32. F. Holsboer, Curr. Opin. Investig. Drugs 4, 46
33. We thank M. Vitek and H. Dawson for tau knockout mice;
P. Seubert and P. Davies for antibodies; H. Solanoy,
X. Wang, and Y. Zhou for technical assistance;
C. McCullough for advice on statistics; D. McPherson and
L. Manuntag for administrative support; and G. Howard
and S. Ordway for editorial review. L.M. received
consulting fees from Merck and honoraria from Amgen,
Elan, and Pfizer. E.D.R. received consulting fees from
Rinat Neuroscience. Supported by NIH grants AG011385
and AG022074 (L.M.), MH070588 (K.S.L), and
NS054811 (E.D.R); the Giannini Foundation (E.D.R.); the
S.D. Bechtel Jr. Young Investigator Award (E.D.R.); and
the NIH National Center for Research Resources grant
Supporting Online Material
Materials and Methods
Figs. S1 to S7
26 February 2007; accepted 27 March 2007
Regulation of NF-kB Activation in
T Cells via Association of the Adapter
Proteins ADAP and CARMA1
Ricardo B. Medeiros,1* Brandon J. Burbach,1* Kristen L. Mueller,1Rupa Srivastava,1
James J. Moon,2Sarah Highfill,1Erik J. Peterson,3Yoji Shimizu1†
The adapter protein ADAP regulates T lymphocyte adhesion and activation. We present evidence for a
previously unrecognized function for ADAP in regulating T cell receptor (TCR)–mediated activation of
the transcription factor NF-kB. Stimulation of ADAP-deficient mouse T cells with antibodies to CD3 and
CD28 resulted in impaired nuclear translocation of NF-kB, a reduced DNA binding, and delayed
degradation and decreased phosphorylation of IkB (inhibitor of NF-kB). TCR-stimulated assembly of the
CARMA1–BCL-10–MALT1 complex was substantially impaired in the absence of ADAP. We further
identified a region of ADAP that is required for association with the CARMA1 adapter and NF-kB
activation but is not required for ADAP-dependent regulation of adhesion. These findings provide new
insights into ADAP function and the mechanism by which CARMA1 regulates NF-kB activation in T cells.
during an adaptive immune response (1). In T
lymphocytes, the adhesion- and degranulation-
promoting adapter protein (ADAP) regulates T
cell receptor (TCR)–dependent changes in the
function of integrin adhesion receptors (2, 3).
ADAP-deficient (ADAP−/−) T cells also exhibit
impaired proliferation and cytokine production
after stimulation of the TCR and the CD28 co-
stimulatory receptor (2, 3). Stimulation of these
receptors leads to activation of the NF-kB family
consisting of the membrane-associated adapter
protein CARMA1 (5, 6), the caspase-like protein
kinase complex and subsequent NF-kB nuclear
Like ADAP-deficient T cells, protein kinase
Cq (PKCq)–deficient T cells exhibit defective
TCR-mediated proliferation, even though prox-
imal TCR signaling events, such as extracellu-
dapter proteins nucleate multimolecular
complexes that are essential for effec-
tive transmission ofintracellular signals
lar signal–regulated kinase (ERK) activation,
are normal (11). Therefore, we examined PKCq-
dependent signaling in ADAP−/−T cells (12).
Membrane localization of PKCq was similar in
ADAP+/−and ADAP−/−Tcells upon stimulation
with antibodies to CD3 and CD28 (Fig. 1, A
and B). Stimulated ADAP+/−and ADAP−/−T
cells also showed similar levels of PKCq
phosphorylation (Fig. 1C). Thus, ADAP is not
required for TCR signaling events leading to
and including PKCq activation. Because PKCq
regulates NF-kB activation downstream of the
TCR (11, 13), we next examined NF-kB signal-
ing in ADAP−/−T cells. Image scanning flow
cytometry (14, 15) (fig. S1) revealed a striking
defect in p65 nuclear translocation after stimu-
lation of ADAP−/−lymph node T cells (Fig. 2,
A and B) or CD4 T cells (fig. S2) by CD3 and
CD28 (CD3/CD28). In contrast, no impairment
in NF-kB activation was detected after stimula-
tion with tumor necrosis factor–a (TNF-a), which
activates NF-kB independently of the TCR.
