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1 Department of Anatomy and Cell Biology and Neuroscience Program, George Washington University Medical Center, Washington DC 20037; 2 Institut National de la Santé et de la Recherche Médicale U114, College de France, 75005 Paris, France
Submitted 27 January 2003; accepted in final form 18 April 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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50% for
EPSCs and 23% for IPSCs. These data indicate that the spontaneous synaptic
activity recorded in the principal cells at E16 is primarily inhibitory,
action potential-independent, and based on the activation of GABAA
receptors that can be modulated by presynaptic GABAB receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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). From intracellular current-clamp recordings at
embryonic day 16 (E16), the postsynaptic responses generated in the principal
cells on vestibular-nerve stimulation were blocked by the co-application of
specific glutamate receptor antagonists,
D-2-amino-5-phosphonovaleric acid (D-APV) and
6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX)
(Peusner and Giaume 1994).
However, the spontaneous synaptic potentials were largely unaffected,
suggesting that such basic activity could be important under physiological
conditions. In neurons, spontaneous spiking activity can be produced either by
synaptic events reaching threshold for firing action potentials or by
nonsynaptic events generated from the intrinsic membrane properties of the
neuron. For example, we have shown that in embryonic vestibular nucleus
neurons, spontaneous synaptic activity does not necessarily reach the
threshold for firing action potential
(Peusner and Giaume 1994).
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Investigations on spontaneous activity in vestibular nucleus neurons have
been performed mainly using extracellular recordings to characterize the
spontaneous discharge rates from single units in the medial vestibular nucleus
(MVN) that were not identified as to morphological neuron type (for review,
see Darlington et al. 1995;
Dutia et al. 1992;
Johnston et al. 2001;
Yamanaka et al. 2000). In
these studies, GABA and/or GABAA and GABAB receptor
agonists (e.g., muscimol, baclofen, respectively) inhibited the spontaneous
discharge, whereas the GABAA receptor antagonists (e.g.,
bicuculline) typically diminished the inhibitory effects of GABA. The effects
of GABA on spontaneous discharge rate in MVN neurons was age dependent with
greater inhibition recorded in the older postnatal rats
(Giardino et al. 2002;
Him et al. 2001).
Using immunolabeling techniques, glycine has been identified in first-order
vestibular neurons (cat, Godfrey et al.
1977; rat and frog,
Reichenberger and Dieringer
1994). However, so far there have been no electrophysiological
studies demonstrating monosynaptic inhibitory responses from vestibular
nucleus neurons on vestibular-nerve stimulation (e.g.,
Straka et al. 1997). In
diverse systems, electrophysiological studies have indicated that GABA and
glycine may be coreleased at the same synapse (e.g., spinal cord interneurons,
Jonas et al. 1998; hypoglossal
motor neurons, O'Brien and Berger
1999; cerebellar Golgi cells,
Dumoulin et al. 2001; abducens
motor neurons, Russier et al.
2002). Further, a developmental shift from GABA to glycine
transmission was reported in the developing auditory system
(Kotak et al. 1998) and spinal
cord (Gao et al. 2001).
Accordingly, we suspect that GABA and glycine may play important and possibly
changing roles in the developing and mature central vestibular system.
Finally, there is mounting evidence for a crucial role of GABA
receptor-mediated events in vestibular nucleus neurons during the recovery of
function that follows unilateral peripheral vestibular lesions, known as
vestibular compensation (e.g., Cameron and
Dutia 1997; Giardino et al.
2002; Johnston et al.
2001; Tighilet and Lacour
2001; Yamanaka et al.
2000).
To our knowledge, there have been no studies on spontaneous postsynaptic
currents in embryonic vestibular nucleus neurons. Under physiological
conditions, we do not know what rules govern spontaneous excitatory and
inhibitory receptor-mediated events in vestibular nucleus neurons, although a
link is expected between input and output. In the present study, we
characterized spontaneous and miniature synaptic activity using whole cell
patch-clamp recordings on identified principal cells of the tangential nucleus
at E16. One objective was to define the characteristics of spontaneous
synaptic activity at a critical age (E16) when developmental change occurs in
the morphological and electrophysiological properties of the principal cells
(Peusner and Giaume 1997). The
second objective relates to lesion studies where some cells re-express
developmentally regulated genes during recovery and then repeat certain
aspects of their development (e.g., Han et
al. 1996; Iwahashi et al.
