Fos-like Immunoreactive Cells Research Articles

Effects of SR140333, a selective non-peptide NK1 receptor antagonist, on trigemino-thalamic nociceptive pathways in the rat.

Trigeminal stimulation of C-fibers increased c-fos expression within the trigeminal nucleus caudalis (NtV) and thalamic neuronal activity which both reflect the transmission of a nociceptive message. We examined the effects on both these phenomena of the selective NK1 and NK2 receptor antagonists, SR140333 and SR48968. SR140333 (0.3, 1 and 3 micrograms/kg intravenously [i.v.]) dose-dependently, reversibly and stereoselectively antagonized the increase of contralateral thalamic activity. This compound, when given i.v. (30 micrograms/kg) or orally (10 mg/kg), also reduced the number of Fos-like immunoreactive cells particularly at the medial and caudal level of the NtV. In contrast, SR48968 did not exert any antagonistic effect either on thalamic activity or on Fos-like immunoreactivity. The data strongly suggest a preferential involvement of NK1 vs NK2 receptors in nociceptive transmission following trigeminal ganglion stimulation. Taken together, our results indicate that SR140333 could provide a potent drug for the relief of pain occurring under excessive activity of sensory trigeminal fibers.

Distinct mechanisms for expression of Fos-like immunoreactivity and synaptic potentiation in telencephalic hyperstriatum of the quail chick

In the intermediate and medial hyperstriatum ventrale (IMHV), a telencephalic region essentially involved in the initial processes of early learning tasks in poultry chicks, induction of an immediate early gene c- fos correlates significantly with the degree of learning (K.V. Anokhin, R. Mileusnic, I.Y. Shamakina, S.P.R. Rose, Effects of early experience on c- fos gene expression in the chick forebrain, Brain Res. 544 (1991) 101–107; B.J. McCabe, G. Horn, Learning-related changes in Fos-like immunoreactivity in the chick forebrain after imprinting, Proc. Natl. Acad. Sci. USA 91 (1994) 11417–11421). In slices of IMHV in vitro, on the other hand, tetanic stimulation at a low frequency induces a potentiation of synaptic responses (P.M. Bradley, B.D. Burns, A.C. Webb, Potentiation of synaptic responses in slices from the chick forebrain, Proc. R. Soc. Lond. B. 243 (1991) 19–24; T. Matsushima, K. Aoki, Potentiation and depotentiation of DNQX-sensitive fast excitatory synaptic transmission in telencephalon of the quail chick, Neurosci. Lett. 185 (1995) 179–182). In this study, we have examined a possible causal link between these two forms of activity-dependent processes, c- fos expression and synaptic potentiation. C- fos was visualized immunohistochemically using antibody raised against the Fos-protein, and potentiation was evaluated on the basis of field potential responses to local electrical stimulation. Tetanic stimulation (5 Hz×300 pulses) was required for potentiation, but not for c- fos expression. Conversely, a negative correlation appeared between them, and slices with relatively high density of Fos-like immunoreactive cells around the stimulation site failed to show potentiation. Furthermore, drugs similarly effective in blocking potentiation (such as AP5 (NMDA receptor antagonist) and bicuculline (GABA A receptor antagonist)) had different effects on the c- fos induction. While AP5 had minor, if any, effects on c- fos expression, bicuculline enhanced it selectively around the site of stimulation. Our results suggest that these two processes are basically distinct, and could represent different aspects in the formation of memory traces in IMHV.

Copulation activates Fos-like immunoreactivity in the male quail forebrain

It has been demonstrated using Fos immunocytochemistry that copulation activates specific cell populations in the mammalian brain. Prior to this study, no similar work has been carried out in birds. In mammals, Fos has identified brain circuits activated by genital (penile)/somatosensory and by olfactory/vomeronasal stimuli. Such inputs, of course, should play little or no role in birds (no penis, little or no role for olfaction) and a differential responsiveness could therefore be expected. Male Japanese quail ( Coturnix japonica) were allowed to interact freely with adult females and the presence of active sexual behavior, including cloacal contact movements, was confirmed in each case. Control subjects were exposed to a domestic chick (same size as an adult quail) and no sexual behavior was observed. Copulation induced the appearance of Fos-like immunoreactive (FLI) cells in the preoptic area, the hyperstriatum ventrale, parts of the archistriatum, and the nucleus intercollicularis. Induction of FLI cells was observed throughout the rostral to caudal extent of the preoptic region of males from the level of the tractus septomesencephalicus to the level of the anterior commissure, and in the rostral part of the hypothalamus to the level of the supraoptic decussation. The FLI cells did not lie directly adjacent to the third ventricle, but were located 500–1000 μm from the ventricle wall at the level of the lateral edge of the medial preoptic nucleus or, in more caudal sections, in a position ventrolateral to the bed nucleus striae terminalis. It is unlikely that the Fos induction in males resulted from copulation-induced endocrine changes because copulation did not affect plasma levels of luteinizing hormone or testosterone. It is concluded that the responses were due to copulation-associated somatosensory inputs and/or to stimuli originating from the female.

