Central circuit controlling thermoregulatory inversion and torpor-like state.

To maintain core body temperature in mammals, the CNS thermoregulatory networks respond to cold exposure by increasing brown adipose tissue and shivering thermogenesis. However, in hibernation or torpor, this normal thermoregulatory response is supplanted by "thermoregulatory inversion", an altered homeostatic state in which cold exposure causes inhibition of thermogenesis and warm exposure stimulates thermogenesis. Here we demonstrate the existence of a novel, dynorphinergic thermoregulatory reflex pathway between the dorsolateral parabrachial nucleus and the dorsomedial hypothalamus that bypasses the normal thermoregulatory integrator in the hypothalamic preoptic area to play a critical role in mediating the inhibition of thermogenesis during thermoregulatory inversion. Our results indicate the existence of a neural circuit mechanism for thermoregulatory inversion within the CNS thermoregulatory pathways and support the potential for inducing a homeostatically-regulated, therapeutic hypothermia in non-hibernating species, including humans.

Centrally Projecting Edinger-Westphal Nucleus in the Control of Sympathetic Outflow and Energy Homeostasis.

The centrally projecting Edinger-Westphal nucleus (EWcp) is a midbrain neuronal group, adjacent but segregated from the preganglionic Edinger-Westphal nucleus that projects to the ciliary ganglion. The EWcp plays a crucial role in stress responses and in maintaining energy homeostasis under conditions that require an adjustment of energy expenditure, by virtue of modulating heart rate and blood pressure, thermogenesis, food intake, and fat and glucose metabolism. This modulation is ultimately mediated by changes in the sympathetic outflow to several effector organs, including the adrenal gland, heart, kidneys, brown and white adipose tissues and pancreas, in response to environmental conditions and the animal's energy state, providing for appropriate energy utilization. Classic neuroanatomical studies have shown that the EWcp receives inputs from forebrain regions involved in these functions and projects to presympathetic neuronal populations in the brainstem. Transneuronal tracing with pseudorabies virus has demonstrated that the EWcp is connected polysynaptically with central circuits that provide sympathetic innervation to all these effector organs that are critical for stress responses and energy homeostasis. We propose that EWcp integrates multimodal signals (stress, thermal, metabolic, endocrine, etc.) and modulates the sympathetic output simultaneously to multiple effector organs to maintain energy homeostasis under different conditions that require adjustments of energy demands.

Median preoptic area neurons are required for the cooling and febrile activations of brown adipose tissue thermogenesis in rat.

Within the central neural circuitry for thermoregulation, the balance between excitatory and inhibitory inputs to the dorsomedial hypothalamus (DMH) determines the level of activation of brown adipose tissue (BAT) thermogenesis. We employed neuroanatomical and in vivo electrophysiological techniques to identify a source of excitation to thermogenesis-promoting neurons in the DMH that is required for cold defense and fever. Inhibition of median preoptic area (MnPO) neurons blocked the BAT thermogenic responses during both PGE2-induced fever and cold exposure. Disinhibition or direct activation of MnPO neurons induced a BAT thermogenic response in warm rats. Blockade of ionotropic glutamate receptors in the DMH, or brain transection rostral to DMH, blocked cold-evoked or NMDA in MnPO-evoked BAT thermogenesis. RNAscope technique identified a glutamatergic population of MnPO neurons that projects to the DMH and expresses c-Fos following cold exposure. These discoveries relative to the glutamatergic drive to BAT sympathoexcitatory neurons in DMH augment our understanding of the central thermoregulatory circuitry in non-torpid mammals. Our data will contribute to the development of novel therapeutic approaches to induce therapeutic hypothermia for treating drug-resistant fever, and for improving glucose and energy homeostasis.

Thermoregulatory inversion: a novel thermoregulatory paradigm.

