Temporal Regulation of the Expression Locus of Homeostatic Plasticity

Article (PDF Available)inJournal of Neurophysiology 96(4):2127-33 · October 2006with24 Reads
DOI: 10.1152/jn.00107.2006 · Source: PubMed
Abstract
Homeostatic plasticity of excitatory synapses plays an important role in stabilizing neuronal activity, but the mechanism of this form of plasticity is incompletely understood. In particular, whether the locus of expression is presynaptic or postsynaptic has been controversial. Here we show that the expression locus depends on the time neurons have spent in vitro. In visual cortical cultures < or =14 days in vitro (DIV), 2 days of TTX treatment induced an increase in miniature excitatory postsynaptic current (mEPSC) amplitude onto pyramidal neurons, without affecting mEPSC frequency. However, in cultures > or =18 DIV, the same TTX treatment induced a large increase in mEPSC frequency, whereas the amplitude effect was reduced. The increased mEPSC frequency was associated with an increased density of excitatory synapses and increased presynaptic vesicle release in response to electrical stimulation. This indicates a shift from a predominantly postsynaptic response to TTX in < or =14 DIV cultures, to a coordinated pre- and postsynaptic response in > or =18 DIV cultures. This shift was not specific for cortical cultures because a similar shift was observed in cultured hippocampal neurons. Culturing neurons from older animals showed that the timing of the switch depends on the time the neurons have spent in vitro, rather than their postnatal age. This temporal switch in expression locus can largely reconcile the contradictory literature on the expression locus of homeostatic excitatory synaptic plasticity in central neurons. Furthermore, our results raise the intriguing possibility that the expression mechanism of homeostatic plasticity can be tailored to the needs of the network during different stages of development or in response to different challenges to network function.
Report
Temporal Regulation of the Expression Locus of Homeostatic Plasticity
Corette J. Wierenga, Michael F. Walsh, and Gina G. Turrigiano
Department of Biology and Center for Complex Systems, Brandeis University, Waltham, Massachusetts
Submitted 31 January 2006; accepted in final form 31 May 2006
Wierenga, Corette J., Michael F. Walsh, and Gina G. Turrigiano.
Temporal regulation of the expression locus of homeostatic plasticity.
J Neurophysiol 96: 2127–2133, 2006. First published June 7, 2006;
doi:10.1152/jn.00107.2006. Homeostatic plasticity of excitatory syn-
apses plays an important role in stabilizing neuronal activity, but the
mechanism of this form of plasticity is incompletely understood. In
particular, whether the locus of expression is presynaptic or postsyn-
aptic has been controversial. Here we show that the expression locus
depends on the time neurons have spent in vitro. In visual cortical
cultures 14 days in vitro (DIV), 2 days of TTX treatment induced an
increase in miniature excitatory postsynaptic current (mEPSC) ampli-
tude onto pyramidal neurons, without affecting mEPSC frequency.
However, in cultures 18 DIV, the same TTX treatment induced a
large increase in mEPSC frequency, whereas the amplitude effect was
reduced. The increased mEPSC frequency was associated with an
increased density of excitatory synapses and increased presynaptic
vesicle release in response to electrical stimulation. This indicates a
shift from a predominantly postsynaptic response to TTX in 14 DIV
cultures, to a coordinated pre- and postsynaptic response in 18 DIV
cultures. This shift was not specific for cortical cultures because a
similar shift was observed in cultured hippocampal neurons. Culturing
neurons from older animals showed that the timing of the switch
depends on the time the neurons have spent in vitro, rather than their
postnatal age. This temporal switch in expression locus can largely
reconcile the contradictory literature on the expression locus of
homeostatic excitatory synaptic plasticity in central neurons. Further-
more, our results raise the intriguing possibility that the expression
mechanism of homeostatic plasticity can be tailored to the needs of
the network during different stages of development or in response to
different challenges to network function.
