J Neurophysiol 111: 1008 1015, 2014.

First published December 11, 2013; doi:10.1152/jn.00554.2013.

BDNF-endocannabinoid interactions at neocortical inhibitory synapses require

phospholipase C signaling
Liangfang Zhao and Eric S. Levine
Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut
Submitted 2 August 2013; accepted in final form 9 December 2013

Zhao L, Levine ES. BDNF-endocannabinoid interactions at neocortical inhibitory synapses require phospholipase C signaling. J
Neurophysiol 111: 1008 1015, 2014. First published December 11,
2013; doi:10.1152/jn.00554.2013.Endogenous cannabinoids (endocannabinoids) and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), are potent synaptic modulators that are
expressed throughout the forebrain and play critical roles in many
behavioral processes. Although the effects of BDNF at excitatory
synapses have been well characterized, the mechanisms of action of
BDNF at inhibitory synapses are not well understood. Previously we
have found that BDNF suppresses presynaptic GABA release in layer
2/3 of the neocortex via postsynaptic tropomyosin-related kinase
receptor B (trkB) receptor-induced release of endocannabinoids. To
examine the intracellular signaling pathways that underlie this effect,
we used pharmacological approaches and whole cell patch-clamp
techniques in layer 2/3 pyramidal neurons of somatosensory cortex in
brain slices from juvenile Swiss CD1 mice. Our results indicated that
phospholipase C (PLC) is involved in the CB1 receptor-mediated
synaptic effect of BDNF, because the BDNF effect was blocked in the
presence of the broad-spectrum PLC inhibitors U-73122 and edelfosine, whereas the inactive analog U-73343 did not alter the suppressive
effect of BDNF at inhibitory synapses. Endocannabinoid release can
also be triggered by metabotropic glutamate receptor (mGluR)-mediated activation of PLC, and BDNF has been shown to enhance
spontaneous glutamate release. An mGluR antagonist, E4CPG, however, did not block the BDNF effect. In addition, the effect of BDNF
was independent of other signaling pathways downstream of trkB
receptor activation, namely, mitogen-activated protein kinase and
phosphoinositide 3-kinase pathways, as well as protein kinase C
signaling.
BDNF; cannabinoid; cerebral cortex; phospholipase C
BRAIN-DERIVED NEUROTROPHIC FACTOR

(BDNF) is as an important
modulator of excitatory and inhibitory synaptic transmission.
During the past 20 years, much has been learned about the
mechanism of action of BDNF at excitatory synapses (reviewed in Carvalho et al. 2008; Gottmann et al. 2009). However, the current knowledge of the role and underlying mechanisms of BDNF in modulating inhibitory synaptic transmission is much less clear. The diverse effects of BDNF at
inhibitory synapses depend on a variety of factors, including
age of the animal, tissue preparation (slice vs. cell culture), and
brain region and cell type being studied, as well as BDNF
treatment time course. For example, BDNF has been found to
suppress inhibitory postsynaptic currents (IPSCs) in cerebellar
granule cells (Cheng and Yeh 2003, 2005) and to enhance
postsynaptic GABA receptor responsiveness in Purkinje cells
(Cheng and Yeh 2005). In cortical and hippocampal cell

Address for reprint requests and other correspondence: E. S. Levine, Dept.

of Neuroscience, MC-3401, Univ. of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030 (e-mail: [email protected]).
1008

cultures, acute application of BDNF rapidly enhances miniature IPSC amplitude, followed by a prolonged suppression.
This switch in the direction of effect was concurrent with
protein kinase C (PKC)-mediated phosphorylation (Jovanovic
et al. 2004). In terms of the locus of BDNF effect, Frerking et
al. (1998) found that the suppressive effect of BDNF on IPSCs
is expressed presynaptically in the hippocampal CA1 region,
whereas several other studies indicated the involvement of
postsynaptic tropomyosin-related kinase receptor B (trkB) receptors (Hewitt and Bains 2006; Tanaka et al. 1997).
We recently showed that at inhibitory synapses onto layer
2/3 cortical pyramidal neurons, acute application of BDNF
rapidly suppresses GABAergic transmission via release of
endocannabinoids from the postsynaptic pyramidal cell, which
act in a retrograde manner to suppress presynaptic transmitter
release (Lemtiri-Chlieh and Levine 2010). This effect of
BDNF is initiated by postsynaptic trkB signaling, because the
effect is blocked when postsynaptic trkB receptors are selectively inhibited by intracellular loading of a tyrosine kinase
inhibitor or when endocannabinoid synthesis and release from
the postsynaptic cell is prevented. The suppression of inhibitory transmission is expressed as a presynaptic decrease in
GABA release probability, because it is associated with
changes in the paired-pulse ratio, the coefficient of variation,
and the frequency of miniature IPSCs, and the BDNF effect is
also blocked by antagonists to the predominantly presynaptically expressed CB1 receptor (Lemtiri-Chlieh and Levine
2010). However, the signaling pathway linking BDNF-trkB
activation to endocannabinoid mobilization is not known.
The trkB receptor is the major receptor for BDNF signaling
in the brain and mediates most of the effects of BDNF on
synaptic transmission and plasticity. Upon binding to trkB
receptors, BDNF stimulates at least three major downstream
intracellular signaling pathways via tyrosine phosphorylation,
namely, Ras/mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol 3-kinases (PI3K)/Akt pathway, and
phospholipase C (PLC) pathway. Activation of PLC leads
to cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2)
into second messengers inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG). IP3 induces Ca2 release from intracellular Ca2 stores upon binding to its receptor and thus increases intracellular Ca2 concentration (reviewed in Huang
and Reichardt 2003). DAG leads to activation of PKC, which
has been found to mediate some of the effects of BDNF at
inhibitory synapses (Henneberger et al. 2002; Jovanovic et al.
2004). Activation of the Ras/MAPK and PI3K/Akt pathways
are generally considered to be critical for neuronal survival and
differentiation (reviewed in Huang and Reichardt 2003; Patapoutian and Reichardt 2001; Segal and Greenberg 1996),

