Phase-and disorder-specific differences in peripheral metabolites of the kynurenine pathway in major depression, bipolar affective disorder and schizophrenia.

OBJECTIVES: Kynurenine, kynurenic and quinolinic acid are important metabolites in tryptophan metabolism. Due to an involvement in glutamatergic neurotransmission and immune response, previous studies have investigated this pathway in mental disorders such as major depressive disorder (MDD), bipolar disorder (BD) or schizophrenia (SCZ). Tryptophan and kynurenine have been shown to be decreased across disorders, hinting at the missing link how inflammation causes neurotoxicity and psychiatric symptoms. The main aim of our study was to investigate if individual catabolites could serve as diagnostic biomarkers for MDD, BD and SCZ. METHODS: We measured plasma levels of tryptophan, kynurenine, kynurenic acid, quinolinic acid and ratio of quinolinic acid/kynurenic acid using mass spectrometry in n = 175 participants with acute episodes and after remission, compared with controls. RESULTS: Decreased levels of all tryptophan catabolites were found in the whole patient group, driven by the difference between BD and HC. Manic and mixed phase BD individuals displayed significantly lower kynurenine and kynurenic acid levels. We could not find significant differences between disorders. Upon reaching remission, changes in catabolite levels partially normalised. CONCLUSIONS: Our data suggests an involvement of the kynurenine pathway in mental disorders, especially BD but disqualifying those metabolites as biomarkers for differential diagnosis.

Tryptophan catabolites and depression in the general population: results from the Gutenberg Health Study.

Previous studies reported significantly altered tryptophan catabolite concentrations in major depression. Thus, tryptophan catabolites were considered as potential biomarkers of depression and their modulators as potential targets for psychopharmacotherapy. However, the results were based mainly on studies with small sample sizes limiting their generalizability. Against this background, we investigated the relationship of peripheral tryptophan catabolites with depression in a population-based sample with n = 3,389 participants (with fasting status ≥ 8 h and C-reactive protein < 10 mg/L). N = 248 had clinically significant depression according to a PHQ-9 score of ≥ 10, n = 1,101 subjects had mild depressive symptoms with PHQ-9 scores between 5 and 9, and n = 2,040 had no depression. After multivariable adjustment, clinically significant depression was associated with lower kynurenine and kynurenic acid. Spearman correlation coefficients of the tryptophan catabolites with the severity of depression were very small (rho ≤ 0.080, p ≤ 0.015). None of the tryptophan catabolites could diagnostically separate depressed from not depressed persons. Concerning linear associations, kynurenine and kynurenic acid were associated only with the severity and the cognitive dimension of depression but not its somatic dimension. Tryptophan catabolites were not associated with persistence or recurrence of depression at the 5 year follow-up. The results replicated the association between kynurenine and kynurenic acid with depression. However, the associations were small raising doubts about their clinical utility. Findings underline the complexity of the relationships between depression and tryptophan catabolites. The search for subgroups of depression with a potentially higher impact of depression might be warranted.

The association between plasma tryptophan catabolites and depression: The role of symptom profiles and inflammation.

BACKGROUND: Tryptophan catabolites ("TRYCATs") produced by the kynurenine pathway (KP) may play a role in depression pathophysiology. Studies comparing TRYCATs levels in depressed subjects and controls provided mixed findings. We examined the association of TRYCATs levels with 1) the presence of Major Depressive Disorder (MDD), 2) depressive symptom profiles and 3) inflammatory markers. METHODS: The sample from the Netherlands Study of Depression and Anxiety included participants with current (n = 1100) or remitted (n = 753) MDD DSM-IV diagnosis and healthy controls (n = 642). Plasma levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KynA), quinolinic acid (QA), C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor (TNF) were measured. Atypical/energy-related symptom (AES), melancholic symptom (MS) and anxious-distress symptom (ADS) profiles were derived from questionnaires. RESULTS: After adjustment for age, sex, education, smoking status, alcohol consumption and chronic diseases, no significant differences in TRYCATs were found comparing MDD cases versus controls. The MS profile was associated (q < 0.05) with lower KynA (ß = -0.05), while AES was associated with higher KYN (ß = 0.05), QA (ß = 0.06) and TRP (ß = 0.06). Inflammatory markers were associated with higher KYN (CRP ß = 0.12, IL-6 ß = 0.08, TNF ß = 0.10) and QA (CRP ß = 0.21, IL-6 ß = 0.12, TNF ß = 0.18). Significant differences against controls emerged after selecting MDD cases with high (top 30%) CRP (KYN d = 0.20, QA d = 0.33) and high TNF (KYN d = 0.24; QA d = 0.39). CONCLUSIONS: TRYCATs levels were related to specific clinical and biological features, such as atypical symptoms or a proinflammatory status. Modulation of KP may potentially benefit a specific subset of depressed patients. Clinical studies should focus on patients with clear evidence of KP dysregulations.

Blood plasma B vitamins in depression and the therapeutic response to electroconvulsive therapy.

A growing body of research has indicated a role for B vitamins in depression, with some previous studies suggesting that B vitamin status in patients with depression can impact on antidepressant response. Here we aimed to investigate B vitamin plasma concentrations in medicated patients with depression (n â€‹= â€‹94) compared to age- and sex-matched healthy controls (n â€‹= â€‹57), and in patients with depression after electroconvulsive therapy (ECT) in a real-world clinical setting. Our results show that nicotinamide (vitamin B3), N1-methylnicotinamide (vitamin B3 metabolite), and pyridoxal 5'-phosphate (PLP; vitamin B6) concentrations were significantly reduced in patients with depression compared to controls. The Cohen's d effect sizes for nicotinamide, N1-methylnicotinamide, and PLP were moderate-large (-0.47, -0.51, and -0.59, respectively), and likely to be of clinical relevance. Functional biomarkers of vitamin B6 status (PAr index, 3-hydroxykynurenine: hydroxyanthranilic acid ratio, 3-hydroxykynurenine: xanthurenic acid ratio, and HKr) were elevated in depressed patients compared to controls, suggestive of reduced vitamin B6 function. Over 30% of the patient cohort were found to have low to deficient PLP concentrations, and exploratory analyses revealed that these patients had higher IL-6 and CRP concentrations compared to patients with PLP levels within the normal range. Treatment with ECT did not alter B vitamin concentrations, and B vitamin concentrations were not associated with depression severity or the therapeutic response to ECT. Overall, reduced plasma PLP, nicotinamide, and N1-methylnicotinamide concentrations could have wide ranging effects on pathways and systems implicated in depression. Further studies are required to understand the reasons why patients with depression present with low plasma B vitamin concentrations.

Tryptophan metabolite concentrations in depressed patients before and after electroconvulsive therapy.

Tryptophan and kynurenine pathway (KP) metabolites are implicated in the pathophysiology of depression. We aimed to investigate their plasma concentrations in medicated patients with depression (n = 94) compared to age- and sex-matched healthy controls (n = 57), and in patients with depression after electroconvulsive therapy (ECT) in a real-world clinical setting, taking account of co-variables including ECT modality and heterogenous psychopathology. Depression severity was assessed using the Hamilton Depression Rating Scale (HAM-D24). Tryptophan (TRP) and kynurenine (KYN) metabolite concentrations [anthranilic acid (AA), 3-hydroxyanthranilic acid (3HAA), picolinic acid (PA), kynurenic acid (KYNA), and xanthurenic acid (XA)] and KYNA/KYN and KYNA/quinolinic acid (QUIN) ratios were lower in patients compared to controls. For the total group there was no significant change in KP metabolites post-ECT or correlations with mood ratings. However, improvements in mood score were correlated with increased KYN, 3-hydroxykynurenine (3HK), 3HAA, QUIN, and KYN/TRP in a subgroup of unipolar depressed patients. Additionally, in remitters baseline KYN, 3HK, and QUIN were associated with baseline HAM-D24 scores, and changes in 3HK and 3HAA concentrations post-ECT correlated with improvement in mood. KYN, KYNA, AA, 3HK, XA, PA, and QUIN were increased in a smaller 3-month follow-up group (n = 19) compared to pre-ECT concentrations. Overall, the results indicate that ECT mobilizes the KP, where a moderate association between selected metabolites and treatment response in unipolar depressed patients is evident.

Altered tryptophan catabolite concentrations in major depressive disorder and associated changes in hippocampal subfield volumes.

