interleukin-6 and nitric oxide synthase expression in the vasopressin and corticotrophin-releasing...

ARTICLE

Interleukin-6 and Nitric Oxide Synthase Expression in theVasopressin and Corticotrophin-releasing Factor Systemsof the Rat Hypothalamus

Tomas Gonzalez-Hernandez, Domingo Afonso-Oramas, Ignacio Cruz-Muros,Pedro Barroso-Chinea, Pedro Abreu, Marıa del Mar Perez-Delgado, Nelida Rancel-Torres,and Marıa del Carmen Gonzalez

Department of Anatomy, Pathology, and Histology (TG-H,DA-O,IC-M,PB-C,MMP-D,NR-T) and Department of Physiology(PA,MCG), Faculty of Medicine, University of La Laguna, La Laguna, Tenerife, Spain

SUMMARY Nitric oxide synthase (NOS) and interleukin-6 (IL-6) are constitutively expressedin hypothalamic cells. However, phenotypic and functional aspects of these cells remainunknown. We have studied the expression pattern of these two molecules in hypothalamiccells expressing corticotropin-releasing factor (CRF) and arginin-vasopressin (AVP), two majorregulatory peptides in the hypothalamus-pituitary system, using immunofluorescence,intracerebroventricular injection of colchicine, and the study in parallel of the labelingpattern of axons in the median eminence. Within AVP cells, we distinguished two differentpopulations: large, intensely stained AVP cells coexpressing IL-6; and large, intensely stainedAVP cells coexpressing IL-6 and NOS. Within the CRF cells, we distinguished three differentpopulations: large, intensely stained CRF cells immunonegative for AVP, NOS, and IL-6; largecells weakly stained for CRF and AVP, immunopositive for NOS and immunonegative for IL-6;and small cells intensely stained for CRF and AVP and immunonegative for IL-6 and NOS. Inaddition, we also found AVP cells containing IL-6 in the suprachiasmatic nucleus. These resultssuggest that neuronal NOS and IL-6 may be involved in different modulatory processes inhypophysiotropic and non-hypophysiotropic cells. (J Histochem Cytochem 54:427–441, 2006)

KEY WORDS

hypophysiotropic

suprachiasmatic nucleus

median eminence

pituitary gland

CORTICOTROPIN-RELEASING FACTOR (CRF) and arginin-vasopressin (AVP) are two major regulatory peptides inthe hypothalamus-pituitary-adrenal (HPA) axis. CRF issynthesized in neurons preferentially localized in theparvocellular region of the paraventricular nucleus(PVN) whose axons reach the median eminence (ME)constituting the main source of CRF in the hypophysialportal vascular system (Rho and Swanson 1987,1989;Sawchenko and Swanson 1990; Lennard et al. 1993).AVP is synthesized in both magno- and parvocellularregions of the PVN, and in the supraoptic nucleus(SON). Large AVPergic neurons in the PVN and SONproject to the neural pituitary lobe, whereas AVPergicneurons lying in the parvocellular region of PVN

project to the external zone of the ME (Vandersandeet al. 1977; Swanson and Sawchenko 1983; Antoniet al. 1990). Furthermore, AVP colocalizes with CRF inparvocellular PVN neurons (Mouri et al. 1993; Grinoand Zamora 1998) and in secretory granules at thenerve endings in the ME, exerting synergistic effects onadrenocorticotropic hormone (ACTH) secretion fromthe anterior pituitary lobe (Gillies et al. 1982). AVP hasalso been detected in the suprachiasmatic nucleus(SCN) (Hornby and Piekut 1989; Hofman et al.1996; Herzog et al. 2000; Abrahamson and Moore2001; Moore et al. 2002), a hypothalamic nucleus thatacts as a pacemaker, controlling different brain andendocrine functions (Saper et al. 2005).

The proinflammatory cytokine interleukin-6 (IL-6)and the short-lived, unstable free radical nitric oxide(NO) have been involved in neuroendocrine functionsplaying regulatory roles on the HPA axis. IL-6 inducesthe synthesis and secretion of CRF (Lyson andMcCann1991,1992; Navarra et al. 1991) and AVP (Mastorakos

Correspondence to: Tomas Gonzalez-Hernandez, Department ofAnatomy, Faculty of Medicine, University of La Laguna, 38207 LaLaguna, Tenerife, Spain. E-mail: [email protected]

Received for publication September 27, 2005; accepted Novem-ber 10, 2005 [DOI: 10.1369/jhc.5A6845.2005].

