Differential effects of haloperidol on neural oscillations during wakefulness and sleep.

The electrical activity of the brain, characterized by its frequency components, reflects a complex interplay between periodic (oscillatory) and aperiodic components. These components are associated with various neurophysiological processes, such as the excitation-inhibition balance (aperiodic activity) or interregional communication (oscillatory activity). However, we do not fully understand whether these components are truly independent or if different neuromodulators affect them in different ways. The dopaminergic system has a critical role for cognition and motivation, being a potential modulator of these power spectrum components. To improve our understanding of these questions, we investigated the differential effects of this system on these components using electrocorticogram recordings in cats, which show clear oscillations and aperiodic 1/f activity. Specifically, we focused on the effects of haloperidol (a D2 receptor antagonist) on oscillatory and aperiodic dynamics during wakefulness and sleep. By parameterizing the power spectrum into these two components, our findings reveal a robust modulation of oscillatory activity by the D2 receptor across the brain. Surprisingly, aperiodic activity was not significantly affected and exhibited inconsistent changes across the brain. This suggests a nuanced interplay between neuromodulation and the distinct components of brain oscillations, providing insights into the selective regulation of oscillatory dynamics in awake states.

Dorsal and median raphe neuronal firing dynamics characterized by nonlinear measures.

The dorsal (DRN) and median (MRN) raphe are important nuclei involved in similar functions, including mood and sleep, but playing distinct roles. These nuclei have a different composition of neuronal types and set of neuronal connections, which among other factors, determine their neuronal dynamics. Most works characterize the neuronal dynamics using classic measures, such as using the average spiking frequency (FR), the coefficient of variation (CV), and action potential duration (APD). In the current study, to refine the characterization of neuronal firing profiles, we examined the neurons within the raphe nuclei. Through the utilization of nonlinear measures, our objective was to discern the redundancy and complementarity of these measures, particularly in comparison with classic methods. To do this, we analyzed the neuronal basal firing profile in both nuclei of urethane-anesthetized rats using the Shannon entropy (Bins Entropy) of the inter-spike intervals, permutation entropy of ordinal patterns (OP Entropy), and Permutation Lempel-Ziv Complexity (PLZC). Firstly, we found that classic (i.e., FR, CV, and APD) and nonlinear measures fail to distinguish between the dynamics of DRN and MRN neurons, except for the OP Entropy. We also found strong relationships between measures, including the CV with FR, CV with Bins entropy, and FR with PLZC, which imply redundant information. However, APD and OP Entropy have either a weak or no relationship with the rest of the measures tested, suggesting that they provide complementary information to the characterization of the neuronal firing profiles. Secondly, we studied how these measures are affected by the oscillatory properties of the firing patterns, including rhythmicity, bursting patterns, and clock-like behavior. We found that all measures are sensitive to rhythmicity, except for the OP Entropy. Overall, our work highlights OP Entropy as a powerful and useful quantity for the characterization of neuronal discharge patterns.

Acute and chronic sleep restriction differentially modify maternal behavior and milk macronutrient composition in the postpartum rat.

BACKGROUNDS: Sleep restriction is considered a stressful condition itself, causing a wide variety of physiological alterations, from cognitive and hormonal to immunological status. In addition, it is established that stress in mother rats can modify milk ejection, milk composition, and maternal care of the pups. Also, sleep disturbances during the early stages of motherhood are a common feature of all studied species. In this context, while the impacts of sleep disruption in non-lactating animals were extensively investigated, its repercussions during the initial phases of motherhood have been poorly explored. Therefore, we wonder if maternal behavior, milk ejection and its macronutrient composition would be disrupted when mother rats are subjected to an additional acute or chronic sleep restriction to the already existing sleep disturbances. METHODS: Lactating rats were implanted with unilateral electrodes for polysomnographic recordings and for deep brain electrical stimulation into mesopontine waking-promoting area (for sleep deprivation). During the early postpartum period (postpartum day 5-9), mother rats were randomly assigned into one of three groups: chronic sleep restriction group (CSR; 6 h of sleep deprivation/day for five consecutive days), acute sleep restriction group (ASR; 6 h of sleep deprivation only for one day), or undisturbed group (control group). Active maternal behaviors (retrievals of the pups into the nest, mouthing, lickings [corporal and anogenital] and sniffing the pups) and passive maternal behaviors (kyphotic and supine nursing postures) were evaluated during a 30 min period without sleep restriction immediately after the sleep restriction or control period. The litter weight gain was assessed every day, and on the last experimental session mothers were milked for posterior macronutrients analysis (protein, carbohydrates and fat). RESULTS: When compared to control group, CSR decreased the amount of milk ejected in the middle days of the sleep restriction period, while ASR did not affect this parameter. Moreover, ASR reduced milk protein content compared to control and CSR groups. Finally, compared to the control group, CSR reduced active maternal behaviors towards the end of the treatment days. CONCLUSIONS: We demonstrated that not only acute but also chronic sleep restriction impacts on the postpartum period, each one affecting different aspects of maternal behavior and lactation. Our results suggest the existence of a homeostatic recovery mechanism in breastfeeding during CSR, possibly ensuring the survival of the litter, while the decline in active maternal behaviors appears to be cumulative.

