Changes in single-channel properties of GlyRs during postnatal development
To determine possible changes in native GlyR subtypes during postnatal development, we studied GlyR single-channel conductances and kinetics in outside-out patches excised from DA neurones taken from animals aged between P7 and P22. It has been shown that all GlyRs exhibit multiple-conductance chloride channels during single-channel recordings (Hamill et al. 1983; Bormann et al. 1993), the conductance state most frequently observed depending on the GlyR subunit combination. These main conductance states are respectively 110 pS, 90 pS and 50 pS for α2 homomeric, α1 homomeric and α/β heteromeric GlyRs (Bormann et al. 1993). In addition, the GlyR subunit composition has a clear influence on channel kinetics: GlyRs containing α2 have a longer mean open time than GlyRs containing α1, and thus the switch from the α2 to the α1 subunit during postnatal development is accompanied by a shortening of the mean open time (Takahashi et al. 1992; Singer et al. 1998).
Unitary currents evoked by glycine in DA neurones exhibited at least five distinct conductance levels during postnatal development (see Fig. 1 and Table 1). Level I (100-110 pS), level II (82-90 pS) and level IV (46-55 pS) were observed in 21, 23 and 61 patches, respectively (n/ 63, see Table 1), and represented 12 %, 25 % and 60 %, respectively, of the total open time recorded. Levels III (65-70 pS) and V (30-36 pS) were observed in 11 and 29 patches, respectively (n= 63, see Table 1), but represented a very small proportion of the total open time (less than 5 %). Levels III and V also occurred as a direct transition from higher conductance levels (see Fig. 1, 1st and 4th traces), suggesting that they were essentially subconductance states, as shown in others studies (Takahashi et al. 1992; Bormann et al. 1993). In contrast, levels I, II and IV were similar to the main conductance states of respectively α2 homomeric, α1 homomeric and α/β heteromeric GlyRs expressed in human embryonic kidney (HEK)-393 cells (Bormann et al. 1993). As shown in Fig. 2, the relative proportion of these three main conductance levels changed during postnatal development. In neonatal rats aged between P7 and P10 (Fig. 2A, 13 patches pooled), level I and level IV were most frequently observed and represented 38 % and 34 % of the total open time, respectively. Level II was also clearly present in recordings and represented 28 % of the total open time. In rats aged between P13 and P16 (Fig. 2B, 11 patches pooled), level II (46 % of the total open time) became predominant relative to level I (3 %), whereas the relative occurrence of level IV increased (51 %). In juvenile rats, between P19 and P22 (Fig. 2C, 5 patches pooled), the relative repartition of GlyR conductance levels was markedly modified with respect to the earlier stages: level IV became predominant and represented 96 % of the total open time, and only a small proportion of level II remained (4 %). This shift from highest conductance levels (levels I and II) to a lower one (level IV) suggests the existence of a developmental regulation of the GlyR functional phenotype in DA neurones.
Figure 1. Conductance states of glycine receptors (GlyRs) expressed by dopamine (DA) neurones of the substantia nigra pars compacta (SNc)
Application of glycine on outside-out patches excised from DA neurones of the SNc evoked unitary currents with multiple-conductance states (holding potential, VH/−70 mV). The conductance levels are marked by dotted lines and labelled from the highest level (I) to the lowest one (V). Chord conductance values indicated for each level were estimated by Gaussian fit of the point-per-point amplitude histograms obtained from the traces displayed. From all recordings, five distinct levels were distinguished, which are classified into five range levels: 100–110 pS (level I), 82–90 pS (level II), 65–70 (level III), 46–55 pS (level IV) and 28–32 pS (level V) according to the classification of Bormann et al. (1993). Traces were sampled at 25 kHz and filtered at 2 kHz, and correspond to epochs from longer recordings obtained from different patches. Note the direct transitions from level I to levels III and V (* and ** on the first trace) and from level IV to level V (* on the fourth trace).
Download figure to PowerPoint
Table 1. Occurence of the different conductance levels of glycine receptors during postnatal development
|Postnatal age (days)||Level I 100–110 pS||Level II 82–90 pS||Level III 65–70 pS||Level IV 46–55 pS||Level V 28–32 pS|
Figure 2. Developmental changes in the repartition of GlyR conductance levels
Point-per-point amplitude histograms of unitary currents evoked by 30 μm of glycine applied on outside-out patches excised from DA neurones of neonatal (A, postnatal day (P)7-P10), intermediate (B, P13-P16) and juvenile rats (C, P19-P22). The amplitude histograms shown in A, B and C were obtained by pooling recording epochs of 25–50 s from 13, 11 and 5 patches, respectively. Amplitude histograms were best fitted to the sum of three or four Gaussian functions, and mean amplitude and relative area (in parentheses) were indicated for each Gaussian function including the baseline (see Methods). Conductance levels were determined from the mean amplitude of each Gaussian function with ECl/−2 mV (VH=−70 mV). Amplitude histograms were normalized using the current amplitude distribution corresponding to the baseline. Analysed recordings were sampled at 25 kHz and filtered at 2 kHz. The bin width is 0.1 pA.
