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

There are several means of applying drugs to a neuron to examine their actions. During in vivo recording, drugs may be administered systemically (i.e., intravenously, intraperitoneal^, subcutaneously, intraventricularly, intramuscularly) or directly to the neuron by microiontophoresis or pressure ejection. (A) Systemic administration of a drug is useful for determining how a drug affects neurons in the intact organism, regardless of whether the action is direct or indirect. In this case, intravenous administration of the V-aminobutyric acid (GABA) agonist muscimol (solid arrows) causes a dose-dependent increase in the firing rate of this dopamine-containing neuron. (B) In contrast, direct administration of a drug to a neuron will provide information about the site of action of the drug, at least as it concerns the discharge of the neuron under study. In this case, GABA is administered directly to a dopamine neuron by microiontophoresis. In this technique, several drug-containing pipettes are attached to the recording electrode. The pH of the drug solutions is adjusted to ensure that the drug molecules are in a charged state (e.g., GABA is used at pH = 4.0 to give it a positive charge), and the drug is ejected from the pipette tip by applying very small currents to the drug-containing pipette. Because the total diameter of the microiontophoretic pipette tip is only about 5 U-m, the drugs ejected typically affect only the cell being recorded. In this case, GABA is applied to a dopamine neuron by microiontophoresis; the horizontal bars show the time during which the current is applied to the drug-containing pipette, and the amplitude of the current (indicated in nA) is listed above each bar. Note that, unlike the excitatory effects produced by a systemically administered GABA agonist in (A), direct application of GABA will inhibit dopamine neurons. This has been shown to be caused by inhibition of a much more GABA-sensitive inhibitory interneuron by the systemically administered drug and illustrates the need to compare systemic drug administration with direct drug administration to ascertain the site of action of the drug of interest.

Source. Adapted from Grace AA, Bunney BS: "Opposing Effects of Striatonigral Feedback Pathways on Midbrain Dopamine Cell Activity." Brain Research 333:271-284, 1985. Copyright 1985, Elsevier. Used with permission.

A technique that overcomes this shortcoming is the use of combined microdialysis and intracellular recording. In this approach, a microdialysis probe is used for delivering a drug to the region surrounding the neuron (Figure 5-8). To preserve the tissue surrounding the probe, the probe is lowered at a rate of 3-6 microns per second using a micromanipulator (West et al. 2002b). The microdialysis probe is then allowed to equilibrate for approximately 2-3 hours, and sharp-electrode intracellular recordings are conducted within 500 micrometers of the active surface of the probe. Provided that the probe has been inserted with care, the passive membrane properties, spontaneous spike activity, and spike characteristics of striatal and cortical neurons recorded during perfusion of artificial cerebrospinal fluid are found to be similar to those of neurons recorded in animals without microdialysis. The viability of neurons recorded proximal to the microdialysis probe is further evidenced by the increase in membrane excitability and spontaneous activity occurring within minutes after introduction of excitatory amino acid agonists (glutamate, W-methyl-D-aspartate [NMDA]) or the GABAA receptor antagonist bicuculline into the perfusate. Conversely, local reverse dialysis of tetrodotoxin eliminates action potentials and the spontaneous plateau depolarizations in prefrontal cortex neurons, indicating that these properties are dependent on synaptic inputs to these neurons. Given these findings, it is clear that this combination method has unique properties in comparison with local application via microiontophoresis. Thus, the site of application will span several hundred micrometers and thereby affect a microcircuit, including the distal dendrites and neighboring neurons of the neuron impaled. Moreover, by applying a neurotransmitter antagonist, one can ascertain the baseline effect of spontaneous neurotransmitter action on a neuron being recorded (West and Grace 2002). This approach has recently been applied to single-unit extracellular recordings (West et al. 2002a) and field potential recordings (Lavin et al. 2005; Goto and Grace 2005a, 2005b).

FIGURE 5-8. Use of a microdialysis probe for delivering drugs locally during in vivo recordings to affect local circuits.

FIGURE 5-8. Use of a microdialysis probe for delivering drugs locally during in vivo recordings to affect local circuits.

(A) In this schematic diagram, the relationship between the microdialysis probe and the intracellular recording electrode is depicted. In this case, the neuron recorded is in the striatum. The active surface of the microdialysis probe is shown in gray; this is the area through which the compound is delivered. The probe is implanted very slowly so as not to disrupt the tissue (i.e., 3-6 Urn per second) and is perfused with artificial cerebrospinal fluid for 2-4 hours to allow equilibration and settling of the tissue prior to recording. The intracellular recording electrode is then advanced, and a neuron is impaled. After recording baseline activity for 10 minutes, the perfusate is changed to a drug-containing solution to examine the effects on the neuron. (B) The histology taken after the recording shows the track of the microdialysis probe; the termination site of the probe tip is indicated by a dashed arrow. To confirm that the neuron recorded was near the probe, the neuron is filled with a stain (in this case, biocytin) so as to allow visualization of the neuron. In this case, the neuron was confirmed to be a medium spiny striatal neuron (magnified in insert). ac = anterior commissure. (C) Recordings taken from the neuron labeled in B. The top trace shows the activity of the neuron while the microdialysis probe is being perfused with artificial cerebrospinal fluid. The neuron demonstrates a healthy resting membrane potential, and spontaneously occurring postsynaptic potentials are evident. The lower trace shows the same neuron 15 minutes after switching to a perfusate containing the dopamine D2 antagonist eticlopride. The neuron shows a strong depolarization of the resting potential (by 12 mV) as well as increased postsynaptic potential activity and spontaneous spike firing. Since the eticlopride is blocking the effects of dopamine that is being released spontaneously from dopamine terminals in this region, we can conclude that basal levels of dopamine D2 receptor stimulation cause a tonic hyperpolarization of the neuronal membrane and suppress spontaneous excitatory postsynaptic potentials.

Source. Adapted from West AR, Grace AA "Opposite Influences of Endogenous Dopamine D1 and D2 Receptor Activation on Activity States and Electrophysiological Properties of Striatal Neurons: Studies Combining In Vivo Intracellular Recordings and Reverse Microdialysis." Journal of Neuroscience 22:294-304, 2002. Copyright 2002, Society for Neuroscience. Used with permission.

On the other hand, the properties that confer distinct advantages on the in vivo preparation with respect to examining how drugs act in the intact organism also limit the type of data that may be collected. Regarding drug administration, some drugs are not easily applied via microdialysis or do not readily cross the blood-brain barrier, or they may actually produce their direct actions outside of the brain via an effect on peripheral organs. Thus, although dopamine cells can be excited by microiontophoretic administration of cholecystokinin (Skirboll et al. 1981), the excitation produced by systemic administration of this peptide is mediated peripherally and affects the brain via the vagus (Hommer et al. 1985). In addition, the inability to control the microenvironment of the neuron restricts the analysis of the ionic mechanisms underlying cell firing or drug action because the researcher cannot readily control the precise drug concentration or the ionic composition of the fluid surrounding the neuron. There is also difficulty in segregating local actions of drugs versus those imposed on afferent neurons or their local axon terminals. Therefore, whereas the in vivo preparation affords many advantages with respect to examining how behaviorally or therapeutically effective drugs may exert their actions through defined neuronal systems, examination of the site of action or the membrane mechanisms underlying these responses is more readily accessible with in vitro systems.

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