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The Journal of Neuroscience, July 1, 2000, 20(13):4864-4870
Long-Term Specification of AMPA Receptor Properties after Synapse
Formation
J. Josh
Lawrence1 and
Laurence O.
Trussell2
1 Neuroscience Training Program and
2 Department of Physiology, University of
Wisconsin-Madison, Madison Wisconsin 53711
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ABSTRACT |
AMPA receptors expressed at auditory nerve synapses in the
mammalian and avian cochlear nuclei display exceptionally rapid channel
gating, an adaptation well suited for acoustic processing. We examined
whether cellular interactions during development might determine the
subunit composition of these receptors. After synapse formation in the
avian nucleus magnocellularis (nMag) in vivo, the rate
of receptor desensitization increased threefold, sensitivity to channel
block by polyamines increased, and sensitivity to cyclothiazide, an
inhibitor of desensitization, increased, indicating a reduction in
glutamate receptor subunit 2 and of flip splice variants. This phenotypic switch was prevented, but not reversed, by isolating nMag
neurons in a cell-culture environment. We propose that the switch in
receptor kinetics is an outcome of cellular interactions during a
critical period that result in the long-term determination of receptor phenotype.
Key words:
cochlear nucleus; auditory nerve; glutamate; development; ion channels; avian
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INTRODUCTION |
The activation of AMPA
receptors by synaptically released glutamate mediates rapid excitatory
transmission in diverse neural pathways in the CNS (Collingridge
and Lester, 1989 ). The assembly of glutamate receptor subunits 1-4
(GluR1-GluR4) into a heteromultimeric complex, as well as the
editing and alternative splicing of the subunit RNA, generates many
possible receptor isoforms for use at a given synapse (Borges and
Dingledine, 1998). The molecular diversity of these receptors underlies
their functional diversity and is an essential element for tailoring
synaptic transmission on a pathway- or synapse-specific basis
(Trussell, 1998).
Although the molecular refinement of neurons is key to the coding of
information in complex neural systems, there is relatively little known
about the cellular mechanisms that direct the cell-specific expression
of AMPA receptor subunits and their processing. One common theme is the
developmental control of expression of different subtypes of receptor,
often through the action of presynaptic signals. Such control has been
posited for acetylcholine, glutamate, glycine, and GABA receptors
(Monyer et al., 1991; Carmignoto and Vicini, 1992; Farrant et al.,
1994; Tia et al., 1996; Fischbach and Rosen, 1997; Ozaki et al., 1997).
Subunits and splice variants of AMPA receptors also differ with brain
region and over development. However, there remains little evidence
linking specific cellular interactions in vivo to the
expression of particular AMPA receptor isoforms. Factors limiting such
analyses include heterogeneity of cell types within a given brain
region, the diverse sources of excitation for any one cell type, the
large number of synaptic contacts, and the broad time period
encompassed by synaptogenesis. Moreover, it has not been possible to
separate intrinsic and determinative programs of gene expression.
Finally, it is unclear whether such determinative cellular interactions
are transitory or whether they must continue throughout the life of the synapse.
Neurons of the avian cochlear nucleus magnocellularis (nMag), which
contain the avian homologs of mammalian spherical bushy cells, offer a
unique opportunity for exploring the immediate consequences of
innervation on the expression of AMPA receptors in vivo. The
total excitatory innervation consists of only two to three auditory
nerve axons that form morphological and functional synaptic contact
between embryonic days 10 (E10) and E11 (Rubel and Parks, 1988). During
the period after innervation, the neurons undergo profound synaptic
rearrangements that result in the formation of somatic end-bulb
synapses by E16. Like many auditory brainstem nuclei, the passaging of
high-frequency, well timed signals is mediated by synapses that express
AMPA receptors having unusually brief kinetics of channel gating (Raman
et al., 1994). We have taken advantage of this well described
preparation to examine the control of the fast-acting AMPA receptors
expressed in nMag neurons. Our results indicate that the channel gating
properties and pharmacological sensitivity of AMPA receptors change
soon after innervation. Neurons cultured before this change took place did not acquire fast-desensitizing AMPA receptors. In contrast, once
this transition in expression took place, cells maintained fast-desensitizing receptors even after 1 month in low-density cultures. Thus, the mature complement of AMPA receptors may be permanently specified by cellular interactions that take place during development.
