Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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Jacobsen, Richard B.,
Daniel Ulrich, and
John R. Huguenard.
GABAB and NMDA Receptors Contribute to Spindle-Like
Oscillations in Rat Thalamus In Vitro.
J. Neurophysiol. 86: 1365-1375, 2001.
Thalamic
slice preparations, in which intrathalamic connectivity between the
reticular nucleus and relay nuclei is maintained, are capable of
sustaining rhythmic burst firing activity in rodents and ferret. These
in vitro oscillations occur spontaneously in the ferret and have
frequencies (6-10 Hz) within the range of sleep spindles observed in
vivo. In the rat, mainly lower frequency (2-4 Hz) oscillations, evoked
under conditions of low bath [Mg2+] and/or
GABAA receptor blockade, have been described.
Here we show that faster rhythms in the range of 4-9 Hz can be evoked in rat thalamic slices by electrical stimulation of the internal capsule and also occur spontaneously. When bath
[Mg2+] was 2 mM, these spindle-like
oscillations were most common in a brief developmental time window,
peaking at postnatal day 12 (P12). The
oscillations were almost completely blocked by the GABAA receptor antagonist picrotoxin, and, in
some cases, the frequency of oscillations was increased by the
GABAB receptor antagonist CGP-35348. The
selective blockade of N-methyl-D-aspartate (NMDA) or -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors by the antagonists 2-amino-5-phosphonovaleric acid or
1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), respectively, significantly shortened oscillations but did not completely block them. A combination of the two drugs was
necessary to abolish oscillatory activity. The barbituate pentobarbital, which enhances GABAAR responses,
initially slowed and synchronized oscillations before completely
blocking them. When bath [Mg2+] was reduced
from 2 to 0.65 mM, evoked oscillations became more robust and were
often accompanied by spontaneously arising oscillations. Under these
conditions, GABAA receptor blockade no longer
inhibited oscillations, but instead converted them into the slow,
synchronous rhythms that have been observed in other studies. The
effects of GABAB or NMDA receptor blockade were
more pronounced in 0.65 mM than in 2 mM external
[Mg2+]. Thus spindle-like oscillations occur in
rat thalamic slices in vitro, and we find that, in addition to the
previously demonstrated contributions of GABAA
and AMPA receptors to these oscillations, NMDA and
GABAB receptors are also involved. The strong
influence of external [Mg2+] on GABAergic
pharmacology and a contribution of NMDA receptors during oscillations
suggest a link between the excitability of NMDA receptors and
the activation of GABABR-mediated inhibitory postsynaptic currents.
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INTRODUCTION |
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A
well-established property of the thalamus is the generation of
transient 7- to 14-Hz oscillations that arise in a periodic manner
during the early stages of slow-wave sleep (Steriade and Deschênes 1984; Steriade and Llinás
1988
). These oscillations have been termed sleep spindles and
are one of several rhythms arising in the thalamus and cortex during
non-rapid eye movement (non-REM) sleep (McCormick and Bal
1997
; Steriade and Deschênes 1984
;
Steriade et al. 1993
). The extensive reciprocal
connectivity between thalamic and cortical neurons results in
propagation of spindles through the cortex (Steriade and
Llinás 1988
), but the inability of cortex to produce
these rhythms when disconnected from thalamus (Burns
1950
), and their persistence in decorticated thalamus
(Morison and Basset 1945
), indicate a thalamic origin for spindle oscillations.
While the function of sleep spindles remains a mystery, the cellular
mechanisms leading to their generation have been intensively studied
and largely elucidated over the past several decades. Nearly 20 yr ago,
electrophysiological recordings from thalamic neurons in vitro
(Llinás and Jahnsen 1982) and in vivo
(Deschênes et al. 1982
; Roy et al.
1984
) provided the basis for a mechanistic understanding of the
state-dependent oscillatory behavior of the thalamus. Reciprocal
connections between the inhibitory, GABAergic nucleus reticularis (nRt)
and excitatory relay nuclei appear to provide the essential circuitry
for spindle generation in the thalamus (von Krosigk et al.
1993
; but see Steriade et al. 1987
). During
sleep onset, thalamic neurons undergo a membrane hyperpolarization (Hirsch et al. 1983
; Steriade et al.
1986
) that fundamentally alters their firing properties,
primarily through the deinactivation of a low-threshold calcium current
(Coulter et al. 1989
; Crunelli et al.
1989
; Hernandez-Cruz and Pape 1989
;
Jahnsen and Llinás 1984
; Suzuki and
Rogawski 1989
). The biophysical properties of this calcium
current in nRt and relay nuclei (Huguenard and Prince 1992
) are such that it can be activated by incoming excitatory potentials in the former and on repolarization following inhibitory potentials in the latter (Bal et al. 1995a
,b
;
Huguenard and Prince 1994a
; Warren et al.
