Laboratoire de Neurophysiologie, Faculté de Médecine, Université Laval, Quebec G1K 7P4, Canada
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ABSTRACT |
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Steriade, M.. Impact of Network Activities on Neuronal Properties in Corticothalamic Systems. J. Neurophysiol. 86: 1-39, 2001. Data from in vivo and in vitro experiments are discussed to emphasize that synaptic activities in neocortex and thalamus have a decisive impact on intrinsic neuronal properties in intact-brain preparations under anesthesia and even more so during natural states of vigilance. Thus the firing patterns of cortical neuronal types are not inflexible but may change with the level of membrane potential and during periods rich in synaptic activity. The incidences of some cortical cell classes (defined by their responses to depolarizing current pulses) are different in isolated cortical slabs in vivo or in slices maintained in vitro compared with the intact cortex of naturally awake animals. Network activities, which include the actions of generalized modulatory systems, have a profound influence on the membrane potential, apparent input resistance, and backpropagation of action potentials. The analysis of various oscillatory types leads to the conclusion that in the intact brain, there are no "pure" rhythms, generated in simple circuits, but complex wave sequences (consisting of different, low- and fast-frequency oscillations) that result from synaptic interactions in corticocortical and corticothalamic neuronal loops under the control of activating systems arising in the brain stem core or forebrain structures. As an illustration, it is shown that the neocortex governs the synchronization of network or intrinsically generated oscillations in the thalamus. The rhythmic recurrence of spike bursts and spike trains fired by thalamic and cortical neurons during states of decreased vigilance may lead to plasticity processes in neocortical neurons. If these phenomena, which may contribute to the consolidation of memory traces, are not constrained by inhibitory processes, they induce seizures in which the neocortex initiates the paroxysms and controls their thalamic reflection. The results indicate that intact-brain preparations are necessary to investigate global brain functions such as behavioral states of vigilance and paroxysmal activities.
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INTRODUCTION |
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This article emphasizes that network synaptic activities modulate, and often overwhelm, intrinsic neuronal properties. I certainly wish that this claim would become a truism for neuroscientists. However, with the advent of in vitro preparations, which provided not only some technical advantages over the work in vivo but also helped to achieve a better understanding of brain operations, a climate arose in which simplistic concepts sometimes appeared, such as the idea that the firing patterns induced by current pulses, taken to define the electrophysiological properties of a given neuronal type, are inflexible. This belief stands in contrast with data showing that patterns of neuronal activity change at various levels of membrane potential and with synaptic activity during shifts in behavioral states. There is also a tendency toward obtaining pure rhythms arising in simple circuits, whereas the intact brain displays oscillations of different types that are grouped together within complex wave sequences due to interactions between a variety of structures. Some investigators working in brain slices use their data to infer normal and pathological processes that require global operations in an intact brain.
Clearly both simplified preparations and normally operating networks are needed, but so far there are too few attempts to regard isolated networks within the context of the whole brain. The development of methods has succeeded in dissecting the brain and transforming it into reduced neuronal circuits. While behavioral and system neuroscience stands to gain from the achievements of biophysics and molecular biology in simplified preparations, the logic of life requires orchestration of the different parts composing the whole. The goal is to apply the information obtained from studies of isolated neurons and simple networks within the context of an intact brain. I will discuss the impact of synaptic activities on neuronal properties, as well as these effects during normal and paroxysmal oscillations in corticothalamic neuronal loops.
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IMPACT OF NETWORK ACTIVITY ON INTRINSIC NEURONAL PROPERTIES |
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The intrinsic properties of neocortical and thalamic neurons were
first revealed in brain slices. The major advantages of these
simplified preparations are the control of the extracellular ionic
environment, the simultaneous exploration of different neuronal compartments, and the possibility of investigating the actions of
neurotransmitters on identified neuronal types after blockage of
synaptic transmission. Presently, some of these techniques are not
possible in vivo. In contrast with the earlier view of nerve cells
acting in a purely reflex way, with little consideration for the role
of their intrinsic properties, the host of voltage- and
transmitter-gated conductances discovered in vitro (Gutnick and
Crill 1995; Huguenard 1996
; Llinás
1988
) have provided new insights into the functions of
different brain structures and changed our thinking on the electrical
properties of central neurons. The ionic nature of different types of
conductances has been investigated in cortical and thalamic neurons
(Crill 1996
; Gutnick and Mody 1995
; Llinás 1988
; Schwindt et al.
1988a
,b
, 1989
). Multiple intracellular recordings from various
cell types in neocortical slices, and from different compartments of
single neurons, have revealed single-axon excitatory and inhibitory
postsynaptic potentials (EPSPs and IPSPs) between identified
neurons, investigated the mechanisms for coupling the inputs reaching
various cortical layers, and demonstrated that synaptic transmission is
differentially exerted by the same axon of a pyramidal neuron
innervating another pyramidal cell and a local inhibitory interneuron,
with synaptic depression in the former case and facilitation in the
latter (Markram 1997
; Markram et al. 1997
,
1998
; Thomson and Deuchars 1997
;
Thomson et al. 1993
). These effects are important for
understanding the rules underlying frequency-dependent plasticity.
Paired-cell recordings revealed networks of electrically and chemically
coupled inhibitory interneurons (Galarreta and Hestrin
1999
; Gibson et al. 1999
).
However, investigators in vitro recommended that the enthusiasm for
work in slices must be tempered with caution (Connors and
Gutnick 1990). They emphasized the biological and
physical reactions occurring in the traumatized tissue and concluded
that some neuronal properties described in vitro may be different from those seen in the living organism. Moreover, the overwhelming majority
of studies have been conducted on slices from one structure, leaving
all related systems aside. The disadvantages of brain slices arise not
only because of absence of long-range connectivity but also from the
fact that different research groups use animals at various early
developmental stages, with different temperature and dissimilar
extracellular bathing milieu, which, as shown in the following text
(see Neocortex: changing firing patterns during different
functional states), may drastically change neuronal properties.
This article addresses the effects exerted by synaptic activity on intrinsic neuronal properties with emphasis on normal and paroxysmal oscillations. In this first section, I will discuss the properties of cortical and thalamic neurons, as investigated in brain slices, when these neurons are embedded in intact-bain circuits and are subject to spontaneous shifts in behavioral states. This part mainly refers to the actions of synaptic activities arising in corticothalamic and generalized modulatory systems on the firing patterns and incidence of some neuronal classes in different types of experiments, membrane potential, apparent input resistance, backpropagation of action potentials, plateau potentials, and regularity of firing patterns. Next I will compare different oscillatory types occurring in the simplified circuits of cortical and thalamic slices with rhythmic activities during natural events that occur in brains with preserved connectivity.
Neocortex: changing firing patterns during different functional states
The morphological diversity of neocortical neurons has been
recognized since Ramón y Cajal (1911), and their
electrophysiological properties are quite complex (reviewed in
Gutnick and Crill 1995
). Since the early 1980s, the
electrophysiological properties of neocortical neurons were
characterized intracellularly by their responses to depolarizing
current pulses, first in slices maintained in vitro (Connors et
al. 1982
; McCormick et al. 1985
), thereafter in
acutely prepared animals under deep anesthesia (Gray and
McCormick 1996
; Nuñez et al. 1993
;
Steriade et al. 1996a
, 1998b
) and, finally, during
chronic experiments in awake cats (Steriade et al. 2001
; Timofeev et al. 2001b
).
Four cellular types are usually described. 1) Regular-spiking (RS) neurons constitute the majority of cortical neurons. They display trains of single spikes that adapt quickly or slowly to maintained stimulation. 2) Intrinsically bursting (IB) neurons generate clusters of action potentials, with clear spike inactivation, followed by hyperpolarization and neuronal silence. During prolonged depolarizing current pulses, the spike bursts of IB neurons may recur rhythmically at 5-10 Hz. 3) Fast-rhythmic-bursting (FRB) neurons give rise to high-frequency (300-600 Hz) spike bursts recurring at fast rates (generally 30-50 Hz). And 4) fast-spiking (FS) neurons fire thin action potentials and sustain tonically very high firing rates without frequency adaptation.
Generally, RS and IB neurons are pyramidal-shaped neurons, while FS
firing patterns are conventionally regarded as local GABAergic cells
(but see following text). Neurons displaying FRB firing patterns are
either pyramidal-shaped neurons or local-circuit, sparsely spiny or
aspiny interneurons. The firing patterns described in cats under
anesthesia are similar to those described in vitro or in awake animals.
As to the duration of intracellularly recorded action potentials at
half-amplitude, measured during the state of natural waking in
chronically implanted cats, RS neurons show modes between 0.6 and 1 ms
(slightly longer spikes are fired by IB neurons); in contrast, both FRB
and FS neurons demonstrate much shorter action potentials, with modes
at about 0.3 ms (Steriade et al. 2001).
The preceding classification in four neuronal types had a temporarily
heuristic value. However, data discussed below show that this
systematization does not implicate strict, distinctly different,
categories. Initially, electrophysiologists were impressed by the
peculiar properties of some cortical neurons. Because of the virtual
absence of spontaneous activity in slices, the characterization of
these neurons could not take into consideration the role of synaptic
activities generated in neocortex and/or thalamus in modifying the
firing patterns resulting from intrinsic cellular properties. The
morphological correlate, laminar location, and electrophysiological
feature of IB neurons have been thought to be so precise that they were
qualified as the signature of layer V and having a unique physiology
(Connors and Amitai 1995). This is possibly why
consciousness was thought by some theoreticians to result from the
activity of a special (bursting) neuronal type (Crick
1994
; Crick and Koch 1998
). These authors
wondered whether the visual representation is largely confined to
certain neurons in deep cortical layers and further suggested that
there are special sets of awareness neurons in the cortex, specifying
layer V bursting cells. It would be a mystery why a peculiar cell
class, IB neurons, which do not exceed 5% of cortical neurons in awake
preparations (Steriade et al. 2001
), would be so
privileged that their activity gives rise to global states of awareness
and consciousness.
