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Journal of Physiology (2001), 536.2, pp. 429-437
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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In the developing brain, neuronal gene expression (LoTurco et al. 1995), differentiation (Marty et al. 1996), migration (Komuro & Rakic, 1993) and synapse maturation (Spoerri, 1988) are regulated by transient changes in [Ca2+]i. Remarkably, these processes occur at a developmental stage at which in many regions of the brain the excitatory glutamatergic connectivity is sparse and weak, or even not yet established (e.g. Durand et al. 1996). Instead, GABA, which acts as the main inhibitory neurotransmitter in the mature brain, seems to provide the dominating excitatory drive in at least some regions of the developing central nervous system (for review see Leinekugel et al. 1999). Clear evidence of such a depolarising role of GABA has been obtained in the hippocampus, cortex, striatum, spinal cord, hypothalamus, retina, olfactory bulb (for review see Cherubini et al. 1991) and, more recently, the brainstem (Brockhaus & Ballanyi, 1998). In some instances, GABA-mediated excitation appears to be sufficiently strong to trigger increases in [Ca2+]i (Yuste & Katz, 1991; Wang et al. 1994; Owens et al. 1996; Kulik et al. 2000) and thereby perhaps to provide the cellular signals needed for normal development (Barker et al. 1998). Despite a considerable amount of information concerning this apparently paradoxical property of GABAergic function, the accurate time course of the postnatal switch in GABA responsiveness has not yet been established in any type of neurone. Thus, related studies either focused only on the transition (e.g. Wu et al. 1992) or were performed on cultured neurones (e.g. Wang et al. 1994), a preparation that does not allow a faithful correlation with the postnatal development in vivo.
Moreover, little is known about the action of GABA on the various cell types in the developing cerebellum (Brickley et al. 1996). The cerebellum, which is remarkably immature at birth (Woodward et al. 1971), undergoes a striking period of maturation that lasts for about 2-3 weeks. This includes, as its most prominent features, the migration of granule cells from the outer germinal layer to the inner granule layer and a massive formation of synapses between the various cell types of the cerebellar cortex (for overview see Altman & Bayer, 1996). Purkinje neurones (PNs), the principal neurones of the cerebellar cortex, also undergo marked morphological and functional changes during the first 2 postnatal weeks. They merge from two to three irregular rows into a single layer, evolve an extensive dendritic tree, and undergo a characteristic cycle of elimination of redundant climbing fibre (CF) synapses, followed by the formation of a large number of parallel fibre synapses (Woodward et al. 1971; Ito, 1984). In the study described here, we quantified the responsiveness of PNs to GABA during this early stage of massive synaptic reorganisation by using two-photon fluorescence microscopy (Denk et al. 1990) and perforated-patch recordings (Abe et al. 1994; Reichling et al. 1994; Owens et al. 1996).
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METHODS |
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Electrophysiological and fluorometric recordings were performed at room temperature (21-22 °C) on visually identified PNs in cerebellar slices taken from 2- to 22-day-old Wistar rats. During recordings, slices were perfused with artificial cerebrospinal fluid (ACSF) containing (mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 20 glucose, bubbled with 95 % O2 and 5 % CO2, pH 7.3-7.4.
Tissue preparation
The animals were decapitated and the cerebella rapidly removed, in accordance with the rules of the institutional ethics committee. The tissue was placed in cold (0-2 °C) ACSF and 200 µm-thick parasagittal slices were cut with a vibratome. These slices were kept for 45 min at 37 °C and then at 20 °C in ACSF.
Ca2+ imaging
Changes in [Ca2+]i in response to focal applications of GABA, the GABAA agonist muscimol, or glutamate (all 100 µM in ACSF) were recorded in PNs loaded with the membrane-permeant Ca2+ indicator dye acetoxymethyl ester fura-2 (fura-2 AM, Molecular Probes). The slices were loaded for 15 min at 37 °C with 15 µM fura-2 AM (from a 5 mM stock solution in DMSO with 10 % Pluronic; Molecular Probes). After wash-out of fura-2 AM, the Ca2+-dependent fluorescence was monitored with a custom-built two-photon imaging system (Denk et al. 1990) consisting of a Ti:sapphire laser system (Millenia and Tsunami, both from Spectra Physics; 780 nm excitation, < 100 fs pulse width) and a scanhead (MRC 1024, BioRad) mounted on an upright microscope (BX 50 WI, Olympus). During the experiments, 500 nM tetrodotoxin (TTX) was added to the bath solution. Fluorescence data were taken from the somatic region excluding the nucleus (see Fig. 1D and F) and are expressed as the background-corrected decrease in fluorescence divided by the prestimulus fluorescence (i.e. -
F/F). Fluorescence images (Fig. 1A-C and F) were obtained by performing a maximum projection of 4-16 images taken at different z positions.
