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[44,48,51,54,56,57] and P. Monmaur, unpublished observa- u duration and spectral values collected during 0 to 15 min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.
An essential clue to the correct understanding of the 4.1. ACh release
relationship between u recorded from the dorsal hippocam- pus and ACh collected in the ventral hippocampus lies in
ACh contents found in the present experiment are the general topography and the functions of the choliner-
approximately of the same order of magnitude as those gic septal efferents to the whole hippocampus. Both the
previously reported by Cao et al. [9] in the ventral dorsal and ventral hippocampus receive most of their
hippocampus of the urethane-anaesthetised rat, but greater septal cholinergic inputs via the fornix, the fimbria, and the
3- to 4-fold than those measured in the ventral hippocam- supracallosal stria dorsal route [21]. The ventral hip-
pus during our previous dialysis study also using rats pocampus, but not the dorsal, receives additional choliner-
anaesthetised with urethane [38]. This difference in ACh gic inputs 10–30 from the septum via the amygdaloid
contents between our two experiments might essentially be area ventral route [17,34]. Since selective lesion of the
attributable to the fact that time between probe insertion fornix-fimbria fibres abolishes u in both the dorsal and
and ACh collection period were significantly larger in the ventral hippocampus [47], the involvement of the ventral
present experiment, which is consistent with the finding of route in u production is unlikely, although this is not yet
Damsma et al. [12] that, at least in the freely moving rat, conclusively demonstrated. Based on these data, it can be
striatal release of ACh gradually increased with time of argued that the activity of the ventral cholinergic route
perfusion.The lowest ACh basal concentration measured in may contribute to changes in ACh level selectively in the
the present study was 65.52 fmol, which is close to the ventral hippocampus, irrespective of the generation and or
detection limit of the present HPLC method, thus demon- modulation of hippocampal u. Such changes may introduce
25
strating that addition of 10 M neostigmine to the
a bias in our attempts to correlate subtle u changes with perfusate was necessary when defining the ACh baseline.
concomitant variations in ACh release from septohip- On the other hand, it might be argued that the use of a
pocampal cholinergic pathway. In fact, observations by cholinesterase inhibitor at this concentration in the perfu-
Nilsson et al. [40] reduces this possibility. On the basis of sate may induce both biochemical and electrophysiological
dialysis experiments in the freely moving rat, these authors changes in the hippocampus from which the EEG was
reported that following transection of the fornix and recorded. Such changes might seriously interfere with
fimbria which normally abolish u in both the dorsal and attempts to correlate subtle EEG changes with real physio-
ventral hippocampus [47], residual ACh about 25 of logical variations in ACh release from the septohippocam-
baseline level was detected not only in the ventral as pal cholinergic pathway. It has been shown that neostig-
expected but also in the dorsal hippocampus, as a possible mine significantly increases the extracellular ACh content
consequence of ACh diffusion from structures adjacent to in rat brain [12]. This increased ACh level might, in turn,
the hippocampus [40]. This finding is of importance for the affect ACh efflux from cholinergic terminals via action on
present study since it can be taken to mean that the whole presynaptic nicotinic and the M2 subtype of muscarinic
hippocampus can nearly be considered as a homogeneous receptors which mediate a feedback activation [45] and
structure in terms of cholinergic activity when evaluated inhibition [23,41,45,46], respectively, of ACh release. In
by the HPLC method. fact, micromolar neostigmine concentrations in the perfu-
sate appear to inhibit only a small portion of the acetyl- 4.2. ACh and u duration
cholinesterase activity in the perfused area [12], and the extracellular ACh changes induced by the feedback mecha-
The present data show that the duration of u recorded nism arising from ACh’s effect upon presynaptic receptors
from the dorsal hippocampus was not correlated with appeared to be rather modest [11,52]. Increased ACh
output of ACh collected ipsilaterally in the ventral hip- elicited by anticholinesterase inhibition is also known to
pocampus. They confirm our previous dialysis data [38]
M .S. Keita et al. Brain Research 887 2000 323 –334
331
and provide further support for the view that the u rhythm anaesthetised animal, is also of relevance to the above
might be generated even though hippocampal ACh release hypothesis. Collectively, these observations are in line with
does not increase, provided that sufficient synchronising the hypothesis that ascending cholinergic systems are
activity occurs by an alternative system, the GABAergic critically involved in the regulation of EEG state.
