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Lesion of the lateral parabrachial nucleus attenuates the anorectic effect of peripheral amylin and CCK
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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
Lesion of the lateral parabrachial nucleus attenuates the anorectic effect of peripheral amylin and CCK Csilla Becskei a,⁎, Valérie Grabler a , Gaylen L. Edwards b , Thomas Riediger a , Thomas A. Lutz a a
Institute of Veterinary Physiology and Centre of Integrative Human Physiology, University of Zurich, 8057 Zurich, Switzerland Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Amylin and CCK activate the area postrema (AP)/nucleus of the solitary tract (NTS) – lateral
Accepted 7 June 2007
parabrachial nucleus (LPBN) – central amygdala (CeA) pathway. However, except for the
Available online 16 June 2007
brainstem structures the role of these nuclei for the anorectic effect of these peptides is not yet well characterized. The current study investigated the role of the LPBN in mediating the
Keywords:
inhibitory effect of peripheral amylin and CCK on feeding behavior. Rats with electrolytic
c-Fos
lesions in the LPBN (LPBN-X) were used in behavioral as well as in immunohistological c-Fos
Food intake
studies. LPBN-X significantly reduced the anorectic effect of amylin (5 μg/kg, i.p.). The effect
Immunohistochemistry
of a higher amylin dose (10 μg/kg, i.p.) was only slightly attenuated in the LPBN-X rats. In
Area postrema
agreement with previous studies, LPBN lesions also reduced the inhibitory effect of CCK on
Central amygdala
food intake. In the immunohistological experiments, amylin and CCK induced c-Fos expression in the AP, NTS, LPBN and CeA in the SHAM rats. Both the amylin- and CCKinduced activation of the CeA was completely abolished in the animals with a LPBN lesion. These results clearly suggest that the signal transduction pathway between the AP/NTS and CeA has been disrupted by the LPBN ablation. We conclude that the LPBN is a crucial brain site mediating the anorectic effect of amylin and CCK. Furthermore, an intact LPBN seems to be essential for the c-Fos response in the CeA induced by these peptides. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
A well-established neuronal axis regulating ingestive behavior comprises the area postrema (AP), the nucleus of the solitary tract (NTS), the lateral parabrachial nucleus (LPBN) and the central nucleus of the amygdala (CeA). Based on functional cFos studies, this pathway is hypothesized to mediate the satiating effect of the pancreatic hormone amylin (Rowland and Richmond, 1999; Rowland et al., 1997; Riediger et al., 2004). Cholecystokinin (CCK), which is released prandially from the endocrine cells of the small intestine, activates a similar central pathway when administered peripherally (Day et al.,
1994; Li and Rowland, 1994, 1995; Mönnikes et al., 1997; Mayne et al., 1998). Despite the similarities, there is a fundamental difference between the action of amylin and CCK at the level of their primary target sites. While the AP in the brainstem is believed to function as the sensory interface for circulating amylin (Lutz et al., 1998b, 2001; Rowland and Richmond, 1999; Riediger et al., 2004), CCK at least in rats seems to act mainly in a paracrine manner on gastrointestinal vagal afferents to reduce food intake (for review see e.g. Ritter et al., 1999). The vagal afferents make their initial synapses in the dorsal vagal complex of the brainstem, involving mainly the NTS but also
⁎ Corresponding author. Institute of Veterinary Physiology, University of Zurich, Wintethurerstrasse 260, 8057 Zurich, Switzerland. Fax: +41 44 635 8932. E-mail address: [email protected] (C. Becskei). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.06.016
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the AP (Shapiro and Miselis, 1985a,b). Thus in the ascending pathway of CCK-induced satiation, the NTS represents the primary relay station in the brain. CCK seems to modulate AP activity indirectly, through vagal inputs and through the NTS (for review see Ritter et al., 1999). The facts that vagotomy abolishes the CCK-induced c-Fos expression in the brain (Day et al., 1994; Li and Rowland, 1995) and that a specific AP lesion does not block the anorectic effect of CCK (Edwards et al., 1986) strongly support that notion. On the other hand, the anorectic effect of amylin seems to be independent of vagal or other sensory abdominal inputs (Morley et al., 1994; Lutz et al., 1994, 1995, 1998a). Hence, the amylin-induced activation of the NTS is believed to be secondary, due to neuronal inputs from the AP (Riediger et al., 2001, 2004). Several studies have shown that both AP and NTS neurons project to the LPBN (Norgen, 1978; Voshart and Van Der Kooy, 1981; Hermann and Rogers, 1985; Shapiro and Miselis, 1985a, b), and the latter to the CeA (Block et al., 1989). Since peripheral amylin and CCK both activate the LPBN and CeA (Day et al., 1994; Li and Rowland, 1994, 1995; Rowland and Richmond, 1999; Rowland et al., 1997; Mayne et al., 1998; Riediger et al., 2004) it seems plausible that these nuclei are linearly connected forming an AP/NTS–LPBN–CeA neuronal axis that processes the amylin and CCK signal. However, the role of the nuclei downstream from the AP/NTS region in the feeding-related effects of amylin and CCK is not clear. Concerning CCK, Trifunovic and Reilly (2001, 2003) have shown that the effect of LPBN ablation (LPBN-X) on CCKinduced satiation depends on the experimental conditions, most importantly on the texture of the food. In these studies, LPBN-X blocked the effect of CCK only when solid rodent chow was presented to the rats, but not when a liquid milk diet was offered. Studies investigating the role of the LPBN in the anorectic effect of amylin are missing. Therefore, the aim of our study was first, to investigate the role of the LPBN in mediating the anorectic effect of amylin. Second, we wanted to test whether LPBN-X blocks the satiating effect of CCK under our experimental conditions, i.e. when rats are fed powdered medium-fat diet. Furthermore, we hypothesized that the flow of afferent information is directed from the AP/NTS region via the LPBN towards the CeA. Thus, if the LPBN as the intermediate relay station is eliminated from this pathway through ablation, the amylin- and CCK-induced neuronal activation would not be transmitted to the CeA. This should result in an attenuated c-Fos expression in the CeA in amylin- or CCK-treated LPBN-lesioned animals. Further, assuming that the neuronal activation in the AP/NTS-LPBNCeA pathway correlates with the anorectic action of amylin and CCK, the disruption of this pathway should also reduce the anorectic effect of these peptides. To test these hypotheses, we conducted feeding and c-Fos immunohistochemical studies with LPBN-lesioned and sham-operated (SHAM) rats, injected peripherally with amylin or CCK.
2.
Results
Based on the histochemical verification of the lesions, fifteen rats had a complete ablation of that part of the LPBN that is activated by peripheral amylin or CCK in intact rats. This area
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comprises the external, the central and the dorsal subnuclei (Mayne et al., 1998). Fig. 1 shows the area of common damage at different levels in the brain. All SHAM rats (n = 26) had an intact LPBN.
2.1.
Feeding studies
There was no difference in food intake after 24 h fasting between the LPBN-X and SHAM groups after saline injection. Lesioning the LPBN significantly attenuated the anorectic effect of a low dose of amylin (5 μg/kg) 1 h post-injection (twoway ANOVA, lesion × treatment interaction: F(1,78) = 5.4, p b 0.05). This attenuation was not significant after 2 h. Overall, amylin reduced food intake at both time points (two-way
Fig. 1 – The areas of common damage in the LPBN of the lesioned rats at different Bregma levels of coronal brain sections. The different patterns of shading represent the number of animals in which the corresponding area was damaged as follows: horizontal lines: 1–3, right hatched area: 4–6, left hatched area 7–9, light gray area: 10–12, dark gray area 13–15 animals. Modified images reprinted from The Rat Brain in Stereotaxic Coordinates, Paxinos, G. and Watson, C., Copyright Paxinos, G. and Watson, C. (1998), with permission from Elsevier.
