Vagal Interoceptive Modulation of Motivated Behavior.

In addition to regulating the ingestion and digestion of food, sensory feedback from gut to brain modifies emotional state and motivated behavior by subconsciously shaping cognitive and affective responses to events that bias behavioral choice. This focused review highlights evidence that gut-derived signals impact motivated behavior by engaging vagal afferents and central neural circuits that generally serve to limit or terminate goal-directed approach behaviors, and to initiate or maintain behavioral avoidance.

Cocaine self-administration and extinction alter medullary noradrenergic and limbic forebrain cFos responses to acute, noncontingent cocaine injections in adult rats.

Central noradrenergic (NA) signaling contributes critically to multiple behavioral effects of cocaine administration, particularly stress- and anxiety-related effects. The present study examined the ability of acute cocaine to induce the immediate early gene product, cFos, in NA neurons and stress-related neural circuits in rats that were cocaine-naïve, or had a history of cocaine self-administration with or without extinction. Rats implanted with jugular catheters were trained to self-administer cocaine (0.5-mg/kg/infusion), with a subset subsequently trained on extinction. Cocaine-naïve controls were handled daily. After a final day of self-administration, extinction, or handling, rats received an i.p. injection of either cocaine (20-mg/kg) or saline, and 90min later were anesthetized and perfused. Tissue sections were processed for immunoperoxidase labeling of nuclear cFos with either immunoperoxidase or immunofluorescent cytoplasmic labeling of dopamine beta hydroxylase or tyrosine hydroxylase. Acute cocaine increased the number of activated NA neurons within the caudal nucleus of the solitary tract (NTS; A2 cell group) in cocaine-naïve and extinguished rats, but not in rats that only self-administered. Extinction attenuated cocaine-induced cFos activation in NA neurons of the caudal ventrolateral medulla (A1/C1 cell groups), and attenuated cFos within the paraventricular nucleus of the hypothalamus, the apex of the central neuroendocrine stress axis. Cocaine consistently increased cFos in the bed nucleus of the stria terminalis, regardless of history. NA neurons of the locus coeruleus (A6 cell group) were not activated after cocaine administration in any experimental group. Thus, the ability of cocaine to activate central stress circuitry is altered after cocaine self-administration. Our results suggest a unique role for the NTS in cocaine-induced reinstatement, as extinction training enhanced the ability of cocaine to activate NA neurons within this region. These findings suggest central NA systems originating in the caudal brainstem as potential targets for the treatment of cocaine addiction.

Yohimbine anxiogenesis in the elevated plus maze is disrupted by bilaterally disconnecting the bed nucleus of the stria terminalis from the central nucleus of the amygdala.

The α2 adrenergic receptor antagonist yohimbine (YO) is a sympathomimetic drug that crosses the blood-brain barrier after systemic administration. YO promotes increased transmitter release from noradrenergic (NA) axon terminals in the central nucleus of the amygdala (CEA), bed nucleus of the stria terminalis (BST), hypothalamus, and other brain regions implicated in physiological and behavioral responses to stressful and threatening stimuli. YO is potently anxiogenic in humans and experimental animals, including rats. To determine whether direct connections between the CEA and anterolateral group of BST nuclei (algBST) are necessary for YO anxiogenesis in rats, neurotoxic ibotenate lesions of the CEA in one hemisphere and the ipsi- or contralateral algBST were conducted to disrupt CEA-algBST communication uni- or bilaterally. Sham-lesioned controls received microinjections of vehicle into the CEA and algBST. Two weeks later, behavior was assessed in the elevated plus maze (EPMZ) in rats after i.p. saline or YO (1.0mg/kg). Central ibotenate lesion placement and extent was assessed post-mortem in NeuN-immunolabeled tissue sections. The ability of YO to increase anxiety-like behavior in the EPMZ was similarly robust in rats with sham lesions or ipsilateral CEA-algBST lesions. Conversely, YO anxiogenesis in the EPMZ was disrupted in rats with asymmetric lesions designed to bilaterally disconnect the CEA and algBST, whereas neither unilateral nor bilateral disconnecting lesions altered EPMZ behavior in rats after i.p. saline. We conclude that the anxiogenic effects of increased NA signaling in rats after YO require direct CEA-algBST interactions that do not shape EPMZ behavior under baseline conditions.

