312
Brain Research, 531 (1990)312 3i7 ffl~c~it:v
BRES 24353
Sexual dimorphisms in the vocal control system of a teleost fish: ultrastructure of neuromuscular junctions Amy Fluet* and Andrew Bass Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853 (U.S.A.)
(Accepted 24 July 1990) Key words: Neuromuscular junction; Synaptic vesicle density; Myelination; Terminal bouton
In the sound-generating fish, Porichthys notatus, large, nest-guarding 'Type I' males use their swimbladder 'drumming' muscles to produce acoustic communication signals. Females and another group of smaller sexually mature males ('Type II') have not been observed to produce sounds. Electron microscopywas used to compare the morphologyof the neuromuscular junctions in vocalizingType I males to those of Type II males and females. Significant differences were seen in synaptic vesicle density, terminal size, degree of terminal invagination below the muscle fiber surface, number of Schwann cell processes along the non-synapticboundary of boutons, and the number of boutons per innervation
site. The midshipman, Porichthys notatus, is a marine teleost fish which is of interest here for two reasons. First, it exhibits a sex-specific vocal (sonic) behavior, and second, there are two classes of sexually mature males 5"6. One class of large males (12.5 cm or greater in standard length) is referred to as 'Type I'. A second class of smaller males (6.5-12.5 cm) has been designated 'Type II'. In their summer breeding season, Type I males build nests in the intertidal zone and, by contracting a set of striated muscles attached to the lateral walls of their swimbladder, produce a variety of sounds 3. Sounds have never been heard from either Type II males or females, both of which are found in the nests of the Type I males during the breeding season 7. How are dramatic sex differences in sonic behavior reflected in the organization of the sonic system within the 3 sexually mature classes? Light and electron microscopic studies have already characterized dramatic interand intra-sexual dimorphisms in the structure of both the central motor pathway and the sonic muscle 5'6. Despite this, midbrain stimulation has also shown that all males (Type I and II) and all females can generate a rhythmic sonic discharge, although it is 20% higher in Type 1 males 4. The physiological demonstration of functionality in the sonic motor system of all 3 adult classes, in contrast with the absence of reported vocal behavior in Type II males and females, suggested that a peripheral component could act as a limiting factor in sound production. To address this, we examined the sonic neuromuscular
junction to describe its presynaptic ultrastructural characteristics in the 3 sexually mature classes. A preliminary report of these findings has appeared elsewhere 1°. Two hundred and fifty-eight terminal boutons were examined from 28 animals: 9 Type I males (15.4-20.5 cm), 5 Type II males (7.0-10.5 cm), and 14 females (10.5-16.5 cm). All analyses were made among fish taken directly from the nest during the breeding season, Tissue was prepared for transmission electron microscopy as previously described 5. Animals were sacrificed within 4-8 h of capture. They were peffused transcardially with a teleost Ringer's containing 1% sodium nitrite and heparin (10 units/ml), followed by a solution of 4.0% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (PB). Plastic embedded (Polybed 812) sections were first cut at 0.85/~m and stained with 1% Toluidine blue and then at intervals of 0.1 /~m. Thin sections were placed on copper grids, treated with 50 mM EDTA (to remove calcium precipitate9), and then stained with 1% uranyl acetate and Reynold's lead citrate. Stained thin sections were examined under a Philips 201 electron microscope. Terminal boutons were photographed at magnifications of 5000x and 10,000x and printed at a final magnification of 26,000x and 52,000x (resp.). These prints were the basis for all analyses. A planimeter (LASICO) was used to determine terminal area and perimeter, and contact widths. 'Contact width' was defined as the synaptic length along which a distinct pre- and postsynaptic membrane and a basal
* Present Address: Graduate Program in Neuroscience, Yale University, New Haven, CT 06510, U.S.A. Correspondence: A. Bass, Section of Neurobiology and Behavior, Cornell University, Seeley G. Mudd Hall, Ithaca, NY 14853, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
313 lamina were visible (see Figs. lb and 2b,c). Micrographs chosen for vesicle counts were not always delineated by a clear membrane border around their entire circumference. However, a visible contact area and boundary membrane were required for their inclusion. Comparisons were made using a grid (each square represented an area of 0.1069/~m 2) printed onto acetate. The grid was laid so that: (1) one edge bordered the area of synaptic contact, and (2) one box was centered over the active zone (or in the cases where no active zone was present, the area most heavily populated with vesicles). Data were collected from this centered grid in order to compare vesicle density. The degree of invagination was expressed as a percentage derived from the depth of the terminal beneath the muscle fiber surface (indicated by arrows in Fig. lb) as a fraction of the total maximal depth of the bouton. Terminal numbers were counted from micrographs. Schwann cell processes were quantified by counting clearly delineated layers around the non-synaptic region of the terminal. Glycogen density was only studied qualitatively: terminals were classified as having no glycogen, scattered glycogen granules, or clumped deposits (10 or more contiguous granules) of glycogen. A random subset of equal sample sizes from each animal was chosen for tests of statistical significance using an ANOVA. A test of least significant difference (LSD) was done to compare groups with significant F values.