1994
). In both cases, activation leads to a regenerative
calcium spike that depolarizes the cells above Na+-channel threshold long enough to cause a
high-frequency burst of action potentials (Deschênes et
al. 1982
; Llinás and Jahnsen 1982
). As a
result of the feedback circuitry between nRt and relay nuclei, bursts
in relay neurons produce glutamate receptor-dependent bursts in nRt
neurons, which in turn inhibit relay cells. The relay neurons do not
fire until they have recovered from the inhibitory input of
nRt cells, meaning that the inter-burst duration, and thus the
frequency of spindles, is largely dependent on the duration of
inhibitory postsynaptic currents (IPSCs) in relay neurons (Bal et al. 1995a
).
The generation of spindles is strongly influenced by inhibitory
connections between nRt neurons, as shown by studies in which these
predominantly GABAAR-mediated connections
(Ulrich and Huguenard 1996) are removed from the circuit
either through pharmacological blockade (Bal et al.
1995a
,b
; Huguenard and Prince 1994a
;
Sanchez-Vives and McCormick 1997
; Sanchez-Vives
et al. 1997
; von Krosigk et al. 1993
) or genetic
manipulation (Huntsman et al. 1999
). The findings show
that the absence of intra-nRt inhibition can enhance the output of nRt
(Huguenard and Prince 1994b
; Sanchez-Vives and McCormick 1997
) and result in hyper-synchronous oscillatory
activity (Huntsman et al. 1999
). The slow, paroxysmal
oscillations that result from GABAAR blockade in
ferret (Steriade et al. 1993
; von Krosigk et al.
1993
) and rat (Huguenard and Prince 1994a
)
thalamic slices resemble those seen during some forms of absence epilepsy.
The recent development of thalamic brain slice preparations in which
spindle-like oscillations can be recorded has been an essential tool
for working out the detailed molecular mechanisms responsible for
oscillatory activity in the thalamus (reviewed in McCormick and
Bal 1997). Thalamic slices from a number of species including
rat (Huguenard and Prince 1994a
; Leresche et al.
1991
), mouse (Warren et al. 1994
), and ferret
(Bal et al. 1995a
,b
; von Krosigk et al.
1993
) have demonstrated oscillatory behavior. The basic
mechanisms responsible for spindle-like oscillations appear to be
similar, although some differences between preparations are also
apparent. Fast (5-10 Hz), spindle-like oscillations occur spontaneously in ferret slices under normal physiological conditions (~1.2 mM bath [Mg2+]) (Bal et al.
1995a
,b
). In contrast, the pro-oscillatory conditions of
GABAA receptor blockade and/or low bath
[Mg2+] (0.2 mM) have been necessary to observe
robust evoked oscillations in thalamic slices from rat (Cox et
al. 1997
; Huguenard and Prince 1994a
;
Ulrich and Huguenard 1995
). Consistent with findings in ferret, oscillations evoked in rat slices in the presence of
GABAAR antagonism are slower (2-4 Hz). Here we
show that oscillations with frequencies in a range closer to that
expected for spindles can be evoked in rat slices, even under
physiological conditions that would not be considered pro-oscillatory.
The spindle-like oscillations in rat differ from those described in
ferret (Bal et al. 1995a
,b
; von Krosigk et al.
1993
) and mouse (Warren et al. 1994
) in being
sensitive to pharmacological blockade of GABAB and N-methyl-D-aspartate (NMDA) receptors.
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METHODS |
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Slice preparation and recording
Thalamic slices were prepared as previously described
(Huguenard and Prince 1994a). Briefly, rat pups of
either sex, 11-15 days old were anesthetized (50 mg/kg ip
pentobarbital sodium) and decapitated. The brain was removed
and transferred into ice-cold slicing solution containing (in mM) 234 sucrose, 11 glucose, 24 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2
bubbled with 95% O2-5%
CO2. The whole brain was glued onto a cover slip, and 400-µm horizontal slices were made using a vibratome (TPI, St.
Louis, MO). The thalamus and parts of the adjacent striatum were dissected out and incubated in artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 26 NaHCO3,
2.5 KCl, 1.25 NaHPO4, 2 MgCl2, 2 CaCl2, and 10 glucose at 32°C for 1 h prior to recording. The solution was
continuously bubbled with 95% O2-5%
CO2, and temperature was allowed to return to
room temperature after the initial hour.
For recordings, slices were placed in an interface type recording chamber and perfused with ACSF (described above) at 34°C. In reduced [Mg2+] experiments, the [MgCl2] in the perfusion solution was reduced to 0.65 mM. Intra-thalamic oscillations were evoked by a 20- to 100-V, 60- to 80-µs stimulus to the internal capsule (IC) through a bipolar tungsten electrode (FHC, Bowdoinham, ME) positioned at the border of the IC and the thalamic reticular nucleus (nRt). Stimulus interval was varied depending on the duration of an oscillation (to avoid inter-sweep variability presumably due to rundown) but was in the range of one stimulus every 15-60 s. Extracellular multi-unit activity was recorded using monopolar tungsten electrodes positioned in the ventrobasal (VB) somatosensory relay nucleus and/or nRt. The amplitude of action potentials ranged from 10 to 15 µV at threshold for spike detection (see Data analysis) to maximum potentials in the 100- to 200-µV range. Although only recordings from VB are presented in this study, spindle-like oscillations could be recorded simultaneously from VB and nRt, demonstrating that both nuclei were participating in oscillatory activity. Signals were band-pass filtered (10 Hz to 3 kHz) and recorded at different sample rates depending on the software used (1.7 kHz, Axotape, or 5-10 kHz, Axoscope, both from Axon Instruments, Foster City, CA).
