IL-27 abrogates receptor activator of NF-kappa B ligand-mediated osteoclastogenesis of human granulocyte-macrophage colony-forming unit cells through STAT1-dependent inhibition of c-Fos.

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression and hypothermia in the adult mouse.

Toll-like receptor 2 (TLR2) recognizes a wide range of microbial molecules and plays critical roles in the initiation of innate immune responses. In the present study, we aimed to investigate whether the depletion of microglia and macrophages with clodronate liposomes (Clod-Lips) attenuates the activation of mouse brain circuits for TLR2-mediated inflammation and hypothermia. The peripheral administration of the TLR2 agonist zymosan induced nuclear factor-κB activation in microglia and macrophages and Fos expression in astrocytes/tanycytes and neurons in the circumventricular organs (CVOs). The depletion of microglia and macrophages with Clod-Lips markedly decreased zymosan-induced Fos expression in astrocytes/tanycytes and neurons in the CVOs. The treatment with Clod-Lips significantly attenuated zymosan-induced hypothermia. These results indicate that microglia and macrophages in the CVOs participate in the initiation and transmission of inflammatory responses after the peripheral administration of zymosan.

c-Fms and the alphavbeta3 integrin collaborate during osteoclast differentiation.

beta(3) integrin-null osteoclasts are dysfunctional, but their numbers are increased in vivo. In vitro, however, the number of beta(3)(-/-) osteoclasts is reduced because of arrested differentiation. This paradox suggests cytokine regulation of beta(3)(-/-) osteoclastogenesis differs in vitro and in vivo. In vitro, additional MCSF, but not receptor activator of NF-kappaB ligand (RANKL), completely rescues beta(3)(-/-) osteoclastogenesis. Similarly, activation of extracellular signal-regulated kinases (ERKs) and expression of c-Fos, both essential for osteoclastogenesis, are attenuated in beta(3)(-/-) preosteoclasts, but completely restored by additional MCSF. In fact, circulating and bone marrow cell membrane-bound MCSFs are enhanced in beta(3)(-/-) mice, correlating with the increase in the osteoclast number. To identify components of the MCSF receptor that is critical for osteoclastogenesis in beta(3)(-/-) cells, we retrovirally transduced authentic osteoclast precursors with chimeric c-Fms constructs containing various cytoplasmic domain mutations. Normalization of osteoclastogenesis and ERK activation, in beta(3)(-/-) cells, uniquely requires c-Fms tyrosine 697. Finally, like high-dose MCSF, overexpression of c-Fos normalizes the number of beta(3)(-/-) osteoclasts in vitro, but not their ability to resorb dentin. Thus, while c-Fms and alpha(v)beta(3) collaborate in the osteoclastogenic process via shared activation of the ERK/c-Fos signaling pathway, the integrin is essential for matrix degradation.

Reduced central c-fos expression and febrile response to repeated LPS injection into periodontal tissue of rats.

Systemic injection of repeated doses of Escherichia coli lipopolysaccharide (LPS) results in attenuation of the febrile response, i.e. endotoxin tolerance, which has been fairly well characterized in rats. In the present study, we tested the hypothesis that endotoxin tolerance also occurs after repeated local injection of LPS into periodontal protection tissue. Male Wistar rats were given a gingival intra-pouch injection of sterile saline or LPS at dose of 100 microg/kg on three consecutive days. Body core temperature (Tb) was measured with a miniature datalogger. Another group of animals were used for Fos immunohistochemistry 3 h after the injections in both non-tolerant and tolerant animals. On day one we observed a polyphasic febrile response after LPS injection. The increase in body temperature started about 2 h after LPS administration and lasted 5 h. On day two this response was sensitized and on day three the febrile response was completely abolished. These data suggest that rats develop endotoxin tolerance after repeated LPS administrations into tissues within the oral cavity. Moreover, immunohistochemistry detected a reduction in LPS-induced Fos-like immunoreactivity expression in the subnucleus caudalis of spinal trigeminal nucleus and in the preoptic area of the hypothalamus (POA) in tolerant rats compared with non-tolerant animals, indicating that the endotoxin tolerance may be locally mediated in the periodontal protection tissues of rats.