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MATERIALS AND METHODS |
Slice recordings. Coronal, 100-350-µm-thick slices
containing nMag were made from chick embryos as described previously
(Zhang and Trussell, 1994). Slices were transferred to a bath chamber perfused with room temperature, oxygenated saline [containing (in
mM): 140 NaCl, 20 glucose, 10 HEPES, 5 KCl, 3 CaCl2, and 1 MgCl2]
flowing at 5 ml/min. Neurons were visualized with an upright microscope
using Nomarski optics and a 40× water-immersion objective. Intracellular solution contained (in mM): 70 Cs2SO4, 85 sucrose, 4 NaCl,
1 MgCl2, 10 HEPES, and 5 BAPTA, buffered to pH
7.3 with CsOH. Resistances of patch pipettes ranged from 3 to 5 M .
Outside-out patches were excised from nMag neurons on the surface of
the slice. Action potentials were elicited in whole-cell mode just
before patch excision to confirm that the recordings were from neurons
and not glial cells. For rapid application of 10 mM
glutamate to patches, a theta pipette attached to a piezoelectric translator was used as described previously (Raman et al., 1994), except that solution delivery through the theta pipette was driven by
nitrogen pressure. Control and glutamate solutions contained normal
Ringer's solution with 200 µM
D,L-2-amino-5-phosphonovaleric acid and 10 µM 7-chlorokynurenic acid, so that no NMDA receptors would be activated during glutamate applications. The 10-90% solution exchange time was typically ~150 µsec, as measured in each
recording using the junction potential between Ringer's solution and
10% Ringer's solution (Jonas, 1995). An example of the exchange time is shown in Figure 1. Electrophysiological data were Bessel
filtered (Frequency Devices, Haverhill, MA) at 10 kHz and
digitized at 20-50 kHz with pClamp software (Axon Instruments, Foster
City, CA).
Preparation of nMag cultures. nMag neurons were
enzymatically dissociated from tissue chunks microdissected from brain
slices as described previously (Raman et al., 1994), with minor
modifications for sterile conditions. Briefly, brain slices prepared as
above were soaked in an oxygenated solution containing papain
(Worthington, Freehold, NJ) for 10-20 min and then rinsed. nMag was
then dissected out of each slice using fine tungsten needles, and the
tissue chunks were gently triturated. After dissociation, aliquots of the cell suspension were seeded onto
poly-D-lysine (Sigma, St. Louis, MO)-laminin
(Worthington)-coated glass coverslips on which a confluent monolayer of
flat astrocytes had been established. Cultures were kept at
37°C and 5% CO2 in MEM-based media containing 10% fetal bovine serum (Sigma), 10% horse serum (Atlanta Biological), 20 mM glucose, and 1%
penicillin- streptomycin (Life Technologies, Gaithersburg,
MD). After 24 hr, the media was changed to one that replaced
fetal bovine serum with horse serum. Cells for acute recordings were
plated onto poly-D-lysine-coated coverslips
without glia and used for up to 2 hr after plating.
Retrograde labeling of nMag. A freshly isolated brainstem
was transferred to a dish containing oxygenated saline and pinned ventricle-side up with insect pins. A pipette of ~10-20 µm tip diameter containing 10-40% 3-10 kDa molecular weight
tetramethylrhodamine (TMR) dextran or biotinylated dextran
(Molecular Probes, Eugene, OR) and 5-10% methyl green dissolved in
0.1 M phosphate buffer, adjusted to pH 2 with
HCl, was inserted into the ipsilateral brainstem at the level of nMag
and nucleus laminaris. Dye was injected until the methyl green was
visible in the tissue by eye. The intact brainstem was then incubated
at 37-40°C for 30-60 min and continually oxygenated. nMag neurons
on the uninjected side were then prepared for culture as described.
