Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination

Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler phenotype in mice, the first described cerebellar mutation. In homozygous mutants missing the Reelin gene (reln-/-), neurons are incapable of correctly positioning themselves in layered brain areas such as the cerebral and cerebellar cortices. We here demonstrate that by employing ex vivo cultured cerebellar slices one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs). Methods: We generated mouse hybrids (L7-GFPrelnF1/) with green fluorescent protein (GFP)-tagged PNs, directly visible under fluorescence microscopy. We then cultured the slices obtained from mice with different reln genotypes and demonstrated that when the slices from reln-/- mutants were co-cultured with those from reln+/- mice, the Reelin produced by the latter induced migration of the PNs to partially rescue the normal layered cortical histology. We have confirmed this observation with Voronoi tessellation to analyze PN dispersion. Results: In images of the co-cultured slices from reln-/- mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than those generated from slices of reln+/- animals. The mean roundness factor, area disorder, and roundness factor homogeneity were different when slices from reln-/- mice were cultivated singularly or co-cultivated, supporting mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied ex vivo. Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it for the possibility to study and manipulate ex vivo the activity of a brain-secreted or genetically engineered protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the total avoidance of severe surgery (3Rs perspective)

Caspase-3 Mediated Cell Death in the Normal Development of the Mammalian Cerebellum

Caspase-3, onto which there is a convergence of the intrinsic and extrinsic apoptotic pathways, is the main executioner of apoptosis. We here review the current literature on the intervention of the protease in the execution of naturally occurring neuronal death (NOND) during cerebellar development. We will consider data on the most common altricial species (rat, mouse and rabbit), as well as humans. Among the different types of neurons and glia in cerebellum, there is ample evidence for an intervention of caspase-3 in the regulation of NOND of the post-mitotic cerebellar granule cells (CGCs) and Purkinje neurons, as a consequence of failure to establish proper synaptic contacts with target (secondary cell death). It seems possible that the GABAergic interneurons also undergo a similar type of secondary cell death, but the intervention of caspase-3 in this case still remains to be clarified in full. Remarkably, CGCs also undergo primary cell death at the precursor/pre-migratory stage of differentiation, in this instance without the intervention of caspase-3. Glial cells, as well, undergo a process of regulated cell death, but it seems possible that expression of caspase-3, at least in the Bergmann glia, is related to differentiation rather than death

Ghrelin in Central Neurons

Ghrelin, an orexigenic peptide synthesized by endocrine cells of the gastric mucosa, is released in the bloodstream in response to a negative energetic status. Since discovery, the hypothalamus was identified as the main source of ghrelin in the CNS, and effects of the peptide have been mainly observed in this area of the brain. In recent years, an increasing number of studies have reported ghrelin synthesis and effects in specific populations of neurons also outside the hypothalamus. Thus, ghrelin activity has been described in midbrain, hindbrain, hippocampus, and spinal cord. The spectrum of functions and biological effects produced by the peptide on central neurons is remarkably wide and complex. It ranges from modulation of membrane excitability, to control of neurotransmitter release, neuronal gene expression, and neuronal survival and proliferation. There is not at present a general consensus concerning the source of ghrelin acting on central neurons. Whereas it is widely accepted that the hypothalamus represents the most important endogenous source of the hormone in CNS, the existence of extra-hypothalamic ghrelin-synthesizing neurons is still controversial. In addition, circulating ghrelin can theoretically be another natural ligand for central ghrelin receptors. This paper gives an overview on the distribution of ghrelin and its receptor across the CNS and critically analyses the data available so far as regarding the effects of ghrelin on central neurotransmission

L'io postumo: Autobiografia e narrazione filosofica del sé in Friedrich Nietzsche

La strana coincidenza di autore e personaggio nell’autobiografia costituisce uno strano “paradosso” che impedisce una definizione precisa di questo genere letterario. Accanto al complesso mondo autobiografico è possibile individuare un’altra modalità di auto-narrazione, connessa all’autobiografia: la narrazione filosofica del sé di Friedrich Nietzsche (1844-1900) mette in opera una strategia che coinvolge l’“io” in maniera riflessiva, fisiologica e psicologica e costituisce un tratto saliente del suo stesso filosofare. Questa originalità stilistico-narrativa caratterizza già i primi scritti, ma è difficilmente catalogabile come “costruzione”, “ritrovamento” o “invenzione” del sé, tipici dell’autobiografia, avvicinandosi semmai ad una particolare forma di identità narrativa. I tentativi autobiografici del giovane Nietzsche si modificano man a mano in testi che, sulla scorta delle riflessioni filologico-filosofiche, giungono con esiti diversi a Ecce Homo. Soprattutto a partire dallo scritto del 1885 Al di là del bene e male l’approccio stilistico-narrativo evidenzia l’esigenza autoriale di riproporre attraverso nuovi paratesti, le prefazioni del 1886, una filosofia che non è altro che l’«autoconfessione involontaria» del proprio autore che si è sempre compreso come inattuale. Questi tratti culmineranno in Ecce Homo, un testo narrativamente difficile da definire, in cui tuttavia si ritrovano i motivi tipici dell’autonarrazione filosofica di Nietzsche

Association of caspase 3 activation and h2ax γ phosphorylation in the aging brain: Studies on untreated and irradiated mice

Phosphorylation of H2AX is a response to DNA damage, but γH2AX also associates with mitosis and/or apoptosis. We examined the effects of X-rays on DNA integrity to shed more light on the significance of H2AX phosphorylation and its relationship with activation of caspase 3 (CASP3), the main apoptotic effector. After administration of the S phase marker BrdU, brains were collected from untreated and irradiated (10 Gray) 24-month-old mice surviving 15 or 30 min after irradiation. After paraffin embedding, brain sections were single- or double-stained with antibodies against γH2AX, p53-binding protein 1 (53BP1) (which is recruited during the DNA damage response (DDR)), active CASP3 (cCASP3), 5-Bromo-2-deoxyuridine (BrdU), and phosphorylated histone H3 (pHH3) (which labels proliferating cells). After statistical analysis, we demonstrated that irradiation not only induced a robust DDR with the appearance of γH2AX and upregulation of 53BP1 but also that cells with damaged DNA attempted to synthesize new genetic material from the rise in BrdU immunostaining, with increased expression of cCASP3. Association of γH2AX, 53BP1, and cCASP3 was also evident in normal nonirradiated mice, where DNA synthesis appeared to be linked to disturbances in DNA repair mechanisms rather than true mitotic activity