In Vivo Patch-Clamp Analysis of IPSCs Evoked in Rat Substantia Gelatinosa Neurons by Cutaneous Mechanical Stimulation

Keita Narikawa,1,2 Hidemasa Furue,1 Eiichi Kumamoto,1 and Megumu Yoshimura1

 1Department of Physiology and  2Department of Otolaryngology-Head and Neck Surgery, Saga Medical School, Saga 849-8501, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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.


    INTRODUCTION
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INTRODUCTION
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Noxious information is carried through fine myelinated Adelta 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 Adelta 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.


    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 MOmega 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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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 MOmega (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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Fig. 1. Inhibitory postsynaptic currents (IPSCs) evoked in a substantia gelatinosa (SG) neuron by cutaneous mechanical stimuli. A brush produced a barrage of IPSCs that persisted during the stimulus (left), while a pinch evoked IPSCs only at its beginning and end (right). In this and subsequent figures, bars given above the traces show the duration of the stimuli or drug application; VH = 0 mV.

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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Fig. 2. Glycine- or GABA-mediated spontaneous and evoked IPSCs. A and B: strychnine (4 µM; A) or bicuculline (20 µM; B) blocked spontaneous IPSCs. Note that the strychnine-sensitive IPSC (inset in A) exhibits a shorter duration by about 3-fold compared with the bicuculline-sensitive one (inset in B). C and D: brush-evoked IPSCs (top in C and D) were blocked by strychnine (4 µM; bottom in C) or bicuculline (20 µM; bottom in D). Each of the records in A-D was obtained from a different SG neuron; VH = 0 mV.

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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Fig. 3. IPSCs and excitatory postsynaptic currents (EPSCs) evoked by mechanical stimuli and possible neuronal circuitry in the SG of the spinal dorsal horn. A and B: a brush evoked a sustained activity of IPSCs during the stimulus (left in A), whereas a pinch elicited IPSCs only at its beginning and end (right in A) at 0 mV in a SG neuron; when examined at -70 mV in the same neuron (B), both elicited a persistent barrage of EPSCs during the stimuli. C: possible neuronal circuitry in the SG. Although excitatory information of pinch or touch is tentatively supposed to be monosynaptically transmitted through Adelta or C fibers to the SG, polysynaptic transmission also exists. DRG, dorsal root ganglia.


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INTRODUCTION
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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 Adelta 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 Adelta fibers. Figure 3C demonstrates our hypothesis that a touch, whose information is conveyed through Adelta 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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