1
Published in Brain Research 1339 (2010) 26 - 40
DOI information: 10.1016/j.brainres.2010.03.104
Research Report

Muscarinic receptor type 1 (M1) stimulation, probably through KCNQ/Kv7
channel closure, increases spontaneous GABA release at thedendrodendritic synapse in the mouse accessory olfactory bulb**#

Yoshito Takahashi , and Hideto Kaba

* Department of Physiology, Kochi Medical School, Nankoku, Kochi 783-8505, Japan# Division of Adaptation Development, Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki444-8585, Japan
Address correspondence to: Yoshito Takahashi, Department of Physiology, Kochi Medical School, Nankoku, Kochi 783-8505, Japan; Telephone
and FAX: +81-88-880-2307; E-mail: yuutarot@yahoo.co.jp
Number of text pages (including figure pages): 68
Number of figures: 8
With supplementary text (16 pages) and a supplementary figure
Abstract
Cholinergic modulation of spontaneous GABAergic currents (mIPSC) was studied using whole-cell patch methods in mouse accessory olfactory
bulb slices. Carbachol (above 100 !M) administration produced an increase in the mIPSC frequency in mitral cells, but did not affect the
responses of mitral cells to GABA. The carbachol effect persisted in the presence of combined ionotropic and metabotropic glutamatergic
receptor antagonists. The carbachol effect was reduced by the muscarinic receptor type-1 and -4 (M1, M4) antagonist pirenzepine (10 !M), but
not by the M2 and M4 antagonist himbacine (10 !M). The KCNQ/Kv7 potassium channel openers retigabine (80 !M) and diclofenac (300 !M)
blocked the carbachol action, while the KCNQ potassium channel blocker XE-911 (20 !M) increased the mIPSC frequency. XE-911’s action
persisted in the presence of glutamate receptor blockers. In the presence of carbachol, mIPSCs were abolished by Ni (200 !M), while being
insensitive to the calcium channel blocker nimodipine (30 !M), suggesting a role for R-type calcium channels in the GABA release. These
results suggest that carbachol closed KCNQ channels by stimulating M1 receptors on granule cell dendrites, and the resulting depolarized andunstable membrane promoted calcium influx, thus increasing the GABA release. The possible role of acetylcholine in facilitating formation of apheromone memory in mice is also discussed.
Classification Terms
Section: Neurophysiology
Key words: M-current, Pheromone, mIPSC, Mitral cell, Granule cellAbbreviations
AC, adenylate cyclase; ACh, acetylcholine; AOB, accessory olfactory bulb; AP3, DL-2-amino-3-phosphonopropionic acid; BAPTA,
1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; D-AP5,
D-2-amino-5-phosphonovaleric acid; CNS, central nervous system; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; DMSO,
dimethyl sulfoxide; IP3, inositol 1,4,5-trisphosphate; K-S test, Kolmogorov-Smirnov test; mIPSC, miniature inhibitory post-synaptic current; PLC,
phospholipase C; MCPG, (RS)-!-methyl-4-carboxyphenylglycine; OB, olfactory bulb; TEA, tetra-ethyl ammonium; TTX, tetrodotoxin
Introduction
The accessory olfactory bulb (AOB) is the first central nervous system(CNS) relay for processing pheromonal sensory information (Luo et al.,2003). Although AOB is not present in humans (Meisami et al., 1998), it is
an attractive region for study because of its simple structure, consisting of
only three types of neurones (Mori, 1987) and its implicated functions suchas information integration (Xu et al., 2005; Hendrickson et al., 2008) or
memory storage (Kaba et al., 1994; Matsuoka et al., 2004; Binns andBrennan, 2005).
The mitral cell, the relay and output neurone in the AOB, has
several long dendrites that receive sensory input. Along the length of thedendrites, numerous dendrodendritic reciprocal synapses are made with thedendrites of interneurones (granule cells). Glutamate is released from themitral cell, while GABA is released from the granule cell (Jia et al., 1999;
Taniguchi and Kaba, 2001). Spontaneous release of GABA from granulecells can be recorded as chloride currents in mitral cells using whole-cellrecording; neuromodulators can increase the spontaneous mIPSC frequency
via stimulation of noradrenergic !1 receptors (Araneda and Firestein, 2006)
or mGluR1 metabotropic glutamate receptors (Castro et al., 2007).
The neuromodulator acetylcholine (ACh) has been
implicated in attention and memory formation (Furey et al., 2000).
Anatomical studies in rodents suggest that cholinergic centrifugal fibres
terminate in the AOB (Carson and Burd, 1980; Macrides et al., 1981).
These most likely originate from basal forebrain magnocellular neurones in
the nucleus of the horizontal limb of the diagonal band (HDB; Macrides etal., 1981) or hypothalamic neurones in the bed nucleus of stria terminalis(BNST; de Olmos et al., 1978). These two regions are in close proximity.
Further, intrinsic cholinergic neurones, though rare, are found in the granulecell layer (Ojima et al., 1988) of the AOB. Muscarinic receptor mRNAs are
expressed in the AOB (strong M1 and M4; weak M3: Buckley et al., 1988).
Confirmed actions of ACh in the AOB, however, have not been
demonstrated. Though cholinergic action increases spontaneous mIPSCs in
the olfactory bulb (Castillo et al., 1999; Ghatpande et al., 2006), this has notbeen shown in the AOB. As the effects of some cholinergic receptors arelinked to the phospholipase C (PLC) pathway via the Gq11G-protein (seeCaulfield and Birdsall, 1998 for review), we hypothesized that cholinergic
stimulation has actions on mIPSCs similar to mGluR1 and noradrenergic !1
receptors stimulations that are also linked to Gq11G-protein. The current
work tests this hypothesis and examines the mechanisms that underlie
increases in spontaneous mIPSC frequency occurring via cholinergic
stimulation in the AOB.
Results
Carbachol administration increased the frequency of
spontaneous mIPSCs in mitral cells
Spontaneous inward currents (ranging from 0 to 3 Hz in frequency, mean ofcells employed for analysis, n = 30, 0.8 ± 0.8 [SD] Hz) were observed inwhole-cell recordings made from mitral cells. Cells with a very low
frequency of spontaneous currents (fewer than one per 30 s) were notincluded in the analysis. When the holding potential was changed from –70
mV to +30 mV, the current reversed polarity around 0 mV, which is the2chloride equilibrium potential under the following recording conditions:
extracellular Cl: 137 mM, intracellular Cl: 162 mM, 58log(137/162) = -4.39
mV (Fig. 1A). Bicuculline methiodide (20 !M), a GABA A receptor blocker,
nearly abolished the spontaneous currents (n = 4, Fig. 1B) completely.
These findings suggest that the spontaneous currents are mediated by the
opening of GABAA chloride channels. Thus, they are the so-called mIPSCs(GABA mediated currents recorded in the presence of TTX, though they
were not miniature; see below).
Carbachol (200 !M) application produced a marked increase inspontaneous mIPSC frequency. Eleven out of 21 cells had at least one time
window (see Experimental procedures) with significant frequency increase
by Kolmogorov-Smirnov test of inter-event interval cumulative plots (Fig.
1Ca, c). However in some carbachol applied cells (6 out of 21 cells), thefrequency did not show any increase or even showed a run down (comparedwith 7 out of 9 cells in control vehicle applied cells). Bicuculline abolishedthe spontaneous currents (10 out of 10 cells), suggesting that the
spontaneous currents increasing in frequency were also GABAergic andwere not contaminated by others such as glutamatergic currents. The extent
of increase and the latency of response varied among cells (Fig.1Ca). The
responding cells tended to show long-lasting responses. Without any drug
application, the mIPSC frequency tended to increase gradually in the first 5
min after the recording patch formation and run down slowly thereafter (Fig.
1Cb). To avoid this run-down, the carbachol applications and the datacollection for analysis were done in the most stable phase, which occurred
between 10 and 30 min after the patch formation.
The average baseline current frequency of recorded cells before
any drug application was 0.74 ± 0.69 Hz in the carbachol-applied group,and 1.04 ± 0.95 Hz in the vehicle applied control group; these were notsignificantly different (two tailed t-test P = 0.23). The average current
frequencies following carbachol or vehicle application were (in Hz ± SD):
carbachol group 0.67 ± 0.70 (0-3 min after), 1.00 ± 1.07 (3-6 min), 1.07
