visceral afferent and efferent columns in the spinal cord of the teleost,ictalurus punctatus

THE JOURNAL OF COMPARATIVE NEUROLOGY 371:437447 (1996)

Visceral Afferent and Efferent Columns in the Spinal Cord of the Teleost,

Ictalurus punctatus

LISA E. GOEHLER AND THOMAS E. FINGER Department of Cellular and Structural Biology, University of Colorado Health Sciences

Center, Denver, Colorado 80262

ABSTRACT In tetrapod vertebrates, neural circuitries subserving visceral and somatic reflexes are each

represented in distinct columns of cells within the gray area of the spinal cord. To determine the location of visceral elements of the spinal cord of a teleost fish, crystals of the carbocyanine dye 1,l‘dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI), were placed on either the abdominal sympathetic (mesenteric) nerves, the coeliac ganglia, or on the rostral three somatic spinal nerves, in fixed specimens of the channel catfish, Zctalurus punctatus. In fish in which DiI had been placed on the mesenteric nerves, labeled fibers coursed along the lateral margin of the dorsal horn within the first and second spinal segments, and appeared to terminate in a region at the base of the dorsal horn. In contrast, when DiI crystals were placed on the somatic spinal nerves, labeled primary afferents terminated in the dorsalmost two thirds of the dorsal horn, as well as in ventral and ventromedial areas of the medial funicular nuclear complex. Labeled somata (motor neurons) were situated in the ventral horn. When DiI crystals were placed bilaterally on the coeliac ganglia, labeled piriform and fusiform preganglionic neurons occurred in intermediate positions adjacent to the central canal, corresponding to the paracentral nucleus of Herrick, and in the lateral funiculus. These results demonstrate that somatic and visceral afferent and efferent functional columns are distinct in a teleost fish as they are in amniote vertebrates. D 1996 Wiley-Liss, Inc.

Indexing terms: autonomic nervous system, coeliac ganglion, preganglionic neuron, sympathetic chain

The autonomic nervous system (ANS) coordinates bodily functions concerned with the maintenance of optimal physiological conditions. This coordination requires control over diverse tissues including those of the cardiovascular, pulmo- nary, gastrointestinal, endocrine, and immune systems. The ANS exerts its control via its three peripheral components: the sympathetic, parasympathetic, and enteric nervous systems.

Afferent signals from abdominal tissues are carried by sensory fibers of the vagus (parasympathetic) and the splanchnic and mesenteric (sympathetic) nerves, respec- tively, to the brainstem and spinal cord. These signals are processed locally and serve to modulate visceral reflexes; they are also relayed to more rostral brain regions for integration with information about cognitive, affective, and motivational states (for review, see Loewy, 1990). Both local reflex and rostral, integrative, processing influence physiological functioning via autonomic “preganglionic” motor neurons.

Preganglionic neurons of the sympathetic system in mammals are situated in four cell columns (central autonomic, interomediolateral, intercalated, and lateral funicu-

lar) in or near the lateral horn of the thoracic and lumbar spinal cord (for review, see Cabot, 1990). Sympathetic preganglionic neurons that control abdominal visceral functions send axons via the splanchnic nerves to the abdominal (prevertebral; e.g., coeliac and superior mesenteric; some- times also termed preaortic) sympathetic ganglia. The axons of the ganglionic neurons residing in the prevertebral ganglia constitute the motor component of the mesenteric nerves (Gibbons, 1993), which traverse the coeliac and mesenteric arteries to innervate the upper abdominal viscera.

This general plan appears to obtain, with some variation, in birds (pigeon) as well as mammals. In pigeons, the principal preganglionic cell column (column of Terni) lies in an area dorsal to the central canal, rather than in the interomediolateral region as it does in mammals. However, some preganglionic neurons in pigeons are also located in

Accepted March 27, 1996. Address reprint requests to Dr. T.E. Finger, Department of Cellular and

Structural Biology UCHSC, 4200 East Ninth Ave Denver, CO 80262. Email: [email protected]

O 1996 WILEY-LISS, INC.

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the lateral pericentral gray, a region similar to that of intercalated neurons of mammals (Cabot and Bogan, 1987). Thus, in two different classes of amniote vertebrates, sympathetic preganglionic neurons are situated in an intermediate position, i.e., in the vicinity of the sulcus limitans of the neural tube.