Breaking biological barriers with a toothbrush.

Toothbrushing exposes epithelia and other tissues of the oral cavity to mechanical stress. Here, we investigated whether brushing induces cell wounding--plasma membrane disruption--in epithelial and other cell types in the oral cavity. Brushing of the gingivae and tongues of rats resulted in a striking increase in the number of cells positive for a marker of disruption injury. These cells included those in all strata of the gingival epithelium, and in the skeletal muscle of the tongue. Additionally, we found that brushing resulted in an increase in c-fos expression by junctional epithelial and skeletal muscle cells. Epithelial barrier function, however, was not overtly affected by brushing, despite the observed individual injuries to cells. We concluded that brushing disrupts cell plasma membrane barriers in the oral cavity and activates gene expression events that may lead to local adaptive changes in tissue architecture beneficial to gingival health.

FosB regulates expression of miR-22 during PMA induced differentiation of K562 cells to megakaryocytes.

Expression of many miRNAs is altered in different cancers and these changes are thought to play a key role in formation and progression of cancer. In chronic myelogenous leukemia (CML) a number of miRNAs are known to be down regulated as compared to normal cells. In this report we have investigated the mechanism of this down regulation by using PMA induced differentiation of CML cell line K562 to megakaryocytes as an experimental system. On treatment with PMA, expression of many down regulated miRNAs including miR-22 is induced. PMA also induces expression of several transcription factors, including FosB, EGR1 and EGR2. Our results using a number of approaches, such as promoter reporter assay, FosB knock down and Chip assay, suggest that the expression of miR-22 is regulated transcriptionally by FosB.

Osteoprotegerin diminishes advanced bone cancer pain.

Bone cancer pain most commonly occurs when tumors originating in breast, prostate, or lung metastasize to long bones, spinal vertebrae, and/or pelvis. Primary and metastatic cancers involving bone account for approximately 400,000 new cancer cases per year in the United States alone, and >70% of patients with advanced breast or prostate cancer have skeletal metastases. Whereas pain resulting from bone cancer can dramatically impact an individual's quality of life, very little is known about the mechanisms that generate and maintain this pain. To begin to define the mechanisms that give rise to advanced bone cancer pain, osteolytic 2472 sarcoma cells or media were injected into the intramedullary space of the femur of C3H/HeJ mice, and the injection hole was sealed using dental amalgam, confining the tumor cells to the bone. Twelve days after injection of 2472 tumor cells, animals showed advanced tumor-induced bone destruction of the injected femur, bone cancer pain, and a stereotypic set of neurochemical changes in the spinal cord dorsal horn that receives sensory inputs from the affected femur. Administration of osteoprotegerin, a naturally secreted decoy receptor that inhibits osteoclast maturation and activity and induces osteoclast apoptosis, or vehicle was begun at 12 days, when significant bone destruction had already occurred, and administration was continued daily until day 21. Ongoing pain behaviors, movement-evoked pain behaviors, and bone destruction were assessed on days 10, 12, 14, 17, and 21. The neurochemistry of the spinal cord was evaluated at days 12 and 21. Results indicated that osteoprotegerin treatment halted further bone destruction, reduced ongoing and movement-evoked pain, and reversed several aspects of the neurochemical reorganization of the spinal cord. Thus, even in advanced stages of bone cancer, ongoing osteoclast activity appears to be involved in the generation and maintenance of ongoing and movement-evoked pain. Blockade of ongoing osteoclast activity appears to have the potential to reduce bone cancer pain in patients with advanced tumor-induced bone destruction.

[Expression of c-fos, OPG, OPGL in rabbit mandibular distraction osteogenesis zone].

