THE JOURNAL OF COMPARATIVE NEUROLOGY 321:112-123 (1992)
Two Types of Motoneurons Supplying Dorsal Fin Muscles in Lamprey and Their Activity During Fictive Locomotion OLEG SHUPLIAKOV, PETER WALLEN, AND STEN GRILLNER Nobel Institute for Neurophysiology ( 0 3 ,P.W., S.G.) and Department of Anatomy (O.S.), Karolinska Institute, S-104 0 1 Stockholm, Sweden
ABSTRACT The location and dendritic morphology of motoneurons supplying the dorsal fin muscles were studied in the lamprey spinal cord (Ichthyomyzon unicuspis). Motoneurons were retrogradely labelled after injection of HRP into the fin muscles or after its application on the cut ends of the ventral roots. HRP-labelled cells were subsequently reconstructed, in the horizontal and/or transverse planes. Fin motoneurons were also injected intracellularly with Lucifer Yellow and their detailed three-dimensional structure was analysed by confocal laser-scanning microscopy. Unlike myotomal motoneurons, which are closely spaced in the lateral cell column, fin motoneurons were distributed along the spinal cord separately or in pairs. They could be distinguished from motoneurons supplying trunk muscles by having a limited number of dendrites in the lateral part of the spinal cord. In addition, some fin motoneurons extend their dendrites into the dorsal column. The motor cells innervating fin muscles were divided into two types based on their dendritic morphology. Type I have a widespread dendritic tree in the rostrocaudal direction and, with few exceptions, completely restricted to the ipsilateral side. A proportion (25%) of these cells have dendrites extending into the dorsal column. Type I1 fin motoneurons extend their dendrites both ipsi- and contra-laterally. The contra-lateral dendrites pass below and above the central canal. The dendrites send off branches into the dorsal columns on both the ipsi- and the contra-lateral sides. Electron microscopic analysis showed that both type I and type I1 fin motoneurons receive numerous synaptic contacts from dorsal column axons. During fictive locomotion both types of motoneurons are active in antiphase in relation to myotomal motoneurons and to the main locomotor burst. D 1992 Wiley-Liss, Inc. Key words: motoneuron,dendritic morphology, sensory-motorconnections, spinal cord, fin
There are two main groups of motor cells which are involved in the generation of locomotor behaviour in lampreys: motoneurons innervating muscles of the body wall and motoneurons supplying fin muscles (Tretjakoff, '09; Birnberger and Rovainen, '71; Teravainen and Rovainen, '71a). During swimming, myotomal motoneurons receive alternating excitatory and inhibitory synaptic input from the central pattern generator (Russell and Wallen, '83). The swimming motor pattern is characterized by alternating contractions of myotomal motor units on the opposite sides of one segment and a delayed activation of more caudal segments, resulting in an undulatory wave travelling down the body (Williams et al., '89). The detailed morphology of different types of myotomal motoneurons, their synaptic inputs and activity during fictive locomotion, have been characterized in the in vitro preparation of the lamprey spinal cord (Teraviiinen and Rovainen, '71a; W a l l h et al., '85). In contrast, our knowledge about the morphology and the synaptic control of fin motoneurons is limited to a few O
1992 WILEY-LISS, INC.
studies (Tretjakoff, '09; Rovainen, '67; Birnberger and Rovainen, '71; Buchanan and Cohen, '82; Rovainen and Dill, '84). This is a serious gap since fin motoneurons play an important role in the compensatory adjustments necessary for the control of equilibrium. The present study describes the detailed morphology of motoneurons supplying the dorsal fin muscles and we suggest a classification of these into two distinct types. The pattern of activity of these two types of fin motoneurons during fictive locomotion is also described.
MATERIALS AND METHODS Experiments were performed on 19 adult silver lampreys (Ichthyonyzon unicuspis) obtained from Iowa. Before dissection the animals were anesthetized with MS-222 (tricaine methane sulphonate, Sandoz, 100 mg/l water). Accepted February 6,1992.
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Fig. 1. Fin motoneurons retrogradely labelled with HRP. A Fin motoneurons (mn) labelled after HRP injection into the musculature of the caudal portion of the dorsal fin. VR1 indicates the position of the ventral root (vr) containing HRP-labelled motoneuron axons, and VR2 the position of the neighbouring ventral root. r,c, Rostra1 and caudal direction, respectively. B: High-power micrograph of the area corresponding to the rectangle in A. af, Primary aEerents labelled after injection of HRP into the musculature on the ipsi- and contra-lateral
sides of the fin. dc, Dorsal cell. ax,Axons of fin motoneurons. Interrupted line indicates spinal cord midline. C: Type I fin motoneurons (mn) innervating the rostral portion of the dorsal fin. ax, Labelled axons of fin motoneurons. D,E: Type I1 fin motoneurons (mn) labelled after injection of HRP into the fin musculature at the level of the rostral division (D) and after HRP application on the cut end of the ventral root (E). d, Indicates dendrites crossing the midline above the central canal. Scale bars: A, 100 pm; E, 100 K r n (also applies to E D ) .
Fin motoneurons were labelled using three different procedures:
Their separation from the trunk musculature allows selective injections of horseradish peroxidase (HRP) into the fin muscles without spread of theknzyme to the trunk muscles. HRP (Sigma Chemical Co., St. Louis, MO, type VI) in physiological solution (20% HRP) was injected unilaterally or, in two cases bilaterally, into the muscles of the dorsal fin. The animals were left to survive for 7 days after the injection. The part of the spinal cord containing retro-
Injection of horseradish peroxidase into fin muscles In the adult lamprey the dorsal fin muscles are represented by several layers of fibers surrounding the cartilaginous skeleton of the fin (Rovainen and Birnberger, '71).
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TYPES OF FIN MOTONEURONS gradely labelled motoneurons was then dissected out and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The HRP was visualized according to standard Graham-Karnovsky or Hanker-Yates protocols (Graham and Karnovsky, '66; Hanker and Yates, '82 ). The preparations were cleared in methylsalicylate, mounted as whole mounts, and photographed. Some of them were then embedded in paraffin, sectioned in 20 pm sections, and mounted on glass slides. The labelled motoneurons were reconstructed with the aid of a camera lucida. For electron microscopy four pieces of the spinal cord, each containing an HRP-labelled fin motoneuron, were fixed in 3% glutaraldehyde (4 hours), osmicated, dehydrated in acetone, and embedded in Vestopal-W. After curing, regions with labelled neurons were cut in serial transverse 7 pm thick sections with a dry glass knife on a pyramitome. Sections were then immersed in glycerol, mounted on glass slides, and examined under the light microscope. Selected sections were remounted on newly faced blank Vestopal blocks. They were then resectioned on an LKB Ultratome with a diamond knife. Ultrathin sections were collected on formvar-coated slot grids, doublestained in uranyl acetate and lead citrate, and examined with a Philips 201 electron microscope.
Application of HRP on the cut ventral roots Isolated spinal cord preparations (11segments long) were used in these experiments. They were pinned down with the ventral side up in a Sylgard-lined dish. Care was taken to fix the spinal cord a t the same length it had in situ. The preparations were perfused with cooled (7-9°C) physiological solution of the following composition (in mM): NaC1, 91; KCl, 2.1; CaC12, 2.6; glucose, 4; NaHC03, 20. The solution was bubbled with 95% 0 2 + 5%COZ to pH 7.4. The cut ends of the ventral roots were drawn into suction electrodes filled with 10% HRP in physiological solution (Shupliakov, '85). Depolarizing current pulses of 2-10 nA and of 80-100 ms duration were applied for a period of 1 hour. After a period of 24 hours the preparations were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer and then processed for light- andlor EM- analysis as decribed above. Cells were identified as fin motoneurons on the basis of criteria elaborated in the first group of experiments.