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ANOVA, main effect of injection: F(1,78) = 23.4, p b 0.001 at 1 h and F(1,78) = 8.2, p b 0.01 at 2 h). However, the post hoc test revealed that amylin significantly reduced feeding in the SHAM (p b 0.001 and p b 0.01, 1 and 2 h after injection, respectively) but not in the LPBN-X rats (Fig. 2A). There was a main effect of lesion only 1 h after injection (two-way ANOVA: F(1,78) = 8.8, p b 0.01). Injection of a higher dose of amylin (10 μg/kg) reduced food intake at both time points (two-way ANOVA: F(1,76) = 19.8, p b 0.001 at 1 h and F(1,76) = 11.4, p b 0.01 at 2 h). The post hoc test revealed that food intake was reduced in both the LPBN-X (p b 0.05) and SHAM rats (p b 0.001) 1 h after amylin injection (Fig. 2B). However, at the 2-h time point, this effect was only significant in the SHAM group (p b 0.05). In the twoway ANOVA, the main effect of lesion and the lesion × treatment interaction did not reach significance at this dose of amylin. LPBN-X significantly reduced the anorectic effect of CCK (3 μg/kg) 1 h after injection (two-way ANOVA, lesion × treatment interaction: F(1,78) = 7.1, p b 0.01). This effect just failed to reach significance at the 0.5 h time point (F(1,78) = 3.7, p = 0.058). Overall, two-way ANOVA revealed a main effect of treatment at both time points (F(1,78) = 81.8, p b 0.001 at 0.5 h and F(1,78) = 26.1, p b 0.001 at 1 h). However, the post hoc test
Fig. 3 – Cumulative food intake in rats with lesion in the lateral parabrachial nucleus (LPBN-X) or with sham lesion (SHAM) 0.5 and 1 h after i.p. CCK administration. (A) CCK 3 μg/kg; n = 15 for LPBN-X and n = 26 for SHAM; (B) CCK 5 μg/kg; n = 6 for LPBN-X and n = 12 for SHAM. Lesioning the LPBN reduced the anorectic effect of CCK (3 μg/kg) 1 h post-injection (two-way ANOVA; p < 0.01). **p < 0.01; ***p < 0.001 indicate significant differences between CCK and saline within SHAM and LPBN-X groups (Holm–Sidak posthoc test).
showed that CCK reduced food intake in both groups 0.5 h after injection (p b 0.001) but only in the SHAM (p b 0.001) rats at 1 h (Fig. 3A). There was no main effect of lesion on food intake (two-way ANOVA). A higher dose of CCK (5 μg/kg) reduced food intake at both time points (two-way ANOVA: F(1,32) = 59.2, p b 0.001 at 0.5 h and F(1,32) = 12.4, p = 0.001 at 1 h). Post hoc test revealed that this CCK dose reduced food intake in both groups 0.5 h following injection (p b 0.001), but only in the SHAM rats (p b 0.01) after 1 h (Fig. 3B). In the two-way ANOVA, the lesion × treatment interaction did not reach significance at any time point and there was a main effect of lesion only 0.5 h after injection (F (1,32) = 11.4, p b 0.01). Fig. 2 – Cumulative food intake in rats with lesion in the lateral parabrachial nucleus (LPBN-X; n = 15) or with sham lesion (SHAM; n = 26) 1 and 2 h after i.p. amylin (A), 5 μg/kg (B) 10 μg/kg injection. Lesioning the LPBN reduced the anorectic effect of amylin (5 μg/kg) 1 h post-injection (two-way ANOVA; p < 0.01). *p < 0.05; **p < 0.01; ***p < 0.001 indicate significant differences between amylin and saline within SHAM and LPBN-X groups (Holm–Sidak post-hoc test).
2.2.
Immunohistochemistry
Amylin treatment induced a strong c-Fos expression in the AP of both the LPBN-X and SHAM rats (Fig. 4). There was no difference in the number of c-Fos-positive cells between the two groups (47 ± 3 and 48 ± 3 cells/section respectively; Fig. 5). In the LPBN of the SHAM rats, abundant c-Fos response was detected after amylin administration (Fig. 4). In the LPBN-X rats with
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Fig. 4 – Amylin (20 μg/kg) induces c-Fos expression in the area postrema (AP; upper panel), lateral parabrachial nucleus (LPBN; middle panel) and in the central nucleus of the amygdala (CeA; lower panel) in the SHAM (left column) animals. In the LPBN-X (right column) rats c-Fos expression is visible in the AP, but not in the CeA. Fluorescent-conjugated secondary antibody (Cy3) was used and the photomicrographs were converted to grayscale images. Scale bars: 200 μm.
complete lesion, the c-Fos-expressing subnuclei were destroyed. A representative photomicrograph of a complete LPBN lesion is shown in Fig. 4. While in the CeA the SHAM animals displayed a strong c-Fos expression after amylin injection (39 ± 7 cells/section), hardly any c-Fos-positive cells could be detected in the LPBN-X rats (5 ± 2 cells/section; Figs. 4 and 5). CCK administration induced a moderate c-Fos expression in the AP of both the lesioned and the SHAM group (Fig. 6).
Quantification of the c-Fos-positive cells showed no significant difference between the two groups (17 ± 2 and 8 ± 1 cells/section, respectively; Fig. 5). Similar to amylin, a strong c-Fos signal was detected in the LPBN and the CeA (23 ± 5 cells/section; Fig. 6) of the SHAM rats after CCK injection. However, CCK-induced neuronal activation in the CeA was blocked in rats with LPBN-X (3 ± 2 cells/section; Figs. 5 and 6).
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Fig. 5 – Quantification of Fos ir cells in the area postrema (AP) and in the central amygdala (CeA) after s.c. application of 20 μg/kg amylin (upper row), 20 μg/kg CCK (middle row) or saline (bottom row). ***p < 0.001, *p < 0.05 compared to the SHAM group.
In the saline-treated groups, very low numbers of c-Fos-ir cells were present in any of the investigated nuclei (Fig. 5).
3.
Discussion
The role of the LPBN in mediating the anorectic action of the satiating peptides amylin and CCK was investigated in LPBNlesioned rats. Ablation of the LPBN reduced the anorectic effect of amylin and CCK and blocked the induction of c-Fos expression in the CeA by both peptides. Lesioning the LPBN significantly reduced the anorectic effect of amylin at a dose of 5 μg/kg, but had no apparent effect at a higher amylin dose (10 μg/kg). The LPBN thus seems to be essential in mediating the anorectic effect of exogenous peripheral amylin at least when low, near-physiological doses are used. It is plausible that a high amylin dose activates neurons in the AP/NTS region which do not project via the LPBN to the higher brain centers. This might account for the remaining anorectic effect of a higher dose of amylin in the lesioned animals. In our previous studies (Lutz et al., 2001), ablation of the AP completely blocked the anorectic action of a
low amylin dose (5 μg/kg) but only attenuated that of a higher dose (50 μg/kg). Based upon these results, it cannot be excluded that higher doses of amylin might additionally activate an alternative pathway, different from the AP– LPBN–CeA axis. Autoradiographic studies have shown abundant amylin binding sites in several forebrain structures involved in the regulation of food intake. These nuclei, in addition to the presumed primary target site, the AP, might be potential targets for the anorectic action of peripheral amylin (Van Rossum et al., 1994; Sexton et al., 1994). A direct effect of amylin on these brain nuclei may also be possible, because amylin can cross the blood–brain barrier by a saturable transport mechanism (Banks et al., 1995). It has to be noted that in spite of the thorough anatomical and functional (c-Fos) evaluation, the possibility that a few neurons and fibers were left intact in the AP-X (former study, Lutz et al., 2001) or LPBN-X (present study) animals cannot be excluded. In the immunohistological study, the SHAM animals displayed a similar c-Fos-ir pattern in the AP, LPBN and the CeA after amylin application as has been reported previously (Rowland and Richmond, 1999; Rowland et al., 1997; Riediger et al., 2004). All SHAM and LPBN-X rats treated with amylin
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Fig. 6 – CCK (20 μg/kg) induces c-Fos expression in the area postrema (AP; upper panel), lateral parabrachial nucleus (LPBN; middle panel) and in the central nucleus of the amygdala (CeA; lower panel) in the SHAM (left column) animals. In the LPBN-X (right column) rats c-Fos expression is visible in the AP, but not in the CeA. Immunoperoxidase method was used. Scale bars: 200 μm.
