FINE STRUCTURE OF THE SWlMMING PADDLE OPENER MUSCLE AND ITS INNERVATION IN THE BLUE CRAB, Callinectes sapidus by Katya Jennifer Honsa A thesis subrnitted in confotmity wiih the requirements for the degree of Mater of Science in Zoology Graduate Department of Zoology, University of Toronto O Copyright by Katya Jennifer Honsa, 200 1.
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FINE STRUCTURE OF THE SWlMMING PADDLE
OPENER MUSCLE AND ITS INNERVATION IN THE
BLUE CRAB, Callinectes sapidus
by Katya Jennifer Honsa
A thesis subrnitted in confotmity wiih the requirements for the degree of Mater of Science in Zoology
Graduate Department of Zoology, University of Toronto
O Copyright by Katya Jennifer Honsa, 200 1.
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FINE S T R U m OF THE SWiMMiNG PADDLE OPENER MUSCLE AND ES
INNERVATION IN THE BLüE CRAB. Callinectes sapidus
An abstract submitted in conformity with the requirements for the degree of Master of Science in Zooiogy
in the Graduate Department of Zoology University of Toronto
Katy a Jennifer Honsa, 200 L
ABSTRACT
The fifth pereiopod of the bIue crab is a specialized swimming limb, the dactyl of
which acts as a rowing paddle. Abduction of this paddle is brought about by contractions of
the opener muscle that has assumed a specialized roIe as a fairly equal antagonist to the
closer muscle (the adductor of the daccyl). Elecuon microscopic examinations of the cenual
region of the opener muscle reveaIed typically slow fibres with long sarcomere lengths and a
high actin to myosin ratio. Muscle fibres were highly innervated with a majonty having
multiple innervation sites each with an infiibitory and excitatory terminal profile. This large
mount of innervation may piay a role in ensunng fatigue resistance and fine control for the
specialized muscle. Inhibitory tenninds originated synapses that were two thirds
neuromuscular, inhibiting the muscle directiy, and one third am-axonal, inhibiting the
excitatory axon itself. This is an unusually high degree of presynaptic inhibition possibly
associated with the fine conuol of the paddle limb. The ovenvhelming rnajonty of synapses
fonned by the excitatory terminal were neuromuscular, in keeping with its primary function
of making the muscLe contract. A very unusual synapse was also recorded in every serially
sectioned area examined: axo-axond synapses polarized from the excitatory terminal to the
inhibitory terminal, suggesting putative presynaptic excitation of the inhibitory nerve
tenninals. Altogether, the innervation of the blue crab swimming paddle opener muscie
indicated specializations for its function as a highly active muscle rapidly contracting for
prolonged periods of t h e .
ACKNOWLEDGEMENTS
It is with great gratitude that 1 thank Dr. C.K. Govind for giving me the guidance
and opportunity to teach, learn and grow beyond what 1 had ever anticipated. To Joanne
Pearce for her incredible patience and brilliance that has helped to make this expenence a
wonderful one. To Raymond who was there when something wasn't quite working, but
was aiways able to fix it. To the people who were with me in the lab, Nadine, Ros,
Asheer and Rahim, who listened and shared. Also, to other fellow students who made me
srnile and laugh, Najeeb, Allison, Lori, Joanne, Manav, Christine, Andrea, Andrea, and
Sandi. And to Herman, Scott, CC, Rhoda, Shintaro, and Richard who helped me grow
spiritually. Thank-you aiso to the students that 1 was so fonunate to teach, 1 have learned
many things from you.
To Jaykob, what sunshine you have brought me; thank-you Robena and Yola for
tnisting me with your beautiful (great-) grandson. To my absolutely temfic hiends who 1
love, thank-you for everything, without you, laughter would not come so easily,
Meaghan, Todd, Carlene, Stacey, Shane, Sam, Thane, Kevin, Kevin, and Ferris, and the
beautiful Angela and Laura who have loved me for 20 years. To the people 1 have shared
a house with Amy, Ray, Jess, Tina, Kevin, Cristiane and Leigh, thank-you. Also, thank-
you [O the Welch's for always listening, and to Chris and Scott for taking such good care
of me.
To my beautiful farnily who have shown me so much and have Listened to me
dream. Thank-you especially mum, dad, Ali and Graham, you are incredible, and
brilliant and so much more patient than 1 cm ever hope CO be. And thank-you God for
answering my many prayers. Thank-you for absolutely everything.
active zones for trammitter release (Atwood and Lnenicka, 1986). Dense bars were
present at most of the synapses although 37% of excitatory and 36% of inhibitory
synapses that were completely sectioned lacked a dense bar.
Excitatory neuromuscular synapses contained 190 dense bars ranging in length
from 0.04 to 0.195 Fm with a mean length of 0.092 Pm (Table 3). However only 1 14 of
these dense bars were located in complete synapses, The number of dense bars per
complete synapse ranged from zero to six with the majority having at Ieast one dense bar
and 21% of these having three or more (Fig. 12).