Gramicidin perforated-patch recordings
GABAergic currents were recorded with an EPC9 patch-clamp amplifier (HEKA, Lambrecht, Germany) using the perforated-patch technique. The chloride-impermeable ionophore gramicidin (gramicidin D, Dubos, Sigma) was chosen so as not to influence [Cl-]i (Abe et al. 1994; Reichling et al. 1994). The bath solution consisted of ACSF to which 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione, 50 µM D-2-amino-5-phosphopentanoic acid and 500 nM TTX were added. The standard pipette solution into which the tip of the pipette was immersed for 5-7 s contained 150 mM KCl and 10 mM Hepes. The pipette was then back-filled with the same solution to which 25 µg ml-1 of gramicidin had been added (from a 25 mg ml-1 stock solution in DMSO). In one-third of the recordings, the fluorescent dye lucifer yellow (0.5 mg ml-1) was added to the pipette solution and standard epifluorescence imaging was used to rule out spontaneous rupturing of the patch membrane.
Throughout the experiments, the series resistance (Rs) was monitored by measuring the peak current in response to a small hyperpolarising voltage pulse. When Rs reached 100 M
, the resting potential of the cell was determined by estimating the zero-current potential. Recordings of GABAergic responses were started when Rs was lower than 25 M. GABA (10 µM in ACSF) was applied focally by brief pressure application (5-20 ms, ~70 kPa; Picospritzer, General Valve, Fairfield, NJ, USA) from a pipette with a resistance of 4-6 M that was positioned close to the soma. The resulting currents, filtered at 3 kHz, were sampled at 5 kHz. Two additive components of the liquid junction potential need to be considered during such recordings: one component between the pipette and the bath solution (while the pipette was in the bath and the amplifier was zeroed), and the second one between the pipette solution and the intracellular environment (during the actual recording). The first one was measured to be 3 mV and was subtracted offline, the second one was calculated to be less than 1 mV (assuming 140 mM [K+]i and 10 mM [Na+]i) and was neglected. Rs correction of the membrane potential was performed offline. Unless otherwise noted, all chemicals were purchased from Sigma. Data analysis was performed using Igor Pro software (Wavemetrics, Lake Oswego, OR, USA). Data are expressed as means ± S.E.M.
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RESULTS |
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GABAA receptor-mediated Ca2+ transients in immature PNs
For investigating GABA-mediated Ca2+ signalling in intact cerebellar PNs, we used the membrane-permeable fluorometric Ca2+ indicator dye fura-2 AM and two-photon imaging (Denk et al. 1990). Immediately after staining, PNs near the top surface of acute cerebellar slices from immature rats were readily detected (Fig. 1A-D). They were identified unambiguously even in the absence of the elaborate dendritic tree, their characteristic feature later in development (compare Fig. 1C and F), based on their relative position within the slice, their size and their typical shape (Altman & Bayer, 1996). The two-photon imaging approach ensured that fluorescence signals were sampled exclusively from the neurone of interest and not from surrounding cells. It should be noted that staining of PNs, even within the immature cerebellar tissue, was less effective than that of other cell types, like for example that of cerebellar granule cells (Fig. 1B).
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Figure 1. GABA-mediated Ca2+ transients in immature PNs A-C, two-photon fluorescence images of a cerebellar slice from a 5-day-old rat (P5) loaded with the Ca2+ indicator dye fura-2 AM. The images were taken at increasingly higher magnification ( | ||
Brief (100-1000 ms) applications of GABA (100 µM in the application pipette), pressure-ejected focally onto individual cells, evoked transient increases in [Ca2+]i in immature PNs (Fig. 1D). The rise time of the transients was confined to the duration of the GABA application. The transients decayed exponentially with a time constant of about 2-4 s. Repetitive stimulation produced responses that were stable in both their amplitude and their kinetics (see Fig. 2A and D) when the interval between GABA applications was at least 2 min. GABA-receptor activation reliably evoked Ca2+ transients in PNs from 3- to 6-day-old (P3-6) animals. While the amplitudes of GABA-evoked Ca2+ transients were dependent on many factors including the relative position of the tip of the application pipette and the parameters of the application pulse, most responses had an amplitude of more than ~5 % -F/F and were clearly resolved from the intrinsic noise of the fluorescence recordings (root-mean-square value < 2.5 %). Due to the spectral properties of two-photon-excited fura-2 (Xu et al. 1996), ratiometric recordings, as routinely performed with single-photon excitation, were not possible. Thus, no absolute quantification of the changes in [Ca2+]i was feasible under our recording conditions. Nevertheless, from the in vitro Ca2+ sensitivity of fura-2 (Grynkiewicz et al. 1985; Xu et al. 1996) we estimated that the largest Ca2+ transients (> 50 % -F/F, Fig. 1E) reached micromolar values and that even the 'small' responses reached peak levels of [Ca2+]i of several hundred nanomolar.
