Gene Therapy for Chronic Pain: How to Manipulate and Unravel Pain Control Circuits from the Brain? - Gene Delivery and Therapy for Neurological Disorders - page 337

Tris, pH 8; 10 mM EDTA; 0.6 % SDS; 0.25 mg/ml proteinase
K) overnight at 37 °C. Extract twice with equal volumes of phe-
nol-HCl 3 -IAA (25:24:1), remove the organic phase, and extract
twice with CHCl 3 -IAA (24:1). Transfer the aqueous phase to a
clean tube and add 2 vols of cold isopropanol. Spool the precipi-
tated DNA onto a glass Pasteur pipette, allow it to dry, and
transfer to a microfuge tube. Add 300 µl of TE buffer and resus-
pend the DNA by gently pipetting the solution with a wide-bore
pipette tip. Measure DNA concentration spectrophotometri-
cally (O.D. 260 nm; 1:50 dilution).

2. Incubate the slides in acid acetone for 5 min. Rinse with dis-
tilled water. Stain in formol-thionin for 20-30 s. Rinse with
distilled water. Dry at 37 °C. Dehydrate in xylene for 5 min
and coverslip with Eukitt.
3. At 22 days p.i., EGFP carried by the second expression cassette
of THZ (Fig. 2b ) is no longer expressed since its expression is
driven by the HCMV promoter which is only active for 2 weeks
in the brain [ 6 ]. Therefore, the green labeling in this double
immunoreaction is only due to endogenous TH expression.

5 Conclusion

The genetic approach described here consisted of using the natural
ability of a replication-defective HSV-1 vector to be retrogradely
transported in order to manipulate the DRt afferents. By coupling
this feature of HSV-1 with a cell-specifi c promoter, we could selec-
tively target noradrenergic DRt afferents and thereby study the
effect of the noradrenergic input to the DRt during chronic pain.
Using other cell-specifi c promoters combined with this strategy
will allow studying other DRt brain afferents or other pain control
circuits in the brain.
Further extending the usefulness of this approach, this vector
can also be used for optogenetic neuronal activation or inhibition
[ 19 - 21 ]. Optogenetics uses light-sensitive ion channels and
pumps, typically from the microbial opsin gene family, to control
neural activity with high spatiotemporal precision [ 22 - 24 ]. Opsin
delivery and targeting to specifi c neurons are achieved by using
viral vectors, derived from HSV-1, lentivirus, and adeno-associated
virus, which carry cell-specifi c promoters [ 23 ]. The application of
optogenetics to study pain pathways has recently received much
interest. At the periphery, studies using light delivered transder-
mally for the optical control of nociceptive fi bers provided a proof-
of-concept demonstration of the potential of optogenetics in basic
and translational pain research [ 25 - 27 ]. At the supraspinal level, it
was recently shown that optogenetic targeting of the locus coeruleus,
a major pain control area, allowed to establish that this area exerts