BACKGROUND AND PURPOSEδ Opioid receptor agonists are being developed as potential treatments for depression and alcohol use disorders. This is particularly interesting as depression is frequently co-morbid with alcohol use disorders. Yet we have previously shown that δ receptor agonists range widely in their ability to modulate alcohol intake; certain δ receptor agonists actually increase alcohol consumption in mice. We propose that variations in β-arrestin 2 recruitment contribute to the differential behavioural profile of δ receptor agonists. EXPERIMENTAL APPROACHWe used three diarylmethylpiperazine-based non-peptidic δ receptor selective agonists (SNC80, SNC162 and ARM390) and three structurally diverse δ receptor agonists (TAN-67, KNT127 and NIH11082). We tested these agonists in cAMP and β-arrestin 2 recruitment assays and a behavioural assay of alcohol intake in male C57BL/6 mice. We used β-arrestin 2 knockout mice and a model of depression-like behaviour to further study the role of β-arrestin 2 in δ receptor pharmacology. KEY RESULTSAll six tested δ receptor agonists were full agonists in the cAMP assay but displayed distinct β-arrestin 2 recruitment efficacy. The efficacy of δ receptor agonists to recruit β-arrestin 2 positively correlated with their ability to increase alcohol intake (P < 0.01). The effects of the very efficacious recruiter SNC80 on alcohol intake, alcohol place preference and depression-like behaviour were β-arrestin 2-dependent. CONCLUSIONS AND IMPLICATIONSOur finding that δ receptor agonists that strongly recruit β-arrestin 2 can increase alcohol intake carries important ramifications for drug development of δ receptor agonists for treatment of alcohol use disorders and depressive disorders.

Stroke is the most common fatal neurological disease in the United States 1 . The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2 . Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3 , many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4 . The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6 . It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8 . To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9 . In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining. Video LinkThe video component of this article can be found at https://www.jove.com/video/2761/ Protocol MCAO MethodThis protocol was approved by the Institutional Animal Care and Use Committees at UCSF and Kent State University, and abides by the National Institutes of Health guidelines for the use of experimental animals.1. Cut a 5-0 monofilament suture (Harvard Apparatus, Holliston, MA) into 20 mm segments. Round the tip of each segment by heating it near a cauterizer (Braintree Scientific, Inc., Braintree, MA). Measure the diameter of the tip using a micrometer (Applied Image Inc., Rochester, NY). We use a suture with a final tip diameter of 0.21-0.22 mm for a mouse with body weight of 25-30 g. 2. Sterilize all surgical tools by autoclaving (minimum 121 °C, 15 PSI, for 15 min). Sanitize the surgery table and associated equipment using 70% ethanol. 3. Anesthetize an 8-12 week-old mouse (25-30 g) with 5% isoflurane (Aerrane, Baxter, Deerfield, IL) in 30% O 2 / 70% N 2 O using the V-10 Anesthesia system (VetEquip, Inc., Pleasanton, CA). Following induction of anesthesia, reduce the level of isoflurane and maintain it at 1.5%. 4. Place the mouse in the supine position on a heating pad. Insert a rectal probe, and monitor and maintain body temperature between 36.5-37.5 °C using the TR-200 homeothermic temperature system (Fine Science Tools Inc., Foster City, CA). 5. Shave the fur on the ventral neck region with electric clippers (Braintree Scientific) to expose the skin. Disinfect the surgica...

Background and Purpose: Stroke disrupts neuronal functions in both local and remotely connected regions, leading to network-wide deficits that can hinder recovery. The thalamus is particularly affected, with progressive development of neurodegeneration accompanied by inflammatory responses. However, the complexity of the involved inflammatory responses is poorly understood. Herein we investigated the spatiotemporal changes in the secondary degenerative thalamus after cortical stroke, using targeted transcriptome approach in conjunction with histology and flow cytometry. Methods: Cortical ischemic stroke was generated by permanent occlusion of the left middle cerebral artery in male C57BL6J mice. Neurodegeneration, neuroinflammatory responses, and microglial activation were examined in naive and stroke mice at from poststroke days (PD) 1 to 84, in both ipsilesional somatosensory cortex and ipsilesional thalamus. NanoString neuropathology panel (780 genes) was used to examine transcriptome changes at PD7 and PD28. Fluorescence activated cell sorting was used to collect CD11c + microglia from ipsilesional thalamus, and gene expressions were validated by quantitative real-time polymerase chain reaction. Results: Neurodegeneration in the thalamus was detected at PD7 and progressively worsened by PD28. This was accompanied by rapid microglial activation detected as early as PD1, which preceded the neurodegenerative changes. Transcriptome analysis showed higher number of differentially expressed genes in ipsilesional thalamus at PD28. Notably, neuroinflammation was the top activated pathway, and microglia was the most enriched cell type. Itgax (CD11c) was the most significantly increased gene, and its expression was highly detected in microglia. Flow-sorted CD11c + microglia from degenerative thalamus indicated molecular signatures similar to neurodegenerative disease–associated microglia; these included downregulated Tmem119 and CX3CR1 and upregulated ApoE, Axl, LpL, CSF1, and Cst7. Conclusions: Our findings demonstrate the dynamic changes of microglia after stroke and highlight the importance of investigating stroke network-wide deficits. Importantly, we report the existence of a unique subtype of microglia (CD11c + ) with neurodegenerative disease–associated microglia features in the degenerative thalamus after stroke.

