Postnatal Rat Spinal Cord Research Articles

Growth Cones of Dorsal Root Ganglion but Not Retina Collapse and Avoid Oligodendrocytes in Culture

Axons in the central nervous system of mature mammals generally fail to regenerate following injury. Although the reason for this regenerative failure remains unknown, several lines of evidence suggest that it is due to nonpermissiveness of oligodendrocytes for axonal elongation. However, most of the in vitro experiments carried out so far used neural-crest-derived peripheral neurons to test the permissiveness of oligodendrocytes, although studying the interactions between central neurons and oligodendrocytes is crucially important for elucidating their roles in vivo. In this study we cultured retinas and dorsal root ganglia of the chick embryo with oligodendrocytes obtained from postnatal rat spinal cord and performed time-lapse analysis. Oligodendrocytes were identified with galactocerebroside antibody. Retinal growth cones readily grew over oligodendrocytes, while growth cones of the dorsal root ganglion collapsed and grew away on contacting the oligodendrocytes. Correspondingly, neurite-free areas centered by oligodendrocytes were formed behind growth cones in DRG-oligodendrocyte but not in retina-oligodendrocyte co-culture. These observations suggest the possibility that responsiveness of growth cones to oligodendrocytes is dependent on neuronal type.

Ontogeny of [ 125I]iodocyanopindolol-labelled 5-hydroxytryptamine 1B-binding sites in the rat CNS

Little is known about the ontogeny of the 5-hydroxytryptamine 1B (5-HT 1B) receptor, a putative terminal autoreceptor in the CNS. To learn more about the regional development of 5-HT 1B sites and how early these sites may become functional, we studied [ 125I]iodocyanopindolol ([ 125I]ICYP)-labelled central 5-HT 1B sites in developing postnatal rat brain and spinal cord. Significant ontogenic changes in B max were found in neocortex, hippocampus, striatum and brainstem. Maximum binding-site density was reached by 21 days in hippocampus and cortex, but not until 28 days in striatum. The rank order of binding site density changed with rat age, but B max was consistently high in brainstem and low in cerebellum. The percent increases in B max also varied with brain region, ranging from a 2-fold increase in brainstem up to 8-fold increases in cortex and striatum. In competition studies, the K i for 5-HT and for RU 24969 was the same in 5-day-old and adult rat brain. These data indicate regional differences in the ontogeny of 5-HT 1B-binding sites and suggest that 5-HT 1B receptors are functional early in the postnatal period.

Distribution and differentiation of A2B5+ glial precursors in the developing rat spinal cord

In many regions of the rat central nervous system, oligodendrocytes develop from migratory A2B5+ precursor cells. In the rat spinal cord, during early embryonic development the capacity for oligodendrogenesis appears to be restricted to ventral regions of the spinal cord, while cultures of postnatal rat spinal cord contain a distinct population of A2B5+ astrocyte precursors. To determine if, as in other regions of the CNS, spinal cord A2B5+ cells give rise directly to oligodendrocytes and astrocytes, the initial distribution, and subsequent dispersion, proliferation, and differentiation of spinal cord A2B5+ cells have been examined in both explant and dissociated cell cultures. Spinal cord oligodendrocytes develop from A2B5+ cells. At E14, A2B5+ cells are restricted to ventral regions of the spinal cord and as development proceeds they become more uniformly distributed throughout the spinal cord. In explant cultures, greater than 95% of the explants that contain oligodendrocytes also contain A2B5+ cells and a proportion of mature oligodendrocytes retain detectable A2B5 immunoreactivity briefly on their surface. The maturation of spinal cord oligodendrocyte precursors occurs in a number of distinct stages characterized by the expression of O4 immunoreactivity, which first appears at E16, and GC immunoreactivity, which first appears at E18. As spinal cord oligodendrocyte precursors acquire O4 immunoreactivity they appear to lose the ability to proliferate in response to PDGF but retain the ability to proliferate in response to bFGF, suggesting that the control of proliferation of oligodendrocyte precursors is, in part, dependent on their maturational state. In the presence of high serum, spinal cord A2B5+ cells fail to develop in isolated E14 dorsal spinal cord cultures, while in ventral cultures they subsequently differentiate into A2B5+ astrocytes suggesting that A2B5+ astrocyte precursors are also initially ventrally located. Unlike oligodendrocyte differentiation, however, the differentiation of spinal cord A2B5+ cells into astrocytes is delayed in early embryonic-derived cultures compared to those from older animals. These observations suggest that local influences may regulate the timing of spinal cord A2B5+ astrocyte development, but not spinal cord oligodendrocyte development.

