James L. Larimer

Entrainment of the circadian locomotor activity rhythm in crayfish

Summary1.Crayfish (Procambarus clarkii) on a light cycle (LD 12∶12) exhibit a bimodal locomotor activity rhythm. One activity maximum, the “lights-on” peak, is synchronized with the onset, while the other, the “lights-off” peak, occurs shortly after the offset of light (Figs. 1–5).2.When placed in constant darkness (DD), these animals maintain a unimodal, free running circadian rhythm, involving only the “lights-off” peak of activity (Fig. 1).3.Removal, or isolation from the CNS, of the sixth abdominal ganglion (the site of the caudal photoreceptor) has no observable effect on activity (Figs. 2, 3), indicating that the caudal photoreceptor is not necessary for entrainment or initiation of either activity maximum.4.Removal of the ommatidia of both eyes, or bilateral section of the optic lobes between the lamina ganglionaris and medulla externa, obliterates the “lights-on” peak but does not affect entrainment of the “lights-off” response. Thus, the retina provides the necessary pathway for generating the “lights-on” activity, but is not required for entrainment of the circadian rhythm (Figs. 4, 5).5.Finally, ablation of both the caudal ganglion and the retina does not abolish entrainment. It is assumed, therefore, that crayfish possess an extraretinal-extracaudal photoreceptor which provides a sufficient pathway for the entraining signal (Figs. 4, 5).

Sensory-induced modifications of ventilation and heart rate in crayfish

Abstract 1. 1. The responses to several types of stimuli have been examined for their effects on the ventilation system and the heart of the crayfish. 2. 2. It is demonstrated that tactile stimuli applied either unilaterally or bilaterally induce a bilateral acceleration of the scaphognathites and may or may not produce a change in heart rate. 3. 3. Chemical stimuli such as CO 2 , low oxygen and solutions of L -glutamic acid and sodium chloride produce an inhibition of the homolateral scaphognathite. This reflex is usually accompanied by a simulataneous bradycardia or cardiac arrest. No response was obtained from solutions of CaCl 2 , d (−)levulose or sucrose. 4. 4. Stimulation with water currents produced a reflex inhibition similar to that obtained from the effective chemicals, but the flow reflex is shown to be distinct. A sudden change in temperature also produces a response similar to that obtained from chemical stimulation. 5. 5. The chemoreceptors involved in the observed reflexes appear to be located somewhere within the gill chambers. The receptors mediating the flow response are less well localized. The nature of the remaining neuronal connections forming the reflex circuits is discussed. 6. 6. The short-term changes in ventillation and circulation observed here may greatly influence gas transport, but such modifications in activity do not necessarily reflect the internal demands for oxygen. A central control mechanism must be present which compensates for the random sensory induced changes according to the long-term requirements of the animal.

The CNS Photoreceptor of crayfish: Morphology and synaptic activity

SummaryMorphological and physiological evidence is presented which adds to our understanding of the responses exhibited by the caudal photoreceptor (CPR) interneuron of the crayfish. Illumination of a restricted area of the 6th abdominal ganglion showed that the CPR responds to light from only a part of the ipsilateral half of the ganglion. From Procion Yellow injections, the CPR neuron was found to consist of a contralateral cell body and an extensive ipsilateral dendritic tree. Significantly, the ganglionic area of light sensitivity was found to correspond closely to the dendritic domain. Electrical responses were recorded intracellularly from the CPR within the neuropil, both as a result of naturally evoked photic and mechanoreceptive input and of direct electrical stimulation of the ganglionic roots. When light was the predominant input, membrane depolarization of the CPR was gradual and smooth, reaching 8–10 mV and triggering trains of spikes. Electrical stimuli applied to the ipsilateral 1st through 5th roots of abdominal ganglion six produced monosynaptic EPSPs while stimuli to the corresponding contralateral roots evoked IPSPs. Recruitment of excitatory units was seen with increasing intensity; trains of stimuli showed summation. These inputs apparently originated from tactile hairs on the caudal appendages which were shown in the present study to be directionally sensitive. Several unidentified interneurons in abdominal ganglion six were also seen to respond to directional fluid movements over the caudal appendages, in much the same manner as did the CPR. Evidence for the presence of other light-sensitive interneurons is also presented. The data suggest that the CPR itself is transducing the light input.

PROPERTIES OF HEMOCYANINS. I. THE EFFECT OF CALCIUM IONS ON THE OXYGEN EQUILIBRIUM OF CRAYFISH HEMOCYANIN.

Abstract 1. 1. The oxygen affinity of hemocyanin from the crayfish, Procambarus simulans , is greatly decreased by dialysis between pH 6·5 and 8·0. This effect is largely reversed by 10 mM calcium or by 60 mM magnesium. 2. 2. The data do not conform to Hills equation, since the value of n increases with degree of oxygenation and is a function both of the calcium concentration and of pH. At low degrees of oxygenation n ≅1·5 and is independent of pH or calcium, while at high oxygenation levels n increases with either pH or calcium. 3. 3. The importance of calcium in controlling the degree of dissociation of hemocyanin into subunits is known. Since the calcium concentration in crayfish blood changes during the molting cycle, it seems possible that calcium may play a role in modifying the oxygen transport function of hemocyanin by changing the degree of aggregation.