Noribogaine acute administration in rats promotes wakefulness and suppresses REM sleep.

Ibogaine is a potent atypical psychedelic that has gained considerable attention due to its antiaddictive and antidepressant properties in preclinical and clinical studies. Previous research from our group showed that ibogaine suppresses sleep and produces an altered wakefulness state, which resembles natural REM sleep. However, after systemic administration, ibogaine is rapidly metabolized to noribogaine, which also shows antiaddictive effects but with a distinct pharmacological profile, making this drug a promising therapeutic candidate. Therefore, we still ignore whether the sleep/wake alterations depend on ibogaine or its principal metabolite noribogaine. To answer this question, we conducted polysomnographic recordings in rats following the administration of pure noribogaine. Our results show that noribogaine promotes wakefulness while reducing slow-wave sleep and blocking REM sleep, similar to our previous results reported for ibogaine administration. Thus, we shed new evidence on the mechanisms by which iboga alkaloids work in the brain.

5-HT2A Receptor Knockout Mice Show Sex-Dependent Differences following Acute Noribogaine Administration.

Noribogaine (noribo) is the primary metabolite from ibogaine, an atypical psychedelic alkaloid isolated from the root bark of the African shrub Tabernanthe iboga. The main objective of this study was to test the hypothesis that molecular, electrophysiological, and behavioral responses of noribo are mediated by the 5-HT2A receptor (5-HT2AR) in mice. In that regard, we used male and female, 5-HT2AR knockout (KO) and wild type (WT) mice injected with a single noribo dose (10 or 40 mg/kg; i.p.). After 30 min., locomotor activity was recorded followed by mRNA measurements by qPCR (immediate early genes; IEG, glutamate receptors, and 5-HT2AR levels) and electrophysiology recordings of layer V pyramidal neurons from the medial prefrontal cortex. Noribo 40 decreased locomotion in male, but not female WT. Sex and genotype differences were observed for IEG and glutamate receptor expression. Expression of 5-HT2AR mRNA increased in the mPFC of WT mice following Noribo 10 (males) or Noribo 40 (females). Patch-clamp recordings showed that Noribo 40 reduced the NMDA-mediated postsynaptic current density in mPFC pyramidal neurons only in male WT mice, but no effects were found for either KO males or females. Our results highlight that noribo produces sexually dimorphic effects while the genetic removal of 5HT2AR blunted noribo-mediated responses to NMDA synaptic transmission.

Sleep disrupts complex spiking dynamics in the neocortex and hippocampus.

Neuronal interactions give rise to complex dynamics in cortical networks, often described in terms of the diversity of activity patterns observed in a neural signal. Interestingly, the complexity of spontaneous electroencephalographic signals decreases during slow-wave sleep (SWS); however, the underlying neural mechanisms remain elusive. Here, we analyse in-vivo recordings from neocortical and hippocampal neuronal populations in rats and show that the complexity decrease is due to the emergence of synchronous neuronal DOWN states. Namely, we find that DOWN states during SWS force the population activity to be more recurrent, deterministic, and less random than during REM sleep or wakefulness, which, in turn, leads to less complex field recordings. Importantly, when we exclude DOWN states from the analysis, the recordings during wakefulness and sleep become indistinguishable: the spiking activity in all the states collapses to a common scaling. We complement these results by implementing a critical branching model of the cortex, which shows that inducing DOWN states to only a percentage of neurons is enough to generate a decrease in complexity that replicates SWS.