1996; MacFarlane and
Sontheimer 1997). Accordingly, our second objective was to add to
our knowledge of the developmental schedule of synaptic properties in
vestibular nucleus neurons, so that this data can be available for studying
synaptic events during vestibular compensation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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All of the observations were made on 16-day-old White Leghorn chick embryos
(Gallus gallus) obtained from CBT Farms (Chesterton, MD) as eggs that
were incubated in the laboratory until the desired age. The age of the embryos
was established by reference to the staging criteria of Hamburger and Hamilton
(1951). The animal protocols
were approved by the Institutional Animal Care and Use Committee of George
Washington University. Briefly, the embryos were removed from the egg and
decapitated, and the part of the cranium containing the brain stem and
cerebellum was placed in a dissecting dish containing oxygenated artificial
cerebrospinal fluid (ACSF) chilled to 1–2°C. The cerebellum, choroid
plexus, periotic capsule, and vestibular ganglia were removed from the brain
stem. The brain stem was glued to a vibroslicer tray, immersed in ice-cold
ACSF, and viewed under a dissection microscope while sections (300 µm
thickness) containing the tangential nucleus were cut on a vibroslicer
(VT1000S, Leica) using feather blades (Ted Pella). Because the anteroposterior
extent of the tangential nucleus is 450–500 µm at E16
(Peusner and Morest 1977), the
tangential nucleus was contained in two transverse slices of the brain stem.
ACSF solution contained (in mM) 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 1.3
MgCl2, 2 CaCl2, 26 NaHCO3, and 10
D-glucose. The pH of this solution was 7.2–7.4 after
saturation with 95% O2-5% CO2 at room temperature. The
osmolarity of the ACSF solution was 310 mOsm.
Brain slice superfusion and microscopy
After 1 h recovery at room temperature, the slices were transferred to
a small glass-bottom recording chamber (180 µl, Warner Instruments) and
kept in place by nylon threads glued to a U-shaped, flattened platinum wire.
The slices were superfused with heated ACSF (30–31°C) at a rate of
2–3 ml/min except for some experiments where the slices were perfused
with room temperature ACSF (21–23°C). The slices were viewed on a
fixed-stage microscope (Axioskop FS1, Zeiss Instruments), equipped with
Nomarski differential interference contrast optics and a x40
water-immersion lens (NA, 0.75). The recorded neuron and pipette movement were
viewed using an infrared light source (filter, 770 nm) that was detected by an
infrared-sensitive camera (Vidicon C2400-01, Hamamatsu) and observed on a
monitor (Sony). The microscope image was further magnified by placing a
x4 lens between the microscope and the camera. Image contrast and
shading were adjusted with a camera controller (C2400-01, Hamamatsu).
All of the drugs, except for CNQX and phaclofen, were prepared daily by dissolving in ACSF and then adding them to the superfusing ACSF to achieve the final concentrations. Tetrodotoxin (TTX, RBI) was applied in the voltage-clamp experiments to block voltage-dependent Na+ currents. Other drugs used included DL-2-amino-5-phosphonovaleric acid (AP-5; Sigma), bicuculline methochloride (Tocris), strychnine (Sigma), and baclofen (Sigma). CNQX (RBI) and phaclofen (RBI) were dissolved daily in dimethyl sulfoxide (DMSO; Fisher Scientific) at a concentration of 20 and 500 mM, respectively, which were then added to the ACSF solution to achieve the final concentrations.
Electrophysiology
Recording pipettes were pulled from borosilicate glass tubing (1.5 mm OD,
1.12 mm ID, thin-walled, World Precision Instruments) with a Brown/Flaming
horizontal pipette puller (P-87, Sutter Instruments). The pipette resistance
was 1–3M
. KCl pipette solution contained (in mM) 130 KCl, 10
EGTA, 10 HEPES, 1.0 CaCl2, and 2.0 Mg-ATP
(Gamkrelidze et al. 1998). The
pH of the solution was adjusted to 7.2 with KOH (8.0 N). Cesium gluconate
pipette solution contained (in mM) 135 Cs-gluconate, 1 EGTA, 10 HEPES, 0.1
CaCl2, and 2 MgCl2 The pH was adjusted to 7.2 with CsOH
(Cossart et al. 2000). The
osmolarity for both pipette solutions was adjusted to 270–290 mOsm.
Finally, 0.5% biocytin was made fresh daily and added to the pipette
solution.
Whole cell recordings were obtained using an amplifier in voltage-clamp
mode (Axopatch-1D, Axon Instruments). The recording pipette was advanced
through the slice under visual guidance using a piezo-electric manipulator
(PCS-5000, Burleigh Instruments). The Rs compensation was
set at 80% and lag 10 µs. Grounding was performed with an Ag-AgCl reference
electrode. Using KCl pipette solution, the membrane potential of the principal
cells was –66 ± 4 mV (range: –54 to –75 mV) and the
input resistance was 63 ± 5 M (range: 45–83 M;
n = 10). Using Cs-gluconate pipette solution, the input resistance of
the principal cells averaged 78 ± 7 M (range: 50–100
M; n = 12).
Biocytin processing of slices
During the recordings, the living principal cells were visualized using the
infrared camera, and their typical oval cell bodies were apparent between the
primary vestibular fibers in the lateral medulla oblongata
(Fig. 1, B and
C). In this study, we intentionally selected to record
from the principal cells and avoided recording from other neuron populations
to accumulate a maximal number of recordings from a homogeneous class of
neurons. Biocytin was loaded into the recording pipette so that all of the
recorded neurons were filled passively with biocytin during the recording
session. After recording, the slices were processed for conventional
fluorescence staining to reconfirm the morphology and neuronal type of the
recorded neuron. At E16, the principal cell exhibited three to seven primary
dendrites that arborized extensively in the dorsoventral plane. The cell's
axon coursed in a medial direction, as already described (e.g.,
Peusner and Giaume 1997).
Due to proximity of the dye-injected neuron to the surface of the slice (50–100 µm), no further sectioning of the slice was necessary before processing for biocytin staining. After the recording, the intact slice was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (overnight, refrigerated), rinsed with 0.1 M phosphate buffered saline containing 0.1% Triton X-100 (PBS-T) (3 times for 20–60 min), incubated in 50% ethanol in PBS-T (20 min), and placed in Streptavidin Alexa Fluor 647 (1:200) in PBS-T (overnight, room temperature). After rinsing in PBS-T (20 min), the slice was counter-stained with DiO (2 mg/ml dissolved in N,N-dimethyl formamide; Molecular Probes; 40 s), to enhance the appearance of the primary vestibular fibers. The slices were mounted on gelatin-coated slides, coverslipped with Fluoromount G, and observed on a Nikon Optiphot fluorescent microscope equipped with x4 (NA, 0.2) and x20 (NA, 0.65) objectives. Fluorescent images were captured with a slow-scan CCD camera (Spot RT, Diagnostic Instruments) connected to a personal computer. Alexa Fluor 647 was excited with a light of 600 to 650 nm wavelength, and the emission was filtered from 670 to 740 nm (filter set XF110–2NS32; Omega Optical). Immunostaining for Alexa Fluor 647 was visualized on the red channel, while DiO was visualized on the green channel.
Data acquisition and analysis
No correction was applied for the voltage offset observed after withdrawing
the pipette from the cell (
±3 mV) or for the liquid junction
potential (–3 mV). The reversal potentials were determined by
identifying the holding potential at which no synaptic current was observed.
During the experiment, the input resistance and series resistance were
determined by giving 5 mV hyperpolarizing pulses. If the membrane exhibited a
peak amplitude change in the capacitance currents >25% in response to the 5
mV pulse, the neuron was excluded from study. All of the data were obtained
using pClamp program (version 6.0.3; Axon Instruments), recorded with a DAT
tape recorder (DTR-1201, Biologic Instruments), and acquired with Axoscope 8.1
software (Axon Instruments). Synaptic currents were analyzed off-line using
Minianalysis program (version 5.6.3, Synaptosoft) with a minimal acceptable
amplitude set at 15 pA. Analysis of the postsynaptic currents included peak
amplitude, rise time (from 10 to 90% peak current), decay time (from 90 to 37%
amplitude return to baseline) and half-width (duration of event at 50% of peak
amplitude). The voltage-clamp data were digitized at 10 kHz and filtered at 2
kHz (pClamp program, Axon Instruments). All of the data are given as the means
± SE. Frequency differences were analyzed with the Student's
t-test for independent samples and considered significant with
P < 0.05. Amplitude distributions were compared with the
Kolmogoroff-Smirnoff (K-S) test with the significance level set at p
< 0.05. Correlations were analyzed using Prism software program (Graphpad),
with a two-tailed distribution and 95% confidence level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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The whole cell recordings presented here were obtained from a total of 94 identified principal cells at E16. First, spontaneous currents were recorded in voltage-clamp mode at different holding potentials with pipettes containing KCl solution and in control ACSF (Fig. 2, A1 and A2). In these experiments, the mean reversal potential for all of the spontaneous synaptic activity was +4.2 ± 1.3 mV (n = 4) because the excitatory and inhibitory events had similar expected reversal potentials due to chloride loading of the cell when using KCl electrodes (Fig. 2B). The mean frequency for these spontaneous postsynaptic currents (sPSCs) was 3.1 ± 0.9 Hz at –60 mV (n = 13). It was apparent that the frequency of spontaneous synaptic activity was temperature dependent because at –60 mV, the mean frequency recorded at room temperature (21–23°C) was 1 ± 0.4 Hz (n = 5), which was significantly less than that recorded at 30–31°C (p < 0.05).
