Linda Rinaman

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 μg/kg) or CCK (100 μg/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.

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‐β‐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 T3–T12 bilaterally; numerous infected interneurons were also found in the thoracic spinal cord (T1–T13). Infected neurons in the brain were first observed in the A5 region, ventromedial medulla, rostral ventrolateral medulla, paraventricular hypothalamic nucleus, Barringtons 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. J. Comp. Neurol. 439:1–18, 2001.

Ascending projections from the caudal visceral nucleus of the solitary tract to brain regions involved in food intake and energy expenditure.

Metabolic homeostasis reflects the complex output of endocrine, autonomic, and behavioral control circuits that extend throughout the central nervous system. Brain regions that control food intake and energy expenditure are privy to continuous visceral sensory feedback signals that presumably modulate appetite, satiety, digestion, and metabolism. Sensory signals from the gastrointestinal tract and associated digestive viscera are delivered to the brain primarily by vagal afferents that terminate centrally within the caudal nucleus of the solitary tract (NST), with signals subsequently relayed to higher brain regions by parallel noradrenergic and peptidergic projection pathways arising within the NST. This article begins with an overview of these ascending pathways identified in adult rats using a standard anterograde tracer microinjected into the caudal visceral sensory region of the NST, and also by immunocytochemical localization of glucagon-like peptide-1. NST projection targets identified by these two approaches are compared to the distribution of neurons that become infected after inoculating the ventral stomach wall with a neurotropic virus that transneuronally infects synaptically-linked chains of neurons in the anterograde (i.e., ascending sensory) direction. Although the focus of this article is the anatomical organization of axonal projections from the caudal visceral NST to the hypothalamus and limbic forebrain, discussion is included regarding the hypothesized role of these projections in modulating behavioral arousal and coordinating endocrine and behavioral (i.e., hypophagic) responses to stress.

Medullary c-Fos activation in rats after ingestion of a satiating meal

The distribution and chemical phenotypes of hindbrain neurons that are activated in rats after food ingestion were examined. Rats were anesthetized and perfused with fixative 30 min after the end of 1-h meals of an unrestricted or rationed amount of food, or after no meal. Brain sections were processed for localization of the immediate-early gene product c-Fos, a marker of stimulus-induced neural activation. Hindbrain c-Fos expression was low in rats that ate a rationed meal or no meal. Conversely, c-Fos was prominent in the medial nucleus of the solitary tract (NST) and area postrema in rats that ate to satiety. There was a significant positive correlation between postmortem weight of gastric contents and the proportion of NST catecholaminergic neurons expressing c-Fos. Cells in the ventrolateral medulla (VLM) were not activated in rats after food ingestion, in contrast with previous findings that stimulation of gastric vagal afferents with anorexigenic doses of cholecystokinin activates c-Fos expression in both NST and VLM catecholaminergic cells. These findings indicate that anatomically distinct subsets of hindbrain catecholaminergic neurons are activated in rats after food ingestion and that activation of these cells is quantitatively related to the magnitude of feeding-induced gastric distension.

Oxytocinergic inputs to the nucleus of the solitary tract and dorsal motor nucleus of the vagus in neonatal rats

The paraventricular nucleus of the hypothalamus (PVN) modulates vagal digestive motor functions via oxytocinergic projections to the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMV) in adult rats. Little is known regarding the structural or functional maturation of these projections. The present study examines the postnatal development of immunocytochemically identified oxytocinergic fibers in gastric subregions of the medial NST‐DMV. For this purpose, a monoclonal antibody (PS36) that recognizes both oxytocin (OT)‐neurophysin and its prohormone was used to identify oxytocinergic fibers. PS36‐positive fibers already were present within the NST‐DMV in rats on the day of birth. Retrograde transport of cholera toxin neural tracer from the NST‐DMV in newborn rats confirmed that PVN neurons were the sole source of these oxytocinergic fibers. The cumulative length of PS36‐positive fibers in sampled subregions of the medial NST and DMV increased approximately 23‐fold and 94‐fold, respectively, between birth and adulthood. The observed postnatal increases in PS36 immunolabeling could reflect increased delivery of immunoreactive antigen from hypothalamic perikarya to distal axons and/or increasing oxytocinergic innervation of the NST‐DMV. Additional work will be needed to address these questions and to determine the time course during which central oxytocinergic pathways become mature in their ability to influence vagally mediated digestive functions. J. Comp. Neurol. 399:101–109, 1998.

Hindbrain noradrenergic A2 neurons: diverse roles in autonomic, endocrine, cognitive, and behavioral functions.

Central noradrenergic (NA) signaling is broadly implicated in behavioral and physiological processes related to attention, arousal, motivation, learning and memory, and homeostasis. This review focuses on the A2 cell group of NA neurons, located within the hindbrain dorsal vagal complex (DVC). The intra-DVC location of A2 neurons supports their role in vagal sensory-motor reflex arcs and visceral motor outflow. A2 neurons also are reciprocally connected with multiple brain stem, hypothalamic, and limbic forebrain regions. The extra-DVC connections of A2 neurons provide a route through which emotional and cognitive events can modulate visceral motor outflow and also a route through which interoceptive feedback from the body can impact hypothalamic functions as well as emotional and cognitive processing. This review considers some of the hallmark anatomical and chemical features of A2 neurons, followed by presentation of evidence supporting a role for A2 neurons in modulating food intake, affective behavior, behavioral and physiological stress responses, emotional learning, and drug dependence. Increased knowledge about the organization and function of the A2 cell group and the neural circuits in which A2 neurons participate should contribute to a better understanding of how the brain orchestrates adaptive responses to the various threats and opportunities of life and should further reveal the central underpinnings of stress-related physiological and emotional dysregulation.

