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. 2005 Jul 7;568(Pt 1):111–122. doi: 10.1113/jphysiol.2005.091371

Enhancement of spontaneous synaptic activity in rat Purkinje neurones by ATP during development

Diana Casel 1, Johannes Brockhaus 1, Joachim W Deitmer 1
PMCID: PMC1474765  PMID: 16002445

Abstract

The establishment of functional synaptic connections and activity is a pivotal process in the development of neuronal networks. We have studied the synaptic activity in the developing rat cerebellum, and the contribution mediated by purinergic receptors. The mean frequency of the spontaneous postsynaptic currents (sPSCs) recorded with the whole-cell patch-clamp technique from Purkinje neurones in acute brain slices at room temperature, increased fourfold from 4.4 ± 0.8 Hz at postnatal day 9/10 (n = 23) to 17.8 ± 1.6 Hz at postnatal day 17–20 (p17–p20; n = 113; P < 0.01). ATP, which increased the frequency of sPSCs by up to 100% (EC50= 18 μm) in the third postnatal week, started to modulate the synaptic activity during the second postnatal week, which was determined by three processes: (1) the appearance of functional ATP receptors during p10–p12, (2) the enhancement of the sPSC frequency by endogenous ATP release becoming apparent after inhibition of ecto-ATPases by 6-N,N-diethyl-β,γ-dibromomethylene-d-adenosine-5-triphosphate (ARL67156; 50 μm) at p11–p12, and (3) with tonic stimulation of purinoceptors at p14, as revealed by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μm). ATP had a similar effect at later stages (p24–p27) and at 35°C. Our results suggest that endogenous release of ATP starts to enhance the synaptic activity in Purkinje neurones by the end of the second postnatal week.

The neuronal network of the rat cerebellar cortex develops during the first 3 weeks of life. Purkinje cells in cerebellar brain slices receive spontaneous synaptic input mainly from local inhibitory interneurones, the basket and stellate cells. In a recent study, we described purinergic stimulation of the inhibitory input to Purkinje neurones, primarily mediated by activation of P2X receptors, and presumably located on somatodendritic regions of the interneurones (Brockhaus et al. 2004). A reduction of the frequency of spontaneous synaptic events in Purkinje neurones, when purinergic P2 receptors were inhibited by pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), indicated that ATP is tonically released in the cerebellar tissue. Purinergic modulation of synaptic activity has been described for several CNS regions, including the habenula nucleus (Edwards et al. 1992), the dorsal root ganglia (Gu & MacDermott, 1997) and hippocampus (Inoue, 1998; Mendoza-Fernandez et al. 2000; Koizumi et al. 2003). Little is known about the development of purinergic modulation and the sequence in which the purinoceptors, the release of ATP and the ecto-ATPases appear in these brain areas. In presynaptic nerve terminals of spinal cord substantia gelatinosa neurones, α,β-methyleneATP-sensitive P2X receptors, which enhance the frequency of spontaneous glycinergic activity, are expressed rather late (later than postnatal day 10–12 (p10–12); Jang et al. 2001). In locus coeruleus neurones in the rat, an inward current was induced by α,β-methylene-ATP at p18–p23, which was not observed at p10–p14, suggesting developmental regulation of functional P2X receptors in this brain region in the third postnatal week (Wirkner et al. 1998).

In the present study, we have focused on the development of the purinergic system that contributes to the synaptic activity in Purkinje neurones, which carry the output of the cerebellar cortex, in order to get insight into the role of purinoceptors for the synaptic activity in the cerebellum. Our results indicate that the purinergic system matures during the second postnatal week in the rat cerebellum, and it starts to contribute to the modulation of the synaptic activity in Purkinje neurones by the end of the second week. Some preliminary results have been published in abstract form elsewhere (Dressel et al. 2004; Deitmer et al. 2004).

