Metabolic cues impact non-oscillatory intergeniculate leaflet and ventral lateral geniculate nucleus: standard versus high-fat diet comparative study.

The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) are subcortical structures involved in entrainment of the brain's circadian system to photic and non-photic (e.g. metabolic and arousal) cues. Both receive information about environmental light from photoreceptors, exhibit infra-slow oscillations (ISO) in vivo, and connect to the master circadian clock. Although current evidence demonstrates that the IGL/VLG communicate metabolic information and are crucial for entrainment of circadian rhythms to time-restricted feeding, their sensitivity to food intake-related peptides has not been investigated yet. We examined the effect of metabolically relevant peptides on the spontaneous activity of IGL/VLG neurons. Using ex vivo and in vivo electrophysiological recordings as well as in situ hybridisation, we tested potential sensitivity of the IGL/VLG to anorexigenic and orexigenic peptides, such as cholecystokinin, glucagon-like peptide 1, oxyntomodulin, peptide YY, orexin A and ghrelin. We explored neuronal responses to these drugs during day and night, and in standard vs. high-fat diet conditions. We found that IGL/VLG neurons responded to all the substances tested, except peptide YY. Moreover, more neurons responded to anorexigenic drugs at night, while a high-fat diet affected the IGL/VLG sensitivity to orexigenic peptides. Interestingly, ISO neurons responded to light and orexin A, but did not respond to the other food intake-related peptides. In contrast, non-ISO cells were activated by metabolic peptides, with only some being responsive to light. Our results show for the first time that peptides involved in the body's energy homeostasis stimulate the thalamus and suggest functional separation of the IGL/VLG cells. KEY POINTS: The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) of the rodent thalamus process various signals and participate in circadian entrainment. In both structures, cells exhibiting infra-slow oscillatory activity as well as non-rhythmically firing neurons being observed. Here, we reveal that only one of these two groups of cells responds to anorexigenic (cholecystokinin, glucagon-like peptide 1 and oxyntomodulin) and orexigenic (ghrelin and orexin A) peptides. Neuronal responses vary depending on the time of day (day vs. night) and on the diet (standard vs. high-fat diet). Additionally, we visualised receptors to the tested peptides in the IGL/VLG using in situ hybridisation. Our results suggest that two electrophysiologically different subpopulations of IGL/VLG neurons are involved in two separate functions: one related to the body's energy homeostasis and one associated with the subcortical visual system.

High-Fat-Diet-Evoked Disruption of the Rat Dorsomedial Hypothalamic Clock Can Be Prevented by Restricted Nighttime Feeding.

Obesity is a growing health problem for modern society; therefore, it has become extremely important to study not only its negative implications but also its developmental mechanism. Its links to disrupted circadian rhythmicity are indisputable but are still not well studied on the cellular level. Circadian food intake and metabolism are controlled by a set of brain structures referred to as the food-entrainable oscillator, among which the dorsomedial hypothalamus (DMH) seems to be especially heavily affected by diet-induced obesity. In this study, we evaluated the effects of a short-term high-fat diet (HFD) on the physiology of the male rat DMH, with special attention to its day/night changes. Using immunofluorescence and electrophysiology we found that both cFos immunoreactivity and electrical activity rhythms become disrupted after as few as 4 weeks of HFD consumption, so before the onset of excessive weight gain. This indicates that the DMH impairment is a possible factor in obesity development. The DMH cellular activity under an HFD became increased during the non-active daytime, which coincides with a disrupted rhythm in food intake. In order to explore the relationship between them, a separate group of rats underwent time-restricted feeding with access to food only during the nighttime. Such an approach completely abolished the disruptive effects of the HFD on the DMH clock, confirming its dependence on the feeding schedule of the animal. The presented data highlight the importance of a temporally regulated feeding pattern on the physiology of the hypothalamic center for food intake and metabolism regulation, and propose time-restricted feeding as a possible prevention of the circadian dysregulation observed under an HFD.