In reality, each of the aforementioned firing patterns does not necessarily apply to a single class of neurons; the electrophysiological characteristics of different cortical cell types are much more flexible than conventionally thought; their location is far from being exclusively confined to distinct cortical layers; and their relative proportions vary with the type of preparation (intact cortex or isolated cortical slabs, anesthetized or nonanesthetized animals).
Thus although FS-firing neurons were previously equated to GABAergic
interneurons, it is now known that some local-circuit inhibitory
neurons fire like RS or bursting cells (Thomson et al.
1996). That the firing pattern of one neuronal type may be transformed, under certain physiological conditions, into another type
became obvious from investigations on various neuronal classes. A
reorganization of firing patterns may occur with shifts in the state of
vigilance, from deafferented to brain-active behavioral states. The
maintained depolarization of IB neurons results in burst inactivation
(Mason and Larkman 1990
; Timofeev et al.
2000
) (Fig. 1A). It
was then proposed that thick layer V neurons could operate in two
modes, switching between bursts and tonic discharges, as a function of
modulatory neurotransmitters (Mason and Larkman 1990
).
Indeed, the enhanced synaptic activity during brain activation by
setting into action the ascending brain stem reticular systems (Steriade et al. 1993a
), and in vitro application of
some neurotransmitters (Wang and McCormick 1993
)
released in the intact brain by generalized activating systems
(Steriade and McCarley 1990
), are all conditions that
may transform IB into RS firing patterns (Fig. 1B).
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The idea of transformation from IB into RS firing patterns, based on
previous results from anesthetized animals (Steriade et al.
1993a), is now substantiated by a similar transformation during
shifts from the natural state of slow-wave sleep to either wakefulness
or rapid-eye-movement (REM) sleep when the membrane potential of
cortical neurons is slightly depolarized (Steriade et al.
2001
). Figure 2 shows different
(IB and RS) firing patterns of the same neuron, evoked by depolarizing
current pulses applied during slow-wave and REM sleep, respectively.
Also, the mode of interspike intervals during the spontaneous activity
in slow-wave sleep was at 3-3.5 ms, reflecting the presence of spike
bursts, while this mode was absent in REM sleep, and there were many
more longer intervals (20-100 ms) during REM sleep, reflecting the single spike firing in the latter state.
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Moreover, antidromically identified and intracellularly stained
corticothalamic (glutamatergic and excitatory) neurons, recorded in
vivo, may fire like FRB neurons in response to depolarizing current
pulses, but below that level they fire like RS neurons and, at more
depolarized levels, like FS neurons (Fig.
3A). Similar voltage-dependent
changes, from RS to FRB and further to FS firing patterns, are observed
in formally identified local-circuit basket cells (Fig. 3B)
(Steriade et al. 1998b). Work in cortical slices also
showed that RS neurons may develop their type of discharges into those
of FRB neurons by repeated application of depolarizing current pulses
(Kang and Kayano 1994
). The transformation of output pattern, from RS single-spike firing to FRB burst discharges (Fig. 3),
may render unreliable cortical synapses reliable (Lisman
1997
). In vitro, FRB neurons are not seen in animals that are
<4 mo of age (Brumberg et al. 2000
). An additional
factor that complicates the recording of this neuronal type in cortical
slices is the composition of the ionic medium that requires 1.2 mM
[Ca2+]o, while most in
vitro studies use [Ca2+]o
of 2 mM or more. Therefore a certain level of increased excitability in
neuronal tissue by decreasing
[Ca2+]o (Hille
1992
) may lead to the transformation of neuronal firing patterns, from an RS into an FRB type. With an ionic composition in
vitro closer to that in the intact brain, FRB neurons could eventually
be recorded in slices (Brumberg et al. 2000
).
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The difficulty in maintaining the strict classification in four
distinctly separate cortical cell classes (RS, IB, FRB, and FS) also
stems from the fact that neurons with thin (<0.5 ms) action potentials
and tonic firing without frequency adaptation (like FS-firing cells),
conventionally regarded as local GABAergic neurons, were actually
identified as corticothalamic cells (see Fig. 3A). The
transformation from FRB to FS patterns was similarly demonstrated in
intracellularly stained corticothalamic and local-circuit aspiny or
sparsely spiny basket (presumably inhibitory) neurons (Fig. 3,
A and B). Note that these transformations in
discharge patterns, from those defining the firing of a cell type into
another, are not just the result of delivering current pulses because
similar changes in membrane potential occur spontaneously when an
animal passes from natural slow-wave sleep, characterized by prolonged hyperpolarizing episodes, to either waking or REM sleep
(Steriade et al. 2001) (see also following text, Fig.
16). The difficulty of considering a simple dichotomy between the two
major cell groups, long-axoned pyramidal (RS) and GABAergic
local-circuit (FS) neurons, arises not only from the diversity of
inhibitory interneurons in the neocortex (Jones 1988
,
1995
), expressing different electrophysiological features in at
least five anatomical classes (Gupta et al. 2000
), but
also from the fact that intracellularly stained cells, with the same
FRB firing pattern, proved to be either deeply lying pyramidal cells or
local-circuit basket cells (Steriade et al. 1998b
) (Fig.
3). As remarked in a study by Markram's group working in cortical
slices (Gupta et al. 2000
), the usual classification of
RS, IB, and FS neurons, stemming from previous in vitro experiments (Connors et al. 1982
; McCormick et al.
1985
), is too vague to encompass the diversity of responses.
Network activity during various functional states is decisive in
altering the firing patterns generated by intrinsic neuronal properties. Thus typical FRB patterns, which are evoked during the
silent background activity of interspindle lulls (as in slices), are
dramatically changed during epochs with rich synaptic activity produced
by intracortical or thalamocortical volleys (Fig.
4) and develop into patterns resembling
the FS firing (Steriade et al. 1998b).
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To fully realize the importance of synaptic activity in a living
animal, compared with slices, and the striking difference in
connectivity as well as incidence of synaptic potentials between slightly different sizes of slices, here are the results of two in
vitro studies on sensorimotor neocortex (Thomson 1997;
Thomson et al. 1996
). Out of 595 dual recordings in
which an interneuron was recorded simultaneously with a pyramidal
neuron in slices 400-µm thick, 39 yielded monosynaptic, single axon
IPSPs, i.e., an average probability of 1:15 of each recorded inhibitory
interneuron contacting a neighboring pyramidal cell; however, with
slices 500-µm thick, the probability rose about three times. Thomson also reported a significantly higher incidence of connections and an
increase in spontaneous activity in 500-µm, compared with 400-µm,
slices. The dramatic increase in connectivity on increasing the slice
thickness by just 0.1 mm may explain the differences between some
results from slices, compared with those from the intact brain. Other
dissimilarities, from works in the cerebral cortex, thalamus and
related systems, are fully discussed elsewhere (Steriade
2001
).
The preceding data show that changes in membrane potential and a high
degree of synaptic activity in the intact brain decisively modulate,
and even transform, the firing patterns due to intrinsic neuronal
properties and expressed by responses to direct depolarization. The
ideas on the discrete laminar localization of various neuronal types in
neocortex also evolved. IB neurons were initially found in layer V, but
IB neurons were later recorded also from layers IV and III
(Connors and Amitai 1995; Montoro et al.
1988
; Steriade et al. 1993e
). The FRB neurons
(also termed "chattering") were described as exclusively located in
supragranular layers II-III of the visual cortex (Gray and
McCormick 1996
), but the same type of rhythmic bursting neurons
was found in all cortical layers, from II to VI, of sensory-motor and
association areas (Steriade et al. 1998b
); their deep
location is corroborated by antidromic identification as
corticothalamic neurons (see Fig. 3A).
The proportions of FS and IB firing patterns in nonanesthetized, awake
animals (Steriade et al. 2001) are quite different from
those found in anesthetized animals with intact cortex
(Nuñez et al. 1993
; Steriade et al.
1998b
) or small isolated slabs in vivo (Timofeev et al.
2000
); the latter type of experiments partially reproduce the
in vitro condition. Neurons displaying the firing patterns of FS
neurons are much more numerous in naturally alert animals (24%) than
in the intact cortex of anesthetized animals (12%) or in small
isolated cortical slabs (4%). The FS (putative inhibitory) neurons
have been implicated in the generation of fast (20-40 Hz) rhythms
(Buzsáki and Chrobak 1995
; Llinás et al. 1991
; Lytton and Sejnowski 1991
;
Traub et al. 1999
) that characterize the spontaneous
activity in the waking state and dreaming mentation in humans and
animals (Llinás and Ribary 1993
; Steriade
et al. 1996a
,b
). These states of network activity, accompanied
by relatively depolarized levels of membrane potential, may transform
neurons with other firing patterns (i.e., FRB) into FS-type neurons
(see Fig. 3). This would result in an increased proportion of neurons identified as FS. On the contrary, neurons displaying IB firing patterns are found in <5% of neurons of awake animals
(Steriade et al. 2001
), whereas they represent ~15%
of neurons in anesthetized animals (Nuñez et al.
1993
; Steriade et al. 1998b
) and may
reach 40% of neurons in isolated cortical slabs in vivo
(Timofeev et al. 2000
) or in some studies in vitro that
reported proportions of
64% IB neurons (Yang et al.
