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Bilateral projection of canine tooth pulps to neurons of the cat sensory trigeminal complex
EXPERIMENTAL
NEUROLOGY
69, 183-195 (1980)
Bilateral Projection of Canine Tooth Pulps to Neurons of the Cat Sensory Trigeminal Complex SAMUELG.NORD
AND DAVIDE.ROLINCE~
Department of Neurology, Upstate Medical Center, State University of New York, Syracuse, New York 13210 Received December 17, 1979
The sensory trigeminal nuclei of cats were explored bilaterally with tungsten microelectrodes until neurons responsive to electrical stimulation of each canine tooth pulp were located. Then, the right trigeminal ganglion was transected and exploration was resumed. Stimulation of the left teeth generated 124 single-unit responses in 34 penetrations into the brain stem and evoked field potentials in 10 others after ganglionectomy. Although both left and right nuclear complexes yielded evoked activity, unit responses were obtained more frequently and field potentials attained higher amplitudes on the left. Stimulation of the right teeth, in contrast, triggered no unit discharges and evoked no field potentials in 51 postganglionectomy nuclear penetrations. Similarly, conditioning stimuli applied to the right teeth after ganglionectomy had no effect upon the responses to test stimuli applied to the left teeth. The data corroborate previous observations that many neurons in the trigeminal complex are innervated by bilateral projections from the canine dental pulps. In addition, they provide evidence that the projection from the contralateral canines is composed of primary and higher order atferent fibers which cross the midline exclusively within the brain stem to form a medullary-pontine pulpal decussation.
INTRODUCTION There is considerable experimental evidence that the cat canine tooth pulps are represented bilaterally in the various nuclei of the trigeminal sensory complex. Electrophysiological investigations demonstrated, for example, that neurons in the complex respond with consistent discharges 1 The authors are grateful to N. J. Horton for histological assistance and to A. Chorazy for typing the manuscript. The research was supported by National Institutes of Health grant NS 10814 and a grant from the Dr. Merrill H. Ross Medical Fund, Inc. 183 00144886’80/070183-13$02.00/O Copyrighi 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
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during either ipsi- or contralateral pulpal stimulation (13, 15, 18, 24). In addition, anatomical studies revealed that unilateral destruction of the canine pulp results in axonal and terminal degenerative changes in the trigeminal nuclei on both sides of the brain stem (3, 9, 21, 22). The projection pathway of pulpal atTerent fibers to the ipsilateral brain stem is apparent. However, the course taken by afferent fibers from the dental pulps to the contralateral trigeminal complex is uncertain and this has been the subject of debate in the recent literature. One very likely possibility is that the fibers project from the pulp to the ipsilateral ganglion and that these (and/or higher order afferents) cross the midline within the brain stem to synapse with neurons on the other side. Although this type of fiber crossing was demonstrated anatomically after lesions of trigeminal nuclear regions (7, 8, 19, 20), there is no direct evidence that pulpal afferent fibers follow such a course. A second possibility is that some pulpal afferents cross the midline peripherally as a transmedian projection to the contralateral ganglion. Several anatomical (2, 3, 5, 16) and physiological (1, 3) investigations of dental projections to the ganglia and/or trigeminal nuclei produced evidence which supports this possibility. However, findings reported in related anatomical (4) and physiological studies (11,12) failed to corroborate this evidence and, in some instances, were in direct conflict with it. For example, unilateral application of horseradish peroxidase to the canine pulps resulted in both bilateral (3, 5) and exclusively ipsilateral(4) labeling in the trigeminal ganglia. Similarly, electrical stimulation of the contralateral canines activated 30% of the pulpal neurons encountered in one investigation of the ganglion (3) but had no effect upon the ganglionic pulpal units found in another (11). The present investigation evolved from these discrepancies. Experiments were designed to determine whether or not neurons in the trigeminal sensory complex which respond to contralateral pulpal stimulation are activated via: (i) a projection of fibers which crosses the midline within the brain stem, (ii) a transmedian, peripheral projection, or (iii) both a peripheral and a central projection. METHODS Data were obtained from nine lightly anesthetized (pentobarbital sodium), paralyzed (gallamine triethiodide) cats which were artificially ventilated via tracheal cannulas. Body temperature, arterial blood pressure, and end-tidal COZ were monitored and were maintained at adequate physiological levels (13). Most of the preparatory, surgical, and experimental procedures were described in our previous study of bilateral pulpal projections to the trigeminal nucleus caudalis (13) and, consequently, are noted only briefly. Methods or procedures which differ from those used previously are presented in more detail.
