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CELLULAR |
1 Department of Anesthesiology, Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA
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Abstract |
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-Aminobutyric acid A receptor (GABAAR)-mediated postsynaptic currents (IPSCs) were recorded from dopaminergic neurons of the ventral tegmental area of young rats in acute brain slices and from mechanically dissociated neurons. Low concentrations (0.1–0.3 µM) of muscimol, a selective GABAAR agonist, increased the amplitude, and reduced the paired pulse ratio of evoked IPSCs. Moreover, muscimol increased the frequency but not the amplitude of spontaneous IPSCs (sIPSCs). These data point to a presynaptic locus of muscimol action. It is interesting that 1 µM muscimol caused an inhibition of sIPSCs, which was reversed to potentiation by the GABAB receptor antagonist CGP52432. Isoguvacine, a selective GABAAR agonist that belongs to a different class, mimicked the effects of muscimol on sIPSCs: it increased them at low (
0.5 µM), and decreased them at a higher concentration (1 µM). Hence, the activation of presynaptic GABAARs facilitates GABA release, which is limited by presynaptic GABABRs. Furthermore, facilitation of sIPSCs by muscimol was eliminated in a medium containing tetrodotoxin or cadmium. It is noteworthy that sIPSC frequency was greatly increased by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol(gaboxadol, or THIP), an agonist with preferential effects on extrasynaptic GABAARs containing 
4
subunits, or by guvacine, a GABA transport blocker, which increases ambient GABA levels. In addition, sIPSC frequency was attenuated by furosemide, a selective antagonist of 6 subunits. Thus, the presynaptic GABAARs may be situated at extrasynaptic sites and may contain 4/6 subunits. Given the marked sensitivity of extrasynaptic GABAARs to ambient GABA, alcohols and anaesthetics, these receptors may present a critical site for regulating synaptic function in the developing brain in both physiological and pathological situations.
(Received 1 November 2006;
accepted after revision 30 January 2007;
first published online 15 February 2007)
Corresponding author J-H. Ye: New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103, USA. Email: ye{at}umdnj.edu
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Introduction |
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-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the adult mammalian brain. In immature neurons, however, GABA is the primary excitatory neurotransmitter, has a depolarizing action, and is pivotal in control of neuronal firing, intracellular calcium signalling and neuronal development (Ben-Ari et al. 1997; Delpire, 2000; Ben-Ari, 2002; Owens & Kriegstein, 2002). The ventral tegmental area (VTA), one of the key brain regions for reward, is rich in dopaminergic (DA) neurons that project to the nucleus accumbens and prefrontal cortex (Beckstead et al. 1979). The DA neurons in the midbrain receive GABAergic inputs from local GABAergic neurons (Spanagel & Weiss, 1999; Diana & Tepper, 2002), medium spiny GABAergic projection neurons in nucleus accumbens (Waddington & Cross, 1978), and GABAergic neurons in ventral pallidum (Groenewegen et al. 1993). Many neurotransmitter and peptide receptors are expressed in GABAergic axon terminals which synapse onto VTA DA neurons (Mansour et al. 1991; Cameron & Williams, 1993; Wu et al. 1995; Lu et al. 1997; Bonci & Malenka, 1999; Mansvelder & McGehee, 2000; Grillner et al. 2000; Svingos et al. 2001; Bergevin et al. 2002; Garzon & Pickel, 2002; Zheng et al. 2002; Szabo et al. 2002). The activation of these receptors modulates GABA release. Recent work from this laboratory demonstrated the existence of glycine receptors on the GABAergic terminals synapsing onto VTA DA neurons, and showed that activation of these receptors increased GABA release in young rats (Ye et al. 2004). Presynaptic GABAARs were found to modulate neurotransmitter release in both peripheral and central synapses. Classic studies of the neuromuscular junction (Dudel & Kuffler, 1961; Takeuchi & Takeuchi, 1966) and spinal motor neurons (Eccles, 1964) indicate that the activation of presynaptic GABAARs inhibits transmitter release. Nevertheless, recent studies in rats showed that the activation of presynaptic GABAARs increases the release of glutamate or glycine (Jang et al. 2002, 2005, 2006; Koga et al. 2005). However, it remains unknown whether GABA receptors exist on GABAergic axon terminals in the VTA, and how these presynaptic GABAA and GABAB receptors regulate GABAergic transmission.
In the present study, using a combination of pharmacological and electrophysiological approaches, we have provided evidence indicating that functional GABAA and GABAB receptors are located on the GABAergic axon terminals which synapse onto VTA DA neurons. Activation of these GABAA or GABAB receptors, respectively, facilitates or suppresses GABAergic transmission in the immature neurons. We have also provided evidence supporting an extrasynaptic locus for these presynaptic GABAARs. In view of the marked sensitivity of the extrasynaptic GABAARs to ambient GABA, alcohols and anaesthetics, these receptors are likely to play a critical role in the regulation of synaptic function in the developing brain in physiological and pathological situations. Some of these results have been reported previously in abstract form (Xiao & Ye, 2005; Ye et al. 2006a).