Methods

Cerebellar brain slices were obtained from juvenile rats by following a standard procedure (Edwards et al. 1989). The rats were bred and housed in our facility in accordance with the current German animal protection laws. The killing protocol was approved by the regional animal care and use committee (Landesuntersuchungsamt Rheinland-Pfalz, Germany). In short, after decapitation, the cerebellum was isolated in ice-cold bath solution (see below) with reduced CaCl2 (0.5 mm; MgCl2 increased to 2.5 mm). Sagittal slices of the vermis (250 μm thick) were obtained by use of a vibratome (VT 1000; Leica, Darmstadt, Germany) and stored in the same solution, gassed with carbogen (95% O2/5% CO2) for 1 h at 30°C, and later at room temperature (21–24°C). Slices from older rats (p24–p27) were stored in the same solution, but prepared in a sucrose-based ice-cold solution containing (mm): sucrose 230, KCl 2.5, NaH2PO4 1.25, glucose 10, MgSO4 10, CaCl2 0.5, Hepes 10, pH adjusted to 7.4 with NaOH (modified from Aghajanian & Rasmussen, 1989).

For electrophysiological recordings, slices were fixed in a recording chamber with a U-shaped platinum wire and nylon grid on the stage of an upright microscope (Axioscope, Zeiss, Oberkochen, Germany). The chamber (volume <0.5 ml) was continuously perfused (2–3 ml min−1) with carbogen-gassed bath solution containing (mm): NaCl 125, KCl 2.5, NaHCO3 26, NaH2PO4 1.25, MgCl2 1, CaCl2 2, glucose 25, d-lactate 0.5. Recordings were controlled with pCLAMP software (Axon Instruments, Union City, CA, USA) with a digidata 1322 A interface and an Axopatch-1D amplifier (Axon Instruments). Pipettes were pulled with a horizontal puller (P-87; Brown and Sutter, Novato, CA, USA) and heat polished to a tip resistance of 2–3 MΩ, filled with an intracellular solution containing (mm): CsCl 120, tetraethylammonium chloride 20, MgCl2 2, Na2ATP 2, EGTA 0.5, Hepes 10; pH adjusted to 7.3 with CsOH. This solution strongly reduced the background K+ conductance, and allowed a better observation of spontaneous synaptic events (Konnerth et al. 1990). Series resistance was 4–12 MΩ (compensation 80–90%).

All drugs were applied via bath solution: ATP, PPADS, adenosine and the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were obtained from Sigma-Aldrich (Taufkirchen, Germany); the inhibitor of ATP-degrading enzymes, 6-N, N-diethyl-β,γ-dibromomethylene-d-adenosine-5-triphosphate (ARL67156, also known as FPL67156), was purchased from Tocris (Bristol, UK). The experiments were carried out at room temperature (21–24°C), and at 35°C, where indicated.

The spontaneous postsynaptic current (sPSC) frequency of Purkinje neurones was calculated from continuous recordings (sampling rate, 4–5 kHz; filter, 2 kHz; duration, 50 s) with pClamp software with a threshold in the first derivative of the current (see Brockhaus & Deitmer, 2002). Threshold values were set at twice the noise maximum, normally 10–15 pA ms−1. The first derivative was used to minimize problems with slow changes of the baseline level, and allowed the counting of postsynaptic events that occurred during the decay phase of a previous sPSC. Further evaluation was done with software for analysis of synaptic currents (MiniAnalysis; Synaptosoft, Inc., Decatur, GA, USA).

Data are given as arithmetic means ± standard error of the mean (s.e.m.) of n number of cells. Significance was calculated with Student's t-test, and was determined as significant at P < 0.05 (*) or ‘highly significant’ at P < 0.01 (**). The curve in Fig. 1B is a Boltzmann fit, y = A2 + (A1–A2)/(1 + exp(x – x0/dx)), and the curve in Fig. 3B is a Hill approximation, f(x) = (Vmaxxn)/(knxn).

Figure 1. Spontaneous postsynaptic currents (sPSCs) as recorded in Purkinje neurones of cerebellar slices during the second and third postnatal week.

Figure 1

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A, recordings of synaptic currents on postnatal day 9 (p9) showing single events with very large amplitude, burst-like activity on p10, synaptic currents on p16, and at 35°C on p26. B, the sPSC frequency increases steeply between p12 and p16, as also shown in C where the sPSC frequency was classified into different developmental stages; hatched bars show the sPSC frequencies at 35°C. D and E, at room temperature (RT, 21–24°C) and at 35°C (hatched bars), the mean amplitude of the sPSC was reduced during maturation, as summarized in the histogram. The curve fit in B was obtained with the Boltzmann equation: y = A2+ (A1–A2)/(1 + exp(x – x0/dx)), where A1 is 326.3, A2 is 925.8, and x0 is 13.8.

