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J Physiol (2003), 547.2, pp. 497-507
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.033415
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ABSTRACT |
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Tottering, a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q-type) Ca2+ channels, the dominant exocytotic Ca2+ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild-type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca2+ channels (N-type), in contrast to the P/Q-channels that dominate release from wild-type terminals. Since N-channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q-channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein-coupled receptors than in their wild-type counterparts. GABAB receptors and2-adrenergic receptors (activated by bath application of transmitters) produced a three- to fivefold more potent inhibition of transmission in tottering than in wild-type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo, since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABAB receptor-mediated presynaptic inhibition in tottering as compared to wild-type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.
(Resubmitted 27 september 2002; accepted after revision 10 December 2002; first published online 24 January 2003)
Corresponding author K. Dunlap: Department of Neuroscience, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. Email: kathleen.dunlap{at}tufts.edu
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INTRODUCTION |
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Epilepsy is a term used to describe neurological disorders characterised by abnormal synchronised electrical activity in the brain. A condition that was recognised in antiquity, it has become clear that epilepsy has many molecular aetiologies. A number of mouse lines harbouring spontaneous mutations exhibit epileptiform activity, and one such line, tottering, is an excellent model for human spinocerebellar ataxia and absence epilepsy (Buchhalter, 1993; Burgess & Noebels, 1999). Tottering is characterised by its abnormal gait, frequent episodes of fixed-gaze arrest, and intermittent focal myoclonic seizures, and is accompanied by the electroencephalographic hallmarks of absence epilepsy (Green & Sidman, 1962).
In an effort to identify the genetic basis of tottering, Fletcher et al. (1996) used positional cloning strategies to isolate and identify the mutant allele of tottering, CACN2, the gene encoding the class A (P/Q) voltage-dependent Ca2+ channel. This gene contains a point mutation (a cytosine to thymidine switch at position 1802) that results in a non-conservative substitution of leucine for proline in the second transmembrane repeat of the protein (Fletcher et al. 1996). Since it has been predicted that the mutation lies near the outer mouth of the ion-conducting pore, it was expected to significantly alter channel function. Surprisingly, when tottering P/Q currents were compared to their wild-type counterparts in cerebellar Purkinje neurone somata, few and subtle biophysical differences were observed. A 40 % reduction in current density in tottering Purkinje cell somata was reported, however (Wakamori et al. 1998). The relationship between these changes and the aberrant motor behaviour in tottering mutants remains obscure.
As the P/Q-channel is the dominant exocytotic Ca2+ channel in the vertebrate central nervous system (Dunlap et al. 1995), the tottering phenotype may be related to alterations in synaptic physiology. Reported differences between mutant and wild-type animals are few, however (Helekar & Noebels, 1991, 1994; Caddick et al. 1999; Qian & Noebels, 2000), and lead to speculation that synaptic networks in the tottering mutant may exhibit a subtle shift in the overall balance of excitation and inhibition. Accordingly, we have begun to explore the mechanisms underlying transmitter release and synaptic transmission in tottering and wild-type animals using acute brain slices from the cerebellum. Specifically, we have focussed on the excitatory synapse between parallel fibres (PFs) and Purkinje cells (PCs) to determine whether the transmitter-induced modulation of PF-PC transmission is altered in tottering.
We have chosen this synapse for study because: (1) the synaptic circuitry is well defined; (2) the efficacy of transmission at this synapse is known to be controlled by presynaptic G protein-coupled receptors (Dittman & Regehr, 1996); and (3) the proliferation of adrenergic innervation from the locus coeruleus, a region of known modulatory function, appears to be correlated with (and exacerbates) the tottering phenotype (Levitt & Noebels, 1981; Noebels, 1984).
Our results demonstrate that cerebellar glutamate release and synaptic transmission between PFs and PCs are dominated by N-type Ca2+ channels in tottering (as opposed to P/Q-channels in wild-type), and that these synapses are more susceptible to inhibitory modulation by G protein-coupled receptors than are their wild-type counterparts. This increased susceptibility prolongs the natural inhibition of PF transmission produced via GABAergic interneurones in the cerebellum and implies that the complement of Ca2+ channels in PF terminals is an important factor in controlling the efficacy of transmission through the excitatory PF-PC synapse. The reduced excitation at this key synapse predicts a reduction in PC output from the cerebellum and may contribute to altered electrical excitability in tottering.
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METHODS |
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All experiments conform to the guidelines established by the National Institutes of Health and by the Institutional Animal Care and Use Committee at Tufts University School of Medicine.
