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Dernière publication 16/01/2024

Opioid-driven disruption of the septal complex reveals a role for neurotensin-ex

Each day for 3 days prior to tissue collection, all animals were habituated to the procedure room for ~2 hours to control for...

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    [title] => Opioid-driven disruption of the septal complex reveals a role for neurotensin-ex
    [paragraph] => Opioid-driven disruption of the septal complex reveals a role for neurotensin-expressing neurons in withdrawal
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Authors
Rhiana C Simon, Weston T Fleming, Pranav Senthilkumar, Brandy A Briones, Kentaro K Ishii, Madelyn M Hjort, Madison M Martin, Koichi Hashikawa, Andrea D Sanders, Sam A Golden, Garret D Stuber

 Lab

Journal
bioRxiv
Abstract
Each day for 3 days prior to tissue collection, all animals were habituated to the procedure room for ~2 hours to control for environmentally-induced immediate early gene activity. Our procedure for tissue collection has been detailed previously30,31. On the day of sacrifice, mice were sequentially euthanized with sodium pentobarbital (> 90 mg/kg Somnasol) and transcardially perfused with ice-cold NMDG (96 mM NMDG, 2.5 mM KCl, 1.35 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na+ ascorbate, 3 mM Na+pyruvate, 0.6 mM glutathione-ethyl-ester, 2 mM N-acetyl-cysteine, 0.5 mM CaCl2, 10 mM MgSO4; pH 7.35-7.40, 300-305 mOsm, continuously oxygenated with 95% O2 and 5% CO2). This NMDG solution was also used for tissue sectioning and for a collection bath. Both the perfusion solution and recovery bath included an inhibitor cocktail containing 500 nM TTX, 10 M APV, 10 M DNQX, 5 M actinomycin, and 37.7 M anysomycin, as described previously30. 250 m thick coronal sections containing septum were rapidly harvested using a Leica Vibratome (VT1200). The septal complex was then rapidly microdissected, flash-frozen on powdered dry ice, and stored at to80 C. We intended to reduce nucleus accumbens contamination as much as possible. To this end, our tissue sections primarily ranged from +1.10 mm through 0.0 mm with respect to Bregma. We also excluded the medial septum and diagonal band level to and ventral to the accumbens. Once tissue from all animals were collected and stored, we proceeded with nuclear isolation, a protocol we adapted from previously-published papers68,69. The following steps were all performed on ice. Also note that the PBS used here was Ca2+and Mg2+free. For each experimental group, we pooled all mice into the same tube containing a hypertonic lysis buffer (10mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal, 0.2 U/l Lucigen NxGen Rnase inhibitor). Nuclei were extracted using a glass dounce homogenizer (DWK, #885300-0002) via 5-10 reps of pestle A and 5 reps of pestle B. Nuclei were then incubated in lysis buffer for 5 min. Next, the nuclear extract was moved into a clean tube, diluted to ~5-6 ml using a wash buffer (1X PBS, 1% BSA, 0.2 U/l Rnase inhibitor), and strained into a new 50 ml conical tube using a 40 m cell strainer (pluriSelect, # 43-50040-51). Strained nuclei were centrifuged for 10 min, 500 x g at 4 C. The resulting pellet was then retained. To purify our nuclei, we used density gradient centrifugation, where we prepared a two-layer column containing a 25% and 30% iodixanol (OptiPrep, Sigma-Aldrich, #D1556) gradient. Briefly, we suspended the nuclear pellet in a homogenate solution (0.25M Sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.8-8.0, and 0.2 U/l Rnase inhibitor), and combined the resuspended pellet 1:1 with a 50% iodixanol solution (50% OptiPrep, 25 mM KCl, 5 mM MgCl2, 20 mM Tris-HCl, 1% BSA, 0.2 U/l Rnase inhibitor, pH 7.8-8.0) in a clean 15-ml conical tube. This creates a 25% iodixanol layer. Next, we prepare a 30% iodixanol layer (30% OptiPrep, 25 mM KCl, 5 mM MgCl2, 20 mM Tris-HCl, 1% BSA, 0.2 U/l Rnase inhibitor) and underlaid the 25% iodixanol layer with this 30% layer using a 18G needle. This density gradient column was then centrifuged for 15 min, 3000 x g at 4 C. The resulting purified and invisible pellet was then resuspended in wash buffer. Nuclei were stained with propidium iodide and assessed for yield and quality using a disposable Neubauer hemocytometer (INCYTO, #DHC-N01) on a fluorescent microscope (Zeiss ApoTome.2). Final nuclear concentration was adjusted to 1,000 nuclei per l in preparation for single nucleus capture.
Keywords/Topics
opioid-driven;disruption;septal;complex;reveals;neurotensin-expressing;neurons;withdrawal;prior;tissue
BIOSEB Instruments Used:
Cold Hot Plate Test (BIO-CHP)
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