an alternative surgical treatment of refractory urge incontinence and/or fecal incontinence. Despite its clinical efficacy, the mechanisms of action of SNS remain poorly understood. The aim of this experimental study was to evaluate the effect of SNS on visceral mechanosensitivity in rats. Anesthetized Sprague-Dawley rats were treated with SNS or sham stimulation. SNS was performed by implanting an electrode close to the sacral nerve root S1. Rats were administered either a non-selective opioid receptor antagonist (naloxone) or a nitric oxide synthase inhibitor (L-NAME). Colonic mechanosensitivity was evaluated using the variation of arterial blood pressure as a spino-bulbar reflex in response to graded isobaric colorectal distension (CRD). C-fos immunoreactive neurons were quantified in spinal and supraspinal sites. μ-opioid receptor (MOR) internalization was counted in the sacral spinal cord with sham or effective SNS in response to CRD. SNS reduced visceral mechanosensitivity in response to CRD. This effect was reversed by intrathecal and intraveinous naloxone administration. In both models, CRD induced increased c-fos immunoreactivity in the dorsal horn neurons of the sacral spinal cord and supraspinal areas. This increase was prevented by SNS. MOR internalization was significantly higher in stimulated group. SNS impacts on visceral mechanosensitivity by decreasing the spino-bulbar reflex in response to CRD. Spinal opioid receptors are likely involved in this effect.

Blockade of spinal glutamate recycling produces paradoxical antinociception in rats with orofacial inflammatory pain.

In our current study, we investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain. DL-threo-β-benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block spinal glutamate transporter and glutamine synthetase activity in astroglia. Intracisternal administration of high dose TBOA (10 μg) produced thermal hyperalgesia in naïve rats but significantly attenuated the thermal hyperalgesia in rats that had been pretreated with interleukin (IL)-1β or Complete Freund's Adjuvant (CFA). In contrast, intracisternal injection of MSO produced anti-hyperalgesic effects against thermal stimuli in CFA-treated rats only. To confirm the paradoxical antinociceptive effects of TBOA and MSO, we examined changes in c-Fos expression in the medullary dorsal horn produced by thermal stimulation in naïve, IL-1β-, or CFA-treated rats, after intracisternal injections of TBOA and MSO. Intracisternal administration of TBOA significantly increased c-Fos immunoreactivity in naïve rats. In contrast, intracisternal administration of TBOA significantly decreased the up-regulation of c-Fos immunoreactivity in the medullary dorsal horn of IL-1β- and CFA-treated rats. However, intracisternal injection of MSO blocked the up-regulation of c-Fos immunoreactivity in CFA-treated rats only. We also investigated the effects of botulinum toxin type A (BoNT-A) on TBOA-induced paradoxical antinociception in CFA-treated rats, as BoNT-A inhibits the release of neurotransmitters, including glutamate. BoNT-A treatment reversed behavioral responses produced by intracisternal administration of TBOA in CFA-treated rats. These results suggest that the paradoxical responses produced by blocking glutamate transporters under inflammatory pain conditions are mediated by the modulation of glutamate release from presynaptic terminals. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic inflammatory pain conditions.

N-Methyl-d-Aspartate Receptor Agonism and Antagonism Within the Amygdaloid Central Nucleus Suppresses Pain Affect: Differential Contribution of the Ventrolateral Periaqueductal Gray

