Real-Time Small-Scale Measurements Provide Insights Into Biological Systems

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Real-Time Small-Scale Measurements Provide Insights Into Biological Systems

Introduction

The very small scale in terms of both time and space in which biological processes occur presents one of the greatest challenges in biological research. In the brain neurotransmission occurs on the 0.3-100 mSec time scale (synaptic delay) and in a space of 30-50 nm (synaptic cleft). This has created significant analytical problems.

Rapid advances in real time measurement have been made in recent years through the use of electrochemical techniques that detect molecules by reacting them at an electrode. In one such technique the oxidized material gives up electrons which are collected by the working electrode, generating a current. The biological compounds that are electrochemically active can be detected by this method. Small electrodes can be used to monitor concentration changes down to the scale of a few cells in intact animals. This article will provide an overview of recent developments in this area and summarize work of two leading researchers.

Traditional analytical methods are slow

Figure 1. Real-time nitric oxide (NO) responses of mollusk mussel nervous tissue elicited by injection (arrow) of 1uM morphine-6-glucuronide. Graph illustrates excellent sensitivity and a clear noise-free recording (sensitivity was found to be 15 pA/1nM NO). Data provided courtesy Prof. G. Stefano & K. Mantione, SUNY Neuroscience Research Institute Old Westbury, N.Y 11568 (April 2006). Click to enlarge.

Analytical methods such as immunoassays and instrumental techniques such as high performance liquid chromatography (HPLC) have long provided the gold standard in biochemical analysis because of their ability to detect infinitesimal amounts of nearly any compound. The problem is that many cellular processes take place on such a brief time scale that the molecule of interest would have disappeared long before the sample could be collected and prepared. Another reason why cellular processes are difficult to measure is that the goal is often to perform measurements in intact animals in order to find correlations between behavior and molecular events.

A variety of different methods have been used traditionally to address this problem. In some cases a trapping molecule may be used that reacts with the short-lived compound, such as hemoglobin for nitric oxide. These methods are often complicated, lack sensitivity and specificity and do not provide real-time measurements. Microdialysis is a process which involves extracting a sample using a specially constructed probe for subsequent analysis with a laboratory instrument. This method has the weakness that the probe is large and impinges on a large number of cells and that the results are averaged over minutes, which means that many cellular chemical reactions that occur on the millisecond time scales may escape detection.

Emergence of fast cyclic voltammetry

Advances in microelectrodes and in electrochemical techniques are providing the potential for improvements in the ability to perform much faster measurements in small areas. One important advance has been the development of voltammetric microelectrodes with dimensions on the micrometer scale that sense substances on the basis of their oxidation or reduction. Dynamic devices that allow control of chemical environments as well as chemical sensing have been used in applications such as the investigation of chemical fluctuations on the surface of individual cells and in the living brain. The small double-layer capacitance of the microelectrodes enables rapid monitoring of chemical events occurring on the submicrosecond time scale.

In one technique these electrodes are used in conjunction with a form of linear sweep voltammetry which has become known as fast cyclic voltammetry (FCV). A two electrode system is used that includes a working electrode and reference electrode. These electrodes are placed in an electrolyte solution, which can consist of aqueous fluid in a laboratory vessel or the extracellular fluid in the brain. The FCV instrument powers the working electrode through a voltammetric scan, a series of anodic and cathodic voltage sweeps in the millisecond time range. This pattern of voltages generates a background current through the interface of the electrode and electrolyte.

When electroactive materials are released into the solution, the current flow increases at specified areas of the waveform. Compounds can be identified by the resulting cyclic voltammogram. This current flow, known as the faradaic current, is proportional to the amount of oxidizable material in the solution, enabling quantitative measurements.

Electrodes provide specificity

Figure 2. Stimulated release of dopamine in rat brain determined by FCV with a carbon fiber electrode. Data is displayed using TH-I software, which is now commercially available.

Specificity can be improved by using an electrode that is selective to the material being measured. Electrodes have been developed that use gas permeable membranes such as nitrocellulose, silicon rubber and Teflon. A variety of pretreatments and coatings are employed. For example, a carbon fiber electrode coated with Nafion and an NO-selective, gas permeable membrane provided high selectivity against ascorbate, dopamine, and nitride while detecting Nitric Oxide. The emergence of new and more sensitive electrodes has put pressure on the levels of sensitivity and resolution required of amplifiers/potentiostats in the measurement of low levels of substances.

