Key pointsr Spinally-projecting neurons of the rostral ventrolateral medulla (RVLM) determine sympathetic outflow to different territories of the body.r Previous studies suggest the existence of RVLM neurons with distinct functional classes, such as neurons that target sympathetic nerves bound for functionally-similar tissue types (e.g. muscle vasculature). The existence of RVLM neurons with more general actions had not been critically tested.r Using viral tracing, we show that a significant minority of RVLM neurons send axon collaterals to disparate spinal segments (T 2 and T 10 ).r Furthermore, optogenetic activation of sympathetic premotor neurons projecting to lumbar spinal segments also produced activation of sympathetic nerves from rostral spinal segments that innervate functionally diverse tissues (heart and forelimb muscle).r These findings suggest the existence of individual RVLM neurons for which the axons branch to drive sympathetic preganglionic neurons of more than one functional class and may be able to produce global changes in sympathetic activity. AbstractWe investigate the extent of spinal axon collateralization of rat rostral ventrolateral medulla (RVLM) sympathetic premotor neurons and its functional consequences. In anatomical

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Sympathetic preganglionic neurons are functionally specialised, each belonging to one of a limited number of functional classes (e.g. muscle vasoconstrictor, cutaneous vasoconstrictor, sudomotor). How far this functional specificity applies to the premotor neurons that drive them is uncertain. We devised an optogenetic test for the existence of premotor neurons that are collateralised to drive more than one functional class of sympathetic neuron.Bulbospinal neurons were transduced by a retrogradely transported viral vector (canine adenovirus) encoding Cre recombinase (CAV‐CMV‐Cre; provided by Eric Kremer, Montpellier). This was injected around sympathetic preganglionic neurons at the T12‐L1 spinal levels. At the same time, a Cre‐activated adeno‐associated virus (AAV) encoding Channelrhodopsin‐2 (ChR2) and the fluorescent label mCherry (AAV1.EF1.dflox.hChR2(H134R)‐mCherry.WPRE.hGH; provided by Deisseroth, Stanford via UPenn) was injected into the left RVLM. Only neurons transduced by both viral vectors should then express the ChR2 and mCherry. Spinal injections were made at T12‐L1 because they were considered unlikely to overlap with the preganglionic supply to the heart. The bulbospinal presynaptic terminals infected with CAV‐Cre were thus presumably on preganglionic neurons that supplied end organs such as hindlimb muscle, but not the heart.Terminal functional experiments under urethane anaesthesia were performed 2–8 weeks after the viral injections. Activity was simultaneously recorded from the left cardiac and lumbar sympathetic nerves, whilst pulses of blue laser light (473 nm, 10–12 mW, 20 ms) were flashed onto the left RVLM through a 100 μm diameter fibre‐optic probe.In about one‐third of experiments, we were able to show functional activation of sympathetic nerves that was time‐locked to the flashed laser light. In all successful experiments, we found cardiac sympathetic activation as well as lumbar sympathetic activation (peak response latencies 135–150 ms and 215–230 ms, respectively). Subsequent fluorescence microscopy on brainstem sections confirmed the presence of small numbers of mCherry expressing bulbospinal neurons in those rats.This study demonstrates that at least some bulbospinal neurons send excitatory synaptic inputs to preganglionic neurons of more than one functional type. It also demonstrates the feasibility of using anatomically‐based optogenetic transduction as a test for functional collateralisation in central pathways.Support or Funding InformationAustralian Research Council DP13014661