BILATERAL
PROJECTION
OF TOOTH
PULPS
185
The animal was mounted in a modified stereotaxic arrangement which left the face and oral cavity unobstructed. A large, right temporal craniectomy and a partial right hemispherectomy were carried out so that the underlying trigeminal ganglion could be visualized. Then, a suboccipital craniectomy was made to expose the medulla oblongata and the caudal cerebellum. The cavities resulting from these procedures were filled with saline-agar. Neural activity was recorded via tungsten microelectrodes (tip diameter = 1 pm) and was amplified and monitored by conventional methods. Caudal to the obex, recording commenced for each penetration with the electrode positioned at the brain surface and continued while it was advanced through the tissue by means of a remote microdrive. More rostrally, the electrode was first advanced through the cerebellum with a coarse drive until its tip was approximately 2 mm above the trigeminal complex. Then, recording and micropositioning of the electrode were initiated. The four canine pulps were stimulated sequentially with single, isolated, rectangular pulses (duration = 0.5 to 3.0 ms; amplitude = 10 to 90 V) at 0.3 pulses/s via bipolar, concentric electrodes embedded in the teeth (13). Current spread to nonpulpal nervous tissue was limited by the design and application of the electrodes (14,15). Field potentials, enhanced through the use of a signal averager, typically were detected as the microelectrode approached responsive neurons. Spike discharges, recorded extracellularly from these neurons usually appeared as the electrode penetrated more deeply. When spikes were triggered, slow-wave recording was discontinued, the functional characteristics of the spike discharge were recorded, and the stereotaxic coordinates of the electrode tip were noted. Then, each remaining canine was tested for its ability to drive the response. Periodically, gallamine triethiodide was administered during recording to ensure that the response was not secondary to reflex activation of the jaw muscles. Selected data were stored on magnetic tape for postexperimental study and analysis. In each preparation, the trigeminal nuclei were explored for regions which were responsive to stimulation of all four canine pulps. After locating such a region and recording unit-response characteristics, the microelectrode tip was elevated above the active site and the exposed trigeminal ganglion was completely transected near the sensory root with a thermocautery. Then the electrode was returned to the previously responsive tissue and dental stimulating and recording procedures were resumed. In addition, the face and oral cavity were stimulated periodically with manual probes. Subsequently, additional penetrations were made into each side of the brain stem. Electrode placements were guided by measurements obtained from the preparation under study prior to
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ganglionectomy, by the data of earlier experiments (13-19, and by Berman’s atlas of the cat brain stem (6). Microlesions were made at the deepest points of many penetrations and at the positions of several responsive cells by passing current through the recording electrode (4 to 5 PA for 10 to 15 s). Conditioning experiments were carried out with 12 neurons which responded with spike discharges to stimulation of one of the left teeth after transection of the ganglion. In these experiments, a conditioning pulse applied to one of the right teeth preceded a test pulse applied to one of the left teeth by an interstimulus interval (ISI) of 90 to 150 ms. In our previous study of bilateral pulpal projections (13), intervals in this range resulted in suppression of the response to a test pulse in each of 18 conditioning experiments. In the present investigation the technique was used to test for the possibility of an inhibitory transmedian projection which might not be detected using our other procedures. The animal was killed at the termination of each experiment. The tissue surrounding the right ganglion was carefully dissected away and the completeness of the transection was verified. A brain stem block containing the regions studied during the experiment was excised and fixed in buffered 10% Formalin. After fixation, the tissue was frozen, and sections cut at 40 pm were stained with cresyl violet. RESULTS Preablation. Stimulation of each canine pulp generated responses in the trigeminal nuclei of every animal prior to ganglionectomy. Spike potentials were recorded from 37 neurons in 18 penetrations and field potentials without spikes were obtained in one penetration. Twenty-four neurons could be activated only by stimuli applied ipsilaterally, 12 responded to stimulation of either tooth, and one was excited by stimulation of a contralateral tooth alone. When field potentials were evoked by either ipsior contralateral stimulation, those which were triggered by stimuli applied to an ipsilateral tooth typically had higher amplitudes and they usually appeared first as the recording electrode was lowered through the responsive tissue. Conversely, the potentials produced by contralateral stimulation tended to persist longer as the electrode was advanced into deeper tissue. These relationships are suggested in Figs. 1 and 2. The neurons activated in the present experiments exhibited response characteristics which were extremely similar to those described in our previous studies of pulpal projections to the trigeminal nuclei (13, 15). Consequently, these characteristics will not be considered in detail. In general, thresholds were slightly lower and initial spike latencies were
BILATERAL
PROJECTION
OF TOOTH PULPS
187
PRE - ABLATION RIGHT
LEFT
LLower
Lower
Upper
Upper
l-------
IL-----
-k-----
IL-.---
L-
k
IOOpVL
POST -ABLATION LEFT Lower
30ms
RIGHT Lower
Upper
Upper
L+----
kk kk
i-----!-L-e-
iI_:i_ tF
FIG. 1. Pulpal field potentials evoked at the same loci in the left brain stem before (preablation) and after (postablation) trigeminal ganglionectomy . The recording electrode was positioned 7.0 mm rostral to the obex and 3.8 mm lateral to the midline. The responses in the first two columns were produced by stimulation of the left lower and upper canine teeth, respectively, and those in the remaining columns by stimulation of the right canines. Each trace in a row was obtained from the same point in the brain and each is an average of 32 consecutive responses. Successive rows (top to bottom) of both pre- and postablation data were recorded at 300~pm intervals as the microelectrode was lowered through the brain. Each of the five postablation rows was obtained at the same locus as the corresponding preablation row.
briefer for ipsilateral than for contralateral stimulation. One or two spikes were triggered in each neuron at threshold. As intensity was raised to maximally effective values, the number of spikes increased in most cells and interspike intervals decreased. Initial spike latencies varied considerably at these values (range = 3 to 22 ms for ipsilateral and 5.5 to 45 ms for contralateral responses), but were always briefer after ipsilateral stimulation.
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PRE - ABLATION RIGHT
LEFT v------v
POST -ABLATION Lower -hf---
L
IOOpV RIGHT
LEFT Lower
Upper
u------b-f----
-----l----r------
+------bf----
-r---
-P-+----
-------I---
30ms Upper
FIG. 2. Pulpal field potentials evoked at the same loci in the right brain stem before (preablation) and after (postablation) trigeminal ganglionectomy. The recording electrode was positioned 6.5 mm rostral to the obex and 4.5 mm lateral to the midline. The responses in the first two columns were produced by stimulation of the left lower and upper canine teeth, respectively, and those in the remaining columns by stimulation of the right canines. Each trace in a row was obtained from the same point in the brain and each is an average of 32 consecutive responses. Successive rows (top to bottom) of both pre- and postablation data were recorded at 500~km intervals as the microelectrode was lowered through the brain. Each of the four postablation rows was obtained at the same locus as the corresponding preablation row.
