Maria Bykhovskaia

Submillimeter-wave Fourier transform spectroscopy of biological macromolecules

In this article we report experimental results on Fourier-transform infrared spectroscopy of deoxyribonucleic acid (DNA) macromolecules and related biological materials in the submillimeter range (i.e., ∼10–500 cm−1). Film samples made from commercial DNA fibers, polyadenylic acid potassium salt, and cellular agents such as the spore form of Bacillus subtillis have been prepared and measured. A broad series of measurements carried out in the low frequency region (10–50 cm−1) with a higher resolution of 0.2 cm−1 revealed fine features—multiple dielectric resonances in the submillimeter-wave spectra obtained from DNA samples. These long-wave absorption features are shown to be intrinsic properties of biological materials determined by phonon modes. The emphasis is on reproducibility of experimental spectra and on receiving reliable results. The effects of differences in sample preparation, including sample geometry, orientation, and aging are studied and separated from the phonon effects that determine the ...

Evaluation of quantal neurosecretion from evoked and miniature postsynaptic responses by deconvolution method

A new deconvolution algorithm has been developed for evaluation of quantal content and its variability at high-output synapses. The algorithm derives the distribution of the number of neurosecretory quanta released in a trial (M) from the measured sizes of evoked postsynaptic responses. The deconvolution employs the distribution of quantal sizes obtained by measuring sizes of miniature postsynaptic responses. The distribution of quantal content M is derived by ridge regression method from the distributions of sizes of the responses and of quantal sizes. The deconvolution method was applied to postsynaptic responses from the excitory innervation of lobster dactyl opener muscle obtained by focal extracellular recordings. The obtained solution (distribution of M) had six to eight components and was stable. The method was tested by the analysis of simulated multiquantal responses. For the simulated responses, the ridge regression solution reproduced the imposed distribution of M within the limits of the calculated confidence intervals. To further test the algorithm, the distribution of M at a low-output synapse was obtained both by deconvolution method and by the method of direct quantal counts. The results of these two methods were found to be in a very good agreement.

An algorithm for high-resolution detection of postsynaptic quantal events in extracellular records

A software package has been developed for the detection and measurement of extracellularly recorded postsynaptic quantal events evoked by neural stimulation. The algorithm is based on the identification of monotonic regions of a differentiated current signal and detects the small inflections and peaks of a postsynaptic response that result from the asynchronous presynaptic release of individual packets of neurotransmitter. Recorded and simulated postsynaptic responses have been used to verify the accuracy of the algorithm and to determine its resolution. The algorithm can accurately detect up to six individual quanta in a time period of 20 ms, with a resolution of 0.5-1.0 ms.

Hyperosmolarity reduces facilitation by a Ca2+ -independent mechanism at the lobster neuromuscular junction: possible depletion of the releasable pool

1 At the crustacean neuromuscular junction, action potential‐evoked neurosecretion increases in proportion to stimulation frequency, a process termed frequency facilitation. In the present study we examined how frequency facilitation is affected by osmotic pressure. 2 Hypertonic solution (HS) was applied by local superfusion of the synaptic area. Quantal release was monitored by focal extracellular recordings of postsynaptic potentials. Several stimulation frequencies (f) in the range from 1 to 10 Hz were employed, and quantal content (m) together with the number of releasable units (n) and release probability (p) was evaluated for each frequency. 3 Osmotic pressure enhanced quantal release at the lowest f tested (1 Hz) but suppressed neurosecretion at higher f (7‐10 Hz). Thus, hyperosmolarity enhanced action potential‐evoked release but suppressed frequency facilitation. 4 Chelation of intracellular calcium by BAPTA showed that the effect of HS was calcium independent. 5 Binomial analysis of quantal content revealed that HS suppressed the increase in the number of releasable units, which was very pronounced during facilitation under control conditions. Since HS also stimulated asynchronous quantal release, the observed effect of HS on facilitation can be explained by the depletion of the releasable pool of quanta caused by the asynchronous neurosecretion. 6 To test this hypothesis we increased the available pool of vesicles using serotonin and demonstrated that the suppressing effect of HS on facilitation was reversed. 7 The observed effects of HS on facilitated neurosecretion could be described quantitatively using our model for mobilization of vesicles into the releasable pool enhanced by action potentials.

