Research report
Spinal interneurons infected by renal injection of pseudorabies virus in the rat

https://doi.org/10.1016/j.brainres.2004.01.016Get rights and content

Abstract

The potency of spinal sympathetic reflexes is increased after spinal injury, and these reflexes may result in life-threatening hypertensive crises in humans. Few, if any, primary afferents project directly to sympathetic preganglionic neurons (SPN). Therefore, spinal sympathetic interneurons (IN) must play a major role in generating dysfunctional sympathetic activity after spinal cord injury. Furthermore, these IN are potentially aberrant targets, either for ascending and descending axons that may sprout after spinal cord injury or for axons that regenerate after spinal cord injury. We identified IN via the transsynaptic retrograde transport of pseudorabies virus (PRV) injected into the kidneys of rats. The proportion of infected IN ranged from approximately 1/3 to approximately 2/3 of the number of infected SPN. IN were heavily concentrated among the SPN in spinal lamina VII. However, IN were located in all lamina of the dorsal horn. The longitudinal distribution of infected IN was closely correlated with the longitudinal distribution of infected SPN. Few infected IN were found rostral or caudal to the longitudinal range of infected SPN. Infected IN were heterogeneous in both their sizes and the extent of their dendritic trees. The strong correlation between longitudinal distributions of infected IN and SPN supports physiological data demonstrating a segmental organization of spinal sympathetic reflexes. The paucity of infected IN in segments distant from SPN suggests that multisegmental sympathetic reflexes are mediated by projections onto IN rather than onto SPN themselves. The morphological heterogeneity of IN probably manifests the variety of systems that affect spinal sympathetic regulation.

Introduction

Brainstem projections to sympathetic preganglionic neurons (SPN) have received substantial attention because levels of ongoing and reflex-elicited sympathetic activity are largely regulated by those synaptic antecedents in spinally intact mammals (see [3], [20] for review). A further motivation for emphasizing brainstem inputs to SPN is the observation that, although primary afferents project into the region of the intermediolateral column of thoracic spinal cord (see, for instance, Ref. [19]), there is little evidence for synapses of these afferents directly onto SPN.

However, SPN do receive input from spinal sympathetic interneurons (IN). These IN have been identified anatomically by transsynaptic infection with pseudorabies virus (PRV) or herpes simplex virus injected into the kidney [21], [29] or spleen [6], by their transsynaptic uptake of the beta fragment of cholera toxin injected into the superior cervical ganglion [4], by their infection with herpes simplex injected into the adrenal gland [15], [21], [36], and by the location of their somas and the morphology and trajectories of their axons [12]. Physiological evidence suggests that spinal IN play a role in spinal sympathetic processing [2], [7], [8], [27]. However, several laboratories, including ours, have shown that spinal sympathetic reflexes are usually tonically inhibited, probably by brainstem systems [9], [11], [16], [28].

Although the role of spinal sympathetic reflexes in normal autonomic regulation may be uncertain, at least three motivations exist for studying spinal sympathetic circuitry. Firstly, experimental spinal cord transection exposes or accentuates spinal sympathetic reflexes [9], [11], [17], [25], and, in humans with spinal cord injury, these reflexes may cause severe, life-threatening hypertensive crises [22], [35]. Secondly, although monosynaptic projections of brainstem autonomic systems onto SPN are well documented [24], [26], almost nothing is known about projections of these same systems onto spinal sympathetic IN. Thirdly, regenerating or sprouting [38] spinal cord pathways after spinal cord injury in humans may make inappropriate synapses on spinal sympathetic IN, leading to autonomic dysfunction.

Recently, we identified a population of spinal neurons whose ongoing and reflex-evoked activities were closely correlated with ongoing renal sympathetic nerve activity (RSNA), and we hypothesized that they were IN that belong to spinal systems capable of increasing or decreasing RSNA [7], [8], [18], [23]. Significantly, the receptive fields from which both noxious and innocuous stimuli excited these neurons were enlarged after chronic spinal injury [18]. We have precisely located and morphologically characterized a small population of these sympathetically correlated neurons using extracellular recording followed by juxtacellular labeling [34]. Although juxtacellular labeling of sympathetically correlated spinal neurons resulted in both excellent physiological and anatomical characterization, this method was arduous for the identification of large numbers of neurons. Furthermore, the yield of juxtacellularly labeled neurons too very small to combine with simultaneous anterograde tracing of brainstem or spinal pathways in future experiments.

Therefore, in the present study, we identified, localized, and anatomically characterized sympathetic IN by the retrograde, transsynaptic transport of PRV from the kidney. PRV injected into the kidney is transported retrogradely within the axons of sympathetic postganglionic neurons and infects those neurons [13], [29], [31], [32]. Virus produced in sympathetic postganglionic neurons passes transsynaptically to the axons of SPN, which are infected in turn. Virus produced in SPN subsequently infects synaptic antecedents to those SPN, among them spinal sympathetic interneurons. Therefore, only IN that project to SPN that, in turn, potentially play a role in regulating the renal circulation or renal function become infected. This method identified hundreds of IN in each rat. This was a large enough sample to determine the cross-sectional and longitudinal distributions and the somatic and proximal dendritic morphology of these IN.

Section snippets

Materials and methods

All procedures used in these experiments were approved by the Johns Hopkins University Committee on Animal Care and Use. Thirty-four male, Sprague–Dawley rats (Charles River), weighing between 275 and 350 g were used in these experiments. As reported previously [29], renal injection of PRV resulted in a range of severity of infection. Our criteria for an acceptable severity of infection were (1) that rats exhibit infection of both SPN and IN, and (2) that rats exhibit no infected α-motoneurons.

Results

Interneurons were identified as neurons that were infected by PRV but were not labeled with HSB (see Fig. 1 and Materials and methods). In the four rats reported herein, we observed 252, 205, 356, and 217 infected IN and 366, 625, 518, and 473 infected SPN. Therefore, the ratio of infected IN to infected SPN was 0.68, 0.33, 0.68, and 0.46, respectively.

As reported previously [29], the proportion of the total number of SPN infected by PRV varied greatly along the longitudinal course of each

Discussion

Although several studies of spinal sympathetic IN have been conducted after tracer injections into a sympathetic ganglion [4] or the adrenal medulla [15], and the presence of sympathetic IN has been noted after injections of pseudorabies virus into the spleen [6] and kidney [5], [13], [29], [30], the present experiments are the first systematic examination of spinal, renal sympathetically related IN. Our use of the term “sympathetically related” does not imply that these neurons are employed

Acknowledgements

We thank Dr. Arthur Loewy, both for supplying the pseudorabies virus and antibodies used in this study and for numerous consultations. We thank Ms. Esther Kim, Ms. Julie Schafer, and Ms. Alexis Webb and Mr. Anand Veeravagu for valuable technical assistance. This research was supported by NIH grant HL16315.

References (38)

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