Postablation. After ganglionectomy, 51 penetrations were made into the brain stem (25 on the left, 26 on the right). Left-tooth stimulation activated 124 neurons (85 on the left; 39 on the right) in 34 penetrations and evoked field potentials in 10 others. Figure 3 illustrates the positions of the responsive penetrations in the horizontal plane. Stimulation of the right tooth after transection generated no observable responses in any of the penetrations, even at locations which had yielded right tooth responses previously and which continued to be activated by left tooth stimulation (e.g., Figs. 1 and 2). The face was stimulated with mechanical probes intermittently after ganglionectomy, particularly when the recording electrode was in tissue in which pulpal responses were being generated. Single or multiunit activity
BILATERAL
PROJECTION
OF TOOTH PULPS
DISTANCE FROM OBEX (mm) FIG. 3. Schematic representation of the brain stem in the horizontal postganglionectomy electrode penetrations which yielded responses canine pulps were plotted with reference to the obex. Solid circles which spike potentials were recorded; triangles indicate penetrations potentials.
plane. The locations of to stimulation of the left indicate penetrations in which yielded only field
frequently was triggered from left orofacial fields during exploration of the left trigeminal complex. In contrast, no clearly defined responses were ever produced by stimulation of the right facial or oral regions during penetrations into the right brain stem, even at points from which pulpal activity was being recorded. Conditioning stimuli (590 V) applied to the right teeth had no effect upon the responses to test stimuli applied to the left teeth in any of the 12 conditioning experiments which were carried out after transection of the ganglion. Examination of appropriate brain stem sections revealed that postganglionectomy recording penetrations entered the left principal sensory nucleus and each of the three subdivisions of the left and right spinal nuclei of the trigeminal complex. The penetrations illustrated in Fig. 3 either entered the complex or passed along its medial border. Field potentials were obtained throughout the nuclear complex. However, the largest were recorded 5 to 8 mm rostra1 to the obex in the nucleus interpolaris and in the nucleus oralis. Spike potentials were generated in neurons which were situated in each of the trigeminal nuclei, as Figs. 3 and 4 suggest. At the level of the obex and caudally, all but 1 of 39 neurons were isolated deep in the nucleus caudalis [laminae IV through VI (lo)]. The remaining unit lay more dorsally, presumably in lamina I (10).
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FIG. 4. Photomicrographs of brain stem cross sections illustrating loci in, or in the vicinity of, the trigeminal complex which yielded responses to stimulation of the left canine teeth after right ganglionectomy. The arrows indicate microlesions made through the recording electrode. Spike potentials were recorded at the lesions and at each of the solid circles in A, C, and D. Field potentials without spikes were recorded at, and immediately dorsal to, the lesion in B. Each photomicrograph was made at the same magnification. The bracketed line in D = 500 pm; A-9 mm rostra1 to obex, left side, B-7 mm rostra1 to obex, left side, C-3 mm rostra1 to obex, left side, D-at the level of the obex, right side.
5, cL
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Rostral to the obex, responsive neurons were found either within the obvious limits of the nuclei interpolaris, oralis, and principalis, or along their medial borders. The loci of several of the units are illustrated in Fig. 4. DISCUSSION The results of the present experiments confirm previous observations that (i) neurons in the sensory trigeminal complex receive afferent input from both the ipsi- and the contralateral tooth pulps (13,15,18,23,24); (ii) a few receive input from the contralateral canines only (13); and (iii) the ratio of such neurons to those which are innervated exclusively by ipsilateral pulpal afferent fibers is approximately 1:3 (13). As in other studies (13, 15, 24), the great majority of neurons were situated deep in the complex with “ipsilateral units” generally lying dorsal or dorsolateral to those which received contralateral projections. After ganglionectomy, neither electrical stimulation of the previously effective right canines or mechanical stimulation of the right side of the face elicited responses in histologically verified penetrations into the right trigeminal complex. These results and post experimental examinations of the right ganglia confirmed that all our transections were complete and that, consequently, each right trigeminal complex was deprived of its normal afferent input. It is apparent, therefore, that the postganglionectomy responses generated at so many loci in the right complex by left tooth stimulation were effected via a projection of afferent fibers which crossed the midline within the brain stem. Thus, our results provide rather strong evidence that there is a decussation of pulpal fibers in the medulla and pons. Although many of the fibers are undoubtedly second and higher order, our latency measurements suggest that some are primary afferents. The existence of a pulpal decussation in the brain stem is not remarkable. Anatomical studies of the cat (7, 19) and monkey (8, 20) showed that discrete lesions made in the trigeminal tract or nuclei on one side of the brain result in evidence of degeneration in, or in the vicinity of, trigeminal structures on the other side. Moreover, unilateral destruction of the dental pulps in cats produced axonal and terminal degenerative changes in the contralateral trigeminal nuclei in several investigations (3, 9, 21, 22). As on the opposite side of the brain stem, right tooth stimulation triggered no responses in the left trigeminal sensory nuclei after ganglionectomy, even though spike and field potentials continued to be elicited when the left teeth were stimulated. These results imply that, in addition to deafferentiating the right trigeminal complex, transection of the
BILATERAL
PROJECTION
OF TOOTH
PULPS
193
ganglion resulted in a massive, if not total, interruption of the excitatory afferent projection from the right canine pulps to the left trigeminal nuclei. In a previous study of the nucleus caudalis (13), a conditioning stimulus applied to a contralateral tooth always suppressed the response to a test stimulus delivered to an ipsilateral tooth when the stimuli were separated by appropriate intervals. Yet, this procedure produced no neuronal response alterations in the present experiments when conditioning stimuli were applied to right teeth and test stimuli to left teeth after ganglionectomy. Thus, the pathway by which the right dental pulps normally exert inhibitory influences upon neurons in the left trigeminal nuclei also appeared to have been completely interrupted by right ganglion transection. In short, postganglionectomy stimulation of the right tooth neither activated any of the 85 neurons in the left complex which were fired by left tooth stimulation nor produced response inhibition in any of the 12 conditioning experiments performed with these neurons. These findings argue strongly against the likelihood that either excitatory or inhibitory pulpal afferent fibers reach the contralateral trigeminal complex via a peripheral, transmedian projection, and they lend further support to the view that the afferents pass to the complex via a brain stem decussation. On the other hand, our results do not exclude the possibility that a transmedian pulpal projection reaches the ganglion, as many of the relevant anatomical (2, 3, 5, 16) and physiological (1, 3) investigations suggested. If this projection exists as described, however, it is difficult to understand why supportive evidence was not obtained in related anatomical (4) and physiological (11, 12) studies. Furthermore, it is not clear where the apparently sizeable number of postganglionic fibers project or what their function(s) might be. It was reported (3) that about 0.6 times as many ganglion cells originate in a contralateral canine tooth as in an ipsilateral one and it would appear that a substantial centripetal projection of “transmedian fibers” must leave the ganglion. As noted above, however, the results of the present experiments provide evidence that this projection does not terminate in the sensory nuclei and one can assume, therefore, that its function is not related to dental sensation. Moreover, it was demonstrated recently (17) that pulpal afferent fibers which participate in the contralateral jaw opening reflex also cross the midline within the lower brain stem rather than as a transmedian projection and, hence, this function must be excluded as well. In an earlier investigation of bilateral pulpal units in the medulla (13), we proposed that caudalis neurons which responded to contralateral dental stimulation might be higher-order components of a transmedian pulpal projection to the trigeminal ganglion which were described previously (1). The data of the present experiments demonstrate that this proposal is no longer tenable.
