Fos-immunoreactive Cells Research Articles

Repeated amphetamine administration induces Fos in prefrontal cortical neurons that project to the lateral hypothalamus but not the nucleus accumbens or basolateral amygdala

The development of sensitization to amphetamine (AMPH) is dependent on increases in excitatory outflow from the medial prefrontal cortex (mPFC) to subcortical centers. These projections are clearly important for the progressive enhancement of the behavioral response during drug administration that persists through withdrawal. The objective of this study was to identify the mPFC subcortical pathway(s) activated by a sensitizing regimen of AMPH. Using retrograde labeling techniques, Fos activation was evaluated in the predominant projection pathways of the mPFC of sensitized rats after a challenge injection of AMPH. There was a significant increase in Fos-immunoreactive cells in the mPFC, nucleus accumbens (NAc), basolateral amygdala (BLA), and lateral hypothalamus (LH) of rats treated repeatedly with AMPH when compared to vehicle-treated controls. The mPFC pyramidal neurons that project to the LH but not the NAc or BLA show a significant induction of Fos after repeated AMPH treatment. In addition, we found a dramatic increase in Fos-activated orexin neurons. The LH, a region implicated in natural and drug reward processes, may play a role in the development and persistence of sensitization to repeated AMPH through its connections with the mPFC and possibly through its orexin neurons.

Peripheral corticotropin releasing factor (CRF) and a novel CRF1 receptor agonist, stressin1‐A activate CRF1 receptor expressing cholinergic and nitrergic myenteric neurons selectively in the colon of conscious rats

Intraperitoneal (i.p.) corticotropin releasing factor (CRF) induced a CRF(1) receptor-dependent stimulation of myenteric neurons and motility in the rat proximal colon. We characterize the colonic enteric nervous system response to CRF in conscious rats. Laser capture microdissection combined with reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry in longitudinal muscle myenteric plexus whole-mount colonic preparations revealed CRF(1) receptor expression in myenteric neurons. CRF (i.p., 10 microg kg(-1)) induced Fos immunoreactivity (IR) (cells per ganglion) selectively in myenteric plexus of proximal (18.3 +/- 2.4 vs vehicle: 0.0 +/- 0.0) and distal colon (16.8 +/- 1.2 vs vehicle: 0.0 +/- 0.0), but not in that of gastric corpus, antrum, duodenum, jejunum and ileum. The selective CRF(1) agonist, stressin(1)-A (i.p., 10 microg kg(-1)) also induced Fos IR in myenteric but not in submucosal plexus of the proximal and distal colon. Fos IR induced by CRF was located in 55 +/- 1.9% and 53 +/- 5.1% of CRF(1) receptor-IR myenteric neurons and in 44 +/- 2.8% and 40 +/- 3.9% of cholinergic neurons with Dogiel type I morphology, and in 20 +/- 1.6% and 80 +/- 3.3% of nitrergic neurons in proximal and distal colon respectively. CRF and stressin(1)-A elicit defecation and diarrhoea. These data support that one mechanism through which peripherally injected CRF ligands stimulate colonic function involves a direct action on colonic cholinergic and nitrergic myenteric neurons expressing CRF(1) receptor.

Differential c‐fos expression in the brain of male Japanese quail following exposure to stimuli that predict or do not predict the arrival of a female

We investigated the effects of presenting a sexual conditioned stimulus on the expression of c-fos in male Japanese quail. Eight brain sites were selected for analysis based on previous reports of c-fos expression in these areas correlated with sexual behaviour or learning. Males received either paired or explicitly unpaired presentations of an arbitrary stimulus and visual access to a female. Nine conditioning trials were conducted, one per day, for each subject. On the day following the ninth trial, subjects were exposed to the conditional stimulus (CS) for 5 min. Conditioning was confirmed by analysis of rhythmic cloacal sphincter movements (RCSM), an appetitive sexual behaviour, made in response to the CS presentation. Subjects in the paired condition performed significantly more RCSM than subjects in the unpaired group. Brains were collected 90 min following the stimulus exposure and stained by immunohistochemistry for the FOS protein. Significant group differences in the number of FOS-immunoreactive (FOS-ir) cells were found in two brain regions, the nucleus taeniae of the amygdala (TnA) and the hippocampus (Hp). Subjects in the paired condition had fewer FOS-ir cells in both areas than subjects in the unpaired condition. These data provide additional support to the hypothesis that TnA is implicated in the expression of appetitive sexual behaviours in male quail and corroborate numerous previous reports of the involvement of the hippocampus in conditioning. Further, these data suggest that conditioned and unconditioned sexual stimuli activate different brain regions but have similar behavioural consequences.

