Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit-Specific Antibody

Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N -glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.     

Membrane-bound neuraminidase in the developing mouse brain

The specific activity of membrane-bound neuraminidase towards exogenously added gangliosides in the developing mouse brain was investigated. While whole brain was examined in fetuses, studies were carried out comparatively in cortex and cerebellum of postnatal stages. Considerable differences were found among brain areas. There was a rapid increase in forebrain neuraminidase activity before birth. This high level of activity was maintained throughout the first postnatal week, followed by a slow leveling-off to adult values on the 30th day. In the cerebellum a rapid increase in neuraminidase activity occurred between the 10th and 16th postnatal day. After having reached the maximum, enzyme activity declined rapidly, with adult values being reached on the 21st day. Neuraminidase activity in the adult cerebellum exceeds that in the cortex by 65.9 %. The results are discussed in reference to developmental changes in ganglioside metabolism and the possible involvement of neuraminidase in its regulation is pointed out.     

αγ-Enolase in the Rat: Ontogeny and Tissue Distribution

Abstract: The rat brain enolases are dimers composed of α and γ subunits. At pH 8.6 αγ-enolase seemed to be stable, and no evidence was found for the possible formation of αγ-enolase from αα-enolase and γγ-enolase in the course of rat brain homogenization. During ontogeny of the rat forebrain, αγ-enolase was formed before γγ-enolase. The half-maximal specific concentrations were reached at postnatal days 14 and 23, respectively. The distribution of αγ- and γγ-enolase in various rat brain areas was also investigated. In all areas both forms were present. In neuroendocrine tissues αγ-enolase was present at a much higher concentration than γγ-enolase. The ratio between γγ-enolase and αγ-enolase may be indicative of the degree of neuronal maturation, a conclusion further substantiated by the high ratio observed in cerebellum and the low ratio observed in olfactory bulbs, both compared with the ratio in forebrain.     

Postnatal Development of a Granule Cell-Enriched, Neurone-Specific Glycoprotein, gp50, in Normal and Thyroid-Deficient Rats

Glycoprotein gp50 is a neurone-specific, granule cell-enriched glycoprotein that is also a major component of isolated synaptic membranes. Here, we describe the use of a monoclonal antibody, mab SM gp50, to study the postnatal development of gp50 in the brain of normal and thyroid-deficient rats. Radioimmunoassay, enzyme-linked immunosorbent assay, and Western blotting show that gp50 is not detectable in brain until postnatal day 4 (P4) in both forebrain and cerebellum. In forebrain, the rate of increase of gp50 levels is maximal between P12 and P20. It is somewhat later in cerebellum, where peak levels are attained between P30 and P35. Immunocytochemical studies show little detectable gp50-like immunoreactivity before P16, and the staining is still weak, relative to adult tissue, at P25. The intense staining of the granule cell layer characteristic of adult cerebellum predominantly appears after P25. Development of gp50 is severely retarded in the cerebellum of thyroid-deficient rats, particularly during the second and third postnatal weeks. However, by the fourth postnatal week, gp50 levels in normal and hypothyroid animals are comparable. The results indicate that significant alterations in the pattern of gp50 expression continue to occur at a late stage of cerebellar development. In particular, the increase in immunocytochemical staining of the granule cells after P25 is striking in that by this time most major events associated with cerebellar development are essentially complete.     

Differentiation-Dependent Sialylation of Individual Neural Cell Adhesion Molecule Polypeptides During Postnatal Development

The postnatal sialylation of individual neural cell adhesion molecule (N-CAM) polypeptides by a developmentally regulated sialyltransferase in Golgi-enriched fractions isolated from rat brain is described. The 120-kilodalton polypeptide of N-CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140- and 180-kilodalton N-CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N-CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated "embryonic" form of N-CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The "embryonic" form of N-CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N-CAM was virtually undetectable in the immunoblots of postnatal material.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP: phosphatidate cytidylyltransferase and CDPdiacylglycerol: inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7–10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP: phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

THE DEVELOPMENT OF D-AMINO ACID OXIDASE IN RAT CEREBELLUM

D-Amino acid oxidase (D-amino acid: O₂ oxidoreductase (deaminating), EC 1.4.3.3; D-AAO) activity is biochemically undetected in rat brain stem, cerebellum and forebrain until 14 days after birth. Adult levels are attained by day 30 in the brain stem, and by day 36 in the cerebellum. At adulthood, forebrain D-AAO activity per g wet weight of tissue is less than 2% that of the cerebellum. In contrast to the pattern in the CNS, substantial D-AAO activity is present in both liver and kidney 2 days before birth and adult levels are approached within 2 weeks of birth. Nonetheless, D-AAO activities in rat liver, kidney, brain stem and cerebellum are likely to be due to a single enzyme which has properties very similar to the purified hog D-AAO. The late ontogenesis of D-AAO activity in cerebellum and brain stem relative to that in liver and kidney parallels reported phylogenetic data. Histochemical staining for D-AAO in rat cerebellar cortex is absent until 15 days after birth when activity is first observed in some cells of the external germinal zone and adjacent molecular layer. These cells appear to migrate to a final destination around the Purkinje cell soma and leave processes at the pial surface. By 21 days of age an adult pattern of staining is manifest throughout the cerebellum but it is of weak intensity. The adult pattern includes some staining in the granular layer which seems to be associated with mossy fibers and certain cerebellar glomeruli, and strong staining at the pial surface, in the molecular layer, and in cells surrounding, but not within, the Purkinje cell soma. The data suggest that the biochemical appearance of D-AAO in developing cerebellum derives from two sources: one associated with differentiation of one of the last cell types to form from the external germinal zone, and the other with maturation of mossy fibers and their synapses (cerebellar glomeruli).     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Pre-and Postnatal Developmental Changes of Adrenoceptor Subtypes in Rat Brain

beta-Adrenergic receptor subtypes, beta 1 and beta 2, were studied during pre- and postnatal development in the rat brain. [125I]Iodocyanopindolol (6-300 pmol/L) binding assays in the presence of 5-hydroxytryptamine (0.6-6 mumol/L) were used to measure exclusively beta-adrenergic receptors. In forebrain tissue, saturable and stereoselective binding was detected on gestational day 13. The amount of beta-adrenergic binding increased until postnatal day 23, when adult values were reached. The dissociation constants of [125I]iodocyanopindolol binding remained the same throughout development, as did the affinity of several beta-adrenergic and non-beta-adrenergic compounds. The proportion of the beta 2-adrenergic receptors was determined using the beta 1-selective antagonist ICI-89406 (7-150 nmol/L) and was found to change from 65% in prenatal forebrain tissue to 28% in adulthood. In cerebellum/medulla pons tissue, however, the proportion of beta 2-receptor binding (80%) remained unchanged during the whole developmental period.     

Cholesterol Esters of the Human Brain During Fetal and Early Postnatal Development: Content and Fatty Acid Composition

Abstract: The content and fatty acid composition of cholesterol esters of the human brain during development from 13 weeks' gestation up to 26 months of age was studied. The three major brain areas, the forebrain, cerebellum, and the brain stem, were studied separately. The concentration of the esters in each brain region was the highest at the earliest fetal age of 13 weeks and fell during growth. However, transient rises in the concentration were observed, at about birth in the forebrain and at 4–5 months after birth in the cerebellum The peak concentration during the transient period (125–150 μg/g fresh tissue of forebrain and 100–125 μg/g of cerebellum) was similar to the concentrations observed in the two parts respectively during early fetal ages. The brain stem also showed similar transient peak at about a few weeks before birth, but only when the esters were expressed as amount per cell. In absolute terms, a clear transient period was evident in the forebrain between birth and 9 months, while in the cerebellum or the brain stem, the total amount of the esters increased up to about 1 year of age and then remained almost unchanged. The major fatty acids of the esters were palmitic, palmitoleic, stearic, oleic, linoleic and arachidonic acid. Most of these fatty acids showed certain changes in relative proportions during development. Thus, in the forebrain, palmitic and oleic acid decreased from about 32% and 40% (weight percentages) at 13–15 weeks of gestation to about 20% and 25% respectively at 26 months of age. During this period, linoleic and arachidonic acid increased from about 3% and S% to about 10% and 24%, respectively. Most of these changes occurred after birth. The cerebellum and the brain stem differed only slightly from the forebrain in either the fatty acid composition or the pattern of the developmental changes in the composition.     

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.     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

Postnatal Changes in Cathepsin D in Rat Neural Tissue

Cathepsin D, an aspartyl endopeptidase, was analyzed in cortex from forebrain and cerebellum, spinal cord, and optic and sciatic nerves, and in the liver of rats from 1 to 120 days of age. Cathepsin D was quantitated in tissue extracts by measurement of enzyme specific activity on a substrate of [methyl-¹⁴C]-methylated hemoglobin and by radioimmunoassay. Immunocytochemistry was used to ascertain the identity of the mixed cell types that contributed to the cathepsin D detected. As quantitated by radioimmunoassay, immunoreactive cathepsin D varied between 0.2 and 1 ng/μg of total protein. Maximum activity occurred at approximately the 15th postnatal day; the least amount of immunoreactive cathepsin D was found at 30 or 60 days of age. A subsequent increase of varying magnitude occurred at postnatal day 120. There was good correspondence between immunoreactive enzyme and enzyme specific activity, which ranged from 1 to 4 ng/μg of total protein, and the activities determined by the two methods provided similar, but not identical, developmental profiles. Cathepsin D was demonstrated by immunocytochemistry to be present in most neurons, in all choroid plexus epithelium, and in certain oligodendrocytes from the first postnatal day. Cathepsin D was present in oligodendrocytes in cord lateral funiculi and optic nerve by the first postnatal day, and by the sixth postnatal day many oligodendrocytes were abundantly stained. In contrast, oligodendrocytes in the corpus callosum and in the cerebellar white matter did not contain demonstrable cathepsin D until postnatal days 10 and 15, respectively. These results indicate a role for cathepsin D during the postnatal development of rat CNS and suggest that this proteinase may be involved in the steps of myelination.     

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.     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

Developmental regulation of somatostatin gene expression in the brain is region specific

Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.     

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.     

Localization of the Extracellular Matrix Protein SC1 Coincides with Synaptogenesis During Rat Postnatal Development

SC1 is an extracellular matrix protein that belongs to the SPARC family of matricellular molecules. This anti-adhesive protein localizes to synapses in the adult rat brain and has been postulated to modulate synapse shape. In this study, increased levels of SC1 were detected from postnatal days 10–20, with a peak at postnatal day 15, a period of intense synaptogenesis. During this time, increased colocalization of SC1 with the synaptic marker synaptophysin was observed in synapse-rich regions of the cerebellum and the cerebral cortex. These findings indicate that the pattern of SC1 localization coincided with synaptogenesis during rat postnatal development.     

Novel Kv3 glycoforms differentially expressed in adult mammalian brain contain sialylated N-glycans.

The N-glycan pool of mammalian brain contains remarkably high levels of sialylated N-glycans. This study provides the first evidence that voltage-gated K+ channels Kv3.1, Kv3.3, and Kv3.4, possess distinct sialylated N-glycan structures throughout the central nervous system of the adult rat. Electrophoretic migration patterns of Kv3.1, Kv3.3, and Kv3.4 glycoproteins from spinal cord, hypothalamus, thalamus, cerebral cortex, hippocampus, and cerebellum membranes digested with glycosidases were used to identify the various glycoforms. Differences in the migration of Kv3 proteins were attributed to the desialylated N-glycans. Expression levels of the Kv3 proteins were highest in cerebellum, whereas those of Kv3.1 and Kv3.3 were much lower in the other 5 regions. The lowest level of Kv3.1 was expressed in the hypothalamus, whereas the lowest levels of Kv3.3 were expressed in both thalamus and hypothalamus. The other regions expressed intermediate levels of Kv3.3, with spinal cord expressing the highest. The expression level of Kv3.4 in the hippocampus was slightly lower than that in cerebellum, and was closely followed by the other 4 regions, with spinal cord expressing the lowest level. We suggest that novel Kv3 glycoforms may endow differences in channel function and expression among regions throughout the central nervous system.