Ovariectomy reduces cholinergic modulation of excitatory synaptic transmission in the rat entorhinal cortex

Abstract

Estrogens are thought to contribute to cognitive function in part by promoting the function of basal forebrain cholinergic neurons that project to the hippocampus and cortical regions including the entorhinal cortex. Reductions in estrogens may alter cognition by reducing the function of cholinergic inputs to both the hippocampus and entorhinal cortex. In the present study, we assessed the effects of ovariectomy on proteins associated with cholinergic synapses in the entorhinal cortex. Ovariectomy was conducted at PD63, and tissue was obtained on PD84 to 89 to quantify changes in the degradative enzyme acetylcholinesterase, the vesicular acetylcholine transporter, and muscarinic M1 receptor protein. Although the vesicular acetylcholine transporter was unaffected, ovariectomy reduced both acetylcholinesterase and M1 receptor protein, and these reductions were prevented by chronic replacement of 17β-estradiol following ovariectomy. We also assessed the effects of ovariectomy on the cholinergic modulation of excitatory transmission, by comparing the effects of the acetylcholinesterase inhibitor eserine on evoked excitatory synaptic field potentials in brain slices obtained from intact rats, and from ovariectomized rats with or without 17β-estradiol replacement. Eserine is known to prolong the effects of endogenously released acetylcholine, resulting in an M1-like mediated reduction of glutamate release at excitatory synapses. The reduction in excitatory synaptic potentials in layer II of the entorhinal cortex induced by 15-min application of 10 μM eserine was greatly reduced in slices from ovariectomized rats as compared to intact rats and ovariectomized rats with replacement of 17β-estradiol. The reduced modulatory effect of eserine is consistent with the observed changes in cholinergic proteins, and suggests that reductions in 17β-estradiol following ovariectomy lead to impaired cholinergic function within the entorhinal cortex.

Fig 1. Ovariectomy results in reductions in proteins associated with cholinergic synaptic function in the entorhinal cortex.

Lysates were obtained from the medial and lateral entorhinal cortex (MEC and LEC), in groups of animals that received either sham surgery (Sham), ovariectomy (OVX), or ovariectomy and a subdermal implant containing 17-β estradiol (E2; OVX+E). A. Representative immunoblots of acetylcholinesterase (AChE) and the β-actin loading control are shown (A₁), and the bar graph shows relative expression of AChE protein (A₂; n = 6 per group). Note that the reduction in AChE induced by ovariectomy is prevented by administration of E2. Asterisks indicate levels of statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). B. No significant changes were observed in immunoblots (B₁) or normalized protein expression (B₂) for the vesicular acetylcholine transporter (VAChT; n = 6 per group; vinculin was the loading control). C. Representative immunoblots (C₁) and relative protein expression (C₂) indicate that the reduction in M1 receptor protein induced by ovariectomy was prevented by administration of E2.

Fig 2. Ovariectomy reduces cholinergic modulation of excitatory synapses.

The acetylcholinesterase inhibitor eserine differentially modulates field excitatory postsynaptic potentials (fEPSPs) in the lateral entorhinal cortex in brain slices obtained from intact rats (A), ovariectomized rats (B), and ovariectomized rats with E2 capsules (C). A. Representative averaged fEPSPs from a slice obtained from an intact rat (A₁) are shown for the baseline period (trace 1), 28 min after onset of application of 10 μM eserine (trace 2), and at the end of the recording period (trace 3). Traces are averages of five consecutive responses. In slices from intact rats, mean fEPSP amplitude was reduced following application of eserine (black bar; n = 13) (A₂). The inset diagram shows typical locations of the stimulating (*) and recording (circle) electrodes in horizontal slices of the lateral entorhinal cortex. Numbers indicate the times at which traces in A₁ were obtained, and bars indicate ± one standard error of the mean. B. Representative averaged traces are shown for a slice obtained from an ovariectomized rat (B₁), and changes in mean amplitude of fEPSPs in the group of slices are shown (B₂; n = 10). C. Representative traces (C₁) and averaged changes in mean fEPSP amplitude (C₂, n = 10) are shown for a group of slices obtained from ovariectomized rats that received E2 replacement. Reductions in fEPSP amplitude induced by eserine were significantly smaller in ovariectomized rats versus either intact rats or ovariectomized rats that received E2 replacement, both 25–30 min and 50–55 min after eserine application (p <0.05).