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Rescue of developing spinal motoneurons from programmed cell death by the GABAA agonist muscimol acts by blockade of neuromuscular activity and increased intramuscular nerve branching

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Abstract

Blockade of neuromuscular activity in the chick embryo during the period of programmed cell death of motoneurons results in a complete rescue of these cells. Understanding the cellular mechanisms that mediate this counterintuitive effect is of considerable interest with respect to the regulation of motoneuron survival during development as well as for understanding why motoneurons die pathologically. Although considerable evidence supports the role of a peripheral site of action at the neuromuscular junction in mediating the rescue of motoneurons following activity blockade, some evidence also supports a role for central nervous system (CNS) neurons. For example, the rescue of motoneurons by curare has been reported to be blocked by the GABAA agonist muscimol via its actions on CNS neurons. We have carried out a series of studies to further investigate this interesting observation. Surprisingly, we find that: (1) muscimol blocks activity and rescues MNs in a dose-dependent manner, similar to curare; (2) muscimol’s effects on MN survival appear to be mediated by its action on intramuscular nerve branching, similar to curare; and (3) although muscimol acts centrally, the effects of muscimol on MN survival and axon branching are mediated peripherally at the neuromuscular junction, similar to curare. Because muscimol reduces MN depolarization these data also suggest that the depolarization of MNs by afferents is not required for promoting MN survival. Taken together, these data provide further evidence in support of a peripheral site of action of activity blockade in rescuing motoneurons from developmental cell death.

Introduction

During most of their development, vertebrate embryos and fetuses exhibit spontaneous neuromuscular activity manifested as overt movements (Oppenheim, 1974). For example, in the chick embryo these movements begin on embryonic day (E) 3.5 (st.23) and continue throughout the 21-day incubation period Hamburger 1963, Hamburger and Oppenheim 1967. Although embryonic and fetal neural activity may be required for appropriate neurobehavioral development and nervous system organization (e.g., connectivity), it is also involved in the regulation of motoneuron (MN) survival during development. Blocking embryonic activity during the period of naturally occurring programmed cell death (PCD, E6–E10) rescues virtually all MNs that would otherwise have degenerated Pittman and Oppenheim 1978, Pittman and Oppenheim 1979. One explanation for the rescue of MNs by activity blockade is that the synthesis or secretion of muscle-derived MN trophic factors is increased by activity blockade (the production hypothesis, see Oppenheim et al., 1989). An alternative explanation is that activity blockade somehow facilitates the ability of MNs to access muscle-derived trophic factors (the access hypothesis, see Oppenheim et al 1989, Landmesser 1992. One version of the access hypothesis, for which there is considerable support Dahm and Landmesser 1998, Dahm and Landmesser 1991, Tang and Landmesser 1993, D’Costa et al 1998, Oppenheim et al 2000, Usiak and Landmesser 1999, is that in the absence of neuromuscular activity, developing intramuscular axons branch more and form increased nerve–muscle contacts which provides greater access for the uptake of muscle-derived trophic factors. The increased branching appears to be regulated in part by an activity-dependent, muscle-derived retrograde signal that promotes axon branching independent of any direct action on MN survival D’Costa et al 1998, Rind and von Bartheld 2002.

Regardless of which of these two hypotheses is correct, for both it has generally been assumed that the major site of action of the neuromuscular blocking agents used experimentally to inhibit embryonic activity [e.g., curare, α-bungarotoxin (α-BTX)] is the neuromuscular junction (NMJ). However, the absence of a blood–brain barrier and the presence of nicotinic acetylcholine receptors (nAChRs) on neurons in the developing spinal cord have raised the possibility that the effects of activity blockade by curare or α-BTX on MN survival may be mediated centrally by neuronal rather than muscle (NMJ) nAChRs (e.g., Hory-Lee and Frank, 1995). However, several recent attempt to test this idea have been unsuccessful and instead have considerably strengthened the argument in favor of a peripheral site of action at the NMJ Oppenheim et al 2000, Banks et al 2001, Terrado et al 2001. A notable exception, however, is the recent study by Usiak and Landmesser (1999) in which the GABAA agonist muscimol, by its direct actions on spinal neurons, was reported to block the curare-induced rescue of MNs, from which the authors inferred that the rescue effects of curare-induced activity blockade must be mediated, at least in part, by a CNS site of action. Because we have also reported observations using a paralytic chicken mutant that could be interpreted as support for a CNS contribution to MN rescue by activity blockade (Oppenheim et al., 2000), a further examination of the phenomenon reported by Usiak and Landmesser seemed warranted. In preliminary studies we were unable to repeat some of the effects of muscimol reported by Usiak and Landmesser (Ayala et al., 2000), and accordingly in the present study we have carried out additional studies in an attempt to further examine this issue. Because activity blockade rescues virtually all MNs from PCD and therefore is the most effective means reported so far for promoting MN survival, understanding the cellular and molecular mechanisms involved is of considerable interest in the context of normal development and may also be relevant for elucidating pathological MN degeneration.

Section snippets

Motility

Muscimol had a dose-dependent inhibitory effect on hindlimb movements beginning within the first 6–10 h of the initial treatment on E5.5 (not shown) and doses of 0.1 mg/day or greater reduced activity by more than 95% for the duration of each 24-h period between E6 and E10 (Fig. 1A). Within 36–48 h following the cessation of muscimol treatment hindlimb movements had returned to near-control values (Fig. 1B). The activity blockade attained by muscimol was comparable to that observed after

Discussion

Following the initial discovery that blocking embryonic motility in the chick embryo with curare or α-BTX rescues MNs from PCD Pittman and Oppenheim 1978, Pittman and Oppenheim 1979, this observation has been independently replicated in several laboratories. Following confirmation of the original findings, the issue then became one of determining how these agents act to prevent PCD. A significant breakthrough occurred when Dahm and Landmesser 1998, Dahm and Landmesser 1991 observed that

Eggs and embryos

Fertilized chicken eggs were obtained from two different sources: Tyson Foods, Inc., Wilkesboro, NC, USA (White Leghorn breed), and COPAGA, Lleida, Spain. Although many of the studies reported here were carried out independently using eggs and embryos from these two sources, because there were no statistically significant differences in the data generated, where appropriate the results have been combined. Embryos were staged in ovo or at the time of sacrifice using the chick embryo stage series

Acknowledgements

We thank Carol FloresDeValgaz, Ester Vazquez and Carme Guerris for technical assistance. This work was supported by NIH Grant NS20402 to Ronald W. Oppenheim and grants from the Ministerio de Ciencia y Technologia (SAF 2000-0168) and The Fundació La Caixa.

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