Molecular determinants of emerging excitability in rat embryonic motoneurons

Abstract

Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na(+) current is supported by Nav1.2 and/or Nav1.3 alpha-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Nav beta 2.1 transcript was not detected, whereas the Nav beta 1.1 transcript was present. Even in the absence of Nav beta 2.1, alpha-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K(+) currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K(+) current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv alpha-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K(+) current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na(+) and K(+) channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.

Figure 1. RT-PCR analysis of sodium channel transcripts in cultured motoneurons

A, cDNA obtained from four different motoneuron cultures (1-4) at DIV 0 (0) or DIV 7 (7) was amplified by PCR with primer pairs Na1-1, Na2-1, Na3-1 and Na6-1 (specific for Nav1.1, Nav1.2, Nav1.3 and Nav1.6, respectively) and with primer pair GAPDH. Simultaneous amplification of gDNA was carried out. Positive controls on cDNA from adult rat brain (Ad brain) were also performed (equivalent to 1 ng total RNA for α-subunits or 100 pg for GAPDH amplification). PCR products after 34 (no asterisk) and 39 cycles (asterisk) of amplification are shown for each cDNA and for gDNA. B, relative expression of different Na⁺ channel α-subunits in cultured motoneurons. Semi-quantification of the signal amplified from motoneuron cDNA was achieved after normalization to the corresponding signal amplified from genomic DNA. Experimental conditions were the same as illustrated in A. Measurements of the signal were performed at 34 cycles (linear zone). Results are expressed as mean + s.e.m. of normalized values obtained from three (DIV 0, ▪) or two (DIV 7, □) independent cultures. C, top: cDNA obtained from cultured motoneurons at day 7 (MN 7) of culture (corresponding to 25 cells) was amplified by PCR with primer pairs GNB1-1 (specific for Navβ1) and with primer pair GAPDH. Simultaneous amplification of cDNA from adult rat brain (adult brain) was performed (2 and 0.25 ng of total RNA). Bottom: PCR amplification of Nav β2.1 with GNB2.1 primer set. cDNA from 10 motoneurons on day 0 (MN 0) or day 7 (MN 7) of culture are compared with cDNA from adult brain (Ad, 50 pg). PCR products after 34 (no asterisk) and 39 cycles (asterisk) of amplification are shown. D, cDNA obtained from cultured motoneurons at day 0 (MN 0) and day 7 (MN 7) of culture (corresponding to 25 cells) was amplified by PCR with primer pairs CHAT (specific for ChAT) and with primer pair GAPDH. Simultaneous amplification of genomic DNA (gDNA) was carried out.

Figure 2. Developmental regulation of alternative splicing of Nav1.6 and Nav1.1 transcripts in regions homologous to exon 18 of the mouse SCN8A gene

A, Nav1.6 alternative splicing: PCR was performed in parallel on cDNA obtained from motoneurons (50 cells) at 0 (MN 0) or 7 days (MN 7) of culture and on cDNA from 1 and 0.1 ng (100 pg) total RNA from adult rat brain (adult brain). PCR was conducted with the Na6A primer pair specific for the adult form of exon 18 (exon 18A) or with the Na6N primer pair specific for the neonatal form of exon 18 (exon 18N). PCR were performed as indicated in Fig. 1 legend with 34 and 39 cycles (asterisk). B, Nav1.1 alternative splicing in a region homologous to Nav1.1 exon 18: Comparative PCR (39 cycles) on cDNA from cultured motoneurons at 0 and 7 days of culture (MN 0 and MN 7) and on adult rat brain cDNA (adult brain), using primer pair Na-ex 18, clearly demonstrates a different amplification profile. The major band in adult brain cDNA had the expected size (331 bp) according to the primer pair position on Nav1.1 sequence. Conversely, PCR from motoneuron cDNA generated a preponderant band slightly above 400 bp.

Figure 5. Immunocytochemical characterization of Kv1.6, Kv1.4, Kv4.3 and α-subunit of sodium channels in cultured motoneurons

A, different optical sections (from left to right and top to bottom) obtained by confocal microscopy illustrate strong labelling of the different neuronal compartments with anti-Kv1.6 antibody. The bottom left image corresponds to the maximal projection of the different sections. B, anti-Kv1.4 faintly stains the intra-cellular compartment. C, immunolabelling of motoneurons with anti-Kv4.3 was granular and present in the different compartments including the axon (maximal projection from confocal microscopy). D, pan anti-sodium channel α-subunit clearly stains the initial segment of the axon (arrow) (DIV 7). E, anti-Nav1.2 labelling of axonal initial segment of a motoneuron at DIV 7 (arrow). F, on DIV 12, motoneuron initial segment is labelled by anti-Nav1.6 antibody.

