Fig 1.
The isolated corpus callosum (CC) for suction electrode (SE) recording of compound action potentials (CAPs).
(A) 400 μm-thick coronal slice of mouse brain before (A1) and after (A2) dissecting out the CC. The isolated CC is shown in A2 above the slice. (B) Field microelectrode (ME) recording of CC CAPs within brain slices, showing two negative peaks conventionally referred to as N1 and N2. (C) Our novel “whole” CC recording of CAPs from isolated CC using SE stimulation and recording, showing two positive peaks 1 and 2. The CAPs in B and C were maximal and were averaged from 6 consecutive traces taken at 10 s intervals. Stimulus artifacts are marked with (*) in (B) and (C). Dashed lines on CAPs explain the measurements of peak amplitudes from their projected bases. (D) Amplitudes of CAP peaks recorded at 1.5–2 mm conduction distances in different slices using ME (black circles) or in isolated CCs using SE (open circles) at room temperature. Horizontal bars in (D) indicate mean amplitudes of ME and SE recorded CAP peaks. Note larger amplitudes of SE-recorded “whole” CC CAP peaks compared to ME recordings. The “whole” CC SE recording more adequately characterizes the axonal population of CC spanning between the stimulated and recorded sites compared to ME recording.
Fig 2.
Stimulus-response relationship and refractoriness of “whole” CC compound action potential peaks.
This figure shows further details on “whole” CC CAP from in Fig 1C. (A) Superimposed CAPs recorded at varying stimulus intensities. (B) stimulus-response plot of amplitudes of peaks 1 and 2 for CAPs shown in (A). (C) Superimposed CAPs evoked by paired stimuli of varying intervals, with 0.5 ms interval increments. Individual traces in (B) and (C) are averaged from 6 consecutive recordings at each stimulation intensity, taken at 10 s intervals. (D) Plot of peak amplitudes of 2nd CAP in the pair as a function of inter-stimulus interval, normalized to peak amplitudes of the 1st CAP in the pair. The absolute refractory period of both peaks is 1.5 ms, while the relative refractory period of both peaks exceeded 15 ms. Conduction distance 1.5 mm; room temperature.
Fig 3.
Rostro-caudal patterns of “whole” CC CAPs compound action potentials.
(A) Seven consecutive 400 μm-thick coronal slices from a single mouse brain. (B) CCs isolated from slices shown on (A). The rostral-most coronal slice was the first from rostral end of the brain where the CC had a continuous appearance across the midline, and its corresponding isolated CC is referred to hereafter as CC1. The rostro-caudal position of the slice from which CC3 was isolated corresponds approximately to bregma 0, identified by the presence of anterior commissure in the lower part of the slice, continuously running across the midline. (C) Suction electrode (SE) recordings from CCs shown in (B). Maximal CAPs from each CC, recorded at 4 mm (dashed traces) and 2 mm (solid traces) conduction distance. The recordings started at 4 mm, followed by drawing the CCs deeper into the SE’s to reach a 2 mm conduction distance, while keeping the arrangement symmetrical around the CC mid-point. The smaller amplitude of CAPs recorded at 4 mm distance likely reflects a lower number of axons in continuity between the stimulated and recording sites, in agreement with decreased CAP areas. Each trace represents an average of 6 consecutive recordings taken at 10 s intervals. All recordings shown here were done at room temperature.
Fig 4.
Detailed characteristics of “whole CC CAPs recorded at 4 mm conduction distance from CC3 and CC6.
(A1, A2) Digitally averaged “whole” CC CAPs recorded from 14 CC3s (A1) and 29 CC6s (A2) from different mouse brains. The CC3 and CC6 were identified as explained in Fig 3. Note different voltage scales in A1 and A2. (B) Detailed statistical comparison of “whole” CC3 and CC6 CAP parameters: ampl.—amplitude; CV—conduction velocity; peak2/peak1 ampl.—ratio of peak amplitudes; peak2/peak1 area—ratio of peak areas. ** p<0.01; *** p<0.001. All recordings were performed at room temperature.
Fig 5.