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Volume 54(4): 427–441, 2006

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et al. 1994; Raber et al. 1997) at the hypothalamic leveland ACTH and glucocorticoid secretions directly at thepituitary (Naitoh et al. 1988; Lyson and McCann1992) and adrenal levels, respectively (Salas et al.1990). In addition, a recent study shows that IL-6 iscoexpressed with AVP in SON and PVN cells (Ghorbelet al. 2003). On the other hand, nitric oxide synthase(NOS), the enzyme responsible for the synthesis of NO,has been detected at different levels of the HPA axis,including oxytocin, AVP, and CRF cells in the PVN andSON (Bredt et al. 1990; Arevalo et al. 1992; Calka andBlock 1993; Miyagawa et al. 1994; Sanchez et al. 1998;Nylen et al. 2001), axon terminals in the posteriorpituitary lobe (Sagar and Ferriero 1987; Ceccatelli et al.1993), and gonadotrophs and folliculostellate cells inthe anterior pituitary lobe (Ceccatelli et al. 1993;Gonzalez-Hernandez and Gonzalez 2000); and also inthe SCN (Decker and Reuss 1994; Reuss et al. 1995;Wang and Morris 1996; Chen et al. 1997; Caillol et al.2000), where NO has been involved in the transductionof retinal inputs (Ding et al. 1994; Watanabe et al.1994; Weber et al. 1995; Amir and Edelstein 1997;Mitome et al. 2001; Golombek et al. 2004). Theadministration of NO-related drugs in different exper-imental models has suggested that it exerts a tonicmodulatory action on the basal activity of the HPA axis(Chiodera et al. 1996; Srisawat et al. 2000; Givaloiset al. 2002; Stern and Zhang 2005), so that IL-6 andNOS appear as two modulatory molecules constitu-tively expressed in hypothalamic cells and potentiallyexerting antagonistic actions. It would be interesting toknow whether all hypothalamic AVP cells, includingthose in the SCN, express IL-6, whether IL-6 is alsoexpressed in hypothalamic cells containing other pep-tides, and whether IL-6 and NOS are coexpressed in thesame hypothalamic cell subpopulation.

To improve our knowledge of the role of IL-6 andthe neuronal isoform of NOS in the hypothalamus, andparticularly in the AVP and CRF systems, we haveinvestigated the expression of IL-6 and NOS in CRF-and AVPergic neurons in different rat hypophysiotro-pic regions (PVN, SON, and ME) and the SCN. Wehave used immunohistochemistry, double immunoflu-orescence, and intracerebroventricular injection ofcolchicine, a chemical that disrupts the assembly ofmicrotubulin and blocks the axonal transport, and thatis frequently used to increase the immunostaining signalof peptides in neuron somata (Norstrom 1975; Banks1976; Lantos et al. 1995)

Materials and Methods

Animals and Tissue Preparation

A total of 22 adult male Sprague-Dawley rats weighing 270–290 g were housed under conditions of controlled tempera-

ture (246 1C) and light (8:00 AM–8:00 PM) with free access torat chow and water. Experiments were carried out inaccordance with the European Communities Council Direc-tive of 24 November 1986 (86/609/EEC) regarding the careand use of animals for experimental procedures, and adequatemeasures were taken to minimize pain and discomfort. Theanimals were heavily anesthetized with chloral hydrate andtranscardially perfused with 150 ml of 0.9% saline and 350ml of 4% paraformaldehyde in 0.1 M phosphate-bufferedsaline (PBS), pH 7.4. Under normal conditions, the peptidelevels in the soma of hypothalamic cells are too low to bedetected with immunohistochemistry; therefore, 24 hr beforekilling, 16 animals were anesthetized (80 mg/kg ketamine 1

12 mg zylazine, i.p.) and injected in the lateral ventricle with10 ml colchicine (10 mg/ml; Sigma, St. Louis, MO) dissolvedin saline, an appropriate procedure for blocking the axonaltransport and increasing the immunoreactivity in somata(Norstrom 1975; Lantos et al. 1995). Brains were removedand stored in the same fixative at 4C for 4 hr andcryoprotected in 30% sucrose in PBS overnight. Diencepha-lons were then cut at 30 mm with a freezing microtome.

Tissue Processing

Sections were collected in four to six parallel series andprocessed for single immunostaining or double immunofluo-rescent labeling. Floating sections for single immunostainingwere immersed for 20 min in 3% H2O2 to inactivate endo-genous peroxidase, and incubated for 60 min at room tem-perature (RT) in the preincubation solution (PIS): 4% normalgoat serum (NGS) (Jackson ImmunoResearch; West Grove,PA) or 4% normal donkey serum (NDS) (Jackson Immuno-Research) in 0.1 M PBS, pH 7.4, containing 0.05% TritonX-100 (Sigma), and incubated overnight in PIS containing oneof the following primary antibodies: a rabbit anti-AVP poly-oclonal antibody (AVPr), 1:4000 (INC; Aurora, Ohio); arabbit polyclonal antibody against amino acids 25–41 withN-terminally added lysine of the C terminus fragment ofhuman CRF (CRFr), 1:2000 (Sigma); a goat anti-CRF poly-oclonal antibody against the C terminus fragment of humanCRF (CRFg), 1:300 (Santa Cruz Biotechnology; Santa Cruz,CA); a goat anti-IL-6 polyclonal antibody (IL-6 g), 1:600(Santa Cruz Biotechnology); a mouse anti-neuronal NOSmonoclonal antibody (NOSm), 1:2000 (Sigma); or a rabbitanti-neuronal NOS polyclonal antibody (NOSr), 1:2000(Zymed; San Francisco, CA). After several rinses, sectionswere incubated for 2 hr in either biotinylated goat anti-mouseantiserum (1:1200; Jackson ImmunoResearch), biotinylatedgoat anti-rabbit antiserum (1:1200; Jackson ImmunoRe-search), or biotinylated donkey anti-goat antiserum (1:1000;Jackson ImmunoResearch) and 1:200 NGS or 1:200 NDSin PBS. Immunoreactions were visible after incubation for 1 hrat RT in ExtrAvidin (1:5000; Sigma) in PBS, and after 10 minin 0.005% DAB and 0.001% H2O2 in cacodylate buffer,pH 4.5. After several rinses in PBS, slides were dehydrated,cleared in xylene, and coverslipped with DPX (BDH Chemi-cals; Poole, England).