Inhibitory neuromuscular synapses also contained dense bars, however these
tended to be longer than excitatory neuromuscular dense bars (Table 3). In inhibitory
neuromuscular synapses, 74 dense bars were located ranging in length from 0.04 to 0.295
pm with a mean length of 0.13 lpm, however only 53 of these dense bars were located in
complete synapses. The number of dense bars per synapse ranged from zero to eight with
the majority of inhibitory neuromuscular synapses having at least one dense bar and 18%
of these having three or more (Fig. 12).
In conclusion, more dense bars were found in the excitatory neuromusculiu
synapses than in inhibitory neuromuscular synapses, however, these had a smaIler range
in size and were 30% smaller than the dense bars from inhibitory neuromuscular
synapses (Table 3). When cornparhg the number of dense bars per synapse for the
Table 3. Quantitative analysis of dense bars from excitatory and inhibitory axons to the
opener muscle in the blue crab swSmmiag paddle.
Excitatory (E)
Terminal length ( p ) 1 18.177
Total number of dense bars 190
Total dense bar length 17.425
Mean dense bar length (pn) *0.092IO.W0
Dense bar lengthl pn terminal O. 147
Number of dense bars in complete synapses I 14
Dense bar number/complete synapse 1.32W.8 16
Dense bar length/p2 of synapse O. 146
Inhibitory (1) E/i nt io
* statistically significant (p < .05)
Figure 12. Histojpm of percentage of complete synapses containhg different numbets of dense
bars frorn O to 5 in excitatory and inhibitory neurornuscular synapses of the blue crab swimming
padde opener muscle.
~xciwto; 0 Lnhibitory
-
--
O 1 2 3 4 5+
Nuniber of dense bars per complete synapse
excitatory neuromuscular and the inhibitory neurornuscular synapses the two had sirnilac
values of 1.33 and 1.36 respectiveiy. Also, when dense bar length per terminal length
was compared the value was 12% smaller in the excitatory tenninds than in the
inhibitory terminals (O. 146 Pm for excitatory neuromuscular terminah and 0.165 pm for
inhibitory neuromuscular terrninals).
E. Axo-axonal Synapses
Two types of axo-axonal synapses were documented, the more cornmon type
k ing the inhibitory axo-axonal synapse. At inhibitory axo-axond synapses, the synapse
is polarized from the inhibitory terminal while the target ceil is the excitatory mon.
Inhibitory axo-axonal synapses occurred in each of the inhibitory terminals examined. A
very few of these synapses occurred on interdigitations of the excitatory mon into the
inhibitory axon (Fig. 8). The majority of inhibitory axo-axonal synapses occurred when
the two terminals were simply adjacent to each other. The second type of axo-axonai
synapse was the excitatory axo-axonal synapse and occurred h m the excitatory terminai
ont0 the inhibitory terminal (Fig. 9D-F, 10). At least one excitatory axo-axonal synapse
was found in each of the four serially-sectioned innervation sites examined.
Quantiîative analysis was done for the inhibitory and excitatory axo-axonai
synapses from the serial micrographs of the fout innervation sites (Table 4). There were 5
excitatory and 25 inhibitory axo-axonal synapses. Of the synapses that were identified, 4
synapses were comple~ely serially sectioned for the excitatory axon and 22 for the
inhibitory mon. The excitatory synapses ranged in size between 0.013 to 0.109 pm2 with
a mean area of 0.016 The inhibitory axo-axonal synapses ranged in size from 0.008
Table 4. Quantitative analysis of excitatory and inbibitory axo-axonal synapses in the
opener muscle to the blue crab swimming paddle
Excitor Inhibitor En ratio
Terminal length (p)
Total nurnber of synapses
~d synaptic ana (pn2)
Synaptic aredterminal length
Nurnber of complete synapses
2 Mean synaptic area (pm ) for complete synapses
to 2.41 pm2 with a mean area of 0.136 lnhibitory axo-axonal synapses were
therefore on average 6.5 times larger than excitatory axo-axonal synapses with the largest
inhibitory axo-axonal synapse approximately twenty tirnes the size of the largest axo-
axonal synapses from the excitatory terminal. When the surface area of synapses was
normalized to the terminal length sampled, the excitatory terminals had a very much
smaller synaptic area per terminal length (0.001 pm) than the inhibitory terminals (O. 15
prn) as reflected in the ratio between the two types (0.007).
Following the definition of Shepherd (1974), that a reciprocal synapse refers to an
arrangement between neural eiements in which chernical transmission is in opposite
directions at two adjacent or separate synapses, the excitatory axo-axonal synapses were
identified as one half of a reciprocai synapse, since each of the inhibitory terminals had
synaptic contact ont0 the excitatory terminals. Thus at each site of excitatory axo-axonal
synapses, a reciprocai synapse wiu occuning.
