Detailed investigations have been performed on only a small number of spinal interneurons. Earlier studies focused on analysis of input to interneurons in various pathways (see Jankowska, 1992) and, with the exception a limited number of types of interneuron, very little is known about their postsynaptic actions. We have combined electrophysiological methods and immunocytochemistry to investigate the organization of commissural interneurons in midlumbar spinal segments and the transmitter content of their axons.

Experiments were performed on three adult cats which were deeply anaesthetised (with chloralose, up to 5 mg kg^-1 h^-1 I.V., after induction with 40 mg kg^-1 I.P. of sodium pentobarbital). If any increase occurred in the heart rate or blood pressure or if the pupils dilated, an additional dose of anaesthetic was given. Neuromuscular transmission was blocked (Pavalon 0.2 mg kg^-1). Cells in laminae VII-VIII were activated monosynaptically by stimulation of muscle nerves at intensities consistent with activation of group II muscle afferent fibres or by descending fibres from the reticular formation. Stimuli were then applied within contralateral motor nuclei in lower lumbar segments to select neurons which were activated antidromically and thus to identify them as commissural interneurons. Cells were labelled intracellularly with a mixture of rhodamine dextran and Neurobiotin. At the end of experiments, animals were killed humanely and fixed by perfusion. Four neurons had well labelled axons that could be followed into the contralateral grey matter where they formed clusters of boutons in laminae VII, VIII and IX. Labelled structures were scanned with a confocal laser scanning microscope and reconstructed. Sections containing axonal processes were incubated with antibodies raised against the vesicular glutamate transporters 1 and 2 (VGLUT1 and 2), which are markers for glutamatergic terminals (Varoqui et al. 2002). Further sections were reacted with antibodies raised against molecules associated with inhibitory transmitters: glycine transporter molecule 2 (GlyT2) to reveal glycine-containing axons and glutamic acid decarboxylase (GAD) to reveal GABAergic axons.

Analysis of individual boutons indicated that two neurons reacted positively for GlyT2 and that one was positive for VGLUT2. The fourth cell was negative for all four markers. These findings provide the first firm evidence that feline commissural interneurones include both excitatory and inhibitory neurons. Since all of the neurons investigated were antidromically activated from motor nuclei, these findings also support the hypothesis that the earliest crossed inhibitory actions of group II afferents may be evoked disynaptically via commissural neurones (Arya et al. 1991).

This work was supported by the NIH.