Intracellular recording and labelling of fin motoneurons Intracellular labelling of motoneurons was performed in the isolated spinal cord-notochord preparation mounted in
Fig. 2. Close appositions between HRP-labelled fin motoneuron dendrites and dorsal column and reticulospinal axons. A: Light micrograph of transverse section through the lamprey spinal cord illustrating location of dendrites of a type I fin motoneuron labelled retrogradely with HRP. B-D: High-power micrographs of the areas correspondingto squares in A. B,C: Close appositions (someof them indicated by arrows) between dorsal column axons and the dendrites (d) of the motoneuron. D: Close appositions (some are indicated by arrows) between dendrites of the fin motoneuron and reticulospinal axons (rs). E. Light micrograph of transverse section from the same segment of the spinal cord containing dendrites of a type I1 fin motoneuron. F-H High-power micrographs from the regions indicated in E. F,G: Close appositions (some indicated by arrows) between dendrites (dj of the motoneuron and axons within the dorsal column on the ipsi- (G) and contra-lateral (F) sides of the spinal cord. H: Close appositions between a contralatera1 reticulospinal axon (ax)and a crossing dendrite (d) of the type 11 motoneuron. Scale bar in E, 100 Km (also applies to A), and scale bar in H, 20 pm (also applies to B-D,F,Gj.
a cooled, Sylgard-lined experimental chamber. Motor cells were impaled with Lucifer-Yellow-filled (5% in 0.1 mM LiC1; Stewart, '78) or HRP-filled (10% HRP in 2M KAc) electrodes and the dye or enzyme was pressure injected (Wallen et al., '85). Cells were identified as motoneurons if an action potential could be recorded in an adjacent ventral root on intracellular stimulation (Russell and Wallen, '83). The fictive locomotor motor pattern was elicited by adding NMDA (100 pM) to the bath (Grillner et al., '87;Brodin and Grillner, '90) and recorded from ventral roots using glass suction electrodes. Intracellular recordings were made using an Axoclamp 2A microelectrode amplifier. In one experiment neurons were injected with HRP and subsequently embedded in Vestopal-W for EM studies as described above. In five experiments the motoneurons were labelled with Lucifer Yellow. These preparations were fixed in formaldehyde (10%) in physiological solution for 2 hours, dehydrated in ethanol, and cleared in methylsalicylate (Wallen et al., '85). They were mounted as whole mounts and labelled neurons were reconstructed in three dimensions using a confocal laser scanning microscope (Sarastro 1000, Molecular Dynamics, Inc.; Wallen et al., '88). As in the previous case labelled motor cells were considered to be fin motoneurons using the criteria established in the first group of experiments.
RESULTS Light microscopic observations on fin motoneurons HRP was injected into dorsal fin muscles in six experiments. Labelled motoneurons were revealed in the spinal cord only on the side of injection. All fin motoneurons studied (n = 148) were located in the lateral cell column. The size of the soma in fin motoneurons did not exceed 35 x 55 pm (width of the spinal cord 0.6-1 mm). They had in general smaller somata than myotomal motoneurons, which may reach 100pm in the longest dimension. Along the spinal cord fin motoneurons were distributed both rostrally and caudally with respect to the parent ventral root (Fig. 1A). Cell bodies labelled through one ventral root could be located as far as the end of the entry zone of the neighbouringventral root. The maximum number of fin motoneurons labelled through one ventral root was 17. Fin motoneurons had one axon which emerged from the axon hillock located on the cell body or a proximal dendrite. Along their course within the spinal cord they were mixed with axons of motoneurons innervating trunk muscles, and they did not form separate bundles of fibres. All axons had a characteristic thin initial segment after which their diameter subsequently increased to reach 3 to 8 p,m in the ventral roots. Within the spinal cord no axonal collaterals could be identified. Thin processes sometimes emerged from the axon hillock, but they could not be distinguished from dendrites. Unlike myotomal motoneurons, which form a column of cells lying close to each other, fin motoneurons can lie separately or in pairs. In the latter case one cell in a pair was usually located more medially (Fig. 1). The distance between single cells or pairs of fin motoneurons was between 110 and 450 pm, mean 230 82 pm (n = 54). Two types of fin motoneurons that differed with respect to their somadendritic morphology were distinguished in this group of experiments. Both types innervate the muscle fibres in the rostra1 and the caudal divisions of the dorsal fin. Within the gray matter type I1 motoneurons lay separately or in pairs
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together with another type I1 cell or with a cell of type I (Fig. lD,E). Pairs consisting of two type I cells were not encountered.
Type I fin motoneurons The motoneurons included in the first group had elongated triangular or oval shaped somata with 3-4 stem dendrites (See Figs. 1B,C, 5, 3A-D), and a wide dendritic tree extending in the rostro-caudal direction and in general restricted to the ipsilateral side. The dendritic width calculated as a fruction of the width of the spinal cord varied 0.11, n = 40). In this respect from 0.1 to 0.7 (mean 0.32 I type I cells resemble motoneurons innervating the dorsal part of the body wall, which have a rather broad dendritic tree (up to 50% of the spinal cord width; Wallen et al., '85). In frontal sections the dendrites could be seen to ramify in different parts of the gray matter and fibre tracts. Characteristically there were relatively few dendrites in the lateral fibre tract area (Figs. 2A, 3). In some cells a high density of dendritic branching was present in the dorsal part of the spinal cord lateral to the dorsal column (Figs. 2A, 3). An interesting finding was that about 25% of the cells in this group (a total of 82 cells were analyzed) directed their dendrites into the ipsilateral dorsal column. Numerous close oppositions with axons within the dorsal column were found in frontal sections (Fig. 2A-C). In some cases these appositions were seen between primary fierents that had been labelled by HRP injection into the dorsal fin and dendrites of the fin motoneurons (Fig. 2C), which suggests the existence of monosynaptic contacts between these elements. About 60% of the type I motoneurons had dendrites directed ventrally towards the midline. In the ventromedial part of the spinal cord single dendrites were found in close apposition to large and medium sized axons (Fig. 2D), most probably belonging to giant reticulospinal neurons (Rovainen, '78, '79). In a few cells (3 out of 82) dendrites crossed the midline. On the contralateral side they were not seen to extend further than the level of the medial group of reticulospinal axons (See Figs. 3C,D, 5C,D).
Type I1 fin motoneurons The main morphological characteristics of type I1 motoneurons are shown in Figures 1-3 and 6. Their soma was fusiform (Figs. l E , 3E). All type I1 motor cells investigated (n = 18) had long dendrites crossing the midline of the spinal cord both above and below the central canal. Dendrites crossing the midline above the central canal passed around the dorsal column, in some cases branching inside the column (Figs. 2E-G, 3F-H). Dendrites formed close appositions with axons in the dorsal column on both the ipsi- and contra-lateral sides (Fig. 2E-G ). Dendrites crossing the midline ventral to the central canal made close appositions with large reticulospinal axons both ipsi- and contra-laterally (Fig. 2H, 4C,D). In some cases they could be traced far into the contralateral side (Fig. 3E-H, 6). Ipsilateral dendrites were oriented in different directions. Like type I cells, some type I1 motoneurons had extensive branching of dendrites within the dorsal part of the spinal cord lateral to the dorsal column (Fig. 3G). Usually the number of dendrites directed to the lateral part of the white matter was less than in other regions. Some type I1 motoneurons did not direct any dendrites into that field (Fig. 3H). Previous studies of myotomal motoneurons (Wallen et al., '85) did not reveal any cells with dendrites crossing the
midline of the spinal cord above the central canal. This criterion could therefore be used for identification of type I1 fin motoneurons in experiments with HRP application on the cut ventral roots (Fig. 1E). Motoneurons were labelled through the ventral roots in eight segments in four lampreys. Forty-one to seventy-eight motor cells were labelled through each ventral root, i.e., more than 60% of the total number of motor cells in one hemisegment in the fin region of the silver lamprey (Rovainen and Dill, '84). The maximum number of type I1 fin motoneurons labelled through a single ventral root was 5.
Electron microscopy To find out whether or not the close appositions between dendrites of fin motoneurons and axons within the dorsal column (Fig. 2B,C,F,G) as well as in the ventro-medial part of the spinal cord (Fig. 2D,H) contained synaptic specializations, an ultrastructural study was carried out. Figures 4 shows examples of close appositions between HRP-labelled dendrites of fin motoneurons and axons within the dorsal column (A) and ventro-medial reticulospinal axons (E), as revealed by light microscopy. Sections containing such appositions were reembedded in Vestopal and cut in serial ultrathin sections, which were then examined on the EM level (see Methods). The EM analysis showed that all close appositions found in the dorsal column (n = 34) had the typical ultrastructural characteristics of a chemical synapse (Gray and Guillery, '66). Thus, presynaptic axons contained spherical vesicles with a diameter of 50 i- 2 nm (mean r+ S.E.M; n = 100) as well as active zones (Fig. 4C,D). Despite heavy labelling of dendrites, post-synaptic densities could be also seen (Fig. 4C,D, arrows). Each close apposition contained one to four active zones. In the case of type I1 motoneurons, synapses were found between axons and dendrites on both ipsi- and contra-lateral sides of the dorsal column. In contrast, most close appositions found between large diameter axons in the ventro-medial region of the spinal cord and dendrites of fin motoneurons appeared not to represent synaptic contacts. The dendrites were often separated from the axon by a very thin glial process. Out of 15 close appositions studied in serial sections (some of them are indicated by arrows in Fig. 4E) synaptic contacts were found only in two cases. One of these synapses is shown in Figure 4F.