INTRODUCTION
Neurons can adapt to prolonged changes in synaptic drive in
a homeostatic manner, so that firing rates remain relatively
stable (Burrone et al. 2002; Turrigiano et al. 1998). Such
homeostatic plasticity likely plays an important role in prevent-
ing the destabilizing effects of developmental and learning-
related changes in synapse number or strength (Davis and
Bezprozvanny 2001; Turrigiano 1999; Turrigiano and Nelson
2004). One important form of homeostatic plasticity in verte-
brate central neurons is the activity-dependent regulation of
excitatory synapses, where prolonged (1 to 2 days) elevation of
activity reduces all of a neuron’s excitatory synaptic strengths
and vice versa (Turrigiano and Nelson 2004). Although ho-
meostatic plasticity of excitatory central synapses has now
been identified in a number of preparations (Turrigiano and
Nelson 2004), the cellular and molecular mechanisms of this
plasticity remain largely obscure. In particular, the expression
locus has been controversial, with some studies supporting
exclusively postsynaptic changes and others supporting both
pre- and postsynaptic changes. Here we show that the expres-
sion locus of homeostatic plasticity is temporally regulated
and suggest that this regulation can comprehensively account
for the differences in expression mechanism reported in the
literature.
In young neocortical neurons, 2 days of tetrodotoxin (TTX)
treatment increases miniature excitatory postsynaptic current
(mEPSC) amplitude, but not frequency. This arises predomi-
nantly, if not exclusively, from postsynaptic changes: the
number of postsynaptic AMPA receptors and amplification of
dendritic currents by Na
channels are increased (Wierenga et
al. 2005). Changes in postsynaptic receptor accumulation after
activity manipulations have also been observed in cultured
spinal (O’Brien et al. 1998) and young hippocampal (Liao et al.
1999; Lissin et al. 1998) neurons, cerebellar slices (Liu and
Cull-Candy 2000, 2002), and the ventral nerve cord of Cae-
norhabditis elegans (Grunwald et al. 2004). In addition, re-
duced sensory drive in vivo increases excitatory synaptic
strength onto visual cortical neurons without affecting mEPSC
frequency and with only a minor effect on short-term plasticity,
suggesting mainly postsynaptic changes (Desai et al. 2002;
Maffei et al. 2004).
In contrast, other studies have suggested a presynaptic locus
of homeostatic plasticity. At the Drosophila neuromuscular
junction, an increase in presynaptic release probability was
observed after postsynaptic activity blockade (Paradis et al.
2001) and, in older hippocampal cultures, activity blockade
induced a modest increase in mEPSC amplitude and a large
increase in mEPSC frequency (Bacci et al. 2001; Burrone et al.
2002; Thiagarajan et al. 2002). This was associated with an
increased size of the presynaptic terminal and increased release
probability (Murthy et al. 2001; Thiagarajan et al. 2005). Thus
even within similar experimental preparations from different
labs (i.e., hippocampal cultures), the locus of change has been
controversial. To date, there has not been any satisfactory
explanation for these contradictory findings (Burrone and Mur-
thy 2003; Turrigiano and Nelson 2004).
Besides inherent differences between brain regions, one
major difference between many of the culture studies men-
tioned above is the time the neurons were kept in vitro. We
show here that the time neurons have spent in vitro—rather
than neuronal age—is a key factor determining the expression
locus of homeostatic plasticity. Our data suggest that there
exists an array of potential expression sites for homeostatic
synaptic plasticity, and which particular mechanism is re-
cruited depends either on the age of the synapse or on other
factors that change with time in vitro.
Address for reprint requests and other correspondence: G. Turrigiano,
Biology MS 008, Brandeis University, 415 South Street, Waltham MA 02454
(E-mail: turrigiano@brandeis.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
J Neurophysiol 96: 2127–2133, 2006.
First published June 7, 2006; doi:10.1152/jn.00107.2006.
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METHODS
Cultures and mEPSC recordings
Visual cortical and hippocampal cultures were prepared from P3
rats, as previously described (Turrigiano et al. 1998; Watt et al. 2000).
Culture medium was changed three times per week during the first
week and once a week thereafter. Cell density declined with time in
vitro: 47 3 neurons/mm
2
at DIV 7, 35 17 neurons/mm
2
at DIV
14, and 22 6 neurons/mm
2
at DIV 21. The 2-day TTX treatment did
not affect cell density (P 0.1; paired t-test). Cultures from P9 rats
were prepared in the same way, but were plated at a twofold higher
initial density. The initial survival rate of neurons from P9 rats was
lower than that from P3 rats, but after the first week in vitro cell
density stayed fairly constant (21 4 neurons/mm
2
at DIV 8 and
22 8 neurons/mm
2
at DIV 21).