A Role for Thalamic Projection GABAergic Neurons in Circadian Responses to Light.

The thalamus is an important hub for sensory information and participates in sensory perception, regulation of attention, arousal and sleep. These functions are executed primarily by glutamatergic thalamocortical neurons that extend axons to the cortex and initiate cortico-thalamocortical connectional loops. However, the thalamus also contains projection GABAergic neurons that do not extend axons toward the cortex. Here, we have harnessed recent insight into the development of the intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (LGv) to specifically target and manipulate thalamic projection GABAergic neurons in female and male mice. Our results show that thalamic GABAergic neurons of the IGL and LGv receive retinal input from diverse classes of retinal ganglion cells (RGCs) but not from the M1 intrinsically photosensitive retinal ganglion cell (ipRGC) type. We describe the synergistic role of the photoreceptor melanopsin and the thalamic neurons of the IGL/LGv in circadian entrainment to dim light. We identify a requirement for the thalamic IGL/LGv neurons in the rapid changes in vigilance states associated with circadian light transitions.SIGNIFICANCE STATEMENT The intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (LGv) are part of the extended circadian system and mediate some nonimage-forming visual functions. Here, we show that each of these structures has a thalamic (dorsal) as well as prethalamic (ventral) developmental origin. We map the retinal input to thalamus-derived cells in the IGL/LGv complex and discover that while RGC input is dominant, this is not likely to originate from M1ipRGCs. We implicate thalamic cells in the IGL/LGv in vigilance state transitions at circadian light changes and in overt behavioral entrainment to dim light, the latter exacerbated by concomitant loss of melanopsin expression.

Rhythmic neuronal activities of the rat nucleus of the solitary tract are impaired by high-fat diet - implications for daily control of satiety.

Temporal partitioning of daily food intake is crucial for survival and involves the integration of internal circadian states and external influences such as the light-dark cycle and dietary composition. These intrinsic and extrinsic factors are interdependent with misalignment of circadian rhythms promoting body weight gain, while consumption of a calorie-dense diet elevates the risk of obesity and blunts circadian rhythms. Recently, we defined the circadian properties of the dorsal vagal complex of the brainstem, a structure implicated in the control of food intake and autonomic tone, but whether and how 24 h rhythms in this area are influenced by diet remains unresolved. Here we focused on a key structure of this complex, the nucleus of the solitary tract (NTS). We used a combination of immunohistochemical and electrophysiological approaches together with daily monitoring of body weight and food intake to interrogate how the neuronal rhythms of the NTS are affected by a high-fat diet. We report that short-term consumption of a high-fat diet increases food intake during the day and blunts NTS daily rhythms in neuronal discharge. Additionally, we found that a high-fat diet dampens NTS responsiveness to metabolic neuropeptides, and decreases orexin immunoreactive fibres in this structure. These alterations occur without prominent body weight gain, suggesting that a high-fat diet acts initially to reduce activity in the NTS to disinhibit mechanisms that suppress daytime feeding. KEY POINTS: The dorsal vagal complex of the rodent hindbrain possesses intrinsic circadian timekeeping mechanisms In particular, the nucleus of the solitary tract (NTS) is a robust circadian oscillator, independent of the master suprachiasmatic clock Here, we reveal that rat NTS neurons display timed daily rhythms in their neuronal activity and responsiveness to ingestive cues These daily rhythms are blunted or eliminated by a short-term high-fat diet, together with increased consumption of calories during the behaviourally quiescent day Our results help us better understand the circadian control of satiety by the brainstem and its malfunctioning under a high-fat diet.

Daily changes in neuronal activities of the dorsal motor nucleus of the vagus under standard and high-fat diet.

