1Pediatric Regional Epilepsy Program and Joseph Stokes Research Institute of the Children's Hospital of Philadelphia and 2Department of Pediatrics, Division of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 3Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0599
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ABSTRACT |
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Cohen, Akiva S., Dean D. Lin, and Douglas A. Coulter. Protracted Postnatal Development of Inhibitory Synaptic Transmission in Rat Hippocampal Area CA1 Neurons. J. Neurophysiol. 84: 2465-2476, 2000. In the CNS, inhibitory synaptic function undergoes profound transformation during early postnatal development. This is due to variations in the subunit composition of subsynaptic GABAA receptors (GABAARs) at differing developmental stages as well as other factors. These include changes in the driving force for chloride-mediated conductances as well as the quantity and/or cleft lifetime of released neurotransmitter. The present study was undertaken to investigate the nature and time course of developmental maturation of GABAergic synaptic function in hippocampal CA1 pyramidal neurons. In neonatal [postnatal day (P) 1-7] and immature (P8-14) CA1 neurons, miniature inhibitory postsynaptic currents (mIPSCs) were significantly larger, were less frequent, and had slower kinetics compared with mIPSCs recorded in more mature neurons. Adult mIPSC kinetics were achieved by the third postnatal week in CA1 neurons. However, despite this apparent maturation of mIPSC kinetics, significant differences in modulation of mIPSCs by allosteric agonists in adolescent (P15-21) neurons were still evident. Diazepam (1-300 nM) and zolpidem (200 nM) increased the amplitude of mIPSCs in adolescent but not adult neurons. Both drugs increased mIPSC decay times equally at both ages. These differential agonist effects on mIPSC amplitude suggest that in adolescent CA1 neurons, inhibitory synapses operate differently than adult synapses and function as if subsynaptic receptors are not fully occupied by quantal release of GABA. Rapid agonist application experiments on perisomatic patches pulled from adolescent neurons provided additional support for this hypothesis. In GABAAR currents recorded in these patches, benzodiazepine amplitude augmentation effects were evident only when nonsaturating GABA concentrations were applied. Furthermore nonstationary noise analysis of mIPSCs in P15-21 neurons revealed that zolpidem-induced mIPSC augmentation was not due to an increase in single-channel conductance of subsynaptic GABAARs but rather to an increase in the number of open channels responding to a single GABA quantum, further supporting the hypothesis that synaptic receptors may not be saturated during synaptic function in adolescent neurons. These data demonstrate that inhibitory synaptic transmission undergoes a markedly protracted postnatal maturation in rat CA1 pyramidal neurons. In the first two postnatal weeks, mIPSCs are large in amplitude, are slow, and occur infrequently. By the third postnatal week, mIPSCs have matured kinetically but retain distinct responses to modulatory drugs, possibly reflecting continued immaturity in synaptic structure and function persisting through adolescence.
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INTRODUCTION |
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During postnatal development in central neurons,
there is a regionally distinct progression in cellular expression
patterns of GABAA receptor
(GABAAR) subunit mRNAs (Brooks-Kayal et
al. 1998a; Killisch et al. 1991
;
Laurie et al. 1992
). Furthermore significant changes in
the kinetic properties and pharmacology of
GABAA-mediated currents are also evident
(Oh et al. 1995
; Rovira and Ben-Ari
1993
). All of these shifts, either individually or in concert,
may contribute to alterations in inhibitory synaptic function evident
during maturation of the nervous system (Hollrigel and Soltesz
1997
). For example, in hippocampal dentate granule cells
(DGCs), the frequency of spontaneous inhibitory synaptic currents
increases, while rise and decay times decrease until adult values are
achieved. In addition, spontaneous inhibitory synaptic currents in DGCs
from developing animals exhibit an increased sensitivity to zinc and
reduced or absent benzodiazepine (BDZ) sensitivity (Hollrigel
and Soltesz 1997
). A lack of BDZ sensitivity is also evident in
developing CA3 pyramidal neurons (Rovira and Ben-Ari
1993
). These functional changes contrast dramatically with
GABAAR properties in adult animals and are most
probably attributable to changes in GABAAR
subunit composition. Thus a developmental, cell-specific shift in the
expression of GABAAR subunits may play an
essential role in transitions in the function of inhibitory circuitry
in the brain (Brooks-Kayal et al. 1998a
; Killisch
et al. 1991
; Laurie et al. 1992
).
However, the impact that different levels of expression of postsynaptic
receptor subunits may have on synaptic function in the developing brain
is difficult to infer. Synaptic currents are shaped in part by
postsynaptic channel kinetics, which are determined in turn by receptor
subunit composition. However, in addition to channel-gating properties,
other factors play a prominent role in shaping inhibitory receptor
currents, including the time course of transmitter release and the
concentration and lifetime of neurotransmitter in the cleft. These
latter properties are determined by variations in specific physiologic
and anatomic features of synapses, including the amount of
neurotransmitter released, possible cooperation between release sites,
and the size and shape of the synaptic cleft. These properties may also exhibit systematic fluctuations during development of the nervous system and therefore could also result in ontogenetic alterations in
inhibitory synaptic function. For example, in developing neurons, the
synaptic area (measured as postsynaptic density length) appears to be
larger (Blue and Parnavelas 1983; Markus and
Petit 1989
). Furthermore synaptic cleft width may be greater in
immature inhibitory synapses. Both of these anatomical changes could
increase the volume of the synaptic cleft during early development.
