Constitutive activity of dopamine receptor type 1 (D1R) increases CaV2.2 currents in PFC neurons.

Alterations in dopamine receptor type 1 (D1R) density are associated with cognitive deficits of aging and schizophrenia. In the prefrontal cortex (PFC), D1R plays a critical role in the regulation of working memory, which is impaired in these cognitive deficit states, but the cellular events triggered by changes in D1R expression remain unknown. A previous report demonstrated that interaction between voltage-gated calcium channel type 2.2 (CaV2.2) and D1R stimulates CaV2.2 postsynaptic surface location in medial PFC pyramidal neurons. Here, we show that in addition to the occurrence of the physical receptor-channel interaction, constitutive D1R activity mediates up-regulation of functional CaV2.2 surface density. We performed patch-clamp experiments on transfected HEK293T cells and wild-type C57BL/6 mouse brain slices, as well as imaging experiments and cAMP measurements. We found that D1R coexpression led to ∼60% increase in CaV2.2 currents in HEK293T cells. This effect was occluded by preincubation with a D1/D5R inverse agonist, chlorpromazine, and by replacing D1R with a D1R mutant lacking constitutive activity. Moreover, D1R-induced increase in CaV2.2 currents required basally active Gs protein, as well as D1R-CaV2.2 interaction. In mice, intraperitoneal administration of chlorpromazine reduced native CaV currents' sensitivity to ω-conotoxin-GVIA and their size by ∼49% in layer V/VI pyramidal neurons from medial PFC, indicating a selective effect on CaV2.2. Additionally, we found that reducing D1/D5R constitutive activity correlates with a decrease in the agonist-induced D1/D5R inhibitory effect on native CaV currents. Our results could be interpreted as a stimulatory effect of D1R constitutive activity on the number of CaV2.2 channels available for dopamine-mediated modulation. Our results contribute to the understanding of the physiological role of D1R constitutive activity and may explain the noncanonical postsynaptic distribution of functional CaV2.2 in PFC neurons.

Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango.

Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.