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The effects of ventromedial tegmental lesions on the biosynthesis of catecholamines in the striatum
Life Sciences Vol . 5, pp . 211-216, 1966 " Printed in Great Britain.
Pergamon Press Ltd .
TH6 BFFRCTS OF VENTBOISDIAL TEGMdNTAL LSSIONB ON T~ BIOSYN1~iSI8 OF GTSCHOLAIQDSS IN THB S1RLITUM M. Goldstein, B . Anagnoste, W . 3 . Oven and A. F . Battista Departments of Psychiatry and Neurosurgery Hew York University Medical Center Neurochemistry Laboratory Iiew York, N . Y . 10016
(Received 9 August 1966 ; in final form 22 September 1966) Poirier and Sourkes (1) have shown that several swaths after Lesions had been placed in the ventrosedial tegsrntal area of the brain rtes is the soakey there was a decrease in the dopaaine and norepinephrine concentrations in the corresponding striatum .
The results of these experi:tents in the swokey era cosy
parable to the neurochemical and neuropathological findings in hose Parkinsonism .
Hornykiewics et al . (2) have reported that the dopasdne concentration is
lower in the basal ganglia of patients who died Eros Parkinwn's disease .
It
has been suggested that the decrease in the dopasd,na contenC of the striates in Parkinson's patients is due to a degeneration of the nigro-neostriatal dopasdas neurons .
Nith the use of histochemical methods evidence . has bun presented
that a pathway of dopamine neurons exists which originates in the substaatia nigra, ascends to the internal capsule and terminates in the naostriatua (3) . The catecholamines are most likely synthesised intraneuroaally (4) and therefore, a degeneration of the nigro-neostriatal neurons could is4air their formation .
Thh present study elucidates the effects of vantrossdial tsgasntal
Lesions on the in vitro and in vivo forenoon o~ catec~olasinas Eras the precursor tyrosine in the at~ciatusu
2172
BIOSY'AT~BIB OF CA
Yol. 5, fto . 23
liethods Surgical procedures " Craan monkeys (cercopithecus sabaaus) neighing 2-3.5 kg mere used in all experiments.
After aaesthetiution, unilateral radiofrequency lesions here
placed is the left ventromedial tegmental region of the roetral pons .
Postop
eratively, all the monkeys exhibited contralateral hypokinesia and ipsilateral third cranial nerve palsies .
The tremors persisted until the end of the exper-
iments in all monkeys . In some experiments, 4-6 weeks after the lesion nee placed, the animals mere injected with tyrosine-C14 (25 ~c, 11 I+g) into both lateral ventricles of the brain by a stereotazic technique. pith Nembutal (15 mB/kg) .
The animals nets lightly anesthetised
The radioactive amino acid ws diluted with saline
and a volume of 0.3 ml was injected into each lateral ventricle .
Two hours
after the administration of tyrosine-C 14 the animals were killed, the brains wre removed gad then carefully dissected. A~sa~
of
tyrosine hydrozylase activity :
Ia separate experiments tyrosine hydro:ylase activity of the striatum was determined in the control monkeys ae well as in the monkeys with lesions in the vnntromedial tegmental region of the brain stem. Tissues ware neighed imvediately after dissection and homgeni:ed with three vormes of 0.32 K sucrose, using glass homogenisers .
The homogenates
were incubated at 37'C for 30 minutes with 200 Nmoles of acetate buffer, pS 6. 0, 4 ~tmolea of 2-amino-4-hydroxy-ó,7-dimethylpteridine, 100 pmoles of mercaptoathaaol, 0.4 ~rmolas of p-bromo-m-hydroxybnnzoxyamine and 100 s~curies of Ltyrosine-C 14 (S . A. 2.05 me/mg) in a total volume of 2 ml . stopped by addition of 3Z trictiloroacetic acid .
The reaction was
After centrifugation, to re-
move the protein precipitate, the catechols formed during incubation were adsorbed on alumna colamos (5) and the radioactivity was determined in a liquid scintillation spectrometer .
Yol .
5, No . 23
BIOSYNTHE8I8 OF CA
213
Determination of endo¢enous and exo¢enous catecholamnea : Tissues were neighed immediately after dissection and hosiogenired in 10 mi of 0 .4 N perchloric acid .
