M .S. Keita et al. Brain Research 887 2000 323 –334
327
ACh levels varied markedly between rats see Section 3. Therefore, statistical relations between ACh release and u
spectral parameters were determined by means of the exacting two-tailed Spearman rank-order correlation coeffi-
cient
r. The Wilcoxon sign test was also used to compare values of measurements made between 0 and 15 min and
those of measurements made during the subsequent 15–30 min of the collection period effect of time. A P value
0.05 was accepted as evidence of a statistically signifi- cant effect. As spectral values were calculated on the basis
of averages as mentioned above, present data were pre- sented as means and standard error of the mean 6S.E.M..
3. Results
The data presented were derived from 19 rats. 3.1. Histology
Fig. 1 shows the diagrammatic representation of the expected positions of the recording electrodes in the dorsal
hippocampus and the typical placement of dialysis probes in the ipsilateral ventral hippocampus of the rat. In general,
the exposed dialysis membrane unshaded area was in contact with most strata of the CA1-dentate regions due to
the slight inclination of the ventral hippocampus at the chosen co-ordinates for probe insertion. As in our previous
work [38], it was unclear whether or not the probes were in contact with the CA3 layers. Moreover, in two rats, the
probe was in an area overlapping CA1 and the lateral adjacent corpus callosum deep cortex regions. In two
other rats, about 50 of the full length of the exposed membrane was inserted in the CA1 area; the remaining
50 was in the extrahippocampal overlying neural tissues. No clear relationship between probe placement and ACh
release emerged from our histological examination.
Fig. 1. Schematic drawing according to the atlas of Albe-Fessard of the
3.2. ACh release
expected position of the recording electrodes in the dorsal hippocampus A and the typical placement of the microdialysis probe with the 4 mm
Basal ACh outputs were detectable in all animals and in
membrane exposed unshaded area in the ipsilateral ventral hippocampus B. Probe shaded areas indicate portions of the probe that were covered
all collection intervals. ACh levels were quite variable
with glue epoxy resin. LE, long electrode; SE, short electrode; MP,
from rat to rat range: 65.52 to 1768.22 fmol but were
microdialysis probe; cc, corpus callosum; cx, cortex; CA1, Ammon’s
relatively stable in the same animal over time. Mean values
horn subfield 1; CA3, Ammon’s horn subfield 3; DG, dentate gyrus.
and the standard error of the means of basal ACh output were 419.876110.53 fmol n519 and 401.816102.97
fmol n519 for 0–15 min and 15–30 min of the 3.3. Electrophysiology and spectral analysis
collection period, respectively. The mean output of ACh calculated over the collection period 0–30 min was
Visual inspection of records revealed that, immediately 410.84674.52 fmol n538. A one-tailed Wilcoxon sign
after probe insertion, hippocampal EEG was desynchron- test failed to reveal any statistically significant difference
ised and wave amplitude was small as compared to EEG z50.68, P.0.1, n519 between the two 15-min intervals,
amplitude seen before probe lowering, although individual clearly confirming the above qualitative observation that
variations were
apparent. No
hippocampal seizures
ACh output was essentially stable over time. emerged from the traces. EEG amplitude gradually in-
328 M
creased and reached an asymptote by 60 to 120 min which 15-min interval and serving for correlation calculation,
was, however, lower than the pre-probe amplitude level. were placed within the u band. This is summarized in
Preliminary experiments indicated that small two-direc- Table 2: the mean values6S.E.M. of the frequency at peak
tional electrode moving in the vertical plane across the power were: 3.4960.15 Hz n519 and 3.6460.17 Hz
hippocampal layers, at the end of the observation period, n519 for the 0–15 min and 15–30 min intervals of the
generally failed to improve significantly EEG including u recording period, respectively. The mean6S.E.M. calcu-
waves amplitude, thus reducing the possibility that incom- lated over the 0–30 min of the recording period was
plete recovery of signal amplitude was only due to shifting 3.5760.11 Hz n538.
of hippocampal tissues relative to electrode tips as a Clear-cut theta rhythm and desynchronised electrophy-
possible consequence of probe implantation. About 30 min siological activities coexisted during each 15-min interval
following probe placement, rhythmicity was generally of the collection period. The means of u duration for the
detectable on traces and 30 to 60 min later trains of u 0–15 min and 15–30 min intervals were 118.62617.82 s
waves alternated with generally longer trains of de- n517 and 116.83621.10 s n517, respectively mean
synchronised activity although individual variations be- over the 0–30 min of the collection period: 117.72615.60
tween animals were observed. These EEG patterns formed s, n534, which are consistent with our previous mi-
the background activity of the dorsal hippocampus of most crodialysis study [38].
