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Effects of secretin on neuronal activity and feeding behavior in central amygdala of rats
Peptides 66 (2015) 1–8
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Peptides journal homepage: www.elsevier.com/locate/peptides
Effects of secretin on neuronal activity and feeding behavior in central amygdala of rats Ya-Yan Pang a , Xin-Yi Chen b , Yan Xue a , Xiao-Hua Han a , Lei Chen a,∗ a b
Department of Physiology, Faculty of Medicine, Qingdao University, Qingdao 266071, China Department of Neurology, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China
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Article history: Received 3 October 2014 Received in revised form 15 December 2014 Accepted 8 January 2015 Available online 16 February 2015 Keywords: Amygdala Secretin Single unit recording Feeding
a b s t r a c t Previous studies have shown that secretin and secretin receptors are expressed in central amygdala neurons. By using both in vivo extracellular recording as well as behavioral test, we investigated the direct electrophysiological effects of secretin in the central amygdala and its involvement in feeding behavior. Micro-pressure ejection of secretin increased the spontaneous firing rate by 104.22 ± 26.18% in 13 out of the 27 central amygdala neurons. In other 6 out of the 27 neurons, secretin decreased the firing rate by 68.80 ± 12.10%. Firing patter analysis showed that secretin did not change the firing pattern significantly. Further electrophysiological recordings revealed that secretin decreased the firing rate of glucose-sensitive neurons. In behavioral test, microinjection of secretin into the central amygdala significantly reduced cumulative food intake through cAMP-activated protein kinase activation. Based on the present electrophysiological and behavioral findings, we hypothesized that secretin may suppress food intake by its modulation of spontaneous firing of central amygdala neurons. © 2015 Elsevier Inc. All rights reserved.
Introduction Secretin is a 27-amino acid peptide which belongs to the secretin/glucagon superfamily. Since its discovery in 1902 [1], secretin has been extensively investigated for its peripheral effects on digestion. In the last three decades, investigators have made great strides in understanding the neurophysiological functions of secretin in central nervous system. The secretin receptor is a G protein coupled receptor with 7 transmembrane domains. In different regions of brain, activation of the secretin receptor activates cAMP pathway to produce diverse potential functions [2–5]. Secretin and secretin receptors are widely expressed in central nervous system. In human brain, secretin immunoreactivity was observed in cerebellum, motor cortex, hippocampus and amygdala [6]. Furthermore, in rat brain, secretin and secretin receptor mRNA expression in central amygdala, hippocampus, nucleus of tractus solitary and cerebellum was age-related [7]. The amygdala consists of two different functional subdivisions, the basolateral complex of the amygdala and the central nucleus of the amygdala. It is well known that the amygdala plays
∗ Corresponding author at: Department of Physiology, Faculty of Medicine, Qingdao University, Qingdao 266071, Shandong, China. Tel.: +86 532 83780051; fax: +86 532 83780136. E-mail address: [email protected] (L. Chen). http://dx.doi.org/10.1016/j.peptides.2015.01.012 0196-9781/© 2015 Elsevier Inc. All rights reserved.
important roles including fear conditioning, reward learning, cognitive functions, cardiovascular functions. Central amygdala neurons connect extensively to extrinsic structures, such as the hypothalamus and brainstem areas [8–10]. Previous extracellular single neuron recordings revealed that there are glucose-sensitive neurons in both rat and monkey amygdala. Local administration of glucose suppressed the basal firing rate in a small part of amygdala neurons [11]. The central amygdala is essentially involved in the regulation of feeding behavior [12]. Several peptides, including enkephalin, neurotensin and corticotropin-releasing hormone, are expressed in central amygdala neurons [13]. In addition, morphological studies have revealed the expression of secretin and secretin receptors in central amygdala neurons [6,7]. By using in vitro autoradiography, moderate secretin binding was observed in amygdala [14]. Peripheral administration of secretin induced Fos expression in central amygdala of rats [15,16]. Furthermore, central injection (intracerebroventricular) of secretin induced Fos immunoreactivity in medial and central amygdala but attenuated Fos immunoreactivity in lateral amygdala [17]. In humans, administration of secretin increased fMRI activation in the right central nucleus of amygdala [18]. Based on the findings that secretin and secretin receptors are expressed in the central amygdala, together with both central and peripheral secretin induced Fos expression, we aim to study the direct in vivo electrophysiological effects of secretin on neuronal activity of central amygdala neurons as well as the possible involvement in feeding behavior.
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Fig. 1. Confirmation of the sites of single unit recordings in central amygdala. (A) A typical photomicrograph revealing the traces of microelectrodes (arrow) in central amygdala. (B) High-magnification photomicrograph showing the pontamine sky blue (arrow) ejected from the recording electrode tip in central amygdala. (C) Brain structure diagrams of coronal sections revealing the single unit recording sites. The distances from Bregma were shown on the right. Circles: sites of neurons with secretin-induced increase in firing rate (n = 13); squares: sites of neurons with secretin-induced decrease in firing rate (n = 6).
