The Effect of Intrahippocampal Insulin Injection on Scopolamine-induced Spatial Memory Impairment and Extracellular Signal-regulated Kinases Alteration

Abstract

Introduction: It is well documented that insulin has neuroprotective and neuromodulator effects and can protect against different models of memory loss. Furthermore, cholinergic activity plays a significant role in memory, and scopolamine-induced memory loss is widely used as an experimental model of dementia. The current study aimed at investigating the possible effects of insulin against scopolamine-induced memory impairment in Wistar rat and its underlying molecular mechanisms.

Methods: Accordingly, animals were bilaterally cannulated in CA1, hippocampus. Intrahippocampal administration of insulin 6 MU and 12 MU in CA1 per day was performed during first 6 days after surgery. During next four days, the animal's spatial learning and memory were assessed in Morris water maze test (three days of learning and one day of retention test). The animals received scopolamine (1 mg/kg) Intraperitoneally (IP) 30 minutes before the onset of behavioral tests in each day. In the last day, the hippocampi were dissected and the levels of MAPK (mitogen-activated protein kinases) and caspase-3 activation were analyzed by Western blot technique.

Results: The behavioral results showed that scopolamine impaired spatial learning and memory without activating casapase-3, P38, and JNK, but chronic pretreatment by both doses of insulin was unable to restore this spatial memory impairment. In addition, scopolamine significantly reduced Extracellular signal-Regulated Kinases (ERKs) activity and insulin was unable to restore this reduction. Results revealed that scopolamine-mediated memory loss was not associated with hippocampal damage.

Conclusion: Insulin as a neuroprotective agent cannot restore memory when there is no hippocampal damage. In addition, the neuromodulator effect of insulin is not potent enough to overwhelm scopolamine-mediated disruptions of synaptic neurotransmission.

Keywords: Alzheimer Disease; Apoptosis; Caspase-3; Cholinergic neurons; Mitogen-Activated Protein Kinases; Scopolamine.

Figure 1.

The effect of vehicle, scopolamine and or insulin treatment on water maze spatial learning and memory A) The learning patterns of the animals treated by vehicle, scopolamine, and or insulin during training sessions; and B) The escape latency to the hidden platform during days 1–3 of training. Data are expressed as Mean±SEM. *** P<0.001 represents the significant difference between control and other groups in each day.

Figure 2.

Superimposed maps of animal’s swimming paths in each group during three days of training in the MWM These heatmap figures show a comparable superimposed map of animal’s movement paths in the MWM apparatus. As it is evident, with learning trials during 3 days, it took less time to find the platform and animals swam mainly in the vicinity of the platform, but in animals treated with scopolamine, the pattern of movement does not show such improvement. The figures are obtained by merging the pattern of all trails of animals in each day for each group (Noldus, EthoVision XT 11).

Figure 3.

The effects of treatments on swimming speed Data are expressed as Mean±SEM. ***P<0.001 shows significant difference between the control and other groups.

Figure 4.

The effects of treatments on probe (retention) test Data are expressed as Mean±SEM. *P<0.05 and **P<0.01 represents significant difference between the control and other groups.

Figure 5.

Comparative amount of caspase-3 cleavage in different groups Western blot analysis showing the effects of IP administration of saline or scopolamine on animals receiving or not receiving intrahippocampal insulin for 6 days, on hippocampal caspase-3 cleavage; immunoblots were probed with caspase-3 and β-actin antibodies. Caspase-3 activity did not show significant differences between the groups. Representative immunoblots are shown in the upper panel.

Figure 6.

The ratio of phosphorylated to total form of JNK in the hippocampus Western blot analysis showing the effects of IP administration of saline or scopolamine on animals receiving or not receiving intrahippocampal insulin for 6 days, on phosphorylated JNK protein in the hippocampi of rats. Immunoblots were probed with antibodies against phosphorylated JNK, total JNK, and β-actin. JNK activity did not show significant differences between the groups. Representative immunoblots are shown in the upper panel.

Figure 7.

Western blot analysis on the activity of p38 as the ratio of phosphorylated to total form of p38 Western blot analysis showing the effects of IP administration of saline or scopolamine on animals receiving or not receiving intrahippocampal insulin for 6 days, on phosphorylated P38 protein in the hippocampi of rats. Immunoblots were probed with antibodies against phosphorylated p38, total p38, and β-actin. p38 activity did not show significant differences between the groups. Representative immunoblots are shown in the upper panel.

Figure 8.

Western blot analysis on phosphorylated (activated) ERK Western blot analysis showing the effects of IP administration of saline or scopolamine on animals receiving or not receiving intrahippocampal insulin for 6 days, on phosphorylated ERK protein in the hippocampi of rats. Immunoblots were probed with antibodies against phosphorylated ERK, total ERK, and β-actin. *P<0.05 represents significant difference between the control and other groups. Representative immunoblots are shown in the upper panel.