Brain Research 887 2000 323–334 www.elsevier.com locate bres
Research report
Acetylcholine release in the hippocampus of the urethane anaesthetised rat positively correlates with both peak theta frequency and relative
power in the theta band
a ,1 a ,1
b c
a , ,1
M.S. Keita , L. Frankel-Kohn
, N. Bertrand , L. Lecanu , P. Monmaur
a
´ ´
` Laboratoire de Neurosciences Comportementales
, Universite Paris V-Rene Descartes, 45 rue des Saints Peres, H468, 75270 Paris Cedex 06, France
b
´ Laboratoire de Pharmacodynamie
, Universite de Bourgogne, 7 bd J. d’Arc, 21033 Dijon, France
c
´ ´ ´
Laboratoire de Pharmacologie , Groupe Circulation Cerebrale, Universite Paris V, 4 rue de l’Observatoire 75006 Paris, France
Accepted 19 September 2000
Abstract
The need to achieve a clearer understanding of relations between hippocampal theta characteristics and cholinergic septohippocampal neuron activity, prompted us to re-examine, in the urethane-anaesthetised rat, the statistical relationships between the electrophysiological
and neurochemical variables using a procedure which is believed to enhance significantly the degree of confidence with which parameters of theta recorded with classic macroelectrodes can be related to concomitant acetylcholine output measured by high-performance liquid
chromatography with electrochemical detection. Firstly, the theta rhythm and the acetylcholine content were derived from the same hippocampus. Secondly, the hippocampal electroencephalogram was quantified using spectral analysis which permits the more objective
quantitative evaluation of selected electroencephalogram samples. Thirdly, a larger number of rats than in our previous study was used here, thus enhancing the validity of statistical results. This procedure yielded, in our time-course determination, two main findings. The
first finding is that acetylcholine release was positively correlated with frequency at the peak power of the theta band which reflects the frequency of the theta signal. This finding had not been reported yet. The second finding is that hippocampal acetylcholine outflow also
covaried with relative power of the theta band which reflects the amplitude of the theta signal. This finding is consistent with our previous study in which EEG was quantified by means of a traditional method. These findings suggest that the cholinergic component of the
septohippocampal system, which is the main source of hippocampal acetylcholine, and neurophysiological mechanisms involved in the modulation of both the amplitude and the frequency of theta are functionally related. The possibility that, at least in the urethane-
anaesthetised rat, hippocampal acetylcholine is involved in these modulator mechanisms is discussed.
2000 Elsevier Science B.V. All
rights reserved.
Theme : Neurotransmitters, modulators, transporters, and receptors
Topic : Acetylcholine
Keywords : Microdialysis; Spectral analysis; Theta rhythm; Septohippocampal system; Septum
1. Introduction activity of neural components of the hippocampus, this
rhythm has been used to build various theories relative to The theta u rhythm which can be recorded from the
hippocampal functions see references in Refs. [21,42]. hippocampus occurs within the 3–12 Hz frequency band
Acetylcholine ACh is considered to play an important [54] in the brain of most mammalian species [6,7]. Since it
role in the control of hippocampal u. Several pharmaco- is generally believed that u is a reliable index of the
logical studies have shown that procholinergic drugs cause hippocampal synchronisation in both anaesthetised and
freely moving animals, an electroencephalogram EEG
Corresponding author. Tel. fax: 133-1-4286-3364.
effect which was blocked by antimuscarinic agents such as
E-mail address : monmaurbiomedicale.univ-paris5.fr P. Monmaur.
1
atropine [25,54,56]. The latter also abolishes u which
Permanent address: Laboratoire de Psychopharmacologie et Processus ´
Cognitifs, Universite Paris VII, 2 place Jussieu, 75005 Paris, France.
occurs spontaneously or after peripheral or central applica-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 2 1 - 3
324 M
tion of stimuli to animals anaesthetised with ether or septal source [47] and, consequently, would occur at the
urethane [25,26,36]. In addition, atropine treatment sup- same frequency and the same amplitude modulation
presses u oscillations seen with alert immobility and alters, throughout the hippocampus. In fact, it could be argued
without suppressing, those that accompany voluntary that attempts to correlate electrophysiological parameters
motor behaviour in the freely moving animal [25,54,56]. and ACh output may be problematic under certain ex-
Many data support the view that the medial septum, the perimental conditions of this acute study. For example,
hippocampus and the reciprocal connections between these damage to hippocampal tissues due to electrode lowering
structures form a crucial network for u generation. For and probe insertion are not equivalent for the left and right
example, lesions of the medial septum or transection of hippocampus. Moreover, neostigmine, an anticholinester-
both the fimbria and the fornix permanently abolish ase agent added to the perfusate [38] and capable of
hippocampal u [3,35,49,47]. Moreover, reversible func- influencing hippocampal EEG [44,51,54,56,57], likely
tional blockade of the medial septum by local infusion of diffused to the adjacent layers in the right but not left
anesthetic results in transient abolition of this rhythm hippocampus. It is possible, therefore, that the physiologi-
[7,22,19,58]. Conversely, electrical or chemical activation cal activity of the left hippocampus and that of the right
of the medial septum elicits u [24,26,32,36,37,39]. His- one, although probably similar, were not identical in this
tological, histochemical and ACh release studies have previous study and that this factor, at least in part,
shown that the medial septum, which projects cholinergic interfered with attempts to correlate changes in u rhythm
fibres to all hippocampal subfields, is the main source of and changes in ACh outflow derived, respectively, from
hippocampal cholinergic innervation [2,27,29,33] and ACh separate hippocampi. It is important to clarify this point
outflow in the hippocampi [15,16,40]. Specifically, hip- 1 to obtain clearer understanding of the neurochemical
pocampal ACh release is increased by stimulation of the mechanisms underlying u, and 2 to interpret more
medial septum [15,16] and strongly reduced following correctly both previous and future investigations using
fimbria fornix pathway transection [40]. Taken together, EEG and or dialysis methods, particularly since, for
the above data suggest that cholinergic components of the technical reasons, ‘hippocampal EEG’ is classically de-
septohippocampal system are of importance for hippocam- rived from the dorsal part of the hippocampus and ‘hip-
pal synchronisation. This suggestion is strongly supported pocampal ACh’ is often collected from the ventral part of
by a study of Lee et al. [27]. These authors have shown this structure.
that, in both the drugged and normal rat, complete and One way to test and clarify this point is to repeat the
selective destruction of cholinergic septal cells, by means EEG microdialysis experiment and calculate the statistical
of immunotoxin 192 IgC-saporin, dramatically reduced the relationships between characteristics of spontaneously
amplitude of hippocampal u, in addition to clearing occurring u in one hippocampus and concomitant ACh
acetylcholinesterase-positive fibres in the hippocampus. output derived from the same hippocampal structure. The
These findings suggest that u amplitude is almost entirely strategy of the present paper, therefore, was to record
dependent on the anatomophysiological integrity of the hippocampal EEG from the dorsal part of the hippocampus
cholinergic component of the septohippocampal system. and concomitantly record ACh output from the ventral part
This is consistent with the suggestion of authors made on of the same hippocampus of the rat. Moreover, ACh output
the basis of a procaine mapping study [19,22] and also was assessed by means of a steadier electrochemical
tallies with our recent investigation using an approach detector than previously [38], and EEG was quantified
combining hippocampal microdialysis with hippocampal u using spectral analysis, which permits the more objective
recording in the urethane-anaesthetised rat [38]. Keeping quantification of selected EEG samples. Finally, more rats
in mind that continuous dialysis measurement of extracel- were used here than in our previous study. This strategy
lular ACh content of the hippocampus is the only method and procedure were believed to enhance significantly the
capable of directly evaluating the dynamic activity of the degree of confidence with which u parameters can be
septohippocampal population of neurons which synthesise related to concomitant ACh output. However, to obtain a
this neurotransmitter [40] we have demonstrated that clearer understanding of relations between hippocampal u
statistically significant positive correlation exists between and cholinergic septohippocampal neuron activity, it is
the amplitude of u recorded from the left dorsal hippocam- essential to determine the degree to which both electro-
pus and scored with a ruler, and the ACh content of the physiological and neurochemical parameters are influenced
contralateral dorsal hippocampus, but not with the fre- by nonspecific behavioural factors. In this respect, it is of
quency of this rhythm. These findings were taken as direct interest that, in the freely moving animal, both sensory
neurochemical evidence for the hypothesis that the u stimulation and motor behaviour were accompanied by
amplitude, as opposed to the u frequency, is cholinergical- increased release of ACh [13,15,16,30,31,40]. It is unclear
ly mediated [38]. However, ACh content of the ventral whether hippocampal ACh release observed in these
hippocampus was not significantly correlated with u studies is causally related to changes in arousal level per se
parameters. This finding was unexpected if one considers or to changes in behavioural processes, or further to the
that u in all hippocampal fields is paced from the same general behavioural state of the animal. It is also of interest
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-