Materials and methods Adult male Wistar rats (200–250 g) were used in the present study. The rats were housed in a temperature controlled (25 ± 2 ◦ C) room and maintained on a 12 h light/dark cycle (lights on at 06:00 a.m.). Food and water were available throughout the experiment. The experiment was approved and all procedures were performed in strict accordance with institutional guidelines of the Animal Care and Use Committee at Qingdao University. Electrophysiological recordings Rats were anesthetized with 20% urethane (1 g/kg, i.p.) in the morning and supplemental anesthetics were administered as needed during the experiments. The anesthetized rats were placed in a stereotaxic frame (Narishige SN-3, Tokyo, Japan). A small hole was drilled in the skull to expose the cortex and the dura was cut. Open part of the brain was covered with warm agar (3% in saline) to improve stability for neuronal recording. According to a standard stereotaxic atlas [19], the recording area of the central amygdala was: 2.3–2.8 mm posterior to bregma, 4.1–4.3 mm lateral to the midline and 7.7–8.2 mm below the outer surface of the skull. Rectal temperature was monitored and maintained at 36–38 ◦ C by use of a heating pad. The microelectrodes of three-barrel (total tip diameter: 3–10 m, resistance: 15–20 M) were prepared using a Stoelting pipette puller (IL, USA). The electrodes were stereotaxically
positioned within the central amygdala for single-unit recordings and micro-pressure ejection. The recording barrel of the electrode was filled with 0.5 M sodium acetate and 2% pontamine sky blue dye. The other two micropressure ejection barrels connected to a four-channel pressure injector (PM2000B; Micro Data Instrument, Inc., USA) contained secretin and vehicle, or secretin and 0.5 M glucose, respectively. Neurons were identified as central amygdala neurons on the basis of their location and electrophysiological features [19,20]. Drugs were ejected onto the surface of firing cells with short gas pressure pulse (1500 ms, 5.0–15.0 psi). The recorded electrical signals were amplified by a microelectrode amplifier (MEZ-8201; Nihon Kohden, Tokyo, Japan) and displayed on a memory oscilloscope (VC-11; Nihon Kohden) while being fed to an audiomonitor. The amplified electrical signals were passed through low and high pass filters into a bioelectricity signal analyzer and computer. Spike data acquisition and analysis were preprocessed with a Micro1401 and spike 2 software (Cambridge Electronic Design, UK). Firing parameters including firing rate, coefficient of variation (CV) of the interspike interval and Fano Factor (FF) were calculated. The basal spontaneous firing was determined by the average of 2–5 min stable recording. The maximal change within 50 s following drug application was considered as drug effect. The threshold of significance of firing rate was established based on deviations from a mean firing rate. An increase in firing rate was considered significant if the elevation in firing rate exceeded 2 standard deviations (SDs). A significant decrease in firing rate was defined as activity falling below a threshold of mean
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Fig. 2. Secretin increased the spontaneous firing rate of central amygdala neurons. (A) Typical frequency histograms showing that 0.1 M secretin increased the firing rate of a central amygdala neuron by 310.53%. In this neuron, firing rate started to increase 170 s after secretin administration and recovered to normal 390 s after secretin administration. The firing of the neuron, displayed at a faster time base, at different stages of experiment are shown in lower trace. (B) Pooled data summarizing the effects of secretin on the firing rate of central amygdala neurons. ***P < 0.001.
rate – 2 SDs of this mean. The CV is defined as the ratio of the standard deviation of interspike interval to its mean. The FF is defined as the ratio of the variance of the spike count to its mean evaluated for long time windows. Drug application was performed only once for each recording and only one recording was allowed in the same track.
of drugs (secretin or H89 or H89 with secretin) or vehicle per side was microinjected into the central amygdala of awake rats over 2 min. At the end of injection, the injector was left in place for an additional 1 min before removal and then replaced by a stylet. Then the rats were allowed to re-feed. The cumulative food intake was measured from 18:00 at different time points up to 12 h and calculated as g/300 g body weight [21].
Feeding behavior Histological controls Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic frame gently. Two guide cannulae constructed from stainless steel (o.d., 0.4 mm; i.d., 0.3 mm) were implanted into a region above the central amygdala on both sides. The cannulae were fixed to the skull with stainless steels crews and dental acrylic. Stainless steel stylets were used to keep the cannulae sealed. Following at least 5 days of recovery, rats were placed in metabolic cages which they had already become habituated. Food was withdrawn from the rats at 15:00. At 18:00, a volume of 0.5 l
To confirm the position of a single-unit recording, pontamine sky blue was ejected from the recording electrode tip by iontophoresis (10 A, 20 min). To assess the extent of drug spread in behavioral test, 0.5 l pontamine sky blue was microinjected into the central amygdala. All the rats used in electrophysiological and behavioral experiments were anesthetized with chloral hydrate (600 mg/kg, i.p.) and perfused with 4% paraformaldehyde solution transcardially. Then, the brains were dissected out and incubated in paraformaldehyde overnight. After that, the brains were frozen
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Fig. 3. Secretin decreased the spontaneous firing rate of central amygdala neurons. (A) Typical frequency histograms showing that 0.1 M secretin decreased the firing rate of a central amygdala neuron by 86.25%. In this cell, firing rate started to increase 120 s after secretin administration and recovered to normal 430 s after secretin administration. The firing of the neuron, displayed at a faster time base, at different stages of experiment are shown in lower trace. (B) Pooled data summarizing the effects of secretin on the firing rate of central amygdala neurons. *P < 0.05.