showed a similar level of activation in the AP, suggesting that the amylin-induced c-Fos expression in this area does not depend on the integrity of the LPBN. The activation of the AP is specific to amylin because no significant c-Fos-ir was detected in the AP of the saline-treated control group. In the LPBN-X rats, c-Fos expression was abolished in the CeA after amylin administration, indicating a crucial role for the LPBN in transmitting neuronal signals to the CeA. Previous studies have shown that the ablation of the AP/NTS region
blocks the amylin-induced c-Fos expression in the LPBN and CeA (Rowland and Richmond, 1999; Riediger et al., 2004). This suggests that the activation of both the LPBN and CeA relies on ascending inputs from the AP/NTS region. These results together with the current observations support the hypothesis that the nuclei which are activated by amylin – AP–NTS–LPBN– CeA – constitute a linearly connected pathway. We recently demonstrated that peripheral amylin injection, similar to refeeding, reversed the fasting-induced c-Fos
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expression in the lateral hypothalamic area (LHA) (Riediger et al., 2004). Furthermore, peripheral amylin has been reported to reduce the mRNA expression of the orexigenic neuropeptide orexin in the LHA (Barth et al., 2003). Nevertheless, a direct effect of amylin on the LHA is not likely, because no amylin binding has been reported in this nucleus (Sexton et al., 1994). On the other hand, the NTS (Ricardo and Koh, 1978), the LPBN (Bester et al., 1997) and the CeA (Berk and Finkelstein, 1981) send neuronal projections to the LHA. It is plausible that the direct brainstem projections, which circumvent the LPBN, convey at least some of the anorectic amylin signal to the LHA and might be responsible for the maintained anorectic effect of high amylin doses. However, the involvement of the LHA in the feeding effect of amylin is still to be confirmed. The results of the feeding studies confirmed the involvement of the LPBN in mediating CCK-induced satiation. The anorectic effect of CCK was significantly attenuated in the LPBN-X animals after 30 min and it was completely blocked after 60 min of feeding, when near-physiological doses were used. The latter finding is in good concert with the results of Trifunovic and Reilly (2001), who demonstrated a complete blockade of the inhibitory effect of CCK (4 and 8 μg/kg, i.p.) on 1 h cumulative food intake in rats with ibotenic acid-induced LPBN lesions when fed pelleted chow. However, the same group also reported that the anorectic action of CCK (2 and 8 μg/kg, i.p.) was unchanged in lesioned animals presented a palatable milk diet (Trifunovic and Reilly, 2003). The authors speculate that the texture or palatability of the food might influence the effect of the lesion on CCK's inhibitory action on feeding (Trifunovic and Reilly, 2003). Nevertheless, it is also plausible that the nutrient composition of the diet might have affected the CCK action, which likely differed between the studies. In our study, the ablation of the LPBN attenuated the anorectic effect of a higher dose of CCK (5 μg/kg), implying that there may be other brain nuclei besides the LPBN that play a role in processing CCK-induced satiation. There is accumulating evidence that a potential hypothalamic candidate to mediate the anorectic effect of CCK could be the paraventricular nucleus of the hypothalamus (PVN; Crawley and Kiss, 1985; Day et al., 1994; Rinaman et al., 1995). The NTS, which is the main terminal field of the vagal afferents conveying the peripheral CCK signal, has been shown to have direct projections to this nucleus (Rinaman et al., 1995). This might also provide an explanation for the partial persistence of the anorectic potency of CCK in our LPBN-X rats. It has to be noted that in contrast to amylin, CCK is unable to cross the blood– brain barrier (Passaro et al., 1982). Thus, a direct effect of peripheral CCK on brain nuclei with a tight blood–brain barrier, like the PVN, seems unlikely. Similar to amylin, the c-Fos expression was abolished in the CeA of the LPBN-X rats after CCK administration. Thus the LPBN seems to play an essential role in mediating the CCKinduced neuronal activation to the CeA. These results strongly support the notion that, similar to amylin, there is a linear pathway involving the AP/NTS–LPBN–CeA nuclei that is activated by peripheral CCK. Taken together, our results suggest that a functionally intact LPBN is important in the amylin- and CCK-induced satiation. Our immunohistological studies provide evidence
that the effect of amylin and CCK on the neuronal activation of the CeA relies completely on ascending information from the LPBN. However, whether the reduced anorectic effect of amylin and CCK in the LPBN-X animals is due to the lack of activation in the CeA and whether the CeA plays a role in the feeding effects of these peptides requires further studies.
4.
Experimental procedure
4.1.
Animals
Adult male Wistar rats housed in individual cages in a temperature-controlled room (22 °C) were used. They had free access to water and were kept under a 12-h light/dark cycle. All animal procedures were approved by the Veterinary Office of the Canton of Zurich's Health Directorate. At least for a week before the surgery and during the whole period of recovery phase after surgery, the animals were handled daily. Prior to the feeding studies, animals were injected with saline daily for 2–3 days to adapt them to the experimental procedure.
4.2.
Electrolytic LPBN lesions
Rats (n = 56) weighing 290–350 g were randomly allocated into two groups, one to be electrically lesioned in the lateral parabrachial nucleus (n = 30) and one of sham-operated controls (n = 26). The procedure was adapted according to Edwards and Ritter (1989). Briefly, after anaesthetizing the rats with ketamine (60 mg/kg) and xylazine (7 mg/kg), clipping and disinfecting the top of the head, the animals were fixed in a stereotaxic apparatus. Access to the skull was gained by a 2- to 3-cm midline skin incision from the level of the orbita to approximately 1 cm caudally from the occipital crest. The periosteum was detached and retracted laterally. A rectangular piece of the skull was removed above the area of the LPBN (coordinates: 9.4 mm caudal, and bilaterally 1.9 mm lateral from Bregma; Paxinos and Watson, 1998). After pulling the transverse sinus slightly aside, the dura mater was incised. An insect pin served as the electrode and was attached to the anode of the electrolytic lesion-making device (Ugo Basile, Italy). The pin was lowered 6.5 mm ventrally from dura and an anodal current (0.5 mA for 10 s) was passed on each side to produce the electrolytic lesions. Sham lesions were made by the same procedure but without passing the current. After surgery, the rats were allowed to recover for at least 2 weeks. The histological verification of the LPBN-X was combined with the immunohistochemical studies (see below). Only animals with complete bilateral lesions of the LPBN were admitted to the statistical evaluation (n = 15). All 26 SHAM animals were included in the analysis.
4.3.
Feeding studies
The feeding trials were performed using a crossover design so that each animal served as its own control. All the animals were fed a powdered medium-fat diet ad libitum (18% fat and 16.5 kJ/g; Lutz et al., 1994). Both the lesioned and the SHAM rats
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were divided into two groups. At dark onset, following 24-h food deprivation, one group received an intraperitoneal (i.p.) injection of amylin (5 μg/kg or 10 μg/kg). The second group was injected with saline (1 ml/kg). Immediately after injection, the rats were given access to food. Cumulative food intake, corrected for spillage, was measured manually after 1 and 2 h. The same experimental protocol was used to investigate the anorectic effect of CCK (3 μg/kg, i.p.). Food intake measurements were performed 30 and 60 min after treatment, because the effect of CCK on food intake lasts generally shorter than that of amylin. Half of the animals were also tested with a higher dose of CCK (5 μg/kg, i.p.; n = 6 for LPBN-X and n = 12 for SHAM).
4.4.
