EP3 and EP4 receptor mRNA expression in peptidergic cell groups of the rat parabrachial nucleus.

This study examines the distribution of prostaglandin E2 receptors of subtype EP3 and EP4 among brain stem parabrachial neurons that were characterized with respect to their neuropeptide expression. By using a dual-labeling in situ hybridization method, we show that preprodynorphin mRNA expressing neurons in the dorsal and central lateral subnuclei express EP3 receptor mRNA. Such receptors are also expressed in preproenkephalin, calcitonin gene related peptide and preprotachykinin mRNA positive neurons in the external lateral subnucleus, whereas preprodynorphin mRNA expressing neurons in this subnucleus are EP receptor negative. In addition, EP3 receptor expression is seen among some enkephalinergic neurons in the Kölliker-Fuse nucleus. Neurons in the central part of the cholecystokininergic population in the regions of the superior lateral subnucleus express EP4 receptor mRNA, whereas those located more peripherally express EP3 receptors. Taken together with previous findings showing that discrete peptidergic cell groups mediate nociceptive and/or visceral afferent information to distinct brain stem and forebrain regions, the present results suggest that the processing of this information in the parabrachial nucleus is influenced by prostaglandin E2. Recent work has shown that prostaglandin E2 is released into the brain following peripheral immune challenge; hence, the parabrachial nucleus may be a region where humoral signaling of peripheral inflammatory events may interact with neuronal signaling elicited by the same peripheral processes.

Activation of prostanoid EP(3) and EP(4) receptor mRNA-expressing neurons in the rat parabrachial nucleus by intravenous injection of bacterial wall lipopolysaccharide.

Systemic inflammation activates central autonomic circuits, such as neurons in the pontine parabrachial nucleus. This activation may be the result of afferent signaling through the vagus nerve, but it may also depend on central prostaglandin-mediated mechanisms. Recently, we have shown that neurons in the parts of the parabrachial nucleus that are activated by immune challenge express prostaglandin receptors of the EP(3) and EP(4) subtypes, but it remains to be determined if the prostaglandin receptor-expressing neurons are identical to those that respond to immune stimuli. In the present study, bacterial wall lipopolysaccharide was injected intravenously in adult male rats and the expression of c-fos mRNA and of EP(3) and EP(4) receptor mRNA was examined with complementary RNA probes labeled with digoxigenin and radioisotopes, respectively. Large numbers of neurons in the external lateral parabrachial subnucleus, a major target of vagal-solitary tract efferents, expressed c-fos mRNA. Quantitative analysis showed that about 60% (range 40%-79%) of these neurons also expressed EP(3) receptor mRNA. Conversely, slightly more than 50% (range 48%-63%) of the EP(3) receptor-expressing neurons in the same subnucleus coexpressed c-fos mRNA. In contrast, few EP(4) receptor-expressing neurons were c-fos positive, with the exception of a small population located in the superior lateral and dorsal lateral subnuclei. These findings show that immune challenge activates central autonomic neurons that could be the target of centrally produced prostaglandin E(2), suggesting that synaptic signaling and paracrine mechanisms may interact on these neurons.

Feeding-related immune responsive brain stem neurons: association with CGRP.

Using dual-labeling in situ hybridization histochemistry, the neurotransmitter expression of immune-responsive neurons in the pontine parabrachial nucleus, a major relay for interoceptive information, was investigated. Intravenous injection of bacterial wall lipopolysaccharide resulted in dense c-fos mRNA expression in the external lateral parabrachial nucleus, and a majority of the c-fos expressing cells also expressed calcitonin gene-related peptide (CGRP) mRNA. In contrast CGRP-positive cells in the adjoining external medial subnucleus were c-fos negative. Taken together with previous hodological and behavioral studies, these data suggest that CGRPergic parabrachial neurons may mediate lipopolysaccharide-induced anorexia by means of their projection to central nucleus of the amygdala.

Analysis of adrenocortical secretory responses during acute an prolonged immune stimulation in inflammation-susceptible and -resistant rat strains.