The intermediate location of sympathetic preganglionic neurons in birds and mammals suggests that this arrangement may be a common feature of the vertebrate ANS. An intermediate location for visceral motor neurons in the spinal cord is consonant with the description of “functional columns’’ of sensory and motor nuclei described by Gaskell (1886, 18891, Herrick (18991, and Johnston (1902). These authors proposed that in the caudal central nervous system (CNS) of vertebrates, the neural apparati supporting somatic and visceral reflexes are anatomically separate. Somatic sensory systems are represented dorsally while somatic motor neurons lie ventrally, whereas visceral sensory and motor components are situated in an intermediate position.

This description has been shown to be accurate for brainstem somatic and visceral structures in vertebrate species as diverse as fish (Sperry and Boord, 1993; Finger, 1993) and mammals, and is accurate for these structures in the spinal cord of mammals as well. However, less information is available regarding functional columns in the spinal cord of non-mammalian vertebrates, especially fishes.

The goals of the experiments reported are threefold: first, to locate the abdominal visceral primary afferent terminations in the spinal cord of a teleost fish; second, to locate the preganglionic neurons innervating the coeliac ganglia (which innervate the abdominal viscera); and third, to determine whether the location of the preganglionic neurons is anatomically separate from spinal somatic neurons. For these experiments, the carbocyanine dye DiI was used in fixed catfish to trace the CNS pathways of the visceral (splanchnic and mesenteric) and somatic components of the three rostra1 spinal nerves that carry abdominal visceral information.

MATERIALS AND METHODS Catfish (n=47, 10-25 cm in length) were anesthetized in

water containing 3-aminobenzoic acid (Sigma Chemical Co., St. Louis, MO) and perfused through the heart with

ac ANS

CG cg cm CNS DiI dh dlf dlfv DMNX DRG GVn IML iml If

marg MFn

cc

lfpg

anterior commissure of the CG autonomic nervous system central canal coeliac ganglia central gray centimeter central nervous system dioctadecyl tetramethylindocarbocyanine dorsal horn dorsolateral fasciculus ventral dorsolateral fasciculus dorsal motor nucleus of the vagus dorsal root ganglia general visceral nucleus of the vagus interomediolateral preganglionic neurons of mammals interomediolateral preganglionic neurons of catfish lateral funiculus lateral funicular preganglionic neurons preganglionic neurons in marginal lateral funiculus medial funicular nucleus

L.E. GOEHLER AND T.E. FINGER

teleost Ringer’s solution (Forster and Taggert, 1950) followed by 4% paraformaldehyde/ 0.1 M phosphate buffer (4% PFA/PB). The fish were post-fixed overnight in 4% PFA/PB. The following day, the fish were rinsed in PB and crystals of the carbocyanine dye, 1,l’ dioctadecyl- ,3,3,3’ ,3 ’ ’ - tetramethylindacarhocyanine (DiI, Sigma) were placed with forceps on one of the five locations indicated on Figure 2: 1) mesenteric nerves (n= 17), 2) bilaterally on the coeliac (sympathetic) ganglia (n= 5), 3) spinal occipital nerve (n= 14), 4) first spinal nerve (n= 111, or 5) second spinal nerve (n= 10). For DiI labeling of spinal nerves, DiI crystals were placed on the whole nerve distal to the point of junction with the rami communicantes to the coeliac ganglia. In this way, DiI labeling of spinal nerves reflected only the somatic component of that nerve. After DiI application, the entire area was covered with 3% agar/ dH20, placed in 4% PFAIPB, and incubated at 37°C for 6-2 weeks, depending on the distance the dye was to travel. After incubation, the first four segments of the spinal cord and caudal brainstem, and in two cases, the coeliac ganglia and dorsal root ganglia were removed and sectioned on either a cryostat (20-30 pm) or Vibratome (50-60pm). For cryostat sections, the tissue was first cryoprotected in 30% sucrose/ 4% PFA/PB for at least a week. Consecutive transverse, or in two coeliac ganglia labeled cases, horizontal, cryostat sections were thaw-mounted onto gelatin coated slides, and coverslipped with glycerine/PB (1%) immediately to prevent the tissue from drying out. For Vibratome sections, the brains were placed in 1-2% glutaraldehyde /PB for 24 hour prior to sectioning. The higher background fluorescence produced by the glutaraldehyde facilitated visualization of unlabeled structures. Consecutive transverse Vibratome sections were collected into PB, mounted, and immediately coverslipped with glycerin/PB. The slides were viewed with a Zeiss epifluorescence microscope using a rhodamine filter set.