Accompanying synaptic cleft volume changes are possible changes in
subsynaptic receptor density. If CNS synapse development mirrors that
present in the peripheral nervous system, then postsynaptic receptor
density may gradually increase during synaptogenesis as receptors
coalesce and become concentrated directly under the presynaptic
terminal (Frank and Fischbach 1979
). In addition,
vesicle number and/or vesicular content could be different in
developing synapses. These factors, individually or in concert, could
decrease the level of postsynaptic receptor occupancy during
development, in contrast to full receptor occupancy of subsynaptic
inhibitory receptors during synaptic transmission present in the adult
CNS (see Mody et al. 1994
for review).
The functional relationship between ligand-gated receptors and their
agonists has most often been studied using isolated cells and
steady-state concentration-response kinetics (cf., Oh et al. 1995; Rovira and Ben Ari 1993
). Extrapolating
the significance of these data to synapses, where agonist concentration
rises and falls rapidly, is difficult. The present study combining
synaptic recording and rapid agonist application to perisomatic patches was undertaken to examine how the aforementioned developmental transitions in GABAAR function and subunit
composition interact to determine inhibitory synaptic function in
hippocampal CA1 neurons from developing and adult brain. Preliminary
reports of this work have appeared (Cohen and Coulter
1998
; Lin et al. 1998
).
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METHODS |
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Tissue preparation
Male Sprague-Dawley rats were used in all experiments.
Recordings were obtained from visually identified pyramidal neurons in
stratum pyramidale of area CA1 of the rat hippocampus. For the purpose
of this study, animals were divided into four groups: neonatal
postnatal day [(P) 1-7], immature (P8-14), adolescent (P15-21), and adult (P56+ days). Brain slices were prepared using previously reported methods (Rafiq et al. 1993). In
brief, rats were anesthetized with halothane and decapitated, and the
brain was quickly removed and chilled for 1-2 min in a modified
sucrose-based artificial cerebrospinal fluid (aCSF) composed of (in mM)
201 sucrose, 3.2 KCl, 1.25 NaHPO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose (equilibrated with 95%
O2-5% CO2 at 32.5°C).
The brain was glued, frontal side down, to a glass platform with
cyanoacrylate cement, and coronal whole brain slices (350 µM for
neonatal, immature and adolescent animals, 225 µM for adult animals)
were sectioned using a Vibratome (Lancer 1000, St. Louis, MO). Brain
slices were subsequently hemisected, transferred to a holding chamber,
and incubated in warm (35°C) normal aCSF containing 126 mM NaCl
substituted for sucrose and allowed to equilibrate for at least 2 h before being transferred to the recording chamber.
Patch recording in slices
Whole cell voltage-clamp recordings were conducted at room
temperature from visually identified CA1 pyramidal neurons using infrared differential interference contrast or Hoffman modulation contrast video microscopy (cf. Stuart et al. 1993).
Cells were voltage clamped at
60 mV, and signals were recorded and
amplified with an Axopatch 1D (Axon Instruments, Foster City, CA),
filtered at 2 kHz, digitized, sampled at 44 kHz with a PCM digitizer
(Neuro-Corder DR-890, Neurodata Instruments, NY), and stored on
videotape for off-line analysis. Electrodes were fabricated from
thick-wall borosilicate glass (World Precision Instruments, Sarasota,
FL) and pulled to a resistance between 2 and 6 M
when filled with an
internal solution composed of (in mM) 135 CsCl, 10 HEPES, 2 MgCl2, and 4 MgATP, pH 7.25 (CsOH) on a two-stage
puller (Narishige PP-83, East Meadow, NY). A 2 mV junction potential
was measured between this solution and the bath aCSF. All data were
left uncorrected.
Rapid agonist application
Fast application of agonists was performed as described by
Jonas (1995). Theta glass was mounted on a piezoelectric
transducer (Burleigh, Fishers, NY). Waveform protocols were generated
using Clampex 7.0 software (Axon Instruments). Agonists were applied at
10 to 20 s intervals, and traces shown in figures are averaged from at
least five applications.
On excision of an outside-out patch, the tip of the patch electrode was positioned in the control solution, approximately 20 µm from the interface separating the control and drug streams, which was visualized by the addition of 25 mM sucrose to the drug solution. The patches yielded GABA (1 mM) currents between 10 and 250 pA. In experiments in which zolpidem and GABA were co-applied, zolpidem was included in control solutions. After rupturing the patch, the 20-80% exchange times of the liquid junction currents between control and a 90% control/10% distilled H20 solution was typically between 200 and 250 µs (see open tip responses in Fig. 7).
Analysis of mIPSCs
Recorded mIPSCs were reacquired using Dempster software
(Strathclyde, Glasgow, UK), which collects events using a manually controlled threshold detector and is capable of detecting events as
small as two to three times the baseline noise. To attempt to minimize
cases of inadequate space clamp, neurons were used for analysis only
when series resistance (Rs) was 20
M
, and
80% series resistance compensation was achieved.
Rs was checked frequently throughout
experiments, and neurons in which Rs
increased >20% were discarded. In addition, event amplitudes were
plotted against rise times and examined for a possible correlation,
where a significant correlation (r2 > 0.5) was assumed to signify inadequate space clamp. Neurons in which
this occurred were discarded. This occurred in <0.2% of neurons. The
kinetics of mIPSCs i.e., amplitude, rise, and decay times were analyzed
using cumulative probability histograms. mIPSC frequency was determined
using Mini Analysis software (Synaptosoft, Leonia, NJ).
