TITLE: Alfaxalone is an Effective Anesthetic for the Electrophysiological Study of Anoxia-Tolerance Mechanisms in Western Painted Turtle Pyramidal Neurons  
AUTHORS: Haushe Suganthan, Domenic Di Stefano, Leslie Buck

[DESCRIPTION]
Many anesthetics have long-term effects on ion channels and are not appropriate for same day experimentation. Using whole-cell electrophysiological techniques, we examine the effects of the anesthetic, Alfaxalone, on pyramidal cell action potential amplitude, threshold, rise and decay time, width, frequency, whole cell conductance, and evoked GABAA receptors currents to determine if any of these characteristics are altered with the use of Alfaxalone for animal sedation. 


[STEPS]
SECTION: Animal Care and Cortical Sheet Preparation
1. 
 
Obtain the adult turtles (Chrysemys picta bellii) from Niles Biological and are then housed in a large aquarium equipped with a freshwater recirculating filter system set at 20 °C, a non-aquatic basking platform, and a heat lamp.
SECTION: Animal Care and Cortical Sheet Preparation
2. Maintain the turtles on a photoperiod of 12 hours of light:12 hours of dark and given access to food three times a week.
SECTION: Animal Care and Cortical Sheet Preparation
3. Give the turtles an intramuscular injection of Alfaxalone 10 2897 stock in hydroxypropyl-beta-cyclodextrin [HP-β-CD] and dissolve in a multi-dose preservative solution (Alfaxan®) (Jurox Pty Ltd; Rutherford, Australia) at 20 1573.

SECTION: Animal Care and Cortical Sheet Preparation
4. Once deep anesthesia was achieved, the legs were flaccid, and the eyes were unresponsive to gentle touch, decapitate the animals with a guillotine, and excise the whole brain from the cranium in about 1 minute.
SECTION: Animal Care and Cortical Sheet Preparation
5. Dissect the entire dorsal cortex free and bathed in 3 °C–5 °C artificial turtle cerebrospinal fluid (aCSF) composed of (in mmol L –1): 97 NaCl, 2.6 KCl, 1.2 CaCl2, 1.0 MgCl2, 2.0 NaH2PO4, 26.5 NaHCO3, 20.0 glucose, 5.0 imidazole (adjusted to pH 7.4, and osmolarity 290–300 mOsM).

SECTION: Animal Care and Cortical Sheet Preparation
6. Cut two cortical sheets medially from the visual cortex of each cerebral hemisphere and subdivide into a total of six cortical sheets.
SECTION: Animal Care and Cortical Sheet Preparation
7. Lift the sheets out of the chamber and store them in vials of aCSF for no longer than 48 hrs.
SECTION: Whole-Cell Electrophysiology Techniques
8. Place the turtle cortical sheets on a cover slip that forms the bottom of an RC-26 open bath perfusion chamber system, with a P1 platform (Harvard Apparatus, Saint-Laurent, QC, Canada).
 

SECTION: Whole-Cell Electrophysiology Techniques
9. Conduct the experiments at room temperature (20 °C - 22 °C).

SECTION: Whole-Cell Electrophysiology Techniques
10. Perform the whole-cell recordings of neurons from the dorsal cortex and dorsal medial cortex using fire-polished 5-8 MΩ micropipettes produced from borosilicate glass capillary tubes using a P-97 micropipette puller model (Sutter Instruments, Novato, CA, USA).
 

SECTION: Whole-Cell Electrophysiology Techniques
11. Fill the electrode and insert into a 1-HL-U electrode holder attached to a CV-4 headstage (gain: 1/100 U, Axon Instruments, Sunnyvale, CA, USA).
SECTION: Whole-Cell Electrophysiology Techniques
12. Obtain the cell-attached 1-20 GΩ seals using the blind-patch technique.
 

SECTION: Whole-Cell Electrophysiology Techniques
13. To break into the cell, apply a soft pulse of negative pressure to break through the cell membrane, while the holding potential was voltage-clamped to -70 mV.
SECTION: Whole-Cell Electrophysiology Techniques
14. Once the whole-cell configuration was established, give the cells at least 2 minutes to acclimate to experimental conditions before access resistance was measured, which normally ranged from 20-30 MΩ.
SECTION: Whole-Cell Electrophysiology Techniques
15. Discard the patches if access resistance varied by >25% over the course of an experiment.
SECTION: Whole-Cell Electrophysiology Techniques
16. Collect the data at 5-10 kHz using a MultiClamp 700B digital amplifier, a CV-7B head stage, and a Digidata 1550B interface (Molecular Devices, Sunnyvale, CA, USA), and store on a computer using Clampex 10 software (Molecular Devices, Sunnyvale, CA, USA).
 

SECTION: Electrophysiological Identification and Measurement of Action Potential Parameters
17. 
 
