Spreading depression appeared during orthodromic-HFS with monophasic but not biphasic pulses
To investigate the different effects between biphasic and monophasic pulses, HFS sequences of pulses were delivered through the stimulation electrodes placed at the Schaffer collaterals for the orthodromic-HFS (O-HFS) and at the alveus fibers for the antidromic-HFS (A-HFS), respectively (Fig. 1a). The stimulation electrodes were a concentric bipolar structure (Fig. 1b). For the stimulations of a single pulse with an identical current intensity, the mean amplitude of orthodromically-evoked population spike (OPS) induced by a monophasic pulse (8.62 ± 1.97 mV, n = 5) was not significantly different from that induced by a biphasic pulse (8.40 ± 2.16 mV, n = 6; t-test, P = 0.95. Fig. 1c and 1d), which indicated a similar excitation action of the two types of pulses.
During the O-HFS of 1-min 200 Hz of biphasic pulses, OPS events only appeared in the initial several seconds of O-HFS (Fig. 2a). After the disappearance of OPS, multiple unit activity (MUA) continued to the end of the O-HFS with a firing rate of unit spikes higher than baseline. A silent period (10 - 30 s) without MUA appeared immediately following the end of O-HFS, indicating that the unit spikes during the late O-HFS period were induced by the stimulation. During the period of O-HFS, an antidromic-test (A-test) pulse was applied every 5 seconds (i.e., 0.2 Hz) at the alveus fibers to evaluate the excitability of the CA1 neurons. The single A-test pulses and orthodromic-test (O-test) pulses were also applied before and after O-HFS to evaluate the baseline and the recovery state of neuronal activity, respectively. Large antidromically-evoked population spike (APS) evoked by A-test pulses persisted throughout the 1-min O-HFS, and the mean amplitude of these APS (7.26 ± 5.59 mV, n = 6) was ~17% greater than the corresponding baseline level (6.19 ± 3.19 mV, n = 6; paired t-test, P < 0.05). The results indicated that the sustained O-HFS increased the excitability of the CA1 neurons. About 4 min after the end of O-HFS, both test APS and test OPS evoked by single pulses recovered to the baseline level. In addition, no SD event appeared in all of the 6 rats with the 200 Hz biphasic O-HFS.
However, SD events appeared in 4 of the 5 rats with the 200 Hz monophasic O-HFS (Fig. 2b). The initial neuronal responses induced by the monophasic O-HFS was similar to that induced by the biphasic O-HFS: large OPS appeared at first, then OPS disappeared and dense MUA appeared. However, an SD appeared later with a slow waveform lasting 4.31 ± 3.11 s (n = 4). At the same time, the MUA disappeared completely and the A-test pulses were no longer able to induce an APS, indicating a silence of neuronal activity. The MUA did not appear until 3.51 ± 2.47 min (n = 4) after the end of O-HFS (Fig. 2b, bottom). By this time, the amplitude of test APS recovered to ~ 80% of the baseline level. However, the test OPS did not recover even ~25 min after the end of O-HFS, while the test APS had almost recovered to baseline level (89.5 ± 9.7%, n = 4. Fig. 2b, middle). The results indicated that 1-min persisted 200 Hz of monophasic O-HFS generated abnormal reactions of the neuronal population.
To show the spread of SD waveforms, we used the recording electrode array with four shanks and total 16 contacts (Fig. 1a & Fig. 3a). The waveforms of OPS and APS along the shanks in baseline recordings indicated the locations of each recording contact in the different stratums of CA1 region [17]. Because the signal recordings in the study were AC-coupled (0.3 - 5000 Hz), the SD waveform appeared as a trough similar to previous reports [18]. The SD trough appeared first in the stratum radiatum (S. rad.) of hippocampal CA1 region (Fig. 3b) and accompanied by a burst of population spikes (60 - 80 spikes/s) in the stratum pyramidale (S. pyr.). The burst was obvious in the filtered signals greater than 10 Hz (Fig. 3b right). Then, the SD trough propagated slowly to the CA1 layers of S. pyr. and stratum oriens (S. ori.) at a speed of 90 ± 51 μm/s (n = 4) in the perpendicular direction, characterized by the movement of the negative peak of SD trough along the recording shanks (Fig. 3b, hollow triangles). Also, the SD trough moved at a speed of 826 ± 627 μm/s (n = 4) in the S. pyr. layer transversely among the recording shanks (Fig. 3a right, blue dotted line). The characteristics of the SD events, including the waveform, the accompanied burst of population spikes, the slow travelling speed and the silence of neuronal electrical activity, were consistent with previous reports [18, 19].
