1Dipartimento di Neuroscienze, Università degli Studi di Roma `Tor Vergata', 00173 Rome; 2Dipartimento di Scienze Biomediche, Università degli Studi di Modena e Reggio Emilia, 41100 Modena, Italy; 3Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada; 4Centre P. Broca and Institut National de la Santé et de la Recherche Médicale U109, 75014 Paris; and 5Institut National de la Santé et de la Recherche Médicale U398, 67000 Strasbourg, France
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ABSTRACT |
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Tancredi, Virginia, Giuseppe Biagini, Margherita D'Antuono, Jacques Louvel, René Pumain, and Massimo Avoli. Spindle-Like Thalamocortical Synchronization in a Rat Brain Slice Preparation. J. Neurophysiol. 84: 1093-1097, 2000. We obtained rat brain slices (550-650 µm) that contained part of the frontoparietal cortex along with a portion of the thalamic ventrobasal complex (VB) and of the reticular nucleus (RTN). Maintained reciprocal thalamocortical connectivity was demonstrated by VB stimulation, which elicited orthodromic and antidromic responses in the cortex, along with re-entry of thalamocortical firing originating in VB neurons excited by cortical output activity. In addition, orthodromic responses were recorded in VB and RTN following stimuli delivered in the cortex. Spontaneous and stimulus-induced coherent rhythmic oscillations (duration = 0.4-3.5 s; frequency = 9-16 Hz) occurred in cortex, VB, and RTN during application of medium containing low concentrations of the K+ channel blocker 4-aminopyridine (0.5-1 µM). This activity, which resembled electroencephalograph (EEG) spindles recorded in vivo, disappeared in both cortex and thalamus during application of the excitatory amino acid receptor antagonist kynurenic acid in VB (n = 6). By contrast, cortical application of kynurenic acid (n = 4) abolished spindle-like oscillations at this site, but not those recorded in VB, where their frequency was higher than under control conditions. Our findings demonstrate the preservation of reciprocally interconnected cortical and thalamic neuron networks that generate thalamocortical spindle-like oscillations in an in vitro rat brain slice. As shown in intact animals, these oscillations originate in the thalamus where they are presumably caused by interactions between RTN and VB neurons. We propose that this preparation may help to analyze thalamocortical synchronization and to understand the physiopathogenesis of absence attacks.
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INTRODUCTION |
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The neocortex and
thalamus are able to generate brain rhythms that are observed during
sleep and in some pathological conditions such as absence seizures
(Avoli 1997; Steriade 1993
). Mechanisms of thalamocortical synchronization have been extensively studied in
vivo (cf. Steriade et al. 1997
) and in vitro (cf.
Huguenard et al. 1995
). However, few of the in vitro
studies published to date have analyzed the function of reciprocal
thalamocortical connections (e.g., Golshani and Jones
1999
; Zhang et al. 1996
). Most often, attention
has focused on the network properties and intrinsic excitability of
either cortical (Agmon and Connors 1991
) or thalamic
neurons (Crunelli et al. 1987
; Kim and McCormick
1998
).
In this study, we used most of the dissecting procedures described in
the mouse by Agmon and Connors (1991) to obtain slices of the rat brain thalamus and cortex in which reciprocal
thalamocorticothalamic connectivity is preserved. Here, we report that
in this preparation of low concentrations of the
K+-channel blocker 4-aminopyridine (4AP) induce
rhythmic, coherent oscillatory field potentials that occur
synchronously in both thalamus and cortex and resemble the
electroencephalograph (EEG) spindles recorded in vivo during non-REM
sleep or following barbiturate treatment (Andersen and Andersson
1968
; Steriade et al. 1993
).
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METHODS |
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Wistar rats (15-28 day old) were decapitated under halothane
anesthesia, and their brains were quickly removed and placed in cold,
oxygenated artificial cerebrospinal fluid (ACSF). Combined coronal
thalamocortical slices were obtained with a Lancer vibratome using the
procedures described by Agmon and Connors (1991). We, however, used thicker slices (550-650 µm versus 400 µm) and a hybrid coronal plane forming a 45° angle with the sagittal plane. Two
slices were obtained from each hemisphere at the level of the
ventrobasal complex (VB). Both were then transferred to a tissue
chamber where they laid in an interface between oxygenated ACSF and
humidified gas (95% O2-5%
CO2) at 32-35°C (pH = 7.4). ACSF
composition was as follows (mM): 124 NaCl, 2 KCl, 1.25 KH2PO4, 0.5 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose. Chemicals were acquired
from Sigma (St. Louis, MO).
Extracellular field potentials were recorded with ACSF-filled
electrodes (resistance = 2-8 M) positioned under visual
control in cortex and thalamus [VB and/or reticular nucleus (RTN)].
Signals were fed to high-impedance amplifiers, processed through
second-stage amplifiers with filtering capability, and displayed on an
oscilloscope and/or on a Gould recorder. A bipolar stainless steel
electrode was used to deliver extracellular stimuli (50-150 µs;
<200 µA) to selected areas of the thalamocortical slice.
