Genetically encoded calcium indicators (GECIs) have gained widespread use for measurement of neuronal activity but their low expression levels in transgenic mice tend to limit sensitivity. We have developed a transgenic mouse line (SyG37) that expresses a ratiometric calcium sensor, SyGCaMP2-mCherry, that is expressed throughout the brain but targeted to presynaptic terminals. Within the CA1 and CA3 regions of hippocampus of male and female mice, SyGaMP2 fluorescence responds linearly up to 10 electrical stimuli at frequencies up to 100 Hz and it can detect responses to a single stimulus. Responses in single boutons can be measured using multiphoton microscopy. The ensemble amplitude of SyGCaMP2 responses is a function of the number of stimuli applied and the number of contributing boutons. The peak responses and initial rates of calcium influx in single boutons in CA1 and CA3 were similar but the rate of calcium clearance from CA3 boutons after stimulation was significantly faster. In CA1, DNQX reduced SyGCaMP2 responses to Schaffer collateral stimulation to 86% of baseline indicating that 14% of the total response originated from presynaptic terminals of neurones synaptically driven via AMPA receptors. Theta burst stimulation induced long-term potentiation (LTP) of both SyGCaMP2 and fEPSP responses in both young and 18-month-old mice. The proportion of postsynaptically connected terminals increased significantly to 76% of the total after LTP induction. The SyG37 mouse allows stable optical detection of synaptic activation and connectivity at the single bouton level and can be used to characterize the contributions of presynaptic calcium to synaptic transmission and plasticity.

Alzheimer’s disease (AD) is a progressive age-related neurodegenerative disorder characterized by progressive impairment of memory and cognitive functions. Oxidative stress, neuroinflammation, and neurotransmitters disturbance may play an important role. This study aimed to evaluate the effect of donepezil alone and in combination with nitric oxide synthase (NOS) substrate L-arginine, and nitric oxide synthase inhibitor (NOSi) Ng-nitro-l-arginine methyl ester hydrochloride (L-NAME) and 7-nitroindazole against aluminum chloride (AlCl3) induced neurotoxicity. A total of 36 male albino rats were divided to six groups: control normal saline, AlCl3. ,Donepezil, Donepezil+ L-arginine, Donepezil+ L-NAME, and Donepezil+7-nitroindazole. Neurotoxicity was induced by AlCl3 in a dose of 10 mg/Kg subcutaneously for 30 days. After the experimental period, brain was removed for determination of acetylcholinesterase activity (AChE), levels of fatty acids fractions by high performance liquid chromatography (HPLC), interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α). AlCl3 significantly increase brain levels of AChE, IL-1β, and TNF-α, and arachidonic acid (AA) and linoleic acid (LA). Treatment with Donepezil alone and in combination with L.NAME and 7-nitroindazole significantly attenuates these changes. In conclusion, regulation of nitric oxide (NO) level is the key for stability and prevention of neurodegenerative diseases. Combination of Donepezil with nitric oxide synthase inhibitors especially 7-nitroindazole attenuates the oxidant and inflammatory induced by AlCl3.

Keywords: AlCl3, L. NAME, Donpezil, fatty acids, HPLC

In this project we used a combination of electrophysiology and fluorescent imaging to monitor synaptic transmission and calcium signalling in synaptic terminals. The study as a whole intended to examine how presynaptic calcium contributes to normal synaptic transmission within different hippocampal neuronal pathways. To this end, we used a transgenic mouse strain known as SyG37 that stably expresses a calcium sensor, SyGCaMP2-mCherry that is expressed in subsets of CNS neurones under the control of the Thy1 promoter. Our findings indicate that this new ratiometric sensor, in the SyG37 mouse strain, provides an excellent tool for detecting neural activity in acute brain slices. First, we showed that evoked calcium transients can be detected in acute brain slices prepared from SyG37 mice where electrical activation of Schaffer collaterals or mossy fibres elicited large calcium transients in area CA1 and CA3, respectively. Using immunohistochemical techniques, SyGCaMP2-mCherry co-localised with presynaptic proteins such as Bassoon, VGLUT1 and VGAT, confirming that it is expressed presynaptically in both excitatory and inhibitory terminals. Blocking fast glutamatergic and GABA/Glycinerergic transmission reduced the size of calcium transients in CA1 and CA3 by only 25 and 20% respectively indicating that the majority of the signals originated from first order presynaptic terminals. Pharmacologically, manipulating the adenosine receptor signalling pathway showed that the actions of adenosine, via the A1 receptor subtype, were different in the CA3 region compared to those in CA1. Forskolin also caused a small, concentration dependent effect on SyGCaMP2 fluorescence in response to electrical stimulation within both CA1 and CA3 regions with pronounced effects on field potential recordings. Together, with this SyG37 strain of transgenic mouse, it is possible to detect neuronal activity with fast temporal and high spatial resolution without the need for pre-incubation with organic calcium dyes or invasive viral transduction procedures.