Fos Immunohistochemistry Research Articles

Dissection of corticotropin-releasing factor system involvement in locomotor sensitivity to methamphetamine

Sensitivity to the euphoric and locomotor-activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin-releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF-R1, CRF-R2, CRF, or the CRF-related peptide Urocortin 1 (Ucn1). CRF-R1 KO mice did not differ from wild-type mice in sensitization to MA, and pharmacological blockade of CRF-R1 with CP-154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA-induced sensitization. Deletion of either of the endogenous ligands of CRF-R1 (CRF, Ucn1) either enhanced or had no effect on MA-induced sensitization, providing further evidence against a role for CRF-R1 signaling. Interestingly, deletion of CRF-R2 attenuated MA-induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF-R2-dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF-R2 involvement in neural processes associated with MA addiction.

P.3.f.006 Deletion of CB2 cannabinoid receptor induces schizophrenia-related behaviours in mice

The tachykinin NK1 receptor was suggested to be involved in psychiatric disorders, but its antagonists have failed to be effective as antidepressants in clinical trials. Hemokinin-1 (HK-1), the newest tachykinin, is present in several brain regions and activates the NK1 receptor similarly to substance P (SP), but acts also through other mechanisms. Therefore, we investigated the roles of the Tac4 gene-derived HK-1 in comparison with SP and neurokinin A (NKA) encoded by the Tac1 gene, as well as the NK1 receptor in anxiety and depression-like behaviors in mice.Mice lacking SP/NKA, HK-1 or the NK1 receptor (Tac1−/−, Tac4−/−, Tacr1−/−, respectively) compared to C57Bl/6 wildtypes (WT), and treatment with the NK1 antagonist CP99994 were used in the experiments. Anxiety was evaluated in the light-dark box (LDB) and the elevated plus maze (EPM), locomotor activity in the open field (OFT) tests. Hedonic behavior was assessed in the sucrose preference test (SPT), depression-like behavior in the tail suspension (TST) and forced swim (FST) tests. FST-induced neuronal responsiveness was evaluated with Fos immunohistochemistry in several stress-related brain regions.In the LDB, Tac4−/− mice spent significantly less, while Tacr1−/− and CP99994-treated mice spent significantly more time in the lit compartment. In the EPM only Tac4−/− showed reduced time in the open arms, but no difference was observed in any other groups. In the OFT Tac4−/− mice showed significantly reduced, while Tac1−/− and Tacr1−/− animals increased motility than the WTs, but CP99994 had no effect. NK1−/− consumed markedly more, while Tac4−/− less sucrose solution compared to WTs. In the TST and FST, Tac4−/− mice showed significantly increased immobility. However, depression-like behavior was decreased both in cases of genetic deletion and pharmacological blockade of the NK1 receptor. FST-induced neuronal activation in different nuclei involved in behavioral and neuroendocrine stress responses was significantly reduced in the brain of Tac4 −/− mice.Our results provide the first evidence for an anxiolytic and anti-depressant-like actions of HK-1 through a presently unknown target-mediated mechanism. Identification of its receptor and/or signaling pathways might open new perspectives for anxiolytic and anti-depressant therapies.

Effect of Complete Maternal and Littermate Deprivation on Morphine-Induced Fos-Immunoreactivity in the Adult Male Rat Brain

Previous research has demonstrated that rats reared in isolation from their dam and littermates show altered behavioral responsiveness to both natural and drug-mediated rewards. This study examined the effects of complete maternal deprivation through the use of artificial rearing on neural activation after acute morphine exposure in adulthood. Male rats were either artificially reared (AR) or maternally reared (MR) from postnatal day 5 to 21. In adulthood (4 mo old), rats received a single injection of morphine sulfate (10 mg/kg) or equivolume saline 2 h before perfusion and brain extraction. Neural activation was quantified using Fos immunohistochemistry. Analyses of several brain regions revealed a consistent pattern of differences between AR and MR rats. Specifically, relative to MR rats, AR rats showed significantly greater morphine-induced Fos-immunoreactivity in brain regions associated with the mesocorticolimbic "reward" pathway. These results support the hypothesis that functional activity in reward neurocircuitry can be altered by early life experience.

