Biomedical subjects

S Karunanithi

Publications and source records attributed to S Karunanithi.

15 recordsLinked to original sources

PubMed2006

The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation.

Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cac(ts2) mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cac(ts2) mutation occurs in the cytoplasmic carboxyl tail of the alpha1-subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+-imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cac(ts2) mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Ca(v)2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 degrees C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Ca(v)2.1 channel inactivation.

Amino Acid Sequence↗

PubMed2004

Turning behavior in Drosophila larvae: a role for the small scribbler transcript.

The Drosophila larva is extensively used for studies of neural development and function, yet the mechanisms underlying the appropriate development of its stereotypic motor behaviors remain largely unknown. We have previously shown that mutations in scribbler (sbb), a gene encoding two transcripts widely expressed in the nervous system, cause abnormally frequent episodes of turning in the third instar larva. Here we report that hypomorphic sbb mutant larvae display aberrant turning from the second instar stage onwards. We focus on the smaller of the two sbb transcripts and show that its pan-neural expression during early larval life, but not in later larval life, restores wild type turning behavior. To identify the classes of neurons in which this sbb transcript is involved, we carried out transgenic rescue experiments. Targeted expression of the small sbb transcript using the cha-GAL4 driver was sufficient to restore wild type turning behavior. In contrast, expression of this sbb transcript in motoneurons, sensory neurons or large numbers of unidentified interneurons was not sufficient. Our data suggest that the expression of the smaller sbb transcript may be needed in a subset of neurons for the maintenance of normal turning behavior in Drosophila larvae.

Age Factors↗

PubMed2001

Effect of chronic morphine treatment on alpha(2)-adrenoceptor mediated autoinhibition of transmitter release from sympathetic varicosities of the mouse vas deferens.

1. The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on alpha(2)-adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry. 2. In chronically saline treated (CST) preparations, morphine (1 microM) and the alpha(2)-adrenoceptor agonist (clonidine, 1 microM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81+/-8% (n=16) and 92+/-6% (n=7) respectively. In CMT preparations, morphine (1 microM) and clonidine (1 microM) decreased mean EJP amplitude by 68+/-8% (n=7) and 79+/-8% (n=7) respectively. 3. When stimulating the sympathetic axons at 0.03 Hz, the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7+/-3.8 mV, n=7 compared with 9.9+/-0.3 mV, n=7). 4. Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle. 5. Results from the present study indicate that the effectiveness of alpha(2)-adrenoceptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and alpha(2)-adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.

Adrenergic alpha-Agonists↗

PubMed1999

Neuroprotection at Drosophila synapses conferred by prior heat shock.

Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Here we show that synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock that strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, we found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties.

Analysis of Variance↗

PubMed1999

Quantal unit populations at the Drosophila larval neuromuscular junction.

Focal extracellular recording at visualized boutons of the Drosophila larval neuromuscular junction was used to determine frequency and time course of the spontaneously occurring quantal events. When simultaneous intracellular recordings from the innervated muscle cell were made, more than one class of quantal event occurred at some of the individual boutons. "True" signals (arising at the bouton within the focal macropatch electrode) were often contaminated by additional signals generated outside the lumen of the focal electrode. Inclusion of these contaminating signals gave spuriously low values for relative amplitude, and spuriously high values for spontaneous quantal emission, for the synapses within the focal electrode. The contaminating signals, which appeared to be conducted along the subsynaptic reticulum surrounding the nerve terminals, generally were characterized by relatively small extracellular signals associated with normal intracellular events in the muscle fiber. From plots of simultaneous extracellular and intracellular recordings, the individual data points were classified according to the angles they subtended with the x axis (extracellular signal axis). Statistical procedures were developed to separate the true signals and contaminants with a high level of confidence. Populations of quantal events were found to be well described by Gaussian mixtures of two or three components, one of which could be characterized as the true signal population. Separation of signals from contaminants provides a basis for improving the estimates of quantal size and spontaneous frequency for the synapses sampled by the focal extracellular electrode.

Animals↗

PubMed1998

Motor nerve terminals on abdominal muscles in larval flesh flies, Sarcophaga bullata: comparisons with Drosophila.

