Peripheral injection of CCK-8S induces Fos expression in the dorsomedial hypothalamic nucleus in rats

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Peripheral injection of CCK-8S induces Fos expression in the dorsomedial hypothalamic nucleus in rats Peter Kobelt a , Stephan Paulitsch a , Miriam Goebel a , Andreas Stengel a , Marco Schmidtmann b , Ivo R. van der Voort a , Johannes J. Tebbe d , Rüdiger W. Veh c , Burghard F. Klapp b , Bertram Wiedenmanna , Yvette Taché e , Hubert Mönnikesa,b,⁎ a

Department of Medicine, Division Hepatology, Gastroenterology, and Endocrinology, Charité, Campus Virchow, Humboldt-Universität zu Berlin, Germany b Department of Medicine, Division Psychosomatic Medicine and Psychotherapy, Charité, Campus Mitte, Humboldt-Universität Berlin, Germany c Institute of Anatomy, Section of Electron Microscopy and Neuroanatomy, Charité, Campus Charité Mitte, Humboldt-Universität Berlin, Germany d Department of Medicine, Division Gastroenterology and Endocrinology, Philipps-Universität Marburg, Germany e Department of Medicine, Division of Digestive Diseases, CURE Digestive Diseases Research Center and Center for Neurovisceral Sciences, UCLA, Los Angeles, CA 90095-1763, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Peripheral cholecystokinin (CCK) plays a physiological role in the regulation of food intake.

Accepted 1 August 2006

The dorsomedial hypothalamic nucleus (DMH) has been implicated in the brain regulation

Available online 26 September 2006

of food intake and satiety. The aim of this study was to determine if peripherally administered CCK affects neuronal activity in the DMH, as assessed by Fos expression.

Keywords:

Density of Fos-positive neurons was determined in the DMH, paraventricular nucleus of the

CCK

hypothalamus (PVN), arcuate nucleus of the hypothalamus (ARC) and ventromedial

Dorsomedial hypothalamic nucleus

hypothalamic nucleus (VMH) in non-fasted Sprague-Dawley rats in response to

Fos

intraperitoneally (ip) injection of CCK-8S (2 μg/kg, n = 6) or vehicle (0.15 M NaCl; n = 6). CCK-

Brain

8S increased Fos immunoreactivity in the DMH (mean ± SEM; cells/section: 108 ± 10 versus

Rat

54 ± 6, p < 0.001) and PVN (120 ± 12 versus 20 ± 3, p < 0.001) compared to the vehicle group while not influencing Fos expression in the ARC and VMH. Double labeling showed that 27.4 ± 6.4% (n = 3) of Fos-positive neurons induced by CCK-8S were positive for corticotropin-releasing factor immunoreactivity, that were mainly localized in the ventral part of the DMH, and encircled in a network of tyrosine-hydroxylase-immunoreactive positive fibers. These data indicate that in addition of the PVN, peripheral CCK increases neuronal activity in the DMH suggesting a possible role in this hypothalamic nucleus in the satiating effect of the peptide. © 2006 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Department of Medicine, Division Hepatology and Gastroenterology, Charité – University Medical Center and School of Medicine, Campus Virchow-Klinikum, Medical Faculty of Freie Universität and Humboldt-Universität at Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Fax: +49 30 450553991. E-mail address: [email protected] (H. Mönnikes). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.08.092

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1.

