Spinothalamic projections in the pigeon

Brain Research, 484 (1989) 139-149 Elsevier

139

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Spinothalamic projections in the pigeon Andreas Schneider and Reinhold Necker lnstitut far Tierphysiologie, Ruhr- Universit?it Bochum, Bochum (F.R. G.) (Accepted 6 September 1988) Key words: Somatosensory system; Thalamus; Spinal cord; Anatomy; Pigeon

The spinothalamic projection in an avian species, the pigeon, was studied both with anterograde and retrograde means. Anterograde transport of wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) was used in order to determine the termination of the spinothalamic tract in the thalamus. Application to the lumbar enlargement of the spinal cord resulted in a dense terminal field in a thalamic nucleus now known as n. dorsointermedius ventralis anterior (DIVA). Less dense labeling was found in the thalamic nuclei n. intercalatus thalami (ICT), n. subrotundus (SRt) and possibly stratum cellulare externum and internum (SCE/SCI). After application of WGA-HRP to the cervical enlargement there was no labeling in the above-mentioned nuclei and only one distinctly labeled terminal in n. dorsolateralis posterior (DLP). Under electrophysioiogical control the fluorescent tracer Fast blue was applied to the DIVA. A considerable number of retrogradely labeled neurons was found in the lumbar enlargement only (contralateral intermediate grey). These results show that there is a substantial direct spinothalamic projection from the hindlimbs (legs) but not from the forelimbs (wings) in pigeons.

INTRODUCTION Whereas the mammalian spinothalamic tract is well investigated 27 there is only little information as to the avian counterpart. Karten 11 and Karten and Revzin 12 described a diffuse projection to the ventral portion of the anterior and posterior dorsolateral nuclei of the thalamus of pigeons as revealed by means of the degeneration technique (hemisection at the cervical level). After HRP-injections into the dorsal thalamus of the pigeon few retrogradely labeled neurons could be demonstrated in the cervical and especially in the lumbar enlargements of the spinal cord 22'24 confirming the existence of a direct spinothalamic pathway. It was the aim of the present study to re-examine the projection of the spinal cord to the thalamus with an anterograde tracing method, namely transport of W G A - H R P , which has been successfully applied to the mammalian spinothalamic tract 18"19, and with Fast blue as a sensitive fluorescent dye for retrograde labeling. Since the study of Necker 22 indicates a predominance of the lumbar cord for spinothalamic projec-

tion the question of a difference between the projection of lumbar and of cervical parts of the spinal cord was addressed. MATERIALS AND METHODS Eleven adult pigeons (Columba livia forma domestica) weighing about 400-500 g were used for W G A - H R P treatment. Under halothane anesthesia in 6 pigeons a laminectomy was performed at the lumbar enlargement of the spinal cord. W G A - H R P (Sigma) dissolved in Ringer solution was pressureinjected by means of a glass capillary connected to a 1/A Hamilton syringe. Five to 6 injections separated by about 1 m m were done along the rostral half of the enlargement. On the opposite side of the spinal cord a comparable number of implants of crystalline W G A - H R P was made. After a survival time of 4-5 days the animals were anesthetized with urethane (2.5 g/kg) and transcardially perfused with warm Ringer solution which was followed by cold fixative (2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer at p H 7.3) and finally by a

Correspondence: R. Necker, Institut ffir Tierphysiologie, Ruhr-Universit~it Bochum, Postfach 102148, D-4630 Bochum 1, F.R.G. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

141) solution of 15% sucrose. The brain and the spinal cord were removed and stored in 15% sucrose in the refrigerator. After embedding in egg yolk the specimens were sectioned at 60 /~m on a freezing microtome. The sections were mounted on gelatincoated slides and processed for HRP with tetramethyl benzidine 2°. Two of 3 parallel series were counterstained with Neutral red. In another 5 pigeons a comparable experimental protocol was applied for the cervical enlargement. Five further pigeons were anesthetized with halothane and fixed in a stereotaxic apparatus 13 for the electrophysiological localization of the nucleus dorsointermedius ventralis anterior (DIVA) and subsequent injection of Fast blue. Either airpuffs directed to various parts of the plumage or electrical excitation of cutaneous nerves were used for stimulation. Responses were recorded with glass capillaries filled with 10% NaCI (impedance 2-10 MY2), amplified and processed by a computer system. After localization of the somatosensory area in the thalamus the recording electrode was replaced by a glass capillary filled with Fast blue. A volume of 0.5-1/A was pressure-injected at the site of best evoked neuronal activity in 3 animals. In two cases only very few dye left the capillary. These latter experiments served for an exact localization of the recording site. After a survival time of 8 days the animals were again deeply anesthetized and perfused with Ringer solution followed by formalin and finally by 15% sucrose. The brain and the spinal cord were removed, embedded in egg yolk and sectioned at 80 ktm on a freezing microtome. The sections were mounted on gelatin-coated glass slides and examined under a Zeiss epifluorescence system. One of 3 parallel series was counterstained with Cresyl violet.

