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Keywords:

  • reticulospinal neurons;
  • hindbrain segmentation;
  • cranial motor nuclei;
  • hindbrain of adult teleost fishes;
  • rhombomere

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

The vertebrate hindbrain develops as a series of well-defined neuroepithelial segments or rhombomeres. While rhombomeres are visible in all vertebrate embryos, generally there is not any visible segmental anatomy in the brains of adults. Teleost fish are exceptional in retaining a rhombomeric pattern of reticulospinal neurons through embryonic, larval, and adult periods. We use this feature to map more precisely the segmental imprint in the reticular and motor basal hindbrain of adult goldfish. Analysis of serial sections cut in three planes and computer reconstructions of retrogradely labeled reticulospinal neurons yielded a segmental framework compatible with previous reports and more amenable to correlation with surrounding neuronal features. Cranial nerve motoneurons and octavolateral efferent neurons were aligned to the reticulospinal scaffold by mapping neurons immunopositive for choline acetyltransferase or retrogradely labeled from cranial nerve roots. The mapping corresponded well with the known ontogeny of these neurons and helps confirm the segmental territories defined by reticulospinal anatomy. Because both the reticulospinal and the motoneuronal segmental patterns persist in the hindbrain of adult goldfish, we hypothesize that a permanent “hindbrain framework” may be a general property that is retained in adult vertebrates. The establishment of a relationship between individual segments and neuronal phenotypes provides a convenient method for future studies that combine form, physiology, and function in adult vertebrates. J. Comp. Neurol. 522:2446–2464, 2014. © 2014 Wiley Periodicals, Inc.

The neuronal architecture of the vertebrate brainstem is strongly influenced by embryonic segmental patterning (Tümpel et al., 2009; Nieuwenhuys, 2011). This is most obvious in the rostral hindbrain, much of which develops from five well-delineated neuroepithelial segments, rhombomeres (r) 2–6 (Gilland and Baker, 1993, 2005). In contrast, the caudal part of the hindbrain in embryos is not morphologically patterned as distinct neuroepithelial segments but has been shown to contain segment-like neuronal and genetic domains, pseudorhombomeres, organized in register with adjacent occipital somites and numbered as r7–r11 in avian embryos (Cambronero and Puelles, 2000; Marin et al., 2008). With r0 and r1 lying between the midbrain–hindbrain boundary and r2, the vertebrate hindbrain thus comprises 12 segmental regions, depending on terminology and, possibly, species differences in the number of subdivisions in the caudal hindbrain.

The origin, migration, and early synaptic connections of neurons in a number of functional classes consistently map to particular positions within the basic rhombomeric framework. Among brainstem neuronal groups, reticular, vestibular and motor nuclei, in particular, have been shown to develop in register with rhombomeres. The reticular and vestibular nucleus neurons most commonly studied with retrograde dye labeling are long projection neurons that target cranial and spinal motor centers (Straka et al., 2001; Díaz and Glover, 2002). These have been demonstrated in developing lampreys (Swain et al., 1993; Murakami et al., 2004), teleosts (Kimmel, 1982; Trevarrow et al., 1990), amphibians (Straka et al., 2001), birds (Díaz et al., 1998), and mammals (Pasqualetti et al., 2007). Along with cranial motoneurons, which have very stable patterns of segmental origins in vertebrates (Gilland and Baker, 2005), these groups are distributed throughout the basal and in parts of the ventral alar region and make up the basic motor backbone of the vertebrate brainstem.

Although rhombomeres are visible features of the hindbrain in all vertebrate embryos, macroscopic identification of hindbrain segments in postembryonic brains is possible only in particular species with extended larval periods (Straka et al., 2001, 2006), after immunohistochemical staining of glial cells (Yoshida and Colman, 2000) or by surgical or genetic fate mapping (Cambronero and Puelles, 2000; Marín et al., 2008; Gray, 2008, 2013). The overall segmental pattern is masked in most adult vertebrates by differential growth of brainstem nuclei and tracts and by longitudinal and/or radial translocation of numerous neuronal groups during embryonic and larval periods (Gilland and Baker, 1993; Bruce et al., 1997; Chandrasekhar et al., 1997). Segmental organization of adult brainstem anatomy can be inferred indirectly by analyzing distribution patterns of specifically marked neuronal populations, whether by retrograde labeling (Straka et al., 2006) or immunohistochemical methods (see, e.g., Morona and González, 2009). Nevertheless, based on the known segmental origins of motor and reticular neurons, the considerable anatomical conservation of their segmental organization in adult frog (Straka et al., 2006), zebrafish (Lee and Eaton, 1991), and goldfish (Nakayama and Oda, 2004) suggests that a permanent “rhombomeric framework” (Straka et al., 2006) may be a general property that is retained in adult vertebrates.

Spatial location is a major aspect of neuronal identity, so identification of segmental territories in adult vertebrates would help to distinguish aspects of the locations and interconnections of identified hindbrain neuronal populations that reflect their rhombomeric origins from those that are dominated by other patterning guides, whether suprasegmental, dorsoventral, or otherwise. Linking anatomical position to segmental identity in adult brains is difficult because of the absence of visible rhombomeric boundaries and the likely distortion of individual segmental territories along the rostrocaudal, dorsoventral, and mediolateral axes during growth. The segmental arrangement of reticulospinal neurons in embryonic, larval, and adult cyprinids (Metcalfe et al., 1986; Trevarrow et al., 1990; Lee and Eaton, 1991; Lee at al., 1993) prompted us to map the locations of other likely segmental hindbrain neuronal populations in an attempt to demarcate the three-dimensional (3D) shape of rhombomeric territories in adult brains. In the present study, retrograde neuronal tracing, choline acetyltransferase (ChAT) immunohistochemistry, and 3D computer reconstructions were used to correlate the locations of segmentally arrayed reticulospinal and cranial motor nuclei in order to define better the rhombomeric imprint found in an adult brain. We show that the segmental organization of reticulospinal and motoneurons known in developing cyprinid fishes is fully retained in the adult goldfish brain and that the locations of the somata and main dendritic fields of these neurons provide evidence for the overall shapes of the rhombomeric territories of the basal hindbrain.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

Retrograde labeling of reticulospinal and/or cranial nerve motoneurons was successful in 20 adult goldfish, Carassius auratus, with a length of 10–12 cm (standard length). All experiments complied with the Principles of animal care, publication No. 86-23, revised 1985, from the National Institute of Health, and protocols were approved by the Williams College IACUC. Goldfish were initially anesthetized in 0.03% ethyl 3-aminobenzoate methanesulfonate salt (MS-222; Sigma-Aldrich, St. Louis, MO) until respiration ceased. The fish were then transferred to a holding chamber in which 0.012% of the anesthetic was passed through their mouth and over their gills.