Recordings of mEPSCs were done at room temperature with a
KMeSO
4
-based pipette solution (Wierenga et al. 2005). Data points in
Fig. 1 were obtained from cultures that were kept 7–9 (DIV 8), 13–16
(DIV 14), 17–18 (DIV 18), and 21–24 (DIV22) days in vitro.
FM1-43 labeling
Presynaptic terminals were labeled with 15
M FM1-43 by either
superfusion with 40 mM K
solution or by electrical stimulation, in
either case in the presence of 10
M DNQX, 50
M APV, and 20
M
bicuculline, as described previously (Wierenga et al. 2005). All
experiments were done at room temperature. In most cases random
puncta were selected, representing a combination of excitatory
(roughly 70%) and inhibitory (roughly 30%) terminals. In a few cases,
we selected only FM1-43 puncta on distal apical dendrites of pyra-
midal neurons (labeled with DsRed transfection; three control and
three TTX neurons). These puncta are primarily excitatory (Wierenga
et al. 2005). Results were qualitatively the same for puncta on
pyramidal neurons and randomly selected puncta and we have pooled
data from all puncta in Fig. 3.
To estimate the destaining kinetics of individual FM1-43 puncta,
the puncta intensity at each time point was measured relative to the
local background and then normalized to the total intensity loss during
the stimulation. For the stimulated destaining experiments, only
puncta that destained to 50% of their initial intensity were selected.
For the spontaneous destaining experiments, only puncta that were
destained by a subsequent 5-min 40 mM K
solution application were
selected for analysis.
Immunostaining
Immunostaining experiments were performed as previously de-
scribed (Wierenga et al. 2005). Measurements of cell densities were
done as described previously (Rutherford et al. 1997), with the
difference that anti-NeuN labeling (1:500, Chemicon) was used to
visualize neuronal nuclei.
Statistics
Statistical analyses were performed using unpaired Student’s t-test,
unless indicated otherwise. For testing differences between distribu-
tions the Kolmogorov–Smirnov (K-S) test was used.
RESULTS
Miniature EPSCs
Two days of activity blockade induces homeostatic changes
in excitatory synaptic transmission. In neocortical, spinal, or
hippocampal cultures 14 days in vitro (DIV), activity block-
ade increases mEPSC amplitude, with no effect on mEPSC
frequency (Ju et al. 2004; O’Brien et al. 1998; Turrigiano et al.
1998), whereas in hippocampal neurons 14 DIV, activity
blockade results in an increase in mEPSC frequency, with only
a small increase in amplitude (Burrone et al. 2002; Thiagarajan
et al. 2002). We examined whether the difference in time in
vitro could explain these contradictory findings.
Neurons from visual cortex from 3-day-old (P3) rat pups
were kept in culture for 8, 14, 18, and 22 DIV and for each time
point half of the dishes were treated with 0.5
M TTX for 2
days before the experiments. The frequency of mEPSCs in
pyramidal neurons increased only slightly during the first 2 wk
in vitro and reached steady state after 14 DIV. The amplitude
FIG. 1. Effect of a 2-day tetrodotoxin
(TTX) treatment depends on time in vitro. A
and B: recordings of miniature excitatory
postsynaptic currents (mEPSCs) in control
and TTX-treated pyramidal neurons at 8 and
18 days in vitro (DIV). C and D: amplitude
and frequency of mEPSCs in control and
TTX-treated neurons as a function of DIV.
Inset in C: CV of mEPSC amplitudes at DIV
22. Data from 8, 18, 18, and 13 control
neurons and 10, 21, 25, and 12 TTX-treated
neurons at DIV 8, 14, 18, and 22, respec-
tively. Asterisks indicate significant differ-
ences (*P 0.05; **P 0.01; ANOVA).
(*) indicates that data were pooled over DIV
14 –22.
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of mEPSCs remained fairly constant over this time period
(Fig. 1).
