fmrfamide-like immunoreactivity in the brain of the pacific hagfish,eptatretus stouti (myxinoidea)

Cell Tissue Res (1992) 270:443 449 Cell&Tissue

Research �9 Springer-Verlag 1992

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish, Eptatretus stouti (Myxinoidea) Helmut Wicht 1 and R. Glenn Northcutt 2

1 Zentrum der Morphologie, Abteilung Neurobiologie, Klinikum der Johann Wolfgang Goethe-Universitfit, Theodor-Stern-Kai 7, W-6000 Frankfurt/Main 70, Federal Republic of Germany 2 Neurobiology Unit, Scripps Institution of Oceanography and Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA

Received April 30, 1992 / Accepted July 13, 1992

Summary. The distribution of FMRFamide- l ike immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish, Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral gangli- onic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinct terminal arborizat ion occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that F M R F a m i d e may play a role in the central control of branchiomotor activity.

Key words: F M R F a m i d e Hypotha lamus - Brain stem - Sensomotor system Eptatretus stouti (Agnatha, Cyc- lostomata, Myxinoidea)

Antibodies directed against the tetrapeptide F M R F - amide ( P h e - M e t - A r g - PheNH2), which was original- ly extracted f rom molluscs (Price and Greenberg 1977), label a variety of brain structures in craniates, most notably and consistently the terminal nerve and its compo- nents (Stell et al. 1984; Bonn and K6nig 1988, 1989a, b; Muske and Moore 1988; Ostholm et al. 1990; Wirsig- Wiechmann 1990) and groups of cells in the hypothalamus that are thought to participate in neuroendocrine mechanisms (Ohtomi et al. 1989; Chiba et al. 1991). Nu- merous other nuclei in different craniate brains also con- tain immunoposi t ive neurons, but the pat tern is inconsis- tent and reveals extensive interspecific differences. Thus, cerebellar Purkinje cells are immunoposi t ive in three species of teleosts (Bonn and K6nig 1988, 1989a, b) but not in other craniates, including another teleost

Correspondence to: H. Wicht

(Ostholm et al. 1990). While positive cell bodies are re- portedly common in various mesencephalic and rhomb- encephalic structures in teleosts (Bonn and K6nig 1988, 1989a, b; CIstholm et al. 1990) and mammals (Williams and Dockray 1983), they are apparently confined to the forebrain in sharks (Chiba et al. 1991), lampreys (Ohto- mi etal . 1989) and amphibians (Muske and Moore 1988). Until now, such cell bodies also appeared re- stricted to (hypothalamic) forebrain structures in myxin- oids (Jirikowski et al. 1984). We began our re-examination of the FMRFamide- l ike immunoreactive system in the Pacific hagfish (Eptatretus stouti, Myxinoidea) primarily because of our interest in the terminal nerve. De- spite some recent discussions concerning the true nature of the terminal nerve (see Discussion), F M R F a m i d e ap- pears to be a reliable marker for the terminal nerve system, in particular for the retinopetally projecting subunit of that system (Stell et al. 1984). Since hagfishes do possess a retinopetal projection (Wicht and Northcut t 1990), but apparently lack a terminal nerve (Northcutt 1985), we decided to re-investigate the distribution of F M R F - amide-like immunoreact ivi ty in their brains.

Materials and methods

Adult Pacific hagfish (Eptatretus stouti) were trapped off the coast of La Jolla, California, at depths of around 200 m. Four animals, females, ~400 mm body length, were used for this study. The animals were anesthetized with tricaine methane sulfonate (MS 222, 1:10000 in sea water), perfused via the heart with 0.1 M phosphate buffer (pH 7.4) followed by a freshly prepared solution of 4% paraforrnaldehyde in phosphate buffer. The brains were dissected from the skulls, postfixed for 2 h, embedded in gelatin and fixed for an additional 12 h. Sections were cut in the transverse plane on a freezing microtome at 40 gm and collected in phosphate buffer containing 0.001% sodium azide to prevent bacterial degra- dation. Immunhistochemistry was carried out on free-floating sections using Sternberger's (1979) peroxidase-antiperoxidase (PAP) protocol. The primary antibody (rabbit anti-FMRFamide, Incstar, lot~ 8630014) was applied at dilutions of 1:4000 and 1:6000 for 48 h at 4 ~ C. After a 20-min wash in phosphate buffer, the sections were incubated in the secondary antibody (goat anti-rabbit IgG)

