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Williams College, Williamstown, MA
When used as a general anesthetic, tricaine significantly alters physiological parameters recorded from supramedullary/dorsal cells of the cunner, Tautogolabrus adspersus. Specifically, tricaine reduces spike height, increases the current needed to elicit an action potential, and blocks afferent input (1). In contrast, equivalent recordings from locally anesthetized fish are not altered in this way. However, although the use of local anesthetic is a better alternative to tricaine general anesthesia for physiological recordings (1), there are limitations. Local anesthetics have a limited lifetime, are difficult to reapply while recording, and can enter the bloodstream.
As in other vertebrates, the telencephalic hemispheres of fish are considered to be the "highest" brain centers. For example, the telencephalon of goldfish has been implicated in spatial and avoidance learning (e.g., 2,3). The removal of the telencephalon under transient tricaine anesthesia is used in lieu of general anesthesia in many laboratories (e.g., 4,5). However, no studies have been conducted to determine whether tricaine—after its removal—affects physiological parameters of neurons whose somata lie within the central nervous system. We have therefore studied whether either the transient use of tricaine or telencephalon removal have any residual effects on resting potential, spike height, and current needed to elicit a spike.
Responses of supramedullary/dorsal cells to depolarizing current pulses and to electrical stimulation of the skin on the right operculum were recorded from cunner, 10.5 ± 1.2 cm (mean ± SD; n = 22) in body length. The fish were either transiently anesthetized with tricaine during the removal of the telencephalic hemispheres, or were not anesthetized.
In the first condition, the fish were initially anesthetized in tricaine (ethyl-m-aminobenzoate; 300 mg/l, Sigma-Aldrich) in seawater adjusted to pH 8. When respiration ceased, the fish were transferred to an operating chamber where tricaine (100 mg/l) in chilled seawater adjusted to pH 8 was recirculated through the mouth and over the gills. Ice packs were placed in the operating chamber on either side of the fish. The skull was removed to expose the telencephalic hemispheres, and these structures were removed. The fish were injected with tubocurarine chloride (0.1 mg/kg) to block neuromuscular transmission, and then the seawater with anesthetic was replaced with anesthetic-free, chilled seawater. Finally, the rostral spinal cord was exposed. In the second condition no anesthetic was used. Fish were injected with tubocurarine chloride and placed in an operating chamber. Their telencephalic hemispheres and the rostral spinal cord were exposed. In seven experiments, the telencephalon was stimulated to determine whether any input to the dorsal cells could be elicited.
Single microelectrode recordings (3 M KCl-filled, 5–20 M
initial resistance) were made from somata of supramedullary/dorsal cell neurons in both anesthetic and anesthetic-free conditions. The recordings were all made within 78 ± 43.4 µm (mean ± SD; n = 26) of the surface of the brain.
After tricaine was used transiently to remove the telencephalic hemispheres, action potentials 102.1 ± 9.4 mV (mean + SD, n = 16) in amplitude could be evoked by 4.5 ± 2.9 nA current (initial recordings were started 68 ± 55 min after removal of tricaine). In anesthetic-free experiments neither the spike height (103.3 ± 12.5 mV, n = 11) nor the current needed to evoke the spike (2.4 ± 1.7 nA) was significantly different (P > 0.05; Bonferroni’s Multiple Comparison Test). In addition, there were no significant differences in resting membrane potential (transient use of tricaine = -74.1 ± 5.8 mV; anesthetic free = -72 ± 7.1 mV). Post-synaptic potentials (PSPs) were readily evoked by stimulation of the skin ofthe right operculum in both conditions. This was not the case in animals under general anesthesia (1; Fig. 1).
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General anesthetics, such as tricaine, are known to reduce sodium currents, and their effects are reversible (6). The transient use of tricaine and the removal of the telencephalic hemispheres in this study appear to have had no residual effect on spike height, on the current needed to elicit an action potential, or on the ability to elicit PSPs from the supramedullary/dorsal cells.
Although Rose (7) provides a compelling argument that it is implausible for fish to experience pain, implausible is not conclusive. Nociceptors have been identified in the trout (8,9). Endogenous opioid peptides (10) and opioid receptors in fish (e.g., 11,12) may serve an anti-nociceptive function as in mammals (11). If there is no telencephalic influence on the system being studied, then the transient use of tricaine followed by removal of the telencephalon serves as a reasonable precaution against the possibility that fish experience pain.
This work was supported in part by Howard Hughes Medical Institute and Essel Foundation grants to Williams College. All experimental procedures were approved by the MBL IACUC.
Literature Cited
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