Journal oJ Neurocbemlsrr!.
1977 VoI. 29. pp. 161 165 Prrganlon Press. Prinled in Grcal Britain
SHORT COMMUNICATION
Tyrosine hydroxylase activity in the catecholamine nerve terminals and cell bodies of the rat brain (Rrceired 6 July 1976. Reoised I7 January 1977. Accepted 21 Jammry 1977)
TYROSINE hydroxylase (TH : EC I . 14.161. r-phcnylalaninc 4-monooxygenase), the rate-limiting enzyme in the biosynthesis of dopamine (DA) and noradrenaline (NA). is present in catecholamine (CA) ncrvc terminals and cell bodies in the brain (MCGEER& McGII'H. 1973). T o understand the physiological roles of CA neuron systcms in brain it is important to study T H activities in the discrete areas, which contain CA nerve tcrminals and cell bodies. Recently. following the quantitative histochemical methods of LOWRY(1953. 1962). a technique for the microdisscction of individual nuclei from freeze-dried sections C I al.. 1974; was developed in our laboratory (UCHIMURA e r al., 1975; SAITO ef al., 1975). 1975: HIRANO In the present paper. T H activities are reported for individual nuclei of the hypothalamus. containing inccrtohypothalamic DA cell groups, mesolimbic DA neuron systems (nucleus accumbens and tubereulum olfactorium-A, cell group). nigro-striatal DA neuron system (nucleus caudatus-putamen: C P ---A, cell group) and some of the pontinc NA cell groups (A, A s cell groups). using our microdissection technique and a n isotopic micromethod to assay the enzyme.
trapping micromethod of FAIRMAN et a / . (1976). Five pl of 50 mu-Tris-acetate buffer (pH 6.0) containing 0.29;; Triton X-100 was added to the dry sample in a microtube. Each microtube stood for 30 rnin in ice-water bath without hornogcnirdtion. then 7 pl of cold buffer substrate were added. All these procedurcs wcrc carried out in a n iccwater bath. The final concentrations of the incubation mixture wcre as follows: 0.17 wsodium phosphate buffer (pH 6.2). 0.8 mu-FeSO,. 0.3 mu-pyridoxal-5-phosphate. 30 units/pl catalase, 0.12 units/pl DOPA decarboxylase ( I unit corresponds to I nmol of I4CO2 formed/min) prepared et al. (1971). from pig kidney by the method of WAYMIRE 8.0 ~ M - ~ M P (2-amino-4-hydroxy-6-methylH, tetrahydrop teridine), 30 rn~-2-rnercaptoethanoIand 200 pM-I.-[I-14C]tyrosine (specific activity: 27.3 mCi/mmol) (New England Nuclear). The incubation tube was connected by rubber tubing to another microtube which contained 40p1 of I M-hyamine solution in mcthanol. The two tubes connected to each other were incubated for 30min a t 37'C. The reaction was stopped by immersing the bottom of the microtube into boiling water for I min (MOSKAL& BAR;. 1975). The procedure gave the same T H activity as that determined by the method using H,SO, for termination of the reaction. T h e tubes were replaced in the water bath MATERIALS AND METHODS for 30min at 37°C in order to obtain complete diffusion Wistar-King male rats (17-week-old) housed in a group of I4CO2. The tube containing hyamine was separated were used for the experiment. The animals were killed by from the connection and was transferred into a counting decapitation a t 4:OO p.m. The whole brain was immediately vial containing 10 ml of toluene scintillator. After shaking removed and carefully cut with a razor blade over the the counting vial for 30min. the radioactivity of the ice-cooled glass-plate into three portions, containing dien- trapped 14C02 was counted in a Packard Tricarb model cephalon, mesencephalon and pons. The tissue blocks were 3320 liquid scintillation spcctrorneter. The counting effiimmediately frozen in liquid nitrogen and a series of fron- ciency was 759/,. tal sections of the thrcc tissue blocks were made at 200 pm In the preliminary study of T H assay using the total thickness in a cryostat .at - 15'C. The sections were rat brain homogenate, the formation of 14C02 from evacuated for about 15 h at -30°C and lo-' mm Hg, and I.-[ 1 -I4C]tyrosine demonstrated a linear relationship durfrcezc-dried completely. The freeze-dricd sections were ing the first 40 min. and was proportional to the tissue stored in evacuated glass-tubes at -20°C until use. concentrations up to 600 pg of wet weight. The apparent The individual nuclei and discrete areas in CA neuron K , for the substrate and the cofactor (6MPH,) were 48 p~ systcms wcrc carcfully dissected freehand. undcr a stcreo- and 3.1 mM respectively. In the case of the frceze-dried microscope with the guide of the rat brain atlases 01 KONIG samples. the formation of '"CO, increased linearly a t least & KLIPPEL (1963). B J ~ R K L L N &DNOBIN(1973) and PALKO- during the first 30 min