Journal: PLoS ONE

Article Title: Muscarinic modulation of M and h currents in gerbil spherical bushy cells

doi: 10.1371/journal.pone.0226954

Figure Lengend Snippet: (A) Calretinin (CR) staining in a parasagittal slice of gerbil cochlear nucleus. The filled arrows point to strong calretinin staining corresponding to big auditory nerve fiber terminals, the endbulb of Held. Scale: 100 μm. (B) M1 muscarinic staining was found on all AVCN cell bodies. (C) Merged image of A and B. Filled arrows show M1-positive SBCs. Empty arrows show M1-positive cells which are not SBCs. (D) Digital magnification of the white square in C. Scale: 50 μm. (E) Calretinin staining in a parasagittal slice of gerbil cochlear nucleus in which endbulb of Held could be identified. Scale: 100 μm. (F) M2 muscarinic staining was mostly present in the anterior part of AVCN, around SBCs. Filled arrowheads indicate sparse but strong M2 staining close to cell bodies. (G) Merged image of E and F. (H) Digital magnification of the white square in G. Empty arrowheads point to M2-positive puncta on SBC cell bodies. Scale: 50 μm (magnification: 63x). D = dorsal; P = posterior; NR = nerve root.

Article Snippet: The primary antibodies used were: goat anti-calretinin antibody (1/500, Merck Millipore, Germany), rabbit anti-M1 muscarinic receptor (443–458) antibody (1/200, #AMR-010, RRID: AB_2340994, Alomone labs, Israel) and rabbit anti-M2 muscarinic receptor antibody (1/200, #AMR-002, RRID: AB_2039995, Alomone labs, Israel).

Techniques: Staining

Journal: PLoS ONE

Article Title: Muscarinic modulation of M and h currents in gerbil spherical bushy cells

doi: 10.1371/journal.pone.0226954

Figure Lengend Snippet: On one hand, acetylcholine (Ach) binds the M1/M3 muscarinic receptor which induces the activation of the phospholipase C (PLC) via the Gα q/11 protein. The activated PLC depletes the phosphatidylinositol biphosphate (PIP 2 ) into diacylglycerol (DAG) and inositol triphosphate (IP 3 ). IP 3 concentration increase releases calcium from the internal cell storage. Directly or indirectly, one or several of these 2 nd messengers trigger the closure of Kv7 channels thereby depolarizing the cell membrane. On the other hand, acetylcholine may bind the M2 muscarinic receptor inhibiting the adenylate cyclase (AC) via the Gα i protein which induces a reduction of cyclic AMP. Thus, for the same membrane potential, more HCN channels are closed inducing the hyperpolarization of the cell. Cell membrane and ion channels were designed by “ Servier Medical Art ” ( https://smart.servier.com ) provided by Les Laboratoires Servier ( https://servier.com/en ), licensed under Creative Commons Attribution 3.0 Unported License .

Article Snippet: The primary antibodies used were: goat anti-calretinin antibody (1/500, Merck Millipore, Germany), rabbit anti-M1 muscarinic receptor (443–458) antibody (1/200, #AMR-010, RRID: AB_2340994, Alomone labs, Israel) and rabbit anti-M2 muscarinic receptor antibody (1/200, #AMR-002, RRID: AB_2039995, Alomone labs, Israel).

Techniques: Activation Assay, Concentration Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility

doi: 10.1152/ajpgi.00155.2018

Figure Lengend Snippet: Primary antibodies used

Article Snippet: Fluorescent labeling was visualized by confocal imaging through the Plan-Apochromat 60X oil immersion objective (1.42 numerical aperture) of an inverted Olympus Fluoview FV-1000 microscope (Olympus, Center Valley, PA). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source Dilution Cat. No. Resource ID No. Chicken anti-GFAP Abcam, Cambridge, MA 1:1,000 AB-4674 AB_304558 Biotinylated mouse anti-human HuC/D Invitrogen, Carlsbad, CA 1:200 {"type":"entrez-nucleotide","attrs":{"text":"A21272","term_id":"514140","term_text":"A21272"}} A21272 AB_2535822 Rabbit anti-M1 muscarinic receptor Alomone, Jerusalem, Israel 1:200 AMR-010 AB_2340994 Rabbit anti-M2 muscarinic receptor Alomone 1:200 AMR-002 AB_2039995 Rabbit anti-M3 muscarinic receptor Alomone 1:200 AMR-006 AB_2039997 Rabbit anti-M4 muscarinic receptor Alomone 1:200 AMR-004 AB_11219338 Rabbit anti-M5 muscarinic receptor Alomone 1:200 AMR-005 AB_10658757 Open in a separate window GFAP, glial fibrillary acidic protein.

