Mouse Spinal Cord-dorsal Root Ganglia Research Articles

Actions of capsaicin on mouse dorsal root ganglion cells in vitro

The effects of capsaicin were investigated on different populations of dorsal root ganglion cells in the in vitro mouse spinal cord-dorsal root ganglion preparation using intracellular electrodes. Dorsal root ganglion cells were characterised by the conduction velocity of their propagated action potential evoked by electrical stimulation of the dorsal root, and by the shape of their action potential. All cells with C-fiber characteristics (conduction velocity < 0.6 m/s; broad action potential with shoulder on the descending slope) were depolarised and generated action potentials when capsaicin (100–700 nM) was added to the bathing solution for 30 s. At these concentrations the membrane potential of DRG cells with myelinated fibers (conduction velocity > 2.0 m/s) was unaffected. Concentrations of capsaicin of 1.0–5.0 μM depolarised 50% of cells with conduction velocity > 10 m/s. During the depolarization of the membrane no action potentials were generated. In 50% of the capsaicin-sensitive neurons with conduction velocity faster than 10 m/s there was an initial hyperpolarization. Electrical stimulation of the dorsal root failed to evoke action potentials during the depolarization in 38% of the DRG cells with myelinated fibers and in all C-fibers tested within 10 min of the onset of the capsaicin effect. Passive depolarization of the membrane by intrasomal current injection mimicked the conduction block in neurons with large myelinated fibers. These observations confirm that capsaicin applied directly to the dorsal root ganglion affects, in a dose-dependent manner, both myelinated and unmyelinated primary afferents with a higher potency for C-neurons. Capsaicin evoked action potentials in C-neurons but not in neurons with myelinated fibers. Action potential block by capsaicin was a common feature in all types of fibers, indicating that capsaicin may impair the propagation of sensory signals generated from the periphery.

Antesera to the ganglioside GM 1 do not have anti-myelin or anti-axon activities in vitro

Four antisera to the ganglioside GM 1 were tested effects on myelin and axons when applied to mouse spinal cord-dorsal root ganglia explant cultures. None of the antisera to GM 1 caused myelination inhibition or demyelination, while an antiserum tto galactocerebroside caused both. Antisera to GM 1 did not axonal outgrowth or destroy mature outgrowth zone axons, while an anteserum to a rat brain axolemma-enriched fraction did both. These results suggest that antibodies to GM 1 do not have significant anti-myelin or anti-axon activity.

Non-selective afferent innervation develops in embryonic mouse spinal cord-dorsal root ganglia explants chronically exposed to GM1 ganglioside

Projection patterns of dorsal root ganglion afferents within organotypic expiants of fetal mouse spinal cord were examined histologically using a horseradish peroxidase staining method. All expiants were also examined electrophysiologically and showed spontaneous and evoked bioelectric discharges, which were evenly distributed across the cord explants. Explants chronically grown in a serum-free medium containing the monosialoganglioside GM1 failed to develop dorsal cord selectivity as was previously observed when a mixture of gangliosides was used. It therefore appears that not all gangliosides can induce selectivity in this model.

Gangliosides as cell adhesion factors in the formation of selective connections within the nervous system

This chapter describes the gangliosides as cell adhesion factors in the formation of selective connections within the nervous system. Gangliosides are glycosphingolipids, containing sialic acid, which are normal constituents of all plasma membranes in vertebrate cells, but which are particularly enriched in neural tissues. Gangliosides consist of a hydrophobic cerarnide portion that is embedded in the outer leaflet of the plasma membrane, and a hydrophilic oligosaccharide chain extending into the extracellular environment. Gangliosides are synthesized in the Golgi apparatus of the cell body through the sequential addition of single saccharide units and sialic acid to the ceramide moiety. Recent work using organotypic explants of fetal mouse spinal cord-dorsal root ganglia (SC-DRG) has provided evidence that gangliosides induce the formation of selective sensory afferent projection patterns within the cord, even in the absence of bioelectric activity. Gangliosides are synthesized in the Golgi apparatus of the cell body through the sequential addition of single saccharide units and sialic acid to the ceramide moiety.

