Oxygen-induced retinopathy induces short-term glial stress and long-term impairment of photoentrainment in mice.

BACKGROUND: Retinopathy of prematurity is a serious potentially blinding disease of pre-term infants. There is extensive vascular remodeling and tissue stress, but data concerning alterations in retinal neurons and glia, and long-term functional sequelae are still incomplete. METHODS: ROP was induced using the oxygen-induced retinopathy (OIR) mouse model. Postnatal day 7 (P7) 129SVE mice were exposed to hyperoxia (75 ± 0.5 % oxygen) for 5 days, and then returned to normoxia to induce OIR. Exposed animals were euthanized at 5 (P17-OIR) and 14 days (P26-OIR) after return to normal air, together with corresponding age-matched control mice (P17-C and P26-C respectively) raised only in room air. Their retinas were examined by immunohistochemistry using a battery of antibodies against key glial and neuronal proteins. A further group of OIR mice and controls were examined at 10 weeks of age for their ability to re-entrain to changing 12 h light/12 h dark cycles, assayed by wheel-running actimetry. In this protocol, animals were subjected to three successive conditions of 300 lux, 15 lux and 1 lux ambient light intensity coupled with 6 hours of jetlag. Animals were euthanized at 4 months of age and used in immunoblotting for rhodopsin. RESULTS: Compared to P17-C, immunohistochemical staining of P17-OIR sections showed up-regulation of stress-related and glutamate-regulatory proteins in astrocytes and Müller glial cells. In contrast, glial phenotypic expression in P26-OIR retinas largely resembled that in P26-C. There was no loss in total retinal ganglion cells (RGC) at either P17-OIR or P26-OIR compared to corresponding controls, whereas intrinsically photosensitive RGC showed significant decreases, with 375 ± 13/field in P26-OIR compared to 443 ± 30/field in P26-C (p < 0.05). Wheel actimetry performed on control and OIR-treated mice at 4 months demonstrated that animals raised in hyperoxic conditions had impaired photoentrainment at low illuminance of 1 lux, as well as significantly reduced levels of rhodopsin compared to age-matched controls. CONCLUSIONS: OIR leads to transient up-regulation of retinal glial proteins involved in metabolism, and partial degeneration of intrinsically photosensitive RGC and rod photoreceptors. OIR affects circadian photo-entrainment at low illuminance values, possibly by affecting the rod pathway and/or intrinsically photosensitive RGC input to the circadian clock. This study hence shows that retinopathy of prematurity affects light-regulated circadian behavior in an animal model, and may induce similar problems in humans.

Setting the main circadian clock of a diurnal mammal by hypocaloric feeding.

Caloric restriction attenuates the onset of a number of pathologies related to ageing. In mammals, circadian rhythms, controlled by the hypothalamic suprachiasmatic (SCN) clock, are altered with ageing. Although light is the main synchronizer for the clock, a daily hypocaloric feeding (HF) may also modulate the SCN activity in nocturnal rodents. Here we report that a HF also affects behavioural, physiological and molecular circadian rhythms of the diurnal rodent Arvicanthis ansorgei. Under constant darkness HF, but not normocaloric feeding (NF), entrains circadian behaviour. Under a light­dark cycle, HF at midnight led to phase delays of the rhythms of locomotor activity and plasma corticosterone. Furthermore, Per2 and vasopressin gene oscillations in the SCN were phase delayed in HF Arvicanthis compared with animals fed ad libitum. Moreover, light-induced expression of Per genes in the SCN was modified in HF Arvicanthis, despite a non-significant effect on light-induced behavioural phase delays. Together, our data show that HF affects the circadian system of the diurnal rodent Arvicanthis ansorgei differentially from nocturnal rodents. The Arvicanthis model has relevance for the potential use of HF to manipulate circadian rhythms in diurnal species including humans.

Melatonin-independent Photoperiodic Entrainment of the Circannual TSH Rhythm in the Pars Tuberalis of the European Hamster.

