Ncm'oscie#lce Letters, 142 (1992) 191 195 :~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92,/$ 05.00

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Miniature postsynaptic currents recorded from identified rat spinal dorsal horn projection neurons in thin-slice preparations Yuuichi H o r i ~' and K a t s u a k i E n d o h 'Department of Physiolo,~y, l£vorin b~uversity School ol' Medicine. To!,yo (Japan) and *'Department of Physiolo,ey, Kvoto University Faculty ,l Medicine, Kvoto (Japan) (Received 6 March 1992: Revised version received 30 April 1992: Accepted 4 May 1992)

Key words: Spinothalamic neuron: Spinomcsencephalic neuron: Thin slice preparation: Miniature postsynaptic current: Patch-clamp recording: 2-Methyl-serotonin Whole-cell vohage-clamp recordings were made from spinothalamic and spinomesencephalic tract neurons in thin-slice preparations of rat spinal cord. In the presence of tetrodotoxin, spontaneous inward and outward postsynaptic currents were observed near the resting membrane potential. These currents were divided into miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate, and miniature inhibitory postsynaptic currents (mlPSCs) mediated by glycine or ?'-aminobutyric acid (GABA). Glutamatergic mEPSCs had two components mediated by NMDA and non-NMDA receptors. Analyzing these miniature synaptic currents, valuable information concerning the pre- and postsynaptic mechanisms underlying modulation of synaptic transmission in the spinal dorsal horn could be obtained.

Somatosensory information from the periphery enters the spinal cord dorsal horn, is modulated there and conveyed to the supraspinal structures including the thalamus, midbrain regions and brainstem reticular formation [25]. The dorsal horn includes non-projection interneurons and projection neurons. Different subclasses of neurons in the dorsal horn have different intrinsic membrane properties and different synaptic inputs. which in turn determine the role of particular neurons in somatosensory processing. Therefore, it seems of particular interest to apply electrophysiological techniques. such as the patch-clamp recording method, to an identified neuron. Since the patch-clamp technique requires the electrode to form a tight seal with cell membrane, most studies of mammalian central neurons that use this recording technique have been carried out on cultured [8, 17] or acutely isolated neurons [6, 12, 13]. Recently, Edwards et al. have described how to perform patch-clamp recording from neurons in thin-slice preparations of the central nervous system [5]. Modifying this method, Chen and Huang applied the patch-clamp technique to ascending tract neurons in thin slices of rat medulla identified by

Corre.q~ondence: Y. Hori, Department of Physiology, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181. Japan.

retrograde labelling with fluorescent dye, and described ionic currents in trigeminothalamic tract neurons [3]. In the present study, we made whole-cell recordings from dorsal horn projection neurons, including spinothalamic tract (STT) and spinomesencephalic tract (SMT) neurons, identified in thin slices of rat spinal cord. These ascending tract neurons are thought to play a key role in some aspects of pain sensation [14, 25]. Since local neuronal circuitry is undamaged in the slice preparation, we could record postsynaptic currents which occur spontaneously and those evoked by electrical stimulation of single neighboring internuncial neurons. This paper describes the characteristics of spontaneous miniature postsynaptic currents. A 7 14-day-old Wistar rat was anesthetized with ether and then positioned in a stereotaxic apparatus. To label STT neurons, Fast blue (FB, 4% in normal saline, total volume of 1- 2/J1) [1] or a rhodamine bead suspension (RB, 1-2/all [16] was injected into the ventrolateral part of the thalamus on both sides. For SMT neurons, a total of 1 -5/,1 of FB or RB was iniected into the periaqueductal gray and the adjacent mesencephalic structures. RB is considered superior to FB as a fluorescent label for dissociated neurons [6, 12], since FB leaks out of cells during an isolation procedure. In slice preparations, however, FB-labeled neurons were as easily detectable tinder the fluorescence microscope as RB-labeled neurons.