Genotyping animals
Pairs of mice (bred on a C57BL/6 background) heterozygous at the tottering locus were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and bred on-site at the Tufts University School of Medicine animal facility. The offspring were genotyped using PCR amplification of specific alleles (Sommer et al. 1992), which employs allele-specific upstream primers with the 3' base ending at nucleotide T1802 of the coding sequence (the mutated base in tottering). The sequence of wild-type-specific forward primer is 5'-TTTGATGAAGGGACTCT-3', and the sequence of tottering-type-specific forward primer is 5'-TTTGATGAAGGGACTCC-3'. The specific primers were each paired with a downstream primer (5'-CAAAATGACGGTGTTTCAGGTACAG-3') common to both alleles and included in a PCR using tail DNA as template. DNA was extracted from tail snips using standard methods of high salt followed by ethanol precipitation. Amplification was carried out in an Eppendorf MasterGradient thermocycler under the following conditions: template 50 ng, primers 1 µM, Mg2+ 1.5 mM, dNTP 0.8 µM. Products were separated electrophoretically on 3 % agarose gels.
Synaptosomal [3H]glutamate release assay
Adult mice (15-30 g) were killed by decapitation and cerebella and/or cortices were removed and homogenised in 0.32 M sucrose, 1 mM K3-EDTA, pH 7.4 in a 2 ml Wheaton glass-Teflon homogeniser. Synaptosomes were prepared using discontinuous Percoll (Pharmacia) gradients (Dunkley et al. 1986). The final pellet (~1 mg protein) was resuspended in basal buffer ((mM): 145 NaCl, 2.7 KCl, 2.4 MgCl2, 10 glucose, 10 Hepes-Tris at pH 7.4) that contained 0.2 mg ml-1 bovine serum albumin. The suspension was adjusted to 2.5 mg ml-1 protein, divided into 20 µl portions, and each portion was combined with 20 µl of concentrated Ca2+ channel toxin solution and incubated on ice for 45 min prior to the beginning of the release experiment, allowing for toxin binding to approach equilibrium (Turner et al. 1993).
To initiate the loading reaction, each 40 µl portion of the synaptosome-toxin suspension was combined with [3H]glutamate (56.0 Ci mM-1 (where 1 Ci = 3.7 
1010 Bq), Amersham International; 15 µCi in a volume of 10 µl) that had been prepared by evaporation of the aqueous stock solution (2 % ethanol) under a stream of N2, and resuspended in basal buffer. The final concentration of exogenous glutamate was 5.4 µM, and the total amount of radioactivity transported was ~106 c.p.m. per 50 µg of synaptosomal protein. Metabolic labelling proceeded for 12 min, at which time the reaction was stopped by adding 800 µl of basal buffer. Synaptosomes were then immobilised on a filter sandwich composed of cellulose ester and glass fibre filters and transferred to a superfusion device, as described previously (Turner et al. 1989).
Release was measured by superfusing stimulating solutions across the synaptosomes and the effluent was collected on a circular fraction collector that had been modified from a phonograph turntable (Forbush, 1984; Turner et al. 1989). Synaptosomes were depolarised with a stimulus buffer containing an elevated concentration of KCl ((mM): 87.7 NaCl, 60 KCl, 0.46 CaCl2, 1.94 MgCl2, 10 glucose, 10 Hepes-Tris at pH 7.4) and collecting the effluent stream in 35 ms fractions. Radioactivity in each fraction and the amount remaining on the filter at the end of the experiment were determined by adding 1.5 ml liquid scintillation cocktail (BioSafe II, Research Products, Mt Prospect, IL, USA) and counting the samples in a Beckman LS7000 scintillation spectrometer.
Ca2+-dependent release was calculated by measuring release in the presence of Ca2+ and subtracting release evoked with a stimulus buffer that contained no added Ca2+ (substituted with Mg2+, free [Ca2+] ~3 µM). Data were analysed using Microsoft Excel. Results are expressed as the ratio of the level of radioactivity (c.p.m.) in each fraction to the total radioactivity remaining on the filter ( 100 %). Peak release rates were calculated by summing the Ca2+-dependent release over a 70 ms period immediately after switching solutions, and are expressed as a percentage of the total [3H]glutamate content per second (% s-1). Data reported are the average of at least three separate experiments performed on different days with freshly prepared synaptosomes. Standard deviations (error bars omitted from kinetic plots for clarity) were generally less than 10 %. 
-Agatoxin IVA was purchased from Peptides International (Louisville, KY, USA), and -conotoxin GVIA was purchased from Bachem (Torrence, CA, USA). Other reagents were obtained from Fluka (Sigma-Aldrich, St Louis, MO, USA).
Slice preparation
The cerebellar vermis dissected from mice (6-54 weeks of age) was glued to the cutting block of a Lancer 1000 vibratome and 300 µm transverse slices were cut in a bath of ice-cold saline ((mM): 120 NaCl , 3.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose). Slices were then incubated at room temperature for at least 1 h in saline saturated with 95 % O2-5 % CO2 before being transferred to a recording chamber on the stage of a Zeiss ACM compound microscope. The chamber was continuously perfused with oxygenated saline at a rate of 2 ml min-1. Drugs were applied to the preparation through the bath solution and exchange of solutions in the bath was achieved with a time constant of ~70 s. Slices were maintained at room temperature for the duration of the experiment.
