Hyperstriatal-tectal projections in the pigeon (Columba livia) as demonstrated by the retrograde double-label fluorescence technique. DOM MICELI* and ...
Brain Research, 276 (1983) 147-153 Elsevier
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Hyperstriatal-tectal projections in the pigeon (Columba livia) as demonstrated by the retrograde double-label fluorescence technique DOM MICELI* and JACQUES REPERANT Groupe de recherche en neuropsychologie exp~rimentale, Universit~du Quebec, C.P. 500, Trois-Rivi~res, Qu& G9A 5H7 (Canada) and Laboratoire de neuromorphologie, Unitd1NSERM 106 HOpital Foch, Suresnes, 92150 (France)
(Accepted May 31st, 1983) Key words: hyperstriatal-tectal projections - - pigeon - - fluorescent retrograde tracers
Hyperstriatal-tectal connections were investigated in the pigeon by mapping retrogradely labeled neurons in the hyperstriatum following the injection of different fluorescent tracers (Fast Blue, Nuclear Yellow, Evans Blue) either bilaterally or unilaterally into distinct portions of the tectum. The projections were found to arise from the hyperstriatum accessorium (HA) of the Wulst, were essentially ipsilateral and topographically organized with respect to the dorsoventral and lateromedial plane of HA. No double-labeled neurons which would indicate bilateral axon branching via collaterals were observed. Some interspecies differences in the organization of the avian hyperstriatal-tectal pathways are discussed. Efferent projections of the hyperstriatum upon the optic tectum have been investigated in various avian species with electrophysiological methods4, 5Aoa5 and anatomical techniques using orthograde cell degenerationl,14,ts, autoradiography18 and the retrograde axonal transport of H R P 3,6.9. Bilateral connections have been demonstrated in the owl and the pigeon 3,9A4 and, in the latter species, have been shown to arise from restricted portions of the visual Wulst: hyperstriatum accessorium (HA) and superficial hyperstriatum intercalatus superior (HIS) 3. That the descending hyperstriatal system is involved in the processing of binocular information is suggested on the basis of features related to the existence of bilaterally organized connections and to the distribution either of efferent neurons in the binocular portion of the visual Wulst9 or of afferents to that part of the rectum coinciding with the representation of the binocular field 18. However, various discrepancies have appeared in the literature regarding the exact location of the efferent hyperstriatal neurons, the distribution of the terminal projection within the rectum and the relative size of the crossed component3,6.9A4,18. The aim of the present study was to determine the sites of origin of hyperstriatal-tectal projections in * To whom reprint requests should be addressed. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
the pigeon using the method of retrograde transport of various fluorescent tracers (Fast Blue, FB; Nuclear Yellow, NY; Evans Blue, EB). A double-labeling technique was employed whereby combinations of different fluorescent substances were injected separately into the left and right tectum. By this procedure, it was possible to identify individual neurons, within the hyperstriatum of either hemisphere, exhibiting either crossed or uncrossed descending projections and to determine whether any cells distributed divergent axon collaterals bilaterally. In other experiments, combinations of the various fluorescent tracers were injected unilaterally within different regions of the tectum in order to examine topographical interrelations between this structure and the hyperstriatum. Fourteen pigeons, anesthetized with Hemineurine (Debat Laboratories, France) received bilateral injections within the tectum of 0.1-0.4/d of either two of the following fluorescent tracers dissolved or suspended in distilled water: FB (5% w/v), NY (3%) and EB (10% solution containing 1% poly-L-ornithine). The EB tracer was injected into the lateral aspect of one tectum whereas a different tracer, either FB or NY was injected symmetrically into the oppo-
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Fig. 1. Microphotographs of labeled hyperstriatal EB (A: x 1280, C: x 360) and NY (B: x 1280) neurons as viewed with the N2 (550 rim) and A (360 nm) filter-mirror systems respectively. D: schematic representation of transverse sections taken at different anteriorities of the pigeon telencephalon. The differential distribution of retrogradely labeled cells within HA is shown for EB (open circles), NY (filled circles) and EB-NY double-labeled cells (crosses) following large anterior and posterior injections within the ipsilateral rectum (illustration at top left) of NY (horizontal lines) and EB (vertical lines) tracers respectively. The actual number of labeled cells counted/section was approx. 5 times that shown in the figure.
