Rohit Manchanda

A computational model of urinary bladder smooth muscle syncytium

Certain smooth muscles, such as the detrusor of the urinary bladder, exhibit a variety of spikes that differ markedly in their amplitudes and time courses. The origin of this diversity is poorly understood but is often attributed to the syncytial nature of smooth muscle and its distributed innervation. In order to help clarify such issues, we present here a three-dimensional electrical model of syncytial smooth muscle developed using the compartmental modeling technique, with special reference to the bladder detrusor. Values of model parameters were sourced or derived from experimental data. The model was validated against various modes of stimulation employed experimentally and the results were found to accord with both theoretical predictions and experimental observations. Model outputs also satisfied criteria characteristic of electrical syncytia such as correlation between the spatial spread and temporal decay of electrotonic potentials as well as positively skewed amplitude frequency histogram for sub-threshold potentials, and lead to interesting conclusions. Based on analysis of syncytia of different sizes, it was found that a size of 21-cube may be considered the critical minimum size for an electrically infinite syncytium. Set against experimental results, we conjecture the existence of electrically sub-infinite bundles in the detrusor. Moreover, the absence of coincident activity between closely spaced cells potentially implies, counterintuitively, highly efficient electrical coupling between such cells. The model thus provides a heuristic platform for the interpretation of electrical activity in syncytial tissues.

Modulation of synaptic potentials and cell excitability by dendritic KIR and KAs channels in nucleus accumbens medium spiny neurons: A computational study

The nucleus accumbens (NAc), a critical structure of the brain reward circuit, is implicated in normal goal-directed behaviour and learning as well as pathological conditions like schizophrenia and addiction. Its major cellular substrates, the medium spiny (MS) neurons, possess a wide variety of dendritic active conductances that may modulate the excitatory post synaptic potentials (EPSPs) and cell excitability. We examine this issue using a biophysically detailed 189-compartment stylized model of the NAc MS neuron, incorporating all the known active conductances. We find that, of all the active channels, inward rectifying K+ (KIR) channels play the primary role in modulating the resting membrane potential (RMP) and EPSPs in the down-state of the neuron. Reduction in the conductance of KIR channels evokes facilitatory effects on EPSPs accompanied by rises in local input resistance and membrane time constant. At depolarized membrane potentials closer to up-state levels, the slowly inactivating A-type potassium channel (KAs) conductance also plays a strong role in determining synaptic potential parameters and cell excitability. We discuss the implications of our results for the regulation of accumbal MS neuron biophysics and synaptic integration by intrinsic factors and extrinsic agents such as dopamine.

Post‐ and prejunctional consequences of ecto‐ATPase inhibition: electrical and contractile studies in guinea‐pig vas deferens

At sites of purinergic neurotransmission, synaptic ecto‐ATPase is believed to limit the actions of ATP following its neural release. However, details of the modulation by this enzyme of the ATP‐mediated conductance change and the possible mechanisms mediating this modulation remain unelucidated. We have addressed these issues by studying the effect of ARL 67156, a selective ecto‐ATPase inhibitor, on ATP‐mediated electrical and contractile activity in the sympathetically innervated guinea‐pig vas deferens. ARL 67156 at 100 μm significantly potentiated the amplitude of spontaneous excitatory junction potentials (SEJPs) by 81.1% (P < 0.01) and prolonged their time courses (rise time by 49.7%, decay time constant by 38.2%; P < 0.01). Moreover, the frequency of occurrence of SEJPs was strikingly increased (from 0.28 ± 0.13 to 0.90 ± 0.26 Hz; P < 0.01), indicating an additional, primarily presynaptic, effect of ecto‐ATPase inhibition. The frequency of occurrence of discrete events (DEs), which represent nerve stimulation‐evoked quantal release of neurotransmitter, was also increased (∼6‐fold; P < 0.01), along with the appearance of DEs at previously ‘silent’ latencies. Purinergic contractions of the vas deferens were potentiated significantly (P < 0.01) by ARL 67156; these potentiated contractions were suppressed by the A1 agonist adenosine (P < 0.01) but left unaffected by the A1 antagonist 8‐phenyltheophylline (8‐PT). Our results indicate (i) that ecto‐ATPase activity, in addition to modulating the ATP‐mediated postjunctional conductance change, may regulate transmitter release prejunctionally under physiological conditions, and (ii) that the prejunctional regulation may be mediated primarily via presynaptic P2X, rather than A1, receptors.

