In vivo imaging of zebrafish reveals differences in the spinal networks for escape and swimming movements.

Most studies of spinal interneurons in vertebrate motor circuits have focused on the activity of interneurons in a single motor behavior. As a result, relatively little is known about the extent to which particular classes of spinal interneurons participate in different behaviors. Similarities between the morphology and connections of interneurons activated in swimming and escape movements in different fish and amphibians led to the hypothesis that spinal interneurons might be shared by these behaviors. To test this hypothesis, we took advantage of the optical transparency of zebrafish larvae and developed a new preparation in which we could use confocal calcium imaging to monitor the activity of individual identified interneurons noninvasively, while we simultaneously filmed the movements of the fish with a high-speed digital camera. With this approach, we could directly examine the involvement of individual interneurons in different motor behaviors. Our work revealed unexpected differences in the interneurons activated in swimming and escape behaviors. The observations lead to predictions of different behavioral roles for particular classes of spinal interneurons that can eventually be tested directly in zebrafish by using laser ablations or mutant lines with interneuronal deficits.

The zebrafish space cadet gene controls axonal pathfinding of neurons that modulate fast turning movements.

All vertebrates depend on neural circuits to produce propulsive movements; however, the contribution of individual neural cell types to control such movements are not well understood. We report that zebrafish space cadet mutant larvae fail to initiate fast turning movements properly, and we show that this motor phenotype correlates with axonal defects in a small population of commissural hindbrain neurons, which we identify as spiral fiber neurons. Moreover, we demonstrate that severing spiral fiber axons produces space cadet-like locomotor defects, thereby providing compelling evidence that the space cadet gene plays an essential role in integrating these neurons into the circuitry that modulates fast turning movements. Finally, we show that axonal defects are restricted to a small set of commissural trajectories, including retinal ganglion cell axons and spiral fiber axons, and that the space cadet gene functions in axonal pathfinding. Together, our results provide a rare example in vertebrates of an individual neuronal cell type that contributes to the expression of a defined motor behavior. Movies available on-line

A confocal study of spinal interneurons in living larval zebrafish.

We used confocal microscopy to examine the morphology of spinal interneurons in living larval zebrafish with the aim of providing a morphological foundation for generating functional hypotheses. Interneurons were retrogradely labeled by injections of fluorescent dextrans into the spinal cord, and the three-dimensional morphology of living cells was reconstructed from confocal optical sections through the transparent fish. At least eight types of interneurons are present in the spinal cord of larval zebrafish; four of these are described here for the first time. The newly discovered cell types include classes of commissural neurons with axons that ascend, descend, and bifurcate in the contralateral spinal cord. Our reexamination of previously described cell types revealed functionally relevant features of their morphology, such as undescribed commissural axons, as well as the relationships between the trajectories of the axons of interneurons and the descending Mauthner axons. In addition to describing neurons, we surveyed their morphology at multiple positions along the spinal cord and found longitudinal changes in their distribution and sizes. For example, some cell types increase in size from rostral to caudal, whereas others decrease. Our observations lead to predictions of the roles of some of these interneurons in motor circuits. These predictions can be tested with the combination of functional imaging, single-cell ablation, and genetic approaches that make zebrafish a powerful model system for studying neuronal circuits.

Paralytic zebrafish lacking acetylcholine receptors fail to localize rapsyn clusters to the synapse.

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato, reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed, showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

Laser ablations reveal functional relationships of segmental hindbrain neurons in zebrafish.

Segmentation of the vertebrate brain is most obvious in the hindbrain, where successive segments contain repeated neuronal types. One such set of three repeated reticulospinal neurons--the Mauthner cell, MiD2cm, and MiD3cm--is thought to produce different forms of the escape response that fish use to avoid predators. We used laser ablations in larval zebrafish to test the hypothesis that these segmental hindbrain cells form a functional group. Killing all three cells eliminated short-latency, high-performance escape responses to both head- and tail-directed stimuli. Killing just the Mauthner cell affected escapes from tail-directed but not from head-directed stimuli. These results reveal the contributions of one set of reticulospinal neurons to behavior and support the idea that serially repeated hindbrain neurons form functional groups.

Zebrafish as a model system for studying neuronal circuits and behavior.

