Identification of Functionally Interconnected Neurons Using Factor Analysis.

The advances in electrophysiological methods have allowed registering the joint activity of single neurons. Thus, studies on functional dynamics of complex-valued neural networks and its information processing mechanism have been conducted. Particularly, the methods for identifying neuronal interconnections are in increasing demand in the area of neurosciences. Here, we proposed a factor analysis to identify functional interconnections among neurons via spike trains. This method was evaluated using simulations of neural discharges from different interconnections schemes. The results have revealed that the proposed method not only allows detecting neural interconnections but will also allow detecting the presence of presynaptic neurons without the need of the recording of them.

Electrophysiology for biomedical engineering students: a practical and theoretical course in animal electrocorticography.

The major challenge in laboratory teaching is the application of abstract concepts in simple and direct practical lessons. However, students rarely have the opportunity to participate in a laboratory that combines practical learning with a realistic research experience. In the Biomedical Engineering career, we offer short and optional courses to complement studies for students as they initiate their Graduation Project. The objective of these theoretical and practical courses is to introduce students to the topics of their projects. The present work describes an experience in electrophysiology to teach undergraduate students how to extract cortical information using electrocorticographic techniques. Students actively participate in some parts of the experience and then process and analyze the data obtained with different signal processing tools. In postlaboratory evaluations, students described the course as an exceptional opportunity for students interested in following a postgraduate science program and fully appreciated their contents.

Functional specificity of rat vibrissal primary afferents.

In this study, we propose to analyze the peripheral vibrissal system specificity through its neuronal responses. Receiver operating characteristics (ROC) curve analyses were used, which required the implementation of a binary classifier (artificial neural network) trained to identify the applied stimulus. The training phase consisted of the observation of a predetermined amount of vibrissal sweeps on two surfaces of different texture and similar roughness. Our results suggest that the specificity of the peripheral vibrissal system easily permits the discrimination between perceived stimuli, quantified through neuronal responses, and that it can be evaluated through an ROC curve analysis. We found that such specificity makes a linear binary classifier capable of detecting differences between stimuli with five sweeps at most.

In Vivo evaluation of adipogenic induction in fibrous and honeycomb-structured atelocollagen scaffolds.

Nowadays, soft tissue restoration techniques are mainly focused on volume regeneration instead of function recovering. So far, autologous fat transplant has been the most popular method although its multiple reported problems like volume and function loss. Adipose tissue engineering therefore emerges as a solution for development of biological substitutes for soft tissue which promotes not only volume regeneration but also function restoration with minimal consequences. Here we tested fibrous-structured atelocollagen (FSA) scaffolds and honeycomb atelocollagen (HCA) scaffolds for their ability to induce adipogenesis in vivo. Implants were subjected to histological and immunohistochemical assessment after 1, 2, and 4 weeks of implantation. Our studies showed that FSA scaffolds induced in vivo a markedly adipogenic response, whereas an acute inflammatory process was observed at HCA scaffolds without tissue regeneration detected within them. Our histological findings concerning FSA scaffolds clearly showed the presence of adipose-like tissue surprisingly composed by a mixture of brown-like and white-like adipocytes at week 2 whereas only white-like adipocytes at week 4. Subsequent positive Pax7 immunostaining at weeks 1 and 2 suggested the existence of a common myogenic progenitor shared by brown-like and white-like adipocytes observed. Then, in this work we present FSA scaffolds as a promising structure for brown and white adipose tissue engineering.

Discriminability measures and time-frequency features: an application to vibrissal tactile discrimination.