To maintain core body temperature in mammals, the normal central nervous system (CNS) thermoregulatory reflex networks produce an increase in brown adipose tissue (BAT) thermogenesis in response to skin cooling and an inhibition of the sympathetic outflow to BAT during skin rewarming. In contrast, these normal thermoregulatory reflexes appear to be inverted in hibernation/torpor; thermogenesis is inhibited during exposure to a cold environment, allowing dramatic reductions in core temperature and metabolism, and thermogenesis is activated during skin rewarming, contributing to a return of normal body temperature. Here, we describe two unrelated experimental paradigms in which rats, a nonhibernating/torpid species, exhibit a "thermoregulatory inversion," which is characterized by an inhibition of BAT thermogenesis in response to skin cooling, and a switch in the gain of the skin cooling reflex transfer function from negative to positive values. Either transection of the neuraxis immediately rostral to the dorsomedial hypothalamus in anesthetized rats or activation of A1 adenosine receptors within the CNS of free-behaving rats produces a state of thermoregulatory inversion in which skin cooling inhibits BAT thermogenesis, leading to hypothermia, and skin warming activates BAT, supporting an increase in core temperature. These results reflect the existence of a novel neural circuit that mediates inverted thermoregulatory reflexes and suggests a pharmacological mechanism through which a deeply hypothermic state can be achieved in nonhibernating/torpid mammals, possibly including humans.

α2 Adrenergic receptor-mediated inhibition of thermogenesis.

α2 adrenergic receptor (α2-AR) agonists have been used as antihypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since α2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat brown adipose tissue (BAT) and shivering thermogenesis by α2-AR agonists. Nanoinjection of the α2-AR agonist clonidine (1.2 nmol) into the rostral raphe pallidus area (rRPa) inhibited BAT sympathetic nerve activity (SNA) and BAT thermogenesis. Subsequent nanoinjection of the α2-AR antagonist idazoxan (6 nmol) into the rRPa reversed the clonidine-evoked inhibition of BAT SNA and BAT thermogenesis. Systemic administration of the α2-AR agonists dexmedetomidine (25 µg/kg, i.v.) and clonidine (100 µg/kg, i.v.) inhibited shivering EMGs, BAT SNA, and BAT thermogenesis, effects that were reversed by nanoinjection of idazoxan (6 nmol) into the rRPa. Dexmedetomidine (100 µg/kg, i.p.) prevented and reversed lipopolysaccharide-evoked (10 µg/kg, i.p.) thermogenesis in free-behaving rats. Cholera toxin subunit b retrograde tracing from rRPa and pseudorabies virus transynaptic retrograde tracing from BAT combined with immunohistochemistry for catecholaminergic biosynthetic enzymes revealed the ventrolateral medulla as the source of catecholaminergic input to the rRPa and demonstrated that these catecholaminergic neurons are synaptically connected to BAT. Photostimulation of ventrolateral medulla neurons expressing the PRSx8-ChR2-mCherry lentiviral vector inhibited BAT SNA via activation of α2-ARs in the rRPa. These results indicate a potent inhibition of BAT and shivering thermogenesis by α2-AR activation in the rRPa, and suggest a therapeutic potential of α2-AR agonists for reducing potentially lethal elevations in body temperature during excessive fever.

Spinal projections of the A5, A6 (locus coeruleus), and A7 noradrenergic cell groups in rats.

The pontine noradrenergic cell groups, A5, A6 (locus coeruleus), and A7, provide the only noradrenergic innervation of the spinal cord, but the individual contribution of each of these populations to the regional innervation of the spinal cord remains controversial. We used an adeno-associated viral (AAV) vector encoding green fluorescent protein under an artificial dopamine beta-hydroxylase (PRSx8) promoter to trace the spinal projections from the A5, A6, and A7 groups. Projections from all three groups travel through the spinal cord in both the lateral and ventral funiculi and in the dorsal surface of the dorsal horn, but A6 axons take predominantly the dorsal and ventral routes, whereas A5 axons take mainly a lateral and A7 axons a ventral route. The A6 group provides the densest innervation at all levels, and includes all parts of the spinal gray matter, but it is particularly dense in the dorsal horn. The A7 group provides the next most dense innervation, again including all parts of the spinal cord, but is it denser in the ventral horn. The A5 group supplies only sparse innervation to the dorsal and ventral horns and to the cervical and lumbosacral levels, but provides the densest innervation to the thoracic intermediolateral cell column, and in particular to the sympathetic preganglionic neurons. Thus, the pontine noradrenergic cell groups project in a roughly topographic and complementary fashion onto the spinal cord. The pattern of spinal projections observed suggests that the locus coeruleus might have the greatest effect on somatosensory transmission, the A7 group on motor function, and the A5 group on sympathetic function.