Electrophysiological recording and data analysis
Extracellular recording was employed to monitor PF-induced synaptic activity in PCs. PFs were stimulated with an extracellular, glass electrode (tip diameter 3-5 µm) filled with normal bath saline, pH 7.4. Rectangular stimuli (150 µs) were delivered via a Grass Instruments S-88 stimulator. Field recordings were carried out with blunt-tipped glass pipettes (3-5 µm in diameter) filled with normal saline (1-3 M
resistance).
Heterosynaptic depression was evaluated with field recording and stimulation methods using two electrodes to stimulate PFs in the molecular layer. One electrode was used to deliver test pulses to PFs (and field EPSPs (fEPSPs) recorded in the PC layer), and the other was used to deliver a 10-pulse, 100 Hz tetanus to a non-overlapping PF pathway (to stimulate the release of GABA from interneurones in the region from which the recording was made; Dittman & Regehr (1997)). To assure that the two electrodes stimulated distinct sets of PFs, we placed the electrodes in a such way that stimulation of one pathway did not produce any detectable paired-pulse facilitation in the other pathway. To allow comparison between different slices, we placed the test electrode exactly 500 µm from one side of the recording electrode and moved the tetanus electrode on the opposite side of the recording electrode to a position that maximised depression without noticeable paired-pulse facilitation (Dittman & Regehr, 1997).
Bicuculline (30 µM) was always added to bath saline to eliminate inhibitory synaptic transmission through GABAA receptors. Potentials were measured using a Biodyne AM-2 amplifier (Santa Monica, CA, USA). The records were filtered at 3 kHz with a Frequency Devices eight-pole Bessel filter, digitised using an ITC-16 interface (Instrutech, Great Neck, NY, USA) under the control of Pulse software (HEKA Electronik), and analysed with PulseFit (HEKA Electronik), IgorPro (WaveMetrics), Excel (Microsoft) or R statistical computing environment (R-project under GNU General Public License). Results are presented as the arithmetic mean ± S.E.M. Differences between group means were evaluated by Student's t test. Differences between concentration-response curves were determined by F statistics on separate sum-of-squares and combined sum-of-squares resulting from curve fitting.
Immunoblot analysis of GABAB receptor
Cerebellar tissue obtained from wild-type or tottering mice was homogenised and solubilised in detergent (1 % Triton X100, 0.5 % Lubrol). Protein samples (10, 40 and 100 µg) were separated electrophoretically on SDS-polyacrylamide gels, transferred to nitrocellulose membrane and GABAB immunoreactivity assayed with an anti-peptide antiserum directed against the N-terminus of GBR1a (the primary GABAB receptor subunit in brain; Kaupmann et al. 1997). Binding of the primary antiserum was detected using HRP-conjugated goat anti-mouse IgG followed by electrochemiluminescence-mediated visualisation with photographic film.
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RESULTS |
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Glutamate release from nerve terminals
On the basis of conflicting reports that somatic Ca2+ channel density is either unchanged (Lorenzon et al. 1998) or diminished (Wakamori et al. 1998; Qian & Noebels, 2000) in tottering neurones, we sought to determine whether any changes in relative channel density occurred at the glutamatergic nerve terminals in the tottering brain. In order to evaluate the relative contributions of P/Q- and N-channels to excitation-secretion coupling, we prepared synaptosomes from homozygous tottering, heterozygous, or wild-type animals to assess the sensitivity of glutamate release to toxins that selectively block P/Q- (-agatoxin IVA) or N- (-conotoxin GVIA) channels.
Depolarisation of wild-type synaptosomes with saline containing 60 mM KCl evoked a rapid, transient, Ca2+-dependent release of [3H]glutamate, reaching a peak release rate of ~1.61 % s-1 (Fig. 1A). Similar maximal rates of [3H]glutamate release were also observed for synaptosomes from heterozygous (1.33 % s-1) and homozygous tottering (1.32 % s-1) animals, suggesting that the mutation has no fundamental effect on the synaptic vesicle fusion apparatus. Incubation of synaptosomes in saturating concentrations of -agatoxin IVA (300 nM) or -conotoxin GVIA (100 nM) significantly inhibited [3H]glutamate release. P/Q-channels predominate at glutamatergic terminals, with -agatoxin IVA producing an average 49 % inhibition of Ca2+-dependent [3H]glutamate release. The N-channel antagonist, -conotoxin GVIA, inhibited [3H]glutamate release by 32 % on average. In contrast to these results from wild-type synaptosomes, [3H]glutamate release from tottering synaptosomes was found to be controlled more by Ca2+ influx through N-channels than through P/Q-channels (Fig. 1B); -conotoxin GVIA blocked 53 % of release, whereas -agatoxin IVA blocked only 13 % in tottering synaptosomes. Synaptosomes from heterozygous animals showed an intermediate biochemical phenotype, with -conotoxin GVIA and -agatoxin IVA blocking approximately equal portions of [3H]glutamate release (33 and 23 %, respectively, data not shown).
