Ron P.A. Gaykema

Vagal immune-to-brain communication: a visceral chemosensory pathway

The immune system operates as a diffuse sensory system, detecting the presence of specific chemical constituents associated with dangerous micro-organisms, and then signalling the brain. In this way, immunosensation constitutes a chemosensory system. Several submodalities of this sensory system function as pathways conveying immune-related information, and can be classified as either primarily brain barrier associated or neural. The vagus nerve provides the major neural pathway identified to date. The initial chemosensory transduction events occur in immune cells, which respond to specific chemical components expressed by dangerous micro-organisms. These immune chemosensory cells release mediators, such as cytokines, to activate neural elements, including primary afferent neurons of the vagal sensory ganglia. Primary afferent activation initiates local reflexes (e.g. cardiovascular and gastrointestinal) that support host defense. In addition, at least three parallel pathways of ascending immune-related information activate specific components of the illness response. In this way, immunosensory systems represent highly organized and coherent pathways for activating host defense against infection.

Reversible inactivation of the dorsal vagal complex blocks lipopolysaccharide-induced social withdrawal and c-Fos expression in central autonomic nuclei.

Peripheral administration of lipopolysaccharide (LPS), a potent activator of the immune system, induces symptoms of behavioral depression, such as social withdrawal, concommitant with increases in c-Fos expression in central autonomic network nuclei. Previous studies implicated vagal visceral sensory nerves in transduction of immune-related signals relevant to for the induction of social withdrawal, a symptom of behavioral depression. Vagal sensory nerves terminate in the dorsal vagal complex (DVC) of the brainstem, a region that functions to integrate visceral signals and may also play a role in modulating arousal and affect. The objective of the current study was to determine whether the DVC contributes to immunosensory pathways driving symptoms of social withdrawal associated with LPS-induced behavioral depression, using a reversible lesion technique to temporarily inactivate the DVC. To assess the effects of DVC inactivation on LPS-induced social withdrawal and the subsequent changes in brain activation, we used behavioral assessment of social withdrawal, and analyzed c-Fos expression, a marker of neuronal activation, in the central nucleus of the amygdala (CEA), bed nucleus of the stria terminalis (BST), hypothalamic paraventricular nucleus (PVN), and ventromendial preoptic area (VMPO). Two hours following intraperitoneal LPS injection, there was a significant increase in c-Fos immunoreactivity in forebrain regions in animals treated with LPS. DVC inactivation completely blocked LPS-induced social withdrawal and dramatically reduced LPS-induced Fos expression in all four forebrain regions assessed. Collectively, these findings support the idea that the DVC acts as an immune-behavior interface between the peripheral stimuli and brain areas involved in modulating social behavior.

Bacterial Endotoxin Induces Fos Immunoreactivity in Primary Afferent Neurons of the Vagus Nerve

Subdiaphragmatic vagotomy inhibits brain-mediated illness responses to peripherally administered bacterial endotoxin, including fever, hyperalgesia, sickness behavior, and activation of the hypothalamic-pituitary-adrenal axis. However, direct evidence implicating vagal afferents specifically in conveying information about peripheral immune activation to the brain is still lacking. This study assessed whether (1) endotoxin induces the expression of the functional activation marker Fos in the vagal sensory ganglia, and (2) vagotomy abrogates endotoxin-induced Fos expression in these ganglia. Male rats, which had previously received vagotomy or sham surgery, were injected intraperitoneally or intravenously with either endotoxin or saline. Fos immunolabeling was absent in saline-treated rats. In contrast, scattered cells within the vagal sensory ganglia showed Fos immunoreactivity after both intraperitoneal and intravenous endotoxin administration in sham-operated rats. Vagotomy abolished Fos expression after intraperitoneal endotoxin administration, whereas after intravenous administration Fos expression was strongly attenuated, but not eliminated. These findings implicate vagal afferents as a potential signaling pathway to brain regions that generate illness responses to pro-inflammatory mediators.

