Chemogenetic Receptors Research Articles

Involvement of a lateral entorhinal cortex engram in episodic-like memory recall

Episodic memory relies on the entorhinal cortex (EC), a crucial hub connecting the hippocampus and sensory processing regions. This study investigates the role of the lateral EC (LEC) in episodic-like memory in mice. Here, we employ the object-place-context-recognition task (OPCRT), a behavioral test used to study episodic-like memory in rodents. Electrophysiology in brain slices reveals that OPCRT specifically induces a shift in the threshold for the induction of synaptic plasticity in LEC superficial layer II. Additionally, a dual viral system is used to express chemogenetic receptors coupled to the c-Fos promoter in neurons recruited during the learning. We demonstrate that the inhibition of LEC neurons impairs the performance of the mice in the memory task, while their stimulation significantly facilitates memory recall. Our findings provide evidence for an episodic-like memory engram in the LEC and emphasize its role in memory processing within the broader network of episodic memory.

Dissociable dorsal medial prefrontal cortex ensembles are necessary for cocaine seeking and fear conditioning in mice

The dorsal medial prefrontal cortex (dmPFC) plays a dual role in modulating drug seeking and fear-related behaviors. Learned associations between cues and drug seeking are encoded by a specific ensemble of neurons. This study explored the stability of a dmPFC cocaine seeking ensemble over 2 weeks and its influence on persistent cocaine seeking and fear memory retrieval. In the first series of experiments, we trained TetTag c-fos-driven-EGFP mice in cocaine self-administration and tagged strongly activated neurons with EGFP during the initial day 7 cocaine seeking session. Subsequently, a follow-up seeking test was conducted 2 weeks later to examine ensemble reactivation between two seeking sessions via c-Fos immunostaining. In the second series of experiments, we co-injected viruses expressing TRE-cre and a cre-dependent inhibitory PSAM-GlyR into the dmPFC of male and female c-fos-tTA mice to enable “tagging” of cocaine seeking ensemble or cued fear ensemble neurons with inhibitory chemogenetic receptors. These c-fos-tTA mice have the c-fos promoter that drives expression of the tetracycline transactivator (tTA). The tTA can bind to the tetracycline response element (TRE) site on the viral construct, resulting in the expression of cre-recombinase, which enables the expression of cre-dependent inhibitory chemogenetic receptors and fluorescent reporters. Then we investigated ensemble contribution to subsequent cocaine seeking and fear recall during inhibition of the tagged ensemble by administering uPSEM792s (0.3 mg/kg), a selective ligand for PSAM-GlyR. In both sexes, there was a positive association between the persistence of cocaine seeking and the proportion of reactivated EGFP+ neurons within the dmPFC. More importantly, inhibition of the cocaine seeking ensemble suppressed cocaine seeking, but not recall of fear memory, while inhibition of the fear ensemble reduced conditioned freezing but not cocaine seeking. The results demonstrate that cocaine and fear recall ensembles in the dmPFC are stable, but largely exclusive from one another.

Astrocytic P2X7 receptor in retrosplenial cortex drives electroacupuncture analgesia.

P2X7 receptor (P2X7R) has been found to contribute to the peripheral mechanism of acupuncture analgesia (AA). However, whether it plays an important role in central mechanism remains unknown. In this study, we aimed to reveal the role of astrocytic P2X7R in retrosplenial cortex (RSC) in AA and provide new evidence for underlying the central mechanism of AA. We applied the chemogenetic receptors hM3Dq to stimulate or hM4Di to inhibit astrocytes ligand clozapine-N-oxide (CNO) following injection of adeno-associated virus (AAV) into the bilateral RSC, or pharmacologically intervened in the activity of the purinergic receptor P2X7R. Current data indicated that chemogenetic inhibition of astrocytes or injection of P2X7R agonist Bz-ATP in the bilateral RSC significantly reverses the analgesic effect of electroacupuncture (EA) in formalin tests while the bilateral injection of the P2X7R antagonist A438079 alleviated formalin-induced nociceptive behavior. Additionally, chemogenetic suppression of astrocytic P2X7R by injection of AAV in the bilateral RSC decreased hind paw flinches induced by formalin in the mice. These findings indicate the participation of both astrocytes and P2X7R in the RSC in EA analgesic. Moreover, P2X7R on astrocytes in the RSC appears to play a critical role in the ability of EA to attenuate formalin-induced pain responses in mice.

