María C. Santa-Cruz

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.

Analysis of steroidogenic pathway key transcripts in interrenal cells isolated by laser microdissection (LMD) in stressed rainbow trout

An assessment of the key transcripts expression of the steroidogenesis-related genes in rainbow trout subjected to either acute or chronic stress was performed in both interrenal cells and whole head kidney tissue. The analysis of interrenal cells was possible thanks to the use, for the first time in this specific type of cells, of the technique of laser microdissection (LMD) which allows to isolate specific cells and process them independently of other surrounding cells in the tissue. The results indicated that both acute and chronic stressors induced a significant up-regulation of the steroidogenesis-related genes with a higher but expected degree in the isolated cells. In addition, under acute stress a delay between cortisol levels and transcript expression was found. Under chronic stress a clear relation between plasma cortisol levels, mRNA transcription and interrenal tissue area was observed, since all parameters were concomitantly increased at day 5 after stress. Moreover results indicated that the LMD technique allowed ascertaining with more precision and accuracy whether and when the steroidogenesis-related genes were significantly expressed, disregarding the noise produced by other cells present in the head kidney. Results also showed a typical physiological response in plasma parameters and a positive relationship between plasma cortisol data and transcript abundance in isolated cells. The present results may help to better understand the mechanisms behind the interrenal response to stress challenges in fish.

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.

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 ([3H]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 [3H]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 [3H]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 [3H]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 [3H]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 +/wv0, +/wv1 and +/wv2, whereas two sub-phenotypes designated as wv0/wv1 and wv0/wv2 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 wv0/wv1 and wv0/wv2 sub-phenotypes had the most altered cytoarchitectonics, followed by the +/wv2 sub-phenotype and then by the +/wv1 one. The sub-phenotype +/wv0 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.

Progressive redistribution of alcohol dehydrogenase during vitellogenesis in Drosophila melanogaster: characterization of ADH-positive bodies in mature oocytes

SummaryThe use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S7), ADH appears to be located mainly in the nurse cells. From stage S9 onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S13–14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or β-spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.

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.

GIRK2 and neuronal pattern of generation and settling in homozygous weaver mice

Correspondence: Joaquin Marti Unidad de Citologia e Histologia, Facultad de Biociencias, Universitat Autonoma de Barcelona, 08193 Barcelona, Spain Tel +34 9 3581 1666 Fax +34 9 3581 3357 email joaquim.marti.clua@uab.es Abstract: G-protein-activated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Several GIRK channel subunits have been found in the central nervous system. The weaver mutation has been identified as a single base-pair substitution in the gene encoding for a GIRK channel subunit, GIRK2. The cerebellum and the mesencephalon are predominately affected in the homozygous weaver mouse (wv/wv). In this article, we review our main findings about the patterns of cell generation, survival, and settling of two neuronal types in the wv/wv: Purkinje cells in the cerebellar cortex and dopaminergic neurons in the ventral midbrain. Moreover, we examine if the time of neuron origin determines the degree of cell vulnerability to the lethal action of mutated GIRK2. The possible involvement of other GIRK channel subunits is also considered within the context of earlier and more recent studies in the field.