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The Journal of Neuroscience, October 15, 2000, 20(20):7846-7854
Evidence for Spatial Modules Mediated by Temporal Synchronization
of Carbachol-Induced Gamma Rhythm in Medial Entorhinal Cortex
Clayton T.
Dickson,
Gerardo
Biella, and
Marco
de Curtis
Department of Experimental Neurophysiology, Istituto Nazionale
Neurologico "Carlo Besta", Milan 20133, Italy
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ABSTRACT |
Fast ( ) oscillations in the cortex underlie the rapid temporal
coordination of large-scale neuronal assemblies in the processing of
sensory stimuli. Cortical rhythm is modulated in
vivo by cholinergic innervation from the basal forebrain and
can be generated in vitro after exogenous cholinergic
stimulation. Using the isolated guinea pig brain, an in
vitro preparation that allows for the study of an intact
cerebrum, we studied the spatial features of activity evoked by the
cholinomimetic carbachol (CCh) in the medial entorhinal cortex (mEC).
activity induced by either arterial perfusion or intraparenchymal
application of CCh showed a phase reversal across mEC layer II and was
reduced or abolished in a spatially localized region by focal infusions
of atropine, bicuculline, and CNQX. In addition, a spatially restricted
zone of activity could be induced by passive diffusion of CCh from
a recording pipette. Finally, oscillations recorded at multiple
sites across the surface of the mEC using array electrodes during
arterial perfusion of CCh demonstrated a decline in synchronization
(coherence) as the interelectrode distance increased. This effect was
independent of the signal amplitude and was specific for as opposed
to theta-like activity induced by CCh in the same experiments.
These results suggest that CCh-induced oscillations in the mEC are
mediated through direct muscarinic excitation of a highly localized
reciprocal inhibitory-excitatory network located in superficial
layers. We propose that functional cortical modules of highly
synchronous oscillations may organize incoming (cortical) and
outgoing (hippocampal) information in the mEC.
Key words:
rhythm; 40 Hz oscillation; synchrony; muscarinic; entorhinal cortex; perforant path; limbic system; inhibitory
interneuron; GABA; integration; , binding; memory
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INTRODUCTION |
Rhythmic oscillatory activity,
through its ability to temporally synchronize the activity of large
subsets of cortical neurons, has been thought to underlie both
fundamental (Schurmann et al., 1997 ) and higher brain functions such as
perception (Eckhorn et al., 1988; Gray et al., 1989), arousal or
attention (Tiitinen et al., 1993; Maloney et al., 1996), sensorimotor
integration (Murthy and Fetz, 1992), and even cognition or
consciousness (Llinás and Ribary, 1993). Fast activity in the range (25-80 Hz) has been observed throughout the cortical mantle,
including limbic cortices, where it has been suggested to play a role
in memory processes (Bragin et al., 1995; Lisman and Idiart, 1995;
Chrobak and Buzsaki, 1998a). A nodal portion of the limbic area
is the entorhinal cortex (EC), which sits at the interface between the neocortex and the hippocampus (Van Hoesen, 1982; Witter et al., 1989)
and, as such, is an integral part of the temporal lobe memory system
(Squire and Zola-Morgan, 1991; Zola-Morgan and Squire, 1993). A
proportion of superficially located EC neurons, the cells of origin of
the perforant path input to the hippocampal formation, are endowed with
endogenous oscillatory properties (Alonso and Llinás, 1989) and
show rhythmic and synchronized activity in vivo at both slow
(theta) and fast () frequencies (Mitchell and Ranck, 1980; Alonso
and García-Austt, 1987; Dickson et al., 1995; Chrobak and
Buzsaki, 1998a). These oscillatory rhythms are presumably important for information processing and memory formation and retrieval
(Winson, 1978; Buzsáki, 1989; Ahissar et al., 1992; Lisman and
Idiart, 1995; Shulz et al., 2000). As in the neocortex (Steriade et
al., 1991; Metherate et al., 1992; Munk et al., 1996; Cape and Jones,
1997; Herculano-Houzel et al., 1999), the expression and modulation of
these rhythms are under the control of cholinergic inputs from the
basal forebrain (Dickson et al., 1994; Jeffery et al., 1995; Ma and
Leung, 1999), which profusely innervates the EC (Alonso and
Köhler, 1984). In vitro studies have shown that
cholinergic activation exerts profound modulatory effects on the
membrane properties of superficial EC neurons, which allow for the
expression of oscillatory behavior (Dickson and Alonso, 1997; Klink and
Alonso, 1997b).
oscillatory activity has been described in in vitro
slice preparations of hippocampus (Fisahn et al., 1998; Fellous and Sejnowski, 2000) and somatosensory cortex (Buhl et al., 1998) treated
with the cholinergic agonist carbachol (CCh). Recently, we have
demonstrated that CCh perfusion elicits activity in the medial EC
(mEC), but not lateral EC (lEC) of the isolated guinea pig brain
preparation (van der Linden et al., 1999). In this preparation the
planar horizontal connections in cortical structures such as the EC are
intact and thus allow for a controlled study of large-scale networks in
the generation of synchronized oscillatory activity. The primary
objective of the present study was to examine the circuit mechanisms
and the spatial distribution of CCh-induced oscillations within the
mEC. Through a series of localized pharmacological and multisite
electrophysiological experiments we show that these oscillations are
dependent on the local muscarinic synchronization of spatially
restricted pools of reciprocally connected superficial inhibitory and
excitatory neurons. A physiological role for these oscillations may be
to spatially and temporally organize into functional modules the output
of mEC projection cells to their targets in the hippocampus.
