Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

AM-251, A Cannabinoid Antagonist, Modifies the Dynamics of Sleep-Wake Cycles in Rats.

Study Objectives: (a) To describe the microarchitecture of wakefulness and sleep following administrations of 5- and 10-mg/kg AM-251 in rats. (b) To develop a new statistical method to follow bout-to-bout dynamics. Method: Wistar rats (n = 6) had been equipped with electroencephalography (EEG) and electromyography (EMG) electrodes. Following their recovery and habituation after the surgery, the animals were injected with vehicle and 5- and 10-mg/kg AM-251 intraperitoneally and EEG, EMG, and motor activity were analyzed for the subsequent 3 h. Results: AM-251 induced a dose- and time-dependent increase in the number of bouts in active wake (AW), and it decreased this number in all other vigilance states except in passive wake (PW). In contrast, the bout duration in PW compensatory decreased. The effect of AM-251 on the sleep transition dynamics was monitored with a new tool we call "transition heatmap." The analysis of bout trajectories with transition heatmaps reveals a highly organized pattern. Conclusion: AM-251 selectively influences the frequency of vigilance state transitions, but it has no direct impact on the state lengths. AM-251 markedly changed the state transition dynamics, which was visualized with the help of state transition heatmaps.

Additive effect of 5-HT2C and CB1 receptor blockade on the regulation of sleep-wake cycle.

BACKGROUND: Previous data show that serotonin 2C (5-HT2C) and cannabinoid 1 (CB1) receptors have a role in the modulation of sleep-wake cycle. Namely, antagonists on these receptors promoted wakefulness and inhibited rapid eye movement sleep (REMS) in rodents. The interaction of these receptors are also present in other physiological functions, such as the regulation of appetite. Blockade of 5-HT2C receptors modulat the effect of CB1 receptor antagonist, presumably in consecutive or interdependent steps. Here we investigate, whether previous blockade of 5-HT2C receptors can affect CB1 receptor functions in the sleep-wake regulation. RESULTS: Wistar rats were equipped with electroencephalography (EEG) and electromyography (EMG) electrodes. Following the recovery and habituation after surgery, animals were injected intraperitoneally (ip.) with SB-242084, a 5-HT2C receptor antagonist (1.0 mg/kg) at light onset (beginning of passive phase) followed by an injection with AM-251, a CB1 receptor antagonist (5.0 or 10.0 mg/kg, ip.) 10 min later. EEG, EMG and motor activity were analyzed for the subsequent 2 h. Both SB-242084 and AM-251 increased the time spent in active wakefulness, while decreased the time spent in non-REMS and REMS stages in the first 2 h of passive phase. In combination, the effect of the agents were additive, furthermore, statistical analysis did not show any interaction between the effects of these drugs in the modulation of vigilance stages. CONCLUSIONS: Our results suggest that 5-HT2C receptor blockade followed by blockade of CB1 receptors evoked additive effect on the regulation of sleep-wake pattern.

Interleukin-6 promoter polymorphism interacts with pain and life stress influencing depression phenotypes.

Interleukin-6 (IL-6) has emerged as a potent biomarker for depression as its elevated plasma levels in patients with clinical depression have been confirmed by meta-analyses. Increased plasma IL-6 concentration was associated with various psychological stress factors and physical disorders accompanied by pain. Another modulator of the IL-6 level is rs1800795, a promoter polymorphism in the IL-6 gene which is able to influence its expression rate. Therefore, we examined in a Hungarian population sample of 1053 volunteers with European origins if rs1800795 polymorphism can affect depression symptoms measured by Zung Self-rating Depression Scale (ZSDS), and Brief Symptom Inventory (BSI). We also investigated the interactions of the polymorphism with reported painful physical conditions and Recent Negative Life Events (RLE) measured by the List of Life Threatening Experiences. Rs1800795 significantly interacted with both RLE and painful condition on depressive symptoms measured by ZSDS and BSI using different heritability models, while no main effects of the polymorphism were identified. After correction for multiple testing only the rs1800795 × RLE interaction effect (recessive model) remained significant on the BSI score, while both RLE and painful conditions significantly interacted on the ZSDS. In conclusion, the functional IL-6 rs1800795 polymorphism in interaction with various stress factors increases the risk of depression and has a greater impact on symptoms measured by the ZSDS. Thus, IL-6 and other cytokines may be more relevant in the development of somatic symptoms compared to affective signs of depression, delineating a specific genotype-phenotype relationship in this heterogeneous disorder.

Nesfatin-1/NUCB2 as a potential new element of sleep regulation in rats.

