Group Title: Molecular Pain 2007, 3:26
Title: The activation of nicotinic acetylcholine receptors enhances the inhibitory synaptic transmission in the deep dorsal horn neurons of the adult rat spinal cord
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Title: The activation of nicotinic acetylcholine receptors enhances the inhibitory synaptic transmission in the deep dorsal horn neurons of the adult rat spinal cord
Series Title: Molecular Pain 2007, 3:26
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Creator: Takeda D
Nakatsuka T
Gu JG
Yoshida M
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Molecular Pain BioMed



Research

The activation of nicotinic acetylcholine receptors enhances the
inhibitory synaptic transmission in the deep dorsal horn neurons of
the adult rat spinal cord
Daisuke Takeda*1,2, Terumasa Nakatsuka3, Jianguo G Gu4 and
Munehito Yoshidal


Address: 'Department of Orthopaedic Surgery, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8510, Japan, 2Department of
Physiology, Kansai University of Health Sciences, Osaka 590-0482, Japan, 3Department of Physiology, Faculty of Medicine, Saga University, Saga
849-8501, Japan and 4Brain Institute and Department of Oral Surgery, Division of Neuroscience, College of Dentistry, University of Florida,
Gainesville, Florida 32610, USA
Email: Daisuke Takeda* dtakeda@kansai.ac.jp; Terumasa Nakatsuka nakatsuk@cc.saga-u.ac.jp; Jianguo G Gu jgu@dental.ufl.edu;
Munehito Yoshida myoshida@wakayama-med.ac.jp
* Corresponding author


Published: 25 September 2007
Molecular Pain 2007, 3:26 doi:10.1 186/1744-8069-3-26


Received: 17 July 2007
Accepted: 25 September 2007


This article is available from: http://www.molecularpain.com/content/3/1/26
2007 Takeda et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Somatosensory information can be modulated by nicotinic acetylcholine receptors (nAChRs) in the
superficial dorsal horn of the spinal cord. Nonetheless, the functional significance of nAChRs in the
deep dorsal horn of adult animals remains unclear. Using whole-cell patch-clamp recordings from
lamina V neurons in the adult rat spinal cord, we investigated whether the activation of nAChRs
could modulate the inhibitory synaptic transmission in the deep dorsal horn. In the presence of
CNQX and APV to block excitatory glutamatergic synaptic transmission, bath applications of
nicotine (100 lIM) significantly increased the frequency of spontaneous inhibitory postsynaptic
currents (slPSCs) in almost all neurons tested. The effect of nicotine was mimicked by N-methyl-
4-(3-pyridinyl)-3-butene-l-amine (RJR-2403, 100 lIM), an a432-nAChR agonist, and was also
mimicked by choline (10 mM), an a7-nAChR agonist. The effect of nicotine was completely blocked
by the nAChR antagonist mecamylamine (5 lIM). In the presence oftetrodotoxin (0.5 lIM), nicotine
(100 lIM) significantly increased the miniature IPSC frequency. On the other hand, RJR-2403 (100
lIM) or choline (10 mM) did not affect miniature IPSCs. The application of nicotine (100 lIM) also
evoked a large inward current in all lamina V neurons tested when cells were held at -60 mV.
Similarly, RJR-2403 (100 lIM) induced inward currents in the majority of lamina V neurons
examined. On the other hand, choline (10 mM) did not elicit any detectable whole-cell currents.
These results suggest that several nAChR subtypes are expressed on the presynaptic terminals,
preterminals, and neuronal cell bodies within lamina V and that these nAChRs are involved in the
modulation of inhibitory synaptic activity in the deep dorsal horn of the spinal cord.


Background
Neuronal nAChRs are a larger family of ligand-gated ion
channels widely expressed in both the central and the


peripheral nervous system. At least 12 different subunits
of nAChRs, including a2-al0, P2-P4, have been identi-
fled so far and these subunits form many different sub-


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types of nAChRs with pentameric structures consisting of
homomers or heteromers [1]. Homomeric nAChRs are
made up of a7, a8 or a9 subunits, while heteromeric
nAChRs comprise various combinations of a2-a6 with
P2-P4 subunits, a9 with al0 subunits [2,3]. These sub-
types of nAChRs have different pharmacological and bio-
physical properties [1]. It has been shown that nAChRs
are involved in a variety of physiological functions includ-
ing learning, reinforcement, development, aging and
nociception [4].