Electronic Imaging: Primary images were acquired on film, which was then scanned at a resolution of 1,200-1,800 dpi into a MacIntosh Quadra 800 system through a Nikon Coolscan. Contrast and brightness were adjusted in Adobe Photoshop and small imperfections in the images, e.g., dust eliminated by replacement with adjacent pixels. In addition, graininess of some images was reduced by use of a 1 pixel diameter Gaussian blur.

Abbreviations

mlf NIX N. mes N. spinalis N. SpO

N. Sp-2 N. Sp-3 N. Sp-4 NXb NXC PB PC PFA rC rCx RF SG vh

N. sp-1

medial longitudinal fasciculus glossopharyngeal nerve mesenteric nerve spinal nerve spinal occipital nerve first spinal nerve second spinal nerve third spinal nerve fourth spinal nerve gill nerve (branchial) branches of the vagus coelomic branch of the vagus phosphate buffer posterior commissure of the CG paraformaldehyde ramus communicante ramus communicante from coelomic vagus reticular formation sympathetic chain ganglia ventral horn

439 SPINAL VISCERAL COMPONENTS IN CATFISH

RESULTS Terminology

The spinal cord of fishes is somewhat different from that of most other vertebrates. First, the dorsal horn is not clearly laminated (Fig. 3B). Rather, different areas of the dorsal horn appear to form nuclei, or nuclear columns (Ariens-Kappers et al., 1936). In addition, many fishes, including channel catfish, possess dorsal specializations of the caudal medulla, including the lateral and medial funicular nuclei (Fig. 3A, Herrick, 1906, 1907). The medial funicular nucleus as defined by Herrick encompasses an intermixed complex including the spinal trigeminal nucleus, the inferior gustatory nucleus, and elements of the visceral sensory system described below. Accordingly we will use the term medial funicular nuclear complex (MFn) when refer- ring to this area. The MFn receives inputs from brainstem visceral (especially gustatory) and somatic sensory areas, as well as ascending inputs from the spinal cord and primary afferent information from the spinal occipital nerve (Her- rick, 1905, 1906; Finger, 1976; Finger and Kalil, 1985). Herrick has suggested that these nuclei subserve integrative functions related to feeding behavior and bodily orientation. Delineation of the various territories within the complex is well beyond the scope of the present work.

In tetrapod vertebrates, the sympathetic ganglia that control abdominal visceral functions (prevertebral) are located in the abdominal cavity at the origins of the main ventral branches from the aorta (e.g., coeliac artery; Gib- bons, 1993); the preganglionic neurons that innervate these ganglia are situated at thoracic and lumbar levels of the spinal cord. In many fish, the sympathetic ganglia innervating the abdominal cavity are also located in association with the ventral branches of the aorta, e.g., coeliaco-mesenteric artery (Gibbins, 1993). However, in fish, the coeliac artery lies rostrally and quite near the paravertebral sympathetic chain. As a result, the coeliac ganglia lie in series with the rest of the sympathetic chain, and thus are “paravertebral” rather than “prevertebral” (Fig. 1). In view of the inconsis- tencies in nomenclature this arrangement has engendered, we have adopted terminology from Gibbons (1993). Accord- ing to this scheme, the “rami communicantes” connect the spinal cord to the sympathetic chain; “splanchnic nerves” connect the sympathetic chain to remote prevertebral ganglia; and “mesenteric nerves” carry postganglionic fibers to peripheral visceral targets. As mentioned above, in many fishes including catfish, the coeliac ganglia are not remote from the sympathetic chain, but are in series with it (Figs. 1, 2). Thus, strictly speaking, no splanchnic nerve exits in these species.

