cAMP-Dependent Presynaptic Regulation of Spontaneous Glycinergic IPSCs in Mechanically Dissociated Rat Spinal Cord Neurons

Shutaro Katsurabayashi, Hisahiko Kubota, Zhi Ming Wang, Jeong Seop Rhee, and Norio Akaike

Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Katsurabayashi, Shutaro, Hisahiko Kubota, Zhi Ming Wang, Jeong Seop Rhee, and Norio Akaike. cAMP-Dependent Presynaptic Regulation of Spontaneous Glycinergic IPSCs in Mechanically Dissociated Rat Spinal Cord Neurons. J. Neurophysiol. 85: 332-340, 2001. Spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with intact glycinergic presynaptic nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were investigated using nystatin-perforated patch and conventional whole cell recording modes under the voltage-clamp conditions. Trans-ACPD (tACPD) reversibly reduced the mIPSC frequency without affecting the mean amplitude. The effect was mimicked by a specific metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory effects on mIPSC frequency were blocked by the specific antagonists for mGluRII, alpha -methyl-1-(2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine and (RS)-alpha -cyclopropyl-4-phosphonophenylglycine. In the slice preparation, eIPSC amplitude and mIPSC frequency were decreased reversibly by L-CCG-I (10-6 M) and L-AP4 (10-6 M). In K+-free or K+-free external solution with Ba2+ and Cs+, Ca2+-free or Cd2+ external solution, the inhibitory effect of tACPD on mIPSC frequency was unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic glycine release, and prevented the inhibitory action of tACPD on mIPSC frequency. Sp-cAMP, however, did not prevent the inhibitory action of tACPD on mIPSC frequency. It was concluded that the activation of mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glutamate is one of major excitatory neurotransmitters in the CNS. The receptors are classified as ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) (Nakanishi 1992). The iGluRs mediate fast synaptic transmission through ligand-gated ion channels, and the mGluRs are coupled to second-messenger systems that are responsible for slower synaptic events. Recently eight mGluR subtypes have been cloned (mGluR1-mGluR8) (Pin and Duvosin 1995; Riedel 1996). The mGluRs have been further divided into three main groups according to their sequence homology, signal transduction mechanism, and agonist selectivity. The mGluR subgroup I includes mGluR1 and mGluR5, which are coupled to phosphoinositide hydrolysis (Abe et al. 1992). Subgroup II (mGluRII) contains mGluR2 and mGluR3, and subgroup III (mGluRIII) includes mGluR4, mGluR6-mGluR8. Both mGluRII and mGluRIII are negatively coupled to adenylyl cyclase (AC) (Tanabe et al. 1992, 1993). The modulations of these mGluRs on synaptic transmission enhance or suppress the release of inhibitory and excitatory neurotransmitters, such as GABA and glutamate, from nerve terminals (Glitsch et al. 1996; Poncer et al. 1995). Several reports show that these actions involve multiple mechanisms. For example, mGluRs inhibit Ca2+ channels or activate the K+ channels of presynaptic terminals (Cochilla and Alford 1998; Takahashi et al. 1996). Also, the mGluRs have direct effects on the vesicle releasing mechanism (Poncer et al. 1995) and elicit Ca2+ release from presynaptic Ca2+ stores (Peng 1996).

Most mGluRs, with exception of mGluR6 and mGluR8, are distributed throughout the spinal cord (Valerio 1997; Watkins and Evans 1981). These mGluRs have dual effects on the synaptic transmission in the spinal cord, potentiating or suppressing the release of excitatory or inhibitory neurotransmitters (Bond and Lodge 1995; Cao et al. 1997; Jane et al. 1996; King and Liu 1996). The sacral dorsal commissural nucleus (SDCN) of spinal cord surrounding the central canal receives abundant afferent inputs from both the visceral and somatic organs. The convergence of the visceral and somatic inputs on neurons in the SDCN has been demonstrated electrophysiologically and anatomically (Honda 1985). Also, the SDCN is known to be involved in nociceptive, analgesic transmission and autonomic functions of visceral and somatic inputs (Ding et al. 1994; Honda 1985; Vizzard et al. 1995). In the SDCN area, glycine is one of the major inhibitory neurotransmitters (Basbaum 1988; Yoshimura and Nishi 1995). However, the releasing mechanism of glycine from glycinergic presynaptic terminals projecting to the SDCN neurons is poorly understood. The intracellular signaling mechanisms underlying the mGluR modulation of glycine release are also not known.

