Fos-like Immunoreactivity Research Articles

Lipoxygenase inhibitors suppressed carrageenan-induced Fos-expression and inflammatory pain responses in the rat.

Lipoxygenase (LO) metabolites are generated in inflamed tissues. However, it is unclear whether the inhibition of the LO activity regulates the expression of c-Fos protein, a pain marker in the spinal cord. Here we used a carrageenan-induced inflammation model to examine the role of LO in the development of c-Fos expression. Intradermally injected carrageenan caused elevated number of cells exhibiting Fos-like immunoreactivity (Fos-LI) in the spinal dorsal horn, and decreased the thermal and mechanical threshold in Hargreaves and von Frey tests. Pretreatment with an inhibitor of phospholipase A2, that generates the LO substrate, prior to the carrageenan injection significantly reduced the number of Fos-(+) cells. A general LO inhibitor NDGA, a 5-LO inhibitor AA-861 and a 12-LO inhibitor baicalein also exhibited the similar effects. Moreover, the LO inhibitors suppressed carrageenan-induced thermal and mechanical hyperalgesic behaviors, which inidcates that the changes in Fos expression correlates with those in the nociceptive behaviors in the inflamed rats. LO products are endogenous TRPV1 activators and pretreatment with BCTC, a TRPV1 antagonist inhibited the thermal but not the mechanical hypersensitivity. Overall, our results from the Fos-LI and behavior tests suggest that LO products released from inflamed tissues contribute to nociception during carrageenan-induced inflammation, indicating that the LO pathway is a possible target for modulating inflammatory pain.

Intracerebroventricular (ICV) injections of nesfatin‐1 induces c‐fos expression in the rat forebrain.

The forebrain contains neuronal circuits of critical importance in the regulation of food intake. Nesfatin has been identified as a satiety peptide within central neurons. ICV injection of nesfatin‐1 decreases food intake in a dose‐dependent manner and result in a decrease in body weight. However, the central pathways activated by nesfatin‐1 are not known. In this study, the distribution of neurons expressing fos‐like immunoreactivity following ICV infusions of nesfatin‐1 (5 pmol/µl; 2 µl followed by two 1 µl injections every 30 min) was identified in the rat forebrain. Fos labeled neurons were found within the hypothalamus: paraventricular, supraoptic, arcuate, lateral, dorsomedial and posterior nuclei. In addition, the organum vasculosum of the lamina terminalis, median preoptic nucleus and subfornical organ expressed fos labeling. Finally, fos labeling was observed within the midline thalamic nuclei, lateral septum, islands of Calleja, medial preoptic nucleus and medial and central nuclei of amygdala. These data suggest that nesfatin‐1 exerts its anorexigenic effects through the activation of forebrain structures previously implicated in the regulation of food intake and energy expenditure.Supported by Heart and Stroke Foundation of Ontario.

Scratching Behavior and Fos Expression in Superficial Dorsal Horn Elicited by Protease-Activated Receptor Agonists and Other Itch Mediators in Mice

Protease-activated receptor (PAR)-2 and PAR-4 are implicated in nonhistaminergic itch. We investigated dose dependence, tachyphylaxis, and cross-tachyphylaxis of itch-associated scratching elicited by intradermal injections of PAR-2 and PAR-4 agonists, serotonin (5-hydroxytryptamine, 5-HT), and histamine in ICR mice, as well as mu-opioid modulation of PAR-2 agonist-evoked scratching. Each agent elicited dose-related increases in scratch bouts. Scratching elicited by the PAR-4 agonist and histamine both exhibited significant tachyphylaxis but no cross-tachyphylaxis with each other. Scratching evoked by 5-HT did not exhibit significant tachyphylaxis but did exhibit significant cross-tachyphylaxis to scratching evoked by the PAR-2 and PAR-4 agonists and histamine. Naltrexone and high-dose morphine (10 mg/kg) attenuated PAR-2 agonist-evoked scratching, whereas lower dose morphine (1 mg/kg) had no effect. High-dose morphine also significantly increased circling behavior, which may have interfered with scratching. The PAR-2 agonist and 5-HT produced overlapping distributions of Fos-like immunoreactivity in the superficial dorsal horn. These results indicate that PAR-2 and PAR-4 agonists, histamine, and 5-HT elicit itch-related scratching and activate superficial dorsal horn neurons that may participate in scratch reflex and ascending itch signaling pathways.

Bright light produces Fos-positive neurons in caudal trigeminal brainstem

Excessive discomfort after exposure to bright light often occurs after ocular injury and during headache. Although the trigeminal nerve is necessary for light-evoked discomfort, the mechanisms underlying this phenomenon, often referred to generally as photophobia, are not well defined. Quantitative Fos-like immunoreactivity (Fos-LI) was used to determine the pattern of neuronal activation in the caudal brainstem after bright light stimulation and, secondly, whether a neurovascular mechanism within the eye contributes to this response. Under barbiturate anesthesia, male rats were exposed to low (1×10 4 lx) or high intensity (2×10 4 lx) light delivered from a thermal neutral source for 30 min (30 s ON, 30 s OFF) and allowed to survive for 90 min. Intensity-dependent increases in Fos-LI were seen in laminae I–II at the trigeminal caudalis/cervical cord junction region (Vc/C1) and nucleus tractus solitarius (NTS). Fos-LI also increased at the trigeminal interpolaris/caudalis transition (Vi/Vc vl) and dorsal paratrigeminal (dPa5) regions independent of intensity. Intravitreal injection of norepinephrine greatly reduced light-evoked Fos-LI at the Vc/C1, dPa5 and NTS, but not at the Vi/Vc transition. Lidocaine applied to the ocular surface had no effect on Fos-LI produced in trigeminal brainstem regions. These results suggested that multiple regions of the caudal trigeminal brainstem complex integrate light-related sensory information. Fos-LI produced at the dPa5 and NTS, coupled with norepinephrine-induced inhibition, was consistent with the hypothesis that light-evoked activation of trigeminal brainstem neurons involves an intraocular neurovascular mechanism with little contribution from neurons that supply the ocular surface.

The effects of synthetic and endogenous imidazoline binding site ligands on neuronal activity in discrete brain regions of naive and restraint-stressed rats

We have mapped Fos expression to investigate brain regions activated by synthetic selective I 2 (BU224) and endogenous (harmane) imidazoline binding site ligands in naive and restraint-stressed rats. Systemic administration of BU224 or harmane to naive rats increased Fos-like immunoreactivity (FLI) in the hypothalamic paraventricular nucleus (PVN), hippocampal dentate gyrus (DG), central and medial nuclei of the amygdala (CeA, MeA) and the locus coeruleus (LC). FLI in restraint-stressed rats was increased in all 5 regions by harmane, and in the CeA, MeA and LC by BU224. Dual-labelling of FLI cells in the PVN of naive rats showed an increase in the number of corticotrophin-releasing-factor-containing cells (CRF) activated by BU224 and harmane. Several CRF-containing neurons in the PVN expressed α 1-adrenoceptors and were densely surrounded by catecholaminergic axons and terminals. Our results provide a functional neuroanatomical framework which may explain the stimulatory effects of imidazoline ligands on basal and stress-induced neuronal and neuroendocrine activity.

Boosting cholinergic activity in gustatory cortex enhances the salience of a familiar conditioned stimulus in taste aversion learning.

