Descending projections to the hindbrain and spinal cord in the paddlefish Polyodon spathula

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Descending projections to the hindbrain and spinal cord in the paddlefish Polyodon spathula Michael G. Metzen b , Mwaka Chambwa a , Lon A. Wilkens a , Michael H. Hofmann a,b,⁎ a

Center for Neurodynamics, Department of Biology, University of Missouri, St Louis, MO 63121, USA Institute for Zoology, University of Bonn, 53115 Bonn, Germany

b

A R T I C LE I N FO

AB S T R A C T

Article history:

In vertebrates, almost all motor neurons innervating skeletal muscles are located in the

Accepted 22 December 2009

hindbrain and spinal cord, and all brain centers that control behavior have descending

Available online 4 January 2010

projections into these parts of the central nervous system. With tracer injections into the spinal cord and hindbrain, we have studied cell groups with descending projections in the

Keywords:

paddlefish. Spinal cord injections reveal retrogradely labeled cells in all reticular and raphe

Descending projections

nuclei, as well as the nucleus of the medial longitudinal fascicle. Additional cell groups with

Hindbrain

projections to the spinal cord are the nucleus of the fasciculus solitarius, descending

Spinal cord

trigeminal nucleus, several octavolateral nuclei, the dorsal hypothalamic nucleus, and the

Paddlefish

pretectum. The only primary sensory fibers with descending projections are trigeminal

Midbrain

fibers. Hindbrain injections reveal a number of additional cell groups in di- and mesencephalon. The most prominent source is the mesencephalic tectum. Other descending cells were found in the dorsal posterior thalamic nucleus, ventral thalamus, torus semicircularis, lateral mesencephalic nucleus, and the central gray of the mesencephalon. Our data show that descending spinal projections are comparable to those of other vertebrates and that the tectum is the most important motor control center projecting to the hindbrain. A surprising result was that the dorsal posterior thalamic nucleus also projects to the hindbrain. This nucleus is thought to be a center that relays

⁎ Corresponding author. University of Bonn, Institute for Zoology, Poppelsdorfer Schoss, 53115 Bonn, Germany. E-mail address: [email protected] (M.H. Hofmann). Abbreviations: aur, auricles; BDA, biotinylated dextran amine; bo, bulbus olfactorius; cer, cerebellum; cpth, central posterior thalamic nucleus; DAB, diaminobenzidine; don, dorsal octavolateral nucleus; dpth, dorsal posterior thalamic nucleus; fr, fasciculus retroflexus; gcm, griseum centrale mesencephali; lmn, lateral mesencephalic nucleus; Mth, Mauthner cell; mIII, oculomotor nucleus; mes, mesencephalon; meV, nucleus mesencephalicus nervi trigemini; mon, medial octavolateral nucleus; nI, olfactory nerve; nII, optic nerve; nIII, oculomotor nerve; nIV, abducens nerve; nIX, glossopharyngeal nerve; nV, trigeminal nerve; nVII, facial nerve; nVIII, octaval nerv; nLLa, anterior lateral line nerve; nLLp, posterior lateral line nerve; npe, nucleus preeminentialis; nuflm, nucleus of the fasciculus longitudinalis medialis; nufs, nucleus fasciculus solitarius; nX, vagal nerve; oli, inferior olive; PB, phosphate buffer; pd, dorsal periventricular hypothalamus; pl, lateral periventricular hypothalamus; pp, preoptic nucleus; pt, posterior tuberal nucleus; pv, ventral periventricular hypothalamus; prtc, central pretectal nucleus; prtp, periventricular pretectum; rai, raphe inferior; ram, raphe medialis; ri, nucleus reticularis inferior; rm, nucleus reticularis medius; rs, nucleus reticularis superior; sac, stratum album centrale; sc, spinal cord; sch, nucleus suprachiasmaticus; sco, supracommissural organ; sfgs, stratum fibrosum et griseum superficiale; sgc, stratum griseum centrale; sm, stratum marginale; so, stratum opticum; spv, stratum periventriculare; tel, telencephalon; tl, torus longitudinalis; tla, torus lateralis; tm, mesencephalic tectum; ts, torus semicircularis; Vd, nucleus descendens trigemini; vem, magnocellular vestibular nucleus; VIIId, descending octaval nucleus; visc, nucleus visceralis secundarius; Vm, trigeminal motor nucleus; vmth, nucleus ventromedialis thalami; Xm, vagal motor nucleus 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.12.078

