Whole-brain mapping of monosynaptic afferents to GABAergic neurons in the sublaterodorsal tegmental nucleus of mice

The sublaterodorsal tegmental nucleus (SLD) is a critical hub for regulating rapid eye movement (REM) sleep and muscle atonia, with its dysfunction linked to various disorders such as REM sleep behavior disorder (RBD) and cataplexy. Despite its physiological significance, the presynaptic patterns influencing SLD γ-aminobutyric acid (GABA)ergic neurons—one of the primary neuronal subtypes within the SLD—remain poorly understood. This study applied a modified rabies virus tracing system combined with a Cre/loxP-based genetic approach to map and quantify the whole-brain monosynaptic afferents to SLD GABAergic neurons in mice. In total, 139 anatomically distinct nuclei were identified as sources of direct input, with predominant projections originating from the midbrain, pons, and medulla. Ipsilateral contributions accounted for 67.99% of all traced inputs, while 32.01% were contralateral. Prominent sources included the mesencephalic reticular nucleus, superior colliculus, oral part of the pontine reticular nucleus, gigantocellular reticular nucleus, lateral hypothalamic area, and zona incerta neurons. Several nuclei displayed contralateral projection biases. Immunofluorescence staining revealed molecular diversity among input neurons, suggesting that SLD GABAergic neurons integrate signals from anatomically and functionally distinct neuronal populations. These findings provide a comprehensive anatomical framework for understanding how SLD GABAergic neurons integrate multisource inputs and offer new perspectives for investigating their involvement in regulating complex physiological functions, including sleep and motor control.