Journal: IBRO Neuroscience Reports

Article Title: Adolescent administration of Δ 9 -THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice

doi: 10.1016/j.ibneur.2021.09.005

Figure Lengend Snippet: M1R immunogold in the cytoplasm of small dendritic shafts receiving synaptic input from unlabeled (A–E) and CB1R immunoperoxidase labeled (B–F) axon terminals within the PL-PFC of adult male mice. These mice received 14 consecutive daily injections of vehicle (A,B black border) or ∆9-THC (C,E no border) through adolescence. Panel A shows, M1R immunogold (small arrows) in the cytoplasm and in contact with the perisynaptic plasma membrane contacted by an unlabeled terminal forming a symmetric synapse (block arrows) like the junction formed by a CB1-labeled terminal in B. Panels C-D and E-F respectively show unlabeled and CB1R-labeled terminals presynaptic to dendritic profiles with a mainly cytoplasmic distribution of M1R gold particles in the PL-PFC of mice that received ∆9-THC during adolescence. M1R-De = Immunogold-labeled dendrite; CB1R-Te = immunoperoxidase CB1R-labeled axon terminal; white block arrows = symmetric synapses; U-Ax = unlabeled axon; CB1-Ax = CB1R-labeled axon; curved black arrows = asymmetric excitatory-type synapses; small black arrows = cytoplasmic M1R gold particles; white arrowheads = perisynaptic plasmalemmal M1R immunogold; U-Te = unlabeled terminal; U-mvb = unlabeled multivesicular body; mt = mitochondrion = Scale bar = 500 nm.

Article Snippet: These sections were incubated for 24 h at room temperature in a solution containing both CB1R and M1R antibodies (Alomone Labs, Jerusalem, Israel Cat # AMR-001).

Techniques: Labeling, Blocking Assay

Journal: IBRO Neuroscience Reports

Article Title: Adolescent administration of Δ 9 -THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice

doi: 10.1016/j.ibneur.2021.09.005

Figure Lengend Snippet: Bar graphs showing a significant reduction in M1R immunogold in PL-PFC dendrites of adult mice receiving Δ9-THC (THC) as adolescents. As compared to vehicle (VEH) mice, animals receiving Δ9-THC had a lower mean density of M1R immunogold particles on the plasma membrane (A) and in the cytoplasm (B) of small, but not medium or large sized dendrites of adult mice chronically exposed to Δ9-THC during adolescence. In (C) the reduction in M1R density in small dendrites of Δ9-THC pretreated mice is statistically significant only when including the total of both plasmalemmal and cytoplasmic labeling ( p < 0.05; Welch’s correction for unequal variance). Values on top of the bars depict absolute number of dendrites in each category.

Article Snippet: These sections were incubated for 24 h at room temperature in a solution containing both CB1R and M1R antibodies (Alomone Labs, Jerusalem, Israel Cat # AMR-001).

Techniques: Labeling

Journal: IBRO Neuroscience Reports

Article Title: Adolescent administration of Δ 9 -THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice

doi: 10.1016/j.ibneur.2021.09.005

Figure Lengend Snippet: Separable locations of M1R immunogold and CB1R-immunoperoxidase labeling at axo-spinous synapses in the PL-PFC of adult male mice receiving vehicle (A) or Δ9-THC (B–D) as adolescents. In A, M1R immunogold is located on (arrowhead) and near the extrasynaptic plasmalemma of a small dendritic spine (M1-Sp) that receives excitatory-type input from an unlabeled axon terminal (U-Te). Panel B shows M1R immunogold in the cytoplasm near endomembranes (em) beneath an asymmetric synapse formed by an unlabeled axon terminal. M1R gold is seen in axon terminals forming asymmetric synapses with large dendritic spine heads containing M1R immunogold particles (M1-Sp in C) or without immunoreactivity (U-Sp in D). In the upper right corner of panel C, a CB1R peroxidase labeled terminal apposes an axospinous synapse between unlabeled profiles. Small arrows = cytoplasmic M1R immunogold; curved arrows = asymmetric synapses; U-Te = unlabeled axon terminal (U-Te). Scale bar = 500 nm.

Article Snippet: These sections were incubated for 24 h at room temperature in a solution containing both CB1R and M1R antibodies (Alomone Labs, Jerusalem, Israel Cat # AMR-001).

Techniques: Labeling

Journal: IBRO Neuroscience Reports

Article Title: Adolescent administration of Δ 9 -THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice

doi: 10.1016/j.ibneur.2021.09.005

Figure Lengend Snippet: M1R-labeled axon terminals forming either symmetric or asymmetric synapses with dendritic profiles defined by their expression of M1R in the PL-PFC of adult male mice pre-exposed to vehicle or Δ9-THC during adolescence.

Article Snippet: These sections were incubated for 24 h at room temperature in a solution containing both CB1R and M1R antibodies (Alomone Labs, Jerusalem, Israel Cat # AMR-001).

Techniques: Expressing

Journal: IBRO Neuroscience Reports

Article Title: Adolescent administration of Δ 9 -THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice

doi: 10.1016/j.ibneur.2021.09.005

Figure Lengend Snippet: Subcellular localization of M1R immunogold and CB1R-immunoperoxidase labeling at axo-dendritic synapses in the PL-PFC of adult mice receiving vehicle (A) or Δ9-THC (B–D) as adolescents. Panel A shows a dually labeled axon terminal (Du-Te) with one M1R gold particle on the presynaptic membrane of an axon terminal containing peroxidase immunoreactivity for CB1R and one large mitochondrion (mt). The symmetry (block arrow) of this inhibitory-type junction contrasts with the thickened asymmetric synapse (curved arrow) formed by an unlabeled terminal (U-Te) convergent on the same unlabeled dendritic profile (UDe). Panel B shows a similar convergent labeling of a single CB1R-immunoreactive terminal and an excitatory-type synapse on a dendrite (M1R-De) in which the M1R gold is aligned on outer membranes contacted by an unlabeled terminal. Cytoplasmic M1R immunogold is associated with a mitochondrion (mt) and on a multivesicular body (mvb) in B and C, respectively. In D, M1R immunogold is localized to endomembranes (em) near the plasma membrane in a dendritic profile (M1-De 1 ) and to the perisynaptic plasma membrane in a second dendrite (M1-De 2 ). One gold particle (arrowhead) opposes the membrane specialization of an asymmetric synapse formed by an unlabeled axon terminal. This dendrite also receives convergent input from an axon terminal containing CB1R peroxidase reaction product. Cytoplasmic M1R gold is shown by small black arrows in all images. Curved arrows = asymmetric synapses; white block arrow = symmetric synapses. Scale bar = 500 nm.

Article Snippet: These sections were incubated for 24 h at room temperature in a solution containing both CB1R and M1R antibodies (Alomone Labs, Jerusalem, Israel Cat # AMR-001).

Techniques: Labeling, Blocking Assay

Journal: IBRO Neuroscience Reports

Article Title: Adolescent administration of Δ 9 -THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice

doi: 10.1016/j.ibneur.2021.09.005

Figure Lengend Snippet: CB1R-terminals forming symmetric or asymmetric synapses with M1R-labeled or unlabeled dendritic profiles in the PL-PFC of adult mice that received vehicle or Δ9-THC during adolescence.

Article Snippet: These sections were incubated for 24 h at room temperature in a solution containing both CB1R and M1R antibodies (Alomone Labs, Jerusalem, Israel Cat # AMR-001).

Techniques:

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