Brain Research, 113 (1976) 435-440
435
© Elsevier ScientificPublishing Company,Amsterdam- Printed in The Netherlands
Neural regulation of acetylcholinesterase in the superior cervical ganglia and the pineal gland of the rat
AMANDA PELLEGRINODE IRALDI and GEORGINA RODRIGUEZ DE LORESARNAIZ Institute de Biologla Celular, Faeultad de Medicina, Universidad de Buenos Aires, Buenos Aires (Argentina)
(Accepted May 17th, 1976)
Structural, pharmacological and biochemical studies have given evidence of the non-cholinergic nature of pineal nerves coming from the cervical superior ganglia1,14,t5. Furthermore the coexistence of ACHE* and catecholamines in several nervous structures, such as the pineal gland nerves6,t6, has been reported s. In a previous paper we have observed that at 7 days after denervation of the pineal gland (by ganglionectomy of the cervical superior ganglia) and decentralization of the cervical superior ganglia, the activity of AChE decreased about 50 ~o in both organs t6. With a similar approach, experiments at shorter and longer periods were carried out. The results suggest a neural regulation of AChE. Wistar rats weighing about 200 g were used. The rats were divided in three groups; in one of them, afferent fibers to the superior cervical ganglia were sectioned. This is referred to as the 'decentralized' group. In a second group, bilateral removal of the cervical superior ganglia was performed. This is referred to as the'ganglionectomized' group. A third group without surgical procedure was used as 'control'. At different times after operation rats were killed by decapitation and the pineal gland and both superior cervical ganglia were dissected. In the case of the ganglia, special care was taken to eliminate the sheath of connective tissue. Homogenates of the pineal gland were prepared by gentle homogenization in a Potter-Elvejhem microhomogenizer; 4-5 glands in 100 #1 of bidistilled water were used for each homogenate. To prepare the homogenates of the ganglia, 4 ganglia were vigorously homogenized in 200 #1 of bidistilled water; the ganglia homogenates were submitted to a brief centrifugation (10 rain at 1500 rev./min) to remove the connective tissue which resisted the homogenization. Cholinesterase was assayed in triplicate aliquots of the homogenates, with acetylthiocholine or butyrylthiocholine as substrate, according to Ellman et al. 4. ChAc was assayed in triplicate samples of each homogenate with the radio-
* Abbreviationsused: ACHE,acetylcholinehydrolase(EC 3.1.1.7); ChE,acetylcholineacyl-hydrolase (EC 3.1.1.8); ChAt, acetyl CoA: choline O-acetyltransferase(EC 2.3.1.6).
436 TABLE I
Changes in AChE of the pineal gland by ganglionectomy and decentroJizatwn The actwity of AChE was determined m homogenates containing 4-5 pineal glands each; the results were expressed as/~moles of substrate hydrolyzed per mg protein/h at 37 °C. Ganglionectomize.d refers to the bilateral removal of the superior cervical ganglia; decentralized refers to the section of afferent fibers of the superior cervical ganglia. In parentheses are the number of homogenates assayed. Data at 7 days from ref. 16
Time Control (#moles/ after mg protein) operation
Izmoles/mg protein
% variatton f~moles/mgprotein
4h 24 h 7 days 32 days 60 days
0.16 0.15 0.10 0.13 0.12
44* --19" --14"
0.14 0.14 0.18 0.16 0.14
5= 0.018 -- 0.018 -+- 0.040 4, 0.008 4. 0.015
(4) (4) (17) (4) (4)
Ganglionectomized
4. 0.014 4- 0.021 4- 0.021 4-0.019 4. 0.009
(2) (4) (15) (5) (5)
Decentrahzed
0.19 4- 0.038 (14) 0.20 -4- 0.026 (5) 0.19 4, 0.022 (5)
% variatton
4-6 +25* +36*
* P < 0.05. TABLE II
The activity of AChE in the cervical superior ganglia after decentralization The activity of AChE was determined in homogenates containing 4 ganglia each; the results are expressed in/~mole of substrate hydrolyzed per mg protein/h at 37 °C. In parentheses are the number of homogenates assayexl. Data at 7 days from ref. 16.