Data analysis
A software Schmidt trigger was used to detect spikes. The "duration" of an oscillation was measured as the time from stimulus to last burst (defined as 4 spikes occurring within 50 ms). The "total spike activity" of an oscillation was defined as the number of spikes occurring between the time of stimulation and the end of the recording sweep. Autocorrelation analysis was performed on spike output, typically with a bin size of 5-10 ms, to estimate the synchrony of oscillations. The results from five consecutively evoked oscillations were summed for each plot presented in the results. Comparisons of synchrony were made using the height of the central peak relative to the adjacent valley of the autocorrelogram as an estimate of the proportion of synchronous versus asynchronous spike activity, respectively, during an oscillation. For comparisons of oscillation synchrony where total unit activity varied between pharmacological conditions within an experiment, autocorrelograms were normalized to the height of the central peak.
Fast Fourier transforms (FFTs) were also performed on spike output starting from the time of stimulation with bin sizes no larger than 20 ms. As summarized by the three-dimensional contour plots of FFT analysis presented in Figs. 1B, 2B, and 5C, FFTs were performed separately on each evoked spindle during an experiment. The oscillation frequency was calculated by determining the dominant spectral component between 2 and 10 Hz for each FFT spectra and averaging over at least five sweeps. Sweeps in which a dominant frequency could not be determined, defined as those in which the peak power of the dominant frequency comprised <40% of the summed peak power of the three most dominant frequencies, were not used.
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Drugs
CGP-35348 (CGP, p-3-aminopropyl-p-diethoxymethyl
phosphoric acid) was a gift from Ciba-Geigy (Basel). All other
chemicals were from Sigma (St. Louis, MO). Picrotoxin,
1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), and CGP stock solutions were made in DMSO such that the final concentration of DMSO in the extracellular recording solution was
0.5%. Unless otherwise noted, drug concentrations were chosen to be
1-2 orders of magnitude greater than previously reported IC50s.
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RESULTS |
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Spindle-like oscillations recorded from rat thalamic slices
Previous studies have demonstrated that 2- to 4-Hz
oscillatory activity can be evoked in rat thalamic slices using a
combination of GABAA receptor
(GABAAR) blockade and reduced
Mg2+ concentration in the extracellular recording
solution ([Mg2+]O)
(Cox et al. 1997; Huguenard and Prince
1994a
; Ulrich and Huguenard 1995
). We found that
higher-frequency oscillations can occasionally be evoked in rat
thalamic slices by electrical stimulation in the IC in the presence of
2 mM [Mg2+]O [which is
slightly higher than the ~1-1.3 mM found in physiological conditions
(Hansen 1985
)]. The frequency of these
oscillations, usually above 4 Hz and as high as 9 Hz, is in the range
observed for sleep spindles in vivo (Steriade and Llinás
1988
) and in numerous in vitro studies using LGN slices from
ferret (Bal et al. 1995a
; Kim et al.
1995
; von Krosigk et al. 1993
). We thus refer to
these 4- to 9-Hz oscillations in rat as "spindle-like oscillations," or SLOs.
Multi-unit, extracellular recordings of SLOs evoked by electrical
stimulation of the internal capsule were made in the VB of rat thalamic
slices, as detailed in METHODS. Our initial
characterization of SLOs was done in 2 mM
[Mg2+]O to allow
comparison with previous studies in rat in which this concentration was
used (Cox et al. 1997; Huguenard and Prince 1994a
). We found, however, that reducing
[Mg2+]O resulted in more
robust spindle-like oscillations, as described below (see
Effects of
[Mg2+]O on
SLOs). Slices were selected for pharmacological manipulation based on the duration and frequency of evoked SLOs. Our selection criteria was for a minimum duration of five resolvable cycles, or an
oscillation lasting 1 s or more. Often, however, evoked oscillations were of much longer duration (up to 10 s). We found that the ability of a thalamic slice to support evoked SLOs in 2 mM
[Mg2+]O was strongly
dependent on age. The proportion of preparations (each yielding 6 400-µm slices) that exhibited SLOs by age group was as follows:
postnatal day 11 (P11), 2/2; P12,
13/14; P13, 3/4; P14, 2/7. Rats younger than
P11 tended to have signal amplitudes that were near the
threshold for resolving multi-unit extracellular activity, making the
occurrence of SLOs difficult to assess. At age P14 and
older, this type of oscillation was very rarely observed in 2 mM
[Mg2+]O. We focused our
experiments on P12 rats due to the ease of obtaining evoked
SLOs at this age and the strength of the signal as compared with
P11 animals. For consistency, only data from this age group
is presented in the remainder of this report.
