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Tryptophan accumulation in discrete areas of the rat brain
Pharmacologica/ Research Communications, VoL 12, No. 9, 1980
TRYPTOPHAN
877
+
ACCUMULATION IN DISCRETE AREAS OF THE .RAT BRAIN
Valzelli L., Bernasconi S., and Sala A, IStituto d~ Ricerche FarmacoloEiche "Mario Negri" Via Eritrea, 62 - 20157 Milano Italy
Rece~edmfinal~rm 26June t980 INTRODUCTION There is growing interest in the biochemical and behavioral
signifi-
cance of brain tryptophan (TRP), since this essential aminoaeid is oseof the rate-limiting steps for serotonin (5-HT) formation in the brain. The concentration of tryptophan at the bra~.n sites where 5-HT is synthesized
is
thus
an essential point in central serotonergic control. In this regard,
it
was
shown that changes in serum TRP levels ire associated with p~rallel
changes
in brain concentrations of TRP a~d 5-HT (Tgglia~ionte et aZ., 1973). However, the blood concentration of other aminoac~ds (tyroslne, phenylala~line, valine, leucine, and isoleucine) may differently modify the blood concentration
of
free TRP (Fernstr~m e~ aZ., 1974; Gessa et ~Z., 1974). In addition, the brain 5-HT concentration is known to be significantly increased by doses
of
TRP
even less than one-twentieth of the daily dietary intake (Fernstrom and Wurt man, 1971). Thus, either the dietary intake of TRP, or any disturbance of TRF ut111zation are likely to have important implications in the regulation behavior (Gibbons et oZ., 1979), and i- psychla~ric diseases
(Wurtman
of and
Partridge, 1979). Several studies have dealt with TRP t~ansport into syna~tosomes
~e]~
and Pujol, 1973; Grahame-Smith and Parfitt, 1970; Knapp and ~qndell,
1973);
and TRP accumulation by glial cells (Bauman e~ ~Z., 1974) and
slices
5rain
(K{ely and Sourkes, 1972; Vahveiainen and oja~ 1972); however, so information + Supported No.
by C.N.R.
79,O1815,O4,
(Consigl~o
Nazionale
Ricerche,
Roma)
grant
1979.
O0,31-6989~o~go877-O6/~p02.00~
© 1980ThehallanPftarm~otog~calSoclety
Pharmacological Research Commun~ations, Vol. 12, No. 9, 1980
878
has focused on possible differences in regional ugtake of TRP. This point was taken into consideration more recently ( D e n l z ~ and Sourkes, 1977;
Martinet
et oZ., 1979) in studies of TRP uptake in brain slices and synaptosomes prepared from brain regions
known to vary in the number of serotonergic
cell
bodies and nerve endings. However, TRP is delivered to the interior of brain cells depending on the transport of the amlnoacid through, first,
the brain
capillary walls (blood-braln barrier), and, second, the
membrane.
neuronal
Since TRP transport through the membrane of neuronal cells and nerve endings is much faster than through the blood-brain barrier (Partridge, 1977),
the
rate-limiting step in brain uptake of plasma TRP is its transport through the capillary walls (Wurtman and Partridge, 1979). This consideration prompted us to study the regional distribution of an orally administered tryptophan load into discrete areas of the rat brain.
MATERIALS AND METHODS Adult CD-COBS male rats (Charles River, Italy), weighing 200+15 g
(54
days old), housed 5 per cage (transparent Makrolon R cages, 42x26x15 cm)
were
used. The animals were maintained at a constant room temperature (21°C~I) and relative humidity (60%), with controlled light-dark cycles (light on from 8:00 a.m. to 8:00 p.m.) and were given laboratory chow (Altromln D~)and
water
ad
Zibitum. L-tryptophan, 25 mg/kg, was administered by oral route, dissolved inluk~ warm (35°C) distilled water, and the rats, ten for each time,
were killed by
decapitation O, 30, 60, and 90 mins after TRP administration. The brains were quickly removed, dissected into the different areas as
described
elsewhere
(Valzelli and Garattlnl, 1968), and stored on frozen CO 2 at -22°C until
bio-
chemical assay. Brain tryptophan was extracted according to a slightly modifi m
Pharmaco/ogicat Research Communications, VoL 12, No. 9, 1980 ed version Dewey
of the revised
(1967).
method
The resulting
sed using an A m i n c o - B o w m a n
(Bloxam and Warren,
samples were
879 1974)
of Denckla and
then s p e c t r o f l u o r i m e t r i e a l l y
analy~
apparatus.
