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Biomedical subjects

A O Stretton

Publications and source records attributed to A O Stretton.

At least 37 records · Page 2Linked to original sources

Generation of monoclonal antibodies against a nematode peptide extract: another approach for identifying unknown neuropeptides.

Monoclonal antibodies that cross-react with Ascaris neural antigens were generated in mice immunized with a conjugate made with keyhole limpet hemocyanin (KLH) linked to a crude peptide extract from Caenorhabditis elegans. The response to KLH was suppressed by injection of cyclophosphamide 3 days after immunization with a gamma-aminobutyric acid (GABA)-KLH conjugate. Screening of hybridomas was carried out by enzyme-linked immunosorbent assay and whole mount immunocytochemistry. Two similar clones produced antibodies that recognized a small subset of Ascaris neurons. This result suggests that the monoclonal antibody technique might be useful for identifying new neuropeptides since the antibodies can be used for localization of the neuropeptidelike substances and, potentially, for immunoaffinity chromatography. As a by-product of this experiment, monoclonal antibodies that recognize GABA-like immunoreactivity in whole mounts and plastic sections were also obtained.

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Slow active potentials in ventral inhibitory motor neurons of the nematode Ascaris.

The ability of ventral inhibitory motor neurons of the nematode Ascaris to generate slow depolarizing potentials was investigated using intracellular recording and current injection. In quiescent cells, regenerative depolarizations with peak amplitudes of approximately 20 mV and durations of several 100 ms were evoked in response to brief depolarizing current pulses. Repetitive slow potentials were produced in response to sustained depolarizing currents in a threshold-dependent manner. Repetitive slow potentials also occurred spontaneously, exhibiting cycle periods of about 700 ms. The ability of inhibitory motor neurons to generate slow potentials was blocked by addition of Co++, Cd++, or other Ca-channel blockers to the saline but not by TTX or substitution of Na+ with Tris. The amplitude and duration of slow potentials were increased in the presence of Ba++, Sr++, and TEA. Spontaneous slow potentials exhibited characteristics expected of intrinsically generated oscillations, including frequency modulation by injection of prolonged offset currents, phase resetting by brief current pulses, and suppression by strong hyperpolarization. Slow potentials appear to be generated in the ventral nerve cord processes and/or cell body of the motor neuron, and they produce rhythmic inhibitory postsynaptic potentials in ventral muscle cells. Slow potentials may therefore contribute to locomotory or other motor behaviors of the animal.

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AF1, a sequenced bioactive neuropeptide isolated from the nematode Ascaris suum.

An FMRFamide-like neuropeptide, named AF1, was isolated from head extracts of the nematode Ascaris suum using five steps of HPLC. AF1 is a heptapeptide with the amino acid sequence Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2. Synthetic AF1 (10(-9) to 10(-7) M) rapidly and reversibly abolished slow membrane potential oscillations of identified ventral and dorsal inhibitory motoneurons and selectively reduced their input resistances. Synaptic transmission was not blocked. In intact Ascaris, AF1 inhibited locomotory movements. This study indicates a potential physiological role for an endogenous neuropeptide in nematodes.

Action Potentials↗

Signaling properties of Ascaris motorneurons: graded active responses, graded synaptic transmission, and tonic transmitter release.

The commissural motorneurons of the nematode Ascaris are capable of transmitting signals passively over long distances with little decrement. This ability is due to the high resistivities of their membranes (Davis and Stretton, 1989). Although these cells rely on their passive properties for long-distance signaling, voltage-sensitive channels are present in commissural membranes. These channels underlie the graded active responses that can be elicited at the offset of abrupt hyperpolarizing and depolarizing intracellular current pulses. The inhibitory motorneurons generate membrane potential oscillations when they are strongly depolarized. All-or-none action potentials have never been observed to occur spontaneously, nor has it been possible to evoke them even when the cells have been strongly hyperpolarized to remove any possible channel inactivation. Our findings indicate that the typical all-or-none action potentials so commonly used in nerve cells throughout the animal kingdom do not occur in these cells. Synaptic transmission is therefore mediated without spikes and is graded. The resting potentials of Ascaris motorneurons lie where the synaptic input-output curves are steepest, above the threshold for release of neurotransmitter. Tonic transmitter release from commissural motorneurons may be the neural mechanism underlying the hydrostatic skeleton of Ascaris.