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 μg dose) or completely blocked (20 μg dose) the inhibition of food intake produced by subsequent ICV infusion of GLP-1-(7-36) amide (5 μg). In the second experiment, rats were infused with 0, 10, or 20 μg 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.

Central c-Fos expression in neonatal and adult rats after subcutaneous injection of hypertonic saline

Centrally-mediated responses to plasma hyperosmolality include compensatory drinking and pituitary secretion of vasopressin and oxytocin in both adult and neonatal rats. However, the anorexia that is produced by plasma hyperosmolality in adult rats is not evident in neonates, perhaps due to functional immaturity of osmoresponsive hindbrain circuits. To examine this possibility, the present study compared treatment-induced brain expression of the immediate-early gene product c-Fos as a marker of neural activation in adult and two-day-old rats after subcutaneous injection of 2 M NaCl (0.1 ml/10 g body weight). This treatment produced marked hypernatremia in adult and two-day-old rats without altering plasma volume. Several brain regions (including components of the lamina terminalis, the paraventricular and supraoptic nuclei of the hypothalamus, and the area postrema) were activated to express c-Fos similarly in adult and two-day-old rats after 2 M NaCl injection, consistent with previous reports implicating a subset of these regions in osmotically-stimulated drinking and neurohypophyseal secretion. In contrast, other areas of the brain that were activated to express c-Fos in adult rats after 2 M NaCl injection were not activated in neonates: these areas included the central nucleus of the amygdala, the parabrachial nucleus and catecholamine cell groups within the caudal medulla. This study demonstrates that certain brain regions that are osmoresponsive in adult rats (as defined by induced c-Fos expression) are not osmoresponsive in two-day-old rats. When considered in the context of known differences between the osmoregulatory capacities of adult and neonatal rats, our results are consistent with the idea that osmoresponsive forebrain centres are primarily involved in osmotically-stimulated compensatory drinking and neurohypophyseal secretion, whereas osmoresponsive regions of the hindbrain are important for concomitant inhibition of feeding and gastric emptying.

The organization of vagal innervation of rat pancreas using cholera toxin—horseradish peroxidase conjugate

The present study was initiated to address the current controversy concerning the parasympathetic innervation of the pancreas, using a more sensitive tracer. The location of retrogradely labeled neurons within the dorsal motor nucleus of the vagus (DMV) was examined 48 h following injections of cholera toxin-horseradish peroxidase (CT-HRP) into designated areas of the rat pancreas. The brainstem and spinal cord were searched for any additional labeled neurons located outside of the DMV. Separate groups of animals were used for control injections into the adipose tissue of the greater omentum, the spleen, abdominal musculature, and the diaphragm. In addition, CT-HRP was dripped over the surfaces of the abdominal viscera in another group of animals. These control cases were designed to indicate whether diffusion of the neural tracer away from injection sites had occurred and had resulted in labeling of neurons which did not innervate the injected areas. Following injection of CT-HRP into the right lobe of the pancreas, labeled neurons were found primarily within the medial region of the left DMV. Injection of CT-HRP into the left lobe of the pancreas resulted in retrogradely labeled neurons predominantly within the medial region of the right DMV. Following injections into the entire pancreas, neural labeling was seen bilaterally within the DMV and was concentrated within the medial regions, with a slightly higher degree of labeling within the right DMV. No labeled neurons were seen within the nucleus ambiguus or other areas of the brainstem or spinal cord following pancreatic injections. Furthermore, no afferent labeling within the nucleus of the solitary tract (NTS) was observed, although a very small number of neurons within the nodose ganglia were labeled. The dendrites of backfilled DMV neurons could be seen extending across the midline to the contralateral DMV as well as dorsally into certain subnuclei of the NTS, and to the borders of the area postrema and the fourth ventricle. These results indicate that both the motor and sensory innervation of the rat pancreas are more restricted than has been previously suggested.

Early Experience Modifies the Postnatal Assembly of Autonomic Emotional Motor Circuits in Rats

Rat pups that are repeatedly handled and separated from their dam exhibit altered adult behavioral, endocrine, and autonomic responses to stress, but the extent to which early handling and/or maternal separation (H/S) alters the development of circuits that underlie these responses is unknown. The present study tested the hypothesis that early H/S alters the postnatal assembly of synapses within preautonomic emotional motor circuits. Circuit development was traced by synapse-dependent retrograde transneuronal transport of pseudorabies virus (PRV) from the stomach wall. Control and H/S rats were analyzed between postnatal day 6 (P6) and P10, a period of rapid synaptic assembly among preautonomic circuit components. Pups in H/S groups were removed from their dam daily for either 15 min or 3 h beginning on P1, and were injected with virus on P8 and perfused on P10. Quantitative analyses of primary and transsynaptic PRV immunolabeling confirmed an age-dependent assembly of hypothalamic, limbic, and cortical inputs to autonomic nuclei. Circuit assembly was significantly altered in H/S pups, in which fewer neurons in the central amygdala, the bed nucleus of the stria terminalis, and visceral cortices were infected compared with age-matched controls. In contrast, H/S did not alter the assembly of paraventricular hypothalamic inputs to gastric autonomic neurons. H/S-related reductions in limbic and cortical transneuronal infection were similar in pups exposed daily to 15 min or 3 h maternal separation. These findings support the view that environmental events during early postnatal life can influence the formation of neural circuits that provide limbic and cortical control over autonomic emotional motor output.