% variation Time Control Decentralized ( l~moles/mg protein) ( t~moles/mgprotein) after operation 4 24 7 32 60
h h days days days
9.02 9.02 10.76 8.02 10.18
i i + 44.
0.372 0.372 1.401 0.962 1.377
(3) (3) (16) (5) (5)
7.65 ± 0.465 5.41 i 0.766 5.10 -4- 0.965 4.60 ± 1.207 7.24 4, 0.842
(4) (5) (16) (5) (4)
--15" --40* --53* --43* --29*
* P = 0.05.
micromethod of McCaman and Hunt 13 with [14C]acetyl coenzyme A (New England Co., 58 mCi/mmole) and choline as substrates. Protein was determined according to the method of Lowry et al. 11 with bovine plasma albumin as standard. The assay of AChE in the pineal gland at short times after ganglionectomy indicated no differences with respect to the control. As previously shown 16 at 7 days the enzyme was reduced almost 50%. Thereafter the differences from the control were less marked; the values were --19 and --14 % at 32 and 60 days, respectively (Table I). Decentralization produced no changes in pineal AChE at 7 days16; an increase of 25 % was found at 32 days and of 36 % at 60 days (Table I). In the pineal gland, at 7 days after bilateral removal of the superior cervical
437 TABLE III
The activity of ChE in the pinea~ gland and cervical superior ganglia of the rat The activity of ChE was determined in homogenates containing 4-5 pineal glands or 4 ganglia each; the results are expressed in #mole of butyrylthiocholine hydrolyzed per mg protein/h at 37 °C. In parentheses are the number of homogenates assayed Data at 7 days from ref 16.
Tissue
Time after Control operation (#moles~me (days) protein)
Ganglionectomized #moles/me protein
Decentralized % variation #moles/me protein
Pineal gland
7 32 60
0.09 + 0.013(5) 0.06 4- 0.006(4) --33* 0.07 4- 0.011(5) 0.06 4- 0.005(5) --15 0.07 4- 0.011(5) 0.06 4- 0.020(5) --15
Superior cervical ganglia
32 60
2.534-0.473(4) 2.53 -4- 0.473(4)
% variation
0.08 -4- 0.009(4) --11 0.07 4- 0.014(4) 0 0.07 4- 0.005(5) 0
0.994-0.412(4) --451" 1.27 4- 0.176(5) --50*
* P < 0.002. TABLE IV
The activity of ChAc in the cervical superior ganglia of the rat after decentralization The activity of ChAc was expressexl as the amount of m o l e s of labeled acetylcholine produced per mg protein/h at 37 °C. In parentheses are the number of homogenates assayed.
Time after decentralization (h)
nmoles/mg protein
-4 24
127.1 -4-55.84 (3) 135.5 + 17.37 (4) 41.9 + 8.98 (5)
% variation
+7 --67*
* P < 0.05.
ganglia, ChE decreased by 33 %t6; at longer times the activity was almost normal since the reduction o f - - 1 5 % was not significant. After sectioning the afferent fibers of the superior cervical ganglia no changes were observed either at 7 days or at 32 and 60 days (Table III). Section of the afferent fibers produced a significant reduction in AChE of the cervical superior ganglia, being 15 % at 4 h and 40 % at 24 h. The reduction observed at 24 h was very similar to that previously observed at 7 days (--53 %). At longer periods it was observed that the difference between control and decentralized was less marked; 32 days after operation the decrease was 43 ~o and at 60 days it was 29 % (Table II). At variance, Ch.E did not change at 7 days 16 or at 32 and 60 days (Table III). On the other hand, decentralization produced no change in ganglion ChAc at 4 h but a significant reduction of--67 % at 24 h (Table IV). The complete disappearance at 7 days was previously described t6.