examined. both in the DA-containVITS & JACOROWITL(1974). Schematic drawings of the dising areas (CP and A, cell group) and in the NA-containing sected nuclei and areas are shown in Fig. I (a-h). Each areas (A, cell group). In addition, to ensure that the rnaxisample was weighed by a n electronic microbalance (Type mum T H activity in these DA and NA containing areas 4125. Sartorius Co.) with a digital voltmeter (Type EO-12. was obtained under the standard assay conditions, T H acEto Co.). The sensitivity of this balance is 0.1 p g . tivities at the higher concentration of the substrate (up T H activity was assayed by a modification of the '"CO, to 300 p ~ o r) the cofactor (up to 10 m u ) were determined in both areas. However, the T H activities under these con.Ahhreuiatiotis used: NA. noradrenaline: DA, dopamine; ditions were the same as the values under the standard CA, catecholamine; TH. tyrosine hydroxylase. assay conditions in both DA and NA containing areas. 161
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The amount of ' ' C 0 2 formation. furthermore. was proportional to the quantities of the dry samples up to the maximum used Tor each tissue examined; 23 pg of median eminence (ME), 31 p g of A, cell group, S3 jig of C'P, 61 pg of area lateralis hypothalami (L) and 200 pg of the tissue containing nucleus preopticus medialis (POM). area p r e o p ticus lateralis (POL)and nucleus anterior hypothalami (A). These amounts of the tissues were about 3 times larger than those of the tissues used in the usual assay. The evacuated tubes containing the freeze-dried sections were retained for about 3-6 months in the refrigerator ( -20' C) until use, and TH activities of these s;-inipIes rcmained stable during at least I2 months.
RESULTS AKD DISCUSSION The results are summarized in Tahle I . There is 186-fold difkrence between the highest 1H activity measured and the lowest one. In the hypothalamic nuclei. the highest
activity was found in the ME, wherc the dense collections of D A and S A nerve terminals exist (JONSSOS et a/., 1972; LiiwrKBhl t'i ( I / . . 1976). Relatively high activities were found i n the anterior part of the nucleus perivenlricularis (PE: A I cell group), medial zona incerta (MZI: A cell group) and nucleus arcuatus (medialis) (AR: A , ? cell group). which contain incerto-hypothalamic D A cell bodies (BJBKKLLND & NOBIN.1973; BJBRKLliNI> t't a/., 1975). In the incerto-hypothalamic CA cell groups, the nucleus posterior hypothalami (P: A , I cell group, comprising both D A and N A cell bodies (BIBRKLLND & NOBIY,1973)) only revealed low TH activity. POM. A. nucleus ventromedialis (VM). nucleus premammillaris ventralis (PMV) and nucleus prernammillitris dorsalis ( P M D ) showed the lowest activities. In the meso-limbic D A neuron system, the A , , cell group had 2- to 5-fold higher T H activity than that of its nerve terminals, nucleus accumhens (AC) and tubercu1971). In the nigrolum olfactorium (TU) (UNGEKSTEDT, striatal D A neuron system, the A, cell group showed about
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FIG. I . Schematic drawings of the dissected nuclei and areas. Frontal sections through the preoptic area (a.b). the anterior (c,d), medial (e) and posterior (0 hypothalamus, the lower brain stem (g,h), and medial part of the median eminence (i). Thc dissected nuclei and cell groups represented by hatched area are; A. n. anterior hypothalami; AC, n. accumbens; AR. n. arcuatus; CP, n. caudatusputamen; DM, n. dorsomedialis; ER, external region of median eminence; IR, internal region of median eminence; IS, infundibular stem; L, area lateralis hypothalami; ME, median eminence; MZI. medial zona incerta; P. n. posterior hypothalami; PA, n. paraventricularis; PE, n. periventricularis; PMD. n. premammillaris dorsalis; PMV. n. premammillaris ventralis; POL, area preopticus lateralis; POM, n. preopticus medialis; RC. area retrochiasmaticus; TC', tubcrculum olfactorium; VM, n. ventromedialis; AIO. A9 dopaminergic cell groups and A6. A5 noradrenergic ccll groups. The other abbreviations used: CA. commisura anterior; CO. chiasma opticus; F, fornix; FMT. fasciculus mammillothalamicus; ip, n. interpeduncularis; LM, lemniscus medialis; ncu, n. cuneiformis; ntd. n. tegmenti dorsalis; os, n. olivaris superior; pcs. pedunculus cerebellaris superior; PS, tructus corticospinalis; r, n. ruber; rpoc, 11. rcticiihris pnntis c:iiid:ilis: snc. siihztantiii n i p 7ona compacta: mr. suhstantia n i p 7ona reticularis: TO. tractus opticus; TSV. tractus spinalis n e n i trigemiIii; 1 1 1 . third ventricle; VII, nervus facialis.