Hypocretins, sleep, and maternal behavior.

The postpartum period is a demanding time during which mothers experience numerous physiological adaptations that enable them to care for their offspring while maintaining their wellbeing. Hypocretins, also known as orexins, are neuropeptides synthesized by hypothalamic neurons that play a fundamental role in several functions, including the promotion of wakefulness and motivated behaviors, such as maternal care. In this regard, several findings suggest that the activity of the hypocretinergic system increases in the early postpartum period and begins to decline as weaning approaches. In particular, hypocretins within the medial preoptic area, a crucial region during this period, modulate both maternal behavior and sleep. Although further studies are necessary to obtain a comprehensive understanding of the role of hypocretins in lactating females, current research suggests that this system participates in promoting active components of maternal behavior and regulating wakefulness and sleep adjustments during the postpartum period, potentially leading to increased wakefulness during this stage. These adaptive adjustments enable the mother to cope with the continuously changing demands of the pups.

Mechanisms and functions of respiration-driven gamma oscillations in the primary olfactory cortex.

Gamma oscillations are believed to underlie cognitive processes by shaping the formation of transient neuronal partnerships on a millisecond scale. These oscillations are coupled to the phase of breathing cycles in several brain areas, possibly reflecting local computations driven by sensory inputs sampled at each breath. Here, we investigated the mechanisms and functions of gamma oscillations in the piriform (olfactory) cortex of awake mice to understand their dependence on breathing and how they relate to local spiking activity. Mechanistically, we find that respiration drives gamma oscillations in the piriform cortex, which correlate with local feedback inhibition and result from recurrent connections between local excitatory and inhibitory neuronal populations. Moreover, respiration-driven gamma oscillations are triggered by the activation of mitral/tufted cells in the olfactory bulb and are abolished during ketamine/xylazine anesthesia. Functionally, we demonstrate that they locally segregate neuronal assemblies through a winner-take-all computation leading to sparse odor coding during each breathing cycle. Our results shed new light on the mechanisms of gamma oscillations, bridging computation, cognition, and physiology.

The cerebral cortex is the most recently evolved region of the mammalian brain. There, millions of neurons can synchronize their activity to create brain waves, a series of electric rhythms associated with various cognitive functions. Gamma waves, for example, are thought to be linked to brain processes which require distributed networks of neurons to communicate and integrate information. These waves were first discovered in the 1940s by researchers investigating brain areas involved in olfaction, and they are thought to be important for detecting and recognizing smells. Yet, scientists still do not understand how these waves are generated or what role they play in sensing odors. To investigate these questions, González et al. used a battery of computational approaches to analyze a large dataset of brain activity from awake mice. This revealed that, in the cortical region dedicated to olfaction, gamma waves arose each time the animals completed a breathing cycle ­ that is, after they had sampled the air by breathing in. Each breath was followed by certain neurons relaying olfactory information to the cortex to activate complex cell networks; this included circuits of cells known as feedback interneurons, which can switch off weakly activated neurons, including ones that participated in activating them in the first place. The respiration-driven gamma waves derived from this 'feedback inhibition' mechanism. Further work then examined the role of the waves in olfaction. Smell identification relies on each odor activating a unique set of cortical neurons. The analyses showed that gamma waves acted to select and amplify the best set of neurons for representing the odor sensed during a sniff, and to quieten less relevant neurons. Loss of smell is associated with many conditions which affect the brain, such as Alzheimer's disease or COVID-19. By shedding light on the neuronal mechanisms that underpin olfaction, the work by González et al. could help to better understand how these impairments emerge, and how the brain processes other types of complex information.

Is sleep critical for lactation in rat?