cuneatus externus (CE; Fig. 2B) in the medulla oblongata and parts of the thalamus. Furthermore substantial labeling was found in the cerebellum and in the n. intercollicularis (ICo). Faint diffuse labeling occurred in the reticular formation. Only the terminal fields in the thalamus will be described here. In the thalamus at least 4 regions with more or less intensive anterograde labeling may be discerned. Among these the area of densiest labeling occupies the DIVA 14"25. It is essentially a flat band extending from n. ovoidalis (Ov) medially to the lateral parts of n. superficialis parvocellularis (SPC) laterally with the n. rotundus (Rt), n. triangularis (T) and lateral forebrain bundle (FPL) as ventral border and an ill defined dorsal border. In the rostrocaudal direction the area of HRP-labeling, which seems to be

RESULTS

Anterograde transport of WGA-HRP In the case of treatment of the lumbar cord the area of W G A - H R P uptake covered large parts of the grey substance (Figs. 1B, 2A). In 5 of the 6 animals with lumbar W G A - H R P treatment aside from many retrogradely labeled neurons at all levels of the brainstem, two prominent regions of distinct anterograde terminal fields could be observed: the nuclei gracilis et cuneatus (GC-complex) as well as n.

Fig. 1. A: composite diagram of two experiments showing the thalamic terminal fields after WGA-HRP injections into the lumbar spinal cord~ Dots represent terminals, arrows point to axons. Stereotaxic coordinates according to the atlas ofthe brain of the pigeont3. B: example of the injection site in the lumbar spinal cord. Black area, needle track; stippled area, WGA-HRP diffusion zone. CL, Clarke's column, DLPc, caudal part of n. dorsolateralis posterior; FPL, fascicu!us prosencephali lateralis; ICT, n. intercalatus thalami; MOT, motoneurons; Ov, n. ovoidalis; Rt, n. rotundus; SC, stratum cellulare; SPC, n. superficialis parvocellularis; SRt, n. subrotundus; T, n. triangularis.

141

Fig. 2. A: brightfield photomicrograph of an injection site in the lumbar spinal cord. Arrows point to needle tracks. Bar = 1 mm. B,C,E: darkfield photomicrographs of anterograde labeling in the GC-complex and CE (B), in the dorsolateral thalamus (DIVA and ventral to SPC) at rostral levels (C) and at caudal levels (E). Arrows point to dense labeling. D,F: brightfield photomicrographs of terminals in the rostrat part (D) and in the caudal part of the terminal field, respectively (F, box in E, magnified). Bars in B - F = 100/~m. Axes point to dorsal and medial.

142 identical to the outline of the D I V A , extends from A 7.0 to A 5.5 (all coordinates according to the stereotaxic atlas of the brain of the pigeon of Karten and Hodos13). Looking in this direction, as depicted in Fig. 1A a composite diagram of two experiments with similar labeling, its rostralmost extension is at the emergence of Rt, being placed in the angle formed by Rt and FPL, medially covering the area dorsal to the FPL. More caudally the area increases mostly by expanding to the above mentioned mediolateral borders, At A 6.5/6.25 the band becomes thicker and forms a dorsal bulge in its middle part. More caudally, not yet at the level of Ov, a region of labeling extending along the ventromediai border

of SPC and into the lateral extent of the bulge can be seen (Figs. 1A, 2C). A t the level of the rostral Ov (A 6.0 and more caudal) the medial part of this angle-like area disappears and the decreasing bulge is shifted a bit laterally (Figs. 1A, 2E). At the level of the rostral part of the caudal n. dorsolateralis posterior (DLP, magnocellutar portion or D L P c according to Gamlin and Cohen 8) or the caudal half of Ov the terminal field is small, lying close to the ventral border of SPC where it fades out before H I P (fasciculus habenulae interpeduncularis = fasciculus retroflexus) appears. It should be noticed that there is a clear separation between this field and D L P c by an area of smaller cells than those of D L P c (Fig. 3).

