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The development of powerful molecular reagents for biology have propelled our understanding of neural circuit functionality. Precisely expressing these tools in well-defined cellular sub-populations has generally been limited to single-component cellular definitions (e.g. neurons defined by a single gene or projection). Intersectional expression approaches combine multiplexed recombinases, including Cre, Flp, and VCre to enable viral expression of molecular payloads in target populations defined by multiple parameters.
Examples of intersectional cell population definitions using recombinases. A) Cre and Flp are multiplexed to enable intersectional viral targeting of populations expressing only Cre AND Flp, Cre NOT Flp, or Flp NOT Cre. B) This strategy has been expanded to incorporate VCre in order to enable three-feature cell targeting. C) Example of viral targeting of cells expressing Cre AND Flp, here expressed through a double transgenic animal strategy (PV-2a-Cre; SOM-IRES-Flp). Image from Fenno et al., 2014.
INTRSECT
INTRSECT (intronic recombinase sites enabling combinatorial targeting) is a synthetic molecular targeting strategy that allows adeno-associated virus (AAV)-borne payloads to be expressed in cells based on a doubly-specified combination of genetic and/or anatomical-defined parameters, by placing two orthogonal recombinase (Cre and Flp) recognition sequences within synthetic introns. INTRSECT was first shown as a proof-of-concept targeting approach in 20141 (using EYFP and ChR2-EYFP as payloads). This approach has been broadly applied using multiple recombinase-expression ssitrategies to define cellular sub-populations of interest, including dual-transgenic recombinase-expressing mouse lines2-9 and combinations of transgenic recombinase-expressing animal lines and retro-grade expressing viruses delivering additional recombinases10-14.
INTRSECT works by inserting short, intronic sequences into the open reading frame (ORF) of a molecular tool and adding recombinase recognition sequences (e.g. Lox sites, FRT sites) inside of the introns (A,D). The addition of these recombinase recognition sequences enables directional control of the portion of the ORF that is sandwiched between the sites; the starting direction of these ORF fragments (which have become exons) determines the logical expression requirements of Cre and Flp (B,E). When the correct combinations of recombinases are present, the introns are excised during mRNA processing, producing a functional molecular tool (C,F).
Implementation
The following resources may be of interest for groups interesting in implementing intersectional experimental design.
The Deisseroth Lab maintains a Standard Operating Procedure with general principles for working with INTRSECT recombinases, viruses, and important controls to consider as part of experimental design.
How-to guide for the molecular design and testing of novel INTSRECT plasmids for groups interested in producing their own INTRSECT plasmids.
Online spreadsheet of Flp-recombinase-expressing transgenic mouse and rat lines.
Plasmids
In addition to EYFP and ChR2-EYFP, a large number of additional, validated molecular payloads in the INTRSECT configuration have been developed, including additional fluorophores, excitatory and inhibitory opsins, genetically-encoded calcium indicators, and rabies targeting genes.
Fenno LE, Mattis J, Ramakrishnan C, Hyun M, Lee SY, He M, Tucciarone J, Selimbeyoglu A, Berndt A, Grosenick L, Zalocusky KA, Bernstein H, Swanson H, Perry C, Diester I, Boyce FM, Bass CE, Neve R, Huang ZJ, Deisseroth K. 2014. Targeting cells with single vectors using multiple-feature Boolean logic. Nat Methods 11(7):763-72. PMID:24908100
Chuhma N, Mingote S, Yetnikoff L, Kalmbach A, Ma T, Ztaou S, Sienna AC, Tepler S, Poulin JF, Ansorge M, Awatramani R, Kang UJ, Rayport S. 2018. Dopamine neuron glutamate cotransmission evokes a delayed excitation in lateral dorsal striatal cholinergic interneurons. Elife. 7. pii: e39786. PMID:30295607
Cummings KA, Clem RL. 2020. Prefrontal somatostatin interneurons encode fear memory. Nat Neurosci. 23(1):61-74. PMID:31844314
Hafner G, Witte M, Guy J, Subhashini N, Fenno LE, Ramakrishnan C, Kim YS, Deisseroth K, Callaway EM, Oberhuber M, Conzelmann KK, Staiger JF. 2019. Mapping Brain-Wide Afferent Inputs of Parvalbumin-Expressing GABAergic Neurons in Barrel Cortex Reveals Local and Long-Range Circuit Motifs. Cell Rep. 28(13):3450-3461. PMID:31553913
Mingote S, Amsellem A, Kempf A, Rayport S, Chuhma N. 2020. Dopamine-glutamate neuron projections to the nucleus accumbens medial shell and behavioral switching. Neurochemistry International 129:104482. PMID:31170424
Poulin JF, Caronia G, Hofer C, Cui Q, Helm B, Ramakrishnan C, Chan CS, Dombeck DA, Deisseroth K, Awatramani R. 2018. Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat Neurosci. 21(9):1260-1271. PMID:30104732
Rovira-Esteban L, Gunduz-Cinar O, Bukalo O, Limoges A, Brockway E, Müller K, Fenno L, Kim YS, Ramakrishnan C, Andrási T, Deisseroth K, Holmes A, Hájos N. 2019. Excitation of diverse classes of cholecystokinin interneurons in the basal amygdala facilitates fear extinction. eNeuro. 6(6). PMID:31636080
Wick ZC, Tetzlaff MR, Krook-Magnuson E. 2019. A novel population of long-range inhibitory neurons. bioRxiv:554360
Yu K, Ahrens S, Zhang X, Schiff H, Ramakrishnan C, Fenno L, Deisseroth K, Zhao F, Luo MH, Gong L, He M, Zhou P, Paninski L, Li B. 2017. The central amygdala controls learning in the lateral amygdala. Nat Neurosci. 20(12):1680-1685. PMID:29184202
Gao ZR, Chen WZ, Liu MZ, Chen XJ, Wan L, Zhang XY, Yuan L, Lin JK, Wang M, Zhou L, Xu XH, Sun YG. 2019. Tac1-Expressing Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Article Tac1-Expressing Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Descending Regulation. Neuron 101(1):45-59. PMID:30554781
Lazaridis I, Tzortzi O, Weglage M, Märtin A, Xuan Y, Parent M, Johansson Y, Fuzik J, Fürth D, Fenno LE, Ramakrishnan C, Silberberg G, Deisseroth K, Carlén M, Meletis K. 2019. A hypothalamus-habenula circuit controls aversion. Mol. Psychiatry 24(9):1351-1368. PMID:30755721
Mandelbaum G, Taranda J, Haynes TM, Hochbaum DR, Huang KW, Hyun M, Umadevi Venkataraju K, Straub C, Wang W, Robertson K, Osten P, Sabatini BL. 2019. Distinct Cortical-Thalamic-Striatal Circuits through the Parafascicular Nucleus. Neuron 102(3):636-652. PMID:30905392
Marcinkiewcz CA, Mazzone CM, D'Agostino G, Halladay LR, Hardaway JA, DiBerto JF, Navarro M, Burnham N, Cristiano C, Dorrier CE, Tipton GJ, Ramakrishnan C, Kozicz T, Deisseroth K, Thiele TE, McElligott ZA, Holmes A, Heisler LK, Kash TL. 2016. Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala. Nature 537(7618):97-101. PMID:27556938