The catecholamnes were adsorbed on alumna and en-
dogenous dopamine levels were determined by the fluoronetric procedure (5 ) . The Labeled catecholamnes were determined and separated by previously described procedures involving adsorption on alumna, acetylation and paper chromatography (6) . Results and Discussion A)
Bffect of ventromedial te¢meatal lesions on tyrosine hvdroxylaae activity in vitro In prelimnary experiments it waa established that there was no difference
in tyrosine hydroxylase activity between the right or left sides of the caudate nucleus and the puta~n (Table I) .
Following ventrooedial tagaental lesions,
the tyrosine hydroxylase activity was reduced by approxistately 50Z on tha lesion aide of the caudate nucleus and potamen as compared with the corresponding intact side (Table I) .
The partial disappearance of tyrosine hydrozylaaa
activity following ventromedial tegmental lesions appears to be a consequence of degeneration of catecholamne containing neurons .
The finding that tyro-
sine hydroxylase activity did not disappear completely 4-6 days after the leeions were placed, suggest either that the catecholamne neurons were not cor pletely degenerated in this short period of time, or that the norepiaephriae containing neurone were not affected by these lesions .
Tha latter possibility
is also supported by the recent findings that following ventromsdial tagsrntal lesions, the storage of dopamine, but not of norepinephrine, is iapaired ia the corresponding striatum (7) .
It is also conceivable that the decraaw in
tyrosine hydroxylase activity is due to a decrease in ensysn content followins changes in impulse activity on the lesion side of the stsiatus.
21~4
B)
HI08YR~FBIB OB CA
Yol . 5, No . 23
$ffects of ventromedial teemental Lesions on the in viv o biosynthesis of catacholaaines from tyrosine-C 14 The effects of the ventraandial teemental lesions on the formation of cat-
acholaaines from tyrosine and on the endogenous dopamine content in the striatun are suaoarised in Table II.
Following intrayentricular injection of tyro
sine-CL 4 , the major part of the radioactivity in the catecholamine fraction was associated with dopamine in the caudate nucleus and the putamen .
However,
the radioactivity in the putaman was much lower than in the caudate nucleus . TABLE I The Effects of Veatromedial Teemeatal Lasion~ on Tyrosine Hydroxylase Activity in the Striatum
Caudate Nucleus
I
Potamen
DOPA -C 14 Formed In Vitro (c .p .m./g) CONTROL MDI~YS Left side
24,500 ± 1,500
35,000 ± 2,000
Right side
25,200 ± 1,500
34,000 ± 2,000
Lesion side
12,000 ~ 800
14,000 ± 800
Intact side
24,800 ± 1,500
32,500 ± 1,800
I~1~Y3 AITH LESIONB~
* Results are the means f S .E .M . far 3 experiments . ** The yentromedial teemental lesion vas placed on the Left side and the animals vase sacrificed 4-6 days after the lesion ws placed . Thars vas ao difference in the accuaulation of the labeled compounds betvean the left or right sides of the caudate uuclaus sad putaasn .
It is widest
that the forrtian of catecholaainas Eros tyrosine-Cl 4 is decreased by appro :iaataly 90x on the lesion side of the caudate nucleus and putaaen .
The
voi. 5, No . 23
Bzosn~~ESZS oF cA
21~5
concentration of endogenous dopamine on the lesion side is also reduced by approximately 90Z in these two regions of the CNS . The specific activity is much higher in the caudate nucleus than in the putamen.
These findings suggest that intrayentricularly injected tyrosine
does not penetrate to the putamen to the same extent as to the caudate auclnus, and it is conceivable that the highly myelinated internal capsule reprnsnnts a barrier for the penetration of tyrosine to the putamen. TABLE II The ßffects of Ventromedial TeAmental Lesions on the In Vivo Biosynthesis of Catecholamines from Tvroaine-C 14 and on the Dopamine Content in the ~txi+ltum
Caudate Nucleus
I+8/g
Dopamine c .p .m./8
Put
k8/8
n
Dopamine c.p .m ./8
OONTROL IYDNI~YS Left side
4.10 ± 0 .50
6,800 ± 400
4.65 ± 0.50
950 f 100
Right side
4.05 ± 0.50
7,000 ± 500
4 .50 ± 0 .50
900 ± 100
?DNKEYS WITH LESIONS*
f
Lesion side
0 .50
0.05
750 ± 100
0.60 ± 0.05
Intact aide
3 .95 ± 0.40
6,500 ± 500
4.40 ± 0.50
N .D . 950 ± 100
* Results ara the ereas ± B.S .M. f~e ~ sxparirants . The wntrosrdial tag"sn- , tal lasion maa placed oa the loft side and tha.anisrls were sacrificed 4-6 weeks after the lesion was placed . N.D ., not detectable < 100 c.p .~./g. Summarv Following ventromediaL tegmental lesions in the brain stem of monkeys, tyrosine hydroxylase activity is significantly lower on the lesioa side of the caudate nucleus and putamen.