Electrophysiologic responses and adenylate cyclase activities of mouse spinal cord-dorsal root ganglion explants rendered tolerant by chronic exposure to morphine or pertussis toxin.

Explant cultures of fetal mouse spinal cord with attached dorsal-root ganglia (DRGs) (Fig. 1: Crain 1976) provide a valuable in vitro system for study of neurotransmitter modulation in the CNS. We have previously developed and utilized this system extensively for analysis of the actions of opioids on the electrophysiologic responses of these neurons and for study of the development of tolerance to opioid depressant effects (see review by Crain, 1984). Exposure of fetal mouse spinal cord-DRG explants to opioid alkaloid or peptide agonists resulted in stereospecific, naloxone-reversible, dose-dependent depression of sensory-evoked dorsal-horn synaptic-network responses within a few minutes (e.g. Fig. 2A; Crain et al., 1977, 1978). After chronic exposure to opioids, e.g. 2–3 days in 1 μM morphine (at 35°C), sensory-evoked dorsal-horn responses recovered, and they could then be elicited by DRG stimuli in the presence of opioids at concentrations 10- to 100-fold higher than required to depress a naive expiant (Fig. 2B; (Crain et al., 1979). In addition, these opioid-tolerant explants developed significant cross-tolerance to serotonin (5HT) (Crain et al., 1982). The tolerant state did not develop if the explants were exposed to morphine at lower temperatures (e.g. 20°C for as long as 7 days; Fig. 2C). The data suggest that the sustained decrease in opioid sensitivity observed during chronic opioid exposure at 35°C is mediated by a temperature-dependent metabolic change in these neurons (Crain et al., 1979; Crain, 1984).

Some functional effects of suppressing bioelectric activity in fetal mouse spinal cord-dorsal root ganglion explants

Organotypic explants of fetal mouse spinal cord-dorsal root ganglia were grown for 3 weeks in the presence of 10 m M magnesium ion, which effectively eliminated all recordable bioelectric activity throughout the culturing period. When tested in minimal essential medium, the chronically silenced explants had significantly fewer points from which spontaneous neuronal activity could be recorded. In addition, fewer points could be found that showed dorsal root ganglion-evoked responses, resulting from a greater tendency for the spinal cord activity to be restricted to the vicinity of the dorsally entering DRG fibers. These findings, therefore, support the hypothesis that spontaneous bioelectric activity is required for functional as well as structural maturation of neural networks.

Gangliosides restore the specificity of afferent projection patterns in spinal cord explants chronically exposed to tetrodotoxin

Sensory afferent projection patterns within organotypic explants of fetal mouse spinal cord-dorsal root ganglia (SC-DRG) were mapped out histologically using an HRP-staining method. Cultures grown in tetrodotoxin-containing medium with added gangliosides, in contrast to those grown without the addition of these compounds, showed preferential DRG innervation of the dorsal half of the cord. It therefore appears that gangliosides can compensate for the absence of functional activity during the development of selective innervation patterns.

Taxol effects on GLIA in organotypic mouse spinal cord-DRG cultures

Exposure of organotypic mouse spinal cord-dorsal root ganglion cultures to 1–2 μM taxol for up to 6 days results in a remarkable increase in cytoplasmic microtubules in differentiated oligodendroglia. In contrast, there is a notably smaller change in the microtubule compliment of astroglia. This suggests that the regulatory and/or synthetic systems for tubulin in these glial types may differ, and that oligodendroglia appear more responsive to modulation in the levels of tubulin subunits as a result of the action of taxol.

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 opioid networks in organotypic cultures of fetal mouse spinal corddorsal root ganglion (drg) explants

Development of opioid networks in organotypic cultures of fetal mouse spinal corddorsal root ganglion (drg) explants

Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures

Co-cultures of mouse spinal cord with dorsal root ganglion (DRG) cultures were grown either in horse serum (HS)-supplemented medium or in a serum-free, chemically defined medium (CDM). The cytoarchitecture of cord-DRG explants was fully retained in CDM, with little or no distortion due to flattening of the explant, as is invariably observed in HS-supplemented cultures. Functional properties such as bioelectric activity and DRG-spinal cord interconnectivity were well sustained in CDM.