Effects of vagotomy on lipopolysaccharide-induced brain interleukin-1β protein in rats

The production of interleukin-1beta (IL-1beta) in brain is thought to be a critical step in the induction of central manifestations of the acute phase response, and the vagus nerve has been implicated in immune-to-brain communication. Thus, this study examined the effects of intraperitoneal (i.p.) injections of lipopolysaccharide (LPS) on brain IL-1beta protein levels in control and subdiaphragmatically vagotomized rats. In the first experiment, vagotomized and sham-operated male Sprague-Dawley rats were injected i.p. with one of three doses (10, 50, 100 microg/kg) of LPS or vehicle (sterile, pyrogen-free saline) and sacrificed 2 h after the injection. In the second experiment, vagotomized and sham-operated rats were injected i.p. with 100 microg/kg LPS or vehicle and sacrificed 1 h after the injection. The i.p. injection of LPS dose-dependently increased IL-1beta protein levels in the hypothalamus, hippocampus, dorsal vagal complex, cerebellum, posterior cortex, and pituitary 2 h after the injection. Brain and pituitary IL-1beta levels were also significantly increased 1 h after the injection of 100 microg/kg LPS. There were no significant differences in brain IL-1beta levels between sham-operated and vagotomized rats at either the 2 h or 1 h time points. The current data are consistent with previous studies showing increases in brain IL-1beta after peripheral injections of LPS, and support the notion that brain IL-1beta is a mediator in the illness-induction pathway. Furthermore, these data indicate that, at the doses and times tested, subdiaphragmatic vagal afferents are not crucial for LPS-induced brain IL-1beta protein.

Subdiaphragmatic vagotomy does not block intraperitoneal lipopolysaccharide-induced fever.

Several recent findings, including the inability of subdiaphragmatic vagotomy to block lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta) protein in brain, have made it necessary to reexamine the role of the subdiaphragmatic vagal afferents in immune-to-brain communication. In this study, we examined the effects of intraperitoneal (i.p.) injections of LPS on core body temperature in control and subdiaphragmatically vagotomized rats. Vagotomized and sham-operated male Sprague-Dawley rats were injected i.p. with vehicle (pyrogen-free saline) on the control day and LPS (1, 10 or 50 microg/kg) on the experimental day, and core body temperature was monitored by telemetry for 6 h after the injection. At this time, rats were sacrificed, and serum, liver, and pituitary samples were collected. The i.p. injection of LPS increased core body temperature in both sham-operated and vagotomized rats compared to the saline injection. In addition, LPS significantly increased IL-1beta levels in serum, liver, and pituitary compared to saline-injected controls. There were no significant differences in the magnitude of the fever or in the levels of IL-1beta in serum, liver, or pituitary between sham-operated and vagotomized rats. Thus, the current data indicate that, at the doses tested, subdiaphragmatic vagal afferents are not crucial for i.p. LPS-induced fever. Because several effects of vagotomy have been shown to be dependent on dose, we are currently investigating whether vagal afferents are involved in lower-dose i.p. LPS-induced fever.

Subdiaphragmatic vagotomy does not prevent fever following intracerebroventricular prostaglandin E2: further evidence for the importance of vagal afferents in immune-to-brain communication.

Brain-mediated sickness responses can be blocked by subdiaphragmatic vagotomy, suggesting that vagal afferents signal peripheral inflammation or infection. This study tested whether subdiaphragmatic vagotomy disrupts sickness responses by interrupting effector pathways. If this explanation is correct, intracerebroventricular prostaglandin E2-induced fever should be blocked by this procedure. Fever was unaffected by subdiaphragmatic vagotomy, thus these data provide support for the conclusion that vagal afferents signal the brain during immune activation.

Vagotomy does not inhibit high dose lipopolysaccharide-induced interleukin-1β immunoreactivity in rat brain and pituitary gland

In the present study, we examined whether the vagus nerve is involved in mediating lipopolysaccharide (LPS)-induced appearance of IL-1beta immunoreactive cells in the brain and pituitary gland. Rats were either sham-operated or subjected to subdiaphragmatic vagotomy. Four weeks later, pyrogen free saline or 400 microg/kg LPS was administered to the rats intraperitoneally. Four and 8 h later, the animals were intracardially perfused with 4% paraformaldehyde and tissues were prepared for IL-1beta immunocytochemistry. IL-1beta positive cells were observed at both time-intervals after LPS administration in the choroid plexus, meninges, circumventricular organs and pituitary gland of both sham-operated and vagotomized rats. We conclude that under the conditions studied, the vagus nerve does not mediate LPS-induced appearance of IL-1beta in the rat brain and pituitary gland.

