reticulospinal and vestibulospinal pathways in the snake python regius

Anat Embryol (1983) 168 : 277-289 Anatomy and Embryology �9 Springer-Verlag 1983

Reticulospinal and Vestibulospinal Pathways in the Snake Python regius

H.J. ten Donkelaar, G.C. Bangma, and R. de Boer-van Huizen Department of Anatomy and Embryology, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen (The Netherlands)

Summary. In the present HRP study extensive reticulospinal projections and more modestly developed vestibulospinal pathways have been demonstrated in the snake Python regius. The funicular trajectories of the main reticulospinal pathways have been shown: via the lateral funiculus pass spinal projections of the nucleus reticularis superior pars lateralis, the nucleus reticularis inferior and nucleus raphes inferior; via the ventral funiculus fibers arising in the nucleus reticularis superior and nucleus reticularis medius. Spinal projections of the locus coeruleus and subcoeruleus area reach their targets via both the lateral and ventral funiculi. Two vestibulospinal pathways have been demonstrated: an ipsilateral tractus vestibulospinalis lateralis arising in the ventrolateral vestibular nucleus, and a contralateral tractus vestibulospinalis medialis from the descending and ventromedial vestibular nuclei. After HRP gel implants into the vestibular nuclear complex direct vestibulocollic projections to motoneurons in the rostral spinal cord were observed.

Spinal projections from the ventral part of the nucleus reticularis inferior and the descending and ventromedial vestibular nuclei are mainly aimed at the thin "neck area" (approximately the first 50 spinal segments). This area is extensively used in such acts as orientation and prey-catching, requiring a rather delicate brain stem control.

Key words: Brain stem - Reticulospinal - Vestibulospinal - Snake - Axial motoneurons

Introduction

Throughout terrestrial vertebrates a basic pattern in the organization of descending pathways is present (ten Donkelaar 1976 b, 1982). With the clas-

Offprint requests to: Dr. H.J. ten Donkelaar, Department of Anatomy and Embryology, Uni- versity of Nijmegen, Faculty of Medicine, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

278 H.J. ten Donkelaar et al.

sical degeneration techniques rubrospinal, reticulospinal and vestibulospinal tracts, terminating in comparable areas of the spinal gray matter, have been demonstrated in quadrupedal vertebrates. A rubrospinal pathway seems to be related to the presence of limbs or limb-like structures (ten Donkelaar 1976b, 1982; Smeets and Timerick 1981; ten Donkelaar and Bangma 1983). Horseradish peroxidase (HRP) studies in terrestrial vertebrates (see ten Donkelaar et al. 1980; ten Donketaar 1982 for references) showed that also the following descending pathways appear to belong to the basic equipment of such vertebrates: hypothalamospinal, cerebello- spinal, solitariospinal and spinal projections from somatosensory nuclei as the descending nucleus of the trigeminal nerve and the dorsal funicular nuclei.

In the present study attention is focussed on reticulospinal and vestibulospinal projections in the snake Python regius, a limbless reptile which lacks a rubrospinal pathway (ten Donkelaar 1982; ten Donkelaar and Bangma 1983). With the HRP technique the cells of origin of reticulospinal and vestibulospinal fibers have been studied; by applying HRP to the vestibular nuclear complex the vestibulospinal projections have also been ana- lysed anterogradely. Reticulospinal and vestibulospinal pathways are func- tionally related to postural activities and progression and constitute a basic system by which the brain exerts control over movements (Kuypers 1981). The brain stem reticular formation is particularly well developed in snakes and limbless lizards (Stefanelli 1944; ten Donkelaar and Nieuwenhuys 1979), but the reptilian homologue of the mammalian nucleus of Deiters, i.e. the nucleus vestibularis ventrolateralis, is only modestly developed. So far, in snakes experimental data on descending pathways to the spinal cord are limited to a study with the classical degeneration techniques (ten Donkelaar 1976 a, b) and some preliminary HRP-data (ten Donkelaar 1982). The termi- nology employed in the present study is mainly based on a recent review of the reptilian brain stem (ten Donkelaar and Nieuwenhuys 1979); for the reticular formation the subdivision of Newman and Cruce (1982), based on Golgi studies, is largely adopted.

