Populations of Hindbrain Glucagon-Like Peptide 1 (GLP1) Neurons That Innervate the Hypothalamic PVH, Thalamic PVT, or Limbic Forebrain BST Have Axon Collaterals That Reach All Central Regions Innervated by GLP1 Neurons.

Neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt) that express the glucagon gene (Gcg) give rise to glucagon-like peptide 1 (GLP1)-immunopositive axons in the spinal cord and many subcortical brain regions. Central GLP1 receptor signaling contributes to motivated behavior and stress responses in rats and mice, in which hindbrain GLP1 neurons are activated to express c-Fos in a metabolic state-dependent manner. The present study examined whether GLP1 inputs to distinct brain regions arise from distinct subsets of Gcg-expressing neurons, and mapped the distribution of axon collaterals arising from projection-defined GLP1 neural populations. Using our Gcg-Cre knock-in rat model, Cre-dependent adeno-associated virus (AAV) tracing was conducted in adult male and female rats to compare axonal projections of IRt versus cNTS GLP1 neurons. Overlapping projections were observed in all brain regions that receive GLP1 input, with the caveat that cNTS injections produced Cre-dependent labeling of some IRt neurons, and vice versa. In additional experiments, specific diencephalic or limbic forebrain nuclei were microinjected with Cre-dependent retrograde AAVs (AAVrg) that expressed reporters to fully label the axon collaterals of transduced GLP1 neurons. AAVrg injected into each forebrain site labeled Gcg-expressing neurons in both the cNTS and IRt. The collective axon collaterals of labeled neurons entered the spinal cord and every brain region previously reported to contain GLP1-positive axons. These results indicate that the axons of GLP1 neural populations that innervate the thalamic paraventricular nucleus, paraventricular nucleus of the hypothalamus, and/or bed nucleus of the stria terminalis collectively innervate all central regions that receive GLP1 axonal input.

Central and peripheral GLP-1 systems independently suppress eating.

The anorexigenic peptide glucagon-like peptide-1 (GLP-1) is secreted from gut enteroendocrine cells and brain preproglucagon (PPG) neurons, which, respectively, define the peripheral and central GLP-1 systems. PPG neurons in the nucleus tractus solitarii (NTS) are widely assumed to link the peripheral and central GLP-1 systems in a unified gut-brain satiation circuit. However, direct evidence for this hypothesis is lacking, and the necessary circuitry remains to be demonstrated. Here we show that PPGNTS neurons encode satiation in mice, consistent with vagal signalling of gastrointestinal distension. However, PPGNTS neurons predominantly receive vagal input from oxytocin-receptor-expressing vagal neurons, rather than those expressing GLP-1 receptors. PPGNTS neurons are not necessary for eating suppression by GLP-1 receptor agonists, and concurrent PPGNTS neuron activation suppresses eating more potently than semaglutide alone. We conclude that central and peripheral GLP-1 systems suppress eating via independent gut-brain circuits, providing a rationale for pharmacological activation of PPGNTS neurons in combination with GLP-1 receptor agonists as an obesity treatment strategy.

Ghrelin signaling contributes to fasting-induced attenuation of hindbrain neural activation and hypophagic responses to systemic cholecystokinin in rats.

In rats, overnight fasting reduces the ability of systemic cholecystokinin-8 (CCK) to suppress food intake and to activate cFos in the caudal nucleus of the solitary tract (cNTS), specifically within glucagon-like peptide-1 (GLP-1) and noradrenergic (NA) neurons of the A2 cell group. Systemic CCK increases vagal sensory signaling to the cNTS, an effect that is amplified by leptin and reduced by ghrelin. Since fasting reduces plasma leptin and increases plasma ghrelin levels, we hypothesized that peripheral leptin administration and/or antagonism of ghrelin receptors in fasted rats would rescue the ability of CCK to activate GLP-1 neurons and a caudal subset of A2 neurons that coexpress prolactin-releasing peptide (PrRP). To test this, cFos expression was examined in ad libitum-fed and overnight food-deprived (DEP) rats after intraperitoneal CCK, after coadministration of leptin and CCK, or after intraperitoneal injection of a ghrelin receptor antagonist (GRA) before CCK. In fed rats, CCK activated cFos in ~60% of GLP-1 and PrRP neurons. Few or no GLP-1 or PrRP neurons expressed cFos in DEP rats treated with CCK alone, CCK combined with leptin, or GRA alone. However, GRA pretreatment increased the ability of CCK to activate GLP-1 and PrRP neurons and also enhanced the hypophagic effect of CCK in DEP rats. Considered together, these new findings suggest that reduced behavioral sensitivity to CCK in fasted rats is at least partially due to ghrelin-mediated suppression of hindbrain GLP-1 and PrRP neural responsiveness to CCK.