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Methods |
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All procedures abided by the UK Animals (Scientific Procedures) Act 1986. The care and use of animals and the experimental protocol of this study were approved by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey. All efforts were made to minimize animal suffering, to reduce the number of animal used, and to utilize alternatives to in vivo techniques. All experiments were done on brains from young Sprague–Dawley rats (at postnatal day (P) 4–10). Most experiments were performed using midbrain slices, which were prepared as previously described (Ye et al. 2004, 2006b). In brief, rats were anaesthetized with ketamine and xylazine (80 and 10 mg kg–1, respectively, administered intraperitoneally) and then killed by decapitation. The midbrain was isolated and sliced in the coronal plane (250–300 µm) with VF-100 or VF-200 slicers (Precisionary Instruments, Greenville, NC, USA), while kept in ice-cold modified glycerol-based artificial cerebral spinal fluid (GACSF) saturated with 95% O2–5% CO2 (carbogen) containing (mM): glycerol 250, KCl 2.5, NaH2PO4 1.2, MgCl2 1.2, CaCl2 2.4, NaHCO3 26 and glucose 11. Slices (two per animal) were allowed to recover at 31°C for at least 1 h in a holding chamber, before they were placed in the recording chamber and superfused (1.5–2.0 ml min–1) with carbogen-saturated ACSF. The recovering bath was filled with standard carbogenated ACSF containing (mM): NaCl 125, KCl 2.5, NaH2PO4 1.2, MgCl2 1.2, CaCl2 2.4, NaHCO3 26 and glucose 11. Some experiments were done on single neurons isolated from VTA slices using an enzyme-free mechanically dissociation procedure, as previously described (Ye et al. 2004). Briefly, midbrain slices (300 µm thick) were kept in carbogenated ACSF at room temperature (21–24°C) for at least 1 h before mechanical dissociation. The slices were then transferred to a 35 mm culture dish (Primaria 3801; Becton Dickinson, Rutherford, NJ, USA). These dishes were filled with standard external solution containing (mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 2, Hepes 10 and glucose 10; 320 mosmol l–1, pH adjusted to 7.4 with NaOH. Under an inverted microscope (Nikon, Tokyo, Japan), the VTA was identified medial to the accessory optic tract and lateral to the fasciculus retroflexus. A heavily fire-polished glass pipette, lightly touching the surface of the VTA, was vibrated horizontally at 20 Hz for 2 min using a homemade device. The slice was then removed. The isolated neurons adhered to the bottom of the dish within 20 min and were then ready for electrophysiological experiments.
Electrophysiological recording
Whole-cell and loose-patch cell-attached configurations were used to record electrical activity with MultiClamp 700A or Axopatch 200B amplifiers (Axon Instruments, Molecular Devices, Union City, CA, USA), Digidata 1320A or 1322A analog-to-digital converters (Axon Instruments), and pClamp 9.2 software (Axon Instruments). Data were filtered at 2 kHz and sampled at 10 kHz. Analyses were limited to data from cells having initial series resistance of 15–25 M
and the changes of which were less than 20% during recording.
The patch electrodes had a resistance of 3–5 M, when filled with (mM): CsF 135, CsCl 5, EGTA 5, CaCl2 0.5, Hepes 10, Mg-ATP 2 and GTP 0.1 (for brain slice recording, Turecek & Trussell, 2001); or CsCl 140, MgCl2 2, EGTA 4, CaCl2 0.4, Hepes 10, Mg-ATP 2 and GTP 0.1 (for recordings from mechanically dissociated neurons); pH was adjusted to 7.2 with Tris base, and the osmolarity to 300 mOsmol l–1 with sucrose. Throughout the experiments, the bath was continuously perfused with the standard external solution (for the isolated neurons) or ACSF (for brain slices). All recordings were made in these solutions at an ambient temperature of 20–23°C.
In brain slices, cells were visualized with an upright microscope (E600FN; Nikon) and near infrared illumination. To evoke monosynaptic IPSCs, a glass stimulation electrode with a tip of 10 µm in diameter was placed 50–100 µm away from the recording site in the VTA. Paired stimuli (100–200 µs depolarizing pulses, 50 ms apart) were given at the rate of 0.05 Hz.
Chemicals and applications
Most of the chemicals including D(–)-2-amino-5-phospho-pentanoate (AP5), 6,7-dinitroquinoxaline-2,3-dione (DNQX), bicuculline (BIC), cadmium chloride, isoguvacine, furosemide, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (gaboxadol, or THIP), guvacine hydrochloride, Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin (DAMGO) and tetrodotoxin (TTX), were obtained from Sigma (St Louis, MO, USA). Muscimol and CGP52432 were from Tocris (Ellisville, MO, USA). The solutions, prepared before the experiment, were applied to a dissociated neuron with a Y-tube. This exchanged the external solution surrounding the neurons within 40 ms (Zhou et al. 2006). In experiments on brain slices, chemicals were added in known concentrations to the superfusate. The fact that 10 µM bicuculline blocked most IPSCs (in slices) within 90 s is an indication of the effective bath exchange time.
Data analysis
Spontaneous discharges and inhibitory postsynaptic currents (sIPSCs) were counted and analysed with Clampfit 9.2 (Axon Instruments); sIPSCs were screened automatically (5 pA amplitude threshold), checked visually and accepted or rejected according to their rise and decay times. The frequency and amplitude of all events, during and after drug applications, were normalized to the mean of the values observed during the initial control period. Cumulative probability plots of the incidence of various inter-event intervals and amplitudes (for 100–1500 sIPSCs), recorded under different conditions from the same neuron, were compared with the Kolmogorov–Smirnov (K-S) test. For other plots, data obtained over a 1–2 min period at the peak of a drug response were normalized to the average values of the frequency and amplitude of sIPSCs or miniature IPSCs (mIPSCs) during the initial control period (4–5 min). The amplitude of eIPSCs was measured with Clampfit 9.2. For each experimental condition, we averaged the amplitudes of eIPSCs or paired pulse ratio (PPR = IPSC2/IPSC1) from 10 to 20 trials. IPSC1 and IPSC2 are the IPSCs in response to the first and second stimulus of the paired pulses, respectively. In the figures, single eIPSCs or paired eIPSCs are averages of > 10 successive traces. Data were expressed as means (± S.E.M.). The statistical significance of drug effects was assessed by Student's paired two-tailed t test. Values of P < 0.05 were considered significant.
