Brain Research, 140 (1978) 33-42 :.t2) Elsevier/North-Holland Biomedical Press
33
O R G A N I Z A T I O N OF THE T R I G E M I N A L MOTOR NUCLEUS BEFORE A N D A F T E R METAMORPHOSIS IN LAMPREYS
SHINJI HOMMA*
Department of Physiology and Biophysics, Washington University School of Medichw, St. Louis, Mo., 63110 (U.S.A.) (Accepted May 4th, 1977)
SUMMARY
During metamorphosis of the lamprey the simple muscles of the mouth of the larvae are replaced with the elaborate muscles of the sucker, rasper and pharynx in adults. The purposes of the present experiments were the identification of motoneurons to mouth muscles and the determination of their patterns in the trigeminal nucleus before and after metamorphosis. (1) M otoneurons were identified physiologically by observation of contractions in motor units following intracellular stimulation and by antidromic stimulation. Locations of motoneurons were also determined morphologically by retrograde transport of horseradish peroxidase. (2) The trigeminal motor nucleus was subdivided into regions containing motoneurons to specific groups of ipsilateral muscles both in larvae and in adults. The medial-caudal portion of the nucleus contained motoneurons innervating muscles of the oral hood in larvae and the annularis muscles of the sucker in adults. The simple ventral muscles of the larva were replaced by the elaborate muscles of the rasper and piston of adults, yet motoneurons were located in rostral portion of the nucleus in both stages. Likewise, the dissimilar muscles innervated through the basilar nerve had motoneurons in the lateral part of the nucleus in larvae and adults. The velum becomes much smaller during metamorphosis, but motoneurons innervating it in larvae and adults had the same lateral location. (3) It is concluded that the pattern of cells in the trigeminal motor nucleus is not changed during metamorphosis of the lamprey. Perhaps the same motoneurons make new peripheral connections during and after metamolphosis, or new neurons form in the same general regions of the trigeminal nucleus and innervate the new muscles forming in the corresponding regions.
* Present Adress: Department of Physiology, Faculty of Medicine Toyama Medical and Pharmaceutical University Toyama City 930, Japan,
34 INTRODUCTION In the lamprey many anatomical, physiological and behavioral changes occur during metamorphosis3. One of the most pronounced is the change from filter feeding by larvae to parasitic feeding on fish by adults with corresponding changes in the structure and function of the mouth. Although brook lampreys are not parasitic~ similar changes occur in the mouth, which is used during nest-building and spawning. The gross anatomy of the mouth muscles in larvae and adults is well known2,8, x.~ 17 and the morphological changes in these muscles during metamorphosis has been describedZ,16,17. The trigeminal nerve innervates most of the mouth muscles of the lamprey7,16,t7, but aside from the marked enlargement of trigeminal nerve motoneurons, nothing is known about the changes of these cells during metamorphosis. In the present experiments, patterns of innervation of mouth muscles by trigeminal motoneurons are determined in larval lampreys and in parasitic and non-parasitic adults. METHODS Adult small silver lampreys (10-12 cm) lchthyomyzon unicuspis, larval and adult least brook lampreys (9-13 cm) Lampetra aepyptera and larval and adult American brook lampreys (12-15 cm) Lampetra lamottei were maintained in aerated fresh water at 4 °C. They were initially anesthetized by immersion in cold water with t0-4-10 -3 g/ml Tricaine (Ayerst) and dissected in cold physiological solution. Larvae were cut transversely at the gill region and prepared by slitting the ventral midline. Mouth, pharynx and gill walls of one or both sides were extended laterally and fixed on the bottom of a chamber with minutien pins (Fig. 1E). In case of adults, one side of the head was slit laterally and trimmed to expose dorsomedially the piston muscles, basilar muscles and velum (Fig. 1A). The brain was exposed dorsally in both larval and adult lampreys. Preparations were superfused with fluid of the following compositions: 115 mM NaC1, 2 mM KCI, 2.6 mM CaCI2, 2 mM MgCI2, 6 mM NaHCO3 or 4 mM Tris.maleate buffer at pH 7.6 and 3 mM glucose, equilibrated with 2 ~o COz-98 o~ O2 at 7-12 °C. Glass microelectrodes were filled with 3 M potassium acetate by boiling under vacuum and were selected for resistences over 50 Mr2 for larvae. Current was passed through recording electrodes with M4 A electrometers (WPI Instrument, Hamden, Conn.). Positions of the tip of the glass microelectrodes were determined through micrometers in the ocular of the stereoscopic microscope. In preparing maps of the trigeminal nucleus, only the first cell which produced visible contractions when stimulated at 10-20/sec was plotted. Electromyographic activities were monitored with a chlorided silver bipolar electrode placed over the muscles and with a Grass p t5 AC amplifier. The bipolar electrode was also used for stimulation with pulses of 0.2 msec and below 10 V. Cell bodies of trigeminal motoneurons were marked by retrograde labeling after application of horseradish peroxidase to target muscles of the head. For all operations
35 the skin and overlying tissues were cut, and the exposed muscle was damaged mechanically with forceps, since this improved the subsequent uptake and labeling of motoneurons. Horseradish peroxidase, 30~,, w/v (type VI, Sigma, St. Louis) was pipetted onto the muscle and the skin was pulled together over the pool of the enzyme. The lampreys anesthetized with 10-3 g/ml Tricaine were left covered with ice, except for the wound, which was exposed to air, and after about one hour the animals were placed in chilled Ringer fluid for recovery from the anesthetic. After 3-8 days (or 4-6 days after double applications) at 15 °C, the lampreys were again anesthetized by immersion in Tricaine, and the brain was dissected in cold Ringer fluid and fixed for I 2 h at 4 °C in 2 °/o glutaraldehyde in 100 m M Na phosphate buffer pH 7. Reaction product was developed in 0.5 mg/ml 3,3'-diaminobenzidine and 3 × 10 ~ H202 in 50 glM Tris.maleate at pH 7.6 for 30 rain and the whole brain was dehydrated in graded ethanols and cleared for observation in methylbenzoate 6. RESULTS
Physiological identification of motoneurons in adults The trigeminal motor nucleus in adult lampreys is recognized as a prominent mound on the rostrolateral floor of the fourth ventricle. The cell bodies of motoneurons lie less than 100 #m from the surface, are easily impaled with microelectrodes, and can be distinguished from axons by action potential configuration, injury discharges and synaptic potentials. Motoneurons were usually identified by visible contractions in their motor units following intracellular stimulation with current pulses through the recording electrode. Both contractions in the motor unit and surface electromyographic potentials up to 20/~V followed intracellular stimulation one-to-one. In some cells extracellular stimulation in the region of the motor units produced antidromic action potentials, which had latencies of 4-10 msec, followed repetitive stimulation at least to 10/sec, and collided with action potentials generated in the cell bodyll, ~. Motoneurons innervating the annularis muscle of the sucker (A in Fig. 1A) were encountered most frequently, about 30-70 per preparation. Contractions in their motor units produced indentations in the dorsal surface of the ipsilateral sucker or movements of the underlying tissues in any of several directions. The cell bodies of these motoneurons were located in the mediocaudal portion of the trigeminal nucleus and are indicated by open circles in Fig. 1B, C and D. The basilaris muscle (B in Fig. l A) is large in adult lampreys, and only its medial and dorsal aspects were visible in the isolated preparation. Nevertheless, motor units in this region were commonly observed, about 15-28 per preparation. Basilar motoneurons were located in the lateral parts of Lhe trigeminal nucleus and are indicated by open squares in Fig. 1 B, C and D. Motoneurons to the muscles of the piston, for example, the styloapicalis, were not found so frequently but were located in the rostral portion of the nucleus (filled circles in Fig. 1B, C and D). Motoneurons to another piston muscle, the cardioapicalis, were also located rostrally (not illustrated). The velum is an important respiratory muscle of the larva 4, but it decreases in size during development of
36 bidirectional ventilation of the gills in adults. Velar motoneurons were the least common of the identified trigeminal motoneurons in adults and were located along the extreme lateral edge of the nucleus against the sulcus limitans (filled triangles, Fig. 1B, C and D), as in larvae 4. The same patterns of motoneurons were observed in adults of all three species examined: silver lampreys (3 preparations), least brook lampreys (2 preparations), and American brook lampreys (2 preparations).
