Morphological identification and biochemical characterization of isolated brain cell nuclei from the developing rat cerebellum

Zagon, Ian S.; McLauglin, Patricia J.

doi:10.1016/0006-8993(79)90963-6

Cited by 23 publications

(5 citation statements)

References 37 publications

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“…In the rat, macroneurons such as the Purkinje cells proliferate prenatally, whereas the ontogeny of microneurons (e.g., internal granule cells) is largely confined to the first 3 postnatal weeks (Altman, 1972). Indeed, between birth and day 2 1, cell number increases from about 6 to 94 million (Zagon and McLaughlin, 1979). Thus, in this study, macroscopic, histological, and morphometric analyses of 2 1 -d-old rats subjected to temporary (1 mg/kg naltrexone) or continuous (50 mg/kg naltrexone) receptor blockade throughout the preweaning period were performed in order to evaluate cerebellar size, cell concentration and distribution, and cellular differentiation.…”

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confidence: 99%

Opioid antagonist (naltrexone) modulation of cerebellar development: histological and morphometric studies

Zagon¹,

Pj²

1986

J. Neurosci.

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The role of endogenous opioid systems in preweaning cerebellar development was explored in rats utilizing naltrexone, a potent opioid antagonist. Sprague-Dawley rats were given daily subcutaneous injections of either 1 or 50 mg/kg naltrexone to invoke a temporary or complete blockade, respectively, of opioid receptors throughout the first 3 weeks of postnatal life; animals injected with sterile water served as controls. At weaning (day 21), macroscopic, morphometric, and histological determinations were conducted. In general, 50 mg/kg naltrexone had a stimulatory action on cerebellar development, whereas 1 mg/kg naltrexone had an inhibitory influence. Both sexes were affected comparably. Limits to naltrexone's ability to modulate cerebellar ontogeny were noted, with more latitude existing toward growth enhancement than impairment. The temporal course of ontogeny was generally unaltered in naltrexone-treated rats. Rather, only events that transpired over this period were dramatically affected. The most notable effects in 1 mg/kg naltrexone rats were marked decreases in cerebellar areal dimensions, the content of internal granule neurons, and cellular and tissue differentiation. Characteristics of the 50 mg/kg naltrexone group included increases in cerebellar areal dimensions, neural cell number, content, and size, and structural changes consistent with acceleration in growth and differentiation. Naltrexone influenced both neurons and glia, but only neural cells still being generated during the first 21 d after birth were altered in regard to quantity. Previous evidence has shown the presence of peak levels of endogenous opioids and opioid receptors in the cerebellum during the first weeks of life, as well as demonstrating the presence of enkephalin immunoreactivity on germinative cerebellar cells during postnatal neurogenesis but not in adult counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

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confidence: 99%

Opioid antagonist (naltrexone) modulation of cerebellar development: histological and morphometric studies

Zagon¹,

Pj²

1986

J. Neurosci.

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“…This switch may represent a general mechanism of cellular differentiation since a global shift of mRNA sizes toward lower-molecular-weight species has been previously reported during cerebellar development [Soreq et al, 1982]. The second week of postnatal development is marked by cellular events that have been precisely described [Altman, 1972a[Altman, , 1972bZagon and McLaughlin, 1979]. A comparison of these events with the different levels of ·6 subunit mRNA expression shows that expression and splicing events that produce the 2.7-kb transcript are switched on when cells of the external granule layer are still dividing and some postmitotic granule cells are in the stage of elaborating parallel fibers and migrating in the molecular layer.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to most brain regions, the rat cerebellum undergoes profound morphological and biochemical transformations after birth [Altman, 1969;Zagon and McLaughlin, 1979]. Late cerebellar ontogeny is marked by large-scale neuronal migration, differentiation, synaptogenesis and myelination [Altman, 1969;Fifkova and Delay, 1982].…”

Section: Introductionmentioning

confidence: 99%

Transient Increase in Cerebellar Transcriptional Activity Precedes the Expression of GABA<sub>A</sub> Receptor α6 Subunit mRNA during Postnatal Maturation

et al. 1998

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The purpose of this study was to investigate the postnatal expression of GABA_A receptor α6 subunit genes in the context of cerebellar differentiation. We examined steady-state levels of GABA_A receptor α1 and α6 subunit mRNAs, polyadenylated (polyA⁺) mRNA and β-actin mRNA in7-, 14-, 21-, 28-, 35-, 49- and 120-day-old rats. Messenger RNA expression and splicing were evaluated in parallel using Northern blot analysis and in situ hybridization histochemistry. The expression of mature GABA_A receptor α6 subunit mRNA species (2.7 kb) was found 1 week after birth in cerebellar granule cells. Prior to stable expression of the mature α6 subunit gene, we detected large α6 subunit premessengers (3.8 and 3.5 kb) by Northern blot analysis. These premessenger species were detected in prenatal day (PND) 15 and neonatal rat cerebellum, when the mature α6 subunit mRNAs (2.7 kb) were not yet expressed. The maximal expression of mature α6 subunit mRNA species was observed at PND 21 when the peak level of cerebellar transcriptional activity was measured by polyA⁺ RNA levels. In contrast, β-actin mRNA expression was decreased at PND 21 compared to birth levels. These major transcriptional events take place during a period of about 1 week (between PND 14 and 21), immediately following the most active phase of cell division in the external granule layer and migration of granule cells to the internal granule cell layer. Comparison between the relative abundance of these genes shows that differential regulation of each gene occurs during postnatal development. The induction of GABA_A receptor α6 subunit gene expression is preceded by a reduction in β-actin mRNA levels and a transient increase in total transcriptional activity. The expression of α6 subunit mRNA is maintained at the PND 21 level through adulthood, but the α1 subunit mRNA levels decrease drastically within the following week (from PND 21 to 28). These results suggest that tissue-specific expression of the GABA_A receptor α6 subunit gene is correlated with a series of developmentally regulated morphologic and transcriptional events.

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“…Because the nuclei of some brain cells may be larger than 15 μm (Zagon and McLaughlin,1979; Gussoni et al,1996,1997), some of our 15‐μm sections inevitably contained regions of nuclei that may be missing the BAG plasmid DNA, causing no hybridization signal to appear in that particular nucleus (Gussoni et al,1996,1997). However, cutting larger sections would compromise FISH detection because multiple nuclei from multiple cells may be situated one on top of the other, resulting in false positives.…”

Section: Methodsmentioning

confidence: 99%

The dynamics of long‐term transgene expression in engrafted neural stem cells

Lee

Tsai

Park

et al. 2009

J of Comparative Neurology

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To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as 3) inserting redundant copies of the transgene into the prospective donor NSC (the preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and insured ex vivo prior to transplantation). The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages.

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Morphological identification and biochemical characterization of isolated brain cell nuclei from the developing rat cerebellum

Cited by 23 publications

References 37 publications

Opioid antagonist (naltrexone) modulation of cerebellar development: histological and morphometric studies

Opioid antagonist (naltrexone) modulation of cerebellar development: histological and morphometric studies

Transient Increase in Cerebellar Transcriptional Activity Precedes the Expression of GABA<sub>A</sub> Receptor α6 Subunit mRNA during Postnatal Maturation

The dynamics of long‐term transgene expression in engrafted neural stem cells

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