The chemogenetic technology Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) affords remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-Noxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities, and potential offtarget effects of CNO represent areas for improvement. Here we provide a new high affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μg/kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 μg/kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents the most potent, selective, metabolically stable and fast-acting DREADD agonist reported with utility in both mice and non-human primates for a variety of applications.

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The primate prefrontal cortex (PFC) is situated at the core of higher brain functions via neural circuits such as those linking the caudate nucleus and mediodorsal thalamus. However, the distinctive roles of these prefronto-subcortical pathways remain elusive. Combining in vivo neuronal projection mapping with chemogenetic synaptic silencing, we reversibly dissected key pathways from dorsolateral part of the PFC (dlPFC) to the dorsal caudate (dCD) and lateral mediodorsal thalamus (MDl) individually in single monkeys. We found that silencing the bilateral dlPFC-MDl projections, but not the dlPFC-dCD projections, impaired performance in a spatial working memory task. Conversely, silencing the unilateral dlPFC-dCD projection, but not the unilateral dlPFC-MDl projection, altered preference in a decision-making task. These results revealed dissociable roles of the prefronto-subcortical pathways in working memory and decision-making, representing the technical advantage of imaging-guided pathway-selective chemogenetic manipulation for dissecting neural circuits underlying cognitive functions in primates.

The chemogenetic technology, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), affords reversible and remote control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, the sluggish kinetics, metabolic liabilities, and potential for off-target effects of CNO represent areas for improvement. Here we provide a new agonist deschloroclozapine (DCZ), which displays high affinity and selectivity for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μ g/kg) enhanced neuronal activity via hM 3 Dq within minutes in mice and monkeys. Intramuscular injection of DCZ (100 μ g/kg) reversibly induced behavioral deficits in hM 4 Di-expressing monkeys.DCZ represents the most potent, selective, metabolically stable and fast-acting DREADD agonist reported with utility in both mice and non-human primates for a variety of applications. 7 at dopaminergic (D 1 and D 2 ) and serotonergic (5-HT 2A and 5-HT 2C ) receptors compared with clozapine 14 . We determined that DCZ has 100-fold improved affinity and stronger agonist potency for hM 3 Dq and hM 4 Di relative to CNO or C21, while reducing off-target binding compared with clozapine in vitro. Using positron emission tomography (PET), we demonstrated that DCZ rapidly penetrates into the brain and selectively binds to DREADDs, and that DCZ doses for DREADD occupancy are 20-and 60-fold smaller than CNO and C21,respectively. Finally, we demonstrated that DCZ is capable of rapidly (<10 min post-injection) activating hM 3 Dq and hM 4 Di in both mice and monkeys without discernible off-target action. Thus, DCZ represents a potent and selective chemogenetic actuator for muscarinic-based DREADDs in mice and primates that is useful for a variety of in vitro and in vivo contexts with high translational potential. 8 RESULTS DCZ selectively binds to DREADDs in vitro and in vivoWe first assessed the binding affinities of DCZ to hM 3 Dq and hM 4 Di, and compared them to those of clozapine, CNO and C21 using radioligand competition binding assays. DCZ inhibited [ 3 H]quinuclidinyl benzilate (QNB) binding to hM 3 Dq and hM 4 Di with nanomolar affinity (inhibition constants; hM3Dq K i = 6.3 nM; hM4Di K i = 4.2 nM), which was comparable to clozapine ( hM3Dq K i = 5.9 nM; hM4Di Ki = 0.89), while CNO and C21 were about 100-and 50-fold weaker, respectively (CNO, hM3Dq K i = 680 nM; hM4Di K i = 360 nM; C21, hM3Dq K i = 850 nM; hM4Di K i = 180 nM)( Table 1). Unlike clozapine ( Fig. 1c and Supplementary Table 1), DCZ had negligible affinities for a large number of tested G protein-coupled receptors (GPCRs), ion channels or transporters (K i 's > 100 nM) and relatively low affinities for a few endogenous receptors such as muscarinic acetylcholine ( hM1 K i = 83 nM; hM5 K i = 55 nM) and serotonin receptors ( 5-HT2A K i = 87 nM)( Fig. 1b and Supplement...

