Activation of the parabrachio-amygdaloid pathway by immune challenge or spinal nociceptive input: a quantitative study in the rat using Fos immunohistochemistry and retrograde tract tracing

Activation of the immune system by e.g. bacteria induces the acute-phase-response and sickness behaviour. The latter encompasses among other things fever, lethargy, anorexia and hyperalgesia. An often used model to study sickness behaviour is the intravenous injection of the gram negative bacterial endotoxin lipopolysaccharide (LPS). LPS induces the production of inflammatory mediators, such as cytokines and prostaglandins, which in turn can interact with the central nervous system (CNS) to affect behaviour. The CNS also memorises substances that have made us sick in the past to avoid future harm, a phenomenon called conditioned taste aversion (CTA). An often used model to study CTA is the intraperitoneal injection of LiCl. The pontine parabrachial nucleus (PB) is an autonomic relay nucleus situated in the rostral brain stem that integrates afferent somatosensory and interoceptive information and forwards this information to the hypothalamus and limbic structures. PB is crucial for the acquisition of CTA and PB neurons are activated by many anorexigenic substances. Further, PB neurons express neuropeptides, among those calcitonin gene related peptide (CGRP) and enkephalin, both of which have been implicated in immune signalling, nociception, food intake, and aversion. By using a dual-labelling immunohistochemical/in situ hybridization technique we investigated if enkephalinergic neurons in PB are activated by systemic immune challenge. While there were many neurons in the external lateral parabrachial subnucleus (PBel) that expressed the immediate early gene fos after intravenous injection of LPS and while a large proportion of the PBel neurons expressed preproenkephalin, there were very few double-labelled cells. The fos-expressing cells were predominantly located to the outer part of the PBel (PBelo), whereas the preproenkephalin-expressing PBel neurons were located closest to the peduncle. Thus we conclude that although enkephalin has been implicated in autonomic and immune signalling, enkephalinergic neurons in PB do not seem to be activated by immune stimulation (paper I). To further characterise the PBelo neurons activated by immune challenge we investigated if these neurons expressed CGRP. Dual-labelling in situ hybridisation showed that PBelo neurons that expressed fos after intravenous injection of LPS to a large extent co-expressed CGRP mRNA, indicating that CGRP may be involved in the regulation of the sickness response in immune challenge (paper II). Using dual-labelling immunohistochemistry we examined if PBel neurons activated by an immune stimulus projected to the amygdala, a limbic structure implicated in the affective response to homeostatic challenge. Animals were injected with the retrograde tracer substance cholera toxin b (CTb) into the amygdala and subsequently subjected to immune challenge. We found that approximately a third of the neurons that expressed fos after the intravenous injection of LPS also were labelled with CTb. Thus PBel neurons activated by immune challenge project to the amygdala. The PBel-amygdala pathway has earlier been suggested to be important in nociceptive signalling. To investigate if amygdala-projecting PBel neurons are activated by nociceptive stimuli we again injected animals with CTb into the amygdala. After recovery the animals were injected with formalin into a hindpaw. Dual-labelling immunohistochemistry against fos and CTb showed that very few noxiously activated PB neurons projected to the amygdala. Thus, the PBel-amygdala projection seems to be important in immune challenge but not in nociceptive signalling (paper III). Many PBel neurons express fos after intraperitoneal injection of LiCl. Melanocortins are neuropeptides that recently have been implicated in metabolism, food intake and aversive mechanisms. The PB is known to express melanocortin receptor-4 (MC4-R) mRNA. Using dual-labelling in situ hybridization we investigated if PB neurons activated by intravenous injection of LPS or intraperitoneal injection of LiCl expressed MC4-R mRNA. We found that many PBelo neurons were activated by either LPS or LiCl and that a large proportion of such activated neurons expressed MC4-R mRNA. Further, using dual-labelling in situ hybridization against MC4-R mRNA and CGRP mRNA, we found that a large proportion of the CGRP positive PBelo neurons also expressed MC4-R mRNA. In summary, this thesis shows that CGRP-expressing neurons in the PBel are activated by peripheral immune challenge, that lipopolysaccharide-activated PBel neurons project to the amygdala, that the amygdala-projecting neurons in the PBel are CGRP-positive, and that PBel neurons activated by immune or aversive challenge express MC4-R. Taken together, these data suggest the presence of a melanocortin-regulated CGRP-positive pathway from the PBel to the amygdala that relays information of importance to certain aspects of sickness behaviour.