- Email: [email protected]
Parabrachial nucleus neurons projecting to the lower brain stem and the spinal cord. A study in the cat by the Fink-Heimer and the horseradish peroxidase methods
EXPERIMENTAL
NEUROLOGY
70,403-413
(1980)
Parabrachial Nucleus Neurons Projecting to the Lower Brain Stem and the Spinal Cord. A Study in the Cat by the Fink-Heimer and the Horseradish Peroxidase Methbds YOSHIKI
TAKEUCHI, MASANORI UEMURA, KOJYURO MATSUDA, RYOTARO MATSUSHIMA.ANDNOBORU MIZUNO'
Department of Oral Anatomy, Department of Anatomy,
School Faculty
of Dentistry, of Medicine,
Received
April
Hiroshima University, Hiroshima, Kyoto University, Kyoto, Japan
and
22, 1980
Direct projections from the parabrachial nucleus (PBN) to the lower brain stem and the spinal cord were examined in the cat by the Fink-Heimer and the horseradish peroxidase (HRP) methods. After placing lesions in the PBN, many fine degenerated fibers were seen contralaterally in the ventromedial portions of the caudal pontine reticular formation, and ipsilaterally in the lateral portions of the facial nucleus, the regions around the hypoglossal nucleus, and the regions around the ambiguus nucleus; some degenerated fibers were traced ipsilaterally down to the spinal cord. Subsequently, HRP injections were attempted into these regions where many fine degenerated fibers were observed. In cats injected with HRP into the lateral portions of the facial nucleus, the regions around the hypoglossal nucleus, the regions around the ambiguus nucleus, or the first cervical cord segment, many HRP-labeled neurons were seen in the ventral portions of the PBN. The mean of the average soma diameters of the PBN neurons labeled with HRP injected into the regions around the hypoglossal nucleus or the first cervical cord segment was significantly larger than that of the PBN neurons labeled with HRP injected into the lateral portions of the facial nucleus or the regions around the ambiguus nucleus.
INTRODUCTION The dorsolateral pontine region around the brachium conjunctivum was reported to contain the “pontine taste area” in the rat; this region, Abbreviations: HRP-horseradish peroxidase, PBN-parabrachial nucleus. r Dr. Mizuno is at the Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. 403
0014-4886/80/l 10403-l 1$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved
404
TAKEUCHI
ET AL.
particularly the parabrachial nucleus (PBN), receives gustatory inputs from the rostra1 portions of the nucleus of the solitary tract and projects to the thalamic taste area as well as to the basal forebrain regions (19-24). The PBN in the cat was also reported to receive afferent fibers from the nucleus of the solitary tract (15, 17), and to give ascending fibers to the parvocellular portion of the posteromedial ventral nucleus of the thalamus as well as to the basal forebrain structures (17, 18). However, the PBN was also shown to project many descending fibers to the lower brain stem regions (8, 17, 26, 27, 35), the spinal cord (1, 14, 36), and the cerebellum (2, 5, 32). In the present study, first the sites of termination of descending fibers from the PBN were surveyed in the cat by the Fink-Heimer method, and second the distribution of the PBN neurons projecting the descending fibers was examined by the horseradish peroxidase (HRP) method. METHODS The experiments were carried out on adult cats (1.8 to 3.5 kg) anesthetized with intramuscular ketamine hydrochloride (15 to 40 mg/kg) or intraperitoneal sodium pentobarbital (40 mg/kg). In 12 cats an electrolytic lesion was produced stereotaxically in the region around the PBN; the electrode was inclined caudally at an angle 40” in the sagittal plane. After 4 to 6 days, the cats were anesthetized deeply and perfused through the ascending aorta with 10% neutral Formalin. After further fixation for 2 weeks, frontal frozen sections were made serially at 25 pm and impregnated by the Fink-Heimer method. In 36 cats a single injection of 0.01 to 0.03 ~1 30% HRP (Toyobo Grade-I-C, RZ: 3.3) dissolved in sterile 0.9% saline containing 2% dimethyl sulfoxide was attempted within the facial, hypoglossal, or ambiguus nucleus during 30 to 40 min with a glass micropipet (tip diameter: 60 pm) attached to a l-p1 Hamilton microsyringe. In five cats a single injection of 0.1~130% HRP was made in the first cervical cord segment. The injection needle was inserted stereotaxically into the facial or ambiguus nucleus at an angle of 39” to the frontal plane to avoid striking the cerebellar tentorium, and the injections into the hypoglossal nucleus or the spinal cord were under direct vision. After 2 days, these cats were again anesthetized deeply and perfused through the ascending aorta with 1500 ml 7% Formalin in 0.9% saline adjusted topH 7.3 with sodium bicarbonate. The brains were removed immediately and placed 3 to 4 days in a 30% sucrose solution in 0.1 M phosphate buffer (PH 7.4) at 4°C. Subsequently, the brain stem was cut serially at 60 pm in the frontal plane on a freezing microtome. For the histochemical demonstration of HRP, the sections were treated with
PARABRACHIALNUCLEUSNEURONS
405
diaminobenzidine (34) or benzidine (9). After the histochemical reaction was completed, the sections were washed in 0.9% saline, mounted on gelatinized slides, counterstained with 0.1% cresyl violet for the diaminobenzidine reaction or with 1% neutral red for the be&dine reaction, and examined microscopically under bright-field and dark-field illumination. Cell soma diameters of HRP-labeled PBN neurons were measured by averaging the major diameter with the minor diameter; the major diameter was the longest axis, and the minor diameter was taken at right angle to a point halfway on the longest axis. The comparison between the means of the average soma diameters was made using the standard deviation (SD) in the differences (D) of the standard error of the mean; when the D/SD was larger than 2.72, a difference of the means was considered to be significant (P c 0.01).
