Abdominal positioning interneurons in crayfish: participation in behavioral acts

Summary 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.Abbreviations FPI flexion-producing interneuron - EPI extension-producing interneuron - MOI mixed output interneuron - SFMN superficial flexor motoneuron - SEMN superficial extensor motoneuron     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

Effects of leg movements on the synaptic activity of descending statocyst interneurons in crayfish,Procambarus clarkii

Crustacean postural control is modulated by behavioral condition. In this study, we investigated how the responses of descending statocyst interneurons were affected during leg movements. Intracellular recording was made from an animal whose statoliths had been replaced with ferrite grains so that statocyst receptors could be activated by magnetic field stimulation. We identified 14 morphological types of statocyst-driven descending interneurons. Statocyst-driven descending interneurons always showed an excitatory response to statocyst stimulation on either ipsilateral or contralateral side to the axon. The response of each statocyst-driven descending interneuron to statocyst stimulation was differently modulated by leg movements in different conditions. During active leg movements, six statocyst-driven descending interneurons were activated regardless of whether a substrate was provided or not. In other two statocyst-driven descending interneurons, the excitatory input during leg movements was stronger when a substrate was provided than when it was not. One statocyst-driven descending interneuron received an excitatory input only during leg movements on a substrate, whereas another statocyst-driven descending interneuron did not receive any input during leg movements both on a substrate and in the air. These results suggest that the descending statocyst pathways are organized in parallel, each cell affected differently by behavioral conditions.Abbreviations EMG electromyogram - NGI nonspiking giant interneuron - SDI statocyst-driven descending interneuron     

Modification of statocyst input to local interneurons by behavioral condition in the crayfish brain

Posture control by statocysts is affected by leg condition in decapod crustaceans. We investigated how, in the crayfish brain, the synaptic response of local interneurons to statocyst stimulation was affected by leg movements on and off a substratum. The magnetic field stimulation method permitted sustained stimulation of statocyst receptors by mimicking body rolling. The statocyst-driven local interneurons were classified into four morphological groups (Type-I–IV). All interneurons except Type-IV projected their dendritic branches to the parolfactory lobe of the deutocerebrum where statocyst afferents project directly. Type-I interneurons having somata in the ventral-paired lateral cluster responded invariably to statocyst stimulation regardless of the leg condition, whereas others having somata in the ventral-unpaired posterior cluster showed response enhancement or suppression, depending on the cell, during leg movements on a substratum, but no response change during free leg movements off the substratum. The synaptic responses of Type-II and IV interneurons were also affected differently by leg movements depending on the substratum condition, whereas those of Type-III remained unaffected. These findings suggest that the statocyst pathway in the crayfish brain is organized in parallel with local circuits that are affected by leg condition and those not affected.     

Stress protein synthesis by crayfish CNS tissue in vitro

Some crustacean axons remain functional for months after injury. This unusual property may require stress proteins synthesized by those neurons or provided to them by glial cells. To begin to explore this hypothesis, we examined the conditions that stimulated stress protein synthesis by crayfish CNS tissue in vitro. Incubation for 1–15 h with arsenite or at temperatures about 15°C higher than the acclimation temperature of 20°C induced transient expression of several stress proteins. The heat stress response was blocked by Actinomycin D, suggesting that synthesis of new mRNA was required. In addition, the major crayfish 66 kD stress protein and its mRNA had sequence identities with the 70 kD stress proteins of mammals. Since the crayfish stress response has much in common with that of other organisms, the unique advantages of the crayfish nervous system can be used to study the impact of stress proteins on glial and neuronal function.     

Mechanosensory interneurons (MSIs) in the crayfish 6th abdominal ganglion are inhibited by activation of other MSIs

1. Identified mechanosensory interneurons (MSIs) in the 6th abdominal ganglion of the crayfish Procambarus clarkii have been shown to inhibit other projecting MSIs. 2. Interneurons sensitive to water-current stimulation of the tailfan, and which inhibited the tactile response of other MSIs when activated by depolarizing currents, were identified by iontophoresis of fluorescent dye. 3. Ten inhibitory interneurons have been identified, including both non-adapting, directional cells and phasic "touch" cells. 4. Inhibition triggered by activation of the identified cells was not widespread among fibers in the connectives. 5. Inhibition recorded intracellularly was mediated by compound inhibitory postsynaptic potentials of long duration (300-400 msec) and latencies of 13-15 msec, and therefore was apparently polysynaptic. 6. Depolarization and/or activity in MSIs, which modulates the stimulus response characteristics of related cells is a possible mechanism for contrast enhancement among directional or frequency-selective interneurons.     