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sensory information to the telencephalon. Further studies are needed to determine the complete set of projections of the dorsal thalamus in paddlefish and other fishes to gain insights into its functional role. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

The aim of this study was to identify the major neuronal tracts that send descending axons to the spinal cord and hindbrain in the paddlefish (Polyodon spathula), a sister group to the sturgeons representing a more basal group of actinopterygian fishes. The actinopterygian radiation of ray-finned fishes consists of five major groups: Cladistia, which are reedfishes and the most basal group; Chodrostei, which are the sturgeons and paddlefishes; Ginglimodi, the gars; Halecomorphi, of which only the bowfin Amia is extant; and the large radiation of Teleostei, the bony fishes. The teleosts consist of four major groups, of which the largest is the Euteleostei, and that group includes a number of the species discussed herein, such as the ostariophysans (goldfishes, carp, and catfishes), trout, and the very large radiation of percomorph fishes that includes perciforms (Nelson, 2006). Cell groups with descending connections to the spinal cord are the sole source of neurons that can modulate or initiate trunk movements, since all the motor neurons innervating trunk muscles are located in the spinal cord (Landmesser, 1980; Westerfield et al., 1986). Motor neurons innervating cranial muscles are all located in the rhombencephalon (Nieuwenhuys et al., 1982), with the exception of the oculomotor nucleus that innervates eye muscles (Butler and Hodos, 2005). Much of the motor control is organized within the spinal cord or hindbrain, but forebrain and midbrain centers that modulate behavior must ultimately send fibers to the hindbrain. Olfactory and visual information enters the forebrain, and most information from the electrosensory, the mechanosensory lateral line, and the auditory system is relayed to the midbrain. To understand information processing in these modalities and identify the major pathways that transform sensory information into motor activity, it is important to get an overview of all pathways descending to motor centers in the hindbrain and spinal cord. Descending afferents of the spinal cord are well investigated in a number of species (Corvaja and d'Ascanio, 1981; d'Ascanio and Corvaja, 1981; Prasada Rao et al., 1987, Prasada Rao et al., 1993; ten Donkelaar, 1976, 1982), but there are currently no studies that describe the complete set of cell groups with descending projections to the hindbrain. Many studies are focused on tectal efferents, which constitute probably the most prominent descending pathway, but little is known about additional sources. In this study, we investigated descending pathways in the paddlefish as part of a larger project to understand the basic functional organization of the fish brain. Paddlefish, like sturgeons, have a relatively simple brain organization that is similar to that in bichirs, reedfish, and garfish. Hagfish, lampreys, elasmobranchs, and teleosts have different specializations with many features that are probably highly derived

and therefore do not represent the ancestral condition. The paddlefish brain, on the other hand, may provide us with information on how major descending pathways are organized in a brain that probably has not changed much in over 100 million years following the appearance of fossils in the family Polyodontidae (Grande et al., 2002).

2.

Results

2.1.

Spinal cord injections

In eight animals, tracer injections were made into the spinal cord at a level just caudal to the large hematopoietic organ situated on top of the spinal cord. Fig. 1 shows a dorsal view of the brain of the paddlefish and the level of the sections for the following figures. We adopted the nomenclature of Nieuwenhuys et al. (1998). Depending on the exact location of the injection site, various cell groups were retrogradely labeled. The results of a combination of all eight cases are shown in Fig. 2. Most of the cells were located in the hindbrain. Within the hindbrain, the most caudally located cell group is the nucleus descendens of the trigeminus (Vd), which is labeled bilaterally (Figs. 2A and 3A) just anterior to the obex. At the more rostral level of this nucleus, some cells of the inferior reticular nucleus (ri) were

Fig. 1 – Dorsal view of the brain with cranial nerves of Polyodon spathula. The dashes indicate the location of cross sections shown in the following figures. Scale bar = 3 mm (modified after Pothmann et al. (2009)).

