Developmental Injury to the Cerebellar Cortex Following Hydroxyurea Treatment in Early Postnatal Life: An Immunohistochemical and Electron Microscopic Study.

Postnatal development of the cerebellar cortex was studied in rats administered with a single dose (2 mg/g) of the cytotoxic agent hydroxyurea (HU) on postnatal day (P) 9 and collected at appropriate times ranging from 6 h to 45 days. Quantification of several parameters such as the density of pyknotic, mitotic, BrdU-positive, and vimentin-stained cells revealed that HU compromises the survival of the external granular layer (EGL) cells. Moreover, vimentin immunocytochemistry revealed overexpression and thicker immunoreactive glial processes in HU-treated rats. On the other hand, we also show that HU leads to the activation of apoptotic cellular events, resulting in a substantial number of dying EGL cells, as revealed by TUNEL staining and at the electron microscope level. Additionally, we quantified several features of the cerebellar cortex of rats exposed to HU in early postnatal life and collected in adulthood. Data analysis indicated that the analyzed parameters were less pronounced in rats administered with this agent. Moreover, we observed several alterations in the cerebellar cortex cytoarchitecture of rats injected with HU. Anomalies included ectopic placement of Purkinje cells and abnormities in the dendritic arbor of these macroneurons. Ectopic granule cells were also found in the molecular layer. These findings provide a clue for investigating the mechanisms of HU-induced toxicity during the development of the central nervous system. Our results also suggest that it is essential to avoid underestimating the adverse effects of this hydroxylated analog of urea when administered during early postnatal life.

Hydroxyurea Treatment and Development of the Rat Cerebellum: Effects on the Neurogenetic Profiles and Settled Patterns of Purkinje Cells and Deep Cerebellar Nuclei Neurons.

The current paper analyzes the development of the male and female rat cerebellum exposed to hydroxyurea (HU) (300 or 600 mg/kg) as embryo and collected at postnatal day 90. Our study reveals that the administration of this drug compromises neither the cytoarchitecture of the cerebellar cortex nor deep nuclei (DCN). However, in comparison with the saline group, we observed that several cerebellar parameters were lower in the HU injected groups. These parameters included area of the cerebellum, cerebellar cortex length, molecular layer area, Purkinje cell number, granule cell counts, internal granular layer, white matter and cerebellar nuclei areas, and number of deep cerebellar nuclei neurons. These features were larger in the rats injected with saline, smaller in those exposed to 300 mg/kg of HU and smallest in the group receiving 600 mg/kg of this agent. No sex differences in the effect of the HU were observed. In addition, we infer the neurogenetic timetables and the neurogenetic gradients of PCs and DCN neurons in rats exposed to either saline or HU as embryos. For this purpose, 5-bromo-2'-deoxyuridine was injected into pregnant rats previously administered with saline or HU. This thymidine analog was administered following a progressively delayed cumulative labeling method. The data presented here show that systematic differences exist in the pattern of neurogenesis and in the spatial location of cerebellar neurons between rats injected with saline or HU. No sex differences in the effect of the HU were observed. These findings have implications for the administration of this compound to women in gestation as the effects of HU on the development of the cerebellum might persist throughout their offsprings' life.

Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally variable and not related to their neurogenetic timetables.

Generation and vulnerability of deep cerebellar nuclei neurons in the weaver condition along the anteroposterior and mediolateral axes.

Production and death of deep cerebellar nuclei (DCN) neurons were investigated in the weaver condition at appropriate anatomical levels throughout the mediolateral (medial, intermediate and lateral) and rostrocaudal (rostral, middle and caudal) axes of three DCN-cell groups: the fastigial, the interposed and the dentate nuclei. Current results have denoted that the deficit of DCN neurons is always more important in the homozygous weaver than in the heterozygous weaver mice. No loss of neurons was found in the dentate nucleus. In the mediolateral axis, an intranuclear gradient of depletion was observed in the mutant mice; in a given deep nucleus, neurodegeneration was more prominent in the medial pars than in lateral ones. In the rostrocaudal axis, on the other hand, when each deep nucleus was studied and compared as a whole, neuron loss was higher in the fastigial nucleus than in the interposed nucleus, which, in turn, was more important than in the dentate nucleus. These data suggest that, in the weaver condition, an internuclear gradient of neurodegeneration exists. Moreover, neurons located in rostral parts of a given nucleus appear to be more vulnerable than those settled in middle parts and these, in turn, are more than the caudal ones. These results seem to indicate the presence of an intranuclear gradient of depletion. Current autoradiographic results have revealed that, in the rostrocaudal axis, deep neurons are settled in the weaver cerebellum following three neurogenetic gradients. The first of these is internuclear; if each deep nucleus is analyzed and compared as a whole, the fastigial nucleus has more late-generated neurons than the interposed nucleus, and this, in turn, has more than the dentate nucleus. The second gradient is also internuclear; if the proportion of late-born neurons is compared throughout the rostral levels from each deep nucleus, it is observed that proportions increase from the fastigial to the dentate nucleus. A similar picture emerges when the middle and caudal regions are taken into account. The third gradient is intranuclear; in a given deep nucleus, the rostral region always presents more late-produced neurons than the middle region and these, in turn, more than in the caudal level.

