As the MS/DBB contains two major cell types (cholinergic and GABAergic), which exert markedly different effects on their target cells, it is a matter of interest as to whether the two types can be distinguished by the waveforms recorded in the extracellular compartment. It is of especial interest as to whether classifying MS/DBB cells using this method yields differences in bursting behaviour or the phase of theta at which the cells fire.
Intracellular studies of GABAergic and cholinergic MS/DBB cells have revealed a number of differences, most notably the presence of a slow
afferhyperpolarisation (AHP) in cholinergic and a fast AHP in GABAergic cells (Griffith and Matthews, 1986; Markram and Segal, 1986; Griffith, 1988;
Markram and Segal, 1990; Gorelova and Reiner, 1996). Comparison of intra- and extracellular recordings from MS/DBB neurons in a slice preparation
(Matthews and Lee, 1991) has demonstrated that the slow repolarisation phase in cholinergic cells causes a "hump" in the action potential's derivative with respect to time. Extracellularly-recorded potentials from cells which had similar firing characteristics (slow versus fast rhythmic firing) and a similar response to apamin (a blocker of the calcium-activated potassium channel responsible of the long afterhyperpolarisartion) also show a very similar "hump" (figure 6.1).
Matthews and Lee also distinguished MS/DBB cells on the basis of their action potential (AP) length: those cells with a “hump” also had an AP length of 1.8- 5.0ms measured at the baseline (these experiments were conducted at a temperature of 32°C, thus slowing down physiological processes), whereas those without had an AP length of 0.6-2.4ms. Cells with a “hump” also fired at a
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S-AHPCELL F-AHP CELL
30«V Imi INTRA -60mV dV/dt EXTRA
Figure 6.1. The action potential shapes of MS/DBB cells with slow (s-AHP) and fast (f-AHP) afterhyperpolarisations respectively. (A) The intracellular record of an action potential triggered by a 3 ms threshold depolarising pulse from a holding potential of -60mV (B) The first derivative of the action potential trace, displaying the "hump" produced by the slow afterhyperpolarisation. (C) Extracellular records from slow rhythmic firing (left) and fast rhythmic firing (right) MS/DBB neurons, which display a close resemblance to the derivatives of the s-AFIP and f-AHP cells respectively. Taken from Matthews and Lee (1991)
"Cholinergic"
GABAergic"
Figure 6.2. Examples of mean extracellular waveforms from putative cholinergic cells (top left group) which display a "hump" in the repolarisation phase of their action potential (see text), and putative GABAergic cells (bottom right group) which do not have such a hump. Each record comprises four waveforms, one from each electrode in the tetrode bundle, representing the average o f several thousand action potentials recorded over a 32 minute period. Calibration bars: 1msec, lOOpV
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Figure 6.3. Examples of two cells which could not easily be classified as G ABA or cholinergic by subjective examination o f their wave forms. Each record comprises four waveforms, one from each electrode in the tetrode bundle, representing the average o f several thousand action potentials recorded over a 32 minute period. Calibration bars: 1msec, lOOpV.
low rate, typically 5 to 6 Hz with a maximum of 20Hz. N on-”hump” cells fired at rates of 15 to lOOHz.
R esults
A similar pattern of action potential shapes was found in this study. Figure 6.2 shows cells which can clearly be classified into the "hump" (putative
cholinergic) and "no-hump" (putative GABAergic) classes. Unfortunately, many of the cells produced waveforms that could not easily be assigned to one or the other group, as shown in figure 6.3. Given this I attempted to find an objective method of classifying the waveforms.
The first method involved simply measuring the length of the action potential. As the computerised waveform sampling system used only stores the first millisecond, the full waveform including the return to resting potential was not available for analysis. The length was determined, therefore, by finding the point in the repolarisation phase at which the largest of the four waveforms from the tetrode closed to within a certain value of the final sample. This value was set at l/50th of the full range. This value was selected from several other candidates as the one yielding the best spread of values. Even so, it did not yield good separation, as shown in figure 6.4. Figure 6.5 plots action potential length against the mean preferential phase of the corresponding cell, and shows no clear relationship.
An alternative, and more successful method involved computing the power spectrum of the extracellular waveform. As those waves which included a "hump" would necessarily incorporate higher frequencies, it was thought that
Page 190 trigger point
I
1 /SQth of full range thresholdAction potential length
16 14
1 2
0 . 3 2 5 0 . 37 5 0 . 425 0 . 4 7 5 0 . 525 0 . 5 7 5 0 . 625 0 . 67 5 0 . 725 0 . 77 5 0 . 82 5 0 . 8 7 5 0 . 925
length (msec)
Figure 6.4. Attempts to separate cells on the basis o f the length o f their action potential did not yield fniithil results. At the top is a diagram demonstrating the method used to determine action potential length. As the length was measured from the start of the record, which was always 0.2msec before the wave crossed the threshold to trigger the start o f recording, this method is potentially sensitive to variations in the height o f the action potential. T he histogram below plots the distribution o f action potential lengths produced by this method. As can be seen, it is difficult to separate the cells into two classes by this criterion.
300 - 1 50 - 0 X a 2 ^ A ° 01 200 - ♦ X « n
%
□ X 1 0 0 - □ A t e ^ □ ♦ 4 X X A X 50 - ^ ^ X 350 - X ^ X ♦Type la XX X ♦ oType 1b ^ □ □ A ^ y p e lc □ xTypeZ 0 0.2 0.4 0.6 0.8Action Potential Length (msec)
Figure 6.5. Action potential length as measured by the method elaborated in figure 6.4 plotted against the preferential phase of the respective cell. T his method yields no clear distinction in terms of either firing behaviour o f the cell or its preferential phase.
Page 192 o Ln IS. 00 Ln Ln Ln - Î5 m O nj Is. m "4- m nj O O O Ln r\i Frequency (Hz)