Inhalation anesthetics suppress the expression of c-Fos protein evoked by noxious somatic stimulation in the deeper layer of the spinal cord in the rat

The effects of inhalation anesthetics, nitrous oxide (N 2O) and halothane, on the expression of c-Fos protein evoked by formalin injection were studied in the spinal cord in the rat. The expression of c-Fos protein was detected by immunocytochemistry following the injection of formalin (5%, 100 μl) into the plantar surface of the left hindpaw. After 15 min of halothane (F) anesthesia, the anesthetics was switched to 40% or 70% of N 2O, 0.5% or 1.5% of F or room air (for control) immediately following the formalin injection. Two hours later the rats were sacrificed and perfused. Sections of the L4 level of spinal cord were immunostained with anti c-Fos antibody. We counted the number of Fos-like immunoreactive (FLI) cells in every specific lamina as follows; superficial layer (laminae I and II), nucleus proprius (laminae III and IV), neck of the dorsal horn (laminae V and VI) and ventral gray (laminae VII–X). Then we compared the results of each category of sample. Both N 2O and halothane suppressed the expression of c-Fos in the neck of the dorsal horn and ventral gray in a dose-dependent manner, but no effects were seen at the superficial layer or nucleus proprius. Suppression of c-Fos expression was greater under N 2O than halothane anesthesia. This finding suggests that N 2O had a stronger analgesic effect than halothane. The current study indicates that inhalation anesthetics do not act equally on every kind of spinal neurons. Both N 2O and halothane have effects on spinal neurons in the deeper layers but not on the neurons existed in laminae I–II, some of which directly receive noxious inputs. Pretreatment with 2 mg/kg of naloxone, which completely reversed the effects of morphine, did not alter the effect of 70%N 2O, suggesting that the analgesic effect of N 2O is not mediated by an intrinsic opioid mechanism at the spinal cord level. ©1977 Elsevier Science B.V. All rights reserved.

Distribution of basal-expressed c-fos-like immunoreactive cells of the periaqueductal grey matter of the rat.

The distribution of c-fos proto-oncogene expression has been studied in the periaqueductal grey matter (PAG) of non-intentional-stimulated rats by immunohistochemistry. A mean number of 53 +/- 9 Fos-like immunoreactive cells per hemiPAG, distributed into three groups, dorsolateral, lateral and ventrolateral, was found. The dorsolateral and the lateral groups appeared along the entire rostrocaudal PAG, whereas the ventrolateral group appeared only in the caudal half. These results reveal new data on the number and extent of the longitudinal columns within the hypothetical organization of the PAG. They also indicate a continuous activation of a significant population of neurones in the PAG, in agreement with the suggested role of the PAG in the modulation of internal information.

Pontine cholinergic neurons show Fos-like immunoreactivity associated with cholinergically induced REM sleep.

Recently, we showed c-fos expression in pontine nuclei in association with cholinergically induced REM sleep (REMc). Pontine cholinergic mechanisms have been implicated in the orchestration of the phasic and tonic events underlying REM sleep. Therefore, in the present study, we examined whether pontine cholinergic neurons demonstrate Fos-like immunoreactivity (Fos-LI) following cholinergically induced, sustained rapid-eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (n = 2) or carbachol (n = 3; 2.0 micrograms/0.25 microliter) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep, and with durations ranging from 27 to 40.1 min. Animals were killed immediately after the end of REMc. Compared to vehicle treated animals (0.9% saline), the animals with REMc showed a significantly higher number of Fos-LI cells in pontine regions implicated in REM sleep generation. More importantly, 11.2% (SEM +/- 0.83) of cholinergic neurons in the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei were determined to be also Fos-LI positive. In the vehicle treated animals very few Fos-LI cells were found and none of these were found to be cholinergic. These findings indicate that during REMc a transcriptional cascade involving c-fos occurs in a subpopulation of pontine cholinergic neurons.