The amygdala contributes to the generation of pain affect, and the amygdaloid central nucleus (CeA) receives nociceptive input that is mediated by glutamatergic neurotransmission. The present study compared the contribution of N-methyl-d-aspartate (NMDA) receptor agonism and antagonism in the CeA to generation of the affective response of rats to an acute noxious stimulus. Vocalizations that occur following a brief tail shock (vocalization afterdischarges) are a validated rodent model of pain affect and were preferentially suppressed, in a dose-dependent manner, by bilateral injection into the CeA of NMDA (.1, .25, .5, or 1 μg/side) or the NMDA receptor antagonist d-(–)-2-amino-5-phosphopentanoic acid (AP5; 1, 2, or 4 μg/side). Vocalizations that occur during tail shock were suppressed to a lesser degree, whereas spinal motor reflexes (tail flick and hind limb movements) were unaffected by injection of NMDA or AP5 into the CeA. Injection of NMDA, but not AP5, into the CeA increased c-Fos immunoreactivity in the ventrolateral periaqueductal gray, and unilateral injection of the μ-opiate receptor antagonist H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; .25 μg) into ventrolateral periaqueductal gray prevented the antinociception generated by injection of NMDA into the CeA. These findings demonstrate that although NMDA receptor agonism and antagonism in the CeA produce similar suppression of pain behaviors, they do so via different neurobiologic mechanisms. PerspectiveThe amygdala contributes to production of the emotional dimension of pain. NMDA receptor agonism and antagonism within the CeA suppressed rats' emotional response to acute painful stimulation. Understanding the neurobiology underlying emotional responses to pain will provide insights into new treatments for pain and its associated affective disorders.

Diazepam improves aspects of social behaviour and neuron activation in NMDA receptor-deficient mice.

NR1 knockdown (NR1KD) mice are genetically modified to express low levels of the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors, and show deficits in affiliative social behaviour. In this study, we determined which brain regions were selectively activated in response to social stimulation and asked whether differences in neuronal activation could be observed in mice with reduced sociability. Furthermore, we aimed to determine whether brain activation patterns correlated with the amelioration of social deficits through pharmacological intervention. The cingulate cortex, lateral septal nuclei, hypothalamus, thalamus and amygdala showed an increase in c-Fos immunoreactivity that was selective for exposure to social stimuli. NR1KD mice displayed a reduction in social behaviour and a reduction in c-Fos immunoreactivity in the cingulate cortex and septal nuclei. Acute clozapine did not significantly alter sociability; however, diazepam treatment did increase sociability and neuronal activation in the lateral septal region. This study has identified the lateral septal region as a neural substrate of social behaviour and the GABA system as a potential therapeutic target for social dysfunction.

The effects of atomoxetine and methylphenidate on the prepulse inhibition of the acoustic startle response in mice.

Atomoxetine (ATM) and methylphenidate (MPD) have been used for the treatment of attention deficit hyperactivity disorder (ADHD). ATM is a selective norepinephrine reuptake inhibitor, whereas MPD is a psychostimulant and acts as a norepinephrine and dopamine reuptake inhibitor. In the present study, we investigated the effects of ATM (1, 3 or 10mg/kg) and MPD (5, 10 or 20mg/kg) on pharmacological mouse models of sensorimotor gating measured by prepulse inhibition (PPI) using the acoustic startle response test. MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist, or apomorphine, a non-competitive dopamine receptor agonist, was used to induce PPI deficits. ATM (3 or 10mg/kg, s.c.) significantly attenuated the MK-801-, but not apomorphine-, induced PPI deficits. In contrast to ATM, MPD did not reverse the PPI deficits induced by either MK-801 or apomorphine. Immunostaining revealed that the number of c-Fos-immunopositive cells was increased in the nucleus accumbens following MK-801 treatment, and this was reversed by the administration of ATM (3mg/kg), but not MPD (10mg/kg). However, neither ATM nor MPD reversed the increased number of c-Fos-immunopositive cells in the nucleus accumbens following apomorphine treatment. These results suggest that the attenuating effect of ATM on the increased c-Fos immunoreactivity in the nucleus accumbens induced by MK-801 may be attributed to the PPI deficit-ameliorating effects of ATM and that ATM would be useful to treat sensorimotor gating-related disorders by improving the patient's attention span or cognitive function.

