Extracellular Acetylcholine Levels Research Articles

Intrinsic dopamine and acetylcholine dynamics in the striatum of mice.

External rewards such as food and money are potent modifiers of behaviour1,2. Pioneering studies established that these salient sensory stimuli briefly interrupt the tonic discharge of neurons that produce the neuromodulators dopamine (DA) and acetylcholine (ACh): midbrain DA neurons (DANs) fire a burst of action potentials that broadly elevates DA in the striatum3,4 at the same time that striatal cholinergic interneurons (CINs) produce a characteristic pause in firing5,6. These phasic responses are thought to create unique, temporally limited conditions that motivate action and promote learning7-11. However, the dynamics of DA and ACh outside explicitly rewarded situations remain poorly understood. Here we show that extracellular DA and ACh levels fluctuate spontaneously and periodically at a frequency of approximately 2 Hz in the dorsalstriatum of mice and maintain the same temporal relationship relative to one another as that evoked by reward. We show that this neuromodulatory coordination does not arise from direct interactions between DA and ACh within the striatum. Instead, we provide evidence that periodic fluctuations in striatal DA are inherited from midbrain DANs, while striatal ACh transients are driven by glutamatergic inputs, which act to locally synchronize the spiking of CINs. Together, our findings show that striatal neuromodulatory dynamics are autonomously organized by distributed extra-striatal afferents. The dominance of intrinsic rhythms in DA and ACh offers new insights for explaining how reward-associated neural dynamics emerge and how the brain motivates action and promotes learning from within.

SUVN-D4010: A serotonin-4 receptor partial agonist for the treatment of cognitive disorders.

Memory impairments are clinical symptoms of a wide range of neurodegenerative diseases, and natural physiological process such as ageing. SUVN-D4010 is an investigational clinical candidate which acts by modulating 5-HT4 receptors. Experimental evidences from animals and humans suggest that 5-HT4 receptor activation holds promise for the treatment of cognitive impairment. Additionally, activation of 5-HT4 receptor also modulates mood. The present investigation is to characterize SUVN-D4010 and evaluate its drug-like properties. SUVN-D4010 has been studied for its affinity and functional activity at 5-HT4 receptor using in vitro methods. ADME properties were evaluated using in vitro and in vivo methods. SUVN-D4010 was studied for its effects on learning and memory using social and object recognition tasks. The effects on the neurotransmitters were monitored using the brain microdialysis technique in rats. The effects on the beta amyloid processing were studied using in vitro and in vivo systems. The safety and tolerability was evaluated in both animals and humans. SUVN-D4010 showed potent affinity towards 5-HT4 receptor when tested using radioligand binding and cell based reporter gene assay. SUVN-D4010 has minimal binding over 70 target sites (at 1 µM, Caliper) including GPCRs, transporters, ion channels, peptides, steroids, second messengers, growth factors and prostaglandins. SUVN-D4010 is orally bioavailable with good brain penetration and CSF concentrations. The likelihood of drug-drug interaction of SUVN-D4010 as an inhibitor/ inducer is remote. No unique metabolites were identified in non-clinical and clinical samples. SUVN-D4010 showed procognitve effects at doses ranging from 0.3 to 3 mg/kg, p.o. SUVN-D4010 significantly increased extracellular acetylcholine levels as a standalone, or treatment adjunct to donepezil. SUVN-D4010 promoted non-amyloidogenic APPα processing and reduced toxic Aβ load. SUVN-D4010 was found to be safe and well tolerated in healthy human volunteers. SUVN-D4010 is a potent, selective, orally bioavailable and brain penetrant 5-HT4 receptor partial agonist with potential utility in treatment of cognitive disorders.

A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents.

