Ascending caudal medullary catecholamine pathways drive sickness-induced deficits in exploratory behavior: brain substrates for fatigue?

Immune challenges can lead to marked behavioral changes, including fatigue, reduced social interest, anorexia, and somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. Part of the neurocircuitry influencing behavior associated with illness likely includes viscerosensory nuclei located in the caudal brainstem, based on findings that inactivation of the dorsal vagal complex (DVC) can prevent social withdrawal. These brainstem nuclei contribute multiple neuronal projections that target different components of autonomic and stress-related neurocircuitry. In particular, catecholaminergic neurons in the ventrolateral medulla (VLM) and DVC target the hypothalamus and drive neuroendocrine responses to immune challenge, but their particular role in sickness behavior is not known. To test whether this catecholamine pathway also mediates sickness behavior, we compared effects of DVC inactivation with targeted lesion of the catecholamine pathway on exploratory behavior, which provides an index of motivation and fatigue, and associated patterns of brain activation assessed by immunohistochemical detection of c-Fos protein. LPS treatment dramatically reduced exploratory behavior, and produced a pattern of increased c-Fos expression in brain regions associated with stress and autonomic adjustments paraventricular hypothalamus (PVN), bed nucleus of the stria terminalis (BST), central amygdala (CEA), whereas activation was reduced in regions involved in exploratory behavior (hippocampus, dorsal striatum, ventral tuberomammillary nucleus, and ventral tegmental area). Both DVC inactivation and catecholamine lesion prevented reductions in exploratory behavior and completely blocked the inhibitory LPS effects on c-Fos expression in the behavior-associated regions. In contrast, LPS-induced activation in the CEA and BST was inhibited by DVC inactivation but not by catecholamine lesion. The findings support the idea that parallel pathways from immune-sensory caudal brainstem sources target distinct populations of forebrain neurons that likely mediate different aspects of sickness. The caudal medullary catecholaminergic projections to the hypothalamus may significantly contribute to brain mechanisms that induce behavioral “fatigue” in the context of physiological stressors.     

Immediate‐early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c‐fos mRNA to adaptation

Stress exposure resulted in brain induction of immediate‐early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c‐fos and arc (activity‐regulated cytoskeleton‐associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c‐fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos‐like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO‐induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double‐labelling in the LS of IMO‐exposed rats revealed that arc was expressed in only one‐third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c‐fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.     

Combined mesencephalic and hypothalamic transplants reverse lesion-induced sexual behavior deficits in the male rat

Studies of sexual behavior in rodent animal models have provided evidence about the relevant role played by the medial preoptic area of the anterior hypothalamus and the central tegmental field within the mesencephalon in the control of this behavior. Bilateral lesions of the anterior hypothalamus or central tegmental field as well as combined unilateral lesions of both these regions result in sexual behavior deficits. Studies using fetal hypothalamic transplants have been shown to reverse sexual behavior deficits induced either by lesions or aging. However, no previous study has evaluated the effect of combined homotopic transplants into both the anterior hypothalamus and the mesencephalon. In the present study male Wistar animals received two electrolytic lesions, one aimed at the ipsilateral medial preoptic area of the anterior hypothalamus and the other at the contralateral central tegmental field. Following these lesions, unilateral homotopic fetal hypothalamic and mesencephalic transplants were placed into the lesioned areas. Sexual behavior recovered gradually and by weeks 14-15 after transplantation, above 90% of animals with bilateral transplants showed mounts, intromissions, and ejaculations. Only animals with viable transplants located within both lesioned areas showed recovery. These results indicate that the behavioral deficits induced by combined unilateral lesions of hypothalamic and mesencephalic regions can be reversed by homotopic fetal transplants and that this recovery could be the result of the restoration of a behavioral relevant circuit between transplants and host brain nuclei separated by as much as 5 mm, which makes this an excellent model to study mechanisms underlying behavioral recovery after transplantation.     

A negative correlation between the induction of long-term potentiation and activation of immediate early genes.

In the present study we examined the relationship between the induction of long-term potentiation (LTP) in the dentate gyrus of anesthetized rats and activation of immediate early genes (IEGs; c-fos and zif/268) using several different high-frequency stimulation paradigms. Stimulation parameters that effectively induced LTP were not associated with IEG activation. Conversely, stimulation parameters that failed to induce LTP consistently resulted in IEG activation. These results suggest that there is a negative correlation between IEG activation and LTP, and that activation of IEGs is neither necessary nor sufficient for the induction of LTP.     