Developmental distribution of vestibular nuclear neurons responsive to different speeds of horizontal translation

To examine whether subgroups of vestibular nuclear neurons encode different frequency oscillation of horizontal linear motion, Fos immunohistochemistry was used to document neuronal subpopulations that were functionally activated by such otolithic stimulations. Conscious rats at P7, P14 and adult were subjected to sinusoidal linear acceleration along the transverse axis on the horizontal plane. Labyrinthectomized and/or stationary controls showed only sporadically scattered Fos-labeled neurons in the vestibular nuclei, confirming otolithic origin of c-fos expression. In each age group, Fos-labeled neurons responsive to high frequency stimulation (> 1.5 Hz) were clustered in the lateral region of the medial vestibular nucleus while those to low frequency stimulation (0.5–1.0 Hz) were found in the medial portion of the medial vestibular nucleus. The number of these neurons increased with age. No apparent frequency-related distribution pattern of Fos-labeled neurons was observed in other vestibular nuclei and subgroups. Our findings therefore reveal subpopulations of central vestibular neurons responsive to different stimulus frequencies that correspond to head motions ranging from tilt to translation.

Schwann cell p75NTR prevents spontaneous sensory reinnervation of the adult spinal cord

Schwann cells are attractive candidates for repair of the injured spinal cord. Transplanted Schwann cells are permissive to regeneration, but their ability to promote regeneration into distal spinal cord remains weak despite their production of growth-promoting neurotrophins. Schwann cell activation such as that which accompanies peripheral nerve injury results in massive upregulation of the p75(NTR) pan-neurotrophin-receptor. Here we test the hypothesis that this p75(NTR) upregulation following dorsal root injury limits availability of endogenous neurotrophin to axons and restricts regeneration of injured axons into the spinal cord. We injured dorsal roots (fourth cervical to second thoracic) in mice lacking the neurotrophin-binding domain of p75(NTR) and in wild-type littermates. Axonal regeneration was assessed by selective tracing of neurotrophin-responsive and non-responsive dorsal root ganglion neurons. Functional reinnervation of the spinal cord was assessed in behavioural experiments and via Fos immunohistochemistry following formalin injection into the forepaw. We also measured levels of nerve growth factor and neurotrophin-3 following nerve injury in knockout and wild-type mice, and used Trk-Fc receptor chimeras to block nerve growth factor and neurotrophin-3 signalling in dorsal root ganglion/Schwann cell co-cultures and following dorsal root injury in vivo. The roles of neuronal and glial p75(NTR) were assessed in transplant experiments in vivo and in co-cultures. We found that nerve growth factor and neurotrophin-3-responsive axons regenerated into the spinal cord of p75(NTR) knockout mice where they made functional connections with dorsal horn neurons. Despite equivalent levels of nerve growth factor and neurotrophin-3 in wild-type and knockout mice, successful regeneration in knockouts was neurotrophin-dependent. Transplantation of p75(-/-) neurons into a wild-type environment, p75(-/-) peripheral nerve grafts into the injured p75(+/+) spinal cord, and dissociated sensory neuron/Schwann cell co-cultures showed that the absence of p75(NTR) from glia, not from neurons, promotes regeneration. These findings indicate that Schwann cell p75(NTR) restricts neurotrophin availability to the extent that it prevents spontaneous sensory axon regeneration into the spinal cord. The implication is that inactivating p75(NTR) in Schwann (or olfactory ensheathing) cells may enable axons to grow beyond transplants, improving the outcome of spinal cord injury.

