Department of Experimental Neurophysiology, Istituto Nazionale
Neurologico, 20133 Milan, Italy
 |
INTRODUCTION |
Several pieces of evidence indicate that the lateral
entorhinal cortex (LERC) and the medial entorhinal cortex (MERC) can be
distinguished according to their general cytoarchitectonic features
(Insausti et al. 1997
), their connectivity
(Deadwyler et al. 1975
; Hjorth-Simonsen and Jeune
1972
; Kosel et al. 1982
; Liu and Bilkey
1997
; Shipley 1975
; Swanson and
Köhler 1986
; Wu et al. 1998
), and
their pattern of activation (van der Linden et al. 1999
). The
possibility that these two cortical regions represent functionally
independent structures will be further tested here by analyzing their
functional activation in response to olfactory input. The olfactory
projection to the limbic cortices has been extensively studied using
anatomic techniques (Krettek and Price 1978
;
Luskin and Price 1983
; Room et al. 1984
;
Schwerdtfeger et al. 1990
; Wilson and Steward
1978
). The fibers of the lateral olfactory tract (LOT) rise
from the olfactory bulbs and project to the piriform cortex (PC) and
the rostral part of the entorhinal cortex (ERC). Both LOT fibers and
corticocortical associative fibers that originate in the PC terminate
principally in the superficial layers of the LERC. Electrophysiological
studies have confirmed the selective olfactory projection to the
rostrolateral ERC (Boeijinga and Van Groen 1984
;
Chapman and Racine 1997
; Deadwyler et al. 1975
; Liu and Bilkey 1997
; Mouly et al.
1998
; Van Groen et al. 1987
). These reports
suggested that olfactory inputs do not project directly to the MERC.
Moreover, olfactory afferents cannot be transmitted to the MERC via the
LERC because the lateral and medial ERC are not interconnected (M. de
Curtis, G. Biella, and T. Iijima, unpublished observations;
Dolorfo and Amaral 1998
). The LERC projects via the
lateral perforant path to the hippocampus (Canning and Leung
1997
; Hjorth-Simonsen and Jeune 1972
;
Leung et al. 1995
), from which a diffusely distributed
projection returns to the ERC (Lopes da Silva et al.
1990
; Witter 1993
).
To verify the existence of selective projection of olfactory afferents
to ERC subregions, we used in vitro isolated guinea pig brain
preparation to map the responses evoked by LOT stimulation in the
medial and lateral portions of the entorhinal region. Isolated brain
preparation is an ideal preparation with which to perform such a study
because the position of the recording electrodes can be easily and
rapidly moved under direct visual control in different sites of the
exposed ERC (Biella and de Curtis 1995
; Biella et al. 1996
; de Curtis et al.
1991
; Muhlethaler et al. 1993
). We show that the
most lateral aspect of the rostral ERC receives monosynaptic olfactory
input whereas an associative polysynaptic response is observed in the
entire LERC. The deep layers of the MERC were exclusively activated
polysynaptically via the hippocampus when a large population spike was
generated in the CA1 region by increased stimulation intensity.
Preliminary results were reported in abstract form (Biella and
de Curtis 1999
).
 |
METHODS |
Adult guinea pigs (150-250 g) were anesthetized with sodium
pentothal (20 mg/kg i.p.). During anesthesia, an intracardiac perfusion
with cold, oxygenated saline solution was performed before the brain
isolation procedure was started (for details see de Curtis et
al. 1991
, 1998
; Muhlethaler et al. 1993
). The brain was perfused through the basilar artery with a complex saline solution composed 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, and 3%
dextran M. W. 70.000 (SIFRA, Isola della Scala, Italy) and was
saturated with a 95% O2-5%
CO2 gas mixture. The brain was maintained in
vitro in an incubation chamber at 15°C during the dissection and the
temperature of the chamber was slowly increased (0.2°C/min) to 32°C
before the experiment was started. The experimental protocol was
reviewed and approved by the Committee on Animal Care and Use and by
the Ethical Committee of the Istituto Nazionale Neurologico.
Extracellular recordings were performed with tungsten electrodes, glass
micropipettes filled with 1 M NaCl, stainless steel electrodes, and
multichannel silicon probes featuring 16 iridium recording sites 100 µm apart and vertically assembled in a single shaft (obtained from
the Center of Neural Communication Technology, University of Michigan,
Ann Arbor, MI). These probes have been proven to be ideal for
recording laminar profiles in cortical structures (Bragin et al.
