Department of Psychology, University of Colorado, Boulder, Colorado 80309-0345
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ABSTRACT |
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Sukov, William and Daniel S. Barth. Cellular Mechanisms of Thalamically Evoked Gamma Oscillations in Auditory Cortex. J. Neurophysiol. 85: 1235-1245, 2001. The purpose of this study was to clarify the neurogenesis of thalamically evoked gamma frequency (~40 Hz) oscillations in auditory cortex by comparing simultaneously recorded extracellular and intracellular responses elicited with electrical stimulation of the posterior intralaminar nucleus of the thalamus (PIL). The focus of evoked gamma activity was located between primary and secondary auditory cortex using a 64-channel epipial electrode array, and all subsequent intracellular recordings and single-electrode field potential recordings were made at this location. These data indicate that PIL stimulation evokes gamma oscillations in auditory cortex by tonically depolarizing pyramidal cells in the supra- and infragranular layers. No cells revealed endogenous membrane properties capable of producing activity in the gamma frequency band when depolarized individually with injected current, but all displayed both sub- and supra-threshold responses time-locked to extracellular fast oscillations when the population was depolarized by PIL stimulation. We propose that cortical gamma oscillations may be produced and propagated intracortically by network interactions among large groups of neurons when mutually excited by modulatory input from the intralaminar thalamus and that these oscillations do not require specialized pacemaker cells for their neurogenesis.
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INTRODUCTION |
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Brain activation, as
registered in the extracranial electroencephalogram (EEG), is typically
regarded as a state in which the electrical activity of large neural
networks is desynchronized compared with higher-amplitude rhythmic EEG
patterns thought to characterize the cerebral cortex when not actively
engaged in information processing. However, these conclusions have been
recently challenged by results obtained from intracranial measurements, indicating the presence of locally synchronous oscillations in the
gamma frequency band (~40 Hz) associated with vigilant states (Franken et al. 1994; Hamada et al. 1999
;
Jones and Barth 1997
) and with sensory stimulation
(Basar and Bullock 1992
; Eckhorn et al.
1988
; Franowicz and Barth 1995
; Freeman
1978
; Jones and Barth 1997
; MacDonald and
Barth 1995
; Singer and Gray 1995
). Fast rhythms
appear as a substantial part of the background intracranial EEG and are
synchronized in both intracortical and thalamocortical networks during
activation induced by stimulation of the mesopontine cholinergic nuclei
or by behavioral tasks in chronic experiments (Bouyer et al.
1981
; Canu et al. 1994
; Steriade et al.
1993b
, 1996a
,b
).
While gamma oscillations appear to reflect transient synchronization of
neural populations during information processing, their cellular
mechanisms are still being elucidated. Several recent reports have
implicated specific subtypes of cortical neurons with endogenous
membrane properties that may produce sub- and suprathreshold activity
in the gamma frequency band when sufficiently depolarized (Gray
and McCormick 1996; Llinás et al. 1991
;
Silva et al. 1991
; Steriade et al. 1998
),
suggesting that dedicated cells may serve as the neural generators or
pacemakers of gamma oscillations in sensory cortex. However, both
computer modeling (Lumer et al. 1997
; Lytton and
Sejnowski 1991
; Traub et al. 1999
) and in vitro
physiological investigations (Buhl et al. 1998
;
Plenz and Kitai 1996
; Whittington et al.
1995
) have demonstrated that gamma oscillations may also appear
as an emergent property within networks of mutually connected and
tonically excited inhibitory and excitatory neurons without the
requirement of specialized cells for gamma neurogenesis.
In vivo studies of the rodent reveal gamma oscillations in a focal
region of ventrotemporal cortex, overlapping primary and secondary
auditory zones, that occur spontaneously (Brett and Barth
1996; Franowicz and Barth 1995
; MacDonald
and Barth 1995
; MacDonald et al. 1996
) and in
response to sensory stimulation (Franowicz and Barth
1995
; MacDonald and Barth 1995
). These
oscillations may also be reliably evoked by electrical stimulation of
the posterior intralaminar nucleus of the thalamus (PIL) (Barth
and MacDonald 1996
; Brett and Barth 1997
;
Sukov and Barth 1998
). While these studies were
conducted in ketamine anesthetized animals, where both spontaneous and
evoked gamma oscillations may be enhanced, we have used PIL stimulation
in this preparation to study the spatiotemporal organization of gamma
oscillations in large neuronal networks at the extracellular level,
recording from high spatial resolution multi-electrode arrays placed on
the cortical surface (Barth and MacDonald 1996
;
Brett and Barth 1997
) and linear electrode arrays
spanning the cortical laminae (Sukov and Barth 1998
).
