Fos-immunoreactive Neurons Research Articles

Maturation of locomotor and Fos responses to the NMDA antagonists, PCP and MK-801

Antagonists at the N-methyl- d-aspartate (NMDA)-type glutamate receptor, such as phencyclidine (PCP) and dizocilpine (MK-801), are well-known to evoke increases in locomotor activity in adult rats and mice. However, little is known about the effects of NMDA antagonists on locomotor activity as a function of development. The present study examined locomotor responses to PCP or MK-801 in male rats of varying ages and found that prepubertal rats were more sensitive to the locomotor-elevating effects of PCP (1.5 mg/kg and 3.0 mg/kg, s.c.) than were adults. Locomotor responses to MK-801 (0.1 and 0.2 mg/kg, s.c.) were not dependent on age. The age-dependent response to PCP may be related to developmental events in the motor cortex, since more Fos-immunoreactive neurons were observed in the motor cortex of prepubertal animals after PCP administration relative to adult animals. An opposite pattern of age-dependent Fos responses was observed in the posterior retrosplenial cortex. The results suggest that locomotor responses to NMDA antagonists can be influenced in an age- and drug-dependent manner and that maturational events in the motor cortex may modify responses to PCP.

Effect of Lesions of the Ventrolateral Preoptic Nucleus on NREM and REM Sleep

Neurons in the ventrolateral preoptic nucleus (VLPO) in rats show c-fos activation after sleep and provide GABAergic innervation of the major monoamine arousal systems, suggesting that they may be a necessary part of the brain circuitry that produces sleep. We examined the effects on sleep behavior in rats of cell-specific damage to the VLPO by microinjection of ibotenic acid. Severe lesions of the central cell cluster of the VLPO ( approximately 80-90% cell loss bilaterally) caused a 60-70% decrease in delta power and a 50-60% decrease in nonrapid-eye-movement (NREM) sleep time (p < 0.001). The number of remaining Fos-immunoreactive neurons in the VLPO cell cluster was linearly related to NREM sleep time (r = 0.77; p < 0.001) and total electroencephalogram delta power (r = 0. 79; p < 0.001) but not to rapid-eye-movement (REM) sleep (r = 0.35; p > 0.10). Lesions in the region containing scattered VLPO neurons medial or dorsal to the cell cluster caused smaller changes in NREM sleep time (24.5 or 15%, respectively) but were more closely associated with loss of REM sleep (r = 0.74; p < 0.01). The insomnia caused by bilateral VLPO lesions persisted for at least 3 weeks. Lesions of the VLPO caused no change in mean body temperature or its circadian variation; after small lesions of the ventromedial preoptic nucleus, body temperature showed normal circadian variation but a wider temperature range, and sleep behavior was not affected. These experiments delineate distinct preoptic sites with primary effects on the regulation of NREM sleep, REM sleep, and body temperature.

The effects of binocular suture and dark rearing on the induction of c-fos protein in the rat visual cortex during and after the critical period.

It has been demonstrated in kittens that binocular lid suture has more deleterious and irreversible effects on plasticity of the developing visual system than rearing in complete darkness. The present study using immunocytochemistry focuses on the effects of the two types of visual deprivation on the inducibility of c-fos protein in visual cortical neurons of rats. Rats were subjected to binocular suture or dark rearing for 1 week during (postnatal days 14-21; P14-P21) and after (P50-P57) the critical period for activity-dependent modifiability of cortical ocular dominance. In rats of both age groups reared in the normal light-dark condition, only a small number of Fos-immunoreactive neurons was obtained in the visual cortex. By contrast, in dark-reared pups and adult rats, numerous c-fos neurons were detected in the layers II-IV and VI of the visual cortex following a brief light exposure (1 h). In rats of both ages subjected to binocular suture, Fos neurons were detected in the same layers as in the dark-reared rats, but significantly less in number. We speculate that the reduced plasticity of the visual cortex in the rats subjected to binocular suture may be due partly to the repressed AP-1 activity in visual cortical neurons. No significant difference was detected in c-fos expression in the visual cortex between visually manipulated pups and adult rats.