Cholinergic neurons utilize choline (Ch) to synthetize acetylcholine (ACh) and contain a high-affinity Ch transporter, Ch acetyltransferase (ChAT), ACh receptors, and acetylcholinesterase (AChE). As the depletion or malfunction of each component of the cholinergic system has been reported in patients with dementia, many studies have sought to evaluate whether treatment candidates affect each of the cholinergic components. The associated changes in the cholinergic components may be reflected by intra- or extra-cellular ACh levels, with an increase in extracellular ACh levels occurring following AChE inhibition. We hypothesized that increases in intracellular ACh levels can be more sensitively detected than those in extracellular ACh levels, thereby capturing subtle effects in the cholinergic components other than AChE. The objective of this study was to test this hypothesis. We developed an in vitro model to measure both extracellular and intracellular ACh levels using the human cholinergic neuroblastoma cell line, LA-N-2, which have been reported to express Ch transporter, ChAT, muscarinic ACh receptor (mAChR), and AChE. With this model, we evaluated several drug compounds and food constituents reported to improve cholinergic function through various mechanisms. In addition, we conducted western blotting to identify the subtype of mAChR that is expressed on the cell line. Our cell-based assay system was capable of detecting increases in extracellular ACh levels induced by an AChE inhibitor at relatively high doses, as well as increases in intracellular ACh levels following the administration of lower AChE-inhibitor doses and an mAChR agonist. Moreover, increases in intracellular ACh levels were observed even after treatment with food constituents that have different mechanisms of action, such as Ch provision and ChAT activation. In addition, we revealed that LA-N-2 cells expressed mAChR M2. The findings support our hypothesis and indicate that the developed assay model can broadly screen compounds from drugs to food ingredients, with varying strengths and mechanisms of action, to develop treatments for ACh-relevant phenomena, including dementia and aging-related cognitive decline.

CSF and Plasma Cholinergic Markers in Patients With Cognitive Impairment.

IntroductionAlzheimer’s disease (AD) is the most prevalent form of dementia with symptoms of deteriorating cognitive functions and memory loss, partially as a result of a decrease in cholinergic neurotransmission. The disease is incurable and treatment with cholinesterase inhibitors (ChEIs) is symptomatic. Choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine (ACh), has been proven recently to be present in both cerebrospinal fluid (CSF) and plasma. As ChAT plays a role in regulating the extracellular ACh levels, it may have an impact on prognosis and cognitive performance in AD patients.ObjectivesTo measure ChAT activity and its protein concentration in CSF and plasma from patients with AD, mild cognitive impairment (MCI), or Subjective cognitive impairment (SCI).MethodsPlasma and CSF samples were obtained from 21 AD, 32 MCI, and 30 SCI patients. The activity and protein levels of ChAT and acetylcholinesterase (AChE), the enzyme catalyzing the hydrolysis of ACh, were analyzed using an integrated activity and protein concentration ELISA-like assay. A Cholinergic Index was calculated as the ratio of ChAT to AChE activities in CSF. The data were analyzed in relation to dementia biomarkers and cognitive performance of the patients.ResultsThe CSF ChAT activity was significantly higher (55–67%) in MCI patients compared to AD and SCI cases. The CSF Cholinergic Index was 41 and 22% lower in AD patients than in MCI and SCI subjects, respectively. This index correlated positively with the Aβ42/p-tau ratio in CSF in SCI but negatively with that in AD and MCI. The ChAT activity and protein levels in plasma exhibited significant differences with the pattern of AD>>MCI>SCI.ConclusionThis is the first study investigating soluble levels of the key cholinergic enzyme, ChAT, in both plasma and CSF of individuals at different clinical stages of dementia. Although further validation is needed, the overall pattern of the results suggests that in the continuum of AD, the cholinergic signaling exhibits an inverse U-shape dynamic of changes in the brain that greatly differs from the changes observed in the plasma compartment.

Stimulating GABAergic Neurons in the Nucleus Accumbens Core Alters the Trigeminal Neuropathic Pain Responses in a Rat Model of Infraorbital Nerve Injury.