For double labeling, sections were immersed for 60 min inPIS, and overnight in PIS containing a mixture of two of theprimary antibodies used in the single immunostaining madein different species and at double concentration. They were

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combined in six different pairs according to the followingschedule: AVPr/CRFg, AVPr/IL-6 g, AVPr/NOSm, CRFr/IL-6 g, CRFr/NOSm, and NOSr/IL-6 g. After several rinses, im-munofluorescent labeling was visible after incubation for 3 hrin a mix containing either 1:150 lissamine rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch)and 1:150 fluorescein isothiocyanate-conjugated goat anti-mouse IgG in PBS containing 1:200 NGS, or 1:150 Cy2-conjugated donkey anti-goat (Jackson ImmunoResearch) inPBS containing 1:200 NDS followed by 1:150 lissaminerhodamine-conjugated goat anti-rabbit IgG (Jackson Immu-noResearch) in PBS containing 1:200 NGS. After severalrinses, sections were mounted on gelatinized slides, air dried,coverslipped with Vectashield (Vector Laboratories; Burlin-game, CA), and examined under epifluorescence microscopyusing appropriate filters. For each type of immunohistochem-istry and immunofluorescence, control experiments wereperformed by removing the primary antibodies, resulting innegative staining. The contrast in fluorescent images wasimproved by using the Adobe Photoshop program.

Analysis

The topographic distribution of stained neurons was studiedin immunohistochemical and immunofluorescent materialfollowing the hypothalamic division of Nylen et al. (2001)and Xiao et al. (2005) for the hypophysiotropic nuclei andMoore et al. (2002) for the SCN. To get a semiquantitativeestimate of the topographical distribution and the neuro-chemical profile of the different cell populations, single- anddouble-labeled neuronal profiles were counted in seven rats.In each rat, eight randomly selected sections 120–160 mmapart in the rostroacudal axis were examined. Only cellprofiles including nucleus were analyzed, and profile countswere corrected using Abercrombie’s formula (Abercrombie1946; Guillery 2002). Quantitative data are expressed asmean 6 SEM.

Results

Because our results involve hypothalamic nuclei withdifferent functional meanings, to better explain ourfindings, we will first describe those concerning hypo-physiotropic nuclei, including mostly PVN, SON, andME, and those found in the SCN.

Hypophysiotropic Nuclei

AVP and CRF Immunostaining in Cells and Fibers.Under normal conditions (colchicine-untreated rats),AVP-positive cells were present in the PVN (Figure 1A),SON, and, in a lower number, throughout the lateralhypothalamic region (see Table 1). These neurons werelarge in size (diameter of 22.6 6 2.7 mm), and most ofthem localized in the posterior magnocellular divisionof the PVN. In the ME, axons showed an intense AVPimmunoreactivity (Figure 1C). They formed a densetransversally cut bundle running to the posterior lobeof the pituitary gland in its internal layer, whereas in the

external layer, they formed a separate plexus of vari-cose terminals. No CRF-positive cells were detected inthe hypothalamus of these animals (Figure 1B), but CRFimmunoreactivity was evident in the terminal plexusof the external zone of the ME (Figure 1D). AVP cellsincreased in number (approximately 260%; Figure 1E;Figures 2A, 2G) and CRF cells became evident in col-chicine-treated rats (Figure 1F; Figure 2H; Table 1). Wedistinguished three types of AVP cells according to theirsize and labeling intensity: large (diameter of 22.6 6

2.7 mm), intensely stained cells, which constituted 396

5% of the total number of AVP cells; large, weaklystained cells (48 6 6%); and small (diameter of 14.2 6

