11/13/2022 0 Comments Blinkk bar los angeles![]() After hybridization, the sections were rinsed in 4× SSC, digested with 20 μg/ml RNase A at 37☌ for 30 min, and washed through descending concentrations of 1× SSC to 0.1× SSC at 60–70☌. The hybridization solution also contains 50% formamide, 10% dextran sulfate, 300 m m NaCl, 0.5 mg/ml yeast RNA, 10 μ m dithiothreitol, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin, 1 m m EDTA, and 10 m m Tris-HCl (pH 8.0). Then, the tissue sections were incubated overnight at 50☌ with 35S-labeled riboprobe (5 × 10 6 cpm/ml) prepared from a pGEM-4Z plasmid containing a 460-bp insert of rat BDNF cDNA clone ( Phillips et al. The sections were fixed with 4% p-formaldehyde in 0.1 m PBS for 30 min and pretreated with 0.25% acetic anhydride and 0.1 m triethanolamine for 10 min. The mutant and wild-type brain sections were mounted on the same slides and processed identically for optimal comparison. In brief, brains from both wild-type and mutant adult mice were excised after decapitation, frozen on dry ice, sliced on a cryostat at 16 μm, and mounted on subbed slides. The procedures for in situ hybridization were the same as in Qiao et al. The tissue was then sectioned at 40 μm and the slices were mounted onto subbed slides and stained with cresyl violet. The brains were removed and postfixed for at least 24 hr at 4☌. The adult mutant and wild-type mice were overdosed with sodium pentobarbital and transcardially perfused with 0.9% saline, followed by 10% buffered formalin. Unlike stargazer, the waggler mutant arises from C57BL/6 inbred strain, therefore allowing us to study potential roles of BDNF in cerebellar functions and learning with minimal interference from strain differences. 1991 Sweet 1993), waggler, also exhibited selective deficit in cerebellar granule cell BDNF expression without apparent abnormality in cerebellar cytoarchitecture. In the present study, we found that another mutant mouse allelic to stargazer ( Sweet et al. 1996), provided a feasible model to study the function of BDNF in the cerebellum ( Chen et al. A recently discovered spontaneous mutant mouse stargazer, with localized BDNF deficit in the cerebellum ( Qiao et al. But they are not as useful in behavioral studies because mutant mice have severe sensory deficits and die during early postnatal development ( Crowley et al. Various neurotrophin and tyrosine kinase (Trk)-receptor knockout mice provided animal models to study functions of neurotrophins in neuronal development and synaptic modulation. 1994, 1996), administrations of anti-NGF antibodies impair passive avoidance learning ( Ricceri et al. Although intraventricular nerve growth factor (NGF) infusion improves spatial memory in aged rats ( Markowska et al. Parallel with its roles in synaptic modulation, neurotrophins also affect behavioral learning. These results strongly suggest an acute function of neurotrophins in synaptic function in adult brain. 1996), and some of the deficits can be rescued by culturing the slices with BDNF ( Korte et al. Furthermore, null mutation of the BDNF gene impairs both basal synaptic transmission and long-term potentiation in the hippocampus ( Korte et al. 1996), whereas induction of long-term potentiation increases brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) expression in vitro ( Patterson et al. 1995) and long-term potentiation ( Figurov et al. Acute application of neurotrophins enhances synaptic transmission ( Lohof et al. In the adult brain, neurotrophins and synaptic transmission exhibit mutual regulation. During early development, neurotrophins promote neuronal proliferation, differentiation, maturation, and survival. Neurotrophins are a class of molecules that regulate both developing and adult nervous systems. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning. However, they were massively impaired in classic eye-blink conditioning. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. ![]()
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