Adaptation of biological rhythms to a seasonal environment in circannual mammals is achieved via the synchronization of intrinsic circannual rhythms to the external year by photoperiod. In mammals, the photoperiodic information is integrated to seasonal physiology via the pineal hormone melatonin regulation of pars tuberalis (PT) TSHß expression and its downstream control of hypothalamic dio2 gene expression. In the circannual European hamster, however, photoperiodic entrainment of the circannual clock is possible in pinealectomized animals. The present study explores whether the TSHß expression in the PT and the downstream hypothalamic pathways are regulated by photoperiod in European hamsters in the absence of melatonin. All animals were kept on an accelerated photoperiodic regime, which compressed the natural year to a 6-month cycle. Sham-operated European hamsters and half of the pinealectomized European hamsters entrained their annual cycle in reproduction, body weight, and activity pattern to this cycle, whereas the other half of the pinealectomized animals followed only each second cycle. In all animals, PT TSHß and hypothalamic dio2 expressions were higher in hamsters displaying a summer physiological state than in those in winter state. Moreover, in agreement with their seasonal state, reproductive animals (summer state) showed higher expression of rfrp and lower expression of kiss1-genes encoding central regulators of the reproductive axis-than those animals in reproductive quiescence (winter state), indicating the hypothalamic integration of the photoperiodic signal even in pinealectomized animals. The appropriate occurrence of a well-characterized activity pattern indicative of a so-called sensitive phase to short photoperiod suggested that the SCN constructs the melatonin-independent photoperiodic message. This message is sufficient to entrain the circannual rhythm in TSHß expression in the PT and the downstream hypothalamic neuroendocrine pathway through a yet unknown pathway. These results reinforce the hypothesis that the PT is the site for the integration of circannual and photoperiodic information.

The lateral habenula interacts with the hypothalamo-pituitary adrenal axis response upon stressful cognitive demand in rats.

The lateral habenula (LHb) is involved in emotional and cognitive behaviors. Recently, we have shown in rats that blockade of excitatory inputs to the LHb not only induced deficits of memory retrieval in the water maze, but also altered swim strategies (i.e., induced excessive thigmotaxis). The latter observation, although consistent with the occurrence of memory deficits, could also possibly be the consequence of an excessive level of stress, further suggesting a role for the LHb in the stress response in our behavioral paradigm. To test this hypothesis we performed in rats intra-LHb infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 267 ng/side in 0.3 µL), or vehicle, and assessed the responsiveness of the hypothalamo-pituitary adrenal (HPA) axis to environmental stressful or non-stressful situations. We have measured plasma corticosterone (CORT) concentrations at different time points before and following intra-LHb infusion of CNQX - or of the same volume of vehicle - in three conditions: during the probe test of a water maze experiment; in an anxiety test, the elevated plus maze; and in a home cage condition. Whereas there were no differences in the home cage condition and in the elevated plus maze, in the water maze experiment we observed that CNQX-treated rats presented, along with memory deficits, a higher level of blood CORT than vehicle-treated rats. These results suggest that perturbations of the modulation of the HPA axis are consecutive to the alteration of LHb function, whether it is the result of a defective direct control of the LHb over the HPA axis, or the consequence of memory deficits.

Arvicanthis ansorgei, a Novel Model for the Study of Sleep and Waking in Diurnal Rodents.

STUDY OBJECTIVES: Sleep neurobiology studies use nocturnal species, mainly rats and mice. However, because their daily sleep/wake organization is inverted as compared to humans, a diurnal model for sleep studies is needed. To fill this gap, we phenotyped sleep and waking in Arvicanthis ansorgei, a diurnal rodent widely used for the study of circadian rhythms. DESIGN: Video-electroencephalogram (EEG), electromyogram (EMG), and electrooculogram (EOG) recordings. SETTING: Rodent sleep laboratory. PARTICIPANTS: Fourteen male Arvicanthis ansorgei, aged 3 mo. INTERVENTIONS: 12 h light (L):12 h dark (D) baseline condition, 24-h constant darkness, 6-h sleep deprivation. MEASUREMENTS AND RESULTS: Wake and rapid eye movement (REM) sleep showed similar electrophysiological characteristics as nocturnal rodents. On average, animals spent 12.9 h ± 0.4 awake per 24-h cycle, of which 6.88 h ± 0.3 was during the light period. NREM sleep accounted for 9.63 h ± 0.4, which of 5.13 h ± 0.2 during dark period, and REM sleep for 89.9 min ± 6.7, which of 52.8 min ± 4.4 during dark period. The time-course of sleep and waking across the 12 h light:12 h dark was overall inverted to that observed in rats or mice, though with larger amounts of crepuscular activity at light and dark transitions. A dominant crepuscular regulation of sleep and waking persisted under constant darkness, showing the lack of a strong circadian drive in the absence of clock reinforcement by external cues, such as a running wheel. Conservation of the homeostatic regulation was confirmed with the observation of higher delta power following sustained waking periods and a 6-h sleep deprivation, with subsequent decrease during recovery sleep. CONCLUSIONS: Arvicanthis ansorgei is a valid diurnal rodent model for studying the regulatory mechanisms of sleep and so represents a valuable tool for further understanding the nocturnality/diurnality switch.