RESULTS AND D I S C U S S I O N The results are shown
in Table
I.
Table Tryptophan
uptake by discrete
areas
after oral a d m i n i s t r a t i o n
Brain areas
1 of rat brain at v a r i o u s
of the aminoaeid
Brain
tryptophan
times
(25 mg/kg).
ug/giS.E,
after
Omln
Ch %
30 min
Ch %
60 min
Ch %
90 min
Ch %
Cerebellum
4.56 ±O.19
-
6.49 ±0.36 °
+ 42
7.22 ±O.29 °
+ 58
5.36 -+0.45
+ 18
C. Q u a d r i g e m i n a
6.80 ±0.33
-
13.49 ±0.54 °
+ 92
13.58 ±O.48 °
+I00
9.03 -+0.48 °
+ 33
Diencephalon
4.96 ±0.26
-
9.02 ±O.25 °
+ 82
9.58 ±O.23 °
+ 93
7.67 +-0.34 °
+ 55
Mesencephalon
4.04 ±0.23
-
7.60 ±O.30 °
+ 88
7.88 ±O.38 °
+ 95
5.91 /O.23 °
+ 46
A m y g d a l a + Hyppocampus
4.95 ±0.25
-
10.20 ±O.57 °
+106
9.84 ±O.23 °
+ 99
7.26 ±0.22 °
+ 47
Corpora
6.87 ±O.17
-
15.36 ±0.53 °
+123
14.29 ±O.47 °
+108
10.33 ±O.49 °
+ 51
Hemispheres
4.65 ±0.15
-
9.07 ±0.37 °
+ 95
8.78 iO.25 °
+ 89
6.64 iO.3b °
+ 43
Whole b r a i n (without c e r e b e l l u m
4.26 ±0.08
-
9.71 ±0.43 °
+127
0.42 +0.29 °
+ 98
5.79 ±0.36 °
+ 36
striata
Ch % - Change %. o .
p < 0.01
Statistical
compared
evaluation
to
basal
value.
by D u n n e t t t s
test.
Pham~Tacolog~al ResearchCommun~ations, VoL 12, No. 9, 1980
880
In basal conditions (0 time), significant1~ higher (p < 0.01) concentr~ tions of TRP were found in the corpora striata and corpora quadrigemina than in the other brain areas considered. In general, peak concentrations
were
reached between 30 and 60 mln from l-tryptophan administration, with
lower
values at 90 m i ~ and in the cerebellum TRP had already \ returned to normal by '\
this time. The most intense uptake of administered TRP was seen in the corp~ ra striata, followed by the llmbic areas pf amygdala and hippocampus, and by the corpora quad.rigemina, even if the apparent differences do not raise significant value.
Of the rat brain areas considered the cerebellum
any
showed
the lowest and most short-lasting TRP incorporation. This may agree with pr~ vious observations that synaptosomes fromlthis area are scarcely effective in accumulating TRP (Denizeau and Sourkes, 1977), and that serotonergic
nerve
endings are present only in small numbers in the cerebellum. In previous studies dealing with brain TRP distribution in rats
(Knott
and Curzon, 1974), the changes induced by an intraperitoneal load of TRP do~ ble that here employed (50 mg/kg) were measured at only one time, 2hrs, after administration. Others (Smith et aZ., 1976) examined the changes of TRP in 4 brain areas of pigeons engaged in multiple operant behavior schedules an intramuscular TRP load of 300 mg/kg. These studies are thus not
after
directly
comparable with the present experiment. Findings ~n vit~,o show that an acute injection or a constant infusion of TRP to rats leads to greater serotonin release in the corpus striatum
and
cortex/hippocampus than in~ehypothalamusand brainstem (Ternaux et aZ.,1976). In addition, a 5-HT receptor blocker such as methiothepln facilitates TRP ul take by brain slices, suggesting that changes of TRP uptake could depend the functional
state
been proposed that ters
represented Thus,
of serotonergic regional
(Hamon e t aZ,,
u p t a k e o f TRP may c o r r e l a t e
by s e r o t o n i n
the functional
neurons
u p t a k e and r e l e a s e
level
of the brain
and J t h a s
w i t h t h e two p a r a m e -
(Martinet
serotonerglc
1976),
on
et aZ.,
1979).
s y s t e m and t h e b e -
Pharmacological Research Communicat~n~ VoL 1~ No. ~ 1980
881
havloral activity of animals probably regulate the passage of TRP
into
the
brain through the blood-brain barrier and penetration of the amlnoacid into nerve cells. Further experiments are obviously required to clarify thispoin~
REFERENCES Bauman, A., Bourgoin, S., Panda, P., Glowlnski, J., and Hamon, H. 3rein Re~. 66, 253.
