Glutamate-like immunoreactivity in ascending spinofugal afferents to the rat periaqueductal grey

The midbrain periaqueductal gray is a key structure for the mediation of an integrated defence behaviour. Although a prominent role for glutamate in PAG mechanisms is supported by both behavioural and morphological studies, whether PAG afferents conveying somatosensory information constitute a source of glutamatergic input to the PAG remains unknown. Here, we have compared the projection pattern of orthogradely-labelled spinoannular fibres with the distribution of glutamate-like immunoreactivity in the PAG at the light microscopic level. Transaxonal labelling was observed throughout the whole rostrocaudal axis of the PAG except for the dorsolateral regions. Cell-processes and terminal-reminiscent puncta were strongly immunoreactive in all PAG regions, including the dorsolateral areas. To ascertain whether glutamate-immunoreactive puncta observed at light microscopy indeed constituted axon terminals of the spinoannular system, glutamate-like immunoreactivity was assessed in orthogradely-labelled synaptic terminals using a post-embedding immunogold procedure for electron microscopy. Quantitative analysis of gold particle densities revealed over twice as strong an immunoreactivity in anatomically-identified spinoannular axon terminals as in dendrites postsynaptic to them, perikarya and inhibitory Gray II synapses, as well as an over 5-fold heavier immunolabelling than in glial profiles. These findings reveal that glutamate is accumulated in synaptic terminals of the spinoannular system, supporting a neurotransmitter role for this acidic amino acid in spinofugal afferents to the PAG.