Prenatal ethanol exposure, generalized learning impairment, and medial prefrontal cortical deficits in rats.

Prenatal ethanol exposure may cause neurological damage and subsequent mental retardation in humans, with learning deficits similar to those following damage to the prefrontal cortex. This study examined cognitive dysfunction and cortical damage after prenatal exposure to ethanol using a chronic administration model. Pregnant Sprague-Dawley rats received one of three diets during gestation: a liquid diet containing 35% ethanol-derived calories (ETOH), an isocaloric liquid diet (ISO), or standard chow (CHOW). Subjects were obtained from ETOH dams with blood alcohol concentrations (BACs) above 90 mg/dl and corresponding ISO and CHOW controls (one male pup/litter; n=6 pups/group). At approximately 90 days of age, subjects began training on a series of unique auditory discrimination problems using a successive go/no-go procedure. A criterion of 85% accuracy determined when a rat continued to the next problem. Subjects completed a varying number of problems within a 30-session limit, after which all rats were tested on a tone/click discrimination and reversal. Subjects were then sacrificed and neuronal number in the medial prefrontal cortex (mPFC) was estimated by the optical fractionator method. Prenatal ethanol exposure induced significant cell loss in the mPFC, which was associated with significantly impaired reversal learning. Poor performance by ETOH subjects on the tone/click reversal indicates a transfer of training deficit that may reflect failures of inhibitory control.

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb.

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.

Restriction of high CD15 expression to a subset of rat cerebellar astroglial cells can be overcome by transduction with adenoviral vectors expressing the rat alpha 1,3-fucosyltransferase IV gene.

Glycoconjugates bearing the epitope 3-fucosyl-N-acetyllactosamine (CD15) are believed to be involved in cell-cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells. In dissociated cerebellar cell cultures, few glial cells normally express CD15. However, transduction of these cells with an adenoviral vector (AdGFPCMVFucT) that expressed both green fluorescent protein (GFP) and FLAG-tagged rat alpha 1, 3-fucosyltransferase IV (rFuc-TIV) resulted in high CD15 expression on the surface of all transduced glial cells. Likewise, infection of cerebellar slice cultures caused the appearance of CD15-positive transduced cells of glial cell morphology in the internal granule cell layer. Thus, enhancement of Fuc-T activity caused robust CD15 expression in cerebellar glial cells that normally show little expression of CD15, suggesting a role for Fuc-T levels in regulating CD15 expression in this cell type. The manipulation of levels of glycosyltransferases using adenoviral vectors may prove a useful tool to investigate questions of glycoconjugate regulation in glial cells in the developing rodent cerebellum.

Callosal axon guidance defects in p35(-/-) mice.

Mice lacking p35, an activator of cdk5 in the central nervous system (CNS), exhibit defects in a variety of CNS structures, most prominently characterized by a disruption in the laminar structure of the neocortex (Chae et al., 1997). In addition, alterations of certain axonal fiber tracts are found in the cortex of p35 mutant mice. Notably, the corpus callosum appears bundled at the midline, but dispersed lateral to the midline. Tracer injection experiments in adult p35 mutant mice reveal that projecting cortical axons fail to assimilate into the corpus callosum, and take oblique paths to the midline. After crossing the midline, cortical axons defasciculate prematurely from the corpus callosum and take similarly oblique paths through the cortex. This callosal phenotype is not detected in reeler mice, which also exhibit defects in cortical lamination, suggesting that the lack of fasciculation of callosal axons is not an inherent manifestation of a disruption of cortical lamination. The embryonic callosal axon tract is defasciculated before crossing the midline, suggesting that axon guidance may be affected during embryonic development of the corpus callosum. In addition, embryonic thalamocortical afferents also exhibit a defasciculated phenotype. These results suggest that defective axonal fasciculation and guidance may be primary responses to the loss of p35 in the cortex. Furthermore, this study postulates a role for the p35/cdk5 kinase in molecular signaling pathways necessary for proper guidance of selective axons during embryonic development.

Polysialic acid facilitates migration of luteinizing hormone-releasing hormone neurons on vomeronasal axons.

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the olfactory placode to the forebrain in association with vomeronasal nerves (VNN) that express the polysialic acid-rich form of the neural cell adhesion molecule (PSA-NCAM). Two approaches were used to investigate the role of PSA-NCAM: injection of mouse embryos with endoneuraminidase N, followed by the analysis of LHRH cell positions, and examination of LHRH cell positions in mutant mice deficient in the expression of NCAM or the NCAM-180 isoform, which carries nearly all PSA in the brain. The enzymatic removal of PSA at embryonic day 12 significantly inhibited the migration of nearly half of the LHRH neuron population, without affecting the VNN tract itself. Surprisingly, the absence of NCAM or NCAM-180 did not produce this effect. However, a shift in the route of migration, resulting in an excess number of LHRH cells in the accessory olfactory bulb, was observed in the NCAM-180 mutant. Furthermore, it was found that PSA expressed by the proximal VNN and its distal branch leading to the accessory bulb, but not the branch leading to the forebrain, was associated with the NCAM-140 isoform and thus was retained in the NCAM-180 mutant. These results provide two types of evidence that PSA-NCAM plays a role in LHRH cell migration: promotion of cell movement along the VNN tract that is sensitive to acute (enzymatic), but not chronic (genetic), removal of PSA-NCAM, and a preference of a subset of migrating LHRH cells for a PSA-positive axon branch over a PSA-negative branch in the NCAM-180 mutant.

Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A.

The NMDA (N-methyl-D-aspartate) subclass of glutamate receptor is essential for the synaptic plasticity thought to underlie learning and memory and for synaptic refinement during development. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or chi-1), which is expressed primarily during brain development. NR3A co-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.

Rapid growth of parallel fibers in the cerebella of normal and staggerer mutant mice.

The growth of cerebellar granule cell axons was examined by placing focal implants of 1,1',dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) in the cerebella of normal and staggerer mutant mice at a series of developmental ages between postnatal day 2 (P2) and P30. Parallel fibers contacting the implant site were brightly labeled by the fluorescent dye, as were the associated granule cell bodies located principally in the internal granule layer. The extent of parallel fiber labeling in the molecular layer and the distance from the implant to the most extreme labeled granule cells were measured in sectioned material. Two additional measures describing the distribution of labeled granule cells about the implant site suggest length bounds for most parallel fibers. Parallel fiber growth is surprisingly rapid; all measures approached peak values at P3-P5, only a few days after the earliest postmitotic granule cells differentiate and migrate. At intermediate ages (P8 and P10), parallel fiber lengths appeared to decrease transiently. At later ages (P15 and beyond), the measures of fiber length increased to their mature values. These values differed little from lengths measured at P3-P5, suggesting that most parallel fiber growth occurs within a few days of cell birth. At early and intermediate ages, parallel fiber lengths in staggerer mice were comparable to controls, suggesting that an interaction with normal healthy Purkinje cells is not essential for parallel fiber outgrowth.

Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors.

Recent reports have suggested that delivery of genes flanked by AAV ITRs may be useful for gene therapy of diseases that involve the brain. We have compared the efficiency of gene expression in vitro in CNS-derived cells from four different promoters when the transgene is flanked by AAV ITRs, using both transfection via cationic liposomes, and infection via rAAV. The human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, and the chicken beta-actin promoter coupled to the CMV enhancer were able to drive expression of the reporter gene beta-galactosidase in all tumor and primary brain cell cultures tested. Although the relative order of efficiency differed between cell types, the CMV promoter was always the strongest, generally by at least one order of magnitude. A comparison of the relative levels of expression seen between different cell types on transfection and infection suggest that not all CNS-derived cells are infected equally efficiently by rAAVs. High level of expression were seen within 24 h of transgene delivery by either transfection or infection, dropping dramatically within days. All cell types and promoters showed the same decline, suggesting that transient expression by rep-rAAVs may be efficient, but stable expression as detected in this system is a low frequency event. In vivo studies using the CMV promoter also suggest that although rep-rAAVs are able to infect efficiently CNS cells and produce high levels of gene expression shortly after transduction, the majority of such infections do not lead to stable high-level expression of transgenes.

The migration of luteinizing hormone-releasing hormone neurons in the developing rat is associated with a transient, caudal projection of the vomeronasal nerve.

Luteinizing hormone-releasing hormone (LHRH) neurons originate in the olfactory placode and vomeronasal organ and migrate to the brain from embryonic day 14 (E14) in the rat. We investigated the development of the vomeronasal nerve and its role as a guide for migrating LHRH neurons. Using fluorescent, lipophilic dye tracing methods, we observed axons that emerge from the vomeronasal organ and cross the nasal septum as several large fascicles. At E14-15, these fascicles converge as they enter the region of the cribriform plate and subsequently disperse, projecting dorsally and caudally across the olfactory bulb and rostral forebrain. At E16, the dorsal branch of the vomeronasal nerve forms a more tightly fasciculated projection; the caudal fibers remain dispersed, extending along the medial forebrain. The number of caudally directed axons decreases during development, leaving four or five present at postnatal day 4 (P4). Immunohistochemical studies indicate that the vomeronasal nerve can be divided into four spatially distinct subpopulations of fibers. One subset, composed of caudal fibers that terminate in the lamina terminalis, selectively expresses TAG-1, a transient axonal surface glycoprotein and PSA-N-CAM, a highly polysialated form of neural cell adhesion molecule. The extension and subsequent retraction of this branch of the vomeronasal nerve corresponds spatially and temporally with the migration of LHRH neurons from the nasal cavity to the brain. Our studies show that between E14 and E18, LHRH neurons migrate in contact with the TAG-1+, PSA-N-CAM+ caudal branch of the vomeronasal nerve.

Can gonadal steroids influence cell position in the developing brain?

The preoptic area/anterior hypothalamus (POA/AH) is a site where hormones dramatically influence development. The POA/AH is comprised of multiple subgroups, but little is known about the derivation of these subgroups during development. Results from several laboratories suggest that some cells in the POA/AH originate from progenitor cells in other regions of the developing nervous system. We are exploring pathways for migration in the developing POA/AH in two ways. First, we are examining the distribution of radial glial processes as potential migratory guides using immunocytochemistry. We have identified a transient pattern of radial glial processes from the lateral ventricles to the pial surface at the base of the POA/AH. Additionally, the expression of a molecule in radial glial processes originating in the third ventricle was decreased by prenatal treatment with testosterone. Second, we are utilizing time-lapse video microscopy in vitro to assess the extent and direction of movements of fluorescent dye-labeled cells at different ages in brain slice preparations from the POA/AH of developing rats. Data from these studies indicate that cell migration in the POA/AH includes movements along dorsal-ventral routes and from lateral to medial positions, in addition to the predicted medial to lateral pathway away from the third ventricle. Several researchers have examined effects of gonadal steroids on neurite outgrowth, cell differentiation, cell death, and synaptogenesis. The determination of cell position, however, may be a key event influenced by gonadal steroids earlier in development. The characterization of migratory pathways that contribute to permanent changes in brain structure and ultimately function is essential for unraveling the process of sexual differentiation.

Purkinje cell compartments in the reeler mutant mouse as revealed by Zebrin II and 90-acetylated glycolipid antigen expression.

The cerebellum is organized into a series of parasagittally aligned bands that may be revealed histologically in the adult mouse by largely complementary immunostaining of Purkinje cells sets with the monoclonal antibodies Zebrin II (ZII; antigen:aldolase C) and P-path (PP; antigen:90-acetyl glycolipids). We compared the normal staining pattern using these markers and an antibody to calbindin with that found in the reeler mutants (rl/rl), in which most Purkinje cell migration is halted beneath the cerebellar white matter. The results revealed that Purkinje cells in reeler mutants, despite their ectopic location in large subcortical masses, show a clear tendency to distribute into alternating zones that either stain for Zebrin II or for P-path, with variable transition zones of mixed labeling. However, the estimated number of zones was fewer than in the normal adult cortex: roughly 7-9 zones are revealed per side in the mutant compared with 14 major divisions in wild type mice. These results raise the possibility that neurons destined to express these markers are segregated during their migration and that the final phase of migration into the cortex might involve further splitting or interdigitation between cell sets expressing the two antigens.

Altered 9-O acetylation of disialogangliosides in cerebellar Purkinje cells of the nervous mutant mouse.

Some gangliosides in the nervous system are developmentally down-regulated, but many other gangliosides continue to be expressed in the adult nervous system. We have previously demonstrated that the 9-O-acetylated gangliosides recognized by a monoclonal antibody, P-path, confer unique compartmentation among Purkinje cell groups in the normal adult cerebellum. We have continued to explore the role of this group of gangliosides in cerebellar organization by investigating the biochemical and cellular expression of this unique epitope in the cerebellum of the mutant mouse, nervous, where postnatally, most Purkinje cells degenerate. Overall ganglioside composition of nervous cerebellum is similar to wild type cerebellum. However, quantitative analysis of gangliosides by TLC-immunostaining shows that the relative concentration of 9-O-acetylated gangliosides varies considerably. In nervous cerebellum, there is more than a three-fold increase in the concentration of 9-O-acetyl disialolactosyl ceramide (GD3), and 9-O-acetyl disialolactoneotetraosyl ceramide (LD1) is decreased to 25% of wild type. In addition, GD3 ganglioside, the immediate precursor of 9-O-acetyl GD3, is detected at 1/3 of the level of wild type cerebellum, and LD1 ganglioside, the precursor of 9-O-acetyl LD1, is virtually absent from nervous cerebellum. Thus, in nervous cerebellum the ratio of 9-O-acetyl GD3 to its disialoganglioside precursor is dramatically increased compared to wild type cerebellum. In accord with the altered expression of 9-O-acetyl gangliosides, immunoelectron microscopy demonstrates a change in the subcellular distribution in mutant Purkinje cells. Instead of being associated with the somatic and dendritic membranes, P-path immunoreactivity is located internally, in the cytoplasm of Purkinje cell bodies and their dendrites. In addition to the changes in the cerebellum, the other regions of the brain decreased in size by about 15% in the nervous mutant. In the ganglioside composition of these regions of nervous brain, 9-O-acetyl GD3 nearly doubled, but 9-O-acetyl LD1 and other gangliosides did not differ. Our findings of significant changes in 9-O-acetylated gangliosides, accompanied by the overall decrease in brain size, suggest that carbohydrate or glycolipid metabolism is abnormal in the nervous mutant mouse brain.

A unique neuronal glycolipid defines rostrocaudal compartmentalization in the accessory olfactory system of rats.

A monoclonal antibody, CC6, reacts with a complex glycolipid whose expression in the rat is restricted to the olfactory system. Structural analysis reveals that the glycolipid contains two alpha-fucose branches; one each at internal and external beta-galactose residues. Immunocytochemistry demonstrates in rat embryos that CC6 glycolipid expression is restricted to a subset of neurons in the vomeronasal organ (VNO) and their corresponding axon projections in the caudal accessory olfactory bulb (AOB). This pattern of expression in the accessory olfactory system is the converse of the pattern revealed by a previously characterized antiglycolipid antibody that reacts with VNO neurons projecting to the rostral AOB. The CC6-reactive glycolipid is also expressed on a subset of neurons in the main olfactory epithelium. Postnatally, axons from these CC6 positive sensory neurons converge to form a limited number of axon bundles running longitudinally through the nerve layer of the main olfactory bulb. These data provide further evidence that groups of vomeronasal and olfactory neurons expressing unique surface molecules project axons that terminate in selected targets in the AOB and OB, respectively.

Effects of nervous mutation on Purkinje cell compartments defined by Zebrin II and 9-O-acetylated gangliosides expression.

The cerebellum is organized into a series of parasagittally aligned bands which are well delineated in the adult mouse by the largely complementary immunostaining of Purkinje cell groups with the monoclonal antibodies Zebrin II (ZII; antigen: aldolase C) and P-path (antigen: 9-O-acetyl gangliosides). We examined the effect of nervous mutation on compartmental organization using these markers and an antibody to calbindin. In nervous mutant, up to 90% of Purkinje cells die in late postnatal development. The size of the cerebellum is about half that of normal, and caudal lobules appear to decrease in size more than anterior ones. Surviving Purkinje cells corresponded to P-path positive ones that were concentrated in two bilateral bands in the vermis and in medial portions of the hemispheres. Only small numbers of ZII positive cells remained, confirming the report by Wassef et al. with Zebrin I antibody. They were primarily located in caudal lobules IX, X and a portion of lobule IV, paraflocculus and flocculus, and their immunoreactivity was weak compared to that of normal. ZII positive cells are dominant in these caudal lobules, while P-path positive cells dominate in rostral lobules in normal mice, and the similar tendency remains in mutant. Thus, the nervous gene action respects not only sagittal compartments delineated by two antibodies, but also rostro-caudal gradient. The cause of the dominant survival of P-path positive cells awaits future study.

Cellular organization in rat somatosensory cortex: effects of sex and laterality.

Layer IV of rodent somatosensory cortex contains distinct arrangements of cells characterized as barrels. When barrels first form in rats, each barrel consists of a cell-dense "wall" and a cell-sparse "hollow." With age, the distinction of the boundary between barrel walls and hollows diminishes. Cellular arrangements within barrels were quantified to test whether the barrels are influenced by sex and laterality during cortical development. A computer-assisted method was developed to measure cell densities in relation to barrel boundaries. The boundaries between barrel walls and hollows were determined in tissue double-stained for Nissl substance and cytochrome oxidase histochemistry. The distinction between barrel walls and hollows revealed by Nissl stains differed significantly between anterior and posterior barrels. This distinction declined significantly in anterior barrels from Postnatal Day 10 (P10) to P30. The area of cortex containing barrels was estimated from composites of Nissl-stained sections. At P20 the detectable barrel cortex area was larger on the right in females and on the left in males resulting in a significant sex difference in barrel cortex asymmetry. This sex difference in barrel cortex laterality was detected only in Nissl-stained tissue; there were no differences attributable to sex or side in barrel cortex area analyzed for cytochrome oxidase reactivity. We hypothesize that sex-dependent differences in barrel cortex structure result from lateralized differences in cellular organization.

Relationship of migrating luteinizing hormone-releasing hormone neurons to unique olfactory system glycoconjugates in embryonic rats.

Following their birth in olfactory placode, luteinizing hormone-releasing hormone (LHRH)-containing neurons migrate across the developing cribriform plate and form a dispersed population in the mammalian basal forebrain. The present study reveals the colocalization of unique glycoconjugate antigens (detected with monoclonal antibody CC2) on a subset of LHRH-immunoreactive (LHRHir) cell bodies and growth cones in the rostral forebrain during embryonic development in rats. In addition, LHRHir neurons were found along CC2-immunoreactive (CC2ir) fibers in the nasal cavity, across the cribriform plate, and in the rostral forebrain. At embryonic Day 16 (E16) approximately 20% of the LHRHir neuronal population in the forebrain had the CC2 epitope on surfaces of cell bodies. This percentage fell as the number of LHRHir neurons in the forebrain increased. Prior to the detection of LHRH-containing neurons, beginning on E14, CC2ir glycoconjugates were observed on vomeronasal cells and axons and also on a dorsomedial subset of olfactory neurons and axons. As early as E14 CC2ir fibers extended into the rostral forebrain. LHRHir neurons were seen in close apposition to CC2ir fibers in both the nasal cavity and rostral forebrain. These studies raise the possibility that CC2ir glycoconjugates provide a specific chemical guide for a subset of LHRH neurons along a part of their migratory pathways. The small percentage of LHRHir neurons which have CC2ir on their surfaces prenatally may constitute a selective homogenous functional subgroup within the population of LHRH neurons.

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons.

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with beta-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.

Subsets of olfactory and vomeronasal sensory epithelial cells and axons revealed by monoclonal antibodies to carbohydrate antigens.

Cell surface glycoconjugates are believed to play an important role in cell-cell interactions during development of CNS pathways. In order to identify developmentally regulated glycoconjugates in the nervous system, monoclonal antibodies were raised and selected for reactivity with carbohydrate antigens. Three monoclonal antibodies were identified, each of which reacts with a defined carbohydrate epitope and reveals a unique pattern of immunoreactivity within the olfactory sensory epithelia, vomeronasal and olfactory nerves and their terminal regions in rats. Antibody CC1 reacts with a globoside-like glycolipid which contains a terminal N-acetylgalactosamine residue. CC1-immunoreactivity is present in just the vomeronasal organ, vomeronasal nerve and in the rostral half of the accessory olfactory bulb. Antibody CC2 reacts with a complex glycolipid which contains a branched chain oligosaccharide terminating with alpha-galactose and alpha-fucose. CC2-immunoreactivity is seen throughout the vomeronasal organ, in dorsomedial regions of the olfactory sensory epithelia, in the vomeronasal and olfactory nerves, the accessory olfactory bulb and dorsomedial glomeruli of the main olfactory bulb. Antibody 1B2 reacts with lacto-N-glycosyl ceramides. 1B2-immunoreactivity is highest at the luminal surfaces of receptor cells throughout the vomeronasal organ and in portions of the olfactory sensory epithelia. 1B2 is also expressed on the surface of a subset of receptor cell bodies, their dendrites and the proximal region of their axons in dorsomedial regions of the main olfactory epithelium.

Ultrastructural localization of stage-specific neurite-associated proteins in the developing rat cerebral and cerebellar cortices.

SNAP/TAG-1 is a glycoprotein of 135 kDa and is expressed on the surface of a subset of growing axons in the developing rodent CNS. The ultrastructural localization of this antigen was analysed in embryonic day 17 cerebral cortex and postnatal days 4 and 8 cerebellar cortex of rats using immunoelectron microscopy with a monoclonal antibody which recognizes SNAP/TAG-1 (4D7), and peroxidase-conjugated secondary antibody. In the embryonic cortex, immunoreactivity was associated with the plasma membranes of restricted groups of axons, neuronal somata and their leading processes located in the intermediate zone, subplate and cortical plate. Immunoreactive axons were bundled together in groups of 10-20 and were separated from non-immunoreactive axons. Some growth cones were immunoreactive; however, not all growth cones of 4D7-immunoreactive axons showed staining. In the postnatal cerebellum, immunoreactivity was associated with the somata and axons of granule cells that are located in the most internal portion of the external granule cell layer. In cerebral and cerebellar cortices, immunoreactivity appeared in corresponding points of adjacent cell membranes in punctuate fashion and with a regular periodicity of 100-200 nm. The possibility that SNAP/TAG-1 is acting as an adhesion molecule among specific subgroups of axons in the developing CNS is discussed.