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Results |
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Experiments were done in acute midbrain slices (Fig. 1A) or mechanically dissociated neurons (Fig. 1B). All recordings were obtained from putative DA neurons identified by their pharmacological and physiological properties. Specifically, spontaneous firing of VTA neurons was first recorded with the loose-patch cell-attached configuration (Fig. 1C). The depression of spontaneous firing by 0.2 µM quinpirole (QP), a dopamine D2/D3 receptor agonist (Fig. 1Ca) (in slices and isolated neurons), and facilitation by 1 µM DAMGO, a µ-opioid receptor agonist (Fig. 1Cb) (in slices), is probably due to DAMGO-induced disinhibition; this is a characteristic feature of VTA DA neurons (Margolis et al. 2006). We then changed the recording into the whole-cell configuration by applying further suction. The existence of a prominent inward current (Ih) activated by hyperpolarizing voltage steps (between –60 and –160 mV) recorded under voltage clamp (Fig. 1Da), or a voltage-sag in response to a hyperpolarizing current pulse (–100 pA) recorded under current clamp (Fig. 1Db) (Lacey et al. 1989), was used to further identify the putative VTA DA neurons (in slices and isolated neurons). Ih is present in 70% of VTA DA neurons, and it is well documented that GABAergic neurons do not have Ih (Cameron et al. 1997; Jones & Kauer, 1999; Neuhoff et al. 2002; Margolis et al. 2006; Schilstrom et al. 2006). Although Ih currents are not present exclusively in the DA cells, it is unlikely that the Ih-positive tyrosine hydroxylase-negative cells made a significant contribution to our observations.
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In midbrain slices, eIPSCs were recorded from VTA DA neurons at a holding potential (VH) of 0 mV in the presence of AP5 (50 µM) and DNQX (20 µM). These eIPSCs were completely eliminated by 10 µM bicuculline (BIC), a specific GABAAR antagonist, indicating that they were mediated by GABAARs (Fig. 2Aa). As illustrated in Fig. 2Aa and Ab, 0.3 µM muscimol (Mus), a specific agonist for GABAARs, prominently potentiated the amplitude of eIPSCs. The potentiation was reversible; eIPSCs recovered to control level after washout of muscimol. In seven experiments, 0.3 µM muscimol significantly enhanced eIPSCs by 35 ± 7% (control, 76 ± 6; muscimol, 103 ± 11 pA, P = 0.02; Fig. 2C).
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Muscimol presynaptically modifies sIPSCs
To further test this possibility, we compared sIPSCs recorded from slices in the absence and presence of muscimol. In the presence of AP5 (50 µM) and DNQX (20 µM), the application of 10 µM bicuculline completely eliminated all sIPSCs (data not shown), indicating that they were mediated by GABAARs. Muscimol (0.3 µM) prominently increased sIPSC frequency (Fig. 3A) by 28 ± 8% (muscimol, 1.62 ± 0.38; control, 1.28 ± 0.31 Hz, n = 6, P < 0.01; Fig. 3C). This was further illustrated by cumulative plots of the incidence of various intervals between successive sIPSCs (Fig. 3B): 0.3 µM muscimol induced a marked shift towards shorter intervals (Fig. 3Ba, K-S test, P < 0.01), but no significant change in amplitude (Fig. 3Bb, K-S test, P > 0.05), and in the mean values (101 ± 6% of control: muscimol, 17 ± 3; control, 17 ± 3 pA, n = 6, P = 0.49; Fig. 3Cb). These data further support a presynaptic locus of action of muscimol. Figure 3Ca illustrates the dose dependence of the action of muscimol on sIPSC frequency. While 0.1 µM muscimol enhanced sIPSC frequency by 35 ± 7% (muscimol, 1.84 ± 0.40; control, 1.39 ± 0.30 Hz, n = 8, P < 0.01), unexpectedly, 1 µM muscimol decreased sIPSC frequency by 40 ± 12% (muscimol, 0.82 ± 0.09; control, 1.60 ± 0.30 Hz, n = 5, P < 0.05; Fig. 3Ca; also see Fig. 4A). On the other hand, at all these concentrations, muscimol did not alter sIPSC amplitude, as shown by the histogram in Fig. 3Cb (0.1 µM: muscimol, 18 ± 3; control, 17 ± 3 pA, n = 8, P = 0.19; 1 µM: muscimol, 13 ± 1; control, 15 ± 2 pA, n = 5, P = 0.11). These results are consistent with a presynaptic mechanism of muscimol action.
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Most GABAergic synapses contain presynaptic GABABRs (Misgeld et al. 1995). These presynaptic GABABRs act as autoreceptors, and they limit transmitter release by reducing Ca2+ entry (Misgeld et al. 1995). To assess an involvement of GABABRs, we compared the effect of muscimol on sIPSCs, recorded from slices, in the absence and presence of CGP52432 (1 µM), a selective GABABR antagonist. Figure 4Aa–c shows that when applied alone, 1 µM muscimol prominently suppressed sIPSC frequency, and induced significant rightward shift of the cumulative probability plot of inter-event intervals (K-S test, P < 0.01). By contrast, in the presence of 1 µM CGP52432, 1 µM muscimol prominently increased sIPSC frequency (Fig. 4Ba and Bb), by 147 ± 38% (muscimol, 0.49 ± 0.18; control, 0.20 ± 0.07 Hz, n = 4, P < 0.01; Fig. 4C), and induced significant leftward shift of the cumulative probability plot of inter-event intervals (K-S test, P < 0.01, Fig. 4Bc). CGP52432 also enhanced the action of muscimol at lower concentrations. In the presence of 1 µM CGP52432, 0.3 µM muscimol increased sIPSC frequency by 138 ± 24% (muscimol, 0.47 ± 0.16; control, 0.19 ± 0.03 Hz, n = 4, P < 0.01), which was significantly greater than that in the absence of CGP52432 (28 ± 8%: muscimol, 1.62 ± 0.38; control, 1.28 ± 0.31 Hz, n = 6, P < 0.01; Fig. 4C). These data indicate the existence of presynaptic GABABRs on the GABAergic terminals synapsing onto VTA DA neurons, and that activation of these GABABRs inhibits GABA release.
Muscimol (10 µM) was found to inhibit synaptic transmission through the activation of presynaptic GABABRs in a pontine auditory nucleus (Yamauchi et al. 2000). To determine whether 1 µM muscimol-induced inhibition of sIPSCs in VTA is due to muscimol-dependent activation of presynaptic GABABRs, we examined the effects of isoguvacine, a selective GABAAR agonist from a different class, on sIPSCs. Note that a direct action of isoguvacine on GABABRs has not been reported. As can be seen in Fig. 4D, 0.1 and 0.5 µM isoguvacine, respectively, increased sIPSC frequency by 20 ± 7% (control, 1.35 ± 0.36; 0.1 µM isoguvacine, 1.62 ± 0.30 Hz, n = 3, P < 0.05), and by 25 ± 5% (control, 1.33 ± 0.30; 0.5 µM isoguvacine, 1.67 ± 0.39 Hz, n = 10, P < 0.01), whereas, 1 µM isoguvacine decreased sIPSC frequency by 36 ± 7% (control, 2.05 ± 0.32; 1 µM isoguvacine, 1.47 ± 0.26 Hz, n = 8, P < 0.01). Thus, it is unlikely that a direct activation of GABABRs by muscimol plays a major role in 1 µM muscimol-induced inhibition of sIPSCs.
Muscimol acts in a tetrodotoxin- and cadmium-sensitive manner
We then studied the mechanism underlying muscimol-induced enhancement of sIPSC frequency on VTA DA neurons freshly isolated using an enzyme-free procedure. These isolated neurons retain attached functional excitatory and inhibitory synaptic boutons (Ye et al. 2004; Zhou et al. 2006).
The sIPSCs were recorded at a VH of –60 mV with CsCl-containing pipette solution (see Method) in the presence of AP5 (50 µM) and DNQX (20 µM). Bicuculline (10 µM) completely blocked these sIPSCs, indicating that they were mediated by GABAA receptors (data not shown). Muscimol (0.3 µM) robustly enhanced sIPSC frequency and induced significant leftward shift of the cumulative probability plot of inter-event intervals (K-S test: P < 0.01; Fig. 5Aa and Ab). The enhancement (mean ± S.E.M.) from eight cells was 82 ± 22% (muscimol, 3.49 ± 0.96; control, 1.93 ± 0.53 Hz, n = 8, P = 0.004; Fig. 5Da). Note that the enhancement induced by 0.3 µM muscimol in the isolated neurons is larger than that observed in slices (also see Fig. 6). The mechanism underlying this difference warrants further study. The simplest interpretation is that the solution around an isolated neuron was much better controlled. Muscimol (0.3 µM) did not change the frequency of the sIPSCs either in the presence of a sodium channel blocker, 0.5 µM tetrodotoxin (TTX) (99 ± 3% of that before muscimol application, from 0.65 ± 0.18 Hz in TTX to 0.64 ± 0.18 Hz in TTX + muscimol, n = 6, P = 0.40, Fig. 5Ba, Bb and Da), or when voltage-gated calcium channels (VGCCs) were blocked by 100 µM CdCl2 (99 ± 6% of that before muscimol application, from 0.3 ± 0.1 Hz in CdCl2 to 0.3 ± 0.1 Hz in CdCl2 + muscimol, n = 7, P = 0.33; Fig. 5Ca, Cb and Da). In addition, muscimol did not alter the mean amplitude of the sIPSCs in the absence (96 ± 4% of that before muscimol application, from 57 ± 9 pA in control to 54 ± 8 pA, n = 8, P = 0.18) and presence of TTX (103 ± 6% of that before muscimol application, from 23 ± 4 pA in TTX to 28 ± 5 pA in TTX + muscimol, n = 6, P = 0.33) or CdCl2 (101 ± 5% of that before muscimol application, from 14 ± 3 pA in CdCl2 to 14 ± 2 pA in CdCl2 + muscimol, n = 7, P = 0.39) (Fig. 5Db). These results suggest that enhancement of GABA release by muscimol depends on both Na+ channels and VGCCs.
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4/6 subunitsTo identify the subunits of presynaptic GABAARs that mediate muscimol-induced facilitation of GABAergic transmission, we conducted experiments on VTA DA neurons freshly isolated using an enzyme-free procedure, as previously mentioned.
The application of 1 µM gaboxadol, a selective agonist for extrasynaptic GABAARs containing 4 subunits (Brown et al. 2002), induced a prominent increase in sIPSC frequency by 47 ± 7% (control, 1.32 ± 0.05; gaboxadol, 1.90 ± 0.33 Hz, n
= 7, P < 0.01), and a significant leftward shift of cumulative probability plot of inter-event intervals of sIPSCs (K-S test, P < 0.001; Fig. 6Aa and Ab).
Next, we compared muscimol-induced facilitation in the absence and presence of furosemide, a selective blocker for GABAARs containing the 6 subunit (Hamann et al. 2002). As described earlier, when muscimol (0.3 µM) was applied alone, it enhanced sIPSC frequency robustly, by 82 ± 18% (n
= 7, P < 0.01, Fig. 6Ba), and induced a significant leftward shift of cumulative probability plot (K-S test, P < 0.001 Fig. 6Bb). By contrast, furosemide (100 µM) significantly decreased sIPSC frequency by 29 ± 6% (n
= 7, P < 0.01, data not illustrated). After the response to furosemide had stabilized, a mixture of 100 µM furosemide and 0.3 µM muscimol was applied. As illustrated in Fig. 6Ca–Cb, under this condition, 0.3 µM muscimol increased sIPSC frequency by only 44 ± 10% (n
= 7, P < 0.01), which is significantly smaller than the muscimol-induced facilitation observed in the absence of furosemide (82 ± 18%, n
= 7, P < 0.05, paired t test). These results indicate that enhancement of GABA release by muscimol might be mediated by GABAARs containing 4 or 6 subunits. Nevertheless, furosemide is a blocker of some Cl– transporters, such as the K+ Cl– cotransporter (KCC2). By blocking KCC2, furosemide could induce a shift of Cl– reversal potential towards less negative potentials. In immature neurons, it will increase the driven force for Cl– efflux (Zhu et al. 2005), and enhance presynaptic GABAAR function. This will counteract the inhibitory effect of furosemide on GABA release. If this were the case, furosemide-induced inhibition of GABA release observed in our experiments would be underestimated.