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Introduction

Peptide hormones from the gastrointestinal (GI) tract play an important role in the short-term regulation of food intake and satiety by influencing the initiation and termination of meals (Woods et al., 1998). Evidence for the existence of GI ‘satiety factors’ came in the 1970s, when it was found that peripheral cholecystokinin (CCK) administered before the time of food availability causes a dose-dependent decrease in meal size (Gibbs et al., 1973). CCK is synthesized within a discrete population of endocrine cells in the upper small intestine (Liddle, 1994) and release of the peptide is stimulated by intraluminal nutrients (Mossner et al., 1992; Guimbaud et al., 1997). The satiating effect of peripheral CCK depends on afferent signaling via the vagus nerve (Smith et al., 1981; Ritter and Ladenheim, 1985). Most of the mapping of brain neuronal circuits recruited by satiety signals has been obtained by assessing changes in Fos expression, as determined by detection of Fos immunoreactivity (Fos-ir), which allows to identify activated neurons at the cellular level (Sagar et al., 1988). Numerous studies have shown that intraperitoneally (ip) injected sulfated CCK octapeptide (CCK-8S) induces Fos-ir in the paraventricular nucleus of the hypothalamus (PVN), locus coeruleus (LC), nucleus of the solitary tract (NTS), and the area postrema (AP) (Olson et al., 1992; Chen et al., 1993; Day et al., 1994; Rinaman et al., 1995; Mönnikes et al., 1997a). In addition, the release of endogenous CCK is involved in the induction of Fos in these brain sites caused by entry of nutrients into the intestine (Mönnikes et al., 1997b; Wang et al., 1999). There is evidence that changes in GI function, e.g., gastric emptying, are neither necessary nor sufficient for CCK-induced satiety (reviewed in Moran and Kinzig, 2004). Therefore, it has been proposed that the activation of specific brain nuclei is important for the integrative regulation of meal termination and GI function by peripheral CCK. Another hypothalamic nucleus which is involved in the regulation of feeding behavior is the dorsomedial nucleus of the hypothalamus (DMH) (Bellinger and Bernardis, 1984; Bellinger et al., 1983; Bi et al., 2001). In particular, previous reports using lesioning of the DMH indicated that the DMH plays a role in mediating the satiety effects caused by peripheral administration of CCK (Bellinger et al., 1983; Bellinger and Bernardis, 1984). Further, it has been shown that microinjection of CCK-8S into the DMH suppresses food intake in rats (Blevins et al., 2000). However, so far it is not known whether peripheral CCK in fact modulates neuronal activity in the DMH. In previous studies it has been demonstrated that the PVN is richly innervated by afferent nerve fibers originating from the DMH (Ter Horst and Luiten, 1986; Ter Horst and Luiten, 1987), and that some of these projections contain the neuropeptide corticotropin-releasing factor (CRF) (Champagne et al., 1998). CRF immunoreactivity (CRF-ir)-positive cells have been found to be located in the parvocellular PVN and in the DMH (Daikoku et al., 1985). In addition, it has been shown that peripherally administered CCK increases serum adrenocorticotropic hormone and corticosterone levels in rodents (Kamilaris et al., 1992) and humans (Calogero et al., 1993). Thus, it

seems reasonable to assume that, in addition to such neurons in the PVN, CCK may also activate neurons in the DMH containing CRF. Therefore, CRF was chosen as a candidate for the neurochemical analysis performed in this study. It has also been reported that activated neurons in the PVN after peripheral CCK injection contain CRF or oxytocin (Verbalis et al., 1991). There is convincing evidence that these neurons are innervated by noradrenergic projections from the A2-cell group in the NTS (Rinaman et al., 1995). In addition, previous studies showed that the DMH receives noradrenergic projections coming from the NTS (Sawchenko and Swanson, 1982). Thus, the aim of the present study was to determine if CCK injected intraperitoneally (ip) influences neuronal activity in the DMH, as assessed by induction of Fos expression and to compare the Fos response in the DMH with that of other hypothalamic nuclei, namely the PVN, ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC) that are also known to influence food intake (reviewed in Williams et al., 2001). Further, we investigated whether DMH neurons activated by CCK-8S ip involve CRF containing neurons. Lastly, tyrosine-hydroxylase immunoreactivity (TH-ir) in the DMH was determined to assess the distribution of noradrenergic fibers in relation to Fos-ir using confocal microscopy.

2.

Results

CCK-8S (2 μg/kg, ip) induced a robust increase in the density of Fos-ir-positive neurons compared to the vehicle group in the PVN (mean ± SEM: 120 ± 12 versus 20 ± 3 neurons/section, p < 0.001; Fig. 1) and DMH (108 ± 10 versus 54 ± 6, p < 0.001; Figs. 1 and 2). CCK-8S had no effect compared to vehicle-treated animals on the Fos expression in the ARC (13 ± 2 versus 12 ± 1, p > 0.05) and in the VMH (25 ± 2 versus 23 ± 2, p > 0.05) (Fig. 1). Interestingly, the Fos-ir-positive neurons induced by ip injection of CCK-8S, were mainly localized within the ventral part of the DMH (Fig. 2). Confocal analysis by laser scanning microscope (y-projection of a z-stack) at high magnification revealed that Fos-positive neurons were encircled within a dense network of TH-ir-positive fibers (Fig. 3). Additional double staining with anti-Fos and anti-CRF revealed that 27.4 ± 6.4% of the Fos-positive neurons in the ventral part of the DMH were positive for CRF (Fig. 4).