Statistical significance was determined by two-tailed t
test, and errors are given as SEM.
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RESULTS |
Developmental control of time course of desensitization
The time course of desensitization of AMPA receptors, which varies
markedly with subunit composition (Mosbacher et al., 1994), was used to
probe developmental changes in AMPA receptors. Application of 10 mM glutamate for 100 msec resulted in activation and nearly complete desensitization of AMPA receptors (Raman and Trussell, 1992;
Trussell et al., 1993) (see Materials and Methods). The time course of
desensitization, measured by fitting the onset of desensitization with
a single exponential, was determined for patches excised from nMag
neurons in brain slices at various stages of development. In Figure
1, the time constant of desensitization ( des) is plotted versus embryonic age and
shows a progressive speeding up of desensitization with development.
For example, at E9, des was 4.45 ± 0.80 msec (n = 6), whereas at E18,
des was 1.56 ± 0.12 msec
(n = 8, significance at p < 0.002).
The inset shows representative glutamate-activated currents
from patches excised from somata at E11, E13.5, and E18. The
deactivation time constant, measured from the decay of responses after
1 msec pulses of glutamate, did not differ significantly over this time
span (E9-E12, 1.02 ± 0.3 msec, n = 13; E17-E18,
0.67 ± 0.13 msec, n = 13; p = 0.29). Similarly, the 10-90% rise times of glutamate responses were
unchanged over this period (E10-E11, 0.26 ± 0.06 msec,
n = 10; E17-E18, 0.21 ± 0.06 msec,
n = 14; p = 0.57). The data indicate
that, in the time period immediately after innervation by the auditory
nerve, fast-desensitizing AMPA receptors replaced receptors having
slower channel kinetics.
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Figure 1.
Developmental acceleration in AMPA receptor
desensitization kinetics. Single exponential time constant of
desensitization (des) versus embryonic age
(E) of development. Glutamate (10 mM)
was rapidly applied to patches excised from nMag neurons in slices at
various stages of development. Shown below the axis are
major developmental events during this time period. From
left, points are mean ± SEM of 6, 3, 9, 10, 8, 11, and 8 patches. Horizontal bars denote variation in
developmental time points pooled for each data point.
Inset, Scaled representative current traces excised from
neurons at E11 (gray), E13.5
(dotted), and E18 (black).
Traces are averages of 10-30 events. Time course of
exchange (junction potential change) is illustrated
above the current traces.
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Spermine block of AMPA receptors is greatest in older neurons
Intracellular spermine blocks AMPA receptor-channels according to
the fractional contribution of GluR2 to the receptor complex, such that reduction in GluR2 content increases susceptibility to
channel block (Washburn et al., 1997). We examined channel block by
spermine in patches isolated from E11 and E18 embryos. AMPA
receptor-mediated currents obtained at holding potentials from  75 to
+60 mV are shown in Figure 2,
A and B. All data were recorded in the continuous
presence of 1 mM intracellular spermine. Sixty
micromolar extracellular cyclothiazide (CTZ) was included to reduce
desensitization during the response. Figure 2C shows overlaid current-voltage (I-V) relationships and
illustrates that, at positive potentials, there was less inward
rectification in E11 than in E18 neurons at positive potentials. The
extent of rectification was summarized in Figure 2D
by using a rectification index [the ratio of conductance at +30 mV to
conductance at 60 mV (Kamboj et al., 1995)]. In
accordance with previous results (Raman and Trussell, 1995), the
I-V relationship for receptors from E18 cells
(gray bar) is slightly outwardly rectifying
(rectification index above 1) in the absence of intracellular spermine.
In the presence of spermine, the rectification index was <1 for both E11 and E18 but significantly smaller for the E18 neurons
(p < 0.001), indicating a decrease in the
expression of GluR2 during the period from E11 to E18.
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Figure 2.
