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A secretin i.v. infusion activates gene expression in the central amygdala of rats
Neuroscience 118 (2003) 881– 888
LETTER TO NEUROSCIENCE A SECRETIN I.V. INFUSION ACTIVATES GENE EXPRESSION IN THE CENTRAL AMYGDALA OF RATS M. GOULET,a P. J. SHIROMANI,b C. M. WARE,a R. A. STRONG,a R. BOISMENUa AND J. R. RUSCHEa*
al., 1983; Nozaki et al., 2002; O’Donohue et al., 1981). Electrophysiology measurements using cerebellum slices show that evoked and spontaneous inhibitory postsynaptic currents (IPSCs) as well as the frequency of miniature IPSCs recorded from Purkinje cells are increased in the presence of secretin (Yung et al., 2001). However, no study is available to document whether or not secretin can function as a neuroactive hormone after i.v. infusion. A putative influence on neuronal activity is suggested from reports of improved social and communicative behavior in autistic children after secretin infusion (Horvath et al., 1998). While some controversy exists over reproduction of the original clinical observation in subsequent small studies (Owley et al., 2001; Sandler et al., 1999), a larger study of young children receiving three doses of secretin measured significant behavioral improvements in reciprocal–social interactions (J. R. Rusche, unpublished observation). Here, we show the effect of i.v. administration of secretin on neuronal activity in discrete brain regions as measured by Fos immunohistochemistry. Peptide levels in blood were determined in order to compare secretin blood concentration in rats to those reported in human use of secretin. Brain regions activated by peptides functionally and/or structurally related to secretin are also presented for comparison.
a Repligen Corporation, Building 1, Suite 100, 41 Seyon Street, Waltham, MA 02453, USA b VA Medical Center and Harvard Medical School, West Roxbury, MA 02132, USA
Abstract—For the last 100 years secretin has been extensively studied for its hormonal effects on digestion. Recent observations that the deficits in social reciprocity skills seen in young (3– 4-year-old) autistic children are improved after secretin infusions suggest an additional influence on neuronal activity. We show here that i.v. administration of secretin in rats induces Fos protein expression in the neurons of the central amygdala as well as the area postrema, bed nucleus of the stria terminalis, external lateral parabrachial nucleus and supraoptic nucleus. However, secretin infusion did not induce Fos expression in the solitary tract nucleus or paraventricular nucleus, regions normally activated by related peptides such as cholecystokinin. The peak blood levels of secretin that induce Fos protein expression in rat brain are similar to the peak blood levels observed during i.v. treatment with secretin in humans. The amygdala is known to be critical for developing reciprocal social interaction skills and abnormalities in this brain region have been demonstrated in autistic children. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: peptide, autism, area postrema, neuronal transcription, Fos, vasoactive intestinal polypeptide.
EXPERIMENTAL PROCEDURES
Secretin belongs to a family of peptides that includes vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and glucagonlike peptide. Secretin family peptides are ligands for known G protein-coupled receptors. Most peptides in this group, including secretin, have been located both peripherally and in the CNS reflecting their known hormonal role and a possible function in modulating neuronal activity. In particular, secretin and secretin receptor are expressed in mammalian brain tissue, including the cerebellum (Fremeau et
Peptides Peptides used were synthetic human secretin (Repligen Corporation, Waltham, MA, USA), cholecystokinin (CCK 26-33, Peninsula Laboratories, San Carlos, CA, USA), PACAP (PACAP27, Calbiochem, La Jolla, CA, USA) and VIP (Calbiochem).
Animals Male Sprague–Dawley albino rats (n⫽64, 7– 8 weeks old, 210 – 300 g) implanted with a catheter into the jugular vein were purchased from Taconic, Germantown, NY, USA. Young rats were used to be more relevant to secretin studies in children. Animals were housed under controlled temperature and lighting (12-h light/ dark cycle; lights on at 0700 h) with food and water available freely unless otherwise noted. In experiments using fasted rats, food was withheld 18 –20 h before treatment. Protocols were approved by the Institutional Animal Care and Use Committee of the New England Medical Research Institute, Brockton-West Roxbury VA Medical Center. All efforts were made to minimize the number of animals used and their suffering.