Central neural responses to restraint stress are altered in rats with an early life history of repeated brief maternal separation.

Repeated brief maternal separation (i.e. 15 min daily, MS15) of rat pups during the first one to two postnatal weeks enhances active maternal care received by the pups and attenuates their later behavioral and neuroendocrine responses to stress. In previous work, we found that MS15 also alters the developmental assembly and later structure of central neural circuits that control autonomic outflow to the viscera, suggesting that MS15 may alter central visceral circuit responses to stress. To examine this, juvenile rats with a developmental history of either MS15 or no separation (NS) received microinjection of retrograde neural tracer, FluoroGold (FG), into the hindbrain dorsal vagal complex (DVC). After 1 week, FG-injected rats and surgically intact littermates were exposed to either a 15-min restraint stress or an unrestrained control condition, and then perfused 1 h later. Brain tissue sections from surgically intact littermates were processed for Fos alone or in combination with phenotypic markers to examine stress-induced activation of neurons within the paraventricular nucleus of the hypothalamus (PVN), bed nucleus of the stria terminalis (BNST), and hindbrain DVC. Compared to NS controls, MS15 rats displayed less restraint-induced Fos activation within the dorsolateral BNST (dBNST), the caudal PVN, and noradrenergic neurons within the caudal DVC. To examine whether these differences corresponded with altered neural inputs to the DVC, sections from tracer-injected rats were double-labeled for FG and Fos to quantify retrogradely labeled neurons within hypothalamic and limbic forebrain regions of interest, and the proportion of these neurons activated after restraint. Only the dBNST displayed a significant effect of postnatal experience on restraint-induced Fos activation of DVC-projecting neurons. The distinct regional effects of MS15 on stress-induced recruitment of neurons within hypothalamic, limbic forebrain, and hindbrain regions has interesting implications for understanding how early life experience shapes the functional organization of stress-responsive circuits.

Repeated brief postnatal maternal separation enhances hypothalamic gastric autonomic circuits in juvenile rats.

Maternal separation of rat pups for 15 min each day over the first one to two postnatal weeks (MS15) has been shown to increase the active maternal care received by pups and to decrease their later neuroendocrine and behavioral stress reactivity compared to non-separated (NS) controls. Stress responses prominently feature altered gastric secretion and motility, and we previously reported that the developmental assembly of forebrain circuits underlying gastric autonomic control, including gastric responses to stress, is delayed by MS15 in neonatal rats [Card JP, Levitt P, Gluhovsky M, Rinaman L (2005) J Neurosci 25(40):9102-9111]. To determine how this early delay affects the later organization of central gastric autonomic circuits, the present study examined the effects of neonatal MS15 on central pre-gastric circuits assessed in post-weaning, juvenile rats. For this purpose, the retrograde transynaptic viral tracer, pseudorabies virus (PRV), was microinjected into the stomach wall of 28-30 day old male rats with an earlier developmental history of either MS15 or NS. Rats were perfused 72 h later and tissue was processed to reveal PRV-positive cells. Transynaptic PRV immunolabeling was quantified in selected preautonomic brainstem and forebrain regions, including the area postrema, bed nucleus of the stria terminalis, central nucleus of the amygdala, paraventricular nucleus of the hypothalamus (PVN), and visceral cortices. Compared to NS controls, MS15 rats displayed a significantly greater amount of PRV labeling within the PVN, including both the dorsal cap and ventral subnuclei. There were no postnatal group differences in the amount of PRV labeling within any other brain region examined in this study. This effect of MS15 to enhance hypothalamic preautonomic circuit structure indicates a strengthening of this pathway and may provide insight into how early life experience produces differential effects on later stress reactivity, including gastric secretory and motor responses to stress.

Noradrenergic inputs to the paraventricular hypothalamus contribute to hypothalamic-pituitary-adrenal axis and central Fos activation in rats after acute systemic endotoxin exposure.