3.

Discussion

The data in the present study show for the first time that peripheral CCK affects the activity of DMH neurons in conscious non fasted rats. We observed a two-fold increase in Fos expression in the DMH in response to CCK-8S injected ip. The dose used (2 μg/kg) was reported to suppress food intake without causing unspecific behavioural aversive effects (e.g., nausea, taste aversion) in rats (Morley, 1987) suggesting that the observed effect on Fos induction in DMH neurons is specific to the peptide and not induced by stress. Functional studies have established that the DMH is involved in the integrative regulation of food intake including the satiety effect of CCK (Bellinger and Bernardis, 2002). In rats

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Fig. 1 – Effects of CCK-8S injected ip on the number of Fos-ir-positive neurons in various hypothalamic nuclei in fed rats. Activation of PVN and DMH neurons induced by ip CCK-8S as shown by Fos expression. Fos-ir-positive neurons in the paraventricular nucleus of the hypothalamus (PVN), arcuate nucleus of the hypothalamus (ARC), ventromedial hypothalamus (VMH) and in the dorsomedial hypothalamic nucleus (DMH) were quantified 90 min after intraperitoneal injection of CCK-8S and vehicle injection. Data are means ± SEM of 6 rats/group. #p < 0.01 versus vehicle; *p < 0.01 versus vehicle.

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with electrolytic lesioning of the DMH, there is no inhibition of food intake induced by peripheral injection of CCK while the attenuation of feeding in response to peripheral bombesin was not altered (Bellinger et al., 1983; Bellinger and Bernardis, 1984). Taken together these data indicate that intestinal CCK takes part in the control of meal size by modulating the activity of DMH neurons. Peripheral CCK-8S modulates neuronal activity in other brain areas, in particular the NTS, AP, LC and the PVN (Chen et al., 1993; Olson et al., 1992; Day et al., 1994; Rinaman et al., 1995; Mönnikes et al., 1997a), suggesting that a network of autonomic brain nuclei is affected by peripheral CCK. The DMH receives input from different brain areas including the PVN, the VMH, the lateral hypothalamic area, ARC, and the NTS (Thompson and Swanson, 1998; Sawchenko and Swanson, 1982) which are known to be involved in the regulation of energy homeostasis (reviewed in Williams et al., 2001). A wealth of evidence showed that catecholaminergic and non-catecholaminergic neurons in the NTS project to various hypothalamic brain areas (Ricardo and Koh, 1978; Sawchenko and Swanson, 1981, 1982; Sawchenko et al., 1988). In particular, neurons in the PVN activated by peripheral injection of CCK receive noradrenergic input from the A2-cell

Fig. 2 – CCK-8S injected ip induces Fos-ir in the dorsomedial hypothalamic nucleus in freely fed rats. The dorsomedial hypothalamic nucleus showed Fos-ir (green staining) 90 min after the ip injection of CCK-8S (B) while after ip saline injection (A) only few Fos-ir-positive neurons were observed. Cell nuclei are stained red as a result of the counterstaining with propidium iodide in the same slice of CCK-8S (D)- and saline-treated (C) animals. The white outer line delineates the area of the dorsomedial hypothalamic nucleus. The white scale bar represents 100 μm. 3V = third ventricle, DMHD = dorsomedial hypothalamic nucleus, dorsal part; DMHC = dorsomedial hypothalamic nucleus, compact part; DMHV = dorsomedial hypothalamic nucleus, ventral part.

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group in the NTS (Rinaman et al., 1995). Peripheral CCK is well established to activate noradrenergic neurons in the NTS, ventrolateral medulla and LC (Rinaman et al., 1995; Buller and

Day, 1996; Mönnikes et al., 1997a). Retrograde tracing experiments revealed direct projections from noradrenergic A2-cells of the NTS to the DMH (Sawchenko and Swanson, 1982).

