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The Journal of Neuroscience, July 1, 2002, 22(13):5374-5386
Somatostatin Inhibits Thalamic Network Oscillations In
Vitro: Actions on the GABAergic Neurons of the Reticular
Nucleus
Qian-Quan
Sun,
John R.
Huguenard, and
David A.
Prince
Department of Neurology and Neurological Science, Stanford School
of Medicine, Stanford, California 94305
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ABSTRACT |
We examined the effects of somatostatin (SST) on neurons in the
thalamic reticular nucleus (RT) using whole-cell patch-clamp techniques
applied to visualized neurons in rat thalamic slices. SST, acting via
sst5 receptors and pertussis toxin-sensitive G-proteins, activated an inwardly rectifying K+ (GIRK) current
in 20 of 28 recorded cells to increase input conductance 15 ± 3%
above control and inhibited N-type Ca2+ currents in
17 of 24 neurons via voltage-dependent mechanisms. SST reversibly
depressed evoked EPSCs (eEPSCs) to 37 ± 8% of control without
altering their kinetics. SST-mediated inhibition of eEPSCs showed
short-term relief from block during 25 Hz stimulus trains. SST also
reduced the frequency (33 ± 8%) but not the amplitude of
miniature EPSCs (mEPSCs). These data indicate that SST mediates presynaptic inhibition of glutamate release onto RT neurons. In current-clamp recordings, SST preferentially inhibited burst discharges mediated by near-threshold corticothalamic EPSPs and intracellularly applied depolarizing currents. SST had powerful effects on in vitro intrathalamic rhythms, which included a shortening of the duration and a reduction in spike count within each oscillatory event.
Furthermore, there was a paradoxical increase in the synchrony of
epileptiform oscillations, likely mediated by a suppression of the
responses to weak synaptic inputs in RT. We conclude that SST
suppresses discharges in RT neurons, via presynaptic inhibition of
glutamate release and postsynaptic activation of GIRK channels, leading
to the dampening of both spindle-like and epileptiform thalamic network
oscillations. SST may act as an important endogenous regulator of
physiological and pathological thalamocortical network activities.
Key words:
somatostatin; EPSCs; GIRK channels; CA2+ channels; neural network; epilepsy
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INTRODUCTION |
The thalamocortical network
underlies major brain rhythms activated during sleep, wakefulness, and
dreaming (Llinas and Ribary, 1993 ; Steriade et al., 1993) and during
3-4 Hz spike-wave discharge (SWD) as seen in absence seizures
(Williams, 1953; Prince and Farrell, 1969; Steriade and Llinas, 1988;
von Krosigk et al., 1993; Huguenard and Prince, 1994) (for
review, see Huguenard and Prince, 1997). It has been shown that
classical neurotransmitters such as norepinephrine, released from
neurons in the brainstem, suppress the generation of synchronized
activities and promote sensory processing by depolarizing neurons of
thalamic reticular and relay nuclei (McCormick and Prince, 1987, 1988).
Anatomical studies have demonstrated abundant peptidergic projections
into mammalian thalamus; however, very little is known about the
physiological roles of these inputs, with the exception of
cholecystokinin (CCK) (Cox et al., 1995, 1997). Unlike classical
neurotransmitters, release of neuropeptides is generally thought to
depend on high-frequency neuronal discharges (5-40 Hz) (Jan and Jan,
1982; Cropper et al., 1990; Wagner et al., 1993; Weisskopf et al.,
1993; Williams and Johnston, 1996). Thus peptides may be preferentially
released from neurons during certain forms of rhythmic oscillations or elevated neuronal activity (Vezzani et al., 1999), such as occur in
absence seizures and spindle waves within the thalamocortical circuit.
Somatostatin (SST) is a peptide neurotransmitter present in the
terminals of inhibitory interneurons in many brain regions, including
cortex (Kawaguchi and Kubota, 1996; Chow et al., 1999), hippocampus
(Buckmaster and Jongen-Relo, 1999), and striatum (Kubota and Kawaguchi,
2000). In human, primate, cat, and mouse thalamus, SST is colocalized
with the inhibitory neurotransmitter GABA in neurons of the thalamic
reticular nucleus (RT) that project to thalamocortical relay cells
(Graybiel and Elde, 1983; Bendotti et al., 1990; Burgunder and Young,
1992). SST activates five distinct G-protein-coupled receptors
(sst1-5) (Hoyer et al., 1995), which are widely
expressed in the mammalian CNS (Dournaud et al., 1996; Stroh et al.,
1999). SST has been linked to many complex brain functions
(Matsuoka et al., 1991) and CNS disorders such as Alzheimer's
(Epelbaum, 1986) and Huntington's diseases (Epelbaum, 1986). Studies
in hippocampus suggest that a loss of SSTergic interneurons occurs in
epilepsy (Sloviter, 1987; Buckmaster and Jongen-Relo, 1999). Recent
immunocytochemical studies in rat indicate a high level of
sst5 receptor expression in RT with lower levels of other SST receptor subtypes in this and other thalamic nuclei (Dournaud et al., 1996; Stroh et al., 1999). SST has recently been shown to suppress GABA release from terminals of RT neurons (Leresche et al., 2000). These findings are of particular interest, because RT provides major GABAergic inputs to thalamocortical neurons
and is a key structure in the generation of intrathalamic oscillations
and the spike-wave discharge (SWD) of absence seizures (von Krosigk et
al., 1993; Huguenard and Prince, 1994; Huntsman et al., 1999). We
examined the effects of exogenous activation of SST receptors on RT
cells and on network oscillations elicited in an in vitro
thalamic slice preparation.
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MATERIALS AND METHODS |
Slice preparation. All experiments were performed
using a protocol approved by the Stanford Institutional Animal Care and Use Committee. Young Sprague Dawley rats [10-16 d old (P10-16)] were deeply anesthetized with pentobarbital sodium (55 mg/kg) and
decapitated. The brains were quickly removed and placed into cold
(~4°C) oxygenated slicing medium containing (in
mM): 2.5 KCl, 1.25 NaH2PO4, 10.0 MgCl2, 0.5 CaCl2, 26.0 NaHCO3, 11.0 glucose, and 234.0 sucrose. Tissue
slices (300-400 µm) were cut in the horizontal plane using a
Vibratome (TPI, St. Louis, MO), transferred to a holding chamber, and
incubated (35°C) for at least 1 hr before recording. Individual
slices were then transferred to a recording chamber fixed to a modified
microscope stage and allowed to equilibrate for at least 30 min before
recording. Slices were minimally submerged and continuously superfused
with oxygenated physiological saline at 4.0 ml/min. Recordings of
G-protein-coupled inward rectifier potassium (GIRK) currents were
obtained at 35 ± 1°C, whereas Ca2+
currents were recorded at room temperature (23°C). The physiological perfusion solution contained (in mM): 126.0 NaCl,
2.5 KCl, 1.25 NaH2PO4, 2.0 MgCl2, 2.0 CaCl2, 26.0 NaHCO3, and 10.0 glucose. These solutions were
gassed with 95% O2/5% CO2
to a final pH of 7.4.