BACKGROUND: Tryptophan depletion is a well-replicated biological finding in Major Depressive Disorder (MDD). The kynurenine pathway (KP) and its rate-limiting tryptophan degrading enzyme, indolamine 2,3 dioxygenase (IDO), have been implicated in the pathogenesis of depression. IDO expression is driven by inflammatory cytokines, providing a putative link between inflammation and neuropathology. This study examined circulating concentrations of C-reactive protein (CRP), plasma tryptophan, kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) and whole blood mRNA expression of IDO in patients with major depressive disorder (MDD) compared with healthy controls (HC). METHODS: A diagnosis of major depression was made according to DSM-IV. Depression severity was assessed using the Hamilton depression (HAM-D) rating scale. 74 MDD patients, 39 with a first presentation of MDD (fpMDD) and 35 with chronic or recurrent episodes (rMDD), and 37 HC were recruited to the study. Whole blood and plasma samples were collected. Expression of markers in whole blood were measured by PCR, circulating CRP by ELISA and KP metabolites by LC-MS/MS. Hippocampal cornu ammonis (CA) and subiculum volumes were determined by MRI and calculated using FreeSurfer. RESULTS: Tryptophan concentrations were significantly reduced in MDD compared to HC. There was a positive correlation between QUIN and both CRP concentrations and whole blood IDO1 in MDD. KYNA concentrations were reduced in MDD patients presenting with a first episode (fpMDD) compared to those presenting with recurrent depression (rMDD) and HC. By contrast QUIN concentrations were elevated in rMDD compared to fpMDD and HC. KYNA/QUIN was reduced in MDD and rMDD but not fpMDD compared to HC. Hippocampal subfield volumes were smaller in MDD patients than HC for CA1 (left only), CA2/3 (left and right) and CA4 (right only). CRP and CA1 volumes were negatively correlated bilaterally in MDD patients. KYNA and subiculum volume were positively correlated bilaterally. DISCUSSION: This study found evidence of KP metabolism imbalance in MDD patients in addition to tryptophan reduction and mild immune activation. Relationships between CRP and KYNA with some hippocampal subfield volumes in MDD patients suggest that this inflammatory signature may be associated with reduced hippocampal subfield volumes in depression.

Flibanserin-Stimulated Partner Grooming Reflects Brain Metabolism Changes in Female Marmosets.

INTRODUCTION: Female sexual interest and arousal disorder is personally distressing for women. To better understand the mechanism of the candidate therapeutic, flibanserin, we determined its effects on an index of brain glucose metabolism. AIM: We hypothesized that chronic treatment with flibanserin would alter metabolism in brain regions associated with serotonergic function and female sexual behavior. METHODS: In a crossover design, eight adult female common marmosets (Calithrix jacchus) received daily flibanserin or vehicle. After 7-12 weeks of treatment, the glucose metabolism radiotracer [(18) F]fluorodeoxyglucose (FDG) was administered to each female immediately prior to 30 minutes of interaction with her male pairmate, after which females were anesthetized and imaged by positron emission tomography. Whole-brain normalized images were analyzed with anatomically defined regions of interest. Whole-brain voxelwise mapping was used to explore treatment effects. Correlations were examined between alterations in metabolism and pairmate social grooming. MAIN OUTCOME MEASURES: Changes in metabolism associated with flibanserin were determined for dorsal raphe, medial prefrontal cortex (mPFC), medial preoptic area of hypothalamus (mPOA), ventromedial nucleus of hypothalamus, and field cornu ammonis 1 (CA1) of the hippocampus. RESULTS: In response to chronic flibanserin, metabolism in mPOA declined, and this reduction correlated with increases in pairmate grooming. A cluster of voxels in frontal cortico-limbic regions exhibited reduced metabolism in response to flibanserin and overlapped with a voxel cluster in which reductions in metabolism correlated with increases in pairmate grooming. Finally, reductions in mPOA metabolism correlated with increases in metabolism in a cluster of voxels in somatosensory cortex. CONCLUSIONS: Taken together, these results suggest that flibanserin-induced reductions in female mPOA neural activity increase intimate affiliative behavior with male pairmates.

Brain region-specific transcriptomic markers of serotonin-1A receptor agonist action mediating sexual rejection and aggression in female marmoset monkeys.

INTRODUCTION: In a marmoset model of hypoactive female sexual function, we have shown that repeated administration of the serotonin (5-HT)-1A agonist R-(+)-8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) inhibits sexual receptivity in female marmoset monkeys and increases aggression toward the male pairmate. AIM: The aims of this study are to investigate gene expression changes induced by 8-OH-DPAT in laser-microdissected brain areas that regulate female sexual function and to identify genes, functional gene classes, and pathways associated with 8-OH-DPAT-mediated inhibition of female sexual receptivity. METHODS: Gene expression was measured in the medial prefrontal cortex (mPFC), medial preoptic area (mPOA), cornu ammonis-1 (CA1) area of the hippocampus (CA1), and dorsal raphé nucleus (DRN) of four 8-OH-DPAT-treated (0.1 mg/kg; daily administration for 16 weeks) and four vehicle-treated female marmosets using a marmoset-specific microarray (European Marmoset Microarray [EUMAMA]) and validated by real-time quantitative polymerase chain reaction (RTqPCR). Enriched functional gene classes were determined. In a parallel candidate gene approach, the expression of serotonergic candidate genes, i.e., the 5-HT1A, 5-HT2A, and 5-HT7 receptors and the 5-HT transporter (5-HTT), was measured by RTqPCR. MAIN OUTCOME MEASURES: The main outcome is the differential expression of genes between 8-OH-DPAT- and vehicle-treated marmosets. RESULTS: 8-OH-DPAT affected the gene classes important to neural development (mPFC, mPOA, and DRN), neurotransmission (mPOA), energy production (mPFC and mPOA), learning and memory (CA1), and intracellular signal transduction (DRN). Oxytocin (OXT) in the mPOA and 5-HTT in the DRN were strongly increased by 8-OH-DPAT. 5-HT1A tended to increase in the mPFC, while 5-HT7 was decreased in the CA1. CONCLUSIONS: Brain region-specific alterations of gene expression regulating neural circuitries, energy demands, and learning processes are associated with 8-OH-DPAT-induced decrease in female sexual receptivity and increase in pairmate aggression. The role of OXT in the serotonergic regulation of female sexual behavior and partner interactions warrants attention in future studies.