Gonadal steroid receptors colocalize with central nervous system neurons projecting to the rat prostate gland

Mating-induced Fos-immunoreactive (-ir) cells are colocalized with androgen receptors (AR), estrogen receptors (ER), or both in limbic and hypothalamic areas known to mediate male rat mating behavior. A steroid-responsive neural network might govern copulatory behavior in male laboratory rats that is analogous to the network described in female rats that governs the lordosis response. This hypothesized network in males may synchronize and coordinate sexual behavioral responses with physiological responses of the genitals and the internal organs of reproduction. Therefore, the pseudorabies virus (PRV; Bartha strain), a transneuronal, viral retrograde tract tracer, was microinjected into the prostate gland to label this network. After 7 days, brains from infected animals were processed for immunohistochemical labeling of AR, ER, and PRV. The majority of PRV-ir cells exhibited either AR or ER immunoreactivity in the medial preoptic area, median preoptic nucleus, bed nucleus of stria terminalis, hypothalamic paraventricular nucleus, and zona incerta, areas known to play roles in male rat mating behavior. Other structures such as the central tegmental field/subparafascicular nucleus of the thalamus, central nucleus of the amygdala, and medial amygdala, also important in the display of male copulatory behavior, were less reliably labeled. Collectively, a steroid receptor-containing neuronal circuit, largely contained in the diencephalon, was revealed that likely is involved in the autonomic control of the prostate gland and the consummatory aspects of male rat mating behavior.

Fos and glutamate AMPA receptor subunit coexpression associated with cue-elicited cocaine-seeking behavior in abstinent rats

Cocaine-associated cues acquire incentive motivational effects that manifest as craving in humans and cocaine-seeking behavior in rats. We have reported an increase in neuronal activation in rats, measured by Fos protein expression, in various limbic and cortical regions following exposure to cocaine-associated cues. This study examined whether the conditioned neuronal activation involves glutamate AMPA receptors by measuring coexpression of Fos and AMPA glutamate receptor subunits (GluR1, GluR2/3, or GluR4). Rats trained to self-administer cocaine subsequently underwent 22 days of abstinence, during which they were exposed daily to either the self-administration environment with presentations of the light/tone cues previously paired with cocaine infusions (Extinction group) or an alternate environment (No Extinction group). All rats were then tested for cocaine-seeking behavior (i.e. responses without cocaine reinforcement) and Fos and AMPA glutamate receptor subunits were measured postmortem using immunocytochemistry. The No Extinction group exhibited increases in cocaine-seeking behavior and Fos expression in limbic and cortical regions relative to the Extinction group. A large number of Fos immunoreactive cells coexpressed GluR1, GluR2/3, and GluR4, suggesting that an action of glutamate at AMPA receptors may in part drive cue-elicited Fos expression. Importantly, there was an increase in the percentage of cells colabeled with Fos and GluR1 in the anterior cingulate and nucleus accumbens shell and cells colabeled with Fos and GluR4 in the infralimbic cortex, suggesting that within these regions, a greater, and perhaps even different, population of AMPA receptor subunit-expressing neurons is activated in rats engaged in cocaine-seeking behavior.