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REFERENCES 1. ANDERSON, K. V., AND G. S. PEARL. 1974. Transmedian innervation of canine tooth pulp in cats. Exp. Neurol. 44: 35-40. 2. ANDERSON, K. V., AND H. S. ROSING. 1977. Location of feline trigeminal ganglion cells innervating maxillary canine teeth: a horseradish peroxidase analysis. Exp. Neurol. 57: 302-306. 3. ANDERSON,
4.
5.
6. 7.
8. 9.
K. V., H. S. ROSING, AND G. S. PEARL. 1977. Physiological and anatomical studies revealing an extensive transmedian innervation of feline canine teeth. Pages 149- 160 in D. J. ANDERSON AND B. MATTHEWS, Eds., Pain in the Trigeminal Region. Elsevier/North-Holland, Amsterdam. ARVIDSSON, J. 1975. Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of horseradish peroxidase. Brain Res. 99: 135-139. AVERY, J. K., AND C. F. Cox. 1977. Role of nerves in teeth relative to pain and dentinogenesis. Pages 37-48 in D. J. ANDERSON AND B. MATTHEWS, Eds., Pain in the Trigeminal Region. ElseviedNorth-Holland, Amsterdam. BERMAN, A. L. 1968. The Bruin Stem Atlas of the Car. Univ. of Wisconsin Press, Madison. CARPENTER, M. B., AND G. R. HANNA. 1961. Fiberprojectionsfrom the spinal trigeminal nucleus in the cat. J. Comp. Neurol. 117: 117-125. DUNN, J. D., AND H. A. MATZKE. 1968. Efferent fibre connections of the marmoset (Oedipoimidas Oedipus) trigeminal nucleus caudalis. .I. Comp. Neurol. 133: 429-438. GOBEL S., AND J. M. BRINCK. 1977. Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat. Brain
Res. 10. GOBEL,
11. 12. 13. 14.
132: 347-354. S., W. M. FALLS,
AND S. HOCKFIELDS. 1977. The division of the dorsal and ventral horns of the mammalian caudal medulla into eight layers using anatomical criteria. Pages 443-453 in D. J. ANDERSON AND B. MATTHEWS, Eds., Pain in the Trigeminal Region. Elsevier/North-Holland, Amsterdam. LISNEY, S. J. W. 1978. Some anatomical and electrophysiological properties of tooth-pulp afferents in the cat. .I. Physiol. (London) 284: 19-36. MATTHEWS, B., AND S. J. W LISNEY. 1978. Do primary afferents from tooth-pulp cross the midline? Bruin Res. 158: 303-312. NORD, S. G. 1976. Bilateral projection of the canine tooth pulp to bulbar trigeminal neurons. Brain Res. 113: 517-525. NORD, S. G. 1976. Electrical stimulation of the tooth pulp in the study of pain. Brain Res. Bull.
1: 251-254.
15. NORD, S. G. 1976. Responses of neurons in rostral and caudal trigeminal nuclei to tooth pulp stimulation. Bruin Res. Bull. 1: 489-492. 16. PEARL, G. S., K. V. ANDERSON, AND H. S. ROSING. 1977. Anatomic evidence revealing extensive transmedian innervation of feline canine teeth. Exp. Neurol. 54: 432-443. 17. SAAG, M., AND K. H. REID. 1979. Surgical determination of the site of crossing ofjaw opening reflex in the cat. Fed. Proc. 38: 1250. 18. SESSLE, B. J., R. DUBNER, L. F., GREENWOOD, AND G. E. LUCIER. 1976. Descending influences of periaqueductal gray matter and somatosensory cerebral cortex on neurons in trigeminal brain stem nuclei. Cannd. J. Physiol. Pharmacol. 54: 66-69. 19. STEWART, W. A., AND R. B. KING. 1963. Fiber projections drom thenucleus caudalis of the spinal trigeminal nucleus. J. Comp. Neurof. 121: 271-286.
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OF TOOTH PULPS
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20. TIWARI, R. K., AND R. B. KING. 1974. Fiber projections from trigeminal nucleus caudalis in primate (squirrel monkey and baboon). J. Camp. Neural. 158: 191-206. 21. WESTRUM, L. E., AND R. C. CANFIELD. 1977. Light and electron microscopy of degeneration in the brain stem spinal trigeminal nucleus following booth pulp removal inadult cats. Pages 171-180inD. J. ANDERSON AND B. MATTHEWS, Eds.,Pain in the Trigeminal Region. ElsevieriNorth-Holland, Amsterdam. 22. WESTRUM, L. E., R. C. CANFIELD, AND R. G. BLACK. 1976. Transganglionic degeneration in the spinal trigeminal nucleus following removal of tooth pulps in adult cats. Brain Res. 101: 137-140. 23. YOKOTA, T. 1975. Excitation of units in marginal rim of trigeminal subnucleus caudalis elicited by tooth pulp stimulation. Brain Res. 95: 154-158. 24. YOKOTA, T. 1976. Two types of tooth pulp units in the bulbar lateral reticular formation. Brain Res. 104: 325-329.
