| Background The olfactory nerve fibers project anatomically to the hippocampus through entorhinal cortex. The populations with olfactory dysfunction always show hippocampus-dependent episodic memory deficit. The bilateral olfactory bulbectomy (OBX) rat always show hippocampus-dependent episodic memory deficit, which serves as a perfect animal model for studying the mechanism of olfactory deficits-induced hippocampal involvements. The OBX rats have been widely used in the pathogenesis research for Alzheimer’s disease (AD) and depression. Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that participate in many kinds of neurodegeneration disease and mood disorder generally, such as AD and depression. The activation of GSK-3 plays a key role in synaptic plasticity, and is closely associated with the hippocampus-dependent learning and memory deficits. But the molecular mechanism how olfactory deficits induces hippocampal impairment are still not clear.Objective It was to explore the molecular mechanism underlying the olfactory deficits-induced hippocampal impairments by using OBX rats, to provide new information for the pathogenesis, early diagnosis and molecular targets for drug development of AD.Methods The Sprague Dawley rats were assigned into two groups:sham-operated rats, SO, and olfactory bulbectomized rats, OBX and tested with the Morris water paradigm, step-down passive avoidance test and step-through passive avoidance test 2 weeks after the surgery. Then the rats were taken an in vivo electrophysiology test and sacrificed for Western blotting, immunohistochemistry to detect the learning and memory function and level of synaptic proteins. To verify the role of GSK-3 in the OBX-induced hippocampus-dependent memory deficits and the underlying mechanisms, we infused the rats by vena caudalis of SB216763 (SB), a specific inhibitor of GSK-3, to the rats. The rats were assigned to three groups:SO, OBX and OBX plus SB and tested with the Morris water paradigm, step-down passive avoidance test and step-through passive avoidance test 2 weeks after the surgery. Then the rats were also taken an in vivo electrophysiology test and sacrificed for Western blotting, immunohistochemistry to detect the learning and memory function and level of synaptic proteins. The morphology of synapses in the tetanized CA3 region of the hippocampus was examined by electromicroscopy and Golgi staining.Results(1) OBX impairs learning and memory and synaptic function.Morris water maze test showed that the latency to find the hidden platform and the distance to reach the platform were longer in OBX team compared with the SO team. In the step-down and step-through passive avoidance test, the OBX rats also showed a shorter latency and an increased number of errors. The result of these behavior tests indicate that the hippocampus-dependent learning and memory is impaired in the OBX rats. We also found that OBX induced memory deficits with activation of several protein kinases, including glycogen synthase kinase-3β(GSK-3β), protein kinase A (PKA), extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB, or Akt) by Western blotting. The results by Western blot showed that the immunoreaction of Tau at Ser9 epitopes of GSK-3βwas weakened significantly in hippocampus when comparing the OBX group to sham-operated controls, while no obvious change was observed in the total GSK-3β. Immunoreaction at pT205, pT231 epitopes were enhanced, suggesting the activity of GSK-3βwas elevated. Similar results were observed by immunohistochemistry staining, in which hyperphosphorylated Tau was mainly detected in cortex and mossy fibers of hippocampal CA3 region. The OBX rats show synapse damages, such as a suppressed long term potentiation (LTP); reduction of synapsin I, synaptophysin, NR2A/B and PSD95. These data suggest the activation of GSK-3 maybe involved in the learning and memory deficits and synaptic malfunction.(2) Simultaneous inhibition of the activated GSK-3βameliorates OBX-induced memory deficits with recuperations of synaptic functions and tau phosphorylation.The result of behavior tests indicated that the hippocampus-dependent learning and memory is impaired in the OBX rats. The OBX group and OBX plus SB group were tail vein injected respectively with 0.5% DMSO or SB216763 (0.6 mg/kg) every other day from the next day after OBX surgery throughout the behavioral testing. Morris water maze test showed that the latency to find the hidden platform and the distance to reach the platform were shorter in SB team compared with the DMSO team after OBX. Western blot show SB-216763 can reverse the tau hyperphosphorylation, memory defects and synaptic deficits.(3) Simultaneous inhibition of GSK-3 ameliorates the OBX-induced synapse functionally and structurally.To verify the structural alterations of the synapses, we examined the morphology of synapses in the tetanized CA3 region of the hippocampus by electromicroscopy. Compared with the SO rats, prominent synaptic impairments, including the reduced size of thinner PSD, and broader synaptic cleft (SC), were viewed in OBX rats, the PSD size was almost restored to the control level when SB was simultaneously administered. By using Golgi stain, we found that both the spine density and the percentage of mushroom-like spines were significantly decreased in the hippocampal CA3 region in the OBX rats, while simultaneous infusion of SB restored the spine density and the mushroom-like spines, suggesting that OBX impairs the postsynaptic morphology and inhibition of GSK-3 preserves the synaptic morphology. Conclusions The activation of GSK-3 plays a key role in the molecular mechanism of olfactory deficits-induced hippocampal-dependent learning and memory impairment. Simultaneous inhibition of the activated glycogen synthase kinase-3 rescues bilateral olfactory bulbectomy-induced hippocampal impairments. |
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