Increasing ambient GABA increases sIPSC frequency in VTA DA neurons in brain slices
According to the localization, GABAARs are classified as synaptic and extrasynaptic receptors. These two kinds of receptors seem different in subunit composition (Farrant & Nusser, 2005; but see Santhakumar et al. 2006). GABAARs containing , 4, 5 or 6 subunits appear to be predominantly extrasynaptic. Therefore, our data suggest that muscimol might enhance GABA release by the activation of extrasynaptic GABAARs expressed on GABAergic axon terminals.
Because extrasynaptic GABAARs can be activated by GABA escaping from the synaptic cleft, we tested whether an increase in ambient GABA levels could enhance sIPSC frequency on VTA DA neurons in brain slices. In this series of experiments, we recorded sIPSCs on VTA DA neurons (VH, –50 mV) in the presence of 50 µM AP5, 20 µM DNQX and 1 µM CGP52432, using CsCl-based pipette solution.
The application of guvacine (20 µM), a GABA transporter blocker, induced a robust and reversible increase in the frequency of sIPSCs recorded in slices (Fig. 7A and C) without inducing a significant postsynaptic current (5 ± 1 pA). Guvacine (20 µM) induced a large leftward shift in the cumulative probability plot of inter-event intervals between successive sIPSCs (K-S test, P < 0.001; upper panel in Fig. 7B) and an increase of 72 ± 14% (n = 7, P = 0.001, upper panel inset in Fig. 7B) in sIPSC frequency, while it did not alter sIPSC amplitude (K-S test, P > 0.5, lower panel in Fig. 7B; 0 ± 3%, n = 7, P = 0.46, lower panel inset in Fig. 7B). These results suggest that an increase in ambient GABA level enhances GABAergic sIPSCs via a presynaptic mechanism in the developing brain.
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Discussion |
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4/6 subunits. In the immature neurons, the activation of these GABAARs enhances GABA release, which is normally limited by the presynaptic GABABR activity. The interaction between these two presynaptic GABA receptors may play a critical role in the regulation of VTA DA neuronal excitability in the developing brain. Presynaptic GABAA receptors facilitate GABA release onto VTA DA neurons of young rats
Low concentrations of muscimol enhanced GABAergic synaptic transmission in the VTA, as evidenced by the increase in the amplitude, the decrease in the paired pulse ratio of eIPSCs, and the increase in the frequency but not the amplitude of spontaneous IPSCs. These results lend support for a presynaptic locus of muscimol action. Muscimol enhances also the frequency of sIPSCs recorded from mechanically dissociated neurons. This finding indicates that functioning GABAARs exist on, or near, GABAergic axon terminals on VTA DA neurons. Several mechanisms may underlie presynaptic GABAAR modulation of neurotransmitter release (Vitten & Isaacson, 2001). In developing and injured neurons, nerve terminals contain high intracellular Cl– levels (Ben-Ari, 2002; Nabekura et al. 2002). Activation of GABAARs causes depolarization, which elevates the intracellular calcium concentration via activation of VGCCs, and increases neurotransmitter release.
Our previous studies showed that intracellular Cl– concentration is high in VTA neurons of young rats, and activation of glycine receptors, opening also a Cl– channel, induces membrane depolarization (Ye, 2000; Wang et al. 2005). Furthermore, activation of presynaptic glycine receptors increases GABA release in neonatal rats (Ye et al. 2004). In keeping with these findings, we showed here that several GABAAR agonists enhanced GABA release onto VTA DA neurons of neonatal rats.
In the VTA, a low concentration of muscimol enhanced sIPSC frequency. The enhancement was not seen in the presence of tetrodotoxin or cadmium. These data indicate that enhancement by muscimol depends on Na+ channel and VGCCs. In nerve terminals of CNS, N- and P/Q- type VGCCs mediate most of the calcium-dependent transmitter release (Takahashi & Momiyama, 1993; Poncer et al. 1997). Based on our data, we postulate that at low concentrations, muscimol induces a slight depolarization, which is enough to activate Na+ channels, but not the VGCCs. Following the activation of Na+ channels, VGCCs are activated, leading to an increase in intraterminal calcium concentration, and an increase in transmitter release. Similar mechanisms have been implicated in presynaptic glycine receptor activation-induced enhancement of GABA release (Ye et al. 2004), and also in presynaptic GABAAR activation-induced enhancement of glutamate release (Koga et al. 2005; Jang et al. 2006).
Nevertheless, in the VTA, 1 µM muscimol (or isoguvacine) reduced sIPSC frequency. Several possible mechanisms could account for this observation. First, the opening of the presynaptic GABAARs decreases the input resistance of the terminal and acts as an electrical shunt, which leads to a reduction in the amplitude of the action potential as it invades the nerve ending. This results in a reduction of neurotransmitter release (Vitten & Isaacson, 2001). Second, strong depolarization can inactivate Na+ channels, causing inhibition of invasion of action potentials (Segev, 1990; Stuart & Redman, 1992). Third, a higher concentration of muscimol (and isoguvacine) may indirectly activate presynaptic GABABRs, which results in a reduction of GABA release. The results shown in Fig. 4A–C support the latter mechanism as a major contributing factor (see below).