149 site tectum. The injections were performed using a direct lateral approach after surgically exposing the tectal lobes between the ocular globe and the external auditory canal. The tracers were injected along the central horizontal axis of the tectal lobe, at coordinates varying between A: 2.5-5.5 mm and at depths of 1.0-2.0 mm from the tectal surface 13. Nine other birds received similar injections of EB and either FB or NY into topographically distinct portions of the rectum on the same side. In some experiments, the first and second tracers were injected respectively within the anterior and posterior halves of the tectal lobe, at stereotaxic coordinates on either side of A: 3.5 mm. Other animals received injections of the first and second tracers respectively within the dorsal and ventral halves of the structure and near the central vertical axis of the tectum (A: 3.5 mm). After a 1-7 day survival period, the animals were anesthetized and perfused transcardially with saline, followed by 10% formol and finally with a solution containing 30% sucrose. The brains were removed and sectioned at 40/~m in the frontal plane on a freezing microtome. The sections were immediately mounted onto slides, air-dried and cover-slipped using a low fluorescence mounting medium. Alternate sections were counterstained with cresyl violet. The fluorescent labeled neurons were observed using a Leitz Ploemopack fluorescence microscope equipped with filter-mirror systems N2 (550 nm) and A (360 nm) for viewing either EB or FB and NY respectively. Neurons labeled with FB displayed a pale blue cytoplasm often containing yellow fluorescent granules and little nuclear labeling. NY-labeled cells showed a bright silver-yellow nuclear labeling and little cytoplasmic fluorescence (Fig. 1B). Lastly, the neurons containing EB exhibited a bright homogeneous red fluorescence (Fig. 1A, C). By injecting a different tracer into each tectum, those neurons in the hyperstriatum providing uncrossed tectal projections would be specifically labeled with the corresponding ipsilaterally injected tracer, whereas those with crossed connections would contain the tracer injected contralateraUy. Moreover, single hyperstriatal neurones providing bilateral projections to the tectum via collateral axon branching, if present, would be double-labeled and display the combined features of both fluorescent
tracers usedT,8. The labeled neurons were mapped using either a video camera and monitor or photographed on colour slides which were then projected o n t o a screen for analyses. Since the pairs of tracers required observation under both N2 and A filter-mirror conditions, double-exposure photographs were taken at both wavelengths. The resulting photographic image provided a superposition of the fluorescent labeling produced by each tracer21. The nomenclature adopted for the different nervous structures is essentially that of Karten and Hodos 13. Bilateral tectal injections. The injection sites in all cases extended from the surface of the rectum to include the deeper layers (stratum griseum centrale: SGC and stratum album centrale: SAC). In some instances, the more rostral injection sites also extended medially invading some pretectal structures (nucleus lentiformis mesencephali and nucleus principalis precommissuralis). Fluorescent-labeled neurons were identified bilaterally within the hyperstriatum and although the labeling was homotopic, the mirror-image pattern of fluorescence was reversed on opposite sides. The labeling was found widespread throughout the rostro-caudal extent of HA and appeared to be restricted to this layer of the Wulst (Fig. 2A-D). No labeling was detected in either adjacent HIS or in the hyperstriatum dorsale (HD). No precise anterior-posterior topographical relations were apparent in these experiments. In any one hemisphere, the labeled neurones contained essentially the tracer material injected ipsilaterally. Cells containing the contralaterally injected tracer were extremely rare. Cell counts indicated 0-3 contralaterally labeled cells per section, whereas the number of ipsilateral labeled neurons reached as high as 325 cells/section. Moreover, the concentration of cells labeled with the contralateral tracer remained relatively unchanged regardless of the length of the survival period. These cells were mainly situated anteriorly and close to the medial border of HA overlying the ventricle. Although cells containing the contralateral label were found interspersed among numerous ipsilaterally labeled neurons, none of these appeared double-labeled. Finally no differences in the distribution of labeling was noted in those cases where the injection sites extended beyond the tectum to include portions of the pretectum. Unilateral tectal injections. The labeling observed
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Fig. 2. Neuronal labeling in H A on the side ipsilateral to the injection of tracers within the tectum. A: EB-labeled cells near the dorsomedial edge of HA (× 265). B: NY-labeling more ventrally in H A ( x 265). C: the dispersed pattern of N ¥ labeling is shown at lower magnification ( x 106), D: extending to the ventral limit of HA between the ventricle (V) and the hemispheric rnidline (M). E: histogram showing the number of labeled EB (striped bars) and NY (open bars) cells counted/section at different anteriorities of H A following injections of NY and EB respectively within the anterior and posterior tectum.