Physiological identi#cation of motoneurons in larvae A simpler preparation could be used for the identification of trigeminal motoneurons in larvae (Fig. 1E) because the lateral parts of the head were more transparent than in adults and contractions of both superficial and deep muscles could lcm
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37 be o b s e r v e d . H o w e v e r , i n d i v i d u a l muscles w e r e m o r e difficult to d i s t i n g u i s h in larvae, a n d t h e i r n o m e n c l a t u r e has b e e n d i s p u t e d 2,7,t7. F o u r r e g i o n s were defined in the p r e s e n t w o r k : the a n t e r i o r b u c c a l m u s c l e s o f the oral h o o d , the superficial b u c c a l muscle, the v e n t r o l a t e r a l muscles, a n d v e l u m (Fig. 1E). T h e t r i g e m i n a l m o t o r nucleus is n o t so p r o m i n e n t in l a r v a e as in adults but consists o f a b a n d o f cells m e d i a l to the sulcus l i m i t a n s b e t w e e n the i s t h m i c r e g i o n r o s t r a l l y a n d the large M a u t h n e r cell caudally. T r i g e m i n a l m o t o n e u r o n s in l a r v a e are m u c h s m a l l e r t h a n in adults a n d were easily d a m a g e d , e v e n by high resistence electrodes. A c t i o n p o t e n t i a l s were usually p r o d u c e d as o f f - r e s p o n s e s f o l l o w i n g h y p e r p o l a r i z a t i o n s . 10-20/sec p r o d u c e d visible m o v e m e n t s in m o t o r
R e p e t i t i v e s t i m u l a t i o n at
units, w h e r e e l e c t r o m y o g r a p h i c
p o t e n t i a l s c o u l d also be r e c o r d e d . A n t i d r o m i c s t i m u l a t i o n p r o d u c e d s m a l l spikes, as p r e v i o u s l y o b s e r v e d in d a m a g e d velar m o t o n e u r o n s 4. In e a c h larval p r e p a r a t i o n it was usually possible to identify a b o u t 50 m o t o neurons
which,
when
stimulated
intracellularly,
elicited visible c o n t r a c t i o n s
in
ipsilateral muscles o f the head. T h e m o s t c o m m o n o f these, 2 0 - 3 0 p e r p r e p a r a t i o n , were m o t o n e u r o n s to the a n t e r i o r b u c c a l muscles o f the o r a l h o o d . In a c c o r d a n c e with the m e s h w o r k a r r a n g e m e n t o f m u s c l e fibers in this r e g i o n `), m o t o r units c o u l d p r o d u c e l o n g i t u d i n a l o r t r a n s v e r s e c o n t r a c t i o n s , d o r s o - v e n t r a l d i m p l i n g , o r s h o r t e n i n g o f the lip. M o t o n e u r o n s
to the a n t e r i o r buccal muscles were l o c a t e d in the m e d i o c a u d a l
Fig. 1. Patterns of physiologically identified motoneurons in the trigeminal nucleus. A : diagram of an isolated preparation of a small adult silver lamprey. The skin has been removed, and most of the cartilage and muscles have been dissected from the left side of the head. The preparation is tilted to the right to expose both the muscles along the ventral midline and the brain. Visible muscles include the annularis (A, ,~) of the sucker disk, the medial portion of the basilaris (B, []), the piston muscles including the styloapicalis (S, O), and the pigmented velum (V, A). After removal of the choroid plexus from the dorsal surface of the brain, the prominent trigeminal motor nucleus (Vm) is exposed for intracellular recordings in the rostral floor of the fourth ventricle. Other landmarks include the right eye (e), nostril (n), pineal body (p), otic capsule (oc), and gill sacs (g) which have been cut open. B : schematic diagram of the ,ight trigeminal motor nucleus in an adult least brook lamprey. The upper curved line indicates the sulcus limitans laterally and the caudal lip of the isthmic tectum rostrally (left). The lower straight line is the ventral midline. Motoneurons were identified by visible contractions in ipsilateral motor units after intracellular stimulation. Locations of motoneurons are indicated in B, C and D as follows: ~-5, annularis; [~, basilaris; O, styloapicalis; and A , velum. C: patterns of trigeminal motoneurons in an adult American brook lamprey. D: patterns of motoneurons in an adult silver lamprey. Note that in adults of all three species the motoneurons to the annularis muscle are located caudally and medially in the trigeminal nucleus, basilar motoneurons are located laterally, styloapicalis motoneurons are clustered rostrally, and the few velar motoneurons are found laterally and caudally. E: diagram of an isolated preparation of a larval least brook lamprey viewed dorsally. The skin was removed, the ventral midline slit, and the right side spread laterally. Muscles observed during transillumination include the anterior buccal (BA, •)) superficial buccal (BS, [Z]), various ventrolateral muscles (VL, O), the large velum (V, A) on the underside of the preparation, and myotomes (Myo) innervated by spinal roots. The right trigeminal motor nucleus (Vm) is located in the rostral floor of the fourth ventricle of the exposed brain. Other landmarks are the nostril (n), diminutive eye (e), otic capsule (oc), and the first external gill opening on the right side l . Same scale as A. F: pattern of physiologically identified motoneurons in the right trigeminal nucleus of a larval least brook lamprey. Same orientation and scale as B. The locations of motoneurons are indicated in F and G as follows: ~ , anterior buccal; 3 , superficial buccal; O, ventrolateral; and A , velum. G: pattern of motoneurons in a larval American brook lamprey. Note the similarities of locations of motoneurons in the two species of larvae and the homologies with adults.