In young neocortical cultures (8 DIV), blocking activity for
2 days resulted in an increase in mEPSC amplitude, without an
effect on mEPSC frequency, as previously described (Turri-
giano et al. 1998; Watt et al. 2000; Wierenga et al. 2005). In
neurons that were kept 14 DIV TTX treatment also induced
an increase in mEPSC amplitude, but the magnitude of the
increase was reduced from nearly 100% at 8 DIV to approxi-
mately 25% at 14 DIV (Fig. 1, AC). The coefficient of
variation (CV) of mEPSC amplitudes was similar in all exper-
imental groups, indicating that the shape of the amplitude
distribution was not significantly affected by the TTX treat-
ment at any age, consistent with an activity-dependent scaling
up of mEPSC amplitude (Turrigiano et al. 1998). TTX treat-
ment did not affect mEPSC frequency in neurons that were
kept in vitro for 14 days, but after 18 DIV the same 2-day
TTX treatment induced a robust increase in mEPSC frequency
(Fig. 1D). Resting membrane potentials and mEPSC kinetics
were not significantly affected by the TTX treatment at any
time point (mean resting membrane potential, 65 1 mV;
mean rise time, 1.3 0.02 ms; mean decay time constant,
5.0 0.2 ms). However, input resistance was increased in
TTX-treated cultures 14 DIV (control: 266 25 M; TTX:
434 25 M; P 0.01). There was no correlation between
input resistance and mEPSC amplitude or frequency (Pearson’s
r values of 0.18 and 0.03, respectively), suggesting that the
increase in input resistance after TTX treatment reflects addi-
tional changes induced by TTX treatment, which are unrelated
to the effect on mEPSCs.
The observed increase in mEPSC frequency in neocortical
cultures 18 DIV is in good agreement with the results
obtained in hippocampal cultures of the same age (Burrone et
al. 2002; Murthy et al. 2001; Thiagarajan et al. 2005). To test
whether the switch we observed in our visual cortical cultures
is a general phenomenon of central neurons, we recorded
mEPSCs in hippocampal neurons and compared the effect of a
2-day TTX treatment at 10 and 18 DIV. As in the visual
cortical cultures (Fig. 1), TTX-treated hippocampal neurons
showed an increase in mEPSC amplitude at 10 DIV, whereas
at 18 DIV both mEPSC frequency and amplitude were in-
creased after TTX treatment (Fig. 2). This suggests that the
observed shift in response to activity blockade with time in
vitro is not specific for neocortical cultures but may be a
general response to prolonged time in vitro. This strongly
suggests that the apparent contradiction between previous ex-
perimental reports on the locus of homeostatic plasticity can be
explained chiefly by differences in the time the neurons have
spent in vitro.
FM1-43 labeling and destaining
Our previous results indicated no change in presynaptic
release properties after TTX treatment in neocortical cultures
14 DIV (Wierenga et al. 2005). To begin to address the
mechanism underlying the increase in mEPSC frequency in-
duced by TTX in cultures 18 DIV, we examined presynaptic
vesicle recycling in DIV 18 cultures by labeling terminals with
the fluorescent styryl dye FM1-43. When presynaptic vesicle
turnover was induced by superfusion for 2 min with a HEPES-
buffered solution containing 40 mM K
, FM1-43 puncta
intensities were higher in TTX-treated cultures than in control
cultures [mean intensity was 122 6% of control (1,000
puncta per condition); P 0.001, K-S test; Fig. 3, A and B].
The increased puncta intensity was also observed when pre-
synaptic vesicle turnover was induced by electrical stimulation
[600 stimuli at 20 Hz; mean TTX-treated puncta intensity was
increased to 139 7% of control (144 control, 239 TTX-
treated puncta); P 0.001, K-S test].
To examine whether the kinetics of destaining was altered
by TTX treatment, we monitored the destaining time course of
individual FM1-43 puncta every 10 s during application of
1,500 stimuli at 10 Hz. FM1-43 puncta in TTX-treated cultures
showed faster destaining kinetics than that of control puncta
(Fig. 3, C and D). These experiments were performed in the
presence of synaptic blockers, but action potentials were not
blocked to allow us to study evoked release. As a consequence,
destaining reflects not only exocytosis of vesicles in response
to electrical stimulation, but also release of synaptic vesicles by
spontaneous action potentials and stochastic release. Poten-
FIG. 2. A similar shift occurs in hippocampal
cultures. A: recordings of mEPSCs in control
hippocampal neurons at 10 DIV. Inset: average
mEPSC. B: summary of mEPSC amplitudes and
frequency in hippocampal cultures at 8 –12 DIV
(10 DIV). Data from 7 control and 8 TTX-treated
neurons. C: summary of mEPSC amplitudes and
frequency in hippocampal cultures at 18 –19 DIV
(18 DIV). Data from 9 control and 10 TTX-
treated neurons. Asterisks indicate significant dif-
ferences (**P 0.01).