A learned odor decreases the number of Fos-immunopositive granule cells in the olfactory bulb of young rats

Olfactory stimulation evokes a column of activity within the olfactory bulb extending from the glomerular layer to the granule cell layer that can be visualized with 2-deoxyglucose autoradiography, optical imaging, Fos protein immunohistochemistry and c- fos mRNA in situ hybridization. The Fos response to odors is typified by the activity of relatively few juxtaglomerular cells, which often occur in foci, and a large number of granule cells extending through much of the bulb. In this study, we characterized the granule cell response to an odor for which young rats had acquired a preference. Fos-like immunoreactive granule cells were quantified by image analysis, and densely stained cells were counted in a region previously shown to be responsive to peppermint odor. We found that odor-trained pups have about half the number of Fos-immunopositive superficial granule cells which respond to a learned odor than do control pups. We then determined whether there was a correlation between the juxtaglomerular cell response and the response of the superficial granule cells deep to those glomerular layer cells. We found a positive correlation between the number of juxtaglomerular cells and the number of granule cells demonstrating Fos immunoreactivity in both control and trained pups, a relationship that changed with early olfactory training.

Common patterns of increased and decreased fos expression in midbrain and pons evoked by noxious deep somatic and noxious visceral manipulations in the rat.

Immunohistochemical detection of the protein product (Fos) of the c-fos immediate early gene was used to study neuronal activation in the rostral pons and midbrain of halothane-anesthetised rats following noxious deep somatic or noxious visceral stimulation. In animals exposed only to halothane anesthesia, Fos-like immunoreactive (IR) neurons were located in the midbrain periaqueductal gray matter, tectum, and parabrachial nucleus. Following noxious stimulation of hindlimb muscle, knee joint, vagal cardiopulmonary, or peritoneal nociceptors, there was, compared to halothane-only animals, a significant increase in the numbers of Fos-like (IR) cells in the caudal ventrolateral periaqueductal gray and the intermediate gray lamina of the superior colliculus. Given the general agreement that increased Fos expression is a consequence of increased neuronal activity, the finding that a range of noxious deep somatic and noxious visceral stimuli evoked increased neuronal activity in a discrete, caudal ventrolateral periaqueductal gray region is consistent with previous suggestions that this region is an integrator of deep noxious evoked reactions. The noxious deep somatic and noxious visceral manipulations also evoked, compared to halothane-only animals, reductions in the numbers of Fos-like IR cells in the stratum opticum of the superior colliculus and the unlaminated portion of the external subnucleus of the inferior colliculus. To our knowledge this is the first report of reductions in Fos-expression in the tectum evoked by noxious stimulation. In separate experiments, the effects of noxious deep somatic and noxious visceral manipulations on arterial pressure and heart rate were measured. The noxious visceral manipulations evoked substantial and sustained falls in arterial pressure (15-45 mmHg), and heart rate (75-100 bpm), whereas the depressor and bradycardiac effects of the noxious deep somatic manipulations were weaker, not as sustained, or entirely absent. As similar distributions and numbers of both increased and decreased Fos-like IR cells were observed after each of the deep noxious manipulations, it follows that the deep noxious evoked increases and decreases in Fos expression were not secondary to the evoked depressor or bradycardiac effects.

