Mechanism of progesterone neuroprotection of rat cerebellar Purkinje cells following oxygen-glucose deprivation

Abstract

The survival of rat Purkinje cell (PCs) cerebellar cultures was used to test the hypothesis that progesterone is protective against oxygen-glucose deprivation through potentiation of GABA(A) receptor activity. Electrophysiological recordings confirm that PCs develop robust excitatory and inhibitory synapses in culture. Exposure of cultured PCs to increasing concentrations of progesterone during oxygen-glucose deprivation revealed a concentration-dependent protection by progesterone, with significant protection observed at physiological concentrations, as low as 10 nm. The concurrent application of the GABA(A) receptor antagonist picrotoxin (100 microm) completely abolished the neuroprotection afforded by progesterone, indicating that progesterone is neuroprotective through activation of GABA(A) receptors. Progesterone potentiates GABA(A) receptor activity indirectly through its metabolites, such as allopregnanolone (ALLO). Therefore, ALLO was applied to PC cultures and was observed to produce significant protection at all concentrations tested, from 10 to 1000 nm. Finally, the inhibition of progesterone metabolism with finasteride abolished the protection afforded by progesterone without having any effect on the neuroprotection caused by ALLO. These data indicate that progesterone protects cerebellar PCs at physiological concentrations through a GABA-active metabolite.

Fig. 1

Cultured cerebellar PCs generate intact inhibitory and excitatory synapses. (A) Representative trace of sEPSCs recorded in the presence of 100 µm picrotoxin. (B) Average of multiple events fitted to determine rise and decay time constants. (C) Representative trace of spontaneous inhibitory postsynaptic currents recorded in the presence of 5 µm CNQX. Note different timescale from trace A. (D) Average of multiple events fitted to determine rise and decay time constants. (E) Trace recorded in the presence of both picrotoxin and CNQX.

Fig. 2

Exposure of cultured cerebellar PCs to 2 h OGD resulted in significant cell death as indicated by TUNEL staining. (A) Representative images of control cultures double-stained with anticalbindin (red) to identify PCs and TUNEL (green) to indicate DNA damage. (B) Representative images of the significant damage observed in PCs following 2 h OGD, as indicated by the high number of yellow (merged) cells.

Fig. 3

Male and female PCs were equally sensitive to OGD. (A) Quantification of the differential sensitivity of cerebellar neurons exposed to 2 h of OGD, indicating that PCs are significantly more sensitive to OGD than non-PC cerebellar neurons. (B) Quantification of damage observed in male and female PCs exposed to 1 or 2 h of OGD. The percentage of TUNEL-positive PCs was assessed following 3 h reoxygenation. Numbers in each bar represent number of experiments performed, each on a different culture. Data are mean ± SEM. *P < 0.05 vs. vehicle-treated cells (2 h OGD).

Fig. 4

Progesterone protected PCs in a concentration-dependent manner. Male and female PCs were exposed to the various concentrations of progesterone for 15 min prior to OGD and maintained throughout reoxygenation. Data are mean ± SEM. *P < 0.05 vs. vehicle-treated cells (2 h OGD) from the same sex.

Fig. 5

Progesterone and its metabolite ALLO protected PCs via activation of GABA_A receptors. (A) Quantification of the neuroprotective effect of exposure to 1 µm progesterone (PROG) for 15 min prior to OGD and maintained throughout reoxygenation. The additional presence of 100 µm picrotoxin prevented the protection afforded by progesterone. (B) Quantification of the neuroprotective effect of exposure to 1 µm ALLO for 15 min prior to OGD and maintained throughout reoxygenation. The additional presence of 100 µm picrotoxin prevented the protection afforded by ALLO. Data are mean ± SEM. *P < 0.05 vs. vehicle-treated cells (2 h OGD).

Fig. 6

Progesterone neuroprotection required its metabolism. Quantification of the ability of finasteride to prevent progesterone neuroprotection. PCs were preincubated with 10 µm finasteride for 15 min, then exposed to either 1 µm progesterone or ALLO for another 15 min prior to OGD and maintained throughout reperfusion. Data are mean ± SEM. *P < 0.05 vs. vehicle-treated cells (2 h OGD).

Fig. 7

Progesterone and ALLO effectively protected PCs when applied after OGD. Progesterone and ALLO were applied to PC cultures concurrent with reoxygenation (PROG or ALLO @reoxygenation). Data are mean ± SEM. *P < 0.05 vs. vehicle-treated cells (2 h OGD).