These results were confirmed with electrophoretic
mobility shift assays (Fig. 2C). ADAP−/−T cells
also displayed defective NF-kB translocation after
treatment with phorbol 12-myristate 13-acetate
(PMA), which activates PKC (Fig. 2, A and B).
1Department of Laboratory Medicine and Pathology, Center
for Immunology, Cancer Center, University of Minnesota
Medical School, Minneapolis, MN 55455, USA.
ment of Microbiology, Center for Immunology, University
of Minnesota Medical School, Minneapolis, MN 55455,
Cancer Center, University of Minnesota Medical School, Min-
neapolis, MN 55455, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
3Department of Medicine, Center for Immunology,
Fig. 1. TCR-dependent membrane localization and activation of PKCq in ADAP
Localization of PKCq (bottom) in ADAP+/−and ADAP−/−T cells to the contact site with beads coated with
antibodies to CD3 and CD28. Differential interference contrast (DIC) images are shown in top panels.
(B) Quantification of PKCq localization. T cell–bead conjugates (minimum 90 per group) were scored
for PKCq polarization from two independent experiments. Graph shows the average percent of T cell–
bead conjugates with polarized PKCq (±SD). (C) Phosphorylation of PKCq after CD3/CD28 stimulation
of ADAP+/−and ADAP−/−T cells for the indicated time points was assessed by Western blotting of whole-
cell lysates with antibody to phosphorylated PKCq (Thr538) (top panels). Blots were also probed with
antibody to b-actin (bottom panels).
−/−T cells. (A)
4 MAY 2007VOL 316
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CD3/CD28 stimulation of ADAP−/−T cells also
resulted in reduced induction of intercellular ad-
hesion molecule–1 (ICAM-1), which is encoded
by a NF-kB–regulated gene (16) (Fig. 2D).
We also examined signaling events proximal
to nuclear translocation of the p65 NF-kB
subunit in ADAP−/−T cells. In resting T cells,
NF-kB subunits are sequestered in the cyto-
plasm via interactions with IkBa (4). Relative
to ADAP+/−T cells, CD3/CD28 stimulation of
ADAP−/−T cells led to a delay in IkBa deg-
radation and decreased IkB phosphorylation
(Fig. 2E and fig. S3). Consistent with previous
results (2, 3), the kinetics of ERK phosphoryl-
ation after CD3/CD28 stimulation were not
affected by loss of ADAP (Fig. 2E). Activa-
tion of the IkB kinase (IKK) complex, which
phosphorylates IkBa, was also impaired after
CD3/CD28 stimulation of ADAP−/−T cells (fig.
S4). Thus, ADAP acts as a positive regulator of
TCR-dependent NF-kB activation, downstream
of PKCq yet upstream of IKK activation, IkBa
degradation, and NF-kB subunit translocation.
We next examined the role of ADAP in in-
ducible membrane localization of BCL-10,
MALT1, and CARMA1 (17, 18). In unstimu-
lated ADAP+/−or ADAP−/−T cells, only low
levels of BCL-10 and MALT1 were detected in
membrane fractions (Fig. 3A). CD3/CD28 or
PMA stimulation of ADAP+/−T cells, but not
ADAP−/−T cells, resulted in enhanced mem-
brane localization of both BCL-10 and MALT1
(Fig. 3A). ADAP also localized to the mem-
brane in stimulated ADAP+/−T cells (Fig. 3A).
The levels of CARMA1 in membrane fractions
were comparable between ADAP+/−and ADAP−/−
T cells both before and after stimulation (Fig.
3A), consistent with previous results (17). Thus,
ADAP is critical for the activation-dependent
membrane localization of BCL-10 and MALT1.
To define potential interactions between
ADAP and the CARMA1–BCL-10–MALT1
signaling complex, we performed coimmunopre-
cipitation experiments. ADAP could be immuno-
precipitated only from membrane fractions
isolated from activated T cells (Fig. 3B). Co-
immunoprecipitation of CARMA1, BCL-10, and
MALT1 with ADAP was observed from stimu-
lated membrane fractions of either lymph node T
cells (Fig. 3B) or purified CD4 T cells (fig. S5).
noprecipitated with CARMA1 only after T cell
stimulation (Fig. 3B). However, in activated
ADAP−/−T cells, MALT-1 and BCL-10 did not
coimmunoprecipitate with CARMA1 (Fig. 3B),
and CARMA1 and MALT-1 did not coimmuno-
confirm that ADAP is required for inducible
complex assembly, we used resting T cells ex-
pressing the hCAR adenovirus receptor to permit
adenoviral-mediated expression of ADAP (19).