Immunohistochemistry
In the immunohistochemical studies the same LPBN-X and SHAM rats were used as in the feeding trials. These studies served as a histological and functional verification of the LPBN lesions as well. The ad libitum fed animals received an injection of peptide (20 μg/kg amylin or CCK) or vehicle (saline) at dark onset. The substances were applied subcutaneously (s.c.), because in previous studies we found that the variation in hormone-induced c-Fos expression is lower after s.c. application compared to i.p. application. Moreover, a 20 μg/kg s.c. dose of amylin produced a similar c-Fos expression as an i.p. injection of 5 μg/kg in the rat AP (approx. 45 cells/section; see Riediger et al., 2001 and current results). 2 h after injection, the rats were anesthetized with pentobarbital (Nembutal, Abbott Laboratories USA 80 mg/kg, i.p.) and perfused transcardially with ice-cold phosphate buffer (PB 0.1 M), followed by 4% paraformaldehyde (in 0.1 M PB). The brains were removed and kept in paraformaldehyde for an additional 2 h to achieve proper tissue fixation. After 48 h of incubation in 20% sucrose solution (in 0.1 M PB) at 4 °C, the samples were snap frozen in CO2 gas. Coronal sections (20 μm) were cut in a cryostat (CM 3050 Leica, Nussloch, Germany) throughout the AP, PBN and CeA. Every slice was thaw mounted on microscopic glass slides (SuperFrost Plus, Faust, Schaffhausen, Switzerland) and was stored at − 20 °C until further processing. For the detection of c-Fos expression, the brain sections of the amylin-treated animals were processed by an immunofluorescent staining procedure. This involved drying the sections at room temperature for 1 h and rehydrating them in 0.1% PBST (phosphate-buffered saline solution (PBS) + Triton X-100; Sigma). Unspecific binding sites were blocked by 1.5% normal donkey serum. The primary antibody (polyclonal rabbit–anti-c-Fos IgG, Oncogene, Ab-5) was applied for 48 h at 4 °C. The unbound antibody was removed by washing in PBST. The sections were then incubated with the secondary antibody (donkey–anti-rabbit, Cy3 conjugated, Milan Analytica, Switzerland) for 75 min at room temperature. The first and the second antibody were diluted to 1:1000 and 1:225, respectively, in 0.3% PBST. After a final washing in 0.1% PBST, the slides were covered and stored at 4 °C until evaluated using a fluorescent microscope (Axioscop 2, Carl Zeiss AG, Feldbach, Switzerland). Due to a general change of the visualization method in our laboratory from fluorescent to immunoperoxidase method, the avidin–biotin immunoperoxidase method was used to
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visualize c-Fos expression in the CCK- and saline-treated groups. Because the lesioned and SHAM animals are only compared within the same treatment group, the different techniques do not compromise the interpretation of the results. The histological sections were air-dried at room temperature for 1 h and rehydrated in PBS. Thereafter the sections were incubated in PBS containing 0.3% H2O2 for 30 min. Unspecific binding was blocked by a 2 h incubation in 1.5% normal rabbit serum. The primary antibody (polyclonal goat anti-c-Fos, Santa Cruz; 1:10,000) was applied for 48 h at 4 °C. The unbound antibody was removed by washing in PBS and the sections were incubated with the secondary antibody (biotinylated rabbit–anti-goat, Vectastain®-Elite ABC Kit, Vector Laboratories; 1:200) for 2 h at room temperature. After incubation in ABC (Vectastain®-Elite ABC Kit, Vector Laboratories), followed by DAB solution (0.04% in PBS with 0.02% H2O2 and for color enhancement 0.08% NiCl2·6H2O and 0.01% CoCl2·6H2O), the sections were dehydrated in graded alcohols, cleared in xylenes and coverslipped. The localization of the c-Fos-expressing neurons was identified according to the rat brain atlas by Paxinos and Watson (1998). Only animals with successful bilateral lesions of the LPBN were admitted to the quantification of the c-Fos-expressing cells (n = 6 for amylin, n = 4 for CCK and n = 3 for saline tretment). The lesion was considered complete when at immunohistochemistry no intact tissue could be seen in the area corresponding to the central, dorsal and external LPBN. For representative histological photographs of complete lesions see Figs. 4 and 6. In each rat, 30 consecutive corresponding sections of both the CeA and the AP were counted for c-Fos-positive cells. Photomicrographs were taken by a digital camera (AxioCam, Carl Zeiss AG). Two rats with complete lesions and eleven SHAM animals, which were included in the feeding studies, were used in other immunohistological experiments. For this reason, the number of animals in the feeding and immunohistological studies presented here is not identical.
4.5.
Statistics
In the feeding studies the effect of lesion on the anorectic effect of amylin and CCK was analyzed by two-way ANOVA, followed by Holm–Sidak post hoc test. In the immunohistological studies, the cell counts for all the sections in a certain brain area were averaged in each individual animal and used for statistical analyses. The LPBN-X rats were compared with the SHAM animals by t-test within each treatment group (amylin, CCK and saline). P b 0.05 was considered significant. Results are presented as means ± SEM.
Acknowledgments This project was supported by the Swiss National Science Foundation, the Research Committee and Young Academics Support Committee of the University of Zurich and by the Stiftung für wissentschaftliche Forschung of the University of Zurich. Cs.B. is a recipient of a fellowship grant from the Centre of Integrative Human Physiology (University of Zurich).
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REFERENCES
Banks, W.A., Kastin, A.J., Maness, L.M., Huang, W., Jaspan, J.B., 1995. Permeability of the blood–brain barrier to amylin. Life Sci. 57, 1993–2001. Barth, S.W., Riediger, T., Lutz, T.A., Rechkemmer, G., 2003. Differential effects of amylin and salmon calcitonin on neuropeptide gene expression in the lateral hypothalamic area and the arcuate nucleus of the rat. Neurosci. Lett. 341, 131–134. Berk, M.L., Finkelstein, J.A., 1981. Afferent projections to the preoptic area and hypothalamic regions in the rat brain. Neuroscience 6, 1601–1624. Bester, H., Besson, J.M., Bernard, J.F., 1997. Organization of efferent projections from the parabrachial area to the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. J. Comp. Neurol. 383, 245–281. Block, C.H., Hoffman, G., Kapp, B.S., 1989. Peptide-containing pathways from the parabrachial complex to the central nucleus of the amygdala. Peptides 10, 465–471. Crawley, J.N., Kiss, J.Z., 1985. Paraventricular nucleus lesions abolish the inhibition of feeding induced by systemic cholecystokinin. Peptides 6, 927–935. Day, H.E., McKnight, A.T., Poat, J.A., Hughes, J., 1994. Evidence that cholecystokinin induces immediate early gene expression in the brainstem, hypothalamus and amygdala of the rat by a CCKA receptor mechanism. Neuropharmacology 33, 719–727. Edwards, G.L., Ritter, R.C., 1989. Lateral parabrachial lesions attenuate ingestive effects of area postrema lesions. Am. J. Physiol. 256, R306–R312. Edwards, G.L., Ladenheim, E.E., Ritter, R.C., 1986. Dorsomedial hindbrain participation in cholecystokinin-induced satiety. Am. J. Physiol. 251, R971–R977. Hermann, G.E., Rogers, R.C., 1985. Convergence of vagal and gustatory afferent input within the parabrachial nucleus of the rat. J. Auton. Nerv. Syst. 13, 1–17. Li, B.H., Rowland, N.E., 1994. Cholecystokinin and dexfenfluramine-induced anorexia compared using devazepide and c-Fos expression in the rat brain. Regul. Pept. 50, 223–233. Li, B.H., Rowland, N.E., 1995. Effects of vagotomy on cholecystokinin- and dexfenfluramine-induced Fos-like immunoreactivity in the rat brain. Brain Res. Bull. 37, 589–593. Lutz, T.A., Del Prete, E., Scharrer, E., 1994. Reduction of food intake in rats by intraperitoneal injection of low doses of amylin. Physiol. Behav. 55, 891–895. Lutz, T.A., Del Prete, E., Scharrer, E., 1995. Subdiaphragmatic vagotomy does not influence the anorectic effect of amylin. Peptides 16, 457–462. Lutz, T.A., Althaus, J., Rossi, R., Scharrer, E., 1998a. Anorectic effect of amylin is not transmitted by capsaicin-sensitive nerve fibers. Am. J. Physiol. 274, R1777–R1782. Lutz, T.A., Senn, M., Althaus, J., Del Prete, E., Ehrensperger, F., Scharrer, E., 1998b. Lesion of the area postrema/nucleus of the solitary tract (AP/NTS) attenuates the anorectic effects of amylin and calcitonin gene-related peptide (CGRP) in rats. Peptides 19, 309–317. Lutz, T.A., Mollet, A., Rushing, P.A., Riediger, T., Scharrer, E., 2001. The anorectic effect of a chronic peripheral infusion of amylin is abolished in area postrema/nucleus of the solitary tract (AP/ NTS) lesioned rats. Int. J. Obes. Relat. Metab. Disord. 25, 1005–1011. Mayne, R.G., Armstrong, W.E., Crowley, W.R., Bealer, S.L., 1998. Cytoarchitectonic analysis of Fos-immunoreactivity in brainstem neurons following visceral stimuli in conscious rats. J. Neuroend. 10, 839–847.
