Images of fluorescently tagged fusion proteins were captured at room temperature in live C. elegans. Mid-L4 through young adult stage hermaphrodite animals were anesthetized using 10 mM levamisole (Sigma-Aldrich) or 50 mM muscimol (Abcam) in M9 buffer, mounted on 2–5% agar pads and imaged as follows: Images in Figures 1 , 2 and 3B -Hwere taken using a 60x CFI Plan Apochromat VC, NA 1.4, oil objective (Nikon) on an UltraView VoX spinning-disc confocal microscope (PerkinElmer). Images in Figures 4A–C and and 7A were taken using a Zeiss LSM710 confocal microscope (Carl Zeiss) with a Plan-Apochromat 63x/1.4 NA objective. Images in Figures 3L , 4D–E and and 7D were taken with a Zeiss Axio Observer Z1 microscope equipped with a Plan-Apochromat 63 × 1.4 objective and a Yokagawa spinning-disk unit. Maximum-intensity projections were generated using ImageJ (NIH) or ZEN 2009 software and used for all the confocal images. Quantification was performed on maximal projections of raw data.
Muscimol
Manufactured by Abcam
Sourced in United States, United Kingdom
Muscimol is a chemical compound that acts as a gamma-aminobutyric acid (GABA) receptor agonist. It is a naturally occurring substance found in certain mushroom species. Muscimol binds to and activates GABA receptors in the central nervous system, which can have various effects on neurological processes and behaviors.
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Lab products found in correlation
Levamisole, by Merck Group (2 mentions)
Plan apochromat 63x 1.4 na objective, by Zeiss (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Ultraview vox spinning disc confocal microscope, by PerkinElmer (1 mentions)
Pregabalin, by Merck Group (1 mentions)
D ap5, by Abcam (1 mentions)
Ab120094, by Abcam (1 mentions)
Spinning disc confocal microscope, by PerkinElmer (1 mentions)
Nodularin, by Enzo Life Sciences (1 mentions)
Phallacidin, by Merck Group (1 mentions)
α ama, by Enzo Life Sciences (1 mentions)
Microcystin lr, by Enzo Life Sciences (1 mentions)
β ama, by Enzo Life Sciences (1 mentions)
γ ama, by Enzo Life Sciences (1 mentions)
Prism 7, by GraphPad (1 mentions)
Ibotenic acid, by Abcam (1 mentions)
Octadecyltrimethoxysilane, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Toluene, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
12 protocols using muscimol
1
Live Cell Imaging of C. elegans
2
Muscimol Infusion in Parietal Cortex
3
Iontophoretic Delivery of Muscimol in Cerebellar Cortex
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Borosilicate glass pipettes (tip diameter approximately 1 μm) were filled with 50 mM muscimol (Abcam, Cambridge, UK) diluted in a citric acid buffer (pH:3.7) and 20 μM of Alexa 594 dye. They were connected by Pt electrodes to an iontophoretic drug ejection system (MVCSC, npi electronic, Tam, Germany). A retaining current of −20 nA was applied to avoid leak of muscimol from the pipette; muscimol was ejected by current pulses of 100 nA applied during 1–2 s. Based on previous diffusion measurements in the cerebellar cortex (Nicholson and Phillips, 1981 (link); Rice et al., 1993 (link)) we estimated that the muscimol concentration released from the tip of the pipette in the present experiments ranged from 50 to 100 μM for a radius of 90 μm.
4
Behavioural Assessment of Mice after Viral Injection
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For behavioural studies, mice 4–5 weeks after viral injection were used. Mice were placed in clear plastic containers (11 cm in diameter, 18 cm high) for observation and allowed to acclimate for 30 min prior to behavioural assessments. All assays were recorded, and subsequently scored time of licking and biting of hind paw on one side, or number of paw flinching (rapid flicking of the limb) on one side after CNO (10 mg kg−1 in saline, Enzo Life Sciences) or saline intraperitoneal injection for 1 hr. To analyse the effect of analgesic drugs, we intrathecally injected muscimol (1 or 2 μg/5 μL in PBS, Abcam), D-AP5 (5 μg/5 μL in PBS, Abcam), pregabalin (10 μg/5 μL in PBS, Sigma), or PBS (for control) under isoflurane (2%) anaesthesia32 (link) 10 min before CNO (10 mg kg−1) injection. To analyse the effect of morphine, we subcutaneously injected morphine (3 mg kg−1) or saline (for control) 10 min before CNO (10 mg kg−1) injection. To investigate the expression of c-Fos after CNO injection, we quickly perfused the mice 90 min after CNO injection as described below.
5
Transient Pharmacological Inactivation of Mouse Somatosensory Cortex
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For pharmacological inactivation, ≈500-μm-diameter craniotomies were drilled over cool (32–22 °C)-responsive areas in S1 and pIC in mice anaesthetized with isoflurane (1.5–2% in O2). Both regions were identified functionally using the widefield calcium imaging response to thermal stimuli. Following the craniotomy, the dura was covered with transparent silicone gel (3-4680, Dow Corning). A 300 nl volume of muscimol or Ringer’s solution was injected at a rate of 100 nl min−1 300–500 μm below the pial surface using a pulled glass pipette and a hydraulic injection system (MO-10, Narashige). muscimol (Abcam, ab120094) was dissolved in Ringer’s solution to a concentration of 5 mM. Imaging sessions were carried out >10 min following the end of Ringer’s solution or muscimol injection (Fig. 1f,g ).