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Passive membrane properties of motorneurons and their role in long-distance signaling in the nematode Ascaris.

In the motornervous system of the large parasitic nematode, Ascaris suum, the dorsal and ventral nerve cords are connected by a repeating pattern of single identified motorneuron processes, called commissures (Stretton et al., 1978). By making microelectrode penetrations of the commissures, we here report the first successful intracellular recordings of nematode neurons. These cells, like muscle cells of Ascaris, exhibit resting potentials of approximately -30 to -40 mV. Several tests indicate that these are the normal resting potentials of the cells and are not low due to damage. Using 2 intracellular microelectrodes (one for stimulation and one for recording), we have determined the input resistance and cable properties of commissural motorneurons. Over the physiological voltage range, the steady-state I-V plots are linear with little indication that voltage-sensitive conductances are contributing substantially to signaling. The membrane capacitance is comparable to that of single biological membranes (range, 0.4-0.9 microF/cm2) and the internal resistivity (range, 79-314 omega cm) is similar to that found in other cells. Because of unusually large membrane resistances (range, 61-251 k omega cm2), the space constants, lambda, are high (range, 4-10 mm). Such membrane properties produce cells that are well-designed for conducting passive signals over long distances. This long-distance signaling ability appears to be due to the intrinsic properties of the motorneuron membrane itself.

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GABA-immunoreactivity in inhibitory motor neurons of the nematode Ascaris.

We have used GABA-specific antisera to detect GABA-immunoreactivity in the motor neurons of the ventral nerve cord of Ascaris. We find that a subset of the individually identifiable commissures of motor neurons is specifically stained. On the basis of the location and morphology of stained commissures and of the location of stained cell bodies in the ventral nerve cord, we conclude that the labeled neurons comprise all members of the VI (inhibiting ventral muscle; 13 cells) and DI (inhibiting dorsal muscle; 6 cells) classes of inhibitory motor neurons (Stretton et al., 1978; Walrond et al., 1985). This result supports previous suggestions (e.g., del Castillo et al., 1964b) that GABA is the neurotransmitter released by the inhibitory motor neurons of nematodes. In the anterior part of the animal, the inhibitory motor neuron commissures have small branches in the sublateral nerve cords that have not been previously described: VI commissures have dorsal sublateral branches, while DI cells have ventral branches. Posterior VI neurons have branches in the lateral nerve cords.

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Identification of excitatory and inhibitory motoneurons in the nematode Ascaris by electrophysiological techniques.

A physiological preparation in which it is possible to record responses in muscle to stimulation of single motoneurons of the nematode Ascaris lumbricoides is described. With this preparation we have determined the physiological sign (E or I; excitatory or inhibitory) of the neuromuscular synapses of 21 identified motoneurons--12 are excitatory and 9 inhibitory. Ascaris motoneurons had previously been classified by morphological criteria into seven classes (Stretton, A. O. W., R. M. Fishpool, E. Southgate, J. E. Donmoyer, J. P. Walrond, J. E. R. Moses, and I. S. Kass (1978) Proc. Natl. Acad. Sci. U. S. A. 75: 3493-3497). Physiological studies were performed on members of five of these classes. Three classes of neurons (DE1, DE2, and DE3) are excitatory to dorsal muscle cells. Two classes (DI and VI) are inhibitory neurons which innervate the dorsal and ventral muscle cells, respectively. The motoneurons in Caenorhabditis elegans (White, J. E., E. Southgate, J. N. Thomson, and S. Brenner (1976) Philos. Trans. R. Soc. Lond. (Biol.) 275: 327-348) can be divided into seven morphological classes which are very similar to those in Ascaris. Based upon the structure-function correlation in Ascaris, we have predicted which motoneurons are excitatory and which are inhibitory in C. elegans.