In the present study therefore, we investigated the mechanism of the mGluR modulation of the spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs). The preparations made from mechanically dissociated rat SDCN neurons attached with native presynaptic boutons were used that combine the advantages of a simple, reduced system while maintaining native synaptic functions. The activation of metabotropic glutamate receptors on the presynaptic glycinergic terminals reduces transmitter release by interfering with cAMP/PKA pathway.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanical dissociation

Ten- to 14-day-old Wistar rats were decapitated under pentobarbital anesthesia. The spinal cord was quickly removed from the vertebral canal and was sliced at a thickness of 400 µm with a microslicer (DTK-1000, Dosaka, Kyoto, Japan). The slices were kept in the incubation medium saturated with 95% O2-5% CO2 at room temperature (22-25°C) for >= 1 h. Thereafter the slices were transferred into a 35-mm culture dish (Primaria3801, Becton Dickinson, NJ) and the SDCN of the spinal cord was identified under a binocular microscope (SMZ-1, Nikon, Tokyo). A fire-polished glass pipette was touched lightly onto the surface of the SDCN region and was vibrated horizontally at 3-5 Hz for ~2 min by apparatus developed by our laboratory. Then the slices were removed from the dish. The mechanically dissociated SDCN neurons adhered to the bottom of the dish within 10 min. These neurons, which were dissociated without using any enzymes, retained their original morphological features, including proximal dendritic processes.

Slice preparation

Thirteen- to 15-day-old Wistar rats were decapitated under pentobarbital anesthesia. The spinal cord was quickly removed from the skull and was sliced at a thickness of 230 µm with a microslicer (VT-1000S, Leica, Germany) in cold Na+-free (including sucrose) medium. Then the slices were kept in incubation medium saturated well with 95% O2-5% CO2 at 30-35°C for ~1 h. Thereafter the slices were transferred into a recording chamber, and the SDCN of the spinal cord was identified under an upright microscope (Axioscope, Zeiss, Germany). The bath solution was perfused at 8-10 ml/min.

All experiments conformed the Guiding Principles for the Care and Use of Animals approved by the Council of the physiological Society of Japan, and all efforts were made to minimize the number of animals used and their suffering.

Electrical measurements

Electrical measurements were performed in the dissociated neurons attached with synaptic boutons using the nystatin-perforated-patch recording mode and in the slice preparation using the whole cell patch recording mode under voltage-clamp conditions. The nystatin-perforated-patch technique but not whole cell patch technique was applied to dissociated neuron. When the nystatin-perforated-patch recording mode was applied to the dissociated neurons, the technique allows a long time recordings not by like conventional whole cell patch recording mode. However, the conventional whole cell patch recording mode was used for slice preparation. In this preparation, either nystatin- or whole cell patch technique is enough to record cell response for >= 3 h because the conventional whole cell patch technique was much easier and convenient to record the responses from slice preparation. Patch pipettes were made from borosilicate glass tubes (1.5 mm OD, 0.9 mm ID; G-1.5, Narishige, Tokyo) in two stages on a vertical pipette puller (PB-7, Narishige). The resistance of the recording electrode was 5-7 MOmega . The neurons were visualized with phase-contrast equipment on an inverted microscope (Diapot, Nikon). The current and voltage were measured with a patch-clamp amplifier (EPC-7, List-Electronic, Germany), monitored on both an oscilloscope (Tektronix 5111A, Sony, Tokyo) and a pen recorder (Recti-Horiz 8K, Nippondenki San-ei, Tokyo), and stored on videotapes (PCM-501 ES, Sony). The membrane currents were filtered at 1 kHz (E-3201A Decade Filter, NF Electronic Instruments, Tokyo), and data were digitized at 4 kHz. The evoked obtained were performed by applying short (100 µs) voltage pulses at 0.1 Hz through glass pipette (phi : 10 µm), which was placed around central canal and filled with the incubation solution, using the PULSE program on a Macintosh computer (HEKA). The signals were filtered at 3 kHz and digitized at 10 kHz.

Data analysis

Events were counted and analyzed using DETECTiVENT (Ankri et al. 1994) and IGOR PRO software (Wavemetrics, Lake Oswego, OR), and the time to peak and decay time of individual mIPSCs were analyzed using pCLAMP software (Axon Instruments). Analysis of mIPSCs was performed with cumulative probability plots. Cumulative amplitude histograms were compared using the Kolmogorov-Smirnov test for significant difference (P < 0.05). Numerical values are provided as means ± SE. Differences in amplitude and frequency distribution were tested by paired two-tailed t-test.

Solutions

The ionic composition of the incubation medium was (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 24 NaHCO3, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose. The pH of the incubation medium was adjusted to 7.4 with 95% O2-5% CO2. The ionic composition of the external standard solution was (in mM) 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES. Ca2+-free external solution contained (in mM) 150 NaCl, 5 KCl, 3 MgCl2, 10 glucose, 10 HEPES, and 2 EGTA. The ionic composition of the Na+-free medium was (in mM) 230 sucrose, 2.5 KCl, 1.25 Na2HPO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 30 glucose. The composition of K+-free external solution was (in mM) 147 NaCl, 5 CsCl, 5 BaCl2, 1 MgCl2, 10 glucose, and 10 HEPES. These external solutions were adjusted pH 7.2 with Tris-OH.

In recording mIPSCs, these solutions routinely contained 3 × 10-7 M tetrodotoxin (TTX) to block voltage-dependent Na+ channels, 3 × 10-6 M bicuculline to block the GABAA response, and 10-6 M 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and 10-5 M DL-2-amino-5-phosphovaleric acid (DL-AP5) to block glutamatergic responses. The ionic composition of the internal (patch pipette) solution for the nystatin-perforated-patch recording was (in mM) 20 N-methyl-D-glucamine methanesulfonate, 20 Cs-methanesulfonate, 5 MgCl2, 100 CsCl, and 10 HEPES. The pH of internal solution was adjusted to 7.2 with Tris-OH. Nystatin was dissolved in acidified methanol at 10 mg/ml. The stock solution was diluted with internal solution just before use to a final concentration of 100-200 µg/ml. The ionic composition of the internal (patch pipette) solution for the whole cell patch recording was (in mM) 43 CsCl, 92 Cs-methanesulfonate, 5 TEA-Cl, 2 EGTA, 4 ATP-Mg, and 10 HEPES. The pH of internal solution was adjusted to 7.2 with Tris-OH.

Drugs

Drugs used in the present study were AP5, bicuculline, CNQX, EGTA, nystatin, forskolin, 1,9-dideoxy-forskolin, 8Br-cAMP, Sp-cAMP, Rp-cAMP, and alpha -bungarotoxin (Sigma); ATP (Yamasa, Japan); TTX and strychnine (Wako Pure Chemicals, Japan); and 2-amino-4-phosphonobutyrate (L-AP4), (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), alpha -methyl-1- (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (MCCG), and (RS)-alpha -cyclopropyl-4-phosphonophenylglycine (CPPG) (Tocris, UK).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Spontaneous glycinergic mIPSCs

The spontaneous postsynaptic currents were recorded from the acutely dissociated rat SDCN neurons attached with the native presynaptic nerve endings, namely "synaptic bouton preparation." The recording utilized the nystatin-perforated-patch recording mode at a holding potential (VH) of -60 mV. Strychnine abolished the spontaneous inhibitory currents in a reversible and a dose-dependent manner in the presence of 3 × 10-7 M TTX, 10-5 M 2-amino-5-phosphonovaleric acid, 10-6 M CNQX, and 10-6 M bicuculline (Fig. 1A). Since it is reported that strychnine is also a potent competitive antagonist of nicotinic acetylcholine receptors (nAChRs) having alpha 7 subunits in the rat hippocampal neurons (Matsubayashi et al. 1998), we examined whether SDCN neurons have nicotinic components or not. Thus the effect of alpha -bungarotoxin (alpha -BTx) (10-7 M), a selective antagonist of alpha 7-containing nAChRs, was tested on the spontaneous postsynaptic current. The alpha -BTx did not alter the frequency or amplitude of the mIPSCs, indicating that nicotinic component does not exist (data not shown).



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Fig. 1. Spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs). A: in the presence of 3 × 10-7 M TTX, 10-5 M 2-amino-5-phosphonovaleric acid (APV), 10-6 M 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), and 3 × 10-6 M bicuculline (Bic.), strychnine (Str.) blocked the mIPSCs in a dose-dependent manner at a VH of -60 mV. B: current-voltage (I-V) relationships for mIPSCs. a: representative spontaneous mIPSCs at various VHs. b: I-V relationship of mIPSCs. The average current amplitude represents the peak amplitude during the 3-min sampling period.

Representative spontaneous glycinergic mIPSCs were also investigated at various VHs (Fig. 1B). The average current amplitude of the mIPSCs during the 3-min sampling period was plotted against to each VHs. The reversal potential of mIPSCs estimated from the current-voltage (I-V) relationship was about -10 mV. The value was almost identical to the theoretical Cl- equilibrium potential (ECl) of -10.4 mV calculated from Nernst equation using 161 mM [Cl-]o and 110 mM [Cl-]i (Fig. 1Bb). These data clearly indicate that the spontaneous miniature postsynaptic currents are glycinergic mIPSCs.

Inhibitory effect of tACPD on glycinergic mIPSCs

The effect of trans-ACPD (tACPD), a metabotropic glutamate agonist, on glycinergic mIPSCs was examined. In the present study, SDCN neurons were subdivided to three groups according to the cell size: i.e., small (<15 µm), middle (15-30 µm), and large (>30 µm). Among 92 dissociated SCDN neurons in the presence of 10-5 M tACPD, the mIPSC frequency decreased in 69 neurons, which were middle- and large-size neurons. The tACPD increased the mIPSC frequency in 13 small neurons. There was no change in 10 neurons, which were small- and middle-size neurons. In the following experiments, therefore we used the middle-size neurons.

