Journol of Mcurochrmurry. 1978. Vol 30. pp. 269-272.
Pergamon Press. Prlntcd in Great Brilaln
SHORT COMMUNICATION Choline acetyltransferase and acetyblinesterase activities in limbic nuclei of the rat brain (Receiued 1 June 1977. Accepted I 1 July 1977)
THE HISTOCHEMICAL distribution of acetylcholinesterase cubated at 38°C for IOmin. The [14C]a~etylcholinepro(AChE, acetylcholine acetyl-hydrolase, EC 3.1.1.7) in the duced was extracted with kalignost in acetonitrile into limbic system has been illustrated by SHUTE& LEWIS toluene scintillator fluor and the radioactivity was counted (1967). LEWIS& SHUTE(1967) and JACOBOWITZ& PALKO- in a Packard Tricarb model 3320 liquid scintillation specv m (1974). However, choline acetyltransferase (ChAT, ace- trometer. The counting efficiency was 68%. The amount tyl-CoA-choline 0-acetyltransferase, EC 2.3.1.6) is a more of the radiolabelled product was linear for 30 min and up spenfic marker for cholinergic neurons than AChE to 200 pg wet weight of whole brain tissue. Furthermore. (SILVER,1967; FONNUM, 1970; CHUPPMELLI et ol., 1976). under standard assay conditions, enzyme activity was also In addition, the histochemical staining method has been proportional to the quantities of the dry samples up to used to obtain relative AChE values (STORM-MATHISEN,the maximum used for each tissue examined: 6 S p g nu1970). For study of physiological functions of the limbic cleus amygdaloideus lateralis, pars posterior; 7.0 pg tubersystem, therefore, it is very important to determine O A T culum olfactorium; 14.0 pg pyramidal cell layer in hippocampus (CA2CA3a field) and 8.0 pg nucleus periventricuand AchE activities in limbic nuclei and areas. This paper describes the quantitative distribution of laris hypothalami. These amounts of tissues were about ChAT and AChE in limbic regions, using the microdissec- 2-3 times larger than those used in the usual assay. AChE was assayed by use of a modification of the tion technique of individual nuclei with freeze-dried sec(1969). Four pl of a solution containing tions (UCHIMURAet a!., 1974, 1975) and a radiochemical method of FONNUM 0.25% Triton X-100, 0.05% BSA and 1 x 10-5M-isomicromethod to assay the enzymes. OMPA(tetraisopropy1pyrophosphoramide)were added to dry samples in microtubes. After preincubation for 30 min MATERIALS AND METHODS at O”C, 2 0 4 cold buffer substrate were added (final conWistar-King male rats (17-weeks-old)housed in a group centrations: 85 mwphosphate buffer, pH 7.2, 0.05% BSA, were used for the experiment. The rats were killed by deca- 1 x lo-’ M-iso-OMPA and 1.0m~[I-”C]acetylcholine, pitation at 4 9 0 p.m. The part of the brain containing the 3.84 mCi/mmol, New England Nuclear). Incubation was limbic regions was isolated and frozen in liquid nitrogen. for 1 0 m h at 30°C. After extraction of acetylcholine with A series of frontal sections of the tissue block were made kalignost, the radioactivity of [“qacetate produced was at 100 pm thickness in a cryostat at - 15°C. The sections counted in 10 ml toluene scintillator containing 4% Triton X-100 and 5% naphthalene (TuQK, 1973). The counting were completely freeze-dried overnight at -30°C and mm Hg. The evacuated tubes containing the freeze-dried efficiency was 68% The preliminary experiments indicated sections were stored in the refrigerator (-20°C) for about that AChE activity was proportional to the amount of tissue examined under the present experimental conditions. 