Ontogeny of GABAA receptor subunit mRNAs in rat spinal cord and dorsal root ganglia

Relatively little is known about the development of GABAA receptor subunits and their gene expression in mammalian spinal cord. The expression of mRNAs encoding 13 GABAA receptor subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) in embryonic, postnatal, and adult rat spinal cord and dorsal root ganglia (DRG) cells were studied by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Both techniques revealed the presence of all subunit mRNAs originally found in the rat brain, except for alpha 6, which was not detectable, and delta, which was weakly detected only by RT-PCR. Two anatomically distinctive sets of subunit mRNAs were found by in situ hybridization within the ventricular zone (VZ) and mantle zone (MZ). The trio of alpha 4, beta 1, and gamma 1 subunit mRNAs emerged exclusively in neuroepithelial cells at embryonic day 13 (E13) and remained detectable in the VZ until E17. In the MZ, beta 3 subunit mRNA was first detected at E12, while alpha 2, alpha 3, alpha 5, beta 2, gamma 2, and gamma 3 transcripts appeared at E13. Expressions of the subunit mRNAs in the MZ rapidly increased and expanded in a ventrodorsal sequence from motoneurons to dorsal horn neurons before reaching a peak in the late embryonic/early postnatal period. The mRNA expressions declined during postnatal development, by region-selective depletion, with alpha 4, alpha 5, beta 1, beta 2, gamma 1, and gamma 3 subunit mRNAs becoming barely detectable. In contrast, alpha 2, alpha 3, beta 3, and gamma 2 transcripts persisted into adulthood with distinct anatomical distributions. RT-PCR analysis revealed unique developmental patterns in the intensities of PCR products, most of which were in good agreement with developmental changes in the densities of hybridized mRNA signals. However, RT-PCR amplified minute amounts of mRNAs for alpha 1, alpha 4, alpha 5, beta 1, beta 2, gamma 1, gamma 3, and delta subunits in adults, which were not found in film autoradiograms, but could be detected in a few grain-positive cells in emulsion-dipped sections. DRG cells expressed alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2 subunit mRNAs during embryogenesis but only alpha 2, beta 3, and gamma 2 subunit mRNAs were reliably detected in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative analysis of transient GABA expression in embryonic and early postnatal rat spinal cord neurons

GABA expression was investigated using biochemical analysis of spinal cord homogenates and immunocytochemical analysis of cells acutely dissociated from the embryonic and postnatal rat spinal cord. γ-Aminobutyric acid (GABA) was detected by both methods as early as embryonic day 13 (E13). At E13, the percentage of neurons that were GABA + was 0.5%. This value increased during embryogenesis, peaked during the first two postnatal weeks to just over 50%, and declined to approximately 20% by the third postnatal week emphasizing the transient nature of GABA expression. At E17 there was a pronounced, positive ventro-dorsal and rostro-caudal gradient of GABA + cells that persisted until just before birth. At this time the gradients reversed in cervical and lumbosacral regions indicating that GABA immunoreactivity in discrete anatomical regions is also a transient phenomenon. During the embryonic period GABA immunoreactivity was diffusely distributed throughout cell bodies and proximal processes. At E21, both GABA and synaptophysin were present in the same cells. However the two antigens did not co-localize point for point. By postnatal day 21 GABA immunoreactivity appeared in puncta that co-localized entirely with puncta of synaptophysin immunoreactivity. The sizable percentage of neurons that transiently express GABA during development, and the fact that it can be detected prior to the synaptic form of glutamic acid decarboxylase (GAD 65), suggest that the amino acid may play a significant role during differentiation before it functions as an inhibitory neurotransmitter.

