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1 Cellular and System Physiology
2 Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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Department of Physiology, Kurume University School of Medicine, Fukuoka Japan
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Research Division for Life Sciences, Kumamoto Health Science University, Kumamoto, Japan
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Abstract |
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(Received 23 April 2004;
accepted after revision 11 August 2004;
first published online 19 August 2004)
Corresponding author N. Akaike: Research Division for Life Sciences, Kumamoto Health Science University, Kumamoto, Japan. Email: akaike{at}kumamoto-hsu.ac.jp
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Introduction |
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The sacral dorsal commissural nucleus (SDCN) is located in the dorsal area of the central canal in the lower lumbar and sacral spinal cord, and is known to receive glycinergic, GABAergic, and mixed synaptic inputs (Katsurabayashi et al. 2001; Jang et al. 2002; Wu et al. 2002). To study the mechanisms involved in the three different synaptic inputs into the SDCN neurones, we recorded spontaneous miniature inhibitory post synaptic currents (mIPSCs), before and after the application of bafilomycin A1, a vacuolar-type H+/ATPase inhibitor, to examine the refilling profile of glycine and GABA into the synaptic vesicles in acutely isolated SDCN neurones with functional synaptic boutons remaining (so called ‘synaptic bouton’ preparations) (Rhee et al. 1999; Katsurabayashi et al. 2001; Jang et al. 2002; Akaike & Moorhouse, 2003). We report the differential profiles for glycine and GABA refilling into the synaptic vesicles in SDCN neurones after pretreatment with bafilomycin A1.
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Methods |
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The spinal cords of 8- to 12- day old (P8–12) Wistar rats were quickly removed during deep anaesthetization by intraperitioneal (I.P.) injection of pentobarbital (50 mg kg–1). Then spinal cord slices of 370 µm thickness were prepared from the lumbosacral (L5–S4) segment. Single sacral dorsal commissural nucleus (SDCN) neurones were mechanically dispersed from fthe resh spinal cord slice preparation to preserve functional presynaptic terminals as previously described (Katsurabayashi et al. 2001). The ionic composition of the internal (patch pipette) solution was (mM): 43 CsCl, 92 Cs-methanesulphonate, 5 TEA-Cl, 2 EGTA, 4 ATP-Mg, and 10 Hepes, which was adjusted to pH 7.2 with Tris-OH. The ionic composition of the external standard solution was (mM): 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes, which was adjusted to pH 7.4 with Tris-OH.
Focal stimulation of a single synaptic terminal
Focal electrical stimulation of a single bouton adherent to a mechanically isolated neurone has been previously described (Akaike et al. 2002). Briefly, 100 µs voltage pulses (30 V) were applied to a glass stimulating pipette (inner diameter about 0.5 µm) at a frequency of 0.2 Hz, using a stimulator (SS-202 J, Nihon Koden, Tokyo, Japan). The stimulating pipette was filled with standard external solution and was placed close to the surface of a single SDCN neurone from which a whole-cell recording was being made. The stimulating pipette was then carefully moved along the surface membrane of the soma or dendrites until an inward current appeared in an all-or-nothing fashion, indicating that the stimulating pipette was positioned just above a single bouton. It was confirmed that the current was evoked from a single bouton by observing whether it disappeared in an all-or-nothing fashion when the stimulus strength was reduced or when the stimulation pipette was shifted horizontally (see Akaike et al. 2002; Akaike & Moorhouse, 2003). When the evoked responses were glycinergic or GABAergic only, they were discarded from the present study.
Data analysis
Synaptic currents were counted and analysed using Mini-Analysis software (version 5.01, Synaptosoft Inc., GA, USA). The threshold for detection of mIPSCs was set at 3 pA above the background noise. The currents were filtered at 2 kHz and digitized at 10 kHz. Events were rejected or accepted on the basis of their rise and decay times. Rise times were determined between 10% and 90% of the peak amplitude of the IPSCs. The decay time constant of mIPSCs was initially obtained by fitting a double exponential function to the mIPSC decay from the time period corresponding to between 90% and 10% of the peak mIPSC amplitude. Individual events were fitted (with >200 iterations) to the function:
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fast and slow are decay time constants. When the relative contribution of one of the exponential distributions was <1%, the mIPSCs were considered to have a single exponential decay. Thus, the decision on whether a single mIPSC decayed with a single or with dual components was completely objective. Decay time constants of pure glycinergic and pure GABAergic mIPSCs pharmacologically isolated by 5 µM bicuculline and 100 nM strychnine were best fitted by a single exponential equation (Fig. 2) (See also Jonas et al. 1998; Nabekura et al. 2004). Differences in the amplitude distribution of mIPSCs were compared by non-parametric statistical analysis (Kolmogorov–Smirnov test). Differences were considered significant if P < 0.05. All results are expressed as means ± S.E.M.
