Cornelia Flachskamm

The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1-/-) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1-/- mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1-/- mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and preproorexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1-/- mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.

Forced swim stress activates rat hippocampal serotonergic neurotransmission involving a corticotropin-releasing hormone receptor-dependent mechanism

Serotonin is important for adequate coping with stress. Aberrant serotonin function is implicated in the aetiology of major depression and anxiety disorders. Dysregulation of the hypothalamic–pituitary–adrenocortical axis, involving elevated corticotropin‐releasing hormone (CRH) activity, also plays a role in these stress‐related illnesses. Here we studied the effects of stress on hippocampal serotonin and the role of the CRH system using in vivo microdialysis. First, rats were subjected to a forced swim stress, resulting in a dramatic increase in hippocampal serotonin (1500% of baseline), which was associated with the occurrence of diving behaviour. The diving‐associated increase in serotonin depended on activation of CRH receptors, as it was antagonized by intracerebroventricular pretreatment with D‐Phe‐CRH12−41. Secondly, the effects of intracerebroventricular administration of CRH and urocortin (0.03–1.0 µg) were studied. Both CRH and urocortin caused a dose‐dependent rise in hippocampal serotonin (maximally 350% of baseline) and 5‐hydroxyindoleacetic acid levels, suggesting the involvement of CRH receptor type 1. Because the effects of urocortin were prolonged, CRH receptor type 2 could play a role in a later phase of the neurotransmitter response. Experiments using adrenalectomized rats showed that CRH‐induced serotonin changes were adrenally independent. These data suggest that the raphe‐hippocampal serotonin system is able to mount, CRH receptor‐dependent, responses to specific stressful situations that surpass the usually observed maximal increases of about 300% of baseline during stress and enhanced vigilance.

Activation of serotonergic and noradrenergic neurotransmission in the rat hippocampus after peripheral administration of bacterial endotoxin: involvement of the cyclo-oxygenase pathway

An endotoxic challenge produces pronounced effects on the immune, endocrine and central nervous systems. However, information on the brain structures and neurotransmitter systems participating in the physiological responses after stimulation of the immune system is still scarce. Using an in vivo microdialysis method is conscious, freely moving rats, the present study describes the effects of an endotoxic challenge on hippocampal serotonergic and noradrenergic neurotransmission. Rats were equipped with a microdialysis probe in the hippocampus, which enables the stress-free measurement of extracellular concentrations of serotonin, noradrenaline and their respective metabolites 5-hydroxyindoleacetic acid and 3-methoxy-4-hydroxyphenylglycol. The behavioral activity was scored by measurement of the time during which rats were active (locomotion, grooming, eating, drinking). In the control rats a significant, positive relationship between the behavioral activity and hippocampal extracellular levels of serotonin, noradrenaline and 3-methoxy-4-hydroxyphenylglycol was found. Intraperitoneally injected bacterial endotoxin (lipopolysaccharide; 100 micrograms/kg body weight) increased extracellular concentrations of serotonin, 5-hydroxyindoleacetic acid, noradrenaline and 3-methoxy-4-hydroxyphenylglycol, whereas the behavioral activity was largely reduced, thus disrupting the correlation between behavioral activity and hippocampal levels of serotonin, noradrenaline and 3-methoxy-4-hydroxyphenylglycol. Intraperitoneal pretreatment of rats with the cyclo-oxygenase inhibitor indomethacin attenuated, but did not completely abolish, the endotoxin-induced increases in hippocampal extracellular levels of serotonin, noradrenaline and their metabolites. From these results it may be concluded that the hippocampal serotonin and noradrenaline neurotransmitter systems are part of the brain circuitry responsive to an endotoxic challenge. Moreover, arachidonic acid metabolites seem to represent important, but not the sole, mediators of the endotoxin-induced changes in hippocampal neurotransmission.