Decreases in urinary pheromonal activities in male mice after exposure to 3-methylchoranthrene

Many classes of environmental pollutants, which are found at significant levels in the environment, affect the reproductive functions. The gonadal functions of various animals are regulated by pheromones excreted from mating partners. Pheromones in male urine play essential roles in the sexual maturation of female mice. Pheromones are received by sensory neurons in the vomeronasal organ, which innervate to the accessory olfactory bulb (AOB). The effects of a typical aromatic environmental pollutant (3-methylchoranthrene) on excretion of pheromones from male mice were explored based on neuronal Fos responses of the AOB of female mice. On days 1 and 3 after intraperitoneal administration of 3-methylchoranthrene (3-MC), the density of Fos-immunoreactive (Fos-ir) cells in the AOB of female mice after exposure to urine excreted from the administered males was lower than that after exposure to urine from non-administered males. These results suggest that 3-MC blocks chemical communication from male to female mice by reducing pheromonal activities.

Developmental changes in odor-evoked activity in rat piriform cortex

In adult rats, odor-evoked Fos protein expression is found in rostrocaudally-oriented bands of cells in anterior piriform cortex (APC), likely indicating functionally distinct subregions, while activated cells in posterior piriform cortex (PPC) lack apparent spatial organization. To determine whether these patterns are present during early postnatal life, and whether they change during development, Fos expression was assessed following acute exposure to single aliphatic acid odors in developing rats beginning at postnatal day 3 (P3). In the olfactory bulb, Fos-immunoreactive cells were present in the granule cell, mitral cell and glomerular layers at the earliest ages examined. Cells immunopositive for Fos were clustered in areas previously reported as active in response to these odors. In piriform cortex, activation in layers II/III shared some features with that seen in the adult; in APC, rostro-caudally oriented bands of Fos-positive cells alternated with bands relatively free of label, while labeled cells were found dispersed throughout PPC. However, in P3–P7 animals, Fos-positive cells in APC were found in a central rostro-caudally oriented band that was flanked by two bands relatively free of Fos-positive cells. This contrasted with the adult pattern, a central cell-poor band flanked by cell-rich bands, which was observed beginning at P10. These results suggest that subregions of APC visualized by odor-evoked Fos expression are active and functionally distinct shortly after birth. Changes in activity within these subregions during early postnatal development coincide with a shift toward adult-like olfactory learning behavior in the second postnatal week, and may play a role in this behavioral shift.

Contribution of the Suprachiasmatic Nucleus to the Formation of a Time Memory for Heat Exposure in Rats

We have reported that after rats were acclimated to heat for about 5 h daily at a fixed time, the pattern of day-night variations of core temperature (T(cor)) altered, i.e., their T(cor) fell, especially during the period when they had previously been exposed to heat. The suprachiasmatic nucleus (SCN) of the hypothalamus is known to be indispensable for the genesis of circadian rhythms of T(cor). We therefore investigated the involvement of the SCN in the characteristic fall in T(cor) in heat-acclimated rats. The rats were exposed to an ambient temperature of 33 degrees C only in the last half of the dark phase for 10 consecutive days. After the heat exposure schedule, the nocturnal pattern of T(cor) variations and Fos expression in the dorsomedial SCN altered so that the T(cor) and the number of Fos immunoreactive cells decreased in the last half of the dark phase. The bilateral lesions of the SCN of rats were made electrically, and the electrical lesions of the SCNs abolished the daily cycle of T(cor). In the SCN-lesioned rats, theT(cor) levels were significantly lowered after the 10-day heat exposure schedule. However, their T(cor) did not specifically drop during the period when they had previously been exposed to heat. These findings suggest that the SCN is crucial for establishing a time memory for heat stress, and it plays a minimal role in heat acclimation-induced changes in T(cor) in rats.