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Also, 11o significant differences in spontaneous or evoked postsynaptic currents were noted between FB- and RBlabeled neurons. After 2 5 days, the rat was reanesthetized with ether, and the lumbar spinal cord was removed from the animal. Using a Microslicer (DTK-1000), 120-150 /am cross-sections were made in cold oxygenated Krebs solution [5, 22]. A slice was placed in a recording chamber under a fluorescent microscope equipped with Nomarski optics. The slice was continuously perfused with Krebs solution. The solution was composed of (in raM) NaC1 113, KC1 3, NaHCO3 25, NaH2PO4 1, CaCL 2, MgC12 1 and D-glucose 11. The pH of the solution was 7.4 when saturated with 95% 02/5% CO2. First, the slice was checked for labeled cells by viewing it under fluorescent optics (Fig. 1A). Then, a labeled neuron was viewed under Nomarski optics. Debris covering the cell was removed by applying positive pressure through a pipette (10/am in orifice diameter) filled with Krebs solution [5]. Conventional patch pipettes were used for tight-seal whole-cell recording from the neurons. The pipettes were filled with an internal solution composed of (in mM) Cesium acetate 126, KC1 14~ sodium gluconate 2, EGTA 1 and HEPES 10, neutralized to pH 7.4 with KOH. In some experiments, Cesium acetate was replaced by CsCI to explore the involvement of C1- ions in the generation of synaptic currents. The DC resistance of the pipette was ~ 1 0 Mr2. The perfusates routinely contained tetrodotoxin (TTX, 0.34).5/aM) to block the generation of action potentials. Strychnine (STR, 0.5-1/aM), bicuculline (BIC, 10/aM), kynurenic acid (KA, 1-2 mM), (+)-2-amino-5phosphonovalerate (APV, 50/aM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 /aM), and 2-methyt-serotonin meleate (2-Me-5-HT, 1 /aM) were dissolved in Krebs solution and applied by switching the perfusion lines. All experiments were carried out at room temperature (20-23°C). The recorded data were stored in a PCM tape recorder (sampling frequency: 10 kHz) and analyzed by computer. With action potentials blocked by TTX, the spontaneous postsynaptic currents were recorded at a holding potential of -40 mV (Fig. 1B1). These synaptic currents were either inward or outward in direction. Since they were recorded in the presence of TTX, they were independent of action potentials and can be identified as the miniature postsynaptic currents (mPSCs). Application of the glycine receptor blocker STR and the GABAA receptor blocker BIC abolished outward mPSCs (Fig. I B2). The remaining inward mPSCs were abolished by the non-specific glutamate receptor antagonist KA (Fig. 1B3). The KA-sensitive mPSCs were inward when

clamped near the resting potential but reversed polarit 5 when clamped above 0 mV (Fig. 1B4). Iheir average reversal potential was -6.9+_7.1 mV (mean+S.l)., n:-61, and they can be identified as miniature excitator? postsynaptic currents (mEPSCs) mediated by glutamate receptors. The frequency of mEPSCs was 0.85_+0.5 Hz (n=21). Their mean amplitude at -70 mV was 24.1_+ 14.5 pA (n= 16). In the presence of TTX and KA, only outward mPSCs were observed at -40 mV (Fig. 1CI). STR depressed the mPSC frequency (Fig. 1C2). The remaining outward mPSCs were abolished by BIC (Fig. IC3). The reversal potential of mPSCs recorded in the presence of KA was -49.5_+10.2 mV (n=5) when the electrode solution contained 14 mM C1 , and +8.2-+9.5 mV (n=4) with 140 mM CI-. The CI equilibrium potential is estimated to be -55 mV and +4 mV in these two conditions, respectively. Therefore, it seems clear that they were mediated mainly by C1 ions and can be identified as miniature inhibitory postsynaptic currents (mlPSCs) elicited by either glycine or GABA [21]. The sensitivity of mlPSCs to BtC indicates the involvement of GABAA receptors, although GABAu as well as GABA A receptors have been localized in the spinal cord [20]. The involvement of Cl ions in the presently recorded mlPSCs is consistent with the previous reports that glycine and GABAA receptors mediate the increase in CI- conductance [8]. The frequency of the STR- and BIC-sensitive mlPSCs was 1.4-+0.5 Hz (n= 12) and 0.8_+0.5 Hz (n=8). Their mean amplitude at -40 mV was 18+6.5 pA (n=10) and 20_+11 pA (n=7), respectively. In the vertebrate central nervous system, the actions of glutamate are known to be mediated by several different types of receptors, such as N-methyl-D-aspartate (NMDA) and quisqualate/kainate (non-NMDA) receptors [24]. We studied whether NMDA receptors were involved in the recorded mEPSCs, mEPSCs at -70 mV and +50 mV in the presence of Mg > (1 mM) are shown in Fig. I DI and D2. When the membrane was depolarized. a slow component to the mEPSC became apparent (Fig. I D2). The slow component was also clearly seen in Mg2+-free bathing solution (Fig. 1D3). The selective NMDA receptor antagonist APV abolished the slow component (Fig. 1D4). In contrast, the selective nonNMDA receptor antagonist CNQX abolished the early component (Fig. ID5). These observations indicate the involvement of NMDA receptors in the generation of the late component [4, 17-19]. The frequency of mPSCs reflects the condition of the presynaptic membrane, and their amplitude is mainly related with the condition of the postsynaptic membrane [15]. Analyzing the effects of opiates on the frequency and the amplitude of mEPSCs recorded from superficial dorsal horn neurons, we have recently shown that opi-

