Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival

Neural activity is significantly reduced in the spinal cords of SMA mice.

<p><b>A</b>. Example of a slice from the lumbar spine with only the ventral horn positioned over the electrodes of the MED probe. <b>B</b>. Two littermates from the Smn^−/−;SMN2 strain of mice at P4 showing the difference in size of the wild type and mutant pups. <b>C</b>. Average number of active electrodes recorded for each slice. One Way ANOVA, p<0.0001 <b>D</b>. Average spike frequency recorded by each active electrode. <i>p</i> = 0.46, One way ANOVA.</p

Excitatory and inhibitory input in motor neurons cultured from wild type and SMA mice.

<p><b>A</b>. Sample traces of the sEPSCs recorded on day 7 and day 17 in control and SMA cells. <b>B</b>. Summary data of the frequency of sEPSCs during the 2^nd and 3^rd week of culture in control and SMA cells. <b>C</b>. Summary data of the amplitude of sEPSCs during the 2^nd and 3^rd week culture in control and SMA cells. <b>D</b>. Sample traces of the sIPSCs at day7 and day17 in control and SMA mice. <b>E</b>. Summary data of the frequency of sIPSCs during the 2^nd and 3^rd week of culture in control and SMA cells. <b>F</b>. Summary data of the amplitude of sIPSCs during the 2^nd and 3^rd week culture in control and SMA mice. We did not find any significant difference in the frequency or amplitude of either excitatory or inhibitory PSPs between the control and SMA cultures (<i>p</i>>0.05, One-way ANOVA).</p

In SMA slices the overall distribution of spike frequencies is not significantly changed by serotonin.

<p>Histograms of spike frequency comparing slices from control and SMA mice. The number of observations was normalized to the total number of observations in each slice and divided into. 0.5 Hz bins. The distributions were fitted with mixed Gaussian approximations using the Expectation-Maximization (EM) algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011696#pone.0011696-Alpaydin1" target="_blank">[26]</a>. Serotonin significantly shift the frequency distribution in control slices (p<0.0001), but the shift is not significant in the SMA slices (p = 0.1433; Kruskal-Wallis).</p

Sample traces show the reduced activity recorded from the ventral horns of SMA mice.

<p>Sample traces showing spike activity recorded from all active electrodes in a single slice from a control and an SMA mouse showing activity in both basal and serotonin-stimulated conditions. Slices from SMA mice had many fewer active electrodes than controls, and showed fewer neurons active after serotonin treatment.</p

Motor neuron activity in both control and SMA slices is stimulated by serotonin.

<p>50 µM of serotonin was added to the bath and neuronal activity was recorded after 2 minutes. For the wash condition, slices were perfused with aCSF for 10 minutes before recording resumed. <b>A</b>. Average number of active electrodes/slice (active electrodes are those that recorded more than 50 spikes in 50 seconds). One way ANOVA, p<0.0001 for both control and SMA. <b>B</b>. Average frequency of spikes recorded on active electrodes. One way ANOVA, p<0.0001 for control, p = 0.137 for SMA.</p

Pharmacological agents cause little change in activity of control or SMA slices.

<p>Acetylcholine (100 µM), bicuculline (10 µM) or GABA (20 µM) were bath applied to the slice and recording was resumed after 2 minutes. For wash, slices were perfused with aCSF for 10 minutes before recording was resumed. Averages are calculated for 12 slices from 3 control mice and 11 slices from 3 SMA mice.</p><p>*p<0.05 (One-way ANOVA). Active electrodes are those electrodes recording spiking activity of at least 1 Hz over a 50 second period. Number of spikes per minute totals the number of spikes recorded on all electrodes recording neural activity from a slice.</p