396
Btatn Reseulth, 106 (1976) 396-402 c, Elsevier Scientific Pubhshlng Company, Amsterdam - Printed m The Netherlands
Acetylcholinesterase and choline acetyltransferase activity in the amygdala of rat brain after septal lesions
BARBARA ODERFELD-NOWAK, OLGIERD NARKIEW1CZ, ANDRZEJ WIERASZKO MALGORZATA GRADKOWSKA
AND
Department of Biochemistry of Nervous System and Muscle, Nencki Institute of Experimental Bzology, Warsaw and Department of Anatomy, Institute of Medical Bwlogy, School of Medicine, Gdahsk (Poland)
(Accepted January 12th, 1976)
For some years special attention has been paid to cholinergic transmission within the hmblc system. In particular, the interaction between septum, hippocampus and amygdala, and their presumed roles in some aspects of behaviour, have been emphasized (cf. ref. 12). It is well established that AChE and ChAc* activity decreased in the hippocampus after interruption of the fimbria 13,14,24 or after septal lesions16,17,2°. On the other hand, while the septum and amygdala are rich in cholinergic components 1,19,2~, little is known about cholinergic interactions between them. After septal lesions of the cat brain, McGeer et al. 16 found a small decrease in the ChAc activity in amygdala, whereas in the rat brain Herink et al. 9 reported that after septal lesions the AChE activity in amygdala varied between 80 and 120% of the control values. The localization of the lesion within the septum was, however, not reported in their papers. In the present study we were interested in the effect of differently localized septal lesions upon the level of AChE and ChAc in amygdala. Part of this work has already been described briefly21. The subjects were 61 operated and 61 control (unoperated and sham operated) male rats of the Wistar strain bred in the Nencki Institute. At the time of surgery the animals weighed between 220 and 280 g and were 2-3 months of age. The rats were housed in groups of 6-8 preoperatively, and in single cages after surgery. The animals were anaesthetized with sodium pentobarbital (55 mg/kg) intramuscularly. Bilateral electrolytical lesions of the septum were made stereotaxically by passing anodal current (2-3 mA) for 10-30 sec through a platinum electrode (0.1-0.3 mm diameter) insulated except for 0.4 mm of its tip. The animals were killed 2-42 days after surgery by decapitation while awake or slightly anaesthetized with ether. * Abbreviations used: ACHE, acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7); ChAc, choline acetyttransferase (O-acetyltransferaseacetyl-CoA:choline, EC 2.3.1.6.).
397 The brains were removed quickly from the skull and were kept frozen at ----4 °C until the dissection was performed. The amygdalae were dissected bilaterally. A cut was made with a knife inserted in a dorsomedial direction between the amygdaloid body and the optic tract. Laterally, a sagittal, slightly oblique, section was placed about 1.5 mm medially to the lateral surface of the pyriform lobe through the external capsule separating the amygdala from most of the entorhinal cortex. Dorsally, a horizontal cut was made at the level of the posterior rhinal sulcus (see Fig. 1). The rostral and caudal cuts in the frontal planes were made according to the limits of the amygdaloid body: rostral, through the terminal part of the olfactory tract; caudal, through the posterior end of amygdaloid tubercle. The histological verification and the consistency in sample weight (18.4 -4- 0.5 mean (mg) ± S.E.M., n ---- 61) indicate that the removal of amygdala was reproducible. The anterior part of each brain including the septum and the parts of the hemispheres surrounding the amygdaloid nuclei were placed in 10~o formalin and later embedded in paraffin. Sections (30/~m) were stained with cresyl violet or by the Klfiver-Barrera method. Histological verification of the extent and place of the septal lesion was made in each brain and histological examination of the removal of amygdalae was performed at random in several brains. The control and the operated animals were examined simultaneously in each experiment. Both left and right amygdalae were analyzed in one sample. The activities of both enzymes were estimated in the same homogenates. The AChE activity was determined by the method of Ellman 5 with slight modifications ~0. The ChAc activity was determined using the coupled incubation system introduced by Fonnum 6 with some modifications 20.
