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Chronically activated microglia and the resulting cascade of neuroinflammatory mechanisms have been postulated to play a critical role in neurodegenerative disorders. Microglia are the main component of the brain's innate immune system and become activated by infection, injury, misfolded proteins or a multitude of other stimuli. Activated microglia release pro-inflammatory and cytotoxic factors that can damage neurons and transform astrocytes to become toxic to neurons as well. Therapeutic approaches aiming to modulate microglia activation may be beneficial to mitigate the progression of inflammatory-mediated neurodegenerative diseases. In this literature review, we provide an overview of recent progress on key microglia targets and discovery of small molecule compounds advancing in clinical trials to minimize neuroinflammation.

PAC1 receptor blockade using a monoclonal antibody inhibits nociceptive neuronal activity in the trigeminocervical complex., Pituitary adenylate cyclase activating polypeptide-38 (PACAP38) may play an important role in primary headaches. Preclinical evidence suggests that PACAP38 modulates trigeminal nociceptive activity mainly through PAC1 receptors while clinical studies report that plasma concentrations of PACAP38 are elevated in spontaneous attacks of cluster headache and migraine and normalize after treatment with sumatriptan. Intravenous infusion of PACAP38 induces migraine-like attacks in migraineurs and cluster-like attacks in cluster headache patients. A rodent-specific PAC1 receptor antibody Ab181 was developed, and its effect on nociceptive neuronal activity in the trigeminocervical complex was investigated in vivo in an electrophysiological model relevant to primary headaches. Ab181 is potent and selective at the rat PAC1 receptor and provides near-maximum target coverage at 10 mg/kg for more than 48 hours. Without affecting spontaneous neuronal activity, Ab181 effectively inhibits stimulus-evoked activity in the trigeminocervical complex. Immunohistochemical analysis revealed its binding in the trigeminal ganglion and sphenopalatine ganglion but not within the central nervous system suggesting a peripheral site of action. The pharmacological approach using a specific PAC1 receptor antibody could provide a novel mechanism with a potential clinical efficacy in the treatment of primary headaches.

The term ‘cognitive structures’ is used to describe the fact that mental models underlie thinking, reasoning and representing. Cognitive structures generally improve the efficiency of information processing by providing a situational framework within which there are parameters governing the nature and timing of information and appropriate responses can be anticipated. Unanticipated events that violate the parameters of the cognitive structure require the cognitive model to be updated, but this comes at an efficiency cost. In reversal learning a response that had been reinforced is no longer reinforced, while an alternative is now reinforced, having previously not been (A+/B− becomes A−/B+). Unanticipated changes of contingencies require that cognitive structures are updated. In this study, we examined the effect of lesions of the orbital frontal cortex (OFC) and the effects of the selective serotonin reuptake inhibitor (SSRI), escitalopram, on discrimination and reversal learning. Escitalopram was without effect in intact rats. Rats with OFC lesions had selective impairment of reversal learning, which was ameliorated by escitalopram. We conclude that reversal learning in OFC-lesioned rats is an easily administered and sensitive test that can detect effects of serotonergic modulation on cognitive structures that are involved in behavioural flexibility.

Background To further understand the role of pituitary adenylate cyclase-activating polypeptide 1 (PAC1) receptors in headache disorders, we mapped their expression in tissues of the trigemino-autonomic system by immunohistochemistry and in situ hybridization. Methods To optimize screening for monoclonal antibodies suitable for immunohistochemistry on formalin-fixed, paraffin-embedded tissues, we developed a new enzyme-linked immunosorbent assay using formalin-fixed, paraffin-embedded cells overexpressing human PAC1 receptors. 169G4.1 was selected from these studies for analysis of rat and human tissues and chimerized onto a mouse backbone to avoid human-on-human cross-reactivity. Immunoreactivity was compared to PAC1 receptor mRNA by in situ hybridization in both species. Results 169G4.1 immunoreactivity delineated neuronal cell bodies in the sphenopalatine ganglion in both rat and human, whereas no staining was detected in the trigeminal ganglion. The spinal trigeminal nucleus in both species showed immunoreactivity as especially strong in the upper laminae with both cell bodies and neuropil being labelled. No immunoreactivity was seen in either rat or human dura mater vessels. In situ hybridization in both species revealed mRNA in sphenopalatine ganglion neurons and the spinal trigeminal nucleus, a weak signal in the trigeminal nucleus and no signal in dural vessels. Conclusion Taken together, these data support a role for PAC1 receptors in the trigemino-autonomic system as it relates to headache pathophysiology.

