Orexin A excites the rat olivary pretectal nucleus via OX2 receptor in a daily manner

Pronounced environmental changes between the day and night forced living organisms to evolve specialised mechanisms organising their daily physiology, named circadian clocks. Currently, it has become clear that the master clock in the suprachiasmatic nuclei of the hypothalamus is not an exclusive brain site to generate daily rhythms. Indeed, several brain areas, including the subcortical visual system have been recently shown to change their neuronal activity across the daily cycle. Here we focus our investigation on the olivary pretectal nucleus (OPN) - a retinorecipient structure primarily involved in the pupillary light reflex. Using the multi-electrode array technology ex vivo we provide evidence for OPN neurons to elevate their firing during the behaviourally quiescent light phase. Additionally, we report the robust sensitivity to orexin A via the identified OX2 receptor in this pretectal centre, with higher responsiveness noted during the night. Interestingly, we likewise report a daily variation in the response to PAC1 receptor activation, with implications for the convergence of orexinergic and visual input on the same OPN neurons. Altogether, our report is first to suggest a daily modulation of the OPN activity via intrinsic and extrinsic mechanisms, organising its temporal physiology.

Neuronal Responses to Short Wavelength Light Deficiency in the Rat Subcortical Visual System

The amount and spectral composition of light changes considerably during the day, with dawn and dusk being the most crucial moments when light is within the mesopic range and short wavelength enriched. It was recently shown that animals use both cues to adjust their internal circadian clock, thereby their behavior and physiology, with the solar cycle. The role of blue light in circadian processes and neuronal responses is well established, however, an unanswered question remains: how do changes in the spectral composition of light (short wavelengths blocking) influence neuronal activity? In this study we addressed this question by performing electrophysiological recordings in image (dorsal lateral geniculate nucleus; dLGN) and non-image (the olivary pretectal nucleus; OPN, the suprachiasmatic nucleus; SCN) visual structures to determine neuronal responses to spectrally varied light stimuli. We found that removing short-wavelength from the polychromatic light (cut off at 525 nm) attenuates the most transient ON and sustained cells in the dLGN and OPN, respectively. Moreover, we compared the ability of different types of sustained OPN neurons (either changing or not their response profile to filtered polychromatic light) to irradiance coding, and show that both groups achieve it with equal efficacy. On the other hand, even very dim monochromatic UV light (360 nm; log 9.95 photons/cm2/s) evokes neuronal responses in the dLGN and SCN. To our knowledge, this is the first electrophysiological experiment supporting previous behavioral findings showing visual and circadian functions disruptions under short wavelength blocking environment. The current results confirm that neuronal activity in response to polychromatic light in retinorecipient structures is affected by removing short wavelengths, however, with type and structure – specific action. Moreover, they show that rats are sensitive to even very dim UV light.

Genetic mapping of etiologic brain cell types for obesity

The underlying cell types mediating predisposition to obesity remain largely obscure. Here, we integrated recently published single-cell RNA-sequencing (scRNA-seq) data from 727 peripheral and nervous system cell types spanning 17 mouse organs with body mass index (BMI) genome-wide association study (GWAS) data from >457,000 individuals. Developing a novel strategy for integrating scRNA-seq data with GWAS data, we identified 26, exclusively neuronal, cell types from the hypothalamus, subthalamus, midbrain, hippocampus, thalamus, cortex, pons, medulla, pallidum that were significantly enriched for BMI heritability (p<1.6×10−4). Using genes harboring coding mutations associated with obesity, we replicated midbrain cell types from the anterior pretectal nucleus and periaqueductal gray (p<1.2×10−4). Together, our results suggest that brain nuclei regulating integration of sensory stimuli, learning and memory are likely to play a key role in obesity and provide testable hypotheses for mechanistic follow-up studies.

The Olivary Pretectal Nucleus Receives Visual Input of High Spatial Resolution

In the mouse, retinal output is computed by over 40 distinct types of retinal ganglion cells (RGCs) (Baden et al., 2016). Determining which of these many RGC types project to a retinorecipient region is a key step in elucidating the role that region plays in visually-mediated behaviors. Combining retrograde viral tracing and single-cell electrophysiology, we identify the RGC types which project to the olivary pretectal nucleus (OPN), a major visual structure. We find that retinal input to the OPN consists of a variety of intrinsically-photosensitive and conventional RGC types, the latter a diverse set of mostly ON RGCs. Surprisingly, while the OPN is most associated with the pupillary light reflex (PLR) pathway, requiring information about absolute luminance, we show that the majority of the retinal input to the OPN is from single cell type which transmits information unrelated to luminance. This ON-transient RGC accounts for two-thirds of the input to the OPN, and responds to small objects across a wide range of speeds. This finding suggests a role for the OPN in visually-mediated behaviors beyond the PLR.Significance statementThe olivary pretectal nucleus is a midbrain structure which receives direct input from retinal ganglion cells (RGC), and modulates pupil diameter in response to changing absolute light level. In the present study, we combine viral tracing and electrophysiology to identify the RGC types which project to the OPN. Surprisingly, the majority of its input comes from a single type which does not encode absolute luminance, but instead responds to small objects across a wide range of speeds. These findings are consistent with a role for the OPN apart from pupil control and suggest future experiments to elucidate its full role in visually-mediated behavior.