The cholinergic system is important for learning, memory, and responses to novel stimuli. Exposure to novel, but not familiar, tastes increases extracellular acetylcholine (ACh) levels in insular cortex (IC). To further examine whether cholinergic activation is a critical signal of taste novelty, in these studies carbachol, a direct cholinergic agonist, was infused into IC before conditioned taste aversion (CTA) training with a familiar taste. By mimicking the cholinergic activation generated by novel taste exposure, it was hypothesized that a familiar taste would be treated as novel and therefore a salient target for aversion learning. As predicted, rats infused with the agonist were able to acquire CTAs to familiar saccharin. Effects of carbachol infusion on patterns of neuronal activation during conditioned stimulus-unconditioned stimulus pairing were assessed using Fos-like immunoreactivity (FLI). Familiar taste-illness pairing following carbachol, but not vehicle, induced significant elevations of FLI in amygdala, a region with reciprocal connections to IC that is also important for CTA learning. These results support the view that IC ACh activity provides a critical signal of taste novelty that facilitates CTA acquisition.

The Antinociceptive Effect of Sigma-1 Receptor Antagonist, BD1047, in a Capsaicin Induced Headache Model in Rats

Intracranial headaches, including migraines, are mediated by nociceptive activation of the trigeminal nucleus caudalis (TNC), but the precise mechanisms are poorly understood. We previously demonstrated that selective blockage of spinal sigma-1 receptors (Sig-1R) produces a prominent antinociceptive effect in several types of pain models. This study evaluates whether the Sig-1R antagonist (BD1047) has an antinociceptive effect on capsaicin (a potent C-fiber activator) induced headache models in rats. Intracisternal infusion of capsaicin evoked pain behavior (face grooming), which was significantly attenuated by BD1047 pretreatment. BD1047 consistently reduced capsaicin-induced Fos-like immunoreactivity (Fos-LI), a neuronal activator, in the TNC in a dose-dependent manner. Moreover, capsaicin-induced phosphorylation of N-methyl-D-aspartate receptor subunit 1 was reversed by BD1047 pretreatment in the TNC. These results indicate that the Sig-1R antagonist has an inhibitory effect on nociceptive activation of the TNC in the capsaicin-induced headache animal model.

Ocular surface-evoked Fos-like immunoreactivity is enhanced in trigeminal subnucleus caudalis by prior exposure to endotoxin

Endotoxin-induced uveitis (EIU) is a common animal model for anterior uveitis in humans that causes long-term changes in trigeminal brain stem neurons. This study used c-fos immunohistochemistry to assess the effects of different routes of administration of endotoxin on activation of trigeminal brain stem neurons produced by ocular surface stimulation. A single dose of endotoxin (lipopolysaccharide (LPS)) given to male rats by systemic (i.p., 1 mg/kg) or intraocular (ivt, 20 μg) routes increased the number of Fos-positive neurons in rostral (trigeminal subnucleus interpolaris/subnucleus transition (Vi/Vc)) and caudal portions of trigeminal subnucleus caudalis (trigeminal subnucleus caudalis/upper cervical spinal cord transition (Vc/C 1–2)) by 20% mustard oil (MO) applied to the ocular surface 7 days, but not at 2 days, after LPS compared with naïve rats. I.c.v. (20 μg) LPS did not affect MO-evoked Fos. To determine if the pattern of enhanced Fos expression after systemic LPS also depended on the nature of the ocular surface stimulus, additional groups received ocular stimulation by 10% histamine or dry eye conditions. Seven days, but not 2 days, after i.p. LPS both histamine- and dry eye–evoked Fos was increased at the Vi/Vc transition, while smaller effects were seen at other regions. These results suggested that EIU modulation of trigeminal brain stem neuron activity was mediated mainly by peripheral actions of LPS. Enhancement of Fos at the Vi/Vc region after MO, histamine and dry eye conditions supports the hypothesis that this region integrates innocuous as well as noxious sensory information, while more caudal portions of Vc process mainly nociceptive signals from the eye.

