Brain, Vol. 123, No. 3, 463-471,
March 2000
© 2000 Oxford University Press
Invited review |
Abnormal transmitter release at neuromuscular junctions of mice carrying the tottering 
1A Ca2+ channel mutation
1 Departments of Physiology, 2 Neurology and 3 Human Genetics, Leiden University Medical Centre, Leiden,The Netherlands
Correspondence to:
Dr P.C. Molenaar, Department of Physiology, Leiden University Medical Centre, Wassenaarseweg 62, PO Box 9604, 2300 RC Leiden, The Netherlands E-mail: p.c.molenaar{at}physiology.medfac.leidenuniv.nl
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Abstract |
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Neurotransmitter release at many synapses is regulated by P/Q-type Ca2+ channels containing the
1A pore-forming subunit. Mutations in 1A cause cerebral disorders including familial hemiplegic migraine (FHM) and ataxia in humans. Tottering (tg) 1A mutant mice display ataxia and epilepsy. It is not known whether 1A mutations induce impairment of synaptic function, which could underlie the symptoms of these cerebral disorders. To assess whether 1A mutations influence neurotransmitter release, we studied P-type Ca2+ channel-mediated acetylcholine (ACh) release at tg neuromuscular junctions (NMJs) with micro-electrode measurements of synaptic potentials. We found a Ca2+-, Mg2+- and K+-dependent increase of spontaneous ACh release at both homo- and heterozygote tg NMJs. Furthermore, there was increased run-down of high-rate evoked release at homozygous tg NMJs. In isotonic contraction experiments this led to block of synaptic transmission at lower concentrations of the ACh antagonist tubocurarine than were needed in wild-type muscles. Our results suggest that in tg motor nerve terminals there is increased influx of Ca2+ under resting conditions. This study shows that functional consequences of 1A mutations causing cerebral disorders can be characterized at the NMJ. acetylcholine release; migraine; P/Q-type calcium channel; neuromuscular junction; tottering mouse
ACh = acetylcholine; EPP = endplate potential; FHM = familial hemiplegic migraine; MEPP = miniature endplate potential; NMJ = neuromuscular junction; PCR = polymerase chain reaction; tg = tottering
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Voltage-gated Ca2+ channels in nerve, muscle and secretory cells consist of low voltage-activated T-type channels and high voltage-activated channels, which can be subdivided on a pharmacological basis into P/Q-, N-, L- and R-type (Catterall, 1995
; Perez-Reyes and Schneider, 1995). Ca2+ channel variants are formed by complexes of pore-forming 1 and auxiliary ß, 2
and 
subunits (Mori et al., 1991; Isom et al., 1994; Stea et al., 1994; Catterall, 1995).
P- and Q-type channels are splice variants of the same gene (Bourinet et al., 1999) and are discriminated by the block of Ca2+ current by 
-agatoxin-IVA or -conotoxin-MVIIC in the nanomolar range (Stea et al., 1994; Randall and Tsien, 1995; Hilaire et al., 1996; Mori et al., 1996; Berrow et al., 1997). P/Q-type channels are involved in transmitter secretion from nerve terminals, at which synaptic vesicles undergo exocytosis following the action of Ca2+ influx on the release machinery (Takahashi and Momiyama, 1993; Geppert et al., 1994; Wheeler et al., 1994; Augustine et al., 1996).
The 1A ion-conducting pore of P/Q-type channels is encoded by the CACNA1A gene, which is widely expressed in the CNS (Mori et al., 1991; Stea et al., 1994; Diriong et al., 1995; Westenbroek et al., 1995; Fletcher et al., 1996). In the mammalian peripheral nervous system, P/Q-type channels are relatively rare and mainly present at the neuromuscular junction (NMJ) where P-type channels, probably encoded by the CACNA1A gene, regulate transmitter release (Uchitel et al., 1992; Bowersox et al., 1995; Hong and Chang, 1995; Day et al., 1997; Lin and Lin-Shiau, 1997).