Interaction between presynaptic GABAA and GABAB receptors
In the VTA, muscimol-induced enhancement of GABA release was observed only at low concentrations (0.1 and 0.3 µM). At a higher concentration (1 µM), muscimol suppressed GABA release. However, application of the GABABR antagonist CGP52432 reversed the effect of 1 µM muscimol, from inhibition to potentiation. Furthermore, CGP52432 potentiated 0.3 µM muscimol-induced enhancement. These data suggest the existence of both GABAA and GABAB receptors on the GABAergic axon terminals, which synapse onto the DA neurons in the VTA. The activation of presynaptic GABAA and GABAB receptors increases and decreases GABA release, respectively. The net effect of muscimol is determined by the balance between the activities of these two types of receptors.
It is possible that at high concentrations, the release of GABA induced by muscimol (and isoguvacine) is high enough to activate presynaptic GABABRs and therefore opposes the effect of presynaptic GABAARs. The higher EC50 value of muscimol (and isoguvacine) for presynaptic GABABRs may be due to their extrasynaptic location. Recent evidence indicates that GABABRs are located extrasynaptically, and that both pre- and postsynaptic GABABRs are located some distance from the site of release. This means that to recruit GABABRs, the GABA concentration must be high, so that more GABA can escape from the synaptic cleft (Nicoll, 2004). In response to low concentrations of muscimol (and isoguvacine), the escaped GABA may not be enough to activate presynaptic GABABRs. Only at much higher concentrations, can muscimol (and isoguvacine) induce enough spillover of GABA to activate GABABRs, overcome the effects of GABAARs, and consequently inhibit GABA release. In a recent study in hippocampal neurons, it was found that potentiation by ethanol of GABAergic synaptic transmission is limited by presynaptic GABABRs (Ariwodola & Weiner, 2004). It will be interesting to determine how the interaction between the presynaptic GABAA and GABAB receptors contributes to the effects of ethanol in the VTA.
Presynaptic GABAA receptors on GABAergic terminals on VTA DA neurons may be at extrasynaptic sites and may contain 4/6 subunits
As mentioned above, VTA DA neurons receive abundant GABAergic inputs from several brain areas. Previous histological studies indicated expression of various subunits of GABAARs in these areas of adult rats. The 1, 3, 2, 1 and 3 subunits were found in medium-sized neurons in nucleus accumbens. Neurons in ventral pallidum express 1, 3, 5, 1–3, 1, 3 and subunits. In the VTA, the 1–3, 5, 1, 3 and subunits were found in the neurons, while the 1–3, 1–3, 2 and subunits were found in the processes (Pirker et al. 2000; Schwarzer et al. 2001). Using a combination of single-cell PCR and in situ hybridization, Okada et al. (2004) found that 2–4, 1, 3 and 2 subunits are expressed on VTA DA neurons. These studies have not examined expression of the 6 subunit.
In the VTA, we observed that a low concentration of gaboxadol induced a robust increase of sIPSC frequency and furosemide attenuated enhancement by muscimol of sIPSC frequency. These findings suggest that functional GABAARs containing 4 or 6 subunits exist on GABAergic axon terminals on VTA DA neurons. To the best of our knowledge, this is the first evidence showing the possibility of the existence of functional 4 and 6 subunits in VTA GABAergic axon terminals.
Extrasynaptic GABAARs were found in several cell types, such as cerebellar granule cells, hippocampal cells and forebrain neurons. The subunit forms extrasynaptic receptors with 6 and 2/3 in cerebellar granule cells, and with the 4 and 1–3 subunits in thalamus, neostriatum and dentate gyrus. In hippocampal pyramidal cells, the 5 subunit forms extrasynaptic receptors probably with 3 and 2 subunits. In view of the accumulating evidence that GABAARs containing , 4, 5 or 6 subunits seem to be predominantly extrasynaptic (Farrant & Nusser, 2005; but see Santhakumar et al. 2006), we postulated that some of the presynaptic GABAARs may be situated at extrasynaptic sites on or near the GABAergic axon terminal. This notion was supported by our observation that increasing ambient GABA levels by application of guvacine, a GABA transport blocker, increased the frequency of sIPSCs.
Physiological role of the presynaptic GABAA receptors in the VTA of young rats
Our finding that functional GABA receptors exist at GABAergic terminals on VTA DA neurons provides a new mechanism for the regulation of the excitability of these neurons. In young rats, GABA induces membrane depolarization and an increase in calcium concentration, which is critical for normal growth of neurons and their synaptic connections. However, this action must be balanced by other mechanisms. Our findings indicate that release of GABA facilitates further GABA release through the activation of presynaptic GABAARs, which is attenuated by presynaptic GABABRs. This integrated action will ensure the accuracy of signal transmission and may protect neurons from over-excitation and neuroexcitotoxicity.
Based on our data, we further propose that the presynaptic GABAARs are situated probably, at least in part, at extrasynaptic sites. Accumulating evidence indicates that extrasynaptic GABAARs are highly sensitive to, and tonically activated by, ambient GABA levels. Hence, by activating these receptors, ambient GABA levels might alter the presynaptic resting potential, which has been shown recently to be a powerful means for regulating synaptic function (Awatramani et al. 2005). Furthermore, in view of emerging evidence that extrasynaptic GABAARs are particularly sensitive to alcohols and anaesthetics (Orser et al. 2002; Carta et al. 2004; Hanchar et al. 2005; Wallner et al. 2006), these receptors may represent a critical site in the regulation of synaptic function in the developing brain in both physiological and pathological situations.
