Specific distribution of sodium channels in axons of rat embryo spinal motoneurones

Abstract

1. The distribution of Na+ channels and development of excitability were investigated in vitro in purified spinal motoneurones obtained from rat embryos at E14, using electrophysiological, immunocytochemical and autoradiographical methods. 2. One hour after plating the motoneurones (DIV0), only somas were present. They expressed a robust delayed rectifier K+ current (IDR) and a fast-inactivating A-type K+ current (IA). The rapid neuritic outgrowth was paralleled by the emergence of a fast-activating TTX-sensitive sodium current (INa), and by an increase in both K+ currents. 3. The change in the three currents was measured daily, up to DIV8. The large increase in INa observed after DIV2 was accompanied by the onset of excitability. Spontaneous activity was observed as from DIV6. 4. The occurrence of axonal differentiation was confirmed by the fact that (i) only one neurite per motoneurone generated antidromic action potentials; and (ii) 125I-alpha-scorpion toxin binding, a specific marker of Na+ channels, labelled only one neurite and the greatest density was observed in the initial segment. Na+ channels therefore selectively targeted the axon and were absent from the dendrites and somas. 5. The specific distribution of Na+ channels was detectable as soon as the neurites began to grow. When the neuritic outgrowth was blocked by nocodazole, no INa developed. 6. It was concluded that, in spinal embryonic motoneurone in cell culture, Na+ channels, the expression of which starts with neuritic differentiation, are selectively addressed to the axonal process, whereas K+ channels are present in the soma prior to the neuritic outgrowth.

Figure 1. Na⁺ and K⁺ currents in motoneuronal cell cultures purified from rat embryos at E14

A, representative whole-cell currents recorded at DIV1 and DIV5. Currents were elicited by applying depolarizing pulses from -50 to 30 mV in 10 mV incremental steps from a holding potential V_h = -60 mV. B, fast transient sodium (I_Na) and outward potassium currents elicited at DIV4 in response to voltage pulses at -50, -40 and -30 mV, from V_h = -60 mV, under control conditions and in the presence of 30 nM TTX. C, delayed rectifier K⁺ currents (I_DR) at 20 mV recorded from V_h = -60 mV. The majority (76%) of this component was blocked by 10 mM TEA (mean: 66 ± 5%, n = 5). The K⁺ current is poorly sensitive to toxins specific of Kv1 α-subunits: NTX, KTX, DTX and BgK were all applied at 200 nM (n = 46). Currents were elicited at 20 mV from a holding potential V_h = -60 mV (TEA, KTX, DTX) or V_h = -80 mV (NTX, BgK).

Figure 2. Evolution of I_Na, I_A and I_DR during the first 8 days of cell culture

A, recordings performed in the presence of 30 nM TTX. Outward currents elicited at DIV2 by voltage pulses from -40 to 20 mV applied from two holding potentials, V_h = -80 mV (a) and V_h = -60 mV (b). The fast transient A-current was isolated by subtracting series b from series a. This current was partially blocked (64%) by 1 mM 4-AP. B, amplitude (mean ±s.e.m., n = 4 to 10 experiments) of I_Na, I_DR and I_A peaks at various days of growth. I_A and I_DR were recorded at +20 mV, and I_Na at -30 mV.

Figure 3. Excitability and spontaneous activity of motoneurones in vitro

A, action potentials in the whole-cell configuration resulting at DIV5 from a suprathreshold depolarizing current pulse of 3 nA applied for 200 ms. A longer stimulation pulse of 3 nA applied for 1.25 s (right-hand trace) induced a tonic discharge at 14 Hz. B, spontaneous action potentials recorded at DIV6 in the cell-attached configuration. Inset: detail of one action potential. C, spontaneous action potentials recorded at DIV7 in the whole-cell configuration, just after seal rupture.

Figure 4. Sharply activating sodium current

A, I_Na elicited at DIV8 by applying voltage pulses from -50 to -47 mV in 1 mV incremental steps, from V_h = -80 mV. The Na⁺ current was almost completely activated by changing the voltage pulse level from -49 to -48 mV. B, left panel: Na⁺ and K⁺ currents elicited at DIV2 by applying voltage pulses from -40 to 0 mV (10 mV increment) from a holding potential V_h = -60 mV. The intracellular pipette contained CsCl, which reduced the K⁺ currents. Right panel: same procedure performed after crushing the longest and thinnest neurite (presumed to be the axon).

Figure 5. Antidromic stimulation of the various neurites of a motoneurone

All the neurites of this motoneurone (DIV7) were stimulated with extracellular bipolar electrodes in order to generate an antidromic action potential. Somatic recordings were carried out in the cell-attached mode. A stimulation pulse of 0.1 ms, 8 V generated an antidromic potential in only one neurite (inset 1). When the stimulation was applied to the same neurite at a point approximately 600 μm from the soma, the antidromic potential reached the soma with a latency of 2.3 ms (inset 2), which corresponds to a propagation velocity of ≈0.3 m s⁻¹. In insets 3 and 4, stimulation pulses of 0.3 ms, 15 V applied to the other processes were ineffective.

Figure 6. Distribution of ¹²⁵I-α-scorpion toxin binding sites at the surface of motoneurones in vitro

A-D, motoneurones cultured for 18 h (D), 2 days (B) and 6 days (A and C) were incubated with 1 nM ¹²⁵I-α-scorpion toxin and 100 nM TTX, in the absence (A, B and D) or presence (C) of 100 nM native α-scorpion toxin. A high density of silver grains was observed at the initial segment of the axons (arrows), whereas the other neurites and the somata were not labelled. Growth cones (arrowheads) were also labelled. E, motoneurones cultured for 18 h were immunolabelled with a monoclonal anti-GAP 43 antibody. Growth cones (arrowheads) were heavily labelled. Scale bar in A also applies to B-D; scale bar in E, 20 μm.

Figure 7. I_Na is dependent on neuritic outgrowth

A, ionic currents recorded 1 h after the plating, when the motoneurones were devoid of neurites. I_Na was absent, whereas I_A and I_DR were already expressed. Voltage pulses: -50 to 10 mV in 20 mV incremental steps. B, same conditions as in A. Experiment performed 18 h after the plating when neurites had developed. Note the expression of I_Na.

Figure 8. Effect of nocodazole on Na⁺ channel expression

A-C, motoneurones grown for 18 h in the absence (A) or presence (B and C) of 30 μM nocodazole, labelled with 1 nM ¹²⁵I-α-scorpion toxin in the presence of 100 nM TTX and processed for autoradiography. D, experiments performed at DIV1. Ionic currents elicited by voltage pulses at -40 to 0 mV in 10 mV incremental steps, under control conditions (left panel) and after treatment with nocodazole (30 μM, right panel). Nocodazole was added to the culture medium immediately after the plating. The nocodazole treatment abolished the Na⁺ current expression and partly reduced the delayed K⁺ current. V_h = -60 mV. Scale bar in A also applies to B and C.