Taurine activates excitatory non-synaptic glycine receptors on dopamine neurones in ventral tegmental area of young rats
1 Departments of Anaesthesiology, Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA
Abstract
The physiological and pharmacological properties of taurine-induced responses were investigated in dopaminergic (DA) neurones from the ventral tegmental area (VTA) of young rats aged 1–13 postnatal days, either in acute brain slices or acutely dissociated neurones. When whole-cell responses were recorded from current-clamped neurones using the gramicidin-perforated technique, the application of taurine (0.01–30 mM) accelerated firings and induced membrane depolarization. In voltage-clamped neurones, taurine induced a current which was antagonized by strychnine and by picrotoxin, but not by bicuculline. In addition, taurine-induced current showed complete cross-desensitization with glycine-activated currents but not with -aminobutyric acid (GABA)-activated currents. Thus, taurine is a full agonist of the glycine receptors (GlyRs) in the VTA. Further studies found that taurine acted mainly on non-synaptic GlyRs. The application of 20 μM bicuculline abolished the spontaneous inhibitory post-synaptic currents (IPSCs) in 40/45 neurones, and 93% of the evoked IPSCs. The addition of 1 μM strychnine completely eliminated the remaining IPSCs. These results suggest that GABAergic IPSCs predominate, and that functional glycinergic synapses are present in a subset of the VTA neurones. The application of 1 μM strychnine alone induced an outward current, suggesting that these neurones were exposed to tonically released taurine/glycine. In conclusion, by activating non-synaptic GlyRs, taurine may act as an excitatory extra-synaptic neurotransmitter in the VTA during early development.
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Introduction
Taurine, a 2-aminoethane-sulphonic acid, has many physiological actions in the nervous system. These actions include serving as a trophic factor in the development of the CNS (Chen et al. 1998), protection against excitotoxic cell death, depressing pyramidal cell excitability (Tang et al. 1996), regulating calcium homeostasis (Lazarewicz et al. 1985) and serving as a neurotransmitter or neuromodulator (Lombardini, 1992) as well as an anticonvulsant. Taurine has long been known as a major constituent of many parts of the CNS, including the ventral tegmental area (VTA) (Huxtable, 1989). Taurine concentration in the immature brain is (in the millimolar range within most parts of the brain) 4- to 5-fold higher than in the adult (Kaczmarek, 1976). Having a strychnine-sensitive, glycine-like action on many cells (Curtis et al. 1968; Krnjevi & Puil, 1976; Legendre, 2001; Mori et al. 2002; Jiang et al. 2004), taurine has been proposed as a candidate inhibitory transmitter (Curtis et al. 1968; Davison & Kaczmarek, 1971; Kaczmarek, 1976).
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In the spinal cord and brainstem, glycine is a major inhibitory neurotransmitter that mediates fast synaptic inhibition by activating glycine receptors (GlyRs) (Werman et al. 1968; Krnjevi, 1974; Nicoll et al. 1990; Kuhse et al. 1995). Recent studied found that GlyRs are widely expressed throughout the mammalian CNS (Werman et al. 1968; Krnjevi, 1974; Nicoll et al. 1990; Betz, 1991; Kuhse et al. 1995), not restricted to the spinal cord and brainstem (Legendre, 2001). Glycine and other GlyR agonists can elicit Cl–-mediated responses in neurones from many brain regions, including the VTA (Ye et al. 1998; Ye, 2000). The VTA is a part of the midbrain stem rich in dopaminergic (DA) neurones that project to the nucleus accumbens and the prefrontal cortex, and this DA pathway plays a pivotal role in mediating the rewarding effects of abused drugs, including alcohol. Previous studies from our laboratory have showed that exogenous application of glycine could elicit strychnine-sensitive currents in most of the rat VTA neurones (Ye et al. 1998), indicating the existence of functional GlyRs in this area. However, the VTA has a relatively sparse glycinergic innervation (Rampon et al. 1996), suggesting that other endogenous ligand(s) might activate GlyRs. To determine whether taurine might be the endogenous agonist of the GlyRs in the VTA, in this study we investigated the physiological and pharmacological properties of the receptors taurine triggers in VTA DA neurones from young rats aged 1–13 of postnatal days (P).
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Methods
Preparations
All procedures conformed with 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. Sprague-Dawley rat pups aged 1–30 postnatal days (P1–30) were decapitated and the brain was quickly excised, placed into ice-cold saline saturated with O2, glued to the chilled stage of a Vibroslice (Campden Instruments, Leicester, UK) or a VF100 slicer (Precisionary Instruments, Greenville, NC, USA) and sliced to a thickness of 300–400 μm. Slices were incubated in standard external solution (see below for composition) saturated with O2 at room temperature (21–23°C) for at least 1 h before use. To obtain neurones with functional terminals, we used a method similar to that described by Akaike & Moorhouse (2003) with modifications. Briefly, following incubation, slices containing the VTA were transferred to a 35 mm dish and the VTA was identified medial to the accessory optic tract and lateral to fasciculus retroflexus under an inverted microscope (Nikon). A heavily fire-polished glass pipette, lightly touching the surface of the VTA, was vibrated horizontally at 20 Hz for 2 min by a home-made apparatus. The slice was then removed and the neurones adhered to the bottom of the dish within 20 min. These neurones, which were dissociated without using any enzymes, retained some of their original morphological features including the proximal dendritic processes (Ye et al. 2004). Some experiments were conducted on neurones acutely dissociated with enzymes with a method described earlier (Ye et al. 1998). Briefly, slices were digested with pronase (1 mg (12 ml)–1) and thermolysin (1 mg (12 ml)–1) and then incubated in oxygen-saturated standard external solution at room temperature (21–23°C).
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Some experiments were conducted in brain slices, which were obtained by a procedure similar to that described above except that the midbrain was sliced in the horizontal plane (300 μm) in an artificial cerebrospinal fluid (ACSF) solution saturated with 95% O2–5% CO2 (carbogen). Slices (two per animal) were transferred to a holding chamber with carbogen-saturated ACSF and allowed to recover for at least 1 h before being placed in the recording chamber, superfused with carbogen-saturated ACSF at a rate of 1.5 ml min–1 at room temperature (Ye et al. 2004).
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Electrophysiological recording
Membrane currents and potentials were recorded under voltage-clamp and current-clamp modes, respectively, with Axopatch MultiClamp 700A or Axopatch 200B amplifiers (Axon Instruments, Forster City, CA, USA), interfaced to a desktop IBM compatible computer via Digidata 1320A or Digidata 1320 (Axon Instruments) analog-to-digital converters and directly digitized with pCLAMP 9.0 software for off-line analysis. Whole-cell recordings were low-pass filtered at 2 kHz and digitized at 10 kHz. When filled with the pipette solutions (see below), the pipettes had a resistance of 3–5 M. In most experiments, the series resistance before compensation was 10–20 M. Routinely, 80% of the series resistance was compensated; hence, there was a 2–4 mV error for every 1 nA of current. Liquid junction potentials were calculated with the generalized Henderson equation using the Axoscope junction potential calculator and corrected post hoc. Neurones with an access resistance < 20 M were selected for further tests.
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For the gramicidin-perforated patch electrodes, the gramicidin stock solution (10 mg ml–1 in methanol; J.T. Baker, Inc., Phillipsburg, NJ, USA) was diluted in the pipette solution to a final concentration of 50–100 μg ml–1 just before the experiment. After establishing a giga-seal in the cell-attached configuration by gentle suction, no further negative pressure was applied. Access resistance was carefully monitored during perforation and throughout the experiment, and any neurones in which a sudden drop in access resistance occurred were discarded. Perforated-patch recordings were rejected if there was a rapid decrease in series resistance, or a loss of sodium currents (see ‘Solutions’ below for further information). Throughout the experiment, the bath was continually perfused with standard saline. All glycine-induced responses were elicited in this solution at an ambient temperature of 21–23°C.
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The brain slices were prepared with an experimental procedure described previously (Ye et al. 2004). Briefly, cells were visualized using an upright microscope with infrared illumination (E600FN, Nikon). To evoke monosynaptic inhibitory post-synaptic currents (eIPSCs) in brain slices, a bipolar nichrome wire-stimulating electrode was placed 50–100 μm rostral to the recording electrode. Stimuli (a 100 μs depolarizing pulse) were given at a frequency of 0.1 Hz. Stimulation intensity was set at the lowest level that evoked stable responses with no failures. Input resistance was calculated from the change in membrane potential in response to small 500-ms hyperpolarizing pulses (–10 or –20 pA) given every 3 s or otherwise as indicated.
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Solutions
The standard external solution contained (mM): NaCl 140, KCl 5.0, MgCl2 1.0, CaCl2 2.0, glucose 10 and Hepes 10. The pH was adjusted to 7.4 with 1 N NaOH. ACSF contained (mM): NaCl 126, KCl 1.6, NaH2PO4 1.2, MgCl2 1.2, CaCl2 2.4, NaHCO3 18 and glucose 11. The osmolarity of these solutions was adjusted to 310 mosmol l–1 with sucrose. For gramicidin-perforated patch-clamp experiments, initially we used a pipette solution containing (mM): K2SO4 60.5, KCl 40, CaCl2 0.5, EGTA 5 and Hepes 10. In the later experiments, we used a pipette solution containing (mM): 140 KCl, 10 Hepes and 2 lidocaine N-ethylbromide (QX-314). QX-314 is a sodium channel blocker; it is a large molecule and can enter the cell only when the membrane is ruptured. Voltage-activated sodium current was checked at the beginning and the end of each cell recording. The disappearance of sodium current is an indication of accidental rupture of the membrane. When this happened, this cell was discarded. Pipette solution for conventional whole-cell voltage-clamp experiments was (mM): KCl 140, Hepes 10, EGTA 4.0, CaCl2 0.4, MgCl2 1.0 and Mg-ATP 2.0. The pH of the pipette solutions was adjusted to 7.2 with 1 N KOH. The osmolarity of these pipette solutions was adjusted to 290 mosmol l–1 with sucrose.
