Administration of 5-bromo-2'-deoxyuridine interferes with neuroblast proliferation and promotes apoptotic cell death in the rat cerebellar neuroepithelium.

The current study was conducted to assess whether a single administration of 5-bromo-2'-deoxyuridine (BrdU) interferes with cell proliferation and leads to the activation of apoptotic cellular events in the prenatal cerebellum. BrdU effects across a wide range of doses (25-300 µg/g b.w.) were analyzed using immunohistochemical and ultrastructural procedures. The pregnant rats were injected with BrdU at embryonic day 13, and their fetuses were sacrificed from 5 to 35 hr after exposure. The quantification of several parameters such as the density of mitotic figures, and BrdU and proliferating cell nuclear antigen (PCNA)-reactive cells showed that, in comparison with the saline injected rats, the administration of BrdU impairs the proliferative behavior of neuroepithelial cells. The above-mentioned parameters were significantly reduced in rats injected with 100 µg/g b.w. of BrdU. The reduction was more evident using 200 µg/g b.w. The most severe effects were found with 300 µg/g b.w. of BrdU. The present findings also revealed that high doses of BrdU lead to the activation of apoptotic cellular events as evidenced by both terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and immunohistochemistry for active caspase-3. In comparison with saline rats, many apoptotic cells were found in rats injected with 100 µg/g b.w. of BrdU. The number of dying cells increased with 200 µg/g b.w. The most important number of apoptotic cells were observed in animals injected with 300 µg/g b.w. of BrdU. Ultrastructural studies confirmed the presence of neuroblasts at different stages of apoptosis.

An immunocytochemical approach to the analysis of the cell division cycle in the rat cerebellar neuroepithelium.

Cerebellar neurons are generated from the rhombic lip and the neuroepithelium. In this study, we analyze the histogenesis of the cerebellar neuroepithelium in terms of cellular kinetics. The experimental animals are the offspring of pregnant dams injected with 5-bromo-2'-deoxyuridine (BrdU) on embryonic day 13. We infer the fraction of S-phase cells by examining a range of survival times after a single BrdU-exposure and a cumulative BrdU-labeling sequence, which allow for the derivation of cell-cycle parameters and phase durations. The current results indicate that the dose of BrdU employed (35 mg/kg) provides saturation S-phase labeling from at least 1 h after marker delivery. The duration of G2, mitotic phase, and G1 are 1.2, 0.5, and 6.9 h, respectively. The duration for the S-phase, growth fraction, and the whole cycle are obtained on the basis of two proliferative models, steady-state and exponential growth. Both models provided similar results. In conclusion, our results indicate that the steady-state and the cumulative S-phase labeling paradigms can be adopted to analyze cell cycle parameters in the cerebellar neuroepithelium. Current results can help in understanding the regulatory mechanisms of cerebellar histogenesis and the cell biological mechanisms of the proliferative cycle of the neuroepithelium.

Patterns of Apoptosis and Autophagy Activation After Hydroxyurea Exposure in the Rat Cerebellar External Granular Layer: an Immunoperoxidase and Ultrastructural Analysis.

The time courses of apoptosis and autophagy activation were investigated in neuroblasts of the cerebellar external granular layer (EGL) following the treatment with a single dose (2 mg/g) of hydroxyurea (HU), a cytotoxic agent. The rats were examined at postnatal day 9 and sacrificed at appropriate times ranging from 10 to 60 h after drug administration. We used the Feulgen method, the TUNEL assay, immunohistochemistry for active caspase-3, and LC3B and p62/SQSTM1 immunoperoxidase procedures. The resulting data indicated that the administration of HU leads to the activation of apoptotic cellular events that began to increase 10 h after HU exposure, peaked at 30 h, and decrease thereafter. It also showed that apoptosis was followed by autophagy activation. Interestingly, LC3B and p62/SQSTM1-stained cells, as well as mitotic cells, started to appear 20 h after the HU injection and their counts increased until 40 h. Afterwards, the values remained stable. The current results highlight an important role of the apoptotic and autophagic processes in the EGL after HU administration. Moreover, they provide a clue for studying the mechanism of chemoresistance triggered by proliferating cells exposed to anticancer agents.

Hydroxyurea Exposure and Development of the Cerebellar External Granular Layer: Effects on Granule Cell Precursors, Bergmann Glial and Microglial Cells.