septohippocampal fibres, for instance [10,50]. However, it can be argued that in the previous study and the present
4.4. ACh and frequency at peak u power relationships investigation as well, u duration was calculated with a ruler
and, as a consequence, the resulting measures could be An additional and important finding brought about by
subjective. Therefore, further study using an automated the present study is that ACh output was positively
method to measure u duration is needed to decide this correlated with the frequency at peak power and that this
issue. correlation persisted throughout the experiment. The fre-
quency at peak power is considered to be a reflection of 4.3. ACh and power relationships
the dominant frequency of the electrophysiological signal. In this context, the above data that ACh output and the
The spectral method used here revealed that the relative frequency at peak power covaried can be taken to suggest
power of the u band was positively correlated with ACh that the septohippocampal cholinergic cells and the neuro-
output. However, peak u power and relative peak u power physiological mechanism involved in u frequency modula-
as well was not statistically related to ACh output. In fact, tion are functionally related. Based on a large compilation
as far as the present spectral method is concerned, the peak of literature data, Kirk [21] recently concluded that both
u power value, which represents only the power related to the amplitude and frequency of hippocampus u likely
a quarter of 1 Hz of the 3–12 Hz u band see Section 2, is resulted from septal autorhythmic bursting cells, the activi-
less suited than the relative power of u band which ty of which was modulated by ascending rhythmic and or
includes peak u power to represent adequately the am- non-rhythmic inputs to the septum. Interestingly, using
plitude of a signal as complex as hippocampal field immunotoxin 192 IgC-saporin, Apartis et al. [4] recently
activity. Therefore, the present finding, that the relative reported that, in the urethane-anaesthetised rat, vast dam-
power of the u band of EEG recorded from the dorsal age of septal cholinergic neurons projecting to the hip-
hippocampus covaries with ACh collected in the ipsilateral pocampus resulted in a substantial reduction in the number
ventral hippocampus, extends our previous microdialysis and activity of rhythmically bursting septal cells. Im-
findings that u amplitude as traditionally measured with a portantly, damage also drastically reduced the frequency of
ruler and ACh release collected in the left and right dorsal u, in addition to the amplitude of this rhythm. These data
hippocampus, respectively, covaried positively. Further- were taken as evidence that a large majority of rhythmical-
more, the present data provide further support for the ly bursting septal cells projecting to the hippocampus are
assumption of involvement of ACh as a neurotransmitter in cholinergic, while the remaining small number of rhythmi-
the control of hippocampal u amplitude, which is con- cally bursting cells spared by saporin use a noncholinergic
sistent with the early hypothesis of Brazhnik and Vin- transmitter, possibly GABA, although this was not conclu-
ogradova [8] and the subsequent conclusions of other sively established from the study. When considered in
authors [19,22,27]. Spectral analysis also revealed that in relation with the above data our present dialysis results are
the delta band, the relative power and ACh release were consistent with the view that, in the urethane-anaesthetised
negatively correlated. This observation, together with the rat, in addition to a role in u amplitude control, the activity
finding that the relative u band power and ACh were of the cholinergic component of the septohippocampal
positively related, suggests some functional interdepen- system may be also involved in the modulation of fre-
dence between neurochemical mechanisms underlying quency of this rhythm. This suggestion is further supported
delta activity and those involved in u rhythm. In this by the pharmacological findings [36,48] that, as already
respect, the most parsimonious hypothesis is that hip- noted above, in the urethane-anaesthetised rat, u could
pocampal cholinergic activation and inactivation are re- either be elicited or blocked with infusions of cholinergic
spectively of importance for amplitude modulation of u agents such as carbachol and atropine, respectively, into
and delta components of hippocampal EEG. This sug- the septum or directly into the hippocampus. The fre-
gestion is further supported by the pharmacological find- quency of the u elicited by intraseptal or intrahippocampal
ings [36,48] that, in the urethane-anaesthetised rat, sys- infusion of carbachol was dependent on the amount of
temic or intracerebral infusion of procholinergic agents drug infused. Thus, collectively, the above data and
elicited u whereas atropine suppressed this rhythm, which observations suggest that cholinergic components, includ-
was then replaced by large amplitude slow activity delta ing receptors, of the septohippocampal pathway are direct-
type waves. The previous finding [14] that ACh, in ly involved in the mechanisms underlying modulation of
addition to other neurotransmitters, is essential for the both the amplitude and the frequency of u. This hypothesis
neocortical activation which generally accompanies hip- does not exclude the possibility proposed previously by
pocampal u in both the freely moving rat and the urethane- Lee et al. [27] that, in both the freely moving and
332 M
urethane-anaesthetised rat, the modulation of u frequency modulation of frequency of this rhythm. They also confirm
and of u amplitude may depend on the activity of that the activation inactivation of the cholinergic septohip-
GABAergic and cholinergic septal neurons projecting to pocampal neurons is of importance for the modulation of
the hippocampus, respectively. However, the degree to both the u and delta components of the hippocampal EEG,
which the action of septohippocampal GABA contributes which underly normal alert immobility and automatic
to the control of u will be conclusively established only behaviour of the waking state, respectively, of the freely
after studying the EEG effects of selective and complete moving rat. These findings and conclusions are derived
inactivation of GABAergic components of the septohip- from the urethane-anaesthetised model which offers advan-
pocampal pathway, which is critically involved in u tages for this study as mentioned in Section 1. However,
production. anaesthesia might modify the activity of other neural
In terms of septohippocampal functions, it is of interest systems involved in EEG regulation, such as serotoniner-
that, in addition to relative u power, hippocampal ACh gic ascending fibres arising from the raphe nucleus and
content is related to frequency at peak u power. This innervating both septum and hippocampus [20,58], a
observation is consistent with the previous hypothesis that phenomenon which may interfere with the present study.
septohippocampal cholinergic activity as well as shifts in Therefore, studies in undrugged animals are necessary to
both hippocampal u amplitude and u frequency are critical determine the degree to which the cholinergic afferents to
for hippocampal role in behavioural modulation, notably the hippocampus modulate u relative to the serotoninergic
when the experimental animal is involved in learning and system, which is believed to cooperate with the septoch-
memory tasks [18,43,53]. olinergic pathway in controlling u which accompanies
voluntary movements in the freely moving rat [55]. 4.5. Technical remarks
Our previous observations [38] indicated that the am-
Acknowledgements
plitude, but not frequency, of u traditionally scored with a ruler and recorded from the dorsal hippocampus of the
We thank Doctor Laurent Pezard for valuable remarks urethane-anaesthetised rat was positively correlated with
regarding spectral analysis of signals. We would also like ACh output measured in the contralateral dorsal hippocam-
to thank Jean-Yves Bertholet for his pertinent suggestions pus. No significant correlation was found between ACh
regarding the discussion. This study was supported by the output in the ventral hippocampus and u parameters. It is
Direction des Recherches et Etudes Techniques D.R.E.T. therefore notable that, in preparations similar to that used
95 108. in the present study, ACh output measured within the
ventral hippocampus was positively correlated not only with relative power in the u band but also the frequency at
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5. Conclusions
