Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord.

Transplantation approaches using cellular bridges, fetal central nervous system cells, fibroblasts expressing neurotrophin-3 (ref. 6), hybridoma cells expressing inhibitory protein-blocking antibodies, or olfactory nerves ensheathing glial cells transplanted into the acutely injured spinal cord have produced axonal regrowth or functional benefits. Transplants of rat or cat fetal spinal cord tissue into the chronically injured cord survive and integrate with the host cord, and may be associated with some functional improvements. In addition, rats transplanted with fetal spinal cord cells have shown improvements in some gait parameters, and the delayed transplantation of fetal raphe cells can enhance reflexes. We transplanted neural differentiated mouse embryonic stem cells into a rat spinal cord 9 days after traumatic injury. Histological analysis 2-5 weeks later showed that transplant-derived cells survived and differentiated into astrocytes, oligodendrocytes and neurons, and migrated as far as 8 mm away from the lesion edge. Furthermore, gait analysis demonstrated that transplanted rats showed hindlimb weight support and partial hindlimb coordination not found in 'sham-operated' controls or control rats transplanted with adult mouse neocortical cells.

Peering into early neurogenesis with embryonic stem cells.

Embryonic stem (ES) cells transplanted into the early mouse embryo have the capacity to differentiate into all cell types of the nervous system. A simplified culture system has been developed in which single ES cells transform into neural progenitor cells that go on to form neurospheres. This system is ideally suited for mechanistic studies of the earliest stages of neurogenesis. In this model, signaling via fibroblast growth factor and bone morphogenetic protein family members is important for the first steps of neural progenitor differentiation.

An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat.

The dentate gyrus of the rat contains about 600,000 granule cells. These small neurons are generated over a prolonged period from the 14th day of gestation until some time after the second postnatal week. The majority of the cells pass through their last phase of DNA synthesis in the postnatal period, and during the peak period of cell generation, between the fifth and seventh days after birth, up to 50,000 granule cells are formed each day. Contrary to earlier reports, most of the cells pass through their last mitotic division either within the stratum granulosum itself, or within the hilar region of the developing gyrus. The precursor population of cells in the hilar region must therefore constitute a pool of true neuroblasts. The origin of this pool of cells has not been definitely established but it seems probable that its cells are derived from the neuroepithelium lining the lateral ventricle adjacent to the region from which the hippocampal pyramidal cells are generated. Examination of the final location of granule cells labeled at different stages reveals three distinct morphogenetic gradients in the gyrus. The cells in the dorsal blade tend to be formed earlier than those in the ventral blade; cells in the more caudal (or temporal) portions of the gyrus are generated earlier than those in more rostral (or septal) regions; and in all regions the more superficial neurons in the stratum granulosum are formed earlier than the deeper granule cells. The bearing of some of these findings on the development and organization of the connections of the dentate gyrus is discussed.

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues.

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.

Expression of the genes coding for glutamic acid decarboxylase in pluripotent cell lines.

The expression of glutamic acid decarboxylase (GAD) is a basic characteristic of a wide array of inhibitory neurons the use gamma-aminobutyric acid as a neurotransmitter. Clonal cell models will be essential for investigating the mechanisms which are responsible for the selective expression of GAD. P19 embryonal carcinoma cells are an important model for the analysis of neuronal gene expression. Depending on culture conditions, undifferentiated cells can be induced to form cells as widely divergent as cardiac muscle-like cells and neuron-like and glial-like cells. P19 cells are amendable to a number of powerful genetic manipulations including transformation with foreign DNA and selection of mutants. In this study we used nuclease protection assays and Northern blot analysis to determine if P19 cells express the GAD1 and GAD2 genes. The results show that uninduced P19 cells express these genes at very low but easily detectable levels. When the cells are induced to differentiate along the neuronal pathway with retinoic acid, the levels of transcripts for both GAD genes rise dramatically. At least some RNA transcripts of both genes from induced cells comigrate with the corresponding mRNA from the brain and thus probably represent processed mRNA. The expression of GAD genes in undifferentiated cultures of embryonal stem (ES) cells was also investigated. These cultures express levels of GAD1 transcripts that are higher than uninduced P19 cells. In contrast, expression of the GAD2 gene was barely detectable. These results indicate that P19 EC cells and ES cells will be useful for the investigation of the mechanisms that regulate the expression of the GAD1 and GAD2 genes.