BACKGROUND: Often, the first problem that the neuroscientist must face is to determine if a specific stimulus set applied to biological system produces specific, precise and well differentiated responses. NEW METHOD: In the present study we have proposed four discriminability measures to evaluate the feasibility of differentiating experimental conditions: information measures based on information theory, percentage overlap based on Linacre method, Bhattacharyya distance and univariate standard distance. All discriminability measures were evaluated on experimental protocols related to vibrissal tactile discrimination. RESULTS: Time-frequency features were extracted from afferent discharges and then, pairwise comparisons were realized by using the proposed discriminability measures. Our results reveal the existence of time-frequency patterns which allows differentiating of sweep conditions from multifiber recordings. COMPARISON WITH EXISTING METHODS: Currently, statistical methods used to justify significant differences in experimental conditions have rigorous criteria that must be met for correct validation of results. Discriminability measures proposed here are robust and can be adjusted to different experimental conditions (time series, repeated measures, specific variables and other). CONCLUSIONS: Discriminability measures allowed determining the time intervals where two sweep situations have the highest probability to be differentiated from each other. High discriminability percentages were observed into protraction phase, although to a lesser degree, it was also observed in retraction phase. It was demonstrated that sensibility of discriminability measures are different. This revealing a greater ability to highlight percentage changes of pairwise comparisons. Finally, the methods here proposed can be adapted to other features of biological responses.

Fast nonlinear region localisation for nonlinear dielectric spectroscopy of biological suspensions.

The nonlinear properties of biological suspensions have been previously presented as a bulk phenomenon without the influences of the electrodes. However, some authors have showed that the behaviour of a biological suspension is due to the nonlinear characteristics of the electrode-electrolyte interface (EEI), which is modulated by the presence of yeast cells. We have developed a method, complementary to the nonlinear dielectric spectroscopy (NLDS) which is used for the study of the behaviour of EEI with resting cell suspensions of Saccharomyces cerevisiae. The method allows researchers to detect simply and quickly the voltage and frequency ranges where the metabolic activity of yeasts is detectable. This method does not replace NLDS, and aims to reduce the time during which the electrodes are exposed to corrosion by high voltages. In this paper we applied AC overpotentials (10-630 mV) with frequencies in the range from 1 to 1000 Hz. Also, we measured current harmonic distortion produced by the nonlinearity of the interface. Changes in the transfer function were observed when yeast suspension was used. Apart from the nonlinear response typical of the EEI, we also observed the biological nonlinear behaviour. The changes in the transfer functions were assessed using the overlapping index which was defined in terms of the conditional probability. The methodology was contrasted favourably with Fourier analysis. This novel strategy has the advantages of simplicity, sensitivity, reproducibility and involves basic tools such as the usual measurement of current.

Neural encoding schemes of tactile information in afferent activity of the vibrissal system.

When rats acquire sensory information by actively moving their vibrissae, a neural code is manifested at different levels of the sensory system. Behavioral studies in tactile discrimination agree that rats can distinguish different roughness surfaces by whisking their vibrissae. The present study explores the existence of neural encoding in the afferent activity of one vibrissal nerve. Two neural encoding schemes based on "events" were proposed (cumulative event count and median inter-event time). The events were detected by using an event detection algorithm based on multiscale decomposition of the signal (Continuous Wavelet Transform). The encoding schemes were quantitatively evaluated through the maximum amount of information which was obtained by the Shannon's mutual information formula. Moreover, the effect of difference distances between rat snout and swept surfaces on the information values was also studied. We found that roughness information was encoded by events of 0.8 ms duration in the cumulative event count and event of 1.0 to 1.6 ms duration in the median inter-event count. It was also observed that an extreme decrease of the distance between rat snout and swept surfaces significantly reduces the information values and the capacity to discriminate among the sweep situations.

Electrophysiological characterization of texture information slip-resistance dependent in the rat vibrissal nerve.