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tially, the observed difference in destaining kinetics could be
explained by differences in spontaneous release, rather than
differences in evoked release. We therefore measured the
spontaneous destaining rates in control and TTX-treated cul-
tures by repeating the destaining experiments while omitting
the destaining stimulation. The average spontaneous destaining
was well fit with a linear function. Spontaneous destaining
rates were similar in control and TTX-treated cultures [control:
0.17 0.004%/s (197 puncta) and TTX: 0.16 0.003%/s
(169 puncta); data not shown]. This indicates that the differ-
ence in destaining kinetics between TTX-treated and control
FM1-43 puncta arises from a different response to the electrical
stimulation.
We also fit the individual destaining time courses of FM
1– 43 puncta to obtain the distribution of destaining time
constants (Fig. 3D). TTX-treated cultures showed a larger
fraction of fast destaining puncta, although fast and slowly
destaining FM1-43 puncta are present in both conditions.
These experiments suggest that, in contrast to young (DIV
7–10) cultures, where the same experimental protocol did not
induce any changes in destaining kinetics (Wierenga et al.
2005), activity blockade in DIV 18 cultures increases vesicle
release and/or recycling in presynaptic terminals.
Number of excitatory synapses
Changes in mEPSC frequency could reflect increased rates
of spontaneous vesicle fusion, changes in the number of
functional synapses, or both. We previously showed that TTX
treatment in cultures 14 DIV does not affect the number of
excitatory synapses (Wierenga et al. 2005). To determine
whether this is also true after DIV 18 –20 when TTX treatment
increases mEPSC frequency, we performed triple immunola-
beling experiments against excitatory synaptic markers. Excit-
atory synapses on apical dendrites of pyramidal neurons were
visualized using antibodies directed against two postsynaptic
markers [the
-amino-3-hydroxy-5-methyl-4-isoxazolepropi-
onic acid receptor (AMPAR) subunit GluR1 and scaffolding
protein PSD95] and one presynaptic marker [the vesicular
glutamate transporter 1 (Vglut1); Fig. 4A]. GluR1 labeling was
performed before permeabilization so that only surface recep-
tors were labeled (Wierenga et al. 2005).
Two days of TTX treatment in DIV 18–20 cultures induced
an increase in the density of puncta for all three excitatory
synaptic markers (Fig. 4B). The density of puncta that had any
combination of two or three of the markers was increased about
twofold (Fig. 4C). However, the percentage of puncta that were
co-localized with other synaptic markers was not different in
control and TTX-treated cultures (Fig. 4D), suggesting that the
number of excitatory synapses was increased, whereas the
proportion of synaptic and extrasynaptic puncta was main-
tained.
Time in vitro versus postnatal age
The results described above indicate that the response to a
period of activity blockade depends on time in vitro. In older
cultures, where both mEPSC amplitude and frequency are
increased after TTX treatment, neurons are both older and have
spent more time in vitro than in the younger cultures, where
only mEPSC amplitude is increased. To distinguish between
the effect of postnatal age of the neurons and the time spent in
vitro we prepared visual cortical cultures from P9 rats. After 2
wk in vitro, cultured neurons from P9 rats have the same
postnatal age as that of neurons from a P3 rat that have been in
vitro for 20 days, whereas they have spent the same time in
vitro as 14 DIV neurons from a P3 culture (Fig. 5A). If the
postnatal age of the neurons determines the response to activity
blockade, one would expect to find an increased mEPSC
frequency after TTX treatment in P9/DIV 14 cultures. How-
FIG. 3. TTX treatment increases FM1-43
labeling in DIV 18 neocortical cultures. A:
examples of FM1-43 puncta in control and
TTX-treated visual cortical cultures. Presyn-
aptic terminals were labeled with superfu-
sion of a 40 mM K
solution. Scale bar 10
m. B: distribution of puncta intensities la-
beled with high K
. Data from 1,348 puncta,
21 images (control) and 1,484 puncta, 20
images (TTX-treated). C: destaining time
course in control and TTX-treated cultures,
normalized to the total loss of fluorescence
during the stimulation. Puncta were labeled
with 600 stimuli at 20 Hz and stimulated
destaining was induced by applying 1,500
stimuli at 10 Hz. Single exponential fits are
shown with time constants indicated. D: dis-
tribution of destaining time constants of in-
dividual puncta (P 0.001, Kolmogorov–
Smirnov test). Data from 132 puncta, 7 im-
ages (control) and 197 puncta, 9 images
(TTX-treated).
