Hindbrain neurovascular anatomy of adult goldfish (Carassius auratus)

Abstract

The goldfish hindbrain develops from a segmented (rhombomeric) neuroepithelial scaffold, similar to other vertebrates. Motor, reticular and other neuronal groups develop in specific segmental locations within this rhombomeric framework. Teleosts are unique in possessing a segmental series of unpaired, midline central arteries that extend from the basilar artery and penetrate the pial midline of each hindbrain rhombomere (r). This study demonstrates that the rhombencephalic arterial supply of the brainstem forms in relation to the neural segments they supply. Midline central arteries penetrate the pial floor plate and branch within the neuroepithelium near the ventricular surface to form vascular trees that extend back towards the pial surface. This intramural branching pattern has not been described in any other vertebrate, with blood flow in a ventriculo‐pial direction, vastly different than the pial‐ventricular blood flow observed in most other vertebrates. Each central arterial stem penetrates the pial midline and ascends through the floor plate, giving off short transverse paramedian branches that extend a short distance into the adjoining basal plate to supply ventromedial areas of the brainstem, including direct supply of reticulospinal neurons. Robust r3 and r8 central arteries are significantly larger and form a more interconnected network than any of the remaining hindbrain vascular stems. The r3 arterial stem has extensive vascular branching, including specific vessels that supply the cerebellum, trigeminal motor nucleus located in r2/3 and facial motoneurons found in r6/7. Results suggest that some blood vessels may be predetermined to supply specific neuronal populations, even traveling outside of their original neurovascular territories in order to supply migrated neurons.

Introduction

Precise vascular network regulation has long been implicated as a necessary metabolic support for differential neuronal activity in vertebrates (Rahmat & Gilland, 2014). Nevertheless, the anatomy and development of the vascular supply to specific brain regions and individual brain nuclei remain scarcely documented in humans or in mammalian and non‐mammalian model species (De Vriese, 1905; Duvernoy, 1999). Although some aspects of mature hindbrain vasculature have been described in a variety of teleost fishes (Allen, 1905; Allis, 1912; Grodzinski, 1946; von Zwehl, 1961), studies correlating brain blood supply and neuroanatomy in adult fish are almost non‐existent. Because of their large size and long use as a neurophysiological model, goldfish (Carassius auratus) are among the best‐studied species of teleosts in terms of brainstem anatomy and defined neuronal circuits (Gilland et al., 2014). Zebrafish (Danio rerio), a cyprinid species closely related to goldfish, is a widely used model for developmental biology. The early development of cerebral vasculature, especially of the hindbrain, has been described in zebrafish (Isogai et al., 2001; Fujita et al., 2011; Ulrich et al., 2011), as has the basic neuroanatomy of the embryonic/larval brainstem (Ma et al., 2009; Gilland et al., 2014). However, knowledge of adult neuroanatomy in zebrafish lags considerably behind that of goldfish and the adult brainstem vasculature has not been described for either species.

Similar to other vertebrates, the goldfish hindbrain develops from a segmented (rhombomeric) neuroepithelial scaffold (Gilland & Baker, 2005). Central nuclei such as motor, reticular and other neuronal groups develop in specific segmental locations within this rhombomeric framework (Fig. 1). Only the rostral portion of the hindbrain that corresponds to the mammalian pons is clearly segmented into distinct rhombomeres (r) in cyprinids, where r2–r6 are distinct anatomically and exhibit segmental gene expression patterns and clear segmentation of motor nuclei (Gilland & Baker, 2005). Segmental reticular neurons are found in clusters in the central part of each rhombomere from r1 to r7. Cranial motor nuclei are distributed throughout the hindbrain segments: CN IV in r0; CN V in r2/r3; CN VI in r5/r6; CN VII in r6/r7; CN IX and CN X in r8 (Fig. 1). The portion of the caudal hindbrain corresponding to the mammalian medulla oblongata develops from r7 and r8 and comprises a length equivalent to three or four pontine rhombomeres. Although this portion of the hindbrain is not overtly segmented into rhombomeres, segmental bands of expression of hox and other genes are present and some caudal hindbrain nuclei are distributed in segment‐like patterns (Bass et al., 2008; Ma et al., 2009). The segmental neuronal pattern of larval goldfish is preserved in the adult hindbrain, with reticular, motor and vestibular nuclei retaining the rhombomeric organization seen in earlier stages (Lee & Eaton, 1991; Lee et al., 1993; Gilland et al., 2014; Rahmat & Gilland, 2014). The persistence of segmental patterning throughout life in cyprinid hindbrains allows direct comparison of neurovascular anatomy between larval and adult stages.

Figure 1.

