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Intracellular accumulation of tau protein is a hallmark of Alzheimer’s Disease and Progressive Supranuclear Palsy, as well as other neurodegenerative disorders collectively known as tauopathies. Despite our increasing understanding of the mechanisms leading to the initiation and progression of tau pathology, the field still lacks appropriate disease models to facilitate drug discovery. Here, we established a novel and modulatable seeding-based neuronal model of full-length 4R tau accumulation using humanized mouse cortical neurons and seeds from P301S human tau transgenic animals. The model shows specific and consistent formation of intraneuronal insoluble full-length 4R tau inclusions, which are positive for known markers of tau pathology (AT8, PHF-1, MC-1), and creates seeding competent tau. The formation of new inclusions can be prevented by treatment with tau siRNA, providing a robust internal control for use in qualifying the assessment of potential therapeutic candidates aimed at reducing the intracellular pool of tau. In addition, the experimental set up and data analysis techniques used provide consistent results in larger-scale designs that required multiple rounds of independent experiments, making this is a versatile and valuable cellular model for fundamental and early pre-clinical research of tau-targeted therapies.

Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred as tauopathies, including frontal temporal dementia and Alzheimer’s disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reporti.molecular proximity of physiological full-length tau at microtubules;ii.changes in tau conformation and self-interaction associated with tau phosphorylation, as well asiii.tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e.full-length or short fragments, wild-type or the disease-associated tau(P301L) variant) further insights in the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.

Histaminergic H3 inverse agonists, by stimulating central histamine release, represent attractive drug candidates to treat cognitive disorders. The present studies aimed to describe the mechanistic profile of S 38093 a novel H3 receptors inverse agonist. S 38093 displays a moderate affinity for rat, mouse and human H3 receptors (Ki=8.8, 1.44 and 1.2 µM, respectively) with no affinity for other histaminergic receptors. In cellular models, the compound was able to antagonize mice H3 receptors (KB=0.65 µM) and to suppress cAMP decrease induced by an H3 agonist via human H3 receptors (KB=0.11 µM). The antagonism properties of the compound were confirmed by electrophysiological studies on rat hippocampal slices (from 0.1 μM). In cells expressing a high H3 density, S 38093 behaved as a moderate inverse agonist at rat and human H3 receptors (EC50=9 and 1.7 µM, respectively). S 38093 was rapidly absorbed in mouse and rat (Tmax=0.25–0.5 h), slowly in monkey (2 h), with a bioavailability ranging from 20% to 60% and t1/2 ranging from 1.5 to 7.4 h. The compound was widely distributed with a moderate volume of distribution and low protein binding. The brain distribution of S 38093 was rapid and high. In mice, S 38093 significantly increased ex vivo N-tele-Methylhistamine cerebral levels from 3 mg/kg p.o. and antagonized R-α-Methylhistamine-induced dipsogenia from 10 mg/kg i.p. Taken together, these data suggest that S 38093, a novel H3 inverse agonist, is a good candidate for further in vivo evaluations, in particular in animal models of cognition.

This study evaluated the procognitive effects of S 38093 (a new inverse agonist of the histaminergic H3 receptor) and S 47445 (a new α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) in 2-3-month-old Swiss mice as compared with donepezil and memantine, two main reference compounds in the treatment of Alzheimer's disease. The object recognition task allows the study of natural forgetting and is classically used in assessing drug effects on memory. Here, we show that mice exhibit significant object recognition at short (15 min) but not long (24 h) retention intervals separating the familiarization and recognition phases. S 47445 (1.0, 3.0, and 10.0 mg/kg) and S 38093 (0.3, 1.0, and 3.0 mg/kg), both administered postoperatively, 1 h before familiarization and recognition sessions, rescued memory at the long retention interval; their memory-enhancing effects were as powerful as those obtained with donepezil or memantine (1.0 and 3.0 mg/kg for both compounds). Thus, S 38093 and S 47445, detected as positive controls in the object recognition task, are promising compounds for the treatment of amnesic syndromes.