Peak scaled nonstationary noise analysis (NSNA) of mIPSCs (De
Koninck and Mody 1994; Perrais and Ropert 1999
;
Traynelis et al. 1993
) was conducted by averaging 50 events to form a single mean ensemble mIPSC time course for a given
cell. Fifty to 75 mIPSCs were then randomly selected from the same cell
and used for further analysis. The ensemble average was scaled up or
down to the size of each original trace and then subtracted.
Subtraction of the average ensemble current left a noise trace which
fluctuated around the zero current level (see RESULTS and
Fig. 6B). The variance at each time point of every
individual trace was calculated, and the mean variance was plotted
against the mean current amplitude. Data plotted in this manner were
fit by a parabolic curve with the following equation
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Reagents and statistical tests
Reagents were purchased from the following vendors: all salts and zolpidem, diazepam, furosemide and bicuculline methiodide from Sigma (St Louis, MO); D-2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from Research Biochemicals International (Natick, MA); tetrodotoxin (TTX) from Calbiochem (La Jolla, CA). All drugs were made as stock solutions and then diluted to their final concentration in the bathing medium. Control cells (n = 3) received sham drug administration, i.e., the flow line was switched for 30 min to the same reservoir used in the drug experiments, but in this case the reservoir contained only normal aCSF (data not shown). This procedure controlled for any switching-induced pressure artifacts that may have affected recording parameters in addition to those caused by the drugs. No switching artifacts were apparent, however.
Statistical significance between cumulative probability distributions
in control and drug conditions in individual neurons was assessed at
the P < 0.05 confidence level using the
Kolmogorov-Smirnov nonparametric statistical test. Two-tailed unpaired
Student's t-tests were performed to determine statistical
significance at the P < 0.05 confidence level when
comparing different treatment groups. Diazepam concentration-response
curves were best fitted employing a nonlinear least-squares method
assuming a monophasic sigmoidal diazepam concentration-response
relationship with the use of the following equation
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RESULTS |
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Spontaneous miniature inhibitory currents are present at all postnatal stages
To investigate how developmental changes might affect synaptic
function in the developing brain, we examined the postsynaptic responses elicited by the spontaneous release of GABA from single presynaptic vesicles. These events persist in the presence of TTX and
are resistant to blocking Ca2+ entry into the
terminal and therefore, by analogy to miniature endplate potentials
first studied by Fatt and Katz (1952) have been termed
mIPSCs. The advantage of studying mIPSCs is that they are due to
activation of single synapses, in contrast to stimulation studies,
which can activate tens or hundreds of synapses simultaneously. Therefore mIPSC studies may provide additional insight into the normal
functioning of GABAergic synapses. In the presence of TTX (400 nM) and
the excitatory amino acid antagonists D-AP5 (50 µM) and
CNQX (6 µM), spontaneous inward currents were evident at all ages
(Fig. 1). Events occurring with a
frequency of
0.1 Hz were only detected in 13 (5/38) and 15% (6/41)
of P1-7 and P8-14 neurons, respectively (Fig. 1C). mIPSC
frequency increased markedly and reached adult values by the third
postnatal week (see Fig. 1), which may represent increased innervation
by inhibitory interneurons (Dupuy and Houser 1996
). The
GABAergic identity of these events was confirmed by their blockade by
the GABAA antagonist bicuculline methiodide (30 µM, data not shown). Furthermore the reversal potentials (Erev) for adolescent and adult
neurons were 3.1 ± 2.6 and 1.2 ± 4.1 mV, respectively
(n = 3 for both populations, not significantly different, P > 0.05). Both values were close to the
theoretical value of EGABA (0.2 mV) as
calculated by the Goldman-Hodgkin-Katz equation for a GABA conductance
(Hodgkin and Katz 1949
), assuming a bicarbonate to
chloride permeability ratio of 0.025 and an activity coefficient of
0.75 for the chloride solution (Bormann et al. 1987
).
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Biophysical properties of miniature IPSCs from neonatal (P1-7), immature (P8-14), adolescent (P15-21), and adult (P > 56) CA1 neurons
Examples of averaged mIPSCs from neonatal, immature, adolescent and adult neurons are depicted in Fig. 2. Analysis of mIPSCs recorded from P1-7 and P8-14 pups revealed significantly larger mIPSC amplitudes, longer decay times, and slower rise times compared with those present in the adult (P > 0.05, see Fig. 2, B-D, and Table 1). Conversely, the decay and amplitude of mIPSCs recorded from adolescent (P15-21) neurons were similar to those present in adult neurons (Fig. 2, B and C; Table 1). However, 10-90% rise times measured in adolescent CA1 neurons were still significantly slower when compared with adult values (P < 0.05, Fig. 2D; Table 1). Because the frequency of mIPSCs in neonatal and immature (P1-14, Fig. 1, B and C; Table 1) neurons was so low, conducting pharmacological studies on these age groups was extremely difficult. Therefore we were confined to investigate further potential differences in inhibitory GABAA receptor pharmacology in adolescent (P15-21) and adult CA1 neurons.
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Differential effects of zolpidem on mIPSCs from adolescent and adult neurons
GABAARs are believed to be pentameric
(Chang et al. 1996; Tretter et al. 1997
)
and composed of subunits from several related subunit families.
GABAAR subunit composition varies
developmentally, regionally, and in specific neuronal subtypes. This in
turn influences the conductance, channel kinetics, and pharmacological
sensitivity of GABA-evoked responses (Macdonald and Olsen
1994
; Wisden et al. 1992
). For example BDZ
sensitivity is conferred by the presence of a
2 subunit
(Pritchett et al. 1989
), and given that a
2 subunit is present, it has been shown that the
subunits in the receptor dictate the specific BDZ ligand that binds to and modulates the GABAAR complex (Lüddens and Wisden
1991
; Pritchett et al. 1989
). For example,
GABAARs in which
1 subunits are expressed
together with
x
2 exhibit high affinity for
BDZ type 1 (BDZ1) agonists like zolpidem (Arbilla et al.