Flibanserin treatment increases appetitive sexual motivation in the female rat.

INTRODUCTION: Flibanserin is a mixed 5-HT1A agonist/5-HT2A antagonist that has been developed for the treatment of hypoactive sexual desire disorder in women. AIM: To assess the acute and chronic dose-response effects of flibanserin on measures of sexual desire and copulation in ovariectomized rats primed with estradiol benzoate (EB) alone or in combination with progesterone (P). METHODS: In Experiment 1, sexually experienced ovariectomized (OVX) rats at one testing site were rendered fully sexually receptive with EB + P priming and tested weekly with a sexually active male in bi-level pacing chambers following daily flibanserin treatment for 28 days. In Experiment 2, sexually experienced OVX rats at a different testing site received EB alone and were tested weekly with sexually active males following daily flibanserin treatment. MAIN OUTCOME MEASURES: Female appetitive behaviors (solicitations, hops and darts, anogenital investigations), defensive behaviors, pacing, lordosis, and male copulatory responses (intromissions and ejaculations) were measured during each 30-minute copulation test. RESULTS: Acute flibanserin or 1 week of chronic flibanserin treatment did not modify sexual responses in fully (EB + P) or partially (EB-alone) primed females. After 2 weeks of chronic treatment, fully primed females displayed significantly more solicitations than the three other groups. After 3 weeks of chronic treatment, a significant increase in female solicitations was observed in both hormone-treatment groups. CONCLUSION: This study shows the first evidence that chronic, but not acute, flibanserin treatment augments appetitive sexual behaviors in OVX female rats primed with EB + P or EB alone. Given the positive effect of flibanserin in clinical trials, these results confirm previous reports that solicitations in the female rat are a predictive animal model of human female sexual desire.

Chronic systemic administration of serotonergic ligands flibanserin and 8-OH-DPAT enhance HPA axis responses to restraint in female marmosets.

BACKGROUND: Flibanserin, a novel serotonin (5-HT)(1A) agonist and 5-HT(2A) antagonist, has been shown to increase sexual desire and reduce distress in women with Hypoactive Sexual Desire Disorder (HSDD). In marmoset monkeys, flibanserin has demonstrated pro-social effects on male-female pairmates, while the classic 5-HT(1A) agonist 8-OH-DPAT suppresses female sexual behavior and increases aggressive interactions between pairmates. Activation of 5-HT(1A) and 5-HT(2A) receptors is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis. This study aims to characterize the effects of repeated flibanserin and 8-OH-DPAT administration on the marmoset HPA axis and to elucidate endocrine correlates of altered marmoset pair behavior. METHODS: Adrenocorticotropic hormone (ACTH) and cortisol were examined at baseline and during 5-HT(1A) agonist and restraint challenges in 8 female marmoset monkeys receiving daily flibanserin (15mg/kg) and an additional 8 female marmosets receiving 8-OH-DPAT (0.1mg/kg) for 15-16weeks. Corresponding vehicle treatments were administered in a counterbalanced, within-subject design. All females were housed in stable male-female pairs. Treatment-induced changes in ACTH and cortisol levels were correlated with previously assessed marmoset pair behavior. RESULTS: While morning basal cortisol levels and HPA responses to a 5-HT(1A) agonist challenge were not altered by chronic flibanserin or 8-OH-DPAT, both treatments increased the responsiveness of the marmoset HPA axis to restraint. Enhanced ACTH responses to restraint correlated with reduced sexual receptivity and increased aggression in 8-OH-DPAT-, but not in flibanserin-treated female marmosets. CONCLUSIONS: Unaltered HPA responses to a 5-HT(1A) agonist challenge after chronic flibanserin and 8-OH-DPAT treatments indicate little or no de-sensitization of the HPA axis to repeated 5-HT(1A) manipulation. Chronic 8-OH-DPAT, but not flibanserin, leads to aggravated ACTH responses to stress that may contribute to anti-sexual and anti-social behavior between 8-OH-DPAT-treated females and their male pairmates. Despite similar flibanserin and 8-OH-DPAT induced ACTH responses to restraint stress, flibanserin-treated females show unchanged cortisol profiles. This is possibly due to flibanserin's regional selectivity in 5-HT(1A) activation and concurrent 5-HT(2A) inhibition. The contrasting restraint-related cortisol responses emulate contrasting behavioral phenotypes of diminished pair-bond of 8-OH-DPAT-treated females compared to the more affiliative pair-bond of flibanserin-treated females.