Materials and Techniques

In the present study a total of 15 snakes (Python regius), varying in length from 90 to 125 cm and in weight from 900 to 1,150 grams, has been used. All experiments were carried out under surgical anaesthesia (endotracheal administration of a mixture of oxygen and nitrous oxide with 1/4-1/2 vol.% halothane). The operative procedures (a laminectomy in the spinal cases; in the vestibular experiments an opening was drilled in the skull at the level of the vestibular nucIear complex) were carried out under aseptic conditions with the aid of a Zeiss binocular operation microscope.

In six cases three to six mostly unilateral injections of HRP (Boehringer; 0.1 gl, a 30% solution, dissolved in distilled water) were made into the spinal cord with a glass micropipette attached to a Hamilton syringe placed in a micromanipulator. In four cases two to three HRP slow-release gels prepared after Griffin et al. (1979), containing 20% HRP and 0.5% dimethylsulfoxide (DMSO), were implanted into the spinal cord with a fine-tipped forceps. In five cases HRP slow-release gels were implanted into the vestibular region.

Following surgery the animals were kept at an environmental temperature of 27-30 ~ and killed after postoperative survival times of five to ten days. The snakes were perfused transcar-

Reticulo- and Vestibulospinal Pathways in Snake 279

dially under deep Nembutal anaesthesia with physiological saline followed by a mixture of 1% formaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After their removal the brain and part of the spinal cord were further fixed in the perfusion mixture for one to three hours. Thoroughly fixed the material was stored for three to five hours in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose at room temperature. The brain and selected parts of the spinal cord were embedded in a 30% sucrose-phosphate solution containing 15% gelatine. These blocks were stored overnight at room temperature in a 10% formaldehyde solution. The embedded material was frozen in dry ice and cut into transverse sections of 40 gm on a freezing microtome. Every sixth section was stained according to a slightly modified Mesulam technique (1978) using tetramethylbenzidine (TMB), and counter- stained with neutral red. In addition, adjacent sections were incubated using the heavy metal intensification of 3',3-diamidinobenzidine tetrahydrochloride (DAB)-based HRP reaction product (Adams 1981).

Results

Cells of Origin of Reticulospinal and Vestibulospinal Projections

Two spinal cases will be discussed, experiment 6142 (Fig. 1) in which three HRP-injections were made into the lateral funiculus of the sixth spinal segment, and case P 82-47 (Figs. 2, 3) in which two HRP slow-release gels were implanted into the fourth segment, mainly into the gray matter and ventral funiculus. In both cases extensive reticulospinal projections were found. Due to the application of HRP to the lateral and ventral funiculus, respectively, also data on the funicular trajectory of reticulospinal and vestibulospinal pathways were obtained. Cells of origin of vestibulospinal fibers were found particularly after an HRP deposit in the ventral funiculus.

In case 6142 labeled neurons were found predominantly in two parts of the rhombencephalic reticular formation: 1) in its rostral part, predominantly contralaterally in the lateral part of the nucleus reticularis superior (Fig. 1 D, E), but also ipsilaterally, in a slightly more dorsal position, as well as in the locus coeruleus and subcoeruleus area (Fig. 1 D, E); 2) in its caudal part (Fig. 1, sections H - K ; see also insert) a large amount of HRP-positive neurons was found in the nucleus reticularis inferior, particularly in its ventral part (nucleus reticularis inferior pars ventralis of Newman and Cruce 1982), and in the adjacent nucleus raphes inferior. Labeled cells were also observed in the dorsal part of the caudal brain stem, lateral to the nucleus of the solitary tract, i.e. in the nucleus dorsalis myelencephali (Molenaar 1977). In the middle part of the rhombencephalic reticular formation (Fig. 1, sections F, G) only relatively few labeled neurons (small and medium-sized cells) were found, e.g. at the level of the sixth cranial nerve a group of small cells was labeled. None of the magnocellular elements of the middle part of the rhombencephalic reticular formation was found labeled. In the midbrain some of the large neurons of the interstitial nucleus of the fasciculus longitudinalis medialis (tim) were labeled, as well as some cells scattered in the midbrain tegmentum (Fig. 1 B, C), presumably part of the midbrain reticular formation.