twice the TH activity of CP, which is mainly innervated from the A, cell group (~:iiGERSTEI).r, 1971). In addition to the mesencephalic DA cell groups, T H activities of A, and AS NA cell groups in the medulla oblongata and the pons were cxamined in this study. The A, cell group. which gives rise to NA nerve terminals mainly in cerebrum and cerebellar cortex and hippo1971). revealed T H activity as high campus (UNGERSTEIJI., as that of mesencephalic DA cell groups. On the other hand, the A s cell group. one of the cell groups which give rise to the ventral ascending NA neuron system (UKGERSTEDT, 1971). showed extremely low T H activity compared to that of A, cell group. This may rellect mainly the difference in cell densities in each cell group. Recently, using the micropunch technique with frozen ei a/. reported the dissections of the rat brain, SAAVEDRA tributions of the TH activities in the hypothalamic (1974) and limbic (1976) nuclei. Compared to their results. A, VM and L in our present study showed about 2- to 5-fold lower T H activities. while AC and TIJ had 2- to 3-fold
higher v;ilucs. Regarding thc T H iictikitics in A,,. A c, and A l 0 cell groups, Ltwis L'I ( I / . (1976) and BCDAC I a / . (1975) reported the values per structure (lo00 pm thickness x 0.9 mm dia.) in these areas. In the present study, the weights of A,, A 9 and Al,, cell groups dissected from the sections of 100jcm thickness were ahout 4.5. 10 and IOpp dry weight. respectively. The total TH activities in these areas. estimated as the values per structure (1000pm thickness), were 400, 1100 and 1400 p molih. These values are 1.5- to 3-fold higher than their values. O n the other hand, the weights of A6. A9 and A l o structures in their studies were approx 0.64 mg wet weight. When the T H activities of these areas in their studies were expressed as the values per mg dry weight, therefore, they appear 5- to 8-fold lower than our values. For example, in this study, the weight of A, cell group was about 6-fold lower than their values. The sample of A, cell group obtained by the micropunch technique seems to have contamination with large amounts of the surrounding tissue, which has lower T H activity than that
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Short communication TABLEI . TI1 AcrIvi'rY Nuclei and areas
IS n I E
CA
N E R V E T ~ ~ K M I N A LASI) S CELL RODIES INT i l l : H A . I BRAIN
TH activity
Anterior part of hypothalamus POM 0.26 f 0.03 (3) POL 0.64 f 0.05(4) PE (A14) 2.55 f 0. I5 (4) A 0.28 f 0.02(4) L 0.96 f 0.10(4) PA 1.61 f 0.12(4) RC 1.92 f 0.1 l ( 4 ) Medial part of hypothalamus MZI (A13) 4.20 f 0.39(4) VM 0.15 f 0.04(3) DM 0.89 f 0.07 (4) PE 1.49 f 0.09 (5) L 1.57 f 0.06(4) AR (AI2) 4.15 f 0.53 (4) ME 9.X7 f 0.24(5) IR 8.40 k 1.24(3) ER 11.39 f 2.18(3)
Nuclei and areas
T H activity
Posterior part of hypothalamus P (A 11) 0.76 f 0.05 (5) PMV 0.2 I f 0.02 (4) PMD 0.24 f 0.07 (3) L 0.37 f 0.03 (4) AR 0.50 f 0. I I (4) IS 10.99 f 0.26 (4) Mew-limbic DA neuron system A10 27.91 f 2.80(3) AC 5.91 0.24 (4) TU 10.54 & 0.21 (4) Nigro-striatal DA ncuron system A9 22.59 f 2.53 (4) CP 10.98 f 0.27 (41 Pontine NA cell groups A6 18.12 f 0.91 ( 3 ) A5 0.46 f 0.04 (4)
The results are expressed as values of mean activity (nmol I4CO1 formed/mg dry w ~ h ) W.M. (number of animals).
