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21 Departments of Anesthesiology, Pharmacology, and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103-2714; 2 Anesthesia Research Unit and Physiology Department, McGill University, Montreal, Quebec H3G 1Y6, Canada
Submitted 4 February 2003; accepted in final form 19 July 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1 mM) showed complete cross-desensitization with IGly, but none with 
-aminobutyric acid (GABA)-induced currents (IGABA). However, ITau elicited by very concentrated taurine (10 mM) showed partial cross-desensitization with IGABA, and it was substantially antagonized by 10 µM bicuculline. These results indicate that taurine binds mainly to GlyRs in NAcc, but it could be a partial agonist of GABAA receptors. By activating GlyRs, taurine may play an important physiological role in the control of NAcc function, especially during development. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1974
; Kuhse et al. 1995; Nicoll et al. 1990; Werman et al. 1968). The results of research over the last 15 yr suggest that glycinergic synapses are not restricted to the spinal cord and brain stem (Legendre 2001). It is now clear that Gly-containing fibers and cell bodies and Gly receptors (GlyRs) are widely distributed in the CNS (Betz 1991; Malosio et al. 1991; Rampon et al. 1996). Gly and other GlyR agonists can elicit Cl–-mediated responses in neurons from many brain regions, including the cerebral cortex (Kilb et al. 2002), striatum (Sergeeva 1998), nucleus accumbens (Martin and Siggins 2002), amygdala (McCool and Botting 2000), hippocampus (Chattipakorn and McMahon 2002; Krishtal et al. 1988; Mori et al. 2002; Ye et al. 1999b), hypothalamus (Akaike and Kaneda 1989; Tokutomi et al. 1989; Ye and McArdle 1996), and VTA (Ye et al. 1998), indicating a wide distribution of functional GlyRs in the CNS.
Nucleus accumbens (NAcc), an area of ventral striatum, is important because it receives a rich dopaminergic innervation from the VTA of the brain stem and mediates rewarding effects of drugs of abuse, such as ethanol (Leshner and Koob 1999). Functional GlyRs have been found in the striatum (Sergeeva 1998) and the mature NAcc (Martin and Siggins 2002), although these areas contain few Gly-immunoreactive cell bodies and fibers (Rampon et al. 1996). However, taurine (Tau), a putative agonist of GlyRs, is abundant in NAcc (Dahchour et al. 1996; Madsen et al. 1987). Because striatal neurons express Tau transporters at a very high level (Clarke et al. 1983; Liu et al. 1992), these cells accumulate Tau in millimolar concentrations (Puka et al. 1991; Trachtman 1992). It has long been known that Tau is a major constituent of many parts of the CNS (Huxtable 1992; Jacobsen and Smith 1968). A notable feature is its high concentration in the immature brain, four-to fivefold higher than in the adult (Kaczmarek 1976; Sturman and Gaull 1976). Having a strychnine-sensitive, Gly-like action on many cells (Curtis et al. 1968; Krnjevi and Puil 1976; Legendre 2001; Mori et al. 2002), Tau has been proposed as a candidate inhibitory transmitter (Curtis et al. 1968; Davison and Kaczmarek 1971; Kaczmarek 1976; Padjen et al. 1989). In a recent study on NAcc neurons isolated from adult rats, Martin and Siggins (2002) found that Gly and Tau elicited similar Cl– current in many cells; but these currents were relatively insensitive to strychnine and quite resistant to picrotoxin (PTX).
Because of the involvement of NAcc in forebrain mechanisms underlying drug addiction, the repeated finding that ethanol promotes the release of Tau in NAcc (Dahchour et al. 1994, 1996, 2000; De Witte et al. 1994) is especially pertinent in the present context. It suggests that ethanol may influence NAcc function by indirectly activating neuronal GlyRs. To obtain more information about the potentially important role of Tau, especially during development, in the present study, we examine the effects of Tau on the electrical activity of NAcc neurons acutely isolated from young rats (12- to 21-day old) as well as the properties of the Tau-activated currents.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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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 (protocol 00074). The neurons were isolated as described earlier (Ye et al. 1999a). Briefly, Sprague-Dawley rats (12- to 21-day old) were decapitated, and the brain was quickly excised and sliced to a thickness of 350 µm. Slices were digested with pronase (1 mg/12 ml) and thermolysin (1 mg/12 ml) and then incubated in standard external solution at room temperature of 20–24°C for 2 h before use. Micropunches of the NAcc region were isolated under a dissecting microscope and transferred to a 35-mm culture dish. Mild trituration through heat-polished pipettes of progressively smaller tip diameters dissociated single neurons. Within 20 min after trituration, isolated neurons attached to the bottom of culture dish and were ready for electrophysiological experiments.