An orexinergic projection from perifornical hypothalamus to raphe pallidus increases rat brown adipose tissue thermogenesis.

Orexin (hypocretin) neurons, located exclusively in the PeF-LH, which includes the perifornical area (PeF), the lateral hypothalamus (LH), and lateral portions of the medial hypothalamus, have widespread projections and influence many physiological functions, including the autonomic regulation of body temperature and energy metabolism. Narcolepsy is characterized by the loss of orexin neurons and by disrupted sleep, but also by dysregulation of body temperature and by a strong tendency for obesity. Heat production (thermogenesis) in brown adipose tissue (BAT) contributes to the maintenance of body temperature and, through energy consumption, to body weight regulation. We identified a neural substrate for the influence of orexin neurons on BAT thermogenesis in rat. Nanoinjection of orexin-A (12 pmol) into the rostral raphe pallidus (rRPa), the site of BAT sympathetic premotor neurons, produced large, sustained increases in BAT sympathetic outflow and in BAT thermogenesis. Activation of neurons in the PeF-LH also enhanced BAT thermogenesis over a long time course. Combining viral retrograde tracing from BAT, or cholera toxin subunit b tracing from rRPa, with orexin immunohistochemistry revealed synaptic connections to BAT from orexin neurons in PeF-LH and from rRPa neurons with closely apposed, varicose orexin fibers, as well as a direct, orexinergic projection from PeF-LH to rRPa. These results indicate a potent modulation of BAT thermogenesis by orexin released from the terminals of orexin neurons in PeF-LH directly into the rRPa and provide a potential mechanism contributing to the disrupted regulation of body temperature and energy metabolism in the absence of orexin.

Parallel preoptic pathways for thermoregulation.

Sympathetic premotor neurons in the rostral medullary raphe (RMR) regulate heat conservation by tail artery vasoconstriction and brown adipose tissue thermogenesis. These neurons are a critical relay in the pathway that increases body temperature. However, the origins of the inputs that activate the RMR during cold exposure have not been definitively identified. We investigated the afferents to the RMR that were activated during cold by examining Fos expression in retrogradely labeled neurons after injection of cholera toxin B subunit (CTb) in the RMR. These experiments identified a cluster of Fos-positive neurons in the dorsomedial hypothalamic nucleus and dorsal hypothalamic area (DMH/DHA) with projections to the RMR that may mediate cold-induced elevation of body temperature. Also, neurons in the median preoptic nucleus (MnPO) and dorsolateral preoptic area (DLPO) and in the A7 noradrenergic cell group were retrogradely labeled but lacked Fos expression, suggesting that they may inhibit the RMR. To investigate whether individual or common preoptic neurons project to the RMR and DMH/DHA, we injected CTb into the RMR and Fluorogold into the DMH/DHA. We found that projections from the DLPO and MnPO to the RMR and DMH/DHA emerge from largely separate neuronal populations, indicating they may be differentially regulated. Combined cell-specific lesions of MnPO and DLPO, but not lesions of either one alone, caused baseline hyperthermia. Our data suggest that the MnPO and DLPO provide parallel inhibitory pathways that tonically inhibit the DMH/DHA and the RMR at baseline, and that hyperthermia requires the release of this inhibition from both nuclei.

Neural circuitry of stress-induced insomnia in rats.

Sleep architecture is often disturbed after a stressful event; nevertheless, little is known about the brain circuitry responsible for the sleep perturbations induced by stress. We exposed rats to a psychological stressor (cage exchange) that initially causes an acute stress response, but several hours later generates a pattern of sleep disturbances similar to that observed in stress-induced insomnia in humans: increased sleep latency, decreased non-REM (nREM) and REM sleep, increased fragmentation, and high-frequency EEG activity during nREM sleep. We examined the pattern of Fos expression to identify the brain circuitry activated, and found increased Fos in the cerebral cortex, limbic system, and parts of the arousal and autonomic systems. Surprisingly, there was simultaneous activation of the sleep-promoting areas, most likely driven by ongoing circadian and homeostatic pressure. The activity in the cerebral cortex and arousal system while sleeping generates a novel intermediate state characterized by EEG high-frequency activity, distinctive of waking, during nREM sleep. Inactivation of discrete limbic and arousal regions allowed the recovery of specific sleep components and altered the Fos pattern, suggesting a hierarchical organization of limbic areas that in turn activate the arousal system and subsequently the cerebral cortex, generating the high-frequency activity. This high-frequency activity during nREM was eliminated in the stressed rats after inactivating parts of the arousal system. These results suggest that shutting down the residual activity of the limbic-arousal system might be a better approach to treat stress-induced insomnia, rather than potentiation of the sleep system, which remains fully active.

Homeostatic, circadian, and emotional regulation of sleep.

A good night's sleep is one of life's most satisfying experiences, while sleeplessness is stressful and causes cognitive impairment. Yet the mechanisms that regulate the ability to sleep have only recently been subjected to detailed investigation. New studies show that the control of wake and sleep emerges from the interaction of cell groups that cause arousal with other nuclei that induce sleep such as the ventrolateral preoptic nucleus (VLPO). The VLPO inhibits the ascending arousal regions and is in turn inhibited by them, thus forming a mutually inhibitory system resembling what electrical engineers call a "flip-flop switch." This switch may help produce sharp transitions between discrete behavioral states, but it is not necessarily stable. The orexin neurons in the lateral hypothalamus may help stabilize this system by exciting arousal regions during wakefulness, preventing unwanted transitions between wakefulness and sleep. The importance of this stabilizing role is apparent in narcolepsy, in which an absence of the orexin neurons causes numerous, unintended transitions in and out of sleep and allows fragments of REM sleep to intrude into wakefulness. These influences on the sleep/wake system by homeostatic and circadian drives, as well as emotional inputs, are reviewed. Understanding the pathways that underlie the regulation of sleep and wakefulness may provide important insights into how the cognitive and emotional systems interact with basic homeostatic and circadian drives for sleep.

Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in rat.

The neural control of renal function is exerted by the central nervous system via sympathetic innervation of the kidneys. To determine the extent to which the control of the two kidneys is provided by the same brain neurons, the central circuitry involved in the innervation of both kidneys was characterized in individual rats by dual viral transneuronal tracing using isogenic recombinant strains (PRV-152 and BaBlu) of pseudorabies virus. Prior to dual tracing, the neuroinvasive properties of PRV-152 and BaBlu were characterized by conducting parametric studies, using the two kidneys as an anatomical model, and comparing the pattern of infection with that obtained following injection of the parental strain, PRV-Bartha, into the left kidney. Once the optimal concentrations of virus required to obtain equivalent infection were established, PRV-152 and BaBlu were injected into the left and right kidney, respectively, in the same rats. Immunocytochemical localization of viral reporter proteins at different postinoculation times allowed us to determine the sequence of infection in the brain, as well as to quantify dual- and single-labeled neurons in each infected area. Neurons that influence autonomic outflow to one or both kidneys coexist in all brain areas involved in the control of the sympathetic outflow to the kidneys at every hierarchical level of the circuit. The proportions of dual-infected neurons with respect to the number of total infected neurons varied across regions, but they were maintained at different survival times. The pattern of infection suggests that the activity of each kidney is controlled independently by organ-specific neurons, whereas the functional coordination of the two kidneys results from neurons that collaterize to modulate the sympathetic outflow to both organs. The advantages of using an anatomical symmetrical system, such as the two kidneys, as an experimental approach to characterize PRV recombinants in general are also discussed.

Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure.

The thermogenic activity of interscapular brown adipose tissue (IBAT) in response to physiologic stimuli, such as cold exposure, is controlled by its sympathetic innervation. To determine which brain regions might be involved in the regulation of cold-evoked increases in sympathetic outflow to IBAT, the present study compared central nervous system (CNS) areas activated by cold exposure with brain regions anatomically linked to the sympathetic innervation of IBAT. Immunocytochemical localization of Fos was examined in the brains of rats exposed to 4 degrees C for 4 hours. In a separate group of rats, the neural circuit involved in IBAT control, including the location of sympathetic preganglionic neurons in the spinal cord, was characterized with pseudorabies virus, a retrograde transynaptic tracer. Central noradrenergic and serotonergic groups related to the sympathetic outflow to IBAT also were identified. Localization of viral antigens at different survival times (66-96 hours) revealed infection in circumscribed CNS populations, but only a subset of the regions comprising this circuitry showed cold-evoked Fos expression. The raphe pallidus and the ventromedial parvicellular subdivision of the paraventricular hypothalamic nucleus (PVH), both infected at early survival times, were the main areas containing sympathetic premotor neurons activated by cold exposure. Major cold-sensitive areas projecting to spinal interneurons or to regions containing sympathetic premotor neurons, which became infected at intermediate intervals, included lateral hypothalamic, perifornical, and retrochiasmatic areas, anterior and posterior PVH, ventrolateral periaqueductal gray, and Barrington's nucleus. Areas infected later, most likely related to reception of cold-related signals, comprised the lateral preoptic area, parastrial nucleus, dorsomedial hypothalamic nucleus, lateral parabrachial nucleus, and nucleus of the solitary tract. These interconnected areas, identified by combining functional and retrograde anatomic approaches, likely constitute the central circuitry responsible for the increase in sympathetic outflow to IBAT during cold-evoked thermogenesis.

The locus coeruleus, Barrington's nucleus, and neural circuits of stress.

Much attention has focused on the role of the locus coeruleus (LC) as a component of the central neural circuitry involved in stress. Many, though not all, stressful stimuli produce activation of LC neurons, as reflected by increased Fos expression in these neurons. Stimulation of the LC elicits many stress-like responses, including increased ACTH secretion, though not all responses to LC stimulation are readily interpretable in the context of stress. In particular, stimulation of the LC, at least in anesthetized rats, elicits a decrease in blood pressure and heart rate. Inhibition of the LC has been reported to inhibit certain responses to stress, including inhibition of ACTH release in response to certain stressors. Furthermore, local inhibition of the LC prevents foot shock-evoked Fos expression in certain brain areas. In the studies of the role of the LC in stress, one complicating factor has been the inadequate attention given to Barrington's nucleus (BN), which is located adjacent to the LC. Although BN is best recognized for its role in the control of micturition, the fact that it is activated by a great variety of stressful stimuli, and that it is anatomically connected to multiple output systems involved in stress responses, suggest that it may play a role in the neural circuitry subserving responses to stress.

Manganese poisoning reduces strychnine-insensitive glycine binding sites in the globus pallidus of the mouse brain / Reducción de los receptores glicinérgicos insensibles a estricnina en el globo pálido del cerebro de ratones intoxicados con manganeso

Manganese (Mn) poisoning is characterized by central nerous system manifestations, including psychiatric disturbances and estrapyramidal disorders. This metal is thought to produce neuronal degeneration due to cytotoxic products originated by oxidative stress and through an indirect excitotoxic process. In previous studies, we have found a reduction in the density of N-methyl-D-aspartate (NMDA) recognition sites in some brain areas of Mn-treated mice. Due to the close relationship between NMDA receptor complex, the [3H]-glycine ([3H]/Gly) binding was manganese choride for 8 weeks. Among all analyzed areas, only the globus pallidus showed a significant reduction in [3H]-Gly binding (27-28). The Gly binding decrease, focalized in the globus pallidus, could reflect a degeneration of structures containing strychnine-insensitive Gly receptors, since this area is the most frequently reported damaged brain region in Mn intoxication. Howerer, it might also be due to a Gly receptor down-regulation to control NMDA complex activation during Mn poisoning.