38 p o r t i o n o f the trigeminal nucleus, as illustrated by open circles in Fig. 1F and G. M o t o n e u r o n s to the superficial buccal muscle, which extends d i a g o n a l l y from the skull to the p o s t e r i o r lip, were n o t c o m m o n but were located laterally in the nucleus (open squares in Fig. 1F a n d G). M o t o n e u r o n s to the various muscles o f the ventrolateral headZ, 17 were observed frequently, 10-30 per p r e p a r a t i o n , a n d were located in the rostral p a r t o f the trigeminal nucleus (filled circles in Fig. 1F a n d G). However, some o f these cells were also distributed laterally and caudally, reflecting the multiplicity o f v e n t r o l a t e r a l muscles. Finally, m o t o n e u r o n s to the velum, up to 12 per p r e p a r a t i o n , were l o c a t e d laterally a l o n g the sutcus limitans, as previously described ~. These p a t t e r n s o f m o t o n e u r o n s within the trigeminal nucleus were the same in larval least b r o o k l a m p r e y s (6 p r e p a r a t i o n s ) a n d in larval A m e r i c a n b r o o k l a m p r e y s (2 preparations). T w o least b r o o k l a m p r e y s in the middle o f m e t a m o r p h o s i s were also p r e p a r e d for identification o f trigeminal m o t o n e u r o n s . Their nerve cells were larger than in y o u n g e r larvae, as j u d g e d by less d a m a g e d u r i n g i m p a l e m e n t and larger action potentials. However, s t i m u l a t i o n o f most cells in the trigeminal nucleus did not p r o d u c e c o n t r a c t i o n s in the head. This was p a r t i c u l a r l y evident in the two regions which change m o s t d r a m a t i c a l l y d u r i n g m e t a m o r p h o s i s , the oral h o o d a n d the ventral muscles. The p r e p a r a t i o n s a p p e a r e d to be in g o o d condition, and m o t o n e u r o n s were identified in otherwise inactive areas: 21 m e d i o c a u d a l m o t o n e u r o n s to the oral hood, 4 c a u d a l m o t o n e u r o n s to the superficial buccal muscle, 5 rostral m o t o n e u r o n s to the ventral muscles, a n d 4 m o t o n e u r o n s near the sulcus limitans innervating the velum.
Retrograde marking of motoneurons with horseradish peroxidase SHRP / M o t o n e u r o n s in the trigeminal nucleus were labeled after a p p l i c a t i o n o f H R P to v a r i o u s muscles o f the h e a d (Fig. 2). The positions o f cells were c o n g r u e n t with the physiological m a p s j u s t described. In larval l a m p r e y s cell bodies in the medial p a r t o f the nucleus b e c a m e labeled after H R P was a p p l i e d to the tissues inside the ipsilateral oral h o o d (Fig. 2A). In 5 p r e p a r a t i o n s 20-40 such cells were m a r k e d . A f t e r H R P was injected into ventrolateral muscles next to the m o u t h in 4 larvae, nerve cells were
Fig. 2. Trigeminal motoneurons marked morphologically by retrograde transport of horseradish peroxidase (HRP) in larval American brook lampreys (A and B) and in adult silver lampreys (C and D). As in Fig. l, the upper curved lines indicate the sulcus limitans laterally and the caudal lip of the isthmic tectum rostrally (left). The lower straight line in C and D represents the midline. Cell bodies of motoneurons appear as dark spots about 10/~m in diameter in larvae and as larger dark profiles with processes in adults. Scales in A and B and in C and D are the same. A: motoneurons labeled by prior application of HRP to the ipsilateral anterior buccat region. Compare with open circles in Fig. 1F and G. B: two groups of motoneurons labeled after application of HRP to the velum (V) and the adjacent ventrolaterat muscles (VL) of the pharyngeal wall. Compare to filled circles in Fig. 1F and G. Some intrinsically HRP-positive cells of blood vessels on the ventral surface of the brain are faintly visible. C: motoneurons in the medial portion of the trigeminal nucleus labeled after application of HRP to the ipsilateral annularis muscles. Compare with open circles in Fig. 1B and C. The intense dark band above the motor nucleus is produced by labeling of sensory axons in the descending trigeminal tract. D: motoneurons in the lateral portion of the nucleus labeled after application of HRP to the lateral basilaris muscle on the same side. Compare to open squares in Fig. 1B and C.