Application of DiI to whole nerves results in the entire nerve becoming pink as the dye diffuses, both in the anterograde and retrograde directions. This feature greatly aids the tracing of peripheral pathways, especially of unmy- elinated nerves (such as visceral nerves) that are otherwise difficult to distinguish from the surrounding tissue. Applica- tion of DiI to the mesenteric nerves labels fibers innervating visceral structures such as the stomach, intestine, and pancreas (L. Goehler, unpublished observations) Thus, cell bodies in ganglia labeled by the same application are presumed to also innervate these abdominal visceral structures. The following is a description of the sympathetic ganglia associated with the mesenteric nerves as revealed by DiI tracing, and their relationship with the spinal nerves with which they enter the spinal cord.

S p C

ac

PC

Dorsal Aorta

Fig. 1. Semi-schematic drawing of a ventral view of the relationship between the sympathetic ganglia and major blood vessels in the channel catfish. Anterior is upward. As in other teleost fish, the ganglionated sympathetic chains extend into the cranial region, rostral to the coeliac ganglia. Note that the coeliac ganglia are located in series with the other ganglia along the chain.

The coeliac and dorsal root ganglia When DiI is applied to the mesenteric nerves of catfish,

two pairs of fused ganglia lying on either side of the dorsal aorta, immediately rostral to the coeliaco-mesenteric artery (Figs. 1, 2) are labeled. Their location is similar to the mammalian coeliac ganglia (as well as the coeliac ganglia of Gadus; Holmgren and Nilsson, 19821, which also innervate abdominal viscera, so this nomenclature, coeliac ganglia (CG), is adopted for the ganglionic complex in catfish. However, in this catfish, these ganglia lie in series with the other ganglia of the sympathetic chain.

The anterior coeliac ganglia are smaller than the posterior coeliac ganglia, which appear to be two or three fused ganglia forming a complex around the base of the celiaco- mesenteric artery. In fish in which DiI was applied to the mesenteric nerves, labeled nerve fibers exit the ganglia to run rostrally and caudally in the sympathetic chain for an undetermined distance, or in rami communicates that join spinal nerves. The labeled rami join the spinal occipital, first and second spinal nerves 1-3 mm distal to the dorsal root ganglia (Fig. 2). In addition, the right anterior CG is connected by a ramus (rCx) to the right caudal vagal (general visceral) ganglion. When DiI is applied to either the mesenteric nerves or the CG, scattered pseudounipolar neurons are labeled in the right caudal vagal ganglion, and bilaterally in the dorsal root ganglia associated with the spinal occipital, first and second spinal nerves (Fig. 4).

440 L.E. GOEHLER AND T.E. FINGER

C- Sympathetic I ld Chain

Fig. 2. Schematic diagram of a dorsal view of the relationship between the coeliac ganglia and the spinal and cranial nerves. Sympa- thetic nervous system elements are shaded as in Figure 1. The location of DiI placements are indicated by heavy circled numbers, as specified in the methods section of the text. Letters A-D at left indicate the approximate level of the chartings shown in Figure 5.

Two commissures connect the CG of the two sides (Fig. 1). The anterior commissure connects the two anterior ganglia; the posterior commissure, forming a band around the ventral base of the celiaco-mesenteric artery, connects the larger posterior ganglia. The posterior commissure is situated caudal to the ganglia, at the point of exit of the right and left mesenteric nerves.

Central organization of visceral and somatic elements of rostral spinal nerves

Visceral and somatic afferent systems Abdominal visceral primary afferent fibers. After DiI

application to the mesenteric nerves or CG, labeled nerve fibers enter the dorsolateral aspect of the spinal cord with the first and second dorsal roots (Fig. 5-CG). A portion of these fibers continues medially to terminate in the most superficial portion of the dorsal horn. The majority of labeled fibers join the dorsolateral fasciculus, where they either ascend or descend, and finally terminate in a ventral region of the dorsal horn (Figs. 6, 7B). This region extends from slightly caudal to the entrance of the second dorsal root, to somewhat rostral to the entrance of the first dorsal root. No label was observed caudal to the second spinal segment. At its most caudal levels, the region of termina- tion is slightly smaller and is located immediately dorsolat- era1 to the central gray. At more rostral levels, the region widens, and is situated more laterally and dorsally in the dorsal horn. Most of the DiI labeled fibers terminate in the ipsilateral ventral dorsal horn, but a few cross the midline in a commissure located in the dorsal central gray (Fig. 6A).