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Chemicals and applications
Taurine, glycine, -aminobutyric acid (GABA), strychnine, picrotoxin, bicuculline, QX-314, DL-2-amino-5-phosphono-valeric acid (DL-APV), 6,7-dinitroquinoxaline-2, 3-dione (DNQX) and other standard chemicals were purchased from Sigma-Aldrich. The solutions were prepared on the day of the experiment. Chemicals were prepared at the final concentrations and were applied to a dissociated neurone via a multi-barrelled pipette, whose tip was placed 50–100 μm from the neurone. With this system, solutions in the vicinity of a neurone could be completely exchanged within 20 ms, without loss of mechanical stability (Ye et al. 1998). In slice experiments, chemicals were prepared at the final concentration in ACSF and were applied via bath perfusion (Ye et al. 2004).
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Immunocytochemistry
Cell staining was done using a method described by Brodie et al. (1999). Briefly, after a 15 min rinse with phosphate-buffered saline (PBS, pH 7.4), cells were fixed for 1 h in 4% paraformaldehyde in PBS at 4°C, and then transferred to PBS. The immunolabelling procedure was conducted at 21–23°C, rotating at 30 r.p.m. After three 10-min rinses in Tris-buffered saline (TBS, pH 7.4) that contained 0.25% Triton X-100 (TBST), cells were blocked in 3% normal goat serum (NGS) in TBST for 30 min, and rinsed for 15 min in TBST. Cells were then incubated in rabbit anti-tyrosine hydroxylase (AB152; Cheicon, Temecula, CA, USA) diluted 1: 250 in 1% NGS in TBST overnight at 4°C, followed by 2 h at room temperature. After five 10-min rinses in 1% NGS in TBS, cells were incubated for 60 min in goat anti-rabbit biotinylated antibody (Vector Laba, Burlingame, CA, USA) for 60 min in PBS, and reacted for 30 min with diaminobenzidine that contained nickel ammonium sulphate. Cells were then rinsed with TBS and double-distilled water, air-dried and mounted on slides with Permount. Cells of interest were examined and photographed with a digital camera on a Nikon inverted microscope.
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Data analysis
All data are presented as means ± S.E.M.; when appropriate, the means were compared by Student's t test. Statistical data were considered significant when P 0.05.
Results
Identification of DA neurones
Figure 1 shows example of large (35–60 μm somatic diameter) and small (20–40 μm) neurones acutely dissociated from the VTA. Most of the cells were tyrosine hydroxylase-positive (Fig. 1A–F), in keeping with a previous report (Brodie et al. 1999). The large neurones are multipolar with three to five major dendrites (Fig. 1A and B). The majority were small neurones, which were bipolar with one to three dendrites (Fig. 1C–F). The tyrosine hydroxylase-negative cells were small (Fig. 1G). In current-clamp condition, the resting membrane potentials of all large neurones and most of the smaller neurones fired spontaneously in a regular pacemaker fashion (Fig. 2Aa). The mean frequency of spontaneous action potentials was 1–13 Hz (4.7 ± 2.6 Hz, n = 10). The spike activity was driven by a slow depolarization, depolarizing the membrane from its resting state to that of the characteristic high spike threshold of DA neurones (–40 mV). The action potential was followed by a prominent afterhyperpolarization. The duration of the action potentials (measured from the point of initiation of its fastest-rising phase) was 5.1 ± 2.6 ms (n = 10). Since VTA neurones are known to express dopamine D2-like receptors, we tested the effects of dopamine on VTA neurones. As shown in Fig. 2Ab, dopamine (1 μM) hyperpolarized many DA neurones and reduced the discharge frequency. In some neurones, dopamine completely blocked the ongoing discharges (data not shown). The spontaneous firings recovered immediately after washout of dopamine. Membrane responses to injection of hyperpolarizing currents were prominent ‘voltage sag’ – one of the distinctive physiological membrane properties of VTA DA neurones (Fig. 2Ac) (Johnson & North, 1992). Assessed from the voltage jumps induced by negative hyperpolarized current injection, the input resistance was 474 ± 96 M (n = 12) measured on the peak of the ‘voltage sag’ and 281 ± 42 M (n = 12) measured at the end of the voltage steps (the steady state, Fig. 2Ac). The input resistance tended to increase with the reduction in cell size. Figure 2B shows the current traces recorded from some of the small neurones of the VTA. These neurones fired irregularly and at a higher frequency (12 ± 3 Hz, n = 5). They tended to have a more negative resting membrane potential (–68.5 ± 4.5 mV, n = 10), comparing with that (–52.3 ± 4.6 mV, n = 10, P < 0.01) of DA neurones. The duration of the spikes was 1.8 ± 0.5 ms (n = 10). They did not respond to dopamine application (Fig. 2Bb). There was no ‘voltage sag’ in response to an injection of hyperpolarizing current and they had a lower spike threshold (Fig. 2Bc). The input resistance of these neurones varied significantly and tended to be higher (1196 ± 227 M, n = 10) compared with that of DA neurones. As judged by the above electrophysiological properties, these neurones were identified as GABAergic neurones (Steffensen et al. 2001). In the following experiments, we focused on DA neurones, which were identified by the presence of a prominent ‘voltage sag’ that was assayed immediately after break-in, using a series of incremental –25 pA steps from the resting membrane potential.
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Light micrographs of the VTA neurones after processing for tyrosine hydroxylase immunohistochemistry. These neurones were enzymatically dissociated from the VTA. A–F are positively stained. A and B are large cells. C–F are small neurones. G is a negatively stained small neurone. Calibration bar is 50 μM.
All responses illustrated in this figure were recorded from neurones in brain slices under current-clamp conditions. The voltage changes in Aa and c were recorded from putative VTA DA neurones of a P13 rat and in Ab from a P10 rat. The voltage changes in Ba and b were recorded from a putative VTA GABAergic neurone from a P18 rat, and from a P10 rat in Bc. Aa, 10 s voltage trace shows the slow and regular spontaneous action potentials of a DA neurone. Note the prominent post-action potential hyperpolarization, after which a slow spontaneous depolarization follows. Ba, 10 s trace shows typical irregular discharges of a GABAergic neurone. Ab, 60 s trace shows that the application of 1 μM dopamine (Dopa) slowed down the action potentials of a DA neurone. Bb, 15 s trace collected at the end of 8 min continuous bath perfusion of 20 μM dopamine, showing that 20 μM dopamine did not alter the firings of a GABAergic neurone. Ac, a DA neurone has a prominent time-dependent ‘sag’ in membrane potential in response to –130 pA current, which was applied as 25 pA steps from 0 pA (wave form shown in d). Bc, a GABAergic neurone has no time-dependent ‘sag’.
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Taurine increases the excitability of VTA DA neurones of young rats
This series of experiments was carried out using the gramicidin-perforated patch clamp to study the effect of taurine without altering the intracellular Cl– concentration (Ye, 2000). As illustrated in Fig. 3, in response to application of taurine, almost all (47/50) of the DA neurones younger than P13 were depolarized (Fig. 3A); 21/40 neurones from P14 to about P19 rats were depolarized and the other 19/40 neurones were hyperpolarized; while 17/20 neurones from rats older than P20 were hyperpolarized (Fig. 3D). According to the extracellular solution and the K2SO4-based pipette solution, the Cl– equilibrium potential is close to –33 mV. To minimize the possibility that the reversal potential of taurine-induced current (ITau) was affected by the K2SO4-based pipette solution, in the later experiments, pipettes contained 140 mM KCl, 10 mM Hepes and 2 mM QX-314. With this pipette solution, 10/10 neurones from young rats (P7 to about P10) were depolarized by taurine. The mean depolarization observed in 14 VTA neurones, from rats younger than P13, were plotted as a function of taurine concentration (Fig. 3B). Using a non-linear least-squares method, the taurine dose–response data were plotted via the logistic equation:
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where V represents the depolarization, Vmax the maximum effect of taurine, n the Hill efficient, and EC50 represents the taurine concentration that evoked a half-maximal response, which was 0.97 mM.
A, currents were recorded from a putative VTA DA neurone of a P10 rat. Aa–e, different dosages of taurine had different effects on the action potentials, with low concentrations of taurine increasing the frequency of action potentials, and higher concentrations of taurine blocking action potentials. The dashed line shows the resting membrane potential, which was –60 mV. B, concentration dependence of taurine-induced depolarization (n = 9), with EC50 = 0.97 mM. The curve was fitted using the Hill equation. C, scatter plot of the changes of membrane potential induced by 1 mM taurine in rats as a function of age (n = 110). The fitted line shows the reversal point was at about P17. Da–e, dosage dependence of taurine-induced hyperpolarization in a VTA DA neurone from a P19 rat. The dashed line shows the resting membrane potential of –60 mV. E, voltage ramps (from +50 to –80 mV) were applied at a rate of 0.13 V s–1 before and during application of taurine. Currents induced by 1 mM taurine in VTA DA neurones of P10 and P20 rats, respectively. Holding potential was –50 mV. F, current–voltage plots are substantially different, with the reversal potentials at –30 mV and –55 mV for P10 and P20 rats, respectively.
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The sustained taurine-induced depolarization had a very intense effect on the action potentials. In rats under P13, low doses of taurine induced a small depolarization, which in turn facilitated the frequency of action potentials (Fig. 3Aa and b). Taurine-induced membrane depolarization was associated with an increase in membrane conductance (data not shown). Higher doses of taurine induced greater depolarization, which suppressed the ongoing discharges (Fig. 3Ac–e). This might have resulted from a combination of the shunting effect of GlyR activation (Staley & Mody, 1992) and Na+ current inactivation. In rats older than P18, taurine-induced hyperpolarization could suppress the ongoing discharges (Fig. 3D). Thus, taurine can regulate the membrane potential and increase the excitability of VTA neurones of young rats.
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The membrane potentials were voltage ramped from +50 to –80 mV to assess the reversal potential of ITau (Fig. 3E). After subtracting the currents obtained under control conditions (the first ramp), we obtained the current–voltage (I–V) relationships for taurine. As illustrated by the I–V curves in Fig. 3F, the reversal potential of ITau shifted to more negative as the rats aged (ETau = –29.3 ± 9.1 mV at P10; –57.8 ± 13.4 mV at P20, n = 8). The above data were collected with a K2SO4-based pipette solution. Virtually identical results were obtained when the pipette solution containing 140 mM KCl (data not shown). In further experiments, we investigated the properties of taurine-activated receptors in rats younger than P13.
