Osvaldo D. Uchitel

Altered properties of quantal neurotransmitter release at endplates of mice lacking P/Q-type Ca2+ channels

Transmission at the mouse neuromuscular junction normally relies on P/Q-type channels, but became jointly dependent on both N- and R-type Ca2+ channels when the P/Q-type channel α1A subunit was deleted. R-type channels lay close to Ca2+ sensors for exocytosis and IK(Ca) channel activation, like the P/Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca2+ channels in the hierarchy P/Q>R>N. The α1A−/− neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca2+/Mg2+ ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters.

Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions

1 The effects of different calcium channel blockers (ω‐agatoxin IVA (ω‐Aga IVA), ω‐conotoxin GVIA (ω‐CgTx GVIA) and dihydropyridines) were tested on spontaneous and evoked transmitter release at embryonic and newborn rat neuromuscular junctions (NMJs). 2 The nerve‐evoked transmitter release quantal content (m) was strongly reduced by the P/Q‐type voltage‐dependent calcium channel (VDCC) blocker ω‐Aga IVA (100 nM) at newly formed endplates of embryos and 0‐ to 11‐day‐old rats, in agreement with the effect of this blocker on transmitter release at mature and reinnervating muscles. 3 ω‐CgTx GVIA (1–5 μm), the N‐type VDCC blocker, also caused a significant reduction in m at newly formed NMJs early in development (embryos and 0‐ to 4‐day‐old rats), while it was ineffective in more mature animals (5‐ to 11‐day‐old rats). 4 L‐type channel blockers, nitrendipine (1 μm) and nifedipine (1 μm), did not significantly affect neurally evoked release at developing NMJs. However, nifedipine (10 μm) was able to increase m significantly at 0‐ to 4‐day‐old rat NMJs. 5 At developing NMJs, K+‐evoked transmitter release was dependent on Ca2+ entry through VDCCs of the P/Q‐type family (100 nmω‐Aga IVA reduced 70% of the K+‐evoked miniature endplate potential frequency). N‐ and L‐type VDCC blockers did not affect this type of release. 6 We conclude that at rat neuromuscular junctions the presynaptic calcium channel types involved in transmitter release undergo developmental changes during the early postnatal period.

Transmitter release and presynaptic Ca2+ currents blocked by the spider toxin ω-Aga-IVA

Mammalian neuromuscular transmission is resistant to L and N type calcium channel blockers but very sensitive to a low molecular weight funnel web spider venom toxin, FTX, which selectively blocks P type calcium channels. To further characterize the calcium channels involved in neuromuscular transmission we studied the effect of omega Agatoxin (omega-Aga-IVA) a polypeptide P type channel blocker from the same spider venom. We show that omega-Aga-IVA is a potent and irreversible inhibitor of the presynaptic Ca2+ currents and of acetylcholine release induced by electrical stimulation or by K+ depolarization. This provides further evidences that transmitter release at the mammalian neuromuscular junction is mediated by P type Ca2+ channels.

Calcium channel blockers and transmitter release at the normal human neurornuscular junction

Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and Ω-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or Ω-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.

Evaluation of antioxidants, protein, and lipid oxidation products in blood from sporadic amiotrophic lateral sclerosis patients

Several parameters indicators of oxidative stress were evaluated in blood from individuals with the sporadic form of amiotrophic lateral sclerosis (SALS) and compared to healthy controls. Plasma levels of 2-thiobarbituric-reactive substances (TBARS), products of lipid peroxidation, were significantly higher (p < 0.03) in the SALS patients compared to controls. The concentration of plasma antioxidants (α-tocopherol, β-carotene, ubiquinol-10 and glutathione) and the activity of red blood cell CuZn superoxide dismutase were not significantly different between the groups. The ratio TBARS/α-tocopherol was 47% higher in the SALS individuals than in controls. Protein thiols and protein-associated carbonyls in red blood cell membranes and supernates were similar for both groups. A positive correlation (r2 = 0.91) was found between the concentration of protein-associated carbonyls in red blood cells and the onset of clinical symptoms. These findings are in agreement with several reports showing higher levels of oxidative damage to cell components in ALS.

