GoKawiilAnimals
All animal procedures were performed in accordance with guidelines set forth and protocols approved by the UCSD Institutional Animal Care and Use Committee and the US National Institutes of Health, as well as by the Government of Upper Bavaria, Germany (animal protocols 55.2-1-54-2532-168-2014, 55.2-1-54-2532-19-2015, 55.2-2532.Vet_02-20-05 and 55.2-2532.Vet_02-19-83). R6/2 mice16 transgenic for the 5′ end of the human huntingtin gene were obtained from Jackson Laboratories (stock no. 002810) and maintained by crossing R6/2 males to F1 C57BL/6–CBA females. Knock-in zQ175DN19,20 mice were obtained from Jackson Laboratories (stock no. 029928) and maintained on a C57BL/6 background. The presence of the transgene or knock-in was verified by PCR with the following primers: R6/2: forward, 5′CCGCTCAGGTTCTGCTTTTA-3′, reverse, 5′-TGGAAGGACTTGAGGGACTC-3′. zQ175DN: forward, 5′- GCGGGCTTATACCCCTACAG-3′, reverse, 5′-TCCAGGACAGCCAGAGCTAC-3′. CAG repeat length was determined by Laragen for all experimental groups. Spontaneous behavioural experiments on the wheel were performed at the Max Planck Institute for Biological Intelligence, and motorized ladder experiments were performed at the UCSD. Separate batches of R6/2 mice were used for these two sets of experiments, and the CAG repeat lengths were different between these two groups (202 ± 13 and 157 ± 6 for the Max Planck Institute and UCSD, respectively, mean ± s.d.), which led to different speeds of disease progression. Therefore, the stages of disease progression were matched across these batches of mice by monitoring their body weights (Extended Data Figs. 1a and 5b). Specifically, for R6/2 mice at the Max Planck Institute, early, middle and late stages were defined as postnatal days 49–56, 57–69 and 70–84, respectively. For R6/2 mice at the UCSD, early, middle and late stages corresponded to postnatal days 40–47, 48–55 and 56–65, respectively. Movement metrics were not used to define stages, avoiding circular logic. CAG repeat length for zQ175DN mice was 165 ± 5. Mice were group housed in cages with standard bedding in a temperature-controlled room (approximately 21 °C) with a reversed 12-h light–12-h dark cycle. Mice were allowed ad libitum access to food and water. Both male and female mice were used for all experiments.
Surgery
Surgical procedures were performed as previously described26,41,42. In brief, 3.5-week-old R6/2 mice or 5-month-old zQ175DN mice were anaesthetized with an intraperitoneal injection of ketamine–xylazine (130 and 8 mg kg−1 body weight, respectively) and a low dose of isoflurane (0.5% with constant flow rate of 1 l min−1 at 0.1 bar). After reaching a deep plane of anaesthesia, enrofloxacin (10 mg kg−1) and dexamethasone (5 mg kg−1) were injected subcutaneously to prevent infection and brain swelling, respectively.
For imaging experiments, a craniotomy (4 mm in diameter) was performed, as previously described26,41,42, over the right caudal forelimb area of M1 centred at 0.5 mm anterior and 1.5 mm lateral from bregma. For imaging S1 (0 mm anterior and 2 mm lateral) and V1 (3 mm posterior and 3 mm lateral) cortices, a 5-mm craniotomy spanning S1 and V1 was performed. For imaging and manipulating cortical inhibitory neurons, viruses (calcium sensors: AAV9-hSyn-FLEX-jGCaMP7f or AAV2/1-hSyn-FLEX-GCaMP6f, titre of approximately 1012 vg ml−1; opsin or control: AAV5-Syn-FLEX-rc[ChR-tdT] or AAV2/1-CAG-FLEX-tdT, titre of approximately 1013 vg ml−1; Addgene) were injected into the caudal forelimb area of M1 (or S1 or V1) using a beveled glass pipette (inner diameter of approximately 12–25 µm). Each injection consisted of an approximately 200 nl volume centred at a depth of approximately 400 µm below the pial surface. Three injections, separated by at least 500 µm horizontally, were performed in each craniotomy. For imaging CStr neurons, retrograde virus (rgAAV-hSyn-jGCaMP8s, titre of approximately 1013 infecting units per ml; Addgene) was injected into the dorsolateral striatum at 0.5 mm anterior and 3.17 mm lateral from bregma. Two injections, each consisting of an approximately 300 nl volume were performed at a 22° angle and at 2.2 mm and 2.0 mm below the pial surface, respectively. After virus injections, the glass pipette was left in place for 5 min to avoid backflow. Following injections, a round coverslip (VWR) was implanted into the craniotomy and affixed to the skull using histoacryl glue (B.Braun) and dental acrylic cement.
For longitudinal optogenetic stimulation experiments, viruses (AAV5-Syn-FLEX-rc[ChR-tdT] or AAV2/1-CAG-FLEX-tdT, titre of approximately 1013 vg ml−1; Addgene) were injected at two sites per hemisphere through small burr holes. Each injection consisted of an approximately 200 nl volume. Injection sites were at 0.5 anterior, ±1.5 lateral and 2.0 anterior, ±1.3 lateral from bregma. Fibre optic cannulas (Doric; core diameter of 600 µm and 0.22 NA diffuser tip) were implanted at a 15° angle onto the cortical surface of both hemispheres at 1.0 anterior, ± 1.0 lateral from bregma.
A custom-built head bar was glued and cemented to the skull to allow stable head fixation. An analgesic (buprenorphine (0.1 mg kg−1) or carprofen (5 mg kg−1)) was injected approximately 1 h before the end of the surgery to manage postoperative pain. Following surgery, mice were administered daily with Baytril, dexamethasone and analgesic for up to 3 days to manage postoperative infection, swelling and pain, respectively.
Behaviour
Handling and training
At the age of 5 weeks (R6/2) or 6 months (zQ175DN), mice were handled on 4 consecutive days for 10 min until they were familiarized with the trainer and routinely ran from hand to hand. In the subsequent behavioural task training, mice got adjusted to the experimental setup and head fixation. Training sessions (30 min each, 2 and 4 consecutive days for ladder and wheel, respectively) were performed in the dark with an IR light source for the camera. Thus, mice were never trained for more than several weeks, minimizing the possibility that the motor cortex disengages due to long-term training43,44.
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