Like neuromuscuiar synapses, the majority of am-axonal synapses contain dense
bars. These dense bars are sirnilar in appearance to the neurornuscular dense bars,
however respective of terminai types the dense bars tend to be longer in the axo-axonal
synapses. Thirty dense bars were Located in inhibitory axo-axonal synapses with a range
in length from 0.07 to 0.41 pn and a mean Iength of 0.18 1 Pm (Table 5). Although 32%
of the inhibitory axo-axonal synapses Iacked a dense bar, 33% had between two and five
dense bars per synapse (Fig. 13). In the rnuch smaller excitatory axo-axonal synapses
four dense bars were found that ranged in length from 0.05 to 0.15 prn and had a mean
length of 0.1 16 Pm. The range and mean dense bar length is smaller for the excitatory
axo-axonai synapses than the inhibitory axo-axonal synapses. Twenty-five percent of the
excitatory axo-axonal synapses lacked a dense bar with the remainder having either one
or two (Fig. 13). Moreover, the dense bar number per synapse is twenty-eight percent
smdler in the excitatory terminals than the inhibitory terminais, with a value of 1.00 for
the excitatory terminals and 1.38 for the inhibitory terrninals (Table 5). Also the dense
bar length per terminal length is 10 times smailer in the excitatory terminals, with a value
of 0.0 1 1 for the excitatory terminals and 0.13 1 for the inhibitory terminals. Synaptic
strength based on dense bar distribution was four times greater in excitatory synapses,
with dense bar length per synapse values of 2.73 for excitatory synapses and 0.88 for
inhibitory synapses.
Although the axo-axonal synapses are smailer than the neurornuscular synapses,
the dense bar data indicates that the axo-axond synapses have a high capacity for
transrnitter release.
Table 5. Quantitative analysis of dense bars in excitatory and inbibitory axo-axonal
synapses in the opener muscle to the blue crab swimming paddle
Tenninal length ( p n )
Total number of dense bars
Total dense bar length (pn)
Mean dense bar length (pm)
Dense bar lengtfv pn terminai
Number of dense bars in complete synapses
Dense bar nurnber/synapse
Dense bar length/pd of synapse
Excitor
118.177
4
0.365
O. 1 16iO.055
0.01 12
4
l.00I 1 A83
2.7252
Inhibitor
41.4
30
5.420
0.18 1 d M O
O. 1309
30
1.381 1.678
0.8790
En ratio
3.855
0.133
0.086
0.64 1
0.086
0.133
0.725
4.176
Figure 13. Histogram of percentage of complete synapses containing different numbers of dense
bars h m O to 3 in excitatory and inhibitory axo-axonal synapses of the biue cnb swimming
paddle opener muscle.
O 1 2 3+
Number of dense bars per romplete synapse
Discussion
A. Opener Muscle
in the crayfish walking limb, the opener muscle is smaller than the antagonistic
closer muscle, indicating that the focal role in movement of the dacryl is played by the
closer and not the opener. On examination of the size of the opener and closer muscles in
the blue crab swimming paddle. it was found that when the two run concurrently, the
opener was 1.5 times larger in girth than the closer. Hypertrophy of the opener muscle is
an indication of the function it pIays as a fairly equal antagonist in the
abductionhdduction of the swimming paddle. This is seen in the swimrning motion of
the blue crab where the dactyl is rapidly abducting and adducting for prolonged penods
of time. Electron microscopy analysis on these fibres revealed properties typicai of slow
muscle: long sarcomeres, thick, wavy and irregularly aligned Z-lines, and a high thin-to-
thick filament ratio. These findings corroborate unpubkished enzyme histochemistry and
light microscopy data (Govind and Pearce. unpubtished). Enzyme histochemistry with
NADH-diaphorase and ATPase on the opener fibres revealed staining intensities typicai
of slow fibres, while light microscopy examinations revealed long irregularly aligned
sarcomeres in the opener fibres.
That the opener muscle in the bIue crab swimming paddle is a slow muscle is not
surpnsing. Previously studied opener muscles from other crustaceans have typically
showed slow properties (Bittner, 1968). This is an indication that the muscle fibres of the
blue crab swimming paddle opener muscle are weii-suited for their function in prolonged
contraction. Slow muscle fibres are typicaiiy found in musde used in postural control
and locomotion as they have the abiiity to support extended pends of tension.
Furthemore, slow fibres fire over a large range of frequencies displaying graded
contraction strength.
B. Innervation
The innervation pattern and synaptic morphology O lf the crayfish opener muscle
have been thoroughly examined using freeze-fracture microscopy (Govind et al., 1994;
Govind et al., 1995), transmission electron microscopy (Atwood and Morin, 1970;
Jahromi and Atwood, 1974) and electrophysiology (Atwood, 1967; Atwood and Bittner.
197 1; Wiens and Atwood, 1975). The opener muscle in crayfish is innervated by three
axons and the blue crab opener has the same innervation pattern (Wiens, 1984;
Rathrnayer and Bevengut, 1986). A cornmon inhibitory axon that innervates d l of the
limb muscles is located in the proximal region of the opener muscle, while the specific
inhibitory axon innervates every muscle fibre in the opener. The excitatory innervation is
a shared axon that innervates every muscle fibre in the opener muscle and in the stretcher
muscle (Wiens, 1984). Although the opener muscle is innervated by three axons, the
centrai and distal regions receive innervation by only two, the excitor and the specific
inhibitor. This then allows for positive identification of the inhibitory axon when
examinations are restricted to the centrai and distal regions. As a result, the data from the
present study was coilected fiom central fibres.