TO
~
CE
Fig. 1. Planes of removal of the amygdaloid body demonstrated by broken lines on the diagram of frontal section showing position of various amygdaloid nuclei. Diagram according to Nltecka et al.lL Abbreviations: BV, basal ventral nucleus; BD, basal dorsal nucleus; C, central nucleus; CE, entorhinal cortex; CO, cortical nucleus; L, lateral nucleus; M, medial nucleus; S, striatum; TO, optic tract; R, rhinal sulcus. Most of the amygdala was included in the sample. The inclusion of the striatum, a structure high in AChE and ChAc activity, was avoided.
398
TYPE I
R-850
R-303
~S
R-449
tR-784
Fig. 2. Examples of type I septal lesions. Frontal sections, left: rostral; right: caudal. The extent of damage is marked in black. Abbreviations: CA, anterior commissure; D, dorsal nucleus; F, fornix; L, lateral nucleus; NA, nucleus accumbens septl; NB, nucleus of the diagonal band (vertical limb); NC, nucleus of the anterior commissure; NF, nucleus fimbriatus septi; S, striatum; ST, stria termlnahs and its nucleus.
399 Small scatter of enzymatic activity (see footnote of Table I) in control animals indicates that, in spite of the known enzymatic variations in the individual nuclei of amygdalaa,19, z2, the investigated material was uniform. The analysis of the experimental data showed that only ventrocaudal lesions caused a decrease of activity of AChE and ChAc. Other lesions, even very large, were ineffective. Therefore, the lesioned animals were divided into two groups according to types of septal lesions: type I, having the ventrocaudal area undamaged and type II, having the ventrocaudal area and also anterior commissure and neighbouring cellular structures damaged. Type I (Fig. 2) involves the lesions which destroyed most of the septal nuclei almost completely, i.e., dorsal, lateral, medial, anterior, fimbriatus septi, nucleus of diagonal band and nucleus triangularis septi (R 850), or which were restricted to the particular groups of nuclei within the septal area: dorsal lesions (R 303), ventromedial lesions (R 449), and ventrolateral lesions (R 784). In rats with these lesions there were no significant changes in AChE and ChAc activity regardless of the time after surgery (Table I). Type II, ventrocaudal (Fig. 3), involves the lesions which damaged the anterior commissure and area laying above it, including nucleus of the anterior commissure, partially the nucleus of the stria terminalis and portions of the columnae fornicis. Some other nuclei of the septum, including the ventral part of the nucleus fimbriatus septi, lateral nucleus and vertical limb of the diagonal band, were also partly affected. Two days postoperatively only ChAc activity decreased up to 85 % of the control value (Table I). AChE activity remained unchanged at that time. Later, 6-42 days after surgery, both enzymes showed decreased activity; ChAc activity in most cases was more affected than that of ACHE. It must be stressed that during the interval between 6 and 42 days after the operation (at 6 ,I0, 14, 28 and 42 days) no time deTABLE I A C h E AND C h A c ACTIVITIES IN AMYGDALA AFTER VARIOUS SEPTAL LESIONS AT DIFFERENT TIMES OF SURVIVAL
Type of leston
AChE
ChAc
2 Days
Type I T y p e II
6-42 Days
2 Days n
n
% of the control values§ (mean ± S.E.M.)
n
% of the control values§ (mean 4. S.E.M.)
6 5
101.6 4- 6.0 98.0 4- 6.3
35 15
97.0 q- 2.8 76.6 4- 2.3** 5
6-42 Days % of the control values§ (mean 4S.E.M.)
n
% of the control values§ (mean 4S.E.M.)
85.0 4- 3.9*
11 10
95.9 4- 4.8 65.0 4- 7.6**
§ C o n t r o l values ( u n o p e r a t e d or s h a m operated animals): ACHE: 818 ± 23 ~ m o l e A T h C h / g wet tissue/h, (n = 61); C h A c : 5.2 4. 0.5/~mole A C h / g wet tissue/h, (n = 31). * 0.01 < P < 0.001, with respect to control values; Student's t-test, two-tailed. ** P < 0.001, with respect to control values; S t u d e n t ' s t-test, two-tailed.
