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. 2015 Nov;64(11):3927-36.
doi: 10.2337/db15-0596. Epub 2015 Jul 27.

Hippocampal Insulin Resistance Impairs Spatial Learning and Synaptic Plasticity

Claudia A Grillo  1 , Gerardo G Piroli  2 , Robert C Lawrence  3 , Shayna A Wrighten  2 , Adrienne J Green  2 , Steven P Wilson  2 , Randall R Sakai  4 , Sandra J Kelly  3 , Marlene A Wilson  5 , David D Mott  2 , Lawrence P Reagan  5
Affiliations

Hippocampal Insulin Resistance Impairs Spatial Learning and Synaptic Plasticity

Claudia A Grillo et al. Diabetes. 2015 Nov.

Abstract

Insulin receptors (IRs) are expressed in discrete neuronal populations in the central nervous system, including the hippocampus. To elucidate the functional role of hippocampal IRs independent of metabolic function, we generated a model of hippocampal-specific insulin resistance using a lentiviral vector expressing an IR antisense sequence (LV-IRAS). LV-IRAS effectively downregulates IR expression in the rat hippocampus without affecting body weight, adiposity, or peripheral glucose homeostasis. Nevertheless, hippocampal neuroplasticity was impaired in LV-IRAS-treated rats. High-frequency stimulation, which evoked robust long-term potentiation (LTP) in brain slices from LV control rats, failed to evoke LTP in LV-IRAS-treated rats. GluN2B subunit levels, as well as the basal level of phosphorylation of GluA1, were reduced in the hippocampus of LV-IRAS rats. Moreover, these deficits in synaptic transmission were associated with impairments in spatial learning. We suggest that alterations in the expression and phosphorylation of glutamate receptor subunits underlie the alterations in LTP and that these changes are responsible for the impairment in hippocampal-dependent learning. Importantly, these learning deficits are strikingly similar to the impairments in complex task performance observed in patients with diabetes, which strengthens the hypothesis that hippocampal insulin resistance is a key mediator of cognitive deficits independent of glycemic control.