*Corresponding author. E-mail address: [email protected] (J. Rusche). Abbreviations: AP, area postrema; AUC, area under the curve; BST, bed nucleus of the stria terminalis; CCK, cholecystokinin; CeA, central amygdala; CeL, central amygdala, lateral division; CeM, central amygdala, medial division; Cmax, maximum blood concentration; DMVN, dorsal motor vagal nucleus; IPSC, inhibitory postsynaptic current; NTS, solitary tract nucleus; PACAP, pituitary adenylate cyclase-activating polypeptide; PBe, external lateral parabrachial nucleus; PBS, phosphate-buffered saline; PVN, paraventricular nucleus; SON, supraoptic nucleus; VIP, vasoactive intestinal polypeptide.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00782-0
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Rats were assigned to groups and injected daily for 5– 8 days with the vehicle solution (10 mM sodium citrate, 2.0 mg/ml Dmannitol and 0.004% Tween 80, pH 6.8, sterile) before experimental treatment to reduce the amount of Fos immunoreactivity induced by handling stress and to keep the canula patent. At various time points after the injection of synthetic peptides, rats were anesthetized and perfused transcardially with normal saline solution followed by 4% formaldehyde in 0.1 M phosphate-buffered saline (PBS). Brains were removed, placed overnight in the same fixative and transferred to a buffer containing 30% sucrose– 0.1 M PBS. Animals used in studies of secretin clearance from blood were 35-day-old Sprague–Dawley rats (Charles River Laboratories). Eighteen rats/sex per dose group were used to measure the maximum blood concentration (Cmax) and elimination rate of human synthetic secretin using a radio-immunoassay (Peninsula Laboratories, San Carlos, CA, USA). Fasted animals were infused i.v. with secretin at 0.4, 4.0 or 40 g/kg. Blood samples were collected from the descending aorta at 2, 5, 10, 15, 20, or 30 min post-dosing with six animals/time point per dose group. The plasma fraction was stored at ⫺20 °C.
Histochemistry Fos immunohistochemistry was carried out on every sixth 30 m section of the whole rostra-caudal extent of each brain. In brief, sections were incubated sequentially with a rabbit anti-c-Fos antibody (AB5, Oncogene Science, San Diego, CA, USA), a biotinylated donkey anti-rabbit secondary antibody (AP182B, Chemicon, Temecula, CA, USA) and an avidin-biotinylated peroxidase complex (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Colorometric detection was with nickel-enhanced diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA). Some sections were counterstained with neutral red.
Data acquisition and analysis Neuroanatomical boundaries of brain nuclei were identified based on the published atlas of rat brain regions (Paxinos et al., 1999a,b). Brain-section images were captured using a digital camera attached to a bright-field microscope. Fos-labeled cells were counted in the outlined brain nuclei manually by at least one person blind to the treatment groups or in an automated fashion using the NIH Image program software (Version 1.62, National Institutes of Health). The number of cells containing Fos immunoreactivity was counted bilaterally (except for the area postrema [AP]) in selected nuclei in consecutive sections: four to five for the central amygdala (CeA), more than five for the supraoptic nucleus (SON), solitary tract nucleus (NTS), and dorsal motor vagal nucleus (DMVN) and two to four for the AP and paraventricular nucleus (PVN). Data are expressed as mean⫾S.E.M., and represent the mean number of Fos-positive cells/section determined from all sections per rat in all animals per treatment group. Peptide and dose effects were analyzed by a t-test or an analysis of variance (ANOVA) followed by a Fisher’s probability of least significant differences with a probability level of ⬍0.05.
RESULTS Fos protein expression in neurons represents a readily measured and well-established early marker for cell-specific gene activation (Dragunow and Faull, 1989; Sagar et al., 1988). Rats received an i.v. infusion of secretin (40 g/ kg) or vehicle, were killed 60 min post-infusion, and their brains were processed for Fos immunohistochemistry. Evaluation of sections representing the entire rostra-caudal brain revealed increased Fos immunoreactivity in the
CeA, bed nucleus of the stria terminalis (BST), AP, external lateral parabrachial nucleus (PBe) and SON in secretin-treated rats compared with vehicle-treated animals (Fig. 1). The CeA showed Fos-positive neurons whereas the lateral, basolateral and medial amygdala regions showed no increase in Fos-positive neurons (Fig. 1B). A trend toward an increase in Fos-positive neurons was seen in the NTS, predominantly in the region proximal to the AP (Fig. 1D). The brain regions with secretin-induced Fos expression were the same in both 35-day- (not shown) and 60-day-old rats (data shown here). The same pattern of Fos expression was seen in fed and fasted rats with the exception that fasted rats did not display Fos-positive cells in the SON (data not shown). In a time-course study rats dosed with 40 g/kg secretin were killed at 60, 120, and 240 min post-infusion and brain Fos expression was detected as previously described. At 120 min, the number of Fos-positive neurons in the CeA of secretin-treated rats was increased seven-fold over vehicle-treated animals (Fig. 2). The left and right CeA displayed an equivalent response to secretin (data not shown). Fos staining in the CeA and SON was similar at 60 and 120 min but absent by 240 min. In contrast, Fos expression in the AP was highest at 60 min, decreased by 120 min and was completely absent by 240 min. This suggests that neuron activation in the AP may precede that in the CeA. At all time points, secretin did not induce Fos expression in the PVN, DMVN, NTS, or dorsal lateral parabrachial nucleus. Next, the effect of secretin dose (0.4 – 40 g/kg) on the number of Fos-positive neurons in the CeA was examined at the 120-min time point (Fig. 3). We found that the increase in Fos-positive neurons was significantly related to dose (ANOVA, P⫽0.001). The secretin-induced increase in Fos-positive cells reached significance at a dose of 4.0 g/kg (t-test, P⫽0.026, n⫽6). A similar dose response was seen for the AP (data not shown). Since Fos expression in the CeA after secretin infusion was dose dependent, it was relevant to determine if this effect was correlated with peptide blood levels. The plasma level of secretin was measured using a radioimmunoassay with a limit of quantitation of 20 pg/ml. Therefore, the baseline concentration of circulating secretin in rat, 6 pg/ml, (Li et al., 1995) was not detectable in this assay. The Cmax, area under the curve (AUC), and elimination rate were determined after a single dose of 0.4, 4, or 40 g/kg of secretin (Table 1). The AUC and Cmax both showed a linear increase with the number of Fos-positive neurons (R2⫽0.9998 and 0.9926, respectively). Thus the increase in Fos-positive cells in the CeA correlated well with secretin blood levels. Comparison with functionally and structurally related proteins CCK, like secretin, is a gut hormone involved with the regulation of pancreatic exocrine secretion. It is well established that CCK induces Fos expression in the CNS after peripheral administration (Day et al., 1994; Monnikes et al., 1997). We compared the pattern of brain Fos ex-
M. Goulet et al. / Neuroscience 118 (2003) 881– 888
Fig. 1. (Caption overleaf).
