Medial Giant Fiber Research Articles

Coupled sensory interneurons mediate escape neural circuit processing in an aquatic annelid worm, Lumbriculus variegatus.

The interneurons associated with rapid escape circuits are adapted for fast pathway activation and rapid conduction. An essential aspect of fast activation is the processing of sensory information with limited delays. Although aquatic annelid worms have some of the fastest escape responses in nature, the sensory networks that mediate their escape behavior are not well defined. Here, we demonstrate that the escape circuit of the mud worm, Lumbriculus variegatus, is a segmentally arranged network of sensory interneurons electrically coupled to the central medial giant fiber (MGF), the command-like interneuron for head withdrawal. Electrical stimulation of the body wall evoked fast, short-duration spikelets in the MGF, which we suggest are the product of intermediate giant fiber activation coupled to MGF collateral dendrites. Since these contact sites have immunoreactivity with a glutamate receptor antibody, and the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dion abolishes evoked MGF responses, we conclude that the afferent pathway for MGF-mediated escape is glutamatergic. This electrically coupled sensory network may facilitate rapid escape activation by enhancing the amplitude of giant axon depolarization.

Biochemical- and neuro-toxicity of silver nanoparticle and silver nitrate in soil to Aporrectodea calginosa earthworms

Silver nanoparticles (AgNPs) are now widely used in many industry applications. Because there are no regulations on discharge limits, improper discharge of waste from these industries can lead to environmental contamination and damage to ecosystem organisms. In this study Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.3, 3, 30, 300 mg/kg Ag as AgNPs and 0, 0.03, 0.3, 3, 10 mg/kg of Ag as AgNO3 in soil for 4 weeks and select biochemical and neurotoxicity studies were conducted weekly. The lipid peroxidation (measured using thiobarbituric acid reactive substances; TBARS) and activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase, glutathione S transferase, lipid peroxidation), and nerve conduction velocity (NCV) of the medial giant fibers (MGF) using a novel non-invasive electrophysiological technique were measured in earthworms weekly. The TBARS and antioxidant enzyme activities were elevated by both AgNO3 and AgNPs and this was most evident in earthworms at 4 weeks >3 > 2 > 1. In neurotoxicity studies, MGF NCV progressively decreased in A. caliginosa exposed to both AgNPs and AgNO3. The findings highlight oxidative stress and neurotoxic effects of Ag compounds on earthworms and the importance of government authorities to have legislations in place to prevent excessive soil contamination by AgNPs produced by the expanding nanotechnology industries.

Investigation of the efficiency of the shape of chopped pulses using earthworm model.

In neural electrical stimulation, limiting the charge delivered during a stimulus pulse is essential to avoid nerve tissue damage and to save power. Previous experimental and modeling studies indicated that waveforms such as non-rectangular continuous pulses or rectangular chopped pulse were able to improve stimulation efficiency. The goal of this study is to evaluate if non-rectangular chopped pulses such as quarter sine and ramp are more charge efficient than rectangular chopped pulse. We performed in vivo study on 17 lumbricus terrestris and compared the charge per stimulating phase needed to activate lateral giant fibers (LGF) and medial giant fiber (MGF) using chopped non-rectangular pulses and rectangular pulse, varying stimulation duration parameters. Results indicated that non rectangular chopped pulses activated MGF and LGF with less charge than rectangular chopped pulses. For MGF (respectively LGF), the gain of charge was up to 33.9\% (resp. 17.8\%) using chopped ramp, and up to 22.8\% (resp. 18.1\%) using chopped quarter sine.

Nerve conduction velocity as a non-destructive biomarker in the earthworm Aporrectodea caliginosa exposed to insecticides.

