 |
INTRODUCTION |
Although neuronal
discharge can be quite low during early development, spontaneous and
evoked activity has a profound impact on the selective loss or survival
of synaptic contacts (Sanes et al. 2000a
). Manipulations
of excitatory transmission can disrupt the normal elimination of motor
axons onto muscle fibers, and prevent the refinement of excitatory
connections in the CNS (Cline et al. 1987
; Ichise
et al. 2000
; Kleinschmidt et al. 1987
;
O'Brien et al. 1978
; Scherer and Udin
1989
; Simon et al. 1992
; Thompson et al.
1979
). There is now evidence that inhibitory terminals also
become refined during development. In the gerbil lateral superior olive
(LSO), the inhibitory afferent fibers from the medial nucleus of the
trapezoid body (MNTB) become restricted anatomically during postnatal
development (Sanes and Siverls 1991
).
Stimulation of MNTB afferents at a low rate leads to a long-lasting
depression of synaptic inhibition in LSO neurons (Kotak and
Sanes 2000
). This form of inhibitory synaptic plasticity
declines with age, and we have hypothesized that it contributes to the activity-dependent reorganization of MNTB arbors within the LSO (Sanes and Takács 1993
). Although long-term
inhibitory synaptic depression has been reported in this and other
systems (Komatsu 1994
; Morishita and Sastry
1991
; Oda et al. 1998
), the signaling pathway that initiates this form of plasticity has not been examined. In contrast, co-activation of glutamatergic and GABAergic afferents can
produce inhibitory depression through an
N-methyl-D-aspartate (NMDA) receptor mechanism
(Caillard et al. 1999
).
This present study focuses on two candidate signaling systems. First,
the MNTB-evoked inhibitory response recorded in the gerbil LSO is
predominantly GABAergic before hearing onset and switches to a
predominantly glycinergic input postnatally (Kotak et al.
1998
). This finding suggested that GABAergic transmission could
play a significant role during inhibitory synaptogenesis. Second, MNTB
neurons express neurotrophins, and LSO neurons express their cognate
receptors during development (Hafidi 1999
; Hafidi et al. 1996
). Since neurotrophin/Trk signaling pathways have
been shown to modulate synaptic transmission (Kang and Schuman
1995
; Kim et al. 1994
; Levine et al.
1998
), they may be relevant to the plasticity displayed by MNTB
synapses. Therefore we have tested whether signals mediated by
GABAB and neurotrophin receptors are involved in
the long-lasting depression of inhibitory synapses in the LSO.
 |
METHODS |
Gerbils (Meriones unguiculatus) aged postnatal
days 8-12 (P8-12) were used to make 300-µM coronal
brain slices through the LSO and MNTB. The artificial cerebrospinal
fluid (ACSF) contained (in mM) 125 NaCl, 4 KCl, 1.2 KH2PO4, 1.3 MgSO4, 26 NaHCO3, 15 glucose, 2.4 CaCl2, and 0.4 L-ascorbic acid (pH
7.3 when bubbled with 95% O2-5%
CO2). The ACSF was continuously superfused in the recording chamber at 4-5 ml per min at room temperature (22-24°C). Whole cell current-clamp recordings were obtained from LSO neurons (Warner PC-501A), and 200-µs electrical pulses were delivered directly to the MNTB, as described previously (Kotak and Sanes 2000
). The internal patch solution contained (in mM) 127.5 potassium gluconate, 0.6 EGTA, 10 HEPES, 2 MgCl2,
5 KCl, 2 ATP, and 0.3 GTP (pH 7.2). To block tyrosine kinase in the
postsynaptic LSO neuron, K-252a (200 nM) was added to the internal
pipette solution. To examine inhibitory synaptic depression,
MNTB-evoked maximum amplitude inhibitory postsynaptic potentials
(IPSPs) were first acquired during a 15-min baseline period initially
every minute for the first 5 min and then at the 10th and 15th min
(Kotak and Sanes 2000
). The MNTB was then activated with
low-frequency stimulation (LFS: 1 Hz for 15 min). Immediately following
LFS, MNTB-evoked IPSPs were recorded every min for the first 5 min and
every 5 min thereafter. To block GABAB receptors,
SCH-50911 (5-10 µM, Tocris) was bath-applied throughout the
experiment beginning 5 min before recording the first IPSP.
