No abstract

Hypothalamic proopiomelanocortin (POMC) neurons release the endogenous opioid beta-endorphin and POMC neuron activity is inhibited by opioids, leading to the proposal that beta-endorphin acts to provide feedback inhibition. However, both intrinsic properties and synaptic inputs contribute to the regulation of POMC neurons such that attributing an autoregulatory role to opioids must include consideration of opioid receptor localization and sensitivity at both presynaptic and postsynaptic sites. In the present study, whole-cell recordings were made in POMC cells in mouse brain slices and the presynaptic and postsynaptic regulation of POMC neurons was examined using selective agonists for mu, kappa, and delta opioid receptors. Activation of mu, but not kappa or delta, receptors induced a direct postsynaptic outward current. Agonists for each of the receptors inhibited the frequency of spontaneous IPSCs. Mu and kappa, but not delta, agonists reduced the amplitude of evoked IPSCs and appeared to colocalize in a significant portion of GABAergic terminals onto POMC neurons. The presynaptic inhibition caused by the mu agonist DAMGO had an EC 50 of 80 nM, whereas the EC 50 was 350 nM when measuring the postsynaptic outward current. This differential sensitivity adds an unexpected component of opioid-dependent feedback regulation, where low levels of opioid receptor activation would likely disinhibit POMC neuron activity and higher concentrations would result in an overall inhibition. The results may help explain why it has been difficult to clearly discern the role that opioids play in the regulation of food intake and other processes involving POMC neurons.

Acute desensitization is a common property of Gi/o-coupled receptors. Recent data, however, suggest that unlike mu opioid receptors (MORs) located somato-dendritically in neurons or expressed in heterologous systems, MORs in the presynaptic compartment of neurons are resistant to acute desensitization. It is not yet clear whether this differential desensitization is a shared property of many Gi/o-coupled receptors nor whether receptors located pre- and postsynaptically in a single cell type display differential desensitization. Here, whole-cell recordings were made from proopiomelanocortin (POMC) neurons in mouse brain slices. Agonists for mu opioid, nociceptin, and GABAB receptors induced postsynaptic currents that desensitized within minutes, whereas inhibition of presynaptic transmitter release mediated by these receptors was maintained throughout agonist exposure. Expression of channelrhodopsin2 in POMC neurons allowed for light-evoked transmitter release from POMC neuron terminals which was detected by recording postsynaptic currents in downstream neurons. Light-evoked currents were inhibited throughout the application of all agonists tested. Thus, the same receptors that desensitize when expressed in the postsynaptic compartment of POMC neurons resist desensitization when located in the presynaptic compartment. Pharmacologic knockdown of MORs revealed that depletion of receptor reserve does not account for presynaptic resistance to desensitization. In ~25% of recordings with GABAB agonist application, presynaptic GABAB receptors desensitized suggesting that resistance to desensitization is not due to an intrinsic property of the terminals themselves. Altogether the results indicate that a variety of presynaptic receptors can continue to function after their postsynaptic counterparts desensitize and suggest that a compartment-specific modification may confer resistance to desensitization.

Agonist binding to the mu opioid receptor (MOR) results in conformational changes that allow recruitment of G-proteins, activation of downstream effectors and eventual desensitization and internalization, all of which could affect receptor mobility. The present study employed single particle tracking (SPT) of quantum dot labeled FLAG-tagged MORs to examine shifts in MOR mobility after agonist binding. FLAG-MORs on the plasma membrane were in both mobile and immobile states under basal conditions. Activation of FLAG-MORs with DAMGO caused an acute increase in the fraction of mobile MORs, and free portions of mobile tracks were partially dependent on interactions with G-proteins. In contrast, 10-minute exposure to DAMGO or morphine increased the fraction of immobile FLAG-MORs. While the decrease in mobility with prolonged DAMGO exposure corresponded to an increase in colocalization with clathrin, the increase in colocalization was present in both mobile and immobile FLAG-MORs. Thus, no single mobility state of the receptor accounted for colocalization with clathrin. These findings demonstrate that SPT can be used to track agonist-dependent changes in MOR mobility over time, but that the mobility states observed likely arise from a diverse set of interactions and will be most informative when examined in concert with particular downstream effectors.