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Throughout all the experiments, the external bath solution contained 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 10 µM D-2-amino-5-phosphonopentanoic acid (D-AP5) to block AMPA- and NMDA-type glutamatergic responses, respectively. All drugs were applied to the recording cell via a Y-tube microperfusion system. With this technique, the external solution surrounding a neurone could be exchanged within 20–50 ms (Murase et al. 1990; Katsurabayashi et al. 2001). Bafilomycin A1 (Tocris, USA) was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 500 µM, and this was added to the solution to give a final concentration of 1 µM. In order to almost exhaust the transmitters in the synaptic vesicles (Williamson & Neale, 1994; Zhou et al. 2000), some slices were incubated with bafilomycin A1 for 2 h at 37°C. The incubation solution contained (mM): 124 NaCl, 5 KCl, 1.2 KH2PO4, 24 NaHCO3, 2.4 CaCl2, 1.3 MgSO4 and 10 glucose, at pH 7.4 (equilibrated with 95% O2 and 5% CO2).
All experiments conformed to 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.
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Results |
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To record mIPSCs from the SDCN neurone, a whole-cell patch recording technique was used at a holding potential (VH) of –60 mV in the presence of 1 µM TTX, 10 µM CNQX and 10 µM D-AP5. Strychnine (100 nM), a glycine receptor antagonist, halved the mean amplitude and slightly decreased the frequency of mIPSCs (before strychnine: amplitude: 42.0 ± 4.1 pA, frequency: 0.8 ± 0.1 Hz; during strychnine: amplitude: 18.9 ± 3.4 pA, frequency: 0.6 ± 0.1 Hz, n = 4, Fig. 1Aa and Ab). Additional application of the selective GABAA receptor antagonist, bicuculline (5 µM), completely suppressed these remaining mIPSCs (Fig. 1Aa). After washing out strychnine and bicuculline, 5 µM bicuculline was again applied in order to pharmacologically isolate the pure glycinergic mIPSCs in the same neurone. Bicuculline also reduced the mean amplitude and the frequency of mIPSCs (before bicuculline: amplitude: 50.8 ± 5.4 pA, frequency: 0.6 ± 0.1 Hz; during bicuculline: amplitude: 33.9 ± 1.9 pA, frequency: 0.4 ± 0.1 Hz, Fig. 1Ac), and additional application of 100 nM strychnine completely blocked the remaining mIPSCs (data not shown). Decrease in the frequency of mIPSCs observed in the present experiments on the application of strychnine (Fig. 1Ab) or bicuculline (Fig. 1Ac) implies that glycine or GABA are accumulated into separate synaptic vesicles, and are spontaneously released from the presynaptic nerve terminal(s).
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gly = 13.9 ± 0.3 ms, n = 11) and were sensitive to strychnine, whereas, in the presence of 100 nM strychnine, all mIPSCs exhibited slow decay time constants (GABA = 39.2 ± 0.4 ms, n = 11) and were sensitive to bicuculline. In addition, mIPSCs with biphasic fast and slow decay time constants were also observed in the control (Fig. 1Aa). The decay time constants of these biphasic mIPSCs were well fitted by double exponential equations (fast and slow were 13.1 ± 0.6 ms and 41.0 ± 1.2 ms, respectively, Fig. 1Bb). The fast and slow time constants were very similar to those of the pharmacologically isolated gly and GABA, and the biphasic mIPSCs were abolished by the combined application of strychnine and bicuculline. The biphasic mIPSCs were still observed at a low concentration of extracellular Ca2+ solution (0.3 mM) without changing the proportion of mIPSCs (2 mM [Ca2+]o: 28.0 ± 1.9%, 0.3 mM [Ca2+]o: 29.1 ± 3.1%, n = 11, Fig. 1C). These data rule out the possibility that the biphasic mIPSCs could have arisen from superimposition of the individual fast- and slow-decaying mIPSCs. Therefore, it seems that the biphasic mIPSCs are mediated by corelease of both glycine and GABA from a single vesicle. There is strong evidence from these observations that the recorded mIPSCs are composed of three types of mIPSCs: i.e. pure glycinergic, pure GABAergic and mixed (glycinergic/GABAergic) mIPSCs.
