Comprehensive analysis of two Shank3 and the Cacna1c mouse models of autism spectrum disorder.

To expand, analyze and extend published behavioral phenotypes relevant to autism spectrum disorder (ASD), we present a study of three ASD genetic mouse models: Feng's Shank3tm2Gfng model, hereafter Shank3/F, Jiang's Shank3tm1Yhj model, hereafter Shank3/J and the Cacna1c deletion model. The Shank3 models mimick gene mutations associated with Phelan-McDermid Syndrome and the Cacna1c model recapitulates the deletion underlying Timothy syndrome. This study utilizes both standard and novel behavioral tests with the same methodology used in our previously published companion report on the Cntnap2 null and 16p11.2 deletion models. We found that some but not all behaviors replicated published findings and those that did replicate, such as social behavior and overgrooming in Shank3 models, tended to be milder than reported elsewhere. The Shank3/F model, and to a much lesser extent, the Shank3/J and Cacna1c models, showed hypoactivity and a general anxiety-like behavior triggered by external stimuli which pervaded social interactions. We did not detect deficits in a cognitive procedural learning test nor did we observe perseverative behavior in these models. We did, however, find differences in exploratory patterns of Cacna1c mutant mice suggestive of a behavioral effect in a social setting. In addition, only Shank3/F showed differences in sensory-gating. Both positive and negative results from this study will be useful in identifying the most robust and replicable behavioral signatures within and across mouse models of autism. Understanding these phenotypes may shed light of which features to study when screening compounds for potential therapeutic interventions.

Developing new pharmacotherapies for autism.

Developing new pharmacotherapies for autism spectrum disorder (ASD) is a challenge. ASD has a complex genetic architecture, several neurobiological phenotypes and multiple symptom domains. However, new opportunities are emerging that could lead to the development of 'targeted' and individualized pharmacological interventions. Here, first we review these important new insights into the aetiology and neurobiology of ASD with particular focus on (i) genetic variants mediating synaptic structure and functioning and (ii) differences in brain anatomy, chemistry and connectivity in this condition. The characterization of the genotypic and phenotypic differences underlying ASD might in the future be invaluable for stratifying the large range of different individuals on the autism spectrum into genetically and/or biologically homogeneous subgroups that might respond to similar targeted interventions. Secondly, we propose a strategic framework for the development of targeted pharmacotherapies for ASD, which comprises several different stages in which research findings are translated into clinical applications. The establishment of animal models and cellular assays is important for developing and testing new pharmacological targets before initiating large-scale clinical trials. Finally, we present the European Autism Interventions - A Multicentre Study for Developing New Medications (EU-AIMS) Initiative, which was set up in the context of the EU Innovative Medicines Initiative as the first European platform for integrated translational research in ASD. The EU-AIMS Initiative consists of academic and industrial partners working in collaboration to deliver a more 'personalized' approach to diagnosing and treating ASD in the future.

Translational approaches to the biology of Autism: false dawn or a new era?

Discovering novel treatments for Autism Spectrum Disorders (ASD) is a challenge. Its etiology and pathology remain largely unknown, the condition shows wide clinical diversity, and case identification is still solely based on symptomatology. Hence clinical trials typically include samples of biologically and clinically heterogeneous individuals. 'Core deficits', that is, deficits common to all individuals with ASD, are thus inherently difficult to find. Nevertheless, recent reports suggest that new opportunities are emerging, which may help develop new treatments and biomarkers for the condition. Most important, several risk gene variants have now been identified that significantly contribute to ASD susceptibility, many linked to synaptic functioning, excitation-inhibition balance, and brain connectivity. Second, neuroimaging studies have advanced our understanding of the 'wider' neural systems underlying ASD; and significantly contributed to our knowledge of the complex neurobiology associated with the condition. Last, the recent development of powerful multivariate analytical techniques now enable us to use multi-modal information in order to develop complex 'biomarker systems', which may in the future be used to assist the behavioral diagnosis, aid patient stratification and predict response to treatment/intervention. The aim of this review is, therefore, to summarize some of these important new findings and highlight their potential significant translational value to the future of ASD research.

