393 Lipopolysaccharide has selective actions on sub-populations of catecholaminergic neurons involved in activation of the hypothalamic–pituitary–adrenal axis and inhibition of prolactin secretion

Jacob H Hollis, Stafford L Lightman and Christopher A Lowry Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (HWLINE), University of Bristol, Dorothy Hodgkin Building (DHB), Whitson Street, Bristol BS1 3NY, United Kingdom (Requests for offprints should be addressed to J H Hollis; Email: [email protected])

Abstract Immune activation results in adaptive neuroendocrine concentrations of plasma corticosterone and decreased responses, including activation of the hypothalamic– concentrations of plasma prolactin compared with vehicle- pituitary–adrenal axis, which are dependent on the integ- injected controls. LPS-injected mice had increased num- rity of medullary catecholaminergic (CA) systems. In bers of c-Fos-positive CA neurons within the medullary contrast, although specific roles of pontine, midbrain, and (A1, A2, C1, C2), pontine (A6) and midbrain (A10) cell hypothalamic CA systems in neuroendocrine function groups when compared with vehicle-injected controls. have been described, the functional roles of these CA Among hypothalamic CA cell groups, LPS had differential systems in modulating neuroendocrine function during effects on the numbers of c-Fos-positive CA neurons in immune responses have not been investigated. We have, topographically organised subdivisions of the arcuate nu- therefore, investigated the effects of immune activation on cleus (A12). Changes in plasma prolactin concentrations the various CA systems of the central nervous system correlated with the numbers of c-Fos-positive CA neurons (CNS) and explored this relationship with changes in within the area postrema, the medullary CA cell groups, plasma corticosterone and plasma prolactin. Male BALB/c the medial posterior division of the arcuate, and the zona mice were injected with lipopolysaccharide (LPS, incerta. The present study identifies topographically or- 500 µg/kg i.p.) and 2 h later cardiac blood was taken and ganised, anatomically distinct CA systems that are likely mice were perfused with fixative. Immunostaining proce- to modulate some of the neuroendocrine responses to dures were performed using antibodies raised against c-Fos immune activation, and may provide novel targets for the and tyrosine hydroxylase, a marker of CA neurons, and relief of symptoms associated with illness and disease. detailed topographical analysis of the CA systems within Journal of Endocrinology (2005) 184, 393–406 the CNS was performed. LPS-injected mice had increased

Introduction tality (Curtis & Patrick 2003). A better understanding of the neural systems regulating neuroendocrine responses The changes in neuroendocrine activity in response to to peripheral immune activation may lead to novel thera- peripheral immune system activation are an important peutic strategies for the treatment of chronic illness and adaptive response. Neuroendocrine responses to immune immune-related disease. activation include increases in plasma concentrations of Previous studies have implicated brainstem catecho- corticosterone and either increases or decreases in plasma laminergic (CA) systems in the neuroendocrine responses concentrations of prolactin (Rettori et al. 1994, Rivest to peripheral immune activation. In particular, brainstem 2001, De Laurentiis et al. 2002). These hormones, in turn, CA systems are integral to the increased hypothalamic– influence homeostatic functions and provide feedback to pituitary–adrenal (HPA) activity. Noradrenaline within regulate the immune system (Buckingham et al. 1996, the paraventricular nucleus of the hypothalamus (Pa) Freeman et al. 2000). Although these changes are adaptive stimulates corticotrophin-releasing factor gene expression, during acute states of immune activation, chronic sickness leading to activation of the HPA axis (Itoi et al. 1994), and immune-related disease can lead to decreases in while depletion of noradrenaline and adrenaline can block quality of life and increases in both morbidity and mor- increases in plasma corticosterone concentrations following

Journal of Endocrinology (2005) 184, 393–406 DOI: 10.1677/joe.1.05839 0022–0795/05/0184–393  2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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immune activation (Chuluyan et al. 1992, Swiergiel et al. to seek darkness during the light phase. All animal 1996). The brainstem CA systems within the nucleus of procedures were approved by the University of Bristol the solitary tract (nTS) and ventral lateral medulla (VLM), Ethical Review Group and were conducted in accordance via direct noradrenergic and adrenergic fibre projections to with Home Office guidelines and the UK Animals the Pa, influence the HPA axis response to peripheral (Scientific Procedures) Act 1986. In addition, all studies immune activation (Sawchenko & Swanson 1981, were consistent with the NIH Guide for the Care and Use Cunningham et al. 1990, Ericsson et al. 1994). Chemical of Laboratory Animals (NIH Publication No. 85A23) and or surgical ablation of the medullary CA cell bodies or were covered by Animal Welfare Assurance #A5057-01. disruption of their hypothalamic projection systems attenuates the increased HPA activity associated with immune system activation (Chuluyan et al. 1992, Ericsson LPS administration and tissue processing et al. 1994). At 15-min intervals between 1200 h and 1330 h, time- Other CA systems within the midbrain and hypothala- matched pairs of mice were given i.p. injections (100 µl) of mus may be involved in neuroendocrine responses to either sterile 0·05 M sodium phosphate-buffered saline immune system activation. Immune activation results in (PBS), pH 7·4 (PBS; n=5), or 12·5 µg LPS dissolved in increased extracellular concentrations of dopamine within PBS (n=7; Escherichia coli LPS, 026:B6, Sigma, UK). After various regions of the brain that may be involved in injections, animals were put back into their home cage. neuroendocrine regulation, including the arcuate nucleus Two hours post-injection mice were deeply anaesthetised (Arc), Pa and hippocampus (MohanKumar et al. 1999). with 150 µl Sagatal (sodium pentobarbital, 60 mg/ml i.p.) These regions are known to be innervated by a variety of and thoracic cavities were exposed. Approximately 0·5 ml potential sources of dopamine, including the ventral teg- blood was taken by cardiac puncture, mixed with 50 µl mental area (VTA, A10) and zona incerta (ZI, A13) 0·5 M ethylenediamine tetraacetic acid (EDTA), and kept (Swanson 1982, Wagner et al. 1995). The secretion of on ice until centrifugation at 10 000 r.p.m. for 10 min; some neuroendocrine hormones such as prolactin is regu- plasma was stored at 20 C until used for radioimmuno- lated by dopamine (Reymond & Porter 1985), but the assay. Immediately after cardiac puncture mice were effects of immune activation on prolactin are controversial perfused through the ascending aorta of the heart with (Rettori et al. 1994, De Laurentiis et al. 2002) and deserve 150 ml PBS at 4 C followed by 250 ml 4% paraformal- further attention. dehyde in 0·1 M sodium phosphate buffer, pH 7·4 (PB), at The aim of the present study was to characterise the 4 C. Brains were post-fixed in the same paraformalde- responses of topographically organised subpopulations of hyde solution overnight at 4 C, followed by two 12 h CA neurons within the central nervous system (CNS) to washes in PB at 4 C. Brains were then placed into PB i.p. administration of an acute peripheral immune stimu- containing 30% sucrose at 4 C for 3 days. Brains were lus, lipopolysaccharide (LPS; cell-wall component of blocked using a mouse brain matrix (RBM-2000C, ASI Gram-negative bacteria), by using double immunostaining Instruments, Warren, MI, USA) to ensure a consistent for the protein product of the immediate-early gene c-fos coronal plane in each brain. Brains were then frozen on and tyrosine hydroxylase (TH), a marker of CA neurones. dry ice and stored at 80 C until sectioning. Brains were The second aim of the study was to correlate changes in sectioned in the coronal plane at a thickness of 30 µm and c-Fos expression within medullary, pontine, midbrain and collected in a series of six consecutive wells containing hypothalamic CA cell groups with neuroendocrine re- cryoprotectant (0·05 M phosphate buffer, 30% ethylene sponses as measured by changes in plasma corticosterone glycol w/w, 20% glycerol w/w, pH 7·4) and stored at and plasma prolactin, in order to identify CA cell groups 20 C until immunostaining. that are likely to be involved in neuroendocrine function during states of sickness or disease. Immunohistochemistry Every sixth section of the brain (and to obtain higher Materials and Methods resolution every third section for the hypothalamus) was used for double immunostaining using an antibody di- Animals rected against the protein product of the immediate-early Adult male BALB/c mice (6–8 weeks, 21–23 g) were gene, c-fos (rabbit anti-c-Fos polyclonal antibody, PC-38 housed in a group of 12 in a temperature-controlled room (Ab-5), 1:12 000; Merck Biosciences, Nottingham, Notts, (232 C) on a 14 h:10 h light:darkness cycle (lights on UK), and an antibody directed against TH (rabbit anti-TH at 0500 h), with food and water available ad libitum. Mice polyclonal antibody, AB152, 1:4000; Chemicon, Chan- were allowed to acclimate under these conditions for one dlers Ford, Hants, UK). Free-floating tissue was incubated week before the experiment. Small cardboard ‘huts’ were in 24-well tissue culture plates and washed in plastic tubs kept in the home cage at all times prior to and throughout using mesh wells (Corning Costar, Sunderland, Tyne & the experiment to enrich the environment and allow mice Wear, UK), and gently shaken on an orbital shaker