400
IYPEII
R-412
s
"
R-446
R-423
R-785
Fig. 3. Examples of type II septal lesions. Abbreviations and explanations as in Fig. 2.
pendence in enzymatic activities was observed. After the septal lesions neither changes in the weight of amygdala nor changes in the protein content (measured by the Lowry method 15) were observed as compared with the control values. Therefore the reported data show the real alterations in the enzymatic activity in amygdala after the septal lesions.
401 In contrast to the dramatic effects of the lesions damaging a large part of the septum on the activity of AChE and ChAc in hippocampus z°, there was no effect on the activity of these enzymes in the amygdala. In the light of the absence of massive connections between the septum and amygdala3 as opposed to those between the septum and the hippocampus 23 these differences in the response to septal damage should not be surprising. Only damage involving structures lying most ventrocaudally in the septum produced a significant decrease in the activity of both enzymes in the amygdala. This critical region includes the anterior commissure and structures lying nearby on its dorsal and rostral side; nucleus of the anterior commissure, nucleus of the stria terminalis and most ventral parts of the other septal nuclei. In the nucleus of the anterior commissure there are a few cells with high AChE activity as well as some AChE-containing fibres which run through this area; much more abundant cholinergic fibres, running probably to the amygdaloid body, pass through the nucleus of stria terminalisis. The observed decrease in the enzymatic activity in amygdala may be due to a secondary degeneration following a damage to cholinergic fibres or cells situated in this critical region. This assumption is based on the fact that only slight decrease of ChAc was observed after 2 days, whereas larger enzymatic changes appeared after the next few days. It is to be noted that there was no further change between 6 and 42 days when the fibres had probably already degenerated. One cannot exclude, however, some transsynaptic effects without degeneration of amygdalopetal fibres. Although the decrease in both enzymes was similar, in most cases ChAc was more affected than AChE (Table I). This might be due to differences in the localization of both enzymes in different substructures of the cholinergic neurones. In summary, our results suggest that most of the septum has no direct cholinergic projection to amygdala but that probably there exists a cholinergic projection arising from or passing through areas lying in its ventrocaudal part. It is possible that the changes observed in the present studies after the damage of the most ventrocaudal part of the septum cause an imbalance in the amygdalar cholinergic system, which may have behavioural consequences. This could, for instance, represent a biochemical correlate of the septal syndrome. Some authors ( c f ref. 2) have suggested that the part of the septal complex lying ventrocaudally may be the responsive area that produces aggression. On the other hand, lesions of the amygdala block this and other aspects of the septal syndrome11 and there is some evidence that the cholinergic system of amygdala is involved in aggressive behaviour4,7,1°. We are greatly indebted to Dr. J. Dobrowska for her help and advice during this study. Our grateful thanks are due to Professor Stella Niemierko and Professor H. Ursin for their criticism and advice during the preparation of the manuscript. Mrs. B. Heiler-Golebiewska's skilful technical assistance is greatly appreciated.
1 BIA~LOWAS,J., AND NARKIEWICZ,O., Acetylcholinesteraseactivityin the septum of the rat: a histochemicaltopography,Acta neurobioL exp. (Warszawa), 34 (1974) 573-582. 2 CAPLAN, i . , An analysis of the effects of septal lesions on negativelyreinforced behaviour, Behav. BioL, 9 (1973) 129-167.