The tACPD (10-5 M) reversibly suppressed mIPSC frequency but had no effect on the distribution of the current amplitudes (Fig. 2, A and C), indicating that the probability of glycine release decreased. In fact, the mIPSC frequency significantly decreased to 72.7 ± 1.7% of control (P < 0.01, n = 69) in the presence of tACPD, whereas the mean mIPSC amplitude was 100.8 ± 1.3% of control (n = 69; Fig. 2B). Figure 2C indicates the superimposed amplitude histograms of mIPSCs with () or without () tACPD for 3 min. The amplitude distribution of the mIPSCs was skewed toward large-amplitude events. There was a clear decrease in the number of mIPSC events at all amplitudes in the presence of tACPD. However, no significant difference in the distribution of mIPSC amplitudes was detected between control and tACPD-treated neurons (P = 0.4328 by Kolmogorov-Smirnov test, number of events 297 for control and 187 for tACPD). Figure 2C, inset, shows the superimposed typical mIPSCs with or without tACPD. The decay time, tau , was measured and averaged for 38 individual events in each neuron for control and tACPD-treated neurons. Both decay times of each group were fitted by a single-exponential function. The means of tau  values for control and tACPD-treated neurons were 19.6 ± 1.3 and 18.8 ± 1.1 ms (n = 9), respectively. There was no significant difference in the time to peak and tau  values between control and tACPD-treated neurons. These results suggest that mGluR exists in the glycinergic nerve terminal and exerts an inhibitory effect on the presynaptic glycine release process without affecting the sensitivity of postsynaptic glycine receptors.



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Fig. 2. Inhibitory effect of trans-ACPD (tACPD) on mIPSCs. Aa: mIPSCs before, during, and after the application of 10-5 M tACPD. b: cumulative amplitude distribution (right) and cumulative frequency distribution (left) of mIPSCs with or without 10-5 M tACPD. The number of events used for cumulative distributions was 297 for control, 187 for tACPD, and 293 for after control during 3 min. B: statistical summary of results from the 69 neurons responded to 10-5 M tACPD. All amplitudes and frequencies are normalized to those of control mIPSCs. The vertical bar shows ±SE. **, statistically significant difference at P < 0.01. C: amplitude histograms from the Aa trace for control () and 10-5 M tACPD (). Inset: individual mIPSCs in the same neuron before and after the application of tACPD were superimposed.

Presynaptic mechanism of tACPD in glycinergic nerve terminal

Three kinds of inhibitory mechanisms could account for the effect of tACPD on glycine release. First, tACPD might suppress Ca2+ influxes through voltage-dependent Ca2+ channels (VDCCs) in the nerve terminal, resulting in decrease in exocytosis (Morishige et al. 1996; Takahashi et al. 1996). Second, tACPD might open K+ channels, thus hyperpolarizing the presynaptic membrane and reducing synaptic release (Cochilla and Alford 1998). Third, the agonist might activate intracellular messenger pathways that inhibit the release mechanism in presynaptic terminals (Trudeau et al. 1996a,b).

We, therefore examined the possible contribution of presynaptic VDCCs in the mediation of tACPD action on mIPSC. The removal of Ca2+ from external solution significantly decreased mIPSC frequency without affecting the distribution of their current amplitudes. Figure 3Aa shows a typical response of mIPSCs before, during, and after the application of tACPD in Ca2+-free external solution. tACPD decreased the mIPSCs frequency in a reversible manner without affecting the distribution of the current amplitudes (Fig. 3Ab). The inhibition of mIPSC frequency by tACPD was 77.8 ± 4.3% of control even in the Ca2+-free solution (P < 0.05, n = 5).



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Fig. 3. Effect of tACPD in either Ca2+-free or Cd2+-external solution. Aa: recording of mIPSCs before, during, and after the application of 10-5 M tACPD in Ca2+-free external solution. b: cumulative amplitude distribution (right) and cumulative frequency distribution (left) of mIPSCs in Ca2+-free external solution. B: averaged inhibition ratios of amplitude (right) and frequency (left) with tACPD, Ca2+-free plus tACPD, and Cd2+ plus tACPD. Each column is the mean of 5 neurons (**P < 0.01). The vertical bar shows ±SE.

Moreover, the application of 100 µM Cd2+, which completely blocks VDCCs, also failed to remove the inhibitory effect of tACPD on mIPSCs frequency (Fig. 3B). The inhibition was almost the same as that in Ca2+-free solution (78.3 ± 4.5% of control, n = 5). The data suggest that the inhibitory effect of mGluRs in the presence of tACPD is independent of Ca2+ influxes passing through VDCC.