Some of these results have previously appeared in abstract form (de
Curtis et al., 1999).
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MATERIALS AND METHODS |
The isolated whole brain preparation has been previously
documented in detail (de Curtis et al., 1991, 1998; Muhlethaler et al.,
1993) and will only briefly be described here. After barbiturate anesthesia (thiopentothal sodium, 20 mg/kg), young adult guinea pigs
(150-300 gm) were perfused through the heart with a cold (4-10°C)
carbogenated (95% O2 and 5%
CO2) solution composed of (in mM):
126 NaCl, 3 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, 15 glucose, 2.1 HEPES, 0.4 thiourea, 0.5 ascorbic acid, and 3% dextran (molecular weight 70,000). After
rapid and careful removal, brains were submerged in a recording chamber
filled with the same solution maintained at a temperature of 16°C.
Cerebral perfusion was achieved by cannulating the vertebral artery and perfusing through the existing brain vasculature at a rate of 5.5 ml/min. Leaky vessels were ligated, and the brain was gradually (0.2°C/min) warmed to a final temperature of 32°C (unless otherwise specified). This protocol was reviewed and approved by the Committee on
Animal Care and Use and by the Ethics Committee of the Istituto Nazionale Neurologico.
Extracellular field recordings were made with (1) glass micropipettes
with a 10-µm-diameter tip and filled with a NaCl solution (0.9%) in
addition to any pharmacological agent (see below), (2) 16-site linear
silicon probes (50-100 µm contact separation; kindly provided by
Jamille Hetke of the Center for Neural Communication Technology,
University of Michigan, Ann Arbor, MI), or (3) tungsten 4 × 1 matrix microelectrode arrays (410 µm tip separation; Frederick Haer
Corporation, Bowdoinham, ME). All recordings, unless otherwise noted, were made at a depth of 500 µm from the pial surface.
Recordings at multiple sites were performed simultaneously. Signals
amplified at a gain of 1000 using an AC amplifier (Biomedical
Engineering, Thornwood, NY) were high-pass filtered at 0.2 Hz and
low-pass filtered at 1000 Hz. They were digitized on-line using
customized software (Clampview) developed by G. Biella in collaboration
with SIDeA (alliance member of National Instruments, Milan,
Italy) and were stored on digital tape (DTR 2602 Biological,
Claix, France) for off-line analysis. Single and dual channel spectral
analysis was conducted off-line using Matlab (Mathworks, Natick, MA).
After digitally filtering with a fourth order Butterworth function in a
2.5 Hz bandwidth around line frequency (50 Hz) to minimize coherence artifacts, the auto power, cross power, phase, and coherence spectra were computed for all signals and all signal pair combinations. We
empirically determined by comparison of the same signals with and
without filtering that this manipulation was adequate in eliminating line frequency artifacts without producing major alterations in the
frequency band of interest which, in this study, was substantially lower than this (25-30 Hz). Stimulation of the lateral olfactory tract
(LOT) was conducted with bipolar silver wires, insulated along their
length except at the tips. Intracortical stimulation was conducted
using thin insulated tungsten bipolar electrodes (Frederick Haer
Corporation). In all preparations, before any experimental
procedures, the response of the piriform or lateral entorhinal cortex
to electrical stimulation of the LOT was assessed to verify the
viability of the preparation. When metal electrodes were used for
intracortical recording and stimulation, electrolytic lesions induced
by 10 mA current application for 5-10 sec were made to mark the
location of the electrode sites. Subsequently, the brains were fixed
overnight with a 4% paraformaldehyde solution in a 0.1 M
sodium phosphate buffer and then sectioned by vibratome at a thickness
of 100 µm. Sections were mounted, stained with thionin, and inspected
for the location of electrode sites.
activity was elicited in the mEC by either local pressure injection
(5-50 mM in 0.9% NaCl for 1-5 sec) or arterial perfusion (25-100 µM in perfusion solution) of CCh. As with
microinjections of CCh, injections of all other substances was
conducted at a depth of 500 µm using a modified recording
pipette that allowed for simultaneous recording and drug injection.
Other pharmacological agents used included the muscarinic antagonist
atropine sulfate, the GABA receptor antagonist bicuculline methiodide
(BMI), and the AMPA receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in concentrations
ranging from 5-10 µM (arterial perfusion) and 0.1-5
mM (local injection). CNQX was predissolved in
dimethylsulfoxide (DMSO) at a concentration of 5-25 mM
before being added to the saline solution. Specifics of the diverse
experimental procedures for intraparenchymal infusions can be found in Results.