Neural control of circadian rhythmicity in the crayfish

SummaryGross locomotor activity, as well as the movements of single walking legs, were monitored in intact and surgically altered crayfish (Procambarus clarkii). Section of the circumesophageal connectives (CEC) abolished the circadian activity rhythm in both the first and fourth pair of walking legs (Figs. 1–3), while section of the nerve cord between the third and fourth thoracic ganglia abolished rhythmicity in only the fourth pair (Fig. 4). Bilateral ablation of the eyestalks led to a several fold increase in total daily activity and caused the animal to be continually active; however, quantitative measurements of the locomotor activity revealed that a circadian rhythm in the level of activity persists for 6–12 days following eyestalk removal (Figs. 7, 8, 13, 14). These results suggest that the circadian oscillation for the locomotor rhythm originates in the supraesophageal ganglion and is coupled to thoracic locomotor centers via axons in the CEC. The activity rhythm of eyestalkless animals could also be entrained to a light cycle even after ablation of the caudal photoreceptor; however, certain features of entrainment were altered (Figs. 9–12). Thus, although the eyestalks are not required for rhythmicity, eyestalk structures apparently do participate in entrainment.

The command hypothesis: a new view using an old example

Abstract The interneurons that underlie abdominal flexion and extension behaviors in crustaceans were among the first to be called command neurons. They fit the original operational definition as cells that produce a well-defined movement or behavior when stimulated. Many examples of these cells are now known to influence behaviors throughout the animal kingdom. The early observation that stimulation of a single command neuron in a crustacean was sufficient to generate an apparently complete behavior led to the erroneous belief that one neuron might be responsible for one behavior. We now know that the strong stimulation of one command element is sufficient to recruit synaptically a group of similar neurons. In addition to the synaptic recruitment of agonists there is also a synaptic inhibition of their antagonists, resulting in what appears to be a complete behavior with reciprocity. Importantly, there is also evidence for the operation of similar functional groups in behaving animals. During animal-initiated behavior, each neuron in the functional group apparently makes only a minor contribution to the total motor output with the result that no single neuron in the group is necessary to generate the behavior. If the necessity criterion is a requirement to define a command neuron, then abdominal positioning interneurons can no longer be considered command neurons. Instead, they are cells with lesser roles, perhaps command elements in larger command systems. In spite of their diminished status, command elements occupy key positions in this and other motor systems.

Motor programs for abdominal positioning in crayfish

SummaryUsing chronically implanted suction electrodes (Fig. 2), records were obtained from the tonic abdominal flexor motor neurons of crayfish while they were undergoing various self-generated movements (Fig. 3). The main behavior examined in this study was one of abdominal extension (Fig. 1), a response which could be evoked repeatedly. Other stereotyped movements were also observed. Each class of behavior we examined has been evoked previously in dissected preparations by stimulating command interneurons, allowing comparison of selfgenerated and electrically evoked motor patterns.During abdominal extension, the flexor inhibitor neuron was observed to fire in a characteristic way (Fig. 4 left, Fig. 5) that was not materially altered even if the associated movements were prevented by rigid restraint (Fig. 4 right). These self-generated motor programs resembled those obtained from command fiber stimulation, both in detail and reproducibility, suggesting that the normal means of executing such stereotyped behavior in these animals is via selected command interneurons.Central reciprocity between the tonic flexor motor neurons and the flexor inhibitor was observed routinely in self-generated programs (Figs. 3, 6, 7), as was seen in dissected animals under command fiber control. The incidence of failure of reciprocity, however, appears to be more common in natural programs than in those evoked by direct stimulation of command interneurons.

Interneurons involved in abdominal posture in crayfish: structure, function and command fiber responses

