Department of Experimental Neurophysiology, Istituto Nazionale
Neurologico, 20133 Milan, Italy
 |
INTRODUCTION |
Fast oscillatory activity in the gamma range
(25-80 Hz) has been described in several cortical regions (for review
see Farmer 1998
; Jefferys et al. 1996
;
Singer and Gray 1995
) and has been interpreted as an
attention and arousal mechanism that promotes associative binding
between large ensembles of neurons in the neocortex (Gray et al.
1989
; Llinas and Ribary 1993
; Murthy and Fetz 1996
; Steriade et al. 1996
). The presence
of fast oscillations in the gamma range has been demonstrated in vivo
in the hippocampus (Bragin et al. 1995
; Penttonen
et al. 1998
; Traub et al. 1996
) and in the
entorhinal cortex (ERC) of the rat (Chrobak and Buzsaki 1998
; Eeckman and Freeman 1990
), the cat
(Boeijinga and Lopes da Silva 1988
), and the guinea pig
(Charpak et al. 1995
) and can be reproduced in vitro
either by orthodromic high-frequency stimulation (Funahashi and
Stewart 1998
; Stanford et al. 1998
;
Whittington et al. 1995
, 1997
) or by pharmacological
cholinergic activation (Fisahn et al. 1998
). It is
commonly accepted that the cholinergic system plays a crucial role in
determining fast activity and in sustaining propagation of such
activity within the cortex. Indeed, fast cortical activity at ~40 Hz
is enhanced during states of cortical arousal (Llinas and Ribary
1993
; Maloney et al. 1996
), increases after
stimulation of the brainstem cholinergic ascending system
(Steriade et al. 1991
, 1996
; Munk et al.
1996
) and after pharmacological activation of the basal
forebrain cholinergic nuclei (Cape and Jones 1998
), and
can be induced by muscarinic agonists (Fishan et al.
1998
), as mentioned earlier.
Gamma activity in limbic cortices has been proposed to provide a
functional setting that facilitates a condition during which synaptic
plasticity occurs (Traub et al. 1998
). A differential function and possibly a hierarchic organization between cortical structures in the control of limbic fast oscillation is suggested by
the observation that surgical removal of the ERC has a modulatory influence on gamma activity in the hippocampus (Bragin et al. 1995
; Charpak et al. 1995
). The demonstration
that fast activity in the ERC leads to or entrains gamma hippocampal
oscillations led us to study gamma activity in this cortical region and
to evaluate its relation with other limbic structures in an in vitro isolated guinea pig brain preparation (de Curtis et al.
1991
; Muhlethaler et al. 1993
). The regional
distribution of fast oscillations in the ERC was evaluated after
pharmacological stimulation of the preparation with the muscarinic
receptor agonist, carbachol. Surprisingly, during our study the fast
oscillations induced by carbachol were observed exclusively in the
medial-septal region of the ERC and not in its lateral-temporal portion
that borders the rhinal sulcus. Preliminary results have been presented
in abstract form (van der Linden and de Curtis 1998
).
 |
METHODS |
Guinea pig brains were isolated from young adult animals
(150-250 g) and were maintained in vitro according to a technique previously described in detail (de Curtis et al. 1991
, 1994
,
1998
; Llinas et al. 1981
;
Muhlethaler et al. 1993
). The animals were anesthetized with 20 mg/kg thiopentotal sodium (Pentothal) and were
perfused intracardially with ice-cold (10°C), carbogenated (95%
O2-5% CO2) saline solution consisting of 126 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 1.3 mM
MgSO4, 2.4 mM CaCl2, 26 mM NaHCO3, 15 mM glucose, 2.1 mM HEPES, 0.4 mM thiourea, and 3% dextran, molecular weight 70,000 (SIFRA, Isola della Scala, Italy). The brain was removed from the skull after an extensive craniotomy performed under hypothermic conditions and was positioned ventral side
up in a perfusion chamber cooled at 15°C. A polyethylene cannula was
inserted and secured to one vertebral artery, and perfusion with the
solution described was started at a rate of 5.5 ml/min. The
contralateral vertebral artery, the carotid artery, and the hypophysial
arteries were tied with silk threads to establish the perfusion of the
whole brain through the basilar system through the circle of Willis.