1995
). The input resistance of the extracellular recording
electrodes varied between 2 and 4 MOhm. The multichannel electrodes
were positioned perpendicular to the cortical lamination at different
sites in the medial and lateral parts of the ERC. The placement of the
electrodes was performed under direct visual control via a stereoscopic
microscope. LOT-evoked responses were used to verify that all recording
sites along the shaft of the silicon probe were inserted in the cortex;
the most superficial contact was positioned at the pial surface. The
positions of the recording electrodes were verified by identifying the
lesions by passing a 20 µAmp current between the two deepest iridium
contacts for 10-20 s at a depth of 1500-1600 µm.
Histological controls were performed on 100-µm coronal sections cut
from brains fixed with 4% paraformaldehyde. Stimulating bipolar
electrodes (custom-made twisted silver wires or tungsten electrodes,
FHC, Bowdoinham, ME) were positioned either on the LOT or in the
molecular layer of the posterior PC.
Averages of 5-7 responses were used to build field potential laminar
profiles recorded with the 16-channel silicon probes. Current source
density (CSD) analysis was performed on 100-µm per step profiles with
a 400-µm separation grid, as previously described (Biella and
de Curtis 1995
; de Curtis et al. 1994
).
The data were recorded with a 16-channel extracellular amplifier
(Biomedical Engineering, Thornwood, NY) and were stored on a digital
tape recorder (Biologic, Claix, France). Online and offline
analyses were performed with CLAMPVIEW (SIDeA, Milan, Italy). Specific
subroutines for CSD data analysis were developed in our laboratory by
G. Biella in collaboration with SIDeA.
 |
RESULTS |
This study was performed on 31 isolated guinea pig brains. In the
first set of experiments, the responses evoked by LOT stimulation in
the olfactory-limbic region were characterized. Responses were mapped
by making recordings, within the same session, from 10-20 sites in the
piriform, entorhinal, insular, and perirhinal cortices and in the
amygdala, with simultaneous recordings from as many as seven
electrodes. One electrode was permanently positioned in the anterior
piriform cortex to monitor the stability of the LOT-evoked response
when the other recording electrodes were moved around during the
experiment. Figure 1 illustrates the typical pattern activated by a LOT stimulus (70% of the intensity necessary to
induce a maximal monosynaptic response in electrodes 1 and 2 from the
anterior PC). The positions of the cortical recording electrodes, shown in the ventral view of a guinea pig brain in Fig. 1,
left, were reproduced between experiments by using surface brain structures as reference points. A monosynaptic response was
observed in the piriform cortex (electrodes 1, 2, and 3), the
periamygdaloid cortex (electrode 4), the basolateral amygdala (electrode 5), and the rostral part of the LERC (electrode 9). A
large-amplitude polysynaptic response was recorded in all cortical sites analyzed, with the exception of the MERC (electrodes 12 and 13)
and the caudal perirhinal cortex (electrode 8), where small-amplitude,
possibly volume-conducted, responses were observed (see Fig.
3). The latencies of the monosynaptic peak amplitude potentials
in the posterior piriform cortex (PPC) and the LERC were 12.38 ± 1.92 (SD) and 15.59 ± 1.52 ms, respectively (n = 12). The polysynaptic responses in the PPC, the rostral LERC (electrode 9), and the caudal LERC (electrode 11) peaked at 21.45 ± 2.93, 29.18 ± 2.47, and 37.33 ± 2.66 ms, respectively
(n = 11). Current source density analysis performed on
laminar field responses recorded with multichannel silicon probes
demonstrated that the mono- and disynaptic responses in the PPC
(n = 22) and the LERC (n = 20) were
generated by current sinks located in the superficial layers (Fig.
2; see also Biella and de Curtis
1995
; Biella et al. 1996
; Boeijinga and
Van Groen 1984
; de Curtis et al. 1991
); no
locally generated sinks were observed in the perirhinal cortex
(PRC) (n = 7; not shown) and MERC (Fig.
3; n = 6). We could not
detect a monosynaptic sink in the caudal two-thirds of the LERC, where the field profile in Fig. 2 was performed. Two electrode tracks determined by the 16-channel silicon probes in the LERC and the MERC
are illustrated in Fig. 3C. As in other mammals,
MERC in the guinea pig was characterized by six distinct layers whereas the LERC cytoarchitecture featured 1) clusters or islands of
neurons in layer II, 2) a thinner lamina dissecans (layer
IV), and 3) a less well-defined distinction between deep
layers V and VI (Insausti et al. 1997
). The CSD results
confirmed that the small-amplitude field responses recorded in the MERC
and the PRC represent far fields generated in the LERC and passively
volume-conducted through the tissue.

