The interneurons of the abdominal positioning system of the crayfish. How these neurons were established and their use as identified cells and command elements.

Arthropods with segmented abdomens show similar abdominal positioning behaviors. It has been possible to gain some understanding of the neural basis of these behaviors in lobsters and crayfish using standard intracellular and dye-filling techniques. Typically crayfish and lobsters have six abdominal segments each controlled by a set of flexor and extensor tonic muscles. Each segment has a dozen tonic motor neurons controlled in turn by a large number of interneurons. A similar set of phasic muscles, motor neurons and interneurons control a fast system. The fast components underlie such behaviors as escape and swimming. Lucifier-filled microelectrodes were used to stimulate, record and dye-fill the motor neurons and interneurons of the tonic systems. It was soon apparent that all of these neurons are identifiable. These data allowed us to determine how many interneurons served in a circuit generating a behavior, while the use of pairs of electrodes permitted the study of synaptic interactions between interneurons. Interneurons involved in abdominal positioning produced either flexion (flexion producing interneurons or FPI), extension (EPI) or inhibition (I). Significantly, FPIs tended to synaptically excite other FPIs and inhibit EPIs. In turn EPIs excited other EPIs and inhibited FPIs. As a result, impaling and stimulating an FPI, for example, tended to recruit others and their combined activity evoked a natural-looking behavior. The inhibition between FPI and EPI and vice versa tended to account for the reciprocity seen between the two behaviors in all experiments. Finally the synaptic connections between EPI-EPI on FPI-FPI were found to be essentially invariable. Thus repeated stimulation of an FPI or the stimulation of this same FPI in another preparation, at another time, gave essentially the same overall behavior such that the stimulation of one FPI or EPI could evoke a wide spread output resembling a normal behavior.

Complementation of physiological and behavioral defects by a slowpoke Ca(2+) -activated K(+) channel transgene.

The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo(4) mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types.

Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.

The Drosophila slowpoke gene encodes a BK-type calcium-activated potassium channel. Null mutations in slowpoke perturb the signaling properties of neurons and muscles and cause behavioral defects. The animals fly very poorly compared with wild-type strains and, after exposure to a bright but cool light or a heat pulse, exhibit a "sticky-feet" phenotype. Expression of slowpoke arises from five transcriptional promoters that express the gene in neural, muscle, and epithelial tissues. A chromosomal deletion (ash2(18)) has been identified that removes the neuronal promoters but not the muscle-tracheal cell promoter. This deletion complements the flight defect of slowpoke null mutants but not the sticky-feet phenotype. Electrophysiological assays confirm that the ash2(18) chromosome restores normal electrical properties to the flight muscle. This suggests that the flight defect arises from a lack of slowpoke expression in muscle, whereas the sticky-feet phenotype arises from a lack of expression in nervous tissue.

Consistency of interneuronal group formation in response to stimulation of identified cells.

In crayfish stimulation of abdominal positioning interneurons (APIs) recruits other interneurons producing various abdominal movements. We investigated whether: (1) the same API from different preparations activated a similar number or group of interneurons, (2) different APIs activated different groups, and (3) repeated stimulation of an API consistently affected a similar set of interneurons. To quantify the similarities and differences of the recruited interneuronal groups we compared the number of interneurons affected, their firing frequencies, and motor outputs. Three types of APIs (Curly Q, L and T) were identified and each type was stimulated in three preparations. Our results showed that for the Curly Q and L cells, each cell type activated interneuronal groups that were statistically similar in number and firing frequency. The T cell activated interneuronal groups that were more variable. Some APIs generally provided a repeatable motor output; all did not. The interneuronal groups activated by the Curly Q, L and T cells were very different from each other. Repeated stimulation of one Curly Q cell affected similar although not identical sets of interneurons. These data suggest that repeated motor outputs could be produced by a similar but not identical group of cells.

Estimation of the size and directional output of functional groups of interneurons underlying abdominal positioning behaviors in crayfish.

