Designer Receptors Exclusively Activated By Designer Drugs Agonist Research Articles

Dorsal raphe to basolateral amygdala corticotropin-releasing factor circuit regulates cocaine-memory reconsolidation.

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. Next, they were briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) immediately after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed the glutamatergic neuronal marker, vesicular glutamate transporter 3. Together, these findings suggest that the DRCRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF and/or glutamate release in the BLA.

DREADD agonist compound 21 causes acute diuresis in wild-type mice.

The targeted activation or inhibition of specific cell populations using chemogenetics allows the precise dissection of cellular signaling and function. Designer receptors exclusively activated by designer drugs (DREADDs) is a chemogenetic platform initially developed by mutating human muscarinic receptors to be unresponsive to endogenous acetylcholine but exclusively activated by an "inert" designer drug. Compound 21 (C21) is a new and potent DREADD agonist; however, radioligand assays from a recent report indicated its ability to bind to endogenous G protein-coupled receptors (GPCRs), including muscarinic M1-3 receptors. Whether this binding causes off-target effects is unclear. Renal innervation is important for the regulation of renal function, and the advent of chemogenetic tools provides significant opportunities for the mechanistic understanding of renal innervation and function. GPCRs such as adrenergic and muscarinic receptors play a role in renal function; thus, a careful pharmacological characterization of C21 in renal function is a prerequisite for this approach. Unexpectedly, an infusion of 1.0mg/kg C21 in anesthetized mice caused an ∼4-fold increase in urine output and correspondingly increased the glomerular filtration rate (GFR), suggesting a C21-mediated acute diuretic effect. This acute diuresis effect was further confirmed in awake mice using voiding spot assays. The exact molecular mechanism for C21-mediated diuresis is unclear; however, we demonstrated by in vitro myography that C21 can effectively inhibit bladder smooth muscle contraction by antagonizing M3 receptors at the micromolar level, causing increased voiding size in vivo. In summary, C21 functions as a GPCR antagonist and has significant dose-dependent off-target effects in the renal system.

Deschloroclozapine exhibits an exquisite agonistic effect at lower concentration compared to clozapine-N-oxide in hM3Dq expressing chemogenetically modified rats

IntroductionWithin the realm of chemogenetics, a particular form of agonists targeting designer receptors exclusively activated by designer drugs (DREADDs) has emerged. Deschloroclozapine (DCZ), a recently introduced DREADDs agonist, demonstrates remarkable potency in activating targeted neurons at a lower dosage compared to clozapine-N-oxide (CNO).MethodsWe conducted a comparative analysis of the effects of subcutaneously administered CNO (1 mg/kg) and DCZ (0.1 mg/kg) in our transgenic rats expressing hM3Dq and mCherry exclusively in oxytocin (OXT) neurons.Results and DiscussionNotably, DCZ exhibited a swift and robust elevation of serum OXT, surpassing the effects of CNO, with a significant increase in the area under the curve (AUC) up to 3 hours post-administration. Comprehensive assessment of brain neuronal activity, using Fos as an indicator, revealed comparable effects between CNO and DCZ. Additionally, in a neuropathic pain model, both CNO and DCZ increased the mechanical nociceptive and thermal thresholds; however, the DCZ-treated group exhibited a significantly accelerated onset of the effects, aligning harmoniously with the observed alterations in serum OXT concentration following DCZ administration. These findings emphasize the remarkable efficacy of DCZ in rats, suggesting its equivalent or potentially superior performance to CNO at considerably lower dosages, thus positioning it as a promising contender among DREADDs agonists.

Targeted stimulation of the vagus nerve reduces renal injury in female mice with systemic lupus erythematosus