General features of sonic neuromuscular junctions (NMJs). Examination of the NMJ's revealed some generalized features in all boutons (Figs. 1,2). Mitochondria and round, clear vesicles with diameters of 0.04-0.06 /zm were always present; dense core vesicles with diameters of 0.08/~m were found less frequently (not shown). In all classes, clear pre- and postsynaptic membranes were seen along with a basal lamina and active zones (sometimes more than one per bouton). These NMJ's, unlike those of other vertebrates, do not show postjunctional folds ~9. A number of traits were dimorphic, as described below. Glycogen deposits. Glycogen deposits (e.g. G, Fig. la) were found in 100% of the Type I (n = 57), 62% of the Type II (n = 34), and 90% of the female (n = 88), terminals analyzed. Type I males had a greater number of clumped deposits (32%) than Type II males (0%) or females (14%) which usually showed glycogen scattered throughout the terminals. Multiple boutons. Synaptic sites in both Type II males and females were more likely to have multiple boutons than Type I males ()~2 = 16.48 and 10.92, respectively, P < 0.001). When multiple boutons occurred, Type II males had more sites with greater than two boutons (33%) compared with female (19%) and Type I males (5%) (see Fig. 3a).
Terminal myelination. Typically, all boutons are covered along their outer, non-synaptic boundary by at least a single layer of Schwann cell cytoplasm (Figs. lb; 2b,c). Some boutons had 2-4 cytoplasmic layers in close apposition to one another, although they occurred more often in Type I males than in either females or Type II males (X2 = 24.84 and 35.91, respectively, P < 0.001). Also, these layers appeared more often in females than in Type II males (X2 = 4.15, P < 0.05). Terminal invaginations (Figs. lb, 2a, c; Table I). The extent to which terminals were invaginated into the surface of the muscle fiber was significantly different (F2,13o = 15.624; P < 0.001) among the 3 groups. A test of LSD showed that Type I males were significantly (P < 0.05) more invaginated than Type II males or females, which were not significantly different from each other. Terminal size (Figs. 1, 2a, c; Table I). There was a significant difference among the 3 groups for all measures of terminal size: perimeter (F2,12 3 = 5.49, P < 0.006), area (FE,x30 = 15.968; P < 0.0001), the ratio of contact width/perimeter (F2,n6 = 5.330, P < 0.007). Type I male terminals were significantly (LSD, P < 0.05) larger than those of either Type II males or females, which were not significantly different from each other. Vesicle density (Figs. lb, 2b-t~ Table I). The density of vesicles near the area of synaptic contact was significantly different (F2,92 = 25.376; P < 0.0001) among the 3 groups. Type I males had significantly (LSD, P < 0.05) fewer vesicles than either Type II males or females, which were also significantly different (P < 0.05) from each other. As with other morphological traits of the midshipman's sonic motor system, the NMJs of Type II males and females were similar to each other, but distinctly different from those of Type I males. The large increase in terminal area seen among Type I males was correlated with a smaller increase in terminal perimeter and relative contact width. Thus, terminals appear more spherical in Type I males. The trend for smaller terminal size going from Type I males to females to Type II males parallels the decrease in muscle fiber diameters. Smaller NMJ size is correlated with smaller fiber diameter in other motor systems as well n. Increased invagination of the terminals in Type I males could arise developmentally from a coincident expansion of the terminal and the muscle fiber which subsequently envelops the terminal. The presence of multiple boutons in both females and Type II males suggests a less mature system; multiple terminals at a site is considered to be an early juvenile characteristic 14,~s. Juvenile (i.e. sexually immature) midshipman do in fact have a high incidence of multiple boutons at innervation sites (A. Fluet and A. Bass, unpublished observations).
315
Fig. 2. Low (a), medium (b,c), and high (d) power electron micrographs showing two representative examples of the neuromuscular junction in Type II males (a,h) and females (c,d). Mitochondria ('M', b) and clear, round vesicles ('V', b,d) occur in all terminals. Type II males and females often have two or more terminals (1-3; a,b) at an innervation site (also see Fig. 3a). Terminals from both of these groups are less likely to be invaginated below the outer surface of the muscle fiber (MU; a-c). Note the lack of glycogen deposits (see Fig. la) and singular layer of Schwann cell wrapping (SC; h,c). The Z-line (Z; a) is indicated for comparison with the greatly enlarged band shown in Fig. 1B that characterizes Type I males 5. See text for dails. Other abbreviations: AZ, active zone. Bar scales: a = 1/tm; b,c = 0.5/~m; d = 0.1/~m.
