Points to consider when initiating clinical investigations in autistic paediatric populations-A White Paper.

Many individuals with autism spectrum disorder (ASD) experience various degrees of impairment in social interaction and communication, restricted, repetitive behaviours, interests/activities. These impairments make a significant contribution to poorer everyday adaptive functioning. Yet, there are no pharmacological therapies to effectively treat the core symptoms of ASD. Since symptoms of ASD likely emerge from a complex interplay of vulnerabilities, environmental factors and compensatory mechanisms during the early developmental period, pharmacological interventions arguably would have the greatest impact to improve long-term outcomes when implemented at a young age. It is essential therefore, that clinical development programmes of investigational drugs in ASD include the paediatric population early on in clinical trials. Such trials need to offer the prospect of direct benefit (PDB) for participants. In most cases in drug development this prospect is supported by evidence of efficacy in adults. However, the effectiveness of treatment approaches may be age-dependent, so that clinical trials in adults may not provide sufficient evidence for a PDB in children. In this white paper, we consolidate recommendations from regulatory guidelines, as well as advice from the Food and Drug Administration, USA (FDA) and the Committee for Human Medicinal Products (CHMP) consultations on various development programmes on: 1) elements to support a PDB to participants in early paediatric clinical trials in ASD, including single-gene neurodevelopment disorders, 2) aspects of study design to allow for a PDB. This white paper is intended to be complementary to existing regulatory guidelines in guiding industry and academic sponsors in their conduct of early paediatric clinical trials in ASD.

Big data and its impact on the 3Rs: a home cage monitoring oriented review.

Undisturbed home cage recording of mouse activity and behavior has received increasing attention in recent years. In parallel, several technologies have been developed in a bid to automate data collection and interpretation. Thanks to these expanding technologies, massive datasets can be recorded and saved in the long term, providing a wealth of information concerning animal wellbeing, clinical status, baseline activity, and subsequent deviations in case of experimental interventions. Such large datasets can also serve as a long-term reservoir of scientific data that can be reanalyzed and repurposed upon need. In this review, we present how the impact of Big Data deriving from home cage monitoring (HCM) data acquisition, particularly through Digital Ventilated Cages (DVCs), can support the application of the 3Rs by enhancing Refinement, Reduction, and even Replacement of research in animals.

A minimal metadata set (MNMS) to repurpose nonclinical in vivo data for biomedical research.

Although biomedical research is experiencing a data explosion, the accumulation of vast quantities of data alone does not guarantee a primary objective for science: building upon existing knowledge. Data collected that lack appropriate metadata cannot be fully interrogated or integrated into new research projects, leading to wasted resources and missed opportunities for data repurposing. This issue is particularly acute for research using animals, where concerns regarding data reproducibility and ensuring animal welfare are paramount. Here, to address this problem, we propose a minimal metadata set (MNMS) designed to enable the repurposing of in vivo data. MNMS aligns with an existing validated guideline for reporting in vivo data (ARRIVE 2.0) and contributes to making in vivo data FAIR-compliant. Scenarios where MNMS should be implemented in diverse research environments are presented, highlighting opportunities and challenges for data repurposing at different scales. We conclude with a 'call for action' to key stakeholders in biomedical research to adopt and apply MNMS to accelerate both the advancement of knowledge and the betterment of animal welfare.

Advancements in the use of xenopus oocytes for modelling neurological disease for novel drug discovery.

INTRODUCTION: Introduced about 50 years ago, the model of Xenopus oocytes for the expression of recombinant proteins has gained a broad spectrum of applications. The authors herein review the benefits brought from using this model system, with a focus on modeling neurological disease mechanisms and application to drug discovery. AREAS COVERED: Using multiple examples spanning from ligand gated ion channels to transporters, this review presents, in the light of the latest publications, the benefits offered from using Xenopus oocytes. Studies range from the characterization of gene mutations to the discovery of novel treatments for disorders of the central nervous system (CNS). EXPERT OPINION: Development of new drugs targeting CNS disorders has been marked by failures in the translation from preclinical to clinical studies. As progress in genetics and molecular biology highlights large functional differences arising from a single to a few amino acid exchanges, the need for drug screening and functional testing against human proteins is increasing. The use of Xenopus oocytes to enable precise modeling and characterization of clinically relevant genetic variants constitutes a powerful model system that can be used to inform various aspects of CNS drug discovery and development.

Selective and brain-penetrant HCN1 inhibitors reveal links between synaptic integration, cortical function, and working memory.