In a separate set of experiments, IPSPs were recorded for about 1 h at a very low rate of acquisition that does not produce synaptic
depression (0.03 Hz), and the slices were exposed to either a
GABAB receptor agonist (baclofen, 100 µM, Sigma
Chemicals), or a neurotrophin [brain derived neurotrophic factor
(BDNF), 50-100 ng/ml, Sigma Chemicals or Alamone Laboratories;
NT-3, 25-50 ng/ml, Sigma Chemicals]. In many of these experiments,
contaminating glutamatergic activity was blocked with
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM) or kynurenic acid
(4 mM). This was done for control LFS experiments (n = 3), baclofen exposure (n = 2), BDNF exposure (n = 7), NT-3 exposure (n = 6), and
SCH-50911 treatment (n = 3).
Data were collected using a Macintosh PPC running a custom-designed
IGOR (WaveMetrics, v3.14) macro called SLICE. The data were analyzed
off-line using a second IGOR macro called SLICE ANALYSIS. Each macro is
available with complete documentation on-line at
http://www.cns.nyu.edu/~sanes/slice_software. The SLICE macro
controls the stimulus isolation units and patch-clamp amplifier via an
ITC-18 Computer Interface (Instrutech Corporation) using an IGOR
external operation commands (XOP version 2.6, Instrutech). Data were
sampled and stored at 10 kHz. Analyses of peak IPSP amplitude, rising
slope, and duration were performed off-line. Data are presented as
means ± SE or as a percent of the normalized IPSP amplitudes as
indicated in RESULTS and the figure legends. All analyses
were performed with the Student's t-test.
 |
RESULTS |
The data reported here are drawn from whole cell current-clamp
recordings from 74 LSO neurons. Each recording was obtained from a
separate brain slice. In the initial experiments, MNTB-evoked maximum
amplitude IPSPs were recorded without any pharmacological agents in the
ACSF. As shown for a control P9 neuron in Fig.
1, the MNTB-evoked IPSP was about 11 mV
during the pre-LFS period, but decreased to about 6.5 mV following LFS
treatment (top). The average IPSP amplitude reduction was
43% at 1 h following LFS, as compared with the baseline IPSP
amplitude prior to LFS (n = 10). In three recordings,
ionotropic glutamate receptors were blocked with kynurenic acid (4 mM),
and this did not alter the magnitude of depression (a 45% reduction in
IPSP amplitude was observed). To assess the role of
GABAB receptors during the initiation of
inhibitory synaptic depression, we applied the
GABAB receptor antagonist SCH-50911 (5-10 µM)
throughout the experiment, beginning 5 min before the first IPSP was
recorded. As shown in Fig. 1, when LFS was delivered in the presence of
SCH-50911, the magnitude of long-lasting depression was blocked as
compared with the untreated controls.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Long-lasting depression of inhibitory transmission was mediated by
GABAB receptors. A: medial nucleus of the
trapezoid body (MNTB)-evoked maximum inhibitory postsynaptic
potentials (IPSPs) were recorded from the lateral superior olive (LSO)
before and after low-frequency stimulation (LFS) of the MNTB. Example
IPSPs are shown for a postnatal day 9
(P9) neuron recorded in control (top) or
SCH-5091-containing ACSF (bottom).
Erest was 53 and 52 mV, respectively.
B: summary for all recorded LSO neurons at
P8-12 in the absence and presence of SCH-50911
(mean ± SE). Synaptic depression was robust (43%) at 50-60 min
following LFS when compared with pre-LFS IPSPs ( ). Age-matched
neurons treated with SCH-50911 ( ) displayed a marginal
change in IPSP amplitude following LFS. The mean percent change was
calculated by comparing the average normalized IPSP amplitude recorded
at 50-60 min post LFS with the normalized mean initial IPSP amplitude
(0%) during 1st 5 min of the recording session (for control neurons:
t = 5.1, df = 18, P < 0.0001; for SCH-50911-treated neurons: t = 0.56,
df = 18, P = 0.57).
|
|
The second experimental strategy to assess GABAB
receptor involvement in inhibitory depression was an extension of our
previous finding that baclofen reversibly depressed IPSPs following a
single exposure (Kotak et al. 1998
). As shown in Fig.
