non-invasive-strategies-for-chronic-manipulation-dreadd-controlled

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by ...

National Institute of Mental Health

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice.

Many biochemical, physiological, and behavioral processes such as glucose metabolism, body temperature, and sleep-wake cycles show regular daily rhythms. These circadian rhythms are adjusted to the environmental light-dark cycle by a central pacemaker located in the suprachiasmatic nucleus (SCN) in order for the processes to occur at appropriate times of day. Here, we investigated the expression and function of a synaptic organizing protein, C1QL3, in the SCN. We found that C1ql3 is robustly expressed in the SCN. C1ql3 knockout mice have a reduced density of excitatory synapses in the SCN. In addition, these mice exhibited less consolidated activity to the active portions of the day and period lengthening following a 15-minute phase-delaying light pulse. These data identify C1QL3 as a signaling molecule that is highly expressed in SCN neurons, where it contributes to the formation and/or maintenance of glutamatergic synapses and plays a role in circadian behaviors, which may include circadian aftereffects.

Anatomical and Behavioral Investigation of C1ql3 in the Mouse Suprachiasmatic Nucleus.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

Artificial light in modern society has led to the ubiquity of light exposure at night as individuals work night shifts and use light-emitting electronic devices before bedtime. These aberrant light conditions have detrimental consequences on cognitive and mental health, compelling the need to understand the mechanisms by which light affects brain functions. Although it was believed that aberrant light impairs health by first disrupting circadian rhythms and sleep, we showed that chronic exposure to a light cycle termed T7 (3.5 h of darkness, 3.5 h of light) caused mood and learning dysfunction in adult mice independent of sleep deprivation or circadian arrhythmicity, suggesting the direct effects of aberrant light on brain function. However, the mechanisms by which light directly causes mood and learning dysfunction remain poorly understood. In this study, we sought to determine whether exposure to the T7 cycle disrupts adult hippocampal neurogenesis, given that suppressed neurogenesis has been correlated with mood and learning dysfunction. After exposing adult mice to the T7 light cycle, we analyzed adult hippocampal neurogenesis by examining cellular proliferation and number of adult-born neurons. Contrary to our hypothesis that T7 would suppress neurogenesis, we found that adult mice exposed to 2 or 10 weeks of the T7 light cycle did not exhibit deficits in hippocampal neurogenesis. Our findings suggest that the direct effects of light on mood and learning do not depend on adult hippocampal neurogenesis.

Chronic Circadian Misalignment without Circadian Arrhythmicity or Sleep Deprivation Does Not Impair Adult Hippocampal Neurogenesis.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are an important tool for modulating and understanding neural circuits. Depending on the DREADD system used, DREADD-targeted neurons can be activated or repressed  following a dose of the DREADD agonist clozapine--oxide (CNO). Because DREADD experiments often involve behavioral assays, the method of CNO delivery is important. Currently, the most common delivery method is intraperitoneal (IP) injection. IP injection is both a fast and reliable technique, but it is painful and stressful particularly when many injections are required. We sought an alternative CNO delivery paradigm, which would retain the speed and reliability of IP injections without being as invasive. Here, we show that CNO can be effectively delivered topically via eye-drops. Eye-drops robustly activated DREADD-expressing neurons in the brain and peripheral tissues and does so at the same dosages as IP injection. Eye-drops provide an easier, less invasive and less stressful method for activating DREADDs .

Eye-Drops for Activation of DREADDs.

To develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies.

A Robust Optomotor Assay for Assessing the Efficacy of Optogenetic Tools for Vision Restoration.

Newborn neurons follow molecular cues to reach their final destination, but whether early life experience influences lamination remains largely unexplored. As light is among the first stimuli to reach the developing nervous system via intrinsically photosensitive retinal ganglion cells (ipRGCs), we asked whether ipRGCs could affect lamination in the developing mouse retina. We show here that ablation of ipRGCs causes cone photoreceptors to mislocalize at different apicobasal positions in the retina. This effect is partly mediated by light-evoked activity in ipRGCs, as dark rearing or silencing of ipRGCs leads a subset of cones to mislocalize. Furthermore, ablation of ipRGCs alters the cone transcriptome and decreases expression of the dopamine receptor D4, while injection of L-DOPA or D4 receptor agonist rescues the displaced cone phenotype observed in dark-reared animals. These results show that early light-mediated activity in ipRGCs influences neuronal lamination and identify ipRGC-elicited dopamine release as a mechanism influencing cone position.

Melanopsin Retinal Ganglion Cells Regulate Cone Photoreceptor Lamination in the Mouse Retina.

Light exerts a range of powerful biological effects beyond image vision, including mood and learning regulation. While the source of photic information affecting mood and cognitive functions is well established, viz. intrinsically photosensitive retinal ganglion cells (ipRGCs), the central mediators are unknown. Here, we reveal that the direct effects of light on learning and mood utilize distinct ipRGC output streams. ipRGCs that project to the suprachiasmatic nucleus (SCN) mediate the effects of light on learning, independently of the SCN's pacemaker function. Mood regulation by light, on the other hand, requires an SCN-independent pathway linking ipRGCs to a previously unrecognized thalamic region, termed perihabenular nucleus (PHb). The PHb is integrated in a distinctive circuitry with mood-regulating centers and is both necessary and sufficient for driving the effects of light on affective behavior. Together, these results provide new insights into the neural basis required for light to influence mood and learning.

Light Affects Mood and Learning through Distinct Retina-Brain Pathways.

The master circadian pacemaker in mammals resides in the hypothalamic suprachiasmatic nuclei (SCN) and is synchronized to ambient light/dark cycles (i.e., photoentrainment). Melanopsin (Opn4) and classical rod-cone photoreceptors are believed to provide all the photic input necessary for circadian photoentrainment. Although the UVA-sensitive photopigment Opn5 is known to be expressed in retinal ganglion cells, its physiological role remains unclear and a potential role for Opn5 in the photoentrainment of the master clock has not been addressed. Here we report impaired photoentrainment and phase shifting to UVA light in Opn5-null mice. However, triple-knockout mice lacking all known functional circadian photoreceptors (i.e., rods, cones, and melanopsin) failed to entrain to UVA-light/dark cycles, despite the presence of Opn5, demonstrating that Opn5 alone is not sufficient for photoentrainment of the SCN clock. Since Opn5 is involved in the regulation of the retinal circadian clock, disrupted retinal function may cause impaired circadian photoentrainment in Opn5-null mice.

Impaired Circadian Photoentrainment in Opn5-Null Mice.

The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone-degrading and -activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.

Samer Hattar

Section on Light and Circadian Rhythms (SLCR)

National Institute of Mental Health

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity