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Patch Clamps Insertion in Brain
Renu Bist*
Department of Zoology, Centre of Advanced Studies, University of Rajasthan, Jaipur, India
Submission:September 04, 2023;Published:March 12, 2024
*Corresponding author:Renu Bist, Department of Zoology, Centre of Advanced Studies, University of Rajasthan, Jaipur, India Email: renu_bisht22@yahoo.co.in
How to cite this article:Renu B. Patch Clamps Insertion in Brain. J Tumor Med Prev. 2024; 4(3): 555639. DOI: 10.19080/ JTMP.2024.04.555639
Abstract
The patch clamp technique allows for precise measurement and manipulation of ion channel currents and membrane potentials in neurons. By using a glass pipette to form a tight seal with the cell membrane, researchers can obtain high-resolution recordings and study neuronal activity at the cellular and molecular levels. This technique has provided invaluable insights into neuronal excitability, synaptic transmission, and ion channel properties. It enables current clamp recordings to study the intrinsic electrical properties of neurons, such as resting membrane potential and action potentials. Voltage clamp recordings allow for the investigation of ion channel kinetics, conductance, and synaptic currents. Patch clamp is also instrumental in pharmacological studies, where the effects of drugs and compounds on ion channels and receptors can be examined.
Keywords:Patch Clamp; Neurons; Ion Channels; Synaptic Transmission; Signaling; Neuroscience
Introduction
Patch clamp is a widely used technique in Neuroscience research to investigate the electrical properties and activity of individual neurons or small groups of neurons. It allows for precise measurement and manipulation of ion channel currents and membrane potentials. The versatility and precision of patch clamp recordings have contributed significantly to our understanding of the fundamental principles of neuronal function and the mechanisms underlying various neurological disorders. If there is any damage to neurons, it can affect their ability to communicate, which may lead to a change in thinking, behaviour and feelings [1]. So, in order to identify a receptor on a membrane patch clamps could be useful.
Patch clamp allows researchers to measure the electrical activity of neurons in two primary configurations, namely current clamp and voltage clamp. The patch pipette is used to measure the membrane potential of a neuron. It provides information about the resting membrane potential, action potentials, synaptic potentials, and other intrinsic electrical properties of the neuron. In voltage clamp mode, the membrane potential is artificially controlled by injecting current through the patch pipette [2]. This enables researchers to measure and manipulate ion channel currents responsible for generating and shaping neuronal activity. Voltage clamp recordings are used to study synaptic transmission, ion channel properties, and other membrane conductance. Beside this, patch clamp experiments allow researchers to investigate the effects of pharmacological agents on neuronal activity. By applying specific drugs or compounds to the bath solution or directly through the patch pipette, researchers can study the modulation of ion channels, receptors, and signaling pathways involved in neuronal function. Recordings of patch clamp enable the study of synaptic transmission, including excitatory and inhibitory synaptic currents. Researchers can measure postsynaptic currents and analyze their kinetics, amplitude, and frequency. By stimulating presynaptic neurons or applying specific drugs, researchers can investigate synaptic plasticity mechanisms such as long-term potentiation (LTP) and long-term depression (LTD). Patch clamp techniques are essential for characterizing the properties of ion channels, such as their conductance, gating kinetics, and pharmacology [3]. Researchers can record and analyze ion channel currents to study their voltage dependence, activation and inactivation properties, and responses to different modulators and drugs. This information is crucial for understanding the contribution of specific ion channels to neuronal excitability and signaling.
Intracellular signaling and second messenger pathways: Patch clamp recordings can be combined with intracellular injections of signaling molecules or second messengers to investigate their effects on neuronal activity. This allows researchers to study the role of various signaling pathways, such as cyclic nucleotides, calcium signaling, or protein kinases/phosphatases, in modulating ion channel activity and neuronal excitability. Patch clamp recordings provide valuable information about the electrophysiological properties of individual neurons, allowing for their identification and classification. By examining action potential patterns, firing rates, and response properties to specific stimuli, researchers can determine the cell type (e.g., excitatory or inhibitory) and study the diversity of neuronal subtypes within a brain region. For example, AChE inhibition, leads to accumulation of acetylcholine at synapse, resulting in the over stimulation of the neurons [4], for which patch clamps certainly can provide information.
Operational Steps
These surgeries are specialized procedures that require expertise and should be performed by trained researchers or veterinarians. Ethical considerations and animal welfare guidelines must be followed to ensure the well-being of the animals involved.
Pre-operative preparation
The animal is typically anesthetized to ensure it remains still and does not experience pain during the procedure. The specific anesthesia protocols may vary depending on the experimental requirements and the veterinarian’s judgment. The animal’s head is typically secured in a stereotaxic frame to immobilize it and provide stability during the surgery.