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Fig. 4 – Double staining of Fos-positive neurons in the DMH with anti-CRF induced by ip CCK. Overview of double staining with anti-CRF (red staining) and anti-Fos (green staining) in the DMH (A). Double staining revealed by high magnification that numerous of these Fos-positive neurons in the ventral part of the DMH were positive for CRF (arrow). The image displays the relationship between Fos in the nuclei and CRF in the cytoplasm of cells and their axons (B). The white scale bar represents 100 μm in panel A and 10 μm in panel B. DMHV = dorsomedial hypothalamic nucleus, ventral part.

Therefore, one can speculate that CCK-8S-induced activation of neurons in the DMH is also mediated via signals from noradrenergic brainstem neurons activated by peripheral CCK. This contention is supported by the present observation that Fos-positive neurons in the DMH activated by CCK-8S are embedded in a network of noradrenergic fibers. This observation suggests that CCK-8S exerts its anorexigenic effects at least partially by activating this noradrenergic NTS-DMH pathway. However, further tracing and pharmacological studies would be needed to support the hypothesis that the activation of DMH neurons by peripheral CCK-8S is mediated via activation of A2-cells in the NTS. It has been shown that the ARC and the VMH project to the DMH (Thompson and Swanson, 1998). Therefore, it is also possible that these brain sites modulate neuronal activity of DMH neurons induced by CCK-8S administration. However, in the present study we could not detect an increased number of cells expressing Fos in these brain nuclei. Therefore, it is unlikely that the increased Fos expression in neurons of the DMH induced by peripheral CCK-8S is mediated via projections arising from the ARC and/or VMH. Previous reports indicate that the peripheral administration of leptin also induces neuronal activation in the DMH (Elmquist et al., 1997; Elias et al., 2000). Some studies suggest that CCK promotes leptin secretion in the stomach (Tsunoda et al., 2003). Thus, it can be speculated that intraperitoneal injection of CCK-8S increases neuronal activity in the DMH by releasing leptin from the stomach. Furthermore, it is known that leptin not only induces neuronal activity in the DMH but also in the ARC and in the VMH (Elmquist et al., 1997; Elias et al., 2000). However, in the present study we could not detect an

elevated level of Fos expression neither in the ARC nor the VMH. Therefore, it is unlikely that the increase of Fos expression in the DMH that we observed does result from an effect of CCK on leptin release. DMH cell bodies contain α-melanocyte-stimulating hormone and several other neuropeptides including CRF. Microinjection of CRF into the PVN or into the lateral brain ventricle reduces food intake (Krahn et al., 1988; Kochavi et al., 2001). Furthermore, peripheral injection of CCK activates CRF neurons in the PVN (Verbalis et al., 1991). In the present study, ip CCK induced a 6-fold increase in Fos expression in the PVN compared to vehicle injection consistent with our previous study (Kobelt et al., 2005). Parkes et al. found that intracerebroventricular administration of CRF triggers the synthesis of CRF mRNA and Fos mRNA in the PVN (Parkes et al., 1993) suggesting a positive CRF-CRF auto feedback system in the PVN supporting the positive CRF-CRF feedback as part of the stress response (Ono et al., 1985). Retrograde tracing identified CRF-positive neurons within the DMH projecting to the PVN (Champagne et al., 1998). In the present study, we found that ∼ 27% of neurons, activated by peripheral CCK-8S mainly localized within the ventral part of the DMH are CRFpositive. Therefore, it can be speculated that CRF neurons in the DMH project to the PVN where they can exert an excitatory influence on the positive CRF-CRF feedback circuit which in turn has inhibitory effects on food intake. In this context, it is of interest that microinjection of norepinephrine into the DMH stimulates corticosteroid secretion in rats (Leibowitz et al., 1989) indicating that catecholaminergic fibers may play a role. A recent study by Kawaguchi et al. reported that the majority of the CRF neurons in the DMH are localized in the

Fig. 3 – Double staining of Fos-positive neurons with anti-tyrosine-hydroxylase in the DMH. Overview of double staining with anti-tyrosine-hydroxylase (TH) and anti-Fos in the DMH (A). Double staining revealed that Fos-ir-positive neurons (green staining) were embedded in a network of TH-ir-positive fibers (red staining) mainly localized in the ventral part of the DMH (B). Confocal analysis by laser scanning microscope (y-projection of a z-stack) demonstrated that Fos-positive neurons were clearly encircled by TH-ir-positive fibers (arrow) (C). The white scale bar represents 50 μm in panel A, 10 μm in panel B and 5 μm in panel C. DMHV = dorsomedial hypothalamic nucleus, ventral part, 3V = third ventricle.

