Number Of Fos-immunoreactive Neurons Research Articles

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Amygdala rapid kindling impairs breathing in response to chemoreflex activation

Temporal lobe epilepsy is often accompanied by behavioral, electroencephalographic and autonomic abnormalities. Amygdala kindling has been used as an experimental model to study epileptogenesis. Although amygdala kindling has been extensively investigated in the context of its clinical relevance to the epilepsies, potential associated respiratory alterations are not well known. Here, our main objective was to better investigate the mechanisms involved in respiratory physiology impairment in the amygdala rapid kindling (ARK) model of epileptogenesis. Male Wistar rats with electrodes implanted into the amygdaloid complex were used. After recovery from surgery, the rats were subjected to electrical stimulation of basolateral amygdala for 2 consecutive days (10 stimuli/day). The ventilatory parameters were evaluated by whole body plethysmography. Thereafter, animals were also exposed to hypercapnia (7% CO2) for 3 h to evaluate fos protein expression in several nuclei involved in respiratory control. We observed a significant reduction in ventilation during the ictal phase elicited by ARK. We also found that 10 days after ARK, baseline ventilation as well as the hypercapnia ventilatory response (7% CO2) were reduced compared to control rats. The number of fos-immunoreactive neurons in the retrotrapezoid nucleus, raphe magnus and nucleus of the solitary tract were also reduced after ARK. Our results showed that ARK was able to impair breathing function, demonstrating a strong coupling between amygdala and the respiratory neurons in the brainstem, with potential impact in respiratory failures, frequently fatal, during or after epileptic seizures in chronic animal models and in patients.

Suckling induced activation pattern in the brain of rat pups

Objectives: The aim of the study was to understand the effects of suckling on the brain of the pups by mapping their brain activation pattern in response to suckling.Methods: The c-fos method was applied to identify activated neurons. Fasted rat pups were returned to their mothers for suckling and sacrificed 2 hours later for Fos immunohistochemistry. Double labeling was also performed to characterize some of the activated neurons. For comparison, another group of fasted pups were given dry food before Fos mapping.Results: After suckling, we found an increase in the number of Fos-immunoreactive neurons in the insular and somatosensory cortices, central amygdaloid nucleus (CAm), paraventricular (PVN) and supraoptic hypothalamic nuclei, lateral parabrachial nucleus (LPB), nucleus of the solitary tract (NTS), and the area postrema. Double labeling experiments demonstrated the activation of calcitonin gene-related peptide-ir (CGRP-ir) neurons in the LPB, corticotropin-releasing hormone-ir (CRH-ir) but not oxytocin-ir neurons in the PVN, and noradrenergic neurons in the NTS. In the CAm, Fos-ir neurons did not contain CRH but were apposed to CGRP-ir fiber terminals. Refeeding with dry food-induced Fos activation in all brain areas activated by suckling. The degree of activation was higher following dry food consumption than suckling in the insular cortex, and lower in the supraoptic nucleus and the NTS. Furthermore, the accumbens, arcuate, and dorsomedial hypothalamic nuclei, and the lateral hypothalamic area, which were not activated by suckling, showed activation by dry food.Discussion: Neurons in a number of brain areas are activated during suckling, and may participate in the signaling of satiety, taste perception, reward, food, and salt balance regulation.

A Blocker of NO Synthase Intensifies c-fos Expression in Spinal Neurons of Rats Realizing Stereotypic Movements