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Excitatory and inhibitory activity in the dorsal musculature of the nematode Ascaris evoked by single dorsal excitatory motonerons.

A physiological preparation, in which identified motoneurons of the nematode Ascaris lumbricoides can be individually stimulated, was used to map the response evoked by single dorsal excitatory (DE) motoneurons in muscle cells innervated along the length of the dorsal nerve cord. As previously reported (Walrond, J. P., I. S. Kaas, A. O. W. Stretton, and J. E. Donmoyer (1985) J. Neurosci. 5: 1-8), stimulation of a DE cell produces excitatory responses in muscle cells which it directly innervates. Excitatory activity propagates along the most strongly activated region of muscle at a velocity of approximately 28 cm/sec, then relaxes into a slower velocity of approximately 12 cm/sec. When either the DE1 or DE3 neurons were stimulated, excitatory responses were also observed in muscle cells not directly innervated by the neuron. These signals propagate in the opposite direction from the fast-propagating activity at a velocity of approximately 13 cm/sec. Injection of hyperpolarizing current into muscle cells blocks this slower propagation but fails to block the faster conduction. We conclude that the fast-conducting responses result from signals propagating in the motor axon, whereas the slow responses are conducted through gap junctions which connect Ascaris muscle cells. Stimulating a single DE motoneuron also evokes hyperpolarizing muscle responses in regions adjacent to the zones of fast and slow excitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

Reciprocal inhibition in the motor nervous system of the nematode Ascaris: direct control of ventral inhibitory motoneurons by dorsal excitatory motoneurons.

In previous physiological experiments (Stretton, A. O. W., R. M. Fishpool, E. Southgate, J. E. Donmoyer, J. P. Walrond, J. E. R. Moses, and I. S. Kass (1978) Proc. Natl. Acad. Sci. U. S. A. 75: 3493-3497), we have shown that the dorsal cord of the nematode Ascaris lumbricoides includes the processes of three types of dorsal excitatory (DE) motoneurons and one type of ventral inhibitory (VI) motoneuron. Ultrastructural studies have revealed that the axons of the DE motoneurons make monosynaptic contacts with the dorsal processes of VI motoneurons. In this paper, we describe a physiological preparation with which to investigate the properties of these synapses. We show that activation of a DE neuron can excite a VI neuron producing inhibition in ventral muscle cells shortly after dorsal muscle cells are excited, thus mediating reciprocity between dorsal and ventral muscles. Each VI dendrite receives input from four or five DE neurons; activation of any one of these DE neurons is sufficient to activate the VI neuron.

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Localization of choline acetyltransferase within identified motoneurons of the nematode Ascaris.

Choline acetyltransferase (CAT) activity has been measured in the nematode Ascaris lumbricoides. Strips of hypodermal tissue which contained branches of single identified motoneurons (Stretton, A.O.W., R.M. Fishpool, E. Southgate, J.E. Donmoyer, J.P. Walrond, and I.S. Kass (1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3493-3497) as well as control strips containing no neural tissue were assayed. In strips of hypodermis which contained branches of excitatory motoneurons (classes DE1, DE2, and DE3), CAT activity was elevated 5 to 10 times compared to control levels. Branches of inhibitory motoneurons (classes D1 and V1) did not add to the level of CAT observed in the controls. These data provide additional support for the suggestion (e.g., del Castillo, J., W.C. deMello, and T. Morales (1963) Experientia 20: 141) that acetylcholine is an excitatory neurotransmitter at nematode neuromuscular junctions. The function of the low level of CAT observed throughout the hypodermis is unknown.

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The effects of avermectin and drugs related to acetylcholine and 4-aminobutyric acid on neurotransmission in Ascaris suum.

