Enhanced G protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel-mutant mouse, tottering

Abstract

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 and alpha2-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.

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 (○), 100 nmω-conotoxin GVIA (▵), 300 nmω-agatoxin IVA (□), both toxins (⋄). The Ca²⁺-independent release (+) was measured by switching to 60 mm K⁺ and nominal Ca²⁺ saline. C, concentration-response relationship for ω-agatoxin IVA; [³H]glutamate release measured from control (wt, ○) or tottering (tg, •) 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 K_i of 10 nm. Data points represent means of six independent measurements.

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 (○) and tottering (•) slices.

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).

Figure 4. Tottering synapses are dominated by N-type Ca²⁺ channels

The time course for ω-conotoxin GVIA (ω-CTx-GVIA)-induced inhibition of synaptic transmission between PFs and PCs. The toxin was bath applied at 100 nm for the time indicated by the horizontal bar during field recording from control (○) or tottering (•) slices. Data represent the mean ±s.e.m. from three control and three tottering slices. **P < 0.01.

Figure 5. Tottering synapses are more susceptible to presynaptic inhibition by GABA_B 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 (○) and eight tottering slices (•) recorded in control saline (in the absence of Ca²⁺ channel toxins). B and C, baclofen concentration-response data taken from wild-type slices (B) or tottering slices (C) in which N-channels were blocked with 100 nmω-conotoxin GVIA (GVIA; squares, n= 4 in B and 5 in C), or in which P/Q-channels were blocked with 500 nmω-agatoxin IVA (IVA; triangles, n= 3 in both B and C). Arrowheads on the abcissae indicate IC₅₀ values for baclofen action in the absence of toxins (data from A). The two data sets in both B and C are statistically different (P < 0.05), as determined by F statistics on separate sum-of-squares and combined sum-of-squares resulting from curve fitting. All data points represent the mean ±s.e.m.

Figure 6. GABA_B 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 GABA_B 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).

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 (Δt) 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 Δt= 10 ms and 300 ms, as marked. Scale bar: 50 μV (+/+), 100 μV (tg/tg) and 5 ms. C, heterosynaptic depression in wild-type slices (left panel) and tottering slices (right panel) plotted as a function of the time interval following the tetanus. Data were taken in control saline (circles), in saline containing 100 μm of the GABA_B receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid 35348 (triangles) and following washout of the antagonist (squares). Data are from nine wild-type and four tottering slices; each point represents the mean ±s.e.m. for 6–9 wild-type recordings and 3–4 tottering recordings.

Figure 8. Tottering synapses are more susceptible to inhibition by α₂-adrenergic receptors

A, concentration-response relationship for noradrenaline-induced inhibition of PF-PC transmission, measured in wild-type (○) and tottering (•) slices during bath-application of noradrenaline. Data sets are statistically different (P < 0.001), as determined by F statistics on separate sum-of-squares and combined sum-of-squares resulting from curve fitting. B, histogram of the pharmacology of receptors mediating noradrenaline-induced inhibition in wild-type (white bars) or tottering (grey bars) slices. Slices were incubated in yohimbine (+YOH, 10 μm), prazosin (+PRAZ, 10 μm) or propranolol (+PROP, 10 μm) and tested with 10 μm noradrenaline. Data represent the means ±s.e.m. of the number of slices noted on or above the bars.*P < 0.05, **P < 0.001; Student's two-tailed t test.