Steroid Hormone Receptors · July 11, 2022

Annu Rev Neurosci

Annu Rev Neurosci. notable perforations, suggesting the involvement of these receptors in activity-dependent synaptic plasticity. DORs were more frequently detected than were MORs within axon terminals that formed either asymmetric synapses with spine heads or symmetric synapses with spine necks. Our results suggest that in striatal patches, DORs, often in cooperation with MORs, play a direct modulatory role in controlling the postsynaptic excitability of spines, whereas presynaptic neurotransmitter release onto spines is mainly influenced by DOR activation. In comparison with MOR, the prevalent association of DOR with cytoplasmic organelles that are involved in intracellular trafficking of cell surface proteins suggests major differences in availability of these receptors to extracellular opioids. A guinea pig polyclonal antiserum against a peptide corresponding to SDZ 220-581 Ammonium salt the extracellular N terminal [amino acids (aa) 34C47] of the mouse DOR (Immuno-Dynamics, Inc., La Jolla, CA) and a rabbit polyclonal antiserum raised against a synthetic peptide corresponding to the intracellular C terminal (aa 384C398) of the rat MOR1 (DiaSorin, Stillwater, MN) were used in this study. The high specificities for antisera against DOR or MOR have been shown previously (Arvidsson et al., 1995; Cheng et al., 1995). The selective cellular distributions of these antisera also have been demonstrated in the rat CPN (Wang and Pickel, 1998; Wang et al., 1999) and in other brain regions (Commons and Milner, 1996, 1997; Svingos et al., 1998). All procedures involving animals and their care were conducted in conformity with the National Institutes of Health and were approved by the Research Animal Resource Center at Weill Medical College of Cornell University. The methods used for fixation and dual immunolabeling were based on those described by Chan et al. (1990). Seven adult male Sprague Dawley rats (250C350 gm; Taconic, Germantown, NY) were anesthetized with sodium AFX1 pentobarbital (100 mg/kg, i.p.). The anesthetized animals were perfused through the ascending aorta with 10C15 ml of heparin saline (1000 SDZ 220-581 Ammonium salt U/ml), followed by 50 ml of 3.8% acrolein and 2% paraformaldehyde in 0.1 m phosphate buffer (PB), pH 7.4, and then by 200 ml of 2% paraformaldehyde. The brains were removed and dissected into 5-mm-thick coronal blocks. The SDZ 220-581 Ammonium salt blocks containing the CPN were post-fixed in 2% paraformaldehyde for an additional 30 min. Sections of 40 m thickness were cut on a Leica Vibratome VT1000 S (Leica Instruments GmbH, Nussloch, Germany) in chilled 0.1m PB. These sections were then incubated for 30 min in 1% sodium borohydride in 0.1 m PB to remove excess aldehydes and rinsed in 0.1 m PB until no bubbles emerged from the tissue. To enhance the penetration of immunoreagents, sections for electron microscopy were incubated for 15 min in a cryoprotectant (25% sucrose and 2.5% glycerol in 0.05 m PB), frozen rapidly in liquid Freon followed by liquid nitrogen, and thawed in PB at room temperature. The sections were incubated for 30 min in 0.5% bovine serum albumin (BSA) in 0.1 m Tris-buffered saline (TBS), pH 7.6, to reduce nonspecific staining and then processed for dual immunocytochemical labeling. For immunocytochemical localization of DOR and MOR, previously prepared sections through the CPN were processed for combined immunoperoxidase and immunogoldCsilver labeling before plastic embedding. All incubations were performed with continuous agitation. Because of known differences in sensitivity and resolution, immunogold and immunoperoxidase were used reversibly for DOR and MOR labeling in sections from all seven rats. Sections were incubated in primary antisera solutions for 1 d at room temperature and the consecutive day at 4C. The solutions were prepared in a 0.1% BSA and TBS solution containing (1) guinea pig polyclonal antiserum for DOR (1:1000 for immunoperoxidase; 1:500 for immunogold) and (2) rabbit polyclonal antiserum for MOR (1:12,000 for immunoperoxidase; 1:4000 for immunogold). After the primary antisera incubation, the sections were processed sequentially for immunoperoxidase and then for immunogold detection. For immunoperoxidase labeling, the sections were incubated for 30 min in biotinylated IgG (1:400 in TBS and 0.1% BSA), either goat anti-guinea pig (Vector Laboratories, Burlingame, CA) or donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA), and then in avidinCbiotinCperoxidase complex (Vectastain Elite kit; Vector Laboratories; 1:100 in TBS) for SDZ 220-581 Ammonium salt 30 min. The immunoperoxidase that was bound to the sections was visualized by.