and sectioned at 50 m, and all the recording and microinjection sites were identified under a light microscope. If the recording sites were outside of the central amygdala, the data were excluded from the analysis (Fig. 1). Drugs and statistical analysis Secretin and H89 dihydrochloride were obtained from Tocris (Avonmouth, UK). All data are expressed as means ± S.E.M. Paired t test was used to compare the difference of firing rate before and after treatment. Statistical comparisons between or among groups were determined with Student’s t test and one-way ANOVA. The level of significance was preset by using a P-value of 0.05. Results Secretin modulated the spontaneous firing of central amygdala neurons The basal spontaneous firing rate of central amygdala neurons ranged from 0.025 Hz to 13.23 Hz, with the average firing rate of
1.91 Hz. Consistent with previous studies, about 55% recorded neurons displayed spontaneous firing at a rate lower than 1 Hz. To observe the direct in vivo electrophysiological effects of secretin in the central amygdala, micro-pressure administration of secretin was performed in normal rats. As shown in Fig. 2, micro-pressure ejection of 0.1 M secretin significantly increased the spontaneous firing rate from 2.30 ± 0.91 Hz to 3.20 ± 1.07 Hz (P < 0.001) in 13 out of the 27 central amygdala neurons. The average increase of firing rate was 104.22 ± 26.18%, which was significantly higher than that of normal saline (P < 0.01). However, in other 6 out of the 27 neurons, secretin significantly decreased the spontaneous firing rate from 6.03 ± 2.13 Hz to 2.20 ± 1.39 Hz (P < 0.05, Fig. 3), with the average decrease of 68.80 ± 12.10% (P < 0.001 compared with normal saline). In the remaining 8 neurons, secretin did not change the firing rate significantly. Further studies were performed to observe the effects of a higher concentration of secretin on central amygdala neurons. Secretin at 0.1 mM caused a 228.42 ± 48.40% increase in the firing rate in 15 neurons, which was significantly stronger than that of 0.1 M secretin (P < 0.05). Two major firing patterns of central amygdala neurons (i.e. bursting firing with moderate and variable firing rate, and
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Fig. 4. Firing patterns observed in central amygdala neurons. The representative recordings showing neurons with bursting firing (A) and irregular firing (B). Left panel: spike train of the example neuron; middle panel: ISI histogram; right panel: autocorrelation using a time window of ±1 s.
irregular firing with low and variable firing rate) were classified in the present recordings (Fig. 4). To observe the effects of secretin on the firing pattern, we further analyzed the CV and FF before and after secretin application. In the neurons with secretin-induced increase in firing rate, micro-pressure ejection of secretin did not change the CV (basal: 1.19 ± 0.16; secretin: 1.17 ± 0.16; P > 0.05) and the FF (basal: 2.74 ± 0.80; secretin: 1.79 ± 0.52; P > 0.05) significantly. In the neurons with secretin-induced decrease in firing rate, secretin did not change the CV (basal: 1.03 ± 0.08; secretin: 1.29 ± 0.12; P > 0.05), as well as the FF (basal: 0.64 ± 0.43; secretin: 2.60 ± 1.53; P > 0.05) significantly. Modulation of spontaneous firing by secretin on glucose-sensitive neurons We next observed the effects of secretin on glucose-sensitive neurons in the central amygdala. In total 117 central amygdala neurons, micro-pressure application of 0.5 M glucose significantly decreased the basal firing rate in 16 neurons. Therefore, in the present study, 13.7% of central amygdala neurons belong to glucose-sensitive neurons. In 7 out of the 16 glucose-sensitive neurons (basal: 4.15 ± 1.28 Hz, glucose: 2.15 ± 0.79 Hz, P < 0.05, n = 7) with long time stable recording, further application of secretin (0.1 M) decreased the firing rate from 5.31 ± 1.48 Hz to 1.74 ± 1.01 Hz (P < 0.01, Fig. 5). The average decrease was 75.32 ± 10.47% (P < 0.001 compared with normal saline). Feeding behavior induced by bilateral microinjection of secretin into the central amygdala Next, feeding behavioral tests were performed to study the possible involvement of secretin in the central amygdala. Bilateral microinjection of 0.1 M secretin into the central amygdala significantly decreased cumulative food intake. As shown in Fig. 6A, food intake was significantly lower in rats treated with secretin as
compared to vehicle-treated rats. The secretin-induced decrease in food intake at the time points of 1 h, 2 h, 4 h and 12 h was 46.08 ± 8.54% (P < 0.001), 44.41 ± 8.87% (P < 0.01), 54.00 ± 6.76% (P < 0.001) and 35.66 ± 9.49% (P < 0.01), respectively. In another group of experiment, we observed the effects of H89 (an inhibitor of cAMP-activated protein kinase) on secretin-induced change of cumulative food intake. Compared with H89 (10 M) alone, co-injection of H89 and secretin (0.1 M) did not change the cumulative food intake significantly (P > 0.05 at the time points of 1 h, 2 h, 4 h and 12 h, n = 11, Fig. 6B), suggesting the possible involvement of cAMP-PKA pathway in secretin-induced decrease of food intake.
Discussion The present in vivo studies showed that secretin modulated the spontaneous electrical activity of central amygdala neurons. Local administration of secretin increased the firing rate in most central amygdala neurons. Previous morphological studies have demonstrated that secretin and secretin receptors are widely expressed in central nervous system, including cerebellum, motor cortex, hippocampus and amygdala [4]. By using quantitative real-time PCR, Tay et al. [7] have reported that secretin and secretin receptor mRNA are detected in the central amygdala, hippocampus, area postrema, nucleus of the tractus solitary and cerebellum. Furthermore, both secretin and secretin receptor expression are age-related in most brain regions. In humans, administration of secretin increased amygdala fMRI activation [18], suggesting amygdala as a possible functional point of secretin. Goulet et al. [15] have reported that i.v. infusion of secretin induced neuronal activation in central amygdala and other defined brain regions. It is known that secretin can transfer from blood to brain [22]. Therefore, at high peripheral concentrations, secretin may enter the brain to influence gene expression in the central nucleus of amygdala. The present single unit recordings identified that secretin may directly activate
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Fig. 5. Effects of secretin on the firing rate of glucose-sensitive neurons in central amygdala. (A) Typical frequency histograms showing that 0.1 M secretin decreased the firing rate of a central amygdala neuron which was inhibited by 0.5 M glucose. In this cell, firing rate started to decrease 120 s after glucose administration, partial recovered to stable after 500 s. Glucose and secretin decreased the firing rate by 43.86% and 44.99%, respectively. (B) Pooled data summarizing the effects of secretin on glucose-sensitive neurons. *P < 0.05, **P < 0.01.
secretin receptor and modulate the spontaneous firing activity of central amygdala neurons. In addition to excitation, secretin also inhibited the basal firing rate in a small portion of central amygdala neurons. Recently, Keshavarzi et al. [23] reported that the posteroventral division of the medial amygdala contained three types of GABAergic neurons and two types of non-GABAergic neurons. The recorded basal firing rate ranging from 0.025 Hz to 13.23 Hz suggests that the central nucleus of amygdala may contain several different subpopulations of neurons. Therefore, the present secretin-induced different electrophysiological effects, excitation and inhibition, may be caused by different subpopulations of neurons. However, for the technical limitation, it was impossible to isolate the recorded neurons and define the types. Consistent with previous studies, the basal firing rate of central amygdala neurons are very low, which could be due to the tonic activity of numerous GABAergic interneurons in the nucleus [24,25]. Lénárd et al. [11] reported that physiological concentration of glucose decreased the basal firing rate in a subpopulation of rat amygdala neurons, subsequently named glucose-monitoring neurons. Consistently, early studies have revealed that the activity of some amygdala (mostly located in the centromedial part of the amygdala) neurons in monkey was inhibited by glucose. These glucose-sensitive cells are also responded to morphine, which may
be involved in the control of food acquisition behavior [26]. The present single unit recording showed that the central amygdala contains glucose-sensitive neurons with the basal firing activity inhibited by glucose. Many studies indicated that glucose-sensitive neurons located in hypothalamus and other brain regions are involved in glucoprivic feeding and homeostatic control of blood glucose [27,28]. Further experiments showed that secretin mainly exerted inhibitory effects on the spontaneous firing rate of glucosesensitive neurons. Being an important functional subdivision in amygdala, the central nucleus of amygdala is essentially involved in the regulation of feeding behavior. For example, Anderberg and the colleagues [29] recently reported that dopamine reduced food intake by activation of D2 receptors in central nucleus of amygdala. Microinjection of RFRP-1 into central nucleus of amygdala decreased liquid food consumption in rats [30]. Administration of ghrelin into the central amygdala increased food intake which may be related with the activation of arcuate nucleus [31]. The central nucleus of amygdala is also involved in melanocortin-induced inhibition of food intake [32]. Cheng et al. [33] first demonstrated that intracerebroventricular microinjection of secretin suppressed food intake dose-dependently in wide type mice but not in secretin receptor-deficient mice, suggesting secretin as an anorectic peptide. The present feeding behavioral studies showing that local
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activated by diverse anorexigenic signals. Optogenetic activation of these neurons in vivo strongly reduced food intake in mice. It is well known that the central nucleus of amygdala contains several neurotransmitters or neuromodulators which are involved in the control of feeding behavior. It has been shown that specific catecholaminergic microlesions reducing the norepinephrine content in amygdala produced hyperphagia and weight increase, while dopamine depletion caused hypophagia and weight decrease [40,41]. Endogenous melanocortin receptor agonists in the central amygdala exerted a tonic inhibitory influence on food consumption by stimulating MC4 receptors [32]. Therefore, secretin may control feeding behavior by its modulation of other neurotransmitter systems. Consistently, Cheng et al. [33] demonstrated that intracerebroventricular secretin-induced suppression of food intake is mediated by melanocortin system. In conclusion, the present studies indicated that secretin increased the neuronal activity in most central amygdala neurons. However, in glucose-sensitive neurons, secretin decreased the firing rate. Bilateral microinjection of secretin into the central amygdala significantly reduced cumulative food intake through cAMP-activated protein kinase activation. Based on the present electrophysiological and behavioral findings, we hypothesized that secretin may suppress food intake by its modulation of neuronal activity of central amygdala neurons. Conflict of interest statement The authors declare no conflict interest. Acknowledgements
Fig. 6. Feeding related effects of secretin in the central amygdala. (A) Cumulative food intake after bilateral microinjection of secretin (0.1 M, 0.5 l/side) or normal saline into the central amygdala. **P < 0.01, ***P < 0.001. (B) Cumulative food intake induced by bilateral microinjection of H89 and H89 with secretin. No significant difference between H89 and H89 with secretin at different time points.