2.5 mm), weakly stained cells (13 6 5%). Large, in-tensely AVP-stained cells were preferentially localizedin the dorsolateral region of the posterior magnocellulardivision of the PVN, where they were densely clustered(Figure 1E; Figure 2A) and, to a lower degree, in theventral aspect of the anterior magnocellular divison(Figure 2G; Figure 3A; Figure 4E). Large, weakly stainedcells displayed an intermedio-lateral distribution par-ticularly conspicuous just medial and dorsal to the dor-solateral and rostroventral clusters of intensely stainedcells (Figures 2A, 2G; Figure 3A; Figure 4E). Small,weakly stained cells were present in the anterior anddorsal parvocellular divisions of the PVN (Figure 2G). Inthe SON, intensely and weakly stained large cells wereintermingled, and in the lateral hypothalamic region,they were also close to each other and arranged insmall clusters around vessels (Figure 3C). The terminalplexus in the external zone of the ME lost its AVPimmonoreactivity after colchicine injection (Figure 1G;Figure 2E). We also distinguished three types of cells inCRF immunostaining: large, intensely stained cells (516

4%); large, weakly stained cells (38 6 5%); and small,intensely stained cells (116 2%; see Table 1). Small cellslocalized preferentially in the anterior and dorsal parvo-cellular regions of the PVN (Figure 2H), as in AVP, butthe distribution pattern of large cells was different fromthat in AVP. The intensely stained cells localized in themedial aspect of the posterior magnocellular division,just lateral to the small cells, so that both cell populationsformed an area occupying the two dorsomedial thirdsof the PVN, whereas the weakly stained cells localizedin the lateral third of its anterior and posterior magno-cellular divisions (Figure 1F; Figure 5G). Intensely andweakly stained large cells were also present around ves-sels in the lateral hypothalamic region (Figure 5K),and a few cells (approximately 3%) also showed CRFimmunoreactivity in SON (Figures 5C, 5I). Doubleimmunohistochemistry for AVP and CRF in colchicine-untreated rats showed that in the external layer of theME, 50–65% of CRF axons contain AVP (Figures 2K,2L), and after colchicine injection, approximately 86%of small and weakly stained cells coexpress both peptides

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IL-6 and NOS Expression in the Rat Hypothalamus 429



(Figures 2I, 2J; Table 1). According to these data, wecan distinguish four different cell types: large AVP cells,large CRF cells, large AVP/CRF cells, and small AVP/CRF cells.

IL-6 and NOS Expression in AVP Cells. Most AVPcells (approximately 96%) contained IL-6 in colchicine-untreated rats. After colchicine injection, we observedthat in PVN (Figures 2A–2C; Figures 3A, 3B), SON

Figure 1 Immunohistochemistry forarginin-vasopressin (AVP) (A,C,E,G)and corticotropin-releasing factor(CRF) (B,D,F,H) in the paraventricularnucleus (PVN) (A,B,E,F) and the me-dian eminence (ME) (C,D,G,H) of sham-(A–D) and colchicine-injected (E–H)rats. Under normal conditions, a fewAVP cells (A) but no CRF cells (B) aredetected in the PVN; but in theME, theinternal layer (IL) shows an intenselabeling for AVP (C), and the externallayer (EL) shows immunoreactivity forAVP (C) and CRF (D). After colchicineinjection, AVP cells increase in number(E), CRF cells become positive (F), andimmunoreactivity for AVP (G) and CRF(H) decreases in the external layer ofthe ME. Bars: in F (for A,B,E,F) 5 150mm; in H (for C,D,G,H) 5 300 mm.

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(Figure 2D), and lateral hypothalamus (Figures 3C,3D), only large, intensely stained cells, which matchedthose identified in intact rats, but not small and largeweakly stained cells, coexpressed IL-6. In terms of cellcount, they corresponded to 36 6 5% of the totalnumber of AVP cells. The bundle of AVP axonsrunning through the internal zone of the ME showedan intense IL-6 immunoreactivity (Figure 2E), whereasin the external zone, no IL-6 immunoreactive terminalswere detected. It should be noted that some neuronssparsely distributed through the PVN, lateral hypotha-lamic region, and SON contained IL-6 but not AVP(Figure 2D, arrows).

With respect to the expression pattern of NOS inAVP cells, a low-magnification view of the PVN andSON in colchicine-untreated animals revealed thatmany NOS-positive cells colocalized with those immu-noreactive for AVP (Figure 4) or IL-6 (Figures 6A–6F);however, at a higher magnification, we saw that insome regions, particularly in the anterior third of thePVN, only a few (3 6 2%) large, intensely AVP- or IL-6-stained cells coexpressed NOS (Figures 4A, 4B),whereas in other regions, such as the dorsolateralregion of the posterior magnocellular division of PVNand SON, 326 3% of the cells contained NOS (Figures4C, 4D; Figures 6A–6F). When weakly stained cellsbecame evident after colchicine injection, we observedthat approximately 80% of large, weakly AVP-stainedcells (immunonegative for IL-6 and immunopositive forCRF) in the PVN and around vessels in the lateralhypothalamic region coexpressed NOS (Figure 2M;Figures 4E, 4F). Therefore, in colchicine-treated ani-mals, approximately 48% of the total number of AVP