Modern digital signal processors (DSPs) can be used to meet the stringent requirements of these super-sensitive electrodes. For example, they enable the mathematical processing of large samples of data so that signals can be detected and enhanced. This allows detection of changes at very low, physiological levels with small electrodes. Not only does a DSP system offer the advantages of real-time signal filtering and data handling, it also provides greater intrinsic accuracy of measurement and a greater dynamic range for the signals being measured facilitating use in real samples. The intrinsic low noise of the electronics allows low limits of detection.

Stefano investigates Alzheimer’s

Dr. George B. Stefano, Director of the Neuroscience Research Institute at the State University of New York, Old Westbury, New York, is using these methods to detect oxidizable compounds released by cells under the influence of pharmaceutical agents. Stefano has been heavily involved in efforts to measure the spontaneous release of compounds by various tissues for the purpose of developing diagnostic methods and evaluating potential pharmaceutical agents. For example, Stefano, along with Theodore Pak, Patrick Cadet, and Kirk J. Mantione, all of the Neuroscience Research Institute, recently determined that nitric oxide produced by morphine inhibits the production of -amyloid peptide, which is a pathologic feature of Alzheimer’s disease and which in turn inhibits the production of NO. This suggests that a deficiency of basal NO or endogenous morphine may trigger drastically reduced levels of basal NO. The outcome is chronic vascoconstriction and brain hypoperfusion and eventual neuronal death. This novel theorized mechanism for Alzheimer’s disease supports an increasingly-accepted vascular pathological hypothesis.

Stefano has recently switched to a real-time measurement system of the type described above and uses it to measure Nitric Oxide and other compounds. This particular instrument provides for real-time amperomeetric, voltammetric and temperature measurements to be made independently and simultaneously on up to four channels. By exploiting DSP technology, accuracy of 0.1% in amperometric and voltammetric applications is achieved with an input signal dynamic range of 100,000. Signals in the femtoamp range (10-15 Amp) can be detected.

Wightman investigates drug abuse mechanisms

Dr. R. Mark Wightman at the Department of Chemistry, University of North Carolina at Chapel Hill, is a pioneer in the field of real-time chemical sensing of individual biological cells. Dr. Wightman has developed techniques for measuring dopamine neurotransmission during behavior. Neurotransmitter lifetimes in the extracellular space are very brief because metabolism and cellular reuptake rapidly restores neurotransmitter concentrations to their pre-release low levels. Monitoring neurotransmission requires the ability to detect changes in concentration over the millisecond time frame, high chemical specificity, and the ability to limit detection to the micrometer scale.

Cocaine and other drugs of abuse appear to tap into the brain reward pathway by releasing dopamine, a neurotransmitter. Wightman said that FCV offers the necessary temporal resolution and specificity for correlating in vivo release of dopamine with specific behaviors. He used these methods recently to study changes in dopamine concentration in the brains of rats that have been trained to self-administer cocaine. He discovered that dopamine is released even prior to administering the cocaine as the rat begins to approach the lever. Another dopamine transient occurs when the lever is pushed. When the animal presses the lever but dopamine is withheld, the dopamine transient associated with the lever pushed is substantially reduced. Wightman designs his own microelectrodes and the FCV instrument in order to provide the high signal to noise ratio required by this application.

The wide spread use of this technique has been additionally hampered by the lack of suitable software. Recently the software for FCV developed in Dr. Wightman’s has been made available. Now with the software, the instrument and electrodes being offered on a commercial basis, researchers are beginning to take advantage of this technology.

More information is available from:
ESA Biosciences, Inc.800-959-5095 www.esabio.com

References

Finlay, Janet M., Smith, Gwenn S. A Critical Analysis of Neurochemical Methods for Monitoring Transmitter Dynamics in the Brain. 11th International Conference on In Vivo Methodes.

Pak, T., Cadet, P., Mantione, K.J. and Stefano GB. Morphine via nitric oxide modulates beta-amyloid metabolism: a novel protective mechanism for Alzheimer's disease. Med Sci Monit 11(10): BR357-BR366 (2005).

Wightman, R. Mark, Probing Cellular Chemistry in Biological Systems with Microelectrodes. Science 311:1570-1574 (2006).

Stuber, Garret D., Wightman, R. Mark, Carelli, Regina M., Extinction of Cocaine Self-Administration Reveals Functionally and Temporally Distinct Dopaminergic Signals in the Nucleus Accumbens. Neuron 46:661-669 (2005).