Biomedical subjects

F Moody-Corbett

Publications and source records attributed to F Moody-Corbett.

At least 19 recordsLinked to original sources

PubMed2001

Cloning and expression of three K+ channel cDNAs from Xenopus muscle.

Embryonic Xenopus muscle cells grown in culture express voltage-gated K+ currents with inactivating and non-inactivating kinetics. Here we report the cloning of three K+ channel cDNAs, designated XKv1.2', XKv1.4 and XKv1.10, from muscle which may underlie these currents. XKv1.2' cDNA appears to be an allelic variant of the XKv1.2 previously cloned from Xenopus. The second cDNA encodes a homologue of Kv1.4 that has not been previously cloned from Xenopus. The predicted XKv1.4 protein shows 73% overall similarity to mouse and chick Kv1.4, but shows significant divergence in the region corresponding to the chain of the inactivating 'ball and chain' domain. The third K+ channel cDNA isolated from Xenopus muscle is a novel Kv1 isoform designated XKv1.10. The predicted protein shares about 70% similarity with other members of the Kv1 subfamily, and about 40% with members of the Kv2, Kv3 and Kv4 subfamilies. XKv1.4 mRNA appears as early as stage 10.5 in whole embryos and is prominent in muscle throughout development from stage 14 to adult. XKv1.2' mRNA is detected by stage 11.5 in whole embryos, but remains at low levels in embryonic skeletal muscle (stages 14 and 21), and is absent from adult muscle. XKv1.10 mRNA is first detected at stage 21 in whole embryos, and is present in muscle from this stage onwards. All three transcripts are present in spinal cord at stage 21. The results support the notion that channels encoded by XKv1.4 contribute to the inactivating K+ current observed in embryonic muscle cells in culture.

Animals↗

PubMed2001

Electrophysiological properties of CA1 neurons protected by postischemic hypothermia in gerbils.

BACKGROUND AND PURPOSE: Recent studies show that prolonged (eg, 24-hour) postischemic hypothermia confers lasting histological and behavioral protection against severe global cerebral ischemia. However, functional abnormalities may be compensated for by undamaged brain regions and thus not detected by behavioral tests. To determine whether hypothermia preserves CA1 functional integrity, we measured synaptic and membrane properties of CA1 neurons in ischemic gerbils treated with postischemic hypothermia. METHODS: Gerbils were subjected to 5 minutes of forebrain ischemia and were either left untreated or exposed to 2 days of hypothermia (32 degrees C for 24 hours and then 34 degrees C for 24 hours). Sham animals were operated on but not made ischemic, then either allowed to recover at room temperature or subjected to hypothermia for 2 days. Approximately 5 weeks after ischemia or sham surgery, patch-clamp recordings were obtained from the CA1 region of hippocampal slices. RESULTS: There was approximately 95% CA1 cell loss in untreated ischemic animals, whereas ischemic gerbils treated with hypothermia had cell counts similar to sham animals. Resting membrane potential, action potential amplitude and duration, input resistance, and synaptic currents evoked by Schaffer collateral stimulation were similar between pyramidal cells obtained from ischemic gerbils treated with hypothermia and sham-operated animals (P:>0.05). CONCLUSIONS: These data demonstrate that postischemic hypothermia preserves the measured electrophysiological properties of CA1 neurons in the absence of any apparent functional abnormalities. This study provides further support for the use of hypothermia as a treatment for cerebral ischemia.

Animals↗

PubMed1999

Localization of sodium and potassium currents at sites of nerve-muscle contact in embryonic Xenopus muscle cells in culture.

Macro-patch current recordings were used to determine the spatial distribution of INa, inactivating (IIK) and non-inactivating (IK) outward potassium currents on nerve-contacted Xenopus muscle cells grown in culture. Sites of nerve contact were identified under 400x magnification and macroscopic currents recorded from cell-attached membrane patches ranging in diameter from 2 microm to 6 microm. Membrane patch (n=133) recordings revealed no detectable current, or INa and/or IIK, or just IK. More than 85% of the membrane patches from which INa (n=35) could be recorded occurred within 40 microm of the position of nerve contact. In addition, more than 90% of the membrane patches which contained INa also contained IIK. The density of IIK in these membrane patches that also contained INa was significantly higher than the density of IIK or IK in membrane patches without INa. Sites of ACh receptor localization at nerve-muscle contacts, identified by labelling with rhodamine-alpha-bungarotoxin, were also found to contain both INa (4/5 cells) and IIK (5/5 cells). These results suggest that in Xenopus muscle cells in culture the channels mediating IIK along with sodium channels are clustered at sites of nerve contact and synaptic specializations.