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Downloaded from Bioscientifica.com at 09/29/2021 05:37:38PM via free access LPS actions on catecholamine systems · J H HOLLIS and others 395 throughout double immunostaining. Tissue was first dilution; kindly supplied by Prof. G Makara, Institute of washed in PBS for 15 min, then incubated in 1% hydrogen Experimental Medicine, Budapest, Hungary) in B buffer peroxide in PBS for 15 min, washed again for 15 min in was added to each tube followed by 50 µl 125I- PBS, pre-incubated in PBS containing 0·3% Triton X-100 corticosterone tracer with a specific activity of 2–3 (PBST) for 15 min, and then incubated for 12–16 h with mCi/µg (MP Biomedicals, Irvine, CA, USA). Tubes were rabbit anti-c-Fos antibody in PBST. Tissue was then mixed well and left to incubate overnight at 4 C. The B washed twice for 15 min in PBST followed by incubation buffer (0·5 ml) containing 0·5% charcoal and 0·05% dex- with a biotinylated swine anti-rabbit IgG polyclonal anti- tran T70 (Sigma) was then added to each tube, mixed body (E0353, 1:200; DAKO, Ely, Cambridgeshire, UK) well, and centrifuged at 4000 r.p.m. at 4 C for 15 min; in PBST for 90 min. Tissue was again washed twice for the supernatant was siphoned off and the total radio- 15 min in PBST followed by incubation with Elite ABC activity in the remaining pellet was determined using a reagent (PK-6100, 1:200; Vector, Orton Southgate, Cobra II gamma counter (Packard Bioscience, UK). Peterborough, UK) in PBST for 90 min. Finally, tissue was Plasma prolactin concentrations were determined by the washed for 15 min in PBST, 15 min in PBS, and then National Hormone and Peptide Program (Dr A F Parlow, incubated in SG substrate (Vector, UK; diluted as recom- Harbor-UCLA, CA, USA) using a radioimmunoassay. mended by Vector) in PBS for 10 min. Tissue was immediately washed in PBS for 15 min, incubated in Imaging 1% hydrogen peroxide in PBS for 15 min, washed in PBS All images were captured using a Leica DMLB microscope for 15 min followed by PBST for 15 min, then incubated fitted with an Insight digital camera (Leica Microsystems, with rabbit anti-TH antibody in PBST for 12–16 h. All Germany) and SPOT image capture software v4.0.2 subsequent steps were identical to those described (Diagnostic Instruments, Sterling Heights, MI, USA), and above for the immunoperoxidase localisation of c-Fos- all figures were designed and assembled in CorelDRAW immunoreactivity, except for the substrate reaction. Sec- 12·0 (Corel Corp., Eden Prairie, MN, USA). tions were incubated in a solution containing 0·01% 3,3-diaminobenzidine tetrahydrochloride (DAB) and Statistics 0·0015% hydrogen peroxide in PBS for 17 min, and then All reported values are mean values and standard error of washed in PBST for 15 min and PBS for 15 min. Brain the mean (S.E.M.). For the cell count data, each of the three slices were then mounted on gelatine-coated glass slides, dependent variables was analysed using a single multifac- dehydrated and cleared with xylene, then coverslipped torial analysis of variance (ANOVA) with repeated using DPX mounting medium (R A Lamb, London, UK). measures, using TREATMENT as the between subject The colour reaction product of the TH immunostaining factor and REGION (e.g. each CA group at a specific was a golden-brown colour and localised to the cytoplasm bregma level) as the within subject factor for repeated while the colour reaction product of the c-Fos immuno- measures analysis. The left and right hemispheres of the staining was a dark blue-black colour and localised to the brain were not distinguished so the cell count data nucleus. Immunohistochemical controls in which the pri- represents the total of the respective left and right sides. mary antibodies were omitted resulted in lack of positive Regions in which the cell counts of the treatment groups staining on a random sample of experimental tissue. had mean values of zero were excluded from the multi- factorial ANOVA with repeated measures. Missing values Cell counting were replaced by the method of Peterson (1985) prior to the multifactorial ANOVA with repeated measures, but Cell counting was performed in a blind manner with the original data were used for post-hoc analysis and for respect to treatment groups after randomisation of slides. representation of the data in the tables and figures. When Cell counts were made to determine (1) the number of TREATMENT effects or TREATMENT*REGION in- c-Fos-positive CA (TH-positive) neurons, (2) the total teractions were observed, multiple pair-wise comparisons number of CA neurons and (3) the number of c-Fos- were made using Fischer’s Protected Least Significant positive non-catecholaminergic (non-CA, TH-negative) Difference tests. Corticosterone and prolactin data were nuclei. The catecholaminergic cell groups, brain regions, analysed using Student’s t-test for independent samples. and bregma levels analysed are listed in Table 1. Correlations between (1) plasma corticosterone or prolac- tin and cell count data and (2) cell count data across Corticosterone and prolactin measurements different regions were determined using Pearson corre- lation. In all cases, significance was accepted at P<0·05. For measurement of corticosterone, plasma was thawed immediately before use and diluted 1:100 in a 0·025 M Results citrate buffer, pH 3·0 (B buffer; to denature the binding globulin) and aliquoted into three 100 µl volumes. Rabbit CA cell groups and regions studied are depicted in Fig. 1, anti-corticosterone polyclonal antiserum (50 µl, 1:124 final and a detailed list of regions that were counted and their www.endocrinology-journals.org Journal of Endocrinology (2005) 184, 393–406

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Table 1 Catecholaminergic groups and regions studied, with abbreviations

