Spontaneously hypertensive rats: further evaluation of age-related memory performance and cholinergic marker expression

Abstract

Objective: The spontaneously hypertensive rat (SHR), often used to study cardiovascular disease processes, may also be utilized to model certain central nervous system changes associated with memory disorders. Previous work in our laboratory indicated that central nicotinic acetylcholine receptors are markedly diminished and that memory-related task performance is impaired in this rodent phenotype. Due to the well-documented importance of the central cholinergic system to memory processes and its vulnerability to the effects of aging, it was of interest to measure other cholinergic markers and to further evaluate memory function in older SHRs.

Method: Radial arm maze performance was used to assess working memory, quantitative receptor autoradiography with [3H]-pirenzipine, [3H]-AFDX-384 and [3H]-epibatidine (combined with cytisine) was used to determine the densities of muscarinic-M1 and -M2 and nicotinic cholinergic alpha3 receptors, respectively. Immunoblotting experiments were also used to determine the expression of the presynaptic cholinergic markers, choline acetyltransferase and the vesicular acetylcholine transporter.

Results: Radial arm maze performance was impaired in hypertensive (compared with normotensive Wistar and Wistar-Kyoto) rats, regardless of age. M1 binding was increased in frontal and prefrontal cortical areas in SHR (p < 0.05), whereas M2 densities were higher in the hypertensive phenotype in the caudate putamen. A lower expression of alpha3-containing nicotinic receptors was observed in the superior colliculus in SHRs. Age-related differences in the expression of the vesicular acetylcholine transporter were noted in the hippocampus.

Conclusion: The SHR may be useful to model some aspects (particularly hypertension-related) of memory disorders, especially those in which cholinergic function is altered.

Fig. 1A: Performance of a win-shift task in an 8-arm radial arm maze across 7 consecutive days of testing (2 trials per day per animal) by the various rat groups (n = 9–12). Each point represents the mean percent correct (and SEM) of the total arms entered during the 5-min trial. * = Wistar performance significantly different from 1 or more of the other groups. B: Overall efficiency (collapsed across all days) defined as the number of correct (i.e., reinforced) entries of the first 8 arms entered.

Fig. 2: Mean (and SEM) systolic blood pressure of young and old 15-month-old WKY and SHR rats obtained via a tail cuff method after behavioural training (n = 10–12 rats per group). * = significant difference between the 2 rat phenotypes (p < 0.05); # = significant age-related difference (p < 0.05).

Fig. 3: Representative autoradiograms illustrating receptors labeled by [³H]-pirenzipine (PRZ, M1 muscarinic cholinergic receptors), [³H]-AFDX-384 (AFX, M2 muscarinic cholinergic receptors) and [³H]-epibatidine + 150 nM cytisine (EBCYS, α3-containing nicotinic cholinergic receptors) in coronal sections of brains from 15-month-old SHR and age-matched controls (WKY). Fr = frontal cortex, AON = anterior olfactory nucleus, DG = dentate gyrus, CPu = caudate putamen, SC = superior colliculus. Sections in which significantly different binding densities between SHR and WKY were found are presented. * = significant difference between the 2 rat phenotypes (p < 0.05).

Fig. 4: Choline acetyltransferase (ChAT) immunoreactivity in the cortex and hippocampus of younger (4-month-old) and older (15-month-old) SHR (black bars) and WKY (white bars) rats (n = 3 per group). A: Quantitative densitometry results from ChAT immunoblotting experiments. B: Representative immunoblots of ChAT in the cortex. C: Representative immunoblots of ChAT in the hippocampus. AU = arbitrary densitometric units.

Fig. 5: Vesicular acetylcholine transporter (VAChT) immunoreactivity in the cortex and hippocampus of younger (4-month-old) and older (15-month-old) SHR (black bars) and WKY (white bars) rats (n = 3 per group). * = younger SHR > older SHR (p < 0.05); # = younger WKY > older WKY (p < 0.05). A: Quantitative densitometry results from VAChT immunoblotting experiments. B: Representative immunoblots of VAChT in the cortex. C: Representative immunoblots of VAChT in the hippocampus.