0022-3077/14 Copyright 2014 the American Physiological Society

www.jn.org

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

although activation of MAPK may also mediate some of the

acute effects of BDNF on long-term potentiation and excitatory
synaptic transmission (Jovanovic et al. 2000; Ying et al. 2002).
The current study examines the potential intracellular signaling
pathways that underlie BDNF-endocannabinoid interactions at
inhibitory synapses onto layer 2/3 cortical pyramidal neurons.
MATERIALS AND METHODS

Animal handling and slice preparation. All animal procedures were

conducted using protocols approved by the University of Connecticut
Health Center Animal Care Committee. Postnatal day 1527 Swiss
CD-1 mice (Charles River, Wilmington, MA) were anesthetized by
3.5% isoflurane inhalation, followed by decapitation. Whole brains
were removed and immersed in ice-cold slicing solution containing
(in mM) 110 choline chloride, 2.5 KCl, 1.25 NaH2PO4H2O, 25
NaHCO3, 0.5 CaCl2, 7 MgCl26H2O, 25 dextrose, 11.6 sodium
ascorbate, and 3.1 sodium pyruvate, equilibrated with 95% O2-5%
CO2 (pH 7.3, 310 5 mosmol/kg). Transverse slices (350 m)
containing somatosensory cortex were cut with a Dosaka EM DTK1000 vibratome (Kyoto, Japan) and transferred to an incubating
chamber. Slices were then incubated for 30 min at 3335C in
carboxygenated incubating solution containing (in mM) 125 NaCl, 2.5
KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 3.5 MgCl26H2O, 4
sodium lactate, 2 sodium pyruvate, 25 dextrose, and 0.4 ascorbic acid
(pH 7.3, 310 5 mosmol/kg) before being transferred to room
temperature. Slices were then individually transferred to a recording
chamber (room temperature) fixed to the stage of an Olympus
BX51WI upright microscope fitted with a 40 water-immersion
objective lens (0.8 NA). The recording chamber was continuously
perfused at 1.52 ml/min with carboxygenated artificial cerebrospinal
fluid (aCSF) consisting of (in mM) 125 NaCl, 2.5 KCl, 1.25
NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl26H2O, and 25 dextrose (pH
7.3, 305 5 mosmol/kg).
Electrophysiology. Whole cell recordings were obtained from layer
2/3 somatosensory cortex pyramidal neurons. Neurons were visually
identified by their morphology and position under infrared differential
interference contrast video microscopy. Patch electrodes (2 4 M)
were pulled from borosilicate glass capillaries using a Flaming/Brown
P-97 micropipette puller (Sutter Instrument, Novato, CA). Pipette
internal solution contained (in mM) 130 CsCl, 10 HEPES, 1 EGTA,
0.1 CaCl2, 1.5 MgCl2, 4 Na2-ATP, 0.3 Na-GTP, 10 di-tris-phosphocreatine, and 5 QX-314 (pH 7.3, 290 5 mosmol/kg). A bipolar
tungsten electrode (1 M; WPI, Sarasota, FL) was positioned 100
150 m lateral to the patched pyramidal neuron to elicit electrically
evoked IPSCs (eIPSCs). Extracellular stimuli consisted of individual
square-wave current pulses (170 s, 4 30 A) and were delivered
every 15 s.
The chloride equilibrium potential (ECl) with the use of the above
internal and external solutions was close to 0 mV; thus IPSCs were
recorded as inward currents. Ionotropic glutamate receptor antagonists
6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 M) and 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP; 3 M) were
used to isolate inhibitory activity. Cells were voltage clamped at 70
mV during recording. All electrical events were filtered at 2.9 kHz and
digitized at 6 kHz using a HEKA EPC9 amplifier and ITC-16
digitizer (HEKA Elektronik, Darmstadt, Germany). Series resistance
(Rs) was compensated up to 50% at 10- to 100-s lag. Input resistance
(Ri) was monitored with 10-mV (50 ms) hyperpolarizing voltage steps
at the end of each sweep. Cells were rejected from analyses if Ri
changed by 15% or fell below 50 M during the course of an
experiment.
Chemicals. Unless otherwise stated, all drugs were obtained from
Tocris Biosciences (Bristol, UK) and were delivered by bath perfusion. Drugs were first prepared as concentrated stock solution in
solvents and stored at 20C. The stock solutions of DNQX,
U-73122, U-73343, GF-109203X, PD-98059, LY-294002, and (S)-