438 • ..o ....... = u
• 0 = ,~
AChE AChE AChE ChAc
Decentrohzed pineal gland Ganghonectomlzed pmeol gland Decentrahzed cervical superior ganglia Decentrahzed cervical supermr gangha
15(; ,........4 ,....,
,.....-
>I--
I00, :"
......0
l-
< 5(;
,\ I
7
I 20
[ 40
I 60
DAYS
Fig. 1. Pattern of changes of AChE and ChAc in the cervical superior ganglia and the pineal gland of the rat. Data from TablesI, II and IV. The changes in enzymes presented here cannot be attributed to changes in protein content by denervation since the amount of protein per organ did not change significatively. In a previous paper we have reported that AChE activity diminished about 50 % in pineal gland and cervical superior ganglia after ganglionectomy and decentralization, respectively16. The pattern of changes in AChE at short times is different for the pineal gland and ganglia; after deprivation of their afferents a rapid decrease was found in the ganglia but not in the pineal. This difference suggests the existence of distinct regulatory mechanisms or different subcellular localizations. The observed increase of AChE in the pineal gland after decentralization and the partial recovery in pineal and ganglia by ganglionectomy and decentralization, respectively, followed a similar pattern (Fig. 1). Considering the values at 7 days as minimum those increases attained 40-50 ~o at 60 days. It is interesting to compare our findings after denervation with those reported on skeletal muscle; results indicating AChE decrease3,7,s,12 or increase a,17 after denervation have been reported. This apparent discrepancy seems to be accounted for by the different times elapsed between surgical procedure and the assays. In those papers which report a time course variation it was observed that decrease in AChE preceded the further increase. This resembles the variation found in our preparation. The recovery of the activity of AChE in muscle has been attributed to reinnervation 9. In our experiments a similar explanation can not be considered since previous studies have not demonstrated reinnervation of the pineal gland after ganglionectomy 15. The greatest decrease (53 %) in AChE in the ganglia was at 7 days, that is, when
439 the nerve endings have disappeared. This result implies an extra nerve ending localiration for the remaining ACHE. The presence of AChE at both the pre- and postsynaptic membranes was demonstrated histochemically in the cat cervical superior ganglia 10. The results found in the ganglia suggest a presynaptic localization for ChAt. The decay in ChAc is faster than that of ACHE. Similar results have been reported after denervation of musclelL In the pineal gland, the data also suggest the presence of AChE outside the nerve endings. This is in line with previous studies of Eranko et al 6. who showed, histochemically, the existence of AChE in relation to nerve endings and pinealocytes. The increase of AChE in the pineal gland after decentralization could be due to an increase in the activity of the enzyme in the adrenergic neuron or to an increase in the activity of the enzyme in other structures of the gland. Since ChE did not change in the same way as AChE at 32 and 60 days in the pineal gland in both experimental conditions, it suggests that the pattern of changes of the total activity is not affected by ChE. In the superior cervical ganglia, the changes were similar for ChE and ACHE; both enzymes share the same localization in the ganglia but not in the pineal gland. Another possible interpretation may be attributed to the existence of different neural regulatory mechanisms for both structures. In summary, denervation of the pineal gland results in a decrease with further recovery of ACHE, suggesting the existence of neural regulatory mechanisms of unknown nature. Deprivation of the afferent fibers of the superior cervical ganglia might also intervene in this regulatory mechanism as suggested by the increase in AChE in the pineal gland after decentralization. This work has been supported by Grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina and the National Institutes of Health (5-R01 NS 06953-09) U.S.A.