Locomotor activity to nicotine and Fos immunoreactivity in the paraventricular nucleus of the hypothalamus in adolescent socially-stressed rats

We reported previously that social stressors in adolescence (SS: one-hour isolation and new cage partners daily for 16 days) increased locomotor activity to nicotine and to amphetamine in females, but not in males, when tested as adults. Here, we investigated whether effects of stressors in adolescence on locomotor responses to nicotine would be observed in both sexes if tested closer in time to the stressor exposure. We also tested whether social instability was necessary to alter nicotine′s effects on locomotor activity by including a group that underwent daily isolation but was housed with the same partner (ISO). The locomotor-activating effects of nicotine were lower in SS rats compared to ISO and non-stressed control rats. In males, but not in females, there were effects of nicotine treatment and of stress condition on Fos immunoreactive (Fos-ir) cell counts in the paraventricular nucleus (PVN) of the hypothalamus: SS males had higher Fos-ir counts than did ISO and non-stressed control males, and higher Fos-ir counts in the PVN were found in repeated-nicotine groups than in acute-nicotine and saline groups. These results add to evidence that adolescents are uniquely vulnerable to stressors due to ongoing brain development, and also indicate that effects are sex- and stressor-specific.

Acute muscle inflammation enhances the monosynaptic reflexes and c-fos expression in the feline spinal cord

The aim of this research was to study the changes of the motor reflex activity (monosynaptic reflex (MSR) of the flexor and extensor muscles) and Fos immunoreactivity in lumbo-sacral spinal cord after acute induced myositis of m. gastrocnemius-soleus (GS). The experiments were carried out on ischaemic decerebrated, spinalized in C1 cats. After infiltration of the GS muscle with carrageenan (2%) MSRs of flexors and extensors showed a significant increase in amplitude +127 ± 24.5% and +155 ± 28.5%, respectively, p < 0.05. The exposed effect was initiated within 30 min and achieved a maximum 2.8 h after the intramuscular injections of carrageenan. After analysis of dynamics of the MSRs, animals were perfused and c-fos expression in the spinal segments L6–S1 was evaluated. In comparison to sham-operated animals, the number of Fos-immunoreactive (Fos-ir) cells was noticeably increased in the lumbar cord of cats with carrageenan-induced myositis. The labeled cells were concentrated in the ipsilateral laminae I/II, neck of the dorsal horn (V/VI) and intermediate zone (VII), however, clear predominance of their concentration was found in the deep laminae. The effect of muscle inflammation was also expressed as a significant decline in the number of NADPH-d-reactive cells ( p < 0.05) in ipsilateral laminae I/II of L6/L7. The results show that the input from acutely inflamed muscles may induce an increase of the reflex responsiveness of flexors and extensors which is not mediated via the gamma-spindle-loop and which coincides with a significant increase in c-fos expression in the deep laminae of the lumbar spinal cord.

Lasting changes in neuronal activation patterns in select forebrain regions of aggressive, adolescent anabolic/androgenic steroid-treated hamsters

Repeated exposure to anabolic/androgenic steroids (AAS) during adolescence stimulates high levels of offensive aggression in Syrian hamsters. The current study investigated whether adolescent AAS exposure activated neurons in areas of hamster forebrain implicated in aggressive behavior by examining the expression of FOS, i.e., the protein product of the immediate early gene c-fos shown to be a reliably sensitive marker of neuronal activation. Adolescent AAS-treated hamsters and sesame oil-treated littermates were scored for offensive aggression and then sacrificed 1 day later and examined for the number of FOS immunoreactive (FOS-ir) cells in regions of the hamster forebrain important for aggression control. When compared with non-aggressive, oil-treated controls, aggressive AAS-treated hamsters showed persistent increases in the number of FOS-ir cells in select aggression regions, namely the anterior hypothalamus and lateral septum. However, no differences in FOS-ir cells were found in other areas implicated in aggression such as the ventrolateral hypothalamus, bed nucleus of the stria terminals, central and/or medial amygdala or in non-aggression areas, such as the samatosensory cortex and the suprachiasmatic nucleus. These results suggest that adolescent AAS exposure may constitutively activate neurons in select forebrain areas critical for the regulation of aggression in hamsters. A model for how persistent activation of neurons in one of these brain regions (i.e., the anterior hypothalamus) may facilitate the development of the aggressive phenotype in adolescent-AAS exposed animals is presented.

