TITLE: TRIO tracing
AUTHORS: Hongwei Dong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> TRIO (Tracing the Relationship of Inputs and Outputs) experiments systematically inject complementary viruses into different target brain regions to monosynaptically label inputs to a specific projection neuron population. TRIO experiments use 3 different viruses (AAVretro-Ef1a-Cre, AAV8-hSyn-FLEX- TVA-P2A-GFP-2A-oG helper virus, and EnvA pseudotyped G-deleted Rabies mCherry (EnvA RVdG-4mCherry).</div></div>

[STEPS]
?. [Day 1 (after habituation)]
Instruments are washed with soap, wiped down with alcohol and autoclaved for the first surgery. Instruments are wiped down with alcohol and sterilized via a glass bead sterilizer in between surgeries.
?. [Day 1 (after habituation)]
Animal is deeply anesthetized. 4% lidocaine gel is applied to the ear bars of the stereotaxic frame, and the animal is mounted to the stereotax on top of a homeothermic blanket
?. [Day 1 (after habituation)]
Ketoprofen (5 mg/kg) or buprenorphine-SR (1 mg/kg) is administered subcutaneously
?. [Day 1 (after habituation)]
Bland ophthalmic ointment is placed on the eyes
?. [Day 1 (after habituation)]
The scalp is prepared with three alternating betadine swabs and alcohol wipes
?. [Day 1 (after habituation)]
A midline incision approximately 1.5 cm is made above the scalp with a sterile scalpel blade, the underlying periosteum is dissected using blunt dissection techniques, and the skull is cleaned using sterile saline
?. [Day 1 (after habituation)]
Bregma is marked under a surgery microscope and a dental drill is used to carefully drill a small hole on the bone over the desired brain nuclei (positions determined from x, y, z coordinates of the Allen Reference Atlas for each region of interest)
?. [Day 1 (after habituation)]
A glass micropipette (tip diameter 10-20 µm) filled with AAVretro-Ef1a-Cre is placed stereotaxically through the hole into the desired brain nucleus. AAVretro-Ef1a-Cre is iontophoretically injected by applying a positive current (5 μA, 7 seconds on/off intervals) for 5 mins, before the glass micropipette is removed. Following AAVretro-Ef1a-Cre injection, 500nl of AAV8-hSyn-FLEX- TVA-P2A-GFP-2A-oG helper virus is filled into a micropipette and pressure-injected into a different target brain region using a Nanoject II injector.
?. [Day 1 (after habituation)]
To avoid backflush, micropipettes are left in situ for an additional 10 minutes following injection
?. [Day 1 (after habituation)]
After tracer infusions, the skin incision (~1.5 cm) is closed using Nylon sutures
?. [Day 1 (after habituation)]
A nystatin-neomycin sulfate-thiostrepton-triamcinolone acetonide ointment is applied to the wound to provide anti-inflammatory, antipruritic, antifungal, and antibacterial protection
?. [Day 1 (after habituation)]
The animal is then released from the stereotaxic frame and put back to the host cage on a warm pad (one animal per cage)
?. [Day 1 (after habituation)]
The animal is closely observed until it fully recovers from anesthesia (i.e., achieves sternal recumbancy and is independently mobile) before it is returned into the host vivarium
?. [Day 14 (2 weeks after AAV injection)]
Instruments are washed with soap, wiped down with alcohol and autoclaved for the first surgery. Instruments are wiped down with alcohol and sterilized via 10% bleach and a glass bead sterilizer in between surgeries.
?. [Day 14 (2 weeks after AAV injection)]
Animal is deeply anesthetized. 4% lidocaine gel is applied to the ear bars of the stereotaxic frame, and the animal is mounted to the stereotax on top of a homeothermic blanket
?. [Day 14 (2 weeks after AAV injection)]
Ketoprofen (5 mg/kg) or buprenorphine-SR (1 mg/kg) is administered subcutaneously
?. [Day 14 (2 weeks after AAV injection)]
Bland ophthalmic ointment is placed on the eyes
?. [Day 14 (2 weeks after AAV injection)]
The scalp is prepared with three alternating betadine swabs and alcohol wipes
?. [Day 14 (2 weeks after AAV injection)]
A midline incision approximately 1.5 cm is made above the scalp with a sterile scalpel blade, the underlying periosteum is dissected using blunt dissection techniques, and the skull is cleaned using sterile saline
?. [Day 14 (2 weeks after AAV injection)]
Bregma is marked under a surgery microscope and a dental drill is used to carefully drill a small hole on the bone over the desired brain nuclei (positions determined from x, y, z coordinates of the Allen Reference Atlas for each region of interest)
?. [Day 14 (2 weeks after AAV injection)]
Within the BSL2 biosafety cabinet, a glass micropipette (tip diameter 10-20 µm) attached to a Nanoject II injector is filled with EnvA RVdG-4mCherry  and placed stereotaxically through the hole into the desired brain nucleus. 200nl of EnvA RVdG-4mCherry is pressure-injected using the Nanoject II injector.
?. [Day 14 (2 weeks after AAV injection)]
To avoid backflush, micropipettes are left in situ for an additional 10 minutes following injection
?. [Day 14 (2 weeks after AAV injection)]
After tracer infusions, the skin incision (~1.5 cm) is closed using Nylon sutures
?. [Day 14 (2 weeks after AAV injection)]
A nystatin-neomycin sulfate-thiostrepton-triamcinolone acetonide ointment is applied to the wound to provide anti-inflammatory, antipruritic, antifungal, and antibacterial protection
?. [Day 14 (2 weeks after AAV injection)]
The animal is then released from the stereotaxic frame and put back to the host cage on a warm pad (one animal per cage)
?. [Day 14 (2 weeks after AAV injection)]
The animal is closely observed until it fully recovers from anesthesia (i.e., achieves sternal recumbancy and is independently mobile) before it is returned to a BSL2 room within the host vivarium
?. [Day 14 (2 weeks after AAV injection)]
Following all rabies surgeries, experimental surfaces and tools are sprayed down with 10% bleach solution and left for 15 mins of contact time before wiping up with ethanol.
?. [Day 21 (one week after rabies injection)]
TRIO mice are transcardially perfused with saline and 4% PFA by rabies vaccinated personnel within a BSL 2 biosafety cabinet. Brains are dissected and removed from the skull and post-fixed within 4% PFA for 1-2 days.
?. [Day 14 (2 weeks after AAV injection)]
Animals that received AAV injections undergo surgical injections of EnvA RV dG-4mCherry. All surgeons and study personnel are vaccinated against rabies virus. Aliquot of rabies virus is thawed from -80°C storage and kept on ice. During surgery, rabies virus is kept in a BSL2-certified biosafety cabinet until needed.