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Fig. 2 – (A–G) Series of transverse sections from caudal to rostral through the hindbrain and midbrain of Polyodon spathula after injection of tracer into the spinal cord. The injected side is indicated with an asterisk in panel A. Retrograde labeled cells are represented by solid dots. Scale bar = 1 mm.

also labeled bilaterally. The inferior reticular nucleus extends caudal and moves ventrally, providing space for the motor nucleus of the vagus (Xm). Neither vagal motor cells nor any other cranial nerve motor neurons were labeled in any of the eight cases. At the caudal end of the vagal motor nucleus, all the dorsal sensory areas, e.g., the dorsal and medial octavo-

lateral nuclei (don, mon), the octaval area, and the nucleus of the fasciculus solitarius (nufs), are well developed (Fig. 2B). At this level, retrogradely labeled cells were seen only in the descending trigeminal nucleus (Vd) (Fig. 3D), the descending octaval nucleus (VIIId), and the nucleus of the fasciculus solitarius (nufs) (Fig. 3C), ipsilateral to the injection but not in

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Fig. 3 – Photographs of different cell groups in the brain of the paddlefish labeled after BDA injections into the spinal cord. (A) Nucleus descendens of the trigeminus. (B) Section through the caudal hindbrain showing cells in the inferior reticular nucleus (arrowhead) and inferior raphe (asterisk). (C) Cells in the ipsilateral nucleus of the fasciculus solitarius. (D) Two cells in the descending trigeminal nucleus. (E) Median reticular nucleus. (F) Superior reticular nucleus. (G) Mauthner cell. (H) Medial raphe. (I) Pretectum. (J) Dorsal periventricular hypothalamus. Scale bars = (A) 100 μm; (B) 250 μm; (C, D, E) 100 μm; (F) 50 μm; (G) 200 μm; (H–J) 100 μm.

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the lateral line areas. In addition, some cells of the inferior reticular nucleus (ri) and inferior raphe (rai) were stained (Figs. 2B and 3B). Further rostrally, at the level of the octaval nerve, the large Mauthner cell (Mth) is stained on the contralateral side (Figs. 2C and 3G). It has mostly ventrally directed dendrites and a special lateral dendrite that extends into the area of incoming acoustic fibers. In this area, some cells of the median reticular nucleus (rm) were also stained bilaterally (Figs. 2C and 3E). Retrogradely labeled cells were also present in the medial raphe (ram) (Figs. 2C and 3H). In some cases, cells in the magnocellular vestibular nucleus (vem) were labeled ipsilaterally (Fig. 2C). At the level of the trigeminal nerve (nV), many cells in the superior reticular nucleus (rs) were labeled bilaterally (Figs. 2D and 3F). This cell group extends up to the isthmic area where it shifts laterally and is displaced by the central grey of the mesencephalon (gcm) (Fig. 2E). Many labeled fibers were found in the trigeminal nerve (nV). Since there were no trigeminal motor neurons stained, these fibers are probably primary afferent fibers that descend into the spinal cord. At the rostral end of the superior reticular nucleus, cells in the secondary gustatory nucleus (visc) were also stained ipsilateral to the injection site. In some cases, large, weakly labeled cells could be identified bilaterally in the nucleus of the medial longitudinal fascicle (nuflm). In addition, smaller cells lateral to this nucleus were stained bilaterally (Fig. 2F). Some of the mesencephalic trigeminal cells (meV) were also lightly stained (Fig. 2G). In the diencephalon, retrogradely labeled cells were present in the contralateral central pretectum (prtc) (Figs. 2G and 3I). Some cells were also stained in the ventral thalamus (vmth), bilaterally, and in the ipsilateral dorsal periventricular hypothalamus (pd) (Figs. 2F and 3J).

2.2.

Hindbrain injections

Most tracer injections (17) were made into areas of the hindbrain caudal to the entrance of the anterior lateral line nerve (nLLa). Of these, 13 injections were made from the dorsal part of the brain and 4 injections were made from the ventral part through the roof of the mouth. No injection was more rostral than the entrance of the trigeminal nerve (nV). Some of the injections were restricted to more lateral areas (don, mon) that are dominated by sensory input. These injections revealed no retrogradely labeled cells in the midbrain or forebrain. Only more medial injections resulted in a large number of labeled cells. There was considerable variation in the results depending on which fiber tract was most heavily labeled in the hindbrain. Fig. 5 summarizes the results of all cases showing all cell groups that send descending projections to the hindbrain. The majority of cells that send fibers into the hindbrain are located in the mesencephalic tectum (tm), which is organized relatively simply. Fig. 4 shows the cell and fiber layers of the paddlefish tectum. All of the tectal layers previously described in Acipenser were found (Yamamoto et al., 1999). The optic tectum of the paddlefish has a relatively thin stratum marginale (sm) and stratum opticum (so) (Fig. 4). Retrogradely labeled neurons were found throughout all layers of the