Although Davis et al. (1932) first reported that nicotine
has analgesic effects [5], high dosages of nicotine were
required to produce antinociception and its effect was rel-
atively modest with a short duration [6-8]. Epibatidine, a
potent nAChR agonist isolated from the skin of an Ecua-
dorian frog, was about 100-fold more potent than mor-
phine in rodents [9-12]. Unfortunately, the dosage of
epibatidine to produce antinociception was near that to
cause seizure, death, and other side effects [12]. The intol-
erable toxic effects of epibatidine were due to its actions
on a broad range of nAChR subtypes. Therefore, the key to
the development of safe and effective nicotinic agonists as
analgesics is to first understand which nAChR subtypes
are involved in modulating nociceptive transmission.

The spinal dorsal horn is the first site in the central nerv-
ous system where somatosensory information is proc-
essed and integrated. Multiple subtypes of nAChRs are
expressed in the spinal dorsal horn and these receptors
have been indicated to modulate sensory inputs from the
periphery. Genzen and McGehee (2003) have demon-
strated that the activation of a7 nAChRs located at the
central terminals of primary afferents enhances the gluta-
matergic excitatory transmission in the spinal dorsal horn
[13]. Several subtypes of nAChRs have been shown to
exert tonic or phasic control on the descending inhibitory
serotonergic transmission [14]. Multiple subtypes of
nAChRs are found to be expressed on both inhibitory and
excitatory intemeurons in the spinal dorsal horn [15]. The
activation of presynaptic nAChRs facilitates GABAergic
and glycinergic inhibitory synaptic transmission in the
superficial dorsal horn [16-19 Although the roles of pre-
synaptic nAChRs were extensively studied in the superfi-
cial dorsal horn, it is unclear whether nAChRs also
mediate sensory modulation in the deep dorsal horn of
the spinal cord in adult animals. A variety of sensory
inputs, including nociceptive and non-nociceptive inputs,
are transmitted into deep dorsal horn [20]. Deep dorsal
horn neurons, especially those in the lamina V region, can
generate long-lasting afterdischarges in response to nocic-
eptive inputs and this hyperactivity has important impli-
cations in pathological pain states [21]. Inhibitory
modulation in this region is critical in preventing the cen-
tral hyperactivity and hyperalgesia. The aim of this study


was to evaluate the effects of nAChR activation on the
inhibitory synaptic transmission in deep dorsal horn neu-
rons.

Results
Effects of nicotine and nAChR agonists on spontaneous
IPSCs in the lamina V neurons
Whole-cell patch-clamp recordings were performed from
lamina V neurons of spinal cord slices prepared from
adult rats. Stable recordings could be obtained from slices
maintained in vitro for more than 12 hours. Glutamatergic
excitatory postsynaptic transmission was blocked by
CNQX (20 iM) and APV (50 iM). All lamina V neurons
tested exhibited spontaneous inhibitory postsynaptic cur-
rents (sIPSCs) when cells were held at -10 mV. In the pres-
ence of bicuculline (20 iM) and strychnine (2 iM),
sIPSCs were completely abolished in all lamina V neurons
tested (n = 3; data not shown), indicating these sIPSCs
were mediated by GABA and/or glycine receptors. Per-
fusion of nicotine (100 iM) for 1 min resulted in a rapid
and significant increase in sIPSC frequency in all neurons
tested (Fig. 1A-C). The average sIPSC frequency in con-
trols was 2.1 + 0.6 Hz (0.4 5.1 Hz, n = 9) and the fre-
quency increased to 15.8 2.3 Hz (4.2 28.1 Hz, n = 9, P
< 0.05) following the application of 100 iM nicotine (Fig.
1C); the sIPSC frequency increased to 1330 + 310% of the
control (n = 9, P < 0.05). The nicotine-induced increase in
sIPSC frequency was completely blocked in the presence
of nAChR antagonist mecamylamine (5 iM) adminis-
trated 5 min prior to the application of nicotine (n = 3;
Fig. 1D, E). After the washout ofmecamylamine, a second
application of nicotine (100 iM) increased the sIPSC fre-
quency in all neurons tested (Fig. 1D, E).

