Retardation of neuritic outgrowth and cytoskeletal changes accompany acetylcholinesterase inhibitor treatment in cultured rat dorsal root ganglion neurons

Over the past two decades acetylcholinesterase (AChE) has been shown to be present in numerous non-cholinergic and non-cholinoceptive tissues. Interestingly, transient expression of AChE in developing nervous tissue corresponds temporally with neuronal migration and neuritic outgrowth. This observation has led our laboratory to investigate a possible novel, non-cholinergic role for AChE in the development of the nervous system. In a previous study, we demonstrated that the activity of AChE in cultured dorsal root ganglion neurons (DRGN) can be modulated by the substratum. In our current study, we have examined the effects of AChE inhibitor treatment on neuritic outgrowth on the highly permissive substratum Matrigel^TM and the less permissive substratum Collagen Type I. DRGN received serial dilutions of the AChE-specific inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) ranging from 10^?4 to 10^?7 M. Results showed that neuritic outgrowth was significantly reduced in DRGN grown on Matrigel^TM at 10^?5 and 10^?4 M BW284c51, while outgrowth on Collagen Type I was significantly reduced at 10^?6, 10^?5, and 10^?4 M concentrations of BW284c51. Inhibitor treatment did not affect cell survival and neuritic outgrowth from BW284c51-treated cells recovered to control levels after removal of the inhibitor from the medium. In addition, massive spiraling accumulations of 10 nm filaments were observed in the cell bodies of treated neurons, which resemble neurofibrillary inclusions observed in neuropathological diseaes such as Pick's disease. This study demonstrates that AChE inhibitor treatment retards neuritic outgrowth and neuronal migration of cultured DRGN which is accompanied by cytoskeletal abnormalities in the cell body. These data further suggest a novel, non-cholinergic role for AChE in neural development and regeneration. © 1994 Wiley-Liss, Inc.     

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

Recognition of specific targets by cultured dorsal root ganglion neurons

We have assessed the effects of different target cell populations on axonally transported proteins by the use of compartmental cell culture systems that separate the soma from the growing axons of rat sensory neurons. The labeling of 3 rapidly transported proteins diminishes when the growing axon contacts spinal cord cells (which are normal in vivo targets), and remains unaffected by contact with fibroblasts or heart cells. Medium conditioned by spinal cord cells does not exert this effect. Thus, specific classes of cells may be distinguished as target tissue by sensory neurons in vitro. Such recognition is accompanied by specific molecular changes in axonally transported proteins.     

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons

Intracellular magnesium concentration ([Mg²⁺]_i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg²⁺]_i was 0.48±0.08 mM (mean±SEM, n=23) at rest, and it increased 3-fold by depolarization with a 60-mM K⁺ solution. The [Mg²⁺]_i increase was observed in the absence of extracellular Mg²⁺, but the increase disappeared in the absence of extracellular Ca²⁺. 50 μM cadmium or 100 μM verapamil, a Ca²⁺ channel blocker, also diminished the rise of [Mg²⁺]_i. The additional measurement of an intracellular Ca²⁺ concentration ([Ca²⁺]_i) indicated that the [Mg²⁺]_i rise requires a threshold concentration of [Ca²⁺]_i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca²⁺-influx through voltage dependent Ca channels and this causes the release of Mg²⁺ from intracellular stores into the cytoplasm.     

Anti-ganglioside antibodies reveal subsets of cultured rat dorsal root ganglion neurons

Subsets of cultured dorsal root ganglion neurons were identified by using the anti-ganglioside monoclonal antibodies A2B5, D1.1, R24 and JONES. A2B5 and D1.1 labelled a population of cells that was relatively stable between 2 and 20 days in vitro, while the population of cells labeled with both R24 and JONES decreased with time, suggesting that the gangliosides recognized by Jones and R24 are developmentally regulated. Given the observation that the relative proportions of ganglioside species changes with time in culture, it is very important to carefully define the stability of ganglioside antigens before using them as cell markers.     

Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures

Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury.     