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Fig. 4 – Photomicrograph of a cross-section through the optic tectum of the paddlefish stained with cresyl violet (sm, stratum marginale; so, stratum opticum; sfgs, stratum fibrosum et griseum superficiale; sgc, stratum griseum centrale; sac, stratum album centrale; spv, stratum periventriculare). Scale bar = 100 μm.

mesencephalic tectum, except for the stratum marginale and the stratum opticum (Figs. 5A, B). We could distinguish several types of cells depending on their size, location, and dendritic arborization. The largest cells were multipolar and found in the stratum album centrale (sac) (Fig. 6A). These cells had the largest dendrites, which extended throughout all layers but only in the contralateral tectum. Large cells were also labeled in the stratum griseum centrale (sgc) (Figs. 5A, B and 6C). The most numerous of these cells were labeled contralateral to the injection site, whereas the ipsilateral tectum revealed only a few labeled cells in the stratum griseum centrale (Figs. 5A, B). Similar to the large multipolar cells found in the stratum album centrale, cells of the stratum griseum centrale also had large dendrites and their arborizations extended mostly into the upper layers of the tectum. In some cases, fusiform cells were labeled bilaterally in the stratum griseum centrale. Small cells were labeled in the stratum griseum periventriculare (spv) (Fig 6B). This stratum consists of several sublayers of small cells (Fig. 4). We found stained cells in each of the layers of the stratum periventriculare and their fine dendrites extended vertically, sometimes to the marginal layer of the tectum (Fig. 6B). Another cell type in the stratum periventriculare was somewhat larger with a pair of dendrites that diverge horizontally for about 100 μm before abruptly turning upwards and extending to the surface of the tectum (Fig. 6D). All of these cell types occurred only in the contralateral tectum. On the ipsilateral side, mostly periventricular cells

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Fig. 5 – Series of transverse sections from caudal to rostral through the midbrain of Polyodon spathula after tracer injection into the hindbrain. The injected side is indicated with an asterisk in panel A. Retrograde labeled cells are represented by solid dots. Scale bar = 1 mm.

were found and, occasionally, somewhat larger cells in the stratum griseum centrale. In nearly all cases, labeled cells were present in the torus semicircularis (ts) (Figs. 5A, B and 6E). These were always found bilateral to the injection site, although the ipsilateral projections revealed a much heavier staining. In some cases, larger numbers of cells were found, indicating that their axons form a distinct tract in the hindbrain that we did not always hit with our tracer injections. Some labeled cells were also found

bilaterally in the lateral mesencephalic nucleus (lmn) (Figs. 5A and 6E). These cells were larger and had dendrites extending laterally and medially over a long distance. Similar cells were found in the ipsilateral central grey of the mesencephalon (gcm) (Fig. 5A). Some of these cells had dendrites extending into the torus lateralis (tla) (Fig. 6F). In addition, a few smaller periventricular cells were labeled in the central grey. Further rostrally, cells of the nucleus of the medial longitudinal fascicle (nuflm) were labeled bilaterally (Fig. 5B).

Fig. 6 – Photographs of different cell groups and types in the tectum mesencephali (A–D) and the midbrain tegmentum (E–I) labeled by BDA injections into the hindbrain of the paddlefish. (A) Multipolar cells in the stratum album centrale of the optic tectum with large dendrites extending throughout all tectal layers. (B) Multipolar cells of the stratum periventriculare with thin dendrites running up to the startum marginale (arrowheads) and small periventricular cells (asterisk). (C) Large cells in the stratum griseum centrale. (D) Cell in the stratum periventriculare (cell body marked with an asterisk) with a pair of primary dendrites running to the surface of the optic tectum (arrowheads). (E) Torus semicircularis (arrowhead) and lateral mesencephalic nucleus (asterisk). (F) Cell in the central grey of the mesencephalon with dendrites extending to the torus lateralis. (G) Dorsal posterior thalamic nucleus. (H) Periventricular pretectum. (I) Pretectum. Scale bars = (A–C) 250 μm; (D, E) 100 μm; (F) 500 μm; (G–I) 100 μm.