The present study used PIL stimulation to investigate the cellular mechanisms of thalamically evoked gamma oscillations in auditory cortex
by relating measurements of extracellular field potentials to
simultaneously recorded intracellular responses in the ketamine anesthetized rat. Our specific objectives were to identify cells in
specific cortical laminae that participate in evoked gamma oscillations, to characterize participating cells using depolarizing current injection to determine if they define a specific cell class
displaying endogenous membrane properties conducive to oscillations in
the gamma frequency range, and to determine the influence PIL inputs
have on cortical neurons that gives rise to the evoked gamma response.
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METHODS |
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Surgical preparation
All procedures were performed in accordance with University of Colorado Institutional Animal Care and Use Committee guidelines for the humane use of laboratory animals in biological research. Male Sprague-Dawley rats (250-350 g) were anesthetized to surgical levels using intramuscular injections of ketamine HCl (100 mg/kg) and xylazine (25 mg/kg) and secured in a stereotaxic frame. A unilateral craniotomy extending from bregma to lambda and lateral to the temporal bone was performed over the right hemisphere, exposing a wide region of parietotemporal cortex from which the dura was reflected. The exposed cortical surface was regularly bathed with physiological saline and body temperature maintained using a regulated heating pad. Additional ketamine and xylazine were administered as required to maintain a level of anesthesia such that the corneal reflex could barely be elicited. Animals were killed without regaining consciousness by anesthesia overdose at the conclusion of the experiment.
Stimulation
Auditory click stimuli were presented using a high-frequency piezo-electric speaker placed ~15 cm lateral to the contralateral ear. Clicks were generated by computer-controlled monophasic square-wave pulses (0.3 ms), which were shown in previous studies to activate most of auditory cortex in the rat. Subcortical electrical stimulation consisted of 500-ms trains of current pulses (10-15 µA; 0.5-ms pulse-width; 1,000 Hz) delivered with a stainless steel bipolar electrode positioned in the PIL (4.8 mm posterior to bregma, 3.0 mm lateral to midline, 6.4 mm ventral to the cortical surface).
Field potential recording
Epipial maps of the auditory evoked potential (AEP) complex were
recorded using a flat multi-channel electrode array consisting of 64 silver wires arranged in a 8 × 8 grid (tip diameter: 100 µm;
inter-electrode spacing: 500 µm) covering a 3.5 × 3.5 mm area of the cortical surface in a single placement (see Fig.
1A). Click stimulation resulted in a highly repeatable averaged surface response (n = 100) whose spatial distribution was used to
consistently position the electrode array across animals. Epipial
potentials were referenced to a silver ball electrode secured over the
contralateral frontal bone and were simultaneously amplified (×1,000),
analog filtered (band-pass cutoff = 6 dB at 0.001-5000 Hz,
roll-off = 5 dB/octave) and digitized at 10 kHz. Following AEP
mapping, the surface response during electrical stimulation of the PIL was recorded to determine the locus of maximum-evoked gamma
oscillations. The electrode array was then removed, and a small
Plexiglas stabilizing plate was brought in contact with the pia that
contained a 0.5-mm access hole centered over the region of PIL-evoked
gamma oscillations for subsequent intracellular recording. A single
silver-wire electrode was mounted in the immediate periphery of the
access hole to simultaneously monitor evoked responses at the cortical
surface.
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Intracellular recording
Intracellular recordings were obtained using glass micropipettes
pulled from thin-walled (1.0 mm) aluminosilicate glass (tip diameter = 0.05 µm). In most penetrations, electrodes were
filled with 2.0 M K+-acetate so that both action
potentials (APs) and postsynaptic potentials (PSPs) could be recorded.
In some penetrations, electrodes were filled with 100 mM lidocaine and
N-ethyl bromide quaternary salt (QX-314; Research
Biochemicals, Natick, MA) in 2.0 M K+-acetate
that was iontophoretically ejected into the cell by applying 100-ms
depolarizing pulses of 2.0 nA at 2.0 Hz until all APs were blocked
(Connors and Prince 1982). The in vivo impedance of
electrodes ranged from 110 to 140 M
. Recording and current injection
was performed using an Axoclamp 2-A amplifier (Axon Instruments)
equipped with a 0.1 gain head-stage (Axon Instruments Model HS-2A).
