Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C086

Oral Communications

Calcium homeostasis is significantly disrupted in dentate gyrus neurons of acute, ex vivo brain slices of a mouse model of Alzheimer's disease.

M. B. Vaughan1, A. Kaar1, G. Mauleon2, D. T. Eddington2, M. G. Rae1

1. Physiology, University College Cork, Cork, Cork, Ireland. 2. Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, United Kingdom.

Recent work suggests that disruption of neuronal calcium homeostasis may be one of the early initiators of Alzheimer's disease (AD)1,2. However many of these findings have been generated using primary neuronal cultures3 which are a relatively poor representation of intact brain tissue. To this end, the aim of the present study was to determine if previous work in our lab, which has demonstrated that intracellular calcium regulation was disrupted in primary hippocampal neurons from a transgenic mouse model of AD (3xTgAD mouse), relative to wildtype (non-Tg) controls could be replicated using more physiological ex vivo acute hippocampal brain slices. Acute hippocampal brain slices (250mM) were prepared from age-matched (18.7±10.3 days, n=61) 3xTgAD and non-Tg mice using a McIlwain tissue chopper and stored in a slice holder containing aCSF for 1 hour prior to experimentation. Slices were then loaded with the calcium-sensitive dye, fluo 2AM (8μM) in a microfluidic oxygenator4, before being placed into a perfusion chamber and continuously superfused with aCSF (2ml/min) containing TTX (1μM). Intracellular calcium (Ca2+i) responses of neurons in the dentate gyrus (DG) to the group 1 metabotropic glutamate receptor agonist (S)-dihydroxyphenylglycine (DHPG), in the absence and presence of elevated K+ (7.5mM) extracellular aCSF (control K+=2.5mM), and/or absence and presence of the cyclic ADP ribose inhibitor, nicotinamide (5mM), were monitored and recorded using WinFluor software. Data were analysed by unpaired Student's t-test and are expressed as mean±S.E.M. Under control conditions (i.e. K+=2.5mM), DHPG (50μM)-evoked Ca2+i signals were significantly (P<0.05) larger in 3xTgAD neurons relative to those in control neurons (123±29%, n=20-53). However, in the presence of elevated (7.5mM) K+ aCSF although the magnitude of DHPG-evoked Ca2+i signals in the non-Tg neurons significantly increased (20±12%; n=53, 5 animals, P<0.05) relative to control, those evoked in the 3xTgAD neurons significantly decreased (32±7%; n=20, 4 animals, P<0.05) relative to controls. Further, we also found that there was no significant difference between mouse genotypes in the degree of attenuation of mGluR - mediated Ca2+ release by nicotinamide (non-Tg by 11±5.2%; n=30 cells, 4 animals, P<0.05; 3xTgAD by 23±6.2%; n=22 cells, 3 animals, P<0.01). In conclusion, we have shown that, as with primary hippocampal neuronal cultures, significant differences in calcium handling exist between 3xTg and non-Tg neurons at an early age. This supports the suggestion that AD pathogenesis may occur well before more overt, neurohistopathological alterations. Additionally, we have also demonstrated that calcium imaging using acute hippocampal slices is a versatile, less time consuming and, arguably, more physiological methodology than primary neuronal culture.

Where applicable, experiments conform with Society ethical requirements