Rhombomeric neuronal organization in adult goldfish. The segmental divisions of the hindbrain (red dashed lines) as well as the rhombomeric neuronal pattern of reticular and motor nuclei are shown in a projection of 11 consecutive parasagittal sections from one side of the brain of a specimen retrogradely labeled with dextran‐conjugated biotin from the contralateral spinal cord (black precipitate) and immunostained for choline acetyl transferase (ChAT; brown reaction product). Rhombomeres are numbered with black Arabic numerals (1–8), cranial nerve roots with black roman numerals (e.g. IIIr) and cranial motor nuclei with white (e.g. III). The ChAT staining deposits brown reaction product in cholinergic neurons; here, mostly cranial motor neurons. Reticulospinal neuronal clusters are located in the centers of each hindbrain segment. Rhombomere 8, as in most other vertebrates, comprises the entire lower medulla and may represent three or four cryptic segments. The front of the hindbrain (rhombomere 1) is just caudal to the oculomotor motor nucleus (III). The facial nerve root (VIIr) can be seen entering the brain in rhombomere 4 and projecting back to the facial motor nucleus in r6/7 (VII). III, oculomotor nucleus; IIIr, oculomotor root; IV, trochlear nucleus; IVr, trochlear root; IX, glossopharyngeal motor nucleus; IXr, glossopharyngeal root; M, Mauthner neuron; MB, midbrain; OLE, octavolateral efferent nucleus; V, trigeminal motor nucleus; VI, abducens nucleus; VII, facial motor nucleus; VIIr, facial nerve root; Vr, trigeminal root; X, vagus motor nucleus. Scale bar: 1 mm.

Since the basic pattern of carotid and basilar arteries is highly conserved in vertebrates (Rahmat & Gilland, 2014), most aspects of goldfish vascular anatomy can be readily interpreted based on studies in other teleosts and more basal actinopterygian fish. Cerebral blood supply in teleosts, as in most other vertebrates, comes from paired internal carotid arteries (ICA) that extend rostrally from the dorsal aortae (Allen, 1905; Allis, 1912; Grodzinski, 1946; von Zwehl, 1961; Rahmat & Gilland, 2014). In many teleosts such as trout, perch and tilefish, the paired carotids join to form a median unpaired carotid trunk that enters the braincase just caudal to the hypophysis (Allen, 1905; Grodzinski, 1946; Rahmat & Gilland, 2014). The ICA continue intracranially as paired cerebral carotid arteries (CCA) that pass rostrally within the hypophyseal fossa and divide into anterior (ACC) and posterior cerebral carotid (PCC) arteries near the forebrain‐midbrain junction (Fig. 2; Rahmat & Gilland, 2014). The ACC continue rostrally, sending vessels to supply the forebrain and parts of the midbrain. Paired efferent pseudobranchial arteries (EPA) carry hyperoxygenated blood from the pseudobranch and either join the CCA, as in elasmobranchs and some basal actinopts, to supply the brain and eye, or remain separate and supply only the specialized choroid rete mirabile seen in most teleosts (Goodrich, 1930; Holmgren, 1943; Rahmat & Gilland, 2014).

Figure 2.

Overall pattern of brain arteries in teleost fishes. Lateral (A) and medial (B) views of a Salmo brain modified after Grodzinski (1946). (A) The cerebral carotid artery (CCA) divides into anterior (ACC) and posterior (PCC) cerebral carotid arteries. The PCC travels caudally and medially and fuses with the corresponding vessel from the other side to form a single midline basilar artery (BA). Prior to fusion, the PCCs give off mesencephalic arteries (Mes) and cerebellar arteries (Cer). (B) As the basilar travels down the hindbrain, multiple rhombencephalic penetrating branches, called central arteries (CAs), are given off. This posterior cerebral/basilar system of arteries is responsible for supplying the brainstem and cerebellar regions of the teleostean brain. II, optic nerve; III, oculomotor nerve; IX, glossopharyngeal nerve; V, trigeminal nerve; X, vagus nerve.

The PCC arc caudally and join just rostral to the trigeminal nerve (CN V) to form a single midline basilar artery (BA) that sends central penetrating arteries (CAs) to supply the hindbrain and cerebellum. The PCC in cyprinids are joined across the midline near their origins by a short commissural vessel, the basal communicating artery (von Zwehl, 1961; Isogai et al., 2001), from which the main mesencephalic central arteries arise to penetrate the midbrain floor and spread throughout the subventricular parts of the tectum, tegmentum and valvula cerebelli (von Zwehl, 1961; Rahmat & Gilland, 2014). The brainstem‐penetrating arteries in most vertebrates branch off main vessels not only in the ventral midline, but also from circumferential vessels extending transversely around the lateral and dorsal sides of the brain (Rahmat & Gilland, 2014). In contrast, the midbrain‐ and hindbrain‐penetrating arteries in teleosts are restricted almost completely to major midline stems arising directly from the PCC and BA. These ‘central arteries’ penetrate the brain near or at the ventral midline and branch extensively within the neuroepithelium to supply the brainstem (Grodzinski, 1946; Rahmat & Gilland, 2014). The hindbrain central arteries (‘rhombencephalic arteries’ of Grodzinski) arise as a segmental series of median stems branching directly from the basilar artery (Grodzinski, 1946; Isogai et al., 2001; Rahmat & Gilland, 2014).

