Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions

Abstract

1. The effects of different calcium channel blockers (omega-agatoxin IVA (omega-Aga IVA), omega-conotoxin GVIA (omega-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 omega-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. omega-CgTx GVIA (1-5 microM), 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 microM) and nifedipine (1 microM), did not significantly affect neurally evoked release at developing NMJs. However, nifedipine (10 microM) 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 nM omega-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.

Figure 4. Correlation between the effect of different VDCC blockers and the maturation of the neuromuscular junctions

The plots show the correlation between the percentage of inhibition (of quantal content) and the time of development (age of the animals) in ω-CgTx GVIA (1 μm; A); ω-Aga IVA (100 nM; B) and 1 μm nitrendipine (▴) and 1 μm nifedipine (▵;C) treated preparations. Each value represents the mean ±s.e.m. of the data pooled from a nerve-muscle preparation (at least 12 endplates per muscle). The lines represent the best linear fit of the data of ω-CgTx GVIA (r²= 0.8, P < 0.01), ω-Aga IVA (r²= 0.37, P= 0.27), nitrendipine (r²= 0.01, P= 0.79) and nifedipine (r²= 0.01, P= 0.74).

Figure 1. Effect of ω-conotoxin GVIA (ω-CgTx GVIA) on synaptic transmission at a 2-day-old rat neuromuscular junction

A, representative recordings from the same neuromuscular junction of 100 successive stimuli delivered at 0.5 Hz recorded before (Control) and after treatment with 1 μmω-CgTx GVIA. Note that the toxin reduced the amplitude of EPPs and significantly inhibited the quantal content (increasing the number of failures) to 50 % of the control value. Stimulation artifacts were reduced for clarity. B, extracellular recordings of compound nerve action potential before (Control) and after application of the toxin. This blocker showed no effect on the conduction of the nerve impulse. Each trace represents an average of 30 successive traces. C, temporal course plot of the effect of ω-CgTx GVIA on quantal content. The inhibition began to be apparent 10-20 min after bath application of this blocker (indicated with the bar) reaching a plateau within 35 min. This effect lasted after washout of the preparation with toxin-free saline solution (data not shown).

Figure 3. Effect of calcium channel blockers on nerve-evoked release at neuromuscular junctions of neonatal rats

The bar diagrams show the effect of the drugs on quantal content (m) expressed as a percentage of the control value. A, effect of calcium channel blockers in embryonic and 0- to 4-day-old rats. The toxins ω-Aga IVA (□) and ω-CgTx GVIA (▪) significantly reduced the evoked response while the dihydropyridines nifedipine () and nitrendipine () lacked any effect. Nifedipine (10 μm), however, significantly increased quantal content. In control fibres, m= 0.85± 0.08 (mean ±s.e.m., n= 64 endplates from 16 muscles). B, effect of calcium channel blockers in 5- to 11-day-old rats. The toxin ω-Aga IVA (□) maintained its strong effect on the evoked response shown at early stages while ω-CgTx GVIA (▪) and the dihydropyridines nifedipine () and nitrendipine () lacked any effect. In control fibres, m= 1.27± 0.16 (mean ±s.e.m., n= 43 endplates from 13 muscles). Control fibres were assayed in a low Ca²⁺/high Mg²⁺ (0.7-1 mM/5-8 mM) saline solution. Treated fibres were assayed in the same muscles after a 1 h incubation with the respective drug. Each column represents the mean ±s.e.m. of data pooled from 2-5 nerve-muscle preparations (at least 12 endplates per muscle). m was calculated by the failure method. Stimulation frequency, 0.5 Hz. **P < 0.0001, *P < 0.05 compared with the values obtained in the same muscles before addition of the calcium channel blockers.

Figure 2. Effect of ω-conotoxin GVIA (ω-CgTx GVIA) on synaptic transmission at an 11-day-old rat neuromuscular junction

A, representative recordings from the same neuromuscular junction of 100 successive stimuli delivered at 0.5 Hz recorded before (Control) and after treatment with 1 μmω-CgTx GVIA. The toxin reduced neither the amplitude of EPPs nor the quantal content of the synaptic transmission. Stimulation artifacts were reduced for clarity. B, temporal course plot of the effect of ω-CgTx GVIA on quantal content. At this age of development, the N-type VDCC blocker showed no effect on quantal content within 70 min after its bath application (indicated with the bar).

Figure 5. Effect of calcium channel blockers on K⁺-evoked release at neuromuscular junctions of neonatal rats

A, 0- to 4-day-old rat NMJs. a, effect of the calcium channel blockers on MEPP frequency (min⁻¹). ω-Aga IVA (100 nM; ▪) strongly inhibited the frequency of MEPPs but not the other blockers, ω-CgTx GVIA (1 μm; ) and nifedipine (10 μm; ). b, effect of the calcium channel blockers on MEPP amplitudes. None of the blockers showed any significant effect on MEPP amplitude. B, 5- to 11-day-old rat NMJs. a, at this stage of development a similar effect of these blockers was observed on MEPP frequency (min⁻¹). ω-Aga IVA (100 nM; ▪), but not ω-CgTx GVIA (1 μm; ) or nifedipine (10 μm; ), significantly inhibited MEPP frequency. b, calcium channel blocker action on MEPP amplitudes. Similar to 0- to 4-day-old rat NMJs, none of the blockers showed any effect on MEPP amplitude. In A and B, control fibres (□) were assayed in high K⁺ (20 mM) saline solution and treated fibres were assayed in the same muscles after 1 h incubation with the respective drug. Mean amplitude per fibre was calculated and corrected assuming a membrane potential of -70 mV. Each column represents the mean ±s.e.m. of data pooled from 2 nerve- muscle preparations (at least 12 endplates per muscle). *P < 0.001 compared with its control value.