1986
; reviewed in Barnard et al. 1998
;
Macdonald and Olsen 1994
). GABAARs
containing
2,
3, or
5 subunits demonstrate lower affinity for
BDZ1-specific ligands, with
4- and
6-containing receptors
completely lacking BDZ agonist sensitivity (Wafford et al.
1996
). Immunohistochemical (Killisch et al.
1991
) and in situ hybridization (Wisden et al.
1992
) studies demonstrated that
1 can be detected shortly
after birth (P6) in forebrain neurons, and its expression continues to
increase significantly until adulthood.
If an apparent developmental up-regulation of the 1 subunit was
occurring in CA1 neurons, we hypothesized that mIPSCs recorded in adult
CA1 neurons would demonstrate higher BDZ1 sensitivity relative to
adolescent neurons. Therefore, we examined the effects of zolpidem
(ZOL), a specific BDZ1 receptor agonist (Biggio et al.
1989
; Wafford et al. 1993
). Bath application of
ZOL (200 nM) affected both the amplitude and decay of mIPSCs recorded
from P15-21 CA1 pyramidal neurons (Fig.
3A, 1 and
2, and inset). The median amplitude was
significantly enhanced from
34.7 ± 6.8 (n = 15)
to
44.8 ± 7.1 pA (a 30.6% enhancement, n = 6, P < 0.05). The median T50 decay time was also significantly
increased from 12.2 ± 2.5 to 24.3 ± 5.0 ms (Fig.
3A2 and inset). Furthermore, the slow component
of the decay,
slow, was significantly
augmented as well from 19.5 ± 2.8 to 32.1 ± 3.2 ms. The
contribution of
slow to the entire decay was
significantly enhanced (from 51 ± 11 to 90 ± 4%,
P < 0.05). The fast component of the decay was unaffected
by ZOL application (
fast = 4.8 ± 1.9;
6.0 ± 3.5 ms for control and ZOL, respectively). These results
contrast with ZOL effects on mIPSCs recorded from adult pyramidal
CA1 neurons, where bath application of ZOL significantly slowed only
the decay of the mIPSCs (Fig. 3B, 1, 2 and inset 1, T50 increased from 10.8 ± 1.5 to 16.9 ± 3.9 ms, n = 6, 4 respectively, and
slow increased from 19.7 ± 3.4 to
28.4 ± 3.1 ms, P < 0.05). Interestingly, mIPSC amplitude and contribution of
slow to the
entire decay were not altered (Fig. 3B1 and
inset). As has been previously shown, mIPSC amplitudes
recorded in adult neurons in the presence of BDZs were unchanged from
that recorded in control solution (Otis and Mody 1992
;
Poncer et al. 1996
). The total mIPSC charge transfer was significantly enhanced by ZOL application from
654.7 ± 289.0 to
1013.5 ± 187.2 pC (a 54.8% augmentation, P < 0.05).
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The mean increase in mIPSC amplitude observed during ZOL exposure to
P15-21 CA1 pyramidal neurons profoundly affected total mIPSC charge
transfer in these cells, relative to ZOL effects on mIPSCs in adult
neurons. The mean charge transfer was augmented 110.1% (from
675.5 ± 134.7 to
1419.1 ± 237.1 pC, Fig. 3A2
and B2, insets). When the amplitude effects were normalized
by scaling the control sweep to the ZOL sweep, the ZOL-induced
enhancement was markedly smaller (56.3%; range 41.2-70%, normalized
ZOL-Cont, Fig. 3A2, inset), and comparable to the
augmentation induced by ZOL on mIPSCs in adult neurons (50.5%; range
32.1-71.8%, Fig. 3B2, inset). Thus the large increase in
charge transfer during ZOL application demonstrates that ZOL is more
efficacious in enhancing inhibitory neurotransmission in P15-21 CA1
neurons primarily due to the specific mIPSC amplitude effects in these
neurons. Furthermore the mean mIPSC frequency was not significantly
altered in the presence of ZOL, suggesting that the ZOL-induced mIPSC
enhancement is due to a postsynaptic mechanism in both adolescent and
adult CA1 neurons. The mIPSC frequency in the adolescent group was
3.1 ± 1.2 and 3.6 ± 2.0 Hz for control and ZOL,
respectively, while the frequency in the adult group was 3.0 ± 1.8 and 3.7 ± 1.2 Hz in control and ZOL (unpaired
t-test P > 0.05).
Diazepam, like ZOL, differentially modulates mIPSCs from adolescent and adult CA1 neurons
What factor(s) could possibly be mediating differential mIPSC amplitude effects of BDZ agonists present in adolescent inhibitory synapses? Could it simply be due to enhanced ZOL sensitivity in the adolescent mIPSCs due to dissimilar expression levels of BDZ-sensitive subunits in the adolescent receptors? To examine this possibility, we pharmacologically probed adolescent and adult GABAARs with a second drug, the broad-spectrum BDZ diazepam (DZ). Concentration-response curves were constructed for effects of DZ on CA1 mIPSCs in both populations. It was technically difficult to record in control aCSF and then bath apply each DZ concentration to each cell due to the necessarily long duration of the recordings. Therefore we preincubated slices in varying diazepam concentrations (1-300 nM) and plotted the mean values of the median amplitudes and T50s of mIPSCs in neurons for each individual concentration. When plotted as log [DZ] versus percentage increase in T50, the decay time was found to increase in sigmoidal manner as greater concentrations of diazepam were applied to both adolescent and adult slices (Fig. 4A).
