Rat Motoneurons In Culture Research Articles

Trk and cAMP-dependent survival activity of adenosine A2A agonist CGS21680 on rat motoneurons in culture

The survival activity of adenosine A2A agonist CGS21680 on motoneurons in culture through the transactivation of neurotrophin receptor TrkB has been reported previously; however, since adenosine A2A receptor belongs to a Gs-protein-coupled receptor, we investigated the involvement of the cAMP pathway in the survival activity of CGS21680 using purified motoneurons in culture. CGS21680 alone showed only small survival activity, but the activity was significantly enhanced by the addition of a phosphodiesterase inhibitor, IBMX. This survival activity was partially inhibited by a protein kinase A inhibitor H89 or a neurotrophin receptor tyrosine kinase inhibitor K252a, and was completely inhibited by their combination. These results indicate that the survival activity of CGS21680 on motoneurons is exerted by the mixed effect of the adenylate cyclase–cAMP–PKA pathway and transactivation of Trk neurotrophin receptor. Under conditions in which the maximum survival of motoneurons was supported by sufficient concentrations of brain-derived neurotrophic factor (BDNF), a TrkB ligand, the addition of 100μM AMPA for 3 days led to significant cell death. Treatment with CGS21680 and IBMX protected motoneurons from the toxicity of AMPA, further supporting the presence of a TrkB-independent pathway of CGS21680 activity and suggesting a novel therapeutic approach to motoneuron diseases such as amyotrophic lateral sclerosis.

Neuroprotective activity of adenosine A 2A agonist in the presence of phosphodiesterase inhibitors in rat motoneurons in culture

Neuroprotective activity of adenosine A 2A agonist in the presence of phosphodiesterase inhibitors in rat motoneurons in culture

Survival‐promoting activity of pituitary adenylate cyclase‐activating polypeptide in the presence of phosphodiesterase inhibitors on rat motoneurons in culture: cAMP–protein kinase A‐mediated survival

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be neurotrophic or neuroprotective in various neurons in culture. It is expressed in spinal motoneurons in vivo and its expression is increased markedly after axotomy, suggesting a neuroprotective role via an autocrine mechanism. However, neurotrophic activity of PACAP has not been reported for motoneurons. In the present study, we investigated the effects of PACAP on rat motoneurons in culture. In the presence of a phosphodiesterase inhibitor, PACAP showed significant neurotrophic activity at concentrations as low as 0.01 nM. Previously, we found that glutamate was excitotoxic to motoneurons even in the presence of brain-derived neurotrophic factor, which is neurotrophic for motoneurons. PACAP with a phosphodiesterase inhibitor protected motoneurons against this excitotoxicity. The activity of PACAP was inhibited by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, as was the case with the activity of forskolin, suggesting downstream involvement of a cAMP-protein kinase A signaling pathway. The present results may suggest a physiological role of PACAP in vivo, and implicate the PACAP-cAMP signaling pathway for the possible therapeutic target of amyotrophic lateral sclerosis as glutamate excitotoxicity was suggested in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis patient IgG alters voltage dependence of Ca 2+ channels in dissociated rat motoneurons

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) have detected IgG within motoneurons of patients and these IgGs have been shown to alter voltage dependent calcium channel activity in various cell types. The current study investigates whether IgG from categorized SALS patients alter voltage dependent calcium currents in rat motoneurons in culture. Patients were categorized based on onset and progression pattern. IgG yields were 38% higher in SALS patients compared to control subjects. Incubation with 1 mg/ml IgG from SALS patients did not cause visible toxicity, alter input resistance, capacitance or the maximal calcium conductance in rat motoneurons when compared to motoneurons incubated with control IgG. However, the activation curve of calcium current was shifted to the left in motoneurons treated with SALS IgG compared to control IgG.

Survival-promoting activity of nimodipine and nifedipine in rat motoneurons: implications of an intrinsic calcium toxicity in motoneurons.

L-type calcium channel antagonists, nimodipine and nifedipine, were tested for effects on the survival of purified rat motoneurons in culture. They showed significant activity, with maximum survival at 30 microm after 3 days in culture as high as 75%, which was comparable to the maximum effect obtained with brain-derived neurotrophic factor, a potent neurotrophic factor for rat motoneurons. It was also found that depolarizing conditions with a high potassium concentration (30 mm) were toxic to motoneurons. This toxicity was blocked by co-treatment with nimodipine. These results implicate a pre-existing calcium burden through calcium channels in motoneurons; they may offer further insights into understanding the selective death of motoneurons and have therapeutic implications in amyotrophic lateral screlosis.