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Fig. 1.
Field responses evoked by lateral olfactory tract (LOT) stimulation in
the olfactory cortex and limbic structures of isolated guinea pig brain
preparation. Positions of electrodes (left) used to
record responses (right) are shown on a schematic
ventral view of the guinea pig brain. Cortical recordings were
performed at depths 500-700 µm from the pial surface. Arrowheads,
LOT stimulus. SE, stimulation electrode.
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Fig. 2.
Current source density (CSD) analysis of field potential laminar
profiles performed with 16-channel silicon probes (100 µm separation
between recording sites) in the posterior piriform cortex (PPC) and the
lateral entorhinal cortex (LERC) after LOT stimulation. Laminar field
profiles (averages of 5 LOT-evoked responses, A),
three-dimensional (B), and contour (C)
plots of the CSD profiles are shown. "Mountains" in
B and solid lines in C identify current
sinks. "Valleys" and dotted lines mark current sources. The
direction of sinks and sources is arbitrary. Asterisks and stars,
monosynaptic and disynaptic sinks, respectively. Current values in
B are mV/mm2. Contour intervals in
C are 1.3 and 0.6 V/mm2 for PPC and LERC,
respectively.
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Fig. 3.
CSD analysis of field potential laminar profiles performed with
16-channel silicon probes (100 µm separation between recording sites)
in the medial entorhinal cortex (MERC) after LOT stimulation.
A: laminar field profiles (average of 5 LOT-evoked
responses); B: three-dimensional plots of the CSD
profiles. See Fig. 2 for details. C: electrode tracks
formed by 16-channel probes in MERC (right) and LERC
(left) are shown in 100-µm coronal sections stained
with thionine. LERC track was obtained in one of the experiments in
Fig. 2. Cortical layers are indicated on the left side of each
photograph. Typical cytoarchitectonic features of the 2 ERC regions are
shown in the representative sections.
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In nine of 13 experiments, the MERC was activated when the intensity of
LOT stimulation was increased above the threshold for induction of a
large and synchronous population spike in the hippocampus. In Fig.
4, simultaneous recordings were performed in
the anterior piriform cortex (APC) (electrode 1), at two sites in the
LERC (electrodes 2 and 3) and the MERC (electrodes 4 and 5), in the
dentate gyrus (DG) (electrode 6), and in the CA1 region of the
hippocampus (electrode 7). The positions of the recording electrodes
are illustrated in Fig. 4, left. The location of the hippocampal electrodes was confirmed histologically by
identifying the electrolytic lesions formed by stainless steel
electrodes at the end of electrophysiological recording (not shown). A
large field response was observed in the MERC when the stimulus
intensity was increased to generate a population spike in the DG and
CA1 recording sites (arrows in Fig. 4, right). A population
spike was consistently observed in the MERC response (asterisk in Fig. 4, right). The latency between the CA1 spike and the
population spike in the MERC response was 10.6 ± 0.9 ms
(n = 11). As illustrated in Fig.
5, late posthippocampal responses (asterisks)
were observed in the caudal and medial parts of the entorhinal region
(dark gray area) whereas no late responses were recorded in the lateral and rostral parts (light gray area), in which large short-latency LOT
responses were observed. Small-amplitude posthippocampal responses in
the LERC were observed in nine experiments (see Fig. 7A).
Such responses probably represent far fields because they were not associated with locally generated current sinks (not shown;
n = 3). Predictions of the recording electrode
location in the LERC or MERC on the basis of electrophysiological
responses were consistently confirmed by morphological controls
performed on Nissl-stained (100 µm) coronal sections after
electrocoagulation of the recording electrode tip (see
DISCUSSION).

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Fig. 4.
Field responses recorded simultaneously in limbic cortices and evoked
by 2 intensities of LOT stimulation (7 and 20 µA, 0.1 ms). Site 1, anterior piriform cortex (APC); sites 2 and 3, rostral and caudal LERC;
sites 4 and 5, rostral and caudal MERC; sites 6 and 7, dentate gyrus
(DG) and CA1. Arrowhead, stimulus delay; arrows, DG and CA1 population
spikes; asterisk, late response in the MERC evoked by high-intensity
LOT stimulation. Piriform cortex (PC) and entorhinal cortex (ERC)
recordings were performed at depths of 500-700 µm.
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Fig. 5.
Map of late response in the ERC in a typical experiment. Histological
identification of typical MERC and LERC cytoarchitectonic features of
the region in which the different recordings were performed was
verified. LERC and MERC are light gray and dark gray, respectively.