Background Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated. Methods Here we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy. Results Using this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model. Conclusion These data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.

The discovery of rare familial monogenic forms of early-onset Parkinson's disease has led to the identification of a mitochondrial quality control process as a key player in this disease. Loss-of-function mutations in the genes encoding PINK1 or Parkin result in insufficient removal of dysfunctional mitochondria through autophagy, a process termed mitophagy. Understanding the mechanism of this process and the function of its two key players, PINK1 and Parkin, has led to the discovery of new therapeutic approaches. Small molecule activators of mitophagy, either activating PINK1 or Parkin directly or inhibiting Parkin's counterplayer, the ubiquitin-specific protease USP30, are in preclinical development. To enable clinical success of future small molecule mitophagy enhancers, biomarkers for mitochondrial integrity and mitophagy are being developed. Only a few years after the discovery of mitophagy deficits in Parkinson's disease, research of the underlying mechanisms, drug discovery of modulators for this mechanism and identification of biomarkers provide new avenues towards the development of disease-modifying therapies.

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.

We report the discovery of a novel series of 2-(3-alkoxy-1-azetidinyl) quinolines as potent and selective PDE10A inhibitors. Structure-activity studies improved the solubility (pH 7.4) and maintained high PDE10A activity compared to initial lead compound 3, with select compounds demonstrating good oral bioavailability. X-ray crystallographic studies revealed two distinct binding modes to the catalytic site of the PDE10A enzyme. An ex vivo receptor occupancy assay in rats demonstrated that this series of compounds covered the target within the striatum.

We report the discovery of novel imidazo[4,5-b]pyridines as potent and selective inhibitors of PDE10A. The investigation began with our recently disclosed ketobenzimidazole 1, which exhibited single digit nanomolar PDE10A activity but poor oral bioavailability. To improve oral bioavailability, we turned to novel scaffold imidazo[4,5-b]pyridine 2, which not only retained nanomolar PDE10A activity but was also devoid of the morpholine metabolic liability. Structure-activity relationship studies were conducted systematically to examine how various regions of the molecule impacted potency. X-ray cocrystal structures of compounds 7 and 24 in human PDE10A helped to elucidate the key bonding interactions. Five of the most potent and structurally diverse imidazo[4,5-b]pyridines (4, 7, 12b, 24a, and 24b) with PDE10A IC50 values ranging from 0.8 to 6.7 nM were advanced into receptor occupancy studies. Four of them (4, 12b, 24a, and 24b) achieved 55-74% RO at 10 mg/kg po.

Vortioxetine (Lu AA21004) is an investigational novel antidepressant with multimodal activity that functions as a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A receptor agonist and inhibitor of the 5-HT transporter in vitro. Here we explore its anxiolytic and antidepressant potential in adult mice. Vortioxetine was assessed in BalB/cJ@RJ mice using the open-field and forced-swim tests (acute: p.o. 1 h, repeated: daily p.o. 21 days), and in 129S6/SvEvTac mice using the novelty suppressed feeding paradigm (acute: p.o. 1 h, sustained: daily p.o. 14 or 21 days). Fluoxetine and diazepam were controls. Acute and repeated dosing of vortioxetine produced more pronounced anxiolytic- and antidepressant-like activities than fluoxetine. Vortioxetine significantly increased cell proliferation and cell survival and stimulated maturation of immature granule cells in the subgranular zone of the dentate gyrus of the hippocampus after 21 days of treatment. After 14 days, a high dose of vortioxetine increased dendritic length and the number of dendrite intersections, suggesting that vortioxetine accelerates the maturation of immature neurons. Vortioxetine displays an antidepressant and anxiolytic profile following repeated administration associated with increased neurogenesis at several stages. Vortioxetine effects were observed at low levels of 5-HT transporter occupancy, suggesting an alternative mechanism of action to 5-HT reuptake inhibition.