administration of secretin decreased food intake provides further evidence that the central nucleus of amygdala may be one of the brain regions involved in secretin-induced inhibition of food intake. Consistently, previous studies revealed that intracerebroventricular administration of glucagon-like peptide 1 inhibited feeding by its activity on central nucleus of the amygdala [29,34]. Similarly, peripheral administration of glucagon-like peptide 1 and glucagon decreased food intake and increased c-fos expression in the central nucleus of amygdala [35]. Based on present secretin-induced in vivo electrophysiological effects, we hypothesized that secretin in the central amygdala suppressed food intake by its excitation on the spontaneous firing of central amygdala neurons. In support of our hypothesis, early studies have shown that amygdaloid lesion induced hyperphagia and obesity in both female and male rats [36]. Bovetto and Richard [37] reported that lesion of central nucleus of amygdala promotes food intake and fat gain in rats. Furthermore, Levine et al. [38] reported that activation of mu opioid receptors in the central nucleus of amygdala increased feeding behavior, probably by inhibiting the activity of selected amygdala neurons. More recently, Cai et al. [39] demonstrated that the lateral part of central amygdala nucleus contains a subpopulation of PKC-␦(+) GABAergic neurons, which were
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Peptides journal homepage: www.elsevier.com/locate/peptides
Effects of secretin on neuronal activity and feeding behavior in central amygdala of rats Ya-Yan Pang a , Xin-Yi Chen b , Yan Xue a , Xiao-Hua Han a , Lei Chen a,∗ a b
Department of Physiology, Faculty of Medicine, Qingdao University, Qingdao 266071, China Department of Neurology, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China
a r t i c l e
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Article history: Received 3 October 2014 Received in revised form 15 December 2014 Accepted 8 January 2015 Available online 16 February 2015 Keywords: Amygdala Secretin Single unit recording Feeding
a b s t r a c t Previous studies have shown that secretin and secretin receptors are expressed in central amygdala neurons. By using both in vivo extracellular recording as well as behavioral test, we investigated the direct electrophysiological effects of secretin in the central amygdala and its involvement in feeding behavior. Micro-pressure ejection of secretin increased the spontaneous firing rate by 104.22 ± 26.18% in 13 out of the 27 central amygdala neurons. In other 6 out of the 27 neurons, secretin decreased the firing rate by 68.80 ± 12.10%. Firing patter analysis showed that secretin did not change the firing pattern significantly. Further electrophysiological recordings revealed that secretin decreased the firing rate of glucose-sensitive neurons. In behavioral test, microinjection of secretin into the central amygdala significantly reduced cumulative food intake through cAMP-activated protein kinase activation. Based on the present electrophysiological and behavioral findings, we hypothesized that secretin may suppress food intake by its modulation of spontaneous firing of central amygdala neurons. © 2015 Elsevier Inc. All rights reserved.
Introduction Secretin is a 27-amino acid peptide which belongs to the secretin/glucagon superfamily. Since its discovery in 1902 [1], secretin has been extensively investigated for its peripheral effects on digestion. In the last three decades, investigators have made great strides in understanding the neurophysiological functions of secretin in central nervous system. The secretin receptor is a G protein coupled receptor with 7 transmembrane domains. In different regions of brain, activation of the secretin receptor activates cAMP pathway to produce diverse potential functions [2–5]. Secretin and secretin receptors are widely expressed in central nervous system. In human brain, secretin immunoreactivity was observed in cerebellum, motor cortex, hippocampus and amygdala [6]. Furthermore, in rat brain, secretin and secretin receptor mRNA expression in central amygdala, hippocampus, nucleus of tractus solitary and cerebellum was age-related [7]. The amygdala consists of two different functional subdivisions, the basolateral complex of the amygdala and the central nucleus of the amygdala. It is well known that the amygdala plays
∗ Corresponding author at: Department of Physiology, Faculty of Medicine, Qingdao University, Qingdao 266071, Shandong, China. Tel.: +86 532 83780051; fax: +86 532 83780136. E-mail address: [email protected] (L. Chen). http://dx.doi.org/10.1016/j.peptides.2015.01.012 0196-9781/© 2015 Elsevier Inc. All rights reserved.
important roles including fear conditioning, reward learning, cognitive functions, cardiovascular functions. Central amygdala neurons connect extensively to extrinsic structures, such as the hypothalamus and brainstem areas [8–10]. Previous extracellular single neuron recordings revealed that there are glucose-sensitive neurons in both rat and monkey amygdala. Local administration of glucose suppressed the basal firing rate in a small part of amygdala neurons [11]. The central amygdala is essentially involved in the regulation of feeding behavior [12]. Several peptides, including enkephalin, neurotensin and corticotropin-releasing hormone, are expressed in central amygdala neurons [13]. In addition, morphological studies have revealed the expression of secretin and secretin receptors in central amygdala neurons [6,7]. By using in vitro autoradiography, moderate secretin binding was observed in amygdala [14]. Peripheral administration of secretin induced Fos expression in central amygdala of rats [15,16]. Furthermore, central injection (intracerebroventricular) of secretin induced Fos immunoreactivity in medial and central amygdala but attenuated Fos immunoreactivity in lateral amygdala [17]. In humans, administration of secretin increased fMRI activation in the right central nucleus of amygdala [18]. Based on the findings that secretin and secretin receptors are expressed in the central amygdala, together with both central and peripheral secretin induced Fos expression, we aim to study the direct in vivo electrophysiological effects of secretin on neuronal activity of central amygdala neurons as well as the possible involvement in feeding behavior.
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Fig. 1. Confirmation of the sites of single unit recordings in central amygdala. (A) A typical photomicrograph revealing the traces of microelectrodes (arrow) in central amygdala. (B) High-magnification photomicrograph showing the pontamine sky blue (arrow) ejected from the recording electrode tip in central amygdala. (C) Brain structure diagrams of coronal sections revealing the single unit recording sites. The distances from Bregma were shown on the right. Circles: sites of neurons with secretin-induced increase in firing rate (n = 13); squares: sites of neurons with secretin-induced decrease in firing rate (n = 6).