Characterization of a cDNA coding for rat glutamic acid decarboxylase.

cDNA clones have been isolated for rat glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) and 3216 bp of the sequence have been determined. This sequence extends the previously reported feline GAD cDNA sequence both in the 5' (67 bp) and 3' (887 bp) directions and contains the polyadenylation signal and tail. The cDNA codes for a 67 kDa mol. wt. protein beginning from the putative initiator methionine found in the feline sequence. Extensive homology to feline GAD was identified at the amino acid level (97% identity) within the coding region. This interspecies homology is high compared to other neurotransmitter synthesizing enzymes and suggests selective pressure to maintain the primary sequence throughout the full length of the protein. Homology is found 5' to the putative initiator methionine. Extensive stretches of homology are also found in the 3' non-coding region. These conserved non-coding regions may play a role in GAD mRNA regulation. The rat cDNA sequence will facilitate investigations into the structure and regulation of the GAD gene.

Localization of the NGFI-A protein in the rat brain.

Antibodies are used to localize the NGFI-A protein in the rat brain. The protein is found in a wide variety of neurons. However, not all neurons are stained. The protein is either absent or present at undetectable levels in glial cells. Neuronal nuclei stain intensely, cytoplasmic staining is lighter. Seizures cause a detectable increase in the intensity of staining.

Time of appearance and tissue distribution of a cell surface antigen in early chick development.

The distribution of the antigen defined by monoclonal antibody 224-1A6-A1 in the early chick embryo was studied. The antigen is unequivocally present in the rudimentary neural tube as early as the 12 somite stage (approximately 40 h of incubation). The antigen appears on some but not all derivatives of the ectoderm and mesoderm, but appears to be absent on endodermal derivatives. Monoclonal antibody 224-1A6-A1 therefore serves as a marker for early events in the developmental sequence leading toward the formation of the nervous system.

A monoclonal antibody which binds to the surface of chick brain cells and myotubes: cell selectivity and properties of the antigen.

A monoclonal antibody has been obtained which binds to the cell surface of cultured chick myotubes and retinal and tectal neurons but not fibroblasts, myoblasts and embryonic liver cells. Indirect immunocytochemistry reveals that antigen is present in all layers of the chick neural retina. The antibody therefore recognizes an antigen common to most, if not all, chick neurons. The antigen has been identified by staining SDS gels with [125I]monoclonal antibody and appears to be a polydisperse collection of polypeptides with molecular weights centered about 250 kdalton.

Opiate binding sites in the chick, rabbit and goldfish retina.

The characteristics of opiate binding sites in the retina of the chick, rabbit and goldfish have been investigated. In the newly hatched chick retina, 131 fmol/mg of binding sites for [D-Ala2-D-Leu5]-[3H]enkephalin are present; competition studies with the delta selective peptide [D-Thr-Leu5]-enkephalin (DTLET) and the mu selective peptide morphiceptin show that all of the [D-Ala2-D-Leu5]-[3H]-enkephalin binding sites are of the delta subtype. Dihydro[3H]morphine binds poorly to the chick retina; 13.2 fmol/mg of this binding is displaceable by morphiceptin and corresponds to mu binding sites. Benzomorphan sites are defined as sites occupied by [3H]diprenorphine which is displaceable by low concentrations of ethylketocyclozacine but not by high concentrations of D-Ala2-D-Leu5-enkephalin and morphiceptin. At least 88 fmol/mg of benzomorphan sites are present in the chick retina. [3H]diprenorphine binding to the rabbit and fish retina was measured. The rabbit retina bound 60 fmol/mg, and the fish retina 42 fmol/mg of [3H]diprenorphine. These findings are discussed in the light of the studies on the localization and physiological effects of enkephalin in the retina.

Developmental changes in glycoproteins of the chick nervous system.

Temporal changes have been noted previously in retinal glycoproteins that bind to wheat germ agglutinin by a technique in which the denatured glycoproteins are first separated according to size by polyacrylamide gel electrophoresis, and are then localized on the gel using [125I]lectin. As reported here this technique will also detect differences between dorsal and ventral halves of the neural retina from 8-day chick embryos, and using other lectins will detect temporal changes in the glycoprotein pattern of the optic tectum. Some of the glycoproteins detected by wheat germ agglutinin in the neural retina appear to be represented on the surface of the retinal cells since: (a) the temporal changes in retinal glycoproteins can also be observed in a plasma membrane enriched fraction prepared from neural retina cells; and (b) antibodies prepared in mice against various size categories of wheat germ lectin binding glycoproteins bind to intact retinal cells.

GABAergic neurons.

One tends to think of the nervous system in terms of chains of excitatory signals that tell neurons to fire. The signals that say "Don't fire" also have a major role. Inhibitory signals damp overall neural activity and also fine-tune the responses of particular circuits. The primary carrier of these signals is the inhibitory transmitter known as GABA.

A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays.