We tested RJR-2403, a selective a4p2 nAChR agonist, to
see if it also increased sIPSC frequency. Similar to nico-
tine, application of 100 iM RJR-2403 for 1 min markedly
increased sIPSC frequency in 13 out of 14 neurons
recorded (Fig. 2Aa-c). The average sIPSC frequency in the
control and following the application of RJR-2403 was 5.8
1.0 Hz (0.5 11.6 Hz, n = 14) and 15.7 1.9 Hz (3.7-
25.2 Hz, n = 14), respectively (Fig. 2Ac). The sIPSC fre-
quency following the applications of RJR-2403 signifi-
cantly increased to 573 + 189% of the control (n = 14, P <
0.05; Fig. 2C). While RJR-2403 alone produced a signifi-
cant increase in sIPSC frequency, the effects of RJR-2403
was completely blocked in the presence of dihydro-beta-
erythroidine (DhpE, 1 gtM), an a4p2 nAChR antagonist
(97 2% of control, n = 3). Perfusion of choline (10 mM),
a selective a7 nAChR agonist, for 1 min also increased the
sIPSC frequency in 11 out of the 13 neurons examined
(Fig. 2Ba-c). The average sIPSC frequency in the control
and following the application of choline was 7.4 + 1.2 Hz
(2.9 16.9 Hz, n = 13) and 15.2 2.3 Hz (5.2 27.6 Hz,
n = 13), respectively (Fig. 2Bc). The sIPSC frequency fol-


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Nic


50 pA
60s
C n=9


500 600


Control / Mec
I I I


Nic / Mec


Control



Nic I'
.,L^'WLJ'^~J ^W Lj~k-^.''


200ms


15

N c0
Mec

2hWasho t
0 100 200 300 400 500 60s) 0


Figure I
Effects of nicotine on spontaneous IPSCs in lamina V
neurons. A, A continuous recording of slPSCs in the con-
trol and following the application of nicotine (Nic, 100 jIM).
B, A histogram shows the time course of the changes in
slPSC frequency following the application of nicotine; time
bin is 10 s. C, The graph shows the individual result from 9
lamina V neurons. D. Effects of mecamylamine (Mec) on nic-
otine-induced increase in slPSC frequency. The consecutive
traces on the left are slPSCs in the control (upper panel) and
following the application of nicotine (lower panel) in the
presence of mecamylamine (5 jiM). The consecutive traces
on the right are slPSCs in the control (upper panel) and fol-
lowing the application of nicotine (lower panel) after the
washout of mecamylamine. Note that the bath application of
nicotine did not affect the slPSCs in the presence of
mecamylamine, but it markedly increased slPSC frequency
after the washout of mecamylamine. E, Two histograms
show time courses of changes in slPSC frequency following
the application of nicotine in the presence of mecamylamine
(left) and after the washout of mecamylamine (right); time bin
is 10 s.


li1 .1i L ~lU 1 i.IIIL ii .1 ..


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lowing the applications of choline significantly increased
to 221 22% of control (n = 13, P < 0.05; Fig. 2C). While
choline alone produced a significant increase in sIPSC fre-
quency, choline did not produce any significant increase
in sIPSC frequency in the presence of methyllycaconitine
(MLA, 50 nM), an a7 nAChR antagonist (98 2% of con-
trol, n= 4).