39
40 labeled in the rostral portion of the trigeminal nucleus, and in one preparation a few additional cells were marked in the lateral-caudal portion. The velum was exposed through an incision through the lateral buccal wall, and application of HRP subsequently produced labelifig of as many as 70 cells along the sulcus limitans in 2 preparations. Since the ventrolateral muscles were also exposed to HRP, many nerve cells in the rostral portion of the nucleus were also labeled (Fig. 2B). All motoneurons labeled in larvae were small, about 10 #m in diameter, and lacked prominent dendritic or axonal processes. Trigeminal motoneurons of adult silver lampreys, in contrast to those of larvae, were large (20 × 30 #m-30 × 50/tm), had prominent dendrites, and their axons could be traced into the fifth nerve. Application of HRP to the annularis muscles of the sucker produced labeling of cells in the mediocaudal portion of the ipsilateral trigeminal nucleus (Fig. 2C); in 3 preparations 20-40 such cells were marked. In contrast to the medial region of the basilaris muscle where motor units were observed physiologically, HRP was applied to lateral aspect of the basilaris muscle. Nevertheless, motoneurons were labeled in the same lateral region of the ipsilateral trigeminal nucleus (Fig. 2D) in 2 preparations. In order to expose the basilaris muscle, the overlying subocularis muscle, a myotomal derivative, was also cut and exposed to HRP; presumed subocularis motoneurons were marked via ipsilateral ventral roots in the caudal end of the medulla below the central canaP,l~L In one adult the ventral midline was cut to expose the piston muscles to HRP. Subsequently, nerve cells were labeled in the rostral portions of the trigeminal nuclei on both sides; some cells located laterally were also marked, probably due to spread of H R P to the medial basilaris muscle. In both adults and larvae, sensory as well as motor pathways were labeled after peripheral applications of H RP. The one trigeminal ganglion which was dissected after injection of the sucker in an adult contained many heavily labeled ganglion cells. The descending tract of the trigeminal was prominently labeled in the lateral medulla and in the spinal cord up to 2 mm beyond the obex after ipsilateral applications of HRP. Axons of the tract were about 1-2 #m in diameter in larvae and 1-5 #m in adults and were associated with enlargements, perhaps varicosities or cell bodies of dorsal cells 9. DISCUSSION The muscles innervated by the trigeminal nerve are quite d~fferent in larval and in adult lampreys2,17. During metamorphosis, a period of a few months, larval muscles progressively degenerate, and at the same time primordia of adult muscles appear and gradually grow to fill the spaces of the enlarging head. For instance, in the oral hood, the anterior buccal muscle fibers of the larva are situated dorsally, and the annularis muscle of the adult forms below them. Similarly, separate primordia of the various piston muscles appear early in tissues ventral to the throat and pharynx. It ~s not known in detail when individual larval muscles lose their ability to contract and when adult muscles become functional. Perhaps in some regions neither group of muscles can be active at some stage, while in other regions larval and adult muscle may contract
41 at the same time. The only muscle innervated by the trigeminal nerve which does not disappear during metamorphosis is the velum, although it does become considerably smaller and is no longer so important for respiration. The three principal motor nerves of the trigeminal are recognizable in both larvae and in adults, although their sizes and detailed branching are different "/. The apical nerve innervates the anterior buccal muscles of the larva and the annularis muscle of adults. The basilaris nerve innervates the superficial buccal muscle of larvae and the large basilaris muscle of adults. Finally, the mandibular nerve innervates the velum and some of the ventral muscles in larvae and many of the piston muscles of adults. Thus, the motor nerves of the larval lamprey could simply change their connections to developing adult muscles during metamorphosis, although degeneration and regeneration of axons within the nerves are possible and have not been investigated. Trigeminal motoneurons could be altered in two different ways during metamorphosis. First, new populations of neurons might differentiate. This may occur in two other parts of the lamprey brain: reticulospinal neurons of the V group 10 and oculomotoneurons became more prominent in adults than in larvae. Although no examples of neuronal degeneration during metamorphosis of the lamprey have been reported, such would be expected by analogy with the frog, in which the Mauthner cell of the tadpole atrophies 12, reduction in cell number in lateral motor column occurs ~ and the entire spinal cord of the tail is resorbed at metamorphosis. Second, the peripheral and central synapses could be changed in accord with the structure and function of newly formed adult muscles. During metamorphosis of the tobacco hornworm a few motoneurons are lost, some new ones appear, but most are retained to innervate the new abdominal muscles of the adult moth ~a. Dendrites of one identified motoneuron change very markedly during metamorphosis, and the new dendritic branches presumably make synaptic connections appropriate for adult behavior 18. In the lamprey, identification of individual trigeminal motoneurons is probably not possible, but a similar retention of cells and a reorganization of their connections might be plausible. The present experiments indicate that specific groups of muscles of the head are innervated by motoneurons in particular regions of the trigeminal nucleus in larvae and in adults. Two larvae at mid-metamorphosis had similar patterns of innervation, but many nerve cells in the trigeminal nucleus did not produce obvious muscle contractions. Perhaps these cells might be in an intermediate stage between innervation of larval and adult muscles. ACKNOWLEDGEMENTS This work was supported by USPHS Grant NS 09367. The author expresses his sincere thanks to Dr. C. M. Rovainen for valuable suggestions and criticism throughout this work. American brook lampreys, larvae and adults, were kindly provided by Patrick Manion and Harry Moore of the U.S. Bureau of Sport Fisheries. Photomicrographic assistance was provided by William DePalma.
42 REFERENCES 1 Addens, J. L., The motor nuclei and roots of the cranial and first spinal nerves of vertebrates, Part 1. Introduction. Cyclostomes, Z. Anat. Emwiekl.-Gesch., 101 (1933) 307 410. 2 Damas, H., Contribution 5. 1'6tude de la m6tamorphose de la t6te de la lamproie, Arch. Biol., 46 (1935) 171 227. 3 Hardisty, M. W. and Potter, I. C., The general biology of adult lampreys. In M. W. Hardisty and I. C. Potter (Eds.), The Biology of Lampreys, Vol. I, Academic Press, London and New York, 1971, pp. 127-206. 4 Homma, S., Velar motoneurons of lamprey larvae, J. comp. Physiol., 104 (1975) 175 183. 5 Kollros, J. J., Order and control of neurogenesis (As exemplified by the lateral motor column), Develop. Biol., Suppl. 12 (1968) 274-305. 6 LaVail, J. H. and LaVail, M. M., Retrograde axonal transport in the CNS, ScieJwe, 176 (1972) 1416-1417. 7 Lindstr6m, T., On the cranial nerves of the cyclostomes with special reference to N. trigeminus, Acta Zool. (Stoekh.), 30 (1949) 315--458. 8 Marinelli, W. and Strenger, A., Vergleichende Anatomie und Morphologie der Wirbeltiere, Kla,s:s'e: Cyclostomata, Franz Deuticke, Wien, 1954, pp. 3-80. 9 Nieuwenhuys, R., Topological analysis of the brain stem of the lamprey, Lampetra fluvial#is, J. comp. Neurol., 145 (1972) 165-177. 10 Rovainen, C. M., Physiological and anatomical studies on large neurons of central nervous system of the sea lamprey (Petromyzon marinus). 1. M011er and Mauthner cells, J. Neurophysiok, 30 (1967) 1000-1023. 11 Rovainen, C. M., Respiratory motoneurons in lampreys, J. comp. Physiol., 94 (1974) 57-68. 12 Stefanelli, A., The Mauthnerian apparatus in the ichthyopsida; its nature and function and correlated problems of neurohistogenesis, Quart. Rev. Biol., 26 (1951) 17-34. 13 Taylor, H. M. and Truman, J. W., Metamorphosis of the abdominal ganglia of the tobacco hornworm, Manduca sexta, J. comp. Physiol., 90 (1974) 367-388. 14 Ter/ivainen, H. and Rovainen, C. M., Fast and slow motoneurons to body muscle of the sea lamprey, J. Neurophysiol., 34 (1971) 990-998. 15 Tretjakoff, D., Das Skelett und die Muskulatur im Kopfe des Flusneunauges, Z. Wiss. Zool., 128 (1926) 267-403. 16 Tretjakoff, D., Das periphere Nervensystem des Flusneunauges, Z. Wiss. Zool., 129 (1927) 359 452. 17 Tretjakoff, D., Die schleimknorpeligen Bestandteil im Kopfskelet yon Ammocoetes, Z. Wiss. Zool., 133 (1929) 470-516. 18 Truman, J. W. and Reiss, S. E., Dendritic reorganization of an identified motoneuron during metamorphosis of the tobacco hornworm moth, Science, 192 (1976) 477 479.