5-8 months until use. Limbic nuclei and areas were carefully dissected freehand under a stereomicroscope with the guide of the rat RESULTS AND DISCUSSION brain atlases of KONIG& KLIPPEL (1963) and JACOBOWTZ The results are shown in Table 1. There was approx. & PALKOVITS (1974). In the hippocampal region, the layers in CAI, CA2-CA3a. CA3b and CA4 fields, which were a 30-fold difference between the highest and lowest ChAT activity in limbic nuclei examined. In the hippocampal defined by LORENTE DE N b (1934), were dissected. Scheregion. the highest ChAT activity was found in the pyramimatic drawings of the dissected nuclei and areas are shown in Fig. 1. Each sample was weighed using an electronic dal cell layer (p) of the CAI field. FONNUM (1970) has shown microbalance (Type 4125, Sartorius Co.) with a digital volt that, in CAI field, ChAT activity was concentrated around meter (Type EO-12, Eto Co.). The sensitivity of this the pyramidal cell layer and the highest ChAT activity balance is 0.1 pg. was found in the infrapyramidal zone of stratum oriens. ChAT was assayed by the method of FONNUM (1975). In the present study, the areas around pyramidal cell Three p1 of 0.5% Triton X-100 solution containing 0.05% bodies were not subdivided, so the pyramidal cell layer BSA were added to dry samples in microtubes After prein- had the highest ChAT activity in the CAI field. Furthercubation for 30min at O”C, 10 4 cold buffer substrate were more, in other fields of hippocampus, the highest activity added (final concentrations: 50 mM-phosphate buffer, pH was also found in the pyramidal cell layers. However, the 7.4, 300 mM-Naa, 10 mM-choline, 0. I mweserine, 10 mMpyramidal cell layers in CA3b and CA4 fields contained EDTA, 0.05% BSA and 0.2 m~-[I-’~CJacetylcoenzyme A, lower amounts of ChAT than those in CAI and CA2-CA3a 55.0 mCi/mmole, New England Nuclear). Samples were in- fields. In amygdaloid nuclei, the posterior lateral nucleus (alp) Abbreviations used: ChAT, choline acetyltransferase; contained the highest concentration of ChAT, while the AChE, acetylcholinesterase. medial nucleus (am) had the lowest concentration in the
270
Short communication a)
A 8620
I
TU
e)
CA 1
A4110
d)
A4380
f)
A 25 80
CE
FIG. 1. Schematic drawings of microdissection of limbic and some hypothalamic nuclei. Frontal sections through the rostra1 limbic nuclei and limbic cortex (a, b, c, f), some hypothalamic nuclei and amygdala (d), and hippocampus (e) with the coordinates (in pm) after atlas of K ~ N I G & KLIPPEL(1963). The stippled areas represent the dissected nuclei. Abbreviations: ala, n. amygdaloideus lateralis pars anterior; ap. n. amygdaloideus posterior; CA, commissura anterior; CAA, commissura anterior pars anterior; CO, chiasma opticus; cp, caudatus putamen; DM. n. dorsomedialis (hypothalami); F, fornix; FMP, fasciculus medialis prosenccphali; FMT, fasciculus mammillothalamicus; FR. fasciculus retroflexus; GD, gyrus dentatus; H,hippocampus; LM,lemniscus medialis; M, mammillary body; POL, n. preopticus lateralis; POM, n. preopticus medialis; snr, substantia nigra zona reticularis; TD, tractus diagonalis; TO, tractus opticus; TOL. tractus olfactorius lateralis; VM, n. ventromedialis (hypothalami) The remainder of the abbreviations used appear in Table 1. Limbic nuclei examined. ChAT activity in the anterior lateral nucleus (ala: 113 pmol/g dry wt./h, mean activity of two animals) was about 5 times lower than the value in the posterior lateral nucleus. Relatively high ChAT activity was also found in nuclei of basolateralis (abl), basomedialis (abm) and corticalis (aco). In the limbic cortex, the third and fifth layers of the cingulate cortex (Ci3, Cis) had relatively low ChAT activity. On the other hand, piriform cortex (Pi) and entorhinal cortex (CE) contained relatively high amounts of ChAT. In tuberculum olfactorium (TU) and nucleus