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To distinguish between excitatory and inhibitory events in the same cell
without applying drug treatments that could interfere with the presynaptic
circuitry, we carried out further experiments using electrodes filled with
Cs-gluconate solution. In these experiments, the spontaneous excitatory and
inhibitory postsynaptic currents (sEPSCs and sIPSCs) recorded in the principal
cells could be separated by their voltage dependencies (Figs.
3 and
4). The currents were measured
at –60 and +10 mV, respectively, because these voltages approximated
their reversal potentials under our experimental conditions. The small inward
currents recorded in the principal cells at –60 mV had a reversal
potential around +15 ± 2mV(n = 5). Due to the difficulty in
detecting the small inward currents from background noise starting around
–40 mV, our estimated value for the reversal potential of the sEPSCs is
likely higher than actual. Accordingly, it was not possible to plot reliably
the I-V relations for the reversal potential of these sEPSCs.
However, according to the Nernst equation, these currents were likely sEPSCs
generated by glutamate (Hille
2001). In addition, the principal cells exhibited outward currents
at a holding potential of +10 mV, which had a mean reversal potential at
–72 ± 2.7 mV (n = 7), which is close to the chloride
equilibrium potential according to the Nernst equation
(Fig. 4B). Thus it is
likely that these currents represented sIPSCs generated by a chloride
conductance. In our subsequent experiments, the cells were recorded primarily
at –60 or +10 mV to distinguish the excitatory and inhibitory
spontaneous synaptic activity, respectively
(Cossart et al. 2000).
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Spontaneous synaptic activity is mainly inhibitory at E16
At a holding potential of –60 mV, the frequency of the inward currents was 0.6 ± 0.1 Hz (n = 27), whereas at +10 mV holding potential, the frequency of the outward currents was 2.3 ± 0.4 Hz (n = 30; Fig. 3, A1 and A2, respectively). Thus the ratio for excitatory and inhibitory events was about 1:4, indicating that at E16 the spontaneous synaptic activity in the principal cells was primarily inhibitory (Fig. 3B).
sEPSCs are mediated by the AMPA receptor
At –60 mV holding potential, the inward currents were blocked
completely by 10 µM CNQX, a specific AMPA/kainate (KA) receptor antagonist
(n = 5; Fig.
3C). Typically, these sEPSCs exhibited the fast kinetics
characteristic of AMPA events. However, in some cells (5/27) the
CNQX-sensitive events included some currents with slower kinetics, which in
other systems have been identified as KA receptor-mediated events
(Fig. 3A1,
arrow) (Cossart et al. 1998,
2002;
Frerking et al. 1998;
Kidd and Isaac 1999).
Accordingly, sEPSCs were identified as primarily AMPA receptor-mediated events
with a few KA currents present.
