Brain-derived neurotrophic factor enhances GABA release probability and nonuniform distribution of N- and P/Q-type channels on release sites of hippocampal inhibitory synapses.
ABSTRACT Long-lasting exposures to brain-derived neurotrophic factor (BDNF) accelerate the functional maturation of GABAergic transmission in embryonic hippocampal neurons, but the molecular bases of this phenomenon are still debated. Evidence in favor of a postsynaptic site of action has been accumulated, but most of the data support a presynaptic site effect. A crucial issue is whether the enhancement of evoked IPSCs (eIPSCs) induced by BDNF is attributable to an increase in any of the elementary parameters controlling neurosecretion, namely the probability of release, the number of release sites, the readily releasable pool (RRP), and the quantal size. Here, using peak-scaled variance analysis of miniature IPSCs, multiple probability fluctuation analysis, and cumulative amplitude analysis of action potential-evoked postsynaptic currents, we show that BDNF increases release probability and vesicle replenishment with little or no effect on the quantal size, the number of release sites, the RRP, and the Ca2+ dependence of eIPSCs. BDNF treatment changes markedly the distribution of Ca2+ channels controlling neurotransmitter release. It enhances markedly the contribution of N- and P/Q-type channels, which summed to >100% ("supra-additivity"), and deletes the contribution of R-type channels. BDNF accelerates the switch of presynaptic Ca2+ channel distribution from "segregated" to "nonuniform" distribution. This maturation effect was accompanied by an uncovered increased control of N-type channels on paired-pulse depression, otherwise dominated by P/Q-type channels in untreated neurons. Nevertheless, BDNF preserved the fast recovery from depression associated with N-type channels. These novel presynaptic BDNF actions derive mostly from an enhanced overlapping and better colocalization of N- and P/Q-type channels to vesicle release sites.
- SourceAvailable from: Pietro Baldelli[Show abstract] [Hide abstract]
ABSTRACT: The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ∼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.Cerebral cortex (New York, N.Y. : 1991). 01/2015;
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ABSTRACT: Microglia-neuron interactions play a crucial role in several neurological disorders characterized by altered neural network excitability, such as epilepsy and neuropathic pain. While a series of potential messengers have been postulated as substrates of the communication between microglia and neurons, including cytokines, purines, prostaglandins, and nitric oxide, the specific links between messengers, microglia, neuronal networks, and diseases have remained elusive. Brain-derived neurotrophic factor (BDNF) released by microglia emerges as an exception in this riddle. Here, we review the current knowledge on the role played by microglial BDNF in controlling neuronal excitability by causing disinhibition. The efforts made by different laboratories during the last decade have collectively provided a robust mechanistic paradigm which elucidates the mechanisms involved in the synthesis and release of BDNF from microglia, the downstream TrkB-mediated signals in neurons, and the biophysical mechanism by which disinhibition occurs, via the downregulation of the K(+)-Cl(-) cotransporter KCC2, dysrupting Cl(-)homeostasis, and hence the strength of GABAA- and glycine receptor-mediated inhibition. The resulting altered network activity appears to explain several features of the associated pathologies. Targeting the molecular players involved in this canonical signaling pathway may lead to novel therapeutic approach for ameliorating a wide array of neural dysfunctions.Neural Plasticity 01/2013; 2013:429815. · 2.86 Impact Factor
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ABSTRACT: A heterogeneous population of inhibitory neurons controls the flow of information through a neural circuit. Inhibitory synapses that form on pyramidal neuron dendrites modulate the summation of excitatory synaptic potentials and prevent the generation of dendritic calcium spikes. Precisely timed somatic inhibition limits both the number of action potentials and the time window during which firing can occur. The activity-dependent transcription factor NPAS4 regulates inhibitory synapse number and function in cell culture, but how this transcription factor affects the inhibitory inputs that form on distinct domains of a neuron in vivo was unclear. Here we show that in the mouse hippocampus behaviourally driven expression of NPAS4 coordinates the redistribution of inhibitory synapses made onto a CA1 pyramidal neuron, simultaneously increasing inhibitory synapse number on the cell body while decreasing the number of inhibitory synapses on the apical dendrites. This rearrangement of inhibition is mediated in part by the NPAS4 target gene brain derived neurotrophic factor (Bdnf), which specifically regulates somatic, and not dendritic, inhibition. These findings indicate that sensory stimuli, by inducing NPAS4 and its target genes, differentially control spatial features of neuronal inhibition in a way that restricts the output of the neuron while creating a dendritic environment that is permissive for plasticity.Nature 11/2013; 503(7474):121-5. · 42.35 Impact Factor
of action has been accumulated, but most of the data support a presynaptic site effect. A crucial issue is whether the enhancement of
Here, using peak-scaled variance analysis of miniature IPSCs, multiple probability fluctuation analysis, and cumulative amplitude
analysis of action potential-evoked postsynaptic currents, we show that BDNF increases release probability and vesicle replenishment
with little or no effect on the quantal size, the number of release sites, the RRP, and the Ca2?dependence of eIPSCs. BDNF treatment
ates the switch of presynaptic Ca2?channel distribution from “segregated” to “nonuniform” distribution. This maturation effect was
accompanied by an uncovered increased control of N-type channels on paired-pulse depression, otherwise dominated by P/Q-type
novel presynaptic BDNF actions derive mostly from an enhanced overlapping and better colocalization of N- and P/Q-type channels to
During neuronal development, brain-derived neurotrophic fac-
tor (BDNF) promotes the formation, maturation, and stabiliza-
tion of both glutamatergic and GABAergic synapses in the CNS,
regulating the balance between excitatory and inhibitory trans-
mission. This is a fundamental step for neural circuit formation
(Poo, 2001; Lessmann et al., 2003). To date, one of the most
unclear aspects of the action of BDNF on the CNS concerns the
molecular mechanism underlying the chronic effect on synaptic
to BDNF can induce strong potentiation and increased connec-
tivity of inhibitory synapses in various brain regions (Vicario-
Abejon et al., 2002). The available data on inhibitory synapses
indicate that BDNF causes net increases in the frequency of min-
IPSCs (eIPSCs) (Rutherford et al., 1997; Vicario-Abejon et al.,
1998; Huang et al., 1999; Bolton et al., 2000; Baldelli et al., 2002;
Yamada et al., 2002). Some of the above effects could be ascribed
to an increased number of inhibitory neurons and to an en-
hanced arborization of dendritic and axonal processes (Vicario-
Abejon et al., 1998; Kohara et al., 2003). This response, however,
was clearly observed in hippocampal and cerebellar slices (Marty
et al., 2000; Seil and Drake-Baumann, 2000) and hippocampal
and Lo, 1999; Bolton et al., 2000; Baldelli et al., 2002).