Fig. 1. Low (a) and high (b) power electron micrographs of Type I male neuromuscular junctions (NMJs). NMJs are characterized by the presence of mitochondria ('M'; a,b) and clear, round vesicles ('V', b). Terminals are usually larger compared to those of Type II males and females (Fig. 2). Type I male NMJs often have large deposits of glycogen ('G'; a) and more than two layers of Schwann cell processes ('SC', b) along their non-synaptic border (also see Fig. 3b). Terminals in Type I males were more highly invaginated below the muscle fiber surface (MU) than those in Type II males or females (Fig. 2). The enlarged Z-line that characterizes the fibers of Type I males is also indicated ('Z', b). See text for details. Bar scales: a,b = 1/tm.
316
n=65
n=42
100
n=109
80 percent of terminal sites
60
•
1 terminal
40
• •
> 2 terminals 2 terminals
1
> 3 layers
1 i
3 layers 2 layers
2O
a
Type I
Type II
Female
100
80 percent of 60 terminals 40
| b
0
Type I
Type II
Female
Fig. 3. Histogram plots of measured characteristics of neuromuscular junctions in Type I males. Type 11 males and females, a: frequency histogram of the number of terminal boutons at innervation sites. 'n' equals the number of innervation sites examined, b: frequency histogram of the number of layers of Schwann cell wrappings for individual terminals. Type I males had a greater number of 3 or more layers. 'n' equals number of terminals. T h e g r e a t e r glycogen content in the NMJs of Type I males m a y b e related to the high metabolic d e m a n d s associated with sound production during the breeding
TABLE I Mean + S.D. for all significant characters (n = number of animals) Character
Type I
Invagination (%) Perimeter (/zm) Area ~m) z
78.18 + 15.09 35.45 + 18.22 39.01 + 17.0 (n = 7) (n = 4) (n = 9) 8.773 + 1.24 5.91 + 0.96 6.39 + 1.10 (n = 7) (n = 4) (n = 9) 34.97 + 8.10 14.29+ 4.74 16.59+ 4.58 (n = 7) (n = 40) (n = 11) 0.342 + 0.59 0.246 + 0.84 0.276 + 0.72 (n = 7) (n = 4) (n = 11) 13.07 + 2.67 19.34+ 4.01 21.92+ 3.37 (n = 7) (n = 4) (n = 11)
Contact width/ perimeter (%) Vesicle density (vesicles//~m2)
Type H
Female
season. In particular, Type I males g e n e r a t e ' h u m s ' which m a y last for up to 1 h, the longest duration of any r e p o r t e d sound from a fish 3'13. T h e unique properties of this sound, especially its long d u r a t i o n , m a y also explain the functional significance of o t h e r features of this species' sonic muscle fibers including its high mitochondrial density, enlarged Z-lines a n d extensive branching of the sarcoplasmic reticulum 5. Type I males have significantly fewer vesicles at synaptic sites than Type II males or females. Differences in vesicle n u m b e r m a y be indicative of synaptic efficacy l' s.12 or levels of use 15't6. W i t h o u t correlative neurophysiological d a t a synaptic efficacy cannot yet be evaluated. Given the known sex difference in vocal o u t p u t , this vesicle d i m o r p h i s m m a y be a t t r i b u t e d , at least in part, to differences in levels of activity. T h e r e also a p p e a r s to be a m o r e f u n d a m e n t a l , and p e r h a p s p e r m a n e n t , aspect of
317 this dimorphism. We c o m p a r e d nesting and non-nesting fish (captive specimens well fed and held in the laboratory for 1 - 4 months), which were not o b s e r v e d to b e vocal when h a n d held (unlike Type I males t a k e n from nests). A l t h o u g h too few specimens were e x a m i n e d for a valid statistical test, the m e a n vesicle densities of nonnesting fish were uniformly higher than those nesting fish of the same group (Type I = 23.1 + 1.56, n = 2 animals; Type II -- 33.2, n = 1; female = 25.9 + 4.36, n = 3). T h e decrease in vesicle density for all nesting groups suggests that the sonic m o t o r system of Type II males and females is in fact m o r e active during the breeding season, like that of Type I males. Activity d e p e n d e n t or seasonal changes in vesicle density highlights the i m p o r t a n c e of additional c o m p a r a t i v e studies b e t w e e n nesting and non-nesting fish to distinguish those traits linked to increased muscle fiber d i a m e t e r from those r e l a t e d to levels of use. O t h e r sexual d i m o r p h i s m s in the vocal control system of Porichthys include the d i a m e t e r of m o t o n e u r o n so-