+
of the A, cell group. Indeed. the TI1 activity in the sur. microisotopic assay for neurotransmitter related enzymes. rounding tissue of A b cell group was below 1.0-2.0nmol has become a useful tool for understanding the physiologiI4CO, formedidry wtih. The diHerences in the TH values cal roles of the neurotransmitters in the small discrete between the rosults obtained by the micropunch technique areas of the hrain. and ours, therefore. may be explaincd mainly by the difference in the niethod for dissecting samples. Ackfionlrdyefnefit~~-This work was supported by a grant It has been reported that there was a highly significant from the Science and Technology Agency of Japan. correlation between TH activity and DA levels (SAAVEDRA er d..1974; SAAVEDKA& Z I V I N1976). Since in the median L h o r a t o r y qf Neurochernisfry. M. SAITO eminence (ME). the external layer contains mainly DA HiZen Narionul Menrul Hospital. M. H I R A N 0 nerve terminals while the internal layer contains mainly Kanzoki, Suyu. 842-01. Japan H. U C H I M I X A er M NA nerve terminals (JONSWK t'r ul., 1972; L ~ F S T R ~ O Departmenr of Cheniisfry, T. NAKAHAHA ul., 1976). we examined. the TH activities in the external Faculty of Science, region and the internal region of M E (divided as shown with stippled lines in Fig. li). to determine whether the Kyushu Unitwsiry, Hakozaki, Fukuoka, 812, Japun TH activity in M E is mainly due to the DA or NA nerve terminals. Although the external region showed slightly Department of Neuropsycliiutry, Faculty qf Mcdicinc,. higher TH activity than that in the internal region, there wiis n o significant diHerence between the two regions. NA K ytrshu Unitvrsiry. Karakasu. F1rkrrnko. 8 I?.J u p u n neuron systems have been considered to have low T H activity (McG~.I.K& MCGI~ER. 1973). Our results obtained by the microdissection technique suggest that some disREFERENCES crete areas which contain dense NA nerve terminals and cell bodies also have high TH activity, as well a s the areas N U& NOBINA. (1973) Brain Res. 51. 193 205. which contain high D A levels. However, VERSTEEC er al. B J ~ K K L U A. A.. LINUVALL 0.& NOBINA. (1975) Bruin Res. (1976) reported that DA neurons might be present in the BJORKLUNU 89, 2 9 4 2 . A, cell group since this region had a relatively high DA BllDA M.. ROUSSELB., RENAUDB. & PUJOLJ. F. (1975) level. So. the possibility that the high TH value of Ah cell Bruin Res. 93. 564569. group may reflect the TH activity of the DA neuron sysFAIRMAN K.. G I A ( . O I ~E.I ~&I CHIAPPINELLI V. (1976) Bruin tems in this region cannot be ruled out. Rrs. 102. 301 -312. In summary. the present study has shown the TH activiHIMNO M., L'CHIMCRA H. & SAITT) M. (1975) Brain Rrs. ties in the mesencephalic and pontine cell groups in the 93. 558 -565. CA neuron systems obtained by precise dissection. and by JONSSONG . . F'I:xE K. & H ~ K F E LT.T (1972) Bruin Res. 40. the simultaneous assay of TH activities in several CA cell 271-28 1. groups and their nerve terminals. It is noteworthy that KONIG J. F. R. & KLIPPEI.R. A,. (1963) The Rur Brain; in the brain nuclei examined. the highest activity was found a Srtwotaxic Arlm of rhr Furubruin m d lower Parts of in A*,. A l , I>A and A, NA cell groups. Further examinthe Brain Stcvn. Williams & Wilkins,. Baltimore. ations of T H activities in other lower brain stem NA and LEWIS B. D.. RENAUI) B.. BUDAM. & Pt;JOl, J. F. (1976) DA cell groups. such as A , , A?. A3. A,. A, and A, are Brain Res. 108. 339 349. therefore desirable. O u r method. using the microdissection L o W R Y 0.H. (1953) J . Hisrocliem. Cyrochem. I . 420428. technique with freeze-dried sections of the rat brain and
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LOWRY0. H. (1962) Haaey Lecture 58, 1-19. M. & UCHIML'RA H. (1975) Broiii Res. SAITOM., HIRANO L ~ F S T RA., ~M JONSON G. & F u x ~K. (1976) J. Histochem. 99. 4 1 W 1 4 . Cyiochern. 24. 41 5 4 2 9 . UCHIMUKAH., HIRANOM.. S A I K I M., MUKAI A. & MCGEERP. L. & MCGEERE. G. (1973) in Progress in HAZAMA H. (1974) Brain atid Nerve (in Japanese) 26, Neurohiology (KERKL'TG. A. & PHILLIPS J. W., eds.) 341 345. Vol. 2, pp. 69--l17. Pergamon Press. Oxford. UCtiIMUR4 H., SAITOM. & HIRANO M. (1975) Brain Res. MOSKALJ. R. & BASU S. (1975) Arialyr. Biochern. 65. 91, 161-164. 449-457. UNGERSTEDT I ; . (I97 I ) Acra physiol. scarid. 82. Suppl. 367. PALKOVITS M. & JACOBOWITZ D. M. (1974) J. cornp. Neur. 1 --48. 157. 29-42. VERSTEECD. H. G.. VAN DER GUGTEN J., DE JONG W. SAAVEDRA J. M.. BROWNSTEIN M., PALKOVITS M., KIZER & PALKOVITS M. (1976) Brain Res. 113. 563-574. S. & AXELROD J. (1974) J. Neurochem. 23. 869-871. WAYMIRE J. C., BJURR. & WEINERN. (1971) A n d y ? . BioJ. M. & ZIVINJ. (1976) Brairi Rrs. 105, 517. 524. SAAVEORA chern. 43. 588 -600.