The in vivo formation of dopamine-C 14 from
21~6
BzosYx~szs
voi . 5, xo. 23
oF cA
tyrosine-C 14 ie impaired on the lesion side in these two regions of the CNS . The present findings support the idea that yentrosiedial tegmental lesions produce a~.degeneration of dopamine neurone in the ipeilateral caudate nucleus and putanan . AcknovledAments - This work was supported by USPHS Grants MH 02717, NB 5480 and NB 04257 and by NSF Grant GB 3176 .
References 1.
L . POI$ISR and T . L . SOURKES, Brain . 88, 181 (1965) .
2.
0 . HORNYKISWICZ, Deutch med . Wochschr . 87, 1807 (1962) .
3.
N . -S . ANDBN, A . CARLSSON, A . DAHLSTROM, K . FUXE, N .
-A . HILLARP and
K . LARSSON, Life Sci . 3, 523 (1964) . 4.
N . -A. HILLARP, K . FU7~ and A . DAHLSTROèI, Pharmscol . Rev . 18, 727 (1966) .
5.
A . H . ANTON and D . F . SAYRE, J . Phars~acol . Exptl . Therap . 145, 326 (1964) .
6.
M. GOLDSTEIN, Int . J . Neuroyharmscol . 3, 37 (1964) .
7.
K. GOLDSTEIN, B . ANAGNOSTE, W . S . OWEN and A . F . BATTISTA, The Phvsiolo giet . ~, 191 (1966) .
Pergamon Press Ltd .
TH6 BFFRCTS OF VENTBOISDIAL TEGMdNTAL LSSIONB ON T~ BIOSYN1~iSI8 OF GTSCHOLAIQDSS IN THB S1RLITUM M. Goldstein, B . Anagnoste, W . 3 . Oven and A. F . Battista Departments of Psychiatry and Neurosurgery Hew York University Medical Center Neurochemistry Laboratory Iiew York, N . Y . 10016
(Received 9 August 1966 ; in final form 22 September 1966) Poirier and Sourkes (1) have shown that several swaths after Lesions had been placed in the ventrosedial tegsrntal area of the brain rtes is the soakey there was a decrease in the dopaaine and norepinephrine concentrations in the corresponding striatum .
The results of these experi:tents in the swokey era cosy
parable to the neurochemical and neuropathological findings in hose Parkinsonism .
Hornykiewics et al . (2) have reported that the dopasdne concentration is
lower in the basal ganglia of patients who died Eros Parkinwn's disease .
It
has been suggested that the decrease in the dopasd,na contenC of the striates in Parkinson's patients is due to a degeneration of the nigro-neostriatal dopasdas neurons .
Nith the use of histochemical methods evidence . has bun presented
that a pathway of dopamine neurons exists which originates in the substaatia nigra, ascends to the internal capsule and terminates in the naostriatua (3) . The catecholamines are most likely synthesised intraneuroaally (4) and therefore, a degeneration of the nigro-neostriatal neurons could is4air their formation .
Thh present study elucidates the effects of vantrossdial tsgasntal
Lesions on the in vitro and in vivo forenoon o~ catec~olasinas Eras the precursor tyrosine in the at~ciatusu
2172
BIOSY'AT~BIB OF CA
Yol. 5, fto . 23
liethods Surgical procedures " Craan monkeys (cercopithecus sabaaus) neighing 2-3.5 kg mere used in all experiments.
After aaesthetiution, unilateral radiofrequency lesions here
placed is the left ventromedial tegmental region of the roetral pons .