A typical evoked SLO recorded in 2 mM [Mg2+]O is shown in the top two traces of Fig. 1A (labeled "control"). The frequency of this activity was ~4.3 Hz, as determined by FFT of the unit activity that followed each stimulus (see METHODS). The FFT spectra for all control recording sweeps in this experiment are shown by the contour plot in Fig. 1B (portion labeled "control"). Note the cluster of darker contours (representing frequencies with the highest FFT power) in the 4- to 5-Hz range during this period.
Effects of GABAB receptor blockade on SLOs
Studies in mouse (Warren et al. 1994) and ferret
(Bal et al. 1995a
; von Krosigk et al.
1993
) found that the blockade of GABABRs did not affect evoked oscillatory activity in thalamic slices. We
tested the contribution of GABABRs to SLOs in rat
by applying the antagonist CGP-35348 (CGP) to the bath solution during
multi-unit, extracellular recording of evoked oscillations. Potential
drug effects were quantified by measuring the total spike activity, duration, frequency, and synchrony of SLOs, as detailed in
METHODS. An example of the effects of CGP is shown in Fig.
1. The effects were difficult to discern from raw recording traces
(shown in Fig. 1A), but Fourier transform revealed a
reversible increase (by ~25%) in the dominant frequency of
oscillations following drug application (Fig. 1, B and
C). An autocorrelation was performed on unit activity under
control, CGP, and wash conditions to assess oscillation synchrony (see
METHODS). The plots in Fig. 1C show a slight
enhancement in the overall number of spikes detected during the
oscillation in the presence of CGP relative to control and wash
conditions. However, when the control and CGP autocorrelograms are
normalized, there is no difference in the peak to valley ratio between
the two plots (Fig. 1C, inset), suggesting no
change in synchrony between these conditions.
In general, the effects of CGP on our measures of oscillation
robustness (total spike activity and duration) were quite variable, ranging from a slight enhancement to a 50% reduction in these parameters (Fig. 1D). Although the population averages
showed a slight suppression in both parameters, neither varied
significantly between control and CGP conditions (P = 0.18, total spikes; P = 0.11, duration; 2-tailed
t-test). In five of eight experiments, CGP caused a
reversible increase in oscillation frequency (Fig. 1E). The
magnitude of increase ranged from 10 to 30% of control and did not
correlate with the frequency under control conditions. In two of eight
experiments, CGP (800 µM) caused an increase in oscillation synchrony
(based on autocorrelation analysis; see METHODS). In both
of these experiments, CGP strongly suppressed the total spike activity
and duration of oscillations while having little effect on frequency
(Fig. 1, D and E, and
). An
example of an experiment in which CGP application increased oscillation synchrony is presented in the section describing experiments with reduced [Mg2+]O.
Effects of pharmacological manipulation of GABAARs on SLOs
The GABAAR antagonist picrotoxin strongly suppressed SLOs recorded in 2 mM [Mg2+]O. An example is shown in the experiment in Fig. 1. The application of 50 µM picrotoxin reduced evoked activity from robust, high-frequency (~4.3 Hz) oscillations to brief, low-frequency (~2.3 Hz) events consisting of only one or two cycles (Fig. 1, A and B). A combination of picrotoxin and CGP abolished oscillatory activity (e.g., Fig. 1A; n = 3).
The suppressive effect of picrotoxin on SLOs is summarized for all
experiments in Fig. 1D. In five of five experiments, the total spike activity and duration of oscillations in the presence of
the drug was reduced by ~50-90%, differing significantly from control conditions across the population (P < 0.001, 2-tailed t-test). When more than one cycle of an oscillation
remained after picrotoxin application, the frequency calculated from
the inter-burst interval was greatly reduced (2.5 ± 0.2 Hz;
mean ± SE, n = 3). These findings are
consistent with observations made in young ferret, where spindles from
animals up to P39, recorded in 1.2 mM
[Mg2+]O, were largely
blocked by the GABAAR antagonist bicuculline (McCormick et al. 1995).
Effects of [Mg2+]O on SLOs
Conditions of reduced extracellular magnesium can greatly enhance
the ability of thalamic slices to support oscillations (Cox et
al. 1997; Huguenard and Prince 1994a
;
Ulrich and Huguenard 1995
). However, we found that
decreasing [Mg2+]O to 0.2 mM, the concentration used in these earlier studies, resulted in
oscillations that were less stable over the extended periods of time
required to perform the pharmacological manipulations described here.
Instead, we adopted a protocol using an intermediate [Mg2+]O of 0.65 mM.