PEGylation enhances the therapeutic potential for insulin-like growth factor I in central nervous system disorders.

OBJECTIVE: Due to its potent neurotrophic activity, insulin-like growth factor I (IGF-I) has been proposed many times for therapeutic application in disorders of the central nervous system (CNS). However, insufficient brain delivery to yield beneficial central without peripheral side effects have prevented clinical development in most instances. DESIGN: We recently reported the generation of a polyethylene-glycol modified IGF-I variant (PEG-IGF-I) with prolonged half-life and less acute side effects, but with fully maintained slow anabolic activity. Here we investigated if these beneficial properties result in improved brain availability of the drug, thereby reaching therapeutically relevant steady-state concentrations to elicit beneficial effects on neuronal function. RESULTS: After a single subcutaneous injection, PEG-IGF-I reached much higher steady-state levels in brain tissue and cerebrospinal fluid compared with IGF-I. Two weeks treatment with PEG-IGF-I was sufficient to modulate brain plasticity processes, as judged by changes in synaptic proteins and related animal behavior. Furthermore, chronic treatment of a mouse model of brain amyloidosis with PEG-IGF-I reverted deficits in insulin/IGF-I signaling, synaptic proteins and cognitive performance. CONCLUSIONS: Our data generate the therapeutic potential for PEG-IGF-I to treat CNS disorders by systemic drug application, and in addition scientifically support its application in disorders of synaptic function and neuronal development.

Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK1 and NK3 receptors.

BACKGROUND AND PURPOSE: Clinical results of osanetant and talnetant (selective-NK3 antagonists) indicate that blocking the NK3 receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK1/NK3 antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative). EXPERIMENTAL APPROACH: RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe7]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK1) and senktide-induced tail whips in mice (MTW; NK3). KEY RESULTS: RO4583298 has a high-affinity for NK1 (human and gerbil) and NK3 (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK3, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW. CONCLUSIONS AND IMPLICATIONS: RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK1/NK3 antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK1 and NK3 receptors in psychiatric disorders.

Dissociable roles of mGlu5 and dopamine receptors in the rewarding and sensitizing properties of morphine and cocaine.

RATIONALE: Drugs of abuse are initially used because of their rewarding properties. As a result of repeated drug exposure, sensitization to certain behavioral effects of drugs occurs, which may facilitate the development of addiction. Recent studies have implicated the metabotropic glutamate receptor 5 (mGlu5 receptor) in drug reward, but its role in sensitization is unclear. Stimulation of dopamine receptors plays an important role in drug reward, but not in the sensitizing properties of cocaine and morphine. OBJECTIVE: This study aims to evaluate the role of mGlu5 and dopamine receptors in the development of cocaine- and morphine-induced conditioned place preference (CPP) and psychomotor sensitization. MATERIALS AND METHODS: Rats were treated with the mGlu5 receptor antagonist MTEP (0, 1, 3, and 10 mg/kg, i.p.) or the dopamine receptor antagonist α-flupenthixol (0, 0.125, 0.25, and 0.5 mg/kg, i.p.) during place conditioning with either morphine (3 mg/kg, s.c.) or cocaine (15 mg/kg, i.p.). Furthermore, MTEP (1 mg/kg, i.p.) or α-flupenthixol (0.5 mg/kg, i.p.) was co-administered during cocaine (30 mg/kg, i.p.) or morphine (3.0 mg/kg, s.c.) pretreatment and psychomotor sensitization was tested 3 weeks post-treatment. RESULTS: MTEP attenuated the development of morphine- but not cocaine-induced CPP. In contrast, MTEP suppressed the development of cocaine- but not morphine-induced psychomotor sensitization. α-Flupenthixol blocked the development of both cocaine- and morphine-induced CPP but did not affect the development of sensitization to either drug. CONCLUSION: Dopamine receptor stimulation mediates cocaine and morphine reward but not sensitization. In contrast, the role of mGlu5 receptors in reward and sensitization is drug-specific.

Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one as a positive allosteric modulator of GABAB receptors.

BACKGROUND AND PURPOSE: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABAB receptors. EXPERIMENTAL APPROACH: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Galpha16-hGABA(B1a,2a) cells by Fluorometric Imaging Plate Reader and GTPgamma[35S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stress-induced hyperthermia (SIH) models. KEY RESULTS: In GTPgamma[35S]-binding assays, 0.3 microM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration-response curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3- and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg(-1) p.o. rac-BHFF (100 mg kg(-1) p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice. CONCLUSIONS AND IMPLICATIONS: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABAB receptors in the central and peripheral nervous systems.

Cognitive performance in neurokinin 3 receptor knockout mice.

RATIONALE: The neurokinin 3 (NK(3)) receptor is a novel target under investigation for improvement of the symptoms of schizophrenia due to its ability to modulate dopaminergic signaling. However, research on effects of NK(3) antagonism with animal models has been hindered because of species differences in the receptor between humans, rats, and mice. OBJECTIVES: The aim of the present study is to further knowledge on the role of NK(3) in cognitive functioning by testing the effect of knockout of the NK(3) receptor on tests of working memory, spatial memory, and operant responding. MATERIALS AND METHODS: NK(3) knockout mice generated on a C57Bl/6 background were tested in delayed matching to position (DMTP), spontaneous alternation, Morris water maze, and active avoidance tasks. RESULTS: NK(3) knockout mice showed better performance in the DMTP task, though not delay dependently, which points to an effect on operant performance but not on working memory. No differences were seen between the groups in spontaneous alternation, another indication that working memory is not affected in NK(3) knockouts. There was no impairment in knockout mice in Morris water maze training, and the mice also showed faster response latency in the active avoidance task during training. CONCLUSIONS: Collectively, these results support a role for the NK(3) receptor in performance of operant tasks and in spatial learning but not in working memory.

Metabotropic glutamate receptor modulation, translational methods, and biomarkers: relationships with anxiety.

RATIONALE: The increasing awareness of the need to align clinical and preclinical research to facilitate rapid development of new drug therapies is reflected in the recent introduction of the term "translational medicine". This review examines the implications of translational medicine for psychiatric disorders, focusing on metabotropic glutamate (mGlu) receptor biology in anxiety disorders and on anxiety-related biomarkers. OBJECTIVES: This review aims to (1) examine recent progress in translational medicine, emphasizing the role that translational research has played in understanding of the potential of mGlu receptor agonists and antagonists as anxiolytics, (2) identify lacunas where animal and human research have yet to be connected, and (3) suggest areas where translational research can be further developed. RESULTS: Current data show that animal and human mGlu(5) binding can be directly compared in experiments using the PET ligand (11)C-ABP688. Testing of the mGlu(2/3) receptor agonist LY354740 in the fear-potentiated startle paradigm allows direct functional comparisons between animals and humans. LY354740 has been tested in panic models, but in different models in rats and humans, hindering efforts at translation. Other potentially translatable methods, such as stress-induced hyperthermia and HPA-axis measures, either have been underexploited or are associated with technical difficulties. New techniques such as quantitative trait loci (QTL) analysis may be useful for generating novel biomarkers of anxiety. CONCLUSIONS: Translational medicine approaches can be valuable to the development of anxiolytics, but the amount of cross-fertilization between clinical and pre-clinical departments will need to be expanded to realize the full potential of these approaches.

Effects of aripiprazole/OPC-14597 on motor activity, pharmacological models of psychosis, and brain activity in rats.