Noradrenergic (NA) neurons within the nucleus of the solitary tract (NST) and caudal ventrolateral medulla (VLM) innervate the hypothalamic paraventricular nucleus (PVN) to initiate and modulate hypothalamic-pituitary-adrenal (HPA) axis responses to interoceptive stress. Systemic endotoxin (i.e. bacterial lipopolysaccharide, LPS) activates NA neurons within the NST and VLM that project to the PVN and other brain regions that receive interoceptive signals. The present study examined whether NA neurons with axonal inputs to the PVN are necessary for LPS to activate Fos expression within the PVN and other interoceptive-related brain regions, and to increase plasma corticosterone. Male Sprague-Dawley rats received bilateral stereotaxic microinjections of DSAP (saporin toxin conjugated to an antibody against dopamine-beta-hydroxylase, DbH) into the PVN to destroy NA inputs. Control rats were microinjected with vehicle into the PVN or received no PVN injections. Two weeks later, DSAP and control rats were injected i.p. with LPS (200 microg/kg BW) or saline vehicle, and perfused with fixative 2.5-3 h later. Brain tissue sections were processed to reveal nuclear Fos protein and cytoplasmic DbH immunolabeling. DSAP lesions depleted NA terminals in the PVN and bed nucleus of the stria terminalis, reduced the number of NA cell bodies in the NST and VLM, attenuated PVN Fos activation after LPS, and attenuated LPS-induced increases in plasma corticosterone. These findings support the view that NA projections from hindbrain to hypothalamus are necessary for a full HPA axis response to systemic immune challenge.

Oxytocin gene deletion mice overconsume palatable sucrose solution but not palatable lipid emulsions.

We previously reported that oxytocin knockout (OT KO) mice display markedly enhanced intake of sweet and nonsweet carbohydrate solutions compared with intake by wild-type (WT) mice of the same background strain. The present study was conducted to determine whether OT KO mice demonstrate enhanced intake of Intralipid, a palatable lipid emulsion. Male or female mice of both genotypes that were naive to the test solution were given continuous two-bottle access to Intralipid and water with food available ad libitum for 3 days. Throughout the experiment, mice of both genotypes showed a marked preference for Intralipid over water. On the 1st day, OT KO mice displayed twofold greater preference and consumed nearly twice as much Intralipid compared with WT cohorts. However, on subsequent days of exposure, Intralipid preference and intake did not differ between genotypes over a range of lipid concentrations presented in descending or ascending order. Daily and hourly measures of lipid vs. sucrose intake confirmed that OT KO mice consumed more sucrose solution, but not lipid emulsion, than WT mice. During ad libitum access to Intralipid, both genotypes consumed significantly more calories from the emulsion as concentration increased. Both genotypes maintained consistent total daily caloric intake (lipid plus chow) and compensated by decreasing chow intake over the course of the study. These findings, coupled with prior reports from our laboratory, support the view that OT signaling pathways participate in limiting intake of palatable carbohydrate-containing solutions, but do not appear to play a role in limiting intake of Intralipid.

Noradrenergic axon terminals contact gastric preautonomic neurons in the paraventricular nucleus of the hypothalamus in rats.

Hypothalamic neural activity is modulated by viscerosensory signals that are carried in large part by noradrenergic (NA) inputs to the paraventricular nucleus of the hypothalamus (PVN). The present study examined the ultrastructural relationship of NA axon varicosities with the somata and dendrites of identified gastric preautonomic PVN neurons in adult male rats. NA varicosities were visualized by immunoperoxidase labeling of dopamine beta hydroxylase (DbH), and gastric preautonomic PVN neurons were identified by immunogold labeling of pseudorabies virus (PRV) transported retrogradely and transneuronally from injection sites in the stomach wall. Among 1,136 DbH-positive varicosities identified within the parvocellular PVN in four rats, approximately 36% formed either a close apposition or a synaptic contact with a somatic or dendritic profile. The majority of identified contacts between DbH- and PRV-positive profiles were classified as close appositions that lacked clear synaptic specializations. Approximately 65% of identified synaptic contacts between DbH- and PRV-positive profiles were classified as symmetric (Gray's type II) synapses. DbH-positive terminals formed close appositions and synaptic contacts with dendritic and somatic compartments of PRV-positive neurons, although dendrites were contacted nearly five times more often than somata. These findings invite continued work to delineate the functional role of NA signaling pathways in conveying interoceptive signals to preautonomic PVN neurons under normal and pathophysiological conditions.