It was investigated that the possible role of presynaptic K+ channels in the mediation of tACPD action on mIPSC frequency. Figure 4A shows the relationship between external K+ concentration ([K+]o) and spontaneous IPSCs frequency with or without TTX in the standard solution. The removal of K+ from the control solution slightly increased mIPSC frequency (P < 0.05, n = 5; Fig. 4A) but did not affect the distribution of the current amplitudes (data not shown).



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Fig. 4. Effect of tACPD in K+-free external solution. A: dependence of mIPSCs on extracellular K+ concentration ([K+]o). All points were normalized to the respective control mIPSCs frequency at 5 mM [K+]o with () or without () TTX. Each point is the average from 5 neurons. Ba: continuous recordings of mIPSCs before, during and after the application of tACPD in K+-free external solution at a VH of -60 mV. b: cumulative amplitude distribution (right) and cumulative frequency distribution (left) of mIPSCs in the K+-free external solution. C: pooled data of amplitude (right) and frequency (left) with tACPD and K+-free plus tACPD from 6 neurons (**P < 0.01).

Figure 4Ba shows a typical response of mIPSCs before, during, and after the application of tACPD in K+-free external solution, containing both Ba2+ and Cs+ to block the K+ conductance. Even in this K+-channel-blocking solution, tACPD reversibly suppressed mIPSC frequency to 78.6 ± 6.8% of control (P < 0.05, n = 6) but had no effect on the distribution of current amplitudes (Fig. 4B, b and c). The results suggest that the inhibitory effect of tACPD on mIPSC frequency is not due to the increase of presynaptic K+ conductance.

The last possibility is a presynaptic inhibition mediated through an intracellular signaling mechanism. The mGluRs could alter synaptic transmission by coupling to phosphoinositide hydrolysis or to adenylyl cyclase (AC) pathways via heterotrimeric G protein (Tanabe et al. 1992, 1993). Forskolin (10-5 M), a membrane permeable AC activator, nearly doubled the mIPSC frequency (195.5 ± 16.3% of control, P < 0.01, n = 9) without affecting the mean mIPSC amplitude (111.8 ± 8.4% of control, n = 9, Fig. 5A, a and b). After application of forskolin, it took 5 min for the frequency to become stable (Fig. 5Ab). The result indicates that forskolin is acting at a glycinergic presynaptic site (Fig. 5, A and B). The data also suggest that either cAMP or PKA might be related to the inhibition of glycine release from the presynaptic terminals.



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Fig. 5. Facilitation of mIPSC frequency by forskolin. Aa: representative recording of mIPSCs before and after application of 10-5 M forskolin. b: time course of event frequency of the top trace. The number of events in every 10 s duration was plotted. c: cumulative frequency distribution (left) and amplitude distribution (right). B: pooled data from 9 neurons. Note that forskolin increased the mIPSCs frequency without affecting the current amplitude (**P < 0.01).

Forskolin (10-5 M) completely prevented tACPD action on the mIPSCs frequency (Fig. 6A, a and b). Before the treatment of forskolin, tACPD reduced mIPSC frequency without affecting the mean mIPSC amplitude. During the treatment of forskolin in the same neuron, the inhibitory effect of tACPD on mIPSC frequency disappeared (102.4 ± 8.7% to control, n = 7) without altering the current amplitude (106.8 ± 3.4% to control, n = 7; Fig. 6, Ac and B). Recent reports indicate that forskolin enhances glutamate release by not only activating AC but also by promoting a Ca2+ influx mechanism (Hoshi et al. 1988; Lonart et al. 1998; Wagoner and Pallotta 1988). Thus it was examined whether 1-9-dideoxy-forskolin, an inactive form of forskolin, has an inhibitory effect on glycine release from the nerve terminals. The inactive form itself potentiated the frequency of mIPSCs (191.0 ± 33.0% of control, n = 5) without altering the current amplitude distribution. But tACPD still had an inhibitory effect on mIPSCs frequency (91.4 ± 5.3% of control, n = 5, figure not shown). Therefore these results suggest that this change of synaptic efficacy by tACPD is elicited via AC-cAMP/PKA pathway.



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Fig. 6. tACPD action on mIPSC-related cAMP/protein kinase A (PKA) pathway. Aa: recordings of mIPSCs before, during, and after the application of tACPD in the presence of forskolin at a VH of -60 mV. b: time course of event frequency of the top trace. The number of events in every 5-s duration was plotted. c: cumulative frequency distribution (left) and amplitude distribution (right). B: average ratios of mIPSCs frequency in the presence of forskolin, 8-Br-cAMP, Sp-cAMP, and Rp-cAMP with or without tACPD. Each column is the mean of 5 neurons (*P < 0.05).