In case 6142 in the vestibular nuclear complex almost no HRP-positive cells were observed. Only in the magnocellular ventrolateral vestibular nucle-

6142

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Fig. 1. The distribution of labeled neurons in the brain stem after HRP injections into the 6th spinal segment of Python regius. Asterisks indicate labeled cells in the interstitial nucleus of the tim, dots labeled cells in other brain stem structures. Insert, photomicrograph showing labeled neurons in the nucleus reticularis inferior and nucleus raphes inferior (section J), TMB- technique, x 35. Abbreviations: cereb cerebellum; Dm nucleus dorsalis myelencephali; f lm fasciculus longitudinalis medialis; Iflm nucleus interstitialis of the tim; Lc locus coeruleus; nIII, nV, nVI nervus oculomotorius, - trigeminus, - abducens; Pb parabrachial nucleus; Prm nucleus profundus mesencephali; Rai, Ras nucleus raphes inferior, superior; Ri, Rm, Rs nucleus reticularis inferior ,- medius , - superior; Riv ventral part of nucleus reticularis inferior; Rsl lateral part of nucleus reticularis superior; Sc subcoeruleus area; Sol nucleus of the solitary tract; tm tectum mesencephali; Tot1 laminar nucleus of the torus semicircularis; Vevl, Vevm ventrolateral and ventromedial vestibular nuclei; III oculomotor nucleus; Vds, Vm descending and motor trigeminal nuclei; ZI I hypoglossal nucleus


us (Fig. 1 G) a few labeled neurons were found. So, in cases as 6142 in which HRP was applied selectively to the lateral funiculus, cells of origin of spinal projections from the brain stem were found mainly in the rostral and caudal parts of the rhombencephalic reticular formation.

In contrast, in cases in which HRP was applied to the ventralfuniculus (e.g. P82-47, Figs. 2, 3), many more labeled cells were observed in the middle part of the rhombencephalic reticular formation and in the vestibular nuclear complex. Particularly the labeling in the magnocellular nucleus reticularis medius (Figs. 2H-J, 3 A) should be emphasized. Labeled axons could be traced retrogradely via the medial longitudinal fascicle (see e.g. Figs. 2H, I, K; 3 A, C) to their parent cell bodies. In the more laterally situated nucleus reticularis superior which extends from the level of the motor trigeminal nucleus rostralwards, also HRP-positive cells were found, including some magnocellular elements. This reticular field extends into the most caudal part of the midbrain tegmentum (Fig. 2D). Labeled cells were also found in the locus coeruleus and nucleus reticularis superior pars lateralis (Fig. 2 E). It should be noted that both gels passed via the most dorsal part of the lateral funiculus. In the caudal part of the rhombencephalic reticular formation a distinct number of labeled neurons was observed in the nucleus reticularis inferior, however, only sparsely in its ventral part (Fig. 2 K, L; compare with Fig. 1, sections I, J).

In the midbrain a distinct spinal projection arising in the interstitial nucleus of the tim (Figs. 2A, B; 3 B) was found. In addition, some scattered HRP-positive cells were present in the midbrain tegmentum, e.g. lateral to the laminar nucleus of the torus semicircularis (Fig. 2 C, D), but particularly contralaterally in and around the nucleus profundus mesencephali.