The serotonergic system plays an important role in cognitive functions via various 5-HT receptors. Vortioxetine (Lu AA21004) in development as a novel multimodal antidepressant is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, a 5-HT1B receptor partial agonist, a 5-HT1A receptor agonist and a 5-HT transporter (5-HTT) inhibitor in vitro. Preclinical studies suggest that 5-HT3 and 5-HT7 receptor antagonism as well as 5-HT1A receptor agonism may have a positive impact on cognitive functions including memory. Thus vortioxetine may potentially enhance memory. We investigated preclinical effects of vortioxetine (1–10mg/kg administered subcutaneously [s.c.]) on memory in behavioral tests, and on cortical neurotransmitter levels considered important in rat memory function. Contextual fear conditioning and novel object recognition tests were applied to assess memory in rats. Microdialysis studies were conducted to measure extracellular neurotransmitter levels in the rat medial prefrontal cortex. Vortioxetine administered 1h before or immediately after acquisition of contextual fear conditioning led to an increase in freezing time during the retention test. This mnemonic effect was not related to changes in pain sensitivity as measured in the hotplate test. Rats treated with vortioxetine 1h before training spent more time exploring the novel object in the novel object recognition test. In microdialysis studies of the rat medial prefrontal cortex, vortioxetine increased extracellular levels of acetylcholine and histamine. In conclusion, vortioxetine enhanced contextual and episodic memory in rat behavioral models. Further demonstration of its potential effect on memory functions in clinical settings is warranted.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology.

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

The serotonin6 (5-HT(6)) receptor has received attention for its proposed role in cognitive impairments associated with schizophrenia and Alzheimer's disease. This has lead to a search for selective 5-HT(6) receptor ligands useful for in vivo imaging in animals and humans. The novel 5-HT(6) receptor antagonist Lu AE60157 (8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline) displays high affinity for the human (h) 5-HT(6) receptor (K(d) 0.2nM), and broad profiling in 60 additional binding and enzyme assays showed that Lu AE60157 displays 16-fold selectivity to the h5-HT(2A) receptor (K(i) 3.2nM) and >100-fold selectivity to all other evaluated targets. Lu AE60157 was labeled with tritium in the N-methyl group and evaluated as a radioligand in vitro as well as in vivo in rats and mice. Autoradiography experiments showed that [(3)H]Lu AE60157 bound preferentially to rat brain regions with expected high 5-HT(6) receptor density. Furthermore, [(3)H]Lu AE60157 showed good brain penetration after systemic administration and high (about 75%) specific in vivo binding to the striatal 5-HT(6) receptor in rats. The striatal binding of [(3)H]Lu AE60157 was fully displaced by selective 5-HT(6) receptor antagonists (SB-742457; Lu AE58054) and antipsychotics known to inhibit the binding of 5-HT(6) receptors in vitro (clozapine; olanzapine; sertindole), but was not displaced by antipsychotics lacking high 5-HT(6) receptor affinities (risperidone; haloperidol; quetiapine). No specific binding to mouse brain tissue in vivo could be obtained. In conclusion, [(3)H]Lu AE60157 is suitable for measuring in vivo occupancies of 5-HT(6) receptor ligands in rat brain regions in which 5-HT(2A) receptors do not interfere.