Earthworms are important and useful soil organisms, but in agricultural soils, they are potentially exposed to a wide variety of pesticides. Insecticides represent the highest threat to earthworms and many are neurotoxic. There is a need for a reliable, relevant, simple biomarker to assess the sub-lethal effects of neurotoxic insecticides on earthworms under laboratory or field conditions. The Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.5×, 1× (normal field application rate), and 5× concentrations of a carbamate (Pirimor®) and an organophosphate (Lorsban®) insecticides. The nerve conduction velocity (NCV) of the medial giant fibers of A. caliginosa earthworm was recorded on days 0, 1, 2, 3, 4, and 7 to quantify sub-lethal neurotoxic effects. Acetylcholinesterase (AChE) enzyme activity of A. caliginosa homogenates was measured at the conclusion of the experiment. Pirimor® but not Lorsban® induced a significant decrease in NCV on days 3, 4, and 7 at 1× and 5× doses. A significant dose-dependent decrease was observed on AChE activity to Pirimor® at the doses used but not Lorsban®. A clear relationship is observed between AChE activity and NCV in the case of Pirimor®. This study showed that NCV is a sensitive biomarker that correlates well with classical biomarker measurements such as AChE enzyme activity. This technique could be used to study the impact of insecticides on earthworms and also their recovery.

Biochemical- and neuro-toxicity of silver nanoparticle and silver nitrate in soil to Aporrectodea calginosa earthworms

Silver nanoparticles (AgNPs) are now widely used in many industry applications. Because there are no regulations on discharge limits, improper discharge of waste from these industries can lead to environmental contamination and damage to ecosystem organisms. In this study Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.3, 3, 30, 300 mg/kg Ag as AgNPs and 0, 0.03, 0.3, 3, 10 mg/kg of Ag as AgNO3 in soil for 4 weeks and select biochemical and neurotoxicity studies were conducted weekly. The lipid peroxidation (measured using thiobarbituric acid reactive substances; TBARS) and activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase, glutathione S transferase, lipid peroxidation), and nerve conduction velocity (NCV) of the medial giant fibers (MGF) using a novel non-invasive electrophysiological technique were measured in earthworms weekly. The TBARS and antioxidant enzyme activities were elevated by both AgNO3 and AgNPs and this was most evident in earthworms at 4 weeks >3 > 2 > 1. In neurotoxicity studies, MGF NCV progressively decreased in A. caliginosa exposed to both AgNPs and AgNO3. The findings highlight oxidative stress and neurotoxic effects of Ag compounds on earthworms and the importance of government authorities to have legislations in place to prevent excessive soil contamination by AgNPs produced by the expanding nanotechnology industries.

Novel neurobiological properties of elements in the escape circuitry of the shrimp.

Escape behaviors in penaeid shrimp are mediated by large myelinated medial giant fibers which course from the brain to the last abdominal ganglion in the ventral nerve cord. In each abdominal segment, the medial giant axons make synaptic connections with paired myelinated motor giant axons that excite the abdominal deep flexor muscles and drive the tailflips that constitute the escape behavior. I examined (1) anatomical features of the abdominal motor giant fibers and (2) electrical properties of both the medial and motor giant axons in the pink shrimp, Farfantepenaeus duorarum The motor giant axons in the paired third roots of shrimp abdominal ganglia emerge from a single fused neurite that originates from two clusters of cell bodies within the ganglion. Injection of large positive currents into the abdominal medial giant fibers generates action potentials that are transmitted to the opposite medial giant axon through putative collateral synapses within the ganglia. Transmission across the medial-to-motor giant synapse is fast and resistant to fatigue, with synaptic delays equal to or less than those previously documented at the lateral-to-motor giant electrical synapse in crayfish. Transmission was found to be extremely reliable even with presynaptic spike frequencies as high as 250 Hz. While action potentials within the medial giant fibers are transmitted across the medial-to-motor giant synapse with a large safety factor, neither prolonged positive nor prolonged negative currents pass through the synaptic nexus, irrespective of the site of injection. The lack of DC current passage along with the inability of neurobiotin or biocytin to spread through the synaptic nexus raises the possibility that the synaptic mechanism may be capacitative.

Easy method to examine single nerve fiber excitability and conduction parameters using intact nonanesthetized earthworms.

