Journal: Journal of Neuroinflammation

Article Title: Selective vulnerability of the aging cholinergic system to amyloid pathology revealed by induced APP overexpression

doi: 10.1186/s12974-025-03682-2

Figure Lengend Snippet: Selective vulnerability of the aged mouse cholinergic system to induced mutant APP expression. a Whole-brain RNA sequencing identified genes with a main effect of mutant APP expression, a main effect of age, and/or a significant age × APP interaction (FDR < 0.05). Samples used for differential gene expression analyses included 2 male and 2 female 18mo controls, 2 male and 2 female APP 6 → 18mo, 2 male and 1 female 24mo controls, and 2 male and 2 female APP 12 → 24mo. Volcano plots showing differentially expressed genes driven by ( b ) mutant APP expression, ( c ) age, and ( d ) an age × APP interaction in the 24-month-old mice (APP 12 → 24mo vs. 24-month-old control). e Correlation analysis of APP effect sizes on gene expression between 24-month-old and 18-month-old animals. f Overview of age and APP-related changes in brain cell lineage marker genes. mRNA levels of ( g ) Chat , ( h ) Slc5a7 , and ( i ) Slc18a3 were measured by qPCR in 18mo control (4 males and 4 females), APP 6 → 18mo (4 males and 4 females), 24mo control (4 males and 3–4 females), and APP 12 → 24mo (4 males and 3–4 females). j Representative western blot images of cholinergic marker proteins. Protein levels of ( k ) ChAT and ( l ) SLC5A7 were specifically reduced in APP-induced 24-month-old mice. Sample size corresponds to 5 male and 5 female 18mo controls, 5 male and 5–6 female APP 6 → 18mo, 4–5 male and 5 female 24mo controls, and 4–5 male and 6 female APP 12 → 24mo. Individual data points and group means ± S.E.M. are presented. ** p < 0.01, **** p < 0.0001

Article Snippet: Gene expression of Chat (Mm01221882_m1), Slc5a7 (Mm00452075_m1), and Slc18a3 (Mm00491465_s1) was quantified using TaqMan Gene Expression Assays (Applied Biosystems, Darmstadt, Germany) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Darmstadt, Germany).

Techniques: Mutagenesis, Expressing, RNA Sequencing, Gene Expression, Control, Marker, Western Blot

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: Primary Antibodies Used

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Sequencing, Clone Assay

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: Neuronal distribution of M2 and/or VAchT immunolabeled cellular profiles in the rat mesopontine PPT/LDT

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Immunolabeling, Labeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: M2 immunolabeling in cholinergic and non-cholinergic neuronal cell bodies. A: A somatodendritic profile (M2-som) identified by Golgi complex cisterns (G) shows some gold particles for M2 (black straight arrows) associated to tubulovesicles (tv) or endoplasmic reticulum (RER) within the cytoplasm, or also attached to the plasma membrane. One M2-immunogold particle is also observed in a small unmyelinated axon in the near neuropil (M2-a). Dense immunoperoxidase precipitate por VAchT (white straight arrows) rims small synaptic vesicles (ssv) within a cholinergic axon terminal (VAchT-t). B: An M2-immunoreactive soma (M2-som) shows prominent M2-immunogold particles (black straight arrows) on membranes of tubulovesicles and on the plasma membrane. A dendrite (M2-d) showing prominent gold particles for M2 (black straight arrows) is seen in the adjoining neuropil, where two axon terminals (VAchT-t1,2) exhibit immunoperoxidase reaction product for VAchT. C: Intense VAchT-immunoperoxidase deposits (white straight arrows) are present on cisterns of the Golgi complex (G) and endomembranes within the cytoplasm in a soma (VAchT+M2-som) showing also M2-immunogold particles (black straight arrows) mainly on the plasma membrane. A M2-labeled dendrite (M2-d) showing immunogold particles for M2 (black straight arrows) is seen in the surrounding neuropil. D: A somatodendritic profile (VAchT+M2-som) showing major cytoplasmic distribution for M2-immunogold particles (black straight arrows) also displays more restricted dense VAchT-immunoperoxidase product (white straight arrows) mainly on Golgi cisterns or some adjacent endomembranes. Asterisks, glial profiles. Abbreviation: N, nucleus. Scale bar = 0.5 μm in A-D.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Immunolabeling, Labeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: Numbers of Asymmetric and Symmetric axodendritic synapses having VAchT- and/or M2R- immunoreactivity at pre- and/or post-synaptic sites within the rat mesopontine PPT and LDT nuclei.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques:

