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

Abstract

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.

Figure 1

Retrograde targeting of brainstem noradrenergic neurons using spinal injection of AVV. a: Schematic of experimental approach. AVV-PRS-EGFP is injected into the dorsal horn of the lumbar spinal cord. The AVV is taken up by axon terminals of the pontospinal noradrenergic neurons and retrogradely transported. EGFP is expressed under the control of the catecholaminergic selective promoter PRS to enable visualization of the neuronal anatomy. b: The location and depth of the injection site is indicated by hexon protein-immunoreactivity (Cy3 fluorescence) in the spinal cord (1 day after unilateral AVV-injection). c: Overlaid brightfield and fluorescence images shows the spread of the AAV to be limited to the dorsal horn. Inset shows Rexed's laminae, red line indicates the injection trajectory. Scale bar = 100 μm.

Figure 2

Retrograde labeling of pontospinal neurons in A5, LC, and A7. Retrogradely labeled pontine neurons following transduction of the L4-5 dorsal horn with AVV-PRS-EGFP. Fluorescent neurons are seen in locus coeruleus (a), A5 (b), and A7 (c) regions. Overlaid brightfield and fluorescence images (a–c) with inset overlaid fluorescence confocal stacks. d: Distribution of pontospinal neurons projecting to L4–5 dorsal horn. Following administration of AVV-PRS-EGFP to the lumbar dorsal horn an average of 145 ± 12 (n = 14) retrogradely labeled neurons were found in the pons. The majority of the cell somata were found in the locus coeruleus with the remainder in A5 and A7 clusters. (Data shown mean ± SEM). 4V, 4th ventricle, 7n, facial tract. Scale bars = 1,000 μm in a,c; 500 μm in b; 80 μm in all insets.

Figure 3

Retrogradely labeled neurons are catecholaminergic. Confocal stack images showing (a) EGFP-positive retrogradely labeled neurons in rostral A7 region, (b) DBH-ir showing the cluster of A7 neurons (Cy3 red fluorescence), and (c) overlay of confocal stack images confirming all EGFP-positive neurons are DBH-positive (d) darkfield image with overlaid fluorescence showing relative position of A7 neurons (bregma −8.4 mm). m5, motor root of the trigeminal nerve, mcp, middle cerebellar peduncle. Magenta-green copy available as Supplementary Figure 1. Scale bars = 80 μm in a–c; 100 μm in d.

Figure 4

Factors influencing efficiency of retrograde labeling. a: The titer of AVV-PRS-EGFP injected to the spinal cord was varied from 3–1,000 × 10⁸ TU/mL (n = 3 in each group, except 300 where n = 14). Efficient retrograde labeling required higher viral titers (>100 × 10⁸ TU/mL). However, at the highest AVV titer (1,000 × 10⁸ TU/mL) the numbers of EGFP-positive neurons fell sharply. b: Time-course of retrograde transduction by AVV-PRS-EGFP was examined by varying the duration of survival following spinal AVV injection. Significant numbers of fluorescent retrogradely labeled pontine neurons were first seen at day 2 after AVV injection and there was a steady increase in the numbers until day 7, when a plateau was reached (n = 3 for each group except day 7 [n = 14] and day 14 [n = 8]). (Means ± SEM, one-way ANOVA with Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.0001.)

Figure 5

Locus coeruleus neurons with bilateral projections to the spinal cord. Ipsi- (AVV-PRS-EGFP) and contra- (AVV-PRS-mRFP) lateral injections of AVV and were made into the dorsal horn of L4/L5 to examine the topography of the projection of the pontospinal NA neurons. The colocalization of fluorophores (a) EGFP and (b) mRFP indicated neurons which projected bilaterally (white arrows, 4% of total) from the locus coeruleus to the spinal cord ((c), overlay). Magenta-green copy available as Supplementary Figure 2. Scale bars = 50 μm.

Figure 6

Demonstration of fine processes. Confocal stack images of a sagittal section showing an EGFP-positive neuron in the ventral pole of the LC (a) native EGFP fluorescence (b) GFP immunoreactivity (Cy3 labeling) produced an enhanced visualization of the fine dendritic and axonal processes of the neuron (c) overlay image; inset shows position of neuron within LC (DBH-ir AMCA, blue). Magenta-green copy available as Supplementary Figure 3. Scale bars = 80 μm, inset 100 μm.

Figure 7

Axonal labeling in spinal cord. In the spinal cord, terminal labeling was revealed using anti-GFP IHC (Cy3 fluorophore, red). a: Lumbar dorsal horn showing a predominance in the superficial dorsal horn. b: Longitudinal sections (plane indicated by lines on inset) revealed axons projecting in the lateral and ventral funiculi (white arrows). Bifurcations were occasionally observed projecting into the gray matter of the spinal cord. Scale bars = 80 μm, inset 20 μm.

Figure 8

Pontospinal NA neurons can be double-labeled from the ventral posterolateral thalamic nucleus. Animals received microinjections of AVV-EGFP to the dorsal horn at L4/L5 and red fluorescent beads (4 × 100 nL) targeted to the thalamic VPL. a: Representative VPL injection site (overlaid fluorescence and darkfield image) and schematic (adapted from Paxinos and Watson, 2005) showing injection track (dashed line). b: Within the LC (shown overlaid on darkfield image) there were numerous labeled neurons identified retrogradely from spinal cord (EGFP) and/or thalamus (red beads). c–e: Confocal stack images of the LC (from boxed area in b) showing examples of retrogradely labeled EGFP-positive and red beaded neurons (arrows), with two neurons showing colocalization (e, white arrows). Magenta-green copy available as Supplementary Figure 4. VPL, ventral posterolateral nucleus; LC, locus coeruleus; CB, cerebellum; 4V, 4th ventricle. Scale bars = 500 μm in a,b; 25 μm in c–e.

Figure 9

Activation of pontine lumbospinal NA neurons during the formalin test. a: Assessment of the nociceptive behavioral response to formalin (red squares) or control saline (blue circles) injected to the hindpaw in rats transfected with spinal AVV-PRS-EGFP. The response was quantified by counting the number of flinches and foot lifts each minute (every 2 minutes for the first 10 minutes, then every 5 minutes for the remainder of the test). Transfected animals showed typical biphasic behavioral response to the formalin injection. b: Pontine-lumbospinal NA neurons express c-fos following the formalin test. After the completion of the formalin test pontine tissue was processed to reveal c-fos-ir and DBH-ir. (i) A5 neurons revealed by the DBH stain (AMCA, blue). (ii) Annotated figure indicating location of cells (blue box, bregma −10.32 mm; Paxinos and Watson, 2005). (iii) One of the A5 neurons is retrogradely labeled (EGFP). (iv) Several neurons show nuclear c-fos-ir (Cy3, red) and from the overlay. (v) It is seen that the pontospinal neuron and several other DBH-positive neurons (white arrows), are c-fos-positive. 7n, facial nerve tract, SO, superior olive. c: Distribution of pontine-lumbospinal NA neurons expressing c-fos following the formalin test. Although the majority of c-fos-positive neurons were located in LC the proportional distribution was relatively even across the cell groups (A5, 21%, LC, 16%, and A7, 26%, n = 8). Scale bars = 100 μm.