Hyperpolarization-activated and cyclic-nucleotide-gated 1 (HCN1) ion channels are proposed to be critical for cognitive function through regulation of synaptic integration. However, resolving the precise role of HCN1 in neurophysiology and exploiting its therapeutic potential has been hampered by minimally selective antagonists with poor potency and limited in vivo efficiency. Using automated electrophysiology in a small-molecule library screen and chemical optimization, we identified a primary carboxamide series of potent and selective HCN1 inhibitors with a distinct mode of action. In cognition-relevant brain circuits, selective inhibition of native HCN1 produced on-target effects, including enhanced excitatory postsynaptic potential summation, while administration of a selective HCN1 inhibitor to rats recovered decrement working memory. Unlike prior non-selective HCN antagonists, selective HCN1 inhibition did not alter cardiac physiology in human atrial cardiomyocytes or in rats. Collectively, selective HCN1 inhibitors described herein unmask HCN1 as a potential target for the treatment of cognitive dysfunction in brain disorders.

Striatum-projecting prefrontal cortex neurons support working memory maintenance.

Neurons in the medial prefrontal cortex (mPFC) are functionally linked to working memory (WM) but how distinct projection pathways contribute to WM remains unclear. Based on optical recordings, optogenetic perturbations, and pharmacological interventions in male mice, we report here that dorsomedial striatum (dmStr)-projecting mPFC neurons are essential for WM maintenance, but not encoding or retrieval, in a T-maze spatial memory task. Fiber photometry of GCaMP6m-labeled mPFC→dmStr neurons revealed strongest activity during the maintenance period, and optogenetic inhibition of these neurons impaired performance only when applied during this period. Conversely, enhancing mPFC→dmStr pathway activity-via pharmacological suppression of HCN1 or by optogenetic activation during the maintenance period-alleviated WM impairment induced by NMDA receptor blockade. Moreover, cellular-resolution miniscope imaging revealed that >50% of mPFC→dmStr neurons are active during WM maintenance and that this subpopulation is distinct from neurons active during encoding and retrieval. In all task periods, neuronal sequences were evident. Striatum-projecting mPFC neurons thus critically contribute to spatial WM maintenance.

Depression of Accumbal to Lateral Hypothalamic Synapses Gates Overeating.

Overeating typically follows periods of energy deficit, but it is also sustained by highly palatable foods, even without metabolic demand. Dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) of the nucleus accumbens shell (NAcSh) project to the lateral hypothalamus (LH) to authorize feeding when inhibited. Whether plasticity at these synapses can affect food intake is unknown. Here, ex vivo electrophysiology recordings reveal that D1-MSN-to-LH inhibitory transmission is depressed in circumstances in which overeating is promoted. Endocannabinoid signaling is identified as the induction mechanism, since inhibitory plasticity and concomitant overeating were blocked or induced by CB1R antagonism or agonism, respectively. D1-MSN-to-LH projectors were largely non-overlapping with D1-MSNs targeting ventral pallidum or ventral midbrain, providing an anatomical basis for distinct circuit plasticity mechanisms. Our study reveals a critical role for plasticity at D1-MSN-to-LH synapses in adaptive feeding control, which may underlie persistent overeating of unhealthy foods, a major risk factor for developing obesity.

Homeostasis Meets Motivation in the Battle to Control Food Intake.

Signals of energy homeostasis interact closely with neural circuits of motivation to control food intake. An emerging hypothesis is that the transition to maladaptive feeding behavior seen in eating disorders or obesity may arise from dysregulation of these interactions. Focusing on key brain regions involved in the control of food intake (ventral tegmental area, striatum, hypothalamus, and thalamus), we describe how activity of specific cell types embedded within these regions can influence distinct components of motivated feeding behavior. We review how signals of energy homeostasis interact with these regions to influence motivated behavioral output and present evidence that experience-dependent neural adaptations in key feeding circuits may represent cellular correlates of impaired food intake control. Future research into mechanisms that restore the balance of control between signals of homeostasis and motivated feeding behavior may inspire new treatment options for eating disorders and obesity.

SHANK3 controls maturation of social reward circuits in the VTA.

Haploinsufficiency of SHANK3, encoding the synapse scaffolding protein SHANK3, leads to a highly penetrant form of autism spectrum disorder. How SHANK3 insufficiency affects specific neural circuits and how this is related to specific symptoms remains elusive. Here we used shRNA to model Shank3 insufficiency in the ventral tegmental area of mice. We identified dopamine (DA) and GABA cell-type-specific changes in excitatory synapse transmission that converge to reduce DA neuron activity and generate behavioral deficits, including impaired social preference. Administration of a positive allosteric modulator of the type 1 metabotropic glutamate receptors mGluR1 during the first postnatal week restored DA neuron excitatory synapse transmission and partially rescued the social preference defects, while optogenetic DA neuron stimulation was sufficient to enhance social preference. Collectively, these data reveal the contribution of impaired ventral tegmental area function to social behaviors and identify mGluR1 modulation during postnatal development as a potential treatment strategy.

Accumbal D1R Neurons Projecting to Lateral Hypothalamus Authorize Feeding.

Feeding satisfies metabolic need but is also controlled by external stimuli, like palatability or predator threat. Nucleus accumbens shell (NAcSh) projections to the lateral hypothalamus (LH) are implicated in mediating such feeding control, but the neurons involved and their mechanism of action remain elusive. We show that dopamine D1R-expressing NAcSh neurons (D1R-MSNs) provide the dominant source of accumbal inhibition to LH and provide rapid control over feeding via LH GABA neurons. In freely feeding mice, D1R-MSN activity reduced during consumption, while their optogenetic inhibition prolonged feeding, even in the face of distracting stimuli. Conversely, activation of D1R-MSN terminals in LH was sufficient to abruptly stop ongoing consumption, even during hunger. Direct inhibition of LH GABA neurons, which received input from D1R-MSNs, fully recapitulated these findings. Together, our study resolves a feeding circuit that overrides immediate metabolic need to allow rapid consumption control in response to changing external stimuli. VIDEO ABSTRACT.

Synaptic basis of social dysfunction: a focus on postsynaptic proteins linking group-I mGluRs with AMPARs and NMDARs.

Most of us engage in social interactions on a daily basis and the repertoire of social behaviors we acquire during development and later in life are incredibly varied. However, in many neurodevelopmental disorders, including autism spectrum disorders (ASDs), social behavior is severely compromised and indeed this represents a key diagnostic component for such conditions. From genetic association studies, it is increasingly apparent that genes identified as altered in individuals with ASDs often encode synaptic proteins. Moreover, these synaptic proteins typically serve to scaffold group-I metabotropic glutamate receptors (group-I mGluRs) and ionotropic glutamate receptors (iGluRs; AMPARs and NMDARs), or to enable group-I mGluR to iGluR crosstalk via protein synthesis. Here we aim to explore the possibility of a causal link between altered function of such synaptic proteins and impaired social behaviors that feature in neurodevelopmental disorders, such as ASDs. We review the known synaptic function and role in social behaviors of selected post-synaptic structural proteins (Shank, SAPAP and neuroligin) and regulators of protein synthesis (TSC1/2, FMRP and PTEN). While manipulations of proteins involved in group-I mGluR and iGluR scaffolding or crosstalk frequently lead to profound alterations in synaptic function and one or more components of social behavior, the neuronal circuits responsible for impairments in specific social behaviors are often poorly defined. We argue for an improved understanding of the neuronal circuits underlying specific social behaviors to aid the development of new ASD therapies.

Expression of cocaine-evoked synaptic plasticity by GluN3A-containing NMDA receptors.

Drug-evoked synaptic plasticity in the mesolimbic dopamine (DA) system reorganizes neural circuits that may lead to addictive behavior. The first cocaine exposure potentiates AMPAR excitatory postsynaptic currents (EPSCs) onto DA neurons of the VTA but reduces the amplitude of NMDAR-EPSCs. While plasticity of AMPAR transmission is expressed by insertion of calcium (Ca(2+))-permeable GluA2-lacking receptors, little is known about the expression mechanism for altered NMDAR transmission. Combining ex vivo patch-clamp recordings, mouse genetics, and subcellular Ca(2+) imaging, we observe that cocaine drives the insertion of NMDARs that are quasi-Ca(2+)-impermeable and contain GluN3A and GluN2B subunits. These GluN3A-containing NMDARs appear necessary for the expression of cocaine-evoked plasticity of AMPARs. We identify an mGluR1-dependent mechanism to remove these noncanonical NMDARs that requires Homer/Shank interaction and protein synthesis. Our data provide insight into the early cocaine-driven reorganization of glutamatergic transmission onto DA neurons and offer GluN3A-containing NMDARs as new targets in drug addiction.

Ventral tegmental area GABA projections pause accumbal cholinergic interneurons to enhance associative learning.