In crustacean systems, such as the crayfish opener muscle, the excitatory and
inhibitory axons are located dose together as offshore axons and under the muscle basal
Lamina as terminal regions (Msghina and Atwood, 1997). Atwood and Kwan (1976)
found that the excitatory and inhibitory axons grow simultaneously into the crayfish limb
opener muscle during early development. Further support for the parallel branching of the
inhibitory and excitatory axons comes from a study by Atwood and Bittner (1971). These
researchers exarnined both the crayfish opener muscle and crab stretcher muscle and
confirmed that parallel branching of the inhibitory and excitatory axons occurred within
both animai systems. My study of the blue crab swimming paddle opener muscle has
produced the same conclusion. When offshore axons were located, one excitatory and
one inhibitory axon profile were found together, giving further evidence that two axons
innervate the central region of the opener muscle. Furthemore, at most terminal regions,
both inhibitory and excitatory innervation coutd be identified with the majority of
innervation sites showing one excitatory and one inhibitory terminal. This would
indicate that two axons innervate the centrai region of the opener muscle, one excitatory
and one inhibitory.
In crayfish and other crustacean pereiopod muscles it is uncornmon to locate
terminai regions on muscle fibres in the first viewing of a sampled section (Govind and
Wiens, 1985; Read and Govind, 1993; Govind et al., 1995). In the blue crab swimming
paddle opener muscle however, innervation sites were found on every viewing of thin
sections taken from randomly sampled muscle fibres. More imponantly, fibres viewed in
a single thin section displayed an average of two sites per fibre, suggesting that the axons
branch profusely. Thus the opener muscle in the blue crab is very highly innervated in
cornparison with other crustacean muscles. This may be an indication of the high level of
fine muscle control and prolonged contraction required in this muscle in order to
maintain rapid abduction of the dactyl for prolonged periods of time. Quantitative
cornparisons of the terminals showed that the excitatory terminais had 3 times the
terminai Length and 1.5 times the terminal volume of the inhibitory terminals, indicating
widespread excitatory innervation in the blue crab opener muscle. This rich innervation
by the excitatory motoneuron may ensure that fatigue or exhaustion of neurouansmitter
does not occur in the highly active opener muscle. Thus this feature of the blue crab
opener muscle rnay be a specialization to its denved function (swirnrning).
In my exarnination of the blue crab swimrning paddle opener muscle, three
instances occurred where excitatory and inhibitory terminais had branches into each
other. That is, a finger of the excitatory terminai was found in an adjacent inhibitory
terminal and fingers of inhibitory terminais were located in adjacent excitatory terminais.
Some of these 'fingers' possessed synapses. suggesting direct communication between
these terminals. This type of branching could be similar to axo-axonai synapses located
at bottlenecks or axonai branch points where the diameter of the terminal is small
(Atwood, 1976). These axo-axonal synapses are theoretically well placed to effectively
block transmission of an impulse. The interdigirations in the blue crab muscle could
serve a similar purpose. A smail synapse with at ieast one dense bar that contacts or is
concacted by an interdigitation may affect the transmission of an impulse differently from
a synapse located on a branch that is adjacent to (as opposed to within) a given terminal.
Fwther shidies would be required to determine the effect and purpose of this type of
branching.
C. Terminal Components
Anaiysis of the innervation to the blue crab swimrning paddle opener muscle
indicated stnicnual adaptations that contribute to the high performance activity of the
neurons. Specialized sites of exocytosis and endocytosis were identified on several
synapses in the muscle indicating that the terminals are very active with membrane
recycling occumng. Exocytosis is the release of neurotransmitter from presynaptic
vesicles into the synaptic cleft. Exocytosis of dear vesicles occurs adjacent to active
zones and is ~ a " dependent. In highly active nerve terminais. membrane recycling is
accomplished via endocytosis, which allaws for efficient disposai of the vesicle
membrane. Dunng endocytosis. coated vesicles are brought into the nerve terminal, and
transponed in membrane bound sacs to cistemae within the golgi apparatus where the
vesicles are recycled and filled with neurotransmitter. The vesicles are then released and
reused.
During clear vesicle recycling, as opposed to coated vesicle recycling, vesicle
membranes are retrieved and taken up into membrane bound sacs, or cistemae. The
vesicle membranes are then recycled and filled wiih glutamate From the axoplasm.
During high levels of activity and neurotransmitter release, there is an increase in the
number of vesicles to be recycled and therefore a demand for vesicle recycling pathways.
In the blue crab swimming paddle opener muscle membrane bound sacs constituted 4%
of the cytoplasm in excitatory and inhibitory terminals, and rnay represent a step in
vesicle recycling which reflects a high level of vesicle release.
Another indicator that the terminal regions were specialized for high-energy
expendinues was the large amount of mitochondria and glycogen present in both
inhibitory and excitatory terminais. Like the tonic terminals of the crayfish limb extensor
muscle (King et al. 1996), the mitochondria in ihe present siudy were cornplex and
branched.
In a study by Sharman er al. (2000), the mitochondria1 content of four stomach
muscles in the blue crab was examined and it was found that inhibitory terminals had a
Iower content (1 1%) than the excitatory terminals (25%). In my snrdy, where the total
energy substrate content was 20.8% for the excitatory termind and 16.1 % for the
inhibitory terminal, the mitochondrial content was 8.7% (excitatory) and 7.7%
(inhibitory) while the glycogen content was 12.1% (excitatory) and 8.6% (inhibitory).