Sleep deprivation is a feature shared by most studied mammals at some point during the postpartum period. Unlike the rabbit, the pig, or the human mother, sleep has been claimed as an essential state for milk ejection in mother rats, where sleep deprivation using gentle handling (GH) prevents milk ejection and pup weight gain. Though sleep deprivation is a stressful situation itself, most common methodologies used in laboratory animals, including GH, usually involve aversive stimulus to prevent sleep, adding further stress to the animal. Deep brain electrical stimulation (DBES) of the brainstem reticular formation is a less common technique used to prevent sleep, and while this methodology may also carry unwanted effects, it avoids stressful conditions. In the present study, we examined the relationship between sleep and nursing, and how different sleep deprivation methodologies impact nursing and lactation. For this purpose, we carried out two sets of experiments. First, we correlated sleep and waking states with different nursing parameters of lactating rats under undisturbed conditions. Second, we slept deprived another group of mother rats using two different techniques: GH and DBES. Our main findings show that sleeping time was positively correlated with the time devote to nurse the pups, but not either with milk ejection or pup weight gain. When mother rats were sleep deprived, maternal behavior was fragmented using both methods, but was substantially more disrupted when using GH. Additionally, lactating dams were capable of ejecting milk and their pups gained weight despite of being sleep deprived using both techniques, but these parameters were significantly reduced using GH compared to control values, while DBES did not differ from control group. Overall, these results suggest that sleep and nursing are behaviorally compatible, but in disagreement with previous findings, we concluded that sleep is not necessary for milk ejection. These observations have critical implications for using the rat as a model to explore sleep loss during the postpartum period.

Breathing modulates gamma synchronization across species.

Nasal respiration influences brain dynamics by phase-entraining neural oscillations at the same frequency as the breathing rate and by phase-modulating the activity of faster gamma rhythms. Despite being widely reported, we still do not understand the functional roles of respiration-entrained oscillations. A common hypothesis is that these rhythms aid long-range communication and provide a privileged window for synchronization. Here we tested this hypothesis by analyzing electrocorticographic (ECoG) recordings in mice, rats, and cats during the different sleep-wake states. We found that the respiration phase modulates the amplitude of cortical gamma oscillations in the three species, although the modulated gamma frequency bands differed with faster oscillations (90-130 Hz) in mice, intermediate frequencies (60-100 Hz) in rats, and slower activity (30-60 Hz) in cats. In addition, our results also show that respiration modulates olfactory bulb-frontal cortex synchronization in the gamma range, in which each breathing cycle evokes (following a delay) a transient time window of increased gamma synchrony. Long-range gamma synchrony modulation occurs during quiet and active wake states but decreases during sleep. Thus, our results suggest that respiration-entrained brain rhythms orchestrate communication in awake mammals.

Electrophysiological characterization of medial preoptic neurons in lactating rats and its modulation by hypocretin-1.

The medial preoptic area (mPOA) undergoes through neuroanatomical changes across the postpartum period, during which its neurons play a critical role in the regulation of maternal behavior. In addition, this area is also crucial for sleep-wake regulation. We have previously shown that hypocretins (HCRT) within the mPOA facilitate active maternal behaviors in postpartum rats, while the blockade of endogenous HCRT in this area promotes nursing and sleep. To explore the mechanisms behind these HCRT actions, we aimed to evaluate the effects of juxta-cellular HCRT-1 administration on mPOA neurons in urethane-anesthetized postpartum and virgin female rats. We recorded mPOA single units and the electroencephalogram (EEG) and applied HCRT-1 juxta-cellular by pressure pulses. Our main results show that the electrophysiological characteristics of the mPOA neurons and their relationship with the EEG of postpartum rats did not differ from virgin rats. Additionally, neurons that respond to HCRT-1 had a slower firing rate than those that did not. In addition, administration of HCRT increased the activity in one group of neurons while decreasing it in another, both in postpartum and virgin rats. This study suggests that the mechanisms by which HCRT modulate functions controlled by the mPOA involve different cell populations.

Low frequency oscillations drive EEG's complexity changes during wakefulness and sleep.

