SUPPLEMENTARY INFORMATION
Supplementary Material & Methods:
Reversible inactivation of anterior cingulate cortex by muscimol (Experiment 1)
Guide cannula implantation.Animals were placed in vaporization chambers, anesthetized with isoflurane gas (5% for induction and 1-3% for maintenance) and placed in a stereotaxic apparatus. and secured to the skull with bone screws and dental acrylic. Wire obturators were inserted into the guides to seal the openings, maintain cannula patency and reduce risk of infection. Animals were administered Rimadyl (Carprofen 5mg/kg s.c.) both pre- and post-surgery and allowed to recover first in thermostatically controlled cages and then in individual home cages for a period of 7 days. Intracranial guide cannula (26 gauge; Bilaney Consultants Ltd, Sevenoaks, UK) were implanted bilaterally, aiming at the ACC(from bregma: AP + 2.0mm, ML ± 0.5mm, DV: - 1.0mm). At the time of injection, the obturators were removed and internal cannulae (33 gauge; Bilaney Consultants Ltd, Sevenoaks, UK) inserted into the guides. Each cannula protruded 1 mm beyond the tip of the guide (for final injection target of AP + 2.0mm, ML ± 0.5mm, DV: - 2.0mm). One minute after insertion of the cannulae, either physiological saline or muscimol (GABAA receptor agonist (1 μg/μl dissolved in 0.9% physiological saline; Sigma-Aldrich, UK) was used to induce reversible inactivation of the ACC (Ragozzino and Rozman. 2007)was slowly infused at a total volume of 0.5 μl per side, over a period of 2 min. Using this protocol, it is conceivable that cortical tissue near the cannula tips was also inactivated, although the infusion protocol was designed to inactivate no more than a 1 mm radius of the targeted brain region, based on previous diffusion and 2-DG measurements (Martin. 1991).
In vivo oxygen amperometry (Experiments 2 and 3)
Electrode construction.Carbon Paste Electrodes (CPE) were constructed from 8T (200µm bare diameter, 270µm coated diameter) Teflon®-coated silver wire (Advent Research Materials, Suffolk, UK) as described previously (Francois et al. 2012). The Teflon insulation was slid along the wire to create an approximately 2-mm deep cavity, which was packed with carbon paste. Carbon paste was prepared by thoroughly mixing 7.1 g of carbon graphite powder and 2.5 ml of silicone oil (both from Sigma-Aldrich, UK). Reference and auxiliary electrodes were also prepared from 8T Teflon®-coated silver wire by removing the Teflon from the tip. All electrodes were soldered to gold connectors, which were cemented into six-pin plastic sockets (both from Plastic One, Roanoke, VA) during surgery.
Electrode calibration.Prior to implantation, all CPEs were calibrated in vitro in a standard three-electrode glass electrochemical cell (BASi C3 cell stand, Bioanalytical Systems, USA) with an Ag/AgCl reference electrode, and a BASi platinum auxiliary electrode. Calibrations were performed in a 15 ml phosphate buffer saline solution, pH 7.4 saturated with nitrogen (N2) gas, atmospheric air (from a RENA air pump), or pure O2 at room temperature providing a three-point calibration of known concentrations of 0 μM (N2-saturated), 240 μM (air-saturated) and 1,260 μM (O2-saturated) oxygen. CPEs were chosen for implantation if their calibration curves were linear and the measured O2 values from the saturated solutions were not greatly different from those expected (least square linear regression, R2≥ 0.98).
Electrode implantation.Animals were placed in vaporization chambers, anesthetized with isoflurane gas (5% for induction and 1-3% for maintenance) and placed in a stereotaxic apparatus. CPEs were implanted in the ACC (from bregma: AP + 2.0mm, ML ± 0.5mm, DV: - 2.0mm). A reference electrode was inserted into the posterior cortex to a depth of 2 mm and secured with cement. An auxiliary electrode was wrapped round a skull screw positioned over posterior cortex. All electrodes were then cemented into place; the gold sockets of the electrodes were inserted into a six-pin plastic socket. All animals were administered Rimadyl (Carprofen 5mg/kg s.c.) and were allowed to recover in thermostatically controlled cages and then placed back in individual home cages for a period of 7 days.
In-vivo [O2] signal validation. To demonstrate the oxygen sensitivity and specificity of the electrodes in vivo, mild hyperoxia and hypoxia was induced by applying gaseous O2 (BOC medical, Manchester, UK) or N2 (BOC gases, Guildford, UK), respectively, to the snout of the animal before and after each experiment. Polyurethane tubing, connected to the appropriate gas cylinder, was held approximately 2cm from the snout and the gas delivered for 30s at a flow rate of 1L/min.