Melanin-concentrating hormone (MCH) is an orexigenic and dipsogenic neuropeptide that has been reported to mediate acute behavioral and neuroendocrine stress-related responses via MCH 1 receptor activation in rodents. The purpose of the present investigation was to use the MCH 1 receptor antagonist SNAP 94847 ( N -(3-{1-[4-(3,4-difluoro-phenoxy)-benzyl]-piperidin-4-yl}-4-methyl-phenyl)-isobutyramide) to determine the effects of MCH 1 receptor blockade on MCH-evoked adrenocorticotropic hormone (ACTH) release, chronic mild stress-induced anhedonia, stress-induced hyperthermia and forced swim stress-induced immobility. The appropriate dose range for testing SNAP 94847 was determined by measuring MCH-evoked water drinking. The corresponding occupancy of MCH 1 receptors in rat striatum was also measured across a broad dose range. Orally administered (p.o.) SNAP 94847 (1–10 mg/kg) corresponds to 30–60% occupancy at MCH 1 receptors and significantly blocks water drinking induced by the intracerebroventricular (i.c.v.) injection of MCH. MCH (i.c.v.) significantly elevates plasma levels of ACTH in rats, and SNAP 94847 (2.5 mg/kg, p.o.) blocks MCH-evoked ACTH release. Using the chronic mild stress paradigm, we show that repeated daily exposure to environmental stressors for 5 weeks significantly suppresses sucrose intake in rats, and that SNAP 94847 (1 mg/kg, BID) for 1–5 weeks restores baseline sucrose intake. Moreover, a single administration of SNAP 94847 attenuates stress-induced hyperthermia and the behavioral effects of forced swim stress with minimal effective doses of 2.5 and 30 mg/kg (p.o.), respectively. The regulation of ACTH release and reversal of the effects of chronic and acute stress by SNAP 94847 are suggestive of a role for MCH 1 receptor blockade in the treatment of disorders characterized by high allostatic load.

Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y1, Y2, Y4, Y5, and y6). The Y5 receptor is well known for its role in appetite. Based on expression in the limbic system, we hypothesized that the Y5 receptor might also modulate stress sensitivity. We identified a novel Y5 receptor-selective antagonist, Lu AA33810 [ N -[[ trans -4-[(4,5-dihydro[1]-benzothiepino[5,4- d ]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide], that bound to cloned rat Y5 receptors ( K i = 1.5 nM) and antagonized NPY-evoked cAMP and calcium mobilization in vitro. Lu AA33810 (3-30 mg/kg p.o.) blocked feeding elicited by intracerebroventricular injection of the Y5 receptor-selective agonist [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide in Sprague-Dawley rats. In vivo effects of Lu AA33810 were correlated with brain exposure ≥ 50 ng/g and ex vivo Y5 receptor occupancy of 22 to 95%. Lu AA33810 was subsequently evaluated in models of stress sensitivity. In Fischer 344 rats, Lu AA33810 (30 mg/kg p.o.) attenuated increases in plasma ACTH and corticosterone elicited by intracerebroventricular injection of [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide. In Sprague-Dawley rats subjected to the social interaction test, Lu AA33810 (3-30 mg/kg p.o.) produced anxiolytic-like effects after acute or chronic treatment. In Flinders sensitive line rats, chronic dosing of Lu AA33810 (10 mg/kg/day i.p.) produced anxiolytic-like effects in the social interaction test, plus antidepressant-like effects in the forced swim test. In Wistar rats exposed to chronic mild stress, chronic dosing of Lu AA33810 (3 and 10 mg/kg/day i.p.) produced antidepressant-like activity, i.e., normalization of stress-induced decrease in sucrose consumption. We propose that Y5 receptors may function as part of an endogenous stress-sensing system to mediate social anxiety and reward or motivational deficits in selected rodent models.