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The concentration-response relationships representing DZ-induced
effects on mIPSC decay time and amplitude contrasted markedly with each
other. In the decay plot (Fig. 4A), both curves are sigmoidal in shape and are not significantly different from each other.
The efficacy (or maximal effect) of DZ on mIPSC decay time was not
significantly different in adolescent and adult neurons (P > 0.05, unpaired t-test, 100 and 300 nM
concentrations). Prolongation of mIPSC decay in both populations is not
surprising since both ZOL and midazolam have been reported to slow
mISPC decay time in adult DG (Otis and Mody 1992) and
CA3 (Poncer et al. 1996
) neurons. The Hill coefficients
for these curves (Fig. 4A) were 1.8 and 1.5 for adolescent
and adult cells, respectively.
In the amplitude plot (Fig. 4B), the two curves are notably
dissimilar. In the adult population, there is no significant amplitude potentiation as the concentration of DZ was increased up to 300 nM
(Fig. 4B), consistent with previous studies (Otis and
Mody 1992; Poncer et al. 1996
). On the other
hand, the amplitude plot in the adolescent population is sigmoidal,
i.e., mIPSC amplitude increases with increasing DZ concentrations. In
addition, the efficacy of DZ-induced mIPSC amplitude augmentation in
the P15-21 group is significantly higher than that exhibited in the
adult population (P < 0.05, unpaired
t-test, 100 and 300 nM concentrations). Adolescent mIPSC
amplitude augmentation induced by DZ was similar to that previously
demonstrated with ZOL (Fig. 3), both of which suggest that postsynaptic
GABAARs in adolescent synapses are not fully
occupied by the release of a single quantum.
Possible developmental increases in BDZ-insensitive receptors do not appear to contribute to altered BDZ sensitivity
BDZ-induced mIPSC amplitude effects, which we are hypothesizing
may be due to partial occupancy of postsynaptic
GABAARs in response to quantal GABA release, may
also be due to different receptor subunit composition present in
inhibitory synapses in adolescent neurons. Therefore we next considered
whether overall BDZ sensitivity might be diminished due to an
up-regulation of 4 in the adult population. If
4 was being
substituted for either
1 or
5, then this should result in an
overall decrease in BDZ sensitivity of mIPSCs recorded from adult
neurons because inclusion of
4 in the pentameric receptor leads to
BDZ insensitivity (Wieland et al. 1992
). To test this
hypothesis, we examined mIPSC sensitivity to an
4 and
6
subunit-specific modulator.
The diuretic furosemide has been shown to specifically block
GABAARs composed of 4 or
6 subunits in
addition to
and
(Tia et al. 1996
; Wafford
et al. 1996
). Bath application of furosemide (600 µM) for 40 min significantly altered the amplitude and decay of mIPSCs from both
adolescent and adult neurons (P < 0.05; Fig. 5). Application of furosemide reduced the
median mIPSC amplitude by 33 ± 3 and 28 ± 4% and increased
the median T50 31 ± 5 and 16 ± 4%, respectively, for
adolescent and adult neurons. These effects are very similar to what
has been reported previously in developing CA1 pyramidal neurons
(P14-42) (Banks et al. 1998
). Since
6 is only
expressed in the cerebellum, the similar alterations in mIPSC kinetics
induced by furosemide suggests that BDZ-induced mIPSC amplitude effects
are not due to differential alterations in
4 expression, which is
making a similar contribution to GABAARs involved
in synaptic transmission at both developmental stages.
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Amplitude augmentation of mIPSCs in adolescent neurons by allosteric modulators is due to an increased number of activated synaptic channels
The significant BDZ augmentation of mIPSC amplitude recorded in
adolescent neurons could be a result of either an increase in single
GABAA channel conductance (g), as
reported in cultured hippocampal neurons (Eghbali et al.
1997), or due to an increase in the number of activated
postsynaptic channels (N) or the open probability of an
activated channel (Po). We conducted peak scaled nonstationary noise
analysis to elucidate the mechanism(s) underlying the ZOL-induced
increase in adolescent (P15-21) mIPSC amplitude (see Fig.
6). According to nonlinear least-squares
fitting of the resulting noise curves under control and ZOL-exposed
conditions (Fig. 6, C and D, respectively), the
mean single-channel conductance g for neurons under control
conditions was 26.7 ± 1.8 pS, not significantly different from
g for the cells in the presence of ZOL (25.0 ± 0.9 pS)
or g of control adult neurons (25.6 ± 1.0 pS,
n = 4, Table 2). This is
similar to the predominant conductance state of 30 pS measured in
single-channel studies (Schonrock and Bormann 1993
).
Hence ZOL-induced augmentation of mIPSC amplitude is probably not due
to an increase in subsynaptic receptor single-channel current. Since
two main variables determine peak amplitude, g and
NPo, and g has not changed, then NPo
must have. Accordingly, NPo (the number of activated
channels) during ZOL application was 32 ± 0.5 (n = 3), significantly larger than NPo in control conditions
(21 ± 0.5, n = 3). Because peak scaling was used
to minimize some of the inherent variability in mIPSC amplitudes, it is
not possible to determine whether N or Po was enhanced by ZOL in this analysis; however, see following text.