Melatonin inhibits cortical spreading depression-evoked trigeminal nociception

This study was conducted to determine the effect of melatonin on cortical spreading depression-evoked trigeminovascular nociception. Melatonin (20 or 40 mg/kg) or saline was given to Wistar rats. KCl was placed on the cortical surface to elicit the cortical spreading depression. Cortical blood flow was monitored and ultrastructure of cerebral microvessels was studied. The medulla and cervical cord were removed for Fos and nitric oxide synthase immunohistochemical study. The results showed that melatonin pretreatment significantly minimized the cortical spreading depression-evoked cerebral hyperaemia and attenuated the cortical spreading depression-induced microvascular changes. Pretreatment with melatonin also reduced the number of Fos and nitric oxide synthase immunoreactive cells in the trigeminal nucleus caudalis. The results of this study suggest that melatonin can attenuate the process of trigeminovascular nociception induced by cortical spreading depression.

Increased Fos Immunoreactivity in Suprachiasmatic Nucleus before Luteinizing Hormone Surge in Estrogen-Treated Ovariectomized Female Rats

Background/Aims: The suprachiasmatic nucleus (SCN) is thought to control the timing of luteinizing hormone (LH) surges. The present study was designed to examine temporal patterns of Fos expression in the dorsomedial and ventrolateral parts of the SCN (SCNdm and SCNvl) of female rats during an LH surge. It also included examination of temporal changes in plasma LH levels and temporal changes in Fos levels in the anteroventral periventricular nucleus (AVPV) and gonadotropin-releasing hormone (GnRH) neurons. Methods: Ovariectomized rats injected with 20 µg estradiol benzoate (EB) or vehicle were sacrificed at various times from Zeitgeber time (ZT) 8:00 to 16:30 h (ZT8–16.5; ZT0 = lights on; ZT12 = lights off) on the 2nd day after the injection. Immunohistochemical analyses for Fos and GnRH and enzyme-linked immunosorbent assays for LH were then performed. Results: In both the SCNdm and SCNvl of EB rats, the number of Fos-immunoreactive cells significantly increased between ZT9.5–10.5 and ZT11–12. On the other hand, in EB rats there were significant peaks of LH levels and Fos levels in GnRH neurons and the AVPV between ZT11–12 and ZT13–14. There was no significant difference in the number of Fos-immunoreactive cells between EB and control rats in either the SCNdm or SCNvl at ZT9.5–10.5, or in the SCNdm at ZT11–12, whereas the SCNvl of EB rats contained more Fos-immunoreactive cells than that of control rats at ZT11–12. Conclusion: These results suggest that in female rats during an LH surge, a peak in the Fos level in the SCN precedes peaks in Fos levels in the AVPV and GnRH neurons.

Accessory olfactory neural Fos responses to a conditioned environment are blocked in male mice by vomeronasal organ removal

The ability of an anesthetized estrous female to induce a conditioned place preference (CPP) response was assessed in male mice from which the vomeronasal organ (VNO) had either been removed (VNOx) or left intact (VNOi) in an initial effort to assess the possible contribution of VNO-accessory olfactory inputs to the intrinsically rewarding properties of opposite-sex body odorants. Both VNOi and VNOx male mice acquired a CPP after repeated pairing of an initially non-preferred test chamber with an anesthetized estrous female mouse, suggesting that odorants detected by the main olfactory system and/or visual and tactile cues from the anesthetized estrous female can compensate for absent VNO inputs to establish a CPP. Subsequent exposure to this conditioning chamber alone caused significant increases in the number of Fos-immunoreactive cells in the mitral and granule cell layers of the accessory olfactory bulb as well as in the medial amygdala and ventral tegmental area of VNOi but not of VNOx males. These results suggest that activity in distal segments of the VNO-accessory olfactory pathway, in addition to the mesolimbic dopamine reward system, can be conditioned to respond to non-odor cues.