2402 ACTH release from the anterior pituitary cultures in diabetes rats

Reproductive events in the female rat can be influenced by exposure to the odors of conspecific males. Much evidence indicates that these pheromonal effects are mediated by the accessory olfactory system (AOS); however, individual cells within the AOS that are stimulated following exposure to male odors have not yet been visualized. The present experiment was designed to determine the effect of exposure to conspecific males and male odors on signal transduction in central AOS neurons as measured by immunohistochemical detection of the induction of the fos-like protein. AOS structures examined included the accessory olfactory bulb (AOB), medial amygdala (mAMYG), and bed nucleus of the stria terminalis (BNST). Due to its importance in the control of reproductive activities and its direct link to the AOS, the ventromedial nucleus of the hypothalamus (VMH) was also examined. Adult, ovariectomized rats were injected with estradiol benzoate (EB) and 48 h later were placed in cages containing bedding material soiled by conspecific males or placed in cages containing clean bedding material. After exposure durations ranging from 10 to 180 min, the animals were sacrificed and the brains were immunohistochemically processed for detection of fos-like immunoreactivity. Another group of ovariectomized, EB-injected females was repeatedly paired with conspeciflc males for 15 min followed by 15 min of rest. Repeated matings were conducted over a 60-, 120-, or 180-min period while control animals were repeatedly exposed to clean bedding material. Quantitative analysis of the number of fos-immunopositive cells in the AOB revealed that continuous exposure to male-soiled bedding or repeated mating resulted in significant induction of foslike immunoreactivity compared to controls. Both treatments produced similar numbers of fos-like immunoreactive cells in the mitral and granule cell layers of the AOB. Fos induction was apparent after 60 min of treatment but was more prominent at 120 and 180 min. In the mAMYG, BLAST, and VMH, differences between the two treatments were noted. Exposure to male-soiled bedding for 60 min produced scattered staining in the mAMYG, BLAST, and VMH, whereas 60 min of repetitive mating resulted in a more dense distribution of fos-like immunoreactive cells in these areas. Strikingly distinct patterns of fos-like immunoreactive cells were observed in the mAMYG, BLAST, and VMH following 120 or 180 min of repetitive mating. These patterns were not present in animals exposed to male odors. The findings indicate that exposure of female rats to reproductively relevant stimuli resulted in induction of fos-like immunoreactivity within the AOS and that both olfactory and nonolfactory cues probably contributed to this effect.

Fos expression induced by changes in arterial pressure is localized in distinct, longitudinally organized columns of neurons in the rat midbrain periaqueductal gray.

The distribution of neurons expressing Fos within the periaqueductal gray (PAG) following pharmacologically induced high or low blood pressure was examined to determine (1) if PAG neurons are responsive to changes in arterial pressure (AP) and (2) the relationship of these cells to the functionally defined hypertensive and hypotensive columns in PAG. Changes in AP differentially induced robust Fos expression in neurons confined to discrete, longitudinally organized columns within PAG. Increased AP produced extensive Fos-like immunoreactivity within the lateral PAG, beginning at the level of the oculomotor nucleus. At the level of the dorsal raphe, Fos expression induced by increased AP shifted dorsally, into the dorsolateral division of PAG; this pattern of Fos labeling was maintained throughout the caudal one-third of PAG. Double-labeling for Fos and nicotinamide adenine dinucleotide phosphate diaphorase confirmed that Fos-positive cells induced by increased AP were located in the dorsolateral division of PAG at these caudal levels. Fos positive cells were codistributed, but not colocalized, with nicotinamide adenine dinucleotide phosphate diaphorase-positive cells. Decreased AP evoked a completely different pattern of Fos expression. Fos-positive cells were predominantly located within the ventrolateral PAG region, extending from the level of the trochlear nucleus through the level of the caudal dorsal raphe. Double-labeling studies for Fos and serotonin indicated that only 1-2 double-labeled cells per section were present. Saline infusion resulted in very few Fos-like immunoreactive cells, indicating that volume receptor activation does not account for Fos expression in PAG evoked by changes in AP. These results indicate that (1) substantial numbers of PAG neurons are excited by pharmacologically induced changes in AP and (2) excitatory barosensitive PAG neurons are anatomically segregated based on their responsiveness to a specific directional change in AP.

Distribution of Fos-like immunoreactivity in the caudal brainstem of the rat following noxious chemical stimulation of the temporomandibular joint.