The organization of the hypothalamic pathways mediating affective defense behavior in the cat

The purpose of this study was to describe the hypothalamic pathways which mediate affective defense in the cat utilizing the methods of [14C]2-deoxyglucose (2-DG) and [3H]leucine radioautography in concert with the technique of electrical brain stimulation. The feline affective defense response, characterized by pupillary dilatation, piloerection, ear retraction, hissing, growling and striking with the forepaws, was elicited consistently by stimulation of sites within the ventromedial hypothalamus and anterior aspect of the medial hypothalamus. In one series of experiments, 2-DG autoradiography was employed to describe the brain regions activated following stimulation of sites in the region of the ventromedial hypothalamus from which affective defense had been elicited. Ventromedial hypothalamic stimulation produced activation primarily in forebrain regions situated rostral to the level of the stimulating electrode. These structures included principally the anteromedial hypothalamus and medial preoptic area, as well as the bed nuclei of the stria terminalis and anterior commissure, diagonal band and lateral septal area. The caudal extent of activation included only the dorsal and perifornical hypothalamus at the level of the stimulation site. In a second series of experiments, affective defense sites in the anteromedial hypothalamus were stimulated and the regional distribution of 2-DG label was identified. In contrast to the results obtained from ventromedial hypothalamic stimulation, these experiments revealed a marked descending distribution of label within the posterior hypothalamus, midbrain central gray and ventral tegmental area. Results obtained from studies in which tritiated amino acids were injected into affective defense sites in both the ventromedial nucleus and anteromedial hypothalamus confirmed the general findings observed with 2-DG autoradiography. From these observations, we have concluded that the organization of the pathway mediating affective defense behavior from the ventromedial hypothalamus to the midbrain involves an initial synapse within the region of the anteromedial hypothalamus and a second synapse in the midbrain central gray substance. The significance of the anteromedial hypothalamus for the expression of affective defense behavior was considered in the Discussion.     

The retrograde tracer fluoro-gold interferes with the expression of fos-related antigens

The retrograde tracer, fluoro-gold (FG) has been used in combination with immediate early gene (IEG) immunohistochemistry to identify neural circuits activated by pharmacological, physiological or behavioral manipulations. However, since FG has been shown to be toxic to cell bodies, axons and terminals at the injection site, the question arises as to whether FG alters the detection of IEG products. To examine this question, FG was microiontophoresed unilaterally into the nucleus accumbens (NAc) of rats and Fos-related antigens (FRAs) were examined in both hemispheres 12 days later. Approximately half as many FRA-positive nuclei were observed in the tracer-injected NAc as were found in the contralateral NAc. Similar results were observed in the ventral subiculum of the hippocampus and the basolateral and central amygdaloid nuclei, but not in the lateral septum or lateral habenula. These results suggest that FG microiontophoresed into the NAc interferes with the expression of FRAs at the injection site and also at other ipsilateral limbic sites.     

Lesions to the medial preoptic nucleus affect immediate early gene immunolabeling in brain regions involved in song control and social behavior in male European starlings

The medial preoptic nucleus (POM) is a brain region outside of the song control system of songbirds. It has been implicated in song production, sexual motivation, and the integration of both sensory and hormonal information with appropriate behavioral responses. The POM is well positioned neuroanatomically to interact with multiple regions involved in song, social behavior, and motivation. However, little is known about the brain regions with which the POM directly or indirectly communicates to influence song. To gain insight into the neuronal circuits normally activated in association with POM activity during male song, we compared activity within multiple brain regions using immunolabeling for protein products of immediate early genes (IEGs) zenk (aka egr-1 ) and c-fos (indirect markers of neuronal activity) in sham and POM-lesioned male European starlings ( Sturnus vulgaris ). As compared to sham lesions, POM lesions disrupted song and interest in a nest box, and females responded less to POM-lesioned males. POM lesions reduced numbers of IEG-labeled cells and disrupted correlations between numbers of IEG-labeled cells and song within several song control, limbic, hypothalamic and midbrain regions. These results are consistent with the possibility that the POM integrates activity among nuclei involved in song control, social behavior and motivational state that work in concert to promote sexually motivated communication.     