151 following the injection of different fluorescent tracers into the same tectal lobe was similar to that obtained for any one tracer in the case of bilateral tectal injections. Also, the bilateral labeling pattern within H A was constant regardless of the length of the survival period. However, the topographical organization of the hyperstriatal-tectal connections was more clearly evident using this paradigm. This was because the relative distribution of differentially labeled neurons could be mapped directly in the same hemisphere rather than relying on less accurate reconstructions based on different experimental cases using the bilateral injection procedure. Injections of different fluorescent tracers into the anterior and posterior portions of the tectum produced a corresponding labeling of cells within the ventral and dorsal regions of H A respectively. Fig. 1D shows the distribution of NY and EB labeled neurons within the hyperstriatum following large injections of these tracers respectively into the anterior and posterior portions of the tectum. The differential distribution of labeled neurons in H A was not as clearcut following dorsal and ventral injections within the tectal lobe. Nevertheless, whereas the former resulted in somatic labeling which was localized more laterally within H A and in proximity to the HIS border, the latter produced a more medially situated labeling, adjacent to and along the hemispheric midline. The areas of differential labeling overlapped in the intermediate regions, however, double-labeled neurons were not detected within these areas. Only in those cases where the tectal injection sites for the two different tracers were extensive and overlapped were some double-labeled neurons identified. The topographical relations in the hyperstriatal-tectal projection with respect to the frontal plane of H A appeared to be maintained over wide areas along the anteriorposterior axis of the hyperstriatum, but were more clearly evident in the rostral portion of H A in front of A: 12.0 mm. Conversely, as in the case of bilateral tectal injections, no clearcut interrelations between the tectum and H A were noted with respect to the anterior-posterior axis of HA. This was mainly due to the fact that the labeling within the latter structure was always very extensive in this particular plane. The latter is illustrated in the histogram of Fig. 2E which shows the relatively similar proportions of NY and EB cells counted at different anteriorities follow-
ing rostral and caudal injections of the tracers respectively within the tectum. The results of the present fluorescent study confirmed the existence of hyperstriatal-tectal projections in the pigeon3,6,14A8, and showed this pathway to arise from H A of the visual Wulst. Some cells, although very rare, exhibit contralateral projections, however the connections appear to be essentially ipsilateral. No double-labeled neurons, indicating collateralized bilateral connections upon the tectum were observed. The relatively more even proportions of ipsi- and contralateral projections reported in a previous study in the pigeon using the H R P method 3 were not confirmed here with the fluorescence technique. The discrepancy is at present difficult to explain and may be linked to differences in the areas of tectum sampled with the various retrograde tracers. Nevertheless, in the present experiment, the injection sites were rather extensive and only some extreme peripheral zones of each tectal quadrant were spared. Very long survival periods were also employed in order to assure maximal uptake and transport of the fluorescent substances from the tectum, but this did not result in an increase in the number of cells labeled contralaterally. Another explanation may be linked to the greater distances between the injected and labeled sites covered by the contralateral projections. However, this would seem unlikely as the distances are comparable to those traced in a previous investigation of the thalamo-hyperstriatal pathway where bilateral labeling with the same fluorescent tracers was observed following survival periods as short as 24 h 21. A final explanation which cannot be completely discounted is that not all cells take up or transport the various fluorescent substances used in the present study. However, in a recent study using the geniculo-striate system of the rat as a model to measure the relative sensitivity of various retrograde fluorescent tracers and HRP, it was shown that FB labeled an equal number, and NY a greater number of cells retrogradely in comparison to HRp2. In contrast to the previous HRP results 3, the virtually unique ipsilateral hyperstriatal-tectal pathway demonstrated here is compatible with previous data obtained in the pigeon using the Fink-Heimer and autoradiographic techniques which failed to clearly demonstrate the existence of a contralateral projection of the hyperstriatum upon