ADAP reexpression in ADAP−/−Tcells restored
with BCL-10 after CD3/CD28 or PMA stimula-
tion (Fig. 3C). Thus, ADAP is a component of the
CARMA1–BCL-10–MALT1 complex and is
required for normal complex formation.
We next examined interactions between
ADAP and purified BCL-10, MALT1, and
CARMA1. A glutathione S-transferase (GST)–
ADAP fusion protein interacted in vitro with
purified CARMA1, but not with purified BCL-10
or MALT1 (Fig. 3D). A truncated form of
CARMA1 (CARMA1/651-1147) containing just
typical of membrane-associated guanylate kinase
(MAGUK)–family proteins (20) (Fig. 3E) also in-
teracted with GST-ADAP in vitro(Fig.3D).Thus,
the interaction of ADAP with CARMA1 is not de-
pendent on the caspase-recruiting domain that me-
diates the interaction of CARMA1 with BCL-10.
Colocalization of ADAP with CARMA1 was
also observed at the contact site between wild-
type T cells and beads coated with antibodies to
CD3 and CD28 (fig. S7).
Truncation and deletion mutants of ADAP
were used to define sites within ADAP critical for
its interaction with CARMA1 (Fig. 4A). Wild-
type and mutant forms of hemagglutinin (HA)
epitope–tagged ADAP were immunoprecipitated
from transiently transfected Jurkat T cells after
PMA stimulation, and these immunoprecipitates
were analyzed for the presence of coimmuno-
precipitating BCL-10, MALT1, and CARMA1
proteins (Fig. 4B). Wild-type ADAP and an
Fig. 2. Activation of the NF-kB pathway is impaired in ADAP−/−T cells. (A) ADAP+/−and ADAP−/−T
cells were unstimulated (unstim.) or stimulated for 10 min with antibodies to CD3 and CD28, PMA, or
TNF-a, stained with 7-AAD and fluorescein isothiocyanate–conjugated antibody to p65, and analyzed
on a multispectral imaging flow cytometer (15). Histograms show the NF-kB–7-AAD similarity index,
which reflects colocalization of the 7-AAD and p65 signals in the population of T cells analyzed.
Marker values indicate the percentage of T cells in each sample with translocated p65. (B) Nuclear
localization of p65 in unstimulated T cells was set to 1; results show the average increase (±SD) in p65
nuclear translocation in stimulated T cells relative to unstimulated T cells for four independent
experiments. (C) Electrophoretic mobility shift assay (EMSA) of NF-kB in ADAP−/−T cells. ADAP+/−or
ADAP−/−lymph node T cells were stimulated with antibodies to CD3 and CD28 for the indicated time
periods (in minutes), or for 10 min with PMA and ionomycin (P/I) or with TNF-a. Nuclear extracts were
prepared and EMSA was performed with a biotin-labeled NF-kB probe. (D) ADAP+/−and ADAP−/−
lymph node T cells were stimulated in vitro with antibodies to CD3 and CD28 for 24 hours. Cells were
harvested and ICAM-1 expression on CD4 T cells was determined by flow cytometry. (E) IkBa
degradation and phosphorylation. ADAP+/−and ADAP−/−T cells were either unstimulated or stimulated
as in (C) before lysis. Lysates were analyzed by Western blotting with antibodies specific for IkBa,
phosphorylated IkBa, phosphorylated ERK, ERK2, or b-actin.
VOL 316 4 MAY 2007
ADAP mutant lacking the N-terminal 327
amino acids (ADAPD1-327), but not an ADAP
mutant containing only the N-terminal 426 amino
acids (ADAP-426), coimmunoprecipitated the
CARMA1–BCL-10–MALT1 complex (Fig.