Fig. 3. Photomicrographs of Nissl-stained sections through the A) Funicular complex, and B) first spinal segment of the channel catfish, Ictuluruspunctutus. The dorsal horn is not obviously laminated and is replaced at more rostral levels by the medial funicular nuclear complex. Section A is approximately at the level of chartingA in Figure 5; section B is approximately at the level of charting C in Figure 5.

DiI applications to the CG label fibers bilaterally in the dorsal roots and dorsolateral fasciculus.

DiI labeled visceral fibers also enter the spinal cord with the spinal occipital dorsal roots. These fibers immediately enter the ventral dorsolateral fasciculus (dlf, Fig. 7A,C). Some fibers descend a short distance, then turn medially, curving around and through the medial funicular nuclear complex (MFn) to terminate in a dorsomedial territory of the complex (Fig. 5-CG, 6B, 7A). Other fibers also turn medially, but continue ventromedially to terminate in a more caudal territory of the MFn , located dorsolateral to the central canal (Figs. 6B,C, 7A). Some fibers also are present in the “reticular formation” (as named by Herrick, 1906, but it is probably not equivalent to the medullary reticular formation of modern usage) dorsolateral to the central canal (Fig. 6B,C) and adjacent to the caudal-most reaches of the dorsal motor nucleus of the vagus (DMNX; Figs. 6C, 7A). The remainder of DiI labeled visceral fibers continue in the dorsolateral fasciculus, finally terminating in the ventral visceral region of the dorsal horn.

SPINAL VISCERAL COMPONENTS IN CATFISH 44 1

Fig. 4. DiI label in sensory neurons after application to the coeliac ganglia. Scale bar, 100 pm. A: Dorsal root ganglion of spinal nerve 1. B: Caudal vagal ganglion. Because these ganglia had to be sectioned frozen, the DiI diffused somewhat to cells adjacent to the heavily labeled ganglion cells.

Following DiI application to the mesenteric nerves or CG, a few labeled pseudounipolar neurons occur in the right caudal (general visceral) vagal ganglion (Fig. 4B). The axons of these neurons either project to the general visceral nucleus of the brainstem, or join the dorsolateral fasciculus to descend to the MFn or dorsal horn.

After DiI application to the first and second spinal nerves (distal to the entry of, and therefore excluding, visceral nerve fibers), DiI labeled nerve fibers enter the spinal cord with the dorsal roots, and

Somatic primary afferents.

terminate in the most superficial region of the dorsal horn and a large region immediately subjacent (Figs. 5-NSp2, 7D). A few fibers travel ventrally along the lateral margin of the dorsal horn, where they appear in the region lateral to the central canal. Many fibers ascend to the level of the MFn, where they terminate in several dorsal territories (Fig. 5-NSp2). A few fibers are also present in a ventromedial territory of the MFn.

Following DiI application to the distal spinal occipital nerve, labeled fibers enter with the dorsal root (Fig. 5-NSpO). A group of labeled fibers ascends in a tract adjacent to the spinal trigeminal tract, while another group descends in the dorsal dorsolateral fasciculus. The remain- ing labeled root fibers terminate in dorsal, dorsomedial, and ventromedial territories of the MFn (Fig. 5-NSpO, 7C). Ascending fibers travel at least as far rostral as the facial lobe, whereas descending fibers terminate in both the dorsal and ventral regions of the spinal dorsal horn.

Visceral and somatic efferent neurons Preganglionic neurons. Preganglionic neurons were la-

beled only by application of DiI to the CG. DiI application to the mesenteric nerves labels only sensory fibers and ganglionic neurons. After DiI was applied to the CG, labeled somata occurred in the pericentral gray (paracentral nucleus, pc; interomediolateral, iml; Figs. 5-CG; 8) and dorsal margin of the ventral horn of the spinal cord (lateral funicular, lf; Fig. 8B) at levels between the first and second spinal segments (Fig. 5-CG). These neurons occur singly or in clusters of two to six neurons. In contrast to the spinal autonomic motor neurons of tetrapods, these neurons are not aggregated into limited nuclei. However, the clusters in the catfish spinal cord occur in identifiable regions, namely: 1) the dorsolateral central gray area, (paracentral nucleus, pc; Fig. 8A), 2) the lateral and ventrolateral central gray, (iml, Fig. 8B,C), and 3) the dorsal margin of the ventral horn and lateral funiculus (lf, Fig. 8B).