Prolonged expression of zinc finger immediate-early gene mRNAs and decreased protein synthesis following kainic acid induced seizures

In the present study in situ hybridization was used to study the effect of kainic acid induced seizures on the expression of the zinc finger immediate-early genes (IEGs) NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3 and Nurr1. Kainic acid markedly induced these IEGs especially in hippocampus, cortex and amygdala by 30 min. This induction gradually decreased and returned to baseline by 24 h in most regions. However, in the CA1 and CA3 subfields of hippocampus known to be damaged by kainic acid the expression of all the IEGs except egr-2 remained elevated for 24 h. NGFI-A, NGFI-B, NGFI-C and to a lesser extent, Nurr1, remained elevated also in the subcortical region of the temporal lobe. By 24 h incorporation of ¹⁴C-leucine decreased in the piriform cortex, amygdala, and in the CA1 and CA3 subfields, but not in CA2 and dentate gyrus. These areas showing decreased protein synthesis in the hippocampus by 24 h showed prolonged IEG induction, whereas IEG expression returned to control levels in areas showing normal protein synthesis. In the temporal lobe decreased protein synthesis coexisted with decreased IEG expression, whereas areas in the vicinity of the region showing decreased protein synthesis demonstrated elevated IEG expression. The decreased protein synthesis was localized in areas where extensive neuronal death has occurred. This prolonged IEG induction in the hippocampus, which has been linked with neuronal death, could solely represent a prolonged mRNA turnover caused by disrupted protein synthesis. The prolonged IEG expression in the temporal lobe appeared to be localized in regions where the cells are in stress, but still viable. The sustained IEG expression might therefore either represent a stress response by which the neurons are trying to protect themselves or, alternatively, the IEG response may be an early sign indicating that these cells are initiating a pathway leading to programmed cell death.     

Neural pathways mediating hypothalamically elicited flight behavior in the cat

This study has sought to identify hypothalamic pathways mediating flight behavior in the cat. Flight behavior, characterized by an initial pupillary dilatation and followed by vigorous attempts to leap out of the observation chamber, was elicited primarily by electrical stimulation of the medial preoptic region and dorsomedial hypothalamus, and to a lesser extent from the perifornical region. A [14C]-2-deoxyglucose analysis was utilized to examine brain regions functionally activated by stimulation of hypothalamic sites which elicited flight behavior. In a second series of experiments, [3H]leucine injected into regions surrounding electrode tips from which flight had previously been elicited, permitted identification of pathways arising from such functionally characterized sites. We describe for the first time pathways arising from the hypothalamus which mediate flight behavior. In spite of individual variation in placement of electrodes eliciting flight, a consistent pattern of labeling was observed following injection of either [14C]-2-deoxyglucose systemically or [3H]amino acids into the hypothalamus. The primary rostral target structures receiving inputs from flight electrode sites included the nuclei of the diagonal band, bed nucleus of the stria terminalis, medial amygdaloid nucleus, lateral septal nucleus, and anterior medial preoptico-hypothalamus. Caudal to the level of stimulation, the principal target nuclei involved the centrum medianum-parafascicular complex and the midbrain central gray substance. Possible roles of these nuclear regions in organization and regulation of flight behavior is discussed.     

Deep brain stimulation of hypothalamus for narcolepsy-cataplexy in mice

BackgroundNarcolepsy type 1 (NT1, narcolepsy with cataplexy) is a disabling neurological disorder caused by loss of excitatory orexin neurons from the hypothalamus and is characterized by decreased motivation, sleep-wake fragmentation, intrusion of rapid-eye-movement sleep (REMS) during wake, and abrupt loss of muscle tone, called cataplexy, in response to sudden emotions.ObjectiveWe investigated whether subcortical stimulation, analogous to clinical deep brain stimulation (DBS), would ameliorate NT1 using a validated transgenic mouse model with postnatal orexin neuron degeneration.MethodsUsing implanted electrodes in freely behaving mice, the immediate and prolonged effects of DBS were determined upon behavior using continuous video-electroencephalogram-electromyogram (video/EEG/EMG) and locomotor activity, and neural activation in brain sections, using immunohistochemical labeling of the immediate early gene product c-Fos.ResultsBrief 10-s stimulation to the region of the lateral hypothalamus and zona incerta (LH/ZI) dose-responsively reversed established sleep and cataplexy episodes without negative sequelae. Continuous 3-h stimulation increased ambulation, improved sleep-wake consolidation, and ameliorated cataplexy. Brain c-Fos from mice sacrificed after 90 min of DBS revealed dose-responsive neural activation within wake-active nuclei of the basal forebrain, hypothalamus, thalamus, and ventral midbrain.ConclusionAcute and continuous LH/ZI DBS enhanced behavioral state control in a mouse model of NT1, supporting the feasibility of clinical DBS for NT1 and other sleep-wake disorders.     