402 3 COWAN, W. M , RAISMAN,G., AND POWELL, T P. S, The connections of the amygdala, J. Nemo/ Neurosurg. Psychiat, 28 (1965) 137 - 15 l 4 EBEL, A., MACK, G., STEEANOVIC,V , AND MANDEL, P , Activity of chohne acetyltransferase and acetylchohnesterase in the amygdala of spontaneous mouse killer rats and m rats after olfactory lobe removal, Brain Research, 57 (1973) 248-251 5 ELLMAN, G L , COURTNEY, K. D., ANDERS, V., AND FEATHERSTONE, R., A new rapid colommetrJc determination of acetylchohnesterase activity, Blochem. Pharmacol., 7 (1961) 88-95. 6 FONNUM, F., A radlochemical method for estimation of chohne acetyltransferase, Biochem J , IO0 (1966) 479~,84 7 GIRGIS, M., The distribution of acetylcholinesterase enzyme m the amygdala and its role in aggressive behavlour, Advanc. Behav Bzol, 2 (1972) 283-293 8 HALL,E, GENESER-JENSEN, F A., D~stribution of acetylchohnesterase and monoamine oxldase m the amygdala of the guinea pig, Z. Zellforsch., 120 (1971) 204-221. 9 HERINK, J , BAJGAR, J., AND PATOCK.~, J., Changes in acetylchohnesterase m some parts of the limbic system following septal lesions m rats, J. Neurochem., 24 (1975) 187-188. 10 IGI(~, R., STERN, P., AND BASAGIC, E., Changes m emotional behavlour after application of chohnesterase mhlbltors m the septal and amygdala region, Neuropharmacology, 9 (1970) 73-75 11 KING, F. A., AND MEYER, P M., Effects of amygdaloid lesions upon septal hyperemotionalJty in the rat, Science, 128 (1958) 655-656. 12 LEATON, R. L., The hmbic system and its pharmacological aspects, in R RECH AND K. E. MOORE (Eds.), An Introduction to Psychopharmacology, Raven Press, New York, 1971, pp. 137-174. 13 LEwis, P. R., AND SHUTE, C. C. O., The chohnergic hmblc system, projection to hippocampal formation, medial cortex nuclei of ascending chohnergic reticular system, and the subfornical organ and supra-optic crest, Brain, 90 (1967) 521-540. 14 LEWIS, P. R., SHUTE, C C. D., AND SILVER, A., Confirmation from chohne acetylase analyses of a massive cholinerglc innervation to the rat hlppocampus, J. Physiol. (Lond.), 191 (1967) 215-224. 15 LowRY, O. H., ROSENBROUGH,J. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Fohn phenol reagent, J biol Chem, 193 (1951) 265-275 16 MCGEER, E. G , WADA, J A., TERAO, A , AND JUNG, E., Amine synthes~s m various brain regions with caudate or septal lesions, Exp. Neurol, 24 (1969) 277-284 17 MELLGREN, S. J., AND SREBRO, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the h~ppocampal region after septal lesions in the rat, Brain Research, 52 (1973) 19-36. 18 NITECKA, L., NARK1EWICZ, O., AND JAK1EL, O., The organization of amygdalopetal projections from the hypothalamus and preoptic area, m preparation. 19 NITECKA, L , NARKIEWICZ, O , AND ZAWISTOWSKA, H., Acetylcholinesterase actlwty in the nucle~ of the amygdaloid complex in the rat, Acta neurobiol exp (Warszawa), 31 (1971) 383-388. 20 ODERFELD-NOWAK, B., NARKIEWICZ, O., BIALOWAS, Y., DABROWSKA,J., W1ERASZKO,A., AND GRADKOWSKA, M., The influence of septal nuclei lesions on activity of acetylcholinesterase and choline acetyltransferase m the hlppocampus of the rat, Acta neurobiol, exp. (Warszawa), 34 (1974) 583-601. 21 ODERFELD-NOWAK, B., NARKIEWICZ, O., AND GRADKOWSKA, M., An effect of septal les~ons on acetylchohnesterase and acetylcholine level in amygdala of rat brain. In Abstracts of Symposium Dlfferentianon of Neuron and Glia and Their Metabohc and Functional Relationships, Castle Liblice, Czechoslovak, 1974, pp. 40~41 22 PALKOVITS, M., SAAVEDRA, M., KOaAYASHI, R. M., AND BROWNSTEIN, M., Choline acetyltransferase content of IimbJc nuclei of the rat, Brain Research, 79 (1974) 443-450. 23 RAISMAN, G., The connections of the septum, Brain, 89 (1966) 317-348. 24 STORM-MATHISEN, J., AND FONNUM~ F , Localization of transmitter candidates in the rat hippocampal reglon In P. B BRADLEYAND R. W. BRIMBLECOMBE(Eds.), Biochemical andPharmacological Mechanisms Underlying Behaviour, Progress in Brain Research, Vol. 36, Elsevier, Amsterdam, 1972, pp 41-57.
