INTRODUCTION: Pain is a highly prevalent and debilitating condition affecting a large proportion of the human population, highlighting the need for improved understanding of the neural circuits underlying nociceptive processing. Importantly, not all retrograde tracers provide sufficient signal quality for targeted electrophysiological recordings.

AIM(S): Here, we aimed to evaluate different retrograde labeling approaches to enable patch-clamp recordings from anatomically defined spinal projection neurons.

METHOD(S): We compared several tracers (fluorescent pink and green dyes, and Alexa Fluor 488–conjugated cholera toxin) in adult rats. We stereotaxically injected 100 nl volumes of these compounds into the brainstem nucleus lateralis parabrachialis (coordinates: AP −8.9; ML ±2.1; DV −6.8 from bregma) in adult (10-week-old) Wistar rats of both sexes. Surgery was performed under general anesthesia and followed by a recovery period of 10–14 days, after which animals were either perfused for fixation and imaging or killed by decapitation for patch-clamp electrophysiological recordings.

RESULTS: We observed efficient retrograde tracing from the brainstem injection site, with numerous labeled spinal neurons located in laminae I–V and X at 12–14 days post-injection. Furthermore, we successfully obtained patch-clamp recordings from these neurons and determined that red-shifted (pink) fluorescent dyes are the most suitable for this purpose, most likely due to reduced light scattering.

CONCLUSIONS: Red-shifted (pink) fluorescent dyes enable reliable retrograde labeling of spinal projection neurons from the nucleus lateralis parabrachialis and are optimal for targeted patch-clamp recordings. This approach provides a robust tool for studying defined nociceptive circuits.

FINANCIAL SUPPORT: This research was supported by the National Center for Science grant nr 2024/08/X/NZ4/00622 awarded to MK.