Transduction of virus

Surgical transduction of the lentivirus in the A1 was performed as follows. Ketamine-xylazine was administered once intraperitoneally as described above and the temporal bone was exposed via a single 5 mm cut from in front of right ear to behind the right eye and gentle removal of the temporal chewing muscle by scraping it down off the bone for 2 mm. Three ~0.5 mm holes were created above the A1 1 mm below the temporal suture through trepanation, 1 mm apart (Figure 1G). Virus, containing Cry2olig, was injected 300 and 600 μm deep at each of the three sites. Each of the 6 injection sites received 23 nl of virus 9 times every 3 s, totaling 207 nl of virus at each site and 1,242 nl across the A1. This virus, an LV-CAG which contains the sequence: CIBN-Xm233-EOS-CCR5, did not contain a fluorescent protein and could not be stained in immunohistochemistry to show the spread (see Rieder et al., 2015, Figure 5). Lentiviruses have been shown as efficient in their role of infecting neurons in vivo in, amongst other species, rats (Naldini et al., 1996a,b). While the spread of the virus is limited, it has been shown that 200 nl injection volumes diffuse within a spherical region with a diameter around 200–600 μm (Desmaris et al., 2001; Osten et al., 2006 see Figure 13.3). Therefore, between each subject, we can assume a large coverage of the A1 down the depth of the cortical column. The vehicle control group underwent the same procedure and injected with an equal volume of a lentivirus not containing Cry2olig. The naïve control group received no surgery prior to electrode implantation (see below). After surgical injection, the muscle was gently placed over the trepanated temporal bone, and the skin was sutured. Metacam (2 mg/ml, Boehringer Ingelheim, Ingelheim am Rhein, Germany) was administered at the onset of surgery subcutaneously for a dose of 1 ml per 1 kg of bodyweight and two consecutive days peri-operation for a dose of 0.5 ml per 1 kg of bodyweight.

The virus was allowed to express for 4 weeks before auditory recording. The implantation and recording were carried out on the same day, per subject, so it was necessary to perform two surgeries to allow time for expression.