The temperatures of both the chamber and the perfusate were gradually
increased to 30°C at a rate of 0.1°C/min. The experimental protocol
was reviewed and approved by the Committee on Animal Care and Use and
by the Ethics Committee of the Istituto Nazionale Neurologico.
Extracellular recordings were performed either with glass micropipettes
filled with a 0.9% NaCl solution (~10 M
input resistance) or with
stainless steel microelectrodes (5-10 M
; FHC, Brunswick, ME). The
signals were amplified and high-pass filtered at 0.2 Hz through an
extracellular amplifier (Biomedical Engineering, Thornwood, NY) and
were stored by means of a digital tape recorder (DTR 2602, Biologic,
Claix, France) for off-line analysis with a Pentium computer.
The software for acquisition and analysis was developed in our
laboratory by Gerardo Biella in collaboration with Marco Fiorentini
(SIDeA, Milan, an alliance member of National Instruments, Italy). In
general, recordings were performed at 4-5 different sites
simultaneously. The location of the recording electrodes in different
ERC regions were decided and reproduced in different experiments with
reference to brain surface markers, using as a guide the guinea pig
brain atlas by Luparello (1978)
. A bipolar silver wire
electrode was used to stimulate the lateral olfactory tract (LOT) with
0.1-ms current pulses of variable amplitudes delivered at low
frequencies (<0.2 Hz). LOT stimulation evoked large-amplitude field
potentials in the lateral ERC (LERC), whereas in the medial ERC (MERC)
small responses that showed a decreasing amplitude gradient from
rostral to caudal were recorded. The potentials evoked by LOT
stimulation were used to evaluate the viability of the preparation
during the experiment. When stainless steel electrodes were used for
recordings, the location of the electrode tip was marked by
electrolytic lesions performed by passing a 100-µA current for 2-5 s
at the tip of the electrode after the electrophysiological experiment.
The brains were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4) for 1 wk and 100-µm coronal sections were cut, mounted on
slides, and stained with thionine to verify the location of the lesions.
Carbachol (Sigma) was applied either by local pressure injection or by
arterial perfusion. Local applications at a cortical depth of 500 µm
in either the LERC or the MERC were achieved by a single 10-s
microinjection of 5-50 mM carbachol in 0.9% NaCl through a glass
pipette (10 µm external tip diameter) connected to a graduated
syringe. These parameters corresponded to the injection of ~50 µl
of solution. Extracellular recordings were performed from the pipette
used for the injection of carbachol. When applied arterially, carbachol
was dissolved in the perfusate at a concentration of 100 µM. The
muscarinic antagonist atropine (Sigma) was dissolved in the Ringer
solution (5 µM) and administrated by arterial perfusion.
To better characterize the presence of gamma activity in the ERC, power
spectral density was estimated by means of a bivariate autoregressive
(AR) parametric model, based on a regression method (Dumermuth
and Molinari 1991
; Marple 1986
). The main
advantages of the AR method are that it does not need the averaging
procedure required by the more commonly applied fast Fourier transform
and that the frequency resolution of the spectrum does not depend on
the duration of the analyzed epoch (Panzica et al.
