In addition to sensory disturbances, neuropathic pain is associated with an ongoing and persistent negative affective state. This condition may be reflected as altered sensitivity to rewarding stimuli. We examined this hypothesis by testing whether the rewarding properties of morphine are altered in a rat model of neuropathic pain. Neuropathic pain was induced by chronic constriction of the common sciatic nerve. Drug reward was assessed using an unbiased, three-compartment conditioned place preference (CPP) paradigm. The rats underwent two habituation sessions beginning 6 days after surgery. Over the next 8 days, they were injected with drug or vehicle and were confined to one CPP compartment for 30 min. On the following test day, the rats had access to all three compartments for 30 min. Consistent with the literature, systemic administration of morphine dose-dependently increased the CPP in pain-naive animals. In rats with neuropathic pain, however, the dose-dependent effects of morphine were in a bell-shaped curve, with a low dose of morphine (2 mg/kg) producing a greater CPP than a higher dose of morphine (8 mg/kg). In a separate group of animals, acute administration of morphine reversed mechanical allodynia in animals with neuropathic pain at the same doses that produced a CPP. The increased potency of systemic morphine to produce a CPP in animals with neuropathic pain suggests that the motivation for opioid-induced reward is different in the two states.

Otoferlin, an integral membrane protein implicated in a late stage of exocytosis, has been reported to play a critical role in hearing although the underlying mechanisms remain elusive. However, its widespread tissue distribution infers a more ubiquitous role in synaptic vesicle trafficking. Glutamate, an excitatory neurotransmitter, is converted to its inhibitory counterpart, γ-aminobutyric acid (GABA), by L-glutamic acid decarboxylase (GAD), which exists in soluble (GAD67) and membrane-bound (GAD65) forms. For the first time, we have revealed a close association between otoferlin and GAD65 in both HEK293 and neuronal cells, including SH-SY5Y neuroblastoma and primary rat hippocampus cells, showing a direct interaction between GAD65 and otoferlin's C2 domains. In primary rat hippocampus cells, otoferlin and GAD65 co-localized in a punctate pattern within the cell body, as well as in the axon along the path of vesicular traffic. Significantly, GABA is virtually abolished in otoferlin-knockdown neuronal cells whereas otoferlin overexpression markedly increases endogenous GABA. GABA attenuation in otoferlin-knockdown primary cells is correlated with diminished L-type calcium current. This previously unknown and close correlation demonstrates that otoferlin, through GAD65, modulates GABAergic activity. The discovery of otoferlin-GAD65 functional coupling provides a new avenue for understanding the molecular mechanism by which otoferlin functions in neurological pathways.

Delta opioid receptor (DOR) agonists alleviate nociceptive behaviors in various chronic pain models, including neuropathic pain, while having minimal effect on sensory thresholds in the absence of injury. The mechanisms underlying nerve injury-induced enhancement of DOR function are unclear. We used a peripheral nerve injury (PNI) model of neuropathic pain to assess changes in the function and localization of DORs in mice and rats. Intrathecal administration of DOR agonists reversed mechanical allodynia and thermal hyperalgesia. The dose-dependent thermal antinociceptive effects of DOR agonists were shifted to the left in PNI rats. Administration of DOR agonists produced a conditioned place preference in PNI, but not in sham, animals, whereas the DOR antagonist naltrindole produced a place aversion in PNI, but not in sham, mice, suggesting the engagement of endogenous DOR activity in suppressing pain associated with the injury. GTPγS autoradiography revealed an increase in DOR function in the dorsal spinal cord, ipsilateral to PNI. Immunogold electron microscopy and in vivo fluorescent agonist assays were used to assess changes in the ultrastructural localization of DORs in the spinal dorsal horn. In shams, DORs were primarily localized within intracellular compartments. PNI significantly increased the cell surface expression of DORs within lamina IV-V dendritic profiles. Using neonatal capsaicin treatment, we identified that DOR agonist-induced thermal antinociception was mediated via receptors expressed on primary afferent sensory neurons but did not alter mechanical thresholds. These data reveal that the regulation of DORs following PNI and suggest the importance of endogenous activation of DORs in regulating chronic pain states.

Chronic pain attenuates midbrain dopamine (DA) transmission, as evidenced by a decrease in opioid-evoked DA release in the ventral striatum, suggesting that the occurrence of chronic pain impairs reward-related behaviors. However, mechanisms by which pain modifies DA transmission remain elusive. Using in vivo microdialysis and microinjection of drugs into the mesolimbic DA system, we demonstrate in mice and rats that microglial activation in the VTA compromises not only opioid-evoked release of DA, but also other DA-stimulating drugs, such as cocaine. Our data show that loss of stimulated extracellular DA is due to impaired chloride homeostasis in midbrain GABAergic interneurons. Treatment with minocycline or interfering with BDNF signaling restored chloride transport within these neurons and recovered DA-dependent reward behavior. Our findings demonstrate that a peripheral nerve injury causes activated microglia within reward circuitry that result in disruption of dopaminergic signaling and reward behavior. These results have broad implications that are not restricted to the problem of pain, but are also relevant to affective disorders associated with disruption of reward circuitry. Because chronic pain causes glial activation in areas of the CNS important for mood and affect, our findings may translate to other disorders, including anxiety and depression, that demonstrate high comorbidity with chronic pain.

Opioid dependence is accompanied by neuroplastic changes in reward circuitry leading to a negative affective state contributing to addictive behaviors and risk of relapse. The current study presents a neuroimmune mechanism through which chronic opioids disrupt the ventral tegmental area (VTA) dopaminergic circuitry that contributes to impaired reward behavior. Opioid dependence was induced in rodents by treatment with escalating doses of morphine. Microglial activation was observed in the VTA following spontaneous withdrawal from chronic morphine treatment. Opioid-induced microglial activation resulted in an increase in brain-derived neurotrophic factor (BDNF) expression and a reduction in the expression and function of the K(+)Cl(-) co-transporter KCC2 within VTA GABAergic neurons. Inhibition of microglial activation or interfering with BDNF signaling prevented the loss of Cl(-) extrusion capacity and restored the rewarding effects of cocaine in opioid-dependent animals. Consistent with a microglial-derived BDNF-induced disruption of reward, intra-VTA injection of BDNF or a KCC2 inhibitor resulted in a loss of cocaine-induced place preference in opioid-naïve animals. The loss of the extracellular Cl(-) gradient undermines GABAA-mediated inhibition, and represents a mechanism by which chronic opioid treatments can result in blunted reward circuitry. This study directly implicates microglial-derived BDNF as a negative regulator of reward in opioid-dependent states, identifying new therapeutic targets for opiate addictive behaviors.

Pain is a multidimensional experience and negative affect, or how much the pain is "bothersome", significantly impacts the sufferers' quality of life. It is well established that the κ opioid system contributes to depressive and dysphoric states, but whether this system contributes to the negative affect precipitated by the occurrence of chronic pain remains tenuous. Using a model of persistent pain, we show by quantitative real-time-PCR, florescence in situ hybridization, Western blotting and GTPgS autoradiography an upregulation of expression and the function of κ opioid receptors (KORs) and its endogenous ligand dynorphin in the mesolimbic circuitry in animals with chronic pain compared with surgical controls. Using in vivo microdialysis and microinjection of drugs into the mesolimbic dopamine system, we demonstrate that inhibiting KORs reinstates evoked dopamine release and reward-related behaviors in chronic pain animals. Chronic pain enhanced KOR agonist-induced place aversion in a sex-dependent manner. Using various place preference paradigms, we show that activation of KORs drives pain aversive states in male but not female mice. However, KOR antagonist treatment was effective in alleviating anxiogenic and depressive affective-like behaviors in both sexes. Finally, ablation of KORs from dopamine neurons using AAV-TH-cre in KOR^loxP mice prevented pain-induced aversive states as measured by place aversion assays. Our results strongly support the use of KOR antagonists as therapeutic adjuvants to alleviate the emotional, tonic-aversive component of chronic pain, which is argued to be the most significant component of the pain experience that impacts patients' quality of life.SIGNIFICANCE STATEMENT We show that KORs are sufficient to drive the tonic-aversive component of chronic pain; the emotional component of pain that is argued to significantly impact a patient's quality of life. The impact of our study is broadly relevant to affective disorders associated with disruption of reward circuitry and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response to treatment of many patients, debilitating affective disorders (other disorders including anxiety and depression that demonstrate high comorbidity with chronic pain) and substance abuse. Indeed, coexisting psychopathology increases pain intensity, pain-related disability and effectiveness of treatments (Jamison and Edwards, 2013).

Pain is a multidimensional experience and negative affect, or how much the pain is "bothersome", significantly impacts the sufferers' quality of life. It is well established that the κ opioid system contributes to depressive and dysphoric states, but whether this system contributes to the negative affect precipitated by the occurrence of chronic pain remains tenuous. Using a model of persistent pain, we show by quantitative real-time-PCR, florescence in situ hybridization, Western blotting and GTPgS autoradiography an upregulation of expression and the function of κ opioid receptors (KORs) and its endogenous ligand dynorphin in the mesolimbic circuitry in animals with chronic pain compared with surgical controls. Using in vivo microdialysis and microinjection of drugs into the mesolimbic dopamine system, we demonstrate that inhibiting KORs reinstates evoked dopamine release and reward-related behaviors in chronic pain animals. Chronic pain enhanced KOR agonist-induced place aversion in a sex-dependent manner. Using various place preference paradigms, we show that activation of KORs drives pain aversive states in male but not female mice. However, KOR antagonist treatment was effective in alleviating anxiogenic and depressive affective-like behaviors in both sexes. Finally, ablation of KORs from dopamine neurons using AAV-TH-cre in KOR^loxP mice prevented pain-induced aversive states as measured by place aversion assays. Our results strongly support the use of KOR antagonists as therapeutic adjuvants to alleviate the emotional, tonic-aversive component of chronic pain, which is argued to be the most significant component of the pain experience that impacts patients' quality of life.SIGNIFICANCE STATEMENT We show that KORs are sufficient to drive the tonic-aversive component of chronic pain; the emotional component of pain that is argued to significantly impact a patient's quality of life. The impact of our study is broadly relevant to affective disorders associated with disruption of reward circuitry and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response to treatment of many patients, debilitating affective disorders (other disorders including anxiety and depression that demonstrate high comorbidity with chronic pain) and substance abuse. Indeed, coexisting psychopathology increases pain intensity, pain-related disability and effectiveness of treatments (Jamison and Edwards, 2013).