Differential expression of Fos and Zif268 in the nigrostriatal system after methamphetamine administration in a rat model of Parkinson's disease

The goal of this study was to examine the topological specificity of methamphetamine-induced activation of the immediate-early gene proteins, Fos and Zif268, in the nigrostriatal system in a unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease with or without intrastriatal grafts of fetal ventral mesencephalon. Methamphetamine (3 mg/kg, i.p.) induced Fos-like immunoreactivity (FLI) dominantly in the striatum and the globus pallidus (GP) on the intact side as well as in the substantia nigra pars reticulata (SNr) on the lesioned side in the 6-OHDA rats. Lower levels of methamphetamine-induced FLI in the striatum and GP on the lesioned side were restored by intrastriatal grafts which could completely suppress the methamphetamine-induced rotation. In the striatum, a similar tendency could be observed between Fos and Zif268 immunoreactivity following methamphetamine. However, sparse immunoreactivity of Zif268 could be detected in the GP and SNr on both sides in the 6-OHDA rats. Intrastriatal grafts had little influence on Zif268 expression in these two regions. The differential expression of Fos and Zif268 was observed among the three regions of the nigrostriatal system following methamphetamine in the 6-OHDA rats. This may suggest that Fos and Zif268 therefore possess gene-specific and region-specific functions in the basal ganglia nuclei.

Glutamatergic Stimulation of Luteinising Hormone Secretion in Relatively Refractory Male Songbirds

Seasonal breeding in two Sonoran desert passerines, the Cassin's (Aimophila cassinii) and Rufous-crowned (Aimophila ruficeps) Sparrows is thought to be terminated by the development of a decrease in responsiveness to photostimulation, a condition known as relative photorefractoriness. It was predicted that the development of relative refractoriness is a consequence of a decrease in gonadotrophin-releasing hormone (GnRH) synthesis and associated stores of releasable GnRH. This hypothesis was tested by determining the luteinising hormone (LH) responses to the excitatory amino acid glutamate agonist N-methyl-D,L-aspartate (NMA) in males of the two species subjected to photomanipulations aimed at generating five groups: Fully photosensitive with undeveloped testes on short days (8L : 16D); fully photosensitive with developed testes on 13L : 11D; relatively photorefractory with regressed testes on 13L : 11D, and groups with developed testes held on 15L : 9D or 16L : 8D. LH release was stimulated in the Cassin's Sparrow by NMA most in the 8L group; to a lesser, but similar extent in the two 13L groups; and not at all in the 15L and 16L groups. LH release was not stimulated by NMA in any of the photoperiodic regimes in the Rufous-crowned Sparrow. In both species, NMA induced Fos-like immunoreactivity in the anterior and basal hypothalamus, but not in GnRH cell bodies. It is concluded that the development of relative photorefractoriness in Cassin's Sparrows is a consequence of reduced GnRH synthesis, reflected in a reduction in releasable GnRH. The lack of LH response of the Rufous-crowned Sparrows to NMA administration may be a consequence of high responsiveness to handling stress.

Evidence for the involvement of the spinoparabrachial pathway, but not the spinothalamic tract or post-synaptic dorsal column, in acute bone nociception

We have previously reported that acute noxious mechanical stimulation of bone activates neurons throughout the dorsal horn of the lumbar spinal cord, and argued that the spinal mechanisms that mediate bone nociception are different to those that mediate cutaneous and visceral nociception. In the present study, we provide evidence that the ascending spinal pathways that mediate acute bone nociception also differ to those that mediate acute cutaneous and visceral nociception. Injections of a retrograde tracer (Fluorogold) were made into the thalamus, gracile nucleus or lateral parabrachial nucleus to identify spinothalamic, post-synaptic dorsal column or spinoparabrachial projection neurons respectively (n=4 in each group). Spinal dorsal horn neurons activated by acute noxious mechanical stimulation of bone (bone drilling) were identified in these animals using Fos immunohistochemistry. Fluorogold and Fos-like immunoreactivity was not colocalized in any dorsal horn neurons projecting to the thalamus or gracile nucleus. In contrast, a total of 12.2+/-1.1% (mean+/-S.E.M.) of the spinoparabrachial projection neurons contained Fos-like immunoreactive nuclei following bone drilling and this was significantly greater than the percentage (3.4+/-0.5%) in animals of a sham surgery group (n=4) that were not exposed to bone drilling (Mann-Whitney; p<0.05). These data provide evidence for the involvement of the spinoparabrachial pathway, but not the spinothalamic or post-synaptic dorsal column pathways, in the relay of information regarding acute noxious mechanical stimuli applied to bone, and suggest that spinal pathways that mediate acute bone nociception may be different to those that mediate acute nociception of cutaneous and visceral origin.