Animals↗

PubMed1996

Debate: a tool for teaching graduate students.

The introduction of a formal debate was used to stimulate discussion of neuroscience in our graduate program. The debate had several advantages for encouraging student participation. Several students actively participated in the debate: four debaters and five judges. Faculty coaches helped provide background knowledge and guidance before the debate but were not debaters. Faculty directed the judges in methods of evaluation but were not judges. It was felt that this method of teaching was not only fun, but also provided an opportunity to learn current topics in neuroscience, method of presentation, and critical evaluation.

Education, Medical, Graduate↗

PubMed1995

Former neuritic pathways containing endogenous neural agrin have high synaptogenic activity.

When Xenopus spinal cord (SC) neurons are grown on an appropriate substrate of basal lamina molecules, the agrin they externalize along their neuritic outgrowth remains bound to the substrate even after the neurons are removed. Here we demonstrate that these former neuritic pathways containing substrate-bound, neural agrin cause an accumulation of acetylcholine receptors (AChR) and cholinesterase (ChE) at sites of contact with muscle cells and inhibit AChR aggregation over the rest of the muscle cell surface. These local and global synaptogenic effects were not triggered by former neuritic pathways that were agrin-negative. The length of AChR accumulation along the agrin pathways contacted by individual muscle cells corresponded to a saturation process, in agreement with the notion that muscle cells have a limited capacity to cluster AChR. The AChR accumulation caused by the agrin pathways was almost twice as extensive as that induced by living neurites. It is concluded that agrin and possibly other synaptogenic molecules externalized by competent SC neurons bind to the culture substrate in quantities which are more than sufficient to account fully for the local and global changes in AChR and ChE distribution associated with embryonic nerve-muscle synaptogenesis.

Agrin↗

PubMed1994

Neuritic deposition of agrin on culture substrate: implications for nerve-muscle synaptogenesis.

Recent experiments have indicated that neural agrin is deposited at newly forming nerve-muscle synapses and has a primary synaptogenic role there. As a step toward assessing how the spatial arrangement of new synaptic sites is regulated, we compared the pattern of agrin deposition by Xenopus neurites on culture substrate and on muscle cells. The neurons were grown on a substrate that bound their externalized agrin so tightly that it remained bound even when the neurites retracted spontaneously or were eliminated experimentally. By contrast, the neural cell adhesion molecule, NCAM, was not left behind on the substrate when the neurites were eliminated. Agrin, visualized by immunofluorescent staining, was deposited on the culture substrate in a continuous fashion along virtually the entire neuritic arbor of many spinal cord (SC) neurites. The pattern of agrin deposition by the same neurites changed from continuous to discontinuous when the neurites contacted muscle cells, and it became continuous again when the neurites returned to the culture substrate. The sites of agrin deposition on muscle cells were also sites of accumulation of ACh receptors (AChRs). Dorsal root ganglion (DRG) neurons and some SC neurons did not deposit agrin along their neuritic outgrowth, either on the culture substrate or on the muscle cells, and did not induce AChR accumulation at sites of contact with muscle cells. Besides adding to the evidence in support of agrin's synaptogenic role, the findings indicate that muscle cells significantly influence how neural agrin and synaptic sites become distributed along paths of neurite-muscle contact.

Agrin↗

PubMed1992

A K+ current in Xenopus muscle cells which shows inactivation.

The purpose of this study was to describe an inactivating potassium current that is present in Xenopus muscle cells in culture. Whole cell currents were recorded from muscle cells grown in culture for 16-30 h. The majority of muscle cells had an outward current which inactivated during a 100 ms maintained depolarization. The outward current was reduced by tetraethylammonium or the aminopyridines, but not completely eliminated by either agent. Depolarizing prepulses reduced both the peak current and the late component of the current. The results suggest that by one day in culture these skeletal muscle cells develop an inactivating outward potassium current.

Animals↗

PubMed1992

Two classes of potassium currents in Xenopus muscle cells in young cultures.

