Structure of the giant fibers of earthworms.

The median giant fiber and the pair of lateral giant fibers that run the length of the ventral nerve cord in earthworms were thought to arise by fusion of the axons of several nerve cells in each segment. The structure of these giant fibers has now been examined with a fluorescent dye injected into single fibers. Each giant axon connects to one cell body in each segment; the giant fibers are not fused axons. In each segment, the median giant fiber has three branches and each lateral giant fiber has five branches. These branches are presumably dendritic. No structural differences between the giant fibers in anterior and posterior regions of the worm seem to account for the functional polarity of the giant fiber system observed in behavioral studies.

Motor pattern production in reciprocally inhibitory neurons exhibiting postinhibitory rebound.

Pairs of neurons which inhibit each other can produce regular alternating bursts of impulses if they also exhibit postinhibitory rebound (PIR). Computer studies show that stable patterns occur spontaneously in systems of pacemaker neurons with PIR, and can be triggered in systems of nonpacemakers without requiring tonic excitation. The repetition rates of these patterns are determined largely by the PIR parameters. The patterns resist perturbation by phasic synaptic inputs, but can be modulated or turned off by tonic inputs. One pair of PIR neurons can be entrained by another pair with a different repetition rate to produce more complex firing patterns.

Antidromic action potentials fail to demonstrate known interactions between neurons.

An identified motor neuron in the stomatogastric ganglion of Panulirus interruptus inhibits four other motor neurons when it fires spontaneously or in response to depolarization of its soma. It does not inhibit these neurons when it is fired antidromically, although the attenuated antidromic spike is visible at its soma. These findings point out the difficulty of interpreting negative results from antidromic stimulation experiments and the importance of neuronal structure to the integrative activities of nervous systems.

State-changes in the swimmeret system: a neural circuit that drives locomotion.

The crayfish swimmeret system undergoes transitions between a silent state and an active state. In the silent state, no patterned firing occurs in swimmeret motor neurons. In the active state, bursts of spikes in power stroke motor neurons alternate periodically with bursts of spikes in return stroke motor neurons. In preparations of the isolated crayfish central nervous system (CNS), the temporal structures of motor patterns expressed in the active state are similar to those expressed by the intact animal. These transitions can occur spontaneously, in response to stimulation of command neurons, or in response to application of neuromodulators and transmitter analogues. We used single-electrode voltage clamp of power-stroke exciter and return-stroke exciter motor neurons to study changes in membrane currents during spontaneous transitions and during transitions caused by bath-application of carbachol or octopamine (OA). Spontaneous transitions from silence to activity were marked by the appearance of a standing inward current and periodic outward currents in both types of motor neurons. Bath-application of carbachol also led to the development of these currents and activation of the system. Using low Ca(2+)-high Mg(2+) saline to block synaptic transmission, we found that the carbachol-induced inward current included a direct response by the motor neuron and an indirect component. Spontaneous transitions from activity to silence were marked by disappearance of the standing inward current and the periodic outward currents. Bath-application of OA led promptly to the disappearance of both currents, and silenced the system. OA also acted directly on both types of motor neurons to cause a hyperpolarizing outward current that would contribute to silencing the system.

Intersegmental coordination in invertebrates and vertebrates.

How does the CNS coordinate muscle contractions between different body segments during normal locomotion? Work on several preparations has shown that this coordination relies on excitability gradients and on differences between ascending and descending intersegmental coupling. Abstract models involving chains of coupled oscillators have defined properties of coordinating circuits that would permit them to establish a constant intersegmental phase in the face of changing periods. Analyses that combine computational and experimental strategies have led to new insights into the cellular organization of intersegmental coordinating circuits and the neural control of swimming in lamprey, tadpole, crayfish and leech.

Local specification of relative strengths of synapses between different abdominal stretch-receptor axons and their common target neurons.

Stretch-receptor (SR) axons form a parallel array of 20 excitatory synapses with target neurons in the crayfish CNS. In each postsynaptic neuron, EPSPs from different SR axons differ significantly in size. These amplitudes are correlated with the segment in which each axon originates and form a segmental gradient of synaptic excitation in individual postsynaptic neurons. These differences might arise postsynaptically because of differential postsynaptic attenuation or presynaptically because of local regulation of the strength of each synapse. To examine these possibilities, we stimulated each SR axon separately and studied integration of its EPSPs in an identified neuron, Flexor Inhibitor 6 (FI6). Transmission from SR axons to FI6 was chemical and direct: EPSPs were accompanied by an increased postsynaptic conductance, were affected by extracellular Ca(2+), and showed frequency-dependent depression. EPSPs from different SR axons summed linearly. The rise times of EPSPs from different SR axons were not significantly different. We also filled individual SR axons and FI6 neurons and mapped and counted their points of contact. Each SR axon contacted each FI6 bilaterally, and contacts of SR axons from different segments were intermingled on FI6. SR axons that made the strongest synapses made more points-of-contact with FI6. These results imply that differences in strength do not arise because of differential postsynaptic attenuation of EPSPs, but rather because certain SR axons predictably make more points of contact with FI6 than do others. Thus, this gradient in excitation requires that each synapse be regulated by an exchange between the SR axon and its target neuron.