4AP (0.5-1 µM, Sigma) was bath-applied and kynurenic acid (5 mM) was pressure-applied to restricted areas of the thalamocortical slice either as a drop delivered from a broken pipette or using a
perfusion system similar to that described by Behr et al.
(1998). To avoid diffusion of the drug from the area of
application to distant regions, slices were positioned in the recording
chamber in such a way that the treated area was downstream with respect to the flow of ACSF. Our work is based on the use of 75 slices. Measurements are expressed as mean ± SE and n
indicates the slice number. Statistical analysis of the data obtained
under control conditions and during any experimental manipulation was
performed with the Student's t-test. Data were considered
significantly different if P < 0.05.
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RESULTS |
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Single-shock stimuli delivered in VB, in the internal capsule or in the white matter, elicited in layers IV-V of the frontoparietal cortex (n = 32) a complex series of field potential waves that were in most cases characterized by: 1) an early, fixed peak-latency (~2.0 ms) negative field potential (amplitude = 0.5-2.3 mV; single arrow in Fig. 1A) that was capable of following high-frequency (>100 Hz) repetitive stimuli (Fig. 1B); and 2) a late negative field potential whose peak-latency depended on stimulus strength and showed synaptic fatigue during prolonged stimulation (double arrows in Fig. 1A). The early component, which disappeared with stimuli of lower intensity (not illustrated), likely represented the antidromic population response of cortical neurons following the activation of corticothalamic fibers. By contrast, the late component resulted from the synaptic activation of cortical neurons in response to thalamocortical afferents.
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An additional response at latency = 12-18 ms could also be observed in the frontoparietal cortex (Fig. 1A, curved arrow). This component was presumably caused by re-activation of thalamocortical afferents due to thalamocortical firing generated in response to descending corticothalamic inputs since kynurenic acid application to VB (n = 4) abolished it without influencing the antidromic or the initial orthodromic response (Fig. 1A). Orthodromic field potential responses were also recorded in the thalamus (n = 18 and 9 slices for VB and RTN, respectively) following electrical stimuli delivered in cortical layers V-VI (not illustrated).
Spontaneous synchronous field potentials were not seen in
thalamocortical slices bathed in normal ACSF (n = 29).
We reasoned that this lack of spontaneous activity was probably due to
the low degree of spontaneous action potential firing observed in most
in vitro preparations. Hence, we used low doses of the
K+ channel blocker 4AP to enhance transmitter
release at both excitatory and inhibitory synapses and thus the
background activity of cortical and thalamic neurons. Such an effect
has been documented in the hippocampus (Perreault and Avoli
1992). Indeed, under these conditions, rhythmic oscillatory
field potential activity occurred either spontaneously or following
electrical stimuli delivered in either thalamic or cortical areas
(n = 25; Figs. 1C and 2). These oscillations were recorded at both cortical (500-1200 µm from the pia) and thalamic recording sites (VB or RTN) and were characterized by bursts
of field potential waves of negative-positive polarity at frequencies
ranging from 9 to 16 Hz. The overall duration of each of these rhythmic
bursts was 0.4-3.5 s and they occurred at intervals of 6-24 s.
As shown in the experiment illustrated in Fig. 2A, the rhythmic field potential activities recorded in cortex and VB were synchronized. However, a small (<500 µm) change in the position of the recording electrode (in either cortex or thalamus) could abolish such synchronization (Fig. 2A, compare traces in a1 and a2 with those in a3). Field potential rhythmic oscillations, which could be time-locked with those seen in cortex and VB, were also recorded in RTN (not shown). Stimulus-induced oscillatory activity had features similar to those of spontaneous events and occurred with a good degree of synchronization in cortex, VB, and RTN (Fig. 2B).
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Next, we used local applications of kynurenic acid in either VB or cortex to establish the contribution of thalamic and cortical networks to the 4AP-induced rhythmic oscillations. Kynurenic acid was applied close to the site from which field potential recordings were obtained. Moreover, to limit possible activity-dependent variabilities in oscillation shape and/or duration, we used stimuli delivered every 10-30 s, and we avoided the occurrence of stimuli immediately after a spontaneous oscillatory sequence. As shown in Fig. 3A, kynurenic acid application in VB abolished (n = 6) the stimulus-induced rhythmic oscillatory responses recorded in both cortex and thalamus, as well as the spontaneous rhythmic oscillations (n = 6). Under these conditions, electrical stimuli induced in both structures a long-lasting (up to 300 ms) negative field potential that was at times followed by a late event at a latency = 200-500 ms (Fig. 3A, arrows in Cx and VB traces during kynurenic acid). In four slices, we also studied the effects induced by kynurenic acid application in the cortex. This procedure abolished the rhythmic oscillations recorded in the cortex while spontaneous and stimulus-induced oscillations continued to occur in VB (Fig. 3C). In addition, the frequency of the rhythmic oscillations elicited in VB during application of kynurenic acid in the cortex was higher than under control conditions (Fig. 3D).
