Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals

Fenno, Lief E.; Ramakrishnan, Charu; Kim, Yoon Seok; Evans, K; Lo, Maisie; Vesuna, Sam; Inoue, Masatoshi; Cheung, Kathy Y.M.; Yuen, Elle; Pichamoorthy, Nandini; Hong, Alice S. O.; Deisseroth, Karl

doi:10.1016/j.neuron.2020.06.003

Cited by 129 publications

(114 citation statements)

References 105 publications

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“…To access the different types of VTA neurons, we used newly developed INTRSECT2.0 viral vectors ( Fenno et al, 2020 ), which under the regulation of Cre and Flp recombinases (together or separately) encoded fluorescent reporter molecules (mCherry, eYFP, or BFP), GCaMP, or ChR2. We used these different viral vectors separately or as a cocktail for VTA cell-type-specific transduction in vglut2-Cre/vgat-Flp mice and validated the VTA cell-specific targeting of the viral vectors by a series of anatomical evaluations and ex vivo recordings.…”

Section: Discussionmentioning

confidence: 99%

“…A third recombinase under the control of the TH promoter would be necessary to gain genetic access to these two cell types. Although recently developed INTRSECT2.0 vectors allow neuronal access based on three genetic characteristics ( Fenno et al, 2020 ), further research will be necessary to genetically dissect VTA neurons based on more than two genetic characteristics.…”

Section: Discussionmentioning

confidence: 99%

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Distinct Signaling by Ventral Tegmental Area Glutamate, GABA, and Combinatorial Glutamate-GABA Neurons in Motivated Behavior

et al. 2020

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SUMMARY Ventral tegmental area (VTA) neurons play roles in reward and aversion. We recently discovered that the VTA has neurons that co-transmit glutamate and GABA (glutamate-GABA co-transmitting neurons), transmit glutamate without GABA (glutamate-transmitting neurons), or transmit GABA without glutamate (GABA-transmitting neurons). However, the functions of these VTA cell types in motivated behavior are unclear. To identify the functions of these VTA cell types, we combine recombinase mouse lines with INTRSECT2.0 vectors to selectively target these neurons. We find that VTA cell types have unique signaling patterns for reward, aversion, and learned cues. Whereas VTA glutamate-transmitting neurons signal cues predicting reward, VTA GABA-transmitting neurons signal cues predicting the absence of reward, and glutamate-GABA co-transmitting neurons signal rewarding and aversive outcomes without signaling learned cues related to those outcomes. Thus, we demonstrate that genetically defined subclasses of VTA glutamate and GABA neurons signal different aspects of motivated behavior.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Distinct Signaling by Ventral Tegmental Area Glutamate, GABA, and Combinatorial Glutamate-GABA Neurons in Motivated Behavior

et al. 2020

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“…Therefore, V2a neurons, at least those located in the Gi nucleus, likely represent a divergent population of excitatory reticular neurons. Addressing the function and endogenous activity of non-V2a excitatory neurons with a dedicated intersectional viral strategy ( 51 ) will be needed to further dissect the functional specialization in the complex brainstem RF. Furthermore, our experiments are dedicated to V2a neurons located in the rostral medulla, i.e., in the Gi and adjacent nuclei.…”

Section: Discussionmentioning

confidence: 99%

Deep imaging in the brainstem reveals functional heterogeneity in V2a neurons controlling locomotion

et al. 2020

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V2a neurons are a genetically defined cell class that forms a major excitatory descending pathway from the brainstem reticular formation to the spinal cord. Their activation has been linked to the termination of locomotor activity based on broad optogenetic manipulations. However, because of the difficulties involved in accessing brainstem structures for in vivo cell type–specific recordings, V2a neuron function has never been directly observed during natural behaviors. Here, we imaged the activity of V2a neurons using micro-endoscopy in freely moving mice. We find that as many as half of the V2a neurons are excited at locomotion arrest and with low reliability. Other V2a neurons are inhibited at locomotor arrests and/or activated during other behaviors such as locomotion initiation or stationary grooming. Our results establish that V2a neurons not only drive stops as suggested by bulk optogenetics but also are stratified into subpopulations that likely contribute to diverse motor patterns.

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“…In our experiments, we utilized AAVs possessing a neuron-specific promoter to limit the expression of sensors to neurons. This cell-type specific genetic labelling could be further refined using either transgenic animals or viruses to express tools such as Cre and Flp recombinases in cells defined by multiple molecular features 27,28 . This approach would also allow multiple biosensors to be expressed in distinct neuronal populations, allowing multiple biochemical activities to be tracked simultaneously, in distinct cell types.…”

Section: Discussionmentioning

confidence: 99%

Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

Jones-Tabah

Martin

Tanny

et al. 2021

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Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional “bulk” analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.

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Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals

Cited by 129 publications

References 105 publications

Distinct Signaling by Ventral Tegmental Area Glutamate, GABA, and Combinatorial Glutamate-GABA Neurons in Motivated Behavior

Distinct Signaling by Ventral Tegmental Area Glutamate, GABA, and Combinatorial Glutamate-GABA Neurons in Motivated Behavior

Deep imaging in the brainstem reveals functional heterogeneity in V2a neurons controlling locomotion

Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

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