Social cognitive mechanisms are central to understanding developmental abnormalities, such as autistic spectrum disorder. Peer relations besides parent-infant or pair-bonding interactions are pivotal social relationships that are especially well developed in humans. Cognition of familiarity forms the basis of peer socialization. Domestic chick (Gallus gallus) studies have contributed to our understanding of the developmental process in sensory-motor cognition but many processes remain unknown. In this report, we used chicks, as they are precocial birds, and we could therefore focus on peer interaction without having to consider parenting. The subject chick behavior towards familiar and unfamiliar reference peers was video-recorded, where the subject and the reference were separated by either an opaque or transparent wall. Spectrogram and behavior correlation analyses based on principal component analysis, revealed that chicks elicited an intermediate contact call and a morphologically different distress call, more frequently towards familiar versus unfamiliar chicks in acoustic only conditions. When both visual and acoustic cues were present, subject chicks exhibited approaching and floor pecking behavior, while eliciting joyful (pleasant) calls, irrespective of whether reference peers were familiar or unfamiliar. Our result showed that chicks recognized familiarity using acoustic cues and expressed cognition through modified distress calls. These finding suggests that peer affiliation may be established by acoustic recognition, independent of visual face recognition, and that eventually, both forms of recognition are integrated, with modulation of acoustic recognition.

Recent progress in affective neuroscience and social neurobiology has been propelled by neuro-imaging technology and epigenetic approach in neurobiology of animal behaviour. However, quantitative measurements of socio-emotional development remains lacking, though sensory-motor development has been extensively studied in terms of digitised imaging analysis. Here, we developed a method for socio-emotional behaviour measurement that is based on the video recordings under well-defined social context using animal models with variously social sensory interaction during development. The behaviour features digitized from the video recordings were visualised in a multivariate statistic space using principal component analysis. The clustering of the behaviour parameters suggested the existence of species-and stage-specific as well as cross-species behaviour modules. These modules were used to characterise the behaviour of children with or without autism spectrum disorders (ASDs). We found that socio-emotional behaviour is highly dependent on social context and the cross-species behaviour modules may predict neurobiological basis of ASDs. The development of social communication is a complex process 2-4 including sensory-motor, limbic, and cognitive system maturation and we are still uncertain of neurobiological basis of the symptom irrespective of great progress in neuroimaging 5-9 and epigenetic and molecular study of social neurobiology [10][11][12][13][14][15][16][17][18][19][20] . The comparative behavioural study has contributed to improve our concept in affective neuroscience and understanding of cross species neuronal basis of socio-emotional development 10,21-25 as briefly described below. A human infant is born with biased ''preference'' toward con-specific facial features. The study of domestic chick (Gallus gallus domesticus) provided clear evidence that facial preference developed through two acquisition steps, predisposition and imprinting [26][27][28][29] . The predisposition is released without any sensory experience of con-specific features, but required non-specific motor activity 24,28 . The imprinting stabilizes the predisposition, thus guides sensory attention toward con-specific individuals 30 . The sensory ''preference'' bias is not only for facial cognition 31 but also includes cognition in vocalization 32,33 , olfaction 34 , self-propelled movement 35 , and biological motion 36,37 . Motion perception becomes the bases of mimicking other's behaviour and understanding other's goal-directed behaviour, eventually leading to acquisition of theory of mind [38][39][40][41] . Another animal model, the common marmoset (Callithrix jacchus) has several merits in neurobiological study of socio-emotional development. The marmoset recognizes biological motion (female only among adults) 42 and is a monogamous species. A pair, as well as young family members, cooperates in raising their offspring together. Furthermore, the marmoset exhibits altruistic behaviour which is a rare among primate species 43,44 . These so...

To interrogate particular neuronal pathways in nonhuman primates under natural and stress-free conditions, we applied designer receptors exclusively activated by designer drugs (DREADDs) technology to common marmosets. We injected adeno-associated virus vectors expressing the excitatory DREADD hM3Dq into the unilateral substantia nigra (SN) in four marmosets. Using multi-tracer positron emission tomography imaging, we detected DREADD expression in vivo, which was confirmed in nigrostriatal dopamine neurons by immunohistochemistry, as well as by assessed activation of the SN following agonist administration. The marmosets rotated in a contralateral direction relative to the activated side 30-90 min after consuming food containing the highly potent DREADD agonist deschloroclozapine (DCZ) but not on the following days without DCZ. These results indicate that non-invasive and reversible DREADD manipulation will extend the utility of marmosets as a primate model for linking neuronal activity and natural behavior in various contexts.