Chronic Suppression of Glucagon-Like Peptide-1 Receptor (GLP1R) mRNA Translation in the Rat Bed Nucleus of the Stria Terminalis Reduces Anxiety-Like Behavior and Stress-Induced Hypophagia, But Prolongs Stress-Induced Elevation of Plasma Corticosterone.

The anterior lateral bed nucleus of the stria terminalis (alBST) expresses glucagon-like peptide-1 receptors (GLP1Rs) and receives input from caudal brainstem GLP1 neurons. GLP1 administered centrally reduces food intake and increases anxiety-like behavior and plasma corticosterone (cort) levels in rats, whereas central GLP1R antagonism has opposite effects. Anxiogenic threats and other stressors robustly activate c-fos expression in both GLP1-producing neurons and also in neurons within alBST subregions expressing GLP1R. To examine the functional role of GLP1R signaling within the alBST, adult male Sprague Dawley rats received bilateral alBST-targeted injections of an adeno-associated virus (AAV) vector expressing short hairpin RNA (shRNA) to knock down the translation of GLP1R mRNA (GLP1R-KD rats), or similar injections of a control AAV (CTRL rats). In situ hybridization revealed that GLP1R mRNA is expressed in a subset of GABAergic alBST neurons, and quantitative real-time PCR confirmed that GLP1R-KD rats displayed a significant 60% reduction in translatable GLP1R mRNA. Compared with CTRL rats, GLP1R-KD rats gained more body weight over time and displayed less anxiety-like behavior, including a loss of light-enhanced acoustic startle and less stress-induced hypophagia. Conversely, while baseline plasma cort levels were similar in GLP1R-KD and CTRL rats, GLP1R-KD rats displayed a prolonged stress-induced elevation of plasma cort levels. GLP1R-KD and CTRL rats displayed similar home cage food intake and a similar hypophagic response to systemic Exendin-4, a GLP1R agonist that crosses the blood-brain barrier. We conclude that GLP1R expressed within the alBST contributes to multiple behavioral responses to anxiogenic threats, yet also serves to limit the plasma cort response to acute stress.SIGNIFICANCE STATEMENT Anxiety is an affective and physiological state that supports threat avoidance. Identifying the neural bases of anxiety-like behaviors in animal models is essential for understanding mechanisms that contribute to normative and pathological anxiety in humans. In rats, anxiety/avoidance behaviors can be elicited or enhanced by visceral or cognitive threats that increase glucagon-like peptide-1 (GLP1) signaling from the caudal brainstem to the hypothalamus and limbic forebrain. Data reported here support a role for limbic GLP1 receptor signaling to enhance anxiety-like behavior and to attenuate stress-induced elevations in plasma cort levels in rats. Improved understanding of central GLP1 neural pathways that impact emotional responses to stress could expand potential therapeutic options for anxiety and other stress-related disorders in humans.

Psychogenic Stress Activates C-Fos in Nucleus Accumbens-Projecting Neurons of the Hippocampal Ventral Subiculum.

Background: The ventral subiculum is known to be activated by the presentation of novel stressors. It has been hypothesized that neuronal ensembles at the ventral aspect of the hippocampal formation are involved in context-dependent processing and can guide the learning of appropriate action selections in response to threatening contexts. Artificial activation of the ventral subiculum can excite medium spiny neurons of the nucleus accumbens and can increase the excitability of mesolimbic dopamine neurons via a polysynaptic pathway through the basal ganglia. However, it remains unknown whether this circuit can be activated by aversive experience, and if so, whether ventral subiculum engages nucleus accumbens monosynaptically. Methods: To address this, the retrograde tracer fluorogold was used in rats to label neurons projecting to the caudomedial nucleus accumbens. One to 2 weeks later, the same rats were exposed to psychogenic stress (i.e., acute restraint in a novel test room) or served as nonhandled controls, followed by dual immunocytochemical localization of retrogradely transported tracer and nuclear Fos. Results: Compared with controls, rats exposed to psychogenic stress displayed more fluorogold-positive ventral subiculum neurons that were double-labeled for Fos. Conclusion: This study establishes that the direct pathway from ventral subiculum to the caudomedial nucleus accumbens is activated by stressful experience.