for BDNF that results in an increased size and intensity of GAD
This project was supported by the Cavalieri-Ottolenghi foundation, by Italian Ministero dell’Istruzione,
dell’Universita ` e della Ricerca Grant 2003/249, and by a Marie Curie Fellowship to J.M.H.-G. (contract HPMF-CT-
2000-00899). We thank Fabio Benfenati and Egidio D’Angelo for helpful discussions. We thank also Claudio
3358 • TheJournalofNeuroscience,March30,2005 • 25(13):3358–3368
depletion (Baldelli et al., 2002). However, an increase in GABAA
receptor density has been also observed, suggesting the involve-
ment of postsynaptic mechanisms (Yamada et al., 2002). Despite
these well established findings, there is an impressive lack of in-
formation on how BDNF affects the elementary parameters con-
trolling neurotransmitter release: probability of release [release
probability (Pr) and vesicle release probability (Pves)], GABAA
forming the readily releasable pool (RRP), and the distribution
Here, using the variance analysis of mIPSCs (Traynelis and
and Silver, 2000), we show that in inhibitory GABAergic syn-
apses, BDNF primarily produces an increased Prand Pves, with
little change in the number of release sites, the RRP, quantal
content, and unitary conductance of GABAAreceptors. In line
with previous reports indicating a BDNF-induced increase in
Ca2?channel synthesis and KCl-evoked synaptic activity
a BDNF-induced increase in Prand Pvesassociated with an en-
hanced contribution and “nonuniform” distribution of N- and
P/Q-type channels to action potential-evoked IPSCs together
with a new role of N-type channels in PPD. This represents a
novel mechanism of the action of BDNF on the maturation of
inhibitory synapses, which may result from a better coupling of
presynaptic Ca2?channels with vesicle release sites.
Cell culture and BDNF treatment. All experiments were performed in
accordance with the guidelines established by the National Council on
University. Pregnant Sprague Dawley rats were killed by inhalation of
CO2, and E18 rats were removed immediately by cesarean section. Re-
moval and dissection of the hippocampus, isolation of neurons, and
culturing procedures were as described previously (Baldelli et al., 2000).
The isolated hippocampal neurons were plated at low density (200 cells/
mm2) and allowed to establish for 4 d, after which 15 ng/ml BDNF
(Sigma, St. Louis, MO) was added every 3 d for the 3–4 weeks of the
culture. When required, the cultured neurons were exposed to the ty-
rosine kinase B (TrkB)-specific inhibitor anti-TrkB IgG1 (10 ?g/ml)
(clone 47; Transduction Laboratories, Lexington, KY), which effectively
of the experiments were recorded in 14–21 d in vitro (DIV) neurons.
ricated from thick borosilicate glasses (Hilgenberg, Mansfield, Ger-
many), were pulled and fire-polished to a final resistance of 2–4 M?.
Patch-clamp recordings were performed in whole-cell configuration us-
ing an EPC-9 amplifier (HEKA Electronic, Lambrecht, Germany). mIP-
at one-half the acquisition rate with an eight-pole low-pass Bessel filter.
Recordings with leak currents of ?100 pA or a series resistance of ?20
M? were discarded. Data acquisition was performed using Pulse pro-
grams (HEKA Elektronic). All of the experiments were performed at
room temperature (22–24°C). Data are expressed as mean ? SEM for
number of cells (n). Unpaired Student’s t tests were used, and p ? 0.05
was considered significant.
taining (in mM): 2 CaCl2, 150 NaCl, 1 MgCl2, 10 HEPES, 4 KCl, and 10
glucose, pH 7.4. When required, solutions with higher Ca2?concentra-
tions (5–10 mM) were obtained by lowering the NaCl content. The uns-
elective glutamate receptor antagonist kynurenic acid (1 mM) (Sigma)
was added to the Tyrode’s solution to block excitatory transmission.
Tetrodotoxin (0.3 ?M) was added to block spontaneous action potential
propagation. When recording eIPSCs, the external solution was supple-
mented with the selective GABAB inhibitor (2S)-3-[[(1S)-1(3,4-
dichlorophenyl) ethyl] amino-2-hydroxypropyl] (phenylmethyl) phos-
phinic acid (5 ?M). The neuron was constantly superfused through a
gravity system, as described previously (Baldelli et al., 2002). The perfu-
trolled periods. The tip of the perfusion pipette (100–200 ?m) was
placed 40–80 ?m to the soma. The standard internal solution was (in
mM): 90 CsCl, 20 TEA-Cl, 10 EGTA, 10 glucose, 1 MgCl2, 4 ATP, 0.5
GTP, and 15 phosphocreatine, pH 7.4. K?was substituted for Cs?and
TEA?in the pipette solution to block outward K?currents. N-(2,6-
was added to block Na?currents activated during an eIPSC. Solutions
?-agatoxin-IVA (?-Aga-IVA) were prepared as indicated previously
(Magnelli et al., 1998). BDNF (Sigma) was dissolved in distilled water
containing 1 mg/ml BSA and maintained in stock solutions at ?80°C.