Micropipettes were advanced perpendicularly into the cortex in 0.5-µm
increments (100 mm/s) using a piezo translator compensated with a motor
drive (Märzhäuser PM-10) equipped with a micrometer which
indicated the depth of the electrode tip. Criteria for an acceptable
cell impalement were a resting membrane potential of at least
60 mV maintained for
30 min required to collect single trial data and obtain satisfactory characterization of the cell using depolarizing current pulses (McCormick et al. 1985
) and action
potential heights of
70 mV and half-amplitude action potential widths
of no more than 2.0 Ms. In cells receiving QX-314, characterization was
performed immediately on penetration prior to AP blockade. In cells not receiving QX-314, characterization was performed after all other responses had been recorded. Hyperpolarizing currents up to
0.5 nA
were applied on initial cell penetration and were maintained for ~5
min, until the membrane potential stabilized. All subsequent recordings
were performed with no hyperpolarizing holding currents. When a stable
cell impalement was obtained, 1-s trials (n = 50-100) were recorded during PIL stimulation (250-ms baseline followed by
500-ms stimulus train). Intracellular and surface records were low-pass
filtered at 3 kHz (
6 dB at 3 kHz, roll-off = 5 dB/octave), digitized at 10 kHz, and stored for subsequent analysis.
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RESULTS |
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Figure 1 depicts methods for mapping the AEP complex at the
cortical surface to determine the locations of primary (area 41) and
secondary (areas 36 and 20) (Krieg 1946) auditory cortex
prior to intracellular recording (Fig. 1A). The AEP in all
animals began with a positive/negative fast wave (Fig. 1C;
P1/N1) that was of largest amplitude and shortest poststimulus latency
over area 41 (Fig. 1C, solid trace; Fig.
1B), and smaller amplitude with a delay of ~5 ms over area
36 (Fig. 1C, dashed trace; Fig. 1B). This spatial and temporal pattern was similar to previous studies (Barth and Di 1990
) and permitted consistent alignment
of the electrode array across animals.
Similar to the AEP, electrical stimulation of the PIL also produced an
initial biphasic response at the cortical surface. However, this was
followed by large-amplitude (~0.5 mV) oscillations in the gamma
frequency band [37.9 ± 0.16 (SE) Hz, n = 15]
that lasted the duration of the 0.5-s stimulus train (Fig. 1,
D and E). As reported in previous studies
(Barth and MacDonald 1996; Brett and Barth
1997
; Sukov and Barth 1998
), gamma oscillations evoked by PIL stimulation were typically superimposed on a negative offset [
0.2 ± 0.08 (SE) mV, n = 15] compared
with the prestimulus baseline (Fig. 1E). While evoked gamma
oscillations were evident in all areas of auditory cortex, they were of
largest amplitude in a region straddling the border of areas 41 and 20 (Fig. 1D, dashed box), slightly caudal to the locus
of the maximum amplitude of the AEP. Following surface mapping, the
array was removed and the location of maximum evoked gamma oscillations
stereotaxically targeted for subsequent single channel intra- and
extracellular recording.
Stable intracellular recordings were obtained from 46 cells in 21 animals. Most of these cells (n = 45) characterized as
regular spiking (RS). A cell was classified as RS if in response to the injection of a depolarizing current pulse (0.5-1.0 nA; 500 ms), it
fired a train of APs exhibiting strong spike frequency adaptation with
half-amplitude AP width >0.5 ms [average half-amplitude spike width
was 1.2 ± 0.2 (SD) ms; average spike height was 78 ± 12 (SD) mV] (Connors and Gutnick 1990; McCormick et
al. 1985
). One cell was classified as a fast spiking (FS)
neuron. This classification was based on its distinctly narrow APs
(<0.5-ms half-amplitude width) (Connors and Gutnick
1990
; McCormick et al. 1985
; Simons 1978
; Simons and Woolsey 1979
) and capacity for
very high-frequency spontaneous and evoked AP discharge (>200 Hz)
(Jones et al. 2000
; Simons 1978
;
Simons and Woolsey 1979
). No cells displayed firing patterns typical of intrinsic bursting (IB) cells during current injection. However, while all cells receiving QX-314 characterized as
RS cells, their identity cannot be established with absolute certainty
due to possible subtle effects of QX-314 immediately on penetration. RS
cells were distributed uniformly throughout the supra
(n = 21)- and infragranular (n = 24)
layers. The FS cell was located at a depth of 550 µm below the
cortical surface.
All RS cells responded to PIL stimulation with a steady depolarization
[+13.3 ± 1.8 (SE) mV] from resting membrane potential (Fig.
2B). This began abruptly at
stimulus onset, remained approximately flat for the 500-ms stimulus
duration, and usually required 50-200 ms to return to baseline
following the stimulus. Of the 26 RS cells not receiving QX-314, 22 displayed multiple APs [6.2 ± 0.98 (SE)] per trial during
PIL-evoked depolarization (the first 50 ms following stimulus onset,
associated with the extracellular P1/N1 wave, were excluded from
analysis). Visual examination of individual trials suggested that the
APs of a given cell maintained a consistent phase relationship with the
evoked gamma oscillations recorded at the cortical surface (Fig.