Neurons with the presence of c-Fos protein, the product of expression of the “early’ c-fos gene, were detected in the cervical region of the spinal cord of rats performing stereotypic operant food-procuring movements by the forelimb. An immunohistochemical technique was used; the presence of c-Fos was interpreted as a correlate of the activated state of the respective neurons. Effects of suppression of the activity of neuronal NO synthase (nNOS) provided by systemic injections of a selective blocker of this enzyme, 7-nitroindazole (7-NI), were studied under the above conditions. In the absence of 7-NI, the main foci of Fos-immunoreactivity in the spinal segments C6 and C7 were observed in laminae 2i, 3, and 4 of the gray substance, while after injection of this blocker these were found in laminae 3, 4, and 6 and also in the motor nuclei (lamina 9). In animals that performed repetitive food-procuring movements and were preliminarily injected with 7-NI, significantly greater mean numbers of Fos-immunoreactive neurons were found in slices of the gray matter, as compared with the respective figures in rats with no suppression of nNOS activity (54.4 ± 0.7 cells in a 40 μm-thick slice ipsilaterally with respect to the working limb, as compared with 31.7 ± 1.1 cells, P < 0.05). Thus, expression of the c-fos gene in certain regions of the cervical part of the spinal cord increases significantly under conditions of 7-NI-induced suppression of NO production; this is observed in rats realizing repeated movements for a long time.

Chronic activation of sigma-1 receptor evokes nociceptive activation of trigeminal nucleus caudalis in rats

Primary headache disorders, including migraine, are thought to be mediated by prolonged nociceptive activation of the trigeminal nucleus caudalis (TNC), but the precise mechanisms are poorly understood. Our past studies demonstrated that sigma-1 receptors (Sig-1R) facilitate spinal nociceptive transmission in several pain models. Based on these findings, this study asked if chronic activation of Sig-1R by intracisternal administration of the selective Sig-1R agonist, PRE084, produced TNC neuronal activation as a migraine trigger in rats. A single infusion of PRE084 (10, 50, 100, 500nmol) significantly increased the number of Fos immunoreactive neurons (Fos-IR) in TNC, which BD1047 (a Sig-1R antagonist) reversed. Chronic infusion of PRE084 (100nmol for 1, 3, 7 and 14days) time-dependently elevated Fos-IR in TNC. The number of Fos-IR elevation from day 7 of infusion was comparable with a single capsaicin infusion as a headache model. Increase in face grooming/scratching behavior was evident from day 7, and peaked at day 14 of chronic PRE084 infusion, which was correlated with ΔFosB elevation and phosphorylation of extracellular signal-regulated kinase, and the NMDA receptor NR1 subunit in TNC. Following 14days of PRE084 infusion, the number of Fos-IR increased until day 7 after final infusion. Moreover, by day 14, Fos-IR associated with PRE084 infusion was significantly reversed by NMDA receptor antagonist MK801, rather than BD1047. These findings indicated that chronic activation of Sig-1R could evoke prolonged neuronal activation in the trigeminovascular system.

Differential effects of electrical stimulation of the central amygdala and lateral hypothalamus on fos-immunoreactive neurons in the gustatory brainstem and taste reactivity behaviors in conscious rats.

Projections from the central amygdala (CeA) and lateral hypothalamus (LH) modulate the activity of gustatory brainstem neurons, however, the role of these projections in gustatory behaviors is unclear. The goal of the current study was to determine the effects of electrical stimulation of the CeA or LH on unconditioned taste reactivity (TR) behaviors in response to intra-oral infusion of tastants. In conscious rats, electrical stimulation of the CeA or LH was delivered with and without simultaneous intra-oral infusion of taste solutions via an intra-oral cannula. Immunohistochemistry for the Fos protein was used to identify neurons in the gustatory brainstem activated by the electrical and/or intra-oral stimulation. In the absence of intra-oral infusion of a tastant, electrical stimulation of either the CeA or the LH increased the number of ingestive, but not aversive, TR behaviors performed. During intra-oral infusions of taste solutions, CeA stimulation tended to increase aversive behaviors whereas LH stimulation dramatically reduced the number of aversive responses to quinine hydrochloride (QHCl). These data indicate that projections from the CeA and LH alter TR behaviors. A few of the behavioral effects were accompanied by changes in the number of Fos-immunoreactive neurons in the gustatory brainstem, suggesting a possible anatomical substrate for these effects.