The standard external solution contained (in mM) 140 NaCl, 5.0 KCl, 1.0 MgCl2, 2.0 CaCl2, 10 glucose, and 10 HEPES. The pH was adjusted to 7.4 with 1 N NaOH and the osmolarity to 310 mosM with sucrose. Most experiments were performed with gramicidin perforated-patch recording using a pipette solution that contained (in mM) 150 KCl and 10 HEPES. In some later experiments, a low-Cl– pipette solution contained 60.5 K2HPO4, 40 KCl, 0.5 CaCl2, 5 EGTA, and 10 HEPES. For conventional whole cell voltage-clamp experiments, the pipette solution contained (in mM) 140 KCl, 10 HEPES, 4.0 EGTA, 0.40 CaCl2, 1.0 MgCl2, and 2.0 Mg-ATP. The pH of the pipette solutions was adjusted to 7.2 with 1 N KOH, and the osmolarity to 290 mosM with sucrose.
Membrane currents and potentials were recorded under voltage- and current-clamp modes, respectively, with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), interfaced to a desktop computer via a Digidata 1320A (Axon Instruments) A/D converter and directly digitized with pCLAMP 8 software for off-line analysis. When filled with the preceding solution, the pipettes had a resistance of 3 
5 M
. The 4.7-mV junction potential between patch pipette and bath solutions, calculated from the generalized Henderson equation using the Axoscope junction potential calculator, was nulled just before forming a giga seal. 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- to 4-mV error for 1 nA of current. Neurons with an access resistance <20 M were selected for further tests.
For the gramicidin perforated-patch electrodes (Ye 2000), the gramicidin stock solution [10 mg/ml in methanol (J.T. Baker, Phillisburg, NJ)] was diluted in the pipette solution to a final concentration of 50–100 µg/ml just before an experiment. After establishing a giga seal in the cell-attached configuration by gentle suction, no further negative pressure was applied. Progressive membrane perforation was monitored by the decrease in membrane resistance, measured with repeated 10-mV hyperpolarizing voltage steps from a holding potential (VH) of –50 mV. Entry into the perforated-patch mode was signaled by a larger capacitive transient. The access resistance reached a steady level of 20 M within 30 min after making a giga seal. When conditions stabilized, recording began. Throughout the experiment, the bath was continually perfused with the standard saline. All ligand-induced responses were elicited at an ambient temperature of 20–24°C.
Chemicals and applications
Gly, Tau, GABA, strychnine, picrotoxin, bicuculline methiodide, and other standard chemicals were purchased from Sigma-Aldrich (St Louis, MO). Solutions of agonists and antagonists were prepared on the day of experiment and applied to a dissociated neuron via a multi-barreled pipette, the tip of which was placed 50–100 µm away from a neuron. As described previously (Ye et al. 2001), with this perfusion system, solutions in the vicinity of a neuron can be completely exchanged within 20 ms without loss of mechanical stability.
Statistics
All data are presented as means ± SE; and, when appropriate, the means were compared by Student's t-test. Statistical data were considered significant when P 0.05.
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RESULTS |
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These NAcc neurons varied greatly in size and shape: 73% had a small soma (10–15 µm diam) and the rest (27%) were larger (>20 µm) with bipolar or multipolar processes. Most of the bipolar cells were ovoid and many multipolar cells were triangular.
