1Department of Physiology and 2Department of Otolaryngology-Head and Neck Surgery, Saga Medical School, Saga 849-8501, Japan
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ABSTRACT |
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Narikawa, Keita, Hidemasa Furue, Eiichi Kumamoto, and Megumu Yoshimura. In Vivo Patch-Clamp Analysis of IPSCs Evoked in Rat Substantia Gelatinosa Neurons by Cutaneous Mechanical Stimulation. J. Neurophysiol. 84: 2171-2174, 2000. To know a functional role of inhibitory synaptic responses in transmitting noxious and innoxious information from the periphery to the rat spinal dorsal horn, we examined inhibitory postsynaptic currents (IPSCs) elicited in substantia gelatinosa (SG) neurons by mechanical stimuli applied to the skin using the newly developed in vivo patch-clamp technique. In the majority (80%) of SG neurons examined, a brush stimulus applied to the ipsilateral hind limb produced a barrage of IPSCs that persisted during the stimulus, while a pinch stimulus evoked IPSCs only at its beginning and end. The pinch-evoked IPSCs may have been caused by a touch that occurs at the on/off time of the pinch. The evoked IPSCs were blocked by either a glycine-receptor antagonist, strychnine (4 µM), or a GABAA-receptor antagonist, bicuculline (20 µM). All SG neurons examined received inhibitory inputs from a wide area throughout the thigh and lower leg. When IPSCs were examined together with excitatory postsynaptic currents (EPSCs) in the same neurons, a brush evoked a persistent activity of both IPSCs and EPSCs during the stimulus while a pinch evoked such an activity of EPSCs but not IPSCs. It is suggested that innoxious mechanical stimuli activate a GABAergic or glycinergic circuitry in the spinal dorsal horn. This inhibitory transmission may play an important role in the modulation of noxious information in the SG.
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INTRODUCTION |
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Noxious information is
carried through fine myelinated A and unmyelinated C fibers to the
superficial laminae (Light and Perl 1979
;
Yoshimura and Jessell 1989
), particularly substantia gelatinosa (SG; laminae II) of the spinal dorsal horn where the information is modulated. Such a modulation is expected to partly occur
through the activation of an inhibitory circuitry. Evidence from recent
immunocytochemical studies indicates that glycine- and GABA-containing
neurons are present at a high proportion in the lamina I-III
(Mitchell et al. 1993
; Todd and McKenzie
1989
). In vivo analyses of unit activities elicited by
cutaneous stimuli have been conducted with respect to the inhibition
(Cervero et al. 1979
; Steedman et al.
1985
). In these studies, however, it was difficult to analyze
in detail the inhibitory transmission. In vitro spinal cord slice
experiments thus have been developed and demonstrate that the
activation of primary A
afferents evokes inhibitory (GABAergic or
glycinergic) postsynaptic potentials or currents (IPSPs or IPSCs,
respectively) in SG neurons (Yoshimura and Jessell 1989
,
1990
; Yoshimura and Nishi 1993
,
1995
). In such in vitro experiments, however, it was
impossible to know what kinds of stimuli applied to the skin elicit
these inhibitory transmissions. To address this issue, we have
developed a patch-clamp technique applicable to SG neurons in an in
vivo rat preparation (Furue et al. 1999
). The aim of the
present work was to know whether IPSCs are evoked in SG neurons in
response to cutaneous mechanical stimulation and if so to reveal what
kinds of neurotransmitters are involved in the IPSCs.
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METHODS |
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The methods used for the current experiment were similar
to those in our preceding study (Furue et al. 1999).