To investigate whether the three types of mIPSCs are generated at specific synapses independently or concomitantly, we performed focal stimulation of a single synaptic bouton onto the SDCN neurone (Akaike et al. 2002; Jang et al. 2002; Murakami et al. 2002). Stimulation of a single synaptic bouton at 0.2 Hz elicited evoked IPSCs (eIPSCs) at a VH of –60 mV. The eIPSCs were all-or-nothing in response to a gradual increase in the stimulus intensity (see also Akaike & Moorhouse, 2003). As shown in Fig. 2A and B, three types of eIPSCs with distinct decay time constants were elicited irregularly, which were pharmacologically classified as glycinergic, GABAergic and mixed, as in the case of mIPSCs. The decay time constants for glycinergic and GABAergic eIPSCs (gly and GABA) were 9.0 ± 0.2 ms and 41.1 ± 3.2 ms, respectively, whereas those of the mixed eIPSCs (fast and slow) were 8.4 ± 0.6 ms and 42.2 ± 4.6 ms (n = 4, Fig. 2C), which were consistent with the values obtained with mIPSCs. We repeated the similar experiments and observed three types of eIPSCs in four other synaptic boutons, thereby indicating that this is a general feature at SDCN inhibitory synapses. In addition, application of 5 µM bicuculline or 500 nM strychnine also increased the failure rate of eliciting eIPSCs from 0.55 ± 0.02 (control) to 0.86 ± 0.03 and 0.75 ± 0.02, respectively (Fig 2D, n = 4). These observations, taken together, indicate that a single synaptic bouton contains three types of vesicles filled with pure glycine, pure GABA and a mixture of glycine and GABA, and could release them, in an independent manner.
Profiles of glycine and GABA refilling into the synaptic vesicles after bafilomycin A1
In order to study mechanisms involved in the three types of vesicles, corresponding to the three types of mIPSCs and eIPSCs, mIPSCs were recorded before and after the application of bafilomycin A1, a vacuolar-type H+/ATPase inhibitor.
Direct application of bafilomycin A1 to the synaptic bouton preparation dramatically increased the frequency of all mIPSCs, which then slowly decreased. However, the treatment with bafilomycin A1 for up to 1 h did not abolish the mIPSCs completely (data not shown). Therefore, the acute spinal slice preparations including SDCN were incubated in a solution containing bafilomycin A1 for 2 h at 37°C, so that the uptake of neurotransmitters into the synaptic vesicles would be expected to be completely inhibited (Williamson & Neale, 1994; Zhou et al. 2000). After incubation for 2 h, we obtained synaptic bouton preparations of SDCN neurones from these slices. Figure 3Aa shows a typical trace of mIPSCs recorded from a SDCN neurone, just after setting up the neurone in the recording system in normal external solution. During the recording period, over 50 min, the input membrane resistance of neurones was not changed. As shown in Fig. 3A, prolonged treatment with bafilomycin A1 had almost completely suppressed the mIPSCs, and then the frequency and amplitude of the mIPSCs gradually recovered during rinsing with normal external solution.
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To further study the mechanisms for the partial recovery of the mixed mIPSCs, we divided the mixed mIPSCs into two components (yfast and yslow) and estimated the amplitude of each component during the washing out of bafilomycin A1, according to the method reported previously (Jonas et al. 1998; Nabekura et al. 2004). As shown in Fig. 3Cb, the mean amplitude of the yfast component at 30–35 min after washing out bafilomycin A1 showed almost complete recovery to the control level (control: 32.1 ± 2.3 pA, 30–35 min: 23.6 ± 3.6 pA, P = 0.16, Fig. 3Cb), whereas the amplitude of yslow at 30–35 min showed only partial recovery (control: 21.2 ± 1.6 pA, 30–35 min: 8.4 ± 1.9 pA, P = 0.004, Fig. 3Cb).
