The development and molecular characterization of muscle spindles from wildtype and mutant mice

Muscle spindles are complex stretch-sensitive mechanoreceptors that
consist of 4-12 specialized muscle fibers. These intrafusal muscle
fibers are innervated in the central (equatorial) region by an
afferent sensory axon and in both peripheral (polar) regions by
efferent γ-motoneurons. Until now little is known about muscle
spindle development at the molecular level, especially about the
development of cholinergic specializations. My study shows that
nicotinic acetylcholine receptors (AChR) are concentrated at the
γ-motoneuron endplate as well as in the equatorial region.
Moreover, enzymes required for the synthesis and removal of
acetylcholine, including choline acetyltransferase (ChAT) and
acetylcholinesterase (AChE), as well as vesicular acetylcholine
transporter (VAChT) and the AChR-associated protein rapsyn are all
concentrated at the polar γ-motoneuron endplate and (with the
exception of AChE) also at the equatorial region. Finally, the
presynaptic protein bassoon, involved in synaptic vesicle
exocytosis, is also present at the γ-motoneuron endplate and at the
annulospiral sensory nerve ending. During postnatal development,
the AChR subunit composition at the γ-motoneuron endplate changes
from the γ-subunit containing fetal AChR to the ε-subunit
containing adult AChR. This is similar to the postnatal change at
the neuromuscular junction. In the equatorial region the ε-subunit
expression starts around postnatal week two; however the γ-subunit
persists in the central region despite the onset of the ε-subunit
expression. Therefore, the γ- and ε-subunits are simultaneously
present in the equatorial region. This result was confirmed using a
mouse line in which the AChR γ-subunit was genetically labelled by
green fluorescence protein (GFP). In this mouse, the GFP-labelled
AChR γ-subunits are concentrated at the contact site of the
intrafusal fiber with the sensory nerve ending. This result
indicates different AChR maturation occurs within two areas of the
same intrafusal fiber. I also show that agrin and the agrin
receptor complex (consisting of LRP4 and MuSK) are present in
muscle spindles in the region of the sensory and motor innervation.
Moreover, agrin, MuSK, and LRP4 are expressed by proprioceptive
neurons in dorsal root ganglia but only agrin and LRP4 were
detected in the cell body of γ-motoneurons in the spinal cord. In
mice with a targeted deletion of agrin, AChR aggregates are absent
from the polar region and γ-motoneuron endplates do not form. By
contrast, AChR aggregates remain detectable in the central part of
intrafusal fibers. Moreover, muscle-specific re-expression of
mini-agrin is sufficient to restore the formation of synaptic
specializations at γ-motoneuron endplates. These results show an
unusual AChR maturation at the annulospiral endings and confirm
that agrin is a major determinant for the formation of γ-motoneuron
endplates. Agrin on the other hand appears dispensable for the
aggregation of AChRs in the central region of intrafusal fibers.