Intoxicación crónica con manganeso: cuantificación autorradiográfica de los receptores colinérgicos muscarínicos en el cerebro de ratón / Chronic manganese poisoning: autoradiograohic cuantitation of muscarinic cholinergic receptors in mouse brain

El tratamiento crónico con cloruro de manganeso (5 mg Mn/kg/día), durante 9 semanas, no afectó la unión del radioligando [3H]-quinuclidinil benzilato a los receptores colinérgicos muscarínicos en el cerebro de ratón. Mediante técnica autorradiográfica se determinó la localización anatómica precisa de los receptores y se procedió a la cuantificació de los mismos en los cortes coronales del bulbo olfatorio y del cerebro medio. A la luz de los resultados obtenidos podemos concluir que, en nuestras condiciones experimentales, no se producen alteraciones en la densidad de los receptores colinérgicos muscarínicos en el cerebro de ratones intoxicados con manganeso

Estrongiloidiasis hiperinfectiva fatal: reporte de tres casos / Hyperinfective strongyloidiasis: report of three cases

Se presentan tres casos fatales de estrongiloidiasis hiperinfectiva. Caso 1. Edad 37 años, femenino, con antecedentes de ingestión de esteroides, murió en shock. La necropsia reveló infección intestinal masiva por S. stercoralis con migración de larvas a pulmones, hígado, bazo y tejido adiposo alrededor de las cápsulas suprarenales. Caso 2. Doce años de edad, femenino, desnutrida, murió por peritonitis. El exámen anatomopatológico mostró litiasis con perforación de yeyuno, estrongiloidiasis hiperinfectiva que afectó intestinos y pulmones y peritonitis aguda y crónica. Caso 3. Siete años de edad, femenino, desnutrida, recibió esteroides. Murió por paro cardiorrespiratorio. La necropsia reveló hiperinfección por S. stercoralis que afectó estomago, intestinos, pulmones, hígado y cerebro. Como en Venezuela, prácticamente no existen publicaciones de estrongiloidiasis masiva fatal, se hace la presente comunicación para señalar la necesidad de pensar en esta parasitosis en todo paciente con riesgo a la diseminación para tratar de prevenir desenlaces fatales

Quiste hidatídico pulmonar. Presentación de un caso / Pulmonary hydatid cyst. Report of a case

Se presenta un caso de quiste hidatídico pulmonar en un extranjero con más de 10 de años de residencia en Maracaibo, Estado Zulia. Se trata de un paciente de 54 años de edad, quien consulta por presentar disnea, fiebre y pérdida de peso. El estudio radiológico de tórax revela una imagen de aspecto tumoral en campo pulmonar izquierdo y se diagnostica Carcinoma Broncogénico. Los exámenes de laboratorio son normales; con esputo negativo para BK, citologías seriadas negativas y biopsia no concluyente. En vista de lo anterior se realiza lobectomía inferior izquierda. El estudio anatomopatológico muestra un quiste de 7x5 cm., con un contenido líquido amarillento. El exámen histológico revela cápsulas prolígeras y escólices en la cavidad quística, haciéndose el diagnóstico de quiste unilocular de Echinococcus granulosus. La presencia de los escólices en alvéolos y vasos sanguíneos pulmonares sugiere la posibilidad de una diseminación del parásito. Por la rareza de esta patología en el país, presentamos el caso, para que se piense en esta afección y pueda ser diagnósticada y tratada en forma adecuada