Mutations in the 1A gene have been found to be responsible for the human episodic neurological disorders familial hemiplegic migraine (FHM) and episodic ataxia type 2 and also for chronic spinocerebellar ataxia type 6 (Ophoff et al., 1996; Zhuchenko et al., 1997). The 1A gene is likely to be also involved in (non-hemiplegic) typical migraine (Terwindt et al., 1997, 1998; Nyholt et al., 1998). Likewise, natural 1A mutations have been reported for the tottering (tg) and leaner (tgla) mice, of which the homozygous animals exhibit symptoms of ataxia and epilepsy (Fletcher et al., 1996; Doyle et al., 1997). The location of the tg mutation P601L (Fletcher et al., 1996) corresponds to proline at position 647 in the human sequence in the extracellular loop between transmembrane domains S5 and S6 of repeat II of the 1A subunit. This is just before the pore-forming loop in IIS5S6 (position 651–675) where one of the FHM mutations (T666M) is located (Ophoff et al., 1996). The tgla mutation is a splice-site mutation leading to a truncated carboxy-terminus (Fletcher et al., 1996).
Each of the four known FHM mutations seems to induce specific changes in Ca2+ current density and/or kinetics. This follows from studies in which rabbit and human 1A genes with introduced FHM mutations were expressed in, respectively, Xenopus oocytes and human embryonic kidney cells (Kraus et al., 1998; Hans et al., 1999). The tg and tgla mutations appear to decrease whole-cell Ca2+ current in Purkinje cells, whereas the single Ca2+ channel conductances are unchanged (Dove et al., 1998; Lorenzon et al., 1998; Wakamori et al., 1998). Thus, either the number of tg Ca2+ channels is decreased or a channel parameter, such as the likelihood of channel opening, is changed. Glutamatergic synaptic potentials in tg/tg mouse thalamus slices are decreased by 40% (Caddick et al., 1999).
We hypothesized that pathological 1A gene mutations will change neurotransmitter release at NMJs from tg mice, because mammalian motor nerve terminals are endowed with P-type channels, supposedly encoded by the CACNA1A gene. Quantal acetylcholine (ACh) release at the NMJ can be studied electrophysiologically with relative ease and great precision. From a clinical point of view it is also of interest whether there is disturbed ACh release in skeletal muscles in inherited 1A cerebral disorders. For instance, reduced ACh release could decrease the safety factor of neuromuscular transmission, rendering patients abnormally sensitive to muscle relaxants.
Here we describe profound changes in ACh release at NMJs from both homo- and heterozygous tg mice.
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Material and methods |
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Mice
Breeding pairs of heterozygous C57BL/6J-tg mice were obtained from the Jackson Laboratory, Bar Harbor, Me., USA. Litters were genotyped after weaning. The experiments were carried out under permission of the Leiden Animal Ethics Committee, approval number 97008.
Genotyping
Genomic DNA was isolated from 4–6 mm portions of the tail. These were rotated overnight at 55°C in a solution containing 50 mM Tris pH 8.9, 0.45% Nonidet P-40 (Sigma Chemical Co., St Louis, Mo., USA) and 100 µg proteinase K (Boehringer Mannheim, Mannheim, Germany) in a total volume of 250 µl. After incubation, proteinase K was inactivated for 10 min at 95°C. A polymerase chain reaction (PCR), spanning the predicted intron 14 and the tg mutation in exon 15 (Fletcher et al., 1996), was performed on 1 µl genomic tail DNA (diluted 1 : 20 in H2O) in a total volume of 30 µl containing 60 mM Tris–HCl, pH 8.5; 15 mM (NH4)2SO4; 2.5 mM MgCl2; 200 µM of each dNTP; 15 pmol of each primer (1729f: 5'-CTGTTCATCGTTGTCTTTGC-3', 1831r: 5'-CTGCTGGAAAAGTGTCGAAG-3'); 0.6 U AmpliTaq (Perkin Elmer Cetus, Foster City, Calif., USA) and 3 µg bovine serum albumin (Pharmacia Biotech, Uppsala, Sweden). After an initial denaturation step of 3 min at 94°C, the products were amplified for 34 cycles (30 s at 94°C, 30 s at 56°C, 45 s at 72°C) followed by a final elongation step of 5 min at 72°C. Subsequently, the 650 bp PCR product was cloned into the pCR® 2.1-TOPO vector of the TOPO TA Cloning kit (Invitrogen Co., Carlsbad, Calif., USA) according to the manufacturer. Several clones were subjected to the above-mentioned PCR and clones containing the product were sequenced by dideoxy sequence analysis (T7 sequencing kit, Pharmacia Biotech, Uppsala, Sweden), allowing the design of primer int1778f.