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: M2-immunoreactivity in dendrites without VAchT. A: Abundant M2-immunogold particles (black straight arrows) are nearly exclusively localized on the plasma membrane of a dendrite (M2-d) making a symmetric synapse (white curved arrow) with an unlabeled terminal (ut1) and appositional contacts (white arrowheads) with other unlabeled terminals (ut2,3). B: A transversely-sectioned large dendrite (M2-d1) exhibiting mainly cytoplasmic M2-immunogold particles (black straight arrows) receives multiple contacts from unlabeled terminals (ut1-3) whereas a small nearby dendrite (M2-d2) showing more obvious plasmalemmal M2-immunogold particles (black straight arrows) unveils no evident contact in the plane of section. Intense immunoperoxidase labeling for VAchT is seen in an adjoining axon terminal (VAchT-t). C: Prominent plasmalemmal localization of M2-immunogold particles (black straight arrows) in a dendrite (M2-d) that establishes an apparent symmetric contact (white curved arrow) with an axon terminal (M2-t) having also conspicuous plasmalemmal M2-immunogold (black straight arrows). The inset in the lower left corner shows an enlargement of the contact area illustrating localization of M2-immunogold particle to tubulovesicles attached to the postsynaptic membrane. VAchT-immunoperoxidase product is observed in some small cholinergic axons localized in the near immediacy (VAchT-a1) or more distant (VAchT-a2) to the M2-immunolabeled profiles. D: A longitudinally-sectioned dendrite (M2-d2) showing M2-immunogold (black straight arrows) in the cytoplasm forms extensive contact showing multiple synaptic junctions, most of them with unambiguous symmetric morphology (white curved arrow), with an axon terminal (M2-t) containing M2-immunogold particles, some of which are attached to the presynaptic side of the synapse. The M2-d2 also received a convergent asymmetric synapse (black curved arrow) from an unlabeled terminal (ut). M2-immunogold (black straight arrows) is also seen mainly associated to tubulovesicles (tv) within a nearby larger dendrite (M2-d1) contacting both M2-t and M2-d2. Scale bars = 0.5 μm in A-D.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Labeling, Immunolabeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: Bar graph summarizing the mean±s.e. immunogold densities (number of gold particles per profile) in M2R-single and VAchT+M2R-dual large (>1μm) or small (<1μm) dendrites within the PPT/LDT. Mean densities were calculated based on the numbers obtained from 1345 VTA dendrites (407 large and 938 small) taken from ultrathin sections from 18 Vibratome sections in 4 rats (2874 total profiles) processed for dual labeling. *p<0.05, Fisher test for cytoplasmic versus plasmalemmal subcellular localization.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Labeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: M2-immunolabeling in dendrites with VAchT. A: A dually labeled large dendrite (VAchT+M2-d) containing M2-immunogold particles (black straight arrows) mostly localized within the cytoplasm attached to vesicular endomembranes (tv) or to a multivesicular body (mvb) and VAchT-immunoperoxidase labeling (white straight arrows) also condensed especially to tv or mvb has large glial ensheathing (asterisks) but also receives input from unlabeled terminals (ut1,2), one of which is clearly asymmetric (black curved arrow). B: A dendrite (M2-d1) showing both VAchT-immunoperoxidase (white straight arrow) and M2-immunogold particles (black straight arrows) makes an asymmetric synapse (black curved arrow) with an unlabeled axon terminal (ut1) and a quite more symmetric synapse (white curved arrow) with another unlabeled axon