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Figure 1. The dominant exocytotic Ca2+ channel is altered in tottering terminals [3H]glutamate release rates from synaptosomes isolated from +/+ (wild-type, A) or tg/tg (tottering mutant, B) cerebellum, evoked with 60 mM K+ with or without 0.46 mM Ca2+ (at t = 0) and plotted as a percentage of the total [3H]glutamate in each sample. Synaptosomes were preincubated for 45 min (on ice) in 2.7 mM K+ and nominal Ca2+ in the presence or absence of saturating concentrations of Ca2+ channel toxins: control ( | ||
These pharmacological results suggest a significant reduction in the involvement of P/Q-channels in controlling glutamate release in the tottering cerebellum. This conclusion is only valid, however, if the toxins are used at saturating concentrations (i.e. if the potency of -agatoxin IVA is unaltered by the tottering mutation). To address this issue, a concentration-inhibition relationship was explored for the toxin in synaptosomes prepared from wild-type and tottering animals. The two data sets were superimposable (Fig. 1C), with apparent Ki values for -agatoxin IVA of 8.4 nM, consistent with our previous results (Turner & Dunlap, 1995). Taken together, these data support the idea that the complement of exocytotic Ca2+ channels is altered in glutamatergic terminals in tottering mutants, consistent with that reported for the hippocampus (Qian & Noebels, 2000) and forebrain (Leenders et al. 2002).
Glutamatergic synaptic transmission between PFs and PCs in the cerebellum
In order to evaluate further the significance of the increased reliance of glutamatergic nerve terminals on N-type channels, we used acute brain slices to investigate synaptic function in a more intact preparation. We chose to study the PF-PC synapse of the cerebellum; this synapse has been characterised in great detail and is representative of many excitatory synapses in the CNS. Transverse tissue slices (300 µm) were cut from the cerebellar vermis of adult wild-type or tottering mice. PFs were stimulated extracellularly with a blunt glass electrode (~3 µm tip diameter) filled with normal bath solution, and excitatory, glutamatergic synaptic activity was measured using extracellular field recording via a saline-filled glass electrode placed in the PC layer (Fig. 2).
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Figure 2. The presynaptic release mechanism is unaltered in tottering parallel fibre (PF)-Purkinje cell (PC) synapses A, superimposed extracellular field recordings from the PC layer of a cerebellar slice, demonstrating the excitatory glutamatergic synaptic response (field EPSP, fEPSP) under control conditions or following bath application of the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione at 10 µM (*). B, scatter plot showing the relationship between the amplitude of the fEPSP and the stimulus amplitude for wild-type ( | ||
To characterise synaptic function at this synapse, we measured synaptic responses evoked by single stimuli and found that the fEPSP amplitudes in wild-type and tottering slices were indistinguishable (Fig. 2), suggesting that basic presynaptic release mechanisms are unchanged by the tottering mutation. To evaluate this further, paired-pulse stimulation (Fig. 3) was used to measure the degree of presynaptic facilitation, a phenomenon that is dependent upon residual Ca2+ concentration in the terminal (Zucker, 1989; Atluri & Regehr, 1996). The degree of paired-pulse facilitation (measured as the ratio of the second to the first synaptic potential) was identical in tottering and wild-type slices (Fig. 3). Taken together, these results confirm the biochemical results shown in Fig. 1, that the primary presynaptic release mechanisms in PFs are little changed by the tottering mutation.
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Figure 3. The paired-pulse facilitation of PF-PC transmission is unaltered in tottering mutants The upper panel shows field recordings of synaptic responses from the PC layer of cerebellar slices (+/+ and tg/tg, as marked) evoked by two extracellular stimuli (20 V, 150 µs, delivered at a 20 ms interval). The stimulus artefact and PF volley have been blanked out for clarity. The lower panel is a histogram of the facilitation ratio (the ratio of the second synaptic response divided by the first) plotted for field recordings from wild-type (white bars) or tottering (grey bars) slices. Ratios were calculated using measurements of amplitude or slope (as marked). Data are plotted as the mean ± S.E.M. of the number of slices noted in each bar. The data sets were not different (P > 0.05). | ||
Given that the biochemical measurements showed N-channels dominating the release of [3H]glutamate in tottering synaptosomes, we investigated whether PF-PC transmission also relies more heavily on N-channels. PFs were stimulated and fEPSPs recorded before and during bath application of 100 nM -conotoxin GVIA for 20 min (sufficient for saturation blockade of N-channels in the nerve terminals; Luebke et al. 1993; Takahashi & Momiyama, 1993; Mintz et al. 1995). In three wild-type slices tested, the toxin blocked an average 20 % of transmission, whereas in tottering slices the toxin was much more efficacious, blocking transmission by about 60 % (n = 3, Fig. 4; P < 0.01). Thus, results from electrophysiological and biochemical assays each indicate that while the complement of Ca2+ channels in PF nerve terminals is altered in tottering (N substituting for P/Q), little change is produced in total Ca2+-dependent exocytosis evoked by either low-frequency stimulation of PFs or elevated KCl. These results are consistent with the studies of Qian & Noebels (2000), which demonstrated no alterations in excitatory transmission between CA3 and CA1 neurones in the hippocampus. The results of Matsushita et al. (2002), however, reported a decrease in PF-PC transmission in both tottering and rolling mice, suggesting variation in transmission as a function of strain and age.