Ectopic sympathetic preganglionic neurons maintain proper connectivity in the reeler mutant mouse.

The location of sympathetic preganglionic neurons (SPN) in the spinal cord of the reeler mouse mutant is abnormal. Instead of their normal location in the intermediolateral column, the majority of SPN in the reeler cluster around the central canal. To determine whether ectopically located SPN in the reeler form appropriate synaptic connections with their pre- and postsynaptic partners, we examined 1). whether the axons of descending neural pathways that normally terminate on SPN follow them to their ectopic location, and 2). whether the central autonomic neural circuit that controls sympathetic output to the kidney is organized normally in the reeler. Using antibodies against tyrosine hydroxylase, serotonin, neuropeptide Y, substance P and calcitonin gene-related peptide as markers for adrenergic, serotonergic and peptidergic terminals, we found that axons which normally innervate SPN follow these neurons to their ectopic spinal location in the reeler. Injection of pseudorabies virus into the kidney of wild type and reeler mutant mice revealed similar patterns of renal sympathetic and pre-sympathetic control circuits in the spinal cord, brainstem and forebrain. These results indicate that the presynaptic inputs and postsynaptic targets of SPN in the reeler are normal, despite the ectopic spinal location of their cell bodies.

Identification of lingual motor control circuits using two strains of pseudorabies virus.

First-order interneurons that project to hypoglossal motoneurons are distributed within reticular formation subdivisions in the pons and medulla in areas thought to control licking, swallowing, chewing, and respiration. Movement of the tongue in each of these functions is achieved by the coordinated action of both intrinsic and extrinsic lingual muscles. Interneuron populations that project to these different lingual motoneuronal pools appear to be largely overlapping in the reticular formation. Because of the functional coupling between intrinsic and extrinsic muscles during most tongue movements, one might predict that individual pre-hypoglossal interneurons project to multiple motoneuronal pools. To test this hypothesis, one strain of pseudorabies virus was injected into the styloglossus muscle (an extrinsic lingual muscle) and a second strain of pseudorabies virus was injected into the intrinsic lingual muscles of the anterior tongue in the same preparation. Rats were perfused with fixative 84-96 h later, and dual-labeling immunohistochemistry was performed to reveal populations of single- and double-labeled brainstem neurons. Motoneurons innervating the different lingual muscles were spatially segregated within the hypoglossal motor nucleus, and no double-labeled motoneurons were observed. In contrast, pre-hypoglossal neurons projecting to each lingual motoneuron pool were highly overlapping in the reticular formation, and many were double-labeled. These observations suggest that coactivation of lingual muscles can be achieved, at least in part, through divergent projections of first-order interneurons to anatomically and functionally distinct pools of lingual motoneurons in the hypoglossal nucleus.

Characterization of the central nervous system innervation of the rat spleen using viral transneuronal tracing.

Splenic immune function is modulated by sympathetic innervation, which in turn is controlled by inputs from supraspinal regions. In the present study, the characterization of central circuits involved in the control of splenic function was accomplished by injecting pseudorabies virus (PRV), a retrograde transynaptic tracer, into the spleen and conducting a temporal analysis of the progression of the infection from 60 hours to 110 hours postinoculation. In addition, central noradrenergic cell groups involved in splenic innervation were characterized by dual immunohistochemical detection of dopamine-beta-hydroxylase and PRV. Infection in the CNS first appeared in the spinal cord. Splenic sympathetic preganglionic neurons, identified in rats injected with Fluoro-Gold i.p. prior to PRV inoculation of the spleen, were located in T(3)-T(12) bilaterally; numerous infected interneurons were also found in the thoracic spinal cord (T(1)-T(13)). Infected neurons in the brain were first observed in the A5 region, ventromedial medulla, rostral ventrolateral medulla, paraventricular hypothalamic nucleus, Barrington's nucleus, and caudal raphe. At intermediate survival times, the number of infected cells increased in previously infected areas, and infected neurons also appeared in lateral hypothalamus, A7 region, locus coeruleus, subcoeruleus region, nucleus of the solitary tract, and C3 cell group. At longer postinoculation intervals, infected neurons were found in additional hypothalamic areas, Edinger-Westphal nucleus, periaqueductal gray, pedunculopontine tegmental nucleus, caudal ventrolateral medulla, and area postrema. These results demonstrate that the sympathetic outflow to the spleen is controlled by a complex multisynaptic pathway that involves several brainstem and forebrain nuclei.