Local Morphine Withdrawal Increases c-fosGene, Fos Protein, and Oxytocin Gene Expression in Hypothalamic Magnocellular Neurosecretory Cells

We measured stimulation of c-fos and oxytocin gene expression during excitation of oxytocin cells induced by systemic or local morphine withdrawal. Female rats were made morphine-dependent by intracerebroventricular morphine infusion over 5 d. Morphine withdrawal, induced by systemic injection of the opioid antagonist naloxone (5 mg/kg) in conscious or anesthetized rats, increased the density of c-fos messenger RNA and of oxytocin heterogeneous nuclear RNA in supraoptic nucleus cells compared with those of nonwithdrawn rats; c-fos messenger RNA was also increased in the magnocellular and parvocellular paraventricular nuclei of withdrawn rats. Morphine withdrawal increased the number of Fos-immunoreactive cells in the supraoptic and magnocellular paraventricular nuclei of conscious or pentobarbitone-anesthetized rats. Morphine withdrawal also increased Fos-immunoreactive cell numbers in the parvocellular paraventricular nucleus of conscious but not anesthetized rats. Central administration of the alpha(1)-adrenoreceptor antagonist benoxathian (5 microg/min) did not prevent morphine withdrawal-induced increases in the numbers of Fos-immunoreactive neurons in the supraoptic or magnocellular paraventricular nucleus. Unilateral microdialysis administration of naloxone (10(-5) M) into the supraoptic nucleus of anesthetized morphine-dependent rats increased Fos-immunoreactive cell numbers compared with the contralateral nucleus. Finally, we investigated whether dependence could be induced by chronic unilateral infusion of morphine into a supraoptic nucleus; systemic naloxone (5 mg/kg) increased Fos-immunoreactive cell numbers in the morphine-infused nucleus compared with the contralateral nucleus. Thus, morphine withdrawal excitation increases c-fos and oxytocin gene expression in supraoptic nucleus neurons. This occurs independently from excitation of their ascending noradrenergic inputs, and both dependence and withdrawal can be induced within the supraoptic nucleus.

Relationship of EP(1-4) prostaglandin receptors with rat hypothalamic cell groups involved in lipopolysaccharide fever responses.

The action of prostaglandin E(2) (PGE(2)) in the preoptic area is thought to play an important role in producing fever. Pharmacologic evidence suggests that, among the four subtypes of E-series prostaglandin (EP) receptors, i.e., EP(1), EP(2), EP(3), and EP(4), the EP(1) receptor mediates fever responses. In contrast, evidence from mice with EP receptor gene deletions indicates that the EP(3) receptor is required for the initial (<1 hour) fever after intravenous (i.v.) lipopolysaccharide (LPS). To investigate which subtypes of EP receptors mediate systemic infection-induced fever, we assessed the coexpression of Fos-like immunoreactivity (Fos-IR) and EP(1-4) receptor mRNA in nuclei in the rat hypothalamus that have been shown to be involved in fever responses. Two hours after the administration of i.v. LPS (5 microg/kg), Fos-IR was observed in the ventromedial preoptic nucleus, the median preoptic nucleus, and the paraventricular hypothalamic nucleus. In these nuclei, EP(4) receptor mRNA was strongly expressed and the Fos-IR intensely colocalized with EP(4) receptor mRNA. Strong EP(3) receptor mRNA expression was only seen within the median preoptic nucleus but Fos-IR showed little coexpression with EP(3) receptor mRNA. EP(2) receptor mRNA was not seen in the PGE(2) sensitive parts of the preoptic area. Although approximately half of the Fos-immunoreactive neurons also expressed EP(1) receptor mRNA, EP(1) mRNA expression was weak and its distribution was so diffuse in the preoptic area that it did not represent a specific relationship. In the paraventricular nucleus, EP(4) mRNA was found in most Fos-immunoreactive neurons and levels of EP(4) receptor expression increased after i.v. LPS. Our findings indicate that neurons expressing EP(4) receptor are activated during LPS-induced fever and suggest the involvement of EP(4) receptors in the production of fever.

