Umair Gaines
The nervous system functions by sending electrical signals between neurons, which are essentially electrical devices. Neurons communicate with each other via electrical events called 'action potentials' and chemical neurotransmitters. To understand how neurons generate and send these electrical signals, it is important to measure the electric potential in neurons. This can be done using electrode-based techniques, optical imaging-based methods, and fluorescent-based voltage-sensitive dyes.
| Characteristics | Values |
|---|---|
| Method | Intracellular microelectrode |
| Microelectrode Description | Glass tubing pulled to a very fine point (less than 1 μm diameter) and filled with a good electrical conductor |
| Voltage Range | A fraction of a volt (typically −40 to −90 mV) |
| Alternative Methods | Optical imaging-based methods, fluorescence-based methods, Brain Mesh Electronics, voltage-sensitive dyes |
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What You'll Learn
Using an intracellular microelectrode to record electrical potential
Intracellular recordings are a group of techniques used to measure the voltage across or electrical currents passing through neuronal or other cellular membranes. This is done by inserting an electrode inside the neuron. The intracellular recording technique was invented by Gerard and Ling in 1949, using glass microelectrodes for this purpose.
Intracellular recordings require impaling a neuron or axon with a microelectrode to measure the potential difference between the tip of the intracellular electrode and the reference electrode positioned outside the cell. A typical microelectrode is a piece of glass tubing pulled to a very fine point (with an opening of less than 1 μm in diameter) and filled with a good electrical conductor, such as a concentrated salt solution. The conductive core can then be connected to a voltmeter, such as an oscilloscope, to record the transmembrane voltage of the nerve cell.
The microelectrode method has been expanded upon by the invention of the patch clamp by Neher and Sakmann in 1976, and the adaptation of electronics for single-electrode voltage clamp by Brennecke and Lindeman in 1974. The use of microelectrodes has also been improved by the development of 3D electrode arrays, which enable long-term, multiplexed intracellular recording and stimulation from cells in cellular networks.
One novel type of microelectrode is the nanovolcano, which spontaneously fuses with the cell membrane and permits stable intracellular access. This microelectrode has a high aspect ratio, which permits in-cell access without limiting the active area of the recording electrode.
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Optical imaging-based methods
Optical dyes are a non-invasive method that can measure membrane potential from multiple cell sites and multiple cells simultaneously. They provide a high spatial resolution compared to microelectrode-based techniques. However, they may be limited by the weak intrinsic signal, especially in mammalian neurons, which are smaller and more transparent than invertebrate neurons.
Fluorescence-based techniques, such as fluorescence lifetime imaging (FLIM), have been used to optically estimate membrane potential. FLIM techniques include voltage-sensitive fluorescent dyes (VSDs) and genetically encoded voltage indicators. VSDs can be used to monitor membrane potential changes and have strong signals and fast-response kinetics, allowing for large-scale monitoring of neural activity. Genetically encoded voltage indicators, on the other hand, offer the advantage of targeting specific cell populations and low-invasive staining of neurons.
Another optical imaging-based method is full-field interferometric imaging, which detects cellular action potentials by observing deformations in the cell membrane during electrical spikes. This technique is non-invasive and can be used to analyze neuronal networks and other cellular activities.
Other optical techniques mentioned include hyperspectral SRS imaging, quantitative phase microscopy, and label-free optical imaging of membrane potential. These techniques offer various advantages, such as high sensitivity and speed, but may also face challenges due to weak intrinsic signals or limitations in imaging deep tissues.
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Fluorescent-based voltage-sensitive dyes
The basic principle of voltage-sensitive dye imaging (VSDI), or optical recording technology, is to use voltage-sensitive dyes to transform changes in membrane potential into a fluorescent signal. This allows scientists to gauge the global electrical state of a neuron. The fluorescence signal can be detected using specialised microscopes and imaging devices, which may include technical lamps or lasers.
VSDI was first applied to electrophysiological research on giant axons of squid in 1968 by Tasaki et al. and has since been widely used to record the electrical activity of myocardial cells and brain tissues.
One example of a voltage-sensitive dye is FMP, which has been used to image the membrane potential of cultured cells using an epifluorescence-based single-cell imaging system. FMP is suitable for this type of imaging as it has a high signal-to-noise ratio, robust signals, and can detect bidirectional changes in fluorescence resulting from hyper- or depolarising conditions.