KEY POINTS: Recently, we found that the dorsal vagal complex displays autonomous circadian timekeeping properties  The dorsal motor nucleus of the vagus (DMV) is an executory part of this complex - a source of parasympathetic innervation of the gastrointestinal tract  Here, we reveal daily changes in the neuronal activities of the rat DMV, including firing rate, intrinsic excitability and synaptic input - all of these peaking in the late day  Additionally, we establish that short term high-fat diet disrupts these daily rhythms, boosting the variability in the firing rate, but blunting the DMV responsiveness to ingestive cues  These results help us better understand daily control over parasympathetic outflow and provide evidence on its dependence on the high-fat diet ABSTRACT: The suprachiasmatic nuclei (SCN) of the hypothalamus function as the brain's primary circadian clock, but circadian clock genes are also rhythmically expressed in several extra-SCN brain sites where they can exert local temporal control over physiology and behaviour. Recently, we found that the hindbrain dorsal vagal complex possesses strong daily timekeeping capabilities, with the area postrema and nucleus of the solitary tract exhibiting the most robust clock properties. The possibility that the executory part of this complex - the dorsal motor nucleus of the vagus (DMV) - also exhibits daily changes has not been extensively studied. The DMV is the source of vagal efferent motoneurons that regulate gastric motility and emptying and consequently influence meal size and energy homeostasis. We used a combination of multi-channel electrophysiology and patch clamp recordings to gain insight into effects of time of day and diet on these DMV cells. We found that DMV neurons increase their spontaneous activity, excitability and responsiveness to metabolic neuromodulators at late day and this was paralleled with an enhanced synaptic input to these neurons. A high-fat diet typically damps circadian rhythms, but we found that consumption of a high-fat diet paradoxically amplified daily variation of DMV neuronal activity, while blunting the neurons responsiveness to metabolic neuromodulators. In summary, we show for the first time that DMV neural activity changes with time of day, with this temporal variation modulated by diet. These findings have clear implications for our understanding of the daily control of vagal efferents and parasympathetic outflow.

Intrinsic circadian timekeeping properties of the thalamic lateral geniculate nucleus.

Circadian rhythmicity in mammals is sustained by the central brain clock-the suprachiasmatic nucleus of the hypothalamus (SCN), entrained to the ambient light-dark conditions through a dense retinal input. However, recent discoveries of autonomous clock gene expression cast doubt on the supremacy of the SCN and suggest circadian timekeeping mechanisms devolve to local brain clocks. Here, we use a combination of molecular, electrophysiological, and optogenetic tools to evaluate intrinsic clock properties of the main retinorecipient thalamic center-the lateral geniculate nucleus (LGN) in male rats and mice. We identify the dorsolateral geniculate nucleus as a slave oscillator, which exhibits core clock gene expression exclusively in vivo. Additionally, we provide compelling evidence for intrinsic clock gene expression accompanied by circadian variation in neuronal activity in the intergeniculate leaflet and ventrolateral geniculate nucleus (VLG). Finally, our optogenetic experiments propose the VLG as a light-entrainable oscillator, whose phase may be advanced by retinal input at the beginning of the projected night. Altogether, this study for the first time demonstrates autonomous timekeeping mechanisms shaping circadian physiology of the LGN.

Daily coordination of orexinergic gating in the rat superior colliculus-Implications for intrinsic clock activities in the visual system.

The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence elucidates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.

Short Wavelengths Contribution to Light-induced Responses and Irradiance Coding in the Rat Dorsal Lateral Geniculate Nucleus - An In vivo Electrophysiological Approach.

The dorsal lateral geniculate nucleus (dLGN) is the main neuronal station en route to higher visual areas. It receives information about environmental light from retinal photoreceptors whose sensitivity peaks are distributed across a visible spectrum. Here, using electrophysiological multichannel recordings in vivo combined with different light stimulations, we investigated short wavelength contribution to the dLGN responses to light and irradiance coding. The results showed that the majority of dLGN cells responded evenly to almost all wavelengths from the 340 to 490 nm spectrum; however, some cells representing extremes of unimodal distribution of Blue-UV index were specialised in the reception of blue or UV light. Moreover, by using alternate yellow and monochromatic light stimuli from blue - UV range, we also assessed the relative spectral contribution to rat dLGN responses to light. Finally, we observed no clear changes in the irradiance coding property of short wavelength-deficient light stimuli, however we noticed a distortion of the coding curves manifested by a significant drop in measure of fit after using short wavelength blocking filter. In conclusion, our data provide the first electrophysiological report on dLGN short wavelength-induced responses under changing light conditions and suggest the presence of colour opponent cells in the rat dLGN.