Materials and methods Adult male Wistar rats (200–250 g) were used in the present study. The rats were housed in a temperature controlled (25 ± 2 ◦ C) room and maintained on a 12 h light/dark cycle (lights on at 06:00 a.m.). Food and water were available throughout the experiment. The experiment was approved and all procedures were performed in strict accordance with institutional guidelines of the Animal Care and Use Committee at Qingdao University. Electrophysiological recordings Rats were anesthetized with 20% urethane (1 g/kg, i.p.) in the morning and supplemental anesthetics were administered as needed during the experiments. The anesthetized rats were placed in a stereotaxic frame (Narishige SN-3, Tokyo, Japan). A small hole was drilled in the skull to expose the cortex and the dura was cut. Open part of the brain was covered with warm agar (3% in saline) to improve stability for neuronal recording. According to a standard stereotaxic atlas [19], the recording area of the central amygdala was: 2.3–2.8 mm posterior to bregma, 4.1–4.3 mm lateral to the midline and 7.7–8.2 mm below the outer surface of the skull. Rectal temperature was monitored and maintained at 36–38 ◦ C by use of a heating pad. The microelectrodes of three-barrel (total tip diameter: 3–10 m, resistance: 15–20 M) were prepared using a Stoelting pipette puller (IL, USA). The electrodes were stereotaxically
positioned within the central amygdala for single-unit recordings and micro-pressure ejection. The recording barrel of the electrode was filled with 0.5 M sodium acetate and 2% pontamine sky blue dye. The other two micropressure ejection barrels connected to a four-channel pressure injector (PM2000B; Micro Data Instrument, Inc., USA) contained secretin and vehicle, or secretin and 0.5 M glucose, respectively. Neurons were identified as central amygdala neurons on the basis of their location and electrophysiological features [19,20]. Drugs were ejected onto the surface of firing cells with short gas pressure pulse (1500 ms, 5.0–15.0 psi). The recorded electrical signals were amplified by a microelectrode amplifier (MEZ-8201; Nihon Kohden, Tokyo, Japan) and displayed on a memory oscilloscope (VC-11; Nihon Kohden) while being fed to an audiomonitor. The amplified electrical signals were passed through low and high pass filters into a bioelectricity signal analyzer and computer. Spike data acquisition and analysis were preprocessed with a Micro1401 and spike 2 software (Cambridge Electronic Design, UK). Firing parameters including firing rate, coefficient of variation (CV) of the interspike interval and Fano Factor (FF) were calculated. The basal spontaneous firing was determined by the average of 2–5 min stable recording. The maximal change within 50 s following drug application was considered as drug effect. The threshold of significance of firing rate was established based on deviations from a mean firing rate. An increase in firing rate was considered significant if the elevation in firing rate exceeded 2 standard deviations (SDs). A significant decrease in firing rate was defined as activity falling below a threshold of mean
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Fig. 2. Secretin increased the spontaneous firing rate of central amygdala neurons. (A) Typical frequency histograms showing that 0.1 M secretin increased the firing rate of a central amygdala neuron by 310.53%. In this neuron, firing rate started to increase 170 s after secretin administration and recovered to normal 390 s after secretin administration. The firing of the neuron, displayed at a faster time base, at different stages of experiment are shown in lower trace. (B) Pooled data summarizing the effects of secretin on the firing rate of central amygdala neurons. ***P < 0.001.
rate – 2 SDs of this mean. The CV is defined as the ratio of the standard deviation of interspike interval to its mean. The FF is defined as the ratio of the variance of the spike count to its mean evaluated for long time windows. Drug application was performed only once for each recording and only one recording was allowed in the same track.
of drugs (secretin or H89 or H89 with secretin) or vehicle per side was microinjected into the central amygdala of awake rats over 2 min. At the end of injection, the injector was left in place for an additional 1 min before removal and then replaced by a stylet. Then the rats were allowed to re-feed. The cumulative food intake was measured from 18:00 at different time points up to 12 h and calculated as g/300 g body weight [21].
Feeding behavior Histological controls Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic frame gently. Two guide cannulae constructed from stainless steel (o.d., 0.4 mm; i.d., 0.3 mm) were implanted into a region above the central amygdala on both sides. The cannulae were fixed to the skull with stainless steels crews and dental acrylic. Stainless steel stylets were used to keep the cannulae sealed. Following at least 5 days of recovery, rats were placed in metabolic cages which they had already become habituated. Food was withdrawn from the rats at 15:00. At 18:00, a volume of 0.5 l
To confirm the position of a single-unit recording, pontamine sky blue was ejected from the recording electrode tip by iontophoresis (10 A, 20 min). To assess the extent of drug spread in behavioral test, 0.5 l pontamine sky blue was microinjected into the central amygdala. All the rats used in electrophysiological and behavioral experiments were anesthetized with chloral hydrate (600 mg/kg, i.p.) and perfused with 4% paraformaldehyde solution transcardially. Then, the brains were dissected out and incubated in paraformaldehyde overnight. After that, the brains were frozen
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Fig. 3. Secretin decreased the spontaneous firing rate of central amygdala neurons. (A) Typical frequency histograms showing that 0.1 M secretin decreased the firing rate of a central amygdala neuron by 86.25%. In this cell, firing rate started to increase 120 s after secretin administration and recovered to normal 430 s after secretin administration. The firing of the neuron, displayed at a faster time base, at different stages of experiment are shown in lower trace. (B) Pooled data summarizing the effects of secretin on the firing rate of central amygdala neurons. *P < 0.05.