All salts were obtained from BDH (Poole, UK) and all drugs from Sigma
(St. Louis, MO), except CNQX, which was obtained from Tocris Cookson
(Bristol, UK). Dextran was obtained from SIFRA (Isola della
Scala, Italy).
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RESULTS |
As previously shown (van der Linden et al., 1999), either arterial
perfusion (25-100 µM) or local injections of CCh (5-50 mM) elicited fast oscillatory activity (25 ± 4 Hz;
n = 18; mean ± SD) that was localized to the mEC
(Fig. 1A,B). In a
subset of experiments (n = 5), the frequency of this
activity increased from 29 ± 3 to 45 ± 2 Hz as the
temperature of the preparation was raised to more physiological levels
(from 30 to 36°C; data not shown), suggesting that it most likely
represents physiological activity.
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Figure 1.
Carbachol-evoked activity is generated in
superficial layer II of mEC but not in lEC. A, Schematic
diagram of a ventral view of the brain with an expansion of the
entorhinal region showing the relative positions of recording
electrodes. B, Extracellular field recordings from sites
shown in A before (left panel) and
30 min after arterial perfusion of 50 µM CCh
(right panel). Continuous activity was
observed in the three mEC sites, but not in the lEC. C,
Simultaneous recording of activity at multiple depths in a
different experiment using a 16 site linear silicon probe positioned in
the mEC at a location between sites 2 and 3 in A. A
reversal of the polarity of the oscillation was observed at a cortical
depth of 300 µm. In the middle portion of panel
C the power, phase, and coherence of frequency (25 Hz) as a function of cortical depth is shown for the data illustrated
in the left-most panel. These values were obtained by
averaging values in a 2 Hz frequency bandwidth surrounding the peak
frequency of . The amplitude of the signal was largest in
superficial layers, with a minimum value at 300 µm, which
corresponded to the location of a 180° phase shift of the
oscillation. Note that the coherence of remained constant and
elevated (~1.0) across all depths except at the site of reversal. The
probe tract for this experiment is shown in the right-most
panel in C. Note that the reversal point of the
oscillation (arrowhead) appears at the level of
layer II.
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To characterize the specific layers responsible for the generation of
this activity, a laminar profile analysis was undertaken. Recordings
were made in the mEC using a 16-contact linear probe oriented
orthogonally to the pial surface and therefore, to the laminar cortical
arrangement of the mEC. The contacts were spaced evenly at an interlead
distance of 50 µm and spanned a total distance of 750 µm. Unlike
laminar profiles performed by advancing a single electrode (van der
Linden et al., 1999), recordings with multisite silicon probes enabled
activity to be recorded simultaneously at multiple depths in the
same "column" of entorhinal tissue. The depth of the probe was
regulated to leave one or two of the proximal channels outside the
brain. In this way, the first proximal contact inside the brain was
assumed to be at a depth of 50 µm. In all experiments
(n = 5), a reversal of the raw field potential rhythm appeared across the superficial layers of the mEC (Fig. 1C, arrow). These results were further analyzed by
dual-channel spectral methods, using a comparison channel corresponding
to the second-to-last contact on the probe itself as a reference for
all other channels. This approach provided a precise and easily comparable method for analyzing the isolated component of the signal as a function of depth across experiments. Values corresponding to a 1-2 Hz bandwidth centered around the frequency corresponding to
the spectral peak were extracted from the power, phase, and coherence spectra for each channel and were plotted as a function of
depth in the cortex (Fig. 1C, central columns). The power of showed a bimodal distribution, with maxima at average depths of
120 ± 27 and 470 ± 27 µm and a minimum between these
depths at an average level of 240 ± 42 µm. This latter depth
also corresponded to the location of the 180° phase shift of as
shown by phase analysis. As shown in the experiment illustrated in
Figure 1C and in all other experiments, the phase shift
occurred abruptly (within 50 µm) at an average depth of 250 ± 35 µm (n = 5). Subsequent histological analysis of
the probe tracts in this and two other experiments after lesions made
at contacts corresponding to the most superficial and deepest sites
within the brain confirmed that the depth of reversal corresponded to
layer II of the mEC (Fig. 1C, right-most panel, histology).
At depths >500 µm, the power decreased monotonically, and no
further maxima or phase reversals were observed. This was confirmed in
two further experiments by recording at even greater depths in the
cortex using a 16 site silicon probe with a 100 µm interelectrode
spacing that spanned a total distance of 1550 µm (data not shown). In
contrast to the changes observed as a function of depth for power and
phase, coherence as a function of depth showed marked uniformity in
three of five experiments. In these three examples, coherence values were only marginally different from a value of 1.0 (signifying almost
perfect synchrony) across all depths except at those locations corresponding to the reversal point and sites outside brain tissue (Fig. 1C). In the two remaining experiments, the coherence
plots showed less uniformity, decreasing monotonically from a value close to 1.0 to minimum values ranging between 0.7 and 0.8 at locations
superficial to the reference channel. The electrode track in one of
these cases was examined histologically and was found to have taken an
oblique course through the cortical laminations. This lateral deviation
may explain the variation in coherence observed.