Excitatory Hindbrain-Forebrain Communication Is Required for Cisplatin-Induced Anorexia and Weight Loss.

Cisplatin chemotherapy is commonly used to treat cancer despite severe energy balance side effects. In rats, cisplatin activates nucleus tractus solitarius (NTS) projections to the lateral parabrachial nucleus (lPBN) and calcitonin-gene related peptide (CGRP) projections from the lPBN to the central nucleus of the amygdala (CeA). We demonstrated previously that CeA glutamate receptor signaling mediates cisplatin-induced anorexia and body weight loss. Here, we used neuroanatomical tracing, immunofluorescence, and confocal imaging to demonstrate that virtually all NTS→lPBN and lPBN→CeA CGRP projections coexpress vesicular glutamate transporter 2 (VGLUT2), providing evidence that excitatory projections mediate cisplatin-induced energy balance dysregulation. To test whether lPBN→CeA projection neurons are required for cisplatin-induced anorexia and weight loss, we inhibited these neurons chemogenetically using a retrograde Cre-recombinase-expressing canine adenovirus-2 in combination with Cre-dependent inhibitory Designer Receptors Exclusive Activated by Designer Drugs (DREADDs) before cisplatin treatment. Inhibition of lPBN→CeA neurons attenuated cisplatin-induced anorexia and body weight loss significantly. Using a similar approach, we additionally demonstrated that inhibition of NTS→lPBN neurons attenuated cisplatin-induced anorexia and body weight loss significantly. Together, our data support the view that excitatory hindbrain-forebrain projections are necessary for cisplatin's untoward effects on energy intake, elucidating a key neuroanatomical circuit driving pathological anorexia and weight loss that accompanies chemotherapy treatment. SIGNIFICANCE STATEMENT: Chemotherapy treatments are commonly used to treat cancers despite accompanying anorexia and weight loss that may limit treatment adherence and reduce patient quality of life. Strikingly, we lack a neural understanding of, and effective treatments for, chemotherapy-induced anorexia and weight loss. The current data characterize the excitatory nature of neural projections activated by cisplatin in rats and reveal the necessity of specific hindbrain-forebrain projections for cisplatin-induced anorexia and weight loss. Together, these findings help to characterize the neural mechanisms mediating cisplatin-induced anorexia, advancing opportunities to develop better-tolerated chemotherapies and adjuvant therapies to prevent anorexia and concurrent nutritional deficiencies during cancer treatment.

Nicotine Enhances Footshock- and Lithium Chloride-Conditioned Place Avoidance in Male Rats.

INTRODUCTION: Numerous studies have shown that nicotine (NIC) can enhance the reinforcing effects of non-NIC stimuli through a nonassociative mechanism. To date, it is unclear whether NIC reinforcement enhancement serves to increase behaviors motivated by rewarding stimuli only, or whether NIC potentiates behavior motivated by all stimuli, regardless of valence. METHODS: The current study used a place conditioning procedure to examine whether acute NIC injection modulates avoidance of an environment previously associated with an aversive stimulus. Separate groups of rats underwent place conditioning using either lithium chloride (125mg/kg/ml, i.p.) or footshock (0.75 mA) as the aversive stimulus. Other rats served as nonconditioned controls. The magnitude of place avoidance was assessed after acute NIC (0.1 or 0.4mg/kg/ml, s.c.) or saline. RESULTS: Rats avoided chambers previously paired with either lithium chloride or footshock, and conditioned place avoidance was significantly enhanced by NIC pre-treatment. CONCLUSIONS: These results demonstrate that the ability of NIC to enhance motivated behavior extends to behaviors elicited by aversive stimuli, evidence that NIC affects behavior motivated by a broader range of stimuli than previously appreciated. IMPLICATIONS: The current study examined whether the reinforcement enhancement properties of NIC apply to aversive stimuli by testing NIC enhancement of conditioned place avoidance in rats. The results demonstrate that NIC enhances the motivational impact of these distinct aversive stimuli, providing novel evidence that NIC affects behavior motivated by a broader range of stimuli than has previously been demonstrated.