FIG. 1. Diagrammatic representation of the distribution pattern of degenerated fibers (black dots) after lesion (black) in the ventral portion of the parabrachial nuclei. Arrows (A-D) point to the degeneration in the lateral part of the facial nucleus, the region around the ambiguus nucleus, the region around the hypoglossal nucleus, and the first cervical cord segment, respectively. Abbreviations in this and subsequent figures: Amb-nucleus ambiguus, Bc-brachium conjunctivum, Cu-nucleus cuneatus, G-nucleus gracilis, Ic-nucleus intercalatus, Lc-locus ceruleus, Llv-nucleus lemnisci lateralis ventralis, Oli-nucleus olivaris inferior, Ols-nucleus olivaris superior, Pbl-nucleus parabrachialis lateralis, Pbm-nucleus parabrachialis medialis, SC-nucleus subceruleus, Ts-tractus solitarius, Vm-nucleus motorius nervi trigemini, Vp-nucleus sensibilis principalis nervi trigemini, Vsp-nucleus tractus spinalis nervi trigemini, VII-nucleus nervi faciais, X-nucleus dorsalis nervi vagi, and XII-nucleus nervi hypoglossi.
406
TAKEUCHI
ET AL.
RESULTS In 12 cats with lesions involving the PBN, degenerated fibers were distributed contralaterally in the ventromedial portions of the caudal pontine reticular formation, ipsilaterally in the lateral portions of the pontine and medullary reticular formation, the regions around the hypoglossal nucleus, the regions around the ambiguus nucleus, the nucleus of the solitary tract, and the spinal cord, and bilaterally in the regions around the motor trigeminal nucleus and the main sensory trigeminal nucleus with dominance of the ipsilateral distribution, in the lateral portions of the facial nucleus with a predominance of the ipsilateral distribution, in the ventral and lateral portions of the interpolar subnucleus of the spinal trigeminal nucleus, and in the juxtatrigeminal regions (16) (Fig. 1). It could not be determined, however, if these degenerated fibers were fibers arising from the PBN, or fibers descending through the PBN regions involved in the lesion. Therefore, HRP injections were attempted into the regions where many fine degenerated fibers were seen in the cats with lesions involving the PBN, and resulting HRP-labeling of PBN neurons was examined. In 13 cats in which the HRP injection had its center in the lateral part of the facial nucleus (Fig. 2A), HRP-labeled neurons were seen in the PBN regions ipsilateral to the injection; the pattern of distribution of HRP-labeled PBN neurons in a representative cat is shown in Fig. 3 (cat 1109). HRP-labeled neurons were seen through the entire rostrocaudal extent of the PBN, especially in the ventral portions of the lateral PBN. A considerable number of HRP-labeled neurons were also seen in the neighboring regions of the PBN, especially in the locus subceruleus. Most HRP-labeled neurons clustered in the ventral portions of the PBN were fusiform or ovoid in shape (Fig. 4A). The mean of the average soma diameter of the labeled neurons in the ventral portions of the PBN was 14.6 k 0.39 pm (N = 166) (Fig. 5A). In 12 cats in which the HRP injection was centered on the regions around the ambiguus nucleus (Fig. 2B), both the morphologic features and the pattern of distribution of HRP-labeled neurons in the PBN regions were similar to those observed in the cats injected with HRP into the lateral portions of the facial nucleus (compare cat 1109 with cat 1114 in Fig. 3, and Fig. 4A with Fig. 4B). The mean of the average soma diameter of labeled FIG. 2. Photomicrographs of cross sections through the HRP injection sites in the facial nucleus (A), the region around the ambiguus nucleus (B), the region around the hypoglossal nucleus (C), and the first cervical cord segment (D). Scale bars indicate 1 mm. For abbreviations see Fig. 1.
PARABRACHIALNUCLEUSNEURONS
407
408
TAKEUCHI
ET AL.
1114
Cat
~~~~ Cat
1109
Cat
1110
Cat
1095
FIG. 3. Distribution of HRP-labeled neurons (black dots) in the PBN regions of four representative cats injected with HRP (shaded areas) in the facial nucleus (cat 1109), the ambiguus nucleus (cat 1114), the hypoglossal nucleus (cat 11lo), and the first cervical segment (cat 1095). Arrows (A-D) point to the cluster of HRP-labeled neurons in the ventral portions of the PBN.
neurons clustered in the ventral portions of the PBN was 14.9 + 0.3 1 pm (N = 205) (Fig. 5B). In 11 cats the HRP injection was centered on the hypoglossal nucleus at the levels of the obex (Fig. 2C). In the representative cat illustrated in Fig. 3 (cat 11 lo), spread of the injected HRP involved the dorsomedial portions of the medullary reticular formation around the medial nucleus of the solitary tract. In that cat, HRP-labeled PBN neurons were mainly clustered in the ventral portions of the PBN; labeled neurons were also seen in the nucleus subceruleus, the regions around the motor trigeminal nucleus, and the locus ceruleus. In that cat, the distribution of labeled PBN neurons was almost confined in the ventral portions of the PBN. Some of these labeled neurons in the ventral portions of the PBN were larger than the labeled PBN neurons seen in the cats injected with HRP into the lateral portions of
PARABRACHIAL
NUCLEUS
NEURONS
409
FIG. 4. Dark-field photomicrographs of HRP-labeled neurons in the ventral portions of the parabrachial nucleus. Each subfigure corresponds to the region indicated, respectively, with the arrows A-D in Fig. 3. Scale bars indicate 50 pm.