±1.35 (6-9 min); control group 0.70 ± 0.74 (0-3 min), 0.57 ± 0.56 (3-6 min),
0.48 ± 0.48 (6-9 min).
The distribution of current frequency change values was skewed
with a subgroup of non-responsive cells and was not Gaussian (skewness,
2.54, Fig. 1D). Therefore, the nonparametric Mann-Whitney U-test wasused to detect significance (see Experimental procedures). This method foranalysis demonstrated that carbachol (200 !M) significantly increased themIPSC frequency compared with vehicle application (carbachol, n = 21,
control, n = 9; frequencies increased by 0.4 ! 1.1 vs. -0.5 ! 0.5 Hz, P <0.0001, U-test; Fig 1E). The 200 !M concentration seemed not to saturate
the carbachol action because a higher concentration (up to 1 mM) in later
experiments resulted in fewer unresponsive cells and reduced latencies (9out of 11 cells with 1mM application had at least one time window with asignificant increase in frequency).
Though rarely, gigantic currents were observed (Fig 1F). Less
than 3% of currents in any recording were giant currents with both
amplitude larger than 100 pA and width greater than 500 ms. The giants
appeared independently of carbachol applications (carbachol n = 21, 0.6 ±
0.8 (SD) % of the total events; control n = 9, 1.1 ± 1.5 (SD) %: t-test p =0.39). They were probably caused by synchronous GABA release, but this
could not be tested because their generations were rare and unpredicted.
Carbachol also increased the current amplitudes in some cells
The mean current amplitude in each base time window was 108 ± 46 pA in
the carbachol group (n = 21), and 98 ± 22 pA in the control group (n = 9),
and these values were not significantly different (P = 0.48, t-test). The
current amplitude change ratios (amplitude mean value in any time
windows/amplitude mean value in base time window) were: carbacholgroup (n = 21), 1.6 ± 2.6 (0-3 min), 1.6 ± 2.6 (3-6 min), 1.7 ± 3.0 (6-9 min);control group (n = 9), 1.1 ± 0.3 (0-3 min), 0.9 ± 0.2 (3-6 min), 1.0 ± 0.2(6-9 min). Though large amplitude increases were occasionally seen (Fig.
2A), t-tests did not show any significant differences (carbachol n = 21,
control n = 9; 0-3 min, P = 0.32; 3-6 min, P = 0.30; 6-9 min, P = 0.26).
Also U-tests, by pooling the amplitude change ratios in each group as in thefrequency analysis, did not show any significance (carbachol, n = 21,
control, n = 9; amplitude change ratios 1.6 ! 2.7 vs. 1 ! 0.2, P = 0.60,
U-test).
Cumulative plot analysis of mIPSC amplitudes was done on
selected cells whose recordings were the most stable (carbachol applicationn = 10, control n = 6). Carbachol group cells were those with good (morethan two time windows with increased current frequency compared with thebase time window) response to carbachol. For each cell, four curves wereplotted for the time windows of base, 0-3, 3-6, and 6-9 min. In each cell,
curve shifts in respect to the base time window were diagnosed by the K-S
test (P < 0.05, two tailed) as: leftward significant, rightward significant with
P < 0.05, or no significant shift (leftward significant shift indicates adecrease in current amplitude while rightward significant shift indicates anincrease in current amplitude). In the ten cells that had carbachol
application, a total of 30 curves were assessed for a curve shift; 14 curves
showed a leftward shift, 5 curves showed a rightward shift, 1 curve showeda crossing (Fig. 2B) with both significant left and right-ward shifts, and 10
curves did not show any significant shift (Fig. 2B). In the 6 control cells, a
total of 18 curves were assessed for the shift; 7 curves showed a leftward
shift and the remaining 11 curves did not show a significant shift (Fig. 2B).
Of interest is the observation that curves for both the carbachol and control
cells showed a similar frequency of leftward shift, while the rightward shift
was only observed in the carbachol-exposed cells (4 cells out of 10
carbachol-exposed cells, one curve each from 3 cells and 2 curves from 1cell). The leftward shifts (decreases in mIPSC amplitude) werecarbachol-independent, as they were observed in both carbachol and control
cells with similar frequency (47% vs. 39%).
We considered two mechanisms to explain the increase incurrent amplitude resulting from carbachol administration. The firstmechanism considered was the selective action of carbachol on
dendrodendritic synapses near the recording site resulting in increases inlarge currents that were not reduced in amplitude by electronic attenuation.
This is possible because more cholinergic fibres are seen in the mitral cellbody side than in the cell’s dendritic tip side (Carson and Burd, 1980). The
second mechanism consists of a quantal increase in the GABA released
affecting postsynaptic currents. This increase may be achieved byincreasing synaptic vesicle content or by increasing the number of vesicles
released for a mIPSC. Analysis of current shapes suggested that a single
synapse could handle multiple levels of transmission (see below). It is also
possible that greatly increased presynaptic calcium works on sites otherthan the known release triggers to increase the pool of readily releasable
vesicles (Sakaba, 2008). However, frequency change value to amplitudechange ratio plots did not show a correlation (Fig. 2C), suggesting
amplitude and frequency changes are independent processes. Thus,
amplitude changes are probably dependent on the current profiles of a
subpopulation of granule cells that are activated by carbachol, but notdependent on presynaptic calcium availability.
Carbachol administration did not change the sensitivity of
mitral cells to GABA
The rise in the amplitude of mIPSCs by carbachol administration posed thepossibilities that carbachol either acted at presynaptic sites (on granulecells) to increase quantal GABA release, or at post synaptic sites (on mitralcells) to increase their sensitivity to the released GABA. To clarify thismatter, we next tested whether or not carbachol application increased the
response of mitral cell to puff-applied GABA (Fig. 3A). The amplitude
increase ratios (see Methods) for the carbachol group (n = 7) were 1.08 ±0.17 (mean ± SD) at 0-3 min, 1.10 ± 0.25 at 3-6 min, and 1.16 ± 0.45 at 6-9
min, whereas the ratios for the control group (n = 6) were 0.85 ± 0.34 at 0-3
min, 1.09 ± 0.32 at 3-6 min, and 0.96 ± 0.71 at 6-9 min. No significant
differences were seen in the ratios of any time windows between the
carbachol and control groups (t-test: 0-3 min, P = 0.67; 3-6 min, P = 0.57;
6-9 min, P = 0.81; Fig. 3B). Non-parametric analysis of the puff current
amplitude, using the same method as the frequency analysis (replacing
frequency change values with the amplitude change ratio values) also failed
to show a significant rise by carbachol application compared withvehicle-only application (1 mM carbachol, n = 7, control, n = 6; amplitudechange ratios 1.1 ! 0.3 vs. 1.0 ! 0.5, P = 0.37, U-test), while thebackground frequency (see Methods) of mIPSCs showed a significant
increase in the carbachol group compared with the vehicle group (1 mM
carbachol, n = 7, control, n = 6; frequencies increased by 2.4 ! 2.1 vs. 0.5! 0.6 Hz, P = 0.001, U-test). In some cells, increases in both thefrequency and amplitude of mIPSCs were robust even without an increasein the amplitude of the GABA puff current (Fig. 3C). We can thus conclude
that the influence of carbachol upon release sites on granule cells and notupon the sensitivity of mitral cells to GABA underlies the observed
increases in spontaneous mIPSC frequency in mitral cells.
The carbachol action was mediated by M1 receptors
Scopolamine (100 !M), a muscarinic antagonist, blocked the action of 1mM carbachol (scopolamine, n = 7, control, n = 7; frequencies increased by-0.3 ! 0.6 vs. 0.5 ! 0.6 Hz, P = 0.001, U-test; Fig. 4A). Application of
pilocarpine (300 !M), a muscarinic agonist, resulted in increases in themIPSC frequency (pilocarpine, n = 4, control, n = 5; frequencies increasedby 3.9 ! 3.5 vs. -0.1 ! 1.2 Hz, P = 0.0004, U-test; Fig. 4A). These
results demonstrate the carbachol action to be muscarinic.
We next tested which muscarinic receptor subtypes (M1~M5)
were involved. Pirenzepine at 10 !M (an antagonist for M1 and M4)
blocked the action of 100 !M carbachol (pirenzepine, n = 7, control, n = 6;
frequencies increased by -0.4 ! 0.7 vs. 0.8 ! 1.5 Hz, P = 0.001, U-test;
Fig. 4A).

