Chemogenetic inhibition of the lateral hypothalamus effectively reduces food intake in rats in a translational proof-of-concept study.

Despite the therapeutic potential of chemogenetics, the method lacks comprehensive preclinical validation, hindering its progression to human clinical trials. We aimed to validate a robust but simple in vivo efficacy assay in rats which could support chemogenetic drug discovery by providing a quick, simple and reliable animal model. Key methodological parameters such as adeno-associated virus (AAV) serotype, actuator drug, dose, and application routes were investigated by measuring the food-intake-reducing effect of chemogenetic inhibition of the lateral hypothalamus (LH) by hM4D(Gi) designer receptor stimulation. Subcutaneous deschloroclozapine in rats transfected with AAV9 resulted in a substantial reduction of food-intake, comparable to the efficacy of exenatide. We estimated that the effect of deschloroclozapine lasts 1-3 h post-administration. AAV5, oral administration of deschloroclozapine, and clozapine-N-oxide were also effective but with slightly less potency. The strongest effect on food-intake occurred within the first 30 min after re-feeding, suggesting this as the optimal experimental endpoint. This study demonstrates that general chemogenetic silencing of the LH can be utilized as an optimal, fast and reliable in vivo experimental model for conducting preclinical proof-of-concept studies in order to validate the in vivo effectiveness of novel chemogenetic treatments. We also hypothesize based on our results that universal LH silencing with existing and human translatable genetic neuroengineering techniques might be a viable strategy to affect food intake and influence obesity.

Procognitive profiling of a serotonin 5-HT6 receptor antagonist in a complex model system in rats: A novel translational approach for clinical prediction.

INTRODUCTION: The serial clinical failures of novel cognitive enhancer candidates point out the lack of predictive power in the preceding animal experimentation. For a more predictive profiling of putative procognitive drugs in rodents, we recently elaborated a methodical approach which consists of three fundamental steps: 1. teaching various learning tasks representing different cognitive domains to the same cohort of animals with the aim to create a population with 'widespread knowledge'. 2. Applying a cognitive deficit-inducing intervention to transform this cohort of animals to a 'patient population'. 3. Testing putative procognitive drugs with a 'clinical trial-like' design on the wide spectrum of cognitive (dys)functions in the actual 'patient population'. The present study has been the first trial to test the feasibility and utility of the proposed system. METHODS: The population with 'widespread knowledge' consisted of 2 year old male Long-Evans rats with a learning history in five-choice serial reaction time task (5-CSRTT, attentional paradigm), Morris water maze (MWM, spatial learning), a cooperative task carried out in pairs (social learning), and a skill-learning task, "pot-jumping". For inducing cognitive deficit, thus creating a 'patient population' we increased the difficulty of the tasks. For the cognitive enhancer mechanism to test in the system we chose a serotonin 5-HT6 receptor antagonist compound, RO4368554. Animals were randomly assigned to vehicle- and drug treated groups based on their baseline learning performance and their response in a pilot test of increase in task difficulty. During the 13-day long treatment with 3 mg/kg ip. RO4368554 all the learning paradigms were repeatedly run with increased difficulty supplemented with a novel object recognition test (NOR, episodic memory). RESULTS: In the 5-CSRTT, reducing the stimulus duration from 1 s to 0.25 s caused a significant decrease in the percentage of correct responses (from 52 % to 31 % in the control group) which was not affected by the 5-HT6 receptor antagonist treatment (correct responses decreased from 58 % to 31 %). In the MWM, replacing the escape platform to a new location did not mean a hard challenge for the rats. Members of both groups could find it within a relatively short time: mean escape latencies were 83 s and 65 s at the first replacement trial and 58 s and 74 s at the second one in the control and drug-treated groups, respectively. In the cooperation paradigm, where the rats had to perform simultaneous nose-pokes to get a reward, task difficulty was increased by requiring two consecutive simultaneous nose-poking from the animals. This caused a fall in the percentage of successful trials in both groups (from 48 % to 12 % and from 50 % to 20 % in the saline - and drug-treated group, respectively), however, by the end of the treatment RO4368554-treated animals showed significantly higher performance (29 %) than saline treated rats (2%). The NOR test, carried out with a 5 -h delay, revealed poor recognition memory in both groups (discrimination index (DI) values were 0.13 and 0.06 for saline and RO4368554, respectively). Performance in the pot jumping test was also not improved by the drug-treatment. CONCLUSIONS: The applied study design allowed parallel measurements of the action of the test compound on several cognitive functions and to follow its time course. RO4368554 did not show notable effects on impaired attention and visual recognition; nor did it affect spatial and procedural learning, but it exerted beneficial effect on cooperative behaviour. The revealed activity pattern highlight the cognitive domain most sensitive to the particular drug effect and may give hints for further target validating and clinical studies.