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In voltage-clamped VTA DA neurones, ITau and IGly had similar I–V curves and reversal potentials
In approximately 98% of VTA DA neurones (voltage held at –50 mV), taurine- and glycine-evoked currents (ITau and IGly) had similar desensitizing kinetics. Figure 4Aa and b shows superimposed traces of inward currents evoked by 30 s application of glycine and taurine at five concentrations (as indicated) in a VTA DA neurone. Averaged data for ITau and IGly from 9 and 12 neurones are presented in the concentration–response curves in Fig. 4B: EC50 values for glycine and taurine were 124 μM and 1.07 mM, respectively. The mean maximal ITau and IGly evoked by 3 mM glycine (869 ± 104 pA) and 30 mM taurine (904 ± 136 pA) were not significantly different (Student's paired t test, P > 0.05, n = 31). In comparison with glycine, 10-fold higher concentrations of taurine were required to obtain similar currents. These results show that activation, desensitization and deactivation of both ITau and IGly occurred faster when agonist concentrations were higher (Fig. 4Aa and b). The membrane potentials were voltage-ramped from +50 to –80 mV to assess the voltage dependence of ITau (Fig. 4C). After subtracting the currents measured under control conditions (the first ramp), we obtained virtually the same I–V relationships for ITau and IGly. Based upon the I–V plots and that of another six neurones, the reversal potentials of both ITau and IGly were near –30 mV (Fig. 4D).
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All traces in this, as well as in the following figures, were recorded at a holding potential of –50 mV from dissociated neurones. Aa, currents recorded by varying the indicated concentrations of glycine in a putative VTA DA neurone of a P11 rat at 2 min. intervals. Ab, comparable series of currents evoked by taurine in a VTA DA neurone of the same rat. B, glycine- and taurine-evoked peak currents as a function of concentration. Currents evoked by glycine (n = 9) and taurine (n = 12) were normalized to the responses induced with 1 mM glycine and 30 mM taurine, respectively. EC50 values were 117 μM for glycine and 1.07 mM for taurine. C and D show that glycine- and taurine-evoked currents produced similar current–voltage (I–V) relations. Voltage ramps were applied before and during application of glycine and taurine. C, initial small ramp currents during resting condition and the largely increased ramp currents during inward shift obtained either by 100 μM glycine (a) or by 1 mM taurine (b) are shown. The data were obtained from the same VTA DA neurone of a P9 rat. D, I–V relations for glycine and taurine elicited by subtracting the control current (first of each pair of ramps) from the currents induced by a second ramp and normalizing values to a maximal outward current. Note, I–V plots for glycine- and taurine-induced currents are virtually identical and both have a reversal potential at –30 mV.
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ITau was sensitive to both picrotoxin and strychnine, but not to bicuculline
To determine if GlyRs in VTA were homomeric, taurine was applied at a concentration near EC50 (1 mM) with different concentrations of strychnine, a selective antagonist of GlyRs, and picrotoxin, an antagonist of homomeric GlyRs (Fig. 5). As the concentrations of strychnine and picrotoxin were increased, ITau became progressively smaller. Strychnine (1 μM) or picrotoxin (30 μM) almost completely abolished ITau. From the concentration–response relationships, we obtained IC50 values of 8.5 μM for picrotoxin (Fig. 5C and D) and 95.9 nM for strychnine (Fig. 5A and B) for ITau. Thus, ITau is sensitive to strychnine and picrotoxin. However, as shown in Fig. 5E and F, the addition of 10 μM bicuculline had no significant effect on ITau induced by 1 mM taurine.
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A, currents evoked by 1 mM taurine in a dissociated VTA DA neurone of a P10 rat were concentration-dependently blocked by strychnine (STR). B, concentration–response relation of strychnine blockage of ITau. Data were normalized to the currents elicited by 1 mM taurine (control); IC50 was 95.9 nM (n = 8). C, currents evoked by 1 mM taurine in a VTA DA neurone of a P11 rat were concentration-dependently blocked by picrotoxin (PTX). D, concentration–response relation of picrotoxin blockage of ITau. Data were normalized similar to B; IC50 was 8.5 μM (n = 7). E, currents elicited in a VTA neurone from a P11 rat had little, if any, sensitivity to 10 μM bicuculline. F, mean antagonistic effects of strychnine, picrotoxin and bicuculline (Bic) on ITau of 7 VTA neurones. Data were normalized to control currents elicited by 1 mM taurine. **P < 0.01.
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Selective cross-desensitization between the currents elicited by the application of glycine, taurine and GABA
The currents induced by these amino acids desensitized. When 100 μM glycine and 1 mM taurine were applied in rapid succession (either glycine first, or taurine first, Fig. 6A), the decay of the obtained currents was almost identical to when glycine or taurine was applied alone. This indicated that a full cross-desensitization existed between glycine and taurine. In comparison, ITau (induced by 1 mM taurine) and IGly (induced by 100 μM glycine) showed no cross-desensitization with IGABA (induced by 100 μM GABA) (Fig. 6B and C). In the case when ITau and IGly were preceded by IGABA, peak ITau and IGly were 98.2 ± 2.1 and 99.3 ± 3.2% of the control, respectively (Fig. 6F, Student's paired t test, P > 0.05, n = 6). In the cases when IGABA was preceded by IGly or ITau, IGABA was 97.3 ± 4.6 and 99.7 ± 4.1% of the control, respectively (Fig. 6F, Student's paired t test, P > 0.05, n = 6). These data suggest that 1 mM taurine has little, if any, effect on GABAARs of immature VTA neurones. However, 10 μM bicuculline blocked 32.2 ± 8.1% (n = 4, P < 0.05) of the peak ITau induced by 10 mM taurine (data not shown). In addition, when ITau (induced by 10 mM taurine) was preceded by IGABA induced by 200 μM GABA, the peak ITau was 79.7 ± 3.9% of control (P < 0.05, n = 6; Fig. 6D and F). On the other hand, when IGABA (induced by 200 μM GABA) was preceded by ITau induced by 10 mM taurine, the peak IGABA was 72.3 ± 4.5% of control (P < 0.05, n = 6; Fig. 6D and F). These results suggest that very concentrated taurine is also a partial agonist of GABAARs, in keeping with a previous report (Jiang et al. 2004). An asymmetrical cross-inhibition between GABAARs and GlyRs of spinal neurones occurred for glycine concentrations > 100 μM (Li et al. 2003). We found similar asymmetrical cross-talk between GABAARs and GlyRs occurring in the VTA neurones. As illustrated in Fig. 6E, IGABA (induced by 300 μM GABA) was significantly reduced when it was preceded by IGly (induced by 300 μM glycine). The mean peak IGABA was 45.0 ± 2.4% of control (n = 4, P < 0.01; Fig. 6D and E). By contrast, IGly (induced by 300 μM glycine) was not significantly changed when it was preceded by IGABA (induced by 300 μM GABA). The mean peak IGly was 93.1 ± 4.7% of control (n = 7, P = 0.17; Fig. 6D and E).
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A, currents induced by 100 μM glycine and 1 mM taurine alone or in sequence (as indicated) in a VTA DA neurone of a P12 rat. B, in similar experiments, currents were evoked by 100 μM GABA and 1 mM taurine in a VTA DA neurone of a P12 rat. C, in similar experiments, currents were evoked by 100 μM glycine and 100 μM GABA in a VTA DA neurone of a P11 rat. D, in similar experiments, currents were evoked by 200 μM GABA and 10 mM taurine in a VTA DA neurone of a P13 rat. The dotted line indicates the control level of IGABA, and the dashed line indicates the control level of ITau. E, in similar experiments, currents were evoked by 300 μM glycine and 300 μM GABA in a VTA DA neurone of a P10 rat. The dashed line indicates the control level of IGly, and the dotted line indicates the control level of IGABA. F, ordinates represent the mean peak amplitude (+ S.E.M.) of currents induced by the agents indicated. These currents were preceded by prepulses of the indicated agents. The letters before each prepulse agent indicate the corresponding data shown in A–E. Note the much stronger reduction of IGABA (induced by 300 μM GABA) by 300 μM glycine than in the opposite order of application. All current traces in this figure were recorded from acutely dissociated neurones at a holding potential of –50 mV. The number of experiments is shown in parentheses. *P < 0.05; ** P < 0.01.
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GABAergic synapses predominate; functional glycinergic/taurinergic synapses are present in a subset of VTA neurones
We next examined the properties of spontaneous inhibitory post-synaptic currents (sIPSCs) in brain slices. With the bath solutions containing the glutamatergic receptor antagonists 20 μM DNQX and 100 μM APV, the remaining spontaneous post-synaptic currents (PSCs) were completely blocked by 20 μM bicuculline in 40/45 neurones tested (Fig. 7A). In the other cells, a mixture of 20 μM bicuculline and 1 μM strychnine completely abolished the sIPSCs (data not shown). As illustrated in Fig. 7B and C, in the presence of 20 μM DNQX and 100 μM APV, 20 μM bicuculline blocked the evoked IPSCs to 7.3 ± 4.4% (n = 4) of control. The addition of 1 μM strychnine completely eliminated the remaining component (to 2.3 ± 1.5% of control, n = 4). Several studies have shown that strychnine (1 μM) had no significant effect on GABA-evoked responses (Hussy et al. 1997; Mori et al. 2002; Mangin et al. 2002). Thus, these results suggest that GABAergic IPSCs predominate, and that a subset of VTA DA neurones receive a functional glycinergic innervation.
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A, representative current traces recorded in a VTA DA neurone in a brain slice from a P13 rat. Parts of the traces are shown below on an expanded time scale. B, the blocking effects of 20 μM bicuculline and 20 μM bicuculline + 1 μM strychnine on evoked IPSCs of a VTA DA neurone in a brain slice from a P12 rat. C, mean area of evoked IPSCs from brain slices were significantly reduced by bicuculline (P < 0.01, n = 4), and completely eliminated by 20 μM bicuculline + 1 μM strychnine (n = 4).