accumbens (a) high ChAT was found especially in tuberculum olfactorium. It is indicated that dopaminergic systems interact with cholinergic neurons in nucleus caudatus and nucleus accumbens (TUBUCCHI et al., 1975; MARCOel al., 1976) Large amounts of ChAT have been found in tuberculum olfactorium, some amygdaloid nuclei (alp, abl) and entorhinal cortex containing dopamine nerve terminals in high density E AND^ et a/., 1966; FUXEet 01.. 1974; HOKFELT et al., 1974). Therefore, dopaminergic-cholinergic nerve interaction m a y also exist in these areas
Short communication TABLE1. ChAT
AND
Nucleus and area Limbic cortex: Cingulate cortex, 3rd layer (Ci3) Cingulate cortex, 5th layer (Ci5) Piriform cortex (Pi) Entorhinal cortex (CE) Hippocampus: CAI field Stratum oriens ( 0 ) Stratum pyramidale (p) Stratum radiatum (r) Stratum lacunosum-moleculare (Im) CA2 and CA3a field Stratum oriens Stratum pyramidale Stratum radiatum CA3b field Stratum oriens Stratum pyramidale Stratum radiatum CA4 field Stratum pyramidale Rostra1 limbic nuclei: Tuberculum olfactorium (TU) Nucleus tractus diagonalis (td) Nucleus accumbens (a) Nucleus septalis dorsalis (xi) Nucleus septalis medialis (sm) Nucelus septalis lateralis (sl) Nucleus septalis fimbrialis (sf) Amygdaloid nuclei: Nucleus amygdaloideus medialis (am) Nucleus amygdaloideus centralis (ac) Nucleus amygdaloideus corticalis (aco) Nucleus amygdaloideus basalis, pars medialis (abm) pars lateralis (abl) Nucleus amygdaloideus lateralis, pars posterior (alp) Hypothalamic nuclei: Nucleus periventricularis, pars medialis @e) Median eminence (ME)
AChE
271
ACTIVITIES IN LIMBIC A N D SOME HYWTHALAMIC NUCLEI
ChAT
AChE
Ratio AChE/ChAT
38.89 k 0.68 (4) 26.17 f 0.88 (5) 102.22 f 3.00 (5) 118.16 f 7.51 (4)
15.98 f 0.80 (6) 12.52 f 0.96 (5) 34.00 f 1.93 (5) 33.14 2.36(5)
24.7 28.7 20.0 16.8
36.13 f 2.54 (4) 76.39 f 2.26 (5) 37.84 f 1.22 (5) 41.16 f 1.94 (4)
16.55 f 1.31 (5) 37.34 f 2.88 (5) 18.53 f 0.91 (4) 14.06 f 1.03 (5)
27.5 29.3 29.4 20.5
48.05 f 3.56 (5) 67.64 f 6.49 (5) 40.32 f 3.64 (5)
22.33 f 2.24 (5) 36.59 f 1.78 (5) 19.37 & 1.16 (5)
27.9 32.5 28.8
23.32 f 2.05 (5) 50.02 f 1.90 (4) 30.67 2.91 (5)
14.95 f 1.16 (6) 25.77 f 1.39 (5) 14.90 f I .46 (4)
38.5 30.9 29.1
51.01 f 1.97 (4)
21.00 f 1.78 (5)
24.7
255.07 f 19.99 (5) 199.62 f 14.37 (5) 64.00 f 1.76 (5) 30.40 f 3.66 (4) 41.65 f 3.27 (4) 26.72 f 4.06 (6) 48.09 f 4.29 (6)
232.61 f 29.51 (5) 87.79 f 10.69 (4) 52.46 f 3.54 (5) 21.08 f 2.58 (5) 32.91 2.39 (5) 24.22 f 3.94 (5) 28.49 f 2.05 (5)
54.7 26.4 49.2 41.6 47.4 54.4 35.6
16.88 f 1.26 (5) 57.11 f 5.07 (5) 91.14 f 2.71 (6)
16.25 f 0.83 (4) 24.51 f 1.83 (4) 32.23 f 1.68 (5)
57.8 25.8 21.2
83.84 f 6.85 (5) 100.88 f 7.10 (5)
30.48 f 1.42 (5) 35.43 f 2.20 (5)
21.8 21.1
509.22 f 7.30 (6)
151.37 _+ 12.20 (5)
17.8
14.99 0.97 (5) 129.16 f 11.23 (4)
19.72 f 1.28 (5) 10.60 f 0.79 (5)
78.9 4.9
The results are expressed as mean values f S.E.M.with number of animals in brackets. ChAT: pmol acetylcholine synthesizedlg dry wt./h. AChE: pmol acetylcholine hydrolysed/g dry wt./min. With regard to the ratio between O A T activity and with frozen sections. They showed that limbic cortical AChE activity in individual nuclei or areas, GOLDBERG & areas (Ci, Pi, CE) and amygdaloid nuclei had small differMCCAMAN(1967) reported that there was no correlation ences in the amount of ChAT compared with each other. between relative levels of ChAT and AChE in the various However, in the present study, large ditlerences in ChAT cerebellar layers. By contrast, FONNUM(1970) reported that activity for these nuclei were found. On the other hand, in layers (CAI field and area