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Madsen et al (2013) Supplementary materials
Measurements of evoked potentials in response to stimuli.
Fig. 1: Experimental setup for recordings of evoked potentials
To measure evoked potentials, anaesthetized turtles (injection of 60 mg kg-1 Ketaminol and 4 mg kg-1
Rompun, into the subcarapacial sinus) were fitted with subcutaneous stainless steel electrodes and
placed in a test chamber (15x15x30 cm) and ventilated manually with 50 ml air once every 10 min to
avoid hypoxemia due to ventilatory depression. The anoxic turtles were ventilated with N2, and the
chamber could be made anoxic by a flow of N2. Temperature could be controlled by circulating water
with the desired temperature (Struers 03T623 cooling and heating bath) through a water jacket
surrounding the chamber. Body temperature was monitored with a thermo probe placed in the cloaca.
Data was collected with a PC via a National Instruments USB 6008 system.
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To establish that artificial ventilation with N2 rendered the turtles anoxic, a catheter was placed in the
carotid artery of three turtles anaesthetised with pentobarbital (50 mg kg-1, i.m.), so arterial blood
samples (0.5 ml) could be withdrawn and measured on GEM Premier 3500 Blood Gas Analyzer. These
turtles were not used for evoked potential experiments; results are shown in the red trace in article fig.
1.
Vibration Experiments. The turtles were placed on a metal plate (10x10 cm) fixed to a mini-shaker
(Brüel & Kjær type 4810) controlled by a Digital to Analog RM2 Mobile Processor (Tucker-Davis),
via a Brüel & Kjær Amplifier type 2706 (Fig. 1). One subcutaneous electrode was placed above the
brain stem, another above the inner ear, while the reference electrode was inserted in one of the front
legs. The stimulus consisting of a 600 Hz single pulse with root mean square (RMS) amplitude of 0.2 g
measured over 12 msec. This stimulus type was chosen because it elicits a consistent and substantial
evoked potential response (1). The evoked potentials were amplified 20x with a RA4PA 4-Channel
Tucker-Davis amplifier and stored on a laptop via a RM2 Mobile Processor (Tucker-Davis) analogue to
digital converter (24 kHz sampling, 16 bit). Each stimulus pre-triggered a 50 ms recording window of 5 to +45 ms around the start of the stimulus. The synchronized averaging of 500 measurements made
up each individual recording. Vibration recordings were filtered with a 4th order, 50 Hz – 1 kHz, bandpass Butterworth filter applied in MATLAB.
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Light stimulation. Anaesthetised turtles were equipped with subcutaneous electrodes placed above the
eye and at the posterior end of the head to measure the differential activity across the optic nerve and
the visual cortex. Light stimulus was provided by an LED unit, flashing at 4 Hz with a light intensity of
3350 lux for 170 ms. The averaging window for light experiments was 500 ms from -5 ms. to 495 ms
around stimulus onset, because of the longer duration of electroretinographs (2). Light recordings were
filtered with a 4th order, 50 Hz low-pass Butterworth filter applied in MATLAB.
Effects of anaesthesia. Control experiments were conducted to estimate the effects of the anaesthetics
by exposing six anesthetized turtles to vibration and another six turtles to light stimuli every 15 min
while measuring the responsiveness until the turtles recovered. These control experiments showed no
significant effects of the anaesthetics used, on the nervous activity.
Hibernation experiments
The animals were fasted for a minimum of 3 days and placed at 3°C for 24 h before being placed into
water-filled, air tight boxes with clear plastic lids. There was mud in the bottom of each tank to ensure
that microbial respiration rendered the water anoxic to avoid cutaneous O2 uptake (3). PO2
measurements of the tank water were made using a Firesting O2 logger, which recorded PO2 levels of
average 2.0 ± 0.5 mmHg after 14 days of hibernation. The turtles were left undisturbed in complete
darkness for 14 days to ensure sufficient time to reach the possible comatose state in cold anoxia.
Different types of stimuli were used to test for turtle vigilance:
A) Water temperature was increased by 0.36 °C every 10 min throughout the recording period using an
aquarium heater placed in the tank (300W Jäger 3609), whereas oxygenation (B) was achieved by
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bubbling O2 and allowing the oxygenated water to enter the boxes enclosing the turtles through holes in
the lids. C) Light stimulation was performed by turning on the lights in the laboratory resulting in an
underwater light intensity within the boxes enclosing the turtles of 240 lux (measured by a LI-COR LI250 Underwater PAR sensor. C) Vibration stimulation was achieved by a shaker (BOSCH GST 120
jigsaw clamped to the water tank) resulting in vibration frequencies at both 40 and 620 Hz > 20 dB
above the thresholds of detection for slider turtles (1).
Activity during hibernation was quantified by establishing a differentiated brightness score for every
10th frame of the video recording. An area of interest corresponding to the size of the box was defined
for analysis, such that all movements made by the turtles altered brightness in the area of interest.
Video data was spilt into 30 min segments, 4 control segments and 9 exposure segments. Specific
movement events were classified from RMS values of the differential brightness in the individual video
segments and the number of movement events was used to quantify turtle activity in each 30 min
segment. The number of movement events for each 30 min period was normalized to the mean number
of movement events during the control periods for each stimulus. Most turtle movements were caused
by the turtles trying to surface by raising their heads against the lids. All video was analysed with a
custom made LabVIEW (National Instruments, version 2011) program.
Statistics
The mean of the normalized response amplitudes was subtracted from each measurement interval. For
temperature experiments, this was done for the normalized responses at each of the measurement
temperatures, 25°C through to 3°C. For hypoxia this was done for each ventilation step, 0 min through
to the start of air ventilations (96 min for light and 80 min for vibration). These data were then pooled
to generate two distributions for each stimulus type, which, subsequently, were tested for equal means
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in a permutation test. First, the actual difference of the means was compared to an estimate of the
distribution of the differences of means, calculated as the means in each of two groups of randomly
selected measurements from the pooled data sets 10,000 times. The null hypothesis was that there
would be no difference in means between vibration and light responses, during both temperature
experiments and during hypoxia experiments and thus, no difference in the decline of responsiveness to
the two types of stimuli during those conditions. The same was done for reheating data and
reoxygenation data.
Changes in activity in response to light, O2, and vibration stimuli of hibernating turtles were tested
using a Wilcoxon signed rank test to determine whether the normalized mean of movement events
upon application of the stimulus was significantly higher than 1 (i.e. increased movement compared to
the preceding control period). The response to elevated temperature was tested using a linear regression
analysis. For all 6 heating turtles normalized movement data were tested for a linear relationship
between turtle movement and increasing temperature.
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References
1. Christensen-Dalsgaard, J., Brandt, C., Willis, K. L., Christensen, C. B., Ketten, D., Edds-Walton,
P., Fay, R. R., Madsen, P. T. and Carr, C. E. (2012) Specialization for underwater hearing by the
tympanic middle ear of the turtle, Trachemys scripta elegans. Proc. Biol. Sci., 279, 2816-24.
2. Stensløkken, K. O., Milton, S. L., Lutz, P. L., Sundin, L., Gillian, M. C. R., Stecyk, J. A. W., Nilsson
G. E. (2008) Effect of anoxia on the electroretinogram of three anoxia-tolerant vertebrates. Comp.
Biochem. Physiol. 150A, 395–403.
3. Ultsch, G. R. & Jackson, D. C. (1982). Long-term submergence at 3°C of the turtle Chrysemys picta bellii, in
normoxic and severely hypoxic water: II. Extracellular ionic responses to extreme lactic acidosis. J. Exp. Biol.,
96, 29-43.
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