1977 VoI. 29. pp. 161 165 Prrganlon Press. Prinled in Grcal Britain
SHORT COMMUNICATION
Tyrosine hydroxylase activity in the catecholamine nerve terminals and cell bodies of the rat brain (Rrceired 6 July 1976. Reoised I7 January 1977. Accepted 21 Jammry 1977)
TYROSINE hydroxylase (TH : EC I . 14.161. r-phcnylalaninc 4-monooxygenase), the rate-limiting enzyme in the biosynthesis of dopamine (DA) and noradrenaline (NA). is present in catecholamine (CA) ncrvc terminals and cell bodies in the brain (MCGEER& McGII'H. 1973). T o understand the physiological roles of CA neuron systcms in brain it is important to study T H activities in the discrete areas, which contain CA nerve tcrminals and cell bodies. Recently. following the quantitative histochemical methods of LOWRY(1953. 1962). a technique for the microdisscction of individual nuclei from freeze-dried sections C I al.. 1974; was developed in our laboratory (UCHIMURA e r al., 1975; SAITO ef al., 1975). 1975: HIRANO In the present paper. T H activities are reported for individual nuclei of the hypothalamus. containing inccrtohypothalamic DA cell groups, mesolimbic DA neuron systems (nucleus accumbens and tubereulum olfactorium-A, cell group). nigro-striatal DA neuron system (nucleus caudatus-putamen: C P ---A, cell group) and some of the pontinc NA cell groups (A, A s cell groups). using our microdissection technique and a n isotopic micromethod to assay the enzyme.
trapping micromethod of FAIRMAN et a / . (1976). Five pl of 50 mu-Tris-acetate buffer (pH 6.0) containing 0.29;; Triton X-100 was added to the dry sample in a microtube. Each microtube stood for 30 rnin in ice-water bath without hornogcnirdtion. then 7 pl of cold buffer substrate were added. All these procedurcs wcrc carried out in a n iccwater bath. The final concentrations of the incubation mixture wcre as follows: 0.17 wsodium phosphate buffer (pH 6.2). 0.8 mu-FeSO,. 0.3 mu-pyridoxal-5-phosphate. 30 units/pl catalase, 0.12 units/pl DOPA decarboxylase ( I unit corresponds to I nmol of I4CO2 formed/min) prepared et al. (1971). from pig kidney by the method of WAYMIRE 8.0 ~ M - ~ M P (2-amino-4-hydroxy-6-methylH, tetrahydrop teridine), 30 rn~-2-rnercaptoethanoIand 200 pM-I.-[I-14C]tyrosine (specific activity: 27.3 mCi/mmol) (New England Nuclear). The incubation tube was connected by rubber tubing to another microtube which contained 40p1 of I M-hyamine solution in mcthanol. The two tubes connected to each other were incubated for 30min a t 37'C. The reaction was stopped by immersing the bottom of the microtube into boiling water for I min (MOSKAL& BAR;. 1975). The procedure gave the same T H activity as that determined by the method using H,SO, for termination of the reaction. T h e tubes were replaced in the water bath MATERIALS AND METHODS for 30min at 37°C in order to obtain complete diffusion Wistar-King male rats (17-week-old) housed in a group of I4CO2. The tube containing hyamine was separated were used for the experiment. The animals were killed by from the connection and was transferred into a counting decapitation a t 4:OO p.m. The whole brain was immediately vial containing 10 ml of toluene scintillator. After shaking removed and carefully cut with a razor blade over the the counting vial for 30min. the radioactivity of the ice-cooled glass-plate into three portions, containing dien- trapped 14C02 was counted in a Packard Tricarb model cephalon, mesencephalon and pons. The tissue blocks were 3320 liquid scintillation spcctrorneter. The counting effiimmediately frozen in liquid nitrogen and a series of fron- ciency was 759/,. tal sections of the thrcc tissue blocks were made at 200 pm In the preliminary study of T H assay using the total thickness in a cryostat .at - 15'C. The sections were rat brain homogenate, the formation of 14C02 from evacuated for about 15 h at -30°C and lo-' mm Hg, and I.-[ 1 -I4C]tyrosine demonstrated a linear relationship durfrcezc-dried completely. The freeze-dricd sections were ing the first 40 min. and was proportional to the tissue stored in evacuated glass-tubes at -20°C until use. concentrations up to 600 pg of wet weight. The apparent The individual nuclei and discrete areas in CA neuron K , for the substrate and the cofactor (6MPH,) were 48 p~ systcms wcrc carcfully dissected freehand. undcr a stcreo- and 3.1 mM respectively. In the case of the frceze-dried microscope with the guide of the rat brain atlases 01 KONIG samples. the formation of '"CO, increased linearly a t least & KLIPPEL (1963). B J ~ R K L L N &DNOBIN(1973) and PALKO- during the first 30 min examined. both