OBJECTIVE: To evaluate the possible signal transduction mechanism of the mechanical stress induced by the distraction procedure in osteocytes. METHODS: An animal model of mandibular distraction osteogenesis in rabbits was established. The expressions of c-fos, OPG and OPGL were detected by ultrasensitive S-P immunohistochemical method. RESULT: At 4 and 8 days after distraction, distraction zone showed strong positive staining of c-fos, which were apparently higher than that in distraction zone of 2, 4 and 6 weeks after consolidation. At 4 and 8 days after distraction and 2 weeks after consolidation, the expression of OPG was strong, and then wore off gradually at 4 and 6 weeks after consolidation. Weak signals of OPGL could be detected at 6 weeks after consolidation only. CONCLUSION: c-fos, OPG and OPGL are important regulators in distraction osteogenesis. c-fos is interrelated with the mechanical stress induced by the distraction procedure closely, OPG promotes new bone formation, while OPGL plays a more active role in bone remodeling.

Effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of EphA2 in osteoblasts and osteoclasts.

Porphyromonas gingivalis (Pg) as the major pathogenic bacterium of chronic periodontitis can cause alveolar bone resorption. Lipopolysaccharide (LPS) is its main virulence factor. The Eph family plays an important role in maintaining bone homeostasis. In this study, the effects of P. gingivalis lipopolysaccharide (Pg-LPS) on the expression of EphA2 in osteoblasts and osteoclasts were investigated. MC3T3-E1 cells and RAW264.7 cells were separately cultured in osteoblast-conditioned medium and osteoclast-conditioned medium to induce their differentiation into osteoblasts and osteoclasts, respectively. MC3T3-E1 cells were treated with 1 µg/mL of Pg-LPS 3, 7, and 14 d later, while RAW264.7 cells were treated with 10 µg/mL of Pg-LPS 1, 3, and 5 d later. The results have shown that Pg-LPS increased the expression of EphA2 both in osteoblasts and osteoclasts, decreased the expression of osteogenic-related genes (ALP, Sp7), and increased the expression of osteoclast-related genes (MMP9, c-fos, ACP5, CtsK, and NFATc1). Tartrate-resistant acid phosphatase (TRAP) staining illustrated that Pg-LPS promoted osteoclast differentiation and decreased the activity of alkaline phosphatase. Therefore, analysis indicates that, when treated with Pg-LPS, the expression of EphA2 is upregulated while the activity of osteoblasts and osteoclasts was reduced and increased, respectively. Our data suggest that EphA2 is closely related to the formation of osteoblasts and resorption of osteoclast and is likely to play an role in bone resorption induced in chronic periodontitis. These findings may provide information on new targets for prevention and treatment of chronic periodontitis.

Effects of pre-emptive morphine, ibuprofen or local anesthetic on fos expression in the spinal trigeminal nucleus following tooth pulp exposure in the rat.

In the present study, we used Fos expression as an index of nociceptive input to the spinal trigeminal nucleus after exposure of the coronal pulp tissue of maxillary right first molars and examined the effects of pretreatment with an opioid, a nonsteroidal anti-inflammatory drug or a local anesthetic before pulp exposure. Exposure of the tooth pulp produced a significant increase in Fos-like immunoreactivity in the superficial laminae of subnucleus caudalis; pretreatment with a control infiltration injection of saline directly above the maxillary molar 30 min before pulp exposure had no effect on Fos expression. Pretreatment with morphine 30 min before pulp exposure dose-dependently (2.5, 5, and 10 mg/kg subcutaneously) reduced Fos expression in subnucleus caudalis whereas pretreatment with ibuprofen (10-100 mg/kg subcutaneously) did not significantly affect Fos expression. Local anesthetic pretreatment was effective in reducing Fos expression only for the long acting bupivacaine; lidocaine without and with epinephrine (1:100,000) failed to significantly affect Fos expression. These results suggest that pre-emptive opioid treatment can decrease postoperative central nervous system changes associated with tooth pulp injury, and therefore, may decrease postoperative pain. Given the effects of local anesthetic on Fos expression, a combination of long acting local anesthetic with pre-emptive opioid would likely be most efficacious in decreasing postoperative dental pain.

Interactions between limbic, thalamo-striatal-cortical, and central autonomic pathways during epileptic seizure progression.