1 Bertler, A., Falck, B. and Owman, C., Cellular localization of hydroxytryptamine in the rat pineal gland, Kungl. Fysiografiska Siillskapets Lund Fi~rhandlingar., 33 (1963) 13-16. 2 Brzin, M. and Majcen-Tkaccv, Z., Cholinesteras¢ in denervated end plates and muscle fibers, J. Cell BioL, 19 (1963) 349-358. 3 Couteaux, R. and Nachmansohn, D., Changes of choline esterase at end plates of voluntary muscle following section of sciatic nerve, Prec. Soc. exp. Biol. (N. Y.), 43 (1940) 177-181. 4 Ellman, G. L., Courtney, K. D., Andros, V. Jr. and Featherstone, R. M., A new and rapid coiorimetric determination of acetylcholinosteras¢ activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 Erank6, O., Histocbemistry of nervous tissues: catecholamincs and cholinosterasos, Ann. Rev. Pharmacol., 7 (1969) 203-222. 6 Eriink6, O., Rvchardt, L., Erink6, L. and Cunningham, A., Light and electron microscopic histochemical observations on cholinosterasc-containing sympathetic nerve fibres in the pineal body of the rat, Histochem. J., 2 (1970) 479-489. 7 Guth, L., Albers, R. W. and Brown, W. C., Quantitative changes in cholinosteras¢ activity of denervated muscle fibers and sole plates, Exp. Neurol., 10 (1964) 236-250. 8 Guth, L. and Brown, W. C., The sequence of changes in cholinosteras¢ activity during reinnervation of muscle, Exp. Neurol., 12 (1965) 329-336. 9 Guth, L. Brown, W. C., and Watson, P. H. K., Studios on the role of nerve impulses and acetylcholine release in the regulation of the cholinosteras¢ activity of muscle, Exp. Neurol., 18 (1967) 443-452.
440 10 Koelle, G B., Davis, R., Koelle, W. A., Smyrl, E. G. and Free, A V, The electron microscopic localization of acetylcholinesterase and pseudochohnesterase m autonomic gangha. In P. G Waser (Ed.), Cholinergic Mechanisms, Raven Press, New York, 1975, pp. 251-255 11 Lowry, O. H , Rosebrough, N. J., Farr, A L. and Randall, R. J , Protein measurement with the Fohn phenol reagent, J. biol. Chem., 193 (1951) 265-275 12 McCaman, M. W , Biochemical effects of denervatlon on normal and dystrophic muscle; acetylchohnesterase and choline acetyltransferase, LiJe Sci, 5 (1966) 1459-1465 13 McCaman, R. E. and Hunt, J M., Microdetermination of choline acetylase m nervous t~ssue, J Neurochem., 12 (1965) 253-259. 14 Pellegrmo de Iraldi, A. and Suburo, A. M., Two compartments m the granulated vesicles of the pineal nerves. In G. E. W. Wolstenholme and J. Kmght (Eds.), A Ctba Foundation Symposium" The Pineal Gland, Churchill, Livingstone, Edinburgh, 1971, pp. 177-195. 15 Pellegrino de Iraldl, A., Zieher, L. M. and De Robertis, E , Ultrastructure and pharmacological studies of nerve endings in the pineal organ. In J. Anens Kappers and J. P. Schad6 (Eds.), Structure and Function of the Epiphysis CerebrL Progress in Brain Research, Vol 10, Elsevier, Amsterdam, 1965, pp. 389-422 16 Rodriguez de Lores Arnalz, G. and Pellegrmo de Iraldi, A , Chohnesterase in cholinerglc and adrenergic nerves: a study of the superior cervical ganglia and the pineal gland of the rat, Brain Research, 42 (1972) 230--233. 17 Wilson, B. W , Kaplan, M. A., Merhoff, W. C. and Mort, S. S., Innervatlon and regulation of acetylcholinesterase actwity during the development of normal and dystrophic chick muscle, J. exp. ZooL, 174 (1970) 39-54.