Effects of nicotine and nAChR agonists on mlPSCs in
lamina V neurons
We examined the effects of nicotine or nAChR agonists on
mIPSC frequency in the presence of tetrodotoxin (TTX,
0.5 riM) to determine whether nAChRs might be localized
at the presynaptic terminals of GABAergic and/or glyciner-
gic inhibitory intemeurons. Application of 0.5 jiM TIX
itself blocked the action potential-driven synaptic trans-
mission and decreased the amplitude of IPSCs from 149.9
125.6 pA to 30.0 18.4 pA (n = 4). Under this condi-
tion, bath application of nicotine (100 jiM) largely
increased mIPSC frequency in all neurons recorded, but
there was no effect on mIPSC amplitude (n = 10; Fig. 3A).
The average mIPSC frequency in the control and follow-
ing the applications of nicotine was 1.7 0.4 Hz (0.4 5.3
Hz, n = 10) and 15.0 2.3 Hz (1.4 25.2 Hz, n = 10),
respectively. The mIPSC frequency following the applica-
tions of nicotine significantly increased to 1043 153%
of control (n = 10, P < 0.05, Fig. 3D). On the other hand,
perfusion of 100 jiM RJR-2403 (n = 6) or 10 mM choline
(n = 7) did not affect mIPSC frequency and amplitude
(Fig. 3B, 3C). The average mIPSC frequency following the
application of RJR-2403 and choline was 95 3% of con-
trol (n = 6) and 98 2% of control (n = 7), respectively
(Fig. 3D).

Whole-cell currents directly evoked by nicotine or nAChR
agonists in lamina V neurons
We determined whether nicotine, RJR-2403 and choline
could evoke whole-cell currents in lamina V neurons. In
this set of experiments, cells were held at -60 mV and
recordings were conducted in the presence of 20 jiM
CNQX, 50 jiM APV, 20 jiM bicuculline and 10 jiM PMBA
(3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-yl]alanine).
Under this condition, both excitatory and inhibitory post-
synaptic currents were completely disappeared. The bath
application of nicotine (100 jiM) for 1 min evoked an
inward current in all neurons tested (Fig. 4A). The average
peak amplitude of the inward currents evoked by nicotine
was 95 + 19 pA (n = 8; Fig. 4D). The bath application of
RJR-2403 (100 jiM) for 1 min also evoked large inward
currents in 6 out of 8 neurons examined (Fig. 4B). The
average peak amplitude of the inward currents induced by
RJR-2403 was 119 42 pA (n = 6; Fig. 4D). In contrast to
nicotine and RJR-2403, choline (10 mM) did not elicit
any detectable currents (n = 6; Fig. 4C).


Molecular Pain 2007, 3:26








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Choline

.,L ..I J.l L pin di.i',l.ul


RJR2403



I 100 opA
(60s


200 pA
60S






300 pA
100 ms
C


i- 100 pA
500ms


RJR2403


S25
20
15

10 s


30 n=14
251
20
15:


0
/ /

le0


Cholne


30 n=13
25

15

0 0


C; C,


0 1C: "'01:, P.,6 i: ) :00 iX s


2000


1500



1000
*

500


100 .. .....
0


Figure 2
Effects of nicotinic receptor agonists on sIPSCs in lamina V neurons. A, (a) A continuous recording of slPSCs in the
control and following the application of the selective a4p2 nAChR agonist RJR-2403 (100 iM). (b), A histogram shows the
time course of changes in slPSC frequency following the application of RJR-2403; time bin is 10 s. (c), The graph shows the
individual result from 14 lamina V neurons. B, The experiment was similar to that shown in (A) except that the selective a7
nAChR agonist choline (10 mM) was tested. Similar results were obtained in I I out of 13 neurons. C, A histogram shows rel-
ative slPSC frequency following the application of nicotine (n = 9), RJR-2403 (n = 14), or choline (n = 13). slPSC frequency
before the applications of testing drugs is used as control and is scaled at 100%. Data represent Mean SEM; *P < 0.05.