2A). This relationship was examined by computing cumulative
time histograms (CTH) of AP latency time-locked to successive gamma
waves in the surface record. The positive amplitude peaks of surface
gamma waves were determined automatically using a peak-seeking
algorithm (Barth and MacDonald 1996; Sukov and
Barth 1998
) and used as a reference for computing time-locked
average extracellular gamma oscillations and associated CTH. To limit
noise, only gamma waves exceeding 1 SD from the mean, computed across
all single trials for a given animal, were included (Fig.
2A;
). Figure 2C depicts a single cycle of
time-locked gamma oscillation averaged in this way (n = 1,059 gamma waves) and superimposed on the associated CTH
(n = 992 APs) for an RS cell located at a depth of 950 µm below the cortical surface. The averaged extracellular gamma
oscillation is displayed as a solid trace peaking at 0.0 ms, and again
as a dashed trace, shifted to align with peaks of the CTH for
comparison. The CTH indicated that APs were tightly time-locked to the
surface oscillations with a similar periodicity. The cross-correlation function between the averaged gamma oscillation and CTH (Fig. 2D) revealed a dominant period of 24 ms (42 Hz), APs delayed
by 8 ms relative to the positive amplitude peak of the surface
response, and a maximum cross-correlation coefficient
(
xy) of 0.96 (P < 0.01; based on the
Fisher z transform of
xy referred to a normal distribution) (Otnes and Enochson 1978
). Figure 2,
E-H, shows results for two additional cells at depths of
110 and 800 µm, respectively, indicating similar periodicity and
phase-locking between the surface and intracellular records and
suggesting no laminar specificity to this relationship. In all, 17 of
the 22 RS cells that produced APs in response to stimulation of the PIL did so in a periodic fashion that was significantly correlated with
extracellular gamma oscillations (
xy = 0.51-0.96;
P < 0.01; Table 1). The
laminar distribution of correlated cells was uniform with no apparent
predominance in either the supragranular or infragranular layers. Time
lags between the CTH and averaged gamma oscillations were generally
earlier in the supragranular (
4 ms) compared with the infragranular
layers (+1.1 ms) layers, but this difference was not significant
(P > 0.05).
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Figure 3 depicts FS cell responses during
PIL stimulation. Similar to RS cells, the FS cell responded with a
steady depolarization but of lower amplitude (~6.0 mV) and building
slowly throughout the stimulation period. Superimposed on the steady
depolarization were trains of thin (<0.5 ms) APs that were far more
numerous per trial than those observed in RS cells (Fig.
3B). While observation of raw records suggested that APs
were equally probable at all phases of the averaged surface gamma
oscillations (Fig. 3, A and B), the CTH indicated
a distinct periodicity that was time-locked to the averaged surface
gamma oscillation (Fig. 3C) with a significant correlation
(xy = 0.64; P < 0.01) at a lag of
7 ms.
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During most trials, depolarizing steady potentials in RS cells during
PIL stimulation were also accompanied by smaller-amplitude (~5-10
mV) deflections, suggestive of synchronized postsynaptic potentials
(PSP) recordable at the soma (Fig.
4A; dashed box). To examine
the relationship between these subthreshold potentials (STP) and
extracellular gamma oscillations, each trace was median-filtered (Fig.
4B) and digitally smoothed (Fig. 4C).
Median-filtering is a nonlinear digital technique that was used in the
present context to truncate APs, leaving an approximation of the
depolarizing potentials that trigger them (Gray and McCormick
1996). This method has little effect on the morphology of STPs
and permits combined averaging without distortion due to APs. STPs,
derived in this way, appeared phase-locked to surface gamma
oscillations in the raw record (Fig. 4D). This was confirmed
by computing averaged STPs, time-locked to the peaks of surface gamma
waves (Fig. 4E). The data shown in Fig. 4 are from the same
cell (c950) depicted in Fig. 2, A-D. The
averaged STP (Fig. 4E; dark trace) maintained an
8-ms delay in relationship to the averaged gamma oscillation (Fig.
4E; light trace) with a cross-correlation
function (Fig. 4F; solid trace) that was nearly
identical to that computed for the CTH alone in this cell (Fig.
4F; dashed trace). The averaged STPs of 24 cells
were significantly correlated with averaged extracellular gamma
oscillations (
xy = 0.51-0.96; P < 0.01; Table 1). Seventeen of these cells also had CTHs that were
significantly correlated with extracellular gamma oscillations (Table
1). Similar to the CTH, time lags between the averaged STPs and
averaged gamma oscillations were earlier in the supragranular (
1.27
ms) compared with the infragranular layers (+2.38 ms) layers; but,
again, this difference was not significant (P > 0.05).
However, phase lags of the CTH and averaged STPs in these cells were
significantly correlated (rxy = 0.96;
P < 0.01).