Efficacy of weight loss intervention can be predicted based on early alterations of fMRI food cue reactivity in the striatum.

Increased fMRI food cue reactivity in obesity, i.e. higher responses to high- vs. low-calorie food images, is a promising marker of the dysregulated brain reward system underlying enhanced susceptibility to obesogenic environmental cues. Recently, it has also been shown that weight loss interventions might affect fMRI food cue reactivity and that there is a close association between the alteration of cue reactivity and the outcome of the intervention. Here we tested whether fMRI food cue reactivity could be used as a marker of diet-induced early changes of neural processing in the striatum that are predictive of the outcome of the weight loss intervention. To this end we investigated the relationship between food cue reactivity in the striatum measured one month after the onset of the weight loss program and weight changes obtained at the end of the six-month intervention. We observed a significant correlation between BMI change measured after six months and early alterations of fMRI food cue reactivity in the striatum, including the bilateral putamen, right pallidum, and left caudate. Our findings provide evidence for diet-induced early alterations of fMRI food cue reactivity in the striatum that can predict the outcome of the weight loss intervention.

Acute 5-HT2C Receptor Antagonist SB-242084 Treatment Affects EEG Gamma Band Activity Similarly to Chronic Escitalopram.

Serotonin 2C receptors (5-HT2CRs) are implicated in the pathomechanism and treatment of anxiety and depression. Recently, as a new biomarker of depression, alterations in the gamma power of the electroencephalogram (EEG) have been suggested. Chronic treatment with the selective serotonin reuptake inhibitor (SSRI) antidepressant escitalopram has been shown to cause sleep-wake stage-dependent alterations in gamma power. However, despite the antidepressant potency of 5-HT2CR-antagonists, there is no data available regarding the effects of selective 5-HT2CR-antagonists on gamma activity. Therefore, we investigate the acute effect of the 5-HT2CR-antagonist SB-242084 on gamma power in different vigilance stages when given in monotherapy, or in combination with chronic escitalopram treatment. We administered SB-242084 (1 mg/kg, intraperitoneally) or vehicle to EEG-equipped rats after a 21-day-long pretreatment with escitalopram (10 mg/kg/day, via osmotic minipumps) or vehicle. Frontoparietal EEG, electromyogram, and motor activity were recorded during the first 3 h of passive phase, after the administration of SB-242084. Quantitative EEG analysis revealed that acute SB-242084 increased gamma power (30-60 Hz) in light and deep slow-wave sleep, and passive wakefulness. However, in active wakefulness, rapid eye movement sleep, and intermediate stage, no change was observed in gamma power. The profile of the effect of SB-242084 on gamma power was similar to that produced by chronic escitalopram. Moreover, SB-242084 did not alter chronic escitalopram-induced effects on gamma. In conclusion, the similarity in the effect of the 5-HT2CR-antagonist and chronic SSRI on gamma power provides further evidence for the therapeutic potential of 5-HT2CR-antagonists in the treatment of depression and/or anxiety.

Differential regulation of metabolic parameters by energy deficit and hunger.

AIMS: Hypocaloric diet decreases both energy expenditure (EE) and respiratory exchange rate (RER), affecting the efficacy of dieting inversely. Energy deficit and hunger may be modulated separately both in human and animal studies by drug treatment or food restriction. Thus it is important to separate the effects of energy deficit and hunger on EE and RER. METHODS: Three parallel and analogous experiments were performed using three pharmacologically distinct anorectic drugs: rimonabant, sibutramine and tramadol. Metabolic parameters of vehicle- and drug-treated and pair-fed diet-induced obese mice from the three experiments underwent common statistical analysis to identify effects independent of the mechanisms of action. Diet-induced obesity (DIO) test of tramadol was also performed to examine its anti-obesity efficacy. RESULTS: RER was decreased similarly by drug treatments and paired feeding throughout the experiment irrespective of the cause of reduced food intake. Contrarily, during the passive phase, EE was decreased more by paired feeding than by both vehicle and drug treatment irrespective of the drug used. In the active phase, EE was influenced by the pharmacological mechanisms of action. Tramadol decreased body weight in the DIO test. CONCLUSIONS: Our results suggest that RER is mainly affected by the actual state of energy balance; conversely, EE is rather influenced by hunger. Therefore, pharmacological medications that decrease hunger may enhance the efficacy of a hypocaloric diet by maintaining metabolic rate. Furthermore, our results yield the proposal that effects of anorectic drugs on EE and RER should be determined compared to vehicle and pair-fed groups, respectively, in animal models.