Limb movements during locomotion: Tests of a model of an intersegmental coordinating circuit.

During normal forward swimming, the swimmerets on neighboring segments of the crayfish abdomen make periodic power-stroke movements that have a characteristic intersegmental difference in phase. Three types of intersegmental interneurons that originate in each abdominal ganglion are necessary and sufficient to maintain this phase relationship. A cellular model of the intersegmental coordinating circuit that also produces the same intersegmental phase has been proposed. In this model, coordinating axons synapse with local interneurons in their target ganglion and form a concatenated circuit that links neighboring segmental ganglia. This model assumed that coordinating axons projected to their nearest-neighboring ganglion but not farther. We tested this assumption in two sets of experiments. If the assumption is correct, then blocking synaptic transmission in an intermediate ganglion should uncouple swimmeret activity on opposite sides of the block. We bathed individual ganglia in a low Ca(2+)-high Mg(2+) saline that effectively silenced both motor output from the ganglion and the coordinating interneurons that originated in it. With this block in place, other ganglia on opposite sides of the block could nonetheless maintain their normal phase difference. Simultaneous recordings of spikes in coordinating axons on opposite sides of the blocked ganglion showed that these axons projected beyond the neighboring ganglion. Selective bilateral ablation of the tracts in which these axons ran showed that they were necessary and usually sufficient to maintain coordination across a blocked ganglion. We discuss revisions of the cellular model of the coordinating circuit that would incorporate these new results.

Acetylcholinesterase activity in neurons of crayfish abdominal ganglia.

Acetylcholine is known to be a neurotransmitter in crustacean central nervous systems, but the numbers and distribution of cholinergic neurons in the segmental ganglia have not been described. To begin a census of cholinergic neurons in these ganglia, we used a histochemical assay for acetylcholinesterase to map neurons that contained this enzyme in the six abdominal ganglia of crayfish. In each abdominal ganglion, about 47 cell bodies were stained. The distributions of these stained cells in individual ganglia were similar, and the numbers were not significantly different. None of these stained cell bodies could be identified from their structures or locations as previously identified motor neurons or sensory neurons with central cell bodies. The process of one unpaired midline neuron that occurred only in the first three abdominal ganglia divided to send a pair of axons anteriorly into both halves of the connective. The central projections of afferent axons from many peripheral sensory neurons stained clearly as they entered each ganglion. Terminals of these axons were heavily stained in the horseshoe neuropil and the lateral neuropils. We labeled both gamma-aminobutyric acid (GABA) and acetylcholinesterase in individual ganglia. Only a few neurons in each ganglion were double-labeled. The unpaired midline neurons in the three anterior ganglia that stained for acetylcholinesterase did not show GABA-like immunoreactivity, but cells with similar shapes did label with the GABA antiserum. Acetylcholinesterase is not a definitive marker of cholinergic neurons, but its presence is often associated with the cholinergic phenotype. These stained cells should be considered as putative cholinergic neurons.

GABA-ergic neurons in the crayfish nervous system: an immunocytochemical census of the segmental ganglia and stomatogastric system.

We used an antiserum directed against gamma-aminobutyric acid (GABA) fixed with glutaraldehyde (Hoskins et al., Cell Tissue Res. 244:243-252, '86) to label neurons with GABA-like immunoreactivity (GLI) in wholemounts of the stomatogastric ganglion and each segmental ganglion of crayfish, except the brain. Each abdominal ganglion had an average of 63 labeled neurons, or 10% of all their neurons. Each peripheral nerve of each abdominal ganglion except the last contained labeled axons. Within each segment, the first peripheral nerve, N1, had five axons; the second peripheral nerve, N2, had at most four; and the third peripheral nerve, N3, had two. In the last ganglion, N2 had one labeled axon, N3 had two and N6 had two; the other nerves contained no labeled axons. A tabulation of the identified inhibitory neurons in the abdominal ganglia revealed that 40% of these GABA-ergic neurons have been identified. The subesophageal ganglion had many labeled neurons in clusters that formed a repeating pattern; it also had labeled neurons near its dorsal midline. The thoracic ganglia contained more labeled neurons than did the abdominals, but their patterns of labeling were similar. The commissural ganglia contained three clusters of labeled neurons and sent labeled axons to the esophageal ganglion. The esophageal ganglion contained four labeled neurons and many labeled axons. The stomatogastric ganglion contained labeled axon terminals but not labeled neurons.