Estradiol-17β-responsive A1 and A2 noradrenergic cells of the brain stem project to the bed nucleus of the stria terminalis in the ewe brain: A possible route for regulation of gonadotropin releasing hormone cells

We have studied brain stem cells in the ewe brain that project to the bed nucleus of the stria terminalis (BNST) and determined if these cells are activated by estradiol-17β. This would predicate an indirect role in the estradiol-17β regulation of gonadotropin releasing hormone (GnRH) cells, since these receive input from the BNST. Ovariectomized ewes received 50 μg estradiol-17β benzoate (i.m.) 1 h prior to brain collection, so that activated cells could be identified by Fos immunohistochemistry. Retrograde tracer (FluoroGold; FG), was injected into the three divisions of the BNST and labeled cells were mapped to the A1 and A2 regions and the parabrachial nucleus (PBN) of the brain stem. With FG injection into the dorsal and lateral BNST, all FG-containing cells in the caudal A1 and 45% of those in A2 stained for dopamine-β-hydroxylase (DBH), indicating noradrenergic type. No FG-labelled cells in the PBN were DBH-positive. In A1 and A2 respectively, 42% and 46% of FG-labelled cells were Fos-positive, with no double-labeling in cells of the PBN. In ewes receiving FG injections into the ventral BNST, estrogen receptor (ER)α-immunoreactive nuclei were found in 82% of A1-FG labeled and 38% of A2-FG labeled cells. No FG-labelled cells of the PBN were ERα-positive. Anterograde tracing from A1 with microruby injection identified projections to the PBN, BNST and preoptic area (POA). Thus, A1 and A2 noradrenergic neurons project to the BNST in the ewe brain, express ERα and are activated by estradiol-17β. These noradrenergic, estrogen-responsive cells may provide indirect input to GnRH cells, via the BNST.

Different antipsychotics elicit different effects on magnocellular oxytocinergic and vasopressinergic neurons as revealed by Fos immunohistochemistry

Acute administration of antipsychotics elicits regionally distinct patterns of Fos expression in the rat brain. Stimulation of oxytocin (OXY) and vasopressin (AVP) release in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei indicates that antipsychotics may play a role in autonomic, neuroendocrine, and behavioral processes. This study was focused to reveal the responsiveness of hypothalamic OXY- and AVP- producing magnocellular neurons, in terms of quantitative and topographical distinctions, to antipsychotics displaying different pharmacological profiles. Naive male Wistar rats were injected intraperitoneally with haloperidol (1 mg/kg), clozapine (30 mg/kg), olanzapine (30 mg/kg), risperidone (2mg/kg), and vehicle (5% chremophor) and were sacrificed 60 min later by a fixative. Fos, Fos/OXY, and Fos/AVP labelings were visualized by immunohistochemistry in the SON, 5 accessory (ACS) cell groups, and 4 distinct PVN subdivisions using a computerized light microscope. Most apparent activation of single Fos, Fos/OXY, and Fos/AVP cells was induced by clozapine and olanzapine; effects of risperidone and haloperidol were substantially lower; no colocalizations were revealed in naive or vehicle treated control rats. The data indicate the existence of a substantial diversity in the stimulatory effect of the selected antipsychotics on quantity of Fos, Fos/OXY, and Fos/AVP immunostainings with the preferential action of the atypicals clozapine over olanzapine and little effects of risperidone and haloperidol. Variabilities in Fos distribution in the PVN, SON, and ACS induced by antipsychotics may be helpful to understand more precisely the extent of their extra-forebrain actions with possible presumption of their functional impact and side effect consequences.

Selective activation of dorsal raphe nucleus-projecting neurons in the ventral medial prefrontal cortex by controllable stress.