Although the values for mitochondria1 content were Lower than those obtained for tonic
excitatory axons in the crayfish limb extensor muscle (10% to 27%) (King er al., 1996),
and in blue crab stomach muscles (16% to 2 1 %) (Pate1 and Govind, L997), the total high-
energy content was sirniiar between the excitatory axon to the opener muscle and these
other two excitatory tonic axons.
Glycogen is included in the high-energy content and provides a much more rapid
source of energy than mitochondria (Morin md McLaughlin 1973). Although glycogen
is common in the terminals of the blue crab opener, very little has been found in the
terminals of the stomach muscle of the blue crab swimming paddie and it is typically
absent in terminals of freshwater crayfish. The relative abundance of the glycogen in the
nerve terminals of the blue crab opener muscle in comparison to nerve terminals of other
rhythmically moving muscks strongly suggests that the opener fires rapidly for sustained
periods of time. This suggestion is verified by the findings of Wood and Derby (1995),
that the frequency of firing in the motoneuroos of the swimming paddle opener muscle is
between 50-100 Hz while the motonems of the stomach muscles fire at a much slower
frequency (5-1OHz) (Selverston and Modins, 1987). Therefore, in order to maintain the
high frequency of iirhg, a high percentage of the terminal volume would have to 'be
composed of rapidly usable energy substrates and glycogen is better suited to this role
than are mitochondria (Morin and McLaughlin, 1973).
Generaily, tonic terminals contain more clear vesicles at the presynaptic
membrane than phasic terminais (Atwood, 1976). This higher availability of
neurotransmitter allows tfie tonic terminais to respond to elevated levels of motoneuron
activity for long periods of time without becorning fatigued. King et al. (1996) found
that the crayfish limb extensor muscle had a higher percentage of clear vesicles in phasic
terminals (21%) than in tonic terminals (16%). yet the ovemll ciear vesicle content was
higher in the tonic terminals however, because the tonic terminals were greater in volume
that the phasic terminais. In the tonic muscles of the blue crab stomach muscle the clear
vesicle content ranges from 22% to 34% (Patel and Govind, 1997). A similar content
was found in the inhibitory terminal of the blue crab stomach musde (Sharman et al.,
2000). In the swimming paddle opener muscle, my data show the percent composition of
clear vesicles in the tonic terminal was slightly lower than that found in the stomach
muscles, but higher than the value obtained in the crayfish tonic lirnb extensor
motoneuron. Like the stomach muscles, the inhibitory and excitatory terminals had
similar compositions of clear vesicles in the blue crab swimming paddle opener muscle
(22.5% for the excitatory terminals and 24.5% for the inhibitory terminais). The high
clear vesicle content indicates that a large amount of neurotransrnitter is available at the
site of the synapse, as is cornmon in tonic axons. Therefore clear vesicle content
indicates that the tenninals of the blue crab opener muscle are adapted to high levels of
prolonged activity.
Dense core vesicles are generally thought to contain neuromodulatory hormones
that, when released, can enhance the efficacy of excitatory neuromuscular synapses
(Lloyd, 1986). Dense core vesicles are commonly found in the nerve terminals of
stomach muscles (Atwood et al., 1978; Patel and Govind, 1997; Sharman et al., 2000)
and are thought to play a role in the repetitive conüactions of the stomach muscles. In
the crayfish limb extensor muscle, less than 1% of the tonic and phasic terminals were
composed of dense core vesicles (King er al., 1996). In blue crab stomach muscles dense
core vesicle content ranged from 2% to 3.6% (Patel and Govind, 1997). while Sharman er
al. (2000) found chat the blue crab stornach muscles had a dense core vesicle content of
47% in neuromodulatory terminals, 9% in excitatory terminals and 17% in inhibitory
terminais. In my examination of the blue crab swirnming paddle opener muscle. the
percent composition of dense core vesicles was approxirnately 1% in both the inhibitory
and excitatory terminals. This is slightly lower than the findings of the crayfish limb
muscle and much lower than values obtained for the blue crab stomach muscles. The low
percentage of dense core vesicles would indicate that regulation of muscle fibre
contraction is not brought about by neuromodulatory mechanisms, but rather through
precise control of the inhibitory and excitatory terminals.
O. Neuromuscular Synapses and Dense Bars
The anaiysis of synapse size and distribution reveaied that the majority of the
synapses were excitatory neuromuscular, this is in accordance with previous studies on
other crustacean muscles (Atwood and Morin, 1970; Jahrorni and Atwood, 1974; Atwood
and Kwan, 1976). Though there were more than *ce as many excitatory neuromuscular
synapses than inhibitory neuromuscuiar synapses the excitatory neuromuscular synapses
were on average twenty-five percent smaller than the inhibitory neuromuscular synapses.
