Animal handling and maintenance
Larval bullfrogs, Rana catesbeiana, 4–8 cm total length, were obtained from Carolina Biological Supply (Miami, FL). Animals were maintained in water tanks (≤ 20 larvae per tank) at 22°C and under a 12:12 light:dark cycle. They were fed twice per week with commercial tadpole chow (Carolina Biological Supply, Miami, FL). Larvae were handled, maintained, used and disposed of according to the standards described in the NIH Guide for the Care and Use of Laboratory Animals and the Guidelines for the Use of Animals in Neuroscience Research. All animal handling procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Universidad Central del Caribe.
In larval Rana catesbeiana approaching metamorphosis, the head increases in diameter as the tail decreases in length. We measured tail length and head diameter, either manually or digitally with the program Scion Image for Windows (Scion Corp., Release Beta 4.0.2 MD). These measurements allowed us to calculate the ratio between tail length and head diameter, developmental index DI, which was used to classify the larvae on a linear scale. Tail and head measurements were obtained for 740 larval bullfrogs.
Behavioral experiments were conducted at the Behavioral Testing Facility of the Universidad Central del Caribe (http://www.btfucc.org
). Measurements were made at 22–24°C in an appropriately lighted room during the light cycle. Test animals were placed inside an acoustic chamber to avoid external noise or any other perturbation. The experimental chamber consisted of four open opaque acrylic cylinder containers (19 cm diameter, 1.5 L total volume). A single larva was placed in each container, which had been filled with 500 mL of distilled water - enough water to completely cover the larva’s body but allowing almost no vertical movement (Z≈0). We assumed that only very minor alterations in the acquisition trajectory would result from displacement in the Z coordinate, therefore, trajectory analyses were carried out only in the X and Y coordinates (2D) to yield net body movement over time.
Effect of T3 on buccal activity
T3 was externally administered to larval R. catesbeiana by adding a fixed amount of a stock solution to the bath water for a final concentration of 250 nM T3.
We measured buccal movement frequency in control conditions and after exposure to T3 in order to estimate the time needed to see an effect. In brief, tadpoles were placed in containers as described above and allowing for a 30 min acclimatization period. Buccal movements were then carefully recorded using a video camera attached the stereoscope microscope or by direct visualization for 15 sec periods per minute for a total of 30 min. After this time T3 was added to the water in the container and rapidly mixed to a final concentration of 250 nM, with little or no larval perturbation. Buccal movements were then evaluated for an additional 30 min. All larvae used in this experiment had DI < 2.8.
Protocols for T3 exposure
Two experimental protocols were implemented to evaluate T3 action on larval locomotion: exposure for 24 h to T3, or exposure for 2 h to T3. All tests were performed in groups of four larvae since that was the maximum number of animals that we could record simultaneously. In the first protocol, four larvae classified by their DI were placed individually in four containers for 2 h of acclimatization followed by 24 h of continuous control recording of their locomotion. Following acquisition of the control data, three of the four larvae were treated with 250 nM of T3 (Sigma Aldrich, St Louis, MO, USA) and patterns of locomotion were again measured for 24 h. After the first 24 h control period the fourth larva was exposed to the vehicle only, and served as the sham control for the next 24 h recording. Thus, each test larva served as its own control. The second protocol was identical to the first, except for the duration of the control period (2 h) and exposure period (2 h).
Larvae in the stages prior to metamorphosis were classified according to DI. Of the 187 larvae examined, 67 had a DI ≥2.8 and 120 had a DI < 2.8. This latter group included at least 8 animals with apparent complete limb bud development. These groups were evaluated in terms of locomotor activity after exposure to T3.
Assessing locomotor activity
Locomotor activity of the larvae was assessed by a video tracking system designed specifically for the automation of behavioral experiments (Ethovision©, v. 3.0 Noldus, Netherlands). Larval locomotion was recorded using a digital camera connected to a computer, which detected each larval movement as a displacement with a sampling rate of 9 positions/sec (inter-point interval=111 ms).