Quantitative studies were made of a large population of interneurons that controls postural flexion and extension of the crayfish abdomen. The number of interneurons needed to produce a motor program was estimated by stimulating a single abdominal positioning interneuron and recording interneuronal activity that was evoked from rostral and caudal connectives in an isolated abdominal nerve cord. We also examined the role that these functional groups have in producing a stronger motor output in either a rostral or caudal direction and thus specifying various abdominal geometrics. The average number of interneurons responding to stimulation of a single abdominal positioning interneuron was 32 (range: 3-50; n = 27). The average number of interneurons that decreased activity was 10 (range: 2-32). Of 653 activated interneurons from 20 preparations, approximately 43% fired between 2 and 5 Hz, 33% fired between 6 and 15 Hz, and 25% fired > 15 Hz. The size of a recruited group was usually but not always correlated with the strength of its motor response or with the direction of motor bias. Therefore, the contribution of a group may depend upon the number of active elements as well as synaptic efficacy.

Interneurons involved in the control of multiple motor centers in crayfish.

A number of studies have suggested that abdominal positioning interneurons (APIs) in the crayfish Procambarus clarkii can influence activity in multiple motor centers. Previous work on this population of neurons has demonstrated that they control the activity of tonic abdominal motor neurons (MNs) which generate postural movements of the abdomen and, to some extent, the activity of swimmeret MNs. This investigation demonstrates that many identified APIs also affect the activity of two populations of MNs which open and close the uropods and, in some cases, that of the swimmeret MNs as well. The majority (64%) of APIs examined in this study have an excitatory effect on both populations of uropod MNs. A smaller number (23%) increase the level of activity in one population of uropod MNs, and suppress, or have no effect on, the activity of the other population of uropod MNs. Approximately 25% of the APIs which were examined influence the output of swimmeret MNs, in addition to affecting the activity of uropod MNs. There are also indications that previous estimates of the number of APIs may have been too low. This is based on the observation that many APIs possess what appear to be similar morphologies but generate different patterns of motor output. Taken together, these findings support the idea that APIs influence the output of multiple motor centers which play a role in the control of general body posture and balance in crayfish.

Cyclic postural behavior in the crayfish, Procambarus clarkii: properties of the pattern-initiating network.

Crayfish exhibit complex cyclical adjustments in abdominal posture during certain forms of backward walking. An isolated nerve cord preparation was used to investigate the properties of the interneurons which direct this alternation of abdominal flexion and extension. The command function for this cyclic postural behavior appears to be the domain of a distributed network of multiple pattern-initiating interneurons: each interneuron may be viewed as a command element within a command system. The cyclic pattern may be elicited by stimulation of small axon bundles pulled from the ventrolateral margins of any of the abdominal connectives. As few as one stimulus pulse to the axon bundle can elicit a single cycle of patterned output, although more pulses are generally necessary. This suggests some convergence or amplification step in the pattern-initiating interneurons. The amplification may be accomplished by several pattern-initiating interneurons that are coupled to one another and converge on pattern generating circuits in each ganglion. Evidence supporting this interpretation is presented. Experiments involving resection of the cord reveal that the pattern-initiating signals transfer laterally across all of the abdominal ganglia, but the network contains a bias for descending signal conduction once a lateral transfer is made. This finding agrees with other results. For example, recordings from pattern-initiating axon bundles at rostral and caudal locations in the abdominal nerve cord show several descending but only one ascending unit activated during cyclic pattern generation. We also show that an isolated ganglion is capable of producing the cyclic motor program, although the outputs are much weaker than those elicited in the intact abdominal cord. Therefore, the pattern-initiating system is both central and distributed.

Specific second messengers activate the caudal photoreceptor of crayfish.