We used immunocytochemistry to determine the regional and temporal distribution of Fos protein expression in awake and unrestrained rats after a unilateral stereotaxic microinjection of a cholinergic agonist, carbachol, in the thalamic ventroposterolateral and reticular nuclei, previously shown to cause limbic and generalized convulsive seizures. The microinjection of carbachol elicits behavioral alterations including immobilization, staring, facial and jaw clonus, rearing, and falling, followed by recurrent generalized convulsive seizures, and a pattern of c-fos expression throughout the brain. In addition to the hypothalamic paraventricular and supraoptic nuclei, the initial induction of c-fos expression was observed as early as 15 minutes after the carbachol microinjection, in the piriform and entorhinal cortices, the thalamic paraventricular, the supramammilary, the lateral parabrachial nuclei, and the central gray. From 30 minutes to 2 hours, corresponding to the occurrence of motor expression of limbic and recurrent generalized convulsive seizures, Fos immunoreactivity was seen in a number of functionally related brain regions including the hippocampus, the amygdala, and the anterior thalamic nucleus (limbic system); the thalamus, the basal ganglia, and the cortex (thalamo-striatal-cortical system); and the hypothalamus, the central nucleus of the amygdala, the pons, and the medulla (central autonomic system). On the basis of the present results showing regional and temporal c-fos expression and well known neuroanatomical connections, we have constructed a neural network relating the limbic, thalamo-striatal-cortical, and central autonomic systems. This analysis provides, for the first time, neuronal circuits and pathways relating epilepsy-elicited behavioral expression of convulsive seizures and adaptive homeostatic responses and could serve as a basis for studying central autonomic regulation during epileptic disorders.

Innocuous jaw movements increase c-fos expression in trigeminal sensory nuclei produced by masseter muscle inflammation.

Muscle tenderness and pain during movements are prominent symptoms associated with persistent jaw muscle pain. However, there is virtually no information on how trigeminal neurons respond to jaw movements (JM) or muscle palpation in the presence of muscle tissue injury or myositis. In this study, we investigated the effects of innocuous JM in the presence of acute masseteric inflammation on postsynaptic responses in the trigeminal brainstem nuclei by examining the expression of c-fos. In one group of rats, unilateral injections of an inflammatory substance, mustard oil (MO: 20%, 25 microl) were made into a masseter muscle. In another group, controlled and systematic JM were provided following MO injection. Three additional groups of rats were used to control for anesthetic, JM, and injection procedure. MO injected in the masseter muscle induced a high level of Fos protein expression in four principal trigeminal regions: the subnucleus caudalis (Vc), the ventral and dorsal regions of the Vc/Vi (subnucleus interpolaris) transition zone, and the paratrigeminal nucleus (PTN). Movements following MO injection consistently produced a significantly greater level of Fos expression in all these areas, especially in the Vc/Vi transition region and caudal Vc on the ipsilateral side. Importantly, movements also induced a significantly greater level of Fos expression in the caudal Vc on the contralateral side. The present results provide the first documentation that innocuous JM in the presence of muscle inflammation significantly increase the MO-induced c-fos expression in the trigeminal brainstem nuclei, which may explain the greater pain experienced during movement of inflamed or injured muscles.

Trigeminal c-Fos expression and behavioral responses to pulpal inflammation in ferrets.