Pharmacological stimulation of the vagus nerve has been shown to suppress inflammation and reduce blood pressure in a murine model of systemic lupus erythematosus (SLE) that is characterized by hypertension, inflammation, renal injury and dysautonomia. The present study aims to directly stimulate vagal nerves at the level of the dorsal motor nucleus of the vagus (DMV) using designer receptors exclusively activated by designer drugs (DREADDs) to determine if there is similar protection and confirm mechanism. Female NZBWF1/J (SLE) mice and NZW/LacJ mice (controls, labeled as NZW throughout) received bilateral microinjections of pAAV-hSyn-hM3D(Gq)-mCherry or control virus into the DMV at 31 weeks of age. After two weeks of recovery and viral transfection, the DREADD agonist clozapine-N-oxide (CNO; 3 mg/kg) was injected subcutaneously for an additional 14 days. At 35 weeks, mean arterial pressure (MAP; mmHg) was increased in SLE mice compared to NZW mice, but selective activation of DMV neurons did not significantly alter MAP in either group. SLE mice had higher indices of renal injury including albumin excretion rate (μg/day), glomerulosclerosis index, interstitial fibrosis, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) compared to NZW mice. Selective DMV neuronal activation reduced albumin excretion rate, glomerulosclerosis, interstitial fibrosis, and NGAL in SLE mice but not NZW mice. Together, these data indicate that selective activation of neurons within the DMV by DREADD protects the kidney suggesting an important role of vagus-mediated pathways in the progression of renal injury in SLE.

Clozapine N-oxide, compound 21, and JHU37160 do not influence effortful reward-seeking behavior in mice

RationaleClozapine N-oxide (CNO) has been developed as a ligand to selectively activate designer receptors exclusively activated by designer drugs (DREADDs). However, previous studies have revealed that peripherally injected CNO is reverse-metabolized into clozapine, which, in addition to activating DREADDs, acts as an antagonist at various neurotransmitter receptors, suggesting potential off-target effects of CNO on animal physiology and behaviors. Recently, second-generation DREADD agonists compound 21 (C21) and JHU37160 (J60) have been developed, but their off-target effects are not fully understood.ObjectivesThe present studies assessed the effect of novel DREADD ligands on reward-seeking behavior.MethodsWe first tested the possible effect of acute i.p. injection of low-to-moderate (0.1, 0.3, 1, 3 mg/kg) of CNO, C21, and J60 on motivated reward-seeking behavior in wild-type mice. We then examined whether a high dose (10 mg/kg) of these drugs might be able to alter responding.ResultsLow-to-moderate doses of all drugs and a high dose of CNO or C21 did not alter operant lick responding for a reward under a progressive ratio schedule of reinforcement, in which the number of operant lick responses to obtain a reward increases after each reward collection. However, high-dose J60 resulted in a total lack of responding that was later observed in an open field arena to be due to a sedative effect.ConclusionsThis study provides definitive evidence that commonly used doses of CNO, C21, and J60 have negligible off-target effects on motivated reward-seeking but urges caution when using high doses of J60 due to sedative effects.

A head-to-head comparison of two DREADD agonists for suppressing operant behavior in rats via VTA dopamine neuron inhibition.

Designer receptors exclusively activated by designer drugs (DREADDs) are a tool for "remote control" of defined neuronal populations during behavior. These receptors are inert unless bound by an experimenter-administered designer drug, commonly clozapine-n-oxide (CNO). However, questions have emerged about the suitability of CNO as a systemically administered DREADD agonist. Second-generation agonists such as JHU37160 (J60) have been developed, which may have more favorable properties than CNO. Here we sought to directly compare effects of CNO (0, 1, 5, & 10mg/kg, i.p.) and J60 (0, 0.03, 0.3, & 3mg/kg, i.p.) on operant food pursuit. Male and female TH:Cre + rats and their wildtype (WT) littermates received cre-dependent hM4Di-mCherry vector injections into ventral tegmental area (VTA), causing inhibitory DREADD expression in VTA dopamine neurons of TH:Cre + rats. All rats were trained to stably lever press for palatable food on a fixed ratio 10 schedule, and doses of both agonists were tested on separate days in counterbalanced order. All three CNO doses reduced operant rewards earned in rats with DREADDs, and no CNO dose had behavioral effects in WT controls. The highest J60 dose tested significantly reduced responding in DREADD rats, but this dose also increased responding in WTs, indicating non-specific effects. The magnitude of CNO and J60 effects in TH:Cre + rats were correlated and were present in both sexes. Findings demonstrate the usefulness of directly comparing DREADD agonists when optimizing behavioral chemogenetics, and highlight the importance of proper controls, regardless of the DREADD agonist employed.