Cell death atlas of the postnatal mouse ventral forebrain and hypothalamus: Effects of age and sex

Naturally occurring cell death is essential to the development of the mammalian nervous system. Although the importance of developmental cell death has been appreciated for decades, there is no comprehensive account of cell death across brain areas in the mouse. Moreover, several regional sex differences in cell death have been described for the ventral forebrain and hypothalamus, but it is not known how widespread the phenomenon is. We used immunohistochemical detection of activated caspase‐3 to identify dying cells in the brains of male and female mice from postnatal day (P) 1 to P11. Cell death density, total number of dying cells, and regional volume were determined in 16 regions of the hypothalamus and ventral forebrain (the anterior hypothalamus, arcuate nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and ventromedial nucleus of the hypothalamus; the basolateral, central, and medial amygdala; the lateral and principal nuclei of the bed nuclei of the stria terminalis; the caudate‐putamen; the globus pallidus; the lateral septum; and the islands of Calleja). All regions showed a significant effect of age on cell death. The timing of peak cell death varied between P1 to P7, and the average rate of cell death varied tenfold among regions. Several significant sex differences in cell death and/or regional volume were detected. These data address large gaps in the developmental literature and suggest interesting region‐specific differences in the prevalence and timing of cell death in the hypothalamus and ventral forebrain. J. Comp. Neurol. 521:2551–2569, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of phencyclidine on immediate early gene expression in the brain

The N-methyl-D-aspartate receptor antagonist phencyclidine (PCP) is a psychotomimetic drug which produces schizophrenia-like psychosis. In animal studies it is toxic to neurons in the posterior cingulate and retrosplenial cortex and to cerebellar Purkinje cells. To find clues about the mechanism and pathways of PCP action, we studied the effect of systemic PCP administration (10 and 50 mg/kg, intraperitoneal) on the expression of immediate-early genes (IEGs) (c-fos, c-jun, egr-2, egr-3, NGFI-A, NGFI-B, NGFI-C, and Nurr1) using in situ hybridization histochemistry. PCP, 50 mg/kg, produced a biphasic IEG induction: an early induction in the hippocampus, cerebral cortex, and cerebellar granule cell layer, and a delayed induction in the posterior cingulate cortex and cerebellar Purkinje cell layer. The early induction of all eight IEGs was observed 30 min after drug treatment in the cerebral cortex and in the hippocampus. c-fos, NGFI-A, and NGFI-B were also induced in thalamic nuclei, and c-fos was also induced in the cerebellar granule cell layer. In contrast, a delayed induction of c-fos, c-jun, NGFI-A, NGFI-B, NGFI-C, and Nurr1 in the posterior cingulate cortex was observed 2–6 hr after PCP, 50 mg/kg. egr-2 and egr-3 were not induced in the posterior cingulate cortex. c-fos induction in the cerebellar Purkinje cell layer peaked 2 hr after PCP, 50 mg/kg. In addition, PCP induced c-fos, egr-3, NGFI-A NGFI-B, NGFI-C, and Nurr1 in the inferior olivary nucleus. PCP-induced IEG expression returned to baseline by 24 hr. A lower PCP dose, 10 mg/kg, induced lower levels of IEG expression, with similar anatomical and biphasic temporal pattern as with the higher PCP dose of 50 mg/kg. However, no IEG induction was observed in the hippocampus following 10 mg/kg PCP. These results demonstrate that PCP produces neural activation not only in the cingulate and retrosplenial cortex, but also in many other regions of forebrain and cerebellum. Moreover, prolonged IEG expression in the posterior cingulate cortex and cerebellar Purkinje cells, the sites of PCP toxicity, suggests that IEGs could mediate neurotoxic/neuroprotective effects in these brain regions. © 1996 Wiley-Liss, Inc.