Injury to peripheral dental tissues evokes dynamic alternations in central sensory pathways. We have previously reported that transient stimulation of the dental pulp with noxious heat evokes the induction of the immediate early gene product Fos in the transitional region between subnucleus interpolaris and caudalis (Vi/Vc) and subnucleus caudalis (Vc). A question arises as to whether similar changes occur in response to inflammation to the tooth pulp. In this study, the effects of pulpal inflammation produced by bacterial lipopolysaccharide (LPS) on face-grooming behavior and trigeminal Fos expression were examined. Face-grooming behaviors were recorded daily for 3 days pre- and 24, 48 and 72 h post- LPS or saline application. All animals were perfused 72 h post- LPS or saline application. Brainstems were processed for Fos-like immunoreactivity (Fos-LI). Teeth were processed for H&E staining. Histological examination of LPS-treated teeth revealed features of an acute pulpitis. Moreover, LPS-treated animals showed greater face-grooming activity (i.e. tongue protrusions) directed to the injured tooth than the sham-operated group. The number of Fos-positive neurons was greater in the trigeminal subnucleus caudalis (Vc) and the transitional regions (Vi/Vc) in LPS-treated animals compared with sham-operated animals, and greater in the deeper laminae than the superficial laminae of each trigeminal region. LPS treatment did not evoke Fos expression in the rostral trigeminal regions above Vi/Vc. These results demonstrate that LPS-induced pulpal inflammation results in significant alterations in the Vi/Vc and Vc, and such changes may underlie the observed nociceptive behavioral responses and may play an important role in producing a symptomatic pulpitis in humans.

The effect of fentanyl on c-fos expression in the trigeminal brainstem complex produced by pulpal heat stimulation in the ferret.

We have previously shown that Fos-like immunoreactivity (Fos-LI) is evoked in the brainstem of ferrets following stimulation of pulpal A delta and C fibers originating from the maxillary canine. This study evaluated the effects of the mu-opioid receptor agonist fentanyl on Fos expression evoked by noxious thermal stimulation of the right maxillary and mandibular canines in pentobarbital/chloral hydrate anesthetized adult male ferrets. Pulpal heating evoked Fos expression in two distinct regions of the spinal trigeminal nuclear complex: the transitional region between subnucleus interpolaris and caudalis (Vi/Vc) and within the subnucleus caudalis (Vc). More Fos positive cells were expressed in both regions ipsilateral to the site of stimulation compared with the contralateral side (P < 0.05, ANOVA). Pretreatment with fentanyl significantly and dose-dependently suppressed the number of Fos positive cells in both the Vi/Vc transitional region and Vc (P < 0.05, ANOVA). The suppressive effect of fentanyl on Fos expression was blocked by the intravenous administration of naloxone, an opioid antagonist, indicating a specific opioid receptor effect. In addition, opioid receptor antagonism with naloxone alone enhanced Fos expression in Vi/Vc and Vc in response to heat stimulation. The administration of naloxone without heat stimulation failed to evoke Fos expression in Vi/ Vc and Vc. These findings suggest that the activation of trigeminal Vi/Vc and Vc neurons by noxious dental heat stimulation is controlled by a naloxone sensitive endogenous opioid system as indicated by Fos expression. Collectively, these results suggest that neuronal populations in Vi/Vc and Vc regions may contribute to pain responses to noxious dental stimulation and these responses can be modulated by both endogenous and exogenous opioids.

The effect of the NK1 receptor antagonist CP-99,994 on emesis and c-fos protein induction by loperamide in the ferret.

The site of the anti-emetic action of the neurokinin1 receptor antagonist CP-99,994 was studied in the ferret using the centrally acting opiate receptor agonist loperamide at a dose (0.5 mg/kg s.c.) which induced emesis in all animals tested. CP-99,994 (1 mg/kg, s.c.x2) abolished the emetic response (retching and vomiting) and the behaviours (licking, wet dog shakes, mouth scratching and gagging) induced by loperamide over a 2-h observation period. The enantiomer of this compound CP-100,263 (1 mg/kg, s.c.x2) did not have any significant effect on emesis or related behaviours. Loperamide (0.5 mg/kg s.c.) administration (but not its vehicle) resulted in dense fos-like immunoreactivity (FLI) mainly throughout the rostro-caudal extent of the nucleus tractus solitarius but not the area postrema. Although CP-99,994 (1 mg/kgx2) abolished the loperamide-induced emesis, it did not have any statistically significant effect on FLI in the brainstem. In loperamide and CP-100,263 (1 mg/kg, s.c.x2) treated animals FLI was comparable to that in animals treated with loperamide and CP-99,994. The results from this study taken together with those from previous studies indicate that loperamide exerts its emetic effect via nucleus tractus solitarius dendrites projecting into the area postrema. The lack of significant effect of CP-99,994 on the FLI induced by loperamide in this nucleus suggests that it is acting at a site "deep" in the nucleus tractus solitarius or elsewhere. The marked reduction in behaviours associated with loperamide administration by CP-99,994 provides a preliminary indication that NK1 receptor antagonist (as represented by CP-99,994) may in the clinic have effects on behaviours induced by emetic agents in addition to their previously described effects on retching and vomiting.