Btatn Reseulth, 106 (1976) 396-402 c, Elsevier Scientific Pubhshlng Company, Amsterdam - Printed m The Netherlands
Acetylcholinesterase and choline acetyltransferase activity in the amygdala of rat brain after septal lesions
BARBARA ODERFELD-NOWAK, OLGIERD NARKIEW1CZ, ANDRZEJ WIERASZKO MALGORZATA GRADKOWSKA
AND
Department of Biochemistry of Nervous System and Muscle, Nencki Institute of Experimental Bzology, Warsaw and Department of Anatomy, Institute of Medical Bwlogy, School of Medicine, Gdahsk (Poland)
(Accepted January 12th, 1976)
For some years special attention has been paid to cholinergic transmission within the hmblc system. In particular, the interaction between septum, hippocampus and amygdala, and their presumed roles in some aspects of behaviour, have been emphasized (cf. ref. 12). It is well established that AChE and ChAc* activity decreased in the hippocampus after interruption of the fimbria 13,14,24 or after septal lesions16,17,2°. On the other hand, while the septum and amygdala are rich in cholinergic components 1,19,2~, little is known about cholinergic interactions between them. After septal lesions of the cat brain, McGeer et al. 16 found a small decrease in the ChAc activity in amygdala, whereas in the rat brain Herink et al. 9 reported that after septal lesions the AChE activity in amygdala varied between 80 and 120% of the control values. The localization of the lesion within the septum was, however, not reported in their papers. In the present study we were interested in the effect of differently localized septal lesions upon the level of AChE and ChAc in amygdala. Part of this work has already been described briefly21. The subjects were 61 operated and 61 control (unoperated and sham operated) male rats of the Wistar strain bred in the Nencki Institute. At the time of surgery the animals weighed between 220 and 280 g and were 2-3 months of age. The rats were housed in groups of 6-8 preoperatively, and in single cages after surgery. The animals were anaesthetized with sodium pentobarbital (55 mg/kg) intramuscularly. Bilateral electrolytical lesions of the septum were made stereotaxically by passing anodal current (2-3 mA) for 10-30 sec through a platinum electrode (0.1-0.3 mm diameter) insulated except for 0.4 mm of its tip. The animals were killed 2-42 days after surgery by decapitation while awake or slightly anaesthetized with ether. * Abbreviations used: ACHE, acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7); ChAc, choline acetyttransferase (O-acetyltransferaseacetyl-CoA:choline, EC 2.3.1.6.).
397 The brains were removed quickly from the skull and were kept frozen at ----4 °C until the dissection was performed. The amygdalae were dissected bilaterally. A cut was made with a knife inserted in a dorsomedial direction between the amygdaloid body and the optic tract. Laterally, a sagittal, slightly oblique, section was placed about 1.5 mm medially to the lateral surface of the pyriform lobe through the external capsule separating the amygdala from most of the entorhinal cortex. Dorsally, a horizontal cut was made at the level of the posterior rhinal sulcus (see Fig. 1). The rostral and caudal cuts in the frontal planes were made according to the limits of the amygdaloid body: rostral, through the terminal part of the olfactory tract; caudal, through the posterior end of amygdaloid tubercle. The histological verification and the consistency in sample weight (18.4 -4- 0.5 mean (mg) ± S.E.M., n ---- 61) indicate that the removal of amygdala was reproducible. The anterior part of each brain including the septum and the parts of the hemispheres surrounding the amygdaloid nuclei were placed in 10~o formalin and later embedded in paraffin. Sections (30/~m) were stained with cresyl violet or by the Klfiver-Barrera method. Histological verification of the extent and place of the septal lesion was made in each brain and histological examination of the removal of amygdalae was performed at random in several brains. The control and the operated animals were examined simultaneously in each experiment. Both left and right amygdalae were analyzed in one sample. The activities of both enzymes were estimated in the same homogenates. The AChE activity was determined by the method of Ellman 5 with slight modifications ~0. The ChAc activity was determined using the coupled incubation system introduced by Fonnum 6 with some modifications 20.