Anorexigenic effects of central adrenomedullin are associated with hypothalamic changes in juvenile Gallus gallus

Adrenomedullin (AM), a 52 residue neuropeptide, is associated with anorexia in mammals and has a poorly understood central mechanism of action. Thus, this study focused on elucidating AM’s central mechanism of action in an alternative vertebrate model, the chick (Gallus gallus). In Experiment 1, chicks centrally injected with AM dose-dependently reduced food but not water intake. In Experiment 2, those chicks that received central AM had increased c-Fos immunoreactivity in the magnocellular division of the paraventricular nucleus (PaMC), ventromedial hypothalamus (VMH) and doromedial hypothalamus (DM). The lateral hypothalamic area, parvocellular division of the paraventricular hypothalamus and the arcuate nucleus were not affected. In Experiment 3, antagonism of corticotrophin releasing factor (CRF) receptors did not affect AM-associated anorexia. In Experiment 4, a comprehensive behavior analysis was conducted and AM-treated chicks pecked less, moved more, jumped more and spent more time in deep rest. In conclusion, exogenous AM induced anorexia is associated with activation of the PaMC, VMH and DM of the hypothalamus, is not CRF dependent, and affects behaviors unrelated to food intake in chicks.

Differences between the nonselective adenosine receptor antagonists caffeine and theophylline in motor and mood effects: Studies using medium to high doses in animal models

RationaleCaffeine and theophylline are methylxanthines that are broadly consumed, sometimes at high doses, and act as minor psychostimulants. Both are nonselective adenosine antagonists for A1 and A2A receptors, which are colocalized with dopamine D1 and D2 receptors in striatal areas. Adenosine antagonists generally have opposite actions to those of dopamine antagonists. Although the effects of caffeine are widely known, theophylline has been much less well characterized, especially at high doses. MethodsAdult male CD1 mice were used to study the effect of a broad range of doses (25.0, 50.0 or 100.0mg/kg) of caffeine and theophylline on measures of spontaneous locomotion and coordination, as well as the pattern of c-Fos immunoreactivity in brain areas rich in adenosine and dopamine receptors. In addition, we evaluated possible anxiety and stress effects of these doses. ResultsCaffeine, at these doses, impaired or suppressed locomotion in several paradigms. However, theophylline was less potent than caffeine at suppressing motor parameters, and even stimulated locomotion. Both drugs induced corticosterone release, however caffeine was more efficacious at intermediate doses. While caffeine showed an anxiogenic profile at all doses, theophylline only did so at the highest dose used (50mg/kg). Only theophylline increased c-Fos immunoreactivity in cortical areas. ConclusionTheophylline has fewer disruptive effects than caffeine on motor parameters and produces less stress and anxiety effects. These results are relevant for understanding the potential side effects of methylxanthines when consumed at high doses.

Serotonin2Creceptors modulate dopamine transmission in the nucleus accumbens independently of dopamine release: behavioral, neurochemical and molecular studies with cocaine

In keeping with its ability to control the mesoaccumbens dopamine (DA) pathway, the serotonin2C receptor (5-HT2C R) plays a key role in mediating the behavioral and neurochemical effects of drugs of abuse. Studies assessing the influence of 5-HT2C R agonists on cocaine-induced responses have suggested that 5-HT2C Rs can modulate mesoaccumbens DA pathway activity independently of accumbal DA release, thereby controlling DA transmission in the nucleus accumbens (NAc). In the present study, we assessed this hypothesis by studying the influence of the 5-HT2C R agonist Ro 60-0175 on cocaine-induced behavioral, neurochemical and molecular responses. The i.p. administration of 1 mg/kg Ro 60-0175 inhibited hyperlocomotion induced by cocaine (15 mg/kg, i.p.), had no effect on cocaine-induced DA outflow in the shell, and increased it in the core subregion of the NAc. Furthermore, Ro 60-0175 inhibited the late-onset locomotion induced by the subcutaneous administration of the DA-D2 R agonist quinpirole (0.5 mg/kg), as well as cocaine-induced increase in c-Fos immunoreactivity in NAc subregions. Finally, Ro 60-0175 inhibited cocaine-induced phosphorylation of the DA and c-AMP regulated phosphoprotein of Mr 32 kDa (DARPP-32) at threonine residues in the NAc core, this effect being reversed by the selective 5-HT2C R antagonist SB 242084 (0.5 mg/kg, i.p.). Altogether, these findings demonstrate that 5-HT2C Rs are capable of modulating mesoaccumbens DA pathway activity at post-synaptic level by specifically controlling DA signaling in the NAc core subregion. In keeping with the tight relationship between locomotor activity and NAc DA function, this interaction could participate in the inhibitory control of cocaine-induced locomotor activity.

