Fluorescent proteins driven in mice by the S100B promoter give variegated labeling of glia, neurons, macrophages, and dendritic cells of the immune system.
Zuo Y, Lubischer JL, Kang H, Tian L, Mikesh M, Marks A, Scofield VL, Maika S, Newman C, Krieg P, Thompson W. (2004) Journal of Neuroscience 24(49):10999-11009.

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Neonatal partial denervation results in nodal, but not terminal, sprouting and a decrease in efficacy of remaining neuromuscular junctions in rat soleus muscle.
Jane L. Lubischer and Wesley J. Thompson (1999) Journal of Neuroscience 19(20):8931-8944.

Mature motoneurons respond to partial denervation of their target muscle by sprouting to reinnervate denervated fibers, thus maintaining muscle strength in the face of motoneuronal loss due to injury or disease. Neonatal motoneurons, however, do not expand to innervate more muscle fibers. The present work seeks to understand this developmental change in motoneuron response to partial denervation. It has been suggested that neonatal motor units cannot increase in size because they are already at their maximum size (about 5 times larger than in adulthood). We ruled out this explanation by showing that after partial denervation on postnatal day 14 (P14), when motor units have decreased to their adult size, motoneurons still did not sprout to reinnervate as many fibers as in adulthood. Instead, we found evidence supporting an alternative explanation involving terminal Schwann cells. After partial denervation of neonatal (but not adult) muscles, terminal Schwann cells at denervated endplates undergo apoptosis. We found that terminal (but not nodal) sprouting was absent in partially denervated neonatal muscles. This finding suggests that terminal Schwann cells, previously reported to guide terminal sprouts to denervated endplates in adult muscles, are necessary for the formation and growth of terminal sprouts. Moreover, partial denervation on P14 severely weakened the remaining, uninjured synapses, suggesting that neonatal motoneurons may withdraw terminals after the denervation of nearby fibers. These findings have implications for the interpretation of previous studies on synapse elimination, and offer insight into the failure of young motor units to expand after partial denervation.

Regulation of terminal Schwann cell number at the adult neuromuscular junction.
Jane L. Lubischer and David M. Bebinger (1999) Journal of Neuroscience 19(24):46RC.

Terminal Schwann cells (TSCs), neuroglia that cover motoneuron terminals, play a role in regulating the structure and function of the neuromuscular junction. In rats, the number of TSCs at each junction increases rapidly in early postnatal life and more slowly in young adults. It is possible that TSC number increases to match increasing endplate area. Alternatively, the increase in TSC number may reflect a developmental process independent of endplate size or terminal function. To experimentally test the relationship between endplate size and TSC number, we manipulated endplate area in an androgen-sensitive muscle of the rat, the levator ani (LA), by castration and by androgen replacement. We found that TSC number not only increased as endplates enlarged, but also decreased when endplates shrank. Ninety days after castration, TSC number decreased by approximately 20% (1 cell per junction) as endplate size decreased by 30%. These effects were reversed by testosterone. Testosterone levels did not affect TSC number in the extensor digitorum longus (EDL) muscle, where endplate area was unaffected by castration or testosterone treatment. TSC number was, however, significantly correlated with endplate area in both LA and EDL muscles. Furthermore, the relationship between endplate size and TSC number, as defined by the slope of the regression line, was the same in LA and EDL muscles, indicating that this relationship is not a unique feature of the LA muscle. These data suggest that TSC number is a dynamic property of the neuromuscular synapse that is actively regulated throughout life.

A neuregulin, glial growth factor (GGF2), reduces SNB motoneuron death during normal development.
Lubischer JL, Thompson WJ (1998) Soc. Neurosci. Abstr. 24:1790.

The neuromuscular junction is characterized by complex trophic interactions among nerve terminals, muscle fibers, and Schwann cells (SCs). For example, neonatal denervation causes SC apoptosis, and this can be prevented by treatment with glial growth factor 2 (GGF), a member of the neuregulin family. We have begun studies to address the possible role of SCs in motoneuron death during normal development. The spinal nucleus of the bulbocavernosus (SNB) is convenient for these studies for 2 reasons: (1) much of SNB motoneuron (MN) death occurs postnatally, making manipulations easier, and (2) SNB MN death is influenced by testosterone (T), providing a means of experimentally regulating this process. More SNB MNs die during development in females than in males, due to a perinatal sex difference in T levels. SNB MNs innervate 3 muscles, including the bulbocavernosus (BC) and levator ani (LA) muscles. Female rats (n=3) received GGF injections twice a day (approx. every 12 h) starting on the day of birth and continuing until postnatal day 12 (P12). Injections of 0.4 ug recombinant human GGF in 5 ul vehicle were targeted for the surface of the left BC and LA muscles. On P12, spinal cords were removed and processed to count SNB MNs ipsilateral and contralateral to GGF injections. The SNB contralateral to GGF injections contained the same number of MNs as normal P13 females, but there were almost twice as many SNB MNs innervating BC/LA muscles that had been treated with GGF (25.7 +/- 1.78 contra vs 48.7 +/- 2.27 ipsi; p < .001). Thus, early postnatal treatment of BC/LA muscles with GGF caused a reduction in SNB MN death through P13. The cellular site of GGF action and whether SNB MNs survive after the cessation of GGF treatment are not known.

