Neuronal responses to intragastric administration of amino acids in the rat amygdala and lateral hypothalamic area. 1 System Emotional Science, Grad Sch. of Med. Pharma. Sci., Univ. Toyama, Toyama, Japan, 2 AJINOMOTO Integrative Research for Advanced Dieting, Grad Sch. of Agriculture, Kyoto Univ., Kyoto, Japan, 3 Inst. for Innovation , Ajinomoto Co., Inc., Kawasaki, Japan, 4 Dept. Judo Neurophysiotherapy, Grad Sch. of Med. Pharma. Sci., Univ. Toyama, Toyama, Japan Syringe pump solution 30 cm 30 cm Video image Red lamp CCD camera Neuronal Activity Mirror Figure 1. Experimental setup A rat was placed on a stainless mesh floor in a transparent acrylic chamber (30 x 30 x 39 cm). The one side of chamber was made of stainless steel mesh. The chamber was placed in the dark room and illuminated by a red lamp. Test solutions were administrated into its stomach by a syringe pump (CFV-3100; Nihon Kohden, Japan). The rat’s behavior during an experiment was captured by a CCD camera in front of the chamber. To capture both sides of the rat’s body, an mirror was placed behind the chamber. Materials & Methods Subjects 5 adult male Wistar rats weighing 240-300 g (SLC, Hamamatsu, Japan) were used for this study. Housing temperature was maintained at 23 ±1°C with a 12-h light/dark cycle (light phase: 7:00 am – 7:00 pm). Surgery Rats were anesthetized intraperitoneally with sodium pentobarbital (40 mg/kg), and a recording electrode assembly [comprised 4 tetrodes, each of which included 4 tungsten microwires (20 μm in diameter; California Fine Wire, Grover Beach, CA, USA), which were encased in a stainless steel cannula (30 gauge; Hakko, Nagano, Japan), and a microdrive. The tip impedance was approximately 200 kΩ at 1 kHz] was implanted in the CeA (2.1 mm caudal from the bregma, 4.0 mm lateral from the midline, and 8.0- 8.5 mm below the brain surface) and / or LH (2.1 mm caudal from the bregma, 2.0 mm lateral from the midline, and 8.5 mm below the brain surface) after referring to the atlas of Paxinos and Watson (2007). Then, for intragastric cannulation, one end of a silicon tube was passed from abdomen under the back skin and held on the skull and the other end was inserted into the gastric fundus and ligated with a silk thread. Behavioral and recording procedure The experimental setup used in this experiment is shown Figure 1. The procedure below were conducted daily during the dark phase after 12-hr fasting. First, the male rat was placed in the large compartment of the test chamber and neuronal activity was checked. If stable neuronal signals over 10 min were found, the recording was started. If no signal was found, the electrode assembly was lowered by about 22–88 μm and the recording was not conducted on the day. Ten min from recording started, one of chemical solutions [60 mM monosodium L-glutamate (MSG), 60 mM arginine HCl (Arg- HCl), 60 mM arginine-glutamine (Arg-Glu), 154 mM NaCl (i.e. saline, NaCl154) and 60 mM NaCl (NaCl60)] were delivered into the stomach via the implanted tube for 10 min at a rate of 1 ml/min/kg body weight. After the 10- min administration, recording was further continued for 40 min. In a daily recording, only one solution was tested for one time. After finishing recordings for all the solutions from a recording position (i.e. 5 days of recording), the electrode assembly was lowered by at least 88 μm to prevent recording from the same neuron in the next recording. Introduction Recent studies indicate that visceral information from the gut is involved in emotional states as well as homeostasis (Mayer 2011). The glutamate signals from the guts to the brain is suggested to be one of such signals. Our behavioral study suggests that the glutamate signal via the vagus nerve affects social behaviors in mice. Tsurugisawa et al. (2008, 2009) reported that activity in the rat amygdala, implicated in emotion, increased by intragastric administration of monosodium L-glutamate (MSG) and that vagotomy strongly suppressed this activation. Furthermore, the rat central nucleus of the amygdale (CeA) receives strong inputs from the nucleus of solitary tract (NTS) that receives afferent inputs from the vagus nerve (Berthoud et al. 2000). These results suggest that glutamate signals from the gut may activate the amygdala to change the emotional state, and consequently affect social behavior. However, it is still unclear how the glutamate signals from the gut is encoded and processed in the amygdala. In this study, we investigated the change of neuronal activity in the rat CeA after intragastric administration of glutamate. In the gut, in addition to mGluR1, which is a glutamate specific receptor, T1R1+T1R3, which is a broadly tuned amino acid receptor (Nelson et al. 2002), is identified (Bezençon et al. 2007; San Gabriel et al. 2007). Furthermore, in the central nervous system, it is reported that arginine suppressed glutamate-induced neuronal activity (Kondoh et al. 2010). Thus, the other amino acids and mixture of glutamate and the other amino acids may differentially activate amygdala, compared to the glutamate alone. Therefore, we investigated the neuronal responses to arginine and a mixture of arginine and glutamate. We also recorded neuronal activity from the lateral hypothalamic area (LHA), which has intimate anatomical connections with the amygdale while it also receives inputs from the NTS. Figure 2. Examples of responses of 5 CeA neurons to intragastric administration of one of tested solutions. Perievent histograms (bin width = 20 s) of CeA neurons’ responses to intragastric administration of the tested solutions (A: MSG, B: Arg-HCl, C: Arg- Glu, D: NaCl154, E: NaCl60). A solution was administrated during 0-10 min (blue line). The red solid line and the red dashed line indicate mean and mean + 2SD of the activity in the baseline period (-10-0 min), respectively. Results A total of 79 (MSG: 16, Arg-HCl: 15, Arg-Glu: 16, NaCl154: 22, NaCl60: 10) and 6 (Arg-HCl: 3, Arg-Glu: 3) neurons were recorded from the CeA and LHA, respectively. Of these, activity of 54 (68 %) of the CeA and 6 (100 %) of the LHA neurons changed significantly during the post-administration period compared with the pre-administration period. The results of analysis of these responsive neurons are shown in following figures LH Figure 3. Response patterns of all responsive neurons Each line indicates response pattern of one neuron. Orange and blue indicate the excitatory (higher than mean baseline firing rate + 2S.D) and inhibitory (lower than mean baseline firing rate - 2S.D) responses, respectively. Black stars indicate a onset of the response (the time when successive 2 bins first deviated from mean baseline firing rate by ±2S.D) of each neuron. Fig. 4 Comparison of the response latencies. A: Bar graphs comparing the mean response latencies to the 5 solutions. Error bars indicate s.e.m. No significant difference between the solutions was found (Kruskal- Wallis test, p = 0.198). B: Bar graphs comparing the latencies of the amino acid solutions (MSG, AHCl and AG) and NaCl solutions (N154 and N60). Error bars indicate s.e.m. The latency to the amino acid solutions were significantly faster than those of the NaCl solutions (Mann-Whitney U test, p = 0.04). Discussion In the CeA, the mean latencies to the amino acid solutions was significantly faster than those to the NaCl solutions. In the LHA, the mean total response duration tended to be longer than those in the CeA. ⇒ The first result suggests that the CeA is more specifically involved in information processing of amino acid signals from the gut than that of NaCl. ⇒ These results provide neurophysiological evidence that the CeA and LHA receive interoceptive information. ⇒ These results further suggest that the CeA might detect amino acids in the gut, and modulate activity of the LHA that plays an important role in various behaviors. Future works 1. Recording more neurons to confirm whether the tendencies found in the current data are statistically significant. 2. Improving the procedure for recording stable neuronal signals across 2-3 days to compare responses of a single neuron to multiple solutions and investigate how the signals from the gut are encoded in the brain. 3. Testing neurons, that are responsive to intragastric administration, with the other emotional and social stimuli to investigate functions of the neurons in emotion and social behavior. Reference Berthoud HR & Neuhuber WL (2000) Functional and chemical anatomy of the afferent vagal system. Auton Neurosci. 85(1-3), 1-17 Bezençon C, le Coutre J & Damak S (2007) Taste-signaling proteins are coexpressed in solitary intestinal epithelial cells. Chem Senses. 32(1), 41–9. Kondoh T & Torii K (2010) Forebrain Activation by Postoral Nutritive Substances. In Victor RP, Ronald RW & Coline RM (eds) Handbook of Behavior, Food and Nutrition, Vol. 1, Springer, Berlin, pp 469-487 Mayer EA (2011) Gut feelings: the emerging biology of gut-brain communication. Nat Rev Neurosci. 12(8), 453-466 Nelson G, Chandrashekar J, Hoon MA, Feng L, Zhao G, Ryba NJ & Zuker CS (2002) An amino-acid taste receptor. Nature. 416(6877) 199–202. San Gabriel AM, Maekawa T, Uneyama H, Yoshie S & Torii K (2007) mGluR1 in the fundic glands of rat stomach. FEBS Lett. 581(6), 1119–23. Tsurugizawa T, Kondoh T & Torii K (2008) Forebrain activation induced by postoral nutritive substances in rats. Neuroreport 19(11), 1111–5. Tsurugizawa T, Uematsu A, Nakamura E, Hasumura M, Hirota M, Kondoh T, Uneyama H & Torii K (2009) Mechanisms of neural response to gastrointestinal nutritive stimuli: the gut-brain axis. Gastroenterology. 