Mönnikes, H., Lauer, G., Arnold, R., 1997. Peripheral administration of cholecystokinin activates c-Fos expression in the locus coeruleus/subcoeruleus nucleus, dorsal vagal complex and paraventricular nucleus via capsaicin-sensitive vagal afferents and CCK-A receptors in the rat. Brain Res. 770, 277–288. Morley, J.E., Flood, J.F., Horowitz, M., Morley, P.M., Walter, M.J., 1994. Modulation of food intake by peripherally administered amylin. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 267, R178–R184. Norgen, R., 1978. Projections from the nucleus of the solitary tract in the rat. Neuroscience 3, 207–218. Passaro Jr., E., Debas, H., Oldendorf, W., Yamada, T., 1982. Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation. Brain Res. 241, 335–340. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic, New York. Ricardo, J.A., Koh, E.T., 1978. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Brain Res. 153, 1–26. Riediger, T., Schmid, H.A., Lutz, T.A., Simon, E., 2001. Amylin potently activates AP neurons possibly via formation of the excitatory second messenger cGMP. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 281, R1833–R1843. Riediger, T., Zuend, D., Becskei, C., Lutz, T.A., 2004. The anorectic hormone amylin contributes to feeding-related changes of neuronal activity in key structures of the gut–brain axis. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 286, R114–R122. Rinaman, L., Hoffman, G.E., Dohanics, J., Le, W.W., Stricker, E.M., Verbalis, J.G., 1995. Cholecystokinin activates catecholaminergic neurons in the caudal medulla that innervate the paraventricular nucleus of the hypothalamus in rats. J. Comp. Neurol. 360, 246–256. Ritter, R.C., Covasa, M., Matson, C.A., 1999. Cholecystokinin: proofs and prospects for involvement in control of food intake and body weight. Neuropeptides 33, 387–399. Rowland, N.E., Richmond, R.M., 1999. Area postrema and the anorectic actions of dexfenfluramine and amylin. Brain Res. 820, 86–91. Rowland, N.E., Crews, E.C., Gentry, R.M., 1997. Comparison of Fos induced in rat brain by GLP-1 and amylin. Regul. Pept. 71, 171–174. Sexton, P.M., Paxinos, G., Kenney, M.A., Wookey, P.J., Beaumont, K., 1994. In vitro autoradiographic localization of amylin binding sites in rat brain. Neuroscience 62, 553–567. Shapiro, R.E., Miselis, R.R., 1985a. The central neural connections of the area postrema of the rat. J. Comp. Neurol. 234, 344–364. Shapiro, R.E., Miselis, R.R., 1985b. The central organization of the vagus nerve innervating the stomach of the rat. J. Comp. Neurol. 238, 473–488. Trifunovic, R., Reilly, S., 2001. Medial versus lateral parabrachial nucleus lesions in the rat: effects on cholecystokinin- and D-fenfluramine-induced anorexia. Brain Res. 894, 288–296. Trifunovic, R., Reilly, S., 2003. Excitotoxic lesions of the lateral parabrachial nucleus do not prevent cholecystokinin-induced suppression of milk intake in rats. Neurosci. Lett. 348, 109–112. Van Rossum, D., Menard, D.P., Fournier, A., St-Pierre, S., Quirion, R., 1994. Autoradiographic distribution and receptor binding profile of [125I] Bolton Hunter-rat amylin binding sites in the rat brain. J. Pharmacol. Exp. Ther. 270, 779–787. Voshart, K., Van Der Kooy, D., 1981. The organization of the efferent projections of the parabrachial nucleus to the forebrain in the rat: a retrograde fluorescent double-labeling study. Brain Res. 212, 271–286.
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
Lesion of the lateral parabrachial nucleus attenuates the anorectic effect of peripheral amylin and CCK Csilla Becskei a,⁎, Valérie Grabler a , Gaylen L. Edwards b , Thomas Riediger a , Thomas A. Lutz a a
Institute of Veterinary Physiology and Centre of Integrative Human Physiology, University of Zurich, 8057 Zurich, Switzerland Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Amylin and CCK activate the area postrema (AP)/nucleus of the solitary tract (NTS) – lateral
Accepted 7 June 2007
parabrachial nucleus (LPBN) – central amygdala (CeA) pathway. However, except for the
Available online 16 June 2007
brainstem structures the role of these nuclei for the anorectic effect of these peptides is not yet well characterized. The current study investigated the role of the LPBN in mediating the
Keywords:
inhibitory effect of peripheral amylin and CCK on feeding behavior. Rats with electrolytic
c-Fos
lesions in the LPBN (LPBN-X) were used in behavioral as well as in immunohistological c-Fos
Food intake
studies. LPBN-X significantly reduced the anorectic effect of amylin (5 μg/kg, i.p.). The effect
Immunohistochemistry
of a higher amylin dose (10 μg/kg, i.p.) was only slightly attenuated in the LPBN-X rats. In
Area postrema
agreement with previous studies, LPBN lesions also reduced the inhibitory effect of CCK on
Central amygdala
food intake. In the immunohistological experiments, amylin and CCK induced c-Fos expression in the AP, NTS, LPBN and CeA in the SHAM rats. Both the amylin- and CCKinduced activation of the CeA was completely abolished in the animals with a LPBN lesion. These results clearly suggest that the signal transduction pathway between the AP/NTS and CeA has been disrupted by the LPBN ablation. We conclude that the LPBN is a crucial brain site mediating the anorectic effect of amylin and CCK. Furthermore, an intact LPBN seems to be essential for the c-Fos response in the CeA induced by these peptides. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
A well-established neuronal axis regulating ingestive behavior comprises the area postrema (AP), the nucleus of the solitary tract (NTS), the lateral parabrachial nucleus (LPBN) and the central nucleus of the amygdala (CeA). Based on functional cFos studies, this pathway is hypothesized to mediate the satiating effect of the pancreatic hormone amylin (Rowland and Richmond, 1999; Rowland et al., 1997; Riediger et al., 2004). Cholecystokinin (CCK), which is released prandially from the endocrine cells of the small intestine, activates a similar central pathway when administered peripherally (Day et al.,
1994; Li and Rowland, 1994, 1995; Mönnikes et al., 1997; Mayne et al., 1998). Despite the similarities, there is a fundamental difference between the action of amylin and CCK at the level of their primary target sites. While the AP in the brainstem is believed to function as the sensory interface for circulating amylin (Lutz et al., 1998b, 2001; Rowland and Richmond, 1999; Riediger et al., 2004), CCK at least in rats seems to act mainly in a paracrine manner on gastrointestinal vagal afferents to reduce food intake (for review see e.g. Ritter et al., 1999). The vagal afferents make their initial synapses in the dorsal vagal complex of the brainstem, involving mainly the NTS but also
⁎ Corresponding author. Institute of Veterinary Physiology, University of Zurich, Wintethurerstrasse 260, 8057 Zurich, Switzerland. Fax: +41 44 635 8932. E-mail address: [email protected] (C. Becskei). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.06.016
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the AP (Shapiro and Miselis, 1985a,b). Thus in the ascending pathway of CCK-induced satiation, the NTS represents the primary relay station in the brain. CCK seems to modulate AP activity indirectly, through vagal inputs and through the NTS (for review see Ritter et al., 1999). The facts that vagotomy abolishes the CCK-induced c-Fos expression in the brain (Day et al., 1994; Li and Rowland, 1995) and that a specific AP lesion does not block the anorectic effect of CCK (Edwards et al., 1986) strongly support that notion. On the other hand, the anorectic effect of amylin seems to be independent of vagal or other sensory abdominal inputs (Morley et al., 1994; Lutz et al., 1994, 1995, 1998a). Hence, the amylin-induced activation of the NTS is believed to be secondary, due to neuronal inputs from the AP (Riediger et al., 2001, 2004). Several studies have shown that both AP and NTS neurons project to the LPBN (Norgen, 1978; Voshart and Van Der Kooy, 1981; Hermann and Rogers, 1985; Shapiro and Miselis, 1985a, b), and the latter to the CeA (Block et al., 1989). Since peripheral amylin and CCK both activate the LPBN and CeA (Day et al., 1994; Li and Rowland, 1994, 1995; Rowland and Richmond, 1999; Rowland et al., 1997; Mayne et al., 1998; Riediger et al., 2004) it seems plausible that these nuclei are linearly connected forming an AP/NTS–LPBN–CeA neuronal axis that processes the amylin and CCK signal. However, the role of the nuclei downstream from the AP/NTS region in the feeding-related effects of amylin and CCK is not clear. Concerning CCK, Trifunovic and Reilly (2001, 2003) have shown that the effect of LPBN ablation (LPBN-X) on CCKinduced satiation depends on the experimental conditions, most importantly on the texture of the food. In these studies, LPBN-X blocked the effect of CCK only when solid rodent chow was presented to the rats, but not when a liquid milk diet was offered. Studies investigating the role of the LPBN in the anorectic effect of amylin are missing. Therefore, the aim of our study was first, to investigate the role of the LPBN in mediating the anorectic effect of amylin. Second, we wanted to test whether LPBN-X blocks the satiating effect of CCK under our experimental conditions, i.e. when rats are fed powdered medium-fat diet. Furthermore, we hypothesized that the flow of afferent information is directed from the AP/NTS region via the LPBN towards the CeA. Thus, if the LPBN as the intermediate relay station is eliminated from this pathway through ablation, the amylin- and CCK-induced neuronal activation would not be transmitted to the CeA. This should result in an attenuated c-Fos expression in the CeA in amylin- or CCK-treated LPBN-lesioned animals. Further, assuming that the neuronal activation in the AP/NTS-LPBNCeA pathway correlates with the anorectic action of amylin and CCK, the disruption of this pathway should also reduce the anorectic effect of these peptides. To test these hypotheses, we conducted feeding and c-Fos immunohistochemical studies with LPBN-lesioned and sham-operated (SHAM) rats, injected peripherally with amylin or CCK.