Application of tracer to the spinal cord and motor or mixed cranial nerves

Application of tracer (dextran biotin; 10,000 MW; Molecular Probes, Eugene, OR) to the rostral spinal cord and to cranial (i.e., IV, V, VII, IX, X) and the first spinal nerve roots was made by a dorsal approach. Accordingly, the skull above the hindbrain was opened and the area caudal to the vagal lobe at the level of the first spinal root was identified. After hemi- or whole spinal transection, dextran biotin (10,000 MW) recrystallized on the tip of a 45-gauge stainless steel wire was placed into the wound and allowed to dissolve. Cranial nerve motoneurons were labeled by placing a small piece of Parafilm under the nerve, cutting the nerve with iridectomy scissors, and placing dextran biotin that had been recrystallized on the tip of a 45-gauge stainless steel wire against the cut end of the nerve. After the label had dissolved on the cut surface of the nerve, surplus tracer was carefully removed from the Parafilm. After application of tracer to a transected spinal cord and/or cranial nerves, the brain was covered with a Vaseline–paraffin oil mixture, and the skull opening was sealed as described previously (Zottoli and Freemer, 2003). Prior to removing the anesthetic, a gel containing 20% benzocaine (Ultradent) was applied to the skin around the rim of the skull opening as an analgesic precaution.

Postoperative preparation of tissue

After a survival time of 2–7 days, fish were anesthetized in 0.03% MS-222 until respiration ceased, and ∼40–60 ml of fixative (4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) was perfused transcardially. Brains were removed and fixed for another 1 hour in fresh fixative. After fixation and rinsing in 0.1 M phosphate buffer (PB), brains were stored overnight in 15% sucrose in 0.1 M PB and then placed in 30% sucrose in 0.1 M PB for another 12–24 hours. Brains were placed on a Peltier platform (Bailey Instruments) covered with Tissue-Tek (Sakura Fine Technical), frozen, and cut at a thickness of 60 μm in horizontal, sagittal, or transverse planes. The endogenous peroxidase activity of the tissue was inhibited before visualizing the reaction product by immersing the sections for 10 minutes in 0.1 M PB containing 10% methanol and 3% hydrogen peroxide. After thorough washing, sections were incubated for 3 hours in 0.1 M PB containing 0.5% Triton X-100 and the avidin-biotin-peroxidase complex (ABC Kit; Vector, Burlingame, CA). After rinsing the sections in 0.1 M PB and transfer into 0.05 M Tris buffer (pH 8.4), they were incubated between 8 and 10 minutes in 0.05 M Tris buffer containing 0.04% diaminobenzidine (DAB; Sigma-Aldrich), 0.4% nickel–ammonium sulfate, and 0.015% hydrogen peroxide until the reaction product was visualized as a black precipitate. After the DAB reaction, all sections were washed in cold Tris buffer, mounted on gelatin-coated slides, dried overnight, counterstained with cresyl violet, and coverslipped.

ChAT immunohistochemistry and antibody specification

ChAT immunoreactivity (ChAT-IR) was revealed in five adult goldfish that had been transcardially perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PB (pH 7.4). Brains were removed, carefully cleaned of surrounding tissue, and transferred into 20% sucrose in 0.1 M PB at 4°C for 1–3 days. Brains were cut at a thickness of 60 μm on a freezing microtome in the frontal, horizontal, or sagittal plane. The immunohistochemical detection of ChAT was performed by using either a rat monoclonal antibody, AB8 (courtesy of Dr. Bruce Wainer), or a goat ChAT antibody (AB144P; Chemicon, Temecula, CA) and the peroxidase–antiperoxidase method of Sternberger (1979). The specificity of AB8 for ChAT (Table 1) has been demonstrated via immunoaffinity chromatography and immunoblotting experiments (Levey et al., 1983). The use of AB8 antiserum against the enzyme ChAT reveals cholinergic neurons in all cranial motor nuclei of not only the goldfish (this study) but also other teleost fishes such as midshipmen, Poricthys notatus (Brantley and Bass, 1988). The specificity of the AB144P (Chemicon) antiserum for ChAT (Table 1) has been tested previously with Western blot analysis (Anadón et al., 2000), and this antiserum has been shown to label motoneurons in all cranial motor nuclei of goldfish (this study) and of other teleost fishes such as trout, Oncorhynchus mykiss (Pérez et al., 2000). The anti-ChAT sera were diluted (AB8, 1:500; AB144P, 1:100), and a double-bridge procedure was used to increase the sensitivity of the immunohistochemical method. Control material for the immunohistochemical procedure was obtained by either eliminating the antibody incubation or substituting a nonspecific rat IgG for the antibody (Rhodes et al., 1986). Retrograde labeling of reticulospinal neurons in the rostral spinal cord with dextran biotin was combined with ChAT immunoreactivity in four adult goldfish. The brain sections were first reacted to visualize the dextran biotin as described above. Then, the sections were processed for ChAT immunoreactivity (Rhodes et al., 1986).

Table 1. Primary Antibodies Used for Labeling Motoneurons
AntigenImmunogenManufacturer, species in which antibody was raised, mono- vs. polyclonal, catalog or lot numberDilution used
Choline acetyltransferaseHuman placental enzymeChemicon, goat polyclonal, AB144P1:100
Choline acetyltransferaseBovine caudate and rat enzymeGift of Bruce Wainer, monoclonal, AB81:500

Data analysis and image processing

For visualization, reconstruction, and spatial positioning of retrogradely labeled neuronal populations within the hindbrain, sections were analyzed and photographed with transmitted illumination and an Axiocam camera mounted on a Zeiss Axiophot microscope. Photomontage mosaics of each section were photographed and “stitched” in Zeiss Axiovision software. Stacks of photomontaged sections were registered, with rigid-body algorithms, and projections were constructed in ImageJ (http://rsb.info.nih.gov/ij/). Sharpening and level adjustments of final images were performed in Adobe Photoshop.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

Reticulospinal neurons help to define rhombomeric territories in the adult goldfish hindbrain

A segmental arrangement of hindbrain reticulospinal neurons has been demonstrated for adult zebrafish (Lee and Eaton, 1991) and goldfish (Lee et al., 1993; Nakayama and Oda, 2004). The various individually identifiable subgroups are arranged in clusters that closely match the embryonic and larval organization of these neurons (Kimmel et al., 1982; Metcalfe et al., 1986; Trevarrow et al., 1990; Kamali et al., 2009). However, previous studies offered only a two-dimensional (i.e., rostrocaudal, mediolateral placed on a horizontal plane) demonstration of the spatial arrangement of reticulospinal neurons within the hindbrain. Here, we provide an initial, qualitative description of the 3D shape of hindbrain territories occupied by reticulospinal neurons by comparing two-dimensional projections of their somata and dendritic fields in horizontal and parasagittal sections and by processing the serial sections (Suppl info. Movie A) into 3D stereo rotations shown in Supporting Information Movies B and C.