The effect of cAMP analogues was further examined to test the direct action of cAMP/PKA pathway. Both 8-Br-cAMP, membrane permeable cAMP activator and Sp-cAMP, membrane permeable PKA activator, increased the frequency of glycine release (126.8 ± 6.9 and 133.1 ± 5.4% of control, respectively, n = 5). 8-Br-cAMP blocked completely the inhibitory action of tACPD (132.0 ± 2.0% of control, n = 5). Sp-cAMP, however, did not block the inhibitory effect of tACPD (96.0 ± 1.7% of control, n = 5). Rp-cAMP, a membrane permeable PKA inhibitor, facilitated the mIPSCs frequency (127.5 ± 4.02% to control, n = 5) without changing the current amplitude distribution (98.9 ± 2.57% to control, n = 5). Rp-cAMP, however, did not block the inhibitory effect of tACPD (96.7 ± 2.91% of control, n = 5; Fig. 6B). Thus these results could be suggested that the inhibitory action of tACPD related to cAMP but not PKA.

Subtype of mGluRs

To determine which subtypes of mGluRs participated in the inhibition of mIPSCs, the effects of mGluR agonists and antagonists were examined. Figure 7Aa shows the mIPSCs before, during, and after the application of a mGluRIII specific agonist, L-AP4 (10-5 M). L-AP4 mimicked the action of tACPD, and the inhibitory effect of L-AP4 disappeared in the presence of 10-4 M CPPG, a mGluRIII specific antagonist (Conn and Pin 1997). The L-AP4 application decreased reversibly the mIPSC frequency to 74.1 ± 4.2% of control (P < 0.01, n = 12) without affecting the mean amplitude (Fig. 7B). CPPG at 10-4 M itself did not affect cumulative distributions of mIPSCs (data not shown). mGluRII-specific agonist, 10-5 M L-CCG-I, also reduced mIPSC frequency (75.0 ± 4.6% to control) without affecting the amplitude distribution in the same cells (Fig. 7B). The inhibitory effect of L-CCG-I was blocked fully by 3 × 10-4 M MCCG, a specific mGluRII antagonist. These results indicate that the inhibitory effect of tACPD on the mIPSC frequency is mediated by at least mGluRII and/or mGluRIII in these glycinergic nerve terminals.



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Fig. 7. Subtypes of metabotropic glutamate receptors (mGluRs) in glycinergic presynaptic terminals. Aa: recording of mIPSCs before, during, and after the application of 10-5 M 2-amino-4-phosphonobutyrate (L-AP4) with (bottom) or without (top) 10-4 M (RS)-alpha -cyclopropyl-4-phosphonophenylglycine (CPPG) at a VH of -60 mV. b: cumulative frequency distribution (left) and amplitude distribution (right) of mIPSCs with or without L-AP4. B: averaged inhibition ratios of frequency of mGluR agonist without (left) or with antagonist (right). Each column is the mean of 11 neurons (**P < 0.01). C: averaged inhibition ratios of mIPSCs frequency and amplitude of tACPD with or without mGluR antagonists. Each column is the mean of 4 neurons (*P < 0.05).

We also examined the effect of mGluR antagonists on tACPD action. CPPG (10-4 M) reduced the inhibitory effect of tACPD (from 73.0 ± 3.5 to 82.5 ± 2.0% of control, n = 4), and 3 × 10-4 M MCCG also relieved the tACPD action in the same cells (to 79.0 ± 3.2% of control, n = 4; Fig. 7C). However, each antagonist did not completely block the inhibitory action of tACPD. The percent of inhibition by L-AP4 and L-CCG-I varied considerably among neurons. Some cells were only sensitive to one agonist, suggesting that the nerve endings of SDCN neurons have only one type of mGluRs. However, most cells were sensitive to both L-AP4 and L-CCG-I. Thus these data suggest that the mGluR subtypes such as mGluRII and mGluRIII in the glycinergic nerve terminals projecting on SDCN neuron exist heterogeneously.

We also examined the existence of mGluRII and/or mGluRIII on glycinergic nerve terminal in the slice preparation using the whole cell patch recording technique at VH of -60 mV.

SDCN neurons had also been subdivided to three groups according to the cell size as that observed in dissociated SDCN neurons. Therefore the experiment was performed on the same middle size neurons. Figure 8Aa shows that the glycinergic eIPSC before, during, and after the application of 10-6 M L-CCG-I and 10-6 M L-AP4, respectively. The application of L-CCG-I decreased reversibly the eIPSC amplitude (47.9 ± 3.9% of control, n = 6). After wash out, when the eIPSC amplitude recovered on control baseline, L-AP4 was applied. L-AP4 also decreased reversibly the eIPSC amplitude (50.0 ± 4.4% of control, n = 6, Fig. 8Ab).