Next, we examined whether prolonged treatment with bafilomycin A1 affects the postsynaptic glycine and/or GABAA receptors. The sensitivities of the SDCN neurone to exogenously applied glycine or GABA were not affected by treatment with bafilomycin A1 (Fig. 4: glycinecontrol: 255.7 ± 19.0 pA, glycineBaf. 263.7 ± 14.5 pA; GABAcontrol: 278.7 ± 27.5 pA, GABABaf. 277.4 ± 24.1 pA, n = 5). These observations indicate that the effect of bafilomycin A1 is only on the presynaptic ending and that refilling of GABA into the synaptic vesicles is hampered after pretreatment with bafilomycin A1.
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Discussion |
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Cotransmission of GABA and glycine was first demonstrated by showing biphasic decay kinetics of mIPSCs in the spinal cord and brainstem (Jonas et al. 1998; O'Brien & Berger, 1999), in addition to pure glycinergic and pure GABAergic transmission (Curtis et al. 1968; Schneider & Fyffe, 1992; Yang et al. 1997). In the rat cerebellum, it was also reported that a new connection exists between the cerebellar Lugaro cells and Golgi cells (Dieudonne & Dumoulin, 2000), and that Golgi cells receive both pure GABAergic inhibition from basket cells and mixed (GABAergic/glycinergic) inhibition from Lugaro cells (Dumoulin et al. 2001). Biphasic mIPSCs or eIPSCs observed in these tissues, which were sensitive to the combined application of bicuculline and strychnine, also indicate that GABA and glycine were coreleased from a single synaptic vesicle. These observations indicate that there exist three types of synaptic vesicles filled with pure GABA, pure glycine and a mixture of GABA and glycine at presynaptic nerve terminals in these preparations. It is generally considered that three types of mIPSCs, namely pure glycine, pure GABA and corelease of glycine and GABA, occur at individual synaptic contacts.
The present experiments confirmed the existence of three types of mIPSCs in SDCN neurones and showed that focal stimulation of a single synaptic bouton elicited three types of eIPSCs. This result indicates that three types of synaptic vesicles exist in a single synaptic bouton, which generates the three types of mIPSCs and eIPSCs. The focal stimulation used in the present experiments evoked eIPSCs in an all-or-nothing manner in response to the change in the stimulus intensity as reported previously (Akaike & Moorhouse, 2003), indicating that the focal stimulation activated a single synaptic contact. Therefore, it seems that one synaptic bouton in an SDCN neurone contains three types of vesicles, which exhibit three types of mIPSCs and eIPSCs.
In the present experiments, we found that the recovery of the amplitude of glycinergic mIPSCs was complete, while that of GABAergic and mixed mIPSCs was incomplete after pretreatment with bafilomycin A1. Bafilomycin A1 inhibits vacuolar H+/ATPase (Williamson & Neale, 1994; Zhou et al. 2000) and reduces the driving force for the uptake of neurotransmitters into the synaptic vesicle through VIAAT. Bafilomycin A1 greatly suppressed the amplitude of the three types of mIPSCs after prolonged treatment in the present experiments. The incomplete recovery of GABAergic and mixed mIPSCs could therefore be explained in two ways. First, cytosolic GABA concentration ([GABA]i) may be reduced by an unknown action of bafilomycin A1, since the loading of glycine and/or GABA into vesicles depends on their cytosolic concentrations (Dumoulin et al. 1999; Gasnier, 2000; 2004,). GABA may require synthesis after treatment with bafilomycin A1, but it is known that glutamic acid decarboxylase 65 (GAD65), one of the GABA synthetic enzymes that resides mainly in GABAergic terminals, is low during development (Dumoulin et al. 1999). The fact that content of the vesicular transmitter depends on the presynaptic cytosolic concentration in the calyx of Held in rat brainstem (Ishikawa et al. 2002) and in cultured synapses (Dan et al. 1994) also supports this view. In addition, Christensen et al. (1991) reported that GABA has a higher affinity to the VIAAT than glycine, and therefore GABA would have appeared first if it was present in the cytoplasm (Christensen et al. 1991). Secondly, the packing process of GABA into the vesicles may be specifically hampered after pretreatment with bafilomycin A1 without any changes in [GABA]i and [glycine]i.