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at a dilution of 1:100 for 1 h at room temperature, followed by another 20-rain wash in phosphate buffer. The PAP-complex (rabbit-PAP) was diluted 1:200; again, incubation was for 1 h at room temperature, followed by a 20-rain wash in phosphate buffer. Per- oxidase was visualized by the method of Adams (1981), utilizing a phosphate-buffered solution of diaminobenzidine. Afterwards the sections were mounted, dehydrated, and coverslipped.

For controls, the primary antibody was omitted from the procedure in one experiment. In another experiment, the primary antibody was preincubated for 24 h at 4 ~ C with 100 gg of synthetic FMRFamide (Sigma) per ml of working solution, according to the instructions given by the manufacturer of the primary antibody. In neither case was labelling detected in the tissue after completion of the immunhistochemical procedure.

The terminology for the forebrain is taken from Wicht and Northcutt (1992), for the hindbrain we have followed Jansen (1930), Kusunoki et al. (1982), and Ronan (1988).

Figs. 1--4. Abbreviations are as follows: aol Octavo-lateral area; BO olfactory bulb; BOci internal cellular layer of olfactory bulb; BOg glomerular layer of olfactory bulb; BOrn mitral layer of olfactory bulb; BOp periglomerular layer of olfactory bulb; coh habenu- lar commissure; copo postoptic commissure; Di diencephalon;farc arcuate commissure; fr fasciculus retrofiexus; gc central mesencephalic grey; Ggl utr utricular ganglion; HA habenula; HYinfinfun- dibular hypothalamic nucleus; lern lemniscus and tecto-bulbar tract; NCd, vl, rn dorsal, ventrolateral, medial subnuclei of the central prosencephalic nucleus; nhyp neurohypophysis; P1-P3 pal- lial layers; POe, irn, p external, intermediate, periventricular subdivisions of the preoptic area; Ret reticular formation; sp spinal nerves; St striatum; Tel telencephalon; THa anterior thalamic nucleus; THdi diffuse thalamic nucleus; THe external thalamic nucleus; THi internal thalamic nucleus; THpco paracommissural thalamic nucleus; THsh subhabenular thalamic nucleus; THt triangu- lar thalamic nucleus; T M tectum mesencephali; V/VIIrnot motor nuclei of nn. V and VII; vIV fourth ventricle; Vsens sensory nucleus ofn. V

Results

Fig. l shows a dorsal view of the brain of the Pacific hagfish and also indicates the levels of sections in Fig. 2, which illustrates the overall distribution of FMRF- amide-like immunoreactivity in the brain. Figs. 3 and 4 illustrate some details of our findings. Labelled cells were found in two regions. In the first, a large number of mainly bipolar cells was observed in the infundibular hypothalamic nucleus (Figs. 2C, 4A). Occasionally, sin- gle labelled cells were seen in closely adjacent structures : the preoptic region, and the paracommissural and diffuse nuclei of the thalamus (Fig. 2B, C). None of the labelled cells displayed any spatial relationship with the ependyma, the ventricle or the neurohypophysis. In particular, there were no cerebrospinal fluid-contacting neurons and no immunoreactive fibers extending into the region of the neurohypophysis or the median eminence.

A second group of labelled cell bodies was found in the ventrolateral part of the rhombencephalon at the level of the utricular ganglion (Figs. 2F, 3 C). The cells were located in the ventrolateral division of the trigeminal sensory nucleus. Some immunopositive cells were found even further ventral in a nucleus wedged between the branchial motor column and the descending trigeminal nucleus (Figs. 2E, F, 3). Fig. 3A shows this nucleus, which was termed nucleus A by Kusunoki et al. (1982), and the adjacent structures in a cresyl-violet stained preparation. It consists of intermingled large and small cells and can therefore be distinguished from the dorsally adjacent, small-celled trigeminal sensory nucleus and the ventromedially adjacent, large-celled nuclei of the branchial motor column. Caudally it is continuous with the lobus vagi of Jansen (1930), which lacks the large cells typical of Kusonoki's nucleus A.