Molecular determinants of emerging excitability in rat embryonic motoneurons.

Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT-PCR, we have shown here that the spike-generating Na(+) current is supported by Nav1.2 and/or Nav1.3 alpha-subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Nav beta 2.1 transcript was not detected, whereas the Nav beta 1.1 transcript was present. Even in the absence of Nav beta 2.1, alpha-subunits were correctly inserted into the initial segment. RT-PCR (at semi-quantitative and single-cell levels) and immunocytochemistry showed that transient K(+) currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K(+) current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv alpha-subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K(+) current. In conclusion, this study has established the first correlation between the molecular nature of voltage-dependent Na(+) and K(+) channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.

Oxidant treatment causes a dose-dependent phenotype of apoptosis in cultured motoneurons.

Evidence is growing that reactive oxygen species (ROS), by-products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose-dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose).

Synergistic Effects of Schwann- and Muscle-Derived Factors on Motoneuron Survival Involve GDNF and Cardiotrophin-1 (CT-1)

The survival of central neurons depends on multiple neurotrophic factors produced by different cell types. We demonstrate that media conditioned by muscle and Schwann cell lines show strong synergistic effects on survival of purified embryonic day 14.5 rat motoneurons in culture. Different lines of evidence implicate glial cell line-derived neurotrophic factor (GDNF) and cardiotrophin-1 (CT-1) in this synergy. Their expression in the environment of the motoneuron is compartmentalized: gdnf transcripts are expressed principally in Schwann cell lines, whereas ct-1 mRNA is present in myotubes. Blocking antibodies to GDNF inhibit the trophic activity of Schwann cell line-conditioned media by 75%, whereas CT-1 antibodies diminish the myotube-derived activity by 46%. CT-1 and GDNF act synergistically to enhance motoneuron survival in vitro. In vivo, individual motoneurons coexpress both GDNF and CT-1 receptor components. GDNF and CT-1, therefore, are major components of the trophic support provided by the Schwann and muscle cells, respectively. The possibility that they act together on individual motoneurons suggests that the motoneuron must integrate distinct signals from different cellular partners when deciding whether to die or to survive.

Prevention of apoptotic motoneuron death in vitro by neurotrophins and muscle extract

In this study, it is shown that rat motoneurons in culture are highly dependent on trophic support and die in the absence of such support via active cell death, or apoptosis. This apoptotic death occurs in their ` in vitro' life (within 24 h) and can be partially prevented by treating the cultures with neurotrophic substances. The most effective support comes from an extract prepared from embryonic chick muscle: without muscle extract, no healthy, neurite-bearing motoneurons are present, but with 0.3 and 1.2% muscle extract, their numbers increase dose-dependently. Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), cannot replace muscle extract and keep motoneurons alive on their own, but in the presence of muscle extract (0.3%) they have a significant effect on survival (increase up to four times, compared to 0.3% muscle extract). At the same time, they prevent apoptotic cell death. Selective motoneuron death has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Despite strong evidence that motoneurons in ALS die via apoptosis, this has not been shown unequivocally in post mortem material. This study shows that motoneurons are very sensitive to conditions that initiate apoptosis, and that cell death can be prevented to a large degree with relatively low concentrations of neurotrophic factors. In view of the fact that several patient trials with neurotrophic factors already are underway, studies with motoneurons in culture may prove important in understanding the mechanism of cell death and the efficacy of drugs such as neurotrophic factors, but also other types of drug.

GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.

For survival, embryonic motoneurons in vertebrates depend on as yet undefined neurotrophic factors present in the limb bud. Members of the neurotrophin family are currently the best candidates for such neurotrophic factors, but inactivation of their receptor genes leads to only partial loss of motoneurons, which suggests that other factors are involved. Glial cell line-derived neurotrophic factor (GDNF), originally identified as a trophic factor specific for dopaminergic neurons, was found to be 75-fold more potent than the neurotrophins in supporting the survival of purified embryonic rat motoneurons in culture. GDNF messenger RNA was found in the immediate vicinity of motoneurons during the period of cell death in development. In vivo, GDNF rescues and prevents the atrophy of facial motoneurons that have been deprived of target-derived survival factors by axotomy. GDNF may therefore be a physiological trophic factor for spinal motoneurons. Its potency and specificity in vitro and in vivo also make it a good candidate for treatment of motoneuron disease.