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dorsomedial part of the DMH (Kawaguchi et al., 2005). Interestingly, in the present study, we found that CRF neurons are also localized in the ventral part of the DMH. However, it has been observed in a tracing study using the anterograde marker Phaseolus vulgaris leucoagglutinin that all subnuclei of the DMH send projections to the PVN (Mihaly et al., 2001). As PVN neurons also receive direct excitatory input by noradrenergic neurons from the NTS (Sawchenko and Swanson, 1981), this may explain the 6-fold versus 2-fold increase of Fospositive neurons induced by ip CCK in the PVN versus DMH. In this context, it is of interest that changes in GI function, e.g., gastric emptying, are neither necessary nor sufficient for CCKinduced satiety (reviewed in Moran and Kinzig, 2004). Recently, it has been reported that expression of CRF mRNA in the DMH was upregulated in rats during running wheel activity (Bi et al., 2004; Kawaguchi et al., 2005). Interestingly, under this experimental condition no change in the expression pattern of CRF was observed in the PVN. The authors speculated that the rise in CRF expression occurring selectively in the DMH could lead to the inhibitory effect of running wheel exercising on feeding behavior (Bi et al., 2004; Kawaguchi et al., 2005). Taken together, these findings support a role for CRF in the DMH in the regulatory control of feeding behavior. In summary, we demonstrated that peripheral CCK-8S induces neuronal activation in the DMH, as assessed by changes in Fos expression 90 min post injection. These activated neurons were encircled by a network of TH-irpositive fibers. Further, ~27% of the DMH neurons activated by peripheral CCK-8S are CRF-positive. These results suggest that activation of CRF-positive neurons in the DMH may play role in CCK-induced inhibition of food intake.

4.

Experimental procedures

4.1.

Animals

Male Sprague-Dawley rats (Harlan-Winkelmann Co., Borchen, Germany) weighing 300–350 g were housed in groups of 4 rats/cage under conditions of controlled illumination (12:12-h light/dark cycle, lights on/off: 6:30 a.m./6:30 p.m.), humidity, and temperature (22 ± 2 °C). Animals were fed with a standard rat diet (Altromin®, Lage, Germany) and tap water ad libitum. All animals were trained daily to accustom them to the experimental conditions for 14 days before starting the experiments. During the handling phase, the back position was practiced to make animals familiar with receiving an intraperitoneal (ip) injection. Animal care and experimental procedures followed institutional ethic guidelines and conformed to the requirements of the state authority for animal research conduct.

4.2.

Peptide and treatment

CCK-8S (Bachem AG, Heidelberg, Germany) was dissolved in distilled water and stored at −20 °C. Immediately before starting the experiments, peptides were diluted in vehicle solution consisting of sterile 0.15 M NaCl (Braun, Melsungen, Germany) to reach the final concentration of 2 μg/kg