Exposure to uncontrollable stressors produces a variety of behavioral consequences (e.g. exaggerated fear, reduced social exploration) that do not occur if the stressor is controllable. In addition, an initial experience with a controllable stressor can block the behavioral and neural responses to a later uncontrollable stressor. The serotonergic (5-HT) dorsal raphe nucleus (DRN) has come to be viewed as a critical structure in mediating the behavioral effects of uncontrollable stress. Recent work suggests that the buffering effects of behavioral control on the DRN-dependent behavioral outcomes of uncontrollable stress require ventral medial prefrontal cortex (mPFCv) activation at the time of behavioral control. The present studies were conducted to directly determine whether or not controllable stress selectively activates DRN-projecting neurons within the mPFCv. To examine this possibility in the rat, we combined retrograde tracing (fluorogold iontophoresed into the DRN) with Fos immunohistochemistry, a marker for neural activation. Exposure to controllable, relative to uncontrollable, stress increased Fos expression in fluorogold-labeled neurons in the prelimbic region (PL) of the mPFCv. Furthermore, in a separate experiment, a prior experience with controllable stress led to potentiation of Fos expression in retrogradely labeled PL neurons in response to an uncontrollable stressor 1 week later. These results suggest that the PL selectively responds to behavioral control and utilizes such information to regulate the brainstem response to ongoing and subsequent stressors.

The anterior cingulate cortex is a target structure for the anxiolytic-like effects of benzodiazepines assessed by repeated exposure to the elevated plus maze and Fos immunoreactivity

Prior experience with the elevated plus maze (EPM) increases the avoidance of rodents to the open arms and impairs the anxiolytic-like effects of benzodiazepines on the traditional behaviors evaluated upon re-exposure to the maze, a phenomenon known as one-trial tolerance. Risk assessment behaviors are also sensitive to benzodiazepines. During re-exposure to the maze, these behaviors reinstate the information-processing initiated during the first experience, and the detection of danger generates stronger open-arm avoidance. The present study investigated whether the benzodiazepine midazolam alters risk assessment behaviors and Fos protein distribution associated with test and retest sessions in the EPM. Naive or maze-experienced Wistar rats received either saline or midazolam (0.5 mg/kg i.p.) and were subjected to the EPM. Midazolam caused the usual effects on exploratory behavior, increasing exploratory activity of naive rats in the open arms and producing no effects on these conventional measures in rats re-exposed to the maze. Risk assessment behaviors, however, were sensitive to the benzodiazepine during both sessions, indicating anxiolytic-like effects of the drug in both conditions. Fos immunohistochemistry showed that midazolam injections were associated with a distinct pattern of action when administered before the test or retest session, and the anterior cingulate cortex, area 1 (Cg1), was the only structure targeted by the benzodiazepine in both situations. Bilateral infusions of midazolam into the Cg1 replicated the behavioral effects of the drug injected systemically, suggesting that this area is critically involved in the anxiolytic-like effects of benzodiazepines, although the behavioral strategy adopted by the animals appears to depend on the previous knowledge of the threatening environment.

In sexually naive anestrous ewes, male odour is unable to induce a complete activation of olfactory systems

Exposure of anestrous females to a ram or his odour induces rapid secretion of LH that can lead to ovulation. This response is mediated by the main olfactory system (MOS) in sexually experienced ewes. The accessory olfactory system (AOS) has a minor, but unclear, role in this response. In sexually naive ewes, male odour is less effective than in experienced ewes, but the neural pathway involved is not known. In our experiment, we investigated the brain regions activated by the male or his odour in young and sexually naive anestrous ewes using immunohistochemistry for Fos alone, Fos double-labeled with Gonadotropin-Releasing Hormone (GnRH) or with Tyrosine Hydroxylase (TH). Ram odour caused neural activation in the main olfactory bulb (MOB) and not the AOS. Exposure to a male induced significant activation in the MOB, the cortical and medial nuclei of the amygdala and the ventromedial hypothalamus but not in cortical areas, or in GnRH or TH positive neurons. These results confirm the predominant role of the MOS in the detection of olfactory signals in sheep and underline the importance of learning processes.