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ever, mEPSC frequency was not affected by TTX treatment in
these cultures (Fig. 5B). Consistent with our results from
P3/DIV 14 cultures (Fig. 1C), mEPSC amplitude was slightly
but significantly increased in P9/DIV 14 cultures after TTX
treatment [control: 17.9 1.0 pA (19 neurons) and TTX:
19.0 1.1 pA (20 neurons); P 0.05, paired t-test]. To rule
out the possibility that cultures derived from P9 animals
generally lack the ability to respond to activity deprivation by
altering mEPSC frequency, we kept P9 cultures in vitro for
another week (DIV 21). In P9/DIV 21 cultures, the same TTX
treatment induced a significant increase in mEPSC frequency
(Fig. 5B, P 0.05). Taken together these experiments indicate
that the time neurons have spent in vitro, rather than their
postnatal age, determines their response to activity blockade.
DISCUSSION
Synaptic scaling of excitatory synapses is an important form
of homeostatic plasticity that operates both in vitro and in vivo
(Turrigiano and Nelson 2004). Understanding when and how
this form of plasticity is expressed has important consequences
for the function of cortical circuits because a purely postsyn-
aptic expression locus will leave the short-term dynamics and
filtering properties of synapses unaffected, whereas presynap-
tic changes in release probability will fundamentally change
these short-term dynamics (Abbott et al. 1997; Markram et al.
1998; Wierenga et al. 2005). Here we show that the expression
locus of excitatory homeostatic plasticity depends on the time
the neurons have spent in vitro. In young cultures (14 DIV),
mEPSC amplitude is increased by activity blockade, whereas
mEPSC frequency is not affected. However, in neurons that
were kept in vitro for longer (18 DIV), activity blockade
increased both mEPSC amplitude and frequency. This increase
in mEPSC frequency was accompanied by a doubling in the
density of excitatory synapses and increased evoked release
from presynaptic terminals. Finally, we found that this change
in response to activity blockade depends not on the postnatal
age of the neurons, but on the time the neurons have spent in
vitro. These data indicate that the same synapses can use
different homeostatic plasticity mechanisms under different
circumstances.
In the literature different effects of activity blockade have
been reported, even within the same experimental preparation.
In postnatal hippocampal neurons, it has been controversial
whether activity blockade affects only mEPSC amplitude (Lis-
sin et al. 1998; Stellwagen and Malenka 2006) or primarily
affects mEPSC frequency with a smaller effect on amplitude
(Burrone et al. 2002; Thiagarajan et al. 2002, 2005). Our data
indicate that the discrepancy between these results can be
explained by a shift in expression locus with time in vitro
because the former studies were done in cultures at 15 DIV,
whereas the latter were performed after 2– 4 wk in vitro.
The finding that hippocampal and neocortical neurons in
culture show a similar shift in expression locus of homeostatic
plasticity does not necessarily mean that the detailed expres-
FIG. 4. TTX treatment increases the number of excitatory synapses in DIV 18 –20 neocortical cultures. A: excitatory synapses on the apical dendrite of a
pyramidal neuron at DIV18 were labeled by postsynaptic markers GluR1 and PSD95, and presynaptic marker Vglut1. B: density of GluR1 puncta in control and
TTX-treated visual cortical cultures. C: density of puncta that were labeled with double or triple markers in TTX-treated cultures as percentage of control. D:
co-localization between the 3 markers was not affected by the TTX treatment. Data from 7 control and 8 TTX-treated neurons. Asterisks indicate significant
differences (P 0.05).
FIG. 5. Time spent in culture, rather than postnatal age of neurons, deter-
mines response to TTX treatment. A: cultures of visual cortical neurons from
P9 rats that are kept in vitro for 14 days have the same postnatal age as DIV
20 neurons from P3 rats, and have spent the same time in vitro as DIV 14
neurons from P3 rats. B: frequency of mEPSCs in pyramidal neurons from a
P9 culture. DIV 14: 19 control and 20 TTX-treated neurons; DIV 21: 12
control and 11 TTX-treated neurons. Asterisk indicates significant difference
(P 0.05).