Intracellular labelling and recording of fin motoneurons In seven experiments motoneurons were labelled intracellularly after recording their activity during fictive locomotion evoked by bath application of NMDA. Three cells were labelled with HRP and 28 with Lucifer Yellow (see Methods). Four motoneurons were identified as fin motoneurons. Two of them were identified as type I and two cells as type I1 fin motoneurons, based on the morphological criteria given above. Figure 5 B-D shows a three-dimensional reconstruction of a type I motoneuron, obtained with confocal laser-scanning microscopy. Dendrites project to the dorsal column and cross the midline of the spinal cord. This area was rescanned at a higher magnification (Fig. 5B-D). The 3D-image volume obtained could be rotated and viewed from different angles (Wall& et al., '88). The location of the dendrites in relation to the central canal and the dorsal column can be seen after a 90" rotation in Figure 5D. A three-dimensional reconstruction of a type I1 mo-
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Fig. 3. Camera lucida reconstructions of the dendritic morphology of type I and type I1 fin motoneurons. A-D: Type I fin motoneurons. A Type I fin motoneuron in a whole-mount preparation of the spinal cord. The lateral margin of the spinal cord is marked with a solid line and the midline with a dashed line. Arrow indicates rostrocaudal direction. B-D: Reconstructions from transverse sections of three different type I
motoneurons. E-H Type I1 fin motoneurons. E: Reconstruction of two type I1 motoneurons in whole-mount. F-H: Reconstructions of three different type I1 motoneurons in the transverse plane. mn, Fin motoneuron; dc, dorsal column; gm, gray matter; cc, central canal; ax, axon of the motoneuron. Scale bars: A-H, 100 pm.
toneuron is shown in Figure 6B-D. This cell had long dendrites crossing the midline of the spinal cord above and below the central canal (Fig. 6C,D).
Fin motoneurons of both type I and type I1 displayed membrane potential oscillations during fictive locomotion in antiphase with the activity of myotomal motoneurons
Figure 4
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midline above and below the central canal. It is thus likely that type I1 neurons receive more synaptic inputs from the contralateral side than type I cells. The different morphologies of type I and type I1 motoneurons is most likely related to differences in function with regard to the geometrical arrangement of muscle fibers and/or the muscle fiber composition of the muscles they innervate. This is the case for myotomal motoneurons in lamprey (Wallen et al., '85) and also for spinal motoneurons of higher vertebrates (Szekely, '76; Ulfhake and Cullheim, '81; Cullheim et al., '87). Our present data do not allow a conclusion in this respect for fin motoneurons. Additional studies are needed DISCUSSION to clarify this issue. Morphology of fin and myotomal motoneurons Whereas myotomal motoneurons send numerous denStudies of the dendritic morphology of motor cells inner- dritic branches to the lateral part of the spinal cord, fin vating the trunk muscles in lamprey have shown that motoneurons have few dendrites in this region. This sugmotoneurons innervating different parts of the body wall gests that they have less synaptic input from the area in have different morphology (Wallen et al., '85).Motoneurons which, for instance, intraspinal stretch receptor neurons supplying the ventral third of the myotome have a dense are located (Grillner et al., '84; Viana Di Prisco et al., '90). fan-like dendritic tree extending to the midline, whereas Rich plexa of GABA-, somatostatin-and neurotensin- immumotoneurons innervating the dorsal third of the myotome noreactive fibres are also located in this area of the lamprey have a more widespread and less dense dendritic tree, with spinal cord (Brodin et al., '89, '90; Brodin and Grillner, '90). The majority of myotomal motoneurons receive monosynfew ramifications near the midline. Motoneurons supplying the most ventral or dorsal parts of the myotome may also aptic input from descending reticulospinal axons located in have dendrites crossing the midline ventral to the central the ventral part of the spinal cord (Rovainen, '78; '79; Brodin and Grillner, '90). In contrast, we found very few canal. On the basis of morphological criteria two distinct classes synaptic contacts between fin motoneuron dendrites and of motoneurons supplying dorsal fin muscles can be also large to medium sized axons in this region. Thus, for fin distinguished. Type I motoneurons is by far the largest motoneurons these connections are probably mainly group, constituting about 20-25% of the total number of all polysynaptic (cf. Rovainen, '78). motor axons in one ventral root in this region of the spinal Dorsal column input to fin motoneurons cord of Ichthyomyzon (cf. Rovainen and Dill, '84). The dendrites of type I motoneurons are mainly located on the Myotomal motoneurons do not have dendrites in the ipsilateral side of the spinal cord. One main characteristic is dorsal column (WallBn et al., '85),whereas about 25% of the a widespread dendritic tree in the rostrocaudal direction. In type I fin motoneurons were found to direct their dendrites that respect they are similar to dorsal myotome motoneu- into the ipsilateral side of the dorsal column. All type I1 fin rons, which also have a widespread dendritic tree (Wall& et motoneurons direct their dendrites to the dorsal column al., '85). So far, there is no strict explanation why certain both ipsi- and contra-laterally. The present EM studies types of motoneurons in lamprey have a widespread den- have shown that most of the close appositions in this region dritic tree. One possible explanation could be that the represent synaptic contacts. dendritic tree of some motoneurons develops later in ontoRovainen and Birnberger ('71) were the first to suggest genesis and during the process when the dendrites grow to that fin motoneurons may receive monosynaptic input from their targets they have to circumvent cellbodies and other primary afferents. It was subsequently shown in unidentielements in the cord to reach certain fiber tracts (Wallh et fied motoneurons that unitary EPSPs evoked by intracellual., '85). For fin motoneurons this explanation would lar stimulation of dorsal cells can be monosynaptic (Batuappear probable since the fins develop relatively late during eva, '83; Shapovalov and Batueva, '84). Shapovalov and metamorphosis (Birnberger and Rovainen, '7 1). Batueva ('84)assumed these motoneurons to be of the The dendritic tree of type I1 fin motoneurons differs from myotomal type. Since, however, myotomal motoneurons do that of type I cells by having long dendrites crossing the not direct their dendrites into the dorsal column (Wallen etal., '851, and since only polysynaptic EPSPs could be evoked in these cells by intracellular stimulation of single dorsal cells and by skin stimulation (Teravainen and Fig. 4. Ultrastructure of close appositions between fin motoneuron Rovainen, '71b), the motor cells studied by Shapovalov and dendrites and spinal axons. A: Light micrograph of a dendrite of an HRP-labelled fin motoneuron within the dorsal column. af, Merent Batueva ('84) were most likely fin motoneurons. It should fibres. Square indicates the region taken for EM analysis. B: Electron be noted that myotomal motoneurons receive monosynapmicrograph of an ultrathin section from the same dendrite (d) making tic input from another type of "sensory neuron," the edge close oppositions (marked by arrows) with two primary afferent axons (af). sv, Synaptic vesicles. C,D: Synapses established by dorsal column cell, which is an intraspinal stretch-receptor (Grillner et al., '84; Viana Di Prisco et al., '90). axons (an on the labelled dendritic shaft (d) at the close oppositions marked by arrows in B, found on neighbouring ultrathin sections. The present finding of monosynaptic connections beArrows indicate postsynaptic densities. E: Light micrograph of an tween primary afferents and fin motoneurons suggests the HRP-labelled fin motoneuron dendrite located in close apposition existence of specific receptors in the fin that probably (arrow) to large diameter axons (rs, reticulospinal axons) in the provide sensory feedback. Primitive muscle receptors, called ventro-medial part of the spinal cord. F: Synaptic contact between presumed reticulospinal axon (rs) and dendrite (d) of the labelled fin Poloumordwinoff bodies have been described in the fin motoneuron from the region marked by rectangle in E. sv, synaptic muscles of rays (Poloumordwinoff, 1898; Cavalie, '02; vesicles. Scale bars: A,E, 10 prn; B,F, 1 pm; C,D, 0.5 pm. Barets, '56; Bone and Chubb, '75). "Stretch" responses can
located in the same hemisegment. Examples of intracellular activity in fin motoneurons of type I and type I1 are shown in Figures 5A and 6A, respectively. The membrane potential of both types of fin motoneurons oscillated with a peak depolarization occurring at a phase value of 0.64 to 0.66 in relation to the burst cycle in myotomal motoneurons recorded in the same ventral root. The amplitude of oscillations in the two type I motoneurons recorded from was 2 and 6 mV, and in the two type I1 cells corresponding values were 4 and 11 mV.