Like GABA and Gly, Tau can depolarize NAcc neurons of young rats and thus change their excitability
To study the effects of Tau without altering the intracellular Cl– concentration, most of the recordings were carried out using the gramicidin perforated-patch method (n = 100) except where indicated. The resting membrane potential varied greatly between neurons (–35 to –80 mV) with a mean of –55 ± 5 mV (n = 20), tending to become more negative in cells from older animals. Most (89/100) of the NAcc neurons from 12- to 21-day-old rats responded to Tau (0.1–10 mM) with depolarization (unresponsive neurons were of both large and small type). With standard KCl-containing pipettes, a relatively positive ECl might result from partial rupture of the patch. To minimize this possibility in the later experiments, the pipettes contained K2HPO4 and only 41 mM Cl–, close to the intracellular [Cl–] of neurons at this age (see Fig. 3 for details). With this pipette solution, 18 of 20 neurons were depolarized by Tau (see following text).
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) and Na current inactivation. As illustrated in Fig. 1B, in a quiescent neuron, Tau evoked an action potential before the plateau of depolarization; its action was similar to that of a 0.15-nA depolarizing pulse. Thus like GABA and Gly, Tau can alter the membrane potential and the excitability of NAcc neurons of young rats. In further experiments, we investigated the question whether these effects of Tau are mediated by GlyRs or by GABAA receptors (GABAARs). Gly and Tau elicit currents in voltage-clamped NAcc neurons
Gly and Tau evoked comparable, fast-desensitizing currents in 98% of NAcc neurons (held at –50 mV). In Fig. 2A are superimposed traces of inward currents elicited by 12-s applications of Gly and Tau at five increasing concentrations (as indicated). Averaged data from six neurons for Gly-induced current (IGly) and five neurons for Tau-induced current (ITau) are summarized by the concentration-response curves in Fig. 2B: EC50 for Gly was 117 µM (with Hill coefficient of 1.3) and EC50 for Tau 1.25 mM (Hill coefficient: 1.3). The mean maximal currents activated by 3 mM Gly and 30 mM Tau (1,397 ± 160 and 1,285 ± 132 pA, respectively) did not differ significantly (P > 0.05). Compared with Gly, 10-fold higher concentrations of Tau were needed to elicit similar currents. These results indicate that functional GlyRs are abundant in the NAcc of young rats and that, as in cells from adult rats (Martin and Siggins 2002), these GlyRs are less sensitive to Tau than to Gly. In addition, it was clear that activation, desensitization and deactivation of both IGly and ITau were more rapid when agonist concentrations were higher (Fig. 2A).
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The voltage dependence of similar IGly and ITau was measured in a NAcc neuron of a 14-day-old rat with voltage-ramps from +50 to –80 mV (Fig. 3A). After subtracting the currents obtained under control conditions (elicited by the 1st ramp in each trace), we obtained two almost identical I-V relations for IGly and for ITau (Fig. 3B). According to these I-V plots (and those from another 6 neurons), the reversal potentials of IGly and ITau were both near 0 mV—as expected for currents recorded in whole cell mode with similar [Cl–] in the recording pipette and in the bathing medium (see following text),
In five other NAcc neurons, ITau-V plots were obtained twice from the same cell: first while recording in the gramicidin perforated-patch mode and then in the conventional whole cell mode, as illustrated in Fig. 3C. These I-V relations (Fig. 3D) showed a reversal potential of –30 mV in the perforated-patch mode and a steeper slope and a reversal potential near 0 mV in the whole cell mode. Such a clear shift of reversal potential is further evidence that Gly and Tau activate channels that are selective for Cl– because the transmembrane gradients of Cl– differed markedly in these two recording conditions. During whole cell recording, the Cl– concentrations [Cl–] of the standard extracellular and intrapipette solutions were almost equal and the steady-state reversal potential for ICl should be near –1.4 mV—as calculated by the Nernst equation with extracellular [Cl–] ([Cl–]out) at 151 mM and intracellular[Cl–] ([Cl–]in) at 143 mM. The observed reversal potential near 0 mV for both IGly and ITau is thus in good agreement with previous evidence that Gly and Tau open Cl– channels. In gramicidin perforated patches, the membrane holes are not permeable to Cl–, so [Cl–]out and [Cl–]in remain different. With our standard [Cl–]out of 151 mM, the Nernst equation gives a relatively high [Cl–]in of 46.5 mM, consistent with the immature state of these NAcc neurons. The threefold rise in [Cl–]in after the change to whole cell recording may also account for the increase in I-V slope, which is especially marked (also about threefold) in the left-hand portion of the plot (Fig. 3D), where the inward currents should be particularly sensitive to internal [Cl–].