Download figure to PowerPoint
The kinetic properties of the highest conductance level (level I) predominantly observed in neonatal rats differed greatly from those of the lower conductance level (level IV) predominantly observed in juvenile rats, as illustrated for two individual patches in Fig. 3. Similarly to what was observed in individual patches, mean time constants of the openings (τo) differed in neonatal and in juvenile rats: τo1/ 0.17 ± 0.16 ms and τo2= 18.9 ± 2.7 ms for level I in neonates (6 patches), and τo1= 0.5 ± 0.3 ms and τo2= 9.6 ± 4 ms for level IV observed in juveniles (7 patches). Relative amplitudes of τo1 and τo2 were 15 ± 4 % and 85 ± 4 %, respectively, in neonates (level I), and 31 ± 9 % and 69 ± 9 %, respectively, in juveniles (level IV). This corresponds to a decrease in the mean open time (τm) from 16 ± 2.3 ms in neonates to 6.8 ± 3.3 ms in juveniles. These τm values, together with their decrease with age, are similar to what is observed for GlyRs in spinal cord and brainstem neurones, and probably reflect the switch from the α2 to the α1 GlyR subunit (Takahashi et al. 1992; Singer et al. 1998; Ali et al. 2000).
Figure 3. Gating properties of GlyRs expressed by SNc DA neurones in neonatal and juvenile rats
A1 and B1, examples of recordings obtained at P8 (A1) and P20 (B1) in response to 100 μm glycine (VH/−70 mV). Corresponding point-per-point amplitude histograms were obtained from the epochs of 10 s displayed. Recordings were digitized at 25 kHz and filtered at 2 kHz. Currents larger than 5 pA in histogram B1 correspond to superimposed openings, as shown in the inset. A2 and B2, dwell-time histograms of the main open state observed in response to 100 μm glycine in neonatal (A2) and juvenile rats (B2). Histograms were obtained by analysing 50 s epochs obtained from the representative patches shown in A1 for neonatal rats and in B1 for juvenile rats. Histograms were best fitted with the sum of two exponential curves with time constants (τ) and proportions as indicated (see Methods). Outside-out recordings were digitized at 25 kHz and filtered at 3 kHz.
Download figure to PowerPoint
The observation of a large main conductance level in neonates suggests strongly the presence of α homomeric GlyRs. To confirm this hypothesis, we have measured the effect of picrotoxin on GlyR-channel activity. Picrotoxin is known to preferentially block homomeric GlyRs at concentrations below 100 μm (Legendre & Korn, 1994; Pribilla et al. 1994; Legendre, 1997; Tapia & Aguayo, 1998; Yoon et al. 1998). The effect of 30 μm picrotoxin was tested on the unitary currents evoked by 30 μm glycine applied to outside-out patches from DA neurones. At this picrotoxin concentration, the discrimination between homomeric and heteromeric states is thought to be optimal, considering the near-complete block reported for homomeric GlyRs compared to the small flickering observed for heteromeric GlyRs (Legendre & Korn, 1994; Lynch et al. 1995; Yoon et al. 1998). In neonatal rats, high-conductance levels I and II were indeed blocked by 30 μm picrotoxin (Fig. 4), the total charge transferred being decreased by 66 ± 15 % (n/ 6). In contrast, in juvenile rats, picrotoxin induced a flickering of the main conductance level (level IV) corresponding to a 24 ± 8 % decrease of the total charge transferred, significantly different from the decrease observed in neonates (n= 4, unpaired t test, P < 0.01). Picrotoxin also significantly decreased conductance level IV by 15 ± 7 % from 52 ± 4 pS to 43 ± 3 pS (n= 4, paired t test, P < 0.01). The picrotoxin sensitivity of high-conductance levels confirms that they represent homomeric GlyR activation, whereas the relative insensitivity of the low-conductance levels suggests that they probably correspond to heteromeric GlyRs.
Figure 4. Differential effects of 30 μm picrotoxin on unitary currents evoked by glycine in neonatal and juvenile rats
Point-per-point amplitude histograms of single-channel currents evoked by 30 μm glycine alone (A1 and B1) and in the presence of 30 μm picrotoxin (A2 and B2) in neonatal (A1 and A2) and juvenile rats (B1 and B2). Amplitude histograms were obtained from 50 s recordings in each condition (bin width / 0.1 pA). Example traces of each recording are illustrated in the insets (2 kHz cut-off frequency; VH=−70 mV). Note that in the presence of 30 μm picrotoxin, the unitary conductance of the GlyR was decreased from 54 to 40 pS in the patch excised from a P22 rat (B1 and B2). Currents larger than 5 pA in histogram B2 correspond to superimposed openings as shown in the inset.
