Differential regenerative response of purkinje cell and inferior olivary axons confronted with embryonic grafts: Environmental cues versus intrinsic neuronal determinants

Abstract

Regeneration of severed central axons is supposed to depend on two factors: a permissive local environment and the particular intrinsic properties of axotomized neurones. To assess the role of each of these factors in axonal regeneration, the capability of two particular axon populations of the adult mouse cerebellum to grow into target-specific (cerebellum) and target-unspecific (neocortex) embryonic grafts was determined. Purkinje cell and inferior olivary axons were transected by passing a microscalpel through the axial white matter of the cerebellar folia, particularly those of the anterior lobe. Immediately after the injury, solid transplants were placed in the lesion cavity. Purkinje cell axons were labelled by using anticalbindin immunocytochemistry, and olivocerebellar fibres were visualized by biotinylated dextran amine anterograde axonal tracing. Following axotomy, Purkinje cell axons appeared as thickened processes ending with large terminal clubs. Their morphology and number did not change up to the longest survival time considered (2 months), thereby confirming previous demonstrations that Purkinje cells survive axon injury (I. Dusart and C. Sotelo, 1994, J. Comp. Neurol. 347:211-232). Inferior olivary axons were thinner and bore smaller terminal bulbs. When embryonic cerebellar grafts, containing cortical and deep nuclear precursors, were placed close to the injured axons, olivocerebellar fibres vigorously regenerated into the transplants and ended in new climbing fibres along the dendrites of grafted Purkinje cells. By contrast, host Purkinje cell axons never showed any outgrowth towards the graft. Similarly, these axons failed to regenerate into grafts containing solely the rostromedial portion of the cerebellar anlage, mostly consisting of deep nuclear neurones, their main targets. Comparable results were obtained by transplanting embryonic neocortical tissue: inferior olivary axons also regenerated into the grafts, although with distinct terminal arbours without the climbing fibre phenotype, whereas Purkinje cell axons always failed to grow. These results provide the first direct demonstration that severed inferior olivary axons are able to regenerate. In addition, they show that the growth-permissive/-promoting conditions created by embryonic nervous tissue are not sufficient to induce the regeneration of every axonal type and allow us to hypothesise that successful regeneration depends on the interplay between environmental cues and intrinsic properties of the axotomized neurones.

Purkinje Cell Survival and Axonal Regeneration Are Age Dependent: AnIn VitroStudy

“…In none of these cocultures do severed Purkinje cell axons regenerate. These negative results confirmed a previous conclusion obtained with an in vivo approach (Rossi et al, 1995): permissive environmental cues and absence of inhibitory factors are necessary (Schwab et al, 1993), but not sufficient, to promote regeneration of adult Purkinje cell axons. Thus, the concept that only the local milieu in adult CNS prevents regeneration cannot be generalized.…”

“…Adult Purkinje cells are among the most resistant neurons to axotomy and the most refractory to axonal regeneration: they do not regenerate their axons even in the presence of their embryonic targets (Rossi et al, 1995). These cells in addition are one of the rare categories of neurons whose axons have never been observed to grow into grafted peripheral nerves (Dooley and Aguayo, 1982).…”

“…By reexamining the problem of the failure of Purkinje cell axonal regeneration in mature rat cerebellum Rossi et al, 1995), we have validated this in vitro system, which closely resembles the in vivo conditions. The results reported in this study provide evidence that during the period of active synaptogenesis between Purkinje cells and their target neurons [postnatal day (P) 1-5], the period during which these neurons follow a complete regressive remodeling of their dendritic arbors (Armengol and Sotelo, 1991), the Purkinje cells are extremely susceptible to in vitro conditions and degenerate when cultured.…”

“…Furthermore, the application of blocking antibodies against CNS myelin inhibitory proteins favors regeneration of axotomized corticospinal axons after spinal cord injury (Schnell and Schwab, 1990;Schwab et al, 1993). These observations strongly support the hypothesis that environmental factors rather than the intrinsic inability of adult CNS axons to regenerate are the limiting factor in the failure of axonal regeneration.Adult Purkinje cells are among the most resistant neurons to axotomy and the most refractory to axonal regeneration: they do not regenerate their axons even in the presence of their embryonic targets (Rossi et al, 1995). These cells in addition are one of the rare categories of neurons whose axons have never been observed to grow into grafted peripheral nerves (Dooley and Aguayo, 1982).…”

Degradation of Chondroitin Sulfate Proteoglycans Induces Sprouting of Intact Purkinje Axons in the Cerebellum of the Adult Rat

“…Despite their poor regenerative potential (Rossi et al, 1995b;Dusart et al, 1997;Carulli et al, 2004), Purkinje axons are capable of extensive sprouting at long-term after injury (Dusart and Sotelo, 1994;Dusart et al, 1999;Gianola and Rossi, 2002), when the scar environment acquires growth-promoting/permissive properties, including disappearance of CSPGs (Dusart et al, 1999(Dusart et al, , 2005Morel et al, 2002). In addition, similar to other systems (Thallmair et al, 1998;Bareyre et al, 2002), sprouting from intact Purkinje axons, with aberrant growth into the deep granular layer, can be induced by application of anti-Nogo antibodies in both adult (Buffo et al, 2000) and developing (Gianola et al, 2003) cerebella.…”