Reducing [Mg2+]O from 2.0 to 0.65 mM markedly increased the occurrence of evoked and
spontaneously arising SLOs in thalamic slices from P12 rats (presented in this study) and in older animals (up to P15,
data not shown). Figure 2A
shows an example of the effects of reducing [Mg2+]O.
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The effects of GABAergic blockade on SLOs evoked in 0.65 mM [Mg2+]O were notably different from those seen in 2 mM [Mg2+]O. In reduced [Mg2+]O, the application of CGP caused either no change or an enhancement in the total spikes and duration of SLOs, as summarized in Fig. 2D. The example shown in Fig. 2 proved to be the most pronounced instance of an increase in these parameters. In addition, the influence of CGP on the frequency and synchrony of SLOs became more pronounced in 0.65 mM [Mg2+]O such that clear effects were seen in every experiment. The application of CGP resulted in an increase in frequency (by 15-22%) in six of six experiments (as compared with 5/8 experiments in 2 mM [Mg2+]O), as shown by the example in Fig. 2 and population summary in Fig. 2E. Synchrony was also enhanced in every experiment (compared with only 2/8 experiments in 2 mM [Mg2+]O), as in Fig. 2C.
The most striking difference between oscillations recorded in high
versus low [Mg2+]O was in
the response to GABAAR blockade by picrotoxin. In
2 mM [Mg2+]O, picrotoxin
nearly abolished SLOs (see Fig. 1), while in 0.65 mM
[Mg2+]O oscillations
became slow and hypersynchronous without being attenuated (Fig. 2,
A and B). In the three experiments performed under these conditions, total spike activity showed a trend toward enhancement while duration remained unchanged (Fig. 2D,
right side). The frequency of oscillations in the presence of
picrotoxin was reduced to 2.7 ± 0.4 Hz (e.g., Fig. 2B;
n = 3). These observations are consistent with results
from earlier studies using older rats and 0.2 mM
[Mg2+]O (Cox et
al. 1997; Huguenard and Prince 1994a
;
Ulrich and Huguenard 1995
).
Contribution of NMDA receptors to oscillations
The enhancement of SLOs in reduced
[Mg2+]O raised the
possibility that at least part of this effect might be explained by the inhibitory interaction of Mg2+ with NMDA
receptors (NMDAR; Nowak et al. 1984). We tested this by
applying the selective NMDAR antagonist 2-amino-5-phosphonovaleric acid
(APV) to SLOs recorded in 2 mM
[Mg2+]O. In eight of
eight experiments, the application of APV attenuated both the total
spike activity and the duration of SLOs by 15-75% (Fig.
3C). The reduction was
significant across the population (P < 0.001, 2-tailed
t-test). An example is provided in Fig. 3A. In
this experiment, control oscillations were evoked by near-threshold (20 V) stimulation of the internal capsule, and bath application of 200 µM APV reduced the duration and total spike activity of oscillations
by ~30%. A slight slowing of oscillation frequency was seen in the
presence of APV in three of five experiments where frequency could be
quantified before and after drug application (e.g., Fig. 3,
A and B, summarized in Fig.
3D).
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The inhibitory actions of APV on evoked SLOs could occur at excitatory inputs activated by the initial stimulation of the internal capsule (at corticothalamic and/or thalamoreticular synapses) and/or at thalamoreticular synapses during the recurrent excitation of nRt cells necessary for the maintenance of oscillations. If NMDAR blockade only reduced the efficacy of the initial stimulus event, the effects of APV might be overcome by an increase in stimulus intensity. As demonstrated in Fig. 3, A and B, increasing stimulus intensity from 20 to 70 V greatly enhanced the total spike activity of oscillations in the presence of APV (to levels comparable with control) but was ineffective in recruiting additional cycles to the oscillation. Similar results were seen in two additional slices using stimulus intensities ranging from threshold to 100 V (not shown). While the correlation between increased unit activity and nRt output using this paradigm cannot be determined directly from extracellular recordings, these results are consistent with an influence of APV at thalamoreticular synapses during recurrent excitation, in addition to the likely effects on stimulus efficacy.
The inhibitory effect of APV was also tested in 0.65 mM [Mg2+]O (n = 3, not shown). On average, the APV-induced reduction in total spike activity of SLOs was more pronounced in 0.65 mM than in 2 mM [Mg2+]O (58 ± 4% vs. 39 ± 6% block, respectively). In addition, APV was slightly more effective at inhibiting the total spike activity of SLOs recorded in 0.65 mM [Mg2+]O than was increasing [Mg2+]O to 2 mM when the two conditions were tested sequentially in the same slice (58 ± 4% vs. 44 ± 4%, respectively, n = 3). These observations are consistent with a role for NMDAR excitability in explaining the strong influence of [Mg2+]O on oscillatory activity in thalamic slices.
We next blocked -amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA)/kainate receptors using the selective antagonist NBQX and
found that oscillations could still be evoked. The addition of a high
concentration of NBQX (2 µM) altered the organization and reduced the
duration of oscillations but had little effect on the total spike
activity (see the example in Fig. 4).
Oscillations evoked in the presence of NBQX could be completely blocked
by the subsequent addition of 50 µM APV, confirming that they were mediated largely by NMDARs (n = 3, e.g., Fig. 4). In
the example shown, NMDAR-mediated oscillations had a lower frequency
than control oscillations (3.3 vs. 5.5 Hz), prolonged bursts and more synchronous burst activity (Fig. 4B). The frequency of
NMDAR-mediated oscillations was dramatically reduced in three of four
experiments using AMPA receptor (AMPAR) antagonists (Fig.
4D). In one of these experiments (indicated in Fig. 4,
C and D,
), the AMPA/kainate receptor
antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was
tested on a rare SLO recorded from a P15 animal with
effects on frequency and burst structure similar to the example in
Fig. 4.
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Effects of pentobarbital on SLOs
We also tested the effects of the barbiturate pentobarbital
(pb) on SLOs evoked in slices from P12 rats. This drug has
been shown to modulate GABAARs by prolonging
channel open times (Macdonald and Olsen 1994). As shown
in Fig. 5, 100 µM pb reversibly
abolished SLOs. During bath application of the drug, a progressive
lengthening of inter-burst intervals gradually reduced the oscillation
frequency from 6-7 Hz to ~4 Hz, accompanied by a pronounced
synchronization of the remaining cycles. Eventually a complete loss of
recurrent activity, including the initial rebound burst normally
induced by the stimulus, was seen. The complete experiment is presented as a spike-rate contour plot in Fig. 5B. The progressive
changes in oscillation frequency following pb application can be seen in the contour plot of FFTs in Fig. 5C. Similar effects,
including the eventual loss of all oscillatory activity in the presence of 100 µM pb, were reproduced in two additional experiments (not shown).
|
Spontaneous SLOs
Spontaneous spindles were often seen in P12 slices using 0.65 mM [Mg2+]O but were observed much more rarely in 2 mM [Mg2+]O. Several examples of spindles recorded in the latter condition from two separate sites in VB are shown in Fig. 6. In these recordings, spontaneous spindles arose every 10-60 s, lasted 1-7 s, and had frequencies of 5-6 Hz.
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DISCUSSION |
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Role of GABAB receptors in spindle oscillations
We have shown that SLOs with frequencies in the 4- to 9-Hz range
can be evoked in thalamic slices from young rats. The oscillations resemble those seen in extracellular recordings from ferret lateral geniculate nucleus (LGN) slices (Bal et al. 1995a;
McCormick et al. 1995
; von Krosigk et al.
1993
), but pharmacological manipulations revealed several
differences between the two preparations. In ferret, the blockade
of GABABRs had no apparent effect
on spontaneous (Bal et al. 1995a
) or evoked
(Sanchez-Vives and McCormick 1997
) spindles recorded in
1.2 mM [Mg2+]O. We found
that the GABABR antagonist CGP-35348 had several effects on evoked SLOs when bath applied to rat thalamic slices, and
that these effects were sensitive to
[Mg2+]O. An increase in
oscillation frequency was seen in five of eight experiments where
[Mg2+]O was 2 mM (range,
13-32% increase) and in all six experiments where
[Mg2+]O was 0.65 mM
(range, 13-24%; compare Figs. 1E and 2E). In
addition, GABABR blockade caused an increase in
the synchrony of oscillations that was much less consistent in 2 mM
than in 0.65 mM [Mg2+]O
(observed in 2/8 and 6/6 experiments, respectively). Finally, based on
our measures of oscillation robustness (total spike activity and
duration), CGP could have enhancing or suppressing effects in 2 mM
[Mg2+]O but tended to
leave unchanged or cause a slight enhancement in 0.65 mM
[Mg2+] (compare Figs. 1D and
2D). These results indicate that in the rat preparation
GABABRs are activated during evoked SLOs where they may have slowing and/or desynchronizing effects on oscillatory activity.
The actions of CGP could be pre- or postsynaptic, as has been
described in several thalamic studies in rat (Ulrich and
Huguenard 1996) and ferret (Sanchez-Vives and McCormick
1997
; Sanchez-Vives et al. 1997
). The increase
in frequency seen following CGP application in the present
study is likely the result of postsynaptic blockade of
GABABRs in relay neurons, as relief
of presynaptic inhibition would be expected to increase GABA output
and, if anything, prolong postsynaptic inhibitory postsynaptic
potentials (IPSPs). The loss of slow,
GABAB-mediated IPSPs would result in more rapid
rebound bursting, since the remaining IPSPs would be shorter duration, GABAA-mediated events. In addition, a homogeneous
group of GABAAR-mediated IPSPs in relay cells
might synchronize rebound activation and result in more organized
oscillations, as was seen in some experiments with CGP (and also
transiently during pentobarbital application; see Effects of
pentobarbital on oscillations). The complex effects of CGP
on oscillation robustness (see Fig. 1D and 2D)
could be due to a mixture of pre- and postsynaptic effects at both
reticulo-thalamic and intra-nRt synapses. Variations in connectivity
and/or membrane properties between preparations might favor the
activation of different sets GABABRs in different experiments.
A study using paired recordings between synaptically coupled
neurons of the perigeniculate nucleus (PGN) and lateral geniculate nucleus (LGN) in ferret slices examined the efficacy of high-frequency IPSCs in promoting GABABR activation during
bursts induced in single PGN neurons (Kim and McCormick
1998; Kim et al. 1997
). This study found that
only prolonged, high-frequency bursts in a PGN neuron, such as those
seen following GABAAR blockade of intra-nRt
connections by picrotoxin or bicuculline, were capable of significantly
activating postsynaptic GABABRs in a
monosynaptically coupled LGN neuron. Our results in rat suggest that
GABABR activation can occur under more normal
bursting conditions. One possible explanation is convergence of
GABAergic input from multiple nRt cells onto single relay neurons. The
increase in GABABR activation in 0.65 mM
[Mg2+]O, as indicated by
the more pronounced effects of CGP and the maintenance of
GABAB-mediated oscillations in the presence of picrotoxin, suggests an increase in the amount of GABA released by nRt
neurons under these conditions. The possible contribution of NMDARs to
an enhancement in nRt output is discussed in the next section.
While this is the first study to implicate
GABABRs in normal SLOs in vitro, intracellular
recordings in mouse (Warren et al. 1997), rat
(Huguenard and Prince 1994a
) and ferret (Bal et
al. 1995b
; Kim et al. 1997
; Sanchez-Vives
and McCormick 1997
; von Krosigk et al. 1993
)
have demonstrated that GABABRs can be activated at synapses between nRt and relay neurons. However, the studies in
ferret and mouse found no effect of GABABR
antagonists on in vitro oscillations. Slice preparations of mouse
ventroposterior thalamus, for example, were unable to support
GABABR-mediated oscillations following the
blockade of GABAARs in 1.3 mM (Warren et
al. 1994
) or 2 mM
[Mg2+]O (Huntsman
et al. 1999
), despite the functional presence of postsynaptic
GABABRs (Warren et al. 1997
). In
contrast, robust GABABR-mediated oscillations can
occur in the rat ventrobasal nucleus (Huguenard and Prince
1994a
) and ferret LGN (von Krosigk et al. 1993
)
under similar conditions. These discrepancies could be explained by
species-specific and/or anatomical differences in preparations.
NMDA receptors contribute to SLOs at thalamoreticular synapses
A number of studies in rodent have shown an NMDAR component to
excitatory potentials evoked at corticothalamic synapses in both the
reticular (de Curtis et al. 1989; Golshani and
Jones 1999
) and relay neurons (Deschênes and Hu
1989
; Golshani et al. 1998
; Kao and
Coulter 1997
; Scharfman et al. 1990
;
Turner and Salt 1998
). An immunocytochemical electron
microscopy study identified the NMDAR1 subunit at putative
corticothalamic and thalamoreticular type synapses in nRt of cat and
rat (Liu 1997
). One previous report has attempted to
specifically identify an NMDAR component to excitatory postsynaptic
potentials (EPSPs) at thalamoreticular synapses during oscillatory
activity (Huguenard and Prince 1994a
). Consistent with
the present results, it was concluded that NMDARs at thalamoreticular synapses contributed to recurrent oscillatory activity.
The finding that the NMDAR antagonist APV reduces the total spike activity and duration of oscillations (see Fig. 3C) evoked by electrical stimulation of both corticothalamic and thalamocortical axons in the internal capsule could be explained in two ways (that are not mutually exclusive). NMDAR activation could be reduced at one or both synapses during stimulation, and/or at thalamoreticular synapses during the maintenance of oscillations following initiation. Several observations strongly suggest that NMDARs are functionally present at thalamoreticular synapses and that they contribute to the maintenance of SLOs. The most convincing argument for this is the persistence of oscillations after AMPAR blockade. It is possible that a small fraction of the AMPAR population remained unblocked in 2 µM NBQX, but the complete suppression of the remaining oscillatory activity by APV shows that NMDARs are playing at least a major facilitative role. Support for an NMDAR contribution to SLO maintenance comes from experiments showing that increasing stimulus intensity does not overcome NMDAR blockade, despite the recruitment of additional unit activity during stimulation (Fig. 3, A-C). In addition, the frequency of oscillations was reversibly reduced in the presence of APV in several experiments. Since stimulus intensity did not influence the frequency of evoked oscillations (unpublished observations), we conclude that the changes in frequency arise from the influence of NMDAR blockade on the internal structure of oscillations, i.e., at thalamoreticular excitatory synapses, rather than through a reduction in stimulus efficacy.
Effects of [Mg2+]O on GABABR activation: a possible link with NMDAR excitability
When [Mg2+]O was 2 mM, SLOs were largely blocked by the GABAAR antagonist picrotoxin, suggesting that postsynaptic GABABR activation in relay neurons was insufficient to induce rebound burst generation and sustain the oscillation. In 0.65 mM [Mg2+]O, SLOs were converted into slow, hyper-synchronous oscillations in the presence of picrotoxin. This suggests that GABABR-mediated IPSPs were sufficiently enhanced under these conditions to drive rebound burst firing in relay neurons, presumably due to an increase in the output of nRt neurons. Also consistent with increased GABABR activation in reduced [Mg2+]O was the more pronounced effect of CGP on SLO frequency under these conditions.
A developmental study in ferret LGN found that bicuculline nearly
abolished spindle oscillations in thalamic slices from animals up to
postnatal day 39 but slowed and synchronized them in older animals (McCormick et al. 1995). The authors concluded
that the ability of PGN neurons to generate sustained high-frequency
bursts of action potentials, which are necessary to activate
postsynaptic GABABRs, was a limiting factor
during development. The changes in
[Mg2+]O in our study are
unlikely to influence action potential firing in the same way, although
there may be other nonspecific effects on excitability, such as changes
in ion channel inhibition, charge screening effects, or
Mg2+ block of NMDARs. Our findings provide some
support for the importance of NMDAR excitability in mediating
Mg2+-related changes in oscillatory activity. The
inhibitory effect of APV and 2 mM
[Mg2+]O on SLOs recorded
in 0.65 mM [Mg2+]O was
similar when compared in the same slice. In addition, the inhibitory
effect of APV was, on average, more pronounced in 0.65 mM than 2 mM
[Mg2+]O, suggesting that
the NMDAR contribution to oscillations is enhanced in lower
[Mg2+]O. These findings
support a link between NMDAR excitability and the sensitivity of SLOs
to [Mg2+]O.
A recent study in the amygdala found that NMDAR-mediated EPSPs were
more effective than AMPAR currents at activating a low-threshold Ca2+ current (Calton et al. 2000).
This raises the interesting possibility that NMDAR activation could
enhance the bursting capabilities of nRt neurons by influencing the
activation of a thalamic low-threshold Ca2+
channel. The apparent increase in GABABR
activation in reduced [Mg2+]O supports the
notion that the output of nRt neurons is enhanced under these
conditions and this could be due, at least in part, to the influence of
NMDAR activation on the bursting properties of nRT.
Effects of pentobarbital on oscillations
The barbiturate pentobarbital completely blocked SLOs in
P12 slices. A gradual increase in inter-burst duration
(slowing of frequency) and a synchronization of the remaining cycles in
the oscillation was followed by complete loss of oscillatory activity. These findings are consistent with the slowing of IPSC decay kinetics caused by barbiturates (Macdonald and Olsen 1994), as
observed in hippocampal neurons (Otis and Mody 1992
;
Thompson and Gähwiler 1992
). The initial rebound
burst, caused by direct activation of nRt cells by the stimulus, was
also abolished by 100 µM pentobarbital. This could be explained by an
enhancement in the GABAergic inhibitory interactions between nRt cells,
which would have a shunting effect on the stimulus and thus reduce the
output of nRt cells to a level insufficient to induce rebound burst
generation in relay neurons. Another likely possibility is that the
GABAergic response of relay neurons was directly prolonged by
pentobarbital such that the time course of repolarization during IPSPs
was too slow to activate T-type Ca2+ channels
(Crunelli et al. 1989
). An increase in the duration of
GABAAR-mediated IPSPs in relay neurons would
explain the slowing of oscillations prior to complete blockade. This is
consistent with the idea that it is primarily relay cell IPSPs that
determine spindle frequency (Bal et al. 1995b
).
In conclusion, the finding that SLOs with frequencies in the 4- to 9-Hz
range occur in rat thalamic slices will allow the extension of previous
observations in this preparation to oscillations that more closely
resemble in vivo spindles. The results presented here outline the
contributions of GABAA,
GABAB, AMPA, and NMDA receptors to SLOs. Combined
with data showing the strong influence of
[Mg2+]O on the
pharmacology of SLOs, our findings suggest several new and important
factors that may be involved in shaping spindle oscillations and
predisposing the thalamic circuit to hyper-synchronous, epileptiform
activity (Huguenard and Prince 1994a; Huntsman et al. 1999
; von Krosigk et al. 1993
).
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-06477 and NS-34774, the Pimley Research Fund, and the Schweizerische Stiftung fur medizinisch-biologische Stipendien.
Present address of D. Ulrich: Institute of Physiology, University of Bern, CH3012 Bern 8006, Switzerland.
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FOOTNOTES |
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Address for reprint requests: J. R. Huguenard, Dept. of Neurology, Stanford University Medical Center, Stanford, CA 94305 (E-mail: john.huguenard{at}stanford.edu).
Received 8 November 2000; accepted in final form 17 May 2001.
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REFERENCES |
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