2, repeated perfusion (100 µM baclofen;
5 × 10 s exposures at 3-min intervals) induced a
long-lasting depression. There was also a significant decrease in the
IPSP rising slope (50% decline, P < 0.01). In three
of four neurons tested, the LSO input resistance decreased by
approximately 30% during baclofen exposure. In two additional
experiments, a single dose exposure of baclofen (100 µM) caused the
MNTB-evoked IPSPs to decrease by about 50% for approximately 10 min.
This baclofen-elicited depression was eliminated when the slice was pretreated for 6 min with 10 µM SCH-50911 (data not shown). This indicated that the synaptic- and agonist-mediated depression involved the same receptor.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Repeated activation of GABAB receptors elicited
long-lasting synaptic depression. A: a maximum amplitude
IPSP is shown for a P10 neuron before and after repeated
baclofen exposure. B: the bar graph compares the percent
change in the normalized IPSP amplitude during a control period, a
15-min drug exposure period, and after a recovery period. There was a
significant decline (asterisk) in IPSP amplitudes during baclofen
treatment, and this depression persisted at 30 min after the last
baclofen exposure. The change in IPSPs was calculated by comparing the
average normalized IPSP amplitude recorded during baclofen exposure and
at 50-60 min of the experiments with the initial IPSP amplitude (0%)
during 1st 10 min of the recording session (comparison between initial
IPSPs and IPSPs during baclofen exposure: t = 4.01, df = 6, P < 0.007; comparison between initial
IPSPs and IPSPs at 50-60 min: t = 4.08, df = 6, P < 0.006).
|
|
While the GABAB receptor antagonist results
suggest that this receptor is necessary for induction of inhibitory
depression, additional mechanisms have not been ruled out. Therefore
two neurotrophin signaling systems (BDNF and NT-3) known to be
localized to the MNTB-LSO pathway were tested as candidates for a
depression mechanism. For these experiments, IPSPs were recorded every
30 s for approximately 1 h. In control recordings, this
stimulus rate did not alter IPSP amplitude significantly. The change in
IPSPs was calculated by comparing the mean IPSP amplitude (±SE)
recorded at 50-60 min with the mean initial IPSP amplitude (±SE)
during first 10 min of the recording session (initial IPSP
amplitude = 8.7 ± 0.7 mV, mean ± SE; IPSP amplitude at
50-60 min following LFS = 8.9 ± 0.8 mV; t =
0.51, df = 10, P = 0.620). In separate
recordings, bath application of BDNF (50-100 ng/ml) for 5-8 min
resulted in a small decrease in IPSP amplitude. Approximately 10 min
after BDNF application, IPSP amplitudes declined, and this attenuation
reached its maximum by about 20-30 min following drug exposure, but
the change did not reach significance (comparison between initial IPSPs
and IPSPs during BDNF exposure: t = 0.36, df = 18, P = 0.72; comparison between initial IPSPs and IPSPs at
50-60 min: t = 0.17, df = 14, P = 0.07). Exposure to NT-3 (25-50 ng/ml) produced a larger and more rapid
decline in IPSP amplitude, and this decline was highly significant
(Fig. 3). Finally, to assess whether
neurotrophin receptors could influence synaptically evoked depression,
a tyrosine kinase antagonist (200 nM K-252a) was added to the internal
patch solution. As shown in Fig. 3B, K-252a prevented LFS
from inducing a significant change in IPSP amplitude (mean initial IPSP
amplitude = 9 ± 1 mV; mean IPSP amplitude at 50-60 min
following LFS = 9.7 ± 0.2 mV).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Neurotrophin signaling depresses inhibitory transmission.
A: a maximum amplitude IPSP is shown for a
P11 neuron before and after NT-3 (25 ng/ml) exposure for
8 min. The IPSP depressed by about 30%. The bar graph compares percent
change in IPSP amplitude before, during, and after NT-3 exposure. The
IPSPs decreased significantly during NT-3 application when compared
with pre-NT-3 treatment IPSPs (asterisk), and remained depressed at
40-50 min (comparison between initial IPSPs and IPSPs during NT-3
exposure: t = 2.58, df = 10, P = 0.02; comparison between initial IPSPs and
IPSPs at 50-60 min: t = 4.65, df = 8, P = 0.001). B: summary for neurons
in the absence and presence of K-252a (control data from Fig. 1).