The labeled somata are multipolar and of medium size (40 pm). Neurons in the central gray are oval or piriform, whereas those located in the lateral funiculus are predomi- nantly fusiform. The dendritic processes of neurons in the central gray extend either dorsally toward the dorsal horn and/or horizontally across the midline. Occasionally dendritic processes of these neurons extend laterally into the lateral funiculus. The dendrites of more laterally situated neurons extend primarily into the marginal area of the lateral funiculus, where they ramify into numerous fine branches. Occasionally, labeled somata are also present in this region of the lateral funiculus (marg, Fig. 6A). For all preganglionic neurons from which axons could be followed, the axons emerged with the ventral roots. These axons form a bundle that courses ventrally between the medial edge of the ventral horn and the ventral funiculus, exiting with the ventral roots (Figs. 5-CG, 8A) of the first and second spinal nerves.

Following DiI application to the somatic components of the first or second spinal nerves (distal to the anastomoses with visceral fibers), labeled somatic motor neurons form two groups in the spinal ventral horn. One group of small (20 pm) cells occupies the dorsomedial region, whereas a second group of larger (50 pm) neurons occupies the ventral and ventromedial portion of the ventral horn (Figs. 5-NSp2, 9A). The dendrites of spinal somatic neurons extend to the adjacent ventral lateral funiculus.

Somatic motor neurons.

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Fig. 5. Chartings of transverse sections through the rostral spinal cord after DiI application to either the coeliac ganglia (CG; column l), somatic portions of the second spinal nerve (SpN-2; column 21, or somatic portions of the spinal occipital nerve (NSpO; column 3). Triangles represent labeled cells; dots represent fibers and terminals.

After DiI application to the spinal occipital nerve, large and medium-sized somatic motor neurons occur in a region extending from the ventral horn of the spinomedullau junction, at the level of the M F ~ ( ~ i ~ . g ~ ) , to the ventral tegmentum of the caudal brainstem, at the level of the

are most numerous in the caudal brainstem. The dendrites of caudal motor neurons extend into the lateral funiculus. Dendrites

Fig. 6. DiI labeled fibers in the dorsal horn and MFn after application to coeliac ganglia. A: DiI labeled fibers occur in superficial dorsal horn, the ventral dorsal horn, and cross the midline in a commissure to the contralateral ventral dorsal horn. Two neurons are labeled; one iml neuron extends dendrites both dorsomedially toward the region of sensory terminations and laterally into the lateral funiculus, the other (marg) is situated at the marginal zone of the lateral funiculus. B: Labeled fibers enter with the spinal occipital nerve, and either enter the

vagal lobe. Dil labeled motor

of large motor neurons in the brainstem extend throu&out ventral dlf or course to medial and ventromedial regions of the MFn. C : the reticular formation, *hereas the dendrites of smaller

of rostra1 spinal occipital motor neurons Course ventrally, then caudally, joining with axons of more caudally situated

Higher power view of the section shown in B, but of a more medial field of view. Numerous closely bundled fibers are visible in the so-called reticular formation (Herrick, 1906) dorsolateral to the central canal, immediately adjacent to the caudal end of the dorsal motor nucleus of the vagus (vagal motor neurons). Scale bar, 100 km.

ramify in a region 'lose the somata. The

Figure 6


Fig. 7. Photomicrographs comparing the pattern of labeled sensory fibers after DiI application to the coeliac ganglia (A, B) or to the spinal occipital nerve (C, D). A and C are transverse sections from the level of the MFn. B is a transverse section from the rostral spinal cord at the level of entry of the first spinal nerve. D is from a level slightly rostral to B. Scale bar, 100 pm.

neurons to exit the spinal cord in the spinal occipital ventral root.

Abdominal visceral afferents terminate in both the most superficial and deepest regions of the dorsal horn, while somatic afferents terminate in the dorsalmost two thirds of the dorsal horn. Sympathetic preganglionic motor neurons occupy the pericentral and lateral regions of the spinal cord, whereas somatic motor neurons occupy the ventral and ventromedial regions of the spinal cord. Figure 10 compares these two patterns, and shows the relationship of visceral and somatic elements with the dorsal root ganglia, sympathetic chain, and spinal nerve.

