Times of neuron origin and neurogenetic gradients in mice Purkinje cells and deep cerebellar nuclei neurons during the development of the cerebellum. A review.

This current review is focused on the generation and settled patterns of mouse Purkinje cells (PCs) and deep cerebellar nuclei (DCN) neurons. By mean of progressively delayed comprehensive labeling procedure, I will show, with the technique of [3H] thymidine autoradiography, the quantitative determination of PCs and DCN neurons production along the mediolateral and rostrocaudal axes of the cerebellum. The procedure consists of injecting groups of pregnant mice, on specific embryonic (E) days, with two doses of [3H] thymidine in an overlapping series with 24 h delays between groups (E11-12, E12-13, E13-14, E14-15). The analysis of the autoradiograms revealed that PCs and DCN neurons are sequentially generated following precise neurogenetic timetables. PCs are born somewhat later than the DCN neurons. Both macroneurons are produced following two gradients. The first of these is mediolateral and the second is rostrocaudal. On the other hand, it will be also shown that PCs and DCN neurons were settled in the cerebellum following accurate neurogenetic gradients. These data have suggested that the chronological sequence of neuron production is a key factor in facilitating, in the adulthood, the cytoarchitecture of the cerebellum, and the establishment of patterns of orderly connections between PCs and DCN neurons.

Natural apoptosis in developing mice dopamine midbrain neurons and vermal Purkinje cells.

Natural cell death by apoptosis was studied in two neuronal populations of BALB/c, C57BL/6 and B6CBA-Aw-j/A hybrid stock mice: (I) dopaminergic (DA) neurons in choosing coronal levels throughout the anteroposterior extent of the substantia nigra pars compacta (SNc), and (II) Purkinje cells (PCs) in each vermal lobe of the cerebellar cortex. Mice were collected at postnatal day (P) 2 and P14 for the midbrain study, and at P4 and P7 for the analysis of the cerebellum. No DA cells with morphologic criteria for apoptosis were found. Moreover, when the combination of tyrosine hydroxylase and TUNEL or tyrosine hydroxylase and active caspase-3 immunohistochemistry were performed in the same tissue section, no DA cells TUNEL positives or active caspase-3-stained DA neurons were seen. On the other hand, when PCs were considered, data analysis revealed that more dying PCs were observed at P4 than at P7. Values of neuron death were highest in the central lobe; this was followed by the posterior and anterior lobes and then by the inferior lobe. To determine if apoptotic death of PCs is linked to their time-of-origin profiles, pregnant dams were administered with [3H]TdR on embryonic days 11-12, 12-13, 13-14 and 14-15. When TUNEL and [3H]TdR autoradiography or active caspase-3 immunohistochemistry and [3H]TdR autoradiography were combined in the same tissue section, results reveal that the naturally occurring PC death is not related to its time of origin but, rather, is random across age.

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling.

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steady-state kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.