rats during the 30-min collection period. Visual scrutiny of From the data of the paragraph before and Tables 1 and
the records indicated that 17 of 19 rats exhibited u rhythm 2 which present the mean6S.E.M. of spectral parameters
whereas clear-cut u waves appeared to be absent from the derived from the spectra collected during the experiment, it
record of the two remaining rats. However, spectral can be seen that, in general, both u duration and spectral
analysis revealed that peak u powers were present in all values collected during 0 to 15 min were comparable to
rats, thus indicating that u waves were, in fact, present those collected during 15 to 30 min of the recording
although not clearly identifiable by visual inspection, in the period. A one-tailed Wilcoxon sign test applied to these
records of all animals. Detailed spectrum by spectrum parameters revealed no statistically significant Ps.0.1
analysis of all spectra carried out during the experiment difference because of their large number, detailed statisti-
indicated that 102 of 114 89.5 spectra had peak power cal data are not presented here between members of pairs,
range: 3 to 6 Hz within the u band peak u power and 12 indicating that u duration and EEG spectral characteristics
of 114 10.5 approximately equally divided between were, as seen for ACh release, essentially stable over the
the 0–15 min and 15–30 min intervals of the collection collection period, in accordance with our previous mi-
period had peak power range: 0.75 to 2.75 Hz within the crodialysis study [38].
delta band peak delta power, thus suggesting that essen- tially u oscillations were included in most spectra. As a
3.4. Correlations probable consequence, all the peak power values resulting
from averaged consecutive spectral values within each The values of the two-tailed Spearman rank-order
Table 1 Correlation coefficient
r between acetylcholine release, on the one hand, and both absolute and relative powers of delta, theta, beta and gamma frequency
a
bands, on the other hand
Collection Two tailed Spearman correlation coefficient
period Delta band 0.25–2.75 Hz
Theta band 3–12 Hz Beta band 12.25–25 Hz
Gamma band 25.25–49.50 Hz Absolute power
Relative Absolute power
Relative Absolute power
Relative Absolute power
Relative mV
power mV
power mV
power mV
power 0–15 min
Mean6S.E.M. 128.5668.49
7.7760.51 327.75621.82
19.8260.49 506.90635.66
30.6661.77 361.88627.02
21.8960.63 r
20.09 20.37
0.15 0.28
0.06 0.16
0.07 0.14
P .0.1
.0.1 .0.1
.0.1 .0.1
.0.1 .0.1
.0.1 n519
15–30 min Mean6S.E.M.
129.73610.59 7.7860.58
327.82621.91 19.6660.39
510.62634.90 30.6161.73
370.67624.29 22.2260.53
r 20.41
20.57 0.29
0.54 0.12
0.14 0.16
0.05 P
.0.1 0.03
.0.1 0.02
.0.1 .0.1
.0.1 .0.1
n519 0–30 min
Mean6S.E.M. 129.1566.69
7.7860.28 327.79615.25
19.7460.31 508.76624.61
30.6561.22 366.27617.93
22.0660.42 pooled
r 20.22
20.48 0.21
0.37 0.1
20.16 0.13
20.09 P
.0.1 0.006
.0.1 0.042
.0.1 .0.1
.0.1 .0.1
n538
a
Correlation was calculated for the 15-min intervals of the collection period and the entire 0–30-min collection period with pooled pairs of values. Also presented are the mean values and standard error of the means 6S.E.M. of the spectral parameters. Statistically significant; n, number of pairs; mV,
millivolt; Hz, hertz.
M .S. Keita et al. Brain Research 887 2000 323 –334
329 Table 2
As in Table 1, except that spectral parameters were total power, peak power, relative peak power, frequency at peak power, phase and coherence, calculated from the 0.25–49.50 Hz band
Collection Two-tailed Spearman correlation coefficient
period Total power mV
Peak power mV Relative peak
Frequency Hz Phase 8
Coherence power
at peak power 0–15 min
Mean6S.E.M. 1652.866103.41
65.0566.96 3.9360.32
3.4960.15 138.82611.75
0.7560.04 r
0.06 0.32
0.27 0.66
0.49 0.02
P .0.1
.0.1 .0.1
0.009 0.072
.0.1 n519
15–30 min Mean6S.E.M.