Fine-labelled fibers were distributed throughout the brain. Typically, these fibers were covered with small

Ggl utr

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Fig. 1. A dorsal view of the brain of the Pacific hagfish, Eptatretus stouti. Roman numerals indicate the cranial nerves. The levels of section in Fig. 2 are indicated. Scale bar: 1 mm

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Fig. 2A-F. Rostral (A) to caudal (F) chartings of the distribution of immunopositive fibers (thin lines) and cells (triangles) in the

brain of a Pacific hagfish. Nucleus A of Kusunoki is indicated by a star. Levels of individual sections are indicated in Fig. 1

varicosities and were sparsely branched (Fig. 4B). The max imum diameter of the thickest fibers was about 1.5 gin, and that of their varicosities was about 5 gm. The majori ty of the fibers was much thinner, however, and some could barely be resolved with the light micro- scope ( < 0.1 gm fiber diameter, with varicosities of less than 1 gm in diameter). There was a general gradient in the density of the labelled fibers, i.e., they were more

numerous in the forebrain than in the hindbrain and more numerous ventrally than dorsally. In the forebrain, only the olfactory nerve, the nervous and the glomerular layers of the bulb, the habenula, the periventricular preoptic nucleus, the neurohypophysis, and the optic nerve were totally free f rom immunoposi t ive fibers. The density of labelled fibers was lowest in the rostral par t of the central prosencephalic nucleus, in the septum, and

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Fig. 3A-D. Photomicrographs (A, C) and accompanying line draw- ings (B, D) of a cresyl-violet-stained transverse section (A) and a section immunostained for FMRFamide (C) from the level of the utricular ganglion. Star indicates nucleus A of Kusunoki. Small

\ v,v "-Lvsons t B g tr

triangles indicate positions of immunopositive cell bodies. Note the cells in the ventrolateral subdivision of the trigeminal sensory nucleus, and the cells and the dense terminal formation in nucleus A of Kusunoki (star). Scale bar = 100 gm for all frames

in the subhabenular and anterior nuclei of the thalamus. All other areas contained many positive fibers, which were especially abundant in the remaining preoptic nuclei, the hypothalamus, and the striatum (see Fig. 2A, B, C).

In the posterior half of the diencephalon, the mid- brain and the hindbrain (Fig. 2D-F) the organization of the immunopositive fiber system was less diffuse, and medial and lateral fiber systems could be distinguished. The lateral system consisted of fibers that followed the course of the postoptic commissure into the mesencepha- lon. More caudally, the lateral system continued into the rhombencephalon by way of the tecto-bulbar tract. In the rhombencephalon, the lateral system formed a conspicuous fiber plexus around the nuclei of the branchial motor column and a distinct terminal formation in nucleus A of Kusunoki (asterisk in Fig. 2E, F; also see Figs. 3 C, 4 C). In the branchiomotor nuclei, the density of labelled fibers was relatively low between the somata of the motor neurons but high at the margins of the nuclei, especially at the ventral margin (Fig. 4 C).

The medial system of immunopositive fibers covered the medial aspect of the diencephalon, and also innervat- ed the central mesencephalic grey. Further caudally, fibers of both the medial and the lateral systems entered the arcuate commissure and the reticular nuclei. Caudal to the level shown in Fig. 2 F, the density of immunola-

belled structures gradually decreased. The fiber plexus around the branchial motor column could be traced to the level of the vagal motor nucleus (Fig. 4 D). Individual fibers in the arcuate commissure and the reticular nuclei were found throughout the posterior rhombencephalon. Still further caudally, at the level of the obex and in the anterior spinal cord, only a few thin fibers (2-3 per section) were found in the dorsal and ventral funiculi.