Recently, the sleep-wake states have been analysed using novel complexity measures, complementing the classical analysis of EEGs by frequency bands. This new approach consistently shows a decrease in EEG's complexity during slow-wave sleep, yet it is unclear how cortical oscillations shape these complexity variations. In this work, we analyse how the frequency content of brain signals affects the complexity estimates in freely moving rats. We find that the low-frequency spectrum - including the Delta, Theta, and Sigma frequency bands - drives the complexity changes during the sleep-wake states. This happens because low-frequency oscillations emerge from neuronal population patterns, as we show by recovering the complexity variations during the sleep-wake cycle from micro, meso, and macroscopic recordings. Moreover, we find that the lower frequencies reveal synchronisation patterns across the neocortex, such as a sensory-motor decoupling that happens during REM sleep. Overall, our works shows that EEG's low frequencies are critical in shaping the sleep-wake states' complexity across cortical scales.

Urethane anaesthesia exhibits neurophysiological correlates of unconsciousness and is distinct from sleep.

Urethane is a general anaesthetic widely used in animal research. The state of urethane anaesthesia is unique because it alternates between macroscopically distinct electrographic states: a slow-wave state that resembles non-rapid eye movement (NREM) sleep and an activated state with features of both REM sleep and wakefulness. Although it is assumed that urethane produces unconsciousness, this has been questioned because of states of cortical activation during drug exposure. Furthermore, the similarities and differences between urethane anaesthesia and physiological sleep are still unclear. In this study, we recorded the electroencephalogram (EEG) and electromyogram in chronically prepared rats during natural sleep-wake states and during urethane anaesthesia. We subsequently analysed the power, coherence, directed connectivity and complexity of brain oscillations and found that EEG under urethane anaesthesia has clear signatures of unconsciousness, with similarities to other general anaesthetics. In addition, the EEG profile under urethane is different in comparison with natural sleep states. These results suggest that consciousness is disrupted during urethane. Furthermore, despite similarities that have led others to conclude that urethane is a model of sleep, the electrocortical traits of depressed and activated states during urethane anaesthesia differ from physiological sleep states.

Ketamine and sleep modulate neural complexity dynamics in cats.

There is increasing evidence that the level of consciousness can be captured by neural informational complexity: for instance, complexity, as measured by the Lempel Ziv (LZ) compression algorithm, decreases during anaesthesia and non-rapid eye movement (NREM) sleep in humans and rats, when compared with LZ in awake and REM sleep. In contrast, LZ is higher in humans under the effect of psychedelics, including subanaesthetic doses of ketamine. However, it is both unclear how this result would be modulated by varying ketamine doses, and whether it would extend to other species. Here, we studied LZ with and without auditory stimulation during wakefulness and different sleep stages in five cats implanted with intracranial electrodes, as well as under subanaesthetic doses of ketamine (5, 10, and 15 mg/kg i.m.). In line with previous results, LZ was lowest in NREM sleep, but similar in REM and wakefulness. Furthermore, we found an inverted U-shaped curve following different levels of ketamine doses in a subset of electrodes, primarily in prefrontal cortex. However, it is worth noting that the variability in the ketamine dose-response curve across cats and cortices was larger than that in the sleep-stage data, highlighting the differential local dynamics created by two different ways of modulating conscious state. These results replicate previous findings, both in humans and other species, demonstrating that neural complexity is highly sensitive to capture state changes between wake and sleep stages while adding a local cortical description. Finally, this study describes the differential effects of ketamine doses, replicating a rise in complexity for low doses, and further fall as doses approach anaesthetic levels in a differential manner depending on the cortex.

Role of Hypocretin in the Medial Preoptic Area in the Regulation of Sleep, Maternal Behavior and Body Temperature of Lactating Rats.