Negative Energy Balance Blocks Neural and Behavioral Responses to Acute Stress by "Silencing" Central Glucagon-Like Peptide 1 Signaling in Rats.

Previous reports indicate that caloric restriction attenuates anxiety and other behavioral responses to acute stress, and blunts the ability of stress to increase anterior pituitary release of adrenocorticotropic hormone. Since hindbrain glucagon-like peptide-1 (GLP-1) neurons and noradrenergic prolactin-releasing peptide (PrRP) neurons participate in behavioral and endocrine stress responses, and are sensitive to the metabolic state, we examined whether overnight food deprivation blunts stress-induced recruitment of these neurons and their downstream hypothalamic and limbic forebrain targets. A single overnight fast reduced anxiety-like behavior assessed in the elevated-plus maze and acoustic startle test, including marked attenuation of light-enhanced startle. Acute stress [i.e., 30 min restraint (RES) or 5 min elevated platform exposure] robustly activated c-Fos in GLP-1 and PrRP neurons in fed rats, but not in fasted rats. Fasting also significantly blunted the ability of acute stress to activate c-Fos expression within the anterior ventrolateral bed nucleus of the stria terminalis (vlBST). Acute RES stress suppressed dark-onset food intake in rats that were fed ad libitum, whereas central infusion of a GLP-1 receptor antagonist blocked RES-induced hypophagia, and reduced the ability of RES to activate PrRP and anterior vlBST neurons in ad libitum-fed rats. Thus, an overnight fast "silences" GLP-1 and PrRP neurons, and reduces both anxiety-like and hypophagic responses to acute stress. The partial mimicking of these fasting-induced effects in ad libitum-fed rats after GLP-1 receptor antagonism suggests a potential mechanism by which short-term negative energy balance attenuates neuroendocrine and behavioral responses to acute stress. SIGNIFICANCE STATEMENT: The results from this study reveal a potential central mechanism for the "metabolic tuning" of stress responsiveness. A single overnight fast, which markedly reduces anxiety-like behavior in rats, reduces or blocks the ability of acute stress to activate hindbrain neurons that are immunoreactive for either prolactin-releasing peptide or glucagon-like peptide 1, and attenuates the activation of their stress-sensitive projection targets in the limbic forebrain. In nonfasted rats, central antagonism of glucagon-like peptide 1 receptors partially mimics the effect of an overnight fast by blocking the ability of acute stress to inhibit food intake, and by attenuating stress-induced activation of hindbrain and limbic forebrain neurons. We propose that caloric restriction attenuates behavioral and physiological responses to acute stress by "silencing" central glucagon-like peptide 1 signaling pathways.

Systemic leptin dose-dependently increases STAT3 phosphorylation within hypothalamic and hindbrain nuclei.

Leptin released peripherally acts within the central nervous system (CNS) to modulate numerous physiological and behavioral functions. Histochemical identification of leptin-responsive CNS cells can reveal the specific cellular phenotypes and neural circuits through which leptin signaling modulates these functions. Leptin signaling elicits phosphorylation of signal transducer and activator of transcription 3 (pSTAT3), making pSTAT3-immunoreactivity (ir) a useful proxy for identifying leptin-responsive cells. Relatively low systemic doses of leptin (i.e., 10-130 µg/kg body wt) are sufficient to decrease food intake, inhibit gastric emptying, and increase sympathetic activity, but there are no histological reports of central pSTAT3-ir following leptin doses within this range. Considering this, we quantified central pSTAT3-ir in rats after intraperitoneal injections of leptin at doses ranging from 50 to 800 µg/kg body wt. Tissue sections were processed to identify pSTAT3-ir alone or in combination with immunolabeling for cocaine- and amphetamine-regulated transcript (CART), glucagon-like peptide-1 (GLP-1), prolactin-releasing peptide (PrRP), or dopamine-ß-hydroxylase (DßH). Leptin doses as low as 50, 100, and 200 µg/kg body wt significantly increased the number of pSTAT3-ir cells in the arcuate nucleus of the hypothalamus (ARC), nucleus of the solitary tract (NTS), and ventromedial nucleus of the hypothalamus, respectively, and also led to robust pSTAT3 labeling in neural processes. The differential dose-dependent increases in pSTAT3-ir across brain regions provide new information regarding central leptin sensitivity. Within the ARC, CART-ir and pSTAT3-ir were often colocalized, consistent with evidence of leptin sensitivity in this neural population. Conversely, within the NTS, pSTAT3 only rarely colocalized with PrRP and/or DßH, and never with GLP-1.