High dose administration of DREADD agonist JHU37160 produces increases in anxiety-like behavior in male rats

Designer receptors exclusively activated by designer drugs (DREADDs) are a promising tool for analyzing neural circuitry, and improved DREADD-selective ligands continue to be developed. Relative to clozapine-N-oxide (CNO), JHU37160 is a selective DREADD agonist recently shown to exhibit higher blood brain barrier penetrance and DREADD selectivity in vivo; however, relatively few studies have characterized the behavioral effects of systemic JHU37160 administration in animals. Here, we report a dose-dependent anxiogenic effect of systemic JHU37160 in male Wistar and Long-Evans rats, regardless of DREADD expression, with no impact on locomotor behavior. These results suggest that high dose (1 mg/kg) JHU37160 should be avoided when performing chemogenetic experiments designed to evaluate circuit manipulation on anxiety-like behavior in rats.

Proopiomelanocortin projections to the nucleus accumbens modulate acquisition and maintenance of operant palatable pellet administration in mice

Obesity is a crisis in the United States, producing many co-morbid diseases that can drastically decrease quality of life. While diet is a major focus for therapeutic intervention, the need to understand underlying appetitive neurocircuitry persists. Proopiomelanocortin (POMC) peptides are well-known for their anorexigenic activity, but also mediate reward and learning. The nucleus accumbens (NAcc) is best known for its role in reward-based learning, but the contribution of POMC projections to NAcc on feeding are controversial since the two major POMC-derived peptides (β-endorphin and α-MSH) have opposite effects on food intake. Our objective was to determine the effect of stimulating POMC projections in the NAcc on acquisition and maintenance of operant self-administration of a palatable food. Adult POMCCre mice were microinjected into the NAcc with a Cre-dependent retrograde adeno-associated viral vector expressing Gq Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Mice were trained to self-administer palatable 20-mg pellets in daily operant sessions. Acquisition of self-administration (fixed ratio 30) and baseline self-administration were measured in daily sessions, with mice receiving injections of either JHU37152 (DREADD agonist) or saline (i.p.) 15 min prior to the sessions. POMC neuron stimulation (JHU injection) before training sessions produced a significant increase in rate of acquisition and accuracy compared to the saline treated group, with no significant effect on rewards earned. Removal of POMC neuron stimulation before sessions initially reduced consumption with a gradual increase in responding for reinforcer over 3 days of saline injections. Reinstatement of POMC neuron stimulation (JHU) before the session resulted in a significant decrease in responding and rewards earned. These results suggest a complex role of POMC peptides within the NAcc that increase reward learning for a novel palatable food while decreasing consumption of the reinforcer following experience with it.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice

ObjectiveInsulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. MethodsWe generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. ResultsRxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. ConclusionThese findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Chemogenetic modulation of sensory afferents induces locomotor changes and plasticity after spinal cord injury.

Neuromodulatory therapies for spinal cord injury (SCI) such as electrical epidural stimulation (EES) are increasingly effective at improving patient outcomes. These improvements are thought to be due, at least in part, to plasticity in neuronal circuits. Precisely which circuits are influenced and which afferent classes are most effective in stimulating change remain important open questions. Genetic tools, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), support targeted and reversible neuromodulation as well as histological characterization of manipulated neurons. We therefore transduced and activated lumbar large diameter peripheral afferents with excitatory (hM3Dq) DREADDs, in a manner analogous to EES, in a rat hemisection model, to begin to trace plasticity and observe concomitant locomotor changes. Chronic DREADDs activation, coupled with thrice weekly treadmill training, was observed to increase afferent fluorescent labeling within motor pools and Clarke's column when compared to control animals. This plasticity may underlie kinematic differences that we observed across stages of recovery, including an increased and less variable hindquarters height in DREADDs animals, shorter step durations, a more flexed ankle joint early in recovery, a less variable ankle joint angle in swing phase, but a more variable hip joint angle. Withdrawal of DREADDs agonist, clozapine-N-oxide (CNO) left these kinematic differences largely unaffected; suggesting that DREADDs activation is not necessary for them later in recovery. However, we observed an intermittent “buckling” phenomenon in DREADDs animals without CNO activation, that did not occur with CNO re-administration. Future studies could use more refined genetic targeted of specific afferent classes, and utilize muscle recordings to find where afferent modulation is most influential in altering motor output.