Intraperitoneal administration of bacterial endotoxin enhances noradrenergic neurotransmission in the rat preoptic area: relationship with body temperature and hypothalamic-pituitary -adrenocortical axis activity

A combined in vivo microdialysis/biotelemetry method in freely moving rats was used to study the effects of an endotoxic challenge on brain neurotransmission, hypothalamic‐pituitary‐adrenocortical (HPA) axis activity, autonomic functions and behaviour. Rats were equipped with a microdialysis probe in the preoptic area and a transmitter for biotelemetry in the peritoneal cavity. Time‐dependent changes in noradrenergic and serotonergic neurotransmission, and HPA axis activity were monitored by measuring noradrenaline, serotonin, their metabolites and free corticosterone concentrations in dialysates. Core body temperature, heart rate and locomotion were measured simultaneously by biotelemetry. In addition, total behavioural activity was scored by measuring the time during which rats were active. Intraperitoneal administration of endotoxin (lipopolysaccharide; 100 μg/kg body weight) caused a pronounced increase in preoptic extracellular concentrations of noradrenaline and its metabolite 3‐methoxy‐4‐hydroxyphenylglycol (MHPG; 500 and 400% of baseline respectively). No effect was found on preoptic concentrations of serotonin, although the levels of its metabolite 5‐hydroxyindoleacetic acid were slightly elevated (120% of baseline). Intraperitoneal lipopolysaccharide caused a marked increase in corticosterone levels, a decline in behavioural activity, and biphasic rises in body temperature and heart rate. Analysis of the time curves revealed that noradrenaline rose in parallel with the first increase in body temperature and the increase in corticosterone levels. Moreover, maximum noradrenaline levels were reached ˜60 min earlier than the peak in body temperature and corticosterone concentrations. Intraperitoneal pretreatment with the cyclo‐oxygenase inhibitor indomethacin prevented the lipopolysaccharide‐induced changes in body temperature, heart rate and behavioural activity, whereas the changes in noradrenaline, MHPG and corticosterone were largely, but not completely, reduced. Taken together, the results show that an endotoxic challenge results in a highly differentiated response in brain neurotransmission. We postulate that the profound increase in preoptic noradrenergic neurotransmission may be related to the lipopolysaccharide‐evoked induction of fever and/or activation of the HPA axis.

Differential monoaminergic, neuroendocrine and behavioural responses after central administration of corticotropin-releasing factor receptor type 1 and type 2 agonists

Corticotropin‐releasing factor (CRF) mediates various aspects of the stress response. To differentiate between the roles of CRF1 and CRF2 receptor subtypes in monoaminergic neurotransmission, hypothalamic–pituitary–adrenocortical axis activity and behaviour we compared the effects of CRF and urocortin 1 with those of the selective CRF2 receptor ligands urocortin 2 and urocortin 3. In vivo microdialysis in the rat hippocampus was used to assess free corticosterone, extracellular levels of serotonin (5‐HT) and noradrenaline (NA), and their metabolites 5‐hydroxyindoleacetic acid (5‐HIAA) and 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), respectively. Intracerebroventricular (i.c.v.) injection of CRF and urocortin 1, 2 and 3 (1.0 µg) increased hippocampal levels of 5‐HT and 5‐HIAA. CRF and urocortin 1 increased NA and MHPG, whereas urocortin 2 and urocortin 3 elevated MHPG, but not NA levels. CRF and the urocortins induced an immediate increase in behavioural activity. CRF and urocortin 1 mainly caused grooming and exploratory behaviour. In contrast, urocortin 2 and urocortin 3 both induced exploratory behaviour, but not grooming, and increased time spent eating food pellets. All urocortins, but not CRF, suppressed food intake 4–6 h after injection. Hippocampal free corticosterone levels were elevated by CRF, urocortin 1 and 3, but not by urocortin 2. The time courses of the CRF‐ and urocortin 1‐induced responses were significantly prolonged as compared to those of the CRF2 receptor ligands. The stimulatory changes evoked by CRF and urocortin 1 were present up to 4–6 h after injection, whereas the effects of urocortin 2 and urocortin 3 returned to baseline within 2.5 h after injection. Pre‐treatment with the selective antagonist antisauvagine‐30 (5.0 µg, i.c.v.) confirmed that the effects of urocortin 3 were CRF2 receptor‐mediated. The differential time course of the monoaminergic, neuroendocrine and behavioural effects of CRF and urocortin 1, as compared to urocortin 2 and urocortin 3, and the specific behavioural pattern induced by the CRF2 receptor ligands, suggest a distinct role for CRF2 receptors in the stress response.

