A spinal cord pathway connecting primary afferents to the segmental sympathetic outflow system
Introduction
Sympathetic fibers entering DRGs and forming “Dogiel's arborizations” around neurons were initially described by Ramón y Cajal (1899). During normal conditions, vessel-associated sympathetic fibers are found between nerve fiber layers and around perikarya of DRG neurons (McLachlan et al., 1993, Olson, 1967). Sympathetic outflow is likely to have a minimal modulatory role on sensory function during normal conditions (Habler et al., 2000, Jänig and Kolzenburg, 1991). However, in the presence of increased levels of NGF (Olson, 1967) with increasing age or in response to nerve injury, sympathetic innervation of DRG neurons tends to increase (Ramer and Bisby, 1998). A connection between sympathetic hyperinnervation of DRGs and behavior associated with neuropathic pain has been suggested (Chung et al., 1993, Kim et al., 1996, McLachlan et al., 1993). In addition, electrophysiological experiments demonstrate a functional sympathetic–sensory coupling following nerve injury (Devor and Janig, 1981, McLachlan et al., 1993, Wall and Gutnick, 1974, Xie et al., 1995). However, whether the sympathetic hyperinnervation is linked to the genesis and/or maintenance of pain is still a matter of controversy (Baron et al., 1999, Desmeules et al., 1995, Perrot et al., 1993, Ringkamp et al., 1999). Little is known about the identity of CNS cell groups that modulate sympathetic outflow to DRGs.
The Bartha strain of pseudorabies virus (PRV-Bartha), a neurotropic alpha herpes virus, has been broadly used to study CNS areas innervating peripheral sympathetic targets (Cano et al., 2001, Jansen et al., 1997, Strack et al., 1989, Weiss and Chowdhury, 1998). PRV-Bartha accesses neurons via axon terminals, but infection may also occur through the cell body (Card et al., 1991, Chen et al., 1999). Subsequently, the virus is transported to the nucleus where it can either elicit a productive infection or enter a latent state. Replication of the virus leads to accumulation of viral particles in the cell cytoplasm and the dendritic tree. Virions become concentrated preferentially at sites of synaptic contacts (Card et al., 1993) and become specifically transferred across synapses to terminals afferent to the infected neuron. PRV-Bartha therefore offers the advantage of being spread in a strictly retrograde fashion through chains of synaptically linked neurons (Pickard et al., 2002) without loss of signaling intensity. Temporal analysis of viral progression through the CNS following inoculation provides information about the hierarchy in complex central neural circuits.
Here, we use the novel approach of intraganglionic delivery of tracers to examine retrograde spread of virus via the sympathetic system to the spinal cord and brainstem following inoculation of lumbar DRGs. We use simultaneous anterograde labeling by intraganglionic injections of cholera toxin B to provide evidence that neurons which regulate sympathetic outflow to DRGs receive primary sensory afferents from corresponding DRGs.
Section snippets
Virus
Recombinant Bartha strain 152 of the pseudorabies virus (PRV) (Billig et al., 2000) was provided by Dr. Lynn Enquist (Princeton University, Princeton, NJ). PRV-152 was constructed to express an enhanced green fluorescent protein (EGFP) and was propagated on PK15 cells to a titer of 1.7 × 108 plaque forming units per milliliter (pfu/ml). The virus was aliquoted at 100 μl/tube and stored at −80°C. Single aliquots were thawed immediately prior to injection and stored on ice during operation.
Statistical analysis
Correlation between viral dose and number of infected DRG neurons was evaluated using the Pearson correlation and Chi-square statistics and between elapsed time and DRG cell numbers using ANOVA. Comparisons of cell volumes were made using the Kolmogorov–Smirnov two-sample test. Significance levels were designated *P < 0.05, **P < 0.01, ***P < 0.001. All values are given as mean ± SEM. Only vital neurons (intact nucleus and continuous cell surface membrane) were counted.
PRV virus does not enter the spinal cord via primary afferent fibers
PRV-infected DRG neurons were detected in the inoculated lumbar DRGs at all times analyzed. Distribution of viral antigen within infected DRG neurons varied significantly at any given time point; in some cells, weak PRV immunoreactivity was detected in the vicinity of the nucleus, while in others, viral particles filled the entire cell body (Fig. 1A). The number of infected DRG neurons was positively correlated with the amount of injected virus (Pearson correlation, r = 0.63; Chi-square
Discussion
In this report, we show that a recombinant variant of the Bartha strain of pseudorabies virus infects DRG neurons and sympathetic postganglionic neurons following direct inoculation of DRGs. In addition, we demonstrate retrograde transsynaptic spread of PRV via preganglionic sympathetic neurons into spinal cord and brainstem areas that regulate the sympathetic outflow to lumbar DRGs.
Although it is clear that alpha herpes viruses exhibit a greater affinity for axon terminals than for neuronal
Acknowledgments
The authors thank Dr. Krister Kristensson for helpful discussion and Eva Lindqvist, Karin Lundströmer, and Karin Pernold for excellent technical assistance. Supported by the Swedish Research Council, AMF, Knut and Alice Wallenberg's Foundation, Hedlunds Stiftelse, and USPH grants.
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