Whole-cell patch-clamp recording. Whole-cell recordings were
obtained using visualized slice patch techniques (Edwards et al., 1989)
and a modified microscope (Zeiss Axioskop) with a fixed stage. A
low-power objective (2.5×) was used to identify the various thalamic
nuclei, and a high-power water immersion objective (40×) with Nomarski
optics and infrared video was used to visualize individual neurons.
Recording pipettes were fabricated from capillary glass obtained from
World Precision Instruments (M1B150F-4), using a Sutter Instrument P80
puller, and had tip resistances of 2-5 M when filled with the
intracellular solutions below. An Axopatch1A amplifier (Axon
Instruments, Foster City, CA) was used for voltage and current-clamp recordings. Access resistance in whole-cell recordings ranged from 4 to
12 M, and 50-75% of this was electronically compensated. Current
and voltage protocols were generated using PCLAMP software (Axon
Instruments). The following software packages were used for data
analysis: Clampfit, PStat (Axon Instruments), SCAN (courtesy of J. Dempster, Strathclyde), Winplot (courtesy of N. Dale, St. Andrews
University), Origin (Microcal), and locally written programs, Metatape
and Detector (J. R. Huguenard).
For recordings of network oscillations (multiunit extracellular and
current clamp, e.g., see Figs. 9, 11), the physiological perfusion
solution listed above was used, but with 1.2 mM
MgCl2. For recording and isolation of
Ca2+ currents in thalamic slices, the
external solution was composed of (in mM): 120 NaCl, 20 tetraethylammonium chloride (TEACl), 3 KCl, 2.5 CaCl2, 2 MgCl2, 10 HEPES, 5 CsCl, 1 4-aminopyridine (4-AP), and 1 µM tetrodotoxin (TTX), pH 7.3, osmolarity
adjusted to 292 mOsm/l. This solution was gassed with 100%
O2. The pipette solution contained (in
mM): 117 Cs-gluconate, 13 KCl, 1.0 MgCl2, 0.07 CaCl2, 0.1 EGTA, 10.0 HEPES, 10 TEACl, 2.0 Na2-ATP,
and 0.4 Na-GTP, pH 7.4, osmolarity adjusted to 280 mOsm/l. For
recording and isolation of GIRK currents, the physiological perfusion
solution contained (in mM): 98.5 NaCl, 30 KCl,
1.25 NaH2PO4, 2.0 MgSO4, 2.0 CaCl2, 26.0 NaHCO3, and 10.0 glucose. This solution was
gassed with 95% O2 and 5%
CO2 to a final pH of 7.4. Pipette saline was modified according to Sodickson and Bean (1996) and was composed of (in
mM): 100 K-gluconate, 13 KCl, 9 MgCl2, 0.07 CaCl2, 10 EGTA, 10.0 HEPES, 2.0 Na2-ATP, and 0.4 Na-GTP, pH
adjusted to 7.4 and osmolarity adjusted to 280 mOsm/l. This
solution was also used as pipette saline for current-clamp recordings.
Network oscillations. Extracellular multiple-unit activities
were recorded using monopolar tungsten electrodes (0.2-2 M; Frederick Haer, Brunswick, ME) and a Grass amplifier (bandwidth, 0.3-3
kHz). All data were digitized (1-2 kHz) and stored using Axotape
software (Axon Instruments). Bipolar extracellular stimuli were
delivered through sharpened tungsten electrodes.
A software Schmidt trigger was used to detect spikes. The
"duration" of an oscillation was measured as the time from stimulus to last spike burst. Bursts were defined as four spikes occurring within 50 msec. The "total spike activity" of an oscillation was defined as the number of spikes occurring between the time of the
stimulus and the end of the recording sweep. To quantify the degree of
synchrony and the duration of the intrathalamic oscillations, autocorrelograms were constructed from the extracelluar multiple-unit data, typically with a bin size of 5-10 msec. Results from five consecutive oscillations were summed for each plot. The oscillatory activity in the autocorrelograms was quantified by three measures: time
constant of oscillation decay, an indication of duration as measured by
the rate of fall-off from the central to the satellite peaks;
oscillatory index (OI), a normalized measure of synchrony estimated
from ratio of the first anti-peak (valley) and peak amplitudes (a
valley to peak ratio of 0 would be completely synchronous and would
have an OI of 100%); and finally the interpeak interval, or period,
which is indicative of the main frequency component of the oscillation
(cf. Cox et al., 1997; Huntsman et al., 1999).
Immunocytochemistry. P15 rats were deeply anesthetized with
sodium pentobarbital and perfused through the heart with 100 ml of 0.1 M PBS 0.9%, pH = 7.4, followed by 300 ml of
4% paraformaldehyde in 0.1 M phosphate buffer.
The brains were removed and postfixed for 1 hr in the same fixative and
then sectioned at 35 µm with the Vibratome. Sections were blocked in
10% normal goat serum and then incubated for 16 hr in a mixture of
rabbit GIRK1 (Oncogene, 1 µg/ml) and mouse SST
antisera (Chemicon, 1:30) diluted in PBS and 0.2% Triton X-100. After
the sections were rinsed in PBS, they were incubated in a mixture of
Alexa goat anti-rabbit Texas Red and Alexa goat anti-mouse fluorescein
(Chemicon; 5 µg/ml each) for 2 hr. Sections were mounted on slides
and coverslipped using Vectashield mounting media (Vector Labs). Double
immunofluorescence was assessed with a laser confocal microscope (model
2010; Molecular Dynamics).
Drugs. Drugs were applied focally through a multibarrel
microperfusion pipette that was positioned within 1 mm of the cell. SST
analogs were made as follows. Concentrated SST (Peninsula Labs,
Belmont, CA) stock solutions were dissolved in ultra-pure water to a
final concentration of 0.1 M and stored in a
 70°C freezer. Stock SST solutions were diluted in physiological
saline to final concentrations of 100 nM to 0.5 µM 1 hr before use. Unless noted otherwise, a
concentration of 100 nM was used. Concentrated cyclo-SST (Peninsula Labs), BIM23052
(D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2), nc8-12
[D-Phe-c(Cys-Tyr-D-Trp-Lys-Abu-Phe),
locally synthesized], and octreotide
(D-Phe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol; American Peptide, Sunnyvale, CA) solutions were also stored at 70°C.
Aliquots were diluted to a final concentration in physiological
solution just before use and applied via multibarrel focal perfusion.
GTP- -S, GDP- -S, and pertussis toxin were purchased from Sigma
(St. Louis, MO). The following ion channel blockers were used:
bicuculline methiodide (BMI; Sigma),  -agatoxin-TK (Sigma),
-conotoxin-GVIA (-CgTx-GVIA, Sigma), CsCl (Sigma), TEACl (GFS
Chemicals, Columbus, OH), TTX (Sigma), ZD7288 (Tocris, Ballwin, MO),
and tertiapin-Q (gift of Dr. Zhe Lu, Department of Physiology,
University of Pennsylvania, Philadelphia, PA).
Statistics. All data are presented as mean ± SEM
unless stated otherwise. Analysis by Student's t test was
performed for paired and unpaired observations unless stated otherwise.
p values of <0.05 were considered statistically significant.
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RESULTS |
As reported previously, GIRK1
immunoreactivity was heterogeneously distributed in the thalamus with
moderate labeling in reticular nucleus (Fig.
1A1) and more intense
staining in the relay neurons of the dorsal thalamus (Fig.