The present study examines the hindbrain arterial anatomy of adult goldfish with respect to segmentally arrayed motor and reticular nuclei. The central brainstem arteries and their main branches were identified in serially sectioned adult goldfish brains retrogradely labeled with dextran‐conjugated biotin applied to the spinal cord and cranial nerve roots. Results provide an initial assessment of the segmental vascular supply of the goldfish brainstem, including descriptions of the robust branching of r3 and r8 arterial stems and the presence of specific branches supplying trigeminal and facial motor nuclei, reticulospinal nuclei and major portions of the cerebellum.

Materials and methods

Analysis of vascular anatomy in serially sectioned adult goldfish brains with specific neuronal populations retrogradely labeled with dextran‐biotin and cholinergic neurons revealed by ChAT‐immunoreactivity

To compare hindbrain neurovascular relationships and the general rhombencephalic anatomic landscape of adult teleosts, brainstem vascular anatomy was examined in a collection of sectioned goldfish brains prepared in a previous study of adult hindbrain neuronal segmentation (Gilland et al., 2014). The collection comprised 20 serially sectioned adult goldfish brains in which different combinations of cranial motor, reticulospinal and vestibulospinal nuclei were retrogradely labeled with dextran‐conjugated biotin. In four cases, specimens with biotin‐dextran‐labeled spinal‐projecting neurons were also processed for ChAT‐immunoreactivity to reveal all hindbrain motor nuclei (brown immunolabel) along with reticulo‐ and vestibulo‐spinal neurons (black retrograde label) in the same specimen. This combined retrograde + immune technique was developed by Dr. S. Zottoli at Williams College (Gilland et al., 2014). In all the sectioned brains, the hindbrain central arterial trunks and their more proximal branches were visible as empty tubes which could be traced in relation to labeled neurons and cell processes as well as unlabeled neurons made visible by cresyl‐violet counterstain.

The experimental methods are described in detail by Gilland et al. (2014). Retrograde labeling was successful in 20 adult goldfish (Carassius auratus) with standard lengths of 10–12 cm. The experiments complied with the Principles of Animal Care (publication No. 86–23, revised 1985) from the National Institutes of Health, and protocols were approved by the Williams College IACUC. Briefly, Goldfish anesthetized in 0.03% ethyl 3‐aminobenzoate methanesulfonate salt (MS‐222; Sigma‐Aldrich, St. Louis, MO, USA) were prepared for surgery through a dorsal approach that exposed the roots of cranial nerves V–X and the occipital and anterior spinal nerves. Crystals of dextran‐biotin conjugate (10 000 MW; Molecular Probes, Eugene, OR, USA, now Thermo Fisher) were applied to the rostral spinal cord following hemi‐ or whole spinal transection and to cranial nerve roots in different ipsi‐ and contralateral combinations to trace retrogradely reticulospinal and vestibulospinal neurons projecting to the spinal cord and cranial nerve motoneurons in the same specimens. After survival times of 2–7 days the fish were terminally anesthetized in MS‐222 followed by fixation by transcardial perfusion of 4% paraformaldehyde, 1% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4. Fixed brains were infiltrated with 30% sucrose, blocked and sectioned on a freezing microtome at 60 μm thickness in horizontal, sagittal or transverse planes. The sections were incubated with an avidin‐biotin HRP complex (ABC kit; Vector, Burlingame, CA, USA). The final diaminobenzidine (DAB; Sigma‐Aldrich) reaction step was supplemented with Nickel toning (0.4% nickel‐ammonium sulfate) to convert the standard DAB brown reaction product into a denser black precipitate for better visualization of biotin‐filled neurons (Straka et al., 2006).