In NAcc neurons of young rats, both IGly and ITau are readily blocked by strychnine
Gly or Tau was applied at a concentration near EC50. Examples of antagonism by strychnine are shown in Fig. 4A. When the concentration of strychnine was raised, IGly and ITau (elicited by 100 µM Gly or 1 mM Tau) became progressively smaller. At 1 µM, strychnine inhibited IGly and ITau almost completely. Mean values of IGly and ITau from such experiments are plotted in Fig. 4B. From these concentration-response relations, we obtained IC50 values for strychnine of 60 nM for IGly (with a Hill coefficient of 0.9) and 40 nM for ITau (with a Hill coefficient of 0.9). Thus both IGly and ITau showed a similar high sensitivity to strychnine.
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In further experiments, IGly and ITau were readily blocked by picrotoxin (PTX). The dose-dependent inhibitory effects of PTX are illustrated in Fig. 5A. Both IGly and ITau (elicited by 100 µM Gly or 1 mM Tau, near EC50 concentrations) gradually diminished when PTX was applied at four increasing concentrations (as indicated). From the plots of the corresponding mean values of IGly and ITau (Fig. 5B), the PTX IC50 was 36.5 µM for IGly (with a Hill coefficient of 1.4) and 42.2 µM for ITau (with a Hill coefficient of 1.1). Thus IGly and ITau were also equally sensitive to PTX.
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Rapid desensitization is a characteristic feature of the currents evoked by these amino acids. The similar rates of decay of IGly and ITau are illustrated in Fig. 6A, a and c. When 100 µM Gly and 1 mM Tau were applied in quick succession (either Gly first, Fig. 6Ab, or Tau first, Fig. 6Ad) for the same total duration, the decay of the evoked currents was virtually identical to that seen when Gly or Tau was applied alone. The superimposed IGly and ITau traces in Fig. 6B indicate full cross-desensitization between the responses. In contrast, Fig. 6C shows no cross-talk between the responses to GABA (100 µM) and Gly (100 µM) or GABA (100 µM) and Tau (100 µM). When IGly and ITau were preceded by IGABA (Fig. 6C), peak IGly and ITau were 99.4 ± 1.6 and 97.5 ± 5.3% of control (Fig. 6D, P > 0.05). Thus 1 mM Tau has little if any effect on GABAARs of these NAcc neurons.
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Negative cross-talk between currents mediated by GABAARs and GlyRs has been observed in rat hippocampal neurons (Grassi 1992; Li and Xu 2002). To further investigate such cross-talk, we compared the maximal currents elicited by Tau (50 mM), Gly (5 mM), and GABA (5 mM) and by mixtures of Gly + GABA, Tau + GABA, and Tau + Gly (at these high concentrations) as illustrated in Fig. 6, G and H. The current evoked in the same cell by coapplication of GABA + Gly produced a much smaller total current than the linear sum of the separate responses. These data indicate that in NAcc neurons, there is occlusion between maximal IGABA and IGly as in hippocampal neurons. Similarly, the total currents induced by Tau + Gly (ITau+Gly) and by Tau + GABA (ITau+GABA) were significantly smaller than the linear sum of the separate currents (ITau + IGly, ITau + IGABA).