Figure 3. Concentration-dependent effect of ATP on the synaptic activity.

Figure 3

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A, averaged plots of the change in sPSC frequency in Purkinje neurones, as induced by ATP applied at different concentrations at p14–p20. B, dose–response curve of the change in sPSC frequency as induced by ATP, indicating a maximal effect of ATP at ≥100 μm, and an EC50 value of 18 μm. The curve fit was obtained with a Hill approximation according to f(x) = (Vmaxxn)/(knxn), where Vmax is 99.6, k is 17.7, and the Hill coefficient n is 1.4 ± 0.6, as calculated from the slope of the fitted curve.

Results

Development of spontaneous synaptic activity

Purkinje neurones in the cerebellum receive synaptic input from the spontaneously active inhibitory basket and stellate cells, the local interneurones of the cerebellar cortex. We recorded sPSCs in Purkinje neurones with a patch-pipette solution containing a high Cl− concentration; this converts inhibitory Cl− currents to inward currents, so that all synaptic currents are inward (Fig. 1A). Earlier studies had shown that the vast majority (>90%) of these events are GABAergic, with a minor number being excitatory from granule cells (Konnerth et al. 1990; Brockhaus et al. 2004). Recordings from younger animals (p9–p12) occasionally showed phases with burst-like activity (Fig. 1A, second trace from top), or currents of large amplitude with larger quiescent intervals (Fig. 1A, upper trace). These bursts and very large synaptic currents were rarely seen in animals older than p13, where the frequency and amplitude of the spontaneous events at room temperature (RT) or at 35°C were more evenly distributed (Fig. 1A, bottom traces). The spontaneous synaptic activity recorded in Purkinje neurones at room temperature greatly increased during the second postnatal week, from 4.4 ± 0.8 Hz (n = 23 cells) and 5.3 ± 1.2 Hz (n = 22) at p9/10 and p11/12, respectively, to 15.6 ± 1.9 Hz (n = 118) at p14–16, to 17.8 ± 1.6 Hz (n = 113) at p17–20, respectively (Fig. 1B and C). These results indicate a fourfold rise of the synaptic activity between day 10 and day 20. The increase in the sPSC frequency was accompanied by a decrease in the sPSC amplitude, which declined from 462 ± 64 pA at p9–p12 (n = 9) to 258 ± 40 pA at p14–p20 (n = 6; P < 0.05; Fig. 1D and E). At later stages (p24–p27), the sPSC frequency was increased even further to 23.6 ± 2.5 Hz (n = 7), while the sPSC amplitude remained at the similar value as at p14–p20 (Fig. 1C and E).

At the physiological temperature of 35°C, the sPSC frequency tended to be larger at earlier stages, being 9.3 ± 1.3 Hz (n = 12) at p9/10, and 9.6 ± 1.4 Hz (n = 7) at p11/12. At later stages, the sPSC frequency was 16.4 ± 3.8 Hz (n = 9) at p14–p16, 18.3 ± 4.3 Hz (n = 10) at p17–p20, and 33.8 ± 4.9 Hz (n = 12) at p24–p27, respectively (Fig. 1C).

Developmental expression of functional purinoceptors

We have previously reported endogenous purinergic modulation of the spontaneous synaptic activity in the cerebellum of rats at p14–p20 (Brockhaus et al. 2004). At this age, the sPSC frequency was increased twofold by bath application of ATP (100 μm; Fig. 2D), resulting to a large extent from the activation of P2X5-like receptors, presumably located at somatodendritic compartments of interneurones presynaptic to Purkinje neurones, and from the activation of P2Y receptors (Brockhaus et al. 2004). We have now looked at earlier stages in development to identify the period when functional purinoceptors are expressed. In younger animals (p9/10), ATP (100 μm, 3 min) failed to increase the sPSC frequency (sPSC frequency 100.8 ± 6.2% of control; Fig. 2A) in 21 out of 28 Purkinje neurones measured. In the remaining seven cells, however, ATP induced a large, but variable increase in the sPSC frequency to 361 ± 93% (Fig. 2B). The effect of ATP as averaged from all 28 cells, amounted to 159 ± 29% at this developmental stage (Fig. 2E), but was not significantly different from control values (P= 0.068) due to the large variance.