BACKGROUND: Studies in tactile discrimination agree that rats are able to learn a rough-smooth discrimination task by actively touching (whisking) objects with their vibrissae. In particular, we focus on recent evidence of how neurons at different levels of the sensory pathway carry information about tactile stimuli. Here, we analyzed the multifiber afferent discharge of one vibrissal nerve during active whisking. Vibrissae movements were induced by electrical stimulation of motor branches of the facial nerve. We used sandpapers of different grain size as roughness discrimination surfaces and we also consider the change of vibrissal slip-resistance as a way to improve tactile information acquisition. The amplitude of afferent activity was analyzed according to its Root Mean Square value (RMS). The comparisons among experimental situation were quantified by using the information theory. RESULTS: We found that the change of the vibrissal slip-resistance is a way to improve the roughness discrimination of surfaces. As roughness increased, the RMS values also increased in almost all cases. In addition, we observed a better discrimination performance in the retraction phase (maximum amount of information). CONCLUSIONS: The evidence of amplitude changes due to roughness surfaces and slip-resistance levels allows to speculate that texture information is slip-resistance dependent at peripheral level.

Evaluation of EMG processing techniques using Information Theory.

BACKGROUND: Electromyographic signals can be used in biomedical engineering and/or rehabilitation field, as potential sources of control for prosthetics and orthotics. In such applications, digital processing techniques are necessary to follow efficient and effectively the changes in the physiological characteristics produced by a muscular contraction. In this paper, two methods based on information theory are proposed to evaluate the processing techniques. METHODS: These methods determine the amount of information that a processing technique is able to extract from EMG signals. The processing techniques evaluated with these methods were: absolute mean value (AMV), RMS values, variance values (VAR) and difference absolute mean value (DAMV). EMG signals from the middle deltoid during abduction and adduction movement of the arm in the scapular plane was registered, for static and dynamic contractions. The optimal window length (segmentation), abduction and adduction movements and inter-electrode distance were also analyzed. RESULTS: Using the optimal segmentation (200 ms and 300 ms in static and dynamic contractions, respectively) the best processing techniques were: RMS, AMV and VAR in static contractions, and only the RMS in dynamic contractions. Using the RMS of EMG signal, variations in the amount of information between the abduction and adduction movements were observed. CONCLUSIONS: Although the evaluation methods proposed here were applied to standard processing techniques, these methods can also be considered as alternatives tools to evaluate new processing techniques in different areas of electrophysiology.

Bio-recognition capability of Streptomyces sp. M7 evaluated in adverse conditions for use as a biological transducer in a Lindane biosensor.

Bio-recognition devices have captured special attention because they combine biological specificity and selectivity with electronics to perform environmental and biomedical analysis. Lindane is a recalcitrant pesticide considered potential carcinogen that has caused serious pollution problems. The purpose of the present study is to evaluate Streptomyces sp. M7 ability to dechlorinate lindane in liquid defined media in adverse culture conditions. Bacterial activity was monitored by electrochemical impedance spectroscopy. These results confirm that the microorganism adhered to a solid support is able to grow and to metabolize the organochlorine pesticide as a sole carbon source. Therefore, Streptomyces sp. M7 can be applied for a future development of a prototype for lindane detection and quantification.

Design and evaluation of a fast Fourier transform-based nonlinear dielectric spectrometer.

Nonlinear dielectric spectroscopy of micro-organism is carried out by applying a moderate electrical field to an aqueous sample through two metal electrodes. Several ad hoc nonlinear spectrometers were proposed in the literature. However, these designs barely compensated the nonlinear distortion derived from the electrode-electrolyte interfaces (EEI). Moreover, the contribution of the suspension is masked by the effect of the nonlinearity introduced by the electrode contacts. Conversely, the nonlinear capability of a commercial tetrapolar analyzer has not been fully investigated. In this paper a new nonlinear tetrapolar spectrometer is proposed based on a commercial linear apparatus and ad hoc control and signal processing software. The system was evaluated with discrete electronic phantoms and showed that it can measure nonlinear properties of aqueous suspension independently of the presence of EEI (ANOVA test, p>0.001). It was also tested with real aqueous samples. The harmonics observed in the current that circulates through the sample reveals useful information about the transfer function of the sample. The total harmonic distortion was computed for linear mediums. Values lower than -60 dB suggest that the system has enough capability to perform nonlinear microbiological analysis. Design specifications, sources of interference, and equipment's limitations are discussed.

