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Brain Stem Involvement in Immune and Aversive Challenge
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2006. , 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 963
Keyword [en]
lipopolysaccharide, Lithium Chloride, anorexia, aversion, parabrachial, brain stem, melanocortin, CGRP, enkephalin
National Category
Physiology
Identifiers
URN: urn:nbn:se:liu:diva-7579ISBN: 91-85643-81-5 (print)OAI: oai:DiVA.org:liu-7579DiVA: diva2:22593
Public defence
2006-11-10, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Note
On the day of the defence date the title of article II was: Feeding-related immune responsive brain stem neurons: association with CGRP. Article II: Erratum for in Neuroreport 2001;12(16):inside back cover. Neuroreport 2001;12(13):inside back cover. Article III: Erratum in: J Comp Neurol. 2005; 483:489-90.Available from: 2006-10-16 Created: 2006-10-16 Last updated: 2012-01-30Bibliographically approved
List of papers
1. Preproenkephalin mRNA expression in rat parabrachial neurons: relation to cells activated by systemic immune challenge
Open this publication in new window or tab >>Preproenkephalin mRNA expression in rat parabrachial neurons: relation to cells activated by systemic immune challenge
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2001 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 316, no 3, 165-168 p.Article in journal (Refereed) Published
Abstract [en]

By using a dual-labeling immunohistochemical/in situ hybridization technique we examined if enkephalin-expressing neurons in the pontine parabrachial nucleus, a major brain stem relay for ascending visceral and homeostatic information, were activated by systemic immune challenge. While rats subjected to intravenous injection of bacterial wall lipopolysaccharide expressed dense labeling for the immediate-early gene product FOS in parts of the parabrachial nucleus that also demonstrated dense preproenkephalin expression, only a small proportion of the enkephalin-positive neurons were FOS-positive. These data indicate that enkephalins, although implicated in a variety of autonomic responses, are not primarily involved in the transmission of immune-related information from the parabrachial nucleus to its different forebrain and brain stem targets.

Place, publisher, year, edition, pages
Elsevier Science B.V., Amsterdam., 2001
Keyword
Parabrachial nucleus, Systemic inflammation, FOS, Enkephalin, In situ hybridization, Feeding
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12954 (URN)10.1016/S0304-3940(01)02393-X (DOI)000173268200010 ()
Available from: 2008-02-25 Created: 2008-02-25 Last updated: 2017-12-13
2. Feeding-related immune responsive brain stem neurons: association with CGRP
Open this publication in new window or tab >>Feeding-related immune responsive brain stem neurons: association with CGRP
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2001 (English)In: Neuroreport, ISSN 0959-4965, Vol. 12, no 11, 2399-2403 p.Article in journal (Refereed) Published
Abstract [en]

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-posi- tive 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14081 (URN)000170141600024 ()
Available from: 2006-10-16 Created: 2006-10-16 Last updated: 2010-09-23
3. 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
Open this publication in new window or tab >>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
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2005 (English)In: Journal of Comparative Neurology, ISSN 0021-9967, E-ISSN 1096-9861, Vol. 481, no 2, 210-219 p.Article in journal (Refereed) Published
Abstract [en]

Peripheral nociceptive stimulation results in activation of neurons in the pontine parabrachial nucleus (PB) of rats. Electrophysiological studies have suggested that noxiously activated PB neurons project to the amygdala, constituting a potential pathway for emotional aspects of pain. In the present study we examined this hypothesis by combining retrograde tract tracing with Fos immunohistochemistry. Cholera toxin subunit B was injected into the amygdala of rats. After a minimum of 48 hours the rats were given a subcutaneous injection of 100 l of 5% formalin into one hindpaw and killed 60-90 minutes later. A dense aggregation of retrogradely labeled neurons was seen in the external lateral PB. Fos-expressing neurons were present preferentially in the central, dorsal, and superior lateral subnuclei as well as in the lateral crescent area, as described previously. There was little overlap between the retrogradely labeled and Fos-expressing populations and double-labeled neurons were rare. In contrast, systemic immune challenge by intravenous injection of bacterial wall lipopolysaccharide resulted in a Fos expression that overlapped the retrograde labeling in the external lateral PB, and many double-labeled neurons were seen. While these data provide direct functional anatomical evidence that nociceptive information from the hindlimb is relayed to the amygdala via the parabrachial nucleus, the number of parabrachio-amygdaloid neurons involved is small. Considering the widespread activation of parabrachio-amygdaloid neurons by a variety of visceral and humoral stimuli, the parabrachio-amygdaloid pathway thus appears to be more involved in the mediation of information related to viscerally and humorally elicited activity than in transmission of spinal nociceptive inputs.

Keyword
external lateral parabrachial nucleus, pain, formalin, immune challenge, cholera toxin subunit b, central nucleus of the amygdala
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14082 (URN)10.1002/cne.20384 (DOI)
Available from: 2006-10-16 Created: 2006-10-16 Last updated: 2017-12-13
4. Expression of melanocortin-4 receptor by rat parabrachial neurons responsive to immune and aversive stimuli
Open this publication in new window or tab >>Expression of melanocortin-4 receptor by rat parabrachial neurons responsive to immune and aversive stimuli
2006 (English)In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 141, no 1, 287-297 p.Article in journal (Refereed) Published
Abstract [en]

The pontine parabrachial nucleus is a major relay area for visceral and other interoceptive information, and has been implicated in mechanisms underlying anorexia and food aversion during disease. Thus, physiological studies have shown that peripheral immune stimuli, as well as the administration of aversive substances such as lithium chloride, evoke a prominent Fos-expression in the lateral parabrachial nucleus and behavioral experiments have demonstrated that this structure is critical for the acquisition of conditioned taste aversion. The present study examined in rats the relationship between parabrachial neurons activated by systemic administration of bacterial cell-wall lipopolysaccharide or lithium chloride and the melanocortin system, a major regulator of feeding and energy homeostasis that also has been implicated in aversive behavior. Dual-labeling in situ hybridization showed melanocortin-4 receptor expression on neurons in the external lateral parabrachial subnucleus that displayed lipopolysaccharide- or lithium chloride-induced expression of c-fos mRNA. Melanocortin-4 receptor mRNA was also co-expressed with mRNA for calcitonin gene-related peptide in this subnucleus. Taken together with previous observations showing that calcitonin gene-related peptide expressing neurons in the external lateral parabrachial subnucleus are activated by peripheral immune challenge, that lipopolysaccharide-activated external lateral parabrachial subnucleus neurons project to the amygdala, and that the amygdala-projecting neurons in the external lateral parabrachial subnucleus are calcitonin gene-related peptide-positive, the present findings suggest the presence of a melanocortin-regulated calcitonin gene-related peptide-positive pathway from the external lateral parabrachial subnucleus to the amygdala that relays information of importance to forebrain responses to certain aspects of sickness behavior. These observations may thus help explain how melanocortins can reduce feeding and influence conditioned taste aversion during inflammation and other disease conditions.

Keyword
external lateral parabrachial nucleus; lipopolysaccharide; lithium chloride; c-fos; in situ hybridization; calcitonin gene-related peptide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14083 (URN)10.1016/j.neuroscience.2006.03.041 (DOI)
Available from: 2006-10-16 Created: 2006-10-16 Last updated: 2017-12-13

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