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Diencephalic catecholamines in chick and pigeon
Vol. 8, Part 1, pp. 889-895, 1989. Life Sciences Printed in Great Britain .
Pergamon Press
DIENCEPHALIC CATECHOLAMINES IN CHKK AND PIGEON Bangt Faldk, Lars Ljunggren and Lars Nordgran Institute of Anatomy and Histology and Departments of Psychiatry II and Zoology, University of Lund, Lund, Sweden
(Received 25 April 1969; in final form 3 June 1969) Despite LOEWI (1,2) having reported that rympathetic stimulation of frog heart released adrenal ine, it was long believed that this amine did not function as neurotransmitter . Not until a fluorescence method for the cellular localization of monaamines was introduced (3,4) was it possible to establ ish that adrenal ine was the introneuronal monoamine in the rympathetic nervous rystem of frog (5) . $mall amounts of adrenaline have been found in mammalian brain, but so far it has not been possible to establish its Cellular locolization and thus its precise function . Recently, however, JUORIO and VOGT (b) discovered high amounts of adrenaline in avian brain suggesting a neuronal function for this amine in the central nervous system, They found, e .g ., adrenaline concentrations of 0 .4Ng/g in the hypothalamus of pigeon and 1 Ng/g in that of chicken . Such high amounts should be favourable for a histochemical localization of adrenal ine on the cellular level . It was also disclosed (b), however, that the distribution of adrenaline followed a pottem similar to that of noradrenaline, implying that the histochemical analysis should preferably be performed on brains after selective depletion of noradrenaline . Picot experiments aimed at finding a suitable drug for this purpose produced conflicting results regard mg the distrbution and concentration of adrenaline and prompted the present inves" igation on diencephalic catecholamines in pigeon and chicken . Material and methods The material consisted of 161 chidkens 4 to 8 weeks of age and 35 pigeons mae than 2 months old . All the animals were killed by decapitation . TFto broth was nnaved rapidly, and the diencephalon was dissected by making two cuts: one just rostrol to the roots of the third cranial nerve through the posterior commiaure, th other just caudal to the anterior commissars . The optic chiasm, optic Croat and adjacent parts of the optic IobK, the pinstol gland, and the choroid plexus were removed . (n chicken, the neurohrpophysls and t~ organon vasculosum were separated for analysis, and the diancepholon w~s dividstd ~
889
890
DIENCEPHALIC CATECHOLANIINES
Vol. 8, No. 15
ventral and dorsal parts roughly corrosponding to the hypothalamus and the thalamus, respectively . In pigeon, the diencephalon was not divided, but the organon vasculosum was separated. The dissected samples wero immediately weighed and homogenized in perchloric acid at 0 too°C. The amounts of dopamine, noradrenaline, and adrenaline in the samples were determined fluorimetrically by the method of BERTLER, CARLSSON, ROSENGREN and WALDECK (~ as modified by HÄGGENDAL (8), in the diencephalic proparotions and the organon vasculosum of both chicken and pigeon and the neurohypophysis of chicken . The diencephalan from 13-15 chickens were pooled for one estimation, wheroas the determination of the amines in pigeon diencephalon was made on two samples obtained from 15 and 20 animals, respectively . Paper chromatography was carried out on material from two extracts of 15 pooled chick diencephalon preparations . The chromatograms were developed in butanol-N HCI and the catecholamines visualized by spraying the paper with potassiumferricyanide according to BERTLER, CARLSSON and ROSENGREN (9) . In addition, the concentration of the catecholamines in whole brain of 1 day old chickens was determined on 5 samples each consisting of 15 pooled brains . Results and Comments Chidken: The results of the spectrofluorimetric determinations on the diencephalic samples appear in Table 1 . The terms ventral and dorsal diencephalon have been used because of the difficulties in finding a precise anatomical border between the hypothalamus and the thalamus . The ventral part of the diencephalon contained 0.70pg/g noradrenaline (NA) and 0 .11 ~g/g adrenaline (A) and