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In addition to these large periventricular cells, smaller cells located more laterally were also stained bilaterally. A few cells were seen in the ipsilateral dorsal periventricular hypothalamic nucleus (pd) (Fig. 5B). The central nucleus of the pretectum (prtc) contained many large cells with dendrites

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oriented horizontally (Figs. 5C and 6I). These cells were found bilaterally, but those in the contralateral pretectum were much more abundant. More medially, many smaller cells of the ipsilateral periventricular pretectum (prtp) were stained, located just above the fasciculus retroflexus (fr) (Figs. 5C and

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6H). Some cells were located ventral to the fasciculus retroflexus, in the ipsilateral dorsal posterior thalamic nucleus (dpth) (Figs. 5C and 6G). Retrogradely labeled cells in the di- and mesencephalon could only be found after tracer injections into the ventral part of the hindbrain. We have extensive material on don injections and never found a single cell labeled in the di- or mesencephalon despite the presence of extensive ascending projections. Two of the hindbrain injections were centered in the acoustic and lateral line center (Figs. 7A, B). Here also, only ascending fibers were labeled in the midbrain, but no cell bodies. Retrogradely labeled cells in the forebrain and midbrain were only present after tracer injections into the medial parts of the hindbrain (Figs. 7C–F). The large multipolar cells in the contralateral tectum were only filled when tracer was applied close to the midline (Figs. 7E, F).

3.

Discussion

3.1.

Cell groups projecting to the spinal cord

In the paddlefish, the descending spinal projections are similar to other vertebrates. The major sources of descending projections are the hindbrain reticular nuclei and the inferior raphe. This is similar to the findings in goldfish and zebrafish, which are ostariophysan euteleosts (Becker et al., 1997; Prasada Rao et al., 1987); the elopomorph Anguilla (Bosch and Roberts, 1994); elasmobranchs (Smeets and Timerick, 1981); lungfishes (Ronan and Northcutt, 1985); the channel catfish, which is also an ostariophysan (New et al., 1998); and terrestrial vertebrates (Cruce and Newman, 1981; ten Donkelaar, 1976; Woodson and Kunzle, 1982). A few labeled cells

Fig. 7 – Photographs of transverse sections at injection sites in the brainstem (A, C, E) and more rostral levels (B, D, F) for three experiments with injections into the octavolateral area (A, B), the lateral basal plate (C, D), and the medial basal plate (E, F). Asterisk in panels A, C, and E marks the center of the injection site. Note that all injections are restricted to one side. Even in the very medial injection (E, F), the tracer deposit is restricted to one side and there is little or no diffusion to the contralateral side. Scale bar = 1 mm.

