Targeted genetic manipulation of pontine noradrenergic neuronal excitability alters thermal nociception in rats in vivo

Anthony Pickering, Louise Hickey, Patrick Howorth, Simon Thornton, Feige Tang, Bridget Lumb, Anja Teschemacher, Sergey Kasparov. University of Bristol, Brisol, United Kingdom.

Pontospinal noradrenergic neurons form part of an endogenous analgesic system. We have tested the principle that genetic manipulation of their excitability can alter nociception. We used viral vectors to express either a leak potassium channel (hKir2.1) or a light sensitive cation channel (ChR2) under the control of a catecholaminergic cell specific promoter (PRS). We expected the expression of Kir2.1 to produce a chronic inhibition of the NA neurons leading to hyperalgesia and conversely that light activation of ChR2-expressing NA neurones would produce hypoalgesia.

LC neurons transduced with hKir2.1 in slice cultures showed a barium-sensitive inward rectification and reduced spontaneous firing. Pontospinal noradrenergic neurons were retrogradely transduced in vivo by injection of an adenoviral vector into the lumbar dorsal horn (L4-5). Rats transduced with hKir2.1 showed thermal but not mechanical hyperalgesia at baseline. Similar selective augmentation of thermal hyperalgesia was seen in the CFA-inflammatory pain model after AVV-PRS-hKir2.1. In the formalin test, rats transduced with hKir2.1 showed enhanced nocifensive behaviors (both Phase I and II) and increased c-fos positive cells in the lumbar dorsal horn.

Catecholaminergic PC12 cells, transfected with ChR2, showed transient depolarisations in response to laser pulses (470nm) confirming the successful expression of the light sensitive channel. Using a lentiviral vector, pontine noradrenergic neurones were transduced in vivo with ChR2-YFP fusion. Expression of the construct was seen in the LC and A7 regions and was also found in projecting axon terminals in the dorsal and ventral horns of the spinal cord. Light activation using an optrode placed over the LC produced both transient and sustained inhibitions (depending on stimulus parameters) of heat-evoked withdrawal in anaesthetised rats.

Thus the selective genetic manipulation of the excitability of pontine noradrenergic neurons can produce both acute and chronic thermal hypo- and hyper-algesia consistent with their role in the mediation of endogenous analgesia. Therefore they may be a viable target for therapeutic genetic manipulation.