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I o"


I'


I '


.L. F.
L








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Control / iX


NIc TTX


Conlrol/TTX


RJR2403ITX


m00





0 00 200 300 400 50(SI 10 200 3 0000 aO 50


Chdne I TIX


P-I A- 0 .I


U rCI~kJL


aChhe a Chne



"- % ."" = 0) 0 00 300 00O 400 5001


o 200ms

RJR203 RJR2R403



0 I00 200 300 400 $( 0 100 200 300 4001
W IS


1200

1O00


600
600



n. n n..

i *


Figure 3
Effects of nicotinic receptor agonists on mlPSCs in lamina V neurons. A, The consecutive traces of mlPSCs are in
the control (left) and following the application of nicotine (100 pM, right). Two histograms on the bottom panel show the time
course of changes in mlPSC frequency (left) and amplitude (right) during the application of nicotine; time bin is 10 s. B, C,
Experiments were similar to that shown in (A) except that 100 pM RJR-2403 or 10 mM choline was tested. D, A histogram
shows relative mlPSC frequency following the applications of nicotine (n = I I), RJR-2403 (n = 6), or choline (n = 7). mlPSC fre-
quency before the applications of testing drugs is used as control and is scaled at 100%. Data represent Mean SEM; *P < 0.05;
n.s., not significant.


Discussion
The present study demonstrated in lamina V neurons of
the adult rat spinal cord that nicotine increased sIPSC fre-
quency when glutamatergic excitatory transmission was
blocked in the presence of CNQX and APV and that nico-
tine also increased mIPSC frequency when action poten-
tial-driven synaptic transmission was not permitted in the
presence of TIX. Interestingly, however, neither the a432
nor a7 nAChR agonists increased mIPSC frequency
although both of them increased sIPSC frequency in lam-
ina V neurons. Together with the findings of our previous
study conducted on superficial laminas of the spinal cord
of adult rats [18], we have provided electrophysiological
evidence showing that inhibitory synaptic activity in both
superficial and deep laminas of the spinal cord dorsal
horn are modulated by different nAChR subtypes.

Nicotinic receptors are abundant in different CNS regions,
where they are shown to regulate the release of various


neurotransmitters, including serotonin, norepinephrine,
glutamate, GABA and glycine [22-24]. In the present
study, the activation of nAChRs enhanced the GABA and/
or glycine release onto lamina V neurons. A similar
enhancement of the inhibitory synaptic transmission by
the activation of nAChRs has been reported in the super-
ficial layers of the spinal dorsal horn [16,18,19]. In neo-
natal rats, a4p2 nAChR subtype has been suggested to be
expressed at presynaptic terminals and these receptors
mediate significantly increases in the glycinergic [16] and
GABAergic inhibitory synaptic transmission in the super-
ficial lamina of the spinal cord dorsal horn [19]. Interest-
ingly, the expression ofnAChR subunits in the spinal cord
changes during development [25]. Consistent with the
development changes of nAChR subunits, our previous
study demonstrated that a non-a4j2 and non-a7 subtype
nAChR mediated an enhancement of both the GABAergic
and glycinergic mIPSC frequency in the superficial lami-
nas of the adult rat spinal dorsal horn [18]. In the deeper



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Contr l/ TTX


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Nic

50 71A


RJR2403


_7 1.7


choline |

50 pA
60,


Figure 4
Whole-cell currents evoked by nicotine and nAChR
agonists in lamina V neurons. A, Whole-cell currents
evoked by bath application of nicotine (100 iM) in a lamina V
neuron. B, Whole-cell currents evoked by RJR-2403 (100
iM) in a different lamina V neuron. C, Choline (10 mM) did
not evoke any membrane current in a neuron. C, The histo-
gram shows the average amplitude of the whole-cell inward
currents induced by nicotine (n = 8), RJR-2403 (n = 6), and
choline (n = 6).


laminas (lamina III-V) of neonatal rats, a previous study
showed that presynaptic a4p2 nAChRs mediate the facili-
tation of GABA release [19], a result similar to those
shown in superficial laminas of neonatal rats [16,19].
However, the present study revealed that the nAChR-
mediated modulation of inhibitory synaptic transmission
in the adult stage is more complicated than that in the
neonatal stage. We showed that nicotine largely increased
both sIPSC frequency and mIPSC frequency in the lamina
V neurons in adult rats. On the other hand, RJR-2403, a
potent activator of a4p2 nAChR, and choline, a selective
agonist for a7 nAChR, significantly increased the sIPSC
frequency, but did not change mIPSC frequency. It has
been demonstrated that nAChRs are expressed at two cel-
lular locations in the central nervous system [26]. One is
presynaptic sites or synaptic boutons where nAChR activa-
tion modulates transmitter release in a TIX-insensitive
manner. The other is preterminals at terminal axon
branches where nAChR activation affects transmitter
release by depolarizing axonal membranes to fire action
potentials. A recent immunohistochemical study revealed
the immunoreactivity of nAChRs in lamina V neurons of
the spinal dorsal horn at both presynaptic and pretermi-
nal sites [27]. At the preterminal sites, nAChR-mediated
regulation of transmitter releases is TTX-sensitive and can
be blocked in the presence of TIX. The block by TIX of the