Figure 2. The effect of ATP on the synaptic activity during development.

Figure 2

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Application of 100 μm ATP for 3 min had either no effect on the sPSC frequency in Purkinje neurones at p9/10 (A), or, in 7 out of 28 experiments, a very large effect (B), but caused a robust increase in the sPSC frequency at p11–p13 (C) and at p14–p20 (D). E, summary of the sPSC frequency in ATP at different ages; hatched bars give the values obtained at 35°C. At p9/10, the data were pooled (left bars), and were presented separately, without ATP effect (middle bars) and with ATP effect (right bars). Asterisks above columns indicate significant increase versus control (100%, dashed line).

At p11–p13, ATP significantly increased the sPSC frequency in all cells, on average to 160 ± 24% (n = 12; P < 0.05; Fig. 2C and E) of control values, which was not significantly different from the frequency increase measured in older animals (at p14–p20: 204 ± 13%; n = 61; at p24–p27: 224 ± 63%; n = 6; Fig. 2D and E). The stimulation of the sPSC frequency by ATP at p14–p20 was suppressed by the P2 receptor antagonist PPADS (10 μm; n = 9; at 21–24°C; not shown), as reported previously (Brockhaus et al. 2004).

At 35°C, the results were qualitatively similar. At stage p9/10, the mean relative sPSC frequency was 132 ± 22% in 100 μm ATP (n = 10) as compared with the control recordings without ATP; in two of the 10 experiments the sPSC frequency was 256 and 266%, while in remaining eight experiments, the sPSC frequency was 100 ± 3% (Fig. 2E) as compared with the control. At stage p11–p13 and at p14–p20, the sPSC frequency was highly significantly increased in ATP to 211 ± 47% (n = 7) and to 249 ± 36% (n = 8; P < 0.01), respectively. At later stages (p24–p27), the ATP-induced increase of the sPSC frequency amounted to 186 ± 24% (n = 5; P < 0.05). None of the mean values of the sPSC frequency in ATP at 35°C was significantly different from those measured in ATP at room temperature (P= 0.17–0.96).

We used different ATP concentrations between 1 and 1000 μm for a dose–response analysis of the effect of ATP on the sPSC frequency in Purkinje neurones in animals at p14–p19 (Fig. 3A and B). ATP at 10 μm was the lowest concentration at which a significant increase in the sPSC frequency by 30% could be detected. The dose–response curve revealed an EC50 value of 18 μm ATP, with a Hill coefficient of 1.4 (Fig. 3B). The maximal effect was achieved with a concentration of 100 μm ATP and above.

ATP effect is neither hidden nor mediated by adenosine A1 receptors

Previous experiments indicated that the ATP effect on the sPSC frequency was not mediated by degradation of ATP to adenosine, which in turn might activate A1 receptors (Brockhaus et al. 2004). In this study, we applied adenosine and ATP in the absence and presence of the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 μm), in slices from p8–p10 and p14–p20 rats (Fig. 4). At p8–p10, neither ATP (100 μm), in the absence or presence of DPCPX, nor adenosine had any effect on the sPSC frequency in Purkinje neurones (Fig. 4A and B), the sPSC frequency being 114 ± 7, 115 ± 18 and 111 ± 8%, respectively (n = 8). At p14–p20, ATP evoked a similar rise of the sPSC frequency, as described above, to 197 ± 33% (n = 8; P < 0.01) and to 185 ± 36% (n = 8; P < 0.01) in the absence and in the presence of DPCPX, respectively, while adenosine was without effect, the sPSC frequency being 89 ± 6% (n = 9; Fig. 4C). These experiments show that A1 receptor activation is not involved, neither in evoking nor in suppressing the rise in the sPSC frequency, as observed in the presence of ATP from p11 onwards.

Figure 4. Adenosine receptors are not involved in modulating synaptic activity in Purkinje neurons.

Figure 4

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A, at early stages, ATP (100 μm) had no effect, even in the presence of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2 μm), indicating that the lack of ATP effect was not obscured by activation of A1 receptors. Summary of the effect of ATP, adenosine and of ATP in the presence of the A1 antagonist DPCPX on the sPSC frequency at the developmental stages p8–p10 (B) and p14–p20 (C).