Previous studies on the crayfish opener muscle have also indicated that the excitatory
neuromuscular synapses are smailer than the inhibitory neuromuscular synapses (Atwood
and Kwan, 1976; Jahromi and Atwood, 1974). The excitatory neuromuscular synapses
found on the adult crayfish opener by Atwood and Kwan (1976) had a mean size of 0.39
pn2 while the inhibitory neuromuscular synapses had a size of 0.50 pn'. These findings
were replicated by Jahromi and Atwood ( 1974), as excitatory neuromuscular synapses
had a rnean size of 0 . 3 9 ~ ' and the inhibitory neuromuscular synapses had a rnean size
of 0.46 pn'. In the blue crab swimming paddle opener muscle, 1 found excitatory
neuromuscular synapses were 0.804 pn' on average, while inhibirory neuromuscular
synapses were 1 .O40 pn' on average. This difference in size between neuromuscular
synapses of the blue crab and the crayfish would make sense as the opener muscle in the
blue crab is larger, and thcrefore larger synapses would be required to have depolarizing
and hyperpolarizing effects on the larger muscle fibres.
Active zones iire the probable sires of ca2' channels required for transmitter
release (Atwood and Lnenicka, 1986). Though the presence of active zones does not
ensure that neurotransmission occurs at that site, dense bar distribution is one method
used to determine synaptic strength. There is a positive correlation between the number
and size of dense bars and the potency of a synapse. Simple synapses, containing zero or
one dense bar, are genedfy recruited at higher frequencies than synapses with more
dense bars (Cooper et al., 1996). When dense bars are within 0.19 pm of each other, the
close proximity of the ca2' channels permit more ~ a ' ' to enter the terminal (Cooper et
al., 1995a). The p a t e r influx of ca2+ allows a greater release of neurotransmitter into
the synaptic cleft (Cooper et al.. 1995a). Cooper et al., (1996) suggested that complex
synapses, with two or more closely associated dense bars, are recruited preferentially to
non-complex synapses.
Govind es al. (1995) showed that there was at least one dense bar in every synapse
of the specific inhibitory terminal in the crafish opener muscle, and in 85-95 % of the
excitatory synapses (Govind et al., 1994). These findings are in concrast to my
examination of the innervation to the opener muscle in the blue crab. Thirty-six percent
of the inhibitory and 39% of the excitatory neuromuscular synapses lacked a dense bar.
This rnay indicate that, though innervation is profuse in the blue crab opener muscle, each
of the synapses may not be as powerful as in other muscle systems. This also may
indicate that many of the synapses analyzed were silent synapses, effectively recruited
only at very high impulse frequencies (Atwood and Wojtowicz, 1999). thus allowing for
synaptic plasticity within the muscle.
In the crayfish opener muscle the proximal region consists of high output
terminals with synapses that are aimost di complex, while the low output terminals in the
central region mainly coniain synapses that are simple, possessing zero or one dense bar
(Govind et al., 1994). An examination of the crayfish opener muscle by Cooper et al.
(1995a) found that terminais containing a larger number of complex synapses had
presynaptic ca2' signals and a quantal content that were higher than in terminais
containing fewer or no complex synapses. Only 0.6% of the synapses frorn the central
terminals of the crayfish opener muscle contained three or more dense bars (Cooper et
al., 1995b). King et al., (1996) found that in the crayfish extensor muscle 2.1% of the
synapses in tonic terminais contained three or more dense bars. In my study 21% of the
excitatory neuromuscular synapses contained three or more dense bars, suggesting
elevated presynaptic caZ+ signds and quantal content. This may be an adaptation of the
blue crab opener muscle to maintain high Levels of activity for prolonged penods, by
ensuring that elevated neurotransmitter release can occur.
In synapses with longer or muitiple dense bars, the ~ a " influx is larger with a
single impulse than it is in simple synapses (Zucker et al., 1991). In a study by
Wojtowicz et al. (1994) neuromuscular terminais of the crayfish opener muscle were
subjected to high frequency stimulation and long term facilitation of neurotransmitter
release resulted. When crayfish that had undergone long-term facilitation were compared
with control animals, a greater nurnber of complex synapses were discovered in the
adapted terminals, dong with a larger number of synapses that were active at transmitter
release during low frequency stimulation. Et was aiso found that as the frequency of
stimulation was increased the number of active synapses releasing neurotransmitter also
increased. With an increase in the intemal ~ a ' + concentration, the interaction of dense
bars further from each other is enhanced, dowing for an additional way in which
synaptic output cm be altered by frequency (Cooper et al., 1996). In my study on the
blue crab opener muscle, approximately one third of the synapses contained two or more
dense bars. This high incidence of complex synapses could be a result of the high
frequency of firing of the nerve terminals. AIso, larger terminals tend to contain more
dense bars (Atwood et al., 1978) and those of the blue crab opener muscle were 2.5 times
larger than those of the crayfish opener muscle.