RJR-2403- and choline-induced increase in sIPSC fre-
quency in our results suggests that a4p2 and a7 nAChRs
are not expressed at presynaptic terminals. These receptors
are likely to be expressed at the preterminals or other parts
of GABAergic and/or glycinergic neurons whose axons
innervate lamina V neurons. Because nicotine could still
increase IPSC frequency in the presence of TIX, it suggests
that a non-a432, non-a7 subtype of nAChR is located at
the presynaptic terminals of GABAergic and/or glycinergic
neurons that innervate lamina V neurons (Fig. 5). The


Primary.


ntrl c
Central .ono'


- ct4f32,


Sa7, o non-a4p2. non-a7.
Inhibitory neuron,

inhibitory or excitatory neuron,


Figure 5
Schematic diagram of nAChR-mediated modulcation
of sensory synaptic transmission in the dorsal horn of
adult rats. In adult rats, a4p2 nAChRs are expressed on the
soma of inhibitory interneurons located in the lamina V
region. These nAChRs may be also expressed on pretermi-
nals but not at presynaptic sites of the lamina V inhibitory
neurons. Lamina V neurons are also synapsed by inhibitory
neurons expressing a7 nAChRs at their preterminals and/or
on their somas and these inhibitory neurons are likely to be
located in other lamina regions. The modulation of inhibitory
activity in lamina V by both a4p2 nAChRs and a7 nAChRs
depends on membrane depolarization and action potentials.
There is a non-a4p2/non-a7 subtype of nAChRs that are
expressed at the presynpatic terminals of inhibitory neurons
in lamina V region. The modulation of inhibitory transmission
in lamina V by non-a4p2/non-a7 subtype of nAChRs is inde-
pendent of membrane depolarization and action potentials.
The distribution of nAChR subtypes in the spinal cord lamina
II region of adult rats [18] is also presented in this diagram
for a comparison.


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presence of a non-a4p2, non-a7 subtype of nAChR in spi-
nal cord dorsal horns are supported by previous studies
using a4p2 nAChR knock-out mouse [28], in situ hybrid-
ization [29] and the combination of patch-clamp record-
ings with single-cell RT-PCR [15]. All these previous
studies pointed to the potential presence of other func-
tional nAChR subtypes in addition to a4p2 and a7 recep-
tors. However, the subunit compositions of the non-a4p32
and non-a7 subtype of nAChRs remain to be identified.

We have shown that both nicotine and the a4p2 nAChR
agonist RJR-2403 directly evoked whole-cell inward cur-
rents in the majority of lamina V neurons. On the other
hand, the a7 nAChR agonist choline did not evoke any
detectable inward currents in lamina V neurons. Since
strychnine has been noted to also be an effective antago-
nist at a7 nAChRs [30,31 ], the effect of choline was exam-
ined in the presence of PMBA, a glycine receptor
antagonist that has no effect on a7 nAChRs [32,33]. The
lack of choline-evoked whole-cell currents raise a possibil-
ity that a7 nAChRs are not expressed on lamina V neu-
rons. If this is the case, then choline-induced increases of
sIPSC frequency might be mediated by a7 nAChRs that
are expressed on the inhibitory intemeurons whose cell
bodies are located in other lamina regions in the spinal
cord. In contrast to a7 nAChRs, our results suggest that
a4p2 nAChR expressing inhibitory intemeurons are
located in lamina V. Consistently, several reports have
indicated the presence of a4p2 nAChR in the deep dorsal
horn and a7 nAChRs in other lamina of the spinal cord
[15,33-35]. Bradia et al. (2002) has reported that a-bun-
garotoxin-sensitive a7 nAChRs are located in the para-
sympathetic preganglionic neurons surrounding the
central canal of the spinal cord (lamina X) [33]. A low
level of a7 transcripts were also detected by in situ hybrid-
ization in the area around the central canal [35]. Moreo-
ver, the single-cell RT-PCR study revealed a more
widespread expression of a7 nAChR subunits in mouse
spinal dorsal horn neurons [15]. These findings support
the idea that a7 nAChR-expressing inhibitory intemeu-
rons innervate lamina V neurons from other lamina
regions in the spinal cord.