In the vestibular nuclear complex a few labeled neurons were observed in the dorsolateral vestibular nucleus (Fig. 2H). Distinct spinal projections were found to arise in the ventrolateral (Fig. 2I), descending (Fig. 2J) and ventromedial (Figs. 2J, K; 3C) vestibular nuclei. In the ventrolateral and descending vestibular nuclei HRP-positive cells were found bilaterally, in the ventromedial nucleus mainly contralaterally. In experiments in which the HRP-deposit was restricted to one ventral funiculus, and did not involve the contralateral spinal gray as in case P82-47, this projection pattern of the vestibular nuclei was further substantiated: the ventrolateral vestibular nucleus was found to project mainly ipsilaterally to the cord, the descending and ventromedial vestibular nuclei predominantly contralaterally. These vestibulospinal projections all pass via the ventral funiculus.

In such experiments in which the HRP injections or implants were restricted to the ventral funiculus in the reticular formation labeled cells were found mainly in the nucleus reticularis superior and nucleus reticularis medius; in both nuclei largely ipsilaterally. In these cases almost no labeled neurons were observed in the contralateral nucleus reticularis superior pars lateralis and sparsely in the locus coeruleus and subcoeruleus area. This indicates that the lateral part of the superior reticular nucleus projects to the spinal cord via the lateral funiculus, the locus coeruleus and subcoeruleus area via both the lateral and ventral funiculus.


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Fig. 2. The distribution of labeled neurons and fibers in the brain stem after the implantat ion of HRP slow-release gels into the 4th spinal segment of Python regius. Asterisks indicate labeled cells in the interstitial nucleus of the tim, dots labeled cells in other brain stem structures, broken lines retrogradely labeled fibers. Abbreviations: Cerm medial cerebellar nucleus; Vedl, Veds dorsolateral and descending vestibular nuclei. For other abbreviations see Fig. I


Fig. 3. Retrogradely labeled neurons in: A the nucleus reticularis medius (see Fig. 2H); B the interstitial nucleus of the tim (see Fig. 2B); C the nucleus vestibulari.s ventromedialis (see Fig. 2K). TMB-technique, x 59

In case P82-47 also distinct spinal projections were found to arise in the medial cerebellar nucleus (Fig. 2 F), in the descending trigeminal nucleus (Fig. 2G-K) and in the nucleus of the solitary tract (Fig. 2L; see also Fig. 1 I-K).

After HRP injections or implants placed more caudally into the spinal cord the number of retrogradely labeled neurons in the brain stem is rapidly declining, especially when made into the intumescentia trunci. This enlarge- ment of the spinal cord is related to the well-developed main part of the snake's trunk and due to a profuse development of propriospinal fibers (Kusuma et al. 1979). Particularly the decline in labeling in the nucleus reticularis inferior (mainly in its ventral part) and in the descending and ventromedial vestibular nuclei is striking, indicating that these reticulospinal and vestibulospinal fibers terminate predominantly in the thin "neck" area (approximately the first 50 spinal segments) of the spinal cord of Python regius. These projections were found at least as far as the 38th segment. After injections into the intumescentia trunci (into the 90th and 104th segments, respectively) almost no labeled cells were observed in the ventral part of the inferior reticular nucleus and no HRP-positive cells in the descending and ventromedial vestibular nucleus. Other parts of the reticular formation including the lateral part of the superior reticular nucleus and the locus coeruleus, as well as the magnocellular ventrolateral vestibular nucleus were found to project at least as far caudally as the 104th spinal segment (in Python regius about 210-220 precloacal segments are present).