Activation of brain neurons following central hypervolaemia and hypovolaemia: contribution of baroreceptor and non-baroreceptor inputs

In the present study we have used the detection of Fos, the protein product of c- fos, to determine the distribution of neurons in the medulla and hypothalamus that are activated by changes in central blood volume. Experiments were conducted in both barointact and barodenervated conscious rabbits, to determine the contribution of arterial baroreceptors to the pattern of Fos expression evoked by changes in central blood volume, induced either by intravenous infusion of an isotonic modified gelatin solution, or by partial occlusion of the vena cava. These procedures resulted in a significant increase and decrease, respectively, in right atrial pressure over a 60 min period. In control experiments, barointact and barodenervated rabbits were subjected to the identical procedures except that no changes in central blood volume were induced. In comparison with the control observations, central hypervolaemia produced a significant increase in the number of Fos-immunoreactive neurons in the nucleus tractus solitarius, area postrema, the caudal, intermediate and rostral parts of the ventrolateral medulla, supraoptic nucleus, paraventricular nucleus, arcuate nucleus, suprachiasmatic nucleus and median preoptic nucleus. The overall pattern of Fos expression induced by central hypervolaemia did not differ significantly between barointact and barodenervated animals. Similarly, the overall pattern of Fos expression induced by central hypovolaemia did not differ significantly between barointact and barodenervated animals, but did differ significantly from that produced by hypervolaemia. In particular, central hypovolaemia produced a significant increase in Fos expression in the same regions as above, but also in the subfornical organ and organum vasculosum lamina terminalis. In addition, compared with central hypervolaemia, hypovolaemia produced a significantly greater degree of Fos expression in the rostral ventrolateral medulla and supraoptic nucleus. Furthermore, double-labelling for tyrosine hydroxylase immunoreactivity demonstrated that neurons in the ventrolateral medulla that expressed Fos following hypovolaemia were predominantly catecholamine cells, whereas following hypervolaemia they were predominantly non-catecholamine cells. Finally, double-labelling for vasopressin immunoreactivity demonstrated that the number of Fos/vasopressin immunoreactive cells in the supraoptic nucleus was approximately 10 times greater following hypovolaemia compared with hypervolaemia, but there were very few such double-labelled neurons in the paraventricular nucleus in response to either stimulus. The results demonstrate that central hypervolaemia and hypovolaemia each induces reproducible and specific patterns of Fos expression in the medulla and hypothalamus. The degree and pattern of Fos expression was unaffected by arterial baroreceptor denervation, indicating that it is primarily a consequence of inputs from cardiac receptors, together with an increase in the level of circulating hormones such as atrial natriuretic peptide, angiotensin II or vasopressin. Furthermore, the pattern of Fos expression produced by central hypervolaemia and hypovolaemia is distinctly different from that evoked by hypertension and hypotension, respectively [Li and Dampney (1994) Neuroscience 61, 613–634], particularly in hypothalamic regions. These findings therefore indicate that the central pathways activated by changes in blood volume are, at least in part, separate from those activated by changes in arterial pressure.

Inferior olivary-induced expression of Fos-like immunoreactivity in the cerebellar nuclei of wild-type and Lurcher mice.

