Molecular recording of calcium signals via calcium-dependent proximity labeling.

Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, calcium transients play a central role in regulating neurotransmitter release and synaptic plasticity. However, it is challenging to capture the molecular events associated with these localized and ephemeral calcium signals. Here we present an engineered biotin ligase that generates permanent molecular traces in a calcium-dependent manner. The enzyme, calcium-dependent BioID (Cal-ID), biotinylates nearby proteins within minutes in response to elevated local calcium levels. The biotinylated proteins can be identified via mass spectrometry and visualized using microscopy. In neurons, Cal-ID labeling is triggered by neuronal activity, leading to prominent protein biotinylation that enables transcription-independent activity labeling in the brain. In summary, Cal-ID produces a biochemical record of calcium signals and neuronal activity with high spatial resolution and molecular specificity.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near-Infrared Light-Triggered Release and Optical Sensing.

Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell-based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin-14 (SST) can be rapidly released under near-infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 µm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

GIRK currents in VTA dopamine neurons control the sensitivity of mice to cocaine-induced locomotor sensitization.

GABABR-dependent activation of G protein-gated inwardly rectifying potassium channels (GIRK or KIR3) provides a well-known source of inhibition in the brain, but the details on how this important inhibitory pathway affects neural circuits are lacking. We used sorting nexin 27 (SNX27), an endosomal adaptor protein that associates with GIRK2c and GIRK3 subunits, to probe the role of GIRK channels in reward circuits. A conditional knockout of SNX27 in both substantia nigra pars compacta and ventral tegmental area (VTA) dopamine neurons leads to markedly smaller GABABR- and dopamine D2R-activated GIRK currents, as well as to suprasensitivity to cocaine-induced locomotor sensitization. Expression of the SNX27-insensitive GIRK2a subunit in SNX27-deficient VTA dopamine neurons restored GIRK currents and GABABR-dependent inhibition of spike firing, while also resetting the mouse's sensitivity to cocaine-dependent sensitization. These results establish a link between slow inhibition mediated by GIRK channels in VTA dopamine neurons and cocaine addiction, revealing a therapeutic target for treating addiction.

Dopamine neuron morphology and output are differentially controlled by mTORC1 and mTORC2.

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties, and whether they have similar or distinct functions is unknown. Here, we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. Disruption of both mTOR complexes leads to pronounced deficits in dopamine release demonstrating the importance of balanced mTORC1 and mTORC2 signaling for dopaminergic function.

Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.