2.
Results
Based on the histochemical verification of the lesions, fifteen rats had a complete ablation of that part of the LPBN that is activated by peripheral amylin or CCK in intact rats. This area
77
comprises the external, the central and the dorsal subnuclei (Mayne et al., 1998). Fig. 1 shows the area of common damage at different levels in the brain. All SHAM rats (n = 26) had an intact LPBN.
2.1.
Feeding studies
There was no difference in food intake after 24 h fasting between the LPBN-X and SHAM groups after saline injection. Lesioning the LPBN significantly attenuated the anorectic effect of a low dose of amylin (5 μg/kg) 1 h post-injection (twoway ANOVA, lesion × treatment interaction: F(1,78) = 5.4, p b 0.05). This attenuation was not significant after 2 h. Overall, amylin reduced food intake at both time points (two-way
Fig. 1 – The areas of common damage in the LPBN of the lesioned rats at different Bregma levels of coronal brain sections. The different patterns of shading represent the number of animals in which the corresponding area was damaged as follows: horizontal lines: 1–3, right hatched area: 4–6, left hatched area 7–9, light gray area: 10–12, dark gray area 13–15 animals. Modified images reprinted from The Rat Brain in Stereotaxic Coordinates, Paxinos, G. and Watson, C., Copyright Paxinos, G. and Watson, C. (1998), with permission from Elsevier.
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ANOVA, main effect of injection: F(1,78) = 23.4, p b 0.001 at 1 h and F(1,78) = 8.2, p b 0.01 at 2 h). However, the post hoc test revealed that amylin significantly reduced feeding in the SHAM (p b 0.001 and p b 0.01, 1 and 2 h after injection, respectively) but not in the LPBN-X rats (Fig. 2A). There was a main effect of lesion only 1 h after injection (two-way ANOVA: F(1,78) = 8.8, p b 0.01). Injection of a higher dose of amylin (10 μg/kg) reduced food intake at both time points (two-way ANOVA: F(1,76) = 19.8, p b 0.001 at 1 h and F(1,76) = 11.4, p b 0.01 at 2 h). The post hoc test revealed that food intake was reduced in both the LPBN-X (p b 0.05) and SHAM rats (p b 0.001) 1 h after amylin injection (Fig. 2B). However, at the 2-h time point, this effect was only significant in the SHAM group (p b 0.05). In the twoway ANOVA, the main effect of lesion and the lesion × treatment interaction did not reach significance at this dose of amylin. LPBN-X significantly reduced the anorectic effect of CCK (3 μg/kg) 1 h after injection (two-way ANOVA, lesion × treatment interaction: F(1,78) = 7.1, p b 0.01). This effect just failed to reach significance at the 0.5 h time point (F(1,78) = 3.7, p = 0.058). Overall, two-way ANOVA revealed a main effect of treatment at both time points (F(1,78) = 81.8, p b 0.001 at 0.5 h and F(1,78) = 26.1, p b 0.001 at 1 h). However, the post hoc test
Fig. 3 – Cumulative food intake in rats with lesion in the lateral parabrachial nucleus (LPBN-X) or with sham lesion (SHAM) 0.5 and 1 h after i.p. CCK administration. (A) CCK 3 μg/kg; n = 15 for LPBN-X and n = 26 for SHAM; (B) CCK 5 μg/kg; n = 6 for LPBN-X and n = 12 for SHAM. Lesioning the LPBN reduced the anorectic effect of CCK (3 μg/kg) 1 h post-injection (two-way ANOVA; p < 0.01). **p < 0.01; ***p < 0.001 indicate significant differences between CCK and saline within SHAM and LPBN-X groups (Holm–Sidak posthoc test).
showed that CCK reduced food intake in both groups 0.5 h after injection (p b 0.001) but only in the SHAM (p b 0.001) rats at 1 h (Fig. 3A). There was no main effect of lesion on food intake (two-way ANOVA). A higher dose of CCK (5 μg/kg) reduced food intake at both time points (two-way ANOVA: F(1,32) = 59.2, p b 0.001 at 0.5 h and F(1,32) = 12.4, p = 0.001 at 1 h). Post hoc test revealed that this CCK dose reduced food intake in both groups 0.5 h following injection (p b 0.001), but only in the SHAM rats (p b 0.01) after 1 h (Fig. 3B). In the two-way ANOVA, the lesion × treatment interaction did not reach significance at any time point and there was a main effect of lesion only 0.5 h after injection (F (1,32) = 11.4, p b 0.01). Fig. 2 – Cumulative food intake in rats with lesion in the lateral parabrachial nucleus (LPBN-X; n = 15) or with sham lesion (SHAM; n = 26) 1 and 2 h after i.p. amylin (A), 5 μg/kg (B) 10 μg/kg injection. Lesioning the LPBN reduced the anorectic effect of amylin (5 μg/kg) 1 h post-injection (two-way ANOVA; p < 0.01). *p < 0.05; **p < 0.01; ***p < 0.001 indicate significant differences between amylin and saline within SHAM and LPBN-X groups (Holm–Sidak post-hoc test).
2.2.
Immunohistochemistry
Amylin treatment induced a strong c-Fos expression in the AP of both the LPBN-X and SHAM rats (Fig. 4). There was no difference in the number of c-Fos-positive cells between the two groups (47 ± 3 and 48 ± 3 cells/section respectively; Fig. 5). In the LPBN of the SHAM rats, abundant c-Fos response was detected after amylin administration (Fig. 4). In the LPBN-X rats with
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Fig. 4 – Amylin (20 μg/kg) induces c-Fos expression in the area postrema (AP; upper panel), lateral parabrachial nucleus (LPBN; middle panel) and in the central nucleus of the amygdala (CeA; lower panel) in the SHAM (left column) animals. In the LPBN-X (right column) rats c-Fos expression is visible in the AP, but not in the CeA. Fluorescent-conjugated secondary antibody (Cy3) was used and the photomicrographs were converted to grayscale images. Scale bars: 200 μm.
complete lesion, the c-Fos-expressing subnuclei were destroyed. A representative photomicrograph of a complete LPBN lesion is shown in Fig. 4. While in the CeA the SHAM animals displayed a strong c-Fos expression after amylin injection (39 ± 7 cells/section), hardly any c-Fos-positive cells could be detected in the LPBN-X rats (5 ± 2 cells/section; Figs. 4 and 5). CCK administration induced a moderate c-Fos expression in the AP of both the lesioned and the SHAM group (Fig. 6).