152 the tectum ~s. The avian Wulst receives direct bilateral projections from the visual dorsal thalamus 12,14,17,19.21-25. Furthermore, it has been shown that the HA component of the pigeon's visual Wulst receives ipsilateral afferents from the dorsolateralis anterior (DLA) complex of the thalamus whereas the deeper strata receive fibers from topographically distinct portions of the D L A on the contralateral side 19. Taken together with the present results, this implies that HA both receives input from the contralateral eye via the thalamofugal system and correspondingly transmits information to the ipsilateral tectum which essentially receives a direct crossed retinal pojection. Within the rectum, there is also evidence that the terminal field of the descending hyperstriatal projection largely overlaps that of the retinal arborizations 6. The essentially ipsilateral pattern of organisation of the HA-tectal system demonstrated in the present study is also compatible with previous electrophysiological data obtained in either the pigeon or the chick, two species which show striking similarities regarding the afferent and efferent projections of the visual Wulst 1~-2°.24 and the functional properties of single neurons within the latter structurelS,26.2L Cells in HA have been shown to be responsive essentially to visual stimulation of the contratateral eye ~s.26. Indeed, in the chick, the cells which responded to visual stimuli appeared to be localized in the H A segment of the Wulst 27. Moreover, of the 267 units sampled, with the exception of 2 binocular neurons the remaining (265 units) were driven exclusively by the contralateral eye and none were found which responded only to ipsilateral eye stimulation. Elsewhere, evoked responses have been recorded in the tectum following electrical stimulation of the visual Wulst and confined to the H A portion of this structure 4. The data of the present study support the hypothesis of the existence of a topographical organisation in the hyperstriatal-tectal connections. Although no interrelations were observed with respect to the rostro-caudal plane of HA, some topographical organisation was apparent along the other planes of the
structure. Here, neurons situated along the dorsoventral and latero-medial axis of HA provided projections respectively upon the posterior-anterior and dorsoventral axis of the rectum. Furthermore, because of the topographically organised HA-tectal projection and the fact that the retina projects in an orderly manner upon the tectum~l,16, the retinotopic organisation of the different areas of H A might be expected to coincide with the visual field representation of their corresponding target sites within the tectum, Some evidence in support of this is provided by data regarding the receptive field distribution of neurons in HA. Consistent with the present results, the location of receptive fields for individual cells lying along different electrode penetrations made perpendicular to the Wulst surface have been shown to shift sequentially through the different visual field quadrants in both the pigeon and the chick 1s.26. Moreover, this particular retinotopic pattern appears to be maintained throughout extensive regions along the rostro-caudal plane of H A 2~. The interspecies differences which have been reported regarding the organisation of the hyperstriatal-tectal pathway in birds and related either to the distribution of the efferent neurons 3,9 or to the relative proportions of the crossed and uncrossed components 9`t4`1s may be linked to the corresponding degree of bilateralisation and to the distribution upon the Wulst of direct afferent input arising from the visual dorsal thalamus. The latter, in turn, may be related to differences in the degree of binocularity such as the greater extent of overlap of the binocular fields found in the owl compared to the chick and pigeon. More comparative studies of the anatomical organisation of hyperstriatal afferent and efferent systems as a function of binocular overlap and using these different bird models are needed in order to further elucidate these relationships.
1 Adamo, N. J., Connections of efferent fibers from hyperstriatal areas in chicken, raven, and African lovebird, J. comp. Neurol.. 131 (1967) 337-356. 2 Aschoff, A. and HolRinder, H., Fluorescent compounds as retrograde tracers compared with horseradish peroxidase
(HRP). I. A parametric study in the central visual system of the albino rat, J. Neurosci. Meth., 6 (1982) 179-197. 3 Bagnoli, P., Grassi, S. and Magni, F., A direct connection between visual wulst and tectum opticum in the pigeon (Columba livia) demonstrated by horseradish peroxidase,
This work was supported by CRSNG and D G R S T research grants. The authors thank M. L. Marchand, Mme. L. Tremblay and Mme. H. Boisclair for their excellent technical and secretarial assistance.