in the DA-containVITS & JACOROWITL(1974). Schematic drawings of the dising areas (CP and A, cell group) and in the NA-containing sected nuclei and areas are shown in Fig. I (a-h). Each areas (A, cell group). In addition, to ensure that the rnaxisample was weighed by a n electronic microbalance (Type mum T H activity in these DA and NA containing areas 4125. Sartorius Co.) with a digital voltmeter (Type EO-12. was obtained under the standard assay conditions, T H acEto Co.). The sensitivity of this balance is 0.1 p g . tivities at the higher concentration of the substrate (up T H activity was assayed by a modification of the '"CO, to 300 p ~ o r) the cofactor (up to 10 m u ) were determined in both areas. However, the T H activities under these con.Ahhreuiatiotis used: NA. noradrenaline: DA, dopamine; ditions were the same as the values under the standard CA, catecholamine; TH. tyrosine hydroxylase. assay conditions in both DA and NA containing areas. 161
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The amount of ' ' C 0 2 formation. furthermore. was proportional to the quantities of the dry samples up to the maximum used Tor each tissue examined; 23 pg of median eminence (ME), 31 p g of A, cell group, S3 jig of C'P, 61 pg of area lateralis hypothalami (L) and 200 pg of the tissue containing nucleus preopticus medialis (POM). area p r e o p ticus lateralis (POL)and nucleus anterior hypothalami (A). These amounts of the tissues were about 3 times larger than those of the tissues used in the usual assay. The evacuated tubes containing the freeze-dried sections were retained for about 3-6 months in the refrigerator ( -20' C) until use, and TH activities of these s;-inipIes rcmained stable during at least I2 months.
RESULTS AKD DISCUSSION The results are summarized in Tahle I . There is 186-fold difkrence between the highest 1H activity measured and the lowest one. In the hypothalamic nuclei. the highest
activity was found in the ME, wherc the dense collections of D A and S A nerve terminals exist (JONSSOS et a/., 1972; LiiwrKBhl t'i ( I / . . 1976). Relatively high activities were found i n the anterior part of the nucleus perivenlricularis (PE: A I cell group), medial zona incerta (MZI: A cell group) and nucleus arcuatus (medialis) (AR: A , ? cell group). which contain incerto-hypothalamic D A cell bodies (BJBKKLLND & NOBIN.1973; BJBRKLliNI> t't a/., 1975). In the incerto-hypothalamic CA cell groups, the nucleus posterior hypothalami (P: A , I cell group, comprising both D A and N A cell bodies (BIBRKLLND & NOBIY,1973)) only revealed low TH activity. POM. A. nucleus ventromedialis (VM). nucleus premammillaris ventralis (PMV) and nucleus prernammillitris dorsalis ( P M D ) showed the lowest activities. In the meso-limbic D A neuron system, the A , , cell group had 2- to 5-fold higher T H activity than that of its nerve terminals, nucleus accumhens (AC) and tubercu1971). In the nigrolum olfactorium (TU) (UNGEKSTEDT, striatal D A neuron system, the A, cell group showed about
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FIG. I . Schematic drawings of the dissected nuclei and areas. Frontal sections through the preoptic area (a.b). the anterior (c,d), medial (e) and posterior (0 hypothalamus, the lower brain stem (g,h), and medial part of the median eminence (i). Thc dissected nuclei and cell groups represented by hatched area are; A. n. anterior hypothalami; AC, n. accumbens; AR. n. arcuatus; CP, n. caudatusputamen; DM, n. dorsomedialis; ER, external region of median eminence; IR, internal region of median eminence; IS, infundibular stem; L, area lateralis hypothalami; ME, median eminence; MZI. medial zona incerta; P. n. posterior hypothalami; PA, n. paraventricularis; PE, n. periventricularis; PMD. n. premammillaris dorsalis; PMV. n. premammillaris ventralis; POL, area preopticus lateralis; POM, n. preopticus medialis; RC. area retrochiasmaticus; TC', tubcrculum olfactorium; VM, n. ventromedialis; AIO. A9 dopaminergic cell groups and A6. A5 noradrenergic ccll groups. The other abbreviations used: CA. commisura anterior; CO. chiasma opticus; F, fornix; FMT. fasciculus mammillothalamicus; ip, n. interpeduncularis; LM, lemniscus medialis; ncu, n. cuneiformis; ntd. n. tegmenti dorsalis; os, n. olivaris superior; pcs. pedunculus cerebellaris superior; PS, tructus corticospinalis; r, n. ruber; rpoc, 11. rcticiihris pnntis c:iiid:ilis: snc. siihztantiii n i p 7ona compacta: mr. suhstantia n i p 7ona reticularis: TO. tractus opticus; TSV. tractus spinalis n e n i trigemiIii; 1 1 1 . third ventricle; VII, nervus facialis.