Data recording. Rats were connected to a four channel potentiostat (Biostat, ACM Instruments, Cumbria, UK) through a 6-pin socket (Plastics One, Roanoke, VA, USA) via a flexible screened 6-core cable (Plastics One). A PowerLab 8/30 was used for analogue/digital conversion and data were collected on a PC running Chart_v5 software (AD Instruments, Oxford, UK). A negative potential (-650mV) was applied to the CPE to allow the electrochemical reduction of dissolved oxygen to occur at the tip of the electrode during the whole recording session; the O2 signal being recorded at a sample rate of 200Hz. This potential is in the mass-transport limited region after the peak potential for O2 reduction (ca. -500 mV) and has previously been shown to be appropriate for CPA O2 detection using cyclic voltammetry (Lowry et al. 1997). Moreover, since the dimension of CPEs (typically 100-200μm) is greater than the scale of a capillary zone (< 100μm), an average extracellular tissue O2 level is detected regardless of the orientation of the electrode relative to the blood vessels and metabolically active sites, or the depth of penetration into the tissue. Filtering and down sampling procedure: Current (nA) recordings from each channel were analysed separately. Linear interpolation was used to replace occasional missing data points and a bi-quad Butterworth filter (high-pass 0.1 Hz) was used to suppress fast events representing noise artefacts. Also, a boxcar-averaging algorithm was used to down-sample the data, keeping a single average from multiple 60-second non-overlapping windows for the global signal and a single average from multiple 0.5-second non-overlapping windows for the task-related signals.
Histology
For all experiments, histological verification of guide cannula or CPE location was performed after behavioural testing. Rats were deeply anesthetized with pentobarbital and perfused transcardiacally with 0.9% saline followed by 10% buffered paraformaldehyde. Brains were removed and placed in 10% buffered paraformaldehyde solution and shipped for histological processing which involved coronal sectioning (40 μm) through relevant brain areas using Multi-Brain® Technology (Neuroscience Associates Inc., Knoxville, USA) and staining with thionin for Nissl substance. Slides were viewed microscopically to assess placement of guide tips.
Supplementary Figures:
Supplementary Figure 1: Representative slice and reconstructions of cannula and electrode placements from all studies.
A.Intendedand B. Representative cannula placements in the anterior cingulate cortex (Experiment 1), C. Reconstruction of CPE placements in the anterior cingulate cortex (Experiment 2), D. Reconstruction of CPE placement in the anterior cingulate cortex (Experiment 3). The tip of each CPE is represented by a black circle. Coronal sections for reconstructions are taken from the atlas of Paxinos & Watson (1998).
Supplementary Figure 2: Replication of baseline SRT performance and ACC O2 signals (Experiment 3). A. Illustration of the mean extracted parameters: average area under the curve (AUC), maximum amplitude (Y Peak) and time at which the maximum amplitude occurs (X Peak). B. Left: Average curves of ACC O2 signals associated with animals completing (black circle or bars) or omitting (white circle or bars) a response during the SRT task. Right: Extracted parameters; ***p<0.001 in comparison to response type. C. Left: Average curves of ACC O2 signals for different RT ranges. Three RT ranges are depicted: Fastest RT range (<0.2s, white circles and bars); Medium RT range (0.4<RT<0.7s, grey circles and bars); Slowest RT range (RT>2s black circles and bars). Right: Extracted parameters; **p<0.01, ***p<0.001 in comparison to the Fastest RT range. All data are presented as mean±SEM.
Supplementary References:
Francois J, Conway MW, Lowry JP, Tricklebank MD, Gilmour G (2012) Changes in reward-related signals in the rat nucleus accumbens measured by in vivo oxygen amperometry are consistent with fMRI BOLD responses in man. Neuroimage 60:2169-2181. DOI: 10.1016/j.neuroimage.2012.02.024 [doi].
Lowry JP, Boutelle MG, Fillenz M (1997) Measurement of brain tissue oxygen at a carbon past electrode can serve as an index of increases in regional cerebral blood flow. J Neurosci Methods 71:177-182. DOI: S0165027096001409 [pii].
Martin JH (1991) Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat. Neurosci Lett 127:160-164.
Ragozzino ME, Rozman S (2007) The effect of rat anterior cingulate inactivation on cognitive flexibility. Behav Neurosci 121:698-706. DOI: 2007-11112-007 [pii].
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