435
© Elsevier ScientificPublishing Company,Amsterdam- Printed in The Netherlands
Neural regulation of acetylcholinesterase in the superior cervical ganglia and the pineal gland of the rat
AMANDA PELLEGRINODE IRALDI and GEORGINA RODRIGUEZ DE LORESARNAIZ Institute de Biologla Celular, Faeultad de Medicina, Universidad de Buenos Aires, Buenos Aires (Argentina)
(Accepted May 17th, 1976)
Structural, pharmacological and biochemical studies have given evidence of the non-cholinergic nature of pineal nerves coming from the cervical superior ganglia1,14,t5. Furthermore the coexistence of ACHE* and catecholamines in several nervous structures, such as the pineal gland nerves6,t6, has been reported s. In a previous paper we have observed that at 7 days after denervation of the pineal gland (by ganglionectomy of the cervical superior ganglia) and decentralization of the cervical superior ganglia, the activity of AChE decreased about 50 ~o in both organs t6. With a similar approach, experiments at shorter and longer periods were carried out. The results suggest a neural regulation of AChE. Wistar rats weighing about 200 g were used. The rats were divided in three groups; in one of them, afferent fibers to the superior cervical ganglia were sectioned. This is referred to as the 'decentralized' group. In a second group, bilateral removal of the cervical superior ganglia was performed. This is referred to as the'ganglionectomized' group. A third group without surgical procedure was used as 'control'. At different times after operation rats were killed by decapitation and the pineal gland and both superior cervical ganglia were dissected. In the case of the ganglia, special care was taken to eliminate the sheath of connective tissue. Homogenates of the pineal gland were prepared by gentle homogenization in a Potter-Elvejhem microhomogenizer; 4-5 glands in 100 #1 of bidistilled water were used for each homogenate. To prepare the homogenates of the ganglia, 4 ganglia were vigorously homogenized in 200 #1 of bidistilled water; the ganglia homogenates were submitted to a brief centrifugation (10 rain at 1500 rev./min) to remove the connective tissue which resisted the homogenization. Cholinesterase was assayed in triplicate aliquots of the homogenates, with acetylthiocholine or butyrylthiocholine as substrate, according to Ellman et al. 4. ChAc was assayed in triplicate samples of each homogenate with the radio-
* Abbreviationsused: ACHE,acetylcholinehydrolase(EC 3.1.1.7); ChE,acetylcholineacyl-hydrolase (EC 3.1.1.8); ChAt, acetyl CoA: choline O-acetyltransferase(EC 2.3.1.6).
436 TABLE I
Changes in AChE of the pineal gland by ganglionectomy and decentroJizatwn The actwity of AChE was determined m homogenates containing 4-5 pineal glands each; the results were expressed as/~moles of substrate hydrolyzed per mg protein/h at 37 °C. Ganglionectomize.d refers to the bilateral removal of the superior cervical ganglia; decentralized refers to the section of afferent fibers of the superior cervical ganglia. In parentheses are the number of homogenates assayed. Data at 7 days from ref. 16
Time Control (#moles/ after mg protein) operation
Izmoles/mg protein
% variatton f~moles/mgprotein
4h 24 h 7 days 32 days 60 days
0.16 0.15 0.10 0.13 0.12
44* --19" --14"
0.14 0.14 0.18 0.16 0.14
5= 0.018 -- 0.018 -+- 0.040 4, 0.008 4. 0.015
(4) (4) (17) (4) (4)
Ganglionectomized
4. 0.014 4- 0.021 4- 0.021 4-0.019 4. 0.009
(2) (4) (15) (5) (5)
Decentrahzed
0.19 4- 0.038 (14) 0.20 -4- 0.026 (5) 0.19 4, 0.022 (5)
% variatton
4-6 +25* +36*
* P < 0.05. TABLE II
The activity of AChE in the cervical superior ganglia after decentralization The activity of AChE was determined in homogenates containing 4 ganglia each; the results are expressed in/~mole of substrate hydrolyzed per mg protein/h at 37 °C. In parentheses are the number of homogenates assayexl. Data at 7 days from ref. 16.
% variation Time Control Decentralized ( l~moles/mg protein) ( t~moles/mgprotein) after operation 4 24 7 32 60
h h days days days
9.02 9.02 10.76 8.02 10.18
i i + 44.
0.372 0.372 1.401 0.962 1.377
(3) (3) (16) (5) (5)
7.65 ± 0.465 5.41 i 0.766 5.10 -4- 0.965 4.60 ± 1.207 7.24 4, 0.842
(4) (5) (16) (5) (4)
--15" --40* --53* --43* --29*
* P = 0.05.