33
O R G A N I Z A T I O N OF THE T R I G E M I N A L MOTOR NUCLEUS BEFORE A N D A F T E R METAMORPHOSIS IN LAMPREYS
SHINJI HOMMA*
Department of Physiology and Biophysics, Washington University School of Medichw, St. Louis, Mo., 63110 (U.S.A.) (Accepted May 4th, 1977)
SUMMARY
During metamorphosis of the lamprey the simple muscles of the mouth of the larvae are replaced with the elaborate muscles of the sucker, rasper and pharynx in adults. The purposes of the present experiments were the identification of motoneurons to mouth muscles and the determination of their patterns in the trigeminal nucleus before and after metamorphosis. (1) M otoneurons were identified physiologically by observation of contractions in motor units following intracellular stimulation and by antidromic stimulation. Locations of motoneurons were also determined morphologically by retrograde transport of horseradish peroxidase. (2) The trigeminal motor nucleus was subdivided into regions containing motoneurons to specific groups of ipsilateral muscles both in larvae and in adults. The medial-caudal portion of the nucleus contained motoneurons innervating muscles of the oral hood in larvae and the annularis muscles of the sucker in adults. The simple ventral muscles of the larva were replaced by the elaborate muscles of the rasper and piston of adults, yet motoneurons were located in rostral portion of the nucleus in both stages. Likewise, the dissimilar muscles innervated through the basilar nerve had motoneurons in the lateral part of the nucleus in larvae and adults. The velum becomes much smaller during metamorphosis, but motoneurons innervating it in larvae and adults had the same lateral location. (3) It is concluded that the pattern of cells in the trigeminal motor nucleus is not changed during metamorphosis of the lamprey. Perhaps the same motoneurons make new peripheral connections during and after metamolphosis, or new neurons form in the same general regions of the trigeminal nucleus and innervate the new muscles forming in the corresponding regions.
* Present Adress: Department of Physiology, Faculty of Medicine Toyama Medical and Pharmaceutical University Toyama City 930, Japan,
34 INTRODUCTION In the lamprey many anatomical, physiological and behavioral changes occur during metamorphosis3. One of the most pronounced is the change from filter feeding by larvae to parasitic feeding on fish by adults with corresponding changes in the structure and function of the mouth. Although brook lampreys are not parasitic~ similar changes occur in the mouth, which is used during nest-building and spawning. The gross anatomy of the mouth muscles in larvae and adults is well known2,8, x.~ 17 and the morphological changes in these muscles during metamorphosis has been describedZ,16,17. The trigeminal nerve innervates most of the mouth muscles of the lamprey7,16,t7, but aside from the marked enlargement of trigeminal nerve motoneurons, nothing is known about the changes of these cells during metamorphosis. In the present experiments, patterns of innervation of mouth muscles by trigeminal motoneurons are determined in larval lampreys and in parasitic and non-parasitic adults. METHODS Adult small silver lampreys (10-12 cm) lchthyomyzon unicuspis, larval and adult least brook lampreys (9-13 cm) Lampetra aepyptera and larval and adult American brook lampreys (12-15 cm) Lampetra lamottei were maintained in aerated fresh water at 4 °C. They were initially anesthetized by immersion in cold water with t0-4-10 -3 g/ml Tricaine (Ayerst) and dissected in cold physiological solution. Larvae were cut transversely at the gill region and prepared by slitting the ventral midline. Mouth, pharynx and gill walls of one or both sides were extended laterally and fixed on the bottom of a chamber with minutien pins (Fig. 1E). In case of adults, one side of the head was slit laterally and trimmed to expose dorsomedially the piston muscles, basilar muscles and velum (Fig. 1A). The brain was exposed dorsally in both larval and adult lampreys. Preparations were superfused with fluid of the following compositions: 115 mM NaC1, 2 mM KCI, 2.6 mM CaCI2, 2 mM MgCI2, 6 mM NaHCO3 or 4 mM Tris.maleate buffer at pH 7.6 and 3 mM glucose, equilibrated with 2 ~o COz-98 o~ O2 at 7-12 °C. Glass microelectrodes were filled with 3 M potassium acetate by boiling under vacuum and were selected for resistences over 50 Mr2 for larvae. Current was passed through recording electrodes with M4 A electrometers (WPI Instrument, Hamden, Conn.). Positions of the tip of the glass microelectrodes were determined through micrometers in the ocular of the stereoscopic microscope. In preparing maps of the trigeminal nucleus, only the first cell which produced visible contractions when stimulated at 10-20/sec was plotted. Electromyographic activities were monitored with a chlorided silver bipolar electrode placed over the muscles and with a Grass p t5 AC amplifier. The bipolar electrode was also used for stimulation with pulses of 0.2 msec and below 10 V. Cell bodies of trigeminal motoneurons were marked by retrograde labeling after application of horseradish peroxidase to target muscles of the head. For all operations
35 the skin and overlying tissues were cut, and the exposed muscle was damaged mechanically with forceps, since this improved the subsequent uptake and labeling of motoneurons. Horseradish peroxidase, 30~,, w/v (type VI, Sigma, St. Louis) was pipetted onto the muscle and the skin was pulled together over the pool of the enzyme. The lampreys anesthetized with 10-3 g/ml Tricaine were left covered with ice, except for the wound, which was exposed to air, and after about one hour the animals were placed in chilled Ringer fluid for recovery from the anesthetic. After 3-8 days (or 4-6 days after double applications) at 15 °C, the lampreys were again anesthetized by immersion in Tricaine, and the brain was dissected in cold Ringer fluid and fixed for I 2 h at 4 °C in 2 °/o glutaraldehyde in 100 m M Na phosphate buffer pH 7. Reaction product was developed in 0.5 mg/ml 3,3'-diaminobenzidine and 3 × 10 ~ H202 in 50 glM Tris.maleate at pH 7.6 for 30 rain and the whole brain was dehydrated in graded ethanols and cleared for observation in methylbenzoate 6. RESULTS
Physiological identification of motoneurons in adults The trigeminal motor nucleus in adult lampreys is recognized as a prominent mound on the rostrolateral floor of the fourth ventricle. The cell bodies of motoneurons lie less than 100 #m from the surface, are easily impaled with microelectrodes, and can be distinguished from axons by action potential configuration, injury discharges and synaptic potentials. Motoneurons were usually identified by visible contractions in their motor units following intracellular stimulation with current pulses through the recording electrode. Both contractions in the motor unit and surface electromyographic potentials up to 20/~V followed intracellular stimulation one-to-one. In some cells extracellular stimulation in the region of the motor units produced antidromic action potentials, which had latencies of 4-10 msec, followed repetitive stimulation at least to 10/sec, and collided with action potentials generated in the cell bodyll, ~. Motoneurons innervating the annularis muscle of the sucker (A in Fig. 1A) were encountered most frequently, about 30-70 per preparation. Contractions in their motor units produced indentations in the dorsal surface of the ipsilateral sucker or movements of the underlying tissues in any of several directions. The cell bodies of these motoneurons were located in the mediocaudal portion of the trigeminal nucleus and are indicated by open circles in Fig. 1B, C and D. The basilaris muscle (B in Fig. l A) is large in adult lampreys, and only its medial and dorsal aspects were visible in the isolated preparation. Nevertheless, motor units in this region were commonly observed, about 15-28 per preparation. Basilar motoneurons were located in the lateral parts of Lhe trigeminal nucleus and are indicated by open squares in Fig. 1 B, C and D. Motoneurons to the muscles of the piston, for example, the styloapicalis, were not found so frequently but were located in the rostral portion of the nucleus (filled circles in Fig. 1B, C and D). Motoneurons to another piston muscle, the cardioapicalis, were also located rostrally (not illustrated). The velum is an important respiratory muscle of the larva 4, but it decreases in size during development of
36 bidirectional ventilation of the gills in adults. Velar motoneurons were the least common of the identified trigeminal motoneurons in adults and were located along the extreme lateral edge of the nucleus against the sulcus limitans (filled triangles, Fig. 1B, C and D), as in larvae 4. The same patterns of motoneurons were observed in adults of all three species examined: silver lampreys (3 preparations), least brook lampreys (2 preparations), and American brook lampreys (2 preparations).