al. (1993), analysis of mIPSCs was preferred to the analysis of eIPSCs to
determine the conductance of postsynaptic receptor channels. Briefly,
is immediately apparent that the rise time of evoked currents could also
be twofold slower than miniature synaptic currents. Two possible expla-
nations for this are (1) that inhibitory fiber stimulation activates several
ual sites, even when evoked release is apparently synchronous. In either
case, asynchronous transmitter release will increase the deviation of in-
aptic single-channel conductance. On the contrary, the mIPSCs that re-
from nearly synchronous release of transmitter and receptor activation
and are ideal for nonstationary fluctuation analysis
The variance analysis of mIPSCs was performed as described by
Traynelis et al. (1993) using MiniAnalysis programs (version 6.0.1, Syn-
aptosoft, Leonia, NJ). The requirements for such analysis include the
stability of the current decay time course throughout the recording and
the absence of any correlation between decay time course and peak am-
40 and 120 events, eliminating all of the mIPSCs with the decay time
uous stationary activity was recorded in control and BDNF-treated
To isolate current fluctuations associated with the random channel-
gating properties from those arising from variation in neurotransmitter
release and number of postsynaptic receptors, the mean waveform ob-
tained from the average of 40–120 traces was scaled to the peak of each
individual mIPSC and subtracted (Traynelis et al., 1993, Nusser et al.,
1997). The current–variance relationship for the decay of mIPSCs was
calculated by dividing the decay phase (10 ? ?decay) into 10–30 sections
based on equal fractional reductions in the amplitude. This resulted in
peak-scaled variance ?2(t) and the mean amplitude I(t) with the follow-
ing equation: ?2(t) ? iI(t) ? (I2(t)/Nch) ? ?B
probability of postsynaptic channel opening. Scaling the average to the
peak of mIPSCs provides a better estimate of the unitary postsynaptic
current. However, this method is based on the assumption that the in-
trinsic stochastic variance is null at the peak of individual mIPSCs [i.e.,
(1993) and De Koninck and Mody (1994), several factors make the vari-
ance ?0 at the peak of the synaptic current. In this case, the current–
2, where i is the weighted
Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampusJ.Neurosci.,March30,2005 • 25(13):3358–3368 • 3359
variance relationship is skewed, and the only parameter that can be reli-
ably estimated is the unitary current i (initial slope of the parabola).
eIPSCs and PPD. Monosynaptic GABAergic eIPSCs were investigated
in pairs of 14–21 DIV cultured neurons. Presynaptic stimuli were deliv-
ered through a glass pipette of 1 ?m tip diameter filled with Tyrode’s
solution placed in contact with the soma of the GABAergic interneuron
in a loose-seal configuration (Baldelli et al., 2002). Current pulses of 0.1
ms and variable amplitude (6–24 ?A) delivered by an isolated pulse
stimulator (model 2100; A-M-System, Carlsburg, WA) were required to
only the synaptic contacts of the selected presynaptic neuron were stim-
ulated by the extracellular stimulating pipette, for the analysis we used
only those eIPSCs that were completely lost after a few micrometer dis-
placements from the soma of the presynaptic neuron and preserved an
all-or-none response to stimuli of graded intensity. The evoked currents
ulation intensity was set at 1.5 times the threshold for all experiments.
ing potential of ?70 mV. The current artifact produced by the presyn-
During long-lasting experiments in which the extracellular solution was
centrations of extracellular calcium ([Ca2?]o), a correction factor of
5–14% was used to compensate for the slight amplitude rundown of
pulse stimulation were studied by recording the eIPSCs associated with
two consecutive stimuli separated by 20–800 ms interpulse intervals.
if spontaneous activity altered the recordings. PPD was calculated as
follows: PPD ? 1 ? (A2/A1), with A1and A2indicating the amplitude of
the first and second eIPSCs. According to the activity-dependent deple-
tion model of available release sites (Betz, 1970), A1is equal to the prod-
uct of the number of release sites (N) multiplied by Prand the quantum
size (q): A1? NqPr. Immediately after the first pulse, the number of
release sites that remain effective is N2? N ? NPr, and the amplitude of
the second eIPSCs is A2? N2qPr? NqPr(1 ? Pr), if no facilitation is
present and Prremains unchanged during the two eIPSCs. Although
these assumptions are often not valid, it follows that the PPD at brief
interpulses furnishes an estimate of Pr: PPD ? 1 ? (A2/A1) ? Pr.
We made no systematic measurements to verify these hypotheses in
multiple probability fluctuation analysis illustrated below. Nevertheless,
we found that variations in PPD within the same cell followed rather
closely the variations in Pr. For instance, by changing extracellular Ca2?
