María del Carmen Basualdo

Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior

Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstrate by means of c-Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and, simultaneously, neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA-containing neurons in the DMH that project to the SCN. Next, we show that DMH lesions result in a loss or diminution of FAA, simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN, thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH–SCN interaction may serve as an intrahypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns.

Shift Work or Food Intake during the Rest Phase Promotes Metabolic Disruption and Desynchrony of Liver Genes in Male Rats

In the liver, clock genes are proposed to drive metabolic rhythms. These gene rhythms are driven by the suprachiasmatic nucleus (SCN) mainly by food intake and via autonomic and hormonal pathways. Forced activity during the normal rest phase, induces also food intake, thus neglecting the signals of the SCN, leading to conflicting time signals to target tissues of the SCN. The present study explored in a rodent model of night-work the influence of food during the normal sleep period on the synchrony of gene expression between clock genes and metabolic genes in the liver. Male Wistar rats were exposed to forced activity for 8 h either during the rest phase (day) or during the active phase (night) by using a slow rotating wheel. In this shift work model food intake shifts spontaneously to the forced activity period, therefore the influence of food alone without induced activity was tested in other groups of animals that were fed ad libitum, or fed during their rest or active phase. Rats forced to be active and/or eating during their rest phase, inverted their daily peak of Per1, Bmal1 and Clock and lost the rhythm of Per2 in the liver, moreover NAMPT and metabolic genes such as Pparα lost their rhythm and thus their synchrony with clock genes. We conclude that shift work or food intake in the rest phase leads to desynchronization within the liver, characterized by misaligned temporal patterns of clock genes and metabolic genes. This may be the cause of the development of the metabolic syndrome and obesity in individuals engaged in shift work.

Reciprocal interaction between the suprachiasmatic nucleus and the immune system tunes down the inflammatory response to lipopolysaccharide.

Several studies have shown circadian variations in the response of the immune system suggesting a role of the suprachiasmatic nucleus (SCN). Here we show that lipopolysaccharide (LPS) administration in the beginning of the active period induced more severe responses in temperature and cytokines than LPS given in the rest period. Moreover night administered LPS increased SCN basal neuronal activity indicating a direct influence of inflammation on the SCN. Bilateral lesions of the SCN resulted in an increased inflammatory response to LPS demonstrating that an interaction between the SCN and the immune system modulates the intensity of the inflammatory response.

The suprachiasmatic nucleus changes the daily activity of the arcuate nucleus α-MSH neurons in male rats.

Timing of metabolic processes is crucial for balanced physiology; many studies have shown the deleterious effects of untimely food intake. The basis for this might be an interaction between the arcuate nucleus (ARC) as the main integration site for metabolic information and the suprachiasmatic nucleus (SCN) as the master clock. Here we show in male rats that the SCN influences ARC daily neuronal activity by imposing a daily rhythm on the α-MSH neurons with a peak in neuronal activity at the end of the dark phase. Bilateral SCN lesions showed a complete disappearance of ARC neuronal rhythms and unilateral SCN lesions showed a decreased activation in the ARC at the lesioned side. Moreover light exposure during the dark phase inhibited ARC and α-MSH neuronal activity. The daily inhibition of ARC neuronal activity occurred in light-dark conditions as well as in dark-dark conditions, demonstrating the inhibitory effect to be mediated by increased SCN (subjective) day neuronal activity. Injections into the SCN with the neuronal tracer cholera toxin B showed that α-MSH neurons receive direct projections from the SCN. The present study demonstrates that the SCN activates and possibly also inhibits depending on the moment of the circadian cycle ARC α-MSH neurons via direct neuronal input. The persistence of these activity patterns in fasted animals demonstrates that this SCN-ARC interaction is not necessarily satiety associated but may support physiological functions associated with changes in the sleep-wake cycle.