Axotomy transiently down-regulates androgen receptors in motoneurons of the spinal nucleus of the bulbocavernosus. Lubischer JL, Arnold AP (1995) Brain Research 694:61-68.

Testosterone is an important trophic factor for motoneurons in the spinal nucleus of the bulbocavernosus (SNB), and SNB motoneurons are more responsive to testosterone than are other motoneurons. Axonal injury during early postnatal life prevents the normal development of steroid-sensitivity by adult SNB motoneurons. Axonal injury also causes changes in the expression by motoneurons of a wide range of proteins, including the up-regulation of trophic factor receptors. We have used a polyclonal antibody (PG-21; G.S. Prins) to study the expression of androgen receptors in SNB motoneurons after axonal injury. PG-21 labeled motoneuronal nuclei in the lower lumbar spinal cord of rats in a pattern that matched autoradiographic reports of androgen accumulation in this region of the nervous system. A population of numerous, small cells located dorsal to the central canal also showed evidence of androgen receptor expression. Cutting the axons of SNB motoneurons in adulthood or in development caused a decrease in androgen receptor immunoreactivity in SNB motoneurons. This is the first report that a trophic factor receptor in motoneurons is down-regulated after axonal injury, and is interesting in light of reports that testosterone treatment can facilitate motoneuronal regeneration after nerve cut. Androgen receptor levels subsequently returned to normal, regardless of the age at axotomy, providing no evidence for a lasting effect of developmental axotomy on androgen receptor levels in SNB motoneurons. Thus, axotomy-induced down-regulation of androgen receptors does not underlie the inability of SNB motoneurons to respond to androgen treatment several months after pudendal nerve cut in development.

Evidence for target regulation of the development of androgen sensitivity in rat spinal motoneurons. Lubischer JL, Arnold AP (1995) Developmental Neuroscience 17:106-117.

Specific neuronal circuits within the vertebrate nervous system express high levels of steroid receptors and are sensitive to the effects of steroid hormones. The mechanisms by which these neuronal circuits develop their unique steroid sensitivity are unknown. One intriguing hypothesis is that retrograde influences during early postnatal life play a role in determining which central nervous system (CNS) neurons become sensitive to steroids. We now present evidence that during a critical period in early postnatal development, axonal injury disrupts the normal development of steroid sensitivity. The spinal nucleus of the bulbocavernosus (SNB) is a neuromuscular system that is highly androgen-sensitive at the level of both the motoneurons and their target muscles. Testosterone levels regulate the size of SNB motoneurons and their muscles in adult rats. Cutting the axons of SNB motoneurons on postnatal day 14 (P14) caused permanent decreases in SNB motoneuronal soma size, as well as in SNB target muscle weight. Interestingly, SNB motoneurons that survived axotomy on P14 failed to develop their normal ability to respond to testosterone in adulthood. That is, they did not respond to changes in testosterone levels with changes in soma size. The same effect was not seen after axotomy 1 week later in development, suggesting a critical period for this effect. Thus, separation from the target muscles during an early critical period in development blocked the differentiation of androgen sensitivity by SNB motoneurons, consistent with a role for the target in the normal development of steroid sensitivity by CNS neurons.

Axotomy of developing rat spinal motoneurons: cell survival, soma size, muscle recovery, and the influence of testosterone.
Lubischer JL, Arnold AP (1995) Journal of Neurobiology 26:225-240.

During the period of synapse elimination, motoneurons are impaired in their ability to generate or regenerate axonal branches: following partial denervation of their target muscle, young motoneurons do not sprout to nearby denervated fibers and after axonal injury, they fail to reinnervate the muscle. In the rat levator ani (LA) muscle, which is innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB), synapse elimination ends relatively late in development and can be regulated by testosterone. We took advantage of this system to determine if the end of synapse elimination and the development of regenerative capabilities by motoneurons share a common mechanism, or, alternatively, if these two events can be dissociated in time. Axotomy on or before postnatal day 14 (P14) caused the death of SNB motoneurons. By P21, toward the end of synapse elimination in the LA muscle, SNB motoneurons had developed the ability to survive axonal injury. Altering testosterone levels by castration on P7 followed by 4 weeks of either testosterone propionate or control injections did not change the ability of SNB motoneurons to survive axonal injury during development, although these same treatments alter the time course of synapse elimination in the LA muscle. Thus, we dissociated the inability of SNB motoneurons to recover from axonal injury from their developmental elimination of synaptic terminals. We also measured the effect of early axotomy on motoneuronal soma size and on target muscle weight. Axotomy on P14 caused a long-lasting decrease in the soma size of surviving SNB motoneurons, whereas motoneurons axotomized on P28 recovered their normal soma size. Axotomy on or before P7 caused severe atrophy of the target muscles, matching the extensive loss of motoneurons. However, target muscle recovery after axotomy on P14 was as good as recovery after axotomy at later ages, despite greater motoneuronal death after axotomy on P14. This result may reflect an increase in motor unit size, a decrease in polyneuronal innervation by SNB motoneurons that survive axotomy on P14, or a combination of the two.