Immunoreactivity for choline acetyltransferase (ChAT‐IR) was revealed in sectioned brains of five adult goldfish, four of which were first reacted to visualize the dextran biotin as described above and then processed for ChAT immunoreactivity using the peroxidase‐antiperoxidase method of Sternberger (1979; Rhodes et al., 1986). Two ChAT antibodies were tested, both giving good results: a rat monoclonal antibody, AB8 (courtesy of Dr. Bruce Wainer), and a goat ChAT antibody (AB144P; Chemicon, Temecula, CA, USA, now MilliporeSigma). Specificity for ChAT has been demonstrated for both AB8 (Levey et al., 1983) and AB144P (Anadón et al., 2000) and both have served to reveal cholinergic neurons in goldfish and other teleost fishes (Brantley & Bass, 1988; Anadón et al., 2000; 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 sensitivity. Eliminating the antibody incubation or substituting a nonspecific rat IgG for the antibody was used for controls (Rhodes et al., 1986). In all four cases of dual retrograde/immune treatment, the ChAT‐IR (brown) neurons can be distinguished from the retrograde dextran‐biotin (black) neurons and from surrounding unlabeled (violet) neurons (Figs 1, 6 and 7).

Microscopy and data analysis

Brightfield and DIC micrographs of sectioned adult goldfish hindbrain specimens were obtained using a Zeiss AxioCam HR on an upright AxioImager. Panoramic mosaics were created by shooting overlapping fields of sections too large to be captured in single images. In addition, series of images were shot throughout the depth of 50‐ to 70‐μm‐thick sections (z‐series) in order to computationally create an extended depth of focus image wherein structures through the entire depth of the section are in focus.

Stitching of the montage fields, computation of extended focus, registration of adjacent sections, three‐dimensional reconstructions of blood vessels and hindbrain nuclei and image editing were performed using Zeiss axiovision, imagej and adobe photoshop applications.

Results

Overall rhombomeric central arterial pattern in adult goldfish

Horizontal sections of adult goldfish brains (Fig. 3A,B) reveal hindbrain central arteries (visible as clear tubes) as a segmental series of 8–10 median stems branching from the basilar artery. Each rhombomere from r3 to r7 possesses one central artery (CA) stem, whereas r8, which is known to form from multiple embryonic segments (Gilland et al., 2014) and is hypertrophic in goldfish due to enlarged vagal lobes, has multiple central artery stems (often three to five in the 10 specimens examined). Rhombomeres 1 and 2 generally do not have segmental central arteries but are instead supplied by branches from the r3 CA and from branches coming off of the posterior cerebral carotids anterior to their fusion to form the basilar artery. Each CA is located in the midline of the neuroepithelium, between paired reticulospinal (RS) neuronal clusters. All specimens examined (n = 10) show large midbrain CA and small r0 CA, as well as r3 and r8 stems that are significantly larger than the other hindbrain CA.

Figure 3.

Hindbrain rhombomeric central arterial organization. The segmental patterns of midline central arterial stems (CA) from rhombomere (r) 3 to r8 are shown in single horizontal sections of the hindbrain in two different adult goldfish specimens retrogradely labeled with dextran‐conjugated biotin and counterstained with cresyl violet (B) or ChAT immunostain (A). The reticulospinal neurons (RS) are located at the center of each rhombomere (best seen in B, a bilateral spinal cord label), on either side of the midline central arterial stems. A contralateral label of dextran‐conjugated biotin followed by immunostaining with ChAT (A) shows the RS neuronal clusters, midline CA stems, and the projecting axons from the spinal cord on one side of the brain. All specimens examined showed large midbrain CAs as well as r3 and r8 stems that were significantly larger than the other hindbrain CAs. Although one large r8 stem was observed in all of these specimens, several smaller midline central arteries arise in r8 as well (A,B) in order to assist in supplying specific regions of this vast segment. Sagittal projection (C) of several dextran‐conjugated biotin‐labeled adult goldfish sections showing central arteries penetrating the midline of each rhombomere. CAs, central arterial stems; MB, midbrain; r, rhombomere; RS, reticulospinal neurons; VII mot, facial motor tract. Scale bars: 500 μm (A,B), 1 mm (C).

Sagittal projections of adult goldfish brains demonstrate the intra‐rhombomeric pathway these central arteries travel, through the midline of each rhombomere and each reticulospinal cluster (Fig. 3C). This differs vastly from the transverse, circumferential hindbrain‐penetrating arteries located at inter‐rhombomeric borders in most other vertebrates (Rahmat & Gilland, 2014).

General intramural branching pattern of central arteries

Each main CA stem penetrates the pial midline and ascends through the floorplate (septum medullae), giving off short transverse paramedian branches. Paired paramedian vessels arise directly from midline CA stems in each rhombomere and extend a short distance into the adjoining basal plate to supply ventromedial areas of the brainstem, including direct supply of reticulospinal neurons present as dark neuronal clusters in r1–r7 (Fig. 4).

Figure 4.