Brain region Bregma level (mm) CA group CVO Area postrema (AP) 7·48 A1 Ventrolateral medulla (VLM) 7·48, 7·32 A2 Nucleus of the solitary tract (nTS) 7·48, 7·32 A1/C1, C1 Ventrolateral medulla (VLM) 7·20, 6·96, 6·84 A2/C2, C2 Nucleus of the solitary tract (nTS) 7·20, 6·96, 6·84 C3 Dorsal midline of rostral medulla oblongata (RMO) 6·96, 6·84 A5 Ventrolateral periolivary area 5·40 A6 Locus coeruleus (LC) 5·80, 5·68, 5·52, 5·34 Dorsal subcoeruleus (SubCD) 5·20, 5·02, 4·96 Ventral subcoeruleus (SubCV) 5·20, 5·02, 4·96 Medial parabrachial nucleus (MPB) 5·20 Central part of the lateral parabrachial nucleus (LPBC) 5·20, 5·02 A7 Lateral pons 4·96 A8 Pedunculopontine tegmental nucleus (PPTg; caudal part of A8) 4·24 Retrorubral field (RRF; dorsal part of A8) 4·16, 4·04, 3·88 Pedunculopontine tegmental nucleus (PPTg; ventral part of A8) 4·16, 4·04 A9 Substantia nigra, compact (SNC) 3·80, 3·64, 3·52, 3·16, 3·08 Substantia nigra, lateral (SNL) 3·80, 3·64, 3·52, 3·16 Substantia nigra, reticular (SNR) 3·88, 3·80, 3·64, 3·52 A10 Ventral tegmental area (VTA) 3·88, 3·80, 3·64, 3·52 Caudal linear nucleus (CLi) 4·24, 4·16, 4·04 Rostral linear nucleus (RLi) 3·88, 3·80, 3·64, 3·52 A11 Dorsal raphe nucleus, caudal part (DRC) 4·96 Dorsal raphe nucleus (DR) 4·24, 4·16, 4·04 Periaquaductal gray (PAG) 4·24, 4·16, 4·04, 3·88, 3·80, 3·64, 3·52, 3·16, 3·08, 2·70, 2·54 Periventricular field (pv) 2·46, 2·30, 2·18, 2·06 A12 Medial posterior arcuate nucleus (ArcMP) 2·70, 2·54, 2·46, 2·30, 2·18 Dorsal arcuate nucleus (ArcD) 2·06 Lateral posterior arcuate nucleus (ArcLP) 2·54, 2·46, 2·30, 2·18 Lateral arcuate nucleus (ArcL) 2·06 Arcuate nucleus (Arc) 1·58, 1·46, 1·34, 1·22 A13 Zona incerta (ZI) 1·58, 1·46, 1·34, 1·22 Dorsomedial nucleus of the hypothalamus (DM) 2·18, 2·06, 1·58, 1·46, 1·34 Anterior hypothalamic area (AHP) 1·22 A14 Periventricular hypothalamic nucleus (Pe) 1·58, 1·46, 1·34, 1·22, 1·06, 0·94, 0·46, 0·10, 0, +0·14 Paraventricular hypothalamic nucleus, posterior part (PaPo) 1·06 Paraventricular hypothalamic nucleus, magnocellular part (PaM) 0·94 Paraventricular hypothalamic nucleus, medial parvocellular part (PaMP) 1·06, 0·94 Suprachiasmatic nucleus (SCh) 0·46 Bed nucleus of the stria terminalis, medial division, posteromedial part 0·46 (BSTMPL) Anterodorsal preoptic nucleus (ADP) and bed nucleus of the stria terminalis, 0·10 medial division, ventral part (BSTMV) Medial preoptic nucleus, medial and central part (MPOM, MPOC) 0·00, +0·14

Catecholaminergic cell groups, brain regions and abbreviations of the mouse were defined based on the work in the rat by Everitt et al. (1992), in multiple species by Smeets and Gonzalez (2000) and in the mouse by Paxinos and Franklin (2001). CVO, circumventricular organ.

abbreviations is presented in Table 1. Out of all the ANOVA with repeated measures. Multifactorial different regions studied (Table 1), only the cell count ANOVA with repeated measures revealed a data in which post-hoc analysis revealed a significant TREATMENT*REGION interaction for the number of ff < TREATMENT e ect is presented schematically (Fig. 1) c-Fos-positive CA neurons (F(50,500)=2·815, P 0·001), and in detail (Table 2). Seventy-two different cell and revealed a TREATMENT*REGION inter- counts (from CA cell groups at specific bregma levels) had action for the number of c-Fos-positive non-CA nuclei < no double immunostaining in either treatment group, (F122,1220=6·864, P 0·001). There was neither a and these regions were not used in the multifactorial TREATMENT effect nor a TREATMENT*REGION

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administration of LPS increased the number of c-Fos- positive CA neurons within the AP and several medullary CA groups.

Pontine noradrenergic neurons (A5/A6/A7) Analysis of pontine regions containing noradrenergic neurons revealed an increase in the numbers of c-Fos- positive CA neurons within subdivisions of the A6 cell group (Table 2, Fig. 3). LPS-injected mice had increased numbers of c-Fos-positive CA neurons within the rostral levels of the locus coeruleus (LC), the medial parabrachial nucleus (MPB), central parts of the lateral parabrachial nucleus (LPBC) and ventral subcoeruleus (SubCV) re- gions when compared with vehicle-injected mice. There were no differences in the numbers of c-Fos-positive CA neurons or c-Fos-positive non-CA nuclei within the A5 and A7 regions between treatment groups, and no differ- ences in the numbers of c-Fos-positive non-CA nuclei within the A6 subdivisions between treatment groups. In summary, i.p. administration of LPS increased the number of c-Fos-positive CA neurons within subdivisions of the A6 cell group including the LC, MPB, LPBC and SubCV.

Midbrain catecholaminergic neurons (A8, A9, A10, A11)

Figure 1 Sagittal (A) and coronal (B) diagrams depicting Analysis of midbrain regions containing putative dopamin- catecholaminergic cell groups of mouse brain used in analysis and ergic neurons revealed an increase in the numbers of affected by lipopolysaccharide treatment. The effects of LPS are c-Fos-positive CA neurons solely within the rostral por- depicted by the degree of shading: no shading, no change in tion of the VTA, but not other A10 cell groups (Table 2, c-Fos; light grey, a change in c-Fos-positive non-CA nuclei; dark Fig. 4). The main dopaminergic cell groups of the mid- grey, a change in c-Fos-positive CA neurons; black, a change in both c-Fos-positive CA and non-CA nuclei. The diagram is adapted brain are those within the substantia nigra (SN, A9) and from a mouse brain stereotaxic atlas (Paxinos & Franklin 2001). VTA (A10); additional putative dopaminergic cells are 4 V, fourth ventricle; LV, lateral ventricle. For additional present in the pedunculopontine tegmental nucleus abbreviations see Table 1. (PPTg, A8), retrorubral field (RRF, A8), rostral linear nucleus (RLi, A10) and periaqueductal grey (PAG, A11). There was virtually no double immunostaining in any of interaction in the total numbers of CA neurons between the divisions of A8, A9 or A11. Mice given injections of treatment groups (F1,10=2·925, P=0·118; F122,1220= LPS had decreased numbers of c-Fos-positive non-CA 0·598, P=0·948). nuclei within the regions of the ventral subdivision of A8, the substantia nigra lateral (SNL; A9), the VTA (A10) and the PAG (A11) cell groups compared with vehicle- Medullary noradrenergic (A1/A2) and adrenergic injected mice. In summary, i.p. administration of LPS (C1/C2/C3) neurons increased the number of c-Fos-positive CA neurons LPS-injected mice had increased numbers of c-Fos- within the rostral portion of the VTA and decreased positive CA neurons along the rostral–caudal axis of the the numbers of c-Fos-positive non-CA nuclei within medulla compared with vehicle-injected controls (Table 2, subdivisions of the A8, A9, A10 and A11 regions. Fig. 2). LPS administration resulted in increased numbers of c-Fos-positive CA neurons within the area postrema Hypothalamic catecholaminergic neurons (A11, A12, A13, (AP), A1, A1/C1, A2/C2 and C1 regions compared with A14, A15) vehicle-injected mice. LPS administration also resulted in increased numbers of c-Fos-positive non-CA nuclei Analysis of hypothalamic and thalamic regions containing within the AP, A1/C1, A2 and A2/C2 regions. There putative dopaminergic neurons revealed differential were no differences in the numbers of c-Fos-positive responses in the numbers of c-Fos-positive CA neurons CA neurons or c-Fos-positive non-CA nuclei in the that were restricted to subdivisions of the Arc (Table 2, C3 region between treatment groups. In summary, i.p. Fig. 5). LPS-injected mice had increased numbers of www.endocrinology-journals.org Journal of Endocrinology (2005) 184, 393–406