and sectioned at 50 m, and all the recording and microinjection sites were identified under a light microscope. If the recording sites were outside of the central amygdala, the data were excluded from the analysis (Fig. 1). Drugs and statistical analysis Secretin and H89 dihydrochloride were obtained from Tocris (Avonmouth, UK). All data are expressed as means ± S.E.M. Paired t test was used to compare the difference of firing rate before and after treatment. Statistical comparisons between or among groups were determined with Student’s t test and one-way ANOVA. The level of significance was preset by using a P-value of 0.05. Results Secretin modulated the spontaneous firing of central amygdala neurons The basal spontaneous firing rate of central amygdala neurons ranged from 0.025 Hz to 13.23 Hz, with the average firing rate of
1.91 Hz. Consistent with previous studies, about 55% recorded neurons displayed spontaneous firing at a rate lower than 1 Hz. To observe the direct in vivo electrophysiological effects of secretin in the central amygdala, micro-pressure administration of secretin was performed in normal rats. As shown in Fig. 2, micro-pressure ejection of 0.1 M secretin significantly increased the spontaneous firing rate from 2.30 ± 0.91 Hz to 3.20 ± 1.07 Hz (P < 0.001) in 13 out of the 27 central amygdala neurons. The average increase of firing rate was 104.22 ± 26.18%, which was significantly higher than that of normal saline (P < 0.01). However, in other 6 out of the 27 neurons, secretin significantly decreased the spontaneous firing rate from 6.03 ± 2.13 Hz to 2.20 ± 1.39 Hz (P < 0.05, Fig. 3), with the average decrease of 68.80 ± 12.10% (P < 0.001 compared with normal saline). In the remaining 8 neurons, secretin did not change the firing rate significantly. Further studies were performed to observe the effects of a higher concentration of secretin on central amygdala neurons. Secretin at 0.1 mM caused a 228.42 ± 48.40% increase in the firing rate in 15 neurons, which was significantly stronger than that of 0.1 M secretin (P < 0.05). Two major firing patterns of central amygdala neurons (i.e. bursting firing with moderate and variable firing rate, and
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Fig. 4. Firing patterns observed in central amygdala neurons. The representative recordings showing neurons with bursting firing (A) and irregular firing (B). Left panel: spike train of the example neuron; middle panel: ISI histogram; right panel: autocorrelation using a time window of ±1 s.
irregular firing with low and variable firing rate) were classified in the present recordings (Fig. 4). To observe the effects of secretin on the firing pattern, we further analyzed the CV and FF before and after secretin application. In the neurons with secretin-induced increase in firing rate, micro-pressure ejection of secretin did not change the CV (basal: 1.19 ± 0.16; secretin: 1.17 ± 0.16; P > 0.05) and the FF (basal: 2.74 ± 0.80; secretin: 1.79 ± 0.52; P > 0.05) significantly. In the neurons with secretin-induced decrease in firing rate, secretin did not change the CV (basal: 1.03 ± 0.08; secretin: 1.29 ± 0.12; P > 0.05), as well as the FF (basal: 0.64 ± 0.43; secretin: 2.60 ± 1.53; P > 0.05) significantly. Modulation of spontaneous firing by secretin on glucose-sensitive neurons We next observed the effects of secretin on glucose-sensitive neurons in the central amygdala. In total 117 central amygdala neurons, micro-pressure application of 0.5 M glucose significantly decreased the basal firing rate in 16 neurons. Therefore, in the present study, 13.7% of central amygdala neurons belong to glucose-sensitive neurons. In 7 out of the 16 glucose-sensitive neurons (basal: 4.15 ± 1.28 Hz, glucose: 2.15 ± 0.79 Hz, P < 0.05, n = 7) with long time stable recording, further application of secretin (0.1 M) decreased the firing rate from 5.31 ± 1.48 Hz to 1.74 ± 1.01 Hz (P < 0.01, Fig. 5). The average decrease was 75.32 ± 10.47% (P < 0.001 compared with normal saline). Feeding behavior induced by bilateral microinjection of secretin into the central amygdala Next, feeding behavioral tests were performed to study the possible involvement of secretin in the central amygdala. Bilateral microinjection of 0.1 M secretin into the central amygdala significantly decreased cumulative food intake. As shown in Fig. 6A, food intake was significantly lower in rats treated with secretin as
compared to vehicle-treated rats. The secretin-induced decrease in food intake at the time points of 1 h, 2 h, 4 h and 12 h was 46.08 ± 8.54% (P < 0.001), 44.41 ± 8.87% (P < 0.01), 54.00 ± 6.76% (P < 0.001) and 35.66 ± 9.49% (P < 0.01), respectively. In another group of experiment, we observed the effects of H89 (an inhibitor of cAMP-activated protein kinase) on secretin-induced change of cumulative food intake. Compared with H89 (10 M) alone, co-injection of H89 and secretin (0.1 M) did not change the cumulative food intake significantly (P > 0.05 at the time points of 1 h, 2 h, 4 h and 12 h, n = 11, Fig. 6B), suggesting the possible involvement of cAMP-PKA pathway in secretin-induced decrease of food intake.
Discussion The present in vivo studies showed that secretin modulated the spontaneous electrical activity of central amygdala neurons. Local administration of secretin increased the firing rate in most central amygdala neurons. Previous morphological studies have demonstrated that secretin and secretin receptors are widely expressed in central nervous system, including cerebellum, motor cortex, hippocampus and amygdala [4]. By using quantitative real-time PCR, Tay et al. [7] have reported that secretin and secretin receptor mRNA are detected in the central amygdala, hippocampus, area postrema, nucleus of the tractus solitary and cerebellum. Furthermore, both secretin and secretin receptor expression are age-related in most brain regions. In humans, administration of secretin increased amygdala fMRI activation [18], suggesting amygdala as a possible functional point of secretin. Goulet et al. [15] have reported that i.v. infusion of secretin induced neuronal activation in central amygdala and other defined brain regions. It is known that secretin can transfer from blood to brain [22]. Therefore, at high peripheral concentrations, secretin may enter the brain to influence gene expression in the central nucleus of amygdala. The present single unit recordings identified that secretin may directly activate
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Fig. 5. Effects of secretin on the firing rate of glucose-sensitive neurons in central amygdala. (A) Typical frequency histograms showing that 0.1 M secretin decreased the firing rate of a central amygdala neuron which was inhibited by 0.5 M glucose. In this cell, firing rate started to decrease 120 s after glucose administration, partial recovered to stable after 500 s. Glucose and secretin decreased the firing rate by 43.86% and 44.99%, respectively. (B) Pooled data summarizing the effects of secretin on glucose-sensitive neurons. *P < 0.05, **P < 0.01.