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Figures

Figure 1
Figure 1
GFP is expressed in the Ammon’s horn and DG of rats infused with lentivirus. A: Representative immunohistochemical (IHC) labeling for GFP shows a consistent expression of the reporter protein across all the hippocampus subfields, without labeling in adjacent areas such as the cortex. B: Representative IHC labeling for GFP in the hypothalamus exhibits lack of expression in this area. C: Representative IHC labeling for GFP in the CA3 subfield of the hippocampus. D: Representative IHC labeling for GFP in the CA1 subfield of the hippocampus. E: Representative IHC labeling for GFP in the DG subfield of the hippocampus. Scale bar, 200 µm for panel A and 100 µm for panels B–E.
Figure 2
Figure 2
IR expression and insulin-stimulated phosphorylation of the IR and Akt are decreased in the hippocampus of LV-IRAS–treated rats. A: Representative immunoblot of IR levels in the hippocampus of LV-Con– and LV-IRAS–treated rats. Normalization for protein loading was performed using a monoclonal antibody for actin. Autoradiographic analysis determined that IR levels are significantly decreased in the hippocampus of the LV-IRAS–treated rats compared with LV-Con–treated rats. Data, representing mean ± SEM (n = 8 per group), were analyzed by Student t test (**P < 0.01). B: Representative immunoblot of phosphorylated IR (pIR) levels in the hippocampus of LV-Con– and LV-IRAS–treated rats. Normalization for protein loading was performed using a monoclonal antibody for actin. Autoradiographic analysis determined that pIR levels are significantly decreased in the hippocampus of the LV-IRAS–treated rats compared with LV-Con–treated rats. Data, representing mean ± SEM (n = 8 per group), were analyzed by Student t test (**P < 0.01). +, Insulin/ATP treatment; −, buffer. C: Representative immunoblot of phosphorylated Akt (pAkt) levels in the hippocampus of LV-Con– and LV-IRAS–treated rats. Normalization for protein loading was performed using an antibody for Akt. Autoradiographic analysis determined that the pAkt-to-Akt ratio is significantly decreased in the hippocampus of the LV-IRAS–treated rats compared with LV-Con–treated rats. Data, representing mean ± SEM (n = 8 per group), were analyzed by Student t test (**P < 0.01). Ins, insulin.
Figure 3
Figure 3
Physiological and endocrine parameters are not affected by downregulation of the hippocampal IRs. A: LV-IRAS rats did not exhibit changes in body weight compared with LV-Con rats (n = 10 per group). B: LV-IRAS rats do not exhibit changes in body fat, lean mass, or water content compared with LV-Con rats (n = 10 per group). C: Plasma leptin levels did not differ between LV-IRAS and LV-Con rats (n = 10 per group). D: In response to an OGTT, LV-IRAS rats exhibited similar profiles in plasma glucose (n = 6 per group). E: Plasma insulin levels were similarly stimulated after the OGTT in both groups. Areas under the curve results, for glucose (D) and insulin (E), were also similar between the groups (n = 6 per group). F: Basal, stress-induced increase and poststress levels of plasma CORT do not differ between the groups (n = 4 per group).
Figure 4
Figure 4
Performance of rats with hippocampal IR deficiency (LV-IRAS) and control rats (LV-Con) in the water maze reference memory task. A: Downregulation of hippocampal IRs increases the time needed to reach the hidden platform in the second and third session. Data, representing mean ± SEM (n = 9 per group), were analyzed by two-way ANOVA followed by Bonferroni post hoc test, showing that there were significant time and treatment effects (*P < 0.05). B–E: Daily performance of rats with hippocampal IR deficiency (LV-IRAS) and control rats (LV-Con) in the water maze reference memory task. Downregulation of the hippocampal IRs increased the time needed to reach the hidden platform in the first trial of the second and third sessions compared with the control group. Data, representing mean ± SEM (n = 9 per group), were analyzed by two-way ANOVA followed by a Bonferroni post hoc test (*P < 0.05). F–H: Probe trial of the water maze reference memory task. Downregulation of the hippocampal IRs (LV-IRAS) does not affect the swimming velocity compared with LV-IRAS rats (F). However, LV-IRAS rats needed more time (G) and exhibited longer paths (H) to reach the position where the platform was placed 1 h after the training compared with LV-Con rats. Data, representing mean ± SEM (n = 9 per group), were analyzed by Student t test (*P < 0.05).
Figure 5
Figure 5
Downregulation of hippocampal IRs impairs LTP. Electrophysiological analysis was performed 21 days after administration of LV-Con or LV-IRAS in the CA1 region of the hippocampus. A: The input-output relation shows a similar fEPSP slope in both LV-Con–treated (n = 9) and LV-IRAS–treated (n = 7) animals over a range of stimulus intensities. B: Paired pulse facilitation (PPF) at a 50-ms interstimulus interval was similar in animals treated with LV-Con (n = 10) and LV-IRAS (n = 7). A and B, top: Superimposed fEPSPs from LV-Con and LV-IRAS animals were scaled to the amplitude of the first fEPSP to show the similarity in PPF. C: LV-Con–treated rats developed LTP in response to HFS (100 Hz, 1 s) of the Schaffer collaterals (arrow). In contrast, LTP, but not short-term potentiation, was blocked in LV-IRAS–treated rats. Inset shows sample fEPSPs collected at the times indicated on the graph. D: LV-IRAS treatment blocked LTP of the fEPSP in both CA1 (LV-Con treated, n = 7; LV-IRAS treated, n = 7; **P < 0.01) and DG (LV-Con treated, n = 6; LV-IRAS treated, n = 4; **P < 0.01).
Figure 6
Figure 6
A: Phosphorylation of GluA1 subunit is decreased in the hippocampus of LV-IRAS–treated rats. Representative immunoblot of phosphorylated and total GluA1 levels in the hippocampus of LV-Con– and LV-IRAS–treated rats. Autoradiographic analysis determined that the pGluA1-to-GluA1 ratio is significantly decreased in the hippocampus of the LV-IRAS–treated rats compared with LV-Con–treated rats. Data, representing mean ± SEM (n = 10 per group), were analyzed by Student t test (*P < 0.05). B: GluN2B subunit expression is decreased in the hippocampus of LV-IRAS–treated rats. Representative immunoblot of GluN2B in the hippocampus of LV-Con– and LV-IRAS–treated rats. Normalization for protein loading was performed using a monoclonal antibody for actin. Autoradiographic analysis determined that expression of GluN2B is significantly decreased in the hippocampus of the LV-IRAS–treated rats compared with LV-Con–treated rats. Each lane represents hippocampal membrane fractions from individual rats that were run on the same gel at the same time. Data, representing mean ± SEM (n = 10 per group), were analyzed by Student t test (*P < 0.05).
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