Differential activation of chemically identified neurons in the caudal nucleus of the solitary tract in non-entrained rats after intake of satiating vs. non-satiating meals.

Satiety signals arising from the gastrointestinal (GI) tract and related digestive organs during food ingestion and digestion are conveyed by vagal sensory afferents to the hindbrain nucleus of the solitary tract (NST). Two intermingled but chemically distinct NST neuronal populations have been implicated in meal size control: noradrenergic (NA) neurons that comprise the A2 cell group, and glucagon-like peptide-1 (GLP-1)-positive neurons. Previous results indicate that A2 neurons are activated in a meal size-dependent manner in rats that have been acclimated/entrained to a feeding schedule in order to increase meal size, whereas feeding under the same conditions does not activate GLP-1 neurons. The present study was designed to test the hypothesis that both A2 and GLP-1 neuronal populations are recruited in non-entrained rats after voluntary first-time intake of an unrestricted, satiating volume of liquid Ensure. DBH-positive A2 neurons within the caudal visceral NST were progressively recruited to express cFos in rats that consumed progressively larger volumes of Ensure. Among these DBH-positive neurons, the prolactin-releasing peptide (PrRP)-positive subset was more sensitive to feeding-induced activation than the PrRP-negative subset. Notably, significant activation of GLP-1-positive neurons occurred only in rats that consumed the largest volumes of Ensure, corresponding to nearly 5% of their BW. We interpret these results as evidence that progressive recruitment of NA neurons within the caudal NST, especially the most caudally-situated PrRP-positive subset, effectively "tracks" the magnitude of GI satiety signals and other meal-related sensory feedback. Conversely, GLP-1 neurons may only be recruited in response to the homeostatic challenge of consuming a very large, unanticipated meal.

Delineation of vagal emetic pathways: intragastric copper sulfate-induced emesis and viral tract tracing in musk shrews.

Signals from the vestibular system, area postrema, and forebrain elicit nausea and vomiting, but gastrointestinal (GI) vagal afferent input arguably plays the most prominent role in defense against food poisoning. It is difficult to determine the contribution of GI vagal afferent input on emesis because various agents (e.g., chemotherapy) often act on multiple sensory pathways. Intragastric copper sulfate (CuSO4) potentially provides a specific vagal emetic stimulus, but its actions are not well defined in musk shrews (Suncus murinus), a primary small animal model used to study emesis. The aims of the current study were 1) to investigate the effects of subdiaphragmatic vagotomy on CuSO4-induced emesis and 2) to conduct preliminary transneuronal tracing of the GI-brain pathways in musk shrews. Vagotomy failed to inhibit the number of emetic episodes produced by optimal emetic doses of CuSO4 (60 and 120 mg/kg ig), but the effects of lower doses were dependent on an intact vagus (20 and 40 mg/kg). Vagotomy also failed to affect emesis produced by motion (1 Hz, 10 min) or nicotine administration (5 mg/kg sc). Anterograde transport of the H129 strain of herpes simplex virus-1 from the ventral stomach wall identified the following brain regions as receiving inputs from vagal afferents: the nucleus of the solitary tract, area postrema, and lateral parabrachial nucleus. These data indicate that the contribution of vagal pathways to intragastric CuSO4-induced emesis is dose dependent in musk shrews. Furthermore, the current neural tracing data suggest brain stem anatomical circuits that are activated by GI signaling in the musk shrew.

Overnight food deprivation markedly attenuates hindbrain noradrenergic, glucagon-like peptide-1, and hypothalamic neural responses to exogenous cholecystokinin in male rats.