micromethod of McCaman and Hunt 13 with [14C]acetyl coenzyme A (New England Co., 58 mCi/mmole) and choline as substrates. Protein was determined according to the method of Lowry et al. 11 with bovine plasma albumin as standard. The assay of AChE in the pineal gland at short times after ganglionectomy indicated no differences with respect to the control. As previously shown 16 at 7 days the enzyme was reduced almost 50%. Thereafter the differences from the control were less marked; the values were --19 and --14 % at 32 and 60 days, respectively (Table I). Decentralization produced no changes in pineal AChE at 7 days16; an increase of 25 % was found at 32 days and of 36 % at 60 days (Table I). In the pineal gland, at 7 days after bilateral removal of the superior cervical
437 TABLE III
The activity of ChE in the pinea~ gland and cervical superior ganglia of the rat The activity of ChE was determined in homogenates containing 4-5 pineal glands or 4 ganglia each; the results are expressed in #mole of butyrylthiocholine hydrolyzed per mg protein/h at 37 °C. In parentheses are the number of homogenates assayed Data at 7 days from ref 16.
Tissue
Time after Control operation (#moles~me (days) protein)
Ganglionectomized #moles/me protein
Decentralized % variation #moles/me protein
Pineal gland
7 32 60
0.09 + 0.013(5) 0.06 4- 0.006(4) --33* 0.07 4- 0.011(5) 0.06 4- 0.005(5) --15 0.07 4- 0.011(5) 0.06 4- 0.020(5) --15
Superior cervical ganglia
32 60
2.534-0.473(4) 2.53 -4- 0.473(4)
% variation
0.08 -4- 0.009(4) --11 0.07 4- 0.014(4) 0 0.07 4- 0.005(5) 0
0.994-0.412(4) --451" 1.27 4- 0.176(5) --50*
* P < 0.002. TABLE IV
The activity of ChAc in the cervical superior ganglia of the rat after decentralization The activity of ChAc was expressexl as the amount of m o l e s of labeled acetylcholine produced per mg protein/h at 37 °C. In parentheses are the number of homogenates assayed.
Time after decentralization (h)
nmoles/mg protein
-4 24
127.1 -4-55.84 (3) 135.5 + 17.37 (4) 41.9 + 8.98 (5)
% variation
+7 --67*
* P < 0.05.
ganglia, ChE decreased by 33 %t6; at longer times the activity was almost normal since the reduction o f - - 1 5 % was not significant. After sectioning the afferent fibers of the superior cervical ganglia no changes were observed either at 7 days or at 32 and 60 days (Table III). Section of the afferent fibers produced a significant reduction in AChE of the cervical superior ganglia, being 15 % at 4 h and 40 % at 24 h. The reduction observed at 24 h was very similar to that previously observed at 7 days (--53 %). At longer periods it was observed that the difference between control and decentralized was less marked; 32 days after operation the decrease was 43 ~o and at 60 days it was 29 % (Table II). At variance, Ch.E did not change at 7 days 16 or at 32 and 60 days (Table III). On the other hand, decentralization produced no change in ganglion ChAc at 4 h but a significant reduction of--67 % at 24 h (Table IV). The complete disappearance at 7 days was previously described t6.
438 • ..o ....... = u
• 0 = ,~
AChE AChE AChE ChAc
Decentrohzed pineal gland Ganghonectomlzed pmeol gland Decentrahzed cervical superior ganglia Decentrahzed cervical supermr gangha
15(; ,........4 ,....,
,.....-
>I--
I00, :"
......0
l-
< 5(;
,\ I
7
I 20
[ 40
I 60
DAYS
Fig. 1. Pattern of changes of AChE and ChAc in the cervical superior ganglia and the pineal gland of the rat. Data from TablesI, II and IV. The changes in enzymes presented here cannot be attributed to changes in protein content by denervation since the amount of protein per organ did not change significatively. In a previous paper we have reported that AChE activity diminished about 50 % in pineal gland and cervical superior ganglia after ganglionectomy and decentralization, respectively16. The pattern of changes in AChE at short times is different for the pineal gland and ganglia; after deprivation of their afferents a rapid decrease was found in the ganglia but not in the pineal. This difference suggests the existence of distinct regulatory mechanisms or different subcellular localizations. The observed increase of AChE in the pineal gland after decentralization and the partial recovery in pineal and ganglia by ganglionectomy and decentralization, respectively, followed a similar pattern (Fig. 1). Considering the values at 7 days as minimum those increases attained 40-50 ~o at 60 