Earlier behavioural studies have shown that the expression of the immediate-early gene c-fos, as visualized by the immunohistochemical detection of Fos, in the inferior olive (IO) correlated closely with expression in related areas of the cerebellar nuclei. It has been speculated that the expression of c-fos within the cerebellar nuclei may be induced by enhanced spiking activity of the immunopositive neurons in the inferior olive. Two potential mechanisms may play a role in this process: a direct induction by way of the collaterals of the olivary climbing fibres to the cerebellar nuclei, or indirectly, by climbing fibre activity-induced depression of mossy fibre-parallel fibre-induced simple spike frequency of the Purkinje cells resulting in a subsequent disinhibition of the related parts of the cerebellar nuclei. In an attempt to distinguish between these possible mechanisms, we analysed Fos immunoreactivity in the olivocerebellar system of wild-type mice and in the mutant mouse Lurcher which lacks Purkinje cells. The tremorgenic agent harmaline, which is known to induce enhanced and rhythmic firing of olivary neurons was given intraperitoneally to anaesthetized mice of both genotypes. Harmaline application coincides with the induction of Fos-immunoreactive neurons in most areas of the IO in both wild-type and Lurcher mice. Both types of mice also showed enhanced expression in the larger neurons of the cerebellar nuclei. However, in the smaller, GABAergic nucleo-olivary neurons, increased c-fos expression was only observed in the wild-type mice. We conclude that: (i) increased olivary activity indeed may result in increased c-Fos expression in related areas of the cerebellar nuclei; (ii) because the indirect mode of induction is not operative in Lurcher mice, the olivary collateral innervation of the cerebellar nuclei is sufficient for c-fos induction in the larger nucleobulbar neurons in Lurcher and potentially also in wild-type mice; however (iii) for the nucleo-olivary cells an intact cerebellar cortical input is necessary to evoke increased expression of c-fos following harmaline application.

Polyamine deprivation alters formalin-induced hyperalgesia and decreases morphine efficacy

Although the exact functions of polyamines in the nervous system remain still unclear, they are thought to have a physiological role in intracellular signal processing and neurotransmission. Polyamine deprivation which consists in the reduction of both the endogenous and exogenous sources of polyamines is a promising treatment for cancer. In a previous study we have shown that this treatment provokes an analgesic effect in rats submitted to brief phasic nociceptive tests. The present study examined the effect of polyamine deprivation on pain-related behaviors and spinal c-fos expression evoked in the formalin test presumed to better reflect clinical pain, using morphine as analgesia control. Polyamine deprivation per se altered the characteristic pain-related behaviors, reducing the interphase depression of pain, without inducing changes in the spinal Fos staining. In addition this treatment prevented the antinociceptive effect of morphine both on behavioral responses and on spinal c-fos expression. In polyamine-deprived rats, despite morphine injection, nociceptive scores remained dramatically high during the intermediate and the late phases of the response and the number of Fos immunoreactive neurons remained largely higher in deeper layers than in morphine control rats. Altogether these data support a modulatory role of polyamines both on the neuronal circuitry mediating sensory information, and on mechanisms underlying morphine analgesia.

Laryngeal afferent stimulation enhances fos immunoreactivity in periaqueductal gray in the cat

The main functions of the larynx are protection of the airways, respiration, and vocalization. Previous studies have suggested a link between the mechanisms controlling vocalization and afferent feedback from the larynx. We inquired whether stimulation of the laryngeal afferents that run in the internal branch of the superior laryngeal nerve (ISLN) activates neurons of the periaqueductal gray (PAG), a midbrain region implicated in vocalization. We counted the number of neurons expressing Fos, the protein product of the immediate early gene c-fos, in the PAG. The counts were done both in experimental cats after electrical stimulation of the ISLN and nonstimulated controls. We also investigated the possible presence of nitric oxide synthase, an enzyme that synthesizes nitric oxide, in PAG neurons that respond to laryngeal afferent stimulation by double labeling for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and Fos. Fos expression was significantly greater (P < or = 0.00714) in the lateral and dorsolateral regions of the PAG in the experimental group than in the controls. The Fos-immunoreactive neurons did not contain NADPH-diaphorase, a marker for nitric oxide synthase. Our study suggests that laryngeal afferent stimulation activates neurons in discrete longitudinal columns of the PAG including the regions that have previously been shown to be involved in vocalization, and that these neurons do not contain nitric oxide synthase.