Figure 3. RT-PCR analysis of potassium channel transcripts in cultured motoneurons

A, K⁺ channel α-subunits: RT-PCR was performed on cDNA from cultured motoneurons (corresponding to 8 cells) on day 0 (0) or 7 (7), on an equivalent amount of RNA (negative control) from the same cells and on 140 pg of rat gDNA (used as a standard allowing relative comparison of different transcripts in the same experiment). Amplification was performed as described in Fig. 1A legend (same amount of initial target and same number of cycles; asterisk indicates the 39th cycle). The transcripts corresponding to Kv1.1 (primer pair 11-2), Kv1.2 (primer pair 12 1) were not detected in motoneurons. A comparison of the intensity of cDNA and gDNA products indicates that Kv1.6, Kv4.2 and Kv4.3 are the most abundant transcripts. The weak signal with Kv3.4 on day 7 was not reproduced in other experiments in different cultures. B, K⁺ channel β-subunits: RT-PCR with specific primers for GAPDH, Kv β1 and Kv β2 was performed on cDNA prepared from 25 motoneurons from three different cultures on day 7 (MN 7) (1 to 3) and on rat adult brain cDNA (adult brain, 2 ng and 500 pg).

Figure 4. Detection of Kv4.2 and Kv4.3 transcripts in single cultured motoneurons

Single cell RT-PCR was performed as described in Methods. The same cells were analysed for Kv4.2 and Kv4.3 mRNA expression. The first round of PCR was conducted with multiplexed primers and the second round with specific primers for Kv4.3 (A) or Kv4.2 (B). Out of 5 cells analysed (numbered 1 to 5), 4 were positive for Kv4.2 (signal for cell 1 is faint) and 5 were positive for Kv4.3.

Figure 6. Activation and inactivation of the transient K⁺ current in cultured motoneurons

A, left traces: transient K⁺ currents obtained by subtracting the currents elicited by depolarizing pulses (-50 to 60 mV, in 10 mV step) from a holding potential of −60 mV from the currents elicited from −80 to 60 mV (10 mV step) from a holding potential of −100 mV. Right traces: transient K⁺ currents evoked by a depolarizing step to 20 mV following successive pre-pulses (1.4 s) from −120 to −10 mV. The test pulse recorded after a conditioning pulse of −10 mV was subtracted from each current trace. Plots: plot of the relative peak conductance (▪, right) as a function of the step voltage, and relative peak current (•, left) as a function of the conditioning potential. The continuous lines are Boltzmann functions. Each point is the mean of n = 5-8 experiments and error bars indicate s.e.m. The bath solution contained 3 μM TTX. B, plot of mean ± s.e.m. inactivation time constant (τ) as a function of step voltage from −50 to 60 mV (n = 12). The transient K⁺ currents were obtained using the procedure described in Fig. 6A. C, inset: the recovery from inactivation was determined by increasing the length of the interpulse (to −100 mV) applied between two depolarizing pulses. The conditioning and test pulses were elicited at 50 mV. The test pulse current recorded with an inter-pulse of 1 ms was subtracted from each recording. Plot: plot of the peak of the test pulse as a function of the inter-pulse duration. Data were fitted to the sum of two exponentials with time constants of 28 and 353 ms.

Figure 7. Pharmacological characterization of the transient K⁺ current

A, upper traces: outward currents evoked by a series of depolarizing steps from −40 to 20 mV from a holding potential of −100 mV. Currents were recorded in control conditions and after application of TEA (20 mm). Lower traces: transient K⁺ currents (I_A) resulting from the subtraction of currents evoked by various steps from −40 to 20 mV from two holding potentials −100 and −60 mV. Currents were recorded in control conditions and after application of 4 AP (1 mm). B, effects of ETYA on a global K⁺ current evoked by a step to 20 mV from a holding potential of −100 mV. C, plot of the normalized peak current as a function of the conditioning voltage, in the presence of CdCl₂ (500 μM) in the bath saline. Each point was the mean ± s.e.m. of n = 4 experiments. The continuous curve was determined as in Fig. 6A and the inactivation curve of Fig. 6A was also drawn. D, A-type current obtained from the subtraction of K⁺ currents evoked from two holding potentials of −100 and −60 mV, in control conditions and after application of PaTX₂ (100 nm). E, single spike elicited by current stimulation from −74 mV. Application of PaTX₂ (100 nm) increased overshoot and spike duration.