Astressin 2b

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Astressin-2B is a selective corticotropin-releasing factor 2 (CRF2) receptor antagonist. It is a laboratory research tool used to study the role of CRF2 receptors in various biological processes.

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Astressin 2B (Ast2B,

10 protocols using astressin 2b

Pharmacological Manipulation of CRF2 Signaling

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Sprague-Dawley rats were purchased from Harlan Laboratories. CRF₂ homozygous KO mice (Stock number: 010842; Strain name: B6; 129-crhr2^tm1jsp/J) and WT mice (from the same colony) were bought from Jackson Laboratories. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of North Dakota (0702-2). All efforts were made to minimize suffering. CRF was purchased from American Peptide Company (Sunnyvale, CA). The following reagents were products of TOCRIS (Ellisville, MO): K41498, astressin 2B, NBI 27914, CP 154526, MDL 12330A, SQ 22536, forskolin, 3,7-dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dione (IBMX), KT 5720, Rp-cAMPS. The other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Kurada L., Yang C, & Lei S. (2014). Corticotropin-Releasing Factor Facilitates Epileptiform Activity in the Entorhinal Cortex: Roles of CRF2 Receptors and PKA Pathway. PLoS ONE, 9(2), e88109.

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Cocaine Addiction CRF Receptor Antagonism

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Cocaine HCl was provided by the National Institute on Drug Abuse (Research Triangle Park, NC), and prepared in sterile 0.9% saline solution for intravenous administration. CRF (rat/human; Sigma Aldrich), CP376395 (Tocris Bioscience) and Astressin-2B (Tocris Bioscience) were prepared in aCSF, and stored in 20µL aliquots at −20°C until use. CRF doses were selected based on previous in vivo studies in rats (Kalivas et al. 1987 (link); Blacktop et al., 2011 (link); Wanat et al. 2013 (link)). A lower dose of CRF (50ng) was initially used in order to characterize the dose-response relationship, however, because the 500ng dose of CRF was ineffective in modulating cocaine-taking behavior during the binge-access procedure, only the 50ng dose was further examined (i.e. Experiments 2 and 3). Doses of antagonists were determined based on previous studies conducted in our laboratory (Boyson et al. 2014 (link); Holly et al. 2015 (link); 2016a (link))

Leonard M.Z., DeBold J.F, & Miczek K.A. (2017). Escalated cocaine “binges” in rats: enduring effects of social defeat stress or intra-VTA CRF. Psychopharmacology, 234(18), 2823-2836.

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Establishing Alcohol Drinking Solution for Mice

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Alcohol for mouse drinking solution (20% v/v) was prepared using 95% ethyl alcohol (Pharmco Products Inc., Brookfield, CT) in tap water. DNQX, AP-5, CGP55845A, CRF peptide, and Astressin-2B were purchased from Tocris Bioscience (Ellisville, MO). SR-95531 (gabazine, GBZ; 100 μM) was purchased from Sigma-Aldrich. The CRF1 antagonist R121919 was supplied by Neurocrine Biosciences, Inc. (San Diego, CA). Stock solutions were prepared in ultra-pure water or DMSO (DNQX only), stored at 20°C, and diluted to final experimental concentration in aCSF on the day of testing. Doses for electrophysiology experiments were chosen based on their ex vivo effects in CeA neurons from prior reports (10 (link), 25 ). CP-154,526 was purchased from Tocris Bioscience and was selected for use in behavioral pharmacology experiments due to a robust literature describing its effects on alcohol drinking in mice (32 (link)–34 (link)).

Agoglia A., Zhu M., Quadir S., Bluitt M., Douglass E., Hanback T., Tella J., Ying R., Hodge C, & Herman M. (2021). Sex-specific plasticity in CRF regulation of inhibitory control in central amygdala CRF1 neurons after chronic voluntary alcohol drinking. Addiction biology, 27(1), e13067.