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Fig. 3. Effects of different doses of secretin on c-Fos expression in the central amygdala. Secretin (0.4 – 40 g/kg, i.v.) dose-dependently induced an increase in neuronal activity, as determined by the average number of Fos positive neurons/section. The number of rats per time point ⱖ4 and the bars represent means⫾SEM. *, P⬍0.05; ***, P⬍0.001 vs. Vehicle, T-test.
Fig. 2. Time course of secretin (40 g/kg) induced Fos expression in A) CeA, B) AP, C) PVN and D) SON, as determined by the average number of Fos positive neurons/section. The number of rats per time point is 3– 4 and the bars represent means⫾SEM. *, P⬍0.05; **, P⬍0.01; ***, P⬍0.001 vs. Vehicle. #, P⬍0.05 and ##, P⬍0.01 vs. 60 min, ANOVA followed by a Fisher probability of least significant difference test (PLSD).
pression 120 min after an equimolar dose of CCK (15 g/ kg) or secretin (40 g/kg). CCK induced Fos-positive neurons in the AP and CeA (Fig. 4). However, Fos expression was clearly more pronounced in the CeA (P⬍0.005) and the AP (P⬍0.001) after secretin versus after CCK treatment. Secretin induced a significant increase of Fos-positive neurons in the lateral division of the bed nucleus of the stria terminalis corresponding to the oval subnucleus (BST, Fig. 4B). No changes were observed in the ventral and posterior BST divisions (data not shown). In contrast to secretin, CCK treatment led to a pronounced increased in Fos expression in the NTS, DMVN and PVN (Fig. 4D–F) in agreement with previous reports (Day et al., 1994; Monnikes et al., 1997). Several other peptides administered peripherally, such as interleukin-1 (Brady et al., 1994) can induce a Fos expression pattern similar to that seen for CCK where NTS, PVN, CeA, and AP neurons are activated. It should be noted that in this experiment, a small but significant (P⬍0.05) increase in Fos-positive neurons was seen in the DMVN after secretin treatment (Fig. 4F) that had not been seen in other experiments (see Fig. 1). The structurally related peptides secretin, VIP and PACAP are expressed in the peripheral and CNS (Larson et al., 1976; Vaudry et al., 2000). We compared the effects of infusion of VIP and PACAP to the Fos expression pattern seen with secretin. VIP was infused at 44 g/kg, the molar equivalent to secretin, which induced slowed locomotion and muscular spasms in rats for the first 20 min after infusion. PACAP was infused at 6.3 g/kg in order to limit previously described vascular hypotensive activity (Kawai et al., 1994). VIP but not PACAP induced Fospositive cells in the CeA and AP (Fig. 4A, C). Under the conditions of this experiment, the secretin effect was more pronounced than VIP in Fos protein induction within the
Fig. 1. Photomicrographs of coronal brain sections taken from rats treated with vehicle (A, C, E, G) or secretin (B, D, F, H). Secretin induced Fos immunoreactive neurons in the amygdala (A, B), area postrema (C, D), supraoptic nucleus (E, F), and parabrachial nucleus (G, H). AP, area postrema; CeA, central amygdala; BLA, basolateral amygdala; MeA, medial amygdala; NTS, solitary tract nucleus; DMVN, dorsal motor vagal nucleus; SON, supraoptic nucleus; PBe, external lateral parabrachial nucleus; PBd, dorsal lateral parabrachial nucleus. Magnification 100⫻.
