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Electrical Paired Pulse Stimulation. Two electrical stimuli (identified by the presence of a stimulus artifact) evoke two inward currents. Compared to baseline (dark trace), the addition of drug (light trace) increased the amplitude of both pulses without changing the paired pulse ratio.
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The purpose of the stimulus isolator is to allow the patch-clamp system to deliver high voltage to the electrode. The current of the stimulator is set with the knob on the top right of the stimulus isolation unit. According to Ohm’s Law, the voltage the stimulator will produce when triggered is a function of the current (set by the knob) and the resistance (a function of the type of stimulator being used). Although there is a “unipolar/bipolar” switch, but unipolar should virtually always be selected. There is a “polarity select” button, which can be toggled to invert the polarity of the outputs. The preferred output polarity is that which produces the cleanest waveform on the recorder and should be determined at recording time by the experimenter. Often, one polarity will be more effective at reliably stimulating a slice than the other.
Copy file name to clipboardExpand all lines: content/pages/experiments.md
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The membrane test protocol is a 40 sweep voltage-clamp protocol which rapidly steps between two voltages. From this protocol, many passive membrane properties can be determined: steady state current (Is), membrane resistance (Rm), access resistance (Ra), and capacitance (Cm).
One of the most common experiments records a membrane test repeatedly as a drug is applied, and passive properties (Ih, Rm, Ra, and Cm) are continuously assessed. What each of these features mean is described in the previous chapter on membrane tests.
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Many advanced experiments (studying spontaneous sIPSC/sEPSCs, I/V ramps, evoked synaptic currents, etc.) include a voltage clamp step at the start of every sweep. A membrane test analysis similar to the one displayed here can be performed on any voltage-clamp sweep with a square pulse voltage step in it, so you will find most voltage-clamp experiments contain this voltage step at the beginning of every sweep.
These protocols use fast voltage steps (0202) and slower voltage ramps (0203) to investigate current/voltage relationships in neurons. The voltage step protocol is best for revealing fast voltage-dependent current transients (e.g., visualizing an excitatory current due to a hyperpolarization-activated channel). While the purpose of voltage steps is to maximize transient currents, the purpose of voltage ramps is the opposite – minimizing transients to produce the smoothest sweeps ideal for averaging to create an I/V plot.
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-**Half-width (ms)** – the width of the AP (in time units). Since APs are triangular shaped (wider at the base than the top), this width is measured half-way between the base of the AP and its peak (hence the term half-width).
We saw earlier that injection of excitatory current through the patch pipette can produce action potentials. The plot where AP frequency is plotted against the current applied is called an AP gain curve and can be used to distinguish different classes of neurons.
Spontaneous excitatory and inhibitory synaptic currents can be visualized in voltage-clamp confirmation as sEPSCs and sIPSCs. Analysis is best performed when only one current is **isolated**, either by clamping at the reversal of other currents or by blocking other currents pharmacologically. When currents are isolated, they can be individually detected based on parameters (like their amplitude). The **parametric event detection** of spontaneous events can be used to quantify how drugs influence their frequency (usually an indication that a drug acts to change AP frequency of pre-synaptic neuron) or amplitude (usually an indication that a drug acts to change post-synaptic membrane resistance).
> 🤓 **Nerd Alert:** Access resistance (Ra) is calculated from the theoretical instantaneous peak of the capacitive transients. In practice just measuring the peaks allows often a good-enough estimation of Ra (especially if your goal is just to access Ra stability over a time course), but deriving this value perfectly involves curve-fitting (to the simple exponential decay curve) and back-calculating the actual peak (which was partially degraded by the based low-pass filter). This is not really important for Ra itself, but since Ra is used to calculate Rm it is critically important that Ra be calculated in this way if Cm is to be accurate. In this case, Cm is calculated using the decay constant (Tau, τ) of the capacitive transient (this is what ClampEx does to calculate Cm), but this is highly sensitive to small changes in Ra and also highly sensitive to the hardware lowpass filter. An alternative method of Cm determination made from voltage-clamp ramps is shown here after the voltage-clamp step. This form of Cm determination is often more accurate, especially for small cells. It is also highly insensitive to the presence of sIPSCs and sEPSCs. For more information see [Exploring the Voltage-Clamp Membrane Test](https://swharden.com/blog/2020-10-11-model-neuron-ltspice/).