Chemogenetic Tools and their Use in Studies of Neuropsychiatric Disorders.

Chemogenetics is a newly developed set of tools that allow for selective manipulation of cell activity. They consist of a receptor mutated irresponsive to endogenous ligands and a synthetic ligand that does not interact with the wild-type receptors. Many different types of these receptors and their respective ligands for inhibiting or excitating neuronal subpopulations were designed in the past few decades. It has been mainly the G-protein coupled receptors (GPCRs) selectively responding to clozapine-N-oxide (CNO), namely Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), that have been employed in research. Chemogenetics offers great possibilities since the activity of the receptors is reversible, inducible on demand by the ligand, and non-invasive. Also, specific groups or types of neurons can be selectively manipulated thanks to the delivery by viral vectors. The effect of the chemogenetic receptors on neurons lasts longer, and even chronic activation can be achieved. That can be useful for behavioral testing. The great advantage of chemogenetic tools is especially apparent in research on brain diseases since they can manipulate whole neuronal circuits and connections between different brain areas. Many psychiatric or other brain diseases revolve around the dysfunction of specific brain networks. Therefore, chemogenetics presents a powerful tool for investigating the underlying mechanisms causing the disease and revealing the link between the circuit dysfunction and the behavioral or cognitive symptoms observed in patients. It could also contribute to the development of more effective treatments.

Distinct roles of monkey OFC-subcortical pathways in adaptive behavior

Primates must adapt to changing environments by optimizing their behavior to make beneficial choices. At the core of adaptive behavior is the orbitofrontal cortex (OFC) of the brain, which updates choice value through direct experience or knowledge-based inference. Here, we identify distinct neural circuitry underlying these two separate abilities. We designed two behavioral tasks in which two male macaque monkeys updated the values of certain items, either by directly experiencing changes in stimulus-reward associations, or by inferring the value of unexperienced items based on the task’s rules. Chemogenetic silencing of bilateral OFC combined with mathematical model-fitting analysis revealed that monkey OFC is involved in updating item value based on both experience and inference. In vivo imaging of chemogenetic receptors by positron emission tomography allowed us to map projections from the OFC to the rostromedial caudate nucleus (rmCD) and the medial part of the mediodorsal thalamus (MDm). Chemogenetic silencing of the OFC-rmCD pathway impaired experience-based value updating, while silencing the OFC-MDm pathway impaired inference-based value updating. Our results thus demonstrate dissociable contributions of distinct OFC projections to different behavioral strategies, and provide new insights into the neural basis of value-based adaptive decision-making in primates.

The Projection-Specific Noradrenergic Modulation of Perseverative Spatial Behavior in Adult Male Rats.

Adaptive behavior relies on efficient cognitive control. The anterior cingulate cortex (ACC) is a key node within the executive prefrontal network. The reciprocal connectivity between the locus ceruleus (LC) and ACC is thought to support behavioral reorganization triggered by the detection of an unexpected change. We transduced LC neurons with either excitatory or inhibitory chemogenetic receptors in adult male rats and trained rats on a spatial task. Subsequently, we altered LC activity and confronted rats with an unexpected change of reward locations. In a new spatial context, rats with decreased noradrenaline (NA) in the ACC entered unbaited maze arms more persistently which was indicative of perseveration. In contrast, the suppression of the global NA transmission reduced perseveration. Neither chemogenetic manipulation nor inactivation of the ACC by muscimol affected the rate of learning, possibly due to partial virus transduction of the LC neurons and/or the compensatory engagement of other prefrontal regions. Importantly, we observed behavioral deficits in rats with LC damage caused by virus injection. The latter finding highlights the importance of careful histological assessment of virus-transduced brain tissue as inadvertent damage of the targeted cell population due to virus neurotoxicity or other factors might cause unwanted side effects. Although the specific role of ACC in the flexibility of spatial behavior has not been convincingly demonstrated, our results support the beneficial role of noradrenergic transmission for an optimal function of the ACC. Overall, our findings suggest the LC exerts the projection-specific modulation of neural circuits mediating the flexibility of spatial behavior.

Perturbing cortical networks: in vivo electrophysiological consequences of pan-neuronal chemogenetic manipulations using deschloroclozapine.