Bilateral application of dextran biotin to the spinal cord at the level of the first ventral root in adult goldfish retrogradely labeled reticulospinal neurons throughout the rostrocaudal extent of the hindbrain (Figs. 1, 2). Slicing the brain in the horizontal plane and projecting multiple consecutive sections into a single image allowed for simultaneous visualization of the different subgroups within an extended volume of brain (Fig. 1A). This procedure confirmed the impression of an organization into separate, rostrocaudally iterated neuronal groups. Based on similar cell morphology and somatodendritic arrangement within the brain between juvenile (Suwa et al., 1996) and adult (Lee et al., 1993; Nakayama and Oda, 2004) goldfish, the different reticulospinal subgroups can be matched to the rhombomeric designations of the developing brain (r1–r8) as a reference frame (Figs. 1A,C, 2). In particular, segments r1–r7 give rise to distinct groupings, and what appears to be a continuous column of neurons, r8, can be seen caudal to r7, even though a precise delineation is difficult to establish (see below). The correlation between the different reticulospinal groups and the original rhombomeric structure is based on the assumption that the neuronal clusters are centered within the actual segments, a supposition supported by their developmental relations to intrasegmental hindbrain central arteries (Ulrich et al., 2011) and intersegmental commissural axons tracts (Marcus and Easter, 1995), but this is not directly demonstrated in the present study. Because the lateral dendritic trees of neurons in these clusters project caudolaterally, it is likely that the segments projected into a horizontal plane are not rectangular but, rather, give the appearance of parallelograms whose lateral ends are warped caudally to form a series of curved, nested arcs (as seen in Figs. 1A,B, 2H–J, and summarized in 8G). A similar chevron-shaped arrangement of reticulospinal dendrites in the horizontal plane has been described for the larval frog hindbrain (Straka et al., 2001).

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Figure 1. Overall pattern of the reticulospinal segmental framework. Three-dimensional dendritic patterns of reticulospinal (RS) neurons within a hindbrain segment form a parallelepiped. Retrograde labeling from the rostral spinal cord revealed neuronal cell clusters in the hindbrain that partition into eight segments (r1–r8). A: Minimum-intensity projection of three registered 60-μm-thick horizontal sections showing the laterocaudal projection of RS dendrites (dRS) in r3–r5. Thus, in this plane, segments as defined by the position of RS neuron somata and their dendritic projections form a parallelogram, as highlighted for r4. The pair of Mauthner cells (M) is a landmark for identification of r4. B: Detail of RS neurons in the right half of a horizontal section. From left to right: the midline, the medial longitudinal fasciculus (MLF), somata of RS in r3–r5, their dendrites sweeping in a laterocaudal direction and the lateral edge of the brain. C: Projection of three 60-μm-thick parasagittal sections showing the ventrorostral orientation of a different subset of RS neuronal dendrites in r1–r5. D: Detail of RS neurons in a parasagittal section. From left to right (i.e., dorsal to ventral): the MLF, somata of RS in r3–r5, their dendrites sweeping in a ventrorostral direction, and the ventral edge of the brain. Combining the horizontal and sagittal views shows that adult RS segments form three-dimensional parallelograms or parallelepipeds. E: Transverse cresyl violet section (15 μm) illustrates the consequence of the oblique arrangement of the rhombomeres for identifying individual segments on transverse sections: axons of the facial tract (axVII) in r4 are visible dorsally, and the rostral abducens nucleus (nVI), located in r5, is visible ventrolaterally to RS neurons (RS) on the same section. Scale bars = 0.5 mm

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Figure 2. Characterization of the rhombomeric scaffold by mapping reticulospinal neurons. A–L: Dorsal (A) to ventral (L) series of 12 horizontal sections at 60 μm thickness depicting the location of spinal cord-projecting neurons retrogradely labeled with dextran biotin in the ventral half of the r1–r7 hindbrain region. The tracer was applied to the transected spinal cord about 2 cm caudal to the caudal edge of the vagal lobes. Hindbrain segments (1–7) were defined by the characteristics of the segmentally iterated groups of retrogradely labeled reticulospinal neurons. The locations of several characteristic biotin-labeled or Nissl-stained neurons are indicated. A, anterior; L, lateral; M, Mauthner cell; Ma, Mauthner cell axon; Md, Mauthner cell lateral dendrites; MLF, medial longitudinal fascicle; nV, rostral (segment 2 in C) and caudal (segment 3 in F) subgroup of the trigeminal nucleus; nVI, caudal subgroup (segment 6 in J) of the abducens nucleus; nVII, facial nucleus; nIX, glossopharyngeal nucleus; REN, rostral mechanoreceptor efferent nucleus; TAN, tangential vestibular nucleus; VIIg, genu of the VIIth cranial nerve; VIII, root of the VIIIth cranial nerve; VS, vestibulospinal neurons. Scale bar = 0.5 mm.

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Some dendrites of reticulospinal neurons as viewed in the parasagittal plane (Fig. 1C,D) project ventrorostrally, which is particularly obvious for reticulospinal neurons located in r1–r5. Thus, based on the combination of horizontal and sagittal reconstructions of reticulospinal neurons, the putative segmental scaffold in the adult goldfish hindbrain appears to adopt in 3D the form of a rostrally slanted parallelepiped. The distortion of segments away from the more regular shape of embryonic rhombomeres, which have faces roughly orthogonal to the main anatomical axes, has a major consequence for the correlation between specific neuronal locations on transverse sections of the brainstem and their segmental location. Borders between adjacent segments will form complex curved surfaces in the transverse plane, and, because of the mediolateral and dorsoventral slant, single transverse sections will often include neurons from more than one segment. As an example, Figure 1E shows a Nissl-stained transverse section at the level of segments r4 and r5. The dorsal part of the section contains neuronal components located in r4, such as the transversely running axons of the VIIth nerve, whereas the ventral portion of this section illustrates abducens motoneurons in the adjacent r5 segment.

Spatial refinement of basal hindbrain segmental organization

The known embryonic segmental origin of cranial motor nuclei in zebrafish (Chandrasekhar et al., 1997; Higashijima et al., 2000) and other vertebrates (see Straka et al., 2006) along with the subsequent migration of several specific cell groups allowed verification and fine tuning of the 3D segmental territories (Fig. 3). In addition, caudal migration of some cranial motoneuron somata such as those in the VIIth and IXth nerve nuclei offered the possibility to trace the axonal pathways from the final segmental position of the cell bodies back to the segmental level of the nerve root. Mapping the spatial position of cranial nerve efferent neurons (i.e., motoneurons and octavolateralis efferents) with respect to the segmental reticulospinal neurons, as shown in Figure 3, allowed extending the 3D rhombomeric boundaries to more dorsal parts of the hindbrain. We now use the parasagittal views (Fig. 3) to describe the relationships between reticulospinal neurons and motoneurons in more detail, utilizing views from the horizontal and transverse planes (Figs. 3-8).

