Identification of lamina V and VII interneurons presynaptic to adrenal sympathetic preganglionic neurons in rats using a recombinant herpes simplex virus type 1
Section snippets
Infection of rats with US5gal herpes simplex virus
All protocols for these experiments were approved by the University of Western Ontario Animal Care Committee in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care. Seventeen male Wistar rats (Charles River, U.S.A.) weighing 200–220 g were sedated with 2.5 mg/kg Diazepam (i.p.) and pretreated with atropine (1 mg/kg, s.c.) 10 min prior to anaesthesia with 25 mg/kg sodium pentobarbital. Supplemental doses of
Results
Infection of spinal cord neurons was observed at three days after adrenal injection of US5galHSV in eight of the 13 inoculated rats. This infection rate was similar to the rate we reported previously in hamsters.26, 29US5galHSV-immunoreactive neurons were found only ipsilateral to the injected adrenal gland and were distributed in and near the sympathetic preganglionic nuclei of the spinal cord at the T4–T13 levels. This infection was limited to neurons as US5galHSV-immunoreactive cells with
Discussion
In the three days following adrenal inoculation, the recombinant virus US5galHSV travelled from the adrenal site of injection to sympathoadrenal SPNs, then crossed at least one synapse to infect interneurons antecedent to the SPNs. Replication of the virus at each site led to clear immunocytochemical evidence for its presence in this pathway. The mutation of the virus that limited its cytotoxicity[26]still permitted a high degree of neurotropism resulting in infection of hundreds of neurons in
Conclusion
A recombinant HSV-1 (US5galHSV) was used in combination with immunocytochemistry for the acetylcholine synthesizing enzyme ChAT to identify interneurons in spinal circuits presynaptic to adrenal SPNs. A large population of non-cholinergic interneurons were found within and close to spinal autonomic nuclei. These smaller interneurons typically had morphological characteristics that distinguished them from the SPNs. The neurotransmitter phenotype of these neurons remains to be determined but they
Acknowledgements
This research was supported by grants from the Heart and Stroke Foundation of Ontario (T2679) and from the Medical Research Council of Canada. L. C. Weaver is a Career Investigator of the Heart and Stroke Foundation of Ontario. G. A. Dekaban is an Ontario Ministry of Health Career Scientist. H. A. Clarke received a John D. Schultz summer student scholarship from the Heart and Stroke Foundation of Ontario. The authors are indebted to Dr Marcia LeVatte for preparation of the US5galHSV used in
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