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Arch. ital. Biol., 118 (1980)72-88. 4 Bagnoli, P., Francesconi, W. and Magni, F., Visual wulst influences on the optic tectum of the pigeon, Brain Behav. Evol., 14 (1977) 217-237. 5 Bagnoli, P., Francesconi, W. and Magni, F., Interaction of optic tract and visual wulst impulses on single units of the pigeon's optic tectum, Brain Behav. Evol., 16 (1979) 19-37. 6 Bagnoli, P., Francesconi, W. and Magni, F., Visual wulst-Optic tectum relationships in birds: a comparison with the mammalian corticotectal system, Arch. ital. Biol., 120 (1982) 212-235. 7 Bentivoglio, M., Kuypers, H. G. J. M. and Catsman-Berrevoetz, C. E., Retrograde neuronal labeling by means of bisbenzimide and nuclear yellow (Hoechst $769121). Measures to prevent diffusion of the tracers out of retrogradely labeled neurons, Neurosci. Lett., 18 (1980) 19-24. 8 Bentivoglio, M., Kuypers, H. G. J. M., Catsman-Berrevoetz, C. E., Loewe, H. and Dann, O., Two new fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (1980) 25-30, 9 Bravo, H. and Pettigrew, J. D., The distribution of neurons projecting from the retina and visual cortex to the thalamus and tectum opticum of the barn owl, Tyto alba, and the burrowing owl, Speotyto canicularia, J. comp. Neurol., 199 (1981) 419-441. 10 Brito, L. R. G., Hyperstriatal projections to primary visual relays in pigeons: electrophysiological studies, Brain Research, 153 (1978) 383-386. 11 Hamdi, F. A. and Whitteridge, D., The representation of the retina on the optic tectum of the pigeon, Quart. J. Exp. Physiol., 39 (1954) 11-119. 12 Hunt, S. P. and Webster, K. E., Thalamo-hyperstriate interrelations in the pigeon, Brain Research, 44 (1972) 647--651. 13 Karten, H, J. and Hodos, W., A Stereotaxic Atlas of the Brain of the Pigeon (Columba livia), Johns Hopkins Press, Baltimore, MD, 1967. 14 Karten, H. J., Hodos, N., Nauta, W. J. H. and Revzin, A. M., Neural connections of the visual wulst of the avian telencephalon. Experimental studies in the pigeon (Columba livia) and owl (Speotyto canicularia), J. comp. Neurol., 150 (1973) 253-276. 15 Leresche, N., Hardy, O. and Jassik-Gerschenfeld, D., Influence des aires t61enc6phaliques (Wulst) sur la s61ectivit6 directionnelle des cellules tectales chez le pigeon, C. R. Acad. Sci., 294 (1982) S6rie III, 833-836.
16 McGill, J. I., Powell, T. P. S. and Cowan, W. M., The retinal representation upon the optic tectum and isthmo-optic nucleus in the pigeon, J. Anat., 100 (1966) 5-33. 17 Meier, R. E., Mihailovic, J. and Cuenod, M., Thalamic organization of the retino-thalamo-hyperstriatal pathway in the pigeon (Columba livia), Exp. Brain Res., 19 (1974) 351-364. 18 Miceli, D., Gioanni, H., Rep6rant, J. and Peyrichoux, J., The avian visual wulst: I. An anatomical study of afferent and efferent pathways. II. An electrophysiological study of the functional properties of single neurons. In A. M. Granda and J. H. Maxwell (Eds.), Neural Mechanisms of Behavior in the Pigeon, Plenum Press, New York, 1979, pp. 223-254. 19 Miceli, D., Peyrichoux, J. and Rep6rant, J., The retinothalamo-hyperstriatal pathway in the pigeon (Colurnba livia), Brain Research, 100 (1975) 125-131. 20 Miceli, D., Peyrichoux, J., Rep~,rant, J. and Weidner, C., Etude anatomique des aff6rences du t~,lenc6phale rostral chez le poussin de Gallus domesticus L., J. Hirnforsch., 21 (1980) 627-646. 21 Miceli, D. and Rep6rant, J., Thalamo-hyperstriatal projections in the pigeon (Columba livia) as demonstrated by retrograde double-labeling with fluorescent tracers, Brain Research, 245 (1982) 365-371. 22 Nixdorf, B. E. and Bischof, H.-J., Afferent connections of the ectostriatum and visual wulst in the Zebra Finch (Taeniopygia guttata castanotis Gould)-an HRP study, Brain Research, 248 (1982) 9--17. 23 Perisic, M., Mihailovic, J. and Cu~nod, M., Electrophysiology of contralateral and ipsilateral visual projections to the wulst in pigeon, ( Columba livia), Int. J. Neurosci., 2 (1971) 7-14. 24 Rep6rant, J., Raffin, J.-P. and Miceli, D., La voie r6tinothalamohyperstriate chez le poussin (Gallus domesticus), C. R. Acad. Sci., 279 (1974) 279-282. 25 Webster, K. E., Changing concepts of the organisation of central visual pathways in birds. In R. Bellairs and E. G. Gray (Eds.), Essays on the Nervous Systems, Oxford University Press, 1974, pp. 258--298. 26 Wilson, P., The organization of the visual hyperstriatum in the domestic chick. I. Topology and topography of the visual projection, Brain Research, 188 (1980) 319-331. 27 Wilson, P. The organization of the visual hyperstriatum in the domestic chick. II. Receptive field properties of single units, Brain Research, 188 (1980) 333-345.