For mutation analysis, 1 µl of tail genomic DNA (diluted 1 : 20 in H2O) was subjected to PCR in a total volume of 15 µl containing 60 mM Tris–HCl, pH 8.5, 15 mM (NH4)2SO4, 1.5 mM MgCl2, 200 µM dTTP, dATP, dGTP, dCTP, 100 ng of each primer (int1778f: 5'-TTCTGGGTACCAGATACAGG-3', 1831r: 5'- CTGCTGGAAAAGTGTCGAAG-3'), 0.3 U AmpliTaq and 1.5 µg bovine serum albumin. The initial denaturation step (3 min at 94°C) was followed by 33 cycles of amplification (30 s at 94°C, 30 s at 57°C, 60 s at 72°C) and a final elongation step of 5 min at 72°C.
Subsequently, 10 µl of PCR product was digested with 0.5 µl restriction enzyme AciI (5 U/µl, New England Biolabs, Beverly, Mass., USA) for at least 1 h at 37°C according to the recommendations of the manufacturer. Upon digestion, the PCR fragments were separated on a 1.5% agarose gel for genotyping. Fragment lengths of 150/28 or 178 were obtained for a wild-type (+) or tg allele, respectively. Figure 1 shows the position of the tg mutation (proline to leucine mutation as a result of a base-pair substitution at position 1802) and an example of the tg and wild-type bands on agarose gel.
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In vitro electrophysiology of the neuromuscular junction
The mice were killed by ether inhalation. The left phrenic nerve-hemidiaphragm was dissected and mounted in a 2 ml bath with Ringer's medium at 26–28°C. The Ringer's solution contained (in mM): NaCl 116; KCl 4.5; MgSO4 1; CaCl2 2; NaH2PO4 1; NaHCO3 23; glucose 11, pH 7.4. The concentrations of Ca2+, Mg2+ and K+ were varied as indicated.
Intracellular recordings of miniature endplate potentials (MEPPs) and endplate potentials (EPPs) were made from the endplate region, using standard micro-electrode equipment as described previously (Plomp et al., 1992). At least 25 MEPPs and EPPs were recorded from each fibre. To be able to measure EPPs, muscle action potentials were blocked by treatment with 2.3 µM µ-conotoxin GIIIB (Scientific Market Associates, Barnet, UK) which blocks Na+ channels of mouse muscle but not those of nerve and does not influence the quantal release of ACh or its effect on ACh receptors (Cruz et al., 1985; Di Gregorio et al., 1989; Hong and Chang, 1989; Plomp et al., 1992). For the generation of the EPPs the phrenic nerve was stimulated supramaximally at either 0.3 or 40 Hz. The amplitudes of MEPPs and EPPs were normalized to –75 mV, assuming 0 mV as the reversal potential for ACh-induced current (Magleby and Stevens, 1972). The normalized EPPs were corrected for non-linear summation with the formula of McLachlan and Martin (1981) using a value for f of 0.8. The quantal content was calculated by dividing the normalized and corrected EPP amplitude by the normalized MEPP amplitude.
-Agatoxin-IVA (Scientific Market Associates) was dissolved to a concentration of 20 µM in distilled water containing 0.1 mg/ml bovine serum albumin. Aliquots of 10 µl were stored at –70°C until further dilution to 200 nM in Ringer's solution just before the experiment. In the experiments where the reducing effect of -agatoxin-IVA on quantal contents was measured after 1 h incubation of nerve–muscle preparations, the effect partially waned during the subsequent 45 min measuring period in spite of the continuous presence of the toxin, as described by others (Hong and Chang, 1995), and possibly due to instability of the toxin or its binding to non-specific sites.
Contraction experiments
In right phrenic nerve-hemidiaphragms of mice, isotonic contractions were recorded with a force transducer. The phrenic nerve was stimulated supramaximally once every 5 min with a train of 150 stimuli at 50 Hz. The muscles were incubated in Ringer's solution to which tubocurarine was added in increasing concentrations (1 h incubation at each concentration). The amplitude of the contractions was measured after 100 stimuli (i.e. 2 s) after the onset of each train.
Statistics
The data are presented as the grand muscle mean ± standard error of the mean. Possible statistical differences were analysed with a paired or unpaired Student's t-test, wherever appropriate. To correct for multiple testing in Table 1 and Figs 2, 3 and 6, a P value of <0.01 was considered to be significant in all other cases P < 0.05 was considered to be significant.