cells express NOS. NOS activity was also detected inboth the varicose plexus of the external layer and thebundle of transverse fibers of the internal layer in theME of intact rats (Figures 6G, 6H). Numerous NOS-positive, AVP-negative cells were present throughoutthe PVN and in neighboring regions; however, becausethe distribution of NOS neurons in the hypothalamushas been extensively studied using NOS immunohisto-chemistry and NADPH-diaphorase histochemistry(Bredt et al. 1990; Arevalo et al. 1992; Calka andBlock 1993; Miyagawa et al. 1994; Sanchez et al. 1998;Nylen et al. 2001; Xiao et al. 2005), we have omittedthis description here.

IL-6 and NOS Expression in CRF Cells. Immuno-fluorescence for CRF and IL-6 in colchicine-treated ratsrevealed that although CRF and IL-6 cells colocalizedin different hypothalamic regions, no cells coexpressingboth markers were found (Figures 5A–5D). In addition,CRF localized in the axonal plexus of the external layerof the ME, whereas IL-6 was largely restricted to theaxon bundle in the internal layer, with only a discreteoverlap at the border between the layers (Figures 6E,6F), supporting the idea that most CRF cells do notcontain IL-6.

With respect to NOS, a low-magnification view of thedistribution pattern of CRF and NOS cells in the PVNshowed a fairly complementary distribution, becauseintensely stained CRF cells occupy the dorsomedial halfand NOS cells occupy the ventrolateral half in the pos-terior magnocellular division (Figures 5G, 5H). Howev-er, at a higher magnification, we saw that the lateralthird of this division also contained large, weakly stained

Table 1 Quantification of AVP and CRF cells expressing IL-6 and NOS in the rat hypothalamus

No colchicine Colchicine

AVP AVP CRFLocation Total 413 6 26 CRF Total 1,076 6 167 Total 1,231 6 149

PVN 256 6 18 (62)a — LI 420 6 51 (39)a LI 628 6 75 (51)b

CRF-AVP/AVP — AVP-CRF/CRF —IL6-AVP/AVP 544/567 (96)c IL6-CRF/CRF —NOS-AVP/AVP 86/477 (18)c NOS-CRF/CRF —

SON 124 6 11(30)a — LW 516 6 64 (48)a LW 468 6 51 (38)b

CRF-AVP/AVP 687/808 (85)c AVP-CRF/CRF 548/622 (88)c

IL6-AVP/AVP — IL6-CRF/CRF —NOS-AVP/AVP 363/459 (81)c NOS-CRF/CRF 542/638 (85)c

LHR 33 6 8 (8)a — SW 149 6 22 (13)a SI 135 6 17 (11)b

CRF-AVP/AVP 206/260 (79)c AVP-CRF/CRF 184/204 (89)c

IL6-AVP/AVP — IL6-CRF/CRF —NOS-AVP/AVP — NOS-CRF/CRF —

aPercent with respect to the total number of AVP cells.bPercent with respect to the total number of CRF cells.cPercent with respect to the total number of single-labeled (AVP or CRF) cells.Data correspond to the number of cells in eight randomly selected sections 120–160 mm apart in the rostroacudal axis (n55 in colchicine-untreated rats; n57 incolchicine-treated rats). LHR, lateral hypothalamic region; LI, large, intensely stained cells; LW, large, weakly stained cells; NOS, nitric oxide synthase; PVN,paraventricular nucleus; SI, small, intensely stained cells; SON, supraoptic nucleus; SW, small, weakly stained cells.

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CRF cells and that most of them (83 6 6%) expressedNOS (Figure 2N; Figures 5G, 5H). Furthermore, most(91 6 5%) CRF cells around vessels in the lateralhypothalamic region contained NOS (Figures 5K, 5L),whereas those in the SON were NOS immunonegative(Figures 5I, 5J). CRF and NOS colocalized in most fibers(786 6%) forming the plexus of the external layer of theME in colchicine-untreated rats (Figure 2O). NOS wasalso present in the bundle of the internal layer, but itsaxons did not contain CRF.

Suprachiasmatic Nucleus

As shown in immunohistochemistry for AVP, CRF, andIL-6 (Figure 7), the rat SCN is a well-delimited nucleuscomposed of densely packed small cells (diameter of9.3 6 2.7 mm), which, according to their topographicaldistribution and neurochemical profile, may be dividedinto two different cell populations. One of these, local-

ized dorsomedially, close to the ventricular wall, isimmunoreactive for AVP (Figure 7A), and the other,localized ventrolaterally, is immunoreactive for CRF(Figure 7B). Double immunofluorescence for AVP andIL-6 revealed that approximately 65% of AVP cells wereimmunoreactive for IL-6 (Figure 2F; Figures 7C, 7D).However, although there was a certain overlap betweenAVP/IL-6 cells and CRF cells at the border between thedorsomedial and ventrolateral regions, no AVP/CRF orIL-6/CRF double-labeled cells were found. With respectto NOS, a few immunoreactive cells were detected in theneighboring regions, but not in the SCN.

