Afferent convergence to a shared population of interneuron AMPA receptors.

Precise alignment of pre- and postsynaptic elements optimizes the activation of glutamate receptors at excitatory synapses. Nonetheless, glutamate that diffuses out of the synaptic cleft can have actions at distant receptors, a mode of transmission called spillover. To uncover the extrasynaptic actions of glutamate, we localized AMPA receptors (AMPARs) mediating spillover transmission between climbing fibers and molecular layer interneurons in the cerebellar cortex. We found that climbing fiber spillover generates calcium transients mediated by Ca2+-permeable AMPARs at parallel fiber synapses. Spillover occludes parallel fiber synaptic currents, indicating that separate, independently regulated afferent pathways converge onto a common pool of AMPARs. Together these findings demonstrate a circuit motif wherein glutamate 'spill-in' from an unconnected afferent pathway co-opts synaptic receptors, allowing activation of postsynaptic AMPARs even when canonical glutamate release is suppressed.

Climbing Fiber-Mediated Spillover Transmission to Interneurons Is Regulated by EAAT4.

Neurotransmitter spillover is a form of communication not readily predicted by anatomic structure. In the cerebellum, glutamate spillover from climbing fibers recruits molecular layer interneurons in the absence of conventional synaptic connections. Spillover-mediated signaling is typically limited by transporters that bind and reuptake glutamate. Here, we show that patterned expression of the excitatory amino acid transporter 4 (EAAT4) in Purkinje cells regulates glutamate spillover to molecular layer interneurons. Using male and female Aldolase C-Venus knock-in mice to visualize zebrin microzones, we find larger climbing fiber-evoked spillover EPSCs in regions with low levels of EAAT4 compared with regions with high EAAT4. This difference is not explained by presynaptic glutamate release properties or postsynaptic receptor density but rather by differences in the glutamate concentration reaching receptors on interneurons. Inhibiting glutamate transport normalizes the differences between microzones, suggesting that heterogeneity in EAAT4 expression is a primary determinant of differential spillover. These results show that neuronal glutamate transporters limit extrasynaptic transmission in a non-cell-autonomous manner and provide new insight into the functional specialization of cerebellar microzones.SIGNIFICANCE STATEMENT Excitatory amino acid transporters (EAATs) help maintain the fidelity and independence of point-to-point synaptic transmission. Whereas glial transporters are critical to maintain low ambient levels of extracellular glutamate to prevent excitotoxicity, neuronal transporters have more subtle roles in shaping excitatory synaptic transmission. Here we show that the patterned expression of neuronal EAAT4 in cerebellar microzones controls glutamate spillover from cerebellar climbing fibers to nearby interneurons. These results contribute to fundamental understanding of neuronal transporter functions and specialization of cerebellar microzones.

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking.

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.

Desensitization-resistant and -sensitive GPCR-mediated inhibition of GABA release occurs by Ca2+-dependent and -independent mechanisms at a hypothalamic synapse.

Whereas the activation of Gαi/o-coupled receptors commonly results in postsynaptic responses that show acute desensitization, the presynaptic inhibition of transmitter release caused by many Gαi/o-coupled receptors is maintained during agonist exposure. However, an exception has been noted where GABAB receptor (GABABR)-mediated inhibition of inhibitory postsynaptic currents (IPSCs) recorded in mouse proopiomelanocortin (POMC) neurons exhibit acute desensitization in ∼25% of experiments. To determine whether differential effector coupling confers sensitivity to desensitization, voltage-clamp recordings were made from POMC neurons to compare the mechanism by which µ-opioid receptors (MORs) and GABABRs inhibit transmitter release. Neither MOR- nor GABABR-mediated inhibition of release relied on the activation of presynaptic K(+) channels. Both receptors maintained the ability to inhibit release in the absence of external Ca(2+) or in the presence of ionomycin-induced Ca(2+) influx, indicating that inhibition of release can occur through a Ca(2+)-independent mechanism. Replacing Ca(2+) with Sr(2+) to disrupt G-protein-mediated inhibition of release occurring directly at the release machinery did not alter MOR- or GABAB -mediated inhibition of IPSCs, suggesting that reductions in evoked release can occur through the inhibition of Ca(2+) channels. Additionally, both receptors inhibited evoked IPSCs in the presence of selective blockers of N- or P/Q-type Ca(2+) channels. Altogether, the results show that MORs and GABABRs can inhibit transmitter release through the inhibition of calcium influx and by direct actions at the release machinery. Furthermore, since both the desensitizing and nondesensitizing presynaptic receptors are similarly coupled, differential effector coupling is unlikely responsible for differential desensitization of the inhibition of release.