Paramedian branches of central arteries. Retrograde label with dextran‐conjugated biotin from the spinal cord followed by cresyl‐violet counterstain of adult goldfish brains (A–D) show the overall arterial and reticulospinal (RS) neuronal segmental pattern (A,C). The cresyl violet counterstain in (A) was much more intense than that of (C). Higher magnifications of parts of (A) and (C) (red dotted boxes) allowed the visualization of paramedian branches (PM) of the central arteries (B,D). The PM branches of the r4 central artery (CA) are shown in (B) and the PM branches of the r3 and r6 CAs are shown in (D). MB, midbrain; r, rhombomere. Scale bars: 500 μm (A,C), 100 μm (B), 200 μm (D).

As the main central arteries approach the ventricular surface at the midline of each hindbrain segment, they branch into paired left–right primary branches at the level of the medial longitudinal fasciculus (MLF). Each lateral trunk continues branching in the subventricular zone, spreading predominantly in posterior and lateral directions. There appears to be a high degree of arterial branching, with more distal vessels arching within the neuroepithelium towards the pial surface, giving the intramural arterial trees an overall shape similar to branches of a drooping willow tree (Fig. 5). Each intramural tree seems to overlap roughly with the one caudal to it during this branching path. In transverse sections of adult goldfish, subventricular vessels can be identified and tracked to supply various regions of the midbrain, hindbrain and cerebellum. The high degree of branching allows these pseudo‐end arteries to form an interconnected network through microvascular anastomoses only after the vessels have left the subventricular zone and traveled back through the gray and white brain matter.

Figure 5.

Adult goldfish intramural vascular branching of r3 and r8 central arteries. Serial sections (A) and graphic reconstruction (B) of adult goldfish hindbrain with neurons retrogradely filled with dextran‐conjugated biotin from the spinal cord showing vascular branches of the r3 CA stem (r3 CA; white holes). Silver‐stained (Bodian) medulla showing r8 CA stem in serial sections (C) and graphic reconstruction of intramural vascular tree (D). The CA branched into vascular trees within the hindbrain neuroepithelium near the ventricular surface, continued branching back towards the pial surface and drained into a laterally located venous network. V, trigeminal motor nucleus; CA, central arteries; CB, cerebellar branches; C, caudal; R, rostral. Scale bars: 1 mm (B,D).

These intramural arterial trees end as small, unbranched channels that traverse nuclei and fiber tracts before joining a highly interconnected, laterally located network of venous tributaries. The general pattern of venous drainage seems to be conserved in most vertebrates, as anterior, middle and/or posterior cerebral veins are the primary drainage vessels in most taxa (Rahmat & Gilland, 2014). Most vertebrates have an extramural arterial supply that encircles the brain and sends penetrating vessels into the neuroepithelium, with outward venous drainage paralleling incoming arterial flow (Rahmat & Gilland, 2014). This pattern is not seen in the cyprinid fishes goldfish and zebrafish, as there is a laterally located venous plexus that accepts all blood drainage from the intramural vascular arterial tree, supporting a unidirectional ventricular‐to‐pial blood flow that appears unique to teleosts.

Although the r3 and r8 CA stems become the main hindbrain blood suppliers and will be discussed more in detail, the central arteries in r4‐7 were examined as well. Horizontal and sagittal sections of retrogradely labeled adult goldfish hindbrains show similar midline paramedian branches coming directly off each of these smaller stems. Also, the r4–7 central arteries have a higher prevalence of arterial variation, where one main midline stem becomes replaced with dual central arteries (right and left) that then branch within the neuroepithelium. These small stems send branches into the adjacent reticular nuclei and abducens motor nuclei but do not extend as far into the dorsal and lateral neuroepithelium as the larger r3 and r8 intramural vascular trees, which spread over the territories supplied by the smaller vessels.

Main branches and territories of the r3 central artery

Adult goldfish possess robust r3 and r8 central arteries that are significantly larger and form a more interconnected network than any of the remaining hindbrain vascular stems. This intricate anatomic change from the other central arteries is likely due to the increased responsibilities the r3/r8 stems undertake as goldfish mature from larvae to adults. The r3 stem is the largest CA and is the primary brainstem vessel. Horizontal, sagittal and transverse sections of labeled material highlight the location of neuronal populations relative to the rhombomeric organization of the brainstem and provide details of the extensive vascular branching the r3 stem undergoes, including: (1) large‐caliber cerebellar branches; (2) ascending branches into r2 that supply the trigeminal motor nucleus; and (3) unique, descending branches that appear to parallel the migratory path of facial motoneurons in r6/7 (Fig. 6A–H).

Figure 6.