Systematic differences in time of cerebellar-neuron origin derived from bromodeoxyuridine immunoperoxidase staining protocols and tritiated thymidine autoradiography: A comparative study.

As exogenous markers of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU) and tritiated thymidine ([(3)H]TdR) have revolutionized our ability to identify proliferating neuroblasts and follow their fate during the development of the central nervous system. The effect of the incorporation of these molecules into DNA on cell proliferation, migration and differentiation is frequently neglected (Duque and Rakic, 2011. J. Neurosci. 31, 15205-15217). By a progressively delayed cumulative labeling method, the current paper analyzes the development of the cerebellum in mice exposed to either BrdU or [(3)H]TdR as embryos and collected at postnatal day 90. We observed that, in comparison to the saline group, several parameters of the cerebellum such as length of the cerebellar cortex, the area of the molecular layer, Purkinje cell (PCs) number, the areas of the cerebellar nuclei, and the number of the deep cerebellar nuclei (DCN) neurons were lower in the BrdU injected group. No consequence of [(3)H]TdR administration was observed. On the other hand, we also studied whether immunohistochemical methods, including BrdU antibodies from different vendors (Sigma and Dako), partial DNA denaturation procedures and trypsin pretreatments, alter the neurogenetic timetables of PC and DCN neurons that resulted from analysis of these tissue specimens. Our analysis revealed that the generative programs of these macroneurons were unrelated to differences in the sensibility of BrdU antibodies but were dependent on the partial denaturation of DNA and trypsin digestion protocols. Finally, we also compare the generation and spatial distribution of PC and DCN neurons in mice exposed to either BrdU or [(3)H]TdR to assess whether the results obtained by these two markers are quantitatively similar. The data presented here show that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar neurons between mice injected with BrdU or [(3)H]TdR. These findings have implications for the interpretation of results obtained by both exogenous makers as an index of the production, migration and settling of neurons in the developing central nervous system.

Principal component and cluster analysis of morphological variables reveals multiple discrete sub-phenotypes in weaver mouse mutants.

The present study evaluates the usefulness of the principal component analysis-based cluster analysis in the categorization of several sub-phenotypes in the weaver mutant by using several morphological parameters from the cerebellar cortex of control, heterozygous (+/wv) and homozygous (wv/wv) weaver mice. The quantified parameters were length of the cerebellar cortex, area of the external granular layer, area of the molecular layer, number of the external granular layer cells (EGL), and number of Purkinje cells (PCs). The analysis indicated that at postnatal day 8, the genotype +/wv presented three sub-phenotypes tagged as +/wv (0), +/wv (1) and +/wv (2), whereas two sub-phenotypes designated as wv (0)/wv (1) and wv (0)/wv (2) were identified in the genotype wv/wv. The number of PCs for the genotype +/wv and the number of EGL cells for the genotype wv/wv were the variables that discriminated the best among sub-phenotypes. Each one of the sub-phenotypes showed specific abnormalities in the cytoarchitecture of the cerebellar cortex as well as in the foliar pattern. In particular, the wv (0)/wv (1) and wv (0)/wv (2) sub-phenotypes had the most altered cytoarchitectonics, followed by the +/wv (2) sub-phenotype and then by the +/wv (1) one. The sub-phenotype +/wv (0) was the less affected one. Apart from reporting for the first time the coexistence of several sub-phenotypes in the weaver mutant, our approach provides a new statistical tool that can be used to assess cerebellar morphology.

Regional differences in the vulnerability of substantia nigra dopaminergic neurons in weaver mice.