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Patch currents
From the data described in the preceding text, we hypothesized that during synaptic activity, GABA cleft concentration may be lower or Po might be decreased in adolescent synapses compared with that present in adult synapses. An alternate explanation for the preceding data is that there could be some occult difference between the properties of receptors in the two developmental stages that are not manifest in whole-cell synaptic recordings, and these subtle distinctions account for the specific amplitude effects of GABA modulators in adolescent neuron mIPSCs. To further explore this issue, we conducted additional experiments, examining ZOL-induced effects on GABA-evoked currents in perisomatic outside-out patches pulled from adolescent and adult CA1 neurons. In these experiments, saturating concentrations of GABA or GABA + ZOL were applied using ultra-rapid agonist application techniques, to better mimic GABA application kinetics during synaptic responses.
If Po was enhanced by ZOL application, then even under saturating GABA
concentrations, an increase in the current amplitude and/or kinetics
should have been evident. Therefore outside-out patches were exposed to
1-ms pulses of a high concentration (1 mM) of GABA that are thought to
be saturating (Lavoie and Twyman 1996; Mellor and
Randall 1997
; Oh et al. 1995
) in the absence and
presence of 200 nM ZOL to examine ZOL's effects on maximally activated
GABAARs (Fig.
7B). In six patches excised
from adolescent CA1 neurons, no significant alterations were induced by
co-application of ZOL and 1 mM GABA (on peak amplitude or decay time)
when compared with the current transient evoked by 1 mM GABA alone
(P > 0.05). In similar fashion to previous investigations
(Hill et al. 1998
), decay kinetics of the GABA-evoked
currents were best fit using two exponentials. The fast, and slow decay
constants and percentage fast decay component of GABA (1 mM)-evoked
responses measured in patches pulled from adolescent neurons were not
significantly different (P > 0.05 t-test,
see Table 3) from the values obtained when GABA was co-applied with ZOL. In similar fashion the fast and slow
decay constants, as well as the percentage fast decay component
exhibited in patches excised from adult CA1 neurons in response to 1 mM
GABA responses, were not significantly different (see Table 3).
Interestingly,
fast in the adult patches (in GABA alone and GABA + ZOL) was significantly slower than that measured
in adolescent patches (P < 0.05, see Table 3). The
data derived from excised patches exposed to saturating concentrations of GABA co-applied with ZOL clearly showed no effect on either the
amplitude or kinetics of the response (Fig. 7B, Table 3). Therefore, it is unlikely that Po is enhanced by ZOL application, but
rather a single GABA quantum activates a larger number of channels in
the presence of ZOL.
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When low (50 µM), nonsaturating concentration of GABA was
applied to patches pulled from adolescent neurons, this resulted in
peak current amplitudes evoked that were only 36.2 ± 4.6%
(n = 4) of the peak amplitudes evoked during maximal
GABAAR activation (to 1 mM GABA), confirming 50 µM GABA is nonsaturating (Fig. 7A). Figure 7C
depicts the currents evoked by 1-ms pulses of low (nonsaturating) concentrations (50 µM) of GABA on the same patch. When co-applied with 50 µM GABA, 200 nM ZOL induced a 17.6 ± 5.9% augmentation of peak response amplitude (n = 3) with no significant
effects on decay kinetics (GABA: fast = 6.4 ± 0.8 ms,
slow = 149.8 ± 23.1;
GABA+ZOL:
fast = 6.0 ± 0.7
slow = 130.5 ± 5.8 ms, P > 0.05). The potentiation observed with co-application of ZOL and subsaturating concentrations of GABA (Fig. 7C) is similar to
the ZOL-induced mIPSC augmentation observed in whole cell experiments, further supporting the concept that GABA cleft concentrations during
the peak of the mIPSC may be subsaturating in adolescent (P15-21) CA1 neurons.
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DISCUSSION |
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The primary findings of this CA1 pyramidal neuron study are GABAergic inhibitory synapses are functional immediately after birth; mIPSCs are larger and slower in neonatal and immature (P1-14) neurons compared with those present in the adult; mIPSC frequency is extremely low early in development but reaches adult values by the beginning of the third postnatal week; mIPSC amplitudes and decay times in adolescent (P15-21) neurons are not significantly different from adult values; however, 10-90% rise times are still significantly slower; and sub-synaptic GABAARs in adolescent CA1 neurons are differentially sensitive to modulation by BDZs, reflecting a protracted postnatal immaturity of inhibitory synapses in these neurons.
In early postnatal development, GABA functions as an excitatory neurotransmitter
In the hippocampus, GABAergic interneurons mature and cease to
divide before their principal neuron counterparts (i.e., pyramidal and
granule cells) (Amaral and Kurz 1985). Moreover,
hippocampal inhibitory networks are well developed at birth
(Rozenberg et al. 1989
) contrasting markedly with
excitatory inputs, which develop during the first postnatal week of
life (Richter and Wolf 1990
). Previous studies have
demonstrated that GABA application to CA3 (Ben-Ari et al.
1994
; Cherubini et al. 1991
) and CA1
(Janigro and Schwartzkroin 1988
) neurons elicits
depolarizing responses at resting membrane potentials during the first
postnatal week, which subsequently reverse to hyperpolarizing during
the second week of development. The depolarizing responses induced by
GABA are thought to be important in causing sufficient depolarization to activate voltage-gated Ca2+ channels promoting
a rise in intracellular Ca2+ that is essential
for neuronal growth and differentiation (Miller 1988
).