Branching of r3 central artery. A montage of eight consecutive horizontal sections of an adult goldfish brain immunostained for ChAT and with dextran‐conjugated biotin applied to the trigeminal sensory root. Sections (A–H) run from ventral to dorsal. The large midline r3 central arterial stem is seen in the most ventral section (A). As it ascends through the midline, the r3 stem divides into two main trunks (B,C,D). Each trunk sends ascending branches (r3 asc; E) to supply r2 and the cerebellum, as well as long descending branches (r3 desc; F,G,H) that travel down the hindbrain. Coming off the ascending vessels are branches that travel directly to the trigeminal motor nucleus and supply them (V; E). The descending branches give off multiple laterally directed branches (G,H) as they follow the trajectory of the facial motor axons (VII mot; G,H) from r4 to r6/7. The brown ChAT immunohistochemical reaction product allows clear visualization of the facial motor tract (C,D), while the facial sensory tract appears pale (VII sens; E–H). CA, central artery; IV vent, fourth ventricle; r, rhombomere; r3 asc, r3 ascending branches; r3 desc, r3 descending branches; V, trigeminal motor nucleus; VII mot, facial motor tract; VII sens, facial sensory tract. Scale bars: 1 mm (A–H).

Differential interference microscopy (DIC) and brightfield microscopy on sectioned series of several adult goldfish hindbrains detail r3 branches, including a vessel that directly supplies the trigeminal motor nucleus (Fig. 7A–E). This ‘trigeminal artery’ is demonstrated in multiple specimens branching directly off the r3 ascending trunk and coursing to the cluster of trigeminal motor neurons in r2/r3. The trigeminal artery appears to be the main supplier of the trigeminal motor nucleus, as no subsequent branches of supply to this region arise from any of the other central arteries in the specimens examined.

Figure 7.

Trigeminal arterial branch from the r3 ascending vessel. (A) Transverse section of an adult goldfish hindbrain retrogradely labeled with dextran‐conjugated biotin, nickel‐toned, and counterstained with cresyl‐violet showing the trigeminal motor nucleus (white V mot) and the axons, dendrites and cell bodies of reticulospinal neurons and projections from the spinal cord. The main r3 central artery (CA) cannot be seen due to plane of section, but some of its branches are shown, specifically the trigeminal artery (V br) and cerebellar branches (CB br). (B) Higher magnification of the trigeminal motor nucleus and its arterial supply branch (V br). (C) Detail of the same trigeminal nucleus and its main vessel showing overlap of reticulospinal dendrites and motor nucleus. (D) Adult goldfish hindbrain retrogradely labeled with dextran‐conjugated biotin and immunostained with ChAT demonstrating the main ascending branches (r3 asc) of the r3 central arterial trunks (r3 CA). The rhombomere 3 location can be verified by the presence of the trigeminal motor nucleus (V) located in r2/3. The main trigeminal artery is visible as well as the facial sensory tract (VII sens). (E) Differential interference contrast (DIC) images from 10 optical planes within the section were used to produce an extended depth of field projection (AxioVision) of the r3 ascending branch and the trigeminal artery (V br) supplying the trigeminal motor nucleus (V). Note the sharpness of the DIC image, as individual trigeminal motor neurons can be distinguished. CB br, cerebellar branches; IV vent, fourth ventricle; r, rhombomere; r3 asc, r3 ascending branch; V br, trigeminal artery; V mot, trigeminal motor nucleus; VII sens, facial sensory tract. Scale bars: 1 mm (A,D), 500 μm (B), 250 μm (C,E).

The r3 descending branches begin in r3 and give off laterally directed branches as they course down the hindbrain to their final position in r6/7. These r3 descending arteries are situated between the facial sensory and motor tracts along their path. Although the importance of the r3 CA stem has never been examined prior to this study, the parallel courses of descending r3 branches and migrating facial motor neurons to their final location in r6/7 (Fig. 6F–H) suggests that brainstem vasculature can extend along specific neuronal pathways in order to supply designated targets. Confocal studies on midshipman larvae confirmed the presence of descending r3 branches that continue caudally from r3 to r6/7 as well (S. Rahmat & E. Gilland, unpubl. data). Further studies are needed to determine the validity of a parallel vascular supply to migrating motor nuclei.

Robust branching of r8

The r8 CA stem mainly supplies the vagal lobes and caudal end of the hindbrain. Rhombomere 8 has multiple central arterial stems with one (main r8 stem) being larger than the others. Extreme hypertrophy of the vagal lobes in adult goldfish may be an explanation as to the increase in number of CAs in r8, as the enlarged area of this region needs more vascular supply to function properly. Goldfish are considered to be gustatory specialists, possessing more oral and trunk taste buds and much larger vagal lobes than zebrafish. Thus, one main r8 central artery becomes the major blood supplier of the caudal hindbrain, along with multiple smaller midline stems that also supply regions of the large rhombomere 8.