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Results |
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High-rate evoked ACh release is depressed at tg/tg NMJs
The EPP is the postsynaptic response resulting from simultaneous exocytosis of a number of ACh quanta (quantal content) from the motor nerve terminal in response to a single nerve action potential. The MEPP is the response resulting from spontaneous exocytosis of a single ACh quantum. We made micro-electrode recordings of MEPPs and EPPs at NMJs in µ-conotoxin-paralysed diaphragm-phrenic nerve preparations (Table 1
). When supramaximal electric stimulations were delivered to the nerve at a low rate (0.3 Hz), the mean amplitude of EPPs was ~25 mV, and did not differ between tg/tg, tg/+ and wild-type mice. Also, the mean amplitude of MEPPs (~0.8 mV) did not differ between these groups. The quantal content, derived from the corrected and normalized mean amplitudes of EPP and MEPP, was ~45 in all groups. However, the amplitude of EPPs during 40 Hz nerve stimulation showed a run-down at tg/tg NMJs to a level of ~71% of the amplitude of the first EPP, which was greater than the EPP run-down at tg/+ and +/+ NMJs (to, in each case, ~81%, P < 0.001; Fig. 2). Enlarged EPP run-down in tg/tg NMJs predicts a smaller safety factor of neuromuscular transmission (this factor being the ratio of the EPP size and the depolarization at which the muscle fibre starts firing). Changes of the safety factor can be tested for by measuring nerve stimulation-evoked muscle contraction at increasing concentrations of tubocurarine, a blocker of nicotinic ACh receptors. Indeed, contraction experiments showed that diaphragms from tg/tg muscles became paralysed at lower concentrations of tubocurarine than those of +/+ mice when measured at 50 Hz nervous stimulation (Fig. 3; tg/+ muscles were not tested).
At both tg/tg and +/+ NMJs, the quantal content at 0.3 Hz nerve stimulation was reduced by ~70% when measured after 1 h incubation with 200 nM -agatoxin-IVA. The quantal contents decreased from 43.6 ± 1.9 to 14.3 ± 2.4 in a tg/tg muscle (15 NMJs sampled) and from 40.7 ± 3.0 to 11.5 ± 3.4 in a +/+ muscle (10 NMJs sampled).
Increased spontaneous quantal ACh release at tg/tg and tg/+ NMJs
We measured spontaneous quantal release of ACh as the frequency of MEPPs (Table 1). The mean MEPP frequency at NMJs of tg/tg mice was more than twice that found in +/+ mice. The MEPP frequency at tg/+ mice was in between that of tg/tg and +/+ mice.
The effect of the P-type Ca2+ channel blocker -agatoxin-IVA (continuous presence of 200 nM toxin during the 30 min measurement period, starting after 15 min incubation) on spontaneous ACh release was studied at tg/tg and +/+ NMJs. The toxin depressed the MEPP frequency in both groups by ~55%, from 2.26 ± 0.10/s to 0.94 ± 0.07/s and from 1.13 ± 0.14/s to 0.55 ± 0.04/s in the tg/tg and +/+ group, respectively (Fig. 4). The observation that -agatoxin-IVA reduced MEPP frequency at wild-type NMJs conflicts for unknown reasons with studies that failed to demonstrate an effect of -agatoxin-IVA on MEPP frequency at normal mouse and rat NMJs (Protti and Uchitel, 1993; Hong and Chang, 1995; Losavio and Muchnik, 1997).
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The MEPP frequency was also measured in a medium with elevated (10 mM) K+ concentration, which should depolarize the motor nerve terminal to about –65 mV and induce a 4-fold increase of the MEPP frequency at mouse NMJs (Protti and Uchitel, 1993
). High K+ increased the MEPP frequency at tg/tg NMJs 9-fold, which was significantly more than the 5-fold increase found at +/+ NMJs (Fig. 5; P < 0.02). Upon subsequent incubation of the 10 mM K+-depolarized muscles with 200 nM -agatoxin-IVA, the MEPP frequencies at both tg/tg and +/+ NMJs decreased by ~75%, approaching the MEPP frequencies measured in standard 4.5 mM K+ medium (Fig. 5), as observed by others at normal NMJs at high K+ concentration (Protti and Uchitel, 1993; Hong and Chang, 1995; Losavio and Muchnik, 1997).
