One of the primary means of loading the main neuronal calcium store, the endoplasmic reticulum (ER), is via neuronal store-operated calcium entry (nSOCE). As such, nSOCE likely plays an important role in regulating synaptic plasticity and spine growth, and its chronic dysregulation may also play a role in neurodegenerative conditions such as Alzheimer’s disease.

A key feature of nSOCE is that its initiation is dependent upon a rapid and significant degree of intracellular rearrangement between the ER and plasma membrane. However, the mechanisms regulating this rearrangement in neurons remain unclear. Previous studies have examining non-excitable cells have reported a role for microtubules in regulating SOCE activation, but it has not yet been established if a similar SOCE mechanism occurs in neurons. The current study therefore investigated whether microtubule stabilisation or depolymerisation played a role in regulating neuronal SOCE in cultured hippocampal neurons.

Cultured primary hippocampal neurons were prepared from 3-5 day old C57BL/10 mice on glass coverslips, and used for experimentation between 7-12 days in culture. Neurons were. Experiments were conducted by first loading neurons with the calcium-sensitive dye, Calbryte 520 AM (4μM) for 1 hour. Neurons were then superfused (2ml/min) with Ca²⁺-free HEPES-buffered saline solution (HBSS) containing the irreversible SERCA pump inhibitor, thapsigargin (1mM; 15mins), to empty ER calcium stores and prime the nSOCE apparatus. nSOCE was then induced by superfusing with HBSS containing Ca²⁺ (2mM; 15 mins). Somatic calcium responses (F/F₀) of  individual cells were recorded and measured using the calcium imaging software, Winfluor (John Dempster, Strathclyde University). Percentage peak nSOCE calcium responses (relative to baseline) in untreated (control) neurons were compared to those evoked in the presence of either the microtubule depolymeriser colchicine (10mM) or the microtubule stabilizer, paclitaxel (10mM). Statistical analyses were conducted using one way ANOVA with Dunnett’s multiple comparisons test, with values of P < 0.05 considered statistically significant. All data are expressed as mean ± S.D.

Activation of nSOCE increased intracellular calcium levels above basal levels by 10.3 ± 11.7%, n = 243) in untreated neurons. Colchicine (10mM) did not significantly modify peak nSOCE responses relative to control, inducing a peak nSOCE amplitude of 9.7 ± 6.1% (n = 97; P = 0.99) above baseline. However, in the presence of the microtubule stabiliser, paclitaxel (10mM), peak nSOCE amplitude was significantly reduced to 6.1 ± 4.0% (n=62; 40.7% reduction relative to control; P = 0.03) of control nSOCE responses.

The preliminary data presented here suggest that microtubule stabilisation exerts a significant inhibitory effect on nSOCE activation, possibly by inhibiting the dynamic microtubule reconfiguration required to initiate nSOCE. It is unclear however, why microtubule depolymerisation with colchicine did not exert a similar inhibitory effect on nSOCE.