sIPSCs are mediated mainly by GABAA-receptors
At a holding potential of +10 mV, 74 ± 3% of the spontaneous outward
currents were blocked completely by the GABAA-receptor antagonist,
bicuculline (10 µM). The addition of the glycine-receptor antagonist,
strychnine (4 µM), blocked the remaining currents
(Fig. 5A1;
2,755 events, n = 15 cells). Consequently, the ratio for GABA to
glycine receptor-mediated events was 3:1
(Fig. 5B). Indeed, the
ratio for GABA versus glycine receptor-mediated events was similar whether
bicuculline or strychnine was applied first
(Fig. 5A2),
suggesting that strychnine did not affect the postsynaptic GABAA
receptors or presynaptic release in this preparation. To further exclude the
possibility that the high strychnine concentration (4 µM) could affect
GABAA events, we performed experiments using 1 µM strychnine
followed by 10 µM bicuculline (not shown). In the latter experiments, 32
± 7% sIPSCs were sensitive to strychnine and 68 ± 7% events were
sensitive to bicuculline (647 events; n = 4 cells). There was no
statistically significant difference (P > 0.05) between the ratio
for GABA to glycine receptor-mediated events obtained with 1 or 4 µM
strychnine. Accordingly, the doses used for strychnine (1 or 4 µM) and
bicuculline (10 µM) were considered to be specific for glycine and GABA
receptor-mediated events, respectively, and there was no cross-reactivity
detected in this preparation. Altogether, these data indicate that GABA was
the major neurotransmitter mediating the inhibitory spontaneous synaptic
activity in the principal cells at E16.
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Inhibition of sEPSCs and sIPSCs by GABAB receptors
To determine if postsynaptic GABAB receptors are present on the principal cells using KCl pipette solution, we recorded the postsynaptic effects of the potent GABAB receptor agonist, baclofen (10 µM), in the presence of 1 µM TTX and the receptor antagonists for GABAA (10 µM bicuculline), glycine (1 µM strychnine), NMDA (30 µM AP-5), and non-NMDA (10 µM CNQX). In these experiments performed at a holding potential of –65 mV, baclofen was added to the ACSF solution and induced an outward current (n = 6; Fig. 6A). The amplitude of this current was small (range: 10–40 pA) and remained stable throughout the application of the drug. After washout with control ACSF, the current decreased slowly back to baseline. This current was not observed when Cs-gluconate pipette solution was used (n = 3, not shown).
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To test whether activation of presynaptic GABAB receptors can modulate spontaneous synaptic activity, we recorded the presynaptic effects of baclofen using Cs-gluconate pipette solution, which blocked the potassium channels and prevented the activation of postsynaptic GABAB receptors. The effect of baclofen on the frequency and the amplitude of sEPSCs and sIPSCs was determined in the absence of TTX (Fig. 6, B1 and B2). The frequency of sEPSCs decreased from 0.87 ± 0.3 to 0.52 ± 0.2 Hz (40 ± 7%; n = 5) and the frequency of sIPSCs decreased from 3.2 ± 1 to 1.5 ± 0.5 Hz (53 ± 7%; n = 8). Both decreases were significant (p < 0.05). In addition, 3/5 cells exhibited significant change in the amplitude distribution of sEPSCs and 4/8 cells showed significant change in the amplitude distribution of sIPSCs, according to K-S statistical analysis (p < 0.05). When the GABAB receptor antagonist, phaclofen (500 µM) was added alone, no effect was observed on the frequency of sEPSCs (n = 3), but a slight increase in frequency of sIPSCs (6 ± 2%) was detected (n = 5; p < 0.05). Addition of baclofen (10 µM) 10 min after phaclofen application reduced the frequency of sEPSCs (26 ± 1%, n = 3) and sIPSCs (24 ± 4%, n = 5) but not so much as with baclofen alone (not shown). However, there was no statistically significant difference in the baclofen effect on sEPSCs frequency (26 vs. 40%) with or without the preapplication of phaclofen (p > 0.05), but there was a significant difference in the baclofen effect on sIPSCs frequency (24 vs. 53%; p < 0.05). These findings could be attributed to pharmacologically heterogeneous GABAB receptors on the presynaptic terminals (see DISCUSSION). Altogether, both pre- and postsynaptic GABAB receptors appear to modulate the spontaneous synaptic activity observed in principal cells at E16.
Most of the spontaneous synaptic activity consists of miniature events
To test whether the spontaneous synaptic activity was evoked by presynaptic firing, TTX (1 µM) was applied to block the action potentials mediated by the activation of voltage-dependent sodium channels (Fig. 7). At a holding potential of –60 mV, we compared the spontaneous synaptic activity recorded in control conditions and after the addition of TTX to determine the percentage of spontaneous synaptic events that are TTX-resistant. We found that 53 ± 7% of sEPSCs were TTX-resistant, the so-called miniature EPSCs, or mEPSCs (984 events; n = 7 cells; Fig. 7, A1 and A2). When the same protocol was applied while recording at a holding potential of +10 mV, 79 ± 4% of sIPSCs were TTX-resistant, the mIPSCs (3,969 events; n = 17 cells; Fig. 7, B1 and B2). These results indicated that the generation of sIPSCs in the principal cells was more independent of action potentials than sEPSCs.
