Wnt7A identifies embryonic γ-motor neurons and reveals early postnatal dependence of γ-motor neurons on a muscle spindle-derived signal.

Motor pools comprise a heterogeneous population of motor neurons that innervate distinct intramuscular targets. While the organization of motor neurons into motor pools has been well described, the time course and mechanism of motor pool diversification into functionally distinct classes remains unclear. γ-Motor neurons (γ-MNs) and α-motor neurons (α-MNs) differ in size, molecular identity, synaptic input and peripheral target. While α-MNs innervate extrafusal skeletal muscle fibers to mediate muscle contraction, γ-MNs innervate intrafusal fibers of the muscle spindle, and regulate sensitivity of the muscle spindle in response to stretch. In this study, we find that the secreted signaling molecule Wnt7a is selectively expressed in γ-MNs in the mouse spinal cord by embryonic day 17.5 and continues to molecularly distinguish γ-from α-MNs into the third postnatal week. Our data demonstrate that Wnt7a is the earliest known γ-MN marker, supporting a model of developmental divergence between α- and γ-MNs at embryonic stages. Furthermore, using Wnt7a expression as an early marker of γ-MN identity, we demonstrate a previously unknown dependence of γ-MNs on a muscle spindle-derived, GDNF-independent signal during the first postnatal week.

SPP1 expression in spinal motor neurons of the macaque monkey.

In the macaque cerebral cortex, the SPP1 (secreted phosphoprotein 1) gene is mainly expressed in corticospinal neurons. In this study, we found that SPP1 was principally expressed in motor neurons in lamina IX of the macaque spinal cord. The expression level varied among different spinal segments and correlated positively with neuron size. The expression was weak in Errγ-positive neurons, presumably gamma motor neurons, and in neurons in sacral Onuf's nucleus. These results suggest that SPP1 is a molecular characteristic of spinal motor neurons and is preferentially expressed in neurons with high conduction velocities.

Some gamma-motoneurons contain gamma-aminobutyric acid in the rat cervical spinal cord.

Gamma-aminobutyric acid (GABA) is utilized in the peripheral as well as central nervous system. In this study, fibers immunoreactive for 67 kDa isoform of glutamic acid decarboxylase (GAD67), an enzyme which synthesizes GABA, were found to terminate in the intercapsular region of muscle spindles of the upper limb. GABA-containing fibers were also found in the ventral roots of C5 to T5 spinal segments, brachial plexus, and radial nerve. These fibers were thin and immunoreactive for choline-acetyl transferase (ChAT). After transection of the brachial plexus, GABA immunoreactivity disappeared completely in the ipsilateral triceps brachii muscle (TBM). After the injection of fluorogold into the TBM, some retrogradely labeled medium-sized neurons were positive for GAD67, but not VGAT mRNA. All these observations clearly indicate that GABA-containing gamma-motoneurons in the lower cervical spinal cord send their fibers to muscle spindles in the upper extremities. Since we detected neither GABAA nor GABAB receptors in the TBM by RT-PCR, the function of the GABA-containing gamma-motoneurons remains unclear.

Differential sensitivity of skeletal and fusimotor neurons to Bcl-2-mediated apoptosis during neuromuscular development.

Proper development of the nervous system requires that a carefully controlled balance be maintained between both proliferation and neuronal survival. The process of programmed cell death is believed to play a key role in regulating levels of neuronal survival, in large part through the action of antiapoptotic proteins, such as Bcl-2. Consistent with this, Bcl-2 has been shown to be a key regulator of apoptotic signaling in post-mitotic neurons. However, we still know remarkably little regarding the role that Bcl-2 plays in regulating the survival of specific motor neuron populations. In the present study, we have examined somatic motor neurons of the lumbar spinal cord, and branchiomotor neurons of the facial nucleus in bcl-2-null mice to determine the differential dependence among motor neuron populations with respect to Bcl-2-mediated survival. Examination of neuronal and axon number, axonal area, and the distribution of axonal loss in bcl-2-null mice demonstrates that, in contrast to the great majority of alpha motor neurons, gamma motor neurons exhibit a unique dependence upon bcl-2 for survival. These results demonstrate, for the first time, the connection between Bcl-2 expression, motor neuron survival, and the establishment of different motor populations.

Rat alpha- and gamma-motoneuron soma size and succinate dehydrogenase activity are independent of neuromuscular activity level.

The chronic level of neuromuscular activity, that is, activation and loading, strongly influences the morphological, metabolic, phenotypic, and physiological properties of skeletal muscles. The effects on the innervating motoneurons, however, are less established. We determined and compared the effects of 30 days of decreased activity (induced by a complete mid-thoracic spinal cord transection, ST) or near inactivity (induced by spinal cord isolation, SI) on the soma size and succinate dehydrogenase (SDH) activity of motoneurons innervating a predominantly slow ankle extensor (soleus) and a predominantly fast ankle flexor (tibialis anterior) muscle of adult rats. Soleus and tibialis anterior motoneuron pools were labeled retrogradely using nuclear yellow. The alpha- and gamma-motoneurons were classified based on soma size. Mean number of labeled motoneurons, and mean soma size and SDH activity for both alpha- and gamma-motoneurons were similar in control, ST, and SI rats. Compared to previous reports showing significant decreases in muscle fiber size and adaptations toward a "faster" metabolic profile following ST and SI, the results indicate that, unlike the muscles they innervate, the motoneurons are relatively unresponsive to chronic reductions in neuromuscular activity. The implication of these results is that mean size and SDH activity are independent of the number of action potentials generated by both alpha- and gamma-motoneurons and that even the absence of afferent input to the spinal cord has no influence on size and oxidative metabolic potential of the motoneuron soma.