Central Activation of the Cholinergic Anti‐Inflammatory Pathway Improves Outcome in a Murine Model of Systemic Lupus Erythematosus

The cholinergic anti‐inflammatory pathway is an endogenous vagus‐to‐spleen mechanism that regulates the immune system and when activated, results in decreases in systemic inflammation. Previous studies from our group demonstrated that the pharmacological potentiation of the efferent vagus nerve via systemic administration of galantamine reduces renal inflammation and blood pressure in systemic lupus erythematosus (SLE), suggesting an impairment in the cholinergic anti‐inflammatory pathway in this model. In this study, we aimed to delineate specific pathways and we hypothesized that central stimulation of vagal efferents by selective activation of neurons within the dorsal motor nucleus of the vagus (DMVN) would similarly protect SLE mice and confirm the role of the cholinergic anti‐inflammatory in controlling inflammation. For that, we selectively activated DMVN neurons using designer receptors exclusively activated by designer drugs (DREADDs). Female NZBWF1 (SLE) and parental control – NZW (Control) mice (n=3‐8) received bilateral microinjections of pAAV‐hSyn‐hM3D(Gq)‐mCherry or pAAV‐hSyn‐mCherry (control virus) into the DMVN at 31 weeks of age. Two weeks post‐microinjection, DREADD agonist CNO (3mg/kg) was administered subcutaneously for 2 weeks starting at 33 weeks. At 35 weeks, mice were housed in metabolic cages for urine collection and catheters were implanted in the carotid to access mean arterial pressure (MAP) in conscious mice. Mice were subsequently euthanized, spleen was collected for Western blotting, blood for double‐stranded (ds) DNA autoantibody quantification and brain to confirm the site of virus microinjection. Selective activation of DMVN neurons did not alter MAP (mmHg) in SLE mice (SLE: 146 ± 6 vs SLE/Gq DREADD: 142 ± 3) or controls (Control: 126 ±4 vs Control/Gq DREADD: 132 ± 2), and also did not change dsDNA autoantibodies (activity units) in SLE mice (SLE: 4.7e5± 1.1e4 vs SLE/Gq DREADD: 3.7e8 ± 1.3e6) or controls (Control: 1.8e5± 5.3e4 vs Control/Gq DREADD: 2.8e5± 1.3e5). Splenic TNFα protein expression was increased in SLE mice compared to controls (SLE: 1.9e6 ± 3.6e5 vs Control: 6.5e5 ± 8.6e4, p=0.001) and DMVN neuron activation decreased splenic TNFα expression in SLE mice (SLE: 1.9e6 ± 3.6e5 vs SLE/Gq DREADD: 1.1e6 ± 1.1e5, p=0.05) but not controls (Control: 6.1e6 ± 3.1e6 vs Control /Gq DREADD: 1.7e6±4.4e5). Albumin excretion rate (µg/day) was higher in SLE mice compared to controls (SLE: 10210±4015 vs Control 97.1±54.3, p=0.07) and DMVN neuron stimulation decreased albumin excretion rate in SLE mice although not significantly (SLE/Gq DREADD: 207.4±142.1, p=0.07). In addition, selective activation of DMV neurons decreased urinary leukocytes (62.5% vs 50%) and blood in the urine (50% vs 16%) in SLE mice. The present study showed that boosting the cholinergic anti‐inflammatory pathway via selective activation of vagal efferents lowered splenic inflammation and protected the kidney without altering blood pressure suggesting an important role of the cholinergic anti‐inflammatory pathway in regulating inflammation and renal injury in SLE.

Glutamatergic Neurons in the Amygdala Are Involved in Paclitaxel-Induced Pain and Anxiety.

Paclitaxel is widely used as a first-line chemotherapy agent to treat malignant tumors. However, paclitaxel causes peripheral nerve fiber damage and neuropathic pain in some patients. In addition, patients received paclitaxel chemotherapy are often accompanied by negative emotions such as anxiety. The amygdala is critically involved in regulating pain signals, as well as anxiety. The purpose of this study is to clarify the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-positive glutamatergic neurons in the amygdala in paclitaxel-induced pain and negative affective symptoms. Intraperitoneal injection of paclitaxel into mice caused mechanical and thermal allodynia, as measured by Von Frey test and Hargreaves test, and anxiety, as measured by open field test and elevated plus maze test. Immunofluorescence staining revealed that c-fos-positive neurons were significantly more in the basolateral amygdala (BLA) and central amygdala (CeA) in paclitaxel-treated mice than untreated mice. Furthermore, part of c-fos-positive neurons in the BLA were immunoreactive of CaMKII. Engineered Designer receptors exclusively activated by designer drugs (DREADD) receptor hM4Di or hM3Dq was selectively expressed on CaMKII neurons by injection of adeno-associated virus (AAV) vectors containing CaMKII and hM4Di or hM3Dq. Administration of DREADD agonist CNO to selectively inhibit the CaMKII neurons in the BLA significantly increased the paw withdrawal thresholds and paw withdrawal latencies. In addition, selectively inhibition of CaMKII neurons in the BLA alleviated anxiety behavior without affecting the motor activity. In summary, our findings suggest that CaMKII neurons in the amygdala are critical for neuropathic pain and anxiety behaviors induced by paclitaxel chemotherapy.