Vulnerability of midbrain dopaminergic (DA) neurons in the weaver mouse was studied at postnatal (P) days 8 and 90, in chosen coronal levels throughout the anteroposterior (AP) extent of the substantia nigra pars compacta (SNc). Wild-type (+/+) and homozygous weaver (wv/wv) mice used were the offspring of pregnant dams injected in several cases with tritiated thymidine on embryonic days 11-15. DA neurons were identified for their tyrosine hydroxylase immunoreactivity. Data reveal that at P8, the frequency of both +/+ and wv/wv late-generated DA cells increases from rostral to caudal SNc. No apparent DA-cell loss was observed at P8 in the mutant genotype, irrespective of the AP level considered. However, throughout the AP, there was a significant reduction in the number of these neurons at any level in 90-day-old weavers. Comparison of P8 and P90 +/+ SNc suggests that cell death is not a major aspect in the developmental regulation of normal DA neurons, although numerical cell depletion in the postnatal development of weaver SNc probably results from the amplification of a basal cell-death process, which affected all the coronal levels studied.

Purkinje cell age-distribution in fissures and in foliar crowns: a comparative study in the weaver cerebellum.

Generation and settling of Purkinje cells (PCs) are investigated in the weaver mouse cerebellum in order to determine possible relationships with the fissuration pattern. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from control and weaver offspring at postnatal (P) day 90. This makes it possible to assess the differential survival of neurons born at distinct embryonic times on the basis of the proportion of labeled cells located at the two foliar compartments: fissures and foliar crowns. Our data show that throughout the surface contour of the vermal lobes, generative programs of PCs were close between wild type and homozygous weaver. Similar data were found in the lobules of the lateral hemisphere. On the other hand, the loss of PCs in weaver cerebella can be related to foliar concavities or convexities depending on the vermal lobe or the hemispheric lobule studied. Lastly, we have obtained evidence that late-generated PCs of both normal and mutant mice were preferentially located in fissures. These quantitative relationships lead us to propose a model in which the final distribution of PCs through the vermal contour would be coupled to two factors: the cortical fissuration patterning and a "time-sequential effect" of weaver mutation.

Generation and survival of midbrain dopaminergic neurons in weaver mice.

Generation and survival of midbrain dopaminergic (DA) neurons were investigated using tyrosine hydroxylase (TH) immunocytochemistry combined with tritiated thymidine autoradiography at appropriate anatomical levels throughout the anteroposterior (A/P) axes of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). The wild-type (+/+) and homozygous weaver (wv/wv) mice used here were the offspring of pregnant dams injected with the radioactive precursor when the mesencephalic neurons were being produced (gestational days 11-15). Data reveal that, at postnatal day 90, depletion of TH-stained cells in the wv/wv presented an A/P pattern of increasing severity and, therefore, the DA cells located in posterior parts of the SNc or the VTA appear to be more vulnerable than the settled anterior neurons. When the time of neuron origin is inferred for each level of these cell groups, it is found that the neurogenesis span is similar for both experimental groups, although significant deficits in the frequency of wv/wv late-generated neurons were observed in any level considered. On the other hand, it has been found that TH-positive neurons were settled along the extent of the SNc and the VTA following precise and differential neurogenetic gradients. Thus, the acute rostrocaudal increase in the proportion of late-generated neurons detected in both+/+DA-cell groups is disturbed in the weaver homozygotes due to the indicated A/P depletion.

The weaver gene expression affects neuronal generation patterns depending on age and encephalic region.

Cell generation and survival are investigated in three different neuronal populations of weaver mice: Purkinje and fastigial neurons in the cerebellum, and dopaminergic neurons in the substantia nigra pars compacta. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from the control and weaver offspring at postnatal (P) day 8 and 90. This makes it possible to assess the differential survival of neurons that were born at distinct embryonic times on the basis of the proportion of labeled cells at two postnatal ages. When labeling profiles were measured at P8, the inferred time of origin was similar between +/+ and wv/wv genotypes for each neuronal population considered. The same occurred at P90 for Purkinje or fastigial neurons, but the labeling profiles of midbrain neurons were different between wild type and weaver homozygotes. There is already a substantial reduction in the number of Purkinje and fastigial cells at P8, but loss of dopaminergic neurons was only detected in 90-day-old weavers and, therefore, vulnerability is built into this midbrain neural system during its late postnatal development. Our results show that depletion of Purkinje and fastigial cells is random with respect to the time of their birth, whereas the weaver gene seems to be specifically targeting the late-generated dopaminergic neurons.