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Fig. 5. Rhythmic locomotor activity in type I fin motoneuron. A: Intracellularly recorded activity of a type I fin motoneuron (mn) during fictive locomotion evoked by adding 100 FM NMDA to the perfusing solution. vr, Rhythmic locomotor adivity in the ventral root containing the axon of the fin motoneuron. B: Whole mount three dimensional reconstruction of the same type I fin motoneuron (mn). The cell was injected intracellularly with Lucifer Yellow from the recording elec-
trode and scanned in a confocal laser scanning microscope. Arrow indicates rostrocaudal direction. ax, motoneuron axon. C : Area corresponding to rectangle in A scanned at high magnification. D: Frontal projection from the image shown on C, obtained after a 90" rotation of the image volume. mn, Motoneuron; d, dendrite projecting into the dorsal column (dc); rs, reticulospinal axons; cc, central canal. Scale bars: A, 100bm; C,D, 5 0 ~ m .
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Fig. 6. Rhythmic locomotor activity in a type I1 fin motoneuron. A: Activity in a type I1 fin motoneuron during fictive locomotion. vr, Activity in the ventral root containing the axon of the fin motoneuron. B: 3-D reconstruction of the same cell as in A injected intracellularly with Lucifer Yellow and scanned in the confocal microscope. Arrow indicates
rostrocaudal direction. C,D: Frontal projections, scanned at high magnification, obtained after a 90" rotation of the image volume, showing the positions of the dendrites marked d l and d2 in B, in relation to the central canal (cc). mn, Motoneuron; ax, axon; dc, dorsal column; rs, reticulospinal axons. Scale bars: A, 100 pm; C, 25 pm; D, 50 IJ-m.
be evoked in the fin nerves upon stretch applied to the fin in rays (Fessard and Sand, '37) and monosynaptic EPSPs can be recorded in motoneurons during stimulation of the fin
nerves (Leonard et al., '78). It would seem likely that a similar type of receptor may exist in the dorsal fin of the lamprey. Trunk muscles in lower vertebrates do not contain
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muscle spindles (e.g., Gray, ’33; Roberts, ’69), however, in lamprey the intraspinal stretch receptor neurons may serve an analogous function (Viana Di Prisco et al., ’90). Muscle spindles have been found only in the jaw-closing musculature, adductor mandibulae, in the salmon (Maeda et al., ’83). Injection of HRP into dorsal fin muscles revealed close appositions between fin motoneuron dendrites and dorsal column axons, at least some of them belonging to dorsal cells (Figs. 1, 2). Lamprey dorsal cells have been classified into two different groups, “touch” and “pressure,” according to their responses to mechanical stimulation of the skin (Christenson et al., ’88). If monosynaptic effects to fin motoneurons are mediated by dorsal cells, it would appear likely that in the fin region these cells could serve as some type of stretch receptor providing movement feedback.
Activity of type I and type I1 motoneurons during fictive locomotion Dorsal fin muscles in the sea lamprey (Petromyzon) are active in antiphase to the burst activity of the ipsilateral myotomes during fictive locomotion (Buchanan and Cohen, ’82). Figures 5 and 6 show that both type I and type I1 fin motoneurons exhibit oscillation of the membrane potential in antiphase with the ipsilateral ventral root discharge, suggesting that both types of fin motoneurons may be driven by the contralateral part of the segmental locomotor network, and that they may receive similar synaptic input despite the differences in morphology. This antiphasic control may act to maintain the fin in an upright position during lateral movements. Large reticulospinal Muller neurons (11and Ms) provide a polysynaptic excitation of contralateral fin motoneurons in contrast to the monosynaptic excitation of ipsilateral myotomal motoneurons (Rovainen, ’78). It would appear likely, however, that the control of fin motoneurons is critical for rapid compensatory movements during a variety of conditions, and it is then expected that they should be under an efficient brain stem control. This aspect will be dealt with in a forthcoming study.
ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council, projects number: 3026 and 6815, and from Karolinska Institutets Fonder. Dr. 0. Shupliakov was supported from Svenska Institutet and by funds to S.G. We thank Professor G. Orlovski and Dr. S. Cullheim and Dr. L. Brodin for valuable comments on the manuscript.
LITERATURE CITED Barets, P.A. (1956) Les recepteurs intra-musculaires des nageoires chez les selaciens. Archives d’Anatomie Microscopique 45.954-260. Batueva, I.V. (1983) Properties of excitatory postsynaptic potentials elicited by stimulation of single segmental afferents in the spinal motoneurones of the lampreyLanpetra~uurati2zs. J. Evol. Biochem. Physiol. 19;54-61. (in Russian). Birnberger, K.L., and C.M. Rovainen (1971) Behavioral and intracellular studies of a habituating fin reflex in the sea lamprey. J. Neurophysiol, 34:983-989. Bone, Q., and A.D. Chubb (1975) The structure of stretch receptor endings in the fin muscles of rays. J. Mar. Biol. Ass. U.K. 55:939-943. Brodin, L., H Ring, P . Grillner, H. Persson, T. Hokfelt, and S.Grillner (1989) Comparative aspects of some neurotransmitter systems in the lamprey CNS. Eur. J. Neurosci. Suppl. 2:12.1. Brodin, L., and S. Grillner (1990) The lamprey CNS in vitro, an experimen-
tally amenable model for synaptic transmission and integrative functions. In H. Jahnsen (ed): Preparations of Vertebrate Central Nervous System In Vitro. New York: John Wiley & Sons, pp. 103-153. Brodin, L., N. Dale, J. Christenson, J. Storm-Mathisen, T. Hokfelt, and S. Grillner (1990) Three types of GABA-immunoreactive cells in the lamprey spinal cord. Brain Res. 508r172-175. Buchanan, J.T., and.4.H. Cohen (1982) Activities of identified interneurons, motoneurons, and muscle fibers during fictive swimming in the lamprey and effects of reticulospinal and dorsal cell stimulation. J. Neurophysiol. 47:948-960. Cavalie, M. (1902) Sur les terminaisons nerveuses motrices et sensitives dans les muscles stries, chez la Torpille (Torpedo marmorata). Comptes Rendu des Seances de la Societe de Biologie. 54: 1279-1280. Christenson J., A. Boman, P:A. Lagerback, and S. Grillner (1988) The dorsal cell, one class of primary sensory neuron in the lamprey spinal cord. I. Touch, pressure but no nociception: A physiological study. Brain Res. 44O:l-8. Cullheim S., J.W. Fleshman, L.L. Glenn, and R.E. Burke (1987) Membrane area and dendritic structure in type-identified triceps surae alpha motoneurons. J. Comp. Neurol. 255:68-81. Fessard, A., and A. Sand (1937) Stretch receptors in the muscles of fishes. J. Exp. Biol. 14:383-404. Graham, R.C., and M.J. Karnovsky (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 1 4 2 9 1-302. Gray, E.G., and R.M. Guillery (1966) Synaptic morphology in the normal and degenerating nervous system. Int. Rev. Cytol. 19:111-181. Gray, J. (1933) Studies in animal locomotion. 11. The relationship between waves of muscular contraction and the propulsive mechanism of the eel. J. Exp. Biol. I0:386-390. Grillner, S., P. Wallen, L. Brodin , and A. Lansner (1991) Neuronal network generating locomotor behavior in lamprey: Circuitry, transmitters, membrane properties and simulation. Annu. Rev. Neurosci. 14:169-199. Hanker, J.S., P.E. Yates, C.B. Metz, and A.A. Rustioni (1977) New specific sensitive and noncarcinogenic reagent for the demonstration of horseradish peroxidase. J. Histochem. 9:789-792. Leonard, R.B., P. Rudomin, and W.D. Willis (1978) Central effects ofvolleys in sensory and motor components of peripheral nerve in the stingray, Dasyatis sabina. J. Neurophysiol. 41:108-125. Maeda, N., S. Miyoshi, and H. Toh (1983) First observation of a muscle spindle in fish. Nature 3 0 2 6 1 4 2 . Poloumordwinoff, D. (1898) Recherches sur les terminaisons nerveuses sensitives dans les muscles stries volontaires: Travaux des Laboratoires de la Societe Scientifique et Station Zoologique d’Arcachon 3:73-79. Roberts, B.L. (1969) The spinal nerves of the dogfish (Scyliorhinus).J. Mar. Biol. Assn. U.K. 49:51-75. Rovainen, C.M. (1967) Physiological and anatomical studies on large neurons of central nervous system of the sea lamprey (Petromyzonmarinus). 11.Dorsal cells and giant interneurons. 3. Neurophysiol. 30t1024-1042. Rovainen, C.M. (1978) Muller cells, “Mauthner” cells, and other identified reticulospinal neurons in the lamprey. In D. Faber, and H. Korn (eds): Neurobiology of the Mauthner Cell. New York: Raven Press, pp, 245-269. Rovainen, C.M. (1979) Neurobiology oflampreys. Physiol. Rev. 59:1007-1077. Rovainen, C.M., and K.L. Birnberger (1971) Identification and properties of motoneurons to fin muscle of the sea lamprey. J. Neurophysiol. 34:974982. Rovainen, C.M., and D.A. Dill (1984) Counts of axons in electron microscopic sections of ventral roots in lampreys. J. Comp. Neurol. 225:433440. Russell, D.F., and P. Wallen (1983) On the control of myotomal motoneurones during “fictive swimming” in the lamprey spinal cord in vitro. Acta Physiol. Scand. 11 7:161-170. Shapovalov, A.I., and I.V. Batueva (1984) Interaction between segmental primary afferents and motoneurons in spinal cord of the lamprey. Sechenov J. Physiol. U.S.S.R. 70:117&1188 (in Russian). Shupliakov, O.V. (1985) Suction electrode for morpho-physiological studies in thin and short nerve trunks in vitro. Sechenov J. Phisiol. U.S.S.R. 71r804-807 (in Russian). Stewart, W.W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14:741759. Szekely, G. (1976) The morphology of motoneurons and dorsal root fibers in the frog spinal cord. Brain Res. 103:275-290.