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Tonic action of taurine on GlyRs in VTA DA neurones
The PSC experiment showed that there was no phasic synaptic action of taurine on GlyRs in the majority of VTA DA neurones. Taurine-elicited currents might serve to enhance a basal level of endogenous transmitter release and display a tonic activation of GlyRs. Therefore, we asked whether GlyRs in VTA are tonically activated. The application of strychnine (0.5 μM) to brain slices produced reversible outward currents (Fig. 8A), indicating that GlyRs in the VTA slice are tonically activated. By contrast, in out-side out patch (Fig. 8B), and in enzymatically dissociated neurones, strychnine induced no current (Fig. 8C), while taurine (1 mM) induced significant inward current (Fig. 8B and D), which suggested that there is no taurine or glycine in the solution.
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A, application of 0.5 μM strychnine to a neurone in a brain slice of a P12 rat produced an outward current. B, strychnine alone did not gate GlyRs, but completely blocked activation of taurine in an outside-out patch containing GlyRs. C, in a enzymatically dissociated neurone of a P12 rat, strychnine induced no current, while taurine (1 mM) induced significant inward current (D).
Discussion
Our data convincingly show that taurine increased the excitability of VTA DA neurones by activating non-synaptic GlyRs in young rats, in contrast to its conventional hyperpolarizing action and synaptic inhibition. Our present study found bicuculline blocked 93% of the IPSCs, whereas strychnine blocked only 7%. These results suggest that GABAergic IPSCs dominate, and that functional glycinergic synapses are present only in a small subset of the VTA neurones. Therefore, taurine, which might be released from glial cells, accomplishes its role in the development and functional modulation of VTA DA neurones by activating non-synaptic GlyRs.
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Taurine-induced depolarization in VTA DA neurones of young rats
Like glycine and GABA, taurine was first proposed as an inhibitory neurotransmitter because it inhibits neuronal firings. In the present study on dissociated VTA DA neurones from young rats (P1P13), taurine facilitated spontaneous discharges at low dosage. At high dosage, taurine induced depolarization and stopped spontaneous on-going discharges. The depolarization might induce Ca2+ influx, which is related to its role in trophic action in developing neurones (Flint et al. 1998). The depolarization might be ascribed to a relatively high intracellular [Cl–] and correspondingly less negative reversal potential in immature neurones (Cherubini et al. 1991). It is in good agreement with the effect of glycine and GABA on developing neurones of many brain regions (Cherubini et al. 1991; Flint et al. 1998). In contrast, in older rats, the major effect of taurine was hyperpolarization and blockage of ongoing discharges. This is consistent with its conventional inhibitory roles (Taber et al. 1986; Saransaari & Oja, 1997).
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The gradual shift of reversal potentials was tested using voltage ramps before and during application of taurine, and the reversal potentials were found to be at –30 and –55 mV for P10 and P20 rats, respectively (Ye, 2000). Therefore, the reversal potentials for taurine in immature neurones were less negative than the resting membrane potentials. This probably resulted from the relatively high cytoplasmic [Cl–] in immature neurones, which has been ascribed to a family of cation–chloride co-transporters (Delpire, 2000). The Na+–K+–2Cl– co-transporter (NKCC1), which is driven by a sodium and potassium gradient, raised [Cl–]i. The K+–Cl– co-transporter KCC2 couples Cl– transport to the K+ gradient and normally lowers [Cl–]i below that dictated by passive distribution (Rivera et al. 1999). It has been shown that KCC2 is up-regulated while NKCC1 is down-regulated in the CNS during development (Delpire, 2000).
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Taurine activated GlyRs in VTA DA neurones of young rats
Taurine has been shown to be a full agonist of GlyRs in isolated rat neurones of ventromedial hypothalamus and nucleus accumbens (Tokutomi et al. 1989; Jiang et al. 2004), in HEK293 cells (Rajendra et al. 1995), and Xenopus oocytes (De Saint Jan et al. 2001) that express recombinant human GlyR 1 and 2 subunits, respectively. However, in supraoptic magnocellular neurones (Hussy et al. 1997) and Xenopus oocytes that express recombinant human GlyR 1 subunits (Schmieden et al. 1992), taurine was a partial agonist of GlyRs. In our experiments, comparable inward currents were induced by 100 μM glycine and 1 mM taurine, and IGly and ITau had a similar I–V curve and the same reversal potential. These results suggest that, like IGly, ITau is carried predominantly by Cl–.
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Pharmacological properties of taurine-activated receptors are similar to those of GlyRs. Picrotoxin and strychnine were both effective antagonists of ITau, but bicuculline had almost no effect on ITau elicited by 1 mM taurine. The IC50 of 8.5 μM for picrotoxin for taurine is similar to that for IGly reported for immature VTA neurones (Ye et al. 1998), for IGly in mature hippocampal neurones (Chattipakorn & McMahon, 2002), as well as that for ITau in nucleus accumbens of young rats (Jiang et al. 2004), but different from that of adult nucleus accumbens neurones (Martin & Siggins, 2002). The IC50 for strychnine (95.9 nM) for taurine is also consistent with previous observations of that for IGly on neurones of many brain regions, including VTA (Ye et al. 1998), basolateral amygdala (McCool & Botting, 2000), striatum (Sergeeva, 1998), supraoptic magnocellular nucleus (Hussy et al. 1997), and hippocampus (Chattipakorn & McMahon, 2002; Mori et al. 2002). GlyRs in the mammalian CNS are formed by a combination of five membrane-spanning protein subunits, while the antagonist picrotoxin, at concentrations lower than 30 μM, inhibits the function of homomeric receptors, but does not affect heteromeric + receptors (Pribilla et al. 1992; Legendre, 1997; Mangin et al. 2002). 2 homomers are most sensitive to picrotoxin, with an IC50 of 6 μM, while GlyRs of mature neurones consisting mainly of 1 heteromers are sensitive to strychnine. The high picrotoxin affinity observed in the current study suggests that the GlyRs did not contain any subunit. The relative high strychnine affinity of ITau found in the present study suggests the expression of 1 subunits (Schmieden et al. 1992). However, better discrimination between 1 homomeric GlyRs and 2 homomeric GlyRs will require single channel recordings (Mangin et al. 2002).
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Overall all, comparable inward currents were induced by taurine and glycine. ITau and IGly had similar characteristics of desensitization and deactivation, as well as reversal potential. Both were readily blocked by picrotoxin and by strychnine, with similar IC50 values. Moreover, both ITau and IGly showed complete cross-desensitization. All these data suggest that taurine and glycine activate the same receptors. In contrast, the current induced by taurine ( 1 mM) was not blocked by bicuculline and showed no cross talk with IGABA. These results indicate at concentrations 1 mM, taurine has little or no activation of GABAARs. In keeping with a previous study on spinal neurones (Li et al. 2003), an asymmetrical cross-inhibition between GABAARs and GlyRs of VTA neurones occurred for glycine concentrations > 100 μM. These results suggest that GABAARs and GlyRs can interact functionally on the VTA neurones.
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Non-synaptic GlyRs and non-synaptic release of taurine
The above results show that exogenous application of taurine could activate functional GlyRs on VTA DA neurones. Whether the physiological role of taurine-activating GlyRs is through cell–cell communication or just through ambient tonic action is unknown. O'Brien & Berger (1999) showed that glycine could be physiologically co-released with GABA from afferents, thereby activating the GlyRs that were co-localized with GABAARs on the same synapses in brain stem motoneurones.
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However, as bicuculline blocked the IPSCs in the majority of the cells tested, this suggests that GABAergic innervation dominates. This also suggests that many GlyRs in the VTA might be non-synaptically activated by taurine. The non-synaptic activation of GlyRs was studied by Legendre's group (Mangin et al. 2003), who found that 2 homomeric GlyRs in young rats were completely inefficient if post-synaptically located, for the rising phase of the responses evoked by glycine were rather slow. They concluded that 2 homomeric GlyRs, which are known to be expressed prior to synaptogenesis (Legendre, 2001), might be involved in a paracrine function of GlyRs if the concentration of the released agonist is high enough. The concentration of taurine is high enough to fulfil this job. In addition, taurine could be released in the absence of action potentials and extracellular calcium, which are a prerequisite for neurotransmitter release (Chen et al. 1996; Flint et al. 1998). Taurine is stored in glial cells (Almarghini et al. 1991; Hussy et al. 2001), where both the synthesizing enzyme and the transporter for taurine are present (Almarghini et al. 1991; Flint et al. 1998). It is released in high levels in response to conditions that elicit exocytotoxic cell death (Saransaari & Oja, 1997) and is also released in response to hypotonic stimulus (Deleuze et al. 1998) or ischaemia (Saransaari & Oja, 2002). Therefore, taurine is most probably the extra-synaptically endogenous ligand for the VTA GlyRs. Because of the involvement of the VTA in midbrain mechanisms underlying drug addiction, the repeated finding that ethanol promotes the release of taurine in mesolimbic structure (Dahchour et al. 1996; De Witte et al. 1994) is especially pertinent to the current report.
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The excitatory drive of taurine through GlyRs is very important for the early stage of development of the CNS, when excitatory glutamatergic synapses are rather quiescent (Cherubini et al. 1991). Taurine deprivation of kittens leads to low brain weight and an apparent disruption of normal cortical migration and differentiation (Palackal et al. 1986). However, mice lacking the two GABA synthetic enzymes GAD65 and GAD67, and thus also lacking GABA, have normal brain histology and cytoarchitecture at birth (Ji et al. 1999). In addition, taurine is one of the most abundant (e.g. 4–10 mM) and ubiquitous free amino acids in the brain (Sturman et al. 1978; Huxtable, 1989). Furthermore, the taurine reuptake transporter is highly expressed in the rat brain (Smith et al. 1992). In contrast, the presence of glycinergic afferents in the VTA is sparse (Rampon et al. 1996) and the levels of glycine in the brain, especially in the neocortex, are very low (Aprison et al. 1969). Taurine may be the most important trophic factor in brain development. Especially relevant is the finding that taurine levels and its release are particularly high in immature rats (Sturman et al. 1978). According to our studies, the prominent effects of taurine in VTA neurones would make immature animals (and possible humans) particularly vulnerable to detrimental effects of those agents, such as ethanol, that enhance GlyR function and/or activity (Ye et al. 2001; Jiang & Ye, 2003; Zhu & Ye, 2005).