Endogenous corticosterone secreted during immune challenge restricts the inflammatory process and genetic variations in this neuroendocrine-immune dialogue have been suggested to influence an individuals sensitivity to develop chronic inflammatory disorders. We have tested inflammation-susceptible Dark Agouti (DA) rats and resistant, MHC-identical, PVG.1AV1 rats for their abilities to secrete corticosterone in response to acute challenge with bacterial lipopolysaccharide (LPS) or a prolonged activation of the nonspecific immune system with arthritogenic yeast beta-glucan. Intravenous injection of LPS triggered equipotent secretion of corticosterone in both rat strains. Interestingly, peak concentrations of corticosterone did not differ significantly between the strains. Intradermal injection of beta-glucan caused severe, monophasic, polyarthritis in DA rats while PVG.1AV1 responded with significantly milder joint inflammation. Importantly, serial sampling of plasma from glucan-injected DA and PVG.1AV1 rats did not reveal elevated concentrations of plasma corticosterone at any time from days 1-30 postinjection compared to preinjection values, in spite of the ongoing inflammatory process. Interestingly, adrenalectomized, beta-glucan-challenged DA rats responded with an aggravated arthritic process, indicating an anti-inflammatory role for the basal levels of corticosterone that were detected in intact DA rats challenged with beta-glucan. Moreover, substitution with subcutaneous corticosterone-secreting pellets, yielding moderate stress-levels, significantly attenuated the arthritic response. In contrast, adrenalectomized and glucan-challenged PVG.1AV1 rats did not respond with an elevated arthritic response, suggesting that these rats contain the arthritic process via corticosterone-independent mechanisms. In conclusion, the hypothalamic-pituitary-adrenal axis in both rat strains exhibited strong activation after challenge with LPS. This contrasted to the basal corticosterone levels observed strains during a prolonged arthritic process. No correlation between ability to secrete corticosterone and susceptibility to inflammation could be demonstrated. Basal levels of endogenous corticosterone appeared to restrain inflammation in beta-glucan-challenged DA rats whereas resistance to inflammation in PVG.1AV1 rats may be mediated via corticosterone-independent mechanisms.

Distribution of the EP3 prostaglandin E(2) receptor subtype in the rat brain: relationship to sites of interleukin-1-induced cellular responsiveness.

The activation of neurosecretory neurons that express corticotropin-releasing hormone (CRH) in response to increased circulating levels of interleukin-1beta (IL-1beta) depends on prostaglandin E(2) (PGE(2)) acting locally within the brain parenchyma. To identify potential central targets for PGE(2) relevant to pituitary-adrenal control, the distribution of mRNA encoding the PGE(2) receptor subtype EP3 (EP3R) was analyzed in rat brain. Hybridization histochemistry revealed prominent labeling of cells in discrete portions of the olfactory system, iso- and hippocampal cortices, and subcortical telencephalic structures in the septal region and amygdala. Labeling over the midline, intralaminar, and anterior thalamic groups was particularly prominent. EP3R expression was enriched in the median preoptic nucleus and adjoining aspects of the medial preoptic area (MPO) implicated in thermoregulatory/febrile responses and sleep induction. EP3R-expressing cells were also prominent in brainstem cell groups involved in nociceptive information processing/modulation (periaqueductal gray, locus coeruleus (LC), parabrachial nucleus (PB), caudal raphé nuclei), arousal and wakefulness (LC, midbrain raphé and tuberomammillary nuclei); and in conveying interoceptive input, including systemic IL-1 signals, to the endocrine hypothalamus (nucleus of the solitary tract (NTS) and rostral ventrolateral medulla [VLM]). Combined hybridization histochemical detection of EP3R mRNA with immunolocalization of IL-1beta-induced Fos protein expression identified cytokine-sensitive, EP3R-positive cells in the medial NTS, rostral VLM, and, to a lesser extent, aspects of the MPO. These findings are consistent with the view that increased circulating IL-1 may stimulate central neural mechanisms, including hypothalamic CRH neurons, through an EP3R-dependent mechanism involving PGE(2)-mediated activation of cells in the caudal medulla and/or preoptic region.

Distribution of prostaglandin EP(3) and EP(4) receptor mRNA in the rat parabrachial nucleus.

By using in situ hybridization, the distribution of mRNA for the PGE(2) receptors EP(3) and EP(4) was examined in the rat parabrachial nucleus (PB), a major brain stem relay for autonomic and nociceptive processing. EP(3) receptor mRNA was present in most subnuclei, with the densest labeling in the external lateral, dorsal lateral, superior lateral, central lateral and Kölliker-Fuse nuclei. EP(4) receptor mRNA expressing cells had a more restricted distribution, largely being confined to the superior lateral and adjacent parts of the dorsal and central lateral nuclei in a pattern complementary to that for EP(3) receptor mRNA. These findings suggest that EP(3) and EP(4) receptors in PB have distinct functional roles that include nociceptive processing, blood pressure regulation and feeding behavior.

Expression of corticotropin-releasing factor in the peripheral nervous system of the rat.

The occurrence and distribution of corticotropin-releasing factor (CRF) in the rat peripheral nervous system was studied by immunohistochemistry. CRF-positive nerve fibers were identified in the spleen, thymus, synovial membrane of the knee joint and adrenal gland. In general, CRF-positive fibers were seen predominantly in and around the blood vessels; however, many non-vascular thin varicose fibers were also observed. The neuronal character of the immunoreactive fibers was confirmed by staining consecutive tissue sections with a general neuronal marker, protein gene product 9.5. The finding of CRF-positive nerve fibers in the periphery demonstrates a strong anatomical link between the nervous, endocrine and immune systems, and may have pathophysiological implications in the inflammatory and stress-related disorders.