Surgical approach
A small incision is made in the scalp to expose the skull. The surgeon uses a high-speed dental drill or a similar instrument to create a small burr hole in the skull at the desired location for electrode or patch clamp insertion. The burr hole is usually made near the brain region of interest.
Microelectrode or patch clamp insertion
For microelectrodes, a glass or metal electrode is inserted into the brain tissue through the burr hole. The electrode is carefully advanced to the desired depth using a micromanipulator. This allows the recording of electrical signals from individual neurons. For patch clamp experiments, a glass micropipette filled with an electrolyte solution is used. The micropipette is positioned near a neuron of interest, and a gentle suction is applied to create a high-resistance seal between the pipette tip and the neuron’s membrane. This enables the measurement of ion channel currents and other electrical properties of the neuron.
Recording and experimentation
Once the microelectrode or patch clamp is in place, electrical signals or currents from the neurons can be recorded or manipulated using appropriate equipment and amplifiers [5]. Researchers can perform various experiments, such as recording spontaneous or evoked neuronal activity, studying synaptic transmission, or applying specific electrical or pharmacological stimuli to understand the properties of individual neurons or neural networks.
Post-operative care
After the surgery, the animal is closely monitored during the recovery period. This may involve pain management, administration of appropriate post-operative care, and observation for any potential complications. Proper wound closure is ensured to minimize the risk of infection and promote healing.
Experimental challenges with patch clamp insertion
Patch clamp experiments can be technically challenging, and researchers often encounter various troubleshooting issues. Here are some common troubleshooting steps for patch clamp experiments:
Electrical noise
Electrical noise can interfere with the quality of patch clamp recordings. To minimize noise, ensure that all electrical connections and cables are properly shielded. Use high-quality amplifiers, filters, and grounding techniques. Check for any potential sources of interference, such as nearby electronic devices, and try to minimize their impact.
Seal formation
For whole-cell patch clamp recordings, achieving a highquality seal between the patch pipette and the cell membrane is crucial. If you’re having trouble forming a seal, check that the pipette tip is clean and free from debris. Adjust the angle of the pipette to improve contact with the cell membrane. Applying gentle suction while approaching the cell can aid in forming a seal.
Gigaseal formation
In some experiments, researchers aim to achieve a gigaseal between the patch pipette and the cell membrane, allowing for high-resistance recordings. If gigaseal formation is challenging, ensure that the pipette tip is properly fire-polished to minimize its size and improve contact. Adjust the pressure and angle of the pipette during approach. Cleaning the pipette and the cell membrane with a suitable detergent or enzyme solution can also help.
Series resistance compensation
Series resistance arises due to the resistance of the electrode and the pipette solution. It can affect the accuracy of the recorded signals. To compensate for series resistance, adjust the bridge balance or use the appropriate compensation settings on the amplifier. Monitor the series resistance throughout the experiment and periodically adjust compensation values as needed.
Cell stability
Maintaining stable recordings over time can be challenging, especially if the cell moves or the access resistance changes. It is ensured that the experimental setup is stable and that the temperature and pH are well-regulated. If the cell is moving, check the stability of the microscope stage or the stability of the tissue slice or preparation.
Low success rate
If it is consistently experienced that there is a low success rate in achieving whole-cell or gigaseal recordings, then pipette fabrication techniques are reviewed, including fire-polishing and pulling parameters. Optimized the extracellular and intracellular solutions, considering factors such as osmolarity, pH, and ion concentrations. Also, the health and viability of the cells or tissue being used is verified.
Conclusion
The patch clamp technique facilitates investigations into synaptic transmission and plasticity, providing a deeper understanding of how neurons communicate and adapt. By characterizing ion channels, researchers can unravel their roles in neuronal excitability and signaling. Patch clamp recordings combined with intracellular injections offer insights into intracellular signaling pathways and second messenger systems. Its applications span from understanding basic neuronal properties to unraveling complex synaptic mechanisms and exploring pharmacological interventions. By addressing common troubleshooting issues, researchers can overcome experimental challenges and maximize the potential of patch clamp recordings in advancing our knowledge of the brain and neurological disorders.
References
- Bist R, Bhatt DK (2023) Cholinergic Transporters Serve as Potential Targets in Alzheimer's Disease. Curr Mol Med.
- Hille B (2001) Ion channels of excitable membranes (3rd Edition). Sinauer Associates. Sackmann, B, Neher E (2004). Patch clamp techniques for studying ionic channels in excitable membranes. In: Cuthbert AW (Ed.), Ionic Channels and Effect of Taurine on the Heart. Springer, p. 15-37.
- Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391(2): 85-100.
- Agarwal S, Bist R, Kuntal H, Bhatt DK (2023) Effect of dichlorvos on kidney functions with reference to altered AChE gene expression in mice. Advances in Redox Research 9: 100078.
- Neher E, Sakmann B (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260: 799-802.