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Abstract
The physiological and pharmacological properties of taurine-induced responses were investigated in dopaminergic (DA) neurones from the ventral tegmental area (VTA) of young rats aged 1–13 postnatal days, either in acute brain slices or acutely dissociated neurones. When whole-cell responses were recorded from current-clamped neurones using the gramicidin-perforated technique, the application of taurine (0.01–30 mM) accelerated firings and induced membrane depolarization. In voltage-clamped neurones, taurine induced a current which was antagonized by strychnine and by picrotoxin, but not by bicuculline. In addition, taurine-induced current showed complete cross-desensitization with glycine-activated currents but not with -aminobutyric acid (GABA)-activated currents. Thus, taurine is a full agonist of the glycine receptors (GlyRs) in the VTA. Further studies found that taurine acted mainly on non-synaptic GlyRs. The application of 20 μM bicuculline abolished the spontaneous inhibitory post-synaptic currents (IPSCs) in 40/45 neurones, and 93% of the evoked IPSCs. The addition of 1 μM strychnine completely eliminated the remaining IPSCs. These results suggest that GABAergic IPSCs predominate, and that functional glycinergic synapses are present in a subset of the VTA neurones. The application of 1 μM strychnine alone induced an outward current, suggesting that these neurones were exposed to tonically released taurine/glycine. In conclusion, by activating non-synaptic GlyRs, taurine may act as an excitatory extra-synaptic neurotransmitter in the VTA during early development.
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Introduction
Taurine, a 2-aminoethane-sulphonic acid, has many physiological actions in the nervous system. These actions include serving as a trophic factor in the development of the CNS (Chen et al. 1998), protection against excitotoxic cell death, depressing pyramidal cell excitability (Tang et al. 1996), regulating calcium homeostasis (Lazarewicz et al. 1985) and serving as a neurotransmitter or neuromodulator (Lombardini, 1992) as well as an anticonvulsant. Taurine has long been known as a major constituent of many parts of the CNS, including the ventral tegmental area (VTA) (Huxtable, 1989). Taurine concentration in the immature brain is (in the millimolar range within most parts of the brain) 4- to 5-fold higher than in the adult (Kaczmarek, 1976). Having a strychnine-sensitive, glycine-like action on many cells (Curtis et al. 1968; Krnjevi & Puil, 1976; Legendre, 2001; Mori et al. 2002; Jiang et al. 2004), taurine has been proposed as a candidate inhibitory transmitter (Curtis et al. 1968; Davison & Kaczmarek, 1971; Kaczmarek, 1976).
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In the spinal cord and brainstem, glycine is a major inhibitory neurotransmitter that mediates fast synaptic inhibition by activating glycine receptors (GlyRs) (Werman et al. 1968; Krnjevi, 1974; Nicoll et al. 1990; Kuhse et al. 1995). Recent studied found that GlyRs are widely expressed throughout the mammalian CNS (Werman et al. 1968; Krnjevi, 1974; Nicoll et al. 1990; Betz, 1991; Kuhse et al. 1995), not restricted to the spinal cord and brainstem (Legendre, 2001). Glycine and other GlyR agonists can elicit Cl–-mediated responses in neurones from many brain regions, including the VTA (Ye et al. 1998; Ye, 2000). The VTA is a part of the midbrain stem rich in dopaminergic (DA) neurones that project to the nucleus accumbens and the prefrontal cortex, and this DA pathway plays a pivotal role in mediating the rewarding effects of abused drugs, including alcohol. Previous studies from our laboratory have showed that exogenous application of glycine could elicit strychnine-sensitive currents in most of the rat VTA neurones (Ye et al. 1998), indicating the existence of functional GlyRs in this area. However, the VTA has a relatively sparse glycinergic innervation (Rampon et al. 1996), suggesting that other endogenous ligand(s) might activate GlyRs. To determine whether taurine might be the endogenous agonist of the GlyRs in the VTA, in this study we investigated the physiological and pharmacological properties of the receptors taurine triggers in VTA DA neurones from young rats aged 1–13 of postnatal days (P).
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Methods
Preparations
All procedures conformed with 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. Sprague-Dawley rat pups aged 1–30 postnatal days (P1–30) were decapitated and the brain was quickly excised, placed into ice-cold saline saturated with O2, glued to the chilled stage of a Vibroslice (Campden Instruments, Leicester, UK) or a VF100 slicer (Precisionary Instruments, Greenville, NC, USA) and sliced to a thickness of 300–400 μm. Slices were incubated in standard external solution (see below for composition) saturated with O2 at room temperature (21–23°C) for at least 1 h before use. To obtain neurones with functional terminals, we used a method similar to that described by Akaike & Moorhouse (2003) with modifications. Briefly, following incubation, slices containing the VTA were transferred to a 35 mm dish and the VTA was identified medial to the accessory optic tract and lateral to fasciculus retroflexus under an inverted microscope (Nikon). A heavily fire-polished glass pipette, lightly touching the surface of the VTA, was vibrated horizontally at 20 Hz for 2 min by a home-made apparatus. The slice was then removed and the neurones adhered to the bottom of the dish within 20 min. These neurones, which were dissociated without using any enzymes, retained some of their original morphological features including the proximal dendritic processes (Ye et al. 2004). Some experiments were conducted on neurones acutely dissociated with enzymes with a method described earlier (Ye et al. 1998). Briefly, slices were digested with pronase (1 mg (12 ml)–1) and thermolysin (1 mg (12 ml)–1) and then incubated in oxygen-saturated standard external solution at room temperature (21–23°C).
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Some experiments were conducted in brain slices, which were obtained by a procedure similar to that described above except that the midbrain was sliced in the horizontal plane (300 μm) in an artificial cerebrospinal fluid (ACSF) solution saturated with 95% O2–5% CO2 (carbogen). Slices (two per animal) were transferred to a holding chamber with carbogen-saturated ACSF and allowed to recover for at least 1 h before being placed in the recording chamber, superfused with carbogen-saturated ACSF at a rate of 1.5 ml min–1 at room temperature (Ye et al. 2004).
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Electrophysiological recording
Membrane currents and potentials were recorded under voltage-clamp and current-clamp modes, respectively, with Axopatch MultiClamp 700A or Axopatch 200B amplifiers (Axon Instruments, Forster City, CA, USA), interfaced to a desktop IBM compatible computer via Digidata 1320A or Digidata 1320 (Axon Instruments) analog-to-digital converters and directly digitized with pCLAMP 9.0 software for off-line analysis. Whole-cell recordings were low-pass filtered at 2 kHz and digitized at 10 kHz. When filled with the pipette solutions (see below), the pipettes had a resistance of 3–5 M. In most experiments, the series resistance before compensation was 10–20 M. Routinely, 80% of the series resistance was compensated; hence, there was a 2–4 mV error for every 1 nA of current. Liquid junction potentials were calculated with the generalized Henderson equation using the Axoscope junction potential calculator and corrected post hoc. Neurones with an access resistance < 20 M were selected for further tests.
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For the gramicidin-perforated patch electrodes, the gramicidin stock solution (10 mg ml–1 in methanol; J.T. Baker, Inc., Phillipsburg, NJ, USA) was diluted in the pipette solution to a final concentration of 50–100 μg ml–1 just before the experiment. After establishing a giga-seal in the cell-attached configuration by gentle suction, no further negative pressure was applied. Access resistance was carefully monitored during perforation and throughout the experiment, and any neurones in which a sudden drop in access resistance occurred were discarded. Perforated-patch recordings were rejected if there was a rapid decrease in series resistance, or a loss of sodium currents (see ‘Solutions’ below for further information). Throughout the experiment, the bath was continually perfused with standard saline. All glycine-induced responses were elicited in this solution at an ambient temperature of 21–23°C.
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The brain slices were prepared with an experimental procedure described previously (Ye et al. 2004). Briefly, cells were visualized using an upright microscope with infrared illumination (E600FN, Nikon). To evoke monosynaptic inhibitory post-synaptic currents (eIPSCs) in brain slices, a bipolar nichrome wire-stimulating electrode was placed 50–100 μm rostral to the recording electrode. Stimuli (a 100 μs depolarizing pulse) were given at a frequency of 0.1 Hz. Stimulation intensity was set at the lowest level that evoked stable responses with no failures. Input resistance was calculated from the change in membrane potential in response to small 500-ms hyperpolarizing pulses (–10 or –20 pA) given every 3 s or otherwise as indicated.
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Solutions
The standard external solution contained (mM): NaCl 140, KCl 5.0, MgCl2 1.0, CaCl2 2.0, glucose 10 and Hepes 10. The pH was adjusted to 7.4 with 1 N NaOH. ACSF contained (mM): NaCl 126, KCl 1.6, NaH2PO4 1.2, MgCl2 1.2, CaCl2 2.4, NaHCO3 18 and glucose 11. The osmolarity of these solutions was adjusted to 310 mosmol l–1 with sucrose. For gramicidin-perforated patch-clamp experiments, initially we used a pipette solution containing (mM): K2SO4 60.5, KCl 40, CaCl2 0.5, EGTA 5 and Hepes 10. In the later experiments, we used a pipette solution containing (mM): 140 KCl, 10 Hepes and 2 lidocaine N-ethylbromide (QX-314). QX-314 is a sodium channel blocker; it is a large molecule and can enter the cell only when the membrane is ruptured. Voltage-activated sodium current was checked at the beginning and the end of each cell recording. The disappearance of sodium current is an indication of accidental rupture of the membrane. When this happened, this cell was discarded. Pipette solution for conventional whole-cell voltage-clamp experiments was (mM): KCl 140, Hepes 10, EGTA 4.0, CaCl2 0.4, MgCl2 1.0 and Mg-ATP 2.0. The pH of the pipette solutions was adjusted to 7.2 with 1 N KOH. The osmolarity of these pipette solutions was adjusted to 290 mosmol l–1 with sucrose.