1999
). In a bivariate AR parametric model, the two signals are
represented through the following linear relationship
where X(t) = (x1,t,
x2,t) is the bidimensional vector
representing the sample of the two signals at the discrete time
t; A1,
A2, . . .,
Ap are the 2 × 2 matrices of the
model coefficients, E(t) = (e1,t, e2,t) is the vector of the two-dimensional
white-noise process with zero mean and covariance matrix R,
and P is the order of the AR model. Given the set of
parameters describing the AR modeling, the power spectral density can
be estimated by the following formula
where
I is the identity matrix,
is the
complex conjugate of H, the apical T denotes the
transposition of the matrix, and
T is the sampling
interval. The AR model order was determined by using the multichannel
version of the Akaike AIC criterion (Marple 1987
). The
coherence function is defined as
where Sxx(f) and
Syy(f) were the power spectral
densities of the two channels, (x and y
respectively), and Sxy(f) is the cross-spectral density.
 |
RESULTS |
Extracellular recordings before and after carbachol application
were performed from different ERC regions in 29 guinea pig brains
maintained in vitro. Considering the similarities between the cortical
organization of the guinea pig and the rat, we used the
cytoarchitectonic criteria described in the rat to divide the guinea
pig ERC in two areas that we designated MERC and LERC (Insausti
et al. 1997
; Menno Witter, personal communication; see DISCUSSION for details). In the large majority of the
experiments electrodes were positioned (under direct visual control
with a stereoscopic microscope) in the rostral and the caudal part of the LERC (rLERC and cLERC) and in the rostral, the intermediate, and
the caudal part of the MERC (rMERC, iMERC, and cMERC; see inset in Fig. 1A)
at a cortical depth of 400-500 µm. The predicted location of the
electrodes was consistently confirmed after histological verification
in 14 experiments (see Figs. 4 and 5 for ERC locations).

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Fig. 1.
Differential effect of local intracerebral injection of carbachol in
the medial entorhinal cortex (MERC) and the lateral entorhinal cortex
(LERC). A: single 10-s injection of carbachol (50 mM) in
the intermediate MERC (iMERC, *) induced fast rhythmic oscillations
in the gamma range (10 min) that gradually increased in
amplitude and propagated to the rostral and caudal MERC (rMERC, cMERC).
In the rostral and caudal lateral ERC (rLERC, cLERC), no gamma activity
was observed. The locations of the recording electrodes on the ERC
surface are shown in the ventral view of the guinea pig brain
(A, left). B: injection of
carbachol into the rLERC (*) after washout of the iMERC-injection
effect evoked no fast activity. In this figure, Fig. 2, and Fig. 3, ERC
recordings at five sites were performed simultaneously.
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|
In a set of experiments the effect of carbachol (5-50 mM) locally
injected for 10 s at 400-500-µm depth in the LERC and the MERC
was first analyzed (Fig. 1). When injections were performed, no
detectable change in the responses evoked by LOT stimulation was
observed at any of the recording sites. Carbachol injections in the
iMERC generated rhythmic field activity at 26.5-32.0 Hz (n = 18). The gamma activity appeared first in the
iMERC 5-10 min after drug injection and subsequently was observed in
the rMERC and cMERC, but never in the LERC (Fig. 1A). The
involvement of the rMERC in the generation of gamma oscillation was
inconsistent and depended on the location of the electrode (description
follows). The activity had a continuous character, increased
progressively to reach maximal amplitude within 30 min, and persisted
2 h after a single 10-s injection of carbachol. The frequency of the
fast oscillations increased to 41.7-45 Hz when the temperature of the preparation was raised from 32 to 37°C (n = 5). When
carbachol was applied locally to the LERC (either in the rLERC or in
the cLERC; n = 6), no rhythmic activity was observed
(Fig. 1B). Figure 2
illustrates in detail the events that followed iMERC application of 20 mM carbachol. In 8 of 18 experiments field oscillations at 8-12 Hz
transiently appeared in the rMERC at the same time that fast
oscillation was initiated in the iMERC (Fig. 2A, 5 min); 8-12-Hz oscillations lasted 5-15 min. Frequency analysis
demonstrated that such carbachol-induced rhythmic activity (see
DISCUSSION) was transiently observed throughout the ERC, as
illustrated by the frequency peaks of the spectra recorded 5 min after
carbachol injection (dashed lines in Fig. 2B; note the
different scales in the four diagrams). Concurrent with 8-12-Hz
activity, faster oscillations in the gamma range appeared in
the MERC, but not in the LERC. In the illustrated experiment
small-amplitude gamma activity was observed in the rMERC
(inset in the top left panel in Fig.