m a t a , p r i m a r y dendrites and axons of the sonic m o t o r nucleus, which are 2 - 3 fold larger in Type I males than in females and Type II males 2'4"-6. M o r e p e r i p h e r a l l y , this
1 Akster, H.A., A comparative study of fibre type characteristics and terminal innervation in head and axial muscle of the carp (Cyprinus carpio I.): a histochemical and electron-microscopical study, Netherlands J. Zool., 33 (1983) 164-188. 2 Anderson, K.A. and Bass, A.H., Sexual polymorphisms in a 'vocalizing' fish: sonic motor axon number and size, Soc. Neurosci. Abstr., 15 (1989) 1137. 3 Bass, A.H., Sounds from the intertidal zone: vocalizing fish, Bioscience, 40 (1990) 249-258. 4 Bass, A. and Baker, R., Sexual dimorphisms in the vocal control system of a teleost fish: morphology of physiologicallyidentified neurons, J. Neurobiol., in press. 5 Bass, A.H. and Marchaterre, M.A., Sound-generating (sonic) motor system in a teleost fish (Porichthys notatus): sexual polymorphism in the ultrastructure of myofibrils, J. Comp. Neurol., 286 (1989) 141-153. 6 Bass, A.H. and Marchaterre, M.A., Sound-generation (sonic) motor system in a teleost fish (Porichthys notatus): sexual polymorphisms and general synaptology of sonic motor nucleus, J. Comp. Neurol., 286 (1989b) 154-169. 7 Brantley, R.K. and Bass, A.H., Intrasexual dimorphism in a sound producing fish: alternative reproductive morphs?, Soc. Neurosci. Abstr., 14 (1988) 691. 8 Fahim, M.A. and Robbins, N., Ultrastructural studies of young and old mouse neuromuscular junctions, J. Neurocytol., 11 (1982) 641-656. 9 Fifkova, E., Markham, J.A. and Delay, R.J., Calcium in the spine apparatus of dendritic spines in the dentate molecular layer, Brain Research, 266 (1983) 163-168. 10 Fluet, A. and Bass, A., Sexual polmorphisms in a 'vocalizing' fish: sonic motor axon terminals, Soc. Neurosci. Abstr., 15 (1989) 1137.
11 Grinnell, A.D. and Herrera, A.A., Specificity and plasticity of neuromuscular connections: long-term regulation of motoneuron function, Prog. Neurobiol., 17 (1981) 203-282. 12 Herrera, A.A., Grinnell, A.D. and Wolowske, B., Ultrastructural correlates of experimentally altered transmitter release in frog motor nerve terminals, Neuroscience, 16 (1985) 491-500. 13 Ibara, R.M., Penny, L.T., Ebeling, A.W., van Dykhuizen, G. and Cailliet, G., The mating call of the plainfin midshipman fish, Porichthys notatus. In D.L.G. Noakes et al. (Eds.), Predators and Prey in Fishes, The Hague, Dr. Junk Publishers, 1983, pp. 205-212. 14 Lichtman, J.W. and Purves, D., Regulation of the number of axons that innervate target cells. In Garrod and Feldman (Eds.), Development in the Nervous System, Cambridge University Press, 1980, pp. 233-243. 15 Palacios-Pru, E., Mendoza, R.V., Palacios, L. and Colasante, C., Morphological changes in neuromuscular junctions during exercise, J. Neurosci. Res., 9 (1983) 371-380. 16 Palacios-Pru, E. and Colasante, C., Ultrastructural reversible changes in fish neuromuscular junctions after chronic exercise, J. Neurosci. Res., 19 (1988) 245-251. 17 Purves, D., The formation and maintenance of synaptic connections. In G.S. Stent (Ed.), Function and Formation of Neural Systems, Dahlem Konferenzen, 1977, pp. 21-49. 18 Ronnevi, L.-O., Spontaneous phagocytosis of boutons on spinal motoneurons during early postnatal development. An electron microscopical study in the cat, J. Neurocytol., 6 (1977) 487-504. 19 Salpeter, M.M., Vertebrate neuromuscular junctions: general morphology, molecular organization, and functional consequences. In M.M. Salpeter (Ed.), The Vertebrate Neuromuscular Junction, New York, Alan R. Liss, 1987, pp. 1-54.
includes sonic muscle mass, muscle fiber d i a m e t e r , and myofibril dimensions, all of which are g r e a t e r in Type I males 5'6. These differences parallel those of the NMJs. Together, the results suggest that all elements of the ' n e u r o m u s c u l a r c o m p a r t m e n t ' of this m o t o r p a t h w a y are p r o p o r t i o n a t e l y larger in Type I males. A s i d e from dimorphisms in size, the NMJs o f Type II males and females r e s e m b l e those of Type I males. This further implies that the sonic muscle itself m a y be placing limitations on the o u t p u t of the sonic m o t o r system in Type II males and females.
Thanks to M. Marchaterre for help with the electron microscopy, M. Grober and J. Ackerman for statistical advice, R. Brantley, M. Grober for helpful comments, and S. Mancil for typing. Supported by NSF BNS8708559 and NIH NS19942.
Brain Research, 531 (1990)312 3i7 ffl~c~it:v
BRES 24353
Sexual dimorphisms in the vocal control system of a teleost fish: ultrastructure of neuromuscular junctions Amy Fluet* and Andrew Bass Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853 (U.S.A.)
(Accepted 24 July 1990) Key words: Neuromuscular junction; Synaptic vesicle density; Myelination; Terminal bouton
In the sound-generating fish, Porichthys notatus, large, nest-guarding 'Type I' males use their swimbladder 'drumming' muscles to produce acoustic communication signals. Females and another group of smaller sexually mature males ('Type II') have not been observed to produce sounds. Electron microscopywas used to compare the morphologyof the neuromuscular junctions in vocalizingType I males to those of Type II males and females. Significant differences were seen in synaptic vesicle density, terminal size, degree of terminal invagination below the muscle fiber surface, number of Schwann cell processes along the non-synapticboundary of boutons, and the number of boutons per innervation
site. The midshipman, Porichthys notatus, is a marine teleost fish which is of interest here for two reasons. First, it exhibits a sex-specific vocal (sonic) behavior, and second, there are two classes of sexually mature males 5"6. One class of large males (12.5 cm or greater in standard length) is referred to as 'Type I'. A second class of smaller males (6.5-12.5 cm) has been designated 'Type II'. In their summer breeding season, Type I males build nests in the intertidal zone and, by contracting a set of striated muscles attached to the lateral walls of their swimbladder, produce a variety of sounds 3. Sounds have never been heard from either Type II males or females, both of which are found in the nests of the Type I males during the breeding season 7. How are dramatic sex differences in sonic behavior reflected in the organization of the sonic system within the 3 sexually mature classes? Light and electron microscopic studies have already characterized dramatic interand intra-sexual dimorphisms in the structure of both the central motor pathway and the sonic muscle 5'6. Despite this, midbrain stimulation has also shown that all males (Type I and II) and all females can generate a rhythmic sonic discharge, although it is 20% higher in Type 1 males 4. The physiological demonstration of functionality in the sonic motor system of all 3 adult classes, in contrast with the absence of reported vocal behavior in Type II males and females, suggested that a peripheral component could act as a limiting factor in sound production. To address this, we examined the sonic neuromuscular
junction to describe its presynaptic ultrastructural characteristics in the 3 sexually mature classes. A preliminary report of these findings has appeared elsewhere 1°. Two hundred and fifty-eight terminal boutons were examined from 28 animals: 9 Type I males (15.4-20.5 cm), 5 Type II males (7.0-10.5 cm), and 14 females (10.5-16.5 cm). All analyses were made among fish taken directly from the nest during the breeding season, Tissue was prepared for transmission electron microscopy as previously described 5. Animals were sacrificed within 4-8 h of capture. They were peffused transcardially with a teleost Ringer's containing 1% sodium nitrite and heparin (10 units/ml), followed by a solution of 4.0% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (PB). Plastic embedded (Polybed 812) sections were first cut at 0.85/~m and stained with 1% Toluidine blue and then at intervals of 0.1 /~m. Thin sections were placed on copper grids, treated with 50 mM EDTA (to remove calcium precipitate9), and then stained with 1% uranyl acetate and Reynold's lead citrate. Stained thin sections were examined under a Philips 201 electron microscope. Terminal boutons were photographed at magnifications of 5000x and 10,000x and printed at a final magnification of 26,000x and 52,000x (resp.). These prints were the basis for all analyses. A planimeter (LASICO) was used to determine terminal area and perimeter, and contact widths. 'Contact width' was defined as the synaptic length along which a distinct pre- and postsynaptic membrane and a basal
* Present Address: Graduate Program in Neuroscience, Yale University, New Haven, CT 06510, U.S.A. Correspondence: A. Bass, Section of Neurobiology and Behavior, Cornell University, Seeley G. Mudd Hall, Ithaca, NY 14853, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
313 lamina were visible (see Figs. lb and 2b,c). Micrographs chosen for vesicle counts were not always delineated by a clear membrane border around their entire circumference. However, a visible contact area and boundary membrane were required for their inclusion. Comparisons were made using a grid (each square represented an area of 0.1069/~m 2) printed onto acetate. The grid was laid so that: (1) one edge bordered the area of synaptic contact, and (2) one box was centered over the active zone (or in the cases where no active zone was present, the area most heavily populated with vesicles). Data were collected from this centered grid in order to compare vesicle density. The degree of invagination was expressed as a percentage derived from the depth of the terminal beneath the muscle fiber surface (indicated by arrows in Fig. lb) as a fraction of the total maximal depth of the bouton. Terminal numbers were counted from micrographs. Schwann cell processes were quantified by counting clearly delineated layers around the non-synaptic region of the terminal. Glycogen density was only studied qualitatively: terminals were classified as having no glycogen, scattered glycogen granules, or clumped deposits (10 or more contiguous granules) of glycogen. A random subset of equal sample sizes from each animal was chosen for tests of statistical significance using an ANOVA. A test of least significant difference (LSD) was done to compare groups with significant F values.