TYPES OF FIN MOTONEURONS Teraviiinen, H., and C.M. Rovainen (1971a) Fast and slow motoneurons to body muscle of the sea lamprey. J.Neurophysiol.34:990-998. Teravainen, H., and C.M. Rovainen (1971b) Electrical activity of myotomal muscle, motoneurons, and sensory dorsal cells during spinal reflexes of IamDrevs. J.NeuroDhvsiol.34:999-1009. Tretjakoff, D. (1909) Das Newensystem von Ammocoetes. I. Das RCckenmark. Arch. Mikrosk. Anat. 73:607-680. Ulfhake B., and S . Cullheim (1981) A quantitative light microscopic study of the dendrites of cat spinal a-motoneurons after intracellular staining with horseradish peroxidase. J. Comp. Neurol. 202585-596. Viana Di Prisco, G., P. Wallen, and S. Grillner (1990) Synaptic effects of 1
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123 intraspinal stretch receptor neurons mediating movement-related feedback during locomotion. Brain Res. 530:161-166. WallBn, P., S. Grillner, J.L. Feldman, and S. Bergelt (1985) Dorsal and ventral myotome motoneurons and their input during fictive locomotion in lamprey. J. Neurosci. 5:654-661.
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Wallen, p., K. Carlsson, A. Liljeborg, and S. Grillner (1988) Threedimensional reconstruction of neurons in the lamprey spinal cord in whole-mount, using a confocal laser scanning microscope. J. Neurosci. Methods 24:91-100, Williams, T.L., S. Grillner, V.V. Smoljaninov, P. WallBn, S. Kashin, and S. Rossignol (1989) Locomotion in lamprey and trout: The relative timing ofactivation and movement, J. Exp. Biol, 143:559-566.
Two Types of Motoneurons Supplying Dorsal Fin Muscles in Lamprey and Their Activity During Fictive Locomotion OLEG SHUPLIAKOV, PETER WALLEN, AND STEN GRILLNER Nobel Institute for Neurophysiology ( 0 3 ,P.W., S.G.) and Department of Anatomy (O.S.), Karolinska Institute, S-104 0 1 Stockholm, Sweden
ABSTRACT The location and dendritic morphology of motoneurons supplying the dorsal fin muscles were studied in the lamprey spinal cord (Ichthyomyzon unicuspis). Motoneurons were retrogradely labelled after injection of HRP into the fin muscles or after its application on the cut ends of the ventral roots. HRP-labelled cells were subsequently reconstructed, in the horizontal and/or transverse planes. Fin motoneurons were also injected intracellularly with Lucifer Yellow and their detailed three-dimensional structure was analysed by confocal laser-scanning microscopy. Unlike myotomal motoneurons, which are closely spaced in the lateral cell column, fin motoneurons were distributed along the spinal cord separately or in pairs. They could be distinguished from motoneurons supplying trunk muscles by having a limited number of dendrites in the lateral part of the spinal cord. In addition, some fin motoneurons extend their dendrites into the dorsal column. The motor cells innervating fin muscles were divided into two types based on their dendritic morphology. Type I have a widespread dendritic tree in the rostrocaudal direction and, with few exceptions, completely restricted to the ipsilateral side. A proportion (25%) of these cells have dendrites extending into the dorsal column. Type I1 fin motoneurons extend their dendrites both ipsi- and contra-laterally. The contra-lateral dendrites pass below and above the central canal. The dendrites send off branches into the dorsal columns on both the ipsi- and the contra-lateral sides. Electron microscopic analysis showed that both type I and type I1 fin motoneurons receive numerous synaptic contacts from dorsal column axons. During fictive locomotion both types of motoneurons are active in antiphase in relation to myotomal motoneurons and to the main locomotor burst. D 1992 Wiley-Liss, Inc. Key words: motoneuron,dendritic morphology, sensory-motorconnections, spinal cord, fin
There are two main groups of motor cells which are involved in the generation of locomotor behaviour in lampreys: motoneurons innervating muscles of the body wall and motoneurons supplying fin muscles (Tretjakoff, '09; Birnberger and Rovainen, '71; Teravainen and Rovainen, '71a). During swimming, myotomal motoneurons receive alternating excitatory and inhibitory synaptic input from the central pattern generator (Russell and Wallen, '83). The swimming motor pattern is characterized by alternating contractions of myotomal motor units on the opposite sides of one segment and a delayed activation of more caudal segments, resulting in an undulatory wave travelling down the body (Williams et al., '89). The detailed morphology of different types of myotomal motoneurons, their synaptic inputs and activity during fictive locomotion, have been characterized in the in vitro preparation of the lamprey spinal cord (Teraviiinen and Rovainen, '71a; W a l l h et al., '85). In contrast, our knowledge about the morphology and the synaptic control of fin motoneurons is limited to a few O
1992 WILEY-LISS, INC.
studies (Tretjakoff, '09; Rovainen, '67; Birnberger and Rovainen, '71; Buchanan and Cohen, '82; Rovainen and Dill, '84). This is a serious gap since fin motoneurons play an important role in the compensatory adjustments necessary for the control of equilibrium. The present study describes the detailed morphology of motoneurons supplying the dorsal fin muscles and we suggest a classification of these into two distinct types. The pattern of activity of these two types of fin motoneurons during fictive locomotion is also described.
MATERIALS AND METHODS Experiments were performed on 19 adult silver lampreys (Ichthyonyzon unicuspis) obtained from Iowa. Before dissection the animals were anesthetized with MS-222 (tricaine methane sulphonate, Sandoz, 100 mg/l water). Accepted February 6,1992.
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Fig. 1. Fin motoneurons retrogradely labelled with HRP. A Fin motoneurons (mn) labelled after HRP injection into the musculature of the caudal portion of the dorsal fin. VR1 indicates the position of the ventral root (vr) containing HRP-labelled motoneuron axons, and VR2 the position of the neighbouring ventral root. r,c, Rostra1 and caudal direction, respectively. B: High-power micrograph of the area corresponding to the rectangle in A. af, Primary aEerents labelled after injection of HRP into the musculature on the ipsi- and contra-lateral
sides of the fin. dc, Dorsal cell. ax,Axons of fin motoneurons. Interrupted line indicates spinal cord midline. C: Type I fin motoneurons (mn) innervating the rostral portion of the dorsal fin. ax, Labelled axons of fin motoneurons. D,E: Type I1 fin motoneurons (mn) labelled after injection of HRP into the fin musculature at the level of the rostral division (D) and after HRP application on the cut end of the ventral root (E). d, Indicates dendrites crossing the midline above the central canal. Scale bars: A, 100 pm; E, 100 K r n (also applies to E D ) .