Zebrafish are best known as a model system for studies of the genetics of development. They do, however, also offer many advantages for the study of neuronal circuitry because the larvae are transparent, allowing optical studies of neuronal activity and noninvasive photoablations of individual neurons. The combination of these optical methods with genetics through the use of mutant and transgenic lines of fish should make the zebrafish model a unique and powerful one among vertebrates. Here we review the strengths of the model and the possibilities it offers for studies of the neural basis of behavior.

Monitoring activity in neuronal populations with single-cell resolution in a behaving vertebrate.

Vertebrate behaviours are produced by activity in populations of neurons, but the techniques typically used to study activity allow only one or very few nerve cells to be monitored at a time. This limitation has prompted the development of methods of imaging activity in the nervous system. The overall goal of these methods is to image neural activity non-invasively in populations of neurons, ideally with high spatial and temporal resolution. We have moved closer to this goal by using confocal calcium imaging to monitor neural activity in the transparent larvae of zebrafish. Neurons were labelled either by backfilling from injections of the calcium indicator (Calcium Green dextran) into muscle or spinal cord of larvae or by injections into blastomeres early in development. The labelled neurons were bright enough at resting calcium levels to allow the identification of individual neurons in the live, intact fish, based upon their dendritic and axonal morphology. The neurons from the live animal could also be reconstructed in three dimensions for morphometric study. Neurons increased their fluorescence during activity produced by direct electrical stimulation and during escape behaviours elicited by an abrupt touch to the head or tail of the fish. The rise in calcium associated with a single action potential could be detected as an increase in fluorescence of at least 7-10%, but neurons typically showed much larger increases during behaviour. Calcium signals in the dendrites, soma and nucleus could be resolved, especially when using the line-scanning mode, which provides 2-ms temporal resolution. The imaging was used to study activity in populations of motoneurons and hindbrain neurons during the escape behaviour fish use to avoid predators. We found a massive activation of the motoneuron pool and a differential activation of populations of hindbrain neurons during escapes. The latter finding confirms predictions that the activity pattern of hindbrain neurons may help to determine the directionality of the escape. This approach should prove useful for studying the activity of populations of neurons throughout the nervous system in both normal and mutant lines of fish.

Imaging neuronal networks in behaving animals.

Neuronal activity has recently been imaged with single-cell resolution in behaving vertebrates. This was accomplished by using fluorescent calcium indicators in conjunction with confocal or two-photon microscopy. These optical techniques, along with other new approaches for imaging synaptic activity, second messengers, and neurotransmitters and their receptors offer great promise for the study of neuronal networks at high resolution in vivo.

Imaging the functional organization of zebrafish hindbrain segments during escape behaviors.

Although vertebrate hindbrains are segmented structures, the functional significance of the segmentation is unknown. In zebrafish, the hindbrain segments contain serially repeated classes of individually identifiable neurons. We took advantage of the transparency of larval zebrafish and used confocal calcium imaging in the intact fish to study the activity of one set of individually identified, serially homologous reticulospinal cells (the Mauthner cell, MID2cm, and MID3cm) during behavior. Behavioral studies predicted that differential activity in this set of serially homologous neurons might serve to control the directionality of the escape behavior that fish use to avoid predators. We found that the serially homologous cells are indeed activated during escapes and that the combination of cells activated depends upon the location of the sensory stimulus used to elicit the escape. The patterns of activation we observed were exactly those predicted by behavioral studies. The data suggest that duplication of ancestral hindbrain segments, and subsequent functional diversification, resulted in sets of related neurons whose activity patterns create behavioral variability.

Labeling blastomeres with a calcium indicator: a non-invasive method of visualizing neuronal activity in zebrafish.

Injections of the calcium indicator calcium green dextran (CGD) into zebrafish embryos at the 1-4 cell stages were used to monitor the activity of neurons in larval fish. Dye was pressure injected into a single cell and the fish allowed to develop until post-hatching, when they were embedded in agar and viewed under a confocal microscope. Labeled larval cells, including identifiable neuronal classes such as Rohon-Beard cells and olfactory neurons, were clearly visible with extensive labeling of the whole fish following injections at the one cell embryonic stage, and a mosaic labeling pattern following injections at the 2 or 4 cell stages. Activity of neurons in the spinal cord, as indicated by intracellular calcium concentration changes, was observed directly by monitoring fluorescence changes of individual spinal neurons and groups of spinal neurons on a confocal microscope. Fluorescence increases of between 9 and 55% in spinal neurons were seen during escape responses produced when the fish was tapped on the tail. This technique can potentially be used to monitor the activity of any neuron or group of neurons with respect to behavior non-invasively in intact living zebrafish.