Hippocampal neurons grown in the presence of a
GABAA antagonist were stunted with respect to
dendritic length, number of primary neurites, and branch points
(Ben-Ari et al. 1994
). Although whole cell
recording-induced dialysis in the present study precludes examination
of altered intracellular chloride levels, changes in the kinetics of
inhibitory synaptic function were evident during early postnatal
development. mIPSCs recorded from neonatal and immature (P1-14) CA1
neurons were significantly slower and larger compared with adult
values. Since GABAAR subunit composition
undergoes significant changes during the first two weeks of postnatal
development (Killisch et al. 1991
; Laurie et al.
1992
), the kinetic changes most notably observed in the present
study between P1 and P14 are probably due to receptor subunit
rearrangement. This hypothesis could not be further tested because a
detailed pharmacological analysis probing neonatal and immature mIPSCs
was unfeasible due to the low frequency of recordable events in these
populations (see Fig. 1C).
However, in the periphery, at the neuromuscular junction, nicotinic
acetylcholine receptors are diffusely present along the entire myotube,
but on innervation, these receptors migrate and concentrate only in the
area directly underneath the presynaptic terminal (Frank and
Fischbach 1979). If CNS synapses develop in similar fashion,
then developmental transitions in postsynaptic receptor clustering
under the presynaptic terminal from diffuse to highly concentrated
would not be surprising. In addition, having loosely packed subsynaptic
GABAARs encompassing more area might be crucial
in the CNS by generating a wider spread depolarization facilitating
robust Ca2+ entry necessary for neuronal growth
and differentiation. Furthermore it is reasonable to hypothesize that
the larger and slower neonatal and immature mIPSCs may also offer a
longer effective temporal window for the requisite summation of
depolarizing GABAA-mediated signals facilitating
intracellular calcium accumulation essential for the aforementioned
neuronal growth and differentiation.
Age-dependent disparate mIPSC modulation by BDZ agonists
Previous studies have demonstrated age-dependent changes in BDZ
binding sites, GABAAR immunohistochemistry, and
expression of mRNAs encoding GABAAR subunits
during postnatal development (Brooks-Kayal et al. 1998a;
Killisch et al. 1991
; Laurie et al. 1992
). Little is known about how these changes in subunit
composition and density of GABAARs may impact
inhibitory synaptic function during development. In the present study,
when CA1 inhibitory neurotransmission was probed with allosteric
agonists, we found a marked discrepancy between the modulatory
responses of adolescent and adult inhibitory synaptic events. Bath
application of broad spectrum and BDZ1-specific BDZ agonists
significantly increased the mIPSC decay time in both adult and
adolescent populations to an equivalent extent. However, in addition to
the expected observation of slowing mIPSC decay time (De Koninck
and Mody 1994
; Otis and Mody 1992
; Poncer
et al. 1996
), both agonists enhanced mIPSC amplitude at
adolescent synapses. Thus the kinetics and sensitivity of adolescent
and adult mIPSCs to modulation by BDZ agonists undergoes
significant transformation during postnatal development.
Possible mechanisms mediating BDZ-induced mIPSC amplitude effects
What factors could potentially be causing the differential BDZ
sensitivities evident in adolescent and adult neurons; specifically what properties of adolescent (P15-21) synapses could lead to BDZ-induced mIPSC peak effects? First, the subsynaptic receptors in the
two populations could be different i.e., the specific subunit composition of GABAARS could be dissimilar;
thereby, conferring disparate pharmacological sensitivities in
adolescent and mature CA1 pyramidal neurons (reviewed in Barnard
et al. 1998; Macdonald and Olsen 1994
;
Mody et al. 1994
). Second, BDZ application could be
causing modulator-induced alterations in single-channel conductance (Eghbali et al. 1997
). Finally, the anatomical structure
of the synapses could differ, altering receptor clustering dynamics
and/or transmitter concentration in the cleft.
To assess whether subunit composition in the two populations differed,
we assayed several subunit specific modulators. Application of both
broad-spectrum BDZ and BDZ1-specific modulators elicited mIPSC peak
enhancement. Therefore, differential expression of subunits regulating
GABAAR BDZ sensitivities (2,
1, and
5) (see Barnard et al. 1998
) are probably not responsible
for the altered BDZ responses in the two receptor populations.
Furthermore, using furosemide to probe the receptors for potential
increased contributions of BDZ-insensitive subunits (
4)
(Banks et al. 1998
; Tia et al.
1996
; Wafford et al. 1996
) demonstrated no
significant differential sensitivities between adult and adolescent
GABAARs (see RESULTS). Finally, no
significant differences in biophysical parameters (besides 10-90%
rise times) or disparate agonist-induced effects on mIPSC kinetics
(specifically decay times) were evident in the two populations, and the
noise-analysis-derived single-channel conductance in adolescent and
adult synaptic GABAARs was not significantly different (see RESULTS). These data suggest that
GABAAR subunit composition in adolescent and
adult CA1 neurons are not markedly different. This suggests two things:
first, most of the large scale developmental transitions in
GABAAR subunit composition may be occurring prior
to adolescence, and second, other factors may be responsible for the
differential BDZ sensitivity in adolescent versus adult synapses.
Several recent reports have reported similar agonist-induced effects on
mIPSC amplitude. High concentrations of zolpidem (10 µM) enhanced
both mIPSC amplitude and duration in layer V cortical neurons
(Perrais and Ropert 1999), rat and mouse CA1 pyramidal cells and interneurons, as well as mouse dentate granule cells (Hajos et al. 2000
). Diazepam increased autaptic mIPSC
amplitude by 125% in cultured amacrine cells (Frerking et al.
1995
), while flurazepam selectively augmented large mIPSCs in
cerebellar stellate cells (Nusser et al. 1997
).