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Chemicals and applications
Taurine, glycine, -aminobutyric acid (GABA), strychnine, picrotoxin, bicuculline, QX-314, DL-2-amino-5-phosphono-valeric acid (DL-APV), 6,7-dinitroquinoxaline-2, 3-dione (DNQX) and other standard chemicals were purchased from Sigma-Aldrich. The solutions were prepared on the day of the experiment. Chemicals were prepared at the final concentrations and were applied to a dissociated neurone via a multi-barrelled pipette, whose tip was placed 50–100 μm from the neurone. With this system, solutions in the vicinity of a neurone could be completely exchanged within 20 ms, without loss of mechanical stability (Ye et al. 1998). In slice experiments, chemicals were prepared at the final concentration in ACSF and were applied via bath perfusion (Ye et al. 2004).
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Immunocytochemistry
Cell staining was done using a method described by Brodie et al. (1999). Briefly, after a 15 min rinse with phosphate-buffered saline (PBS, pH 7.4), cells were fixed for 1 h in 4% paraformaldehyde in PBS at 4°C, and then transferred to PBS. The immunolabelling procedure was conducted at 21–23°C, rotating at 30 r.p.m. After three 10-min rinses in Tris-buffered saline (TBS, pH 7.4) that contained 0.25% Triton X-100 (TBST), cells were blocked in 3% normal goat serum (NGS) in TBST for 30 min, and rinsed for 15 min in TBST. Cells were then incubated in rabbit anti-tyrosine hydroxylase (AB152; Cheicon, Temecula, CA, USA) diluted 1: 250 in 1% NGS in TBST overnight at 4°C, followed by 2 h at room temperature. After five 10-min rinses in 1% NGS in TBS, cells were incubated for 60 min in goat anti-rabbit biotinylated antibody (Vector Laba, Burlingame, CA, USA) for 60 min in PBS, and reacted for 30 min with diaminobenzidine that contained nickel ammonium sulphate. Cells were then rinsed with TBS and double-distilled water, air-dried and mounted on slides with Permount. Cells of interest were examined and photographed with a digital camera on a Nikon inverted microscope.
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Data analysis
All data are presented as means ± S.E.M.; when appropriate, the means were compared by Student's t test. Statistical data were considered significant when P 0.05.
Results
Identification of DA neurones
Figure 1 shows example of large (35–60 μm somatic diameter) and small (20–40 μm) neurones acutely dissociated from the VTA. Most of the cells were tyrosine hydroxylase-positive (Fig. 1A–F), in keeping with a previous report (Brodie et al. 1999). The large neurones are multipolar with three to five major dendrites (Fig. 1A and B). The majority were small neurones, which were bipolar with one to three dendrites (Fig. 1C–F). The tyrosine hydroxylase-negative cells were small (Fig. 1G). In current-clamp condition, the resting membrane potentials of all large neurones and most of the smaller neurones fired spontaneously in a regular pacemaker fashion (Fig. 2Aa). The mean frequency of spontaneous action potentials was 1–13 Hz (4.7 ± 2.6 Hz, n = 10). The spike activity was driven by a slow depolarization, depolarizing the membrane from its resting state to that of the characteristic high spike threshold of DA neurones (–40 mV). The action potential was followed by a prominent afterhyperpolarization. The duration of the action potentials (measured from the point of initiation of its fastest-rising phase) was 5.1 ± 2.6 ms (n = 10). Since VTA neurones are known to express dopamine D2-like receptors, we tested the effects of dopamine on VTA neurones. As shown in Fig. 2Ab, dopamine (1 μM) hyperpolarized many DA neurones and reduced the discharge frequency. In some neurones, dopamine completely blocked the ongoing discharges (data not shown). The spontaneous firings recovered immediately after washout of dopamine. Membrane responses to injection of hyperpolarizing currents were prominent ‘voltage sag’ – one of the distinctive physiological membrane properties of VTA DA neurones (Fig. 2Ac) (Johnson & North, 1992). Assessed from the voltage jumps induced by negative hyperpolarized current injection, the input resistance was 474 ± 96 M (n = 12) measured on the peak of the ‘voltage sag’ and 281 ± 42 M (n = 12) measured at the end of the voltage steps (the steady state, Fig. 2Ac). The input resistance tended to increase with the reduction in cell size. Figure 2B shows the current traces recorded from some of the small neurones of the VTA. These neurones fired irregularly and at a higher frequency (12 ± 3 Hz, n = 5). They tended to have a more negative resting membrane potential (–68.5 ± 4.5 mV, n = 10), comparing with that (–52.3 ± 4.6 mV, n = 10, P < 0.01) of DA neurones. The duration of the spikes was 1.8 ± 0.5 ms (n = 10). They did not respond to dopamine application (Fig. 2Bb). There was no ‘voltage sag’ in response to an injection of hyperpolarizing current and they had a lower spike threshold (Fig. 2Bc). The input resistance of these neurones varied significantly and tended to be higher (1196 ± 227 M, n = 10) compared with that of DA neurones. As judged by the above electrophysiological properties, these neurones were identified as GABAergic neurones (Steffensen et al. 2001). In the following experiments, we focused on DA neurones, which were identified by the presence of a prominent ‘voltage sag’ that was assayed immediately after break-in, using a series of incremental –25 pA steps from the resting membrane potential.
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Light micrographs of the VTA neurones after processing for tyrosine hydroxylase immunohistochemistry. These neurones were enzymatically dissociated from the VTA. A–F are positively stained. A and B are large cells. C–F are small neurones. G is a negatively stained small neurone. Calibration bar is 50 μM.
All responses illustrated in this figure were recorded from neurones in brain slices under current-clamp conditions. The voltage changes in Aa and c were recorded from putative VTA DA neurones of a P13 rat and in Ab from a P10 rat. The voltage changes in Ba and b were recorded from a putative VTA GABAergic neurone from a P18 rat, and from a P10 rat in Bc. Aa, 10 s voltage trace shows the slow and regular spontaneous action potentials of a DA neurone. Note the prominent post-action potential hyperpolarization, after which a slow spontaneous depolarization follows. Ba, 10 s trace shows typical irregular discharges of a GABAergic neurone. Ab, 60 s trace shows that the application of 1 μM dopamine (Dopa) slowed down the action potentials of a DA neurone. Bb, 15 s trace collected at the end of 8 min continuous bath perfusion of 20 μM dopamine, showing that 20 μM dopamine did not alter the firings of a GABAergic neurone. Ac, a DA neurone has a prominent time-dependent ‘sag’ in membrane potential in response to –130 pA current, which was applied as 25 pA steps from 0 pA (wave form shown in d). Bc, a GABAergic neurone has no time-dependent ‘sag’.
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Taurine increases the excitability of VTA DA neurones of young rats
This series of experiments was carried out using the gramicidin-perforated patch clamp to study the effect of taurine without altering the intracellular Cl– concentration (Ye, 2000). As illustrated in Fig. 3, in response to application of taurine, almost all (47/50) of the DA neurones younger than P13 were depolarized (Fig. 3A); 21/40 neurones from P14 to about P19 rats were depolarized and the other 19/40 neurones were hyperpolarized; while 17/20 neurones from rats older than P20 were hyperpolarized (Fig. 3D). According to the extracellular solution and the K2SO4-based pipette solution, the Cl– equilibrium potential is close to –33 mV. To minimize the possibility that the reversal potential of taurine-induced current (ITau) was affected by the K2SO4-based pipette solution, in the later experiments, pipettes contained 140 mM KCl, 10 mM Hepes and 2 mM QX-314. With this pipette solution, 10/10 neurones from young rats (P7 to about P10) were depolarized by taurine. The mean depolarization observed in 14 VTA neurones, from rats younger than P13, were plotted as a function of taurine concentration (Fig. 3B). Using a non-linear least-squares method, the taurine dose–response data were plotted via the logistic equation:
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where V represents the depolarization, Vmax the maximum effect of taurine, n the Hill efficient, and EC50 represents the taurine concentration that evoked a half-maximal response, which was 0.97 mM.
A, currents were recorded from a putative VTA DA neurone of a P10 rat. Aa–e, different dosages of taurine had different effects on the action potentials, with low concentrations of taurine increasing the frequency of action potentials, and higher concentrations of taurine blocking action potentials. The dashed line shows the resting membrane potential, which was –60 mV. B, concentration dependence of taurine-induced depolarization (n = 9), with EC50 = 0.97 mM. The curve was fitted using the Hill equation. C, scatter plot of the changes of membrane potential induced by 1 mM taurine in rats as a function of age (n = 110). The fitted line shows the reversal point was at about P17. Da–e, dosage dependence of taurine-induced hyperpolarization in a VTA DA neurone from a P19 rat. The dashed line shows the resting membrane potential of –60 mV. E, voltage ramps (from +50 to –80 mV) were applied at a rate of 0.13 V s–1 before and during application of taurine. Currents induced by 1 mM taurine in VTA DA neurones of P10 and P20 rats, respectively. Holding potential was –50 mV. F, current–voltage plots are substantially different, with the reversal potentials at –30 mV and –55 mV for P10 and P20 rats, respectively.
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The sustained taurine-induced depolarization had a very intense effect on the action potentials. In rats under P13, low doses of taurine induced a small depolarization, which in turn facilitated the frequency of action potentials (Fig. 3Aa and b). Taurine-induced membrane depolarization was associated with an increase in membrane conductance (data not shown). Higher doses of taurine induced greater depolarization, which suppressed the ongoing discharges (Fig. 3Ac–e). This might have resulted from a combination of the shunting effect of GlyR activation (Staley & Mody, 1992) and Na+ current inactivation. In rats older than P18, taurine-induced hyperpolarization could suppress the ongoing discharges (Fig. 3D). Thus, taurine can regulate the membrane potential and increase the excitability of VTA neurones of young rats.
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The membrane potentials were voltage ramped from +50 to –80 mV to assess the reversal potential of ITau (Fig. 3E). After subtracting the currents obtained under control conditions (the first ramp), we obtained the current–voltage (I–V) relationships for taurine. As illustrated by the I–V curves in Fig. 3F, the reversal potential of ITau shifted to more negative as the rats aged (ETau = –29.3 ± 9.1 mV at P10; –57.8 ± 13.4 mV at P20, n = 8). The above data were collected with a K2SO4-based pipette solution. Virtually identical results were obtained when the pipette solution containing 140 mM KCl (data not shown). In further experiments, we investigated the properties of taurine-activated receptors in rats younger than P13.