Neurons recorded with K-252a in the pipette solution ( )
displayed no change in IPSP amplitude following LFS (comparison between
initial IPSPs and IPSPs at 50-60 min: t-test;
t = 0.73, df = 8, P = 0.48).
|
|
 |
DISCUSSION |
A number of studies suggest that auditory coding properties mature
postnatally, and that this improvement is due, in part, to
experience-dependent mechanisms (Sanes and Walsh 1997
).
For example, sound localization in the barn owl is influenced by both auditory and visual experience (Knudsen and Brainard
1991
; Mogdans and Knudsen 1993
). In the gerbil
LSO, interaural level difference coding improves with age, and several
anatomical and physiological properties are disrupted by
deafferentation during development (Sanes et al. 2000b
).
We have previously shown that inhibitory projections from MNTB to LSO
become refined during development, and this process is disrupted by
deafferentation (Sanes and Siverls 1991
; Sanes
and Takács 1993
). More recently, we have found that the
strength of these inhibitory synapses depends on activity, and this
phenomenon wanes with age (Kotak and Sanes 2000
). The present results suggest that use-dependent depression of inhibitory synapses requires GABAB receptors, and may also
employ neurotrophin signaling.
Inhibitory synapses in LSO are predominantly GABAergic during the first
two postnatal weeks, and gradually adopt a glycinergic phenotype
(Kotak et al. 1998
). This led us to hypothesize that GABA may provide a metabotropic signal that is important for synapse maturation. In the present study, we found that blockade of
GABAB receptor transduction could eliminate
long-lasting synaptic depression (Fig. 1). This result is consistent
with the ability of a GABAB agonist to initiate
long-lasting depression (Fig. 2). While it is not yet clear how
GABAB receptor activation initiates inhibitory depression, a G protein-linked mechanism has recently been shown to
depress GABAA receptor-gated responses through
alteration of cytoskeletal anchoring proteins (Meyer et al.
2000
). Postsynaptic GABAB receptors
apparently exist in LSO since these neurons exhibited an increased
conductance following baclofen exposure. However, a presynaptic
contribution to inhibitory depression cannot be ruled out. For example,
presynaptic GABAB receptor-coupled mechanisms are known to decrease transmission at both excitatory and inhibitory synapses (Brenowitz et al. 1998
; Lim et
al. 2000
; Takahashi et al. 1998
). However, these
effects commonly last for seconds to minutes and are not as likely to
underlie the long-lasting change we observe in the LSO.
Neurotrophins and their receptors have also been implicated in synapse
development and plasticity. In cerebellar cultures, activity blockade
reduces the number of inhibitory synapses, but inhibitory
synaptogenesis is restored by BDNF or neurotrophin-4 (NT-4), while
antibodies to BDNF and NT-4 reduce inhibitory synapse formation
(Seil and Drake-Baumann 2000
). In addition, NT-3
depresses GABAA receptor-mediated transmission
in developing cortical neurons (Kim et al. 1994
). In the
MNTB-LSO pathway, immunoreactivity for BDNF, NT-3, and their receptors
is quite prominent during the first two postnatal weeks (Hafidi
1999
; Hafidi et al. 1996
). In the present study,
neurotrophin-3 exposure depressed inhibitory synaptic gain (Fig.
3A). IPSP amplitude declined within 10 min of exposure, but
this slow time course may have been due to access to the recording site
within the brain slice. The blockade of use-dependent depression by
K-252a implies that neurotrophin receptors may participate along with
GABAB receptors to induce inhibitory depression.
One possibility is that the neurotrophin signal acts to raise
intracellular free calcium (Kang and Schuman 2000
),
which is required for inhibitory depression to occur in LSO neurons (Kotak and Sanes 2000
). As in excitatory synaptogenesis,
adjustments of inhibitory synaptic strength may thus be regulated by
several receptors and intracellular signaling pathways. Dissection of those mechanisms will be critical to appreciate the functionality of
inhibitory synapses before and after sound-evoked activity (Kotak and Sanes 2000
).
This work was supported by National Institute on Deafness and Other
Communication Disorders Grant DC-00540 to D. H. Sanes.
Address for reprint requests: D. H. Sanes, Center for Neural
Science, 4 Washington Place, New York University, New York, NY 10003 (E-mail: sanes{at}cns.nyu.edu).