Inhibitory effect of lidocaine on pain and itch using formalin-induced nociception and 5′-guanidinonaltrindole-induced scratching models in mice: Behavioral and neuroanatomical evidence

The aim of this study was to establish the effect of lidocaine, a local anesthetic, on pain and itch using formalin-induced nociception and kappa opioid antagonist-induced scratching models in mice. We investigated if local intradermal pretreatment (at − 10 min) with lidocaine N-ethyl bromide (lidocaine, 2%, 0.1 ml) antagonizes behavioral responses and prevents c- fos expression induced by pain and itch. Male, Swiss Webster mice (25–30 g, n = 6–10) were used. Formalin (5%, 20 µl, s.c.) or saline was administered to the right dorsal hindpaw and the time spent licking this paw was recorded at 0–10 min and 20–35 min. For itching, mice were challenged with 5′-guanidinonaltrindole (GNTI, 0.3 mg/kg, s.c., behind the neck) or saline and the number of neck-directed scratches with hindpaws was counted for 30 min. C- fos immunohistochemistry was performed in lumbar (for pain) and cervical (for scratching) spinal sections 2 h after the respective treatments. We found that lidocaine (a) antagonizes both formalin-induced pain and GNTI-induced scratching and (b) prevents c- fos expression evoked by pain (medial side of the superficial layer and deeper layers of the dorsal horn) and itch (lateral side of the superficial layer of the dorsal horn). Additionally, GNTI caused c- fos activation in mice wearing an Elizabethan collar (to prevent scratching of the neck) suggesting that GNTI provokes c- fos expression by inducing an itch sensation. Our results highlight the antipruritic properties of lidocaine and argue for its comprehensive clinical testing against pruritic states.

Stress-induced activity in the locus coeruleus is not sensitive to stressor controllability

An important factor in determining the adverse consequences of a stress experience is the degree to which an individual can exert control over the stressor. Stressor controllability is known to influence brain norepinephrine levels, but its impact on activity in noradrenergic cell bodies is unknown. In the present study we investigated whether noradrenergic neurons within the locus coeruleus (LC), the major source of forebrain norepinephrine, are sensitive to stressor controllability. We exposed adult male Sprague–Dawley rats to escapable or yoked inescapable tailshock and assessed LC activity by measuring changes in the immediate early gene c- fos and the enzyme tyrosine hydroxylase (TH). We used in situ hybridization to measure levels of c- fos mRNA, TH mRNA, and TH primary transcript in the LC. In all three cases stress exposure increased expression relative to an unstressed homecage control group, but expression did not differ between controllable and uncontrollable stress. To further examine whether stressor controllability influences the number of stress-responsive LC neurons we performed double-label immunohistochemistry for TH and Fos. Again we detected an overall effect of stress, which did not differ between controllable and uncontrollable stress. We conclude that exposure to stress robustly increases expression of TH and c- fos in the LC, but this effect is not influenced by stressor controllability. To the extent that the expression of these genes reflects degree of neuronal activation, our results suggest that stress-induced activity of noradrenergic cell bodies in the LC is not sensitive to stressor controllability.

Intrahippocampal cholinesterase inhibition induces epileptogenesis in mice without evidence of neurodegenerative events

The mechanisms of epileptogenesis remain largely unknown and are probably diverse. The aim of this study was to investigate the role of focal cholinergic imbalance in epileptogenesis. To address this question, we monitored electroencephalogram (EEG) activity up to 12 weeks after the injection of a potent cholinesterase (ChE) inhibitor (soman) at different doses (0.53, 0.75, 1, 2, 2.8, 4 and 11 nmol) into the right dorsal hippocampus of C57BL/6 mice. Different parameters were used to choose the dose for a focal model of epileptogenesis (mainly electrographic patterns and peripheral ChE inhibition). The pattern of neuronal activation was studied by Fos immunohistochemistry (IHC). Brain damage was evaluated by hemalun-phloxin, neuronal nuclei antigen IHC and silver staining. Glial fibrillary acidic protein IHC was used to evaluate astroglial reaction. Finally, long-term behavioral consequences were characterized. At the highest dose (11 nmol), soman quickly evoked severe signs, including initial seizures and promoted epileptogenesis in the absence of tissue damage. With lower doses, late-onset seizures were evidenced, after 1–4 weeks depending on the dose, despite the absence of initial overt seizures and of brain damage. Only a weak astroglial reaction was observed. Following injection of 1 nmol, Fos changes were first evidenced in the ipsilateral hippocampus and then spread to extrahippocampal areas. A selective deficit in contextual fear conditioning was also evidenced two months after injection. Our data show that focal hypercholinergy may be a sufficient initial event to promote epilepsy and that major brain tissue changes (cellular damage, edema, neuroinflammation) are not necessary conditions.