The ventral tegmental area (VTA) and nucleus accumbens (NAc) are essential for learning about environmental stimuli associated with motivationally relevant outcomes. The task of signalling such events, both rewarding and aversive, from the VTA to the NAc has largely been ascribed to dopamine neurons. The VTA also contains GABA (γ-aminobutyric acid)-releasing neurons, which provide local inhibition and also project to the NAc. However, the cellular targets and functional importance of this long-range inhibitory projection have not been ascertained. Here we show that GABA-releasing neurons of the VTA that project to the NAc (VTA GABA projection neurons) inhibit accumbal cholinergic interneurons (CINs) to enhance stimulus-outcome learning. Combining optogenetics with structural imaging and electrophysiology, we found that VTA GABA projection neurons selectively target NAc CINs, forming multiple symmetrical synaptic contacts that generated inhibitory postsynaptic currents. This is remarkable considering that CINs represent a very small population of all accumbal neurons, and provide the primary source of cholinergic tone in the NAc. Brief activation of this projection was sufficient to halt the spontaneous activity of NAc CINs, resembling the pause recorded in animals learning stimulus-outcome associations. Indeed, we found that forcing CINs to pause in behaving mice enhanced discrimination of a motivationally important stimulus that had been associated with an aversive outcome. Our results demonstrate that VTA GABA projection neurons, through their selective targeting of accumbal CINs, provide a novel route through which the VTA communicates saliency to the NAc. VTA GABA projection neurons thus emerge as orchestrators of dopaminergic and cholinergic modulation in the NAc.

The predictive validity of the rat self-administration model for abuse liability.

The self-administration model is the primary non-clinical approach for assessing the reinforcing properties of novel compounds. Given the now frequent use of rats in self-administration studies, it is important to understand the predictive validity of the rat self-administration model for use in abuse liability assessments. This review of 71 drugs identifies high concordance between findings from rat self-administration studies and two clinical indicators of abuse liability, namely reports of positive subjective-effects and the DEA drug scheduling status. To understand the influence of species on concordance we compare rodent and non-human primate (NHP) self-administration data. In the few instances where discrepancies are observed between rat data and the clinical indicators of abuse liability, rat self-administration data corresponds with NHP data in the majority of these cases. We discuss the influence of genetic factors (sex and strain), food deprivation state and the study design (acquisition or drug substitution) on self-administration study outcomes and highlight opportunities to improve the predictive validity of the self-administration model.

Modeling appetitive Pavlovian-instrumental interactions in mice.

In appetitive Pavlovian associative learning, a stimulus (conditioned stimulus, CS) that has been associated with the delivery of a reinforcing event (unconditioned stimulus, US; e.g., food) can subsequently elicit or modulate goal-directed instrumental behaviors. For example, a Pavlovian CS can serve to reinforce (novel) instrumental behavior (conditioned reinforcement or CRf), or it can energize and potentiate ongoing instrumental responses when presented non-contingently (Pavlovian-instrumental transfer or PIT). Notably, these different effects of a Pavlovian CS on instrumental behavior are mediated by dissociable psychological and neurobiological mechanisms. Given the critical role that Pavlovian-instrumental interactions play in regulating motivated behavior and maladaptive manifestations of motivation such as eating disorders and addictions, understanding the underlying psychological and neurobiological mechanisms will be important. This unit describes behavioral protocols that produce robust and reliable PIT and CRf in mice and that open the door for future studies using transgenic approaches into the molecular mechanisms underlying associative learning and motivation.

Incentive learning underlying cocaine-seeking requires mGluR5 receptors located on dopamine D1 receptor-expressing neurons.

Understanding the psychobiological basis of relapse remains a challenge in developing therapies for drug addiction. Relapse in cocaine addiction often occurs following exposure to environmental stimuli previously associated with drug taking. The metabotropic glutamate receptor, mGluR5, is potentially important in this respect; it plays a central role in several forms of striatal synaptic plasticity proposed to underpin associative learning and memory processes that enable drug-paired stimuli to acquire incentive motivational properties and trigger relapse. Using cell type-specific RNA interference, we have generated a novel mouse line with a selective knock-down of mGluR5 in dopamine D1 receptor-expressing neurons. Although mutant mice self-administer cocaine, we show that reinstatement of cocaine-seeking induced by a cocaine-paired stimulus is impaired. By examining different aspects of associative learning in the mutant mice, we identify deficits in specific incentive learning processes that enable a reward-paired stimulus to directly reinforce behavior and to become attractive, thus eliciting approach toward it. Our findings show that glutamate signaling through mGluR5 located on dopamine D1 receptor-expressing neurons is necessary for incentive learning processes that contribute to cue-induced reinstatement of cocaine-seeking and which may underpin relapse in drug addiction.