1A1) (cf. Ponce et al., 1996). For these studies we
compared RT with the adjacent ventrobasal (VB) complex.
GIRK1 immunoreactivity was found on somata and
dendrites in both VB and reticular neurons (Fig.
1A1,B, small white arrows). By
contrast, SST immunoreactivity was not detected in somatodendritic regions but only found in linearly distributed puncta, presumably along
fibers, in both VB and RT (Fig. 1A2).
SST-immunoreactive puncta were much more prominent in the reticular
nucleus than VB (Fig. 1A2,B, large
red arrows), suggesting an important role of SST in regulating the
function of RT neurons. High-magnification images showed that these
SST-immunoreactive fibers were closely associated with somata and
dendrites of GIRK1-immunoreactive neurons in both
VB and RT (Fig. 1B, large red arrows).
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Figure 1.
Photomicrographs showing localization of
GIRK1 and SST immunoreactivity (IR) in rat thalamic slices.
A, Confocal laser scan image of a thalamic slice showing
GIRK1-IR (A1, red) and SST-IR
(A2, green) in the reticular
(RT) and adjacent ventrobasal (VB)
nucleus. Dotted line in A1 and
A2 demarcates the border between RT and VB.
B, Superimposed images from A1 and
A2 showing SST-immunopositive puncta
(green pixel clusters indicated by large
red arrows, also in A2) and adjacent
GIRK1-immunopositive somata (B, small
white arrows). Scale bars, 10 µm.
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SST activation of K+ currents in RT neurons
RT neurons, visually identified in slices and recorded with
whole-cell techniques, had characteristic electrophysiological features
(Huguenard and Prince, 1994), including brief action potentials and
high-frequency burst discharges (Figs.
2A,
8A1,B1,C1,D, 9A2,B1-2). The effects of SST on
K+ currents were examined using
voltage-ramp commands from 130 to 50 mV. SST (100-500
nM) increased the magnitude of the ramp-evoked current responses in the majority (20 of 28) (Fig.
2B1) of neurons, indicating that the effects of SST
were mediated through an increase in membrane conductance. The ramp
current amplitude in the remaining neurons was either unaffected (5 of
28) or reduced (3 of 28) by SST, suggesting a closing of
K+ channels and a decrease in membrane
conductance in some cells. The SST-mediated activation of
K+ channels in RT produced a 0.8 ± 0.1 nS conductance change, when measured at a membrane potential near
rest (70 to 50 mV). This represented an increase of 15 ± 3%
above baseline input conductance (5.2 ± 0.2 nS), measured in the
same way.
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Figure 2.
SST activates K+-selective
currents in RT neurons. A1, Current-clamp recording
showing typical responses of an RT neuron to a series of current steps
ranging from 150 to +100 pA. A2, Currents elicited in
a voltage-clamped RT neuron by voltage ramps (120 to 60 mV,
over 1 sec) from a holding potential of 50 mV (holding
current: 100 pA; 30 mM
[K+]out) before, during, and
after application of 500 nM SST application.
B, Currents elicited by voltage ramps in an RT neuron in
the absence (control and wash averaged) and presence of 500 nM SST in 2.5 mM (B1) and 30 mM [K+]out
(B2). Superimposed traces in
B2 also show the lack of effect of 100 nM
Cyclo-SST on K+ currents. Each current trace was
averaged from 10 consecutive responses. Horizontal dashed
lines in insets: 0 pA. Vertical dashed
lines in each panel show expected
K+-dependent reversal potentials for SST-evoked
responses with [K+]in = 113 mM. Insets, SST-sensitive currents obtained
by subtracting normalized control [(control + wash)/2] from SST
traces. Calibration: B1 inset, 10 mV, 150 pA; B2 inset, 30 mV, 300 pA. Vertical dashed
lines in B1 and B2 insets
indicate reversal potential of 87 and 50 mV, respectively.
C1, Normalized current amplitude at 130 mV with 30 mM [K+]out in control
solution and during SST (100 nM) application and washout in
17 RT neurons. In this and the following figures, relative
K+ current indicates the ratio of the holding
currents at 130 mV in drug (e.g., SST, Ba2+, etc.)
and control conditions. Dashed lines connect control and
+SST responses for each cell.  , Inhibition;  , enhancement.
C2, Mean K+ current in control
solution and after perfusion of SST (n = 28;
**p < 0.01 vs control), 100 nM
octreotide (n = 6; p < 0.05 vs
control), 100 nM Bim23052 (n = 5;
**p < 0.01 vs control), or SST + Cyclo-SST
(n = 6; not significant vs control).
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The K+ selectivity of the SST-sensitive
currents was studied by examining their reversal potential and overall
conductance in perfusates containing different
[K+]o
concentrations. Reversal potentials examined in 2.5 mM
[K+]o [98 ± 4 mV vs 99 mV theoretical value (cf. Sodickson and Bean,1996);
n = 20] and 30 mM
[K+]o, (45 ± 3 vs 48 mV theoretical value; n = 7) agreed well
with those calculated from the Nernst equation for potassium. The
relative conductance was larger when
[K+]o was
increased to 30 mM (250 ± 45% increase;
n = 7), a finding consistent with previous studies of
GIRK currents (Sodickson and Bean, 1996). Thus the SST-induced current
was highly K+ selective.
The effects of SST on K+ currents were
assessed in one series of experiments in a perfusate containing 30 mM
[K+]o to maximize
the GIRK current responses, and TTX (1 µM) and Cd2+ (200 µM) to block
inward voltage-gated Na+ and
Ca2+ currents and outward
Ca2+-activated
K+ currents. Under these conditions 100 nM SST increased conductance in 7 of 11 RT cells (Fig.
2A2,B2). In the majority of cells tested, the current-voltage plot of SST-sensitive current in RT neurons in
both 2.5 mM (15 of 20) and 30 mM K+ (4 of 7)
demonstrated inward rectification (Fig. 2B1). In the remaining cells, SST-sensitive currents were more linear (Fig. 2B2). These properties are similar to those reported
previously for baclofen- or neuropeptide Y (NPY)-elicited GIRK currents
in rat thalamic neurons (Sun et al., 2001a,b).
SST activation or inhibition of K+ currents in
VB neurons
For comparison we also examined the effects of SST on GIRK current
in VB neurons. Interestingly, 100 nM SST inhibited
K+ currents in 25 of 41 VB neurons and
activated or had no effect on them in 16 of 41 VB cells. The
SST-induced change in input conductance in the former group of VB cells
was 0.6 ± 0.1 nS, which amounted to a 12 ± 2% decrease.
These results suggest that SST differentially regulates
K+ currents in RT and VB neurons.
The effects of SST on K+ currents in RT neurons
are predominantly mediated by sst5 receptors
To identify the SST receptors involved in modulation of
K+ currents in RT neurons, we applied
selective agonists. NC8-12 (100 nM), a selective
sst2 agonist (cf. Tallent et al., 1996) had no significant effects on K+ currents in RT
neurons (94 ± 6% of control level; n = 7; not significant). Octreotide (100 nM), an agonist for
sst2, sst3, and
sst5 receptors (Hoyer et al., 1995), activated
GIRK currents in RT (130 ± 10% of control; p < 0.05; with six of six neurons responding). Bim23052 (100 nM), a selective sst5
agonist (cf. Tallent et al., 1996), also mimicked the effects of
activation of K+ currents by SST in RT
neurons (129 ± 6% of control; p < 0.01; with
five of five neurons responding). These results suggest that the
sst5 receptor subtype mediates the activation of
K+ currents in RT. Cyclo-SST (100 nM), a specific SST receptor antagonist, blocked
the effects of the peptide on K+ currents
(Fig. 2C2) (n = 5 of 6 cells), suggesting
that these effects were mediated via activation of G-protein-coupled
sst receptors, rather than nonspecific actions on the channels.
In VB neurons, the pharmacological profiles of SST-mediated responses
were more complex. Although NC8-12 (100 nM) decreased resting K+ conductance in 6 of 7 cells
tested (85 ± 7% of control; p < 0.05; data not
shown), bim23052 (100 nM) and octreotide (100 nM) consistently increased membrane conductance
(112 ± 8% of control, n = 12 of 14 cells,
p < 0.01, and 119 ± 12% of control,
n = 7 of 8 cells, p < 0.01, respectively). These results suggest that multiple SST receptors
coexist in VB neurons, with sst2 receptors being
negatively coupled and sst5 and possibly
sst3 positively coupled to GIRK channels.
GIRK channels are the principal targets of SST receptor activation
in RT neurons
We characterized the types of K+
channels modulated by SST by applying SST alone and in the presence of
various ion channel blockers. In these experiments, a fast solution
exchange method was used (cf. Sun et al., 2001a). We found that the
effects of SST on K+ currents occurred
within 1 min (Figs. 2A2, 3A1,
8A1), consistent with direct G-protein-mediated
responses (cf. Leubke and Dunlap, 1994; Sodickson and Bean, 1996; Viana
and Hille, 1996) and similar to responses elicited by
NPY1 receptor-mediated activation of GIRK
channels in these neurons (Sun et al., 2001a). The activation of
K+ currents by SST was inhibited by a low
concentration of Ba2+ (0.1 mM)
(Fig.
3A,B),
which blocks GIRK currents in various CNS neurons (Sodickson and Bean,
1996; Sun et al., 2001a). Cs+ (500 µM), which also blocks GIRK and other
K+ channels, produced a similar block of
SST effects (Fig. 3B2). Tertiapin-Q (20 nM), a very potent peptide inhibitor of
recombinant GIRK1/4 channels (Jin and Lu, 1999),
occluded 80% of SST effects in RT (Fig. 3B2). To exclude
the possibility of an effect of Ba2+ on
constitutive GIRK channels, we applied
Ba2+ (0.1 mM) before
SST application. We found that Ba2+ alone
reduced input conductance by 13 ± 5%, presumably because of
blockade of constitutively active GIRK currents (Fig. 3C)
(n = 5). SST had no effects in the presence
Ba2+ (Fig. 3C). These results
suggest that GIRK channels are the principal targets for SST-mediated
modulation of excitability in RT neurons.
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Figure 3.
Effects of SST on K+ currents
were occluded by 0.1 mM Ba2+, 0.1 mM Cs+, and 10 nM
tertiapin-Q. A1, Continuous current record of experiment
showing reversible effects of local perfusion with control solution,
200 nM SST, and SST + 0.1 mM
Ba2+. Dashed line, Baseline holding
current; vertical lines, responses to 100 msec voltage
steps from 60 to 75 mV, applied at 1 Hz. A2,
Left, Individual current responses in the four
conditions of A1. SST elicited an outward shift of holding currents
(SST), reversed by concurrent
Ba2+ perfusion (SST + Ba2+). A2,
Right, Current responses aligned to baseline level
demonstrate changes in conductance under different conditions.
B1, Currents elicited by voltage ramps (130 to 0 mV, 1 sec) in 30 mM external K+ in an RT
neuron. The increased conductance produced by SST was occluded by 0.1 mM Ba2+. B2, Summary
of effects of various ion channel blockers on SST-evoked currents in RT
neurons. *p < 0.05, **p < 0.01 vs SST alone (n = 5-12 for each treatment).
C1, Continuous current record of experiment showing
occlusion of SST effects by 0.1 mM
Ba2+. Dashed line, Baseline holding
current; vertical lines, responses to 100 msec voltage
steps from 60 to 80 mV, applied at 1 Hz. C2, Summary
of Ba2+ pre-perfusion on SST-evoked currents in RT
neurons. *p < 0.05 (n = 4).
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Modulation of high-voltage-activated but not low-voltage-activated
Ca2+ currents by SST
Reliable whole-cell voltage-clamp
recordings of voltage-gated Ca2+ currents
were obtained in 24 of 58 RT neurons (Figs. 4,
5, 6). Effective voltage and space clamp and the efficacy of voltage control
in these neurons were verified using criteria described in a previous
study (Sun et al., 2001a).
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Figure 4.
Effects of SST receptor agonists and baclofen on
whole-cell Ca2+ currents. A,
Whole-cell voltage-gated Ca2+ currents evoked in an
RT neuron by voltage steps from a holding potential of 90 mV
(A1) or 70 mV (A2) to test potentials
between 80 and +10 mV. A3,
I-V curves measured from peak
Ca2+ currents in A1 and
A2 showing combined LVA and HVA current amplitudes ()
or HVA currents (). B1, LVA () and HVA ()
currents that were elicited in an RT neuron by command steps of 60
and 20 mV from holding potentials of 90 and 70 mV, respectively.
HVA currents (and their associated tail currents) were specifically
reduced by 500 nM SST (arrow).
Inset, HVA Ca2+ tail currents
elicited in control (black trace) and SST
(gray trace). Calibration: 2 msec, 400 pA.
B2, Time-series measurements showing that the effects of
SST were specific to HVA currents () and reversible in the same
neuron. LVA currents () were unaffected. B3, Summary
of effects of SST (100-500 nM), Bim23052 (200 nM), and baclofen (5 µM) on LVA and HVA
currents in RT neurons (n = 29).
**p < 0.01 vs control.
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Figure 5.
Voltage dependence of SST- and baclofen-mediated
inhibition of HVA currents. A1, Leak-subtracted HVA
Ca2+ currents elicited by 500 msec voltage ramps
from 70 to +20 mV in control and in the presence of SST (200 nM) or baclofen (5 µM). A2,
Plot of inhibition of HVA Ca2+ currents by SST and
baclofen versus membrane potential (n = 6).
B1, Whole-cell, leak-subtracted CA2+
currents elicited by a test potential of 0 mV from a holding potential
of 70 mV in control (dark trace) and SST (light
trace) showing slowed activation of Ca2+
currents by SST. Currents were normalized to approximately the same
peak amplitude. Leak currents were obtained in the presence of 200 nM Cd2+. Vertical scale bar applies only
to control currents. B2, Summary of inhibition of HVA
Ca2+ currents by SST measured at 3 and 20 msec from
the onset of voltage steps (n = 6;
**p < 0.01 vs 20 msec). C1,
Voltage-dependent removal of inhibition of HVA Ca2+
currents. Responses to 20 mV test pulses after 1 sec conditioning
steps to between +10 and 140 mV from an initial holding potential of
70 mV in the presence of SST. C2, Plot of voltage
dependence of the relief of inhibition for conditioning prepulses from
+10 to +130 mV obtained from four neurons.
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Figure 6.
Inhibition of HVA currents by SST was mediated by
pertussis-sensitive G-proteins activating via N-type calcium channels.
A1, Time series measurements showing the effects of
-CgTx-GVIA (1 µM) and Cd2+ (200 µM) on SST inhibition of HVA currents in an RT neuron.
ICa was measured at time indicated by in
A2. The inhibition of HVA currents by SST (200 nM) was essentially blocked by -conotoxin-GVIA (1 µM). A2, HVA currents recorded at points
corresponding to letters in A1.
B, Time series measurements (B1) and
current traces (B2) showing effects of intracellular
GTP--S (100 µM) on SST-mediated inhibition of HVA
currents in an RT neuron. Traces a-c in
B2 were taken at times indicated in B1.
C, Example of time series measurements
(C1) and current traces (C2) showing
effects of bath incubation with pertussis-toxin (0.5 mg/ml, >8 hr) on
SST-mediated inhibition of HVA currents
(ICa) in an RT neuron.
Traces in C2 were obtained at points
a and b in C1.
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Whole-cell Ca2+ currents recorded from RT
neurons demonstrate both T-type [low-voltage-activated (LVA)] and
high-voltage-activated (HVA) components (cf. Coulter et al., 1989).
T-type currents were elicited at test potentials positive to 70 mV
and inactivated during a 100 msec test pulse (Fig.
4A1,B1), whereas HVA currents were evoked
at potentials positive to 40 mV and showed less inactivation (Fig.
4A). Using a triple pulse protocol from an initial
holding potential of 90 mV, T-type and HVA currents were elicited,
and the effects of SST on both were examined (Fig.
4B1). In 17 of 24 RT neurons, SST reversibly reduced
HVA Ca2+ currents but had no effects on
T-type currents (Fig. 4B1,B2), results similar to those obtained with the GABAB
receptor agonist baclofen (Fig. 4B3). The mean
steady-state inhibition of HVA currents in responsive RT neurons was
20 ± 5%. These inhibitory effects of SST were mimicked by
bim23052 (200 nM; n = 5) (Fig.
4B3) and octreotide (100 nM;
n = 8), but not by NC8-12 (50 nM, n = 5), suggesting that
sst5 receptors mediated the effects of SST on HVA
Ca2+ currents.
Effects of SST on HVA Ca2+ currents
We examined the voltage-dependence of SST-mediated inhibition of
HVA currents by using voltage ramps (500 msec; 70 to +20 mV) to
elicit these currents (Fig. 5A1). The relative inhibition elicited with either SST or baclofen varied with membrane potential and
was a bell-shaped function of membrane voltage (Fig.
5A1,A2) (n = 7) (cf.
Hille, 1994; Jones and Elmslie, 1997; Dolphin, 1998). Maximum
inhibition occurred at membrane voltages near 0 mV. A small rightward
shift in the I-V curve was detected with either SST or baclofen (Fig. 5A). When steps to 0 mV were applied,
the time course of activation was clearly delayed by SST (Fig.
5B1, arrow) (cf. Bean, 1989; Sahara and
Westbrook, 1993). The degree of inhibition was greater at the onset
(28 ± 7; n = 7) (Fig. 5B2) than at 20 msec (13 ± 2%) (Fig. 5B1,B2) (cf. Sahara
and Westbrook, 1993; Leubke and Dunlap, 1994; Viana and Hille, 1996;
Dolphin, 1998; Sun and Dale, 1998). Relief of SST-mediated inhibition
of HVA currents was voltage dependent, as determined by analyzing the
effect of conditioning prepulses. Half-maximal relief occurred at +50
mV, with maximum effect at voltages >100 mV (Fig.
5C1,C2). In control solutions, these prepulses
had very little effect on HVA calcium currents (n = 4;
data not shown), indicating that the voltage-dependent relief of
inhibition apparent in these nonleak-subtracted traces was not caused
by changes in leak conductance. In summary, these results indicate that
inhibition of HVA Ca2+ currents by SST is
mediated by voltage-dependent mechanisms.
L-, N-, P/Q-, and R-type HVA Ca2+ channels
are present in rat thalamic neurons (Huguenard and Prince, 1992;
Kammermeier and Jones, 1997; Sun et al., 2001a). Because N- and
P/Q-type calcium channels are most susceptible to voltage-dependent
inhibition (for review, see Hille, 1994; Jones and Elmslie, 1997;
Dolphin, 1998), we examined SST modulation of these channels. The
effects of SST alone and in the presence of -CgTx-GVIA (1 µM) or -agatoxin-TK (200 nM) were studied
to determine whether the blocking action of SST and each toxin was
additive or occlusive. Results showed that -CgTx-GVIA virtually
eliminated SST-sensitive currents (5 ± 3% inhibition after
-CgTx-GVIA treatment, p < 0.05, vs 24 ± 3%
effects in control; n = 5) (Fig. 6A),
whereas -agatoxin had no significant effects on SST-mediated
inhibition of HVA currents (22 ± 4% inhibition after
-agatoxin treatment vs 20 ± 5% inhibition in controls; data
not shown; n = 5). These results suggests that N-type
HVA channels are the predominant target for inhibition by SST.
G-proteins are involved in modulation of K+ and
Ca2+ currents
The effects of SST on both HVA Ca2+
currents and K+ currents were blocked by
intracellular loading of GDP--S (100 µM;
n = 6; inhibition 8 ± 3 and 4 ± 1%,
respectively, not significant vs controls; data not shown). By
contrast, the inhibition of HVA Ca2+
currents by SST was irreversibly enhanced in cells loaded with GTP--S (100 µM; 40 ± 5% inhibition;
n = 4) (Fig. 6B). These data suggest
that G-proteins are involved in SST-mediated responses. In slices
treated with pertussis toxin (0.5 mg/ml; 8-11 hr of preincubation),
both T-type and HVA currents were indistinguishable from control
currents (data not shown). However, the effects of SST on both HVA
Ca2+ currents (n = 5;
6 ± 2%; not significant vs controls) (Fig. 6C) and
K+ currents (5 ± 3%;
n = 6; data not shown) were virtually abolished. This
suggests that pertussis toxin-sensitive G-proteins
(Gi/0) (cf. Hille, 1994) were involved.
Effects of SST on excitatory neurotransmission in RT neurons
Isolated EPSCs were evoked in RT neurons by electrical stimuli
delivered to the internal capsule (IC) in the presence of the GABAA receptor antagonist bicuculline (10-20
µM). Typical monosynaptic EPSCs had a reversal potential
~0 mV, an early rapidly decaying component at 70 mV, and both
rapidly and more slowly decaying components at positive potentials
(Fig. 7A1, inset).
The early, fast component exhibited a near linear current-voltage
relationship (Fig. 7A1, inset, ), consistent
with a prominent AMPA/kainate receptor component, whereas the late
phase was outwardly rectifying (Fig 7A1, inset,
 ), consistent with a prominent NMDA receptor contribution. EPSCs
evoked by a train of stimuli (40-80 msec interstimulus interval)
showed short-term plasticity, which was manifest most commonly as
short-term depression (five of eight cells) (Fig. 7A1,A2), occasionally as facilitation (two of
eight; data not shown) or early facilitation followed by late
depression (one of eight). Addition of SST reversibly reduced the
amplitude of evoked EPSCs in RT neurons (Fig.
7A2,A3; A4 for summary) without any
change in the kinetics of individual EPSCs (Fig. 7A2,
inset). This suggests the involvement of presynaptic
mechanisms. Furthermore, the inhibition by SST varied during each
train, with the largest inhibition occurring at first response within
the train (37 ± 8%; n = 8) (Fig. 7
A1; A4 for summary) and progressively less inhibition toward the end of the train (25 ± 5% inhibition at the fourth response in the train; n = 8;
p < 0.001; vs 37 ± 8% inhibition at the first
response; tested by one-way ANOVA analysis with Tukey-Kramer multiple
comparison test). When the relative inhibition within a train response
was normalized, short-term relief of inhibition occurred, with 30%
relief occurring at fourth stimulus compared with the first (Fig.
7A4) (p < 0.001),
suggesting that voltage-dependent modulation mediated by SST occurred
at this synapse (Parker and Dunlap, 1998; cf. Brody and Yue, 1999). The
SST-mediated differential effect on the EPSCs elicited at the start and
end of the train suggests changes in the short-term plasticity. Indeed,
in all eight neurons examined, the paired-pulse ratio increased during
SST application regardless of what forms of short-term plasticity
occurred in control conditions (Fig. 7A2).
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Figure 7.
Effects of SST on eEPSCs and mEPSCs in RT neurons.
A1, Current-voltage relations of initial EPSC of each
train in inset. Current amplitude measured at 6 msec
() and 25 msec () latency, as shown in 100 mV trace of
inset. Inset, Averaged EPSCs evoked by a
train of stimuli (20 Hz) recorded at different holding potentials
between 100 and +70 mV in the presence of bicuculline to block
GABAA receptor-mediated IPSCs. A2,
Representative EPSCs evoked by a 20 Hz stimulus train in control
(a, black traces), SST (200 nM)
(b, gray traces), and washout
(c, darker traces) recorded at a holding
potential of 70 mV in an RT neuron. The inset shows
superimposed, scaled responses of the first EPSCs in the train in
control and SST conditions. A3, Time course of SST-mediated
effects on the amplitude of the first EPSCs from the same experiment as
that depicted in A2. A4, Summary of effects of SST on the
amplitude of various EPSCs within the train responses
(***p < 0.001, *p < 0.05 vs first
EPSC; one-way ANOVA analysis with Tukey-Kramer multiple comparison
test; n = 8). B1, mEPSCs recorded at
Vhold = 80 mV in an RT neuron in the
presence of bicuculline (20 µM) and TTX (1 µM). Traces are control (top), 100 nM SST (middle), and 3 min after
washout (bottom). B2, B3, Cumulative
probability plots of mEPSC inter-events intervals (B2) and
amplitudes (B3) from 3 min epochs before, during, and 3 min
after the application of SST in cell of B1.
Inset, Mean frequency (B2) but not amplitude
(B3) of mEPSCs in RT (n = 6) was
significantly reduced by SST (*p < 0.05 vs control and
washout).
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We examined the effects of SST on mEPSCs recorded in the presence of 1 µM TTX and found that SST reversibly reduced the
frequency (Fig. 7B1,B2) (33 ± 8% decrease;
p < 0.05; n = 6) but not the amplitude
of mEPSCs in RT neurons (Fig. 7B3). These results suggest that SST acts presynaptically to reduce glutamate release.
SST preferentially inhibits burst discharges evoked by
near-threshold inputs
During both normal and epileptiform oscillations, RT neurons fire
bursts of action potentials and provide a major GABAergic input to
relay neurons (von Krosigk et al., 1993; Huguenard and Prince, 1994).
SST has previously been shown to directly inhibit release of GABA from
RT terminals onto thalamic relay neurons (Leresche et al., 2000).
Effects such as those described above that alter either presynaptic
excitatory neurotransmitter release or postsynaptic membrane
excitability could affect burst generation in RT neurons and thus
influence network oscillations. We next tested the possibility that SST
would inhibit RT neuron discharge evoked by excitatory synaptic inputs
onto these neurons.
In experiments with normal artificial CSF (see Materials and Methods),
extracellular stimulus trains (20-50 Hz; three to five stimuli)
applied to the internal capsule (Fig.
8A2) elicited EPSPs in
RT neurons (Fig. 8A1). The EPSPs included those
originating from both corticothalamic and thalamocortical inputs onto
RT neurons (cf. Turner and Salt, 1998; Blumenfeld and McCormick, 2000).
In the majority of cells tested (n = 12/15), we found
that EPSPs elicited by trains of stimuli (25 Hz) showed robust
facilitation characterized by supralinear summation (Fig.
8A1). Increases in stimulus intensity resulted in
increased EPSP amplitude and Na+-dependent
action potentials (Fig. 8A1). The probability of
spike firing, the number of spikes per stimulus, and the integrated area (millivolts × milliseconds) of the depolarization all
increased with stimulus intensity (Fig. 8A1) (cf.
Steriade, 1999). SST preferentially reduced the number of action
potentials evoked by small to moderate amplitude EPSCs (Fig.
8B1,B2). This effect can also be seen in the plot of Figure 8B3, which shows an inverse
relationship between control EPSP magnitude (area) and SST-dependent
spike inhibition (Fig. 8B3) (r = 0.71; p < 0.001; n = 84 responses).
These results suggest that during evoked network oscillations, SST may
preferentially regulate discharges of RT neurons evoked by threshold
EPSP inputs and have weaker effect on bursts generated by strong
inputs.
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Figure 8.
SST decreased the probability of evoked burst
discharge by near-threshold stimulation in RT neurons.
A1, Whole-cell current-clamp recordings of EPSPs and
action potentials elicited in an RT neuron by a train of four stimuli
delivered to the internal capsule. Increasing the stimulation intensity
resulted in larger EPSPs and a progressive increase in the number of
action potentials within each burst. Traces progressing from
left to right are responses evoked by 4, 6, 8, 10, and 12 µA stimulus trains (each pulse 40 µsec),
respectively. Inset, Compound EPSP area as a function of
the number of spikes within each burst, demonstrating the nonlinear
amplification of spike output with larger EPSP amplitudes. Both values
increased as a function of stimulus strength, but spike counts
increased dramatically with the strongest stimuli. A2, A
schematic depicting the thalamic circuit with recording sites.
M, Multibarrel local perfusion pipette;
IC, internal capsule; RT, thalamic
reticular nucleus. B1, B2, Burst
discharge elicited by threshold (10 µA, B1) and more
intense (30 µA, B2) stimuli in control (darker
traces) and SST (lighter traces) in the same
neuron as A. B3, Scatter plot of
percentage depression of spike number versus control depolarization
area in seven RT neurons. The solid line is a linear
regression with r = 0.71, p < 0.0001 (n = 84). C, Current-clamp
recordings of burst and tonic firing in an RT neuron elicited by direct
current injection (1 sec duration, +80 pA) in control condition
(left), during 100 nM SST application
(middle, gray trace), and after SST
washout (right). Note that SST reduced the repetitive
spiking (arrow) but had very little effect on resting
membrane potentials and initial low-threshold bursts (open
arrowhead). Dotted line represents resting
membrane potential level. C2, Time course of
SST-mediated effects on the total spikes per stimulus from the same
experiment as that depicted in A2. C3,
Voltage-current relation for the neuron of C1.
C4, Current-spike relation in control () and during
SST application () for six RT neurons. *p < 0.05 vs controls. D, Burst discharges in an RT neuron
elicited by direct current injection (100 msec duration, 100 pA;
responses on the left) or a train of four stimuli (20 Hz) delivered to the internal capsule (responses on the
right). SST (100 nM; light
traces) did not affect the bursts evoked by direct current
injection (left) but abolished the synaptically evoked
bursts (right). All responses in A, B,
and D were obtained in the presence of 20 µM BMI.
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The effects of SST on synaptic responses were usually accompanied by a
small membrane hyperpolarization (Fig. 8B1). Because SST had both presynaptic effects on glutamate release (Fig. 7) and
postsynaptic effects on K+ currents (Fig.
2) [and membrane potential hyperpolarization (Fig. 8B,C)], we next examined how the
two effects contributed to the SST-dependent suppression of
synaptically evoked bursts. We elicited complex spike (burst + tonic)
discharges in RT neurons with a range of direct depolarizing currents
and found that SST preferentially inhibited tonic firing, especially
that elicited by near-threshold currents (Fig.
8C1,C2,C4). SST increased the
membrane conductance (Fig. 8C3), an effect consistent with
activation of K+ channels. In contrast to
the effect of SST on EPSC-triggered bursts (Fig.
8B1-3), which occurred over a wide range
of EPSPs, the effects on direct-current-triggered bursts were only
manifest with the smallest stimulus intensity (50 pA; n = 4) (Fig. 8C4). When comparable burst discharges
were evoked in the same neuron by applying direct current (100 pA)
(Fig. 8D) and synaptic stimuli, the latter were more
powerfully inhibited. Note in particular how the train of EPSPs failed
to summate to trigger a regenerative burst after SST application in
Figure 8D. Taken together, these results suggest that
the bursts elicited by weak to moderate strength synaptic inputs are
more prone to suppression by SST.
Addition of the GABAA receptor antagonist BMI can
alter thalamic network oscillations such that RT excitability is
increased and epileptiform activity prevails (Fig. 11) (von Krosigk et
al., 1993; Huguenard and Prince, 1994; for review, see Huguenard and Prince, 1997). We next examined the effects of SST on RT cell bursts
under these conditions. To mimic the repetitive activation of
excitatory synapses during epileptiform 3 Hz oscillations, trains of
stimuli were applied to the internal capsule to elicit burst
discharges. Brief trains of 20-25 Hz stimuli were delivered at 3 Hz
with a total two to four trains within a train group. Train groups were
applied once per minute (Fig.
9A1). Repetitive bursts in RT
cells were elicited during the stimuli and were blocked by bath
perfusion of ionotropic glutamate receptor antagonists CNQX and APV
(data not shown; n = 4). In eight neurons tested, SST
produced a 6 ± 1 mV hyperpolarization and concomitantly inhibited the discharges elicited by the late (second through fourth) trains within a train group (75 ± 10% reduction in spike number;
p < 0.05; n = 5) but not the first
train within the cycle (10 ± 8%; p > 0.1;
n = 5) (Fig. 9A2). The inhibitory effects of
SST on firing could not be fully accounted for by SST-induced membrane
hyperpolarization, because direct depolarizing current injection was
unable to reverse the effects of SST on spike generation (60 ± 11% inhibition; n = 5) (Fig. 9, compare
A2c, A2b). In cells in which network
oscillatory responses were recorded, SST had very little effect on the
powerful bursts activated by extracellular stimuli (Fig.
9B1,B2, ) but exerted strong inhibitory
effects on the bursts activated by recurrent network EPSPs (Fig.
9B1,B2, arrows). As a result, both the
duration and spike count of oscillations were reduced by SST.
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Figure 9.
Effects of SST on RT neuronal responses
under conditions of bicuculline methiodide (10 µM)
application. A1, Whole-cell current-clamp recording of
burst discharges in an RT neuron elicited by stimuli to the internal
capsule. The stimulus train parameters are described in
Results. Dotted horizontal line indicates level
of resting membrane potential in control (70 mV). Filled
bar indicates the time when SST (500 nM) was
applied, and open bar indicates the time when
depolarizing direct current was applied to restore membrane potential
to control levels. A2, Expanded time base showing
stimulus train-evoked discharges in Control,
SST, and SST + D.C. injection.
Traces in A2 were obtained at points
a-c in A1. Note that SST
significantly reduced the number of spikes elicited in the second train
but had little effect on the first train. B1,
Intracellular recordings of evoked () and spontaneous
(arrow) bursts and EPSPs from an RT neuron in control
condition (bottom), during 100 nM SST
application (indicated by shaded bar), and after washout
(top). B2, Superimposed recordings from
the same experiment as that depicted in B1. Note that
SST (gray trace) had no effect on the initial
evoked discharges () and small effects on both resting membrane
potential (2 mV) and the initial spontaneous bursts
(first arrow), but it virtually abolished the
late spontaneous bursts (second and third
arrow) and also reduced the amplitude of late subthreshold
EPSPs (third arrow).
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Effects of SST on intrathalamic oscillations
in vitro
We studied the effects of SST on two variants of intrathalamic
oscillatory rhythms (Jacobsen et al., 2001): (1) spindle-like oscillations, with a dominant frequency of 6-8 Hz, that occur in
normal physiological saline, and (2) epileptiform, hypersynchronous activity with a dominant frequency of 3 Hz that results from addition of BMI.
In normal physiological solutions, extracellular activities evoked in
VB and RT by stimulating the IC are composed of repetitive burst
discharges over a period of 1-5 sec, similar to activity occurring
during sleep spindles (Fig.
10A1-4).
Fast Fourier Transform (FFT) analysis indicated that these oscillatory
activities had no predominant peak, although scattered local peaks
ranged from 1 to 7 Hz. During baseline recordings before application of
SST, both the number of spikes in each episode and the duration of the
network oscillations remained relatively constant over a period of
10s of minutes in these slices (Fig. 10A5).
However, when SST (100 nM) was applied, there was
an abrupt and reversible reduction in total spikes evoked per
oscillation to 45 ± 9% of control (Fig. 10A3,B) (p < 0.05). The reduction in neuronal activity was mediated predominantly by
a shortening of episode duration (50 ± 9% reduction) (Fig.
10A3) (n = 5; p < 0.05), because SST had little effect on the activity within the first
few cycles (Fig. 10A3). SST also reduced the total
FFT power throughout the entire frequency range measured (data not
shown). In slices in which oscillations were simultaneously recorded
from both RT and VB nuclei, the total "spindle-like" activity in
both nuclei was reduced to a similar extent (Fig.
10A5), indicating that SST inhibited spindle-like extracellular activities by suppression of reciprocal network activities.
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Figure 10.
Modulation of thalamic spindle-like oscillations
by SST in vitro. A1, A2,
Extracellular multiunit recordings of oscillatory activity in RT
(A1) and VB (A2) in response to single
extracellular stimuli in control solution (top 2 darker
traces; 15 min after the start of recording) and during 100 nM SST application (bottom 2 gray traces; 15 min after the start of SST application). A3,
A4, Contour plot of rate meter output (cf. Jacobsen et
al., 2001) depicting the time course of the same experiment in RT
(A3) and VB (A4) from recordings
as in A1 and A2. The
x-axis represents time within each evoked oscillation,
whereas the y-axis represents the time course throughout
the experiment. Time calibration for y-axis: 2 min.
Bar to left shows time of perfusion with
control solution (clear), SST (black),
and wash (gray). The z-axis
represents the spike intensity during a single evoked oscillation;
darker grays correspond to higher frequencies. Note
decrease in activity and shortening of episode duration during SST
application. A5, Effects of SST on total spikes per
event in the same experiment as in A1-A4.
B, Summary of effects of SST on spike counts in RT in
five experiments in different slices.
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We next tested the effects of SST in slices bathed with 2 µM BMI to block GABAA-mediated
IPSPs and enhance RT cell firing. Under these conditions, the duration,
synchrony, and strength of intrathalamic oscillations were greatly
enhanced (von Krosigk, 1993; Huguenard and Prince, 1994; Cox et al.,
1997; Jacobsen et al., 2001). This epileptiform
oscillatory activity occurred in episodes lasting up to 10 sec (Fig. 11 A, Table
1), with predominant frequency peaks near
3 Hz (Fig. 11D, Table 1). These highly synchronized epileptiform activities depend on the intrinsic properties of thalamic
neurons and GABAB receptor-mediated slow IPSPs
(Crunelli and Leresche, 1991; Steriade et al., 1993; Huguenard and
Prince, 1994; Ulrich and Huguenard, 1996). In these slices, addition of SST (100 nM) reduced the episode duration (65%
of control) (Fig. 11A,B, Table 1),
the time constant of oscillation decay (75% of control) (Table 1), and
the total spike count (65% of control) (Fig. 11A,
Table 1). Therefore, the predominant effect of SST on synchronized
intrathalamic oscillations is inhibitory. Interestingly, SST
paradoxically increased the oscillatory index (see Materials and
Methods) by 25% (Fig. 11C, Table 1), indicating an increase in synchrony. FFT analysis revealed that during baseline activities, the oscillation had multiple frequency components, with the primary peak power frequency in the 3.0-3.3 Hz range (Table 1) and secondary peaks between 2.5 and 4 Hz (Fig. 11D). During SST
application, the main peak frequency was slowed slightly to ~2.9 Hz
(Table 1), and secondary peaks were mostly attenuated (Fig.
11D). This reduced variability could account for the
enhanced synchrony produced by SST. In addition, it could also be
caused by reduction of spontaneous neuronal activities occurring
between burst cycles (data not shown). In summary, our results suggest
that exogenous activation of SST receptors results in novel changes
that include earlier termination of burst episodes and a paradoxical
alteration of the pattern of paroxysmal oscillations evoked in thalamic
slices.
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Figure 11.
Modulation of thalamic epileptiform oscillations
by SST in vitro. A, Extracellular
multiunit recordings of bicuculline-enhanced oscillatory activity in
response to single extracellular stimulus in control solution
(top, blue; 10 min after start of
recording), during 100 nM SST application
(middle, red; 10 min after onset of SST
application), and after SST washout (bottom,
green; 15 min after SST washout). The evoked rhythmic
discharges lasted >10 sec in control but only ~6 sec in SST.
B, Contour plot reflecting rate-meter output of the same
experiment. The z-axis represents the spike intensity
during a single evoked oscillation. x- and
y-axes are as in Figure 10, whereas in this figure the
z-axis is color coded: warmer color levels
(red, yellows) correspond to higher
firing frequencies. The oscillatory phasic activity evoked by internal
capsule stimulation continues throughout the sample period (~27
cycles over 10 sec) in control and washout conditions
(blue and green bars,
left). In the presence of 100 nM SST
(red bar, middle left), the spike
discharge ceased after ~6-7 sec. Time calibration is 5 min for
y-axis. C, Autocorrelograms of the same
experiment illustrating the overall decrease in activity (decreased
amplitude of the central peak) but increased synchrony (higher
peak-to-valley ratio) in SST (red, blue,
and green lines represent control, SST, and washout).
D, Three-dimensional color map of surface plots of FFT
analysis of the same experiment, which demonstrate the frequency
changes. The ordinate represents the time course throughout the
experiment (top blue bar, pre-drug; middle red
bar, SST; bottom blue bar, SST washout), whereas
the abscissa represents frequency and the z-axis
represents Fourier power. Time calibration for y-axis in
D as in B. Warmer color levels
(red, yellows) correspond to higher
Fourier power. In control conditions, rhythmic activity had a main peak
frequency of 3.5 Hz and a second peak at 3.2 Hz. Shortly after SST
application, the amplitude of main peak was reduced, its frequency
shifted to 2.9 Hz, whereas the second peak was virtually abolished.
This shift of frequency recovered toward baseline level in the washout.
In this experiment, the extracellular perfusate contained 2 µM bicuculline and 1.2 mM
Mg2+.
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DISCUSSION |
Modulation of GIRK channels
SST modulates several classes of K+
currents. For example, this peptide increases a linear "leak"
current in rat CA1 neurons (Schweitzer et al., 1998), activates or
blocks an inward rectifier K+ current
(Inoue et al., 1988; Dichter et al., 1990; Tallent et al.,
1996), increases or decreases delayed rectifier currents in cortical
neurons (Wang et al., 1989) and in rod and cone photoreceptors (Akopian
et al., 2000), and increases
Ca2+-activated
K+ currents in chick parasympathetic
neurons (White et al., 1997). By contrast, in rat RT neurons the
effects of SST (via activation of sst5 receptors)
on K+ currents were primarily mediated by
GIRK channels, because they were blocked by GIRK inhibitors, including
Ba2+, Cs+ and
Tertiapin-Q. When expressed in Xenopus oocytes, rat
sst2-5 receptors were activated by octreotide
and coupled to coexpressed GIRK1 channels,
whereas sst1 receptors did not couple to
GIRK1 (Kreienkamp et al., 1997). In the mouse
pancreatic cell line MIN-6, five SST receptor types,
sst1-5, were detected; however, only
sst5 receptor subtypes were found to be coupled
to GIRK channels (Smith et al., 2001). Our results in RT neurons are
thus consistent with these actions of SST in non-neuronal preparations.
The pharmacological characterization of receptor subtypes in the
present experiments was based on agonist effects studied in populations
of thalamic neurons. Octreotide (an sst2,
sst3, sst5 agonist) and the
specific sst5 receptor agonist BIM23052 mimicked
SST effects. It is unlikely that SST3
receptors are involved, because NC8-12, which has a high affinity
(0.09 nM) for SST3
receptors (cf. Tallent et al., 1996) and a low affinity for
SST5 receptors (>1000 nM),
did not mimic the effects of SST on K+
currents. Therefore, sst5 receptors are most
likely involved.
Four currently identified subtypes of G-protein-coupled inward
rectifier K+ channels (GIRK1-4) are
expressed in different regions of the CNS (for revi |
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ABSTRACT
INTRODUCTION