Background Phosphodiesterase 10A (PDE10A) is expressed at high levels in the striatum and has been proposed both as a biomarker for Huntington's disease pathology and as a target for intervention. Objective PDE10A radiotracers have been successfully used to measure changes in binding density in Huntington's disease patients, but little is known about PDE10A binding in mouse models that are used extensively to model pathology and test therapeutic interventions. Methods Our study investigated changes in PDE10A binding using the selective tracer 3H-7980 at specific ages of two Huntington's disease transgenic mouse models: R6/2, a short-lived model carrying exon-1 of mutant HTT and BACHD, a longer-lived model carrying full-length mutant HTT. PDE10A binding was compared to binding of known markers of striatal atrophy in Huntington's disease, e.g. dopamine transporter (DAT) and dopamine receptors D1 and D2. Results We found that in the R6/2 model at 6 weeks of age, mice showed high variability of binding, however binding of all ligands was significantly decreased at 8 and 12 weeks of age. In contrast, no changes were detectable in the BACHD model at 8, 10 or 12 month of age. Conclusions These findings suggest that radiotracer binding of PDE10A, DAT, D1 and D2 receptor in the R6/2 model may be a good indicator of striatal pathological changes that are observed in Huntington's disease patients, and that the first 12 months in the BACHD model may be more reflective of early stages of the disease.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Introduction Phosphodiesterase 10A (PDE10A) is an intracellular enzyme responsible for the breakdown of cyclic nucleotides which are important second messengers for neurotransmission. Inhibition of PDE10A has been identified as a potential target for treatment of various neuropsychiatric disorders. To assist drug development, we have identified a selective PDE10A positron emission tomography (PET) tracer, AMG 580. We describe here the radiosynthesis of [ 18 F]AMG 580 and in vitro and in vivo characterization results. Methods The potency and selectivity were determined by in vitro assay using [ 3 H]AMG 580 and baboon brain tissues. [ 18 F]AMG 580 was prepared by a 1-step [ 18 F]fluorination procedure. Dynamic brain PET scans were performed in non-human primates. Regions-of-interest were defined on individuals' MRIs and transferred to the co-registered PET images. Data were analyzed using two tissue compartment analysis (2TC), Logan graphical (Logan) analysis with metabolite-corrected input function and the simplified reference tissue model (SRTM) method. A PDE10A inhibitor and unlabeled AMG 580 were used to demonstrate the PDE10A specificity. K D was estimated by Scatchard analysis of high and low affinity PET scans. Results AMG 580 has an in vitro K D of 71.9 pM. Autoradiography showed specific uptake in striatum. Mean activity of 121±18MBq was used in PET studies. In Rhesus, the baseline BP ND for putamen and caudate was 3.38 and 2.34, respectively, via 2TC, and 3.16, 2.34 via Logan, and 2.92, and 2.01 via SRTM. A dose dependent decrease of BP ND was observed by the pre-treatment with a PDE10A inhibitor. In baboons, 0.24mg/kg dose of AMG 580 resulted in about 70% decrease of BP ND . The in vivo K D of [ 18 F]AMG 580 was estimated to be around 0.44 nM in baboons. Conclusion [ 18 F]AMG 580 is a selective and potent PDE10A PET tracer with excellent specific striatal binding in non-human primates. It warrants further evaluation in humans.

The serotonin6 (5-HT(6)) receptor has received attention for its proposed role in cognitive impairments associated with schizophrenia and Alzheimer's disease. This has lead to a search for selective 5-HT(6) receptor ligands useful for in vivo imaging in animals and humans. The novel 5-HT(6) receptor antagonist Lu AE60157 (8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline) displays high affinity for the human (h) 5-HT(6) receptor (K(d) 0.2nM), and broad profiling in 60 additional binding and enzyme assays showed that Lu AE60157 displays 16-fold selectivity to the h5-HT(2A) receptor (K(i) 3.2nM) and100-fold selectivity to all other evaluated targets. Lu AE60157 was labeled with tritium in the N-methyl group and evaluated as a radioligand in vitro as well as in vivo in rats and mice. Autoradiography experiments showed that [(3)H]Lu AE60157 bound preferentially to rat brain regions with expected high 5-HT(6) receptor density. Furthermore, [(3)H]Lu AE60157 showed good brain penetration after systemic administration and high (about 75%) specific in vivo binding to the striatal 5-HT(6) receptor in rats. The striatal binding of [(3)H]Lu AE60157 was fully displaced by selective 5-HT(6) receptor antagonists (SB-742457; Lu AE58054) and antipsychotics known to inhibit the binding of 5-HT(6) receptors in vitro (clozapine; olanzapine; sertindole), but was not displaced by antipsychotics lacking high 5-HT(6) receptor affinities (risperidone; haloperidol; quetiapine). No specific binding to mouse brain tissue in vivo could be obtained. In conclusion, [(3)H]Lu AE60157 is suitable for measuring in vivo occupancies of 5-HT(6) receptor ligands in rat brain regions in which 5-HT(2A) receptors do not interfere.