Discussion

In an earlier study of FMRFamide-like immunoreactivity in the brain of a hagfish, Jirikowski et al. (1984) re- ported that "a large number of FMRF-amide-positive cells can be found in the posterior part of the ventrome- dial hypothalamus "; in an accompanying drawing, however, they depicted positive cells in the mesencephalic interpeduncular nucleus, caudal to the ventral encephalic flexure, and not in the hypothalamus. If we assume that their drawing is mistaken, our findings confirm their observations. We also confirm their observation that the hypothalamic FMRFamide-like immunoreactive cells in hagfishes do not contact the cerebrospinal fluid, the median eminence, or the neurohypophysis. In other craniates, most notably in lampreys (Ohtomi et al. 1989) and sharks (Chiba etal. 1991), but also in teleosts

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Fig. 4A-D. Photomicrographs of transverse sections through the brain of a Pacific hagfish showing A labelled cells in the infundibular nucleus of the hypothalamus. Arrow points to the slit-like infundibular ventricle. Note the absence of cerebrospinal fluid-contacting neurons; B typical appearance of labelled fibers in the forebrain (from the posterior part of the central prosencephalic nucleus) note the irregular orientation of the fibers and the presence of varicosities; C labelled fibers and terminals in the vicinity of the motor neurons of the trigeminal/facial nerve. The somata of the (un-

stained) motor neurons are visible as greyish dots. Note dense terminal formation in nucleus A of Kusunoki (right). Arrows point to a terminal field at the ventral margin of the motor nucleus. Also note individual varicose fibers among the cell bodies of the motor neurons; D labelled fibers surrounding the glossopharyngeal/vagal motor nucleus (dashed line). Arrow points to the com- bined root of the glossopharyngeal/vagal nerve. Scale bars= 100 gm

(Ostholm et al. 1990), hypothalamic FMRFamide- l ike immunoreact ive cells display such contacts. Thus, it is unlikely that the diencephalic immunoposit ive cells of hagfishes participate in neuroendocrine mechanisms. Rather, the occurrence of FMRFamide-posi t ive fibers in most parts of the forebrain suggests that this substance may serve primarily as a neurotransmit ter or neu- romodula tor in the brain of hagfishes (Jirikowski et al. 1984).

It is interesting to note the absence of FMRFamide- like immunoreact ivi ty f rom several forebrain structures. The olfactory nerve, the nervous and glomerular layers of the olfactory bulb, and the optic nerve are all free of immunoposi t ive structures. The density of fibers in the septum is very low. In many other craniate species one encounters luteinizing hormone releasing-hormone ( L H R H ) and/or F M R F a m i d e positive cells and fibers dispersed along the course of the olfactory nerve, the ventral aspect of the olfactory bulb, the subpallial forebrain, and the ventral thalamus. Furthermore, subsets o f cells o f this system often display retinopetal projec-,

tions; the best known example is the nucleus olfactoretinalis of teleosts (Mfinz et al. 1981, 1982; Stell et al. 1984; Muske and Moore 1988; Wirsig-Wiechmann and Basinger /988; Rusoff and Hapner /990; Uchiyama et al. 1988; Uchiyama 1990; Chiba et al. 1991; North- cutt and Butler 1991). Classically, this system has been regarded as a par t of the terminal nerve. However, some recent studies indicate that the telencephalic L H R H / FMRFamide system in general and the nucleus olfactoretinalis in particular may not actually be homologous to what has been described as the terminal nerve (it is beyond the scope of the present discussion to summar- ize this confusing debate; see Szabo et al. 1991, and Hof- mann and Meyer 1991, 1992 for a summary and some recent findings). At any rate, an FMRFamide-posi t ive neuronal system comparable to the one described in the olfactory nerve, olfactory bulb and ventral telencephalon of other craniates is lacking in the Pacific hagfish and, in the absence of the entire system, the Pacific hagfish also lacks a retinopetal projection arising f rom that system.

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However, hagfishes possess a retinopetal projection which arises from a rostral tegmental cell group (nucleus of the posterior commissure), which is homologuous to a similar group of retinopetally projecting cells in lampreys (Wicht and Northcut t 1990). However, those cell groups do not display FMRFamide-l ike immunoreactivity in either lampreys (Ohtomi et al. 1989) or hagfishes, and the neurotransmitters in the retinopetal systems of both agnathan groups still need to be identified.

