scholarly journals Various transgenic mouse lines to study proopiomelanocortin cells in the brain stem label disparate populations of GABAergic and glutamatergic neurons

2018 ◽  
Vol 315 (1) ◽  
pp. R144-R152 ◽  
Author(s):  
Andrew R. Rau ◽  
Alexander R. Hughes ◽  
Shane T. Hentges
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Glutamatergic Neurons ◽  
Pomc Mrna ◽  
Low Levels ◽  
Green Fluorescent ◽  
Transgenic Tools ◽  
The Brain ◽  
Mouse Lines ◽  
Transmitter Phenotype

Products of the proopiomelanocortin (POMC) prohormone regulate aspects of analgesia, reward, and energy balance; thus, the neurons that produce POMC in the hypothalamus have received considerable attention. However, there are also cells in the nucleus of the solitary tract (NTS) that transcribe Pomc, although low levels of Pomc mRNA and relative lack of POMC peptide products in the adult mouse NTS have hindered the study of these cells. Therefore, studies of NTS POMC cells have largely relied on transgenic mouse lines. Here, we set out to determine the amino acid (AA) transmitter phenotype of NTS POMC neurons by using Pomc-Gfp transgenic mice to identify POMC cells. We found that cells expressing the green fluorescent protein (GFP) represent a mix of GABAergic and glutamatergic cells as indicated by Gad2 and vesicular Glut2 ( vGlut2) mRNA expression, respectively. We then examined the AA phenotype of POMC cells labeled by a Pomc-Cre transgene and found that these are also a mix of GABAergic and glutamatergic cells. However, the NTS cells labeled by the Gfp- and Cre-containing transgenes represented distinct populations of cells in three different Pomc-Cre mouse lines. Consistent with previous work, we were unable to reliably detect Pomc mRNA in the NTS despite clear expression in the hypothalamus. Thus, it was not possible to determine which transgenic tool most accurately identifies NTS cells that may express Pomc or release POMC peptides, although the results indicate the transgenic tools for study of these NTS neurons can label disparate populations of cells with varied AA phenotypes.

Morphological and Electrophysiological Properties of GABAergic and Non-GABAergic Cells in the Deep Cerebellar Nuclei

2007 ◽  
Vol 97 (1) ◽  
pp. 901-911 ◽  
Author(s):  
Marylka Uusisaari ◽  
Kunihiko Obata ◽  
Thomas Knöpfel
Keyword(s):  
Fluorescent Protein ◽  
Cerebellar Nuclei ◽  
Spike Rate ◽  
Deep Cerebellar Nuclei ◽  
Homogeneous Population ◽  
Electrophysiological Properties ◽  
Glutamatergic Neurons ◽  
Green Fluorescent ◽  
The Brain ◽  
High Firing

The deep cerebellar nuclei (DCN) integrate inputs from the brain stem, the inferior olive, and the spinal cord with Purkinje cell output from cerebellar cortex and provide the major output of the cerebellum. Despite their crucial function in motor control and learning, the various populations of neurons in the DCN are poorly defined and characterized. Importantly, differences in electrophysiological properties between glutamatergic and GABAergic cells of the DCN have been largely elusive. Here, we used glutamate decarboxylase (GAD) 67-green fluorescent protein (GFP) knock-in mice to unambiguously identify GABAergic (GAD-positive) and non-GABAergic (GAD-negative, most likely glutamatergic) neurons of the DCN. Morphological analysis of DCN neurons patch-clamped with biocytin-containing electrodes revealed a significant overlap in the distributions of the soma sizes of GAD-positive and GAD-negative cells. Compared with GAD-negative DCN neurons, GAD-positive DCN neurons fire broader action potentials, display stronger frequency accommodation, and do not reach as high firing frequencies during depolarizing current injections. Furthermore, GAD-positive cells display slower spontaneous firing rates and have a more shallow frequency-to-current relationship than the GAD-negative cells but exhibit a longer-lasting rebound depolarization and associated spiking after a transient hyperpolarization. In contrast to the rather homogeneous population of GAD-positive cells, the GAD-negative cells were found to consist of two distinct populations as defined by cell size and electrophysiological features. We conclude that GABAergic DCN neurons are specialized to convey phasic spike rate information, whereas tonic spike rate is more faithfully relayed by the large non-GABAergic cells.

scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Nuclear localization of angiotensinogen in astrocytes

2005 ◽  
Vol 288 (2) ◽  
pp. R539-R546 ◽  
Author(s):  
Mikhiela Sherrod ◽  
Xuebo Liu ◽  
Xiaoji Zhang ◽  
Curt D. Sigmund
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Endocrine Function ◽  
Astrocytoma Cell ◽  
Protein Encoding ◽  
Localization Signal ◽  
Green Fluorescent ◽  
The Brain

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.

scholarly journals Iba1+/NG2+ Macrophage-Like Cells Expressing a Variety of Neuroprotective Factors Ameliorate Ischemic Damage of the Brain

2009 ◽  
Vol 30 (3) ◽  
pp. 603-615 ◽  
Author(s):  
Anna Smirkin ◽  
Hiroaki Matsumoto ◽  
Hisaaki Takahashi ◽  
Akihiro Inoue ◽  
Masahiko Tagawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescent Protein ◽  
Artery Occlusion ◽  
Brain Lesions ◽  
Ischemic Lesion ◽  
Ischemic Brain ◽  
Green Fluorescent ◽  
High Level ◽  
Cerebral Artery Occlusion ◽  
The Brain

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1+/NG2+Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.

PU.1 determines the self-renewal capacity of erythroid progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3615-3623 ◽  
Author(s):  
Jonathan Back ◽  
Andrée Dierich ◽  
Corinne Bronn ◽  
Philippe Kastner ◽  
Susan Chan
Keyword(s):  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Self Renewal ◽  
Adult Mice ◽  
Erythroid Progenitor Cells ◽  
Low Levels ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

Abstract PU.1 is a hematopoietic-specific transcriptional activator that is absolutely required for the differentiation of B lymphocytes and myeloid-lineage cells. Although PU.1 is also expressed by early erythroid progenitor cells, its role in erythropoiesis, if any, is unknown. To investigate the relevance of PU.1 in erythropoiesis, we produced a line of PU.1-deficient mice carrying a green fluorescent protein reporter at this locus. We report here that PU.1 is tightly regulated during differentiation—it is expressed at low levels in erythroid progenitor cells and down-regulated upon terminal differentiation. Strikingly, PU.1-deficient fetal erythroid progenitors lose their self-renewal capacity and undergo proliferation arrest, premature differentiation, and apoptosis. In adult mice lacking one PU.1 allele, similar defects are detected following stress-induced erythropoiesis. These studies identify PU.1 as a novel and critical regulator of erythropoiesis and highlight the versatility of this transcription factor in promoting or preventing differentiation depending on the hematopoietic lineage.

scholarly journals Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5626-5632 ◽  
Author(s):  
Tamar Alon ◽  
Ligang Zhou ◽  
Cristian A. Pérez ◽  
Alastair S. Garfield ◽  
Jeffrey M. Friedman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Brain Regions ◽  
Ingestive Behavior ◽  
Physiological Processes ◽  
Functional Relevance ◽  
Green Fluorescent ◽  
And Function ◽  
The Brain

Abstract CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

Sign in / Sign up

Export Citation Format

Share Document