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DISCUSSION |
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The field potential responses recorded in this study from
the middle-deep layers of the rat frontoparietal cortex following VB
stimuli bear close resemblance to those described in the mouse barrel
cortex by Agmon and Connors (1991). We could identify an early negative event with fixed peak-latency that likely results from
the antidromic activation of cortical neuron populations. This view is
in line with its ability to follow high-frequency repetitive stimuli.
In addition, we recorded a subsequent negative potential with
peak-latency and amplitude that depend on VB stimulus strength and thus
may reflect a postsynaptic, mainly excitatory, field potential. Hence,
these data indicate that thalamocortical and corticothalamic
connections are functionally operant in our slice preparation. This
conclusion is supported by the presence of an additional cortical
excitatory component with latency = 12-18 ms that was elicited by
stimuli in VB or in the internal capsule. This cortical response is
presumably due to the re-activation of thalamocortical afferents caused
by thalamocortical firing occurring after excitation of thalamic
neurons by corticothalamic inputs. Accordingly, kynurenic acid
application to VB abolished this late response without influencing the
antidromic or the initial postsynaptic excitatory field potential. In
addition, we have recorded orthodromic field potential responses in VB
or RTN following electrical stimuli delivered in the somatosensory
cortex, thus indicating that corticothalamic projections are functional
in our slice preparation (Deschenes et al. 1998
).
When challenged with low doses of 4AP, rhythmic oscillatory
activity appeared in both cortex and thalamic areas. These field potential oscillations have characteristics different from those reported for epileptiform events induced by higher concentrations of
4AP in different cortical preparations (Perreault and Avoli 1992), including the neocortex (Golshani and Jones
1999
). Indeed, they were organized in a rhythmic succession of
negative or negative-positive waves that occurred at frequencies of
9-16 Hz and were reminiscent of non-REM sleep (Avoli and Gloor
1982
; Avoli et al. 1984
; Steriade et al.
1993
) or barbiturate (Andersen and Andersson
1968
) spindles recorded by the EEG in vivo.
These in vitro oscillations appeared to be synchronized in cortex and
thalamus. However, a small change in the position of the recording
electrode (in either cortex or thalamus) could interfere with such
synchronization, suggesting that coherent oscillatory activity between
thalamic and cortical networks in rat brain slices may be restricted to
very small thalamocortical sectors. This may result from the existence
of reduced reciprocal connectivity between VB and RTN, as well as from
a diminished corticothalamic output function secondary to lesioning of
some intracortical connections (Staiger et al. 1999). It
should be noted, however, that even in intact preparations, the action
potential firing generated by cortical and thalamic neurons of the
association system often shows a low degree of correlation during
nonbarbiturate spindles (Avoli et al. 1984
).
A further similarity of our findings with the EEG spindles recorded in
vivo derives from the primary role played by the thalamus in generating
rhythmic oscillations in vitro. Blocking synaptic excitatory
transmission in VB with local application of kynurenic acid caused the
rhythmic oscillations to disappear. By contrast, they continued to
occur in the thalamus after application of this excitatory amino acid
antagonist to the frontoparietal cortex. Experiments performed in
intact animals have demonstrated that spindles can be recorded in the
thalamus of decorticated cats (Avoli and Gloor 1982;
Villablanca and Schlag 1968
). It has also been shown
that spindle oscillations are not observed in thalamic relay neurons
disconnected from RTN (Steriade et al. 1985
). These in
vivo data further support the presence of functional connectivity between VB and RTN in our slice preparation.
We have also discovered that corticothalamic inputs are capable of
modulating the rhythmic oscillations generated by VB neurons in the
presence of 4AP. Depression of cortical excitability by kynurenic acid
resulted in the disappearance of cortical oscillations, but at the same
time caused an increase in the frequency of the oscillations recorded
in the thalamus. Our data are in keeping with recent evidence obtained
in slices of the ferret lateral geniculate nucleus in which
corticothalamic fibers were electrically stimulated (Blumenfeld
and McCormick 1999). These investigators have demonstrated that
the activation pattern of corticothalamic inputs controls the frequency
of the rhythmic oscillations generated by geniculate neurons.
In conclusion, our study demonstrates that rat thalamocortical slices can retain enough reciprocal connectivity to express field potential rhythmic oscillations resembling the non-REM sleep or barbiturate spindles recorded by EEG in vivo. We would like to propose that this preparation represents a powerful tool for studying thalamocortical synchronization and for understanding the physiopathogenesis of absence attacks.
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ACKNOWLEDGMENTS |
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We thank R. Motalli and V. Epp for editorial assistance.
This study was supported by Medical Research Council of Canada Grant MT-8109. G. Biagini was the recipient of a North Atlantic Treaty Organization-Consiglio Nazionale delle Ricerche fellowship (Advanced Fellowship Programme 1998).
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FOOTNOTES |
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Address for reprint requests: M. Avoli, 3801 University St., Montreal, Quebec H3A 2B4, Canada (E-mail: mavoli{at}pobox.mcgill.ca).
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.
Received 27 January 2000; accepted in final form 20 April 2000.
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REFERENCES |
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