Postnatal development of catecholamine inputs to the paraventricular nucleus of the hypothalamus in rats.

Adrenergic and noradrenergic neural projections to the paraventricular nucleus of the hypothalamus (PVN) contribute importantly to viscerosensory modulation of pituitary hormone secretion. Immaturity of ascending catecholamine pathways may partially underlie the documented hyporesponsiveness of PVN neurosecretory cells to certain interoceptive stimuli in rats during the first few weeks of postnatal development. To explore this possibility, the present study compared the distribution and number of dopamine-beta-hydroxylase (DBH)- and phenylethanolamine-N-methyltransferase (PNMT)-positive neurons projecting to the PVN in newborn and adult rats. In addition, a quantitative analysis of DBH- and PNMT-immunoreactive fibers in the medial parvocellular subnucleus, dorsal division (PVNmpd) and posterior magnocellular subnucleus, lateral division (PVNpml) was performed in adult rats and in developing rats on postnatal day (P)1, P7, P14, and P21. The numbers of PVN-projecting neurons in the A1, C1, A2/C2, C3, or A6 catecholamine cell groups were similar in newborn and adult rats, as were the proportions of PVN-projecting neurons in each region that were PNMT-positive. However, fewer PVN-projecting neurons in the C1 and C3 regions expressed DBH immunolabeling in newborn rats compared to adults. DBH immunolabeling increased progressively in the PVNmpd and PVNpml between postnatal days P1 and P21, when adult-like levels were achieved. Conversely, PNMT immunolabeling in the same PVN subdivisions was most dense at P1, gradually decreasing to adult-like levels by P21. These dynamic developmental changes in catecholamine synthetic enzyme immunolabeling densities in the PVN may reflect functional changes in noradrenergic and adrenergic signaling capacity in rats during the first few weeks of postnatal development.

Plasma hormone levels and central c-Fos expression in ferrets after systemic administration of cholecystokinin.

Posterior pituitary hormone secretion and central neural expression of the immediate-early gene product c-Fos was examined in adult ferrets after intravenous administration of CCK octapeptide. Pharmacological doses of CCK (1, 5, 10, or 50 microg/kg) did not induce emesis, but elicited behavioral signs of nausea and dose-related increases in plasma vasopressin (AVP) levels without significant increases in plasma oxytocin (OT) levels. CCK activated neuronal c-Fos expression in several brain stem viscerosensory regions, including a dose-related activation of neurons in the dorsal vagal complex (DVC). Activated brain stem neurons included catecholaminergic and glucagon-like peptide-1-positive cells in the DVC and ventrolateral medulla. In the forebrain, activated neurons were prevalent in the paraventricular and supraoptic nuclei of the hypothalamus and also were observed in the central nucleus of the amygdala and bed nucleus of the stria terminalis. Activated hypothalamic neurons included cells that were immunoreactive for AVP, OT, and corticotropin-releasing factor. Comparable patterns of brain stem and forebrain c-Fos activation were observed in ferrets after intraperitoneal injection of lithium chloride (LiCl; 86 mg/kg), a classic emetic agent. However, LiCl activated more neurons in the area postrema and fewer neurons in the nucleus of the solitary tract compared with CCK. Together with results from previous studies in rodents, our findings support the view that nauseogenic treatments activate similar central neural circuits in emetic and nonemetic species, despite differences in treatment-induced emesis and pituitary hormone secretion.

Progressive postnatal assembly of limbic-autonomic circuits revealed by central transneuronal transport of pseudorabies virus.

