Static magnetic stimulation induces structural plasticity at the axon initial segment of inhibitory cortical neurons

Abstract

Static magnetic stimulation (SMS) is a form of non-invasive brain stimulation that alters neural activity and induces neural plasticity that outlasts the period of stimulation. This can modify corticospinal excitability or motor behaviours, suggesting that SMS may alter the intrinsic excitability of neurons. In mammalian neurons, the axon initial segment (AIS) is the site of action potential initiation and undergoes structural plasticity (changes in length and position from the soma) as a homeostatic mechanism to counteract chronic changes in neuronal activity. We investigated whether the chronic application of SMS (6 and 48 h, 0.5 T) induces structural AIS plasticity in postnatally derived primary cortical neurons. Following 6 h of SMS, we observed a shortening in mean AIS length compared to control, that persisted 24 h post stimulation. In contrast, 48 h of SMS induced an immediate distal shift that persisted 24 h post-stimulation. Pharmacological blockade of voltage gated L/T-type calcium channels during stimulation did not prevent SMS-induced AIS structural plasticity. Our findings provide the foundation to expand the use of chronic SMS as a non-invasive method to promote AIS plasticity.

Figure 1

Maximum intensity z-projection of immunolabelled inhibitory cortical neurons in cell culture at DIV7. Neurons were immunolabeled for (a) the neuronal marker MAP2, (b) AIS marker AnkG, (c) inhibitory neuronal marker GAD65/67and (d) nuclear marker Hoechst. Images were (e) merged and AIS length and distance from soma were quantified. (f) A magnified image of the white box in (e). Filled triangles indicate the approximate start and end of the AIS and the unfilled triangle indicates the approximate edge of the soma used in AIS distance quantification. Images were captured on a C2 confocal microscope at 60 × magnification. Scale bars in (a–e) = 50 µm; (f) = 25 µm.

Figure 2

Cumming estimation plots of AIS lengths obtained from cultured cortical neurons treated with 15 mM KCl or 0.5 T SMS. (a) Raw data of AIS lengths obtained from individual neurons (top) and mean differences of treatment compared to the shared Control (bottom) plotted as bootstrap sampling distributions. Neurons were fixed immediately after (left panel) and 24 h after (right panel) 6 h of stimulation. (b) Raw data (top) of AIS lengths and mean differences (bottom) obtained immediately after (left panel) and 24 h after (right panel) 48 h of stimulation. The number of neurons analysed in each group are presented under the group names. Each mean difference in the bottom graph is depicted as a dot with 95% confidence intervals indicated by the vertical error bars. Data from each treatment group at each time point was collected from at least 5 coverslips over a minimum of 3 separate culture experiments.

Figure 3

Cumming estimation plots of the proximal start of the AIS from the soma obtained from cultured cortical neurons treated with 15 mM KCl or 0.5 T SMS. (a) Raw data of AIS distance from the soma obtained from individual neurons (top) and the mean differences of treatment compared to the shared control (bottom) plotted as bootstrap sampling distributions. Neurons were fixed immediately after (left panel) and 24 h after (right panel) 6 h of stimulation. (b) Raw data (top) of AIS distance from the soma and mean differences (bottom) obtained immediately after (left panel) and 24 h after (right panel) 48 h of stimulation. The number of neurons analysed in each group are presented under the group names. Each mean difference in the bottom graph is depicted as a dot with 95% confidence intervals indicated by the vertical error bars. Data from each treatment group at each time point was collected from at least 5 coverslips over a minimum of 3 separate culture experiments.

Figure 4

Quantification of AIS length and distance from soma immediately after 6 h of stimulation with 3 µM of mibefradil dissolved in the culture medium of all treatment groups. (a) Cumming plots of AIS length and (b) AIS distance from soma. Raw data obtained from individual neurons (top) and the mean differences of treatment compared to shared control (bottom) plotted as bootstrap sampling distributions. Number of neurons analysed in each group presented in brackets under the group names. Data from each treatment group was collected from at least 3 coverslips from 3 separate culture experiments.