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Table 2 Cell counts of c-Fos-positive CA (TH-positive) neurons, total CA (TH-positive) neurons, and c-Fos-positive non-CA (TH-negative) nuclei within medullary, pontine, midbrain and hypothalamic catecholaminergic cell groups in which LPS injection resulted in a significant change compared with vehicle injection. Cell counts are meansS.E.M. (2005) c-Fos-positive TH-positive cells (double-immunostained) TH-positive cells c-Fos-positive TH-negative nuclei P cin nctcoaiesystems catecholamine on actions LPS 184, CA group, brain area Bregma level (mm) Vehicle LPS Vehicle LPS Vehicle LPS 393–406

AP 7·48 0 50·8 (P<0·001) 72·66·7 7213·8 16·64·8 282·337·6 (P<0·001) A1 7·32 10·4 3·41 (P=0·05) 5·42·7 9·61·7 235191·9 A2 7·48 0·20·2 0·30·2 41·4 6·21·4 8·43·5 36·28·7 (P<0·05) 7·32 0·60·2 2·10·9 26·81·4 24·12·3 18·83·9 90·69·4 (P<0·001) A1/C1 7·20 0·80·4 3·91·3 (P=0·05) 5·82·5 8·71·8 202·9 39·44·7 (P<0·01) C1 6·84 0·40·4 3·60·8 (P<0·01) 9·22·7 14·43·1 121·8 17·12·5 A2/C2 7·20 10·8 5·41 (P<0·01) 163·2 9·71·6 23·66·1 52·110·1 (P=0·05) 6·96 0·20·2 3·40·6 (P<0·05) 13·22 16·72·6 5·81·3 6·41·5 A6, LC 5·68 1·41 11·44·2 (P<0·05) 57·811 74·114·6 15·44·6 146·1 5·52 13·69 305·7 (P<0·05) 185·234·1 180·821·6 15·44·6 23·37·5 SubCV 5·20 0·20·2 2·20·9 (P=0·05) 3·60·7 4·31·1 43·615·2 32·810·7 MPB 5·20 0·20·2 2·50·4 (P<0·001) 132·8 9·74·1 25·411 27·88·5 LPBC 5·02 0·40·2 2·20·9 (P<0·05) 4·61 5·72 62·833·4 63·77·3 A8, ventral 4·16 0 0 21·65·7 13·32·1 44·62·4 24·93·4 (P<0·01) 4·04 0 0 43·57·1 34·72·2 43·59 18·32·9 (P<0·05) A9, SNL 3·80 0 0 31·87 24·94·7 20·22·4 10·31·4 (P<0·01) A10, VTA 3·64 4·82·2 14·83·6 (P<0·05) 424·224·3 467·716·8 14·82·6 111·8 3·52 72·3 13·92·7 (P<0·05) 44620 45128·9 26·63·5 122·4 (P<0·01) A11, PAG 2·70 0·20·2 0·30·2 4410·1 253·8 354·255·7 236·726·7 (P<0·05) 2·54 0 0·10·1 28·87·7 31·98·1 40348·5 291·437·1 (P<0·05) A12,ArcMP 2·18 0·80·4 4·31·2 (P<0·05) 697·3 70·68 18·68·3 49·910·8       

Downloaded fromBioscientifica.com at09/29/202105:37:38PM ArcD 2·06 1·8 1 4·3 1·2 (P=0·05) 57 5·5 46·5 3·5 11·4 3·5 12·7 2·7 ArcLP 2·18 4·41·7 0·90·5 (P<0·05) 7611·4 60·12·8 31·68·6 35·17·1 Arc 1·58 2·50·3 5·20·9 (P<0·05) 5510·3 56·87·2 30·512·8 33·84·7 1·46 31·2 7·33·3 (P<0·05) 37·25 41·810·6 19·46·2 34·56 A13, ZI 1·46 43·89·3 20·33·6 251·215·9 228·826 51·410·7 26·85·6 (P<0·05) PaPo 1·06 0·60·2 0 5·81·6 3·80·5 39·810·9 84·514·8 (P<0·05)      271·533·8 < www.endocrinology-journals.org PaM 0·94 0·4 0·4 0 5 1·8 3 1 35·8 9·8 (P 0·05) PaMP 1·06 0·20·2 0 153·9 7·72·1 37·28·1 58·84·7 (P<0·05) 0·94 0·40·2 0·30·2 15·23·5 12·23·1 4810·5 108·722 (P<0·05)

Out of 123 regions analysed (Table 1), the regions in which LPS administration resulted in a significant difference in cell counts of either c-Fos-positive CA neurons or c-Fos-positive non-CA nuclei between treatment groups are presented. Bold text represents a significant treatment effect versus vehicle-injected controls, with the P value in parentheses. Abbreviations are listed in Table 1. via freeaccess LPS actions on catecholamine systems · J H HOLLIS and others 399

Figure 2 Medullary CA neurons are responsive to peripheral LPS injection (vehicle injection: A–C, G–I; LPS injection: D–F, J–L). LPS administration resulted in increased numbers of c-Fos-positive CA neurons within the AP (D, E), A1 (D, F), C1 (J, L) and C2 (J, K) cell groups compared with vehicle-injected controls (A–C, G–I). B,C, E,F, H,I and K,L are high magnifications of A, D, G and J respectively. Open arrows represent c-Fos-negative CA neurons while closed arrows indicate c-Fos-positive CA neurons. Arrowheads indicate c-Fos-positive non-CA nuclei. Scale bar=200
m (A, D, G, J) or 50
m (B,C, E,F, H,I, K,L). For abbreviations see Table 1. c-Fos-positive CA neurons in the medial posterior Arc lation analysis revealed correlations between plasma (ArcMP), dorsal Arc (ArcD) and rostral Arc compared with corticosterone concentrations and (1) the number of vehicle-injected mice. In contrast, LPS administration c-Fos-positive CA neurons within the AP, A1/C1, A2/ resulted in decreased numbers of c-Fos-positive CA C2, MPB and VTA, and (2) the number of c-Fos-positive neurons within the lateral posterior Arc (ArcLP). There non-CA nuclei within the Pa and the region of the A2 cell were no differences in the numbers of c-Fos-positive CA group. Correlation analysis also revealed correlations neurons within the A11, A13 or A14 cell groups between between plasma prolactin concentrations and (1) the treatment groups. Due to lack of tissue containing the A15 numbers of c-Fos-positive CA neurons within the AP, cell group, the region was not included in the analysis. A1/C1, A2/C2, ArcMP and ZI, and (2) the number of Mice given injections of LPS had decreased numbers of c-Fos-positive non-CA neurons within the ArcMP. c-Fos-positive non-CA nuclei within the ZI at one bregma level, and increased numbers of c-Fos-positive non-CA nuclei within all subdivisions of the Pa studied Discussion compared with vehicle-injected controls. In summary, i.p. administration of LPS had differential effects on the num- This study provides a detailed analysis of topographically bers of c-Fos-positive CA neurons within subdivisions of organised CA systems in the mouse brain that are candi- the Arc but not other hypothalamic CA cell groups. dates for components of the neural systems mediating neuroendocrine responses to peripheral immune acti- vation. We have been able to provide indirect evidence Corticosterone, prolactin and correlation analysis that CA neurons within the parabrachial and subcoeruleus LPS-injected mice had increased plasma corticosterone regions play a role in relaying signals of peripheral immune and decreased plasma prolactin concentrations measured activation within the mouse CNS. Although previous 2 h after injection compared with vehicle-injected con- studies have described increases in dopaminergic neuro- trols, and these measurements were correlated with cell transmission in the CNS in response to peripheral immune count data within various brain regions (Fig. 6). Corre- activation, this is the first study to illustrate responses to www.endocrinology-journals.org Journal of Endocrinology (2005) 184, 393–406

