- Email: [email protected]
Acetylcholinesterase concentration in the optic tectum and in the two main cerebellar subdivisions of three freshwater and three marine teleosts
182
Brain Research, 157 11978) 182 1~5 i) Elsevier/North-Holland Bitmmdical Pre~s
Acetylcholinesterase concentration in the optic tectum and in the two main cerebellar subdivisions of three freshwater and three marine teleosts
ANTONIO CONTESTABILE
Institute of Comparative Anatomy, Universityof Bologna, 40126 Bologna (Italy) (Accepted July 20th, 1978)
Acetylcholinesterase (ACHE) content has been measured in the whole brain of some teleosts 1,13,2a. The data from whole brain do not help in understanding the true role of the enzyme in neural mechanisms, because of the marked differences in AChE activity among various nervous regions as suggested by histochemical resultsZ-L The histochemical studies also revealed that the same brain region may possess different AChE activity in different teleost species. This is the case for the cerebellum, in which AChE activity varies greatly not only among different species, but also in different parts of the same cerebellum 4,5. A second highly developed nervous region of teleost brain, the optic tectum, exhibits a more uniform pattern of AChE activity, but here again the histochemical picture sometimes suggests quantitative differences in enzyme concentration among different species 5,6. With these considerations in mind, the present study is concerned with biochemical determination of AChE in the optic tectum and in the two main cerebellar subdivisions of 3 freshwater and 3 marine teleosts. The freshwater species used were: goldfish (Carassius auratus), catfish (lctalurus nebulosus) and rainbow trout (Salmo gairdneri). The marine species were: mullet (Mugil cephalus), mackerel (Scomber scombrus) and goby (Gobius Paganellus). The freshwater species came from fish breeders and the marine ones from the Adriatic sea. The animals were killed by decapitation, the brains were immediately removed from the skull and the different brain regions were dissected out. Enzyme assays were carried out on the two main subdivisions of the teleost cerebellum: they are the basal lobe, including eminentiae granulares and the valvula cerebelli, and the dorsal lobe, made up of the dorsally expanded part of the corpus cerebelli. The optic tectum was dissected free of subtectal structures, in particular of the torus semicircularis. The different specimens of brain tissue were weighed and homogenized in ice-cold 20 mM potassium phosphate buffer at pH 7 (8-20 mg fresh tissue/ml). Aliquots of the homogenates were assayed for AChE by the colorimetric method of Hestrin 12. Samples were preincubated for 15 min in 20 m M potassium phosphate buffer with 2 :x: 10 -5 M iso-OMPA, a selective pseudocholinesterase inhibitor, and then incubated for 10-20 rain in the complete medium containing the same inhibitor concentration and 2.7 m M acetylcholine iodide, 100 m M NaC1, 20 mM MgM12 in 20 m M potassium
183 TABLE I
AChE concentration in the two cerebellar subdivisions Each value is the mean of assays carried out on 4 different animals ± S.E. The results are expressed as /~mole substrate hydrolysed/mg protein/h. P value is the degree of significance of the differences in A C h E concentration between the two cerebellar subdivisions, as determined by the Student t-test. Assay temperature was 28 °C.
Dorsal lobe
Basal lobe
P value
Freshwater species Carassius Salmo lctalurus
13.52 ± 0.69 6.55 ± 0.32 28.45 ± 2.02
14.85 ± 1.27 8.30 ± 0.44 23.65 ± 2.80
> 0.01 < 0.01 > 0.01
Marine species Mugil Scomber Gob±us
10.94 ± 0.74 16.38 ± 0.55 46.22 ± 3.88
14.74 i 1.25 22.62 ± 0.82 32.19 ± 1.73
< 0.01 < 0.01 < 0.01
phosphate buffer at pH 7. Both preincubation and incubation were carried out in test tubes in a thermoregulated water bath at 28 °C with continuous stirring of tubes. The concentration of proteins was determined in each homogenate by the Hartree 1° modification of the method of Lowry et al. 19. The results from enzyme assays in the 3 brain regions of each teleost species are summarized in Tables I and ih In the cerebellum the overall enzyme concentration varies considerably among the different species. This variability appears to be in good agreement with the pictures obtained by histochemical methods under optical and electron microscopy<5, 7. The results seem to suggest that cerebellar AChE is not directly correlated with the swimming performance. The highest concentration is, in fact, present in the sedentary Gob±us, followed by Ictalurus, while the best swimmer of the marine group, the mackerel, has the lowest. Similarly among the freshwater species the rainbow trout, which is the best swimmer, has the lowest AChE value. T A B L E il
AChE concentration in the optic tectum Same indications as for Table I.