At high concentration, Tau was also an agonist at GABAA receptors
Some of the preceding data suggest that 10 mM Tau also activates GABAARs. To further test this hypothesis, we examined the effects of antagonists on currents induced by both low and high concentrations of Tau. As expected, the current elicited by 100 µM GABA was fully blocked by 10 µM bicuculline but only slightly by 1 µM strychnine (Fig. 7A). The mean results obtained from six NAcc neurons are summarized by the histograms in Fig. 7B: IGABA was virtually abolished (to 2.5 ± 1.5% of control) by 10 µM bicuculline and only mildly depressed (to 84.3 ± 5.9%, P = 0.07, n = 4)by1 µM strychnine. Conversely, the current elicited by 300 µM Tau was almost completely suppressed by 1 µM strychnine and only slightly by 10 µM bicuculline (Fig. 7C). In contrast, when Tau was applied at a much higher concentration (10 mM), ITau was significantly depressed by bicuculline and the block by strychnine was less complete (Fig. 7D). As shown by the mean data in Fig. 7E, strychnine virtually abolished the response to 300 µM Tau (to 1.8 ± 1.5% of control, P < 0.01, n = 7) but less completely the response to 10 mM Tau (down to 23.2 ± 4.2%, P < 0.01, n = 7): these effects differed significantly (P < 0.05). When Tau was applied at 300 µM, bicuculline (10 µM) only reduced ITau to 87.3 ± 3.5% (P < 0.05, n = 7); but ITau diminished to 61.5 ± 4.6% (P < 0.01, n = 7) when 10 mM Tau was applied in the presence of 10 µM bicuculline (Fig. 7E). Judging by these results, in the NAcc of young rats, 300 µM Tau binds mainly to GlyRs; but at a much higher concentration (10 mM), it can also activate GABAARs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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By demonstrating functional GlyRs on 98% of NAcc neurons acutely isolated from 12- to 21-day-old rats, the present study extends the previous findings of Martin and Siggins (2002), who observed Gly-induced currents in only 20% of NAcc neurons of adult rats. The Gly concentrations needed to elicit maximal currents (1–3 mM) and the EC50 (117 µM) in the present study were comparable to those reported by Martin and Siggins (2002) and by other authors for neurons isolated from various brain regions, including the basolateral amygdala (McCool and Botting 2000), ventromedial hypothalamus (Tokutomi et al. 1989), striatum (Sergeeva 1998), and the VTA (Ye et al. 1998) as well as for neurons in hippocampal slices (Chattipakorn and McMahon 2002; Mori et al. 2002).
In agreement with previous reports (Akaike and Kaneda 1989; Chattipakorn and McMahon 2002; Hussy et al. 1997; Martin and Siggins 2002; Werman et al. 1968), IGly was carried predominantly by Cl– as indicated by its reversal at potentials close to the equilibrium potential for Cl– currents (calculated with the Nernst equation and the transmembrane [Cl–] gradient). With similar [Cl–] in the extracellular and intrapipette solutions, the reversal potential during whole cell recording was near 0 mV. In recordings in gramicidin perforated mode, the reversal potential was more negative (near –30 mV), in keeping with the high intrapipette [Cl–] (150 mM) and much lower cytoplasmic [Cl–] (calculated to be 47 mM). These values differ substantially from the –50 mV reversal potential and 10 mM estimated [Cl–]i reported by Martin and Siggins (2002) for more mature NAcc neurons. The relatively high cytoplasmic [Cl–] in immature neurons (Alvarez-Leefmans et al. 1988) can be explained by the late developmental expression of the K+/Cl– co-transporter KCC2, mainly responsible for Cl– extrusion from mature neurons (Rivera et al. 1999).
Pharmacological properties of Gly receptors on NAcc neurons of young rats
Strychnine and PTX were both very effective antagonists of Gly. The IC50 for strychnine (60 nM) is consistent with previous observations on isolated neurons from the basolateral amygdala (McCool and Botting 2000), striatum (Sergeeva 1998), VTA (Ye et al. 1998) and supraoptic magnocellular nucleus (Hussy et al. 1997) as well as neurons in hippocampal slices (Chattipakorn and McMahon 2002; Mori et al. 2002). The much higher IC50 (12 µM) reported by Martin and Siggins (2002) for mature NAcc neurons may be ascribed to their use of more concentrated Gly (1 mM). The immature NAcc neurons' high sensitivity to strychnine is further evidence that the currents were indeed mediated by conventional GlyRs.
The IC50 of 36.5 µM for PTX is similar to that reported for immature VTA neurons (Ye et al. 1998) as well as mature neurons in hippocampal slices (Chattipakorn and McMahon 2002). In this respect, however, our observations differ from those of Martin and Siggins (2002), who found no effect of 200 µM PTX on adult NAcc neurons.