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Figure 4. Tottering synapses are dominated by N-type Ca2+ channels The time course for | ||
Modulation of excitatory transmission between PFs and PCs
Given that synaptic efficacy is regulated in part by G protein-coupled presynaptic receptors that target exocytotic Ca2+ channels, we explored whether alterations in presynaptic inhibition accompany the tottering mutation. We hypothesised that the increased reliance of tottering PF terminals on N-channels for exocytosis might render those terminals more susceptible to inhibition, because N-channels are generally known to be more effectively inhibited by G proteins (Zhang et al. 1996; Currie & Fox, 1997; Roche & Triestman, 1998). A number of transmitters (including GABA and adenosine) have been reported to effectively reduce transmission at PF-PC synapses by activating metabotropic receptors in nerve terminals and inhibiting Ca2+ influx through voltage-dependent channels (Dittman & Regehr, 1996). Thus, we tested our hypothesis by measuring the ability of the presynaptic GABAB receptor agonist baclofen to inhibit transmission at the PF-PC synapse.
We focussed on baclofen because: (1) GABA release from stellate interneurones in the molecular layer of the cerebellum has been shown to inhibit PF-PC transmission via presynaptic GABAB receptors (Mody et al. 1994; Dittman & Regehr, 1996, 1997) and (2) baclofen-induced inhibition is mediated largely through an action on terminal Ca2+ channels (Dittman & Regehr, 1996). Glutamate release was evoked by PF stimulation and fEPSPs monitored during bath application of baclofen. Concentration was varied from 30 nM to 10 µM, and recovery was monitored by washing the drug out after each application. In wild-type slices, baclofen inhibited transmission with an IC50 of 1.71 µM (n = 16). In tottering slices, baclofen was ~threefold more potent than in wild-type slices (IC50 of 0.58 µM, n = 8; significant at P < 0.0001; Fig. 5A).
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Figure 5. Tottering synapses are more susceptible to presynaptic inhibition by GABAB receptors Concentration-response relationships for baclofen-induced inhibition of PF-PC transmission. In all cases, the smooth curves represent best fits to a Hill equation, and the vertical dashed lines indicate concentrations at half-maximal inhibition by baclofen. A, data taken from 16 wild-type ( | ||
To further explore whether the increase in the apparent potency of baclofen was a function of the increased involvement of N-type channels, we treated wild-type slices with selective Ca2+ channel toxins to study GABAB receptor action at synapses where Ca2+ influx through N or P/Q-channels was pharmacologically eliminated. To reduce the amount of toxin required for these experiments, slices were first incubated in saturating concentrations of toxin prior to the synaptic recordings, in a chamber of small volume. Incubations were carried out for sufficient time to approach equilibrium for toxin binding; 20 min in 100 nM -conotoxin GVIA or 500 nM -agatoxin IVA. Since these toxins are quasi-irreversible (Luebke et al. 1993), a complete baclofen concentration-response relationship could be established for each of the unblocked Ca2+ channel types.
When N-channels were inhibited by -conotoxin GVIA, the amplitude of the fEPSP in wild-type slices was decreased by 15-20 % (Fig. 5B). Under these conditions, the IC50 for baclofen-induced inhibition was < 2 µM (n = 4), close to that observed with wild-type synapses containing their normal complement of Ca2+ channels (Fig. 5B). By contrast, when P/Q-channels were blocked using -agatoxin IVA, transmission was reduced to 40 % of control (not shown). In order to make reliable measurements of presynaptic modulation in the face of the reduced EPSP amplitude, we increased the extracellular Ca2+ concentration (to 4 mM) to compensate for the loss of Ca2+ influx through P/Q-channels. When PF-PC transmission was under the exclusive control of N-channels, the IC50 for baclofen was decreased to 0.77 µM (n = 3, P < 0.005), which is comparable to that observed for tottering mutants (Fig. 5B).