Glucagon-like peptide-1 of brainstem origin activates dorsomedial hypothalamic neurons in satiated rats

A high number of neurons express c-fos in response to unlimited food intake in fasted rats in the ventral subdivision of the hypothalamic dorsomedial nucleus (DMHv). We report here, that in same conditions, limited food consumption failed to induce Fos expression in DMHv neurons suggesting that satiation should be one of the important signals that activate these neurons. The possible origin of fibers conducting satiation signals to the DMHv could be in the lower brainstem, especially glucagon-like peptide-1 (GLP-1)-containing neurons in the nucleus of the solitary tract (NTS). We demonstrate that GLP-1-immunoreactive fibers and fiber terminals topographically overlap with activated Fos-positive neurons in the DMHv in refed rats. Using immunocytochemistry and in situ hybridization histochemistry, we demonstrated GLP-1 receptors in Fos-expressing neurons of the DMH. Unilateral transections of ascending GLP-1-containing fibers from the NTS inside the pons in refed rats (unlimited food consumption) resulted in a dramatic decrease in the density of GLP-1 fibers and in the number of Fos-immunoreactive neurons in the DMHv, but only on the side of the transection. Contralateral to the transection, neither the GLP-1 fiber density nor the number of Fos-positive cells changed significantly. Meanwhile, the density of GLP-1 immunoreactivity was markedly accumulated in transected nerve fibers caudal to the cuts, as a consequence of the interruption of the ascending GLP-1 transport route. These findings suggest that the solitary-hypothalamic projections may represent the neuronal route through GLP-1 neurons of the NTS activate DMHv neurons via GLP-1 receptors by conveying information on satiety.

Male and female odors induce Fos expression in chemically defined neuronal population

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide urocortin 3, or glutamic acid decarboxylase mRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir in the MEA and PMV comparing fed and fasted animals. In the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 mRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine- and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues.

State-dependent pattern of Fos protein expression in regionally-specific sites within the preoptic area of the cat

Clinical and experimental data have shown that the preoptic area of the hypothalamus (POA) is involved in the generation and maintenance of NREM sleep. However, the activity of specific populations of POA neurons during REM sleep, NREM sleep and different waking conditions is still not firmly established. Consequently, we performed a quantitative, regionally-specific analysis of the Fos immunoreactivity of neurons in the POA of the cat during NREM sleep and REM sleep induced by microinjections of carbachol into the nucleus pontis oralis (REMc), as well as during quiet and alert wakefulness. We observed that while the total number of Fos immunoreactive neurons in the POA did not change as a function of these behavioral states, state-specific differences in neuronal activity were detected in restricted regions of the POA. An increase in the number of Fos+ neurons was observed in the rostral tip of the suprachiasmatic nucleus (SCN) during NREM (83.4 ± 25.6) compared to quiet wakefulness (5.1 ± 1.3, p < 0.05) but not with the other behavioral states. In the median preoptic nucleus (MnPN), the number of Fos immunoreactive neurons was greater during NREM sleep (39.5 ± 6.1) compared with quiet wakefulness (13.5 ± 1.4, p < 0.05) and REMc (16.2 ± 2.0, p < 0.05). State-specific Fos immunoreactive neurons were not observed in the ventro-lateral preoptic nucleus (VLPO). Finally, there was no significant increase in the number of Fos+ neurons during REMc in any of the subregions of the POA. In conclusion, within the POA, a selective neuronal activation during NREM sleep was found only in the MnPN. In addition, our data suggest a potential role of the SCN in NREM sleep. Finally, based on the distribution of Fos+ neurons in the entire POA, we conclude that the neuronal network involved in the regulation of NREM sleep is dispersed and intermingled with waking-related neurons.