Toxins affecting calcium channels in neurons

Calcium enters the cytoplasm mainly via voltage-activated calcium channels (VACC), and this represents a key step in the regulation of a variety of cellular processes. Advances in the fields of molecular biology, pharmacology and electrophysiology have led to the identification of several types of VACC (referred to as T-, N-, L-, P/Q- and R-types). In addition to possessing distinctive structural and functional characteristics, many of these types of calcium channels exhibit differential sensitivities to pharmacological agents. In recent years a large number of toxins, mainly small peptides, have been purified from the venom of predatory marine cone snails and spiders. Many of these toxins have specific actions on ion channels and neurotransmitter receptors, and the toxins have been used as powerful tools in neuroscience research. Some of them (omega-conotoxins, omega-agatoxins) specifically recognize and block certain types of VACC. They have common structural backbones and some been synthesized with identical potency as the natural ones. Natural, synthetic and labeled calcium channel toxins have contributed to the understanding of the diversity of the neuronal calcium channels and their function. In particular, the toxins have been useful in the study of the role of different types of calcium channels on the process of neurotransmitter release. Neuronal calcium channel toxins may develop into powerful tools for diagnosis and treatment of neurological diseases.

Calcium channels involved in synaptic transmission at the mature and regenerating mouse neuromuscular junction.

1. The involvement of the different types of voltage‐dependent calcium channels (VDCCs) in synaptic transmission at the mature and newly formed mammalian neuromuscular junction was studied by evaluating the effects of L‐, P/Q‐ and N‐type VDCC antagonists on transmitter release in normal and reinnervating levator auris preparations of adult mice. 2. Nerve‐evoked transmitter release was blocked by omega‐agatoxin IVA (omega‐AgaIVA), a P/Q‐type VDCC blocker, both in normal and reinnervating endplates (100 nM omega‐AgaIVA caused > 90% inhibition). The N‐type VDCC antagonist omega‐conotoxin GVIA (omega‐CgTX; 1 and 5 microM), as occurs in normal preparations, did not significantly affect this type of release during reinnervation. Nitrendipine (1‐10 microM), an L‐type VDCC blocker, strongly antagonized release in reinnervating muscles (approximately 40‐69% blockade) and lacked any effect in normal preparations. 3. In reinnervating muscles, spontaneous release was not dependent on Ca2+ entry through either P‐ or L‐type VDCCs. Neither 100 nM omega‐AgaIVA nor 10 microM nitrendipine affected the miniature endplate potential (MEPP) frequency or amplitude. 4. At the newly formed endplates, K(+)‐evoked release was dependent on Ca2+ entry through VDCCs of the P‐type family (100 nM omega‐AgaIVA reduced approximately 70% of the K(+)‐evoked MEPP frequency). L‐type VDCCs were found not to participate in this type of release (10 microM nitrendipine lacked any effect). 5. In reinnervating muscles, the L‐type VDCC blocker, nitrendipine (10 microM), provoked a significant increase (approximately 25%) in the latency of the evoked endplate potential (EPP). This drug also caused an increase (approximately 0.3 ms) in the latency of the presynaptic currents. The P/Q‐ and Ny‐type VDCC blockers did not affect the latency of the EPP. 6. These results show that at newly formed mouse neuromuscular junctions, as occurs in mature preparations, VDCCs of the P‐type family play a prominent role in evoked transmitter release whereas N‐type channels are not involved in this process. In addition, signal conduction and transmitter release become highly sensitive to nitrendipine during reinnervation. This suggests that L‐type VDCCs may play a role in synaptic transmission at the immature mammalian neuromuscular junction.