Optic tectum Freshwater .species Carassius Salmo Ictalurus
35.35 ± 1.90 14.18 ± 0.39 33.85 i 3.02
Marine species Mugil Scomber Gob±us
29.41 £ 2.77 22.21 ± 2.61 31.24 ± 1.78
184 The differences between the AChE concentration in the basal lobe plus valvul~ and that in the dorsal lobe vary among the fishes examined, and these quanlitativc differences largely support the previous histochemical observations4, 5. In keeping wJl h the histochemical finding that, in the rainbow trout, the mullet and the mackerel, the AChE staining was less in the dorsal lobe than in the basal lobe, a significantly greater activity was found in the latter region. This higher AChE activity seems linked to the presence of well-developed eminentiae granulates and valvula ccrebelli, ill the goldfish, in which the valvula is well developed but the eminentiae are reduced, the difference in AChE concentration between the two main cerebellar subdivisions is not statistically significant. In the species in which the basal structures are reduced or very small, the difference between the two cerebetlar subdivisions is not significant (lctalurus) or is even reversed (Gobius). The differences found may be functionally important. In teleosts, a clear distinction can be made between the ba~al lobe plus valvula, which almost exclusively receives laterovestibular fibres, and the dorsal lobe, which is predominently linked to spinal, trigeminal and tectal afferentCr.Js,e~L Since the pars vestibulolateralis appears to possess a significantly higher AChE concentration in those species in which the structures constituting the lobe are more developed, this may mean that the laterovestibular input is cholinergic. The AChE concentrations in the optic tectum show a more uniform pattern compared with that in the cerebellum, and 4 species, the goldfish, the catfish, the mullet and the goby give similar values for enzyme activity. It is interesting to note that, in two of these species, the goldfish and the mullet, the eyes and optic tectum are well developed while, in the other two, both structures are much less developed. By contrast, the mackerel, and particularly the trout with highly developed eyes and tecta, show the lowest AChE value. Thus, as in the cerebellum, the enzyme level seems not to be directly proportional to the main function controlled by the nerve center. These different levels of AChE activity appear to support the hypothesis, derived from previous experimental and ultrastructural studies 2,8,~1, that cholinergic mechanisms in the optic tectum are linked to intrinsic control circuits rather than with the input from the retina. The present study confirms the great variability in brain levels of AChE among teleosts. In addition, the AChE concentration in specific brain regions does not appear to be directly correlated with the main functional activity controlled by the nerve center considered, with the possible exception of laterovestibular projection to the cerebellar basal lobe. These observations raise various possibilities: AChE might be connected with subsidiary neural mechanisms which are very different in the various species; different rates of AChE synthesis and utilization might occur; AChE production might be sometimes redundant compared with the actual use for nervous function; AChE might be involved in non-nervous functions. Some of these possibilities may coexist, and experimental support l\~r each of them derives from previous studiesT-9,14 16,21,22.
185 1 Baldwin, J. and Hochachka, P. W., Functional significance of isoenzymes in thermal acclimatization. Acetylcholinesterase from trout brain, Biochem. J., 116 (1970) 883 887. 2 Ciani, F., Contestabile, A. and Villani, L., Acetylcholinesterase activity in the normal and retinodeprived optic tectum of the quail. Light and electron microscope histochernistry and biochemical determination, in preparation. 3 Contestabile, A., Histochemical study on the distribution of some enzyme activities in the vagal and facial lobes of the goldfish, Carassius auratus, Histochemisto', 44 (1975) 123 132. 4 Contestabile, A., Histochemical localization of acetylcholinesterase in the cerebellum of some seawater teleosts, Brain Research, 99 (1975) 425-429. 5 Contestabile, A. and Zannoni, N., Histochemical localization of acetylcholinesterase in the cerebellum and optic tectum of four freshwater teleosts, Histochemistry, 45 (1975) 279-288. 6 Contestabile, A., Laminar acetylcholinesterase localization in the optic tectum of five seawater teleosts, Experientia (Basel), 32 (1976) 625 626. 7 Contestabile, A., Villani, L. and Ciani, F., Ultrastructural analysis on acetylcholinesterase localization in the cerebellar cortex of teleosts, Anat. Embryol., 152 (1977) 15 27. 8 Contestabile, A., Ercolessi, M. and Bellucci, A., Acetylcholinesterase decrease in the optic lobe after unilateral eye deprivation, Experientia (Basel), in press. 9 Flumerfelt, B. A., Lewis, P. R. and Gwyn, D. G., Cholinesterase activity of capillaries in the rat brain. A light and electron microscope study, Histochem. J., 5 (1973) 67 77. 