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Fig. 3. A,B: photomicrographs of frontal sections of Nissl-stained dorsal thalamus at the level of the rostral DIVA (A) and of the caudal DLP (B). Note the difference in cytology of the DIVA and of the caudal DLP. Bar = 1 mm. Axes point to dorsal and medial. C: spike trace of the response to electrical stimulation of the contralateral hindlimb (arrow points to stimulus artifact). Recording site in the DIVA is shown in A by a filled circle.

143 A common pattern of this terminal field is a central region consisting of labeled axons with boutons terminaux embedded in a field of reaction product granules distinct from the less dense background artefacts (Fig. 2D). Terminals contralateral to the crystalline W G A - H R P treatment were more numerous and more intensively labeled. Fibers were often oriented lateromedially. From a cytological point of view the terminal field consists of densely packed, medium-sized, darkly staining cells (Fig. 3) of ovoid shape (especially in the bulge's base and more rostral). This dense package allows a delimination to the overlying, less densely packed nuclei (DLA, DLM, DLP). The larger cells of the DLPc are clearly separated from this terminal field (Fig. 3). This cytologically defined area forms an angle-like shape at its caudal portion. The cells of the lateral part (ventral to the lateral border of SPC) appear a bit smaller and more often clustered. Another thalamic region receiving projections from the lumbar enlargement of the spinal cord is the region medial to Rt. Here the n. intercalatus thalami (ICT) and the stratum cellulare internum and externum (SCI/SCE) are involved (Fig. 1A). Labeling in the latter area consists of faint, far spread fine dust among darkly stained retrogradely labeled cells (see ref. 2) at the stereotaxic coordinates A 6.30 to A 5.50. It is not clear whether labeled fibers belong to the retrogradely labeled cells or to the anterogradely labeled granules which are even finer than those found in the DIVA. The density of the granules increases dorsalwards. The same holds for the anterograde labeling of ICT although there is no confusion with retrogradely labeled neurons. While a concentration of the dust among and around few bigger boutons occurs in the dorsal apex of the nucleus (consisting of relatively small cells) the ventral base is invaded by fibers running from ventrolateral to dorsomedial along the long axis of ICT and ending in a dust-like area at A 6.50, confirming its anterograde character. A fourth area of the thalamus receiving projections from the lumbar enlargement of the spinal cord is the n. subrotundus (SRt), especially its medial part subadjacent to Ov (Figs. 4B,C,D). Terminals at the end of axons could be found here almost at the ventral border but still within the nucleus and among

its typical big multipolar (net-like clustered) cells. Although there are only few terminals (less than 10 per animal) they could be identified with no doubt because of their size and because of labeled fibers which run towards the shelf region of Or. This shelf region may be of interest because caudal to the Ov, where the shelf region may still be present, few fibers could be found running horizontally from lateral to medial and ending with a bouton (Fig. 4A). These terminals are bigger and obviously easier to identify than the dust-like pattern of SCE/SCI and ICT. They are comparable to those in the DIVA area. In 5 pigeons W G A - H R P was applied to the cervical spinal cord. Despite a similar experimental protocol labeling in the thalamus was found in one animal only. In this case one faint fiber with a bouton terminal could be demonstrated in the DLPc.

Retrograde transport of Fast blue Based on the results of the anterograde transport in another 5 pigeons Fast blue injections into DIVA were made under electrophysiological control. Spikes (Fig. 4C) and evoked field potentials could be recorded in a depth of about 7000 to 7400 /zm (reading from the surface of the brain) at the stereotaxic coordinates A 6.3-5.5/L 2.0-1.5. This is at more caudal parts of the DIVA terminal field (see Fig. 1A). Latencies to electrical or airpuff stimulation ranged from about 7 to 40 ms. Receptive fields were usually on the contralateral side. Although receptive fields often covered both wing and leg, responses to leg stimulation were more vigorous. Optic (LED flash) and acoustic stimuli (click) failed to evoke a response. The area of uptake of Fast blue injected into the recording site covered DIVA in its full mediolateral extent but also parts of the overlying nuclei (Fig. 5, and see Fig. 7F). The center of injections, i.e. the recording site, was always in the DIVA which was especially evident in the experiments where Fast blue served to mark the recording site. The number of labeled cells in the spinal cord of the 3 successful experiments (see Materials and Methods) is shown in Table I. A considerable number of retrogradely labeled cells were found in the contralateral lumbar enlargement of the spinal

