A Feeding-Related Mechanoreceptor Identified in the Crab Cancer borealis Shares Similarities and Differences with Homologs in Other Crustaceans.

AbstractSensory feedback plays an essential role in shaping rhythmic animal movements. In the crustacean stomatogastric nervous system, which is responsible for grinding and filtering food particles in the animal's foregut, a number of mechanoreceptors whose activity affects motor output have been characterized. The hepatopancreas duct receptor neurons, which are located in the pyloric region of the foregut that is responsible for filtering, are among the less well understood groups of stomatogastric mechanoreceptors. Although they were first described decades ago in a number of decapod species, many questions remain about their role in shaping the movements produced by the stomatogastric nervous system. Here we provide the first anatomical and physiological evidence that there are also hepatopancreas duct receptors in the crab Cancer borealis, and we demonstrate that hepatopancreas duct receptor spiking produced by mechanical stimulation modifies the properties of an ongoing pyloric motor program.

Dual modulatory effects on feedback from a proprioceptor in the crustacean stomatogastric nervous system.

Neuromodulatory actions that change the properties of proprioceptors or the muscle movements to which they respond necessarily affect the feedback provided to the central network. Here we further characterize the responses of the gastropyloric receptor 1 (GPR1) and gastropyloric receptor 2 (GPR2) neurons in the stomatogastric nervous system of the crab Cancer borealis to movements and contractions of muscles, and we report how neuromodulation modifies those responses. We observed that the GPR1 response to contractions of the gastric mill 4 muscle (gm4) was absent, or nearly so, when the neuron was quiescent but robust when it was spontaneously active. We also found that the effects of four neuromodulatory substances (GABA, serotonin, proctolin, and TNRNFLRFamide) on the GPR1 response to muscle stretch were similar to those previously reported for GPR2. Finally, we showed that an excitatory action on gm4 due to proctolin combined with an inhibitory action on GPR2 due to GABA can allow for larger muscle contractions without increased proprioceptive feedback.NEW & NOTEWORTHY We report that the combination of GABA and the peptide proctolin increases contraction of a stomatogastric muscle while decreasing the corresponding response of the proprioceptor that reports on it. These results suggest a general mechanism by which muscle movements can be modified while sensory feedback is conserved, one that may be particularly well suited for providing flexibility to central pattern generator networks.

Incorporating spike-rate adaptation into a rate code in mathematical and biological neurons.

For a slowly varying stimulus, the simplest relationship between a neuron's input and output is a rate code, in which the spike rate is a unique function of the stimulus at that instant. In the case of spike-rate adaptation, there is no unique relationship between input and output, because the spike rate at any time depends both on the instantaneous stimulus and on prior spiking (the "history"). To improve the decoding of spike trains produced by neurons that show spike-rate adaptation, we developed a simple scheme that incorporates "history" into a rate code. We utilized this rate-history code successfully to decode spike trains produced by 1) mathematical models of a neuron in which the mechanism for adaptation (IAHP) is specified, and 2) the gastropyloric receptor (GPR2), a stretch-sensitive neuron in the stomatogastric nervous system of the crab Cancer borealis, that exhibits long-lasting adaptation of unknown origin. Moreover, when we modified the spike rate either mathematically in a model system or by applying neuromodulatory agents to the experimental system, we found that changes in the rate-history code could be related to the biophysical mechanisms responsible for altering the spiking.

Modifying spiking precision in conductance-based neuronal models.

The temporal precision of a neuron's spiking can be characterized by calculating its "jitter," defined as the standard deviation of the timing of individual spikes in response to repeated presentations of a stimulus. Sub-millisecond jitters have been measured for neurons in a variety of experimental systems and appear to be functionally important in some instances. We have investigated how modifying a neuron's maximal conductances affects jitter using the leaky integrate-and-fire (LIF) model and an eight-conductance Hodgkin-Huxley type (HH8) model. We observed that jitter can be largely understood in the LIF model in terms of the neuron's filtering properties. In the HH8 model we found the role of individual conductances in determining jitter to be complicated and dependent on the model's spiking properties. Distinct behaviors were observed for populations with slow (<11.5 Hz) and fast (>11.5 Hz) spike rates and appear to be related to differences in a particular channel's activity at times just before spiking occurs.

Quantification and analysis of ecdysis in the hornworm, Manduca sexta, using machine vision-based tracking.

We have developed a machine vision-based method for automatically tracking deformations in the body wall to monitor ecdysis behaviors in the hornworm, Manduca sexta. The method utilizes naturally occurring features on the animal's body (spiracles) and is highly accurate (>95 % success in tracking). Moreover, it is robust to unanticipated changes in the animal's position and in lighting, and in the event tracking of specific features is lost, tracking can be reestablished within a few cycles without input from the user. We have paired our tracking technique with electromyography and have also compared our in vivo results to fictive motor patterns recorded from isolated nerve cords. We found no major difference in the cycle periods of contractions during naturally occurring ecdysis compared to ecdysis initiated prematurely through injection of the peptide ecdysis-triggering hormone, and we confirmed that the ecdysis period in vivo is statistically similar to that of the fictive motor pattern.