1009

3,5-dihydroxyphenylglycine (DHPG) were dissolved in 100% dimethyl sulfoxide (DMSO). Edelfosine (ET-18-OCH3) was dissolved
in anhydrous ethanol. CPP, DNQX disodium, chelerythrine chloride
(Sigma-Aldrich, St. Louis, MO), (RS)--ethyl-4-carboxyphenylglycine (E4CPG), and BDNF (PeproTech, Rocky Hill, NJ) were dissolved in 18-M water. Drug stock solutions were diluted into aCSF
on the day of recording to the final concentrations. The final concentration of DMSO did not exceed 0.1%, and the final ethanol concentration was 0.04%, which by themselves had no effect on synaptic
transmission.
Data analysis. Off-line analysis was carried out using Clampfit 10
(Molecular Devices, Sunnyvale, CA) and Prism 5 (GraphPad Software, La Jolla, CA). In individual examples, sweeps of evoked
responses were averaged traces of five consecutive eIPSCs before and
after 10 min of BDNF application. Group data are reported as means
SE. Statistical comparisons were made using one-way ANOVA and
Dunnetts multiple comparison test for post hoc comparison or paired
Students t-test. P 0.05 was taken as a statistically significant effect.
RESULTS

We first examined the effect of BDNF on inhibitory transmission in layer 2/3 pyramidal neurons. As shown in the
individual example in Fig. 1A, bath application of 20 ng/ml
(0.8 nM) BDNF rapidly reduced the peak amplitude of eIPSCs.
This effect persisted during BDNF application and typically
showed little or no recovery up to 15 min after washout of
BDNF. Overall, peak eIPSC amplitude was significantly decreased to 75.6 6.6% of baseline after 10 min of BDNF
exposure, as shown in the group time course in Fig. 1B [F(7,11)
5.698, P 0.05, n 8; baseline, 896.8 181.7 pA; BDNF,
712.4 163.5 pA]. Post hoc tests revealed a significant
decrease after 4 min of BDNF treatment, which likely reflects
penetration time of BDNF in the brain slice. In contrast,
application of the vehicle solution had no significant effect on
eIPSC amplitude (100.6 3.0%, n 3). We also confirmed
that this effect of BDNF required activation of CB1 cannabinoid receptors (CB1R). As shown in Fig. 1, C and D, the
BDNF effect on eIPSC amplitude was completely blocked in
the presence of the CB1R antagonist AM251 (5 M; 100.6
6.5% of baseline, n 5; AM251 baseline, 1,564.0 482.3 pA;
BDNF AM251, 1,539.0 437.9 pA).
We hypothesized that the CB1R-mediated synaptic effect of
BDNF may be dependent on PLC signaling. For example,
PLC signaling has been implicated in the effect of BDNF at
hippocampal and cerebellar inhibitory synapses (Cheng and
Yeh 2005; Tanaka et al. 1997). In addition, the -isoform of
PLC (PLC), which is activated downstream of Gq proteincoupled receptors, is known to be involved in mobilizing
endocannabinoids (Galante and Diana 2004; Hashimotodani et
al. 2005; Varma et al. 2001). We therefore examined the effect
of BDNF in the presence of U-73122 (2 M), a selective
membrane-permeable broad-spectrum PLC inhibitor (Gartner
et al. 2006; Reyes-Harde and Stanton 1998). As shown in Fig.
2, A and D, U-73122 prevented the effect of BDNF (97.3
5.7% of baseline, n 6; baseline, 814.1 148.6 pA; BDNF,
825.8 191.4 pA). In contrast, the inactive analog U-73343 (5
M) did not block the BDNF effect (Fig. 2, B and D). After 10
min of exposure, BDNF reduced eIPSC amplitude to 77.0
6.6% of baseline in the presence of U-73343 (Fig. 2D, P
0.05, n 9), similar to the effect of BDNF alone. The latency
to onset of the BDNF effect in the presence of U-73343 was
also similar to that with BDNF alone.

J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org

1010

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

Fig. 1. Brain-derived neurotrophic factor (BDNF) rapidly suppresses inhibitory transmission via cannabinoid CB1 receptor signaling. A: example time course
showing the effect of bath application of BDNF (20 ng/ml) on evoked inhibitory postsynaptic current (eIPSC) amplitude. Horizontal bar above the trace indicates
BDNF application. Inset shows example sweeps before and after 10 min of BDNF application. Scale bars: 250 pA, 25 ms. B: group time course showing the
effect of BDNF on normalized peak amplitude of eIPSCs (n 8). C: example time course showing the effect of BDNF on eIPSC amplitude in the presence of
the CB1 receptor antagonist AM251 (5 M). Inset shows example sweeps before and after 10 min of BDNF application. Scale bars: 500 pA, 25 ms. D: group
time course illustrating the lack of effect of BDNF on eIPSC amplitude in the presence of AM251 (n 5).

To further confirm the involvement of PLC signaling, we

used another selective PLC inhibitor, edelfosine (ET-18-OCH3,
or ET-18; 5 M). Similar to the result obtained with U-73122,
the BDNF effect was completely abolished in the presence of
ET-18 (Fig. 2, C and D, 102.3 4.9% of baseline). Exposure

to each of the PLC inhibitors alone did not have any significant
effect on baseline eIPSC amplitude (U-73122, 118.6 17% of
baseline, n 6; ET-18, 107.0 17% of baseline, n 4). The
vehicles used to dissolve the PLC inhibitors also did not have
any effect on eIPSC amplitudes (0.08% DMSO for U-73122

Fig. 2. BDNF suppression of IPSC amplitude requires phospholipase C (PLC) signaling. A: group time course of the effect of BDNF on peak eIPSC amplitude
in the presence of U-73122, a broad-spectrum PLC inhibitor (n 6). Horizontal bar above the trace indicates BDNF application. B: group time course showing
the effect of BDNF on peak amplitude of eIPSCs in the presence of U-73343, an inactive analog of U-73122 (n 9). C: group time course showing the effect
of BDNF on peak amplitude of eIPSCs in the presence of the PLC inhibitor edelfosine (ET-18; n 4). D: compiled group data for normalized eIPSC amplitude
under various conditions. WIN, CB1 receptor agonist WIN 55,212-2. *P 0.05 compared with baseline (BL).
J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