The nucleus accumbens core (NAcc) is an important component of brain reward circuitry, but studies have revealed its involvement in pain circuitry also. However, its effect on trigeminal neuralgia (TN) and the mechanism underlying it are yet to be fully understood. Therefore, this study aimed to examine the outcomes of optogenetic stimulation of NAcc GABAergic neurons in an animal model of TN. Animals were allocated into TN, sham, and control groups. TN was generated by infraorbital nerve constriction and the optogenetic virus was injected into the NAcc. In vivo extracellular recordings were acquired from the ventral posteromedial nucleus of the thalamus. Alterations of behavioral responses during stimulation “ON” and “OFF” conditions were evaluated. In vivo microdialysis was performed in the NAcc of TN and sham animals. During optogenetic stimulation, electrophysiological recordings revealed a reduction of both tonic and burst firing activity in TN animals, and significantly improved behavioral responses were observed as well. Microdialysis coupled with liquid chromatography/tandem mass spectrometry analysis revealed significant alterations in extracellular concentration levels of GABA, glutamate, acetylcholine, dopamine, and citrulline in NAcc upon optic stimulation. In fine, our results suggested that NAcc stimulation could modulate the transmission of trigeminal pain signals in the TN animal model.

Kuwanon G protects HT22 cells from advanced glycation end product-induced damage.

The incidence of diabetic encephalopathy is increasing as the population ages. Evidence suggests that formation and accumulation of advanced glycation end products (AGEs) plays a pivotal role in disease progression, but limited research has been carried out in this area. A previous study demonstrated that Kuwanon G (KWG) had significant anti-oxidative stress and anti-inflammatory properties. As AGEs are oxidative products and inflammation is involved in their generation it is hypothesized that KWG may have effects against AGE-induced neuronal damage. In the present study, mouse hippocampal neuronal cell line HT22 was used. KWG was shown to significantly inhibit AGE-induced cell apoptosis in comparison with a control treatment, as determined by both MTT and flow cytometry. Compared with the AGEs group, expression of pro-apoptotic protein Bax was reduced and expression of anti-apoptotic protein Bcl-2 was increased in the AGEs + KWG group. Both intracellular and extracellular levels of acetylcholine and choline acetyltransferase were significantly elevated after KWG administration in comparison with controls whilethe level of acetylcholinesterase decreased. These changes in protein expression were accompanied by increased levels of superoxide dismutase and glutathione peroxidase synthesis and reduced production of malondialdehyde and reactive oxygen species. Intracellular signaling pathway protein levels were determined by western blot and immunocytochemistry. KWG administration was found to prevent AGE-induced changes to the phosphorylation levels of Akt, IκB-α, glycogen synthase kinase 3 (GSK3)-α and β, p38 MAPK and NF-κB p65 suggesting a potential neuroprotective effect of KWG against AGE-induced damage was via the PI3K/Akt/GSK3αβ signaling pathway. The findings of the present study suggest that KWG may be a potential treatment for diabetic encephalopathy.

Central cholinergic function and metabolic changes in streptozotocin-induced rat brain injury.

As glucose hypometabolism in the brain is an early sign of Alzheimer´s dementia (AD), the diabetogenic drug streptozotocin (STZ) has been used to induce Alzheimer-like pathology in rat brain by intracereboventricular injection (icv-STZ). However, many details of the pathological mechanism of STZ in this AD model remain unclear. Here, we report metabolic and cholinergic effects of icv-STZ using microdialysis in freely moving animals. We found that icv-STZ at a dose of 3mg/kg (2×1.5mg/kg) causes overt toxicity reflected in body weight loss. Three weeks after STZ administration, histological examination revealed a high number of glial fibrillary acidic protein reactive cells in the hippocampus, accompanied by Fluoro-Jade C-positive cells in the CA1 region. Glucose and lactate levels in microdialysates were unchanged, but mitochondrial respiration measured ex vivo was reduced by 9%-15%. High-affinity choline uptake, choline acetyltransferase, and acetylcholine esterase (AChE) activities in the hippocampus were reduced by 16%, 28%, and 30%, respectively. Importantly, extracellular acetylcholine (ACh) levels in the hippocampus were unchanged and responded to behavioral and pharmacological challenges. In comparison, extracellular ACh levels and cholinergic parameters in the striatum were unchanged or slightly increased. We conclude that the icv-STZ model poorly reflects central cholinergic dysfunction, an important characteristic of dementia. The icv-STZ model may be more aptly described as an animal model of hippocampal gliosis.

