The organization of the parabigemino-tectal projections in the opossum

Brain Research, 198 (1980) 183-189 © Elsevier/North-Holland Biomedical Press

183

Short Communications

The organization of the parabigemino-tectal projections in the opossum

ROSAL1A MI~NDEZ-OTERO, CARLOS EDUARDO ROCHA-MIRANDA and VICTOR HUGH PERRY* Departamento de Neurobiologia, Instituto de Bwfisica da UFRJ, Centro de Ci~nciasda Sat~de, BIoco G. Cidade Universittiria, Ilha do Fundfio 21910, Rio de Janeiro RJ (Brasi!)

(Accepted June 5th, 1980) Key words: parabigeminal nucleus -- superior colliculus-- opossum

The superior colliculus of the opossum (Didelphis marsupialis aurita, Wied, 1826) can be subdivided into 3 regions according to the terminal pattern of the retinotectal projection: a rostral pole, a direct binocular region and a caudal region. Only the direct binocular region receives the terminals from the ipsilateral ganglion cells and its rostral border corresponds to the vertical meridian representation 14. Neurons in the rostral pole (ipsilateral hemifield18) can be driven by both eyes8. The response to stimulation of the contralateral eye may be conveyed by the direct projection from the temporal retina to this region 10. The pathway from the ipsilateral eye to the rostral pole is unknown. The parabigeminal nucleus has been shown in the cat to be reciprocally and almost restrictively connected with the superior colliculus, suggesting that this nucleus is closely involved with visual processing of the tectum 16. This paper reports a well organized projection from the parabigeminal nucleus to both superior colliculi in the opossum and advances the hypothesis that the crossed parabigemino-tectal pathway may convey the information from the ipsilateral eye to the rostral pole. Experiments were carried out in 11 adult opossums anesthetized with 1-1.5 70 halothane (Ayerst) in a mixture of 70 7oo N20 and 30 70 02, paralyzed with pancuronium bromide (Pavulon, Organon) and artificially respired. Visual response fields to a small flashing spot were mapped on a transparent hemisphere (radius 30 cm) during multi-unit recording from the superficial layers of the superior colliculus. The position of the horizontal and vertical meridians were estimated from the optic disc projection 12. After mapping the response field, horseradish peroxidase (HRP, Sigma type VI) was inj ected electrophorectically6 (positive rectangular pulses of 4 #A with a total on-time of 30-50 min) by means of a double-barrelled glass micropipette. One barrel was filled with a 25 700solution of HRP in 50 mM Tris.HCl at pH 7.6 and the other, used * Present address: Department of Experimental Psychology, University of Oxford, South Parks Road, Oxford OX1 3UD, U.K.

184 for recording, with a 0.9 To NaCl solution. Each animal received a single injection. Between 20 and 24 h after the H R P injections the animals were perfused through the aorta, under deep anesthesia, with a 0.9 o/,, NaCI solution followed by a mixture of 2 '}4; paraformaldehyde and !.25~o glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), following Rosene and Mesulam's technique 15. Histological analysis was performed on serial frozen sections cut frontally at 40 ktm, reacted with tetramethyl benzidine 11 (TMB, Sigma) and mounted on glass slides. Alternate sections were counterstained with neutral red for cytoarchitectonic analysis. Labeled cells were counted under bright-field illumination. The parabigeminal nucleus in the opossum extends along the anteroposterior axis for about 2 mm, from the caudal pole of the medial geniculate nucleus to the level of the dorsal nucleus of the lateral lemniscus. It lies along the lateral margin of the midbrain ventral to the brachium of the inferior colliculus. In Nissl stained sections we found no evidence of the subdivisions described in the rat 17. In the atlas of the opossum brain by Oswaldo-Cruz and Rocha-Miranda la this nucleus was named sagulum, since the term parabigeminal had been reserved by Bodian I for another tegmental nuclear mass. This nomenclature is now revised in view of the present results and the fact that the current usage of the term parabigeminal nucleus is well established in several mammals2,.~, 17. Four animals received large injections of H R P placed near the representation of the vertical meridian in the superior colliculus. The enzyme spread to the deep layers and labeled cells were found bilaterally in the parabigeminal nuclei and, sparsely, in the adjacent tegmentum as described in the cat 5. In the opossum, in contrast to the cat 18, labeled cells are more numerous in the contralateral parabigeminal nucleus. In some sections almost all cells in this nucleus are filled with H R P (Fig. 1A and B). Labeled cells are found along the entire rostro-caudal axis of the nucleus with a peak density at about 0.6 m m from the rostral end (Fig. 1C). The distribution is more uniform in the ipsilateral side except at both ends, where few labeled cells are found (Fig. 1F). The anterograde transport of H R P 11 is restricted to the ipsilateral side (Fig. 1D and E) and encompasses the same levels which show retrogradely labeled cells, as well as the scantily marked rostral segment. The other 7 animals received small injections limited to the superficial layers, at sites representing the following meridians: - - 3 4 °, --28 ° and - - 3 °, in the ipsilateral hemifield, and 17°, 39 °, 40 ° and 53 °, in the contralateral hemifield. Labeling of the parabigeminal nucleus was restricted to the contralateral side after injections placed at