Ketamine suppresses c-fos expression in dorsal horn neurons after acute constrictive sciatic nerve injury in the rat

Fos immunoreactivity within the spinal cord in a model of neuropathic pain was studied. Dorsal horn neurons in laminae I and II exhibited selective expression within the tibial, peroneal and posterior cutaneous nerve territories which, in turn, was suppressed during ketamine but not halothane anesthesia. Fos immunoreactive neurons have a unique response pattern to neuropathic pain which is sensitive to ketamine.

Neuronal Fos activation in olfactory bulb and forebrain of male rats having erections in the presence of inaccessible estrous females

Volatile odors from estrous female rats are necessary and sufficient to induce non-contact penile erections in male rats. It is not known whether these pheromones are detected by the accessory as opposed to the main olfactory system or whether they are processed by forebrain regions that receive olfactory inputs. Using nuclear Fos immunoreactivity as a marker of neuronal activation, we asked how the detection and processing of distal cues from inaccessible estrous females, which elicited non-contact penile erections, compared with the processing of sensory cues from soiled estrous bedding which did not elicit non-contact penile erections. In Experiment 1, groups of sexually experienced males were given one of five treatments. A control group was placed on clean bedding. A second group displayed non-contact penile erections when exposed to the smell, sight and sound of an estrous female restrained behind a permeable barrier. A third group was exposed to the same stimuli as the second (an estrous female) but failed to exhibit non-contact penile erections during the first hour of testing. A fourth group was placed on soiled estrous bedding, and a fifth group was allowed two ejaculations with an estrous female. All males were perfused with 4% paraformaldehyde 2 h after the onset of these respective treatments, and their brains were later processed for Fos immunoreactivity. Non-contact penile erections were observed in males that were exposed to distal cues from an estrous female but not in males exposed to soiled estrous bedding. Males that displayed non-contact penile erections or that were exposed to estrous bedding showed significantly more neuronal Fos immunoreactivity than clean-bedding controls in the nucleus accumbens core and shell, anterior and posterior medial amygdala, bed nucleus of the stria terminalis and the medial preoptic nucleus. Even greater neuronal Fos responses occurred in these regions in mated males. In Experiment 2 these same treatments were given to another cohort of sexually experienced males. Increased neuronal Fos immunoreactivity was observed in the granule and mitral cell layers of the accessory olfactory bulb of males that were either mated or exposed to estrous bedding, but not in males that displayed non-contact penile erections in response to distal cues from an estrous female. The volatile odors which presumably caused non-contact penile erections failed to stimulate significant neuronal Fos immunoreactivity in five main olfactory bulb sites examined. Even so, it seems likely that these pheromones are detected via the main olfactory system and are subsequently processed by the same projection circuit that responds to other pheromones present in estrous bedding that are incapable of eliciting non-contact penile erections.

Colocalization of androgen receptors and mating-induced FOS immunoreactivity in neurons that project to the central tegmental field in male rats.

Bilateral lesions of the central tegmental field (CTF) in male rats virtually eliminate mating behavior. This study examined if mating-induced Fos expression (a measure of neuronal activation) and androgen receptors (AR) are colocalized in brain and spinal cord neurons which project to the CTF. Animals received unilateral injections of the retrograde tracer Fluorogold (FG) in the lateral part of the CTF (CTFl), and 10 days later were killed after ejaculating with females. Brains and spinal cords were examined for FG transport, AR-immunoreactivity (AR-ir), and Fos-immunoreactivity (Fos-ir). AR-ir and Fos-ir were visualized with fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. The CTFl received projections from AR-containing neurons in forebrain structures (bed nucleus of stria terminalis, medial preoptic area, lateral and ventromedial hypothalamus), in the central amygdala and various mid- and hindbrain structures (dorsolateral tegmentum, superior and inferior colliculi, pedunculopontine nucleus), and in the lumbosacral spinal cord (lamina X). Some of the AR-containing neurons in bed nucleus of stria terminalis and in the dorsal part of the medial preoptic area with projections to the CTFl were activated by mating. Most AR-containing neurons in spinal lamina X with projections to the CTFl were also activated by mating. Information from spinal cord and pontine nuclei and from outputs descending from the forebrain may be relayed in the CTFl. Thus, as part of a network of hormone-sensitive neurons linking brain and spinal cord mechanisms for mating, the CTFl could participate in the integration of visceral and somatic information relevant for sexual behavior.