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Figure 2. Pharmacological properties of GABA-mediated Ca2+ transients A, plot of peak amplitudes of Ca2+ responses in an immature PN (P3) during successive GABA applications (100 µM). The transients were reversibly blocked by bath application of the GABAA receptor antagonist bicuculline (100 µM). The dashed line represents the mean amplitude of the responses during the control recordings. B, Ca2+ transients (averages of 3) recorded before, during and after application of bicuculline (same experiment as in A). C, bar graph summarising the effect of bicuculline on GABA-mediated Ca2+ transients (n = 5 cells, P3-6). D and E, Ca2+ transients evoked by muscimol (100 µM) were reversibly blocked by bath application of 1 mM Ni2+. Representative experiment from a P3 rat. F, bar graph summarising the effect of Ni2+ on muscimol-mediated Ca2+ transients (n = 5 cells, P3-6). | ||
Only a few postnatal days later, GABA applications no longer evoked Ca2+ transients. Figure 1F and G shows a representative experiment obtained in a cerebellar slice from a 9-day-old rat. In these experiments we used double-barrelled application pipettes to apply alternately GABA and glutamate (100 µM), the latter serving as a control for the cell's viability. While glutamate applications evoked Ca2+ transients in all cells tested, GABA invariably failed to produce detectable responses at stages later than P8 (Fig. 1G and H).
Mechanisms underlying GABA-evoked Ca2+ transients
The GABAA receptor-specific agonist muscimol (Bowery et al. 1983) was as effective in generating Ca2+ signals as GABA, with no apparent difference in the kinetics of the evoked transients (compare, for example, Fig. 2B and E). Furthermore, as shown in Fig. 2A-C, the competitive GABAA receptor-specific antagonist bicuculline reversibly suppressed GABA-evoked Ca2+ transients (average block of 92 ± 8 % of the initial amplitude; n = 5). Thus, GABA-evoked Ca2+ signalling requires the activation of GABAA receptors. We next tested whether voltage-operated Ca2+ channels (VOCCs) were activated during GABAergic stimulation and, thus, whether they underlie the observed Ca2+ transients. Figure 2D and E shows a representative experiment in which the Ca2+ channel antagonist Ni2+ (1 mM, a concentration that effectively blocks all subtypes of VOCCs in PNs) reversibly abolished muscimol-evoked Ca2+ transients. On average, Ni2+ blocked 90 ± 10 % of the GABA-evoked responses (n = 5, Fig. 2F).
Perforated-patch recordings of GABAergic currents
In the next set of experiments, we performed whole-cell patch-clamp recordings to quantify the developmental changes in GABA responsiveness. In order not to alter the [Cl-]i concentration during these recordings, we used the perforated-patch technique with gramicidin D as the ionophore (Fig. 3A; Abe et al. 1994; Reichling et al. 1994). Figure 3B illustrates the time course of the patch 'perforation'. As in conventional whole-cell recordings, perforation was accompanied by an increase in 'leak' current and an increase in the amplitude of the current transients associated with the test potential pulse. The peak amplitudes of these transients were used to determine Rs. In the experiment illustrated in Fig. 3B, Rs reached a stable value of about 20 M at 35 min after seal formation. The steady-state Rs values of the successful perforated whole-cell recordings ranged from 10 to 25 M.