Briefly, male Sprague-Dawley rats (7-10 wk old) were anesthetized with
urethan (1.2-1.5 g/kg ip) and artificially ventilated; if a withdrawal reflex appeared, a supplemental dose of urethan was given during surgery and data collection. Bilateral pneumothorax was made to reduce
a respiratory movement of thorax. A lumbar laminectomy was performed at
the level of L4 or L5, and
then the animal was placed in a stereotaxic apparatus. After removing
the dura and cutting arachnoid membrane to make a window large enough
to let a patch electrode, the surface of spinal cord was irrigated with 95% O2-5%
CO2-equilibrated Krebs solution (in mM: 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 11 glucose, and 25 NaHCO3) at 38.0 ± 0.5°C. A whole cell
voltage-clamp technique was applied to SG neurons with an electrode
that had a tip resistance of 10-15 M
and was filled with a solution
having the following composition (in mM): 110 Cs2SO4, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 ATP-Mg, and 5 tetraethylammonium; pH 7.2. Data were digitized
with an A/D converter, stored, and analyzed with a personal computer
using pCLAMP6 and Axograph3.5 data acquisition program (Axon
Instruments, Foster City, CA). The recorded neurons were identified as
being in the SG based either on their morphological features revealed by an intrasomatic injection of biocytin or on the depth of the neurons
from the surface of the spinal cord. The mechanical stimuli used were
pinching of skin folds with a toothed forceps and brushing the surface
of skin or the hairs in the ipsilateral hind limb. Drugs used were
bicuculline and strychnine (Sigma, St. Louis, MO); they were given by
superfusion. Average data values are presented as means ± SE.
Statistical significance was determined as P < 0.05 using paired Student's t-test. All the experiments
involving rats were conducted in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Science
of the Physiological Society of Japan.
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RESULTS |
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An animal preparation was maintained in a stable state over
10 h, and patch-clamp recordings of up to 3 h were obtained
from 31 SG neurons having input resistances of 447 ± 26 M
(mean ± SE, n = 25). All of the neurons
examined exhibited spontaneous excitatory postsynaptic currents (EPSCs)
at a holding potential (VH) of
70
mV, as reported by Furue et al. (1999)
, and when
measured at 0 mV where spontaneous EPSCs were invisible,
spontaneous IPSCs could be recorded in all of these cells (see Fig. 2,
A and B).
IPSCs evoked by cutaneous mechanical stimulation
In the majority (80%) of the SG neurons examined
(n = 15), a brush produced a barrage of IPSCs, which
persisted during the stimulus (left panel of Fig.
1; see also Fig. 3A). They
had an average amplitude and frequency, respectively, of
87.9 ± 12.3 pA and 18.3 ± 2.6 Hz (n = 12),
values being significantly larger than those of spontaneous IPSCs
(27.2 ± 3.6 pA and 6.6 ± 0.5 Hz, respectively;
n = 12; P < 0.05). These IPSCs
subsided within 1 s after the stimuli were terminated. A
pinch, on the other hand, evoked IPSCs, the activity of which lasted
for only 1-2 s at its beginning and end, in 12 of the 15 neurons, as
seen in the right panel of Fig. 1 (see also Fig.
3A). This transient response seemed to follow the movement
of forceps. The remaining three neurons (20%) were insensitive to the
brush while exhibiting a barrage of IPSCs without a decline in the
frequency in response to the pinch, as seen for the brush (data
not shown). They had an average amplitude and frequency,
respectively, of 69.1 ± 2.7 pA and 15.3 ± 1.2 Hz
(n = 3), values being significantly larger than those of spontaneous IPSCs (P < 0.05). With respect to
inhibitory receptive fields of SG neurons, the points sensitive to
stimulation were the lower leg, upper leg, and thigh and thus were
unexpectedly wide, as seen for EPSCs (Furue et al.
1999).
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IPSCs mediated by glycine and GABAA receptors
In 10 (71%) of 14 SG neurons examined, spontaneous IPSCs were blocked by a glycine-receptor antagonist, strychnine (4 µM; Fig. 2A). When examined in 6 of the 10 neurons, the strychnine-sensitive IPSCs were comparable in the average of peak amplitude in the absence and presence of a GABAA-receptor antagonist, bicuculline (20 µM; 28.2 ± 2.6 pA and 22.8 ± 2.1 pA, respectively; P > 0.05). In the remaining four neurons (29%), spontaneous IPSCs were blocked by bicuculline, as seen in Fig. 2B. The bicuculline-sensitive IPSCs were comparable in the average of peak amplitude in the absence and presence of strychnine (4 µM; 34.7 ± 6.9 pA and 37.7 ± 4.9 pA, respectively; n = 4; P > 0.05). The strychnine- and bicuculline-sensitive IPSCs had a frequency of 7.0 ± 0.5 Hz (n = 10) and 6.5 ± 1.0 Hz (n = 4), respectively. When compared in duration, the strychnine-sensitive IPSC (11-14 ms) was shorter by about threefold than the bicuculline-sensitive one (26-38 ms; see insets in Fig. 2, A and B).