The distribution of FOS-immunoreactive neurons in the brainstem, midbrain and diencephalon of fetal sheep in response to acute hypoxia in mid and late gestation

FOS immunohistochemistry was used to map the distribution of neuronal pathways activated by hypoxia in fetal sheep. Conscious pregnant sheep were exposed to hypoxia (7–9% O 2, 1–2% CO 2, balance N 2) for 2 h at either 100–105 days ( n=5) or 130–133 days gestation ( n=5); term is ∼147 days. The hypoxia caused cessation of breathing movements at both fetal ages, and increased FOS staining in the medulla (area postrema, dorsal motor nucleus of vagus, nucleus solitary tract, ventrolateral medulla); pons (locus coeruleus and subcoeruleus, lateral and medial parabrachial nuclei); midbrain (habenula, periaqueductal grey, substantia nigra, areas ventrolateral to Red Nucleus); and hypothalamus (anterior and lateral hypothalamic areas, paraventricular and supraoptic nuclei). The results were essentially the same at both gestational ages, except that hypoxia increased FOS-staining in the habenula only in the older fetuses. The presence of FOS protein in pontomedullary cardiorespiratory nuclei at 100–105 days gestation indicates that the peripheral chemoreceptors respond to hypoxia at this early age, and in the subcoeruleus and medial parabrachial regions of the pons is consistent with lesion studies suggesting these areas mediate the inhibition of fetal breathing in response to hypoxia. FOS staining in the ventrolateral periaqueductal grey and habenula was unexpected, and suggests that pathways normally involved in response to noxious stimuli, or which are part of the hypothalamic `defense' response are activated by hypoxia in the fetus. Some FOS-labelling could arise secondarily as a consequence of the cardiovascular and endocrine responses to hypoxia.

C-Fos induction in spinal cord neurons after renal arterial or venous occlusion.

Experiments were done in the anesthetized rat to identify the dorsal root ganglia (DRG) and the spinal cord segments that contain neurons activated by either renal venous occlusion (RVO) or by renal arterial occlusion (RAO). Fos induction, detected immunohistochemically in DRG and the spinal cord neurons, was used as a marker for neuronal activation. RVO induced Fos immunoreactivity in neurons in the DRG of spinal segments T8-L2 on the side ipsilateral to that of occlusion. The largest number of Fos-labeled neurons was found in the T11 DRG. In the spinal cord the largest number of Fos-labeled neurons was found in the ipsilateral dorsal horn of spinal segments T11-T12, predominantly in a cluster near the dorsomedial edge of laminae I-II. A few additional Fos-labeled neurons were observed in laminae IV and V. After RAO Fos-labeled neurons were found in the ipsilateral DRG of spinal segments similar to those observed to contain neurons after RVO. However, most of the Fos-labeled neurons were observed within the T12-L1 DRG. In the spinal cord Fos-labeled neurons were scattered throughout lamina I-II of the ipsilateral dorsal horn of spinal segments T8-L2, although the largest number was observed at the T13 level. Additionally, a distinct cluster of Fos-labeled neurons was observed predominantly in the region of the ipsilateral intermediolateral cell column, although a few neurons were found scattered throughout the nucleus intercalatus, central autonomic areas, and laminae IV and V of the cord bilaterally. No Fos labeling was observed in the complementary contralateral DRG or dorsal horns after either RVO or RAO. In addition, renal nerve transection prevented Fos labeling in the ipsilateral DRG and dorsal horns after RVO or RAO. Taken together, these data suggest that functionally different renal afferent fibers activate DRG neurons that may have distinct projections in the spinal cord.