days. It is interesting to compare our findings after denervation with those reported on skeletal muscle; results indicating AChE decrease3,7,s,12 or increase a,17 after denervation have been reported. This apparent discrepancy seems to be accounted for by the different times elapsed between surgical procedure and the assays. In those papers which report a time course variation it was observed that decrease in AChE preceded the further increase. This resembles the variation found in our preparation. The recovery of the activity of AChE in muscle has been attributed to reinnervation 9. In our experiments a similar explanation can not be considered since previous studies have not demonstrated reinnervation of the pineal gland after ganglionectomy 15. The greatest decrease (53 %) in AChE in the ganglia was at 7 days, that is, when
439 the nerve endings have disappeared. This result implies an extra nerve ending localiration for the remaining ACHE. The presence of AChE at both the pre- and postsynaptic membranes was demonstrated histochemically in the cat cervical superior ganglia 10. The results found in the ganglia suggest a presynaptic localization for ChAt. The decay in ChAc is faster than that of ACHE. Similar results have been reported after denervation of musclelL In the pineal gland, the data also suggest the presence of AChE outside the nerve endings. This is in line with previous studies of Eranko et al 6. who showed, histochemically, the existence of AChE in relation to nerve endings and pinealocytes. The increase of AChE in the pineal gland after decentralization could be due to an increase in the activity of the enzyme in the adrenergic neuron or to an increase in the activity of the enzyme in other structures of the gland. Since ChE did not change in the same way as AChE at 32 and 60 days in the pineal gland in both experimental conditions, it suggests that the pattern of changes of the total activity is not affected by ChE. In the superior cervical ganglia, the changes were similar for ChE and ACHE; both enzymes share the same localization in the ganglia but not in the pineal gland. Another possible interpretation may be attributed to the existence of different neural regulatory mechanisms for both structures. In summary, denervation of the pineal gland results in a decrease with further recovery of ACHE, suggesting the existence of neural regulatory mechanisms of unknown nature. Deprivation of the afferent fibers of the superior cervical ganglia might also intervene in this regulatory mechanism as suggested by the increase in AChE in the pineal gland after decentralization. This work has been supported by Grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina and the National Institutes of Health (5-R01 NS 06953-09) U.S.A.
1 Bertler, A., Falck, B. and Owman, C., Cellular localization of hydroxytryptamine in the rat pineal gland, Kungl. Fysiografiska Siillskapets Lund Fi~rhandlingar., 33 (1963) 13-16. 2 Brzin, M. and Majcen-Tkaccv, Z., Cholinesteras¢ in denervated end plates and muscle fibers, J. Cell BioL, 19 (1963) 349-358. 3 Couteaux, R. and Nachmansohn, D., Changes of choline esterase at end plates of voluntary muscle following section of sciatic nerve, Prec. Soc. exp. Biol. (N. Y.), 43 (1940) 177-181. 4 Ellman, G. L., Courtney, K. D., Andros, V. Jr. and Featherstone, R. M., A new and rapid coiorimetric determination of acetylcholinosteras¢ activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 Erank6, O., Histocbemistry of nervous tissues: catecholamincs and cholinosterasos, Ann. Rev. Pharmacol., 7 (1969) 203-222. 6 Eriink6, O., Rvchardt, L., Erink6, L. and Cunningham, A., Light and electron microscopic histochemical observations on cholinosterasc-containing sympathetic nerve fibres in the pineal body of the rat, Histochem. J., 2 (1970) 479-489. 7 Guth, L., Albers, R. W. and Brown, W. C., Quantitative changes in cholinosteras¢ activity of denervated muscle fibers and sole plates, Exp. Neurol., 10 (1964) 236-250. 8 Guth, L. and Brown, W. C., The sequence of changes in cholinosteras¢ activity during reinnervation of muscle, Exp. Neurol., 12 (1965) 329-336. 9 Guth, L. Brown, W. C., and Watson, P. H. K., Studios on the role of nerve impulses and acetylcholine release in the regulation of the cholinosteras¢ activity of muscle, Exp. Neurol., 18 (1967) 443-452.
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