Although the antidepressant and anxiolytic effects of selective serotonin reuptake inhibitors and serotonin–noradrenaline reuptake inhibitors are well-documented, less is known about their cognitive effects. Escitalopram, a selective serotonin reuptake inhibitor, and atomoxetine, a selective noradrenaline reuptake inhibitor, were used to evaluate the interaction between noradrenergic and serotonergic neurotransmission in the modulation of contextual fear conditioning in rats. Contextual fear-conditioning test was used to investigate the acute effects of escitalopram, alone or in combination with atomoxetine, in different stages of learning and memory in rats. Furthermore, microdialysis in freely moving animals was used to investigate the effect of escitalopram on serotonin, dopamine, and noradrenaline levels in the rat hippocampus. Escitalopram significantly increased conditioned responses when applied before the acquisition, but decreased responses, when applied before the recall test. When administered during memory consolidation, escitalopram dose-dependently enhanced conditioned responding. These effects were blocked by atomoxetine. Escitalopram (at a dose that affects memory consolidation) increased hippocampal serotonin levels fourfold without changing dopamine or noradrenaline. Atomoxetine, at dose levels that blocked the effects of escitalopram on contextual fear conditioning, increased the extracellular levels of noradrenaline eightfold but did not change dopamine or serotonin. A combined treatment of escitalopram and atomoxetine caused a significant attenuation of escitalopram-induced increase in serotonin levels, while noradrenaline levels were not affected. These findings indicate that escitalopram affects fear memory in rats, likely modulated by increases in serotonin levels in the brain. This effect is impaired by atomoxetine, probably due to a noradrenaline-mediated decrease in serotonin levels. Further studies are warranted to study the effects of potential differences among antidepressant therapies on long-term cognitive outcomes.

Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y(1), Y(2), Y(4), Y(5), and y(6)). The Y(5) receptor is well known for its role in appetite. Based on expression in the limbic system, we hypothesized that the Y(5) receptor might also modulate stress sensitivity. We identified a novel Y(5) receptor-selective antagonist, Lu AA33810 [N-[[trans-4-[(4,5-dihydro[1]-benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide], that bound to cloned rat Y(5) receptors (K(i) = 1.5 nM) and antagonized NPY-evoked cAMP and calcium mobilization in vitro. Lu AA33810 (3-30 mg/kg p.o.) blocked feeding elicited by intracerebroventricular injection of the Y(5) receptor-selective agonist [cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPancreatic Polypeptide in Sprague-Dawley rats. In vivo effects of Lu AA33810 were correlated with brain exposureor = 50 ng/g and ex vivo Y(5) receptor occupancy of 22 to 95%. Lu AA33810 was subsequently evaluated in models of stress sensitivity. In Fischer 344 rats, Lu AA33810 (30 mg/kg p.o.) attenuated increases in plasma ACTH and corticosterone elicited by intracerebroventricular injection of [cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPancreatic Polypeptide. In Sprague-Dawley rats subjected to the social interaction test, Lu AA33810 (3-30 mg/kg p.o.) produced anxiolytic-like effects after acute or chronic treatment. In Flinders sensitive line rats, chronic dosing of Lu AA33810 (10 mg/kg/day i.p.) produced anxiolytic-like effects in the social interaction test, plus antidepressant-like effects in the forced swim test. In Wistar rats exposed to chronic mild stress, chronic dosing of Lu AA33810 (3 and 10 mg/kg/day i.p.) produced antidepressant-like activity, i.e., normalization of stress-induced decrease in sucrose consumption. We propose that Y(5) receptors may function as part of an endogenous stress-sensing system to mediate social anxiety and reward or motivational deficits in selected rodent models.