Changes in c-Fos expression induced by noxious stimulation in the trigeminal spinal nucleus caudalis and C1 spinal neurons of rats after hyperbaric exposure.

The present study aims to test the hypothesis that hyperbaric exposure inhibits nociceptive processing in the trigeminal spinal nucleus caudalis and C1 spinal neurons. We investigated the c-Fos-like immunoreactivity of the brainstem and upper cervical spinal cord (C1 region) following an injection of mustard oil (15 microliters of 20%) into the nasal mucosa of pentobarbital anesthetized rats after exposure to hyperbaric (2-atmospheres, 1 h) and normobaric pressures. After the hyperbaric exposure, the mean number of Fos-immunoreactive neurons in the ipsilateral laminae I-II and III-IV of the trigeminal spinal nucleus caudalis were significantly lower than those in the normobaric condition. Similarly, the mean number of c-Fos positive neurons in the superficial layer (I-II) of the ipsilateral C1 segment were significantly reduced as compared with that in the normobaric condition. When treated with the vehicle alone, no significant difference was detected in the numbers of c-Fos positive neurons in the trigeminal spinal nucleus caudalis and C1 regions between hyperbaric and normobaric conditions. These results suggest that hyperbaric exposure may attenuate nociceptive signals from the area innervated by the trigeminal nerves at the level of both the trigeminal spinal nucleus caudalis and C1 dorsal horn.

Distribution of cortical fibers and Fos immunoreactive neurons in ventrolateral medulla and in nucleus tractus solitarius following the motor cortex stimulation in the rat

The present study demonstrates that the motor cortex (MC) stimulation induces expression of Fos-like immunoreactivity (FLI) in the rostro-caudal parts of ventrolateral medulla (VLM) and nucleus tractus solitarius (NTS). The coupling of biotinylated dextran (BD) injections with the MC stimulation also permits to identify cortical labeled fibers in the vicinity of FLI neurons in the VLM. Results suggest that the MC is involved in a direct and an indirect modulation of bulbar cardiovascular nuclei.

Differential time- and dose-related effects of haemorrhage on tyrosine hydroxylase and neuropeptide Y mRNA expression in medullary catecholamine neurons.

Hypotensive haemorrhage induces nuclear Fos expression and upregulates tyrosine hydroxylase (TH) mRNA in catecholamine-containing cell groups of the rat medulla oblongata. To shed light on the significance of the coexistence of neuropeptide Y (NPY) in aminergic neurons, the impact of graded levels of haemorrhage on temporal changes in the expression of TH and NPY mRNAs was compared; concurrent staining for Fos permitted comparisons between cells that ostensibly were and were not targeted by the stimulus. A 15% haemorrhage provoked increased NPY expression in all medullary catecholamine cell groups except the A2; these changes were detected predominantly in Fos-immunoreactive neurons (Fos-ir) at later (2-4 h) time points. Upregulation of TH and NPY mRNAs in Fos-ir neurons followed distinct time courses, with NPY responses peaking more rapidly, particularly in the C1 and C2 cell groups. Adrenergic cell groups displayed greater maximal increases in NPY expression than the A1 noradrenergic cell group while the converse was true of TH mRNA response. Increasing the severity of haemorrhage resulted in more pronounced increases in both mRNA responses in each aminergic region. These findings indicate that haemorrhage differentially affects TH and NPY expression in medullary catecholamine cell groups that participate in the maintenance of cardiovascular homeostasis. The differential nature of these responses suggests them not to be a simple consequence of metabolic alterations pursuant to increased synaptic activity. The prompt and robust NPY mRNA responses in adrenergic neurons suggests a mechanism by which peptide content of these cell groups' terminal projections is defended.