Chemogenetic techniques, specifically the use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), have become invaluable tools in neuroscience research. Yet, the understanding of how Gq- and Gicoupled DREADDs alter local field potential (LFP) oscillations in vivo remains incomplete. This study investigates the in vivo electrophysiological effects of DREADD actuation by deschloroclozapine, on spontaneous firing rate and LFP oscillations recorded from the anterior cingulate cortex in lightly anesthetized male rats. Unexpectedly, in response to the administration of deschloroclozapine, we observed inhibitory effects with pan-neuronal hM3D(Gq) stimulation, and excitatory effects with pan-neuronal hM4D(Gi) stimulation in a significant portion of neurons. These results emphasize the need to account for indirect perturbation effects at the local neuronal network level in vivo, particularly when not all neurons express the chemogenetic receptors uniformly. In the current study, for instance, the majority of cells that were transduced with both hM3D(Gq) and hM4D(Gi) were GABAergic. Moreover, we found that panneuronal cortical chemogenetic modulation can profoundly alter oscillatory neuronal activity, presenting a potential research tool or therapeutic strategy in several neuropsychiatric models and diseases. These findings help to optimize the use of chemogenetic techniques in neuroscience research and open new possibilities for novel therapeutic strategies.

Neuronal Ensembles in the Amygdala Allow Social Information to Motivate Later Decisions.

Social experiences carry tremendous weight in our decision-making, even when social partners are not present. To determine mechanisms, we trained female mice to respond for two food reinforcers. Then, one food was paired with a novel conspecific. Mice later favored the conspecific-associated food, even in the absence of the conspecific. Chemogenetically silencing projections from the prelimbic subregion (PL) of the medial prefrontal cortex to the basolateral amygdala (BLA) obstructed this preference while leaving social discrimination intact, indicating that these projections are necessary for socially driven choice. Further, mice that performed the task had greater densities of dendritic spines on excitatory BLA neurons relative to mice that did not. We next induced chemogenetic receptors in cells active during social interactions-when mice were encoding information that impacted later behavior. BLA neurons stimulated by social experience were necessary for mice to later favor rewards associated with social conspecifics but not make other choices. This profile contrasted with that of PL neurons stimulated by social experience, which were necessary for choice behavior in social and nonsocial contexts alike. The PL may convey a generalized signal allowing mice to favor particular rewards, while units in the BLA process more specialized information, together supporting choice motivated by social information.

Reciprocal activity of AgRP and POMC neurons governs coordinated control of feeding and metabolism

Agouti-related peptide (AgRP)-expressing and proopiomelanocortin (POMC)-expressing neurons reciprocally regulate food intake. Here, we combine non-interacting recombinases to simultaneously express functionally opposing chemogenetic receptors in AgRP and POMC neurons for comparing metabolic responses in male and female mice with simultaneous activation of AgRP and inhibition of POMC neurons with isolated activation of AgRP neurons or isolated inhibition of POMC neurons. We show that food intake is regulated by the additive effect of AgRP neuron activation and POMC neuron inhibition, while systemic insulin sensitivity and gluconeogenesis are differentially modulated by isolated-versus-simultaneous regulation of AgRP and POMC neurons. We identify a neurocircuit engaging Npy1R-expressing neurons in the paraventricular nucleus of the hypothalamus, where activated AgRP neurons and inhibited POMC neurons cooperate to promote food consumption and activate Th+ neurons in the nucleus tractus solitarii. Collectively, these results unveil how food intake is precisely regulated by the simultaneous bidirectional interplay between AgRP and POMC neurocircuits.

Connectivity of the Brain in the Light of Chemogenetic Modulation of Neuronal Activity.