Interactions between the neural networks for escape and swimming in goldfish.

Interactions between neural networks for different motor behaviors occur frequently in nature; however, there are few vertebrate models for studying these interactions. One potentially useful model involves the interactions between escape and swimming behaviors in fish. Fish can produce escape bends while swimming, using some of the same axial muscles for both behaviors. Here we study the interactions between escape and swimming in a paralyzed goldfish preparation in which we can activate the networks for both behaviors. Fictive swimming was elicited by electrical stimulation in the midbrain locomotor region. During the swimming, we fired a single action potential in the reticulospinal Mauthner (M) cell, which initiates the escape behavior (Zottoli, 1977). Firing the M cell overrode the swimming motor output to produce an output appropriate for escape regardless of the phase of swimming at which it was fired. The M cell also could reset the swimming rhythm dramatically in a way that led to a smooth transition from an escape bend to one side into subsequent swimming. Both the override and reset supported predictions based on previous studies of the organization of the M-cell network. They apparently allow for a well coordinated motor output when a fish must produce an escape while swimming. The potent effects of one action potential in a single, identifiable reticulospinal neuron make this an attractive model system for future studies of the cellular basis of interactions between descending pathways and spinal rhythm-generating networks.

Beating the competition: the reliability hypothesis for Mauthner axon size.

The Mauthner cell has an axon that is among the largest in diameter of any vertebrate neuron. It is commonly thought that the large size is needed for short latency escape responses involving a major contraction of the trunk musculature. Previous work, however, has shown that there is nothing unique about the strength of the Mauthner initiated response, compared to responses initiated by other smaller cells, and it is debatable that there is any important improvement in response latency due to Mauthner axon size. In this paper we advance an alternative explanation: although the Mauthner cell has a powerful excitatory influence on motoneurons, the large size of the Mauthner axon is most important in rapidly spreading an inhibitory signal that turns off other competing motor commands. Such competing commands are likely to arise in the presence of ongoing swimming behavior or ambiguous stimuli that could activate a fast turn either toward or away from the stimulus. These stimuli include apparent food items, or lures, presented by predators (such as anglerfish) and escape eliciting sounds which, in the presence of background noise, may have 180 degrees directional ambiguity. Thus, large size of the axon contributes most to the reliable expression of the escape behavior. We base this reliability hypothesis on a retrospective analysis of previous neurophysiological data and new anatomical measurements of the diameters of the large spinal cord axons from which we calculated conduction velocities. Our calculations show that the Mauthner-derived inhibition is fast enough that it allows an escape response to occur even when a conflicting motor command enters the spinal cord at the same time as the Mauthner axon impulse. The rapid spread of inhibitory influence, along with excitation, may be a general feature of motor system cells with large axonal diameters.

Visualization of active neural circuitry in the spinal cord of intact zebrafish.

1. One of the major obstacles in studying vertebrate neural networks is the difficulty in simultaneously monitoring activity in a population of neurons. To take advantage of the transparency of larval zebrafish, we used confocal microscopy to look into the spinal cord of immobilized fish to monitor neural responses during an escape behavior. 2. Populations of identified neurons were labeled with a calcium indicator and neural activity was monitored on a millisecond time scale. The calcium dependent nature of the fluorescent signals was confirmed by monitoring the accumulation, diffusion, and removal of calcium that was introduced by electrical and sensory stimulation. 3. Zebrafish, like most swimming vertebrates, have two major classes of motoneurons: large primary motoneurons thought to be used primarily for rapid movements and smaller secondary motoneurons implicated in slower movements. Our optical approach allowed us to ask how these groups of primary and secondary motoneurons respond during the escape behavior--one of the fastest and most forceful motor behaviors produced by vertebrates. 4. We demonstrate a previously unknown synchrony in the response of populations of primary and secondary motoneurons. This synchrony can account for the massive activation of the axial musculature during powerful escapes. Detection of this synchrony depended on the rapid in vivo imaging of activity in this neuronal population. This optical approach will allow functional studies of neuronal populations in the brain and spinal cord of normal and mutant lines of zebrafish.