1011

Fig. 3. Effect of BDNF on inhibitory transmission does not depend on protein kinase C (PKC) signaling. A: example time course for effect of BDNF on eIPSC
amplitude in the presence of the selective PKC inhibitor chelerythrine (Chel; 10 M). Inset shows example sweeps before and after 10 min of BDNF in the
presence of Chel. Scale bars: 100 pA, 25 ms. B: group time course for the Chel experiments (n 5). C: example time course of BDNF effect on eIPSC amplitude
in the presence of the selective PKC inhibitor GF-109203X (GF; 1 M). Inset shows example sweeps before and after 10 min of BDNF application in the
presence of GF. Scale bars: 50 pA, 50 ms. D: group time course showing the effect of BDNF on normalized peak amplitude of eIPSCs in the presence of GF
(n 5).

and U-73343: 101.4 1.2% of baseline, n 3; 0.04%

anhydrous ethanol for ET-18: 94.7 3.0% of baseline, n 4).
To verify that the effect of PLC inhibition is due to disruption
of BDNF-trkB signaling in the postsynaptic cell, and not a
disruption of presynaptic CB1R signaling, we tested the direct
effect of a CB1R agonist, WIN 55,212-2 (WIN; 5 M), in the
presence of U-73122. As shown in Fig. 2D, exposure to WIN
in the presence of the PLC inhibitor caused a significant
suppression of eIPSC amplitude (74.8 3.1% of baseline,
n 5, P 0.05), which was similar to the effect of WIN alone
(Lemtiri-Chlieh and Levine 2010). Taken together, these results indicate that the BDNF effect on cortical inhibitory
transmission requires PLC signaling downstream of trkB activation.
Activation of PLC leads to the generation of DAG and IP3.
DAG is involved in several intracellular signaling pathways,
including the DAG lipase (DGL)-dependent generation of the
endocannabinoid 2-arachidonoyl glycerol (2-AG) (Stella et al.
1997). Previously, we have shown that the effect of BDNF is
disrupted in the presence of the DGL inhibitor RHC-80267 (50
M) (Lemtiri-Chlieh and Levine 2010), suggesting involvement of 2-AG. However, DAG also leads to the downstream
activation of PKC, which has been found to mediate some of
the effects of BDNF at inhibitory synapses (Henneberger et al.
2002; Jovanovic et al. 2004). Therefore, we also tested the
possible involvement of PKC signaling. We used two different
selective PKC inhibitors, namely, chelerythrine (Chel; 10 M)
and GF-109203X (GF; 1 M). Neither drug blocked the BDNF
effect (Chel, 63.9 12.0% of baseline, P 0.05, n 5; GF,

64.6 9.5% of baseline, P 0.05, n 5), as shown in

individual examples and group data in Fig. 3. The latency and
the magnitude of suppression by BDNF in the presence of
either Chel or GF were not significantly different from the
effect of BDNF alone.
In addition to the above signaling cascades, PI3K and
MAPK pathways are also activated by BDNF-trkB signaling.
We therefore tested whether these pathways were involved in
BDNF modulation of inhibitory transmission. We examined
eIPSC amplitude in the presence of selective PI3K and MAPK
inhibitors. As shown in Fig. 4A, the PI3K inhibitor LY-294002

Fig. 4. BDNF effect on IPSC amplitude does not depend on phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) signaling.
A: group data showing eIPSC amplitude before () and after 10 min of BDNF
exposure () in the presence of the PI3K inhibitor LY-294002 (LY; 10 M;
n 5). B: group data showing eIPSC amplitude before () and after 10 min
of BDNF exposure () in the presence of the MAPK inhibitor PD-98059 (PD;
10 M; n 5). Each point represents the average IPSC amplitude evoked
during the last 2 min of BL or during minutes 9 and 10 of BDNF treatment (8
sweeps). *P 0.05.

J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org

1012

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

(LY; 10 M) did not block BDNF effect. By the end of 10-min

bath application, BDNF significantly reduced eIPSC amplitude
to 80.6 5.8% of baseline (n 5, P 0.05). Similar results
were seen with the MAPK inhibitor PD-98059 (PD; 10 M),
shown in Fig. 4B (74.6 6.7% of baseline, n 8, P 0.05).
Thus the PI3K and MAPK signaling pathways are not required
for the BDNF effect at cortical inhibitory synapses.
We also addressed whether the effect of BDNF could be
mediated by glutamate, rather than a direct effect of BDNFtrkB signaling. BDNF can enhance presynaptic glutamate release (Numakawa et al. 2001, 2002; Zhang et al. 2013), and
glutamate can induce endocannabinoid release via activation of
metabotropic glutamate receptors (mGluRs) acting through
PLC signaling (for reviews see Chevaleyre et al. 2006; Kano
et al. 2009). Thus we examined the BDNF effect in the
presence of 500 M E4CPG, a group I/group II mGluR
antagonist. Blocking mGluR signaling did not prevent the
effect of BDNF or alter its time course (66.8 2.5% of
baseline at the end of 10-min BDNF application, n 4, P
0.05; Fig. 5).
As a positive control to ensure that E4CPG effectively
blocked mGluR activation, we also examined the effect of
DHPG, a selective group I mGluR agonist, on inhibitory
transmission. We found that DHPG (50 M) caused a suppression of eIPSC amplitude, similar to the effect of BDNF (77.2
7.5% of baseline, P 0.05, n 8; Fig. 5C, solid bar). This effect
of DHPG was completely prevented by E4CPG (103.6 11.7%
of baseline, n 4; Fig. 5C, hatched bar). These results confirm
the efficacy of the mGluR antagonist and indicate that the
effect of BDNF on cortical inhibitory synaptic transmission is
not dependent on glutamate-mediated mGluR activation. Because DHPG can also induce endocannabinoid release that is
sensitive to DGL inhibition (Galante and Diana 2004; Gregg et
al. 2012), we examined the interaction between BDNF and
DHPG using the same concentrations as above. DHPG induced
a stable suppression of eIPSC amplitude (73.5 6.0% of
baseline, n 6), and subsequent addition of BDNF had no
significant further effect on eIPSC amplitude (94.3 3.7% of
DHPG baseline, n 6). Similarly, in the presence of BDNF,
there was no effect of subsequent addition of DHPG (BDNF,
67.5 5.3% of baseline; DHPG, 100.9 8.1% of BDNF, n
5). These results indicate that the effects of BDNF and DHPG
occlude each other, suggesting possible saturation of endocannabinoid signaling.
DISCUSSION