Hypocretins (HCRT), also known as orexins, includes two neuroexcitatory peptides, HCRT-1 and HCRT-2 (orexin A y B, respectively), synthesized by neurons located in the postero-lateral hypothalamus, whose projections and receptors are widely distributed throughout the brain, including the medial preoptic area (mPOA). HCRT have been associated with a wide range of physiological functions including sleep-wake cycle, maternal behavior and body temperature, all regulated by the mPOA. Previously, we showed that HCRT in the mPOA facilitates certain active maternal behaviors, while the blockade of HCRT-R1 increases the time spent in nursing. As mother rats mainly sleep while they nurse, we hypothesize that HCRT in the mPOA of lactating rats reduce sleep and nursing, while intra-mPOA administration of a dual orexin receptor antagonist (DORA) would cause the opposite effect. Therefore, the aim of this study was to determine the role of HCRT within the mPOA, in the regulation and integration of the sleep-wake cycle, maternal behavior and body temperature of lactating rats. For that purpose, we assessed the sleep-wake states, maternal behavior and body temperature of lactating rats following microinjections of HCRT-1 (100 and 200 µM) and DORA (5 mM) into the mPOA. As expected, our data show that HCRT-1 in mPOA promote wakefulness and a slightly increase in body temperature, whereas DORA increases both NREM and REM sleep together with an increment of nursing and milk ejection. Taken together, our results strongly suggest that the endogenous reduction of HCRT within the mPOA contribute to the promotion of sleep, milk ejection and nursing behavior in lactating rats.

In vivo brain levels of acetylcholine and 5-hydroxytryptamine after oleoylethanolamide or palmitoylethanolamide administrations are mediated by PPARα engagement.

The peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that has been linked to the modulation of several physiological functions, including the sleep-wake cycle. The PPARα recognizes as endogenous ligands the lipids oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), which in turn, if systemically injected, they exert wake-promoting effects. Moreover, the activation of PPARα by the administration of OEA or PEA increases the extracellular contents of neurotransmitters linked to the control of wakefulness; however, the role of PPARα activated by OEA or PEA on additional biochemicals related to waking regulation, such as acetylcholine (ACh) and 5-hydroxytryptamine (5-HT), has not been fully studied. Here, we have investigated the effects of treatments of OEA or PEA on the contents of ACh and 5-HT by using in vivo microdialysis techniques coupled to HPLC means. For this purpose, OEA or PEA were systemically injected (5, 10 or 30 mg/kg; i.p.), and the levels of ACh and 5-HT were collected from the basal forebrain, a wake-related brain area. These pharmacological treatments significantly increased the contents of ACh and 5-HT as determined by HPLC procedures. Interestingly, PPARα antagonist MK-886 (30 mg/kg; i.p.) injected before the treatments of OEA or PEA blocked these outcomes. Our data suggest that the activation of PPARα by OEA or PEA produces significant changes on ACh and 5-HT levels measured from the basal forebrain and support the conclusion that PPARα is a suitable molecular element involved in the regulation of wake-related neurotransmitters.

EEG Gamma Band Alterations and REM-like Traits Underpin the Acute Effect of the Atypical Psychedelic Ibogaine in the Rat.

Ibogaine is a psychedelic alkaloid that has attracted large scientific interest because of its antiaddictive properties in observational studies in humans as well as in animal models. Its subjective effect has been described as intense, vivid dream-like experiences occurring while awake; hence, ibogaine is often referred to as an oneirogenic psychedelic. While this unique dream-like profile has been hypothesized to aid the antiaddictive effects, the electrophysiological signatures of this psychedelic state remain unknown. We previously showed in rats that ibogaine promotes a waking state with abnormal motor behavior along with a decrease in NREM and REM sleep. Here, we performed an in-depth analysis of the intracranial electroencephalogram during "ibogaine wakefulness". We found that ibogaine induces gamma oscillations that, despite having larger power than control levels, are less coherent and less complex. Further analysis revealed that this profile of gamma activity compares to that of natural REM sleep. Thus, our results provide novel biological evidence for the association between the psychedelic state and REM sleep, contributing to the understanding of the brain mechanisms associated with the oneirogenic psychedelic effect of ibogaine.