Aripiprazole (OPC-14597) is an antipsychotic with a unique pharmacology as a dopamine D2 receptor partial agonist, which has been demonstrated to reduce symptoms of schizophrenia. To further profile this compound in preclinical models, we examined aripiprazole-induced activity changes as measured by pharmacological magnetic resonance imaging (MRI) and characterized the drug in several rodent models of motor behaviors and of psychosis. Continuous arterial spin labeling MRI measuring blood perfusion (as an indirect measure of activity) reveals that aripiprazole dose-dependently decreased brain activity in the entorhinal piriform cortex, perirhinal cortex, nucleus accumbens shell, and basolateral amygdala. While no deficits were observed in the rotarod test for motor coordination in the simpler (8 RPM) version, in the more challenging condition (16 RPM) doses of 10 and 30mg/kg i.p. produced deficits. Catalepsy was seen only at the highest dose tested (30mg/kg i.p.) and only at the 3 and 6h time points, not at the 1h time point. In pharmacological models of psychosis, 1-30mg/kg aripiprazole i.p. effectively reduced locomotor activity induced by dopamine agonists (amphetamine and apomorphine), NMDA antagonists (MK-801 and phencyclidine (PCP)), and a serotonin agonist (2,5-dimethoxy-4-iodoamphetamine (DOI)). However, aripiprazole reversed prepulse inhibition deficits induced by amphetamine, but not by any of the other agents tested. Aripiprazole alters brain activity in regions relevant to schizophrenia, and furthermore, has a pharmacological profile that differs for the two psychosis models tested and does not match the typical or atypical psychotics. Thus, D2 partial agonists may constitute a new group of antipsychotics.

Allosteric modulators for mGlu receptors.

The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions. The potential use of mGluRs as drug targets for various nervous system pathologies such as anxiety, depression, schizophrenia, pain or Parkinson's disease has triggered an intense search for subtype selective modulators and resulted in the identification of numerous novel pharmacological agents capable to modulate the receptor activity through an interaction at an allosteric site located in the transmembrane domain. The present review presents the most recent developments in the identification and the characterization of allosteric modulators for the mGlu receptors.

Apomorphine-induced disruption of prepulse inhibition that can be normalised by systemic haloperidol is insensitive to clozapine pretreatment.

RATIONALE: Prepulse inhibition (PPI) of startle refers to the phenomenon in which a weak prepulse attenuates the startle response to a succeeding intense stimulus. PPI can be disrupted by systemic apomorphine in animals, and reduced PPI has been consistently reported in schizophrenia patients. The ability of the atypical antipsychotic clozapine to reverse apomorphine-induced PPI deficit has been demonstrated in the rat, but has not yet been tested in the mouse. The present study was designed to fill this gap. OBJECTIVE AND RESULTS: We investigated the efficacy of clozapine in reversing apomorphine-induced (2.0 or 2.5 mg/kg, s.c.) PPI deficit in C57BL6 mice. Clozapine failed to restore PPI disruption in apomorphine-treated mice in two independent laboratories across two dose ranges (1-3 mg/kg, i.p., or 3-30 mg/kg, p.o.), whereas the typical antipsychotic haloperidol (1 mg/kg, i.p.) completely normalised PPI performance. CONCLUSIONS: Unlike the rat, apomorphine-induced PPI disruption in mice might be instrumental in distinguishing between typical and atypical antipsychotic drugs. This also lends further support to the suggestion that the neuropharmacology of PPI is not identical in the two rodent species.

Insight into the function of Group I and Group II metabotropic glutamate (mGlu) receptors: behavioural characterization and implications for the treatment of CNS disorders.