Previously we have shown that the suppressive effect of

BDNF on cortical layer 2/3 inhibitory synapses in somatosensory cortex is induced postsynaptically but expressed
presynaptically. We have further shown that this effect
requires endocannabinoid release from the postsynaptic cell
and subsequent presynaptic CB1R activation (LemtiriChlieh and Levine 2010). In the present studies, we investigated the signaling pathways that underlie the CB1Rmediated synaptic effect of BDNF at inhibitory synapses
onto layer 2/3 pyramidal neurons in somatosensory cortex
from juvenile mice. Our results showed that activation of
PLC is necessary for this effect of BDNF, because it was
blocked by two different PLC inhibitors, U-73122 and
ET-18, while maintained in the presence of the inactive

Fig. 5. BDNF suppression of IPSC amplitude does not require metabotropic

glutamate receptor (mGluR) signaling. A: example time course showing BDNF
effect on eIPSC amplitude in the presence of the group I/II mGluR inhibitor
(RS)--ethyl-4-carboxyphenylglycine (E4CPG; 500 M). Inset shows example sweeps before and after 10 min of BDNF application in the presence of
E4CPG. Scale bars: 500 pA, 25 ms. B: group time course showing the effect
of BDNF in the presence of E4CPG (n 4). C: compiled group data for
normalized eIPSC amplitude in the presence of BDNF alone (open bar; n 8),
BDNF in the presence of E4CPG (shaded bar; n 4), the mGluR agonist
(S)-3,5-dihydroxyphenylglycine (DHPG) alone (solid bar; n 8), and DHPG
in the presence of E4CPG (hatched bar; n 4). *P 0.05 compared with BL.

analog U-73343. In addition, PLC inhibition did not alter

the direct suppressive effect of a cannabinoid agonist. Taken
together, these results support a role for PLC signaling
downstream of trkB activation. Furthermore, the CB1Rmediated synaptic effect of BDNF does not require PKC,
MAPK, or PI3K signaling, because inhibition of these
pathways did not prevent the effect of BDNF. Interestingly,
this effect also does not require mGluR signaling, because
the mGluR antagonist E4CPG did not block the BDNF
effect on inhibitory transmission. Taken together with findings of our previous study (Lemtiri-Chlieh and Levine
2010), these results suggest that BDNF requires postsynaptic PLC signaling to induce endocannabinoid release,

J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

which leads to the CB1R-mediated synaptic effect of BDNF

at inhibitory synapses.
We have previously shown that the effect of BDNF was
blocked by the DGL inhibitor RHC-80267, suggesting involvement of 2-AG (Lemtiri-Chlieh and Levine 2010). Therefore, a
potential signaling pathway underlying the BDNF-CB1R interaction could be trkB-induced activation of PLC, leading to
generation of DAG and subsequent synthesis and release of
2-AG. This appears to be similar to the mechanism of mGluRinduced 2-AG release. However, it is important to note that
elevated calcium is not necessary for mGluR-induced 2-AG
production, although it can enhance it (Hashimotodani et al.
2005; Maejima et al. 2005), whereas elevated intracellular
calcium appears to be necessary for the effect of BDNF at
inhibitory cortical synapses (Lemtiri-Chlieh and Levine 2010).
Therefore, different signaling mechanisms might be involved
in mGluR-induced and BDNF-induced 2-AG release, even
though they both require PLC signaling. On the other hand, it
is possible that the other major endocannabinoid, anandamide
(AEA), is also involved. It has been established that elevated
intracellular calcium is needed for AEA synthesis (Cadas et al.
1996; Di Marzo et al. 1994). Moreover, an earlier study
suggested an alternative pathway of AEA synthesis via PLC
signaling (Liu et al. 2008). Further systematic studies are
needed to identify the specific endocannabinoid(s) released by
BDNF.
BDNF-induced endocannabinoid release may play a role in
endocannabinoid-dependent depolarization-induced suppression of inhibition (DSI), a form of short-term synaptic plasticity at inhibitory synapses (Diana et al. 2002; Wilson and Nicoll
2001). During DSI, endocannabinoids are released from postsynaptic sites in response to depolarization-induced calcium
influx and act retrogradely via presynaptic CB1Rs to suppress
GABA release, similar to the effect of BDNF. It will therefore
be interesting to explore whether BDNF contributes to endocannabinoid mobilization during DSI. In addition, BDNFinduced endocannabinoid release may play a role in the induction of long-term potentiation (LTP). Several studies have
indicated an essential role for endogenous BDNF in the induction of LTP in hippocampus (Chen et al. 1999; Gartner and
Staiger 2002; Kovalchuk et al. 2002) and cortex (Abidin et al.
2006; Aicardi et al. 2004; Inagaki et al. 2008; Lu et al. 2010).
However, the underlying mechanisms remain largely unknown. A recent study from Lu et al. (2010) showed that
BDNF facilitates LTP by suppressing GABAergic inhibition
and enhancing pyramidal neuron excitability. Interestingly,
that resembles the mechanism of endocannabinoid-dependent
enhancement of plasticity. Moreover, high-frequency stimulation during LTP induction releases BDNF (Aicardi et al. 2004;
Gartner and Staiger 2002). Endogenously released BDNF can
act at both excitatory and inhibitory synapses, because BDNF
can be released from the somatodendritic region as well as
from axons and glial cells (for review see Edelmann et al.
2013). Thus it is possible that endogenously released BDNF
may enhance LTP at least in part by inducing endocannabinoid
release at inhibitory synapses.
In addition to suppressing inhibitory transmission, endocannabinoids have been shown to directly modulate excitatory
synaptic transmission by suppressing glutamate release in
many brain areas, including cortical layer 5 (Fortin and Levine
2007), amygdala (Kodirov et al. 2010), cerebellum (Kreitzer