Immunohistochemical localization of caffeine-induced c-Fos protein expression in the rat brain.

Although caffeine is the most widely used central nervous system stimulant, the neuronal populations and pathways mediating its stimulant effects are not well understood. Using c-Fos protein as a marker for neuronal activation, the present study investigated the pattern of c-Fos induction at 2 hours after low locomotor-stimulant doses (1, 5, 10, and 30 mg/kg, i.p.) of caffeine and compared them with those after a higher dose (75 mg/kg, i.p.) or saline injection in adult male rats. Fos-immunoreactive neurons were counted in selected nuclei across the entire brain. Caffeine induced an increase in locomotor activity in a dose-dependent manner up to doses of 30 mg/kg and a decline at 75 mg/kg. Quantitative analysis of Fos-immunoreactive neurons indicated that no structures showed significant Fos expression at doses below 75 mg/kg or a biphasic pattern of Fos expression, as in locomotion. In contrast, caffeine at 75 mg/kg induced a significant increase compared with the saline condition in the number of Fos-immunoreactive neurons in the majority of structures examined. The structures included the striatum, nucleus accumbens, globus pallidus, and substantia nigra pars reticulata and autonomic and limbic structures including the basolateral and central nuclei of the amygdala, paraventricular and supraoptic hypothalamic nuclei, periventricular hypothalamus, paraventricular thalamic nuclei, parabrachial nuclei, locus coeruleus, and nucleus of the solitary tract. The locomotor-enhancing effects of low doses of caffeine did not appear to be associated with significant Fos expression in the rat brain.

NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation

In the present study, we examined the distribution of neurons in the parabrachial nucleus (PB), the Kölliker–Fuse nucleus (KF), the spinal trigeminal nucleus caudalis (Sp5C), the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM), which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl- d-aspartate (NMDA) receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c- fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases ( p≤0.001) of Fos-immunoreactive (-ir) neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Bötzinger/pre-Bötzinger complex, and in the ventrolateral and commissural NTS the increases were only moderately significant ( p≤0.05). With respect to the numbers of NR1-/Fos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C ( p≤0.001) and the midlevel KF ( p≤0.01) and moderately significant ( p≤0.05) in the external lateral PB, Bötzinger/pre-Bötzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses.

Manipulations during forced wakefulness have differential impact on sleep architecture, EEG power spectrum, and Fos induction

We propose a hypothesis suggesting that the most prominent experiences occurring during wakefulness activate specific clusters of neurons related to such experiences. These neurons could possibly then evoke the release of various types of sleep-inducing molecules, thereby causing different patterns of sleep architecture. In this study, we therefore sought to determine whether manipulations of behavior during wakefulness, such as forced wakefulness induced by gentle handling, forced wakefulness associated with a stressful condition such as immobilization, or forced wakefulness associated with excess intake of palatable food, could result in a variation of Fos immunoreactivity in selective brain structures and could also result in different sleep and EEG power density patterns. The results showed that the sleep-wake cycle of rats after all the experimental manipulations was different not only with respect to the control group but also among themselves. Additionally, power spectrum analysis showed an increase of 0.25–4.0 Hz in all experimental manipulations, whereas the 4.25–8.0 Hz increase occurred only in the situation of forced wakefulness plus stress. The Fos induction showed activation of cell clusters in cortical areas and telencephalic centers, in several hypothalamic nuclei, in monoaminergic cell groups, and in brain stem nuclei. The density of Fos-immunoreactive neurons varied in relation to the different paradigms of forced wakefulness. These results suggest that activation of cell clusters in the brain are related to the type of manipulation imposed on the rat during wakefulness and that such variation in cell activation prior to sleep may be associated with sleep architecture and EEG power.