144

Fig. 4. A: two examples of terminals with long axons in the caudal Ov shelf region (the upper one is shown as a darkfield photomicrograph also). B-D show terminals within the SRt, indicated by arrowheads. Bar = 100/~m. Axes point to dorsal and medial.

cord (see Fig. 5). Only few cells were found on the ipsilateral side. Nearly all neurons lay in the intermediate grey ventral and lateral to Clarke's column (laminae V - V I according to Leonard and Cohen17). Few cells lay in other parts of the grey substance (laminae I, V I I I and medial IX). In the cervical enlargement only very few contralaterally located cells were found (Fig. 5 and Table I). In the lower brainstem many labeled cells were found in the nn. gracilis et cuneatus (GC-complex), in n. cuneatus externus (CE) and in the trigeminal system (TTD) (Fig. 6). In the spinal cord several cell types may be discerned (Fig. 7). Most cells near Clarke's column were rather large (major axis about

30-40 /~m) and multipolar. A lower number of pyramidal cells was also found at this location. At the lateral border of the grey matter and especially at the medial border smaller fusiform cells often orientated parallel to the borderline were more common. This class could be devided into bipolar and double bipolar cells (Fig. 7D). Very often axons run ventromedially towards the midline and some could be observed to cross it. DISCUSSION This is the first demonstration of a spinothalamic projection in an avian species with anterograde

145

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Fig.5.A,B:twocompositediagramsoflabeledcellsin thecervicalandlumbarenlargementsofthespinalcordafterFastblue injections into the dorsolateral thalamic somatosensory area. In the example shown in B the injection site is depicted also. Lesioned area blackened, diffusion zone stippled (see also Fig. 7F). n, number of labeled neurons in the cervical and lumbar enlargements. For number of sections see Table I.

146

Fig. 6. Photomicrographs of retrogradely labeled neurons in the GC-complex (A) and in CE (B). Note the partial labeling of the trigeminal system (TTD). Bar = 1 mm. Axes point to dorsal and medial.

t r a n s p o r t of W G A - H R P . W h e r e a s labeling in the thalamus was consistently o b s e r v e d after t r e a t m e n t of the l u m b a r cord, most experiments with cervical t r e a t m e n t failed to show such a projection. Only in one e x p e r i m e n t a single fiber occurred in DLPc. This is in a g r e e m e n t with previous r e t r o g r a d e transport studies which disclosed m o r e r e t r o g r a d e l y labeled n e u r o n s in the l u m b a r cord than in the cervical cord 22. A l t o g e t h e r labeling was faint, however. This points to a weakly d e v e l o p e d spinothalamic projection as c o m p a r e d with m a m m a l i a n species l~'19'23. This is c o r r o b o r a t e d by the small n u m b e r of retrogradely labeled cells in this and in previous investigations 22,24. With the d e g e n e r a t i o n m e t h o d K a r t e n " found a spinothalamic projection to ventral portions of the

TABLE I Distribution of labeled cells in the cervical and lumbar enlargements o f the spinal cord after Fast blue injection into the somatosensory thalamus

Each section of the corresponding enlargement was considered for evaluation. Experiment

