Tomokazu Tsurugizawa

Mechanisms of Neural Response to Gastrointestinal Nutritive Stimuli: The Gut-Brain Axis

BACKGROUND & AIMS The gut-brain axis, which transmits nutrient information from the gastrointestinal tract to the brain, is important for the detection of dietary nutrients. We used functional magnetic resonance imaging of the rat forebrain to investigate how this pathway conveys nutrient information from the gastrointestinal tract to the brain. METHODS We investigated the contribution of the vagus nerve by comparing changes of blood oxygenation level-dependent signals between 24 control rats and 22 rats that had undergone subdiaphragmatic vagotomy. Functional data were collected under alpha-chloralose anesthesia continuously 30 minutes before and 60 minutes after the start of intragastric infusion of L-glutamate or glucose. Plasma insulin, L-glutamate, and blood glucose levels were measured and compared with blood oxygenation level-dependent signals. RESULTS Intragastric administration of L-glutamate or glucose induced activation in distinct forebrain regions, including the cortex, hypothalamus, and limbic areas, at different time points. Vagotomy strongly suppressed L-glutamate-induced activation in most parts of the forebrain. In contrast, vagotomy did not significantly affect brain activation induced by glucose. Instead, blood oxygenation level-dependent signals in the nucleus accumbens and amygdala, in response to gastrointestinal glucose, varied along with fluctuations of plasma insulin levels. CONCLUSIONS These results indicate that the vagus nerve and insulin are important for signaling the presence of gastrointestinal nutrients to the rat forebrain. These signal pathways depend on the ingested nutrients.

Brain-gut communication via vagus nerve modulates conditioned flavor preference

It is well known that the postingestive effect modulates subsequent food preference. We previously showed that monosodium L‐glutamate (MSG) can increase flavor preference by its postingestive effect. The neural pathway involved in mediating this effect, however, remains unknown. We show here the role of the vagus nerve in acquiring this learned flavor preference and in the brain’s response to intragastric glutamate infusion. Adult rats with an intragastric cannula underwent total abdominal branch vagotomies (TVX), common hepatic branch vagotomies (HVX), total abdominal branch vagotomies with the common hepatic branch intact (TVXh), or sham operations (Sham). Following recovery, rats were subjected to a conditioned flavor preference paradigm, in which they drank a flavored solution (CS+) paired with intragastric MSG or another flavored solution (CS−) paired with intragastric distilled water. After conditioning, the Sham and HVX groups demonstrated significantly higher intake of CS+ than CS−, whereas the TVXh and TVX groups showed no significant differences. We then conducted an fMRI study to identify the brain areas that responded to the intragastric glutamate in each group. In the Sham, HVX and TVXh groups, intragastric MSG significantly increased the BOLD intensity in the nucleus of the solitary tract. The amygdala, hippocampus and lateral hypothalamus were also activated in the Sham and HVX groups but not in the TVXh and TVX groups. These results indicate that the abdominal vagus nerve is necessary for acquiring preference and that the lateral hypothalamus and limbic system could be key areas for integrating the information on gut glutamate and oronasal stimuli.

Forebrain activation induced by postoral nutritive substances in rats.

Recent studies have shown the nutrient-sensing systems transmitting nutritive information from the gut to the brain. However neural activity evoked by ingested dietary nutrients has not beeninvestigated adequately. Using functional magnetic resonance imaging, we demonstrated that rat forebrain responded to intragastric administration of glucose, L-glutamate, and NaCl. These dietary nutrients led to a significant activation in the forebrain regions including nucleus accumbens, hypothalamic area, and limbic system with different timings. These data indicate that several forebrain regions have important roles on perception and process of postingestive nutrient information.

Effects of isoflurane and alpha-chloralose anesthesia on BOLD fMRI responses to ingested l-glutamate in rats.