the dorsal port contained I .ONg/g NA and 0.19 Ng/g A. In agreement with this, the paper chromatography revealed a distinct spot corresponding to NA, but only a faint spot which had migrated as A. This was in contrast to the findings of JUORIO and VOGT (6), who reported levels of 1 .01 I+g/g A in the hypothalamus and 0.64 Ng/g A in the thalamus of ch icken. On the other hand, there was a better agreement between the data reported here and the findings of JUORIO and VOGT on NA levels in the hypothalamus and thalamus . Because of the dissectional difficulties, we cannot exclude the possibility that certain borderline structures, e.g, the paroventricular and suprooptic nuclei, of which especially the former contains high amounts of catecholamines (10) have, at least partly, been included in the dorsal diencephalic proparotions . Such possble variations caused by the dissection technique cannot explain, however, the discrepancy between our findings and the results achieved by JUORIO and VOGT . In this
Vol. 8, No. 15
891
DIENCEPIiALIC CATECHOLANIINES
connection, it must be emphasized that the ventral and dorsal diencephalic parts used in
this study did not include the organon vasculosum of the neurohypophysis which wero analysed separately . Neither A nor dopamine (DA), however, were found in these structures in agreement with findings of ENEMAR and LJUNGGREN (11) and BAUMGARTEN et al, (12) - but high amounts of NA, in fact the h:~ghest values of NA which were found in the different diencephalic preparations (Table 1) . TABLE I Concentration of adrenaline, noradrenaline, and dopamine (Ng/g t S. D .) in four differont parts of the diencephalon of chicken No . of estimations
adrenaline
noradrenaline
dopamine
Ventral part
0.11 ± 0.04
0.70 + 0.13
0 .08 t 0.05
4
Dorsal port
0.19 ± 0 .03
1 .0
+ 0.06
0.11 + 0 .02
4
Organon vaaculosum
0 .00
2.6
t 0.30
0.00
4
Neurohypophysis
0 .00
3 .6
t 0 .60
0.00
6
In the chicken brain, the concentration of NA was found to be 0.19 ± 0.06Ng/g, and of A, 0.02 t 0.004Ng,/g. This is a lower relative adrenaline content thae in the brain stem of hen where GUNNE (13) found that A amounted to 17 per cent of the total amounts of NA and A. It is thus obvious that the A has an uneven distribution in chicken brain and that it
is not inevitably found in the regions of the diencephalon which are rich in NA.
P~no Concentrations of 0.60 - 0.90 Ng/g NA and 0.08 Ng/g DA were found
in pigeon diencephalon . This compares well with the data reported by JUORIO and VOGT (6) . These authors, however, found significant amounts of A in differont park of pigeon brain (e .g, hypothalamus: 0.38 Ng/g and thalamus: 0.24 Ng/g) distributed similarly to the NA,
twenty per cent or more of the total amount of NA and A being in the methylated form . In
this study,. no significant amounts of A could be found in the diencephalon, not even in the organon vasculosum which contained very high amounts of NA (Table In . The neurohypo-
physic was not separored in the dissections since this structure in pigeon contains only slight
DIENCEPi:iALIC CATECHOLANIINES
892
Vol . 8, No. 15
amounts of catecholamines (14) . TABLE II Concentration of adrenaline, noradrenaline, and dopamine (Na/g) in pigeon organon vasculosum and diencephalon after separation of the organon vasculosum
adrenaline
noradrenaline
dopamine
I`1o, of animals
Diencephalon
0.00
0.60
0.08
15
Diencephalon
0.00
0.90
0.08
20
Organon vasculosum
0.00
2 .9
0.00
15
Organon vasculosum
0.00
2.4
0.00
20
I
A spectrofluorimetric method of the kind applied in this investigation has a high
reliability provided a sufficient amount of tissue is used . In addition, the paper chromatographic analyses of the diencephalic samples from chicken brain indicated a presence of A of the same order as that found in the fluorimetric analyses . The discrepancies between
this study and that of JUORIO and VOGT (b) suggests that the bioassay methods are not
sufficiently specific for brain tissue, at least not with respect to adrenaline . Acknowledgement
This investigation was supported by research grants from the Swedish State Medical Research Council (Projects No . B69-14X-712-04C and B69-14X-56-05C) and the Swedish Natural Science Research Council (Project No . 99-35) . References 1. 2. 3. 4.