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Himbacine, an antagonist for M2 and M4 receptors, could not
block the 100 !M carbachol action (himbacine 1 !M, n = 3, frequencies
increased by 0.0 ± 1.0 Hz; 10 !M n = 1, 1.0 ± 0.5 Hz; Fig. 4B). The M3
antagonist, 4-DAMP, also could not inhibit the 100 !M carbachol action(4-DAMP 30 nM, n = 2, frequencies increased by 1.1 ± 1.4 Hz; 300 nM, n= 2, 0.6 ± 0.9 Hz; Fig. 4B).
The demonstrated sensitivity to pirenzepine suggests M1 or M4
as likely candidates, while insensitivity to himbacine or 4-DAMP suggests
M2, M3, and M4 as unlikely. Taken together, these response profiles tomuscarinic antagonists suggest that the receptor subtype involved in the
carbachol action was M1.
Carbachol acted on granule cells and possibly also on mitralcells to increase the mIPSC frequency
Two possibilities were considered to explain the mechanisms of carbachol’s
actions upon the mIPSCs. The first possibility is that carbachol may act
directly on cholinergic receptors on granule cells to increase GABA release.
The second is that carbachol may act upon mitral cell receptors to increase
glutamate release, which would then stimulate granule cell GABA release(Fig. 5A). The third is that both sites are involved (as in the olfactory bulb;
Ghatpande et al., 2009).
We tested the action of carbachol in the presence ofglutamatergic blockers. Glutamatergic actions can be mediated by either
ionotropic or metabotropic glutamate receptors. Among the metabotropic
glutamate receptors, mGluR1 alone is responsible for increases inspontaneous GABA release from granule cells (Castro et al., 2007). Thus,
we applied carbachol together in the presence of antagonists of ionotropic
glutamate receptors (kynurenic acid, CNQX and D-AP5) and mGluR1
receptors (AP3 and MCPG). Carbachol (1 mM) application, in the presenceof kynurenic acid (5 mM) and AP3 (200 !M), still caused an trend ofincrease in the spontaneous mitral cell current frequency (carbachol, n = 8,
control, n = 5; frequencies increased by 0.9 ! 2.4 vs. 0.1 ! 1 Hz, P =0.06, U-test; Fig. 5B). On the other hand, the carbachol’s action was not
significantly reduced by a mixture of glutamatergic blockers (5 mM
kynurenic acid, 30 !M CNQX, 50 !M D-AP5, and 500 !M MCPG, n = 8,
vehicle, n = 6; direct bath application of 300 !M carbachol in both groups,
frequencies increased by -0.1 ! 0.6 vs. 0.3 ! 1.2 Hz, P = 0.83, U-test;
Fig. 5B). Although the blockers possibly reduced the carbachol effect,apparent mIPSC frequency increases were observed in some cells in thepresence of the glutamatergic blockers ( Fig. 5B).
We considered a role of mitral cells because theirdepolarization to +30 mV increased the mIPSC frequency (Fig. 5C, left),and application of glutamic acid, the transmitter of mitral cell, also
increased the current frequency (Fig. 5D). However, in the presence of themixed glutamatergic blockers, neither the +30 mV mitral cell depolarization(in the presence of 5 mM kynurenic acid + 200 !M AP3 + 50 !M CNQX +50 !M D-AP5, n = 5, Fig. 5C right);) nor the 100 !M glutamic acidapplication (in the presence of 5 mM kynurenic acid, 30 !M CNQX, 50 !M
D-AP5, and 500 !M MCPG, n = 3) increased the mIPSC frequency. Thesesuggest that mitral to granule cell transmission was successfully blocked by
the mixed glutamatergic blockers. Furthermore, loading of recorded mitralcells with intracellular BAPTA to block the cell’s glutamate release failed
to inhibit the effects of 100 !M carbachol (intracellular 2.5 ~ 5 mM
BAPTA, n = 3, frequency increased by 1.9 ! 0.8 Hz). However,glutamate released by other mitral cells may stimulate granule cells.
These results suggest that the carbachol directly acts on
granule cells to stimulate their GABA release. Possibly, as reported in theolfactory bulb (Ghatpande et al., 2009), carbachol acts on mitral cells to
stimulate their glutamate release, which then stimulate GABA release by
granule cells. A role of periglomerular cells (another GABA neuron in
AOB) was not ruled out but is less likely because cholinergic fibres are notseen in the periglomerular layer (Carson and Burd, 1980).
Blockade of L or T-type calcium channels or intracellular
calcium release did not reduce the carbachol’s action on themIPSC frequency
We tested the hypothesis that the carbachol action increased calcium
channel activity in granule cells, thereby increasing calcium influx and
GABA release. The involved calcium channel could be directly coupled, ornot, to vesicular release. Uncoupled calcium channels can increase thevesicle release probability by increasing the calcium concentration in therelease terminal. Directly coupled channels cannot be tested because, if a
blocker is applied, the spontaneous mIPSCs will disappear. We therefore
sought to inhibit calcium channels that are unlikely to be directly coupled tovesicle release.
The L-type calcium channel blocker nifedipine, and the T-type
blocker amiloride, or nickel chloride (100 !M) all failed to significantly
reduce carbachol’s action (30 !M nifedipine, n = 10, vehicle, n = 8; P =0.29; 500 !M amiloride, n = 4, vehicle n = 4; P = 0.75; 100 !M Ni, n =12, control, n = 5; P = 0.84, U-test; see Supplement for details). On theother hand, carbachol significantly increased the current frequency in thepresence of 20 !M mibefradil, a T-type blocker (direct bath application of
300 !M carbachol, n = 4, vehicle only application, n = 3; frequencies
increased by 0.2 ! 0.4 vs. -0.8 ! 1.2 Hz, P = 0.02, U-test).
In quest of the calcium source that enhanced the spontaneous
GABA release, we also tried blockade of intracellular calcium release. At
15 !M, 2-APB blocks inositol 1,4,5-trisphosphate (IP3) receptor-mediatedcalcium release from endoplasmic reticulum calcium stores (Maruyama etal., 1997). However, no significant differences in the carbachol effect wereshown in the presence of 2-APB at 15, 75 or 150 !M (>20 min) comparedwith its vehicle (15 !M 2-APB, n = 7, vehicle ethanol only, n = 4; 1 mM
carbachol application in both wings, frequencies increased by 0.7 ! 1.7 vs.
-0.3 ! 1.5 Hz, P = 0.10, U-test: see also results for 75 or 150 !M 2-APB
in Supplement).
However, the mIPSCs were abolished almost totally by 5 mM
nickel chloride (n = 6; Fig. 6C) even when the mIPSC frequency wasenhanced by carbachol. This suggests that extracellular calcium via calcium
channels other than L or T-type are necessary for generation of the mIPSCs
and of the increased currents resulting from carbachol administrations.
To ascertain the source of calcium responsible for spontaneous
GABA release, blockers of cell calcium entry were investigated (See also
Supplement). The mitral cell currents were almost completely abolished bynickel chloride at 200 !M, while cadmium or cobalt at the same
concentration was not effective (Fig. 6B, C). This suggests the involvementof R or T-type calcium channels, which are sensitive to nickel (Foehring etal., 2000). The L and T-type blockers, nifedipine (30 !M, n = 6) or
nimodipine (30 !M, n = 5; Fig. 6B, C) were not inhibitory upon themIPSCs. This implicate the R-type over the T-type calcium channel
because the R-type channel current is not responsive to block bynimodipine, while the T-type current is responsive (Randall and Tsien,1997). Furthermore, the highly selective T-type calcium channel blocker,(-)-(R) enantiomer efonidipine (at saturating concentration, above 10 !M, n= 6; Fig. 6B, C; Perez-Reyes et al., 2009; Furukawa et al., 2004) failed toshow any notable effect. Mixture of the P/Q-type calcium channel
antagonist, the peptide omega-agatoxin IVA (1 !M), and the N-typeinhibitory peptide omega-conotoxin GVIA (1 !M) had no notable influence
upon the mIPSCs (Fig. 6B). Our findings thus implicate the role for R-type
calcium channels in the generation of mIPSCs.
KCNQ channel antagonists increased the frequency of
spontaneous mIPSCs, while KCNQ channel openers inhibitedthe increase of mIPSC frequency by carbachol
The KCNQ (Kv7) channel is a potassium channel that is closed bymuscarinic action (Delmas and Brown, 2005). The potassium current
mediated via these channels is referred to as an M-current (frommuscarinic). Potassium channel closure excites the neurone by depolarizing
its membrane as well as by making the membrane more susceptible todepolarizing inputs. Barium (1 mM), which blocks KCNQ channels(Constanti et al., 1981), was bath applied and resulted in a marked increase
in mIPSC frequency (n = 10, Fig. 7A). Barium does enter through calcium
channels, but compared with calcium, has negligible actions to releasevesicles (Miledi, 1966). The marked effect of barium was also observed in
the presence of a mixture of glutamatergic receptor inhibitors (5 mM
kynurenic acid, 30 !M CNQX, 50 !M D-AP5, and 500 !M MCPG; n = 3),
which should inhibit all of the glutamatergic receptors that are known tostimulate GABA release from granule cells (Castro et al., 2007).
Furthermore, XE-991, a derivate of linodipine and a more potent andspecific antagonist for the KCNQ channel (Elmedyb et al., 2007), increased
the mIPSCs in the presence of the glutamatergic blockers (5 mM kynurenic
acid, 30 !M CNQX, 50 !M D-AP5, and 500 !M MCPG in perfusate; from10 to 50 !M XE-991, 7 out of 9 cells; Fig. 7A). This suggests that XE-991
closed the KCNQ channels on granule cells, depolarizing their membranes,
and that the glutamatergic drive from mitral cells was then unnecessary forthe increase in current frequency.
Retigabine (80 !M), a KCNQ channel opener (expected toparalyse closure) inhibited the carbachol action (retigabine, n = 6, vehicle
only, n = 5; frequencies increased by -0.1 ! 0.3 vs. 1.6 ! 2 Hz, P =0.03, U-test; Fig. 7B). This concentration of retigabine was expected not toshow an observable effect on calcium channels (Rundfeldt and Netzer,
2000) and is in the range that sufficiently blocks epileptic activity inhippocampus slices (Dost and Rundfeldt, 2000). Retigabine possibly
enhances GABA receptor sensitivity (Otto et al., 2002) and inhibits theexcitability of granule cells. However, in agreement with the retigabine
results, another KCNQ channel opener, diclofenac (Peretz et al., 2005) also
blocked carbachol’s action (300 !M diclofenac, n = 10, vehicle ethanol
only, n = 6; frequencies increased by 0.1 ! 0.5 vs. 0.7 ! 1 Hz, P = 0.02,