Central expression of the protooncogene c-fos was used to examine areas receiving noxious sensory input from the rat temporomandibular joint (TMJ). Fos-like immunoreactivity (Fos-LI) in the caudal brainstem was visualized 2 hours after unilateral injection of the small-fiber-specific excitant/inflammatory irritant mustard oil into the TMJ region. Control animals received injection of either mustard oil into the subcutaneous fascia overlying the masseter muscle or mineral oil vehicle into the TMJ region. In all groups, Fos-LI was consistently observed ipsilaterally in the spinal trigeminal nucleus and cervical dorsal horn and, bilaterally, in the nucleus of the solitary tract and the ventrolateral medulla. The expression of Fos-LI ipsilaterally in the paratrigeminal nucleus was variable. Within the trigeminal sensory complex, Fos-LI was restricted to subnucleus caudalis and the caudal portions of subnucleus interpolaris near the level of the obex. Approximately 12% of Fos-LI cells in subnucleus caudalis and in the cervical dorsal horn were found in laminae III-VI. Compared to TMJ mustard oil injection, mineral oil injection produced less Fos-LI at all rostrocaudal levels, whereas subcutaneous mustard oil injection produced less Fos-LI in caudal subnucleus caudalis but similar amounts in the cervical dorsal horn. Neither of these injections yielded significant ipsilateral responses in subnucleus caudalis, indicating that Fos-LI in this region following TMJ mustard oil injection could be ascribed solely to small-fiber stimulation in the deep TMJ region. The wide rostrocaudal distribution of Fos-LI within the caudal brainstem reflects the distribution of TMJ-responsive nociceptive neurons that may underlie the spread and referral of pain from the TMJ region.

Time course of Fos-like immunoreactivity associated with cholinergically induced REM sleep

Now that the pharmacology and neuronal connectivity underlying REM sleep is beginning to be understood, it is important to begin investigations that elucidate the transcriptional response related to the REM sleep process. The present study focuses on determining the temporal development of Fos-like immunoreactivity (Fos-LI) in the dorsolateral pons following cholinergically induced, sustained rapid eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (n = 3) or carbachol (0.2-4.0 micrograms/0.25 microliters) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep, and with durations from 0 min to 120 min. Animals were killed either immediately or at various intervals after the end of REMc. Compared to vehicle- and carbachol-treated animals without REMc, the animals with REMc showed a significantly higher number of Fos-LI cells in pontine regions that have been implicated in REM sleep generation. Regions with REMc-associated Fos-LI increases included the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei; the locus coeruleus; the dorsal raphe; and the medial pontine reticular formation. More Fos-LI cells were found with longer REMc bouts than with shorter-duration REMc bouts. However, with 2 hr long REMc bouts the number of Fos-LI cells returned to control levels, suggesting that the c-fos transcriptional cascade is turned off once a threshold of REMc has been reached. These findings indicate that pontine neuronal populations implicated in REM sleep express more c-fos in the course of REMc, and that the extent of expression is related to the duration of the state.

Photoperiodic activation of fos-like immunoreactive protein in neurones within the tuberal hypothalamus of Japanese quail.

Photoperiodic stimulation of quail (Coturnix coturnix japonica) resulted in the appearance of a nuclear fos-like protein within neurones of the basal tuberal hypothalamus. On transfer to long days the number of neurones containing this fos-like immunoreactivity increased from about 150 to 700, the neurones being scattered throughout the length of the tubero-infundibular complex. This activation had occurred by early in the second long day and was maintained for at least three long days. Over this period circulating levels of LH increased seven-fold, indicating that photoperiodic induction had taken place in the birds. A similar time-course of fos-like induction occurred in castrated quail exposed to a single long day and then returned to short days. Activation mirrored the long-term changes in LH secretion found in this paradigm and fos-like immunoreactivity showed the same "carry-over" characteristics of photoperiodic induction, being maximal two days after the quail had been exposed to the single long day (and were again on short days) and when LH secretion was at its maximum. Activation of fos-like immunoreactive cells did not take place when long-day quail were transferred to short photoperiods. The evidence supports the view that the neurones being activated are involved in a specific fashion in the avian photoperiodic response.

Autonomic areas of rat brain exhibit increased Fos-like immunoreactivity during opiate withdrawal in rats

We sought to identify the brain areas that might contribute to the increased autonomic activity seen during morphine withdrawal by mapping neuronal expression of c- fos protein (Fos) and Fos-related antigens. Rats were implanted with morphine pellets or placebo pellets over a 5 day regimen and injected on day 6 with either saline or naltrexone (100 mg/kg). After a standard PAP immunocytochemical protocol, Fos-like immunoreactivity (Fos-LIR) was observed in medullary nuclei including the NTS (nucleus of the solitary tract), caudal (CVL) and rostral ventrolateral medulla (RVL). Although some Fos-LIR was seen in these areas in control rats (either morphine-implanted, saline injected, or placebo-implanted, saline or naltrexone injected), a significantly higher number of Fos-LIR-positive cells in NTS, CVL and RVL were seen after morphine withdrawal. Large numbers of Fos-like immunoreactive cells were also seen in the A5 area, the parabrachial nuclei of the pons and the locus coeruleus. Increased Fos-LIR was also detected in the paraventricular nucleus of the hypothalamus and the amygdala of morphine withdrawn rats. The Fos-LIR was co-localized with tyrosine hydroxylase immunoreactivity in many of the cells in caudal and rostral ventrolateral medulla, A5 and locus coeruleus. These data support the conclusion that autonomic areas in brain and noradrenergic/adrenergic cells in these areas are activated during morphine withdrawal and may contribute to the autonomic symptoms of opiate withdrawal.