Functional organization of crayfish abdominal ganglia. III. Swimmeret motor neurons.

Swimmerets are limbs on several segments of the crayfish abdomen that are used for forward swimming and other behaviors. We present evidence that the functional modules demonstrated previously in physiological experiments are reflected in the morphological disposition of swimmeret motor neurons. The single nerve that innervates each swimmeret divides into two branches that separately contain the axons of power-stroke and return-stroke motor neurons. We used Co(++) or biocytin to backfill the entire pool of neurons that innervated a swimmeret, or functional subsets whose axons occurred in particular branches. Each filled cell body extended a single neurite that projected first to the Lateral Neuropil (LN), and there branched to form dendritic structures and its axon. All the motor neurons that innervated one swimmeret had cell bodies located in the ganglion from which their axons emerged, and the cell bodies of all but two of these neurons were located ipsilateral to their swimmeret. Counts of cell bodies filled from selected peripheral branches revealed about 35 power-stroke motor neurons and 35 return-stroke motor neurons. The cell bodies of these two types were segregated into different clusters within the ganglion, but both types sent their neurites into the ipsilateral LN and had their principle branches in this neuropil. We saw no significant differences in the numbers or distributions of these motor neurons in ganglia A2 through A5. These anatomical features are consistent with the physiological evidence that each swimmeret is controlled by its own neural module, which drives the alternating bursts of impulses in power-stroke and return-stroke motor neurons. We propose that the LN is the site of the synaptic circuit that generates this pattern.

Functional organization of crayfish abdominal ganglia: I. The flexor systems.

For insect ganglia, Altman (Advances in Physiological Science, Vol. 23. Neurobiology of Invertebrates. New York: Pergamon Press, pp. 537-555, '81) proposed that individual neuropils control different motor activities. A corollary of this hypothesis is that motor neurons involved in many behavioral functions should branch in more neuropils than those active in fewer behaviors. In crayfish, the abdominal fast-flexor muscles are active only during the generation of the powerstroke for tailflips, whereas the slow-flexor muscles are involved in the maintenance of body posture. The slow flexors are thus active in many of the crayfish's behavioral activities. To test the generality of Altman's idea, we filled groups of crayfish fast-flexor and slow-flexor motor neurons with cobalt chloride and described their shapes with respect to the ganglionic structures through which they pass. Individual fast flexors were also filled intracellularly with HRP. Ganglia containing well-filled neurons were osmicated, embedded in plastic, and sectioned. Unstained sections were examined by light microscopy and pertinent sections were photographed. We found that the paths of the larger neurites were invariant, that the dendritic domains of fast and slow motor neurons occupied distinctive sets of neuropils, and that dendrites of slow motor neurons branched in more ganglionic structures than did those of fast motor neurons. These results are consistent with Altman's hypothesis.

Proctolin and excitation of the crayfish swimmeret system.

The ventral nerve cord of crayfish contains axons of five pairs of excitatory interneurons, each of which can activate the swimmeret system. Perfusion of the ventral nerve cord with the neuropeptide proctolin also activates the swimmeret system. The experiments reported here were conducted to test the hypothesis that one or more of these excitatory interneurons uses proctolin as a transmitter. Each of the five excitatory axons was located and stimulated separately in an individual crayfish, and similar motor activity was elicited by stimulating each of them. Quantitative comparison of spontaneous swimmeret motor patterns with activity caused by stimulating one of these excitatory axons, EC, or by perfusing with proctolin solutions showed that the motor patterns produced under these three conditions were not significantly different (P > 0.05). By using a new, affinity-purified proctolin antiserum, we labeled axons in the connective tissue between the last thoracic and first abdominal ganglion and compared the positions of labeled axons with the previously described positions of the excitatory axons. About 0.3% of the axons in these connective tissues showed proctolin-like immunoreactivity, but heavily labeled pairs of axons did occur bilaterally in the regions of excitatory swimmeret axons. The projections of these labeled axons into the abdominal ganglia were traced in serial plastic sections. Labeled processes were abundant in the lateral neuropils, the loci of the swimmeret pattern-generating circuitry. From this evidence, we propose that three of these excitatory swimmeret interneurons use proctolin as a transmitter, but that a fourth does not. The evidence for the fifth axon is ambiguous.

