MAPPING INPUT-DEFINED MOTOR THALAMUS CIRCUITS USING AAV1-MEDIATED TRANSSYNAPTIC LABELING

The motor thalamus (mThal) is a key node in motor circuits, integrating inputs from basal ganglia, cerebellar and midbrain motor regions. However, how these anatomically and functionally distinct inputs are organized within mThal remains incompletely characterized. Here, we establish an anterograde transsynaptic tracing strategy to systematically map and compare input-defined mThal neuron populations. Using intracranial injections of AAV1-based tracers targeting basal ganglia output nuclei, deep cerebellar nuclei and midbrain motor nuclei, we selectively label mThal neurons receiving input from these different regions. We combine whole-brain slide-scanner imaging with automated cell body detection and three-dimensional computational reconstruction to quantify the spatial distribution of input-defined populations. In parallel, we optimize a brain clearing protocol that preserves endogenous fluorescence, enabling light-sheet imaging and three-dimensional reconstruction of intact brains. To enable simultaneous labeling of multiple input-defined populations, we combine Cre- and Flp-expressing AAV1 vectors with dual-recombinase reporter mice, allowing parallel visualization of distinct mThal populations defined by their upstream inputs. Together, this approach provides a scalable framework for multiplexed, whole-brain mapping of motor thalamic circuits.