As previously described, (van der Linden et al., 1999), CCh-evoked activity was found to be dependent on the activation of muscarinic
receptors because it was completely blocked by arterial perfusion of
atropine (5 µM; n = 4). Given the ability
of local CCh application to evoke activity (present results and van
der Linden et al., 1999), muscarinic receptor excitation presumably takes place at the level of the mEC itself. This hypothesis was tested
using pressure microinjections of atropine (100-500
µM) in the mEC during activity evoked by
arterial perfusion of CCh. As shown in Figure
2 and in all other experiments
(n = 4), this manipulation inhibited activity in
the area directly surrounding the injection site as observed in both
the recording traces (Fig. 2B) and power spectra
(Fig. 2C) for site 2. Such an inhibition was also observed
without pressure ejection, when higher concentrations of atropine (5 mM) were allowed to freely diffuse from recording pipettes (n = 2). Therefore, local muscarinic
excitation appears to provide the driving force for the elicitation of
activity by CCh in the mEC. In contrast to arterial perfusions of
atropine (5 µM) (van der Linden et al., 1999),
the effect of intraparenchymal application of higher concentrations of
atropine did not reverse, even after wash times as long as 2 hr.
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Figure 2.
Local blockade of muscarinic transmission produces
a localized inhibition of activity. A, Expanded
schematic diagram of the entorhinal region with recording electrode
positions. B, Field recordings at positions noted in
A during evoked by arterial perfusion with 100 µM CCh. The left panel shows traces
directly before, during, and after a pressure pulse application of
atropine sulfate through the recording pipette at position 2 (marked by
the bar); the right panel shows the same
series of recordings 5 min after atropine application. This
manipulation evoked a robust inhibition of only at the site in
which the injection was made (expansion of the traces shown below).
C, Power spectra of the signal before and 5 min after
the atropine injection demonstrate a selective depression of the
amplitude of the signal in the recordings at position 2. Note
different frequency peaks at different sites. The "notch"
appearing at 50 Hz in this and all other figures showing power spectra
corresponds to the effects of digital filtering of the signals to
eliminate line frequency noise (see Materials and Methods).
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Previous studies have proposed that activity depends on the
rhythmic synchronization of IPSPs on principal neurons (Whittington et
al., 1995; Buhl et al., 1998; Fisahn et al., 1998; Penttonen et al.,
1998). Blocking fast GABAergic neurotransmission with the receptor
antagonist BMI tested this hypothesis. After the elicitation of by
arterial perfusion of 25-50 µM CCh, coperfusion of BMI
(10 µM) resulted in the abolition of the fast rhythm
evoked by CCh (n = 2). However, systemic blockade of
GABAergic neurotransmission also evoked large-amplitude epileptiform
activity (cf. Librizzi and de Curtis, 1999), which could have masked
activity through other, nonspecific mechanisms such as postictal or
spreading depression. Therefore, brief microinjections of BMI were made
directly within the mEC during produced by arterial perfusion of
CCh (Fig. 3B, left traces;
n = 6). This manipulation completely abolished activity as observed both in the recording traces (Fig. 3B)
and power spectra (Fig. 3C). Similarly to the effects of
locally applied atropine, this blockade occurred in a spatially
localized region directly surrounding the injection site. To test
whether the microinjection procedure per se interfered with the
production of , similar pressure microinjections of saline solution
were made (n = 3). As illustrated in Figure
3B (right panel), after the washout of BMI
effect, a pressure injection of saline from electrode 2 had no effect
on activity. Thus, the suppressive effect seen with drug
application was specific to its pharmacological action.
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Figure 3.
Local blockade of fast GABAergic transmission
produces a localized abolition of activity. A,
Schematic diagram of the recording electrode sites. B,
Field recordings at positions noted in A during activity evoked by arterial perfusion with 50 µM CCh with
expansions of selected portions shown below. The left
panel shows the recordings before, during, and directly after a
pressure pulse application of BMI through the recording pipette at
position 1 (see bar). A selective abolition of activity is observed at the same site at which the injection was made
(middle panel). This effect was reversible after
a 2 hr wash period (right panel). After the
washout of bicuculline, a similar pressure pulse injection of saline
through the recording pipette at site 2 (see bar, right
panel) had no influence on activity at any site.
C, Power spectra of the signal before, 6 min, and 2 hr
after BMI application demonstrate a reversible abolition of the
amplitude of the signal in the recordings at position 1.
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Because fast glutamatergic transmission has been shown to be important
in the generation of CCh-induced activity in hippocampal and
neocortical slices (Buhl et al., 1998; Fisahn et al., 1998) we also
performed brief local pressure applications of CNQX (5 mM)
during activity evoked by arterial perfusions of CCh. In all
experiments performed (n = 6), and as illustrated in
Figure 4, a marked depression of the
amplitude of was observed, again in a spatially localized region
surrounding the injection pipette. Likewise, a depression of the
potential evoked by electrical stimulation of associative fibers was
seen at the same, but not at distant sites in the mEC (Fig. 4C,
right panel). The depression of both and the evoked
potential was partially recovered in three cases over a washout period
of 30-90 min. In contrast, infusions of saline and vehicle (DMSO;
n = 3), as well as lower concentrations of CNQX (500 µM; n = 2), had no effect upon
activity (data not shown). Therefore, it would appear that as for
activity in brain slices, the excitatory network contributes to the
generation of carbachol-generated activity in the mEC.
