Neuroprotective effects of donepezil against cholinergic depletion

Abstract

Introduction: Intraparenchymal injections of the immunotoxin 192-IgG-saporin into medial septum and nucleus basalis magnocellularis causes a selective depletion of basal forebrain cholinergic neurons. Thus, it represents a valid model to mimic a key component of the cognitive deficits associated with aging and dementia. Here we administered donepezil, a potent acetylcholinesterase inhibitor developed for treating Alzheimer's disease, 15 days before 192-IgG-saporin injection, and thus we examined donepezil effects on neurodegeneration and cognitive deficits.

Methods: Caspase-3 activity and cognitive performances of lesioned rats pre-treated with donepezil or saline were analyzed and compared to the outcomes obtained in pre-treated sham-lesioned rats.

Results: Cholinergic depletion increased hippocampal and neocortical caspase-3 activity and impaired working memory, spatial discrimination, social novelty preference, and ultrasonic vocalizations, without affecting anxiety levels and fear conditioning. In lesioned animals, donepezil pre-treatment reduced hippocampal and neocortical caspase-3 activity and improved working memory and spatial discrimination performances and partially rescued ultrasonic vocalizations, without preventing social novelty alterations.

Conclusions: Present data indicate that donepezil pre-treatment exerts beneficial effects on behavioral deficits induced by cholinergic depletion, attenuating the concomitant hippocampal and neocortical neurodegeneration.

Figure 1

Experimental design. Diagram describing the global timing of the experimental design of the four groups. Data derived from pre-treatment (donepezil or saline), lesioned (192-IgG-saporin, Sap, or sham), caspase-3 and choline acetyltransferase analyses, as well asbehavioral testing, are indicated. ChAT, Choline acetyltransferase; Don-Sap, donepezil-treated Sap-lesioned rats; Don-Sham, donepezil-treated sham-lesioned rats; IgG, immunoglobulin G; Sal-Sap, saline-treated Sap-lesioned rats; Sal-Sham, saline-treated sham-lesioned rats.

Figure 2

Choline acetyltransferase immunohistochemical staining. Representative photomicrographs of intraparenchimal Sap injection effects. Images show brain sections of rats that were saline-treated, sham-lesioned (a, b and c); saline-treated, Sap-lesioned (a1, b1 and c1); donepezil-treated, sham-lesioned (d, e and f); and donepezil-treated, Sap-lesioned (d1, e1 and f1). Coronal sections at the level of medial septum (a, a1, d and d1), nucleus basalis magnocellularis (b, b1, e and e1) and striatal (c, c1, f and f1) regions with choline acetyltransferase immunoreactive neurons are shown. Note the substantial absence of cholinergic neurons in both regions of the lesioned animal (a1, b1, d1 and e1) and the substantial preservation of striatal cholinergic interneurons (c1 and f1). Lens objective: 10×. Scale bars: 50 μm.

Figure 3

Choline acetyltransferase immunoblot analysis. Representative choline acetyltransferase (ChAT) immunoblots of hippocampal (hp) and neocortical (cx) total protein extracts and densitometric quantification of changes in gray values expressed as mean ± SD. The relative fold change in the levels of ChAT protein was normalized with respect to the level of actin, which was used as a loading control. Statistical differences were observed between the sham and 192-IgG-saporin (Sap)-lesioned animals (n = 3/group). *P < 0.05 by t-test. Don-Sap, donepezil-treated Sap-lesioned rats; Don-Sham, donepezil-treated sham-lesioned rats; Sal-Sap, saline-treated Sap-lesioned rats; Sal-Sham, saline-treated sham-lesioned rats.

Figure 4

Hippocampal and neocortical caspase-3 activity. Caspase-3 activity was revealed by a fluorometric assay in total hippocampal and neocortical homogenates from sham and lesioned rats (n = 3/group). The fluorometric data are expressed as mean ± SEM. *P < 0.05, ***P ≤ 0.001 (Bonferroni multiple comparisons test). Don-Sap, donepezil-treated Sap-lesioned rats; Don-Sham, donepezil-treated sham-lesioned rats; Sal-Sap, saline-treated Sap-lesioned rats; Sal-Sham, saline-treated sham-lesioned rats.

Figure 5

Open field with objects. Effects of donepezil pre-treatment and 192-IgG-saporin (Sap) lesions on open field with objects contact time with objects during spatial change in session 5 (S5) (a), S6 (b) and novelty (S7) (c). Cholinergic depletion per se (Sal-Sap group) significantly affected spatial discrimination abilities in S5 and S6, and donepezil pre-treatment (Don-Sap group) prevented spatial memory deficits. In S7, all rats detected the presence of the novel object. Behavioral data are expressed as means ± SEM, and asterisks indicate post hoc comparisons between groups. DO, Displaced object; Don-Sap, donepezil-treated Sap-lesioned rats; Don-Sham, donepezil-treated sham-lesioned rats; FO, familiar object; NDO, non-displaced objects; NO, novel object; Sal-Sap, saline-treated Sap-lesioned rats; Sal-Sham, saline-treated sham-lesioned rats. **P < 0.01, ***P < 0.001.

Figure 6

Radial arm maze. Effects of donepezil pre-treatment and 192-IgG-saporin (Sap) lesions on radial arm maze total errors (a), spatial spans (b) and perseverations (c). Donepezil pre-treatment reduced the number of errors and increased spatial span in cholinergically depleted rats. Sal-Sham vs. Sal-Sap: #p < 0.05; ##p < 0.01; ###p < 0.001; Don-Sap vs. Sal-Sap: *p = 0.05, **p = 0.01. Don-Sap, donepezil-treated Sap-lesioned rats; Don-Sham, donepezil-treated sham-lesioned rats; Sal-Sap, saline-treated Sap-lesioned rats; Sal-Sham, saline-treated sham-lesioned rats.

Figure 7

Sociability and preference for social novelty test. Effects of donepezil pre-treatment and 192-IgG-saporin (Sap) lesions on sociability (a) and preference for social novelty test (PSNT) (b). Although no differences among the four groups were found in the sociability test, both groups of cholinergically depleted animals (Don-Sap and Sal-Sap) failed to exhibit an overt social novelty during PSNT. Asterisks indicate comparisons between stranger 1 and empty/stranger 2 chamber behaviors in the four groups. **P < 0.01, ****P < 0.0001. Pound symbols indicate comparisons between stranger 1 chamber and stranger 2 chamber behaviors in the sham groups (Don-Sham and Sal-Sham). #P < 0.05, ##P = 0.01. Don-Sap, donepezil-treated Sap-lesioned rats; Don-Sham, donepezil-treated sham-lesioned rats; Sal-Sap, saline-treated Sap-lesioned rats; Sal-Sham, saline-treated sham-lesioned rats.

Figure 8

Fear conditioning. Effects of donepezil pre-treatment and 192-IgG-saporin (Sap) lesions on percentage of freezing and total number of 22-kHz fear-associated ultrasonic vocalizations (USVs) during fear conditioning (FC) training (a), context (CN) test (b) and tone (TN) test (c). All animals acquired a clear freezing response during FC training and showed mnesic retention of the conditioned freezing response during CN and TN tests. USVs were nearly totally suppressed in saline-treated Sap-lesioned rats, and donepezil pre-treatment tended to increase USV production in donepezil-treated Sap-lesioned rats during FC training. BS, Baseline; Don-Sham, donepezil-treated sham-lesioned rats; ITI, Inter-trial interval; Sal-Sham, Saline-treated sham-lesioned rats. All groups: ****P < 0.00001; Sal-Sham vs. Sal-Sap: #P < 0.05.