Projections from the periaqueductal grey to pontine noradrenergic neurones are both excitatory and inhibitory

Louise Hickey1, Emanuele Sher2, Bridget Lumb1, Anthony Pickering1. 1University of Bristol, Bristol, United Kingdom, 2Eli Lilly, Surrey, United Kingdom.

The periaqueductal grey (PAG) is a midbrain site involved in the modulation of nociception and is thought to control nociception, in part, by engaging pontospinal noradrenergic (NA) neurones. Synaptic connections between the PAG and noradrenergic cell groups have been demonstrated (Bajic et al., 2001), however, the neurotransmitter phenotype is as yet unknown. To address this issue we have used viral vectors to anterogradely label projections from the PAG to pontine noradrenergic (NA) neurones in combination with immunocytochemistry for glutamate (vGlut1&2) and GABA (vGat) transporters.

Male Wistar rats received stereotaxic injections (under anaesthesia, ketamine 60mg.kg-1 and medetomidine 25µg.kg-1 i.p) of an adeno-associated viral vector (AAV-CMV-EGFP, 250nl) to express EGFP in PAG neurones. After 8 days animals were perfusion-fixed with 4% formalin and the tissue was processed to reveal Dopamine ß-hydroxylase (DBH), green fluorescent protein (EGFP) and either VGlut1&2 or VGat immuno-reactivity. Injection sites and terminal labelling were visualised using confocal imaging and 3D reconstruction software (Volocity 4, Improvision).

Injections of AAV-CMV-EGFP produced bright labelling of neurons within the PAG columns and dense, distinctive anterograde labelling in the pons. EGFP containing terminals showing co-localisation with Vglut or Vgat were identified in all pontine NA cell groups indicating excitatory or inhibitory amino acid (EAA or IAA) projections from the PAG. Quantification of terminals closely apposing NA cell bodies showed that A6 and A7 both had a dense innervation of EAA and IAA profiles although the neurons of the A7 received more inputs per soma. In addition, the A5 territory received proportionally more IAA than EAA terminals from the ventrolateral (VL) PAG.

In conclusion, we show that PAG projections to A5, A6 and A7 have terminals closely apposed to NA neurones. These terminals contain vesicular transporters for EAA and IAA indicating that there are both excitatory and inhibitory phenotypes. The A6 and A7 regions are densely innervated by glutamatergic and GABAergic profiles whereas the A5 region receives a strong inhibitory input from the VL PAG, which may be responsible for the depressor response that occurs on activation of VL PAG. These data provide further evidence for specific and differential control of pontine NA neurones by the PAG.