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Figure 4.
Local blockade of fast glutamatergic transmission
produces a localized inhibition of activity. A,
Schematic diagram of the recording and stimulation electrode sites.
B, Field recordings at positions noted in
A during evoked by arterial perfusion with 50 µM CCh. The left panel shows the
recordings directly before, during, and after a pressure pulse
application (see bar) of CNQX through the recording
pipette at position 1, and the middle panel shows the
same series of recordings 5 min after the application. This
manipulation evoked a robust inhibition of only at the site in
which the injection was made (expansion of the traces shown below).
Correspondingly, in the right panel, the potentials
evoked by stimulation of superficial associational fibers before
(continuous lines) and 7 min after the local application
of CNQX (dotted lines) show a selective depression only
at site 1. C, Power spectra of the field signals before
and 5 min after the injection demonstrate a selective depression of the
amplitude of the signal in the recordings at position 1.
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The spatial restriction of the pharmacological effects described above
suggests the existence of multiple independent generators of activity across the planar horizontal extent of mEC superficial layers.
Further support for this idea was garnered when recordings were made
using electrodes with a high (100 mM) concentration of CCh.
Soon (1-5 min) after pipette placement at a depth of 500 µm in the
mEC (but not the lEC; n = 2), developed in a highly circumscribed region of ~500 µm surrounding the electrode site (Fig. 5B; n = 10). This was confirmed by changing the position of another recording
electrode (without CCh) from a distant site to a closer site from
position 1a to 1b (Fig. 5B). This effect was most likely
mediated through passive diffusion of CCh into the mEC because activity disappeared shortly after (5-10 min) removal of the
CCh-filled electrode (Fig. 5B, right panel;
n = 3). evoked in this manner was abolished by
arterial perfusion of atropine (5 µM,
n = 2; data not shown). Sequential recordings taken at
multiple depths at a site proximal to the CCh-filled electrode
demonstrated a phase reversal at ~300 µm, confirming its similarity
to activity evoked by arterial perfusion (n = 2;
data not shown). When two pipettes containing 100 mM CCh were placed in the mEC (Fig.
5C), was generated at both sites (Fig. 5D).
When a large distance separated these electrodes (>1 mm) the coherence
of the signals across both was extremely low (near random levels;
Fig. 5E, dotted line). In fact, the peak frequency of was often different between the two sites. When the separation between
distally spaced electrodes was reduced by moving one electrode closer
to the other, the coherence of increased dramatically (Fig.
5D,E; continuous line, n = 5).
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Figure 5.
Diffusion of carbachol from recording
pipettes evokes spatially localized activity. A,
Schematic diagram of the recording electrode positions. As noted, the
pipette used for recording at site 2 contained 100 mM CCh
and electrode 1 was moved from position 1a to position 1b.
B, Field activity recorded at distant (position
1a, left panel) and adjacent (position 1b,
middle panel) separations of the recording electrodes
and 10 min after the removal of the CCh electrode (right
panel). activity was observed with the saline
pipette only when it was positioned in close proximity to the CCh
pipette (left and middle panels,
respectively) and dissipated shortly after removal of the CCh pipette
(right panel). C, Diagrammatic
representation of an experiment using two CCh-filled electrodes. As
noted, electrode 1 was moved from position 1a to position 1b.
D, Field recordings at distant (left
panel) and near (right panel)
sites. was recorded from both electrodes at both positions.
E, Coherence spectrum of the signals shown in
D. Although amplitude was high for both placements,
the coherence at frequencies (arrow) was negligible
for far separations (continuous line) as compared to
near separations (dotted line).
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In light of the spatial localization observed with focal
pharmacological applications, we elected to study the horizontal spatial distribution and synchronization of activity evoked by
arterial perfusion of CCh by performing multisite recordings across the
surface of the mEC. This was conducted with two electrode arrays, each
having four contacts with interelectrode distances of 410 µm between
adjacent pairs within an array (Fig.
6A). After the
induction of (Fig. 6B), dual-channel spectral
analysis was conducted for every pair of electrodes both within and
between arrays. These data are illustrated in Figure 6C.
Linear coherence was calculated at the peak frequency corresponding to
the peak in the cross spectra power peak. As shown in Figure
6D and in all other experiments, there was a
consistent and significant (p < 0.01) negative
linear relationship between coherence and the inter-recording site
distances (Fig. 6D). Across all experiments, this
relationship was characterized by the following equation:
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However, given the variation in signal amplitude across electrode
locations, a possible confounding element to the coherence measurements
could have been caused by the differences in relative amplitudes of the
signals compared. Therefore, a regression analysis was conducted
between the cross-spectral amplitude (an indication of the relative
amplitude of the signal between two sites) and coherence for
each pair of signals. No significant linear relationship between these
two parameters was observed (Fig. 6E;
p > 0.01; n = 5). Thus, coherence
appeared to be independent of the signal amplitudes measured in this
study.