Connectivity is the coordinated activity of the neuronal networks responsible for brain functions; it is detected based on functional magnetic resonance imaging signals that depend on the oxygen level in the blood (blood oxygen level-dependent (BOLD) signals) supplying the brain. The BOLD signal is only indirectly related to the underlying neuronal activity; therefore, it remains an open question whether connectivity and changes in it are only manifestations of normal and pathological states of the brain or they are, to some extent, the causes of these states. The creation of chemogenetic receptors activated by synthetic drugs (designer receptors exclusively activated by designer drugs, DREADDs), which, depending on the receptor type, either facilitate or, on the contrary, inhibit the neuronal response to received physiological stimuli, makes it possible to assess brain connectivity in the light of controlled neuronal activity. Evidence suggests that connectivity is based on neuronal activity and is a manifestation of connections between brain regions that integrate sensory, cognitive, and motor functions. Chemogenetic modulation of the activity of various groups and types of neurons changes the connectivity of the brain and its complex functions. Chemogenetics can be useful in reconfiguring the pathological mechanisms of nervous and mental diseases. The initiated integration, based on the whole-brain connectome from molecular-cellular, neuronal, and synaptic processes to higher nervous activity and behavior, has the potential to significantly increase the fundamental and applied value of this branch of neuroscience.

Dorsal motor vagal neurons can suppress heart rate in mice

The dorsal motor nucleus of the vagus (DMV) is a functionally heterogeneous vagal motor region which controls a variety of digestive and metabolic functions but also innervates the heart, where its role in controlling heart rate is less clear. We therefore chemogenetically activated DMV neurons in awake behaving mice while monitoring heart rate. To selectively activate DMV neurons, we injected an adeno-associated virus (AAV) which expresses the excitatory chemogenetic receptor, hM3Dq (8 injections, 40nl each), only after recombination by both Cre and Flp recombinases, into the DMV of Chat-Cre::Phox2b-Flp mice. Several months later, we injected the hM3Dq ligand clozapine N-oxide (CNO; 1mg/kg) via intraperitoneal injection (n=2 females, 3 males; mean age ± S.D., 32 ± 2 weeks) to activate hM3Dq+ DMV neurons while measuring heart rate via a non-invasive ECG system (ECGenie). We measured heart rate 20 minutes prior to CNO and 0, 20, 40 minutes and 1, 2, 6, 8, 24 hours later. CNO administration significantly but reversibly decreased heart rate (mean ± standard deviation, S.D.: 20min before CNO, 705 ± 15 bpm; 40min after CNO, 536 ± 86 bpm, p=0.0271; 24hr after CNO, 727 ± 25 bpm, p=0.7347; one-way ANOVA, all timepoints, F 1.660, 6.638 =10.31; Dunnet's post-hoc test, p=0.0107). In order to identify the specific neurons responsible, we repeated these studies in Calb2-Cre::Chat-Flp mice (n=2 females, 3 males; mean age ± S.D., 29 ± 2 weeks) to target Calb2+ DMV neurons, a recently identified DMV molecular subtype. However, in striking contrast to what we observed when activating DMV neurons generally, administering CNO to activate Calb2+ DMV neurons specifically did not significantly affect heart rate (mean ± S.D.: 20min before CNO, 691 ± 69 bpm; 40min after CNO, 718 ± 31 bpm, p=0.6787; 24hr after CNO, 710 ± 54 bpm, p=0.4701; one-way ANOVA, all timepoints, F 2.911, 11.65 =0.6277; Dunnet’s, p=0.6069). A higher dose of CNO (3.5mg/kg) also had no effect on heart rate in Calb2-Cre::Chat-Flp mice (data not shown). Together, our studies indicate that DMV neurons are capable of suppressing heart rate and that the underlying neurons likely do not express Calb2. Uncovering the DMV’s role in heart rate will expand our understanding of how this clinically relevant metric, associated with a host of cardiovascular disorders, is controlled. Ingrassia Family Echols Scholars Research Grant and Schwager Summer Research Scholarship to L.S.K.; ADA Pathway to Stop Diabetes Award 1-18-INI-14 and NIH grant HL153916 to J.N.C. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Efficient viral expression of a chemogenetic receptor in the old-world monkey amygdala.

Genetically encoded synthetic receptors, such as the chemogenetic and optogenetic proteins, are powerful tools for functional brain studies in animals. In the primate brain, with its comparatively large, intricate anatomical structures, it can be challenging to express transgenes, such as the hM4Di chemogenetic receptor, in a defined anatomical structure with high penetrance. Here, we compare parameters for lentivirus vector injections in the rhesus monkey amygdala. We find that four injections of 20μl, infused at 0.5μl/min, can achieve neuronal hM4Di expression in 50-100% of neurons within a 60mm3 volume, without observable damage from overexpression. Increasing the number of hM4Di_CFP lentivirus injections to up to 12 sites per hemisphere, resulted in 30%-40% neuronal coverage of the overall amygdala volume, with coverage reaching 60% in some subnuclei. Manganese Chloride was mixed with lentivirus and used as an MRI marker to verify targeting accuracy and correct unsuccessful injections in these experiments. In a separate monkey we visualized, in vivo, viral expression of the hM4Di receptor protein in the amygdala, using Positron Emission Tomography. Together, these data show efficient and verifiable expression of a chemogenetic receptor in old-world monkey amygdala.