Colocalization of TRH mRNA and Fos-like Immunoreactivity in Limbic Structures Following Amygdala Kindling

Previous studies have demonstrated that the immediate-early gene, Fos, and Fos-related antigens (Fras) are increased with discrete anatomical localization following kindled seizures. Other studies have shown that transcription of several neuropeptide genes, which may be regulated by Fos and Fras, is also altered in circumscribed anatomical structures with kindling. The patterns of Fos and thyrotropin-releasing hormone (TRH) mRNA induction following kindling were found to be remarkably similar. The present study examined whether TRH mRNA and Fos-like immunoreactivity (Fos-LI), with an antibody that recognizes both Fos and Fras, are colocalized following amygdala kindling. Rats were sacrificed 5 h after a generalized kindled seizure and the brains processed for in situ hybridization of TRH mRNA and immunohistochemistry of Fos-LI. The percentage of Fos-LI cells with TRH mRNA was 70% in the entorhinal cortex, 62% in the pyriform cortex, and 79% in the perirhinal cortex. Very dense expression of Fos-LI and TRH mRNA was also found in the granule cell layer of the dentate gyrus, but the colocalization was too numerous to count. Sham-kindled control rats had fewer Fos-LI cells and almost no TRH mRNA in any of these regions. In contrast to the limbic cortices and the dentate gyrus, Fos-LI and TRH mRNA were expressed in the hypothalamus of both sham and kindled rats, but were not colocalized. These results demonstrate that the induction of Fos, Fras, and TRH mRNA following kindled seizures are colocalized in limbic structures known to be important for kindling.

Transfer of Function to a Specific Area of the Cortex After Induced Recovery from Brain Damage.

Different methods of inducing recovery after brain damage and different mechanisms that might mediate the induced recovery have been proposed. One possible mechanism involves the ability of one part of the brain to take over the function of another. We show here in rats that (1) bar-pressing behaviour to eliminate an aversive stimulus, which becomes dramatically impaired after bilateral lesion of the frontal primary motor - sensory cortex, is recovered when the animals receive an electrical intracranial stimulation in the ventral tegmental nucleus of the brain contingent on an adequate response during performance in the behavioural task, (2) in recovered animals an area in the posterior primary motor - sensory cortex, the hindlimb motor - sensory cortex, shows a 35% increase in the number of fos-like immunoreactive cells compared to non-recovered animals, and (3) a bilateral lesion of this area in recovered animals reinstates the impairment in the performance of the behavioural task. These results indicate that an area of the cerebral cortex is able to assume the role of another area after induced recovery from brain damage.

Induction of FOS immunoreactivity in central accessory olfactory structures of the female rat following exposure to conspecific males

