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Comparative Study
, 12 (10), 1395-408

Dizocilpine (MK-801) Induces Distinct Changes of N-methyl-D-aspartic Acid Receptor Subunits in Parvalbumin-Containing Interneurons in Young Adult Rat Prefrontal Cortex

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Comparative Study

Dizocilpine (MK-801) Induces Distinct Changes of N-methyl-D-aspartic Acid Receptor Subunits in Parvalbumin-Containing Interneurons in Young Adult Rat Prefrontal Cortex

Dong Xi et al. Int J Neuropsychopharmacol.

Abstract

N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

Figures

Fig. 1
Fig. 1
Relative mRNA expression of NMDAR subunits in adult rat PFC and parvalbumin-immunoreactive (PV-ir) interneurons. (a) mRNA expression of NMDAR subunits in the PFC tissue. NR2B mRNA expression (31.7±3.91, n=8) is significantly higher than that of the NR2A subunit (1.92±0.66, n=8, p=0.0001). (b, c) High-magnification photographs showing the PV-ir interneurons (arrows) labelled with rapid NovaRED immunostaining before (b) and after (c) the laser cut. Scale bar in (c)=50 μm. (d, e) RNA integrity numbers (RIN) were measured with Agilent Bioanalyzer and the electropherogram of RNA extracted from laser microdissection (LMD)-picked PV-ir interneurons is shown in (d), where 5S covers the small rRNA fragments (5S and 5.8S rRNA and tRNA) and 18S and 28S cover the 18S peak and 28S peak (e). RIN=8.63±0.16 (n=9) with 28S/18S=1.90±0.15 and 18S/baseline=7.23±1.17. (f) RT–PCR amplification of PV (150 bp) showing the expression of PV in LMD-captured PV-ir cells compared to that in PV-negative tissue. The left lane was the molecular weight marker with 100-bp intervals. (g) Relative mRNA expression of NMDAR subunits in PV-ir interneurons. The mRNA level of NR2A subunit was significantly higher than that of NR2B subunit (p=0.031). # Indicates that NR2D mRNA was at an undetectable level (* p<0.05, ** p<0.01).
Fig. 2
Fig. 2
Effect of subchronic MK-801 administration on mRNA expression of NMDAR subunits in adult rat PFC tissue. (a–c) mRNA levels of NR1, NR2A, and NR2B subunits showed similar changes for different doses of MK-801 treatment. Both were significantly increased at very low doses of 0.01, 0.033 and 0.1 mg/kg, significantly decreased at a high dose of 1.0 mg/kg (p<0.05), and increased but not significantly at 0.33 mg/kg (p>0.05). (d, e) the mRNA levels of NR2C subunits were similar to the NR2B subunit except at 0.01 mg/kg, a dose at which the changes were not significant (p=0.095). (e) the mRNA change of NR2D was not significant at the high dose of 1.0 mg/kg (p=0.149), slightly different from other subunits (* p<0.05, ** p<0.01).
Fig. 3
Fig. 3
Effect of subchronic MK-801 administration on mRNA expression of NMDAR subunits in parvalbumin-immunoreactive (PV-ir) interneurons. (a) the mRNA levels of NR1 subunit in response to MK-801 treatment at different doses. NR1 was significantly down-regulated at doses 0.01 and 0.033 mg/kg (p<0.05), returned to control levels at 0.1 and 0.33 mg/kg (p>0.05) and significantly decreased at 1.0 mg/kg (p<0.05). (b–d) NR2 subunits A–C showed a similar biphasic pattern of changes in mRNA expression except NR2C, which did not show significance at 0.01 mg/kg following MK-801 administration. # Indicates that the expression of NR2C at the dose of 0.033 mg/kg was undetectable (* p<0.05, ** p<0.01).
Fig. 4
Fig. 4
Differential protein expression of NR2A and NR2B subunits in response to low and high doses of MK-801 treatment in PFC tissue. (a) Representative samples of Western blots showing the relative protein levels of NR2A and NR2B subunits to β-actin in response to treatment with MK-801 at 0.033 and 1.0 mg/kg, respectively. (b) Summary histogram showing the relative changes of NR2A and NR2B subunit proteins that were normalized to β-actin and then to the control with Tukey’s ANOVA test. It was clear that 0.33 mg/kg MK-801 treatment significantly increased protein expression of NR2A and NR2B subunits, whereas treatment with 1.0 mg/kg significantly decreased the expression of NR2A and NR2B subunits. The β-actin levels were stable without clear changes in both low- and high-dose treatments (* p<0.05).
Fig. 5
Fig. 5
MK-801-induced distinct changes of NR2A and NR2B subunits in parvalbumin (PV)-containing interneurons vs. pyramidal neurons. (a–f) Representative photographs of double immunofluorescent labelling of PV (red) and NMDAR subunits (green) in controls (a, b) and MK-801 treatment at low dose (c, d) and high dose (e, f). The PV-immunoreactive (ir) interneurons (arrows) were double-labelled by PV and NR2A or NR2B, whereas the putative pyramidal neurons (arrowheads) were surrounded by both PV-ir axon terminals (red) and labelled NR2 positive puncta (green). Scale bar in (f)=20 μm for all images in panels (a)–(f). (g, h) Images at high magnification showing the methods used for quantification of NR2A and NR2B puncta in both PV-ir cells (g) and pyramidal neurons (h). Scale bar in (h)=20 μm for all images in panels (g) and (h). (i) Western blot analyses showing the specificities of anti-NR2A and anti-NR2B used for immunostaining. Both antibodies exhibited single molecular weight band. (j, k) Summary histograms showing changes of NR2A and NR2B subunit protein expression (puncta numbers) in the PV-ir interneurons and pyramidal neurons in MK-801-treated rats. Compared to control, 0.033 mg/kg MK-801 treatment significantly decreased the expression of both NR2A and NR2B subunits in PV-ir cells (p<0.05). In contrast, MK-801 significantly increased the puncta numbers of both NR2A and NR2B subunits in pyramidal neurons at low dose (0.033 mg/kg) but dramatically decreased the puncta numbers at high dose (1 mg/kg), suggesting distinct cell-type-specific effects in PFC circuitry (* p<0.05, ** p<0.01).
Fig. 6
Fig. 6
Dose-dependent effects in NMDAR and neuronal activity in pyramidal neurons (P) and interneurons (NP) in a MK-801 model. (a) Low dose (<0.1 mg/kg) selectively blocks NMDARs in the interneurons and thus results in increased NMDARs and excitatory activity in P. (b) High-dose MK-801 (1.0 mg/kg) blocks NMDARs in both P and NP, reduces all activities and even induces apoptosis or cell death of parvalbumin-immunoreactive (PV-ir) interneurons.

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