Physiological identi#cation of motoneurons in larvae A simpler preparation could be used for the identification of trigeminal motoneurons in larvae (Fig. 1E) because the lateral parts of the head were more transparent than in adults and contractions of both superficial and deep muscles could lcm
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37 be o b s e r v e d . H o w e v e r , i n d i v i d u a l muscles w e r e m o r e difficult to d i s t i n g u i s h in larvae, a n d t h e i r n o m e n c l a t u r e has b e e n d i s p u t e d 2,7,t7. F o u r r e g i o n s were defined in the p r e s e n t w o r k : the a n t e r i o r b u c c a l m u s c l e s o f the oral h o o d , the superficial b u c c a l muscle, the v e n t r o l a t e r a l muscles, a n d v e l u m (Fig. 1E). T h e t r i g e m i n a l m o t o r nucleus is n o t so p r o m i n e n t in l a r v a e as in adults but consists o f a b a n d o f cells m e d i a l to the sulcus l i m i t a n s b e t w e e n the i s t h m i c r e g i o n r o s t r a l l y a n d the large M a u t h n e r cell caudally. T r i g e m i n a l m o t o n e u r o n s in l a r v a e are m u c h s m a l l e r t h a n in adults a n d were easily d a m a g e d , e v e n by high resistence electrodes. A c t i o n p o t e n t i a l s were usually p r o d u c e d as o f f - r e s p o n s e s f o l l o w i n g h y p e r p o l a r i z a t i o n s . 10-20/sec p r o d u c e d visible m o v e m e n t s in m o t o r
R e p e t i t i v e s t i m u l a t i o n at
units, w h e r e e l e c t r o m y o g r a p h i c
p o t e n t i a l s c o u l d also be r e c o r d e d . A n t i d r o m i c s t i m u l a t i o n p r o d u c e d s m a l l spikes, as p r e v i o u s l y o b s e r v e d in d a m a g e d velar m o t o n e u r o n s 4. In e a c h larval p r e p a r a t i o n it was usually possible to identify a b o u t 50 m o t o neurons
which,
when
stimulated
intracellularly,
elicited visible c o n t r a c t i o n s
in
ipsilateral muscles o f the head. T h e m o s t c o m m o n o f these, 2 0 - 3 0 p e r p r e p a r a t i o n , were m o t o n e u r o n s to the a n t e r i o r b u c c a l muscles o f the o r a l h o o d . In a c c o r d a n c e with the m e s h w o r k a r r a n g e m e n t o f m u s c l e fibers in this r e g i o n `), m o t o r units c o u l d p r o d u c e l o n g i t u d i n a l o r t r a n s v e r s e c o n t r a c t i o n s , d o r s o - v e n t r a l d i m p l i n g , o r s h o r t e n i n g o f the lip. M o t o n e u r o n s
to the a n t e r i o r buccal muscles were l o c a t e d in the m e d i o c a u d a l
Fig. 1. Patterns of physiologically identified motoneurons in the trigeminal nucleus. A : diagram of an isolated preparation of a small adult silver lamprey. The skin has been removed, and most of the cartilage and muscles have been dissected from the left side of the head. The preparation is tilted to the right to expose both the muscles along the ventral midline and the brain. Visible muscles include the annularis (A, ,~) of the sucker disk, the medial portion of the basilaris (B, []), the piston muscles including the styloapicalis (S, O), and the pigmented velum (V, A). After removal of the choroid plexus from the dorsal surface of the brain, the prominent trigeminal motor nucleus (Vm) is exposed for intracellular recordings in the rostral floor of the fourth ventricle. Other landmarks include the right eye (e), nostril (n), pineal body (p), otic capsule (oc), and gill sacs (g) which have been cut open. B : schematic diagram of the ,ight trigeminal motor nucleus in an adult least brook lamprey. The upper curved line indicates the sulcus limitans laterally and the caudal lip of the isthmic tectum rostrally (left). The lower straight line is the ventral midline. Motoneurons were identified by visible contractions in ipsilateral motor units after intracellular stimulation. Locations of motoneurons are indicated in B, C and D as follows: ~-5, annularis; [~, basilaris; O, styloapicalis; and A , velum. C: patterns of trigeminal motoneurons in an adult American brook lamprey. D: patterns of motoneurons in an adult silver lamprey. Note that in adults of all three species the motoneurons to the annularis muscle are located caudally and medially in the trigeminal nucleus, basilar motoneurons are located laterally, styloapicalis motoneurons are clustered rostrally, and the few velar motoneurons are found laterally and caudally. E: diagram of an isolated preparation of a larval least brook lamprey viewed dorsally. The skin was removed, the ventral midline slit, and the right side spread laterally. Muscles observed during transillumination include the anterior buccal (BA, •)) superficial buccal (BS, [Z]), various ventrolateral muscles (VL, O), the large velum (V, A) on the underside of the preparation, and myotomes (Myo) innervated by spinal roots. The right trigeminal motor nucleus (Vm) is located in the rostral floor of the fourth ventricle of the exposed brain. Other landmarks are the nostril (n), diminutive eye (e), otic capsule (oc), and the first external gill opening on the right side l . Same scale as A. F: pattern of physiologically identified motoneurons in the right trigeminal nucleus of a larval least brook lamprey. Same orientation and scale as B. The locations of motoneurons are indicated in F and G as follows: ~ , anterior buccal; 3 , superficial buccal; O, ventrolateral; and A , velum. G: pattern of motoneurons in a larval American brook lamprey. Note the similarities of locations of motoneurons in the two species of larvae and the homologies with adults.