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The previous analysis averaged true STPs with those derived from
suprathreshold events by median-filtering. The necessary inclusion of
derived STPs in the analysis no doubt influenced the similarity between
temporal characteristics of these averages and previously computed CTHs
in the same cells. However, two cells (Table 1; c70 and
c460) produced no APs during PIL stimulation. Yet they still
displayed averaged STPs that were significantly correlated with surface
gamma oscillations, suggesting that STPs are produced by synchronized
postsynaptic currents and are not dependent on AP discharge in a given
cell. To further explore this possibility, 19 additional cells were
injected with QX-314 to block APs during recording. Figure
5 shows a particularly interesting example because in this cell the QX-314 took effect gradually, making
it possible to study both APs and subsequent STPs during evoked gamma
oscillations in the same cell. Within ~30 s after cell penetration,
APs were already of reduced amplitude (Fig. 5B). Their peaks
appeared to be aligned with the troughs of gamma oscillations in the
surface record, observable on individual trials (Fig. 5A),
and in the CTH (Fig. 5C) and cross-correlation function (Fig. 5D). After ~10 min, the QX-314 had completely
suppressed all APs. STPs were still clear in the raw record (Fig.
5F) and appeared time-locked to the evoked gamma
oscillations at the surface (Fig. 5E). Time-locked averaging
(Fig. 5G) and cross-correlation (Fig. 5H)
indicated a common periodicity and timing between STPs and
extracellular gamma oscillations that was nearly identical to that of
APs before their suppression. Analysis of the remaining 18 cells with
QX-314 was performed after complete AP blockade. All responded to PIL
stimulation with depolarizing shifts [14.2 ± 2.1 (SE) mV] and
17 had averaged STPs significantly correlated with surface gamma
oscillations (xy = 0.56-0.95; P < 0.01; Table 2).
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While PIL stimulation consistently produced steady depolarizing intracellular potentials accompanied by both APs and STPs time-locked to extracellular gamma oscillations, the injection of intracellular current did not produce noticeable sub- or suprathreshold activity in the gamma frequency range. RS cells generated trains of APs with frequencies <100 Hz at large (1.0 nA) levels of current injection and with marked frequency adaptation occurring within the first 100 ms of current onset (Fig. 6A). AP frequency varied with the level of depolarizing current and showed no apparent preference for gamma frequencies, but this was not systematically studied. However, PIL stimulation during smaller current injections (0.75 nA) typically produced an increase in AP frequency with concurrent time-locking of the AP to surface-evoked gamma oscillations and little further depolarization beyond that produced by the injected current (Fig. 6B). When APs were blocked with QX-314, current injections produced equivalent depolarizations but without consistent STPs (Fig. 6C). The occurrence of STPs in these cells invariably coincided with spontaneous surface extracellular gamma oscillations or with PIL-evoked gamma oscillations, suggesting that they were not produced by endogenous oscillatory properties of the depolarized membrane but were instead exogenously derived PSPs, accentuated by movement of the membrane potential away from resting levels during current injection.
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DISCUSSION |
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The PIL is part of a system of intralaminar and midline thalamic
nuclei that provide what have been considered to be nonspecific projections to the cortex (Herkenham 1986; Jones
1985
), a designation inspired by their widespread afferent
input and cortical projection fields (Macchi and Bentivoglio
1986
). However, improved tracing methods have revealed that the
projection fields of certain intralaminar nuclei in the rodent,
including the PIL, are far more specific than previously thought
(Berendse and Groenewegen 1991
; Linke 1999
). The PIL may be considered to be a distinct part of the ascending auditory pathway in the rat, receiving its major afferent input from the inferior colliculus (Arnault and Roger
1987
; Ledoux et al. 1987
) and sharing reciprocal
connections with the ventrotemporal cortex (Arnault and Roger
1987
, 1990
; Linke 1999
). Maps of the epipial
response to electrical stimulation of the PIL presented here replicate
earlier findings (Barth and MacDonald 1996
; Brett and Barth 1997
; Sukov and Barth 1998
) and
provide functional evidence for a localized evoked response in register
with these anatomically defined recipient zones for PIL projections to
primary and secondary auditory cortex.
Despite the specificity of cortical projections from the PIL, its
apparent influence on cortical excitability remains consistent with the
earliest views of the intralaminar nuclei as modulatory (Lorente
de Nó 1949), a view based on anatomical evidence for termination of nonspecific thalamocortical projections in the supragranular cortical laminae (Berendse and Groenewegen
1991
; Cunningham and Levay 1986
;
Herkenham 1980
; Linke 1999
) and on functional evidence for their activating influence as measured in the
electroencephalogram (Dempsey and Morison 1942
).
Prolonged stimulation of the PIL produces a steady and focal increase
in the excitability of cells within auditory cortex. The increased excitability is apparent as a steady depolarization recorded
intracellularly in all cells of the present study as well as a steady
negative potential shift recorded extracellularly at the cortical
surface. Extracellular recordings, performed with laminar electrodes,
demonstrate that the PIL-evoked surface negative shift reverses
polarity in the depth (Sukov and Barth 1998
), a pattern
expected from an equivalent current dipole oriented perpendicular to
the cortical surface. This dipolar pattern indicates that the shift is
produced intracortically, as opposed to reflecting volume conducted
potentials from subcortical structures, and that it is produced by
synchronized PSPs in the parallel apical dendrites of large populations
of cortical pyramidal cells (Barth et al. 1989
;
Mitzdorf 1985
; Sukov and Barth 1998
). Combined with the present demonstration that PIL stimulation results in
prolonged intracellular depolarization, it is likely that the extracellular surface negativity reflects summed excitatory PSPs imposed on the distal ends of apical dendrites, an observation that is
consistent with the predominant synaptic termination of PIL fibers in
layer I of temporal cortex (Linke 1999
).
It is interesting that infragranular RS cells display PIL-evoked
depolarization as consistently as those in the supragranular layers
despite the fact that, at least in primary auditory cortex of the rat,
the apical dendrites of RS cells are shorter than those of
electrophysiologically identified IB cells that have extensive
arborizations in layer I (Hefti and Smith 2000).
There are perhaps three factors contributing to this result. First, infragranular apical dendrites can reach layer I even though their arborizations are far less extensive than IB cells. Second, it is
notable that our focus of maximum PIL-evoked gamma activity is not
directly within primary auditory cortex but is instead at the caudal
and lateral border, a region that may contain infragranular RS cells
with more extensive dendritic projections to layer I and a region that
may receive distinct PIL projections to both layer I and layers III/IV
(Linke 1999
). Finally, as noted earlier, while all cells
receiving injections of QX-314 characterized as putative RS cells
during depolarizing current injection, their identity cannot be
established with absolute certainty due to possible subtle effects of
QX-314 immediately on penetration. Thus some IB cells may have been
included in this group.
Accompanying the steady depolarization evoked in auditory cortex by PIL
stimulation are oscillations of extracellular membrane potential in the
gamma frequency band that suggest a role for the thalamus in their
neurogenesis. There is now abundant evidence that gamma-band
oscillatory rhythms are not a unique property of cortex and may be
found in the lateral geniculate nucleus (Ghose and Freeman
1992; however, also see Gray et al. 1989
), the
ventroposterior and ventrolateral nuclei (Steriade et al.
1996b
), the thalamic reticular nucleus (Pinault and
Deschênes 1992
; Steriade et al. 1996b
), as
well as the centrolateral nucleus of the intralaminar group
(Steriade et al. 1993
). More importantly,
synchronization of gamma oscillations has been reported in
thalamocortical networks during anesthesia, resting sleep, and during
behavioral arousal (Steriade et al. 1996b
). Yet while
thalamocortical and reciprocal corticothalamic circuits can become
synchronized during gamma oscillations, this synchronization is not
required for gamma neurogenesis in cortex. Spontaneous gamma
oscillations persist in auditory cortex after total destruction of the
PIL (MacDonald et al. 1998
) and following widespread
lesioning of the acoustic thalamus capable of completely eliminating
the AEP complex (Brett and Barth 1996
). Similarly, gamma
oscillations in rat auditory cortex have been observed in vitro
following electrical stimulation of the superior thalamic radiation in
thin slices (400 µm) with no intact thalamocortical link
(Metherate and Cruikshank 1999
), in slices of rat cortex with more limited portions of cortical circuits intact
(Chagnac-Amitai and Connors 1989
; Whittington et
al. 1995
), and in cortical cultures with no extrinsic inputs
(Plenz and Kitai 1996
). Thus it is not likely that the
PIL serves as a pacemaker for cortical gamma oscillations but instead
plays a modulatory role, providing a source of excitation that
activates intrinsic cortical oscillatory mechanisms.
There have been several recent reports that have proposed putative
cellular generators of gamma oscillations in the cortex (Gray
and McCormick 1996; Silva et al. 1991
;
Steriade et al. 1998
; for a review see Traub et
al. 1999
). Each has identified a unique pyramidal cell type
that possesses intrinsic membrane properties producing AP bursts with
periodicity in the gamma frequency band. A distinct class of cells in
the supragranular layers of cat visual cortex, termed "chattering
cells" (CH), intrinsically generate 20- to 70-Hz repetitive burst
firing in response to depolarizing current injection and exhibit
similar burst firing associated with membrane oscillations during
visual stimulation (Gray and McCormick 1996
).