dentata) of the hippocampal recently BEN-ARIet 01. (1977), using microdissection techregion, ChAT distributed similarly to AChE. In the present nique with fresh sections, showed that ChAT was unevenly study, the ratios of AChE to ChAT were quite constant distributed in the amygdaloid complex and also within in the different nuclei or areas with few exceptions within some nuclei (alp, abl, Pi). These authors found relatively each gross region (hippocampus, amygdaloid nuclei, rostra1 similar activities to those of present report. However, limbic nuclei, limbic cotex) but not constant in the different ChAT activities in some nuclei (aco, abm, alp, Pi) dissected gross regions In addition, in some hypothalamic nuclei, in the present study were 1.5-2.5 times higher than those it is of interest that the median eminence has high ChAT obtained by them. These discrepancies may reflect the difactivity and very low AchE activity. This suggests that the ferences in the dissection procedures. In the study of PALmedian eminence may have a characteristic cholinergic KOMTS et ul., each nucleus was punched from frozen see structure participating in the regulation of hormonal se- tions (300 pm) with a hollow needle and samples of tissues cretions from 3 rats were pooled for each assay. On the other hand, PALKOWTS et 01. (1974) reported the distribution of in the study of BEN-ARIet al., each nucleus was dissected ChAT in the limbic system, using a micropunch technique from fresh, lightly stained sections (400pm) and samples
Short communication
272
of tissues from several consecutive sections of a single rat BEN-ARIY., ZIGMOND R. E., SHL~TE C. C. D. & LEWISP. were pooled for each assay. In addition, samples of subdiR. (1977) Brain Res. 120. 4 3 5 4 5 . visions of some nuclei (alp, abl, Pi) were dissected from CHIAPPINELLI V., GIACOBINI E., PILARG. & UCHIMURA H. a section. In contrast, in the present study. each nucleus (1976) J. Physiol., Lond. 257. 749-766. was dissected from freeze-dried sections (100 pm) and only FONNUM F. (1969) Biochem J. 115. 465472. one dissected sample was used for each assay. Further- FONNUM F. (1970) J. Neurochem. 17, 1029-1037. more, the present method has some other advantages: FONNUM F. (1975) J. Neurochem. 24, 407409. ChAT and AChE activities in freeze-dried samples in eva- FUXEK., H~KFELT T., JOHANSSON 0.. JONSSON G.. LIDcuated tubes (-20°C) were stable for at least 18 months BRINK P. & ~ J N G D A H L li. (1974) Brain Res. 82,349--355. and it was possible under the microscope to see and dissect GOLDBERG A. M. & MCCAMANR . E. (1967) Lfe- Sci. 6, fine structures of freeze-dried sections as reported by 1493--1500. LOWRY (1953. 1962). H~KFELT T., LJL-GDAHLA., FLXEK. & JOHANSSON 0. In summary, ChAT and AChE were unevenly distri(1974) Science-, N.Y. 184, 177-179. buted in limbic regions. T h e present method, using mic- JACOBOWITZ D. M. & PALKOMTSM. (1974) J . comp. rodissection technique with freeze-dried sections, may help Neurol. 157, 13-28. in understanding the physiological roles of the limbic nu- K ~ N I C J. F. R. & KLIPPEL R. A. (1963) The Rat Brain. clei. a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams & Wilkins, Baltimore. Acknowledgement-This work was supported by a grant LEWISP. R. & SHUTEC. C. D. (1967) Brain 90, 521-540. LORENIT DE N 6 R. (1934) J . Psychol. Neurol., Leipzig 46. from the Science and Technology Agency of Japan. 113-177. Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga, 842-01, Japan
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LOWRY0. H. (1953) J. Histochem. Cytochem. 1, 420428. uCHIMURA s, K~~ LOWRY 0.H. (1962) Harvey Lecture 58. 1-19. J.