With regard to the subunit composition of GlyRs in these NAcc neurons, the relatively high sensitivity to PTX indicates the presence of 
2 homomeric receptors (Laube et al. 2002; Legendre 1997; Pribilla et al. 1992). However, the observed values of IC50 for ITau and IGly (both near 40 µM) were substantially above the 6 µM IC50 reported for homomeric GlyRs (Pribilla et al. 1992). Therefore the receptors of NAcc neurons in 12- to 21-day-old rats cannot be exclusively homomeric GlyRs [the immature form of extrasynaptically located GlyRs (Laube et al. 2002)]; in addition, 
heteromeric receptors are probably expressed as in mature synapses. Thus during this late stage of transition from immaturity to maturity, homomeric and heteromeric GlyRs appear to co-exist in NAcc neurons. Such a progressive switch of GlyRs from neonatal to adult type has also been observed in the spinal cord (Becker 1995; Laube et al. 2002). However, better discrimination between 1 homomeric GlyRs and 2 homomeric GlyRs will require single-channel recordings (Mangin et al. 2002).
Tau-induced potentials and currents in NAcc neurons of young rats
Tau, a sulfonated -amino acid, is abundant in rat brain NAcc (Dahchour et al. 1996; Madsen et al. 1987). Like Gly and GABA, Tau can alter the membrane potential and thus either increase or decrease neuronal excitability. In the present study on NAcc neurons from young rats, Tau mainly depolarized cells in a dose-dependent manner, but the overall effect on excitability depended on the state of background activity. In quiescent NAcc neurons, Tau could initiate an action potential, but ongoing firing was interrupted during Tau's sustained depolarizing effect, presumably by a combination of Na current inactivation and the shunting action of a high GCl.
The consistent depolarization of these developing NAcc neurons by Tau, Gly, and GABA is in good agreement with the effects of these amino acids on immature hippocampal (Cherubini et al. 1991), neocortical (Flint et al. 1998), and many other developing neurons. As discussed in the preceding text, this can be ascribed to a relatively high intracellular [Cl–] and correspondingly less negative Cl– reversal potential (Cherubini et al. 1991; Leinekugel et al. 1999).
Early studies had already shown that Tau can activate both GlyRs and GABAARs (Krnjevi and Puil 1976). Tau is a full agonist of GlyRs in isolated ventromedial hypothalamic neurons of rats (Tokutomi et al. 1989), in HEK293 cells (Rajendra et al. 1995) and Xenopus oocytes (De Saint Jan et al. 2001) that express recombinant human GlyRs 1 subunits and both 1 and 2 subunits, respectively. However, in supraoptic magnocellular neurons (Hussy et al. 1997) and Xenopus oocytes that express recombinant human GlyRs 1 subunits (Schmieden et al. 1992), Tau may be a partial agonist of GlyRs.
In our experiments, comparable inward currents were induced by 100 µM Gly and by Tau at concentrations 1 mM, and ITau and IGly had similar characteristics of desensitization and deactivation as well as the same reversal potential. Both were readily blocked by strychnine and by PTX: the respective IC50s of strychnine (40 nM) and PTX (42 µM) for ITau were similar to those for IGly. Moreover, both ITau and IGly (induced at EC50 concentrations) showed complete cross-desensitization, suggesting that Tau and Gly can activate the same receptors. By contrast, at 1 mM Tau showed no cross-desensitization with GABA, indicating little or no activation of GABAARs. And with low agonist concentrations, there was also no crosstalk between GlyRs and GABAARs. However, when Tau was applied at 10 mM, ITau showed partially cross-desensitization with IGABA. Furthermore, ITau could not be completely blocked by 1 µM strychnine but was significantly reduced by 10 µM bicuculline (Fig. 7). Thus at 10 mM, Tau was also a weak agonist of GABAARs, in agreement with findings in rat supraoptic magnocellular (Hussy et al. 1997) and basolateral amygdala neurons (McCool and Botting 2000). As already discussed, when GABA and Gly were applied at high concentrations, there was clear evidence of cross-talk between GABAARS and GlyRs.