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In addition to decreasing the frequency of spontaneous synaptic activity in the principal cells, the application of TTX decreased the number of large-amplitude events recorded, which is indicated by a shift to the left of the cumulative probability curves for both EPSCs and IPSCs (Fig. 8, A and B). The mean amplitude was 35.2 ± 0.4 pA for sEPSCs and 23.3 ± 0.5 pA for mEPSCs (n = 7 cells), whereas the mean amplitude was 47.2 ± 0.7 pA for sIPSCs and 36.8 ± 0.4 pA mIPSCs (n = 6 cells), with no statistical difference (p > 0.05).
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While TTX did not produce a noticeable change in the kinetics of the mEPSCs and mIPSCs as compared with their controls (Fig. 8, A and B, insets), the mEPSCs exhibited statistically faster rise time, faster decay time, shorter duration, and smaller amplitudes compared with the mIPSCs (p < 0.01; Table 1). Furthermore, the glycinergic mIPSCs exhibited significantly larger amplitudes and faster rise times compared with the GABAergic mIPSCs (p < 0.01). Also, there was a tendency for the glycinergic mIPSCs to exhibit shorter half-width and decay time than the GABAergic mIPSCs, but these differences were not statistically significant (p > 0.05).
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To test whether the lack of space clamp affected the kinetics, correlation
between the 10–90% rise time and 90–37% decay time and between
90–37% decay time and peak amplitude was plotted
(Fig. 9). A lack of correlation
between rise time and decay time, and the positive correlation between decay
time and peak amplitude in all of the principal cells tested (for mIPSCs,
n = 9; for mEPSCs, n = 5) supported the proposal that the
kinetics differences were not due to space-clamp problems
(Weiss et al. 1988).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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). Because part of the phenotypic developmental
schedule could be repeated in neurons during their recovery from injury or
trauma (see INTRODUCTION), the present data could be relevant to
studies of spontaneous synaptic activity in vestibular nucleus neurons during
vestibular compensation. Spontaneous synaptic activity in principal cell is mainly inhibitory and GABAergic
The frequency of spontaneous synaptic activity is determined by preand
postsynaptic factors. Presynaptic factors may include the frequency of action
potentials entering the presynaptic terminals, the number of presynaptic
terminals, and neurotransmitter metabolism. Postsynaptic factors include the
activation of postsynaptic receptor subunits
(Nicholls et al. 2001).
Inhibitory receptors, including GABA and glycine receptors, and the excitatory
receptors, AMPA/KA and NMDA receptors, have been demonstrated in vestibular
nucleus neurons of different species
(Dutia et al. 1992;
Kinney et al. 1994;
Smith et al. 1991). In the
embryonic principal cells, the frequency of excitatory spontaneous synaptic
activity was much less than that of inhibitory events (1:4), and the main
neurotransmitter of the inhibitory receptor-mediated events was GABA. Indeed,
at this age, the entire inhibitory response could be blocked by the
co-application of bicuculline and strychnine, which suggests that
GABAA and glycine receptors are responsible for all of these events
under our experimental conditions. Of course, these findings were obtained
using pipettes containing Cs-gluconate, which blocked the postsynaptic
GABAB receptor-mediated events. A relatively low rate of action
potential firing at vestibular presynaptic terminals at this age could
underlie the low frequency of excitatory spontaneous synaptic activity
recorded. Indeed, in the intact, ketamineanesthesized newborn rat, vestibular
nucleus neurons exhibited a spontaneous discharge rate of about 4.3 Hz
(Lannou et al. 1979).
It is likely that AMPA/KA receptors mediate input from the vestibular nerve
and may at least partially transmit input from the contralateral vestibular
nuclei via brain stem commissural fibers
(Doi et al. 1990;
Kinney et al. 1994) or from
the spinal cord (frog: Knopfel
1987; rat: Doi et al.
1990). Where are the GABAergic inputs coming from? So far, there
is no immunohistochemical evidence directly demonstrating inhibitory
connections to the tangential nucleus. However, from extracellular horseradish
peroxidase injections in the tangential nucleus, we know that there are inputs
originating from the cerebellar flocculus
(Cox and Peusner 1990), which
release GABA in the cat (Fonnum et al.