MARm E., MAO C. C., CHENEY D. L., REVUELTA A. & CO~A E. (1976) Nature, Lond. 264, 363- 365. PALKOVITS M., SMVEDRAJ. M., KOBAYASHI R. M. & Department of Neuropsychiatry, BROWNSTEIN M. (1974) Brain Res. 79, 443450. Faculty of Medicine. Kyushu Unioersity, SILVER A. (1967) Int. Rev. Neurohwl. 10, 57-109. Katakasu, Fukuoka, 812, Japan STORM-MATHISEN J. (1970) J. Neurochem. 17, 739-750. T. NAKAHARA SHUTEC. C. D. & LEWISP. R. (1967) Brain 90, 497-520. Department of Chemistry, TRABUCCHI M., CHENEY D. L., RACAGNIG. & COSTA E. Faculty of Science, Kyushu University, (1975) Brain Res. 85, 13G134. Hakozaki, Fukuoka, 812, Japan TU&K S. (1973) J . Neurochem. 20, 901-903. UCHIMURAH., HIRANOM., S A I K JM., MUKAI A. & REFERENCES HAZAMA H. (1974) Brain & Nerve (in Japanese) 26, 341 345. AND^ N.-E.. DAHLSTR~M A., FUXE K.. LARSSONL., OLSON M. & HIRANOM.(1975) Brain Res. L. & UNGWSTEDTU. (1966) Acto physiol. scand. 67, UCHlrn'RA H., 3 13-326. 91, 161-164.
M, M. H~~~~ M''TI
Pergamon Press. Prlntcd in Great Brilaln
SHORT COMMUNICATION Choline acetyltransferase and acetyblinesterase activities in limbic nuclei of the rat brain (Receiued 1 June 1977. Accepted I 1 July 1977)
THE HISTOCHEMICAL distribution of acetylcholinesterase cubated at 38°C for IOmin. The [14C]a~etylcholinepro(AChE, acetylcholine acetyl-hydrolase, EC 3.1.1.7) in the duced was extracted with kalignost in acetonitrile into limbic system has been illustrated by SHUTE& LEWIS toluene scintillator fluor and the radioactivity was counted (1967). LEWIS& SHUTE(1967) and JACOBOWITZ& PALKO- in a Packard Tricarb model 3320 liquid scintillation specv m (1974). However, choline acetyltransferase (ChAT, ace- trometer. The counting efficiency was 68%. The amount tyl-CoA-choline 0-acetyltransferase, EC 2.3.1.6) is a more of the radiolabelled product was linear for 30 min and up spenfic marker for cholinergic neurons than AChE to 200 pg wet weight of whole brain tissue. Furthermore. (SILVER,1967; FONNUM, 1970; CHUPPMELLI et ol., 1976). under standard assay conditions, enzyme activity was also In addition, the histochemical staining method has been proportional to the quantities of the dry samples up to used to obtain relative AChE values (STORM-MATHISEN,the maximum used for each tissue examined: 6 S p g nu1970). For study of physiological functions of the limbic cleus amygdaloideus lateralis, pars posterior; 7.0 pg tubersystem, therefore, it is very important to determine O A T culum olfactorium; 14.0 pg pyramidal cell layer in hippocampus (CA2CA3a field) and 8.0 pg nucleus periventricuand AchE activities in limbic nuclei and areas. This paper describes the quantitative distribution of laris hypothalami. These amounts of tissues were about ChAT and AChE in limbic regions, using the microdissec- 2-3 times larger than those used in the usual assay. AChE was assayed by use of a modification of the tion technique of individual nuclei with freeze-dried sec(1969). Four pl of a solution containing tions (UCHIMURAet a!., 1974, 1975) and a radiochemical method of FONNUM 0.25% Triton X-100, 0.05% BSA and 1 x 10-5M-isomicromethod to assay the enzymes. OMPA(tetraisopropy1pyrophosphoramide)were added to dry samples in microtubes. After preincubation for 30 min MATERIALS AND METHODS at O”C, 2 0 4 cold buffer substrate were added (final conWistar-King male rats (17-weeks-old)housed in a group centrations: 85 mwphosphate buffer, pH 7.2, 0.05% BSA, were used for the experiment. The rats were killed by deca- 1 x lo-’ M-iso-OMPA and 1.0m~[I-”C]acetylcholine, pitation at 4 9 0 p.m. The part of the brain containing the 3.84 mCi/mmol, New England Nuclear). Incubation was limbic regions was isolated and frozen in liquid nitrogen. for 1 0 m h at 30°C. After extraction of acetylcholine with A series of frontal sections of the tissue block were made kalignost, the radioactivity of [“qacetate produced was at 100 pm thickness in a cryostat at - 15°C. The sections counted in 10 ml toluene scintillator containing 4% Triton X-100 and 5% naphthalene (TuQK, 1973). The counting were completely freeze-dried overnight at -30°C and mm Hg. The evacuated tubes containing the freeze-dried efficiency was 68% The preliminary experiments indicated sections were stored in the refrigerator (-20°C) for about that AChE activity was proportional to the amount of tissue examined under the present experimental conditions. 