S.04.02 Imidazoline2 (I2) binding site ligands increase Fos-like immunoreactivity in the naive and restraint-stressed rat brain

Cannabis is the most widely used illicit drug in the world. Although there is a high prevalence of users who seek treatment for cannabis dependence, no accepted pharmacologic treatment is available to facilitate and maintain abstinence. The hypocretin/orexin system plays a critical role in drug addiction, but the potential participation of this system in the addictive properties of cannabinoids is unknown.We investigated the effects of hypocretins in the intravenous self-administration of the synthetic cannabinoid agonist WIN55,212-2 using hypocretin receptor-1 (Hcrtr-1) and hypocretin receptor-2 antagonists and Hcrtr-1 knockout mice. Additional groups of mice were trained to obtain water to rule out operant responding impairments. Activation of hypocretin neurons was analyzed by using double-label immunofluorescence of FosB/ΔFosB with hypocretin-1. Microdialysis studies were performed to evaluate dopamine extracellular levels in the nucleus accumbens after acute Δ9-tetrahydrocannabinol administration.Systemic administration of the Hcrtr-1 antagonist SB334867 reduced intravenous self-administration of WIN55,212-2, as well as the maximum effort to obtain a WIN55,212-2 infusion, as revealed under a progressive ratio schedule. This role of Hcrtr-1 in the reinforcing and motivational properties of WIN55,212-2 was confirmed in Hcrtr-1 knockout mice. Contingent, but not noncontingent, WIN55,212-2 self-administration increased the percentage of hypocretin cells expressing FosB/ΔFosB in the lateral hypothalamus. The enhancement in dopamine extracellular levels in the nucleus accumbens induced by Δ9-tetrahydrocannabinol was blocked in mice lacking the Hcrtr-1.These findings demonstrate that Hcrtr-1 modulates the reinforcing properties of cannabinoids, which could have a clear therapeutic interest.

Fos expression following activation of the ventral pallidum in normal rats and in a model of Parkinson’s Disease: implications for limbic system and basal ganglia interactions

The circuit-related consequences of activating the ventral pallidum (VP) are not well known, and lacking in particular is how these effects are altered in various neuropathological states. To help to address these paucities, this study investigated the brain regions affected by VP activation by quantifying neurons that stain for Fos-like immunoreactivity (ir). Fos-ir was assessed after intra-pallidal injections of the excitatory amino acid agonist, NMDA, or the GABA(A) antagonist, bicuculline in normal rats and in those rendered Parkinsonian-like by lesioning dopaminergic neurons with the neurotoxin, 6-OHDA. We hypothesized that activation of the VP will alter the activity state of brain regions associated with both the basal ganglia and limbic system, and that this influence would be modified in the Parkinsonian state. Blocking tonically activated GABA(A) receptors with bicuculline (50 ng/0.5 microl) elevated Fos-ir in the VP to 423% above the contralateral, vehicle-injected side. Likewise, intra-VP NMDA (0.23 microg or 0.45 microg/0.5 microl), dose-dependently increased the number of pallidal neurons expressing Fos-ir by 224 and 526%, respectively. At higher NMDA doses, the density of Fos-ir neurons was not elevated above control levels. This inverted U-shaped profile was mirrored by a VP output structure, the medial subthalamic nucleus (mSTN). The mSTN showed a 289% increase in Fos-ir neurons with intra-VP injections of 0.45 microg NMDA, and this response was halved following intra-VP injections of 0.9 microg NMDA. Of the 12 other brain regions measured, three showed VP NMDA-induced enhancements in Fos-ir: the frontal cortex, entopeduncular nucleus and substantia nigra pars reticulata, all regions associated with the basal ganglia. In a second study, we evaluated the NMDA activation profile in a rat model of Parkinson's Disease (PD) which was created by a unilateral injection of 6-OHDA into the rostral substantia nigra pars compacta. Comparisons of responses to intra-VP NMDA between the hemispheres ipsilateral and contralateral to the lesion revealed that Fos-ir cells in the pedunculopontine nucleus was reduced by 62%, whereas Fos-ir for the basolateral amygdala and STN was reduced by 32 and 42%, respectively. These findings support the concept that the VP can influence both the basal ganglia and the limbic system, and that that the nature of this influence is modified in an animal model of PD. As the VP regulates motivation and cognition, adaptations in this system may contribute to the mood and mnemonic disorders that can accompany PD.