The generation and conduction of neuronal action potentials (APs) were the subjects of a cell physiology exercise for first-year medical students. In this activity, students demonstrated the all-or-none nature of AP generation, measured conduction velocity, and examined the dependence of the threshold stimulus amplitude on stimulus duration. For this purpose, they used the median giant nerve fiber (MGF) in the ventral nerve cord of the common earthworm (Lumbricus terrestris). Here, we introduce a specialized stimulation and recording chamber that the nonanesthetized earthworm enters completely unforced. The worm resides in a narrow round duct with silver electrodes on the bottom such that individual APs of the MGF can be elicited and recorded superficially. Our experimental setup combines several advantages: it allows noninvasive single fiber AP measurements taken from a nonanesthetized animal that is yet restrained. Students performed the experiments with a high success rate. According to the data acquired by the students, the mean conduction velocity of the MGF was 30.2 m/s. From the amplitude-duration relationship for threshold stimulation, rheobase and chronaxie were graphically determined by the students according to Lapicque's method. The mean rheobase was 1.01 V, and the mean chronaxie was 0.06 ms. The acquired data and analysis results are of high quality, as deduced from critical examination based on the law of Weiss. In addition, we provide video material, which was also used in the practical course.

Direct detection of a single evoked action potential with MRS in Lumbricus terrestris

Functional MRI (fMRI) measures neural activity indirectly by detecting the signal change associated with the hemodynamic response following brain activation. In order to alleviate the temporal and spatial specificity problems associated with fMRI, a number of attempts have been made to detect neural magnetic fields (NMFs) with MRI directly, but have thus far provided conflicting results. In this study, we used MR to detect axonal NMFs in the median giant fiber of the earthworm, Lumbricus terrestris, by examining the free induction decay (FID) with a sampling interval of 0.32 ms. The earthworm nerve cords were isolated from the vasculature and stimulated at the threshold of action potential generation. FIDs were acquired shortly after the stimulation, and simultaneous field potential recordings identified the presence or absence of single evoked action potentials. FIDs acquired when the stimulus did not evoke an action potential were summed as background. The phase of the background‐subtracted FID exhibited a systematic change, with a peak phase difference of (−1.2 ± 0.3) × 10–5 radians occurring at a time corresponding to the timing of the action potential. In addition, we calculated the possible changes in the FID magnitude and phase caused by a simulated action potential using a volume conductor model. The measured phase difference matched the theoretical prediction well in both amplitude and temporal characteristics. This study provides the first evidence for the direct detection of a magnetic field from an evoked action potential using MR. Copyright © 2011 John Wiley & Sons, Ltd.

Behavioral plasticity and central regeneration of locomotor reflexes in the freshwater oligochaete, Lumbriculus variegatus. I: Transection studies

Abstract. Access to the ventral nerve cord in living specimens of Lumbriculus variegatus, an aquatic oligochaete, is normally impossible because surgical invasion induces segmental autotomy (self‐fragmentation). We show here that nicotine is a powerful paralytic agent that reversibly immobilizes worms, blocks segmental autotomy, and allows experimental access to the nerve cord. Using nicotine‐treated worms, we transected the ventral nerve cord and used non‐invasive electrophysiological recordings and behavioral analyses to characterize the functional recovery of giant nerve fibers and other reflex pathways. Initially, after transection, medial giant fiber (MGF) and lateral giant fiber (LGF) spikes conducted up to, but not across, the transection site. Reestablishment of MGF and LGF through‐conduction across the transection site occurred as early as 10 h (usually by 20 h) after transection. Analyses of non‐giant‐mediated behavioral responses (i.e., helical swimming and body reversal) were also made following nerve cord transection. Immediately after transection, functional reorganization of touch‐evoked locomotor reflexes occurred, so that the two portions of the worm anterior and posterior to the transection site were independently capable of helical swimming and body reversal responses. Similar reorganization of responses occurred in amputated body fragments. Reversion back to the original whole‐body pattern of swimming and reversal occurred as early as 8 h after transection. Thus, functional restoration of the non‐giant central pathways appeared slightly faster than giant fiber pathways. The results demonstrate the remarkable plasticity of locomotor reflex behaviors immediately after nerve cord transection or segment amputation. They also demonstrate the exceptional speed and specificity of regeneration of the central pathways that mediate locomotor reflexes.

Fenestration nodes and the wide submyelinic space form the basis for the unusually fast impulse conduction of shrimp myelinated axons.

Saltatory impulse conduction in invertebrates is rare and has only been found in a few giant nerve fibres, such as the pairs of medial giant fibres with a compact multilayered myelin sheath found in shrimps (Penaeus chinensis and Penaeus japonicus) and the median giant fibre with a loose multilayered myelin sheath found in the earthworm Lumbricus terrestris. Small regions of these nerve fibres are not covered by a myelin sheath and serve as functional nodes for saltatory conduction. Remarkably, shrimp giant nerve fibres have conduction speeds of more than 200 m s-1, making them among the fastest-conducting fibres recorded, even when compared with vertebrate myelinated fibres. A common nodal structure for saltatory conduction has recently been found in the myelinated nerve fibres of the nervous systems of at least six species of Penaeus shrimp, including P. chinensis and P. japonicus. This novel node consists of fenestrated openings that are regularly spaced in the myelin sheath and are designated as fenestration nodes. The myelinated nerve fibres of the Penaeus shrimp also speed impulse conduction by broadening the gap between the axon and the myelin sheath rather than by enlarging the axon diameter as in other invertebrates. In this review, we document and discuss some of the structural and functional characteristics of the myelinated nerve fibres of Penaeus shrimp: (1) the fenestration node, which enables saltatory conduction, (2) a new type of compact multilayered myelin sheath, (3) the unique microtubular sheath that tightly surrounds the axon, (4) the extraordinarily wide space present between the microtubular sheath and the myelin sheath and (5) the main factors contributing to the fastest impulse conduction velocity so far recorded in the Animal Kingdom.

VENTRAL NERVE CORD TRANSECTION IN CRAYFISH: A STUDY OF FUNCTIONAL ANATOMY

ABSTRACT In crayfish, neural degeneration and regeneration in the ventral nerve cord occur in one of two ways, depending on the injured fiber. Most fibers degenerate in 1 or 2 weeks, while giant fibers degenerate slowly. Although degenerative changes are similar in both cases, they do not seem to correlate with motor behavioral alterations. The aim of this work was to characterize the time course of behavioral and anatomical changes following ventral nerve cord (VNC) transection in crayfish. The behavioral analysis was focused on the righting reflex whose changes were correlated with morphological studies performed on longitudinal sections and analyzed with transmission (TEM) and scanning electron microscope (SEM). Latency for the righting reflex increased after VNC transection and then slowly decreased toward control values. Anatomically, degenerative changes began to appear 10 days after VNC transection. Disruption in membrane arrangement, subcellular organelles, and a strong increase in glia appeared in small fibers. To a lesser degree, similar changes could be detected in medial and lateral giant fibers. Glial growth reconnected the transected VNC where regeneration signs were detected in small fibers. Both stumps were reconnected at least by glial tissue 90 days after transection, while giant axons were still degenerating; at this time, the righting reflex returned to control values.

Axolemmal and septal conduction in the impedance of the earthworm medial giant nerve fiber

Ionic conduction in the axolemmal and septal membranes of the medial giant fiber (MGF) of the earthworm (EW) Lumbricus terrestris was assessed by impedance spectroscopy in the frequency range 2.5-1000 Hz. Impedance loci in the complex plane were described by two semi-circular arcs, one at a lower characteristic frequency (100 Hz) and the other at a higher frequency (500 Hz). The lower frequency arc had a chord resistance of 53 k omega and was not affected by membrane potential changes or ion channel blockers [tetrodotoxin (TTX), 3,4-diaminopyridine (3,4-DAP), 4-aminopyridine (4-AP), and tetraethylammonium (TEA)]. The higher frequency arc had a chord resistance of 274 k omega at resting potential, was voltage-dependent, and was affected by the addition of TTX, 3,4-DAP, 4-AP, and TEA to the physiological EW salines. When all four blockers were added to the bathing solution, the impedance locus was described by two voltage-independent arcs. Considering the effects of these and other (i.e., Cd and Ni) ion channel blockers, we conclude that: 1) the higher frequency locus reflects conduction by voltage-sensitive ion channels in the axolemmal membrane, which contains at least four ion channels selective for sodium, calcium, and potassium (delayed rectifier and calcium-dependent), and 2) the lower frequency locus reflects voltage-insensitive channels in the septal membrane, which separates adjacent MGFs.