General features of sonic neuromuscular junctions (NMJs). Examination of the NMJ's revealed some generalized features in all boutons (Figs. 1,2). Mitochondria and round, clear vesicles with diameters of 0.04-0.06 /zm were always present; dense core vesicles with diameters of 0.08/~m were found less frequently (not shown). In all classes, clear pre- and postsynaptic membranes were seen along with a basal lamina and active zones (sometimes more than one per bouton). These NMJ's, unlike those of other vertebrates, do not show postjunctional folds ~9. A number of traits were dimorphic, as described below. Glycogen deposits. Glycogen deposits (e.g. G, Fig. la) were found in 100% of the Type I (n = 57), 62% of the Type II (n = 34), and 90% of the female (n = 88), terminals analyzed. Type I males had a greater number of clumped deposits (32%) than Type II males (0%) or females (14%) which usually showed glycogen scattered throughout the terminals. Multiple boutons. Synaptic sites in both Type II males and females were more likely to have multiple boutons than Type I males ()~2 = 16.48 and 10.92, respectively, P < 0.001). When multiple boutons occurred, Type II males had more sites with greater than two boutons (33%) compared with female (19%) and Type I males (5%) (see Fig. 3a).
Terminal myelination. Typically, all boutons are covered along their outer, non-synaptic boundary by at least a single layer of Schwann cell cytoplasm (Figs. lb; 2b,c). Some boutons had 2-4 cytoplasmic layers in close apposition to one another, although they occurred more often in Type I males than in either females or Type II males (X2 = 24.84 and 35.91, respectively, P < 0.001). Also, these layers appeared more often in females than in Type II males (X2 = 4.15, P < 0.05). Terminal invaginations (Figs. lb, 2a, c; Table I). The extent to which terminals were invaginated into the surface of the muscle fiber was significantly different (F2,13o = 15.624; P < 0.001) among the 3 groups. A test of LSD showed that Type I males were significantly (P < 0.05) more invaginated than Type II males or females, which were not significantly different from each other. Terminal size (Figs. 1, 2a, c; Table I). There was a significant difference among the 3 groups for all measures of terminal size: perimeter (F2,12 3 = 5.49, P < 0.006), area (FE,x30 = 15.968; P < 0.0001), the ratio of contact width/perimeter (F2,n6 = 5.330, P < 0.007). Type I male terminals were significantly (LSD, P < 0.05) larger than those of either Type II males or females, which were not significantly different from each other. Vesicle density (Figs. lb, 2b-t~ Table I). The density of vesicles near the area of synaptic contact was significantly different (F2,92 = 25.376; P < 0.0001) among the 3 groups. Type I males had significantly (LSD, P < 0.05) fewer vesicles than either Type II males or females, which were also significantly different (P < 0.05) from each other. As with other morphological traits of the midshipman's sonic motor system, the NMJs of Type II males and females were similar to each other, but distinctly different from those of Type I males. The large increase in terminal area seen among Type I males was correlated with a smaller increase in terminal perimeter and relative contact width. Thus, terminals appear more spherical in Type I males. The trend for smaller terminal size going from Type I males to females to Type II males parallels the decrease in muscle fiber diameters. Smaller NMJ size is correlated with smaller fiber diameter in other motor systems as well n. Increased invagination of the terminals in Type I males could arise developmentally from a coincident expansion of the terminal and the muscle fiber which subsequently envelops the terminal. The presence of multiple boutons in both females and Type II males suggests a less mature system; multiple terminals at a site is considered to be an early juvenile characteristic 14,~s. Juvenile (i.e. sexually immature) midshipman do in fact have a high incidence of multiple boutons at innervation sites (A. Fluet and A. Bass, unpublished observations).
315
Fig. 2. Low (a), medium (b,c), and high (d) power electron micrographs showing two representative examples of the neuromuscular junction in Type II males (a,h) and females (c,d). Mitochondria ('M', b) and clear, round vesicles ('V', b,d) occur in all terminals. Type II males and females often have two or more terminals (1-3; a,b) at an innervation site (also see Fig. 3a). Terminals from both of these groups are less likely to be invaginated below the outer surface of the muscle fiber (MU; a-c). Note the lack of glycogen deposits (see Fig. la) and singular layer of Schwann cell wrapping (SC; h,c). The Z-line (Z; a) is indicated for comparison with the greatly enlarged band shown in Fig. 1B that characterizes Type I males 5. See text for dails. Other abbreviations: AZ, active zone. Bar scales: a = 1/tm; b,c = 0.5/~m; d = 0.1/~m.