Central artery variations

In most specimens, there was only one midline r3 vessel. However, one specimen showed a variation in which dual r3 CA stems were present, one closely behind the other. As seen in consecutive horizontal sections (Fig. 8), both stems gave off paramedian branches before traveling to opposite sides, with the more rostral stem (r3R) coursing and supplying only the right side of the brainstem and the caudal stem (r3C) only the left. Notably, although the rostral and caudal stems supplied regions on one side of the brainstem during their distal branching, the paramedian‐directed branches given off earlier from both stems were to the contralateral side of their eventual course. This specimen was the only one that demonstrated dual r3 central arterial stems but doubled arterial stems in r4–r7 were more commonly observed in goldfish specimens.

Figure 8.

Dual r3 central artery variation. A consecutive series of horizontal sections of an adult goldfish hindbrain retrogradely labeled from the spinal cord with dextran‐conjugated biotin and counterstained with cresyl violet shows a variation of doubled r3 stems (A) and the rhombomeric organization of the black reticulospinal neurons (RS; B–E). Moving through the montage, the sections continue dorsally, with the more rostral r3 stem (r3R) sending paramedian branches to the right side (B) and the caudal r3 stem (r3C) sending paramedian branches to the left (C). The main stems diverge from each other and course to sides opposite to the paramedian branches, with all further branching limited to one side of the hindbrain (D–H). IV vent, fourth ventricle; PM, paramedian branches; r3C, caudal r3 central artery; r3R, rostral r3 central artery; RS, reticulospinal neurons; VII mot, facial motor tract. Scale bar: 1 mm (A–H).

The long descending r3 branches that parallel the migratory path of facial motor nuclei appear in some specimens to supply the facial motor nucleus in r6/7 directly. However, other specimens show lateral vessels branching directly from r5–7 central arteries that travel to the facial motor nucleus, suggesting that vascular supply to this region may come from multiple potential sources.

Discussion

Teleosts are unique in possessing a segmental series of unpaired, midline central arteries that extend from the basilar artery and penetrate the pial midline of each hindbrain rhombomere (Rahmat & Gilland, 2014). While previous studies (Lee & Eaton, 1991; Lee et al., 1993; Gilland et al., 2014) have shown that many hindbrain nuclei form in a segmental nature, this study demonstrates that the rhombencephalic arterial supply of the brainstem forms in relation to the neural segments they supply. The midline central arteries that penetrate the pial floorplate are the primary entry site of vessels into the goldfish hindbrain, and all subsequent branching of the central arteries occurs intramurally, completely within the neuroepithelium. The overall hindbrain blood flow thus appears to be in a ventriculo‐pial direction. This differs fundamentally from the extramural branching observed in other vertebrates, including humans (Yasargil, 1984) where the main branches of the basilar artery navigate circumferentially around the brain, sending penetrating vessels into the neuroepithelium at numerous locations along the pial surface. Developmental studies in rodent hindbrains have shown that penetrating arteries enter the pial surface, elongate towards the ventricle without branching and then form anastomosing branches immediately upon reaching the subventricular area (Gerhardt et al., 2004; Scremin, 2004). In goldfish, contrary to the rodent pattern, the penetrating arteries do not anastomose until they leave the subventricular area and go back through the gray and white brain matter towards the venous network located just beneath the pial surface (Fig. 5). The central arteries connecting the basilar artery to venous channels in goldfish thus play similar roles to the circumferential and penetrating vessels arising from the vertebral and basilar arteries in mammals, but with a very different vascular geometry (Rahmat & Gilland, 2014). The exact location of the transition from hindbrain basilar to spinal intersegmental neurovascular territories has not been determined in cyprinid fish (zebrafish, goldfish), nor have the homologies between occipital and vertebral arteries in fish been established with similar named arteries in mammals (Padget, 1948; Isogai et al., 2001).

A noteworthy anatomical feature in teleosts is that the central arteries enter the brain intra‐rhombomerically, through the midline of each rhombomere, instead of forming at the inter‐rhombomeric borders as in humans and other vertebrates (Rahmat & Gilland, 2014). Sagittal and horizontal sections of adult goldfish confirm the presence of these intra‐rhombomeric vessels and graphical reconstructions demonstrate that these vessels travel from the pial surface to the ventricle within the midline floorplate of each rhombomere in a segmental manner (Fig. 3). The small lateral branches of the median CA stems that supply the adjacent hindbrain reticulospinal neuronal clusters further demonstrate the rhombomeric nature of the basilar branches (Fig. 4).