We have examined some aspects of the neuropharmacology of the nematode Ascaris suum using a divided chamber and selective stimulation technique to localize the sites of action of drugs. These techniques enabled us to investigate separately excitatory neuromuscular transmission, inhibitory neuromuscular transmission, and transmission from interneurons to excitatory motorneurons. We find that a curare-sensitive mechanism is involved in the excitation of the excitatory motorneuron via interneurons. The anthelmintic avermectin Bla (AVM) also blocks interneuronal stimulation of excitatory motorneurons. This action of AVM can be reversed by picrotoxin. AVM has no effect on excitatory neuromuscular transmission. Two GABAergic agonists in nematodes, muscimol and piperazine, mimic the effects of AVM when applied ventrally. This suggests that the action of AVM is related to a GABAergic mechanism. Ventral inhibitory neuromuscular transmission is also blocked by AVM, but this action is not reversed by picrotoxin. Thus AVM has two distinct sites of action in A. suum.

Acetylcholine↗

Avermectin B1a, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris.

Avermectin B1a (AVM) is an antiparasitic agent that paralyzes nematodes without causing hypercontraction or flaccid paralysis. Using selective stimulation techniques, we have shown that AVM blocks transmission between interneuron(s) and excitatory motorneurons in the ventral nerve cord of Ascaris. It also inhibits transmissin between inhibitory motoneurons and muscle but has little effect on excitatory neuromuscular transmission. Picrotoxin can reverse the AVM-induced block of interneuron-excitatory motoneuron transmission but has no effect on the inhibitory motoneuronal synapse in either the presence or absence of AVM. Our results provide an explanation of how AVM may cause paralysis of nematodes.

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Structure and physiological activity of the motoneurons of the nematode Ascaris.

The nervous system of the nematode worm Ascaris contains about 250 nerve cells; of these, the motoneurons consist of five segmental sets, each containing 11 cells. Morphologically, the motoneurons can be divided into seven different types. Their geometry is simple: some are unbranched, others have one branch point, and the most complex have two. There is no neuropil in the nerve cords; synapses are made by axo-axonal contact or onto short spines. These features enable us to study the anatomy and physiology of the system with a degree of completeness that would be difficult in other systems. The physiological activity of five of the motoneurons has been investigated, three being excitatory and two inhibitory. The excitatory motoneurons receive input from intersegmental interneurons. The inhibitory motoneurons do not receive input from the interneurons; instead they receive their input from the excitatory motoneurons in a circuit that can mediate reciprocal inhibition between the dorsal and the ventral musculature.

Action Potentials↗

Anatomy and development of the somatic musculature of the nematode Ascaris.

The musculature of the nematode Ascaris has been studied by the examination of serial sections by light and electron microscopy. The muscle cells of nematodes are unusual in that they send branches to the neurones in contrast to the more usual situation in other animals where neurones send processes to the muscles. The neuromuscular synapses are made at the ends of the arms. Muscle cells receive multiple innervations and perform integration of the combined inputs. The action potentials are initiated near the ends of the arms so each arm acts as an integrative centre. It is shown that it is common for a muscle cell to have several arms, raising the possibility that each arm may integrate different combinations of neuronal inputs. In the second larval stage the total number of muscle cells is 83. The adult has approximately 5 X 10(4) muscle cells. The very striking increase in cell numbers of the musculature is not matched by a corresponding increase in the number of cells in the nervous system. A model for the way in which a small number of neurones can co-ordinate the activity of an increasing population of muscle cells is presented.

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Neuronal geometry: determination with a technique of intracellular dye injection.

In a study of the specificity of neuronal connections in lobster abdominal ganglia, the dye Procion Yellow M4RS was electrophoretically injected into identified cell bodies. This dye spreads into fine branches of cells, survives fixation and routine histological procedures, and permits the reconstruction of cell shapes through examination of serial sections of ganglia. Certain cells were found to have an internal bilateral symmetry. Repeated injection of the same cells in ganglia from different animals showed that cells have characteristic shapes and that the neuropil is highly structured. This method of dye injection should have general applicability in studies where a knowledge of the geometry of specific cells is important.

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