Summary.

DISCUSSION Despite differences in the anatomical organization of the

sympathetic ganglia in catfish and tetrapods, the location of abdominal visceral sensory and motor elements of the catfish spinal cord is similar. For example, in both groups the visceral motor neurons lie near or lateral to the central canal, whereas somatic motor neurons are situated ventrally. Similarly, visceral sensory fibers terminate ventrally in the dorsal horn, whereas somatic sensory fibers terminate dorsally. This suggests that a general pattern for the organization of the visceral nervous system exists through- out much of the vertebrate lineage.

SPINAL VISCERAL COMPONENTS IN CATFISH 445

Fig. 8. Labeled neurons after DiI application bilaterally to the coeliac ganglia. A: Two neurons in a dorsolateral (paracentral) position (pc) extend dendrites dorsolaterally. Labeled s o n s (arrows) from contralaterally located neurons (cell bodies not in the section) can be seen coursing ventrally toward the ventral root. B: Two groups of neurons are labeled in this transverse section at the level of the second

In contrast to the situation in tetrapods, the catfish coeliac ganglia, although closely associated with the aorta and coeliac and mesenteric arteries, are located in series with the sympathetic chain. Thus preganglionic axons innervate the ganglia via rami communicantes from the spinal roots directly, rather than via splanchnic nerves as is the case in tetrapods. This arrangement is similar to the situation in selachians, in which sympathetic ganglia (“gas- tric ganglia”) that project to the intestine are innervated directly by rami communicantes from the rostra1 spinal nerves (Young, 1933; Nichol, 1952; Campbell, 1970). Short splanchnic nerves may be present in some species of teleosts, possibly similar to the situation in some anuran amphibians, e.g., Rana (Gibbins, 1993).

In the channel catfish, as well as in amniotes studied to date, visceral afferent fibers terminate in both the most superficial and the deepest regions of the spinal dorsal horn. In mammals, fine caliber sensory fibers (A-delta and C fibers) terminate in laminae I and V, corresponding to the most dorsal and ventral areas of the dorsal horn (Cervero and Connell,1984; Kuo and DeGroat,1985). These fine caliber sensory fibers carry both visceral and somatic (nociceptive) information. In catfish, however, few somatic

spinal nerve. An interomedial group (iml) is situated adjacent to the central canal, whereas a more diffuse group of four neurons (10 is distributed laterally in the lateral funiculus. C: Two groups of iml neurons send dendrites through a commissure in the dorsal central gray or laterally into the lateral funiculus. Scale bars, 100 pm.

sensory fibers terminate in deep regions of the dorsal horn. Rather, in catfish, somatic sensory fibers terminate primarily in the dorsal two thirds of the dorsal horn, whereas the ventral region of the dorsal horn receives mostly visceral afferent information. This arrangement suggests that visceral and somatic sensory information may be processed separately within the catfish spinal cord.

In the medial funicular nuclear complex (MFn), unlike the dorsal horn, visceral and somatic sensory terminal fields overlap (in ventromedial regions). The spinal occipital nerve, whose afferents terminate in the MFn, innervates muscles involved in vegetative functions requiring coordination of visceral and somatic systems, e.g., feeding and breathing (McMurrich, 1884). The functions of the ventromedial region of the MFn, and the subjacent reticular formation, are as yet unknown. However, the proximity to this region of dendrites of the caudal-most neurons of the dorsal motor nucleus of the vagus (Goehler, unpublished observations; Herrick, 19061, and the overlap of spinal occipital nerve and abdominal sympathetic afferent projections in this area suggests that it may be important for the integration of information relevant for visceral functions.


SOMATIC

root I \

Fig. 9. DiI application to the second spinal nerve (A) or spinal occipital nerve (B) labels motor neurons in a ventral region of the ventral horn. Scale bars, 100 Fm.

Sensory neurons of the caudal (general visceral) vagal ganglion send fibers to the coeliac ganglia andlor mesenteric nerves. Whether vagal fibers terminate in the ganglia or continue in the mesenteric nerves to innervate the viscera could not be determined. However, intermingling of vagal fibers with sympathetic nerves has been described in other vertebrates species, including other fishes (e.g., Young, 1931). In fish, nerve fibers travelling with the coelomic vagus innervate only the upper gastrointestinal tract (esophagus and stomach), whereas nerve fibers of the mesenteric nerves innervate the intestine as well (Burn- stock, 1959; Goehler, unpublished observations). Thus, a vagal sensory pathway through the mesenteric nerves may provide feedback about digestion directly to vagal nuclei involved in feeding behavior.