Comparison of “whole” CC CAPs of wild type (wt) and dysmyelinated shiverer (shi-/-) mice.
(A1, A2) Superimposed “whole” CC CAPs recorded at varying stimulation intensities in CC3 (A1) and CC6 (A2). Each trace in A1 and A2 represents an average of 6 consecutive recordings taken at 10 s intervals. (B1, B2) Digitally averaged “whole” CC CAPs recorded from wt (thick gray traces) and shi-/- (thin black traces) CC3 (B1, digitally averaged from 8 experiments) and CC6 (B2, digitally averaged from 11 experiments). CAPs from wt CCs, (digitally averaged, from Fig 4A1 and 4A2; brought to same scale with shi-/- CAPs) are shown in B1 and B2 as thick gray traces. Dashed arrows with question marks show hypothetical relationships between peaks of wt and shi-/- CAPs (see text). (C1, C2) Statistical comparison of main parameters of wt and shi-/- “whole” CC CAPs. ** p<0.01; *** p<0.001.
Fig 6.
Temperature dependence of “whole” CC CAPs.
(A) Superimposed “whole” CC CAPs recorded during the increase of the temperature of perfusing solution from 21°C to 36°C. (B) Superimposed CAPs recorded at 21°C (thick gray trace) and 36°C (thin black trace). The dashed arrows show the changes of CAP peaks from room to high temperature. The CAPs shown in this figure were recorded without averaging to reflect the changes during the temperature test. From these experiments, we estimated the Q10 of conduction velocities of peak 1 (myelinated axons) and peak 2 (non-myelinated axons) as 1.33 and 1.37, respectively.
Fig 7.
Pronounced effects of oxygen glucose deprivation (OGD) on “whole” CC CAPs.
(A) Superimposed CC CAPs recorded before (control), during OGD and during washout. (B1, B2) Time course of changes of absolute (B1) and normalized (B2) amplitudes of peaks 1 and 2 during 10-minute OGD and 20-minute washout. (C) Superimposed CAPs before, during and after OGD. (A-C) Representative data from a single experiment on CC6. The CAPs were recorded without averaging to reflect the fast changes during the test. (D) Summary of OGD–induced changes of CAP peaks after 10 min OGD and 20 min washout in CC3 and CC6. Temperature 35.5°C–36.5°C.
Fig 8.
Pronounced effects of potassium channel blocker 4-aminopyridine (4-AP) on “whole” CC CAPs.
(A and B) Superimposed “whole” CC CAPs recorded from CC6 during administration of 0.5 mM 4–AP, shown at two different time scales to illustrate the pronounced prolongation of CAP decay. Note the two visually identifiable slow peaks (peak 2a and peak 2b) at the maximal effect of 4-AP, emerging by gradual changes of peak 2 shape from control to 4-AP. The CAPs were recorded without averaging to reflect the fast changes during the test. (C-E) Time course of changes of CAP area (C) and peak amplitude (D, absolute; E, normalized) during administration of 4-AP. The half-maximum of the effect of 4-AP was reached within 3.5–4 minutes. The effects were reversible (not shown). Room temperature.
Fig 9.
A third peak of “whole” CC CAPs.
(A) Superimposed CAPs evoked by varying stimulation intensities in an experiment on CC6. (B) Same traces as in (A), shown at higher gain and separated vertically to illustrate the pattern of appearance of peak 3 at higher stimulation intensities. Each trace in A and B represents an average of 6 consecutive recordings taken at 10 s intervals. (C) Top (maximal) CAP from (B); dashed lines explain the measurement of the amplitudes of peaks 1, 2 and 3 from their projected bases. (D–E) Stimulus–response plots of the amplitudes of peaks 1, 2 and 3 shown in absolute (D) and normalized (E) values to illustrate different thresholds of CAP peaks, and specifically the higher threshold of peak 3 compared to peak 2. (F) Statistical comparison of the amplitudes of CAP peaks 2 and 3 in CC3 and CC6. (G) Comparison of peak3/peak2 amplitude ratios (peak3/peak2 ampl) and conduction velocities (CVs) of peaks 2 and 3 in CC3 and CC6. Room temperature. *** p<0.001.