1970), chick (du Lac and
Lisberger 1992) and guinea pig
(Babalian 2000). Other major
inputs to the tangential nucleus originate from cervical levels of the spinal
cord and the vestibular nuclei on the ipsi- and contralateral sides
(Cox and Peusner 1990). GABA
and glycine immunoreactive neurons have been stained in the vestibular nuclei
of many species, including frog
(Reichenberger et al. 1997),
rat (Houser et al. 1984;
Nomura et al. 1984), mouse
(Ottersen and Storm-Mathisen
1984), cat (Tighilet et al.
2001; Walberg et al.
1990), guinea pig (Kumoi
1987) and monkey (Holstein et
al. 1996). In addition, GABA immunolabeled fibers and putative
synaptic terminals have been found throughout the vestibular nuclei
(Houser et al. 1984;
Kumoi 1987;
Nomura et al. 1984;
Walberg et al. 1990).
Accordingly, it is likely that some GABA and glycine neurons are located in or
around the chick tangential nucleus or in other vestibular nuclei and could
contact the principal cells. Indeed, neurons that corelease GABA and glycine
in the vestibular nuclei are another possibility
(Dumoulin et al. 2001;
Jonas et al. 1998;
O'Brien and Berger 1999;
Russier et al. 2002).
Inhibitory effect of GABAB receptor
It was reported that both GABAA and GABAB receptor
agonists decreased the frequency of spontaneous spiking activity in vestibular
nucleus neurons in young and old rats (Him
et al. 2001) and after unilateral labyrinthectomy
(Johnston et al. 2001). From
autoradiographic studies performed on the vestibular nuclei of young rats, the
presence of GABAB receptors was confirmed
(Turgeon and Albin 1994). In
the present study using KCl pipette solution, we showed that postsynaptic
GABAB receptors are present on the principal cells by applying the
GABAB agonist baclofen which induced an outward current. The
current could be blocked by intracellular Cs; this allowed us to observe the
presynaptic effects of baclofen. In these experiments, the frequency of sEPSCs
and sIPSCs was decreased by baclofen 40 and 53%, respectively. When
phaclofen, a specific GABAB receptor antagonist, was added
alone, there was no effect on sEPSCs frequency, but there was a
slight increase in sIPSCs frequency (6%). This finding suggests that in our
system sEPSCs and sIPSCs are generated by separate presynaptic terminals. It
seems that the presynaptic GABAB receptors on the excitatory
terminals are not activated tonically by ambient GABA, whereas the presynaptic
GABAB receptors on the inhibitory terminals can be activated.
Finally, when phaclofen was preapplied, baclofen still reduced the frequency
of sEPSCs (26%) and sIPSCs (24%) but to lesser extents. Altogether, these
experiments indicate that there may be different subtypes of GABAB
receptors present at various presynaptic terminals in the tangential nucleus
(for review, see Ong and Kerr
2000). Accordingly, GABAB receptors may play crucial
roles in modulating the input to the principal cells and could be involved in
the compensation process occurring after peripheral vestibular lesions.
GABAB receptors show different functions according to their pre-
and postsynaptic locations. In the case of presynaptic action, baclofen could
regulate the release of GABA, glutamate, and glycine
(Kabashima et al. 1997;
Lim et al. 2000) by either
inhibiting the calcium current necessary for transmitter release
(Deisz and Lux 1985;
Doze et al. 1995), activating
a potassium channel that indirectly inhibits the calcium current
(Doze et al. 1995) or directly
inhibiting quantal release by affecting protein kinase C
(Jarolimek and Misgeld 1997).
For postsynaptic inhibition, GABAB receptors are coupled to G
proteins and mediate postsynaptic effects by increasing the potassium
conductance (Dutar and Nicoll
1988).
Possible significance of inhibitory inputs
In the young postnatal brain, GABA functions primarily as an inhibitory
neurotransmitter, although it may produce depolarizing effects in embryonic
auditory nuclei (Hyson et al.
1995; Lu and Trussell
2001). During embryonic development, GABAergic inhibition may
provide a continuous background level of inhibition to the principal cells,
driving the membrane potential away from firing threshold and maintaining a
relative steady state. Indeed, there was no rhythmic bursting observed in the
embryonic tangential principal cells at E13 and E16 using intracellular and
patch-clamp recordings (Gamkrelidze et al.