EVIDENCE THAT INHIBITION OF SPINAL NITRIC OXIDE PRODUCTION CONTRIBUTES TO THE ANTINOCICEPTIVE EFFECTS OF EMULSIFIED ISOFLURANE ON FORMALIN-INDUCED PAIN IN RATS

The aim of the present study was to investigate the contribution of spinal nitric oxide (NO) to the antinociceptive effects of emulsified isofluane in rats. The formalin test was used to assess nociceptive responses. Immunocytochemistry and histochemistry were performed to determine the effects of emulsified isoflurane on formalin-induced changes in Fos-like immunoreactive (Fos-LI)- and nicotinamide adenine dinucleotide phosphatediaphorase (NADPH-d)-positive neurons, respectively. The results showed that emulsified isofluane, administered intraperitoneally, significantly decreased the formalin-induced paw licking time and that this was attenuated by pretreatment with intrathecal injection of the NO precursor L-arginine. Furthermore, Fos-LI- and NADPH-d-positive neurons were mainly found in the ipsilateral dorsal horn after injection of formalin, some of which were Fos-LI/NADPH-d double-labelled neurons. Administration of emulsified isofluane significantly decreased Fos-LI- and NADPH-d-positive, as well as Fos-LI/NADPH-d double-labelled, neurons. Finally, emulsified isofluane produced a significant reduction of NOS activity and a decrease of NO production in the spinal cord of formalin-treated rats. In conclusion, the results suggest that inhibition of spinal NO production contributes to the antinociceptive effects of emulsified isofluane on formalin-induced pain in rats.

Activation of submucosal but not myenteric plexus of the gastrointestinal tract accompanies reduction of food intake by camostat

It has been shown in the rat that endogenous cholecystokinin (CCK), released in response to the non-nutrient trypsin inhibitor camostat, reduces food intake at meals and increases Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation) in the dorsal vagal complex (DVC) of the hindbrain but not the myenteric plexus of the duodenum and jejunum. Experiment 1: We examined Fos-LI in the myenteric and the submucosal plexuses of the gut in response to orogastric gavage of camostat in rats. As we reported previously, camostat failed to increase Fos-LI in the myenteric plexus. We show here that camostat increased Fos-LI in the submucosal plexus of the duodenum and jejunum. Camostat also increased Fos-LI in the DVC. Experiment 2: Pretreatment with devazepide, a specific CCK 1 receptor antagonist abolished camostat-induced Fos-LI in the submucosal plexus and the DVC. Experiment 3: Bilateral subdiaphragmatic vagotomy reduced camostat-induced Fos-LI in the submucosal plexus approximately 40% and abolished it in the DVC. Conclusions: Activation of the submucosal plexus by cholecystokinin at the CCK 1 receptor accompanies stimulation of the dorsal vagal complex of the hindbrain and inhibition of food intake. Unlike the submucosal plexus, activation of the myenteric plexus is not necessary for cholecystokinin's influence on the dorsal vagal complex and food intake. The lack of activation in the myenteric plexus after camostat stimulation, in contrast to nutrient releasers of CCK such as oleate, suggests that intestinal stimulants can either release different amounts of CCK or cause release of CCK from I cells with different molecular forms of CCK. This would suggest that CCK-8 is released by camostat and is not able to travel to the myenteric plexus while a more stable form of CCK such as CCK-58 can travel to this site that is further away from the I cell.

