Biomedical subjects
B G Wallace
Publications and source records attributed to B G Wallace.
Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes.
A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.
Segmental variation in the arborization of identified neurons in the leech central nervous system.
Mechanosensory and motor neurons in the central nervous system of the leech have been examined by intracellular injection of horseradish peroxidase and electrophysiological mapping of their peripheral fields to determine how the arborizations of homologous cells are influenced by their segmental position. The branching patterns of annulus erector (AE) motoneurons in ganglia near the head and tail were found to be more extensive than those of cells in midbody ganglia. As in midbody ganglia, the peripheral fields of AE motoneurons in adjacent ganglia near the head and tail overlapped extensively, but the subfields innervated by individual branches of a single AE motoneuron showed little or no overlap. No AE motoneurons were found in the head ganglion or in the 20th and 21st free segmental ganglia. The branching pattern of touch-sensitive mechanosensory cells showed a similar segmental variation; touch cells in ganglia near the head and tail had more extensive arborizations than those in midbody ganglia. The rostrocaudal position along the cord at which the branching pattern changed from that characteristic of midbody ganglia to one with a more extensive arborization differed for different types of neurons. These findings demonstrate that a cell's pattern of arborization is not determined by a simple segmental difference between ganglia and suggest that during development neurons respond individually to cues that vary along the length of the cord.
Selective loss of neurites during differentiation of cells in the leech central nervous system.
The arborizations of annulus erector (AE) motoneurons in the central nervous system of the leech, Hirudo medicinalis, have been examined during embryogenesis to determine how segmental differences in their branching patterns arise. Early in development AE motoneurons all along the ganglionic chain had a similar central arborization, with major branches extending both rostrally and caudally along the connectives that link adjacent ganglia. As the embryo grew, the processes in the connectives elongated but failed to increase significantly in caliber and eventually atrophied and were lost. This sequence of events did not occur uniformly along the cord, however. AE motoneurons in midbody ganglia lost both anterior and posterior branches, cells near the head lost only their posterior branch, while cells near the tail lost only their anterior branch. In this way, the selective atrophy of neurites during development produced a systematic segmental difference in the morphology of homologous cells.
Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.
The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component. Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5.5 extracts from the muscle fraction. The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction. This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath. Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina.
Molecular components of the synaptic basal lamina that direct differentiation of regenerating neuromuscular junctions.
Results of experiments outlined here provide evidence that components of the myofiber basal lamina sheath direct the formation of active zones in regenerating motor nerve terminals and the development of infoldings and the aggregation of AChRs in the plasma membrane of regenerating myofibers. As a step toward identifying the basal lamina molecules that aggregate AChRs, we are now studying an ECM fraction from the Torpedo electric organ that causes AChRs to aggregate on cultured myotubes. We have solubilized and purified the electric organ AChR-aggregating molecules over 1000-fold. Only nanogram amounts of the most purified extracts are required to cause detectable AChR aggregation. We have also shown that similar activity can be extracted in relatively small amounts from muscle. Antiserum raised against the partially purified electric organ material completely blocked and immunoprecipitated the AChR-aggregating activity in extracts of the electric organ and muscle and bound to components of the basal lamina of frog muscle fibers. Although several polypeptides are present in our most purified extracts, an antiserum against polypeptides in the range of 80 kD completely blocked AChR aggregation by soluble extracts of the electric organ. These findings demonstrate the feasibility of isolating molecules from the synapse-rich electric organ that cause AChR aggregation and comparing them by immunological techniques with those in basal lamina at the neuromuscular junction.
Characterization of acetylcholinesterase in individual neurons in the leech central nervous system.