The role of nAChRs in modulating pain transmission has
been reported by a number of studies. Using a4p2 knock-
out mice, Marubio et al. (1999) showed a reduced antino-
ciceptive effect in a behavior study [28]. In a neuropathic
mouse model, epibatidine, a potent agonist of nAChRs
showed strong analgesic effects. However, the effects of
epibatidine were not completely prevented by the a432
nAChR antagonist dihydro-B-erythroidine [36]. These
studies suggested that in addition to a4p2 nAChR, other
nAChRs were involved in nAChR-mediated analgesic
effects. Consistent with this idea, the intrathecal injection
of choline, an a7 nAChR agonist, has been reported to


have an antinociceptive effect [37]. Several mechanisms
have been proposed to contribute to nAChR-mediated
analgesic effects, including the desensitization of nAChRs
on nociceptive primary afferent fibers, the increase of
noradrenaline and serotonin release within the spinal
cord, the activation of the descending inhibitory pathways
[14,38], and the increases of GABA and glycine release
from inhibitory intemeurons in the superficial spinal cord
dorsal horn [39]. Our study suggest that a4B2, a7
nAChRs, and another undefined subtype of nAchRs are
involved in regulating GABA and/or glycine release in the
deep lamina of the spinal cord dorsal horn in adult ani-
mals.

In this study, the application of nicotine or nicotinic ago-
nists significantly facilitated GABAergic and/or glycinergic
inhibitory synaptic transmission in the deep dorsal horn
of the spinal cord. This raises a possibility that acetylcho-
line released endogenously may modulcate inhibitory
synaptic transmission in a similar fashion. Recently,
Rashid et al. (2006) suggested that endogenous acetylcho-
line tonically stimulated the GABA and glycine release via
a4p2 subtype of nAChRs in the superficial dorsal horn in
mice [40]. Acetylcholine may be released from the
interneurons in the dorsal horn since the cell bodies of
cholinergic intemeurons have been found in lamina III-V
[41]. It appears that in the deep dorsal horn there are no
descending cholinergic systems in the rat [20,42]. Thus,
cholinergic interneurons in the dorsal horn [43,44] may
play an important role in modulating inhibitory synaptic
transmission.

In the dorsal horn, GABAergic and glicinergic inhibitory
synapses undergo developmental changes [45-47]. In the
present study, we did not separate inhibitory activity
between those of GABAergic synapses and those of glycin-
ergic synapses. It would be interesting to further study
whether nAchR subtype expression on GABAergic and gly-
cinergic neurons is different in the spinal cord dorsal
horn.

Conclusion
We have demonstrated that several nAChR subtypes are
expressed on the presynaptic terminals, preterminals, and
neuronal cell bodies within lamina V and that they are
involved in the facilitation of inhibitory synaptic trans-
mission. Therefore, the activation of nAChRs in the deep
dorsal horn of the spinal cord may be capable of inhibit-
ing nociceptive signaling in physiological and pathologi-
cal pain sensations.

Methods
All the experimental procedures involving the use of ani-
mals were approved by the Ethics Committee on Animal
Experiments, Wakayama Medical University, and were in


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accordance with the UK Animals (Scientific Procedures)
Act 1986 and all associated guidelines.