Download figure to PowerPoint
Postnatal changes in the whole-cell currents evoked by glycine
In order to determine whether this switch concerns all GlyRs in DA neurones, whole-cell currents evoked by 100 μm glycine as well as their inhibition by 30 μm picrotoxin were compared between DA neurones obtained from animals aged P7-P20 (Fig. 5A). The membrane capacitance of DA neurones slightly increased in this period from 26 ± 7 pF (n/ 16) at P7 to 34 ± 6 pF (n= 19) at P15-P17 and further to 37 ± 9 pF (n= 7) at P20. To determine whether any change in GlyR density occurred at the cell membrane during development, we analysed changes in the current density (pA pF−1). The mean current density was 19 ± 8.7 pA pF−1 (n= 16) at P7; this decreased significantly to 7.4 ± 7.3 pA pF−1 (n= 13, ANOVA-DMCT, P < 0.001) at P15 and further to 2.6 ± 1.6 pA pF−1 (n= 6, ANOVA-DMCT, P < 0.001) at P17, but then increased again to 19.6 ± 5 pA pF−1 (n= 7) at P20 (Fig. 5B). This result suggests that a transient decrease in GlyR density occurs around P17.
Figure 5. Developmental changes in the amplitude of glycine-evoked whole-cell currents and in their sensitivity to picrotoxin
A, examples of whole-cell currents evoked by 100 μm glycine and their inhibition by the transient application of 30 μm picrotoxin at three developmental stages (VH/−70 mV). Traces were digitized at 10 kHz and filtered at 1 kHz. B, changes in the current density amplitude measured at the plateau current evoked by 100 μm glycine (Ig) at different developmental stages (mean amplitudes ±s.d., number of neurones tested given above). The current density amplitudes measured at P15 and P17 were significantly lower than those measured at P7 and P20 (ANOVA-DMCT, P < 0.001). C, changes in the percentage inhibition of the current evoked by 100 μm glycine in response to 30 μm picrotoxin applications at different ages. The percentage inhibition is calculated as the ratio of the current decrease in the presence of picrotoxin (Ip) to Ig. Mean ±s.d. The percentage inhibition at P20 was significantly lower than at P7, P15 and P17 (ANOVA-DMCT, P < 0.001).
Download figure to PowerPoint
As illustrated in Fig. 5C, the mean percentage inhibition evoked by 30 μm picrotoxin remains stable up to P17, with values of 48 ± 15 % at P7 (n/ 16), 54 ± 7 % at P15 (n= 13) and 49 ± 20 % at P17 (n= 6). By contrast, at P20, the picrotoxin sensitivity of the glycine current was significantly reduced to only 14 ± 7 % inhibition (n= 7, ANOVA-DMCT, P < 0.001) compared to the three other ages. These data confirm a developmental regulation of GlyR functional characteristics in DA neurones. Such changes in GlyR functional properties have already been described in other preparations where synaptic contacts are present (Takahashi et al. 1992; Kungel & Friauf, 1997; Tapia & Aguayo, 1998; Ali et al. 2000).
Lack of taurine-mediated GlyR activation in DA neurones
Several lines of evidence suggest that taurine could be the endogenous ligand of GlyRs in the SNc and could be co-released with GABA (Clarke et al. 1983; Allen et al. 1986; Bianchi et al. 1998). To test whether GlyRs could be co-activated with GABAA receptors in response to the activation of GABA afferents surrounding DA neurones, IPSCs evoked by electrical stimulation of the GABAergic fibres were recorded in the presence of glutamatergic antagonists (dl-APV 50 μm, CNQX 10 μm). To optimize the taurine co-release with GABA from GABAergic terminals, we looked systematically for the best location around the recorded neurone to obtain the highest evoked synaptic current. Recordings were performed on both neonatal rats between P6 and P9 and on juvenile rats between P19 and P21. Evoked IPSC amplitudes (Fig. 7) were not significantly different between neonates and juveniles (respectively 509 ± 308 pA, n/ 30 and 708 ± 547 pA, n= 14; unpaired t test, P > 0.05). By contrast, the half-width duration of GABAergic IPSCs was significantly shorter in juveniles (11 ± 2 ms, n= 14) compared to neonates (31 ± 7 ms, n= 30; unpaired t test, P < 0.01), due to a decrease in the deactivation time constants. At both ages, deactivation phases could be fitted by a double exponential function (see Methods). In neonatal rats, the deactivation time constants τslow and τfast were 117 ± 79 ms (weight of 35 ± 11 %) and 23 ± 8 ms (n= 30), respectively. In juveniles compared to neonates, the two deactivation time constants were significantly shorter (unpaired t test, P < 0.01): τslow was 46 ± 19 ms and τfast was 11 ± 3 ms (n= 14). However, the relative weights of these two deactivation components were not significantly different between neonates (τslow: 35 ± 11 %) and juveniles (τslow: 23 ± 7 %; unpaired t test, P > 0.05). These observations suggest that a maturation process also occurred at GABAergic synapses during this period. Application of gabazine (1 μm; Fig. 7) almost completely suppressed the evoked IPSCs (97 ± 2 %; n= 28). Even train stimuli (see Methods) failed to reveal any putative GlyR activation in neonates (n= 7) and in juveniles (n= 5). The remaining current was not significantly modified by the subsequent application of strychnine (1 and 2 μm) either in neonates (n= 20) or in juveniles (n= 8).
