Neurogenesis-Dependent and Independent Effects of Fluoxetine in an Animal Model of Anxiety/Depression (original) (raw)
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Mice were anesthetized with ketamine and xylazine (100 mg/ml ketamine; 20 mg/ml xylazine), placed in a stereotaxic frame and exposed to cranial irradiation using a Siemens Stabilopan X-ray system operated at 300 kVp and 20 mA. Animals were protected with a lead shield that covered the entire body, but left unshielded a 3.22 X 11-mm treatment field above the hippocampus (interaural 3.00 to 0.00) exposed to X-Ray. Dosimetry was done using a Capintec Model PR06G electrometer ionization chamber and Kodak Readypack Radiographic XV films. The corrected dose rate was approximately 1.8 Gy per min at a source to skin distance of 30 cm. The procedure lasted 2 min and 47 sec, delivering a total of 5 Gy. Three 5 Gy doses were delivered on days 1, 4 and 8.
Synapse, 1988
Proteolysis of brain spectrin (fodrin), a major compo-were incubated overnight with either Abl (1:lOO) or nent of the neuronal cytoskeleton, has been proposed as AB2 (1:lO) in 1% FCS in TBS with 0.1% Triton-X 100. an event underlying both plasticity (Lynch and Baudry, Sections were then washed, blocked again, incubated 1984) and degeneration in neurons. with 0.3% horseradish peroxidase-conjugated goat anti-It has recently been reported, based on immunocyto-rabbit IgG, washed, reacted with 0.06% 4-chloro-1-naphchemical evidence, that dendrites lack a form of brain tho1 with 0.01% hydrogen peroxide, washed again, and spectrin present in axons and neuronal cell bodies (Ried-finally coverslipped as in . For ease erer et al., 1986). This would suggest a simple means of of processing, two variations were introduced to method distinguishing between pre-and postsynaptic loci of pro-1: glass slides were used instead of coverslips for mountteolytic activation using immunoblot analysis of stable ing of cryostat sections and the avidin-biotin complex spectrin breakdown products. However, we demonstrate (ABC) method (Vector Labs, Burlingame, CA) was used here that the previously reported lack of the brain spec-for visualization of the antibody-antigen complex; neitrin subtype in dendrites was very probably due to anti-ther of these altered the resulting patterns of immunogen washout during processing of sections for immu-reactivity (compare ,B to Riederer et al., 1986, nocytochemistry. Brain spectrin, as originally suggested ). In the second method, rats were perfused with , seems to be present through-PBS plus 1% sodium citrate, followed by buffered 4% out the cytoplasm of mammalian neurons.
The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by realtime qPCR using primers directed against b-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.
European Journal of Neuroscience, 2014
Biochemical analysis of central nervous system proteins and nucleic acids requires fresh-tissue homogenates, whereas immunohistochemistry usually is performed in sections prepared from perfusion-fixed tissue. Post-mortem immersion-fixation is possible, but largely impairs morphological preservation and protein antigenicity. Here, we present a simple, fast and versatile protocol allowing concurrent biochemical and immunohistochemical analysis, including pre-embedding immunoelectron microscopy, using tissue from the same animal. The protocol includes a brief transcardiac perfusion with ice-cold, oxygenated and glucosesupplemented artificial cerebrospinal fluid to maintain brain tissue alive, prior to isolation of regions of interest, followed by homogenisation for biochemistry or immersion-fixation for immunohistochemistry. We provide several examples demonstrating that this protocol allows optimal biochemical and morphological analysis, characterised with optimal sensitivity and preservation of tissue structure, along with a reduction of artefacts typically seen in perfusion-fixed tissue. This protocol should find widespread applications for combining analytical methods in tissue from the same animal, thereby reducing the number of mice required for a given experiment.
International Journal of Molecular Sciences
Recently, a population of “immature” neurons generated prenatally, retaining immaturity for long periods and finally integrating in adult circuits has been described in the cerebral cortex. Moreover, comparative studies revealed differences in occurrence/rate of different forms of neurogenic plasticity across mammals, the “immature” neurons prevailing in gyrencephalic species. To extend experimentation from laboratory mice to large-brained mammals, including humans, it is important to detect cell markers of neurogenic plasticity in brain tissues obtained from different procedures (e.g., post-mortem/intraoperative specimens vs. intracardiac perfusion). This variability overlaps with species-specific differences in antigen distribution or antibody species specificity, making it difficult for proper comparison. In this work, we detect the presence of doublecortin and Ki67 antigen, markers for neuronal immaturity and cell division, in six mammals characterized by widely different brain ...
Journal of Neuroscience Methods, 1995
Biotinyiated dextran amine (BDA) has proven to be an excellent anterograde tracer in adult mammalian brains, having some advantages over other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin. However, results are inferior when BDA is used in neonatal mammals. To improve the sensitivity and quality of BDA labeling in neonatal mammalian brains, the tetramethylbenzidine-sodium tungstate (TMB-ST) method for horseradish peroxidase (WRP) histochemistry was modified and used in BDA histochemistry. After BDA application to the visual cortex of neonatal rat and cat, contralateral and ipsilateral cortical and subcortical regions were examined for BDA-labeled axons and terminals. The modified BDA histochemistry produced corpus callosum (CC) axons in neonatal rat and cat that were heavily and continuously labeled, The distribution, trajectories, branching and termination of individual CC axons, and even possible axon-axon contacts, were clearly identified in exquisite detail, even at low magnification. The quality of BDA labeling in the ipsilateral lateral geniculate nucleus and superior colliculus was similar to that of the CC axonal labeling. These results indicate that the modified BDA histochemistry provides a very sensitive and reliable approach to revealing the detailed distribution and morphology of projecting axons and terminals in the developing mammalian nervous system.
BMC Research Notes, 2015
Background: The heterogeneity of the brain requires appropriate molecular biological approaches to account for its morphological complexity. Laser-assisted microdissection followed by transcript profiling by quantitative determination has been reported to be an optimal methodology. Nevertheless, not all brain regions can be identified easily without staining, restricting the accuracy and efficiency in sampling. The aim of the present study was to validate whether fixation and staining treatments are suitable for quantitative transcript expression analysis in laser microdissection (LMD) samples. Quantitative RT-PCR was used to determine the absolute transcript expression levels and profiles of samples obtained from the hippocampal dentate gyrus from fresh frozen mice brain sections that had been fixed with ethanol and stained with NeuroTrace. The results were compared with those obtained from unfixed and unstained samples. Results: We found that the quantitative relationship of transcript expression levels between various housekeeping genes and immediate early genes was preserved, although the preparation compromised the yield of the transcripts. In addition, histological and molecular integrities of the fixed and stained specimens were preserved for at least a week at room temperature. Based on the lobe specific profiles of transcripts in the anterior and posterior lobes of the pituitary, we confirmed that no cross-contamination on transcription expressions occurred as a result of the fixation and staining. Conclusions: We have provided detailed information of the procedures on ethanol fixation followed by NeuroTrace staining on the absolute quantitative RT-PCR analysis using microdissected fresh frozen mouse brain tissues. The present study demonstrated that quantitative transcript expression analysis can be conducted reliably on stained tissues. This method is suitable for applications in basic and clinical studies on particular transcript expressions in various regions of the brain.