Direct inhibition of hypothalamic proopiomelanocortin neurons by dynorphin A is mediated by the µ-opioid receptor.

It has recently been shown that dynorphin A (Dyn A), an endogenous agonist of the κ-opioid receptor (KOR), directly inhibits proopiomelanocortin (POMC) neurons in the hypothalamus through activation of G-protein-coupled inwardly rectifying K(+) channels (GIRKs). This effect has been proposed to be mediated by the putative κ2-opioid receptor (KOR-2), and has been suggested as a possible mechanism for the orexigenic actions of KOR agonists. Using whole-cell voltage clamp recordings in brain slice preparations, the present study demonstrates that Dyn A (1 or 5 µm) induces an outward current in POMC neurons that is reversed by the highly selective µ-opioid receptor (MOR) antagonist CTAP and is absent in mice lacking MORs. Additionally, the KOR-2-selective agonist GR89696 binds MORs on POMC neurons but fails to induce an outward current. Similar to Dyn A, the KOR-selective antagonist nor-binaltorphimine (nor-BNI) lacked specificity when used at sufficiently high concentrations. Maximal concentrations of the MOR-selective agonist DAMGO induced outward currents in POMC neurons that were completely reversed by a relatively high concentration of nor-BNI. Experiments using a half-maximal concentration of DAMGO demonstrate that nor-BNI must be used at concentrations <100 nm to avoid non-specific actions of the antagonist at MORs located on POMC neurons. These data suggest that direct inhibition of POMC neurons by Dyn A is mediated through the MOR, not the KOR-2, which is consistent with previous studies demonstrating that Dyn A can act at the µ-opioid receptor (MOR) when present in high concentrations.

Multiple inhibitory G-protein-coupled receptors resist acute desensitization in the presynaptic but not postsynaptic compartments of neurons.

Acute desensitization is a common property of G(i/o)-coupled receptors. Recent data, however, suggest that, unlike µ-opioid receptors (MORs) located somatodendritically 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 G(i/o)-coupled receptors nor whether receptors located presynaptically 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 µ-opioid, nociceptin, and GABA(B) 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 GABA(B) agonist application, presynaptic GABA(B) receptors desensitized, suggesting that resistance to desensitization is not due to an intrinsic property of the terminals themselves. Together, 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.

Differential expression and sensitivity of presynaptic and postsynaptic opioid receptors regulating hypothalamic proopiomelanocortin neurons.

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.

Proopiomelanocortin expression in both GABA and glutamate neurons.

Proopiomelanocortin (POMC) neurons have been intensively studied because of their essential role in regulating energy balance and body weight. Many effects of POMC neurons can be attributed to their release of cognate neuropeptides from secretory granules in axon terminals. However, these neurons also synaptically release non-peptide neurotransmitters. The aim of this study was to settle the controversy whether there are separate populations of POMC neurons that release GABA or glutamate. Transgenic mice expressing a red fluorescent protein [Discosoma red (DsRed)] driven by Pomc neuronal regulatory elements (POMC-DsRed) were crossed to mice that expressed green fluorescent protein (gfp) in GABAergic neurons (GAD67-gfp). Approximately 40% of POMC neurons in the arcuate nucleus of the double-transgenic mice expressed the GAD67-gfp transgene. In vitro neurotransmitter release was detected using whole-cell electrophysiologic recordings in cultured GAD67-gfp-positive and GAD67-gfp-negative POMC neurons that had formed recurrent synapses (autapses). Autapses from GAD67-gfp-positive neurons were uniformly GABAergic. In contrast, autapses from the GAD67-gfp-negative POMC neurons exclusively exhibited postsynaptic currents mediated by glutamate. Together, these results indicate that there are two subpopulations of POMC neurons in the arcuate nucleus differentiated by their amino acid neurotransmitter phenotype. Whole-cell voltage-clamp recordings from POMC neurons in live brain slices indicated that GABAergic and glutamatergic POMC neurons are under similar presynaptic and postsynaptic regulation, although the GABAergic POMC neurons are smaller and have higher input resistance. GABAergic and glutamatergic POMC neurons may mediate distinct aspects of POMC neuron function, including the regulation of energy homeostasis.