Methadone is substantially less effective than morphine in modifying locomotor and brain Fos responses to subsequent methadone challenge in rats

Heroin addicts can benefit from methadone substitution therapy. However, little is known about the significance of pre-exposure to opioids for psychoactive effects of methadone. We modeled some behavioral and neurobiological aspects of the opioid abuse-related phenomena in Sprague-Dawley rats, using morphine (10 mg/kg/day) or methadone (1 or 2 mg/kg/day) treatment (14 doses over a 16-day period) followed by 2-week withdrawal and methadone challenge; control rats were given 0.9% NaCl treatment and methadone challenge by the same schedule. Locomotor response to the challenge showed substantial enhancement only after the morphine treatment. Fos immunohistochemistry in selected brain regions including cortex, nucleus accumbens, striatum and some parts of the hippocampus, thalamus and amygdala also revealed marked differences between the effects of the tested treatments. Sensitization of Fos response was found in a few regions of the morphine-treated rats. The rats given the higher methadone dose treatment showed a fairly weak tendency for sensitization that reached significance only in somatosensory cortex layer IV. The rats given the lower methadone dose treatment showed a weak while widespread tendency for an opposite change, which reached significance in cingulate cortex layer II/III and resulted in significant differences in Fos response between these rats and the morphine-treated rats in most regions studied. These results indicate that lasting neuroplastic changes associated with the sensitization caused by (sub)chronic exposure to opioids are relatively mild for methadone as compared to those caused by morphine, and suggest that psychoactive effects of methadone can be notably enhanced by past opiate use.

Cocaine-induced Fos expression is detectable in the frontal cortex and striatum of rats under isoflurane but not α-chloralose anesthesia: Implications for FMRI

The ability of intravenous cocaine to induce Fos protein expression in anesthetized rats was tested. Two anesthetic regimens commonly used for in vivo FMRI of animals, i.v. α-chloralose and gaseous isoflurane, were studied in separate cohorts. The first experiment included three groups that received the following treatments: saline i.v. and no anesthetic; 2mg/kg cocaine i.v. and no anesthetic; and 2mg/kg cocaine i.v. under 36mg/kg/h α-chloralose anesthesia. The second experiment had a factorial design of four groups that were either nonanesthetized or isoflurane-treated and were either given saline or cocaine (2mg/kg, i.v.). Anesthetized rats were maintained for 2h under 2.5–3.5% isoflurane anesthesia, while nonanesthetized rats were kept in an alternative environment for the same time period. Rats were given 2mg/kg cocaine or saline i.v., 30min into the test session. Rats were perfused and their brains were processed for Fos immunohistochemistry 90min after the i.v. treatment. In both experiments, the frontal cortex and striatum of the cocaine-treated nonanesthetized rats expressed Fos in greater amounts than the saline-treated nonanesthetized rats, as expected. The α-chloralose treatment prevented cocaine-induced Fos expression across all eight subregions of the striatum and frontal cortex that were examined. In contrast, isoflurane only partially attenuated Fos expression in the orbital and Cg2 subregions of frontal cortex. These results suggest a strong advantage for using isoflurane, as opposed to α-chloralose, when studying anesthetized rats for in vivo effects of psychostimulants.

Activation of superficial dorsal horn neurons in the mouse by a PAR-2 agonist and 5-HT: potential role in itch.