4B). The CARMA1 binding site in ADAP was
mapped to a region of ADAP between amino
acids 426 and 541 (Fig. 4A), because a deletion
541) was completely unable to coimmunopre-
cipitate the CARMA1–BCL-10–MALT1 complex
(Fig. 4B). This region of ADAP contains the
N-terminal helical SH3 (hSH3) domain(21–23)
region rich in Glu and Lys residues (E/K-rich
deletion mutants lacking either the N-terminal
hSH3 domain (ADAPD482-541) or the E/K-
rich region (ADAPD426-481) (Fig. 4A) showed
weak coimmunoprecipitation of the CARMA1–
BCL-10–MALT1 complex relative to wild-type
ADAP (Fig. 4B). A GST-ADAP fusion protein
expressing both the hSH3 domain and the E/K-
rich region of ADAP was able to associate with
the truncated CARMA1/651-1147 protein in
vitro (fig. S8), which suggests that this region
of ADAP is sufficient for CARMA1 association.
Expression of wild-type ADAP in resting
ADAP−/−T cells restored the ability of PMA
(Fig. 4C) or CD3/CD28 stimulation (Fig. 4D) to
induce nuclear translocation of p65. ADAP ex-
pression in transduced T cells was verified by
intracellular flow cytometry (fig. S9). Expression
of the ADAPD1-327 mutant, but not the ADAP-
426 mutant, in ADAP−/−T cells was also able to
fully restore CD3/CD28-mediated NF-kB trans-
deletion mutant did not restore CD3/CD28-
mediated NF-kB translocation after expression in
ADAP−/−T cells (Fig. 4E). Expression of either
the ADAPD426-481 or ADAPD482-541 deletion
mutants partially restored NF-kB p65 nuclear
translocation (Fig. 4E).
ADAP also regulates TCR-mediated integrin
activation (2, 3) and thus ADAP−/−Tcells exhibit
impaired integrin-dependent conjugate forma-
tion with antigen-pulsed antigen-presenting cells
(24) (Fig. 4F). Expression of either wild-type
T cells restored TCR-induced conjugate forma-
426 and 541 is critical for NF-kB activation but
is not required for the regulation of integrin-
dependent conjugate formation.
We have identified a novel function for ADAP
amino acids 426 and 541 is critical for assembly of
the CARMA1–BCL-10–MALT1 complex at the
membrane. ADAP may provide mechanisms
for membrane localization and stabilization of
C-terminal ADAP hSH3 domain can associate
Fig. 3. ADAP is critical for the assembly of the CARMA1–BCL-10–MALT1 complex and associates
directly with the C-terminal end of CARMA1. (A) ADAP+/−and ADAP−/−T cells were either unstimulated
(U) or stimulated with antibodies to CD3 and CD28 (3/28) or with PMA (P). Cytosolic (Cyto) and
membrane (Memb) fractions were prepared from each cell sample and analyzed by Western blotting
with the indicated antibodies. (B) Membrane fractions were prepared from unstimulated and stimulated
ADAP+/−and ADAP−/−T cells as in (A). ADAP or CARMA1 was immunoprecipitated and the immu-
noprecipitates analyzed as in (A). (C) T cells isolated from ADAP+/−and ADAP−/−transgenic mice
expressing the hCAR adenovirus receptor were transduced with control adenovirus encoding Thy1.1
(ctrl) or adenovirus expressing wild-type mouse ADAP and Thy1.1 (ADAPwt). T cells were then left
unstimulated or were stimulated with antibodies to CD3 and CD28 or with PMA before lysis and
immunoprecipitation with a monoclonal antibody (mAb) to BCL-10. Immunoprecipitates were analyzed
by Western blotting as in (A). (D) Interaction of ADAP with CARMA1 in vitro. GST only or GST-ADAP
fusion proteins were incubated with in vitro transcribed/translated BCL-10, MALT1, CARMA1, or
truncated FLAG epitope–tagged CARMA1/651-1147. GST pull-downs were analyzed by Western blotting
for the presence of the indicated proteins. An equivalent amount of the purified proteins used in the
pull-down assays, along with a sample from in vitro transcription and translation reactions using a
control vector (vec), were analyzed in separate gels (input). (E) Diagram of CARMA1 and CARMA1/651-
1147 constructs used in (D).
4 MAY 2007VOL 316
with membrane phospholipids (22). The inter-
action of ADAP with the MAGUK region of
CARMA1 may also alter intramolecular in-
teractions within CARMA1 (25, 26), thereby
promoting recruitment of BCL-10 and MALT1.
The region of ADAP critical for association with
CARMA1 is not required for ADAP-dependent
regulation of integrins (2, 3), which involves the
association of ADAP with the SKAP-55 and
SLP-76 adapters (27–29). Two biochemically
distinct pools of ADAP can be identified in
CD3/CD28-stimulated T cells: one that interacts
with the CARMA1–BCL-10–MALT1 complex,
and one that interacts with SLP-76 (fig. S10). In
contrast, CARMA1 is required for NF-kB
activation (2, 3, 30, 31) but is not required for
conjugate formation (31). Thus, ADAP serves
distinct roles downstream of the TCR that
promote functions critical to T cell immune
References and Notes
1. L. E. Samelson, Annu. Rev. Immunol. 20, 371 (2002).
2. E. J. Peterson et al., Science 293, 2263 (2001).
3. E. K. Griffiths et al., Science 293, 2260 (2001).
4. J. Schulze-Luehrmann, S. Ghosh, Immunity 25, 701 (2006).
5. J. Bertin et al., J. Biol. Chem. 276, 11877 (2001).
6. L. M. McAllister-Lucas et al., J. Biol. Chem. 276, 30589
7. A. G. Uren et al., Mol. Cell 6, 961 (2000).
8. P. C. Lucas et al., J. Biol. Chem. 276, 19012 (2001).
9. J. Ruland et al., Cell 104, 33 (2001).
10. D. J. Rawlings, K. Sommer, M. E. Moreno-Garcia,
Nat. Rev. Immunol. 6, 799 (2006).
11. Z. Sun et al., Nature 404, 402 (2000).
12. See supporting material on Science Online.
13. C. Pfeifhofer et al., J. Exp. Med. 197, 1525 (2003).
14. T. C. George et al., Cytometry 59, 237 (2004).
15. T. C. George et al., J. Immunol. Methods 311, 117
16. C. C. Chen, A. M. Manning, Agents Actions Suppl. 47,
17. O. Gaide et al., Nat. Immunol. 3, 836 (2002).
18. D. Wang et al., Mol. Cell. Biol. 24, 164 (2004).
19. V. Hurez, R. Dzialo-Hatton, J. Oliver, R. J. Matthews,
C. T. Weaver, BMC Immunol. 3, 4 (2002).
20. S. D. Dimitratos, D. F. Woods, D. G. Stathakis, P. J. Bryant,
Bioessays 21, 912 (1999).
21. K. Heuer et al., J. Mol. Biol. 361, 94 (2006).
22. K. Heuer, A. Arbuzova, H. Strauss, M. Kofler, C. Freund,
J. Mol. Biol. 348, 1025 (2005).
23. K. Heuer, M. Kofler, G. Langdon, K. Thiemke, C. Freund,
Structure 12, 603 (2004).
24. J. N. Wu et al., J. Immunol. 176, 6681 (2006).
25. A. W. McGee et al., Mol. Cell 8, 1291 (2001).
26. G. A. Tavares, E. H. Panepucci, A. T. Brunger, Mol. Cell 8,
27. S. Kliche et al., Mol. Cell. Biol. 26, 7130 (2006).
Fig. 4. The region of ADAP con-
taining the N-terminal helical
SH3 domain and an E/K-rich re-
gion is critical for ADAP-CARMA1
association and TCR-dependent
activation of NF-kB, but is dis-
pensable for ADAP-dependent
regulation of antigen-dependent
conjugate formation. (A) Dia-
gram of the HA-tagged ADAP
truncation and deletion mutants
used in this study. Numbers in-
dicate amino acid position in
mouse ADAP. Asterisks indicate
Tyr residues (amino acids 547/
549, 584, 615, and 687) im-
plicated in ADAP binding to the
SLP-76 adapter protein. (B)
Jurkat T cells were transiently
transfected with the indicated
HA-ADAP constructs and then
stimulated with PMA before im-
munoprecipitation with BCL-10
mAb, followed by Western blot-
ting with antibodies specific
for the indicated proteins. (C)
T cells isolated from ADAP+/−
and ADAP−/−hCAR transgenic
mice were transduced with con-
trol adenovirus encoding Thy1.1
(ctrl) or adenovirus encoding
wild-type ADAP and Thy1.1
(ADAPwt). Cells were either un-
stimulated or stimulated with
PMA or TNF-a before analysis
of NF-kB p65 nuclear trans-
location as in Fig. 2. (D and E)
T cells isolated from ADAP+/+
and ADAP−/−hCAR transgenic
mice were transduced as in (C)
with either a control adenovirus
or adenovirus encoding the in-
dicated ADAP constructs. Cells
were either unstimulated or
stimulated with antibodies to CD3 and CD28 before analysis of NF-kB p65
nuclear translocation. Graphs show the average increase (±SD) in p65 nuclear
translocation in stimulated relative to unstimulated T cells for three [(C) and
(E)] or two (D) independent experiments. (F) T cells isolated from ADAP+/+
and ADAP−/−DO11.10/hCAR transgenic mice were transduced as in (E) with
control adenovirus or the indicated ADAP constructs. T cells were then
analyzed by flow cytometry for their ability to form conjugates with unpulsed
(unstim.) or ovalbumin-pulsed (OVA) splenocytes.
VOL 316 4 MAY 2007
28. H. Wang et al., J. Exp. Med. 200, 1063 (2004).
29. H. Wang et al., Nat. Immunol. 4, 366 (2003).
30. T. Egawa et al., Curr. Biol. 13, 1252 (2003).
31. H. Hara et al., J. Exp. Med. 200, 1167 (2004).
32. We thank T. George, M. Schwartz, G. Veltri, K. Pavlovich,
H.-D. Nguyen, L. Nacusi, K.-S. Tudor, and J. Hanson
Ostrander for technical support; C. Weaver for hCAR
transgenic mice; and M. Jenkins, J. DeGregori,
G. Koretzky, S. Jameson, and P. Holman for reagents
and advice. Supported by NIH grants R01AI038474 (Y.S.)
and R01AI056016 (E.J.P.), the Arthritis Foundation
(E.J.P.), NIH grant T32DE007288 (B.J.B.), American
Heart Association grant 0415400Z (K.L.M.), and NIH
grant F32AI063793 (J.J.M.). Y.S. is supported in part by
the Harry Kay Chair in Biomedical Research at the
University of Minnesota.
Supporting Online Material
Materials and Methods
Figs. S1 to S10
22 November 2006; accepted 26 March 2007
Specialized Inhibitory Synaptic
Actions Between Nearby Neocortical
Ming Ren, Yumiko Yoshimura, Naoki Takada, Shoko Horibe, Yukio Komatsu*
We found that, in the mouse visual cortex, action potentials generated in a single layer-2/3
pyramidal (excitatory) neuron can reliably evoke large, constant-latency inhibitory postsynaptic
currents in other nearby pyramidal cells. This effect is mediated by axo-axonic ionotropic glutamate
receptor–mediated excitation of the nerve terminals of inhibitory interneurons, which connect to
the target pyramidal cells. Therefore, individual cortical excitatory neurons can generate inhibition
independently from the somatic firing of inhibitory interneurons.
action potentials propagating through the axonal
arbor to axon terminals, at which signals are
transmitted to postsynaptic neurons. Action
potential–dependent transmitter release from axon
terminals is modulated by ionotropic glutamate
and g-aminobutyric acid (GABA) receptors
that are present, either synaptically or extrasyn-
aptically, on the axon terminals (1, 2).
We used dual whole-cell recording under mi-
croscopic observation to study synaptic connec-
tions from pyramidal and nonpyramidal neurons
of the mouse visual cortex (3). Single action
potentials in a pyramidal neuron could produce
inhibitory postsynaptic current (IPSC)–like out-
ward currents in another pyramidal neuron held at
the reversal potential (0 mV) of excitatory post-
were evoked by individual action potentials with
relatively constant latencies that were comparable
to those seen in monosynaptic connections (Fig.
1A). The responses were abolished by bath appli-
cation of the GABA type A (GABAA) receptor
antagonist bicuculline methiodide (BMI) (20 mM)
and after reversal could then be abolished again
by application of the non–N-methyl-D-aspartate
(NMDA) glutamate receptor antagonist 2,3-dioxo-
sulphonamide (NBQX) (10 mM, n = 6 neuron
pairs), indicating that they were polysynaptic
IPSCs (Fig. 1B).
n the mammalian brain, neurons integrate
synaptic inputs onto their somatodendritic
domains, which control the generation of
To further characterize these interpyramidal
IPSCs (ip IPSCs), we compared them with two
kinds of monosynaptic currents: unitary EPSCs
(uEPSCs) recorded from pyramidal neuron pairs
at the reversal potential (–70 mV) of IPSCs
(Fig. 1C) and unitary IPSCs (uIPSCs) from non-
In pyramidal neuron pairs, the probability of de-
tecting an ip IPSC [28%; 31 out of 110 (31/110)]
was slightly higher than that for the detection of
an EPSC (22%; 24/110). NBQX blocked ip
IPSCs in all of the tested pairs (n = 27 ). Recip-
rocal interpyramidal inhibitory connections were
never observed. Six pairs had both inhibitory and
excitatory connections. The direction was the
same for three pairs and opposite for three pairs.
In these pyramidal neuron pairs, patch pipettes
containing a Cs+-based internal solution were
used for recording from both pre- and postsyn-
aptic neurons. Similar ip IPSCs were also
recorded with a K+-based internal solution (fig.
S1). Recordings from pairs involving an inhibi-
tory neuron and a pyramidal neuron had a
detection probability for uIPSCs of 32% (19/60),
which was slightly higher than that for ip IPSCs.
The amplitudes of ip IPSCs were signifi-
cantly larger (P < 0.01) than those of uIPSCs
(Fig. 1E), and their time course was similar to
that of uIPSCs (Fig. 1, A and D, and fig. S2).
Although the average latency of ip IPSCs was
significantly (P < 0.02) longer than that of either
uIPSCs or uEPSCs, it was distributed in a wide
range that included latency values for the two
monosynaptic connections (Fig. 1F). If ip IPSCs
resulted from conventional polysynaptic activa-
tion involving action-potential generation at the
somata of inhibitory neurons, the expected vari-
ation in latency for each pair should be far larger
than that in monosynaptic connections. How-
ever, the coefficient of variation of their latency
was indistinguishable (P > 0.2) from those for
either uEPSCs or uIPSCs (Fig. 1G), suggesting
that they were unlikely to be mediated by the gen-
eration of somatic action potentials in inhibitory
failure rate of ip IPSCs was not significantly dif-
ferent (P > 0.1) from that of uIPSCs or uEPSCs
(fig. S3). This interpretation is also supported by
the observation that unitary excitatory inputs
alone induce only small postsynaptic responses
that are subthreshold for action-potential genera-
tion in inhibitory interneurons (4–7). Thus, we
hypothesized that ip IPSCs are generated by
direct excitation of the presynaptic terminals of
inhibitory neurons, which in turn connect to the
target pyramidal neuron (Fig. 2A). This mecha-
nism implies that the axo-axonic synaptic trans-
mission must be strong enough to release GABA
immediately from the inhibitory terminals. If this
synaptic transmission is very strong, extraordi-
narily quick depolarization would occur at the
terminals because of their small volume and lack
of strong filtering effects on input signals seen in
dendrites. This may, at least in part, explain the
short latency of ip IPSCs, together with the ab-
sence of conduction time in interneurons. We
tested this hypothesis, as described below.
If such excitatory axo-axonic synapses are
recorded from pyramidal cells in the presence of
er, may be affected by glutamatergic agents. Bath
0.02) increased the frequency of mIPSCs without
(Fig. 2, B, D, and E). Similar facilitative effects
were produced by the selective activation of
AMPA receptors with AMPA (1 mM) and kainate
receptors with (RS)-2-amino-3-(3-hydroxy-5-terf-
butylisoxazol-4-yl) propanoic acid (ATPA) (1 mM)
(8) or a low dose (200 nM) of domoic acid (9),
suggesting that both AMPA and kainate receptors
contribute to the facilitation of mIPSC frequency
(Fig. 2, D and E). We confirmed this supposition
(fig. S4). The effect of these receptors may be
mediated by the depolarization of nerve terminals,
because the facilitation of mIPSC frequency was
not found in the presence of Co2+, which blocks
voltage-gated Ca2+channels, and because the
metabotropic action of kainate receptors was not
involved in this process (fig. S5).
The application of NBQX significantly (P <
0.02) reduced the frequency of mIPSCs without
any significant (P > 0.9) changes in their ampli-
Department of Neuroscience, Research Institute of Environ-
mental Medicine, Nagoya University, Nagoya 464-8601, Japan.
*To whom correspondence should be addressed. E-mail:
4 MAY 2007VOL 316
DOI: 10.1126/science.1137895 Download full-text
, 754 (2007);
et al. Ricardo B. Medeiros
Adapter Proteins ADAP and CARMA1
B Activation in T Cells via Association of the
Regulation of NF-
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