The cytosolic concentrations of GABA and glycine are controlled by the neuronal GABA transporters GAT1 and GAT4 (Radian et al. 1990; Guastella et al. 1991; Jursky & Nelson, 1996), and the neuronal glycine transporter GLYT2 (Liu et al. 1993) that are the plasma membrane uptake transporters for GABA and glycine, respectively. More recently, it has also been reported that GABA uptake via GAT1 modulates GABAergic transmission in the early postnatal rat hippocampus (Sipilä et al. 2004). Therefore, GABA accumulation into the nerve terminal by GATs may prevent glycine from significantly entering the vesicles by a common VIATT which recognizes both GABA and glycine as substrates. Likewise, the accumulation of glycine into the nerve terminal by GLYT2 may prevent the vesicular loading of other VIAAT substrates including trace amounts of GABA or ß–alanine in the nerve terminal. Corelease of GABA and glycine at mixed inhibitory synapses may be achieved by a balance between the expression and activities of GATs and GLYT2 in the plasma membrane.
According to the current biochemical and immunocytochemical studies, it is generally considered that a common VIAAT is involved in the packing of both GABA and glycine into the synaptic vesicles (Gasnier 2000, 2004). The biochemical studies showed that GABA and glycine compete for uptake into purified synaptic vesicles. Furthermore, similar glycine-to-GABA uptake ratios were observed with vesicles from the brain or from the spinal cord where the glycinergic synapses are enriched (Christensen et al. 1991). These observations suggest the existence of similar, if not identical, transporters for the two amino acids (Burger et al. 1991; Christensen et al. 1991). Furthermore, in situ hybridization with a VIAAT probe showed that high levels of transcript can be detected in superior olive nuclei of the brainstem, which are known to be enriched glycinergic neurones. This indicates that a common VIAAT is responsible for the transport of GABA and glycine (Sagné et al. 1997). Moreover, immunocytochemical studies showed that VIAAT immunoreactivity can be observed in glycinergic axon terminals as well as GABAergic terminals in the brainstem and spinal cord (Chaudhry et al. 1998; Dumoulin et al. 1999).
Quantitative analysis, on the other hand, indicated that about 60% of the VIAAT-positive terminals lacked a detectable immunoreactivity for GAD65 (Dumoulin et al. 1999). VIAAT-positive and GAD-negative boutons were also found apposed to postsynaptic clusters of gephyrin, a protein responsible for clustering glycine receptors (Kuhse et al. 1995), as well as GABAA receptors (Essrich et al. 1998; Kneussel et al. 1999). These observations are considered to indicate that VIAAT-positive and GAD-negative puncta may represent glycinergic nerve terminals, assuming the presence of a common VIAAT (Dumoulin et al. 1999). However, these authors also noticed that a few presynaptic terminals enriched in GABA or glycine were immunonegative for VIAAT, leaving room for the possible existence of subtypes of VIAAT or an additional inhibitory amino acid transporter (Chaudhry et al. 1998; Dumoulin et al. 1999). This might indicate the possible existence of a subtype of VIAAT, which is more sensitive to bafilomycin A1 and has higher affinity for GABA. The existence of such a subtype of VIAAT would explain rationally the existence of three types of vesicles in a synaptic bouton, which induces pure glycinergic, pure GABAergic and mixed mIPSCs in the synaptic contact observed in the present experiments. However, further studies are definitely warranted to help understanding of the subtypes of VIAAT in central synapses.
In conclusion, the present study revealed that focal stimulation of a synaptic bouton results in three types of eIPSCs in SDCN neurones, and distinct refilling of glycine and GABA into the synaptic vesicles occurs after treatment with bafilomycin A1.
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Footnotes |
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P < 0.05, n.s: not significant). Cb, summary data of yfast and yslow of the mixed mIPSCs after washing out bafilomycin A1 for 30–35 min (**P < 0.01 versus control, n.s: not significant).
) or absence (
) of bafilomycin Al. Each column is the mean + S.E.M. of data from five neurones.