2B). The histological verification performed afterward showed that the recording electrode was not precisely positioned in the
rMERC, but at the border with the rLERC (described later).

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Fig. 2.
Effect of local injection of carbachol in the MERC. A:
single 10-s injection of carbachol (20 mM) in the iMERC (*) induced
transient rhythmic sequences at 8-12 Hz in the rMERC and rLERC
simultaneously to gamma activity in the iMERC (5 min).
The 8-12-Hz activity decreased within 10 min after carbachol
injection, and gamma oscillations become larger and propagated to the
entire MERC, but were not observed in the LERC. Positions of the
recording electrodes are shown in the ventral view of the brain
(A, left). Arrowheads indicate the responses evoked by
afferent stimulation of the lateral olfactory tract. B:
power spectra obtained from recordings performed in the same experiment
as in A before (dotted lines) and 5 min (dashed lines)
and 25 min (continuous lines) after carbachol injection into the iMERC.
Peak of 8-12-Hz activity was present in all recording sites at 5 min
(*) and disappeared in all sites except the rLERC at 25 min.
Inset, top left (rMERC): gamma activity when the power
density scale was uniformed to that used for cMERC and iMERC.
|
|
In a subsequent set of experiments, carbachol (50-100 µM) was
administered by arterial perfusion (n = 5). The drug
was diluted in the perfusate and applied for 10 min. As for the local
MERC application, 8-12-Hz activity that showed a continuous pattern at
the onset was observed (Fig. 3A, 16 min); such activity subsequently grouped in prolonged 2-10-s
bursts (Fig. 3A, 25 min) that recurred in 20-30-s
intervals. Eventually, 8-12-Hz activity decreased in amplitude and
disappeared within 10-30 min. When 8-12-Hz activity sequences were
observed, small-amplitude gamma activity also appeared in the MERC. Its
amplitude increased progressively with time and reached maximal values
in the intermediate-caudal MERC ~30 min after perfusion began,
regardless of the duration of the carbachol application, which usually
did not last longer than 10 min (Fig. 3A, 1 h). The mean
peak frequency of the fast activity recorded in different ERC regions
is reported in Fig. 3B (n = 5).

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Fig. 3.
Arterial perfusion of carbachol induced appearance of gamma
oscillations selectively in the MERC. A: 10-min
perfusion with 100 µM carbachol diluted in the perfusate induced
8-12-Hz activity in the rMERC and gamma activity in the iMERC
(16 min; time points after the onset of the carbachol
perfusion). In the next 15 min the 8-12-Hz activity organized in ictal
sequences (25 min) that disappeared within 30 min.
Continuous gamma activity was observed in the MERC exclusively and
persisted for 2 h. B: mean power spectra
(n = 4) 1 h after a 10-min arterial perfusion
of carbachol show gamma activity in the MERC (dashed line, iMERC and
continuous line, cMERC), but not in the LERC (dashed line, cLERC and
continuous line, rLERC).
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Regarding the local carbachol injections, gamma activity was expressed
in the MERC exclusively. When the perfusion with carbachol was
maintained for a period >10 min (n = 4), epileptiform
discharges characterized by ictal bursts of large-amplitude population
spikes that propagated throughout the limbic cortices, the hippocampus, and the amygdala were observed (not shown). Fast oscillatory activity in the MERC was observed also when epileptiform activity was induced. To sort out whether the gamma oscillations recorded simultaneously in
cMERC and iMERC were related to each other, we performed coherence analysis between pairs of recordings. This procedure revealed a very
low degree of coherence between the cortical regions. The correlation
coefficient calculated on the oscillatory activity recorded
simultaneously at different MERC locations was 0.226 for the local
carbachol iMERC injections (n = 6) and 0.155 for the
arterial carbachol perfusions (n = 4).