Furthermore flunitrazepam increased mIPSC amplitude in cultured
cerebellar granule cells (Mellor and Randall 1997
), and
the
-butyrolactone, diethyl-lactam, enhanced mIPSCs in cultured
hippocampal neurons (Hill et al. 1998
). Finally, the
imidazole etomidate increased mIPSC amplitude by 143% in cultured hippocampal neurons (Yang and Uchida 1996
).
Interestingly, all the aforementioned studies were undertaken in
developing animals (P13-26) (Hajos et al. 2000
; Nusser et al.
1997
; Perrais and Ropert 1999
) or in neuronal
cultures where developmental status is difficult to assess
(Frerking et al. 1995
; Hill et al. 1998
;
Mellor and Randall 1997
; Yang and Uchida
1996
). These data all suggest that subsynaptic
GABAARS in developing neurons are not fully
occupied by the release of a single quantum.
If the postsynaptic receptor-containing area is larger in adolescent
neurons, distributing subsynaptic receptors over a wider postsynaptic
area, then transmitter released from a punctate source, e.g., a single
quantum, may not reach saturating levels for those receptors distant
from the vesicular release site. Examining synaptic ultrastructure in
developing (P14) and mature brains (P90), Blue and Parnavelas
(1983) reported a 20% decrease in mean postsynaptic density
length in putative inhibitory Gray's type II synapses accompanying
development. Furthermore, Markus and Petit (1989)
showed
a shift in synaptic curvature from a balance between concave and convex
synapses during development (P15-30) to a predominance of convex
synapses in adult (P60-90). This is intriguing because concave or
"smile" synapses have larger postsynaptic areas compared with
convex or "frown" synapses (Markus and Petit 1989
).
The latter study did not discriminate between type I and type II
synapses. However, both of these findings suggest a consolidation in
subsynaptic area may occur during postnatal development, consistent
with a putative transition from partial to full synaptic receptor
occupancy during activity.
Developmental decreases in synaptic area may be accompanied by
increases in subsynaptic receptor density. Other anatomical and
functional alterations besides subsynaptic area and receptor number
could also impact subsynaptic GABA concentrations. For example,
transmitter clearance mechanisms could be different in adolescent
neurons. This is unlikely, however, since GABA transporters appear to
have matured by the second week of ontogenesis (Draguhn and
Heinemann 1996), and mIPSCs have been shown to be unaffected by
GABA uptake inhibitors (Otis and Mody 1992
;
Thompson and Gähwiler 1992
). An additional
anatomical mechanism could involve alterations in the synaptic cleft,
where the cleft area could diminish in size on maturity, thereby
decreasing cleft volume and increasing the GABA cleft concentration
following quantal release. Interestingly, even though the mean mIPSC
amplitude was not significantly different in the adult and adolescent
populations, there was a tendency demonstrating that mIPSCs recorded in
adult neurons were larger than their adolescent counterparts. Moreover
GABAergic synapses are difficult to identify morphologically in early
postnatal dentate gyrus (Dupuy and Houser 1996
),
supporting a possible increased synaptic cleft distance during
development. Furthermore in the present study, 10-90% rise times in
adolescent mIPSCs were significantly slower than those measured in
adults, suggesting that activation of adolescent postsynaptic
GABAARS is retarded because the effective transmitter cleft concentration is less in these synapses, delaying channel activation.
In summary, these results demonstrate that spontaneous miniature inhibitory currents become faster and smaller as they mature and reach adult values by the third postnatal week. This maturation may be due to alterations in GABAAR subunit composition as well as fine tuning of synaptic ultrastructure/cytoarchitecture and the resultant condensing of receptor clusters apposing presynaptic active zones. Although adolescent synapses appear to function similarly to adults, significant differences in their modulatory responses to BDZs are evident. The pharmacological disparity existing between inhibitory synapses in adolescent and adult CA1 pyramidal neurons may be conferred by anatomical alterations in synaptic structure in the absence of significant receptor changes and may represent a novel mechanism for regulating transmitter efficacy by receptor modulators in adolescent inhibitory CNS synapses. These data demonstrate that inhibitory synaptic transmission undergoes a markedly protracted postnatal development in rat CA1 pyramidal neurons, possibly reflecting continued immaturity in synaptic structure persisting until adolescence.
Alterations in inhibitory function can contribute to the generation
of seizure disorders (Brooks-Kayal et al. 1998b,
1999
; Buhl et al. 1996
; Gibbs et al.
1997
). Moreover, since GABAARs are a
primary site of action for many anti-epileptic drugs, e.g., BDZs and
barbiturates, developmental changes in GABAA
receptor function may have critical implications for the development of treatment regimens in the pediatric population. The data presented here
may offer insight facilitating the development of novel therapeutic strategies for age-specific seizure disorders.
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ACKNOWLEDGMENTS |
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We are grateful to Dr. C. M. Baumgarten for many helpful discussions, to Drs. A. Lavoie and R. Twymann for providing waveform protocols for rapid application experiments, and to Dr. N. A. Cohen and D. D. Limbrick and D. A. Sun for constructive suggestions on the manuscript.
This work was supported by an Epilepsy Foundation postdoctoral fellowship to A. S. Cohen and National Institute of Neurological Disorders and Stroke Grant NS-32403 to D. A. Coulter.
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FOOTNOTES |
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Present address and address for reprint requests: A. S. Cohen, Abramson Research Center, Rm. 707c, 3516 Civic Center Blvd., Philadelphia, PA 19104-4318 (E-mail: cohena{at}emailchop.edu).
Received 19 April 2000; accepted in final form 4 August 2000.
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REFERENCES |
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