All adult goldfish specimens showed identical configurations of central stems supplying the midbrain (Mes and r0 CAs) and rostral hindbrain (r3 CA), where relations are not one‐to‐one between CAs and rhombomeres. Descending branches from the r0 CA, which lies immediately behind the trochlear nucleus, appear to supply most of r1. Ascending branches of the r3 CA supply r2, including a ‘trigeminal artery’ that travels unimpeded to the trigeminal motor nucleus in r2/3. The large r3 CA originated from the most rostral part of the midline basilar artery, immediately caudal to the convergence of the posterior cerebral carotids (PCC). The constancy of a large r3 CA arising at the rostral end of the basilar suggests both a morphogenetic and anatomical importance for this region. Comparison with studies in embryonic and larval zebrafish (Isogai et al., 2001; Ulrich et al., 2011) suggest that only in later stages does the r3 vessel become the main stem supplying the r2–r3 hindbrain region as well as parts of the cerebellum and parts of r4–r7 along the path of migrating CN VII motoneurons.

A similar developmental pattern likely explains the vast territory of the adult goldfish r8 central artery (Fig. 5). Adult goldfish have relatively larger vagal lobes than zebrafish (Morita & Finger, 1985; Wulliman et al., 1996) and possess significantly larger r8 stems (S. Rahmat & E. Gilland, unpubl. obs.). Since goldfish are ‘taste specialists’, it seems that the r8 CA grows larger than its neighbors because it is supplying a hypertrophic sensory area. In the simplest cases, it seems plausible that if a cerebral neuronal region that is supplied by a particular artery undergoes hypertrophy, then branches of that vessel will also undergo hypertrophy in order to provide an adequate supply to the given region. Given the diversity of brain anatomy seen in a large taxonomic group such as the cyprinids, it is possible that in species with other cerebral regions that have undergone hypertrophy, other central arterial stems in the rhombomeric series may become elaborated due to the enlarged target area. The drastically larger sizes of the r3 and r8 stems have been observed in previous studies which described the hindbrain as being supplied mainly by ‘anterior and posterior medullary arteries’ (Allen, 1905). While the nomenclature for these vessels makes sense due to their position within the hindbrain, it is likely that smaller, less noticeable segmental central arteries are present in these other species as well, making these names outmoded.

The r3 stem is also unique due to the presence of long, descending branches that parallel the migratory path of the facial motor neurons from r4 to r6/7 (Fig. 6). The descending branches are not present in the early larval stages of zebrafish, but are clearly visible in adult goldfish, along with a significant subventricular arterial tree that lies over the facial motor tract. Although migrating facial motoneurons are present in many vertebrates (Gilland & Baker, 2005), there has been no reported paralleling path of vasculature that follows the neurons (McArthur & Fetcho, 2017; Han et al., 2018). Abundant blood flow to neurons is necessary for proper functions but, presumably, supply from any nearby source would meet that need. The fact that r3 vessels travel outside of their original neurovascular territories in order to supply migrated neurons is evidence that some blood vessels may be predetermined to supply specific neuronal populations regardless of changing anatomical position.

The segmental rhombencephalic central arteries described here in adult goldfish (Fig. 9), and shown in adult trout (Grodzinski, 1946) and midshipman (Rahmat & Gilland, 2014), likely represent a unique vascular innovation seen only in teleosts. Their development, as seen in zebrafish, involving medial migration of angiogenic cells from ‘primitive’ lateral venous channels mirrors the early embryonic stages in mammals (Padget, 1948; Fujita et al., 2011; Ulrich et al., 2011). However, the adult teleost cerebral vasculature differs so greatly from the vascular patterns seen in other vertebrates that it may comprise a ‘key adaptation’ in the diversification of this group. Given that the cerebral blood supply in fishes operates at low pressure having passed through the gill capillaries, teleosts may also possess unique mechanisms of neurovascular regulation different from those known in air‐breathing vertebrates. If they can be related back to their segmental origins, the brainstem vessels of teleosts should provide a large series of ‘natural experiments’ in neurovascular design, especially when comparing species with different oxygen demands and life histories.

Figure 9.

Overall neurovascular pattern in adult goldfish. Reconstruction of adult goldfish hindbrain retrogradely labeled from spinal cord with dextran‐conjugated biotin. Schematic overlay of blue rhombomeric (1–8) boundaries, green motor nuclei and red central arterial stems and branches. CA, central arteries of the hindbrain; III‐X, cranial motor nuclei; MB, midbrain; Mes, mesencephalic arteries; OLE, octavolateral efferent nucleus; OS, occipito‐spinal motor column; r, rhombomere; SC, spinal cord; VL, vagal lobe. Scale bar: 1 mm.