The surface glycoprotein Thy-1 is excluded from growing axons during development: a study of the expression of Thy-1 during axogenesis in hippocampus and hindbrain.

Thy-1 is a developmentally regulated surface glycoprotein expressed on a number of tissues, including nerve where it is a major surface component of mature neurons. During neural development in the rat and mouse, expression of Thy-1 protein does not necessarily follow appearance of its mRNA, but additionally requires completion of the initial phase of axonal growth. Where there is a substantial lag phase between initial elongation and final axonal outgrowth into a terminal field (e.g. pontine projection to the cerebellum), Thy-1 protein appears at the cell body and dendrites of the neurons, but is excluded from their axons until the terminal phase of axonal growth is completed. In the more complex case of the vestibular ganglion neurons, whose axons project primarily to the vestibular nuclei in the brainstem before birth, and then 1-2 weeks later into the cerebellum, Thy-1 enters the proximal axonal regions where growth is completed, but not the distal growing ends. Thus complex controls govern the initial expression and distribution of Thy-1 so as to exclude it from growing regions of axons.     

mPPP1R16B is a novel mouse protein phosphatase 1 targeting subunit whose mRNA is located in cell bodies and dendrites of neurons in four distinct regions of the brain

We cloned a cDNA encoding a novel mouse protein whose human homolog has been annotated in GenBank as a regulatory subunit of protein phosphatase 1, PPP1R16B. Both the primary protein sequence and the domain structure are highly conserved between PPP1R16B and proteins of unknown function from other species, such as Caenorhabditis elegans and Drosphila melanogaster. Besides a protein phosphatase 1 interaction motif, mouse PPP1R16B (mPPP1R16B) and the related proteins contain ankyrin repeats that may constitute binding sites for other proteins and C-terminal prenylation signals that are likely to target the proteins to the plasma membrane. In the adult mouse, Ppp1r16b mRNA is expressed in most tissues examined, with highest expression levels in kidney and brain. In the brain, Ppp1r16b message is particularly enriched in the olfactory bulb, striatum, dentate gyrus, and cerebellum. During postnatal cerebellar development, Ppp1r16b mRNA expression levels increase gradually and are maximal around postnatal day 30. In situ hybridization revealed that Ppp1r16b message is found in both the cell bodies and the dendrites in Purkinje cells of the cerebellum and granule neurons of the dentate gyrus.     

Developmental expression of the GABAA receptor alpha 1 subunit mRNA in the rat brain

Recent studies have suggested that the GABAA, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABAA) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the alpha 1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that alpha 1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the alpha 1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the alpha 1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of alpha 1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABAA receptor complex. Furthermore, the onset of alpha 1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABAA receptor subunit in the cerebellum.     

Staggerer Mutant Mouse Purkinje Cells Do Not Contain Detectable Calmodulin mRNA

Within the cerebellum calmodulin mRNA is found predominantly in Purkinje cells, with lower levels in granule cells and interneurons. The message shows developmental increases during the first 14 days postnatally. Surviving Purkinje cells of the staggerer (sg/sg) mutant, which are grossly stunted and lack tertiary dendritic spines, contain no detectable calmodulin mRNA, as assayed by Northern blot or an enhanced biotinylated in situ hybridization. This is in contrast to both the Lurcher Purkinje cells and sg/sg granule cells, which express normal levels of this mRNA up until the time they disappear. The sg/sg phenotype can be explained by a defective Purkinje-cell-specific regulatory mechanism for calmodulin.     

Developmental expression of the GABAA receptor α1 subunit mRNA in the rat brain

Recent studies have suggested that the GABA_A, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABA_A) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the α1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that α1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the α1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the α1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of α1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABA_A receptor complex. Furthermore, the onset of α1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABA_A receptor subunit in the cerebellum.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Altered Ontogenesis of Muscarinic Receptors in Agranular Cerebellar Cortex