10 Hartree, E. F., Determination of protein: a modification of the Lowry method that gives a linear photometric response, Analyt. Biochem., 48 (1972) 422 427. 1 I Henke, H. and Fonnum, F., Topographical and subcellular distribution of choline acetyltransferase and glutamate decarboxylase in pigeon optic tectum, J. Neurochem., 27 (1976) 387 391. 12 Hestrin, S., The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application, J. biol. Chem., 180 (1949) 249-261. 13 Hogan, J. W., Brain acetylcholinesterase from cutthroat trout, Trans. Amer. Fish. Soc., 100 (1971) 672 675. 14 Joo, F. and Csillik, B., Topographical correlation between the hematoencephalic barrier and the cholinesterase activity of the brain capillaries, Exp. Brain Res., 1 (1966) 147-151. 15 Koelle, J. B., The histochemical identification of acetylcholinesterase, in cholinergic, adrenergic and sensory neurons, J. Pharmacol. exp. Ther., 114 (1955) 167-184. 16 Kreutzberg, G. W. and Toth, L., Dendritic secretion: a way for the neuron to communicate with the vasculature, Naturwissensehafien, 61 (1974) 37. 17 Kuhlenbeck, H., The Central Nervous System q[' Vertebrates, Vol. 4, Spinal Cord and Deuterencephalon, Karger, Basel, 1975, 1006 pp. 18 Larsell, O., The Comparative Anatomy and Histology of the cerebellum Jkom Myxinids Through Birds, University of Minnesota Press, Minneapolis, 1967. 19 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 20 Nieuwenhuys, R., Comparative anatomy of the cerebellum. In C. A. Fox and R. S. Snyder (Eds.), The Cerebellum, Progr. Brain Res., Vol. 18, Elsevier, Amsterdam, 1967, pp. 1-93. 21 Shen, S. C., Greenfield, P. and Boell, E. J., The distribution of cholinesterase in the frog brain, J. comp. Neurol., 102 (1955) 717-744. 22 Silver, A., Cholinesterase of the central nervous system with special reference to the cerebellum, lut. Rev. Neurobiol., 10 (1967)57-109. 23 Studnicka, M. and Svobodova, Z., Acetylcholine hydrolase activity in fish brain, Bull. VURH Vodn., 9 (1973) 3 ~6.
Brain Research, 157 11978) 182 1~5 i) Elsevier/North-Holland Bitmmdical Pre~s
Acetylcholinesterase concentration in the optic tectum and in the two main cerebellar subdivisions of three freshwater and three marine teleosts
ANTONIO CONTESTABILE
Institute of Comparative Anatomy, Universityof Bologna, 40126 Bologna (Italy) (Accepted July 20th, 1978)
Acetylcholinesterase (ACHE) content has been measured in the whole brain of some teleosts 1,13,2a. The data from whole brain do not help in understanding the true role of the enzyme in neural mechanisms, because of the marked differences in AChE activity among various nervous regions as suggested by histochemical resultsZ-L The histochemical studies also revealed that the same brain region may possess different AChE activity in different teleost species. This is the case for the cerebellum, in which AChE activity varies greatly not only among different species, but also in different parts of the same cerebellum 4,5. A second highly developed nervous region of teleost brain, the optic tectum, exhibits a more uniform pattern of AChE activity, but here again the histochemical picture sometimes suggests quantitative differences in enzyme concentration among different species 5,6. With these considerations in mind, the present study is concerned with biochemical determination of AChE in the optic tectum and in the two main cerebellar subdivisions of 3 freshwater and 3 marine teleosts. The freshwater species used were: goldfish (Carassius auratus), catfish (lctalurus nebulosus) and rainbow trout (Salmo gairdneri). The marine species were: mullet (Mugil cephalus), mackerel (Scomber scombrus) and goby (Gobius Paganellus). The freshwater species came from fish breeders and the marine ones from the Adriatic sea. The animals were killed by decapitation, the brains were immediately removed from the skull and the different brain regions were dissected out. Enzyme assays were carried out on the two main subdivisions of the teleost cerebellum: they are the basal lobe, including eminentiae granulares and the valvula cerebelli, and the dorsal lobe, made up of the dorsally expanded part of the corpus cerebelli. The optic tectum was dissected free of subtectal structures, in particular of the torus semicircularis. The different specimens of brain tissue were weighed and homogenized in ice-cold 20 mM potassium phosphate buffer at pH 7 (8-20 mg fresh tissue/ml). Aliquots of the homogenates were assayed for AChE by the colorimetric method of Hestrin 12. Samples were preincubated for 15 min in 20 m M potassium phosphate buffer with 2 :x: 10 -5 M iso-OMPA, a selective pseudocholinesterase inhibitor, and then incubated for 10-20 rain in the complete medium containing the same inhibitor concentration and 2.7 m M acetylcholine iodide, 100 m M NaC1, 20 mM MgM12 in 20 m M potassium
183 TABLE I
AChE concentration in the two cerebellar subdivisions Each value is the mean of assays carried out on 4 different animals ± S.E. The results are expressed as /~mole substrate hydrolysed/mg protein/h. P value is the degree of significance of the differences in A C h E concentration between the two cerebellar subdivisions, as determined by the Student t-test. Assay temperature was 28 °C.