1996
). The strikingly diminished proportion of IB firing
patterns in the alert condition is likely due to the enhanced synaptic
activity and increased release of some modulatory neurotransmitters,
i.e., conditions that may transform IB into RS firing patterns
(Steriade et al. 1993a
; Wang and McCormick 1993
).
To sum up, the bursting and regular (tonic) firing patterns represent a continuum of discharge properties and the electrophysiological distinctions between various neuronal classes are much less clear-cut in nonanesthetized animals than were conventionally thought in the early studies on cortical slices or in anesthetized preparations.
The impact of spontaneous synaptic activity on intrinsic neuronal properties was further studied with emphases on the membrane potential (Vm), the apparent input resistance (Rin, a measure resulting from passive electrical neuronal properties and balanced changes in excitatory and inhibitory inputs from specific and modulatory pathways), backpropagation of action potentials from the axonal initial segment to dendrites, and plateau potentials after blockage of K+ currents. These issues are discussed below.
MEMBRANE POTENTIAL AND INPUT RESISTANCE.
The isolated cortical slab in vivo (10 × 6 mm) is a new
preparation that was introduced to examine the necessary number of interconnected neurons for the presence of sleep-like oscillations and
that has the advantages of both in vitro and in vivo preparations; that
is, it does not drastically change the milieu of the neurons in the
network (Timofeev et al. 2000). In this preparation,
triple intracellular recordings have been first performed in vivo. The mean Vm in small isolated neocortical
slabs in vivo is
70 mV and the Rin
is 49 M
, whereas in intact (adjacent) cortical areas of the same
animal the values are
62 mV and 22 M
, respectively. In another
study (Paré et al. 1998b
), the
differences between in vivo and in vitro recordings of the same type of
pyramidal neurons are as follows: the standard deviation of the
intracellular signal is 10-17 times lower in vitro than in vivo and
the Rin measured in vivo during
relatively quiescent periods (37 ± 3.9 M
) is reduced by
70%
during epochs associated with intense synaptic activity, and increases
by
70%, approaching the in vitro values (66.14 ± 1.3 M
),
after tetrodotoxin (TTX) application in vivo (Fig.
5).
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BACKPROPAGATION OF ACTION POTENTIALS.
The backpropagation of action potentials (APs) has two aspects. The
first refers to the propagation of APs generated at ectopic sites
(regions remote from the axon hillock) toward the soma. Ectopically
generated APs generally occur in pathological conditions, such as
thalamic (Gutnick and Prince 1972; Schwartzkroin
et al. 1974
) or callosal (Schwartzkroin et al.
1975
) neurons projecting to cortical epileptic foci. The second
aspect of backpropagation, which is still a disputable issue because of
differences in results from in vitro and in vivo experiments (see
following text), is the initiation of APs in the axon hillock and its
propagation into the dendrites of various neuronal types, thus
providing a retrograde signal of neuronal output to the dendritic tree
(Häusser et al. 2000
; Stuart and
Sakmann 1994
; Stuart et al. 1997
). The functional consequences of APs, backpropagated from the axon hillock to
dendrites, may be an influx of Ca2+ without
evoking a Ca2+ AP (Larkum et al.
1999
). This would imply a facilitation of the initiation of
Ca2+ APs when backpropagating APs coincide
(within a time window of ~10 ms) with distal dendritic inputs and is
regarded as a mechanism for coupling inputs reaching cortical neurons
at different layers. The backpropagating APs may signal the level of
neuronal output to the dendritic sites receiving synaptic inputs, thus
serving as a link between output and input.
PLATEAU POTENTIALS.
Plateau potentials in neocortical neurons are elicited after blockage
of K+ currents and are due to a class of
high-voltage-activated Ca2+ channels in dendrites
(Reuveni et al. 1993; Yuste et al. 1994
). The high background activity in vivo may block the
Ca2+ plateau potentials. Synaptic inputs lead to
the termination of plateaus (Paré et al.
1998a
). Using dual intracellular recordings in vivo, with one
pipette filled with potassium acetate to control network activity and
the other pipette filled with cesium acetate to block
K+ currents, we showed that synaptic inputs,
generated by corticipetal volleys during thalamically generated spindle
oscillations, consistently shut off plateaus (Contreras et al.
1997c
). Similarly, PSPs evoked by electrical thalamic
stimulation blocked the Cs+-induced plateaus
(Fig. 7). The dendritic
Ca2+ electrogenesis in cortical neurons
(Kim and Connors 1993
; Llinás 1988
) may play an important role in synaptic plasticity
(Swanson 1989
). The fact that
Cs+-induced plateaus are blocked (but sometimes
triggered) by synaptic inputs suggests that coherent oscillations in
thalamocortical networks may have a role in plasticity by modifying
Ca2+ electrogenesis.
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REGULAR FIRING PATTERNS DURING BRAIN-ACTIVE BEHAVIORAL STATES.
The reliability of firing patterns increases with fluctuating current
waveforms resembling synaptic activity (Mainen and Sejnowski 1995). Suprathreshold current pulses to the soma elicit spike trains with a progressive lack of reliability in precise timing, whereas fluctuating current waveforms evoke precise and stable timing
throughout the length of the trials (Fig.
8). In the latter condition, the action
potentials may be separated by 100 ms, yet they occur with the
precision of most responses in the range of 1-2 ms (see also
Nowak et al. 1997
). These data suggest that currents resembling synaptic inputs may be repeatedly encoded into spike patterns with millisecond precision. The results regarding the regularity of firing evoked by depolarizing current pulses with added
fluctuating waveforms, which simulate synaptic activity, fit well with
the relative regularity of firing seen during behavioral states of
vigilance associated with a high degree of synaptic activity as in
wakefulness and REM sleep (Evarts 1964
; Steriade 1978
; Steriade et al. 1974
). The firing
regularity during these two brain-active states is expressed by
Gaussian-like interspike interval histograms with virtual absence of
very short (<25 ms) and very long (>150 ms) intervals (see Fig. 7 in
Steriade et al. 1974
). During slow-wave sleep, when the
cerebral cortex is disconnected from the outside world because of the
blockade of synaptic transfer within the thalamus (Steriade et
al. 1969
; Timofeev et al. 1996
), there
is a much greater irregularity of firing patterns because the presence
of spike bursts, reflected by very short interspike intervals,
interspersed with long periods of silence.
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Thalamus: effects of synaptic inputs on neuronal properties and local oscillations
There are three main classes of thalamic neurons: thalamocortical
(TC), which are all glutamatergic, thus excitatory; thalamic reticular
(RE), which send their axons to the dorsal thalamus and are all
GABAergic, thus inhibitory; and local-circuit GABAergic neurons whose
axonal domain is confined within the limits of the thalamic nucleus
where their somata are located. With few exceptions, the three types of
thalamic neurons are homogenous in terms of morphology (Jones
1985) and electrophysiological properties (Steriade et
al. 1997b
). This stands in contrast with the variety and
complexity of neocortical neurons.
Most schemes of thalamic functioning include only TC and RE neurons.
However, all dorsal thalamic nuclei of cats and primates (and the
lateral geniculate nucleus of rodents) also possess an important
proportion (25%) of local-circuit inhibitory interneurons (Jones 1985). About 8-10% of RE neurons project to
local thalamic interneurons (Liu et al. 1995
), and
although apparently minor, this GABAergic-to-GABAergic projection may
produce significant effects on the ultimate targets, TC neurons,
eventually leading to their disinhibition. Indeed, a greatly increased
incidence of IPSPs in TC neurons was observed after destruction of RE
neurons, reflecting the release from the inhibition of local
interneurons after the excitotoxic lesion of RE perikarya
(Steriade et al. 1985
). The connection between the two
types of thalamic GABAergic cells, RE and local-circuit interneurons,
may be important for focusing attention to relevant signals
(Steriade 1999
). Figure 9
illustrates this hypothesis. The top RE neuron, part of the RE pool
that is directly connected to the top TC (Th-cx)
neuron, contributes to enhancement of relevant activity by
inhibiting the appropriate pool of local-circuit elements.
Simultaneously, the activity in adjacent RE areas is suppressed by
RE-to-RE GABAergic contacts within the nucleus. The consequence would
be the disinhibition of related local interneurons (bottom
L-circ cell) and the inhibition of weakly excited
TC neurons in areas adjacent to the active focus.
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Here, I will focus on the impact exerted by synaptic activity, arising
locally or in distant structures, on a major intrinsic property of
thalamic neurons (the low-threshold spike, LTS) as well as on related
oscillatory phenomena in these neurons, the network generated sleep
spindles and the intrinsically generated clock-like (delta) rhythm.
Other intrinsic properties of TC and RE neurons, their ionic bases, and
the biophysical models of ionic currents, are discussed in two recent
monographs (Destexhe and Sejnowski 2001; Steriade
et al. 1997b
).
THE LTS.
The ability of thalamic neurons to display a paradoxical form of
excitation resulting from their hyperpolarization was known since the
late 1960s (Andersen and Andersson 1968; see also
Maekawa and Purpura 1967
), but systematic studies on the
postinhibitory rebound and the discovery of the
Ca2+-dependent low-threshold current
(IT) underlying this intrinsic neuronal property were only possible with the advent of slice studies
(Jahnsen and Llinás 1984a
,b
; reviewed in
Huguenard 1996
; Llinás
1988
). More recent studies reported that the LTS of TC neurons
also contains a component mediated by a persistent
Na+ current
(INa(p)) (Parri and Crunelli
1998
).
