Microinjection Technique for Assessment of Gap Junction Function.

Gap junctions are essential for the proper function of many native mammalian tissues including neurons, cardiomyocytes, embryonic tissues, and muscle. Assessing these channels is therefore fundamental to understanding disease pathophysiology, developing therapies for a multitude of acquired and genetic conditions, and providing novel approaches to drug delivery and cellular communication. Microinjection is a robust, albeit difficult, technique, which provides considerable information that is superior to many of the simpler techniques due to its ability to isolate cells, quantify kinetics, and allow cross-comparison of multiple cell lines. Despite its user-dependent nature, the strengths of the technique are considerable and with the advent of new, automation technologies may improve further. This text describes the basic technique of microinjection and briefly discusses modern automation advances that can improve the success rates of this technique.

Phase transition in the economically modeled growth of a cellular nervous system.

Spatially embedded complex networks, such as nervous systems, the Internet, and transportation networks, generally have nontrivial topological patterns of connections combined with nearly minimal wiring costs. However, the growth rules shaping these economical tradeoffs between cost and topology are not well understood. Here, we study the cellular nervous system of the nematode worm Caenorhabditis elegans, together with information on the birth times of neurons and on their spatial locations. We find that the growth of this network undergoes a transition from an accelerated to a constant increase in the number of links (synaptic connections) as a function of the number of nodes (neurons). The time of this phase transition coincides closely with the observed moment of hatching, when development switches metamorphically from oval to larval stages. We use graph analysis and generative modeling to show that the transition between different growth regimes, as well as its coincidence with the moment of hatching, may be explained by a dynamic economical model that incorporates a tradeoff between topology and cost that is continuously negotiated and renegotiated over developmental time. As the body of the animal progressively elongates, the cost of longer-distance connections is increasingly penalized. This growth process regenerates many aspects of the adult nervous system's organization, including the neuronal membership of anatomically predefined ganglia. We expect that similar economical principles may be found in the development of other biological or manmade spatially embedded complex systems.

Direct assessment of tubuloglomerular feedback responsiveness in connexin 40-deficient mice.

Participation of connexin 40 (Cx40) in the regulation of renin secretion and in the tubuloglomerular feedback (TGF) component of renal autoregulation suggests that gap junctional coupling through Cx40 contributes to the function of the juxtaglomerular apparatus. In the present experiments, we determined the effect of targeted Cx40 deletion in C57BL/6 and FVB mice on TGF responsiveness. In C57BL/6 mice, stop-flow pressure (PSF) fell from 40.3 ± 2 to 34.5 ± 2 mmHg in wild-type (WT) and from 31 ± 1.06 to 26.6 ± 0.98 mmHg in Cx40-/- mice. PSF changes of 5.85 ± 0.67 mmHg in WT and of 4.3 ± 0.55 mmHg in Cx40-/- mice were not significantly different (P = 0.08). In FVB mice, PSF fell from 37.4 ± 1.5 to 31.6 ± 1.5 mmHg in WT and from 28.1 ± 1.6 to 25.4 ± 1.7 mmHg in Cx40-/-, with mean TGF responses being significantly greater in WT than Cx40-/- (5.5 ± 0.55 vs. 2.7 ± 0.84 mmHg; P = 0.002). In both genetic backgrounds, PSF values were significantly lower in Cx40-/- than WT mice at all flow rates. Arterial blood pressure in the animals prepared for micropuncture was not different between WT and Cx40-/- mice. We conclude that the TGF response magnitude in superficial cortical nephrons is reduced by 30-50% in mice without Cx40, but that with the exception of a small number of nephrons, residual TGF activity is maintained. Thus gap junctional coupling appears to modulate TGF, perhaps by determining the kinetics of signal transmission.

Simulations of complex and microscopic models of cardiac electrophysiology powered by multi-GPU platforms.

Key aspects of cardiac electrophysiology, such as slow conduction, conduction block, and saltatory effects have been the research topic of many studies since they are strongly related to cardiac arrhythmia, reentry, fibrillation, or defibrillation. However, to reproduce these phenomena the numerical models need to use subcellular discretization for the solution of the PDEs and nonuniform, heterogeneous tissue electric conductivity. Due to the high computational costs of simulations that reproduce the fine microstructure of cardiac tissue, previous studies have considered tissue experiments of small or moderate sizes and used simple cardiac cell models. In this paper, we develop a cardiac electrophysiology model that captures the microstructure of cardiac tissue by using a very fine spatial discretization (8 µm) and uses a very modern and complex cell model based on Markov chains for the characterization of ion channel's structure and dynamics. To cope with the computational challenges, the model was parallelized using a hybrid approach: cluster computing and GPGPUs (general-purpose computing on graphics processing units). Our parallel implementation of this model using a multi-GPU platform was able to reduce the execution times of the simulations from more than 6 days (on a single processor) to 21 minutes (on a small 8-node cluster equipped with 16 GPUs, i.e., 2 GPUs per node).

Assessment of gap junctional intercellular communication.

