Extrinsicregulationofinjury/growth-relatedgeneexpression intheinferioroliveoftheadultrat Annalisa Buffo, Daniela Carulli, Ferdinando Rossi and Piergiorgio Strata Rita Levi-Montalcini Center for Brain Repair, Department of Neuroscience, University of Turin, C. Raffaello 30, 10125 Turin, Italy Keywords: axotomy, cell body response, mouse, rat, retrograde degeneration, target deletion Abstract Successfulaxonregenerationreliesonthecapabilityofthelesionedneuronstoup-regulateaspeci®csetofinjury/growth-associated genes.Intheadultcentralnervoussystem,thestrengthofthecellbodyresponseisgenerallyrelatedtothedistanceoftheinjurysite fromtheperikaryon,beingstrongerforproximallesions.Nevertheless,inferiorolive(IO)cellsreacttoinjuryandregeneratetheiraxons even after distal transections. To investigate the mechanisms that regulate the IO growth properties, we examined the expression of injury/growth markers (nitric oxide synthase, growth-associated protein 43 and c-Jun) after target deletion or axotomy performed at differentsitesalongtheolivocerebellarpathway.BothaxoninjuryandtargetlossdisclosetwosubsetsofIOneuronsdistributedwithin precisesubnuclei:onesubsetup-regulatesallmarkersinallconditions,whereastheothershowsamildc-Junexpressionbutremains unresponsive even after a very proximal axotomy. These observations indicate that distinct subpopulations of IO cells respond to different regulatory strategies. Unresponsive neurons appear insensitive to environmental positive or negative cues, suggesting that theyareintrinsicallyunabletosetupacellularreactiontoinjury.Incontrast,cellbodychangesinreactiveneuronsareelicitedafterthe removalofretrogradelytransportedtarget-derivedinhibitorysignals.TargetlossalsoinducesdegenerationofIOcells,whosesurvival remainspartiallydependentonPurkinjetargetsinadulthood.Thus,theintrinsicregenerativepotentialofafunctionallyhomogeneous population is regulated by multiple mechanisms, speci®c for distinct neuronal subsets. Introduction Axon growth-associated proteins can be constitutively expressed in adultneuronsbut,moreoften,theyareup-regulatedfollowinginjury.It iscurrentlyassumedthatthestrengthoftheirexpressionafteraxotomy correlates with the regenerative potential of a given neuronal popula- tion. In the peripheral nervous system, the growth proteins growth- associated protein 43 (GAP-43) and c-Jun are poorly expressed in motoneurons and dorsal root ganglion cells under physiological con- ditions but are strongly up-regulated following axotomy performed at any distance from the soma (Liabotis & Schreyer, 1995; Kenney & Kocsis,1998;Fernandes etal.,1999).Suchanup-regulationlastsuntil target reinnervation (Baizer & Fishman, 1987; Leah et al., 1991), suggestingthatthegrowthprogrammeiscontrolledbynegativetarget- derived signals. Inthecentralnervoussystem,severalneuronsdonotdisplayabasal growth gene expression. Some reports indicate that these genes are regulated by target-derived cues also in central neurons (Haas et al., 1998; Karimi-Abdolrezaee & Schreyer, 2002). However, in retinal ganglion cells (Doster et al., 1991), rubrospinal (Fernandes et al., 1999) and Purkinje neurons (Zagrebelsky et al., 1998), the activation ofgrowthgenesisrelatedtothedistanceofthelesionsitefromthecell body, being maximally intense for proximal lesions. These observa- tions suggest that signals issued by non-neuronal elements along the axoncontributetoregulatingtheintrinsicgrowthpropertiesofcentral nervoussystemneurons(Skene,1989,1992;Zagrebelsky etal.,1998; Bareyre et al., 2002). In contrast, other central populations show a constitutive expres- sion of GAP-43, which may be associated with their capability to regenerateevenafteraverydistalaxotomy(corticospinalneurons,Li et al., 1998; Brosamle et al., 2000; Ramon-Cueto et al., 2000; substantia nigra pars compacta cells, Anderson et al., 1998; Moon etal.,2000).Amongthese,inferiorolive(IO)cellscanregrowtheir axonsintopermissiveterritoriesevenafterinjuriesclosetoterminal ®elds (Rossi et al., 1995b; Bravin et al., 1997). Such a strong regenerative potential of IO neurons is accompanied by a sustained basal level of growth markers, including GAP-43 (Kruger et al., 1993),N-CAM(Fernandez etal.,1999),EGR-1(Buffo etal.,1998) and a vigorous cell body response to axotomy (Buffo et al., 1998; Florenzano et al., 2002). Most interestingly, however, after a com- plete transection of the olivocerebellar pathway, cell body changes onlyoccurinneuronsubsetsdistributedoverwell-de®nedIOregions, suggesting that the IO contains distinct cell populations with differ- ential sensitivity to regulatory environmental cues (Buffo et al., 1998). To address this issue, here we investigated the regulation of a set ofinjury/growthmarkers[nitricoxidesynthase(NOS),GAP-43and c-Jun]inaxotomisedolivaryneurons.First,weaskedwhetherthese molecules share the same pattern of activation or are independently up-regulated by distinct subsets of IO cells. We then compared neurochemical changes induced by axotomy performed at different positions along the olivocerebellar pathway. Finally, we monitored the selected genes in IO neurons after axonal transport blockade or target degeneration in both Purkinje cell degeneration (pcd) mice and after saporin-induced Purkinje cell (PC) immunolesions (Wiley, 1992). Preliminary data have been published in Strata et al. (2001). European Journal of Neuroscience, Vol. 18, pp. 2146±2158, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02940.x Correspondence: Dr Annalisa Buffo, as above. E-mail: annalisa.buffo@unito.it Received 7 April 2003, revised 26 June 2003, accepted 14 July 2003