Postop
eratively, all the monkeys exhibited contralateral hypokinesia and ipsilateral third cranial nerve palsies .
The tremors persisted until the end of the exper-
iments in all monkeys . In some experiments, 4-6 weeks after the lesion nee placed, the animals mere injected with tyrosine-C14 (25 ~c, 11 I+g) into both lateral ventricles of the brain by a stereotazic technique. pith Nembutal (15 mB/kg) .
The animals nets lightly anesthetised
The radioactive amino acid ws diluted with saline
and a volume of 0.3 ml was injected into each lateral ventricle .
Two hours
after the administration of tyrosine-C 14 the animals were killed, the brains wre removed gad then carefully dissected. A~sa~
of
tyrosine hydrozylase activity :
Ia separate experiments tyrosine hydro:ylase activity of the striatum was determined in the control monkeys ae well as in the monkeys with lesions in the vnntromedial tegmental region of the brain stem. Tissues ware neighed imvediately after dissection and homgeni:ed with three vormes of 0.32 K sucrose, using glass homogenisers .
The homogenates
were incubated at 37'C for 30 minutes with 200 Nmoles of acetate buffer, pS 6. 0, 4 ~tmolea of 2-amino-4-hydroxy-ó,7-dimethylpteridine, 100 pmoles of mercaptoathaaol, 0.4 ~rmolas of p-bromo-m-hydroxybnnzoxyamine and 100 s~curies of Ltyrosine-C 14 (S . A. 2.05 me/mg) in a total volume of 2 ml . stopped by addition of 3Z trictiloroacetic acid .
The reaction was
After centrifugation, to re-
move the protein precipitate, the catechols formed during incubation were adsorbed on alumna colamos (5) and the radioactivity was determined in a liquid scintillation spectrometer .
Yol .
5, No . 23
BIOSYNTHE8I8 OF CA
213
Determination of endo¢enous and exo¢enous catecholamnea : Tissues were neighed immediately after dissection and hosiogenired in 10 mi of 0 .4 N perchloric acid .
The catecholamnes were adsorbed on alumna and en-
dogenous dopamine levels were determined by the fluoronetric procedure (5 ) . The Labeled catecholamnes were determined and separated by previously described procedures involving adsorption on alumna, acetylation and paper chromatography (6) . Results and Discussion A)
Bffect of ventromedial te¢meatal lesions on tyrosine hvdroxylaae activity in vitro In prelimnary experiments it waa established that there was no difference
in tyrosine hydroxylase activity between the right or left sides of the caudate nucleus and the puta~n (Table I) .
Following ventrooedial tagaental lesions,
the tyrosine hydroxylase activity was reduced by approxistately 50Z on tha lesion aide of the caudate nucleus and potamen as compared with the corresponding intact side (Table I) .
The partial disappearance of tyrosine hydrozylaaa
activity following ventromedial tegmental lesions appears to be a consequence of degeneration of catecholamne containing neurons .
The finding that tyro-
sine hydroxylase activity did not disappear completely 4-6 days after the leeions were placed, suggest either that the catecholamne neurons were not cor pletely degenerated in this short period of time, or that the norepiaephriae containing neurone were not affected by these lesions .
Tha latter possibility
is also supported by the recent findings that following ventromsdial tagsrntal lesions, the storage of dopamine, but not of norepinephrine, is iapaired ia the corresponding striatum (7) .
It is also conceivable that the decraaw in
tyrosine hydroxylase activity is due to a decrease in ensysn content followins changes in impulse activity on the lesion side of the stsiatus.
21~4
B)
HI08YR~FBIB OB CA
Yol . 5, No . 23
$ffects of ventromedial teemental Lesions on the in viv o biosynthesis of catacholaaines from tyrosine-C 14 The effects of the ventraandial teemental lesions on the formation of cat-
acholaaines from tyrosine and on the endogenous dopamine content in the striatun are suaoarised in Table II.
Following intrayentricular injection of tyro
sine-CL 4 , the major part of the radioactivity in the catecholamine fraction was associated with dopamine in the caudate nucleus and the putamen .