Decreases in Pheromonal Responses at the Accessory Olfactory Bulb of Mice with a Deficiency of the .ALPHA.1B or .BETA.3 Subunits of Voltage-Dependent Ca2+-Channels

Pheromones affect gonadal functions and sexual behaviors. Information in regard to pheromones is received by the vomeronasal organ (VNO) and transmitted to the accessory olfactory bulb (AOB). We investigated the physiological role of the alpha1B and beta3 subunits of the N (neuronal)-type voltage-dependent Ca2+ channel in the neurotransduction in the accessory olfactory (vomeronasal) system using alpha1B-deficient mice and beta3-deficient mice. RT-PCR studies showed the existence of beta1, beta2, beta3, beta4, alpha1A, alpha1B, and alpha1C subunits of voltage-dependent Ca2+ channels in the mouse VNO. Immunohistochemical studies showed that the alpha1A, alpha1B, and alpha1C subunits of voltage-dependent Ca2+ channels exist in the sensory neurons and supporting cells of the mouse VNO. Exposure of the VNO to urine samples excreted from male mice induced lower Fos-immunoreactivity in the periglomerular (PG) cells of the AOBs in alpha1B-deficient female mice than in those of wild mice. The density of Fos-immunoreactive (Fos-ir) cells after exposure to female urine samples at the periglomerular cell layer of alpha1B-deficient male mice was lower than that of wild mice. Exposure of the VNO of beta3-deficient female mice to male urine samples also induced low Fos-ir cells in the periglomerular cell layer of the AOB. These data suggest the importance of the alpha1B and beta3 subunits of the N-type voltage-dependent Ca2+ channel for the pheromone signal transduction system.

INDUCTION OF FOS-LIKE IMMUNOREACTIVITY IN THE LATERAL HYPOTHALAMIC AREA OF THE RAT AFTER MURICIDE

Serotonergic innervation in the lateral hypothalamic area (LHA) has been implicated in mouse-killing behavior, muricide, of rats. In the present study, functional activation of neurons in the LHA was demonstrated by immunohistochemistry of Fos-protein, a marker of activated neurons, following raphe lesion-induced muricide. Fos-protein induction was seen in the ventral part of the intermediate hypothalamic area (IHA) of the LHA in the muricide rat. Following raphe transplantation into the brain near the bilateral IHA, a significant decrease in the number of Fos-immunoreactive cells was observed in this area, coinciding with decreased muricide behavior. The present results suggest that the decline in Fos-protein expression in this area may be associated with the inhibition of this behavior.

Serotonin Depletion, Cortical Spreading Depression, and Trigeminal Nociception

The attack of migraine has been observed to be associated with low level of serotonin (5-HT). Although the mechanism underlying this relationship is still unclear, change in cortical excitability or susceptibility of trigeminal system is a possible explanation. The aim was to study the effect of 5-HT depletion on the development of cortical spreading depression (CSD) and CSD-evoked trigeminal nociception. Wistar rats were separated into low 5-HT and control groups (eight rats each). 5-HT was depleted by administration of para-chlorophenylalanine, a tryptophan hydroxylase inhibitor. CSD was induced by applying 3 mg of potassium chloride on parietal cortex. Cortical activity was monitored for 1 hour. Trigeminal nociception was determined using number of Fos-immunoreactive (Fos-IR) neurons in trigeminal nucleus caudalis as the indicator. Application of KCl led to the development of series of depolarization shift characteristics for CSD. The development of these CSD waves was enhanced in low 5-HT state. The area under curve of each CSD wave and the number of CSD waves occurring within 1 hour were greater in low 5-HT group. No significant change in peak amplitude and duration of CSD wave was observed. The numbers of Fos-IR cells on ipsilateral and contralateral trigeminal nucleus caudalis were significantly greater in the low 5-HT group than those of the controls. Our findings indicate that 5-HT depletion enhances CSD-induced trigeminal nociception by increasing the cortical excitability and sensitivity of trigeminal nociceptive system. These findings may provide a better understanding regarding the relationship between low 5-HT and clinical headaches.