Dorsal lobe
Basal lobe
P value
Freshwater species Carassius Salmo lctalurus
13.52 ± 0.69 6.55 ± 0.32 28.45 ± 2.02
14.85 ± 1.27 8.30 ± 0.44 23.65 ± 2.80
> 0.01 < 0.01 > 0.01
Marine species Mugil Scomber Gob±us
10.94 ± 0.74 16.38 ± 0.55 46.22 ± 3.88
14.74 i 1.25 22.62 ± 0.82 32.19 ± 1.73
< 0.01 < 0.01 < 0.01
phosphate buffer at pH 7. Both preincubation and incubation were carried out in test tubes in a thermoregulated water bath at 28 °C with continuous stirring of tubes. The concentration of proteins was determined in each homogenate by the Hartree 1° modification of the method of Lowry et al. 19. The results from enzyme assays in the 3 brain regions of each teleost species are summarized in Tables I and ih In the cerebellum the overall enzyme concentration varies considerably among the different species. This variability appears to be in good agreement with the pictures obtained by histochemical methods under optical and electron microscopy<5, 7. The results seem to suggest that cerebellar AChE is not directly correlated with the swimming performance. The highest concentration is, in fact, present in the sedentary Gob±us, followed by Ictalurus, while the best swimmer of the marine group, the mackerel, has the lowest. Similarly among the freshwater species the rainbow trout, which is the best swimmer, has the lowest AChE value. T A B L E il
AChE concentration in the optic tectum Same indications as for Table I.
Optic tectum Freshwater .species Carassius Salmo Ictalurus
35.35 ± 1.90 14.18 ± 0.39 33.85 i 3.02
Marine species Mugil Scomber Gob±us
29.41 £ 2.77 22.21 ± 2.61 31.24 ± 1.78
184 The differences between the AChE concentration in the basal lobe plus valvul~ and that in the dorsal lobe vary among the fishes examined, and these quanlitativc differences largely support the previous histochemical observations4, 5. In keeping wJl h the histochemical finding that, in the rainbow trout, the mullet and the mackerel, the AChE staining was less in the dorsal lobe than in the basal lobe, a significantly greater activity was found in the latter region. This higher AChE activity seems linked to the presence of well-developed eminentiae granulates and valvula ccrebelli, ill the goldfish, in which the valvula is well developed but the eminentiae are reduced, the difference in AChE concentration between the two main cerebellar subdivisions is not statistically significant. In the species in which the basal structures are reduced or very small, the difference between the two cerebetlar subdivisions is not significant (lctalurus) or is even reversed (Gobius). The differences found may be functionally important. In teleosts, a clear distinction can be made between the ba~al lobe plus valvula, which almost exclusively receives laterovestibular fibres, and the dorsal lobe, which is predominently linked to spinal, trigeminal and tectal afferentCr.Js,e~L Since the pars vestibulolateralis appears to possess a significantly higher AChE concentration in those species in which the structures constituting the lobe are more developed, this may mean that the laterovestibular input is cholinergic. The AChE concentrations in the optic tectum show a more uniform pattern compared with that in the cerebellum, and 4 species, the goldfish, the catfish, the mullet and the goby give similar values for enzyme activity. It is interesting to note that, in two of these species, the goldfish and the mullet, the eyes and optic tectum are well developed while, in the other two, both structures are much less developed. By contrast, the mackerel, and particularly the trout with highly developed eyes and tecta, show the lowest AChE value. Thus, as in the cerebellum, the enzyme level seems not to be directly proportional to the main function controlled by the nerve center. These different levels of AChE activity appear to support the hypothesis, derived from previous experimental and ultrastructural studies 2,8,~1, that cholinergic mechanisms in the optic tectum are linked to intrinsic control circuits rather than with the input from the retina. The present study confirms the great variability in brain levels of AChE among teleosts. In addition, the AChE concentration in specific brain regions does not appear to be directly correlated with the main functional activity controlled by the nerve center considered, with the possible exception of laterovestibular projection to the cerebellar basal lobe. These observations raise various possibilities: AChE might be connected with subsidiary neural mechanisms which are very different in the various species; different rates of AChE synthesis and utilization might occur; AChE production might be sometimes redundant compared with the actual use for nervous function; AChE might be involved in non-nervous functions. Some of these possibilities may coexist, and experimental support l\~r each of them derives from previous studiesT-9,14 16,21,22.