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In voltage-clamped VTA DA neurones, ITau and IGly had similar I–V curves and reversal potentials
In approximately 98% of VTA DA neurones (voltage held at –50 mV), taurine- and glycine-evoked currents (ITau and IGly) had similar desensitizing kinetics. Figure 4Aa and b shows superimposed traces of inward currents evoked by 30 s application of glycine and taurine at five concentrations (as indicated) in a VTA DA neurone. Averaged data for ITau and IGly from 9 and 12 neurones are presented in the concentration–response curves in Fig. 4B: EC50 values for glycine and taurine were 124 μM and 1.07 mM, respectively. The mean maximal ITau and IGly evoked by 3 mM glycine (869 ± 104 pA) and 30 mM taurine (904 ± 136 pA) were not significantly different (Student's paired t test, P > 0.05, n = 31). In comparison with glycine, 10-fold higher concentrations of taurine were required to obtain similar currents. These results show that activation, desensitization and deactivation of both ITau and IGly occurred faster when agonist concentrations were higher (Fig. 4Aa and b). The membrane potentials were voltage-ramped from +50 to –80 mV to assess the voltage dependence of ITau (Fig. 4C). After subtracting the currents measured under control conditions (the first ramp), we obtained virtually the same I–V relationships for ITau and IGly. Based upon the I–V plots and that of another six neurones, the reversal potentials of both ITau and IGly were near –30 mV (Fig. 4D).
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All traces in this, as well as in the following figures, were recorded at a holding potential of –50 mV from dissociated neurones. Aa, currents recorded by varying the indicated concentrations of glycine in a putative VTA DA neurone of a P11 rat at 2 min. intervals. Ab, comparable series of currents evoked by taurine in a VTA DA neurone of the same rat. B, glycine- and taurine-evoked peak currents as a function of concentration. Currents evoked by glycine (n = 9) and taurine (n = 12) were normalized to the responses induced with 1 mM glycine and 30 mM taurine, respectively. EC50 values were 117 μM for glycine and 1.07 mM for taurine. C and D show that glycine- and taurine-evoked currents produced similar current–voltage (I–V) relations. Voltage ramps were applied before and during application of glycine and taurine. C, initial small ramp currents during resting condition and the largely increased ramp currents during inward shift obtained either by 100 μM glycine (a) or by 1 mM taurine (b) are shown. The data were obtained from the same VTA DA neurone of a P9 rat. D, I–V relations for glycine and taurine elicited by subtracting the control current (first of each pair of ramps) from the currents induced by a second ramp and normalizing values to a maximal outward current. Note, I–V plots for glycine- and taurine-induced currents are virtually identical and both have a reversal potential at –30 mV.
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ITau was sensitive to both picrotoxin and strychnine, but not to bicuculline
To determine if GlyRs in VTA were homomeric, taurine was applied at a concentration near EC50 (1 mM) with different concentrations of strychnine, a selective antagonist of GlyRs, and picrotoxin, an antagonist of homomeric GlyRs (Fig. 5). As the concentrations of strychnine and picrotoxin were increased, ITau became progressively smaller. Strychnine (1 μM) or picrotoxin (30 μM) almost completely abolished ITau. From the concentration–response relationships, we obtained IC50 values of 8.5 μM for picrotoxin (Fig. 5C and D) and 95.9 nM for strychnine (Fig. 5A and B) for ITau. Thus, ITau is sensitive to strychnine and picrotoxin. However, as shown in Fig. 5E and F, the addition of 10 μM bicuculline had no significant effect on ITau induced by 1 mM taurine.
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A, currents evoked by 1 mM taurine in a dissociated VTA DA neurone of a P10 rat were concentration-dependently blocked by strychnine (STR). B, concentration–response relation of strychnine blockage of ITau. Data were normalized to the currents elicited by 1 mM taurine (control); IC50 was 95.9 nM (n = 8). C, currents evoked by 1 mM taurine in a VTA DA neurone of a P11 rat were concentration-dependently blocked by picrotoxin (PTX). D, concentration–response relation of picrotoxin blockage of ITau. Data were normalized similar to B; IC50 was 8.5 μM (n = 7). E, currents elicited in a VTA neurone from a P11 rat had little, if any, sensitivity to 10 μM bicuculline. F, mean antagonistic effects of strychnine, picrotoxin and bicuculline (Bic) on ITau of 7 VTA neurones. Data were normalized to control currents elicited by 1 mM taurine. **P < 0.01.
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Selective cross-desensitization between the currents elicited by the application of glycine, taurine and GABA
The currents induced by these amino acids desensitized. When 100 μM glycine and 1 mM taurine were applied in rapid succession (either glycine first, or taurine first, Fig. 6A), the decay of the obtained currents was almost identical to when glycine or taurine was applied alone. This indicated that a full cross-desensitization existed between glycine and taurine. In comparison, ITau (induced by 1 mM taurine) and IGly (induced by 100 μM glycine) showed no cross-desensitization with IGABA (induced by 100 μM GABA) (Fig. 6B and C). In the case when ITau and IGly were preceded by IGABA, peak ITau and IGly were 98.2 ± 2.1 and 99.3 ± 3.2% of the control, respectively (Fig. 6F, Student's paired t test, P > 0.05, n = 6). In the cases when IGABA was preceded by IGly or ITau, IGABA was 97.3 ± 4.6 and 99.7 ± 4.1% of the control, respectively (Fig. 6F, Student's paired t test, P > 0.05, n = 6). These data suggest that 1 mM taurine has little, if any, effect on GABAARs of immature VTA neurones. However, 10 μM bicuculline blocked 32.2 ± 8.1% (n = 4, P < 0.05) of the peak ITau induced by 10 mM taurine (data not shown). In addition, when ITau (induced by 10 mM taurine) was preceded by IGABA induced by 200 μM GABA, the peak ITau was 79.7 ± 3.9% of control (P < 0.05, n = 6; Fig. 6D and F). On the other hand, when IGABA (induced by 200 μM GABA) was preceded by ITau induced by 10 mM taurine, the peak IGABA was 72.3 ± 4.5% of control (P < 0.05, n = 6; Fig. 6D and F). These results suggest that very concentrated taurine is also a partial agonist of GABAARs, in keeping with a previous report (Jiang et al. 2004). An asymmetrical cross-inhibition between GABAARs and GlyRs of spinal neurones occurred for glycine concentrations > 100 μM (Li et al. 2003). We found similar asymmetrical cross-talk between GABAARs and GlyRs occurring in the VTA neurones. As illustrated in Fig. 6E, IGABA (induced by 300 μM GABA) was significantly reduced when it was preceded by IGly (induced by 300 μM glycine). The mean peak IGABA was 45.0 ± 2.4% of control (n = 4, P < 0.01; Fig. 6D and E). By contrast, IGly (induced by 300 μM glycine) was not significantly changed when it was preceded by IGABA (induced by 300 μM GABA). The mean peak IGly was 93.1 ± 4.7% of control (n = 7, P = 0.17; Fig. 6D and E).
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A, currents induced by 100 μM glycine and 1 mM taurine alone or in sequence (as indicated) in a VTA DA neurone of a P12 rat. B, in similar experiments, currents were evoked by 100 μM GABA and 1 mM taurine in a VTA DA neurone of a P12 rat. C, in similar experiments, currents were evoked by 100 μM glycine and 100 μM GABA in a VTA DA neurone of a P11 rat. D, in similar experiments, currents were evoked by 200 μM GABA and 10 mM taurine in a VTA DA neurone of a P13 rat. The dotted line indicates the control level of IGABA, and the dashed line indicates the control level of ITau. E, in similar experiments, currents were evoked by 300 μM glycine and 300 μM GABA in a VTA DA neurone of a P10 rat. The dashed line indicates the control level of IGly, and the dotted line indicates the control level of IGABA. F, ordinates represent the mean peak amplitude (+ S.E.M.) of currents induced by the agents indicated. These currents were preceded by prepulses of the indicated agents. The letters before each prepulse agent indicate the corresponding data shown in A–E. Note the much stronger reduction of IGABA (induced by 300 μM GABA) by 300 μM glycine than in the opposite order of application. All current traces in this figure were recorded from acutely dissociated neurones at a holding potential of –50 mV. The number of experiments is shown in parentheses. *P < 0.05; ** P < 0.01.
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GABAergic synapses predominate; functional glycinergic/taurinergic synapses are present in a subset of VTA neurones
We next examined the properties of spontaneous inhibitory post-synaptic currents (sIPSCs) in brain slices. With the bath solutions containing the glutamatergic receptor antagonists 20 μM DNQX and 100 μM APV, the remaining spontaneous post-synaptic currents (PSCs) were completely blocked by 20 μM bicuculline in 40/45 neurones tested (Fig. 7A). In the other cells, a mixture of 20 μM bicuculline and 1 μM strychnine completely abolished the sIPSCs (data not shown). As illustrated in Fig. 7B and C, in the presence of 20 μM DNQX and 100 μM APV, 20 μM bicuculline blocked the evoked IPSCs to 7.3 ± 4.4% (n = 4) of control. The addition of 1 μM strychnine completely eliminated the remaining component (to 2.3 ± 1.5% of control, n = 4). Several studies have shown that strychnine (1 μM) had no significant effect on GABA-evoked responses (Hussy et al. 1997; Mori et al. 2002; Mangin et al. 2002). Thus, these results suggest that GABAergic IPSCs predominate, and that a subset of VTA DA neurones receive a functional glycinergic innervation.
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A, representative current traces recorded in a VTA DA neurone in a brain slice from a P13 rat. Parts of the traces are shown below on an expanded time scale. B, the blocking effects of 20 μM bicuculline and 20 μM bicuculline + 1 μM strychnine on evoked IPSCs of a VTA DA neurone in a brain slice from a P12 rat. C, mean area of evoked IPSCs from brain slices were significantly reduced by bicuculline (P < 0.01, n = 4), and completely eliminated by 20 μM bicuculline + 1 μM strychnine (n = 4).