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EFFECTS OF SYNAPTIC ACTIVITY IN ASCENDING AND CORTICOTHALAMIC
PROJECTIONS ON TERMINATION AND WIDE
SYNCHRONIZATION OF THALAMICALLY GENERATED SPINDLE
OSCILLATIONS.
Spindles (7-14 Hz) arise within the thalamus even after decortication
and high brain stem transection (Morison and Bassett 1945). The RE nucleus is the pacemaker of spindle oscillations as demonstrated by the abolition of spindles in target thalamic nuclei
and corresponding cortical areas after disconnection of TC neurons from
the RE nucleus (Steriade et al. 1985
) and the preservation of spindles in the RE nucleus disconnected from the remaining thalamus (Steriade et al. 1987
). The different
reasons explaining the failure to obtain spindles in the isolated RE
nucleus of thalamic slices (Von Krosigk et al. 1993
),
among them the requirement of a larger and more intact collection of RE
neurons than usually found in thalamic slices (see Steriade et
al. 1993d
) and the absence of some brain stem modulatory
systems (Destexhe et al. 1984
), are discussed elsewhere
(Steriade et al. 1997a
). Although this oscillation was
recorded intracellularly within the thalamus, in the absence of cortex,
both in vivo (Deschênes et al. 1984
; Steriade and Deschênes 1984
;
Timofeev and Steriade 1996
) and in vitro (Bal et
al. 1995a
,b
; Von Krosigk et al. 1993
), the
neocortex contributes to the termination of individual spindle
sequences, but on the other hand, it plays an important role in the
synchronization of spindle sequences over widespread thalamic and
cortical territories. Thus long-range projections in corticothalamic
systems influence a thalamically generated oscillation, and although
spindles arise in local intra-RE and RE-TC circuits, network activities
originating in distant cortical areas are powerful enough to change the
duration and synchronization patterns of this sleep oscillation. These data are discussed below.
|
CORTICAL SYNCHRONIZATION OF AN INTRINSIC (CLOCK-LIKE DELTA)
THALAMIC OSCILLATION.
The other thalamically generated oscillation is the clock-like delta
rhythm (usually 2-4 Hz), due to the interplay between two currents,
IH and
IT, that are activated and
de-inactivated, respectively, by membrane hyperpolarization
(Curró Dossi et al. 1992;
Leresche et al. 1990
, 1991
; McCormick and Pape
1990
; Soltesz et al. 1991
). This oscillation is
modulated by different substances that act on purinergic and adrenergic
receptors and up- or downregulate the H current (Pape
1996
; Pape and Mager 1992
; Pape and
McCormick 1989
; Yue and Huguenard 2001
).
Although intrinsic to TC neurons (Fig.
13A), this oscillation,
which represents only one component of delta waves seen on the EEG
during slow-wave sleep, is subject to influences arising in neocortex.
Corticothalamic volleys synchronize TC neurons (Fig. 13B) by
primarily exciting GABAergic RE neurons that fulfill two basic
requirements: they set the Vm of TC
neurons at the appropriate level of hyperpolarization for the
appearance of the two currents (IH and
IT) and they project to different dorsal thalamic nuclei, thus synchronizing not only nearby but also
distant TC neurons (Steriade et al. 1991
).
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DEVELOPMENT OF NORMAL BRAIN RHYTHMS TO PAROXYSMAL ACTIVITY |
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In this section, I shall first focus on intrinsic cell properties
and network operations underlying the major rhythm that characterizes
sleep, the slow oscillation at 0.5-1 Hz that is present throughout all
(light and deep) stages of natural slow-wave sleep in animals
(Steriade and Amzica 1998) and humans (Achermann and Borbély 1997
; Amzica and Steriade
1997
). Although spontaneously occurring brain rhythms are
sometimes regarded as bearing little or no functional significance, the
spontaneous electrical activity is information-rich, provides signals
that influence neighboring cells, and accounts for changes in evoked
responses (Arieli et al. 1996
; Bullock
1997
). It was proposed that the rhythmic spike bursts or spike
trains fired by thalamic and neocortical neurons during sleep
oscillations lead to reorganization of neocortical networks and
consolidation of memory traces formed during the waking state
(Steriade et al. 1993b
,d
). This hypothesis was tested and will lead me to discuss the synaptic changes, related to
plasticity, during sleep oscillations. Finally, I will elaborate on the
development from normal sleep oscillations into paroxysmal events
mimicking some types of clinical seizures associated with loss of consciousness.
Fast brain rhythms (20-60 Hz), which occur mainly, but not
exclusively, during states of brain alertness (Herculano-Houzel et al. 1999; Llinás and Paré 1991
;
Llinás and Ribary 1993
; Murthy and Fetz
1997
; Rougeul-Buser 1994
; Singer
1993
; Steriade et al. 1996a
,b
), and their
relation with discrete conscious events, are discussed elsewhere
(Steriade 2000
, 2001
).
No pure rhythms, no simple circuits: the slow oscillation groups cortical and thalamic rhythms
The slow oscillation (generally 0.5-1 Hz) was initially described
using intracellular recordings from cortical neurons in cats under
anesthesia and EEG recordings during human natural sleep
(Steriade et al. 1993e). Although the frequency of the
slow oscillation may reach 1 Hz and even slightly exceed this frequency during late periods of natural sleep, the slow oscillation is different
from waves in the delta frequency band (1-4 Hz), as the former groups
both cortically and thalamically generated delta waves into rhythmic
wave-sequences (Steriade et al. 1993e
,f
). This indicates
the distinct nature of the two (slow and delta) oscillations. Human
sleep recordings add further support for the differences between slow
and delta activities: the typical decline in delta waves (2-4 Hz) from
the first to the second episode during EEG-synchronized sleep is not
present at lower frequencies that characterize the slow oscillation
(Achermann and Borbély 1997
). The slow oscillation
was also recorded, with the same characteristics as initially described
in animals, using magnetic (MEG) recordings during natural sleep in
humans (Simon et al. 2000
).
The cortical origin of the slow oscillation was demonstrated by its
presence in neocortex after thalamectomy (Steriade et al.
1993f), the disruption of its long-range synchronization after interrupting corticocortical links (Amzica and Steriade
1995a
), and its absence in the thalamus of decorticated animals
(Timofeev and Steriade 1996
). That the slow oscillation
arises in intracortical networks was confirmed by the presence of this
rhythmic activity in vitro using a bathing milieu more similar to that
present in vivo (Sanchez-Vives and McCormick 2000
) than
the ion concentrations usually employed in cortical slices. Because the
cortex projects to many subcortical structures, the slow oscillation
was also recorded not only in the thalamus (Steriade et al.
1993b
) but also in the caudate nucleus, the
subthalamic-pallidus network, basal forebrain, mesopontine, and
medullary brain stem nuclei (Magill et al. 2000
;
Mariño et al. 2000
; Nuñez
1996
; Steriade et al. 1994a
; Wilson and
Kawaguchi 1996
). In many of these subcortical sites, the slow
oscillation disappeared after functional inactivation of cortex or decortication.
Single and dual intracellular recordings from neocortical neurons in
vivo, together with multi-site field potentials, show that the slow
oscillation is built up by synchronous sequences of prolonged
depolarizations and hyperpolarizations, each lasting 0.4-0.7 s
(Amzica and Steriade 1995a
,b
; Contreras and
Steriade 1995
; Steriade et al. 1993e
,f
, 1994b
)
(Fig. 14, cat). These data, first
obtained under different types of anesthetics, are now confirmed during
the natural slow-wave sleep of chronically implanted cats (Steriade et al. 2001
; Timofeev et al.
2001b
). The long-lasting hyperpolarizations are obliterated on
natural awakening in behaving animals (Fig.
15), and they appear as distinct events
from the very onset of slow-wave sleep.
|
|
All major types of neocortical neurons (RS, IB, FRB, and FS; see
Neocortex: changing firing patterns during different functional states) behave similarly during the slow oscillation. Namely, they
discharge during the depolarizing phase associated with depth-negative field potentials and are silent during the hyperpolarizing phase associated with depth-positive field potentials. The depolarizations are due to a combination of a persistent Na+
current and N-methyl-D-aspartate (NMDA)- as well
as non-NMDA-mediated EPSPs, but they also include IPSPs
(Steriade et al. 1993e). The fact that no cellular type,
including FS neurons, some of them formally identified as short-axoned
basket-type neurons (Contreras and Steriade 1995
),
discharges during the hyperpolarization (Fig. 16B) indicates that this
component of the slow oscillation is not mediated by
GABAergic processes. Also, the major part of the prolonged hyperpolarizing potentials is not affected by recordings with Cl
-filled pipettes (Fig. 16B; see
also Timofeev et al. 2001b
). The hyperpolarizations are
mainly produced by Ca2+-dependent
K+ currents and disfacilitation processes
(Contreras et al. 1996b
; Timofeev et al.