410
TAKEUCHI
ET AL. *m
FIG. 5. Distribution of cell body size of parabrachial neurons labeled with HRP injected into the facial nucleus (A), the regions around the ambiguus nucleus (B), the regions around the hypoglossal nucleus (C), and the first cervical cord segment(D), as represented with major (abscissa) and minor (ordinate) diameters of the somata.
the facial nucleus or the regions around the ambiguus nucleus (compare Fig. 4C with Fig. 4A and B). The mean of the average soma diameter of labeled neurons in cat 1110 was 21.3 k 0.5 pm (N = 124), which was significantly larger than that in cat 1109 or cat 1114 (Fig. 5). In five cats injected with HRP unilaterally in the first cervical cord segment (Fig. 2D), many HRP-labeled neurons were seen in the ventral portions of the PBN ipsilateral to the HRP injection. The findings in a representative cat were shown in Fig. 2 (cat 1095). Here the pattern of distribution of HRP-labeled neurons was similar to that of cat 1110, and larger PBN neurons were also labeled in this cat as in cat 1110 (compare Fig. 4D with Fig. 4C). The mean of the average soma diameter of HRPlabeled PBN neurons in cat 1095 was 21.1 + 0.41 pm (N = 81) (Fig. SD). Thus, the cell morphology and the distribution pattern of HRP-labeled PBN neurons after HRP injections into the lateral portions of the facial nucleus or the regions around the ambiguus nucleus were somewhat different from those labeled with HRP injected into the regions around the hypoglossal nucleus or the first cervical cord segment. DISCUSSION In the present study the regions surrounding the brachium conjunctivum, i.e., the PBN, the nucleus subceruleus, and the locus ceruleus, were shown to be the sites of origin of descending fibers projecting to the lateral portions
PARABRACHIALNUCLEUSNEURONS
411
of the facial nucleus, the regions in the medullary reticular formation around the hypoglossal and ambiguus nuclei, and the spinal cord; these fibers appeared to arise mainly from the ventral portions of the PBN. Because the PBN of the cat was reported to contain catecholamine neurons (12,25), some PBN neurons labeled with HRP in the present study might send catecholaminergic fibers to the lower brain stem regions and/or the spinal cord. Some neurons in the ventral portions of the PBN, which sent their axons to the regions around the hypoglossal nucleus and/or the spinal cord, were found to be larger than other PBN neurons. The ventral portions of the PBN in the cat were also reported to contain many neurons giving ascending fibers to the forebrain regions, especially to the central nucleus of the amygdala (17, 18). Most of these PBN neurons, however, did not seem to be as large as the larger PBN neurons found in the present study. Therefore, some of the larger PBN neurons found in the present study appeared to be different from those having ascending fibers to the forebrain regions. On the other hand, judging from the cell morphology and the distribution pattern, the smaller PBN neurons might contribute both ascending and descending fibers by way of axon collaterals to the forebrain regions and the lower brain stem regions and/or the spinal cord. The PBN of the cat was reported to receive afferent fibers from the central nucleus of the amygdala (11, 13, 17, 18) and other basal forebrain regions (17,28,29,3 1) as well as from the nucleus of the solitary tract (15, 17) and other various regions of the lower brain stem (17, 28, 31). These various afferent inputs to the PBN were considered to be mediated to the lower brain stem regions and the spinal cord through the descending fibers arising from the PBN regions; these connections may play roles in control of respiration (4,6-8, 10,30), locomotor activities (10,33), and/or emotional behaviors (3). REFERENCES 1. BASBAUM, A. I., AND H. L. FIELDS. 1979. The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation. .I. Comp. Neural. 187: 513-532. 2. BATINI, C., C. BIJISSERET-DELMAS, I. CORVISIER, 0. HARDY, AND D. JASSIKGERSCHENFELD. 1978. Brain stem nuclei giving fibers to lobules VI and VII of the cerebellar vermis. Brain Res. 153: 241-261. 3. BERNSTON, G. G. 1972. Attacks, grooming, and threat elicited by stimulation of the pontine tegmentum in cats. Physiol. Behav. 11: 81-87. 4. BERTRAND, F., AND A. HUGELIN. 1971. Respiratory synchronizing function of nucleus parabrachialis medialis: pneumotaxic mechanismus. J. Neurophysiol. 34: 189-207. 5. CHU, N.-S., AND F. E. BLOOM. 1974. The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections. Brain Res. 66: l-21.
412
TAKEUCHI
ET AL.
6. COHEN, M. I. 1971. Switching of the respiratory phases and evoked phrenic responses produced by rostra1 pontine electrical stimulation. J. Physiol. (London) 217, 133- 158. 7. COHEN, M. I., AND S. C. WANG. 1959. Respiratory neuronal activity in pons of cat. J. Neurophysiol. 22: 33-50. 8. DENAVIT-SAUBIP, M., AND D. RICHE. 19’77. Descending input from the pneumotaxic system to the lateral respiratory nucleus of the medulla. An anatomical study with horseradish peroxidase technique. Neurosci. Left. 6: 121-126. 9. DE OLMOS J. S. 1977. An improved HRP method for the study of central nervous connections. Exp. Brain Res. 29: 541-551. 10. EULER, C. VON, AND T. TRIPPENBACH. 1976. Excitability changes of the inspiratory “off-switch” mechanism tested by electrical stimulation in nucleus parabrachialis in the cat. Acta Physiol. Stand. 97: 175- 188. 11. HOPKINS, D. A.. AND G. HOLSTEGE. 1978. Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Exp. Bruin Res. 32: 539-547. 12. JONES, B. E., AND R. Y. MOORE. 1974. Catecholamine-containing neurons of the nucleus locus coeruleus in the cat. J. Comp. Neural. 157: 43-52. 13. KRETTEK, J. E., AND J. L. PRICE. 1978. Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. Comp. Neuro/. 178: 225-254. 14. KUYPERS, H. G. J. M., AND V. A. MAYSKY. 1975. Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the cat. Neurosci. Lett. 1: 9-14. 15. LOEWY, A. D., AND H. BURTON. 1978. Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat. J. Comp. Neural. 181: 421-450. 16. MIZUNO, N. 1970. Projection fibers from the main sensory trigeminal nucleus and the supratrigeminal region. J. Comp. Neurol. 139: 457-472. 17. MIZUNO, N., S. NOMURA, AND Y. TAKEUCHI. 1980. The parabrachial nucleus as an intermediate relay station of the visceral afferent pathways in the cat. In M. IT0 et al. (Eds). integrative Control Functions of the Brain, Vol. 3, Kodansha, TokyoiElsevier, Amsterdam, in press. 18. NOMURA, S., N. MIZUNO, K. ITOH, K. MATSUDA, T. SUGIMOTO, AND Y. NAKAMURA. 1979. Localization of parabrachial nucleus neurons projecting to the thalamus or the amygdala in the cat using horseradish peroxidase. Exp. Neurol. 64: 375-385. 19. NORGREN, R. 1974. Gustatory afferents to ventral forebrain. Brain Res. 81: 285-295. 20. NORGREN, R. 1976. Taste pathways to hypothalamus and amygdala. J. Comp. Neural. 166: 17-30. 21. NORGREN, R. 1978. Projections from the nucleus of the solitary tract in the rat. Neuroscience 3: 207-218. 22. NORGREN. R., AND C. M. LEONARD. 1971. Taste pathways in rat brainstem. Science 173: 1136- 1139. 23. NORGREN, R., AND C. M. LEONARD. 1973. Ascending central gustatory pathways. J. Comp. Neural. 150: 217-238. 24. NORGREN, R., AND C. PFAFFMANN. 1975. The pontine taste areain the rat. Brain Res. 91: 99- 117. 25. POITRAS, D., AND A. PARANT. 1978. Atlas of the distribution of monoamine-containing nerve cell bodies in the brain stem of the cat. J. Comp. Neural. 179: 699-718. 26. RICHE. D.. M. DENAVIT-SAUBI~, AND J. CHAMPAGNAT. 1979. Pontine afferents to the medullary respiratory system: anatomofunctional correlation. Neurosci. Lett. 13: 151-155.
PARABRACHIALNUCLEUSNEURONS
413
27. SAKAI, K., J. P. SASTRE, D. SALVERT, M. TOURET, M. TOHYAMA, AND M. JOUVET. 1979. Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: an HRP study. Brain Res. 176: 233-254. 28. SAKAI, K., M. TOURET, D. SALVERT, L. LEGER, AND M. JOUVET. 1977. Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique. Brain Res. 119: 21-41. 29. SAPER, C. B., A. D. LOEWY, L. M. SWANSON, AND W. M. COWAN. 1976. Direct hypothalamo-autonomic connections. Brain Res. 117: 305-312. 30. SIECK, G. C., AND R. M. HARPER. 1980. Pneumotaxic area neuronal discharge during sleep-waking states in the cat. Exp. Neurol. 67: 79- 102. 31. SMITH, D. A., AND J. P. FLYNN. 1979. Afferent projections related to attack sites in the pontine tegmentum. Brain Res. 164: 103-119. 32. SOMANA, R., AND F. WALBERG. 1979. The cerebellar projection from the parabrachial nucleus in the cat. Brairr Res. 172: 144-149. 33. STEEVES, J. D., L. M. JORDAN, AND N. LAKE. 1975. The close proximity of catecholamine-containing cells to the “mesencephalic locomotor region” (MLR). Brain Res. 100: 663-670. 34. STREIT, P., AND J. C. REUBI. 1977. A new and sensitive staining method for axonal transported horseradish peroxidase (HRP) in the pigeon visual system. Bruin Res. 126: 530-537. 35. TAKEUCHI, Y., K. NAKANO, M. UEMURA, K. MATSUDA, R. MATSUSHIMA, AND N. MIZUNO. 1979. Mesencephalic and pontine afferent fiber system to the facial nucleus in the cat: a study using the horseradish peroxidase and silver impregnation techniques. Exp. Neurol. 66: 330-342. 36. TOHYAMA, M., K. SAKAI, M. TOURET, D. SALVERT, AND M. JOUVET. 1979. Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. II. Projections from the dorsolateral pontine tegmentum and raphe nuclei. Brain Res. 176: 215-231.