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Table 1. Pharmacokinetic parameters of intravenous secretin dosing in ratsa Dose (g/kg)
Cmax (ng/ml)
AUC (ng/ml) min
Elimination rate (min)
CeA Fos Neurons
0.4 4.0 40
0.55 7.46 23.9
2.4 33.1 153.8
2.4 2.3 19.1
3.5 15.1 58.5
a Area under the curve (AUC) and elimination rate were established from secretin concentration measurements in serial blood samples. For each dose, 18 rats were used. The maximum blood concentration (Cmax) is the value at 2 min post-dosing. A separate set of animals was used to count Fos-positive neurons in the central amygdala (CeA) as described in Experimental Procedures
CeA and AP. VIP, like secretin, showed a small increase in Fos-positive neurons in the DMVN. Neither VIP nor PACAP increased the number of Fos-positive neurons in the PVN or NTS (Fig. 4D, E). A more careful comparison of peptide-induced changes in the CeA (Fig. 4A) can be done by examining the number of Fos-positive cells within medial (CeM) and lateral (CeL) subregions of the CeA as well as subdividing the amygdala in a rostral to caudal accounting of Fospositive cells. Fig. 5 shows the number of CeA neurons that are Fos positive at five different rostro-caudal loca-
tions (bar graphs). Fos-immunoreactive cells induced by secretin within the CeA were mostly confined to the CeL (Fig. 5A–D) as only at the more rostral level were Fospositive cells observed in the CeM. VIP-induced Fos-positive cells were also located in the CeL but extended more caudal than with secretin treatment (Fig. 5E). This report establishes that i.v. infusion of secretin results in neuronal activation in defined brain regions as judged from Fos expression. Increases in the number of Fos-positive neurons following secretin administration could be demonstrated in the CeA, BST, SON, PBe, and
Fig. 4. Comparison of structurally or functionally related peptides for Fos protein expression following intravenous administration of vehicle (Veh), secretin (Sec, 40 g/kg), cholecystokinin (CCK, 15 g/kg), PACAP (6.3 g/kg), or vasoactive intestinal polypeptide (VIP, 44 g/kg). The average number of Fos positive neurons/section is presented for A) CeA, B) BST, C) AP D) PVN, E) NTS and F) DMVN. Data are expressed as the mean⫾SEM. *, ** and ***P⬍0.05, P⬍0.01 and P⬍0.001 vs vehicle; ### P⬍0.001 vs the other groups; ANOVA followed by a Fisher PLSD; ND, not done.
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Fig. 5. Quantitative expression and photomicrographs of Fos immunoreactivity in the central amygdala induced by i.v. injection of secretin and related peptides. The comparison of peptides for Fos protein expression was performed at different rostrocaudal levels as indicated for each bar graph. Each column represents the mean⫾SEM of cell number (cells/section, unilateral) from 1–2 sections/rat for 2– 4 rats for each treatment. Photomicrographs of secretin (A–D) and VIP (E) are taken from the rostrocaudal level indicated for the adjacent bar graph. Boundaries for the lateral (CeL) and medial (CeM) divisions of the CeA are indicated. *, P⬍0.05; **, P⬍0.01; ***, P⬍0.001 vs. vehicle. ###, P ⬍0.001 vs. other peptide treatments using ANOVA followed by a Fisher PLSD.
M. Goulet et al. / Neuroscience 118 (2003) 881– 888
AP. Since knowledge of the long-term effects of Fos expression on neuronal activity remains limited, ongoing studies aim to establish changes in expression for markers other than c-fos occurring in these brain regions subsequent to secretin infusion. Additional studies are also required to define the route(s) or mechanisms by which i.v. secretin activates genes in the CeA, AP, BST, SON, and PBe. Endogenous release of secretin affects pancreatic fluid release and reduces gastric motility through a vagal-dependent pathway (Jin et al., 1994). However, supraphysiologic blood levels of secretin (100-fold endogenous levels) affect pancreatic fluid release by a mechanism independent of the vagus (Lu and Owyang, 1995). We have demonstrated in mice that secretin can transfer from blood to brain (Banks et al., 2002). In addition, activation of the CeA may be mediated through activation of the AP, which is a circumventricular organ and could be activated by bloodborne peptide. Therefore, neuronal signaling by supraphysiologic doses of secretin as used in this study or currently in humans may occur by more than one route and may differ from the neuronal signaling that occurs after endogenous secretin release. Autism is a disease characterized by significant social and communication deficits that are usually apparent before the age of 3. Reciprocal social interaction requires recognition of socially relevant information from faces and involves neural processing in the amygdala (Adolphs, 1999; Emery, 2000). Multiple investigators have used functional magnetic resonance imaging to observe abnormal amygdala function in autistic subjects during socially relevant performance tasks (Critchley et al., 2000; Pierce et al., 2001; Schultz et al., 2000). The convergence of behavioral and biological data from both primate and human studies have led to published hypotheses that amygdala dysfunction is central to the etiology of autism (BaronCohen et al., 2000; Howard et al., 2000). Data presented here demonstrate that in rats infusion of secretin at a clinically relevant dose affects gene expression in several brain regions, most notably, the CeA.
