D-amino acid oxidase is expressed in the ventral tegmental area and modulates cortical dopamine

Abstract

D-amino acid oxidase (DAO, DAAO) degrades the NMDA receptor co-agonist D-serine, modulating D-serine levels and thence NMDA receptor function. DAO inhibitors are under development as a therapy for schizophrenia, a disorder involving both NMDA receptor and dopaminergic dysfunction. However, a direct role for DAO in dopamine regulation has not been demonstrated. Here, we address this question in two ways. First, using in situ hybridization and immunohistochemistry, we show that DAO mRNA and immunoreactivity are present in the ventral tegmental area (VTA) of the rat, in tyrosine hydroxylase (TH)-positive and -negative neurons, and in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Second, we show that injection into the VTA of sodium benzoate, a DAO inhibitor, increases frontal cortex extracellular dopamine, as measured by in vivo microdialysis and high performance liquid chromatography. Combining sodium benzoate and D-serine did not enhance this effect, and injection of D-serine alone affected dopamine metabolites but not dopamine. These data show that DAO is expressed in the VTA, and suggest that it impacts on the mesocortical dopamine system. The mechanism by which the observed effects occur, and the implications of these findings for schizophrenia therapy, require further study.

Keywords: D-serine; DAAO; DAO; NMDA receptor; microdialysis; schizophrenia.

Figure 1

Expression of DAO mRNA in rat VTA and cerebellum. (A) Northern blot of rat cerebellar RNA showing that the DAO cRNA probe detects a single band of ~2 kb. (B) In situ hybridization, showing signal for DAO mRNA in the VTA, with clusters of grains over neurons (arrow). (C) VTA, sense strand hybridization control, showing low level of background signal. (D) Cerebellum, showing DAO mRNA concentrated in the Purkinje cell layer (P) with moderate signal in the granule cell layer (G) and low signal in the molecular layer (M). (E) Cerebellum, sense strand hybridization control. Sections are counterstained with cresyl violet.

Figure 2

DAO immunoreactivity in rat VTA and cerebellum. (A) Triple-labeling in VTA showing immunofluorescence for DAO (green), tyrosine hydroxylase (TH, blue), and glial fibrillary acidic protein (GFAP, red). The open head arrows point to two TH-positive neurons immunoreactive for DAO; the closed head arrows show DAO-immunoreactive TH-negative neurons. The inset shows a DAO-immunoreactive GFAP-positive astrocyte. (B) Double-labeling in cerebellum, showing DAO (green), and GFAP (red) immunoreactivity. Punctate labeling for DAO is concentrated in the Purkinje cell layer (P), wherein it surrounds the unlabeled Purkinje cell somata (asterisk), and the immunoreactivity extends into the molecular layer (M) along GFAP-positive processes. G: granule cell layer. Bar: 50 microns.

Figure 3

Dopamine (DA) and its metabolites DOPAC and HVA in medial frontal cortex (mPFC) after injection of sodium benzoate (SB), D-serine (DS), the combination (SB+DS), or vehicle (Veh), into the rat VTA. (A–C) Effects of sodium benzoate, showing increases in DA (A), DOPAC (B), and HVA (C). (D–F) Effects of D-serine, showing no effect on DA (D), but increases in DOPAC (E) and HVA (F). (G–I) Effects of combined sodium benzoate and D-serine, showing increases in DA (G) and no significant change in DOPAC (H) or HVA (I). The arrow in each panel denotes the time of injection. ^*p < 0.05; ^**p < 0.01; ^***p < 0.005.