NEUROLOGY
69, 183-195 (1980)
Bilateral Projection of Canine Tooth Pulps to Neurons of the Cat Sensory Trigeminal Complex SAMUELG.NORD
AND DAVIDE.ROLINCE~
Department of Neurology, Upstate Medical Center, State University of New York, Syracuse, New York 13210 Received December 17, 1979
The sensory trigeminal nuclei of cats were explored bilaterally with tungsten microelectrodes until neurons responsive to electrical stimulation of each canine tooth pulp were located. Then, the right trigeminal ganglion was transected and exploration was resumed. Stimulation of the left teeth generated 124 single-unit responses in 34 penetrations into the brain stem and evoked field potentials in 10 others after ganglionectomy. Although both left and right nuclear complexes yielded evoked activity, unit responses were obtained more frequently and field potentials attained higher amplitudes on the left. Stimulation of the right teeth, in contrast, triggered no unit discharges and evoked no field potentials in 51 postganglionectomy nuclear penetrations. Similarly, conditioning stimuli applied to the right teeth after ganglionectomy had no effect upon the responses to test stimuli applied to the left teeth. The data corroborate previous observations that many neurons in the trigeminal complex are innervated by bilateral projections from the canine dental pulps. In addition, they provide evidence that the projection from the contralateral canines is composed of primary and higher order atferent fibers which cross the midline exclusively within the brain stem to form a medullary-pontine pulpal decussation.
INTRODUCTION There is considerable experimental evidence that the cat canine tooth pulps are represented bilaterally in the various nuclei of the trigeminal sensory complex. Electrophysiological investigations demonstrated, for example, that neurons in the complex respond with consistent discharges 1 The authors are grateful to N. J. Horton for histological assistance and to A. Chorazy for typing the manuscript. The research was supported by National Institutes of Health grant NS 10814 and a grant from the Dr. Merrill H. Ross Medical Fund, Inc. 183 00144886’80/070183-13$02.00/O Copyrighi 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
184
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ROLINCE
during either ipsi- or contralateral pulpal stimulation (13, 15, 18, 24). In addition, anatomical studies revealed that unilateral destruction of the canine pulp results in axonal and terminal degenerative changes in the trigeminal nuclei on both sides of the brain stem (3, 9, 21, 22). The projection pathway of pulpal atTerent fibers to the ipsilateral brain stem is apparent. However, the course taken by afferent fibers from the dental pulps to the contralateral trigeminal complex is uncertain and this has been the subject of debate in the recent literature. One very likely possibility is that the fibers project from the pulp to the ipsilateral ganglion and that these (and/or higher order afferents) cross the midline within the brain stem to synapse with neurons on the other side. Although this type of fiber crossing was demonstrated anatomically after lesions of trigeminal nuclear regions (7, 8, 19, 20), there is no direct evidence that pulpal afferent fibers follow such a course. A second possibility is that some pulpal afferents cross the midline peripherally as a transmedian projection to the contralateral ganglion. Several anatomical (2, 3, 5, 16) and physiological (1, 3) investigations of dental projections to the ganglia and/or trigeminal nuclei produced evidence which supports this possibility. However, findings reported in related anatomical (4) and physiological studies (11,12) failed to corroborate this evidence and, in some instances, were in direct conflict with it. For example, unilateral application of horseradish peroxidase to the canine pulps resulted in both bilateral (3, 5) and exclusively ipsilateral(4) labeling in the trigeminal ganglia. Similarly, electrical stimulation of the contralateral canines activated 30% of the pulpal neurons encountered in one investigation of the ganglion (3) but had no effect upon the ganglionic pulpal units found in another (11). The present investigation evolved from these discrepancies. Experiments were designed to determine whether or not neurons in the trigeminal sensory complex which respond to contralateral pulpal stimulation are activated via: (i) a projection of fibers which crosses the midline within the brain stem, (ii) a transmedian, peripheral projection, or (iii) both a peripheral and a central projection. METHODS Data were obtained from nine lightly anesthetized (pentobarbital sodium), paralyzed (gallamine triethiodide) cats which were artificially ventilated via tracheal cannulas. Body temperature, arterial blood pressure, and end-tidal COZ were monitored and were maintained at adequate physiological levels (13). Most of the preparatory, surgical, and experimental procedures were described in our previous study of bilateral pulpal projections to the trigeminal nucleus caudalis (13) and, consequently, are noted only briefly. Methods or procedures which differ from those used previously are presented in more detail.