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(Accepted 5 October 2002)
LETTER TO NEUROSCIENCE A SECRETIN I.V. INFUSION ACTIVATES GENE EXPRESSION IN THE CENTRAL AMYGDALA OF RATS M. GOULET,a P. J. SHIROMANI,b C. M. WARE,a R. A. STRONG,a R. BOISMENUa AND J. R. RUSCHEa*
al., 1983; Nozaki et al., 2002; O’Donohue et al., 1981). Electrophysiology measurements using cerebellum slices show that evoked and spontaneous inhibitory postsynaptic currents (IPSCs) as well as the frequency of miniature IPSCs recorded from Purkinje cells are increased in the presence of secretin (Yung et al., 2001). However, no study is available to document whether or not secretin can function as a neuroactive hormone after i.v. infusion. A putative influence on neuronal activity is suggested from reports of improved social and communicative behavior in autistic children after secretin infusion (Horvath et al., 1998). While some controversy exists over reproduction of the original clinical observation in subsequent small studies (Owley et al., 2001; Sandler et al., 1999), a larger study of young children receiving three doses of secretin measured significant behavioral improvements in reciprocal–social interactions (J. R. Rusche, unpublished observation). Here, we show the effect of i.v. administration of secretin on neuronal activity in discrete brain regions as measured by Fos immunohistochemistry. Peptide levels in blood were determined in order to compare secretin blood concentration in rats to those reported in human use of secretin. Brain regions activated by peptides functionally and/or structurally related to secretin are also presented for comparison.
a Repligen Corporation, Building 1, Suite 100, 41 Seyon Street, Waltham, MA 02453, USA b VA Medical Center and Harvard Medical School, West Roxbury, MA 02132, USA
Abstract—For the last 100 years secretin has been extensively studied for its hormonal effects on digestion. Recent observations that the deficits in social reciprocity skills seen in young (3– 4-year-old) autistic children are improved after secretin infusions suggest an additional influence on neuronal activity. We show here that i.v. administration of secretin in rats induces Fos protein expression in the neurons of the central amygdala as well as the area postrema, bed nucleus of the stria terminalis, external lateral parabrachial nucleus and supraoptic nucleus. However, secretin infusion did not induce Fos expression in the solitary tract nucleus or paraventricular nucleus, regions normally activated by related peptides such as cholecystokinin. The peak blood levels of secretin that induce Fos protein expression in rat brain are similar to the peak blood levels observed during i.v. treatment with secretin in humans. The amygdala is known to be critical for developing reciprocal social interaction skills and abnormalities in this brain region have been demonstrated in autistic children. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: peptide, autism, area postrema, neuronal transcription, Fos, vasoactive intestinal polypeptide.
EXPERIMENTAL PROCEDURES
Secretin belongs to a family of peptides that includes vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and glucagonlike peptide. Secretin family peptides are ligands for known G protein-coupled receptors. Most peptides in this group, including secretin, have been located both peripherally and in the CNS reflecting their known hormonal role and a possible function in modulating neuronal activity. In particular, secretin and secretin receptor are expressed in mammalian brain tissue, including the cerebellum (Fremeau et
Peptides Peptides used were synthetic human secretin (Repligen Corporation, Waltham, MA, USA), cholecystokinin (CCK 26-33, Peninsula Laboratories, San Carlos, CA, USA), PACAP (PACAP27, Calbiochem, La Jolla, CA, USA) and VIP (Calbiochem).
Animals Male Sprague–Dawley albino rats (n⫽64, 7– 8 weeks old, 210 – 300 g) implanted with a catheter into the jugular vein were purchased from Taconic, Germantown, NY, USA. Young rats were used to be more relevant to secretin studies in children. Animals were housed under controlled temperature and lighting (12-h light/ dark cycle; lights on at 0700 h) with food and water available freely unless otherwise noted. In experiments using fasted rats, food was withheld 18 –20 h before treatment. Protocols were approved by the Institutional Animal Care and Use Committee of the New England Medical Research Institute, Brockton-West Roxbury VA Medical Center. All efforts were made to minimize the number of animals used and their suffering.
