Tara A. LeGates

Melanopsin-Expressing Retinal Ganglion-Cell Photoreceptors: Cellular Diversity and Role in Pattern Vision

Using the photopigment melanopsin, intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light to drive circadian clock resetting and pupillary constriction. We now report that ipRGCs are more abundant and diverse than previously appreciated, project more widely within the brain, and can support spatial visual perception. A Cre-based melanopsin reporter mouse line revealed at least five subtypes of ipRGCs with distinct morphological and physiological characteristics. Collectively, these cells project beyond the known brain targets of ipRGCs to heavily innervate the superior colliculus and dorsal lateral geniculate nucleus, retinotopically organized nuclei mediating object localization and discrimination. Mice lacking classical rod-cone photoreception, and thus entirely dependent on melanopsin for light detection, were able to discriminate grating stimuli from equiluminant gray and had measurable visual acuity. Thus, nonclassical retinal photoreception occurs within diverse cell types and influences circuits and functions encompassing luminance as well as spatial information.

Aberrant light directly impairs mood and learning through melanopsin-expressing neurons

The daily solar cycle allows organisms to synchronize their circadian rhythms and sleep–wake cycles to the correct temporal niche. Changes in day-length, shift-work, and transmeridian travel lead to mood alterations and cognitive function deficits. Sleep deprivation and circadian disruption underlie mood and cognitive disorders associated with irregular light schedules. Whether irregular light schedules directly affect mood and cognitive functions in the context of normal sleep and circadian rhythms remains unclear. Here we show, using an aberrant light cycle that neither changes the amount and architecture of sleep nor causes changes in the circadian timing system, that light directly regulates mood-related behaviours and cognitive functions in mice. Animals exposed to the aberrant light cycle maintain daily corticosterone rhythms, but the overall levels of corticosterone are increased. Despite normal circadian and sleep structures, these animals show increased depression-like behaviours and impaired hippocampal long-term potentiation and learning. Administration of the antidepressant drugs fluoxetine or desipramine restores learning in mice exposed to the aberrant light cycle, suggesting that the mood deficit precedes the learning impairments. To determine the retinal circuits underlying this impairment of mood and learning, we examined the behavioural consequences of this light cycle in animals that lack intrinsically photosensitive retinal ganglion cells. In these animals, the aberrant light cycle does not impair mood and learning, despite the presence of the conventional retinal ganglion cells and the ability of these animals to detect light for image formation. These findings demonstrate the ability of light to influence cognitive and mood functions directly through intrinsically photosensitive retinal ganglion cells.

Rods-cones and melanopsin detect light and dark to modulate sleep independent of image formation

Light detected in the retina modulates several physiological processes including circadian photo-entrainment and pupillary light reflex. Intrinsically photosensitive retinal ganglion cells (ipRGCs) convey rod-cone and melanopsin-driven light input to the brain. Using EEGs and electromyograms, we show that acute light induces sleep in mice during their nocturnal active phase whereas acute dark awakens mice during their diurnal sleep phase. We used retinal mutant mouse lines that lack (i) the ipRGCs, (ii) the photo-transduction pathways of rods and cones, or (iii) the melanopsin protein and showed that the influence of light and dark on sleep requires both rod-cone and melanopsin signaling through ipRGCs and is independent of image formation. We further show that, although acute light pulses overcome circadian and homeostatic drives for sleep, upon repeated light exposures using a 3.5 h/3.5 h light/dark cycle, the circadian and homeostatic drives override the light input. Thus, in addition to their known role in aligning circadian physiology with day and night, ipRGCs also relay light and dark information from both rod-cone and melanopsin-based pathways to modulate sleep and wakefulness.

Lhx1 Controls Terminal Differentiation and Circadian Function of the Suprachiasmatic Nucleus

SUMMARY Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. Here, we show that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. Our results identify a factor required for SCN terminal differentiation. In addition, our in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillators role in physiology and cognition.