Associations between intermediate filament proteins expressed in cultured dorsal root ganglion neurons

The developmental profile of the neurofilament (NF) triplet proteins, α-internexin and peripherin in cultured dorsal root ganglion neurons from gestation day 15 rat embryos was determined by Western blot analysis. At the outset (day 0 in culture), the neurons contained mostly α-internexin. A significant increase in peripherin levels was seen at days 1–2, in the mid-sized (NFM) and low molecular weight (NFL) NF subunits at days 2–3, and in the high molecular weight (NFH) NF subunit at days 5–6. Immunofluorescence microscopy showed that the five intermediate filament proteins were co-localized in all neuronal cell bodies and neurites. Analysis of Triton X-100 extracts from okadaic acid-treated dorsal root ganglion cultures revealed that peripherin and α-internexin followed the same fragmentation pattern observed with NFs. Interactions between the various neuronal intermediate filament proteins in these extracts were assessed by immunoprecipitation under native conditions using antibodies specific for the individual proteins. Co-immunoprecipitation of NFH with NFL, NFM with NFL, NFM with α-internexin, and α-internexin with peripherin demonstrated that the intermediate filament cytoskeleton in cultured sensory neurons is a highly integrated structure. J. Neurosci. Res. 47:300–310, 1997. © 1997 Wiley-Liss, Inc.     

Taurine-induced modulation of voltage-sensitive Na+ channels in rat dorsal root ganglion neurons

The physiological role of taurine, an abundant free amino acid in the neural system, is still poorly understood. The aim of this study was to investigate its effect on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents in enzymatically dissociated neurons from rat dorsal root ganglion (DRG) with conventional whole-cell recording manner under voltage-clamp conditions. A TTX-S Na+ current was recorded preferentially from large DRG neurons and a TTX-R Na+ current preferentially from small ones. For TTX-S Na+ channel, taurine of the concentration > or = 10 mM shifted the activation curve in the depolarizing direction and the inactivation curve in the hyperpolarizing direction. There was no change in the activation curve for TTX-R Na+ channel and the inactivation curve was shifted in the hyperpolarizing direction slightly in the presence of taurine > or = 20 mM. When the recovery kinetics was examined, the presence of taurine resulted in a slower recovery from inactivation of TTX-S currents and no change of TTX-R ones. All the effects of taurine were weakly concentration-dependent and partly recovered quite slowly after washout. Our data indicate that taurine alters the properties of Na+ currents in intact DRG neurons. These may contribute to the understanding of taurine as a natural neuroprotectant and the potential of taurine as a useful medicine for the treatment of sensory neuropathies.     

Warm cells revealed by microfluorimetry of Ca in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca²⁺ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca²⁺]_i from 52.5±9.5 nM (mean±S.D., n=18) to 171.0±15.6 nM in cells between 15 and 25 μm in diameter. The depletion of extracellular Ca²⁺ diminished the Ca²⁺ elevation. The Na⁺-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Opioid regulation of intracellular free calcium in cultured mouse dorsal root ganglion neurons

Opioid agonists induced an increase in the intracellular free calcium concentration ([Ca²⁺]_i) or an inhibition of K⁺ (25 mM)-stimulated increase in [Ca²⁺]_i in different subsets of mouse dorsal root ganglion (DRG) neurons. The total neuronal population was grouped into three classes according to somatic diameter and defined as small (<16 μm), intermediate (16–25 μm), or large (>25 μm) neurons. Substance P-like immunoreactivity was detected mainly in the small and intermediate neurons. The δ, κ, and μ opioid receptor agonists [D-Ser², Leu⁵]enkephalin-Thr (DSLET), U69593, and [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO) each induced a transient increase in [Ca²⁺]_i in a small fraction (<30%) of neurons. The increases in [Ca²⁺]_i were blocked by the opioid antagonist naloxone. The dihydropyridine-sensitive calcium channel blocker nifedipine also blocked the increase in [Ca²⁺]_i induced by 1 μM DSLET. The rank order of potency (percentage of cells responding to each opioid agonist) was DSLET > U69593 > DAMGO. The opioid-induced increase in [Ca²⁺]_i was observed mainly in large neurons, with a low incidence in small and intermediate neurons. Opioid agonists also caused inhibition of K⁺-stimulated increases in [Ca²⁺]_i, which were blocked by naloxone (1 μM). Inhibition of the K⁺-stimulated increase by 1 μM DSLET or U69593 was greater in small and intermediate neurons than in large neurons. © 1996 Wiley-Liss, Inc.     