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Awatramani GB, Price GD & Trussell LO (2005). Modulation of transmitter release by presynaptic resting potential and background calcium levels. Neuron 48, 109–121.[CrossRef][Medline]
Beckstead RM, Domesick VB & Nauta WJ (1979). Efferent connections of the substantia nigra and ventral tegmental area in the rat. Brain Res 175, 191–217.[CrossRef][Medline]
Ben-Ari Y (2002). Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci 3, 728–739.[CrossRef][Medline]
Ben-Ari Y, Khazipov R, Leinekugel X, Caillard O & Gaiarsa JL (1997). GABAA, NMDA and AMPA receptors: a developmentally regulated ‘Menage a trois’. Trends Neurosci 20, 523–529.[CrossRef][Medline]
Bergevin A, Girardot D, Bourque MJ & Trudeau LE (2002). Presynaptic mu-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons. Neuropharmacology 42, 1065–1078.[CrossRef][Medline]
Bonci A & Malenka RC (1999). Properties and plasticity of excitatory synapses on dopaminergic and GABAergic cells in the ventral tegmental area. J Neurosci 19, 3723–3730.
Brown N, Kerby J, Bonnert TP, Whiting PJ & Wafford KA (2002). Pharmacological characterization of a novel cell line expressing human 43 GABAA receptors. Br J Pharmacol 136, 965–974.[CrossRef][Medline]
Cameron DL, Wessendorf MW & Williams JT (1997). A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids. Neuroscience 77, 155–166.[CrossRef][Medline]
Cameron DL & Williams JT (1993). Dopamine D1 receptors facilitate transmitter release. Nature 366, 344–347.[CrossRef][Medline]
Carta M, Mameli M & Valenzuela CF (2004). Alcohol enhances GABAergic transmission to cerebellar granule cells via an increase in Golgi cell excitability. J Neurosci 24, 3746–3751.
Delpire E (2000). Cation-chloride cotranspoters in neuronal communication. News Physiol Sci 15, 344–347.
Diana M & Tepper JM (2002). Electrophysiological pharmacology of mesencephalic dopaminergic neurons. In Handbook of Experimental Pharmacology, ed. Di Chiara G, pp. 1–61. Springer-Verlag, Berlin Heidelberg.
Dobrunz LE & Stevens CF (1997). Heterogeneity of release probability, facilitation, and depletion at central synapses. Neuron 18, 995–1008.[CrossRef][Medline]
Dudel J & Kuffler SW (1961). Presynaptic inhibition at the crayfish neuromuscular junction. J Physiol 155, 543–562.
Eccles JC (1964). Presynaptic inhibition in the spinal cord. Prog Brain Res 12, 65–91.[Medline]
Farrant M & Nusser Z (2005). Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat Rev Neurosci 6, 215–229.[CrossRef][Medline]
Garzon M & Pickel VM (2002). Ultrastructural localization of enkephalin and mu-opioid receptors in the rat ventral tegmental area. Neuroscience 114, 461–474.[CrossRef][Medline]
Grillner P, Berretta N, Bernardi G, Svensson TH & Mercuri NB (2000). Muscarinic receptors depress GABAergic synaptic transmission in rat midbrain dopamine neurons. Neuroscience 96, 299–307.[CrossRef][Medline]
Groenewegen HJ, Berendse HW & Haber SN (1993). Organization of the output of the ventral striatopallidal system in the rat: ventral pallidal efferents. Neuroscience 57, 113–142.[CrossRef][Medline]
Hamann M, Rossi DJ & Attwell D (2002). Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33, 625–633.[CrossRef][Medline]
Hanchar HJ, Dodson PD, Olsen RW, Otis TS & Wallner M (2005). Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity. Nat Neurosci 8, 339–345.[CrossRef][Medline]
Jang IS, Ito Y & Akaike N (2005). Feed-forward facilitation of glutamate release by presynaptic GABAA receptors. Neuroscience 135, 737–748.[CrossRef][Medline]
Jang IS, Jeong HJ, Katsurabayashi S & Akaike N (2002). Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord. J Physiol 541, 423–434.
Jang IS, Nakamura M, Ito Y & Akaike N (2006). Presynaptic GABAA receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons. Neurosci 138, 25–35.[CrossRef][Medline]
Jones S & Kauer JA (1999). Amphetamine depresses excitatory synaptic transmission via serotonin receptors in the ventral tegmental area. J Neurosci 19, 9780–9787.
Koga H, Ishibashi H, Shimada H, Jang IS, Nakamura TY & Nabekura J (2005). Activation of presynaptic GABAA receptors increases spontaneous glutamate release onto noradrenergic neurons of the rat locus coeruleus. Brain Res 1046, 24–31.[CrossRef][Medline]
Lacey MG, Mercuri NB & North RA (1989). Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. J Neurosci 9, 1233–1241.[Abstract]
Lu XY, Churchill L & Kalivas PW (1997). Expression of D1 receptor mRNA in projections from the forebrain to the ventral tegmental area. Synapse 25, 205–214.[CrossRef][Medline]
Mansour A, Meador-Woodruff JH, Zhou QY, Civelli O, Akil H & Watson SJ (1991). A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques. Neuroscience 45, 359–371.[CrossRef][Medline]
Mansvelder HD & McGehee DS (2000). Long-term potentiation of excitatory inputs to brain reward areas by nicotine. Neuron 27, 349–357.[CrossRef][Medline]
Margolis EB, Lock H, Hjelmstad GO & Fields HL (2006). The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons. J Physiol 577, 907–924.
Misgeld U, Bijak M & Jarolimek W (1995). A physiological role for GABAB receptors and the effects of baclofen in the mammalian central nervous system. Prog Neurobiol 46, 423–462.[CrossRef][Medline]
Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A & Akaike N (2002). Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury. J Neurosci 22, 4412–4417.
Neuhoff H, Neu A, Liss B & Roeper J (2002). Ih channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain. J Neurosci 22, 1290–1302.