Fast-rhythmic bursting (FRB) cells, discovered in the supra- and
infragranular layers of cat motor and association cortex, display
dynamic responses to depolarizing current injection, ranging from
rhythmic bursting (30-40 Hz) during moderate levels of stimulation to
higher-frequency tonic firing with increased current levels
(Steriade et al. 1998
). FRB cells were identified as
both pyramidal as well as sparsy spiny interneurons. A separate group
of layer V pyramidal neurons have also been identified in rat
sensorimotor cortex that display intrinsic rhythmicity near gamma
frequencies when sufficiently depolarized (Silva et al. 1991
). Putative inhibitory interneurons have also been
identified with intrinsic oscillatory properties in the gamma frequency
range (Llinás et al. 1991
).
Yet while each of these distinct cell types clearly participates in
cortical gamma oscillations, none have been shown to represent an
essential intracortical generator across species or sensory modalities.
CH and FRB cells have not been observed in in vivo studies of rat
sensory cortex (Jones et al. 2000; Zhu and
Connors 1999
). The present results indicate no cells in any of
the cortical layers that respond to depolarizing current injection with
phasic gamma frequency AP bursts suggestive of intrinsic oscillatory properties. This, despite the fact that these recordings were performed
in a highly active generator of evoked and spontaneous gamma
oscillations in the rodent cortex (Barth and MacDonald
1996
; Brett and Barth 1996
, 1997
;
Franowicz and Barth 1995
; MacDonald and Barth
1995
; MacDonald et al. 1996
). Furthermore when
APs in cells of the supra- and infragranular layers were blocked with QX-314, none revealed endogenous membrane oscillations during depolarizing current injection even at quite large (~2.0 nA) levels of excitation (Fig. 6C). However, CH cells require
suprathreshold activation to exhibit intrinsic oscillations
(Gray and McCormick 1996
) and therefore would not be
detected with QX-314.
It is possible that our failure to identify any cells with endogenous
membrane properties producing AP bursts in the gamma frequency range
can be explained by sampling error. However, this seems unlikely for
three reasons. First, given the percentage of FRB (28%), CH (34%),
and intrinsically bursting layer V cells (59% in deep layer V)
reported previously, we should have encountered a similar percentage
here that belonged to one or more of these classes, if they exist in
rat auditory cortex in same ratio. Second, CH, FRB, and layer V
bursting cells have been described as morphologically similar to RS
pyramidal cells with large soma that should present little challenge to
penetration with sharp electrodes used here. Finally, in a previous
study (Jones et al. 2000), we used similar methods to
examine rodent vibrissa/barrel cortex, also a very active generator of
stimulus-evoked and spontaneous gamma oscillations in the anesthetized
(MacDonald and Barth 1995
; MacDonald et al. 1996
) and unanesthetized animal (Jones and Barth
1997
). Stable in vivo recordings were obtained from RS
(n = 58), FS (n = 4), and IB
(n = 5) cells, none of which produced intrinsic gamma
oscillations or AP bursts in response to a range of depolarizing
currents. However, it should be noted that the subset of FRB cells that are sparsy spiny (Steriade et al. 1998
), and thus
probably interneurons, and intrinsically oscillatory interneurons
(Llinás et al. 1991
), may have been undersampled
by our present recordings given that we were able to capture only a
single FS cell, which has a similarly small soma.
While none of the cells in the present study produced independent gamma
oscillations when depolarized artificially, nearly all displayed APs
and subthreshold PSPs time-locked to epipial gamma oscillations when
the entire population was tonically depolarized by stimulation of the
PIL. The simplest explanation for this result is that gamma
oscillations are an emergent property of circuit interactions between
cortical cells when mutually excited and do not require an intrinsic
pacemaker for their neurogenesis. This conclusion is consistent with
computer models of large-scale neural systems that emphasize the
importance of network interactions in the generation of fast
oscillations (Lumer et al. 1997). Neural modeling
demonstrates that while gamma oscillations may persist in the absence
of cortico-thalamic projections, they are markedly suppressed by
removing either back-projections from supra- to infragranular layers or
forward projections from the infragranular layers to the granular and
supragranular layers. These interlaminar loops provide a mechanism of
high-gain amplification capable of driving local cortical networks into
fast oscillations. The predicted influence of reciprocal excitatory
connections between supra- and infragranular pyramidal cells in the
genesis of gamma oscillations is in agreement with the present
demonstration of APs and PSPs in both laminae that are tightly
time-locked to the population response. It is also in agreement with
previous laminar analysis of PIL-evoked gamma oscillations in auditory
cortex, indicating fast rhythmic interactions between supra- and
infragranular pyramidal cell groups. These appear to be a repetition of
the biphasic P1/N1 wave that characterizes the sensory evoked potential
complex, with an average 6 ms time lag between gamma waves in the
supra- and infragranular layers, producing a 90° phase shift at 40 Hz that is optimal for resonance if the coupled circuits approximate a
driven oscillator (Sukov and Barth 1998
).
