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Cre-dependent hM3Dq-mCherry DREADD expression in the zona incerta and lateral hypothalamus of RXFP3-Cre mice

Addgene #44361-AAV5

Data Submitted By: Brandon Richards and Christina Perry
Submission Date: February 21, 2023
Publication Date: March 14, 2023 (modified May 16, 2023)
DOI: https://doi.org/10.57733/addgene.50ojhh
Funding: NHMRC-ARC Dementia Research Development Fellowship 1107144
Abstract: RXFP3-Cre transgenic male mice received bilateral intracranial injections of either pAAV-hSyn-DIO-hM3D(Gq)-mCherry (AAV5) DREADD virus (200 nl; 5 nl/sec; n = 32) or pAAV-hSyn-DIO-mCherry (AAV2) control virus (200 nl; 5 nl/sec, n = 29) in the medial zona incerta (ZIm). After 6 weeks, mice underwent behavioural testing, where all animals received injections of clozapine-N-oxide (CNO; 3 mg/kg, i.p) when required. Injection of CNO resulted in increased locomotor behaviour in animals harbouring the hM3Dq-mCherry DREADD compared to animals with the control mCherry virus. During tissue processing, the mCherry reporter was immunoamplified, and fluorescent photomicrographs were taken to observe viral spread. We observed that the AAV5 DREADD exhibited greater spread from the ZIm injection site than the AAV2 control virus; however, both the AAV5 DREADD and AAV2 control virus consistently spread beyond the boundaries of the ZIm into rostral zona incerta and the anterodorsal area of the lateral hypothalamus.
Citation: Cre-dependent hM3Dq-mCherry DREADD expression in the zona incerta and lateral hypothalamus of RXFP3-Cre mice. Richards B, Perry C 2023. Addgene Report, https://doi.org/10.57733/addgene.50ojhh

**Caption:** Representative photomicrograph of pAAV-hSyn-DIO-hM3D(Gq)-mCherry (AAV5) injection site in the ZI and LH. Bregma 1.46 mm. Scale bar = 500 μm. Image taken on a Zeiss Axio Imager M2 with epi-fluorescent Colibri 7.7 LED illumination with a NeoFluar 10x/0.45 (WD = 2.1 mm) lens using The Stereo Investigator (MBF Bioscience, USA) imaging software.
**Image attribution:** Brandon Richards

This report is made available under the Creative Commons Attribution 4.0 International License.

Vector Used: pAAV-hSyn-DIO-hM3D(Gq)-mCherry
Addgene #44361-AAV5

Virus & Injection

Virus

Virus Name: pAAV-hSyn-DIO-hM3D(Gq)-mCherry
Serotype: AAV5
Source: Addgene
Catalog Number: 44361-AAV5
Virus Lot Number: v14933
Promoter: Synapsin
Cargo Type: Chemogenetics
Injection Titer / Dose: 1.3 x 10^13 vg/mL
Injection Volume: 200 nl
Injection Rate: 5 nl/sec
Injector Material: Glass capillary tubes (0.53 mm inner diameter; 3-000-203G/X; Drummond Scientific Company, USA) were pulled into needles by a Narishige PE-22 micropipette puller (Narishige Group, Japan) set to 70 ºC. Pulled needles were inserted onto a Nanoject III AutoNanoliter Injector (3-000-207; Drummond Scientific Company, USA) mounted onto the arm of a stereotaxic frame (Kopf Instruments, USA).
Coatings: N/A
Injection Time: After injection completion, the micropipette was left in place for 3 minutes. It was then raised 0.05mm dorsal to the injection site and left in place for a further minute, then removed completely.
Injection Site / Route: medial zona incerta, Neuronames ID: 438
Additional Details: The injection was aimed at the medial zona incerta (coordinates from bregma: A/P: -1.5 mm, M/L: ± 1.0 mm, D/V: -5.0 mm).

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Other Details

Species: Mouse
Strain or Cell Line: Tg(Rxfp3-cre)RS38Gsat/Mmucd
Source: Nathaniel Heintz, Ph.D., The Rockefeller University, GENSAT and Charles Gerfen, Ph.D., National Institutes of Health, National Institute of Mental Health
Stock Number: 036667-UCD
Other Information: The RXFP3-Cre founder strain (Tg(Rxfp3-cre)RS38Gsat) had a mixed FVB/N and Crl:CD1(ICR) background (acquired from MMRRC/Gensat, stock number 036667UCD, RRID: MMRRC_036667-UCD). In these mice, a bacterial artificial chromosome (BAC) clone (RP23-220A13) was inserted immediately after the start ATG codon of the Rxfp3 gene (chromosome 15) to create an Rxfp3-Cre transgene. The regulatory sequences of the BAC clone enabled Cre-recombinase expression in RXFP3+ cells. Hemizygous male and female mice were paired to produce homozygous Cre+/+ offspring. Homozygous Cre+/+ offspring were inbred in all successive generations; males from these litters were used for all behavioural experiments.
Age at Injection: 7-10 weeks
Sex: Male
Time After Injection: 6 weeks
Detection Method: IHC
Assay & Results: RXFP3-Cre mice received bilateral injections of the Cre-dependent hM3D(Gq) DREADD (n = 32) into the medial zona incerta (ZIm). This was to selectively target RXFP3-expressing cells in the ZIm for chemogenetic activation. Fluorescent immunoamplification of the mCherry reporter was performed to visualise viral expression. Spread was not confined to the ZIm; the mCherry reporter was additionally observed in the anterodorsal area of the lateral hypothalamus and the rostral zona incerta, more so than the AAV2-mCherry control virus (see linked report - https://datahub.addgene.org/aav/4kv1xp).
Target Cell Transduction: N/A
Functional Testing: Mice expressing the hM3D(Gq) DREADD were injected with clozapine-N-oxide (CNO; Advanced Molecular Technologies Pty Ltd; Australia, AMTA056-CO16; 34233-69-7; 3 mg/kg; i.p.) to selectively activate RXFP3+ cells in the LH/ZI during behavioural experiments. Chemogenetically activating RXFP3+ LH/ZI cells increased general locomotor behaviour compared to controls.
Other Information: This report is made available under the Creative Commons Attribution 4.0 International License.