Antinociceptive Effect of Amygdalin Isolated from &lt;i&gt;Prunus armeniaca&lt;/i&gt; on Formalin-Induced Pain in Rats

Amygdalin is a plant glucoside isolated from the stones of rosaceous fruits, such as apricots, peaches, almond, cherries, and plums. To investigate the pain-relieving activity of amygdalin, we induced pain in rats through intraplantar injection of formalin, and evaluated the antinociceptive effect of amygdalin at doses of 0.1, 0.5, 1.0, and 10.0 mg/kg-body weight by observing nociceptive behavior such as licking, biting and shaking, the number of Fos-immunoreactive neurons in the spinal cord, and the mRNA expression of inflammatory cytokines in the plantar skin. The intramuscular injection of amygdalin significantly reduced the formalin-induced tonic pain in both early (the initial 10 min after formalin injection) and late phases (10-30 min following the initial formalin injection). During the late phase, amygdalin did reduce the formalin-induced pain in a dose-dependent manner in a dose range less than 1 mg/kg. Molecular analysis targeting c-Fos and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) also showed a significant effect of amygdalin, which matched the results of the behavioral pain analysis. These results suggest that amygdalin is effective at alleviating inflammatory pain and that it can be used as an analgesic with anti-nociceptive and anti-inflammatory activities.

Bee venom injection produces a peripheral anti-inflammatory effect by activation of a nitric oxide-dependent spinocoeruleus pathway

Our recent data, obtained using a zymosan-induced inflammatory air pouch model in mice, have demonstrated that subcutaneous bee venom (BV) injection into the hind limb selectively activates the contralateral brain stem locus coeruleus (LC) and then via a descending noradrenergic pathway and subsequent adrenal medullary catecholamine release induces a potent anti-inflammatory effect. While the efferent limb of this BV-induced neuroimmune anti-inflammatory pathway is well documented, the afferent limb of this pathway is poorly understood. In particular the spinal mechanisms involved with BV activation of the LC are currently unknown. Spinal nitric oxide (NO) and its synthase (NOS) have been shown to play an important role in the transmission and amplification of neuronal information from the spinal cord to the brain stem. In the present study we evaluated whether spinal NO plays a role in BV-induced LC activation, since we have previously shown that LC activation underlies this 'BV-induced anti-inflammatory effect' (BVAI) using the mouse air pouch model. Intrathecal (i.t.) pretreatment with l-nitro arginine methyl ester (l-NAME, non-selective NOS inhibitor), hemoglobin (NO scavenger) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, soluble guanylate cyclase inhibitor) abolished BVAI on zymosan-induced leukocyte migration into the air pouch. Moreover, i.t. injection of l-N-iminoethyl-lysine (l-NIL, inducible NOS inhibitor), but not 7-nitroindazole (7-NI, neuronal NOS inhibitor), also inhibited BVAI. BV injection significantly increased both the number of Fos immunoreactive neurons and tyrosine hydroxylase-Fos double labeling neurons in the contralateral LC in zymosan-induced inflamed mice. Importantly this increase in Fos expression in the LC was also completely inhibited by i.t. injection of l-NIL, but not by i.t. injection of 7-NI. Collectively these results indicate that spinal NO generated from inducible NOS is involved in the BV-induced LC activation that underlies BVAI.

Glucocorticoid modulation of atrial natriuretic peptide, oxytocin, vasopressin and Fos expression in response to osmotic, angiotensinergic and cholinergic stimulation

The regulation of fluid and electrolyte homeostasis involves the participation of several neuropeptides and hormones that utilize hypothalamic cholinergic, alpha-adrenergic and angiotensinergic neurotransmitters and pathways. Additionally, it has been suggested that hypothalamus–pituitary–adrenal axis activity modulates hormonal responses to blood volume expansion. In the present study, we evaluated the effect of dexamethasone on atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) responses to i.c.v. microinjections of 0.15 M and 0.30 M NaCl, angiotensin-II (ANG-II) and carbachol. We also evaluated the Fos protein immunoreactivity in the median preoptic (MnPO), paraventricular (PVN) and supraoptic (SON) nuclei. Male Wistar rats received an i.p. injection of dexamethasone (1 mg/kg) or vehicle (0.15 M NaCl) 2 h before the i.c.v. microinjections. Blood samples for plasma ANP, OT, AVP and corticosterone determinations were collected at 5 and 20 min after stimulus. Another set of rats was perfused 120 min after stimulation. A significant increase in plasma ANP, OT, AVP and corticosterone levels was observed at 5 and 20 min after each central stimulation compared with isotonic saline–injected group. Pre-treatment with dexamethasone decreased plasma corticosterone and OT levels, with no changes in the AVP secretion. On the other hand, dexamethasone induced a significant increase in plasma ANP levels. A significant increase in the number of Fos immunoreactive neurons was observed in the MnPO, PVN and SON after i.c.v. stimulations. Pre-treatment with dexamethasone induced a significant decrease in Fos immunoreactivity in these nuclei compared with the vehicle. These results indicate that central osmotic, cholinergic, and angiotensinergic stimuli activate MnPO, PVN and SON, with a subsequent OT, AVP, and ANP release. The present data also suggest that these responses are modulated by glucocorticoids.