secretin receptor and modulate the spontaneous firing activity of central amygdala neurons. In addition to excitation, secretin also inhibited the basal firing rate in a small portion of central amygdala neurons. Recently, Keshavarzi et al. [23] reported that the posteroventral division of the medial amygdala contained three types of GABAergic neurons and two types of non-GABAergic neurons. The recorded basal firing rate ranging from 0.025 Hz to 13.23 Hz suggests that the central nucleus of amygdala may contain several different subpopulations of neurons. Therefore, the present secretin-induced different electrophysiological effects, excitation and inhibition, may be caused by different subpopulations of neurons. However, for the technical limitation, it was impossible to isolate the recorded neurons and define the types. Consistent with previous studies, the basal firing rate of central amygdala neurons are very low, which could be due to the tonic activity of numerous GABAergic interneurons in the nucleus [24,25]. Lénárd et al. [11] reported that physiological concentration of glucose decreased the basal firing rate in a subpopulation of rat amygdala neurons, subsequently named glucose-monitoring neurons. Consistently, early studies have revealed that the activity of some amygdala (mostly located in the centromedial part of the amygdala) neurons in monkey was inhibited by glucose. These glucose-sensitive cells are also responded to morphine, which may
be involved in the control of food acquisition behavior [26]. The present single unit recording showed that the central amygdala contains glucose-sensitive neurons with the basal firing activity inhibited by glucose. Many studies indicated that glucose-sensitive neurons located in hypothalamus and other brain regions are involved in glucoprivic feeding and homeostatic control of blood glucose [27,28]. Further experiments showed that secretin mainly exerted inhibitory effects on the spontaneous firing rate of glucosesensitive neurons. Being an important functional subdivision in amygdala, the central nucleus of amygdala is essentially involved in the regulation of feeding behavior. For example, Anderberg and the colleagues [29] recently reported that dopamine reduced food intake by activation of D2 receptors in central nucleus of amygdala. Microinjection of RFRP-1 into central nucleus of amygdala decreased liquid food consumption in rats [30]. Administration of ghrelin into the central amygdala increased food intake which may be related with the activation of arcuate nucleus [31]. The central nucleus of amygdala is also involved in melanocortin-induced inhibition of food intake [32]. Cheng et al. [33] first demonstrated that intracerebroventricular microinjection of secretin suppressed food intake dose-dependently in wide type mice but not in secretin receptor-deficient mice, suggesting secretin as an anorectic peptide. The present feeding behavioral studies showing that local
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activated by diverse anorexigenic signals. Optogenetic activation of these neurons in vivo strongly reduced food intake in mice. It is well known that the central nucleus of amygdala contains several neurotransmitters or neuromodulators which are involved in the control of feeding behavior. It has been shown that specific catecholaminergic microlesions reducing the norepinephrine content in amygdala produced hyperphagia and weight increase, while dopamine depletion caused hypophagia and weight decrease [40,41]. Endogenous melanocortin receptor agonists in the central amygdala exerted a tonic inhibitory influence on food consumption by stimulating MC4 receptors [32]. Therefore, secretin may control feeding behavior by its modulation of other neurotransmitter systems. Consistently, Cheng et al. [33] demonstrated that intracerebroventricular secretin-induced suppression of food intake is mediated by melanocortin system. In conclusion, the present studies indicated that secretin increased the neuronal activity in most central amygdala neurons. However, in glucose-sensitive neurons, secretin decreased the firing rate. Bilateral microinjection of secretin into the central amygdala significantly reduced cumulative food intake through cAMP-activated protein kinase activation. Based on the present electrophysiological and behavioral findings, we hypothesized that secretin may suppress food intake by its modulation of neuronal activity of central amygdala neurons. Conflict of interest statement The authors declare no conflict interest. Acknowledgements
Fig. 6. Feeding related effects of secretin in the central amygdala. (A) Cumulative food intake after bilateral microinjection of secretin (0.1 M, 0.5 l/side) or normal saline into the central amygdala. **P < 0.01, ***P < 0.001. (B) Cumulative food intake induced by bilateral microinjection of H89 and H89 with secretin. No significant difference between H89 and H89 with secretin at different time points.
administration of secretin decreased food intake provides further evidence that the central nucleus of amygdala may be one of the brain regions involved in secretin-induced inhibition of food intake. Consistently, previous studies revealed that intracerebroventricular administration of glucagon-like peptide 1 inhibited feeding by its activity on central nucleus of the amygdala [29,34]. Similarly, peripheral administration of glucagon-like peptide 1 and glucagon decreased food intake and increased c-fos expression in the central nucleus of amygdala [35]. Based on present secretin-induced in vivo electrophysiological effects, we hypothesized that secretin in the central amygdala suppressed food intake by its excitation on the spontaneous firing of central amygdala neurons. In support of our hypothesis, early studies have shown that amygdaloid lesion induced hyperphagia and obesity in both female and male rats [36]. Bovetto and Richard [37] reported that lesion of central nucleus of amygdala promotes food intake and fat gain in rats. Furthermore, Levine et al. [38] reported that activation of mu opioid receptors in the central nucleus of amygdala increased feeding behavior, probably by inhibiting the activity of selected amygdala neurons. More recently, Cai et al. [39] demonstrated that the lateral part of central amygdala nucleus contains a subpopulation of PKC-␦(+) GABAergic neurons, which were
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