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sion mechanisms are identical. There may be region-specific
differences in the underlying AMPAR trafficking mechanisms
that adjust mEPSC amplitude and/or differences in presynaptic
vesicle recycling (Ju et al. 2004; Thiagarajan et al. 2005;
Virmani et al. 2006).
Our data suggest that, at cortical synapses, a major contrib-
utor to the increase in mEPSC frequency is the doubling in the
density of excitatory synaptic contacts induced by activity
blockade. In addition, we found an increased synaptic vesicle
release in response to electrical stimulation. Higher puncta
intensities in 18 DIV TTX-treated cultures are consistent with
an increased size of presynaptic terminals and larger vesicle
pools, as reported in hippocampal cultures (Murthy et al.
2001). The faster destaining kinetics we observed after activity
blockade could reflect an increased release probability (Murthy
et al. 2001; Thiagarajan et al. 2005), although neither our data
nor previous reports can exclude the possibility that increased
excitability of TTX-treated neuronal terminals (possibly re-
flected in the observed increase in input resistance) also con-
tributed to the faster destaining kinetics (Desai et al. 1999).
It is not clear what determines the change in expression
locus of homeostatic plasticity. Our results indicate that the
postnatal age of cultured neurons is not crucial, but rather that
the expression locus is determined by the time the neurons
have spent in vitro. The lower cell density at longer times in
vitro is not a determining factor for the response to activity
blockade because the P9/DIV14 cultures had densities similar
to those of P3/DIV 20 cultures, but responded differently to
activity blockade. One possibility is that the altered response to
activity blockade with time in vitro arises from prolonged
exposure to the (artificial) in vitro environment. Alternatively,
the response to activity blockade may be determined by the
maturational state of the synapses because neurons that have
spent longer times in vitro have more mature synaptic contacts.
Maturation of synapses is associated with a multitude of pre-
and postsynaptic changes, including increased vesicle pool size
(Mohrmann et al. 2003), a change in composition of postsyn-
aptic glutamate receptors (Gomperts et al. 2000), increased
scaffolding proteins together with a decreased dependency on
F-actin (Zhang and Benson 2001), a change in vesicular
glutamate transporters (de Gois et al. 2005; Wilson et al. 2005),
and altered expression of
- and
-CaMKII (Fink et al. 2003;
Thiagarajan et al. 2002). Each of these changes has the poten-
tial to alter the effect of activity blockade on synaptic trans-
mission. A recent report suggests that astrocytes are involved
in mediating homeostatic changes in cultures 15 DIV (Stell-
wagen and Malenka 2006), raising the possibility that changes
in neuron–astrocyte interactions could contribute to this time-
dependent switch. The existence of multiple forms of excita-
tory homeostatic plasticity raises the general possibility that the
expression mechanism may be tailored to the needs of the
network during different stages of development or in response
to different challenges to network function.
ACKNOWLEDGMENTS
Present address of C. J. Wierenga: Max Planck Institute for Neurobiology,
Martinsried, Germany.
GRANTS
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-36853.
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    • "These results suggest that the expression of different synaptic vesicle proteins is not closely coregulated, despite the fact that they are all needed only for the formation of one organelle, the synaptic vesicle. It has also been proposed that silencing neurons for a period of time resulted in presynaptic strengthening (Wierenga et al., 2006). This activity dependent adaptation of the synapse was termed synaptic homeostasis . "
    [Show abstract] [Hide abstract] ABSTRACT: Alzheimer's disease (AD) is neuropathologically characterized by aggregates of amyloid-β peptides (Aβ) and tau proteins. The consensus in the AD field is that Aβ and tau should serve as diagnostic biomarkers for AD. However, their aggregates have been difficult to investigate by conventional fluorescence microscopy, since their size is below the diffraction limit (~200 nm). To solve this, we turned to a super-resolution imaging technique, stimulated emission depletion (STED) microscopy, which has a high enough precision to allow the discrimination of low- and high-molecular weight aggregates prepared in vitro. We used STED to analyze the structural organization of Aβ and tau in cerebrospinal fluid (CSF) from 36 AD patients, 11 patients with mild cognitive impairment (MCI), and 21 controls. We measured the numbers of aggregates in the CSF samples, and the aggregate sizes and intensities. These parameters enabled us to distinguish AD patients from controls with a specificity of ~87% and a sensitivity of ~79%. In addition, the aggregate parameters determined with STED microscopy correlated with the severity of cognitive impairment in AD patients. Finally, these parameters may be useful as predictive tools for MCI cases. Two MCI patients who developed AD during the course of the study were correctly identified. The same was observed for MCI patients whose Aβ ELISA values fall within the accepted range for AD. We suggest that super-resolution imaging is a promising tool for AD diagnostics.
