Virus-induced Gene Knockdown in the CA3 and Subsequent Spatial Relocation Test to Assess CA3-dependent Cognitive Function

Summary

Hippocampal cognitive function can be modulated by locally manipulating gene expression. The spatial object relocation test is a short and robust spatial memory test. We combined this test with virus-induced manipulation of gene expression in the CA3 region to assess the impact of the target gene in shaping cognitive performance.

Abstract

Investigating the role of a gene of interest in a specific brain region allows further understanding of brain physiology and pathophysiology. Modulation of gene expression by local injection of adeno-associated virus (AAV) has been proven to be efficient and safe. The stability and long-term expression of the AAV construct allows the use of a battery of behavioral tests to screen the animals for a region-specific involvement of the target gene in shaping performance in different behavioral domains.

The spatial object relocation (SOR) test is a hippocampal-dependent one-trial memory test based on the natural spontaneous exploratory behavior of rodents. This test gives robust information on memory function and can be easily integrated in a battery of behavioral testing for phenotype screening.

In this video-article, we provide a detailed protocol to assess the role of a particular target gene in shaping hippocampus-dependent spatial memory function. The protocol includes stereotactic AAV-induced gene transfer specifically into the mouse hippocampal CA3 region and combines this with the SOR test. Due to the variability in SOR protocols in the literature, we carefully described relevant aspects of the protocol to ensure the optimal behavioral protocol and setup selection. Also, detailed analyses of the results are described to guarantee the proper interpretation of the results.

Introduction

Dissecting the specific function of an individual gene, expressed in a defined brain region, is a key milestone to a better understanding of brain physiology and pathophysiology. One valuable approach is to investigate the behavioral consequences of local gene expression changes in the brain. Adeno-associated virus (AAV) based gene transfer has been proven to be efficient, safe and to induce long-term gene expression in the central nervous system^1–5. In rodents, the stability of AAV-induced gene expression is suitable for extensive behavioral characterization, which usually requires several sessions in different days.

Protocol

C57Bl6/N male mouse (<8 weeks old) were used for all the procedures. Animals were individually housed and kept on a 12-hr light/dark cycle (lights on at 7:00 AM), at room temperature of 23 ± 2 °C with food and water ad libitum. All experiments were conducted in accordance with European Communities Council Directive 2010/63/EU. All efforts were made to minimize animal suffering during the experiments. The protocols were approved by the committee for the Care and Use of Laboratory Animals of the Government of Upper Bavaria, Germany.

1. Stereotactic Adeno-associated Virus Injection in the Mouse CA3

1. Pull capil

Results

The functional role of DRR1 expression was studied using a combination of AAV-induced mRNA changes in the CA3 region and spatial memory assessment using the SOR test in C57Bl/6N mice ²⁸. We used an AAV containing a short hairpin RNA against DRR1 (shDRR1) to knock-down DRR1 expression and a scrambled version (shSCR) as control. Here we present (1) data showing how the SOR test conditions were previously selected, using naive C56Bl/6N mice; (2) the results of the SOR in mice injected in the CA3 with AAV-shDRR1 o...

Discussion

The SOR is an robust and valid test to investigate changes in hippocampus-dependent cognitive function. The test can be easily included in a screening battery of tests because it is short, non-aversive, and does not require food deprivation or repeated training. In the present video article, we combined the SOR test with an AAV-mediated local gene knock-down of DRR1. The results of the study showed that the SOR was efficient in detecting memory impairment induced by DRR1 knock-down locally in the CA3.

Materials

Name	Company	Catalog Number	Comments
LightCycler Capillaries	Roche	4929292001	1-5 µl glass capillar
Micropipette puller	Narishige	PC-10
Warming pad
Oxygen/Isofluran system			General anesthesia
Stereotactic system for mouse	Kofp
Cold light source	Leica/SCHOTT	KL1600LED
Micro drill: Ultimate-XL, UMXL-T + UHRXL-T	NSK	Y141440 + H269
Stereo Microscope	Leica	M80
Panthenol	Bepanthen, Bayer		Humectant, emollient and moisturizer
Lidocain	Xylocain		Local anesthesia
Povidone-Iodine (or propanol)	Betadine (or Kodan)		Antiseptic for desinfection
Meloxicam	Metacam, Heiland		Pain killer
Cotton swabs
Ethilon sutures	Ethicon	EH7500H
Y-maze	Custom made		Three arms (30 x 10 x 15 cm) at 120° from each other made of grey PVC
Objects			Glass salt shakers
AAV-shDRR1/AVV-shSCR	Gene Detect	AAV1/2-U6-DRR1 shRNA-terminator-CAG-EGFP-WPRE-BGH-polyA	DRR1 knock-down virus expression cassette
AAV-shshSCR	Gene Detect	AAV1/2-U6-scrambled shRNA-terminator-CAG-EGFP-WPRE-BGH-polyA	Control virus expression cassette
Videotracking software	ANY-maze		Any videotracking software can be used