5-8 months until use. Limbic nuclei and areas were carefully dissected freehand under a stereomicroscope with the guide of the rat RESULTS AND DISCUSSION brain atlases of KONIG& KLIPPEL (1963) and JACOBOWTZ The results are shown in Table 1. There was approx. & PALKOVITS (1974). In the hippocampal region, the layers in CAI, CA2-CA3a. CA3b and CA4 fields, which were a 30-fold difference between the highest and lowest ChAT activity in limbic nuclei examined. In the hippocampal defined by LORENTE DE N b (1934), were dissected. Scheregion. the highest ChAT activity was found in the pyramimatic drawings of the dissected nuclei and areas are shown in Fig. 1. Each sample was weighed using an electronic dal cell layer (p) of the CAI field. FONNUM (1970) has shown microbalance (Type 4125, Sartorius Co.) with a digital volt that, in CAI field, ChAT activity was concentrated around meter (Type EO-12, Eto Co.). The sensitivity of this the pyramidal cell layer and the highest ChAT activity balance is 0.1 pg. was found in the infrapyramidal zone of stratum oriens. ChAT was assayed by the method of FONNUM (1975). In the present study, the areas around pyramidal cell Three p1 of 0.5% Triton X-100 solution containing 0.05% bodies were not subdivided, so the pyramidal cell layer BSA were added to dry samples in microtubes After prein- had the highest ChAT activity in the CAI field. Furthercubation for 30min at O”C, 10 4 cold buffer substrate were more, in other fields of hippocampus, the highest activity added (final concentrations: 50 mM-phosphate buffer, pH was also found in the pyramidal cell layers. However, the 7.4, 300 mM-Naa, 10 mM-choline, 0. I mweserine, 10 mMpyramidal cell layers in CA3b and CA4 fields contained EDTA, 0.05% BSA and 0.2 m~-[I-’~CJacetylcoenzyme A, lower amounts of ChAT than those in CAI and CA2-CA3a 55.0 mCi/mmole, New England Nuclear). Samples were in- fields. In amygdaloid nuclei, the posterior lateral nucleus (alp) Abbreviations used: ChAT, choline acetyltransferase; contained the highest concentration of ChAT, while the AChE, acetylcholinesterase. medial nucleus (am) had the lowest concentration in the
270
Short communication a)
A 8620
I
TU
e)
CA 1
A4110
d)
A4380
f)
A 25 80
CE
FIG. 1. Schematic drawings of microdissection of limbic and some hypothalamic nuclei. Frontal sections through the rostra1 limbic nuclei and limbic cortex (a, b, c, f), some hypothalamic nuclei and amygdala (d), and hippocampus (e) with the coordinates (in pm) after atlas of K ~ N I G & KLIPPEL(1963). The stippled areas represent the dissected nuclei. Abbreviations: ala, n. amygdaloideus lateralis pars anterior; ap. n. amygdaloideus posterior; CA, commissura anterior; CAA, commissura anterior pars anterior; CO, chiasma opticus; cp, caudatus putamen; DM. n. dorsomedialis (hypothalami); F, fornix; FMP, fasciculus medialis prosenccphali; FMT, fasciculus mammillothalamicus; FR. fasciculus retroflexus; GD, gyrus dentatus; H,hippocampus; LM,lemniscus medialis; M, mammillary body; POL, n. preopticus lateralis; POM, n. preopticus medialis; snr, substantia nigra zona reticularis; TD, tractus diagonalis; TO, tractus opticus; TOL. tractus olfactorius lateralis; VM, n. ventromedialis (hypothalami) The remainder of the abbreviations used appear in Table 1. Limbic nuclei examined. ChAT activity in the anterior lateral nucleus (ala: 113 pmol/g dry wt./h, mean activity of two animals) was about 5 times lower than the value in the posterior lateral nucleus. Relatively high ChAT activity was also found in nuclei of basolateralis (abl), basomedialis (abm) and corticalis (aco). In the limbic cortex, the third and fifth layers of the cingulate cortex (Ci3, Cis) had relatively low ChAT activity. On the other hand, piriform cortex (Pi) and entorhinal cortex (CE) contained relatively high amounts of ChAT. In tuberculum olfactorium (TU) and nucleus