Presynaptic inhibition is mediated by histamine and GABA in the crustacean escape reaction.

1. Presynaptic inhibition of sensory transmission during the escape reaction in Crustacea has been studied using an in vitro preparation of the crayfish thoracic ganglia. Electrical stimulation of the medial giant fiber mediating the escape reaction induced depolarization in sensory afferent terminals of the coxo-basal chordotonal organ (CBCO). This depolarization was associated with an increase of the membrane conductance and was partially blocked by a gamma-aminobutyric acid (GABA) antagonist, picrotoxin, and by a histamine antagonist, cimetidine. 2. Pressure ejection of histamine on CBCO sensory terminals (CBT) recorded intracellularly, induced a depolarization of the membrane potential accompanied by a large increase of the conductance. Histamine-induced depolarization persisted after blockade of synaptic transmission mediated by Na+ spikes by tetrodotoxin. The amplitude of histamine-induced depolarization increased when negative current was injected into the sensory terminal through the recording electrode. Moreover, injection of chloride into the CBT, which shifts the reversal potential of chloride to a more positive value, resulted in an increase of the amplitude of the histamine-induced depolarization. 3. The existence of separate receptors for GABA and histamine on the CB sensory terminals was demonstrated using two complementary sets of experiments. The first one consisted of using specific blockers of GABA and histamine. Picrotoxin blocked selectively the GABA-induced depolarization of the CB sensory terminals, while it was ineffective in blocking the histamine-induced depolarization. Conversely, cimetidine blocked the histamine-induced depolarization totally, but did not affect the GABA response. The second set of experiments tested for of cross-desensitization between GABA and histamine responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionic currents of the nodal membrane underlying the fastest saltory conduction in myelinated giant nerve fibers of the shrimpPenaeus japonicus

The myelinated giant nerve fiber of the shrimp, Penaeus japonicus, is known to have the fastest velocity of saltatory impulse conduction among all nerve fibers so far studied, owing to its long distances between nodal regions and large diameter. For a better understanding of the basis of this fast conduction, a medial giant fiber of the ventral nerve cord of the shrimp was isolated, and ionic currents of its presynaptic membrane (a functional node) were examined using the sucrose-gap voltage-clamp method. Inward currents induced by depolarizing voltage pulses had a maximum value of 0.5 microA and a reversal potential of 120 mV. These currents were completely suppressed by tetrodotoxin and greatly prolonged by scorpion toxin, suggesting that they are the Na current. Both activation and inactivation kinetics of the Na current were unusually rapid in comparison with those of vertebrate nodes. According to a rough estimation of the excitable area, the density of Na current reached 500 mA/cm2. In many cases, the late outward currents were induced only by depolarizing pulses larger than 50 mV in amplitude. The slope conductance measured from late currents were mostly smaller than that measured from the Na current, suggesting a low density of K channels in the synaptic membrane. These characteristics are in good harmony with the fact that the presynaptic membrane plays a role as functional node in the fastest impulse conduction of this nerve fiber.