Alterations of prefrontal cortex GABAergic transmission in the complex psychotic-like phenotype induced by adolescent delta-9-tetrahydrocannabinol exposure in rats

Although several findings indicate an association between adolescent cannabis abuse and the risk to develop schizophrenia later in life, the evidence for a causal relationship is still inconclusive. In the present study, we investigated the emergence of psychotic-like behavior in adult female rats chronically exposed to delta-9-tetrahydrocannabinol (THC) during adolescence.To this aim, female Sprague-Dawley rats were treated with THC during adolescence (PND 35–45) and, in adulthood (PND 75), a series of behavioral tests and biochemical assays were performed in order to investigate the long-term effects of adolescent THC exposure.Adolescent THC pretreatment leads to long-term behavioral alterations, characterized by recognition memory deficits, social withdrawal, altered emotional reactivity and sensitization to the locomotor activating effects of acute PCP.Moreover, since cortical disinhibition seems to be a key feature of many different animal models of schizophrenia and GABAergic hypofunction in the prefrontal cortex (PFC) has been observed in postmortem brains from schizophrenic patients, we then investigated the long-lasting consequences of adolescent THC exposure on GABAergic transmission in the adult rat PFC.Biochemical analyses revealed that adolescent THC exposure results in reduced GAD67 and basal GABA levels within the adult PFC. GAD67 expression is reduced both in parvalbumin (PV)- and cholecystokinin (CCK)-containing interneurons; this alteration may be related to the altered emotional reactivity triggered by adolescent THC, as silencing PFC GAD67 expression through a siRNA-mediated approach is sufficient to impact rats' behavior in the forced swim test. Finally, the cellular underpinnings of the observed sensitized response to acute PCP in adult THC-treated rats could be ascribed to the increased cFos immunoreactivity and glutamate levels in the PFC and dorsal striatum. The present findings support the hypothesis that adolescent THC exposure may represent a risk factor for the development of a complex psychotic-like behavior in adulthood.

Expression of maternal behavior and activation of the bed nucleus of the stria terminalis during predatory threat exposure: Modulatory effects of transport stress

Past work has established that levels of maternal care provided to rat pups during the postpartum period plays an important role in shaping development of the stress response system, such that high levels of pup licking and grooming and active nursing behaviors are associated with more efficient hypothalamic–pituitary–adrenal responses to stressors in adulthood. Furthermore, a prior study from our laboratory has demonstrated facilitation of maternal care for five days following a one-hour predator odor exposure on the day of giving birth. The present study was an investigation of the effects on maternal care during a one-hour predator odor exposure administered on the day of giving birth, with or without the addition of transport stress immediately prior to the odor exposure. Stress-induced activation of the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST), two brain regions involved in regulating maternal behaviors, were also quantified using c-Fos immunohistochemistry. Our results show that predator odor exposure soon after birth does not significantly alter expression of maternal behaviors during the hour-long exposure period, unless the dams are also exposed to transport stress, in which case maternal behaviors are reduced during the first 10min of the exposure but not significantly different during the final 10min. Predator odor exposure (with or without additional transport stress) increased c-Fos immunoreactivity in the BNST, but not the MPOA, relative to control odor exposure, suggesting that the BNST may play an important role in integration of threat cues and transduction of their meaning into long-term effects on expression of maternal care. Future experiments should be designed to test the effects of temporary inactivation of the BNST during postpartum predator odor exposure.