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Fig. 8. Effects of mGluRII and -III agonists in glycinergic presynaptic terminals in the sacral dorsal commissural nucleus (SDCN) slice preparation. A: recording of glycinergic IPSCs evoked (eIPSCs) before, during, and after the application of 10-6 M (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I) and 10-6 M L-AP4 at a VH of -60 mV. a: the time course of relative eIPSCs in the application of L-CCG-I and L-AP4 (n = 6). Inset: the typical trace in each stage. b: mean amplitude of eIPSCs in the presence of L-CCG-I and L-AP4. Each column is the mean of 6 neurons. B: recordings of glycinergic mIPSCs in the presence of TTX at a VH of -60 mV. Typical traces (a) and cumulative frequency and amplitude distribution (b) of glycinergic mIPSCs with or without L-CCG-I and L-AP4.

Figure 8Ba shows effects of 10-6 M L-CCG-I and L-AP4 on glycinergic mIPSCs of the slice preparation in the presence of TTX. Both L-CCG-I and L-AP4 decreased reversibly the mIPSC frequency (47.9 ± 5.9, 54.6 ± 6.4% of control, respectively, n = 5) without affecting its current amplitude (Fig. 8Bb).

These results indicate that the inhibitory effects of mGluR on eIPSC amplitude and mIPSC frequency are mediated by the function of both mGluRII and mGluRIII in these glycinergic nerve terminals. Thus these data suggest that the mGluR subtypes such as mGluRII and mGluRIII in the glycinergic nerve terminals projecting on SDCN neuron also exist heterogeneously in the slice preparation.


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Multisynapses on the dissociated single neuron

In the present study, the slice preparation and the mechanically dissociated neurons without treating any enzymes were used. The "synaptic bouton preparation" preserves native synaptic boutons on isolated SDCN cell bodies. Using pharmacological isolation of the glycinergic IPSCs, the functional modulation of glycine release mediated by mGluRs. The amplitude distribution of the mIPSCs was skewed toward large amplitude events (Fig. 2), indicating that the dissociated single neuron has multiple intact functional synapses. The data are consistent with recordings from slice preparation (Zhou and Hablitz 1997). TTX reversibly decreased mIPSC frequency to 45.5% of control (P < 0.01, n = 52) but had no effect on the distribution of their current amplitudes. The decrease of mIPSCs frequency was not accompanied by a change in the current amplitude, indicating that the probability of glycine release decreases in the presence of TTX. In addition, TTX-sensitive Na+ channels that contribute to spontaneous glycine release in this isolated preparation evidently exist quite near to the nerve terminals.

Inhibitory mechanism of tACPD

It is reported that activation of K+ conductance and/or reduction of voltage-dependent Ca2+ currents induce presynaptic inhibition (Cochilla and Alford 1998; Takahashi et al. 1996). Figure 4A shows the effect of extracellular K+ concentration ([K+]o) in the standard solution. Both [K+]o-free and 10 mM [K+]o solution slightly depolarized the presynaptic terminals by different mechanisms. In [K+]o-free solution, the Na pump could be inhibited, resulting in depolarization (Akaike et al. 1992) while 10 mM [K+]o solution depolarized cell membrane as predicted from the Goldman-Hodgkin-Katz equation. Consequently, both conditions could activate VDCC (Akaike et al. 1992; Haage et al. 1998). Interestingly, although [K+]o-free and 10 mM [K+]o solutions increased the frequency of mIPSCs, the values of frequency change normalized at 5 mM [K+]o were not altered by TTX. The data suggest that TTX-sensitive Na+ channels in or near the presynaptic terminal might contribute little to spontaneous glycine release induced by [K+]o. Postsynaptically, tACPD indirectly activates a Ca2+-activated K+ currents (IKCa) via IP3, Ca/CaM pathway coupled to a GTP-binding protein (G protein) in rat hippocampal neurons (Shirasaki et al. 1994). In the present experiment, using an internal solution with Cs+ instead of K+, the activation of postsynaptic K+ channels could be excluded. In any case, the quantitative and kinetic analysis showed that tACPD suppressed only mIPSC frequency, but not the current amplitude, and that tACPD did not change time to peak or the decay time course of individual mIPSCs. These results indicate that tACPD acts selectively on the presynaptic glycine release process without altering postsynaptic glycine receptor sensitivity.

The VDCCs in the active zone of synapses are found in dense aggregates that have an important role in the triggering or the modulation of transmitter release in presynaptic nerve terminals (Haage et al. 1998; Stanley 1997; Takahashi et al. 1996). VDCC subtypes such as N, P, and/or Q types participate in the inhibitory modulation of excitatory synaptic transmission in nerve endings of CNS (Huang et al. 1996; Poncer et al. 1997). In our experiment, Ca2+ influxes via VDCC at the glycinergic presynaptic terminals did not contribute to the inhibitory effect of tACPD on mIPSCs since the tACPD inhibition of mIPSC frequency did not alter in either Ca2+-free or Cd2+-external solution.