Chemogenetic Seizure Control with Clozapine and the Novel Ligand JHU37160 Outperforms the Effects of Levetiracetam in the Intrahippocampal Kainic Acid Mouse Model

Expression of inhibitory designer receptors exclusively activated by designer drugs (DREADDs) on excitatory hippocampal neurons in the hippocampus represents a potential new therapeutic strategy for drug-resistant epilepsy. To overcome the limitations of the commonly used DREADD agonist clozapine, we investigated the efficacy of the novel DREADD ligand JHU37160 in chemogenetic seizure suppression in the intrahippocampal kainic acid (IHKA) mouse model for temporal lobe epilepsy (TLE). In addition, seizure-suppressing effects of chemogenetics were compared to the commonly used anti-epileptic drug (AED), levetiracetam (LEV). Therefore, an adeno-associated viral vector was injected in the sclerotic hippocampus of IHKA mice to induce expression of a tagged inhibitory DREADD hM4Di or only a tag (control) specifically in excitatory neurons using the CamKIIα promoter. Subsequently, animals were treated with LEV (800 mg/kg), clozapine (0.1 mg/kg), and DREADD ligand JHU37160 (0.1 mg/kg) and the effect on spontaneous seizures was investigated. Clozapine and JHU37160-mediated chemogenetic treatment both suppressed seizures in DREADD-expressing IHKA mice. Clozapine treatment suppressed seizures up to 34 h after treatment, and JHU37160 effects lasted for 26 h after injection. Moreover, both compounds reduced the length of seizures that did occur after treatment up to 28 h and 18 h after clozapine and JHU37160, respectively. No seizure-suppressing effects were found in control animals using these ligands. Chemogenetic seizure treatment suppressed seizures during the first 30 min after injection, and seizures remained suppressed during 8 h following treatment. Chemogenetics thus outperformed effects of levetiracetam (p < 0.001), which suppressed seizure frequency with a maximum of 55 ± 9% for up to 1.5 h (p < 0.05). Only chemogenetic and not levetiracetam treatment affected the length of seizures after treatment (p < 0.001). These results show that the chemogenetic therapeutic strategy with either clozapine or JHU37160 effectively suppresses spontaneous seizures in the IHKA mouse model, confirming JHU37160 as an effective DREADD ligand. Moreover, chemogenetic therapy outperforms the effects of levetiracetam, indicating its potential to suppress drug-resistant seizures.

Off-Target Expression of Cre-Dependent Adeno-Associated Viruses in Wild-Type C57BL/6J Mice.

Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although nonamplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the neocortex of wild-type C57BL/6J mice. While we observed weak but consistent nonamplified off-target double inverted open reading frame (DIO) expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed notable Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype, or promoter. We also evaluated whether Cre-independent expression had functional effects via designer receptors exclusively activated by designer drugs (DREADDs). The DREADD agonist C21 (compound 21) had no effect on contextual fear conditioning or c-Fos expression in DIO-hM3Dq-mCherry+ cells of C57BL/6J mice. Together, our results indicate that DIO constructs have off-target expression in wild-type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.

Nucleus Accumbens Chemogenetic Inhibition Suppresses Amphetamine-Induced Ultrasonic Vocalizations in Male and Female Rats.

Adult rats emit ultrasonic vocalizations (USVs) related to their affective states, potentially providing information about their subjective experiences during behavioral neuroscience experiments. If so, USVs might provide an important link between invasive animal preclinical studies and human studies in which subjective states can be readily queried. Here, we induced USVs in male and female Long Evans rats using acute amphetamine (2 mg/kg), and asked how reversibly inhibiting nucleus accumbens neurons using designer receptors exclusively activated by designer drugs (DREADDs) impacts USV production. We analyzed USV characteristics using “Deepsqueak” software, and manually categorized detected calls into four previously defined subtypes. We found that systemic administration of the DREADD agonist clozapine-n-oxide, relative to vehicle in the same rats, suppressed the number of frequency-modulated and trill-containing USVs without impacting high frequency, unmodulated (flat) USVs, nor the small number of low-frequency USVs observed. Using chemogenetics, these results thus confirm that nucleus accumbens neurons are essential for production of amphetamine-induced frequency-modulated USVs. They also support the premise of further investigating the characteristics and subcategories of these calls as a window into the subjective effects of neural manipulations, with potential future clinical applications.