*Corresponding author. E-mail address: [email protected] (J. Rusche). Abbreviations: AP, area postrema; AUC, area under the curve; BST, bed nucleus of the stria terminalis; CCK, cholecystokinin; CeA, central amygdala; CeL, central amygdala, lateral division; CeM, central amygdala, medial division; Cmax, maximum blood concentration; DMVN, dorsal motor vagal nucleus; IPSC, inhibitory postsynaptic current; NTS, solitary tract nucleus; PACAP, pituitary adenylate cyclase-activating polypeptide; PBe, external lateral parabrachial nucleus; PBS, phosphate-buffered saline; PVN, paraventricular nucleus; SON, supraoptic nucleus; VIP, vasoactive intestinal polypeptide.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00782-0
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Rats were assigned to groups and injected daily for 5– 8 days with the vehicle solution (10 mM sodium citrate, 2.0 mg/ml Dmannitol and 0.004% Tween 80, pH 6.8, sterile) before experimental treatment to reduce the amount of Fos immunoreactivity induced by handling stress and to keep the canula patent. At various time points after the injection of synthetic peptides, rats were anesthetized and perfused transcardially with normal saline solution followed by 4% formaldehyde in 0.1 M phosphate-buffered saline (PBS). Brains were removed, placed overnight in the same fixative and transferred to a buffer containing 30% sucrose– 0.1 M PBS. Animals used in studies of secretin clearance from blood were 35-day-old Sprague–Dawley rats (Charles River Laboratories). Eighteen rats/sex per dose group were used to measure the maximum blood concentration (Cmax) and elimination rate of human synthetic secretin using a radio-immunoassay (Peninsula Laboratories, San Carlos, CA, USA). Fasted animals were infused i.v. with secretin at 0.4, 4.0 or 40 g/kg. Blood samples were collected from the descending aorta at 2, 5, 10, 15, 20, or 30 min post-dosing with six animals/time point per dose group. The plasma fraction was stored at ⫺20 °C.
Histochemistry Fos immunohistochemistry was carried out on every sixth 30 m section of the whole rostra-caudal extent of each brain. In brief, sections were incubated sequentially with a rabbit anti-c-Fos antibody (AB5, Oncogene Science, San Diego, CA, USA), a biotinylated donkey anti-rabbit secondary antibody (AP182B, Chemicon, Temecula, CA, USA) and an avidin-biotinylated peroxidase complex (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Colorometric detection was with nickel-enhanced diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA). Some sections were counterstained with neutral red.
Data acquisition and analysis Neuroanatomical boundaries of brain nuclei were identified based on the published atlas of rat brain regions (Paxinos et al., 1999a,b). Brain-section images were captured using a digital camera attached to a bright-field microscope. Fos-labeled cells were counted in the outlined brain nuclei manually by at least one person blind to the treatment groups or in an automated fashion using the NIH Image program software (Version 1.62, National Institutes of Health). The number of cells containing Fos immunoreactivity was counted bilaterally (except for the area postrema [AP]) in selected nuclei in consecutive sections: four to five for the central amygdala (CeA), more than five for the supraoptic nucleus (SON), solitary tract nucleus (NTS), and dorsal motor vagal nucleus (DMVN) and two to four for the AP and paraventricular nucleus (PVN). Data are expressed as mean⫾S.E.M., and represent the mean number of Fos-positive cells/section determined from all sections per rat in all animals per treatment group. Peptide and dose effects were analyzed by a t-test or an analysis of variance (ANOVA) followed by a Fisher’s probability of least significant differences with a probability level of ⬍0.05.
RESULTS Fos protein expression in neurons represents a readily measured and well-established early marker for cell-specific gene activation (Dragunow and Faull, 1989; Sagar et al., 1988). Rats received an i.v. infusion of secretin (40 g/ kg) or vehicle, were killed 60 min post-infusion, and their brains were processed for Fos immunohistochemistry. Evaluation of sections representing the entire rostra-caudal brain revealed increased Fos immunoreactivity in the
CeA, bed nucleus of the stria terminalis (BST), AP, external lateral parabrachial nucleus (PBe) and SON in secretin-treated rats compared with vehicle-treated animals (Fig. 1). The CeA showed Fos-positive neurons whereas the lateral, basolateral and medial amygdala regions showed no increase in Fos-positive neurons (Fig. 1B). A trend toward an increase in Fos-positive neurons was seen in the NTS, predominantly in the region proximal to the AP (Fig. 1D). The brain regions with secretin-induced Fos expression were the same in both 35-day- (not shown) and 60-day-old rats (data shown here). The same pattern of Fos expression was seen in fed and fasted rats with the exception that fasted rats did not display Fos-positive cells in the SON (data not shown). In a time-course study rats dosed with 40 g/kg secretin were killed at 60, 120, and 240 min post-infusion and brain Fos expression was detected as previously described. At 120 min, the number of Fos-positive neurons in the CeA of secretin-treated rats was increased seven-fold over vehicle-treated animals (Fig. 2). The left and right CeA displayed an equivalent response to secretin (data not shown). Fos staining in the CeA and SON was similar at 60 and 120 min but absent by 240 min. In contrast, Fos expression in the AP was highest at 60 min, decreased by 120 min and was completely absent by 240 min. This suggests that neuron activation in the AP may precede that in the CeA. At all time points, secretin did not induce Fos expression in the PVN, DMVN, NTS, or dorsal lateral parabrachial nucleus. Next, the effect of secretin dose (0.4 – 40 g/kg) on the number of Fos-positive neurons in the CeA was examined at the 120-min time point (Fig. 3). We found that the increase in Fos-positive neurons was significantly related to dose (ANOVA, P⫽0.001). The secretin-induced increase in Fos-positive cells reached significance at a dose of 4.0 g/kg (t-test, P⫽0.026, n⫽6). A similar dose response was seen for the AP (data not shown). Since Fos expression in the CeA after secretin infusion was dose dependent, it was relevant to determine if this effect was correlated with peptide blood levels. The plasma level of secretin was measured using a radioimmunoassay with a limit of quantitation of 20 pg/ml. Therefore, the baseline concentration of circulating secretin in rat, 6 pg/ml, (Li et al., 1995) was not detectable in this assay. The Cmax, area under the curve (AUC), and elimination rate were determined after a single dose of 0.4, 4, or 40 g/kg of secretin (Table 1). The AUC and Cmax both showed a linear increase with the number of Fos-positive neurons (R2⫽0.9998 and 0.9926, respectively). Thus the increase in Fos-positive cells in the CeA correlated well with secretin blood levels. Comparison with functionally and structurally related proteins CCK, like secretin, is a gut hormone involved with the regulation of pancreatic exocrine secretion. It is well established that CCK induces Fos expression in the CNS after peripheral administration (Day et al., 1994; Monnikes et al., 1997). We compared the pattern of brain Fos ex-
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Fig. 1. (Caption overleaf).