TO
~
CE
Fig. 1. Planes of removal of the amygdaloid body demonstrated by broken lines on the diagram of frontal section showing position of various amygdaloid nuclei. Diagram according to Nltecka et al.lL Abbreviations: BV, basal ventral nucleus; BD, basal dorsal nucleus; C, central nucleus; CE, entorhinal cortex; CO, cortical nucleus; L, lateral nucleus; M, medial nucleus; S, striatum; TO, optic tract; R, rhinal sulcus. Most of the amygdala was included in the sample. The inclusion of the striatum, a structure high in AChE and ChAc activity, was avoided.
398
TYPE I
R-850
R-303
~S
R-449
tR-784
Fig. 2. Examples of type I septal lesions. Frontal sections, left: rostral; right: caudal. The extent of damage is marked in black. Abbreviations: CA, anterior commissure; D, dorsal nucleus; F, fornix; L, lateral nucleus; NA, nucleus accumbens septl; NB, nucleus of the diagonal band (vertical limb); NC, nucleus of the anterior commissure; NF, nucleus fimbriatus septi; S, striatum; ST, stria termlnahs and its nucleus.
399 Small scatter of enzymatic activity (see footnote of Table I) in control animals indicates that, in spite of the known enzymatic variations in the individual nuclei of amygdalaa,19, z2, the investigated material was uniform. The analysis of the experimental data showed that only ventrocaudal lesions caused a decrease of activity of AChE and ChAc. Other lesions, even very large, were ineffective. Therefore, the lesioned animals were divided into two groups according to types of septal lesions: type I, having the ventrocaudal area undamaged and type II, having the ventrocaudal area and also anterior commissure and neighbouring cellular structures damaged. Type I (Fig. 2) involves the lesions which destroyed most of the septal nuclei almost completely, i.e., dorsal, lateral, medial, anterior, fimbriatus septi, nucleus of diagonal band and nucleus triangularis septi (R 850), or which were restricted to the particular groups of nuclei within the septal area: dorsal lesions (R 303), ventromedial lesions (R 449), and ventrolateral lesions (R 784). In rats with these lesions there were no significant changes in AChE and ChAc activity regardless of the time after surgery (Table I). Type II, ventrocaudal (Fig. 3), involves the lesions which damaged the anterior commissure and area laying above it, including nucleus of the anterior commissure, partially the nucleus of the stria terminalis and portions of the columnae fornicis. Some other nuclei of the septum, including the ventral part of the nucleus fimbriatus septi, lateral nucleus and vertical limb of the diagonal band, were also partly affected. Two days postoperatively only ChAc activity decreased up to 85 % of the control value (Table I). AChE activity remained unchanged at that time. Later, 6-42 days after surgery, both enzymes showed decreased activity; ChAc activity in most cases was more affected than that of ACHE. It must be stressed that during the interval between 6 and 42 days after the operation (at 6 ,I0, 14, 28 and 42 days) no time deTABLE I A C h E AND C h A c ACTIVITIES IN AMYGDALA AFTER VARIOUS SEPTAL LESIONS AT DIFFERENT TIMES OF SURVIVAL
Type of leston
AChE
ChAc
2 Days
Type I T y p e II
6-42 Days
2 Days n
n
% of the control values§ (mean ± S.E.M.)
n
% of the control values§ (mean 4. S.E.M.)
6 5
101.6 4- 6.0 98.0 4- 6.3
35 15
97.0 q- 2.8 76.6 4- 2.3** 5
6-42 Days % of the control values§ (mean 4S.E.M.)
n
% of the control values§ (mean 4S.E.M.)