Defective response inhibition and collicular noradrenaline enrichment in mice with duplicated retinotopic map in the superior colliculus.

The superior colliculus is a hub for multisensory integration necessary for visuo-spatial orientation, control of gaze movements and attention. The multiple functions of the superior colliculus have prompted hypotheses about its involvement in neuropsychiatric conditions, but to date, this topic has not been addressed experimentally. We describe experiments on genetically modified mice, the Isl2-EphA3 knock-in line, that show a well-characterized duplication of the retino-collicular and cortico-collicular axonal projections leading to hyperstimulation of the superior colliculus. To explore the functional impact of collicular hyperstimulation, we compared the performance of homozygous knock-in, heterozygous knock-in and wild-type mice in several behavioral tasks requiring collicular activity. The light/dark box test and Go/No-Go conditioning task revealed that homozygous mutant mice exhibit defective response inhibition, a form of impulsivity. This defect was specific to attention as other tests showed no differences in visually driven behavior, motivation, visuo-spatial learning and sensorimotor abilities among the different groups of mice. Monoamine quantification and gene expression profiling demonstrated a specific enrichment of noradrenaline only in the superficial layers of the superior colliculus of Isl2-EphA3 knock-in mice, where the retinotopy is duplicated, whereas transcript levels of receptors, transporters and metabolic enzymes of the monoaminergic pathway were not affected. We demonstrate that the defect in response inhibition is a consequence of noradrenaline imbalance in the superficial layers of the superior colliculus caused by retinotopic map duplication. Our results suggest that structural abnormalities in the superior colliculus can cause defective response inhibition, a key feature of attention-deficit disorders.

Photoperiod can entrain circannual rhythms in pinealectomized European hamsters.

In mammals, the pineal hormone melatonin is thought to be essential to process environmental photoperiodic information. In this study, we demonstrate in a circannual species, the European hamster Cricetus cricetus, the existence of a melatonin-independent second pathway. In 4 physiological parameters (reproduction, body weight, activity pattern, body temperature), a large majority of pinealectomized European hamsters were entrained to an accelerated photoperiodic regime. It compressed the natural variations in the photoperiod to a 6-month cycle, which allowed us to record up to 6 complete physiological cycles during the life span of the individuals. We show further that whether a pinealectomized animal is able to entrain to changes in the photoperiod is influenced by the season of pinealectomy. The results do not disprove that melatonin is capable of entraining a circannual rhythm, but they show clearly that melatonin is not necessary, demonstrating another melatonin-independent pathway for circannual entrainment by the photoperiod. In view of these new insights, a revision of the original literature revealed that probably the melatonin-independent pathway plays an important role in most circannual mammals but only a minor role in photoperiodic species. Thus, the present work provides also the first evidence for different synchronization mechanisms in photoperiodic and circannual species.

Aging-like circadian disturbances in folate-deficient mice.

The elderly population shows various circadian disturbances, including dampened amplitude of rhythmicity and decreased responsiveness to light. The common poor folate status in the elderly might account for these aging-related circadian disturbances. To test this hypothesis, we investigated whether folate deficiency in mice affects circadian oscillations of the master clock in the suprachiasmatic nuclei, and the shifting responses to light. Mice fed a diet without folate for 6 weeks displayed markedly reduced (4.5-fold) erythrocyte folate concentration and increased (2.3-fold) homocysteinemia compared with control mice. Folate deficiency decreased the circadian amplitude of vasopressin and the clock protein PERIOD 2 (PER2) in the master clock, slowed the rate of re-entrainment of behavioral rhythms after delayed light-dark cycle and reduced light-induced phase-delays, without detectable morphologic changes in the retina, such as the number of melanopsinergic ganglion cells, that might have impaired photodetection. In conclusion, folate deficiency and consecutive hyperhomocysteinemia led to dampened PER2 and vasopressin oscillations in the master clock and reduced responsiveness to photic resetting, which constitute hallmarks of aging effects on circadian rhythmicity.

Relevance of exocytotic glutamate release from retinal glia.