Abstract MP24: Selective Activation Of DMV Neurons Improves Renal Injury In Systemic Lupus Erythematosus

The cholinergic anti-inflammatory pathway is a vagally-mediated mechanism that controls inflammation. Our published data suggest that an impaired cholinergic anti-inflammatory pathway contributes to hypertension and renal disease in female mice with systemic lupus erythematosus (SLE), since pharmacological potentiation of the efferent vagus via administration of galantamine reduces renal inflammation, mean arterial pressure (MAP) and glomerulosclerosis. The aim of the current study is to selectively target neurons within the dorsal motor nucleus of the vagus (DMV) to stimulate the efferent vagus and the cholinergic anti-inflammatory pathway using designer receptors exclusively activated by designer drugs (DREADDs). We hypothesized that selective activation of DMV neurons would reduce inflammation, eliminating the associated end organ damage in SLE. To study this, female SLE ( NZBWF1 ) and parental control ( NZW ) mice received bilateral microinjections of pAAV-hSyn-hM3D(Gq)-mCherry or pAAV-hSyn-mCherry (control virus) into the DMV at 31 weeks of age using the following coordinates with calamus as reference: 0 mm caudal, 0.25 mm lateral and 0.48 mm ventral. Two weeks post-microinjection, DREADD agonist CNO (3mg/kg) was administered subcutaneously for 2 weeks starting at 33 weeks. At 35 weeks, mice were housed in metabolic cages for urine collection and catheters were implanted in the carotid artery for MAP measurement. Mice were subsequently euthanized and the brain collected to confirm the site of virus microinjection. Selective activation of DMV neurons decreased the incidence of albuminuria [&gt; 300 mg/dL; 66% (4 out of 6) vs. 0% (0 out of 7)], urinary leukocytes [62.5% (5 out of 8) vs. 50% (3 out of 6)] and blood in the urine [50% (4 out of 8) vs. 16% (1 out of 6)] in SLE mice. MAP did not significantly change with the chemogenetic activation of DMV neurons in SLE mice or parental controls (SLE/control virus: 146 ± 6, n=7; SLE/Gq DREADD: 142 ± 3, n=6; NZW/control virus: 126 ± 4, n=4; NZW/Gq DREADD: 132 ± 2, n=5). These results suggests that this timeline of selective activation of DMV neurons in SLE mice reduces renal injury without altering blood pressure, but future studies will confirm the effect on hypertension and renal inflammation in SLE mice

Chemogenetic activation of nigrostriatal dopamine neurons in freely moving common marmosets

SummaryTo 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.

Abnormalities in the composition of the gut microbiota in mice after repeated administration of DREADD ligands

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are known as genetically modified G-protein-coupled receptors (GPCRs), which can be activated by synthetic ligands such as clozapine N-oxide (CNO) and DREADD agonist 21 (compound 21: C21). The brain-gut-microbiota axis has a crucial role in bidirectional interactions between the brain and the gastrointestinal microbiota. In this study, we investigated whether repeated administration of CNO or C21 could influence the gut microbiota and short-chain fatty acids (SCFAs) in feces of adult mice. Repeated administration of CNO or C21 as drinking water did not alter the α- and β-diversity of gut microbiota in mice compared with control mice. However, we found significant changes in relative abundance for several bacteria in the CNO (or C21) group at the taxonomic level compared to the control group. The linear discriminant analysis effect size (LEfSe) algorithm distinguished the family Prevotellaceae, the genus Anaerocolumna, the genus Prevotella, and the genus Frisingicoccus, these four specific microbial markers for the CNO group relative to the control group. In addition, the LEfSe algorithm identified the family Clostridiaceae, the genus Faecalicatena and the genus Marinisporobacter, these three bacteria of different taxonomic as potential microbial markers for the C21 group relative to the control group. In contrast, repeated administration of CNO (or C21) did not alter SCFAs in feces samples of adult mice. The data suggest that repeated administration of CNO or C21 contributes to an unusual organization of the gut microbiota in adult mice. Therefore, abnormalities in the composition of gut microbiota by repeated dosing of DREADD ligands should be taken into consideration for behavioral and biological functions in rodents treated with DREADD ligands.