Motor nerve terminals on abdominal body-wall muscles 6A and 7A in larval flesh flies were investigated to establish their general structural features with confocal microscopy, transmission electron microscopy, and freeze-fracture procedures. As in Drosophila and other dipterans, two motor axons supply these muscles, and two morphologically different terminals were discerned with confocal microscopy: thin terminals with relatively small varicosities (Type Is), and thicker terminals with larger varicosities (Type Ib). In serial electron micrographs, Type Ib terminals were distinguished from Type Is terminals by their larger cross-sectional area, more extensive subsynaptic reticulum, more mitochondrial profiles, and more clear synaptic vesicles. Type Ib terminals possessed larger synapses and more synaptic contact area per unit terminal length. Although presynaptic dense bars of active zones were similar in mean length for the two terminal types, there were almost twice as many dense bars per synapse for Type Ib terminals. Freeze-fractures through the presynaptic membrane showed particle-free areas indicative of synapses on the P-face, within which were localized aggregations of large intramembranous particles indicative of active zones. These particles were similar in number to those found at active zones of several other arthropod neuromuscular junctions. In general, synaptic structural parameters strongly paralleled those of the anatomically homologous muscles in Drosophila melanogaster. In live preparations, simultaneous focal recording from identified varicosities and intracellular recording indicated that the two terminals produced excitatory junction potentials of similar amplitude in a physiological solution similar to that used for Drosophila.

Abdominal Muscles↗

PubMed1997

Imaging of calcium in Drosophila larval motor nerve terminals.

Calcium measurements in the presynaptic terminal are essential in the investigation of mechanisms underlying neurotransmitter release. To enhance the genetic analysis of secretory mechanisms, we have developed Ca2+ imaging techniques for Drosophila larval motor nerve terminals. We studied Ca2+ signals in "big" (type Ib) and "small" (type Is) boutons that innervate ventral longitudinal muscles 6 and 7 in each abdominal segment of Canton-S (CS)-strain 3rd instar larvae. The indicator fluo-3 in conjunction with confocal microscopy was used to detect stimulus-dependent changes in [Ca2+]i. The Ca2+ signals were reliable and reproducible, and the resting fluorescence remained constant throughout the experiments. The Ca2+ signals increased with stimulus frequency from 5 to 20 Hz for both bouton types. No significant differences in the Ca2+ signals were seen between the two bouton types at 5 and 20 Hz, but there was a difference at 10 Hz. The decay of the Ca2+ signal was more prolonged after 20-Hz stimulation than after 5 and 10 Hz. At the single-synapse level, the secretory efficacy of Is synapses is greater than that of Ib synapses, but our data show that factors other than differences in Ca2+ entry may govern the strength of synaptic transmission.

Animals↗

PubMed1996

Quantal components of spontaneous excitatory junction potentials at visualised varicosities.

The electrical signs of spontaneous transmitter release were recorded with an extracellular electrode from single visualized sympathetic varicosities on the mouse vas deferens. Ultrastructural examination of these varicosities with the electron microscope showed that they formed close-contacts with smooth muscle cells. Amplitude-frequency histograms of the spontaneous excitatory junction potentials (SEJPs) were constructed in order to determine the statistical nature of spontaneous transmitter release from a varicosity. SEJP histograms often possessed several peaks. Statistical tests showed that these were separate modes in the histograms indicating that the SEJPs were composed of subunits. The SEJP histograms were described by a mixture of distributions in which the components were identified as quanta and there was a Poisson release of quanta. The second mode in the SEJP histograms was sometimes twice that of the first mode but generally greater, suggesting a potentiation of the effects of one quantum released nearly simultaneously with another quantum. The components in the SEJP histograms were well fitted by either a Gaussian or a gamma distribution indicating that the quantum of transmitter could be described as a Gaussian or gamma variate.

Animals↗

PubMed1995

Statistics of quantal secretion during long trains of sympathetic nerve impulses in mouse vas deferens.