from 2 to 5 mM, we obtained Prchanges of 28 and 29% in control and
BDNF treated-neurons, respectively (see Fig. 3D), and changes in mean
addition to this, we never observed paired-pulse facilitation but rather
homogeneous depressions to paired-pulse stimulations under condi-
tions of high probability of release. Thus, although PPD could not be
taken as an absolute indicator of Pr, changes in PPD appeared to be
line with recent observations on excitatory synapses for which average
PPD is not found to be a reliable indicator of Prin a cell population but
rather to be linearly related to Prwithin the same cell (Oleskevich et al.,
(V–M) plots was used to estimate three main parameters describing the
synaptic function (Clements and Silver, 2000): the average amplitude of
the postsynaptic response to a vesicle of transmitter (Qav), the average
independent release sites (N). The three parameters were derived from
the parabolic relationship between eIPSC variance (?2) and mean
different release probability conditions by assuming that Iav? NQavPrav
(Silver et al., 1998; Reid and Clements, 1999). A and B (the initial slope
and the curvature of the parabola, respectively) are free parameters that
are adjusted to optimally fit the V–M plots and used to calculate a
weighted mean of Pravand Qavand a lower limit for the number of
independent release sites, Nmin:
Qav? A/(1 ? CVi
Prav? Iav(B/A) (1 ? CVi
0.04 (n ? 12) in BDNF-treated cells (see Fig. 1D), indicating that the
correction factor (1 ? CVi
conditions (17–18%). The adjustment, however, introduces a small dis-
crepancy between Qavand mean mIPSC amplitudes (19.3 vs 25.2 pA in
controls) (see Figs. 1E, 3E), which does not limit the conclusions of our
which produces eIPSCs of lower peak amplitudes than the linear sum of
synchronized elementary events. In our case, BDNF did not specifically
affect the mean latency associated with asynchronous release, which
could introduce either delayed activation or prolonged decay phases of
eIPSCs. In fact, BDNF had no effect on both the rising and the decaying
phase of eIPSCs (see Results) and preserved the kinetic properties of
lower value of Qavis that the mIPSC amplitudes are overestimated be-
cause of the occasional coincidence of two release events. Attempts to
filter out double-peak events will not abolish all such paired events.
external solution (0.5, 2, and 6 ?M). Iavand ?2were calculated over a
stable epoch of 30–150 events after the wash-in of each extracellular
solution. Presynaptic stimulation continued during the wash-in at 0.1
Hz. After the solution exchange, the eIPSC amplitude remained stable
throughout the subsequent analysis epoch. The variance attributable to
the recording noise was estimated in the region before the test pulse and
subtracted from the eIPSC variance. A zero point was included in each
the variance was calculated after subtracting a fitted regression line. This
rundown correction was usually required under conditions in which Pr
was high (Oleskevich et al., 2000). Data were rejected if the decrease was
RRP of synchronous release (RRPsyn) and the probability that any given
vesicle in the readily releasable pool will be released. We indicated this
repetitive stimuli applied at a frequency of 10 Hz. This analysis assumes
that depression during the steady-state phase is limited by a constant
replenishment of vesicles and that equilibrium occurs between release
data points to include in the linear fit of the steady-state phase was eval-
(15th stimulus), we calculated the best linear fit including the maximal
number of data points. We observed in all control (n ? 8) and treated
(n ? 8) neurons that the cumulative amplitude profile showed the best
linearity, after the first eight stimuli, in the range of 800–1400 ms. To
estimate the cumulative eIPSC amplitudes, the last seven data points
the first eIPSC amplitude (I1) and RRPsynfurnished Pves. Dividing
RRPsynby the mean amplitude of mIPSCs, we could also determine the
total number of vesicles ready for release (Nsyn).
Rat embryo hippocampal neurons maintained in culture until 3
weeks of age were used to evaluate the effect of long-term expo-
sure to BDNF on miniature and action potential-evoked IPSCs
2) appears as a minor adjustment in both
3360 • J.Neurosci.,March30,2005 • 25(13):3358–3368Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampus
(mIPSCs and eIPSCs). As reported previously (Baldelli et al.,
2002), BDNF treatment (15 ng/ml added 4 d after plating and
readded every 3 d for the 3–4 weeks of culture) enhanced the
because of the high reproducibility and low fatigability of the
synaptic response to action potential stimulation. This is an es-
sential requisite for long-lasting electrophysiological protocols
and for correctly determining the elementary parameters of
DIV neurons (?97%) was characterized by action potential-
evoked IPSCs with fast activation (?activ? 2.8 ? 0.36 ms) and
bled those generated by hippocampal CA1 interneurons, the ac-
tivity of which is controlled by N- and P/Q-type presynaptic
Ca2?channels, and are classified as slow GABAAinhibitory re-
sponses (Wilson et al., 2001). Occasionally, we also observed fast
eIPSCs with decay times in the order of 20 ms that were systemati-
that the GABAergic transmission in 14–21 DIV neurons was con-
trolled by mixtures of N- and P/Q-type channels (see below and
Ohno-Shosaku et al., 1994), suggested that our recordings derived
Figure 1A depicts three typical eIPSCs recorded from 18 DIV
hippocampal neurons that were maintained under control con-
ditions, incubated with BDNF (15 ng/ml), or pretreated with
BDNF plus the selective antibody for TrkB receptors, anti-TrkB
IgG1 (10 ?g/ml), which antagonizes the action of BDNF. BDNF
caused a net increase in mean eIPSC amplitude (from 2.1 to 3.7
nA; n ? 18; p ? 0.01) that was effectively prevented by the anti-
TrkB IgG1 (2.3 vs 2.1 nA; n ? 18). Despite the increase in ampli-
tude, BDNF had no significant effect on the time course of the
eIPSCs, the activation and decay time constants of which re-
61.3 ? 8.4 ms with controls (n ? 6); ?activwas 2.9 ? 0.38 pA and
?decaywas 57.6 ? 7.3 ms with BDNF (n ? 6). The neurotrophin
also induced a net increase in miniature frequency (from 1.0 ?
0.1 to 1.5 ? 0.2 Hz) (Fig. 1B,C) and a weak shift in the profile of
1D), with no significant changes to the mean amplitude of mIP-
SCs (25.2 and 27.3 pA) (Fig. 1E). The action of BDNF on the
amplitude of miniatures was more marked in 7–14 DIV neurons
and probably depended on an accelerated maturation of func-
tional synapses (Wang et al., 1995).