Corticotropin-releasing hormone receptor type 1-deficiency enhances hippocampal serotonergic neurotransmission: An in vivo microdialysis study in mutant mice

Corticotropin-releasing hormone plays an important role in the coordination of various responses to stress. Previous research has implicated both corticotropin-releasing hormone and the serotonergic system as causative factors in the development and course of stress-related psychiatric disorders such as major depression. To delineate the role of the corticotropin-releasing hormone receptor type 1 (CRH-R1) in the interactions between corticotropin-releasing hormone and serotonergic neurotransmission, in vivo microdialysis was performed in CRH-R1-deficient mice under basal (home cage) and stress (forced swimming) conditions. Hippocampal dialysates were used to measure extracellular levels of serotonin and its metabolite 5-hydroxyindoleacetic acid, and free corticosterone levels to monitor the status of the hypothalamic-pituitary-adrenocortical axis. Moreover, behavioural activity was assessed by visual observation and a scoring paradigm. Both wild-type and heterozygous mutant mice showed a clear diurnal rhythm in free corticosterone. Free corticosterone concentrations were, however, lower in heterozygous mutant mice than in wild-type animals and undetectable in homozygous CRH-R1-deficient mice. Homozygous CRH-R1-deficient mice showed enhanced hippocampal levels of 5-hydroxyindoleacetic acid but not of serotonin during the light and the dark phase of the diurnal cycle, which may point to an enhanced synthesis of serotonin in the raphe-hippocampal system. Moreover, the mutation resulted in higher behavioural activity in the home cage during the light but not during the dark period. Forced swimming caused a rise in hippocampal serotonin followed by a further increase after the end of the stress paradigm in all genotypes. Homozygous and heterozygous mutant mice showed, however, a significantly amplified serotonin response to the forced swimming as compared to wild-type control animals. We conclude that CRH-R1-deficiency results in reduced hypothalamic-pituitary-adrenocortical axis activity, in enhanced synthesis of serotonin during basal conditions, and in an augmented response in extracellular levels of serotonin to stress. These data provide further evidence for the intricate relationship between corticotropin-releasing hormone and serotonin and the important role of the CRH-R1 herein.

Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep.

Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

Altered Serotonergic Neurotransmission but Normal Hypothalamic–Pituitary–Adrenocortical Axis Activity in Mice Chronically Treated with the Corticotropin-Releasing Hormone Receptor Type 1 Antagonist NBI 30775

Antagonists of the corticotropin-releasing hormone receptor type 1 (CRH-R1) are regarded as promising tools for the treatment of stress-related psychiatric disorders. Owing to the intricate relationship between CRH and serotonin (5-HT), we studied the effects of chronic oral treatment of C57Bl6/N mice with the CRH-R1 antagonist NBI 30775 (formerly known as R121919) on hippocampal serotonergic neurotransmission during basal (on 15th day of treatment) and stress (forced swimming; on 16th day of treatment) conditions by in vivo microdialysis. Given the important role of CRH in the regulation of hypothalamic–pituitary–adrenocortical (HPA) axis activity and behavior, the effects of NBI 30775 on dialysate-free corticosterone levels, and on home cage and forced swimming-related behavior were also assessed. Chronic administration of NBI 30775 (18.4±0.9 mg/kg/day) did not result in alterations in food consumption and body weight. NBI 30775 caused complex changes in hippocampal serotonergic neurotransmission. Whereas no effects on the diurnal rhythms of 5-HT and its metabolite 5-hydroxyindoleacetic acid were found, the responses of the neurotransmitter and its metabolite to 10 min of forced swim stress were reduced and prolonged, respectively. NBI 30775 did not change free corticosterone levels over the diurnal rhythm. Moreover, NBI 30775-treated mice showed a similar forced swim stress-induced increase in corticosterone as observed in the control group. No effects of NBI 30775 on home cage, and swim stress-related active behaviors (climbing, swimming) and immobility were found. Thus, whereas chronic antagonism of CRH-R1 did not compromise HPA axis performance and behavior, distinct changes in serotonergic neurotransmission developed. Owing to the important role of 5-HT in the pathophysiology of mood and anxiety disorders, the latter observation may contribute to the therapeutical efficacy of CRH-R1 antagonists in these illnesses.