1. A statistical analysis has been made of the occurrence of excitatory junctional currents (EJCs) of similar amplitude recorded with an extracellular electrode during long trains of nerve impulses to the mouse vas deferens. 2. The number of EJCs of similar amplitude that occurred in consecutive impulses during trains of 500-1000 impulses at 0.5-2.0 Hz increased with the number of EJCs evoked during the train. 3. There was no evidence of significant dependence between consecutive EJCs of similar amplitude in sixteen out of eighteen trains in eighteen preparations. 4. The time course of clusters of EJCs of similar amplitude was examined by determining the standard deviation of different groups of EJCs within a cluster throughout their time course. Most EJCs within a cluster could be grouped with a coefficient of variation < 0.1 throughout their time course. 5. The observations on EJCs of similar amplitude leave open the possibility that secretion from single varicosities is, in general, multiquantal.

Animals↗

PubMed1995

Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors.

The time course of most quantal currents recorded with a small diameter electrode placed over visualized varicosities of sympathetic nerve terminals that secrete ATP was determined: these had a time to reach 90% of peak of 1.3-1.8 ms and a time constant of decay of 12-18 ms; they were unaffected by blocking ectoenzymes or the uptake of adenosine. Monte Carlo methods were used to analyze the stochastic interaction between ATP, released in a packet from a varicosity, and the underlying patch of purinoceptors, to reconstitute the time course of the quantal current. This leads to certain restrictions on the possible number of ATP molecules in a quantum (about 1000) and the density of purinoceptors at the junctions (about 1000 microns-1), given the known geometry of the junction and the kinetics of ATP action. The observed quantal current has a relatively small variability (coefficient of variation < 0.1), and this stochastic property is reproduced for a given quantum of ATP. Potentiation effects (of about 12%) occur if two quanta are released from the same varicosity because the receptor patch is not saturated even by the release of two quanta. The simulations show that quantal currents have a characteristically distinct shape for varicosities with different junctional cleft widths (50-200 nm). Finally, incorporation of an ectoenzyme with the known kinetics of ATPase into the junctional cleft allows for a quantal current of the observed time course, provided the number of ATP molecules in a quantum is increased over the number in the absence of the ATPase.

Adenosine Triphosphatases↗

PubMed1994

Effect of yohimbine on the contractile response of the mouse vas deferens to short, low frequency trains of nerve impulses.

A study was undertaken to ascertain why in the mouse vas deferens excitatory junction potentials facilitate whereas contractions depress during low frequency stimulation. In a set of contraction studies, where a conditioning stimulus was followed 2 s later by a test stimulus, depression in the first phase of contraction was observed only at high stimulus strengths when many nerves are activated leading to a large secretion. Low stimulus strengths did not produce depression, suggesting that the autoinhibitory effect due to released noradrenaline is absent. At supramaximal stimulus strengths short, low frequency (0.1-0.5 Hz) trains gave varying degrees of depression of the first phase at each stimulus frequency. This depression was reversed by yohimbine (10 microM), indicating that the level of autoinhibition depends on the total amount of noradrenaline secreted. In the absence of autoinhibition, facilitation could be predicted in terms of the residual Ca2+ hypothesis.

Adrenergic alpha-2 Receptor Antagonists↗

PubMed1993

Evidence that each nerve varicosity on the surface of the mouse vas deferens secretes ATP.

The secretion of ATP from visualised varicosities on the surface of the mouse vas deferens was determined. Small diameter microelectrodes (about 6 microns and 20 microns) were placed over a set of 2 to 5 varicosities visualised with DiOC2(5) in order to record excitatory junctional currents (e.j.c.s') arising from the evoked secretion of quanta. In high external calcium concentrations, [Ca2+]o, the amplitude-frequency distribution of e.j.c.s' was well described by binomial statistics in which binomial parameter n and the number of varicosities were approximately the same. Idazoxan (1 microM) had no effect on the e.j.c.s' or the binomial parameters p and n. Suramin (100 microM) blocked the e.j.c.s' as well as the spontaneous e.j.c.s' (s.e.j.c.s') in all cases, reducing n and p to zero. These results indicate that all varicosities secrete the transmitter blocked by suramin, presumably ATP.

Adenosine Triphosphate↗

PubMed1992

The effect of adenosine on spontaneous and evoked quantal secretion from different release sites of amphibian motor-nerve terminals.

