Transsynaptic viral tools in neural circuit analysis: from anatomical mapping to functional interrogation

The application of transsynaptic viruses has transformed neural circuit analysis, enabling increasingly precise mapping of neuronal connectivity. These approaches can be broadly divided into polysynaptic and monosynaptic strategies. Polysynaptic retrograde tracers based on pseudorabies virus, including the classically selected Bartha strain and genetically engineered retrograde variants have provided important insights into multisynaptic circuit organization in rodents. For polysynaptic anterograde tracing, herpes simplex virus strain H129 and its derivatives have been used to map output pathways, with later genetic modifications improving detectability and experimental control. Over the past few years, growing attention has shifted toward monosynaptic tracing based on engineered rabies virus platforms used in combination with adeno-associated virus helper systems, which enable genetically restricted spread to direct presynaptic partners. Viral tracing is increasingly integrated with functional approaches, including calcium and voltage imaging, optogenetics, chemogenetics, and recombinase-based genetic strategies, thereby extending circuit analysis from anatomical mapping to causal interrogation of the function of defined neuronal populations. Looking forward, improved control of polysynaptic spread, together with large transgene capacity, suitability for tracing hierarchical circuit architecture, and the ability to incorporate functional readouts, is expected to revive interest in multisynaptic tracing.