The modulation of transmitter release by a Ca2+-independent mechanism has been also reported recently (Thomson et al. 1993; Trudeau et al. 1996a,b). Some studies revealed that the increase of cAMP/PKA directly activated presynaptic transmission in rat CNS neurons (Capogna et al. 1995; Chavez-Noriega and Stevens 1994; Chen and Regher 1997; Trudeau et al. 1996a,b). In the present experiment, the effect of tACPD was blocked by forskolin, which activates the AC cascade leading to cAMP activation of PKA (Zhang et al. 1997). tACPD, however, decreased glycine release in the presence of Sp-cAMP, a cAMP analogue that directly activated PKA. On the other hand, 8-Br-cAMP blocked the inhibitory effect of tACPD. Evidently, the cAMP receptor responsible for the increase in transmitter release is much more sensitive to 8-Br-cAMP than to Sp-cAMP. Thus cAMP itself contributes to presynaptic glycine release and the presynaptic inhibitory effect of tACPD could be mediated by decreasing the intracellular cAMP concentration ([cAMP]i) level. Therefore present results suggest that the inhibitory action of mGluR is mediated by the decrease of [cAMP]i but not the reduction of PKA in the nerve terminals.

Distribution of mGluR subtypes

The present study suggests that both mGluRII and mGluRIII exist in the presynaptic terminals of majority of SDCN neurons (Figs. 7 and 8). tACPD, L-AP4, and L-CCG-I reversibly suppressed the mIPSC frequency but had no effect on the distribution of the current amplitude in the dissociated neuron and also suppressed both mIPSC frequency and eIPSC amplitude in the slice preparation. The mean inhibition induced by tACPD, L-AP4, and L-CCG-I was almost the same, and yet mGluR antagonists alone could not block the inhibitory action of tACPD. Thus it was considered the possibility of three models about the various distributions of their mGluRs in the synaptic terminals. First, the postsynaptic neuron may have several presynaptic terminals in which each terminal has only one kind of mGluR subtypes (mGluRII or mGluRIII). Second, one kind of mGluR subtype may have an effect on each terminal, but the terminals with different subtypes are variously distributed on postsynaptic neurons. And last, the mGluR subtypes may coexist in the same presynaptic nerve terminal. In the present experiments, a few cells responded to only one of mGluR subtypes. However, the distribution of the magnitudes of the inhibition by each of the agonists varied considerably among the middle size cells tested, suggesting that most of SDCN neurons may have a heterogeneous distribution of presynaptic mGluR subtypes.

The first and third hypotheses could be supported by the slice experiment because both mGluRII and III agonists modulated the evoked glycine release in all neurons tested. These results suggest that if only the nerve ending coexisting of both mGluR subtypes was stimulated, the evoked glycine release could be modulated by both of agonists. On the other hand, if a few nerve endings in spite of existence of one kind of different mGluR subtype in each terminal were stimulated, the evoked glycine release could be also modulated by both of agonists.

Physiological role

SDCN receives abundant inputs from the raphe, locus coeruleus, and hypothalamus. The innervation of the sensory afferents to the SDCN region is involved in nociceptive transmission. The SDCN neurons could be subdivided into three regions according to the effective somatic and/or visceral inputs (Honda 1985). The size of neurons responsive to somatic stimulation (SOM neurons) was between 15 and 50 µm, while the neurons responsive to visceral (VISC neurons) or to both somatic and visceral stimulation (SOMVISC neurons) was <15 µm (Honda 1985). Thus the middle-size neurons used in the present experiments might be visceral neuron or somatic visceral neurons.

In the present study, tACPD induced presynaptic inhibition in the glycinergic nerve terminal of the dissociated SDCN neurons. The inhibition of glycine release might result in the depolarization of the postsynaptic VISC or SOMVISC neurons. Such a change of synaptic efficacy might enhance the sensation of pain from the visceral and somatic organs, resulting in hyperalgesia.


    ACKNOWLEDGMENTS

The authors thank Dr. M. C. Andresen and Dr. M. Brodwick for advice and critical reading of the manuscript.

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan to N. Akaike (10044301 and 10470009), and by Kyushu University Interdisciplinary Programs in Education and Projects in Research Development and by National Natural Science Foundation of China to Z. M. Wang (39625011 and 39800044).


    FOOTNOTES

Address for reprint requests: N. Akaike (E-mail: akaike{at}mailserver.med.kyushu-u.ac.jp).

Received 5 June 2000; accepted in final form 27 September 2000.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society