The aim of the present study was to determine if both inactivating and non-inactivating potassium currents occur in skeletal muscle cells at early times in culture. Whole cell current was recorded from embryonic muscle cells within 4-10 h of plating. We found that in the cells which had an outward current, half had a current that did not inactivate and half had a current that inactivated during a maintained depolarization (100 ms). The two groups of cells did not differ in their age in culture or resting membrane potential. The results indicate that Xenopus skeletal muscle cells can acquire both types of outward potassium current early in culture life.

Animals↗

PubMed1991

Exposure to nerve does not affect the appearance of calcium currents in embryonic muscle.

Xenopus skeletal muscle cells in culture develop two inward calcium currents, a slow and a transient current, which differ in terms of kinetics, voltage of activation and sensitivity to nifedipine. The slow current was present in many cells by the first day in culture and in all the cells by the second day in culture. In contrast, the transient current was present in only a small proportion of cells on the first day and in the majority of cells by the fifth day. We found that neither the characteristics of the two currents nor the developmental sequence of their appearance were altered by the presence or absence of nerve or by a prior exposure to nerve in-vivo.

Animals↗

PubMed1990

Inward rectifier potassium current on embryonic Xenopus muscle cells at different times in culture.

Whole cell current recordings of Xenopus muscle cells in 1-day-old cultures revealed an inward K+ current, with properties similar to the inward rectifier of adult muscle. However, at the earliest ages (less than 10 h in culture) the time-dependent decline in the current amplitude, indicative of a depletion of extracellular K+, was not present. The change in the time-dependent decline of the current amplitude may result from a change in the channel itself or a change in the distribution of the channel in the membrane.

Animals↗

PubMed1989

Calcium current in embryonic Xenopus muscle cells in culture.

We have investigated the appearance of calcium current in Xenopus muscle cells in 1- to 6-day-old cultures. Whole cell currents were recorded using a patch-clamp amplifier with sodium and potassium replaced with tetraethylammonium and cesium, respectively, and BaCl2 used in place of CaCl2. When the muscle membrane was depolarized above -30 mV, a slow inward current was activated, the current reached a peak amplitude near 0 mV, and an outward current became apparent above +10 mV. This slow current was enhanced by adding barium or Bay K 8644 to the extracellular recording solution and was blocked by the addition of cobalt, cadmium, or the dihydropyridines nifedipine or (+)PN 200-110. Taken together these results indicate the presence of an inward calcium current mediated through L-type channels. Thirty-one percent of the cells examined on the first day in culture showed no discernible slow inward current; however, as the age of the culture increased, all cells showed slow inward current and there was an increase in the amplitude of the current. A small proportion of the muscle cells (5 out of 34) also showed a fast activating and inactivating inward current. This current, which activated at more hyperpolarized potentials (-40 mV) was only present when 5 mM ATP was included in the internal recording solution. It also appeared to be mediated through a calcium channel but not a dihydropyridine, sensitive channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1987

Transcriptional and translational requirements for developmental alterations in acetylcholine receptor channel function in Xenopus myotomal muscle.

Two functionally distinct types of acetylcholine (ACh) receptor channels are present on embryonic Xenopus myotomal muscle. During differentiation of this muscle, both in vivo and in dissociated cell culture, the occurrence of the high conductance "fast" channel type increases relative to the low conductance "slow" channel type. In order to ascertain whether new receptor synthesis is required for this rapid switch in channel types we examined the effects of inhibitors of transcription (alpha-amanitin and actinomycin D) and translation (cycloheximide) on developing muscle in culture. Inhibition of protein synthesis resulted in greater than 95% reduction in ACh receptor incorporation and also reversibly blocked the developmental appearance of the high conductance channel type. Inhibition of mRNA synthesis only slightly reduced the rate of receptor incorporation into muscle membrane over a 24-hr period but reversibly blocked appearance of the high conductance channel. These findings suggest that the high conductance ACh receptor channel type does not result from post-translational modifications of the low conductance type, but rather from transcription of a different mRNA encoding one or more of the ACh receptor subunits.

Amanitins↗

PubMed1987

Acetylcholine reduces inward rectification on thymus-derived macrophage cells in culture.