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Fig. 3. Effects of different doses of secretin on c-Fos expression in the central amygdala. Secretin (0.4 – 40 g/kg, i.v.) dose-dependently induced an increase in neuronal activity, as determined by the average number of Fos positive neurons/section. The number of rats per time point ⱖ4 and the bars represent means⫾SEM. *, P⬍0.05; ***, P⬍0.001 vs. Vehicle, T-test.
Fig. 2. Time course of secretin (40 g/kg) induced Fos expression in A) CeA, B) AP, C) PVN and D) SON, as determined by the average number of Fos positive neurons/section. The number of rats per time point is 3– 4 and the bars represent means⫾SEM. *, P⬍0.05; **, P⬍0.01; ***, P⬍0.001 vs. Vehicle. #, P⬍0.05 and ##, P⬍0.01 vs. 60 min, ANOVA followed by a Fisher probability of least significant difference test (PLSD).
pression 120 min after an equimolar dose of CCK (15 g/ kg) or secretin (40 g/kg). CCK induced Fos-positive neurons in the AP and CeA (Fig. 4). However, Fos expression was clearly more pronounced in the CeA (P⬍0.005) and the AP (P⬍0.001) after secretin versus after CCK treatment. Secretin induced a significant increase of Fos-positive neurons in the lateral division of the bed nucleus of the stria terminalis corresponding to the oval subnucleus (BST, Fig. 4B). No changes were observed in the ventral and posterior BST divisions (data not shown). In contrast to secretin, CCK treatment led to a pronounced increased in Fos expression in the NTS, DMVN and PVN (Fig. 4D–F) in agreement with previous reports (Day et al., 1994; Monnikes et al., 1997). Several other peptides administered peripherally, such as interleukin-1 (Brady et al., 1994) can induce a Fos expression pattern similar to that seen for CCK where NTS, PVN, CeA, and AP neurons are activated. It should be noted that in this experiment, a small but significant (P⬍0.05) increase in Fos-positive neurons was seen in the DMVN after secretin treatment (Fig. 4F) that had not been seen in other experiments (see Fig. 1). The structurally related peptides secretin, VIP and PACAP are expressed in the peripheral and CNS (Larson et al., 1976; Vaudry et al., 2000). We compared the effects of infusion of VIP and PACAP to the Fos expression pattern seen with secretin. VIP was infused at 44 g/kg, the molar equivalent to secretin, which induced slowed locomotion and muscular spasms in rats for the first 20 min after infusion. PACAP was infused at 6.3 g/kg in order to limit previously described vascular hypotensive activity (Kawai et al., 1994). VIP but not PACAP induced Fospositive cells in the CeA and AP (Fig. 4A, C). Under the conditions of this experiment, the secretin effect was more pronounced than VIP in Fos protein induction within the
Fig. 1. Photomicrographs of coronal brain sections taken from rats treated with vehicle (A, C, E, G) or secretin (B, D, F, H). Secretin induced Fos immunoreactive neurons in the amygdala (A, B), area postrema (C, D), supraoptic nucleus (E, F), and parabrachial nucleus (G, H). AP, area postrema; CeA, central amygdala; BLA, basolateral amygdala; MeA, medial amygdala; NTS, solitary tract nucleus; DMVN, dorsal motor vagal nucleus; SON, supraoptic nucleus; PBe, external lateral parabrachial nucleus; PBd, dorsal lateral parabrachial nucleus. Magnification 100⫻.