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Ethanol-Based CRF Antagonist Evaluation

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Ethanol (20% w/v) solutions were prepared in tap water from 95% ethyl alcohol (Pharmaco–AAPER, Brookfield, CT, USA). The selective CRFBP antagonist CRF_6–33 (0.125, 0.25 and 0.5 µg; Tocris Bioscience, Bristol, United Kingdom) and the CRF₂ antagonist Astressin2-B (A2B, 0.25 and 0.5 µg; Tocris Bioscience) were dissolved in artificial cerebrospinal fluid (aCSF).

Albrechet-Souza L., Hwa L.S., Han X., Zhang E.Y., DeBold J.F, & Miczek K.A. (2015). CRF binding protein and CRF2 receptors in the ventral tegmental area: modulation of ethanol binge drinking in C57BL/6J mice. Alcoholism, clinical and experimental research, 39(9), 1609-1618.

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CRHR2 Agonist and Antagonist Effects

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Cells were treated with the CRHR2 specific agonist Urocortin 2 (Bachem, Torrance, CA), the CRHR2 specific antagonist Astressin 2B (Tocris Bioscience, Bristol, UK) and an agonist antibody for Fas, anti-Fas (clone CH11) (Millipore, Temecula, CA) used either as single agents or in combination at the indicated concentrations of each experimental condition.

Pothoulakis C., Torre-Rojas M., Duran-Padilla M.A., Gevorkian J., Zoras O., Chrysos E., Chalkiadakis G, & Baritaki S. (2017). CRHR2/Ucn2 signaling is a novel regulator of miR-7/YY1/Fas circuitry contributing to reversal of colorectal cancer cell resistance to Fas-mediated apoptosis. International journal of cancer, 142(2), 334-346.

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Pharmacological Modulation of Addiction-Related Behaviors

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Methamphetamine hydrochloride (METH: 2mg/kg, IP; Sigma-Aldrich) or cocaine hydrochloride (COC: 15mg/kg, IP; in the mini-pump, the concentration was 22.5mg/ml or 11.25mg/ml; NIDA) were dissolved in sterile 0.9% saline and administered at the designated doses. In some experiments, mice also received injections (IP) of vehicle (0.9% DMSO/25% hydropropylβ-cyclodextrin; HPβCD) or racemic (+/−) Blebbistatin (blebb; Tocris). Blebb was diluted to 1mg/ml in HPβCD vehicle, and administered at the dose of 10mg/kg, as previously described [4 (link),6 (link),8 (link)]. In some experiments, vehicle (sterile saline) or the selective CRF2 antagonist Astressin-2B [AS2B, Tocris; 20 (link),21 (link),22 (link)] was infused into BLA (1ug/0.5ul/side at a rate of 0.25ul/minute) 20 minutes before conditioning, or 15 minutes after the final conditioning session. AS2B dose, rate, and times of infusions were based on previous research [23 (link)–25 (link)]. Additionally, in other experiments, vehicle (sterile saline) or the selective CRF2 agonist Urocortin 3 (UCN3, Bachem) was infused into BLA (2ug/0.66ul/side at a rate of 0.3ul/minute) 15 minutes after the final conditioning session or 20 minutes before each PM conditioning session (CS+). UCN3 dose and rate were based on previous research [26 (link)–29 (link)].

Hafenbreidel M., Briggs S.B., Arza M., Bonthu S., Fisher C., Tiller A., Hall A.B., Reed S., Mayorga N., Lin L., Khan S., Cameron M.D., Rumbaugh G, & Miller C.A. (2023). Basolateral Amygdala Corticotrophin Releasing Factor Receptor 2 Interacts with Nonmuscle Myosin II to Destabilize Memory. bioRxiv.

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CRF Receptor Antagonists and Cell Death

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Six-well plate cultures of either the C-20/A4 cell line or AC were treated for 8 h with individual or combination treatments of: the CRF-R1 specific antagonist CP-154526 (1–50 μM; Tocris), the CRF-R2 specific antagonist astressin 2B (1–50 μM; Tocris), the non-selective cation channel blocker Gd³⁺ (100 μM; Tocris), the adenylate cyclase activator forskolin (0.1 μM; Tocris), the PLC activator m3M3FBS (0.1 μM; Tocris), or the PLA₂ inhibitor OBAA (0.1 μM; Tocris). Cells were then assayed for cell death or photographed using an Olympus IX73 phase contrast microscope and camera.