2001b
).
|
The initiation and termination of the hyperpolarizing phase of the slow
oscillation, which sculpts the firing of neocortical neurons during
slow-wave sleep, may be explained by the following scenario
(Contreras et al. 1996b). During the depolarizing phase of the slow oscillation, synaptic currents
(Isyn) are maintained at a high level
of activity in intracortical circuits. Small decreases in
Isyn will allow the passive leak
current (Ileak) to dominate the scene,
hyperpolarizing the neuron and decreasing its firing probability. As a
consequence, the target cortical cells will reduce their firing rates,
eventually leading, by an avalanche effect, to generalized neuronal
silence. During the prolonged hyperpolarization, the space constant
will increase due to a decrease in
Isyn, the cell will become more
compact, and, toward the end of this phase, neurons may reach firing
threshold. At this stage, firing from any small group of cortical or
thalamic neurons would rapidly bring the whole system back to the
depolarizing state and evoke action potentials, which would explain the
steep uprising phase of the depolarizing phase. Even during abnormally
synchronized EEG activity, with burst-suppression patterns, volleys
applied during prolonged periods of neuronal silence are sufficient to restore neuronal activity and spiking in corticothalamic networks (Steriade et al. 1994a
). The rebound spike bursts fired
by TC neurons, due to IT
de-inactivation toward the end of the hyperpolarizing phase
(Contreras and Steriade 1995
), may be an important
factor in triggering new cycles of the slow oscillation. However,
cortical neurons certainly preserve this ability as the slow
oscillation can be recorded in the absence of the thalamus
(Steriade et al. 1993f
). The relationship between
neurons and glial cells, whose intracellular activities were recorded
simultaneously (Amzica and Neckelmann 1999
;
Amzica and Steriade 1998b
, 2000
), suggests that glial
cells might also play a role in pacing the slow oscillation and, when
sleep oscillations develop to seizures, in triggering paroxysmal events
(see following text).
Three other rhythms, generated in the thalamus and cortex, sleep spindles and delta waves as well as fast rhythms, are grouped by the slow cortical oscillation in complex wave sequences. This defies a strict dissociation of different brain rhythms and justifies the title of this subsection, which maintains that, in the living brain, oscillations are not generated in circumscribed neuronal networks but in interconnected neuronal loops between the cerebral cortex and thalamus, under the control of modulatory systems of the brain stem core, hypothalamus, and basal forebrain.
During the sharp depth-negative field potential of the slow
oscillation, the summated activity of corticothalamic neurons sets into
action thalamic (RE and TC) neurons, thus triggering spindles that are
fed back to cortex (see Fig. 14, cat). The grouping of
thalamically generated spindles by the slow cortical oscillation is at
the origin of the KC, a major element of sleep EEG in humans and
animals, which consists of an ample surface-positive (depth-negative) wave, followed by a spindle sequence (Amzica and Steriade 1997, 1998a
; Contreras and Steriade 1995
). The power
spectrum of human sleep EEG reveals a peak at ~0.7 Hz reflecting the
slow oscillation, a peak at ~13-14 Hz reflecting spindles, and a
spectral content between 1 and 4 Hz reflecting delta waves that are due
to the shape and duration of KCs (Fig. 14, human). KCs may
also be triggered by sensory stimulation during sleep. However, evoked
KCs are rather the exception (compared with the "spontaneously"
occurring ones, in fact elicited by the slow cortical oscillation) as
sleep generally occurs in environments free from sensory stimuli.
The slow oscillation also groups delta waves (1-4 Hz). One type of
delta activity is generated in the thalamus and is due to the interplay
of two currents in TC neurons, IH and
IT, dependent on their
hyperpolarization (Curró Dossi et al. 1992;
Leresche et al. 1990
, 1991
; McCormick and Pape
1990
; Soltesz et al. 1991
). Although
intrinsically generated in single TC neurons, this clock-like delta
activity can be synchronized in pools of TC cells by synaptic activity
in corticothalamic systems (see preceding text, Fig. 13). When pools of
delta-oscillating TC neurons are synchronized, their clock-like
activity is reflected at the cortical level and seen in conjunction
with the slow oscillation (Fig.
17A). The other type of
delta activity is generated in neocortex as it survives thalamectomy
(Steriade et al. 1993f
; Villablanca 1974
)
and can be partially generated through the intrinsic properties of
neocortical neurons, as seen by rhythmic responses of IB cells to
depolarizing current pulses. The depolarizing phase of the slow
oscillation is composed of activity in the delta frequency range
(Amzica and Steriade 1998b
; Steriade
1997
). The combined slow and delta oscillations in a bursting
cell and in two simultaneously recorded cortical neurons are
illustrated in Fig. 17, B and C. Cortical delta
waves are under the control of cholinergic nucleus basalis neurons
(Buzsáki et al. 1988
).
|
Finally, fast rhythms appear during the depolarizing phase of the slow
sleep oscillation (Steriade et al. 1996a,b
). The fact that the slow oscillation groups not only other sleep rhythms but also
generates fast rhythms (20-50 Hz) stands in contrast with the idea
that these rhythms are reliable indicators of alertness and conscious
states. Fast neocortical rhythms are voltage (depolarization) dependent
(Gutfreund et al. 1995
; Llinás et al.
1991
; Nuñez et al. 1992
), and thus they
appear over the depolarizing component of the slow sleep oscillation
but are obliterated during the hyperpolarizing phase (Steriade
et al. 1996a
,b
). The difference between fast oscillations in
the aroused and sleeping brain is that in the former case, these
rhythms are continuous, whereas in the latter, they are cyclically
interrupted by hyperpolarizations. Spontaneous fast rhythms are
distributed without phase reversal throughout the cortical depth
(Steriade and Amzica 1996
). Time lags between surface and depth activities, action potentials superimposed on the negative phase of fast field potentials in superficial and deep layers, and
absence of fast activity in the white matter underlying the cortex,
indicate that the fast fields are not volume-conducted but
locally generated (Steriade et al. 1996a
).
In humans, cortical activity during slow-wave sleep, measured by
regional cerebral blood flow (rCBF), displays more changes in those
areas that are implicated during wakefulness in heteromodal association
processes and the control of emotions and social interactions (Maquet 2000; Maquet et al. 1996
). The
decreased rCBF in the thalamus and cerebral cortex during slow-wave
sleep was reported in a series of studies (Braun et al.
1997
; Fiset et al. 1999
; Hofle et al. 1997
; Kajimura et al. 1999
; Maquet et al.
1997
). The majority of these studies emphasized that, far from
being associated with a global decrease in rCBF, slow-wave sleep is
accompanied by local changes in the cerebrum.
In sum, the complexity of brain electrical activity, with wave-sequences composed of different rhythms, originating in interacting structures as well as the dependence of these rhythms on the activity of generalized activating systems emphasizes the need for studies conducted in intact-brain preparations.
Short-term plasticity follows rhythmic spike trains during sleep oscillations
Plasticity is defined as an activity-dependent alteration in the
strength of connections among neurons and is a mechanism through which
information is stored. Although hippocampal and neocortical mechanisms
of plasticity have received the greatest emphasis in experimental
studies (Martin et al. 2000; Tsumoto 1992
), thalamic neurons also display reorganization after
deafferentation (Jones 2000
) and brain stem cholinergic
stimulation produces a prolonged enhancement of thalamic synaptic
responsiveness (Paré et al. 1990
).
The surprisingly high discharge rates of all cortical cell types during
the depolarizing phase of the slow oscillation in natural slow-wave
sleep (see Figs. 15 and 16) suggests that during this behavioral state
in which the brain is disconnected from the external world because of
synaptic inhibition of messages in the thalamus, neocortical neurons
are actively involved in processing internally generated signals. The
hypothesis that during sleep oscillations neocortical neurons are
implicated in plasticity processes and in consolidating memory traces
acquired during wakefulness (Steriade et al. 1993b,d
)
may be related to a similar idea (Buzsáki 1989
)
that was tested in the hippocampal system (Pavlides and Winson
1989
; Qin et al. 1997
; Wilson and
McNaughton 1994
). In the following text, I will discuss data
from experiments in which a thalamically generated oscillation, sleep
spindles, was mimicked by stimulating the thalamus and neocortex within
the frequency range of this rhythm (~10 Hz) to produce changes in
synaptic responsiveness of neurons and self-sustained events following
stimuli that are reminiscent of "memory" traces in reciprocal
corticothalamic loops.
The experimental model of sleep spindles consists of augmenting (or
incremental) responses (Morison and Dempsey 1942).
Augmenting responses are generally defined as thalamically evoked
cortical potentials that grow in size during the first stimuli at a
frequency of 5-15 Hz, usually ~10 Hz, like the waxing of waves at
the onset of spontaneously occurring spindle sequences. A series of
investigators have attempted to make distinctions between various types
of incremental responses and to emphasize the exclusive role of the
thalamus, or the neocortex, in the generation of augmenting responses.
The view expressed here is that although augmentation occurs in the thalamus of decorticated animals (Steriade and Timofeev
1997
) and in the intact cortex of thalamic preparations
(Steriade et al. 1993f
) or even in cortical slices
(Castro-Alamancos and Connors 1996b
), the full
development of augmenting responses, leading to self-sustained
activities, requires interacting thalamic and cortical networks.
The idea that incremental thalamocortical responses are of two
basically different types, augmenting and recruiting, was suggested on
the basis that augmenting responses are elicited in appropriate localized cortical areas by stimulation of "specific" thalamic nuclei and their polarity is positive at the cortical surface, whereas
recruiting responses are elicited by stimulation of "nonspecific" thalamic nuclei, are negative at the cortical surface, and occur with a
longer latency than that of augmenting responses (Dempsey and
Morison 1942). The longer latency of recruiting responses suggested a "diffuse multineuronal system" (Jasper
1949
). This view continued during the 1950s when recruiting
responses were regarded as implicating a recruitment through a
divergent multineuronal chain with intralaminar nuclei serving as an
intrathalamic association system. We now know that there are virtually
no direct pathways linking different dorsal thalamic nuclei, that the
longer latency of cortical recruiting responses is not due to the
intrathalamic spread of activity but to slower conduction velocities of
axons from some thalamic nuclei projecting directly to the neocortex, and that some recruiting (depth-positive) responses may display latencies as short as those of augmenting (depth-negative) responses. In fact, there are no pure augmenting or recruiting responses. Most are
mixed responses, with augmenting preceding the recruiting or vice versa
(Spencer and Brookhart 1961
) because of the
multi-laminar distribution of thalamic projections to cortex. As an
illustration, thalamocortical incremental responses evoked by rhythmic
stimulation of rostral intralaminar nuclei (conventionally known as
typically inducing recruiting responses) are of the recruiting type in
one cortical area and of the augmenting type in another area of the same gyrus (because intralaminar nuclei project preferentially to layer
I but also to deep layers); moreover, the latencies of both augmenting
and recruiting responses evoked by thalamic intralaminar stimulation
are equally short (<4 ms) (see Fig. 4 in Steriade et al.