Developmental control of subunit composition
indicated by changes in spermine sensitivity.
A, B, Families of AMPA receptor-mediated
currents at potentials from 75 to +60 mV in 15 mV increments in
patches from E11 and E18 neurons. All patches were in the continuous
presence of 1 mM intracellular spermine and 60 µM extracellular CTZ. Slowed deactivation in
A was inconsistently observed. C,
Current- voltage relationship showing, that at positive potentials,
there is less inward rectification in E11 (open circles)
than in E18 (filled circles) neurons. Currents
were normalized to the current amplitude at 60 mV. D,
Rectification index, as defined by the ratio of conductance
(G) at +30 mV relative to conductance at 60 mV
for both E11 (n = 4) and E18 (n = 5)
neurons. I-V relationships in patches from E18 neurons
(gray bar) are slightly outwardly rectifying
(rectification index >1) in the absence of intracellular
spermine (n = 3). * indicates rectification ratio
significantly less at E18 than E11 (p < 0.001). E11 and spermine-free control data also differed significantly
(p < 0.01).
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Reduced expression of flip isoforms
With the exception of the GluR1 subunit (which is weakly expressed
in auditory brainstem nuclei; see Discussion), AMPA receptors composed
of flop splice variants have faster desensitization that those
containing flip subunits (Mosbacher et al., 1994; Koike et al., 2000).
We tested whether a shift in the relative proportion of flip- versus
flop-containing subunits could account for the developmental changes in
AMPA receptor kinetics by measuring the extent of desensitization in
the presence of CTZ. As a result of their higher affinity for CTZ, flip
receptors are more resistant to desensitization in the presence of CTZ
than are flop receptors (Partin et al., 1995; Fleck et al., 1996). Long
pulses (2 sec) of 10 mM glutamate were delivered in the
continued presence of 60 µM CTZ. The extent of
desensitization was expressed as the steady-state/peak current. Of 19 patches taken from either E11 or E17 neurons, all but three
desensitized by >75%, indicating the presence of flop subunits. The
remaining three desensitized by 0-30% and were excluded from further
analysis. Nevertheless, as illustrated in Figure
3, A and B,
desensitization was significantly stronger for receptors in cells from
older animals. These data suggest that flip subunits become less
prevalent during development.
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Figure 3.
Developmental changes in flip-flop splice
variants indicated by changes in effectiveness of cyclothiazide.
A, Long pulses (2 sec) of 10 mM glutamate in
the continued presence of 60 µM CTZ show marked
desensitization in E11 (gray trace) and E18
(black trace) patches. Traces scaled to same peak value.
Double-exponential fits to the traces with the following parameters are
shown: E11, fast 86 msec, slow 588 msec,
28% fast phase, 27% steady-state current; E18, fast 51 msec, slow 462 msec, 38% fast phase, 7% steady-state
current. B, The current remaining after desensitization,
measured as steady-state/peak ratio in which steady-state current was
obtained from best single- or double-exponential fits to the traces
during onset of desensitization. n = 5 and 11 patches for E11 and E18, respectively. *p < 0.05 indicates significant difference.
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Factors in the cellular environment drive changes in
receptor phenotype
To determine whether cellular interactions within nMag are
required for the acquisition of fast-desensitizing AMPA receptors, we
studied the desensitization time course of isolated neurons maintained
in vitro for varying periods of time. Because this experiment requires microdissection of neurons and treatment with proteolytic enzymes (see Materials and Methods), which might alter receptors (Allen et al., 1988), we first showed that enzymatically isolated nMag neurons still retain normal channel gating kinetics. In
initial experiments, nMag was retrogradely labeled with a TMR-dextran conjugate before enzymatic isolation (see Materials and Methods), and
fluorescent neurons in the dissociate were selected for patch-clamp analysis. Figure 4A
shows fluorescently labeled E11 nMag neurons in a slice before
enzymatic treatment and an enzymatically isolated E11 neuron
(inset). Cells taken from animals younger than E15-E16 generally had somatic protrusions and dendrites characteristic of
immature nMag (Jhaveri and Morest, 1982). In Figure
4B, the open circles illustrate the time
constant of desensitization measured in acutely isolated, labeled
neurons isolated from animals of different age. For comparison, the
time constants measured in brain slices (filled
triangles) are also shown. In Figure 4B, the
gray filled circles show three points in which time
constants were measured in neurons from unlabeled nMag dissections.
