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. 2015 Feb 17;10(2):e0117555.
doi: 10.1371/journal.pone.0117555. eCollection 2015.

Preventing effect of L-type calcium channel blockade on electrophysiological alterations in dentate gyrus granule cells induced by entorhinal amyloid pathology

Hamid Gholami Pourbadie  1 , Nima Naderi  2 , Nasrin Mehranfard  3 , Mahyar Janahmadi  4 , Fariba Khodagholi  5 , Fereshteh Motamedi  6
Affiliations

Preventing effect of L-type calcium channel blockade on electrophysiological alterations in dentate gyrus granule cells induced by entorhinal amyloid pathology

Hamid Gholami Pourbadie et al. PLoS One. .

Abstract

The entorhinal cortex (EC) is one of the earliest affected brain regions in Alzheimer's disease (AD). EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG) with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs), nimodipine and isradipine, were investigated. The amyloid beta (Aβ) 1-42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from Aβ treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin) accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days), almost preserved the normal excitability against Aβ. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer's disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Excitatory neurotransmission onto the dentate gyrus granule cells is reduced in the granule cells from Aβ treated rats.
A, Spontaneous EPSCs were less frequent in the EC-Aβ (lower trace) than control (upper trace) neurons with unaltered kinetics. Far right panel shows the analysis of the time constant of rise and decay phases and examples of sEPSCs traces of granule cells from control and Aβ treated groups which were best fit with single exponential function. Cumulative probability plot (B) and mean amplitude (C) of spontaneous EPSCs were unaltered. D, cumulative probability plots showed that spontaneous events in the EC-Aβ neurons shifted to longer inter-event intervals (IEIs) (lower frequencies). E, Daily i.c.v. treatment by nimodipine produced a significant trend (p = 0.07) towards reversing decreased sEPSCs frequency (2.569 ± 0.25 Hz, n = 6 cells/4 rats) (Fig. 1D and E). Values are mean ± SEM. **p < 0.01 and ***p < 0.001 contrasted to the control cells. Control and EC-Aβ group, n = 6 cells/ 5 rats; EC-Aβ + ISR and EC-Aβ + NIM, n = 6 cells/ 4 rats.
Fig 2
Fig 2. Calcium channel blockers, isradipine and nimodipine, prevent abnormalities in spontaneous IPSCs of DG granule cells caused by amyloid pathology in the EC.
A, amplitude of spontaneous IPSCs increased in the EC-Aβ (lower trace) in contrast with the control (upper trace) neurons and there is a significant decrease in monoexponential decay time constant (fast inactivated) compared to the control cells (right plot, p < 0.05). B, cumulative probability plots showed that spontaneous events in the granule cells from Aβ treated rats shifted to larger amplitude (**p = 0.004 by Kolmogorov–Smirnoff test) and the change was blocked by isradipine and nimodipine. C, the amplitude of sIPSC significantly increased in granule cells from Aβ treated rats compared to the control, and isradinpine and nimodipine blocked the change. Cumulative probability plot (D) and mean frequency of spontaneous IPSCs (E) were unaltered between groups. Values are mean ± SEM. ***p < 0.001 contrasted to the control cells, ##p < 0.01 and #p < 0.05 compared to EC-Aβ group. Control group, n = 6 cells/ 4 rats; EC-Aβ group, n = 8 cells/ 6 rats; EC-Aβ + ISR and EC-Aβ + NIM, n = 6 cells/ 4 rats.
Fig 3
Fig 3. CCBs preserve normal Rin against EC-Aβ in the granule cells.
A, Rin was significantly decreased in the granule cells from Aβ treated rats compared to the control group. Treatment by istradipine and nimodipine preserved Rin against Aβ. B, Rin of granule cells in presence of synaptic blockers (CNQX; 10 μM, APV; 50 μM and bicuculline; 20 μM) in different groups. Values are mean ± SEM. *p < 0.05 and ***p < 0.001 compared to the control group, #p < 0.05, ###p < 0.001 contrasted to the EC-Aβ group. Control group, n = 15 cells/ 6 rats; EC-Aβ group, n = 15 cells/ 8 rats; EC-Aβ + ISR, n = 10 cells/ 6 rats; EC-Aβ + NIM, n = 7 cells/ 5 rats.
Fig 4
Fig 4. Changes of excitability of granule cells following treatment with Aβ and the protective effect of CCBs.
A, a small current injection was sufficient to evoke an AP in the control granule cells, but not in the EC-Aβ cells. B, the current required to evoke minimal AP firing in EC-Aβ granule cells (right panel) evoked strong AP firing in the control cells (left panel). C, the minimal current to evoke one AP during one second (rheobase current) is increased in the EC-Aβ granule cells compared to the control group. Treatment with calcium channel blockers, isradipine and nimodipine, preserved rheobase current in the EC-Aβ granule cells. D and E, the rheobase related to the Rin in the control and granule cells from Aβ treated rats, respectively. Note different y and x axis scales in these panels. Values are mean ± SEM. ***p < 0.001 compared to the control group and ##p < 0.01 compared to the EC-Aβ group. Control group, n = 15 cells/ 6 rats; EC-Aβ group, n = 15 cells/ 8 rats; EC-Aβ + ISR, n = 10 cells/ 6 rats; EC-Aβ + NIM, n = 7 cells/ 5 rats.
Fig 5
Fig 5. Firing properties of DG granule cells evoked by current injections.
A, Aβ pathogenesis in the EC produced changes in firing pattern and decreased firing rate of DG granule cells at different current injections. Co-treatment with calcium channel blockers, isradipine or nimodipine, almost restored the normal firing pattern. B, Number of action potentials evoked at different current steps from 50 to 300 pA with intact synaptic transmission. C, Number of action potentials evoked at different current injections after synaptic transmission blockade in the presence of CNQX; 10 μM, APV; 50 μM and bicuculline; 20 μM, the difference of AP numbers still remained between the control and EC-Aβ cells. Values are mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 contrasted to control cells, ##p < 0.01 and ###p < 0.001 compared to EC-Aβ group. Control group, n = 12 cells/ 6 rats; EC-Aβ group, n = 12 cells/ 8 rats; EC-Aβ + ISR, n = 10 cells/ 6 rats; EC-Aβ + NIM, n = 7 cells/ 5 rats.
Fig 6
Fig 6. Firing properties of dentate gyrus granule cells in response to ramp currents.
A, representative spike firings of DG granule cells in response to ramp current clamp. Aβ microinjection into the EC induced: a significant increase in latency of the first AP (B), a significant increase of current required to evoke the first AP (C), and a significant decrease in AP numbers (D). Treatment by isradipine and nimodipine preserved granule cells from these changes. *p < 0.05 and **p < 0.01 compared with the control cells. #p < 0.05 and ##p < 0.01 compared to the EC-Aβ group. Control and EC-Aβ groups, n = 6 cells/ 4 rats; EC-Aβ + ISR, n = 5 cells/ 4 rats; EC-Aβ + NIM, n = 6 cells/ 4 rats.
Fig 7
Fig 7. Dentate gyrus TUNEL positive cells one week after inducing amyloid pathogenesis in the EC.
A, top panel indicates which region has been quantified and middle and bottom panels indicate several TUNEL positive neurons (arrows) in granule cell layer and hilus of the dentate gyrus, respectively, after Aβ microinjection into the EC. Isradipine and nimodipine could decrease TUNEL positive cells in the granule layer of Aβ treated rats. Light microscope (400x) was utilized for taking picture. B, The quantitative analysis of the data. C, no significant difference was observed between groups regarding to TUNEL-positive hilar neurons. Values are mean ± SEM. ***p < 0.001 compared to the control and ###p < 0.001 compared to the EC-Aβ group, n = 6.
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Grants and funding

This work was supported by a grant from Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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