These results imply that the P/Q- to N-channel shift in nerve terminals is sufficient to account for the increased susceptibility of the PF-PC synapse in tottering mutants to modulation by GABAB receptors. To explore this further, we performed a parallel experiment in slices from tottering animals (Fig. 5C). N-channels were first blocked with -conotoxin GVIA and extracellular Ca2+ was raised to 4 mM to bring P/Q-channel-mediated transmission to near-control levels. Under these conditions, the concentration-response relationship was right-shifted (IC50 1.1 µM) as compared with that for tottering slices not exposed to toxin (IC50 0.58 µM). With blockade of P/Q-channels (and a concomitant increase in extracellular Ca2+ to bring the fEPSP to near-control levels), the IC50 was even lower (0.47 µM) than that of untreated tottering slices. These results demonstrate that N-channel-dominated synapses are more potently modulated by baclofen than are P/Q-channel-dominated synapses.
Alterations in modulation are not due to changes in GABAB receptor density
Several mechanisms could account for the increased potency of baclofen at tottering PF-PC synapses. Since the apparent potency of baclofen is directly related to the GABAB receptor density, it was possible that the data shown in Fig. 5 could result from upregulation of GABAB receptors in PF terminals in tottering tissue. In order to test this possibility, we carried out immunblot analysis of cerebellar protein from wild-type and tottering animals using an antiserum specific for the primary GABAB receptor subunit in brain, GBR1a (Fig. 6). Given that granule cells contribute the vast majority of protein to such samples (they represent almost 50 % of all neurones in the CNS, Ghez & Thach, 2000), such immunoblot analysis of cerebellar protein provides a reasonable estimate of GABAB receptor density in these cells. As expected from published studies (Kaupmann et al. 1997; White et al. 1998), there were two prominent immunoreactive bands, migrating at ~140 and ~100 kDa. These represent the glycosylated and non-glycosylated forms of GBR1a, respectively (Kaupmann et al. 1997). Densitometric analysis indicated no difference in the amount of GABAB receptor immunoreactivity between tottering and control membranes, suggesting that increased receptor density is unlikely to account for the change in the apparent potency of baclofen in tottering.
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Figure 6. GABAB receptor density does not change in tottering mutants Immunoblot of protein homogenates made from wild-type (+/+) and tottering (tg/tg) cerebella, probed with an antiserum specific for the primary GABAB receptor subunit in brain, GBR1a. Lanes were loaded with either 100 or 40 µg protein (as marked). The arrowheads at the right indicate the molecular weights of GBR1a in its non-glycosylated (lower) and glycosylated (upper) forms (as in Kaupmann et al. 1997). | ||
Heterosynaptic depression is prolonged in tottering animals
The observation that the potency of baclofen is increased in tottering mutants prompted us to investigate whether modulation of tottering synapses by endogenous GABAergic pathways in the cerebellum was also strengthened. In order to assess endogenous modulation of the PF-PC synapse by GABA, we used a protocol to elicit heterosynaptic depression (Dittman & Regehr, 1997). In this protocol, tetanic stimulation from one electrode elicits GABA release from interneurones in the molecular layer; GABA, in turn, stimulates GABAB receptors on PF terminals and inhibits glutamate release (measured by recording synaptic potentials in the PC layer; Fig. 7A). In control slices, the tetanus decreased PF-PC synaptic strength by ~25 %, and the inhibition lasted ~100 ms (Fig. 7B, left). In tottering slices, maximal inhibition was comparable to wild-type (~30 %), but it was much longer lasting, with some inhibition remaining even 1 s after the tetanus (Fig. 7B, right). This inhibition was mediated by GABAB receptors, since it was blocked by the receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid 35348, bath applied at 100 µM (Fig. 7B). These results are consistent with an enhanced susceptibility to presynaptic modulation of Ca2+ channels in PF terminals of tottering animals.
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Figure 7. Heterosynaptic depression is enhanced in tottering A, stimulation protocol: two extracellular stimulus electrodes were placed in the molecular layer. Single stimuli applied to S1 were separated by a tetanus of 10 pulses at 100 Hz applied to electrode S2, delivered at a variable interval ( | ||
Adrenergic modulation
Other studies have suggested that the locus coeruleus plays a central role in the tottering phenotype through increased noradrenergic innervation in several brain areas, including the cerebellum (Levitt & Noebels, 1981; Noebels, 1984). Thus, we sought to determine whether transmission at PF-PC synapses was also modulated by noradrenaline (NA) and whether such effects were altered in tottering. As with the baclofen experiments described above, NA was bath applied in concentrations ranging from 0.1 to 30 µM, and recovery was monitored during washout after each application. NA inhibited PF-PC transmission with an IC50 of 1.0 µM (n = 5) and a maximal efficacy of 18.6 % at wild-type synapses; NA was approximately fivefold more potent at tottering synapses (IC50 of 220 nM, n = 4, P < 0.001; Fig. 8A), similar to results with baclofen. The efficacy of NA was also increased (~1.5-fold, P < 0.05) at tottering synapses (producing a maximal 28.5 % inhibition). Bath application of the selective 2-adrenergic receptor antagonist yohimbine (10 µM) blocked the inhibitory action of NA (P < 0.001), whereas prazosin (10 µM, an 1-adrenergic receptor antagonist) or propranolol (10 µM, a non-selective 
-adrenergic receptor antagonist) had no measurable effects (Fig. 8B).