85.0 4- 3.9*
11 10
95.9 4- 4.8 65.0 4- 7.6**
§ C o n t r o l values ( u n o p e r a t e d or s h a m operated animals): ACHE: 818 ± 23 ~ m o l e A T h C h / g wet tissue/h, (n = 61); C h A c : 5.2 4. 0.5/~mole A C h / g wet tissue/h, (n = 31). * 0.01 < P < 0.001, with respect to control values; Student's t-test, two-tailed. ** P < 0.001, with respect to control values; S t u d e n t ' s t-test, two-tailed.
400
IYPEII
R-412
s
"
R-446
R-423
R-785
Fig. 3. Examples of type II septal lesions. Abbreviations and explanations as in Fig. 2.
pendence in enzymatic activities was observed. After the septal lesions neither changes in the weight of amygdala nor changes in the protein content (measured by the Lowry method 15) were observed as compared with the control values. Therefore the reported data show the real alterations in the enzymatic activity in amygdala after the septal lesions.
401 In contrast to the dramatic effects of the lesions damaging a large part of the septum on the activity of AChE and ChAc in hippocampus z°, there was no effect on the activity of these enzymes in the amygdala. In the light of the absence of massive connections between the septum and amygdala3 as opposed to those between the septum and the hippocampus 23 these differences in the response to septal damage should not be surprising. Only damage involving structures lying most ventrocaudally in the septum produced a significant decrease in the activity of both enzymes in the amygdala. This critical region includes the anterior commissure and structures lying nearby on its dorsal and rostral side; nucleus of the anterior commissure, nucleus of the stria terminalis and most ventral parts of the other septal nuclei. In the nucleus of the anterior commissure there are a few cells with high AChE activity as well as some AChE-containing fibres which run through this area; much more abundant cholinergic fibres, running probably to the amygdaloid body, pass through the nucleus of stria terminalisis. The observed decrease in the enzymatic activity in amygdala may be due to a secondary degeneration following a damage to cholinergic fibres or cells situated in this critical region. This assumption is based on the fact that only slight decrease of ChAc was observed after 2 days, whereas larger enzymatic changes appeared after the next few days. It is to be noted that there was no further change between 6 and 42 days when the fibres had probably already degenerated. One cannot exclude, however, some transsynaptic effects without degeneration of amygdalopetal fibres. Although the decrease in both enzymes was similar, in most cases ChAc was more affected than AChE (Table I). This might be due to differences in the localization of both enzymes in different substructures of the cholinergic neurones. In summary, our results suggest that most of the septum has no direct cholinergic projection to amygdala but that probably there exists a cholinergic projection arising from or passing through areas lying in its ventrocaudal part. It is possible that the changes observed in the present studies after the damage of the most ventrocaudal part of the septum cause an imbalance in the amygdalar cholinergic system, which may have behavioural consequences. This could, for instance, represent a biochemical correlate of the septal syndrome. Some authors ( c f ref. 2) have suggested that the part of the septal complex lying ventrocaudally may be the responsive area that produces aggression. On the other hand, lesions of the amygdala block this and other aspects of the septal syndrome11 and there is some evidence that the cholinergic system of amygdala is involved in aggressive behaviour4,7,1°. We are greatly indebted to Dr. J. Dobrowska for her help and advice during this study. Our grateful thanks are due to Professor Stella Niemierko and Professor H. Ursin for their criticism and advice during the preparation of the manuscript. Mrs. B. Heiler-Golebiewska's skilful technical assistance is greatly appreciated.
1 BIA~LOWAS,J., AND NARKIEWICZ,O., Acetylcholinesteraseactivityin the septum of the rat: a histochemicaltopography,Acta neurobioL exp. (Warszawa), 34 (1974) 573-582. 2 CAPLAN, i . , An analysis of the effects of septal lesions on negativelyreinforced behaviour, Behav. BioL, 9 (1973) 129-167.
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