Quantification of the c-Fos-positive cells showed no significant difference between the two groups (17 ± 2 and 8 ± 1 cells/section, respectively; Fig. 5). Similar to amylin, a strong c-Fos signal was detected in the LPBN and the CeA (23 ± 5 cells/section; Fig. 6) of the SHAM rats after CCK injection. However, CCK-induced neuronal activation in the CeA was blocked in rats with LPBN-X (3 ± 2 cells/section; Figs. 5 and 6).
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Fig. 5 – Quantification of Fos ir cells in the area postrema (AP) and in the central amygdala (CeA) after s.c. application of 20 μg/kg amylin (upper row), 20 μg/kg CCK (middle row) or saline (bottom row). ***p < 0.001, *p < 0.05 compared to the SHAM group.
In the saline-treated groups, very low numbers of c-Fos-ir cells were present in any of the investigated nuclei (Fig. 5).
3.
Discussion
The role of the LPBN in mediating the anorectic action of the satiating peptides amylin and CCK was investigated in LPBNlesioned rats. Ablation of the LPBN reduced the anorectic effect of amylin and CCK and blocked the induction of c-Fos expression in the CeA by both peptides. Lesioning the LPBN significantly reduced the anorectic effect of amylin at a dose of 5 μg/kg, but had no apparent effect at a higher amylin dose (10 μg/kg). The LPBN thus seems to be essential in mediating the anorectic effect of exogenous peripheral amylin at least when low, near-physiological doses are used. It is plausible that a high amylin dose activates neurons in the AP/NTS region which do not project via the LPBN to the higher brain centers. This might account for the remaining anorectic effect of a higher dose of amylin in the lesioned animals. In our previous studies (Lutz et al., 2001), ablation of the AP completely blocked the anorectic action of a
low amylin dose (5 μg/kg) but only attenuated that of a higher dose (50 μg/kg). Based upon these results, it cannot be excluded that higher doses of amylin might additionally activate an alternative pathway, different from the AP– LPBN–CeA axis. Autoradiographic studies have shown abundant amylin binding sites in several forebrain structures involved in the regulation of food intake. These nuclei, in addition to the presumed primary target site, the AP, might be potential targets for the anorectic action of peripheral amylin (Van Rossum et al., 1994; Sexton et al., 1994). A direct effect of amylin on these brain nuclei may also be possible, because amylin can cross the blood–brain barrier by a saturable transport mechanism (Banks et al., 1995). It has to be noted that in spite of the thorough anatomical and functional (c-Fos) evaluation, the possibility that a few neurons and fibers were left intact in the AP-X (former study, Lutz et al., 2001) or LPBN-X (present study) animals cannot be excluded. In the immunohistological study, the SHAM animals displayed a similar c-Fos-ir pattern in the AP, LPBN and the CeA after amylin application as has been reported previously (Rowland and Richmond, 1999; Rowland et al., 1997; Riediger et al., 2004). All SHAM and LPBN-X rats treated with amylin
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Fig. 6 – CCK (20 μg/kg) induces c-Fos expression in the area postrema (AP; upper panel), lateral parabrachial nucleus (LPBN; middle panel) and in the central nucleus of the amygdala (CeA; lower panel) in the SHAM (left column) animals. In the LPBN-X (right column) rats c-Fos expression is visible in the AP, but not in the CeA. Immunoperoxidase method was used. Scale bars: 200 μm.
showed a similar level of activation in the AP, suggesting that the amylin-induced c-Fos expression in this area does not depend on the integrity of the LPBN. The activation of the AP is specific to amylin because no significant c-Fos-ir was detected in the AP of the saline-treated control group. In the LPBN-X rats, c-Fos expression was abolished in the CeA after amylin administration, indicating a crucial role for the LPBN in transmitting neuronal signals to the CeA. Previous studies have shown that the ablation of the AP/NTS region
blocks the amylin-induced c-Fos expression in the LPBN and CeA (Rowland and Richmond, 1999; Riediger et al., 2004). This suggests that the activation of both the LPBN and CeA relies on ascending inputs from the AP/NTS region. These results together with the current observations support the hypothesis that the nuclei which are activated by amylin – AP–NTS–LPBN– CeA – constitute a linearly connected pathway. We recently demonstrated that peripheral amylin injection, similar to refeeding, reversed the fasting-induced c-Fos
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expression in the lateral hypothalamic area (LHA) (Riediger et al., 2004). Furthermore, peripheral amylin has been reported to reduce the mRNA expression of the orexigenic neuropeptide orexin in the LHA (Barth et al., 2003). Nevertheless, a direct effect of amylin on the LHA is not likely, because no amylin binding has been reported in this nucleus (Sexton et al., 1994). On the other hand, the NTS (Ricardo and Koh, 1978), the LPBN (Bester et al., 1997) and the CeA (Berk and Finkelstein, 1981) send neuronal projections to the LHA. It is plausible that the direct brainstem projections, which circumvent the LPBN, convey at least some of the anorectic amylin signal to the LHA and might be responsible for the maintained anorectic effect of high amylin doses. However, the involvement of the LHA in the feeding effect of amylin is still to be confirmed. The results of the feeding studies confirmed the involvement of the LPBN in mediating CCK-induced satiation. The anorectic effect of CCK was significantly attenuated in the LPBN-X animals after 30 min and it was completely blocked after 60 min of feeding, when near-physiological doses were used. The latter finding is in good concert with the results of Trifunovic and Reilly (2001), who demonstrated a complete blockade of the inhibitory effect of CCK (4 and 8 μg/kg, i.p.) on 1 h cumulative food intake in rats with ibotenic acid-induced LPBN lesions when fed pelleted chow. However, the same group also reported that the anorectic action of CCK (2 and 8 μg/kg, i.p.) was unchanged in lesioned animals presented a palatable milk diet (Trifunovic and Reilly, 2003). The authors speculate that the texture or palatability of the food might influence the effect of the lesion on CCK's inhibitory action on feeding (Trifunovic and Reilly, 2003). Nevertheless, it is also plausible that the nutrient composition of the diet might have affected the CCK action, which likely differed between the studies. In our study, the ablation of the LPBN attenuated the anorectic effect of a higher dose of CCK (5 μg/kg), implying that there may be other brain nuclei besides the LPBN that play a role in processing CCK-induced satiation. There is accumulating evidence that a potential hypothalamic candidate to mediate the anorectic effect of CCK could be the paraventricular nucleus of the hypothalamus (PVN; Crawley and Kiss, 1985; Day et al., 1994; Rinaman et al., 1995). The NTS, which is the main terminal field of the vagal afferents conveying the peripheral CCK signal, has been shown to have direct projections to this nucleus (Rinaman et al., 1995). This might also provide an explanation for the partial persistence of the anorectic potency of CCK in our LPBN-X rats. It has to be noted that in contrast to amylin, CCK is unable to cross the blood– brain barrier (Passaro et al., 1982). Thus, a direct effect of peripheral CCK on brain nuclei with a tight blood–brain barrier, like the PVN, seems unlikely. Similar to amylin, the c-Fos expression was abolished in the CeA of the LPBN-X rats after CCK administration. Thus the LPBN seems to play an essential role in mediating the CCKinduced neuronal activation to the CeA. These results strongly support the notion that, similar to amylin, there is a linear pathway involving the AP/NTS–LPBN–CeA nuclei that is activated by peripheral CCK. Taken together, our results suggest that a functionally intact LPBN is important in the amylin- and CCK-induced satiation. Our immunohistological studies provide evidence
that the effect of amylin and CCK on the neuronal activation of the CeA relies completely on ascending information from the LPBN. However, whether the reduced anorectic effect of amylin and CCK in the LPBN-X animals is due to the lack of activation in the CeA and whether the CeA plays a role in the feeding effects of these peptides requires further studies.
4.
Experimental procedure
4.1.