Identification of Active Central Nervous System Sites in Renal Wrap Hypertensive Rats

To identify central neurons participating in cardiovascular regulation in hypertension, we studied Fos staining, a marker for synaptically activated neurons, in adult male normotensive and hypertensive (HT) rats. At 1 and 4 weeks after induction of unilateral nephrectomy, renal wrap hypertension mean arterial pressure was 138+/-4 mm Hg (n=6) in 1-week HT rats and 159+/-6 mm Hg (n=6) in 4-week HT rats. Mean arterial pressure was 103+/-2 mm Hg (n=6) in sham-operated, normotensive rats. Mean arterial pressure was greater in both HT groups compared with normotensive rats, and the mean arterial pressure in 4-week HT rats was greater than that in 1-week HT rats. Rats were anesthetized and perfused, brains sectioned and processed using a Fos antibody, and the number of Fos immunoreactive neurons counted in sections through various brain regions. Hypertension of 1 or 4 weeks did not alter the number of Fos immunoreactive neurons in the area postrema, the supraoptic nucleus, and the median preoptic nucleus. The number of Fos immunoreactive neurons was increased after 1 and 4 weeks in the nucleus of the solitary tract, both the caudal and ventral lateral medulla, and the organum vasculosum of the lamina terminalis. In addition, after 4 weeks of HT, the number of Fos immunoreactive neurons was increased in the parabrachial nucleus and the paraventricular nucleus of the hypothalamus. The results indicate central regions active in acute and chronic HT rats and suggest certain areas that may be differentially activated depending on the duration of the hypertension.

Correlation of noxious evoked c- fos expression in areas of the somatosensory system during chronic pain: Involvement of spino-medullary and intra-medullary connections

Chronic pain induces functional alterations of the endogenous pain control system namely in the modulation of nociceptive transmission at the spinal cord. We used the c- fos expression as a tool to study correlated neuronal activation, induced by bending the inflamed paw of monoarthritic animals, between the spinal dorsal horn and medullary centers belonging to the endogenous pain control system, namely the lateralmost reticular formation of the ventrolateral medulla (VLMlat), the lateral reticular nucleus (LRt), the dorsal reticular nucleus (DRt), the nucleus tractus solitarius (Sol) and the rostroventromedial medulla (RVM). Awake monoarthritic rats were subjected to 4 min of paw bending followed by anaesthesia and perfusion either immediately or 2 h later. The numbers of Fos immunoreactive neurons in the spinal dorsal horn and in the medulla oblongata were significantly correlated mainly immediately after stimulation: lamina I correlated with the VLMlat, LRt, Sol and RVM; lamina II correlated with the VLMlat, LRt and Sol; and laminae IV-V correlated with the VLMlat and LRt. Between medullary pain control centers significant correlations occurred immediately and 2 h after bending at the VLMlat-Sol and LRt-Sol, at the VLMlat-LRt and VLMlat-RVM in animals perfused immediately, and at the VLMlat-DRt and LRt-RVM in animals perfused 2 h later. These data demonstrate that the mobilization of a chronically inflamed paw triggers intense correlated neuronal activity in several areas of the somatosensory system, indicating functional relevant links in pain control.