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Figure 8. Tottering synapses are more susceptible to inhibition by A, concentration-response relationship for noradrenaline-induced inhibition of PF-PC transmission, measured in wild-type ( | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our results demonstrate a significant functional consequence of the tottering mutation for synaptic transmission at the PF-PC synapse in the cerebellum. This excitatory synapse in tottering mutants was significantly more susceptible to inhibition by GABA and NA, an effect mediated by G protein-coupled GABAB and 2-adrenergic receptors. Such fundamental changes in the regulatory properties of this excitatory synapse in the cerebellum are likely to have significant implications for the dynamics of synaptic strength.
In contrast to the results from the PF-PC synapse reported here (and in Matsushita et al. 2002), electrophysiological experiments in the tottering hippocampus have identified few differences in glutamatergic transmission. As in PF terminals, hippocampal CA3 terminals in tottering demonstrate a similar switch in N-channel complement (Qian & Noebels, 2000), yet no alterations in GABAB or adenosine receptor-mediated inhibition of CA3-CA1 transmission were reported. Similarly, no changes in the effects of GABAB receptor agonists and antagonists were reported for glutamatergic transmission in the lethargic thalamus (Caddick et al. 1999), another Ca2+ channel-mutant mouse with a behavioural phenotype similar to that of tottering. In the two latter studies, however, single saturating concentrations of receptor agonists were employed and the potencies of these drugs were not evaluated.
Our working hypothesis for the increased apparent potency of baclofen in tottering is based on the established direct competition between G protein 
subunits and neuronal Ca2+ channel subunits for the AID domain of the pore-forming Ca2+ channel subunit (DeWaard et al. 1997; Zamponi et al. 1997). If the affinity of the Ca2+ channel subunit for N-type channels is low (or dissociation rate is high) relative to P/Q-type channels, then for a given G concentration, the fraction of N-channels that will be inhibited is higher. Therefore, if most or all parallel fibres express GABAB receptors in sufficient numbers to completely inhibit exocytosis, our hypothesis predicts that the apparent potency of GABAB agonists would be increased without changing efficacy. This prediction was confirmed by the data shown in Fig. 5. Our results indicate that synaptic transmission would be more sensitive to presynaptic receptor occupancy. It is important to explore whether the use of lower drug concentrations will reveal an increased susceptibility of hippocampal and thalamic transmission in tottering or whether this effect is unique to the cerebellum.
Further exploration of NA as a regulator of synaptic transmission in the tottering brain is also warranted. Although our results demonstrate that 2-adrenergic receptors underlie the inhibitory action of bath-applied NA on PF-PC transmission, the synaptic target of NA action remains to be determined. Given the many examples of presynaptic inhibitory regulation via 2-adrenoceptors in both the central and peripheral nervous systems (Hein et al. 1999), it is reasonable to propose that PF terminals are likely to be one site of action for NA in the cerebellum. Furthermore, 2-adrenoceptors, like GABAB receptors, commonly couple to pathways that inhibit voltage-dependent Ca2+ channels (Hille, 1994; Dunlap & Ikeda, 1998) suggesting a possible mechanism for NA-induced inhibition of exocytosis from PF terminals. This idea can be tested by Ca2+-sensitive dye recordings to directly measure Ca2+ influx in PF terminals.
There are several mechanisms that could underlie the observed increase in potency of presynaptic receptor agonists. One possibility that was investigated was that an increase in the density of presynaptic -adrenergic or GABAB receptors would produce an increase in the apparent potency of an appropriate agonist, a reasonable scenario in light of the observation that there is a proliferation of adrenergic fibres in the tottering brain. However, immunoblot analysis of cerebellar tissue indicated that the amount of GABAB receptor protein was identical in tottering and wild-type littermates. While it is possible that changes in the coupling between presynaptic receptors and their target effectors is altered in tottering, the absence of detectable changes in receptor level argues against a scenario in which an increase in receptor number underlies the increase in apparent potency. Likewise, the observed increase in the efficacy of adrenoceptor agonists in tottering is consistent with the enhanced modulation conferred by the switch in N-channel phenotype. An increase in agonist efficacy in tottering suggests that -adrenoceptor density is low relative to the GABAB receptor, accounting for the incomplete inhibition of transmission. Alternatively, -adrenoceptors may be expressed on a larger fraction of terminals in tottering mutants.