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Figure 3 Multiple subpopulations of the A6 cell group in addition to the LC are responsive to peripheral LPS injection (vehicle injection: A,B, E,F; LPS injection: C,D, G,H). LPS administration resulted in increased numbers of c-Fos-positive CA neurons within the LC (C, D) and MPB (G, H) but not the LPB (G, H) or SubCD (G) at 5·20 mm bregma compared with vehicle-injected controls (A, B, E, F). B, D, F and H are higher magnifications of A, C, E and G respectively. Open arrows indicate c-Fos-negative CA neurons while closed arrows indicate c-Fos-positive CA neurons. Scale bar=200
m (A, C, E, G), 50
m (B, D) or 25
m (F, H). PB, parabrachial nucleus; scp, superior cerebellar peduncle. For additional abbreviations see Table 1.

peripheral immune activation within midbrain dopamin- responses at single cell resolution among large populations ergic cell groups. We have also investigated the functional of cells. It is also important to be aware of temporal factors properties of dopaminergic neurons within the Arc, and when comparing the present data with previous physio- for the first time have shown differential c-Fos responses to logical and behavioural studies because the expression of peripheral immune activation in subdivisions of the Arc c-Fos is transient and varies across the circadian cycle which also correlate with plasma prolactin concentrations. (Grassi et al. 1994, Kovacs 1998). Interestingly, we have identified multiple regions within Although omission of the primary antibodies from the the brain where peripheral immune activation results in immunostaining protocol resulted in a lack of positive decreased c-Fos expression (ventral A8, substantia nigra staining, it does not confirm that the antibodies are in fact lateral, VTA, PAG) compared with controls. Thus, the specific for the protein markers of interest. Control pep- present data suggest that there are distinct, topographically tides were not used but the antibodies used in the present organised subsets of medullary, pontine, midbrain and study have been used previously for the identification of hypothalamic CA systems that modulate the neuro- changes in c-Fos-immunoreactivity (Zhang et al. 2003) endocrine responses to peripheral immune system and the identification of CA neurons in mouse tissue activation. (Horger et al. 1998). In this study, we describe TH In order to identify c-Fos-immunoreactivity within immunostaining in several regions not previously de- topographically organised subpopulations of CA neurons scribed in mouse brain (MPB, lateral parabrachial nucleus with a high level of neuroanatomical detail, double (LPB), A8, RLi, PAG); however all of these regions have immunostaining of the protein product of the immediate- been found to contain CA neurons in at least one of the early gene c-fos was used in conjunction with TH immuno- multiple vertebrate species studied to date (Smeets & staining of CA neurons. While a change in the c-Fos- Gonzalez 2000). immmunoreactivity does not necessarily indicate a change in electrophysiological or electrochemical properties of a cell (e.g. firing rate, depolarization, calcium influx) Medullary and pontine CA systems (Morgan & Curran 1986, Luckman et al. 1994), it does The LPS-induced effects we observed in the medulla are provide a useful method to identify functional cellular consistent with previous findings in rats where LPS

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the AP. The AP has long been implicated in rapid homeostatic responses such as those associated with osmo- regulatory and nutrient challenges, and is an important centre for the integration of systemic and visceral sensory information as it is a circumventricular organ and also receives sensory information from peripheral vagal ganglia (Oldfield & McKinley 2004). The projections of the AP have been shown to be primarily restricted to the brain- stem, with dense projections to CA neurons of the nTS (Cunningham et al. 1994) and moderate projections to the external lateral parabrachial nucleus, the dorsal motor nucleus of the vagus (DMN), nucleus ambiguus and VLM (Shapiro & Miselis 1985). The AP has been shown to be critical for brainstem c-Fos responses, hypothalamic para- ventricular c-Fos responses, and HPA axis responses to immune stimuli (Lee et al. 1998). The projections and Figure 4 Dopaminergic midbrain neurons within the ventral functions of the CA neurons within the mouse AP are not tegmental area (VTA) are responsive to peripheral LPS injection. known, although CA projections from the AP to the LPS administration resulted in increased numbers of c-Fos-positive rostral VLM have been found in the rabbit (Blessing et al. CA neurons within the VTA (C, D) but not the SN, RLi or PAG when compared with vehicle-injected controls (A, B). B and D are 1987) so it is feasible that the mouse brain contains similar higher magnifications of A and C respectively. Open arrows connections. indicate c-Fos-negative CA neurons while closed arrows indicate The present study is the first to identify CA neurons of c-Fos-positive CA neurons. Scale bar=200
m (A, C) or 25
m(B, the A6 cell group (MPB, LPBC, SubCV) other than the D). For abbreviations see Table 1. LC that are responsive to peripheral LPS injection. The CA neurons within the LC have repeatedly been shown to be responsive to immune stimuli (Zalcman et al. 1991, administration resulted in increased numbers of c-Fos- Hare et al. 1995, Kaneko et al. 2001), and the present positive CA neurons within specific CA groups (A1, results confirm these previous studies. The external por- A1/C1, A2/C2, C2) along the rostral–caudal extent of the tion of the LPB (LPBE) is known to be especially medulla (Ericsson et al. 1994, Dayas et al. 2001). Our responsive to immune activation (Elmquist et al. 1996), observation that LPS failed to alter the numbers of although no CA cells are located within this region. c-Fos-positive CA neurons within the mouse A2 region Because only regions containing CA cells were focused on (7·48 to 7·32 mm bregma) appears to conflict with a in this study, single c-Fos counts were not performed recent study in rats demonstrating that interleukin-1 within all subdivisions of the parabrachial nucleus. The (IL-1) treatment results in increased numbers of c-Fos- LPBC projects to the Pa in the rat (Bester et al. 1997), positive noradrenergic neurons that are more rostral than although the neuroanatomical projections of PB subdivi- in rats exposed to other stressors such as restraint or sions in the mouse have not been studied and it is not footshock (Dayas et al. 2001). However, the bregma levels known if the neurochemical phenotypes of this presumed in mouse brain containing noradrenergic neurons (7·20 connection includes CA neurons. Reciprocal connections to 6·96 mm bregma) equivalent to the IL-1-responsive between the MPB and subdivisions of the nTS (caudal regions in rat brain described by Dayas and colleagues are region of commissural subdivision, rostral portions of nTS) more rostral than the bregma levels we defined as the A2 have been shown in the rat (Herbert et al. 1990, Krukoff cell group. We defined this region of the mouse nTS et al. 1993), and this may be an important circuit for (7·20 to 6·96 mm bregma) as part of the A2/C2 relaying visceral signals related to immune stimuli within because it contains a mixture of noradrenergic and adren- the mouse CNS as well. Cells of the SubCV are known to ergic neurons that we could not distinguish using an project to the spinal cord in the rat (Clark & Proudfit antibody raised against TH. Taking into account these 1991), although the functional significance of this connec- differences in nomenclature used to describe mouse and tion is not known. This study provides evidence that there rat brain, the present results demonstrating that medullary are further topographical subdivisions of the mouse A6 cell noradrenergic and/or adrenergic CA neurons are respon- group (MPB, LPB, SubCV) that may relay signals of sive to peripheral immune activation are consistent with peripheral immune activation within the CNS. previous reports (Ericsson et al. 1994, Dayas et al. 2001). The existence of CA neurons within the AP has Midbrain CA systems previously been described (Dahlstrom & Fuxe 1964), and to our knowledge this is the first study describing LPS- The A10 dopaminergic neurons within the VTA are induced increases in c-Fos-positive CA neurons within often associated with motivation, incentive, and reward www.endocrinology-journals.org Journal of Endocrinology (2005) 184, 393–406