Fig. 1. Low (a) and high (b) power electron micrographs of Type I male neuromuscular junctions (NMJs). NMJs are characterized by the presence of mitochondria ('M'; a,b) and clear, round vesicles ('V', b). Terminals are usually larger compared to those of Type II males and females (Fig. 2). Type I male NMJs often have large deposits of glycogen ('G'; a) and more than two layers of Schwann cell processes ('SC', b) along their non-synaptic border (also see Fig. 3b). Terminals in Type I males were more highly invaginated below the muscle fiber surface (MU) than those in Type II males or females (Fig. 2). The enlarged Z-line that characterizes the fibers of Type I males is also indicated ('Z', b). See text for details. Bar scales: a,b = 1/tm.
316
n=65
n=42
100
n=109
80 percent of terminal sites
60
•
1 terminal
40
• •
> 2 terminals 2 terminals
1
> 3 layers
1 i
3 layers 2 layers
2O
a
Type I
Type II
Female
100
80 percent of 60 terminals 40
| b
0
Type I
Type II
Female
Fig. 3. Histogram plots of measured characteristics of neuromuscular junctions in Type I males. Type 11 males and females, a: frequency histogram of the number of terminal boutons at innervation sites. 'n' equals the number of innervation sites examined, b: frequency histogram of the number of layers of Schwann cell wrappings for individual terminals. Type I males had a greater number of 3 or more layers. 'n' equals number of terminals. T h e g r e a t e r glycogen content in the NMJs of Type I males m a y b e related to the high metabolic d e m a n d s associated with sound production during the breeding
TABLE I Mean + S.D. for all significant characters (n = number of animals) Character
Type I
Invagination (%) Perimeter (/zm) Area ~m) z
78.18 + 15.09 35.45 + 18.22 39.01 + 17.0 (n = 7) (n = 4) (n = 9) 8.773 + 1.24 5.91 + 0.96 6.39 + 1.10 (n = 7) (n = 4) (n = 9) 34.97 + 8.10 14.29+ 4.74 16.59+ 4.58 (n = 7) (n = 40) (n = 11) 0.342 + 0.59 0.246 + 0.84 0.276 + 0.72 (n = 7) (n = 4) (n = 11) 13.07 + 2.67 19.34+ 4.01 21.92+ 3.37 (n = 7) (n = 4) (n = 11)
Contact width/ perimeter (%) Vesicle density (vesicles//~m2)
Type H
Female
season. In particular, Type I males g e n e r a t e ' h u m s ' which m a y last for up to 1 h, the longest duration of any r e p o r t e d sound from a fish 3'13. T h e unique properties of this sound, especially its long d u r a t i o n , m a y also explain the functional significance of o t h e r features of this species' sonic muscle fibers including its high mitochondrial density, enlarged Z-lines a n d extensive branching of the sarcoplasmic reticulum 5. Type I males have significantly fewer vesicles at synaptic sites than Type II males or females. Differences in vesicle n u m b e r m a y be indicative of synaptic efficacy l' s.12 or levels of use 15't6. W i t h o u t correlative neurophysiological d a t a synaptic efficacy cannot yet be evaluated. Given the known sex difference in vocal o u t p u t , this vesicle d i m o r p h i s m m a y be a t t r i b u t e d , at least in part, to differences in levels of activity. T h e r e also a p p e a r s to be a m o r e f u n d a m e n t a l , and p e r h a p s p e r m a n e n t , aspect of
317 this dimorphism. We c o m p a r e d nesting and non-nesting fish (captive specimens well fed and held in the laboratory for 1 - 4 months), which were not o b s e r v e d to b e vocal when h a n d held (unlike Type I males t a k e n from nests). A l t h o u g h too few specimens were e x a m i n e d for a valid statistical test, the m e a n vesicle densities of nonnesting fish were uniformly higher than those nesting fish of the same group (Type I = 23.1 + 1.56, n = 2 animals; Type II -- 33.2, n = 1; female = 25.9 + 4.36, n = 3). T h e decrease in vesicle density for all nesting groups suggests that the sonic m o t o r system of Type II males and females is in fact m o r e active during the breeding season, like that of Type I males. Activity d e p e n d e n t or seasonal changes in vesicle density highlights the i m p o r t a n c e of additional c o m p a r a t i v e studies b e t w e e n nesting and non-nesting fish to distinguish those traits linked to increased muscle fiber d i a m e t e r from those r e l a t e d to levels of use. O t h e r sexual d i m o r p h i s m s in the vocal control system of Porichthys include the d i a m e t e r of m o t o n e u r o n so-
m a t a , p r i m a r y dendrites and axons of the sonic m o t o r nucleus, which are 2 - 3 fold larger in Type I males than in females and Type II males 2'4"-6. M o r e p e r i p h e r a l l y , this
1 Akster, H.A., A comparative study of fibre type characteristics and terminal innervation in head and axial muscle of the carp (Cyprinus carpio I.): a histochemical and electron-microscopical study, Netherlands J. Zool., 33 (1983) 164-188. 2 Anderson, K.A. and Bass, A.H., Sexual polymorphisms in a 'vocalizing' fish: sonic motor axon number and size, Soc. Neurosci. Abstr., 15 (1989) 1137. 3 Bass, A.H., Sounds from the intertidal zone: vocalizing fish, Bioscience, 40 (1990) 249-258. 4 Bass, A. and Baker, R., Sexual dimorphisms in the vocal control system of a teleost fish: morphology of physiologicallyidentified neurons, J. Neurobiol., in press. 5 Bass, A.H. and Marchaterre, M.A., Sound-generating (sonic) motor system in a teleost fish (Porichthys notatus): sexual polymorphism in the ultrastructure of myofibrils, J. Comp. Neurol., 286 (1989) 141-153. 6 Bass, A.H. and Marchaterre, M.A., Sound-generation (sonic) motor system in a teleost fish (Porichthys notatus): sexual polymorphisms and general synaptology of sonic motor nucleus, J. Comp. Neurol., 286 (1989b) 154-169. 7 Brantley, R.K. and Bass, A.H., Intrasexual dimorphism in a sound producing fish: alternative reproductive morphs?, Soc. Neurosci. Abstr., 14 (1988) 691. 8 Fahim, M.A. and Robbins, N., Ultrastructural studies of young and old mouse neuromuscular junctions, J. Neurocytol., 11 (1982) 641-656. 9 Fifkova, E., Markham, J.A. and Delay, R.J., Calcium in the spine apparatus of dendritic spines in the dentate molecular layer, Brain Research, 266 (1983) 163-168. 10 Fluet, A. and Bass, A., Sexual polmorphisms in a 'vocalizing' fish: sonic motor axon terminals, Soc. Neurosci. Abstr., 15 (1989) 1137.
11 Grinnell, A.D. and Herrera, A.A., Specificity and plasticity of neuromuscular connections: long-term regulation of motoneuron function, Prog. Neurobiol., 17 (1981) 203-282. 12 Herrera, A.A., Grinnell, A.D. and Wolowske, B., Ultrastructural correlates of experimentally altered transmitter release in frog motor nerve terminals, Neuroscience, 16 (1985) 491-500. 13 Ibara, R.M., Penny, L.T., Ebeling, A.W., van Dykhuizen, G. and Cailliet, G., The mating call of the plainfin midshipman fish, Porichthys notatus. In D.L.G. Noakes et al. (Eds.), Predators and Prey in Fishes, The Hague, Dr. Junk Publishers, 1983, pp. 205-212. 14 Lichtman, J.W. and Purves, D., Regulation of the number of axons that innervate target cells. In Garrod and Feldman (Eds.), Development in the Nervous System, Cambridge University Press, 1980, pp. 233-243. 15 Palacios-Pru, E., Mendoza, R.V., Palacios, L. and Colasante, C., Morphological changes in neuromuscular junctions during exercise, J. Neurosci. Res., 9 (1983) 371-380. 16 Palacios-Pru, E. and Colasante, C., Ultrastructural reversible changes in fish neuromuscular junctions after chronic exercise, J. Neurosci. Res., 19 (1988) 245-251. 17 Purves, D., The formation and maintenance of synaptic connections. In G.S. Stent (Ed.), Function and Formation of Neural Systems, Dahlem Konferenzen, 1977, pp. 21-49. 18 Ronnevi, L.-O., Spontaneous phagocytosis of boutons on spinal motoneurons during early postnatal development. An electron microscopical study in the cat, J. Neurocytol., 6 (1977) 487-504. 19 Salpeter, M.M., Vertebrate neuromuscular junctions: general morphology, molecular organization, and functional consequences. In M.M. Salpeter (Ed.), The Vertebrate Neuromuscular Junction, New York, Alan R. Liss, 1987, pp. 1-54.
includes sonic muscle mass, muscle fiber d i a m e t e r , and myofibril dimensions, all of which are g r e a t e r in Type I males 5'6. These differences parallel those of the NMJs. Together, the results suggest that all elements of the ' n e u r o m u s c u l a r c o m p a r t m e n t ' of this m o t o r p a t h w a y are p r o p o r t i o n a t e l y larger in Type I males. A s i d e from dimorphisms in size, the NMJs o f Type II males and females r e s e m b l e those of Type I males. This further implies that the sonic muscle itself m a y be placing limitations on the o u t p u t of the sonic m o t o r system in Type II males and females.
Thanks to M. Marchaterre for help with the electron microscopy, M. Grober and J. Ackerman for statistical advice, R. Brantley, M. Grober for helpful comments, and S. Mancil for typing. Supported by NSF BNS8708559 and NIH NS19942.