Following the molecular cloning in the early 1990s of the metabotropic glutamate receptors (mGlu1-8), research that focused on the physiology, pharmacology and function of these receptors revealed their potential role in CNS disorders. Numerous psychiatric and neurological dis-orders are indeed linked to changes in excitatory processes, in which glutamate plays a key role. In contrast to ligand-gated ion channels [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and kainate], which are responsible for fast excitatory transmission, mGlu receptors have a more modulatory role, by contributing to fine-tuning of synaptic efficacy, and control of the accuracy and sharpness of the transmission. Given the fact that the mGlu receptors are G-protein coupled, they obviously constitute new 'drugable' targets for the treatment of various CNS disorders. Due to the recent emergence of subtype-specific ligands for Group I and II mGlu receptors, this review will concentrate on the molecular characteristics, brain localization, pharmacology and physiological role of these receptors, in order to provide further insights into their therapeutic potential.

NPY-mRNA expressions in the nucleus accumbens, caudate putamen and cerebral cortex of apomorphine-susceptible and apomorphine-unsusceptible rats.

Using the apomorphine-induced stereotyped gnawing response as a selection criterion, two distinct groups of rats can be distinguished, apomorphine-susceptible (APO-SUS) and apomorphine-unsusceptible (APO-UNSUS) rats. These two lines differ in several components of both striatal and extrastriatal areas. This study deals with the expression of neuropeptide Y (NPY)mRNA-expressing neurons in the nucleus accumbens, caudate putamen and cerebral cortex of both rat lines, using non-radioactive in situ hybridisation. The morphology of the neurons in the three regions is similar, viz. oblong, rectangular or triangular, with two or three processes. The neurons are homogeneously distributed in all regions, and in the nucleus accumbens they are particularly numerous ventrally to the anterior commissure. Using automated image analysis, the mean numerical density of NPYmRNA-positive neurons per brain region and the mean NPYmRNA expression level per neuron per brain region were determined. No differences appear in the numerical densities of NPYmRNA-containing neurons in the nucleus accumbens, caudate putamen and cortex between APO-SUS and APO-UNSUS rats. However, distinct differences between the rat lines are present in the level of NPYmRNA expression per neuron in the nucleus accumbens and in the caudate putamen, showing that NPY contributes to the differential neurochemical make-up of these rat lines that is responsible for their obvious differences in behaviour, physiology and immune competence.

In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article.

There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain. We have characterized novel subtype-selective mGlu(5) receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu(5) receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety.

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)).

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Novel allosteric antagonists shed light on mglu(5) receptors and CNS disorders.

Although multiple metabotropic glutamate (mglu) receptor subtypes were cloned in the early 1990s, progress in the characterization of these receptors has been slow because of difficulties in obtaining subtype-selective ligands. However, in the past few years exciting progress has been made on the mglu(5) receptor subtype following the identification of selective non-amino-acid-like ligands that implicate the mglu(5) receptor as a potentially important therapeutic target, particularly for the treatment of pain and anxiety.

Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain.

The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naïve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.

Mouse models of alpha-synucleinopathy and Lewy pathology.

The discovery of two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein alpha-synuclein (alphaSN) that are genetically linked to rare familial forms of Parkinson's disease and its accumulation in Lewy bodies and Lewy neurites has triggered several attempts to generate transgenic mice overexpressing human alphaSN. Analogous to a successful strategy for the production of transgenic animal models for Alzheimer's disease we generated mice expressing wildtype and the A53T mutant of human alphaSN in the nervous system under control of mouse Thy1 regulatory sequences. These animals develop neuronal alpha-synucleinopathy, striking features of Lewy pathology, neuronal degeneration and motor defects. Neurons in brainstem and motor neurons appeared particularly vulnerable. Motor neuron pathology included axonal damage and denervation of neuromuscular junctions, suggesting that alphaSN may interfere with a universal mechanism of synapse maintenance. Thy1-transgene expression of wildtype human alphaSN resulted in comparable pathological changes thus supporting a central role for mutant and wildtype alphaSN in familial and idiopathic forms of diseases with neuronal alpha-synucleinopathy and Lewy pathology. The mouse models provide means to address fundamental aspects of alpha-synucleinopathy and to test therapeutic strategies.