The temporal sequence of morphological and molecular changes in axotomized feline motoneurons leading to the formation of axons from the ends of dendrites

Abstract

At 8-12 weeks post axotomy, unusual distal processes (UDPs) with axon-like structural (uniform diameter, tortuous) and molecular (growth-associated protein [GAP]43, absence of microtubule-associated protein [MAP]2a/b immunoreactivity) features emerge from distal motoneuron dendrites (Rose et al. [2001] Eur J Neurosci 13:1166-1176). In this study, we determine the time course of molecular and morphological changes associated with the formation of axons from dendrites. Motoneurons innervating neck muscles in the adult cat were permanently axotomized for 2, 4, 20, or 35 weeks and intracellularly stained with Neurobiotin. Computer-assisted reconstructions were used to map the location of MAP2a/b and GAP-43 immunoreactivity. At 2 and 4 weeks post axotomy, all UDPs had short appendages, giving them an arboreal appearance. They were immunoreactive for GAP-43 and lacked immunostaining for MAP2a/b. Axon-like UDPs were not seen until 8-12 weeks post axotomy. By 20 and 35 weeks post axotomy, some axon-like UDPs acquired morphological features of axons with synaptic connections (right-angled branching, bouton-like specializations). GAP-43 immunoreactivity was not detected in any axotomized motoneurons by 20 weeks post axotomy, whereas all UDPs remained devoid of MAP2a/b immunoreactivity even at 35 weeks post axotomy. These molecular changes accompanied structural modifications to proximal regions of "dendrites" giving rise to UDPs. The distance from the ends of the UDPs to the soma did not change. Thus, all UDPs begin as simple, arboreal structures with molecular features of growing axons, but over a period of 35 weeks, some UDPs slowly acquire morphological and molecular features of motoneuron axons with synaptic connections. These results suggest a new modus operandi for axonal growth and the establishment of new synaptic connections after injury.

Dendrite-Derived Supernumerary Axons on Adult Axotomized Motor Neurons Possess Proteins That Are Essential for the Initiation and Propagation of Action Potentials and Synaptic Vesicle Release

“…This second sleeve was longer and was pulled as far proximally as possible. The second sleeve was designed to prevent the escape of regenerating axons from the first sleeve as was sometimes observed in previous experiments after post-axotomy periods of 35 weeks (MacDermid et al, 2004). The muscle layers and skin were sutured with Vicryl, and an analgesic, buprenorphine (0.01 mg/kg), was administered as well as an antibiotic, Tribrissen (24%, 0.13 ml/kg).…”

“…ALPs were identified using one or more of the following criteria (based on the study by MacDermid et al, 2004). They followed a tortuous trajectory and formed either simple or complex arborizations that consisted of fine branches that emerged at right angles and had several bouton-like swellings.…”

“…There is considerable evidence that proximal axotomy may trigger a similar reorganization of neuronal polarity both in vitro (Bradke and Dotti, 2000;Gomis-Rüth et al, 2008) and in vivo (Lindå et al, 1985(Lindå et al, , 1992Havton and Kellerth, 1987;Rose and Odlozinski, 1998;Rose et al, 2001;MacDermid et al, 2002MacDermid et al, , 2004. This reorganization is manifested in the emergence of axon-like processes (ALPs) from the terminals of distal dendrites (Lindå et al, 1985(Lindå et al, , 1992Rose et al, 2001;MacDermid et al, 2002MacDermid et al, , 2004Gomis-Rüth et al, 2008).…”

“…This reorganization is manifested in the emergence of axon-like processes (ALPs) from the terminals of distal dendrites (Lindå et al, 1985(Lindå et al, , 1992Rose et al, 2001;MacDermid et al, 2002MacDermid et al, , 2004Gomis-Rüth et al, 2008). With time, these ALPs become myelinated (Lindå et al, 1992;Rose et al, 2001) and develop fine right-angled branches with bouton-like swellings, and the dendrites that give rise to these ALPs undergo a process of apparent axonification (MacDermid et al, 2004). ALPs lack dendritic cytoskeletal proteins and initially express the axonal growth proteins (Rose et al, 2001;MacDermid et al, 2002MacDermid et al, , 2004.…”

“…With time, these ALPs become myelinated (Lindå et al, 1992;Rose et al, 2001) and develop fine right-angled branches with bouton-like swellings, and the dendrites that give rise to these ALPs undergo a process of apparent axonification (MacDermid et al, 2004). ALPs lack dendritic cytoskeletal proteins and initially express the axonal growth proteins (Rose et al, 2001;MacDermid et al, 2002MacDermid et al, , 2004. Thus, the ALPs meet some of the morphological and molecular criteria of axons; however, it is unknown whether they have the functional capacity of an axon.…”