QUANTAL EVOKED DEPOLARIZATIONS UNDERLYING THE EXCITATORY JUNCTION POTENTIAL OF THE GUINEA-PIG ISOLATED VAS DEFERENS

1 The effects of a putative gap junction uncoupling agent, heptanol, on the intracellularly recorded junction potentials of the guinea‐pig isolated vas deferens have been investigated. 2 After the stimulation‐evoked excitatory junction potentials (EJPs) had been suppressed by heptanol (2.0 mm) to undetectable levels, a different pattern of evoked activity ensued. This consisted of transient depolarizations that were similar to EJPs in being stimulus locked and in occurring at a fixed latency, but differed from EJPs in that they occurred intermittently and had considerably briefer time courses. 3 Analysis of the amplitudes and temporal parameters of the rapid residual depolarizations revealed a close similarity with spontaneous EJPs (SEJPs). There was no statistically significant difference between the rise times, time constants of decay and durations of the rapid residual depolarizations and of SEJPs. 4 Selected evoked depolarizations were virtually identical to SEJPs occurring in the same cell. Evoked depolarizations of closely similar amplitude and time course also occurred, usually within a few stimuli of each other. 5 These depolarizations appear to represent the individual quantal depolarizations that normally underlie the EJP and are therefore termed ‘quantal excitatory junction potentials’ (QEJPs) to distinguish them from both EJPs and SEJPs. 6 We examined the possibility that heptanol revealed QEJPs by disrupting electrical coupling between cells in the smooth muscle syncytium. Heptanol (2.0 mm) had no effect on the amplitude distribution, time courses, or the frequency of occurrence of SEJPs. Intracellular input impedance (Rin) of smooth muscle cells was left unaltered by heptanol. 7 ‘Cable’ potentials of the vas deferens, recorded using the partition stimulation method, also remained unchanged in the presence of heptanol. Thus, heptanol appeared not to modify syncytial electrical properties of the smooth muscle in any significant way. 8 Our observations show directly that the quantal depolarizations underlying the EJP in syncytial smooth muscle are SEJP‐like events. However, no unequivocal statement can be made about the mechanism by which heptanol unmasks QEJPs from EJPs.

Effects of cooling and ARL 67156 on synaptic ecto-ATPase activity in guinea pig and mouse vas deferens

We have studied the influence of temperature and ARL 67156 on ATP hydrolysis in mouse and guinea pig vas deferens in order to explore the properties of the enzymatic inactivation mechanism proposed to regulate purinergic neurotransmission at the sympathetic neuromuscular junction of smooth muscle. The ectonucleotidase activity was determined by using the malachite green method to measure the inorganic phosphate (Pi) liberated with ATP used as a substrate. ATP hydrolysis in both species was found to be insensitive to ouabain (100 microM), sodium azide (1 mM), sodium vanadate (100 microM) and beta-glycerophosphate (10 mM) and was also found to depend on Ca2+ and Mg2+. V(MAX) of the ectonucleotidase activity for guinea pig and mouse vas deferens was 958.4+/-66.3 and 79.7+/-8.5 pmol/min/mg, while K(M) was 625.1+/-45.2 and 406.0+/-29.0 microM, respectively. Cooling the tissues from 35 to 25 degrees C reduced the enzyme activity significantly (P<0.01) by 52.7+/-9.2% in guinea pig vas deferens and 34.9+/-5.3% in mouse vas deferens. ARL 67156 (100 microM), the specific ecto-ATPase inhibitor, caused a reduction in enzyme activity in guinea pig and mouse vas of 54.1+/-16.4% and 53.0+/-7.6%, respectively (P<0.01). The degree of inhibition of ATP hydrolysis by lowered temperature and 100 microM ARL 67156 correlates well with the reported potentiation and prolongation of junction potentials under these conditions. It is concluded that ecto-ATPase or a closely related ectonucleotidase plays an important role in the physiological regulation of purinergic neurotransmission.

Classification and analysis of electrical signals in urinary bladder smooth muscle using a modified vector quantization technique

Voiding of the urinary bladder depends on contraction of the smooth muscle of its wall, known as detrusor muscle, and this in turn relies on electrical signals generated in the muscle cells. The exact shapes of these signals contain important information about bladder biophysics, but are poorly understood. We present an analysis of detrusor signals using a vector quantization technique based on a modified k-means clustering algorithm for the automatic detection and classification of the signals. We find that our procedure is able to sort the signals from a mixed pool into three predefined classes with an overall sensitivity of 0.9 and a specificity of 0.97. The various features of the signals belonging to an example class are evaluated for inter-feature correlation, and these correlations appear to be consistent with certain hypotheses about the mechanism of generation of the signals. Our work offers a novel approach to analyzing intracellularly recorded signals and inferring muscle biophysics at the cellular level.