terminal (ut2). Prominent plasmalemmal M2-immunogold particles (black straight arrows) are seen in a near dendrite (M2-d2) devoid of VAchT but receiving also input from unlabeled terminals (ut3,4). VAchT-immunoperoxidase is also detected in some small unmyelinated axons (VAchT-a1,2) in the surrounding area. C and D: Serial sections depicting a dual-labeled dendrite (VAchT+M2-d) showing both VAchT-immunoperoxidase (white straight arrows) and M2-immunogold particles (black straight arrows) receives an asymmetric synapse from an axon terminal lightly-labeled for VAchT (VAchT-t). The VAchT+M2-d is close to a longitudinally-sectioned M2-immunogold labeled dendrite (M2-d1), but intervening astrocytic leaflet keeps apart from apposition each other profile. Several M2-immugold particles (black straight arrows) are seen in some dendrites (M2-d2-4) in one and/or the other of the adjacent serial sections. Abbreviations: asterisks, glial processes. Scale bars = 0.5 μm in A-D.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Immunolabeling, Labeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: VAchT-labeled terminals interactions with M2-labeled profiles. A: Immunoperoxidase reaction product for VAchT rims small synaptic vesicles within a terminal that establishes a symmetric synapse (white curved arrow) onto an M2-immunogold (black straight arrows) labeled dendrite (M2-d). B: A transversely-sectioned dendrite (M2-d) showing intracytoplasmic M2-immunogold particles receives convergent input from a VAchT-immunoperoxidase labeled terminal (VAchT-t) and an axon terminal (M2-t) containing numerous M2-immunogold particles. The appositional contact areas of M2-d with both axon terminals (VAchT-t and M2-t) are marked with white arrowheads. C: A small VAchT-immunoperoxidase labeled terminal (VAchT-t) contacts an astrocytic profile (M2-g1) containing two M2-immunogold particles (black straight arrows). The M2-g1 profile makes a gap junction with another glial astrocytic profile (M2-g2) that also partially contacts the VAcht-t and shows one M2-immunogold particle in the plane of section. D: Axon terminal (VAchT-t1) containing VAchT-immunoperoxidase product contacts another terminal (M2-t) showing plasmalemmal M2-immunogold particles (black straight arrows) that makes a symmetric synapse (white curved arrow) with a longitudinally-sectioned M2-labeled dendrite (M2-d). Two smaller intensely VAchT-labeled terminals (VAchT-t2,3) and a VAchT-labeled small unmyelinated axon (VAchT-a) are seen in the neighboring neuropil. terminal. Scale bars = 0.5 μm in A-D.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Labeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: M2-immunolabeling in axon terminals with VAchT. A: Axon terminal containing VAchT-immunoperoxidase mainly on membranes of small synaptic vesicles (VAchT+M2-t) also shows M2-immunogold particles (black straight arrows) and apposes a large dendrite (M2-d) containing intracytoplasmic M2-immunogold. An adjacent axon terminal showing plasmalemmal M2-immunogold makes a symmetric contact (white straight arrow) with an unlabeled dendrite (ud) that receives convergent asymmetric input (black curved arrow) from an unlabeled terminal (ut). B: A dually labeled axon terminal (VAchT+M2-t) showing both VAchT-immunoperoxidase and plasmalemmal M2-immunogold particles (black straight arrows) makes a symmetric synapse onto an unlabeled dendrite (ud) receiving convergent asymmetric synaptic input from an unlabeled terminal (ut). A nearby dendrite (M2-d) shows also M2-immunogold labeling on the plasma membrane and tubulovesicular (tv) endomembranes. Scale bars = 0.5 μm in A-B.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Immunolabeling, Labeling