Does chronic systemic injection of the DREADD agonists clozapine-N-oxide or Compound 21 change behavior relevant to locomotion, exploration, anxiety, and depression in male non-DREADD-expressing mice?

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are chemogenetic tools commonly-used to manipulate brain activity. The most widely-used synthetic DREADD ligand, clozapine-N-oxide (CNO), is back-metabolized to clozapine which can itself activate endogenous receptors. Studies in non-DREADD-expressing rodents suggest CNO or a DREADD agonist that lacks active metabolites, such as Compound 21 (C21), change rodent behavior (e.g. decrease locomotion), but chronic injection of CNO does not change locomotion. However, it is unknown if chronic CNO changes behaviors relevant to locomotion, exploration, anxiety, and depression, or if chronic C21 changes any aspect of mouse behavior. Here non-DREADD-expressing mice received i.p. Vehicle (Veh), CNO, or C21 (1 mg/kg) 5 days/week for 16 weeks and behaviors were assessed over time. Veh, CNO, and C21 mice had similar weight gain over the 16-week-experiment. During the 3rd injection week, CNO and C21 mice explored more than Veh mice in a novel context and had more open field center entries; however, groups were similar in other measures of locomotion and anxiety. During the 14th-16th injection weeks, Veh, CNO, and C21 mice had similar locomotion and anxiety-like behaviors. We interpret these data as showing chronic Veh, CNO, and C21 injections given to male non-DREADD-expressing mice largely lack behavioral effects. These data may be helpful for behavioral neuroscientists when study design requires repeated injection of these DREADD agonists.

Hypothalamic-vagal oxytocinergic neurocircuitry modulates gastric emptying and motility following stress.

Stress triggers and exacerbates the symptoms of functional gastrointestinal disorders, such as delayed gastric emptying and impaired gastric motility. Understanding the mechanisms by which the neural circuits, impaired by stress, are restored may help to identify potential targets for more effective therapeutic interventions. Oxytocin administration or release ameliorates the stress-induced delayed gastric emptying and motility. However, is it unclear whether the effects are mediated via the hypothalamic-pituitary-adrenocortical axis or the oxytocinergic projections from the paraventricular nucleus of the hypothalamus to brainstem neurones of the dorsal vagal complex. We used Cre-inducible designer receptors exclusively activated by designer drugs to demonstrate the fundamental role of the oxytocinergic hypothalamic-vagal projections in the gastric adaptation to stress. Stress triggers and exacerbates the symptoms of functional gastrointestinal (GI) disorders, such as delayed gastric emptying and impaired gastric motility. The prototypical anti-stress hormone, oxytocin (OXT), plays a major role in the modulation of gastric emptying and motility following stress. It is not clear, however, whether the amelioration of dysregulated GI functions by OXT is mediated via an effect on the hypothalamic-pituitary-adrenocortical axis or the oxytocinergic projections from the paraventricular nucleus of the hypothalamus (PVN) to neurones of the dorsal vagal complex (DVC). In the present study we tested the hypothesis that the activity of hypothalamic-vagal oxytocinergic neurocircuits plays a major role in the gastric adaptation to stress. Cre-inducible designer receptors exclusively activated by designer drugs (DREADDs) were injected into the DVC of rats and retrogradely transported to allow selective expression in OXT neurones in the PVN. Following acute stress and either chronic heterotypic (CHe) or chronic homotypic (CHo) stress, gastric emptying was assessed via the [13 C]-octanoic acid breath test, and gastric tone and motility were assessed via strain gauges sewn on the surface of the stomach. Activation of the hypothalamic-vagal oxytocinergic neurocircuitry, by DREADD agonist clozapine-N-oxide (CNO), prevented the delayed gastric emptying observed following acute or CHe stress, and 4th ventricular administration of CNO increased gastric tone and motility. Conversely, CNO-mediated inhibition of the hypothalamic-vagal oxytocinergic neurocircuitry prevented the CHo-induced adaptation in gastric emptying, and an increase in gastric tone and motility. Taken together, the data support the hypothesis that hypothalamic-vagal oxytocinergic neurocircuits play a major role in the modulation of gastric emptying and motility following stress.