3-week-old hippocampal neurons, it is likely that the neurotro-
phin preserves the unitary conductance and the density of
postsynaptic GABAAreceptors. To examine this possibility, we
estimated the unitary conductance of GABAAchannels by using
the peak-scaled variance analysis (PSVA) of mIPSCs, which al-
lows an estimate of the unitary current carried by single postsyn-
rizes the procedure followed to determine the variance, ?2(t),
from a group of mIPSCs (see Materials and Methods).
In 14–21 DIV neurons, BDNF caused almost no changes in
scaled variance similar to controls (Fig. 2C). The plot was clearly
parabolic in both cases and only slightly skewed toward higher
amplitudes. The initial slope of the parabola yielded an estimate
corresponding to a mean single-channel conductance of 26.0 ?
0.9 and 24.6 ? 1.3 pS. A similar conductance was obtained when
Vhto ?100 mV (24.8 ? 1.2 pS; n ? 4) or decreased by lowering
altered by BDNF (Fig. 1E), we concluded that in 3-week-old
hippocampal neurons, the density of GABAAreceptors was also
obtained for control and treated neurons in younger cultures
(7–14 DIV; data not shown).
BDNFincreases Pr,preservingthequantalsizeof vesicles
We then investigated how BDNF altered the elementary events
responsible for the increased size of eIPSCs. For this, we used the
under control conditions, with BDNF and BDNF plus anti-TrkB IgG1 (Vh? ?70 mV). Right,
Mean amplitude of eIPSCs in the conditions and number of neurons indicated; **p ? 0.01
plitude distributions of mIPSCs from the same two neurons in B. BDNF causes a consistent
Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampusJ.Neurosci.,March30,2005 • 25(13):3358–3368 • 3361
mean-peak fluctuation analysis (MPFA), which gives direct in-
formation about the average quantal release probability (Prav),
the number of independent release sites (Nmin), and the average
plitude of eIPSCs (Iav) recorded under different Prconditions.
We changed Prby either elevating [Ca2?]ofrom 2 to 5 mM or
adding increasing doses of Cd2?(0.5, 2, and 6 ?M) to the extra-
simple parabola that furnished an estimate of Nminand Qav, and
from the initial slope of the parabola and corrected for the factor
1/1 ? (CVi
mIPSCs at an individual site (see Materials and Methods). In the
case of Figure 3C, the V–M plot for control and BDNF-treated
pA, Nmin? 227 and 249, and Prav? 0.53 and 0.78, respectively.
On average, these values were confirmed in control and
BDNF-treated neurons (n ? 8). There was a net 50% increase in
Pravat 5 mM Ca2?(0.43 ? 0.05 vs 0.78 ? 0.04; p ? 0.01) and a
similar increase at 2 mM Ca2?(0.34 ? 0.04 vs 0.60 ? 0.04; p ?
treated neurons (4.0 ? 0.9 vs 2.2 ? 0.7 nA; p ? 0.01) (Fig. 3D),
2), with CViindicating the coefficient of variation of
BDNF-treated neurons (19.3 ? 0.9 vs 19.7 ? 0.9 pA) (Fig. 3E).
Nminwas slightly larger in BDNF-treated with respect to control
neurons (245 ? 27 vs 280 ? 25; p ? 0.05), indicating a small but
significant increase in the number of functioning release sites
(Fig. 3E). It is worth noting that MPFA slightly underestimates
controls). As discussed in Materials and Methods, this depends
and BDNF-treated neurons and, thus, do not introduce serious
errors to the present conclusions.
and the size of the RRP, we analyzed the cumulative amplitude
profile during high-frequency trains of stimuli (10 Hz for 1.5 s).
As shown in Figure 4A, there was a significant depression of
In the two cases, the cumulative profile of repeated eIPSCs
showed a rapid rise followed by a slower linear increase of differ-
ent steepness at later pulses (Fig. 4B). Assuming that the slow
the linear portion to time 0 yielded the total release minus the
total replenishment, corresponding to the RRPsyn(Schneggen-
burger et al., 1999). As shown in Figure 4B, the RRPsynwas
IPSC was almost doubled (2.0 ? 0.3 vs 3.9 ? 0.2 nA) (Fig. 4C).
Because Pvescan be calculated as the ratio between I1and RRPsyn
(see Materials and Methods), this implies that Pveswas also en-
hanced by BDNF (0.39 ? 0.04 vs 0.59 ? 0.05; p ? 0.01). Figure
4D also shows that the number of vesicles (Nsyn) forming the
RRPsyn, obtained by dividing RRPsynby the mean amplitude of
13; p ? 0.05). This gives a number of readily releasable vesicles
comparable with the number of active sites evaluated by MPFA
(245 ? 27 vs 280 ? 25; p ? 0.05). The analysis of cumulative
amplitude profile further confirms the data obtained by MPFA
and suggests that on average, one vesicle is ready for release at
each active site.
The responses to trains of stimuli provide interesting infor-
mation concerning the recovery from depression during repeti-
tive stimulation. The time course of normalized eIPSCs during
a fast and slow time constant (?f? 76 ms; ?s? 720 ms) and a
dramatically changed these parameters. The initial phase of de-
pression was faster, as expected from the increased Pves(?f? 45
was approximately twofold larger with respect to control neu-
rons. The slower rate of depression and the higher asymptotic
eIPSCs are indicative of an increased recovery from depression
with repetitive release, which may be associated with a higher
bly induced by an enhanced level of residual presynaptic Ca2?