Other commonly used voltage-sensitive dyes include substituted aminonaphthylethenylpyridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and RH237. These dyes are used for different experimental procedures depending on their chemical modifications, which change their physical properties.
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Brain Mesh Electronics
The brain is the most unexplored part of our body, and a lack of tools has hindered our understanding of it and its associated diseases. In recent decades, several techniques have been developed to study neurons and neuronal communication. One such technique is Brain Mesh Electronics, which involves injectable mesh electronics that can record signals at a single neuron level. This technique has been demonstrated in mice brains and can record signals for at least eight months without eliciting a chronic immune response.
Mesh electronics probes are designed with structural and mechanical properties that allow them to "look and behave" like neural tissue. They are ultra-flexible and can be functionalized with targeting molecules for in vivo neuron-subtype-specific electrophysiology. The probes are similar in size to the cell body of neurons, and their small active detector areas allow for highly localized detection of both extracellular and intracellular field/action potentials in vivo.
The long-term stability of mesh electronics enables the study of brain circuit evolution and provides insights into the neurological basis of important brain functions and cognitive processes. Mesh electronics can also be combined with multisite injection to achieve stable local field potential and single-unit recordings from multiple brain regions. This has been demonstrated in awake restrained mice over four months, with 32 to 128 channels per probe.
The capabilities of mesh electronics can be expanded by incorporating stimulation electrodes, allowing for simultaneous chronic stimulation and recording at the single-neuron level. This has the potential to be used for chronic neuron/circuit modulation and recording studies. Mesh electronics have also been used to create innervated synthetic neural tissue, where rat hippocampal neurons were cultured within a 3D mesh electronics scaffold, resulting in interpenetrating neural and electronic networks.
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Calculating the ionic basis of electrical potential
The electrical potential in neurons is calculated by measuring the difference in electrical charge across the plasma membrane, also known as the membrane potential. This potential is influenced by the concentration of ions on either side of the membrane, particularly sodium (Na+) and potassium (K+).
To calculate the ionic basis of the electrical potential, we consider the diffusion of ions across a semi-permeable membrane. In a typical setup, a high concentration of KCl is placed on one side of the membrane, while the other side has a low concentration. The membrane is permeable only to K+, allowing it to diffuse and establish an electrical potential difference. This diffusion creates an electrostatic voltage force, which draws some K+ ions back towards the side with the higher concentration.
The movement of K+ ions results in a separation of electrical charge, with a greater number of positively charged ions on the side with the lower concentration of KCl. This charge separation is the basis of the membrane voltage and is known as the potential difference. It is the starting point for all electrical events in nervous systems.
The potential difference can be calculated using the Nernst equation, which accounts for the concentration gradient and the electrical gradient. The concentration gradient describes the movement of a solute from an area of higher concentration to an area of lower concentration. The electrical gradient, on the other hand, influences the movement of charged solutes towards areas with opposite charges.
The Nernst equation is as follows:
E = R * T * ln(Q) / n * F
Where:
- E is the equilibrium potential or Nernst potential
- R is the gas constant
- T is the absolute temperature
- Ln is the natural logarithm
- Q is the ratio of the ion concentrations across the membrane
- N is the ion charge
- F is the Faraday constant
By using this equation, we can calculate the electrical potential at which there is no net flux of a specific ion across the membrane. This calculation provides valuable insights into the ionic basis of electrical potential in neurons.
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Frequently asked questions
Action potentials are transient changes in the resting membrane potential of neurons. They are electrical events that signal the neuron as "active".
There are several methods to measure the electric potential in a neuron, including electrode-based techniques, optical imaging-based methods, and optical-based molecular dyes. The best way to observe an action potential is to use an intracellular microelectrode to record the electrical potential across the neuronal plasma membrane.
A microelectrode is a piece of glass tubing pulled to a very fine point (with an opening of less than 1 μm in diameter) and filled with a good electrical conductor, such as a concentrated salt solution. This conductive core can then be connected to a voltmeter to record the transmembrane voltage of the nerve cell.
Optical-based molecular dyes offer several advantages over microelectrode-based techniques, including the ability to measure membrane potential simultaneously from multiple cell sites and multiple cells simultaneously, without the need for complicated surgical procedures.