NEUROLOGY
70,403-413
(1980)
Parabrachial Nucleus Neurons Projecting to the Lower Brain Stem and the Spinal Cord. A Study in the Cat by the Fink-Heimer and the Horseradish Peroxidase Methbds YOSHIKI
TAKEUCHI, MASANORI UEMURA, KOJYURO MATSUDA, RYOTARO MATSUSHIMA.ANDNOBORU MIZUNO'
Department of Oral Anatomy, Department of Anatomy,
School Faculty
of Dentistry, of Medicine,
Received
April
Hiroshima University, Hiroshima, Kyoto University, Kyoto, Japan
and
22, 1980
Direct projections from the parabrachial nucleus (PBN) to the lower brain stem and the spinal cord were examined in the cat by the Fink-Heimer and the horseradish peroxidase (HRP) methods. After placing lesions in the PBN, many fine degenerated fibers were seen contralaterally in the ventromedial portions of the caudal pontine reticular formation, and ipsilaterally in the lateral portions of the facial nucleus, the regions around the hypoglossal nucleus, and the regions around the ambiguus nucleus; some degenerated fibers were traced ipsilaterally down to the spinal cord. Subsequently, HRP injections were attempted into these regions where many fine degenerated fibers were observed. In cats injected with HRP into the lateral portions of the facial nucleus, the regions around the hypoglossal nucleus, the regions around the ambiguus nucleus, or the first cervical cord segment, many HRP-labeled neurons were seen in the ventral portions of the PBN. The mean of the average soma diameters of the PBN neurons labeled with HRP injected into the regions around the hypoglossal nucleus or the first cervical cord segment was significantly larger than that of the PBN neurons labeled with HRP injected into the lateral portions of the facial nucleus or the regions around the ambiguus nucleus.
INTRODUCTION The dorsolateral pontine region around the brachium conjunctivum was reported to contain the “pontine taste area” in the rat; this region, Abbreviations: HRP-horseradish peroxidase, PBN-parabrachial nucleus. r Dr. Mizuno is at the Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. 403
0014-4886/80/l 10403-l 1$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved
404
TAKEUCHI
ET AL.
particularly the parabrachial nucleus (PBN), receives gustatory inputs from the rostra1 portions of the nucleus of the solitary tract and projects to the thalamic taste area as well as to the basal forebrain regions (19-24). The PBN in the cat was also reported to receive afferent fibers from the nucleus of the solitary tract (15, 17), and to give ascending fibers to the parvocellular portion of the posteromedial ventral nucleus of the thalamus as well as to the basal forebrain structures (17, 18). However, the PBN was also shown to project many descending fibers to the lower brain stem regions (8, 17, 26, 27, 35), the spinal cord (1, 14, 36), and the cerebellum (2, 5, 32). In the present study, first the sites of termination of descending fibers from the PBN were surveyed in the cat by the Fink-Heimer method, and second the distribution of the PBN neurons projecting the descending fibers was examined by the horseradish peroxidase (HRP) method. METHODS The experiments were carried out on adult cats (1.8 to 3.5 kg) anesthetized with intramuscular ketamine hydrochloride (15 to 40 mg/kg) or intraperitoneal sodium pentobarbital (40 mg/kg). In 12 cats an electrolytic lesion was produced stereotaxically in the region around the PBN; the electrode was inclined caudally at an angle 40” in the sagittal plane. After 4 to 6 days, the cats were anesthetized deeply and perfused through the ascending aorta with 10% neutral Formalin. After further fixation for 2 weeks, frontal frozen sections were made serially at 25 pm and impregnated by the Fink-Heimer method. In 36 cats a single injection of 0.01 to 0.03 ~1 30% HRP (Toyobo Grade-I-C, RZ: 3.3) dissolved in sterile 0.9% saline containing 2% dimethyl sulfoxide was attempted within the facial, hypoglossal, or ambiguus nucleus during 30 to 40 min with a glass micropipet (tip diameter: 60 pm) attached to a l-p1 Hamilton microsyringe. In five cats a single injection of 0.1~130% HRP was made in the first cervical cord segment. The injection needle was inserted stereotaxically into the facial or ambiguus nucleus at an angle of 39” to the frontal plane to avoid striking the cerebellar tentorium, and the injections into the hypoglossal nucleus or the spinal cord were under direct vision. After 2 days, these cats were again anesthetized deeply and perfused through the ascending aorta with 1500 ml 7% Formalin in 0.9% saline adjusted topH 7.3 with sodium bicarbonate. The brains were removed immediately and placed 3 to 4 days in a 30% sucrose solution in 0.1 M phosphate buffer (PH 7.4) at 4°C. Subsequently, the brain stem was cut serially at 60 pm in the frontal plane on a freezing microtome. For the histochemical demonstration of HRP, the sections were treated with
PARABRACHIALNUCLEUSNEURONS
405
diaminobenzidine (34) or benzidine (9). After the histochemical reaction was completed, the sections were washed in 0.9% saline, mounted on gelatinized slides, counterstained with 0.1% cresyl violet for the diaminobenzidine reaction or with 1% neutral red for the be&dine reaction, and examined microscopically under bright-field and dark-field illumination. Cell soma diameters of HRP-labeled PBN neurons were measured by averaging the major diameter with the minor diameter; the major diameter was the longest axis, and the minor diameter was taken at right angle to a point halfway on the longest axis. The comparison between the means of the average soma diameters was made using the standard deviation (SD) in the differences (D) of the standard error of the mean; when the D/SD was larger than 2.72, a difference of the means was considered to be significant (P c 0.01).