Enzymatic degradation of endogenously released ATP

ATP is known to be degraded by ecto-ATPases in the extracellular space, which can result in a reduction of the effective concentration of both endogenously released and bath-applied ATP (Zimmermann, 2000). A block of ecto-ATPases with ARL67156 (50 μm) led to an increase in spontaneous synaptic activity in cerebellar brain slices of more mature animals (p14–p20), in line with a tonic release of ATP in the tissue (Brockhaus et al. 2004). We then looked at the influence of the ecto-ATPase inhibitor ARL67156, which specifically inhibits the enzymatic conversion of ATP to ADP (Crack et al. 1995) at earlier stages. At p9/10, ARL67156 (50 μm) had no effect on the sPSC frequency of Purkinje neurones (111.6 ± 5.6% of control; n = 7; Fig. 5A and D). In animals at p11/12, inhibition of ecto-ATPases increased the sPSC on average to 132 ± 10% (n = 17; P < 0.05; Fig. 5B and D). In 5 of 17 cells, however, the sPSC frequency remained unaffected by ARL67156 at this stage (91 ± 2% of control). At p14–p20, blockade of ecto-ATPases increased the spontaneous activity to 157 ± 9% (n = 16; Fig. 5C and D). These results indicate that ATP is already endogenously released at the onset of the functional expression of purinoceptors around p11/12. Since ecto-ATPases in younger animals might degrade ATP more effectively, we applied 300 μm ATP in the presence of the ecto-ATPase inhibitor ARL67156 (50 μm); even at this high concentration, ATP failed to modulate the sPSC frequency at p9/10 (111 ± 13%; n = 8; not shown).

Figure 5. Block of ecto-ATPases reveals endogenous ATP release in the tissue.

Figure 5

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In the presence of the ATP-degrading enzyme (ecto-ATPase) inhibitor 6-N,N-diethyl-β,γ-dibromomethylene-d-adenosine-5-triphosphate (ARL67156; 50 μm), the sPSC frequency in Purkinje neurones was not changed at p9/10 (A), but significantly increased at p11/12 (B) and at p14–p20 (C), as also summarized in D.

Role of endogenous ATP on the enhancement of synaptic activity

In more mature animals (p14–p20), tonic ATP release in the cerebellar slice was indicated both by an increase of the sPSC frequency after inhibiting ecto-ATPases, and by a decrease in the sPSC frequency during blockade of P2 receptors by PPADS (Brockhaus et al. 2004). It remained unresolved, however, whether these processes develop in parallel with the functional expression of P2 receptors. We recorded the spontaneous synaptic activity in Purkinje neurones following the blockade of P2 receptors with PPADS (10 μm) at different ages. At p9–p13, the sPSC frequency was not affected by PPADS (111 ± 13%; n = 18; Fig. 6A). Even at the stage when ATP and ARL67156 already enhanced the synaptic activity in Purkinje neurones (p11–13), PPADS failed to modulate the sPSC frequency (4.4 ± 0.5 Hz in control, and 4.3 ± 0.5 Hz in PPADS; n = 9; Fig. 6C). In rats from p14 onwards (p14–p20 and p24–p27), however, PPADS induced a highly significant decrease in the sPSC frequency to 75 ± 4% at p14–p20 (n = 29), and 61 ± 12% (n = 5) at p24–p27. This suggests that tonic enhancement of synaptic activity by endogenous ATP, as mediated by PPADS-sensitive purinoceptors, does not occur before p14. At 35°C, the decrease of the sPSC frequency by PPADS obtained at p14–p20 was similar to that at room temperature (Fig. 6B), indicating tonic purinergic stimulation also at physiological temperature.

Figure 6. Blocking P2 receptors reveals endogenous purinergic stimulation of synaptic activity.

Figure 6

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A, the relative sPSC frequency in Purkinje neurones in the presence of the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′4′-disulphonic acid (PPADS, 10 μm) averaged for the developmental stages p9–p13, p14–p20 and p24–p27. B, the effect of PPADS on the synaptic activity was similar at room temperature (RT) and at 35°C. C, the sPSC frequency at p9/10, p11–p13, p14–p20 and p24–p27 in the presence and absence (control, open bars) of PPADS at RT. D and E, effect of a shorter exposure to PPADS (5 μm, for 5 min) on the sPSC frequency and its partial reversibility. The inset in D shows averaged sPSC recordings (200 from 4 slices) before the addition of PPADS (control), 9–11 min after addition of PPADS, and 18–21 min after removal of PPADS, as indicated.

