Elevated levels of the NR2C subunit of the NMDA receptor in the locus coeruleus in depression

Abstract

Low levels of the intracellular mediator of glutamate receptor activation, neuronal nitric oxide synthase (nNOS) were previously observed in locus coeruleus (LC) from subjects diagnosed with major depression. This finding implicates abnormalities in glutamate signaling in depression. Receptors responding to glutamate in the LC include ionotropic N-methyl-D-aspartate receptors (NMDARs). The functional NMDAR is a hetero-oligomeric structure composed of NR1 and NR2 (A-D) subunits. Tissue containing the LC and a nonlimbic LC projection area (cerebellum) was obtained from 13 and 9 matched pairs, respectively, of depressed subjects and control subjects lacking major psychiatric diagnoses. NMDAR subunit composition in the LC was evaluated in a psychiatrically normal subject. NR1 and NR2C subunit immunoreactivities in LC homogenates showed prominent bands at 120 and 135 kDa, respectively. In contrast to NRI and NR2C, very weak immunoreactivity of NR2A and NR2B subunits was observed in the LC. Possible changes in concentrations of NR1 and NR2C that might occur in depression were assessed in the LC and cerebellum. The overall amount of NR1 immunoreactivity was normal in the LC and cerebellum in depressed subjects. Amounts of NR2C protein were significantly higher (+ 61%, p = 0.003) in the LC and modestly, but not significantly, elevated in the cerebellum (+ 35%) of depressives as compared to matched controls. Higher levels of NR2C subunit implicate altered glutamatergic input to the LC in depressive disorders.

Figure 1

Relationship between the optical density values of NR1 and NR2C subunit immunoreactivity and total protein concentration in the human LC. Wells were loaded with 40, 20, and 10 µg of total human LC protein.

Figure 2

NMDAR subunit immunoreactivities detected in LC (lanes 1 and 2) and in hippocampus (lanes 3 and 4). In lanes 1 and 3, each well was loaded with 20 µg of total protein; for lanes 2 and 4, 40 µg of total protein was loaded into wells. The polyclonal antibody used against NR2C protein also detects a 180 kDa protein representing NR2A and NR2B subunits in the hippocampus.

Figure 3

(a) NR1 subunit immunoreactivity in LC from a single pair used in the analysis, representing three separate anatomical levels: rostral (R), middle (M), and caudal (C). Each well was loaded with 20 µg of total protein. The bottom panel shows immunoreactive actin probed with anti-actin antibody on the same blots as a control for protein loading and transfer. (b) NR1 immunoreactivity expressed as percentages of values from paired control subjects. Each bar is an average of duplicate comparisons. The overall relative amount of NR1 in the LC was not different comparing depressive subjects to individually matched control subjects.

Figure 4

(a) NR1 subunit immunoreactivity in the cerebellum from a single pair used in the analysis blotted in duplicates. Each well was loaded with 20 µg of total protein. The bottom panel shows immunoreactive actin probed with anti-actin antibody on the same blots as a control for protein loading and transfer. (b) NR1 immunoreactivity expressed as percentages of values from paired control subjects. Each bar is an average of duplicate comparisons. The overall relative amount of NR1 in the cerebellum was normal among depressive subjects compared with individually matched control subjects.

Figure 5

(a) NR2C subunit immunoreactivity in the LC from a single pair used in the analysis, representing pooled tissue from three anatomical levels (rostral, middle, and caudal). Each well was loaded with 20 µg of total protein. The bottom panel shows immunoreactive actin probed with anti-actin antibody on the same blots as a control for protein loading and transfer. (b) NR2C immunoreactivity expressed as percentages of values from paired control. Each bar is an average of duplicate comparisons. The overall relative amount of NR2C was higher in the LC (+61%, p = 0.003) among depressive subjects compared with individually matched control subjects.

Figure 6

(a) NR2C subunit immunoreactivity in the cerebellum from a single pair used in the analysis. Each well was loaded with 20 µg of total protein. The bottom panel shows immunoreactive actin probed with anti-actin antibody on the same blots as a control for protein loading and transfer. (b) NR2C immunoreactivity expressed as percentages of values from paired control. Each bar is an average of duplicate comparisons. The overall relative amount of NR2C was elevated (+35%), but this difference did not reach statistical significance among depressives compared with individually matched control subjects.

Figure 7

Relationships between the amount of NR2C immunoreactivity and PMI for control (a) and depressed subjects (b).