Recordings in the MERC were performed at a depth of 200 µm and
recordings in the LERC at a depth of 500 µm. Asterisks, late MERC
responses. Amplitude calibration bars on the left and right refer to
LERC and MERC potentials, respectively.
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CSD analysis of MERC field potential profiles confirmed the local
origin of the late posthippocampal response. As illustrated in Fig. 6,
B and C (the average of
6 CSD profiles), a fast, large-amplitude sink superimposed on a slower
sink centered at 600-1000 µm (layers III-V in MERC) was observed.
During paired LOT stimulation (10-50% of the intensity necessary for
achieving the hippocampal activation threshold), the
hippocampus-MERC circuit was activated in the second conditioned
response for an interstimulus interval between 100 and 900 ms (Fig.
7B). As illustrated in in Fig.
7C, bottom trace, repeated low-intensity LOT
stimulation at a frequency between 2 and 8 Hz determined MERC response
activation. MERC activation via the hippocampus (Fig. 7C,
arrows) showed a noncontinuous pattern.

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Fig. 6.
CSD analysis of MERC field potential profile (A)
performed with 16-channel probes. A: potentials from a
representative experiment. Three-dimensional (B) and
contour plots (C) of CSD profile (average of 6 profiles
obtained in different experiments) are shown. Asterisk, large sink
generated in deep cortical layers. Current values in B
are mV/mm2. Contour intervals in C are 0.12 V/mm2.
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Fig. 7.
A: sequential activation of the PPC, LERC, hippocampus
(CA1), and MERC on high-intensity LOT stimulation; asterisks, late ERC
responses. B: MERC responses to LOT pairing at stimulus
intensity subthreshold for hippocampal activation. MERC response was
observed for interstimulus intervals between 100 ms and 1 s.
C: repetitive low-intensity LOT stimulation at 3, 5, and
7 Hz (but not at 1 or 10 Hz) induces late activation of the MERC
(arrows). Stimuli are indicated by dots below each trace.
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 |
DISCUSSION |
The present study demonstrates that, in the guinea pig,
1) stimulation of LOT fibers originating in the olfactory
bulbs induces short-latency, monodisynaptic responses in the LERC but
not in the MERC, 2) the MERC is polysynaptically activated
exclusively after hippocampal activation, and 3) the MERC
can be entrained by augmenting LOT stimulation to an intensity above
the threshold for hippocampal activation or by repetitive low-intensity
stimulation at 2-8 Hz.
Anatomic studies with retrograde and anterograde tracers in different
animal species demonstrated that the olfactory bulb projects to the ERC
via the LOT. In most of these studies, the olfactory fibers were
reported to project almost exclusively to the LERC (Haberly and
Price 1977
; Kosel et al. 1981
; Luskin and Price 1983
; Room et al. 1984
;
Schwerdtfeger et al. 1990
), primarily to its most
rostral portion. Only one study showed a diffuse projection that also involved the MERC (Wouterlood and Nederlof
1983
). The prevalent view that olfactory input
projects to the LERC is supported by electrophysiological studies that
demonstrate field responses in the LERC after LOT stimulation
(Boeijinga and Van Groen 1984
; Chapman and Racine
1997
; Liu and Bilkey 1997
; Mouly et al.
1998
; Van Groen et al. 1987
) whereas there are
no reports of olfactory-evoked responses in the MERC. A study that
described the olfactory projections to the hippocampus via the ERC
(Wilson and Steward 1978
) demonstrated that the response
evoked by LOT stimulation in the DG was abolished when the LERC was
lesioned, which suggests that the olfactory path to the hippocampus
does not pass through the MERC. Our data confirmed that the rostral
portion of the LERC mediates a direct olfactory projection to the
hippocampus that does not involve MERC significantly.
LOT stimulation induced prominent polysynaptic responses in the LERC.
The results described here suggest that such a projection is
functionally maintained by the contribution of associative cortical
input from the piriform cortex, which is synchronously and massively
activated by LOT stimulation. This conclusion is supported by
1) the demonstration of high-amplitude polysynaptic responses in the LERC during our experiments and in the rat in vivo
(Mouly et al. 1998
) and 2) the observation of
a larger response evoked by PPC stimulation in comparison with the
LOT-evoked potential in the LERC (de Curtis et al. 1994
;
Habets et al. 1980
; Wilson and Steward
1978
). In our experiments, the monosynaptic response evoked by
LOT stimulation indeed showed small amplitude and was restricted to the
most rostral portion of the LERC whereas a large polysynaptic potential
was found throughout the LERC (see also Mouly et al.