Systemic administration of sulfated cholecystokinin-8 (CCK) activates neurons within the hindbrain nucleus of the solitary tract (NTS) that project directly to the paraventricular nucleus of the hypothalamus (PVN), and these projections underlie the ability of exogenous CCK to activate the hypothalamic-pituitary-adrenal (HPA) stress axis. CCK inhibits food intake, increases NTS neuronal cFos expression, and activates the HPA axis in a dose-dependent manner. While the hypophagic effects of exogenous CCK are attenuated in food-deprived rats, CCK dose-response relationships for NTS and hypothalamic activation in fed and fasted rats are unknown. Within the NTS, noradrenergic A2 and glucagon-like peptide-1 (GLP-1) neurons express cFos after high doses of CCK, and both neuronal populations project directly to the medial parvocellular (mp)PVN. We hypothesized that increasing and correlated proportions of A2, GLP-1, and mpPVN neurons would express cFos in rats after increasing doses of CCK, and that food deprivation would attenuate both hindbrain and hypothalamic neural activation. To test these hypotheses, ad libitum-fed (ad lib) and overnight food-deprived (DEP) rats were anesthetized and perfused with fixative 90min after i.p. injection of 1.0ml saline vehicle containing CCK at doses of 0, 3, or 10µg/kg BW. Additional ad lib and DEP rats served as non-handled (NH) controls. Brain tissue sections were processed for dual immunocytochemical localization of cFos and dopamine-ß-hydroxylase to identify A2 neurons, or cFos and GLP-1. Compared to negligible A2 cFos activation in NH control rats, i.p. vehicle and CCK dose-dependently increased A2 activation, and this was significantly attenuated by DEP. DEP also attenuated mpPVN cFos expression across all treatment groups, and A2 activation was strongly correlated with mpPVN activation in both ad lib and DEP rats. In ad lib rats, large and similar numbers of GLP-1 neurons expressed cFos across all i.p. treatment groups, regardless of CCK dose. Surprisingly, DEP nearly abolished baseline GLP-1 cFos expression in NH controls, and also in rats after i.p. injection of vehicle or CCK. We conclude that CCK-induced hypothalamic cFos activation is strongly associated with A2 activation, whereas the relationship between mpPVN and GLP-1 activation is less clear. Furthermore, activation of A2, GLP-1, and mpPVN neurons is significantly modulated by feeding status, suggesting a mechanism through which food intake and metabolic state might impact hypothalamic neuroendocrine responses to homeostatic challenge.

Immune challenge activates neural inputs to the ventrolateral bed nucleus of the stria terminalis.

Hypothalamo-pituitary-adrenal (HPA) axis activation in response to infection is an important mechanism by which the nervous system can suppress inflammation. HPA output is controlled by the hypothalamic paraventricular nucleus (PVN). Previously, we determined that noradrenergic inputs to the PVN contribute to, but do not entirely account for, the ability of bacterial endotoxin (i.e., lipopolysacharide, LPS) to activate the HPA axis. The present study investigated LPS-induced recruitment of neural inputs to the ventrolateral bed nucleus of the stria terminalis (vlBNST). GABAergic projections from the vlBNST inhibit PVN neurons at the apex of the HPA axis; thus, we hypothesize that LPS treatment activates inhibitory inputs to the vlBNST to thereby "disinhibit" the PVN and increase HPA output. To test this hypothesis, retrograde neural tracer was iontophoretically delivered into the vlBNST of adult male rats to retrogradely label central sources of axonal input. After one week, rats were injected i.p. with either LPS (200 µg/kg BW) or saline vehicle, and then perfused with fixative 2.5h later. Brains were processed for immunohistochemical localization of retrograde tracer and the immediate-early gene product, Fos (a marker of neural activation). Brain regions that provide inhibitory input to the vlBNST (e.g., caudal nucleus of the solitary tract, central amygdala, dorsolateral BNST) were preferentially activated by LPS, whereas sources of excitatory input (e.g., paraventricular thalamus, medial prefrontal cortex) were not activated or were activated less robustly. These results suggest that LPS treatment recruits central neural systems that actively suppress vlBNST neural activity, thereby removing a potent source of inhibitory control over the HPA axis.

Central Fos expression and conditioned flavor avoidance in rats following intragastric administration of bitter taste receptor ligands.

