We demonstrate a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase II␣ in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity.heterooligomerization ͉ SKF83959 ͉ calcium signaling ͉ calcium/calmodulin-dependent protein kinase II␣ D iverse roles for each of the five dopamine receptors (D1-D5) have been shown to be initiated primarily through stimulation or inhibition of adenylyl cyclase (AC) via G s /olf or G i /o signaling proteins, respectively (1). There have been reports, however, of a D1-like receptor in brain that is coupled to G q /11, stimulating phospholipase C (PLC) and intracellular calcium release (2-5). Activation of this G q /11-coupled D1-like receptor by specific receptor agonists does not correlate with the ability of these same agonists to activate AC (4), suggesting that the G q /11-coupled D1-like receptor is a molecular entity distinct from the G s /olfcoupled D1 receptor.Molecular identification of the G q /11-coupled D1-like receptor has proven elusive because D1 receptor coupling to PLC has not been demonstrated in a variety of cell types in which the D1 receptor was expressed. We had postulated that G q /11 activation by D1 receptor agonists in brain could occur by concurrent activation of the D1 receptor and the D2 receptor (6). We have shown that heterologously coexpressed D1 and D2 dopamine receptors formed heterooligomers (7) and that coactivation of these receptors resulted in a PLC-dependent rise in intracellular calcium (6). We also demonstrated that D1 and D2 receptors could be coimmunoprecipitated from striatal membranes (6). These results suggested the possibility of a unique signaling complex in brain composed of PLC-coupled D1-D2 receptor heterooligomers.In this work we report the presence of such a D1-D2 dopamine receptor signaling complex in striatum that is coupled to rapid G q /11 signaling on activation of both receptors and which can be defined by a unique pharmacology. The complex was more readily detected in older mice and could modulate levels of calcium/calmodulin-dependent protein kinase II␣ (CaMKII␣) in the nucleus accumbens, indicating a potential role for the D...

Collections of cells called engrams are thought to represent memories. Although there has been progress in identifying and manipulating single engrams, little is known about how multiple engrams interact to influence memory. In lateral amygdala (LA), neurons with increased excitability during training outcompete their neighbors for allocation to an engram. We examined whether competition based on neuronal excitability also governs the interaction between engrams. Mice received two distinct fear conditioning events separated by different intervals. LA neuron excitability was optogenetically manipulated and revealed a transient competitive process that integrates memories for events occurring closely in time (coallocating overlapping populations of neurons to both engrams) and separates memories for events occurring at distal times (disallocating nonoverlapping populations to each engram).

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the G q pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.The dopamine D1 receptor has been shown to signal via G s and G olf proteins to stimulate adenylyl cyclase (1), and there have been reports of calcium signaling by D1 receptor-mediated phosphatidylinositol hydrolysis by phospholipase C (PLC), 1 presumably through G q coupling. Although PLC activity was not detected in studies on the D1 receptor expressed in COS7 cells (2) or in Chinese hamster ovary and baby hamster kidney cells (3), it has been shown that activation of the D1 receptor in brain tissue (4, 5) or in Xenopus oocytes injected with mRNA taken from striatum (6) resulted in phosphatidylinositol turnover. This discrepancy suggests the involvement of a factor in D1 receptor-mediated PLC activity that is present in native tissues but absent in heterologous expression systems. An examination of the above studies where PLC activation was observed indicates that, although D1 receptor-selective antagonists blocked phosphatidylinositol turnover demonstrating the involvement of the D1 receptor, the amount of agonist used to elicit the response was greater than 100 M, a concentration at which even selective ligands may bind other receptors. For example, SKF 81297, a highly D1-selective agonist, has a K i for the D1 receptor of ϳ1 nM but has a K i for the D2 receptor of ϳ900 nM (7). This analysis suggests the possibility that the D1 agonistmediated PLC stimulation by high concentrations of drugs observed in physiological systems may have resulted from the coincident activation of another receptor.There are considerable data indicating that the dopamine D1 and D2 receptors are functionally linked. For example, behavioral studies have shown that co-stimulation of the D1 receptor is essential for D2 agonists to produce maximal locomotor stim...

Memories are thought to be sparsely encoded in neuronal networks, but little is known about why a given neuron is recruited or allocated to a particular memory trace. Previous research shows that in the lateral amygdala (LA), neurons with increased CREB are selectively recruited to a fear memory trace. CREB is a ubiquitous transcription factor implicated in many cellular processes. Which process mediates neuronal memory allocation? One hypothesis is that CREB increases neuronal excitability to bias neuronal recruitment, although this has not been shown experimentally. Here we use several methods to increase neuronal excitability and show this both biases recruitment into the memory trace and enhances memory formation. Moreover, artificial activation of these neurons alone is a sufficient retrieval cue for fear memory expression, showing that these neurons are critical components of the memory trace. These results indicate that neuronal memory allocation is based on relative neuronal excitability immediately before training.