The caudal photoreceptor (CPR) found in the last abdominal ganglion of crayfish is a well-known example of a non-retinal photosensitive element. In addition to light sensitivity, this cell has been assigned a command role for a walking behavior. The molecular mechanism of transduction in this cell has not been previously studied. The involvement of an intermediate messenger substance is suggested by its long latency to response, its prolonged afterdischarge, and by the requirement for an amplification process for the efficient transduction of light. We tested the effect of some putative second messengers by pressure injecting them into the CPR and noting the physiological response. Here we report that intracellular injection of inositol 1,4,5-trisphosphate (IP3), calcium, and the guanosine nucleotide GTP mimics the light response, while cAMP, IP1 and IP2 have no effect on the firing rate. The key intermediate in transduction in vertebrate photoreceptors, cGMP, was ineffective in this system. This work adds to the growing body of evidence that IP3 plays a role in invertebrate phototransduction.

Synaptic interactions among neurons that coordinate swimmeret and abdominal movements in the crayfish.

1. Many interneurons in the crayfish (Procambarus clarkii) abdominal nervous system influence two behaviors, abdominal positioning and swimmeret movements. Such neurons are referred to as dual output cells. Other neurons which influence either one behavior or the other are single output cells. 2. Extensive synaptic interactions were observed between both dual and single output neurons involved in the control of abdominal positioning and swimmeret movements. Over 60% of all neuron pairs examined displayed interactions. Pairs of agonist neurons displayed excitatory interactions, while pairs of antagonists had inhibitory interactions. This pattern of interaction was observed in about 75% of interactive neuron pairs whether abdominal positioning or swimmeret outputs were considered. 3. Evidence for both serial and parallel connectivity, as well as, reciprocal or looping connections was observed. Looping connections can be found both between the abdominal positioning and swimmeret systems and within each system. 4. Most (28/34) single output neurons were not presynaptic to dual output neurons. No single output neurons were found to excite dual output neurons to spiking, although inhibitory interactions and weak excitations were observed. 5. Abdominal positioning inhibitors displayed properties consistent with a role in mediating some of the coordination between the swimmeret and abdominal positioning systems. 6. None of the dual output neurons examined influenced the swimmeret motoneurons directly.

The effect of various neurotransmitters and some of their agonists and antagonists on the crayfish abdominal positioning system.

1. Crayfish abdominal nerve cords were perfused with selected transmitters or their agonists or antagonists. Motor activity underlying abdominal positioning behavior was monitored. 2. All the neurotransmitters except glycine had a measurable effect on this system. 3. Acetylcholine and its agonists were slightly stimulatory. Both muscarinic and nicotinic receptors were indicated. 4. GABA was weakly inhibitory. Picrotoxin was strongly stimulatory, perhaps as a result of its known ability to block GABA and inhibitory acetylcholine receptors. 5. Histamine was strongly inhibitory. Both H1 and H2 receptors were indicated. 6. Glutamate was found to be slightly inhibitory while its agonist, NMDA, showed no effect. 7. Finally, L-Dopa was stimulatory, but only at a high concentration.

Dual motor output interneurons in the abdominal ganglia of the crayfish Procambarus clarkii: synaptic activation of motor outputs in both the swimmeret and abdominal positioning systems by single interneurons.

Many behavior patterns of the crayfish involve the positioning of the abdomen by the tonic motor system. Movements and positionings of the swimmerets are coordinated with these abdominal movements. Evidence from extracellular analyses suggested that single interneurons of the abdominal nerve cord could produce motor outputs in both the swimmeret and the abdominal positioning systems. Our intracellular investigation has revealed that many single cells can evoke outputs in both motor systems. Interneurons which produced fictive extension or flexion of the abdomen or inhibition of abdominal movement were also able to modulate a variety of swimmeret behavior including cyclic beating and excitation or inhibition of episodic outputs. Although interneurons were discovered that evoked each of the possible classes of dual-output combinations, those that evoked combinations frequently observed in the freely behaving animal were more common than those that evoked infrequently observed combinations. Evidence also indicated that abdominal positioning inhibitors are present in greater numbers than previously suspected and that many are closely associated with the swimmeret circuitry. Interneurons with the ability to start and stop swimmeret cyclic outputs with current injections of opposite polarity are proposed to be higher-order cells, and some are shown to have the properties of trigger neurons. It is proposed that most dual-output cells are presynaptic to single-output cells and that groups of related dual-output cells may function together as command elements.