Capsaicin-induced effects on c-fos expression and NADPH-diaphorase activity in the feline spinal cord

The distribution of c-fos expression and NADPH-diaphorase reactivity in the cervical and lumbar segments after stimulation of the vanilloid receptors in the dorsal neck muscles with capsaicin was studied in cats anaesthetized with α-chloralose. After the unilateral intramuscular injection of capsaicin, the mean number of Fos-immunoreactive neurons detected with an avidin–biotin–peroxidase technique was significantly increased in the superficial laminae (I), neck of the dorsal horn (V), and area around the central canal (VII) within both the cervical and lumbar spinal cord. Most Fos-immunoreactive neurons in the cervical spinal cord were giant and small cells. The widespread distribution of Fos-immunoreactive cells throughout the cervical cord within the intermediate zone (VII) coincided with the sites of localization of last-order premotor interneurons and cells of origin of inter-segmental crossed and uncrossed descending propriospinal pathways to the lumbar spinal cord. Fos-immunoreactive neurons were co-distributed with nitric oxide-generating cells at both levels of the spinal cord, although the double-labeled cells were not observed. In conclusion, the analysis of c-fos expression and NADPH-diaphorase reactivity shows that stimulation of vanilloid receptors in the neck muscles can initiate distinctive neuronal plasticity in the cervical (C1–C8) and lumbar (L1–L7) segments, and confirms the anatomical and functional coupling of both regions during processing of nociceptive signals from the dorsal neck muscles.

Social recognition memory requires two stages of protein synthesis in mice

Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was injected 20 min before, immediately after, or 6 h after sampling. No effect was observed when anisomycin was administered 3 h or 18 h after sampling. Immunohistochemical analysis of Fos expression revealed that sampling-like exposure to a juvenile increased the activity of a subset of cells in the accessory olfactory bulb and the brain areas that are associated with it. Additionally, increased Fos expression was measured in the main olfactory bulb and the piriform cortex, whereas no signs of activation were seen in the cortical nucleus of the amygdala, all components of the main olfactory system. No increases in Fos immunoreactivity were observed after 4 h. Our data suggest that long-lasting olfactory recognition memory requires two stages of protein synthesis. The first stage takes place within 1-2 h and the second stage between 6-7 h after sampling. The first but not the second stage is paralleled by an increase in the number of Fos-immunoreactive cells in brain areas associated with both the main and accessory olfactory systems. It therefore appears that the role of the second stage of protein synthesis in recognition memory depends on the integrity of the first stage of protein synthesis.

Neuromedin S Is a Novel Anorexigenic Hormone

A novel 36-amino acid neuropeptide, neuromedin S (NMS), has recently been identified in rat brain and has been shown to be an endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1. These receptors have been identified as neuromedin U (NMU) receptor type 1 and type 2, respectively. In this study, the physiological role of the novel peptide, NMS, on feeding regulation was investigated. Intracerebroventricular (icv) injection of NMS decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent and persistent than that observed with the same dose of NMU. Neuropeptide Y, ghrelin, and agouti-related protein-induced food intake was counteracted by coadministration of NMS. Icv administration of NMS increased proopiomelanocortin mRNA expression in the arcuate nucleus (Arc) and CRH mRNA in the paraventricular nucleus (PVN). Pretreatment with SHU9119 (antagonist for alpha-MSH) and alpha-helical corticotropin-releasing factor-(9-41) (antagonist for CRH) attenuated NMS-induced suppression of 24-h food intake. After icv injection of NMS, Fos-immunoreactive cells were detected in both the PVN and Arc. When neuronal multiple unit activity was recorded in the PVN before and after icv injection of NMS, a significant increase in firing rate was observed 5 min after administration, and this increase continued for 100 min. These results suggest that the novel peptide, NMS, may be a potent anorexigenic hormone in the hypothalamus, and that expression of proopiomelanocortin mRNA in the Arc and CRH mRNA in the PVN may be involved in NMS action on feeding.