Multispectral Imaging and Automated Laser Capture Microdissection of Human Cortical Neurons: A Quantitative Study of CXCR4 Expression

Pitcher, Jonathan; Würth, Roberto; Shimizu, Saori; Meucci, Olimpia

doi:10.1007/978-1-62703-426-5_3

Cited by 4 publications

(6 citation statements)

References 3 publications

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“…Immunohistochemistry was performed as previously reported, with modifications (Pitcher et al, 2013). Paraffin blocks containing mPFC were sectioned at 5-µm thickness on a microtome.…”

Section: Methodsmentioning

confidence: 99%

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Morphine-Induced Modulation of Endolysosomal Iron Mediates Upregulation of Ferritin Heavy Chain in Cortical Neurons

Nash

Tarn

Irollo

et al. 2019

eNeuro

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HIV-associated neurocognitive disorders (HAND) remain prevalent and are aggravated by µ-opioid use. We have previously shown that morphine and other µ-opioids may contribute to HAND by inhibiting the homeostatic and neuroprotective chemokine receptor CXCR4 in cortical neurons, and this novel mechanism depends on upregulation of the protein ferritin heavy chain (FHC). Here, we examined the cellular events and potential mechanisms involved in morphine-mediated FHC upregulation using rat cortical neurons of either sex in vitro and in vivo. Morphine dose dependently increased FHC protein levels in primary neurons through µ-opioid receptor (µOR) and Gαi-protein signaling. Cytoplasmic FHC levels were significantly elevated, but nuclear FHC levels and FHC gene expression were unchanged. Morphine-treated rats also displayed increased FHC levels in layer 2/3 neurons of the prefrontal cortex. Importantly, both in vitro and in vivo FHC upregulation was accompanied by loss of mature dendritic spines, which was also dependent on µOR and Gαi-protein signaling. Moreover, morphine upregulated ferritin light chain (FLC), a component of the ferritin iron storage complex, suggesting that morphine altered neuronal iron metabolism. Indeed, prior to FHC upregulation, morphine increased cytoplasmic labile iron levels as a function of decreased endolysosomal iron. In line with this, chelation of endolysosomal iron (but not extracellular iron) blocked morphine-induced FHC upregulation and dendritic spine reduction, whereas iron overloading mimicked the effect of morphine on FHC and dendritic spines. Overall, these data demonstrate that iron mediates morphine-induced FHC upregulation and consequent dendritic spine deficits and implicate endolysosomal iron efflux to the cytoplasm in these effects.

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“…Immunohistochemistry was performed as previously reported, with modifications (Pitcher et al, 2013). Paraffin blocks containing mPFC were sectioned at 5-µm thickness on a microtome.…”

Section: Methodsmentioning

confidence: 99%

“…Imaging and analysis was performed as previously described (Pitcher et al, 2013), with modifications. Briefly, fluorescent microscopy coupled with multispectral image analysis was used to identify individual neurons immunostained for NeuN (Cell Signaling Technology 24307, 1:400) within layer 2/3 of the mPFC prelimbic region.…”

Section: Methodsmentioning

confidence: 99%

Morphine-Induced Modulation of Endolysosomal Iron Mediates Upregulation of Ferritin Heavy Chain in Cortical Neurons

Nash

Tarn

Irollo

et al. 2019

eNeuro

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“…Postmortem frontal cortex tissue of both humans and rhesus macaques was formalin fixed and paraffin embedded for immuno histo chemistry analyses. The dual staining immuno histochemistry protocol was previously described in greater detail (72). Tissue was sequentially dual-stained with the neuronal marker MAP2 (Chemicon, 1:250) and the protein of interest: FHC (Abcam, 1:50), pCXCR4 (Abcam, 1:50), or CXCR4 (1:100).…”

Section: Methodsmentioning

confidence: 99%

“…Imaging and analysis of dual-stained human and rhesus macaque cortex was previously described in detail (72). Briefly, brightfield microscopy coupled with multispectral image analysis was performed to identify individual MAP2 + neurons within the cortex and measure the absolute OD of the given antigen of interest (FHC, CXCR4, or pCXCR4) within each individual MAP2 + neuron.…”

Section: Methodsmentioning

confidence: 99%

Neuronal ferritin heavy chain and drug abuse affect HIV-associated cognitive dysfunction

Pitcher¹,

Abt²,

Myers³

et al. 2014

J. Clin. Invest.

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Interaction of the chemokine CXCL12 with its receptor CXCR4 promotes neuronal function and survival during embryonic development and throughout adulthood. Previous studies indicated that μ-opioid agonists specifically elevate neuronal levels of the protein ferritin heavy chain (FHC), which negatively regulates CXCR4 signaling and affects the neuroprotective function of the CXCL12/CXCR4 axis. Here, we determined that CXCL12/CXCR4 activity increased dendritic spine density, and also examined FHC expression and CXCR4 status in opiate abusers and patients with HIV-associated neurocognitive disorders (HAND), which is typically exacerbated by illicit drug use. Drug abusers and HIV patients with HAND had increased levels of FHC, which correlated with reduced CXCR4 activation, within cortical neurons. We confirmed these findings in a nonhuman primate model of SIV infection with morphine administration. Transfection of a CXCR4-expressing human cell line with an iron-deficient FHC mutant confirmed that increased FHC expression deregulated CXCR4 signaling and that this function of FHC was independent of iron binding. Furthermore, examination of morphine-treated rodents and isolated neurons expressing FHC shRNA revealed that FHC contributed to morphine-induced dendritic spine loss. Together, these data implicate FHC-dependent deregulation of CXCL12/CXCR4 as a contributing factor to cognitive dysfunction in neuroAIDS.

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“…For example, Fends et al [ 38 ] demonstrated a rapid immunostaining technique for fresh frozen sections using tissues from neoplasmic lymph nodes, the breast, and the prostate, in combination with LCM, which allowed the recovery of high-quality mRNA. As to paraffin embedded human cortical neurons, Pitcher et al [ 37 ] reported an effective immunohistochemistry protocol for quantitative analysis of protein and RNA expressions using LCM (PALMRobo).…”

Section: Discussionmentioning

confidence: 99%

Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

et al. 2015

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BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1222-9) contains supplementary material, which is available to authorized users.

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Multispectral Imaging and Automated Laser Capture Microdissection of Human Cortical Neurons: A Quantitative Study of CXCR4 Expression

Cited by 4 publications

References 3 publications

Morphine-Induced Modulation of Endolysosomal Iron Mediates Upregulation of Ferritin Heavy Chain in Cortical Neurons

Morphine-Induced Modulation of Endolysosomal Iron Mediates Upregulation of Ferritin Heavy Chain in Cortical Neurons

Neuronal ferritin heavy chain and drug abuse affect HIV-associated cognitive dysfunction

Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

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