Licking and gaping elicited by microstimulation of the nucleus of the solitary tract

Intraoral infusions of bitter tastants activate expression of the immediate-early gene c-Fos in neurons located in the medial third of the rostral nucleus of the solitary tract (rNST). The distribution of these neurons is distinct from that activated by sour or sweet stimuli. Bitter stimuli are also distinctive because of their potency for eliciting gaping, an oral reflex that functions to actively reject potentially toxic substances. Glossopharyngeal nerve transection profoundly reduces, whereas decerebration spares, the bitter-evoked Fos-like immunoreactivity (FLI) pattern and gaping, implicating the medial rNST as a substrate for the sensory limb of oral rejection. The present experiment tested this hypothesis using microstimulation (100 Hz, 0.2 ms, 5-40 microA) to activate the rNST in awake rats. NST microstimulation elicited licking and gaping, and gaping was evoked from a restricted rNST region. The results indicated some topographic organization in sites effective for evoking gaping, but, in direct conflict with the hypothesis, lateral sites farther from bitter-evoked FLI were more effective than medial sites centered closer to FLI-expressing neurons. The gape-effective sites resemble locations of bitter-responsive neurons recently observed in neurophysiological recordings. These results indicate that bitter-responsive rNST neurons critical for triggering gaping may not express FLI and imply an alternate function for bitter-responsive neurons that do.

Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex

The enteric nervous system (ENS: myenteric and submucosal plexuses) of the gastrointestinal tract may have a role in the reduction of food intake by cholecystokinin (CCK). Exogenous cholecystokinin-8 (CCK-8) activates the myenteric plexus and the feeding control areas of the dorsal vagal complex (DVC) of the brainstem. An increasing number of reports, however, have shown that CCK-58 is the sole or the major circulating form of CCK in rat, human and dog, and that it is qualitatively different from CCK-8 in evoking various gastrointestinal physiological responses (e.g., contraction of the gallbladder and exocrine pancreatic secretion). In the current report, we compared the abilities of exogenous CCK-58 to activate the myenteric plexus and the dorsal vagal complex with those of exogenous CCK-8 by quantifying Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). We report that CCK-58 (1, 3, and 5 nmol/kg) increased Fos-LI in the myenteric plexus ( p < 0.001) and in the DVC ( p < 0.001) compared to the saline vehicle. The highest dose of CCK-58 increased Fos-LI more than an equimolar dose of CCK-8 in the myenteric plexus and the area postrema. Thus, CCK-8 and CCK-58 produce the same qualitative pattern of activation of central and peripheral neurons, but do not provoke identical quantitative patterns at higher doses. The different patterns produced by the two peptides at higher doses, in areas open to the circulation (myenteric plexus and area postrema) may reflect endocrine actions not observed at lower doses.

The Effects of Intrathecal Cyclooxygenase-1, Cyclooxygenase-2, or Nonselective Inhibitors on Pain Behavior and Spinal Fos-Like Immunoreactivity