1013

and Regehr 2001), and hippocampus (Hoffman et al. 2010).

Interestingly, BDNF potentiates excitatory synaptic transmission in these same brain areas by enhancing glutamate release
(Madara and Levine 2008; Schinder et al. 2000). Therefore, in
addition to their synergistic action at inhibitory synapses,
BDNF and endocannabinoids may have counteracting effects
at excitatory synapses.
Several other lines of studies have also suggested mutual
interactions between BDNF and endocannabinoid systems. In
addition to the present findings, BDNF also has been shown to
alter sensitivity to endocannabinoids via modulation of CB1Rs.
For example, BDNF has been reported to increase CB1R
expression in cerebellar granule neurons (Maison et al. 2009).
On the other hand, BDNF can inhibit CB1R function in the
striatum (De Chiara et al. 2010). Furthermore, endocannabinoid signaling has been implicated in the regulation of BDNF
expression. For example, kainic acid-induced seizures induce
BDNF expression in a CB1R-dependent manner (Marsicano et
al. 2003), and BDNF has been reported to mediate CB1Rdependent protection against excitotoxicity (Khaspekov et al.
2004) It also has been found that the anxiogenic-like behavioral phenotype of CB1R knockout mice are related to decreased hippocampal BDNF level (Aso et al. 2008). Both
BDNF and endocannabinoids are currently major targets for
the development of novel therapeutics (Ashton and Moore
2011; Nagahara and Tuszynski 2011). Future studies are
needed to further explore BDNF-endocannabinoid interactions
and their role in regulating synaptic plasticity and neurological
diseases.
In summary, the results above indicate that trkB-induced
activation of PLC is required for the CB1R-mediated synaptic
effect of BDNF at cortical inhibitory synapses, whereas
MAPK, PI3K, and PKC signaling pathways are not involved.
Our results also suggest that this effect is independent of
mGluR-mediated signaling.
GRANTS
This work was supported by National Institute of Mental Health Grant
MH-094896 (E. S. Levine).

DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS
L.Z. and E.S.L. conception and design of research; L.Z. performed experiments; L.Z. analyzed data; L.Z. and E.S.L. interpreted results of experiments;
L.Z. prepared figures; L.Z. drafted manuscript; L.Z. and E.S.L. approved final
version of manuscript; E.S.L. edited and revised manuscript.

REFERENCES
Abidin I, Kohler T, Weiler E, Zoidl G, Eysel UT, Lessmann V, Mittmann
T. Reduced presynaptic efficiency of excitatory synaptic transmission impairs LTP in the visual cortex of BDNF-heterozygous mice. Eur J Neurosci
24: 3519 3531, 2006.
Aicardi G, Argilli E, Cappello S, Santi S, Riccio M, Thoenen H, Canossa
M. Induction of long-term potentiation and depression is reflected by
corresponding changes in secretion of endogenous brain-derived neurotrophic factor. Proc Natl Acad Sci USA 101: 15788 15792, 2004.
Ashton CH, Moore PB. Endocannabinoid system dysfunction in mood and
related disorders. Acta Psychiatr Scand 124: 250 261, 2011.