Background Tau is an axon-enriched protein that binds to and stabilizes microtubules, and hence plays a crucial role in neuronal function. In Alzheimer’s disease (AD), pathological tau accumulation correlates with cognitive decline. Substantial visual deficits are found in individuals affected by AD including a preferential loss of retinal ganglion cells (RGCs), the neurons that convey visual information from the retina to the brain. At present, however, the mechanisms that underlie vision changes in these patients are poorly understood. Here, we asked whether tau plays a role in early retinal pathology and neuronal dysfunction in AD. Methods Alterations in tau protein and gene expression, phosphorylation, and localization were investigated by western blots, qPCR, and immunohistochemistry in the retina and visual pathways of triple transgenic mice (3xTg) harboring mutations in the genes encoding presenilin 1 (PS1M146 V), amyloid precursor protein (APPSwe), and tau (MAPTP301L). Anterograde axonal transport was assessed by intraocular injection of the cholera toxin beta subunit followed by quantification of tracer accumulation in the contralateral superior colliculus. RGC survival was analyzed on whole-mounted retinas using cell-specific markers. Reduction of tau expression was achieved following intravitreal injection of targeted siRNA. Results Our data demonstrate an age-related increase in endogenous retinal tau characterized by epitope-specific hypo- and hyper-phosphorylation in 3xTg mice. Retinal tau accumulation was observed as early as three months of age, prior to the reported onset of behavioral deficits, and preceded tau aggregation in the brain. Intriguingly, tau build up occurred in RGC soma and dendrites, while tau in RGC axons in the optic nerve was depleted. Tau phosphorylation changes and missorting correlated with substantial defects in anterograde axonal transport that preceded RGC death. Importantly, targeted siRNA-mediated knockdown of endogenous tau improved anterograde transport along RGC axons. Conclusions Our study reveals profound tau pathology in the visual system leading to early retinal neuron damage in a mouse model of AD. Importantly, we show that tau accumulation promotes anterograde axonal transport impairment in vivo, and identify this response as an early feature of neuronal dysfunction that precedes cell death in the AD retina. These findings provide the first proof-of-concept that a global strategy to reduce tau accumulation is beneficial to improve axonal transport and mitigate functional deficits in AD and tauopathies.

S 38093, a novel histamine H3 receptor inverse agonist, was tested in a series of neurochemical and behavioral paradigms designed to evaluate its procognitive and arousal properties. In intracerebral microdialysis studies performed in rats, S 38093 dose-dependently increased histamine extracellular levels in the prefrontal cortex and facilitated cholinergic transmission in the prefrontal cortex and hippocampus of rats after acute and chronic administration (10mg/kg i.p.). Acute oral administration of S 38093 at 0.1mg/kg significantly improved spatial working memory in rats in the Morris water maze test. The compound also displayed cognition enhancing properties in the two-trial object recognition task in rats, in a natural forgetting paradigm at 0.3 and 1mg/kg p.o. and in a scopolamine-induced memory deficit situation at 3mg/kg p.o. The property of S 38093 to promote episodic memory was confirmed in a social recognition test in rats at 0.3 and 1mg/kg i.p. Arousal properties of S 38093 were assessed in freely moving rats by using electroencephalographic recordings: at 3 and 10mg/kg i.p., S 38093 significantly reduced slow wave sleep delta power and induced at the highest dose a delay in sleep latency. S 38093 at 10mg/kg p.o. also decreased the barbital-induced sleeping time in rats. Taken together these data indicate that S 38093, a novel H3 inverse agonist, displays cognition enhancing at low doses and arousal properties at higher doses in rodents.

Cannabis is the most widely consumed drug associated with 3,4-methylenedioxymethamphetamine (MDMA) use. This study examines whether low doses of MDMA and delta-9-tetrahydrocannabinol (THC) produce synergistic rewarding/reinforcing effects in mice using the conditioned place preference (CPP) and operant self-administration paradigms. Changes in dopamine (DA) outflow were monitored in the nucleus accumbens (NAC) after single or combined administration of these compounds. MDMA induced a significant CPP at the dose of 10 mg/kg but not at the dose of 3 mg/kg. THC (0.3 mg/kg) by itself was also ineffective in this paradigm. The combined administration of the low dose of MDMA (3 mg/kg) and THC (0.3 mg/kg) produced CPP, whereas the combination of MDMA (10 mg/kg) and THC (0.3 mg/kg) significantly decreased CPP. Animals treated with THC self-administered a sub-threshold dose of MDMA (0.06 mg/kg per infusion), while animals receiving vehicle did not. However, THC did not modify the self-administration of an effective dose of MDMA (0.125 mg/kg per infusion). In microdialysis studies, a low dose of THC significantly increased DA outflow in the NAC, while a low dose of MDMA did not. When MDMA was administered before THC, DA levels decreased with respect to THC. However, when THC was administered before MDMA, DA levels were not significantly modified with respect to THC. These results demonstrate that a low dose of THC modifies in different ways (increases and decreases) the sensitivity of animals to the behavioural effects of MDMA and that THC and MDMA converge at a common mechanism modulating DA outflow in the NAC of mice.