Spinal cord slice preparation
The method used to prepare adult rat spinal cord slices
has been described previously [48]. In brief, male adult
Sprague-Dawley rats (6-8 weeks of age, 200-300 g) were
deeply anaesthetized with isoflurane through a nose cone
inhalation, and then lumbosacral laminectomy was per-
formed. The lumbosacral spinal cord (L1-S3) was
removed and placed in pre-oxygenated Krebs solution at
1-3 C. Immediately after the removal of the spinal cord,
the rats were killed by exsanguination. The pia-arachnoid
membrane was removed after cutting all the ventral and
dorsal roots near the root entry zone. The spinal cord was
mounted on a vibratome and then a 600 jim-thick trans-
verse slice was cut. The slice was placed on nylon mesh in
the recording chamber, which had a volume of 0.5 ml,
and then was perfused at a rate of 10-15 ml/min with
Krebs solution saturated with 95% 02 and 5% CO2, and
maintained at room temperature. A platinum grid was
placed on the top of the slice to prevent slice movement.
The Krebs solution contained (in mM) 117 NaC1, 3.6 KC1,
2.5 CaC12, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3 and 11
glucose.

Patch-clamp recordings from lamina V neurons
Blind whole-cell patch-clamp recordings were made from
lamina V neurons with patch-pipette electrodes having a
resistance of 5-10 MQ [48]. The patch-pipette solution
was composed of (in mM) 110 Cs2SO4, 5 Tetraethylam-
monium (TEA), 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5
ATP-Mg, pH 7.2. Signals were acquired with a patch-
clamp amplifier (Axopatch 200B; Axon Instruments, Fos-
ter City, CA, USA). The data were digitized with an A/D
converter (Digidata 1200, Axon Instruments) and stored
and analyzed with a personal computer using the
pCLAMP data acquisition program (Version 8.2, Axon
Instruments). Lamina V neurons were viable for up to 24
h in slices perfused with a pre-oxygenated Krebs solution.
All the recordings described in this study were made
within 12 h. Whole-cell patch-clamp recordings were sta-
ble for up to 4 h. All of the neurons had membrane poten-
tials more negative than -50 mV. Unless otherwise noted,
all the recordings in this study were performed in the pres-
ence of CNQX (20 jiM) and APV (50 jiM).

Drug Applications
Drugs were dissolved in Krebs solution and then were
applied by perfusion via a three-way stopcock without any
change in the perfusion rate or the temperature. The time
necessary for the solution to flow from the stopcock to the
surface of the spinal cord slice was approximately 20 s.
The drugs used in this study were nicotine (Sigma-Aldrich,
St. Louis, MO, USA), RJR2403 (Tocris, Ballowin, MO,


USA), choline (Sigma-Aldrich), mecamylamine (Sigma-
Aldrich), 3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-
yl]alanine (PMBA, Sigma RBI), bicuculline (Sigma-
Aldrich), strychnine (Sigma-Aldrich), 6-cyano-7-nitroqui-
noxaline-2,3-dion (CNQX, Tocris), D(-)-2-Amino-5-
phosphonopentanoic acid (D-APV, Tocris), and tetrodo-
toxin (TIX, Tocris).

Statistical analysis
All numerical data were expressed as the mean + S.E.M.
Statistical significance was determined as P < 0.05 using
paired Student's t-test. For electrophysiological data, n
refers to the number of neurons recorded.

Abbreviations
nACh, nicotinic acetylcholine receptor;

IPSC, inhibitory postsynaptic current;

RJR2-403, N-methyl-4-(3-pyridinyl)-3-butene-l-amine;

TTX, tetrodotoxin;

GABA, gamma-aminobutyric acid;

TEA, tetraethylammonium;

EGTA, ethyleneglycol bis(2-aminoethylether)tetraacetic
acid;

HEPES, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesul-
fonic acid);

ATP-Mg, adenosine triphosphate-magnesium;

CNQX,6-cyano-7-nitroquinoxaline-2,3-dion;

PMBA,3-[2'-Phosphonomethyl[ 1, '-biphenyl]-3-
yl]alanine;

D-APV, D(-)-2-Amino-5-phosphonopentanoic acid;

Competing interests
The authors) declare that they have no competing inter-
ests.

Acknowledgements
This work was supported by The General Insurance Association of Japan,
The Japanese Health Sciences Foundation, and Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan to T.N.

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