In contrast to the paucity of Gly-immunoreactive cell bodies and fibers in NAcc (Rampon et al. 1996), Tau-containing cells are abundant in NAcc (Dahchour et al. 1996; Madsen et al. 1987) perhaps because striatal neurons express a high level of Na-dependent Tau transporter (Clarke et al. 1983; Liu et al. 1992), which allows them to accumulate Tau in millimolar concentrations (Puka et al. 1991; Trachtman 1992). Therefore physiological actions of GlyRs in NAcc are likely to be mediated by Tau rather than Gly.
Functional significance of Tau-induced activation of GlyRs
Although GlyRs are present in most parts of the forebrain, attempts at identifying glycinergic synaptic transmission have met with little success. But even in organotypic hippocampal slices, where Gly-mediated inhibitory postsynaptic potentials could not be detected, Mori et al. (2002) demonstrated an ongoing strychnine-sensitive activation of GlyRs after they selectively suppressed Tau transport. There is much evidence that large amounts of Tau can be released by electrical, chemical, or osmotic stimulation as well as cerebral ischemia (Dahchour et al. 1996; Del Arco et al. 2000; Flint et al. 1998; Jasper and Koyama 1969; Kaczmarek 1976; Saransaari and Oja 2002).
Especially relevant is the finding that Tau levels and release are particularly high in immature rats (and humans and monkeys) and drop markedly during the postnatal period (Kazcmarek 1976; Sturman and Gaull 1976). Accordingly, although glycinergic inhibitory postsynaptic currenss are also not detectable in fetal and neonatal cortical slices, there is clear evidence of Tau-mediated ongoing activation of GlyRs, which is much potentiated by tetanic or osmotic stimulation or by blockage of Tau transport (Flint et al. 1998). As emphasized by Sturman (1993), such tonic excitation by extra-synaptic release of Tau is probably important for normal brain development. The trophic action of Tau may well be mediated by Tau-induced Ca2+ influx (Flint et al. 1998), like the long-lasting potentiation of transmission induced by Tau in mature cortical synapses (Chepkova et al. 2002; del Olmo et al. 2000; Galarreta et al. 1996). Clearly, Tau could have similar long-lasting effects on development and synaptic transmission in NAcc, especially in the Tau-rich immature brain.
Bearing in mind the central role of NAcc in the mediation of rewarding effects of drugs of abuse, notably ethanol (Leshner and Koob 1999), the well-established finding that ethanol promotes Tau release in NAcc (Dahchour et al. 1994, 1996, 2000; De Witte et al. 1994) strongly suggests that Tau is involved in mechanisms of ethanol intoxication and withdrawal (Dahchour and De Witte 2000). According to our observations, the prominent effects of Tau on immature NAcc neurons would make very young animals (and possibly humans) particularly susceptible to undesirable effects of ethanol.
In summary, the present results show that functional GlyRs are abundant in the NAcc of young rats and that Tau, as an endogenous full agonist of GlyRs, may play an important role in the development and functional modulation of NAcc neurons.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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GRANTS
This study was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA-11989 (to J. H. Ye).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. H. Ye, New Jersey Medical School (UMDNJ), 185 S. Orange Ave., Newark, NJ 07103-2714 (E-mail: ye{at}umdnj.edu).
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, periods of drug application. A: voltage traces obtained from NAcc neuron of a 17-day-old rat with pipette containing 40 mM KCl /60 mM K2HPO4 solution; 10 mM Tau, 1 mM Gly, or 300 µM GABA caused large depolarization and interrupted ongoing firing; B: depolarization of another NAcc neuron of a 21-day-old rat by 0.1, 1, and 10 mM Tau and a 0.15-nA depolarizing current pulse; like the depolarizing pulse, 1 and 10 mM Tau initially evoked an action potential. C: similar mean depolarizations (±SE, n = 5) induced by 30 and 100 µM Gly or GABA. D: concentrationdependence of Tau-induced depolarizations (for each point n = 4). The curve was fitted using the Hill equation (see text).
