The Neuroanatomy of Hymenolepis diminuta and H. nana

Abstract

Using a modification of the Koelle-Gomori method for the histochemical localization of cholinesterases, the distribution of a specific acetylcholinesterase has been determined in whole adult Hymenolepis diminuta and Hymenolepis nana. The specificity of the enzyme was established by comparing the rates of cholinesterase activity using esters of acetylthiocholine and butyrylthiocholine. The presence of acetylcholinesterase, and its known association with the mechanism of neurotransmission, has made it possible to define and compare the neuroanatomy of these species. The gross neuroanatomy of H. diminuta and to a lesser extent, that of H. nana, is described. The results suggest that the specific cholinesterase activity observed in these organisms can be correlated either with a neuromuscular function, or with the presence of neurosecretory glands and chemoreceptors. Following the histochemical demonstration (Wilson, 1965) of specific acetylcholinesterase in intact specimens of the hymenolepidid cestodes, Hymenolepis diminuta and H. nana, additional evidence for the occurrence of this enzyme in homogenates of the former species has been reported (Graff and Read, 1967). Specific histochemical resolution of the sites of activity of acetylcholinesterase, based on a further modification of the Koelle and Friedenwald (1949)-Gomori (1952) method as described by Bueding et al. (1967), has enabled visualization and comparison of the more prominent features of the central nervous system in these worms. The gross neuroanatomy of H. diminuta, and to a lesser extent, that of H. nnan, is described herein. MATERIALS AND METHODS The cestodes, H. diminuta and H. nana, were reared in experimentally infected Holtzman (Holtzman Co., Madison, Wisconsin) rats and white mice, respectively. At autopsy, adult worms were expelled from the intestine by cautious syringing with either ice-cold distilled water or Krebs-Ringer solution (Fairbair, et al., 1961) containing 0.02 M glucose and adjusted by saturation for 10 min with a gas mixture of 97% N2-3% CO2, followed by the addition of 2.0 g of NaHCO3 and gassing for another 10 min. Immediately after removal from the host, the worms were fixed by immersion for 20 min in ice-cold formaldehyde that had been diluted to 10% with either distilled water or adjusted Krebs-Ringer solution. Received for publication 23 August 1968. * 30 Travistock Place, London. W. C. I. t Department of Pathobiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland. This investigation was supported by Public Health Service Research Grant AI-01508 from the NIAID of the NIH. Solutions for the histochemical resolution of acetylcholinesterase (AChE) in these worms were prepared as described by Bueding et al. (loc. cit.). Modifications of the procedures reported by these authors are included in the following description. After fixation, the worms were placed in fritted glass Biichner funnels (30 ml capacity, coarse porosity) for purposes of washing and incubation. The worms were washed for 15 min in each of 3 successive changes of ice-cold distilled water. After the final washing, they were preincubated at room temperature in 10 ml of Gomori's stock solution (pH 6.8) for periods ranging from 2 to 10 hr and then incubated for periods of 4, 8, 12, and 16 min in either of the 2 substrate solutions, acetylthiocholine or butyrylthiocholine. Thereafter, the worms were washed for 5 min in each of 3 successive changes of a Na2SO4 solution, saturated at room temperature, and then with 70% ethanol saturated with H2S (room temperature) for 20 sec, in 50% ethanol for 2 to 3 min, and finally in distilled water for 5 min. Subsequently, the worms were removed from the Biichner funnels, incubated in 30% acetic acid for 20 min, and then mounted in polyvinylpyrrolidinone for microscopic examination. During incubation with the substrate, the latter is hydrolyzed by the acetylcholinesterase present in the worm to form copper thiocholine and acetic acid. In the presence of hydrogen sulfide, the cupric salt is converted to cupric sulfide, which precipitates at the sites of acetylcholinesterase activity. The results of a series of preliminary experiments indicated that the sharpest definition of nerve tracts, particularly those of the scolex, was obtained when ice-cold distilled water had been used to expel the worms from the host's intestine and when used as a diluent for formalin fixation. On the other hand, when adjusted Krebs-Ringer solution was used for these purposes, the distribution and intensity of acetylcholinesterase-substrate reactivity observed after incubation was more consistent from one worm to the next. Therefore, both procedures were used: the former for the purpose of elucidating details of the neuroanatomy; and the latter for comparing reaction velocities of cholinesterase