The development of neuronal projections to a target and the establishment of synaptic connections with that target can be temporally distinct events, which typically are distinguished by functional assessments. We have applied a novel neuroanatomical approach to characterize the development of limbic forebrain synaptic inputs to autonomic neurons in neonatal rats. Transneuronal labeling of preautonomic forebrain neurons was achieved by inoculating the ventral stomach wall with pseudorabies virus (PRV) on postnatal day 1 (P1), P4, or P8. In each age group, PRV-positive neurons were present in autonomic and preautonomic regions of the spinal cord and brainstem 62-64 hr after inoculation. Transneuronal forebrain labeling in rats injected on P8 was similar to the transneuronal labeling reported previously in adult rats and included neurons in the medial and lateral hypothalamus, amygdala, bed nucleus of the stria terminalis, and visceral cortices. However, no cortex labeling and only modest amygdala and bed nucleus labeling were observed in rats injected with PRV on P4, and only medial hypothalamic labeling was observed in rats injected on P1. Additional tracing experiments involving central injections of PRV or cholera toxin beta indicated that lateral hypothalamic and telencephalic regions projected to the medullary dorsal vagal complex several days before establishing synaptic connections with gastric-related autonomic neurons. These results demonstrate a novel strategy for evaluating synaptic connectivity in developing neural circuits and show a temporally segregated postnatal emergence of medial hypothalamic, lateral hypothalamic, and telencephalic synaptic inputs to central autonomic neurons.

Connections of Barrington's nucleus to the sympathetic nervous system in rats.

Barrington's nucleus (BN) has been considered a pontine center related exclusively to the control of pelvic parasympathetic activity. The present study demonstrates an anatomical linkage between BN and autonomic outflow to visceral targets innervated exclusively by the sympathetic division of the autonomic nervous system. Temporal analysis of infection after injection of pseudorabies virus (PRV), a retrograde transynaptic tracer, into two sympathetically innervated organs, the spleen and the kidney, revealed the presence of infected neurons in BN at early post-inoculation survival intervals. Immunohistochemical localization of PRV after spleen injections showed that a small subpopulation of BN neurons became labeled in a time frame coincident with the appearance of infected neurons in other brain regions known to project to sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord; a larger number of infected neurons appeared in BN at intermediate intervals after PRV injections into the spleen or kidney. Coinjection of the retrograde tracer Fluoro-Gold i.p. and PRV into the spleen demonstrated that parasympathetic preganglionic neurons in the caudal medulla or lumbo-sacral spinal cord were not infected, indicating that infected BN neurons were not infected via a parasympathetic route. Thus, BN neurons become infected after PRV injections into the spleen or kidney either directly through BN projections to SPNs, or secondarily via BN projections to infected pre-preganglionic neurons. These results demonstrate an anatomical linkage, either direct or indirect, between BN and sympathetic activity. Because BN receives numerous inputs from diverse brain regions, the relation of BN with both branches of the autonomic nervous system suggests that this nucleus might play a role in the integration of supraspinal inputs relevant to the central coordination of sympathetic and parasympathetic activity.

Antagonism of central glucagon-like peptide-1 receptors enhances lipopolysaccharide-induced fever.

Lipopolysaccharide (LPS; a model of systemic bacterial infection) causes fever and activates glucagon-like peptide-1 (GLP-1) neurons in the caudal brainstem. The present study examined whether central GLP-1 receptor signaling plays a functional role in LPS-induced fever. Adult male Sprague-Dawley rats were injected i.p. with LPS (0 or 100 microg/kg), then were infused intracerebroventricularly with GLP-1 receptor antagonist (0 or 10 microg) delivered 2.5 h after injection of LPS or vehicle. Core body temperature was measured at 30-min intervals for 6.5 h after LPS treatment. Consistent with previous reports, body temperature was significantly elevated within 90 min and remained elevated for the remainder of the monitoring period. The pyrogenic effect of LPS was amplified in rats that received central infusion of GLP-1 receptor antagonist, although the antagonist by itself did not alter body temperature. These findings suggest that endogenous GLP-1 acts at central receptors to limit the fever response in rats after i.p. administration of LPS.

A functional role for central glucagon-like peptide-1 receptors in lithium chloride-induced anorexia.