Figures 4 and
5 illustrate the histological
verification of the ERC recording sites. Figure 4 shows the locations
of three electrolytic lesions performed during an experiment in which
carbachol was ejected in the iMERC. Gamma activity was observed in the
iMERC (
) and in the cMERC (
), but not in the cLERC (
; Fig.
4B). In Fig. 5 the distribution of the electrolytic lesions
performed in 14 experiments is illustrated. The location of the lesions (represented by the symbols) were superimposed on sampled coronal ERC
sections representative of the rostrocaudal levels at which the lesions
were observed. The open symbols represent the sites where gamma
activity was recorded. No gamma oscillatory activity was observed in
the sites illustrated by the filled symbols. The caudal, intermediate,
and rostral portions of the LERC and MERC are described by different
symbols (see legend). The results confirm that fast oscillatory
activity was generated exclusively in the caudal and medial part of the
ERC. The lesions corresponding to the recording electrodes directed at
the most rostral part of the MERC were found in a cortical region that
showed the cytoarchitectonic features of the rLERC in three of five
experiments, due to the broadening of the most rostral part of the
LERC, which could be visualized by the medial extension of the patchy
appearance of layer II in Fig. 5, D, E, and
F. In the experiments in which the displacement of the
electrode in the LERC instead of the targeted rMERC was histologically
demonstrated, no gamma activity was observed (filled symbols).

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Fig. 4.
Histological controls of MERC and LERC recording electrodes.
A: electrolytic lesions in the cMERC ( ,
left) and in the iMERC and cLERC ( ,
right) were induced after recording the
activity illustrated in B. Thionine-stained 100-µm
thick coronal section. B: carbachol was ejected in the
iMERC, and gamma oscillations were recorded in the cMERC and in the
iMERC, but not in the cLERC. In A, calibration bar:
1 mm.
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Fig. 5.
A-F: recording sites reconstructed after histological
verification of the position of the electrolytic lesion induced at the
end of the recording session in 14 experiments. Location of the
electrodes was reconstructed on sampled coronal sections of the ERC
counterstained with thionine. Open symbols identify the regions from
which gamma oscillations were recorded. No fast activity was observed
at the sites marked by the filled symbols. , cMERC;
, iMERC; , cLERC; and
, rMERC; , rLERC. Note the patchy
appearance of the LERC where neurons in layer II are grouped in
clusters or islands. D, E, and
F: LERC extends medially in its most rostral portion.
Calibration bar: 1 mm.
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To identify the depth location of the generators of the fast
oscillatory activity in the MERC, depth profiles were performed in the
iMERC by inserting an electrode perpendicular to the cortical surface
and by advancing it in 100-µm steps, while a reference recording
electrode was placed close by at 500 µm. During the penetrations a
phase reversal of the gamma activity was observed at 200-400-µm
depth (Fig. 6A and
B), which corresponds to layer II (n = 5).
The amplitude of the gamma activity was maximal at 400-500 µm.

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Fig. 6.
Laminar profile of gamma activity in the iMERC after local injection of
carbachol. Profile was obtained by penetrating the cortex with a
recording electrode in 100-µm steps (top traces in
each pair of recordings illustrate different cortical depths), while a
reference electrode (ref; bottom traces) was steadily
inserted to a 500-µm depth in the near vicinity. A:
gamma activity showed a depth reversal at 200-300 µm and had maximal
amplitude at 500 µm. B: phase reversal in detail;
pairs a and b are magnified.
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When gamma activity was established in the MERC after local iMERC
injection, simultaneous extracellular recordings were performed from
different rhinencephalic structures in five experiments. No
carbachol-induced gamma activity was found in the anterior and
posterior piriform cortices, in the amygdala, in the perirhinal (PRC)
and postrhinal (PoRC) cortices ipsilateral to the carbachol injection,
or in the contralateral ERC (Fig. 7).
Fast oscillatory activity in the CA1 region of the hippocampus was
observed in three of four experiments. Such activity started earlier in
the MERC and showed higher frequency in the hippocampus (23-30 Hz) than in the iMERC (22-25 Hz), suggesting the existence of independent local gamma activity generators in the two cortical structures (Fig.