J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org

1014

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

Aso E, Ozaita A, Valdizan EM, Ledent C, Pazos A, Maldonado R,

Valverde O. BDNF impairment in the hippocampus is related to enhanced
despair behavior in CB1 knockout mice. J Neurochem 105: 565572, 2008.
Cadas H, Gaillet S, Beltramo M, Venance L, Piomelli D. Biosynthesis of an
endogenous cannabinoid precursor in neurons and its control by calcium and
cAMP. J Neurosci 16: 3934 3942, 1996.
Carvalho AL, Caldeira MV, Santos SD, Duarte CB. Role of the brainderived neurotrophic factor at glutamatergic synapses. Br J Pharmacol 153,
Suppl 1: S310 S324, 2008.
Chen G, Kolbeck R, Barde YA, Bonhoeffer T, Kossel A. Relative contribution of endogenous neurotrophins in hippocampal long-term potentiation.
J Neurosci 19: 79837990, 1999.
Cheng Q, Yeh HH. Brain-derived neurotrophic factor attenuates mouse
cerebellar granule cell GABAA receptor-mediated responses via postsynaptic mechanisms. J Physiol 548: 711721, 2003.
Cheng Q, Yeh HH. PLCgamma signaling underlies BDNF potentiation of
Purkinje cell responses to GABA. J Neurosci Res 79: 616 627, 2005.
Chevaleyre V, Takahashi KA, Castillo PE. Endocannabinoid-mediated synaptic plasticity in the CNS. Annu Rev Neurosci 29: 3776, 2006.
De Chiara V, Angelucci F, Rossi S, Musella A, Cavasinni F, Cantarella C,
Mataluni G, Sacchetti L, Napolitano F, Castelli M, Caltagirone C,
Bernardi G, Maccarrone M, Usiello A, Centonze D. Brain-derived
neurotrophic factor controls cannabinoid CB1 receptor function in the
striatum. J Neurosci 30: 8127 8137, 2010.
Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC,
Piomelli D. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372: 686 691, 1994.
Diana MA, Levenes C, Mackie K, Marty A. Short-term retrograde inhibition
of GABAergic synaptic currents in rat Purkinje cells is mediated by
endogenous cannabinoids. J Neurosci 22: 200 208, 2002.
Edelmann E, Lessmann V, Brigadski T. Pre- and postsynaptic twists in
BDNF secretion and action in synaptic plasticity. Neuropharmacology 2013.
Fortin DA, Levine ES. Differential effects of endocannabinoids on glutamatergic and GABAergic inputs to layer 5 pyramidal neurons. Cereb Cortex
17: 163174, 2007.
Frerking M, Malenka RC, Nicoll RA. Brain-derived neurotrophic factor
(BDNF) modulates inhibitory, but not excitatory, transmission in the CA1
region of the hippocampus. J Neurophysiol 80: 33833386, 1998.
Galante M, Diana MA. Group I metabotropic glutamate receptors inhibit
GABA release at interneuron-Purkinje cell synapses through endocannabinoid production. J Neurosci 24: 4865 4874, 2004.
Gartner A, Polnau DG, Staiger V, Sciarretta C, Minichiello L, Thoenen H,
Bonhoeffer T, Korte M. Hippocampal long-term potentiation is supported
by presynaptic and postsynaptic tyrosine receptor kinase B-mediated phospholipase Cgamma signaling. J Neurosci 26: 3496 3504, 2006.
Gartner A, Staiger V. Neurotrophin secretion from hippocampal neurons
evoked by long-term-potentiation-inducing electrical stimulation patterns.
Proc Natl Acad Sci USA 99: 6386 6391, 2002.
Gottmann K, Mittmann T, Lessmann V. BDNF signaling in the formation,
maturation and plasticity of glutamatergic and GABAergic synapses. Exp
Brain Res 199: 203234, 2009.
Gregg LC, Jung KM, Spradley JM, Nyilas R, Suplita RL 2nd, Zimmer A,
Watanabe M, Mackie K, Katona I, Piomelli D, Hohmann AG. Activation
of type 5 metabotropic glutamate receptors and diacylglycerol lipase-alpha
initiates 2-arachidonoylglycerol formation and endocannabinoid-mediated
analgesia. J Neurosci 32: 94579468, 2012.
Hashimotodani Y, Ohno-Shosaku T, Tsubokawa H, Ogata H, Emoto K,
Maejima T, Araishi K, Shin HS, Kano M. Phospholipase Cbeta serves as
a coincidence detector through its Ca2 dependency for triggering retrograde endocannabinoid signal. Neuron 45: 257268, 2005.
Henneberger C, Juttner R, Rothe T, Grantyn R. Postsynaptic action of
BDNF on GABAergic synaptic transmission in the superficial layers of the
mouse superior colliculus. J Neurophysiol 88: 595 603, 2002.
Hewitt SA, Bains JS. Brain-derived neurotrophic factor silences GABA
synapses onto hypothalamic neuroendocrine cells through a postsynaptic
dynamin-mediated mechanism. J Neurophysiol 95: 21932198, 2006.
Hoffman AF, Laaris N, Kawamura M, Masino SA, Lupica CR. Control of
cannabinoid CB1 receptor function on glutamate axon terminals by endogenous adenosine acting at A1 receptors. J Neurosci 30: 545555, 2010.
Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction.
Annu Rev Biochem 72: 609 642, 2003.
Inagaki T, Begum T, Reza F, Horibe S, Inaba M, Yoshimura Y, Komatsu
Y. Brain-derived neurotrophic factor-mediated retrograde signaling required