Influence of the size of syncytial units on synaptic potentials in smooth muscle

The effect of the size of syncytial bundles of cells on the passive synaptic potentials generated within them has been explored. Computer simulations have been performed of neuronally produced spontaneous excitatory junction potentials (SEJPs) generated in a cubical ‘bidomain’ model of syncytial tissue. It is found that indical properties of SEJPs vary conspicuously in syncytium sizes smaller than about 15–17-cube, but change very little in syncytium sizes greater than this. At the centroid of the cube, the peak amplitude Vp of the SEJP declines from 14.32 mV to 11.70 mV as syncytium size increases from 7-cube to 15-cube, i.e. a decrease of ∼18%, while between system sizes 15-cube (Vp=11.70 mV) and 29-cube (Vp=11.6/ mV), the reduction is only ∼0.3%. Similar trends are observed for the time to peak of SEJPs. These observations indicate a minimum bundle size in smooth muscle below which syncytial function is modified; the implications of this are discussed.

Syncytial Basis for Diversity in Spike Shapes and their Propagation in Detrusor Smooth Muscle

Syncytial tissues, such as the smooth muscle of the urinary bladder wall, are known to produce action potentials (spikes) with marked differences in their shapes and sizes. The need for this diversity is currently unknown, and neither is their origin understood. The small size of the cells, their syncytial arrangement, and the complex nature of innervation poses significant challenges for the experimental investigation of such tissues. To obtain better insight, we present here a three-dimensional electrical model of smooth muscle syncytium, developed using the compartmental modeling technique, with each cell possessing active channel mechanisms capable of producing an action potential. This enables investigation of the syncytial effect on action potential shapes and their propagation. We show how a single spike shape could undergo modulation, resulting in diverse shapes, owing to the syncytial nature of the tissue. Differences in the action potential features could impact their capacity to propagate through a syncytium. This is illustrated through comparison of two distinct action potential mechanisms. A better understanding of the origin of the various spike shapes would have significant implications in pathology, assisting in evaluating the underlying cause and directing their treatment.

Computational studies on bladder small dorsal root ganglion neurons: Modelling BK channels.

The urinary bladder afferent neurons called the dorsal root ganglion (DRG) neurons carry information on diverse modalities such as stretch, pressure and nociception to the spinal cord. This information is carried in the form of electrical activity called action potentials (AP). The bladder small diameter DRG neurons that are considered to be putative nociceptors express several ion channels and active mechanisms which are responsible for generating this electrical activity. One of the channels that has been suggested to play a role in cell excitability is the large conductance calcium activated potassium channel (BK) channel. Its activation is governed by cell membrane potential and intracellular calcium concentration. Here, we present a computational model of the BK channel along with other ion channels and mechanisms present in the bladder small DRG neuron cell body. The BK channel simulations show properties that are similar to those shown by Isolectin B4 (IB4) negative cutaneous small DRG neurons. The bladder small DRG neurons have also been found to show some of these properties. Thus, we hypothesize that the bladder small DRG neurons are IB4 negative. This hypothesis is supported by experimental studies which suggest that about 80% of bladder small DRG neurons are IB4 negative. The model of bladder small DRG neuron also faithfully reproduced some of the electrical properties that have been reported experimentally. This model can thus be used to predict abnormal behaviour of the DRG neuron during pathological conditions.

A mathematical model of the calcium transient in urinary bladder smooth muscle cells

An increase in cytoplasmic calcium (Ca²⁺) concentration ([Ca²⁺]_i) is a prerequisite for the contraction of detrusor smooth muscle (DSM) cells . The increase in [Ca²⁺]_i is accomplished by Ca²⁺ entry mainly via voltage dependent L-type Ca²⁺ channel and Ca²⁺ release from intracellular stores. We report here a simulation of the processes that regulate intracellular Ca²⁺ and their dependence on Ca²⁺ concentration. Based on experimentally recorded data, mathematical equations for Ca²⁺ current (generated mainly by L-type Ca²⁺ channel) are developed along with representation of Ca²⁺ATPase pump currents. The plasma membrane Ca²⁺ATPase (PMCA) pump and sarco/endoplasmic reticulum Ca²⁺ATPase (SERCA) pump are responsible for lowering [Ca²⁺]_i which leads to relaxation of smooth muscle. Our model simulates Ca²⁺ current, action potential and the Ca²⁺ transient response so as to reasonably mimic the experimental recordings. In novel findings, currents produced by PMCA and SERCA along with their amplitude and waveform pattern under voltage clamp condition have been predicted for DSM cells. The model has further been used to produce the Ca²⁺ transient which results because of L-type Ca²⁺ channel, Ca²⁺ release from intracellular store, PMCA, SERCA and presence of buffer in the cytoplasm. To explore the model further, Ca²⁺ transient decay rate in control condition is compared to the decay rate reached when PMCA and SERCA are inhibited. We conclude that this model can be used to describe the Ca²⁺ transient response produced by the DSM cell in response to depolarization of cell membrane.