Asynchronous synapse elimination in neonatal motor units: studies using GFP transgenic mice.

In developing muscle, synapse elimination reduces the number of motor axons that innervate each postsynaptic cell. This loss of connections is thought to be a consequence of axon branch trimming. However, branch retraction has not been observed directly, and many questions remain, such as: do all motor axons retract branches, are eliminated branches withdrawn synchronously, and are withdrawing branches localized to particular regions? To address these questions, we used transgenic mice that express fluorescent proteins in small subsets of motor axons, providing a unique opportunity to reconstruct complete axonal arbors and identify all the postsynaptic targets. We found that, during early postnatal development, each motor axon loses terminal branches, but retracting branches withdraw asynchronously and without obvious spatial bias, suggesting that local interactions at each neuromuscular junction regulate synapse elimination.

Glial cell line-derived neurotrophic factor administration in postnatal life results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) in embryonic muscle fibers causes dramatic hyperinnervation of neuromuscular junctions. However, it is not known whether GDNF induces the extra innervation by regulation of axonal branching and/or synaptic maintenance. To address this issue, high levels of circulating GDNF were established by administering subcutaneous injections starting either at birth or later and continuing for up to 40 d. Treatment with exogenous GDNF beginning in the first week, but not later, increased the number of axons converging at neuromuscular junctions. The effect of GDNF on the branching pattern of individual motor axons was determined by reconstructing labeled axonal arbors from transgenic mice expressing yellow fluorescent protein in subsets of motor neurons. Whereas, at postnatal day 8 (P8) individual axons in control animals branched to sporadically innervate junctions within circumscribed regions of the muscle, motor units from GDNF injected animals had significantly more axonal branches and exhibited a high degree of localized arborization such that adjacent muscle fibers were often innervated by the same axon. Administration beginning at P0 and continuing through P40 prolonged multiple innervation of most fibers throughout the period of injection. Between P30 and P40 there was no net change in multiple innervation, although there was evidence of retraction bulbs, suggesting that axon extension and retraction were in equilibrium. We conclude that GDNF has a developmentally regulated effect on presynaptic branching and that sustained administration of GDNF induces a state of continuous synaptic remodeling.

Widespread elimination of naturally occurring neuronal death in Bax-deficient mice.

The proapoptotic molecule BAX is required for death of sympathetic and motor neurons in the setting of trophic factor deprivation. Furthermore, adult Bax-/- mice have more motor neurons than do their wild-type counterparts. These findings raise the possibility that BAX regulates naturally occurring cell death during development in many neuronal populations. To test this idea, we assessed apoptosis using TUNEL labeling in several well-studied neural systems during embryonic and early postnatal development in Bax-/- mice. Remarkably, naturally occurring cell death is virtually eliminated between embryonic day 11.5 (E11.5) and postnatal day 1 (PN1) in most peripheral ganglia, in motor pools in the spinal cord, and in the trigeminal brainstem nuclear complex. Additionally, reduction, although not elimination, of cell death was noted throughout the developing cerebellum, in some layers of the retina, and in the hippocampus. Saving of cells was verified by axon counts of dorsal and ventral roots, as well as facial and optic nerves that revealed 24-35% increases in axon number. Interestingly, many of the supernumerary axons had very small cross-sectional areas, suggesting that the associated neurons are not normal. We conclude that BAX is a critical mediator of naturally occurring death of peripheral and CNS neurons during embryonic life. However, rescue from naturally occurring cell death does not imply that the neurons will develop normal functional capabilities.

Bcl-xL is an antiapoptotic regulator for postnatal CNS neurons.

Bcl-xL is a death-inhibiting member of the Bcl-2/Ced9 family of proteins which either promote or inhibit apoptosis. Gene targeting has revealed that Bcl-xL is required for neuronal survival during brain development; however, Bcl-xL knock-out mice do not survive past embryonic day 13.5, precluding an analysis of Bcl-xL function at later stages of development. Bcl-xL expression is maintained at a high level postnatally in the CNS, suggesting that it may also regulate neuron survival in the postnatal period. To explore functions of Bcl-xL related to neuron survival in postnatal life, we generated transgenic mice overexpressing human Bcl-xL under the control of a pan-neuronal promoter. A line that showed strong overexpression in brainstem and a line that showed overexpression in hippocampus and cortex were chosen for analysis. We asked whether overexpression of Bcl-xL influences neuronal survival in the postnatal period by studying two injury paradigms that result in massive neuronal apoptosis. In the standard neonatal facial axotomy paradigm, Bcl-xL overexpression had substantial effects, with survival of 65% of the motor neurons 7 d after axotomy, as opposed to only 15% in nontransgenic littermates. To investigate whether Bcl-xL regulates survival of CNS neurons in the forebrain, we used a hypoxia-ischemia paradigm in neonatal mice. We show here that hypoxia-ischemia leads to substantial apoptosis in the hippocampus and cortex of wild-type neonatal mice. Furthermore, we show that overexpression of Bcl-xL is neuroprotective in this paradigm. We conclude that levels of Bcl-xL in postnatal neurons may be a critical determinant of their susceptibility to apoptosis.

