Recording From Individual Neurons

Most of the data under discussion in this chapter were generated using extracellular recording techniques, but a number of much more sophisticated electrophysiologic techniques have been used to great effect in studies of LTP. These types of techniques fall into two broad categories generally referred to as sharp electrode recording and patch clamp recording. The basic difference in the two techniques is that sharp electrode recording impales the neuron with the recording electrode, while patch clamping involves forming a tight seal between the recording electrode and the cell membrane. In general, sharp electrode recording is used for current clamp experiments, or experiments where one monitors the membrane potential passively. Patch clamp experiments in general involve voltage clamp of the membrane potential. In these experiments, one can hold the membrane potential constant while measuring current flow through membrane channels. Alternatively, one can use the electrode to manipulate the membrane potential directly. This latter type of experiment was the sort used to discover pairing LTP as described in the text, and it was also used in key experiments demonstrating that depolarization of the postsynaptic cell is required for LTP induction. More recent applications of patch-clamping approaches have allowed the direct recording of dendritic membrane potential (prior studies had all recorded from the much larger cell body region), and this dendritic-patch-recording technique allowed the seminal finding of back-propagating action potentials in dendrites.

One great strength that single-cell recording techniques have in common is that they allow access to the cytoplasm of a single postsynaptic neuron. This allows the introduction of pharmacologic agents, including large proteins, specifically into the single postsynaptic cell. Application of this approach has led to a number of landmark findings in investigations of LTP, including the discovery of a necessity for postsynaptic calcium for LTP induction, and the necessity of postsynaptic protein kinase activity for LTP induction.

briefly chilled in ice-cold "cutting" saline. For the sake of completeness, I will note that the cutting saline consists of 110 mM sucrose, 60 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 28 mM NaHCO3, 500 yM CaC12, 5 mM D-glucose, 7 mM MgCl2, and 600 yM ascorbate. After this solution is made, it is saturated with 95% O2 and 5% CO2 by bubbling this gas through the solution. A standard aquarium air stone serves quite nicely for this purpose. The high Mg2+ concentration in the cutting solution helps maintain the health of the tissue during subsequent slicing, by decreasing neuronal excitability and lowering neurotransmitter release. As a practical matter, it is important to remove the brain from the animal and get it into the chilled cutting solution as quickly as possible in order to be able to get healthy slices. The current record in my lab is held by Coleen Atkins, who in her prime could get the brain out of a rat and into cutting solution in 15 seconds.

Once the brain is out and cold, one can be a little more deliberate but must still move expeditiously. Removing the hippocampus from the brain and making transverse slices, while still maintaining the tissue in a healthy state, generally involves idiosyncratic maneuvers, high anxiety, and no small amount of superstitious behavior. The principal component to success is clearly practice. Transverse slices approximately 400-^m thick are prepared with a MacIlwain tissue chopper or Vibratome (preferable but more expensive) and maintained at least 45 minutes in a holding chamber containing 50% artificial cerebrospinal fluid (ACSF) and 50% cutting saline. ACSF contains 125 mM NaCl, 2.5 mM KCl, 1.24 mM NaH2PO4, 25 mM NaHCO3, 10 mM D-glucose, 2 mM CaCl2, and 1 mM MgCl2, saturated with 95% O2 and 5% CO2 as described previously and continuously bubbled throughout the experiment.

After slices and experimenter have recovered from the dissection, they are then transferred to an electrophysiology rig and slice recording chamber, respectively, and the slices are perfused with 100% ACSF. Slices are allowed to equilibrate for about 60-90 minutes before recording begins. This is generally the point at which the experimenter eats lunch, if he is a postdoc, or dinner, if he is a graduate student.

B. Measuring Synaptic Transmission in the Hippocampal Slice

As mentioned previously, the main information processing circuit in the hippocampus is the relatively simple trisynaptic pathway, and much of this basic circuit is preserved in transverse slices across the long axis of the hippocampus. Various types of long-term potentiation can be induced at all three of these synaptic sites, and we will discuss later some mechanistic differences among the various types of LTP that can be induced. Most experiments on the basic attributes and mechanisms of LTP have been studies of the synaptic connections between axons from area CA3 pyramidal neurons that extend into area CA1. These are the synapses onto CA1 pyramidal neurons that are known as the Schaffer-collateral inputs.

In a popular variation of the basic LTP experiment, extracellular field potential recordings in the dendritic regions of area CA1 are utilized to monitor synaptic transmission at Schaffer-collateral synapses (see Figure 4). A bipolar stimulating electrode is placed in the stratum radiatum subfield of area CA1 and stimuli (typically constant current pulses ranging from 1-30 ^A) are delivered. Stimuli delivered in this fashion stimulate the output axons of CA3 neurons that pass nearby, causing action potentials to propagate down these axons. Responses are typically recorded through an amplifier coupled to a personal computer, using any of a variety of data acquisition software. Recording electrodes typically are drawn from glass filament microcapillaries using an electrode puller and are filled with ACSF.