Fig. 1. Bright- and dark-field photomicrographs of frontal sections of the parabigeminal nuclei at corresponding levels on the eontralateral (A and B) and ipsilateral side (D and E) of the brain after a large HRP injection in the superior colliculus at 0° of visual field representation. Observe that there are many more retrogradely labeled cells in the contralateral than in the ipsilateral side (compare A with D) and that the anterograde transport of HRP is restricted to the ipsilateral nucleus (compare B with E). Calibration bar = 50 ktm. C and F: distribution of labeled cells along the rostro-caudal axis of the parabigeminal nuclei on the contralateral (C) and ipsilateral side (F) in the same animal. The ordinates represent the number of labeled cells sampled at regular intervals along the rostro-caudal axis (abscissae). The vertical scale indicates 15 labeled cells and the horizontal scale represents 0.2 ram.

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186 --34 ° and --28 ° (rostral pole), while only the ipsilateral nucleus was labeled at 53°. The other injections labeled both ipsilateral and contralateral parabigeminal nuclei. A topographic relationship between the superior colliculus and the parabigeminal nucleus can be found following reconstructions of the distribution of labeled cells in the rostro-caudal axis of the parabigeminal nuclei (Fig. 2). In the ipsilateral side the location of labeled cells shifts from the caudal end to the middle part of the nucleus as the injection site is displaced from 53° to the vertical meridian representation in the colliculus. The injection centered at --3 ° in the rostral pole labeled only a few scattered cells and that at --28 °, none at all (Fig. 2A-E on the right hand side of the figure). There is a projection from the contralateral parabigeminal nucleus to the rostral pole and to that portion of the rectum representing the contralateral hemifield up to at least 40 ° of longitude but we have found no evidence for a projection to the caudal region (Fig. 2A-E on the left hand side of the figure). Moving the injection site forward in the tectum results in a caudal displacement of the labeled cells in the contralateral parabigeminal nucleus, the opposite direction to that found in the ipsilateral nucleus. The tecto-parabigeminal projection is restricted to the ipsilateral side. After injections in the rostral pole or in the region representing the contralateral hemifield, evidence of anterogradely transported HRP was found in the rostral and middle parts of the parabigeminal nucleus, respectively. A similar organization has been described in the cat 4 and tree shrew 8. The existence of a retinotopic organization in the parabigeminal nucleus similar to that of the ipsilateral colliculus has been described in the cat 16. In our data this is suggested by the distribution of the parabigemino-tectal projections described in the preceding paragraphs. The displacement of the maximum number of labeled cells in both nuclei would show the same trend illustrated in Fig. 2 if one segment of the parabigeminal were to be linked with retinotopic homologous regions of both colliculi. An unexpected, albeit not contradictory, finding in the proposed organization is the absence of a projection from the anterior segment of the parabigeminal nucleus to the ipsilateral rostral pole. Furthermore, the presence of a projection to the contralateral rostral pole from the expected region of the parabigeminal nucleus should be remarked. In the cat, Sherk found a somewhat similar organization for the ipsilateral parabigemino-tectal projection. On the other hand, at the contralateral side the parabigemino-tectal projection is retinotopically organized up to the vertical meridian but not at the rostral pole la. We do not know how to interpret this discrepancy. Considering the organization of the ipsilateral tecto-parabigeminal and of the crossed parabigemino-tectal projections it is conceivable that the responses elicited from the ipsilaterat eye within the rostral pole are conveyed through a pathway from the binocular region of the superior colliculus to the middle segment of the ipsilateral parabigeminal nucleus and from this region to the contralateral rostralpole. The optic tectum has for long been considered a homologue of the superior colliculus and Le Gros Clark 9 proposed a homology between the nucleus isthmi of submammals and the parabigeminal nucleus of mammals. In this respect, the recent findings of Grobstein et al.7, namely that the ipsilateral visuotectal responses in the frog depend upon the

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Fig. 2. Distribution of labeled cells in the parabigeminal nuclei of 5 opossums after injections in the superior colliculus at sites indicated on a schematic dorsal-view (fight inset) according to Volchan et al.18. The centers of the corresponding response fields are indicated as dots in the polar representation of the visual field (left inset). The histograms show the number of labeled cells (ordinates) along the rostro-caudal axis (abscissae) of the parabigeminal nuclei on both sides of the brain. Contralateral nucleus is on the left. The vertical scale indicates 15 labeled cells and the horizontal scale represents 0.2 mm.