Preclinical Pharmacology of [2-(3-Fluoro-5-Methanesulfonyl-phenoxy)Ethyl](Propyl)amine (IRL790), a Novel Dopamine Transmission Modulator for the Treatment of Motor and Psychiatric Complications in Parkinson Disease.

IRL790 ([2-(3-fluoro-5-methanesulfonylphenoxy)ethyl](propyl)amine, mesdopetam) is a novel compound in development for the clinical management of motor and psychiatric disabilities in Parkinson disease. The discovery of IRL790 was made applying a systems pharmacology approach based on in vivo response profiling. The chemical design idea was to develop a new type of DA D3/D2 receptor type antagonist built on agonist rather than antagonist structural motifs. We hypothesized that such a dopamine antagonist with physicochemical properties similar to agonists would exert antidyskinetic and antipsychotic effects in states of dysregulated dopaminergic signaling while having little negative impact on physiologic dopamine transmission and, hence, minimal liability for side effects related to dopamine-dependent functions. At the level of in vivo pharmacology, IRL790 displays balancing effects on aberrant motor phenotypes, reducing l-DOPA-induced dyskinesias in the rodent 6-hydroxydopamine lesion model and reducing psychostimulant-induced locomotor hyperactivity elicited by pretreatment with either d-amphetamine or dizocilpine, without negatively impacting normal motor performance. Thus, IRL790 has the ability to normalize the behavioral phenotype in hyperdopaminergic as well as hypoglutamatergic states. Neurochemical and immediate early gene (IEG) response profiles suggest modulation of DA neurotransmission, with some features, such as increased DA metabolites and extracellular DA, shared by atypical antipsychotics and others, such as increased frontal cortex IEGs, unique to IRL790. IRL790 also increases extracellular levels of acetylcholine in the prefrontal cortex and ventral hippocampus. At the receptor level, IRL790 appears to act as a preferential DA D3 receptor antagonist. Computational docking studies support preferential affinity at D3 receptors with an agonist-like binding mode. SIGNIFICANCE STATEMENT: This paper reports preclinical pharmacology along with molecular modeling results on IRL790, a novel compound in clinical development for the treatment of motor and psychiatric complications in advanced Parkinson disease. IRL790 is active in models of perturbed dopaminergic and glutamatergic signaling, including rodent 6-hydroxydopamine l-DOPA-induced dyskinesias and psychostimulant-induced hyperactivity, in a dose range that does not impair normal behavior. This effect profile is attributed to interactions at dopamine D2/D3 receptors, with a 6- to 8-fold preference for the D3 subtype.

Diverse Mechanisms Lead to Common Dysfunction of Striatal Cholinergic Interneurons in Distinct Genetic Mouse Models of Dystonia.