Amylin Acts in the Central Nervous System to Increase Sympathetic Nerve Activity

The pancreatic hormone amylin acts in the central nervous system (CNS) to decrease food intake and body weight. We hypothesized that amylin action in the CNS promotes energy expenditure by increasing the activity of the sympathetic nervous system. In mice, ip administration of amylin significantly increased c-Fos immunoreactivity in hypothalamic and brainstem nuclei. In addition, mice treated with intracerebroventricular (icv) amylin (0.1 and 0.2 nmol) exhibited a dose-related decrease in food intake and body weight, measured 4 and 24 hours after treatment. The icv injection of amylin also increased body temperature in mice. Using direct multifiber sympathetic nerve recording, we found that icv amylin elicited a significant and dose-dependent increase in sympathetic nerve activity (SNA) subserving thermogenic brown adipose tissue (BAT). Of note, icv injection of amylin also evoked a significant and dose-related increase in lumbar and renal SNA. Importantly, icv pretreatment with the amylin receptor antagonist AC187 (20 nmol) abolished the BAT SNA response induced by icv amylin, indicating that the sympathetic effects of amylin are receptor-mediated. Conversely, icv amylin-induced BAT SNA response was enhanced in mice overexpressing the amylin receptor subunit, RAMP1 (receptor-activity modifying protein 1), in the CNS. Our data demonstrate that CNS action of amylin regulates sympathetic nerve outflow to peripheral tissues involved in energy balance and cardiovascular function.

Metformin decreases meal size and number and increases c-Fos expression in the nucleus tractus solitarius of obese mice

Metformin is widely used to treat obese diabetics because of its beneficial effects on body weight, energy intake, and glucose regulation. However, it has not been investigated how oral metformin affects meal patterns, or whether the reduced food intake is associated with neuronal activation in the hindbrain. Accordingly, we investigated how orally administered metformin (150 or 300mg/kg daily for 4 or 7days) reduces body weight in obese mice on a high-fat diet by continuously measuring meal patterns, energy expenditure, and locomotor activity, and whether oral metformin (300mg/kg daily for 3days) increases c-Fos expression in the nucleus tractus solitarius (NTS) and area postrema. Furthermore, we determined whether oral metformin produces a conditioned taste aversion (CTA) in obese mice administered a single dose of metformin (75, 150, or 300mg/kg, p.o.). Metformin (300mg/kg daily for 7days) reduced body weight and adiposity by decreasing nocturnal energy intake but did not significantly change energy expenditure or locomotor activity relative to vehicle, and it transiently decreased nocturnal meal size and reduced meal number throughout the experiments. Furthermore, metformin significantly increased c-Fos immunoreactivity within the NTS of obese mice compared to that in controls and pair-fed group, and induced a CTA at doses of 150 or 300mg/kg. These results indicate that metformin-induced weight loss is associated with a sustained reduction in energy intake maintained by a reduction in meal size and number, and that oral administration of metformin causes visceral illness and neuronal activation in the NTS.

Infant satiety depends on transient expression of cholecystokinin‐1 receptors on ependymal cells lining the third ventricle in mice

Cholecystokinin (CCK) is a hypothetical controller for suckling and infancy body weight, although the underlying mechanisms remain unclear. Therefore, the present study analysed the mechanisms using mice lacking the CCK-1 receptor (CCK1R-/-). Although CCK1R-/- mice displayed normal weights at birth and adulthood, CCK1R-/- pups had enlarged adipocytes and were overweight from the first to second week after birth, regardless of maternal genotype. The lacZ reporter gene assay and/or calcium imaging analysis demonstrated that CCK-1 receptors were abundant in satiety-controlling regions such as the hypothalamus, brainstem, nodose ganglion and pylorus in adults, whereas these signals were few to lacking at pre-weanling stages. At postnatal day (PD) 6, the increase in cFos expression in the medullary nucleus tractus solitarius was similarly triggered by gastrointestinal milk- or saline filling in both genotypes, further indicating immature CCK-1 receptor function in an ascending satiety-controlling system during infancy. Conversely, third ventricle ependymal tanycyte-like cells expressed CCK-1 receptors with expression peaking at PD6. At PD6, wild-type but not CCK1R-/- mice had increased cFos immunoreactivity in ependymal cells following gastrointestinal milk filling whereas the response became negligible at PD12. In addition, ependymal cFos was not increased by saline filling, indicating that these responses are dependent on CCK-1 receptors, developmental stage and nutrients. Furthermore, body weights of wild-type pups were transiently increased by blocking ependymal CCK receptor function with microinjection of a CCK-1 antagonist, but not a CCK-2 antagonist. Hence, we demonstrate de novo functions of ependymal CCK-1 receptors and reveal a new aspect of infant satiety-controlling mechanisms.