Mapping heritability of obesity by brain cell types

The underlying cell types mediating predisposition to obesity remain largely obscure. Here we first integrated recently published single-cell RNA-sequencing (scRNA-seq) data from >380 peripheral and nervous system cell types spanning 19 mouse organs with body mass index (BMI) genome-wide association study (GWAS) data from >450,000 individuals. Leveraging a novel strategy for integrating scRNA-seq data with GWAS data, we identified 22, exclusively neuronal, cell types from the subthalamus, midbrain, hippocampus, thalamus, cortex, pons, medulla, pallidum that were significantly enriched for BMI heritability (P<1.6×10-4). Using genes harboring coding mutations leading to syndromic forms of obesity, we replicate four midbrain cell types from the anterior pretectal nucleus, superior nucleus, periaqueductal gray and pallidum (P<1.7×10-4). Testing an additional set of 347 hypothalamic cell types, ventromedial hypothalamic steroidogenic-factor 1 (SF1) and cholecystokinin b receptor (CCKBR)-expressing neurons (P=4.9×10-5) previously implicated in energy homeostasis and glucose control and three cell types from the preoptic area of the hypothalamus and the lateral hypothalamus enriched for BMI GWAS associations (P<4.9×10-5). Together, our results suggest brain nuclei regulating integration of sensory stimuli, learning and memory are likely to play a key role in obesity and provide testable hypotheses for mechanistic follow-up studies.

Pupillary light reflex circuits in the macaque monkey: the preganglionic Edinger–Westphal nucleus

The motor outflow for the pupillary light reflex originates in the preganglionic motoneuron subdivision of the Edinger–Westphal nucleus (EWpg), which also mediates lens accommodation. Despite their importance for vision, the morphology, ultrastructure and luminance-related inputs of these motoneurons have not been fully described in primates. In macaque monkeys, we labeled EWpg motoneurons from ciliary ganglion and orbital injections. Both approaches indicated preganglionic motoneurons occupy an EWpg organized as a unitary, ipsilateral cell column. When tracers were placed in the pretectal complex, labeled terminals targeted the ipsilateral EWpg and reached contralateral EWpg by crossing both above and below the cerebral aqueduct. They also terminated in the lateral visceral column, a ventrolateral periaqueductal gray region containing neurons projecting to the contralateral pretectum. Combining olivary pretectal and ciliary ganglion injections to determine whether a direct pupillary light reflex projection is present revealed a labeled motoneuron subpopulation that displayed close associations with labeled pretectal terminal boutons. Ultrastructurally, this subpopulation received synaptic contacts from labeled pretectal terminals that contained numerous clear spherical vesicles, suggesting excitation, and scattered dense-core vesicles, suggesting peptidergic co-transmitters. A variety of axon terminal classes, some of which may serve the near response, synapsed on preganglionic motoneurons. Quantitative analysis indicated that pupillary motoneurons receive more inhibitory inputs than lens motoneurons. To summarize, the pupillary light reflex circuit utilizes a monosynaptic, excitatory, bilateral pretectal projection to a distinct subpopulation of EWpg motoneurons. Furthermore, the interconnections between the lateral visceral column and olivary pretectal nucleus may provide pretectal cells with bilateral retinal fields.

Superior Colliculus Lesions Lead to Disrupted Responses to Light in Diurnal Grass Rats (Arvicanthis niloticus)

The circadian system regulates daily rhythms of physiology and behavior. Although extraordinary advances have been made to elucidate the brain mechanisms underlying the circadian system in nocturnal species, less is known in diurnal species. Recent studies have shown that retinorecipient brain areas such as the intergeniculate leaflet (IGL) and olivary pretectal nucleus (OPT) are critical for the display of normal patterns of daily activity in diurnal grass rats ( Arvicanthis niloticus). Specifically, grass rats with IGL and OPT lesions respond to light in similar ways to intact nocturnal animals. Importantly, both the IGL and OPT project to one another in nocturnal species, and there is evidence that these 2 brain regions also project to the superior colliculus (SC). The SC receives direct retinal input, is involved in the triggering of rapid eye movement sleep in nocturnal rats, and is disproportionately large in the diurnal grass rat. The objective of the current study was to use diurnal grass rats to test the hypothesis that the SC is critical for the expression of diurnal behavior and physiology. We performed bilateral electrolytic lesions of the SC in female grass rats to examine behavioral patterns and acute responses to light. Most grass rats with SC lesions expressed significantly reduced activity in the presence of light. Exposing these grass rats to constant darkness reinstated activity levels during the subjective day, suggesting that light masks their ability to display a diurnal activity profile in 12:12 LD. Altogether, our data suggest that the SC is critical for maintaining normal responses to light in female grass rats.