1998; Peusner and Giaume
1994,
1997). This contrasts to the
spontaneous action potential firing recorded from E14 to 18 chick auditory
neurons in the nucleus magnocellularis and nucleus laminaris
(Lippe et al. 1994).
Inhibitory spontaneous synaptic activity could contribute to the typical
response of most embryonic principal cells of a single spike on depolarization
at E16 (Gamkrelidze et al.
1998; Peusner and Giaume
1997). In some systems, GABA acts as a trophic factor during
nervous system development, influencing multiple diverse events including
proliferation, migration, differentiation, synapse maturation, cell death and
GABAA receptor expression
(Owens and Kriegstein 2002).
At E16, the tangential nucleus undergoes a major morphological transition
because the spoon ending retracts and acquires gap junction channels, and
dendritic outgrowth in the principal cells becomes exuberant as compared with
just a few days earlier at E13.
Miniature activity
In this study, most spontaneous synaptic activity was TTX-resistant, the
so-called miniature activity, which is thought to be due to quantal
neurotransmitter release (Nicholls et al.
2001). The quantal release theory attributes each mPSC to the
release of the neurotransmitter content from a single vesicle. The experiments
demonstrating a higher percentage of TTX-resistant inhibitory events than
excitatory events suggest that excitatory events were more dependent on action
potentials than inhibitory events. Because the kinetics of mIPSCs were longer
than those of mEPSCs, it is conceivable that at this age excitatory events are
shunted by inhibitory events and do not reach the threshold for firing action
potentials.
There is accumulating evidence that cotransmission of GABA and glycine
occurs from the same synaptic terminal with distinctive kinetics (e.g.,
Dumoulin et al. 2001;
Jonas et al. 1998;
O'Brien and Berger 1999;
Russier et al. 2002). In our
study, small differences in the kinetics of GABA and glycine (decay time and
duration) were not statistically significant. The differences in the kinetics
between mEPSCs and mIPSCs and between GABAergic mIPSCs and glycinergic mIPSCs
may be attributed to the intrinsic receptor properties, variability in
synaptic structures, including uptake system, barriers for diffusion, and/or
transmitter concentration in the synaptic cleft
(Clements 1996;
Weiss et al. 1988). In our
experiments, we excluded the possibility of inadequate space clamp because
there was no correlation between rise time and decay time and a positive
correlation between decay time and peak amplitude. The reasons for kinetics
differences in our study as compared with other works may be due to variations
in the slice preparations, receptor properties of the neurons, recording
temperatures, pipette solutions, holding potentials and the use of
flunitrazepam or pentobarbital.
What is the significance of spontaneous synaptic activity in second-order vestibular neurons?
While evoked synaptic transmission is accepted as crucial for nervous
system function, the exact physiological significance of spontaneous synaptic
activity is uncertain at present. First described in 1932 by Hoagland,
spontaneous activity has been reported to occur in just about all first- and
second-order sensory neurons, including vestibular sensory neurons (e.g.,
Goldberg and Fernandez 1971).
Excitability is a crucial property of mature vestibular nucleus neurons
because most of them fire action potentials tonically (see
Peusner et al. 1998 for
review). This feature is essential for them to transmit reliably vestibular
stimuli and to perform normal reflex functions
(du Lac and Lisberger 1995).
Even when there is no angular or linear acceleration, vestibular sensory
neurons fire spikes actively so that there is a continual flow of action
potentials along the primary vestibular fibers and vestibular nucleus neurons.
In mature or nearly mature vestibular sensory neurons, spontaneous activity
results in spike firing. However, in the embryonic principal cells, the
spontaneous synaptic activity does not reach firing threshold. Despite small
amplitudes, we suspect that the spontaneous synaptic activity may provide
sufficient inhibitory and possibly excitatory drive to modulate the vestibular
glutamatergic transmission in principal cells as shown for other
glutamate-releasing systems (e.g., Cossart
et al. 2002). Spontaneous synaptic activity may influence many
aspects of developing excitable cells, including formation of their basic
neural circuitry, neurogenesis, generation of space maps, shaping of the
tuning curve in the auditory system, and the expression of myogenic factors
(e.g., Dallman et al. 1998;
Friauf and Lohmann 1999).
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests: K. D. Peusner, Dept. of Anatomy and Cell Biology, George Washington University Medical Center, 2300 I Street NW, Washington, DC 20037 (E-mail address: anakdp{at}gwumc.edu).
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