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Figure 3. Relations between ChAT-immunopositive cranial motor nuclei and retrogradely labeled brainstem neurons are shown in parasagittal planes. Sections are contralateral to the application of dextran biotin to a hemisection of the rostral spinal cord. A–C: Each panel is a minimum-intensity projection of three registred parasagittal sections (60 μm each) and thus shows neurons in a 180-μm-thick volume. Sections in A are more medial and those in B and C, which are progressively more lateral. Segments defined by retrogradely labeled RS neurons are labeled 1–8. A: The medial section contains cranial motor nuclei III, IV, rostral V, and the rostral otic efferent nucleus (REN) as well as the nerve root axons of IIIr, IVr, and VIIr. B: The more lateral section contains cranial motor nuclei VI (rostral and caudal divisions), VII, and IX. C: The most lateral section contains cranial motor nuclei V (both rostral and caudal divisions) and X and root axons of nerves V and VII.

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Midbrain–hindbrain boundary, r0 and r1

ChAT immunolabeling showed that the oculomotor and trochlear motoneuron somata form two nonoverlapping cell populations in the mid-/hindbrain (Fig. 4A). Even though the dendrites of the two ChAT-immunopositive motoneuronal cell groups intermingle (see Bacskai et al., 2008), the somata of the two motor nuclei are separated rostrocaudally by a small gap devoid of labeled neurons (Fig. 4A, dashed line). This gap helps define the boundary between the midbrain and the first hindbrain segment, r0 (Straka et al., 2006). The midbrain oculomotor nucleus forms a relatively long cell column (Fig. 4A), essentially arranged around the medial longitudinal fasciculus (MLF; Fig. 4B). Most oculomotor motoneurons have axons that project ventrally and then laterally to exit the brain ipsilaterally (IIIr in Fig. 4B), whereas those innervating the superior rectus muscles project contralaterally (Graf and McGurk, 1985). Trochlear motoneurons make up a more circular, compact cell group just caudal to the midbrain–hindbrain boundary (Fig. 4C). Their axons follow a dorsal axonal trajectory, cross the midline above the ventricle (IVr in Fig. 4C), and project laterally where they exit the brainstem (not shown). The ChAT-immunopositive cell-free zone between the most caudal trochlear motoneurons and the rostral subgroup of trigeminal motoneurons (Fig. 3A,B; see below) delineates the r1 segment, thus confining the trochlear nucleus to r0 as in bichirs (Lopez et al., 2013), frogs (Straka et al., 2006), and other groups (Nieuwenhuys, 2011). A segmental cluster of reticulospinal neurons (Figs. 1C, 2F–L) and a dorsal group of ChAT-immunopositive nonmotoneurons (Fig. 3B) are also located in r1.

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Figure 4. Midbrain–hindbrain boundary. A: The boundary between the midbrain (MB) and hindbrain (HB) lies between the ChAT-immunostained oculomotor and trochlear motor nuclei as designated by the dashed line in this horizontal section. Some reticulospinal neurons of the nucleus of the medial longitudinal fasciculus (nMLF), backfilled from the rostral spinal cord with dextran biotin, can be seen bilaterally at the rostral end of the oculomotor nucleus (nIII). B,C: Transverse sections taken from either side of the midbrain–hindbrain boundary. B: Oculomotor nucleus (nIII) in the midbrain with dendrites, somata and axons. The axons project ventrolaterally and then laterally to exit the brain (IIIr). C: Trochlear nucleus (nIV) in hindbrain (r0) with dendrites, somata, and axons. The axons project dorsally, cross the midline dorsal to the ventricle, and form a bundle (IVr) that exits the brain laterally (not shown).

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Rhombomeres 2–4

The locations of ChAT-immunopositive and retrogradely labeled trigeminal motoneurons confirm their retention of embryonic segmental organization and, compared with the adjacent reticulospinal neurons, help to outline hindbrain segments r2–r3 (Fig. 5). Portions of retrogradely filled reticulospinal clusters are recognizable in the horizontal section in Figure 5A and provide the framework from which to view the ChAT-immunopositive motoneurons in the transverse sections in Figure 5B,D,F. Trigeminal motoneurons form two distinct subgroups, with a rostral cluster in r2 and a caudal cluster in r3. The two neuronal clusters are also shown after backfilling of the Vth nerve in Figure 5C,E. The rostral trigeminal subgroup is further divided into a subgroup that consists of a few large neurons located more medially and a set of smaller motoneurons located more laterally (Fig. 5C; Luiten, 1976). In general, neurons in the rostral trigeminal nucleus are located dorsomedially in comparison with a more ventrolateral location of the caudal subgroup in r3 (compare Fig. 5B and D). The sharp rostral and caudal delimitation of the ventrolaterally projecting dendritic trees of the trigeminal motoneurons supports the restriction of the two cell groups to r2 and r3 (Fig. 5; Straka et al., 2006). The axons of all trigeminal motoneurons project medially and rostrally to arc around the ascending secondary gustatory tract (sgt) to exit the brain laterally in r2 (Fig. 5B), independent of the segmental position of the parent somata in r2 or r3.

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Figure 5. Motor and reticulospinal nuclei in r2–r4. A: ChAT-immunopositive cranial motor nuclei and retrogradely labeled reticular neurons in a 60-μm-thick horizontal section. Portions of reticulospinal clusters in r1–r5 can be seen. ChAT-immunopositive structures include the rostral trigeminal nucleus (nVr) and motor genu of cranial nerve VII (VIIg). Mauthner cells (M) backfilled from the rostral spinal cord were reconstructed from five horizontal sections (60 μm each) and placed into this horizontal section; note that normally these neurons would lie ventral to the facial motor genu fibers (VIIg). The rostrocaudal levels of transverse sections in B, D, and F are shown. The midline is on the left in B, D, and F. B–E: Rostral (nVr) and caudal (nVc) trigeminal nuclei seen in transverse and horizontal planes. B: Transverse section through nVr. Note that the axons of this division project dorsally and wrap around the secondary gustatory tract (sgt) before exiting the brain laterally. C: Horizontal section of nVr backfilled from the Vth nerve. Vr has neurons with large somata near the midline and smaller ones more laterally. D: Transverse section at the level of the nVc. E: Horizontal section of nVc backfilled from the Vth nerve. F,G: Axons of facial motoneurons seen in transverse (F) and horizontal sections (G) preserve the topography of embryonic motoneuron migration from r4 (genu, VIIg) caudally to r6–r7. A, anterior; D, dorsal; L, lateral.

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Unique markers for segment r4 include the paired Mauther neurons (M-cells) and the VIIth nerve motor genu and its root (Fig. 5A,F,G). The combination of M-cell size and shape, location in the caudal part of r4, and distinct crossed axonal trajectory make this anamniote neuron an excellent and reliable internal landmark for identifying this hindbrain segment. The large lateral (Fig. 5A) and ventral (Fig. 3A) dendrites provide a good example of how the orientation of dendritic trees helps to define the 3D orientation of a segment. That is, an M-cell lateral dendrite projects in a caudolateral direction whereas the ventral dendrite travels in a ventrorostral direction. ChAT-immunopositive cell bodies are absent in r4, compatible with the lack of cranial motoneurons or octavolateralis efferent neurons postembryonically in fish resulting from the translocation of the cell bodies into more caudal rhombomeres during early ontogeny (Higashijima et al., 2000). However, a prominent “central facial genu” formed by VIIth nerve motor axons as they turn toward the nerve root and the octavolateralis axons that project out the VIIIth cranial nerve are ChAT immunopositive (Fig. 5A,F,G). The genu of the large, dorsally located sensory tract of the VIIth nerve offers another obvious internal feature with which to identify this hindbrain segment (Fig. 2A-C).