The distribution of excitatory amino acid receptors on acutely dissociated dorsal horn neurons from postnatal rats

Excitatory amino acid receptor distribution was mapped on acutely dissociated neurons from postnatal rat spinal cord dorsal horn. N-methyl- d-aspartate, quisqualate and kainate were applied to multiple locations along the somal and dendritic surfaces of voltage-clamped neurons by means of a pressure application system. To partially compensate for the decrement of response amplitude due to current loss between the site of activation on the dendrite and the recording electrode at the soma, a solution containing 0.15 M KCl was applied on the cell bodies and dendrites of some cells to estimate an empirical length constant. In the majority of the cells tested, the dendritic membrane had regions of higher sensitivity to excitatory amino acid agonists than the somatic membrane, with dendritic response amplitudes reaching more than seven times those at the cell body. A comparison of the relative changes in sensitivity between each combination of two of the three excitatory amino acid agonists along the same dendrite showed different patterns of agonist sensitivity along the dendrite in the majority of the cells. These data were obtained from dorsal horn neurons that had developed and formed synaptic connections in vivo. They demonstrate that in contrast to observations made on ventral horn neurons, receptor density for all the excitatory amino acid receptors on dorsal horn neurons, including the N-methyl- d-aspartate receptor, are generally higher on the dendrites than on the soma. Further, these results are similar to those obtained from dorsal horn neurons grown in culture.

Ventral and dorsal horn acetylcholinesterase neurons are maintained in organotypic cultures of postnatal rat spinal cord explants

Transverse sections of postnatal rat spinal cord have been cultured using the organotypic roller tube method. These explant cultures retain identifiable anatomical landmarks, allow identification of individual neurons, can be maintained for up to 8 weeks, and undergo maturational changes in vitro. Putative ventral horn motoneurons were identified in these cultures by localization to ventral horn regions analogous to those of motoneurons in vivo and by staining for choline acetyltransferase (ChAT) immunoreactivity and acetylcholinesterase (AChE) activity. Morphometric studies of the photomicrographic areas of cell bodies of these ventralhorn neurons in intact cultures show a range of sizes up to 1635 μm 2 with the average size being245 ± 7 μm 2 ( n = 724) (average ± S.E.M.). The size ranges are roughly comparable to cross-sectional areas determined previously for ventral horn motoneurons in vivo. Dorsal horn regions of these cultures also development AChE activity that was absent at explantation. Biochemical analysis of ChAT and AChE activity in pooled samples of whole cultures showed ChAT activity to be0.48 ± 0.08 ( n = 7) μmol/min/g protein and AChE activity to be2.2 ± 2.0 ( n = 7) μmol/min/g protein at 37°C (averages ± S.E.M.). These values are comparable to previously reported values for neonatal rat spinal cord in situ. Organotypic roller tube cultures of postnatal rat spinal cord provide an attractive system for studies of survival, morphology, growth and differentiation of mammalian ventral horn neurons in vitro.

Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord

The pattern of expression of nerve growth factor (NGF)-receptor-like immunoreactivity during postnatal development in rat central nervous system (CNS) was analyzed using immunohistochemical localization of the receptor. Interestingly, in addition to the expected staining in basal forebrain, several structures were strongly labelled only during specific postnatal developmental stages. These structures included fibers in the thalamus, the external granule cell layer of the cerebellum and motor neurons, indicating that specific neurotrophic mechanisms might play an important role for the labelled cells during a precisely defined period.

Selective expression of endogenous lactose-binding lectins and lactoseries glycoconjugates in subsets of rat sensory neurons.

Cell-surface glycoconjugates and endogenous lectins have been implicated in cellular interactions that contribute to embryonic development. Functional subsets of primary sensory neurons in mammalian dorsal root ganglia (DRG) have been shown recently to express specific cell-surface oligosaccharide structures. We report here that endogenous lectins with affinity for sensory neuron glycoconjugates are also synthesized by subsets of DRG neurons and are present in the dorsal horn of the developing spinal cord. The distribution of two endogenous lactose-binding lectins, RL-14.5 and RL-29 (subunit Mrs of 14,500 and 29,000, respectively), was examined by immunoblotting and by immunocytochemistry in embryonic and postnatal rat DRG and spinal cord. The two lectins appear soon after the formation of the DRG and are present in the cell bodies and terminals of subsets of DRG neurons that also express cytoplasmic and cell-surface lactoseries glycoconjugates. RL-14.5 and RL-29 are present in overlapping, but not coincident, subsets of DRG neurons that project to the superficial dorsal horn of the spinal cord. In addition, RL-14.5, but not RL-29, is expressed in spinal motoneurons from embryonic day 14. The preferential localization of lactoseries glycoconjugates and lactose-binding lectins in the DRG and the dorsal horn of the spinal cord suggests that these complementary molecules contribute to the development and function of primary sensory neurons.