Unexpected divergence among identified interneurons in different abdominal segments of the crayfish Procambarus clarkii.

The command elements that initiate and coordinate the abdominal movements in crayfish show little similarity between the various abdominal segments. Our criteria for similarity among interneurons were based on both cell morphology and electrophysiology. By contrast, previously published evidence shows much greater intersegmental similarity in the skeletal, muscular, motoneuronal, and sensory components of the abdominal system in crayfish, structures that are controlled by or send information to the command elements. Therefore, unlike the command elements, these structures have retained nearly identical form and function in the various segments. We also found in different ganglia examples of interneurons involved with abdominal positioning behavior that have similar morphology but different function and vice versa. Such interneurons could represent divergent pairs of serial homologues. It is unknown why so many of the abdominal positioning interneurons have become different. The various ganglia may perform subtly different functions, requiring differences in the positioning interneurons but not in the motor neurons or muscles. Alternatively, some of the abdominal positioning interneurons underlie more than one behavior; consequently, selection acting on these multiple functions may have changed these interneurons through evolution.

Abdominal positioning interneurons in crayfish: participation in behavioral acts.

Premotor interneurons involved in the abdominal positioning behaviors of the crayfish, Procambarus clarkii, were studied intracellularly, along with motoneuron activity, in semi-intact preparations during episodes of fictive behavior. Each impaled cell was tested by injecting depolarizing current and examining the motor output. If a response was evoked then the cell was classified as a flexion-producing interneuron (FPI), extension-producing interneuron (EPI) or mixed output interneuron (MOI). A platform drop/rise procedure was then used to elicit abdominal extension-like and flexion-like responses. Interneurons that were active during positioning behavior were silenced by hyperpolarization to determine their contribution in generating the underlying motor program. The data were used to assess the degree of participation of these interneurons in abdominal positioning behavior. Fewer than half of the FPIs, EPIs and MOIs became active during the behavioral episodes. Strength of response to depolarizing current was not correlated with the probability that a cell would fire during behavior. Hyperpolarization tests showed that typical FPIs, EPIs and MOIs were only responsible for a small part of the overall motor output. Also, interneurons, regardless of their FPI or EPI classification, were often observed to fire during both flexion-like and extension-like behaviors. Responses of FPIs, EPIs and MOIs to repeated platform movements suggest that these cells may fire according to a probability distribution depending on: (1) strength of the stimulus; (2) location of the stimulus; (3) location of the interneuron. Most identified cells could not readily be assigned to a specific behavior except for the 'T' cell type, which seems intimately involved in most flexion behaviors. The results of this study support the hypothesis that there are few if any 'command neurons', as defined by Kupfermann and Weiss (1978), in the crayfish abdominal positioning system. Abdominal positioning behavior, therefore, is probably under the control of a large network of cells each contributing a small part to the overall motor output.

A quantitative study of command elements for abdominal positioning behavior in the crayfish Procambarus clarkii.

We develop a statistical method to estimate the total number of command elements devoted to abdominal positioning behavior in crayfish. We assumed that all command elements can be identified, that each identified cell is equivalent to a tagged individual in a population, and that the cells were sampled randomly. Samples of 29, 30, 20, and 35 cells from abdominal ganglia A3, A4, A5, and A6, respectively, were taken from our catalog. We characterized each cell using several morphological and physiological criteria, determined how many times each identified cell was present in the sample, and estimated the total number of command elements using both a maximum likelihood method and a modification of the Lincoln index. The larger the proportion of identified cells seen only once in the sample, the more identified cells there were that were unrepresented in the sample. We estimate there are approximately 34, 60, 86, and 98 command elements in ganglia A3, A4, A5, and A6, respectively. Using a slightly different data set we show that the motor output of unipolar cells is more often stronger in the direction of the cell's axonal projection. In bipolar command elements, the output strength was uncorrelated with the relative sizes of the two projecting axons. No two cells in our sample were completely identical, and this morphological variability sometimes made it difficult to determine whether or not two cells obtained from different individuals were the same identified cell. We discuss why caution should be exercised in studies requiring precision in cell identification.