Fin motoneurons were labelled using three different procedures:
Their separation from the trunk musculature allows selective injections of horseradish peroxidase (HRP) into the fin muscles without spread of theknzyme to the trunk muscles. HRP (Sigma Chemical Co., St. Louis, MO, type VI) in physiological solution (20% HRP) was injected unilaterally or, in two cases bilaterally, into the muscles of the dorsal fin. The animals were left to survive for 7 days after the injection. The part of the spinal cord containing retro-
Injection of horseradish peroxidase into fin muscles In the adult lamprey the dorsal fin muscles are represented by several layers of fibers surrounding the cartilaginous skeleton of the fin (Rovainen and Birnberger, '71).
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TYPES OF FIN MOTONEURONS gradely labelled motoneurons was then dissected out and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The HRP was visualized according to standard Graham-Karnovsky or Hanker-Yates protocols (Graham and Karnovsky, '66; Hanker and Yates, '82 ). The preparations were cleared in methylsalicylate, mounted as whole mounts, and photographed. Some of them were then embedded in paraffin, sectioned in 20 pm sections, and mounted on glass slides. The labelled motoneurons were reconstructed with the aid of a camera lucida. For electron microscopy four pieces of the spinal cord, each containing an HRP-labelled fin motoneuron, were fixed in 3% glutaraldehyde (4 hours), osmicated, dehydrated in acetone, and embedded in Vestopal-W. After curing, regions with labelled neurons were cut in serial transverse 7 pm thick sections with a dry glass knife on a pyramitome. Sections were then immersed in glycerol, mounted on glass slides, and examined under the light microscope. Selected sections were remounted on newly faced blank Vestopal blocks. They were then resectioned on an LKB Ultratome with a diamond knife. Ultrathin sections were collected on formvar-coated slot grids, doublestained in uranyl acetate and lead citrate, and examined with a Philips 201 electron microscope.
Application of HRP on the cut ventral roots Isolated spinal cord preparations (11segments long) were used in these experiments. They were pinned down with the ventral side up in a Sylgard-lined dish. Care was taken to fix the spinal cord a t the same length it had in situ. The preparations were perfused with cooled (7-9°C) physiological solution of the following composition (in mM): NaC1, 91; KCl, 2.1; CaC12, 2.6; glucose, 4; NaHC03, 20. The solution was bubbled with 95% 0 2 + 5%COZ to pH 7.4. The cut ends of the ventral roots were drawn into suction electrodes filled with 10% HRP in physiological solution (Shupliakov, '85). Depolarizing current pulses of 2-10 nA and of 80-100 ms duration were applied for a period of 1 hour. After a period of 24 hours the preparations were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer and then processed for light- andlor EM- analysis as decribed above. Cells were identified as fin motoneurons on the basis of criteria elaborated in the first group of experiments.
Intracellular recording and labelling of fin motoneurons Intracellular labelling of motoneurons was performed in the isolated spinal cord-notochord preparation mounted in
Fig. 2. Close appositions between HRP-labelled fin motoneuron dendrites and dorsal column and reticulospinal axons. A: Light micrograph of transverse section through the lamprey spinal cord illustrating location of dendrites of a type I fin motoneuron labelled retrogradely with HRP. B-D: High-power micrographs of the areas correspondingto squares in A. B,C: Close appositions (someof them indicated by arrows) between dorsal column axons and the dendrites (d) of the motoneuron. D: Close appositions (some are indicated by arrows) between dendrites of the fin motoneuron and reticulospinal axons (rs). E. Light micrograph of transverse section from the same segment of the spinal cord containing dendrites of a type I1 fin motoneuron. F-H High-power micrographs from the regions indicated in E. F,G: Close appositions (some indicated by arrows) between dendrites (dj of the motoneuron and axons within the dorsal column on the ipsi- (G) and contra-lateral (F) sides of the spinal cord. H: Close appositions between a contralatera1 reticulospinal axon (ax)and a crossing dendrite (d) of the type 11 motoneuron. Scale bar in E, 100 Km (also applies to A), and scale bar in H, 20 pm (also applies to B-D,F,Gj.
a cooled, Sylgard-lined experimental chamber. Motor cells were impaled with Lucifer-Yellow-filled (5% in 0.1 mM LiC1; Stewart, '78) or HRP-filled (10% HRP in 2M KAc) electrodes and the dye or enzyme was pressure injected (Wallen et al., '85). Cells were identified as motoneurons if an action potential could be recorded in an adjacent ventral root on intracellular stimulation (Russell and Wallen, '83). The fictive locomotor motor pattern was elicited by adding NMDA (100 pM) to the bath (Grillner et al., '87;Brodin and Grillner, '90) and recorded from ventral roots using glass suction electrodes. Intracellular recordings were made using an Axoclamp 2A microelectrode amplifier. In one experiment neurons were injected with HRP and subsequently embedded in Vestopal-W for EM studies as described above. In five experiments the motoneurons were labelled with Lucifer Yellow. These preparations were fixed in formaldehyde (10%) in physiological solution for 2 hours, dehydrated in ethanol, and cleared in methylsalicylate (Wallen et al., '85). They were mounted as whole mounts and labelled neurons were reconstructed in three dimensions using a confocal laser scanning microscope (Sarastro 1000, Molecular Dynamics, Inc.; Wallen et al., '88). As in the previous case labelled motor cells were considered to be fin motoneurons using the criteria established in the first group of experiments.
RESULTS Light microscopic observations on fin motoneurons HRP was injected into dorsal fin muscles in six experiments. Labelled motoneurons were revealed in the spinal cord only on the side of injection. All fin motoneurons studied (n = 148) were located in the lateral cell column. The size of the soma in fin motoneurons did not exceed 35 x 55 pm (width of the spinal cord 0.6-1 mm). They had in general smaller somata than myotomal motoneurons, which may reach 100pm in the longest dimension. Along the spinal cord fin motoneurons were distributed both rostrally and caudally with respect to the parent ventral root (Fig. 1A). Cell bodies labelled through one ventral root could be located as far as the end of the entry zone of the neighbouringventral root. The maximum number of fin motoneurons labelled through one ventral root was 17. Fin motoneurons had one axon which emerged from the axon hillock located on the cell body or a proximal dendrite. Along their course within the spinal cord they were mixed with axons of motoneurons innervating trunk muscles, and they did not form separate bundles of fibres. All axons had a characteristic thin initial segment after which their diameter subsequently increased to reach 3 to 8 p,m in the ventral roots. Within the spinal cord no axonal collaterals could be identified. Thin processes sometimes emerged from the axon hillock, but they could not be distinguished from dendrites. Unlike myotomal motoneurons, which form a column of cells lying close to each other, fin motoneurons can lie separately or in pairs. In the latter case one cell in a pair was usually located more medially (Fig. 1). The distance between single cells or pairs of fin motoneurons was between 110 and 450 pm, mean 230 82 pm (n = 54). Two types of fin motoneurons that differed with respect to their somadendritic morphology were distinguished in this group of experiments. Both types innervate the muscle fibres in the rostra1 and the caudal divisions of the dorsal fin. Within the gray matter type I1 motoneurons lay separately or in pairs
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together with another type I1 cell or with a cell of type I (Fig. lD,E). Pairs consisting of two type I cells were not encountered.