G protein-coupled receptors that signal bitter taste (T2Rs) are expressed in the mucosal lining of the oral cavity and gastrointestinal (GI) tract. In mice, intragastric infusion of T2R ligands activates Fos expression within the caudal viscerosensory portion of the nucleus of the solitary tract (NTS) through a vagal pathway (Hao S, Sternini C, Raybould HE. Am J Physiol Regul Integr Comp Physiol 294: R33-R38, 2008). The present study was performed in rats to further characterize the distribution and chemical phenotypes of brain stem and forebrain neurons activated to express Fos after intragastric gavage of T2R ligands, and to determine a potential behavioral correlate of this central neural activation. Compared with relatively low brain stem and forebrain Fos expression in control rats gavaged intragastrically with water, rats gavaged intragastrically with T2R ligands displayed significantly increased activation of neurons within the caudal medial (visceral) NTS and caudal ventrolateral medulla, including noradrenergic neurons, and within the lateral parabrachial nucleus, central nucleus of the amygdala, and paraventricular nucleus of the hypothalamus. A behavioral correlate of this Fos activation was evidenced when rats avoided consuming flavors that previously were paired with intragastric gavage of T2R ligands. While unconditioned aversive responses to bitter tastants in the oral cavity are often sufficient to inhibit further consumption, a second line of defense may be provided postingestively by ligand-induced signaling at GI T2Rs that signal the brain via vagal sensory inputs to the caudal medulla.

Experimental dissociation of neural circuits underlying conditioned avoidance and hypophagic responses to lithium chloride.

We previously reported that noradrenergic (NA) neurons in the nucleus of the solitary tract (NST) are necessary for exogenous CCK octapeptide to inhibit food intake in rats. To determine whether NST NA neurons also are necessary for lithium chloride (LiCl) to inhibit food intake and/or to support conditioned avoidance behavior, saporin toxin conjugated to an antibody against dopamine beta hydroxylase (DSAP) was microinjected bilaterally into the NST to ablate resident NA neurons. DSAP and sham control rats subsequently were tested for the ability of LiCl (0.15M, 2% body wt) to inhibit food intake and to support conditioned flavor avoidance (CFA). LiCl-induced hypophagia was significantly blunted in DSAP rats, and those with the most extensive loss of NST NA neurons demonstrated the most attenuated LiCl-induced hypophagia. Conversely, LiCl supported a robust CFA that was of similar magnitude in sham control and DSAP rats, including rats with the most extensive NA lesions. A terminal c-Fos study revealed intact LiCl-induced c-Fos expression in the lateral parabrachial nucleus and central amygdala in DSAP rats, despite significant loss of NST NA neurons and attenuated c-Fos activation of corticotropin-releasing hormone-positive neurons in the paraventricular nucleus of the hypothalamus (PVN). Thus, NST NA neurons contribute significantly to LiCl-induced hypophagia and recruitment of stress-responsive PVN neurons but appear to be unnecessary for CFA learning and expression. These findings support the view that distinct central nervous system circuits underlie LiCl-induced inhibition of food intake and conditioned avoidance behavior in rats.

Progressive postnatal increases in Fos immunoreactivity in the forebrain and brain stem of rats after viscerosensory stimulation with lithium chloride.

Interoceptive signals have a powerful impact on the motivation and emotional learning of animals during stressful experiences. However, current insights into the organization of interoceptive pathways stem mainly from observation and manipulation of adults, and little is known regarding the functional development of viscerosensory signaling pathways. To address this, we have examined central neural activation patterns in rat pups after treatment with lithium chloride (LiCl), a malaise-inducing agent. Rat pups were injected intraperitoneally with 0.15 M LiCl or 0.15 M NaCl (2% body wt) on postnatal day (P)0, 7, 14, 21, or 28, perfused 60 to 90 min postinjection, and their brains assayed for Fos protein immunolabeling. Compared with saline treatment, LiCl increased Fos only slightly in the area postrema, nucleus of the solitary tract, and lateral parabrachial nucleus on P0. LiCl did not increase Fos above control levels in the central nucleus of the amygdala, bed nucleus of the stria terminalis (BNST), or paraventricular nucleus of the hypothalamus on P0 but did on P7 and later. Maximal Fos responses to LiCl were observed on P14 in all areas except the BNST, in which LiCl-induced Fos activation continued to increase through P28. These results indicate that central LiCl-sensitive interoceptive circuits in rats are not fully functional at birth, and show age-dependent increases in neural Fos responses to viscerosensory stimulation with LiCl.

The anxiogenic drug yohimbine activates central viscerosensory circuits in rats.