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Figure 5 Dopaminergic neurons within subdivisions of the arcuate nucleus are differentially responsive to peripheral LPS injection (saline injection: A-F, M-R; LPS injection: G-L, S-X). LPS administration resulted in increased numbers of c-Fos-positive CA neurons in the ArcMP at –2·18 mm bregma (K, W), in the ArcD at –2·06 mm bregma (L), and in the Arc at –1·58 and –1·46 mm bregma (S, T), but decreased numbers of c-Fos-positive CA neurons in the ArcLP at –2·18 mm bregma (K, X) compared with vehicle-injected controls (A-F, M-R). Q and R are higher magnifications of E while W and X are higher magnifications of K. Open arrows represent c-Fos-negative CA neurons and closed arrows represent c-Fos-positive CA neurons. Scale bar=100
m(A-P,S-V)or25
m (Q, R, W, X). For abbreviations see Table 1.

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and prefrontal cortex (Lacosta et al. 1994, Hayley et al. 2001), and our results support the hypothesis that the VTA may be the source of limbic dopamine changes in response to peripheral immune activation. Although the role of the VTA during immune responses is not known, it is feasible that the VTA is involved in behavioural reinforcement during states of sickness or disease.

Hypothalamic CA systems To our knowledge, this is the first study reporting differ- ential c-Fos responses within topographically organised subsets of Arc dopaminergic (DA) neurons of the mouse in response to immune activation. The Arc is known to express IL-1 receptors, and peripheral immune activation results in c-fos mRNA induction within the Arc (Brady et al. 1994, Ericsson et al. 1995). The majority of IL-1 receptors within the Arc are within the medial division that contains the majority of the median eminence (ME)- projecting tuberoinfundibular dopaminergic (TIDA) neurons thought to be the primary source of dopamine acting to regulate prolactin secretion (Rethelyi 1985, Reymond & Porter 1985). The lateral divisions of the Arc are not thought to be as important in prolactin regulation, as they are thought to project primarily to the lateral divisions of the ME (Lofstrom et al. 1976). In the rat, these cells within the lateral divisions of the Arc contain putative CA neurons that do not express the enzyme aromatic amino acid decarboxylase that is necessary for dopamine synthesis (Meister et al. 1988), and the functions of these cells have yet to be elucidated. One study reported similar results in the Arc as in the present study after administering a dopamine receptor antagonist instead of LPS; however Figure 6 Peripheral LPS injection results in neuroendocrine this also resulted in increased plasma prolactin concen- hormone changes that are correlated with cell count data from trations (Hentschel et al. 2000). Prolactin is thought various brain regions. LPS administration resulted in increased plasma corticosterone concentrations compared with to be the main negative-feedback regulator of TIDA vehicle-injected controls (A), and corticosterone concentrations neurons because TIDA neurons lack autoreceptor feed- correlated with the numbers of c-Fos-positive CA neurons within back (Demarest & Moore 1979). However, treatment the MPB (B) and VTA (C). LPS administration resulted in with prolactin alone has provided controversial results, decreased plasma prolactin concentrations compared with vehicle-injected controls (D), and prolactin concentrations resulting in increased numbers of c-Fos-positive CA correlated with the numbers of c-Fos-positive CA neurons within neurons within either both subdivisions of the Arc the A1/C1 (E), ArcMP (F) and ZI (G) and with the numbers of (Hentschel et al. 2000) or only the medial subdivision c-Fos-positive non-CA neurons within the ArcMP (H). The slope of (Cave et al. 2001), suggesting prolactin is not the sole each line (correlation coefficient, r) and P value of the correlation regulator of TIDA neurons. The present results add further analysis are shown for each scatter plot. *P<0·05, **P<0·001 versus vehicle-injected controls. For abbreviations see Table 1. evidence that the medial and lateral subdivisions of the Arc are indeed functionally distinct, and are differentially responsive to peripheral immune activation. (Le Moal & Simon 1991), and it is both interesting and The present results are in accordance with a recent novel that LPS administration resulted in increased c-Fos- study showing decreased plasma prolactin concentrations positive CA neurons at the level of the A10 dopaminergic following immune activation (De Laurentiis et al. 2002); cell body. The VTA is the major source of the mesocortico- however there are multiple other studies reporting either limbic dopaminergic system that projects to forebrain increased plasma prolactin concentrations (Rettori et al. structures including the nucleus accumbens, prefrontal 1994, Phelps et al. 2001, Gonzalez et al. 2004) or no cortex and the amygdala (Swanson 1982). Peripheral change in prolactin (Abreu et al. 1994) following immune immune activation results in increased extracellular con- activation. The major difference between these conflicting centrations of dopamine within the nucleus accumbens results is the route of LPS administration or, in the case of www.endocrinology-journals.org Journal of Endocrinology (2005) 184, 393–406