Rhombomeres 5–7

Abducens motoneurons form compact rostral and caudal nuclei with a distinct gap between the two cell clusters that is characterized by the absence of ChAT-immunopositive motoneurons (Fig. 3B in a parasagittal view; Fig. 6A in a horizontal view; Cabrera et al., 1992). The rostral and caudal abducens nuclei align with the somata and ventral dendrites of reticulospinal neurons in segments r5 and r6, respectively (Figs. 3B, 6A). The rostrocaudal tilt of the rhombomeres (see Fig. 3) is evident in transverse sections at this hindbrain level (Fig. 6D). The dorsal portion of a transverse section through this area depicts the mediolaterally traversing central component of the VIIth nerve root in r4 (Fig. 6D, dorsal), whereas the ventral part of this section contains the ChAT-immunopositive neurons in the rostral portion of the abducens nucleus and their dorsolaterally extending dendritic tree in r5 (Fig. 6D, ventral).

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Figure 6. Motor and reticulospinal neurons in hindbrain segments r5 and r6. A,B: Horizontal sections with retrograde staining of segmental reticulospinal neurons (dextran biotin) and cranial motoneurons labeled for ChAT in r5 and r6. The midlines are marked with dashed lines, and the rostrocaudal levels of transverse sections in D–F are shown. A: Ventral, horizontal section with rostral (VIr) and caudal (VIc) abducens motor nuclei visible bilaterally. B: More dorsal horizontal section with the rostral efferent nucleus (REN) and the facial nucleus (nVII) visible. C: Horizontal section of otic efferents in REN backfilled from the anterior branch of the VIIIth nerve. D: Transverse section at the level of VIr. This section has ChAT-immunopositive VII axons visible in r4 dorsally as they form a genu (VIIg) and VIr motorneurons visible in r5 ventrally. E: Transverse section with VIc in r6 ventrally and the sensory (VIIs) and motor (VIIm) divisions of the facial nerve in r5 dorsally. The VIIIth nerve root (VIIIr) is visible laterally. F: Transverse section cut at the level of r6–r7. ChAT-immunopositive REN and nVII neurons can be seen as well as axons of the VII motor tract (axVII) and the IX motor root fibers (IXr).

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The entire population of octavolateralis efferents and facial as well as glossopharyngeal motoneurons translocates caudally from their origins in the segments in which their nerve roots are located (Chandrasekhar et al., 1997; Sapède et al., 2005). Octavolateralis efferents form two nuclear subgroups close to the midline, one in r6 (rostral efferent nucleus [REN]; Figs. 3A, 6B,C) and a second in r7 (caudal efferent nucleus [CEN]; not clearly visible in the plane of Fig. 6B; see Zottoli and van Horne, 1983). The otic efferent somata translocate caudally from r4 to their final destination in either r6 or r7. Retrograde labeling of axons in the anterior branch of the VIIIth nerve reveals the REN and its dendrites in the horizontal plane (Fig. 6C). Some of the octavolateralis efferent axons project rostrally in close association with VIIth motoneuron axons, join labyrinthine afferent fibers exclusively on the ipsilateral side, and exit as part of the VIIIth cranial nerve in r4 (Zottoli and van Horne, 1983). The ChAT-immunopositive otic efferents have one set of dendrites that extends ventrolaterally and another set that projects across the midline to the opposite side of the brain (Fig. 6F), similar to the anatomy of posterior lateral line efferent neurons (Zottoli and Danielson, 1989). Facial motoneurons translocate caudally from r4 where the VIIth nerve root exits. The anterior border of the VIIth nerve motor nucleus is at the same rostrocaudal level as the REN neurons in r6. The facial somata extend caudolaterally throughout r7 and possibly into the rostral portion of r8, forming a more or less continuous column with large somata located more medially and smaller somata laterally (Figs. 3B, 7A,D; Luiten, 1976).

Glossopharyngeal motoneurons undergo a caudal translocation into r8. The IXth motor nucleus is a compact cluster of neurons, located more dorsally than the facial motor nucleus, with dendrites that sweep laterocaudally (partially visible in Fig. 7B,C,E). The axons of these motoneurons form a fascicle near the midline that extends rostrally to the level of the REN, where it turns laterally and forms a “glossopharyngeal genu” before projecting laterocaudally to exit the hindbrain in the caudal portion of r6 (Fig. 7E). The “backward” orientation of this axonal pathway and the caudally displaced exit with respect to the genu is reminiscent of the oblique orientation of segment r6 (see Figs. 1A, 2C,D), suggesting that the IXth nerve axonal trajectory aligns within the segmental orientation. The considerable caudal migration of the IXth motor nucleus to a position caudal to VIIth nerve motoneurons is illustrated by the distinct fiber bundle seen in the horizontal sections of Figure 7E.

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Figure 7. Motor and reticulospinal neurons in hindbrain segments r7 and r8. A: Horizontal section with retrograde staining of reticulospinal neurons (dextran biotin) and ChAT-immunopositive cranial motoneurons encompassing r6–r8. ChAT-immunopositive neurons are labeled in nVII, the rostral border of which is in r6. The ChAT-immunopositive nIX and nX can be seen more caudally in r8. Most of the nIX neurons are dorsal to this plane (see transverse section in C), hence the empty zone on the right side between the caudal edge of nVII and nIX–X. The rostrocaudal levels of the transverse sections in B and C are shown. B: Transverse section highlights the rostral portion of nVII and the axons of nVII cells that form a tract near the midline (axVII). The IXth nerve axons leave the midline tract and project laterally toward the IX root (IXr). C: Transverse section in rostral r8 that shows ChAT-immunopositive nIX neurons and a ventral portion of their dendrites (dIX) at the same level as nX axons projecting ventrolaterally to a rootlet of the Xth nerve (Xr). In B,C, the midline is near the left side of the panels. D: Horizontal section showing REN (arrow) and nVII neurons backfilled from the VIIth nerve. E: Horizontal section with nIX in r8 backfilled from IXth nerve. The trajectory of IX axons (axIX) extends from the nucleus rostrally to REN, where they turn laterocaudally (IXg) to exit (IXr) at the r6–r7 border.

