The Journal of Neuroscience, April 1995, 15(4): 2972-2984 Anterograde Tracing of Trigeminal Afferent Pathways from the Murine Tooth Pulp to Cortex Using Herpes Simplex Virus Type 1 Edward M. Barnett,’ Gregory D. Evans,2 Ning Suq3 Stanley Perlman,is3 and Martin D. CasseW4 ‘Neuroscience Program, %ollege of Dentistry, 3Departments of Microbiology and Pediatrics, and 4Department of Anatomy, University of Iowa, Iowa City, Iowa 52242 Due to its predominantly nociceptive innervation, viral trac- pathways is that arising from the dental pulp, not only because ing from the tooth pulp provides a potential means for trac- of its clinical significance but also becauseother than a small ing central pain pathways. The neural pathways from the complement of postganglionic sympathetic fibers and mecha- tooth pulp to cortex were determined using in situ hybrid- noreceptors,the fibers in pulpal afferents are thought to be en- ization to detect the anterograde transneuronal spread of tirely nociceptive (Sessle,1987). In the rat, intrapulpal applica- herpes simplex virus type 1 strain H129 following inocu- tion of WGA-HRP transganglionically labels afferent terminals lation into the murine mandibular incisor pulp. Virus first throughout the ipsilateral trigeminal sensory complex, including appeared in the brain at day 3 in the dorsomedial region of the principal sensory nucleus, and the partes oralis, interpolaris, all three subnuclei of the spinal trigeminal nucleus and the and caudalis (Marfurt and Turner, 1984). Physiological studies principal sensory nucleus. By days 5-6 virus had spread suggestthat the pars caudalis is most strongly associatedwith to the contralateral medial nucleus of the medial geniculate the transmissionof dental nociceptive information, though the complex, posterior thalamus, and ventroposteromedial other componentsof the trigeminal sensory complex appear to thalamus. At days 7-8 virus was detected in laminae IV and be involved as well. Va of the primary somatosensory cortex and lamina IV of Much of our current understandingof further CNS structures the secondary somatosensory cortex in regions previously involved in relaying and processingdental nociceptive infor- shown to receive input from the lower jaw. Several mice mation comesfrom electrophysiological studies(e.g., Albe-Fes- also showed infection of laminae ll/lll of the ipsilateral dys- sard et al., 1983, though anatomical studies of trigeminothal- granular insular cortex, along with labeling for virus in the amic, trigeminomesencephalic,and trigeminopontoamygdaloid ipsilateral external lateral parabrachial nucleus, posterior pathways have contributed significantly (Peschanski;1984; Ber- thalamus, and posterior basolateral amygdala. nard et al., 1991; Yoshida et al., 1991). A large number of mes- Our results are highly consistent with previous tracing encephalic (e.g., parabrachial complex, central gray), thalamic and electrophysiological studies utilizing the tooth pulp (e.g., ventroposteromedial,centromedian, posterior, submedial, and with studies implicating the infected structures in no- parafascicularnuclei), hypothalamic (e.g., lateral hypothalamus, ciception. Viral spread appeared to define two separate af- arcuate nucleus), and forebrain (e.g., central amygdaloid nucle- ferent systems with infection of structures which have us) areas have been demonstratedphysiologically to contain been implicated in the sensory-discriminative aspects of neuronsresponsive to activation of trigeminal nociceptive path- pain, such as the ventroposteromedial thalamus and so- ways, though the anatomical basesfor these responsesare still matosensory cortex, as well as in the dysgranular insular unclear. Even less clearly understoodare the cortical areasin- cortex and related subcortical nuclei which may have a role volved in high level processingof trigeminal nociceptive infor- in the affective-motivational aspects of pain. mation (Roland, 1992; Snow et al., 1992). Recent anatomical [Key words: herpes simplex virus type 1, anterogracie vi- and physiological studies (Yoshida et al., 1991; Snow et al., ral tracing, trigeminal, nociception, tooth pulp, insular cor- 1992) suggestthat there are two distinctly different representa- tex] tions of nociception in the cortex: one, associatedwith the so- matosensorycortex, is involved primarily in the topographical Trigeminal sensory pathways convey nociceptive information localization of painful or noxious stimuli; the other, associated originating from a wide range of cranial structures,including the with the ventral orbital cortex, is concerned with “affective- cutaneousand mucosalsurfaces, cornea, and meninges,as well motivational” aspectsof pain. How nociceptive information is as the cranial bones and teeth. The most well-studied of these relayed to these separateareas remains largely unknown. The present study has utilized the spread of strain H129 of Received June 13, 1994; revised Oct. 18, 1994; accepted Nov. 1, 1994. herpes simplex virus 1 (HSV-1) following inoculation into the This work was supported by grants from the National Institutes of Health mousemandibular incisor tooth pulp to determinethe nature and (NS24401 and DC01 3 11) and the National Multiple Sclerosis Society (RG- extent of structures which are transneuronally connectedto the 2117Al). S.P. was supported by a Research Career Development Award from tooth pulp and therefore possible recipients of nociceptive in- the NIH, E.M.B. by a Predoctoral M.D.