Non-linear dielectric spectroscopy of microbiological suspensions.

BACKGROUND: Non-linear dielectric spectroscopy (NLDS) of microorganism was characterized by the generation of harmonics in the polarization current when a microorganism suspension was exposed to a sinusoidal electric field. The biological nonlinear response initially described was not well verified by other authors and the results were susceptible to ambiguous interpretation. In this paper NLDS was performed to yeast suspension in tripolar and tetrapolar configuration with a recently developed analyzer. METHODS: Tripolar analysis was carried out by applying sinusoidal voltages up to 1 V at the electrode interface. Tetrapolar analysis was carried on with sinusoidal field strengths from 0.1 V cm(-1) to 70 V cm(-1). Both analyses were performed within a frequency range from 1 Hz through 100 Hz. The harmonic amplitudes were Fourier-analyzed and expressed in dB. The third harmonic, as reported previously, was investigated. Statistical analysis (ANOVA) was used to test the effect of inhibitor an activator of the plasma membrane enzyme in the measured response. RESULTS: No significant non-linearities were observed in tetrapolar analysis, and no observable changes occurred when inhibitor and activator were added to the suspension. Statistical analysis confirmed these results.When a pure sinus voltage was applied to an electrode-yeast suspension interface, variations higher than 25 dB for the 3rd harmonic were observed. Variation higher than 20 dB in the 3rd harmonics has also been found when adding an inhibitor or activator of the membrane-bounded enzymes. These variations did not occur when the suspension was boiled. DISCUSSION: The lack of result in tetrapolar cells suggest that there is no, if any, harmonic generation in microbiological bulk suspension. The non-linear response observed was originated in the electrode-electrolyte interface. The frequency and voltage windows observed in previous tetrapolar analysis were repeated in the tripolar measurements, but maximum were not observed at the same values. CONCLUSION: Contrary to previous assertions, no repeatable dielectric non-linearity was exhibited in the bulk suspensions tested under the field and frequency condition reported with this recently designed analyzer. Indeed, interface related harmonics were observed and monitored during biochemical stimuli. The changes were coherent with the expected biological response.

Laboratory experience for teaching sensory physiology.

The major challenge in laboratory teaching is the application of abstract concepts in simple and direct practical lessons. However, students rarely have the opportunity to participate in a laboratory that combines practical learning with a realistic research experience. In the Bioengineering Department, we started an experiential laboratory physiology to teach graduated students some aspects of sensorial physiology and exposes them to laboratory skills in instrumentation and physiological measurements. Students were able to analyze and quantify the effects of activation of mechanoreceptors in multifiber afferent discharges using equipment that was not overly sophisticated. In consequence, this practical laboratory helps students to make connections with physiological concepts acquired in theoretical classes and to introduce them to electrophysiological research.

Texture discrimination and multi-unit recording in the rat vibrissal nerve.

BACKGROUND: Rats distinguish objects differing in surface texture by actively moving their vibrissae. In this paper we characterized some aspects of texture sensing in anesthetized rats during active touch. We analyzed the multifiber discharge from a deep vibrissal nerve when the vibrissa sweeps materials (wood, metal, acrylic, sandpaper) having different textures. We polished these surfaces with sandpaper (P1000) to obtain close degrees of roughness and we induced vibrissal movement with two-branch facial nerve stimulation. We also consider the change in pressure against the vibrissa as a way to improve the tactile information acquisition. The signals were compared with a reference signal (control)--vibrissa sweeping the air--and were analyzed with the Root Mean Square (RMS) and the Power Spectrum Density (PSD). RESULTS: We extracted the information about texture discrimination hidden in the population activity of one vibrissa innervation, using the RMS values and the PSD. The pressure level 3 produced the best differentiation for RMS values and it could represent the "optimum" vibrissal pressure for texture discrimination. The frequency analysis (PSD) provided information only at low-pressure levels and showed that the differences are not related to the roughness of the materials but could be related to other texture parameters. CONCLUSION: Our results suggest that the physical properties of different materials could be transduced by the trigeminal sensory system of rats, as are shown by amplitude and frequency changes. Likewise, varying the pressure could represent a behavioral strategy that improves the information acquisition for texture discrimination.