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Figure 6.
activity evoked by arterial perfusion of CCh
shows spatially limited synchronization across the surface of the EC.
A, Schematic diagram of the two recording array
positions. B, field recordings from all eight sites
after arterial perfusion with 50 µM CCh.
C, Auto, cross power, and coherence spectrum for signals
at sites 2, 3, 4, and 8 as compared to site 1. The coherence values at
the frequency peak (vertical dotted line) decreased
as the distance between electrode sites increased. D,
Plot of coherence values as a function of interelectrode distance
for all possible pairwise comparisons in the experiment illustrated in
B. The regression of the average coherence (large
squares) on distance demonstrated a significant negative linear
relationship of the equation: coherence = 1.0  0.45 (interelectrode distance in millimeters) (r = 0.995; p < 0.01). E, Plot of coherence as a function of relative amplitude (cross power at frequency) for all possible pairwise comparisons for the same data as
in B. The regression of coherence on cross power
revealed no significant linear relationship
(p > 0.01).
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To characterize the time course of synchronization between different
sites across time as activity developed during arterial perfusion
of CCh, coherence analysis was performed at multiple time points,
before, during and after the appearance of activity in different
experiments (n = 5). As illustrated in Figure
7B, the amplitude of activity was typically low at onset and gradually increased with time.
Figure 7C demonstrates that, despite variation in
cross-spectral power amplitude for activity with time, coherence remained stable for given electrode pairs (far and near pairs
illustrated in the left and right panels, respectively). The coherence
measurements obtained in five experiments between near and far
recording sites showed no significant changes across time (see
regression fittings in Fig. 7D; p > 0.01;
n = 5). This provides further evidence that synchronization depends on the spatial relationship between the sites,
rather than on the amplitude of activity.
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Figure 7.
synchronization between given sites remains
consistent across time, independently of signal amplitude.
A, Schematic diagram of the recording array positions.
B, Field recordings from sites 1, 6, and 7, demonstrating development of at different times during perfusion of
50 µM CCh. was initially observed 10 min after the
start of CCh perfusion. C, Cross and coherence spectra
for channels 1 and 6 (far separation) and channels 6 and 7 (near
separation) during different times after the start of CCh perfusion
(t = 0, continuous line;
t = 10 min, dotted line;
t = 15 min, dashed line;
t = 20, dash-dot line). Despite
differences in amplitude of the signal across time, the coherence
of remained constant within the near and far site comparisons.
D, Scatter plot and regression fittings of coherence
for near (410 µm; filled shapes-continuous lines) and
far (>1230 µm; open shapes-dotted lines) electrode
separations as a function of time. Different experiments are
represented by different geometrical shapes. Coherence values were
consistently high for near electrodes and consistently low for far
electrodes from the onset of . No regression fitting for any
experiment showed a significant linear trend
(p > 0.01).
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As previously reported (van der Linden et al., 1999), both perfusion
and local injection of CCh occasionally induced transient trains of
theta-like oscillations in the mEC, similar in form to activity induced
by CCh in the mEC slice preparation (Dickson and Alonso, 1997).
Isolated theta sequences were composed of rhythmic waves between 3 and
8 Hz that appeared simultaneously across the surface of the mEC (Fig.
8B, left traces) and
often preceded the onset of a stable oscillatory activity (Fig.
8B, right traces). In contrast to coherence
measured in the same experiment, theta coherence showed little
variation with intersite distance (Fig. 8C, compare
left and right panels for theta and
, respectively). In fact, as illustrated in Figure
8D, theta-like oscillations in different experiments
were almost perfectly coherent (average value of 0.93 ± 0.04, n = 5) across the covered extent of the mEC (open
squares), whereas coherence values of decreased with distance
between recording sites (filled squares). There was a significant negative linear relationship between coherence and distance
for (r = 0.995; p < 0.01;
n = 5) but not for theta (r = 0.408;
p > 0.01; n = 5). Thus, the spatial
limitation of coherence would appear to reflect an unique property
of activity itself.
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Figure 8.
Spatial synchronization is specific for , but
not theta-like activity elicited by CCh. A, Schematic
diagram of recording array positions. B, Field
recordings from all electrode sites demonstrating theta-like
(left) and (right) oscillatory
activity that developed after an injection of 5 mM CCh at
location 9. C, Cross and coherence spectra for a series
of electrode comparisons to channel 4 during both theta
(left) and (right). Note that the
coherence values at theta frequencies remained high regardless of
interelectrode distance, whereas coherence decreased with
increasing interelectrode distances. D, Scatter plot of
theta (empty squares) and (filled
squares) coherence as a function of interelectrode distance.
coherence showed a significant negative linear relationship with
distance (r = 0.995; p < 0.01). Theta coherence, however, was uniformly high, and independent of
distance and showed no significant linear trend (r = 0.408; p > 0.01).