    Full-text · Article · Apr 2015
    • "Prolonged activity blockade also increases postsynaptic density of AMPA receptors at excitatory synapses. Immunolabeling revealed a higher intensity for both GluR1 and GluR2 subunits on the cell surface of cultured visual cortical neurons grown with TTX for 2d [42]. At the synapse, both presynaptic mechanisms leading to an increase in the amount of transmitter released and postsynaptic mechanisms increasing the number and sensitivity of glutamatergic receptors are responsible for increased excitation when neurons are pharmacologically silenced by TTX. "
    [Show abstract] [Hide abstract] ABSTRACT: The concept of homeostatic plasticity postulates that neurons maintain relatively stable rates of firing despite changing inputs. Homeostatic and use-dependent plasticity mechanisms operate concurrently, although they have different requirements for induction. Depriving central somatosensory neurons of their primary activating inputs reduces activity and results in compensatory changes that favor excitation. Both a reduction of GABAergic inhibition and increase in glutamatergic excitatory transmission are observed in input-deprived cortex. Topographic reorganization of the adult somatosensory cortex is likely driven by both homeostatic and use-dependent mechanisms. Plasticity is induced by changes in the strengths of synaptic inputs, as well as changes in temporal correlation of neuronal activity. However, there is less certainty regarding the in vivo contribution of homeostatic mechanisms as in vitro experiments rely on manipulations that create states that do not normally occur in the living nervous system. Homeostatic plasticity seems to occur, but more in vivo research is needed to determine mechanisms. In vitro research is also needed but should better conform to conditions that might occur naturally in vivo.
    Full-text · Article · Mar 2015
    • "Although we cannot exclude the possibility that ionic conductance mediating TTX-induced homeostatic intrinsic plasticity may not be equivalent in both high-and low-density cultures, our findings (Figure 6) are consistent with previous studies reporting that the elevation in AP firing frequency, which occurs with TTX treatment, is coupled with reduced K + current density in dissociated cortical neurons [4]. Despite the wide application of dissociated neuronal culture to study homeostatic plasticity [2-4,13,15,29-31, 34,83], the results from this culture system should be interpreted with caution due to the dependence of homeostatic plasticity expression on culture density and age [28,84]. Remarkably, TTX and APV treatments but not Nif or STO-609 treatments led to a homeostatic increase in AP firing rates in both high-density (Figures 1, 3 and 4) and low-density cultures [2]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Homeostatic intrinsic plasticity encompasses the mechanisms by which neurons stabilize their excitability in response to prolonged and destabilizing changes in global activity. However, the milieu of molecular players responsible for these regulatory mechanisms is largely unknown.ResultsUsing whole-cell patch clamp recording and unbiased gene expression profiling in rat dissociated hippocampal neurons cultured at high density, we demonstrate here that chronic activity blockade induced by the sodium channel blocker tetrodotoxin leads to a homeostatic increase in action potential firing and down-regulation of potassium channel genes. In addition, chronic activity blockade reduces total potassium current, as well as protein expression and current of voltage-gated Kv1 and Kv7 potassium channels, which are critical regulators of action potential firing. Importantly, inhibition of N-Methyl-D-Aspartate receptors alone mimics the effects of tetrodotoxin, including the elevation in firing frequency and reduction of potassium channel gene expression and current driven by activity blockade, whereas inhibition of L-type voltage-gated calcium channels has no effect.Conclusions Collectively, our data suggest that homeostatic intrinsic plasticity induced by chronic activity blockade is accomplished in part by decreased calcium influx through N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes.
    Full-text · Article · Jan 2015
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