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studies which found no changes in plasma prolactin i.c.v., when given intraperitoneally, may relay signals to the Arc IL-1 was administered instead of LPS. In studies using through medullary CA cell groups. LPS given intraperitoneally (De Laurentiis et al. 2002), including the present study, LPS injection resulted in decreased serum prolactin concentrations. In contrast, in Conclusions studies using high doses of LPS given i.v. (Rettori et al. 1994, Phelps et al. 2001) or LPS given i.c.v. (Gonzalez The present study provides evidence that the CA systems et al. 2004), LPS injection resulted in increased serum of the CNS are composed of functionally distinct, topo- prolactin concentrations. One explanation is that when graphically organised subgroups that are likely to be LPS is given intravenously or intracerebroventricularly, involved in a variety of neuroendocrine, autonomic and LPS actions at the level of the Arc result in increased behavioural responses to immune activation. Our results release of prolactin. The Arc is highly vascularised provide further evidence for the involvement of medullary and capable of directly sensing circulating signals of (A1, A2, C1, C2) and pontine (LC) CA systems, and immune activation (Brady et al. 1994, Ericsson et al. 1995, identify new CA subpopulations within the medulla (AP), Herkenham et al. 1998). When LPS is given in compart- pons (MPB, LPB, SubCV) and midbrain (VTA) that may ments such as the peritoneal cavity, activation of the Arc be involved in neuroendocrine responses to peripheral may be primarily due to ascending signals arising from immune activation. This study is the first to demonstrate brainstem nuclei such as medullary CA systems. The that peripheral immune activation results in differential medullary neuronal systems innervate the Arc (Ricardo & c-Fos responses within topographically distinct subpopu- Koh 1978) and adrenergic fibres actually terminate on lations of TIDA neurons which are known to have TIDA neurons (Hrabovszky & Liposits 1994). Because important roles in neuroendocrine hormone regulation, LPS administration results in c-Fos responses within including the LPS-induced decrease in the concentration medullary CA systems, this could be a possible mechanism of plasma prolactin. This study has also identified various of relaying signals of peripheral immune activation from brainstem (A1/C1, A2/C2) and hypothalamic (ArcMP, the periphery to the Arc. ZI) CA cell groups where cell counts were correlated with plasma prolactin concentrations. These subpopulations of CA neurons deserve further attention in efforts to define Correlation analyses neural systems contributing to dysregulation of CNS and endocrine systems in acute and chronic illness and Correlation analyses support the involvement of particular immune-related disease. CA systems in both the LPS-induced increases in plasma corticosterone concentrations and the LPS-induced de- creases in plasma prolactin concentrations. The correlation Acknowledgements between the numbers of c-Fos-positive CA neurons within medullary CA systems and plasma corticosterone This work was supported by the Neuroendocrinology concentrations largely confirms previous studies, whereas Charitable Trust, UK (grant no. PMS/VW-00/01-606 to the correlation between the numbers of c-Fos-positive CA S L L and C A L). Dr Christopher A Lowry is a Wellcome neurons in the MPB and VTA are unreported as far as the Trust Research Fellow (RCDF 068558/Z/02/Z). The authors are aware. However, the MPB and VTA have not authors declare that there is no conflict of interest that been shown to project to the Pa or to be directly involved would prejudice the impartiality of this scientific work. in the increased HPA axis activity in response to immune activation (Elmquist & Saper 1996), so the correlations should not be over interpreted. The negative correlation References between the numbers of c-Fos-positive CA neurons in the ArcMP and plasma prolactin concentrations, although not Abreu P, Llorente E, Hernandez MM & Gonzalez MC 1994 strongly correlated, further supports the involvement of Interleukin-1 beta stimulates tyrosine hydroxylase activity in the TIDA neurons in prolactin release in response to immune median eminence. Neuroreport 5 1356–1358. activation. The strong correlation between the numbers of Bester H, Besson JM & Bernard JF 1997 Organization of efferent c-Fos-positive CA neurons within the ZI and plasma projections from the parabrachial area to the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. Journal of prolactin concentrations was unexpected, although the ZI Comparative Neurology 383 245–281. is largely associated with the reproductive axis and is Blessing WW, Hedger SC, Joh TH & Willoughby JO 1987 Neurons known to be involved in the regulation of other neuro- in the area postrema are the only catecholamine-synthesizing cells endocrine hormones (MacKenzie et al. 1988). The corre- in the medulla or pons with projections to the rostral ventrolateral medulla (C1-area) in the rabbit. Brain Research 419 336–340. lation between the numbers of c-Fos-positive CA neurons Brady LS, Lynn AB, Herkenham M & Gottesfeld Z 1994 Systemic within the medullary CA cell groups and plasma prolactin interleukin-1 induces early and late patterns of c-fos mRNA concentrations further supports the idea that LPS injection, expression in brain. Journal of Neuroscience 14 4951–4964.

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Buckingham JC, Loxley HD, Christian HC & Philip JG 1996 Gonzalez MC, Abreu P, Barroso-Chinea P, Cruz-Muros I & Activation of the HPA axis by immune insults: roles and Gonzalez-Hernandez T 2004 Effect of intracerebroventricular interactions of cytokines, eicosanoids, glucocorticoids. Pharmacology, injection of lipopolysaccharide on the tuberoinfundibular Biochemistry and Behavior 54 285–298. dopaminergic system of the rat. Neuroscience 127 251–259. Cave BJ, Wakerley JB, Luckman SM & Tortonese DJ 2001 Grassi ZG, Menegazzi M, Carcereri DP, Vescia S, Ranucci G & Hypothalamic targets for prolactin: assessment of c-Fos induction in Bentivoglio M 1994 Different programs of gene expression are tyrosine hydroxylase- and proopiomelanocortin-containing neurones associated with different phases of the 24 h and sleep-wake cycles. in the rat arcuate nucleus following acute central prolactin Chronobiologia 21 93–97. administration. Neuroendocrinology 74 386–395. Hare AS, ClarkeG&TolchardS1995Bacterial lipopolysaccharide- Chuluyan HE, Saphier D, Rohn WM & Dunn AJ 1992 induced changes in FOS protein expression in the rat brain: Noradrenergic innervation of the hypothalamus participates in correlation with thermoregulatory changes and plasma adrenocortical responses to interleukin-1. Neuroendocrinology 56 corticosterone. Journal of Neuroendocrinology 7 791–799. 106–111. Hayley S, Lacosta S, Merali Z, van RooijenN&Anisman H 2001 Clark FM & Proudfit HK 1991 The projection of locus coeruleus Central monoamine and plasma corticosterone changes induced by neurons to the spinal cord in the rat determined by anterograde a bacterial endotoxin: sensitization and cross-sensitization effects. tracing combined with immunocytochemistry. Brain Research 538 European Journal of Neuroscience 13 1155–1165. 231–245. Hentschel K, Moore KE & Lookingland KJ 2000 Effects of prolactin Cunningham ET Jr, Bohn MC & Sawchenko PE 1990 Organization on expression of Fos-related antigens in tyrosine hydroxylase- of adrenergic inputs to the paraventricular and supraoptic nuclei of immunoreactive neurons in subdivisions of the arcuate nucleus. the hypothalamus in the rat. Journal of Comparative Neurology 292 Brain Research 857 110–118. 651–667. Herbert H, Moga MM & Saper CB 1990 Connections of the Cunningham ET Jr, Miselis RR & Sawchenko PE 1994 The parabrachial nucleus with the nucleus of the solitary tract and the relationship of efferent projections from the area postrema to vagal medullary reticular formation in the rat. Journal of Comparative motor and brain stem catecholamine-containing cell groups: an Neurology 293 540–580. axonal transport and immunohistochemical study in the rat. Herkenham M, Lee HY & Baker RA 1998 Temporal and spatial Neuroscience 58 635–648. patterns of c-fos mRNA induced by intravenous interleukin-1: a Curtis JR & Patrick DL 2003 The assessment of health status among cascade of non-neuronal cellular activation at the blood–brain patients with COPD. European Respiratory Journal Supplement 41 barrier. Journal of Comparative Neurology 400 175–196. 36s–45s. Horger BA, Nishimura MC, Armanini MP, Wang LC, Poulsen KT, Dahlstrom A & Fuxe K 1964 Localization of monoamines in the lower Rosenblad C, Kirik D, Moffat B, Simmons L, Johnson E Jr, brain stem. Experientia 20 398–399. Milbrandt J, Rosenthal A, Bjorklund A, Vandlen RA, Hynes MA Dayas CV, Buller KM, Crane JW, XuY&DayTA2001 Stressor & Phillips HS 1998 Neurturin exerts potent actions on survival and categorization: acute physical and psychological stressors elicit function of midbrain dopaminergic neurons. Journal of Neuroscience distinctive recruitment patterns in the amygdala and in medullary 18 4929–4937. noradrenergic cell groups. European Journal of Neuroscience 14 HrabovszkyE&Liposits Z 1994 Adrenergic innervation of dopamine 1143–1152. neurons in the hypothalamic arcuate nucleus of the rat. Neuroscience De Laurentiis A, Pisera D, Caruso C, Candolfi M, Mohn C, Rettori Letters 182 143–146. V & Seilicovich A 2002 Lipopolysaccharide- and tumor necrosis Itoi K, Suda T, Tozawa F, Dobashi I, Ohmori N, Sakai Y, Abe K & factor-alpha-induced changes in prolactin secretion and Demura H 1994 Microinjection of norepinephrine into the dopaminergic activity in the hypothalamic–pituitary axis. paraventricular nucleus of the hypothalamus stimulates Neuroimmunomodulation 10 30–39. corticotropin-releasing factor gene expression in conscious rats. Demarest KT & Moore KE 1979 Comparison of dopamine synthesis Endocrinology 135 2177–2182. regulation in the terminals of nigrostriatal, mesolimbic, Kaneko YS, Ikemoto K, Mori K, Nakashima A, NagatsuI&OtaA tuberoinfundibular and tuberohypophyseal neurons. Journal of Neural 2001 Expression of GTP cyclohydrolase I in murine locus coeruleus Transmission 46 263–277. is enhanced by peripheral administration of lipopolysaccharide. Brain Elmquist JK & Saper CB 1996 Activation of neurons projecting to the Research 890 203–210. paraventricular hypothalamic nucleus by intravenous Kovacs KJ 1998 c-Fos as a transcription factor: a stressful (re)view lipopolysaccharide. Journal of Comparative Neurology 374 315–331. from a functional map. Neurochemistry International 33 287–297. Elmquist JK, Scammell TE, Jacobson CD & Saper CB 1996 Krukoff TL, Harris KH & Jhamandas JH 1993 Efferent projections Distribution of Fos-like immunoreactivity in the rat brain following from the parabrachial nucleus demonstrated with the anterograde intravenous lipopolysaccharide administration. Journal of Comparative tracer Phaseolus vulgaris leucoagglutinin. Brain Research Bulletin 30 Neurology 371 85–103. 163–172. Ericsson A, Kovacs KJ & Sawchenko PE 1994 A functional anatomical Lacosta S, Merali Z, Zalcman S & Anisman H 1994 Time-dependent analysis of central pathways subserving the effects of interleukin-1 in vivo mesolimbic dopamine variations following antigenic on stress-related neuroendocrine neurons. Journal of Neuroscience 14 challenge. Brain Research 664 225–230. 897–913. Le Moal M & Simon H 1991 Mesocorticolimbic dopaminergic Ericsson A, Liu C, Hart RP & Sawchenko PE 1995 Type 1 network: functional and regulatory roles. Physiological Reviews 71 interleukin-1 receptor in the rat brain: distribution, regulation, and 155–234. relationship to sites of IL-1-induced cellular activation. Journal of Lee HY, Whiteside MB & Herkenham M 1998 Area postrema Comparative Neurology 361 681–698. removal abolishes stimulatory effects of intravenous interleukin-1 Everitt BJ, MeisterB&Hokfelt T 1992 The organization of beta on hypothalamic–pituitary–adrenal axis activity and c-fos monoaminergic neurons in the hypothalamus in relation to mRNA in the hypothalamic paraventricular nucleus. Brain Research neuroendocrine integration. In Neuroendocrinology, 1st edn, pp Bulletin 46 495–503. 87–128. Ed. CB Nemeroff. Boca Raton: CRC Press. Lofstrom A, Jonsson G & Fuxe K 1976 Microfluorimetric quantitation Freeman ME, Kanyicska B, LerantA&NagyG2000 Prolactin: of catecholamine fluorescence in rat median eminence. I. Aspects structure, function, and regulation of secretion. Physiological Reviews on the distribution of dopamine and noradrenaline nerve terminals. 80 1523–1631. Journal of Histochemistry and Cytochemistry 24 415–429. www.endocrinology-journals.org Journal of Endocrinology (2005) 184, 393–406