Fos expression in the ferret trigeminal nuclear complex following tooth pulp stimulation.

The aim of this study was to establish which regions of the trigeminal nucleus are activated by tooth pulp stimulation in the normal ferret. The distribution of Fos-like immunoreactivity was examined following electrical stimulation of the tooth pulp in the awake and anaesthetized ferret. Stimulus-specific labelling was found in subnuclei caudalis and oralis of the trigeminal spinal nucleus. Three groups of chronically prepared animals; conscious, anaesthetized (alphaxolone/alphadolone) and anaesthetized-paralysed (alphaxolone/alphadolone with gallamine triethiodide), received electrical stimuli to both the upper and lower left canine teeth (1 Hz train of 3 x 0.5 ms at 200 Hz) at an amplitude of 10 times the threshold of the jaw opening reflex. Three control groups were treated identically except no stimulus was given. In stimulated anaesthetized and anaesthetized-paralysed animals, Fos-positive profiles were seen in laminae I and II of subnucleus caudalis and in the medial part of subnucleus oralis. There was no labelling evident in subnucleus interpolaris or the main sensory nucleus, or contralaterally in any of the subnuclei. In all conscious stimulated animals there was additional bilateral Fos-positive labelling, mainly in the deeper laminae of subnucleus caudalis. This bilateral labelling was not stimulus-specific as it was also seen in conscious non-stimulated animals. After correction for this bilateral labelling no significant difference was found between conscious, anaesthetized and anaesthetized-paralysed groups of stimulated animals or between the different groups of control animals. These results support the concept that the rostral parts of the trigeminal spinal nucleus are involved in processing of nociceptive input. They also demonstrate that light alphaxolone/alphadolone anaesthesia has no effect on stimulus-specific Fos expression following tooth pulp stimulation. The second aim of this study was to develop a clearly defined model for future studies in which Fos expression is no different to that seen in the conscious state. As in the conscious animal, labelling not associated with the stimulus is difficult to distinguish from stimulus specific labelling, further studies using this model of trigeminal nociceptive pathways would be best carried out in lightly anaesthetized animals.

Fos induction following systemic kainic acid: early expression in hippocampus and later widespread expression correlated with seizure.

We determined the distribution of Fos protein expression in a model of generalised epilepsy caused by excessive neuronal excitation. Fos immunoreactivity was mapped in forebrain in unrestrained rats, previously prepared with an indwelling venous catheter, after the intravenous administration of kainic acid (10 mg/kg). We determined cerebral activation following various periods of exposure to kainic acid by using intravenous administration of pentobarbitone to prevent further activation. Within a few minutes, kainic acid caused episodes of staring, sniffing, wet dog shakes, nodding and chewing. Fos induction occurred initially and simultaneously in hippocampus, subiculum, septum and entorhinal cortex as early as 9.5 min after kainate injection. After up to 40 min of staring, sniffing, wet dog shakes, nodding and chewing, Fos induction was not further increased above levels present within the first 9.5 min. After 56 +/- 6 min a motor convulsion occurred, initially affecting the jaw, head and tail and variably extending to the forelimbs, trunk or hindlimbs. Following the convulsive event, additional Fos was expressed in hippocampus, thalamus, caudate-putamen and other subcortical structures and in the cerebral cortex. Fos induction was sometimes asymmetric in entorhinal, visual, piriform, cingulum, parietal and frontal cortices and in amygdala and dorsal endopiriform area. Electroencephalographic recordings after a few minutes exposure to kainic acid revealed an increased amplitude of fast frequencies in hippocampus which appeared to correlate with Fos induction in this structure. The findings are generally consistent with the reported distribution and slow development of kainic acid-induced seizure activity using electrophysiological and deoxyglucose methods. However, the Fos distribution suggests that (i) hippocampal, possibly dentate, activation precedes significant activation elsewhere, (ii) extensive involvement of other cerebral structures and cerebral cortex occurs simultaneously and correlates with motor seizures and (iii) brain structures can be recruited asymmetrically.