Clinical and experimental data indicate striatal cholinergic dysfunction in dystonia, a movement disorder typically resulting in twisted postures via abnormal muscle contraction. Three forms of isolated human dystonia result from mutations in the TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25) genes. Experimental models carrying these mutations facilitate identification of possible shared cellular mechanisms. Recently, we reported elevated extracellular striatal acetylcholine by in vivo microdialysis and paradoxical excitation of cholinergic interneurons (ChIs) by dopamine D2 receptor (D2R) agonism using ex vivo slice electrophysiology in Dyt1ΔGAG/+ mice. The paradoxical excitation was caused by overactive muscarinic receptors (mAChRs), leading to a switch in D2R coupling from canonical Gi/o to noncanonical β-arrestin signaling. We sought to determine whether these mechanisms in Dyt1ΔGAG/+ mice are shared with Thap1C54Y/+ knock-in and Gnal+/- knock-out dystonia models and to determine the impact of sex. We found Thap1C54Y/+ mice of both sexes have elevated extracellular striatal acetylcholine and D2R-induced paradoxical ChI excitation, which was reversed by mAChR inhibition. Elevated extracellular acetylcholine was absent in male and female Gnal+/- mice, but the paradoxical D2R-mediated ChI excitation was retained and only reversed by inhibition of adenosine A2ARs. The Gi/o-preferring D2R agonist failed to increase ChI excitability, suggesting a possible switch in coupling of D2Rs to β-arrestin, as seen previously in a DYT1 model. These data show that, whereas elevated extracellular acetylcholine levels are not always detected across these genetic models of human dystonia, the D2R-mediated paradoxical excitation of ChIs is shared and is caused by altered function of distinct G-protein-coupled receptors.SIGNIFICANCE STATEMENT Dystonia is a common and often disabling movement disorder. The usual medical treatment of dystonia is pharmacotherapy with nonselective antagonists of muscarinic acetylcholine receptors, which have many undesirable side effects. Development of new therapeutics is a top priority for dystonia research. The current findings, considered in context with our previous investigations, establish a role for cholinergic dysfunction across three mouse models of human genetic dystonia: DYT1, DYT6, and DYT25. The commonality of cholinergic dysfunction in these models arising from diverse molecular etiologies points the way to new approaches for cholinergic modulation that may be broadly applicable in dystonia.

Sleep and Neurochemical Modulation by DZNep and GSK-J1: Potential Link With Histone Methylation Status.

Histone methylation/demethylation plays an important modulatory role in chromatin restructuring, RNA transcription and is essential for controlling a plethora of biological processes. Due to many human diseases have been related to histone methylation/demethylation, several compounds such as 3-deazaneplanocin A (DZNep) or 3-((6-(4,5-Dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoic acid; N-[2-(2-pyridinyl)-6-(1,2,4,5-tetrahydro-3H-3-benzazepin-3-yl)-4-pyrimidinyl]-β-Alanine (GSK-J1), have been designed to inhibit histone methylase or suppress histone demethylase, respectively. In the present study, we investigated the effects on the sleep-wake cycle and sleep-related neurochemical levels after systemic injections of DZNep or GSK-J1 given during the light or dark phase in rats. DZNep dose-dependently (0.1, 1.0, or 10 mg/kg, i.p.) prolonged wakefulness (W) duration while decreased slow wave sleep (SWS) and rapid eye movement sleep (REMS) time spent during the lights-on period with no changes observed in dark phase. In opposite direction, GSK-J1 (0.1, 1.0, or 10 mg/kg, i.p.) injected at the beginning of the lights-on period induced no statistical changes in W, SWS, or REMS whereas if administered at darkness, we found a diminution in W and an enhancement in SWS and REMS. Finally, brain microdialysis experiments in freely moving animals were used to evaluate the effects of DZNep or GSK-J1 treatments on contents of sleep-related neurochemicals. The results showed that DZNep boosted extracellular levels of dopamine, norepinephrine, epinephrine, serotonin, adenosine, and acetylcholine if injected at the beginning of the lights-on period whereas GSK-J1 exerted similar outcomes but when administered at darkness. In summary, DZNep and GSK-J1 may control the sleep-wake cycle and sleep-related neurochemicals through histone methylation/demethylation activity.

Commonalities and differences in extracellular levels of hippocampal acetylcholine and amino acid neurotransmitters during status epilepticus and subsequent epileptogenesis in two rat models of temporal lobe epilepsy

