Interspecific Differences in the Reaction to Atropine and in the Histology of the Esophagi of the Common California Sea Hares of the Genus Aplysia

LINDSAY R. WINKLER and BERNARD E. TILTON!

DURING A STUDY of the effects of certain cho­ through two 5 gal. carboys maintained in a re­ linergic agents on the tissues of Aplysia, it was frigerator. It was thus possible to keep the water noted that the esophagi of the two California clean and cooled to approximately the tempera­ species (A. califarnica and A. vaccaria) reacted ture of the intertidal environment of these ani­ divergently to atropine. This is of interest to mals. Parsley obtained in the local market was both the taxonomy and physiology of the genus eaten in quantity by A. califarnica but was re­ as well as potentially to a better understanding fused by A. vaccaria. Consequently all speci­ of the mode of action of atropine. Other drugs mens of the latter were used as soon as prac­ commonly known to show activity on muscle ticable. tissue were also tested on the two species to Animals were sacrificed by incising the entire determine if any other interspecifically diver­ length of the foot, turning the animal inside out gent reactions existed. These pharmacological and removing the esophagus after tying it at reactions will be reported later. both ends. The esophagi were suspended from Botazzi (1898) observed the periodic con­ a plastic holder in conventional baths using 30 tractions of the esophagus of the European ml. of sea water. The movable end of the esoph­ Aplysia and made a thorough study of its phys­ agus was ligated to a Grass force-displacement iology. The physiology of the crop was studied transducer, which was connected-to a Grass am­ extensively by von B~iicke (1905). Straub plifier-recorder. (1907) reported the reaction of the heart of an To identify the two types of excursions noted unspecified species of Aplysia to muscarine and in the tracings, cross sections of esophagus tis­ noted the lack of atropine antagonism in the sue were tied on opposite sides of the ring thus heart ventricle. Hogben (1924) studied the re­ formed so as to obtain tracings of the contrac­ action of the crop of an European Aplysia to tions of the circular muscle with little or no epinephrine. effect from the longitudinally oriented tissue. At­ ropine was used in 1: 1000 solution (1 mg/ml) MATERIALS AND METHODS in all cases. Small- to medium-size specimens of Aplysia For histological examination both longitudi­ nal and cross sections were made using standard (Neaplysia) califarnica Cooper and Aplysia techniques. These were stained with eosin and (Aplysia) vaccaria Winkler were collected dur­ hematoxylin. Sections were also made of all the ing the summer months at Lunada Bay, Palos noticeably differentiated areas of the digestive Verdes, Los Angeles Co., California. The ani­ tract. mals were packed in wet Pelvetia fastigata and transported to the laboratory where they were EXPERIMENTAL RESULTS maintained in a 10-gal. salt water aquarium until needed. A Cole-Parmer model all-plastic The normal pattern of contraction as re­ impeller-type pump drew the water from the corded from the isolated esophagus consists pri­ aquarium through a filter containing glass wool marily of comparatively rapid short excursions and activated charcoal and pumped it serially which vary in frequency and amplitude. These may be interspersed with occasional contractions 1 Departmenr of Pharmacology, Lorna Linda Uni­ versity School of Medicine, Lorna Linda, California. of greater amplitude, especially in A. califarnica Manuscript received January 13, 1961. (Fig. 1a). Since the latter contractions were ab-