Fictive swimming elicited by electrical stimulation of the midbrain in goldfish.

1. We developed a fictive swimming preparation of goldfish that will allow us to study the cellular basis of interactions between swimming and escape networks in fish. 2. Stimulation of the midbrain in decerebrate goldfish produced rhythmic alternating movements of the body and tail similar to swimming movements. The amplitude and frequency of the movements were dependent on stimulus strength. Larger current strengths or higher frequencies of stimulation produced larger-amplitude and/or higher-frequency movements. Tail-beat frequency increased roughly linearly with current strength over a large range, with plateaus in frequency sometimes evident at the lowest and highest stimulus strengths. 3. Electromyographic (EMG) recordings from axial muscles on opposite sides at the same rostrocaudal position showed that stimulation of the midbrain led to alternating EMG bursts, with bursts first on one side, then the other. These bursts occurred at a frequency equal to the tail-beat frequency and well below the frequency of brain stimulation. EMG bursts recorded from rostral segments preceded those recorded from caudal segments on the same side of the body. The interval between individual spikes within EMG bursts sometimes corresponded to the interval between brain stimuli. Thus, whereas the frequency of tail beats and EMG bursts was always much slower than the frequency of brain stimulation, there was evidence of individual brain stimuli in the pattern of spikes within bursts. 4. After paralyzing fish that produced rhythmic movement on midbrain stimulation, we monitored the motor output during stimulation of the midbrain by using extracellular recordings from spinal motor nerves. We characterized the motor pattern in detail to determine whether it showed the features present in the motor output of swimming fish. The fictive preparations showed all of the major features of the swimming motor pattern recorded in EMGs from freely swimming fish. 5. The motor nerves, like the EMGs produced by stimulating midbrain, showed rhythmic bursting at a much lower frequency than the brain stimulus. Bursts on opposite sides of the body alternated. The frequency of bursting ranged from 1.5 to 13.6 Hz and was dependent on stimulus strength, with higher strengths producing faster bursting. Activity in rostral segments preceded activity in caudal ones on the same side of the body. Some spikes within bursts of activity occurred at the same frequency as the brain stimulus, but individual brain stimuli were not as evident as those seen in some of the EMGs. 6. The duration of bursts of activity in a nerve was positively and linearly correlated with the time between successive bursts (cycle time).(ABSTRACT TRUNCATED AT 400 WORDS)

Excitation of motoneurons by the Mauthner axon in goldfish: complexities in a "simple" reticulospinal pathway.

1. The Mauthner cell in fish and amphibians initiates an escape behavior that has served as a model system for studies of the reticulospinal control of movement. This behavior consists of a very rapid bend of the body and tail that is thought to arise from the monosynaptic excitation of large primary motoneurons by the Mauthner cell. Recent work suggests that the excitation of primary motoneurons might be more complex than a solely monosynaptic connection. To examine this possibility, I used intracellular recording and staining to study the excitation of primary motoneurons by the M cell. 2. Simultaneous intracellular recordings from the M axon and ipsilateral primary motoneurons show that firing the M cell leads to complex postsynaptic potentials (PSPs) in the motoneurons. These PSPs usually have three components: an early, small, slow depolarization (component 1), a later, large, fast depolarization (component 2), and an even later, large, long-lasting depolarization (component 3). The first component has a latency of 0.52 +/- 0.15 (SD) ms, (n = 27) and most probably is a monosynaptic input from the M cell. This study focused on the two subsequent, less-understood parts of the PSP. Motoneurons typically fire off the second part of the PSP. This is usually (27 of 33 cells) the largest component, and it has a mean amplitude of 6.24 +/- 3.33 (SD) mV (n = 33) and a half-decay time of 0.44 +/- 0.18 (SD) ms (n = 27). The mean amplitude of the third component is 3.20 +/- 1.7 (SD) mV (n = 35), and its half-decay is 6.73 +/- 2.66 (SD) ms (n = 35). The latency of the second component averages 0.66 +/- 0.21 (SD) ms (n = 32), indicating that there are few synapses in the pathway mediating it. 3. One candidate pathway for the second component of the PSP involves a class of descending interneurons (DIs) that are monosynaptically, chemically excited by the M cell and appear in light microscopy to contact motoneurons. Simultaneous intracellular recordings from the M axon, a DI, and a primary motoneuron show that the interneurons are electrotonically coupled to motoneurons and produce the fast, second component of the PSP. Direct excitation of an interneuron leads to a very short-latency (less than 0.2 ms), fast PSP in a motoneuron similar to the second component of the PSP produced by the M axon. The short latency and fatigue resistance of this connection indicate it is electrotonic, and this is supported by evidence for DC coupling between the two cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The spinal motor system in early vertebrates and some of its evolutionary changes.