Chemically or electrically induced status epilepticus (SE) in rodents is a commonly used method for induction of epilepsy. Structural and functional changes in the hippocampus play a pivotal role in epileptogenesis induced by SE. Although cholinergic mechanisms have long been thought to play an important role in the onset and propagation of epileptic seizures, not much is known about the potential role of acetylcholine (ACh) in ictogenesis and epileptogenesis in SE models of temporal lobe epilepsy. Here we used in vivo microdialysis to determine extracellular levels of ACh and, for comparison, several amino acid transmitters in the ventral hippocampus during SE, epileptogenesis, and the chronic epileptic state in two rat models of SE-induced epilepsy. SE was either induced by lithium-pilocarpine or by sustained electrical stimulation of the basolateral amygdala (BLA). ACh increased during SE in both models. Pretreatment with the muscarinic receptor antagonist scopolamine before BLA stimulation reduced SE severity and duration. In contrast to ACh, no consistent changes in amino acid levels were found during SE in the two models. During epileptogenesis and the chronic epileptic state, the only commonalities found in both models were a decrease in ACh in epileptic rats during the chronic epileptic state and a decrease in aspartate during epileptogenesis. The data demonstrate complex, model-dependent alterations in extracellular levels of ACh and amino acid neurotransmitters and only few commonalities. Thus, data originating from only one model of post-SE epilepsy should not be generalized but may have a limited translational value for understanding ictogenesis or epileptogenesis.

Systemic Injections of Cannabidiol Enhance Acetylcholine Levels from Basal Forebrain in Rats

Cannabis sativa is a plant that contains more than 500 components, of which the most studied are Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Several studies have indicated that CBD displays neurobiological effects, including wake promotion. Moreover, experimental evidence has shown that injections of CBD enhance wake-related compounds, such as monoamines (dopamine, serotonin, epinephrine, and norepinephrine). However, no clear evidence is available regarding the effects of CBD on additional wake-related neurochemicals such as acetylcholine (ACh). Here, we demonstrate that systemic injections of CBD (0, 5, 10 or 30mg/kg, i.p.) at the beginning of the lights-on period, increase the extracellular levels of ACh collected from the basal forebrain and measured by microdialysis and HPLC means. Moreover, the time course effects on the contents of ACh were present 5h post-injection of CBD. Altogether, these data demonstrate that CBD increases ACh levels in a brain region related to wake control. This study is the first to show the effects of ACh levels in CBD-treated rats and suggests that the basal forebrain might be a site of action of CBD for wakefulness modulation.

Tokishakuyakusan ameliorates spatial memory deficits induced by ovariectomy combined with β-amyloid in rats

Previously, we reported that ovariectomy (OVX) combined with β-amyloid peptide (Aβ) impaired spatial memory by decreasing extracellular acetylcholine (ACh) levels in the dorsal hippocampus. Here, we investigated the effect of tokishakuyakusan (TSS), a Kampo medicine, on the impairment of spatial memory induced by OVX combined with Aβ in rats. Repeated administration of TSS (300 mg/kg, p.o.) significantly decreased the number of errors in the eight-arm radial maze test. Though TSS had no effect on extracellular ACh levels at baseline, TSS significantly increased extracellular ACh levels in the dorsal hippocampus. These results suggest that TSS improves the impairment of spatial memory induced by OVX combined with Aβ by (at least in part) increasing extracellular ACh levels in the dorsal hippocampus.

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

Neurochemical arguments for the use of dopamine D4 receptor stimulation to improve cognitive impairment associated with schizophrenia

BackgroundDopamine (DA) D4 receptors have been implicated in schizophrenia and the ability of some atypical antipsychotic drugs (APDs) to improve the cognitive impairment associated with schizophrenia (CIAS). Systemic administration of a D4 agonist, PD168077, at a sub-effective dose, together with a sub-effective dose of lurasidone, an atypical APD which is a weak D4 receptor antagonist, reversed the deficit in novel object recognition (NOR) in rats treated subchronically with phencyclidine (PCP), a rodent model of CIAS. Atypical APDs potentially stimulate D4Rs via their ability to enhance DA release in key brain areas related to cognition. However, some atypical APDs are relatively potent D4 antagonists at clinical dosages, including clozapine, and risperidone. The D4 antagonist, L745870, blocked the ability of clozapine, but not lurasidone, to reverse the NOR deficit in rats. MethodsThe purpose of this study was to determine the effects of a selective D4 agonist and antagonist, alone, and as pretreatment with lurasidone, on neurotransmitter efflux in mouse medial prefrontal cortex (mPFC) and dorsal striatum (dSTR), using in vivo microdialysis. Results and discussionPD168077 alone, and in combination with sub-effective dose lurasidone, increased DA and acetylcholine (ACh) efflux in mPFC, but only DA efflux in dSTR. L745870 had no effect on neurotransmitter efflux on its own or on the ability of lurasidone to increase cortical or striatal neurotransmitter efflux. These results indicate D4 receptor agonism alone is sufficient to increase cortical DA and ACh efflux without interfering with the effects of lurasidone and possibly other atypical APDs on extracellular cortical DA and ACh levels. A D4 agonist may be useful for treating CIAS, especially as augmentation of those atypical APDs which are not potent D4 antagonists.