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Table 1. Pharmacokinetic parameters of intravenous secretin dosing in ratsa Dose (g/kg)
Cmax (ng/ml)
AUC (ng/ml) min
Elimination rate (min)
CeA Fos Neurons
0.4 4.0 40
0.55 7.46 23.9
2.4 33.1 153.8
2.4 2.3 19.1
3.5 15.1 58.5
a Area under the curve (AUC) and elimination rate were established from secretin concentration measurements in serial blood samples. For each dose, 18 rats were used. The maximum blood concentration (Cmax) is the value at 2 min post-dosing. A separate set of animals was used to count Fos-positive neurons in the central amygdala (CeA) as described in Experimental Procedures
CeA and AP. VIP, like secretin, showed a small increase in Fos-positive neurons in the DMVN. Neither VIP nor PACAP increased the number of Fos-positive neurons in the PVN or NTS (Fig. 4D, E). A more careful comparison of peptide-induced changes in the CeA (Fig. 4A) can be done by examining the number of Fos-positive cells within medial (CeM) and lateral (CeL) subregions of the CeA as well as subdividing the amygdala in a rostral to caudal accounting of Fospositive cells. Fig. 5 shows the number of CeA neurons that are Fos positive at five different rostro-caudal loca-
tions (bar graphs). Fos-immunoreactive cells induced by secretin within the CeA were mostly confined to the CeL (Fig. 5A–D) as only at the more rostral level were Fospositive cells observed in the CeM. VIP-induced Fos-positive cells were also located in the CeL but extended more caudal than with secretin treatment (Fig. 5E). This report establishes that i.v. infusion of secretin results in neuronal activation in defined brain regions as judged from Fos expression. Increases in the number of Fos-positive neurons following secretin administration could be demonstrated in the CeA, BST, SON, PBe, and
Fig. 4. Comparison of structurally or functionally related peptides for Fos protein expression following intravenous administration of vehicle (Veh), secretin (Sec, 40 g/kg), cholecystokinin (CCK, 15 g/kg), PACAP (6.3 g/kg), or vasoactive intestinal polypeptide (VIP, 44 g/kg). The average number of Fos positive neurons/section is presented for A) CeA, B) BST, C) AP D) PVN, E) NTS and F) DMVN. Data are expressed as the mean⫾SEM. *, ** and ***P⬍0.05, P⬍0.01 and P⬍0.001 vs vehicle; ### P⬍0.001 vs the other groups; ANOVA followed by a Fisher PLSD; ND, not done.
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Fig. 5. Quantitative expression and photomicrographs of Fos immunoreactivity in the central amygdala induced by i.v. injection of secretin and related peptides. The comparison of peptides for Fos protein expression was performed at different rostrocaudal levels as indicated for each bar graph. Each column represents the mean⫾SEM of cell number (cells/section, unilateral) from 1–2 sections/rat for 2– 4 rats for each treatment. Photomicrographs of secretin (A–D) and VIP (E) are taken from the rostrocaudal level indicated for the adjacent bar graph. Boundaries for the lateral (CeL) and medial (CeM) divisions of the CeA are indicated. *, P⬍0.05; **, P⬍0.01; ***, P⬍0.001 vs. vehicle. ###, P ⬍0.001 vs. other peptide treatments using ANOVA followed by a Fisher PLSD.
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AP. Since knowledge of the long-term effects of Fos expression on neuronal activity remains limited, ongoing studies aim to establish changes in expression for markers other than c-fos occurring in these brain regions subsequent to secretin infusion. Additional studies are also required to define the route(s) or mechanisms by which i.v. secretin activates genes in the CeA, AP, BST, SON, and PBe. Endogenous release of secretin affects pancreatic fluid release and reduces gastric motility through a vagal-dependent pathway (Jin et al., 1994). However, supraphysiologic blood levels of secretin (100-fold endogenous levels) affect pancreatic fluid release by a mechanism independent of the vagus (Lu and Owyang, 1995). We have demonstrated in mice that secretin can transfer from blood to brain (Banks et al., 2002). In addition, activation of the CeA may be mediated through activation of the AP, which is a circumventricular organ and could be activated by bloodborne peptide. Therefore, neuronal signaling by supraphysiologic doses of secretin as used in this study or currently in humans may occur by more than one route and may differ from the neuronal signaling that occurs after endogenous secretin release. Autism is a disease characterized by significant social and communication deficits that are usually apparent before the age of 3. Reciprocal social interaction requires recognition of socially relevant information from faces and involves neural processing in the amygdala (Adolphs, 1999; Emery, 2000). Multiple investigators have used functional magnetic resonance imaging to observe abnormal amygdala function in autistic subjects during socially relevant performance tasks (Critchley et al., 2000; Pierce et al., 2001; Schultz et al., 2000). The convergence of behavioral and biological data from both primate and human studies have led to published hypotheses that amygdala dysfunction is central to the etiology of autism (BaronCohen et al., 2000; Howard et al., 2000). Data presented here demonstrate that in rats infusion of secretin at a clinically relevant dose affects gene expression in several brain regions, most notably, the CeA.
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(Accepted 5 October 2002)