FIG. 1. Diagrammatic representation of the distribution pattern of degenerated fibers (black dots) after lesion (black) in the ventral portion of the parabrachial nuclei. Arrows (A-D) point to the degeneration in the lateral part of the facial nucleus, the region around the ambiguus nucleus, the region around the hypoglossal nucleus, and the first cervical cord segment, respectively. Abbreviations in this and subsequent figures: Amb-nucleus ambiguus, Bc-brachium conjunctivum, Cu-nucleus cuneatus, G-nucleus gracilis, Ic-nucleus intercalatus, Lc-locus ceruleus, Llv-nucleus lemnisci lateralis ventralis, Oli-nucleus olivaris inferior, Ols-nucleus olivaris superior, Pbl-nucleus parabrachialis lateralis, Pbm-nucleus parabrachialis medialis, SC-nucleus subceruleus, Ts-tractus solitarius, Vm-nucleus motorius nervi trigemini, Vp-nucleus sensibilis principalis nervi trigemini, Vsp-nucleus tractus spinalis nervi trigemini, VII-nucleus nervi faciais, X-nucleus dorsalis nervi vagi, and XII-nucleus nervi hypoglossi.
406
TAKEUCHI
ET AL.
RESULTS In 12 cats with lesions involving the PBN, degenerated fibers were distributed contralaterally in the ventromedial portions of the caudal pontine reticular formation, ipsilaterally in the lateral portions of the pontine and medullary reticular formation, the regions around the hypoglossal nucleus, the regions around the ambiguus nucleus, the nucleus of the solitary tract, and the spinal cord, and bilaterally in the regions around the motor trigeminal nucleus and the main sensory trigeminal nucleus with dominance of the ipsilateral distribution, in the lateral portions of the facial nucleus with a predominance of the ipsilateral distribution, in the ventral and lateral portions of the interpolar subnucleus of the spinal trigeminal nucleus, and in the juxtatrigeminal regions (16) (Fig. 1). It could not be determined, however, if these degenerated fibers were fibers arising from the PBN, or fibers descending through the PBN regions involved in the lesion. Therefore, HRP injections were attempted into the regions where many fine degenerated fibers were seen in the cats with lesions involving the PBN, and resulting HRP-labeling of PBN neurons was examined. In 13 cats in which the HRP injection had its center in the lateral part of the facial nucleus (Fig. 2A), HRP-labeled neurons were seen in the PBN regions ipsilateral to the injection; the pattern of distribution of HRP-labeled PBN neurons in a representative cat is shown in Fig. 3 (cat 1109). HRP-labeled neurons were seen through the entire rostrocaudal extent of the PBN, especially in the ventral portions of the lateral PBN. A considerable number of HRP-labeled neurons were also seen in the neighboring regions of the PBN, especially in the locus subceruleus. Most HRP-labeled neurons clustered in the ventral portions of the PBN were fusiform or ovoid in shape (Fig. 4A). The mean of the average soma diameter of the labeled neurons in the ventral portions of the PBN was 14.6 k 0.39 pm (N = 166) (Fig. 5A). In 12 cats in which the HRP injection was centered on the regions around the ambiguus nucleus (Fig. 2B), both the morphologic features and the pattern of distribution of HRP-labeled neurons in the PBN regions were similar to those observed in the cats injected with HRP into the lateral portions of the facial nucleus (compare cat 1109 with cat 1114 in Fig. 3, and Fig. 4A with Fig. 4B). The mean of the average soma diameter of labeled FIG. 2. Photomicrographs of cross sections through the HRP injection sites in the facial nucleus (A), the region around the ambiguus nucleus (B), the region around the hypoglossal nucleus (C), and the first cervical cord segment (D). Scale bars indicate 1 mm. For abbreviations see Fig. 1.
PARABRACHIALNUCLEUSNEURONS
407
408
TAKEUCHI
ET AL.
1114
Cat
~~~~ Cat
1109
Cat
1110
Cat
1095
FIG. 3. Distribution of HRP-labeled neurons (black dots) in the PBN regions of four representative cats injected with HRP (shaded areas) in the facial nucleus (cat 1109), the ambiguus nucleus (cat 1114), the hypoglossal nucleus (cat 11lo), and the first cervical segment (cat 1095). Arrows (A-D) point to the cluster of HRP-labeled neurons in the ventral portions of the PBN.
neurons clustered in the ventral portions of the PBN was 14.9 + 0.3 1 pm (N = 205) (Fig. 5B). In 11 cats the HRP injection was centered on the hypoglossal nucleus at the levels of the obex (Fig. 2C). In the representative cat illustrated in Fig. 3 (cat 11 lo), spread of the injected HRP involved the dorsomedial portions of the medullary reticular formation around the medial nucleus of the solitary tract. In that cat, HRP-labeled PBN neurons were mainly clustered in the ventral portions of the PBN; labeled neurons were also seen in the nucleus subceruleus, the regions around the motor trigeminal nucleus, and the locus ceruleus. In that cat, the distribution of labeled PBN neurons was almost confined in the ventral portions of the PBN. Some of these labeled neurons in the ventral portions of the PBN were larger than the labeled PBN neurons seen in the cats injected with HRP into the lateral portions of
PARABRACHIAL
NUCLEUS
NEURONS
409
FIG. 4. Dark-field photomicrographs of HRP-labeled neurons in the ventral portions of the parabrachial nucleus. Each subfigure corresponds to the region indicated, respectively, with the arrows A-D in Fig. 3. Scale bars indicate 50 pm.