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were found in the medial raphe, which has been identified by Adrio et al. (1999) in Acipenser. There appears to be no projection from the superior raphe to the spinal cord like in other vertebrates (see Lillesaar et al., 2009). The Mauthner cells are also an important source of descending projections to the spinal cord (Ronan and Northcutt, 1985). In addition to these premotor areas, rhombencephalic cell groups also send sensory information to the paddlefish spinal cord. These include the descending trigeminal nucleus and several octaval nuclei that probably have vestibular function. These projections are also present in hagfish (Ronan, 1989), lampreys (Swain et al., 1993; Swain et al., 1995), elasmobranchs (Cruce et al., 1999; Smeets and Timerick, 1981; Timerick et al., 1992), rayfinned fish (Behrend and Donicht, 1990; Demski and Sloan, 1985; Gregory and Tweedle, 1985; Hlavacek et al., 1984; Northcutt, 1983; Oka et al., 1986; Prasada Rao et al., 1987, 1993; Roberts, 1992; Sharma et al., 1993), and lungfish (Ronan and Northcutt, 1985). The nucleus of the tractus solitarius sends taste information to the spinal cord in lampreys (Ronan, 1989), elasmobranchs (Smeets and Timerick, 1981), lungfish (Ronan and Northcutt, 1985), catfish (Finger, 1978), and the goldfish (Prasada Rao et al., 1987, 1993; Sharma et al., 1993), but apparently not in other teleosts. In catfish and goldfish, it is the facial lobe that projects to the spinal cord, but not the vagal lobe. In the paddlefish, there are no distinct facial and vagal lobes, but cells descending to the spinal cord were found only in the rostral part of the nucleus of the solitary tract. This suggests that also in the paddlefish, only the facial component has descending projections. In the paddlefish, there are also a few cells labeled dorsolateral to the nucleus reticularis inferior that we identified as the secondary gustatory nucleus (visc). It is in a similar location as a nucleus gustatorius secundarius described by Nieuwenhuys and Pouwels (1983). Since a spinal projection of this nucleus was not reported for other vertebrates, further data are necessary to confirm the identity of this cell group. The nucleus of the medial longitudinal fascicle projects to the spinal cord in all vertebrates investigated so far. In other actinopterygian fish, the red nucleus is described as a small but distinct aggregation of cells in the medial part of the tegmentum that receives a crossed projection from the cerebellum and projects to the spinal cord (Nieuwenhuys and Pouwels, 1983). Corrêa et al. (1998) described that electrical stimulation of the red nucleus in the ostariophysan Gymnotus carapo induced bilateral alternating movements of the tail. A contralaterally projecting red nucleus was also found in many other vertebrate groups (Smeets and Timerick, 1981). These include elasmobranchs (Ronan and Northcutt, 1985), lungfish, and several other teleosts (Behrend and Donicht, 1990; Demski and Sloan, 1985; Gregory and Tweedle, 1985; Hlavacek et al., 1984; Oka et al., 1986; Prasada Rao et al., 1987, 1993; Roberts, 1992; Sharma et al., 1993). In elasmobranchs, additional cell groups were found to project from the mesencephalon to the spinal cord, including the optic tectum and nucleus intercollicularis (Smeets and Timerick, 1981). Diencephalic cell groups projecting to the paddlefish spinal cord include the pretectum, ventral thalamus, and dorsal periventricular hypothalamic nucleus. These cell groups also project to the spinal cord in lampreys, but not in hagfish

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(Ronan, 1989). In elasmobranchs (Smeets and Timerick, 1981) and amphibians (Sanchez-Camacho et al., 2001), the ventral thalamus projects to the spinal cord and, in Scyliorhinus, some cells of the periventricular hypothalamic nucleus were also reported to project to the spinal cord (Smeets and Timerick, 1981). Furthermore, some cells were found in the nucleus of the posterior commissure (Smeets and Timerick, 1981), which may be comparable to the pretectal cells in the paddlefish. In teleosts, a projection from the pretectum was reported only in salmon (Oka et al., 1986). But in the salmon, it is a central pretectal cell group that projects to the spinal cord, not a periventricular one. Descending projections from the contralateral pretectum to the spinal cord were also described for African lungfish (Ronan and Northcutt, 1985). In summary, most descending projections to the spinal cord in lower vertebrates are highly preserved and include the reticular formation and raphe nuclei, as well as the trigeminal, vestibular, and taste systems, a pattern that is consistent in the paddlefish. In contrast, spinal projections arising from the red nucleus and optic tectum, which are present in some other vertebrate groups, are absent in the paddlefish.

3.2.

Cells groups projecting to the hindbrain

Brain stem injections were rarely made because the injection site is not restricted to a single nucleus and the interpretation of the results is difficult. Usually a large number of passing fibers are interrupted, and it is not clear where a retrogradely labeled cell in the forebrain or midbrain really projects. Despite this uncertainty, our data are useful and show the cell types that ultimately send information to the hindbrain and/or spinal cord and control behavior. Some of these descending neurons may be involved in sensory feedback loops, but most probably have a motor function since their axons run medially in the basal plate of the hindbrain. Information on the location and morphology of these cells is an important starting point for more detailed investigations of each of the descending systems. Many of the cells labeled after hindbrain injections may actually project directly to the spinal cord. To distinguish between those cells projecting to the spinal cord or to the hindbrain, we did spinal cord injections and compared these results with those from the hindbrain injections. However, due to the much longer distance to the injection site in the spinal cord, we may have missed some of the spinal cord projections. Nevertheless, our spinal cord and hindbrain injections have revealed the major descending pathways and the most important centers that organize or modulate behavior in the paddlefish. Next we will discuss only the cell groups that were not labeled after tracer injections into the spinal cord, i.e., cells stained from injections in the hindbrain. The majority of cells projecting to the paddlefish hindbrain were found in the mesencephalic tectum (Fig. 5). Some of these were among the largest of all cells seen anywhere in the forebrain and midbrain that have descending projections. It has long been recognized that the most prominent target of efferent pathways from the tectum is the reticular formation (Bosch and Paul, 1993; Ebbesson and Vanegas, 1976; Grover and Sharma, 1981; Luiten, 1981; Smeets, 1981; Striedter, 1990; Wullimann and Northcutt, 1990). Both ipsilateral and

