Introduction
Alzheimer's disease (AD) is the most common cause of dementia among the elderly. Acetylcholine and cholinergic signaling are essential for cognitive functions, including learning and memory. Disorders in cholinergic neurons or postsynaptic acetylcholine receptors have been found to be directly associated with cognitive impairment caused by Alzheimer's disease [2]. Dysfunction or lack of cholinergic cell groups in the basal forebrain, including the nucleus basalis magnocellularis (NBM) in rodents or its equivalent in humans, the nucleus basalis of Meynert (NBM), are among the first pathological events in the pathogenesis of Alzheimer's disease [3]. The present study aims to evaluate the synergistic effects of memantine and vitamin D on improving spatial learning and memory impairments in adult male rats of Alzheimer's disease model through the bilateral electrical lesion of NBM.
Materials and Methods
In this experimental study, male Wistar rats were randomly divided into 9 groups (7 rats in each): control group (received no injection or surgery), NBM lesion group (received electrically-induced lesion in NBM), Sham group (the electrode was impaled into the NBM with no lesion), NBM lesion+memantine (saline), NBM lesion+vitamin D (sesame oil), NBM lesion+memantine+vitamin D (saline+sesame oil), and NBM lesion+Vitamin D.
One week after NBM lesion: the last group received 5 μg/kg vitamin D for 10 days, starting 3 days before NBM lesion, by intraperitoneal injection (IP injection) [13]; the group of NBM lesion + memantine received 3 mg/kg memantine for 5 days, half an hour before training, by intraperitoneal injection (IP injection) [14]; the group of NBM lesion+memantine+vitamin D received 5 μg/kg vitamin D for 10 days, starting 3 days before lesion, and 3 mg/kg memantine for 5 days, half an hour before training, by intraperitoneal injection (IP injection). One week later, rats were trained for 5 days with a Y-shaped maze. Twenty-five days after training, a memory recall test was performed to assess long-term memory.
Results 
Comparison of different groups in the 5 consecutive days of training with one-way analysis of variance (ANOVA) showed no significant difference between control and Sham groups in spatial learning in any of the days, whereas bilateral lesion of NBM led to decreased spatial learning (p˂0.001) compared to control and Sham groups. In this study, no change was observed in the spatial learning in vehicle memantine and vitamin D groups compared with the NBM lesion group. On the other hand, the findings of this study showed an improvement in spatial learning and memory of NBM lesion+memantine+vitamin D group (p˂0.001) as compared with the two groups of NBM lesion+vitamin D (p˂0.01) and NBM lesion+memantine (p˂0.05). Besides, no significant difference was observed between the results of the fifth day of training and the 30th-day memory recall test in either group.


Discussion 
In this study, the bilateral electrical lesion of the nucleus basalis magnocellularis (NBM) was used as a model to induce Alzheimer's disease, besides spatial learning and memory impairments in rats. The findings of this study indicated that the combined treatment of vitamin D and memantine through the intraperitoneal (IP) injection of vitamin D for 10 days (starting 3 days before NBM lesion) and induction of Alzheimer's disease model and intraperitoneal injection of memantine for 5 days (starting 7 days after NBM lesion) had a greater effect on the improvement of spatial learning and memory impairments in the Alzheimer's disease model rats as compared to the administration of these two treatments each alone. Similarly, Di et al. 's studies have shown that 17 β-estradiol, which has neuroprotective effects as similar to vitamin D, leads to decreased neuronal degeneration and increased neuronal survival when combined with memantine treatment in traumatic brain injury model [21].
 On the other hand, Ihalainen's studies on rats with Fimbria-fornix lesion have shown a modest increase in acetylcholine levels in neocortex and hippocampus under chronic treatment with memantine and a significant increase under acute treatment with memantine [5]. Therefore, it seems that in the combined treatment of memantine and vitamin D, vitamin D improves memory and learning in Alzheimer's model rats by increasing neuronal protection and inhibiting axonal degeneration by increasing acetylcholine levels.
Ethical Considerations
Compliance with ethical guidelines
This study with ethics code EE/ 97, 24, 3061243/ scu.ac.ir was approved by the Research Ethics Committee of Shahid Chamran University of Ahvaz.
Funding
This study received financial support of Shahid Chamran University of Ahvaz.
Authors' contributions
All authors met standard writing criteria according to the recommendations of the International Committee of Medical Journal Editors (ICMJE).

Conflicts of interest
The authors declare that there is no conflict of interest in the present study.
Acknowledgements
The authors would like to thank the Deputy for Research of the Shahid Chamran University of Ahvaz for their support.