These values were indistinguishable from both labeled neurons and
neurons in slices for these stages of development, indicating that most or all of the cells in the dissociate were nMag neurons.
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Figure 4.
Culture environment arrests the developmental
switch to fast AMPA receptor kinetics. A, Fluorescent
image of contralateral (uninjected) side of slice after retrograde
labeling of nMag neurons with TMR. Arrow, Labeled fibers
in dorsal acoustic commissure. Double arrow, Labeled
nMAG cell bodies in situ. Inset,
Representative fluorescent E11 neuron retrogradely labeled and acutely
dissociated. Scale bar, 10 µm. B, AMPA receptor
desensitization time constant from neurons in slices
(filled triangles; data from Fig. 1), acutely
dissociated TMR-labeled neurons (open circles;
n = 3-9 cells), unlabeled acutely dissociated
neurons (gray filled circles;
n = 8-9), and neurons cultured from E10 embryos
(black filled circles; n = 9-16).
C, Representative scaled traces of desensitizing
currents in patches from E17 neurons either acutely dissociated
(gray trace) or grown for 14-16 d in culture
(black trace). Ten millimolar glutamate was applied
during the time period marked by the horizontal bar.
Inset shows average desensitization time constants of
acutely dissociated (n = 10) or cultured E17
(n = 22) neurons. Time constants were not
significantly different (p = 0.49).
D, Scaled traces from E10 neurons either acutely
dissociated (gray trace) or grown for 14-16 d in
culture (black trace). Inset shows
average desensitization time constants of acutely dissociated
(n = 6) or cultured E10 (n = 20) neurons. *p < 0.05 indicates
significance.
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Having demonstrated the isolation of nMag neurons with normal receptor
properties, E10 neurons were then isolated and grown in low-density
cell culture for varying periods of time (see Materials and Methods),
and the desensitization rate of their AMPA receptors was assayed. The
average time constant of desensitization in E10 neurons after 1 d
in vitro was indistinguishable from that of E10 acutely
dissociated neurons (p = 0.94). Unlike the
acceleration of desensitization observed in vivo, the time
constant of desensitization for neurons isolated at E10 changed
marginally over the first 7.5 d in vitro (Fig.
4B, black filled circles). For example,
the time constant was 4.4 ± 0.3 msec (n = 6) in
freshly isolated E10 cells and 6.0 ± 0.5 msec (n = 16) in E10 cells cultured for 8.5 d (p = 0.07). These results suggest that, in the cell-culture environment,
young neurons are not exposed to signals that direct expression of
fast-gating subtypes of AMPA receptor.
An alternative model, however, is that the cell-culture environment
does not remove an in vivo signal, but itself imposes a
"slow" phenotype on AMPA receptors, as suggested by the slight but
insignificant slowing of desensitization over 8 d in
vitro and by the documented effects of culture conditions of
glutamate receptor expression (see Discussion). To test this
alternative more explicitly, we contrasted the kinetics of
desensitization in cells isolated from E10 and E17 animals and
maintained in culture for over 2 weeks. Remarkably, desensitization
time constants in neurons cultured from E17 animals did not become
slower and indeed were virtually unchanged, even after 14-31 d in
culture (Fig. 4C). In contrast, E10 neurons grown for 14-16
d exhibited slightly slower desensitization rates, with a mean value
significantly longer than the E10 cells in vivo
(p < 0.05) (Fig. 4D). Thus, although the culture environment may cause a slowing of receptor kinetics, only the younger cells responded to such an influence; once
the mature kinetic phenotype developed, it was stable even when cells
were removed from their native environment for long periods of time.