Glial cells release molecules that influence brain development, function, and disease. Calcium-dependent exocytosis has been proposed as potential release mechanism in astroglia, but the physiological relevance of "gliotransmission" in vivo remains controversial. We focused on the impact of glial exocytosis on sensory transduction in the retina. To this end, we generated transgenic mice to block exocytosis by Cre recombinase-dependent expression of the clostridial botulinum neurotoxin serotype B light chain, which cleaves vesicle-associated membrane protein 1-3. Ubiquitous and neuronal toxin expression caused perinatal lethality and a reduction of synaptic transmission thus validating transgene function. Toxin expression in Müller cells inhibited vesicular glutamate release and impaired glial volume regulation but left retinal histology and visual processing unaffected. Our model to study gliotransmission in vivo reveals specific functions of exocytotic glutamate release in retinal glia.

A time-dependent history of mood disorders in a murine model of neuropathic pain.

BACKGROUND: Chronic pain is clinically associated with the development of affective disorders. However, studies in animal models of neuropathic pain are contradictory and the relationship with mood disorders remains unclear. In this study, we aimed to characterize the affective consequences of neuropathic pain over time and to study potential underlying mechanisms. METHODS: Neuropathic pain was induced by inserting a polyethylene cuff around the main branch of the right sciatic nerve in C57BL/6J mice. Anxiety- and depression-related behaviors were assessed over 2 months, using a battery of tests, such as elevated plus maze, marble burying, novelty suppressed feeding, splash test, and forced swimming test. Plasma corticosterone levels were assessed by radioimmunoassay. We also investigated changes in cyclic adenosine monophosphate response element (CRE) activity using CRE-LacZ transgenic mice. RESULTS: Mice developed anxiety-related behavior 4 weeks after induction of the neuropathy, and depression-related behaviors were observed after 6 to 8 weeks. Control and neuropathic mice did not differ for basal or stress-induced levels of corticosterone or for hypothalamic-pituitary-adrenal axis negative feedback. After 8 weeks, the CRE-mediated activity decreased in the outer granule layer of dentate gyrus of neuropathic mice but not in the amygdala or in the anterior cingulate cortex. CONCLUSIONS: Our results demonstrate that the affective consequences of neuropathic pain evolve over time, independently from the hypothalamic-pituitary-adrenal axis, which remains unaffected. CRE-mediated transcription within a limbic structure was altered at later time points of the neuropathy. These experiments provide a preclinical model to study time-dependent development of mood disorders and the underlying mechanism in a neuropathic pain context.

[Mechanisms of structural plasticity associated with photic synchronization of the circadian clock within the suprachiasmatic nucleus]. / Mécanismes de plasticité structurale associés à la synchronisation photique de l'horloge circadienne au sein du noyau suprachiasmatique.

The mammalian circadian clock, whose central component is located in the suprachiasmatic nucleus of the hypothalamus (SCN), orchestrates rhythmic events in metabolism, physiology and behavior. Adaptation of the organism to its environment requires precise adjustment of the clock to the 24 h astronomical time, primarily by the light/dark cycle. Photic synchronization acts on both the molecular loops which trigger circadian oscillations and the phasing of the multiple SCN cellular oscillators whose coordination permits elaboration of the rhythmic message that will be distributed throughout the organism. It is concomitant with structural plastic events characterized by day/night rearrangements of the SCN neuronal-glial network. The two main sources of SCN efferents, namely the VIP (vasoactive intestinal peptide)-synthesizing neurons which are major integrators of photic signals and the AVP (arginine-vasopressin)-synthesizing neurons which are known to importantly contribute to conveying rhythmic messages to brain targets, are involved in these mechanisms. Over the light/dark cycle, they indeed undergo ultrastructural changes in the extent of their membrane coverage by glial, axon terminal and/or somato-dendritic elements. These structural rearrangements appear to be dependent on light entrainment, as the rhythmic expression in SCN of glial fibrillary acidic protein (GFAP), a marker for brain astrocytes whose changing expression has proved to be a reliable index of neuronal-glial plasticity, is disrupted under constant darkness. Glucocorticoid hormones, which are known as important endocrine outputs of the clock, are required to maintain amplitude of the SCN GFAP rhythm to normal values, indicating that they modulate astrocytic plasticity within the SCN and, therefore, nycthemeral changes of the configuration of its neuronal-glial network. The view that such plastic events may subserve synchronization of the clock to the light-dark cycle is reinforced by other data showing that the daily fluctuations of circulating glucocorticoids actually are involved in modulation of light effects, contributing to the resistance of the circadian timing system to variations of the photoperiod. It is thus proposed that the capacity of the clock to integrate cyclic variations of the environment rely on the inherent capacity of the SCN to undergo neuronal-glial plasticity.