Our results also suggest that inhibitory cells participate in the
generation of cortical gamma oscillations. The FS cell we were able to
study during PIL stimulation displayed high-frequency APs, both
spontaneously and during all phases of evoked surface gamma
oscillations. However, CTH and cross-correlation analysis revealed a
periodic fluctuation in AP probability that was time-locked to the
averaged population gamma wave. FS cells in neocortex have been
identified as sparsely-spiny or smooth GABAergic inhibitory interneurons (Kawaguchi 1993; McCormick et al.
1985
). Since these cells receive input from both supra- and
infragranular pyramidal cells, it is not surprising that their APs are
time-locked to the evoked gamma response, a result that does not
necessarily imply direct involvement in its neurogenesis. While our
data include only a single FS cell and are therefore inconclusive on
their own, they are in agreement with in vitro evidence obtained from slices of rat hippocampus (Whittington et al. 1995
),
guinea pig frontal cortex (Llinás 1992
;
Llinás et al. 1991
), mouse somatosensory cortex
(Buhl et al. 1998
), and in cortical cultures
(Plenz and Kitai 1996
), indicating an important role for
inhibitory postsynaptic potentials (IPSPs) in phasing the
suprathreshold activity of pyramidal cells during gamma oscillations.
Of particular interest are the results from somatosensory cortex, which
demonstrate that GABAergic IPSPs phase, rather than evoke, APs in
pyramidal cells (Buhl et al. 1998
); tonic depolarization
of the pyramidal cell population close to threshold is required before
gamma oscillations can be produced. Whereas in vitro, tonic
depolarization must be established artificially, using kainate or
elevated K+, in vivo, the PIL may serve as a key
source of tonic depolarization, inducing reciprocal excitatory and
inhibitory neural networks to rhythmic firing.
The putative cortical circuit responsible for gamma oscillations indicated by the present results, and the findings of our previous laminar analysis of PIL-evoked gamma oscillations, is shown in Fig. 7. Prolonged stimulation of the PIL results in tonic depolarization of the distal apical dendrites of both supra- and infragranular pyramidal cells (Fig. 7A). This produces an extracellular current sink near the cortical surface, recorded as a steady negative potential shift in the epipial population response, accompanied by steady depolarization of both populations of pyramidal cells. As the pyramidal cells begin to fire APs, two reciprocal circuits are engaged to phase these discharges at the gamma frequency. The first involves intralaminar negative feedback between the pyramidal cells and respective inhibitory interneurons (Fig. 7B), producing excitatory/inhibitory interactions that may serve as a pacemaker for gamma oscillations. The second involves reciprocal excitatory interlaminar interactions between the two pyramidal cell populations (Fig. 7C). This may serve as a pacemaker by itself but may also establish spatial and temporal patterns of oscillatory interactions along the vertical cortical axis with outputs capable of synchronizing more distal cell populations in cortex (Fig. 7D) and thalamus (Fig. 7E).
|
This model fits the present results using constituent circuit elements
and connections that are not specific to auditory cortex or to the
rodent. If the neural interactions responsible for cortical gamma
oscillations are indeed a repetitive realization of interactions giving
rise to middle latency components of the classic evoked potential
complex (Basar et al. 1987; Sukov and Barth
1998
), it may be assumed that they possess the same inter-areal
and -species similarities as the archetypal transient evoked response
(Steriade 1984
). However, unlike the transient evoked
response, gamma oscillations require a prolonged source of excitatory
drive to the principle neurons. The source of mutual excitation may be
regional, such as that provided by PIL stimulation, serving the
function of selective arousal of auditory cortex and consistent with a
view of the intralaminar thalamus as focal and probably modality
specific modulator of cortical excitability. Mutual excitation may also
be far more specific, such as that produced when the cells of
functionally related cortical columns are concurrently excited by a
common preferred stimulus, producing locally synchronized gamma
oscillations (Singer and Gray 1995
). This model does not
rule out the participation of cells with specialized membrane
properties favoring synaptic drive in the gamma frequency range. Cells
with endogenous oscillatory characteristics may be seen not as
essential generators of cortical gamma oscillations but as
preferentially tuned recipients, responding to and perhaps enhancing
patterned and transient synchronization of activity in subpopulations
of cells within sensory cortex during information processing.
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ACKNOWLEDGMENTS |
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We thank M. Jones and B. Brett-Green for critical comments on the manuscript.
This research was supported by Whitehall Foundation Grant S-97-06 and National Institute of Neurological Disorders and Stroke Grant 1 R01 NS-36981.
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FOOTNOTES |
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Address for reprint requests: D. S. Barth, Dept. of Psychology, University of Colorado, Campus Box 345, Boulder, CO 80309-0345 (E-mail: dbarth{at}psych.colorado.edu).
Received 30 June 2000; accepted in final form 2 November 2000.
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REFERENCES |
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