137(1) 262–73. Jumpei Matsumoto 1 , Davaasuren Munkhzul 1 , Etsuro Hori 1 , Takashi Kondoh 2 , Akihiko Kitamura 3 , Hideki Matsumoto 3 , Kunio Torii 3 , Taketoshi Ono 4 , Hisao Nishijo 1 CeA LHA MSG Arg-HCl Arg-Glu NaCl 154 NaCl 60 Arg-HCl Arg-Glu -10 0 10 20 30 40 50 ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ Time (min) Fig. 5 Comparison of the total response duration A: Bar graphs comparing the mean total response duration among the 5 solutions. Red, blue and purple bars indicate the total durations of excitatory responses, inhibitory responses and both excitatory and inhibitory responses, respectively. Error bars indicate s.e.m. No significant difference among the solutions was found (Kruskal-Wallis test, p = 0.60, 0.93 and 0.51 for the duration of excitatory responses, inhibitory responses and both excitatory and inhibitory responses, respectively). B: Bar graphs comparing the response latencies to the amino acid solutions (MSG, Arg-HCl and Arg-Glu) and NaCl solutions (NaCl154 and NaCl60). Error bars indicate s.e.m. No significant difference between the solutions was found (Mann-Whitney U test, p = 0.22, 0.92 and 0.91 for the duration of excitatory responses, inhibitory responses and both excitatory and inhibitory responses, respectively). Fig. 6 Comparison between the CeA and LHA A: Bar graphs comparing the mean response latency between the CeA and LHA. Error bars indicate s.e.m. No significant difference between the regions found (Mann-Whitney U test, p = 0.78). B: Bar graphs comparing the mean response duration between the CeA and LHA. Note that excitatory and inhibitory responses were not separately analyzed because of low number of recocded neurons. Error bars indicate s.e.m. There was a tendency that the duration were longer in the LHA than the CeA (Mann-Whitney U test, p = 0.08). Data analysis Spike sorting The digitized neuronal activity was isolated into single units by waveform components using the Offline Sorter program (Plexon Inc.). Superimposed waveforms of the isolated units were drawn to check their consistency throughout the recording sessions, and were then transferred to the NeuroExplorer program (Nex Technology, Littleton, MA) for further analysis. Typically, 1–4 single units were isolated by offline cluster analysis from 4 channels (wires) of 1 tetrode. Spike sorting was performed with the Offline Sorter program (Plexon Inc.) that can plot spikes in two- or three-dimensional feature spaces, in which various features of spike waveforms (waveform projection onto principal components, peak amplitudes of the waveforms, valley amplitudes of the waveforms, peak-valley amplitudes of the waveforms, etc.) can be selected as a dimension. Each cluster in the feature space was then checked manually to ensure that the cluster boundaries were well separated and the waveform shapes were consistent with the action potentials. For each isolated cluster, an interspike interval histogram was constructed and an absolute refractory period of at least 1.0 ms was used to exclude suspected multiple units. Finally, superimposed waveforms of the isolated units were drawn to check waveform consistency. Change of firing rate after intragastric administration The pre and post administration periods were defined as 10 min before and 50 min after the onset of the administration, respectively. A firing rate histogram for each neuron around each administration of solution during these periods (bin size = 20 s) was computed. The baseline firing rate of a neuron was calculated from the bins within the pre administration period. A neuron was considered to be responsive to administration of a given solution if successive 2 bins within the post administration period in the histogram deviated from the baseline firing rate by ±2S.D. For each responsive neuron, response latency (the time when successive 2 bins first deviated from the baseline firing rate by ±2S.D.) and total response duration (the number of bins deviating the baseline firing rate by ±2S.D.) were computed. Note that data within first 1 min of the post administration period was removed from these analysis for eliminating possible artifacts caused by experimental manipulations. 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 0 0.2 0.4 0.6 0.8 0 1 2 3 4 0 1 2 3 4 A. MSG B. Arg-HCl C. Arg-Glu D. NaCl154 (Saline) E. NaCl60 Firing rate (Hz) Firing rate (Hz) Firing rate (Hz) -10 0 10 20 30 40 50 Time (min) -10 0 10 20 30 40 50 Time (min) -10 0 10 20 30 40 50 Time (min) -10 0 10 20 30 40 50 Time (min) -10 0 10 20 30 40 50 Time (min) Firing rate (Hz) Firing rate (Hz) Infusion Infusion Infusion Infusion Infusion A 0 NaCl154 NaCl60 MSG Arg-HCl Arg-Glu Response latency (min) Amino Acids (MSG, Arg-HCl, Arg-Glu) NaCls (NaCl154, NaCl60) 0 10 20 30 Response latency (min) p = 0.04 Excitatory Inhibitory 0 5 10 15 Excitatory Inhibitory Both NaCl154 NaCl60 MSG Arg-HCl Arg-Glu Excitatory Inhibitory Both B A B Total response Duration (min) 0 5 10 15 Amino Acids (MSG, Arg-HCl, Arg-Glu) NaCls (NaCl154, NaCl60) Total response Duration (min) 0 10 20 30 CeA LHA CeA LHA 0 10 20 30 Response latency (min) p = 0.08 A B 10 20 30