Techniques:

Journal: Brain and Behavior

Article Title: Most calbindin‐immunoreactive neurons, but few calretinin‐immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey

doi: 10.1002/brb3.1071

Figure Lengend Snippet: Antibody details

Article Snippet: Anti‐m1 muscarinic acetylcholine receptor , GST fusion protein corresponding to aa227‐353 of human m1 ACh receptor (GSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGESVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRK) , Alomone Labs (Jerusalem, Israel). Rabbit polyclonal. Catalog#AMR‐001 Lot#AN‐05 http://scicrunch.org/resolver/AB_2039993 , 1:1,000.

Techniques: Purification, Recombinant

Journal: Brain and Behavior

Article Title: Most calbindin‐immunoreactive neurons, but few calretinin‐immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey

doi: 10.1002/brb3.1071

Figure Lengend Snippet: Quantification methods. The figure (a) shows m1 acetylcholine receptor immunoreactivity. Cells marked with a circle met counting criteria and were counted. The figure (b) shows immunoreactivity for calbindin‐D28k. Within this population are two subgroups: cells with darkly stained somata (marked with squares) and cells with faintly stained somata (marked with arrows). Only darkly stained cells were quantified. The merged image in (c) shows cells that were counted in both single‐label images, and thus are counted as dual‐labeled cells. Scale bar = 25 µm (all panels)

Article Snippet: Anti‐m1 muscarinic acetylcholine receptor , GST fusion protein corresponding to aa227‐353 of human m1 ACh receptor (GSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGESVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRK) , Alomone Labs (Jerusalem, Israel). Rabbit polyclonal. Catalog#AMR‐001 Lot#AN‐05 http://scicrunch.org/resolver/AB_2039993 , 1:1,000.

Techniques: Staining, Labeling

Journal: Brain and Behavior

Article Title: Most calbindin‐immunoreactive neurons, but few calretinin‐immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey

doi: 10.1002/brb3.1071

Figure Lengend Snippet: Laminar distributions of cells immunoreactive for the m1 acetylcholine receptor (a), calbindin‐D28k (b), and calretinin (c). Layer boundaries are marked to the left of each panel. m1‐immunoreactive neurons are more uniformly distributed throughout the tissue than are CB ‐ and CR ‐immunoreactive neurons, which are mostly expressed in layers II and III . Scale bar = 50 µm (all panels)

Article Snippet: Anti‐m1 muscarinic acetylcholine receptor , GST fusion protein corresponding to aa227‐353 of human m1 ACh receptor (GSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGESVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRK) , Alomone Labs (Jerusalem, Israel). Rabbit polyclonal. Catalog#AMR‐001 Lot#AN‐05 http://scicrunch.org/resolver/AB_2039993 , 1:1,000.

Techniques:

Journal: Brain and Behavior

Article Title: Most calbindin‐immunoreactive neurons, but few calretinin‐immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey

doi: 10.1002/brb3.1071

Figure Lengend Snippet: The figure (a) shows the percentage of the calbindin‐D28k ( CB ) and calretinin ( CR ) immunoreactive populations that are also immunoreactive (ir) for the m1 acetylcholine receptor (m1 AC hR), collapsed across all cortical layers. 55% of CB ‐ir neurons express m1 AC hRs, while 10% of CR ‐ir neurons express m1 AC hRs. The figure (b) shows the same data (52% of CB ‐ir neurons and 11% of CR ‐ir neurons), but for layers II and III only. The figure (c) shows the percentage of cells immunoreactive for m1 AC hRs that express CB or CR collapsed across all cortical layers. 2% of m1‐ir neurons express CB , while 0.5% of m1‐ir cells express CR . The figure (d) shows the same data (4% of m1 AC hR‐ir neurons express CB , while 1% express CR ), but for layers II and III only. Bars indicate standard error, and asterisks indicate statistical significance ( p < 0.01)

Article Snippet: Anti‐m1 muscarinic acetylcholine receptor , GST fusion protein corresponding to aa227‐353 of human m1 ACh receptor (GSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGESVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRK) , Alomone Labs (Jerusalem, Israel). Rabbit polyclonal. Catalog#AMR‐001 Lot#AN‐05 http://scicrunch.org/resolver/AB_2039993 , 1:1,000.