Prostaglandins are synthesized by cyclooxygenase (COX) and are thought to play an important role in nociceptive transmission in the spinal cord. Fos expression is an indicator of spinal neuron activation. We examined the role of intrathecal selective and nonspecific COX inhibitors on spinal C-Fos expression. To evaluate the relative contribution of COX-1 and COX-2 in nociceptive transmission in the spinal cord, we assessed the effects of the selective COX-1 inhibitor SC 560, the selective COX-2 inhibitor celecoxib, and the nonselective COX inhibitor ketorolac on formalin-evoked behavior and spinal c-Fos-like immunoreactivity (FLI). Rats received each of the drugs (30, 60, or 90 microg) intrathecally before the subcutaneous administration of formalin (5%, 50 microL) to the plantar surface of a hindpaw. The control group received vehicle intrathecally before the administration of formalin. Phase 1 flinching behavior decreased in rats given celecoxib or ketorolac 90 mug. Phase 2 flinching behavior decreased in rats given all doses of ketorolac or celecoxib 90 microg (P < 0.05). The FLI was significantly reduced in rats given celecoxib or ketorolac 90 microg for laminae I-II (P < 0.05). By contrast, for laminae V-VI, only the ketorolac 60 or 90 microg treatment group demonstrated a larger decrease in FLI (P < 0.05). The FLI expression in laminae V-VI had a significant correlation with phase 2 flinching behavior (P < 0.05). A dual inhibitor of COX-1 and COX-2 suppressed both responses of formalin-evoked behaviors and FLI expression of whole laminae in the lumbar spinal cord. FLI expression of laminae I-II alone may not be a good indicator of the ability to produce anti-hypersensitivity; however, the FLI of laminae V-VI correlates with phase 2 responses.

The pattern of Fos expression in the spinal dorsal horn following acute noxious mechanical stimulation of bone

Little is known of the spinal mechanisms that mediate bone nociception. The aim of this study was to determine the pattern of neuronal activation in the spinal dorsal horn following acute noxious mechanical stimulation of bone. This was achieved by examining Fos expression in the spinal dorsal horn following acute, noxious mechanical stimulation of the rat tibia. Noxious mechanical stimuli were applied by bone drilling and raising tibial intra-osseous pressure. Control experiments consisted of surgery to expose the tibia. There was a significant increase in the number of Fos-like immunoreactive (Fos-LI) nuclei in the superficial, ipsilateral dorsal horn of animals in the bone drilling and pressure groups relative to animals of the control group at spinal cord segments L3 and L4 ( P < 0.05). The number of Fos-LI nuclei in the deep dorsal horn was always lower than the number in the superficial dorsal horn (significant at L3 but not L4; P < 0.05). Whilst there appeared to be a small increase in the number of Fos-LI nuclei in the ipsilateral deep dorsal horn of bone drilling and pressure groups relative to the ipsilateral deep dorsal horn control group at both L3 and L4 segments, no significant effect was observed ( P > 0.05). The present study implicates the superficial dorsal horn of the spinal cord as a region of interest in studies of acute bone pain, and highlights the notion that spinal mechanisms that mediate bone nociception may be different to those that mediate nociception of cutaneous and visceral origin.

Cholecystokinin-33 is more effective than cholecystokinin-8 in inhibiting food intake and in stimulating the myenteric plexus and dorsal vagal complex

We compared the abilities of cholecystokinin-33 (CCK-33) and CCK-8 to reduce food intake and to activate feeding-related areas of the nervous system. (1) Overnight food-deprived rats were presented with a 10% sucrose solution, and intake was measured at 5-min intervals throughout a 90-min test beginning immediately after intraperitoneal injections of 1, 3, or 5 nMol/kg of CCK-33, CCK-8, or the vehicle control. In the initial 20 min (first meal), both peptides were equally effective, producing large reductions of food intake. Thereafter, however, CCK-33 was more effective than CCK-8, producing much more sustained reductions. Overall, both peptides reduced total food intake, but CCK-33 was more effective than CCK-8. (2) Possible roles for the myenteric plexus of the duodenum and the dorsal vagal complex (DVC) of the brainstem in the differential satiety effects of CCK-33 and CCK-8 were examined by quantifying CCK-33- and CCK-8-stimulated Fos-like immunoreactivity (Fos-LI) in each site. Consistent with the greater ability of CCK-33 to produce sustained inhibitions of food intake, CCK-33 produced more Fos-LI than CCK-8 in nearly every section of the sampled sites. The results demonstrate: (1) Different forms of CCK have different efficacies in reducing food intake; (2) CCK-33 produces a much more prolonged satiety action than CCK-8; and (3) the myenteric plexus and DVC may play roles in these differential satiety actions.