Discovery and Development of 1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (SUVN-502): A Novel, Potent, Selective and Orally Active Serotonin 6 (5-HT6) Receptor Antagonist for Potential Treatment of Alzheimer's Disease.

Optimization of a novel series of 3-(piperazinylmethyl) indole derivatives as 5-hydroxytryptamine-6 receptor (5-HT6R) antagonists resulted in identification of 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole dimesylate monohydrate (5al, SUVN-502) as a clinical candidate for potential treatment of cognitive disorders. It has high affinity at human 5-HT6R (Ki = 2.04 nM) and selectivity over 100 target sites which include receptors, enzymes, peptides, growth factors, ion channels, steroids, immunological factors, second messengers, and prostaglandins. It has high selectivity over 5-HT2A receptor. It is orally bioavailable and brain penetrant with robust preclinical efficacy. The combination of 5al, donepezil, and memantine (triple combination) produces synergistic effects in extracellular levels of acetylcholine in the ventral hippocampus. Preclinical efficacy in triple combination and high selectivity over 5-HT2A receptors are the differentiating features which culminated in selection of 5al for further development. The Phase-1 evaluation of safety and pharmacokinetics has been completed, allowing for the initiation of a Phase-2 proof of concept study.

Effects of the 5-HT6 receptor antagonist idalopirdine on extracellular levels of monoamines, glutamate and acetylcholine in the rat medial prefrontal cortex

Idalopirdine (Lu AE58054) is a high affinity and selective antagonist for the human serotonin 5-HT6 receptor (Ki 0.83nM) in phase III development for mild-to-moderate Alzheimer's disease as an adjunct therapy to acetylcholinesterase inhibitors (AChEIs). We have studied the effects of idalopirdine on extracellular levels of monoamines, glutamate and acetylcholine in the medial prefrontal cortex (mPFC) of freely-moving rats using microdialysis. Idalopirdine (10mg/kg p.o.) increased extracellular levels of dopamine, noradrenaline and glutamate in the mPFC and showed a trend to increase serotonin levels. No effect was observed on acetylcholine levels. The AChEI donepezil (1.3mg/kg s.c.) significantly increased the levels of acetylcholine. Pretreatment with idalopirdine 2h prior to donepezil administration potentiated the effect of donepezil on extracellular acetylcholine levels. The idalopirdine potentiation of donepezil-induced increase in acetylcholine levels was also observed during local infusion of idalopirdine (6µg/ml) into the mPFC by reverse dialysis. The data from the current study may provide a mechanistic model for the pro-cognitive effects observed with administration of idalopirdine in donepezil-treated patients with Alzheimer's disease observed in the phase 2 studies (Wilkinson et al. 2014).

Task- and Treatment Length-Dependent Effects of Vortioxetine on Scopolamine-Induced Cognitive Dysfunction and Hippocampal Extracellular Acetylcholine in Rats.

