Electrophysiology and toxins Flashcards
What is patch clamp physiology?
Patch clamp physiology is a technique used to study the electrical activity of individual cells by measuring ion currents across their membranes.
patch clamp physiology step = 5
- Preparation: Secure a glass pipette with a tiny tip onto the cell membrane.
- Seal Formation: Apply gentle suction to create a tight seal between the pipette tip and the membrane.
- Whole-Cell Configuration: Apply more suction to rupture the membrane patch, allowing access to the cell’s interior.
- Recording: Use an amplifier to measure the ionic currents flowing across the membrane.
- Analysis: Analyze the recorded data to understand the cell’s electrical properties and ion channel behavior.
What are the differences between voltage and current clamp techniques?
- Voltage Clamp:
- involves controlling and maintaining the MEMBRANE VOLTAGE of a cell at a CONSTANT LEVEL while MEASURING THE RESULTING CURRENTS
- It allows researchers to study the behaviour of ion channels. - Current Clamp:
- technique involves CONTROLLING and MAINTAINING the MEMBRANE CURRENT FLOWING through a cell at a CONSTANT LEVEL l while measuring the RESULTING CHANGES IN MEMBRANE POTENTIAL.
- It allows researchers to study the EXCITABILITY AND RESPONSE PROPERTIES OF NEURONS OR OTHER CELLS
What is ‘current clamp’ patch clamp electrophysiology used for, and what parameters can it characterise in neurons?
= 7
- Current clamp patch clamp electrophysiology involves ‘injecting current into cells and is commonly used to characterise the excitability of neurons’
Parameters it can characterise include:
- Resting membrane potential
- Action potential firing frequency
- Action potential threshold
- Current threshold
- Rheobase
- Action potential waveform
What is voltage clamp patch clamp electrophysiology used for, and what properties can it characterise in ion channels and synaptic transmission?
= 4
- Voltage clamp patch clamp electrophysiology ‘applies voltage to cells and is commonly used to characterize ion channel properties and synaptic transmission’.
It can characterize:
- Activation voltage
- Inactivation voltage of ion channels
- Frequency and amplitude of synaptic inputs at the synapse
What is brain slice patch clamp electrophysiology, and what advantages does it offer in studying neuronal activity? = 4
- Brain slice patch clamp electrophysiology involves making slices (~300µm) from the brain,
- preserving local circuitry, and providing direct access to entire neurons.
- Recordings can be made from
- soma,
- dendrites,
- axons,
- or multiple compartments, - offering detailed insights into neuronal function and connectivity.
In vivo patch clamp electrophysiology
- recordings made in vivo in ANESTHETISED OR HEAD FIXED MICE
- POSSIBLE TO RECORD NEURONAL ACTIVITY WITH SINGLE CELL RESOLUTION DURING BEHAVIOUR
What are the steps involved in patch clamp electrophysiology, along with the corresponding light wavelength and pressure settings?
= 5
-
Positioning:
- Light: 550nm
- Pressure: 200mbar
-
Insertion:
- Light: 820nm
- Pressure: 90 to 30mbar
-
Targeting:
- Light: 930nm
- Pressure: 30mbar
-
Sealing:
- Light: 820nm
- Pressure: 30 to -5mbar
-
Whole Cell:
- Light Overlay: 820/930nm
- Pressure: -20 to 0mbar
Describe the setup for in vivo patch clamp electrophysiology, including the equipment and conditions used.
-
Equipment:
- ‘Spherical treadmill’
- Toroidal screen
-
Conditions:
- Membrane potential (Vm) recording
- Rodent model
- Virtual reality (VR) environment
- Air environment
-Additional Information:
- ‘Linear treadmill’ may also be used
- Local field potential (LFP) recording along with Vm recording from rodents.
What are toxins, and how do they function in altering ion channel activity? = 4
- Toxins are substances produced by PLANTS AND ANIMALS to INCAPACITATE prey or defend against predators.
- They MODIFY CHANNEL FLOW BY:
- .BLOCKING THE PORE OF ION CHANNEL
- INTERACTING WITH the channel to ALTER CONFORMATIONAL CHANGES… AFFECTING OPENING AMNF CLOSING DYNAMICS
How does tetrodotoxin (TTX) function, and
how is its sensitivity utilized in classifying sodium ion channels? = 4
- Function of TTX:
Tetrodotoxin (TTX) is a marine toxin that blocks SODIUM ion flow through the pore of sodium channels.
Sensitivity in Channel Classification:
2. TTX sensitivity is used to classify sodium ion channels:
- Nav1.1 to Nav1.4 and Nav1.6 to Nav1.7 channels are blocked by nanomolar (nM) concentrations of TTX.
- Nav1.8 and Nav1.9 channels are inhibited by millimolar (mM) concentrations of TTX.
Function of TTX APPLICATIONS = 2
Nav toxins are commonly used to
- STUDY PRESYNAPTIC PROPERTIES
- DETERMINE OF A PROCESS IS ACTION POTENTIAL DEPENDENT
How does dendrotoxin (DTX) affect potassium ion channels (KV), and what impact does it have on neuronal function?
= 3
Effect on KV Channels:
- Dendrotoxin (DTX), found in snake venom, blocks several Kv channels.
Impact on Neuronal
Function:
- DTX facilitates theRELEASE OF NEUROTRANSMITTERS
- DTX broadens the WAVEFORM OF THE ACTION POTENTIAL
What are agatoxins, and how do they interact with glutamate receptors? =
- Agatoxins are toxins produced by funnel web spiders.
- ## Interaction with Glutamate Receptors:α-agatoxin preferentially blocks NMDA receptors, which are a subtype of glutamate receptors.
What are some examples of bacterial toxins, and what are their targets and lethal doses? = 5
Examples:
1. Diphtheria toxin
2. Botulinum neurotoxins
3. Tetanus neurotoxins
- Targets:
Botulinum neurotoxins target the PRESYNAPTIC TERMINAL OF MOTOR NERVES - Lethal Doses:
Lethal doses range from 0.1ng/Kg to 1ng/Kg in mice.