Effect of sleep and sleep deprivation on serotonergic neurotransmission in the hippocampus: a combined in vivo microdialysis/EEG study in rats.

Brainstem serotonergic neurotransmission is implicated in sleep regulation. However, the role of serotonin (5‐HT) in forebrain regions in sleep–wake mechanisms is still unclear. Here, we have investigated, using a combined in vivo microdialysis/electroencephalogram method, the relationship between hippocampal 5‐HT levels and sleep–wake behaviour in the rat. A clear‐cut relationship was found between hippocampal 5‐HT levels and vigilance state. The highest levels of 5‐HT were observed during wakefulness, whereas a progressive decrease of 5‐HT going from nonrapid eye movement sleep to rapid eye movement sleep was found. Sleep deprivation (SD) causes a transient enhancement of mood in depressed patients. Given the putative role of 5‐HT in the aetiology of depression and the therapeutical efficacy of selective serotonin reuptake inhibitors in this illness, we also studied hippocampal 5‐HT during 4 h of SD and during the subsequent recovery period. During the whole SD period, 5‐HT levels were elevated substantially when compared to 5‐HT levels during basal wakefulness. However, no changes in 5‐HT levels and the relationship between hippocampal 5‐HT and vigilance state were found during the subsequent recovery period. As SD is a potentially stressful experience and glucocorticoids are involved in the regulation of serotonergic neurotransmission and sleep, we investigated the effects of SD on free corticosterone levels. SD caused a marked rise in free corticosterone levels. However, the effects of SD on 5‐HT seem not to be mediated by this hormone, because adrenalectomy did not affect the rise in hippocampal 5‐HT during SD. We hypothesize that the elevated hippocampal 5‐HT levels during SD may participate in the transient mood enhancing properties of forced wakefulness observed in depressed patients.

Effects of exposure to a predator on behaviour and serotonergic neurotransmission in different brain regions of C57BL/6N mice

Clinical studies and animal models have provided evidence that stress and serotonin may play a role in the aetiology of psychiatric diseases such as depression and anxiety. In addition, reciprocal interactions between stress and serotonergic neurotransmission have been demonstrated. However, the relationships between stress, serotonin and behaviour are far from completely understood. In this integrative study, we aimed to elucidate the effect of the psychological stress model predator exposure on behaviour and serotonergic neurotransmission in mice. We used a high time‐resolution microdialysis method to measure extracellular levels of serotonin (5‐hydroxytryptamine, 5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) in the hippocampus, prefrontal cortex, lateral septum and caudate putamen of C57bl/6N mice, before (08:30–10:30 h), during (10:30–11:00 h) and after exposure (11:00–14:00 h) to a rat. Detailed behavioural observations were also made. Rat exposure resulted in behavioural activation, with predominant risk‐assessment activities, and in increases in hippocampal, cortical, septal but not striatal 5‐HT and 5‐HIAA. When rat exposure was repeated on the consecutive day, small behavioural differences and reductions in 5‐HIAA levels, but no differences in the 5‐HT response, as compared with the first exposure were observed. As increases in 5‐HT often coincide with behavioural activation, it was particularly interesting to find that 5‐HT also increased in periods when mice only made minor movements such as sniffing, and that an effect of predator stress was absent in the caudate putamen. Our results indicate that the presence of the rat leads to differential activation of serotonergic neurotransmission in higher brain structures, probably involved in the coping response to this potentially life‐threatening situation.