Type I fin motoneurons The motoneurons included in the first group had elongated triangular or oval shaped somata with 3-4 stem dendrites (See Figs. 1B,C, 5, 3A-D), and a wide dendritic tree extending in the rostro-caudal direction and in general restricted to the ipsilateral side. The dendritic width calculated as a fruction of the width of the spinal cord varied 0.11, n = 40). In this respect from 0.1 to 0.7 (mean 0.32 I type I cells resemble motoneurons innervating the dorsal part of the body wall, which have a rather broad dendritic tree (up to 50% of the spinal cord width; Wallen et al., '85). In frontal sections the dendrites could be seen to ramify in different parts of the gray matter and fibre tracts. Characteristically there were relatively few dendrites in the lateral fibre tract area (Figs. 2A, 3). In some cells a high density of dendritic branching was present in the dorsal part of the spinal cord lateral to the dorsal column (Figs. 2A, 3). An interesting finding was that about 25% of the cells in this group (a total of 82 cells were analyzed) directed their dendrites into the ipsilateral dorsal column. Numerous close oppositions with axons within the dorsal column were found in frontal sections (Fig. 2A-C). In some cases these appositions were seen between primary fierents that had been labelled by HRP injection into the dorsal fin and dendrites of the fin motoneurons (Fig. 2C), which suggests the existence of monosynaptic contacts between these elements. About 60% of the type I motoneurons had dendrites directed ventrally towards the midline. In the ventromedial part of the spinal cord single dendrites were found in close apposition to large and medium sized axons (Fig. 2D), most probably belonging to giant reticulospinal neurons (Rovainen, '78, '79). In a few cells (3 out of 82) dendrites crossed the midline. On the contralateral side they were not seen to extend further than the level of the medial group of reticulospinal axons (See Figs. 3C,D, 5C,D).
Type I1 fin motoneurons The main morphological characteristics of type I1 motoneurons are shown in Figures 1-3 and 6. Their soma was fusiform (Figs. l E , 3E). All type I1 motor cells investigated (n = 18) had long dendrites crossing the midline of the spinal cord both above and below the central canal. Dendrites crossing the midline above the central canal passed around the dorsal column, in some cases branching inside the column (Figs. 2E-G, 3F-H). Dendrites formed close appositions with axons in the dorsal column on both the ipsi- and contra-lateral sides (Fig. 2E-G ). Dendrites crossing the midline ventral to the central canal made close appositions with large reticulospinal axons both ipsi- and contra-laterally (Fig. 2H, 4C,D). In some cases they could be traced far into the contralateral side (Fig. 3E-H, 6). Ipsilateral dendrites were oriented in different directions. Like type I cells, some type I1 motoneurons had extensive branching of dendrites within the dorsal part of the spinal cord lateral to the dorsal column (Fig. 3G). Usually the number of dendrites directed to the lateral part of the white matter was less than in other regions. Some type I1 motoneurons did not direct any dendrites into that field (Fig. 3H). Previous studies of myotomal motoneurons (Wallen et al., '85) did not reveal any cells with dendrites crossing the
midline of the spinal cord above the central canal. This criterion could therefore be used for identification of type I1 fin motoneurons in experiments with HRP application on the cut ventral roots (Fig. 1E). Motoneurons were labelled through the ventral roots in eight segments in four lampreys. Forty-one to seventy-eight motor cells were labelled through each ventral root, i.e., more than 60% of the total number of motor cells in one hemisegment in the fin region of the silver lamprey (Rovainen and Dill, '84). The maximum number of type I1 fin motoneurons labelled through a single ventral root was 5.
Electron microscopy To find out whether or not the close appositions between dendrites of fin motoneurons and axons within the dorsal column (Fig. 2B,C,F,G) as well as in the ventro-medial part of the spinal cord (Fig. 2D,H) contained synaptic specializations, an ultrastructural study was carried out. Figures 4 shows examples of close appositions between HRP-labelled dendrites of fin motoneurons and axons within the dorsal column (A) and ventro-medial reticulospinal axons (E), as revealed by light microscopy. Sections containing such appositions were reembedded in Vestopal and cut in serial ultrathin sections, which were then examined on the EM level (see Methods). The EM analysis showed that all close appositions found in the dorsal column (n = 34) had the typical ultrastructural characteristics of a chemical synapse (Gray and Guillery, '66). Thus, presynaptic axons contained spherical vesicles with a diameter of 50 i- 2 nm (mean r+ S.E.M; n = 100) as well as active zones (Fig. 4C,D). Despite heavy labelling of dendrites, post-synaptic densities could be also seen (Fig. 4C,D, arrows). Each close apposition contained one to four active zones. In the case of type I1 motoneurons, synapses were found between axons and dendrites on both ipsi- and contra-lateral sides of the dorsal column. In contrast, most close appositions found between large diameter axons in the ventro-medial region of the spinal cord and dendrites of fin motoneurons appeared not to represent synaptic contacts. The dendrites were often separated from the axon by a very thin glial process. Out of 15 close appositions studied in serial sections (some of them are indicated by arrows in Fig. 4E) synaptic contacts were found only in two cases. One of these synapses is shown in Figure 4F.
Intracellular labelling and recording of fin motoneurons In seven experiments motoneurons were labelled intracellularly after recording their activity during fictive locomotion evoked by bath application of NMDA. Three cells were labelled with HRP and 28 with Lucifer Yellow (see Methods). Four motoneurons were identified as fin motoneurons. Two of them were identified as type I and two cells as type I1 fin motoneurons, based on the morphological criteria given above. Figure 5 B-D shows a three-dimensional reconstruction of a type I motoneuron, obtained with confocal laser-scanning microscopy. Dendrites project to the dorsal column and cross the midline of the spinal cord. This area was rescanned at a higher magnification (Fig. 5B-D). The 3D-image volume obtained could be rotated and viewed from different angles (Wall& et al., '88). The location of the dendrites in relation to the central canal and the dorsal column can be seen after a 90" rotation in Figure 5D. A three-dimensional reconstruction of a type I1 mo-
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Fig. 3. Camera lucida reconstructions of the dendritic morphology of type I and type I1 fin motoneurons. A-D: Type I fin motoneurons. A Type I fin motoneuron in a whole-mount preparation of the spinal cord. The lateral margin of the spinal cord is marked with a solid line and the midline with a dashed line. Arrow indicates rostrocaudal direction. B-D: Reconstructions from transverse sections of three different type I
motoneurons. E-H Type I1 fin motoneurons. E: Reconstruction of two type I1 motoneurons in whole-mount. F-H: Reconstructions of three different type I1 motoneurons in the transverse plane. mn, Fin motoneuron; dc, dorsal column; gm, gray matter; cc, central canal; ax, axon of the motoneuron. Scale bars: A-H, 100 pm.
toneuron is shown in Figure 6B-D. This cell had long dendrites crossing the midline of the spinal cord above and below the central canal (Fig. 6C,D).