Interactions between the tonic and cyclic postural motor programs in the crayfish abdomen.

1. In the crayfish (Procambarus clarkii) abdomen, the superficial flexor and extensor muscles and the motoneurons that innervate them are employed during two completely different modes of behavior: (1) tonic postural adjustments and (2) cyclic movements associated with backwards terrestrial walking. We have tested the possibility that these two behavioral subsystems share at least some of the same tonic premotor interneurons. 2. Of the 108 tonic flexion- and extension-producing interneurons monitored during cyclic pattern generation, only 25 were recruited while 36 were inhibited. None of the recruited interneurons made a measurable contribution to the cyclic motor output. Similarly, none of the 20 inhibitory interneurons of the tonic subsystem recorded in this study was found to play a role in shaping the cyclic motor pattern. 3. Simultaneous activation of single tonic postural interneurons with the cyclic motor pattern revealed that the two behavioral subsystems interact in complex ways. Some tonic interneurons produced motor outputs that overrode the cyclic motor outputs while the motor outputs of other tonic interneurons were completely overwhelmed by the cyclic motor program. Still other tonic interneurons generated motor outputs that predominated over cyclic patterned outputs in some ganglia but were masked by the cyclic motor pattern in other ganglia. 4. Although weak interactions between the two subsystems occur at the premotor level, they have little effect on the normal generation of the cyclic pattern. Stronger interactions apparently occur at the level of the motoneurons and these interactions presumably may form the basis of switching from one behavior to the other. We conclude, therefore, that each behavioral subsystem relies upon its own unique set of premotor interneurons. Finally, those interneurons contributing to the cyclic motor pattern have not yet been identified.

Neural control of a cyclic postural behavior in the crayfish, Procambarus clarkii: the pattern-initiating interneurons.

As part of its repertoire of defensive behaviors, the crayfish, Procambarus clarkii, may respond to mildly threatening tactile or visual stimuli from the front of its body by walking backwards. During this behavior, the abdomen undergoes complex cyclical movements involving flexion and extension of the postural musculature which cause the tail to alternately contact and withdraw from the substrate. Intracellular neuropil recordings and dye injections were used to search for the interneurons responsible for initiating this postural motor pattern in the crayfish abdomen. Several diverse morphological types of interganglionic pattern-initiating (PI) interneurons were found. Each interneuron, when driven intracellularly, was capable of eliciting the same motor program, in its entirety, throughout the abdominal nerve cord. During pattern generation, PI interneurons exhibited a burst of spikes preceding the motor output. Silencing single PI interneurons with hyperpolarizing current during pattern generation failed to affect the motor program, indicating a redundancy of pattern-initiating function. The observations of extensive dye-coupling with other parallel axons, consistent dye-coupling with other identified cells in the pattern-initiating system, and the presence of multiple spike amplitudes in the bursts suggested electrotonic coupling among the PI interneurons. An additional group of interganglionic interneurons, the partial pattern-initiating (PPI) interneurons, were found to comprise a significant subset of the pattern-initiating system. As with the PI cells, the PPI interneurons exhibited a complex burst of spikes just preceding the patterned motor program. However, the PPI interneurons were only capable of eliciting an incomplete, though recognizable, postural motor pattern. Silencing any PPI interneuron during pattern generation caused a deficit in the motor pattern, indicating either an absence or lesser degree of functional redundancy within the PPI interneuron population compared to that occurring within the PI interneuron group. We conclude that a large number of PI interneurons are presynaptic to a relatively small group of PPI interneurons which, in turn, conduct pattern-initiating signals to the ganglionic oscillators. Our results indicate that pattern-initiation is accomplished through a command system involving multiple command elements organized in a coordinated interganglionic network.