557 558 PACIFIC SCIENCE, Vol. XV, October 1961

FIG. 1. The-normal tracing of the esophagus of A. cali/arnica is indicated in a and the effect produced by 0.2 mg. of atropine solution added to the 30 ml. bath is shown at the arrow: The depressing of the circular contractions of a ring of esophagus by 0.2 mg. atropine is shown in b. Tracings c and d show two types of normal tracings obtained from A. vaccaria esophagus and the effect of 0.4 mg. atropine added at arrow. sent when the rings of esophagus were used (Fig. Ib). This depression was not noticed in (Fig. Ib), they are attributed to longitudinally the usual tracings of A. cali/arnica muscle since oriented muscle in contrast to a primary circular it was obscured by the longitudinal contractions. muscle response. For some unknown reason the Other muscle-active drugs with specific reac­ rapidity of contraction was increased when the tions of interest in themselves (which are to esophagus was arranged in rings. The deep, in­ be reported later) did not produce divergent termittent excursions are not as pronounced in results between the two species. A. vaccaria (Fig. lc) and may be absent (Fig. In an attempt to gain an insight into possi­ Id). The response of circular muscle, however, ble reasons for the divergence of reaction be­ is consistently more rapid and more pronounced tween the two species, histological sections were than is the case in A. cali/arnica. made and stained with eosin and hematoxylin. In A. cali/arnica even very small doses (0.05 Strongly eosinophilic bundles of coarse, cylin­ mg. in some cases) of atropine produce an im­ drical muscle strands were observed in the mediate and unfailing but transitory contraction esophagus of A. cali/arnica (Fig. 2a, b). These of the longitudinal muscle (Fig. la). The esoph­ strands appear coarsely striated in some prep­ agus of Aplysia vaccaria, however, does not react arations (Fig. 2b) and coarsely granular in to atropine except in comparatively large doses others, the differences possibly representing of 0.4 ml. or more (in a 30 ml. bath), in which problems in killing and fixing. The nuclei are case the excursions of the circular muscle are larger and more sparse than those of A. vaccaria depressed (Fig. Id). The circular muscle of A. (Fig. 2c, d) and of the surrounding more conven­ cali/arnica, as demonstrated by the muscular tional muscle of the present species. Not only ring preparations, is noticeably depressed by at­ are the nuclei three times as large but they tend ropine in even smaller doses than that required to be arranged across the muscle strands, thus to depress the circular type muscle in A. vaccaria appearing rectangular in section. This muscle is Differences in Sea Hares-WINKLER and TILTON 559 contrasted with the less eosinophilic, more un­ diate cause of the divergent pharmacological dulatory muscle of the circular (Fig. 2a) and reactions observed, they are better interpreted as A. vaccaria type muscle (Fig. 2c, d), which is visible, easily demonstrable, morphological dif­ very heavily nucleated. While strands of a type ferences which parallel and are closely associated of muscle which seems morphologically to be with neurohumoral and even biochemical dif­ somewhat similar to the former type of muscle ferences which are themselves the underlying appear sparsely in the crop, and to even a lesser mechanistic causes. This is especially true since extent in the area between the gizzard and the effect of atropine on conventional tissue sys­ "stomach" of the digestive tract in A. califarnica, tems is thought to be on the neuromuscular re­ none of the eosinophilic muscles observed in ceptors. The contractatory response observed in other parts of the digestive tract of A. vaccaria A. califarnica appears to be the transitory result possessed the distinctive tubular shape with the of stimulation, since the mechanical stimulation vacuolation, striation or granulation, and lack of washing will produce similar contractions. of undulatory characteristics. However, the sensitivity to this chemical stimu­ lation is very great, being as low as 0.05 mg. DISCUSSION AND CONCLUSIONS atropine placed in the 30 ml. bath-a concen­ 5 While it might be tempting to assume that tration of 1:6xl0 . Such a sensitivity represents the histological differences represent the imme- a very delicately balanced chemical system.

FIG. 2. Ao. cali/arnica: a, cross section; h, longitudinal section. A. t'accaria: c, longitudinal section; d, cross .~ction. The midsection of the esophagi were used for cross sections. 560 PACIFIC SCIENCE, Vo!. XV, October 1961

Moreover, the mechanism is of special interest SUMMARY since it is unique among known muscle prep­ arations in that it is stimulated rather than de­ 1. The normal pattern of esophageal contrac­ pressed by atropine. tions in Aplysia californica consists of short The drug reaction and histological difference rapid contractions attributed to circular muscle, accentuates the divergence present within the interspersed at intervals by deep excursions at­ genus. A. vaccaria belongs to a large and wide­ tributed to the longitudinal musculature. These spread subgenus (Aplysia) , while A. californica secondary contractions are less pronounced and less regular in A. vaccaria. (subgenus Neaplysia) is unique to the Califor­ 2. Atropine causes marked contraction of the nias. When it becomes possible to study com­ esophagus in A. californica, even in low con­ paratively the members of the two other sub­ centration, but in A. vaccaria it depresses circu­ genera2 (Varria and Pruvataplysia) , a taxonomic lar activity in higher concentrations. generalization may be possible. However, Bot­ 3. The esophagus of A. californica has longi­ tazzi (1898) in his physiological study of the tudinal bundles of cylindrical muscle appearing esophagus indicated that A. limacina (almost coarsely striated or granular. These have not certainly A. fasciata of subgenus Varria) was been found in A. vaccaria, and are suggested as much more atonic than the close relative of A. being associated with the mechanistic cause for vaccaria (subgenus Aplysia) with which he also the differences in reaction noted. worked. Since it is thought that A. californica is an offshoot of this subgenus Varria, and since REFERENCES its esophagus is far from atonic by any inter­ pretation, it may De that the musculature of the BOTTAZZI, PHILLIP. 1898. Contributions to the former has diverged considerably from its fore­ physiology of unstr-iated muscular tissue, Part runners. IV. The action of electrical stimuli upon the It would also seem quite probable that more oesophagus of Aplysia depilans and Aplysia comparative studies of the tissues of the species limacina. Jour. Physio!. 22: 481-506. contained in other genera of animals would re­ COOPER, J. G. 1863. On new and rare mollusca veal occasional divergences of at least equal inhabiting the coast of California. Proc. Calif. magnitude. Such a possibility cannot safely be Acad. Sci. 3: 57. overlooked in experimental biology in any of HOGBEN, LANCELOT T. 1924. Studies on inter­ its phases, and accentuates the need for accurate nal secr,etion, III. The action of pituitary ex­ taxonomy before proceeding with studies which tract and adrenaline on contractile tissues of may be of a nontaxonomic nature. certain invertebrata. J. Exp. Bio!. 1: 487-500. STRAUB, WALTHER. 1907. Zur chemischen " Specimens of esophagus tissue from these subgen­ Kinetic der Muscarinwirkung. Pfluger's Ar­ era were kindly supplied from existing museum mate­ chiv. 119. rial on hand by Dr. N. B. Eales but, unfortunately, VON BRUCKE, E. Th. 1905. Zur Physiologie der rhe conventional preservation method employed by the collectors was not adequate for any type of conclu­ Kropfmuskulatur von Aplysia. Pfluger's Ar­ SIOns. chiv. 108.