The present study sought to determine whether central glucagon-like peptide-1 (GLP-1)-receptor signalling contributes to the anorexigenic effects of systemically administered lithium chloride (LiCl). Male Sprague-Dawley rats with chronic intracerebroventricular (ICV) cannulas were acclimated to a feeding schedule that included daily 30-min access to palatable mash. In the first experiment, ICV infusion of a GLP-1-receptor antagonist [exendin-4-(3-39)] significantly attenuated (10 microgram dose) or completely blocked (20 microgram dose) the inhibition of food intake produced by subsequent ICV infusion of GLP-1-(7-36) amide (5 microgram). In the second experiment, rats were infused with 0, 10, or 20 microgram of the GLP-1-receptor antagonist ICV, followed by injection of 0.15 M LiCl (50 mg/kg ip) or the same volume of 0.15 M NaCl. The ability of LiCl treatment to suppress food intake was significantly attenuated in rats that were pretreated with the GLP-1-receptor antagonist. These results support the view that central mechanisms underlying LiCl-induced anorexia include a prominent role for endogenous GLP-1 neural pathways.

Distribution of glucose transporter isoform-3 and hexokinase I in the postnatal murine brain.

The facilitative glucose transporter-3 (GLUT 3) and hexokinase I were examined in postnatal mouse brains using immunohistochemical methods. GLUT 3 demonstrated a polarized distribution limited to neuronal processes of most anatomical regions except the suprachiasmatic nucleus and the cerebellum, where GLUT 3 expression was limited to neuronal cell somata. In contrast, hexokinase I was observed in the cytoplasm of neuronal and non-neuronal (subependymal and choroid plexus epithelial) cell bodies in all regions. In general, while the spatial distribution of GLUT 3 and hexokinase I did not change with age, a temporal increase in intensity was noted in all regions except for the decline in suprachiasmatic nuclear GLUT 3 immunoreactivity.

Lesions of the C1 catecholaminergic neurons of the ventrolateral medulla in rats using anti-DbetaH-saporin.

Phenylethanolamine-N-methyltransferase (PNMT)-containing neurons in the rostral ventrolateral medulla (RVLM) are believed to play a role in cardiovascular regulation. To determine whether injection of anti-dopamine beta-hydroxylase (DbetaH)-saporin directly into the RVLM in rats could selectively destroy these cells and thereby provide an approach for evaluating their role in cardiovascular regulation, we studied rats 2 wk after unilateral injection of 21 ng anti-DbetaH-saporin into the RVLM. There was an approximately 90% reduction in the number of PNMT-positive neurons in the RVLM, although the number of non-C1, spinally projecting barosensitive neurons of this area was not altered. The A5 cell group was the only other population of DbetaH-containing cells that was significantly depleted. The depressor response evoked by injection of tyramine into the RVLM was abolished by prior injection of toxin. The pressor response evoked by injection of glutamate into the RVLM was attenuated ipsilateral to the toxin injection but was potentiated contralateral to the toxin injection. Thus anti-DbetaH-saporin can be used to make selective lesions of PNMT-containing cells, allowing for the evaluation of their role in cardiovascular regulation.

Interoceptive stress activates glucagon-like peptide-1 neurons that project to the hypothalamus.

This study tested the hypothesis that systemic stressors in rats activate glucagon-like peptide-1 (GLP-1)-immunoreactive neurons in the caudal brain stem, including those that project to the paraventricular nucleus of the hypothalamus (PVN). Neural tracer was microinjected into the PVN to retrogradely label brain stem neurons. Seven to ten days later, rats were injected with lithium chloride (LiCl; 50 mg/kg). Additional non-tracer-injected rats were treated with lipopolysaccharide (LPS; 100 microgram/kg) or CCK (100 microgram/kg) or were allowed to consume a very large meal. Rats were killed 90-120 min after drug treatment or 30 min after the meal. Brains were processed for immunocytochemical localization of c-Fos (a marker of neuronal activation), GLP-1, and, when appropriate, neural tracer. The majority of GLP-1 neurons were activated to express c-Fos after LiCl, LPS, or CCK treatment, including (in LiCl-treated rats) those projecting to the PVN. In contrast, GLP-1 neurons rarely expressed c-Fos after ingestion of a large meal, despite prominent activation of other brain stem neurons. These results suggest that GLP-1 neurons are uniquely activated in situations of interoceptive stress, and may participate in adaptive hypothalamic stress responses.