Chronic escitalopram treatment attenuated the accelerated rapid eye movement sleep transitions after selective rapid eye movement sleep deprivation: a model-based analysis using Markov chains.

BACKGROUND: Shortened rapid eye movement (REM) sleep latency and increased REM sleep amount are presumed biological markers of depression. These sleep alterations are also observable in several animal models of depression as well as during the rebound sleep after selective REM sleep deprivation (RD). Furthermore, REM sleep fragmentation is typically associated with stress procedures and anxiety. The selective serotonin reuptake inhibitor (SSRI) antidepressants reduce REM sleep time and increase REM latency after acute dosing in normal condition and even during REM rebound following RD. However, their therapeutic outcome evolves only after weeks of treatment, and the effects of chronic treatment in REM-deprived animals have not been studied yet. RESULTS: Chronic escitalopram- (10 mg/kg/day, osmotic minipump for 24 days) or vehicle-treated rats were subjected to a 3-day-long RD on day 21 using the flower pot procedure or kept in home cage. On day 24, fronto-parietal electroencephalogram, electromyogram and motility were recorded in the first 2 h of the passive phase. The observed sleep patterns were characterized applying standard sleep metrics, by modelling the transitions between sleep phases using Markov chains and by spectral analysis. Based on Markov chain analysis, chronic escitalopram treatment attenuated the REM sleep fragmentation [accelerated transition rates between REM and non-REM (NREM) stages, decreased REM sleep residence time between two transitions] during the rebound sleep. Additionally, the antidepressant avoided the frequent awakenings during the first 30 min of recovery period. The spectral analysis showed that the SSRI prevented the RD-caused elevation in theta (5-9 Hz) power during slow-wave sleep. Conversely, based on the aggregate sleep metrics, escitalopram had only moderate effects and it did not significantly attenuate the REM rebound after RD. CONCLUSION: In conclusion, chronic SSRI treatment is capable of reducing several effects on sleep which might be the consequence of the sub-chronic stress caused by the flower pot method. These data might support the antidepressant activity of SSRIs, and may allude that investigating the rebound period following the flower pot protocol could be useful to detect antidepressant drug response. Markov analysis is a suitable method to study the sleep pattern.

Recent patents on novel MCH1 receptor antagonists as potential anti-obesity drugs.

Today, the 'obesity pandemic' is one of the biggest health issues around the world. Melanin-concentrating hormone (MCH), a hypothalamic neuropeptide, is one of the most potent, central stimulators of feeding and it also attenuates energy expenditure. Inhibitions of the MCH receptor, the melanin-concentrating hormone receptor-1 (MCHR1), has attracted considerable attention as a potential anti-obesity drug, during the last decade. Now, there are a large number of MCHR1 antagonists, pharmacological tools and clinical drug candidates that can provide clues to develop new structures with high potency and good pharmacokinetic profile. The function of MCHR1 in energy homeostasis, obesity, metabolic syndrome, mood disorders and inflammatory bowel disease is discussed. Relevant clinical trials and patent background information of the MCHR1 antagonists over the last 4 years are also reviewed.

Acute escitalopram treatment inhibits REM sleep rebound and activation of MCH-expressing neurons in the lateral hypothalamus after long term selective REM sleep deprivation.