However,
the radioactivity in the putaman was much lower than in the caudate nucleus . TABLE I The Effects of Veatromedial Teemeatal Lasion~ on Tyrosine Hydroxylase Activity in the Striatum
Caudate Nucleus
I
Potamen
DOPA -C 14 Formed In Vitro (c .p .m./g) CONTROL MDI~YS Left side
24,500 ± 1,500
35,000 ± 2,000
Right side
25,200 ± 1,500
34,000 ± 2,000
Lesion side
12,000 ~ 800
14,000 ± 800
Intact side
24,800 ± 1,500
32,500 ± 1,800
I~1~Y3 AITH LESIONB~
* Results are the means f S .E .M . far 3 experiments . ** The yentromedial teemental lesion vas placed on the Left side and the animals vase sacrificed 4-6 days after the lesion ws placed . Thars vas ao difference in the accuaulation of the labeled compounds betvean the left or right sides of the caudate uuclaus sad putaasn .
It is widest
that the forrtian of catecholaainas Eros tyrosine-Cl 4 is decreased by appro :iaataly 90x on the lesion side of the caudate nucleus and putaaen .
The
voi. 5, No . 23
Bzosn~~ESZS oF cA
21~5
concentration of endogenous dopamine on the lesion side is also reduced by approximately 90Z in these two regions of the CNS . The specific activity is much higher in the caudate nucleus than in the putamen.
These findings suggest that intrayentricularly injected tyrosine
does not penetrate to the putamen to the same extent as to the caudate auclnus, and it is conceivable that the highly myelinated internal capsule reprnsnnts a barrier for the penetration of tyrosine to the putamen. TABLE II The ßffects of Ventromedial TeAmental Lesions on the In Vivo Biosynthesis of Catecholamines from Tvroaine-C 14 and on the Dopamine Content in the ~txi+ltum
Caudate Nucleus
I+8/g
Dopamine c .p .m./8
Put
k8/8
n
Dopamine c.p .m ./8
OONTROL IYDNI~YS Left side
4.10 ± 0 .50
6,800 ± 400
4.65 ± 0.50
950 f 100
Right side
4.05 ± 0.50
7,000 ± 500
4 .50 ± 0 .50
900 ± 100
?DNKEYS WITH LESIONS*
f
Lesion side
0 .50
0.05
750 ± 100
0.60 ± 0.05
Intact aide
3 .95 ± 0.40
6,500 ± 500
4.40 ± 0.50
N .D . 950 ± 100
* Results ara the ereas ± B.S .M. f~e ~ sxparirants . The wntrosrdial tag"sn- , tal lasion maa placed oa the loft side and tha.anisrls were sacrificed 4-6 weeks after the lesion was placed . N.D ., not detectable < 100 c.p .~./g. Summarv Following ventromediaL tegmental lesions in the brain stem of monkeys, tyrosine hydroxylase activity is significantly lower on the lesioa side of the caudate nucleus and putamen.
The in vivo formation of dopamine-C 14 from
21~6
BzosYx~szs
voi . 5, xo. 23
oF cA
tyrosine-C 14 ie impaired on the lesion side in these two regions of the CNS . The present findings support the idea that yentrosiedial tegmental lesions produce a~.degeneration of dopamine neurone in the ipeilateral caudate nucleus and putanan . AcknovledAments - This work was supported by USPHS Grants MH 02717, NB 5480 and NB 04257 and by NSF Grant GB 3176 .
References 1.
L . POI$ISR and T . L . SOURKES, Brain . 88, 181 (1965) .
2.
0 . HORNYKISWICZ, Deutch med . Wochschr . 87, 1807 (1962) .
3.
N . -S . ANDBN, A . CARLSSON, A . DAHLSTROM, K . FUXE, N .
-A . HILLARP and
K . LARSSON, Life Sci . 3, 523 (1964) . 4.
N . -A. HILLARP, K . FU7~ and A . DAHLSTROèI, Pharmscol . Rev . 18, 727 (1966) .
5.
A . H . ANTON and D . F . SAYRE, J . Phars~acol . Exptl . Therap . 145, 326 (1964) .
6.
M. GOLDSTEIN, Int . J . Neuroyharmscol . 3, 37 (1964) .
7.
K. GOLDSTEIN, B . ANAGNOSTE, W . S . OWEN and A . F . BATTISTA, The Phvsiolo giet . ~, 191 (1966) .