Synaptic and extrasynaptic receptors
Previous studies have suggested that, in some cases, synaptic and
extrasynaptic receptors for a given transmitter may be structurally and
physiologically different (Li et al., 1998). The present study indicated that the transition from slow to fast-desensitizing AMPA
receptors occurs uniformly over the neuron and not separately at
synaptic or extrasynaptic regions. We reasoned that if synaptic and
extrasynaptic receptors exhibited different rates of desensitization, as might be expected if developmental regulation acted only on synaptic
sites, then there should be a strong correlation between response
amplitude and channel gating kinetics. The somatic excitatory innervation of nMag neurons occupies ~45% of the cell body surface (Parks et al., 1990); some fraction of this area consists of
postsynaptic, receptor-rich densities, so that in mature cells some
patches should have contained extrasynaptic membrane, synaptic
membrane, or mixtures of both. Because AMPA receptors are clustered at
synaptic regions (Jones and Baughman, 1991; O'Brien et al., 1998;
Trussell et al., 1988), patches with the largest responses probably
contained synaptic receptors. Surprisingly, none of our patches from
either slices or freshly isolated cells were without glutamate
responses, suggesting that extrasynaptic receptors contribute, at least
in part, to the smallest responses we obtained. In Figure
5, the time constant of desensitization
is plotted against the response amplitude for patches from E16-E18
cells. Assuming a uniform size of patch (see Materials and Methods),
the data show that there is no correlation between receptor gating
kinetics and receptor density, despite an over 50-fold variation in
response amplitude. Thus, it seems likely that the expression of fast
AMPA receptors occurs at both synaptic and extrasynaptic regions.
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Figure 5.
AMPA receptor kinetics are uniform over the entire
neuron. Amplitude versus time constant of desensitization for 32 patches from E16-E18 nMag neurons. There was no correlation between
patch amplitude and desensitization at these ages, indicating that
synaptic and nonsynaptic receptors were indistinguishable in kinetic
characteristics. Correlation coefficient is 0.05. Inset,
Representative traces from data points, marked by gray
and black circles in main figure, which showed a 50-fold
variation in amplitude (left), are identical in time
course when peak responses are normalized
(right).
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DISCUSSION |
Developmental control of receptor subunits
Rapidly gating AMPA receptors mediate synaptic transmission in a
wide variety of neurons of the central auditory pathway of mammals and
birds (Trussell, 1999). Accordingly, several GluR subunits,
particularly GluR3 and GluR4, have been identified in mammalian and
avian auditory neurons, and glutamate receptor proteins localized to
their subsynaptic regions (Hunter et al., 1993; Sato et al., 1993;
Petralia et al., 1996; Levin et al., 1997; Wang et al., 1998). In rat
medial nucleus of the trapezoid body and the chick nMag, flop splice
variants are prominent (Geiger et al., 1995; Ravindranathan et al.,
2000). Moreover, calcium permeability measurements and molecular
analyses in rat and chick auditory neurons indicate reduced expression
of GluR2 (Geiger et al., 1995; Otis et al., 1995; Zhou et al., 1995;
Wang et al., 1998; Ravindranathan et al., 2000). The enhancement of
spermine sensitivity during development strongly indicates that there
is a reduction in the contribution of GluR2 to functional receptors
(Washburn et al., 1997). What differences in expression pattern might
underlie the slower desensitization observed at earlier developmental
stages? The strongest contributor to fast AMPA receptor kinetics is the expression of flop splice variants, as evidenced by the more rapid desensitization in
GluR2flop-GluR4flop
(Mosbacher et al., 1994; Koike et al., 2000) and the association of
slow desensitization with GluR2flip in vivo
(Geiger et al., 1995; Angulo et al., 1997; Gotz et al., 1997). Given
the strong influence of these splice variants in determining channel
kinetics, the data presented here suggest that a reduction in flip
expression is most likely to explain the developmental changes AMPA
receptor kinetics observed in nMag.