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image

Figure 8. Vagal and occipital motor nuclei in r8. A: Horizontal section of the right side of the brain with r7–r8 ChAT-immunopositive IX and X motoneurons (nIX–X) visible. B: Rostral half of the occipital motor nucleus in the horizontal plane backfilled from the first ventral root. C–E: Rostral to caudal series of nIX and nX motoneurons. Levels of sections are shown in A. C: nIX and a dorsal portion of nX in r7–r8. D: Dorsal–ventral extent of nX as well as some root fibers of nX (Xr). E: Caudal portion of nX with rostral occipital motoneurons (Occ) visible ventromedial to nX. F: Occipital motoneurons backfilled from the first ventral root shown in caudal r8 (F, upper panel) and at the hindbrain–spinal cord junction (F, lower panel). G: The hindbrain segmental framework is schematically overlain onto an adult goldfish brain reconstructed from multiple horizontal sections. Reticulospinal neurons were backfilled with dextran biotin from the rostral spinal cord. The shapes of segments r1–r8 are outlined in blue. The positions of cranial motor nuclei relative to the segments are indicated for somatomotor nuclei (nIII, nIV, nVIr, nVIc, occ) on the left and branchiomotor nuclei (nVr, nVc, nVII, nIX, nX) on the right. The rostral efferent nucleus (REN) and caudal efferent nucleus (CEN) are shown on the right.

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Rhombomere 8

Glossopharyngeal motoneuron somata do not overlap rostrocaudally with those of the facial motor nucleus. Along with the location of this nucleus caudal to r7, this confirms a segmental position in rostral r8. Segment r8 is also the origin of vagal motoneurons. The vagal motoneurons in goldfish form a long motor column consisting of four subpopulations, with branchiomotor neurons located in a lateral, more rostral motor nucleus as well as in the vagal lobe; general visceromotor neurons in a medial, more caudal nucleus; and cardiac neurons in a long paramedian zone (Morita and Finger, 1987a,1987b). The lateral nucleus starts rostrally near the level of the facial lobe (r7) and extends back to the level of the commissural nucleus of Cajal. The medial nucleus extends from the middle of the lateral nucleus back into the rostral spinal cord. The ventral portion of the lateral, branchiomotor nucleus can be seen in the horizontal section in Figure 8A, whereas both vagal lobe and lateral components appear in the transverse sections of Figure 8C–E. The rostral vagal motoneurons are classic branchiomotor motoneurons, whereas the more caudal neurons have more general visceral functions (Finger, 2008, 2009). The first spinal ventral root in cyprinids is a compound nerve that contains axons from hindbrain occipital as well as rostral spinal ventral rootlets and innervates hypobranchial, pectoral, and axial muscles (Ma et al., 2010). Motoneurons backfilled from the first spinal ventral root extend rostrocaudally from the center of the vagal nucleus back into the first segment of the spinal cord, with the hindbrain neurons exiting the occipital rootlets thus forming the rostral pole of the spinooccipital motor column. The rostral portion of these motoneurons is shown in Figure 8B in the horizontal plane. In the transverse plane, they are shown in caudal r8 (Fig. 8F, upper panel) and at the hindbrain/spinal junction (Fig. 8F, lower panel, and G).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

The current study correlates the locations of reticular and motor neurons in the adult goldfish hindbrain to test for retention of the underlying segmental framework throughout ontogeny. The known rhombomeric origin of reticulospinal neurons in embryonic and larval cyprinids served as a reference frame for identifying rhombomeres r1–r8 in the adult goldfish. This provided a framework on which we placed cranial nerve motoneurons and octavolateralis efferent neurons, whose embryonic segmental origins are likewise well documented. The resultant 3D pattern allowed us to infer the shapes of adult rhombomeric territories in the basal portion of the adult brain and assign precise segmental locations for specific brainstem nuclei, including those that are characterized by substantial longitudinal migration.

Reticulospinal neurons in adult goldfish serve as intrinsic neuronal markers of hindbrain segments

The discovery and description of identified nerve cells and equivalent sets of neurons is one of the major contributions of comparative neuroscience to a fundamental understanding of the vertebrate brain (Bullock, 1978, 1984, 2000; Buchanan, 2001). Reticular neurons, present in all vertebrates, form one such equivalence set (Baron van Hoevell, 1911), within which clusters of reticulospinal neurons form specific subsets (Zottoli et al., 2007). In general, the vertebrate reticular formation divides into median, medial, and lateral longitudinal zones, with further rostrocaudal subdivisions in the hindbrain based on anatomical particularities (for review see Nieuwenhuys and Pouwels, 1983; Cruce and Newman, 1984; Wullimann et al., 1996). The medial reticular zone has been further divided into superior, middle, and inferior reticular nuclei that have been used to describe the location of retrogradely labeled reticulospinal neurons in adult teleost fishes (see, e.g., Prasada et al., 1987, 1993; Bosch and Roberts, 1994, 2001; Becker et al., 1997). In addition, these zones have also been used independently as a positional reference for cranial motor nuclei (see, e.g., Luiten and van der Pers, 1977; Oka et al., 1986a) and afferent projections (see, e.g., Oka et al., 1986b; Puzdrowski, 1987-1989).

The discovery of distinct segmental clusters of reticulospinal neurons in the medial rhombencephalic zone of larval zebrafish using whole-mount techniques led to a re-evaluation of the three major divisions in teleosts (i.e., superior, middle, and inferior) as rostral (Ro), middle (Mi), and caudal (Ca) subgroups, each with multiple subdivisions (Kimmel, 1982; Kimmel et al., 1982). Linking the segmental neuronal pattern to the earlier described but largely overlooked rhombomeric pattern of embryonic neuroepithelium helped to establish the current model of hindbrain segmentation. Accordingly, the rhombencephalic medial reticular zone has eight segments (Ro 1–3 [r1–r3]; Mi 1–3 [r4–r6]; Ca 1,2 [r7,8]) that have been identified in embryonic, larval, and juvenile cyprinid fish (Trevarrow et al., 1990; Suwa et al., 1996). Seven of these segments are clearly distinguishable (r1–r7), whereas a much larger but less well-defined eighth appears to be composed of at least four segments (Hanneman et al., 1988; Bass et al., 2008; Ma et al., 2009). A comparison of the present data with those from earlier studies in cyprinids (Lee and Eaton, 1991; Lee et al., 1993) and amphibians (Straka et al., 2001) indicates that somata of identifiable subsets of reticulospinal neurons reside near the centers of segments, whereas the terminations of some of their dendritic fields approximate segmental boundaries. Further analysis of the 3D organization of commissural axon tracts, radial glia, and blood vessels in adult fish brains should allow refinement of the exact configuration of segmental centers and borders known from embryonic stages (Marcus and Easter, 1995; Yoshida and Colman, 2000; Ulrich et al., 2011).