The statistical results showed that the SD incidence during 200 Hz monophasic O-HFS (4/5) was significantly greater than the incidence during 200 Hz biphasic O-HFS (0/6; Fisher’s exact test, P < 0.05). In addition, with a decrease of the O-HFS frequency from 200 to 100 Hz, no SD events were observed with monophasic O-HFS (five rats) or with biphasic O-HFS (six rats). Therefore, for the data of monophasic O-HFS only, the SD incidence during 100 Hz O-HFS (0/5) was significantly lower than that during 200 Hz O-HFS (4/5; Fisher’s exact test, P < 0.05).
These results indicated that O-HFS of monophasic pulses with a higher stimulation frequency can generate SD events in the hippocampal CA1 region and affect the orthodromic pathway persistently. Because the generation of OPS by the stimulation at afferent fibers involves both the axonal conductions and the synaptic transmissions, the non-recovery of OPS after the monophasic O-HFS could have been caused by potential damages in the axons and/or synapses. To verify whether the monophasic HFS could cause damages in axons, we next inspected the responses of CA1 neurons to the A-HFS at their own axons (i.e., the alveus) without involving synaptic transmissions.
No spreading depression but more attenuation of APS amplitudes caused by A-HFS of monophasic pulses
To investigate the neuronal responses to the A-HFS of biphasic and monophasic pulses, we analyzed each APS evoked by each pulse of A-HFS. During the 1-min 200 Hz A-HFS of biphasic pulses, APS was able to follow each stimulation pulse. However, the APS waveforms with a large amplitude only appeared at the initial period. The APS amplitudes decreased rapidly with the proceeding of A-HFS (Fig. 4a), which may be caused by the depolarization block of axons [5, 20]. After the end of A-HFS, the test APS recovered to ~70% of baseline level in ~1 min and to 92.2 ± 21.0% (n = 4) of baseline level in ~2 min.
When the A-HFS was applied with monophasic pulses, the neuronal responses at the initial period were similar to that of biphasic pulses -- each pulse induced a large APS. However, at the late period of A-HFS, the monophasic pulses hardly induced APS (Fig. 4b). In addition, after the end of A-HFS, the test APS only recovered to 34.5 ± 12.1% (n = 5) of baseline level even after ~20 min.
To compare the attenuations of APS during the A-HFS with biphasic and monophasic pulses, we calculated the APS amplitudes normalized to baseline value to evaluate the changes of APS (Fig. 4c). The decrement of APS amplitudes at the initial 1 s (ΔA1s) of A-HFS was significantly greater with monophasic A-HFS (86.9 ± 8.7 %, n = 5) than with biphasic A-HFS (73.5 ± 8.0 %, n = 4; t–test, P < 0.05. Fig. 4d). Also, the decrement of APS amplitudes at the end of A-HFS (ΔA60s) was significantly greater with monophasic A-HFS (98.6 ± 1.2 %, n = 5) than with biphasic A-HFS (94.1 ± 3.4 %, n = 4; t–test, P < 0.05. Fig. 4e).
The faster and larger attenuation of APS amplitudes during monophasic A-HFS indicated that the monophasic pulses can cause more conduction failures than biphasic pulses. The partial recovery of test APS after monophasic A-HFS indicated that the conduction failures in at least a portion of axons can even persist after the A-HFS.
In addition, no SD event was observed in all the 9 rats applied 1-min 200 Hz A-HFS (4 with biphasic pulses and 5 with monophasic pulses). That is, the SD incidence during 200 Hz monophasic A-HFS (0/5) was significantly smaller than that during 200 Hz monophasic O-HFS (4/5; Fisher’s exact test, P < 0.05).