lpsi

Contra

Total number o f ......... Cells Sections

cervical lumbar 2 cervical lumbar 3 cervical lumbar

0 1 0 6 0 1

5 35 0 83 1 74

5 36 0 89 1 75

1

120 106 135 122 130 133

Cells/ section

0.04 0.34 0 0.73 <0.01 0.56

anterior and posterior dorsolateral nuclei of the thalamus and SPC as well as part of I C T medial to Rt 12. This is in partial a g r e e m e n t with the present results. H o w e v e r , this investigation adds to the previous results a detailed description of the extent of thalamic labeling. Karten 11 m a d e hemisections at the cervical spinal cord and was thus not able to distinguish between cervical and l u m b a r projections. With injections of W G A - H R P into both l u m b a r and cervical segments in the present investigation it could be shown that there is only a substantial projection to the thalamus from the l u m b a r cord. The ventral parts of D L P and D L A as described by Karten 11 may now be recognized as a separate somatosensory area denominated D I V A 14"25. W h e r e a s K a r t e n '~'~2 found terminals within the SPC the present investigation suggests that there are terminals ventral to the SPC which are in part in continuity with the D I V A forming an angle-like bending towards the SPC (Fig. 1A). H o w e v e r , it is unclear whether this terminal field near SPC is a dorsolateral extension of the D I V A or a s e p a r a t e terminal field. A schematic reconstruction of the spatial structure of the whole dorsolateral thalamic terminal field and its relation to the neighbouring nuclei is presented in Fig. 8. A further significant spinothalamic p r o j e c t i o n from the hindlimb seems to be to I C T and to SRt, but are so far not identified with electrophysiological means. Wild 25 and F u n k e 6 showed that the D I V A projects to the rostral telencephalic s o m a t o s e n s o r y area first described by Delius and B e n n e t t o 5. The

147

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Fig. 7. Photomicrographs of retrogradely labeled fluorescent neurons in the lumbar spinal cord ( A - E ) and of the injection site (F). A: labeled neurons in the contralateral lumbar cord. B: box in A, magnified. B-E: different types of cells. Arrowheads point to pyramidal cells, large arrows to muitipolar and small arrows to fusiform cells. In D two types of fusiform cells are shown, a double bipolar one and a unipolar one (inset). F: an example of the injection site in the dorsolateral thalamus (see lesioned area; for area of dye spread see Fig. 5). Bars = 1 mm in A and F and 100/~m in B-E. Axes point to dorsal and medial, resp.

148

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Fig. 8. Three-dimensional presentation of the dorsolateral thalamic terminal field (DIVA area, stippled).

retrogradely labeled cells described in these papers are well within the area of anterograde transport shown in the present study. This supports the assumption of a specific spino-thalamo-telencephalic pathway via the DIVA to the telencephalic rostral Wulst, the only cortex-like structure in birds 4. The electrophysiological recordings of the present study suggest that the DIVA may be a specific somatosensory relay because optic and acoustic stimuli were ineffective. In current electrophysiological experiments specificity and receptive fields are being studied in more detail. If such a specific function can be confirmed DIVA may be considered the homologue to the specific mammalian ventrobasal complex. This is supported by a massive projection of the dorsal column nuclei (GC-complex) to the D I V A but not to the DLPc 24. The more caudally placed DLPc, where also somatosensory responses can be recorded has been shown to be largely multimodal and has been compared to the REFERENCES 1 Brauth, S.E., McHale, C.M., Brasher, C.A. and Dooling, R.J., Auditory pathways in the budgerigar. I. Thalamotelencephalic projections, Brain Behav. Evol., 30 (1987) 174-199. 2 Cabot, J.B., Reiner, A. and Bogan, N., Avian bulbospinal pathways: anterograde and retrograde studies of cells of origin, funicular trajectories and laminar termination. In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending

mammalian PO ~s' ~,25. The finding of a mainly lumbar spinothalainic projection is corroborated by the results of retrograde transport of Fast blue. As to the distribution of labeled spinal cells there is a general agreement with a previous investigation using H R P 2e. However, the number of labeled cells was considerably larger in the present investigation. In mammalian species many spinothalamic cells are found in both the cervical and the lumbar enlargement 3"9"m'15. As to the location of the spinothalamic neurons one main difference to mammalian species is a nearly total lack of labeled lamina I neurons. In mammals both lamina I and lamina V neurons (neck of dorsal horn) project to the ventrobasal complex (specific somatosensory thalamus26). The location in laminae V - V I in the pigeon may be compared to the mammalian lamina V neurons projecting to the specific thalamus. This supports the assumption of a specific somatosensory function of the DIVA. Although there is nearly no direct spinothalamic projection from cervical segments of the pigeon, wing stimulation may activate DIVA neurons. This input is probably conveyed via the mechanoreceptive lamina IV neurons in the cervical cord 21 which reach the thalamus via the postsynaptic dorsal column system; a corresponding system for the lumbar enlargement is lacking 7. Aside from the above-mentioned pathways somatosensory information is conveyed by primary afferents via the dorsal column nuclei to the DIVA. This is likely to be the dominant input to this nucleus. ACKNOWLEDGEMENTS This investigation was supported by the Deutsche Forschungsgemeinschaft (Ne 268/1-1). The technical assistance of Christel Schermuly and Silvia Schweer is gratefully acknowledged. Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, 1982, pp. 79-108.

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