Gap junctions are important plasma membrane organelles through which most cells in normal tissues communicate with each other. They exist in two neighboring cells and each cell contributes half of the structure. One gap junction consists of two hexameric connexons that dock with each other to create a channel. Six of the basic subunits referred to as connexins form a connexon. Less than one hundred to several thousand gap junction channels cluster together in the plane of the membrane. The gap junction channels serve as a regulated conduit for the intercellular exchange of small molecules. Maintenance of the integrity of gap junctional intercellular communication (GJIC) is important and required for normal electrical coupling, homeostasis, and embryogenesis. Aberrations of gap junctions have been related to human diseases such as cancer, cardiac arrhythmia, Charcot-Marie-tooth disease, and visceroatrial heterotaxia syndrome. This unit describes methods for measuring gap junctional intercellular communication using primary mouse hepatocytes as a model. Focus is only on functional evaluation based on dye coupling. Other methods, such as intracellular calcium waves and dual patch clamp, have been used to measure gap junctional communication, but these are not described in this unit.

Cells coupled by voltage-dependent gap junctions: the asymptotic dynamical limit.

We study the steady state and dynamical properties of a pair of cells coupled by a voltage-dependent gap junction. The cells have linear membrane properties, and the gap junction is modelled using a simple Markov chain with a voltage-dependent transition matrix. We first show that the voltage-independent case is globally convergent using energy dissipation as a Lyapunov function for the cells, and standard results on the convergence of homogeneous Markov chains for the junction. For the voltage-dependent case, we use the difference in cell and gap junction time scales to reduce the coupled equations for cells and the gap junction to a single equation for the gap junction, but with a transition matrix that depends upon the current gap junction state. We identify cooperativity as key property behind the global convergence of Markov chains and investigate convergence of the voltage-dependent system by establishing some conditions under which cooperativity is preserved.

Gap junctional intercellular communication is not a major mediator in the bystander effect in photodynamic treatment of MDCK II cells.

Photodynamic treatment (PDT) of confluent MDCK II cells resulted in a noticeable clustering of dead cells, consistent with a significant bystander effect. Likewise, PDT of cells in microcolonies resulted in an overabundance of microcolonies that had responded to the treatment as a single unit, that is, in which either all or no cells were dead. Confluent MDCK II cells appeared to communicate via gap junction channels, while cells in microcolonies did not. Monte Carlo simulation models were fitted to the distributions of dead cells in confluent monolayers and in microcolonies. The simulations showed that the degree of the bystander effect was higher in microcolonies than in confluent cells, suggesting that gap junction communication may be involved in the bystander effect. However, when the gap junction hypothesis was tested by treatment of microcolonies with 30 microM dieldrin, an inhibitor of gap junctional intercellular communication, there was no reduction of the bystander effect, indicating that this effect was not mediated by gap junctional intercellular communication. PDT influenced phosphorylation of tyrosine residues in several proteins in the cells. Protein phosphorylation is important in cellular signaling pathways and may be involved in the bystander effect, for example by influencing the mode of cell death.

The biological conditions of assessment of ethylene glycol-induced inhibition of gap junctional intercellular communication by metabolic cooperation assay.

Ethylene glycol (EG) has been previously shown to inhibit gap junctional intercellular communication. In this paper we examine conditions under which the effect of EG on gap junctional communication is assessed by metabolic cooperation assay. The later, after the start of metabolic cooperation assay, EG was added, the lower its inhibitory effect was. If treatment with EG began 12 h or even later after plating of cells, no significant effect on gap junctional communication was observed. Short EG treatments (2-8 h) induced a reversible inhibition of cell-to-cell communication, provided that the cells could communicate freely after the drug was removed. However, if further cell-to-cell communication was excluded, the effect of short exposures was irreversible. Using Scrape loading method we observed that after a 60 min exposure to EG the standard gap junctional intercellular communication was completely restored in a few hours.

Description of interacting channel gating using a stochastic Markovian model.

Single-channel recordings from membrane patches frequently exhibit multiple conductance levels. In some preparations, the steady-state probabilities of observing these levels do not follow a binomial distribution. This behavior has been reported in sodium channels, potassium channels, acetylcholine receptor channels and gap junction channels. A non-binomial distribution suggests interaction of the channels or the presence of channels or the presence of channels with different open probabilities. However, the current trace sometimes exhibits single transitions spanning several levels. Since the probability of simultaneous transitions of independent channels is infinitesimally small, such observations strongly suggest a cooperative gating behavior. We present a Markov model to describe the cooperative gating of channels using only the all-points current amplitude histograms for the probability of observing the various conductance levels. We investigate the steady-state (or equilibrium) properties of a system of N channels and provide a scheme to express all the probabilities in terms of just two parameters. The main feature of our model is that lateral interaction of channels gives rise to cooperative gating. Another useful feature is the introduction of the language of graph theory which can potentially provide a different avenue to study ion channel kinetics. We write down explicit expressions for systems of two, three and four channels and provide a procedure to describe the system of N channels.