-Ph.D. Fellowship (MH10384) from the NIMH, and N.S. by NIH Training Grant T32 A107343. We thank Drs. formation. Strain H129 spreadstransneuronally in an antero- Gerald Gebhardt and Richard Traub for helpful discussions, and Paul Reimann grade direction, and has been used to trace sequential neural for photographic assistance. Correspondence should be addressed to Dr. M. D. Cassell, Department of pathways in the basal ganglia (Zemanick et al., 1991). Anatomy, University of Iowa, Iowa City, IA 52242. A preliminary report of this work has appearedin abstract Copyright 0 1995 Society for Neuroscience 0270.6474/95/152972-13$05.00/O form (Barnett et al., 1993b). The Journal of Neuroscience, April 1995, 75(4) 2973 Materials and Methods from a plasmid encoding the VP5 gene of HSV-1 (kindly provided by Dr. E. Wagner, University of California at Irvine) and in situ hybridiza- Animals tion performed as previously described (Perlman et al., 1988; Barnett Forty-six young adult male (5 weeks old) Balb/c mice purchased from et al., 1993a). Briefly, 35 km coronal brain sections were cut at lOO- Sasco Laboratories (Omaha, NE) were used in these studies. For sur- 200 p,rn intervals on a cryostat, collected on silane-treated slides, fixed, geries, mice were anesthetized by intraperitoneal injections (52.5 mg/ treated with proteinase K, and acetylated. Approximately lo6 cpm of kg) of a sodium pentobarbital solution (Shipley and Geinisman, 1984). probe in hybridization solution was applied to each slide. After an- All surgical procedures were approved by the Institutional Animal Care nealing overnight, slides were treated with RNase and washes of in- and Use Committee at the University of Iowa. creasing stringency. Slides were then placed on film for several days at 4”C, followed by dipping in NTB-2 photographic emulsion (Kodak, Virus Rochester, NY) for a 2 week exposure. After development and staining Strain H129 of HSV-1 (kindly provided by Dr. William Stroop, Baylor with cresyl violet, slides were examined by bright-field and dark-field College of Medicine) was grown on RK13 cells and titered on Vero light microscopy to localize viral nucleic acid in the brain. Infection of cells. The virus was maintained frozen at -70°C in tissue culture media. a cell was evidenced by labeling (i.e., silver grains) overlying the cell body, as this is the location of viral nucleic acid production. Inoculations Results Tooth pulp inoculations. Each mouse was deeply anesthetized and the free gingiva was removed from the left mandibular incisor. The erupted Control inoculations part of the incisor was then removed just coronal to the gingival at- Infection of the mesencephalic nucleus of the trigeminal nerve, tachment using a cutting burr on a high-speed dental handpiece. Any which would be expected to occur with viral leakage from the gingival bleeding encountered during the procedure was either cauter- apical foramen and infection of the innervation to the periodon- ized or blotted to secure hemostasis. Removal of the incisor at the level of the gingival attachment provided adequate access to the pulp canal tal ligament (e.g., Gonzalo-Sanz and Insausti, 1980), was not in mice up to 6 weeks of age. The pulp canal was penetrated and seen (data not shown). Of the five mice which received inocu- enlarged using endodontic files and 6 p,l (containing 6 X lo4 plaque- lations of HSV-1 strain H129 directly into the oral cavity, viral forming units) of HSV-1 strain H129 was delivered into the cavitation nucleic acid was detected by in situ hybridization in only one with a microsyringe. The tooth was subsequently sealed with a tem- porary dental cement. Postsurgically, mastication was not significantly case. This mouse showed a pattern of infection consistent with altered, and supereruption (which is normally continuous in the rodent) infection via both the maxillary and mandibular divisions of the of the surgically treated incisor occurred following the procedure. Four trigeminal nerve and the vagus nerve with heavy labeling in the mice each were killed at