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When examined for evoked IPSCs, strychnine (n = 5) or bicuculline (n = 1) blocked a sustained barrage of IPSCs induced by brush, as seen in Fig. 2, C and D. The pinch-evoked on/off response of IPSCs was also blocked by the antagonists (n = 2 for strychnine; not shown). These effects of antagonists appeared 8-10 s following their superfusion and disappeared 30-40 s after their washout, indicating that the effects were due to a direct action in the spinal cord and were not achieved via blood circulation.
IPSCs and EPSCs evoked by mechanical stimuli
To know a relationship of the inhibitory to excitatory responses
in the SG in response to cutaneous stimuli, IPSCs were analyzed together with EPSCs in the same SG neurons by altering
VHs. In a SG neuron where a sustained
activity of IPSCs was evoked by brush but not pinch (Fig.
3A), both stimuli elicited a
barrage of EPSCs (VH = 70 mV), which
persisted during the stimuli (Fig. 3B), as reported
previously (Furue et al. 1999
). Similar results were
obtained in the other 11 cells. There appeared not to be a difference
in receptive field between the IPSCs and EPSCs, although this was not
examined in detail.
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DISCUSSION |
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The present study revealed in SG neurons of the adult
rat spinal cord in vivo that IPSCs occur spontaneously or in
response to cutaneous mechanical stimuli and that they are
mediated by either glycine or GABA. This is the first report showing
mechanical stimulus-evoked IPSCs in spinal dorsal horn neurons in
vivo, although Light and Willcockson (1999) have
reported spontaneous IPSPs in a similar preparation. Such a glycinergic
or GABAergic transmission has been observed in in vitro slice
preparations (Yoshimura and Nishi 1995
). When compared
between the in vivo and in vitro preparation, the IPSCs were
similar in that glycinergic IPSCs exhibit a shorter duration
than GABAergic ones (see Yoshimura and Nishi 1995
).
On the other hand, the percentage of in vivo SG neurons exhibiting glycine or GABAA receptor-mediated IPSCs was
different from that obtained in slices. Although Yoshimura and
Nishi (1995)
have reported that GABAA-,
glycine-, and both-mediated evoked IPSPs are observed in 47, 37, and 16% of the SG neurons examined, respectively, we could not
note SG neurons exhibiting IPSCs mediated by both;glycinergic IPSCs
were mainly observed. Such a difference would be due to a distinction
in the number of the inhibitory fiber inputs between SG neurons in vivo
and in slices.
Our present results revealed in the majority (80%) of SG neurons that a brush produces a persistent activity of IPSCs during the stimulus while a pinch elicits IPSCs only at its beginning and end. The pinch-evoked activity may have been caused by a touch response that occurs at the on/off time of pinching the skin. Alternatively, it is possible that an activation of peripheral receptors on the skin by pinch is rapidly accommodated. This is, however, unlikely since EPSCs persist during pinch. When examined in the same SG neuron, both brush and pinch evoked a persistent activity of EPSCs during the stimuli, while brush stimulus only induced a similar persistent activity of IPSCs. It is suggested that both excitatory and inhibitory information are transmitted to SG neurons when a brush is applied to the skin, while a pinch evokes excitatory but not inhibitory responses. This idea seems not to be applied to all SG neurons, because pinch evoked persistent IPSCs in 20% of the SG neurons examined.
Our previous studies using spinal cord slices have suggested that
primary-afferent A fibers innervate glycinergic and/or GABAergic
interneurons, the activation of which results in the production of
IPSPs in SG neurons (Yoshimura and Nishi 1995
). So the
IPSCs evoked by cutaneous mechanical stimuli are likely to be due to
the activation of A
fibers. Figure 3C demonstrates our
hypothesis that a touch, whose information is conveyed through A
fibers to the spinal dorsal horn, may activate inhibitory interneurons, resulting in the inhibition of noxious transmission from the periphery to SG neurons. This hypothetical circuitry may explain the well-known behavioral observation that touching near the skin where a pain occurs
leads to its alleviation.
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ACKNOWLEDGMENTS |
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This work was supported in part by the Human Frontier Science Program and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to M. Yoshimura.
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FOOTNOTES |
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Address for reprint requests: K. Narikawa, Dept. of Physiology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan (E-mail: narikawa{at}post.saga-med.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 March 2000; accepted in final form 12 June 2000.
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REFERENCES |
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