Insulin receptor signaling in rat hippocampus: A study in STZ (ICV) induced memory deficit model

Abstract

Brain insulin receptors (IRs) have been suggested as an important regulatory factor for cognitive functions but the involvement of IR signaling in memory deficit associated with neurodegenerative conditions is not yet explored. In the present study, IR gene expression was studied by RT-PCR and signaling pathways by immunoblotting in CA1, DG and CA3 subregions of hippocampus in intracerebroventricular (ICV) administered streptozotocin (STZ, 3 mg/kg twice) induced memory deficit model in rat. The effect of pre- and post-treatment of donepezil (5 mg/kg po) and melatonin (20 mg/kg po) on signaling pathways were studied. Effect of LY294002 (ICV), a PI3 Kinase inhibitor, was also investigated on memory functions and Akt phosphorylation. An increased IR expression (both gene and protein), phosphorylation of Shc, Erk1/2, IRS-1 and Akt in CA1 and CA3 region of P2M fraction was observed after training as compared to control. STZ treated rats showed memory deficit and significant decrease in IR expression, phosphorylation of IRS-1 and Akt only in CA3 region as compared to trained group which were reversed by pre and post-treatment of melatonin but donepezil was effective only against memory deficit. LY294002 (3 mM) treatment showed delayed learning and decrease in Akt phosphorylation. This study suggests that IR expression and its signaling pathways in hippocampal CA1 and CA3 regions are involved in memory functions and STZ (ICV) induced memory deficit. Hippocampal IR system might be playing an important role in regulation of memory functions, however only IR/IRS-1/Akt pathway in CA3 region is associated with STZ induced memory deficit.

Introduction

Recently, the brain insulin/insulin receptors (IRs) system is receiving major attention in regulation of brain functions. Insulin receptors (IR) are distributed in the brain regions and differ from peripheral IR in molecular weight of both α and β subunits (Zhao et al., 1999). The presence of IR in the hippocampus suggests its functional involvement in cognition (Dou et al., 2005). Moreover, deregulation of brain IR has been linked to the pathogenesis of age-related neurodegenerative disorders such as Alzheimer's (Frolich et al., 1998) and Parkinson's disease (Takahashi et al., 1996).

The molecular cascades downstream from IR are composed of a large number of signaling molecules including insulin receptor substrates (IRSs) and Src homology 2 (SH2) and SH2-SH3 domain-containing proteins. Among the diverse signaling pathways of IR, insulin receptor substrate-1 (IRS-1)/PI-3 kinase/phosphoinositide-dependent kinase (PDK)/protein kinase B (PKB/Akt) and the SH2 and collagen containing protein (Shc)/growth factor receptor-bound protein-2 (Grb2)/mitogen-activated protein (MAP) kinase pathways in brain have been suggested for learning and memory functions (Zhao et al., 2004).

A number of signaling molecules have been implicated in learning and memory functions in different species, such as MAPK activity is involved in associative learning (Atkins et al., 1998), spatial learning in the Morris water maze task and arm radial maze (Selcher et al., 1999, Zhao et al., 1999), inhibitory avoidance learning (Izquierdo et al., 2000) and contextual and auditory fear conditioning (Schafe et al., 1999, Selcher et al., 1999). It has been suggested that MAPK mediates changes in the long-term storage of information in the brain, a process that requires participation of gene regulation and expression. The molecular events, such as activation of PKC and Akt/PKB Ser/Thr kinase, may also play a role in memory storage. Taken together, these studies suggest that IR signaling has a role in the regulation of memory but the involvement of IR signaling in memory deficit linked with neurodegenerative conditions is not yet explored.

Intracerebroventricular (ICV) administration of streptozotocin (STZ) in rat has been described as an appropriate experimental model for dementia close to humans as both are characterized by progressive deterioration of memory, cerebral glucose and energy metabolism (Lannert and Hoyer, 1998). Grünblatt et al. (2007) demonstrated brain insulin system dysfunction in this model, which may lead to an increase in hyperphosphorylated tau-protein concentration in hippocampus. Recently, we had reported the decrease in IR protein expression in hippocampus in STZ (ICV) induced memory deficit (Agrawal et al., 2009).

Hippocampus is the key area to regulate memory functions. It has also been reported that STZ (ICV) causes chronic reductions in glucose and glycogen metabolism in hippocampus (Plaschke and Hoyer, 1993). Recent studies have shown the regional specificity of hippocampus in memory processes. The involvement of CA3 seems to be important at the earliest stage of acquisition, presumably for developing instant representation of a context, whereas the CA1 and dentate gyrus (DG) were critically involved in retrieving contextual memory after a long time period (Lee and Kesner, 2004). Inspite of different functional role of hippocampal CA1, DG and CA3 subregions in memory functions, the role of IR and its signaling present in these areas has not been investigated in relation to learning and memory functions.

Zhao et al. (1999) previously reported the training induced changes in IR in subregions of hippocampus which is associated with short-term memory. In our previous study (Agrawal et al., 2009), we had reported the effect of donepezil, an anti-dementia drug and melatonin, an antioxidant on IR in STZ induced memory deficit in brain areas. But studies on IR expression and its signaling in hippocampal subregions associated with state of memory deficit and effect of anti-dementia and antioxidant drugs are lacking.

Therefore, the present study was planned to investigate the involvement of IR and its signaling pathways in CA1, DG and CA3 subregions of hippocampus in memory deficit model of rat induced by STZ (ICV). Further, we have also included the study on pre- and post-treatment of donepezil, an anti-dementia drug and melatonin, an antioxidant on these signaling pathways in STZ (ICV) induced memory impairment.