188 integrity of the isthmo-tectal projection assume a special relevance to our proposition. This work was supported by research grants to C.E.R.-M. from the Conselho N a c i o n a l de Desenvolvimento Cientifico e Tecnol6gico (CNPq, Proc. 2222.1006/'78), F i n a n c i a d o r a de Estudos e Projetos a n d Conselho de Pesquisas e Ensino para G r a d u a d o s da U F R J . R.M.-O. is a fellow of the Conselho N a c i o n a l de Desenv o l v i m e n t o Cientifico e Tecnoldgico. We are grateful to our colleagues who collaborated on some of the experiments a n d gave us helpful suggestions on the manuscript. We extend our thanks to Dr. E. Oswaldo-Cruz, who lent us the hemisphere used in this work, to Mr. R. F. Bernardes a n d Edil S. da Silva Filho for their technical assistance a n d to Ms. M a r i a Luiza da Silva for secretarial help.

1 Bodian, Dr, Studies on the diencephalon of the Virginia opossum. Part 1. The nuclear pattern in the adult, at. comp. Neurol., 71 (1939) 259-323. 2 Castaldi, L., Studi sulla struttura e sullo sviluppo del mesencefalo. Richerche in Carla cobaya, Arch. Ital. Anat. Embriol., 23 (1926) 481-609. 3 Gawryszewski, L. G., Rocha-Miranda, C. E., Volchan, E. and Linden, R., Receptive held properties of single units in the opossum superior colliculus, In C. E. Rocha-Miranda and R. Lent (Eds.), Opossum Neurobiology, Academia Brasileira de Ci6ncias, 1978, pp. 167-191. 4 Graham, J., An autoradiographic study of the efferent connections of the superior colliculus of the cat, J. comp. Neurol., 173 (1977) 629-654. 5 Graybiel, A. M., A satellite system of the superior colliculus: the parabigeminal nucleus and its projections to the superficial collicular layers, Brain Research, 145 (1978) 365 374. 6 Graybiel, A. M. and Devor, M. A., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 7 Grobstein, P., Comer, C., Hollyday, M. and Archer, S. M., A crossed isthmo-tectal projection in Rana pipiens and its involvement in the ipsilateral visuotectal projection, Brain Research, 156 (1978) 117-123. 8 Harting, J. K., Hall, W. C., Diamond, I. T. and Martin, G. F., Anterograde degeneration study of the superior colliculus in Tupaia glis: evidence for a subdivision between superficial and deep layers, J. comp. Neurol., 148 (1973) 361-386. 9 Le Gros Clark, W. E., The medial geniculate body and the nucleus isthmi, J. Anat. (Lond.), 67 (1933) 536-549. 10 Linden, R. and Rocha-Miranda, C. E., Projections from the striate cottex to the superior colliculus in the opossum (Didelphis marsupialis aurita). In C. E. Rocha-Miranda and R. Lent (Eds.), Opossum Neurobiology, Academia Brasileira de Ci6ncias, 1978, pp. 137-150. 11 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 12 Oswaldo-Cruz, E., Hokoq, J. N. and Sousa, A. P. B., A schematic eye for the opossum, Vision Res., 19 (1979) 263-278. 13 Oswaldo-Cruz, E. and Rocha-Miranda, C. E., The Brain of the Opossum (Didelphis marsupialis aurita), Instituto de Biofisica da Universidade Federal do Rio de Janeiro, Rio de Janeiro, 1968, 99 pp. 14 Rocha-Miranda, C. E., Cavalcante, L. A., Gawryszewski, L. G., Linden, R. and Volchan, E., The vertical meridian representation and the pattern of retinotectal projections in the opossum. In C. E. Rocha-Miranda and R. Lent (Eds.), Opossum Neurobiology, Academia Brasileira de Ci~ncias, 1978, pp. 113-126. 15 Rosene D. L. and Mesulam, M.-M., Fixation variables in horseradish peroxidase neurohistochemistry. I. The effects of fixation time and perfusion procedures upon enzyme activity, J. Histochem. Cytochem., 26 (1978) 28-39.

189 16 Sherk, H., Connections and visual.field mapping in cat's tectoparabigeminal circuit, J. Neurophysiol., 42 (1979) 1656-1668. 17 Tokunaga, A. and Otani, K., Neuronal organization of the corpus parabigeminum in the rat, Exp. Neurok, 58 (1978) 361-375. 18 Volchan, E., Rocha-Miranda, C. E., Lent, R. and Gawryszewski, L. G., The retinotopic organization of the superior colliculus in the opossum (Didelphis marsupialis aurita). In C. E. RochaMiranda and R. Lent (Eds.), Opossum Neurobiology, Academia Brasileira de Ci~ncias, 1978, pp. 107-112.