The current paper presents a histological analysis of the cell death in the cerebellar external granular layer (EGL) following the treatment with a single dose (2 mg/g) of hydroxyurea (HU). The rats were examined at postnatal days (P) 5, 10, and 15, and sacrificed at appropriate times ranging from 6 to 48 h after treatment administration. Studies were done in each cortical lobe (anterior, central, posterior, and inferior). The quantification of several parameters, such as density of 5-bromo-2'-deoxyuridine, TUNEL, vimentin, and tomato lectin-stained cells, revealed that HU compromises the viability of EGL cells. Our results indicate that P10 is a time of high vulnerability to injury. We also show here that the anterior and central lobes are the cortical regions most susceptible to the action of the HU. Additionally, our data also indicate that from 6 to 24 h after HU-exposure is a time-window of high sensibility to this agent. On the other hand, our ultrastructural analysis confirmed that HU administration produces the activation of apoptotic cellular events in the EGL, resulting in a substantial number of dying cells. Different stages of apoptosis can be observed in all cortical lobes at all investigated postnatal ages and survival times. Moreover, we observed that dying neuroblasts were covered by laminar processes of Bergmann glia, and that these unipolar astrocytes presented cytological features of phagocytes engulfing apoptotic bodies and cell debris. The electron microscopy study also revealed the participation of ameboid microglial cells in the phagocytosis of apoptotic cells in the regions of the EGL with extensive cell death.

An Animal Model for Assessing the Effects of Hydroxyurea Exposure Suggests That the Administration of This Agent to Pregnant Women and Young Infants May Not Be as Safe as We Thought.

The cytostatic agent hydroxyurea (HU) has proven to be beneficial for a variety of conditions in the disciplines of oncology, hematology, infectious disease and dermatology. It disrupts the S phase of the cell cycle by inhibiting the ribonucleotide reductase enzyme, thus blocking the transformation of ribonucleotides into deoxyribonucleotides, a rate limiting step in DNA synthesis. HU is listed as an essential medicine by the World Health Organization. Several studies have indicated that HU is well tolerated and safe in pregnant women and very young pediatric patients. To our knowledge, only a few controlled studies on the adverse effects of HU therapy have been done in humans. Despite this, the prevalence of central nervous system abnormalities, including ischemic lesions and stenosis have been reported. This review will summarize and present the effects of HU exposure on the prenatal and perinatal development of the rat cerebellar cortex and deep cerebellar nuclei neurons. Our results call for the necessity to better understand HU effects and define the administration of this drug to gestating women and young pediatric patients.

Effects of Hydroxyurea Exposure on the Rat Cerebellar Neuroepithelium: an Immunohistochemical and Electron Microscopic Study Along the Anteroposterior and Mediolateral Axes.

We present a histological study of the cell death of cerebellar neuroepithelial neuroblasts following treatment with the cytotoxic agent hydroxyurea (HU) during the embryonic life. Pregnant rats were treated with a single dose of HU (300 mg/kg) at embryonic days 13, 14, or 15 of gestation, and their fetuses were studied from 5 to 35 h after treatment to elucidate the mechanisms of HU-induced fetotoxicity. Quantification of several parameters such as the density of pyknotic, mitotic, and PCNA-immunoreactive cells indicated that HU compromises the survival of the cerebellar neuroepithelium neuroblasts. On the other hand, our light and electron microscopic investigations during the course of prenatal development indicated that HU leads to two types of cell death: apoptosis and cells presenting cytoplasmic vacuolization, altered organelles, and a recognizable cell nucleus. Both modalities of cell death resulted in a substantial loss of cerebellar neuroepithelium cells. Current results suggest that HU exposure during gestation is toxic to the cerebellar neuroepithelium. Moreover, they allow to examine the mechanisms of HU-induced toxicity during the early development of the central nervous system. Our data also suggest that it is essential to avoid underestimating the adverse effects of HU when administered during early prenatal life.

Cell cycle analysis in the rat external granular layer evaluated by several bromodeoxyuridine immunoperoxidase staining protocols.

An important step in bromodeoxyuridine (BrdU) immunohistochemistry is the production of single-stranded DNA to make the incorporated BrdU accessible to the antibodies. This paper examines the effect of distinct DNA denaturation pretreatments (DNase I, sodium citrate buffer, endonuclease Eco RI and exonuclease III, and HCl hydrolysis) on detection of BrdU. We found that all the methods used in the partial denaturation of DNA combined good nuclear immunostaining with acceptable tissue integrity. We also observed that these immunohistochemical protocols revealed a spatial pattern in the distribution of DNA-synthesizing cells within the cerebellar external granular layer (EGL) of 10-day-old rats, allowing us to estimate the fraction of S-phase cells. Our results indicate that detection of BrdU-stained cells is affected by the distinct histological procedures used in such detection. Additionally, as the duration and phases of the cell cycle in EGL neuroblasts are estimated in accordance with BrdU detection, an effect on this detection can render the measurement of cell cycle inaccurate. The present work shows that DNase I and citrate buffer, at appropriate conditions, may be good alternatives for acid denaturation. However, they are less sensitive than autoradiographic techniques that use 3H-thymidine administration. Finally, current data reveal that short survival times after a single BrdU exposure do not seem to affect the cell cycle progression of the EGL neuroblasts.

Glioblastoma Multiforme and Adult Neurogenesis in the Ventricular-Subventricular Zone: A Review.

Brain cancers account for <1,5% of all new cancer cases reported in the United States each year. Due to their invasive and heterogeneous nature, in addition to their resistance to multimodal treatments, these tumors are usually fatal. Gliomas, and in particular high-grade astrocytomas such as glioblastoma multiforme (GBM), are the most common and lethal primary tumors of the central nervous system. The median survival of most patients is less than 1 year after application of multimodal therapies. The question is why are these cancers so injurious? And above all, how is it possible for a so carefully orchestrated area like the brain to develop such tumors? This brings us to the study of glioma stem cells, their specialized niches (perivascular and hypoxic), and the neurogenic phenomena that takes place within the adult ventricular-subventricular zone: a structure that lies at the intersection between brain development and gliomagenesis. J. Cell. Physiol. 232: 1596-1601, 2017. © 2016 Wiley Periodicals, Inc.

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.

A combined immunohistochemical and autoradiographic method to detect midbrain dopaminergic neurons and determine their time of origin.

The present paper describes an efficient tyrosine hydroxylase immunohistochemical method combined with [3H]thymidine autoradiography to identify, in the same tissue section, substantia nigra pars compacta dopaminergic neurons and quantitatively determine their neurogenetic timetables both in control animals and in homozygous weaver mice. The experimental animals were the offspring of pregnant dams injected with [3H]thymidine on embryonic days 11-12, 12-13, 13-14 and 14-15. Preservation of tyrosine hydroxylase immunoreactivity, as well as the establishment of the best conditions for the [3H]thymidine autoradiography, were attained after many trials in which fixatives, buffers, antibody dilution and conditions for photographic emulsion, developer and fixer were tested. The proposed combined technique reveals that the detection of tyrosine hydroxylase positive neurons is accurate, unambiguous and has a low interassay variability. Moreover, it did not influence posterior [3H]thymidine autoradiography, which was highly reproducible and presented very low background. The method described here allows us to demonstrate that the neurogenetic timetables of midbrain dopaminergic neurons were different between control and homozygous weaver mice in the mesencephalic area studied.

Regional differences in the Purkinje cells settled pattern: a comparative autoradiographic study in control and homozygous weaver mice.

To determine whether Purkinje cells located in the vermis and the lateral hemispheres of weaver mice homozygotes are distributed according to precise neurogenetic gradients, [3H]thymidine autoradiography was applied on sections of homozygous weaver mice and normal controls on postnatal day 90. The experimental animals were the offspring of pregnant dams injected with [3H]thymidine on embryonic days 11-12, 12-13, 13-14, and 14-15. The results indicate that, at the level of the vermis, neurogenetic gradients were similar for wild-type and homozygous weaver in each lobe studied of the cerebellar cortex. The same was found for the lobulus simplex and for the ansiform and paramedian lobules when the lateral hemisphere was considered. In the vermis of both experimental groups, the anterior and inferior lobes have more late-generated Purkinje cells than the central and posterior lobes, while in the lateral hemisphere, the lobulus simplex and the ansiform lobule present more early generated Purkinje cells than the paramedian lobule. In weaver homozygotes, the most important deficit of Purkinje cells, in the region of the vermis, was observed in the central lobe; depletion was less observable in the anterior lobe and least observable in the posterior and inferior lobes. In the lateral hemispheres, the most important loss of Purkinje cells was observed in the paramedian lobule, followed by the lobulus simplex. The ansiform lobule presented values that showed no statistical difference between control and homozygous weaver. When Purkinje cells were registered in the entire sections, no significant differences were observed between the two experimental groups. This was due to a considerable volume of the weaver homozygote cerebellar tissue, which has no counterpart in the control mice, compensating for the neuronal loss observed in the other studied areas of the lateral hemisphere.