In a set of experiments, we applied a lower concentration of PPADS (5 μm) for a shorter time (5 min) to ease reversibility of the PPADS effect (Fig. 6D and E). The sPSC frequency decreased 10 min after addition of PPADS to 83 ± 5% (n = 6; P > 0.05), and slowly started to recover partially after another 10 min; 18–21 min after removal of PPADS, the sPSC frequency had recovered to 98 ± 7% (n = 6), which was not significantly different from the frequency before addition of PPADS. The sPSC frequency was constant before addition of PPADS for about 15 min, showing that spontaneous run-down of the sPSC frequency, which coincided with the application of PPADS, was unlikely. The experiments also showed that the kinetics of the PSC remained the same before and after addition of PPADS (Inset of Fig. 6D). Both parameters, constant frequency and PSC kinetics, indicate fairly stable recording conditions over the entire duration of our experiments.

These results further show that part of the spontaneous synaptic input of Purkinje neurones might be induced by endogenously released ATP, which develops at the same time (>p13) as the increase in the sPSC frequency was observed (see Fig. 1B). Hence, a substantial fraction of the increase in synaptic activity at ≥p14 appears to depend on the endogenous stimulation of purinoceptors (Fig. 6C).

Discussion

The modulation by purinoceptor activation of spontaneous network activity in the cerebellar cortex, recorded as postsynaptic currents in Purkinje neurones, develops in several steps during the second postnatal week in the rat. An ATP-induced increase in the sPSC frequency was observed beginning with p10/11, but tonic endogenous stimulation of purinoceptors became effective at around p14, as indicated by the reduction of the spontaneous synaptic activity following inhibition of P2 receptors with PPADS. At p11/12, the ecto-ATPase inhibitor ARL67156 increased the sPSC frequency without added ATP, revealing tonic release of ATP in the tissue. The ATP release was, however, not sufficient to affect the frequency of sPSCs in Purkinje neurones at p11–p13, presumably due to the activity of ATP-degrading enzymes. The frequency of spontaneous synaptic events increased more than fourfold between p10 and p20, and even further at p24–p27; part of this increase appears to be due to endogenous release of ATP, activating P2 receptors from the end the second postnatal week onwards. A similar pattern for the sPSC frequency and amplitude in the presence of ATP was obtained at physiological temperature of 35°C. The results also indicate that adenosine or adenosine A1 receptor antagonists did not affect the modulation of synaptic activity by ATP.

Maturation of the cerebellar cortex and the purinergic system

During the second and third postnatal week, the cerebellar morphology undergoes some major steps of maturation, and the gross synaptic connectivity is established (Llinas & Walton, 1990; Altman & Bayer, 1997). The inhibitory interneurones mainly proliferate in the second week, with a peak for the basket cells at p7 and for the stellate cells at p11. The outer granule cell layer is reduced as the premature granule cells migrate along Bergmann glial processes to the inner granule cell layer, with each cell leaving a parallel fibre at the distal end of an ascending process, and thus forming the parallel fibre system. As the molecular layer matures, the radial processes of the Bergmann glial cells bud off branches, which get into close contact with the newly formed synapses on the growing dendritic tree of Purkinje neurones and form synaptic microdomains (Grosche et al. 1999, 2002; Yamada et al. 2000). During this period, the frequency of spontaneous activity in Purkinje neurones greatly increases. This boost of activity appears in an almost step-like manner around p14. A comparable increase in spontaneous IPSC frequency was observed in one type of local interneurones in the cerebellum, the stellate cells, where synaptic input increases fourfold between p11–p13 and p21–p25 (Bureau & Mulle, 1998).

