During development, patterned repetitive synaptic activity has been demonstrated in several neuronal systems (for review see Katz & Shatz, 1996). It is conceivable that repetitive activity of Cl− conductances can lead to short-term alterations of [Cl−]i, and thus to changes in Cl−-mediated responses. Indeed, repetitive activation of GABAARs with brief pulses or single activation with a prolonged pulse results in a ‘fading’ of the activated currents due to a shift in ECl and in GABA reversal potential (EGABA); this shift in EGABA is frequency dependent (Huguenard & Alger, 1986; Akaike et al. 1987). These experiments showed that Cl− regulation mechanisms can be challenged and the neuronal Cl− gradient can be manipulated by GABA-evoked activity. To assess whether the same holds for Cl− fluxes through activated GlyRs in LSO neurones, we varied the voltage protocol and glycine application interval (Δt) and investigated whether shifts in EGly occurred. ‘Up’ and ‘down’ voltage protocols were applied and Δt was varied; an ‘up’ protocol was immediately followed by a ‘down’ protocol. In P1-4 neurones with Δt= 10 s, EGly determined with the ‘down’ protocol (EGly(down)) was considerably more positive than EGly determined with the ‘up’ protocol (EGly(up)). In contrast, this effect was much smaller in P8-11 neurones. Examples of our observations are illustrated for a P2 and a P8 neurone in Fig. 6A and B, respectively. We calculated EDiff=EGly(down) - EGly(up) to use it as a measure for shifts in EGly. Group data showed that EDiff was significantly greater in P1-4 than in P8-11 neurones (14.1 ± 1.7 mV, n= 17 vs. 3.8 ± 1.8 mV, n= 12; Student's t test, P < 0.01; Fig. 6C). What could be the reason for the observed effects? We think that Cl− regulation mechanisms are not effective enough to maintain a constant [Cl−]i when Δt is as short as 10 s. As a consequence, EGly(up) and EGly(down) should deviate from the ‘real’ value for EGly in that EGly(up) is more negative and EGly(down) is more positive than the ‘real’EGly. We assume that EGly(up) is generated by Cl− depletion, i.e. an efflux of Cl− during the voltage steps more negative than ECl, which is sufficient to result in a considerable reduction of [Cl−]i and, consequently, in a negative shift of ECl. Likewise, EGly(down) is generated by Cl− loading, i.e. an influx of Cl− during the voltage steps more positive than ECl which results in an increase in [Cl−]i and, thus, a positive shift of ECl. Finally, the finding that EDiff is larger in P1-4 than in P8-11 neurones may reflect less efficient Cl− regulation mechanisms in the younger group. One way to test these ideas is to vary Δt in the ‘up’ and ‘down’ protocol: an increase in Δt should result in a smaller EDiff, whereas a larger EDiff should be observed when Δt is decreased. We analysed P1-4 and P8-11 neurones and found that EDiff indeed depended on Δt in both groups (Fig. 6D). In the younger group, Δt had to be increased to 50 s to yield a small EDiff of 3.5 ± 1.6 mV (n= 5). However, when Δt was decreased to 5 s, EDiff increased to 20.3 ± 2.3 mV (n= 9). In the older group, EDiff was negligible for Δt= 20 s (1.1 ± 1.2 mV, n= 3) but increased to 13.7 ± 3.2 mV when Δt was decreased to 5 s (n= 7). For a given Δt, EDiff was always larger in the younger than in the older group. These results demonstrate that EGly can be shifted in both a frequency-dependent and an age-dependent manner.
In a next step, we determined how much EGly(up) and EGly(down) deviated from the ‘real’EGly in P1-4 neurones when Δt= 10 s. Values for the deviation will be referred to as EShift in the following. An example for a P2 neurone is shown in the left panel of Fig. 7A. Almost identical values for EGly(up) and EGly(down) were found for Δt= 50 s, and the arithmetic mean was therefore taken as the ‘real’EGly. However, when Δt= 10 s, EGly(up) was more negative and EGly(down) was more positive than the ‘real’EGly, resulting in an EShift of −4.8 and +12.4 mV, respectively. When the analysis included all 12 P1-4 neurones tested, the average EShift was −4.3 ± 1.8 mV for EGly(up) and +10.3 ± 0.7 mV for EGly(down) (Fig. 7A, right panel). The results demonstrate that EGly is shifted in the positive and negative direction in a frequency-dependent manner by glycine-evoked activity.
Further evidence that positive and negative shifts of EGly occur in an age-dependent manner was obtained in another series of experiments: glycine was applied repetitively (20 pulses with Δt= 10 s) at a Vs that was 20 mV more negative than the ‘real’EGly. After a waiting period of about 2 min, another 20 pulses were applied at a Vs that was 20 mV more positive than the ‘real’EGly. In both cases, a reduction of successive amplitudes of glycine-evoked currents (‘fading’) was observed in P1-4 neurones (Fig. 7B), amounting to 58 ± 6 % in inward and 60 ± 6 % in outward currents (n= 6). This demonstrates that EGly was shifted in the positive and negative direction. The ‘fading’ effect was larger for Δt= 5, as it amounted to 79 ± 10 % for inward currents and 83 ± 3 % for outward currents (n= 3, not shown), demonstrating frequency dependence. In two P10 neurones, in which Δt was 10 s, the ‘fading’ of glycine-evoked inward and outward currents amounted to 10 ± 4 % and 26 ± 6 %, respectively (Fig. 7C). Thus, the ‘fading’ effect was much smaller in P10 than in P1-4 neurones, again indicating age dependence.