Fin motoneurons of both type I and type I1 displayed membrane potential oscillations during fictive locomotion in antiphase with the activity of myotomal motoneurons
Figure 4
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midline above and below the central canal. It is thus likely that type I1 neurons receive more synaptic inputs from the contralateral side than type I cells. The different morphologies of type I and type I1 motoneurons is most likely related to differences in function with regard to the geometrical arrangement of muscle fibers and/or the muscle fiber composition of the muscles they innervate. This is the case for myotomal motoneurons in lamprey (Wallen et al., '85) and also for spinal motoneurons of higher vertebrates (Szekely, '76; Ulfhake and Cullheim, '81; Cullheim et al., '87). Our present data do not allow a conclusion in this respect for fin motoneurons. Additional studies are needed DISCUSSION to clarify this issue. Morphology of fin and myotomal motoneurons Whereas myotomal motoneurons send numerous denStudies of the dendritic morphology of motor cells inner- dritic branches to the lateral part of the spinal cord, fin vating the trunk muscles in lamprey have shown that motoneurons have few dendrites in this region. This sugmotoneurons innervating different parts of the body wall gests that they have less synaptic input from the area in have different morphology (Wallen et al., '85).Motoneurons which, for instance, intraspinal stretch receptor neurons supplying the ventral third of the myotome have a dense are located (Grillner et al., '84; Viana Di Prisco et al., '90). fan-like dendritic tree extending to the midline, whereas Rich plexa of GABA-, somatostatin-and neurotensin- immumotoneurons innervating the dorsal third of the myotome noreactive fibres are also located in this area of the lamprey have a more widespread and less dense dendritic tree, with spinal cord (Brodin et al., '89, '90; Brodin and Grillner, '90). The majority of myotomal motoneurons receive monosynfew ramifications near the midline. Motoneurons supplying the most ventral or dorsal parts of the myotome may also aptic input from descending reticulospinal axons located in have dendrites crossing the midline ventral to the central the ventral part of the spinal cord (Rovainen, '78; '79; Brodin and Grillner, '90). In contrast, we found very few canal. On the basis of morphological criteria two distinct classes synaptic contacts between fin motoneuron dendrites and of motoneurons supplying dorsal fin muscles can be also large to medium sized axons in this region. Thus, for fin distinguished. Type I motoneurons is by far the largest motoneurons these connections are probably mainly group, constituting about 20-25% of the total number of all polysynaptic (cf. Rovainen, '78). motor axons in one ventral root in this region of the spinal Dorsal column input to fin motoneurons cord of Ichthyomyzon (cf. Rovainen and Dill, '84). The dendrites of type I motoneurons are mainly located on the Myotomal motoneurons do not have dendrites in the ipsilateral side of the spinal cord. One main characteristic is dorsal column (WallBn et al., '85),whereas about 25% of the a widespread dendritic tree in the rostrocaudal direction. In type I fin motoneurons were found to direct their dendrites that respect they are similar to dorsal myotome motoneu- into the ipsilateral side of the dorsal column. All type I1 fin rons, which also have a widespread dendritic tree (Wall& et motoneurons direct their dendrites to the dorsal column al., '85). So far, there is no strict explanation why certain both ipsi- and contra-laterally. The present EM studies types of motoneurons in lamprey have a widespread den- have shown that most of the close appositions in this region dritic tree. One possible explanation could be that the represent synaptic contacts. dendritic tree of some motoneurons develops later in ontoRovainen and Birnberger ('71) were the first to suggest genesis and during the process when the dendrites grow to that fin motoneurons may receive monosynaptic input from their targets they have to circumvent cellbodies and other primary afferents. It was subsequently shown in unidentielements in the cord to reach certain fiber tracts (Wallh et fied motoneurons that unitary EPSPs evoked by intracellual., '85). For fin motoneurons this explanation would lar stimulation of dorsal cells can be monosynaptic (Batuappear probable since the fins develop relatively late during eva, '83; Shapovalov and Batueva, '84). Shapovalov and metamorphosis (Birnberger and Rovainen, '7 1). Batueva ('84)assumed these motoneurons to be of the The dendritic tree of type I1 fin motoneurons differs from myotomal type. Since, however, myotomal motoneurons do that of type I cells by having long dendrites crossing the not direct their dendrites into the dorsal column (Wallen etal., '851, and since only polysynaptic EPSPs could be evoked in these cells by intracellular stimulation of single dorsal cells and by skin stimulation (Teravainen and Fig. 4. Ultrastructure of close appositions between fin motoneuron Rovainen, '71b), the motor cells studied by Shapovalov and dendrites and spinal axons. A: Light micrograph of a dendrite of an HRP-labelled fin motoneuron within the dorsal column. af, Merent Batueva ('84) were most likely fin motoneurons. It should fibres. Square indicates the region taken for EM analysis. B: Electron be noted that myotomal motoneurons receive monosynapmicrograph of an ultrathin section from the same dendrite (d) making tic input from another type of "sensory neuron," the edge close oppositions (marked by arrows) with two primary afferent axons (af). sv, Synaptic vesicles. C,D: Synapses established by dorsal column cell, which is an intraspinal stretch-receptor (Grillner et al., '84; Viana Di Prisco et al., '90). axons (an on the labelled dendritic shaft (d) at the close oppositions marked by arrows in B, found on neighbouring ultrathin sections. The present finding of monosynaptic connections beArrows indicate postsynaptic densities. E: Light micrograph of an tween primary afferents and fin motoneurons suggests the HRP-labelled fin motoneuron dendrite located in close apposition existence of specific receptors in the fin that probably (arrow) to large diameter axons (rs, reticulospinal axons) in the provide sensory feedback. Primitive muscle receptors, called ventro-medial part of the spinal cord. F: Synaptic contact between presumed reticulospinal axon (rs) and dendrite (d) of the labelled fin Poloumordwinoff bodies have been described in the fin motoneuron from the region marked by rectangle in E. sv, synaptic muscles of rays (Poloumordwinoff, 1898; Cavalie, '02; vesicles. Scale bars: A,E, 10 prn; B,F, 1 pm; C,D, 0.5 pm. Barets, '56; Bone and Chubb, '75). "Stretch" responses can
located in the same hemisegment. Examples of intracellular activity in fin motoneurons of type I and type I1 are shown in Figures 5A and 6A, respectively. The membrane potential of both types of fin motoneurons oscillated with a peak depolarization occurring at a phase value of 0.64 to 0.66 in relation to the burst cycle in myotomal motoneurons recorded in the same ventral root. The amplitude of oscillations in the two type I motoneurons recorded from was 2 and 6 mV, and in the two type I1 cells corresponding values were 4 and 11 mV.
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200ms
Fig. 5. Rhythmic locomotor activity in type I fin motoneuron. A: Intracellularly recorded activity of a type I fin motoneuron (mn) during fictive locomotion evoked by adding 100 FM NMDA to the perfusing solution. vr, Rhythmic locomotor adivity in the ventral root containing the axon of the fin motoneuron. B: Whole mount three dimensional reconstruction of the same type I fin motoneuron (mn). The cell was injected intracellularly with Lucifer Yellow from the recording elec-
trode and scanned in a confocal laser scanning microscope. Arrow indicates rostrocaudal direction. ax, motoneuron axon. C : Area corresponding to rectangle in A scanned at high magnification. D: Frontal projection from the image shown on C, obtained after a 90" rotation of the image volume. mn, Motoneuron; d, dendrite projecting into the dorsal column (dc); rs, reticulospinal axons; cc, central canal. Scale bars: A, 100bm; C,D, 5 0 ~ m .
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Fig. 6. Rhythmic locomotor activity in a type I1 fin motoneuron. A: Activity in a type I1 fin motoneuron during fictive locomotion. vr, Activity in the ventral root containing the axon of the fin motoneuron. B: 3-D reconstruction of the same cell as in A injected intracellularly with Lucifer Yellow and scanned in the confocal microscope. Arrow indicates
rostrocaudal direction. C,D: Frontal projections, scanned at high magnification, obtained after a 90" rotation of the image volume, showing the positions of the dendrites marked d l and d2 in B, in relation to the central canal (cc). mn, Motoneuron; ax, axon; dc, dorsal column; rs, reticulospinal axons. Scale bars: A, 100 pm; C, 25 pm; D, 50 IJ-m.
be evoked in the fin nerves upon stretch applied to the fin in rays (Fessard and Sand, '37) and monosynaptic EPSPs can be recorded in motoneurons during stimulation of the fin
nerves (Leonard et al., '78). It would seem likely that a similar type of receptor may exist in the dorsal fin of the lamprey. Trunk muscles in lower vertebrates do not contain
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muscle spindles (e.g., Gray, ’33; Roberts, ’69), however, in lamprey the intraspinal stretch receptor neurons may serve an analogous function (Viana Di Prisco et al., ’90). Muscle spindles have been found only in the jaw-closing musculature, adductor mandibulae, in the salmon (Maeda et al., ’83). Injection of HRP into dorsal fin muscles revealed close appositions between fin motoneuron dendrites and dorsal column axons, at least some of them belonging to dorsal cells (Figs. 1, 2). Lamprey dorsal cells have been classified into two different groups, “touch” and “pressure,” according to their responses to mechanical stimulation of the skin (Christenson et al., ’88). If monosynaptic effects to fin motoneurons are mediated by dorsal cells, it would appear likely that in the fin region these cells could serve as some type of stretch receptor providing movement feedback.
Activity of type I and type I1 motoneurons during fictive locomotion Dorsal fin muscles in the sea lamprey (Petromyzon) are active in antiphase to the burst activity of the ipsilateral myotomes during fictive locomotion (Buchanan and Cohen, ’82). Figures 5 and 6 show that both type I and type I1 fin motoneurons exhibit oscillation of the membrane potential in antiphase with the ipsilateral ventral root discharge, suggesting that both types of fin motoneurons may be driven by the contralateral part of the segmental locomotor network, and that they may receive similar synaptic input despite the differences in morphology. This antiphasic control may act to maintain the fin in an upright position during lateral movements. Large reticulospinal Muller neurons (11and Ms) provide a polysynaptic excitation of contralateral fin motoneurons in contrast to the monosynaptic excitation of ipsilateral myotomal motoneurons (Rovainen, ’78). It would appear likely, however, that the control of fin motoneurons is critical for rapid compensatory movements during a variety of conditions, and it is then expected that they should be under an efficient brain stem control. This aspect will be dealt with in a forthcoming study.
ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council, projects number: 3026 and 6815, and from Karolinska Institutets Fonder. Dr. 0. Shupliakov was supported from Svenska Institutet and by funds to S.G. We thank Professor G. Orlovski and Dr. S. Cullheim and Dr. L. Brodin for valuable comments on the manuscript.
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