C. K. Govind

Developmental Neuroethology: Changes in Escape and Defensive Behavior During Growth of the Lobster

The changes in relative efficacy of two incompatible behaviors was investigated during growth of the lobster, Homarus americanus. In larval and early juvenile stages, physiological and morphological factors favor use of the escape response over defensive behavior. In large animals, defensive behavior is preferred almost exclusively to escape behavior unless the claws are lost. The interaction of escape and defensive behavior is modified by neural and morphological factors, which are dependent on the stage in the life cycle of the organism.

Correlation between presynaptic dense bodies and tranmitter output at lobster neuromuscular terminals by serial section electron microscopy

Lobster neuromuscular terminals releasing comparatively small (low-output type) and large (high-output type) amounts of transmitter but arising from the single excitatory motor axon to the proximal accessory flexor muscle were serially sectioned for electron microscopy. The three-dimensional reconstruction showed the two types of terminals to have a complex branching pattern in which thin branches of the motor axon often enlarged into synapse bearing terminal regions. Quantitative comparison showed that the mean surface area of a synapse is similar in the two types of terminals. However, the low-output terminal has a higher synaptic density and devotes a greater part of its surface area to synapses compared to its high-output counterpart suggesting that transmitter output is not directly related to synaptic area. The mean surface area of a presynaptic dense body is not significantly different between low- and high-output synapses, but there is a significantly greater density of these active zones in the high-output terminal. This results in the ratio of mean dense body area to mean synaptic area being approximately 3 X greater in the high-output synapses than the low-output ones. This significant difference in the surface area of presynaptic dense bodies between low- and high-output synapses correlates with the difference in transmitter output at these two synapses, and implicates the dense bodies in the mechanism of transmitter release at lobster neuromuscular synapses.

DEVELOPMENT OF THE DIMORPHIC CLAW CLOSER MUSCLES OF THE LOBSTER HOMARUS AMERICANUS: I. REGIONAL DISTRIBUTION OF MUSCLE FIBER TYPES IN ADULTS

1. The closer muscles of the dimorphic claws (chelipeds) were studied for the presence and location of fast and slow muscle fibers. 2. Cutter claws were composed of about 60-70% short sarcomere (less than 4 mum) fast fibers; the remainder was longer sarcomere (greater than 6 mum) slow and intermediate (4-6 mum) fibers. 3. Crusher claws were composed of a uniform population of long sarcomere (6-13 mum) slow and intermediate (4-6 mum) fibers. 4. There was a regional distribution of fibers in the cutter claw. Ventral fibers were predominantly slow. Dorsal fibers and central medial fibers were fast. Proximal and distal fibers in the medial section were usually mixed. 5. The regional distribution of cutter fibers correlates with previous physiological studies on the distribution of the fast and slow motor axons to these muscle fibers.

STRUCTURAL FEATURES OF CRAYFISH PHASIC AND TONIC NEUROMUSCULAR TERMINALS

We examined the fine structure of terminals of the phasic and tonic excitatory axon to the crayfish limb extensor muscle. The phasic terminals are known to release 50–100 times more transmitter for a small length of terminal for a single impulse. Phasic terminals labeled with horseradish peroxidase (HRP) were relatively thin and contained a single unbranched mitochondrion; tonic terminals were much thicker, and their varicosities contained several multibranched mitochondria. Tonic terminals devoted a larger proportion of their total volume to mitochondria. The percentage volume of clear synaptic vesicles was slightly higher in phasic axon terminals, but as the tonic axon terminals were fivefold larger in volume, the total synaptic volume is much greater in tonic than phasic terminals. The number of synapses per length of terminal, and the total number of active zones per length of terminal, were greater for tonic terminals, and individual synapses were, on average, slightly larger in surface contact area for tonic terminals. In contrast, individual active zones were, on average, longer in phasic synapses. A higher proportion (50%) of phasic synapses had multiple active zones than was the case for tonic synapses (16%), and pairs of closely spaced active zones were more frequently found on phasic synapses. These findings clearly rule out synapse and active zone number as a factor contributing to higher transmitter output, but suggest that active zone size and synaptic complexity, as evidenced by multiple closely spaced active zones in a single synapse, are likely to play a causal role in the greater transmitter release of the phasic terminal. Even synapse complexity would not be enough to account fully for the large difference in terminal transmitter output, and additional factors may include electrical and biochemical differences.