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DISCUSSION |
Based on our pharmacological experiments, we hypothesize that
CCh-evoked activity in the mEC involves an interaction between reciprocally connected inhibitory interneurons and principal cells in
superficial layers. Previous studies on the mechanisms of activity
generation have proposed that the extracellular field oscillation
reflects the bombardment of rhythmic and synchronized IPSPs on
principal neurons (Whittington et al., 1995; Buhl et al., 1998; Fisahn
et al., 1998; Penttonen et al., 1998) (for review, see Buzsáki
and Chrobak, 1995; Jefferys et al., 1996; Connors and Amitai, 1997;
Ritz and Sejnowski, 1997). The demonstration that activity in our
preparation was crucially dependent on the functional preservation of
fast GABAergic neurotransmission is consistent with this idea.
Synchronization within an inhibitory interneuronal network has been
shown to be mediated either through synaptic (Whittington et al., 1995;
Traub et al., 1996; Wang and Buzsaki, 1996), or gap junction
connections between interneurons (Galarreta and Hestrin, 1999; Gibson
et al., 1999; Mann-Metzer and Yarom, 1999; Fukuda, 2000) or both
(Tamás et al., 2000). Although independent synchronization of an
inhibitory interneuronal network has been suggested to be sufficient in
sustaining oscillations (Whittington et al., 1995; Traub et al.,
1996; Wang and Buzsaki, 1996), our present findings concerning the
dependence of activity on fast glutamatergic transmission support
the recently reported conclusion that recurrent glutamatergic
excitation is necessary for generation (Buhl et al., 1998; Fisahn
et al., 1998; Leung, 1998). Anatomical studies have shown that layer II
principal neurons form autoassociative recurrent contacts within layer
II (Kohler, 1986; Klink and Alonso, 1997a; Dolorfo and Amaral, 1998),
and physiological evidence has demonstrated robust feedforward and recurrent inhibitory mechanisms in principal neurons within this layer
(Jones and Buhl, 1993; Jones, 1994). Moreover, inhibitory inputs have
been shown to reset discharge in principal neurons of the mEC layer II
(Dickson et al., 2000) and hippocampus (Cobb et al., 1995; Chapman and
Lacaille, 1999). Therefore, it is likely that the recurrent excitatory
population discharge of mEC layer II principal cells is entrained at
frequencies by rhythmic IPSPs. In support of this, we have
occasionally observed single-unit activity in superficial layers
showing a phase relation to the extracellular rhythm (cf. Chrobak
and Buzsaki, 1998a).
A novel finding of the present study is that the basic mEC circuit
responsible for muscarinic-dependent activity is highly localized.
Whereas this may not seem surprising based on the finding that activity can be observed in slice preparations in which horizontal
connectivity is highly limited (to often <400 µm) (cf. Whittington
et al., 1995; Buhl et al., 1998; Fisahn et al., 1998), what is
surprising is that despite the fact that horizontal connectivity in the
whole brain preparation is intact, this spatial specificity of generation appears to be preserved. The rhythmic synchronization of
neuronal activity at frequencies in spatially localized regions over the surface of the mEC may confer a functional modular
organization to this structure, in the absence of a typical cortical
columnar arrangement. This conclusion is supported by the following
experimental evidence: (1) local application of pharmacological agents
impaired the generation of oscillations in restricted portions of
the mEC, (2) activity could be induced in highly localized patches of cortex restricted to the immediate zone surrounding a CCh-containing electrode, (3) oscillations induced by two CCh-containing
electrodes at remote positions showed little coherence
(synchronization), and (4) the synchronization of activity at
different mEC locations showed a strict linear decrease with distance,
whereas activity in a "column" of mEC tissue as recorded using
a multidepth probe was consistently and highly coherent. This
relationship was a unique property of activity because, in the same
experiments, theta-like activity showed high synchronization across the
mEC surface, regardless of the interelectrode distance.
Estimations of the minimal size of cortical tissue necessary for the
generation of activity (corresponding to a "functional module")
can be garnered from the evaluation of the linear relationship between
coherence and distance. Because coherence values are mathematically
analogous to the correlation coefficient, the squared value of
coherence describes the proportion of variance predicted in one signal
by the other, i.e., for perfect correspondence
coherence2 = 1 (Bendat and Piersol, 1986;
Armitage and Berry, 1994). Therefore, if we assume that high
synchronization occurs with coherence values of at least 0.75 (corresponding to a minimum relationship between signals of
0.752 * 100 = 56%), it can be
estimated that the upper limit for the radius of the basic cortical
area that generates independently oscillations is 500-600 µm,
according to the regression equation elaborated in Results. This value
may be substantially smaller, given our findings with diffusion of CCh
from pipette tips. Similarly, in the mEC slice preparation, it has been
shown that CCh induces a nonglutamatergic-dependent synchronization
of layer II interneurons in spatially localized "pools" having a
radial distance of ~250-300 µm (Dickson and Alonso, 1997).