Recent studies of the spinal motor systems of vertebrates allow us to begin to infer the organization of the motor apparatus of primitive vertebrates. This paper attempts to define some of the features of the motor system of early vertebrates based on studies of the motor systems in anamniotes and in Branchiostoma. It also deals with some changes in the primitive motor system during evolution. The primitive motor system consisted of myomeric axial muscles, with a functional subdivision of the musculature into non-spiking slow muscle fibers segregated in the myomeres from spiking fast ones. These fibers were innervated by two major classes of motoneurons in the cord-large motoneurons innervating faster fibers and small motoneurons innervating slow fibers. There was not a simple isomorphic mapping of the position of motoneurons in the motor column onto the location of the muscle fibers they innervated in the myomeres. Early vertebrates used these axial muscles to bend the body, and the different types of muscle fibers and motoneurons reflect the ability to produce slow swimming movements as well as very rapid bending associated with fast swimming or escapes. The premotor network producing bending was most likely a circuit composed of a class of descending interneurons (DIs) that provided excitation of ipsilateral motoneurons and other interneurons, and inhibitory commissural interneurons (CIs) that blocked contralateral activity and played an important role in generating the rhythmic alternating bending during swimming. This DI/CI network was retained in living anamniotes. At least two major descending systems linked the sensory systems in the head to these premotor networks in the spinal cord. The ability to turn on swimming by activation of DI/CI premotor networks in the cord resided at least in part in a midbrain locomotor region (MLR) that influenced spinal networks via projections to the reticular formation. Reticulospinal neurons were important not only for initiation of rhythmic swimming but also in the production of turning movements. The reticulospinal cells involved in turns produced their effects in part via monosynaptic connections with motor neurons and premotor interneurons, including some involved in rhythmic swimming. A prominent and powerful Mauthner cell was most likely present and important for rapid escape or startle movements. Some features of this primitive motor apparatus were conserved during the evolution of vertebrate motor systems, and others changed substantially. Many features of the early motor system were retained in living anamniotes; major changes occur among amniotes.(ABSTRACT TRUNCATED AT 400 WORDS)

Axial motor organization in postmetamorphic tiger salamanders (Ambystoma tigrinum): a segregation of epaxial and hypaxial motor pools is not necessarily associated with terrestrial locomotion.

The axial motor column has undergone a major reorganization during the evolution of vertebrates. In aquatic anamniotes including lampreys, goldfish, and mudpuppies, epaxial and hypaxial motoneurons are intermingled in the column. In contrast, epaxial and hypaxial motoneurons are spatially segregated in water snakes, rats, and monkeys, apparently as a consequence of an isomorphic mapping of motoneuron location onto the position of innervated muscle in the embryonic myotome. The presence of these two very different arrangements of motoneurons requires a major restructuring of the motor column during vertebrate evolution. The time of this reorganization is unknown. All amniotes studied to date have an epaxial/hypaxial segregation, and all anamniotes do not, suggesting that the map arose with the origin of amniotes. All the anamniotes examined previously were permanently aquatic, however, and the map might therefore be associated with terrestrial locomotion. If so, we would expect terrestrial anamniotes to have an arrangement of motoneurons like that in amniotes. We studied the organization of motoneurons innervating the trunk muscles of postmetamorphic, terrestrial tiger salamanders and asked whether their motor columns are more like those of amniotes or those of aquatic anamniotes. The motor column in tiger salamanders is similar to that seen in aquatic anamniotes and very like that in mudpuppies--permanently aquatic salamanders. There are several classes of motoneurons with morphological similarities to the primary and secondary motoneurons characteristic of aquatic anamniotes. Epaxial and hypaxial motoneurons show no obvious morphological differences and occupy extensively overlapping positions in the motor column. The only epaxial/hypaxial distinction is the presence of a few, small, relatively undifferentiated motoneurons located subadjacent to the ependymal layer. These motoneurons are filled only by horseradish peroxidase (HRP) applied to hypaxial nerves. They are probably newly born motoneurons, and their presence suggests continued addition of motoneurons, even in adult salamanders. We conclude that the epaxial/hypaxial segregation seen in amniotes is not necessarily associated with terrestrial locomotion. The segregation and the topographic map it reflects may have arisen in conjunction with the origin of amniotes. If they instead arose prior to the origin of extant amphibians, they must have been secondarily lost in those salamanders studied to date. An examination of the motor column of other amphibians should help to resolve this issue.

Spinal network of the Mauthner cell.

Most swimming vertebrates, particularly fishes and amphibians, avoid predators by producing an escape behavior initiated by a single action potential in one of a pair of cells, the Mauthner cells, located in the hindbrain. The most prominent feature of this behavior is a rapid, forceful bend of body and tail which leads to a characteristic C bend (stage 1) early in the escape. The spinal output of the Mauthner cell is largely responsible for this bend. Each Mauthner cell sends an axon down the length of the spinal cord on the side opposite the soma. When one Mauthner axon fires, it massively excites the ipsilateral musculature by (1) monosynaptic excitation of the large primary motoneurons that innervate the fast white muscle fibers and (2) polysynaptic excitation of motoneurons which is most likely mediated through an identified class of descending interneurons. While motoneurons on the side of the C bend are excited, excitation of those on the opposite side is blocked by inhibition of primary motoneurons and descending interneurons. This inhibition is mediated by commissural interneurons that are electrotonically coupled to the Mauthner axon and cross the spinal cord to monosynaptically inhibit cells on the opposite side. They inhibit not only primary motoneurons and descending interneurons, but also the commissural inhibitory interneurons on the opposite side. The inhibition of contralateral primary motoneurons and descending interneurons prevents motor activity on the side opposite the C bend from opposing that bend, while the inhibition of commissural interneurons prevents them from interfering, via their inhibitory connections, with excitation of motoneurons on the side of the bend. The spinal network responsible for the bend has several similarities with the spinal network for swimming in other anamniotic vertebrates, including lampreys and embryonic frogs. These similarities reveal important, primitive features of axial motor networks among vertebrates.

Morphological variability, segmental relationships, and functional role of a class of commissural interneurons in the spinal cord of goldfish.

As part of an attempt to understand the spinal control of the segmented axial musculature in goldfish, commissural spinal interneurons that are electronically coupled to the Mauthner axon (M-axon) were studied with intracellular recording and staining to examine their morphology, segmental relationships, and functional role. Prior studies suggested that these cells might mediate the crossed inhibition that blocks excitation of motoneurons on one side of the body during an escape bend to the opposite side. Simultaneous intracellular recordings from a M-axon, a commissural interneuron coupled to it, and a presumed primary motoneuron show that: (1) the interneurons produce monosynaptic, Cl(-)-dependent IPSPs in contralateral motoneurons, (2) the interneurons are responsible for the short latency, crossed spinal inhibition in the M-cell network, and (3) more than one interneuron terminates on each postsynaptic cell. Reconstructions of interneurons from wholemounts show that they form a fairly homogeneous morphological class of cells. Each one is unipolar, with an axon that crosses the cord and then usually bifurcates into a short, thin ascending branch and a thicker, longer descending one. Neighboring interneurons have overlapping terminal arbors consistent with the physiological data showing convergence of interneurons onto the same postsynaptic cell. The interneurons showed little relationship with body segments as defined by ventral roots. Their axons usually straddled segmental boundaries, with terminals typically occupying parts of two adjacent segments. Thus the functional unit of these cells is probably not a segment or a complete group of segments, but instead includes only parts of two adjacent segments. The presence of interneurons like these suggests that the overt peripheral segmentation of trunk musculature is not necessarily reflected in the organization of neurons that control those segments. A consideration of some functional characteristics of the activation of overlapping, serially repeated arrays of interneurons by descending pathways leads to the conclusion that the high conduction velocity of the M-axon, and the large size and short longitudinal extent of the axons of the inhibitory interneurons promote a strong, brief inhibition that is appropriate for the production of an escape turn that has a rapid bend to one side.