Circadian actions of orexins on the retinorecipient lateral geniculate complex in rat.

KEY POINTS: Rhythmic processes in living organisms are controlled by biological clocks. The orexinergic system of the lateral hypothalamus carries circadian information to provide arousal for the brain during the active phase. Here, we show that orexins exert an excitatory action in three parts of the lateral geniculate nucleus (LGN), in particular upon directly retinorecipient neurons in the non-image forming visual structures. We provide evidence for the high nocturnal levels of orexins with stable circadian expression of predominant orexin receptor 2 in the LGN. Our data additionally establish the convergence of orexinergic and pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems (used by melanopsin-expressing retinal ganglion cells), which directly regulates responses to the retinal input. These results help us better understand circadian orexinergic control over the non-image forming subcortical visual system, forming the animal's preparedness for the behaviourally active night. ABSTRACT: The orexinergic system of the lateral hypothalamus is tightly interlinked with the master circadian clock and displays daily variation in activity to provide arousal-related excitation for the plethora of brain structures in a circadian manner. Here, using a combination of electrophysiological, optogenetic, histological, molecular and neuronal tracing methods, we explore a particular link between orexinergic and visual systems in rat. The results of the present study demonstrate that orexinergic fibre density at the area of subcortical visual system exerts a clear day to night variability, reaching a maximum at behaviourally active night. We also show pronounced electrophysiological activations of neurons in the lateral geniculate nucleus by orexin A through 24 h, via identified distinct orexin receptors, with the ventrolateral geniculate displaying a daily cycle of responsiveness. In addition, for the first time, we provide a direct evidence for orexins to act on retinorecipient neurons with a high convergence of orexinergic and putatively retinal pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems. Altogether, the present study ties orexins to non-image forming visual structures with implications for circadian orexinergic modulation of neurons, which process information on ambient light levels.

Melanopsin: From a small molecule to brain functions.

Melanopsin, a G family coupled receptor, found within the ganglion cell layer in the retina, plays an important role in non-image-forming visual functions, including hormone secretion, entrainment of circadian rhythms, cognitive and affective processes. Diffuse projections of melanopsin-containing cells to many brain areas suggest that different responses may involve different neural projections, thus different melanopsin cells. Considering the complexity of the melanopsin system, its contribution to so many different biological functions is not surprising. In this review, we summarize the current knowledge about melanopsin in terms of its photophysics, photochemistry, mechanisms of activation, cell signaling, morphology, and physiology. In the last part, the role of melanopsin in image and non-image forming processes and cognitive and affective functioning of animals and humans, are discussed. Although in recent years considerable insight has been gained into the melanopsin system, it still remains an open question of how one protein expressed by several thousand cells in the retina, could be responsible for so many diverse functions and what activation mechanism(s) it uses.

Altered oscillation frequencies in the lateral geniculate complex in the rat model of absence epilepsy.

