Surgery Protocol

From UCLA Miniscope
Jump to: navigation, search

Here we will take you through the complete procedure for implanting a GRIN lens above the hippocampus for CA1 imaging.

Virus Injection

Before implanting the GRIN lens, you will need to inject a fluorescent indicator such as GCaMP6. We have generally used AAV1.Syn.GCaMP6f.WPRE.SV40 from Penn Vector (Item number AV-1-PV2822) and had great success in dorsal CA1. You may also be able to use GCaMP6 transgenic mice, but we have not tested whether any of the newer lines are bright enough to be imaged.

Basic Equipment Needed

You will only need basic surgical equipment to perform GRIN lens implantation surgeries. This includes a mouse stereotax, an isoflurane vaporizor, surgical heat pad, stereo surgical microscope, light source, dental drill, and bead sterilizer. For recommended equipment, see the master parts list.

You will the following basic tools and supplies: fine forceps, blunt forceps, fine scissors, scalpel and blade, small skull screws, drill bits, cyanoacrylate glue, dental cement, Kwik-Sil, and cortex buffer

Aspirator and Lens Holder

You will need an aspirator to remove cortex above the hippocampus. This can be build very simply with a vacuum line (or pump), a liquid trap, 1 ml syringe (with a hole to control suction), blunt needles, tubing and connectors, and hot glue.

You can easily build a GRIN lens holder by using a suction system to hold the lens in place as it is dropped into the brain for implantation. To build, start with two 1ml micropipette tips. Cut the first so that the tip is just larger than the diameter of the GRIN lens you will be using. Cut the second tip so that the end is just smaller than the size of the GRIN lens. Connect this end to a 1ml syringe and connect to a vacuum line. This will then hold the lens in place during implantation. You can then use tape to attach this holder to a stereotax. Another solution we have used is a basic drill chuck, but this is only good for large (2mm) lenses.

Skull Preparation

You must first prepare the skull for implantation. We recommend shaving the head area and sterilizing with 3 alternating betadine and ethanol scrubs. Next, remove the scalp with scissors and clean the skull with hydrogen peroxide and saline. Scrape and score the skull to increase the bond with the glue. We also detach the neck muscle from the skull in order to reduce pull on the skull and prevent muscle growth that can make the implant less stable. Finally we insert a skull screw on the opposite side of the skull to enhance the stability of the implant.

Craniotomy and Aspiration