Tramadol acts as a weak reinforcer in the rat self-administration model, consistent with its low abuse liability in humans.

Rodent models of abuse potential are considered to represent a false positive with respect to the low risk of abuse liability associated with the atypical opioid analgesic tramadol. This may reflect either the predictive limitations of the models used to formulate this proposition (drug discrimination and conditioned place preference) or the predictive ability of the rodent per se. To address this concern, we used the rat self-administration model to examine the reinforcing properties of tramadol (0.3-3mg/kg/infusion) under fixed (FR) and progressive-ratio (PR) schedules of reinforcement. Comparisons were made with the typical opioid analgesics morphine (0.03-0.3mg/kg/infusion) and remifentanil (0.001-0.03mg/kg/infusion). All three compounds maintained responding under an FR3 schedule of reinforcement, although clear differences were observed in the rates of responding between compounds. Under a PR schedule, morphine and remifentanil maintained comparable break points, while break points for tramadol did not differ from vehicle. Thus, when examined in the self-administration model, tramadol acts as a relatively weak reinforcer in rodents. These data are consistent with the low risk of tramadol abuse liability in humans and highlight the value of using multiple abuse potential models for assessing abuse liability.

The mGluR5 antagonist MTEP dissociates the acquisition of predictive and incentive motivational properties of reward-paired stimuli in mice.

An environmental stimulus paired with reward (a conditioned stimulus; CS) can acquire predictive properties that signal reward availability and may also acquire incentive motivational properties that enable the CS to influence appetitive behaviors. The neural mechanisms involved in the acquisition and expression of these CS properties are not fully understood. The metabotropic glutamate receptor, mGluR5, contributes to synaptic plasticity underlying learning and memory processes. We examined the role of mGluR5 in the acquisition and expression of learning that enables a CS to predict reward (goal-tracking) and acquire incentive properties (conditioned reinforcement). Mice were injected with vehicle or the mGluR5 antagonist, MTEP (3 or 10 mg/kg), before each Pavlovian conditioning session in which a stimulus (CS+) was paired with food delivery. Subsequently, in the absence of the primary food reward, we determined whether the CS+ could reinforce a novel instrumental response (conditioned reinforcement) and direct behavior toward the place of reward delivery (goal-tracking). MTEP did not affect performance during the conditioning phase, or the ability of the CS+ to elicit a goal-tracking response. In contrast, 10 mg/kg MTEP given before each conditioning session prevented the subsequent expression of conditioned reinforcement. This dose of MTEP did not affect conditioned reinforcement when administered before the test, in mice that had received vehicle before conditioning sessions. Thus, mGluR5 has a critical role in the acquisition of incentive properties by a CS, but is not required for the expression of incentive learning, or for the CS to acquire predictive properties that signal reward availability.

The alpha4beta2 nicotinic acetylcholine-receptor partial agonist varenicline inhibits both nicotine self-administration following repeated dosing and reinstatement of nicotine seeking in rats.

INTRODUCTION: The alpha4beta2 nicotinic acetylcholine receptor partial agonist varenicline has greater efficacy than other pharmacotherapeutic aids for smoking cessation. This presents an opportunity to evaluate the predictive validity of rat models of nicotine taking and relapse. The aim of this study was to evaluate the ability of varenicline to attenuate nicotine self-administration and relapse, as modelled by the reinstatement model of nicotine relapse in rats. MATERIALS AND METHODS: Rats were trained to respond for intravenous nicotine under a fixed ratio schedule of reinforcement. The effects of varenicline (0.3-3.0 mg/kg s.c.) on both nicotine and food self-administration and reinstatement of nicotine seeking were evaluated. RESULTS AND DISCUSSION: Varenicline dose-dependently reduced nicotine self-administration and attenuated both nicotine prime and combined nicotine prime plus nicotine-paired cue-induced reinstatement. Varenicline had no effect on cue-induced reinstatement in the absence of a nicotine prime nor did it induce reinstatement when given alone. CONCLUSION: The effects of varenicline on nicotine-induced reinstatement of drug-seeking are consistent with the demonstrated clinical efficacy of varenicline for smoking cessation.