410
TAKEUCHI
ET AL. *m
FIG. 5. Distribution of cell body size of parabrachial neurons labeled with HRP injected into the facial nucleus (A), the regions around the ambiguus nucleus (B), the regions around the hypoglossal nucleus (C), and the first cervical cord segment(D), as represented with major (abscissa) and minor (ordinate) diameters of the somata.
the facial nucleus or the regions around the ambiguus nucleus (compare Fig. 4C with Fig. 4A and B). The mean of the average soma diameter of labeled neurons in cat 1110 was 21.3 k 0.5 pm (N = 124), which was significantly larger than that in cat 1109 or cat 1114 (Fig. 5). In five cats injected with HRP unilaterally in the first cervical cord segment (Fig. 2D), many HRP-labeled neurons were seen in the ventral portions of the PBN ipsilateral to the HRP injection. The findings in a representative cat were shown in Fig. 2 (cat 1095). Here the pattern of distribution of HRP-labeled neurons was similar to that of cat 1110, and larger PBN neurons were also labeled in this cat as in cat 1110 (compare Fig. 4D with Fig. 4C). The mean of the average soma diameter of HRPlabeled PBN neurons in cat 1095 was 21.1 + 0.41 pm (N = 81) (Fig. SD). Thus, the cell morphology and the distribution pattern of HRP-labeled PBN neurons after HRP injections into the lateral portions of the facial nucleus or the regions around the ambiguus nucleus were somewhat different from those labeled with HRP injected into the regions around the hypoglossal nucleus or the first cervical cord segment. DISCUSSION In the present study the regions surrounding the brachium conjunctivum, i.e., the PBN, the nucleus subceruleus, and the locus ceruleus, were shown to be the sites of origin of descending fibers projecting to the lateral portions
PARABRACHIALNUCLEUSNEURONS
411
of the facial nucleus, the regions in the medullary reticular formation around the hypoglossal and ambiguus nuclei, and the spinal cord; these fibers appeared to arise mainly from the ventral portions of the PBN. Because the PBN of the cat was reported to contain catecholamine neurons (12,25), some PBN neurons labeled with HRP in the present study might send catecholaminergic fibers to the lower brain stem regions and/or the spinal cord. Some neurons in the ventral portions of the PBN, which sent their axons to the regions around the hypoglossal nucleus and/or the spinal cord, were found to be larger than other PBN neurons. The ventral portions of the PBN in the cat were also reported to contain many neurons giving ascending fibers to the forebrain regions, especially to the central nucleus of the amygdala (17, 18). Most of these PBN neurons, however, did not seem to be as large as the larger PBN neurons found in the present study. Therefore, some of the larger PBN neurons found in the present study appeared to be different from those having ascending fibers to the forebrain regions. On the other hand, judging from the cell morphology and the distribution pattern, the smaller PBN neurons might contribute both ascending and descending fibers by way of axon collaterals to the forebrain regions and the lower brain stem regions and/or the spinal cord. The PBN of the cat was reported to receive afferent fibers from the central nucleus of the amygdala (11, 13, 17, 18) and other basal forebrain regions (17,28,29,3 1) as well as from the nucleus of the solitary tract (15, 17) and other various regions of the lower brain stem (17, 28, 31). These various afferent inputs to the PBN were considered to be mediated to the lower brain stem regions and the spinal cord through the descending fibers arising from the PBN regions; these connections may play roles in control of respiration (4,6-8, 10,30), locomotor activities (10,33), and/or emotional behaviors (3). REFERENCES 1. BASBAUM, A. I., AND H. L. FIELDS. 1979. The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation. .I. Comp. Neural. 187: 513-532. 2. BATINI, C., C. BIJISSERET-DELMAS, I. CORVISIER, 0. HARDY, AND D. JASSIKGERSCHENFELD. 1978. Brain stem nuclei giving fibers to lobules VI and VII of the cerebellar vermis. Brain Res. 153: 241-261. 3. BERNSTON, G. G. 1972. Attacks, grooming, and threat elicited by stimulation of the pontine tegmentum in cats. Physiol. Behav. 11: 81-87. 4. BERTRAND, F., AND A. HUGELIN. 1971. Respiratory synchronizing function of nucleus parabrachialis medialis: pneumotaxic mechanismus. J. Neurophysiol. 34: 189-207. 5. CHU, N.-S., AND F. E. BLOOM. 1974. The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections. Brain Res. 66: l-21.
412
TAKEUCHI
ET AL.