Acetylcholinesterase (AChE) activity was measured in cholinergic and non-cholinergic neurons in the central nervous system of the leech. Intracellular AChE was assayed by pretreating intact ganglia with echothiophate to inhibit selectively extracellular enzyme. The concentration of intracellular AChE in cholinergic neurons was 3- to 24-fold higher than that in non-cholinergic cells. The properties of AChE in extracts of leech ganglia were similar to those of "true" acetylcholinesterase, although butyrylthiocholine was almost as good a substrate as acetylthiocholine. There was also cholinesterase activity in leech blood; this enzyme resembled butyrylcholinesterase. Sucrose gradient velocity sedimentation of Triton X-100 extracts of leech ganglia revealed a major peak of AChE activity at 6.5 S and a small peak at 4.3 S. The pattern of activity in the gradient was the same when intact ganglia were pretreated with echothiophate, although the total activity was reduced by 98%. Intact leech ganglia were stained for AChE activity with and without echothiophate pretreatment. In ganglia that had not been exposed to echothiophate, cholinesterase reaction product was deposited primarily on the ganglionic sheath. In pretreated ganglia, on the other hand, cholinesterase activity was concentrated within neuronal cell bodies. Electrophysiological identification and intracellular injection of the fluorescent dye Lucifer Yellow prior to staining were used to confirm that most AChE-positive cells were cholinergic motoneurons. Two previously unidentified neurons staining for AChE were shown to be motoneurons. These results demonstrate that cholinergic motoneurons can be differentiated from other cells in the leech nervous system by their high intracellular concentration of AChE.
Distribution of AChE in cholinergic and non-cholinergic neurons.
Intracellular and surface acetylcholinesterase activities were determined for individual cholinergic and non-cholinergic neurons dissected from the central nervous system of the leech. Echothiophate pretreatment was used to inhibit selectively extracellular enzyme. Cells releasing acetylcholine as a transmitters had approximately 10-fold higher levels of intracellular acetylcholinesterase activity, while all neurons had similar levels of activity associated with the cell surface. These results suggest that intracellular cholinesterase may be a useful marker for cholinergic neurons.
Modulation of transmission at an inhibitory synapse in the central nervous system of the leech.
The synaptic interactions among a group of cells in the leech C.N.S. that regulate the animal's heartbeat exhibit several remarkable features (Thompson & Stent, 1976 a, b, c). We have examined in detail the properties of the inhibitory synapse between two of these cells, the heart interneurone (HN cell) and the heart excitor motoneurone (HE cell). 1. Impulses in the presynaptic HN cell gave rise to monosynaptic i.p.s.p.s in the HE cell that were blocked by high concentrations of Mg and were reversed when the membrane potential of the post-synaptic motoneurone was hyperpolarized beyond--75 m V or when Cl was injected into the cell body. These i.p.s.p.s were chemically mediated, and involved an increase in chloride conductance. 2. In contrast to chemical synapses between sensory and motor cells in the leech C.N.S., little facilitation or depression of transmission occurred when the HN cell was stimulated at frequencies of 0.1--50 Hz. 3. Steady subthreshold depolarization of the presynaptic HN interneurone evoked a maintained hyperpolarization of the post-synaptic HE cell, indicating that currents injected into the HN cell body could spread to the terminals and cause continuous release of transmitter. 4. The size of the i.p.s.p. evoked in the HE motoneurone by an action potential in the HN interneurone varied with the resting membrane potential of the presynaptic cell. An impulse superimposed on a prolonged, subthreshold, depolarizing pulse produced a larger i.p.s.p.; conversely, prolonged hyperpolarization of the HN interneurone reduced the i.p.s.p. amplitude recorded in the HE cell. This effect was most obvious when the natural, rhythmical bursts of activity in the HN interneurone were interrupted by bathing the preparation in leech Ringer fluid containing elevated concentrations of Mg. Under these conditions a 10 mV depolarization of the HN cell increased the size of the i.p.s.p. in the HE cell approximately sixfold. Significant changes in i.p.s.p. amplitude occurred without any noticeable change in the amplitude and duration of the presynaptic action potential. With large presynaptic depolarizations, which produced the biggest i.p.s.p.s, there was some reduction in the amplitude and increase in the duration of the action potential. 5. Following a step depolarization of the presynaptic cell, the size of successive i.p.s.p.s increased with a time constant of about 1 sec. Upon repolarization the i.p.s.p.s decreased in amplitude to the original level. 6. stimulation of one HN cell also gives rise to an i.p.s.p. in its contralateral homologue (Thompson & Stent, 1976c). Trains of i.p.s.p.s produced in this way hyperpolarized at HN cell to such an extent that the size of the synaptic potential it evoked in an HE cell was reduced. 7. Thus, an HN interneurone inhibitis transmission between the contralateral HN and HE cells presynapitcally in addition to inhibiting directly the ipsilateral HE motoneurone.
