Cell signaling involves dynamic changes in protein oligomerization leading to the formation of different signaling complexes and modulation of activity. Spatial intensity distribution analysis (SpIDA) is an image analysis method that can directly measure oligomerization and trafficking of endogenous proteins in single cells. Here, we show the use of SpIDA to quantify dimerization/activation and surface transport of receptor protein kinases--EGF receptor and TrkB--at early stages of their transactivation by several G protein-coupled receptors (GPCRs). Transactivation occurred on the same timescale and was directly limited by GPCR activation but independent of G-protein coupling types. Early receptor protein kinase transactivation and internalization were not interdependent for all receptor pairs tested, revealing heterogeneity between groups of GPCRs. SpIDA also detected transactivation of TrkB by dopamine receptors in intact neurons. By allowing for time and space resolved quantification of protein populations with heterogeneous oligomeric states, SpIDA provides a unique approach to undertake single cell multivariate quantification of signaling processes involving changes in protein interactions, trafficking, and activity.

Although chronic pain is one of the most important medical problems facing society, there has been very limited progress in the development of novel therapies for this condition. Here, we discuss high-impact research priorities to reduce the number of people transitioning from acute to chronic intractable pain.

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.

Type 1 cannabinoid receptors (CB1Rs) are widely expressed in the vertebrate retina, but the role of endocannabinoids in vision is not fully understood. Here, we identified a novel mechanism underlying a CB1R-mediated increase in retinal ganglion cell (RGC) intrinsic excitability acting through AMPK-dependent inhibition of NKCC1 activity. Clomeleon imaging and patch clamp recordings revealed that inhibition of NKCC1 downstream of CB1R activation reduces intracellular Cl(-) levels in RGCs, hyperpolarizing the resting membrane potential. We confirmed that such hyperpolarization enhances RGC action potential firing in response to subsequent depolarization, consistent with the increased intrinsic excitability of RGCs observed with CB1R activation. Using a dot avoidance assay in freely swimming Xenopus tadpoles, we demonstrate that CB1R activation markedly improves visual contrast sensitivity under low-light conditions. These results highlight a role for endocannabinoids in vision and present a novel mechanism for cannabinoid modulation of neuronal activity through Cl(-) regulation.

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.

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We found that hyperalgesia-inducing treatment with morphine resulted in downregulation of the K(+)-Cl(-) co-transporter KCC2, impairing Cl(-) homeostasis in rat spinal lamina l neurons. Restoring the anion equilibrium potential reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor-dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signaling preserved Cl(-) homeostasis and reversed the hyperalgesia. Gene-targeted mice in which Bdnf was deleted from microglia did not develop hyperalgesia to morphine. However, neither morphine antinociception nor tolerance was affected in these mice. Our findings dissociate morphine-induced hyperalgesia from tolerance and suggest the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target for preventing hyperalgesia without affecting morphine analgesia.

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (¹⁰panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.

Although chronic pain is one of the most important medical problems facing society, there has been very limited progress in the development of novel therapies for this condition. Here, we discuss high-impact research priorities to reduce the number of people transitioning from acute to chronic intractable pain.