Methylene blue filling of the dorsal giant fibres of the earthworm by electrophoresis

Multisegmental methylene blue filling of the three dorsal giant fibres in the nervous system of the earthworm was obtained by the use of electrophoresis. There was no difference in aldehyde fixed and unfixed tissue; due to its larger diameter, the median giant fibre could be stained over longer distances than the lateral ones. Accumulations of the dye were detected in the median giant fibre in the dorsal openings of the myelin sheath and at the origins of the ventral branches descending from both fibre systems. Since no significant axonal filling was observed by using several other dyes similar to methylene blue in diffusion and dialysis properties, the staining mechanism obviously depends on the specific binding sites of methylene blue in combination with the current. Therefore, the revealed phenomena provides evidence of a direct correlation between methyelne blue coupling and electrical coupling in the dorsal giant fibres of the earthworm.

MODELING HEAD END ESCAPE BEHAVIOR IN THE EARTHWORM: THE EFFERENT ARC AND THE END ORGAN

The purpose of this study was to begin the task of mathematically modeling earthworm head end escape behavior. Three important mathematical expressions were developed. First of all, a neuronal equation was developed which was capable of predicting information relay along the efferent arc from the medial giant fiber to the neuromuscular junction. Secondly, an intramuscular equation was developed which represented the operation of intramuscular components that initiate the contractile response. Thirdly, the neuronal equation and intramuscular equation were combined into a single expression which was capable of predicting the contractile tension produced at the longitudinal muscle when the medial giant fiber becomes activated by a head end stimulus. This new formulation may be useful as a representative model of a rapid motor response that requires a brief period of repetitive discharge in the neuronal network.

Action potential of the median giant fibre of the earthworm, Lumbricus terrestris, as a pharmacological test of the radio-labelled TTX-derivatives activity

1. 1. The isolated median giant fibre action potential from the earthworm nerve cord was recorded in a double sucrose-gap apparatus used in current-clamp configuration. 2. 2. [ 3H]Lysine-TTX was synthesized and its inhibitory effect on the spike area was measured. Its activity is very similar to that of TTX. 3. 3. The dose-response curve obtained with superthreshold stimuli is shown to be the right-hand side of the K d (K d + [ TTX]) relation. 4. 4. A 3.9nM K d is calculated which agrees with the known values for the frog Ran vier node.

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm, Lumbricus terrestris

The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.

Fine-structural and morphometrical studies on the segmental septa of the giant fibers in the earthworm, Eisenia foetida

The fine structure of junctional specializations on the segmental septa in the median and lateral giant fibers of the earthworm (Eisenia foetida) was examined. Eight morphologically different septal domains were identified; a gap junction, a junction with hemispherical hollow structures, a chemical synapse-like junction, intermediate type and punctum adherens type junctions, a junction with adjoining vesicular layers, an area flanked by flattened membranous sacs, a non specialized area, and an area consisting of widely separated membranes with interposed glial processes. The area of each domain was measured by a cytometrical technique using quasi-serial sections. The gap junction occupied 3% and 0.2% of the septal area of the median and lateral giant fibers, respectively. Junctions with hemisperical hollow structures, characteristic of the earthworm giant fibers, occupied 2.5% and 13.9% of the median and lateral giant fibers, respectively. Various membrane domains except the gap junction, the junction with hemispherical hollow structures, and the chemical synapse-like junction accounted for similar proportional areas in both median and lateral giant fibers. The functional implications of these junctional specializations, especially the gap junction, are discussed.

Rapid escape reflexes in aquatic oligochaetes: variations in design and function of evolutionarily conserved giant fiber systems

This study provides neuroanatomical and electrophysiological evidence that an arrangement of three dorsal giant fibers, functioning as two distinct and dichotomous conduction pathways, has been evolutionarily conserved within the three major orders of aquatic and terrestrial oligochaetes. The medial giant fiber (MGF), activated by afferents of anterior segments, initiates anterior shortening; whereas, the two lateral giant fibers (LGFs), activated in synchrony by afferents of posterior segments, initiate a different response (usually tail withdrawal). Notwithstanding these common features, the design and function of LGF systems differ considerably in aquatic and terrestrial groups. In posterior segments of aquatic species, LGFs are disproportionately larger and conduct faster than MGFs. This contrasts with posterior segments of earthworms in which LGFs are smaller and conduct slower than MGFs.