accumbens (a) high ChAT was found especially in tuberculum olfactorium. It is indicated that dopaminergic systems interact with cholinergic neurons in nucleus caudatus and nucleus accumbens (TUBUCCHI et al., 1975; MARCOel al., 1976) Large amounts of ChAT have been found in tuberculum olfactorium, some amygdaloid nuclei (alp, abl) and entorhinal cortex containing dopamine nerve terminals in high density E AND^ et a/., 1966; FUXEet 01.. 1974; HOKFELT et al., 1974). Therefore, dopaminergic-cholinergic nerve interaction m a y also exist in these areas
Short communication TABLE1. ChAT
AND
Nucleus and area Limbic cortex: Cingulate cortex, 3rd layer (Ci3) Cingulate cortex, 5th layer (Ci5) Piriform cortex (Pi) Entorhinal cortex (CE) Hippocampus: CAI field Stratum oriens ( 0 ) Stratum pyramidale (p) Stratum radiatum (r) Stratum lacunosum-moleculare (Im) CA2 and CA3a field Stratum oriens Stratum pyramidale Stratum radiatum CA3b field Stratum oriens Stratum pyramidale Stratum radiatum CA4 field Stratum pyramidale Rostra1 limbic nuclei: Tuberculum olfactorium (TU) Nucleus tractus diagonalis (td) Nucleus accumbens (a) Nucleus septalis dorsalis (xi) Nucleus septalis medialis (sm) Nucelus septalis lateralis (sl) Nucleus septalis fimbrialis (sf) Amygdaloid nuclei: Nucleus amygdaloideus medialis (am) Nucleus amygdaloideus centralis (ac) Nucleus amygdaloideus corticalis (aco) Nucleus amygdaloideus basalis, pars medialis (abm) pars lateralis (abl) Nucleus amygdaloideus lateralis, pars posterior (alp) Hypothalamic nuclei: Nucleus periventricularis, pars medialis @e) Median eminence (ME)
AChE
271
ACTIVITIES IN LIMBIC A N D SOME HYWTHALAMIC NUCLEI
ChAT
AChE
Ratio AChE/ChAT
38.89 k 0.68 (4) 26.17 f 0.88 (5) 102.22 f 3.00 (5) 118.16 f 7.51 (4)
15.98 f 0.80 (6) 12.52 f 0.96 (5) 34.00 f 1.93 (5) 33.14 2.36(5)
24.7 28.7 20.0 16.8
36.13 f 2.54 (4) 76.39 f 2.26 (5) 37.84 f 1.22 (5) 41.16 f 1.94 (4)
16.55 f 1.31 (5) 37.34 f 2.88 (5) 18.53 f 0.91 (4) 14.06 f 1.03 (5)
27.5 29.3 29.4 20.5
48.05 f 3.56 (5) 67.64 f 6.49 (5) 40.32 f 3.64 (5)
22.33 f 2.24 (5) 36.59 f 1.78 (5) 19.37 & 1.16 (5)
27.9 32.5 28.8
23.32 f 2.05 (5) 50.02 f 1.90 (4) 30.67 2.91 (5)
14.95 f 1.16 (6) 25.77 f 1.39 (5) 14.90 f I .46 (4)
38.5 30.9 29.1
51.01 f 1.97 (4)
21.00 f 1.78 (5)
24.7
255.07 f 19.99 (5) 199.62 f 14.37 (5) 64.00 f 1.76 (5) 30.40 f 3.66 (4) 41.65 f 3.27 (4) 26.72 f 4.06 (6) 48.09 f 4.29 (6)
232.61 f 29.51 (5) 87.79 f 10.69 (4) 52.46 f 3.54 (5) 21.08 f 2.58 (5) 32.91 2.39 (5) 24.22 f 3.94 (5) 28.49 f 2.05 (5)
54.7 26.4 49.2 41.6 47.4 54.4 35.6
16.88 f 1.26 (5) 57.11 f 5.07 (5) 91.14 f 2.71 (6)
16.25 f 0.83 (4) 24.51 f 1.83 (4) 32.23 f 1.68 (5)
57.8 25.8 21.2
83.84 f 6.85 (5) 100.88 f 7.10 (5)
30.48 f 1.42 (5) 35.43 f 2.20 (5)
21.8 21.1
509.22 f 7.30 (6)
151.37 _+ 12.20 (5)
17.8
14.99 0.97 (5) 129.16 f 11.23 (4)
19.72 f 1.28 (5) 10.60 f 0.79 (5)
78.9 4.9
The results are expressed as mean values f S.E.M.with number of animals in brackets. ChAT: pmol acetylcholine synthesizedlg dry wt./h. AChE: pmol acetylcholine hydrolysed/g dry wt./min. With regard to the ratio between O A T activity and with frozen sections. They showed that limbic cortical AChE activity in individual nuclei or areas, GOLDBERG & areas (Ci, Pi, CE) and amygdaloid nuclei had small differMCCAMAN(1967) reported that there was no correlation ences in the amount of ChAT compared with each other. between relative levels of ChAT and AChE in the various However, in the present study, large ditlerences in ChAT cerebellar layers. By contrast, FONNUM(1970) reported that activity for these nuclei were found. On the other hand, in layers (CAI field and area dentata) of the hippocampal recently BEN-ARIet 01. (1977), using microdissection techregion, ChAT distributed similarly to AChE. In the present nique with fresh sections, showed that ChAT was unevenly study, the ratios of AChE to ChAT were quite constant distributed in the amygdaloid complex and also within in the different nuclei or areas with few exceptions within some nuclei (alp, abl, Pi). These authors found relatively each gross region (hippocampus, amygdaloid nuclei, rostra1 similar activities to those of present report. However, limbic nuclei, limbic cotex) but not constant in the different ChAT activities in some nuclei (aco, abm, alp, Pi) dissected gross regions In addition, in some hypothalamic nuclei, in the present study were 1.5-2.5 times higher than those it is of interest that the median eminence has high ChAT obtained by them. These discrepancies may reflect the difactivity and very low AchE activity. This suggests that the ferences in the dissection procedures. In the study of PALmedian eminence may have a characteristic cholinergic KOMTS et ul., each nucleus was punched from frozen see structure participating in the regulation of hormonal se- tions (300 pm) with a hollow needle and samples of tissues cretions from 3 rats were pooled for each assay. On the other hand, PALKOWTS et 01. (1974) reported the distribution of in the study of BEN-ARIet al., each nucleus was dissected ChAT in the limbic system, using a micropunch technique from fresh, lightly stained sections (400pm) and samples
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of tissues from several consecutive sections of a single rat BEN-ARIY., ZIGMOND R. E., SHL~TE C. C. D. & LEWISP. were pooled for each assay. In addition, samples of subdiR. (1977) Brain Res. 120. 4 3 5 4 5 . visions of some nuclei (alp, abl, Pi) were dissected from CHIAPPINELLI V., GIACOBINI E., PILARG. & UCHIMURA H. a section. In contrast, in the present study. each nucleus (1976) J. Physiol., Lond. 257. 749-766. was dissected from freeze-dried sections (100 pm) and only FONNUM F. (1969) Biochem J. 115. 465472. one dissected sample was used for each assay. Further- FONNUM F. (1970) J. Neurochem. 17, 1029-1037. more, the present method has some other advantages: FONNUM F. (1975) J. Neurochem. 24, 407409. ChAT and AChE activities in freeze-dried samples in eva- FUXEK., H~KFELT T., JOHANSSON 0.. JONSSON G.. LIDcuated tubes (-20°C) were stable for at least 18 months BRINK P. & ~ J N G D A H L li. (1974) Brain Res. 82,349--355. and it was possible under the microscope to see and dissect GOLDBERG A. M. & MCCAMANR . E. (1967) Lfe- Sci. 6, fine structures of freeze-dried sections as reported by 1493--1500. LOWRY (1953. 1962). H~KFELT T., LJL-GDAHLA., FLXEK. & JOHANSSON 0. In summary, ChAT and AChE were unevenly distri(1974) Science-, N.Y. 184, 177-179. buted in limbic regions. T h e present method, using mic- JACOBOWITZ D. M. & PALKOMTSM. (1974) J . comp. rodissection technique with freeze-dried sections, may help Neurol. 157, 13-28. in understanding the physiological roles of the limbic nu- K ~ N I C J. F. R. & KLIPPEL R. A. (1963) The Rat Brain. clei. a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams & Wilkins, Baltimore. Acknowledgement-This work was supported by a grant LEWISP. R. & SHUTEC. C. D. (1967) Brain 90, 521-540. LORENIT DE N 6 R. (1934) J . Psychol. Neurol., Leipzig 46. from the Science and Technology Agency of Japan. 113-177. Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga, 842-01, Japan
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