NPY and VGF Immunoreactivity Increased in the Arcuate Nucleus, but Decreased in the Nucleus of the Tractus Solitarius, of Type-II Diabetic Patients

Ample animal studies demonstrate that neuropeptides NPY and α-MSH expressed in Arcuate Nucleus and Nucleus of the Tractus Solitarius, modulate glucose homeostasis and food intake. In contrast is the absence of data validating these observations for human disease. Here we compare the post mortem immunoreactivity of the metabolic neuropeptides NPY, αMSH and VGF in the infundibular nucleus, and brainstem of 11 type-2 diabetic and 11 non-diabetic individuals. α-MSH, NPY and tyrosine hydroxylase in human brain are localized in the same areas as in rodent brain. The similar distribution of NPY, α-MSH and VGF indicated that these neurons in the human brain may share similar functionality as in the rodent brain. The number of NPY and VGF immuno positive cells was increased in the infundibular nucleus of diabetic subjects in comparison to non-diabetic controls. In contrast, NPY and VGF were down regulated in the Nucleus of the Tractus Solitarius of diabetic patients. These results suggest an activation of NPY producing neurons in the arcuate nucleus, which, according to animal experimental studies, is related to a catabolic state and might be the basis for increased hepatic glucose production in type-2 diabetes.

nef/Long Terminal Repeat Quasispecies from HIV Type 1-Mexican Patients with Different Progression Patterns and Their Pathogenesis in hu-PBL-SCID Mice

To examine the genetic features of the long terminal repeat (LTR) derived from six HIV-1-infected individuals enrolled in the Mexico City Cohort, we cloned and sequenced a 505-bp fragment of the proviral LTR from their peripheral blood mononuclear cells (PBMCs). All patients harbored HIV-1 LTR quasispecies corresponding to the B subtype. Three patients with high CD4+ T cell counts (>500/mm3) presented LTR sequences with point mutations in the TAR bulge. The LTR sequence from a patient classified as a long-term nonprogressor (LTNP) presented the most frequent naturally occurring length polymorphism (MFNLP) and two substitutions in the TAR region that were predicted to result in two alternative secondary RNA structures. A novel 18-bp deletion, which eliminates part of the putative binding site for the nuclear factor of activated T cells (NFAT-1), was identified in the overlapping nef/LTR sequence derived from a patient progressing to AIDS. This deletion coincides with the ability of this virus to consistently replicate at low levels in vivo (viral load <500 RNA copies/ml) and in vitro (unsuccessful virus isolation). On one occasion, when virus isolation was successful, the 18-bp deletion was no longer evident and LTR sequences with intact NFAT-1-binding sites were observed. Inoculation of hu-PBL-SCID mice with viruses from several Mexican patients resulted in differential CD4+ T cell depletion patterns 15 days postinfection, which agree with the in vivo CD4+ T cell count data from each patient.

Shift Work in Rats Results in Increased Inflammatory Response after Lipopolysaccharide Administration A Role for Food Consumption

The suprachiasmatic nucleus (SCN) drives circadian rhythms in behavioral and physiological variables, including the inflammatory response. Shift work is known to disturb circadian rhythms and is associated with increased susceptibility to develop disease. In rodents, circadian disruption due to shifted light schedules (jet lag) induced increased innate immune responses. To gain more insight into the influence of circadian disruption on the immune response, we characterized the inflammatory response in a model of rodent shift work and demonstrated that circadian disruption affected the inflammatory response to lipopolysaccharide (LPS) both in vivo and in vitro. Since food consumption is a main disturbing element in the shift work schedule, we also evaluated the inflammatory response to LPS in a group of rats that had no access to food during their working hours. Our results demonstrated that the shift work schedule decreased basal TNF-α levels in the liver but not in the circulation. Despite this, we observed that shift work induced increased cytokine response after LPS stimulation in comparison to control rats. Also, Kupffer cells (liver macrophages) isolated from shift work rats produced more TNF-α in response to in vitro LPS stimulation, suggesting important effects of circadian desynchronization on the functionality of this cell type. Importantly, the effects of shift work on the inflammatory response to LPS were prevented when food was not available during the working schedule. Together, these results show that dissociating behavior and food intake from the synchronizing drive of the SCN severely disturbs the immune response.

Food entrains clock genes but not metabolic genes in the liver of suprachiasmatic nucleus lesioned rats

Hepatic circadian transcription, considered to be driven by the liver clock, is largely influenced by food even uncoupling it from the suprachiasmatic nucleus (SCN). In SCN lesioned rats (SCNx) we determined the influence of a physiological feeding schedule on the entrainment of clock and clock‐controlled (CCG) genes in the liver. We show that clock genes and the CCG Rev‐erbα and peroxisome proliferator‐activated receptor alpha (PPARα) in food‐scheduled intact and SCNx have a robust diurnal differential expression persisting after a 24 h fast. However, hepatic nicotinamide phosphoribosyl transferase (Nampt) shows time dependent changes that are lost in intact animals under fasting; moreover, it is unresponsive to the nutrient status in SCNx, indicating a poor reliance on liver clock genes and highlighting the relevance of SCN‐derived signals for its metabolic status‐related expression.

A role for VGF in the hypothalamic arcuate and paraventricular nuclei in the control of energy homeostasis

The arcuate nucleus is the main receptive area of the brain for peripheral and central metabolic cues and its integrity is essential for the maintenance of energy homeostasis. In the arcuate nucleus, different neuronal populations process metabolic signals and transmit this information to other nuclei of the hypothalamus by means of neurotransmitters and a combination of neuropeptides whose expression is modulated by the nutritional status. Here we investigated the changes in expression and synthesis of the polypeptide VGF in the arcuate nucleus of rats, in relation to the two main categories of neurons that show colocalization with VGF: the orexigenic NPY-expressing cells and the anorexigenic POMC-expressing cells. The results show that fasting is the most important stimulus for VGF expression, and that the up-regulation of VGF mRNA is restricted to the NPY area of the arcuate nucleus. POMC neurons express VGF under all feeding conditions, but especially in ad libitum-fed and fasted-refed animals. We also show that VGF arcuate neurons project to the pre-autonomic neurons of the paraventricular nucleus of the hypothalamus, providing anatomical evidence suggesting VGF as a central modulator of the autonomic nervous system.

The Suprachiasmatic Nucleus Modulates the Sensitivity of Arcuate Nucleus to Hypoglycemia in the Male Rat

The suprachiasmatic nucleus (SCN) and arcuate nucleus (ARC) have reciprocal connections; catabolic metabolic information activates the ARC and inhibits SCN neuronal activity. Little is known about the influence of the SCN on the ARC. Here, we investigated whether the SCN modulated the sensitivity of the ARC to catabolic metabolic conditions. ARC neuronal activity, as determined by c-Fos immunoreactivity, was increased after a hypoglycemic stimulus by 2-deoxyglucose (2DG). The highest ARC neuronal activity after 2DG was found at the end of the light period (zeitgeber 11, ZT11) with a lower activity in the beginning of the light period (zeitgeber 2, ZT2), suggesting the involvement of the SCN. The higher activation of ARC neurons after 2DG at ZT11 was associated with higher 2DG induced blood glucose levels as compared with ZT2. Unilateral SCN-lesioned animals, gave a mainly ipsilateral activation of ARC neurons at the lesioned side, suggesting an inhibitory role of the SCN on ARC neurons. The 2DG-induced counterregulatory glucose response correlated with increased ARC neuronal activity and was significantly higher in unilateral SCN-lesioned animals. Finally, the ARC as site where 2DG may, at least partly, induce a counterregulatory response was confirmed by local microdialysis of 2DG. 2DG administration in the ARC produced a higher increase in circulating glucose compared with 2DG administration in surrounding areas such as the ventromedial nucleus of the hypothalamus (VMH). We conclude that the SCN uses neuronal pathways to the ARC to gate sensory metabolic information to the brain, regulating ARC glucose sensitivity and counterregulatory responses to hypoglycemic conditions.