1998d
). Therefore the distinction between augmenting and recruiting responses is no longer necessary. We can simply designate such responses as augmenting or incremental, describe their polarity, and keep in mind that thalamic neurons may project to middle, deep and
more superficial cortical layers.
In the decorticated thalamus, TC neurons display two types of
augmenting responses to local thalamic stimulation at 10 Hz. One type
of intrathalamic augmenting responses is based on progressively increased LTSs, which are de-inactivated by the increasing
hyperpolarization produced by repetitive stimuli in the train (Fig.
18B).
The other type is associated with progressively decreased IPSPs
elicited by successive stimuli in the train and with progressive
depolarization of neurons leading to high-threshold spike bursts with
increasing numbers of action potentials and spike inactivation (see
below, Fig. 20A). The first type of augmentation (with
progressively increased LTSs and rebound spike bursts) is due to the
parallel excitation in a pool of thalamic RE GABAergic neurons, whereas
the high-threshold form of augmenting is due to decremental responses
in a pool of RE neurons (Timofeev and Steriade 1998).
Then, the relations between RE and TC neurons are essential for the
development of augmenting responses in decorticated animals. As
augmenting responses mimic spindles, and spindles have been recorded in
the deafferented RE nucleus (Steriade et al. 1987
),
augmenting responses as well as spindles were also obtained in models
of isolated RE nucleus with synaptic interconnections including both
GABAA and GABAB components
(Bazhenov et al. 1998a
). Patterns of propagated activity within isolated RE networks can occur if only a small fraction of RE
neurons is hyperpolarized below the GABAA
reversal potential and self-sustained oscillations within the frequency
range of 10-15 Hz are found in two-dimensional network models when
only 25% of RE neurons are hyperpolarized below the
Cl
reversal potential (Houweling et al.
2000
). Thus these models show that
GABAA-mediated excitation in RE neurons can
robustly generate sequences of spindle waves (Bazhenov et al.
1999
), as was experimentally demonstrated in RE neurons
disconnected from the remaining thalamus (Steriade et al.
1987
).
|
Although the thalamus is capable of producing augmenting responses
through its own network activity, we further investigated this
phenomenon using dual intracellular recordings from TC and cortical
neurons (Steriade et al. 1998d) and computational models (Bazhenov et al. 1998b
). These studies revealed that the
augmentation in neocortical neurons is expressed by a selective
increase in the secondary depolarizing component of thalamically evoked
responses and that the secondary cortical depolarization invariably
follows by ~3 ms the rebound burst in simultaneously recorded TC
neurons (Fig. 18C). Thus in intact-brain preparations,
augmenting responses primarily depend on the LTS type of augmentation
and related spike bursts in TC neurons. The comparative analysis of
augmenting responses of neocortical neurons from different layers was
performed by means of dual intracellular recordings of neurons that
were stained and found to be located within deep (layers V-VI) and
more superficial layers (Steriade et al. 1998d
). Deeply
lying pyramidal neurons, and especially FRB cells with thalamic
projections (see Neocortex: changing firing patterns during
different functional states), consistently showed a higher
propensity, shorter latencies, and greater number of action potentials
during augmenting responses, compared with more superficially located
neurons. Other investigators emphasized the role of IB neurons in the
process of augmentation as they investigated layer V in cortical slices
(Castro-Alamancos and Connors 1996a
). The difference
between simultaneously recorded neocortical and TC neurons is that the
former display postaugmenting oscillatory activities in the frequency
range of responses, whereas the latter remained hyperpolarized because
of the pressure from the GABAergic RE neurons (Fig.
19) (Steriade et al.
1998d
). These data show that intracortical circuits have a
major influence on the inputs from TC neurons and can amplify
oscillatory activity arising in the thalamus (Grenier et al.
1998
). Such a view is consistent with the fact that although
spindles are generated in the thalamus, they are not passively
reflected in cortex and cortical synaptic circuitry has a major role in
modifying and amplifying thalamocortical volleys (Kandel and
Buzsáki 1997
).
|
To summarize, because of their high propensity to fire spike bursts, TC neurons trigger incremental responses in target neocortical neurons, but the latter have the ability to maintain and develop self-sustained oscillations.
What is the experimental evidence that such responses are associated
with short-term plasticity processes? Although the augmentation phenomenon characterizes a state of vigilance, resting sleep, during
which brain "utilitarian" processes are apparently suspended, incremental responses are associated with short-term plasticity in both
thalamus and cortex. During repetitive thalamic stimuli at 10 Hz in
decorticated animals, the IPSPs of TC neurons are progressively
diminished and, conversely, the depolarization area of augmenting
responses increases continuously with the repetition of pulse-trains at
10 Hz (Fig.
20A).
Similar data can be elicited by using testing stimuli to the neocortex.
After spontaneously occurring spindle sequences, single-spike responses
of cortical association neurons, evoked by stimulating the same
cortical area, are transformed into greatly increased responses, a
potentiation that lasts for several minutes (Grenier et al.
1999). The same phenomenon occurs when mimicking spontaneously
occurring spindles with pulse-trains within the frequency range (10 Hz)
of spindles. In the cerebral cortex of animals with ipsilateral
thalamectomy, augmenting responses progressively develop with the
depolarization of membrane potential, and the spike bursts acquire more
and more action potentials, eventually developing into self-sustained
paroxysmal discharges as in a seizure (Steriade et al.
1993f
).
|
Thus although cortical augmenting responses mainly depend on spike bursts generated by an intrinsic property (the de-inactivation of IT in TC neurons), in brain-intact animals, the cortex has the necessary equipment to develop some forms of augmentation even after thalamectomy. The rich spontaneous firing of neocortical neurons and their preserved synaptic excitability and self-sustained oscillations following internally generated incoming signals during slow-wave sleep, together suggest that this deafferented behavioral state may sustain mental events. Indeed, repeated spike bursts evoked by volleys applied to corticothalamic pathways as well as occurring during spontaneous oscillations may lead to self-sustained activity patterns, resembling those evoked in the late stages of stimulation (Fig. 20B). Such changes are due to resonant activities in closed loops as in "memory" processes.
These data indicate that slow-wave sleep is not associated
with a global annihilation of consciousness as previously assumed (Eccles 1961) but that some mental processes are taking
place in this state. Indeed, recent studies demonstrate that the
overnight improvement of visual-discrimination tasks requires several
steps, some of them depending on the early night slow-wave sleep
(Stickgold et al. 2000
). The significant improvement of
visual discrimination skills by early stages of sleep (associated with
spindles and slow oscillation) led to the conclusion that procedural
memory formation is prompted by slow-wave sleep (Gais et al.
2000
). It was suggested that the massive
Ca2+ entry in cortical pyramidal neurons during
low-frequency sleep oscillations, such as spindling, activates a
molecular "gate," for example mediated by protein kinase A, opening
the door to gene expression and that this process could allow permanent
changes to subsequent inputs following sleep spindles (Sejnowski
and Destexhe 2000
). That sleep spindles may lead to memory
traces in corticothalamic circuits is indeed shown by experiments using
volleys in the frequency range of spindles (Fig. 20B).
Moreover, it is known that dreaming mentation is not confined to REM
sleep but also appears with a different content (more logical, closer
to real life events) in slow-wave sleep (Foulkes
1967
; Hobson et al. 2000
). The recall rate of dreaming mentation in quiet sleep is quite high (Nielsen 2000
). If plasticity, triggered by volleys within the frequency range of sleep oscillations and resembling memory processes in the
corticothalamic circuit (Steriade 1991
) (Fig.
21B), is not constrained by
inhibitory processes, they could develop into seizures (see
Paroxysmal activities developing from sleep oscillations). This is indeed a puzzling development as memory and epilepsy are antinomies. The basic mechanisms of this relation are not fully elucidated yet, but they probably depend on a delicate balance between
excitatory and inhibitory synaptic activities.
|
Paroxysmal activities developing from sleep oscillations
Our current in vivo studies of mechanisms underlying paroxysmal
activities use simultaneous intracellular recordings from two or three
neocortical neurons or from neocortical and thalamic neurons; for
obvious technical reasons, at this time, these experiments are
conducted on anesthetized and paralyzed animals. The major electrographic aspects that reflect the paroxysms under scrutiny are
spike-wave (SW) or polyspike-wave (PSW) complexes recurring at
frequencies between 2 and 4 Hz, often associated with fast runs at
10-15 Hz (Steriade et al. 1998a), thus resembling the EEG pattern of the Lennox-Gastaut syndrome (see Niedermeyer
1999
). In some instances, seizures consisting of pure SW
complexes at 3 Hz occur during the natural state of drowsiness or light
sleep, using extracellular recordings in behaving monkeys (Fig. 21)
(see also Figs. 1-2 in Steriade et al. 1998a
, for
chronic experiments on cats). For the sake of simplicity, I shall term
all these paroxysms SW or SW/PSW seizures. Needless to say, a disease
entity is not just an electrographic pattern, and this is why I think
that the term epileptic seizures should be limited to
clinical studies. What neurophysiologists usually do is find the aspect
that is closest to the clinical case and search for its neuronal
substrates in terms of both intrinsic and network neuronal properties.