Altered cell proliferation in the spinal cord of mouse neural tube mutants curly tail and Pax3 splotch-delayed.

The mutant mouse strains splotch-delayed (Pax3Sp-d) and curly tail (ct) develop neural tube defects (NTDs) in the lumbosacral region of the neuraxis. Some research has focused on cell proliferation around the time of posterior neuropore closure in these mutants; however, there are little data on the effects of NTDs on cell birth at later stages of development. To investigate the role neural tube closure might play in cytogenesis of the spinal cord, the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was injected into pregnant splotch-delayed and curly tail mice at various stages of gestation. The mean number of labelled cells in the dorsal and ventral halves of spina bifida and control embryos was then calculated per section and per mm2. Mutagenically separated PCR (MS-PCR), was used to ascertain the genotype of splotch-delayed embryos. Our data indicate that the peak proliferation dates, for both the dorsal and ventral regions of the cord, are similar in spina bifida and control embryos. However, the quantity of proliferation is significantly different between affected and unaffected embryos. In general, there are markedly fewer cells born in spina bifida embryos in early neural tube development, followed by a short period of equal proliferation, and culminating in a significant increase in cell proliferation later in gestation. This increase in proliferation results in a greater number of cells being born in spina bifida embryos compared to controls. Several possible explanations for this phenomenon are considered, including the hypothesis that the roof plate, or other factors induced by neural tube closure, might have an anti-mitotic activity.

Patterns of neuronal differentiation in neural tube mutant mice: curly tail and Pax3 splotch-delayed.

A battery of antibodies was used to assess development of the spinal cord and its neurons in mouse embryos with neural tube defects (NTDs). The two mutant strains examined, curly tail (ct) and splotch-delayed (Pax3Sp-d), develop an open neural tube for unrelated reasons, and thus provided for a complementary analysis. Five percent of embryos homozygous for the ct gene and 89% of embryos homozygous for the Pax3Sp-d gene develop spina bifida in the lumbosacral region of the neuraxis. Expression of several neuronal antigens, including Islet-1/2, polysialylated neural cell adhesion molecule (NCAM), neurofilaments, and a neuronal-specific nuclear protein (Neu-N) recognized by monoclonal antibody A60, were used as indicators of the level of differentiation of neuronal tissue. Immunohistochemical labeling suggests that early (embryonic days 12-15) neuronal differentiation in the dorsal and ventral region of the dysraphic neural tube occurs remarkably normally in both of the mutants. Similarly, labeling with antibodies to NCAM and neuroafilaments indicate that axonal development during early neurogenesis is unperturbed. Later stages of neuronal maturation, however, do not occur in the usual manner. Instead, the neuronal tissue begins a prodigious degeneration at embryonic day 17 (E17), so that by E18 only a rudimentary tissue remains. These results suggest that the aberrant morphology of the neural tube does not affect neuronal differentiation. However, the anomalous morphological and chemical environment may contribute to the neuronal degeneration observed at later stages.

Annexin IV is a marker of roof and floor plate development in the murine CNS.

Midline structures, such as the notochord and floor plate, are crucial to the developing central nervous system (CNS). Previously, we demonstrated that annexin IV is an excellent marker of midline structures. In the present study, we explore the possible role of annexin IV in development of the CNS midline. Using immunocytochemistry with an antibody to annexin IV, we have elucidated the temporal and spatial expression of this molecule. Annexin IV is present in the notochord at embryonic day (E) 8.5, prior to its expression in any structures within the neural tube. Subsequently, annexin IV is expressed by floor plate cells at E9.5. Annexin IV is also expressed in the roof plate, but not until E10.5. To determine if normal morphogenesis of these midline structures is essential for annexin IV expression, we analyzed two strains of mutant mice that have defective formation of either the floor or the roof plate. In Danforth's short-tail mice, the floor plate is absent from the caudal spinal cord, and annexin IV immunopositivity disappears at the level where the floor plate is missing. In curly tail mutant mice, there can be a failure of the neural tube to close, and in these regions there is no annexin IV expression in presumptive roof plate cells. Finally, annexin IV immunolabeling is present from the caudal spinal cord, through the brainstem up to the diencephalon and lamina terminalis. Thus, annexin IV is an excellent marker for differentiated midline cells, is temporally and spatially correlated with development of the floor and roof plates, and is expressed in a rostral-caudal manner that supports the hypothesis that the floor plate extends the full length of the original neural tube.