M .S. Keita et al. Brain Research 887 2000 323 –334
325
that in our recent microdialysis study both u and ACh were not met, the rats were excluded from the study. Histologi-
concomitantly and spontaneously detected in the rat given cal results deriving from our prior experiments conducted
an anaesthetic dose of urethane which blocked locomotor on a very large number of rats, and using this hippocampal
activity [38]. Moreover, intraperitoneal administration of EEG recording technique, have shown that in all rats
atropine in this preparation resulted in a strong increase in tested, short electrode tips were located in the corpus
ACh release in the ventral hippocampus [P. Monmaur, callosum or in the overlying deep neocortex, and long
unpublished data], likely via action of the anticholinergic electrode tips were located near the hippocampal fissure of
agent on presynaptic M2 muscarinic subtype receptors the dorsal hippocampal formation [36–38].
[41,45,46]. These observations suggest that under urethane Home-made parallel I-shaped probes of an acrylonit-
anaesthesia, septohippocampal cholinergic neurons of the rile sodium methylsulfonate dialysis copolymer membrane
rat have a spontaneous activity. Taken together, the above molecular weight cut-off of 45,000, 0.24 mm internal
considerations prompted us to use again, for the present diameter, 0.29 mm outer diameter, AN 69, Filtral, Hospal
study, the rat immobilised by an anaesthetic dose of SA, Lyon, France were used in this study. The exposed
urethane ethyl carbamate, Sigma, an experimental model tips of the dialysis membrane were 4 mm. The probe was
which can substantially facilitate the identification of implanted in the ventral hippocampus, ipsilateral to EEG
potential causal relationships between u activity and electrodes. The co-ordinates for microdialysis probes were:
concomitant ACh metabolism in the brain. Here we anterior 2.5 mm, lateral 4.7 mm from the lambda-skull
significantly extend our previous hippocampal EEG mi- surface and ventral 6.9 mm from the meninges surface.
crodialysis data. Following probe implantation, ether, which was essentially
used for the surgical steps including electrode and probe lowering into the brain, was then kept out and urethane,
2. Materials and methods which ensures more stable and longer-lasting light anaes-
thesia with theta than ether, was administered 0.33 g kg 2.1. Animals
via the abdominal cannula until the u occurred sponta- neously or was easily elicited by gentle tail pinch. At this
Experiments were performed on male Wistar rats Jan- stage of the experiment, the rat was lightly anaesthetised.
vier, France weighing 280–320 g. They were housed one If clear-cut vibrissae movements were detected during the
per cage under 12 h light 12 h dark conditions and subsequent minutes of the experiment, an additional in-
25628C. They had free access to food and water. jection of urethane was given at the dose of 0.1 g kg.
Urethane was prepared by dissolving 2.5 g of the drug in 5 2.2. Surgery
ml of saline. Body temperature was maintained at 36.860.58C. Myographic activity and heart rate were
The general surgical and recording techniques were continuously monitored by means of electrodes implanted
similar to those previously described [44]. Under deep in the forepaws of the animals.
volatile ether diethyl ether, Prolabo anaesthesia, a trach- eotomy was performed to facilitate breathing and a cannula
2.3. Microdialysis and ACh assay hypodermic needle was inserted into the peritoneal cavity
for subsequent administration of urethane solution. The rat Immediately after probe insertion in the ventral hip-
was then mounted in a stereotaxic apparatus and both pocampus, the inlet cannula was connected to a microinfu-
superficial and deep us of the right dorsal hippocampus sion syringe pump CMA 100 and the probe was continu-
were reached under light ether anaesthesia by continuously ously perfused with normal saline 0.9, pH 7.0 at a rate of
recording EEG by means of home-made monopolar elec- 2 ml min. A reversible cholinesterase inhibitor neostig-
25
trodes constructed by twisting together two lengths of mine bromide, 10
M, Sigma was included in the Teflon-coated stainless steel wire each 0.12 mm in
perfusion solution in order to ensure detectable dialysate diameter insulated except for the tips which were sepa-
concentrations of ACh [38]. The probes were perfused for rated by 0.8 mm in the vertical plane. According to the
180 min before collection of samples to allow for 1 atlas of Albe-Fessard [1], the anterior-lateral co-ordinates
equilibrium between interstitial fluid and perfusion fluid for electrode implantation were 4.5 mm and 1.7 mm,
and 2 the hippocampus to recover from trauma caused by respectively, relative to lambda. A stainless steel screw
probe insertion. This interval was determined on the basis driven into the interparietal bone served as indifferent
of a previous study combining EEG and the microdialysis electrode and an aural fixation bar was connected to the
technique in the hippocampus sensorimotor cortex of the ground. This procedure allowed us to obtain, in most
freely moving rat [28]. The perfusate samples were animals, well-developed superficial and deep us which
collected at 15-min intervals for 30 min. Samples were were approximately phase reversed accepted range: 150–
immediately frozen in liquid nitrogen and stored at 2808C 1808. Moreover, deep u amplitude was larger than that of
before ACh assay. Following the 30-min ACh collection superficial u. When these electrophysiological criteria were
period, the rats were submitted to various pharmacological
326 M
treatments. These treatments are outside the scope of the hippocampal electrode which usually produced well-de-
present investigation and pharmacological data are there- veloped u in contrast to the short one, and the phase and
fore not presented in this paper. the coherence spectra which were calculated for the
ACh was assayed by high performance liquid chroma- electrode pair. The frequency corresponding to peak power
tography HPLC
with electrochemical
detection produced by the long electrode was chosen as a reference
Coulochem II ESA. Sample volumes of the perfusate for phase and coherence calculations. The peak power was
were injected by means of a microsyringe in a 20-ml loop defined as the largest power value between 0.25 Hz and
of a manually controlled injection valve Rheodyne 7725i. 49.50 Hz. When peak power was in the u band 3–12 Hz
This volume was less than the theoretical 30 ml normally it was named peak u power. The absolute power sum of
collected and was chosen to make sure that equivalent spectral values within a limited frequency band and the
volumes of available sample were submitted to the HPLC relative power 1003absolute power total power sum of
analysis. The ACh and choline were separated on a CC spectral values between 0.25 Hz and 49.50 Hz were
12532 mm, 4 mm Superspher column Macherey-Nagel. calculated for 0.25–2.75 Hz d band, 3–12 Hz u band,
A CC 833 mm, 4 mm Superspher precolumn Macherey- 12.25–25 Hz b band and 25.25–49.50 Hz g band. The
Nagel was always fitted to the column. A home-made relative peak power 1003peak power total power was
enzyme postcolumn reactor containing silanised glass also calculated. Due to marked interindividual differences,
beads with covalently bonded acetylcholinesterase and absolute power appears to be less reliable than relative
choline oxidase [5] was used to convert ACh to hydrogen power in comparing power spectra recorded during the
peroxide which was detected by a platinum electrode at experiment. Therefore, relative powers were always calcu-
300 mV. The temperature of the pre-column, column and lated and used, in addition to absolute powers, for sub-
the post-column was maintained at 258C in a column sequent statistical calculations. One spectral average was
thermostat controller 482 Kontron Instruments. The made for each 5-min interval for the 30 min of the ACh
mobile phase pH 6.6 delivered by a dual piston pump collection period. Within each 5-min interval, attempts
Kontron at 0.350 ml min was 50 mM potassium were made to carry out spectral averaging, preferentially
dihydrogen phosphate KH PO Sigma, 5.5 mM tetra- during u waves or electrophysiological field oscillations
2 4
methylammonium chloride Aldrich, 1.2 mM 1-heptane- resembling u, as identified by visual inspection. The
sulfonic acid Sigma, 50 ml l MBA Eurosep. The spectral measurements were then assembled in two inter-
perfusate ACh concentration was quantified with an vals of 15 min corresponding to two ACh collection
MT450 integrator Kontron using external ACh standards. intervals of 15 min each. Thus, each interval included three
ACh release i.e. efflux of dialysable transmitter was consecutive spectral values which were averaged. Clearly,
expressed as fmol of ACh per 20 ml dialysate sample. The within each 15-min interval, there was one average spectral
main parameters used for amperometric measures of ACh value and one value for ACh release. These values were
were filter510 s, gain50.5 nA V, upper and lower current used for statistical analysis see below.
limits in which measurements were carried out520 and 10 The proportion of time s that u was present in the
nA, respectively. The detection limit, defined as the record u duration was scored from an at least 1-s
amount of standard ACh producing a peak consistently uninterrupted train of u with a ruler at 15-min intervals for
emerging from the basal noise, was 50 fmol per injection the 30 min of the ACh collection period. Here again,
of 20 ml and the accuracy of measurements probe within each 15-min interval, there was one u duration
efficiency of 100 fmol was 23. value and one value for ACh release. Measurements were
made for only the long hippocampal electrode which 2.4. EEG recording and analysis
produced well-developed u in contrast to the short one. Only trains of clear-cut u oscillations, i.e. almost sinusoid-
The EEG from hippocampal electrodes was recorded shaped waves, were considered.
monopolarly by a physiopolygraph Alvar. The recording was made continuously throughout the experiment. Hip-
2.5. Probe placement control pocampal EEG was digitised on-line at 128 Hz by a signal
processor Tektronix 2630 Fourier Analyser installed on a Upon completion of the experiments, methyl blue
computer Elonex 486 spectral measurements. Spectral solution was perfused in the hippocampus at the rate of 2
averages were computed using the fast-Fourier transform ml min for 15 min. The rat was then killed and the brain
FFT on five 4-s segments of 512 points overlapped by removed. Resulting slices of fresh brain tissue were
50. This procedure resulted in one spectral value per checked under a binocular microscope for dialysis probe
0.25 Hz frequency resolution over the range from 0 to 50 placements.
Hz. Data under 0.25 Hz and above 49.50 Hz were omitted from the analysis in order to avoid any possible signal
2.6. Statistics artefact. Three kinds of spectra were routinely considered:
the power spectrum which was calculated for only the long In agreement with our previous data [38], hippocampal
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