LOEWI, O., Arch, ges. Physiol . 189, 239 (1921) . LOEWI, O., Arch, ges. Physiol , 237, 504 (1936) .
FALCK, B . and TORP, A., Med, exp. 5, 429 (1961) .
FALCK, B., Acta Physiol . stand. 56, suppl. 197 (1962) .
Vol. 8, No. 15 5.
DIENCEPHALIC CATECHOLANIINES
893
FALCK, B., HÄGGENDAL, J. and OWMAN, CH ., Quart. J . e~ . Ph~siol . 48, 253 (1963) .
b.
JUORIO, A .V. and VOGT, M., J . Physiol . _189, 489 (1967) .
7.
BERTLER, A., CARLSSON, A., ROSENGREN E . and WALDECK, B., Kungl . Fysiogr. Stitlsk . Lund Förh . ~8, 121 (1958) .
8.
HÄGGENDAL, J., Acta physiol . stand. 59 242 (1963) .
9.
BERTLER, A., CARLSSON, A . and ROSENGREN, E ., Acta physiol, stand. 44, 273 (1958) .
10 .
FOXE, K. and UUNGGREN, L., J. Comp. Neural . 125, 355 (1965) .
11 .
ENEMAR, A . and LJUNGGREN, L., Z . Zellforsch . _91, 496 (1968) .
12 .
BAUMGARTEN, H. G ., BJÖRKLUND, A., FALCK, B. and UUNGGREN, L., to be published (1969) .
13 .
GUNNE, L.-M. , Acta physiol, stand . 56, 324 (1962) .
14 .
BJÜRKLUND, A., FALCK, B. and LJUNGGREN, L ., Z . Zellforsch . 89, 193 (1968) .
Pergamon Press
DIENCEPHALIC CATECHOLAMINES IN CHKK AND PIGEON Bangt Faldk, Lars Ljunggren and Lars Nordgran Institute of Anatomy and Histology and Departments of Psychiatry II and Zoology, University of Lund, Lund, Sweden
(Received 25 April 1969; in final form 3 June 1969) Despite LOEWI (1,2) having reported that rympathetic stimulation of frog heart released adrenal ine, it was long believed that this amine did not function as neurotransmitter . Not until a fluorescence method for the cellular localization of monaamines was introduced (3,4) was it possible to establ ish that adrenal ine was the introneuronal monoamine in the rympathetic nervous rystem of frog (5) . $mall amounts of adrenaline have been found in mammalian brain, but so far it has not been possible to establish its Cellular locolization and thus its precise function . Recently, however, JUORIO and VOGT (b) discovered high amounts of adrenaline in avian brain suggesting a neuronal function for this amine in the central nervous system, They found, e .g ., adrenaline concentrations of 0 .4Ng/g in the hypothalamus of pigeon and 1 Ng/g in that of chicken . Such high amounts should be favourable for a histochemical localization of adrenal ine on the cellular level . It was also disclosed (b), however, that the distribution of adrenaline followed a pottem similar to that of noradrenaline, implying that the histochemical analysis should preferably be performed on brains after selective depletion of noradrenaline . Picot experiments aimed at finding a suitable drug for this purpose produced conflicting results regard mg the distrbution and concentration of adrenaline and prompted the present inves" igation on diencephalic catecholamines in pigeon and chicken . Material and methods The material consisted of 161 chidkens 4 to 8 weeks of age and 35 pigeons mae than 2 months old . All the animals were killed by decapitation . TFto broth was nnaved rapidly, and the diencephalon was dissected by making two cuts: one just rostrol to the roots of the third cranial nerve through the posterior commiaure, th other just caudal to the anterior commissars . The optic chiasm, optic Croat and adjacent parts of the optic IobK, the pinstol gland, and the choroid plexus were removed . (n chicken, the neurohrpophysls and t~ organon vasculosum were separated for analysis, and the diancepholon w~s dividstd ~
889
890
DIENCEPHALIC CATECHOLANIINES
Vol. 8, No. 15