accumulating during repetitive stimuli (Dittman and Regehr,
1998; Stevens and Wesseling, 1998). This is an unexpectedly im-
neurotransmitter release by limiting vesicle depletion during re-
current and peak-scaled variance obtained from a representative control and BDNF-treated
neuron. D, Mean unitary currents in control neurons and BDNF-treated neurons (n ? 15) at
Vh? ?100 mV (n ? 4) and at [Cl?]i? 25 mM (n ? 5) (**p ? 0.01 and *p ? 0.05 vs
3362 • J.Neurosci.,March30,2005 • 25(13):3358–3368Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampus
An increased Prafter BDNF exposure could underlie different
actions on presynaptic mechanisms. A possibility is that BDNF
increases the Ca2?dependence of synaptic transmission by in-
creasing the efficacy by which Ca2?flowing through presynaptic
Ca2?channels stimulates vesicles fusion. To examine this, we
studied the dose–response relationship of eIPSC amplitude ver-
the eIPSCs increased steeply with [Ca2?]oand saturated at ?5
slope coefficient (Ca2?cooperativity) and the dissociation con-
stant (KD) remained unchanged (n ? 3.3 vs 3.2; KD? 1.03 vs
after BDNF treatment remained constant at different levels of
[Ca2?]o, suggesting that potentiation of eIPSCs occurred re-
gardless of the levels of [Ca2?]o(Fig. 5A2).
Having shown that BDNF does not affect the Ca2?depen-
dence of eIPSCs, we then examined whether BDNF could affect
the Ca2?dependence of PPD at different levels of [Ca2?]o(see
Materials and Methods). Under control conditions, with 2 mM
external Ca2?, the PPD at a 50 ms interpulse interval was ?0.49
and decreased to ?25 and 2% when [Ca2?]owas lowered to 1
and 0.5 mM, respectively (Fig. 5B1,B2). At higher levels of
[Ca2?]o, PPD increased moderately. As a result, the PPD of in-
hibitory synapses in cultured hippocampal neurons was steeply
mM [Ca2?]o, a hallmark of central syn-
apses operating near maximal Prunder
physiological conditions. With respect to
controls, BDNF-treated neurons dis-
played higher PPDs at all [Ca2?]olevels
but similar Ca2?dependence (Fig. 5B3).
The increase in Probserved regardless of
the levels of extracellular Ca2?in BDNF-
treated neurons may have different ori-
(Baldelli et al., 2000), it is possible that
pression or changing the proportions of
presynaptic Ca2?channels coupled with
amined how selective Ca2?channel an-
tagonists affect action potential-evoked
IPSCs and PPD in control and BDNF-
treated neurons. As shown below, previ-
ous information on Ca2?channel distri-
bution derived from KCl-evoked IPSCs
(Baldelli et al., 2002) furnishes a rather
ing involved in GABA release and thus
should be taken as mainly qualitative of
the action of BDNF.
An initial series of experiments was
ipine (3 ?M), ?-Ctx-GVIA (1 ?M), and
?-Aga-IVA (0.5 ?M) on the same neuron,
to block L-, N-, and P/Q-type channels.
Figure 6A shows the responses of two representative neurons,
maintained under control conditions (left) or exposed to BDNF
on the eIPSCs in both control and treated neurons (?3%),
whereas ?-Ctx-GVIA and ?-Aga-IVA had irreversible blocking
effects that either preserved 15% of the eIPSCs under control
conditions or fully blocked the response in BDNF-treated neu-
rons. Accounting for the small contribution of L-type channels,
these data indicate that R-type channels contribute partially to
synaptic transmission in control neurons and are absent in
BDNF-treated neurons. Interestingly, by inverting the applica-
tion of the two toxins in control neurons, we obtained compara-
ble blocks of the eIPSCs (total block between 80 and 85%; see
below), suggesting that N- and P/Q-type channels control dis-
tinct release sites in BDNF-untreated neurons and also that pre-
A second series of experiments was performed to evaluate
precisely the contribution of each Ca2?channel type to synaptic
transmission by applying separately the Ca2?channel antago-
nists on control and BDNF-treated neurons. As shown in Figure
6B, under control conditions (open bars), nifedipine induced a
weak inhibition (2.7%; n ? 12), whereas ?-Ctx-GVIA and
?-Aga-IVA blocked 34.7% (n ? 10) and 46.8% (n ? 12) of the
initial synaptic current, respectively. A mean contribution of
10.9% was estimated for the R-channel. In BDNF-treated neu-
n ? 11), whereas the average inhibition by ?-Ctx-GVIA and
each condition) recorded in two representative control and BDNF-treated neurons at 0.1 Hz. Changing [Ca2?] (2–5 mM) or
[Ca2?]o. E, Average quantal size (Qav) and number of release sites (Nmin) in control and BDNF-treated neurons (*p ? 0.05 vs
Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampus J.Neurosci.,March30,2005 • 25(13):3358–3368 • 3363
?-Aga-IVA increased to 49.4% (n ? 10;
p ? 0.01) and 66.5% (n ? 11; p ? 0.01),
respectively, with a net increase of 42% in
type channels increased from 81.5 to
116.9%, and R-type channels no longer
contributed to the eIPSCs. We conclude
that in BDNF-treated neurons, N- and
synaptic inhibitory transmission and that
together they control ?100% the size of
eIPSCs (supra-additivity). This is strong
evidence that in BDNF-treated neurons, a
percentage of release sites are controlled
by mixed populations of N- and P/Q-type
channels that are distributed in a nonuni-
form manner. Notice that supra-additivity
and the switching from segregated to non-
uniform distribution of presynaptic Ca2?
channels with BDNF were totally over-
1 s KCl stimulation overestimated the con-
tribution of distal R-type channels and di-
minished that of N- and P/Q-type channels
ular, P/Q-type channels appeared to con-
der control conditions, whereas with action
potential stimulation, their contribution is
always maximal with and without BDNF
To gain a deeper insight into the action of
BDNF, we next examined how N- and
P/Q-type channels specifically affect PPD
and recovery from depression. We found
tial values (65.7% at ?t ? 20 ms) (Fig.