Animals
Adult male Wistar rats housed in individual cages in a temperature-controlled room (22 °C) were used. They had free access to water and were kept under a 12-h light/dark cycle. All animal procedures were approved by the Veterinary Office of the Canton of Zurich's Health Directorate. At least for a week before the surgery and during the whole period of recovery phase after surgery, the animals were handled daily. Prior to the feeding studies, animals were injected with saline daily for 2–3 days to adapt them to the experimental procedure.
4.2.
Electrolytic LPBN lesions
Rats (n = 56) weighing 290–350 g were randomly allocated into two groups, one to be electrically lesioned in the lateral parabrachial nucleus (n = 30) and one of sham-operated controls (n = 26). The procedure was adapted according to Edwards and Ritter (1989). Briefly, after anaesthetizing the rats with ketamine (60 mg/kg) and xylazine (7 mg/kg), clipping and disinfecting the top of the head, the animals were fixed in a stereotaxic apparatus. Access to the skull was gained by a 2- to 3-cm midline skin incision from the level of the orbita to approximately 1 cm caudally from the occipital crest. The periosteum was detached and retracted laterally. A rectangular piece of the skull was removed above the area of the LPBN (coordinates: 9.4 mm caudal, and bilaterally 1.9 mm lateral from Bregma; Paxinos and Watson, 1998). After pulling the transverse sinus slightly aside, the dura mater was incised. An insect pin served as the electrode and was attached to the anode of the electrolytic lesion-making device (Ugo Basile, Italy). The pin was lowered 6.5 mm ventrally from dura and an anodal current (0.5 mA for 10 s) was passed on each side to produce the electrolytic lesions. Sham lesions were made by the same procedure but without passing the current. After surgery, the rats were allowed to recover for at least 2 weeks. The histological verification of the LPBN-X was combined with the immunohistochemical studies (see below). Only animals with complete bilateral lesions of the LPBN were admitted to the statistical evaluation (n = 15). All 26 SHAM animals were included in the analysis.
4.3.
Feeding studies
The feeding trials were performed using a crossover design so that each animal served as its own control. All the animals were fed a powdered medium-fat diet ad libitum (18% fat and 16.5 kJ/g; Lutz et al., 1994). Both the lesioned and the SHAM rats
BR A IN RE S E A RCH 1 1 62 ( 20 0 7 ) 7 6 –8 4
were divided into two groups. At dark onset, following 24-h food deprivation, one group received an intraperitoneal (i.p.) injection of amylin (5 μg/kg or 10 μg/kg). The second group was injected with saline (1 ml/kg). Immediately after injection, the rats were given access to food. Cumulative food intake, corrected for spillage, was measured manually after 1 and 2 h. The same experimental protocol was used to investigate the anorectic effect of CCK (3 μg/kg, i.p.). Food intake measurements were performed 30 and 60 min after treatment, because the effect of CCK on food intake lasts generally shorter than that of amylin. Half of the animals were also tested with a higher dose of CCK (5 μg/kg, i.p.; n = 6 for LPBN-X and n = 12 for SHAM).
4.4.
Immunohistochemistry
In the immunohistochemical studies the same LPBN-X and SHAM rats were used as in the feeding trials. These studies served as a histological and functional verification of the LPBN lesions as well. The ad libitum fed animals received an injection of peptide (20 μg/kg amylin or CCK) or vehicle (saline) at dark onset. The substances were applied subcutaneously (s.c.), because in previous studies we found that the variation in hormone-induced c-Fos expression is lower after s.c. application compared to i.p. application. Moreover, a 20 μg/kg s.c. dose of amylin produced a similar c-Fos expression as an i.p. injection of 5 μg/kg in the rat AP (approx. 45 cells/section; see Riediger et al., 2001 and current results). 2 h after injection, the rats were anesthetized with pentobarbital (Nembutal, Abbott Laboratories USA 80 mg/kg, i.p.) and perfused transcardially with ice-cold phosphate buffer (PB 0.1 M), followed by 4% paraformaldehyde (in 0.1 M PB). The brains were removed and kept in paraformaldehyde for an additional 2 h to achieve proper tissue fixation. After 48 h of incubation in 20% sucrose solution (in 0.1 M PB) at 4 °C, the samples were snap frozen in CO2 gas. Coronal sections (20 μm) were cut in a cryostat (CM 3050 Leica, Nussloch, Germany) throughout the AP, PBN and CeA. Every slice was thaw mounted on microscopic glass slides (SuperFrost Plus, Faust, Schaffhausen, Switzerland) and was stored at − 20 °C until further processing. For the detection of c-Fos expression, the brain sections of the amylin-treated animals were processed by an immunofluorescent staining procedure. This involved drying the sections at room temperature for 1 h and rehydrating them in 0.1% PBST (phosphate-buffered saline solution (PBS) + Triton X-100; Sigma). Unspecific binding sites were blocked by 1.5% normal donkey serum. The primary antibody (polyclonal rabbit–anti-c-Fos IgG, Oncogene, Ab-5) was applied for 48 h at 4 °C. The unbound antibody was removed by washing in PBST. The sections were then incubated with the secondary antibody (donkey–anti-rabbit, Cy3 conjugated, Milan Analytica, Switzerland) for 75 min at room temperature. The first and the second antibody were diluted to 1:1000 and 1:225, respectively, in 0.3% PBST. After a final washing in 0.1% PBST, the slides were covered and stored at 4 °C until evaluated using a fluorescent microscope (Axioscop 2, Carl Zeiss AG, Feldbach, Switzerland). Due to a general change of the visualization method in our laboratory from fluorescent to immunoperoxidase method, the avidin–biotin immunoperoxidase method was used to
83
visualize c-Fos expression in the CCK- and saline-treated groups. Because the lesioned and SHAM animals are only compared within the same treatment group, the different techniques do not compromise the interpretation of the results. The histological sections were air-dried at room temperature for 1 h and rehydrated in PBS. Thereafter the sections were incubated in PBS containing 0.3% H2O2 for 30 min. Unspecific binding was blocked by a 2 h incubation in 1.5% normal rabbit serum. The primary antibody (polyclonal goat anti-c-Fos, Santa Cruz; 1:10,000) was applied for 48 h at 4 °C. The unbound antibody was removed by washing in PBS and the sections were incubated with the secondary antibody (biotinylated rabbit–anti-goat, Vectastain®-Elite ABC Kit, Vector Laboratories; 1:200) for 2 h at room temperature. After incubation in ABC (Vectastain®-Elite ABC Kit, Vector Laboratories), followed by DAB solution (0.04% in PBS with 0.02% H2O2 and for color enhancement 0.08% NiCl2·6H2O and 0.01% CoCl2·6H2O), the sections were dehydrated in graded alcohols, cleared in xylenes and coverslipped. The localization of the c-Fos-expressing neurons was identified according to the rat brain atlas by Paxinos and Watson (1998). Only animals with successful bilateral lesions of the LPBN were admitted to the quantification of the c-Fos-expressing cells (n = 6 for amylin, n = 4 for CCK and n = 3 for saline tretment). The lesion was considered complete when at immunohistochemistry no intact tissue could be seen in the area corresponding to the central, dorsal and external LPBN. For representative histological photographs of complete lesions see Figs. 4 and 6. In each rat, 30 consecutive corresponding sections of both the CeA and the AP were counted for c-Fos-positive cells. Photomicrographs were taken by a digital camera (AxioCam, Carl Zeiss AG). Two rats with complete lesions and eleven SHAM animals, which were included in the feeding studies, were used in other immunohistological experiments. For this reason, the number of animals in the feeding and immunohistological studies presented here is not identical.
4.5.
Statistics
In the feeding studies the effect of lesion on the anorectic effect of amylin and CCK was analyzed by two-way ANOVA, followed by Holm–Sidak post hoc test. In the immunohistological studies, the cell counts for all the sections in a certain brain area were averaged in each individual animal and used for statistical analyses. The LPBN-X rats were compared with the SHAM animals by t-test within each treatment group (amylin, CCK and saline). P b 0.05 was considered significant. Results are presented as means ± SEM.
Acknowledgments This project was supported by the Swiss National Science Foundation, the Research Committee and Young Academics Support Committee of the University of Zurich and by the Stiftung für wissentschaftliche Forschung of the University of Zurich. Cs.B. is a recipient of a fellowship grant from the Centre of Integrative Human Physiology (University of Zurich).
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