Glutamatergic Projections from the Posterior Complex of the Anterior Olfactory Nucleus to the Amygdala Complexes.

Social buffering is a phenomenon where stress responses are ameliorated by an affiliative conspecific. Our previous findings suggest that the posterior complex of the anterior olfactory nucleus (AOP) is well positioned to participate in the neural mechanisms underlying social buffering. However, the lack of anatomical information prevents us from further estimating the role of the AOP. Here, we obtained anatomical information regarding the AOP in male rats. In Experiment 1 (n = 5), among 4',6-diamidino-2-phenylindole-positive cells in the AOP, the proportion of glutamic acid decarboxylase 67 (GAD67)-positive cells was 13.8% ± 1.2%. In Experiment 2 (n = 5), among the cells that were labeled by a retrograde tracer injected into the basolateral complex of the amygdala (BLA), the proportion of GAD67-positive cells was 18.6% ± 0.8%. In Experiment 3 (n = 5), we demonstrated the existence of cells that were labeled by the retrograde tracer injected into the posterior part of the medial amygdala (MeP), mostly into the ventral part of the MeP. In addition, the proportion of GAD67-positive cells among the tracer-labeled cells was 21.7% ± 1.7%. In Experiment 4 (n = 3), the retrograde tracers were injected into the BLA and MeP, mostly into the ventral part of the MeP. The proportion of double-labeled cells among the tracer-labeled cells was 2.1% ± 1.2%. Taken together, these results suggest that the AOP is predominantly composed of glutamatergic neurons. In addition, the AOP sends mutually independent glutamatergic-predominant projections to the BLA and MeP.

Whole-brain afferent input mapping to functionally distinct brainstem noradrenaline cell types.

The locus coeruleus (LC) is a small region in the pons and the main source of noradrenaline (NA) to the forebrain. While traditional models suggested that all LC-NA neurons project indiscriminately throughout the brain, accumulating evidence indicates that these cells can be heterogeneous based on their anatomical connectivity and behavioral functionality and exhibit distinct coding modes. How LC-NA neuronal subpopulations are endowed with unique functional properties is unclear. Here, we used a viral-genetic approach for mapping anatomical connectivity at different levels of organization based on inputs and outputs of defined cell classes. Specifically, we studied the whole-brain afferent inputs onto two functionally distinct LC-NA neuronal subpopulations which project to amygdala or medial prefrontal cortex (mPFC). We found that the global input distribution is similar for both LC-NA neuronal subpopulations. However, finer analysis demonstrated important differences in inputs from specific brain regions. Moreover, sex related differences were apparent, but only in inputs to amygdala-projecting LC-NA neurons. These findings reveal a cell type and sex specific afferent input organization which could allow for context dependent and target specific control of NA outflow to forebrain structures involved in emotional control and decision making.

The tyrosine capsid mutations on retrograde adeno-associated virus accelerates gene transduction efficiency.

Adeno-associated virus (AAV) vector is a critical tool for gene delivery through its durable transgene expression and safety profile. Among many serotypes, AAV2-retro is typically utilized for dissecting neural circuits with its retrograde functionality. However, this vector requires a relatively long-term incubation period (over 2 weeks) to obtain enough gene expression levels presumably due to low efficiency in gene transduction. Here, we aimed to enhance transgene expression efficiency of AAV2-retro vectors by substituting multiple tyrosine residues with phenylalanines (YF mutations) in the virus capsid, which is previously reported to improve the transduction efficiency of AAV2-infected cells by evading host cell responses. We found that AAV2-retro with YF mutations (AAV2-retroYF)-mediated transgene expression was significantly enhanced in the primary culture of murine cortical neurons at 1 week after application, comparable to that of the conventional AAV2-retro at 2 week after application. Moreover, transgene expressions in the retrogradely labeled neurons mediated by AAV2-retroYF were significantly increased both in the cortico-cortical circuits and in the subcortical circuits in vivo, while the retrograde functionality of AAV2-retroYF was equally effective as that of AAV2-retro. Our data indicate that YF mutations boost AAV2-retro-mediated retrograde gene transduction in vivo and suggest that the AAV2-retroYF should be useful for efficient targeting of the projection-defined neurons, which is suited to applications for dissecting neural circuits during development as well as future clinical applications.

Enhanced synchronization between prelimbic and infralimbic cortices during fear extinction learning.

The ability to extinguish aversive memories when they are no longer associated with danger is critical for balancing survival with competing adaptive demands. Previous studies demonstrated that the infralimbic cortex (IL) is essential for extinction of learned fear, while neural activity in the prelimbic cortex (PL) facilitates fear responding and is negatively correlated with the strength of extinction memories. Though these adjacent regions in the prefrontal cortex maintain mutual synaptic connectivity, it has been unclear whether PL and IL interact functionally with each other during fear extinction learning. Here we addressed this question by recording local field potentials (LFPs) simultaneously from PL and IL of awake behaving rats during extinction of auditory fear memories. We found that LFP power in the fast gamma frequency (100-200 Hz) in both PL and IL regions increased during extinction learning. In addition, coherency analysis showed that synchronization between PL and IL in the fast gamma frequency was enhanced over the course of extinction. These findings support the hypothesis that interregional interactions between PL and IL increase as animals extinguish aversive memories.