Determine the action potential threshold (APth) by current-clamping cells and injecting current in 10 pA increments in a stepwise manner from sub-threshold for 500 ms until a spike was elicited.
SECTION: Electrophysiological Identification and Measurement of Action Potential Parameters
18. Record the threshold at the point at which a sharp elevation in voltage was observed.
SECTION: Electrophysiological Identification and Measurement of Action Potential Parameters
19. Measure the full spike amplitude from the point of the APth to the spike tip, while the half-amplitude spike width was measured as the time elapsed between the two-points of the half-amplitude on the spike.
SECTION: Electrophysiological Identification and Measurement of Action Potential Parameters
20. Calculate the rise time as the time elapsed between 10% of the full spike amplitude and 90% of the full spike amplitude, while the decay time was calculated as the time elapsed between 10% of the full spike amplitude and 90% of the full spike amplitude.
SECTION: Electrophysiological Identification and Measurement of Action Potential Parameters
21. Measure the whole-cell conductance (Gw) as the slope of a voltage-ramp   from -120 to -60 mV for 150 ms.
 

SECTION: Impact of acute Alfaxalone application on evoked naïve tissue GABAA Receptor current
22. To initiate a GABAA receptor current neurons were voltage-clamped at a holding potential of -100 mV, and 2 mM of GABA were applied for 1-2 seconds. These changes resulted in large outward GABAA- receptor currents that were easily detected and differentiated from other currents.


SECTION: Impact of whole-animal Alfaxalone exposure on evoked GABAA Receptor Whole Cell current
28. 
 
Determine the GABAA receptor current decay time, integrated area under the curve, and peak amplitude as described above.
SECTION: Impact of whole-animal Alfaxalone exposure on evoked GABAA Receptor Whole Cell current
29. Normalize the decay time, integrated area under the curve, peak amplitude, and baseline holding current to the whole-cell capacitance.
SECTION: Impact of whole-animal Alfaxalone exposure on evoked GABAA Receptor Whole Cell current
30. Repeat the protocol with cerebral cortex sheets obtained from Alfaxalone-sedated painted turtles at 1 hour, 2 hours, 3 hours, and 5 hours following Alfaxalone administration.
SECTION: Impact of whole-animal Alfaxalone exposure on evoked GABAA Receptor Whole Cell current
31. Bin the timepoints, so that any measurement performed between 1 and 2 hours was considered 1 hour following whole-animal Alfaxalone exposure, any measurement performed between 2 and 3 hours following whole-animal Alfaxalone exposure was considered 2 hours following whole-animal Alfaxalone exposure,  any measurement performed between 3 and 4 hours was considered 3 hours following whole-animal Alfaxalone exposure, and any measurement performed between 5 and 6 hours was considered 5 hours following whole-animal Alfaxalone exposure.
SECTION: Impact of whole-animal Alfaxalone exposure on evoked GABAA Receptor Whole Cell current
32. Make the measurements on the 30-minute mark were placed in the latter time group.
SECTION: Impact of whole-animal Alfaxalone exposure on evoked GABAA Receptor Whole Cell current
33. Replace the aCSF solution bathing the Alfaxalone-treated sheets every 30 minutes following the completion of the dissection with the first washout occurring 0.5 hours after Alfaxalone application.
SECTION: Impact of acute Alfaxalone application on evoked naïve tissue GABAA Receptor current
23. From the GABAA receptor currents produced, measure the peak amplitude, baseline holding current, decay time as the 90%-to-10% decay time, and the area under the curve as the integrated area between the measured current and the baseline using Clampfit software (Molecular Devices, Sunnyvale, CA, USA).
SECTION: Impact of acute Alfaxalone application on evoked naïve tissue GABAA Receptor current
24. Perfuse the sheets with oxygenated aCSF and 1 µM Alfaxalone for 15 min, and the GABAA receptor current decay time, integrated area under the curve, peak amplitude, and baseline holding current were measured again.
SECTION: Impact of acute Alfaxalone application on evoked naïve tissue GABAA Receptor current
25. Repeat this procedure utilizing gabazine (25 µM), a GABAA receptor antagonist, rather than Alfaxalone, to see if the currents could be blocked to confirm we were recording GABAA receptor currents. Additionally, Alfaxalone-free stock Alfaxalone solution (vehicle, a generous gift from Jurox; Rutherford, NSW, Australia) had no significant impact when applied alone (n = 4).
SECTION: Alfaxalone Dose-Response
26. To construct a dose-response curve, GABAA receptor current measurements following acute Alfaxalone treatment were normalized to pre-treatment values to determine the relative change in decay time, area under the curve, and peak amplitude.
SECTION: Alfaxalone Dose-Response
27. The same process to determine relative changes in decay time, area under the curve, and peak amplitude was repeated for tissue sheets perfused with oxygenated aCSF and 0.1 micromolar (µM), 0.5 micromolar (µM), 1 micromolar (µM) or 1.5 micromolar (µM) Alfaxalone for 15 min.