Techniques:

Journal: Brain and Behavior

Article Title: Most calbindin‐immunoreactive neurons, but few calretinin‐immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey

doi: 10.1002/brb3.1071

Figure Lengend Snippet: Neurons immunoreactive for the m1 acetylcholine receptor in layer III . Arrows mark cells that met counting criteria and were quantified. These neurons are characterized by strong somatic labeling appearing intense along the perimeter of the cell body. Scale bar = 20 µm

Article Snippet: Anti‐m1 muscarinic acetylcholine receptor , GST fusion protein corresponding to aa227‐353 of human m1 ACh receptor (GSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGESVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRK) , Alomone Labs (Jerusalem, Israel). Rabbit polyclonal. Catalog#AMR‐001 Lot#AN‐05 http://scicrunch.org/resolver/AB_2039993 , 1:1,000.

Techniques: Labeling

Journal: Brain and Behavior

Article Title: Most calbindin‐immunoreactive neurons, but few calretinin‐immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey

doi: 10.1002/brb3.1071

Figure Lengend Snippet: Comparison of the m1 acetylcholine receptor (m1 AC hR) expression by populations immunoreactive for calretinin ( CR ), calbindin‐D28k ( CB ), and parvalbumin ( PV ) in primary visual area V1 (dark gray) and middle temporal area MT (light gray). In V1, 40% of CR ‐immunoreactive neurons, 60% of CB ‐immunoreactive neurons, and 80% of PV ‐immunoreactive neurons express m1 AC hR. Those numbers in MT are 10%, 55%, and 75%, respectively

Article Snippet: Anti‐m1 muscarinic acetylcholine receptor , GST fusion protein corresponding to aa227‐353 of human m1 ACh receptor (GSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGESVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRK) , Alomone Labs (Jerusalem, Israel). Rabbit polyclonal. Catalog#AMR‐001 Lot#AN‐05 http://scicrunch.org/resolver/AB_2039993 , 1:1,000.

Techniques: Expressing

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Primary antibodies

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Purification

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Mean soma size (in μ m) by cell-type

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques:

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Qualitative comparison of single-label immunoperoxidase reactivity for m1 ACh receptors in V1 (A), and the middle temporal visual area (MT) (B). Immunopositive somata are present in all cortical layers of both areas. Higher levels of neuropil immunoreactivity in area MT (B) make the immunopositive somata more difficult to identify at this magnification (10×) although some strongly labeled cell bodies are evident in layer 5 (see arrow, e.g.). The micrographs present images of the two areas from the same tissue section. Layer boundaries are indicated on the left of each panel. Imaged with a 10× objective. Scale bars = 100 μ m.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Labeling

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Qualitative detail of single-label immunoperoxidase reactivity for m1 ACh receptors, panels A and B) and parvalbumin (C and D) in visual areas V1 (A and C) and the middle temporal visual area (MT) (B and D). The micrograph in panel A shows m1 AChR-immunoreactivity in layer 5 of area V1. Panel B shows m1 AChR-immunoreactivity in layer 3 of MT. In both panels the characteristic stained cytoplasmic ring around an immunonegative nuclear region is evident (asterisks). When immunoreactive dendrites are evident, they are usually (arrowhead in A), but not always (arrowhead in B) visibly attached to a cell body. There was no axonal staining evident in either cortical area. Parvalbumin immunoreactivity (C and D) on the other hand, is evident in cell bodies, dendrites (arrowheads in C and D) and axons (arrows in D). Panel C is a micrograph captured in layer 2 of area V1. The parvalbumin immunoreactivity shown in Panel D is of layer 5 in MT. Scale bar = 20 μ m.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Staining

