Effects of sleep on wake-induced c-fos expression

We investigated the effects of sleep on wake-induced c-fos expression in the cerebral cortex of rats and c-fos-lacZ transgenic mice. In the cortex of rats, the levels of c-Fos, detected both by immunocytochemistry and Western blot, remained high during 6 or 12 hr of enforced wakefulness but declined rapidly (within 1 hr) with increasing time of recovery sleep. Similarly, in the transgenic mice in which lacZ expression is driven from the c-fos promoter, beta-galactosidase activity was high after enforced wakefulness and declined with increasing amounts of sleep. These results suggest that the decrease in c-Fos protein in cortical neurons during sleep may be attributable to cessation of c-fos expression, activation of a process that degrades the wake-induced c-Fos, or both.     

Temporal and spatial specificity of PDGF alpha receptor promoter in transgenic mice

Aberrant expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) has been linked to developmental abnormalities in vertebrate models, and has been implicated in multiple disease states in humans. To identify cis-acting regulatory elements that dictate expression of this receptor, we generated transgenic mice bearing the reporter gene beta-galactosidase (lacZ) under the control of a 6-kb promoter sequence. Expression of lacZ was monitored throughout embryonic development, with special focus on nervous tissue, skeleton, and several organ systems wherein PDGF alpha R expression is thought to play a pivotal role. In several independent transgenic mouse strains, lacZ expression recapitulated predominant features of PDGF alpha R gene expression during mouse development. These results demonstrate that critical tissue-specific regulatory elements for PDGF alpha R expression are located within a 6-kb upstream region of the PDGF alpha R gene.     

Analysis of a multi-mutant herpes simplex virus type 1 for gene transfer into sympathetic preganglionic neurons and a comparison to adenovirus vectors

A non-replicating triple-mutant herpes simplex virus (14H delta 3vhsZ) expressing the bacterial marker enzyme beta-galactosidase, was assessed for neurotropism and cytopathic effects as a vector for gene transfer into differentiated phaeochromocytoma 12 cells in vitro and into spinal sympathetic neurons in vivo. In the in vivo study, the 14H delta 3vhsZ was injected into the adrenal gland of hamsters. For comparison, an evaluation of two adenovirus vectors, AdCA17lacZ and AdCA36lacZ, was performed. Infection of the differentiated phaeochromocytoma 12 cells by 14H delta 3vhsZ resulted in intense beta-galactosidase staining in 80-90% of the cells without changes in cell morphology, detected by light microscopy, after a period of four days. No cytoskeletal disruption was detected by immunocytochemistry for the neurofilament protein and no apoptosis was demonstrated by the Hoescht stain for nuclear chromatin in virus-infected cells in comparison to mock-infected control cells. Twoto three days after adrenal inoculation with 14H delta 3vhsZ, beta-galactosidase was detected in 240 preganglionic neurons per hamster (n = 8), a number equal to about 25% of the population of targeted neurons. The beta-galactosidase reaction product extended throughout the normal kite-shaped neuronal somata and extensive dendritic arbour. The number decreased to 120 by five days (n = 3) and to two by eight days (n = 4). This decrease was presumably due to loss of expression of the marker gene and not to cell death because, at eight days, the number of sympathetic pregnanglionic neurons in the nucleus intermediolateralis, pars principalis, that were immunoreactive for the neurotransmitter enzyme choline acetyltransferase, and demonstrated nicotinamide adenine dinucleotide phosphate-diaphorase activity, were the same on the infected left side of the cord as on the uninfected right side. Inflammatory cells surrounded some of the infected neurons at five days but by eight days the infiltrate was reduced. Infection of differentiated phaeochromocytoma 12 cells by AdCA17lacZ and AdCA36lacZ also resulted in marker gene expression in a large proportion of the cells (80-90%) in the absence of cytopathic effects. In contrast, four days after adrenal injection of AdCA17lacZ or AdCA36lacZ (n = 5 for each) only an average of three preganglionic neurons per hamster expressed beta-galactosidase activity, despite clear adrenal infection. AdCA17lacZ and AdCA36lacZ both produced light patches of staining confined to the neuronal soma. These neurons had normal morphology but sometimes were surrounded by an inflammatory infiltrate. In conclusion, the non-replicating herpes simplex virus, 14H delta 3vhsZ, had minimal cytotoxic effects in neurons, in vitro or in vivo, and was efficiently transported from the adrenal gland to infect many sympathoadrenal pregnanglionic neurons. In contrast, very few neurons demonstrated beta-galactosidase activity after injection into the adrenal gland of AdCA17lacZ and AdCA36lacZ. Therefore, 14H delta 3vhsZ is a more suitable vector than either of the adenovirus vectors tested for eliciting short-term changes in preganglionic neuron gene expression.     

Simultaneous detection of beta-galactosidase activity and surface antigen expression in viable haematopoietic cells

The quantitation of intracellular beta-galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG) in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe beta-galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express beta-galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express beta-galactosidase. The intracellular fluorescence reflecting beta-galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, beta-galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells.     

Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene

The platelet-derived growth factor alpha-receptor (PDGFR-alpha) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5'-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR-alpha mRNA, while they failed to specify reporter expression in PDGFR-alpha expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.     

A 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.     

Olfactory epithelial organotypic slice cultures: a useful tool for investigating olfactory neural development

An in vitro slice culture was established for investigating olfactory neural development. The olfactory epithelium was dissected from embryonic day 13 rats; 400 microns slices were cultured for 5 days in serum-free medium on Millicell-CM membranes coated with different substrates. The slices were grown in the absence of their appropriate target, the olfactory bulb, or CNS derived glia. The cultures mimic many features of in vivo development. Cells in the olfactory epithelium slices differentiate into neurons that express olfactory marker protein (OMP). OMP-positive cells have the characteristic morphology of olfactory receptor neurons: a short dendrite and a single thin axon. The slices support robust axon outgrowth. In single-label experiments, many axons expressed neural specific tubulin, growth-associated protein 43 and OMP. Axons appeared to grow equally well on membranes coated with type I rat tail collagen, laminin or fibronectin. The cultures exhibit organotypic polarity with an apical side rich in olfactory neurons and a basal side supporting axon outgrowth. Numerous cells migrate out of the slices, of which a small minority was identified as neurons based on the expression of neural specific tubulin and HuD, a nuclear antigen, expressed exclusively in differentiated neurons. Most of the migrating cells, however, were positive for glial fibrillary acidic protein and S-100, indicating that they are differentiated glia. A subpopulation of these glial cells also expressed low-affinity nerve growth factor receptors, indicating that they are olfactory Schwann cells. Both migrating neurons and glia were frequently associated with axons growing out of the slice. In some cases, axons extended in advance of migrating cells. This suggests that olfactory receptor neurons in organotypic cultures require neither a pre-established glial/neuronal cellular terrain nor any target tissue for successful axon outgrowth. Organotypic olfactory epithelial slice cultures may be useful for investigating cellular and molecular mechanisms that regulate early olfactory development and function.