Quantal analysis of transmitter release at an inhibitory synapse in the central nervous system of the leech.
The quantal nature of transmitter release has been analysed at central inhibitory synapses in the leech nervous system between an interneurone (HN) and a motoneurone (HE) that regulate the heartbeat. 1. Ganglia were bathed in leech Ringer fluid containing 20 mM-Mg and 1.8 mM-Ca and the membrane of the presynaptic HN interneurone was hyperpolarized by current injection. Under these conditions successive inhibitory potentials in the HE motoneurone, evoked by impulses in the HN interneurone, showed striking fluctuations in amplitude. 2. Assuming a Poisson distribution of the i.p.s.p.s and estimating the number of failures from the amplitude histograms of the observed responses, the mean size of the quantal unit was estimated as 0.25 +/- 0.015 mV (S.E. of mean, n = 26). When m, the mean number of quanta released per trial, was varied by changing the membrane potential of the presynaptic HN cell (Nicholls & Wallace, 1978), the experimentally observed amplitude distributions could be predicted by the Poisson theory. 3. An independent estimate of the unit size was obtained by noise analysis. A long subthreshold depolarizing pulse applied to the presynaptic HN interneurone evoked a sustained hyperpolarization of the HE motoneurone, apparently caused by an increase in the rate of on-going release of quanta by the HN cell terminals. From the mean change in membrane potential and the increase in variance, the size of the unit was calculated as 0.21 +/- 0.039 mV (S.E. of mean, n = 11). For ten pairs of cells an estimate of unit amplitude was made both from the Poisson analysis and the analysis of variance, again with good agreement. For these cells the estimated unit sizes were 0.24 +/- 0.023 mV (S.E. of mean, n = 10) from the failures and 0.21 +/- 0.043 m V (S.E. of mean, n = 10) from the noise. 4. A similar analysis was made of the inhibitory synaptic potentials evoked in one HN interneurone by stimulation of its contralateral homologue. Transmission again appeared to be qualtal; the mean unit amplitude from Poisson analysis was 0.31 +/- 0.022 mV (S.E. of mean, n = 19) and from the noise 0.29 +/- 0.027 mV (S.E. of mean, n = 3). 5. We conclude that transmitter is released from the terminals of the HN interneurone in quantal units that evoke miniature i.p.s.p.s of about 0.25 mV in the post-synaptic cells. Furthermore, modulation of transmission proudced by variation in the presynaptic resting potential and during presynaptic inhibition results from changes in the mean number of quanta released by each impulse.
Regeneration of synaptic connections by sensory neurons in leech ganglia maintained in culture.
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Octopamine neurons in lobsters: location, morphology, release of octopamine and possible physiological role.
Octopamine cells are found along second thoracic roots, where they serve as neurosecretory neurons capable of releasing octopamine at two distinct points: one into the hemolymph immediately before it enters the gills; one into the hemolymph immediately after it leaves the gills. The octopamine cells receive a cholinergic synaptic input. We presume that this input is from processes of peripheral sensory cells bringing information to the CNS. Octopamine can increase the strength of contraction of exoskeletal muscles and, at higher concentrations, can induce contractures in these muscles. These effects can be interpreted as a resetting of the level of ionized calcium within muscle fibers (the contracture) to a higher value or a possible enhanced entry of calcium ions during nerve stimulation (increased strength of contraction). The observed effects are of a prolonged duration, outlasting the time of application of octopamine by some 20-40 minutes. We do not know if this effect on muscle tension production is the normal physiological role of octopamine. Other possible roles will be explored in the future. The pathway involving the octopamine neurons in lobsters may provide a model neurohumoral system that can be studied and understood in detail from the level of sensory input to the level of behavioral output.
Modification and regeneration of synaptic connections in cultured leech ganglia.