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U-test; Fig. 7B). These consistent results suggest that both agents worked
specifically via the opening of KCNQ channels.
KCNQ channel opening in neurones hyperpolarizes theirmembrane potential by 10 mV (Otto et al., 2002). To rule out the possibility
that hyperpolarization per se blocked carbachol’s action, we used low
potassium (1.13 mM instead of 2.5 mM standard) external solution. The
potassium equilibrium potential should be 20 mV lower (58 log 2.5/ [Kin] 58 log 1.13/ [Kin] = 20), thus granule cell will be hyperpolarized to thisextent with the low-potassium solution. However, the influence of
carbachol was apparent even under these conditions (n = 4, frequency
increased by 0.5 ± 0.6 Hz; Fig. 7B).
Taken together, these results suggest that KCNQ openers, by
their specific effect, inhibited carbachol’s actions. And thus suggests
carbachol’s action is mediated by closing KCNQ channels.
Analysis of the mIPSCs suggested that they are constructed bysynchronized multiple quantum currents
The shapes of the mIPSCs varied greatly. Some had multiple small
inward-current peaks, like a saw, while others had a notch when
up-shooting to the peak (Fig.8A). These shapes suggest that a summation ofsmaller events underlie the observed currents. Current amplitude or area
occurrence frequency histograms occasionally showed evenly spaced
multiple peaks and dips (Fig.8B). Using Asertine analysis (Fig.8C), thesewere judged to be quantally distributed. An explanation for this is thatsynchronized multiple small quantum currents were constructing greater
currents.
These results suggest that dendrodendritic synapses in the AOB
were not saturated by a single vesicle and can accommodate multiple levelsof transmission. This is in contrast to findings in other areas of the CNS
cortex (Silver et al., 2003).
Discussion
A role for the KCNQ channel in the enhancement of the mIPSC
by carbachol administration
An increase in the mIPSCs by barium or XE-991 suggested that KCNQ
channel was indeed operating at the synapse. As these actions wereobserved under the glutamatergic inhibitors, the granule cell is the likely
site of action. Inhibition of carbachol’s action by the KCNQ channel
openers retigabine or diclofenac suggested that carbachol acts via thedown-regulation of KCNQ channels. The down-regulation of KCNQ
channels increases transmitter release (Lechner et al., 2003; Martire et al.,
2004; Peretz et al., 2007; Zhang et al., 2009), and the link between
muscarinic stimulation and KCNQ channel down-regulation iswell-established (Delmas and Brown, 2005). It is therefore probable thatcarbachol down-regulates the activity of KCNQ channels in the granulecells and, through increased membrane depolarization, increases the open
probability of membrane calcium channels, resulting in an increase insynaptic vesicle release.
We examined the role of T-type calcium channels, which open
at relatively weak depolarization levels compared with other subtypes. In
the presence of mibefradil, however, the carbachol effect persisted, rulingout the T-type as the sole calcium channel involved in the enhancement of
mIPSCs. The granule cell depolarization thus probably activates a mixtureof calcium channel subtypes. The R-type channel is especially implicated,as the pharmacological profile (Fig. 6B, C) suggests a dominant role for thischannel in the current and as the R-type channel activates at relatively low
depolarization threshold (Soong et al., 1993) compared with other subtypesof high-voltage activated channels.
In this study, actions of carbachol on mitral cell excitability were
not studied. Thus, additional possibility that mIPSC was enhanced throughmitral cell excitation still remains.
Differences between the AOB and the olfactory bulb (OB)Our findings in the AOB are different from previous work in the OB, where
the carbachol–induced increase in spontaneous GABA currents was blocked
by the intracellular calcium store blocker thapsigargin, suggesting thatcalcium released from internal stores stimulated GABA release (Ghatpande
et al., 2006). In contrast, in the AOB, the intracellular calcium store blocker
2-APB did not inhibit the carbachol effect; instead, a different mechanism,
the closure of KCNQ channels by M1 stimulation, was implicated. The
difference in these studies possibly resulted from the ages of the animals.
Ghatpande et al. used young rats (within 3 w), while we used mice older
than 3 w. The expression of KCNQ3, a component of the neuronal KCNQ
channel, continues to increase in the brain as the animal matures (Tinel etal., 1998). It is thus possible that the KCNQ pathway replaces theintracellular calcium pathway as the animal matures. Our result in the AOB
was is in agreement with findings that the M1 ACh receptor is not linked to
intracellular calcium stores in sympathetic neurones (Delmas and Brown,2002). The AOB was also less sensitive to carbachol than the OB (50 !Mis sufficient for 300 !m slice in mouse OB; Castillo et al., 1999), and we
had to use carbachol concentrations higher than 100 !M to see a reliable
effect. This possibly results from fewer cholinergic fibres observed in the
AOB external plexiform layer (site of the dendrodendritic synapses)compared with the OB (Carson and Burd, 1980; Ojima et al., 1988), or
different G protein use downstream of M1 stimulation (Gudermann et al.,1997). These disparate findings constitute another line of evidence that the
AOB and OB are significantly different and are worth investigating
separately.
Acetylcholine may facilitate pheromonal memory formation
The AOB dendrodendritic synapse is implicated in the plasticity ofpheromonal memory (Brennan and Keverne, 1989; Kaba et al., 1994;
Matsuoka et al., 2004). Female mice learn to give up responding to a matedmale’s pheromone at a critical period just after the mating and thereafter
become insensitive to the pheromone (Leinders-Zufall et al., 2004).
Noradrenaline released during mating has been proposed to facilitate thislearning (Rosser and Keverne, 1985; Kaba and Keverne, 1988; Keverne andRiva, 1982). However, the learning is also possible innoradrenaline-deficient mice (Thomas and Palmiter, 1997). This implies theexistence of alternative candidates for the facilitation of the learning. The
carbachol effect in our study was similar to the actions of noradrenaline in
the AOB (Araneda and Firestein, 2006). It is possible that acetylcholine andnoradrenaline cooperate to facilitate learning in the AOB, as they do in thevisual cortex (Kasamatsu, 1991).
A hypothesis of how increases in the spontaneous release of
GABA facilitate synaptic plasticity
Given that GABA is widely accepted as the major inhibitory transmitter, it
is understandable that one could be puzzled in how increases in GABAergic
activity, which increase inhibition, could play a permissive role in plasticity.
However, as long as we are uncertain about the exact local membranepotentials and chloride concentration in the mitral cell dendrites, we cannot
determine whether the inputs from the granule cells are actually inhibitory,
for with lower membrane potential below the chloride equilibrium potential(58 log [Clex]/ [Clin ]) or with higher intracellular chloride, the action of
GABA is excitatory (Szabadics et al., 2006).
Assuming GABA to be inhibitory, we consider two
possibilities to explain its role in promoting plasticity. The first possibility
is that the inhibition by mIPSCs optimizes the sensory input by avoiding
saturation and promising fidelity, so that the properly-transmitted
high-frequency inputs are able to induce plasticity. The second possibility isthat the facilitation of the spontaneous GABA release depletes theready-releasable pool of GABA, and thus may desensitize granule cell
dendrites from secreting GABA in response to trains of action potentials in
the mitral cell dendrite, thus releasing the mitral cell from GABA
inhibition. In any case, further study is needed to determine the role of
spontaneous GABA currents in AOB plasticity.
In our experimental condition with chloride loaded mitral cells,
mIPSCs may depolarize mitral cells and cause their glutamate release,which may facilitate the mIPSCs in a feed forward manner. On the other
hand, mIPSCs should be more abundant in vivo without slicing. Thus ourstudy could not precisely define the extent of the mIPSCs enhanced byacetylcholine in vivo.
Conclusion
The current study suggests that muscarinic stimulation, through thedown-regulation of KCNQ channel activity, increases spontaneous GABA
release from granule cells. This mechanism could play a role in pheromone
memory formation. In a broader sense the AOB, because it has a simple
structure, is a CNS region that may facilitate the study of memory. Since
acetylcholine in the CNS is associated with memory disorders such as
Alzheimer’s disease, and the KCNQ channel in the CNS is associated withepileptic disorders such as benign familial neonatal convulsions (Biervert etal., 1998), understanding the precise mechanism underlying the synaptic
plasticity evident in our system may greatly increase both the understandingof, and the therapeutic options for, these debilitating diseases.
Experimental procedures
Ethical approval
All experiments were carried out in accordance with the Kochi Medical
School guidelines for care and use of laboratory animals. All efforts weremade to minimize animal suffering and to reduce the number of animals
used.
Slice preparation
Adult BALB/c mice (3 weeks to 3 months old) were sacrificed by cervical5
dislocation. Parasagittal AOB slices (275 !m thick) were made with a linear
tissue slicer (Pro7 Dosaka Japan) in ice-cold sucrose-replaced low calcium
saline (sucrose 220 mM replacing NaCl, CaCl2 reduced to 0.25 mM ofexternal solution, see below). The slices were incubated in a holding
chamber, gently bubbled with an O2 (95%) and CO2 (5%) gas mixture for atleast 40 min at room temperature.
Whole cell electrophysiological recording
Methods for whole cell patch recording are similar as described elsewhere(Takahashi, 1990). Briefly, slices in a recording chamber (3 ml volume)
were continuously perfused (2 ml/min during recordings) using a peristaltic
pump (Gilson MINIPULS3, Middleton, USA). In later experiments, agravity drip system with a slower perfusion rate was used. The perfusedRinger’s solution consisted of (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2 , 1.3
MgSO4 , 1 NaH2 PO4 , 26.2 NaHCO3 , and 11 glucose. The pH wasmaintained at 7.4 by saturation of the external solution with an O2 (95%)
and CO2 (5%) gas mixture at 300 Osm. In most experiments unless noted
otherwise, 10 mM tetra-ethyl ammonium (TEA) and 1 !M tetrodotoxin(TTX) were added to the external solution (“standard external solution”).
Recordings were performed at room temperature. In winter, experiments
were heat controlled to 26°C. Slices were viewed under an uprightmicroscope (DM-LFS Leica, Wetzlar, Germany) and a CCD camera(C2400; Hamamatsu Photonics, Hamamatsu, Japan) using a 40" water
immersion lens. Mitral cells were identified on the basis of their size (longaxis greater than 20 !m), polygonal morphology, and their location in the
middle and deep external plexiform layers. Patch pipettes with resistancesof 2 to 8 M# were made from borosilicate glass capillaries (1B150F-4;
WPI, Sarasota, FL, USA) using an electrode puller (P-97; Sutter
Instruments, Novato, CA, USA) and then heat-polished. Pipettes were filledwith an internal solution of (in mM): 140 CsCI, 10 HEPES, 5 EGTA, 3
ATP-Mg, and 0.5 GTP-Na (pH 7.4). Membrane currents were recorded in
the whole-cell configuration (holding potential= -70 mV). Whole cell
current measurements were made using a patch-clamp amplifier (EPC9;Heka, Lambrecht, Germany). The sampling frequencies were 100 Hz (inmost experiments) or 1 kHz (to study current trace morphology). The
filtering frequency was 3 kHz and Rseries was not compensated. Data
sampling and analysis were performed on a personal computer using Pulse(Heka), PowerLab/4SP (AD Instruments, Australia), Mini Analysis(Synaptosoft), Excel (Microsoft) and StatView (SAS institute, Cary, USA).
Chemicals
If not otherwise noted, chemicals were dissolved in water or, if hydrophobic,
in dimethyl sulfoxide (DMSO) at the highest concentration according to the
supplier’s data sheet, and stored in a -20°C freezer. Chemicals (stock
concentration, vehicle, and storage temperature in parenthesis) werepurchased from the following sources: Sigma (St. Louis, USA), carbachol(1 M or 100 mM in water, 4°C prepared daily), ATP-Mg (30 mM in water,
4°C, prepared daily), kynurenic acid (added directly to solution, sonicated),
TEA-Cl (1 M in water, 4°C), amiloride (directly into solution, sonicated),DL-2-Amino-3-phosphonopropionic acid (AP3; 100 mM in 1 N NaOHaq),
scopolamine (100 mM in water), bicuculline methiodide (10 mM in water,
4°C), nifedipine (30 mM in ethanol),
1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA; 25
mM, 50 mM CsOHaq), D-2-amino-5-phosphonovaleric acid (D-AP5; 50mM), mibefradil, himbacine (5 mM in ethanol),
6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 mM in DMSO), XE-991(100 mM in DMSO), glutamic acid (10 mM, 4°C), GABA (1 mM, 4°C),
4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP); Nakarai(Kyoto, Japan), TTX (100 !M in water), GTP Na (5 mM in water, 4°C,
prepared daily), pilocarpine (50 mM in DMSO); Toronto Research
Chemicals (North York, Canada), retigabine hydrochloride (100 or 50 mM
in water, -20°C); Wako (Osaka, Japan), D-AP5, diclofenac (100 mM in
ethanol); Tocris (Bristol, UK), D-AP5 (5 mM in water, 4°C),(RS)-!-methyl-4-carboxyphenylglycine (MCPG; 50 mM in 0.1N NaOH);
Peptide Institute (Osaka, Japan): omega-conotoxin-GVIA (0.5 mM),omega-agatoxin-IVA (0.1 mM). 2-aminoethoxydiphenylborane (2APB; 10mM in ethanol or 100 mM in DMSO, -20°C) was kindly provided by Dr.
Mikoshiba (Brain Science Institute, RIKEN, Japan). Efonidipine and its
enantiomers were kindly provided by Nissan Chemical Industries, Ltd(Tokyo, Japan).
For the preparation of carbachol for application to the chamber,a volume of the 1 M stock was applied to the perfusing solution (before
entering the line tube) to create the desired concentration. The time of
application was defined as the time at which the carbachol-containing
solution began to perfuse the recording chamber (approximately 2 min after
carbachol addition to the perfusing solution). With the chamber volume of 3
ml, and a perfusion rate of 2 ml/min, the concentration in the chamber was
estimated to be around 100 !M at 1 min after the start of 200!M carbachol

application.
A slow perfusion rate (0.2 ml/min) was used for later
experiments to minimize costly reagent use. In these experiments, carbachol
was directly applied into the chamber by a pipette. They are noted as ‘directbath application of carbachol’ in Results. A gravity-flow setup was used tomaintain the slow perfusion rate. The bath volume was estimated to be 2.5
ml (though it varied from 2 to 3 ml by perfusion and vacuuming conditions).
Carbachol concentrates of 7.5 !l (100 mM in water) were mixed with 30 !lof the standard solution (to enhance diffusion in the bath) and gently
bath-applied with a pipette, resulting in a bath carbachol concentration of
300 !M.
Study design and analysis
Most experimental manipulations were assigned either randomly, by
throwing dice, or alternating experimental versus control conditions.
To assess changes in the mIPSC frequency, four consecutive
time windows of three minutes each were introduced to each cell’srecording. These were defined as: base, 0-3 min, 3-6 min, 6-9 min.
Carbachol application began just after the base time window. In each cell,
frequency values (in Hz) from the 0-3, 3-6, 6-9min time windows weresubtracted by the frequency value of base time window, which were definedas frequency change values. The frequency change values from theexperimental cell groups and the control cell groups were each pooled (cellnumber [n] " 3 values for each group) and analysed by Mann-Whitney’s
U-test (U-test) for statistical difference (P < 0.05 was consideredsignificant). The values are expressed as mean ± SD.
We assessed three time windows after the carbachol
application to make the analysis more specific for the detection of carbachol
effects that tended to last for a long duration. Increases in the current
frequency can be caused by occasional transient bursts (Fig. 1Cb, upper)
unrelated to the carbachol effects. If only one time window after carbachol
was assessed to count the number of events, we would not be able todifferentiate between transient spontaneous bursts unrelated to carbachol
and a modest but long-lasting frequency increase in response to carbachol(Fig. 1Ca, middle). The introduction of three time windows to a cell enables
us to confine a transient burst to one time window and get only one value ofincreased frequency out of the cell’s three values. On the other hand, we
can reliably identify a long-lasting response that was specific to thecarbachol action, and get three values of increased frequency out of thecell’s three values. Thus, the introduction of three time windows raises the
specificity of the analysis.
Puff application of GABAGABA (30 !M) dissolved in the perfusing solution was puff applied (2-5psi, 50-500 ms at 30 s intervals) via a glass pipettes which was madesimilarly to that used for whole cell recording (Picospritzer II, General
Valve Corporation, Fairfield, NJ, USA: for electronic pulse generation;
SEN7203, Nihonkoden, Tokyo, Japan). Because mIPSCs often contaminate
puff current peak values, the mean values of the puff currents were assessed.
The puff current was calculated as follows: the averaged current trace of an
850 ms time window beginning 100 ms after the start of the puff, minus abaseline value consisting of the averaged current trace of an 850 ms time
window up to 50 ms before the start of puff (thus positive values are
obtained for the puff current, see Fig. 3Ab). For each cell, means of the puff
current amplitudes were calculated for the base, 0-3, 3-6, and 6-9 min time
windows (one puff at 30 s intervals resulted in 6 amplitude values per 3 mintime window). Ratios relative to the base values were calculated for the 0-3,
3-6, and 6-9 min time windows and these ratios were defined as amplitudechange ratios. The amplitude change ratio values for the carbachol-exposedand control groups were expressed as mean ± SD.
Assessment of peaks in the mIPSC amplitude distribution
We applied a method used by Edwards et al. (1990; Asertine’s analysis) todiagnose the occurrence of peaks in mIPSC amplitude histograms. The
autocorrelation function (ACF) of the amplitude histograms was calculatedusing the expression: ACF(j) = {$i H(i)H(i+j)}/($i H(i)H(i)), where H(i) is
the height of the ith bin which is centred at the amplitude value given by i "bin width. The ACFs were compared with a smoothed autocorrelation
function in which each point was replaced by a five-point rolling average.
The two functions were superimposed, and if the ACF showed at least twoconsecutive and equally spaced peaks and dips, the amplitudes werediagnosed as quantally distributed.
Acknowledgements
We thank Dr. T. Hori (Okinawa institute of Science and Technology) for
providing instructions on patch clamp and puff application methods; Dr. K.
Kato (Kochi Medical School) for advice on slice preparation and providing

6
D-AP5; Dr. GZ Huang (Northwestern Univ. Chicago. USA) for providing
instruction on slice preparation; Dr. M. Taniguchi (Kochi Medical School)
for assistance in setting up experimental apparatuses and animal care, andfor troubleshooting; Dr. K. Mikoshiba (Brain Science Institute, RIKEN) for
providing 2-APB, and Dr. A. Yamamoto (Nissan Chemical Industries, Ltd.)for providing efonidipine and enantiomers. This work was supported in partby research grants from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan and from Kochi University.
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7Figure Legends
FIG. 1. Carbachol increased the frequency of spontaneous mIPSCs. A,
Spontaneous currents reversed polarity around 0 mV, the chloride
equilibrium potential. B, Spontaneous currents were abolished within 5 minby bath application of the GABA A blocker, bicuculline (20 !M). Ca,
Examples of recordings with carbachol application. Uppermost trace:
typical response to carbachol. Middle: delayed response. Lower: a modest
response and rundown. Cb, Examples with vehicle application. Upper:transient spontaneous bursts at the final part of the recording. Middle: run
down. Lower: stable. The scale bar is for both Ca and Cb. The angled
arrows in Ca and Cb show the times at which carbachol or placebo
application started. Cc, An inter-event interval cumulative plots of Ca
uppermost trace. In this cell, the frequency increases in all three 3 min time
windows after carbachol application (3 curves are indistinguishable in theplot, left side curve) were significant compared with the frequency before
carbachol (base, right side curve) by Kolmogorov-Smirnov test. The
numbers in the parentheses are the mean frequencies of each time window.!
D, Distributions of mIPSC frequencies in 3 min time window before and in
3 min time window (from 3 to 6 min) after carbachol or vehicle application.
The distribution of mIPSC frequencies after carbachol application was
skewed and was not Gaussian. E, The U-test showed a significant increase
in mIPSC frequency in the carbachol application group compared with thevehicle application group. F, A giant spontaneous current (arrow) of
unidentified origin. Such currents were seen in the presence of TTX 1 !M.
They were rare, but were more frequently seen in thick slices (500 !m).
FIG. 2. Carbachol enhanced the amplitudes of the mIPSCs in some cells. A,
Amplitude change ratios after carbachol or vehicle applications (any time
window’s amplitude/base time window’s amplitude); b, base time window,
0-3, the time window from 0 to 3 min after the application, etc. B, Upper:
pie charts summarize the observed shifts in the amplitude cumulative plot
curves. For each cell (carbachol application, n = 10; control, n = 6), 3
curves for the 3 time windows were analysed. A rightward shift indicates anincrease in amplitude, while a leftward shift, a decrease. Note that therightward shifts are only observed in the carbachol-applied group. Lower:
example cell amplitude cumulative plots from each pie chart category(arrow) demonstrating the shifts summarized in the chart. C, Absence ofcorrelations among frequency change value and amplitude change ratio.
This implies the amplitude change is not dependent on presynaptic calcium
availability. Values from each time window were plotted as a point (n [cellnumber] " 3 [time windows from each cell] = 63 points), Correl: coefficientvalue.
FIG. 3. Carbachol did not change the mitral cell’s sensitivity to GABA. Aa,
GABA puffs onto a recorded mitral cell evoked currents that reversed
around 0 mV, indicating that the currents were carried by chloride. Ab, The
puff current value was calculated from the mean values in 850 ms time
windows before and after the puff. B, Summary of puff current amplitudes
before (base) and after carbachol or vehicle application (0-3, 3-6, 6-9 min).
No significant changes in amplitude were observed. The means ofamplitudes after the applications are shown by the ratios to the baseamplitudes. Error bars represent SDs. See Results for detailed values. C, An
example of a cell from the carbachol-applied group. The backgroundmIPSC frequency increased independently of increases in the puff current.
Arrows indicate the times of recordings (*1, *2) which are shown below.
FIG. 4. The carbachol effect was mediated by M1 receptors. A, Muscarinic
agonist pilocarpine increased the current frequency while muscarinic
antagonist scopolamine blocked the carbachol effect. Pirenzepine (M1 +M4 antagonist) blocked the carbachol effect. On the right side of the bar
pairs are P values by U-test. Sidebars are ± 1 SD ranges. See results forfurther details. B, The carbachol effect was apparent in the presence of
himbacine (M2 + M4 antagonist) or 4-DAMP (M3 antagonist).
FIG. 5. Carbachol could increase the mIPSC frequency without effect on
glutamate release from mitral cells. A, Schematic drawing of thedendrodendritic synapse in the accessory olfactory bulb. Glutamatergic and
GABAergic synapses reside in close proximity. When a mitral cell releases
glutamate, the granule cell depolarizes and releases GABA back onto themitral cell. B, Carbachol effect remained in the presence of mixtures ofionotropic and metabotropic glutamatergic receptor antagonists. On theright side of the bar pairs are P values by U-test. Sidebars are ± 1 SD
ranges. See results for further details. C, Cells frequently showed robust
increases in the mIPSC frequency when the holding potential was raised
from –70 mV to +30 mV (left). The recorded cell likely releases glutamate
as a result of depolarization and stimulates granule cells to release GABA
reciprocally. In the presence of mixed glutamatergic blockers, the frequency
increase by mitral cell depolarisation was never observed (right, with 5mM
kynurenic acid + 200 !M AP3 + 50 !M CNQX + 50 !M D-AP5). D, Bathapplication of glutamic acid (100 !M) caused a robust increase in themIPSC frequency. Kynurenic acid + AP3 blocked the action (not shown).
An arrow indicates the time of the direct bath application.
FIG. 6. A, Antagonists of L- or T-type calcium channels or intracellular
calcium had no significant effect on the carbachol’s action. B, Actions ofcalcium channel blockers on mIPSCs. Sensitivity to a low concentration of
Ni suggests the involvement of T or R-type channels. An insensitivity tonimodipine and to the highly specific T-type blocker (-)-efonidipine
excludes the involvement of T-type channels. P-, Q-, and N-type channels
were also excluded as the calcium source for the recorded events. Straightarrows indicate the time of direct bath application of the drug concentrateby a pipette. Abbreviations: Ni, nickel chloride; Cd, cadmium chloride; Co,
cobalt chloride; (-)Efo, R(-) enatiomer of efonidipine; Nimo, nimodipine;
AgaIV, omega-agatoxin-IVA; ConoVI, omega-conotoxin-GVIA. C,
Summary of the actions of calcium channel blockers (in !M) on mIPSC.
The profile of inhibition by calcium channel blockers suggests a dominantrole of R-type calcium channel underlying the mIPSC activity.
FIG. 7. A, Barium caused a robust increase in the mIPSC frequency. Thissuggests the involvement, in the AOB, of KCNQ channels which, whenclosed by barium, allow the depolarization of granule cells. The KCNQ
channel-specific antagonist XE-991 also increased the mIPSCs(glutamatergic blockers present). Barium or XE-991 increased mIPSCs
regardless of the presence or absence of glutamate receptor antagonists.!B,
KCNQ channel openers blocked the carbachol effect. On the right side of
the bar pairs are P values by U-test. Sidebars are ± 1 SD ranges. See results
for further details.
FIG. 8. Synchronized multiple quantum currents compose the mIPSCs. A,
Examples of notched currents, which indicate the summation of quantacurrents. B, Amplitude distribution showed some peaks at regular intervals.
C, Asertine analysis of B. The analysis reveals the amplitude peaks to be atregular intervals. A line represents the autocorrelation function (ACF) of
the simulated data shown in panel A. The curve of a dotted line represents
the ‘smoothed’ ACF, i.e. the curve created by averaging each point of theoriginal ACF and the two points on either side of it. Note that the original
ACF crosses the smoothed ACF at somewhat regular intervals.

Supplementary Figure Legend
FIGURE The profile of inhibition by calcium channel blockers. Straightarrows indicate the time of direct bath application of the drug concentrateby a pipette. A modest sensitivity of the currents to high-dose T-type
blockers, was observed probably due to their non-specific actions.
Insensitivity to D-AP5 excluded NMDA channels as the calcium source forthe recorded events. Abbreviations: Ami, amiloride; Mib, mibefradil; Nif,
nifedipine; (+/-)Efo, racemic mixture of efonidipine; Nimo, nimodipine;
AgaIV; omega-agatoxin-IVA; ConoVI, omega-conotoxin-GVIA; SNX,
SNX-482 (tarantula spider venom peptide, R-type selective antagonist).

8
Supplements
Supplementary Results
Unsuccessful attempts to block carbachol actions by calcium
channel blockers
The L-type calcium channel blocker nifedipine, and the T-type blocker
amiloride, or nickel chloride (100 !M) all failed to significantly reduce
carbachol’s action (30 !M nifedipine, n = 10, vehicle ethanol only, n = 8;1mM carbachol application in both groups, frequencies increased by 0.5 ±0.9 vs. 0.5 ± 1.5 Hz, P = 0.29, U-test) (500 !M amiloride, n = 4, vehicle
only, n = 4; 1mM carbachol application in both groups frequencies
increased by 0.2 ± 0.2 vs. 0.4 ± 0.6 Hz, P = 0.75, U-test) (100 !M Ni, n =12, control, n = 5; 200 !M carbachol application in both groups frequencies
increased by 0.0 ± 0.5 vs. 0.0 ± 0.8 Hz, P = 0.84, U-test).
Also, when the calcium channels were non-specifically
blocked (probably partially because mIPSC activity should disappear [thatwas not the case] if calcium channels were completely blocked), bylowering the calcium concentration (0.1 mM) and increasing magnesium(3.6 mM) in the external solution, robust increases in the mIPSC frequency
by 200 !M carbachol application were still observed (n = 12, uncontrolled,
frequency increased by -0.1 ± 0.5 Hz).
Actions of calcium channel blockers on mIPSCs (seeSupplementary figure)
The T-type channel blockers amiloride (1 mM) or mibefradil (30!M) had a modest effect, though the concentrations required for the effectwere higher than their T-type selective range (Suppl. figure; Randall andTsien, 1997; Soong et al., 1993; Viana et al., 1997). The L and T-type
blockers, nifedipine (30 !M, n = 6) or nimodipine (30 !M, n = 5), were notinhibitory upon the mIPSCs while the efonidipine racemic mixture (30 !M,saturated, n = 4; Perez-Reyes et al., 2009; Furukawa et al., 2004) had amodest effect. SNX-482 (1 !M), a peptide that inhibits R-type currents incertain brain regions (Wilson et al., 2000), also failed to show any notableeffect. Another possible calcium entry site, the NMDA channel, was ruledout using D-AP5 (50 !M), which failed to inhibit mIPSCs.
Inhibition of intracellurar pathways such as PLC or inositol
1,4,5-trisphosphate (IP3) receptors did not abolish the carbachol
effectSubtype M1, M3, and M5 muscarinic receptors up-regulate
phospholipase C (PLC) activity while M2 and M4 down-regulate adenylate
cyclase (AC) activity (Caulfield and Birdsall, 1998). The effectiveness ofpirenzepine upon the actions of carbachol suggests an involvement of the
M1 receptor, which signals via PLC. We therefore studied the influence of
PLC pathway blockade upon the carbachol effect. At 15 !M, 2-APB blocks
IP3 receptors of the endoplasmic reticulum (ER) and IP3-mediated calcium
release from ER stores (Maruyama et al., 1997). A higher concentration (75!M) also blocks some store-operated calcium channels and some TRP
channels. No significant differences in the carbachol effect were shown in
the presence of 2-APB at 15, 75 or 150 !M (>20 min) compared with its
vehicle ethanol or DMSO (15 !M 2-APB, n = 7, vehicle ethanol only, n =4; 1 mM carbachol application in both groups, frequencies increased by 0.7± 1.7 vs. -0.3 ± 1.5 Hz, P = 0.10, U-test)(2-APB 75 !M, n = 10, vehicle
DMSO only, n = 5; 200 !M carbachol application in both groups,
frequencies increased by -0.5 ± 0.8 vs. -0.2 ± 0.4 Hz, P = 0.18, U-test) (150
!M 2-APB, n = 9, vehicle DMSO only, n = 6; 1 mM carbachol applicationin both groups, frequencies increased by 0.0 ± 1.2 vs. -0.1 ± 0.8 Hz, P =0.78, U-test). Furthermore, in the presence of gadolinium (10 !M), an
inhibitor of store-operated calcium channels and TRP channels (Trebak etal., 2002), carbachol had a robust effect (n = 1, frequency increased by 0.6± 0.4 Hz). Thus, intracellular calcium, calcium store-operated calcium
channels, and TRP channels are not significantly involved. Application of
the protein kinase C inhibitor, 0.5 !M calphostin (> 20 min) under normal
fluorescent lighting condition (Bruns et al., 1991) could not abolish thecarbachol effect (1 mM carbachol, n = 5, vehicle water only, n = 3;
frequencies increased by 2.3 ± 1.9 vs. 0.4 ± 1.1 Hz, P = 0.005, U-test).
In the presence of the PLC inhibitor U73122 (10 !M, >20 min),carbachol application significantly increased the mIPSC frequency
compared with vehicle-only application (1 mM carbachol, n = 9, vehicle
water only, n = 3; frequencies increased by 1.1 ± 1.1 vs. -0.3 ± 0.6 Hz, P =0.002, U-test).Another PLC inhibitor, 50 !M edelfosine (Horowitz et al.,2005; Wong et al., 2007), also had no significant effect on the carbachol
action (20 !M edelfosine, n = 8, vehicle water only, n = 8; frequencies
increased by -0.1 ± 0.5 vs. 0.2 ± 0.7 Hz, P = 0.80, U-test) (50 !M
edelfosine, n = 6, vehicle water only, n = 5; 200 !M carbachol applicationin both groups, frequencies increased by 1.1 ± 2.9 vs. 0.1 ± 0.3 Hz, P = 0.56,
U-test).
As none of the PLC pathway blockers used had any significant
effect on the action of carbachol, we next examined the involvement of the

adenylate cyclase (AC) pathway. In the absence of carbachol, applicationof the AC activator forskolin resulted in a robust increase in the mIPSC
frequency (n = 4). This may result from activation of protein kinase A(PKA) that works to increase the size of readily releasable pools of synaptic
vesicles by disassociating vesicles from the actin meshwork (Greengard etal., 1993). In the olfactory bulb, muscarinic receptors are known to
up-regulate AC activity (Olianas and Onali, 1992; Onali and Olianas 1995).
The AC inhibitor SQ22536, however, failed to significantly block the actionof 100 !M carbachol (100 !M SQ22536, n = 5, vehicle water only, n = 4;
frequencies increased by 0.2 ± 0.5 vs. 1 ± 1.3 Hz, P = 0.16, U-test). To test
the possibility that muscarinic receptors up-regulate both AC and PLC, a
mixture of the two antagonists, SQ22536 (300 !M) and edelfosine (50 !M),was added to the preparation (preincubation, 100 !M SQ22536 and 10 !Medelfosine for more than an hour). Under these conditions, carbachol still
increased the mIPSC frequency significantly (direct bath application of 300!M carbachol, n = 4, vehicle only application, n = 4; frequencies increasedby 1.7 ± 1.8 vs. -0.5 ± 0.5 Hz, P = 0.001, U-test).
In the absence of carbachol, SQ22536 application (AC inhibitor),
which had been expected to have the opposite effect of forskolin (ACactivator), surprisingly stimulated a modest increase in the mIPSC
frequency (5 out of 6 cells), suggesting the possibility of AC
down-regulation being the mechanism underlying the carbachol action.
PKA down-regulation and thus dephosphorylation may reduce KCNQ
currents (Schroeder et al., 1998) thereby excite presynaptic membrane andincrease GABA release. To test the possibility that muscarinic receptors
down-regulate AC while up-regulating PLC, a mixture of 10 !M himbacine(blocking M2 and M4 receptors, which monopolize the AC pathway;
Caulfield and Birdsall, 1998) and 50 !M edelfosine was used. Nonetheless,carbachol application significantly increased the mIPSC frequency
compared with vehicle (direct bath application of 300 !M carbachol, n = 7,
vehicle only application, n = 11; frequencies increased by 0.3 ± 0.8 vs. -0.6
± 0.9 Hz, P = 0.008, U-test).
An alternative pathway using cyclic ADP ribose as amessenger (Higashida et al., 1996) was next considered. This requires long(> 5 h) incubation of slices with streptozotocin to deplete the NAD+ that isthe precursor of the cyclic ADP messenger. We incubated slices withstreptozotocin (5 mM) in addition to a PLC inhibitor (edelfosine, 10 !M for
5 h and 50 !M for 20 min), without any noticeable effect on the action of
carbachol (direct bath, 300 !M, data not shown). Longer incubation was notpractical in regards to the reliability of the recordings and the health of theslice.
A high concentration of carbachol stimulates Gi in addition to
the Gq linked to M1 receptors (Gudermann et al., 1997). Gi activates
phosphatidylinositol (PI) 3-kinase and mitogen-activated protein (MAP)kinase through Ras (Alberts et al., 2002). To test the possibility thatcarbachol’s action is mediated by PI3 kinase, we applied its inhibitor
LY294002 (50 !M effective in slice; Zhang et al., 2009) combined with the
PLC inhibitor edelfosine. Carbachol application still had apparent actions
under these conditions (20 !M LY294002 + 10 !M edelfosine, for morethan 30 min in the holding chamber and thereafter, 100 !M LY294002 + 50
!M edelfosine for more than 15 min in the recording chamber, n = 9; directbath 300 !M carbachol, frequencies increased by 1.1 ± 1.4 Hz) The
carbachol effect was also apparent in the presence of 50 !M PD98059 (aninhibitor of MAPK pathway; the concentration reported to be effective inslices; Kawasaki et al., 2004, Yuill et al., 2007) combined with edelfosine(10 !M PD98059 + 10 !M edelfosine, for more than 50 min in the holding
chamber and thereafter, 50 !M PD98059 + 50 !M edelfosine for more than
15 min in the recording chamber, n = 4; direct bath 300 !M carbachol,frequency increased by 0.0 ± 0.7 Hz).
Supplementary Discussion
Intracellular signalling pathway downstream of muscarinic
stimulation
We could not find a pathway downstream of muscarinic receptor
stimulation. There are two possible explanations: the first possibility is thatthe inhibitors used did not sufficiently block the intracellular pathways toshow a significant effect. However, the concentrations and durations of
incubation for the PLC or AC blockers were likely sufficient, as they are
effective in similar slice experiments (Herold et al., 2005; Evans et al.,
2001; Woodhall et al., 2007). Though depletion of phosphatidylinositol
4,5-bisphosphate (PIP2) by PLC is the currently acceptable mechanism of
KCNQ channel closure (Delmas and Brown 2005), PLC blockades act only
marginally for the M1 receptor-KCNQ channel pathway (Bofill-Cardona etal., 2000; Haley et al., 2000). The possibility of cyclic ADP involvement
remains viable, since we could not implement with our slices the longincubation times required to deplete the cyclic ADP ribose completely(Higashida et al., 1996). For the second possibility, without a need ofintracellular pathway, G-proteins may directly modify KCNQ channels

9(Marrion, 1997). A much potent PLC inhibitor in the future may help tofurther address this question.
Supplementary experimental procedures
Chemicals
If not otherwise noted, chemicals were dissolved in water or, if hydrophobic,
in dimethyl sulfoxide (DMSO) at the highest concentration according to the
supplier’s data sheet, and stored in a -20°C freezer. Chemicals (stock
concentration, vehicle, and storage temperature in parenthesis) werepurchased from the following sources: Biomol (Plymouth Meeting, USA),calphostin (0.5 mM in DMSO); Cayman (Ann Arbor, USA), PD98059 (50mM in DMSO); Sigma (St. Louis, USA), U73122 (dissolved initially inchloroform to 10 mM, divided into 100 !l aliquots, dried, and stored at-20°C, then resuspended at 10 or 5 mM in DMSO and stored at -20°C);
Nakarai (Kyoto, Japan), gadolinium chloride; Wako (Osaka, Japan),forskolin (10 mM in ethanol), streptozotocin (500 mM in water), LY294002(25 mM in DMSO); Calbiochem (Plymouth Meeting, USA), SQ22536 (100mM in water); Tocris (Bristol, UK), edelfosine (20 mM in water),(RS)-!-methyl-4-carboxyphenylglycine (MCPG; 50 mM in 0.1N NaOH);
Peptide Institute (Osaka, Japan): SNX-482 (50 !M).
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A

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Frequency increase value (Hz)Fbb

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2 sec

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Figure 1

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Spontaneous current frequency(pA) (Hz)600 12400

Carbachol (n = 7)
Control (n = 6)

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Figure 3

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Figure 4
A Stand. sol. / CCh 200
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Application (!M)

P < 0.0001

Stand. sol. / pilocarpine 300

P = 0.0004

Stand. sol. / Vehicle
Scopolamine 100 / CCh 1mM
Vehicle/ CCh 1mM

P = 0.001

Pirenzepine 10 / CCh 100
Vehicle/ CCh 100-2

P = 0.010246Frequency increase value (Hz)8B
CCh 100 !M
Himbacine 10 !M

CCh 100 !M
4-DAMP 300 nM

400 pA
2 min

2 min
400 pA

2 min

400 pA

y912
umm
y912
csum
y912
aphm

A

Figure 5

B
Base condition / Application (!M)
Ky* 5mM+AP3 200 / CCh1mM
P = 0.06

Ky* 5mM+AP3 200 / Vehicle
Glu. antago. mix* / CCh 300

P = 0.83

Vehicle / CCh 300-202Frequency increase value (Hz)4

CCh 300 !M direct bath application
Glu. antago. mix*

2 min

400 pA

Ky*: kynurenic acid
Glu. antago. mix*: Ky* (5 mM) + MCPG (500 !M) + CNQX (30 !M)+ D-AP5 (50 !M)C
Glutamatergic blockers present-70 mV-70 mV+30 mV
1 min+30 mV200 pAD

Glutamate 100 !M
200 pA
2 min

A
Base condition / Application (!M)
P = 0.29

Nifedipine 30 / CCh 1mM
Vehicle / CCh 1mM

P = 0.02

Mibefradil 20 / CCh 300
Mibefradil 20 / vehicle

0
!

$

%

&

'

o
Ef
(-)

o

0
>1
(n

5)

=
6)

=

3)

6)

3)

=

=

(n

(n

=

(n

(n

30

0

0
20

m
Ni

Cd

#

20

"

M

4

100

m

-2
0
2
Frequency increase value (Hz)

200

Ni

-4

%
300

5
Ni

P = 0.10

2-APB 15 / CCh 1 mM
Vehicle / CCh 1mM

mIPSC freq. change (after 3 min)

C

B
100

Ni

Cd

200 µM

200 µM

Nimo 30#M(-)Efo saturated

Co 100 250 500 1000 (µM)
Aga!(1 µM)+Cono"(1 µM)
Mg

10 mM

400 pA
2 min

Nicdcomg
Figure 6

Nimo EfAgacono

4203nimoet30f38
snx1f624103f46aga1cono3
1310 f65efof100cd6551966

8314f1820mu14m50mu0-1048

Figure 7A

Ba 1 mM

XE-991 20 µM

2 min

400 pA

B
Base condition / Application (!M)
Retigabine 80 / CCh 200
VehicleP = 0.03/ CCh 200

Diclofenac 300 / CCh 300
Vehicle / CCh 300
Low K+ / CCh 300(uncontrolled)-2

P = 0.0202Frequency increase value (Hz)4

Figure 8

A

250
ms

Number of events

B

C

)!

80
(!

'!
60
&!
%!
40
$!
#!
20
"!

0!

0

300

"

600

Amplitude

900

ACF (i)

!"%#

ACF

!"%
0.2

Smoothed ACF

!"$#

Dip

!"$
0.1

Peak
Peak

Dip

!"!#

0!
!

0
0

$

%

&

'

#

(

5
300

)

*

(i)

+ $! $$ $% $& $' $# $( $) $*

10
600

15
900

pA

pA

200
pA

Ami 1 mM

Nif 30 µM(+/-)Efo 20 µM

Mib 10 µM

Snx 1 µM

D-AP5 100 200 µM

400 pA
2 min

dap5 snx1
Agacono4203nimoet30f38
snx1f621310

4103f46aga1cono3
f65efof100cd6551966

Supplementary Figure