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The origin of the skewed amplitude distribution of spontaneous excitatory junction potentials in poorly coupled smooth muscle cells.

Young JS, Brain KL, Cunnane TC - Neuroscience (2007)

Bottom Line: Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001).The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC.The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. john.young@pharm.ox.ac.uk

ABSTRACT
The skewed amplitude distribution of spontaneous excitatory junction potentials (sEJPs) in the mouse vas deferens and other electrically-coupled smooth muscle syncytia has been attributed to electrically-attenuated depolarizations resulting from the spontaneous release of quantized packets of ATP acting on remote smooth muscle cells (SMCs). However, in the present investigation surface SMCs of the mouse isolated vas deferens were poorly electrically coupled, with input resistances (176+/-18 MOmega, range: 141-221 MOmega, n=4) similar to those of dissociated cells. Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001). Using simultaneous electrophysiology and confocal microscopy to investigate these poorly-coupled cells, it is shown that alpha-latrotoxin-stimulated sEJPs correlate, in timing (median delay ranged from -30 to -57 ms, P<0.05 in all experiments, n=5) and amplitude (Pearson product moment correlation, rho>0.55 and P<0.001), with purinergic neuroeffector Ca2+ transients (NCTs) in SMCs. The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC. The amplitude correlation of sEJPs and NCTs argues against the attenuation of electrical signal amplitude along the length of a single SMC. The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.

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Amplitudes of sEJPs and NCTs are positively correlated (Pearson product moment correlation, P<0.001 for each of n=4 experiments, unbinned data). The amplitude correlation between sEJPs and NCTs, where both events could be simultaneously measured, suggests that they arise from the same event and that the variability in sEJP amplitude represents variability in the local action of ATP. The amplitudes were normalized to a percentage of the maximum value in each experiment. NCT amplitudes were binned and the mean amplitude of coincident sEJPs for each bin was plotted. sEJP amplitudes are mean±S.E.M. for each preparation. Lines were fitted to the binned data; the solid lines relate to the filled symbols and the dashed lines to the open symbols.
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fig7: Amplitudes of sEJPs and NCTs are positively correlated (Pearson product moment correlation, P<0.001 for each of n=4 experiments, unbinned data). The amplitude correlation between sEJPs and NCTs, where both events could be simultaneously measured, suggests that they arise from the same event and that the variability in sEJP amplitude represents variability in the local action of ATP. The amplitudes were normalized to a percentage of the maximum value in each experiment. NCT amplitudes were binned and the mean amplitude of coincident sEJPs for each bin was plotted. sEJP amplitudes are mean±S.E.M. for each preparation. Lines were fitted to the binned data; the solid lines relate to the filled symbols and the dashed lines to the open symbols.

Mentions: The quantal basis of neurotransmission at the skeletal neuromuscular junction was identified by del Castillo and Katz (1954), in their reanalysis of experiments by Fatt and Katz (1952). They concluded that “transmission at a nerve-muscle junction takes place in all-or-none ‘quanta’ whose sizes are indicated by the spontaneously occurring miniature (end-plate potential) discharges.” The narrow, Gaussian amplitude distribution of the miniature end-plate potentials (their Fig. 7), the invariance of this distribution as the Ca2+ concentration changed and the existence of a multimodal histogram of excitatory postjunctional potentials imply that evoked neurotransmitter release causes integer multiples of a consistent unitary electrical response. The distribution of packet sizes released from sympathetic postganglionic terminals has been much harder to study because the electrical syncytium that arises through the coupling of smooth muscle cells (SMCs) makes it difficult to distinguish local from distant neurotransmitter release sites during intracellular recording. To tackle this problem, several different approaches have been tried. By differentiating the excitatory junction potential (EJP) it is possible to identify intermittent ‘discrete events’ buried within the depolarization phase (Blakeley and Cunnane, 1979), which indicate the intermittent release of neurotransmitter packets. Some of these discrete events had sizes comparable to the amplitude of the derivative of spontaneous excitatory junction potentials (sEJPs), which argues that the same types of packets contribute to both events, but does not indicate that evoked release is an integral multiple of unitary packets. A further approach is to measure local extracellular potential changes arising following both spontaneous and evoked neurotransmitter release: the excitatory junction currents (sEJCs and EJCs; Brock and Cunnane, 1987). This approach demonstrates intermittent neurotransmitter release with a broad packet size. A development of this approach to study visualized varicosities under conditions of low release probability with small electrode tips (Macleod et al., 1994) demonstrated that, at 1 mM Ca2+, the truncated amplitude distribution of EJPs was broad (their Fig. 3B), arguing against packets of uniform size; that packet size was not uniform was implicit in the model they used to fit the results at higher external Ca2+ concentrations, where the basic packet size varied as a gamma variate (Robinson, 1976). Further evidence for a broad amplitude distribution of the fundamental packet size at the autonomic neuroeffector junction has come from the use of laser-scanning confocal microscopy to image purinergic neuroeffector Ca2+ transients (NCTs) on an impulse-to-impulse basis at individual neuroeffector junctions (Brain et al., 2002, 2003). NCTs arise when intermittently-released packets of ATP activate local P2X1 receptors, causing a local increase in smooth muscle Ca2+ concentration. The finding that these local Ca2+ transients are amplified by Ca2+-induced Ca2+ release in the SMC, however, means that the broad amplitude distribution of NCTs at a single junction cannot be used to imply a broad distribution of basic packets size (Brain et al., 2003).