BILATERAL
PROJECTION
OF TOOTH
PULPS
185
The animal was mounted in a modified stereotaxic arrangement which left the face and oral cavity unobstructed. A large, right temporal craniectomy and a partial right hemispherectomy were carried out so that the underlying trigeminal ganglion could be visualized. Then, a suboccipital craniectomy was made to expose the medulla oblongata and the caudal cerebellum. The cavities resulting from these procedures were filled with saline-agar. Neural activity was recorded via tungsten microelectrodes (tip diameter = 1 pm) and was amplified and monitored by conventional methods. Caudal to the obex, recording commenced for each penetration with the electrode positioned at the brain surface and continued while it was advanced through the tissue by means of a remote microdrive. More rostrally, the electrode was first advanced through the cerebellum with a coarse drive until its tip was approximately 2 mm above the trigeminal complex. Then, recording and micropositioning of the electrode were initiated. The four canine pulps were stimulated sequentially with single, isolated, rectangular pulses (duration = 0.5 to 3.0 ms; amplitude = 10 to 90 V) at 0.3 pulses/s via bipolar, concentric electrodes embedded in the teeth (13). Current spread to nonpulpal nervous tissue was limited by the design and application of the electrodes (14,15). Field potentials, enhanced through the use of a signal averager, typically were detected as the microelectrode approached responsive neurons. Spike discharges, recorded extracellularly from these neurons usually appeared as the electrode penetrated more deeply. When spikes were triggered, slow-wave recording was discontinued, the functional characteristics of the spike discharge were recorded, and the stereotaxic coordinates of the electrode tip were noted. Then, each remaining canine was tested for its ability to drive the response. Periodically, gallamine triethiodide was administered during recording to ensure that the response was not secondary to reflex activation of the jaw muscles. Selected data were stored on magnetic tape for postexperimental study and analysis. In each preparation, the trigeminal nuclei were explored for regions which were responsive to stimulation of all four canine pulps. After locating such a region and recording unit-response characteristics, the microelectrode tip was elevated above the active site and the exposed trigeminal ganglion was completely transected near the sensory root with a thermocautery. Then the electrode was returned to the previously responsive tissue and dental stimulating and recording procedures were resumed. In addition, the face and oral cavity were stimulated periodically with manual probes. Subsequently, additional penetrations were made into each side of the brain stem. Electrode placements were guided by measurements obtained from the preparation under study prior to
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NORD
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ROLINCE
ganglionectomy, by the data of earlier experiments (13-19, and by Berman’s atlas of the cat brain stem (6). Microlesions were made at the deepest points of many penetrations and at the positions of several responsive cells by passing current through the recording electrode (4 to 5 PA for 10 to 15 s). Conditioning experiments were carried out with 12 neurons which responded with spike discharges to stimulation of one of the left teeth after transection of the ganglion. In these experiments, a conditioning pulse applied to one of the right teeth preceded a test pulse applied to one of the left teeth by an interstimulus interval (ISI) of 90 to 150 ms. In our previous study of bilateral pulpal projections (13), intervals in this range resulted in suppression of the response to a test pulse in each of 18 conditioning experiments. In the present investigation the technique was used to test for the possibility of an inhibitory transmedian projection which might not be detected using our other procedures. The animal was killed at the termination of each experiment. The tissue surrounding the right ganglion was carefully dissected away and the completeness of the transection was verified. A brain stem block containing the regions studied during the experiment was excised and fixed in buffered 10% Formalin. After fixation, the tissue was frozen, and sections cut at 40 pm were stained with cresyl violet. RESULTS Preablation. Stimulation of each canine pulp generated responses in the trigeminal nuclei of every animal prior to ganglionectomy. Spike potentials were recorded from 37 neurons in 18 penetrations and field potentials without spikes were obtained in one penetration. Twenty-four neurons could be activated only by stimuli applied ipsilaterally, 12 responded to stimulation of either tooth, and one was excited by stimulation of a contralateral tooth alone. When field potentials were evoked by either ipsior contralateral stimulation, those which were triggered by stimuli applied to an ipsilateral tooth typically had higher amplitudes and they usually appeared first as the recording electrode was lowered through the responsive tissue. Conversely, the potentials produced by contralateral stimulation tended to persist longer as the electrode was advanced into deeper tissue. These relationships are suggested in Figs. 1 and 2. The neurons activated in the present experiments exhibited response characteristics which were extremely similar to those described in our previous studies of pulpal projections to the trigeminal nuclei (13, 15). Consequently, these characteristics will not be considered in detail. In general, thresholds were slightly lower and initial spike latencies were
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PRE - ABLATION RIGHT
LEFT
LLower
Lower
Upper
Upper
l-------
IL-----
-k-----
IL-.---
L-
k
IOOpVL
POST -ABLATION LEFT Lower
30ms
RIGHT Lower
Upper
Upper
L+----
kk kk
i-----!-L-e-
iI_:i_ tF
FIG. 1. Pulpal field potentials evoked at the same loci in the left brain stem before (preablation) and after (postablation) trigeminal ganglionectomy . The recording electrode was positioned 7.0 mm rostral to the obex and 3.8 mm lateral to the midline. The responses in the first two columns were produced by stimulation of the left lower and upper canine teeth, respectively, and those in the remaining columns by stimulation of the right canines. Each trace in a row was obtained from the same point in the brain and each is an average of 32 consecutive responses. Successive rows (top to bottom) of both pre- and postablation data were recorded at 300~pm intervals as the microelectrode was lowered through the brain. Each of the five postablation rows was obtained at the same locus as the corresponding preablation row.
briefer for ipsilateral than for contralateral stimulation. One or two spikes were triggered in each neuron at threshold. As intensity was raised to maximally effective values, the number of spikes increased in most cells and interspike intervals decreased. Initial spike latencies varied considerably at these values (range = 3 to 22 ms for ipsilateral and 5.5 to 45 ms for contralateral responses), but were always briefer after ipsilateral stimulation.
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PRE - ABLATION RIGHT
LEFT v------v
POST -ABLATION Lower -hf---
L
IOOpV RIGHT
LEFT Lower
Upper
u------b-f----
-----l----r------
+------bf----
-r---
-P-+----
-------I---
30ms Upper
FIG. 2. Pulpal field potentials evoked at the same loci in the right brain stem before (preablation) and after (postablation) trigeminal ganglionectomy. The recording electrode was positioned 6.5 mm rostral to the obex and 4.5 mm lateral to the midline. The responses in the first two columns were produced by stimulation of the left lower and upper canine teeth, respectively, and those in the remaining columns by stimulation of the right canines. Each trace in a row was obtained from the same point in the brain and each is an average of 32 consecutive responses. Successive rows (top to bottom) of both pre- and postablation data were recorded at 500~km intervals as the microelectrode was lowered through the brain. Each of the four postablation rows was obtained at the same locus as the corresponding preablation row.