Discussion

Our current concept of the peptidergic profile of hy-pothalamic nuclei comes from immunohistochemicalstudies carried out on different mammals since the earlyeighties (Kiss et al. 1984; Sawchenko et al. 1984a,b;

’

Figure 2 A–C: Double labeling for AVP and interleukin-6 (IL-6) in the PVN of a colchicine-injected rat showing that intensely stained AVP cellsin the dorsolateral region of the PVN are IL-6 immunopositive, but those weakly stained lying in the central region are IL-6 immunonegative.D:Double labeling for AVP and IL-6 in the supraoptic nucleus (SON) showing that most cells coexpress both markers, although a few of them areAVP negative (arrows). E: Double labeling for AVP and IL-6 in theME of a colchicine-injected rat showing that most transversely cut fibers in theinternal layer (IL) are immunoreactive for both markers. F: Double labeling for AVP and IL-6 in the dorsomedial region of the suprachiasmaticnucleus (SCN) showing that most AVP cells contain IL-6. G–L: Double labeling for AVP and CRF in the PVN of a colchicine-injected rat (G–J), andthe ME of an intact rat (K,L). We note that small cells in the dorsomedial region (I) and large, weakly stained cells in the lateral region (J) of thePVN, and axons in the external layer (EL) of the ME (K,L) show double labeling, whereas large, intensely stained cells in the PVN (G–I) and fibersin the internal layer (IL) of the ME are only immunoreactive for AVP. L: Boxed area in K. M: Double labeling for AVP and nitric oxide synthase(NOS) in the central region of the PVN. N,O: Double labeling for CRF and NOS in the lateral region of the PVN (N) and the ME. Note that mostaxons in the external layer (EL) of theME are doubly labeled, whereas fibers in the internal layer (IL) are only immunoreactive for NOS. Bars: in F(for E,F) 5 150 mm; in I (for A–D,G–I) 5 100 mm; in K 5 300 mm; in N (for M,N) 5 50 mm; in O 5 120 mm.

Figure 3 Double labeling for AVP (A,C) and IL-6 (B,D) in the PVN (A,B) and the lateral hypothalamic region (C,D) of a colchicine-injected rat.We note that only intensely AVP-stained cells coexpress IL-6. Arrows in A and B, and v in C and D indicate the same vessel, respectively. Bars: inB (for A,B) 5 120 mm; in D (for C,D) 5 100 mm.

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Figure 4 Double labeling for AVPand NOS in the PVN of an intact rat(A–D) and a colchicine-injected rat(E,F). We note that in the rostralregion of the PVN of intact rats, AVPcells and NOS cells colocalize but AVPand NOS are not coexpressed (A,B),whereas in other regions, many cellscoexpress both markers (C,D). Aftercolchicine injection, weakly AVP cellsbecome evident and most of themexpress NOS (E,F). Arrows in A and Bindicate a vessel; in C and D, double-labeled cells. v in E and F indicates avessel. Bars: in B (for A,B)5 150 mm; inD (for C,D) 5 50 mm; in F (for E,F) 5120 mm.

’

Figure 5 A–F: Double labeling for CRF and IL-6 in the PVN (A,B) and SON (C,D) of a colchicine-injected rat, and in theME of an intact rat (E,F). Inthe PVN and SON, CRF cells (A, arrows in C) do not express IL-6 (B, arrows in D), and in the ME, CRF is in the axonal plexus of the external layer(EL) (E), and IL-6 in fibers running through the internal layer (IL) (F).G–L: Double labeling for CRF and NOS in the PVN (G,H), SON (I,J), and lateralhypothalamic region (K,L) of a colchicine-injected rat. We note that intensely stained CRF cells in the dorsomedial region of PVN (G) and thefew cells present in the SON (arrows in I) are not immunoreactive for NOS (H, arrows in J), but those lying in the lateral region of PVN (weaklystained) and those in the lateral hypothalamic region (K) are NOS immunoreactive (H,L). Arrows in C, D, I, and J indicate CRF cells inthe SON that are immunonegative for IL-6 and NOS. v in K and L indicates a vessel. Bars: in F (for A–F) 5 200 mm; in H (for G,H) 5 250 mm; inL (for I–L) 5 150 mm.

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Sawchenko and Swanson, 1985; Whitnall et al. 1985;Alonso et al. 1986; Piekut and Joseph 1986; Saw-chenko 1987; Whitnall 1988; De Goeij et al. 1992;

Mouri et al. 1993; Kikusui et al. 1997; Matz andHofeldt 1999). According to these studies, CRF ispreferentially expressed in parvocellular cells and AVP

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Figure 6 Double labeling for IL-6 andNOS in the PVN (A–D) and SON (E,F) ofan intact rat, and the ME of acolchicine-injected rat (G,H). We cansee that NOS is expressed in many IL-6-positive cells (which are intenselystained for AVP), and that in theinternal layer (IL) of the ME, bothmarkers show a similar labeling pat-tern, whereas in the external layer(EL), only NOS is detected. Arrows inC–F indicate some double-labeledcells. Bars: in B (for A,B) 5 150 mm;in D (for C,D) 5 60 mm; in F (for E,F) 580 mm; in H (for G,H) 5 300 mm.

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(and oxytocin) in the magnocellular cells in the PVN. Inaddition, subpopulations of parvo- and magnocellularcells coexpress both CRF and AVP (or oxytocin).

Our results confirm these findings, and enable us todistinguish subpopulations of AVP- and CRFergic hy-pophysiotropic cells according to their IL-6 and neuro-nal NOS coexpression patterns (Figure 8). Althoughwe have not used triple labeling, the combination ofdifferent pairs of antibodies together with cytoarchitec-tural criteria reveal five different types of hypophysio-tropic cells. Within AVP cells: large, intensely stained

AVP cells coexpressing IL-6; and large, intensely stainedAVP cells coexpressing IL-6 and NOS.Within CRF cells:large, intensely stained CRF cells immunonegative forAVP, NOS, and IL-6; large cells weakly stained for CRFand AVP, immunopositive for NOS and immunonega-tive for IL-6; and small cells intensely stained forCRFandAVP and immunonegative for IL-6 and NOS. Inaddition, we identified two different cell populations inthe SCN, one in its dorsomedial region, which expressesAVP and IL-6, and the other in its ventromedial region,which is immunoreactive for CRF.

Figure 7 Double labeling for AVP and CRF (A,B)and AVP and IL-6 (C,D) in the SCN of a colchicine-injected rat. The dotted lines in A and B indicatethe dorsomedial region of the SCN, and v in A andB, and C and D, indicates the same vessel, re-spectively. We note that AVP cells localize in thedorsomedial region (A) and CRF cells in the ven-trolateral region (B). C and D show that most AVPcells contain IL-6. Bars: in B (for A,B)5 150 mm; in D(for C,D) 5 150 mm.

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In light of previous morphological studies combiningtract-tracing methods (Armstrong and Hatton 1980;Ju et al. 1986; Taniguchi et al. 1988) or partial hypo-thalamic lesion (Vandersande et al. 1977; Antoni et al.1990) with immunohistochemistry, and the study inparallel of axons in theME, the fact that large, intenselyAVP-stained cells coexpress IL-6 and that axonsrunning through the internal layer of the ME coexpressAVP and IL-6 indicates that they correspond to theAVPergic cells recently described by Ghorbel et al.(2003), which corelease AVP and IL-6 in the posteriorpituitary lobe. However, our data reveal that theyactually represent a subpopulation of no more than40% of the total number of AVP cells, that 18% ofthem also coexpress NOS, and that two othersubpopulations that do not express IL-6, small AVP/CRF cells (13%) and large, AVP weakly stained cellsimmunoreactive for CRF and NOS (47%), becomeevident after colchicine injection. In any case, thesepercentages should be taken as tentative, because theyhave been partially obtained from colchicine-treatedrats, and the effect of colchicine can differ from one celltype to another and from one animal to another.

The finding that NOS displays the same labelingpattern as AVP and IL-6 in the internal layer of ME

agrees with the presence of NOS in AVP/IL-6 cells.However, although the thickness and labeling intensityin the ME was similar for the three markers (NOS,AVP, and IL-6), only 35% of AVP/IL-6 cells showedNOS activity. This discrepancy could be explained bythe fact that most oxytocinergic cells (another PVNmagnocellular subpopulation whose axons reach theposterior pituitary lobe through the internal layer of theME) also contain NOS (Nylen et al. 2001; Xiao et al.2005) and consequently contribute to the NOS labelingintensity of this bundle.

With respect to CRF, we identified three cell types:magnocellular cells intensely stained for CRF, whichare immunonegative for AVP, NOS, and IL-6; magno-cellular cells weakly stained for CRF and AVP and alsocontaining NOS; and parvocellular CRF cells, whichcoexpress AVP. In addition, we observed that CRFaxons localize in the external layer of the ME, and thatmany of them contain AVP and NOS. These findingsare consistent with previous studies reporting that CRFaxons running to the anterior pituitary lobe occupy aventral position in the ME (Rho and Swanson1987,1989; Kawano et al. 1988; Antoni et al. 1990;Weiss and Cobbett 1992; Lennard et al. 1993; Reyeset al. 2005), that the external zone of the ME expressesNOS activity (Sagar and Ferriero 1987; Ceccatelli et al.1993), and that approximately 50% of CRF terminalsin the external zone of the ME contain AVP (Whitnalland Gainer 1988). Taken together, these findings sug-gest that in the posterior pituitary lobe, constitutive IL-6is synthesized by AVP cells, and NO by both oxytocinand AVP cells; and that in the hypophysial portalvascular system, NO is synthesized by AVP/CRF cells.However, in light of previous studies reporting that asubpopulation of large CRF cells also contains oxytocin(Dohanics et al. 1990; Imaki et al. 2001) and thatNOS isexpressed in oxytocinergic cells (Miyagawa et al. 1994;Hatakeyama et al. 1996; Yamada et al. 1996; Nylenet al. 2001; Xiao et al. 2005), it is possible that CRF/oxytocin cells also participate in the putative modu-latory role of NO in the hypothalamic-pituitary system(Duvilanski et al. 1995; Ceccatelli 1997; Rivier 2001).