Absence epilepsy (AE) is a neurological disease that manifests in spike-wave discharges not present in healthy neuronal circuits. Mutations in ion channels directly underlying this rhythmic discharge may additionally affect rhythms in multiple brain centres which disturbances contribute to the epileptic phenotype. Malfunctioning of the light detection system (from retina to subcortical visual structures), heavily dependent on oscillatory activities, could partially explain severe problems with sleep and arousal observed in epileptic patients. Therefore, the aim of our study was to evaluate characteristics of retinal-derived oscillations in the lateral geniculate complex of the thalamus; a major gateway for the light information flow for the brain. Extracellular recordings in vivo were performed on urethane-anaesthetised WAG/Rij and Wistar rats from single units in the identified parts of lateral geniculate complex to test their basic oscillatory features as well as reaction to transient and sustained changes in ambient light conditions. Here, we show altered rhythmic activity of the lateral geniculate neurons in the absence epilepsy model with the increase of both the infra-slow and fast oscillatory frequencies. Further, we describe their disturbed reaction to sustain change in ambient light and provide evidence for major changes in the intergeniculate leaflet neuronal firing; a part of the lateral geniculate complex implicated in the circadian timekeeping. Altogether, our results are the first to show a malfunctioning of light detection mechanisms in the absence epilepsy that may in turn underpin sleep-promoting system insufficiencies and other arousal disturbances contributing to epileptic phenotype.

Modulation of Spontaneous and Light-Induced Activity in the Rat Dorsal Lateral Geniculate Nucleus by General Brain State Alterations under Urethane Anesthesia.

The thalamic dorsal lateral geniculate nucleus (dLGN) serves as a gating station for the transfer of light information en route to the primary visual cortex (V1). Although the modulatory input arising from the V1 and several brainstem nuclei to the dLGN is well characterised in higher mammals, little is known about its influence on dLGN activity in rodents. Using simultaneous recordings of electrocorticogram (ECoG) and single unit neuronal activity under urethane anesthesia in Long Evans rats, we managed to show that cyclic changes in the general brain state strongly affect spontaneous activity and light encoding properties of dLGN neurons. First, we characterised several groups of dLGN cells: neurons led by ECoG, neurons in which the spike rate preceded ECoG changes and neurons co-occurring or not correlated with ECoG signal. Secondly, we verified that although the general light responsiveness of the dLGN is not influenced by the state of the brain, modulation of types of photoresponses and differences in ability to encode ambient light levels were observed. Cells responding to light in a sustained manner encoded light intensity more accurately during the cortical activation phase of urethane anesthesia. On the other hand, isoflurane anesthesia does not induce such rhythmic changes in ECoG and shuts down the spontaneous neuronal activity in the dLGN. Together, these data suggest a greater modulation of spontaneous activity and dLGN neurons function, than it was previously reported for the rodent dLGN and highlight the role of anesthesia in interpretations of findings from ongoing acute experiments.

Gamma and infra-slow oscillations shape neuronal firing in the rat subcortical visual system.

KEY POINTS: Neuronal oscillations observed in sensory systems are physiological carriers of information about stimulus features. Rhythm in the infra-slow range, originating from the retina, was previously found in the firing of subcortical visual system nuclei involved in both image and non-image forming functions. The present study shows that the firing of neurons in the lateral geniculate nucleus is also governed by gamma oscillation (∼35 Hz) time-locked to high phase of infra-slow rhythm that codes the intensity of transient light stimulation. We show that both physiological rhythms are synchronized within and between ipsilateral nuclei of the subcortical visual system and are dependent on retinal activity. The present study shows that neurophysiological oscillations characterized by various frequencies not only coexist in the subcortical visual system, but also are subjected to complex interference and synchronization processes. ABSTRACT: The physiological function of rhythmic firing in the neuronal networks of sensory systems has been linked with information coding. Also, neuronal oscillations in different frequency bands often change as a signature of brain state or sensory processing. Infra-slow oscillation (ISO) in the neuronal firing dependent on the retinal network has been described previously in the structures of the subcortical visual system. In the present study, we show for the first time that firing of ISO neurons in the lateral geniculate nucleus is also characterized by a harmonic discharge pattern (i.e. action potentials are separated by the intervals governed by fundamental frequency in the gamma range: ∼35 Hz). A similar phenomenon was recently described in the suprachiasmatic nuclei of the hypothalamus: the master biological clock. We found that both gamma and ISO rhythms were synchronized within and between ipsilateral nuclei of the subcortical visual system and were dependent on the retinal activity of the contralateral eye. These oscillatory patterns were differentially influenced by transient and prolonged light stimulation with respect to both frequency change direction and sustainability. The results of the present study show that the firing pattern of neurons in the subcortical visual system is shaped by oscillations from infra-slow and gamma frequency bands that are plausibly generated by the retinal network. Additionally, the results demonstrate that both rhythms are not a distinctive feature of image or non-image forming visual systems but, instead, they comprise two channels carrying distinctive properties of photic information.