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contralateral projections are present. Several descending cell types are present in the carp, an ostariophysan (Niida et al., 1989). Fusiform, horizontal, and multipolar cells were described predominantly in the stratum album centrale for the catfishes Ictalurus and Bagrus (Schroeder and Vanegas, 1977) and the crucian carp (Niida et al., 1989) as well as in the stratum griseum centrale and stratum album centrale in the mojarra Eugerres plumieri, which is a perciform euteleost (Vanegas et al., 1974). Fusiform, ventrally orientated cells were described in the stratum fibrosum et griseum centrale for the goldfish (Meek and Schellart, 1978). The axons of cells in the stratum album centrale constitute the tectobulbar tract (Niida et al., 1989). Large multipolar cells of the stratum album centrale with large dendritic branches extending to almost all strata were also described in E. plumieri (Vanegas et al., 1974) and in the goldfish (Meek and Schellart, 1978). Large cells in deeper tectal layers with large dendritic fields are known to be motion-sensitive and project to the hindbrain in the carp (Niida et al., 1989). Cells with similar physiological properties have been found in the perch, another perciform euteleost (Guthrie and Banks, 1978), and in the Japanese dace, an ostariophysan (Kawasaki, 1983). Large cells with extensive dendritic trees were also found to project to the hindbrain in frogs (Lázár et al., 1983) and reptiles (Sjostrom and Ulinski, 1985). Cells with similar morphology that are known to be motion-sensitive are present in the tectum of birds (Luksch et al., 1998) and in the superior colliculus of mammals (Major et al., 2000). In these animals, however, a major target of these cells is the nucleus rotundus or pulvinar complex and not the hindbrain as in anamniotes. Further studies have to show whether the large multipolar cells in the tectum of the paddlefish are also motion-sensitive and mediate prey catching behavior. The pretectum also projects to the hindbrain and some fibers descend into the spinal cord, as mentioned above. It may be involved in a wide variety of visual responses and visual-guided behavior, as is described for mammals (Sprague, 1966, 1972; Thompson et al., 1963; Thompson and Massopust, 1960; Thompson and Rich, 1961). The periventricular pretectum and the central nucleus of the pretectum are highly conserved nuclei among actinopterygian fishes (Rupp and Northcutt, 1998). The central nucleus of the pretectum is described as an area with a scattered collection of cells receiving retinal projections in chondrosteans (Ito et al., 1999) and in the cichlid Haplochromis burtoni (Fernald and Shelton, 1985). Folgueira et al. (2008) reported a direct projection to the central nucleus of the pretectum from the contralateral retina in the trout.

and these are the centers through which information must flow through to modulate behavior. It is not surprising that the tectum is an important structure that projects to the hindbrain. Numerous studies have shown that the tectum is involved in orientation and prey capture. What we did not expect to this extent is that the tectum is apparently the most important source of descending fibers to the hindbrain. Only the nucleus of the medial longitudinal fascicle has cells of a size comparable to the larger cells in the tectum, but it contains only a small number of cells. All other areas in the midbrain or forebrain projecting to the hindbrain or spinal cord have only small cells. It appears that most of the motor control is organized in the tectum, and consequently, all necessary information has to go through the tectum. The functional role of the projections from the torus semicircularis is not known. Only small toral cells project to the hindbrain, and until their exact targets can be determined, their role in behavior remains speculative. However, one function of the pretectum is to organize the optokinetic nystagmus (Klar and Hoffmann, 2002); thus a direct projection to the abducens motor nucleus in the hindbrain and to the spinal cord seems plausible. A surprising result was that the dorsal posterior thalamic nucleus also projects to the hindbrain. This nucleus is known to receive afferents from the tectum and torus semicircularis (Northcutt, 2006) and is thought to be a major relay of sensory information to the telencephalon in all vetebrates, including anamniotes. A major projection to the hindbrain challenges this view and shows that the dorsal posterior nucleus is at least as much a descending system as it is an ascending one, providing that further studies in other species corroborate the existence of this descending projection. It should be noted that the dorsal thalamus of mammals is a complex aggregation of nuclei, and not all of them relay sensory information to the cortex. The dorsal posterior thalamic nucleus could be equivalent to the non-specific thalamic motor region described in mammals (Grossman, 1958; Swerdlow and Koob, 1987). A subthalamic motor region is known to be present in probably all vertebrates (Dubuc et al., 2008; Edwards and Maillard, 1988; Ménard and Grillner, 2008; Narita et al., 2002; Sirota et al., 2000). The subthalamic locomotor region includes parts of the ventral thalamus and dorsal hypothalamus, both of which project to the hindbrain and/or spinal cord in the paddlefish. There is only limited information on the organization and connections of the thalamus in anamniotes, and more studies are needed to gain insights into its functional role and possible homologies to amniotes.