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P 8 2 - 5 1 Fig. 4. The distribution of labeled neurons, fibers and terminal structures after the implantation of an HRP slow-release gel into the vestibular nuclear complex. The dark area indicates the HRP gel, the shaded area the distribution of HRP within the vestibular nuclear complex. Abbreviations: Codm dorsal magnocellular cochlear nucleus; n VIII, nXI I vestibulocochlear and hypoglossal nerves; Oli oliva inferior; Phy perihypoglossal nuclear complex; vespl, vespm lateral and medial vestibulospinal tracts; Vdsl lateral descending trigeminal nucleus; VI abdu- cent nucleus; VII facial motor nucleus; VII-VIII , I X spinal gray areas after Kusuma et al. (1979); Z m d nucleus motorius dorsalis nervi vagi. For other abbreviations see Figs. 1, 2


HRP-Gel Implants into the Vestibular Nuclear Complex: Vestibulospinal Projections

After HRP slow-release gel implants into the vestibular nuclear complex vestibulospinal fibers could be traced anterogradely at least as far as the rostral spinal cord. In case P82-51 the HRP deposit involved the ventrolateral and descending vestibular nuclei as well as a small part of the ventromedial vestibular nucleus (Fig. 4A-C). Two vestibulospinal projections could be traced as far as the third spinal segment. A lateral vestibulospinal tract passed through the lateral part of the caudal brain stem, gradually taking in a more medial position (Fig. 4H, I) and continues in the ventral funiculus. This course is in keeping with anterograde degeneration findings in Python reticulatus (ten Donkelaar 1976b). Fibers leave the lateral vestibulospinal tract to terminate in area VII-VIII of the spinal gray (subdivision of Ku- suma et al. 1979). In restricted number labeled terminal structures were also found in the motoneuron area, i.e. area IX (Fig. 4). A contralateral vestibulospinal pathway could be traced via the tim, i.e. the medial vestibulospinal tract (Fig. 4 ~ J ) . This tract was found to terminate selectively in area IX of the first three spinal segments.

In case P82-51 in addition to vestibutospinal projections also com- missural vestibular connections and vestibulomesencephalic projections were found. Vestibulomesencephalic fibers could be traced via the tim to the nuclei innervating the external eye muscle and to the interstitial nucleus of the tim.

Furthermore, the gel presumably damaged olivocerebellar fibers, since retrogradely labeled neurons were also observed in the inferior olive (Fig. 4G-I; see also Bangma and ten Donkelaar 1982).

In experiments in which the HRP implant remained restricted to the ventrolateral vestibular nucleus only the ipsilateral vestibulospinal tract was labeled.

Discussion

In the present study extensive reticulospinal projections have been demonstrated in the snake Python regius as well as more modestly developed vestibulospinal pathways. The present data are in keeping with previous findings in Python reticulatus with the retrograde cell degeneration technique (ten Donkelaar i976a). However, in addition spinal projections of various other reticular cell groups as the nucleus reticularis superior pars lateralis and the subcoeruleus area were demonstrated in Python regius. Furthermore, the pattern of labeling in HRP-experiments is much more impressive than in retrograde cell degeneration studies.

In the vestibular nuclear complex, the spinal projection arising in the ventrolateral vestibular nucleus (ten Donkelaar 1976a) was confirmed. This nucleus gives rise to the lateral vestibulospinal tract (ten Donkelaar 1976b). In the present study also distinct spinal projections were found to arise


in the descending and ventromedial vestibular nuclei. These vestibular nuclei send their axons to the spinal cord via the contralateral flm and give rise to the medial vestibulospinal tract. These findings are in keeping with experimental data in the turtle Pseudernys scripta elegans (ten Donkelaar et al. 1980; Woodson and Kiinzle 1982; Bangma and ten Donkelaar 1983) and the lizard Varanus exanthematicus (ten Donkelaar et al. 1980; ten Donkelaar and de Boer-van Huizen 1983). In addition as in Pseudemys scripta elegans (Woodson and Ktinzle 1982) a small spinal projection of the dorsolateral vestibular nucleus was found.

