Investigate the preganglionic neurons in the intermediolaterol nucleus (IML) that innervate the postganglionic neurons innervating interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT).We have used Adeno-associated virus (AAV) for monosynaptic retrograde tracing and Pseudorabies virus (PRV) for transsynaptic retrograde tracing. However, this experiment is monosynaptic circuit mapping where we combine monosynaptic retrograde tracing and transsynaptic retrograde tracing to only identify the neurons that directly innervate the sympathetic nervous system.In this experiment, we injected 2 new viruses: 1) AAV6-FLEX-TVA-P2A-eGFP-2A-oG) and 2) G-Deleted Rabies-mCherry in the iBAT in two separate minor survival surgeries; we aimed to investigate which preganglionic neurons innervate the postganglionic neurons that innervate the interscapular brown adipose tissue. This was done by rabies guided monosynaptic circuit mapping, which labels exclusively monosynaptic input neurons.We injected one cre-dependent helper AAV that expresses TVA (= receptor for avian sarcoma leucosis virus glycoprotein EnvA) and oG (optimized glycoprotein) AAV6-FLEX-TVA-P2A-eGFP-2A-oG) into the iBAT of cre driver mice (TH-ires-cre). After sufficient viral expression (4 weeks) mice received another iBAT injection with the modified rabies virus G-Deleted Rabies-mCherry that lacks glycoprotein gene (commercially available from Salk Institute GT3 Core) and thus cannot be further propagated into upstream neurons in the absence of TVA and oG expression. TVA and oG expression are required for rabies propagation and infection of upstream neurons and thus retrograde and transsynaptic transport of the mCherry labeled EnvA would only occur in upstream neurons with monosynaptic inputs. Primary infected neurons would be co-labeled with green TVA eGFP and red EnvA mCherry, while monosynaptic input neurons would only show red EnvA mCherry expression. The 1st viral injections were performed in BSL1, and the 2nd viral injections were performed in BSL2 and all animals were then maintained in BSL2 housing until euthanasia followed by perfusion (10 days post-injection of the 2nd virus).Mouse model: TH-ires-creInjection site: iBATMouse numbers: 2 males and 2 females.The injection dose for the 1st virus: around (5.92E^10 GC\/ml)*12.5 ul = 7.4E^8 GC, and the volume would be 12.5 ul.The injection dose for the 2nd virus was (4.89E^7 GC\/ml)*12.5 ul = 6.1E^5 GC, and the volume would be around 12.5 ul.We monitor the body weight of mice every day as we consider an excess weight loss (>20%) as a sign of sickness due to the viral infection. In this case, we would perfuse the animals as soon as we detect the loss of body weight. We do not expect the mice to die throughout the experiment, but only expect the IML labeling. We do the weight monitor because when we do similar experiments in the mice's CNS, we monitor their weight every day.