It is important to investigate the effect of anesthesia on blood oxygenation level-dependent (BOLD) signals in an animal model. Many researchers have investigated the BOLD response to visual, sensory, and chemical stimuli in anesthetized rats. There are no reports, however, comparing the differences in the BOLD signal change between anesthetized and conscious rats when a visceral nutrient signal arises. Here, using functional magnetic resonance imaging (fMRI), we investigated the differences in the BOLD signal changes after intragastric administration of l-glutamate (Glu) under three anesthesia conditions: conscious, alpha-chloralose-anesthetized, and isoflurane-anesthetized condition. Under the conscious and alpha-chloralose condition, we observed the significant BOLD signal increase in the medial prefrontal cortex (mPFC), insular cortex (IC), hippocampus, and several hypothalamic regions including the lateral and ventromedial nucleus. In chloralose group, however, gut Glu stimulation induced BOLD signal increase in the prelimbic cortex and orbital cortex, which did not activate in conscious condition. Meanwhile, under isoflurane-anesthetized condition, we did not observe the BOLD signal increase in these areas. BOLD signal intensity in the nucleus of the solitary tract (NTS), to which vagus nerve transmits the visceral information from the gastrointestinal tract, increased in all conditions. Importantly, under conscious condition, we observed increased BOLD signal intensity in several regions related to the metabolic state (i.e. hunger or satiety), such as the mPFC, ventromedial and lateral hypothalamus (LH). Our results suggest that alpha-chloralose and isoflurane anesthesia caused distinct effects on BOLD response to the gut l-Glu stimulation in several brain regions.

Conditioned flavor preference learning by intragastric administration of L-glutamate in rats

The preference for foods or fluids in rats is partly dependent on its postingestive consequences. Many studies have investigated postingestive effect of high caloric substances, such as carbohydrate or fat. In this study, we examined postingestive effect of L-glutamate at the preferable concentration using conditioned flavor preference paradigm. Adult male rats with chronic intragastric (IG) cannula were trained to drink a flavored solution (conditioned stimulus; CS+) paired with IG infusion of nutrient solution and another flavored solution (CS-) with IG distilled water infusion on alternate days. The nutrient solution was 60mM monosodium L-glutamate, sodium chloride or glucose. Before and after conditioning, rats received 30min two-bottle choice tests for CS+ and CS- solution. All groups exhibited no significant preference for CS+ in pre-test period. By the last half of conditioning period, intake of CS+ solution was significantly higher than that of CS- in MSG group, but not in NaCl and glucose groups. After conditioned, the MSG group showed significantly higher intake and preference for CS+ solution (69.9%), while the NaCl and glucose group did not show any significant intake and preference for CS+ solution (50.9%, 43.5%, respectively). These results indicate that the amino acid L-glutamate at a preferable concentration has a positive postingestive effect as demonstrated by its ability to condition a flavor preference. The mechanism(s) for this positive effect could be through a direct effect on gut Glu receptors rather than the provision of calories or glucose from metabolized Glu; Further studies are needed to test these hypotheses.

Blood oxygenation level-dependent response to intragastric load of corn oil emulsion in conscious rats.

The postingestive actions after intragastric or oronasal stimulation of fat have been well investigated. The blood oxygenation level-dependent (BOLD) signal changes, however, after intragastric load of corn oil emulsion have yet to be elucidated. Here, using functional magnetic resonance imaging, we investigated the BOLD signal response to gut corn oil emulsion in nonanesthetized rats. Intragastrically infused 7% corn oil emulsion induced a BOLD signal increase in several brain regions, including the bilateral amygdala, hippocampus and the ventral tegmental area. These results indicate that the limbic system responds to gut corn oil emulsion and that activation of this system could promote the reinforcing action for food with high fat content.

Functional brain mapping of conscious rats during reward anticipation

Functional magnetic resonance imaging (fMRI) in humans and non-primates has been useful to clarify the brain regions involved in the psychological process such as the reward anticipation. However, there is still no report of the fMRI study on the reward prediction in rodents. This is mainly because of the problem of anesthesia in rodent fMRI. In this study, we first developed awake fMRI method to investigate the brain region involved in reward anticipation in rats. After fMRI adaptation training, rats received light stimulation 1min before intraperitoneal infusion of ethanol solution (4g/kg body weight) in the MRI bore. Five or six days after the start of the experiment, the caudate-putamen, anterior insular cortex, hippocampus, ventral pallidum, nucleus accumbens and medial preoptic area were activated during light presentation. In contrast, no activation was observed in the control group. These results indicate the availability of awake fMRI method to investigate neural plasticity in the psychological process, learning, and memory such as the reward anticipation.