The funicular trajectories of the various reticulospinal and vestibulospinal projections demonstrated appear to be in keeping with previous data in quadrupedal reptiles (ten Donkelaar and de Boer-van Huizen 1978; ten Donkelaar et al. 1980; Wolters et al. 1982; Newman et al. 1983). The following reticular nuclei project via the lateral funiculus: the nucleus reticularis superior pars lateralis, the nucleus reticularis inferior and the nucleus raphes inferior. Reticulospinal pathways by way of the ventral funiculus arise in the nucleus reticularis superior and the nucleus reticularis medius. Vestibu- lospinal and interstitiospinal projections also pass through the ventral funiculus. Spinal projections from the locus coeruleus and subcoeruleus area apparently course through both the lateral and ventral funiculi.

These observations on the funicular trajectories of reticulospinal and vestibulospinal pathways in Python regius show striking resemblances with experimental data in mammals (see e.g. Kuypers and Maisky 1975, 1977; Basbaum and Fields 1979; Zemlan and Pfaff 1979; Martin et al. 1979, 1981 ; Holstege and Kuypers 1982). In the lateral pontine tegmentum of mammals (part of the rostral rhombencephalic reticular formation) a dorsal part including the locus coeruleus and subcoeruleus area projects mainly ipsilaterally via both the lateral and ventral funiculi, a ventral part (the lateral pontine area, Kuypers and Maisky 1975) contralaterally via the lateral funiculus. Other parts of the pontine reticular formation in mammals make use of the ventral funiculus for their spinal projections, the medullary reticular formation of the lateral funiculus. As in reptiles in mammals vestibulospinal and interstitiospinal projections pass via the ventral funiculus.

The impressive labeling in the nucleus reticularis inferior, particularly in its ventral part (see Fig. 1) is only evident after HRP injections or gel implants into the rostral spinal cord. After more caudally applied HRP, particularly when placed in the intumescentia trunci, almost no labeled neurons were observed in the ventral part of the nucleus reticularis inferior. This suggests a role in the control o f ' n e c k ' musculature. In this respect it is interesting to note that in cat, Peterson and co-workers (Peterson et al. 1975, 1978, 1979; Peterson 1979) presented experimental data, indicating that the medial pontomedullary reticular formation can be divided into a number of zones each with a different pattern of connections with limb, neck or trunk motoneurons, respectively. HRP data in opossum (Martin et al. 1981) and cat (Coulter et al. 1979, Huerta and Harting 1982) showed that particular parts of the medullary reticular formation such as the nucleus


reticularis medullae oblongatae ventralis projects most heavily to cervical levels of the spinal cord. It is tempting to conclude that such connections as the spinal projections from the ventral part of the inferior reticular nucleus in Python regius, are involved in the control of head and neck movements.

Also the descending and ventromedial vestibular nuclei project mainly to the "neck area" of the spinal cord (approximately the first 50 spinal segments). Direct projections of vestibular nuclei to motoneurons in the rostral spinal cord were demonstrated, particularly arising in the ventromedial and descending vestibular nuclei. These vestibulocollic projections have also been found in Pseudemys scripta elegans (Bangma and ten Donkelaar 1983) and Varanus exanthernaticus (ten Donkelaar and de Boer-van Huizen 1983) and are in keeping with recent results of Eden and Correia (1982) in the pigeon. With autoradiographic techniques both medial and lateral vestibulospinal fibers were demonstrated to terminate among neck muscle motoneurons.

The relatively thin "neck area" of Python regius is used extensively in acts as prey-searching and orientation, requiring a special brain stem control. The present data indicate that reticulospinal projections arising in the ventral part of the nucleus reticularis inferior and vestibulospinal fibers arising in the ventromedial and descending vestibular nuclei play im- portant roles in such mechanisms.

Acknowledgements. The authors wish to thank Mr. Hendrik Jan Janssen for expert technical assistance, Mr. Joop Russon for the drawing and Mrs. Riet Fliervoet for typing the manuscript.

This investigation was supported by the Foundation for Medical Research FUNGO, which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

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Accepted September 2, 1983

reticulospinal and vestibulospinal pathways in the snake python regius

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