Coordinated activity of a central pathway drives associative opioid analgesic tolerance.

Opioid analgesic tolerance, a root cause of opioid overdose and misuse, can develop through an associative learning. Despite intensive research, the locus and central pathway subserving the associative opioid analgesic tolerance (AOAT) remains unclear. Using a combination of chemo/optogenetic manipulation with calcium imaging and slice physiology, here we identify neuronal ensembles in a hierarchically organized pathway essential for AOAT. The association of morphine-induced analgesia with an environmental condition drives glutamatergic signaling from ventral hippocampus (vHPC) to dorsomedial prefrontal cortex (dmPFC) cholecystokininergic (CCKergic) neurons. Excitation of CCKergic neurons, which project and release CCK to basolateral amygdala (BLA) glutamatergic neurons, relays AOAT signal through inhibition of BLA µ-opioid receptor function, thereby leading to further loss of morphine analgesic efficacy. This work provides evidence for a circuit across different brain regions distinct for opioid analgesic tolerance. The components of this pathway are potential targets to treat opioid overdose and abuse.

Combined alcohol and cannabinoid exposure leads to synergistic toxicity by affecting cerebellar Purkinje cells.

Combined use of cannabis and alcohol results in greater psychoactive toxicity than either substance alone, but the underlying central mechanisms behind this worsened outcome remain unclear. Here we show that the synergistic effect of Δ9-tetrahydrocannabinol (THC) and ethanol on motor incoordination in mice is achieved by activating presynaptic type 1 cannabinoid receptors (CB1R) and potentiating extrasynaptic glycine receptors (GlyR) within cerebellar Purkinje cells (PCs). The combination of ethanol and THC significantly reduces miniature excitatory postsynaptic current frequency in a CB1R-dependent manner, while increasing the extrasynaptic GlyR-mediated chronic chloride current, both leading to decreased PC activity. Ethanol enhances THC actions by boosting the blood-brain-barrier permeability of THC and enriching THC in the cell membrane. Di-desoxy-THC, a designed compound that specifically disrupts THC-GlyR interaction without affecting the basic functions of CB1R and GlyR, is able to restore PC function and motor coordination in mice. Our findings provide potential therapeutic strategies for overcoming the synergistic toxicity caused by combining cannabis and alcohol use.

Simvastatin impairs hippocampal synaptic plasticity and cognitive function in mice.

Lipophilic statins which are blood brain barrier (BBB) permeable are speculated to affect the cholesterol synthesis and neural functions in the central nervous system. However, whether these statins can affect cholesterol levels and synaptic plasticity in hippocampus and the in vivo consequence remain unclear. Here, we report that long-term subcutaneous treatments of simvastatin significantly impair mouse hippocampal synaptic plasticity, reflected by the attenuated long-term potentiation of field excitatory postsynaptic potentials. The simvastatin administration causes a deficiency in recognition and spatial memory but fails to affect motor ability and anxiety behaviors in the mice. Mass spectrometry imaging indicates a significant decrease in cholesterol intensity in hippocampus of the mice receiving chronic simvastatin treatments. Such effects of simvastatin are transient because drug discontinuation can restore the hippocampal cholesterol level and synaptic plasticity and the memory function. These findings may provide further clues to elucidate the mechanisms of neurological side effects, especially the brain cognitive function impairment, caused by long-term usage of BBB-permeable statins.

Structural basis of GABARAP-mediated GABAA receptor trafficking and functions on GABAergic synaptic transmission.

GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABAARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABAARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously unappreciated region in the γ2 subunit of GABAAR. We demonstrate that GABARAP functions to stabilize GABAARs via promoting its trafficking pathway instead of blocking receptor endocytosis. The GABARAPL1-γ2-GABAAR crystal structure reveals the mechanisms underlying the complex formation. We provide evidence showing that phosphorylation of γ2-GABAAR differentially modulate the receptor's binding to GABARAP and the clathrin adaptor protein AP2. Finally, we demonstrate that GABAergic synaptic currents are reduced upon specific blockage of the GABARAP-GABAAR complex formation. Collectively, our results reveal that GABARAP/GABARAPL1, but not other members of the Atg8 family proteins, specifically regulates synaptic localization of GABAARs via modulating the trafficking of the receptor.

18F-Fluorodeoxyglucose positron emission tomography/computed tomography findings in a patient with bilateral macronodular adrenal hyperplasia.

Adrenocorticotropic hormone-independent macronodular adrenal hyperplasia (AIMAH) is a rare bilateral adrenocorticotropic hormone (ACTH)-independent nodular adrenal hyperplastic disease. Most patients with AIMAH are usually asymptomatic and only a small percentage present with subclinical or apparent Cushing's syndrome caused by excessive corticosteroid secretion. Herein, we reported the case of a 51-year-old female with bilateral macronodular adrenal hyperplasia with mild fluorodeoxyglucose uptake based on PET/CT imaging findings. Her symptoms resolved after surgical resection of the right adrenal gland.

Cannabinoids Rescue Cocaine-Induced Seizures by Restoring Brain Glycine Receptor Dysfunction.