ventral and dorsal parts roughly corrosponding to the hypothalamus and the thalamus, respectively . In pigeon, the diencephalon was not divided, but the organon vasculosum was separated. The dissected samples wero immediately weighed and homogenized in perchloric acid at 0 too°C. The amounts of dopamine, noradrenaline, and adrenaline in the samples were determined fluorimetrically by the method of BERTLER, CARLSSON, ROSENGREN and WALDECK (~ as modified by HÄGGENDAL (8), in the diencephalic proparotions and the organon vasculosum of both chicken and pigeon and the neurohypophysis of chicken . The diencephalan from 13-15 chickens were pooled for one estimation, wheroas the determination of the amines in pigeon diencephalon was made on two samples obtained from 15 and 20 animals, respectively . Paper chromatography was carried out on material from two extracts of 15 pooled chick diencephalon preparations . The chromatograms were developed in butanol-N HCI and the catecholamines visualized by spraying the paper with potassiumferricyanide according to BERTLER, CARLSSON and ROSENGREN (9) . In addition, the concentration of the catecholamines in whole brain of 1 day old chickens was determined on 5 samples each consisting of 15 pooled brains . Results and Comments Chidken: The results of the spectrofluorimetric determinations on the diencephalic samples appear in Table 1 . The terms ventral and dorsal diencephalon have been used because of the difficulties in finding a precise anatomical border between the hypothalamus and the thalamus . The ventral part of the diencephalon contained 0.70pg/g noradrenaline (NA) and 0 .11 ~g/g adrenaline (A) and the dorsal port contained I .ONg/g NA and 0.19 Ng/g A. In agreement with this, the paper chromatography revealed a distinct spot corresponding to NA, but only a faint spot which had migrated as A. This was in contrast to the findings of JUORIO and VOGT (6), who reported levels of 1 .01 I+g/g A in the hypothalamus and 0.64 Ng/g A in the thalamus of ch icken. On the other hand, there was a better agreement between the data reported here and the findings of JUORIO and VOGT on NA levels in the hypothalamus and thalamus . Because of the dissectional difficulties, we cannot exclude the possibility that certain borderline structures, e.g, the paroventricular and suprooptic nuclei, of which especially the former contains high amounts of catecholamines (10) have, at least partly, been included in the dorsal diencephalic proparotions . Such possble variations caused by the dissection technique cannot explain, however, the discrepancy between our findings and the results achieved by JUORIO and VOGT . In this
Vol. 8, No. 15
891
DIENCEPIiALIC CATECHOLANIINES
connection, it must be emphasized that the ventral and dorsal diencephalic parts used in
this study did not include the organon vasculosum of the neurohypophysis which wero analysed separately . Neither A nor dopamine (DA), however, were found in these structures in agreement with findings of ENEMAR and LJUNGGREN (11) and BAUMGARTEN et al, (12) - but high amounts of NA, in fact the h:~ghest values of NA which were found in the different diencephalic preparations (Table 1) . TABLE I Concentration of adrenaline, noradrenaline, and dopamine (Ng/g t S. D .) in four differont parts of the diencephalon of chicken No . of estimations
adrenaline
noradrenaline
dopamine
Ventral part
0.11 ± 0.04
0.70 + 0.13
0 .08 t 0.05
4
Dorsal port
0.19 ± 0 .03
1 .0
+ 0.06
0.11 + 0 .02
4
Organon vaaculosum
0 .00
2.6
t 0.30
0.00
4
Neurohypophysis
0 .00
3 .6
t 0 .60
0.00
6
In the chicken brain, the concentration of NA was found to be 0.19 ± 0.06Ng/g, and of A, 0.02 t 0.004Ng,/g. This is a lower relative adrenaline content thae in the brain stem of hen where GUNNE (13) found that A amounted to 17 per cent of the total amounts of NA and A. It is thus obvious that the A has an uneven distribution in chicken brain and that it
is not inevitably found in the regions of the diencephalon which are rich in NA.