The effects of adenosine on the spontaneous quantal secretion from different release sites along terminal branches of toad (Bufo marinus) motor-nerve terminals was studied. Terminal branches were visualized using 3,3-diethyloxardicarbocyanine iodide (DiOC2(5))-fluorescence to assist in the placement of extracellular electrodes along different release sites of terminal branches. The maximum rate of spontaneous secretions (fe) observed with an extracellular electrode within any 10 microns length of terminal branch declined towards the distal end of the terminal branch as does the average number of evoked quantal secretions (mE). Adenosine (1-50 microM) depressed both fe and me. The ED50 of adenosine in depressing me was 5 microM. Adenosine (10 microM) produced on average a 43% decrease in fe and a 63% decrease in me, regardless of the initial values of fe and me. It is suggested that adenosine has qualitatively similar effects on both fe and me, regardless of their initial size, at different release sites within motor-nerve terminals.

Adenosine↗

PubMed1991

Probabilistic secretion of quanta from nerve terminals in toad (Bufo marinus) muscle modulated by adenosine.

1. A study has been made of the effect of blocking endogenous adenosine on the statistics of quantal secretion at nerve terminals in toad (Bufo marinus) muscle during summer and winter. 2. Exogenous adenosine (10-50 microM) reduces the mean quantal content of the endplate potential (EPP) recorded with an intracellular microelectrode (m) by 36 +/- 6% (mean +/- S.E.M.), independent of the control value of m in both summer and winter. The variance of the EPP (S2) was reduced by adenosine in proportion to m, so that the probability of quantal secretion (p) remained relatively constant. 3. Exogenous adenosine reduces the mean quantal content of secretion recorded with an extracellular electrode (m(e)) to a similar extent (47 +/- 6%) at different relatively high secreting sites along nerve terminal branches in both summer and winter. 4. Both theophylline (20-100 microM) and adenosine deaminase (2.5 i.u./ml) increase the amplitude of the EPP in summer in a [Ca2+]o of 0.5 mM or greater; a maximum increase of about 40% is reached at a [Ca2+]o of 1.2 mM. The amplitude of the EPP in winter is usually reduced by theophylline in a [Ca2+]o of 0.5 mM, but is always increased in a [Ca2+]o greater than 0.9 mM to reach a maximum increase of about 40% at high [Ca2+]o. 5. The variance of the EPP (S2) was always increased by theophylline to a greater extent than m in summer, so that p decreased and Poisson rather than binomial statistics could be used to describe the distribution of EPP amplitudes. In winter, theophylline generally increased m and S2 to about the same extent, so that p did not change much. 6. An autocorrelation analysis of the amplitude of successive EPPs in a long train at 0.5 Hz in high [Ca2+]o showed that these are likely to be independently distributed. Adenosine secreted by one impulse is then unlikely to affect secretion by a subsequent impulse in the train. 7. These observations are discussed in terms of the hypothesis that endogenously secreted adenosine at a release site inhibits secretion by nearby release sites in summer; this has the effect of reducing m and to a greater extent S2 so that binomial rather than Poisson statistics can describe the frequency distribution of EPP amplitudes.

Acetylcholine↗

PubMedDate not supplied

Strength of synaptic transmission at neuromuscular junctions of crustaceans and insects in relation to calcium entry.

Crustacean and insect neuromuscular junctions typically include numerous small synapses, each of which usually contains one or more active zones, which possess voltage-sensitive calcium channels and are specialized for release of synaptic vesicles. Strength of transmission (the number of quantal units released per synapse by a nerve impulse) varies greatly among different endings of individual neurons, and from one neuron to another. Ultrastructural features of synapses account for some of the physiological differences at endings of individual neurons. The nerve terminals that release more neurotransmitter per impulse have a higher incidence of synapses with more than one active zone, and this is correlated with more calcium build-up during stimulation. However, comparison of synaptic structure in neurons with different physiological phenotypes indicates no major differences in structure that could account for their different levels of neurotransmitter release per impulse, and release per synapse differs among neurons despite similar calcium build-up in their terminals during stimulation. The evidence indicates differences in calcium sensitivity of the release process among neurons as an aspect of physiological specialization.

Animals↗