Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy

Abstract

Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein, microtubule-associated protein2a/b (MAP2a/b); some had GAP-43-immunoreactive terminals; and nearly all had morphological features typical of axons. Dendrites of other CINs did not give rise to de novo axons. These CINs did, however, each have a long axon-like process (L-ALP) that projected directly from the soma or a very proximal dendrite. L-ALPs were devoid of MAP2a/b immunoreactivity. Some of these L-ALPs projected through the lesion and formed bouton-like swellings. These results suggest that proximally axotomized spinal interneurons have the potential to form new connections via de novo axons that emerge from distal dendrites. Others may be capable of regeneration of their original axon.

Spinal Interneuron Axons Spontaneously Regenerate after Spinal Cord Injury in the Adult Feline

“…All surgical procedures were performed on adult cats (Ͼ11 months of age) under deep general anesthesia using either isoflurane (recovery experiments) or sodium pentobarbital (terminal experiments). Surgical procedures, peripheral nerve stimulation protocols, and postoperative care procedures were identical to those used in previous experiments (Fenrich et al, 2007). Briefly, for the recovery surgeries, the animal was anesthetized, and the dorsal C2 and C3 vertebrae were exposed and removed to access the C3 segment.…”

“…We confirmed that all lesions transected the midline through the central canal after the terminal experiments (see below). The lesion protocol has previously been described in detail (Fenrich et al, 2007) and was performed using a specially designed double-edged scalpel blade that was mounted to a motorized microdrive. Briefly, the knife was inserted to a depth of at least 5 mm (the most ventral aspect of the ventral horns is ϳ4 mm below the dorsal surface of the spinal cord at C3) (supplemental Fig.…”

“…We stained PCIs with multiple iontophoretic injections of Neurobiotin into the extracellular space of the ventral horn as described previously (Fenrich et al, 2007). Briefly, Neurobiotin was injected twice in each electrode track.…”

“…The methods used to identify PCIs are described below. After completion of the electrophysiological recordings, the cats were perfused, and the spinal cords were collected as described previously (Fenrich et al, 2007).…”

“…This reexamination was based on observations of axons arising from spinal interneurons. Previous studies have shown that spinal interneurons survive well after axotomy caused by contusion spinal cord injury (SCI) (Conta and Stelzner, 2004) and, after midsagittal spinal transactions, axotomized spinal interneurons develop axon-like processes that cross the midline (Fenrich et al, 2007). In addition, the axons of spinal interneurons also have a remarkable capacity for the formation of new collateral branches that circumvent injury sites after partial SCIs (Bareyre et al, 2004;Courtine et al, 2008).…”