Figure 3. Prion disease is associated with a disruption in hippocampal cholinergic innervation, a deficit in learning, and memory rescued by donepezil, while maintaining muscarinic receptor expression and signaling.

(A) Cholinergic innervation of the hippocampus was assessed by ChAT (green) immunostaining of the CA1 region of the hippocampus in mice at 9 and 10 w.p.i. Data shown are representative of 3 individual mice per group. Scale bars: 50 μm. (B) Burrowing response of control and prion-infected mice. n = 4–9 mice. *P < 0.05; ***P < 0.001, 1-way ANOVA. (C) Fear-conditioning response of control and prion-infected mice at 9–10 w.p.i. n = 19 mice per group. ***P < 0.001, 2-way ANOVA with Sidak’s multiple comparison test. (D) Pain threshold response of control and prion-infected mice at 9–10 w.p.i. n = 6 mice per group. Unpaired Student’s t test. (E) Anxiety levels of control and prion-infected mice at 9–10 w.p.i. were assessed by elevated plus maze. n = 6 mice per group. Unpaired Student’s t test. (F) Fear-conditioning response of prion-infected mice (9–10 w.p.i.) treated with vehicle or donepezil (0.5 mg/kg) 60 minutes before training. n = 9 (vehicle); n = 15 (donepezil). ***P < 0.001, 2-way ANOVA with Sidak’s multiple comparison test. (G) Determination of the total muscarinic receptor population by [³H]-NMS binding to hippocampal membranes prepared from control or prion-infected mice (10 w.p.i.). Nonspecific binding was determined by the addition of atropine (1 μM). Data are expressed as fmol/mg protein (n = 3). Bmax, maximal binding capacity. (H) Total [³H]-NMS binding to membranes prepared from the frontal cortex of control or AD patients. n = 10. (I) Stimulation of [³⁵S]-GTPγS binding to membranes prepared from control or prion-infected mice (9–10 w.p.i.) in response to oxotremorine-M. Data shown are increases in [³⁵S]-GTPγS binding over basal; mean pEC₅₀ values of 6.45 ± 0.03, 6.63 ± 0.01, and 6.50 ± 0.04, respectively (n = 3). (J) [³⁵S]-GTPγS binding to membranes prepared from the frontal cortex of control or AD patients in response to acetylcholine. Data are the percentage of the maximal [³⁵S]-GTPγS binding stimulated by oxotremorine-M. Mean pEC₅₀ values of 6.00 ± 0.09 and 5.86 ± 0.11, respectively (n = 3).