Neuroanatomical tracing combined with in situ hybridization: Analysis of gene expression patterns within brain circuits of interest

Conte-Perales, Lorena; Barroso‐Chinea, Pedro; Rico, Alberto J.; Gómez-Bautista, Virginia; López, Icíar P.; Roda, Elvira; Wouterlood, Floris G.; Lanciego, José L.

doi:10.1016/j.jneumeth.2009.11.021

Cited by 9 publications

(6 citation statements)

References 42 publications

(42 reference statements)

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“…23,24 As an example, the backfill of neurons with neuroanatomical tracers enabled the isolation of specific insect ( Periplaneta americana ) neurons; 22 distinct FMRFamide-related peptides were detected in these individual neurons using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry (MS). 25 …”

Section: Single Cell Metabolomics and Peptidomicsmentioning

confidence: 99%

Profiling metabolites and peptides in single cells

et al. 2011

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Metabolites and peptides play important roles in almost every aspect of cell function. Their intracellular levels and spatial localizations reflect the state of each cell and its relationship to its surrounding environment. Moreover, their levels and dynamics are indicative of normal or pathological cellular conditions. Bioanalytical technologies for microanalysis are able to qualitatively and quantitatively characterize subsets of peptides and metabolites from individual microorganism, plant and animal cells. Highlighted here are the established and evolving strategies for characterization of the metabolome and peptidome of single cells. Focused studies of the chemical composition of individual cells and their networks promise to provide a greater understanding of cellular fate, function, and homeostatic balance. Single cell bioanalytical microanalysis has also become increasingly valuable for examining cellular heterogeneity, particularly in the fields of neuroscience, stem cell biology, and developmental biology.

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Section: Single Cell Metabolomics and Peptidomicsmentioning

confidence: 99%

Profiling metabolites and peptides in single cells

et al. 2011

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“…The combination of ISH with retrograde tracing can offer important neural connection information, as mentioned in previous reports (Le Moine, 2003 ; Conte-Perales et al, 2010 ). To verify the practicability of SST probe in retrograde tracing, we injected Alexa 555–CTB into the SI (Figure 5A ) to investigate the co-expression of SST+, PKC-δ+, and CTB+ neurons in the CeL.…”

Section: Resultsmentioning

confidence: 76%

A PCR-Based Method for RNA Probes and Applications in Neuroscience

Hua

Liu

et al. 2018

Front. Neurosci.

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In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.

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“…To determine whether HB1.F3.BDNF cells can make neural connections with host cells after transplantation, a retrograde tracer FG (Fluorochrome) [26] was injected into the globus pallidus of the QA-lesioned hemisphere at 6 weeks after transplantation and the animals were sacrificed one week later. The ability of the trans-planted cells to establish a neural network with the host cells can be partly determined by the presence of retrograde transportation to the transplanted cells.…”

Section: Resultsmentioning

confidence: 99%

Intracerebral Transplantation of BDNF-overexpressing Human Neural Stem Cells (HB1.F3.BDNF) Promotes Migration, Differentiation and Functional Recovery in a Rodent Model of Huntington's Disease

et al. 2020

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Short Communication Huntington' s disease (HD) is a dominantly inherited neurodegenerative disorder caused by abnormally expanded CAG repeats in the huntingtin gene. The huntingtin gene mutation leads to the progressive degeneration of striatal GABAergic medium spiny neurons (MSN) and reduces the level of brain-derived neurotrophic factor (BDNF) in HD patient' s brain. BDNF is an essential neurotrophic factor for the cortico-striatal synaptic activity and the survival of GABAergic neurons. In this study, we transplanted BDNF-overexpressing human neural stem cells (HB1.F3.BDNF) into the contra-lateral side of unilateral quinolinic acid (QA)-lesioned striatum of HD rat model. The results of in vivo transplantation were monitored using various behavioral tests, 4.7 T animal magnetic resonance imaging (MRI) and immunohistochemical staining. We observed that the QA-lesioned rats receiving HB1.F3.BDNF cells exhibited significant behavioral improvements in the stepping, rotarod and apomorphine-induced rotation tests. Interestingly, contralaterally transplanted cells were migrated to the QA-lesioned striatum and the size of lateral ventricle was reduced. Histological analyses further revealed that the transplanted cells, which had migrated to the QA lesion site, were differentiated into the cells of GABAergic, MSN-type neurons expressing DARPP-32, and neural networks were established between the transplanted cells and the host brain, as revealed by retrograde tracing. Finally, there was a significant reduction of inflammatory response in HB1.F3.BDNF-transplanted HD animal model, compared with vehicle-transplanted group. Taken together, these results suggest that HB1.F3.BDNF can be an effective therapeutic strategy to treat HD patients in the future.

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Neuroanatomical tracing combined with in situ hybridization: Analysis of gene expression patterns within brain circuits of interest

Cited by 9 publications

References 42 publications

Profiling metabolites and peptides in single cells

Profiling metabolites and peptides in single cells

A PCR-Based Method for RNA Probes and Applications in Neuroscience

Intracerebral Transplantation of BDNF-overexpressing Human Neural Stem Cells (HB1.F3.BDNF) Promotes Migration, Differentiation and Functional Recovery in a Rodent Model of Huntington's Disease

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