Ontogeny of calretinin in chick dorsal root ganglion neurons.

Calretinin immunostaining was performed on chick lumbosacral dorsal root ganglia during embryonic development. Calretinin-immunopositive neurons were first observed at around the 9th day of incubation. Quantitative evaluation revealed a close correlation between the number of immunopositive cells and the duration of incubation. Morphometric measurements disclosed that calretinin-immunoreactive cells belong in the large or intermediate categories of dorsal root ganglion neurons. It was concluded that the appearance of calretinin immunopositivity in spinal ganglion cells during development may be associated with both the morphological and functional maturation of this particular population of primary sensory neurons.     

Immunocytochemical study of phenotypic plasticity of cultured dorsal root ganglion neurons during development

Rat dorsal root ganglia (DRG) were cultured from different stages of development ranging from embryonic day-14 to adult. The expression of eight neurotransmitter phenotypes was examined with immunocytochemical detection and the percentages of each phenotype were calculated with reference to the whole neuronal population defined by the expression of neuron-specific enolase (NSE). The expression of peptides, calcitonin gene-related peptide (CGRP), substance P (SP), cholecystokinin (CCK) and neuropeptide Y (NPY) was always present whatever the age at onset of the cultures. Although the percentage of CGRP remained stable, that of the other peptides declined progressively. Their in-vitro expression did not differ marked from that found in vivo. Another group of neurotransmiiters, including 5-hydroxytryptamine (5-HT), thyrotropin-releasing hormone (TRH) and gamma-aminobutyric acid (GABA) was never expressed in situ in DRG neurons. In culture, they were expressed in a high percentage of neurons, especially for 5-HT and TRH, and they showed a similar evolution, with a decrease at early postnatal ages followed by a further increase. This profile suggests that the expression of these transmitters is strongly environment-dependent and may be repressed in situ. Finally, somatostatin (SOM) was found only in cultures prepared from adult tissues, whereas it was present in situ from the embryo onwards. The expression of this peptide would thus require a stabilization by a long exposure to environmental factors.We can conclude that the great diversity of phenotypic expression found in DRG neurons in situ is the result of a wide variety of influences occurring at different stages of development in a large potential repertory present in these neurons.     

Myelination of cultured dorsal root ganglion neurons by oligodendrocytes obtained from adult rats

Enzymatically dissociated cell suspensions from adult rat spinal cord were added at low densities (5 X 10(3) cells/culture) to cultures of pure dorsal root ganglion neurons. Oligodendrocytes, identified by immunostaining with a monoclonal antibody to galactocerebroside, began to proliferate by 4 days after their addition, forming large colonies of cells by the 14th day. Myelin formation by oligodendrocytes began 4 weeks after addition and myelin was abundant by 6 weeks. Oligodendrocyte proliferation and myelination did not require the immediate presence of astrocytes; the number of astrocytes overall remained low throughout the culture period. Preliminary studies indicated that the specific removal of galactocerebroside-positive cells from the cultures with anti-galactocerebroside antibody and complement 3 days after their addition prevented the subsequent generation of new oligodendrocytes and myelination. These preliminary results suggest that a major source of new myelinating cells in the adult central nervous system (CNS) might be already committed, galactocerebroside-positive, oligodendrocytes rather than uncommitted stem cells. The absence of cellular barriers between the myelinating cells and the medium make these cultures well suited for studies probing cellular and molecular mechanisms of myelination in the CNS.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

Axoplasmic transport of mitochondria in cultured dorsal root ganglion cells.

The movements of individual mitochondria in cultured mouse dorsal root ganglion cells were directly observed by using fluorescent staining with rhodamine 123 in combination with video microscopic techniques. This gives greater spatial and temporal resolution and much higher specificity than possible by conventional methods. The instantaneous velocities were 0.55 +/- 0.11 microns/s anterograde and 0.60 +/- 0.10 microns/s retrograde. Movement of the mitochondria was in fits and starts, and some reversed direction. The number of mitochondria moving retrogradely was 1.5-1.9 times greater than the number moving anterogradely. The average length of mitochondria moving retrogradely was 2.8 microns and of mitochondria moving anterogradely was 4.1 microns. These results suggest that mitochondria increase their numbers by division in the nerve fiber terminal.     

Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons.

The effects of mercuric chloride and methylmercury chloride on the rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch clamp technique. gamma-Aminobutyric acid-induced chloride currents were augmented by mercuric chloride in a potent and efficacious manner; at concentrations of 1 and 10 microM, the current amplitude was increased to 130% and 200% of the control. Methylmercury even at 100 microM did not augment but rather decreased the GABA-induced chloride current. Both mercuric chloride and methylmercury generated slow inward currents by themselves. These currents are not mediated by the GABA-activated chloride channels or by voltage-activated sodium, potassium or calcium channels, and are likely to be due to non-specific cation channels.     

Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes

The [³H]resiniferatoxin (RTX) binding assay using membrane preparations has been used to identify and characterize the vanilloid receptors in the central and peripheral nervous system of different species. In the present study, using cultured adult rat dorsal root ganglion neurons either in suspension or attached to the tissue culture plates, we developed an assay to measure specific [³H]RTX binding by the intact cells. We were able to characterize the vanilloid binding characteristics of the neurons and compared those to the properties of vanilloid binding sites present in rat dorsal root ganglia membrane preparations. We found that [³H]RTX bound with similar affinity and positive cooperatively to attached neurons (cultured for 5 days before being assayed), neurons in suspension (using a filtration assay) and dorsal root ganglion membrane preparations. Dissociation constants obtained in the three assays were 47.6 ± 3.5 pM, 38.4 ± 3.1 pM and 42.6 ± 3.1 pM, respectively. The cooperativity indexes determined by fitting the data to the Hill equation were 1.73 ± 0.11, 1.78 ± 0.12 and 1.78 ± 0.09, respectively. The maximal binding capacity was 0.218 ± 0.026 fmol/10³ cells and 0.196 ± 0.021 fmol/10³ cells in the case of the attached cells and cells in suspension, respectively. Nonradioactive RTX, capsaicin, capsazepine and resiniferonol 20-homovanillylamide fully displaced specifically bound [³H]RTX from cells in suspension with K_i and Hill coefficient values of 42.5 ± 5.3 pM, 2.06 ± 0.16 μM, 3.16 ± 0.21 μM and 32.4 ± 4.1 nM and 1.79 ± 0.17, 1.68 ± 0.06, 1.72 ± 0.11 and 1.81 ± 0.12, respectively. Structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and inducing ⁴⁵Ca uptake in rat dorsal root ganglion neurons. Affinities for receptor binding and stimulation of ⁴⁵Ca uptake of RTX, resiniferonol 20-homovanillylamide, RTX-thiourea, tinyatoxin, phorbol 12,13-dibenzoate 20-homovanillylamide and capsaicin were 38.5 ± 2.9 pM, 25.7 ± 3.0 nM, 68.5 ± 3.8 nM, 173 ± 25 pM, 7.98 ± 0.83 μM and 4.93 ± 0.35 μM as compared to 0.94 ± 0.12 nM, 26.5 ± 3.5 nM, 149 ± 30 nM, 1.46 ± 0.25 nM, 1.41 ± 0.48 μM and 340 ± 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperatively of receptor binding; however, stimulation of ⁴⁵Ca uptake appeared to follow a non-cooperative mechanism of action. The competitive capsaicin antagonist capsazepine inhibited specific binding of [³H]RTX by rat dorsal root ganglion membrane preparations with K_i and Hill coefficient values of 3.89 ± 0.38 μM and 1.74 ± 0.11. On the other hand it inhibited the induction of ⁴⁵Ca uptake into the cells induced by capsaicin and RTX in a non-cooperative fashion with K_i values of 271 ± 29 nM and 325 ± 47 nM. Our results show that the membrane binding assay relates to the reality of receptor function in the intact, cultured neurons, both in terms of affinity and positive cooperatively. However the different vanilloid derivatives displayed markedly distinct structure-activity relations for high affinity receptor binding and stimulation of ⁴⁵Ca uptake into rat dorsal root ganglion neurons. Among various explanations for this discrepancy, we favor the possibility that the two assays detect distinct classes of the vanilloid (capsaicin) receptor present in primary sensory neurons.