Summary1.An intracellular search was undertaken in an effort to locate and characterize the elements involved in command-driven positional behavior in the isolated abdominal nerve cord of the crayfish,Procambarus clarkii. Small bundles containing flexion or extension-evoking command fibers were isolated in the anterior connectives; these bundles are considered here to be equivalent to identified command fibers. A microelectrode filled with Lucifer Yellow CH was used to find elements within the fourth abdominal ganglion that showed synaptic responses to either flexion or extension command fibers; and these elements were further characterized by current and dye injections.2.The interneurons thus encountered have been classified primarily by the form of motor output they evoked when strongly depolarized, and secondarily by their morphologies. We present data in this paper from 12 types of flexion-evoking cells, 4 types of extension-evoking cells and one inhibitory type that we term ‘flexion-antagonistic’.3.With the exception of this latter group, many of the interneurons evoked a fully or nearly fully organized motor output. That is, flexion-evoking interneurons activated the flexor motoneurons and the peripheral inhibitor to the extensor muscles, and usually inhibited much of the activity in the extensor motoneurons. Conversely, the extension-evoking interneurons activated the extensor motoneurons and the peripheral inhibitor to the flexor muscles and usually inhibited the flexor motoneurons. The flexion-antagonists inhibited the flexor motoneurons and the peripheral inhibitor to the extensor muscles, but did not activate any motoneurons.4.Because of the search strategy used, the majority of the cells that were studied showed synaptic connections to one or more of the three flexion and one extension command fiber bundle stimulated. Both apparently monosynaptic and polysynaptic connections were evident, but very few inhibitory connections were found. Thus, while flexion-evoking interneurons were in general depolarized by the flexion CFs, they were not hyperpolarized by the extension CF. Furthermore, we have demonstrated that several flexion-evoking interneurons share inputs from more than one flexion CF, whereas others have inputs from only one of three flexion CFs. Finally, at least two interneurons were found (type 5 and 6) that were apparently independent of the command neurons used in this study.5.Six of the flexion-evoking types were seen to have their somata within ganglion 4 and all had axonal processes extending either anteriorly or posteriorly. The remaining 6 flexor interneurons were seen only as an axonal process running through ganglion 4. We cannot determine from the present data their origins or the extent of their axons in the nerve cord. In several preparations, both forms of flexion-evoking cells were seen to produce motor output in the ganglia adjacent to G4, suggesting that these cells may be either command neurons or driver interneurons. Different experimental approaches will be required to resolve this question.6.While we cannot at this point propose the organization of CF-driven postural behavior, we have encountered several potentially important elements involved in the control of abdominal flexion. The motor output appears to be achieved by a combination of direct synaptic connections from the CF to the motoneurons and through connections with several other interneurons acting in parallel. A combination of these connections may provide for the strong reciprocity between flexor and extensor motoneurons, since individual driver cells are not always capable of this reciprocal output. Finally, redundancy of driver cells is indicated since we have not been able to identify any critical elements that, when functionally removed by hyperpolarization, eliminate a significant portion of the CF driven motor output.

Circadian Rhythm of Retinal Sensitivity in Crayfish: Modulation by the Cerebral and Optic Ganglia

Summary1.Surgical sections or smaller chemical lesions made in the optic tract of one eye of the crayfish,Procambarus clarkii, lead to loss of the ERG amplitude rhythm in the operated eye while the intact eye continued to oscillate in constant darkness (Fig. 2). Apparent recovery of generally lower amplitude oscillations after surgery did, however, occur in 7 out of 17 preparations tested (Fig. 6).2.Only lesions which included the central part of the optic tract resulted in a loss of rhythmicity (Fig. 1). Ultrastructure of this region of the optic tract showed axons with dense core vesicles and blood vessels; however, presumptive neurosecretory axons were not confined to this region (Fig. 3, 4).3.Tests for circulatory patency of the lesioned optic system were made by injecting eyestalk hormone extracts. Such injections caused depression of the ERG amplitude of both the operated and unoperated eye indicating that exogenous (and presumable endogenous) hormone reached the retina. Hormone injections, however, failled to alter the phase of the rhythm (Fig. 5).4.Low level unilateral retinal illumination in an animal with intact optic tracts successfully entrained the ERG rhythm in both eyes (Fig. 7).5.In operated animals unilateral illumination of the neurally isolated retina failed to entrain the intact contralateral side as evidenced by the fact that the intact side free-ran throughout the 13 days of attempted entrainment (Fig. 8A).6.Transfer of the entraining light to the intact retina phase-shifted its rhythm, while the operated eye became phase-locked to and appeared to be driven by the intact side (Fig. 8B).7.A summary diagram is provided which details the known coupling components of this circadian system (Fig. 9). Both hormonal and neural coupling appear to synchronize retinal sensitivity in the two eyes. Although the hormonal contribution is substantial, preliminary evidence indicates that their titers are driven by the underlying clock rather than comprising an integral part of the oscillator itself (Fig. 5).

Synaptic interactions between neurons involved in the production of abdominal posture in crayfish

SummaryPairs of neurons that produce or influence motor outputs in the abdominal positioning system of the crayfish (Procambarus clarkii) were impaled in isolated nerve cords with Lucifer Yellow-filled microelectrodes to determine their morphologies and the nature and extent of the synaptic interactions between them. Although the motor programs for positional adjustments can be produced by directly stimulating single interneurons, we found extensive interactions between these neurons, often involving the recruitment of one interneuron by another. The data indicate that the positioning interneurons do not operate as labelled lines, each independently producing a discrete position. Pairs of interneurons, each producing similar motor outputs when activated, were often found to be connected by unidirectional excitatory synapses. In contrast, central inhibition was commonly found between pairs of interneurons that produced antagonistic motor effects. Finally, the unidirectional interactions between positioning interneurons revealed a hierarchy of at least two tiers in this system. Based on these observations, we suggest that abdominal positioning in crustaceans is produced by constellations of interacting interneurons.