Activity of crayfish abdominal-positioning interneurones during spontaneous and sensory-evoked movements.

The premotor interneurones that produce coordinated abdominal movements in crayfish (Procambarus) when stimulated directly, are also 'sensorimotor'. Sets of these interneurones respond in predictable ways to touching the body surface. One set of interneurones (type I) is activated to spiking by touch, while another (type II) receives only subthreshold influences. Several of these interneurones have overlapping receptive fields on the body surface. Touching areas of overlap activates groups of interneurones which discharge at low to moderate frequencies, rather than producing a high-frequency discharge of a single cell. No single positioning interneurone has been identified which is solely responsible for a "voluntary' (spontaneous) motor programme. When active, the positioning interneurones contribute to the production of the behaviour as a member of a constellation of such cells. The results show that this motor system comprises interneurones with sensory as well as motor properties. Although single cells can produce coordinated movements when stimulated at high frequencies, these positioning interneurones appear to function as 'command elements' within a large 'command system' and not as individual units.

Abdominal positioning interneurons in crayfish: projections to and synaptic activation by higher CNS centers.

Intracellular recording, stimulation, and Lucifer dye injections were used to characterize abdominal positioning interneurons from the neuropile of the second through sixth abdominal ganglia of the crayfish, Procambarus clarkii. Motor outputs of these cells were recorded with extracellular electrodes placed on various flexion and extension roots along the nerve cord. In an effort to assess the functional relationships between the postural interneurons in the abdomen and those known to exist in the circumesophageal connectives ( CECs ), a stimulus pulse train was delivered to each of the CECs while monitoring the intracellular responses of the impaled interneurons. Abdominal positioning interneurons were grouped into four general categories based on their responses to CEC stimulation: 1) those that projected their axons directly through the CECs ; 2) those that were remotely activated to spiking; 3) those locally activated to produce EPSPs or IPSPs; and 4) those that were not affected by CEC stimulation. The majority of abdominal positioning interneurons encountered in this study evoked flexion (N = 82), with relatively fewer evoking extension (N = 29). A major difference appeared between the two classes. Whereas 39% of the flexion interneurons had axons coursing to the brain, only 7% of the axons of extension interneurons coursed rostrally beyond the thoracic level. Finally, the large majority of those flexion and extension interneurons that lacked processes in the CEC received synaptic inputs at various levels along the lower CNS from other CEC neurons. Thus, control of abdominal positioning involves neurons at all levels in the CNS - some sequentially organized, others forming "through" pathways, but all with multiple input sites.

The organization of flexion-evoking interneurons in the abdominal nerve cord of the crayfish, Procambarus clarkii.

Intracellular recording and dye injection was used to investigate the flexion-evoking interneurons in the isolated abdominal nerve cord of the crayfish, Procambarus clarkii. The interneurons described in this study have all of the characteristics of cells called flexion command fibers in earlier works. These interneurons show cell bodies in all of the abdominal ganglia, all are interganglionic, and those with posteriorly directed axons enter the terminal caudal ganglion. Some cells were found in as many as eight different preparations, supporting the idea that these interneurons are identified cells. In addition to presenting evidence for identities, evidence was obtained for serial homology in this premotor system. A summary of the general organization of the flexion premotor apparatus is offered. It consists primarily of parallel elements (command interneurons) that are either bipolar or monopolar whose axonal projections course to the terminal caudal ganglion or rostrally into the thoracic region, perhaps into the cerebral ganglion. When activated singly, these parallel elements do not recruit additional interneurons but there are data to suggest that several of these elements could act in concert and interact in a low-gain fashion to produce coordinated positioning behavior.