Itch, an unpleasant sensation associated with the desire to scratch, is symptomatic of dermatologic and systemic disorders that often resist antihistamine treatment. Histamine-independent itch mediators include serotonin (5-HT) and agonists of the protease-activated receptor-2 (PAR-2). We used behavior, Fos immunohistochemistry, and electrophysiology to investigate if these mediators activate spinal dorsal horn neurons in a manner consistent with itch. Intradermal (i.d.) injection of the PAR-2 agonist SLIGRL-NH(2) in the rostral back evoked bouts of directed hindlimb scratches over 20-30 min. Hindpaw injection of SLIGRL-NH(2) produced Fos staining in superficial dorsal horn which was then targeted for single-unit recording. Small id microinjections of SLIGRL-NH(2) or 5-HT identified responsive single units in the superficial dorsal horn of mice anesthetized with pentobarbital. Thirty-eight units characterized as wide dynamic range, nociceptive specific, or mechanically insensitive exhibited significantly increased firing after i.d. SLIGRL-NH(2) for 9 min, to partial (25%) tachyphylaxis with repeated injection. A majority additionally responded to 5-HT (70%), mustard oil (79%), and capsaicin (71%). Seven units isolated with the 5-HT search stimulus exhibited significant and prolonged responses to 5-HT with tachyphylaxis to repeated injections. The majority also responded to SLIGRL-NH(2), mustard oil, and capsaicin. The prolonged responses of superficial dorsal horn neurons to SLIGRL-NH(2) and 5-HT suggest a role in signaling itch. However, their responsiveness to algogens is inconsistent with itch specificity. Alternatively, such neurons may signal itch, whereas noxious stimulus levels recruit these and a larger population of pruritogen-insensitive cells to signal pain which masks or occludes the itch signal.

Effects of short-term acetaminophen and celecoxib treatment on orthodontic tooth movement and neuronal activation in rat

Non-steroidal anti-inflammatory drugs (NSAIDs) have been used for pain relief in orthodontics, but clinical studies reported that they may reduce tooth movement (TM). By other side, TM seems to activate brain structures related to nociception, but the effects of NSAIDs in this activation have not been studied yet. We analyzed the effect of short-term treatment with acetaminophen or celecoxib in the separation of rat upper incisors, as well as in neuronal activation of the spinal trigeminal nucleus, following tooth movement. Thirty rats (400–420 g) were pretreated through oral gavage (1 ml/dose) with acetaminophen (200 mg/kg), celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance for TM. In controls, this appliance was immediately removed after its introduction. Rats received ground food, and every 12 h, one of the drugs or vehicle. After 48 h, they were anesthetized, maxilla was radiographed, and were perfused with 4% paraformaldehyde. Brains were further processed for Fos immunohistochemistry. TM induced incisor distalization ( p < 0.05) and neuronal activation of the spinal trigeminal nucleus. Treatment with both drugs did not affect tooth movement, but reduced c-fos expression in the caudalis subnucleus. No changes in c-fos expression were seen in the oralis and interpolaris subnuclei. We conclude that neither celecoxib nor acetaminophen seems to affect tooth movement, when used for 2 days, but both drugs are able to reduce the activation of brain structures related to nociception. Short-term treatment with celecoxib, thus, may be a therapeutic alternative to acetaminophen when the latter is contraindicated.