STUDY OBJECTIVES: Millions suffer from sleep disorders that often accompany severe illnesses such as major depression; a leading psychiatric disorder characterized by appetite and rapid eye movement sleep (REMS) abnormalities. Melanin-concentrating hormone (MCH) and nesfatin-1/NUCB2 (nesfatin) are strongly co - expressed in the hypothalamus and are involved both in food intake regulation and depression. Since MCH was recognized earlier as a hypnogenic factor, we analyzed the potential role of nesfatin on vigilance. DESIGN: We subjected rats to a 72 h-long REMS deprivation using the classic flower pot method, followed by a 3 h-long 'rebound sleep'. Nesfatin mRNA and protein expressions as well as neuronal activity (Fos) were measured by quantitative in situ hybridization technique, ELISA and immunohistochemistry, respectively, in 'deprived' and 'rebound' groups, relative to controls sacrificed at the same time. We also analyzed electroencephalogram of rats treated by intracerebroventricularly administered nesfatin-1, or saline. RESULTS: REMS deprivation downregulated the expression of nesfatin (mRNA and protein), however, enhanced REMS during 'rebound' reversed this to control levels. Additionally, increased transcriptional activity (Fos) was demonstrated in nesfatin neurons during 'rebound'. Centrally administered nesfatin-1 at light on reduced REMS and intermediate stage of sleep, while increased passive wake for several hours and also caused a short-term increase in light slow wave sleep. CONCLUSIONS: The data designate nesfatin as a potential new factor in sleep regulation, which fact can also be relevant in the better understanding of the role of nesfatin in the pathomechanism of depression.

Association between the activation of MCH and orexin immunorective neurons and REM sleep architecture during REM rebound after a three day long REM deprivation.

Rapid eye movement (REM) sleep rebound following REM deprivation using the platform-on-water method is characterized by increased time spent in REM sleep and activation of melanin-concentrating hormone (MCH) expressing neurons. Orexinergic neurons discharge reciprocally to MCH-ergic neurons across the sleep-wake cycle. However, the relation between REM architecture and the aforementioned neuropeptides remained unclear. MCH-ergic neurons can be divided into two subpopulations regarding their cocaine- and amphetamine-regulated transcript (CART) immunoreactivity, and among them the activation of CART-immunoreactive subpopulation is higher during the REM rebound. However, the possible role of stress in this association has not been elucidated. Our aims were to analyze the relationship between the architecture of REM rebound and the activation of hypothalamic MCH-ergic and orexinergic neurons. We also intended to separate the effect of stress and REM deprivation on the subsequent activation of subpopulations of MCH-ergic neurons. In order to detect neuronal activity, we performed MCH/cFos and orexin/cFos double immunohistochemistry on home cage, sleep deprived and sleep-rebound rats using the platform-on-water method with small and large (stress control) platforms. Furthermore, REM architecture was analyzed and a triple MCH/CART/cFos immunohistochemistry was also performed on the rebound groups in the same animals. We found that the activity of MCH- and orexin-immunoreactive neurons during REM rebound was positively and negatively correlated with the number of REM bouts, respectively. A negative reciprocal correlation was also found between the activation of MCH- and orexin-immunoreactive neurons during REM rebound. Furthermore, difference between the activation of CART-immunoreactive (CART-IR) and non-CART-immunoreactive MCH-ergic neuron subpopulations was found only after selective REM deprivation, it was absent in the large platform (stress control) rebound group. These results support the role of CART-IR subpopulation of MCH-ergic neurons and the inverse relationship of MCH and orexin in the regulation of REM sleep after REM sleep deprivation.

Medicinal chemistry of 5-HT5A receptor ligands: a receptor subtype with unique therapeutical potential.

Although the 5-HT(5) receptor subfamily was discovered more than 15 years ago, it is unambiguously the least known 5-HT receptor subtype. The G(i)/G(0)-mediated signal transduction and its intensive presence in raphe and other brainstem and pons nuclei suggest mechanisms similar to those of 5-HT(1) receptors, the ligands of which are already applied in the treatment of e.g. anxiety and migraine. In addition, a unique coupling and inhibition of adenosine diphosphate-ribosyl cyclase have also been described. High concentrations of 5-HT(5) receptor in other key regions including, e.g. locus coeruleus, nucleus of the solitary tract, arcuate and suprachiasmatic nuclei of the hypothalamus indicate a wide range of physiological effects, thus its ligands are potential drug candidates in various areas, e.g. anxiety, sleep, incontinence, food intake, learning and memory, pain or chemoreception pathways. These findings have motivated several institutes and pharmaceutical companies to participate in the research of this field. Despite extensive research, no selective agonist and only two selective antagonists have been identified until now. Beyond these compounds, the present review provides a complete overview on all other published 5-HT(5A) receptor ligands as well as on the structure, function, distribution, genetics and possible therapeutic applications of this receptor.