J. Neurochem. (2012) 121, 44–53.AbstractIn this study, we report that spiro[imidazo[1,2‐a]pyridine‐3,2‐indan]‐2(3H)‐one (ST101; previously coded as ZSET1446) targets T‐type voltage‐gated calcium channels in mediating improved cognition in the CNS. We prepared rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiological analyses using various voltage‐gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased calcium/calmodulin‐dependent protein kinase II (CaMKII) autophosphorylation following a bell‐shaped dose–response curve, with 0.1 nM ST101 representing the maximally effective concentration. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice preparations. Consistent with increased cortical CaMKII autophosphorylation, AMPA‐type glutamate receptor subunit 1 (Ser‐831) phosphorylation as a CaMKII post‐synaptic substrate was significantly increased by treatment with 0.1–1 nM ST101, whereas phosphorylation of the pre‐synaptic substrate synapsin I (Ser‐603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre‐treatment with 1 μM mibefradil, a T‐type VGCC inhibitor, but not with N‐type (ω‐conotoxin), P/Q‐type (ω‐agatoxin) or L‐type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long‐term potentiation in cortical but not hippocampal slices. Enhanced long‐term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole‐cell patch‐clamp analysis of Neuro2A cells over‐expressing recombinant human CaV3.1 (α1G) T‐channels and treated with 0.1 nM ST101 showed significant increases in T‐type VGCC currents. These results indicate that T‐type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.