How does immune challenge inhibit ingestion of palatable food? Evidence that systemic lipopolysaccharide treatment modulates key nodal points of feeding neurocircuitry

Immune challenge induces behavioral changes including reduced ingestion of palatable food. Multiple pathways likely contribute to this effect, including viscerosensory pathways controlling hypothalamic feeding circuits or by influence on reward circuitry previously established to control ingestive behavior. To investigate whether the effects of immune challenge may influence this network, we compared brain activation patterns in animals trained to drink a palatable sweetened milk solution and treated systemically with either the immune stimulant lipopolysaccharide (LPS) or saline. Brain sections were processed for localization of the activation marker c-Fos in neurons of regions implicated in regulation of feeding behavior. Sweetened milk ingestion was associated with increased numbers of c-Fos positive neurons in the caudal core and shell of the nucleus accumbens (NAc), the paraventricular thalamus (PVT), central nucleus of the amygdala (CEA), the basal lateral amygdala (BLA), in orexin-A containing neurons of the lateral hypothalamus (LH), and in cocaine and amphetamine regulated transcript (CART) neurons of the arcuate hypothalamus. In LPS-treated animals sweetened milk consumption was significantly reduced, as was c-Fos induction in the hypothalamic orexin-A and CART neurons, and in the BLA. In addition, induction of c-Fos in the rostral regions of the NAc, the PVT, and CEA was increased following LPS treatment, compared to controls. The findings from this study point to a network of brain regions (LH, PVT, NAc, and BLA) previously implicated in the modulation of feeding behavior, reward, and arousal that may also contribute to neural substrates involved in the reorganization of behavioral priorities that occurs during sickness.

Subdiaphragmatic vagotomy blocks interleukin-1β-induced fever but does not reduce IL-1β levels in the circulation

Peripheral interleukin-1β has been implicated in the initiation of fever responses, yet the pathways by which it influences brain function are still unclear. Sectioning the abdominal vagus has been reported to inhibit fever after intraperitoneal administration of interleukin-1β, suggesting that vagal afferents participate in signaling the brain to mount a fever response to interleukin-1β. However, the inhibitory effect of subdiaphragmatic vagotomy could be due to alterations in pharmacokinetics such that the intraperitoneally injected cytokine does not reach the general circulation in sufficient quantities to activate the brain via blood-borne signaling. We measured both fever and plasma levels of interleukin-1β in vagotomized and sham-operated rats after intraperitoneal administration of 1 μg/kg human recombinant interleukin-1β to determine whether vagotomy reduces fever and levels of circulating interleukin-1β after intraperitoneal injection. Plasma levels of human recombinant and endogenous rat interleukin-1β were measured in separate enzyme-linked immunosorbent assays. While intraperitoneal administration of human recombinant interleukin-1β elevated plasma levels of this cytokine similarly in vagotomized and sham-operated animals, only sham-operated rats responded with fever. Plasma levels of endogenous rat interleukin-1β were unchanged by any treatment. These results demonstrate that the blockade of intraperitoneal interleukin-1β-induced fever after subdiaphragmatic vagotomy cannot be accounted for by alterations of interleukin-1β levels in the general circulation.

Neural Pathways Mediating Behavioral Changes Associated with Immunological Challenge

Peripheral infection or inflammation influences behavior by interacting with neural systems in the periphery and central nervous system that mediate pain, arousal and behavior. Multiple parallel pathways convey information relevant to sickness behavior, local inflammation and pain. For instance, vagal sensory neurons seem to contribute to systemic, brain-mediated responses, whereas spinal viscerosensory nerves modulate local inflammation and pain. Neural pathways of immuneto- brain communication drive projection neurons in the brainstem that potently influence forebrain regions, including the paraventricular thalamus, much of the hypothalamus, the basal forebrain, amygdala and bed nucleus of the stria terminalis. Cortical components of the immune-responsive network include the anterior cingulate, medial prefrontal and insular cortices. This immune “sensory system” provides the means by which the brain can monitor peripheral immune challenges and carry out relevant behavioral responses.