Far-infrared phonon modes of selected RNA molecules

Low-frequency phonon modes of DNA and RNA molecules can serve as a signature of their structure, flexibility and, hence, their biological function. To investigate the relationship between RNA structure and far IR absorption spectra, we performed FTIR measurements on RNA molecules with known sequence in the spectral range from 10 cm-1 to 25 cm-1 and calculated their internal vibrations. To understand which phonon modes are determined by a double helical topology of nucleic acids, we compared the spectra of single stranded and double stranded RNA molecules. Homopolymers PolyA, polyU, polyC, and polyG, and double stranded homopolymers PolyA-polyU and polyC-polyG were investigated. Theoretical conformational analysis of the double stranded RNA molecules was performed and utilized to calculate the low-frequency vibrational modes. Conformational energy was minimized in the space of internal coordinates of a molecule using standard A-helical topology as an initial approximation. Normal modes were calculated as eigenfrequencies and eigenvectors of the matrix of energy second derivatives. Oscillator strengths were calculated for all the vibrational modes in order to evaluate their weight in the absorption spectrum of a molecule. The obtained phonon modes were convoluted to derive the far IR spectrum of a molecule. These predicted spectra were compared to those obtained by FTIR spectroscopy. Our results confirm that very far IR absorption spectra of biopolymers reflect specific dynamical properties resulting from their structure and topology and, therefore, can be used as fingerprints for specific molecules.

Stochastic Modeling of Facilitated Neurosecretion

Two models of neurosecretion were evaluated in terms of their ability to predict the dependency of quantal content (m) on the frequency of repetitive stimulation of a lobster motoneuron. First, the hypothesis that neurosecretion is limited by a fixed number of release sites was tested by the fit of the distribution of m by uniform and nonuniform binomial statistics. The obtained release probabilities suggest that frequency facilitation can be due to activation of a group of sites with high release probabilities. However, the fit obtained using this model is not statistically significant due to a large number of fitting parameters. Second, the hypothesis that neurosecretion is limited by the rates of exchange between the releasable pool and the total store of quanta and that each stimulus enhances quantal mobilization was tested. Monte Carlo simulation was carried out in accordance with this model and reproduced the observed distribution of m with very few fitting parameters and therefore with a high level of significance (>0.1). This result demonstrates that mobilization of extra vesicles with each stimulus is a mechanism that allows a very accurate and parsimonious quantitative description of frequency facilitation.

Effect of Ca2+ on presynaptic facilitation

Abstract We investigated and modeled the effect of Ca2+ on presynaptic facilitation. Facilitation of transmitter release from a presynaptic terminal is believed to arise from the local increase of Ca2+ concentration. We measured and simulated the effect of Ca2+ on facilitation at the lobster neuromuscular synapse. Facilitation was measured using quantal analysis of postsynaptic potentials recorded focally during trains of stimuli. We found a facilitation component, which was not affected by Ca2+ concentration. Transmitter release was simulated using a model of two pools of presynaptic vesicles available for release. The simulation reproduced quantitatively Ca2+-dependent and Ca2+-independent components of facilitation.

Presynaptic facilitation: Quantal analysis and simulations ☆

Abstract The time-course of quantal neurosecretion indicated asynchrony in releases of presynaptic vesicles (quanta) in response to a stimulus. This was interpreted as reflecting the sequential release of vesicles from a single release site. We performed Monte-Carlo simulation of facilitated neurosecretion based upon the hypothesis that release sites do not limit transmitter release. The output of this simulation succeeded in reproducing the experimentally obtained distribution of quantal releases. These results support a model for neurosecretion in which each action potential evokes mobilization of synaptic vesicles to a single presynaptic release site followed by probabilistic secretion of releasable vesicles one after another.