Wnt5a is essential for hippocampal dendritic maintenance and spatial learning and memory in adult mice

Significance The maintenance of neuronal morphology in the adult brain is an understudied area. Here, using tissue-specific deletion in mice, we reveal Wnt5a, a member of the Wnt family of developmental morphogens, as an essential factor for the long-term stability of dendritic architecture in the adult hippocampus. Wnt5a influences synaptic plasticity and related cognitive functions in the mature hippocampus through CaMKII-mediated signaling, Rac1-dependent actin dynamics, and cyclic AMP-responsive element binding-mediated NMDA receptor biosynthesis. In the long-term, Wnt5a-mediated regulation of cytoskeletal signaling and excitatory synaptic transmission is responsible for the maintenance of dendritic arbors and spines in adult CA1 pyramidal neurons. These findings provide insight into the poorly understood structural maintenance mechanisms that exist in the adult brain. Stability of neuronal connectivity is critical for brain functions, and morphological perturbations are associated with neurodegenerative disorders. However, how neuronal morphology is maintained in the adult brain remains poorly understood. Here, we identify Wnt5a, a member of the Wnt family of secreted morphogens, as an essential factor in maintaining dendritic architecture in the adult hippocampus and for related cognitive functions in mice. Wnt5a expression in hippocampal neurons begins postnatally, and its deletion attenuated CaMKII and Rac1 activity, reduced GluN1 glutamate receptor expression, and impaired synaptic plasticity and spatial learning and memory in 3-mo-old mice. With increased age, Wnt5a loss caused progressive attrition of dendrite arbors and spines in Cornu Ammonis (CA)1 pyramidal neurons and exacerbated behavioral defects. Wnt5a functions cell-autonomously to maintain CA1 dendrites, and exogenous Wnt5a expression corrected structural anomalies even at late-adult stages. These findings reveal a maintenance factor in the adult brain, and highlight a trophic pathway that can be targeted to ameliorate dendrite loss in pathological conditions.

Abnormal wake/sleep pattern in a novel gain-of-function model of DISC1

Sleep disturbances are common in psychiatric disorders, but the causal relationship between the two and the underlying genetic factors is unclear. The DISC1 gene is strongly linked to mood disorders and schizophrenia in a Scottish pedigree. In an earlier study we found a sleep homeostasis disturbance in a Drosophila model overexpressing wild-type human DISC1. Here we aimed to explore the relationship between sleep and the DISC1 gene in a mammalian model, a novel transgenic mouse model expressing full-length human DISC1. We assessed circadian rhythms by monitoring wheel running activity under normal 24-h light:dark conditions and in constant darkness and found the DISC1 mice to have normal circadian photoentrainment and normal intrinsic circadian period. We also assessed sleep duration and quality in the DISC1 mice and found that they were awake longer than wild-type controls at baseline with a tendency for lower rebound of delta activity during recovery from a short sleep deprivation. Thus we suggest that DISC1 may be involved in sleep regulation.