Brain Functional Changes in Rats Administered with Monosodium l-Glutamate in the Stomach

Recent studies have demonstrated the existence of receptors for l‐glutamate (GLU) and their transduction molecules in the gut mucosa as well as in the oral cavity. Among 20 amino acids, gastric vagal afferent fibers respond only to intragastric administration of GLU. Functional magnetic resonance imaging revealed activation of several forebrain regions in response to intragastric infusion of taste solutions (d‐glucose [sweet], monosodium l‐glutamate [MSG; umami], and NaCl [salty] at 60 mM) in rats. Glucose activated the nucleus accumbens. MSG activated the medial preoptic area, dorsomedial nucleus of the hypothalamus, and habenular nucleus. Both glucose and MSG activated the amygdala. Some areas, such as the insular cortex, anterior cingulate cortex, hippocampus, and caudate–putamen were activated by all three substances. Glucose‐induced brain activation developed slowly and persisted for a long time, whereas activation by MSG developed rapidly during infusion and reduced rapidly after cessation of infusion. NaCl induced only small and transient activation. Thus, both activated areas and temporal response patterns in the brain were distinct between sweet and umami substances delivered in the stomach. Postoral taste substances may activate the brain via neural (vagal) and/or humoral pathways.

Correlation Between Activation of the Prelimbic Cortex, Basolateral Amygdala, and Agranular Insular Cortex During Taste Memory Formation

Conditioned taste aversion (CTA) is a well-established learning paradigm, whereby animals associate tastes with subsequent visceral illness. The prelimbic cortex (PL) has been shown to be involved in the association of events separated by time. However, the nature of PL activity and its functional network in the whole brain during CTA learning remain unknown. Here, using awake functional magnetic resonance imaging and fiber tracking, we analyzed functional brain connectivity during the association of tastes and visceral illness. The blood oxygen level-dependent (BOLD) signal significantly increased in the PL after tastant and lithium chloride (LiCl) infusions. The BOLD signal in the PL significantly correlated with those in the amygdala and agranular insular cortex (IC), which we found were also structurally connected to the PL by fiber tracking. To precisely examine these data, we then performed double immunofluorescence with a neuronal activity marker (c-Fos) and an inhibitory neuron marker (GAD67) combined with a fluorescent retrograde tracer in the PL. During CTA learning, we found an increase in the activity of excitatory neurons in the basolateral amygdala (BLA) or agranular IC that project to the PL. Taken together, these findings clearly identify a role of synchronized PL, agranular IC, and BLA activity in CTA learning.

Different BOLD Responses to Intragastric Load of L-glutamate and Inosine Monophosphate in Conscious Rats

In this study, we compared the blood oxygen level-dependent (BOLD) signal changes between intragastric load of monosodium L-glutamate (MSG) and inosine monophosphate (IMP), which elicit the umami taste. An intragastric load of 30 mM IMP or 60 mM MSG induced a BOLD signal increase in several brain regions, including the nucleus of the solitary tract (NTS), lateral hypothalamus (LH), and insular cortex. Only MSG increased the BOLD signal in the amygdala (AMG). The time course of the BOLD signal changes in the NTS and the LH in the IMP group was different from that of the MSG group. We further compared the brain regions correlated with the BOLD signal change in the NTS between MSG and IMP groups. The BOLD responses in the hippocampus and the orbital cortex were associated with activation of the NTS in both MSG and IMP groups, but the association in the AMG and the pyriform was only in MSG group. These results indicate that gut stimulation with MSG and IMP evoked BOLD responses in distinct regions with different temporal patterns and that the mechanism of perception of L-glutamate and IMP in the gastrointestinal tract differed from that in the taste-sensing system.