Fig. 1. 1,25(OH)₂D enhanced activity-dependent cytosolic Ca²⁺ levels in a subset of PFC neurons.

a Schematic of field stimulation configuration and analysis. b Representative PFC region bulk-loaded with Cal-520, zoomed in (white box) to show difference between unstimulated (c) and stimulated (d) fluorescence intensity in electrically responsive cells. e Bath application of L-VGCC agonist Bay K8644 evoked increased Ca²⁺ ΔF/F in single neurons within 3 min. f Bay K8644 evoked an increase in Ca²⁺ ΔF/F in almost half the imaged neurons (average ΔF/F trace, n = 38), while application of equimolar DMSO (10⁷-fold dilution, equivalent to dilution of 1,25(OH)₂D stock) produced a negligible effect on Ca²⁺ ΔF/F (1/144 cells, 0.7%). g The majority of neurons showed no change in Ca²⁺ ΔF/F following bath application of 1,25(OH)₂D (average ΔF/F trace, n = 427). h Raw (left) and analysed (right) Ca²⁺ imaging data from a single representative VDRN (directly responsive). i Average ΔF/F trace of all detected VDRN (n = 53). j 1,25(OH)₂D induced a smaller change in the mean single cell Ca²⁺ ΔF/F compared to Bay K8644 (p < 0.0001). k Range of response times to 1,25(OH)₂D was significantly larger compared to Bay K8644 (p < 0.0001). l Representative Cal-520 bulk-loaded slice (imaged in the presence of synaptic blockers) labelling detected VDRN with orange arrow heads. m Neither the percentage of VDRNs detected per imaged slice (p = 0.6) nor from the total pool of imaged neurons (n) was significantly altered by the presence (orange) or absence (blue) of synaptic blockers. Data = Mean ± SEM (unpaired two-tailed t test with Welch’s correction, ****p < 0.0001)