Transcription in higher eukaryotes is often studied by the use of transient transfection assays. These experiments are performed by cloning putative cis-acting transcriptional elements (i.e., a promoter or enhancer) with a reporter gene that codes for a protein not expressed by the target cells. Although this approach is useful in many cases, the limited sensitivity of reporter assays can prevent studies in cases where few cells are obtainable or efficiency of transfection is low. We present an alternative approach. Cells are transfected with a plasmid containing a promoter with a human growth hormone (hGH) reporter gene. After an incubation period, RNA is isolated, and DNA complementary to the growth hormone mRNA is produced. The reporter cDNA concentration is measured by quantitative polymerase chain reaction (PCR). We have designed PCR primers that span the mRNA splice sites of the human growth hormone gene; these ensure exclusive amplification of the hGH cDNA and not the reporter plasmid. The assay is sensitive and simple to perform, requires no special equipment, and can quantify reporter cDNA concentration over a broad range.

Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase.

Immunoaffinity columns are prepared from the monoclonal antibody (MAb) GAD-1. These columns are used to enrich glutamic acid decarboxylase (GAD) from the cytosolic fraction of rat brain homogenates and from Triton X-100 extracts of the brain membrane fraction. In each case enzyme activity is enriched over 400-fold. The immunopurified fractions were analyzed by SDS-PAGE. Fractions purified from the cytosol consisted of a quantitatively major band of 59 kDa, and one band of 63 kDa, as well as a group centered around 55 kDa. Fractions purified from membranes consisted primarily of the 59 and 63 kDa components; only traces of the lower-molecular-weight components were present. The entire set of proteins purified on GAD-1 immunoaffinity columns is strongly recognized by 2 widely used antisera to GAD, those described in Saito et al. (1974) and Oertel et al. (1981). The 59 kDa protein from the cytosolic fraction was purified to homogeneity by preparative SDS-PAGE; a partial amino acid sequence of this protein was obtained. The 59 kDa protein has a high degree of sequence homology with the deduced amino acid sequence of the protein that was coded for by a cDNA for feline GAD (Kaufman et al., 1986; Kobayashi et al., 1987). Thus, these proteins are either products of a single gene that diverged during the evolution of rat and cat from a common ancestor, or are members of a closely related set of genes found in both species. The MAb GAD-6 recognizes the 59 kDa band and the group of bands centered around 55 kDa on Western blots. Therefore, these proteins are immunochemically related. GAD-6 does not recognize the 63 kDa band. In Western blots of unfractionated homogenates of the whole brain, the only band recognized by GAD-6 is a 59 kDa band.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary olfactory projection has two chemically distinct zones.

The sensory neurons of the olfactory epithelium form an anatomically uniform population but are differentially excited by odorants. We have discovered an unexpected biochemical heterogeneity within this population that extends to its axonal projection onto the olfactory bulb. This heterogeneity is recognized by a newly generated monoclonal antibody, designated RB-8, that differentially stains the primary olfactory projection in rats and divides it into 2 nonoverlapping zones. With light-microscopic immunohistochemistry, RB-8 densely labels the fascicles of the olfactory nerve from the ventral and lateral parts of the olfactory epithelium, where there is also some epithelial staining. This area, which we designate RB-8-positive, comprises about two-thirds of the epithelial sheet. RB-8 labeling of the other third of the epithelium, which includes the dorsal recess and medial tips of the dorsal turbinals, is not detectable, and the fascicles from these RB-8-negative areas are only weakly stained. These RB-8-negative areas form a contiguous zone on flattened maps of the epithelial sheet. In the olfactory bulb, RB-8 staining of the glomeruli in the ventrolateral part is correspondingly dense, while that in the dorsomedial glomeruli is undetectable or very light. In the labeled glomeruli, the RB-8 staining is precisely coextensive with anti-olfactory marker protein staining, which serves as a marker for the olfactory axons and terminals. In addition, knife-cut lesions of the olfactory nerve totally eliminate the RB-8 staining in the glomeruli where the destruction of the olfactory terminals is complete. There is also a good correlation between the staining patterns in the bulb and epithelium and what is known from tract-tracing studies of the arrangement of the axonal projection of the epithelium onto the bulb. This evidence strongly suggests that, in the olfactory nerve and glomeruli, RB-8 stains the olfactory axons and their terminals. A survey of the CNS and peripheral tissues demonstrates that staining with RB-8 is nervous system-specific; not all components of the CNS and PNS are stained. The antigen recognized by RB-8 was characterized in immunoblots and by use of a direct radioimmunoassay (RIA) which assessed binding of 125I-RB-8. With this assay, the RB-8 binding sites in whole brain are shown to be membrane-associated, saturable, immunologically specific for RB-8, and trypsin-sensitive. In SDS-PAGE immunoblots of membrane proteins, the antigen in rat forebrain and in the olfactory nerve is a protein of 125 kDa Mr, which comigrates in mixtures of membranes from the 2 sources.(ABSTRACT TRUNCATED AT 400 WORDS)