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. 2017 Nov 19:364:130-142.
doi: 10.1016/j.neuroscience.2017.09.019. Epub 2017 Sep 20.

Novel calcium-related targets of insulin in hippocampal neurons

Shaniya Maimaiti  1 , Hilaree N Frazier  1 , Katie L Anderson  1 , Adam O Ghoweri  1 , Lawrence D Brewer  1 , Nada M Porter  1 , Olivier Thibault  2
Affiliations

Novel calcium-related targets of insulin in hippocampal neurons

Shaniya Maimaiti et al. Neuroscience. .

Abstract

Both insulin signaling disruption and Ca2+ dysregulation are closely related to memory loss during aging and increase the vulnerability to Alzheimer's disease (AD). In hippocampal neurons, aging-related changes in calcium regulatory pathways have been shown to lead to higher intracellular calcium levels and an increase in the Ca2+-dependent afterhyperpolarization (AHP), which is associated with cognitive decline. Recent studies suggest that insulin reduces the Ca2+-dependent AHP. Given the sensitivity of neurons to insulin and evidence that brain insulin signaling is reduced with age, insulin-mediated alterations in calcium homeostasis may underlie the beneficial actions of insulin in the brain. Indeed, increasing insulin signaling in the brain via intranasal delivery has yielded promising results such as improving memory in both clinical and animal studies. However, while several mechanisms have been proposed, few have focused on regulation on intracellular Ca2+. In the present study, we further examined the effects of acute insulin on calcium pathways in primary hippocampal neurons in culture. Using the whole-cell patch-clamp technique, we found that acute insulin delivery reduced voltage-gated calcium currents. Fura-2 imaging was used to also address acute insulin effects on spontaneous and depolarization-mediated Ca2+ transients. Results indicate that insulin reduced Ca2+ transients, which appears to have involved a reduction in ryanodine receptor function. Together, these results suggest insulin regulates pathways that control intracellular Ca2+ which may reduce the AHP and improve memory. This may be one mechanism contributing to improved memory recall in response to intranasal insulin therapy in the clinic.

Keywords: aging; diabetes; electrophysiology; excitability; imaging; intranasal.

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Figures

Figure 1
Figure 1
Calcium channel currents are reduced in response to acute insulin application in mixed hippocampal cultured neurons. A. Example of VGCC currents recorded during recording solution perfusion (black) and after 10 min insulin (grey) during maximal step depolarization from −70 mV. B.-C. Quantification across groups of cells following 10 min of insulin perfusion (Insulin) versus recording solution perfusion (Time Control) shows peak and late Ca2+ currents were significantly reduced (p<0.05). D. In a subgroup of cells, current-voltage relationships were not significantly altered. E. Example of the effects of insulin on L-VGCCs recorded from a holding potential of −40 mV during recording solution perfusion (black) and after 10 min insulin perfusion (gray). F.-G. Compared to time control, recordings from −40 mV, max peak (F) and late Ca2+ current (G) were significantly reduced by insulin (p<0.05). The effect of insulin on VGCC does not appear to be L-VGCC selective. Data are presented as mean +/− SEM except for data in F, right, which represent median with interquartile ranges. Data are normalized to cell size (density, pA/pF) and asterisks indicate significance at the p<0.05 level.
Figure 2
Figure 2
Spontaneous Ca2+ transients are not altered in response to acute insulin exposure. A. Example of neurons and ROI selection (green circles and numbered arrows- left). Calibration bar = 10 μm. Right shows spontaneous activity in the three ROIs (used to quantify insulin-mediated changes). Measures were taken during two 10 min periods of imaging solution perfusion (Time Control) or during a 10 min period of control solution followed by 10 min of insulin perfusion (Insulin). B.-D. Insulin perfusion did not change spontaneous calcium activity based on measures of AUC, peak transient amplitude, or the number of events detected. Data are presented as mean +/− SEM except for data in D, which represent median with interquartile ranges.
Figure 3
Figure 3
Effects of insulin on KCl-induced depolarization. A. Pseudocolored images of cells in culture during Fura-2 imaged at rest (left) and during peak depolarization (right). Calibration bar = 10 μm. Red colors indicate greater levels of calcium (higher ratios). Two sequential KCl depolarizations were separated by 10 minutes of either control solution or insulin. Representative calcium transients are shown and highlight measures of resting calcium, peak calcium and AUC. Note the selective effect of insulin on reducing the late phase of the calcium transient, essentially eliminating the “hump”. B. Ratiometric quantification during time control experiments or after insulin shows the AUC resulting from KCl-induced depolarization was significantly decreased by insulin. C.-D. Peak ratio or resting ratio measures were not altered by insulin. Data are presented as mean +/− SEM except for data in C, right, which represent median with interquartile ranges. Asterisks indicate significance at the p<0.05 level.
Fig 4
Fig 4
Ryanodine receptors are likely targets of insulin actions. A. Three sequential KCl-mediated depolarizations were used in these experiments and were triggered under imaging solution perfusion (left) and during perfusion with ryanodine, or insulin (right). Representative ratiometric calcium transients highlight the impact of inhibiting CICR with ryanodine on the “hump”. Addition of insulin did not further reduce the “hump”. B. Peak ratios were not different across groups. C. Quantification across the groups of cells imaged shows ryanodine (Rya) significantly reduced the “hump”, indicating ryanodine and insulin may both be working on inhibition of RyRs. All data represent mean +/− SEM. Asterisks indicate significance at the p<0.05 level.
Fig 5
Fig 5
Anti-insulin affibody peptide and anti-insulin receptor antibody inhibited insulin actions on KCl-mediated Ca2+ transients. Experiments were conducted as in Figure 3, with 2 sequential KCl depolarizations. A. High-dose (1 μg/mL) anti-insulin affibody neutralized the effect of insulin on KCl-induced Ca2+ transients while lower concentrations (100 ng/mL and 500 ng/mL) did not. B. 500 ng/mL insulin receptor antibody also was able to inhibit insulin actions on the KCl-mediated Ca2+ transients. These results indicate the actions reported here on calcium homeostasis are mediated by insulin working on insulin receptors. All data represent mean +/− SEM. Asterisks indicate significance at the p<0.05 level.
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