Microinjections of muscimol into lateral superior colliculus disrupt orienting and oral movements in the formalin model of pain.

An important reaction in rodent models of persistent pain is for the animal to turn and bite/lick the source of discomfort (autotomy). Comparatively little is known about the supraspinal pathways which mediate this reaction. Since autotomy requires co-ordinated control of the head and mouth, it is possible that basal ganglia output via the superior colliculus may be involved; previously this projection has been implicated in the control of orienting and oral behaviour. The purpose of the present study was therefore, to test whether the striato-nigro-tectal projection plays a significant role in oral responses elicited by subcutaneous injections of formalin. Behavioural output from this system is normally associated with the release of collicular projection neurons from tonic inhibitory input from substantia nigra pars reticulata. Therefore, in the present study normal disinhibitory signals from the basal ganglia were blocked by injecting the GABA agonist muscimol into different regions of the rat superior colliculus. c-Fos immunohistochemistry was used routinely to provide regional estimates of the suppressive effects of muscimol on neuronal activity. Biting and licking directed to the site of a subcutaneous injection of formalin (50 microliters of 4%) into the hind-paw were suppressed in a dose-related manner by bilateral microinjections of muscimol into the lateral superior colliculus (10-50 ng; 0.5 microliter/side); injections into the medial superior colliculus had little effect. Bilateral injections of muscimol 20 ng into lateral colliculus caused formalin-treated animals to re-direct their attention and activity from lower to upper regions of space. Muscimol injected unilaterally into lateral superior colliculus elicited ipsilateral turning irrespective of which hind-paw was injected with formalin. Oral behaviour was blocked when the muscimol and formalin injections were contralaterally opposed; this was also true for formalin injections into the front foot. Interestingly, when formalin was injected into the perioral region, injections of muscimol into the lateral superior colliculus had no effect on the ability of animals to make appropriate contralaterally directed head and body movements to facilitate localization of the injected area with either front- or hind-paw. These findings suggest that basal ganglia output via the lateral superior colliculus is critical for responses to noxious stimuli which entail the mouth moving to and acting on the foot, but not when the foot is the active agent applied to the mouth. The data also suggest that pain produces a spatially non-specific facilitation of units throughout collicular maps, which can be converted into a spatially inappropriate signal by locally suppressing parts of the map with the muscimol.

Catecholamine and oxytocin cells respond to hypovolaemia as well as hypotension.

Medullary catecholamine and hypothalamic neurosecretory oxytocin cells are activated by hypotension, but previous studies have provided uncertain outcomes concerning their ability to respond to a purely hypovolaemic stimulus. In the present study, injections of PEG/water and pentolinium were used to elicit non-hypotensive, isosmotic hypovolaemia and isovolaemic, isosmotic hypotension, respectively, in conscious rats. Animals were sacrificed 2 h after treatment. Immunolabelling for Fos, tyrosine hydroxylase and oxytocin established that these two stimuli activate almost identical populations of catecholamine neurons in the ventrolateral and dorsomedial medulla, and very similar populations of oxytocin cells in the supraoptic and paraventricular nuclei of the hypothalamus.

Hypovolaemic and osmotic stimuli induce distinct patterns of c-Fos expression in the rat subfornical organ.

Investigation of the effects of osmotic and hypovolaemic stimuli on the pattern of subfornical organ (SFO) c-fos expression yielded three distinct distributions of activated neurons. Hypertonic saline induced c-fos expression in peripheral SFO only. PEG/water induced c-fos in the central core of SFO and PEG/saline induced c-fos in both the central and peripheral regions. Isotonic saline failed to induce SFO c-fos expression. These results are consistent with the notion of functional segregation within the SFO.