While we cannot exclude this latter possibility, much of our data favour a simpler hypothesis, that both GABAB and -adrenergic receptors are more effective inhibitors because N-type Ca2+ channels, which dominate at tottering synapses, are more susceptible to G protein-dependent inhibition. The baclofen concentration-response relationship is left-shifted in wild-type slices in which P/Q-channels are blocked, and right-shifted in tottering slices in which N-channels are blocked. The fact that the latter experiment does not shift the concentration-response relationship fully to that of wild-type levels suggests that the P/Q- to N-channel shift may not account for all of the difference in presynaptic inhibition seen for tottering and wild-type synapses. It will be particularly important to explore the possibility that the mutation alters G protein-dependent inhibition of tottering P/Q-channels compared with wild-type-channels.
Our model suggests that the tottering phenotype results from enhanced presynaptic modulation of glutamatergic transmission, thereby changing the balance of synaptic excitation and inhibition. Results from synaptic studies of thalamus from tottering and lethargic mice support this idea and suggest that such imbalances extend beyond the cerebellum. Glutamatergic transmission appears to be selectively impaired in the thalamus of the two mutant mouse strains, whereas GABAergic inhibition remains unaffected (Caddick et al. 1999). Similarly, preliminary results comparing [3H]GABA release from tottering and wild-type synaptosomes indicate that the Ca2+ channel complement of GABAergic terminals is unaffected (T. J. Turner, unpublished observations). This predicts that inhibitory transmission in the cerebellum, like the thalamus, may be spared in tottering animals. If so, this would be predicted to work in concert with the enhanced G protein-mediated inhibition of excitatory transmission (through GABAB and 2-adrenergic receptors) to further suppress PC output from the cerebellum, as discussed above.
Such alterations in cerebellar circuit behaviour may underlie the defect that initiates and/or maintains motor seizures in tottering animals. Consistent with this idea, abnormal PC output was recently suggested to promote the intermittent myoclonic seizures observed in tottering animals, because lesions of the anterior cerebellar vermis significantly reduced seizure frequency and duration (Abbott et al. 2000). In addition, the observation that adrenergic fibres proliferate in tottering mutants (Levitt & Noebels, 1981) and that ablation of the locus coeruleus reduces the frequency of absence episodes and the 6 Hz spike-wave activity associated with them (Noebels, 1984), strongly implicates adrenergic innervation in the aetiology of the tottering phenotype. Our results demonstrating an increased susceptibility of PF-PC transmission to inhibition by NA offer one account for how increased adrenergic innervation could effect cerebellar dysfunction in tottering slices.
Given our results, which demonstrate that the potency of presynaptic GABAB receptors at PF-PC synapses is similarly enhanced in the tottering mouse mutant, one might predict that GABA, acting via GABAB receptors, exacerbates the shift in 'synaptic equilibrium' produced by increased basal levels of NA. Under normal conditions, GABAB receptors on PF terminals provide a powerful means of regulating PC activity through their mediation of heterosynaptic depression of PF-PC transmission (Dittman & Regehr, 1997). A central role for GABAB receptors in producing motor deficits similar to those in tottering animals is suggested by experiments in which systemic administration of the GABAB receptor agonist baclofen was found to potentiate (and antagonists to suppress) absence seizures in leaner mice, another P/Q-channel mutant with a motor phenotype similar to, but more severe than, that of tottering (Hosford et al. 1992). Furthermore, direct injection of baclofen into the thalamus of the 'genetic absence epilepsy rats from Strasbourg', rat (another animal model of absence epilepsy) has also been found to enhance absence seizures (Liu et al. 1992). Thus, GABAB receptors clearly play an important role in the development and/or maintenance of bilateral, generalised seizures. Future experiments addressing whether the cerebellum (per se) is a site of action central to baclofen-induced seizures will provide a stringent test of the hypothesis suggested by our data.
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Acknowledgements
The authors are indebted to Drs Joan Schein and Suzanne Roffler-Tarlov for their advice on maintaining the mouse colony. This work was supported by NS16483 and NS41322 (KD), NSF-IBN-0218619 (TJT) and an award from the New England Medical Center Research Fund (TJT).
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), 100 nM 
), 300 nM 
), both toxins (
). The Ca2+-independent release (+) was measured by switching to 60 mM K+ and nominal Ca2+ saline. C, concentration-response relationship for 
) synaptosomes preincubated to achieve near-equilibrium binding of toxin concentrations shown on the abscissa. The smooth line represents the best fit to a single binding site isotherm with a Ki of 10 nM. Data points represent means of six independent measurements.
t) before the second S1 stimulus. The S2 tetanus produced an interval-dependent decrease in S1-evoked synaptic responses, as illustrated by the fEPSPs shown in B. Each panel contains two superimposed traces evoked by S1 before (*) or after the tetanus. Traces represent averaged responses from three wild-type slices (+/+) or three tottering slices (tg/tg) with