The immunoreactive cells in the trigeminal sensory nucleus and in Kusunoki's nucleus A have not been described before. In most groups of anamniotes investigated, except teleosts (Bonn and K6nig 1988, 1989a, b; Ostholm et al. 1990; Rusoff and Hapner 1990), FMRFa- mide-like immunoreactive cell bodies have been observed only in the forebrain. In mammals immunoreactive cells have been found in a variety of structures in the brain stem (Williams and Dockray 1983), including an area ventral to the trigeminal sensory nucleus which is topologically very similar to nucleus A of Kusunoki. Based on double-labelling studies, it has been argued that labelling of the cells in this area, as well as of those in many other areas of the brain stern of mammals, is due to a cross-reaction of the FMRFamide antibody with pancreatic polypeptide (PPP), at least in guinea pigs and rats (Triepel and Grimmelikhuijzen 1984). In hagfishes, however, the immunoreactivity of the rhomb- encephalic cell group does not seem to be due to such a cross-reaction, as an antibody against PPP failed to show any labelled structures in hagfishes (Jirikowski et al. 1984; H. Wicht, unpublished observation).

The location of the immunopositive cell bodies in the trigeminal sensory nucleus and the presence of a terminal arborization in Kusunoki's nucleus A and around the branchiomotor column suggest the FMRFa- mide plays a role in the sensory-motor control of the branchial apparatus, even though many details of this interaction remain obscure. The immunopositive neurons in the trigeminal sensory nucleus are not distributed evenly throughout the nucleus but occupy an area where sensory information from the oral and nasal mucosa, possibly including inputs from taste buds (Nishizawa et al. 1988), is apparently processed. Judging from their position, the positive neurons in the trigeminal sensory nucleus are probably concerned with such processing but it is not clear whether they are interneurons or first order sensory neurons. In a position identical to that of the immunopositive cells described in the present study, Ronan (1988) found a group of large (30 60 ~tm) retrogradely labelled cells after application of horseradish peroxidase to the dental ramus of the fifth nerve in the Pacific hagfish and suggested that they might represent a homologue of the dorsal cells in lampreys or the mesencephalic trigeminal nucleus in other craniates. Our immunopositive cells are much smaller (10-15 gm), however, and cresyl violet-stained material failed to re- veal cells as large as those observed by Ronan. The large size of his cells may therefore represent degeneration artifacts, since their peripheral processes had been cut in order to administer the horseradish peroxidase. In

any case the positive cells observed in our study might be identical to those observed by Ronan.

Nucleus A of Kusunoki is a structure of uncertain significance. Its position and histochemistry suggest " a correlative function in relation to the motor nuclei of the cranial nerves" (Kusunoki et al. 1982), but it is clear- ly not a motor nucleus of either the fifth or the seventh nerve (Kishida et al. 1986; Ronan 1988), even though the neurons are fairly large. The size of the neurons suggests a pre-motor function, which, based on our immunohistochemical data, is under FMRFamidergic control. The cells of the infundibular hypothalamus, or those of the trigeminal sensory nucleus, or both, may be the source of input to nucleus A. The motor neurons of the branchial nerves themselves also appear to receive an FMRFamidergic input. The density of positive fibers is relatively low around the neuronal somata but high on the ventral, medial, and dorsal margins of the motor nuclei. As the main dendrites of the motorneurons ex- tend in these directions, not laterally into the region of nucleus A (Kishida et al. 1986), this FMRFamidergic fiber system may form the basis for axo-dendritic contacts.

In conclusion, the present data suggest that F M R F - amide plays at least a dual role in the brain of the Pacific hagfish. On one hand, as evident from the rather diffuse organization of the immunopositive system in the forebrain, FMRFamide may serve as a neuromodulator or neurotransmitter influencing many, if not most, functional systems. On the other hand, in the central systems concerned with the branchial apparatus, FMRFamide- positive structures may play a more specific functional role.

Acknowledgements. Mary Sue Northcutt assisted throughout the research and the preparation of the manuscript. Supported by a stipend from the DFG (Wi 909/1-2) to H.W. and by a grant from the NIH (NS 24869) to R.G.N.

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Note added in proof: While this paper was in press, and after the final proof-reading by the authors, an article by Chiba and Honma was published: Chiba A, Honma Y (1992) FMRFamide-immunoreactive structures in the brain of the brown hagfish, Paramyxine atami: relationship with neuropeptide Y-immunoreactive structures. Histochemistry 98:33-38. With regard to the distribution of FMRFamide-like immunoreactive structures in the brain, the findings of Chiba and Honma are identical to ours.

fmrfamide-like immunoreactivity in the brain of the pacific hagfish,eptatretus stouti (myxinoidea)

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