Postablation. After ganglionectomy, 51 penetrations were made into the brain stem (25 on the left, 26 on the right). Left-tooth stimulation activated 124 neurons (85 on the left; 39 on the right) in 34 penetrations and evoked field potentials in 10 others. Figure 3 illustrates the positions of the responsive penetrations in the horizontal plane. Stimulation of the right tooth after transection generated no observable responses in any of the penetrations, even at locations which had yielded right tooth responses previously and which continued to be activated by left tooth stimulation (e.g., Figs. 1 and 2). The face was stimulated with mechanical probes intermittently after ganglionectomy, particularly when the recording electrode was in tissue in which pulpal responses were being generated. Single or multiunit activity
BILATERAL
PROJECTION
OF TOOTH PULPS
DISTANCE FROM OBEX (mm) FIG. 3. Schematic representation of the brain stem in the horizontal postganglionectomy electrode penetrations which yielded responses canine pulps were plotted with reference to the obex. Solid circles which spike potentials were recorded; triangles indicate penetrations potentials.
plane. The locations of to stimulation of the left indicate penetrations in which yielded only field
frequently was triggered from left orofacial fields during exploration of the left trigeminal complex. In contrast, no clearly defined responses were ever produced by stimulation of the right facial or oral regions during penetrations into the right brain stem, even at points from which pulpal activity was being recorded. Conditioning stimuli (590 V) applied to the right teeth had no effect upon the responses to test stimuli applied to the left teeth in any of the 12 conditioning experiments which were carried out after transection of the ganglion. Examination of appropriate brain stem sections revealed that postganglionectomy recording penetrations entered the left principal sensory nucleus and each of the three subdivisions of the left and right spinal nuclei of the trigeminal complex. The penetrations illustrated in Fig. 3 either entered the complex or passed along its medial border. Field potentials were obtained throughout the nuclear complex. However, the largest were recorded 5 to 8 mm rostra1 to the obex in the nucleus interpolaris and in the nucleus oralis. Spike potentials were generated in neurons which were situated in each of the trigeminal nuclei, as Figs. 3 and 4 suggest. At the level of the obex and caudally, all but 1 of 39 neurons were isolated deep in the nucleus caudalis [laminae IV through VI (lo)]. The remaining unit lay more dorsally, presumably in lamina I (10).
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FIG. 4. Photomicrographs of brain stem cross sections illustrating loci in, or in the vicinity of, the trigeminal complex which yielded responses to stimulation of the left canine teeth after right ganglionectomy. The arrows indicate microlesions made through the recording electrode. Spike potentials were recorded at the lesions and at each of the solid circles in A, C, and D. Field potentials without spikes were recorded at, and immediately dorsal to, the lesion in B. Each photomicrograph was made at the same magnification. The bracketed line in D = 500 pm; A-9 mm rostra1 to obex, left side, B-7 mm rostra1 to obex, left side, C-3 mm rostra1 to obex, left side, D-at the level of the obex, right side.
5, cL
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Rostral to the obex, responsive neurons were found either within the obvious limits of the nuclei interpolaris, oralis, and principalis, or along their medial borders. The loci of several of the units are illustrated in Fig. 4. DISCUSSION The results of the present experiments confirm previous observations that (i) neurons in the sensory trigeminal complex receive afferent input from both the ipsi- and the contralateral tooth pulps (13,15,18,23,24); (ii) a few receive input from the contralateral canines only (13); and (iii) the ratio of such neurons to those which are innervated exclusively by ipsilateral pulpal afferent fibers is approximately 1:3 (13). As in other studies (13, 15, 24), the great majority of neurons were situated deep in the complex with “ipsilateral units” generally lying dorsal or dorsolateral to those which received contralateral projections. After ganglionectomy, neither electrical stimulation of the previously effective right canines or mechanical stimulation of the right side of the face elicited responses in histologically verified penetrations into the right trigeminal complex. These results and post experimental examinations of the right ganglia confirmed that all our transections were complete and that, consequently, each right trigeminal complex was deprived of its normal afferent input. It is apparent, therefore, that the postganglionectomy responses generated at so many loci in the right complex by left tooth stimulation were effected via a projection of afferent fibers which crossed the midline within the brain stem. Thus, our results provide rather strong evidence that there is a decussation of pulpal fibers in the medulla and pons. Although many of the fibers are undoubtedly second and higher order, our latency measurements suggest that some are primary afferents. The existence of a pulpal decussation in the brain stem is not remarkable. Anatomical studies of the cat (7, 19) and monkey (8, 20) showed that discrete lesions made in the trigeminal tract or nuclei on one side of the brain result in evidence of degeneration in, or in the vicinity of, trigeminal structures on the other side. Moreover, unilateral destruction of the dental pulps in cats produced axonal and terminal degenerative changes in the contralateral trigeminal nuclei in several investigations (3, 9, 21, 22). As on the opposite side of the brain stem, right tooth stimulation triggered no responses in the left trigeminal sensory nuclei after ganglionectomy, even though spike and field potentials continued to be elicited when the left teeth were stimulated. These results imply that, in addition to deafferentiating the right trigeminal complex, transection of the
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PULPS
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ganglion resulted in a massive, if not total, interruption of the excitatory afferent projection from the right canine pulps to the left trigeminal nuclei. In a previous study of the nucleus caudalis (13), a conditioning stimulus applied to a contralateral tooth always suppressed the response to a test stimulus delivered to an ipsilateral tooth when the stimuli were separated by appropriate intervals. Yet, this procedure produced no neuronal response alterations in the present experiments when conditioning stimuli were applied to right teeth and test stimuli to left teeth after ganglionectomy. Thus, the pathway by which the right dental pulps normally exert inhibitory influences upon neurons in the left trigeminal nuclei also appeared to have been completely interrupted by right ganglion transection. In short, postganglionectomy stimulation of the right tooth neither activated any of the 85 neurons in the left complex which were fired by left tooth stimulation nor produced response inhibition in any of the 12 conditioning experiments performed with these neurons. These findings argue strongly against the likelihood that either excitatory or inhibitory pulpal afferent fibers reach the contralateral trigeminal complex via a peripheral, transmedian projection, and they lend further support to the view that the afferents pass to the complex via a brain stem decussation. On the other hand, our results do not exclude the possibility that a transmedian pulpal projection reaches the ganglion, as many of the relevant anatomical (2, 3, 5, 16) and physiological (1, 3) investigations suggested. If this projection exists as described, however, it is difficult to understand why supportive evidence was not obtained in related anatomical (4) and physiological (11, 12) studies. Furthermore, it is not clear where the apparently sizeable number of postganglionic fibers project or what their function(s) might be. It was reported (3) that about 0.6 times as many ganglion cells originate in a contralateral canine tooth as in an ipsilateral one and it would appear that a substantial centripetal projection of “transmedian fibers” must leave the ganglion. As noted above, however, the results of the present experiments provide evidence that this projection does not terminate in the sensory nuclei and one can assume, therefore, that its function is not related to dental sensation. Moreover, it was demonstrated recently (17) that pulpal afferent fibers which participate in the contralateral jaw opening reflex also cross the midline within the lower brain stem rather than as a transmedian projection and, hence, this function must be excluded as well. In an earlier investigation of bilateral pulpal units in the medulla (13), we proposed that caudalis neurons which responded to contralateral dental stimulation might be higher-order components of a transmedian pulpal projection to the trigeminal ganglion which were described previously (1). The data of the present experiments demonstrate that this proposal is no longer tenable.