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Effect of Ca2+ and Mg2+ on MEPP frequency
Spontaneous quantal release of ACh at the NMJ is mainly dependent on the concentration of Ca2+ in the medium. We measured the MEPP frequency at tg/tg and +/+ NMJs at low (0.2 mM), standard (2 mM), and high (5 mM) Ca2+ concentration. The difference in MEPP frequency between tg/tg and +/+ varied with the Ca2+ concentration; it disappeared at 0.2 mM Ca2+ (Fig. 6A
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Mg2+ is a reversible Ca2+ channel blocker. It antagonizes Ca2+-induced neurotransmitter release at the NMJ (Jenkinson, 1957). We measured MEPP frequency at tg/tg and +/+ NMJs at low (0.2 mM), standard (1 mM), and high (10 mM) Mg2+ concentration. The mean MEPP frequency decreased with increasing concentration of Mg2+, but not to the same extent in tg/tg and +/+ NMJs. The difference between mean MEPP frequency of the groups disappeared at 10 mM Mg2+ (Fig. 6B).
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Discussion |
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This study shows that the tg
1A gene mutation can influence neurotransmitter release. We observed two types of changes at tg NMJs. First, an increase of run-down of ACh release during high frequency nerve stimulation, and secondly, an increase in rate of spontaneous quantal release, the extent of which was dependent on the concentrations of Ca2+, Mg2+ and K+ in the extracellular medium. This strongly suggests that the Ca2+ channel involved in ACh release at mouse motor nerve terminals is encoded by the 1A gene, as was predicted on pharmacological grounds (see Introduction). Thus, if the CACNA1A gene encodes endplate P/Q-type Ca2+ channels it would follow that subclinical abnormalities at the NMJ may exist in cerebral diseases such as FHM, possibly leading to an altered sensitivity for non-depolarizing muscle relaxants. Neuromuscular transmission has a substantial safety factor, preventing small decreases in ACh output from affecting the all-or-none process of muscle activation. However, blockade of some of the ACh receptors may unveil subclinical defects at the NMJ, as demonstrated in this study by the effect of tubocurarine in tg/tg muscles.
Evoked ACh release in tg/tg mice
P/Q-type Ca2+ channel expression or the likelihood of Ca2+ channel opening is decreased in tg Purkinje cells (see Introduction). It came as a surprise, therefore, that the quantal content at low-rate (0.3 Hz) nerve stimulation in tg/tg NMJs was unchanged. This suggests that there is no reduced expression of mutant P-type Ca2+ channels at tg motor nerve terminals and that the density of mutant P-type channels, or their subunit composition, in the NMJ is regulated in a different manner, as in Purkinje cell bodies. However, it cannot be excluded that a decrease of P/Q-type Ca2+ channels was masked in NMJs of tg mice by a compensatory increase of the sensitivity to Ca2+ of the ACh release process.
Yet, at high-rate nerve stimulation the quantal content at tg/tg NMJs fell below the level of controls; there was increased run-down of EPPs. This may be caused by a decrease in Ca2+ channel availability due to slowed recovery from inactivation, as has been shown for the nearby T666M FHM mutation (Kraus et al., 1998; Hans et al., 1999). Another factor that could add to extra EPP run-down is reduced availability of ACh quanta at tg/tg NMJs, since it is possible that tg mutant P-type channels have abnormal interactions with active zone proteins involved in recruiting and docking of synaptic vesicles.
The fact that we did not observe increased EPP run-down at tg/+ NMJs indicates that this is a threshold phenomenon and a recessive phenotype with loss of physiological function, i.e. increased EPP rundown requires more than half of the population of P-type channels at the NMJ to be tg mutated.
Spontaneous ACh release and low-voltage-activated Ca2+ channels
MEPP frequency was increased by ~100% in tg/tg and by 40% in tg/+ mice. The increase in MEPP frequency in the tottering mouse was therefore a dominant trait with gain-of-(physiological) function. The difference between tg/tg and control MEPP frequency was abolished at low (0.2 mM) Ca2+ or high (10 mM) Mg2+ in the medium. In tg mice there is upregulation of L-type Ca2+ channels in Purkinje cells (Campbell and Hess, 1999). The question arises as to whether the increased MEPP frequency was due to expression of L-type channels in motor nerve terminals or some other, unrelated secondary adaptive reaction. However, the fact that heterozygous tg mice have no epileptic/ataxic symptoms, but show increased MEPP frequency, seems to argue against this possibility. Furthermore, MEPP frequencies at tg/tg and +/+ NMJs with standard (4.5 mM) as well as elevated (10 mM) K+ concentration in the medium were equally sensitive to -agatoxin-IVA. These findings indicate that P-type Ca2+ channels are responsible for ~50% of the MEPP frequency at wild-type NMJs and for most of the MEPP increase at tg NMJs.