Target cells of the auditory nerve all produce rapidly gating AMPA
receptors and high levels of GluR3 and GluR4 (Hunter et al., 1993;
Raman et al., 1994; Wang et al., 1998; Gardner et al., 1999). Even
auditory nerve targets such as avian nucleus angularis or mammalian
stellate neurons, which may not transmit timing information (and
therefore would not be expected to require brief currents), also
feature fast-acting receptors (Hunter et al., 1993; Raman et al., 1994;
Gardner et al., 1999). Moreover, fusiform cells of the mammalian dorsal
cochlear nucleus, which receive excitatory input from both auditory
nerve and parallel fibers, express a mixture of subunit types, which
are segregated to the different types of synapse, with GluR4-containing
receptors restricted to auditory nerve synapses (Rubio and Wenthold,
1997).
Given this background, our results are consistent with the
hypothesis that cellular interactions associated with synapse formation by auditory nerve fibers directs the production of AMPA receptors suited for fast transmission. Within days after innervation, the rate
of desensitization of AMPA receptors increases, a change that is
completely prevented by isolating cells in tissue culture during the
same period. Apparently, the consequences of this cellular interaction
are irreversible; isolation of cells after receptors kinetics
have become fast did not result in the expression of slowly
desensitizing receptors, even after 2 weeks in vitro. Based on these studies, we suggest that a consequence of the initial innervation by auditory nerve is the imprinting of a unique profile of
coordinated receptor subunit expression. According to this hypothesis,
the maintenance of the mature pattern of receptor expression is
independent of ongoing transmitter release or electrical activity.
Finally, the observation that younger cells in culture acquire slower
receptors suggests that the cell-culture environment does indeed alter
the pattern of receptor expression (Condorelli et al., 1993; Bessho et
al., 1994; Gottmann et al., 1997; Chew and Gallo, 1999). Because older
neurons do not respond to such an influence, there may be a "critical
period" during which young neurons are able to respond to signals
that determine the subtypes of AMPA receptor that a neuron produces.
Changes in receptor subunits are often associated with innervation and
developmental maturation of neural circuitry. Synaptic currents in
neuromuscular junctions of mammals and Drosophila speed up
as synapses mature (Broadie and Bate, 1993; Fischbach and Rosen, 1997).
In mammals, this change is caused by neurally induced replacement of
 with  subunits of the muscle nicotinic acetylcholine
receptor. Alterations in NMDA, GABA, and glycine receptor composition
are observed in various brain regions during early development
(Carmignoto and Vicini, 1992; Takahashi et al., 1992, 1996; Monyer et
al., 1994; Gottmann et al., 1997; Singer et al., 1998). In the hair
cell-spiral ganglion cell synapse, receptors labeled by an antibody to
GluR2/3 are developmentally downregulated, with compensation by the
GluR4 subunit (Knipper et al., 1997). Developmental changes in receptor
composition may be mediated by factors released by presynaptic neurons
(Fischbach and Rosen, 1997). Indeed, secreted proteins have been
shown to cluster or induce expression of glutamate receptors on a
subunit-specific basis (Ozaki et al., 1997; O'Brien et al., 1999). It
is possible that auditory nerve terminals release substances that act
to control expression of AMPA receptor subunits and their alternative splicing.
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FOOTNOTES |
Received June 14, 1999; revised March 21, 2000; accepted April 18, 2000.
This work was supported by National Institutes of Health Grant DC02004.
We are grateful to Stephan Brenowitz, Stephanie Gardner, Donata Oertel,
Indira Raman, Rostislav Turecek, and Ken Tovar for thoughtful comments
on this manuscript.
Correspondence should be addressed to Dr. L. O. Trussell at
his present address: Auditory Neuroscience, L-335A, Oregon
Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR
97201. E-mail: trussell{at}ohsu.edu.
Dr. Lawrence's present address: Unit on Cellular and Synaptic
Physiology, Laboratory of Cellular and Molecular Neurobiology, National
Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, MD 20892.
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ABSTRACT
INTRODUCTION