The transition from non-dependent alcohol use to alcohol dependence involves increased activity of the dorsal striatum. Interestingly, the dorsal striatum expresses a large number of inhibitory G-protein-coupled receptors (GPCRs), which when activated may inhibit alcohol-induced increased activity and can decrease alcohol consumption. Here, we explore the hypothesis that dorsal striatal Gi/o-protein activation is sufficient to reduce voluntary alcohol intake. Using a voluntary, limited-access, two-bottle choice, drink-in-the-dark model of alcohol (10%) consumption, we validated the importance of Gi/o signaling in this region by locally expressing neuron-specific, adeno-associated-virus encoded Gi/o-coupled muscarinic M4 designer receptors exclusively activated by designer drugs (DREADD) in the dorsal striatum and observed a decrease in alcohol intake upon DREADD activation. We validated our findings by activating Gi/o-coupled delta-opioid receptors (DORs), which are natively expressed in the dorsal striatum, using either a G-protein biased agonist or a β-arrestin-biased agonist. Local infusion of TAN-67, an in vitro-determined Gi/o-protein biased DOR agonist, decreased voluntary alcohol intake in wild-type and β-arrestin-2 knockout (KO) mice. SNC80, a β-arrestin-2 biased DOR agonist, increased alcohol intake in wild-type mice; however, SNC80 decreased alcohol intake in β-arrestin-2 KO mice, thus resulting in a behavioral outcome generally observed for Gi/o-biased agonists and suggesting that β-arrestin recruitment is required for SNC80-increased alcohol intake. Overall, these results suggest that activation Gi/o-coupled GPCRs expressed in the dorsal striatum, such as the DOR, by G-protein biased agonists may be a potential strategy to decrease voluntary alcohol consumption and β-arrestin recruitment is to be avoided.

G protein–coupled receptors (GPCRs) are implicated in the regulation of fear and anxiety. GPCR signaling involves canonical G protein pathways but can also engage downstream kinases and effectors through scaffolding interactions mediated by β-arrestin. Here, we investigated whether β-arrestin signaling regulates anxiety-like and fear-related behavior in mice in response to activation of the GPCR δ-opioid receptor (δOR or DOR). Administration of β-arrestin–biased δOR agonists to male C57BL/6 mice revealed β-arrestin 2–dependent activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) in the dorsal hippocampus and amygdala and β-arrestin 1–dependent activation of ERK1/2 in the nucleus accumbens. In mice, β-arrestin–biased agonist treatment was associated with reduced anxiety-like and fear-related behaviors, with some overlapping and isoform-specific input. In contrast, applying a G protein–biased δOR agonist decreased ERK1/2 activity in all three regions as well as the dorsal striatum and was associated with increased fear-related behavior without effects on baseline anxiety. Our results indicate a complex picture of δOR neuromodulation in which β-arrestin 1– and 2–dependent ERK signaling in specific brain subregions suppresses behaviors associated with anxiety and fear and opposes the effects of G protein–biased signaling. Overall, our findings highlight the importance of noncanonical β-arrestin–dependent GPCR signaling in the regulation of these interrelated emotions.

Poststroke optogenetic stimulations can promote functional recovery. However, the circuit mechanisms underlying recovery remain unclear. Elucidating key neural circuits involved in recovery will be invaluable for translating neuromodulation strategies after stroke. Here, we used optogenetic functional magnetic resonance imaging to map brain-wide neural circuit dynamics after stroke in mice treated with and without optogenetic excitatory neuronal stimulations in the ipsilesional primary motor cortex (iM1). We identified key sensorimotor circuits affected by stroke. iM1 stimulation treatment restored activation of the ipsilesional corticothalamic and corticocortical circuits, and the extent of activation was correlated with functional recovery. Furthermore, stimulated mice exhibited higher expression of axonal growth–associated protein 43 in the ipsilesional thalamus and showed increased Synaptophysin+/channelrhodopsin+ presynaptic axonal terminals in the corticothalamic circuit. Selective stimulation of the corticothalamic circuit was sufficient to improve functional recovery. Together, these findings suggest early involvement of corticothalamic circuit as an important mediator of poststroke recovery.

Background As a legal drug, alcohol is commonly abused and it is estimated that 17 million adults in the United States suffer from alcohol use disorder. Heavy alcoholics can experience withdrawal symptoms including anxiety and mechanical allodynia that can facilitate relapse. The molecular mechanisms underlying this phenomenon are not well understood, which stifles development of new therapeutics. Here we investigate whether delta opioid receptors (DORs) play an active role in alcohol withdrawal-induced mechanical allodynia (AWiMA) and if DOR agonists may provide analgesic relief from AWiMA. Methods To study AWiMA, adult male wild-type and DOR knockout C57BL/6 mice were exposed to alcohol by a voluntary drinking model or oral gavage exposure model, which we developed and validated here. We also used the DOR-selective agonist TAN-67 and antagonist naltrindole to examine the involvement of DORs in AWiMA, which was measured using a von Frey model of mechanical allodynia. Results We created a robust model of alcohol withdrawal-induced anxiety and mechanical allodynia by orally gavaging mice with 3 g/kg alcohol for three weeks. AWiMA was exacerbated and prolonged in DOR knockout mice as well as by pharmacological blockade of DORs compared to control mice. However, analgesia induced by TAN-67 was attenuated during withdrawal in alcohol-gavaged mice. Conclusions DORs appear to play a protective role in the establishment of AWiMA. Our current results indicate that DORs could be targeted to prevent or reduce the development of AWiMA during alcohol use; however, DORs may be a less suitable target to treat AWiMA during active withdrawal.

No abstract