Flibanserin and 8-OH-DPAT implicate serotonin in association between female marmoset monkey sexual behavior and changes in pair-bond quality.

INTRODUCTION: Psychopathological origins of personally distressing, hypoactive sexual desire disorder (HSDD) in women are unknown, but are generally attributed to an inhibitory neural regulator, serotonin (5-HT). Flibanserin, a 5-HT(1A) agonist and 5-HT(2A) antagonist, shows promise as a treatment for HSDD. AIM: To test the hypothesis that female marmoset sexual behavior is enhanced by flibanserin and diminished by 8-OH-DPAT, in order to evaluate the efficacy of serotonergic modulation of female sexual behavior in a pairmate social setting comparable to humans. METHODS: Sexual and social behavior were examined in eight female marmoset monkeys receiving daily flibanserin (15 mg/kg), 8-OH-DPAT (0.1 mg/kg), or corresponding vehicle for 15-16 weeks in a counterbalanced, within-subject design, while housed in long-term, stable male-female pairs. MAIN OUTCOME MEASURES: Marmoset pairmate interactions, including sexual and social behavior, were scored during weeks 5-6 of daily flibanserin, 8-OH-DPAT or vehicle treatment. 24-hour pharmacokinetic profiles of the drugs and their metabolites, as well as drug-induced acute symptoms of the 5-HT behavioral syndrome were also assessed. RESULTS: Two-way analysis of variance reveals that flibanserin-treated females attract more male sexual interest (P=0.020) and trigger increased grooming (P=0.001) between partners. In contrast, 8-OH-DPAT-treated females show increased rejection of male sexual advances (P=0.024), a tendency for decreased male sexual interest (P=0.080), and increased aggression with their male pairmates (P=0.049). CONCLUSIONS: While 8-OH-DPAT-treated female marmosets display decreased sexual receptivity and increased aggressive interactions with their male pairmates, flibanserin-treated female marmosets demonstrate increased affiliative behavior with their male pairmates. Such pro-affiliation attributes may underlie flibanserin's effectiveness in treating HSDD in women.

Positron emission tomography assessment of 8-OH-DPAT-mediated changes in an index of cerebral glucose metabolism in female marmosets.

As part of a larger experiment investigating serotonergic regulation of female marmoset sexual behavior, this study was designed to (1) advance methods for PET imaging of common marmoset monkey brain, (2) measure normalized FDG uptake as an index of local cerebral metabolic rates for glucose, and (3) study changes induced in this index of cerebral glucose metabolism by chronic treatment of female marmosets with a serotonin 1A receptor (5-HT(1A)) agonist. We hypothesized that chronic treatment with the 5-HT(1A) agonist 8-OH-DPAT would alter the glucose metabolism index in dorsal raphe (DR), medial prefrontal cortex (mPFC), medial preoptic area of hypothalamus (mPOA), ventromedial nucleus of hypothalamus (VMH), and field CA1 of hippocampus. Eight adult ovariectomized female common marmosets (Callithrix jacchus) were studied with and without estradiol replacement. In a crossover design, each subject was treated daily with 8-OH-DPAT (0.1mg/kg SC daily) or saline. After 42-49 days of treatment, the glucose metabolism radiotracer FDG was administered to each female immediately prior to 30 min of interaction with her male pairmate, after which the subject was anesthetized and imaged by PET. Whole brain normalized PET images were analyzed with anatomically defined regions of interest (ROI). Whole brain voxelwise mapping was also used to explore treatment effects and correlations between alterations in the glucose metabolism index and pairmate interactions. The rank order of normalized FDG uptake was VMH/mPOA>DR>mPFC/CA1 in both conditions. 8-OH-DPAT did not induce alterations in the glucose metabolism index in ROIs. Voxelwise mapping showed a significant reduction in normalized FDG uptake in response to 8-OH-DPAT in a cluster in medial occipital cortex as well as a significant correlation between increased rejection of mount attempts and reduced normalized FDG uptake in an overlapping cluster. In conclusion, PET imaging has been used to measure FDG uptake relative to whole brain in marmoset monkeys. Voxelwise mapping shows that 8-OH-DPAT reduces this index of glucose metabolism in medial occipital cortex, consistent with alterations in female sexual behavior.