It is in this period of prominent increase of the synaptic input to Purkinje neurones when purinoceptors become involved in modulating the sPSC frequency. The development of the effect mediated by the purinergic system comprises three processes that together shape the purinergic modulation of synaptic activity during the course of the second postnatal week. These are: (1) the expression of functional P2 purinoceptors, (2) the endogenous release of ATP, and (3) the activity of ecto-ATPases that regulate the effective concentration of extracellular ATP. Isolation of these processes was obtained by recording the effect of extracellularly applied ATP to indicate the developmental expression of functional purinoceptors, and through the use of the ecto-ATPase inhibitor ARL67156 and the P2 receptor antagonist PPADS to identify endogenous ATP release at different developmental stages (Fig. 7). The functional expression of purinoceptors was reliably observed from p11 onwards, while in a few experiments (25%), a prominent ATP effect was seen already at p9/10. Even 300 μm ATP, in the presence of ARL67156 to inhibit extracellular degradation of ATP, failed to significantly stimulate the sPSC frequency at <p11. This supports the conclusion that the lack of any ATP effect in the majority of the recordings from younger animals is due to the lack of functional purinoceptors at this age, and argues against the possibility that it was enhanced efficacy of ecto-ATPases, which suppressed the effect of bath-applied ATP. In addition, adenosine and the adenosine A1 receptor antagonist DPCPX, had no effect on inducing a rise of synaptic activity in Purkinje neurones, or suppressing the rise evoked by ATP, suggesting that the ATP effect was not mediated by adenosine receptors. These experiments also exclude the possibility that at earlier stages the ATP effect was obscured by a depressing effect of adenosine formed by enzymatic ATP degradation.

Figure 7. Scheme summarizing the development of the purinergic system and the synaptic activity in the cerebellum during the second postnatal week.

Figure 7

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Developmental expression of functional ATP receptors, as indicated by the increase of the sPSC frequency in Purkinje neurones by bath-applied ATP (upper trace). The scheme shows the occurrence of an effect of inhibiting ecto-ATPases with ARL67156, suggesting endogenous release of ATP (second trace down), and of an effect of blocking P2 receptors with PPADS, suggesting effective tonic stimulation of purinoceptors mediating an increase in the sPSC frequency (third trace down), as compared with the increase of the sPSC frequency in Purkinje neurones measured under control conditions in normal saline (bottom trace).

Beginning with p11, an increase in the sPSC frequency was observed after blocking ecto-ATPases with ARL67156, suggesting that ATP was released endogenously in the brain slices at that stage (Fig. 7). Since the sPSC frequency was only affected by inhibiting P2 receptors with PPADS at p14 and later, we conclude that between p11 and p13, the ATP released endogenously was not sufficient to exert an effect on the sPSC frequency in Purkinje neurones, presumably in part due to the activity of ATP-degrading enzymes. At around p14, when blocking P2 receptors alone is sufficient to reduce the sPSC frequency, either the amount of ATP released endogenously is enhanced, or the degrading efficacy of the ecto-ATPases is reduced, in order to provide an extracellular ATP concentration high enough to exert its effect on the sPSC frequency. The purinergic modulation of the spontaneous synaptic activity by ATP or PPADS was not only seen at room temperature, but also at 35°C. This supports the conclusion that, although the processes involved are expected to be temperature sensitive, such as, e.g. the rate of ATP release and the enzymatic degradation of ATP, purinergic modulation of the synaptic activity is similar at room temperature and at physiological temperature.

Purinergic stimulation of spontaneous activity

The frequency of spontaneous synaptic input to Purkinje neurones increases with maturation. At the end of the second postnatal week, the sPSC frequency increased from around 5 Hz at p9–p12 to around 17 Hz at p14–p20, and to nearly 24 Hz at p24–p27. At 35°C, a mean sPSC frequency of as much as 34 Hz was recorded at p24–p27. In contrast to the frequency of the sPSCs, which increased, the average amplitude of the sPSCs decreased with age, both at room temperature and at 35°C. This is in line with the finding that the amplitude of Purkinje neurone IPSCs, induced by single action potentials in cerebellar inhibitory interneurones, decreases several fold between p11 and p31 (Pouzat & Hestrin, 1997).