6. COHEN, M. I. 1971. Switching of the respiratory phases and evoked phrenic responses produced by rostra1 pontine electrical stimulation. J. Physiol. (London) 217, 133- 158. 7. COHEN, M. I., AND S. C. WANG. 1959. Respiratory neuronal activity in pons of cat. J. Neurophysiol. 22: 33-50. 8. DENAVIT-SAUBIP, M., AND D. RICHE. 19’77. Descending input from the pneumotaxic system to the lateral respiratory nucleus of the medulla. An anatomical study with horseradish peroxidase technique. Neurosci. Left. 6: 121-126. 9. DE OLMOS J. S. 1977. An improved HRP method for the study of central nervous connections. Exp. Brain Res. 29: 541-551. 10. EULER, C. VON, AND T. TRIPPENBACH. 1976. Excitability changes of the inspiratory “off-switch” mechanism tested by electrical stimulation in nucleus parabrachialis in the cat. Acta Physiol. Stand. 97: 175- 188. 11. HOPKINS, D. A.. AND G. HOLSTEGE. 1978. Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Exp. Bruin Res. 32: 539-547. 12. JONES, B. E., AND R. Y. MOORE. 1974. Catecholamine-containing neurons of the nucleus locus coeruleus in the cat. J. Comp. Neural. 157: 43-52. 13. KRETTEK, J. E., AND J. L. PRICE. 1978. Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. Comp. Neuro/. 178: 225-254. 14. KUYPERS, H. G. J. M., AND V. A. MAYSKY. 1975. Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the cat. Neurosci. Lett. 1: 9-14. 15. LOEWY, A. D., AND H. BURTON. 1978. Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat. J. Comp. Neural. 181: 421-450. 16. MIZUNO, N. 1970. Projection fibers from the main sensory trigeminal nucleus and the supratrigeminal region. J. Comp. Neurol. 139: 457-472. 17. MIZUNO, N., S. NOMURA, AND Y. TAKEUCHI. 1980. The parabrachial nucleus as an intermediate relay station of the visceral afferent pathways in the cat. In M. IT0 et al. (Eds). integrative Control Functions of the Brain, Vol. 3, Kodansha, TokyoiElsevier, Amsterdam, in press. 18. NOMURA, S., N. MIZUNO, K. ITOH, K. MATSUDA, T. SUGIMOTO, AND Y. NAKAMURA. 1979. Localization of parabrachial nucleus neurons projecting to the thalamus or the amygdala in the cat using horseradish peroxidase. Exp. Neurol. 64: 375-385. 19. NORGREN, R. 1974. Gustatory afferents to ventral forebrain. Brain Res. 81: 285-295. 20. NORGREN, R. 1976. Taste pathways to hypothalamus and amygdala. J. Comp. Neural. 166: 17-30. 21. NORGREN, R. 1978. Projections from the nucleus of the solitary tract in the rat. Neuroscience 3: 207-218. 22. NORGREN. R., AND C. M. LEONARD. 1971. Taste pathways in rat brainstem. Science 173: 1136- 1139. 23. NORGREN, R., AND C. M. LEONARD. 1973. Ascending central gustatory pathways. J. Comp. Neural. 150: 217-238. 24. NORGREN, R., AND C. PFAFFMANN. 1975. The pontine taste areain the rat. Brain Res. 91: 99- 117. 25. POITRAS, D., AND A. PARANT. 1978. Atlas of the distribution of monoamine-containing nerve cell bodies in the brain stem of the cat. J. Comp. Neural. 179: 699-718. 26. RICHE. D.. M. DENAVIT-SAUBI~, AND J. CHAMPAGNAT. 1979. Pontine afferents to the medullary respiratory system: anatomofunctional correlation. Neurosci. Lett. 13: 151-155.
PARABRACHIALNUCLEUSNEURONS
413
27. SAKAI, K., J. P. SASTRE, D. SALVERT, M. TOURET, M. TOHYAMA, AND M. JOUVET. 1979. Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: an HRP study. Brain Res. 176: 233-254. 28. SAKAI, K., M. TOURET, D. SALVERT, L. LEGER, AND M. JOUVET. 1977. Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique. Brain Res. 119: 21-41. 29. SAPER, C. B., A. D. LOEWY, L. M. SWANSON, AND W. M. COWAN. 1976. Direct hypothalamo-autonomic connections. Brain Res. 117: 305-312. 30. SIECK, G. C., AND R. M. HARPER. 1980. Pneumotaxic area neuronal discharge during sleep-waking states in the cat. Exp. Neurol. 67: 79- 102. 31. SMITH, D. A., AND J. P. FLYNN. 1979. Afferent projections related to attack sites in the pontine tegmentum. Brain Res. 164: 103-119. 32. SOMANA, R., AND F. WALBERG. 1979. The cerebellar projection from the parabrachial nucleus in the cat. Brairr Res. 172: 144-149. 33. STEEVES, J. D., L. M. JORDAN, AND N. LAKE. 1975. The close proximity of catecholamine-containing cells to the “mesencephalic locomotor region” (MLR). Brain Res. 100: 663-670. 34. STREIT, P., AND J. C. REUBI. 1977. A new and sensitive staining method for axonal transported horseradish peroxidase (HRP) in the pigeon visual system. Bruin Res. 126: 530-537. 35. TAKEUCHI, Y., K. NAKANO, M. UEMURA, K. MATSUDA, R. MATSUSHIMA, AND N. MIZUNO. 1979. Mesencephalic and pontine afferent fiber system to the facial nucleus in the cat: a study using the horseradish peroxidase and silver impregnation techniques. Exp. Neurol. 66: 330-342. 36. TOHYAMA, M., K. SAKAI, M. TOURET, D. SALVERT, AND M. JOUVET. 1979. Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. II. Projections from the dorsolateral pontine tegmentum and raphe nuclei. Brain Res. 176: 215-231.