Extracellular recording experiments in in vivo animals
Extracellular recording experiments in in vivo animals
Source : Practical Laboratory Techniques in Neurobiology
Operation method
basic program
Principle
Extracellular recording (extracellular recording) is the placement of a guiding electrode on the surface or adjacent parts of a nerve cell or nerve tissue to guide the discharge activity associated with the recording. When excitatory activity occurs in thousands of nerve cells or tissues, the neurons at the active site are depolarized, and the inactive site is in a normal polarized state, and the potentials between the two sites are different in a volumetric conductor, and current flows from one point to the other. Electrodes placed on the surface of a thousand cells then record the potential difference generated between the two. The convenience of extracellular microelectrode recording is that the electrodes are not inserted into the cell. This method is used in neuroscience mainly to detect the site and time of generation of neural action potentials, as well as the frequency and pattern of discharge sequences, and is generally not used to determine the amplitude and waveform of action potentials.
Materials and Instruments
Nerve Cells Move 1. Recording of electrical activity of individual nerve cells: Extracellular guidance can be used when it is necessary to guide and record the electrical activity of individual cells in certain nuclei of the center. The first step is to prepare and select the applicable glass microelectrodes or metal microelectrodes, determine the three-dimensional coordinates of the nuclei to be detected on a cranial orienting device, and then drill a hole in the skull, tear the dura mater, and send the electrode tip to the site of the nucleus under the manipulation of the micro-propellers to search for the guided cells. Accurate positioning of the electrode tip is critical to the success of the experiment and is often determined by repeated tip labeling and examination of sections. Metal microelectrode tips are more or less constant due to their exposure (no insulation), and can sometimes direct the firing activity of multiple nerve cells at the same time. Regarding glass microelectrodes, qualified microelectrodes are important for recording cell potentials. The first step in pulling a glass microelectrode is to place a capillary glass tube that has been cleaned and treated on a microelectrode puller to pull the glass microelectrode. This is done in two passes to produce a pair of microglass tubes with equal tip sizes and tip diameters up to about 0.5µm. Immediately put under the microscope to check the shape and tip opening, as well as the tip diameter to see if it meets the requirements. 2. Spinal cord dorsal horn wide dynamic neuron - (WDR neuron) recording: Also an extracellular recording technique, this is a recording method in which recording electrodes are placed at specific sites within the dorsal horn of the spinal cord under anesthesia of the animal to record in vivo the spontaneous firing activity of the WDR neurons or the electrical response to peripheral stimuli.1 The waveforms of the extracellular potentials vary depending on the site of the cell where they are recorded. Potentials recorded extracellularly are much smaller than those recorded intracellularly, due to shunting of the signal by the low-resistance extracellular fluid pathway. Neurons recorded in the CNS using extracellular recording can develop local currents between the activated site of the recorded neuron and the power source of the rest of the neuron after being activated by excitatory or inhibitory information transmitted from other neurons. Depending on the relationship of where the extracellular recording microelectrodes are located, an electrical wave of varying polarity can be recorded, either downward (positive potential) or upward (negative potential). Due to the limited volume within the CNS, the current lines in the electric field can be compressed and deformed, and for a variety of reasons such as the inconsistent conductivity coefficients of structures such as vascular connective tissues, and the fact that the activities of different neurons are not synchronized, the magnitude of the potentials recorded by the extracellular electrodes can have a wide range of variations in magnitude and waveforms. Therefore, the analysis of extracellularly recorded potentials focuses on the frequency and latency of discharges without comparing the magnitude of discharges. The purpose of the potential analysis is to recognize from which part of a neuron the potential is generated, so that the significance of the experimental results can be easily judged. For example, in the case of electrical stimulation of the optic nerve, in the lateral geniculate body recordings, it can be seen that the potential of the presynaptic axon is an all-or-nothing unidirectional ascending wave, generated by the postsynaptic axon is also a unidirectional ascending wave. However, there are differences with the presynaptic axonal potential: ① the latency is longer, about 1.2-2.2ms; ② for a single stimulus can often cause the rise of a unidirectional wave. (ii) A single stimulus can often cause multiple potentials; (iii) The rising wave is biconcave, forming an M-wave. The cytosolic potential is a long-duration negative front potential with the same latency as that of the postsynaptic axon. For WDR neurons, the neuron types were discriminated as follows according to the characteristics of the neuron's response to various types of stimuli: (i) Non-injurious afferent neurons (non-nociceptive neurons) only respond to non-injurious stimuli (e.g., gentle brushing with soft bristles, etc.) by causing an increase in the frequency of discharge, and there is no significant change in the frequency of discharge in response to injurious stimuli (e.g., pincer stimuli); (ii) Specific injurious afferent neurons (nociceptive-specific neuron) applying injurious stimuli (e.g., pinching the skin with a toothed ingesta) elicited a significant increase in the discharge frequency, whereas there was no response to non-injurious stimuli; (iii) Wide-dynamic range neuron (WDRneuron) elicits an increase in discharge frequency for both non-injurious and injurious stimuli, and the response elicited by injurious stimuli is significantly higher than the response elicited by non-injurious stimuli. Caveat l In extracorporeal cell recording, pay attention to heat preservation for anesthetized animals, and try to avoid blood vessels on the tissue surface when microelectrodes are inserted. 2. Towel in the room temperature is high, the water in the microelectrode perfusion solution is easy to evaporate, can be coated with petroleum jelly at the opening of the end of the microelectrode. Common Problems 1 The signal recorded outside the cell is weak and susceptible to external electromagnetic interference, the waveform, amplitude and time of the recorded electrical signal depend on the position of the recording electrode, the closer the recording electrode is to the neuron, the larger the recorded electrical signal is. The biggest advantage of this method is that it does not damage the recorded neurons, so the activity of neurons is closer to the physiological state than that of intracellular recording, the activity is good, and it can maintain a long period of recording, and the recording method is simple and easy to be practiced, and it is easy to be mastered by beginners. The method does not require a high level of instrumentation and is easy to carry out in the laboratory. In the overall animal experiment, the recording stability requirements are higher. The breathing and physical activity of the animal affects the stability of the recording, which can be alleviated by mechanical ventilation and injection of muscle relaxants. The recording trough can also be covered with agarose to attenuate the effect of respiratory movements on the recording. The glass microelectrodes tend to be blocked by tissue during descent, and can be given a least positive pressure through a thin tube connected to the electrodes. For more product details, please visit Aladdin Scientific website.
Microelectrode materials Glass microelectrode puller Microscope Micromanipulator Microelectrode amplifier Recording instruments Other instruments and equipment