Downloaded from Bioscientifica.com at 09/29/2021 05:37:38PM via free access 406 J H HOLLIS and others · LPS actions on catecholamine systems

Luckman SM, Dyball RE & Leng G 1994 Induction of c-fos Ricardo JA & Koh ET 1978 Anatomical evidence of direct projections expression in hypothalamic magnocellular neurons requires synaptic from the nucleus of the solitary tract to the hypothalamus, activation and not simply increased spike activity. Journal of amygdala, and other forebrain structures in the rat. Brain Research Neuroscience 14 4825–4830. 153 1–26. MacKenzie FJ, James MD & Wilson CA 1988 Changes in dopamine Rivest S 2001 How circulating cytokines trigger the neural circuits activity in the zona incerta (ZI) over the rat oestrous cycle and the that control the hypothalamic–pituitary–adrenal axis. effect of lesions of the ZI on cyclicity: further evidence that the Psychoneuroendocrinology 26 761–788. incerto–hypothalamic tract has a stimulatory role in the control of Sawchenko PE & Swanson LW 1981 Central noradrenergic pathways LH release. Brain Research 444 75–83. for the integration of hypothalamic neuroendocrine and autonomic Meister B, Hokfelt T, Steinbusch HW, Skagerberg G, Lindvall O, responses. Science 214 685–687. ff Ge ard M, Joh TH, Cuello AC & Goldstein M 1988 Do tyrosine Shapiro RE & Miselis RR 1985 The central neural connections of the hydroxylase-immunoreactive neurons in the ventrolateral arcuate area postrema of the rat. Journal of Comparative Neurology 234 nucleus produce dopamine or only L-dopa? Journal of Chemical 344–364. Neuroanatomy 1 59–64. Smeets WJ & Gonzalez A 2000 Catecholamine systems in the brain of MohanKumar SM, MohanKumar PS & Quadri SK 1999 vertebrates: new perspectives through a comparative approach. Brain Lipopolysaccharide-induced changes in monoamines in specific areas Research. Brain Research Reviews 33 308–379. of the brain: blockade by interleukin-1 receptor antagonist. Brain Research 824 232–237. Swanson LW 1982 The projections of the ventral tegmental area and Morgan JI & Curran T 1986 Role of ion flux in the control of c-fos adjacent regions: a combined fluorescent retrograde tracer and expression. Nature 322 552–555. immunofluorescence study in the rat. Brain Research Bulletin 9 Oldfield BJ & McKinley MJ 2004 Circumventricular organs. In The 321–353. Rat Nervous System, 3rd edn, pp 389–406. Ed G Paxinos. London: Swiergiel AH, Dunn AJ & Stone EA 1996 The role of cerebral Elsevier Academic Press. noradrenergic systems in the Fos response to interleukin-1. Brain PaxinosG&Franklin KBJ 2001 The Mouse Brain in Stereotaxic Research Bulletin 41 61–64. Coordinates, 2nd edn. London: Academic Press. Wagner CK, Eaton MJ, Moore KE & Lookingland KJ 1995 Efferent Peterson RG 1985 Design and Analysis of Experiments. New York: projections from the region of the medial zona incerta containing Marcel Dekker, Inc. A13 dopaminergic neurons: a PHA-L anterograde tract-tracing Phelps CP, Dong JM, Chen LT & Menzies RA 2001 Plasma study in the rat. Brain Research 677 229–237. interleukin-1 beta, prolactin, ACTH and corticosterone responses to Zalcman S, Shanks N & Anisman H 1991 Time-dependent variations endotoxin after damage of the anterior hypothalamic area. of central norepinephrine and dopamine following antigen Neuroimmunomodulation 9 340–351. administration. Brain Research 557 69–76. Rethelyi M 1985 Dendritic arborization and axon trajectory of Zhang YH, Lu J, Elmquist JK & Saper CB 2003 Specific roles of neurons in the hypothalamic arcuate nucleus of the rat–updated. cyclooxygenase-1 and cyclooxygenase-2 in lipopolysaccharide- Neuroscience 16 323–331. induced fever and Fos expression in rat brain. Journal of Comparative Rettori V, Dees WL, Hiney JK, LysonK&McCannSM1994 An Neurology 463 3–12. interleukin-1-alpha-like neuronal system in the preoptic– hypothalamic region and its induction by bacterial lipopolysaccharide in concentrations which alter pituitary hormone Received in final form 29 October 2004 release. Neuroimmunomodulation 1 251–258. Reymond MJ & Porter JC 1985 Involvement of hypothalamic Accepted 5 November 2004 dopamine in the regulation of prolactin secretion. Hormone Research Made available online as an 22 142–152. Accepted Preprint 17 November 2004

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