DEVELOPMENTAL PLASTICITY OF RAT CEREBELLAR CORTEX AFTER CISPLATIN INJURY: INHIBITORY SYNAPSES AND DIFFERENTIATING PURKINJE NEURONS M. B. PISU, a E. RODA, a D. AVELLA a AND G. BERNOCCHI a,b * a Dipartimento di Biologia Animale, Laboratorio di Biologia Cellulare e Neurobiologia, Università di Pavia, Piazza Botta 10, I-27100 Pavia, Italy b Istituto di Genetica Molecolare del CNR - Sezione di Istochimica e Citometria, Università di Pavia, Pavia, Italy Abstract—A single injection of cisplatin, a cytostatic agent, (5 g/g body weight) in 10-day old rats leads later to the reorganization of the cerebellar cortex in lobules VI–VIII of the vermis. Double immunofluorescence reaction for glutamate receptor (GluR)2/3, a ionotropic glutamate receptor that la- bels postsynaptically Purkinje neurons, and glutamic acid decarboxylase (GAD)65, an isoform of the GABA synthesis enzyme that labels presynaptically inhibitory terminals in the molecular layer, were employed. Less-differentiated Purkinje cells were present in rats treated on postnatal day (PD)11 at the top of lobule VI and in lobules VII–VIII, in comparison with the deep zones of the same lobules and lobule III. The changes were interpreted as due to loss of trophic factors of Purkinje cell growth, e.g. signaling molecules and granule cells. However, we have shown that a remodelling of Purkinje cell dendrites occurred on PD30 (20 days after cisplatin). In fact, despite of the GluR2/3 labeling of the entire Purkinje cell dendrites, the GAD65 immunofluorescent terminals were ad- jacent to the proximal parts of the dendrite, while they were scarce in the distal dendritic branchlets. The findings were discussed in relation to the changed cytoarchitecture of the cerebellar cortex, which from PD17 to PD30 includes regen- eration of the external germinal layer, reorientation of the main dendritic branches and of the Purkinje cell branchlets, and the presence of ectopic cells. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: postnatal cerebellar development, drug injury, GluR2/3 receptor, GAD65, double immunofluorescence. The critical steps of Purkinje cell differentiation, as well as the influence of their cellular microenvironment, are ana- lytically reported by Altman (1972b, 1982) and Altman and Bayer (1978). Moreover, extensive literature describes neuroanatomical and functional effects of cerebellar devel- opmental disruptions after treatment, exposition to cyto- toxic substances or irradiation (Altman, 1982; Ferguson, 1996; Fonnum and Lock, 2000). Abnormal development in the cerebellum, when treatments are performed around birth or during the first week of life, can lead to the reduc- tion of granule cells and to the appearance of an ectopic granule cell layer (Altman, 1972b; De Barry et al., 1987; Garcia-Ladona et al., 1991); in some cases there is atro- phy of the cerebellum (Fonnum and Lock, 2000), usually accompanied by different forms of ataxia and an unstable gait. Remodelling events, such as alterations in the posi- tioning of the Purkinje cells and the formation of synapses between mossy fibers and Purkinje cells, are also reported as a consequence of MAM (methylazoxymethanol) cyto- toxicity (De Barry et al., 1987; Garcia-Ladona et al., 1991). The study of cerebellum development disorders is an important step toward understanding the mechanisms in- volved in both genetic and epigenetic cortical dysgenesis as well as the mechanisms underlying the determination of the neuronal phenotype and connectivity within the neural circuits. Cisplatin is an antitumoral drug that is cytotoxic to the development of the cerebellar cortex during the postnatal life of the rat, inducing death of the proliferating granule cells and alteration or even degeneration of differentiating Purkinje cells (Scherini and Bernocchi, 1994; Guioli et al., 2002). Recently, we have shown the acute effects of a single injection of cisplatin on the interaction of signaling mole- cules, i.e. glutamate and nitric oxide, through the labeling of glutamate ionotropic receptors (Pisu et al., 2003). Among these, the lower immunocytochemical expression of glutamate receptor (GluR)2, similarly to GluR2/3 (Hafidi and Hillman, 1997; Pisu et al., 2002a), indicates the slow- ing down or alteration of the differentiative process of Purkinje cell dendrites (Pisu et al., 2003). In the last years, the strict link between glutamate and GABA, the major excitatory and inhibitory neurotransmit- ters largely represented in the cerebellum and acting as morphogens in the development of the CNS, has emerged from literature (Nguyen et al., 2001). Alterations of these signaling molecules during development are associated with neurological disorders such as ataxia or epilepsy (Egeberg et al., 2002). On this basis, following cisplatin injury we have exam- ined firstly i) the morphological signs of the remodelling within the cerebellar cortex and then ii) the neurochemical changes in glutamate and GABA in relation to the changed dendritic morphology of Purkinje cells. In fact, there are no available data on possible, concurrent alterations of both *Correspondence to: G. Bernocchi, Dipartimento di Biologia Animale, Laboratorio di Biologia Cellulare e Neurobiologia, Piazza Botta 10, I-27100 Pavia, Italy. Tel: +39-0382-506327; fax: +39-0382-506325. E-mail address: bern@unipv.it (G. Bernocchi). Abbreviations: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propi- onic acid; bf, barrier filter; dm, dichroic mirror; excf, excitation filter; GABA A , GABA receptor A; GAD, glutamic acid decarboxylase; GluR, glutamate receptor; MAM, methylazoxymethanol; ML, molecular layer; PD, postnatal day. Neuroscience 129 (2004) 655– 664 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.08.023 655