The typical waveform consists of a "fiber volley," which is an indication of the presynaptic action potential arriving at the recording site, and the excitatory postsy-naptic potential (EPSP) itself. The EPSP responses are a manifestation of synaptic activation (depolarization) in the CA1 pyramidal neurons. For measuring "field" (i.e., extracellularly recorded) EPSPs, the parameter typically measured is the initial slope of the EPSP waveform (see Figure 4). Absolute peak amplitude of EPSPs can also be measured, but the initial slope is the preferred index. This is because the initial slope is less subject to contamination from other sources of current flow in the slice. For example, currents are generated by feed-forward inhibition due to GABAergic neuron activation. Also, if the cells fire action potentials, this also can contaminate later stages of the EPSP, even when one is recording from the dendritic region.

Extracellular field recordings measure responses from a population of neurons, so EPSPs recorded in this fashion are referred to as population EPSPs (pEPSPs). Note that pEPSPs are downward-deflecting for

Recording in Stratum Pyramidale Stimulating in Area CA1

Recording in Stratum Pyramidale Stimulating in Area CA1

Hippocampus Slice Record

FIGURE 4 Recording configuration and typical physiologic responses in a hippocampal-slice recording experiment. Electrode placements and responses from stratum pyramidale (cell body layer) and stratum oriens (dendritic regions) are shown. In addition, the typical waveform of a population EPSP is illustrated, showing the stimulus artifact, fiber volley, and population EPSP. Figure and data by Joel Selcher.

FIGURE 4 Recording configuration and typical physiologic responses in a hippocampal-slice recording experiment. Electrode placements and responses from stratum pyramidale (cell body layer) and stratum oriens (dendritic regions) are shown. In addition, the typical waveform of a population EPSP is illustrated, showing the stimulus artifact, fiber volley, and population EPSP. Figure and data by Joel Selcher.

stratum radiatum recordings (see Figure 4). If one is recording from the cell body layer (stratum pyramidale), the EPSP is an upward deflection; if the cells fire action potentials, the EPSP has superimposed upon it a downward deflecting "spike", the population spike. As mentioned earlier, for both stratum radiatum and stratum pyramidale recordings, the EPSP slope measurements are taken as early as possible after the fiber volley to eliminate contamination by population spikes.

Test stimuli are typically delivered and responses recorded at 0.05 Hz (once every 20 seconds); every six consecutive responses over a 2-minute period are pooled and averaged. As a prelude to starting an LTP experiment, input/output (I/O) functions for stimulus intensity versus EPSP magnitude are recorded in response to increasing intensities of stimulation (e.g.

from 2.5 to 45.0 ^A, see Figure 5). For the remainder of the experiment, the test stimulus intensity is set to elicit an EPSP that is approximately 35-50% of the maximum response recorded during the I/O measurements. Baseline synaptic transmission at this constant test stimulus intensity is usually monitored for a period of 15-20 minutes to ensure a stable response.

Once the health of the hippocampal slice is confirmed as indicated by a stable baseline synaptic response, LTP can be induced using any one of a wide variety of different LTP induction protocols. Many popular variations include a single or repeated period of 1-second, 100 Hz stimulation (with delivery of the 100-Hz trains separated by 20 seconds or more) where stimulus intensity is at a level necessary for approximately half-maximal

Steps Synaptic Transmission

FIGURE 5 An input-output curve and typical LTP experiment. (A) This panel shows the relationship of EPSP magnitude versus stimulus intensity (in microamps) and the same data converted to an input-output relationship for EPSP versus fiber volley magnitude in order to allow an evaluation of postsynaptic response versus presy-naptic response in the same hippocampal slice. (B) This panel illustrates a typical high-frequency stimulation-induced potentiation of synaptic transmission in area CA1 of a rat hippocampal slice in vitro. The arrow indicates the delivery of 100 Hz (100 pulses/second) synaptic stimulation. Data courtesy of Ed Weeber and Coleen Atkins.

Time (ruin)

FIGURE 5 An input-output curve and typical LTP experiment. (A) This panel shows the relationship of EPSP magnitude versus stimulus intensity (in microamps) and the same data converted to an input-output relationship for EPSP versus fiber volley magnitude in order to allow an evaluation of postsynaptic response versus presy-naptic response in the same hippocampal slice. (B) This panel illustrates a typical high-frequency stimulation-induced potentiation of synaptic transmission in area CA1 of a rat hippocampal slice in vitro. The arrow indicates the delivery of 100 Hz (100 pulses/second) synaptic stimulation. Data courtesy of Ed Weeber and Coleen Atkins.

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