38 p o r t i o n o f the trigeminal nucleus, as illustrated by open circles in Fig. 1F and G. M o t o n e u r o n s to the superficial buccal muscle, which extends d i a g o n a l l y from the skull to the p o s t e r i o r lip, were n o t c o m m o n but were located laterally in the nucleus (open squares in Fig. 1F a n d G). M o t o n e u r o n s to the various muscles o f the ventrolateral headZ, 17 were observed frequently, 10-30 per p r e p a r a t i o n , a n d were located in the rostral p a r t o f the trigeminal nucleus (filled circles in Fig. 1F a n d G). However, some o f these cells were also distributed laterally and caudally, reflecting the multiplicity o f v e n t r o l a t e r a l muscles. Finally, m o t o n e u r o n s to the velum, up to 12 per p r e p a r a t i o n , were l o c a t e d laterally a l o n g the sutcus limitans, as previously described ~. These p a t t e r n s o f m o t o n e u r o n s within the trigeminal nucleus were the same in larval least b r o o k l a m p r e y s (6 p r e p a r a t i o n s ) a n d in larval A m e r i c a n b r o o k l a m p r e y s (2 preparations). T w o least b r o o k l a m p r e y s in the middle o f m e t a m o r p h o s i s were also p r e p a r e d for identification o f trigeminal m o t o n e u r o n s . Their nerve cells were larger than in y o u n g e r larvae, as j u d g e d by less d a m a g e d u r i n g i m p a l e m e n t and larger action potentials. However, s t i m u l a t i o n o f most cells in the trigeminal nucleus did not p r o d u c e c o n t r a c t i o n s in the head. This was p a r t i c u l a r l y evident in the two regions which change m o s t d r a m a t i c a l l y d u r i n g m e t a m o r p h o s i s , the oral h o o d a n d the ventral muscles. The p r e p a r a t i o n s a p p e a r e d to be in g o o d condition, and m o t o n e u r o n s were identified in otherwise inactive areas: 21 m e d i o c a u d a l m o t o n e u r o n s to the oral hood, 4 c a u d a l m o t o n e u r o n s to the superficial buccal muscle, 5 rostral m o t o n e u r o n s to the ventral muscles, a n d 4 m o t o n e u r o n s near the sulcus limitans innervating the velum.
Retrograde marking of motoneurons with horseradish peroxidase SHRP / M o t o n e u r o n s in the trigeminal nucleus were labeled after a p p l i c a t i o n o f H R P to v a r i o u s muscles o f the h e a d (Fig. 2). The positions o f cells were c o n g r u e n t with the physiological m a p s j u s t described. In larval l a m p r e y s cell bodies in the medial p a r t o f the nucleus b e c a m e labeled after H R P was a p p l i e d to the tissues inside the ipsilateral oral h o o d (Fig. 2A). In 5 p r e p a r a t i o n s 20-40 such cells were m a r k e d . A f t e r H R P was injected into ventrolateral muscles next to the m o u t h in 4 larvae, nerve cells were
Fig. 2. Trigeminal motoneurons marked morphologically by retrograde transport of horseradish peroxidase (HRP) in larval American brook lampreys (A and B) and in adult silver lampreys (C and D). As in Fig. l, the upper curved lines indicate the sulcus limitans laterally and the caudal lip of the isthmic tectum rostrally (left). The lower straight line in C and D represents the midline. Cell bodies of motoneurons appear as dark spots about 10/~m in diameter in larvae and as larger dark profiles with processes in adults. Scales in A and B and in C and D are the same. A: motoneurons labeled by prior application of HRP to the ipsilateral anterior buccat region. Compare with open circles in Fig. 1F and G. B: two groups of motoneurons labeled after application of HRP to the velum (V) and the adjacent ventrolaterat muscles (VL) of the pharyngeal wall. Compare to filled circles in Fig. 1F and G. Some intrinsically HRP-positive cells of blood vessels on the ventral surface of the brain are faintly visible. C: motoneurons in the medial portion of the trigeminal nucleus labeled after application of HRP to the ipsilateral annularis muscles. Compare with open circles in Fig. 1B and C. The intense dark band above the motor nucleus is produced by labeling of sensory axons in the descending trigeminal tract. D: motoneurons in the lateral portion of the nucleus labeled after application of HRP to the lateral basilaris muscle on the same side. Compare to open squares in Fig. 1B and C.
