Stereotactic head holder

NSPC grafting was performed as described previously (18 (link)). Mice were mounted onto a stereotactic head holder (Kopf Instruments, Tujunga, CA, USA) under anesthesia with isoflurane (Isoba® vet; Schering-Plough Corp., NJ, USA; 5% for induction, and 2% to 3% for maintenance) in the flat skull position. In order to graft NSPCs into the hippocampus, a 5-μL 26 gauge syringe (Innovative Labor Systeme, Stuetzerbach, Germany) attached to the holder was inserted according to the following coordinates: body weight < 9 g: 0.45 × (distance from lambda to bregma) mm posterior and ± 1.2 mm lateral to bregma, 3.0 mm deep from the skull surface; body weight ≥ 9 g, 0.42 × (distance from lambda to bregma) mm posterior and ± 1.3 mm lateral to bregma, 3.2 mm deep from the skull surface. Before the needle was inserted, a small hole was drilled in the proper position according to the above coordinates. Then, 1 × 10⁵ NSPCs in 2 μL DMEM were injected very slowly over a 2-min period, with a 4-min delay prior to removal of the syringe, and 2 min were allowed for syringe removal. No immunosuppressive drugs were administered.

As the actual injections have been described earlier (Suzuki et al., 2012 (link)), we will only recapitulate the main procedures briefly. The animals, after inducing ketamine/xylazine anesthesia, were placed in a stereotactic head holder (David Kopf Instruments). The vermis and the right side of the posterior cerebellum were exposed by incision of the skin overlying the occipital bone and the neck, separation of the neck muscles and partial removal of the occipital bone. After making a small incision in the dura, a fine micromanipulator driven Hamilton needle was horizontally and under visual guidance, introduced into either the vermis of lobule VII (n = 3), paramedian lobule (n = 4) or crus 2b (n = 2). The Hamilton needle, by way of thin plastic tubing, filled with water, was connected to a motorized Hamilton syringe. A small air bubble was introduced between the injectate and the rest of the water-filled tube and was used to monitor the injection. A volume of 150 or 200 nL of a mixture of 1 part 1% CTb (low salt; List Biological Laboratories, 1% w/v in 0.2 M phosphate buffer (PB), pH 7.4) and 4 parts RABV (Ruigrok et al., 2008 (link)) was injected in every case at a depth of 300–600 μm below the surface. All animals but one (survival time of case 1080 was 50 h) were allowed to survive for 48 h, during this period no behavioral changes of the animals were noted.