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Figure 3. Perforated-patch recordings with gramicidin A, schematic diagram of the experimental arrangement; the composition of the pipette solution is given. B, amplitude of the transient current at the start of membrane hyperpolarisation (which is inversely proportional to the series resistance) as a function of time after gigaseal formation. Representative traces are shown the insets. C, brief applications of 10 µM GABA to the soma elicited current responses in all PNs studied, irrespective of age. Bars indicate the time point of GABA application. Currents represent averages of five responses recorded in a PN from a P7 rat. The currents were reversibly blocked by bicuculline (10 µM). D, bar graph summarising the effect of bicuculline on IGABA (98 ± 2 % inhibition, mean ± S.E.M., P7-8). | ||
Under these conditions, brief somatic applications of GABA (10 µM, 5-20 ms) elicited inward current responses, as in the experiment illustrated in Fig. 3C. Depending on the age of the animals and the holding potential, either inward or outward currents were obtained (see below). The GABA-mediated currents were reversibly abolished (98 ± 2 % inhibition, n = 5) by bicuculline (10 µM; Fig. 3C and D). In agreement with previous work (Sorimachi et al. 1991; Vigot & Batini, 1997), currents mediated by GABAB receptors, which would be expected to be slow outward currents, were not observed.
Developmental shift in EGABA in PNs
The voltage-dependence of the GABA-mediated responses was studied in 45 PNs from rats ranging in age from P2 to P22. Pulse-like applications of GABA to the soma elicited currents that had a similar time course at all ages tested. At a holding voltage near the resting membrane potential (-60 to -65 mV), large inward currents were observed early in the 1st postnatal week (e.g. at P3; Fig. 4A). In contrast, at stages later than the 1st postnatal week, currents at this holding voltage were outward (e.g. at P8; Fig. 4B). EGABA was determined by applying GABA to the cell while it was clamped at different holding voltages. Thus, in the examples shown in Fig. 4, EGABA was found to be -44 mV at P3 and -70 mV at P8.
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Figure 4. Developmental shift in the reversal potential of the GABA-mediated currents (EGABA) A and B, responses to 5 ms applications of GABA (10 µM) to the somata of PNs from a P3 and a P8 rat, respectively, held at the membrane potentials (mV) indicated to the left of the traces. C and D, current-voltage relationship estimated from the peak current during the responses in A and B, respectively. EGABA was determined by the x-intercept of a polynomial fit (continuous lines) of the data points. E, lower part, the values for EGABA at different postnatal ages. Each data point is from one cell. The continuous line represents a sigmoidal fit of the data. The dashed line represents the fit that was extended to P0 and P25 (see text for details). Upper part, plot of the residuals (i.e. the data points after subtraction of the fit). Note that the residuals are distributed randomly, indicating that the fit accurately describes the data. | ||
Figure 4E demonstrates that EGABA becomes progressively more negative during the course of postnatal development, shifting from a value of around -40 mV at P2-3, to values around -80 mV at P9-10 and eventually to about -85 mV after P12. These data could be well fitted with a sigmoidal function that assumes EGABA values of -33 mV at birth and -87 mV after P15. The curve had a maximal slope of -7 mV day-1 at P5.6. The plot of the residuals (i.e. data points after subtraction of the fit; Fig. 4E, upper trace) confirms that the sigmoidal function faithfully describes the data.
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DISCUSSION |
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GABA-mediated Ca2+ signalling in immature PNs
Our results are the first demonstration of GABA-mediated Ca2+ signalling in PNs and in the cerebellum in general. We identified precisely the period of the switch of GABAergic transmission in PNs from depolarising to hyperpolarising. Our findings are in contrast with the conclusions of the only two other studies investigating GABAergic transmission in immature PNs. Both Woodward et al. (1971) and Sorimachi et al. (1991) failed to obtain evidence for a depolarising action of GABA in immature cerebellar PNs. Woodward et al. (1971) performed an extensive study on synapse formation and neurotransmitter responsiveness in developing PNs in vivo. The technical difficulty of those experiments may have precluded the detection of the depolarising action of GABA. Sorimachi et al. (1991) studied PNs from rats at P5-11 and did not find evidence for GABA-mediated Ca2+ transients. However, since these authors did not state the specific age of the rats used for these recordings, it remains unclear whether our results are in conflict with theirs. We presume that they focused on a more mature age group.
Our conclusions concerning the depolarising action of GABA in immature PNs are based on two independent sets of experiments. These experiments involved Ca2+ imaging in fura-2 AM-loaded PNs and perforated-patch recordings. Both approaches delivered robust and highly reproducible results. The variability of the EGABA values during the transition period (around P6) probably resulted from the differences in the stage of maturation of individual PNs. In no case was GABA-mediated Ca2+ signalling detected in PNs from rats older than P8/9, that is at a stage at which PNs have a more elaborate dendritic tree (see for example Fig. 1).