RATIONALE: Selective rapid eye movement sleep (REMS) deprivation using the platform-on-water ("flower pot") method causes sleep rebound with increased REMS, decreased REMS latency, and activation of the melanin-concentrating hormone (MCH) expressing neurons in the hypothalamus. MCH is implicated in the pathomechanism of depression regarding its influence on mood, feeding behavior, and REMS. OBJECTIVES: We investigated the effects of the most selective serotonin reuptake inhibitor escitalopram on sleep rebound following REMS deprivation and, in parallel, on the activation of MCH-containing neurons. METHODS: Escitalopram or vehicle (10 mg/kg, intraperitoneally) was administered to REMS-deprived (72 h) or home cage male Wistar rats. During the 3-h-long "rebound sleep", electroencephalography was recorded, followed by an MCH/Fos double immunohistochemistry. RESULTS: During REMS rebound, the time spent in REMS and the number of MCH/Fos double-labeled neurons in the lateral hypothalamus increased markedly, and REMS latency showed a significant decrease. All these effects of REMS deprivation were significantly attenuated by escitalopram treatment. Besides the REMS-suppressing effects, escitalopram caused an increase in amount of and decrease in latency of slow wave sleep during the rebound. CONCLUSIONS: These results show that despite the high REMS pressure caused by REMS deprivation procedure, escitalopram has the ability to suppress REMS rebound, as well as to diminish the activation of MCH-containing neurons, in parallel. Escitalopram caused a shift from REMS to slow wave sleep during the rebound. Furthermore, these data point to the potential connection between the serotonergic system and MCH in sleep regulation, which can be relevant in depression and in other mood disorders.

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.

Small platform sleep deprivation selectively increases the average duration of rapid eye movement sleep episodes during sleep rebound.

The single platform-on-water (flower pot) method is extensively used for depriving rapid eye movement sleep (REMS). Detailed comparison of sleep-wake architecture, recorded during the rebound period after spending three days on either a small or large platform, could separate the effects of REMS deficit from other stress factors caused by the procedure. A further aim of the study was to find the most characteristic REMS parameter of the rebound originating from REMS deficit. Rats were kept on a small or large platform for 72 h. Their fronto-parietal electroencephalogram, electromyogram and motility were recorded during the 24 h rebound at the beginning of the passive phase. A similar period of a home cage group was also recorded. The most typical differences between the two rebound groups were the increased cumulative time and longer average duration of REMS episodes without significant change in the number of these episodes of the small platform animals during the passive phase. Results obtained by cosinor analysis were in accordance with the findings above. Since we did not find any difference in the average duration of REMS episodes comparing the large platform rebound group and the home cage group, we concluded that the increased mean duration of REMS episodes is a selective marker for the rebound caused by small platform sleep deprivation, while other changes in sleep architecture may be the consequence of stress and also some sleep deficit.

MDMA treatment 6 months earlier attenuates the effects of CP-94,253, a 5-HT1B receptor agonist, on motor control but not sleep inhibition.

The possible long-term effects of the recreational drug "ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) on the function of 5-hydroxytryptamine-1B (5-HT(1B)) receptor in sleep and motor control were investigated using a selective 5-HT(1B) receptor agonist, 5-propoxy-3-(1,2,3,6-tetrahydro-4-pyrinzidyl)-1H-pyrrolo([3,2-b])pyridine hydrochloride (CP-94,253; 5 mg/kg). CP-94,253 or vehicle was administered to freely moving rats pre-treated with MDMA (15 mg/kg) or vehicle 6 months earlier, and polygraphic recording for 24 h and motor activity measurements were performed. Active wake (AW), passive wake (PW), light slow wave sleep (SWS-1), deep slow wave sleep (SWS-2), paradoxical sleep (PS), and diurnal rhythm were analyzed for the whole period. In additional, the EEG power spectrum was calculated for the second hour after the acute treatment for AW, PW, SWS-1, and SWS-2. 5-HT transporter (5-HTT) immunohistochemistry was measured in brain areas related to sleep and motor control 6 months after MDMA treatment. CP-94,253 increased AW and PW, decreased SWS-2 and PS, and altered parameters of diurnal rhythm in control animals. CP-94,253 decreased the EEG power spectra at higher frequencies. The effects of CP-94,253 on AW and diurnal rhythm were reduced or eliminated in MDMA-treated animals. MDMA treatment decreased 5-HTT fibre density in posterior hypothalamus, tuberomammillary nucleus, caudate putamen and ventrolateral striatum. These data suggest that long-term changes in 5-HT(1B) receptor function occur after serotonergic damage caused by a single dose of MDMA.