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Most parvalbumin (PV) neurons (panels A and D) in both V1 (top row, A–C) and the middle temporal visual area (MT) (bottom row, D–F) express m1 AChRs (panels B and E). These images are of layer 3 from areas V1 and MT. The images were taken from the same tissue section using a 40× objective. Roughly the same size area is shown (26,148 μ m 2 in V1 and 27,423 μ m 2 in MT), although as a result of the higher packing density in V1, this results in a larger number of neurons imaged. The impression given, particularly comparing panels B and E, is of a larger proportion of neurons immunoreactive for m1 AChRs in V1 (B) compared with MT (E), but the packing density makes qualitative comparisons misleading. Neuropil staining is similarly strong and punctate for PV (A) and weak for m1 AChRs (B) in both areas. Most PV neurons are immunoreactive for m1 AChRs. In fact in these images all three PV neurons in V1 (+, A, B) and the three PV neurons in MT (+, D, E) are immunoreactive for m1 AChRs (note white appearance of regions of the PV somata, indicative of co-localization, in panels C and F). Scale bar = 50 μ m.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Staining

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Most m1 AChR-immunoreactive neurons (panel B, magenta) in the middle temporal visual area (MT) are not immunoreactive for parvalbumin (PV; panel A, green). This image was captured in layer 5 of area MT using a 40× objective. There are seven PV neurons in the imaged field (+, A). Note that the cell body marked with a ∼ in panel A would not have been counted in this study as it does not have a clearly defined boundary within the imaging plane, although it is a PV neuron. Five of these seven PV neurons are also immunoreactive for the m1 AChR (+ in B and C). Two are m1-immunonegative (arrows in B). A striking feature of this image is the number of neurons that are immunoreactive for m1 AChRs (B, C), but not PV (A). Although these singly labeled m1-ir neurons occasionally appear pyramidal (*), it is generally not possible to determine cell morphology (or, therefore, cell class) from the immunoreactivity for m1 AChRs. The strongly fluorescent regions circled in B and C would not have been counted as they do not appear somatic in shape. These regions appear in only one channel and can thus be distinguished from the lipofuscin autofluorescence (∧ in A, B, and C) which is equally strong but appears in all channels. Scale bar = 100 μ m.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Imaging, Labeling

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Percentage of parvalbumin (PV)-immunoreactive neurons also immunoreactive for m1 acetylcholine receptors in V1 (top) and the middle temporal visual area (MT) (bottom)

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques:

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Quantification of m1 AChR expression by parvalbumin neurons. The graphs show the percentage of parvalbumin (PV) neurons encountered, by cortical layer, that were also immunoreactive for m1 AChRs in areas V1 (A) and the middle temporal visual area (MT) (B). Error bars represent SEM. Number of PV neurons in each graph is the same: V1 = 293 PV neurons, MT = 293 PV neurons.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Expressing

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Percentage of m1 acetylcholine receptors-expressing neurons also immunoreactive for parvalbumin in V1 (top) and middle temporal (MT) (bottom)

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques:

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: Quantification of the parvalbumin-immunoreactive population as a percentage of m1 AChR-expressing neurons. The graphs show the percentage of m1 AChR-expressing neurons encountered, by cortical layer, that were also immunoreactive for parvalbumin in areas V1 (A) and the middle temporal visual area (MT) (B). Error bars represent SEM. Number of m1 AChR-ir neurons: V1 = 520, MT = 1081.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Expressing

Journal: Brain and Behavior

Article Title: Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

doi: 10.1002/brb3.225

Figure Lengend Snippet: The distributions of soma sizes are similar between the populations of singly and dually labeled neurons in V1 (A) or the middle temporal visual area (MT) (B). In these histograms of soma sizes, measured along the long axis, it can be seen that in V1 (A) the distributions for singly-labeled parvalbumin (PV)- and m1 AChR-ir neurons, and for dually immunoreactive neurons are unimodal and centered around 12–14 microns. In MT these distributions are shifted to the right and somewhat skewed, with a long tail at the end representing soma sizes greater than 20 μ m. This tail of the distribution has a very small N , but all three groups (single PV-ir, single m1AChR-ir and dual PV/m1-ir) are represented in the population of neurons with large somata, offering no evidence for a bi-modal distribution of soma size in either cortical area. N for V1: m1 single = 52, PV single = 11, dual = 50 neurons. N for MT: m1 single = 52, PV single = 12, dual = 45 neurons.

Article Snippet: We detected m1 muscarinic ACh receptors (m1 AChRs) using a polyclonal antibody raised in rabbit against amino acids 227–353 of the intracellular loop i3 of the human m1 AChR, obtained from Alomone Labs (Jerusalem, Israel, catalog #AMR-001, lot # AN-05).

Techniques: Labeling