Retrograde viral vector-mediated inhibition of pontospinal noradrenergic neurons causes hyperalgesia in rats.

Pontospinal noradrenergic neurons form a component of an endogenous analgesic system and represent a potential therapeutic target. We tested the principle that genetic manipulation of their excitability can alter nociception using an adenoviral vector (AVV-PRS-hKir(2.1)) containing a catecholaminergic-selective promoter (PRS) to retrogradely transduce and inhibit the noradrenergic neurons projecting to the lumbar dorsal horn through the expression of a potassium channel (hKir(2.1)). Expression of hKir(2.1) in catecholaminergic PC12 cells hyperpolarized the membrane potential and produced a barium-sensitive inward rectification. LC neurons transduced by AVV-PRS-hKir(2.1) in slice cultures also showed barium-sensitive inward rectification and reduced spontaneous firing rate (median 0.2 Hz; n = 19 vs control 1.0 Hz; n = 18, p < 0.05). Pontospinal noradrenergic neurons were retrogradely transduced in vivo by injection of AVV into the lumbar dorsal horn (L4-5). Rats transduced with AVV-PRS-hKir(2.1) showed thermal but not mechanical hyperalgesia. Similar selective augmentation of thermal hyperalgesia was seen in the CFA-inflammatory pain model after AVV-PRS-hKir(2.1). In the formalin test, rats transduced with hKir(2.1) showed enhanced nocifensive behaviors (both Phase I and II, p < 0.05, n = 11/group) and increased c-Fos-positive cells in the lumbar dorsal horn. Transduction with AVV-PRS-hKir(2.1) before spared nerve injury produced no change in tactile or cold allodynia. Thus, the selective genetic inhibition of approximately 150 pontospinal noradrenergic neurons produces a modality-specific thermal hyperalgesia, increased nocifensive behaviors, and spinal c-Fos expression in the formalin test, but not in the spared nerve injury model of neuropathic pain, indicating that these neurons exert a selective tonic restraining influence on in vivo nociception.

Retrograde adenoviral vector targeting of nociresponsive pontospinal noradrenergic neurons in the rat in vivo.

The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4-L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 +/- 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4-L5. These neurons were immunopositive for dopamine beta-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception.