James E. Crandall

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 χ-1), which is expressed primarily during brain development,. NR3Aco-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.

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.

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. J. Comp. Neurol. 415:218–229, 1999.

Axon strata of the cerebral wall in embryonic mice

The stratification of principal fiber systems affiliated with the developing neocortex has been analyzed by means of HRP tracing methods, monoamine histofluorescence and silver impregnations in mouse embryos ranging from the 15th to 16th embryonic day (E15/16) to the end of gestation (E19 = the day of birth). As early as E15/16 a fiber stratum divides the subplate and marks the inferior boundary of the developing cortex. Axons coursing in this fiber plane, termed the external sagittal stratum (ESS), include components of at least 5 identifiable systems: thalamocortical, corticothalamic, ipsilateral corticocortical, callosal and monoaminergic. The neocortical afferents of extrinsic origin, i.e., the thalamocortical, callosal and monoaminergic systems, cross the intermediate zone from their separate directions and converge upon the ESS. After a variable course through this stratum, single fibers ascend from their parent fascicles to ramify densely in the cortical subplate (CSB). Fibers of each of the extrinsic afferent systems mingle with each other and with locally arising axons within the CSB. Axons of the monoaminergic projection as well as fibers of the thalamic projection cross the cortical plate to ramify in the marginal zone. Other axons apparently of local intracortical origin course tangentially through the cortical plate. Otherwise, the cortical plate is devoid of proliferating axons at this early developmental stage. The set of observations illustrates the existence of sharply defined boundaries between axon-rich and axon-poor strata of the developing neocortex. These boundaries also compartmentalize postmigratory neurons with respect to their state of differentiation.

Ontogenesis of microtubule-associated protein 2 (MAP2) in embryonic mouse cortex.

The developing neocortex in mice from embryonic day 13 (E13) until birth (E19) was immunoreacted with a monoclonal antibody for microtubule-associated protein 2 (MAP2) that is highly specific for neuronal somata and dendrites. In E13 neocortex there was no detectable MAP2 immunoreactivity on tissue sections or on gel blots. From E14 to birth the MAP2 immunoreactivity was present in both tissue sections and immunoblots of homogenized cortex. In the neocortex the staining pattern was lamina-specific. The molecular layer and the cortical subplate contained the most dense staining of dendrites and cell somata. The cortical plate showed weak to moderate staining at these ages while the intermediate and ventricular zones were not stained above background control levels. Gel blots correspondingly did not show detectable levels of MAP2 until E14. Ultrastructural data suggest that MAP2 is present in dendrites in each of the laminae. The laminar pattern of MAP2 immunoreactivity may be due to either the higher density of differentiating dendrites in the molecular and subplate layers or to compartmentalization of MAP2 within individual cortical neurons.

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.

Developmental regulation of microtubule-associated protein 2 expression in regions of mouse brain

Abstract: The relative levels of microtubule‐associated protein 2 (MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate‐containing gels and analyzed by im‐munoblotting with MAP2‐specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high‐molecular‐weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low‐molecular‐weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.

The development of radial glia and radial dendrites during barrel formation in mouse somatosensory cortex

The development of the mouse barrel field (the mystacial whisker representation in SI cortex) was examined using immunocytochemical probes for radial glia and neuronal dendrites. The maturing dendrites were revealed using antibodies against microtubule-associated protein 2 (MAP2) and the radial glia were demonstrated with a recently described monoclonal antibody, RC2. By postnatal day 7 both antibodies clearly demonstrated a non-uniform distribution of dendrites and glia that was unique to layer IV of the barrel field. Both MAP2-immunoreactive dendrites and RC2-immunoreactive radial glial fibers were dense near the walls (sides and septae) of barrels than near the hollows (centers) of barrels. In contrast, in other cortical regions, radial glia and dendrites did not appear obviously patterned. Not until postnatal day 4 did the pattern of both radial glial fibers and apical dendrites begin to emerge in a barrel-like distribution. We conclude that the non-uniform distribution of radially oriented dendrites and radial glial fibers appears with a similar developmental time course to that described for the appearance of the cellular barrels themselves.

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 cell 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.

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.