Measured locomotor behavior parameters were determined as follows:
- Displacement (movement): Distance traveled by the larva’s center of gravity of the specimen between two sampling periods:
DM = Distance moved from sample n−1 to sample n.
Xn−1, Yn−1 = X, Y coordinates of objects at sample n−1.
Xn, Yn = X, Y coordinates of objects at sample n.
- Velocity: Distance traveled by the larva per unit time; i.e. the linear velocity in the X,Y plane (Eq. 2). Velocity was determined only during the time intervals in which the larva was actively moving. Thus, we generated the average velocity during displacement. The formula used was:
V = velocity
DM = distance moved
Tail preparations for electrophysiological recording
Larvae were placed in cold (3–5 °C) saline until no movement was apparent and then decapitated. The skin of the tail was carefully removed to expose the underlying muscle fibers and longitudinal slices were obtained for use in the electrophysiological experiments.
Focal recording of miniature endplate currents
MEPCs, resulting from spontaneous neurotransmitter release, were recorded from tail muscles of larval Rana catesbeiana
having DI < 2.8 and DI ≥ 2.8. The caudal muscle fibers used were between 20–50 µm in diameter and 200–500 µm in length, and the fiber surface is relatively clean allowing MEPCs to be focally recorded from discrete spots (Quiñonez, Romero and Rojas, 1996
; Rojas, Bonilla, Báez, and Lasalde, 2003
Miniature endplate current (MEPC) recordings were performed at the ends of the muscle fibers where the nerve terminals and acetylcholine receptors (AChRs) are located (Rojas, Bonilla, Báez, Lasalde-Dominicci, 2003
). Typically, then, myotomes 3 to 4 of the rostral segment were used to record MEPCs. Frog saline composition (in mM) was NaCl 125, KCl 6, CaCl2
1.8, Hepes 10 with 100 nM TTX, adjusted to pH of 7.2 and with osmolarity of 255 mOsm/kg water. Hypertonic sucrose solution was prepared by adding sucrose to the normal frog saline up to a final sucrose concentration of 100 mM. All solutions were prepared daily.
Pipettes for focal recording were pulled using a P-87 puller (Sutter Instruments, Novato, California USA) and fire-polished using a micro-forge (Narashige Scientific Instrument Lab, Tokyo, Japan), to obtain a tip diameter between 9 and 14 µm. The pipettes were constructed using soft glass to avoid the channel rundown encountered when hard glass is used in embryonic muscle cells (Rojas and Zuazaga, 1988
; Rojas, Bonilla, Báez, and Lasalde, 2003
). Pipettes were filled with saline solution.
Focal recordings of MEPCs were obtained using pipette electrodes attached to an amplifier (Quiñones, Romero, and Rojas, 1996; Rojas, Bonilla, Báez, and Lasalde, 2003
). A patch amplifier (GeneClamp 500, Axon Instruments Inc., Foster City, CA, USA) was used with a feedback resistor of 1 GΩ to record MEPCs in the macropatch configuration. The MEPCs were acquired using an A/D converter (Series E, National Instrument, Austin, Texas, USA) in a PC computer. The signals were continuously recorded using the program Electrophysiological Digital Recording WinEDR v2.3.9. (Dempster J., http://spider.science.strath.ac.uk/PhysPharm
Solution exchange during MEPC recording
Experiments were conducted with a continuous superfusion of the solutions, allowing for rapid exchange with a set of valves. Stabilization of MEPC frequency was allowed for 10–15 min, after that a control recording was performed. The first 900 sec of acquisition of control MEPCs in normal saline were followed by 900 sec of acquisition in hypertonic saline (containing 100 mM sucrose). Finally, a 900 sec recovery period was obtained during washout with normal saline solution.
Statistical analyses were performed with SYSTAT (SPSS Inc. v10, Chicago, IL, USA) and GraphPad Prism (v.4 GraphPad Software Inc. San Diego, CA, USA). Mean values ± Standard Deviation (n) are shown. A Kruskal-Wallis test was used to compare populations. A fiduciary level of 0.1% was used for all statistical tests.