The location of the preganglionic neurons seems to correspond to the paracentral nucleus described in fish by

VISCERAL

root I \

Fig. 10. Schematic summary diagram comparing the arrangement of somatic (top) and visceral (bottom) sensory and motor elements in the spinal cord and associated spinal nerves and sympathetic chains.

Herrick (1899). Although Herrick presumed neurons of the paracentral nucleus to be visceral in nature, this could not be confirmed by methods available at the time. The availabil- ity of a carbocyanine dye appropriate for use in fixed tissue has enabled us to confirm these earlier assumptions. The application of DiI to the coeliac ganglia, but not the mesenteric nerve, labels neurons in the pericentral gray area, as well as laterally in the lateral funiculus. The fact that these neurons are retrogradely labeled only when DiI is applied to the ganglia supports their identity as sympathetic preganglionic neurons.

Although catfish do not possess a well-defined lateral horn, the distribution of sympathetic preganglionic neurons in the spinal cord is otherwise similar to that of amniote vertebrates. Preganglionic neurons of the catfish located in the dorsolateral central gray correspond in position to the central autonomic nucleus, column of Terni, and intercalated cell group of other vertebrates. However, we elect not to adopt the amniote terminlogy for the teleost situation, first because the homology is uncertain, and second, because the term “paracentral” applied by Herrick (1899) predates the Terni work, and therefore is the most appropriate term. The ventromedial iml neurons in catfish likely correspond to the IML of mammals. Similarly, the more laterally located, fusiform motor neurons in the catfish may correspond to the lateral funicular neurons of mammals and/or intercalated neurons of birds (Cabot and

SPINAL VISCERAL COMPONENTS IN CATFISH 447

Bogan,1987). Determination of whether the similarly situated preganglionic neurons in these diverse species are homologous must await study of other vertebrate groups.

The mediolateral orientation of the dendrites of many catfish preganglionic neurons may be a characteristic typi- cal of vertebrate preganglionic neurons, because this feature is present in both rats and pigeons (Cabot and Bogan, 1987; Forehand, 1990). This pattern of dendritic extension suggests that the central regulation of abdominal sympathetic function derives both from local reflexes mediated by the spinal visceral areas or primary afferents, and from rostral brain centers via axons in the lateral funiculus. We have preliminary evidence (Goehler and Finger, 1993) that the vagal general visceral nucleus projects to spinal visceral areas through the dorsolateral fasciculus and the lateral funiculus. These pathways through the lateral funiculus appear similar to the projection of the vagal solitary nucleus to the interomediolateral column in mammals (Loewy and Burton, 1978; Amendt et al, 1979; Mtui et.al., 1993).

The segregation of visceral and somatic areas in the spinal cord of the catfish corresponds to the functional columns described for the caudal CNS (brainstem and spinal cord) of vertebrates (Gaskell, 1889; Herrick, 1899; Johnston, 1902). Indeed, although other features of autonomic neurocircuitry (for example, the migration of the coeliac ganglia from the sympathetic chain to the abdominal cavity) seem to have arisen later in vertebrate evolution (for review, see Gibbons, 1993), segregation of visceral and somatic reflex circuitry seems to be an early pattern. The caudal CNS controls basic homeostatic and somatic reflexes, and the orderly representation of sensory and motor components of these reflex systems may facilitate these important functions.

In conclusion, the sensory components of the mesenteric nerves of the catfish terminate in spinal cord regions similar to these elements in amniote vertebrates. Likewise, preganglionic neurons innervating the coeliac ganglia of catfish are located in spinal cord regions analogous to those of amniotes. These results support the idea that the organization of A N S reflex circuitry reflects a fundamental vertebrate design, which evolved early in vertebrate phylog- eny.

ACKNOWLEDGMENTS This work is dedicated to Dr. Sanford Palay whose

exemplary research set new standards for ultrastructural studies on the nervous system. Research reported in this paper was supported by NIH grant DC00147 to T.E.F.

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