Because of the stereotyped pattern of SW complexes, it is likely that the cellular correlates of these seizures in animals are close to those
of corresponding epileptic fits in humans, more so when tonic eyelid
movements are associated with the onset and end of SW seizures (Fig.
21), as in absence epilepsy. Still, I will refrain from calling
epileptic the electrical seizures described in the following
text. Seizure models should be considered distinct from epilepsy models
(Colder et al. 1996
). Regrettably, the term absence (or
petit-mal) epilepsy is used in some experiments conducted in thalamic
slices maintained in vitro.
I shall attempt to define the term seizure because even this simple,
descriptive term is sometime used for forms of rhythmic activity that
are characterized by changed frequencies and amplitudes of oscillations
without however reaching the degree of paroxysms. This is the case with
the slowed spindles induced by bicuculline injections in the thalamus
(Bal et al. 1995a; Steriade and Contreras 1998
) that are not seizures but continuously and
regularly recurring oscillations whose only differences from
barbiturate or natural spindles are the slowed frequency and increased
number of action potentials in spike bursts. I use the term seizure to
describe a transient episode whose electrical signs are in sharp
contrast to the background activity and that, even if it emerges
without apparent discontinuity from the previous sleep-like pattern,
has a sudden end.
In what follows, I shall focus on the site of initiation and cellular mechanisms of SW/PSW seizures. There are many types of SW/PSW seizures. The thalamic or cortical origin of these seizures was, and continues to be, hotly debated. In reality, the corticothalamic system is a unified entity and, although studies on extremely simplified preparations or intact-brain animals pointed to one or another component of this system, experimental studies congruently reached the conclusion that neocortical excitability represents the leading factor in controlling thalamic events during this type of seizures (see following text).
The thalamic (or "centrencephalic") origin of such seizures was
considered since the late 1940s in the light of experiments in which
the medial thalamus was stimulated at 3 Hz (Jasper and Droogleever-Fortuyn 1949). In that study, only SW-like
responses were evoked in cortex but no self-sustained
activity. It is difficult to envisage the presence of a centrencephalic
system as there are no bilaterally projecting thalamic neurons, and, on
the other hand, brain stem core neurons with generalized projections
disrupt, rather than produce, SW seizures (Danober et al.
1995
). The conventional feature of clinical SW seizures, which
was at the basis of the hypothesis pointing to a deeply located
pacemaker, is their suddenly generalized appearance. This might be so
on EEG recordings, but topographical analyses of SW complexes in humans
show that the "spike" component propagates from one hemisphere to
another with time lags as short as 15 ms (Lemieux and Blume
1986
), which cannot be estimated by visual inspection. Earlier
EEG studies and toposcopic analyses have also indicated that some SW
seizures are locally generated and result from multiple, independent
cortical foci (Jasper and Hawkes 1938
; Petsche
1962
). This explains why absence seizures are less detrimental
than grand-mal epilepsy that implicates more widespread neuronal
manifestations. Experiments using multi-site, extra- and intracellular
recordings show that neocortical neurons become progressively entrained
into the seizures, indicating that the buildup of SW seizures obeys the
rule of synaptic circuits, sequentially distributed through short- and
long-range circuits, with time lags as short as 15-20 ms, but also
longer (100-150 ms), the latter being ascribable to inhibition-rebound
sequences (Steriade and Amzica 1994
). This aspect stands
in contrast to the usual definition of "suddenly generalized,
bilaterally synchronous" discharges, ascribed to SW seizures.
Since the earlier concept of thalamically generated SW seizures, views
have changed and another hypothesis proposed that sleep spindles
develop into SW seizures because of an enhanced excitability of
neocortical neurons (Gloor et al. 1990). This was closer
to reality as the major role in the induction of SW seizures was ascribed to the increased excitability of the neocortex. Although SW
seizures may occur in thalamectomized animals, in which spindles are
absent (Steriade and Contreras 1998
), in the intact
brain, spindles might lead to SW seizures. One likely possibility is that sleep spindles are prevalently linked with the occurrence of SW
seizures in humans (Kellaway 1985
), whereas the slow
sleep oscillation distinctly leads to patterns resembling the
Lennox-Gastaut syndrome or hypsarrhythmia (Steriade and
Contreras 1995
; Steriade et al. 1998a
; reviewed
in McCormick and Contreras 2001
).
That focal SW seizures are initiated in circumscribed pools of neurons
within the cortex was initially suggested on the basis of typical SW
complexes at ~3 Hz occurring in the depth of monkey's cortex, even
without reflection at the cortical surface, thus suggesting the
involvement of a local pool of short-axoned interneurons (see Fig. 8 in
Steriade 1974). Seizures with SW/PSW complexes can also
be elicited by electrical stimulation in completely isolated neocortical slabs in vivo (Timofeev et al. 1998
). Thus
the SW/PSW seizures are initiated in neocortex, and multi-site
recordings show that they spread from cortex to thalamus after a few
seconds (Neckelmann et al. 1998
) (Fig.
22).
|
The cortical and thalamic mechanisms underlying different components of
SW/PSW seizures have been investigated using multi-site, including dual
intracellular, recordings in cats under ketamine-xylazine anesthesia.
In some instances, we recorded bicuculline-induced paroxysms, but we
mainly investigated spontaneously occurring and electrically induced
seizures in the absence of any convulsing substance. The high incidence
of spontaneous SW/PSW seizures in acutely prepared cats under
ketamine-xylazine anesthesia (30-50% of animals) is explained by the
highly synchronized corticothalamic activity produced by this
anesthetic in conjunction with the great number of recording and
stimulating macroelectrodes that are inserted for the identification of
input-output organization of neurons. It is known that repeated
electrical stimulation is a favorable factor for the occurrence of
seizures. A similar factor (stimuli applied for physiological
identification of neuronal inputs and targets) is likely responsible
for the appearance of spontaneous SW seizures in chronic experiments on
monkeys (Steriade 1974) and cats (Steriade et al.
1998a
). In view of this high incidence of spontaneous SW/PSW
seizures, it is likely that many SW seizures occur in "normal"
subjects during drowsiness or slow-wave sleep, and they are not
recognized as such as these paroxysms may develop without discontinuity
from the slow sleep oscillation (Steriade et al. 1998a
)
and stimulation within the frequency range of sleep oscillations leads
to SW/PSW seizures (Amzica and Steriade 1999
).
Dual simultaneous intracellular recordings from the cortex and
thalamus, in vivo, show that seizures consisting of SW/PSW complexes at
2-3 Hz, often associated with fast runs (10-15 Hz) originate in the
neocortex. Simultaneously, most (60%) TC neurons display a steady
hyperpolarization as well as phasic IPSPs, closely related to the spike
component of cortical SW complexes (Fig. 23A). At the end of the
cortical seizure, TC neurons fire at high rates as if they were
released from the inhibition that occurred during the seizure (the
source of inhibition is in GABAergic RE neurons). These SW seizures
develop, often without discontinuity, from preceding periods of
sleep-like patterns. Indeed, the phase relations between cortical and
thalamic neurons during sleep are preserved during seizures, but the
amplitude of membrane excursions are accentuated (Fig. 23B).
The similar relations between field potential and intracellular
activities during slow-wave sleep and SW/PSW seizures are due to
similar mechanisms that account for the depolarizing/hyperpolarizing
components of the slow sleep oscillation, on one hand, and the
spike/"wave" components of SW complexes, on the other hand (see
below). Although these data point to cortically initiated SW seizures
and to the steady hyperpolarization in a majority of thalamocortical
neurons during such seizures, the remaining TC neurons are capable of
firing rebound spike bursts during a cortical SW seizure
(Steriade and Contreras 1995) and thus may potentiate
and disseminate cortical seizures. Thus our data point to the
intracortical origin of these seizures. After a few seconds,
they irradiate to the ipsilateral thalamus (Fig. 22) as well as to
other subcortical structures.
|
Subsequent research by other teams (Pinault et al. 1998;
Slaght et al. 2000
) using intracellular recordings from
TC neurons during spontaneous SW discharges in a genetic model of
absence epilepsy in rats similarly demonstrated that the main events
which characterize the activity of an overwhelming majority (>90%) of TC neurons are a tonic hyperpolarization, present throughout the SW
seizure, and rhythmic IPSPs. The results of these in vivo study emphasized that the intracellular activity of TC neurons during SW
seizures does not involve rhythmic sequences of
GABAB receptor-mediated IPSPs, but
GABAA-mediated IPSPs as they appeared as
depolarizing events when recorded with KCl-filled pipettes. The origin
of the GABAA-mediated IPSPs in TC neurons should
be searched for in the GABAergic RE neurons that faithfully fire spike
bursts during SW seizures in response to each paroxysmal depolarizing
shift of cortical neurons (Steriade and Contreras 1995
;
Timofeev et al. 1998
). Modeling studies show that
increasing the inhibitory strength from GABAergic RE neurons onto TC
neurons favors the quiescent mode of the latter (Lytton et al.
1997
). The cortically induced inhibition of TC neurons during
SW seizures, mediated by GABAergic RE neurons, was recently
corroborated by demonstrating that EPSCs elicited in RE neurons by
minimal stimulation of corticothalamic axons are ~2.5 times larger
than in TC neurons and GluR4 receptor subunits in RE neurons outnumber
those in TC neurons by 3.7 times (Golshani et al. 2001
).