185 1 Baldwin, J. and Hochachka, P. W., Functional significance of isoenzymes in thermal acclimatization. Acetylcholinesterase from trout brain, Biochem. J., 116 (1970) 883 887. 2 Ciani, F., Contestabile, A. and Villani, L., Acetylcholinesterase activity in the normal and retinodeprived optic tectum of the quail. Light and electron microscope histochernistry and biochemical determination, in preparation. 3 Contestabile, A., Histochemical study on the distribution of some enzyme activities in the vagal and facial lobes of the goldfish, Carassius auratus, Histochemisto', 44 (1975) 123 132. 4 Contestabile, A., Histochemical localization of acetylcholinesterase in the cerebellum of some seawater teleosts, Brain Research, 99 (1975) 425-429. 5 Contestabile, A. and Zannoni, N., Histochemical localization of acetylcholinesterase in the cerebellum and optic tectum of four freshwater teleosts, Histochemistry, 45 (1975) 279-288. 6 Contestabile, A., Laminar acetylcholinesterase localization in the optic tectum of five seawater teleosts, Experientia (Basel), 32 (1976) 625 626. 7 Contestabile, A., Villani, L. and Ciani, F., Ultrastructural analysis on acetylcholinesterase localization in the cerebellar cortex of teleosts, Anat. Embryol., 152 (1977) 15 27. 8 Contestabile, A., Ercolessi, M. and Bellucci, A., Acetylcholinesterase decrease in the optic lobe after unilateral eye deprivation, Experientia (Basel), in press. 9 Flumerfelt, B. A., Lewis, P. R. and Gwyn, D. G., Cholinesterase activity of capillaries in the rat brain. A light and electron microscope study, Histochem. J., 5 (1973) 67 77. 10 Hartree, E. F., Determination of protein: a modification of the Lowry method that gives a linear photometric response, Analyt. Biochem., 48 (1972) 422 427. 1 I Henke, H. and Fonnum, F., Topographical and subcellular distribution of choline acetyltransferase and glutamate decarboxylase in pigeon optic tectum, J. Neurochem., 27 (1976) 387 391. 12 Hestrin, S., The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application, J. biol. Chem., 180 (1949) 249-261. 13 Hogan, J. W., Brain acetylcholinesterase from cutthroat trout, Trans. Amer. Fish. Soc., 100 (1971) 672 675. 14 Joo, F. and Csillik, B., Topographical correlation between the hematoencephalic barrier and the cholinesterase activity of the brain capillaries, Exp. Brain Res., 1 (1966) 147-151. 15 Koelle, J. B., The histochemical identification of acetylcholinesterase, in cholinergic, adrenergic and sensory neurons, J. Pharmacol. exp. Ther., 114 (1955) 167-184. 16 Kreutzberg, G. W. and Toth, L., Dendritic secretion: a way for the neuron to communicate with the vasculature, Naturwissensehafien, 61 (1974) 37. 17 Kuhlenbeck, H., The Central Nervous System q[' Vertebrates, Vol. 4, Spinal Cord and Deuterencephalon, Karger, Basel, 1975, 1006 pp. 18 Larsell, O., The Comparative Anatomy and Histology of the cerebellum Jkom Myxinids Through Birds, University of Minnesota Press, Minneapolis, 1967. 19 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 20 Nieuwenhuys, R., Comparative anatomy of the cerebellum. In C. A. Fox and R. S. Snyder (Eds.), The Cerebellum, Progr. Brain Res., Vol. 18, Elsevier, Amsterdam, 1967, pp. 1-93. 21 Shen, S. C., Greenfield, P. and Boell, E. J., The distribution of cholinesterase in the frog brain, J. comp. Neurol., 102 (1955) 717-744. 22 Silver, A., Cholinesterase of the central nervous system with special reference to the cerebellum, lut. Rev. Neurobiol., 10 (1967)57-109. 23 Studnicka, M. and Svobodova, Z., Acetylcholine hydrolase activity in fish brain, Bull. VURH Vodn., 9 (1973) 3 ~6.