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REFERENCES 1. ANDERSON, K. V., AND G. S. PEARL. 1974. Transmedian innervation of canine tooth pulp in cats. Exp. Neurol. 44: 35-40. 2. ANDERSON, K. V., AND H. S. ROSING. 1977. Location of feline trigeminal ganglion cells innervating maxillary canine teeth: a horseradish peroxidase analysis. Exp. Neurol. 57: 302-306. 3. ANDERSON,
4.
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K. V., H. S. ROSING, AND G. S. PEARL. 1977. Physiological and anatomical studies revealing an extensive transmedian innervation of feline canine teeth. Pages 149- 160 in D. J. ANDERSON AND B. MATTHEWS, Eds., Pain in the Trigeminal Region. Elsevier/North-Holland, Amsterdam. ARVIDSSON, J. 1975. Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of horseradish peroxidase. Brain Res. 99: 135-139. AVERY, J. K., AND C. F. Cox. 1977. Role of nerves in teeth relative to pain and dentinogenesis. Pages 37-48 in D. J. ANDERSON AND B. MATTHEWS, Eds., Pain in the Trigeminal Region. ElseviedNorth-Holland, Amsterdam. BERMAN, A. L. 1968. The Bruin Stem Atlas of the Car. Univ. of Wisconsin Press, Madison. CARPENTER, M. B., AND G. R. HANNA. 1961. Fiberprojectionsfrom the spinal trigeminal nucleus in the cat. J. Comp. Neurol. 117: 117-125. DUNN, J. D., AND H. A. MATZKE. 1968. Efferent fibre connections of the marmoset (Oedipoimidas Oedipus) trigeminal nucleus caudalis. .I. Comp. Neurol. 133: 429-438. GOBEL S., AND J. M. BRINCK. 1977. Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat. Brain
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AND S. HOCKFIELDS. 1977. The division of the dorsal and ventral horns of the mammalian caudal medulla into eight layers using anatomical criteria. Pages 443-453 in D. J. ANDERSON AND B. MATTHEWS, Eds., Pain in the Trigeminal Region. Elsevier/North-Holland, Amsterdam. LISNEY, S. J. W. 1978. Some anatomical and electrophysiological properties of tooth-pulp afferents in the cat. .I. Physiol. (London) 284: 19-36. MATTHEWS, B., AND S. J. W LISNEY. 1978. Do primary afferents from tooth-pulp cross the midline? Bruin Res. 158: 303-312. NORD, S. G. 1976. Bilateral projection of the canine tooth pulp to bulbar trigeminal neurons. Brain Res. 113: 517-525. NORD, S. G. 1976. Electrical stimulation of the tooth pulp in the study of pain. Brain Res. Bull.
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15. NORD, S. G. 1976. Responses of neurons in rostral and caudal trigeminal nuclei to tooth pulp stimulation. Bruin Res. Bull. 1: 489-492. 16. PEARL, G. S., K. V. ANDERSON, AND H. S. ROSING. 1977. Anatomic evidence revealing extensive transmedian innervation of feline canine teeth. Exp. Neurol. 54: 432-443. 17. SAAG, M., AND K. H. REID. 1979. Surgical determination of the site of crossing ofjaw opening reflex in the cat. Fed. Proc. 38: 1250. 18. SESSLE, B. J., R. DUBNER, L. F., GREENWOOD, AND G. E. LUCIER. 1976. Descending influences of periaqueductal gray matter and somatosensory cerebral cortex on neurons in trigeminal brain stem nuclei. Cannd. J. Physiol. Pharmacol. 54: 66-69. 19. STEWART, W. A., AND R. B. KING. 1963. Fiber projections drom thenucleus caudalis of the spinal trigeminal nucleus. J. Comp. Neurof. 121: 271-286.
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20. TIWARI, R. K., AND R. B. KING. 1974. Fiber projections from trigeminal nucleus caudalis in primate (squirrel monkey and baboon). J. Camp. Neural. 158: 191-206. 21. WESTRUM, L. E., AND R. C. CANFIELD. 1977. Light and electron microscopy of degeneration in the brain stem spinal trigeminal nucleus following booth pulp removal inadult cats. Pages 171-180inD. J. ANDERSON AND B. MATTHEWS, Eds.,Pain in the Trigeminal Region. ElsevieriNorth-Holland, Amsterdam. 22. WESTRUM, L. E., R. C. CANFIELD, AND R. G. BLACK. 1976. Transganglionic degeneration in the spinal trigeminal nucleus following removal of tooth pulps in adult cats. Brain Res. 101: 137-140. 23. YOKOTA, T. 1975. Excitation of units in marginal rim of trigeminal subnucleus caudalis elicited by tooth pulp stimulation. Brain Res. 95: 154-158. 24. YOKOTA, T. 1976. Two types of tooth pulp units in the bulbar lateral reticular formation. Brain Res. 104: 325-329.