1998
). The presence of a strong associative connectivity between the PC and the LERC is suggested by the relative amplitudes of
the mono- and disynaptic components in the PPC and the LERC (see also
Boeijinga and Van Groen 1984
). Although the monosynaptic potential amplitude decreased from rostral to caudal and virtually disappeared in the caudal two-thirds of the LERC, the disynaptic peak
amplitude did not decline with the monosynaptic potential and was
consistently observed throughout the LERC. The disynaptic potential in
the LERC was abolished by interrupting the LOT and the associative
fibers with a superficial coronal section at the PPC-ERC border
(unpublished observations; Biella et al. 1996
), suggesting that the disynaptic potential in the ERC is mediated by the
activation of associative fibers originating in the PC.
The absence of an associative response in the MERC after LOT
stimulation demonstrates that 1) the corticocortical
projections arising from the entire PC do not project to the MERC and
2) the LERC and MERC are completely separate with regard to
olfactory input. Our results not only confirm the pattern of
distribution of the olfactory fibers in the ERC, but also corroborate
anatomic observations that exclude the presence of a lateral-to-medial associative fiber system within the ERC and demonstrate intrinsic associative connections, predominantly in the rostrocaudal dimension (Dolorfo and Amaral 1998
). This conclusion is further
strengthened by preliminary results that confirm the absence of an LERC
response when the MERC is stimulated, and vice versa (unpublished
observations). Incidentally, our results show that there is no
functionally active direct projection from the olfactory areas to the
PRC whereas a polysynaptic response can be recorded in the rostral PRC
and in the insular cortex located just lateral to the rhinal sulcus at
the same rostrocaudal level of the piriform cortex. Based on the
connectivity patterns shown here, it can be concluded that the LERC,
but not the MERC, can be regarded as an associative olfactory area.
Our findings demonstrate that the hippocampus can be activated by
olfactory stimulation. Hippocampal responses to LOT stimulation were
been previously reported in different animal species (Habets et
al. 1980
; Schwerdtfeger et al. 1990
;
Wilson and Steward 1978
). When the hippocampal loop was
activated by strong or repetitive LOT stimulation, an efferent signal
reentered the MERC, as illustrated in Figs. 2, 3, and 6. The latencies
between the hippocampal spike in CA1 and the MERC response were
compatible with a single-synapse transmission. Even if anatomic studies
show that all layers in the ERC receive fibers from the hippocampus
(see Witter 1993
), a large contingent of hippocampal
efferents from the CA1/3 area and the subiculum has been shown to
contact the deep layers in the MERC in rat and guinea pig
(Hjorth-Simonsen and Jeune 1972
; Swanson and
Köhler 1986
). According to our findings, the
hippocampal efferent projection activated by olfactory stimulation
generates a distinct sink 600-1000 µm deep in layers III-V of the
MERC. Electrophysiological studies performed in vivo in the guinea pig demonstrated that stimulation of the dorsal psalterium induced activation of the contralateral hippocampus followed by a
posthippocampal response generated in the contralateral ERC
(Bartesaghi et al. 1989
) that showed general features
and latencies comparable to the MERC potential recorded in our
experiments. In agreement with our findings, in these in vivo studies
the posthippocampal potentials were 1) recorded in the
medial part of the ERC, 2) observed diffusely in the
rostrocaudal dimension of the ERC (Bartesaghi 1994
), and 3) generated between layers VI and III.
The hippocampus and the MERC were activated by repetitive LOT
stimulation in a particular frequency range (2-8 Hz) close to olfactory theta activity (Freeman and Schneider 1982
),
an oscillatory pattern that has been linked to odor discrimination
induced by sniffing in mammals (Macrides et al. 1982
;
Yougentob et al. 1987
). It is tempting to speculate that
repeated, rhythmic olfactory activation at a frequency that mimics
"theta sniffing" might determine a condition that promotes
associative interactions in the MERC between olfactory signals and
nonolfactory cortical inputs. Further evaluation of such interactions
in the MERC will help clarify the role of olfaction in memory formation
and retrieval.
We thank J. Hetcke of the University of Michigan for
generously providing multichannel silicon probes without which this
study would not have been possible. We also thank C. Grassi and D. Brambilla for technical assistance.
This study was sponsored by Human Frontier Science Program Organization
Grant RG 109/96. G. Biella was supported by the same HFSPO grant.
Address for reprint requests: M. de Curtis, Dept. of Experimental
Neurophysiology, Istituto Nazionale Neurologico, via Celoria 11, 20133 Milan, Italy.
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.