7). When carbachol was applied by arterial perfusion (n = 3), fast activity appeared simultaneously in the amygdala, the hippocampus, and the MERC, but not in the LERC, PC, PRC, and PoRC (data
not shown).

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Fig. 7.
Simultaneous recordings from different structures of the rhinencephalic
region during fast oscillations induced by local iMERC injection of
carbachol. Positions of recording electrodes are shown in the guinea
pig brain (left). Arrowheads (left) show
the field response evoked by lateral olfactory tract stimulation at
each recording site. Gamma activity was recorded in the MERC and in the
hippocampus, exclusively. Note the different frequency of the
oscillatory activity in the two structures. APC, anterior piriform
cortex; PPC, posterior piriform cortex; PRC, perirhinal cortex; PoRC,
postrhinal cortex.
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Previous in vitro and in vivo studies have demonstrated that the
effects of carbachol application are abolished by the antagonist of the
cholinergic muscarinic receptor atropine. Accordingly, the fast
oscillations induced by carbachol in the MERC completely disappeared
after arterial perfusion of atropine (5 µM; n = 7), whereas the LOT-evoked field response was not affected or was slightly
increased in amplitude in both the MERC and LERC (Fig. 8C). The atropine effect
reverted after washout in four tests.

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Fig. 8.
Responses evoked in the iMERC and the rLERC by LOT stimulation during
gamma activity induced by a 5-min arterial carbachol perfusion (100 µM). Fast oscillations in iMERC were abolished by coperfusion of
carbachol and 5 µM atropine (bottom traces). Atropine
induced an increase in the amplitude of the LOT-evoked responses.
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 |
DISCUSSION |
Recently Insausti and co-workers (1997)
proposed to
use cytoarchitectonic and connectivity criteria to distinguish ERC
subfields. According to this study, the portions of the ERC composed of
six distinct layers located caudally and medially correspond to the previously defined medial entorhinal area (Hjorth-Simonsen and Jeune 1972
; Steward and Scoville 1976
), whereas
the strip of cortex close to the rhinal sulcus that broadens rostrally
and shows a dense layer II formed by neurons grouped in clusters or
islands separated by acellular bands corresponds to the lateral
entorhinal area. The ERC subdivision in two principal regions on the
basis of cytoarchitectonic features is also present in the guinea pig and was used in our study to identify the regions that are capable of
generating carbachol-induced gamma oscillations. The medial part of the
ERC that corresponds to the medial and caudal ERC of Insausti et
al. (1997)
produced high-frequency oscillations, whereas the
lateral and intermediate parts of the ERC close to the rhinal sulcus
did not generate gamma rhythmic activity after either local or arterial
application of carbachol. It should be noted that the LERC and the MERC
also differ substantially in their patterns of afferent and efferent
connections. Retrograde tracing studies of the cortical
extrahippocampal projections to the ERC demonstrate that in the rat and
the monkey the lateral strip of ERC adjacent to the rhinal sulcus
projects diffusely to those neocortical areas (Kosel et al.
1982
; Swanson and Köhler 1986
) that are
also connected to the PRC (Burwell et al. 1995
; Kosel et al. 1982
), whereas sparse retrograde labeling
was observed in the MERC when neocortical injections were performed.
Moreover, the LERC shares with the PRC the pattern of projection into
the subiculum (van Haeften and Witter 1997
). Further
indications of the functional independence between MERC and LERC are
provided by the existence of separate anatomic projections to either
one of the two major subdivisions of the ERC. For instance, the
presubicular projections to the ERC are confined to the medial and
dorsal portions of the guinea pig ERC (Shipley 1975
) and
the projection from the olfactory bulb and the piriform cortex
terminate preferentially in the LERC (Krettek and Price
1978
). These findings suggest that the MERC and the LERC can be
considered as anatomically and functionally distinct structures.