The segments in the adult goldfish hindbrain adopt the 3D form of a series of rostrocaudally and mediolaterally slanted parallelepipeds

Visualization of adult goldfish brain sections in all three planes (i.e., transverse, horizontal, and parasagittal) illustrated the orientation of dendritic trees of backfilled reticulospinal neurons within each hindbrain segment. The horizontal view highlights dendrites in each segment that project caudolaterally, and the sagittal view demonstrates another set of dendrites that projects ventrorostrally. Accordingly, the dendrites approximately fill a 3D parallelogram (i.e., a parallelepiped) that is slanted such that, 1) the most rostral portion of a segment is located ventrally and 2) the lateral portion is more caudal than the medial portion (Fig. 2; Supp. Info. Movie A). In the parasagittal plane, the caudal to rostral slant is most dramatic in the rostral segments (r1–r5), whereas the caudal segments (r6–r8) appear to be more vertically oriented. Therefore, the slanted orientation of a specific segment must be taken into account when assigning a morphological structure to that segment. This is particularly important when linking neuronal identities to specific rhombomeres on transverse sections, because neuronal subgroups known to be located in adjacent segments can occur in the same cross-section (see Figs. 1C, 6D–F). The difficulty in defining specific structures as part of a particular segment can be compounded by sections that are not cut perpendicular to an axis. Furthermore, brain flexures that secondarily change the shape of the brain during development and thus the orientation of neurons or entire nuclei within individual segments further complicate assignment of structures to a particular segment. For example, unlike that of cyprinids, the trout hindbrain has a shallow, U-shaped flexure in the rostral hindbrain where neuronal processes slant rostrally from dorsal to ventral, whereas neuronal processes in the caudal hindbrain appear to slant in the opposite direction (Salmo fario; Beccari, 1922, Fig. 50).

The location of adult motor nuclei relative to the reticulospinal framework shows that the nuclei retain the positions established by embryonic origin or by embryonic longitudinal migration

Previous studies of the adult fish brain have located cranial motor nuclei with respect to superior, middle, and inferior reticular nuclei of the medial reticular zone (e.g., Luiten, 1976). The ability to identify hindbrain segments in the adult goldfish allows cranial nerve motoneuronal and octavolateralis efferent populations (Gilland and Baker, 2005), their dendritic and axonal projections, and their nerve root exits to be placed within a segmental scaffold. Cranial motoneuron somata and their dendrites appear either to be confined to one hindbrain segment (nVr,nVc, nVIr, nVIc, nIX, nX) or to extend over multiple segmental boundaries (nVII; Fig. 8G). Cranial motor nuclei nVII and nIX, as well as octavolateral efferent neurons (REN, CEN, Fig. 8G), are displaced caudally from their segments of origin, which results in sharp lateral bends as their axon bundles turn to exit nerve roots in the original segments. The axonal genu of nIX motoneurons is at the level of the REN in r6, whereas that of the nVII neurons is at the level of the Mauthner cell in r4. Otic efferent neurons that project to the inner ear of the goldfish exit the brain at the r4/r5 border as part of the VIIIth cranial nerve, but their somata are displaced caudally, forming a rostral (REN) and a caudal (CEN) nucleus in segments r6 and r7, respectively (for larval zebrafish see Sapède et al., 2005). In addition, lateral line efferent neurons (not labeled in this study) are intermingled with inner ear efferents in the two nuclei, and their axons have been traced through two pathways that ultimately enter the posterior lateral line nerve; one pathway exits the brain in r6 while the other pathway exits the brain in r4 and projects caudally outside of the brain to join the posterior lateral line nerve (Zottoli and van Horne, 1983).

The location of cranial motor nuclei within the hindbrain scaffold agrees in general with work on larval trout and larval zebrafish. Beccari (1922, Fig. 50) provides a diagrammatic representation of the larval trout (Salmo fario) hindbrain in parasagittal sections on which he draws reticular neurons and cranial motoneurons stained with a Cajal reduced silver method. His dashed lines approximate the center of segments r2–r4, r6, and r8 (Beccari's labels A–E, respectively). If lines are added to mark the center of r5 and r7, the positions of the cranial motor nuclei are nearly identical to what is described here. Our results also agree, in general, with the position of motor nuclei and migratory patterns described for larval zebrafish (Chandrasekhar et al., 1997; Higashijima et al., 2000; Gilland and Baker, 2005), with a few minor differences. Specifically, in larval zebrafish, the somata of nVII appear to be confined to r6 and the rostral portions of r7, whereas, in the adult goldfish, somata of nVII neurons extend from the center of r6, through segment r7, and possibly into the rostral portion of r8. In addition, the somata of nIX neurons in larval zebrafish have been reported in segment r7, whereas they are located in the rostral portion of r8 in the adult goldfish.

Conservation of hindbrain organization during ontogeny offers the possibility to postulate the developmental origin and physiological function of other neuronal phenotypes

The configuration of reticulospinal neurons in the adult goldfish hindbrain clearly demonstrates the conservation of segmental organization during ontogeny. The reticulospinal scaffold provides robust landmarks to link numerous morphophysiological phenotypes described in the literature with the hindbrain segmental framework. The location of neurons based on their neurochemistry within the reticulospinal framework has recently met that goal for larval zebrafish; the immunoreactivity for tyrosine hydroxylase and serotonin has been specifically correlated with elements of the sensorimotor circuitry involved in the startle response known to originate from specific hindbrain segments (Kinkhabwala et al., 2011; Koyama et al., 2011). In addition, the development of a highly detailed “template coordinate system” for reticulospinal neurons in larval zebrafish (Kamali et al., 2009) will assist in the placement and identification of individual reticulospinal neurons on a hindbrain scaffold when comparable studies of adult neurons are undertaken. The distribution of neurons and their processes containing specific transmitters, enzymes, or related chemicals reported for adult teleost fishes can be aligned to the hindbrain scaffold. In goldfish, these functional phenotypes include neurons containing choline acetyltransferase (Danielson et al., 1988; Zottoli and Danielson, 1989; Giraldez-Perez et al., 2009), nitric oxide (Giraldez-Perez et al., 2009), and substance P (Sharma et al., 1989), among others. In each case, specific neuronal landmarks allow correlation with adult segmental regions described here. Segmental evaluation of the reticular pattern in other well-studied teleosts (e.g., midshipman [Brantley and Bass, 1988] and three spined stickleback [Ekström, 1987]) will allow estimation of the broader taxonomic generality of such correlations and should identify unique variations that are certain to exist in such a diverse group.

The most important test for the usefulness of an adult reticular scaffold will be to see whether detailed segmental homologies can be established with the reticular formation nuclei described for nonteleosts, especially elasmobranchs, basal actinopterygians, and tetrapods. Such studies would reveal the generality, or lack thereof, for segmental reticular anatomy as a primitive vertebrate feature and would perfectly complement the large number of detailed reports on brainstem cholinergic neurons, which have been mapped with segmental interpretations for lamprey (Pombal et al., 2001), bichir (López et al., 2013), gar and bowfin (Morona et al., 2013), lungfish (López et al., 2012), and other critical comparative species (see references in López et al., 2012, 2013).

