Differential Expression of AMPA Subunits Induced by NMDA Intrahippocampal Injection in Rats

Abstract

Glutamate is involved in excitotoxic mechanisms by interacting with different receptors. Such interactions result in neuronal death associated with several neurodegenerative disorders of the central nervous system (CNS). The aim of this work was to study the time course of changes in the expression of GluR1 and GluR2 subunits of glutamate amino-acid-3-hydroxy-5-methyl-isoxazol-4-propionic acid (AMPA) receptors in rat hippocampus induced by NMDA intrahippocampal injection. Rats were submitted to stereotaxic surgery for NMDA or saline (control) microinjection into dorsal hippocampus and the parameters were evaluated 24 h, 1, 2, and 4 weeks after injection. The extension and efficacy of the NMDA-induced injury were evaluated by Morris water maze (MWM) behavioral test and Nissl staining. The expression of GluR1 and GluR2 receptors, glial fibrillary acidic protein (GFAP), and neuronal marker (NeuN) was analyzed by immunohistochemistry. It was observed the impairment of learning and memory functions, loss of neuronal cells, and glial proliferation in CA1 area of NMDA compared with control groups, confirming the injury efficacy. In addition, NMDA injection induced distinct changes in GluR1 and GluR2 expression over the time. In conclusion, such changes may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and in the activation of neuronal plasticity.

Keywords: AMPA receptors; Morris water maze; NMDA; excitotoxicity; hippocampus; immunohistochemistry; neurodegenerative disorders.

Figure 1

Effect of intrahippocampal NMDA in MWM acquisition task 2 and 4 weeks after injection. (A) Escape latency to find the platform during the four training sessions. (B) Time spent in the target quadrant during the probe trial (without the platform). Data represent mean ± SEM (n = 6/group). ^*p < 0.01 (Repeated measures two-way (A) or one-way (B) ANOVA followed by Bonferroni test).

Figure 2

(A) Dorsal hippocampus of the control and NMDA groups 2 weeks after injection. Lower (top) and higher magnification of the bounded areas (bottom). Plates used for neuron quantification (right) showing the injured area (hachured) (modified from Paxinos and Watson, 1986). (B) CA1, CA3, and hilus of the control, after 2 weeks, and NMDA after 2 and 4 weeks. (C) Number of neurons in CA1, CA3, and hilus of the control, after 2 weeks, and NMDA after 2 and 4 weeks. Data represent mean ± SEM of the number of cells quantified bilaterally in three sections/rat (n = 6/group). Nissl staining. ^***p < 0.001 (one-way ANOVA followed by Newman–Keuls test).

Figure 3

Immunohistochemistry for GluR1 in CA1 (A), CA3 (B), and hilus (C) of the control and NMDA groups evaluated at 24 h, 1, 2, and 4 weeks after intrahippocampal injection. Cells labeled with the chromogen 3,3-diaminobenzidine (DAB).

Figure 4

Number of GluR1-immunopositive cells (A,C,E) and optical density (B,D) in CA1, CA3, and hilus of the control and NMDA groups evaluated at 24 h, 1, 2, and 4 weeks after intrahippocampal injection. Data represent mean ± SEM of the number of cells and optical density quantified bilaterally in three sections/rat (n = 6/group). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 (one-way ANOVA followed by Newman–Keuls test).

Figure 5

Immunohistochemistry for GluR2 in CA1 (A), CA3 (B), and hilus (C) of the control and NMDA groups evaluated at 24 h, 1, 2, and 4 weeks after intrahippocampal injection. Cells labeled with the chromogen 3,3-diaminobenzidine (DAB).

Figure 6

Number of GluR2-immunopositive cells (A,C,E) and optical density (B,D) in CA1, CA3, and hilus of the control and NMDA groups evaluated at 24 h, 1, 2, and 4 weeks after intrahippocampal injection. Data represent mean ± SEM of the number of cells and optical density quantified bilaterally in three sections/rat (n = 6/group). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 (one-way ANOVA followed by Newman–Keuls test).

Figure 7

Immunofluorescent labeling for glial cells (A, GFAP–green) and neuronal cells (B, NeuN–red) in CA1 (adjacent area of injection) of the control and NMDA groups 1 and 4 weeks after injection. Nuclei labeled with DAPI (blue).

Figure 8

Immunofluorescent labeling for glial cells (GFAP–green) and neuronal cells (NeuN–red) in CA1 (injected area) 1 week (A) and 4 weeks (B) after injection. Nuclei labeled with DAPI (blue).