The ERC receives a cholinergic input from the basal forebrain
predominantly on layer II and layer V (Alonso and Köhler
1984
; Eckenstein et al. 1988
; Gaykema et
al. 1990
). Cholinergic inputs to the cortex are commonly
interpreted as an arousing signal. In fact, it has been demonstrated
that cortical gamma frequency activity is enhanced either by electrical
stimulation of the mesopontine cholinergic nuclei (Steriade et
al. 1991
, 1996
) or by pharmacological activation of the basal
forebrain (Cape and Jones 1998
). Moreover, functional
brain states such as wakefulness and paradoxical sleep correlate to an
increase of gamma frequencies in the electroencephalogram (Maloney et al. 1996
). The ultimate evidence that
establish the involvement of the cholinergic system in the control of
fast oscillations is the recent demonstration that gamma activity can
be induced by application of the muscarinic receptor agonist carbachol
in slices of hippocampus and neocortex maintained in vitro (Buhl et al. 1998
; Fisahn et al. 1998
). Our findings
confirm that fast oscillations in the gamma range are activated by
muscarinic activation in the MERC, therefore supporting the role of
cholinergic activation in the modulation of fast brain rhythms.
Our understanding of the MERC function is based on a wealth of anatomic
data and physiological studies performed in vivo and in vitro.
According to these studies it can be concluded that even if neurons in
the MERC are able to generate fast oscillations in the gamma range
(Chrobak and Buzsaki 1998
), such activity cannot be
accounted for exclusively by intrinsic membrane properties. Indeed,
neurons that exhibit intrinsic fast oscillatory activity such as those
described in thalamus and neocortex (Gray and McCormick 1996
; Llinas et al. 1991
; Steriade et al.
1996
) were never observed in the MERC neurons of layer II
(Alonso and Klink 1993
; Alonso and Llinas
1989
), layer III (Dickson et al. 1997
; van der Linden and Lopes
da Silva 1998
), and layer V (Jones 1993
; Jones
and Heinemann 1988
). Moreover, the lack of studies that
specifically describe the network and intrinsic properties of LERC
neurons does not allow any conclusion to be drawn about the role of
differential expression of electroresponsive properties in explaining
the different ability of MERC and LERC to generate gamma oscillations
in response to cholinergic activation.
The possibility should be mentioned that the absence of gamma in the
LERC could be due to the experimental conditions we used
that is, the
use of cholinergic agonists to induce fast oscillation. It is feasible
that sustained gamma activity in the hippocampus could synaptically
entrain fast oscillations in the LERC. A careful evaluation of the
coherence relationship between gamma rhythms in limbic structures
performed in vivo will help to clear up this issue.
The demonstration of a low coherence between fast oscillations recorded
simultaneously at different sites in the MERC after either local or
arterial perfusion of carbachol suggests that gamma activity is not a
population event that synchronizes the entire cortical region, but
rather a local event generated at multiple sites within the MERC. As
for CA1 and the subiculum (Bragin et al. 1995
;
Penttonen et al. 1998
; Stanford et al.
1998
; Traub et al. 1996
; Whittington et
al. 1995
), the gamma oscillations in the ERC were proposed to
be elicited in principal neurons by rhythmic inhibitory postsynaptic
potentials (IPSPs) imposed by a tonic excitation on mutually inhibitory
interneurons (Chrobak and Buzsaki 1998
). Interactions
between local interneurons (together with a reciprocal excitation among
pyramidal cells) also play a major role in sustaining the oscillations
induced by carbachol in the CA3 region of the hippocampus
(Fisahn et al. 1998
). If local interneurons are involved
in the generation of fast oscillatory activity in the MERC, our data on
the absence of coherence between gamma activity recorded in different
MERC locations and between MERC and hippocampus support the idea that
segregated pools of interneurons may entrain patches of cortex
independently. This assumption is in agreement with the conclusion
reached in a recent study on carbachol-induced epileptiform discharges
in the MERC, which demonstrated that giant IPSPs activated by
muscarinic stimulation probably result from the postsynaptic effect of
synchronous firing of interneurons on layer II principal neurons
(Dickson and Alonso 1997
). These IPSPs were asynchronous
at sites separated by more than 200 µm, suggesting that the events
were generated by discrete and independent pools of neurons in the ERC.
