Multiple neuromodulatory systems coupled to GPCRs share common signal transduction pathways. In the prefrontal cortex, neuronal activity is regulated by Gα-mediated signaling through receptors for norepinephrine, GABA, dopamine (D2), and acetylcholine (M2, M4). However, it remains largely unknown how individual neurons distinguish between these modulatory inputs and prevent crosstalk between similar biochemical signaling pathways. Here, we found that activation of α2Rs and GABARs selectively inhibits AMPARs and NMDARs, respectively, and that this modulation occurs at single glutamatergic synapses. In both cases, the regulation of glutamate receptors occurs via a Gα-dependent reduction in PKA activity. Our evidence suggests that all four receptors are present in individual spines with a preferential co-localization (<20 nm) of α2Rs/AMPARs and GABARs/NMDARs. Under control conditions, crosstalk between α2R- and GABAR-coupled signaling cascades is prevented by the actions of RGS4, a GTPase Activating Protein (GAP) that targets Gα). Our results provide evidence for a novel mechanism by which biochemical signaling pathways are functionally compartmentalized and highlight the role of RGS4 in regulating synaptic transmission ( Figure S5 ). In future studies, it will be interesting to determine whether other modulatory pathways (e.g., D2 dopamine receptors) obey similar compartmentalization to regulate specific glutamate receptors.

Establishment of Synaptic Microdomains for Neuromodulation

Our data show that, under control conditions, there is no crosstalk between α2R- and GABA B R-mediated modulation of glutamate receptors despite similar actions on cAMP and PKA activity. One explanation for this compartmentalization is that α2Rs and GABA B Rs are located on different dendritic spines. We excluded this possibility by (1) showing that AMPAR- and NMDAR-dependent synaptic responses evoked by stimulation of a single spine are reduced by focal co-application of guanfacine and baclofen, (2) showing that α2Rs and GABA B Rs co-localize with PSD95, and (3) finding that guanfacine and baclofen mutually occlude each other’s modulation of VGCCs. A second explanation for our data is that one or both of the actions of guanfacine and baclofen occur via distinct, non-cell-autonomous mechanisms. However, we find that loading single cells with a membrane impermeable PKA antagonist occludes the modulation of both AMPARs and NMDARs, arguing that the relevant α2Rs and GABA B Rs are localized to the recorded neuron.

B Rs are located in the same spines, and the lack of crosstalk is mediated by functional compartmentalization of signaling cascades. This latter conclusion is strongly supported by our results, which suggest a novel functional microdomain established by the limited lifetime of Gα i , whose signaling is terminated by its endogenous GTPase activity ( Arshavsky and Pugh, 1998 Arshavsky V.Y.

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et al. Assembly of a beta2-adrenergic receptor—GluR1 signalling complex for localized cAMP signalling. A third possibility is that α2Rs and GABARs are located in the same spines, and the lack of crosstalk is mediated by functional compartmentalization of signaling cascades. This latter conclusion is strongly supported by our results, which suggest a novel functional microdomain established by the limited lifetime of Gα, whose signaling is terminated by its endogenous GTPase activity (). The ability of Gαsubunits to hydrolyze GTP is strongly accelerated by Regulators of G protein Signaling (RGS) proteins (). Of this protein family, RGS4 is strongly expressed in layer 5 of the PFC (). Here, we show that blocking RGS4 activity pharmacologically (CCG50014) or by dialyzing the cells with an antibody against RGS4 impairs the selectivity of Gα-coupled neuromodulators, thus enabling crosstalk of second messenger systems and leading to a breakdown in signal specificity in dendritic spines of layer 5 pyramidal neurons ( Figure S8 ). Notably, previous computational work explicitly predicted a role for RGS4 in restricting Gαsignaling to a small microdomain (). In this model, high RGS4 activity can limit diffusion of GTP-bound Gαto <20 nm. This suggestion is supported by our proximity ligation assay data, which suggest preferential postsynaptic coupling of α2Rs/AMPARs and GABARs/NMDARs within 20 nm (). There is mounting anatomical evidence that synaptic proteins, including glutamate receptors, are organized into 70- to 80-nm clusters within the postsynaptic density (). Precedent for such structural links was shown previously for a presynaptic β2-adrenergic-AMPAR signaling complex allowing highly localized cAMP signaling in the hippocampus (). Here, we demonstrate a functional role for these nano-structures and provide a plausible biochemical mechanism for the segregation of signaling domains within a single synapse.

We note that an additional explanation for our results is that both adrenergic and GABAergic activity stimulates an unidentified non-canonical (e.g., not adenylate cyclase-mediated) signaling pathway that inhibits cross-modulation of glutamate receptors. While possible, this explanation seems unlikely given the findings that two independent methods of blocking RGS4 activity lead to cross-modulation. Thus, this alternative explanation would require the existence of an unidentified GPCR-coupled pathway that is also regulated by RGS4.

Surprisingly, we find that blocking RGS4 activity allows both guanfacine and baclofen to modulate the total current through NMDARs in addition to the Ca2+ influx. A similar result was seen when blocking PKA signaling directly with H89. This result suggests a second PKA target on the NMDAR, in addition to GluN2B S1166, such as GluN1 S897, that controls total current magnitude. We propose that modest reduction in PKA signaling (as occurs with activation of GABA B Rs) influences Ca2+ influx by selectively dephosphorylating S1166, while stronger reduction in PKA signaling (either with a pharmacological block or the increased activity of Gα i following RGS4 block) leads to decreased Ca2+ and total current by dephosphorylating multiple targets.