Govind et al. (1994) fond that synapses that produce larger excitatory
postsynaptic potentials had longer rnean deuse bar lengths. A larger number of ca2'
channels, as found in longer dense bars, allowing for a greater influx of ~ a " for a single
impulse (Cooper et al., 1996). In previous studies tonic synapses were found to possess
dense bars with a mean length of 0.1 pn (Cooper et al., 1995b; King et al., 1996;
Coulthard, 1998). Studies on the opener muscle of crayfish walking limbs indicate that
inhibitory synapses had mean dense bar lengths that were longer than those of the
excitatory synapses (Govind et al., 1994; Govind et al., 1995). Govind et al. (1994)
found that the mean length of excitatory dense bars in the central region of the crayfish
opener muscle was between 0.08 p and 0.09 p, while inhibitory synapses in the
central region of the crayfish opener muscle had a mean dense bar length of O. 1 1 pm
(Govind et al., 1995). These findings are very sirnilar to those in my study of the central
region of the blue crab swirnming paddle opener muscle where the dense bars of the
neuromuscular synapses had a mean length of 0.09 pn for the excitatory tenninals and
0.13 pm for the inhibitory terminais. In my examination, excitatory neuromuscular
synapses had two and a half times as many dense bars as inhibitory neuromuscular
synapses for the sarne length of serial examined. Dense bars were thirty percent smdler
in the excitatory neuromuscular synapses than in the inhibitory neuromuscular synapses,
however because there were so many more dense bars in the excitatory neuromuscular
synapses, the dense bar area was still greater in the excitatory terminais. Although the
synapses in the blue crab swirnming paddle opener muscle had a large synaptic area, the
dense bar lengths were sirnilar between the crayf%h opener muscle and the blue crab
opener muscle. The ciifference in dense bar distribution may lie in the fact that one-third
of the blue crab synapses were cornplex, containing two or more dense bars.
E. Axo-axonal Synapses and Dense Bars
Axo-axonal synapses are also common in crustacean neuromuscular systems.
Inhibitory axo-axonal synapses have been found to comprise 10-20% of the synapses of
the specific inhibitory axon in the crayfish opener muscle (Govind et al., 1995).
Presynaptic inhibition is used for precise control of muscle movement. Moreover, once
the presynaptic inhibition is removed from the excitatory terminal a strong excitatory
response is produced as a result of the availability of a large readily releasable pwl of
neurotransmitter (Atwood and Walcott, 1965). In the blue crab swimrning paddle opener
muscle 36% of the inhibitory synapses were axo-axonal, this is in contrast to the 10-20 %
found in the cra$sh opener muscle (Govind et al., 1995). The high incidence of
presynaptic inhibition in the opener muscle of the blue crab swimming paddle may be an
adaptation to the highly specialized swimming motion, The high percentage of
presynaptic inhibition would indicate that precise conuol of the opener muscie rnight be
necessary for it to function effectively as a fairly equal antagonist to the closer muscle.
Like neuromuscular synapses, not al1 of the inhibitory axo-axonal synapses
contained dense bars. This is contrary to what was previously reported by Govind et al.
(1995) in rhe crayfrsh opener muscle, and could indicate that though there are a greater
percentage of axu-axonal synapses in the blue cnb, many may be either silent and could
be recniited at much higher frequencies as is seen in synapses that do not possess dense
bars, or they may be immature and not fuiiy developed (Atwood 1976; Atwood and
Wojtowicz, 1999).
The surprising finding was the presence of excitatory axo-axonal synapses.
Though there were not a large number of these, they were found at least once in every
area sampled and were thus deiennined not to be an artifact. The excitatory axo-axonal
synapses were rnuch smaller than any of the other types of synapses, and three quarters
containeci at least one dense bar. These dense bars were smaller than those of the
inhibitory axo-axonal synapses, but longer than those of the excitatory neuromuscular
synapses. Because the synapses are small, but contain long dense bars, the value for
dense bars per synapcic area is largest in the excitatory axo-axonal synapse. Based on
dense bar distribution, synaptic strength per synaptic area is highest in the excitatory axo-
axonai synapses.
The excitatory axo-axonal synapse is actually one of two correlates of a reciprocal
synapse, tbe second being the inhibitory axo-axonai synapse. In my study, each of the
excitatory axo-axonal synapses were polarized onto an inhibitory terminal that had
adjacent synapses onto the excitatory terminal. Within the serial sections, the excitatory
am-axonal synapses were generally located 50 to 250 nm away h m an inhibitory axo-
axonal synapse, Shepherd (1974) defined a ceciprocd synapse as an arrangement between
neurological elements in which chernical trruismission is in opposite directions at two
adjacent or sepatate synapses. Four cases of reciprocal synapses were seen in the blue
crab opener muscle.
Reciprocal synapses have only been documented in three other insmces in
crustacean limb muscles (Atwood and Kwan, 1979; Pearce and Govind, 1993). Atwood
and Kwan (1979) found the fmt evidence of a reciprocal synapse in the stretcher muscle
of the spider crab, Hyas areneus. In theu ultrastnictural study, it wsts not possible to
conclude whether or not the reciprocal synapses were between the excitatory terminai and
the common or specific inhibitory axon since the NO inhibitory axons innervate the same
regions of the suetcher muscle. Pearce and Govind (1993) also examined the presence of
reciprocai synapses but found it in muscies only receiving inhibition frorn the common
inhibitory axon; the limb closer muscle of the crab Eriphia spinifrons and the distal
accessory flexor muscle in the crayfish Frocambarus clarkii. One third of the axo-axonal
synapses located in the study by Pearce and Govind (1993) were identified as reciprocal,
while10% of the axo-axonal synapses in the study by Atwood and Kwan (1979) were
identified as reciprocal. In the present study, 15% of the axo-axonai synapses in the blue
crab opener muscle were found to be reciprocal, thus within the range of those previousiy
reported.