Multifunctional pharmacology of flibanserin: possible mechanism of therapeutic action in hypoactive sexual desire disorder.

INTRODUCTION: Flibanserin is a novel pharmacologic agent in late-stage clinical testing for hypoactive sexual desire disorder (HSDD) in premenopausal women. AIM: The aim of this article is to review the hypothetical mechanism of action of flibanserin in HSDD. METHODS: A literature review was conducted of all published works on flibanserin and on related studies of serotonin (5-HT)(1A) receptors and 5-HT(2A) receptors, including their actions on monoamines and on sexual function. MAIN OUTCOME MEASURES: The main outcome measures are preclinical pharmacologic actions, especially changes in regional monoamines following treatment with flibanserin. RESULTS: At clinically relevant doses, flibanserin acts predominantly at 5-HT(1A) receptors as an agonist and secondarily at 5-HT(2A) receptors as an antagonist. Additional binding actions within an order of magnitude of its 5-HT(1A) affinity, which are not likely to be clinically relevant, include weaker antagonist actions at 5-HT(2C) and 5-HT(2B) receptors, and less defined activity at dopamine (DA) D4 receptors. The 5-HT(1A) actions of flibanserin are only seen postsynaptically, which is unlike other agents such as buspirone that act at presynaptic 5-HT(1A) receptors. Furthermore, the postsynaptic actions of chronic flibanserin administration appear to demonstrate a preference for some populations of postsynaptic 5-HT receptors, particularly those that are located on the prefrontal cortex (PFC) pyramidal neurons, which regulate monoamine release in certain selective brain regions. CONCLUSIONS: The regional selectivity of flibanserin results in a unique pattern of monoamine modulation. Sustained increases in baseline of DA and norepinephrine (NE) are observed in the PFC, and flibanserin dosing increases DA and NE levels above the basal changes. Conversely, flibanserin induces transient decreases in 5-HT levels in some brain areas such as the PFC, nucleus accumbens, and hypothalamus, but not in other brain areas such as the hippocampus. Therefore, since DA and NE are excitatory and 5-HT is inhibitory to sexual desire and arousal, it is tempting to postulate that the actions of flibanserin on serotonin receptors at the PFC pyramidal neurons, resulting in increased DA and NE yet reduced 5-HT in the PFC, are the mechanistic underpinnings of enhancing sexual desire in HSDD.

Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain.

The melanocortin-4 receptor (MC4-R) plays a critical role in several physiological functions, from food intake, energy homeostasis, neuroendocrine and cardiovascular function, to sexual responses. The brain regions and the central neuronal pathways mediating the different actions of MC4-R remain largely unknown. We aimed to use immunocytochemistry using a specific antibody against rat MC4-R, to establish the detailed neuroanatomical distribution of MC4-R in brain slices of male and estrous female rats. We demonstrated that MC4-R-positive neurons were widely distributed in several brain regions including the cortex, thalamus, hypothalamus, and brainstem. In both male and female brains, MC4-R-positive cells were especially abundant in the hypothalamus, including the paraventricular hypothalamic nucleus, lateral septal nucleus, arcuate nucleus, supraoptic nucleus, medial preoptic area and lateral hypothalamic area. A moderate number of MC4-R-positive neurons were found in the piriform cortex, bed nucleus of the stria terminalis, medial and basolateral nuclei of amygdala, periaqueductal gray, red nucleus and raphe nucleus. A dimorphic sexual difference in the number of MC4-R-positive neurons was observed in some brain regions. In the medial preoptic area and arcuate nucleus, MC4-R-positive neurons were significantly more abundant in female than in males, whereas in the lateral hypothalamus the opposite proportion was observed. This is the first time the neuroanatomical distribution, and sex differences, of brain MC4-R localisation have been described. The distribution of MC4-R is consistent with the proposed roles of MC4-R-positive neurons and provides further information about the circuitry controlling food intake, energy balance and sexual responses in both males and females.

Flibanserin, a drug intended for treatment of hypoactive sexual desire disorder in pre-menopausal women, affects spontaneous motor activity and brain neurochemistry in female rats.