Characterization of Ultrapotent Chemogenetic Ligands for Research Applications in Nonhuman Primates.

Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of "Pharmacologically Selective Actuator Modules" (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent "Pharmacologically Selective Effector Molecules" (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates, it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in in vitro studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus monkeys received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817 (0.064 mg/kg) or uPSEM792 (0.87 mg/kg), and plasma and cerebrospinal fluid samples were collected over 48 h. Both compounds exhibited good brain penetrance, relatively slow washout, and negligible conversion to potential metabolites─varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered the heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

The basolateral amygdala to posterior insular cortex tract is necessary for social interaction with stressed juvenile rats

Vocalizations, chemosignals, and behaviors are influenced by one’s internal affective state and are used by others to shape social behaviors. A network of interconnected brain structures, often called the social behavior network or social decision-making network, integrates these stimuli and coordinates social behaviors, and in-network connectivity deficits underlie several psychiatric disorders such as schizophrenia and autism spectrum disorders. Here, we investigated the role of the basolateral amygdala (BLA) and its projections to the posterior insular cortex, regions independently implicated in a range of sociocognitive processes, in a social affective preference (SAP) test. Viral vectors containing the gene coding for inhibitory chemogenetic receptors (AAV5-hSyn-hM4Di-mCherry) were injected into the BLA. SAP tests, which allow for the observation of unconditioned behavioral responses to the affective states of others, were conducted after inhibition of the BLA by systemic administration of the hM4Di agonist clozapine-n-oxide (CNO), or inhibition of BLA-insula terminals by direct infusion of CNO to the insula. After vehicle infusions, rats displayed preference for interactions with stressed juvenile conspecifics. However, CNO treatment eliminated preference behavior. The current results suggest that social decision making involves the transfer of emotional information from the BLA to the insula which represents a previously unrecognized anatomical substrate for social cognition.

Localized chemogenetic silencing of inhibitory neurons: a novel mouse model of focal cortical epileptic activity.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

Viral Strategies for Targeting and Manipulating Opioidergic Neuronal Populations for Cell Specific Study and Therapeutic Intervention

With concurrent global epidemics of opioid use and chronic pain disorders, there is a critical need to identify, target, observe and manipulate specific cell populations in the peripheral and central nervous systems expressing the mu-opioid receptor (MOR). To gain long-term access to neural cells types and circuits involved in modulating pain, analgesia and addiction, we have developed a catalog of MOR specific promoter constructs (MORsp) for driving transgene expression in MOR+ cells compatible with use in AAV vectors. Working off the mouse Oprm1 promoter, we created a pAAV1-mMORsp-eYFP construct and confirmed its expression in cultured rat cortical neurons. To validate MOR+ cell-type selectivity in vivo, we performed intracranial injections into several structures implicated in pain-processing, opioid analgesia and drug reward in C57BL6/J mice, including: PFC, insula, amygdala, VTA, PAG and spinal cord. Robust expression of the reporter tag was observed in all regions, and in situ hybridization studies demonstrated eYFP transcript signal overlaps heavily with endogenous Oprm1 mRNA expression patterns. Comparable levels of reporter were also observed in the CeA and VTA of injected Sprague-Dawley rats and common tree shrews. Next, MORsp construct variants designed to drive expression of the chemogenetic receptor hM4Di and calcium activity indicator GCaMP6f were injected into mouse spinal cord and amygdala, respectively. Behavioral responses to noxious mechanical and heat stimuli were markedly decreased in hM4Di-expressing animals following DREADD agonist administration, and noxious heat stimuli produced robust GCaMP6f-mediated calcium transients that were morphine-sensitive. In addition to mouse promoter based constructs, we created a human Oprm1 promoter variant and confirmed its selective expression in OPRM1+ neurons in macaque PFC, insula, and amygdala. In total, our new MORsp toolkit provides researchers cell-type specific genetic access to monitor and functionally manipulate opioidergic neural circuits across a range of vertebrate species and translational screening models for pain and addiction. Grant support from Harnessing cortical neuromodulation to disrupt pain perception (1DP2GM140923-01) T32 Translational Addiction Research Fellowship Program (5T32DA028874-11).