39
40 labeled in the rostral portion of the trigeminal nucleus, and in one preparation a few additional cells were marked in the lateral-caudal portion. The velum was exposed through an incision through the lateral buccal wall, and application of HRP subsequently produced labelifig of as many as 70 cells along the sulcus limitans in 2 preparations. Since the ventrolateral muscles were also exposed to HRP, many nerve cells in the rostral portion of the nucleus were also labeled (Fig. 2B). All motoneurons labeled in larvae were small, about 10 #m in diameter, and lacked prominent dendritic or axonal processes. Trigeminal motoneurons of adult silver lampreys, in contrast to those of larvae, were large (20 × 30 #m-30 × 50/tm), had prominent dendrites, and their axons could be traced into the fifth nerve. Application of HRP to the annularis muscles of the sucker produced labeling of cells in the mediocaudal portion of the ipsilateral trigeminal nucleus (Fig. 2C); in 3 preparations 20-40 such cells were marked. In contrast to the medial region of the basilaris muscle where motor units were observed physiologically, HRP was applied to lateral aspect of the basilaris muscle. Nevertheless, motoneurons were labeled in the same lateral region of the ipsilateral trigeminal nucleus (Fig. 2D) in 2 preparations. In order to expose the basilaris muscle, the overlying subocularis muscle, a myotomal derivative, was also cut and exposed to HRP; presumed subocularis motoneurons were marked via ipsilateral ventral roots in the caudal end of the medulla below the central canaP,l~L In one adult the ventral midline was cut to expose the piston muscles to HRP. Subsequently, nerve cells were labeled in the rostral portions of the trigeminal nuclei on both sides; some cells located laterally were also marked, probably due to spread of H R P to the medial basilaris muscle. In both adults and larvae, sensory as well as motor pathways were labeled after peripheral applications of H RP. The one trigeminal ganglion which was dissected after injection of the sucker in an adult contained many heavily labeled ganglion cells. The descending tract of the trigeminal was prominently labeled in the lateral medulla and in the spinal cord up to 2 mm beyond the obex after ipsilateral applications of HRP. Axons of the tract were about 1-2 #m in diameter in larvae and 1-5 #m in adults and were associated with enlargements, perhaps varicosities or cell bodies of dorsal cells 9. DISCUSSION The muscles innervated by the trigeminal nerve are quite d~fferent in larval and in adult lampreys2,17. During metamorphosis, a period of a few months, larval muscles progressively degenerate, and at the same time primordia of adult muscles appear and gradually grow to fill the spaces of the enlarging head. For instance, in the oral hood, the anterior buccal muscle fibers of the larva are situated dorsally, and the annularis muscle of the adult forms below them. Similarly, separate primordia of the various piston muscles appear early in tissues ventral to the throat and pharynx. It ~s not known in detail when individual larval muscles lose their ability to contract and when adult muscles become functional. Perhaps in some regions neither group of muscles can be active at some stage, while in other regions larval and adult muscle may contract
41 at the same time. The only muscle innervated by the trigeminal nerve which does not disappear during metamorphosis is the velum, although it does become considerably smaller and is no longer so important for respiration. The three principal motor nerves of the trigeminal are recognizable in both larvae and in adults, although their sizes and detailed branching are different "/. The apical nerve innervates the anterior buccal muscles of the larva and the annularis muscle of adults. The basilaris nerve innervates the superficial buccal muscle of larvae and the large basilaris muscle of adults. Finally, the mandibular nerve innervates the velum and some of the ventral muscles in larvae and many of the piston muscles of adults. Thus, the motor nerves of the larval lamprey could simply change their connections to developing adult muscles during metamorphosis, although degeneration and regeneration of axons within the nerves are possible and have not been investigated. Trigeminal motoneurons could be altered in two different ways during metamorphosis. First, new populations of neurons might differentiate. This may occur in two other parts of the lamprey brain: reticulospinal neurons of the V group 10 and oculomotoneurons became more prominent in adults than in larvae. Although no examples of neuronal degeneration during metamorphosis of the lamprey have been reported, such would be expected by analogy with the frog, in which the Mauthner cell of the tadpole atrophies 12, reduction in cell number in lateral motor column occurs ~ and the entire spinal cord of the tail is resorbed at metamorphosis. Second, the peripheral and central synapses could be changed in accord with the structure and function of newly formed adult muscles. During metamorphosis of the tobacco hornworm a few motoneurons are lost, some new ones appear, but most are retained to innervate the new abdominal muscles of the adult moth ~a. Dendrites of one identified motoneuron change very markedly during metamorphosis, and the new dendritic branches presumably make synaptic connections appropriate for adult behavior 18. In the lamprey, identification of individual trigeminal motoneurons is probably not possible, but a similar retention of cells and a reorganization of their connections might be plausible. The present experiments indicate that specific groups of muscles of the head are innervated by motoneurons in particular regions of the trigeminal nucleus in larvae and in adults. Two larvae at mid-metamorphosis had similar patterns of innervation, but many nerve cells in the trigeminal nucleus did not produce obvious muscle contractions. Perhaps these cells might be in an intermediate stage between innervation of larval and adult muscles. ACKNOWLEDGEMENTS This work was supported by USPHS Grant NS 09367. The author expresses his sincere thanks to Dr. C. M. Rovainen for valuable suggestions and criticism throughout this work. American brook lampreys, larvae and adults, were kindly provided by Patrick Manion and Harry Moore of the U.S. Bureau of Sport Fisheries. Photomicrographic assistance was provided by William DePalma.
42 REFERENCES 1 Addens, J. L., The motor nuclei and roots of the cranial and first spinal nerves of vertebrates, Part 1. Introduction. Cyclostomes, Z. Anat. Emwiekl.-Gesch., 101 (1933) 307 410. 2 Damas, H., Contribution 5. 1'6tude de la m6tamorphose de la t6te de la lamproie, Arch. Biol., 46 (1935) 171 227. 3 Hardisty, M. W. and Potter, I. C., The general biology of adult lampreys. In M. W. Hardisty and I. C. Potter (Eds.), The Biology of Lampreys, Vol. I, Academic Press, London and New York, 1971, pp. 127-206. 4 Homma, S., Velar motoneurons of lamprey larvae, J. comp. Physiol., 104 (1975) 175 183. 5 Kollros, J. J., Order and control of neurogenesis (As exemplified by the lateral motor column), Develop. Biol., Suppl. 12 (1968) 274-305. 6 LaVail, J. H. and LaVail, M. M., Retrograde axonal transport in the CNS, ScieJwe, 176 (1972) 1416-1417. 7 Lindstr6m, T., On the cranial nerves of the cyclostomes with special reference to N. trigeminus, Acta Zool. (Stoekh.), 30 (1949) 315--458. 8 Marinelli, W. and Strenger, A., Vergleichende Anatomie und Morphologie der Wirbeltiere, Kla,s:s'e: Cyclostomata, Franz Deuticke, Wien, 1954, pp. 3-80. 9 Nieuwenhuys, R., Topological analysis of the brain stem of the lamprey, Lampetra fluvial#is, J. comp. Neurol., 145 (1972) 165-177. 10 Rovainen, C. M., Physiological and anatomical studies on large neurons of central nervous system of the sea lamprey (Petromyzon marinus). 1. M011er and Mauthner cells, J. Neurophysiok, 30 (1967) 1000-1023. 11 Rovainen, C. M., Respiratory motoneurons in lampreys, J. comp. Physiol., 94 (1974) 57-68. 12 Stefanelli, A., The Mauthnerian apparatus in the ichthyopsida; its nature and function and correlated problems of neurohistogenesis, Quart. Rev. Biol., 26 (1951) 17-34. 13 Taylor, H. M. and Truman, J. W., Metamorphosis of the abdominal ganglia of the tobacco hornworm, Manduca sexta, J. comp. Physiol., 90 (1974) 367-388. 14 Ter/ivainen, H. and Rovainen, C. M., Fast and slow motoneurons to body muscle of the sea lamprey, J. Neurophysiol., 34 (1971) 990-998. 15 Tretjakoff, D., Das Skelett und die Muskulatur im Kopfe des Flusneunauges, Z. Wiss. Zool., 128 (1926) 267-403. 16 Tretjakoff, D., Das periphere Nervensystem des Flusneunauges, Z. Wiss. Zool., 129 (1927) 359 452. 17 Tretjakoff, D., Die schleimknorpeligen Bestandteil im Kopfskelet yon Ammocoetes, Z. Wiss. Zool., 133 (1929) 470-516. 18 Truman, J. W. and Reiss, S. E., Dendritic reorganization of an identified motoneuron during metamorphosis of the tobacco hornworm moth, Science, 192 (1976) 477 479.