Functional organization of crayfish abdominal ganglia: II. Sensory afferents and extensor motor neurons.

Abdominal ganglia of crayfish contain identifiable neuropils, commissures, longitudinal tracts, and vertical tracts. To determine the functional significance of this ganglionic framework, we backfilled the following types of neurons with cobalt chloride: sensory hair afferents, slow and fast extensor motor neurons, the segmental stretch receptor neurons, and their inhibitory accessory cells. After the cobalt ions were precipitated and intensified, we studied the central projections of the filled neurons within the ganglionic structures. All of the axons of these neurons exit or enter each of the first five abdominal ganglia through the second pair of nerves. Our description of the central projections of the hair afferents is the first in the literature. These afferents innervate the large ventral horseshoe neuropil (HN) in the core of each ganglion. This neuropil is homologous to the insect ventral association centers, which also process sensory information. Furthermore, we discovered that some of the crayfish afferents innervate glomeruli within the HN. The slow and fast extensor motor neurons, the stretch receptor neurons, and the accessory cells branch mostly in the dorsal part of the ganglion. We reinterpret previous identifications of the extensor neurons that were based largely on soma position. Together with our previous descriptions of the flexor motor neurons, these results allow us to relate both rapid tail-flips and slower postural movements to the structure of the segmental ganglia.

Intersegmental coordination of swimmeret movements: mathematical models and neural circuits.

Swimmerets move periodically through a cycle of power-strokes and return-strokes. Swimmerets on neighboring segments differ in phase by approximately 25%, and maintain this difference even when the period of the cycle changes from < 1 to > 4 Hz. We constructed a minimal cellular model of the segmental pattern-generating circuit which incorporated its essential components, and whose dynamics were like those of the local circuit. Three different intersegmental coordinating units were known to link neighboring ganglia, but their targets are unknown. We constructed different intersegmental circuits which these units might form between neighboring cellular models, and compared their dynamics with the real system. One intersegmental circuit could maintain an approximately 25% phase difference through a range of periods. In physiological experiments, we identified three types of intersegmental interneurons that originate in each ganglion and project to its neighbors. These neurons fire bursts at certain parts of the swimmeret cycle in their home ganglion. These three neurons are necessary and sufficient to maintain normal coordination between neighboring segments. Their properties conform to the predictions of the cellular model.

The PD programs: a method for the quantitative description of motor patterns.

We describe a family of 6 computer programs that measure, analyse and create graphic displays of complex motor patterns. The programs create lists of times at which successive bursts of impulses in different nerves started and stopped, and use these lists to calculate the periods and durations of these bursts and to calculate their phases relative to some specified frame of reference. When calculating phases, the programs take into account missing bursts or extra bursts in each reference interval. Individual programs then calculate descriptive statistics for these parameters, select lists of paired data for plotting and regression analysis, and prepare files for graphical display of statistics as boxplots. A final-program plots these files on a digital plotter. These programs are available for non-commercial use.

The osmium-ethyl gallate procedure is superior to silver impregnations for mapping neuronal pathways.

Ganglia processed through the osmium-ethyl gallate procedure (OEG)19 retain more structural integrity than those processed through various silver impregnation methods. However, the OEG method continues to be neglected by most neuroanatomists. Both types of procedures have been used to trace large neuronal tracts, but during silver impregnation the neuropils lose many of their identifying characteristics. We demonstrate here the advantages of the OEG procedure by comparing it with two silver techniques, Rowell's and Holmes's. The OEG method yields consistent and reliable results and is easier to carry out than silver protocols. Most importantly, the better preservation of the neuropils has led to the discovery and study of regional specializations that were previously undetected from silver preparations.

Motor-pattern production: interaction of chemical and electrical synapses.

A pair of neurons exhibiting postinhibitory rebound, if connected through reciprocally inhibitory chemical synapses, will exhibit a stable pattern of alternating bursts. If two such oscillating pairs, of similar but not identical properties are connected by means of an electrical synapse and an inhibitory chemical synapse between two neurons, one in each pair, the burst patterns may drift, may lock in synchrony, may entrain in antiphase, may entrain at an intermediate phase, or may be suppressed in the inhibited pair. The behavior depends on the strengths of the chemical and electrical coupling as well as on the degree of depression at the chemical synapse. There relationships of the motor patterns are illustrated quantitatively through theoretical calculations.