These data corroborate those in intact-brain animals showing that
stimulation of corticothalamic projections evokes a strong excitation
in RE cells, in parallel with prolonged IPSPs in TC cells that are due
to the activation of GABAergic RE cells (see Fig. 1 in Steriade
2000
).
During cortically generated seizures in vivo, neocortical neurons
display a progressive depolarization on which SW complexes are
superimposed, with paroxysmal spike bursts correlated with the EEG
spike component and hyperpolarization correlated with the EEG wave
component (Steriade and Contreras 1995; Steriade et al. 1998a
). Eventually, the progressive depolarization leads to a state of tonic depolarization accompanied by fast (10-15 Hz) runs
(Fig. 24). The fast runs, which
constitute the polyspike component of PSW complexes, are also generated
intracortically as they are unaffected by thalamic inactivation using
tetrodotoxin (Castro-Alamancos 2000
). This picture
characterizes SW/PSW seizures recorded close to the initial focus. When
SW seizures are recorded far (5-10 mm) from the cortical site where
the seizures were initiated, the large depolarizing envelope may be
absent, but basically the same features are observed: development from
the slow sleep oscillation, even without discontinuity, and SW or PSW
complexes at ~2 Hz, interrupted by short periods when fast runs
(10-15 Hz) occur. While RS neurons fire single action potentials
during the fast runs, FRB neurons fire high-frequency bursts during the
fast runs (Steriade et al. 1998a
). Simultaneous
intracellular recordings from neurons and glial cells show, on one
hand, that the glial membrane potential displays negative events
related to the onset of paroxysmal depolarizing shifts (PDSs) in
cortical neurons and, on the other hand, that the maximal glial
depolarization is reached later than the end of the neuronal
depolarization (Fig. 25). This may
control the pace of paroxysmal oscillations (Amzica and Steriade 2000
).
|
|
Here, an interesting similarity between TC and some neocortical neurons should be mentioned. Although neocortical neurons generally display a tonic depolarization during SW/PSW seizures (Figs. 23-25), whereas most TC neurons are tonically hyperpolarized during these paroxysms (Fig. 23), due to summated IPSPs from RE neurons, we also observed seizures associated with an exclusively hyperpolarizing envelope in neocortical neurons. In such cases, the hyperpolarization of cortical neurons, which lasts throughout the seizure, is initiated from the very onset of the paroxysm, being coincident with the sharp depth-negative EEG spike that reflects summated PDSs in cortical neurons and gives rise to seizures consisting of fast events, 10-20 Hz, followed by PSW complexes at 2 Hz (Fig. 26A). During this prolonged hyperpolarization, cortical neurons exhibit voltage excursions that are synchronous with the SW field activity at 2 Hz but that only rarely reach the firing threshold. The input resistance is dramatically decreased during the hyperpolarizing periods associated with SW/PSW seizures (Fig. 26B). This suggests that such cortical neurons, displaying an exclusive and prolonged hyperpolarization during SW/PSW seizures, are the prevalent targets of local inhibitory neurons. The hyperpolarization of some RS neocortical cells during seizures is homologous to that of TC neurons that are targets of GABAergic RE neurons themselves driven by corticothalamic neurons during SW/PSW seizures.
|
In previous studies on neocortex and hippocampus, the PDS component was
commonly regarded as a giant EPSP (Ayala et al. 1973; Johnston and Brown 1981
, 1984
). While this is partially
true, the PDS also contains GABAA-mediated
inhibitory processes. The role of inhibitory processes in the genesis
of cortical SW complexes was studied in our laboratory using
intracellular recordings and measurements of membrane conductance in
vivo. Briefly, neocortical neurons show a maximal conductance during
the PDS component (EEG spike) of SW complexes and a significantly lower
conductance during the hyperpolarization related to the EEG wave
(Neckelmann et al. 2000
). The difference between the
increased membrane conductance during the PDSs of SW/PSW seizures and
the lower membrane conductance during the wave component of these
paroxysms is supported by the differential intracellular responsiveness
to antidromic and synaptic volleys during these two components of SW
seizures (Steriade and Amzica 1999
). Thus synaptic
responses leading to PDSs can be elicited during the wave
(hyperpolarizing) component of SW complexes and antidromic responses
can also be evoked by steadily depolarizing the neuron during the wave
component, whereas antidromic responses, followed by orthodromic
responses, can only be evoked during the declining (repolarizing) phase
of the spike (PDS) component (Fig. 27).
|
A great part of the increased conductance during the EEG spike (PDS) is
due to an important inhibitory component, as recordings with
Cl-filled pipette reveal depolarizing shifts by
15-30 mV during this part of SW seizures and conventional FS
(presumably local inhibitory) neurons fire at very high rates (500-800
Hz) during the PDSs of SW/PSW complexes; on the other hand, the wave
component of SW seizures is partly due to K+
currents, as shown using recordings with
Cs+-filled pipettes (Steriade et al.
1998c
; I. Timofeev, F. Grenier, and M. Steriade, in
preparation). These and the preceding data on measurements of membrane
conductance (Neckelmann et al. 2000
) indicate that the
major mechanism underlying the wave-related hyperpolarization of SW
seizures does not mainly rely on active GABAergic inhibition as
suggested in many previous studies, but on a mixture of disfacilitation
and K+ currents. Similar mechanisms
(disfacilitation and K+ currents) underlie the
hyperpolarizing component of the slow sleep oscillation as indicated by
measuring the input resistance of cortical neurons (Contreras et
al. 1996
) and recordings with Cs+-filled
pipettes (Steriade et al. 1993e
). This is one of the
factors that explain similar relations between field potential and
intracellular activities during slow sleep oscillation and SW seizures,
the latter developing from the former (see preceding text and Fig. 24B).
To sum up, the aforementioned data lead to the conclusion that SW/PSW seizures are initiated in the cortex, that subsequently they spread to the thalamus, and that a majority of TC neurons are steadily hyperpolarized and display phasic IPSPs in close time relation with cortical PDSs that excite GABAergic RE neurons. The inhibition of TC neurons during SW seizures, and their inability to relay signals from the external world, may contribute in humans to the period of unconsciousness that is associated with petit-mal epilepsy.
The results of in vivo experiments showing that the neocortex is the
leading factor in the generation of SW seizures, that most TC neurons
are silent during these seizures, and that GABAergic RE neurons (driven
by corticothalamic paroxysmal inputs) are responsible for the steady
hyperpolarization and phasic IPSPs in TC neurons (Steriade and
Contreras 1995), were corroborated by in vivo experiments conducted in a genetic model of SW seizures (Slaght et al.
2000
). The same concept is now supported by a series of studies
in slices. Thus corticothalamic stimulation induces bursting at 3 Hz in
thalamic neurons; however, following the removal of cortex, such bursts can no longer be evoked in the thalamus (Kao and Coulter
1997
). In mouse thalamocortical slices, prolonged paroxysmal
depolarizing potentials elicited by
GABAA-receptor antagonists were present in cortex
isolated from the thalamus but not in thalamus isolated from the cortex
(Golshani and Jones 1999
). Finally the idea that corticofugal volleys are decisive in the induction of paroxysmal thalamic activity at 3-4 Hz is now confirmed in work conducted in
vitro (Bal et al. 2000
; Blumenfeld and McCormick
2000
) in contrast to the previous hypothesis that thalamic
networks are alone implicated in the genesis of SW seizures.
Concluding remarks
Several lines of evidence point to the powerful effects exerted by network synaptic activity on intrinsic neuronal properties of neocortical and thalamic neurons. Firing patterns of cortical neurons, as elicited by depolarizing current pulses, are transformed from one type to another with changes in Vm and increased synaptic volleys during shifts in the level of vigilance from disconnected to activated behavioral states. Basic intrinsic properties of thalamic neurons, such as the Ca2+-dependent LTS, are overwhelmed by barrages of PSPs in ascending and descending pathways, and local thalamic oscillations are not exclusively generated by intrinsic properties of TC neurons but rather by long-range synaptic connections involving the pacemaking GABAergic RE neurons. Instead of pure, simple rhythms generated in circumscribed territories, as found in simplified in vitro preparations, the global electrical activity of intact brains in living animals displays complex wave sequences consisting of grouped oscillations consisting of both low- and fast-frequency rhythms due to corticothalamic interactions under the control of generalized modulatory systems. The cerebral cortex controls the shape and synchronization patterns of thalamic neurons during both normally developing and paroxysmal activities. All this complexity of global operations requires investigation in intact-brain preparations.
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ACKNOWLEDGMENTS |
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I thank the following PhD students and postdoctoral fellows for skillful and creative collaboration in experiments performed in recent years (in alphabetical order): F. Amzica, D. Contreras, R. Curró Dossi, F. Grenier, D. Neckelmann, A. Nuñez, D. Paré, and I. Timofeev. Collaboration with T. J. Sejnowski, A. Destexhe, W. W. Lytton, and M. Bazhenov was instrumental in computational studies. The assistance of P. Giguère was essential for the technical development of my laboratory.
Personal experiments discussed in this article are supported by grants from the Canadian Institutes for Health Research (MT-3689 and MOP-36545), Natural Sciences and Engineering Research Council of Canada (170538), Human Frontier Science Program (RG0131), and National Institute of Neurological Disorders and Stroke (1-R01 NS-40522-01).
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FOOTNOTES |
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Author E-mail: mircea.steriade{at}phs.ulaval.ca.
Received 20 November 2000; accepted in final form 7 March 2001.
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REFERENCES |
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