D-serine as a neuromodulator: regional and developmental localizations in rat brain glia resemble NMDA receptors

Abstract

D-Serine is localized in mammalian brain to a discrete population of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared the immunohistochemical localizations of D-serine, glycine, and NMDA receptors in rat brain. In the telencephalon, D-serine is concentrated in protoplasmic astrocytes, which are abundant in neuropil in close vicinity to NMDA receptor 2A/B subunits. Ultrastructural examination of the CA1 region of hippocampus reveals D-serine in the cytosolic matrix of astrocytes that ensheath neurons and blood vessels, whereas NR2A/B is concentrated in dendritic spines. By contrast, glycine immunoreactivity in telencephalon is the lowest in brain. During postnatal week 2, D-serine levels in cerebellum are comparable to those in adult cerebral cortex but fall to undetectable levels by day 26. During week 2, we observe parallel ontogeny of D-serine in Bergmann glia and NR2A/B in Purkinje cells, suggesting a role for astrocytic D-serine in NMDA receptor-mediated synaptogenesis. D-Serine in the radial processes of Bergmann glia is also well positioned to regulate NMDA receptor-dependent granule cell migration. In the inner granule layer, D-serine is found transiently in protoplasmic astrocytes surrounding glomeruli, where it could regulate development of the mossy fiber/granule cell synapse. D-Serine seems to be the endogenous ligand of glycine sites in the telencephalon and developing cerebellum, whereas glycine predominates in the adult cerebellum, olfactory bulb, and hindbrain.

Fig. 1.

P21 serial brain sections stained ford-serine, NR2A/B, or glycine. Am, Amygdala;Cl, claustrum; Cx, cortex;EPL, external plexiform layer; Hb, habenula; Hp, hippocampus; Hy, hypothalamus; PM, pons/medulla; Sn, substantia nigra; Sp, spinal cord; WM, white matter; VNL, vomeronasal nerve layer.

Fig. 2.

P21 amygdala stained for d-serine, NR2A/B, or glycine. Both d-serine and NR2A/B appear concentrated near blood vessels.

Fig. 3.

P21 hippocampus stained for d-serine, NR2A/B, or glycine. DG, Dentate gyrus;Hi, hilus; L, stratum lacunosum molecular; Lu, stratum lucidum of CA3 region;Mol, molecular layer of dentate gyrus; O, stratum oriens; P, stratum pyramidale; R, stratum radiatum; S, subiculum; WM, white matter.

Fig. 4.

Detailed comparison of d-serine and NR2A/B in P21 hippocampal CA1 region. A, B,d-Serine concentrates in the glia of molecular layers, especially near blood vessels (asterisks), whereas NR2A/B is found in pyramidal neurons and all layers of neuropil.C, D, Higher-power magnification of regions near blood vessels.

Fig. 5.

Ultrastructural comparison of d-serine and NR2A/B in hippocampal CA1 region. Brain sections were stained with the immunoperoxidase technique and then processed for electron microscopy. d-Serine concentrates in the cytosolic matrix of astrocytes (Ast) in neuropil and in foot processes ensheathing blood vessels (BV), whereas endothelial cells (En) are unstained. NR2A/B concentrates in dendritic spines (arrows). 10,000× magnification.

Fig. 6.

Levels of free d-serine and glycine in cerebellum during postnatal development. Cerebella were analyzed by HPLC for free amino acids. Values are mean ± SEM;n = 3.

Fig. 7.

Transient staining for d-serine and NR2A/B in developing cerebellum. The cell bodies ofd-serine glia (Ast) are well labeled by P7, when Purkinje cells (P) begin to stain for NR2A/B. One week later, both d-serine and NR2A/B concentrate in the molecular layer, with d-serine in Bergmann glia (BG) and NR2A/B throughout the dendritic tree of Purkinje cells. In the P14 granule layer, many protoplasmic astrocytes stain intensely for d-serine, whereas a few Golgi neurons (Go) are lightly stained for NR2A/B. By P21, staining for both has decreased, but substantial amounts of d-serine persist in the radial process of BG and in the cell bodies of protoplasmic astrocytes (Ast). NR2A/B at P21 has become less prominent in Purkinje cells and has appeared in some basket cell pinceau (Pi). In mature adults, d-serine occurs weakly in Bergmann glia cell bodies, whereas NR2A/B is restricted to basket cell pinceau.

Fig. 8.

High magnification of adult cerebellum near Purkinje cell bodies. d-Serine is restricted to Bergmann glia (BG) in the molecular layer (Mol), especially in glial cell bodies that reside between Purkinje cells (P). The granule layer (Gr) is not stained for d-serine. NR2A/B still occurs in a minority of Purkinje cell dendrites, but the most intense staining occurs in basket cell pinceau (Pi), which also stain for glycine. Golgi neurons (Go), whose cell bodies reside in the granule layer, are the major glycinergic element of the cerebellum.

Fig. 9.

Models depicting the proposed modulatory roles ford-serine and glycine in the CA1 region of hippocampus (left) and the AOB (ACCES. OLF BULB, right). d-Serine is black; glycine is gray. Stars indicate localizations of NMDA receptors. In the hippocampus, d-serine-containing protoplasmic astrocytes (Ast) are localized near NMDA receptors located on pyramidal cell (Py) dendrites, whereas glycinergic cells are rare. In the AOB, bothd-serine and glycine appear concentrated near NMDA receptors located on mitral cells (Mi), with thed-serine found in superficial bulbar glia (SBG) surrounding the vomeronasal nerve (VN) and in protoplasmic astrocytes (Ast) in the plexiform layer. Glycine concentrates in interneurons (I) and periglomerular cells (PG).

Fig. 10.

Models contrasting the proposed roles ford-serine and glycine in developing and adult cerebellum.d-Serine is black; glycine isgray. Stars indicate localizations of NMDA receptors. In developing molecular layer (left), astrocytic d-serine is found in Bergmann glia (BG), which ensheath Purkinje cells expressing NMDA receptors and also guide migrating granule cells (Gr) expressing NMDA receptors. d-Serine released from Bergmann glial processes might synergize with glutamate released by parallel fibers (PF) and climbing fibers (CF). In the developing inner granule layer, protoplasmic astrocytes (Ast) might released-serine near the developing glomerular synapse to synergize with glutamate from mossy fibers (MF), whereas glycinergic basket (Ba) and Golgi neurons (Go) have not yet established connections with NMDA receptor-containing synapses. In contrast, in adult cerebellum (right), no d-serine is present, and NMDA receptors have disappeared from Purkinje cells. NMDA receptor-associated glycine sites located on the basket cell pinceau and granule cells might be modulated exclusively by glycinergic basket (Ba) and Golgi (Go) neurons.