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Tonic action of taurine on GlyRs in VTA DA neurones
The PSC experiment showed that there was no phasic synaptic action of taurine on GlyRs in the majority of VTA DA neurones. Taurine-elicited currents might serve to enhance a basal level of endogenous transmitter release and display a tonic activation of GlyRs. Therefore, we asked whether GlyRs in VTA are tonically activated. The application of strychnine (0.5 μM) to brain slices produced reversible outward currents (Fig. 8A), indicating that GlyRs in the VTA slice are tonically activated. By contrast, in out-side out patch (Fig. 8B), and in enzymatically dissociated neurones, strychnine induced no current (Fig. 8C), while taurine (1 mM) induced significant inward current (Fig. 8B and D), which suggested that there is no taurine or glycine in the solution.
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A, application of 0.5 μM strychnine to a neurone in a brain slice of a P12 rat produced an outward current. B, strychnine alone did not gate GlyRs, but completely blocked activation of taurine in an outside-out patch containing GlyRs. C, in a enzymatically dissociated neurone of a P12 rat, strychnine induced no current, while taurine (1 mM) induced significant inward current (D).
Discussion
Our data convincingly show that taurine increased the excitability of VTA DA neurones by activating non-synaptic GlyRs in young rats, in contrast to its conventional hyperpolarizing action and synaptic inhibition. Our present study found bicuculline blocked 93% of the IPSCs, whereas strychnine blocked only 7%. These results suggest that GABAergic IPSCs dominate, and that functional glycinergic synapses are present only in a small subset of the VTA neurones. Therefore, taurine, which might be released from glial cells, accomplishes its role in the development and functional modulation of VTA DA neurones by activating non-synaptic GlyRs.
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Taurine-induced depolarization in VTA DA neurones of young rats
Like glycine and GABA, taurine was first proposed as an inhibitory neurotransmitter because it inhibits neuronal firings. In the present study on dissociated VTA DA neurones from young rats (P1P13), taurine facilitated spontaneous discharges at low dosage. At high dosage, taurine induced depolarization and stopped spontaneous on-going discharges. The depolarization might induce Ca2+ influx, which is related to its role in trophic action in developing neurones (Flint et al. 1998). The depolarization might be ascribed to a relatively high intracellular [Cl–] and correspondingly less negative reversal potential in immature neurones (Cherubini et al. 1991). It is in good agreement with the effect of glycine and GABA on developing neurones of many brain regions (Cherubini et al. 1991; Flint et al. 1998). In contrast, in older rats, the major effect of taurine was hyperpolarization and blockage of ongoing discharges. This is consistent with its conventional inhibitory roles (Taber et al. 1986; Saransaari & Oja, 1997).
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The gradual shift of reversal potentials was tested using voltage ramps before and during application of taurine, and the reversal potentials were found to be at –30 and –55 mV for P10 and P20 rats, respectively (Ye, 2000). Therefore, the reversal potentials for taurine in immature neurones were less negative than the resting membrane potentials. This probably resulted from the relatively high cytoplasmic [Cl–] in immature neurones, which has been ascribed to a family of cation–chloride co-transporters (Delpire, 2000). The Na+–K+–2Cl– co-transporter (NKCC1), which is driven by a sodium and potassium gradient, raised [Cl–]i. The K+–Cl– co-transporter KCC2 couples Cl– transport to the K+ gradient and normally lowers [Cl–]i below that dictated by passive distribution (Rivera et al. 1999). It has been shown that KCC2 is up-regulated while NKCC1 is down-regulated in the CNS during development (Delpire, 2000).
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Taurine activated GlyRs in VTA DA neurones of young rats
Taurine has been shown to be a full agonist of GlyRs in isolated rat neurones of ventromedial hypothalamus and nucleus accumbens (Tokutomi et al. 1989; Jiang et al. 2004), in HEK293 cells (Rajendra et al. 1995), and Xenopus oocytes (De Saint Jan et al. 2001) that express recombinant human GlyR 1 and 2 subunits, respectively. However, in supraoptic magnocellular neurones (Hussy et al. 1997) and Xenopus oocytes that express recombinant human GlyR 1 subunits (Schmieden et al. 1992), taurine was a partial agonist of GlyRs. In our experiments, comparable inward currents were induced by 100 μM glycine and 1 mM taurine, and IGly and ITau had a similar I–V curve and the same reversal potential. These results suggest that, like IGly, ITau is carried predominantly by Cl–.
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Pharmacological properties of taurine-activated receptors are similar to those of GlyRs. Picrotoxin and strychnine were both effective antagonists of ITau, but bicuculline had almost no effect on ITau elicited by 1 mM taurine. The IC50 of 8.5 μM for picrotoxin for taurine is similar to that for IGly reported for immature VTA neurones (Ye et al. 1998), for IGly in mature hippocampal neurones (Chattipakorn & McMahon, 2002), as well as that for ITau in nucleus accumbens of young rats (Jiang et al. 2004), but different from that of adult nucleus accumbens neurones (Martin & Siggins, 2002). The IC50 for strychnine (95.9 nM) for taurine is also consistent with previous observations of that for IGly on neurones of many brain regions, including VTA (Ye et al. 1998), basolateral amygdala (McCool & Botting, 2000), striatum (Sergeeva, 1998), supraoptic magnocellular nucleus (Hussy et al. 1997), and hippocampus (Chattipakorn & McMahon, 2002; Mori et al. 2002). GlyRs in the mammalian CNS are formed by a combination of five membrane-spanning protein subunits, while the antagonist picrotoxin, at concentrations lower than 30 μM, inhibits the function of homomeric receptors, but does not affect heteromeric + receptors (Pribilla et al. 1992; Legendre, 1997; Mangin et al. 2002). 2 homomers are most sensitive to picrotoxin, with an IC50 of 6 μM, while GlyRs of mature neurones consisting mainly of 1 heteromers are sensitive to strychnine. The high picrotoxin affinity observed in the current study suggests that the GlyRs did not contain any subunit. The relative high strychnine affinity of ITau found in the present study suggests the expression of 1 subunits (Schmieden et al. 1992). However, better discrimination between 1 homomeric GlyRs and 2 homomeric GlyRs will require single channel recordings (Mangin et al. 2002).
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Overall all, comparable inward currents were induced by taurine and glycine. ITau and IGly had similar characteristics of desensitization and deactivation, as well as reversal potential. Both were readily blocked by picrotoxin and by strychnine, with similar IC50 values. Moreover, both ITau and IGly showed complete cross-desensitization. All these data suggest that taurine and glycine activate the same receptors. In contrast, the current induced by taurine ( 1 mM) was not blocked by bicuculline and showed no cross talk with IGABA. These results indicate at concentrations 1 mM, taurine has little or no activation of GABAARs. In keeping with a previous study on spinal neurones (Li et al. 2003), an asymmetrical cross-inhibition between GABAARs and GlyRs of VTA neurones occurred for glycine concentrations > 100 μM. These results suggest that GABAARs and GlyRs can interact functionally on the VTA neurones.
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Non-synaptic GlyRs and non-synaptic release of taurine
The above results show that exogenous application of taurine could activate functional GlyRs on VTA DA neurones. Whether the physiological role of taurine-activating GlyRs is through cell–cell communication or just through ambient tonic action is unknown. O'Brien & Berger (1999) showed that glycine could be physiologically co-released with GABA from afferents, thereby activating the GlyRs that were co-localized with GABAARs on the same synapses in brain stem motoneurones.
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However, as bicuculline blocked the IPSCs in the majority of the cells tested, this suggests that GABAergic innervation dominates. This also suggests that many GlyRs in the VTA might be non-synaptically activated by taurine. The non-synaptic activation of GlyRs was studied by Legendre's group (Mangin et al. 2003), who found that 2 homomeric GlyRs in young rats were completely inefficient if post-synaptically located, for the rising phase of the responses evoked by glycine were rather slow. They concluded that 2 homomeric GlyRs, which are known to be expressed prior to synaptogenesis (Legendre, 2001), might be involved in a paracrine function of GlyRs if the concentration of the released agonist is high enough. The concentration of taurine is high enough to fulfil this job. In addition, taurine could be released in the absence of action potentials and extracellular calcium, which are a prerequisite for neurotransmitter release (Chen et al. 1996; Flint et al. 1998). Taurine is stored in glial cells (Almarghini et al. 1991; Hussy et al. 2001), where both the synthesizing enzyme and the transporter for taurine are present (Almarghini et al. 1991; Flint et al. 1998). It is released in high levels in response to conditions that elicit exocytotoxic cell death (Saransaari & Oja, 1997) and is also released in response to hypotonic stimulus (Deleuze et al. 1998) or ischaemia (Saransaari & Oja, 2002). Therefore, taurine is most probably the extra-synaptically endogenous ligand for the VTA GlyRs. Because of the involvement of the VTA in midbrain mechanisms underlying drug addiction, the repeated finding that ethanol promotes the release of taurine in mesolimbic structure (Dahchour et al. 1996; De Witte et al. 1994) is especially pertinent to the current report.
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The excitatory drive of taurine through GlyRs is very important for the early stage of development of the CNS, when excitatory glutamatergic synapses are rather quiescent (Cherubini et al. 1991). Taurine deprivation of kittens leads to low brain weight and an apparent disruption of normal cortical migration and differentiation (Palackal et al. 1986). However, mice lacking the two GABA synthetic enzymes GAD65 and GAD67, and thus also lacking GABA, have normal brain histology and cytoarchitecture at birth (Ji et al. 1999). In addition, taurine is one of the most abundant (e.g. 4–10 mM) and ubiquitous free amino acids in the brain (Sturman et al. 1978; Huxtable, 1989). Furthermore, the taurine reuptake transporter is highly expressed in the rat brain (Smith et al. 1992). In contrast, the presence of glycinergic afferents in the VTA is sparse (Rampon et al. 1996) and the levels of glycine in the brain, especially in the neocortex, are very low (Aprison et al. 1969). Taurine may be the most important trophic factor in brain development. Especially relevant is the finding that taurine levels and its release are particularly high in immature rats (Sturman et al. 1978). According to our studies, the prominent effects of taurine in VTA neurones would make immature animals (and possible humans) particularly vulnerable to detrimental effects of those agents, such as ethanol, that enhance GlyR function and/or activity (Ye et al. 2001; Jiang & Ye, 2003; Zhu & Ye, 2005).
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