twice the TH activity of CP, which is mainly innervated from the A, cell group (~:iiGERSTEI).r, 1971). In addition to the mesencephalic DA cell groups, T H activities of A, and AS NA cell groups in the medulla oblongata and the pons were cxamined in this study. The A, cell group. which gives rise to NA nerve terminals mainly in cerebrum and cerebellar cortex and hippo1971). revealed T H activity as high campus (UNGERSTEIJI., as that of mesencephalic DA cell groups. On the other hand, the A s cell group. one of the cell groups which give rise to the ventral ascending NA neuron system (UKGERSTEDT, 1971). showed extremely low T H activity compared to that of A, cell group. This may rellect mainly the difference in cell densities in each cell group. Recently, using the micropunch technique with frozen ei a/. reported the dissections of the rat brain, SAAVEDRA tributions of the TH activities in the hypothalamic (1974) and limbic (1976) nuclei. Compared to their results. A, VM and L in our present study showed about 2- to 5-fold lower T H activities. while AC and TIJ had 2- to 3-fold
higher v;ilucs. Regarding thc T H iictikitics in A,,. A c, and A l 0 cell groups, Ltwis L'I ( I / . (1976) and BCDAC I a / . (1975) reported the values per structure (lo00 pm thickness x 0.9 mm dia.) in these areas. In the present study, the weights of A,, A 9 and Al,, cell groups dissected from the sections of 100jcm thickness were ahout 4.5. 10 and IOpp dry weight. respectively. The total TH activities in these areas. estimated as the values per structure (1000pm thickness), were 400, 1100 and 1400 p molih. These values are 1.5- to 3-fold higher than their values. O n the other hand, the weights of A6. A9 and A l o structures in their studies were approx 0.64 mg wet weight. When the T H activities of these areas in their studies were expressed as the values per mg dry weight, therefore, they appear 5- to 8-fold lower than our values. For example, in this study, the weight of A, cell group was about 6-fold lower than their values. The sample of A, cell group obtained by the micropunch technique seems to have contamination with large amounts of the surrounding tissue, which has lower T H activity than that
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Short communication TABLEI . TI1 AcrIvi'rY Nuclei and areas
IS n I E
CA
N E R V E T ~ ~ K M I N A LASI) S CELL RODIES INT i l l : H A . I BRAIN
TH activity
Anterior part of hypothalamus POM 0.26 f 0.03 (3) POL 0.64 f 0.05(4) PE (A14) 2.55 f 0. I5 (4) A 0.28 f 0.02(4) L 0.96 f 0.10(4) PA 1.61 f 0.12(4) RC 1.92 f 0.1 l ( 4 ) Medial part of hypothalamus MZI (A13) 4.20 f 0.39(4) VM 0.15 f 0.04(3) DM 0.89 f 0.07 (4) PE 1.49 f 0.09 (5) L 1.57 f 0.06(4) AR (AI2) 4.15 f 0.53 (4) ME 9.X7 f 0.24(5) IR 8.40 k 1.24(3) ER 11.39 f 2.18(3)
Nuclei and areas
T H activity
Posterior part of hypothalamus P (A 11) 0.76 f 0.05 (5) PMV 0.2 I f 0.02 (4) PMD 0.24 f 0.07 (3) L 0.37 f 0.03 (4) AR 0.50 f 0. I I (4) IS 10.99 f 0.26 (4) Mew-limbic DA neuron system A10 27.91 f 2.80(3) AC 5.91 0.24 (4) TU 10.54 & 0.21 (4) Nigro-striatal DA ncuron system A9 22.59 f 2.53 (4) CP 10.98 f 0.27 (41 Pontine NA cell groups A6 18.12 f 0.91 ( 3 ) A5 0.46 f 0.04 (4)
The results are expressed as values of mean activity (nmol I4CO1 formed/mg dry w ~ h ) W.M. (number of animals).