The increase in the spontaneous sPSC frequency occurs in parallel with the onset of endogenous ATP release stimulating the synaptic activity as indicated by the reduction of the sPSC frequency by PPADS (Fig. 7). This supports the idea that the endogenous ATP release found in preparations from more mature animals significantly contributes to the increase in the sPSC frequency during maturation. In a recent study, we reported that the effect of ATP was predominantly mediated by ionotropic P2X5-like receptors and by P2Y receptors (Brockhaus et al. 2004). Earlier studies on cultured neurones or synaptosomes also showed increased GABAergic transmission in the presence of ATP (Inoue et al. 1999; Hugel & Schlichter, 2000; Gomez-Villafuertes et al. 2001). The dose–response curve for the ATP effect studied here revealed an EC50 value of 18 μm. This value is higher than the 3 μm ATP reported for the EC50 value for isolated homomeric P2X5 receptors (Khakh et al. 2001). This can be expected due to endogenous nucleotidase activity, which reduces the effective ATP concentration seen by the purinoceptors in the tissue. Furthermore, the dose–response curve showed a Hill coefficient of near to 1.4. This suggests that at least two ATP molecules probably bind to the P2X receptors in the cerebellar network, presumably located on somatodendritic regions of the inhibitory interneurones (Brockhaus et al. 2004). A predicted trimeric structure of P2X receptors as reported by Nicke et al. (1998) infers that indeed more than one agonist molecule may bind to these receptors.

The sPSC frequency in Purkinje neurones that was recorded after blocking P2 receptors with PPADS was 75% of that under control conditions (at ≥p14). A 25–35% increase of the sPSC frequency could be induced by the addition of approximately 10 μm ATP, as estimated from the dose–response curve (Fig. 3B). Hence, this ATP level might be expected to be released endogenously in the cerebellar tissue. It should be noted that 1 μm ATP or ADP bath-applied to a cerebellar slice was sufficient to evoke Ca2+ transients in 50% of the Bergmann glial cells via P2Y receptors, as recorded by confocal Ca2+ imaging (Teßmann, 2004). This infers that an ATP level of around 10 μm, as estimated to be released endogenously, would either tonically activate or desensitize P2Y receptors in Bergmann glial cells (and elsewhere in the cerebellum), and that addition of 1 μm ATP is not likely to evoke an additional distinct response. Therefore, it must be contemplated that the ATP is released locally, and is presumably not accessible to all synaptic microdomains in the cerebellar cortex.

Possible sources of endogenously released ATP

There are several possible sources that may contribute to the endogenous release of ATP in the cerebellum. A source of ATP might be dying cells, which could leak ATP into the tissue. Since the brain slices used here were allowed a period of at least 1 h to recover from the dissection, ATP leaking from superficial cells that were injured during the preparation procedure would be expected to be degraded or removed by the superfusate. It has been postulated that parallel fibres release ATP together with glutamate (Rubio & Soto, 2001). Inhibitory neurones may also use ATP as a cotransmitter together with GABA, as reported for cultured neurones from spinal dorsal horn (Jo & Schlichter, 1999) and from the hypothalamus (Jo & Role, 2002). This type of corelease of ATP may be especially important at the interneurone–interneurone synapses in the cerebellum, where an additional excitatory component would be provided, which can be – though GABAergic – excitatory rather than inhibitory due to a high intracellular Cl− concentration in the cells (Chavas & Marty, 2003). The spontaneous activity of the local interneurones in the cerebellar cortex supports this idea. On the other hand, glial cells and glial progenitor cells can also release ATP, and hence be a source for the endogenously released ATP (Guthrie et al. 1999; Cotrina et al. 2000; Newman, 2003; Weissman et al. 2004).

Tonic purinergic stimulation is not only seen in brain slice preparations, but was also found in in vivo experiments using adult rats, where spontaneous action potential firing was reduced in medullary neurones of the respiratory network during a block of P2 receptors with suramine (Gourine et al. 2003). Thus, endogenous, tonic ATP release might be a more widespread physiological process, which could contribute to the modulation of neuronal activity. The present results indicate that the increase of synaptic activity in the cerebellum, starting during the second postnatal week and continuing over and beyond the third postnatal week, is partly due to endogenously released ATP resulting in tonic stimulation of P2 receptors, presumably located on inhibitory interneurones.

Acknowledgments

We thank S. Bergstein for excellent technical assistance. This work was supported by a stipend of the Boehringer-Ingelheim Foundation (to D.C.) and the Deutsche Forschungsgemeinschaft (DE-231/17-1, 19-1).

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