Fetal Mouse Spinal Cord Explants Research Articles

Horseradish peroxidase tracing of dorsal root ganglion afferents within fetal mouse spinal cord explants chronically exposed to tetrodotoxin

Afferent projection patterns within organotypic explants of fetal mouse spinal cord-dorsal root ganglia (DRG) were mapped out histologically using an HRP-staining method. The role of spontaneous bioelectric activity in the development of cord-DRG connections was studied using tetrodotoxin (TTX) to eliminate action potential discharges in the experimental cultures. Cultures grown in TTX-supplemented medium had a significantly lower proportion of DRG afferents within the dorsal half of the cord explant than did untreated cultures. In addition, afferent fiber entrances were made predominantly on the dorsolateral aspect in control cord explants, in contrast with the more diffuse entrance pattern displayed by the experimental group. Comparison of those cultures in both groups where the fiber entrances were chiefly dorsal revealed a greater tendency in the TTX group for DRG afferents to grow ventrally after penetrating the cord. Thus, it appears that bioelectric activity of the target neurons may be required by DRG afferents for the development of selective innervation patterns.

Development of Met-Enkephalin Immunoreactivity in Organotypic Explants of Fetal Mouse Spinal Cord and Attached Dorsal Root Ganglia

Radioimmunoassays of methionine-enkephalin (Met-Enk) in organotypic cultures of 13-day fetal mouse spinal cord explants with attached dorsal root ganglia (DRG) demonstrate a progressive development of immunoreactivity (IR) during 5 weeks in vitro. Met-Enk IR in these cultures increased to levels observed in adult rodent spinal cord. Most of the Met-Enk IR assays were made on cord explants excised from cord-DRG cultures. In smaller numbers of assays performed on entire DRG-cord cultures or on cord cultured in the absence of DRGs, similar levels of Met-Enk IR were obtained. Thus most of the Met-Enk IR appeared to be located within the cord tissue. No Met-Enk IR was detected in DRGs cultured in the absence of cord. In contrast, low levels of Met-Enk IR were present in about 50% of the assays of DRGs cultured attached to the cord. Since these assays included the neuritic outgrowths of the cultures, our data do not preclude possible contamination by Met-Enk immunoreactive cord neurites that may have aberrantly projected into the outgrowth zones. Nevertheless, the data raise the possibility of a trophic influence of cord tissue on the development of Met-Enk IR in DRG neurons. The development of Met-Enk IR in cord regions of cord-DRG explants extends previous binding assays demonstrating development of opiate receptors in these cultures 13,14 and provides further support to electrophysiological analyses suggesting tonic opioid inhibitory networks in these explants 7.

Effects of gangliosides on the development of selective afferent connections within fetal mouse spinal cord explants

Monosynaptic evoked responses were used to localize dorsal root ganglion (DRG) afferent terminals within organotypic fetal mouse spinal cord explants which had been chronically exposed to purified bovine gangliosides during early development. Ganglioside grown cultures showed a highly significant increase in dorsal cord innervation preferences in comparison with control cultures. The amount of evoked activity was significantly lower than was observed in controls, suggesting that the formation of ‘incorrect’ functional connections was blocked by specific chemical factors. Exposure to N-acetylgalactosamine, a major ganglioside amino sugar, also resulted in an increased dorsal cord innervation preference by the DRG afferents.

Influence of growth medium, age in vitro and spontaneous bioelectric activity on the distribution of sensory ganglion-evoked activity in spinal cord explants.

The role of serum added to the culture medium and of spontaneous bioelectric activity in the development of sensory afferent connections was studied, employing fetal mouse spinal cord explants with attached dorsal root ganglia (DRG) as an in vitro model system. Afferent DRG terminals in the cord explants were localized on the basis of 'fixed-latency' DRG-evoked action potentials, which were anatomically verified in several experiments using horseradish peroxidase histology. In serum-supplemented medium (HSSM), but not in chemically defined medium (CDM), those DRG fibers which grew into the dorsal side of the cord terminated predominantly within the dorsal cord region, and remained there throughout the experimental period (18-33 days in vitro). In contrast, ventrally entering fibers terminated equally in both the dorsal and the ventral cord regions in young cultures (18-24 days in vitro) but were no longer observed after 27 days in vitro. Cultures grown in HSSM with the addition of xylocaine, in order to chronically suppress spontaneous bioelectric activity, essentially corresponded (at 25-32 days in vitro) to the picture seen in the control series at the same age. On the basis of polysynaptic DRG-evoked responses in the cord, developmental changes in local neuronal networks were inferred which resulted in less spread of DRG-evoked activity with age in HSSM, and more spread with age in CDM-grown cultures. It is concluded that for the formation of selective DRG connections in the spinal cord: (i) a serum-borne factor plays a role: and (ii) functional activity is not required.