Secondary hyperalgesia in the monoarthritic rat is mediated by GABAB and NK1 receptors of spinal dorsal horn neurons: A behavior and c-fos study

Secondary hyperalgesia in the monoarthritic rat is accompanied by a decrease in nociceptive activation of spinal neurons expressing GABAB receptors and by the opposite effect in the cells expressing neurokinin 1 (NK1)-receptors. In order to ascertain the relative role of each receptor, the effects of intrathecal administration of SP-saporin (SP-SAP), baclofen or both were evaluated, using a model of secondary hyperalgesia that consists of mechanical stimulation of the hindlimb skin close to an inflamed joint. Four days after the induction of monoarthritis by intraarticular injection of Complete Freund’s Adjuvant (CFA), a cannula was implanted at T13–L1 level and 10 μl of saline or SP-SAP (10−6 M) were intrathecally (i.t.) injected. Fourteen days after CFA-injection, half of the animals from each group received i.t. injections of 10 μl saline and the remainder were injected with the same volume of baclofen (1 μg). Ten minutes later, the animals were behaviorally evaluated by the von Frey test or submitted to noxious mechanical stimulation to analyze c-fos expression. The von Frey thresholds increased after the treatments, but more pronouncedly after baclofen or SP-SAP plus baclofen. In segments L2–L3, the spinal area that receives input from the stimulated skin close to the inflamed joint, the numbers of Fos-immunoreactive neurons were reduced after the three treatments both in the superficial and deep dorsal horn. In segments T13–L1, the numbers of Fos-immunoreactive neurons were significantly reduced after treatment with SP-SAP plus baclofen in both dorsal horn regions, and in the deep dorsal horn after baclofen treatment. We conclude that both GABAB and NK1 receptors of spinal dorsal horn neurons participate in secondary hyperalgesia in the monoarthritic rat, although the decrease in GABA inhibition appears to play a more important role than the increase in SP-mediated effects.

Hemorrhage activates proopiomelanocortin neurons in the rat hypothalamus

Severe blood loss lowers arterial pressure through a central mechanism that is thought to include opioid neurons. In this study, we investigated whether hemorrhage activates proopiomelanocortin (POMC) neurons by measuring Fos immunoreactivity and POMC mRNA levels in the medial basal hypothalamus. Hemorrhage (2.2 ml/100 g body weight over 20 min) increased the number of Fos immunoreactive neurons throughout the rostral–caudal extent of the arcuate nucleus, the retrochiasmatic area and the peri-arcuate region lateral to the arcuate nucleus where POMC neurons are located. Double label immunohistochemistry revealed that hemorrhage increased Fos expression by β-endorphin immunoreactive neurons significantly. The proportion of β-endorphin immunoreactive neurons that expressed Fos immunoreactivity increased approximately four-fold, from 11.7 ± 1.4% in sham-operated control animals to 42.0 ± 5.2% in hemorrhaged animals. Hemorrhage also increased POMC mRNA levels in the medial basal hypothalamus significantly, consistent with the hypothesis that blood loss activates POMC neurons. To test whether activation of arcuate neurons contributes to the fall in arterial pressure evoked by hemorrhage, we inhibited neuronal activity in the caudal arcuate nucleus by microinjecting the local anesthetic lidocaine (2%; 0.1 or 0.3 μl) bilaterally 2 min before hemorrhage was initiated. Lidocaine injection inhibited hemorrhagic hypotension and bradycardia significantly although it did not influence arterial pressure or heart rate in non-hemorrhaged rats . These results demonstrate that hemorrhage activates POMC neurons and provide evidence that activation of neurons in the arcuate nucleus plays an important role in the hemodynamic response to hemorrhage.