The crayfish opener muscle has been extensively studied and no evidence of
reciprocai synapses has been documented. Following their finding of reciprocal synapses
in the closer of the crab Eriphia spinifrons and distal accessory flexor muscle of crayfish,
Pearce and Govind (1993) examined the crayfish opener muscle for the presence of
reciprocal synapses. The opener muscle receives inhibitory innervation by both the
common and the specific inhibitory axons, however, the common inhibitory axon only
innervates a very restricted proximal region of this muscle, whereas the specific
inhibitory axon shows much more widespread innervation (Bevengut and Cournil, 1990;
Wiens, 1984). For this reason, if excitatory axo-axonal synapses were discovered in the
distai or centrai regions of the opener muscle it wouid be possible to state that excitatory
axo-axonal synapses were polarized onto the specific inhibitory axon. Thorough
examinations have found no evidence of excitatory axo-axonal synapses, although
inhibitory axo-axonai synapses are a common occurrence in the crayfish opener muscle.
When the entire muscle was sarnpkd in juvenile craflsh, excitatory axo-axonal synapses
were not seen (Pearce and Govind, 1993). The presence of reciprocal synapses in the
blue crab swimming paddle opener muscle suggests that a specidized innervation pattern
is utiiized in the swimming motion of the btue crab paddle.
The function of the reciprocal synapse in the crustacean neuromuscular system is
as yet unknown. Atwood and Kwan (1979) suggested a metabouopic function for this
neural arrangement and Pearce and Govind (1993) suggested that the function could be to
decrease the inhibitory effect of the common inhibitory axon where the reciprocal
synapse is occurring by activating K" channels within the inhibitory membrane. In order
to state for certain the purpose of the excitatory axo-axonal synapse it would be necessary
to determine whether or not the receptors on the postsynaptic inhibitory terminal
membrane are excitatory (GABAe) or inhibitory (GABAA) (Miwa et al., 1990).
The fifth pereiopod in the blue crab exhibits many exoskeletal and rnuscular
adaptations in its function as a swimrning paddle. Fibres of the fifth pereiopod opener
muscle were anaiyzed to examine the presence of neuromuscular adaptations.
Ultrastructural analysis of muscle fibres and terminai regions were used to obtain
quantitative and qualitative data.
In cross-sections of the central regions of the opener and closer muscles, the
opener muscle was found to be 1.5 cimes greater in girth then the closer. The
hypemophy of the opener muscle is indicative of the role that it plays as an equai
antagonist in the abductiodadduction of the blue crab opener muscle. With
ultrastructural examination, the blue crab swimrning paddle opener muscle was
determined to be composed of slow muscle fibres. This was found in data collected using
electron microscopy where the sarcornere length measured between 7 to 9 prn, the
number of actin filaments surrounding one myosin filament was high (13:1), the
sarcomeres were irregularly aiigned, and the 2-lines were thick and wavy. Unpublished
light microscopy and NADH-diaphorase and ATPase enzyme histochemistry results
confirmed that the blue crab opener was composed of slow muscle fibres.
An examination of the opener muscle innervation reveaied that this muscle is very
profusely imervated. Every viewing of thin sections taken from randorniy sampled
muscle fibres revealed innervation sites, with fibres containing an average of two sites.
Each site was composed of one excitatory and one inhibitory terminal profiIe chat were
dosely associated.
Off-shore axons, as well as terminai regions, of the inhibitory and excitatory
neurons were found in parailel. An interesting method of branching was located at several
innervation sites. A branch of one terminal had protruded into an adjacent antagonistic
terminal. On one of these branches was located a reciprocal synapse where the inhibitory
branch had a synapse polarized ont0 the excitatory terminai, and adjacent to this the
excitatory terminai had a synapse polarized onto the inhibitory branch.
An analysis of the percent composition of the inhibitory and exciratory rerminals
reveded that the two terminai types had similar organelle composition. The greatest
percent composition for terminai types was the clear vesicles, occupying approximately
onequarter of the terminals. The high-energy content was also a major component of the
tenninals and was composed of rnitochondria and glycogen. Membrane bound sacs and
dense cored vesicles were located within the terminais as well, but represented only a
smalI percentage of the total composition.
Further andysis of the terminais revealed that neuromuscular and axo-monal
synapses occurred in the inhibitory and excitatory terminais. Like in other crustacean
neuromuscular systems, the excitatory neuromuscular synapses were the greatest in
quantity, but the inhibitory neuromuscular synapses had the largest area. Inhibitory axo-
axonai synapses were present throughout sampled regions and composed 36% of the
inhibitory synapses. Axo-axonai synapses polarized from the excitatory terminal to the
inhibitory terminai were also located. These are very rare in crustacean neuromuscular
systems and have k e n documented in only three other muscles. The inhibitory and
excitatory axo-axonai synapses are the two correlates necessary to make up a recipmd
synapse and ceciprocal synapses were located in each of rhe four senally sectioned areas
examined in the present study of the blue crab swim paddle opener muscle.
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