Motor nerve terminals on abdominal muscles in larval flesh flies, Sarcophaga bullata: Comparisons with Drosophila

Motor nerve terminals on abdominal body‐wall muscles 6A and 7A in larval flesh flies were investigated to establish their general structural features with confocal microscopy, transmission electron microscopy, and freeze‐fracture procedures. As in Drosophila and other dipterans, two motor axons supply these muscles, and two morphologically different terminals were discerned with confocal microscopy: thin terminals with relatively small varicosities (Type Is), and thicker terminals with larger varicosities (Type Ib). In serial electron micrographs, Type Ib terminals were distinguished from Type Is terminals by their larger cross‐sectional area, more extensive subsynaptic reticulum, more mitochondrial profiles, and more clear synaptic vesicles. Type Ib terminals possessed larger synapses and more synaptic contact area per unit terminal length. Although presynaptic dense bars of active zones were similar in mean length for the two terminal types, there were almost twice as many dense bars per synapse for Type Ib terminals. Freeze‐fractures through the presynaptic membrane showed particle‐free areas indicative of synapses on the P‐face, within which were localized aggregations of large intramembranous particles indicative of active zones. These particles were similar in number to those found at active zones of several other arthropod neuromuscular junctions. In general, synaptic structural parameters strongly paralleled those of the anatomically homologous muscles in Drosophila melanogaster. In live preparations, simultaneous focal recording from identified varicosities and intracellular recording indicated that the two terminals produced excitatory junction potentials of similar amplitude in a physiological solution similar to that used for Drosophila. J. Comp. Neurol. 402:197–209, 1998.

Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1—wild‐type Sarcophaga and Drosophila and Drosophila mutants duncem14 and fasIIe76, a hypomorphic allele of the fasciclin II gene; and axon 2—drosophila wild‐type, duncem14, and fasIIe76. These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15–40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T‐shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center‐to‐center distance of each synapse to its nearest neighbor. The mean spacing between nearest‐neighbor pairs ranged from 0.84 μm to 1.05 μm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 μm to 0.75 μm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 μm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely. J. Comp. Neurol. 393:482–492, 1998.

Differential reflex activity determines claw and closer muscle asymmetry in developing lobsters

The paired claws and closer muscles of the lobster, Homarus americanus, are identical in the early juvenile stages, but subsequently differentiate into a stout crusher claw with only slow fibers and a slender cutter with largely fast fibers. Rearing with different substrates or exercise of the claws revealed that claw laterality is determined in the central nervous system by differential reflex activity in the paired claws; the side with greater activity becomes the crusher, while the contralateral side becomes the cutter.

Intramembranous organization of lobster excitatory neuromuscular synapses.

SummaryThe fine structure of identified neuromuscular synapses of the single excitatory axon to the distal accessory flexor muscle in lobster limbs was examined with freeze-fracture and serial thin-section electron microscopy. The latter technique reveals presynaptic dense bars with synaptic vesicles aligned on either side of these bars and often fused to the membrane, suggesting exocytosis and confirming our previous contention that these bars are active zones of transmitter release. The intramembranous organization of these active zones, as revealed in freeze-etched tissue, is a ridge-like elevation of the P-face of the axolemma with a matching trough on the complementary E-face. The ridge on the P-face has rows of large scattered intramembranous particles along the apex and is often bordered by a series of small, circular depressions which are presumed to represent exocytotic vesicles attached to the presynaptic membrane. Complementing these depressions are a few volcano-like protuberances seen occasionally on the E-face membrane. Because such evidence for transmitter release occurred in both stimulated and non-stimulated preparations, it demonstrates that chemical fixatives employing aldehydes induce transmitter release. The postsynaptic receptor sites of these excitatory synapses are characterized by oval-shaped patches of densely packed particles on the E-face, arranged in a random pattern on the sarcolemma. The complementary P-face view exhibits a regular square array of particle imprints or pits.

Proliferation and relocation of developing lobster neuromuscular synapses

Abstract The development of multiterminal innervation from a single identifiable excitatory motoneuron to the lobster distal accessory flexor muscle (DAFM) was studied by serial section electron microscopy. The number, size, and location of neuromuscular synapses and presynaptic dense bars within the peripheral branching pattern of the axon was determined in cross sections of the DAFM in 1st (24-hr-old)-, 4th (2-week-old)-, and 12th (1-year-old)-stage lobsters. The mean size of synapses remains fairly constant in these three stages but synaptic density, i.e., the number of synapses per unit length of fiber, increased more than 20-fold between the 1st and 4th stages and more than 5-fold between the 4th and 12th stages. Synaptic surface area per fiber length showed a parallel increase. Consequently there is a proliferation of synapses along the length of individual muscle fibers during primary development. Furthermore from the 1st stage where only a few fibers are innervated, synapses proliferate to many more fibers in the 4th and to all fibers in the 12th stage. The neuromuscular synapses are distributed in different proportions within the axonal branching pattern in the three stages. Based on the number and size of synapses and presynaptic dense bars, the main axon and primary branches provide almost equal amounts of innervation in the 1st stage. With further branching in the 4th stage, the main axon accounts for only 20–25% of the innervation; the primary branches for 45% and other finer branches the remainder. By the 12th-stage synapses are found only on branches other than the main axon and its primary offshoots. There is therefore a shift in innervation from the main axon to the primary branches and then to the finer branches during primary development. This shift in innervation involves the formation of new synaptic terminals and the restructuring of existing ones into axonal areas. In this way the multiterminal innervation arising from an identifiable motoneuron is remodeled.

Presynaptic dense bars at neuromuscular synapses of the lobster, Homarus americanus

SummaryThe threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.