Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

Summary

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

Abstract

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with naïve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

Introduction

The role of particular miRNAs in nervous system functioning has recently been challenged by lentiviral injection in several studies. MiRNAs have been found to be crucial for maintaining and re-shaping synapse structure ¹, synaptogenesis ² and synapse remodeling and maintenance ³. These studies strongly suggest that miRNAs are engaged, via multileveled regulatory effects in both the shaping up and in maintaining the main output of the nervous system, cognitive function. Stereotactic injection of lentivirus particles into specific regions in the rodent brain enables searching for alterations in synapse morphology and neuronal act....

Protocol

1. Lentivirus Preparation

1. Grow HEK-293FT cells to 90% confluence.
2. On the day of transfection change cell medium to serum-free DMEM supplemented with 1 mM glutamine and 50 mg/ml penicillin-streptomycin.
3. Co-transfect the cells with a pLKO.1-Puro vector and with plasmids coding for the delta R8.2 and VSV-G moieties and the miRNA of interest, using 10 μl of 1 mg/ml polyethylenimine as a carrier ⁹.
4. Collect packaged lentiviruses at 24 hr and 48 hr post-transfection, filter through 0.45 μm filter.
5. Concentrate the lentiviruses using ultracentrifugation in 70,000 x g, for 2 hr and 15 °C, aliquot and store....

Results

Injection of 0.5 μl lentivirus into the CA1 region in the mouse hippocampus, with the flow rate indicated in the protocol section yields an infected sphere of about 1 mm in the rostral-caudal axis, and about 0.5 mm in the medial-lateral and the anterior-posterior axes (Figure 3).

Figure 1. Bregma point and injection apparatus. (a).......

Discussion

Stereotactic injection of a lentivirus is a relatively rapid method for in vivo assessment for both up- or down- regulation of different genes and miRNAs. The main alternative is a genetically engineered mouse which is a much more laborious and time consuming technique then direct lentivirus injection. In addition, the up regulation, in lentivirus injection, occurs at a specific time in the adult mouse and does not include any possibility of leakiness during development, as is most often the case in the genetica.......

Materials

Name	Company	Catalog Number	Comments
			Equipment
Rodent weigh scale	Burtons (UK)	115-455
heating pad	FIRstTechnology	DCT-25
trimming machine	Stoelting	51465
stereotact	Stoelting	51730
Scalpel and blades	Kent scientific	INS500348
Harland syringe	Hamilton	7632-01
driller	Stoelting	51449
digital pump	Harvard apparatus	704507
Water tank and platform	Stoelting	60135

			Reagents
ketamine	Vetoquinol(Lure France)	3055503
domitor	Orion pharma	107140-10
Rimadyl	Pfizer animal health	24751
moisture ointment - Synthomycine 5%	Rekah Pharmaceutical	195
histoacryl	Braun	112101
saline	Sigma Aldrich	D8662