Glial cell-line derived neurotrophic factor-dependent fusimotor neuron survival during development.

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for motor neurons. Previous studies have shown that some motor neurons depend upon GDNF during development but this GDNF-dependent motor neuron subpopulation has not been characterized. We examined GDNF expression patterns in muscle and the impact of altered GDNF expression on the development of subtypes of motor neurons. In GDNF hemizygous mice, motor neuron innervation to muscle spindle stretch receptors (fusimotor neuron innervation) was decreased, whereas in transgenic mice that overexpress GDNF in muscle, fusimotor innervation to muscle spindles was increased. Facial motor neurons, which do not contain fusimotor neurons, were not changed in number when GDNF was over expressed by facial muscles during their development. Taken together, these data indicate that fusimotor neurons depend upon GDNF for survival during development. Since the fraction of cervical and lumbar motor neurons lost in GDNF-deficient mice at birth closely approximates the size of the fusimotor neuron pool, these data suggest that motor neuron loss in GDNF-deficient mice may be primarily of fusimotor neuron origin.

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis.

BACKGROUND: Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. RESULTS: Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppressed not only axon pruning but also the infiltration and engulfing action of the surrounding glia. CONCLUSIONS: These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. We therefore propose that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis.

Glia engulf degenerating axons during developmental axon pruning.

Developmental axon pruning is widely used in constructing the nervous system. Accordingly, diverse mechanisms are likely employed for various forms of axon pruning. In the Drosophila mushroom bodies (MB), gamma neurons initially extend axon branches into both the dorsal and medial MB axon lobes in larvae. Through a well-orchestrated set of developmental events during metamorphosis, axon branches to both lobes degenerate prior to the formation of adult connections. Here, we analyze ultrastructural changes underlying axon pruning by using a genetically encoded electron microscopic (EM) marker to selectively label gamma neurons. By inhibiting axon pruning in combination with the use of this EM marker, we demonstrate a causal link between observed cellular events and axon pruning. These events include changes in axon ultrastructure, synaptic degeneration, and engulfment of degenerating axon fragments by glia for their subsequent breakdown via the endosomal-lysosomal pathway. Interestingly, glia selectively invade MB axon lobes at the onset of metamorphosis; this increase in cell number is independent of axon fragmentation. Our study reveals a key role for glia in the removal of axon fragments during developmental axon pruning.

Quantitative ultrastructural analysis of glycine- and gamma-aminobutyric acid-immunoreactive terminals on trigeminal alpha- and gamma-motoneuron somata in the rat.

Detailed knowledge of the inhibitory input to trigeminal motoneurons is needed to understand better the central mechanisms of jaw movements. Here a quantitative analysis of terminals contacting somata of jaw-closing (JC) and jaw-opening (JO) alpha-motoneurons, and of JC gamma-motoneurons, was performed by use of serial sectioning and postembedding immunogold cytochemistry. For each type of motoneuron, the synaptic boutons were classified into four groups, i.e., immunonegative boutons or boutons immunoreactive to glycine only, to gamma-aminobutyric acid (GABA) only, or to both glycine and GABA. The density of immunolabeled boutons was much higher for the alpha- than for the gamma-motoneurons. In the alpha-motoneuron populations, the immunolabeled boutons were subdivided into one large group of boutons containing glycine-like immunoreactivity only, one group of intermediate size harboring both glycine- and GABA-like immunoreactivity, and a small group of boutons containing GABA-like immunoreactivity only. The percentage of immunolabeled boutons was higher for JC than JO alpha-motoneurons, the most pronounced difference being observed for glycine-like immunoreactivity. In contrast, on the somatic membrane of gamma-motoneurons, the three types of immunoreactive bouton occurred at similar frequencies. These results indicate that trigeminal motoneurons are strongly and differentially controlled by premotoneurons containing glycine and/or GABA and suggest that these neurons play an important role for the generation of masticatory patterns.

Distribution of the high-affinity choline transporter in the human and macaque monkey spinal cord.

The distribution of the high-affinity choline transporter (CHT) was determined in the human and macaque monkey spinal cord using in situ hybridization histochemistry and immunohistochemistry. Signals for CHT mRNA were observed in somatic motor neurons, sympathetic preganglionic neurons, and neurons in the medial part of lamina VII. The mRNA for CHT was co-localized in single neurons with the mRNAs for vesicular acetylcholine transporter and cholineacetyltransferase. These same cholinergic neuronal groups were labeled by immunohistochemistry for human CHT. Of somatic motor neurons, smaller cell bodies of gamma-motor neurons were labeled very intensely, whereas larger cell bodies of alpha-motor neurons showed various degrees of labeling from weak to moderately intense. Human CHT is thus a novel cholinergic marker, which not only labels cholinergic neurons, but also reveals their heterogeneity.

Differentiation of alpha and gamma motoneurons by the retrograde uptake of horseradish peroxidase.

The classification of motoneurons based on size alone may not be an absolute morphological criterion. There appears to be a fair difference in the pattern of horseradish peroxidase uptake between the phrenic and the intercostal motoneurons. Hence we would like to suggest that the gamma and the alpha motoneurons differ in the horseradish peroxidase uptake.

Nonselective motor innervation of intrafusal fibers in muscle spindles of the rat.

Distributions of motor axons to different types of intrafusal fiber were reconstructed from serial 1-micron thick transverse sections of six poles of muscle spindle in the rat soleus. Motor axons innervated (dynamic) bag1 fibers, or (static) bag2 fibers in conjunction with chain fibers. However, approximately forty percent of axons that supplied the spindles synapsed on both bag1 and bag2 or bag1 and chain fibers. The significance of this co-innervation of dynamic and static intrafusal fibers is discussed relative to the general organization and function of mammalian spindles.