Amplifier spurious input current components in electrode-electrolyte interface impedance measurements.

BACKGROUND: In Impedance Microbiology, the time during which the measuring equipment is connected to the bipolar cells is rather long, usually between 6 to 24 hrs for microorganisms with duplication times in the order of less than one hour and concentrations ranging from 10(1) to 10(7) [CFU/ml]. Under these conditions, the electrode-electrolyte interface impedance may show a slow drift of about 2%/hr. By and large, growth curves superimposed on such drift do not stabilize, are less reproducible, and keep on distorting all over the measurement of the temporal reactive or resistive records due to interface changes, in turn originated in bacterial activity. This problem has been found when growth curves were obtained by means of impedance analyzers or with impedance bridges using different types of operational amplifiers. METHODS: Suspecting that the input circuitry was the culprit of the deleterious effect, we used for that matter (a) ultra-low bias current amplifiers, (b) isolating relays for the selection of cells, and (c) a shorter connection time, so that the relays were maintained opened after the readings, to bring down such spurious drift to a negligible value. Bacterial growth curves were obtained in order to test their quality. RESULTS: It was demonstrated that the drift decreases ten fold when the circuit remained connected to the cell for a short time between measurements, so that the distortion became truly negligible. Improvement due to better-input amplifiers was not as good as by reducing the connection time. Moreover, temperature effects were insignificant with a regulation of +/- 0.2 [ degrees C]. Frequency did not influence either. CONCLUSION: The drift originated either at the dc input bias offset current (Ios) of the integrated circuits, or in discrete transistors connected directly to the electrodes immersed in the cells, depending on the particular circuit arrangement. Reduction of the connection time was the best countermeasure.

Comparative analysis of hematocrit measurements by dielectric and impedance techniques.

In a previous paper, a new dielectric technique was used to estimate hematocrit (HTC) in extracorporeal blood circulation systems, independently of plasma conductivity or osmolarity. Although many impedance techniques have been formerly proposed in the literature, none has been evaluated against plasma conductivity and osmolarity. Herein, we estimate HTC based on permittivity changes and also with other four techniques found in the literature. Besides, the error incurred in each is also studied when plasma conductivity and osmolarity changed as much as 1 mS/cm and 50 mOsm/kg, respectively. The dielectric (permittivity) technique has an error close to 5.4%, while the others showed both tendencies, i.e., lower error (2.5%, two of them) and higher error (8.6% and 16.3%, the other two). The dielectric technique, even though did not produce the lowest error, provides a well-described physical model along with simple instrumentation.

Hematocrit measurement by dielectric spectroscopy.

Based on permittivity changes, a new method to measure hematocrit (HCT) in extracorporeal blood systems is presented. Human blood samples were tested at different HCT levels pairing the values of permittivity change, obtained by means of a commercial impedance analyzer, with traditional centrifugation measurements. Data were correlated using both linear and nonlinear regression. When using the lineal model, the comparison yielded a high correlation coefficient (r = 0.99). Theoretical simplifications suggest that the method is independent of changes in the conductivities of the intracellular and extracellular compartments. The influence of osmolarity and conductivity of the extracellular compartment was analyzed. It is shown that HCT can be predicted within an error lower than 5% when those parameters changed as much as 1 mS/cm and 50 mOsm/kg, respectively. Thus, the method appears as valid and viable showing good possibilities in applications such as renal dialysis.