Flibanserin, a 5-HT(1A) receptor agonist and 5-HT(2A) receptor antagonist, is being developed for the treatment of hypoactive sexual desire disorder (HSDD) in pre-menopausal women. Here, we investigated the effects of acute administration of flibanserin (15 and 45 mg/kg, p.o.) and the selective 5-HT(1A) receptor agonist (+)-8-OH-DPAT (1 mg/kg, i.p.) on neurotransmitter levels in brain areas of female rats. Specifically, levels of dopamine (DA) and serotonin (5-HT) and neurotransmitter metabolites were examined in prefrontal cortex (PFC), nucleus accumbens, hypothalamus and brain stem using high performance liquid chromatography coupled to electrochemical detection. In addition, spontaneous motor activity was determined in an automated motor activity system. Flibanserin (45 mg/kg) but not (+)-8-OH-DPAT significantly reduced motor activity, when compared to vehicle controls. Specifically, the DA turnover was significantly increased (279%) in the PFC after flibanserin treatment but less pronounced (159%) after 8-OH-DPAT administration. Serotonin tissue levels were not altered in any of the investigated brain regions upon flibanserin treatment. However, flibanserin produced a significant decrease of the major serotonin metabolite 5-hydroxyindoleacetic acid and 5-HT turnover in the PFC, nucleus accumbens, hypothalamus and brain stem similar to (+)-8-OH-DPAT. In conclusion, the present study indicates that flibanserin is able to modulate dopaminergic and serotonergic activity in distinct brain areas. The observed effects in the PFC on dopaminergic markers are different from those induced by (+)-8-OH-DPAT and may contribute to its therapeutic efficacy in HSDD. The effects of flibanserin on spontaneous motor behaviour are in agreement with its receptor profile and underscore that flibanserin is devoid of any locomotor hyperactivity inducing properties.

Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina.

INTRODUCTION: The clitoris and the vagina are the main peripheral anatomical structures involved in physiological changes related to sexual arousal and orgasm. Their efferent control and, more particularly, the neurochemical phenotype of these descending neuronal pathways remain largely uncharacterized. AIM: To examine if brain neurons involved in the efferent control of the clitoris and the vagina possess melanocortin-4 receptor (MC4-R) and/or contain oxytocin (OT). METHODS: Neurons involved in the efferent control of the vagina and clitoris were identified following visualization of pseudorabies virus (PRV) retrograde tracing. PRV was injected into the vagina and clitoris in adult rats in estrous. On the fifth day postinjection, animals were humanely sacrificed, and brains were removed and sectioned, and processed for PRV visualization. The neurochemical phenotype of PRV-positive neurons was identified using double or triple immunocytochemical labeling against PRV, MC4-R, and OT. Double and triple labeling were quantified using confocal laser scanning microscopy. MAIN OUTCOME MEASURE: Neuroanatomical brain distribution, number and percentage of double-labeled PRV/MC4-R and PRV-/OT-positive neurons, and triple PRV-/MC4-R-/OT-labeled neurons. RESULTS: The majority of PRV immunopositive neurons which also expressed immunoreactivity for MC4-R were located in the paraventricular and arcuate nuclei of the hypothalamus. The majority of PRV positive neurons which were immunoreactive (IR) for OT were located in the paraventricular nucleus (PVN), medial preoptic area (MPOA), and lateral hypothalamus. PRV positive neurons were more likely to be IR for MC4-R than for OT. Scattered triple-labeled PRV/MC4-R/OT neurons were detected in the MPOA and the PVN. CONCLUSION: These data strongly suggest that MC4-R and, to a less extent, OT are involved in the efferent neuronal control of the clitoris and vagina, and consequently facilitate our understanding of how the melanocortinergic pathway regulates female sexual function.

Neuronal activity in the hypothalamic paraventricular nucleus varies across the estrous cycle in anesthetized female rats: effects of dopamine receptor agonism.

PURPOSE: The central nervous system plays a pivotal role in sexual behavior. The role of the paraventricular nucleus (PVN) of the hypothalamus in female sexual behavior is poorly characterized. In males, there is a correlation between PVN neuron activity and erectile activity, and in mediating dopamine receptor agonist-induced sexual arousal. MATERIAL AND METHODS: To understand the role of the PVN in female sexual function, baseline PVN neuronal activity and responses to dopamine receptor agonism were assessed in anesthetized rats. Single unit recordings were used to assess the firing properties of individual PVN neurons; and local field potentials quantified PVN network activity (combined activity of large numbers of PVN neurons). Baseline and apomorphine-stimulated activity was measured across the estrous cycle. RESULTS: Baseline firing rates of single units were found to differ across the stages of the estrous cycle with metestrus showing the highest firing rate (3.7 vs. 0.9 Hz in diestrus). Apomorphine administration caused significant increases in firing rate in 29% of neurons, and significant decreases in 71%. Basal local field potentials also varied between estrous stages and in response to apomorphine; significant differences in the total power of alpha1 and beta1 bands were observed in both cases. CONCLUSIONS: This study shows that PVN neuronal activity varies with hormonal state, and these neurons are differentially affected by apomorphine, suggesting two different populations. These data are indicative of a critical role for the PVN in female sexual function. It is important that the hormonal state should be considered when investigating sexual physiology and the effect of pharmacological agents.