A careful pharmacological characterization of the events observed in
our experiments will reveal whether a similar network organization
could sustain gamma generation and propagation in the MERC.
Studies performed in slices have demonstrated that layer II neurons in
the MERC have the intrinsic capability to produce subthreshold membrane
oscillations in the theta range (Alonso and Klink 1993
; Alonso and Llinas 1989
). Rhythmic oscillations at 5-12
Hz can be induced in vitro by perfusing cortical slices with carbachol (Bland and Colom 1993
; Konopacki et al.
1987
). In our experiments 8-12-Hz activity occurred
simultaneously with gamma activity but was prevalent in most rostral
portions of LERC and MERC, in contrast with the gamma oscillations that
were found exclusively in the MERC and were observed only transiently
(5-15 min), whereas gamma activity lasted several hours. Such
theta-like activity showed peculiar features reminiscent of
carbachol-induced hypersynchronous discharges described in the
hippocampus (Traub et al. 1992
; Williams and
Kauer 1997
), the ERC (Dickson and Alonso 1998
;
Klink and Alonso 1997
), and the neocortex
(Lukatch and McIver 1997
) in vitro. The functional
significance of this activity is still unresolved and its possible
correlation with the theta activity recorded in vivo is controversial.
Early anatomic studies demonstrated that layer II neurons of the MERC
and the LERC in the rat project to different portions of the molecular
layer in the dentate gyrus (i.e., to the middle (proximal) and the
outer (distal) dendrites of granule cells, respectively)
(Hjorth-Simonsen and Jeune 1972
; Steward and
Scoville 1976
; Witter 1993
). Such a distinct
projection pattern was subsequently confirmed electrophysiologically
(Canning and Leung; 1997
; Dickson and Alonso
1998
; Leung et al. 1995
; McNaughton
1980
; Wilson and Steward 1978
). The laminar
segregation of MERC and LERC inputs to the dentate gyrus may be related
to the functional meaning of the differential expression of gamma
oscillations in the two ERC regions. We speculate that if both MERC and
LERC receive simultaneously afferent inputs from different brain
regions, the reciprocal weight of the ERC outputs transmitted to the
hippocampus through the dentate gyrus could be different whether fast
oscillations in the MERC are present or not. The cholinergic modulation
of layer II MERC neurons during gamma oscillations can enhance the
probability that these neurons will reach firing threshold, as
demonstrated in the hippocampus (Fishan et al. 1998
). It
has been demonstrated that layer II neurons in the MERC are able to
endure high-frequency firing at 40 Hz during synaptic activation
(Gloveli et al. 1997
). Repetitive high-frequency firing
of the MERC may lead to a sustained synaptic depolarization of the
proximal dendrites the granule cell, which could heterosynaptically
facilitate a separate synaptic input simultaneously impinging on the
distal dendrites of the dentate neuron through the concomitant
activation of the LERC input.
The present findings represent the first demonstration of a functional
distinction between the MERC and the LERC that may underlie different
mechanisms of information transfer from the neocortex to the
hippocampus. Cholinergic modulation may influence the processes of
memory formation and/or retrieval by way of inducing gamma oscillations
in the MERC that could work as a switch to enhance the strength of the
synapses formed by the medial perforant path on the dentate granular cells.
We thank M. Witter for instructive discussion on the structural
organization of the ERC in the guinea pig, F. Lopes da Silva for
helpful comments on a draft of the manuscript, and G. Biella for
developing acquisition and analysis software programs in Labview.
The study was sponsored by the Human Frontier Science Program
Organization Grant RG0019-1996B. S. van der Linden was partially supported by the Italian National Health Ministry.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.