Cannabinoids are reported to rescue cocaine-induced seizures (CISs), a severe complication in cocaine users. However, the molecular targets for cannabinoid therapy of CISs remain unclear. Here, we report that the systemic administration of cannabinoids alleviates CISs in a CB1/CB2-receptor-independent manner. In HEK293 cells and cortical neurons, cocaine-induced dysfunction of the glycine receptor (GlyR) is restored by cannabinoids. Such restoration is blocked by GlyRα1S296A mutation. Consistently, the therapeutic effects of cannabinoids on CISs are also eliminated in GlyRα1S296A mutant mice. Based on molecular dynamic simulation, the hydrogen-bonding interaction between cocaine and the GlyR is weakened by cannabinoid docking. Without altering cocaine distribution across the brain, cannabinoids significantly suppress cocaine-exaggerated neuronal excitability in the prefrontal cortex (PFC) and hippocampus by rehabilitating extra-synaptic GlyR function. Microinjection of cannabinoids into the PFC and hippocampus restores cocaine-puzzled neural activity and alleviates CISs. These findings suggest that using GlyR-hypersensitive cannabinoids may represent a potential therapeutic strategy for treating CISs.

The synthetic cannabinoid dehydroxylcannabidiol restores the function of a major GABAA receptor isoform in a cell model of hyperekplexia.

The functions of the glycine receptor (GlyR) and GABAA receptor (GABAAR) are both impaired in hyperekplexia, a neurological disorder usually caused by GlyR mutations. Although emerging evidence indicates that cannabinoids can directly restore normal GlyR function, whether they affect GABAAR in hyperekplexia remains unknown. Here we show that dehydroxylcannabidiol (DH-CBD), a synthetic nonpsychoactive cannabinoid, restores the GABA- and glycine-activated currents (IGABA and IGly , respectively) in HEK293 cells coexpressing a major GABAAR isoform (α1ß2γ2) and GlyRα1 carrying a human hyperekplexia-associated mutation (GlyRα1R271Q). Using coimmunoprecipitation and FRET assays, we found that DH-CBD disrupts the protein interaction between GABAAR and GlyRα1R271Q Furthermore, a point mutation of GlyRα1, changing Ser-296 to Ala-296, which is critical for cannabinoid binding on GlyR, significantly blocked DH-CBD-induced restoration of IGABA and IGly currents. This S296A substitution also considerably attenuated DH-CBD-induced disruption of the interaction between GlyRα1R271Q and GABAAR. These findings suggest that, because it restores the functions of both GlyRα1 and GABAAR, DH-CBD may represent a potentially valuable candidate drug to manage hyperekplexia.

Human Hyperekplexic Mutations in Glycine Receptors Disinhibit the Brainstem by Hijacking GABAA Receptors.

Hyperekplexia disease is usually caused by naturally occurring point mutations in glycine receptors (GlyRs). However, the γ-aminobutyric acid type A receptor (GABAAR) seems to be also involved regarding the therapeutic basis for hyperekplexia using benzodiazepines, which target GABAARs but not GlyRs. Here, we show that the function of GABAARs was significantly impaired in the hypoglossal nucleus of hyperekplexic transgenic mice. Such impairment appeared to be mediated by interaction between GABAAR and mutant GlyR. The GABAAR dysfunction was caused only by mutant GlyR consisting of homomeric α1 subunits, which locate primarily at pre- and extra-synaptic sites. In addition, the rescue effects of diazepam were attenuated by Xli-093, which specifically blocked diazepam-induced potentiation on α5-containing GABAAR, a major form of pre- and extra-synaptic GABAAR in the brainstem. Thus, our results suggest that the pre- and extra-synaptic GABAARs could be a potential therapeutic target for hyperekplexia disease caused by GlyR mutations.

Intrathecal lentivirus-mediated RNA interference targeting nerve growth factor attenuates myocardial ischaemia-reperfusion injury in rat.

BACKGROUND: Nerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia-reperfusion injury and explored the underlying mechanisms. METHODS: In adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia-reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. RESULTS: NGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia-reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5'-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. CONCLUSIONS: Spinal NGF contributes to myocardial ischaemia-reperfusion injury by mediating nociceptive signal transmission.

Involvement of glycine receptor α1 subunits in cannabinoid-induced analgesia.

Some cannabinoids have been shown to suppress chronic pain by targeting glycine receptors (GlyRs). Although cannabinoid potentiation of α3 GlyRs is thought to contribute to cannabinoid-induced analgesia, the role of cannabinoid potentiation of α1 GlyRs in cannabinoid suppression of chronic pain remains unclear. Here we report that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, significantly suppresses chronic inflammatory pain caused by noxious heat stimulation. This effect may involve spinal α1 GlyRs since the expression level of α1 subunits in the spinal cord is positively correlated with CFA-induced inflammatory pain and the GlyRs antagonist strychnine blocks the DH-CBD-induced analgesia. A point-mutation of S296A in TM3 of α1 GlyRs significantly inhibits DH-CBD potentiation of glycine currents (IGly) in HEK-293 cells and neurons in lamina I-II of spinal cord slices. To explore the in vivo consequence of DH-CBD potentiation of α1 GlyRs, we generated a GlyRα1S296A knock-in mouse line. We observed that DH-CBD-induced potentiation of IGly and analgesia for inflammatory pain was absent in GlyRα1S296A knock-in mice. These findings suggest that spinal α1 GlyR is a potential target for cannabinoid analgesia in chronic inflammatory pain.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry.

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.