In vivo uptake of a fluorescent conjugate of melanin-concentrating hormone in the rat brain.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide synthesized by posterior hypothalamic and incerto-hypothalamic neurons that project throughout the central nervous system. The MCHergic system modulates several important functions such as feeding behavior, mood and sleep. MCH exerts its biological functions through interaction with the MCHR-1 receptor, the only functional receptor present in rodents. The internalization process of MCHR-1 triggered by MCH binding was described in vitro in non-neuronal heterologous systems with over-expression of MCHR-1. Reports of in vivo MCHR-1 internalization dynamics are scarce, however, this is an important process to explore based on the critical functions of the MCHergic system. We had previously determined that 60 min after intracerebroventricular (i.c.v.) microinjections of MCH conjugated with fluorophore rhodamine (R-MCH), the dorsal and median raphe nucleus presented R-MCH positive labeled neurons. In the present work, we further studied the in vivo uptake process focusing on the distribution and time-dependent pattern of R-MCH positive cells 10, 20 and 60 min (T10, T20 and T60, respectively) after i.c.v. microinjection of R-MCH. We also explored this uptake process to see whether it was receptor- and clathrin-dependent and examined the phenotype of R-MCH positive cells and their proximity to MCHergic fibers. We found a great number of R-MCH positive cells with high fluorescence intensity in the lateral septum, nucleus accumbens and hippocampus at T20 and T60 (but not at T10), while a lower number with low intensity was observed in the dorsal raphe nucleus. At T20, in rats pre-treated with a MCHR-1 antagonist (ATC-0175) or with phenylarsine oxide (PAO), a clathrin endocytosis inhibitor, a robust decrease (> 50 %) of R-MCH uptake occurred in these structures. The R-MCH positive cells were identified as neurons (NeuN positive, GFAP negative) and some MCHergic fibers run in the vicinities of them. We concluded that neurons localized at structures that were close to the ventricular surfaces could uptake R-MCH in vivo through a receptor-dependent and clathrin-mediated process. Our results support volume transmission of MCH through the cerebrospinal fluid to reach distant targets. Finally, we propose that R-MCH would be an effective tool to study MCH-uptake in vivo.

Sleep-wake cycle disturbances and NeuN-altered expression in adult rats after cannabidiol treatments during adolescence.

RATIONALE: The medical uses of cannabidiol (CBD), a constituent of the Cannabis sativa, have accelerated the legal and social acceptance for CBD-based medications but has also given the momentum for questioning whether the long-term use of CBD during the early years of life may induce adverse neurobiological effects in adulthood, including sleep disturbances. Given the critical window for neuroplasticity and neuro-functional changes that occur during stages of adolescence, we hypothesized that CBD might influence the sleep-wake cycle in adult rats after their exposure to CBD during the adolescence. OBJECTIVES: Here, we investigated the effects upon behavior and neural activity in adulthood after long-term administrations of CBD in juvenile rats. METHODS: We pre-treated juvenile rats with CBD (5 or 30 mg/Kg, daily) from post-natal day (PND) 30 and during 2 weeks. Following the treatments, the sleep-wake cycle and NeuN expression was analyzed at PND 80. RESULTS: We found that systemic injections of CBD (5 or 30 mg/Kg, i.p.) given to adolescent rats (post-natal day 30) for 14 days increased in adulthood the wakefulness and decreased rapid eye movement sleep during the lights-on period whereas across the lights-off period, wakefulness was diminished and slow wave sleep was enhanced. In addition, we found that adult animals that received CBD during the adolescence displayed disruptions in sleep rebound period after total sleep deprivation. Finally, we determined how the chronic administrations of CBD during the adolescence affected in the adulthood the NeuN expression in the suprachiasmatic nucleus, a sleep-related brain region. CONCLUSIONS: Our findings are relevant for interpreting results of adult rats that were chronically exposed to CBD during the adolescence and provide new insights into how CBD may impact the sleep-wake cycle and neuronal activity during developmental stages.

Effects of Cannabis Consumption on Sleep.

Despite the fact that medical properties of Cannabis have been recognized for more than 5000 years, the use of Cannabis for medical purposes have recently reemerged and became more accessible. Cannabis is usually employed as a self-medication for the treatment of insomnia disorder. However, the effects of Cannabis on sleep depend on multiple factors such as metabolomic composition of the plant, dosage and route of administration. In the present chapter, we reviewed the main effect Cannabis on sleep. We focused on the effect of "crude or whole plant" Cannabis consumption (i.e., smoked, oral or vaporized) both in humans and experimental animal models.The data reviewed establish that Cannabis modifies sleep. Furthermore, a recent experimental study in animals suggests that vaporization (which is a recommended route for medical purposes) of Cannabis with high THC and negligible CBD, promotes NREM sleep. However, it is imperative to perform new clinical studies in order to confirm if the administration of Cannabis could be a beneficial therapy for the treatment of sleep disorders.