Wild-type P-type Ca2+ channels are high-voltage-activated with minute probability of opening at a membrane potential range of –80 to –60 mV, according to the Boltzmann equation using the constants of rabbit or mouse 1A channels (Kraus et al., 1998; Wakamori et al., 1998). Thus, in spontaneous quantal ACh release at wild-type NMJs, an underlying Ca2+ channel has the -agatoxin-IVA sensitivity but not the gating characteristics of a P-type channel. Interestingly, low-voltage-activated `T-type' channels can be transformed by an as yet unknown mechanism from high-voltage-activated N-, L- and R-type channels (Meir and Dolphin, 1998). Hence, P/Q-type channels may also exhibit dual forms with different activation characteristics, and MEPP frequency may be determined by a fraction of the channels expressed in a low-voltage-activated form (perhaps too low in number to be recognized in whole-cell I–V relationships).
The increase in MEPP frequency at tg NMJs could, in principle, be caused by a shift of activation voltage of high-voltage-activated P-type channels to values in the direction of the resting membrane potential. However, no significant changes of the Boltzmann constants have been found in tg mice and at any rate it would require a much greater change than reported for FHM mutations in rabbit and human 1A (Kraus et al., 1998; Wakamori et al., 1998; Hans et al., 1999). A better explanation seems to be that the tg mutation is shifting activation voltage of the putative 1A gene-encoded low-voltage-activated P-type Ca2+ channel in the negative direction. The observation that slight depolarization by 10 mM K+ increased MEPP frequency more at tg/tg than at +/+ NMJs supports this hypothesis. It has previously been suggested that the 1A gene might in addition encode a low-threshold isoform, or that the tg mutation has an indirect effect on T-type Ca2+ channel function (Fletcher et al., 1996).
It has been suggested that Mg2+ is involved in the pathogenesis of migraine (Welch and Ramadan, 1995). Our observation that high Mg2+ in the medium abolishes the difference in the rate of spontaneous ACh release between tg/tg and +/+ NMJs is therefore of interest in relation to migraine.
Implications for cerebral synaptic dysfunction in tg and human inherited 1A CNS diseases
The tg 1A gene mutation may affect P/Q-type channel behaviour in cell bodies as well as nerve terminals of the CNS, and it remains to be seen which type of dysfunction is causing ataxia and seizures.
Increased spontaneous transmitter release could directly influence dendritic signal integration on postsynaptic neurons since many CNS presynaptic terminals have only one release site and release only a single transmitter quantum per nerve impulse (Redman, 1990; Stevens, 1993). Alternatively, depletion of neurotransmitter vesicles due to the high rate of spontaneous release might block evoked release. Mildly depressed evoked release could already impair impulse transmission, because there is no safety margin as present in the NMJ.
The four known FHM mutations cause different effects on kinetic parameters of whole-cell Ca2+ currents in expression systems (Kraus et al., 1998; Hans et al., 1999). Accordingly, it should be realized that different mutations in the 1A gene may lead to different `synaptic phenotypes', even if the mutations give rise to very similar clinical phenotypes.
CNS synaptic dysfunction may also play a role in progression of symptoms and cerebral atrophy in 1A diseases. Synaptic activity influences gene expression in the postsynaptic neuron, thus regulating protein synthesis for maintenance of synaptic integrity and structural changes accompanying functional synaptic plasticity (Bito et al., 1997; Bito, 1998). Furthermore, increased presynaptic Ca2+ influx via mutated 1A subunits and the overstimulation of postsynaptic receptors by the consequent increase in spontaneous transmitter release may result in pre- and/or postsynaptic Ca2+ overload, triggering apoptotic mechanisms.
In conclusion, the present study strongly suggests that P/Q-type channels in the NMJ are encoded by the same gene as that encoding cerebral P/Q-type Ca2+ channels involved in FHM and episodic ataxia type 2. Accordingly, the NMJ, being relatively easy to study as compared with synapses in the CNS, can be used to study in detail the changes in neurotransmitter release resulting from P/Q-type Ca2+ channel mutations.
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Acknowledgments |
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The authors wish to thank Mr J. A. van der Zwet for breeding the tg mice and Drs A. R. Wintzen, J. J. G. M. Verschuuren, R. J. van den Berg and D. L. Ypey for valuable discussions.
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Received July 2, 1999. Revised August 27, 1999. Accepted September 28, 1999.
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