We also found AVP- and CRF-immunoreactive (-ir)cells in the SCN. These cells are densely packed anddisplay a compartmental distribution, with AVP-ir cellslocalized in the dorsomedial region and CRF-ir cells inthe ventrolateral region. This labeling pattern ispartially supported by previous studies reporting thatAVP cells are the main component of the dorsomedial(or shell) region of the SCN (Hofman et al. 1996;Herzog et al. 2000; Abrahamson and Moore 2001;Moore et al. 2002). In addition, as described for AVPhypophysiotropic cells projecting to the posteriorpituitary lobe, we found that SCN AVP cells containIL-6, indicating that IL-6 may be released from different

Figure 8 Schematic drawing summarizing the different popula-tions of AVP and CRF hypothalamic cells according to the expressionof NOS and/or IL-6 and their projections. PVN, paraventricularnucleus. In light of previous studies (Miyagawa et al. 1994;Hatakeyama et al. 1996; Yamada et al. 1996; Nylen et al. 2001;Xiao et al. 2005), oxytocin/NOS cells have also been included.

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hypothalamic AVPergic cells, and consequently, maybe involved in different regulatory processes, possiblyincluding the control of circadian rhythms.

On the other hand, although the ventrolateral (orcore) SCN is mainly composed of vasoactive intestinalpolypeptide- and gastrin-releasing polypeptide-immu-noreactive cells (Hofman et al. 1996; Herzog et al.2000; Abrahamson and Moore 2001; Moore et al.2002), the presence of CRF-ir cells is supported byprevious immunohistochemical and bioassay studies inthe rat (Hashimoto et al. 1982; Daikoku et al. 1985;Hornby and Piekut, 1989) and goat (Kikusui et al.1997). However, this finding should be interpretedcautiously, because the protein fragments used toobtain the CRF antibodies display structural homologywith the peptide histidine isoleucine (PHI) (Sawchenko1987; Antoni and Linton 1989), which has beendetected in the ventrolateral SCN (Stopa et al. 1988;Denis et al. 1993). Therefore, a cross-reaction betweenCRF antisera and PHI cannot be ruled out.

With respect to NOS expression in the SCN, our dataagree with those reporting that in the rat, SCN neuronsdo not contain NOS (Decker and Reuss 1994; Reusset al. 1995; Wang and Morris 1996; Chen et al. 1997)and suggesting that the source of NO in the signaltransduction of retinal glutamatergic inputs in the SCN(Ding et al. 1994; Watanabe et al. 1994; Weber et al.1995; Amir and Edelstein 1997; Mitome et al. 2001;Golombek et al. 2004) is neighboring neurons or non-neuronal elements. In this respect, the finding thatdifferent parameters of circadian activity were notmodified in neuronal NOS knockout mice (Kriegsfeldet al. 1999) and that SCN astrocytes express endothelialNOS (Caillol et al. 2000) suggested that this isoformcould be the source of NO in the SCN. However, otherstudies have shown that endothelial NOS is notnecessary formaintaining circadian rhythms (Kriegsfeldet al. 2001) and that the inducible isoform of NOS canbe constitutively expressed in the adult SCN (Starkeyet al. 2001). Taken together, these data suggest thatin the SCN, NO can be synthesized by different NOSforms and can arise from different cell types.

In conclusion, the labeling patterns of IL-6 and NOSfound here indicate that IL-6 is coexpressed with AVPin hypophysiotropic cells projecting to the posteriorpituitary gland and in the dorsomedial region of theSCN, and that NOS is coexpressed with AVP and IL-6in a subpopulation of AVPergic cells projecting to theposterior pituitary gland and with AVP and CRF inmagnocellular cells projecting to the external layer ofthe ME. In light of previous functional studies, IL-6 canexert an autocrine/paracrine modulatory effect onposterior pituitary secretion and SCN AVPergic targets,and NO can exert a similar effect on the secretion ofboth pituitary lobes.

Acknowledgments

This work was supported by the Gobierno Autonomo deCanarias (grant no. PI042004/074), the Fundacion Canariade Investigacion y Salud (grant no. PI 86/04), and theMinisterio de Educacion y Ciencia de Espana (grant no.BFU2004-05756).

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