GluN2D N-Methyl-d-Aspartate Receptor Subunit Contribution to the Stimulation of Brain Activity and Gamma Oscillations by Ketamine: Implications for Schizophrenia

Abstract

The dissociative anesthetic ketamine elicits symptoms of schizophrenia at subanesthetic doses by blocking N-methyl-d-aspartate receptors (NMDARs). This property led to a variety of studies resulting in the now well-supported theory that hypofunction of NMDARs is responsible for many of the symptoms of schizophrenia. However, the roles played by specific NMDAR subunits in different symptom components are unknown. To evaluate the potential contribution of GluN2D NMDAR subunits to antagonist-induced cortical activation and schizophrenia symptoms, we determined the ability of ketamine to alter regional brain activity and gamma frequency band neuronal oscillations in wild-type (WT) and GluN2D-knockout (GluN2D-KO) mice. In WT mice, ketamine (30 mg/kg, i.p.) significantly increased [(14)C]-2-deoxyglucose ([(14)C]-2DG) uptake in the medial prefrontal cortex (mPFC), entorhinal cortex and other brain regions, and decreased activity in the somatosensory cortex and inferior colliculus. In GluN2D-KO mice, however, ketamine did not significantly increase [(14)C]-2DG uptake in any brain region examined, yet still decreased [(14)C]-2DG uptake in the somatosensory cortex and inferior colliculus. Ketamine also increased locomotor activity in WT mice but not in GluN2D-KO mice. In electrocorticographic analysis, ketamine induced a 111% ± 16% increase in cortical gamma-band oscillatory power in WT mice, but only a 15% ± 12% increase in GluN2D-KO mice. Consistent with GluN2D involvement in schizophrenia-related neurologic changes, GluN2D-KO mice displayed impaired spatial memory acquisition and reduced parvalbumin (PV)-immunopositive staining compared with control mice. These results suggest a critical role of GluN2D-containing NMDARs in neuronal oscillations and ketamine's psychotomimetic, dissociative effects and hence suggests a critical role for GluN2D subunits in cognition and perception.

Graphical abstract

Fig. 1.

The effect of ketamine on [¹⁴C]-2DG uptake in WT mice and GluN2D-KO mice. (A) Representative autoradiographic images showing the effect of administering saline (left panels) and ketamine (right panels; 30 mg/kg, i.p.) on [¹⁴C]-2DG uptake in horizontal brain sections of WT (top panels) and GluN2D-KO (bottom panels) mice. Red to blue color spectrum indicates high to low activity, respectively, as shown in the calibration bars. (B) [¹⁴C]-2DG uptake expressed as mean relative radioactivity concentration ± S.E.M., in WT and GluN2D-KO mice after saline (Sal.) or ketamine (Ket.) injections, n = 7–9 per group. Statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CP/CPu, caudate putamen; EC/Ent Ctx, entorhinal cortex; H/HC, hippocampus; P/Presub, presubiculum; PFC medial prefrontal cortex; Rspl Ctx, retrosplenial cortex; SSC/Sens. Ctx., somatosensory cortex; Th, thalamus.

Fig. 2.

The effect of ketamine on neuronal oscillations. (A) Electrocorticographic recordings in WT and GluN2D-KO mice before and after administration of ketamine. Representative power spectrum analysis of WT (B) and GluN2D-KO mice (C) ECoG responses over 2 to 200 Hz before (baseline) or after ketamine injection. (D) The average percentage of power increase induced by ketamine-injection as a function of frequency in WT (blue line) and GluN2D-KO mice (red line). S.E.M. is shown by light blue/red shading. The dotted line represents 0% increase, no drug-induced change in power. Results shown represent the mean ± S.E.M. of WT and GluN2D-KO animals (n = 8 and 9, respectively). (E) Average ketamine-induced power increases in the upper gamma frequency band for WT and GluN2D-KO mice. ***P = 0.0002.

Fig. 3.

Reduced ketamine-induced locomotor behavior in the GluN2D-KO mouse. WT and GluN2D-KO mice (n = 7–10 per group) were treated with saline or ketamine, and their motor behavior was monitored for 15 minutes by the open-field test. Locomotor behavior was measured by the average number of grid lines crossed in each of the 5-minute periods (A) or for the total period (B). Also measured were the number of entries into the open, central squares of the arena (C), rearings (D), wall-climbing attempts (E), and walking in circles/rotations (F). Results are represented as mean number ± S.E.M. *P < 0.05; ***P < 0.001.

Fig. 4.

Parvalbumin immunoreactivity in different brain regions of WT and GluN2D-KO mice. (A) Photomicrographs are from representative coronal sections from WT (first and second columns) and GluN2D-KO mice (third and fourth column) stained by PV immunohistochemistry at low magnification (first and third columns). Images from the boxes in the low-magnification photographs are shown at higher magnification in the adjacent column (second and fourth columns). Each row represents the brain region indicated on the left. Scale bars = 500 µm (first photo) or 50 µm (second photo) as indicated; arrows in second photograph indicate representative PV-positive cells. (B) Histograms show the mean density of PV-positive cells for each region ± S.E.M. **P < 0.01, adjusted for multiple comparisons, n = 4 per group. BLA, basolateral/lateral amygdala; CPu, caudate putamen; dHC, dorsal hippocampus; PFC, medial prefrontal cortex; SNR, substantia nigra reticulata; vHC, ventral hippocampus.

Fig. 5.

Spatial memory acquisition in WT and GluN2D-KO mice. Spatial memory was tested in the Morris Water Maze with the time necessary to first reach the submerged platform (latency) measured for (A) each successive trial (four per day, 3 successive days). On the fourth day, the submerged platform was removed and in the subsequent test trial, (B) the percentage of time spent in the correct quadrant outside of the starting quadrant was measured, as was (C) the number of times the mouse passed over the prior location of the removed platform. Both test measures of task acquisition were statistically significant between WT and GluN2D-KO (n = 10 for each group, *P < 0.05; two-tailed t test).