Major depressive disorder (MDD) is a common psychiatric disorder that often features impairments in cognitive function, and these cognitive symptoms can be important determinants of functional ability. Vortioxetine is a multimodal antidepressant that may improve some aspects of cognitive function in patients with MDD, including attention, processing speed, executive function, and memory. However, the cause of these effects is unclear, and there are several competing theories on the underlying mechanism, notably including regionally-selective downstream enhancement of glutamate neurotransmission and increased acetylcholine (ACh) neurotransmission. The current work sought to evaluate the ACh hypothesis by examining vortioxetine’s ability to reverse scopolamine-induced impairments in rodent tests of memory and attention. Additionally, vortioxetine’s effects on hippocampal extracellular ACh levels were examined alongside studies of vortioxetine’s pharmacokinetic profile. We found that acute vortioxetine reversed scopolamine-induced impairments in social and object recognition memory, but did not alter scopolamine-induced impairments in attention. Acute vortioxetine also induced a modest and short-lived increase in hippocampal ACh levels. However, this short-term effect is at variance with vortioxetine’s moderately long brain half life (5.1 hours). Interestingly, subchronic vortioxetine treatment failed to reverse scopolamine-induced social recognition memory deficits and had no effects on basal hippocampal ACh levels. These data suggest that vortioxetine has some effects on memory that could be mediated through cholinergic neurotransmission, however these effects are modest and only seen under acute dosing conditions. These limitations may argue against cholinergic mechanisms being the primary mediator of vortioxetine′s cognitive effects, which are observed under chronic dosing conditions in patients with MDD.

The 5-HT6 receptor antagonist idalopirdine potentiates the effects of acetylcholinesterase inhibition on neuronal network oscillations and extracellular acetylcholine levels in the rat dorsal hippocampus

The 5-HT6 receptor has emerged as a promising target for cognitive disorders and combining a 5-HT6 receptor antagonist with an acetylcholinesterase inhibitor (AChEI) represents a novel approach for the symptomatic treatment of Alzheimer's disease (AD). A recent phase 2 trial showed that the selective 5-HT6 receptor antagonist idalopirdine (Lu AE58054) improved cognition in patients with moderate AD on stable treatment with the AChEI donepezil. Here we investigated the effects of idalopirdine in combination with donepezil on hippocampal function using in vivo electrophysiology and microdialysis. Network oscillations in the hippocampus were recorded during electrical stimulation of the brainstem nucleus pontis oralis (nPO) in the anesthetized rat and hippocampal acetylcholine (ACh) levels were measured in the freely-moving rat. In addition, potential pharmacokinetic interactions between idalopirdine and donepezil were assessed. Idalopirdine alone did not affect hippocampal network oscillations or ACh levels. Donepezil (0.3 and 1.0 mg/kg i.v.) dose-dependently increased hippocampal theta and gamma power during nPO stimulation. Idalopirdine (2 mg/kg i.v.), administered 1 h prior to donepezil, potentiated the theta and gamma response to 0.3 mg/kg donepezil and prolonged the gamma response to 1 mg/kg donepezil. Donepezil (1.3 mg/kg s.c.) increased extracellular ACh levels in the hippocampus and this was further augmented by administration of idalopirdine (10 mg/kg p.o.) 2 h prior to donepezil. These effects could not be attributed to a pharmacokinetic interaction between the compounds. This study demonstrates that idalopirdine potentiates the effects of donepezil on two pharmacodynamic biomarkers associated with cognition, i.e. neuronal oscillations and extracellular ACh levels in the hippocampus. Such potentiation could contribute to the procognitive effects of idalopirdine observed in donepezil-treated AD patients.

Extracellular levels of ATP and acetylcholine during lithium-pilocarpine induced status epilepticus in rats

Acetylcholine (ACh) and ATP are rapidly acting neurotransmitters with a putative role in epileptic seizures. In the present study we investigated extracellular concentrations of both neurotransmitters in parallel by microdialysis in rat hippocampus. We found that infusion of neostigmine increases, while calcium-free perfusion and infusion of tetrodotoxin (TTX) decreases, ACh levels. Calcium-free perfusion also decreased ATP levels which were, however, not affected by neostigmine or TTX. During status epilepticus, ACh levels were increased threefold but returned to baseline after the termination of seizures by diazepam. ATP levels were unchanged during status epilepticus but a several-fold increase was seen when AOPCP, an inhibitor of 5′-endonucleotidase, was infused. The results demonstrate an increase of ATP levels during epileptic seizures which, however, was not of neuronal origin.