The origin of the skewed amplitude distribution of spontaneous excitatory junction potentials in poorly coupled smooth muscle cells.

Young JS, Brain KL, Cunnane TC - Neuroscience (2007)

Amplitudes of sEJPs and NCTs are positively correlated (Pearson product moment correlation, P<0.001 for each of n=4 experiments, unbinned data). The amplitude correlation between sEJPs and NCTs, where both events could be simultaneously measured, suggests that they arise from the same event and that the variability in sEJP amplitude represents variability in the local action of ATP. The amplitudes were normalized to a percentage of the maximum value in each experiment. NCT amplitudes were binned and the mean amplitude of coincident sEJPs for each bin was plotted. sEJP amplitudes are mean±S.E.M. for each preparation. Lines were fitted to the binned data; the solid lines relate to the filled symbols and the dashed lines to the open symbols.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543106&req=5

fig7: Amplitudes of sEJPs and NCTs are positively correlated (Pearson product moment correlation, P<0.001 for each of n=4 experiments, unbinned data). The amplitude correlation between sEJPs and NCTs, where both events could be simultaneously measured, suggests that they arise from the same event and that the variability in sEJP amplitude represents variability in the local action of ATP. The amplitudes were normalized to a percentage of the maximum value in each experiment. NCT amplitudes were binned and the mean amplitude of coincident sEJPs for each bin was plotted. sEJP amplitudes are mean±S.E.M. for each preparation. Lines were fitted to the binned data; the solid lines relate to the filled symbols and the dashed lines to the open symbols.
Mentions: The quantal basis of neurotransmission at the skeletal neuromuscular junction was identified by del Castillo and Katz (1954), in their reanalysis of experiments by Fatt and Katz (1952). They concluded that “transmission at a nerve-muscle junction takes place in all-or-none ‘quanta’ whose sizes are indicated by the spontaneously occurring miniature (end-plate potential) discharges.” The narrow, Gaussian amplitude distribution of the miniature end-plate potentials (their Fig. 7), the invariance of this distribution as the Ca2+ concentration changed and the existence of a multimodal histogram of excitatory postjunctional potentials imply that evoked neurotransmitter release causes integer multiples of a consistent unitary electrical response. The distribution of packet sizes released from sympathetic postganglionic terminals has been much harder to study because the electrical syncytium that arises through the coupling of smooth muscle cells (SMCs) makes it difficult to distinguish local from distant neurotransmitter release sites during intracellular recording. To tackle this problem, several different approaches have been tried. By differentiating the excitatory junction potential (EJP) it is possible to identify intermittent ‘discrete events’ buried within the depolarization phase (Blakeley and Cunnane, 1979), which indicate the intermittent release of neurotransmitter packets. Some of these discrete events had sizes comparable to the amplitude of the derivative of spontaneous excitatory junction potentials (sEJPs), which argues that the same types of packets contribute to both events, but does not indicate that evoked release is an integral multiple of unitary packets. A further approach is to measure local extracellular potential changes arising following both spontaneous and evoked neurotransmitter release: the excitatory junction currents (sEJCs and EJCs; Brock and Cunnane, 1987). This approach demonstrates intermittent neurotransmitter release with a broad packet size. A development of this approach to study visualized varicosities under conditions of low release probability with small electrode tips (Macleod et al., 1994) demonstrated that, at 1 mM Ca2+, the truncated amplitude distribution of EJPs was broad (their Fig. 3B), arguing against packets of uniform size; that packet size was not uniform was implicit in the model they used to fit the results at higher external Ca2+ concentrations, where the basic packet size varied as a gamma variate (Robinson, 1976). Further evidence for a broad amplitude distribution of the fundamental packet size at the autonomic neuroeffector junction has come from the use of laser-scanning confocal microscopy to image purinergic neuroeffector Ca2+ transients (NCTs) on an impulse-to-impulse basis at individual neuroeffector junctions (Brain et al., 2002, 2003). NCTs arise when intermittently-released packets of ATP activate local P2X1 receptors, causing a local increase in smooth muscle Ca2+ concentration. The finding that these local Ca2+ transients are amplified by Ca2+-induced Ca2+ release in the SMC, however, means that the broad amplitude distribution of NCTs at a single junction cannot be used to imply a broad distribution of basic packets size (Brain et al., 2003).

Bottom Line: Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001).The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC.The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. john.young@pharm.ox.ac.uk

ABSTRACT
The skewed amplitude distribution of spontaneous excitatory junction potentials (sEJPs) in the mouse vas deferens and other electrically-coupled smooth muscle syncytia has been attributed to electrically-attenuated depolarizations resulting from the spontaneous release of quantized packets of ATP acting on remote smooth muscle cells (SMCs). However, in the present investigation surface SMCs of the mouse isolated vas deferens were poorly electrically coupled, with input resistances (176+/-18 MOmega, range: 141-221 MOmega, n=4) similar to those of dissociated cells. Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001). Using simultaneous electrophysiology and confocal microscopy to investigate these poorly-coupled cells, it is shown that alpha-latrotoxin-stimulated sEJPs correlate, in timing (median delay ranged from -30 to -57 ms, P<0.05 in all experiments, n=5) and amplitude (Pearson product moment correlation, rho>0.55 and P<0.001), with purinergic neuroeffector Ca2+ transients (NCTs) in SMCs. The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC. The amplitude correlation of sEJPs and NCTs argues against the attenuation of electrical signal amplitude along the length of a single SMC. The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.

Show MeSH
Related in: MedlinePlus