Synaptic inputs onto spiking local interneurons in crayfish are depressed by nitric oxide

We have analyzed the action of nitric oxide on the synaptic inputs of spiking local interneurons that form part of the local circuits in the terminal abdominal ganglion of the crayfish, Pacifastacus leniusculus. Increasing the availability of NO in the ganglion by bath applying the NO donor SNAP, or the substrate for its synthesis, L-arginine, caused a depression of synaptic inputs onto the interneurons evoked by electrically stimulating mechanosensory neurons in nerve 2 of the terminal ganglion. Conversely, reducing the availability of NO by bath application of an NO scavenger, PTIO, and an inhibitor of nitric oxide synthase, L-NAME, increased the amplitude of the evoked potentials. These results suggest that elevated NO concentration causes a depression of the synaptic inputs to spiking local interneurons. To determine whether these effects could be mediated through an NO/cGMP signaling pathway we bath applied a membrane permeable analogue of cGMP, 8-br-cGMP, which decreased the amplitude of the inputs to the interneurons. Bath application of an inhibitor of soluble guanlylyl cyclase, ODQ, produced an increase in the amplitude of the synaptic inputs. Our results suggest that NO causes a depression of synaptic inputs to spiking local interneurons probably by acting through an NO/cGMP signaling pathway. Moreover, application of NO scavengers modulates the inputs to these interneurons, suggesting that NO is continuously providing a powerful and dynamic means of modulating the outputs of local circuits.     

Homeostatic plasticity in the CNS: synaptic and intrinsic forms

The study of experience-dependent plasticity has been dominated by questions of how Hebbian plasticity mechanisms act during learning and development. This is unsurprising as Hebbian plasticity constitutes the most fully developed and influential model of how information is stored in neural circuits and how neural circuitry can develop without extensive genetic instructions. Yet Hebbian plasticity may not be sufficient for understanding either learning or development: the dramatic changes in synapse number and strength that can be produced by this kind of plasticity tend to threaten the stability of neural circuits. Recent work has suggested that, in addition to Hebbian plasticity, homeostatic regulatory mechanisms are active in a variety of preparations. These mechanisms alter both the synaptic connections between neurons and the intrinsic electrical properties of individual neurons, in such a way as to maintain some constancy in neuronal properties despite the changes wrought by Hebbian mechanisms. Here we review the evidence for homeostatic plasticity in the central nervous system, with special emphasis on results from cortical preparations.     

NRG1 and synaptic function in the CNS

Recent genetic evidence indicates that neuregulin 1 (NRG1) and its receptor erbB4 may be susceptibility genes in schizophrenia, but their function in CNS synaptic transmission and circuitry is not well understood. In this issue of Neuron, studies from Li et al. and Woo et al. show that NRG1 and erbB4 regulate transmission at brain glutamate and GABA synapses. These findings raise the possibility of synaptic defects in schizophrenia.     

Evidence that synaptic transmission between giant interneurons and identified thoracic interneurons in the cockroach is cholinergic

In the cockroach, a population of thoracic interneurons (TIs) receives direct inputs from a population of ventral giant interneuons (vGIs). Synaptic potentials in type-A TIs (TI_As) follow vGI action potentials with constant, short latencies at frequencies up to 200 Hz. These connections are important in the integration of directional wind information involved in determining an oriented escape response. The physiological and biochemical properties of these connections that underlie this decision-making process were examined. Injection of hyperpolarizing or depolarizing current into the postsynaptic TI_As resulted in alterations in the amplitude of the postsynaptic potential (PSP) appropriate for a chemical connection. In addition, bathing cells in zero-calcium, high magnesium saline resulted in a gradual decrement of the PSP, and ultimately blocked synaptic transmission, reversibly. Single-cell choline acetyltransferase (ChAT) assays of vGI somata were performed. These assays indicated that the vGIs can synthesize acetylcholine. Further more, the pharmacological specificity of transmission at the vGI to TI_A connections was similar to that previously reported for nicotinic, cholinergic synapses in insects, suggesting that the transmitter released by vGIs at these sypapses is acetylcholine. © 1992 John Wiley & Sons, Inc.     

Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses.

Completely desensitizing excitatory channels were activated in outside-out patches of crayfish muscle membrane by applying glutamate pulses with switching times of approximately 0.2 ms for concentration changes. Channels were almost completely activated with 10 mM glutamate. Maximum activation was reached within 0.4 ms with greater than or equal to 1 mM glutamate. Channel open probability decayed with a time constant of desensitization of 2 ms with 10 mM glutamate and more rapidly at lower glutamate concentrations. The rate of beginnings of bursts (average number of beginnings of bursts per time bin) decayed even faster but approximately in proportion to the glutamate concentration. The dose-response curve for the channel open probability and for the rate of bursts had a maximum double-logarithmic slope of 5.1 and 4.2, respectively. Channels desensitized completely without opening at very low or slowly rising glutamate concentrations. Desensitization thus originates from a closed channel state. Resensitization was tested by pairs of completely desensitizing glutamate pulses. Sensitivity to the second pulse returned rapidly at pulse intervals between 1 and 2 ms and was almost complete with an interval of 3 ms. Schemes of channel activation by up to five glutamate binding steps, with desensitization by glutamate binding from closed states, are discussed. At high agonist concentrations bursts are predominantly terminated by desensitization. Quantal currents are generated by pulses of greater than 1 mM glutamate, and their decay is determined by the duration of presence of glutamate and possibly by desensitization.     

Information processing by nonspiking interneurons: passive and active properties of dendritic membrane determine synaptic integration

Nonspiking interneurons control activities of postsynaptic cells without generating action potentials in the central nervous system of many invertebrates. Physiological characteristics of their dendritic membrane have been analyzed in previous studies using single electrode current- and voltage-clamp techniques. We constructed a single compartment model of an identified nonspiking interneuron of crayfish. Experimental results allowed us to simulate how the passive and active properties of the dendritic membrane influence the integrative processing of synaptic inputs. The results showed that not only the peak amplitude but also the time course of synaptic potentials were dependent on the membrane potential level at which the synaptic activity was evoked. When the synaptic input came sequentially, each individual input was still discernible at depolarized levels at which the membrane time constant was short due to depolarization-dependent membrane conductances. In contrast, synaptic potentials merged with each other to develop a sustained potential at hyperpolarized levels where the membrane behaved passively. Thus, synaptic integration in a single nonspiking interneuron depends on the value of membrane potential at which it occurs. This probably reflects the temporal resolution required for specific types of information processing.     

Evidence that synaptic transmission between giant interneurons and identified thoracic interneurons in the cockroach is cholinergic.

In the cockroach, a population of thoracic interneurons (TIs) receives direct inputs from a population of ventral giant interneurons (vGIs). Synaptic potentials in type-A TIs (TIAs) follow vGI action potentials with constant, short latencies at frequencies up to 200 Hz. These connections are important in the integration of directional wind information involved in determining an oriented escape response. The physiological and biochemical properties of these connections that underlie this decision-making process were examined. Injection of hyperpolarizing or depolarizing current into the postsynaptic TIAs resulted in alterations in the amplitude of the post-synaptic potential (PSP) appropriate for a chemical connection. In addition, bathing cells in zero-calcium, high-magnesium saline resulted in a gradual decrement of the PSP, and ultimately blocked synaptic transmission, reversibly. Single-cell choline acetyltransferase (ChAT) assays of vGI somata were performed. These assays indicated that the vGIs can synthesize acetylcholine. Furthermore, the pharmacological specificity of transmission at the vGI to TIA connections was similar to that previously reported for nicotinic, cholinergic synapses in insects, suggesting that the transmitter released by vGIs at these synapses is acetylcholine.