Acute and repeated flibanserin administration in female rats modulates monoamines differentially across brain areas: a microdialysis study.

INTRODUCTION: Hypoactive sexual desire disorder (HSDD) is defined as persistent lack of sexual fantasies or desire marked by distress. With a prevalence of 10% it is the most common form of female sexual dysfunction. Recently, the serotonin-1A (5-HT(1A)) receptor agonist and the serotonin-2A (5-HT(2A)) receptor antagonist flibanserin were shown to be safe and efficacious in premenopausal women suffering from HSDD in phase III clinical trials. AIM: The current study aims to assess the effect of flibanserin on neurotransmitters serotonin (5-HT), norepinephrine (NE), dopamine (DA), glutamate, and gamma-aminobutyric acid (GABA) in brain areas associated with sexual behavior. METHODS: Flibanserin was administered to female Wistar rats (280-350 g). Microdialysis probes were stereotactically inserted into the mPFC, NAC, or MPOA, under isoflurane anesthesia. The extracellular levels of neurotransmitters were assessed in freely moving animals, 24 hours after the surgery. MAIN OUTCOME MEASURES: Dialysate levels of DA, NE, and serotonin from medial prefrontal cortex (mPFC), nucleus accumbens (NAC), and hypothalamic medial preoptic area (MPOA) from female rats. RESULTS: Acute flibanserin administration decreased 5-HT and increased NE levels in all tested areas. DA was increased in mPFC and MPOA, but not in the NAC. Basal levels of NE in mPFC and NAC and of DA in mPFC were increased upon repeated flibanserin administration, when compared to vehicle-treated animals. The basal levels of 5-HT were not altered by repeated flibanserin administration, but basal DA and NE levels were increased in the mPFC. Glutamate and GABA levels remained unchanged following either repeated or acute flibanserin treatment. CONCLUSIONS: Systemic administration of flibanserin to female rats differentially affects the monoamine systems of the brain. This may be the mechanistic underpinning of flibanserin's therapeutic efficacy in HSDD, as sexual behavior is controlled by an intricate interplay between stimulatory (catecholaminergic) and inhibitory (serotonergic) systems.

II. Slow oscillations in vaginal blood flow: regulation of vaginal blood flow patterns in rat by central and autonomic mechanisms.

INTRODUCTION: A new method for assessing female sexual arousal through changes in slow oscillatory patterns in vaginal blood flow was first described in the previous manuscript [1]. This method was translational and discriminated between normal healthy volunteers and women with female sexual arousal disorder. AIM: These studies addressed the influence of autonomic and central nervous systems on slow vaginal blood flow oscillations in rats. METHODS: Vaginal blood flow oscillations were measured in urethane-anesthetized rodents using laser Doppler flowmetry. Acquired data were filtered for frequency analysis range of 0.013-2.5 Hz. MAIN OUTCOME MEASURES: Data were assessed for changes in a high frequency range (HF = 0.6-2.5 Hz), and low frequency range (LF = 0.013-0.6 Hz). RESULTS: The basal HF oscillatory component of vaginal blood flow was primarily vagally mediated, although could be modulated pharmacologically with p-chloroamphetamine in the absence of vagal innervation. The LF component could be modulated by antagonists of noradrenergic receptors but did not appear to be dependent upon tonic activation of sympathetic circuitry. The non-selective dopamine receptor agonist apomorphine induced changes in vaginal blood flow oscillations consistent with sexual arousal during metestrus in the presence of the peripheral antagonist domperidone but not in the presence of the centrally acting antagonist haloperidol. Electrical stimulation of the paraventricular nucleus (PVN) of the hypothalamus induced an anti-arousal response in vaginal blood flow oscillations. These data demonstrated that manipulation of the central nervous system alone (via centrally acting apomorphine or electrical stimulation of the PVN) could produce either a pro-arousal or an anti-arousal response in vaginal blood flow oscillations. Alterations in the LF/HF ratio measured from vaginal laser Doppler flowmetry were independently regulated from vasculature in the trunk, the tongue, and electrocardiogram-derived heart rate variability, and were independent of overall vasocongestion of the vagina as measured by mean blood flow. CONCLUSIONS: These data indicated that slow oscillations in vaginal blood flow from rodents may be utilized as an animal model of female sexual arousal. Changes in these oscillations are driven by the central nervous system and modulated by the autonomic nervous system.