Angiotensin II AT1Receptors Are Involved in Neuronal Activation Induced by Amphetamine in a Two-Injection Protocol

It was already found that Ang II AT1 receptors are involved in the neuroadaptative changes induced by a single exposure to amphetamine, and such changes are related to the development of behavioral and neurochemical sensitization. The induction of the immediately early gene c-fos has been used to define brain activated areas by amphetamine. Our aim was to evaluate the participation of AT1 receptors in the neuronal activation induced by amphetamine sensitization. The study examined the c-fos expression in mesocorticolimbic areas induced by amphetamine challenge (0.5 mg/kg i.p) in animals pretreated with candesartan, a selective AT1 receptor blocker (3 mg/kg p.o × 5 days), and amphetamine (5 mg/kg i.p) 3 weeks before the challenge. Increased c-fos immunoreactivity was found in response to the amphetamine challenge in the dorsomedial caudate-putamen and nucleus accumbens, and both responses were blunted by the AT1 receptor blocker pretreatment. In the infralimbic prefrontal cortex, increased c-fos immunoreactivity was found in response to amphetamine and saline challenge, and both were prevented by the AT1 receptor blocker. No differences were found neither in ventral tegmental area nor prelimbic cortex between groups. Our results indicate an important role for brain Ang II in the behavioral and neuronal sensitization induced by amphetamine.

Antinociceptive effects of A1 and A2 type botulinum toxins on carrageenan-induced hyperalgesia in rat

We performed a study on the antinociceptive effects of A1 and A2 type (A1LL and A2NTX, respectively) botulinum toxin on carrageenan-induced hyperalgesia in the rat. Both A1LL and A2NTX had antinociceptive effects in the carrageenan-induced inflammatory pain model, reducing the mechanical and thermal hyperalgesia. A2NTX also reduced the increase in c-fos immunoreactivity in L4–L5 spinal segments induced by carrageenan, suggesting that A2NTX inhibits the activation of spinal nociceptive afferent fibers that project to the CNS. Our results indicate that A2NTX may offer a new therapeutic tool to treat inflammatory pain.

C-Fos immunoreactivity in the pig brain following deoxynivalenol intoxication: Focus on NUCB2/nesfatin-1 expressing neurons

Deoxynivalenol (DON), produced by the cereal-contaminating Fusarium fungi, is a major trichothecene responsible for mycotoxicoses in farm animals, including swine. The main effect of DON-intoxication is food intake reduction and the consequent body weight loss. The present study aimed to identify brain structures activated during DON intoxication in pigs. To this goal, we used c-Fos staining which constitutes a useful approach to identify activated neurons. We showed that per os administration of Fusarium graminearum extracts (containing the equivalent of 1mg DON per kg of body weight) induced an increase in c-Fos immunoreactivity in several central structures, including the ventrolateral medulla (VLM), dorsal vagal complex (DVC), paraventricular nucleus of the hypothalamus (PVN), arcuate nucleus (Arc), supraoptic nucleus (SON) and amygdala (CeA). Moreover, we coupled c-Fos staining with phenotypic markers detection in order to specify the neuronal populations activated during DON intoxication. This phenotypic characterization revealed the activation of catecholaminergic but not of serotoninergic neurons in response to the toxin. In this context, we also paid a particular attention to NUCB2/nesfatin-1 positive cells, since nesfatin-1 is known to exert a satiety effect. We report here, for the first time in the pig brain, the presence of NUCB2/nesfatin-1 neurons in the VLM, DVC, PVN, Arc and SON, and their activation during DON intoxication. Taken together, these data show that DON stimulates the main structures involved in food intake in pigs and suggest that catecholaminergic and NUCB2/nesfatin-1 neurons could contribute in the anorexigenic effects of the mycotoxin.