(1.74 nmol/kg). Peptide solutions were kept on ice for the duration of the experiments. The dose of CCK-8S was selected based on a previous studies (Zittel et al., 1999; Kobelt et al., 2005). Freely fed rats were injected ip (final volume 0.5 ml) with vehicle (0.15 M NaCl, n = 6) or with CCK-8S (2 μg/kg body weight, n = 6) at the end of the night phase under very short and weak illumination (dim light source through an opened door) in order to minimize light induced neuronal activity in the DMH. Experiments were performed at the end of the dark phase to avoid confounding factors linked with the onset of the orexigenic systems activated as at the beginning of the night phase and after fasting that also can induce stressrelated Fos expression in the PVN (Cano et al., 2003). Immediately after injection, animals had ad libitum access to food and water for 90 min then brain was processed for Fos immunohistochemistry. The selection of 90-min period post injection to monitor Fos expression is based on a number of time course establishing peak response of Fos within 60– 120 min post stimuli (Hughes and Dragunow, 1995) and our previous studies showing that increased Fos-ir after peripheral CCK-8S injection was only detectable at 60 min and reached its peak at 120 min in 4 brain nuclei (PVN, LC, NTS, AP) (Mönnikes et al., 1997a). At 90 min after the ip injection, animals were deeply anesthetized with ip injections of 100 mg/kg ketamine (Ketanest®, Curamed, Karlsruhe, Germany) and 10 mg/kg xylazine (Rompun® 2%, Bayer, Leverkusen, Germany) and heparinized with 2.500 U heparin ip (Liquemin®, Hoffmann-La Roche, Grenzach-Whylen, Germany). Transcardial perfusion, performed as described before (Kobelt et al., 2004), consisted of a 10-s flush of a plasma substitute (Longasteril® 70; Fresenius, Bad Homburg, Germany) followed by a mixture of 4% w/v paraformaldehyde, 0.05% v/v glutaraldehyde, and 0.2% v/v picric acid in 0.1 M phosphate buffer, pH 7.4, for 30 min and ended with a 5% w/v sucrose solution for 5 min. After dissection, brains were kept in a 5% w/v sucrose solution overnight and then cut into 1.0 to 4.5 mm coronal blocks enclosing the respective hypothalamic regions using a plexiglas brain matrix. For cryoprotection, blocks were moved through a sucrose gradient (15% w/v and 27.3% w/v), then shock-frozen in hexane at −70 °C, and stored at −80 °C until further processing.

4.3.

Immunohistochemistry

4.3.1.

Staining for Fos immunoreactivity (Fos-ir)

The method was essentially as we previously described (Kobelt et al., 2004). First, 25-μm free-floating brain sections were pretreated with 1% w/v sodium borohydride in phosphate-buffered saline (PBS) for 15 min. Subsequently, sections were incubated in a solution containing 5% w/v bovine serum albumin (BSA; Sigma, St. Louis, USA) and 0.3% v/v Triton X-100 in PBS for 60 min for blockade of unspecific antibody binding. Thereafter, the diluted primary antibody solution (rabbit antirat c-Fos, Oncogene Research Products, Boston, USA; 1:4000 in a solution of 5% w/v BSA, 0.3% v/v Triton X-100, and 0.1% w/v sodium azide in PBS) was applied for 42 h at room temperature. Sections were rinsed in PBS three times and incubated in a solution containing 5% w/v BSA and 0.3% v/v Triton X-100 in

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PBS for 60 min, then FITC-labeled goat–anti-rabbit IgG (Sigma) was applied for 12 h at room temperature in an appropriate dilution (1:800 in 5% w/v BSA in PBS). Sections were rinsed in PBS three times and stained with propidium iodide (2.5 μg/ml in PBS; Sigma) for 15 min to counterstain cell chromatin. Tissue sections were finally embedded in 15 μl anti-fading solution (100 mg/ml 1,4-diazabicyclo [2.2.2] octane (Sigma) in 90% v/v glycerin, 10% v/v PBS, pH 7.4), and analyzed using a confocal laser scanning microscope (cLSM 510 Meta, Carl Zeiss, Germany).

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then embedded in anti-fading solution and analyzed using a confocal laser scanning microscope.

4.3.4.

Controls for TH and CRF immunostaining

The following control solutions were used for TH and CRF immunostaining: (1) a solution without the primary antibody, (2) a solution without any antibody, and (3) a solution without the secondary antibody. Compared with the normal solutions, no TH fibers or CRF-positive neurons were detected (data not shown).

4.3.2. Double staining for Fos and CRF immunoreactivity (CRF-ir)

4.4.