Segmental ganglia of the central nervous system of the leech were maintained in culture medium outside the animal for several weeks in order to study the properties of synapses and regeneration by identified sensory and motor nerve cells. A variety of preparations were used, including single ganglia, chains of ganglia and ganglia connected to the areas of skin and muscle that they normally innervate in the animal. (1) For up to 10 weeks after removal from the animal, resting and action potentials recorded from sensory and motor neurons resembled those seen in normal ganglia. The same individual cell in a cultured ganglion could be recorded from with intracellular electrodes on a second occasion after an interval of a few days. (2) Sensory cells, identified as touch, pressure or nociceptive according to their morphology and electrical properties, continued to respond selectively to stimuli of the appropriate modality applied to their receptive fields in the skin; action potentials in motor cells caused contractions in the appropriate muscles. Culture of ganglia for more than 3 weeks caused the disappearance of synaptic potentials and a loss of transparency in ganglia. (3) Certain chemically mediated synaptic interactions between sensory and motor nerve cells became markedly changed in cultured ganglia. These changes appeared over the first 3 weeks and consisted of abnormally large excitatory and inhibitory synaptic potentials. The changes in synaptic transmission observed in culture were in many respects similar to those occurring in ganglia maintained within an animal after lesions have been made in the nervous system (Jansen et al. 1974). (4) The morphological appearances of sensory cells were compared in cultured and normal ganglia after injection of horseradish peroxidase. In cultured ganglia, the branching pattern appeared normal, but varicosities became more conspicuous. (5) When connectives linking cultured ganglia were crushed or cut, regeneration occurred. By 7 days, impulses propagated through the regenerated fibers and evoked synaptic potentials on cells within the next ganglion. The course taken by regenerating axons was observed in cells injected with horseradish peroxidase. The results again resembled those seen in animals with similar lesions. (6) The cultured ganglia provide preparations in which it is possible to analyze the mechanisms that underlie long-term changes similar to those seen in the leech central nervous system in situ.
The biosynthesis of octopamine--characterization of lobster tyramine beta-hydroxylase.
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The association of octopamine with specific neurones along lobster nerve trunks.
Octapamine and its synthetic enzyme, tyramine beta-hydroxylase (TBH), are found in high concentrations at two points along second thoracic nerve roots in lobsters. The first is in the proximal section of the second root between the ventral nerve cord and the bifurcation of the root into medial (to flexor muscles) and lateral (to extensors) branches. The second region of high concentration is within a well known crustacean neurosecretory system, the pericardial organ, located close to the ends of the lateral branches of the roots. 2. With several different staining procedures, small clusters of nerve cell bodies are found within the connective tissue sheath in the proximal regions of the second roots. No cell bodies are seen in the pericardial organ regions. Cell bodies are variable in number and position between corresponding roots in the same animal and homologous roots among different animals. The average numbers of cell bodies, however, correlate well with TBH and octopamine content, and with the synthesis of octopamine in these same regions of roots. 3. Small clusters of root cell bodies dissected from preparations have greater than 500-fold higher activities of TBH than isolated efferent excitatory and inhibitory or afferent sensory axons. 4. Along with octopamine, the preferential synthesis of acetylcholine and serotonin is also seen in proximal segments of roots. Acetylcholine synthesis in these regions may represent transmitter synthesized in the nerve terminals innervating the root cells. The role of serotonin in these regions is not understood at this time but the amounts of endogenous serotonin found are only a tenth of the amounts of octopamine present. 5. Dopamine is not synthesized from tyrosine in second thoracic roots. However, if DOPA or dopamine are used as precursor compounds, then noradrenaline, which is usually not found in lobsters, can be accumulated in proximal segments of roots. 6. Phenolamines are converted to two further metabolites by lobster tissues. The compounds are unidentified and are named fast and slow product on the basis of their migration on electrophoresis at acid pH. Some partial characterization of slow product reveals that it is a mixture of compounds that can be converted on mild acid hydrolysis to fast product and the parent phenolamine. 7. The several lines of evidence presented suggest that nerve cells found in the proximal segments of the second thoracic roots contain and can synthesize octopamine. Since not all the cells in any single root have been analysed for octopamine or TBH, however, the possibility that one or more of the cells contain physiologically interesting substances other than octopamine is not eliminated.
Octopamine: selective association with specific neurons in the lobster nervous system.
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Streptococcal proteinase-catalyzed hydrolysis of some ester and amide substrates.
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Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions.
The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.