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40 msec Fig. 1. A: e x a m p l e of an STT n e u r o n identified by r e t r o g r a d e labeling with FB. Viewed u n d e r a fluorescent microscope. This n e u r o n was located in l a m i n a V of the spinal d o r s a l horn. B: m E P S C s recorded from an S M T neuron. (1 3) at a h o l d i n g p o t e n t i a l of - 4 0 mV, and (4) at +46 mV. C: m I P S C s recorded from an STT n e u r o n at

40 inV. D: N M D A a n d n o n - N M D A c o m p o n e n t s o f m E P S C s recorded from an S M T neuron. Each record is an a v e r a g e of 20 consecutive m E P S C s . F o r t k m h e r details, see text.

ates inhibit spontaneous mEPSCs by the presynaptic mechanisms [11]. The same approach being taken in the present experiment, we have studied the effects of the selective serotonin3 receptor agonist 2-methyl-serotonin (2-Me-5-HT) in an attempt to describe applicability of mPSCs analysis to the study of synaptic transmission onto somatosensory ascending tract neurons. The serotonim receptor subtype has been localized in the rat spi-

nal dorsal horn [9], and its activation leads to modulation of nociceptive transmission [7]. Following bath application of 2-Me-5-HT, the mEPSC frequency increased in all 7 neurons studied (Fig. 2A,B). This stimulatory effect was observed without any clear changes in the amplitude distribution or the mean amplitude of mEPSCs (Fig. 2C). From these observations, we conclude that 2-Me-5-HT increases mEPSC

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Fig. 2. A: sample recordings from an SMT neuron before and during 2-Me-5-HT application. B: time course of 2-Me-5-HT action. The period of drug application (40 s) is indicated by a bar. C: amplitude histograms of mEPSCs before (upper column) and during (lower column) drug application. Arrows indicate the mean amplitude of mEPSCs. The same number of mEPSCs (80 events) were sampled for both histograms. The length ofsampling period was 90 s (control) and 45 s (2-Me-5-HT), corresponding each to 0.89 Hz and 1.78 Hz, respectively.

f r e q u e n c y t h r o u g h a p r e s y n a p t i c m e c h a n i s m . In the n o m i n a l a b s e n c e o f e x t r a c e l l u l a r C a 2÷, 2 - M e - 5 - H T no l o n g e r affected the m E P S C f r e q u e n c y (in 3 neurons, d a t a n o t shown). O n e p o s s i b i l i t y is t h a t 2 - M e - 5 - H T e n h a n c e s v o l t a g e - g a t e d C a 2+ e n t r y by d e p o l a r i z i n g nerve terminals. S e r o t o n i n 3 r e c e p t o r has been r e p o r t e d to elicit a m e m b r a n e d e p o l a r i z a t i o n with a n increase in c a t i o n c o n d u c t a n c e [2, 10, 23]. F u r t h e r s t u d y is n e e d e d to clarify the precise m e c h a n i s m s o f the p r e s y n a p t i c 2 - M e - 5 - H T action. A n d it is expected t h a t the a p p r o a c h d e s c r i b e d here c a n e l u c i d a t e the pre- a n d p o s t s y n a p t i c m e c h a n i s m s o f somatosensory modulation. Finally, s y n a p t i c i n p u t s m e d i a t e d b y N M D A , n o n N M D A , glycine a n d G A B A r e c e p t o r s o n t o a s c e n d i n g t r a c t n e u r o n s in the spinal d o r s a l h o r n seem to p l a y s o m e role in c o n t r o l l i n g s o m a t o s e n s o r y o u t p u t to the s u p r a s p i nal structures.

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