Abstract: The developmental pattern, the agonist binding properties and the cellular origin(s) of muscarinic binding sites were investigated in agranular cerebellum of x-irradiated rats, of Gunn rats with hereditary hyperbilirubinemia, and of staggerer mutant mice. The density of muscarinic binding sites was found to be higher than normal in all of these cerebellar types, indicating that granular neurons do not greatly contribute to binding of acetylcholine in the rodent cerebellum. The total number of muscarinic binding sites as measured by binding of [³H]4NMPB remains unchanged in the agranular cerebellum of x-irradiated rats. However, the number of muscarinic sites is reduced by about 30% in the agranular cerebellum of homozygous Gunn rats (jj), in which fibrous astrocytes and Purkinje cells are also damaged. In the cerebellum of staggerer mice (sg/sg), where a cascade of events leads to massive damage to mossy fibers and Golgi cells in addition to granular neurons and Purkinje cells, the content of muscarinic receptors is reduced by 50%. Thus, the number of muscarinic binding sites in the rodent cerebellum seems to depend on the integrity of the additional cell types and cellular elements, damaged in these agranular models. The ontogenetic variations in the affinity of cerebellar muscarinic sites for binding of carbamylcholine in normal and Gunn rat cerebellum were compared with those observed in x-irradiated and staggerer cerebellum, where elimination of granular neurons induces the formation of ‘heterologous’ synapses. Muscarinic binding affinity increases 10-fold during postnatal development in the cerebellum of normal and Gunn rats. In the immature x-irradiated cerebellum, the affinity of muscarinic binding sites was found to be nearly as high as that detected in the adult normal cerebellum. In contrast, cerebella of 5-month-old staggerer mice display 5-fold lower affinity than their normal counterpart values, as low as that determined in normal immature cerebellum. The characteristic ontogenetic pattern of muscarink binding is therefore indicated to be related to the formation of correct circuitry, but not to the presence of granular neurons, in the developing rat cerebellum.     

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.     

Regulation and developmental expression of the divalent metal-ion transporter in the rat brain.

Divalent metal ion transporter 1 (DMT1) is a recently identified metal-ion transporter that appears to mediate the absorption of iron in the intestine. DMT1 mRNA is also present in discrete areas of the brain. In this study, we examined the expression of DMT1 mRNA in developing rat brain. DMT1 mRNA was found by in situ hybridization in the striatum, cortex, hippocampus and cerebellum. During development, DMT1 mRNA was found in Purkinje and granule cells in the cerebellum at post-natal day (PND) 14 and PND 30. DMT1 mRNA was also expressed in the external granular layer of the cerebellum at PND 14. No change in the level of DMT1 mRNA was observed by Northern analysis in the cerebellum at different ages between PND 1 and 21. DMT1 was found by Northern analysis in cultures of rat astrocytes. Activation of protein kinase C increased the expression of DMT1 in kidney epithelial cells but not astrocytes from newborn rats. Because DMT1 is expressed in a wide variety of types of cells, we suggest that it plays an important role in metal homeostasis in the brain.     

Axonal Membrane-Skeletal Protein A60: Association with a Brain Spectrin-Binding Activity and Entry into Cerebellar Axons at a Stage After the Initiation of Axonal Growth

Abstract: A60 is a 60-kDa component of the axonal cortical cytoskeleton in CNS neurones. It appears to be neurone specific and is tightly bound to brain membranes. In this study the cytoskeletal activities and developmental expression of A60 in rat cerebellum have been examined using the monoclonal antibody DR1. A60 in a partially purified soluble extract of brain membranes interacts selectively with brain but not erythrocyte spectrin. Because erythrocyte spectrin is more closely related to the dendritic form of spectrin than the axonal form, this raises the possibility that AGO localises in axons by interaction with the axonal form of spectrin only. A60 is not found in rat cerebellum before the day of birth. However, during postnatal development of the cerebellum (days 1–13) DR1 reactivity appears progressively. On postnatal day 1, a small population of cells in the mantle layer (presumptive Purkinje cells) is DR1 positive. There is no DR1 reactivity found in Purkinje cell axons during their initial phase of growth. By postnatal day 7, Purkinje cell bodies, initial dendritic segments, and the cerebellar white matter are all positive. This pattern of labelling is strengthened up until postnatal day 13. By contrast, in adult rat cerebellum, the location of A60 has changed so that it is most concentrated in axons, and dendritic staining is lost. These data indicate that A60 is a spectrin-binding component of the adult axonal membrane skeleton, the presence of which is only required in axons after the initial phase of growth.     

Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.     

Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellum were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H]glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-1 glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-beta-galactosidase, 40-45% of the [3H]glucosamine or [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence, while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. At least eight early postnatal rat brain glycoproteins also appear to be anchored to the membrane by phosphatidylinositol. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.     

Chromogranin A in neurons of the rat cerebellum and spinal cord: quantification and sites of expression.

Chromogranin A (CGA) is an abundant protein of dense-cored secretory vesicles in endocrine and neuronal cells. The present study, for the first time, compares CGA of neurons of the central nervous system with the CGA of adrenal origin. By S1 nucleus protection assay, we found that the 3' part of the CGA mRNA between exons 5-8 of the cerebellum and the spinal cord of the rat is homologous to that of the adrenal. In situ hybridization histochemistry revealed that CGA mRNA in the cerebellar cortex is present in cell bodies of Purkinje cells and in neurons of the deep cerebellar nuclei. The perikarya of these cells also exhibit CGA-like immunoreactivity. CGA mRNA and CGA-like immunoreactivity are also present in the motoneurons of the ventral, lateral, and dorsal horns of the rat spinal cord. The amounts of CGA, as determined by radioimmunoassay in cerebellum and spinal cord, were about one tenth of the amounts detected in the adrenal, adenohypophysis, or the olfactory bulb. The sites of CGA expression suggest that CGA may be involved in signal transduction in the motor system.     

Thy-1 is an in vivo and in vitro marker of liver myofibroblasts

Thy-1, a glycophosphatidylinositol-linked glycoprotein of the outer membrane leaflet, has been described in myofibroblasts of several organs. Previous studies have shown that, in fetal liver, Thy-1 is expressed in a subpopulation of ductular/progenitor cells. The aim of this study has been to investigate whether the liver myofibroblasts belong to the Thy-1-positive subpopulation of the adult liver. The expression of Thy-1 has been studied in normal rat liver, in the rat liver regeneration model following 2-acetylaminofluorene treatment and partial hepatectomy (AAF/PH), and in isolated rat liver cells, at the mRNA and protein levels. In normal rat liver, Thy-1 is detected in sparse cells of the periportal area, whereas 7 days after PH in the AAF/PH model, a marked increase of the number of Thy-1-positive cells is detectable by immunohistochemistry. Comparative immunohistochemical analysis has revealed the co-localization of Thy-1 and smooth muscle actin, but not of Thy-1 and cytokeratin-19, both in normal rat liver and in the AAF/PH model. Investigation of isolated rat liver cell populations has confirmed that liver myofibroblasts are Thy-1-positive cells, whereas hepatocytes, hepatic stellate cells, and liver macrophages are not. Thy-1 is the first cell surface marker for identifying liver myofibroblasts in vivo and in vitro. Jozsef Dudas and Tümen Mansuroglu contributed equally to this study. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 402, projects C6, D3, D4).     

Disturbed Purkinje cell migration due to reduced expression of Reelin by X-irradiation in developing rat cerebellum.

The major histogenetic events of the rat cerebellum take place in the early postnatal days. During this period, precursors of microneurons, such as granule cells, form the external granular layer (EGL), extend over the surface of the primordial cerebellum, and actively proliferate. Postmitotic granule cells leave the EOL and migrate to the internal granular layer (IGL). On the other hand, guided by radial glial fibers, immature Purkinje cells migrate from the ventricular zone of the fourth ventricle and settle in the Purkinje cell plate with thickness of several cells. Various cell adhesion molecules are involved in the interaction between the migratory immature Purkinje cells and processes of the radial glia as the basis for contact guidance. The second process is the formation of immature Purkinje cells to the monolayer. This process takes place at the first week after birth of the rat and cell adhesion molecules such as neural cell adhesion molecule (NCAM), fibronectin, tenascin and Reelin are also suggested to play an important role for the cell patterning. When rat fetuses are exposed to X-radiation in the last gestation period, abnormal foliation of the cerebellum develops with ectopic Purkinje cells. The molecular mechanism that contributes to abnormal migration of Purkinje cells and foliar malformation induced by X-irradiation in the cerebellum are not yet clear. This study was undertaken to elucidate the mechanisms of ectopic Purkinje cell formation by examining the expression of cell adhesion molecules.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.