Modulation of ACh release by presynaptic muscarinic autoreceptors in the neuromuscular junction of the newborn and adult rat

We studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release and their relationship with voltage‐dependent calcium channels in the neuromuscular synapses of the Levator auris longus muscle from adult (30–40 days) and newborn (3–6 and 15 days postnatal) rats. Using intracellular recording, we studied how several muscarinic antagonists affected the evoked endplate potentials. In some experiments we previously incubated the muscle with calcium channel blockers (nitrendipine, ω‐conotoxin‐GVIA and ω‐Agatoxin‐IVA) before determining the muscarinic response. In the adult, the M1 receptor‐selective antagonist pirenzepine (10 µm) reduced evoked neurotransmission (≈ 47%). The M2 receptor‐selective antagonist methoctramine (1 µm) increased the evoked release (≈ 67%). Both M1‐ and M2‐mediated mechanisms depend on calcium influx via P/Q‐type synaptic channels. We found nothing to indicate the presence of M3 (4‐DAMP‐sensitive) or M4 (tropicamide‐sensitive) receptors in the muscles of adult or newborn rats. In the 3–6‐day newborn rats, pirenzepine reduced the evoked release (≈ 30%) by a mechanism independent of L‐, N‐ and P/Q‐type calcium channels, and the M2 antagonist methoctramine (1 µm) unexpectedly decreased the evoked release (≈ 40%). This methoctramine effect was a P/Q‐type calcium‐channel‐dependent mechanism. However, upon maturation in the first two postnatal weeks, the M2 pathway shifted to perform the calcium‐dependent release‐inhibitory activity found in the adult. We show that the way in which M1 and M2 muscarinic receptors modulate neurotransmission can differ between the developing and adult rat neuromuscular synapse.

Changes in synaptic transmission properties due to the expression of N‐type calcium channels at the calyx of Held synapse of mice lacking P/Q‐type calcium channels

P/Q‐type and N‐type calcium channels mediate transmitter release at rapidly transmitting central synapses, but the reasons for the specific expression of one or the other in each particular synapse are not known. Using whole‐cell patch clamping from in vitro slices of the auditory brainstem we have examined presynaptic calcium currents (IpCa) and glutamatergic excitatory postsynaptic currents (EPSCs) at the calyx of Held synapse from transgenic mice in which the α1A pore‐forming subunit of the P/Q‐type Ca2+ channels is ablated (KO). The power relationship between Ca2+ influx and quantal output was studied by varying the number of Ca2+ channels engaged in triggering release. Our results have shown that more overlapping Ca2+ channel domains are required to trigger exocytosis when N‐type replace P/Q‐type calcium channels suggesting that P/Q type Ca2+ channels are more tightly coupled to synaptic vesicles than N‐type channels, a hypothesis that is verified by the decrease in EPSC amplitudes in KO synapses when the slow Ca2+ buffer EGTA‐AM was introduced into presynaptic calyces. Significant alterations in short‐term synaptic plasticity were observed. Repetitive stimulation at high frequency generates short‐term depression (STD) of EPSCs, which is not caused by presynaptic Ca2+ current inactivation neither in WT or KO synapses. Recovery after STD is much slower in the KO than in the WT mice. Synapses from KO mice exhibit reduced or no EPSC paired‐pulse facilitation and absence of facilitation in their presynaptic N‐type Ca2+ currents. Simultaneous pre‐ and postsynaptic double patch recordings indicate that presynaptic Ca2+ current facilitation is the main determinant of facilitation of transmitter release. Finally, KO synapses reveal a stronger modulation of transmitter release by presynaptic GTP‐binding protein‐coupled receptors (γ‐aminobutyric acid type B receptors, GABAB, and adenosine). In contrast, metabotropic glutamate receptors (mGluRs) are not functional at the synapses of these mice. These experiments reinforce the idea that presynaptic Ca2+ channels expression may be tuned for speed and modulatory control through differential subtype expression.

Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals

The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; DL50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, omega-conotoxin-GVIA and omega-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 microM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker omega-conotoxin-GVIA (1 microM) inhibited neurotransmitter release by 50-70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker omega-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and omega-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to transmitter release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.