Figure 7. Electrical stimulation of DA neurone afferents fails to evoke any glycinergic IPSCs
Evoked IPSCs obtained in DA neurones recorded in neonatal (A) and juvenile rats (B). In the presence of glutamatergic antagonists (50 μmdl-APV + 10 μm CNQX), note that the addition of a GABAA antagonist (1 μm gabazine) inhibited the evoked IPSCs. Subsequent addition of strychnine (1 μm) had little effect on the remaining current (approximately 5 pA, lower traces). Traces were digitized at 10 kHz and filtered at 1 kHz. Lower traces are a magnification of upper traces (VH/−70 mV).
Download figure to PowerPoint
To avoid possible taurine depletion, slices were pre-incubated in the presence of 1 μm taurine (n/ 7; Hussy et al. 2001). We also used thicker slices (350 μm) in the horizontal plane (n= 5) with the aim of best preserving striatonigral afferents, which have been reported to release taurine in the substantia nigra (Clarke et al. 1983; Bianchi et al. 1998). Finally, some of the thick slices were pre-incubated in a medium containing 1 μm taurine (n= 4). However, both taurine pre-incubation and/or thick horizontal slices failed to reveal a strychnine-sensitive component in response to the electrical stimulation of the GABA afferents surrounding DA neurones.
In addition, we tried to reveal an endogenous but subthreshold taurine release by blocking taurine uptake. Focal application of the taurine uptake blocker GES (300 μm) on DA neurones induced an inward current of 140 ± 64 pA, which was reduced to 18 ± 6 pA in the presence of 1 μm gabazine and was almost fully blocked by 10 μm gabazine (n/ 5, Fig. 8A). Application of 100 μm GES on patches excised from DA neurones induced single-channel openings exhibiting a main conductance of 24 pS that were almost fully blocked by 1 μm gabazine (n= 4, Fig. 8B1 and B2). Our results confirm that GES, in addition to the blockade of taurine uptake, exerts an agonist action on GABAA receptors, as shown previously (Mellor et al. 2000). Furthermore, the current induced by 300 μm GES in the presence of 1 μm gabazine was strychnine-insensitive (1 μm strychnine, n= 8, Fig. 8C). Thus, no GlyR activation can be induced by the blockade of taurine uptake.
Figure 8. The inward current induced by guanidinoethyl sulfonate (GES) in DA neurones is mediated by GABAA receptors
A, example of inward currents evoked by the focal application of 300 μm GES on a DA neurone. Note that this current can be completely inhibited by the application of 10 μm gabazine, a specific GABAA receptor antagonist (see Results). The trace was digitized at 10 kHz and filtered at 500 Hz. B1 and B2, example of single-channel currents evoked by 100 μm GES in a patch excised from a DA neurone (B1). The main conductance level of the single channels was 24 pS. Note that single-channel activity was dramatically reduced by 1 μm gabazine application (B2). Traces were digitized at 25 kHz and filtered at 1 kHz. C, the residual whole-cell current observed in the presence of 300 μm GES + 1 μm gabazine was insensitive to 1 μm strychnine application. The trace was digitized at 10 kHz and filtered at 500 Hz.
Download figure to PowerPoint
Finally, as it has been shown that during fetal and early postnatal development, GlyRs expressed by immature neocortical neurones can be activated via a non-synaptic pathway by taurine release in response to hyposmotic shock (Flint et al. 1998), this possibility was also tested. Hyposmotic stimulation using low-osmolarity PBBS was performed on DA neurones from neonatal rats (P6-P10). In the presence of dl-APV (50 μm), CNQX (10 μm) and gabazine (1 μm), local application of 270 mosmol l−1 PBBS or 200 mosmol l−1 PBBS had no effect on whole-cell current baseline (n/ 5, data not shown).