Interestingly, our identification of a limited cortical element in
which coherence was found to be maximal (i.e., a functional mEC
module) corresponds well with the observation of highly synchronous
fast oscillations within orientation columns of the visual cortex
(Eckhorn et al., 1988; Gray et al., 1989; Gray and Singer, 1989). It is
therefore tempting to speculate that these functional modules
corresponding to separate and independent generators of activity
represent a fundamental hardware processing unit in the mEC.
Interactions between mEC functional modules might be performed by
tangential associational connections within layer II of the mEC (cf.
Gray et al., 1989). As previously discussed, these connections may be
mediated through either or both excitatory and inhibitory connections.
Anatomical findings have shown a robust collateralization of axonal
branches spreading in a tangential plane of both excitatory (Kohler,
1986; Klink and Alonso, 1997a; Dolorfo and Amaral, 1998) and inhibitory
(GABAergic) (Jones and Buhl, 1993; Wouterlood et al., 1995) neurons
within EC layer II. A prevalent contribution of inhibitory collaterals
in connecting functional modules was indicated by the difference in the
efficacy of locally applied BMI versus CNQX. Indeed, whereas activity was completely abolished by GABAa receptor antagonism, AMPA
receptor blockade, although completely eliminating the stimulus-evoked field potential and substantially reducing the amplitude of the oscillation, did not eliminate the spectral peak completely. A
physiological mechanism by which coherence of activity across extremely distant modules within the mEC may occur is via an extrinsic synchronizing input, such as from the medial septum, which has been
shown to be important for the regulation of theta rhythmic activity in
the mEC (Mitchell et al., 1982; Dickson et al., 1995; Jeffery et al.,
1995) and both theta and in the hippocampus (Bland, 1986; Ma and
Leung, 1999). Indeed, there may exist the possibility that the slow
(theta) rhythm itself exerts a synchronizing influence on the
superimposed oscillation (cf. Buzsáki and Chrobak, 1995).
Neocortical oscillations, through the synchronization of neuronal
activity on a fast time scale (tens of milliseconds) have been shown to
be a mechanism for the temporal formation of ensembles that code for
both simple and complex aspects of sensory stimuli (for review, see
Gray, 1994; Singer and Gray, 1995; Laurent, 1996; Connors and Amitai,
1997; Basar et al., 1999; Tallon-Baudry and Bertrand, 1999). This
synchronization is enhanced with manipulations that induce cortical or
behavioral arousal, typically through activation of ascending
cholinergic projections (Steriade et al., 1991; Metherate et al., 1992;
Munk et al., 1996; Cape and Jones, 1997; Herculano-Houzel et al., 1999)
that may correlate with attentional processes (Tiitinen et al., 1993;
Maloney et al., 1996). Accordingly, activity in the EC has been
shown to be modulated in a similar way across behavioral and
sleep-wake states (Chrobak and Buzsáki, 1998b). Because
the EC is considered to represent a supramodal associational cortical
region in which inputs from multiple sensory areas converge (Van
Hoesen, 1982; Amaral and Witter, 1989; Witter et al., 1989), a
state-dependent regulation of synchronization within and between
spatial modules could set a condition that facilitates or impedes
binding between different sensory stimuli. activity is generated by
layer II, which receives associative neocortical inputs and is itself
the source of the perforant path input to the hippocampus (Steward and
Scoville, 1976). The existence of a regulatory function mediated by synchronization in such a nodal element in the cortical-hippocampal
system could be crucial for information processing, storage, and
retrieval in limbic memory circuitry (cf. Buzsáki and Chrobak,
1995).
In summary, the present study demonstrates the uniqueness and
advantages of the isolated whole brain preparation for recording and
comparing activity within and across brain regions with a precision,
stability, and spatial definition otherwise impossible to attain in
either in vivo or in vitro conditions. Muscarinic activation, at the level of the mEC in this preparation, provides a
good model for studying the generation of activity because of its
close correspondence to that described in vivo (Chrobak and
Buzsáki, 1998b). Our findings suggest that activity
synchronizes activity in spatially localized cortical modules and is
sustained by a circuit involving interactions between inhibitory and
excitatory neurons within mEC layer II. We propose that such functional
modules in the mEC represent a fundamental organizing device that
constrains the processing and throughput of information in limbic circuitry.
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FOOTNOTES |
Received June 22, 2000; revised July 28, 2000; accepted Aug. 9, 2000.
This study was supported by the Human Frontier Science Program (HFSP)
Grant RG 19/96. C. Dickson was the recipient of a short term HFSP
fellowship SF 9/99. G. Biella was partially sponsored by the European
Community Grant VSAMUEL (IST-99-1-1-A).
Correspondence should be addressed to Marco de Curtis, Dipartimento di
Neurofisiologia Sperimentale, Istituto Nazionale Neurologico "Carlo
Besta", via Celoria, 11, 20133 Milan, Italy. E-mail:
decurtis{at}istituto-besta.it.
| |
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TOP
ABSTRACT
INTRODUCTION