Stress-opioid interactions: a comparison of morphine and methadone

The utility of methadone and morphine for analgesia and of methadone for substitution therapy for heroin addiction is a consequence of these drugs acting as opioid receptor agonists.We compared the cataleptogenic and antinociceptive effects of single subcutaneous doses of methadone hydrochloride (1–4mg/kg) and morphine sulfate (2.5–10mg/kg) using catalepsy and hot-plate tests, and examined the effects of the highest doses of the drugs on Fos protein expression in selected brain regions in male Sprague-Dawley rats. Methadone had greater cataleptogenic and analgesic potency than morphine. Fos immunohistochemistry revealed substantial effects on the Fos response of both the stress induced by the experimental procedures and of the drug exposure itself. There were three response patterns identified: 1) drug exposure, but not stress, significantly elevated Fos-positive cell counts in the caudate-putamen; 2) stress alone and stress combined with drug exposure similarly elevated Fos-positive cell counts in the nucleus accumbens and cingulate cortex; and 3) methadone and morphine (to a lesser extent) counteracted the stimulatory effect of nonpharmacological stressors on Fos protein expression in the somatosensory cortex barrel field, and Fos-positive cell counts in this region correlated negatively with both the duration of catalepsy and the latency time in the hot-plate test. The overlap between brain regions reacting to nonpharmacological stressors and those responding to exogenous opioids suggests that stress contributes to opioid-induced neuronal activation.

Cortagine, a CRF1 agonist, induces stresslike alterations of colonic function and visceral hypersensitivity in rodents primarily through peripheral pathways

Corticotropin-releasing factor (CRF) 1 receptor (CRF(1)) activation in the brain is a core pathway orchestrating the stress response. Anatomical data also support the existence of CRF signaling components within the colon. We investigated the colonic response to intraperitoneal (ip) injection of cortagine, a newly developed selective CRF(1) peptide agonist. Colonic motor function and visceral motor response (VMR) were monitored by using a modified miniaturized pressure transducer catheter in adult conscious male Sprague-Dawley rats and C57Bl/6 mice. Colonic permeability was monitored by the Evans blue method and myenteric neurons activation by Fos immunohistochemistry. Compared with vehicle, cortagine (10 microg/kg ip) significantly decreased the distal colonic transit time by 45% without affecting gastric transit, increased distal and transverse colonic contractility by 35.6 and 66.2%, respectively, and induced a 7.1-fold increase in defecation and watery diarrhea in 50% of rats during the first hour postinjection whereas intracerebroventricular (icv) cortagine (3 microg/rat) had lesser effects. Intraperitoneal (ip) cortagine also increased colonic permeability, activated proximal and distal colonic myenteric neurons, and induced visceral hypersensitivity to a second set of phasic colorectal distention (CRD). The CRF antagonist astressin (10 mug/kg ip) abolished ip cortagine-induced hyperalgesia whereas injected icv it had no effect. In mice, cortagine (30 microg/kg ip) stimulated defecation by 7.8-fold, induced 60% incidence of diarrhea, and increased VMR to CRD. Stresslike colonic alterations induced by ip cortagine in rats and mice through restricted activation of peripheral CRF(1) receptors support a role for peripheral CRF(1) signaling as the local arm of the colonic response to stress.

Fos expression and hormone changes following electrical stimulation of the posterodorsal amygdala and the effects on food intake in conscious female rats

Previous studies have shown that lesions of the posterodorsal amygdala (PDA) produce hyperphagia and obesity in female rats. To better understand the role of the PDA in the regulation of feeding behavior, the current study examined the effects of electrical stimulation of the PDA of female rats on food intake and identified neurons activated by PDA stimulation using Fos immunohistochemistry. Hormonal levels following stimulation of the PDA were also investigated. Electrical stimulation (100 μA, 0.2 ms, 20 Hz, 10 min) of the PDA remarkably decreased 1 h food intake of rats. Following PDA stimulation, the number of Fos-like immunoreactive neurons increased in the caudal and intermediate parts of the nucleus of the solitary tract (NST), the area postrema (AP), the external lateral subnucleus of the parabrachial nucleus (PBN), the arcuate nucleus of the hypothalamus (Arc), and the central amygdaloid nucleus (CeA). The level of circulating leptin was elevated significantly by PDA stimulation as well. Together with previous studies, the results suggest that the PDA may play an important role in the regulation of feeding behavior, at least partly by modulating the circulating leptin, and that the caudal and intermediate parts of the NST, AP, external lateral PBN, Arc, and CeA probably participate in this regulation.