Two novel mouse models of slow-wave-sleep enhancement in aging and Alzheimer's disease.

Aging and Alzheimer's disease (AD) are both associated with reduced quantity and quality of the deepest stage of sleep, called slow-wave-sleep (SWS). Slow-wave-sleep deficits have been shown to worsen AD symptoms and prevent healthy aging. However, the mechanism remains poorly understood due to the lack of animal models in which SWS can be specifically manipulated. Notably, a mouse model of SWS enhancement has been recently developed in adult mice. As a prelude to studies assessing the impact of SWS enhancement on aging and neurodegeneration, we first asked whether SWS can be enhanced in animal models of aging and AD. The chemogenetic receptor hM3Dq was conditionally expressed in GABAergic neurons of the parafacial zone of aged mice and AD (APP/PS1) mouse model. Sleep-wake phenotypes were analyzed in baseline condition and following clozapine-N-oxide (CNO) and vehicle injections. Both aged and AD mice display deficits in sleep quality, characterized by decreased slow wave activity. Both aged and AD mice show SWS enhancement following CNO injection, characterized by a shorter SWS latency, increased SWS amount and consolidation, and enhanced slow wave activity, compared with vehicle injection. Importantly, the SWS enhancement phenotypes in aged and APP/PS1 model mice are comparable to those seen in adult and littermate wild-type mice, respectively. These mouse models will allow investigation of the role of SWS in aging and AD, using, for the first time, gain-of SWS experiments.

A novel ligand for chemogenetic receptors, Deschloroclozapine, enables rapid and selective modulation of neuronal activity and behavior in living animals

A brain function is manifested by harmonizing some brain regions responsible for the function. Since pathological conditions in neuropsychiatric disorders are induced by the failure of this mechanism, it is crucial to identify the affected function by manipulating the specific brain regions. Designer receptors exclusively activated by designer drugs (DREADDs) are one of the chemogenetic tools that offer a means to repeatedly reversible control of the activity of a target neural population expressing a "designer receptor" by systemic injection of "designer drug," which is biologically inert. The most widely used DREADDs are muscarinic-based receptors, such as hM3Dq (excitatory) and hM4Di (inhibitory), which can be activated by clozapine-N-oxide (CNO). However, CNO has some concerns. First, because CNO has a modest brain penetrability, the effect is slow. Second, since CNO is metabolized to clozapine, an antipsychotic drug that acts on numerous endogenous receptors, the systemic administration may produce off-target actions. Therefore, we developed a new compound, deschloroclozapine (DCZ), to solve these issues. DCZ has a higher affinity and greater agonist potency than CNO with reduced off-target actions and can rapidly modulate the neuronal activity and behavior with muscarinic-based DREADDs in living animals. Given the potential weak point of CNO, DCZ affords clear benefits to many users of muscarinic-based DREADD, with increased reliability by removing concerns about possible off-target responses.

Chemogenetic inactivation reveals the inhibitory control function of the prefronto-striatal pathway in the macaque brain

The lateral prefrontal cortex (LPFC) has a strong monosynaptic connection with the caudate nucleus (CdN) of the striatum. Previous human MRI studies have suggested that this LPFC-CdN pathway plays an important role in inhibitory control and working memory. We aimed to validate the function of this pathway at a causal level by pathway-selective manipulation of neural activity in non-human primates. To this end, we trained macaque monkeys on a delayed oculomotor response task with reward asymmetry and expressed an inhibitory type of chemogenetic receptors selectively to LPFC neurons that project to the CdN. Ligand administration reduced the inhibitory control of impulsive behavior, as well as the task-related neuronal responses observed in the local field potentials from the LPFC and CdN. These results show that we successfully suppressed pathway-selective neural activity in the macaque brain, and the resulting behavioral changes suggest that the LPFC-CdN pathway is involved in inhibitory control.