7A,C), and recovery from depression was
well approximated by a single exponential
sion remained almost unaltered when
P/Q-type channels were mainly responsi-
ble for the eIPSCs (?-Ctx-GVIA applied).
and ?rec? 98.9 ms (Fig. 7A,F, ‚). On the
contrary, the PPD markedly decreased
?recwas shortened (?rec? 49.5 ms) (Fig.
responsible of neurotransmission (?-
Aga-IVA applied). This suggests distinct
roles of N- and P/Q-type channels in con-
trolling GABA release in control neurons. P/Q-type channels
on residual Ca2?at the presynaptic terminals (Dittman and Re-
marked effects on the recovery of PPD, as proven by the faster
rate of recovery supported by these channels.
BDNF had a marked effect on the contribution of N- and
P/Q-type channels to PPD. PPD markedly increased with BDNF
(85.9% at ?t ? 20 ms) (Fig. 7B,D). Recovery from depression
eIPSC (I1) is nearly doubled in treated neurons (filled bars; n ? 8) versus control neurons (open bars; n ? 8) (**p ? 0.01 vs
mean number of vesicles forming the RRPsyn(*p ? 0.05 vs controls). E, Plot of eIPSC amplitude versus time during repetitive
n ? 10). B3, Percentage increase in PPD in the presence of BDNF did not change between 2 and 10 mM [Ca2?]o. Data were
3364 • J.Neurosci.,March30,2005 • 25(13):3358–3368Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampus
was comparable with control neurons (?rec? 101.2 ms) (Fig.
7B,E) but revealed a second, slower component responsible for
the persisting PPD at ?t ? 800 ms (?rec? 3 s). In contrast to
control neurons, N-type channel blockade had a clear effect on
PPD. ?-Ctx-GVIA uniformly depressed the PPD by 25–30% at
all interpulse intervals, causing the block of the slow phase of
recovery from depression (Fig. 7B, Œ). The PPD at ?t ? 20 ms
neurons (59.2%) (Fig. 7D).
BDNF did not affect the rate of recovery from depression
lease: ?recwas 98.9 and 107.6 ms, respectively, for control and
uncovered a PPD associated with N-type channels that was re-
ms) (Fig. 7D), whereas recovery from depression remained sim-
ms) (Fig. 7G). Thus, the increased PPD induced by BDNF was
mainly attributable to a shift of the contribution of N-type chan-
contributed almost equally to the slow phase of depression.
of recovery from depression during paired-pulse stimuli was
poorly affected by BDNF (Fig. 7E), the
same parameter was markedly increased
during trains of repetitive stimulations
(Fig. 4E). This apparent contradiction is
ulus used with PPD is unable to generate
the presynaptic Ca2?accumulation nec-
essary to boost the recovery from depres-
sion achieved during trains (Dittman and
Regehr, 1998; Stevens and Wesseling
1998). It is most likely that the enhance-
ment of Prduring the depression induced
by paired-pulse stimuli predominates,
and that increasing vesicle depletion
ery of RRP.
We have provided evidence that long-
lasting exposures to BDNF produce a
marked enhancement of eIPSCs in devel-
increasing Prand Pves. This, together with
increased contribution of N- and P/Q-
type Ca2?channels to the eIPSCs, un-
equivocally points to a presynaptic site of
action of BDNF.
A presynaptic mechanism of action for
BDNF on inhibitory synapses is suggested
phological data in rat hippocampal neu-
rons that show increments of eIPSC am-
plitude with little change in the size of
mIPSCs and the number of synaptic con-
nections (Vicario-Abejon et al., 1998; Sh-
erwood and Lo, 1999; Bolton et al., 2000;
Baldelli et al., 2002). Our present data in-
dicate that these effects are associated with an increase in Prand
Pves, which most likely derive from a better redistribution of N-
and P/Q-type channels at the sites controlling neurotransmitter
the number of release sites (Rutherford et al., 1997; Huang et al.,
1999; Marty et al., 2000; Seil and Drake-Baumann, 2000; Kohara
al., 2001; Tartaglia et al., 2001; Tyler and Pozzo-Miller, 2001;
Carter et al., 2002).
of action of BDNF on neurotransmission, there is evidence for a
marked increase in GABAAreceptors in young (7–10 DIV) hip-
pocampal cultures chronically exposed to BDNF (Yamada et al.,
2002). An increased density of functional GABAAreceptors
should cause measurable increases in mIPSC amplitude that we
observed only in 5–14 DIV neurons but not in 14–21 DIV neu-
rons (Baldelli et al., 2002). Here, we extended our previous find-
ings and show that the unitary conductance and the density of
GABAAreceptors was unaffected by BDNF in 14–21 DIV neu-
rons. Thus, most likely, overexpression of GABAAreceptors oc-
curs predominantly at early stages of differentiation in vitro.
Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampusJ.Neurosci.,March30,2005 • 25(13):3358–3368 • 3365
BDNF induces a marked increase in Pves,
mented (Fig. 4). This conclusion is in
agreement with the observation that
BDNF does not affect the size of the recy-
cling pool (Yamada et al., 2002) and pos-
sibly of the RRP as well (Murthy and
Stevens, 1999). An action on Pvespreserv-
exposures to BDNF enhance presynaptic
activity by increasing the RRP (Collin et
al., 2001; Tartaglia et al., 2001; Tyler and
Pozzo-Miller, 2001; Carter et al., 2002).
enhance the rate of vesicle release in exci-
tatory and inhibitory synapses, increasing
either the RRP or Pves.