Cultures prepared from dissociated rat thymus were examined 1-2 weeks after plating. Macrophage cells were identified by their adherence, morphological appearance, and ability to phagocytize carbon particles or heat-inactivated Staphylococcus aureus. Whole cell current recordings from macrophage cells revealed an inward current at potentials more negative than the equilibrium potential for potassium and an outward current at potentials more positive than -40 mV in normal recording solution. Acetylcholine or muscarine caused a reduction in inward current but did not alter the outward current. The inward current and acetylcholine effect were seen at less negative potentials by decreasing the potassium equilibrium potential and both were blocked by the addition of cesium to the external recording solution. These results indicated that the inward current was mediated by potassium through the inward or anomalous rectifier. Physiologically, the action of acetylcholine on the inward rectifier of these macrophage cells may be mediated by cholinergic innervation of the thymus.

Acetylcholine↗

PubMed1986

Formation of the vertebrate neuromuscular junction.

The sequence of events leading to the formation of the NMJ based on the data presented in this chapter from rat, chick, and Xenopus muscle can be divided into three developmental stages, as shown in Table I. The essential components of the NMJ are acquired early. Acetylcholine is present and can be released from the growing nerve. Acetylcholine receptors are present in the muscle membrane and are functional even at the earliest times. These components of the junction--ACh release and functional ACh receptors--can develop independently of each other; i.e., cell culture studies have shown that nerve cells are capable of releasing ACh before their growing tips have come into contact with the postsynaptic muscle membrane. Conversely, muscle cells grown without nerve synthesize and incorporate in their membranes functional ACh receptors. This situation ensures that functional (table; see text) contacts can occur even at the earliest times. Local accumulation of ACh receptors is also detected at the earliest times of junction formation. Although cell culture studies have demonstrated that receptors can aggregate in the absence of nerve, it would appear that the nerve plays an important role in directing where the highest density of receptors will be localized. Acetylcholinesterase, identified both histochemically and electrophysiologically, occurs at the presumptive NMJ shortly after synaptic transmission and receptor clustering have begun, suggesting that these events may play a role in localizing cholinesterase. Although the studies on rat and chick muscle support this view, development of AChE on Xenopus muscle does not require prior exposure to nerve or muscle activity. The ultrastructural features characteristic of the adult NMJ also do not become apparent until after synaptic transmission and receptor clustering have been seen. However, detection of small regions of specialization could be easily overlooked at the ultrastructural level, particularly if the tissue has not been serially sectioned. The young tissue is more fragile (Gordon et al., 1974) and may be more susceptible to mechanical damage or alterations from the fixation procedures (Kullberg et al., 1977). For these reasons, results pertaining to when the ultrastructural specializations occur are difficult to interpret and must await identification of these structures by other means. A number of other changes occur at the NMJ late in development: (1) ACh receptors become metabolically more stable, (2) there is a conversion in the kinetics of the ACh receptor channel, and (3) junctional folds become apparent. The extent to which these changes occur varies among the different organisms discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗

PubMed1984

Properties of non-junctional acetylcholine receptor channels on innervated muscle of Xenopus laevis.

Patch-clamp recordings of current through acetylcholine-activated channels were made from non-junctional membrane of innervated myotomal muscle from Xenopus laevis. Two classes of acetylcholine (ACh) receptor channels were identified on the basis of current amplitudes. Both amplitude classes exhibited current-voltage relations which deviated from linearity as the extrapolated reversal potential was approached (-5 to -12 mV). Over the range of greatest linearity the conductances of the two classes were 64 and 44 pS. Both event classes were blocked by alpha-bungarotoxin. At the normal resting membrane potential (approximately -95 mV) the larger conductance channel (gamma) exhibited an apparent mean channel open time of less than 1 ms, compared to approximately 2 ms for the smaller gamma class. The apparent open time was voltage-dependent, changing e-fold with a 63 mV hyperpolarization for the high gamma channel and 93 mV hyperpolarization for the low gamma channel. At low ACh concentrations (0.1-0.3 microM) both amplitude classes exhibited bursts of successive openings separated by brief closures of less than 0.5 ms. Bursts were separated by longer closed intervals of 1 to greater than 100 ms. Closed interval histograms revealed corresponding populations of brief and long closures, indicating that at least two kinetic processes are required to describe the distribution of closed intervals. In the absence of exogenous ACh, channels were observed in an occasional patch which showed a conductance and extrapolated reversal potential similar to ACh-activated channels. In such patches the event frequency could occasionally be altered by adjusting the negative pressure applied to the patch. The two main conductance classes of ACh activated channels were observed to coexist in most patches. However, the most frequent event observed in non-junctional membrane of innervated muscle corresponded to the high gamma class. In this respect, the non-junctional ACh receptors bore a greater similarity to junctional ACh receptors than to non-junctional receptors reported for denervated muscle.