+
of the A, cell group. Indeed. the TI1 activity in the sur. microisotopic assay for neurotransmitter related enzymes. rounding tissue of A b cell group was below 1.0-2.0nmol has become a useful tool for understanding the physiologiI4CO, formedidry wtih. The diHerences in the TH values cal roles of the neurotransmitters in the small discrete between the rosults obtained by the micropunch technique areas of the hrain. and ours, therefore. may be explaincd mainly by the difference in the niethod for dissecting samples. Ackfionlrdyefnefit~~-This work was supported by a grant It has been reported that there was a highly significant from the Science and Technology Agency of Japan. correlation between TH activity and DA levels (SAAVEDRA er d..1974; SAAVEDKA& Z I V I N1976). Since in the median L h o r a t o r y qf Neurochernisfry. M. SAITO eminence (ME). the external layer contains mainly DA HiZen Narionul Menrul Hospital. M. H I R A N 0 nerve terminals while the internal layer contains mainly Kanzoki, Suyu. 842-01. Japan H. U C H I M I X A er M NA nerve terminals (JONSWK t'r ul., 1972; L ~ F S T R ~ O Departmenr of Cheniisfry, T. NAKAHAHA ul., 1976). we examined. the TH activities in the external Faculty of Science, region and the internal region of M E (divided as shown with stippled lines in Fig. li). to determine whether the Kyushu Unitwsiry, Hakozaki, Fukuoka, 812, Japun TH activity in M E is mainly due to the DA or NA nerve terminals. Although the external region showed slightly Department of Neuropsycliiutry, Faculty qf Mcdicinc,. higher TH activity than that in the internal region, there wiis n o significant diHerence between the two regions. NA K ytrshu Unitvrsiry. Karakasu. F1rkrrnko. 8 I?.J u p u n neuron systems have been considered to have low T H activity (McG~.I.K& MCGI~ER. 1973). Our results obtained by the microdissection technique suggest that some disREFERENCES crete areas which contain dense NA nerve terminals and cell bodies also have high TH activity, as well a s the areas N U& NOBINA. (1973) Brain Res. 51. 193 205. which contain high D A levels. However, VERSTEEC er al. B J ~ K K L U A. A.. LINUVALL 0.& NOBINA. (1975) Bruin Res. (1976) reported that DA neurons might be present in the BJORKLUNU 89, 2 9 4 2 . A, cell group since this region had a relatively high DA BllDA M.. ROUSSELB., RENAUDB. & PUJOLJ. F. (1975) level. So. the possibility that the high TH value of Ah cell Bruin Res. 93. 564569. group may reflect the TH activity of the DA neuron sysFAIRMAN K.. G I A ( . O I ~E.I ~&I CHIAPPINELLI V. (1976) Bruin tems in this region cannot be ruled out. Rrs. 102. 301 -312. In summary. the present study has shown the TH activiHIMNO M., L'CHIMCRA H. & SAITT) M. (1975) Brain Rrs. ties in the mesencephalic and pontine cell groups in the 93. 558 -565. CA neuron systems obtained by precise dissection. and by JONSSONG . . F'I:xE K. & H ~ K F E LT.T (1972) Bruin Res. 40. the simultaneous assay of TH activities in several CA cell 271-28 1. groups and their nerve terminals. It is noteworthy that KONIG J. F. R. & KLIPPEI.R. A,. (1963) The Rur Brain; in the brain nuclei examined. the highest activity was found a Srtwotaxic Arlm of rhr Furubruin m d lower Parts of in A*,. A l , I>A and A, NA cell groups. Further examinthe Brain Stcvn. Williams & Wilkins,. Baltimore. ations of T H activities in other lower brain stem NA and LEWIS B. D.. RENAUI) B.. BUDAM. & Pt;JOl, J. F. (1976) DA cell groups. such as A , , A?. A3. A,. A, and A, are Brain Res. 108. 339 349. therefore desirable. O u r method. using the microdissection L o W R Y 0.H. (1953) J . Hisrocliem. Cyrochem. I . 420428. technique with freeze-dried sections of the rat brain and
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LOWRY0. H. (1962) Haaey Lecture 58, 1-19. M. & UCHIML'RA H. (1975) Broiii Res. SAITOM., HIRANO L ~ F S T RA., ~M JONSON G. & F u x ~K. (1976) J. Histochem. 99. 4 1 W 1 4 . Cyiochern. 24. 41 5 4 2 9 . UCHIMUKAH., HIRANOM.. S A I K I M., MUKAI A. & MCGEERP. L. & MCGEERE. G. (1973) in Progress in HAZAMA H. (1974) Brain atid Nerve (in Japanese) 26, Neurohiology (KERKL'TG. A. & PHILLIPS J. W., eds.) 341 345. Vol. 2, pp. 69--l17. Pergamon Press. Oxford. UCtiIMUR4 H., SAITOM. & HIRANO M. (1975) Brain Res. MOSKALJ. R. & BASU S. (1975) Arialyr. Biochern. 65. 91, 161-164. 449-457. UNGERSTEDT I ; . (I97 I ) Acra physiol. scarid. 82. Suppl. 367. PALKOVITS M. & JACOBOWITZ D. M. (1974) J. cornp. Neur. 1 --48. 157. 29-42. VERSTEECD. H. G.. VAN DER GUGTEN J., DE JONG W. SAAVEDRA J. M.. BROWNSTEIN M., PALKOVITS M., KIZER & PALKOVITS M. (1976) Brain Res. 113. 563-574. S. & AXELROD J. (1974) J. Neurochem. 23. 869-871. WAYMIRE J. C., BJURR. & WEINERN. (1971) A n d y ? . BioJ. M. & ZIVINJ. (1976) Brairi Rrs. 105, 517. 524. SAAVEORA chern. 43. 588 -600.