for the induction of long-term potentiation at inhibitory synapses of visual

cortical pyramidal neurons. Neurosci Res 61: 192200, 2008.
Jovanovic JN, Czernik AJ, Fienberg AA, Greengard P, Sihra TS. Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat Neurosci 3: 323329, 2000.
Jovanovic JN, Thomas P, Kittler JT, Smart TG, Moss SJ. Brain-derived
neurotrophic factor modulates fast synaptic inhibition by regulating GABAA
receptor phosphorylation, activity, and cell-surface stability. J Neurosci 24:
522530, 2004.
Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe
M. Endocannabinoid-mediated control of synaptic transmission. Physiol
Rev 89: 309 380, 2009.
Khaspekov LG, Brenz Verca MS, Frumkina LE, Hermann H, Marsicano
G, Lutz B. Involvement of brain-derived neurotrophic factor in cannabinoid
receptor-dependent protection against excitotoxicity. Eur J Neurosci 19:
16911698, 2004.
Kodirov SA, Jasiewicz J, Amirmahani P, Psyrakis D, Bonni K, Wehrmeister M, Lutz B. Endogenous cannabinoids trigger the depolarization-induced
suppression of excitation in the lateral amygdala. Learn Mem 17: 43 49,
2010.
Kovalchuk Y, Hanse E, Kafitz KW, Konnerth A. Postsynaptic induction of
BDNF-mediated long-term potentiation. Science 295: 1729 1734, 2002.
Kreitzer AC, Regehr WG. Retrograde inhibition of presynaptic calcium
influx by endogenous cannabinoids at excitatory synapses onto Purkinje
cells. Neuron 29: 717727, 2001.
Lemtiri-Chlieh F, Levine ES. BDNF evokes release of endogenous cannabinoids at layer 2/3 inhibitory synapses in the neocortex. J Neurophysiol
104: 19231932, 2010.
Liu J, Wang L, Harvey-White J, Huang BX, Kim HY, Luquet S, Palmiter
RD, Krystal G, Rai R, Mahadevan A, Razdan RK, Kunos G. Multiple
pathways involved in the biosynthesis of anandamide. Neuropharmacology
54: 17, 2008.
Lu H, Cheng PL, Lim BK, Khoshnevisrad N, Poo MM. Elevated BDNF
after cocaine withdrawal facilitates LTP in medial prefrontal cortex by
suppressing GABA inhibition. Neuron 67: 821 833, 2010.
Madara JC, Levine ES. Presynaptic and postsynaptic NMDA receptors
mediate distinct effects of brain-derived neurotrophic factor on synaptic
transmission. J Neurophysiol 100: 31753184, 2008.
Maejima T, Oka S, Hashimotodani Y, Ohno-Shosaku T, Aiba A, Wu D,
Waku K, Sugiura T, Kano M. Synaptically driven endocannabinoid
release requires Ca2-assisted metabotropic glutamate receptor subtype 1 to
phospholipase Cbeta4 signaling cascade in the cerebellum. J Neurosci 25:
6826 6835, 2005.
Maison P, Walker DJ, Walsh FS, Williams G, Doherty P. BDNF regulates
neuronal sensitivity to endocannabinoids. Neurosci Lett 467: 90 94, 2009.
Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich
A, Azad SC, Cascio MG, Gutierrez SO, van der Stelt M, LopezRodriguez ML, Casanova E, Schutz G, Zieglgansberger W, Di Marzo V,
Behl C, Lutz B. CB1 cannabinoid receptors and on-demand defense against
excitotoxicity. Science 302: 84 88, 2003.
Nagahara AH, Tuszynski MH. Potential therapeutic uses of BDNF in
neurological and psychiatric disorders. Nat Rev Drug Discov 10: 209 219,
2011.
Numakawa T, Matsumoto T, Adachi N, Yokomaku D, Kojima M, Takei
N, Hatanaka H. Brain-derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca2 and Na in cultured
cerebellar neurons. J Neurosci Res 66: 96 108, 2001.
Numakawa T, Yamagishi S, Adachi N, Matsumoto T, Yokomaku D,
Yamada M, Hatanaka H. Brain-derived neurotrophic factor-induced potentiation of Ca2 oscillations in developing cortical neurons. J Biol Chem
277: 6520 6529, 2002.
Patapoutian A, Reichardt LF. Trk receptors: mediators of neurotrophin
action. Curr Opin Neurobiol 11: 272280, 2001.
Reyes-Harde M, Stanton PK. Postsynaptic phospholipase C activity is
required for the induction of homosynaptic long-term depression in rat
hippocampus. Neurosci Lett 252: 155158, 1998.
Schinder AF, Berninger B, Poo M. Postsynaptic target specificity of neurotrophin-induced presynaptic potentiation. Neuron 25: 151163, 2000.
Segal RA, Greenberg ME. Intracellular signaling pathways activated by
neurotrophic factors. Annu Rev Neurosci 19: 463 489, 1996.
Stella N, Schweitzer P, Piomelli D. A second endogenous cannabinoid that
modulates long-term potentiation. Nature 388: 773778, 1997.

J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org

BDNF-ENDOCANNABINOID INTERACTIONS AT INHIBITORY SYNAPSES

Tanaka T, Saito H, Matsuki N. Inhibition of GABAA synaptic responses by
brain-derived neurotrophic factor (BDNF) in rat hippocampus. J Neurosci
17: 2959 2966, 1997.
Varma N, Carlson GC, Ledent C, Alger BE. Metabotropic glutamate
receptors drive the endocannabinoid system in hippocampus. J Neurosci 21:
RC188, 2001.
Wilson RI, Nicoll RA. Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. Nature 410: 588 592, 2001.

1015

Ying SW, Futter M, Rosenblum K, Webber MJ, Hunt SP, Bliss TV,
Bramham CR. Brain-derived neurotrophic factor induces long-term potentiation in intact adult hippocampus: requirement for ERK activation coupled
to CREB and upregulation of Arc synthesis. J Neurosci 22: 15321540,
2002.
Zhang Z, Fan J, Ren Y, Zhou W, Yin G. The release of glutamate from
cortical neurons regulated by BDNF via the TrkB/Src/PLC-gamma1 pathway. J Cell Biochem 114: 144 151, 2013.

J Neurophysiol doi:10.1152/jn.00554.2013 www.jn.org