Nicoll RA (2004). My close encounter with GABAB receptors. Biochem Pharmacol 68, 1667–1674.[CrossRef][Medline]
Okada H, Matsushita N, Kobayashi K & Kobayashi K (2004). Identification of GABAA receptor subunit variants in midbrain dopaminergic neurons. J Neurochem 89, 7–14.[CrossRef][Medline]
Orser BA, Canning KJ & Macdonald JF (2002). Mechanisms of general anesthesia. Curr Opin Anaesthesiol 15, 427–433.[CrossRef][Medline]
Owens DF & Kriegstein AR (2002). Is there more to GABA than synaptic inhibition? Nat Rev Neurosci 3, 715–727.[CrossRef][Medline]
Pirker S, Schwarzer C, Wieselthaler A, Sieghart W & Sperk G (2000). GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neurosci 101, 815–850.[CrossRef][Medline]
Poncer JC, McKinney RA, Gähwiler BH & Thompson SM (1997). Either N- or P-type calcium channels mediate GABA release at distinct hippocampal inhibitory synapses. Neuron 18, 463–472.[CrossRef][Medline]
Santhakumar V, Hanchar HJ, Wallner M, Olsen RW & Otis TS (2006). Contributions of the GABAA receptor alpha6 subunit to phasic and tonic inhibition revealed by a naturally occurring polymorphism in the alpha6 gene. J Neurosci 26, 3357–3364.
Schilstrom B, Yaka R, Argilli E, Suvarna N, Schumann J, Chen BT, Carman M, Singh V, Mailliard WS, Ron D & Bonci A (2006). Cocaine enhances NMDA receptor-mediated currents in ventral tegmental area cells via dopamine D5 receptor-dependent redistribution of NMDA receptors. J Neurosci 26, 8549–8558.
Schwarzer C, Berresheim U, Pirker S, Wieselthaler A, Fuchs K, Sieghart W & Sperk G (2001). Distribution of the major gamma-aminobutyric acidA receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat. J Comp Neurol 433, 526–549.[CrossRef][Medline]
Segev I (1990). Computer study of presynaptic inhibition controlling the spread of action potentials into axon terminals. J Neurophysiol 63, 987–998.
Spanagel R & Weiss F (1999). The dopamine hypothesis of reward: past and current status. Trends Neurosci 22, 521–527.[CrossRef][Medline]
Stuart GJ & Redman SJ (1992). The role of GABAA and GABAB receptors in presynaptic inhibition of Ia EPSPs in cat spinal motoneurones. J Physiol 449, 551–572.
Svingos AL, Garzon M, Colago EE & Pickel VM (2001). Mu-opioid receptors in the ventral tegmental area are targeted to presynaptically and directly modulate mesocortical projection neurons. Synapse 41, 221–229.[CrossRef][Medline]
Szabo B, Siemes S & Wallmichrath I (2002). Inhibition of GABAergic neurotransmission in the ventral tegmental area by cannabinoids. Eur J Neurosci 15, 2057–2061.[CrossRef][Medline]
Takahashi T & Momiyama A (1993). Different types of calcium channels mediate central synaptic transmission. Nature 366, 156–158.[CrossRef][Medline]
Takeuchi A & Takeuchi N (1966). A study of the inhibitory action of gamma- aminobutyric acid on neuromuscular transmission in the crayfish. J Physiol 183, 418–432.
Turecek R & Trussell LO (2001). Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse. Nature 411, 587–590.[CrossRef][Medline]
Vitten H & Isaacson JS (2001). Synaptic transmission: exciting times for presynaptic receptors. Curr Biol 11, R695–R697.[CrossRef][Medline]
Waddington JL & Cross AJ (1978). Neurochemical changes following kainic acid lesions of the nucleus accumbens: implications for a GABAergic accumbal-ventral tegmental pathway. Life Sci 22, 1011–1014.[CrossRef][Medline]
Wallner M, Hanchar HJ & Olsen RW (2006). Low dose acute alcohol effects on GABAA receptor subtypes. Pharmacol Ther 112, 513–528.[CrossRef][Medline]
Wang F, Xiao C & Ye JH (2005). Taurine activates excitatory non-synaptic glycine receptors on dopamine neurones in ventral tegmental area of young rats. J Physiol 565, 503–516.
Wu YN, Mercuri NB & Johnson SW (1995). Presynaptic inhibition of gamma-aminobutyric acidB-mediated synaptic current by adenosine recorded in vitro in midbrain dopamine neurons. J Pharmacol Exp Ther 273, 576–581.
Xiao C & Ye JH (2005). GABA-induced potentiation of GABAergic synaptic transmission is limited by presynaptic GABAB receptors. Abstr Soc Neurosci 40.13.
Yamauchi T, Hori T & Takahashi T (2000). Presynaptic inhibition by muscimol through GABAB receptors. Eur J Neurosci 12, 3433–3436.[CrossRef][Medline]
Ye J (2000). Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons. Brain Res 862, 74–82.[CrossRef][Medline]
Ye J, Wang F, Krnjevic K, Wang W, Xiong ZG & Zhang J (2004). Presynaptic glycine receptors on GABAergic terminals facilitate discharge of dopaminergic neurons in ventral tegmental area. J Neurosci 24, 8961–8974.
Ye J, Xiao C & Zhou C (2006a). Presynaptic GABAA receptors regulate GABAergic transmission of dopamine neurons in the ventral tegmental
and presence 
of 1 µM CGP52432. D, summary of the effects of isoguvacine (Isog, 0.1, 0.5 and 1.0 µM) on the frequency of sIPSCs. Change of sIPSCs was calculated as [IPSCisog/IPSCcontrol) – 1]
x 100. IPSCisog and IPSCcontrol are the IPSCs recorded in the presence and absence of isoguvacine, respectively. Number of neurons in each group is indicated in parentheses. *P < 0.05, **P < 0.01, paired t test for muscimol (or isoguvacine) versus pre-muscimol (or isoguvacine) control.