Acetylcholine↗

PubMed1984

Acetylcholine receptor channel properties during development of Xenopus muscle cells in culture.

Developmental changes in acetylcholine (ACh) receptor channel function on aneural cultures of embryonic myotomal muscle cells were examined using the patch-clamp technique. At all stages of differentiation two different unitary-event amplitudes were observed, corresponding to high-gamma (single-channel conductance) (64 pS) and low-gamma (46 pS) channel types. No change in conductance occurred for either channel type during the 6-day in vitro period examined. At resting membrane potential (-85 mV) the low-gamma channel exhibited a mean open time of approximately 2 ms which, on the average, was 2-3-fold longer than that measured for the high-gamma channel. Neither the estimated mean channel open time nor the voltage dependence of the open state measured for either channel type changed during development. In recordings with low ACh concentration (0.1-0.25 microM) both high-gamma and low-gamma channel types exhibited non-stationary opening probabilities over the recording period. Usually the opening rate of both channel types decreased with time following seal formation, however, the 'drop-out' rate was faster for the low-gamma channel. A developmental increase in the proportion of high-gamma events occurred between day 1 (16%) and day 5 (56%) in culture, paralleling the time-dependent changes in the channel kinetics based on ACh-activated membrane noise. We conclude that the development of non-junctional muscle membrane is associated with increased expression of high-gamma channels and that this process is primarily responsible for the previously reported developmental alterations in macroscopic ACh receptor channel currents.

Animals↗

PubMed1982

Cholinesterase localization at sites of nerve contact on embryonic amphibian muscle cells in culture.

Cholinesterase (ChE), detected histochemically, was found to be localized at many sites of nerve-muscle contact in cultures of spinal cord and muscle cells derived from Xenopus laevis embryos. Such contacts were often characterized by a corresponding localization of acetylcholine receptors and by synaptic ultrastructure, including aggregates of clear vesicles in the nerve fibre and an 80-100 nm wide intercellular cleft. The ChE reaction product was localized in the cleft. When cultures were grown in the presence of curare many of the nerve-contacted muscle cells still exhibited ChE at the sites of contact. It is concluded that ChE accumulates at synaptic contacts in these cultures even in the absence of muscle action potentials and contraction.

Animals↗

PubMed1982

Influence of nerve on the formation and survival of acetylcholine receptor and cholinesterase patches on embryonic Xenopus muscle cells in culture.

Embryonic Xenopus muscle cells grown in culture develop discrete patches of high acetylcholine receptor (AChR) density. By following identified muscle cells after staining with fluorescent alpha-bungarotoxin, we have found that many of these AChR patches survive in a fixed position for several days. For AChR patches on the lower surface of the cell (the surface apposed to the culture dish), more than 60% of those which were followed beginning on day 2 survived for a further 4 days. The survival rate was greater when patches were followed from day 3 or later and was almost as high for AChR patches on the upper surface. New AChR patches also formed on all of the muscle cells. When muscle cells were cultured together with spinal cord cells, nerve-muscle contacts developed with a characteristic localization of AChRs along the path of contact. AChR patches did not form elsewhere on these contacted cells. Nerve-contacted muscle cells examined 2 to 3 days after adding spinal cord cells to established (2- to 5-day-old) muscle cultures also exhibited a marked reduction of AChR patches away from the site of contact. This reduction was not due to the nerve having contacted pre-existing AChR patches. Rather, the findings indicate that contact by an appropriate nerve inhibits the formation of AChR patches elsewhere on the contracted muscle cells and reduces the survival of pre-existing AChR patches. Nerve contact also inhibited the formation of cholinesterase (ChE) patches remote from the site of contact and appeared to cause some reduction in the survival of pre-existing ChE patches. Spontaneous twitching was not observed in these experiments, thereby indicating that the remote effects of nerve contact were not mediated by muscle action potentials or contraction. Such remote influences of the nerve may play a role in determining the pattern of innervation on individual muscle cells.

Animals↗