Our results indicate that PNs are members of a larger family of neurones that exhibit GABA-mediated depolarisation during early postnatal stages of life (for reviews see Cherubini et al. 1991 and Leinekugel et al. 1999). The functional role(s) of the depolarising action is (are), however, unclear. One of the obstacles for testing earlier hypotheses, including the role of GABA in dendritic growth and synaptic wiring, results from the difficulty of monitoring activity in defined types of neurone. This is a challenging task even in a layered structure like the hippocampus, due to the lack of clear-cut morphological hallmarks for most immature neurones. Our study overcomes these problems by identifying PNs as a cellular model system, which allows the establishment of an unusually good correlation between changes in morphology, as well as changes in the synaptic connectivity, with the rather sharply timed changes in GABA responsiveness.
Mechanisms underlying GABA-mediated Ca2+ signalling
Since GABAA receptor channels (GABAA-Rs) are permeable to both HCO3- and Cl- (Bormann et al. 1987), several mechanisms could potentially underlie the depolarising GABA responses in PNs. Thus, a strengthening of HCO3- efflux through GABAA-Rs would shift EGABA to more positive potentials. This could be accomplished either by a change in the HCO3- permeability of GABAA-Rs (Perkins & Wong, 1996) or by an increase in [HCO3-]i (Sun et al. 1999). A more likely mechanism, however, is an elevated [Cl-]i in immature PNs, which would lead to a reduction in Cl- influx through GABAA-Rs and, thus, to a more positive EGABA. Thus, various immature neurones that lack a Cl- extrusion mechanism (Zhang et al. 1991; Rivera et al. 1999) or even actively accumulate Cl- (Rohrbough & Spitzer, 1996; Kakazu et al. 1999) respond with a depolarisation to GABAergic stimulation. Indeed, the developmental upregulation of the cerebellar K+-Cl- cotransporter (Lu et al. 1999) would be compatible with our findings. Undoubtedly, more detailed studies on the molecular mechanisms controlling Cl- and HCO3- homeostasis in PNs are necessary. However, from our data it seems safe to conclude that as in other brain regions (Yuste & Katz, 1991; Lin et al. 1994; Reichling et al. 1994; Owens et al. 1996; Kulik et al. 2000), GABA-mediated Ca2+ signalling is entirely caused by GABAA receptor-evoked depolarisation and the subsequent activation of VOCCs. Preliminary observations suggest that GABA-mediated Ca2+ signalling is produced through both the subthreshold activation of T-type channels and action potential-evoked activation of P-type Ca2+ channels (A. Konnerth, unpublished observations).
Functional implications and perspectives
The time window during which GABA is depolarising overlaps with a critical period in the development of PNs. Between P1 and P4, PNs merge from two to three irregular rows to a single layer (Ito, 1984) and form at their final destination first GABAergic and then glutamatergic synapses. This is the beginning of a developmental period lasting from P3 until P5/6, during which PNs undergo a dramatic change in their morphology (see Fig. 1) and synaptic connectivity (Altman & Bayer, 1996). In this period, PNs switch from their multipolar dendritic phenotype into the mature form, consisting of a dendritic tree that is usually connected to the PN cell body through a single dendrite. The change in dendritic morphology is paralleled by a switch in the number of afferent CF axons (Mariani & Changeux, 1981). This process of synaptic reorganisation involves a Ca2+-dependent long-term synaptic potentiation of the 'winner' CF input (H. Takechi, J. Eilers & A. Konnerth, unpublished observations). We propose that GABA-mediated synaptic Ca2+ signalling, which is present during the period of most intensive reorganisation, contributes to the activity-dependent maturation of the excitatory CF synaptic system. Another mechanism through which GABA-mediated Ca2+ signalling may control the wiring of the immature cerebellum is a homosynaptic potentiation of the GABAergic synapses themselves. Thus, at GABAergic inputs to PNs, GABA-mediated increases in [Ca2+]i could lead to a self-reinforcement via the previously described calcium-dependent process of rebound potentiation (Kano et al. 1992). Whatever the detailed roles of the depolarising GABA action in PNs are, the narrow time window and rapid shift towards the mature hyperpolarising response make it an attractive model for neuronal development and synaptic physiology. The quantification of [Cl-]i as well as the identification of Cl- transporters will be important next steps in understanding the physiology of the GABAergic system in the immature cerebellum.
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Acknowledgements
This work was supported by grants from the Deutsche Forschungsgemeinschaft to J.E. and A.K.
Corresponding author
J. Eilers: Max-Planck-Institut für Hirnforschung, Abteilung Neurophysiologie, 60528 Frankfurt, Germany.
Email: eilers{at}mpih-frankfurt.mpg.de
Author's present address
T. D. Plant: Institut für Pharmakologie, Freie Universität Berlin, 14195 Berlin, Germany.
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