Accelerated re-entrainment to advanced light cycles in BALB/cJ mice

Circadian rhythms in mammals are coordinated by the suprachiasmatic nuclei (SCN) of the hypothalamus, which are most potently synchronized to environmental light-dark cycles. Large advances in the light-dark cycle typically yield gradual advances in activity rhythms on the order of 1-2h per day until re-entrainment is complete due to limitations on the circadian system which are not yet understood. In humans, this delay until re-entrainment is accomplished is experienced as jetlag, with accompanying symptoms of malaise, decreased cognitive performance, sleep problems and gastrointestinal distress. In these experiments, locomotor rhythms of BALB/cJ mice monitored by running wheels were shown to re-entrain to large 6- or 8-hour shifts of the light-dark cycle within 1-2 days, as opposed to the 5-7 days required for C57BL/6J mice. A single-day 6-hour advance of the LD cycle followed by release to constant darkness yielded similar phase shifts, demonstrating that exaggerated re-entrainment is not explained by masking of activity by the light-dark cycle. Responses in BALB/cJ mice were similar when monitored instead by motion detectors, indicating that wheel-running exercise does not influence the magnitude of responses. Neither brief (15 min) light exposure late during subjective nighttime nor 6-hour delays of the light-dark cycle produced exaggerated locomotor phase shifts, indicating that BALB/cJ mice do not merely experience enhanced sensitivity to light. Fos protein was expressed in cells of the SCN following acute light exposure at ZT10 of their previous light-dark cycle, a normally non-responsive time in the circadian cycle, but only in BALB/cJ (and not C57BL/6J) mice that had been subjected two days earlier to a single-day 6-hour advance of the light-dark cycle, indicating that their SCN had been advanced by that treatment. BALB/cJ mice may thus serve as a useful comparative model for studying molecular and physiological processes that limit responsiveness of circadian clocks to photic input.

An LHX1-Regulated Transcriptional Network Controls Sleep/Wake Coupling and Thermal Resistance of the Central Circadian Clockworks

The suprachiasmatic nucleus (SCN) is the central circadian clock in mammals. It is entrained by light but resistant to temperature shifts that entrain peripheral clocks [1-5]. The SCN expresses many functionally important neuropeptides, including vasoactive intestinal peptide (VIP), which drives light entrainment, synchrony, and amplitude of SCN cellular clocks and organizes circadian behavior [5-16]. The transcription factor LHX1 drives SCN Vip expression, and cellular desynchrony in Lhx1-deficient SCN largely results from Vip loss [17, 18]. LHX1 regulates many genes other than Vip, yet activity rhythms in Lhx1-deficient mice are similar to Vip-/- mice under light-dark cycles and only somewhat worse in constant conditions. We suspected that LHX1 targets other than Vip have circadian functions overlooked in previous studies. In this study, we compared circadian sleep and temperature rhythms of Lhx1- and Vip-deficient mice and found loss of acute light control of sleep in Lhx1 but not Vip mutants. We also found loss of circadian resistance to fever in Lhx1 but not Vip mice, which was partially recapitulated by heat application to cultured Lhx1-deficient SCN. Having identified VIP-independent functions of LHX1, we mapped the VIP-independent transcriptional network downstream of LHX1 and a largely separable VIP-dependent transcriptional network. The VIP-independent network does not affect core clock amplitude and synchrony, unlike the VIP-dependent network. These studies identify Lhx1 as the first gene required for temperature resistance of the SCN clockworks and demonstrate that acute light control of sleep is routed through the SCN and its immediate output regions.

Measuring circadian and acute light responses in mice using wheel running activity.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day). They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

Long-Term Potentiation Requires a Rapid Burst of Dendritic Mitochondrial Fission during Induction

Synaptic transmission is bioenergetically demanding, and the diverse processes underlying synaptic plasticity elevate these demands. Therefore, mitochondrial functions, including ATP synthesis and Ca2+ handling, are likely essential for plasticity. Although axonal mitochondria have been extensively analyzed, LTP is predominantly induced postsynaptically, where mitochondria are understudied. Additionally, though mitochondrial fission is essential for their function, signaling pathways that regulate fission in neurons remain poorly understood. We found that NMDAR-dependent LTP induction prompted a rapid burst of dendritic mitochondrial fission and elevations of mitochondrial matrix Ca2+. The fission burst was triggered by cytosolic Ca2+ elevation and required CaMKII, actin, and Drp1, as well as dynamin 2. Preventing fission impaired mitochondrial matrix Ca2+ elevations, structural LTP in cultured neurons, and electrophysiological LTP in hippocampal slices. These data illustrate a novel pathway whereby synaptic activity controls mitochondrial fission and show that dynamic control of fission regulates plasticity induction, perhaps by modulating mitochondrial Ca2+ handling.