An increased Pr(or Pves) might have
serious drawbacks during high-frequency
stimulation in synapses that function at a
high rate of release and saturating Ca2?
conditions, as in our case. Control eIPSCs
undergo rapid depression during trains of
stimuli (Fig. 4E), which would be wors-
ened after a 50% increase in Pr. However,
BDNF is able to slow down the rate of de-
pression, maintaining high eIPSC ampli-
tudes during trains. The twofold lower
rate of depression observed with BDNF is possibly associated
with an increased rate of vesicle replenishment attributable to an
enhanced level of residual Ca2?accumulating presynaptically
Wesseling, 1998). This new unexpected finding reinforces the
presynaptic nature of the action of BDNF and can be associated
with the BDNF-induced supra-additivity of presynaptic Ca2?
channels in controlling eIPSCs. BDNF may increase the amount
of presynaptic terminals in which N- and P/Q-type channels are
coexpressed and cooperate to control vesicle release, enhancing
the possibility of intraterminal Ca2?accumulation during high-
frequency stimulation. Indeed, other presynaptic mechanisms
could account for the higher rate of vesicle recycling induced by
the neurotrophin. BDNF activation of TrkB receptors and in-
creased intraterminal Ca2?concentration activate mitogen-
activated protein (MAP)-kinase and calmodulin kinase II, which
phosphorylate synapsins at distinct sites (Benfenati et al., 1992;
reserve to the RRP (Greengard et al., 1993). During high-
frequency stimulation, the level of synapsin phosphorylation
strictly correlates with synapsin dispersion and with the amount
of vesicles sustaining repetitive release (Chi et al., 2001). Thus,
BDNF could increase the rate of vesicle replenishment through
direct (TrkB/MAP-kinase pathway) or indirect (Ca2?/calmodu-
icle mobilization from the reserve pool.
Our data show clearly that BDNF changes both the distribution
and contribution of N- and P/Q-type channels controlling the
eIPSCs. The two channels control distinct release sites in BDNF-
untreated neurons (segregated distribution), and their contribu-
tion is limited to 82% of the total eIPSCs. With BDNF, this per-
centage increases to 117%, increasing the percentage of release
sites containing mixtures of the two channels (nonuniform dis-
tribution). This could represent a new form of synapse matura-
tion in developing neurons induced by BDNF.
An appropriate colocalization of presynaptic Ca2?channels
at the vesicle release sites, rather than a generalized increase in
Ca2?channel density as the cause of the higher Prwith BDNF, is
also supported by two other findings. First, neurotransmission is
near saturation under control conditions, and Princreases very
little at ?2 mM Ca2. Because saturation of Ca2?fluxes through
open Ca2?channels occurs at ?50 mM Ca2?(Hess et al., 1986),
it is evident that local [Ca2?] at the nanodomains formed by
Ca2?channels and docked vesicles (Augustine, 2001) is already
maximal during control eIPSCs in 2 mM Ca2?. Additional eleva-
tions of [Ca2?]oor increased densities of distantly located Ca2?
saturation of eIPSCs occurs at values significantly smaller than the
size of the RRPsyn(2.6 vs 5.5 nA in controls and 4.4 vs 6.7 nA with
BDNF) and, thus, the number of available vesicles and Ca2?entry
the main limitation to Pris the quantity of Ca2?ions effectively
entering the terminal through presynaptic Ca2?channels, which
may derive from a better colocalization of Ca2?channels to the
release active zones (Spafford and Zamponi, 2003). Second, BDNF
enhances the expression of syntaxin 1 and synaptotagmin 1 (Wang
et al. 2002). The two proteins bind selectively to the “synprint” re-
Percentage of PPD vs time in control neurons in the absence of toxins and in the presence of 1 ?M ?-Ctx-GVIA and 0.5 ?M
Pharmacological dissection of presynaptic Ca2?channel types supporting PPD and recovery from depression. A,
3366 • J.Neurosci.,March30,2005 • 25(13):3358–3368Baldellietal.•BDNFPotentiationofeIPSCsintheHippocampus
lack the binding sequence (Catterall, 2000). Thus, an increased ex-
pression of vesicle-binding proteins would favor the colocalization
A final consideration concerns the role that N- and P/Q-type
channels play in the control of PPD and recovery from depres-
sion. As for other neurons (Mintz et al., 1995; Reid et al., 1997,
Prafter blockade of either N- or P/Q-type channels. In our case,
type channels to the size of eIPSCs may simply derive from an
purely controlled by the N-type.
A relevant issue of the present study is that BDNF seems par-
ticularly effective in increasing the contribution of N-type chan-
contradiction that N-type channels contribute to the eIPSCs
(35% block) and little to the PPD is attributable to the fact that
vesicle replenishment, which is Ca2?dependent and determines
The inability of ?-Ctx-GVIA to affect PPD suggests that vesicle
depletion induced by Ca2?influx through N-type channels is
well counterbalanced by an effective Ca2?-dependent recovery
from depression by the Ca2?ions flowing through the same
channel types. Notice that recovery from depression is particu-
larly fast when N-types are the only channels supporting GABA
release and BDNF does not affect this feature (Fig. 7G). This is
synapses possessing distinct populations of presynaptic Ca2?
channels (Poncer et al., 2000). The striatum oriens interneurons
expressing only P/Q-type channels show marked PPD at all in-
terpulse intervals, whereas the striatum radiatum interneurons,
possessing only N-type channels, display facilitation at short in-
tervals and weak PPD at longer intervals.
In conclusion, we have brought new evidence in favor of a
a sharp increase in Prand accounts for most of the increase in
inhibitory postsynaptic signaling in developing hippocampal
neurons. Maturation and stabilization of GABAergic synapses
are expected to play a key role in balancing the activity of excita-
tory synapses during neuronal network formation (Vicario-
Abejon et al., 2002). Our data suggest the possibility that a better
colocalization of presynaptic Ca2?channels with the vesicle fu-
itory synapses to increase their strength in the hippocampus and
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