Copy file name to clipboardExpand all lines: content/pages/optogenetics.md
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**Channelrhodopsin-2 (ChR2)** is a light-gated cation channel which can be selectively placed in specific types of neurons thanks to genetic engineering. A flash of blue light transiently opens ChR2, allowing cations (primarily Na+) to rush into the cell, often resulting in action potentials. When combined with genetic engineering to selectively express ChR2 only in certain classes of neurons, blue light can be used to activate specific subpopulations populations of neurons. Optogenetics is the field of study where genetic engineering is used to express light-sensitive ion channels in cells. Light-gated ion channels are called opsins. While ChR2 is the most commonly used opsins in neuroscience, halorhodopsin (light-gated anion channel) is also used to produce light-sensitive inhibitory currents.
Neurons with ChR2 are easy to identify because they will rapidly respond to light. The response to light is always extremely fast (less than 1ms from onset). In this example, a CRH-ChR2+ neuron is shown to produce an excitatory current in voltage clamp in response to blue light stimulation (shaded area).
Although one can record from ChR2+ neurons, typically the most common use of ChR2 in acute brain slice preparations is to facilitate the study of synaptic neurotransmission. In these lines of experiments, pre-synaptic neurons express ChR2 and post-synaptic neurons (targeted for patch-clamp analysis) receive light-evoked EPSCs in response to blue light.
There are two ways to know the neuron you are recording is receiving an EPSC in response to blue light (synaptic in origin) vs. directly responding to blue light itself (what you would expect if the recorded neuron expressed ChR2). First, light-evoked synaptic currents take a few ms to occur (due to the charging of the presynaptic axon, propagation of action potential release of neurotransmitter, etc). Second, pharmacological blockade of the receptors underlying the synaptic current will eliminate the light-evoked current. This result would not be observed if the post-synaptic neuron expressed ChR2.
**Reversal potential of isolated spontaneous GABAA (chloride) currents:** Voltage clamp trace at 3 different voltages demonstrates how cell voltage can determine current direction. In this example, GABAA currents (mediated by Cl- ions) are observed. Pharmacological inhibition of glutamate receptors means these transients are likely all GABA-mediated currents. In these conditions, the reversal potential for Cl- is near -55 mV. Clamping near the Cl- reversal potential minimizes the size of these currents. Clamping below or above the reversal potential magnifies these currents in opposite directions. At negative voltages, opening of chloride channels causes outflow of Cl- ions (inward current). At positive voltages, opening of the same channel causes inflow of Cl- ions (outward current).
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By voltage-clamping a neuron at the reversal potential of a specific ion, an experimenter can effectively silence currents produced by the flow of that ion. This technique is commonly used to measure isolated GABAA (chloride-mediated) currents and isolated AMPA (sodium-mediated) currents in the same cell by clamping at their reversal potentials in tandem. In addition, the experimenter can clamp between two reversal potentials to simultaneously view both currents which will diverge in opposite directions (sometimes called biphasic current transients).
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**Patch-clamp technique** uses a glass pipette filled with a conductive liquid to measure voltage inside the neuron. The pipette is lowered onto the surface of a cell until it forms a seal with the cell membrane (called a **cell-attached** or on-cell configuration), as seen in A. Although cell-attached configuration allows some measurements to be performed, more advanced electrical measurements can be performed by breaking the small patch of cell membrane separating the patch pipette internal solution from the cellular cytoplasm. A small burst of negative pressure (suction) ruptures this patch, turning a cell-attached configuration to a **whole-cell configuration**, as seen in C. Although it is possible to pull part of the cell membrane away from the cell (as in B and D) to perform smaller-scale studies (i.e., single channel recordings), these techniques are best performed using specialized electrical and mechanical equipment which is not discussed by this document.
**Comparison of different cell modes:** (A) When sealed, cells are in cell-attached / on-cell mode. A patch could be ripped-off the cell to study ion channels in a small piece of membrane (B), but usually we proceed with suction to break into cell and establish whole-cell mode (C). Pulling away from the cell at this point could result in an outside-out patch (D).
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