Enhancement of muscle contraction in the stomach of the crab Cancer borealis: a possible hormonal role for GABA.

Gamma-aminobutyric acid (GABA) is best known as an inhibitory neurotransmitter in the mammalian central nervous system. Here we show, however, that GABA has an excitatory effect on nerve-evoked contractions and on excitatory junctional potentials (EJPs) of the gastric mill 4 (gm4) muscle from the stomach of the crab Cancer borealis. The threshold concentration for these effects was between 1 and 10 micromol l(-1). Using immunohistochemical techniques, we found that GABA is colocalized with the vesicle-associated protein synapsin in nearby nerves and hence is presumably released there. However, since these nerves do not innervate the muscle directly, we conclude that these release sites are not the likely source of the GABA responsible for muscle modulation. We also extracted hemolymph from the crab pericardial cavity, which contains the pericardial organs, a major neurosecretory structure. Through reversed-phase liquid chromatography-mass spectrometry analysis we determined the concentration of GABA in the hemolymph to be 3.3 +/- 0.7 micromol l(-1), high enough to modulate the muscle. These findings suggest that the gm4 muscle could be modulated by GABA produced by and released from a distant neurohemal organ.

Characteristic differences in modulation of stomatogastric musculature by a neuropeptide in three species of Cancer crabs.

Stomatogastric musculature from crabs in the genus Cancer provides a system in which modulatory roles of peptides from the FLRFamide family can be compared. The anterior cardiac plexus (ACP) is a neuroendocrine release site within the Cancer stomatogastric nervous system that is structurally identical in C. borealis, C. productus, and C. magister but that appears to contain FLRFamide-like peptide(s) only in C. productus. We measured the effect of TNRNFLRFamide on nerve-evoked contractions of muscles that were nearby, an intermediate distance, or far from the ACP. We found the spatial pattern of FLRFamidergic modulation of muscles in C. productus to be qualitatively different than in C. borealis or C. magister. In C. productus, muscles proximal to the ACP were more responsive than distal muscles. In C. borealis, FLRFamidergic response was less dependent on muscle location. These results suggest that functionally different roles of FLRFamides in modulating stomatogastric muscle movements may have evolved in different Cancer species.

Neuromodulation of spike-timing precision in sensory neurons.

The neuropeptide allatostatin decreases the spike rate in response to time-varying stretches of two different crustacean mechanoreceptors, the gastropyloric receptor 2 in the crab Cancer borealis and the coxobasal chordotonal organ (CBCTO) in the crab Carcinus maenas. In each system, the decrease in firing rate is accompanied by an increase in the timing precision of spikes triggered by discrete temporal features in the stimulus. This was quantified by calculating the standard deviation or "jitter" in the times of individual identified spikes elicited in response to repeated presentations of the stimulus. Conversely, serotonin increases the firing rate but decreases the timing precision of the CBCTO response. Intracellular recordings from the afferents of this receptor demonstrate that allatostatin increases the conductance of the neurons, consistent with its inhibitory action on spike rate, whereas serotonin decreases the overall membrane conductance. We conclude that spike-timing precision of mechanoreceptor afferents in response to dynamic stimulation can be altered by neuromodulators acting directly on the afferent neurons.

Bistable behavior originating in the axon of a crustacean motor neuron.

Both vertebrate and invertebrate motor neurons can display bistable behavior in which self-sustained tonic firing results from a brief excitatory stimulus. Induction of the bistability is usually dependent on activation of intrinsic conductances located in the somatodendritic area and is commonly sensitive to action of neuromodulators. We have observed bistable behavior in a neuromuscular preparation from the foregut of the crab Cancer borealis that consists of the gastric mill 4 (gm4) muscle and the nerve that innervates it, the dorsal gastric nerve (dgn). Nerve-evoked contractions of enhanced amplitude and long duration (>30 s) were induced by extracellular stimulation when the stimulus voltage was above a certain threshold. Intracellular and extracellular recordings showed that the large contractions were accompanied by persistent firing of the dorsal gastric (DG) motor neuron that innervates gm4. The persistent firing could be induced only by stimulating a specific region of the axon and could not be triggered by depolarizing the soma, even at current amplitudes that induced high-frequency firing of the neuron. The bistable behavior was abolished in low-Ca2+ saline or when nicardipine or flufenamic acid, blockers of L-type Ca2+ and Ca2+-activated nonselective cation currents, respectively, was applied to the axonal stimulation region of the dgn. Negative immunostaining for synapsin and synaptotagmin argued against the presence of synaptic/modulatory neuropil in the dgn. Collectively, our results suggest that bistable behavior in a motor neuron can originate in the axon and may not require the action of a locally released neuromodulator.

Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crab Cancer productus.

A club-shaped, tachykinin-immunopositive structure first described nearly two decades ago in the commissural ganglion (CoG) of three species of decapod crustaceans has remained enigmatic, as its function is unknown. Here, we use a combination of anatomical, mass spectrometric and electrophysiological techniques to address this issue in the crab Cancer productus. Immunohistochemistry using an antibody to the vertebrate tachykinin substance P shows that a homologous site exists in each CoG of this crab. Confocal microscopy reveals that its structure and organization are similar to those of known neuroendocrine organs. Based on its location in the anterior medial quadrant of the CoG, we have named this structure the anterior commissural organ (ACO). Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry shows that the ACO contains the peptide APSGFLGMRamide, commonly known as Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). Using the same technique, we show that CabTRP Ia is also released into the hemolymph. As no tachykinin-like labeling is seen in any of the other known neuroendocrine sites of this species (i.e. the sinus gland, the pericardial organ and the anterior cardiac plexus), the ACO is a prime candidate to be the source of CabTRP Ia present in the circulatory system. Our electrophysiological studies indicate that one target of hemolymph-borne CabTRP Ia is the foregut musculature. Here, no direct CabTRP Ia innervation is present, yet several gastric mill and pyloric muscles are nonetheless modulated by hormonally relevant concentrations of the peptide. Collectively, our findings show that the C. productus ACO is a neuroendocrine organ providing hormonal CabTRP Ia modulation to the foregut musculature. Homologous structures in other decapods are hypothesized to function similarly.

Lymnaea stagnalis and the development of neuroelectronic technologies.

The recent development of techniques for stimulating and recording from individual neurons grown on semiconductor chips has ushered in a new era in the field of neuroelectronics. Using this approach to construct complex neural circuits on silicon from individual neurons will require improvements at the neuron/semiconductor interface and advances in controlling synaptogenesis. Although devices incorporating vertebrate neurons may be an ultimate goal, initial investigations using neurons from the pond snail Lymnaea stagnalis have distinct advantages. Simple two-cell networks connected by electrical synapses have already been reconstructed on semiconductor chips. Furthermore, considerable progress has been made in controlling the processes that underlie chemical synapse formation in Lymnaea. Studies of Lymnaea neural networks on silicon chips will lead to a deeper understanding of the long-term dynamics of simple neural circuits and may provide the basis for reliable interfaces for new neuroprosthetic devices.

Neuromodulation in invertebrate sensory systems: from biophysics to behavior.

Neuromodulation may enhance the ability of sensory circuits to respond appropriately to widely variable environmental stimuli. The functional significance of neuromodulation will emerge from understanding the effects of modulators not just on single cells and synapses, but also on networks and the behavior of intact animals. With their relatively simple circuitry and large identifiable cells, invertebrate nervous systems offer insights into the complex roles of neuromodulators in modifying networks to meet the changing needs of the animal. Here we describe the role of neuromodulation in several invertebrate sensory systems that have been studied at a variety of levels, from the biophysical up to the behavioral.

Differential and history-dependent modulation of a stretch receptor in the stomatogastric system of the crab, Cancer borealis.

Neuromodulators can modify the magnitude and kinetics of the response of a sensory neuron to a stimulus. Six neuroactive substances modified the activity of the gastropyloric receptor 2 (GPR2) neuron of the stomatogastric nervous system (STNS) of the crab Cancer borealis during muscle stretch. Stretches were applied to the gastric mill 9 (gm9) and the cardio-pyloric valve 3a (cpv3a) muscles. SDRNFLRFamide and dopamine had excitatory effects on GPR2. Serotonin, GABA, and the peptide allatostatin-3 (AST) decreased GPR2 firing during stretch. Moreover, SDRNFLRFamide and TNRNFLRFamide increased the unstimulated spontaneous firing rate, whereas AST and GABA decreased it. The actions of AST and GABA were amplitude- and history-dependent. In fully recovered preparations, AST and GABA decreased the response to small-amplitude stretches proportionally more than to those evoked by large-amplitude stretches. For large-amplitude stretches, the effects of AST and GABA were more pronounced as the number of recent stretches increased. The modulators that affected the stretch-induced GPR2 firing rate were also tested when the neuron was operating in a bursting mode of activity. Application of SDRNFLRFamide increased the bursting frequency transiently, whereas high concentrations of serotonin, AST, and GABA abolished bursting altogether. Together these data demonstrate that the effects of neuromodulators depend on the previous activity and current state of the sensory neuron.