P~no Concentrations of 0.60 - 0.90 Ng/g NA and 0.08 Ng/g DA were found
in pigeon diencephalon . This compares well with the data reported by JUORIO and VOGT (6) . These authors, however, found significant amounts of A in differont park of pigeon brain (e .g, hypothalamus: 0.38 Ng/g and thalamus: 0.24 Ng/g) distributed similarly to the NA,
twenty per cent or more of the total amount of NA and A being in the methylated form . In
this study,. no significant amounts of A could be found in the diencephalon, not even in the organon vasculosum which contained very high amounts of NA (Table In . The neurohypo-
physic was not separored in the dissections since this structure in pigeon contains only slight
DIENCEPi:iALIC CATECHOLANIINES
892
Vol . 8, No. 15
amounts of catecholamines (14) . TABLE II Concentration of adrenaline, noradrenaline, and dopamine (Na/g) in pigeon organon vasculosum and diencephalon after separation of the organon vasculosum
adrenaline
noradrenaline
dopamine
I`1o, of animals
Diencephalon
0.00
0.60
0.08
15
Diencephalon
0.00
0.90
0.08
20
Organon vasculosum
0.00
2 .9
0.00
15
Organon vasculosum
0.00
2.4
0.00
20
I
A spectrofluorimetric method of the kind applied in this investigation has a high
reliability provided a sufficient amount of tissue is used . In addition, the paper chromatographic analyses of the diencephalic samples from chicken brain indicated a presence of A of the same order as that found in the fluorimetric analyses . The discrepancies between
this study and that of JUORIO and VOGT (b) suggests that the bioassay methods are not
sufficiently specific for brain tissue, at least not with respect to adrenaline . Acknowledgement
This investigation was supported by research grants from the Swedish State Medical Research Council (Projects No . B69-14X-712-04C and B69-14X-56-05C) and the Swedish Natural Science Research Council (Project No . 99-35) . References 1. 2. 3. 4.
LOEWI, O., Arch, ges. Physiol . 189, 239 (1921) . LOEWI, O., Arch, ges. Physiol , 237, 504 (1936) .
FALCK, B . and TORP, A., Med, exp. 5, 429 (1961) .
FALCK, B., Acta Physiol . stand. 56, suppl. 197 (1962) .
Vol. 8, No. 15 5.
DIENCEPHALIC CATECHOLANIINES
893
FALCK, B., HÄGGENDAL, J. and OWMAN, CH ., Quart. J . e~ . Ph~siol . 48, 253 (1963) .
b.
JUORIO, A .V. and VOGT, M., J . Physiol . _189, 489 (1967) .
7.
BERTLER, A., CARLSSON, A., ROSENGREN E . and WALDECK, B., Kungl . Fysiogr. Stitlsk . Lund Förh . ~8, 121 (1958) .
8.
HÄGGENDAL, J., Acta physiol . stand. 59 242 (1963) .
9.
BERTLER, A., CARLSSON, A . and ROSENGREN, E ., Acta physiol, stand. 44, 273 (1958) .
10 .
FOXE, K. and UUNGGREN, L., J. Comp. Neural . 125, 355 (1965) .
11 .
ENEMAR, A . and LJUNGGREN, L., Z . Zellforsch . _91, 496 (1968) .
12 .
BAUMGARTEN, H. G ., BJÖRKLUND, A., FALCK, B. and UUNGGREN, L., to be published (1969) .
13 .
GUNNE, L.-M. , Acta physiol, stand . 56, 324 (1962) .
14 .
BJÜRKLUND, A., FALCK, B. and LJUNGGREN, L ., Z . Zellforsch . 89, 193 (1968) .