6
GABA Neuron Stimulation in Worms
7
Selectivity Assay for Monoclonal Antibodies
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Indirect cELISAs were completed using a panel of inhibitors to determine the selectivity of the mAbs. The cELISA procedure was nearly the same as that described for the serum screening, except for the addition of inhibitors (50 µL), which were mixed with 50 µL of the antibody solution during the primary antibody incubation step. The inhibitors tested were α-AMA (≥90%, Enzo Life Sciences, Farmingdale, NY, USA), β-AMA (≥90%, Enzo), γ-AMA (≥90%, Enzo), microcystin-LR (≥95%, Enzo), nodularin (≥95%, Enzo), phalloidin (>90%, Enzo), phallacidin (≥85%, Sigma), pysilocybin (>99%, Cerilliant, Round Rock, TX, USA), muscimol (>99%, Abcam, Cambridge, MA, USA), ibotenic acid (>98%, Abcam). Each analyte stock was dissolved in dH2O, then serially diluted into TBST, starting at the highest concentration of ,000 ng mL−1, and assessed in triplicate. Data were analyzed using a 4-parameter logistic equation (GraphPad Prism 7 Software, La Jolla, CA, USA) to determine the concentration of inhibition at half of the maximal signal (IC50). Cross-reactivity (%) was calculated as follows: (IC50 α-AMA)/(IC50 test inhibitor) × 100.
8
Muscimol Infusion for Gerbil Social Behavior
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Prior to each exposure session, gerbils were briefly anesthetized (2% isoflurane). Infusion cannulae (33 gauge, 4 mm cannula length, C235IS-5/SP; Plastics One) were inserted and connected to PE-50 tubing for the infusions of either saline or muscimol. The concentration of muscimol (Abcam) used was 1 mg/ml (dose infused: 0.5 µL per hemisphere at a rate of 0.1 μL/min39 (link)). For control infusions, physiological saline solution (0.9% NaCl) was used (dose infused: 0.5 µL per hemisphere at a rate of 0.1 μL/min). Animals were allowed to recover in a recovery cage for 15–20 min before each exposure session.
Prior to starting the social exposure session with a demonstrator, all infused animals were monitored for a few minutes in the test cage to assess their locomotion and general behavior. No animals required a longer recovery period, and all animals were alert and engaged and displayed proper posture and normal motor functions.
Prior to starting the social exposure session with a demonstrator, all infused animals were monitored for a few minutes in the test cage to assess their locomotion and general behavior. No animals required a longer recovery period, and all animals were alert and engaged and displayed proper posture and normal motor functions.
9
Quantitative Analysis of Psilocin and Muscimol
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Muscimol (pKa: 4.8 and 8.4 [1 (link),43 (link)]) was purchased from Abcam (Cambridge, UK). Psilocin (pKa 9.41 [4 ]) was from Sigma-Aldrich (Poznań, Poland). The chemical structures of the investigated analytes are shown in Figure 5 .
A stock solution of the studied analytes was prepared in methanol (100 µg/mL) and stored at 4 °C. Sodium phosphate heptahydrate, toluene, dichloromethane, ethyl acetate, and octadecyltrimethoxysilane (ODTS) were obtained from Sigma-Aldrich (Poznań, Poland). Orthophosphoric acid (85%), octanol, and sodium hydroxide were purchased from POCH. S.A. (Gliwice, Poland). Deionized water was obtained from the Milli-Q system (Millipore, MA, USA).
A stock solution of the studied analytes was prepared in methanol (100 µg/mL) and stored at 4 °C. Sodium phosphate heptahydrate, toluene, dichloromethane, ethyl acetate, and octadecyltrimethoxysilane (ODTS) were obtained from Sigma-Aldrich (Poznań, Poland). Orthophosphoric acid (85%), octanol, and sodium hydroxide were purchased from POCH. S.A. (Gliwice, Poland). Deionized water was obtained from the Milli-Q system (Millipore, MA, USA).
10
Intracerebral Pharmacological Modulation of Cognitive Processes
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Scopolamine (25 μg/kg, Tocris, UK), dissolved in 0.9% saline, was injected intraperitoneally (i.p.). Muscimol (1.25 μg/2 μl, Abcam, USA) dissolved in 0.9% saline was bilaterally injected into the mPFC 1 h prior to Scopolamine. M2-AChR antagonist methoctramine (MCT, GlpBio, USA), dissolved in 0.9% saline (0.5 μg/2 μl, 1 μg/2 μl or 2 μg/2 μl), was injected intracerebroventricularly (i.c.v.) (8 (link)). M3-AChR antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 100 pmol, Cayman, USA) was dissolved in 0.9% saline (1 μg/2 μl) and injected i.c.v (8 (link)). Rapamycin (Solarbio, China), dissolved in dimethyl sulfoxide (0.2 nmol/2 μl), was delivered i.c.v. 30 min prior to MCT injection. Groups of control animals for the above experiments received equal volumes of vehicle alone (0.9% saline or dimethyl sulfoxide). The experimental procedures are illustrated in Figure 1 .
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