Systemic administration of the alpha(2)-adrenoceptor antagonist yohimbine (YO) activates the HPA stress axis and promotes anxiety in humans and experimental animals. We propose that visceral malaise contributes to the stressful and anxiogenic effects of systemic YO and that YO recruits brainstem noradrenergic (NA) and peptidergic neurons that relay viscerosensory signals to the hypothalamus and limbic forebrain. To begin testing these hypotheses, the present study explored dose-related effects of YO on food intake, conditioned flavor avoidance (CFA), and Fos immunolabeling in rats. Systemic YO (5.0 mg/kg BW, i.p.) inhibited food intake, supported CFA, and increased Fos immunolabeling in identified NA neurons in the ventrolateral medulla, nucleus of the solitary tract, and locus coeruleus. YO also increased Fos in the majority of corticotropin releasing hormone-positive neurons in the paraventricular nucleus of the hypothalamus. YO administered at 1.0 mg/kg BW did not inhibit food intake, did not support CFA, and did not increase Fos immunolabeling. Retrograde neural tracing demonstrated that neurons activated by YO at 5.0 mg/kg BW included medullary and pontine neurons that project to the central nucleus of the amygdala and to the lateral bed nucleus of the stria terminalis, the latter region receiving comparatively greater input by Fos-positive neurons. We conclude that YO produces anorexigenic and aversive effects that correlate with activation of brainstem viscerosensory inputs to the limbic forebrain. These findings invite continued investigation of how central viscerosensory signaling pathways interact with hypothalamic and limbic regions to influence interrelated physiological and behavioral components of anxiety, stress, and visceral malaise.

Dehydration anorexia is attenuated in oxytocin-deficient mice.

Evidence in rats suggests that central oxytocin (OT) signaling pathways contribute to suppression of food intake during dehydration (i.e., dehydration anorexia). The present study examined water deprivation-induced dehydration anorexia in wild-type and OT -/- mice. Mice were deprived of food alone (fasted, euhydrated) or were deprived of both food and water (fasted, dehydrated) for 18 h overnight. Fasted wild-type mice consumed significantly less chow during a 60-min refeeding period when dehydrated compared with their intake when euhydrated. Conversely, fasting-induced food intake was slightly but not significantly suppressed by dehydration in OT -/- mice, evidence for attenuated dehydration anorexia. In a separate experiment, mice were deprived of water (but not food) overnight for 18 h; then they were anesthetized and perfused with fixative for immunocytochemical analysis of central Fos expression. Fos was elevated similarly in osmo- and volume-sensitive regions of the basal forebrain and hypothalamus in wild-type and OT -/- mice after water deprivation. OT-positive neurons expressed Fos in dehydrated wild-type mice, and vasopressin-positive neurons were activated to a similar extent in wild-type and OT -/- mice. Conversely, significantly fewer neurons within the hindbrain dorsal vagal complex were activated in OT -/- mice after water deprivation compared with activation in wild-type mice. These findings support the view that OT-containing projections from the hypothalamus to the hindbrain are necessary for the full expression of compensatory behavioral and physiological responses to dehydration.

Trimethylthiazoline supports conditioned flavor avoidance and activates viscerosensory, hypothalamic, and limbic circuits in rats.

Interoceptive stimuli modulate stress responses and emotional state, in part, via ascending viscerosensory inputs to the hypothalamus and limbic forebrain. It is unclear whether similar viscerosensory pathways are recruited by emotionally salient exteroceptive stimuli, such as odors. To address this question, we investigated conditioned avoidance and central c-Fos activation patterns in rats exposed to synthetic trimethylthiazoline (TMT), an odiferous natural component of fox feces. Experiment 1 demonstrated that rats avoid consuming novel flavors that previously were paired with TMT exposure, evidence that TMT supports conditioned flavor avoidance. Experiment 2 examined central neural systems activated by TMT. Odor-naive rats were acutely exposed to low or high levels of TMT or a novel nonaversive control odor and were perfused with fixative 60-90 min later. A subset of rats received retrograde neural tracer injections into the central nucleus of the amygdala (CeA) 7-10 days before odor exposure and perfusion. Brain sections were processed for dual-immunocytochemical detection of c-Fos and other markers to identify noradrenergic (NA) neurons, corticotropin-releasing hormone (CRH) neurons, and retrogradely labeled neurons projecting to the CeA. Significantly greater proportions of medullary and pontine NA neurons, hypothalamic CRH neurons, and CeA-projecting neurons were activated in rats exposed to TMT compared with activation in rats exposed to the nonaversive control odor. Thus the ability of TMT to support conditioned avoidance behavior is correlated with significant odor-induced recruitment of hypothalamic CRH neurons and brain stem viscerosensory inputs to the CeA.