Because their ontogenies are quite similar, the segmental organization of reticulospinal and vestibulospinal neurons in adult cyprinids is likely to be mirrored in the hindbrains of adult amniotes as well. Mapping the neuronal contributions of individual rhombomeres using segmental expression reporters (e.g., r1, Moreno-Bravo et al., 2013; r4, Chen et al., 2012) or of entire brain regions by charting expression of large numbers of transciption factors (Gray, 2013) shows that rhombomeric patterning is likely to prevail in the adult reticular and vestibular anatomy throughout amniotes. It is likely that clusters of reticulospinal neurons, homologous both in developmental history and functional connectivity to those in cyprinids, will be found in adult mammals. For example, about 60 large reticulospinal neurons are found in the ventrocaudal pontine reticular formation in mammals (Davis et al., 1982; Newman, 1985a,b; Lingenhöhl and Friauf, 1994; Yeomens and Frankland, 1996; Martin et al., 2011). These neurons, likely located between r4 and r6 in adult mammalian brains, compose a network that integrates sensory information to initiate startle responses. Their similarity in development and function to reticular neurons in cyprinids indicate that visualization of the adult hindbrain scaffold will provide a useful tool to link the massive amount of anatomical, immunohistochemical, and physiological information already available in the literature (Nieuwenhuys, 2011).

As a practical outcome of the present study, the delineated 3D shape of the goldfish hindbrain segments (parallelepipeds) can be used to map neuronal phenotypes segmentally onto the rhombomeric framework in an adult brain. For example, a vast body of information exists on neuronal identities responsible for vestibulomotor behavior in the adult goldfish (Straka and Baker, 2011, 2013). Accordingly, vestibulo-ocular neurons, retrogradely labeled from the oculomotor nucleus, are located within the limits of the fish anterior octaval nucleus in the rostral hindbrain (Gilland et al., 2004; Straka and Baker, 2013). In the 3D reconstruction in the current study, this nucleus coincides with r1–r3; this segmental identification was facilitated by the overlapping position of the caudal portion of this vestibular subgroup with trigeminal motoneurons in r2 and r3. The r1–r3 hindbrain location of oculomotor nucleus-projecting vestibulo-ocular neurons along with the termination of anterior semicircular canal afferent fibers in this octavolateral region makes r1–r3 a dominant relay area for vertical angular vestibulo-ocular reflexes in adult teleosts (Straka and Baker, 2013). The identical segmental location of this particular population of vestibulo-ocular neurons in adult, embryonic, and larval goldfish (Suwa et al., 1996) suggests a positional persistence of the circuitry during ontogeny. This remarkable stability with respect to hindbrain segmental organization in goldfish also includes known oculomotor-projecting vestibulo-ocular subgroups in more caudal rhombomeres (Torres et al., 1992; Suwa et al., 1999) and likely applies for vestibulomotor pathways in general (Straka and Baker, 2013). The spatiotemporal functionality of the vestibulo-ocular reflex depends not only on vestibular afferent inputs but also on reciprocal commissural inhibitory connections (Straka and Dieringer, 2004). These vestibular commissural neurons, retrogradely labeled from the contralateral vestibular nuclei, form a rostral and a caudal population within the goldfish octavolateral area (Gilland et al., 2004; Straka and Baker, 2013). A comparison of the somal location of these neurons with the established 3D outline of hindbrain segments indicates that the rostral subgroup aligns with r2 and r3 and the caudal subgroup with r5–r7, separated by a zone (r4) devoid of vestibular commissural neurons. The segmental positions of identified subgroups of vestibulo-ocular and vestibular commissural neurons are thus nearly identical to those described for adult frog (Straka et al., 2001, 2003). This suggests a conserved segmental stability of the basic circuitry underlying vestibulomotor behavior. This example of successfully placing specific neuronal subpopulations within segmental domains illustrates the potential to align a large number of other identified neuronal phenotypes in a similar manner in adult goldfish.

CONCLUSIONS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

The 3D hindbrain morphology in the present study demonstrates that the basal rhombencephalon comprises an iterated series of congruent parallelepipeds that form a space-filling mosaic of uniquely shaped neuroepithelial compartments. Mapping the locations of distinctive and highly stable reticular neuronal populations demarcates the intrinsic 3D rhombomeric territories and establishes the relationship between individual segments and cranial nerve motor nuclei in an adult teleost fish. Integrating the adult segmental territories defined by reticular and motor nuclei with those emerging from ongoing efforts to map segmental contributions from alar neurogenetic zones (see, e.g., Wullimann et al., 2011) will eventually yield a complete picture of the interplay between segmental and longitudinal patterning mechanisms in establishing adult brain morphology and neuronal connectivity. The likely broad generality of this framework will provide a convenient method for future studies that combine form, physiology, and function in adult vertebrates. Especially revealing will be studies that contrast hindbrain neuronal patterns in basal and derived taxa or between species with highly specialized body plans and locomotion styles, such as eels and pufferfish. The comparison of segmental origins and potential embryonic and postembryonic longitudinal migration across species should reveal general segment-specific developmental principles of hindbrain circuit formation and phylogenetic conservation of the neuronal phenotypes throughout vertebrate evolution (see, e.g., Bass and Baker, 1997).

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

Greg Corrodi, Alex Peruta, Ken Rhodes, and Tina Zeng contributed to portions of this research.

ROLE OF AUTHORS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: EG, HS, RB, SJZ. Acquisition of data: TWW, SJZ. Analysis and interpretation of data: EG, HS, RB, SJZ. Drafting of the manuscript: HS, SJZ. Critical revision of the manuscript for important intellectual content: EG, HS, RB, SJZ. Obtained funding: HS.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGMENTS
  8. CONFLICT OF INTEREST STATEMENT
  9. ROLE OF AUTHORS
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
cne23544-sup-0001-suppmovie1.mov9461KMovie A. The ventral half of the r1-r7 hindbrain region of the specimen in Figures 1A and 2 is displayed in 20 serial horizontal sections showing neurons retrogradely labeled with biotin dextran 10K from bilateral application to the ventral, rostral spinal cord. Sections were cut at 60 micrometer thickness, processed to yield black reaction product from the label, and counterstained with Cresyl Violet (see methods). The series runs from dorsal to ventral in the movie and rostral is towards the top of the frame. Rigid body registration of the slices was performed using the Stackreg plugin for ImageJ written by P. Thévenaz (http://bigwww.epfl.ch/thevenaz/stackreg/).
cne23544-sup-0002-suppmovie2.mov32901KMovie B. 3D Stereo rotations of maximum value projections (ImgeJ) of the sections shown in movie A with the pixel values inverted, i.e., the dark reaction product appears bright, the background dark. The blue channel of the RGB section stack was used for the projections, which span +/- 20 degrees around the Y-axis and are offset by 10 degrees between the left and right images. The fused image will appear as a dorsal view for crossed gaze and as a ventral view for parallel/divergent gaze. Note that while the reconstructions give the appearance of being tomographic they are projections based on images of discrete thick sections and are thus intended to give essentially macroscopic views of hindbrain neuronal organization.
cne23544-sup-0003-suppmovie3.mov36393KMovie C. The same projections as in Movie B, but with left and right images switched, giving a ventral view for crossed gaze and a dorsal view for parallel/divergent gaze.

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