1666.676103.15 63.9966.83
3.8360.30 3.6460.17
143.09611.23 0.7460.05
r 0.14
0.4 0.38
0.55 0.27
20.02 P
.0.1 .0.1
.0.1 0.037
.0.1 .0.1
n519 0–30 min
Mean6S.E.M. 1659.77672.04
64.5264.81 3.8860.21
3.5760.11 140.9668.02
0.7460.03 pooled
r 0.09
0.35 0.32
0.62 0.36
20.01 P
.0.1 0.066
0.091 0.0004
P50.052 .0.1
n538
correlation r calculated between ACh release and the EEG
during the 30-min period were pooled Table 2. Finally, a spectral parameters are presented in Tables 1 and 2. As can
statistically significant correlation was no longer observed be seen from Table 1, a statistically significant positive
between the cholinergic parameter and the duration of u correlation 0.54, P50.02, n519 exists between the ACh
for each consecutive 15-min interval 0.32, P.0.1, n517 output and the relative power of the theta band when the
and 0.08, P.0.1, n517, respectively and when pairs of pairs of values were collected during the 15–30-min
values collected during the 30-min period were pooled interval of the recording period. Conversely, a statistically
0.08, P.0.1, n534. significant negative correlation 20.57, P50.03, n519
In our previous study [38], in which electrophysiological was found in parallel between the neurochemical parame-
characteristics of u were quantified with a ruler, ACh ter and the relative power of the delta band for pairs of
content and u frequency were not found to be correlated. It measurements taken within the same interval. Both results
could be argued that this apparent discrepancy between the were confirmed by the existence of significant correlations
previous and the present experiments is due to the fact that 0.37, P50.042, n538 for ACh relative power of theta
10.5 of spectra used for correlation calculation in the band pairs and 20.48, P50.006, n538 for ACh relative
present study had peak power out of the u band, as power of delta band pairs when the pairs of values were
mentioned above. Specifically, these spectral values, al- pooled. Consistent with the above data, the relative power
though infrequent, might interfere with attempts to corre- of the u band and that of delta band were negatively
late subtle changes in both u oscillations and ACh release. correlated at least when the pairs of values were pooled
Therefore, in order to come to a clearer understanding of 20.46, P50.009, n538 data not presented in the
ACh release–u frequency, correlations between these tables. No correlation between spectral parameters and
neurochemical and spectral parameters have been recalcu- ACh emerged from any high frequency bands beta and
lated using only spectra having peak power within the u gamma. From Table 2, it can be seen that a statistically
band. Clearly, only the averaged spectral values resulting significant positive correlation exists between ACh and the
from three consecutive spectra with peak u power, col- frequency at peak u power when measurement pairs were
lected during intervals of 15 min see Section 2, were collected in the 0–15 min period 0.66, P50.009, n519.
considered. On the basis of this criterion, we were able to This correlation was conserved between the pairs of values
select 31 pairs of neurochemical spectral values that were collected during the subsequent 15–30-min interval 0.55,
used for correlation calculation. The results revealed that P50.037, n519. This result was confirmed by the
statistically significant correlations exist between ACh existence of a significant positive correlation between the
release and frequency at peak u power when measurement cholinergic parameter and the frequency at peak u power
pairs were collected in the 0–15-min period 0.61, P5 when the pairs of values collected during the 30-min
0.032, n516, the 15–30-min period 0.59, P50.05, n5 period were pooled 0.62, P50.0004, n538. The other
15 and when value pairs collected during the 30-min spectral parameters, i.e. total power, peak power, relative
period were pooled 0.54, P50.006, n531. They are peak power, phase and coherence, were not significantly
comparable to those derived from all the spectra collected correlated with ACh output, although the P value was
during the same recording periods and which are presented close to the significance level of 0.05
r50.36, P50.052, in Table 2 frequency Hz at peak power. Thus, it
n538 when the ACh phase pairs of values collected appears that the difference in results between our two
330 M
experiments regarding ACh release–u frequency relation- influence both the hippocampal EEG and autonomous
ship is more likely due to differences in the two methods nervous system activity. For example, intrahippocampal or
of calculation than inconsistencies in the selection of EEG systemic administration of large but sublethal doses of a
segments for traditional and spectral measurements, re- reversible cholinesterase inhibitor, physostigmine, general-
spectively. ly elicits, in both the normal and urethane-anaesthetised
From Tables 1 and 2 which present the mean6S.E.M. of rat, uninterrupted trains of robust u waves often associated
spectral parameters derived from the spectra collected with
tremors, salivation,
and defecation
Refs. during the experiment, it can be seen that, in general, both
[44,48,51,54,56,57] and P. Monmaur, unpublished observa- u duration and spectral values collected during 0 to 15 min
tion. Rats used in the present experiment exhibited none were comparable to those collected during 15 to 30 min of
of these central and peripheral manifestations which can be the recording period.
considered as clear-cut signs of toxicity. Collectively, these observations suggest that the neostigmine concentration in
the perfusate used in the present dialysis study should be
4. Discussion within or near the limits of physiological conditions.