A dopaminergic switch for fear to safety transitions.

Overcoming aversive emotional memories requires neural systems that detect when fear responses are no longer appropriate so that they can be extinguished. The midbrain ventral tegmental area (VTA) dopamine system has been implicated in reward and more broadly in signaling when a better-than-expected outcome has occurred. This suggests that it may be important in guiding fear to safety transitions. We report that when an expected aversive outcome does not occur, activity in midbrain dopamine neurons is necessary to extinguish behavioral fear responses and engage molecular signaling events in extinction learning circuits. Furthermore, a specific dopamine projection to the nucleus accumbens medial shell is partially responsible for this effect. In contrast, a separate dopamine projection to the medial prefrontal cortex opposes extinction learning. This demonstrates a novel function for the canonical VTA-dopamine reward system and reveals opposing behavioral roles for different dopamine neuron projections in fear extinction learning.

Modular organization of the brainstem noradrenaline system coordinates opposing learning states.

Noradrenaline modulates global brain states and diverse behaviors through what is traditionally believed to be a homogeneous cell population in the brainstem locus coeruleus (LC). However, it is unclear how LC coordinates disparate behavioral functions. We report a modular LC organization in rats, endowed with distinct neural projection patterns and coding properties for flexible specification of opposing behavioral learning states. LC projection mapping revealed functionally distinct cell modules with specific anatomical connectivity. An amygdala-projecting ensemble promoted aversive learning, while an independent medial prefrontal cortex-projecting ensemble extinguished aversive responses to enable flexible behavior. LC neurons displayed context-dependent inter-relationships, with moderate, discrete activation of distinct cell populations by fear or safety cues and robust, global recruitment of most cells by strong aversive stimuli. These results demonstrate a modular organization in LC in which combinatorial activation modes are coordinated with projection- and behavior-specific cell populations, enabling adaptive tuning of emotional responding and behavioral flexibility.

Projection specificity in heterogeneous locus coeruleus cell populations: implications for learning and memory.

Noradrenergic neurons in the locus coeruleus (LC) play a critical role in many functions including learning and memory. This relatively small population of cells sends widespread projections throughout the brain including to a number of regions such as the amygdala which is involved in emotional associative learning and the medial prefrontal cortex which is important for facilitating flexibility when learning rules change. LC noradrenergic cells participate in both of these functions, but it is not clear how this small population of neurons modulates these partially distinct processes. Here we review anatomical, behavioral, and electrophysiological studies to assess how LC noradrenergic neurons regulate these different aspects of learning and memory. Previous work has demonstrated that subpopulations of LC noradrenergic cells innervate specific brain regions suggesting heterogeneity of function in LC neurons. Furthermore, noradrenaline in mPFC and amygdala has distinct effects on emotional learning and cognitive flexibility. Finally, neural recording data show that LC neurons respond during associative learning and when previously learned task contingencies change. Together, these studies suggest a working model in which distinct and potentially opposing subsets of LC neurons modulate particular learning functions through restricted efferent connectivity with amygdala or mPFC. This type of model may provide a general framework for understanding other neuromodulatory systems, which also exhibit cell type heterogeneity and projection specificity.

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.

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic ß-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

The sense of taste in the upper gastrointestinal tract.

Digestion and the absorption of food and nutrients have been considered the only functions of the gastrointestinal (GI) tract. However, recent studies suggest that taste cells in the oral cavity and taste-like cells in the GI tract share many common characteristics (taste receptors and transduction signaling). Over the last two decades, it has been revealed that the GI tract is a chemosensory organ that transfers nutrient information via GI hormone secretion (glucagon-like peptide-1, Peptide YY, oxyntomodulin, glucose-dependent insulinotropic polypeptide and others) and the activation of abdominal vagus afferents. In addition, the information relayed via the abdominal vagus nerve plays an important role in autonomic reflexes. This information, both humoral and neural, contributes to the maintenance of homeostasis (digestion, absorption, metabolism and food intake) in the body. In this review, we provide a brief overview of the following: GI chemosensory molecules, their distribution, the effect of nutrients on GI hormone secretion and the activation of vagus afferent nerves. We also focus on the possibility of clinical applications that control abdominal vagus activity.

Reversible brain response to an intragastric load of L-lysine under L-lysine depletion in conscious rats.

L-Lysine (Lys) is an essential amino acid and plays an important role in anxiogenic behaviour in both human subjects and rodents. Previous studies have shown the existence of neural plasticity between the Lys-deficient state and the normal state. Lys deficiency causes an increase in noradrenaline release from the hypothalamus and serotonin release from the amygdala in rats. However, no studies have used functional MRI (fMRI) to compare the brain response to ingested Lys in normal, Lys-deficient and Lys-recovered states. Therefore, in the present study, using acclimation training, we performed fMRI on conscious rats to investigate the brain response to an intragastric load of Lys. The brain responses to intragastric administration of Lys (3 mmol/kg body weight) were investigated in six rats intermittently in three states: normal, Lys-deficient and recovered state. First, in the normal state, an intragastric load of Lys activated several brain regions, including the raphe pallidus nucleus, prelimbic cortex and the ventral/lateral orbital cortex. Then, after 6 d of Lys deprivation from the normal state, an intragastric load of Lys activated the ventral tegmental area, raphe pallidus nucleus and hippocampus, as well as several hypothalamic areas. After recovering from the Lys-deficient state, brain activation was similar to that in the normal state. These results indicate that neural plasticity in the prefrontal cortex, hypothalamic area and limbic system is related to the internal Lys state and that this plasticity could have important roles in the control of Lys intake.

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.

New therapeutic strategy for amino acid medicine: effects of dietary glutamate on gut and brain function.

The gustatory and visceral stimulation from food regulates digestion and nutrient utilization, and free glutamate (Glu) release from food is responsible for the umami taste perception that increases food palatability. The results of recent studies reveal a variety of physiological roles for Glu. For example, luminal applications of Glu into the mouth, stomach, and intestine increase the afferent nerve activities of the glossopharyngeal nerve, the gastric branch of the vagus nerve, and the celiac branch of the vagus nerve, respectively. Additionally, luminal Glu evokes efferent nerve activation of each branch of the abdominal vagus nerve. The intragastric administration of Glu activates several brain areas (e.g., insular cortex, limbic system, and hypothalamus) and has been shown to induce flavor-preference learning in rats. Functional magnetic resonance imaging of rats has shown that the intragastric administration of Glu activates the nucleus tractus solitarius, amygdala, and lateral hypothalamus. In addition, Glu may increase flavor preference as a result of its postingestive effect. Considering these results, we propose that dietary Glu functions as a signal for the regulation of the gastrointestinal tract via the gut-brain axis and contributes to the maintenance of a healthy life.

Evaluation of the 'liking' and 'wanting' properties of umami compound in rats.

Food reward is neurologically and psychologically divided into at least two properties; 'liking' and 'wanting'. Although umami taste enhances food palatability, the liking and wanting properties of umami taste, and the underlying neural mechanisms for these properties are not clear. Here, we compared sucrose (0, 10, 30, 120 and 480 mM) and monosodium l-glutamate (MSG; 0, 10, 30, 60 and 120 mM) solutions using a taste reactivity test to evaluate liking, and fixed/progressive-ratio operant licking tasks to evaluate wanting. To determine the underlying neural mechanisms, we also conducted systemic blockade of opioid receptors in both tests. In the taste reactivity test, the hedonic reactions to 30, 60 and 120 mM MSG were greater than those to water (0mM) but lower than those to 480 mM sucrose. In the operant task, the intake, number of licks, and breakpoint to MSG reached peaks at around 60mM but they were lower than those to 30-480 mM sucrose. The systemic naloxone treatment decreased the hedonic responses to MSG and sucrose, and reduced the incentive salience of MSG but not sucrose. These findings indicate that the hedonic response and incentive salience of MSG is lower than those of sucrose when compared at the maximum response and that the incentive salience of MSG is lower than sucrose even where the hedonic response is similar. The present study also suggest that the hedonic response and incentive salience of umami compound is modulated by brain opioid signaling.

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.

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.

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.

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.

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.

Maternal approaches to pup ultrasonic vocalizations produced by a nanocrystalline silicon thermo-acoustic emitter.

When infant rodents are isolated from their mother and littermates, they cool rapidly and emit ultrasonic vocalizations (USVs). The effect of pup USVs on the mother has been investigated using models of pup USVs from ultrasonic speakers. We used a nanocrystalline silicon thermo-acoustic emitter (nc-Si emitter) to investigate mothers' responses to digitally reproduced pup USVs in mice. The nc-Si emitter could reproduce ultrasonic sounds more accurately than conventional emitters. We compared the sound properties of pup USVs and reproduced USVs. We then investigated maternal responses to hypothermic pups, which emit USVs, and anesthetized pups, which are silent, as well as maternal responses to pup USVs reproduced by the nc-Si emitter and a silent mode. The nc-Si emitter can reproduce pup USVs very accurately in terms of duration, frequency, and sound pressure level. Mothers approached reproduced digitally recorded pup USVs from the nc-Si emitter and their behavior was similar to their behavior toward hypothermic pups. In contrast, mothers did not approach other synthesized ultrasounds, such as double-duration USVs, double-silence domain ultrasounds nor double-ultrasonic domain ultrasounds, indicating that they approach the specific profiles of pup USVs. These results indicate that the nc-Si emitter can be useful to elucidate the role of ultrasonic acoustic communication in rodents.