3.3.

4.

Experimental procedures

4.1.

Animals

Functional considerations

Our results show that the descending pathways to the spinal cord are very conservative, and the paddlefish is no exception. The most important centers controlling trunk movements are the reticular formation and raphe nuclei. Since all cranial motor nuclei, with the exception of two oculomotor nuclei, are also located in the hindbrain, all centers in the midbrain and forebrain that control behavior have to project to the hindbrain. Thus, with our hindbrain injections, we have identified brain areas that project directly to the hindbrain,

A total of 25 paddlefish (P. spathula) with a length of 15–20 cm were used for this study. The fish were obtained from the Hunnewell Fish Hatchery, Missouri Department of Conservation, and kept in groups in large bio-filtered and aerated tanks. The research reported herein was performed under the guidelines established by the Institutional Animal Care and Use Committee of the University of Missouri at St. Louis.

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4.2.

Retrograde tracing

Before surgery, the tips of small insect pins were dipped into a paste made from biotinylated dextran amine (BDA, molecular weight 10,000, Molecular Probes, Eugene, OR) dissolved in a small drop of distilled water. The BDA paste was allowed to dry on the tip of the pin, which was then inserted into the hindbrain or spinal cord. For surgery, animals were anesthetized with MS-222 (1:10,000; Sigma, St. Louis, MO). The skull was carefully opened with a scalpel, reflecting back a piece of cartilage to expose the brain. BDA-coated pins were inserted at different levels of the hindbrain (rhombencephalon). The skull was then closed and sealed with Vetbond Tissue Adhesive (3M, St. Paul, MN). After surgery, fish were revived by perfusing the gills with aerated water until they restarted respiratory and swimming movements. In four cases, we approached the brain from the roof of the mouth. In eight additional cases, injections were made into the spinal cord just behind the large hematopoietic organ that is situated above the spinal cord. This is approximately the location of the third spinal nerve. After 4 days of survival, the animal was deeply anesthetized with MS-222 (1:5000) and the cartilaginous skull containing the brain was removed. The brain was then exposed and fixed by immersion in 4% paraformaldehyde in phosphate buffer (PB, pH 7.4, 0.1 M). The skull and brain were stored overnight in the fixative in a refrigerator. After removal from the skull the brain was embedded in 4% agar in PB. The agar block was cut into 100-μm sections on a vibratome (OTS-4000, Electron Microscopy Sciences, Fort Washington, PA) and collected into PB. Sections were treated with a 3% H2O2 solution for 7 min to block endogenous peroxidase activity. The sections were then rinsed three times in PB for 10 min and subsequently incubated for 2 h or overnight in ABC-solution (Vectastain ABC Kit, Vector, Burlingame, CA) dissolved in PB containing 0.1 % Triton X-100. After three 10-min washings in PB, sections were stained with diaminobenzidine (DAB, Sigma, St. Louis) containing ammonium nickel sulphate and H2O2. The sections were then washed three times with PB, mounted on slides, dehydrated, and cover slipped. Some sections were counterstained with neutral red (Sigma). Photomicrographs were taken with a digital camera and adjusted for contrast, brightness, and sharpness. No retouching or other image manipulations have been performed.

Acknowledgments We thank the Hunnewell fish hatchery and the Missouri Conservation Department for kindly providing the fishes. This article was supported by a grant from the National Science Foundation (IOB-0524869).

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