Intracellular Recordings

With intracellular recording, an electrode with a much smaller tip and a much higher electrical resistance than that used with extracellular recording is inserted into the membrane of the neuron. Although the tip of the electrode is smaller, the signal measured is much larger than that with extracellular recording, because it measures the potential difference across the membrane directly rather than relying on transmembrane current density changes outside the neuron. As a result, one can measure electrical activity occurring within the neuron that would be nearly impossible to measure extracellularly, such as spontaneously occurring or evoked (via afferent pathway stimulation) electrical potentials generated by neurotransmitter release (or postsynaptic potentials). Furthermore, because the membrane potential of the neuron may be altered by injecting depolarizing or hyperpolarizing current into the cell through the electrode, the equilibrium potential (also known as the reversal potential) of the response may be determined. In addition, the overall conductance of the membrane may be measured by injecting known levels of current and measuring the membrane voltage deflection produced. By applying this information using Ohm's law, the input resistance of the cell can be determined. This could be important in assessing drug effects. A drug could increase the input resistance of the membrane, making it more responsive to current generated by afferent synapses, without changing the membrane potential of the neuron. Indeed, such a condition has been proposed to underlie the mechanism through which norepinephrine exerts a "modulatory" action—that is, increasing the amplitude of the response of a neuron to a stimulus without affecting its basal firing rate (which has also been described as increasing its "signal-to-noise" ratio; Freedman et al. 1977; Woodward et al. 1979).

The intracellular recording electrode can also be used to inject specific substances into the neuron. For example, second messengers or calcium chelators can be introduced into the neuron to examine how they regulate the excitability and modulate the baseline activity of the neuron or how they affect the neuron's response to drugs (Figure

5-4). Furthermore, by injecting the neuron with a fluorescent dye or enzymatic marker, the morphology of the specific cell impaled may be recovered and examined (Figure 5-5). This technique can be combined with immunocytochemistry to examine the neurotransmitter synthesized by the cell under study (e.g., Grace and Onn 1989).

FIGURE 5-4. Effects of intracellular manipulations of cGMP levels on basal activity of striatal medium spiny neurons.

FIGURE 5-4. Effects of intracellular manipulations of cGMP levels on basal activity of striatal medium spiny neurons.

Intracellular application of selective pharmacological agents enables the investigator to examine the direct effects of these agents on the membrane activity of single neurons as well as to manipulate intracellular second-messenger systems. This figure demonstrates that manipulation of intracellular cyclic guanosine monophosphate (cGMP) levels potently and specifically modulates the membrane activity of striatal medium spiny neurons in a manner that cannot be achieved by extracellular application of drugs. Striatal neurons were recorded after intracellular application (~5 minutes) of either A) vehicle (control), a 0.5% solution of dimethylsulfoxide (DMSO); B) the drug 1W-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), which blocks cGMP synthesis by inhibiting the synthetic enzyme guanylyl cyclase; C) ODQ plus cGMP; or D) the drug zaprinast, which inhibits phosphodiesterase enzymes responsible for degrading cGMP. (A) Left: After vehicle injection, striatal neurons exhibited typical rapid spontaneous shifts in steady-state membrane potential and irregular spontaneous spike discharge. Right: Time interval plots of membrane potential activity recorded from control neurons demonstrated bimodal membrane potential distributions indicative of bistable membrane activity. (B) Left: Striatal neurons recorded after ODQ injection exhibited significantly lower-amplitude depolarizing events compared with vehicle-injected controls and rarely fired action potentials. Right: The depolarized portion of the membrane potential distribution of neurons recorded after ODQ injection was typically shifted leftward (i.e., hyperpolarized) compared with controls. (C) Left: Striatal neurons recorded after ODQ and cGMP coinjection rarely fired action potentials but exhibited high-amplitude depolarizing events with extraordinarily long durations. Right: The membrane potential distribution of neurons recorded after ODQ and cGMP coinjection was similar to that of controls, indicating that cGMP partially reversed some of the effects of ODQ. (D) Left: Striatal neurons recorded after intracellular injection of zaprinast exhibited high-amplitude depolarizing events with extraordinarily long durations. Additionally, all of the cells fired action potentials at relatively high rates (0.4-2.2 Hz). Right: The membrane potential distribution of these neurons was typically shifted rightward (i.e., depolarized) compared with controls. Because zaprinast blocks the degradation of endogenous cGMP, we can conclude that basal levels of cGMP depolarize the membrane potential of striatal neurons and facilitate spontaneous postsynaptic potentials. Arrows indicate the membrane potential at its maximal depolarized and hyperpolarized levels.

Source. Adapted from West AR, Grace AA: "The Nitric Oxide-Guanylyl Cyclase Signaling Pathway Modulates Membrane Activity States and Electrophysiological Properties of Striatal Medium Spiny Neurons Recorded In Vivo." Journal of Neuroscience 24:1924-1935, 2004. Copyright 2004, Society for Neuroscience. Used with permission.

FIGURE 5-5. Intracellular staining of neuron recorded intracellularly.

FIGURE 5-5. Intracellular staining of neuron recorded intracellularly.

Copyright © American Psychiatric Publishing, Inc., or American Psychiatric Association, unless otherwise indicated in figure legend, All rights reserved.

During intracellular recordings, the recording pipette is filled with an electrolyte to enable the transmission of membrane voltage deflections to the preamplifier. The electrode may also be filled with substances, such as a morphological stain, for injection into the impaled neuron. In this example, the electrode was filled with the highly fluorescent dye Lucifer yellow. Because this dye has a negative charge at neutral pH, it may be ejected from the electrode by applying a negative current across the electrode, with the result that the Lucifer yellow carries the negative current flow from the electrode and into the neuron. Because this dye diffuses rapidly in water, it quickly fills the entire neuron impaled. The tissue is then fixed in a formaldehyde compound, the lipids clarified by dehydration-defatting or by using dimethylsulfoxide (Grace and Llinas 1985), and the tissue examined under a fluorescence microscope. In this case, a brightly fluorescing pyramidal neuron in layer 3 of the neocortex of a guinea pig is recovered.

Inserting an electrode into the membrane of a cell to measure transmembrane voltage and manipulating its membrane by injecting current are commonly known as current clamp, because the amount and direction of ionic current crossing the membrane of the cell can be controlled by the experimenter, such as when determining the reversal potential of a response. Another technique that is effective in neurophysiological research is the use of voltage clamping. With voltage clamping, the membrane potential of the neuron is maintained at a set voltage level by injecting current into the cell. This is achieved by rapid feedback electronics that adjusts the current injected to accurately offset any factors that may act to change this potential. Thus, when the neuron is exposed to a drug that opens ion channels, the effect of the ionic influx is precisely counterbalanced by altering the current injected into the neuron by the voltage clamp device. The amount of additional current that must be injected into the neuron to maintain the membrane potential at its set point is therefore the inverse of the transmembrane current generated in response to the drug. By using specific ion channel blockers or by altering the extracellular ionic environment of the neuron, the precise ionic mechanism and conductance changes induced in a neuron by a drug or neurotransmitter may be determined.

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