From synapse to gene product: prolonged expression of c- fos induced by a single microinjection of carbachol in the pontomesencephalic tegmentum

Abstract It is not known how the brain modifies its regulatory systems in response to the application of a drug, especially over the long term of weeks and months. We have developed a model system approach to this question by manipulating cholinergic cell groups of the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei in the pontomesencephalic tegmentum (PMT), which are known to be actively involved in the timing and quantity of rapid eye movement (REM) sleep. In a freely moving feline model, a single microinjection of the cholinergic agonist carbachol conjugated to a latex nanosphere delivery system into the caudolateral PMT elicits a long-term enhancement of one distinguishing phasic event of REM sleep, ponto-geniculo-occipital (PGO) waves, lasting 5 days but without any significant change in REM sleep or other behavioral state. Here, we test the hypothesis that cholinergic activation within the caudolateral PMT alters the postsynaptic excitability of the PGO network, stimulating the prolonged expression of c- fos that underlies this long-term PGO enhancement (LTPE) effect. Using quantitative Fos immunohistochemistry, we found that the number of Fos-immunoreactive (Fos-IR) neurons surrounding the caudolateral PMT injection site decreased sharply by postcarbachol day 03, while the number of Fos-IR neurons in the more rostral LDT/PPT increased >30-fold and remained at a high level following the course of LTPE. These results demonstrate a sustained c- fos expression in response to pharmacological stimulation of the brain and suggest that carbachol's acute effects induce LTPE via cholinergic receptors, with subsequent transsynaptic activation of the LDT/PPT maintaining the LTPE effect.

Effects of repeated maternal stress on FOS expression in the hypothalamic paraventricular nucleus of fetal rats

The effects of repeated prenatal stress with different severity (restraint and immobilization) on Fos expression in the maternal and fetal hypothalamic paraventricular nucleus (PVN) were examined in rats. Acute stress treatment was performed for 30 min on gestational day 21, and repeated stress treatment for 30 min daily for 5 days from gestational days 17–21. In the parvocellular region of the maternal PVN, the stress-induced increases in the number of Fos-immunoreactive neurons were smaller in the repeated stress groups than the acute stress groups, indicating an adaptation of Fos expression to repeated stress. The attenuated Fos expression observed in the maternal PVN following repeated mild stress did not occur in the fetal PVN. In contrast, repeated immobilization stress caused a much smaller increase in Fos expression in the fetal PVN than did acute immobilization stress. The reduced Fos expression in the fetal PVN following repeated severe stress was thought to be due to cell death, since the fetal PVN in the chronic immobilization group revealed a reduction in the total number of cells and an increase in the number of apoptotic cells. In the female but not male fetuses, repeated restraint stress induced a significant increase in the number of apoptotic cells in the PVN. These findings suggest that the fetal PVN shows no adaptation of Fos expression to repeated maternal stress, but great vulnerability to cell death, including apoptosis. In addition, stress-induced apoptosis may more easily occur in the fetal PVN in females than males.

Distinctive pattern of c-fos expression in the feline cervico-lumbar spinal cord after stimulation of vanilloid receptors in dorsal neck muscles

In the present study, c- fos expression in the spinal cord has been used as a marker of neuronal activation induced by capsaicin-sensitive sensory afferents from the dorsal neck muscles in cats ( n=6). The number of Fos-immunoreactive neurons, which were revealed using the avidin–biotin-peroxidase method, was significantly increased in the cervical and lumbar spinal cord. In contrast to the control group ( n=3), 2 h after intramuscular capsaicin injection, c- fos expression was more extensive ipsilaterally to the injected side in the C3–C6 segments, and bilaterally in the L4–L6 segments. Most labeled neurons in the cervical spinal cord were small and giant cells, predominantly located in the middle and lateral parts of lamina I and, additionally, at the neck of the dorsal horn (lamina V), i.e., within the zones of termination of high-threshold muscle afferents. The widespread distribution of labeled cells throughout the cervical cord within the intermediate zone (lamina VII) coincided with the sites of last-order premotor interneurons and cells of origin of long crossed and uncrossed descending propriospinal pathways to the lumbar spinal cord. These findings suggest possible mechanisms for spreading of nociceptive signals between cervical and lumbar regions.