Data and statistical analysis

Free-floating brain sections (25 μm) were pretreated with a 1% w/v sodium borohydride solution for 15 min. Subsequently, sections were incubated in a solution containing 5% w/v normal goat serum (NGS; Jackson ImmunoResearch Laboratories Inc., Pennsylvania, USA) and 0.3% v/v Triton X-100 in PBS for 60 min for blockade of unspecific antibody binding. Afterwards, the diluted primary antibody solution (rabbit anti-c-Fos; 1:4000 and guinea pig anti-CRF; Biotrend, Köln, Germany; 1:200 in a solution of 5% w/v NGS, and 0.1% v/v sodium azide in PBS) was applied for 42 h at room temperature. After rinsing the sections in PBS three times and incubation in a solution containing 5% w/v NGS and 0.1% v/v sodium azide in PBS for 2 h, sections were incubated with FITC-labeled goat–anti-rabbit IgG (Sigma) for 12 h at room temperature (1:600 in 5% w/v NGS in PBS). Sections were rinsed again three times in PBS and incubated in a solution containing 5% w/v BSA, 3% w/v rabbit normal serum (Sigma) and 0.1% v/v sodium azide in PBS for 5 hours. Then, the secondary antibody rabbit– anti-guinea pig IgG labeled with TRITC (Sigma) was applied for 12 h at room temperature (1:200 in 5% w/v BSA, 3% w/v rabbit normal serum, and 0.1% v/v sodium azide in PBS). Sections were rinsed in PBS three times and stained with DAPI (2 μg 4′6-diamidino-2-phenylindole/ml in PBS) for 10 min to counterstain cell chromatin. Sections were rinsed in PBS three times again, then embedded in anti-fading solution and analyzed using a confocal laser scanning microscope.

Semi-quantitative assessment of Fos-immunoreactivity (Fosir) was achieved by counting the number of Fos-ir-positive cells as described before (Kobelt et al., 2005). Neurons with green nuclear staining were considered Fos-ir-positive. Every second of all consecutive coronal 25-μm sections was counted bilaterally for Fos-ir-positive staining in the ARC, PVN, VMN, and DMH throughout their rostrocaudal extent. Fos-positive cells were counted in 10 sections per rat of the PVN, and 20 sections per rat of the ARC, VMH and DMH. Anatomic correlations were made according to landmarks given in Paxinos and Watson's stereotaxic atlas (Paxinos and Watson, 1997). The investigator counting the number of Fos-ir-positive cells was blinded to treatments received by the animals. The average number of Fos-ir-positive cells per section for the brain nuclei mentioned above was calculated for each rat. Semi-quantitative assessment of Fos-ir and CRF-ir double staining was achieved by counting the total number of Fos-positive and doubled labeled Fos and CRF neurons in the DMH. Thereafter, the percentage of CRF-positive Fos neurons in the CCK-8S-treated group (n = 3) was calculated. All data are expressed as means ± SEM and analyzed by two-way ANOVA. Differences between groups were evaluated by the Turkey's post hoc test with p < 0.05 was considered significant.

Acknowledgments 4.3.3. Double staining for Fos and tyrosine-hydroxylase immunoreactivity (TH-ir) Free-floating sections (25 μm) were pretreated with a 1% w/v sodium borohydride solution for 15 min. Subsequently, sections were incubated in a solution containing 5% w/v BSA and 0.3% v/v Triton X-100 in PBS for 60 min for blockade of unspecific antibody binding. Afterwards, the diluted primary antibody solution (rabbit anti-c-Fos; 1:4000 and mouse antityrosine-hydroxylase (Clone TA-16); Sigma 1: 6000 in a solution of 5% w/v BSA and 0.1% v/v sodium azide in PBS) was applied for 42 h at room temperature. After rinsing the sections in PBS three times and incubating them in a solution containing 5% w/v NGS and 0.1% v/v sodium azide in PBS for 2 h, FITC-labeled goat–anti-rabbit IgG (Sigma) and TRITC-labeled goat–anti-mouse IgG (Sigma) was applied for 12 h at room temperature (1:400 and 1:200 in 5% w/ v NGS in PBS). Sections were rinsed in PBS three times and stained with DAPI (2 μg/ml in PBS) for 10 min to counterstain cell chromatin. Sections were rinsed in PBS three times again,

We are grateful to Christa Josties for her excellent technical support. This work was supported by a grant from the German Research Foundation (DFG) to H.M. (DFG: Mö 458/4-3), and grants to H.M. (Charité: UFF 2006-251), and Y.T. (Research Career Scientist Award, Department of Veterans Affairs and NIHDK R01 33061).

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