Disinhibition of the intergeniculate leaflet network in the WAG/Rij rat model of absence epilepsy.

The intergeniculate leaflet (IGL) of the thalamus is a retinorecipient structure implicated in orchestrating circadian rhythmicity. The IGL network is highly GABAergic and consists mainly of neuropeptide Y-synthesising and enkephalinergic neurons. A high density of GFAP-immunoreactive astrocytes has been observed in the IGL, with a probable function in guarding neuronal inhibition. Interestingly, putatively enkephalinergic IGL neurons generate action potentials with an infra-slow oscillatory (ISO) pattern in vivo in urethane anesthetised Wistar rats, under light-on conditions only. Absence epilepsy (AE) is a disease characterised by spike-wave discharges present in the encephalogram, directly caused by hypersynchronous thalamo-cortical oscillations. Many pathologies connected with the arousal system, such as abnormalities in sleep architecture and an insufficient brain sleep-promoting system accompany the epileptic phenotype. We hypothesise that disturbances in the function of biological clock structures, controlling this rhythmic physiological process, may be responsible for the observed pathomechanism. To test this hypothesis, we performed an in vitro patch-clamp study on WAG/Rij rats, a well-validated genetic model of AE, in order to assess dampened GABAergic synaptic transmission in the IGL expressed as a lower IPSC amplitude and reduced sIPSC frequency. Moreover, our in vivo extracellular recordings showed higher firing rate of ISO IGL neurons with an abnormal reaction to a change in constant illumination (maintenance of rhythmic neuronal activity in darkness) in the AE model. Additional immunohistochemical experiments indicated astrogliosis in the area of the IGL, which may partially underlie the observed changes in inhibition. Altogether, the data presented here show for the first time the disinhibition of IGL neurons in a model of AE, thereby proposing the possible involvement of circadian-related brain structures in the epileptic phenotype.

Retinal gap junctions are involved in rhythmogenesis of neuronal activity at remote locations - Study on infra-slow oscillations in the rat olivary pretectal nucleus.

A subpopulation of olivary pretectal nucleus (OPN) neurons fire action potentials in a rhythmic manner with an eruption of activity occurring approximately every two minutes. These infra-slow oscillations depend critically on functional retinal input and are subject to modulation by light. Interestingly, the activity of photoreceptors is necessary for the emergence of the rhythm and while classic photoreceptors (rods and cones) are necessary in darkness and dim light, melanopsin photoreceptors are indispensable in bright light. Using pharmacological and electrophysiological approaches in vivo, we show that also blocking retinal gap junctions (GJs), which are expressed by multitude of retinal cells, leads to the disruption of oscillatory activity in the rat OPN. Intravitreal injection of carbenoxolone (CBX) quenched oscillations in a concentration-dependent manner with 1mM being ineffective, 5mM showing partial and 20mM showing complete effectiveness in disrupting oscillations. Moreover, the most effective CBX concentration depressed cone-mediated light-induced responses of oscillatory neurons suggesting that CBX is also acting on targets other than GJs. In contrast, intravitreal injection of meclofenamic acid (MFA, 20mM) led to disruption of the rhythm but did not interfere with cone-mediated light-induced responses of oscillatory neurons, implying that MFA is more specific toward GJs than CBX, as suggested before. We conclude that electrical coupling between various types of retinal cells and resultant synchronous firing of retinal ganglion cells is necessary for the generation of infra-slow oscillations in the rat OPN.