Lawrence K.M., Jones R.C., Jackson T.R., Baylie R.L., Abbott B., Bruhn-Olszewska B., Board T.N., Locke I.C., Richardson S.M, & Townsend P.A. (2017). Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1. Scientific Reports, 7, 5147.

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CRF Receptor Agonists Modulate ATP Release

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Normal and FIC UT cells, grown on collagen-coated cover slips (2–3 DIV), were transferred into a perfusion chamber and superfused with an oxygenated physiological saline solution (flow rate=0.5ml. min⁻¹) until a stable baseline level of ATP release was measured (Control Phase). The perfusate was modified by the addition of the chemical agents (Test Phase). Perfusate was collected (100μl) in the Control and Test phases at 30–60s intervals. The agents used were: CRF (CRF-R1 agonist; 0.01μM; Tocris, Minneapolis, USA) and UCN3 (CRF-R2 receptor agonist; 0.1μM; Sigma-Aldrich, St. Louis, USA). CRF-R1 and CRF-R2 antagonists used were: Antalaramin (0.1μM) and Astressin-2B (Ast2B; 1.0μM), respectively (Tocris, Minneapolis, USA). ATP levels were quantified using a luciferin-luciferase reagent (Adenosine Triphosphate Assay Kit; Sigma-Aldrich, St. Louis, USA) and ATP concentrations were extrapolated from a standard-curve (ATP assay, Sigma-Aldrich, St. Louis, USA). Data are expressed as mean percentage change in ligand-evoked ATP release with respect to basal; taking basal as 100%.

Hanna-Mitchell A.T., Wolf-Johnston A., Roppolo J.R., Tony Buffington C.A, & Birder L.A. (2014). Corticotropin-releasing factor family peptide signaling in feline bladder urothelial cells. The Journal of endocrinology, 222(1), 113-121.

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Bilateral VTA CRF Receptor Modulation

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Mice were implanted with a dual cannulae system (Plastics One, Roanoke, VA) to bilaterally target the mouse posterior VTA (AP-3.2, ML±0.75, DV-4.5mm from bregma and dura). Dummy cannulae and dust caps fitted the length of the cannula while VTA dual injectors protruded 0.1mm past the cannula. Other groups of mice were implanted with a unilateral VTA cannula and an ipsilateral microdialysis guide cannula (CMA7, Harvard Apparatus, Holliston, MA) targeting the NAc shell (AP+1.7, ML-0.7, DV-4.0mm from bregma and dura). On test days, doses of the CRF-R1 antagonist CP376395 (0.3–0.6µg, Tocris, Ellisville, MO) or CRF-R2 antagonist astressin2B (0.25–0.5µg, Tocris, Ellisville, MO) were freshly dissolved in an artificial cerebral spinal fluid (aCSF) vehicle (0.2µl infused at 0.1µl/min). Doses were chosen based on previous microinjection procedures (Hwa et al. 2013 (link), Boyson et al. 2014 (link)). EtOH and H2O were given to animals 10 min post-infusion. After testing, mice were intracardiacally perfused with 0.9% saline and 4% paraformaldehyde, followed by brain removal. Coronal sections were Nissl stained to check placement of guide cannulae. Animals with incorrect placements into target sites were excluded from analysis.

Hwa L.S., Holly E.N., DeBold J.F, & Miczek K.A. (2015). Social stress-escalated intermittent alcohol drinking: modulation by CRF-R1 in the ventral tegmental area and accumbal dopamine in mice. Psychopharmacology, 233(4), 681-690.

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Reagent Procurement for Biochemical Assays

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NBI27914 and Astressin2B were purchased from Tocris. All other reagents were purchased from Sigma-Aldrich unless otherwise noted in the text.

Silberman Y., Fetterly T.L., Awad E.K., Milano E.J., Usdin T.B, & Winder D.G. (2015). Ethanol produces corticotropin releasing factor receptor-dependent enhancement of spontaneous glutamatergic transmission in the mouse central amygdala. Alcoholism, clinical and experimental research, 39(11), 2154-2162.

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