Studies showing psychostimulant-like effects of exogenous neurotensin (NT) infused into the ventral tegmental area (VTA) prompted us to examine the role in the VTA of the endogenous NT in behavioral sensitization to amphetamine. Rats were sensitized to amphetamine by means of a subcutaneous amphetamine (1 mg/kg) injection, and the same dose was injected 7 days later to evaluate the expression of sensitization. The highly selective NT-receptor antagonist SR 142948A was injected into the VTA prior to the first and/or second amphetamine administration. SR 142948A (5 pmol/side) given before the first amphetamine exposure prevented the induction of behavioral sensitization, but did not alter the acute response to amphetamine. SR 142948A given with the second amphetamine administration did not affect the expression of behavioral sensitization. In contrast to administration into the VTA, intraperitoneal administration of SR 142948A (0.03, 0.1, or 0.3 mg/kg) had no detectable effect on the induction of amphetamine sensitization. These results suggest that activation of VTA NT receptors by endogenous NT may contribute to the neuroadaptations underlying behavioral sensitization to amphetamine.

Melanin-concentrating hormone (MCH) 1 receptors are widely expressed in limbic structures and cortex. Their inactivation is associated with anxiolytic and antidepressive properties but little information is available concerning cognition. This issue was addressed using the selective antagonists, SNAP-7941 and GW3430, in a social recognition paradigm in rats. The muscarinic blocker, scopolamine (1.25 mg/kg s.c.), reduced social recognition, an action dose-dependently blocked by SNAP-7941 and GW3430 (0.63-10.0 and 20.0-80.0 mg/kg i.p., respectively) which did not themselves display amnesic properties. Further, in a protocol where a spontaneous deficit was induced by a prolonged inter-session delay, SNAP-7941 and GW3430 dose-dependently enhanced social recognition. In dialysis studies, SNAP-7941 (0.63-40.0 mg/kg i.p.) and GW3430 (10.0-40.0 mg/kg i.p.) elevated extracellular levels of acetylcholine (ACh) in the frontal cortex (FCX) of freely moving rats. The SNAP-7941 effect was specific, as it did not increase levels of ACh in ventral and dorsal hippocampus: moreover, it did not modify levels of noradrenaline, dopamine, serotonin and glutamate in FCX. Active doses of SNAP-7941 and GW3430 corresponded to doses (2.5-40.0 and 10.0-80.0 mg/kg i.p., respectively) exerting anxiolytic properties in Vogel conflict and ultrasonic vocalization tests, and antidepressant actions in forced swim, isolation-induced aggression and marble-burying procedures. In contrast to SNAP-7941 and GW3430, the benzodiazepine, diazepam, decreased social recognition and dialysate levels of ACh, while the tricyclic, imipramine, reduced social recognition and failed to enhance cholinergic transmission. In conclusion, at anxiolytic and antidepressant doses, SNAP-7941 and GW3430 improve social recognition and elevate extracellular ACh levels in FCX. This profile differentiates MCH 1 receptor antagonists from conventional anxiolytic and antidepressant agents.

Though dopaminergic mechanisms modulate cholinergic transmission and cognitive function, the significance of specific receptor subtypes remains uncertain. Here, we examined the roles of dopamine D(3) versus D(2) receptors. By analogy with tacrine (0.16-2.5 mg/kg, s.c.), the selective D(3) receptor antagonists, S33084 (0.01-0.63) and SB277,011 (0.63-40.0), elicited dose-dependent, pronounced and sustained elevations in dialysis levels of acetylcholine (ACh) in the frontal cortex, but not the hippocampus, of freely-moving rats. The actions of these antagonists were stereospecifically mimicked by (+)S14297 (1.25), whereas its inactive distomer, (-)S17777, was ineffective. The preferential D(2) receptor antagonist, L741,626 (10.0), failed to modify levels of ACh. S33084 (0.01-0.63) and SB277,011 (0.16-2.5) also mimicked tacrine (0.04-0.63) by dose-dependently attenuating the deleterious influence of scopolamine (1.25) upon social memory (recognition by an adult rat of a juvenile conspecific). Further, (+)S14297 (1.25) versus (-)S17777 stereospecifically blocked the action of scopolamine. Using an intersession interval of 120 min (spontaneous loss of recognition), S33084 (0.04-0.63), SB277,011 (0.16-10.0) and (+)S14297 (0.63-10.0) likewise mimicked tacrine (0.16-2.5) in enhancing social memory. In contrast, L741,626 (0.16-10.0) displayed amnesic properties. In conclusion, selective blockade of D(3) receptors facilitates frontocortical cholinergic transmission and improves social memory in rats. These data support the pertinence of D(3) receptors as a target for treatment of disorders in which cognitive function is compromised.

Acute and repeated psychostimulant administration induces a long-lasting enhanced behavioural response to a subsequent drug challenge, known as behavioural sensitization. This phenomenon involves persistent neurophysiological adaptations, which may lead to drug addiction. Brain dopaminergic pathways have been implicated as the main neurobiological substrates of behavioural sensitization, although other neurotransmitters and neuromodulators may also participate. In order to investigate a possible involvement of opioid systems in amphetamine (AMPH) behavioural sensitization, we studied the AMPH-induced changes in Proenkephalin (Pro-Enk) mRNA expression in forebrain areas in both drug-naive and AMPH-sensitized rats. Male Sprague-Dawley rats were sensitized to AMPH by means of a single AMPH (1 mg/kg s.c.) injection and the same dose was injected 7 days later to assess the expression of sensitization. Pro-Enk mRNA levels were evaluated by in situ hybridization in coronal brain sections. AMPH injection induced an increase in Pro-Enk mRNA expression in the nucleus accumbens and the medial-posterior caudate-putamen in drug-naive rats. Challenge with AMPH to rats injected 1 week earlier with AMPH induced motor sensitization and increased and decreased Pro-Enk mRNA expression in the prefrontal cortex and the anterior medial caudate-putamen, respectively. Our results suggest that alterations in cortical and striatal enkephalinergic systems could contribute to the expression of AMPH behavioural sensitization.

Since its inception some two decades ago, microdialysis has rapidly assumed a crucial role as an interface between cellular models of drug actions in vitro and studies of their behavioural effects in vivo. Microdialysis provides invaluable information regarding the mechanisms of action of psychotropic agents and their influence upon endogenous modulators implicated in the aetiology and treatment of CNS disorders. In addition, measures of extracellular levels of specific neurotransmitters are complementary to behavioural parameters in the characterisation of experimental models of psychiatric and neurological diseases. Further, microdialysis can be used for quantification of levels of psychotropic agents themselves in specific brain regions. Though microdialysis techniques are increasingly being applied to a broad variety of cellular mediators, most studies have to date focussed on monoamines, acetylcholine and amino acids like glycine, glutamate and GABA; that is, neurotransmitters strongly implicated in the pathogenesis and control of depression, anxiety, schizophrenia and other psychiatric states. Recent years have seen substantial improvements in the sensitivity of systems for their detection, which now permit, for example, the simultaneous quantification of dopamine (DA), serotonin (5-HT) and noradrenaline (NA) levels in single dialysis samples; quantification of acetylcholine (ACh) levels in the absence of acetylcholinesterase inhibitors; and determination of glutamate and GABA levels concurrently with glycine and a miscellany of related amino acids. The present chapter provides an overview of how microdialysis can be applied to the discovery and evaluation of centrally active drugs. Furthermore, it specifically focuses on the application of novel microdialysis techniques to the characterisation of antipsychotic agents for the improved treatment of schizophrenia.

Although dopaminergic mechanisms are known to modulate cognitive function and cholinergic transmission, their pharmacological characterization remains incomplete. Herein, the role of D1 sites was evaluated employing neurochemical and behavioural approaches. By analogy to the acetylcholinesterase inhibitor, galantamine (0.0025-0.63 mg/kg s.c.), the selective and high efficacy D1 receptor agonist, SKF 82958, dose-dependently (0.0025-0.63), robustly and potently enhanced extracellular levels of acetylcholine (ACh) in the frontal cortex and hippocampus of freely moving rats. A further agonist, SKF 81297 (0.04-0.63), mimicked this action whereas the selective antagonist, SCH 23390 (0.00063-0.63), decreased levels of ACh. In the presence of SCH 23390 (0.08), the facilitatory influence of SKF 82958 (0.04) upon ACh levels was abolished. In a model of social memory (recognition of a juvenile by an adult rat), galantamine (0.04-0.63), SKF 82958 (0.01-0.16) and SKF 81297 (0.001-0.16) dose-dependently abrogated amnesic effects of the muscarinic receptor antagonist scopolamine (1.25). Further, under conditions of spontaneous loss of recognition, mimicking the effects of galantamine (0.04-2.5), SKF 82958 (0.01-0.16) and SKF 81297 (0.04-1.25) dose-dependently and specifically facilitated social recognition. Conversely, SCH 23390 (0.0025-0.04) exerted a modest negative influence upon social recognition and, in its presence, the pro-cognitive properties of SKF 82958 were blocked. In conclusion, D1 receptors exert a tonic, facilitatory influence upon cholinergic transmission and social recognition. Although the relationship between these actions awaits further clarification, these data underpin the relevance of D1 receptors to CNS disorders in which cholinergic transmission and social cognition are disrupted.

MDMA is one of the most widely consumed recreational drugs in Europe. However, the mechanisms involved in the reinforcing properties of MDMA are still unclear. In this sense, the establishment of a reliable model of MDMA self-administration in mice could represent an important approach to study the neuronal substrates associated with MDMA reward by using genetically modified mice. To develop a reliable model of operant intravenous MDMA self-administration in drug-naive mice. Mice were trained to acquire intravenous self-administration of MDMA at different doses (0, 0.06, 0.125, 0.25, 0.5 and 1.0 mg/kg/infusion) on a FR1 schedule of reinforcement for 15 consecutive days. The motivational value of different doses of MDMA (0.125, 0.25 and 0.5 mg/kg/infusion) was then tested using a progressive ratio paradigm. Finally, [3H]-mazindol autoradiographic studies were carried out in order to quantitatively assess presynaptic dopamine transporter (DAT) binding sites in the striatum of mice trained to self-administer MDMA (0 and 1.0 mg/kg/infusion) during 15 days. The latency for discrimination between the active and inactive holes, as well as the number of animals acquiring stability criteria, varied as a function of the dose of MDMA. The mice responding for intermediate doses (0.125, 0.25 and 0.5 mg/kg/infusion) discriminated earlier than those responding for low (0.06 mg/kg/infusion) or high (1.0 mg/kg/infusion) doses. The percentage of animals achieving stability criteria increased with days of testing and was inversely proportional to the dose of MDMA. The breaking points achieved for doses of 0.125 and 0.25 mg/kg/infusion were significantly higher than for a dose of 0.5 mg/kg/infusion. No significant DAT neurotoxicity was observed in the striatum of animals self-administering MDMA at a dose of 1 mg/kg/infusion. The present results show that MDMA can be reliably self-administered by drug-naive mice.

Recent findings have given evidence a role for noradrenergic transmission in the mechanisms underlying behavioural sensitization to psychostimulants. This work was undertaken to investigate the possible role of beta-adrenergic receptors in amphetamine-induced behavioural sensitization in rats. Rats were sensitized by a single administration of amphetamine (1 mg/kg s.c.) and challenged with the same dose 7 d later. The beta(1) /beta(2) -adrenergic receptor antagonists timolol (10 mg/kg i.p.) and nadolol (10 mg/kg i.p.), which respectively cross or do not readily cross the blood-brain barrier, were injected prior to the first or second amphetamine administration. Timolol, but not nadolol, prevented the initiation of behavioural sensitization without interfering with the expression of the sensitized response or the acute locomotor response to amphetamine. Since we found amphetamine-induced fos-activated cells closely associated with dopamine beta-hydroxylase immunoreactive varicosities in the bed nucleus of the stria terminalis (BNST), we investigated the effect of a bilateral micro-injection of timolol into this nucleus. Similarly to systemic administration, intra-BNST timolol (2.5 microg/side) prevented the development of behavioural sensitization. These results suggest that central beta-adrenergic receptors could specifically modulate early neuronal changes leading to the development of behavioural sensitization to psychostimulants, and that the BNST could be an important part of the brain circuitry involved in these long-term neuroadaptations.