Reproductive events in the female rat can be influenced by exposure to the odors of conspecific males. Much evidence indicates that these pheromonal effects are mediated by the accessory olfactory system (AOS); however, individual cells within the AOS that are stimulated following exposure to male odors have not yet been visualized. The present experiment was designed to determine the effect of exposure to conspecific males and male odors on signal transduction in central AOS neurons as measured by immunohistochemical detection of the induction of the fos-like protein. AOS structures examined included the accessory olfactory bulb (AOB), medial amygdala (mAMYG), and bed nucleus of the stria terminalis (BNST). Due to its importance in the control of reproductive activities and its direct link to the AOS, the ventromedial nucleus of the hypothalamus (VMH) was also examined. Adult, ovariectomized rats were injected with estradiol benzoate (EB) and 48 h later were placed in cages containing bedding material soiled by conspecific males or placed in cages containing clean bedding material. After exposure durations ranging from 10 to 180 min, the animals were sacrificed and the brains were immunohistochemically processed for detection of fos-like immunoreactivity. Another group of ovariectomized, EB-injected females was repeatedly paired with conspeciflc males for 15 min followed by 15 min of rest. Repeated matings were conducted over a 60-, 120-, or 180-min period while control animals were repeatedly exposed to clean bedding material. Quantitative analysis of the number of fos-immunopositive cells in the AOB revealed that continuous exposure to male-soiled bedding or repeated mating resulted in significant induction of foslike immunoreactivity compared to controls. Both treatments produced similar numbers of fos-like immunoreactive cells in the mitral and granule cell layers of the AOB. Fos induction was apparent after 60 min of treatment but was more prominent at 120 and 180 min. In the mAMYG, BLAST, and VMH, differences between the two treatments were noted. Exposure to male-soiled bedding for 60 min produced scattered staining in the mAMYG, BLAST, and VMH, whereas 60 min of repetitive mating resulted in a more dense distribution of fos-like immunoreactive cells in these areas. Strikingly distinct patterns of fos-like immunoreactive cells were observed in the mAMYG, BLAST, and VMH following 120 or 180 min of repetitive mating. These patterns were not present in animals exposed to male odors. The findings indicate that exposure of female rats to reproductively relevant stimuli resulted in induction of fos-like immunoreactivity within the AOS and that both olfactory and nonolfactory cues probably contributed to this effect.

Change in Fos-like immunoreactivity in the suprachiasmatic nucleus in the adult male rat.

The circadian change in the number of the Fos-like immunoreactive (IR) cells in the suprachiasmatic nucleus (SCN) was examined in the adult male rats, whose eyeballs were enucleated two months before sacrifice. Their circadian rhythms were determined by their locomotor activity. They were sacrificed in the middle of the active phase or inactive phase. Then brain sections were cut for immunocytochemistry for Fos. A marked increase in the number of the Fos-like IR cells was observed in the inactive phase in the SCN, whereas no such increase was observed in the supraoptic nucleus. These results indicate that, in the SCN, Fos expression was changed with endogenous circadian rhythm in the free-running rat.

Cholinergically induced REM sleep triggers Fos-like immunoreactivity in dorsolateral pontine regions associated with REM sleep

We sought to determine the presence of Fos-like immunoreactive (Fos-LI) cells in the pontine brainstem following cholinergically induced sustained rapid-eye movement (REMc) sleep in cats. Microinjections (0.25 μl) of vehicle ( N = 2) or carbachol (2.0 μg/0.25 μl; N = 4) were made into the medical pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep and with durations of 15.2–57.8 min. Compared with vehicle control animals, carbachol treated animals showed a significantly higher number of Fos-LI cells in pontine regions implicated in REM sleep generation, with longer REMc bouts associated with more Fos-LI cells than the short-duration bout. Regions with REMc-associated Fos-LI increases included: the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei, where some Fos-LI cells were immunohistochemically identified as cholinergic; the locus coeruleus, where some of the Fos-LI cells were identified to be catecholaminergic; the dorsal raphe and the pontine reticular formation. These findings suggest immediate early gene activation is associated with the ubiquitous biological state of REM sleep.

Effects of sleep deprivation on fos-like immunoreactivity in the rat brain.

The molecular mechanisms involved in sleep regulation and function are largely unknown, and our understanding of the localization of such mechanisms within specific brain structures is still incomplete. In this work, we explored the consequences of sleep deprivation by the immunocytochemical mapping of the induction of the protein product of the immediate early gene c-fos in the brain of sleep-deprived rats. The expression of Fos protein is an indicator of neuronal activity. In addition, since immediate early genes can function as "third messengers" and regulate the transcription of a number of target genes, their induction could be directly relevant to the homeostasis and functions of sleep. The present results show that, as a result of 24 hours of manual sleep deprivation, Fos-like immunoreactive cells are found in specific brain areas. These areas include the medial preoptic area of the hypothalamus, the nucleus accumbens, the lateral septum, several regions of the dorsal pontine tegmentum (central gray, dorsal raphe, locus coeruleus, pedunculopontine and laterodorsal tegmental nuclei, parabrachial nuclei) and an area medial to the parabigeminal nucleus at the ponto-mesencephalic junction. Some of these areas had already been implicated in slow-wave sleep and desynchronized sleep regulation.