Journal: The Journal of comparative neurology

Article Title: Electron Microscopic Localization of M2-muscarinic receptors in Cholinergic and Non-Cholinergic Neurons of the Laterodorsal Tegmental and Pedunculopontine Nuclei of the Rat Mesopontine Tegmentum

doi: 10.1002/cne.24010

Figure Lengend Snippet: Schematic diagram showing the primary distributions of M2 muscarinic receptors (M2Rs, stars) in neuronal profiles and glial profiles (g) that express (gray filling) or not (white filling) the vesicular acetylcholine transporter (VAchT, black circles) in the rat mesopontine PPT/LDT complex. The M2R is located in a non-VAchT soma (A) whose dendrites receive synaptic inputs from M2R-labeled, VAchT-labeled and/or unlabeled terminals. In these neurons and in VAchT-labeled neurons (B), the M2R is localized mainly to plasma membranes of dendrites, but is also associated with cytoplasmic membranes in both somata and dendrites. These dendrites receive many symmetric (inhibitory-type) and asymmetric (excitatory-type) synapses from terminals that do not contain either M2R or VAchT, both of which are separately located in nearby axonal and glial processes.

Article Snippet: The immunolabeling was further shown to be specific for the antigenic VAchT peptide by absence of immunoreactivity in sections of brain tissue processed for immunolabeling after prior adsorption with cognate peptide but not with the carrier protein ( Arvidsson et al., 1997 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Immunogen Manufacturer Dilution M2R Synthetic fusion protein containing glutathione S-transferase fused to a part of the i3 intracellular loop of human M2R (residues 225-356) Alomone Labs Ltd (AMR-002 [lot AN-08]) RRID: AB_2039995Rabbit polyclonal 1:100 (gold) 1:2000 (peroxidase) VAchT 20 residue C-terminal synthetic peptide sequence corresponding to amino acids 511-530 of the cloned rat VAchT ImmunoStar (cat. 24286) RRID: AB_572269 Goat polyclonal 1:16000 (peroxidase) 1:3000 (gold) Open in a separate window VAchT, vesicular acetylcholine transporter; M2R, M2 muscarinic receptor. caption a8 Primary Antibodies Used The M2R immunoreactivity was detected with a rabbit polyclonal antiserum (product number AMR-002, Lot AN-08, RRID: AB_2039995, Alomone Labs Ltd, Jerusalem, Israel) ( ).

Techniques: Labeling

Journal: The Journal of Neuroscience

Article Title: Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception

doi: 10.1523/JNEUROSCI.1537-15.2015

Figure Lengend Snippet: Differentially expressed genes in the posterior insular cortex of oxaliplatin-treated rats compared with controls using TLDA a

Article Snippet: Rn00581454_s1 , Slc18a3 , Solute carrier family 18 (vesicular acetylcholine), member 3 (VAChT) , 0.38 , 0.401.

Techniques:

Journal: Biochemical pharmacology

Article Title: CDP-choline modulates cholinergic signaling and gut microbiota to alleviate DSS-induced inflammatory bowel disease.

doi: 10.1016/j.bcp.2023.115845

Figure Lengend Snippet: Fig. 3. Effect of CDP-choline on ACh synthesis-related proteins in colonic tissue of mice. (A) The representative image of ChT1, ChAT, VAChT, AChE, TNF-α, and IL-6 expression among the three groups. Histogram of the relative expressions of ChT1 (B), ChAT (C), VAChT (D), AChE (E), TNF-α (F), and IL-6 (G), respectively (n = 6). (H) Histogram of ChAT activity in the colon among the three groups measured by ELISA (n = 8). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were sealed (20 °C, 12 min) in protein-free rapid blocking buffer (EpiZyme, PS108P, China) and incubated overnight at 4 °C in the following primary antibody: β-Actin (1:1000, Cell Signaling Technology, #3700, USA), Occludin (1:1000, Cell Signaling Technology, #91131, USA), High-affinity choline transporter (SLC5A7/ChT1 1:1000, ThermoFisher, PA5-42485, USA), Choline acetyltransferase (ChAT, 1:1000, Cell Signaling Technology, #27269, USA), Acetylcholinesterase (AChE, 1:1500, Abcam, ab183591, UK), Vesicular acetylcholine transporter (SLC18A3/VAChT, 1:1000, Boster Bio, A04891, China), α7 nicotinic acetylcholine receptor (CHRNA7/α7 nAChR, 1:1000, Proteintech, 21379–1-AP, China), Tumor necrosis factor-α (TNF-α, 1:1000, Cell Signaling Technology, #11948, USA), Interleukin 6 (IL-6, 1:1000, HUABIO, R1412-2, China) and ZO-1 (1:1500, Bioswamp, PAB36669, China).

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay