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.

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.

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.

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.

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.

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.

The organization of the motoneurons innervating the axial musculature of vertebrates. I. Goldfish (Carassius auratus) and mudpuppies (Necturus maculosus).

The motoneurons innervating different regions of the myomeres in goldfish and mudpuppies were examined by applying HRP to the musculature or to branches of spinal nerves. In goldfish, the populations of motoneurons innervating epaxial or hypaxial muscle occupied similar positions in the motor column and had similar size distributions. There was no relationship between the size or location of a motoneuron in the motor column and the dorsoventral location of the muscle it innervated in the myomeres. Instead, different populations of motoneurons innervated the functionally different red and white musculature. The red muscle was innervated only by small motoneurons that occupied the ventral portion of the motor column. Their small axons passed lateral to the Mauthner axon in the cord, and most of them traveled in a separate branch of each spinal nerve that ran in the horizontal septum to the red muscle. The white muscle was innervated by a population of motoneurons that did not innervate red. They were large and they occupied a characteristic position in the extreme dorsal part of the motor column. Their large axons traveled medial to the Mauthner axon in the cord and entered branches of spinal nerves running deep in the epaxial or hypaxial muscle. The white muscle was probably also innervated by some smaller motoneurons similar to those innervating red; however, these may have been motoneurons whose axons ran through white muscle to reach other muscle. The large motoneurons innervating only white muscle are similar to the primary motoneurons identified in developmental studies in teleosts (Myers: Soc. Neurosci. Abstr. 9:848, '83); the smaller ones, innervating both red and white, are like secondary motoneurons. Therefore, in goldfish, motoneurons having different morphology and developmental history also innervate different regions in the myomeres. The motor column in mudpuppies was, in general respects, similar to the column in goldfish. There were large primary motoneurons and small secondary ones. Though there were slight differences in the locations of motoneurons filled from nerves entering epaxial and hypaxial muscle, their distributions in the cord overlapped substantially. The motor columns in these two anamniotes differ substantially from the motor columns in those amniotes that have been studied. In amniotes, the motoneurons innervating epaxial and hypaxial muscles are spatially segregated in the cord (Smith and Hollyday: J. Comp. Neurol. 220:16-28, '83; Fetcho: J. Comp. Neurol. 249:551-563, '86).(ABSTRACT TRUNCATED AT 400 WORDS)

The organization of the motoneurons innervating the axial musculature of vertebrates. II. Florida water snakes (Nerodia fasciata pictiventris).

The motor pools of axial muscles in Florida water snakes (Nerodia fasciata pictiventris) were studied by applying horseradish peroxidase (HRP) to branches of spinal nerves innervating individual muscles or groups of muscles. Motor pools of different muscles or muscle groups were located in characteristic positions in both the transverse and the longitudinal extent of the motor column. Epaxial pools were located ventromedially in the column, segregated from most hypaxial ones, which were dorsolateral. The only exception to this general rule was the motoneurons innervating the levator costae muscle. Some of the motoneurons innervating this hypaxial muscle were located in the ventral part of the motor column, like epaxial motoneurons, but they were segregated longitudinally from epaxial ones. The arrangement of the motor pools was strikingly similar to the motor pools of presumptive homologous muscles in rats (Smith and Hollyday: J. Comp. Neurol. 220:29-43, '83), even though the locomotor mechanics in the two animals are very different. The similarities may reflect a comparable relationship between the location of motoneurons in the motor column and the location, in embryonic life, of the muscles they innervate. They also suggest that differences in the locomotor mechanics in the two species are accomplished without any dramatic reorganization of the medial motor column, in marked contrast to the substantial reorganization necessary to account for differences in the motor columns of amniotes and anamniotes.

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.

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.

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.

A review of the organization and evolution of motoneurons innervating the axial musculature of vertebrates.

In most anamniotes the axial musculature is myomeric and is functionally subdivided into superficial red and deep white muscle. In those anamniotes that have been studied the organization of the motor column is related to this functional subdivision. The motoneurons innervating red and white muscle differ in size, distribution in the motor column, and developmental history. There is no obvious topographic relationship between the location of motoneurons in the motor column and the dorsoventral location of the muscle they innervate in the myomeres; epaxial motoneurons are not segregated from hypaxial ones. Among amniotes, the myomeres divide to form a number of discrete muscles that may be complexly arranged. This breakup of the musculature is correlated with a subdivision of the motor column into discrete motor pools serving the different muscles. Unlike anamniotes, the motor pools are topographically organized. The epaxial pools are segregated from hypaxial ones, and within the epaxial and hypaxial pools the location of motoneurons innervating any particular muscle is related to the location of the muscle's precursor in the embryonic muscle masses. Thus adjacent motor pools innervate muscles arising from adjacent positions in the myotome. These dramatic differences between the motor columns in anamniotes and amniotes imply that the medial motor column has undergone a major restructuring during the evolution of vertebrates. The available evidence--which is tentative because of the few species that have been studied--suggests that a topographically organized motor column was absent in early vertebrates. A motor column/myotome map appears to have arisen just prior to, or in conjunction with the origin of amniotic vertebrates. The details of this map were conserved in different amniotes in spite of major structural and functional changes in the musculature. The map may be important for the proper control of the many muscles arising from the myotomes in amniotes because it facilitates the development and evolution of motor systems in which anatomically and functionally different muscles have spatially separate motor pools in the cord.

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)

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)

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.

Identification of motoneurons and interneurons in the spinal network for escapes initiated by the mauthner cell in goldfish.

We used intracellular recording and staining techniques to study the spinal circuitry of the escape behavior (C-start) initiated by the Mauthner axon (M-axon) in goldfish. Simultaneous intracellular recordings from one or both M-axons and a spinal neuron, followed by HRP labeling of the spinal cell, show that each M-axon makes monosynaptic, chemical excitatory synapses onto 2 populations of ipsilateral spinal neurons. The first consists of the large primary motoneurons that, based on earlier work (Fetcho, 1986), innervate exclusively the faster, white muscle fiber types in the myomeres. The second group of cells is formed by previously undescribed descending interneurons with ipsilateral axonal branches that have contacts with primary and secondary motoneurons spread over 2 or more body segments. Indirect evidence suggests that these descending interneurons are excitatory, and they may explain the polysynaptic activation of motoneurons observed in earlier studies of the spinal circuitry (Diamond, 1971). Both classes of neurons excited by the ipsilateral M-axon are disynaptically inhibited by the contralateral one. The morphology and physiology indicate that this inhibition is mediated by interneurons that are electrotonically coupled to one M-axon and have processes that cross the cord to inhibit contralateral neurons in the region where these postsynaptic cells receive excitatory input from the other M-axon. We have identified interneurons with the physiological and morphological features of these predicted crossed inhibitory interneurons. These cells are electrotonically coupled to the ipsilateral M-axon and receive a chloride-dependent disynaptic inhibitory input from the contralateral M-axon. Their very simple somata give rise to a process that crosses the spinal cord between the 2 M-axons. Once on the opposite side of the cord, the crossing process sends myelinated branches that run rostrally and caudally, roughly parallel to the contralateral M-axon. Processes that arise from these longitudinal branches terminate in a striking association with collaterals of the M-axon; nearly every M-axon collateral along the longitudinal course of an interneuron is met by a branch or branches of the interneuron whose terminals are apposed to neurons postsynaptic to the collateral.(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.

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)