(1974).
Belin, M.-F., and Pujol, J.-F. (1973). Experientia 29,~411. Bloxam, D.L.,and
Warren, W.H. (1974). Anal.Biochem. 60, 621.
Denckla, W.D., and Dewey, H.K. (1967). J.Lab.clin.~d.
69, 160.
Denizeau, F., and Sourkes, T.L. (1977). J.Neurochem. 28, 951. Fernstr~m, J.D., b~dras, B.K., ~nro, H.N., and Wurtman, R.J. I n: Aromatic Amino Acid8 in the Brain, Ciba Foundatlon Symposium 22 (new series), Amsterdam, Elsevier, p. 153, 1974. Fernstr~m, J.D. and Wurtman, R.J. (1971). Science 174, 1023. Gessa, G.L., Bigglo, G., Fadda, F., Corsini, G.U., and Tagliamonte, A. (1974). J..Neurochrm. 22, 869. Gibbons, J.L., Barr, G.A., Bridger, W.H., and Lelbowitz, S.F. (1979). Brain Res. 169, 139. Grahame-Smith, D.G., and Parfitt, A.G. (1970). J.Neurochem. I?, 1339. Hamon, M., Bourgoln, S., Hery, F., Ternaux, J.P. and Glowlnski, J. (1976). Nature 2~0, 61. Kiely, M., and Sourkes, T.L. (1972). J.Neurochem. 19, 2863. Knapp, S., and b~ndell, A.J. (1973). Science 180, 645. Knott, P.J., and Curzon, O. (1974). J.Neurochem. 22, I065. Martinet,
hi., F o n l u p t ,
P . , and Pachco, H. (1979). PsyohopharmacoZouH ~6, 63.
P a r d r i d g e , W.M. In: N u t r i t i o n and the Brain, Vol. 1, R . J . Wurtman and J . J . Wurtman ( E d s - ) . New York, Raven P r e s s , p. 141, 1977. Smith, J . E . , Hingten, J . N . , 26, 537.
Lane, J . D . ,
Tagliamonte, A., B i g g i o , a . , Vargiu, L . , 20, 909.
and Aprison, N.H. (1976). J.Neurochem. and Gessa, G.L. (1973). J. Neurochem.
882
Pharmacological Research Communications, Vol. 12, No. 9, 1980
Ternaux, J.P., Boireau, A., Bourgoin, S., Hamon, ~., Hery, F., and Glowinski, J. (1976). Brain Res. I01, 533. Vahvelainen, M.-L., and Oja, S.S. (1972). Brain Res. 40, 477. Valze111, L., and Garattini, S. (1968). J. Neuroche, n. 15, 259. Wurtman, R.J., and Pardridge, W.M. (1979). 227.
J.Neural Transmission suppZ. 15,
TRYPTOPHAN
877
+
ACCUMULATION IN DISCRETE AREAS OF THE .RAT BRAIN
Valzelli L., Bernasconi S., and Sala A, IStituto d~ Ricerche FarmacoloEiche "Mario Negri" Via Eritrea, 62 - 20157 Milano Italy
Rece~edmfinal~rm 26June t980 INTRODUCTION There is growing interest in the biochemical and behavioral
signifi-
cance of brain tryptophan (TRP), since this essential aminoaeid is oseof the rate-limiting steps for serotonin (5-HT) formation in the brain. The concentration of tryptophan at the bra~.n sites where 5-HT is synthesized
is
thus
an essential point in central serotonergic control. In this regard,
it
was
shown that changes in serum TRP levels ire associated with p~rallel
changes
in brain concentrations of TRP a~d 5-HT (Tgglia~ionte et aZ., 1973). However, the blood concentration of other aminoac~ds (tyroslne, phenylala~line, valine, leucine, and isoleucine) may differently modify the blood concentration
of
free TRP (Fernstr~m e~ aZ., 1974; Gessa et ~Z., 1974). In addition, the brain 5-HT concentration is known to be significantly increased by doses
of
TRP
even less than one-twentieth of the daily dietary intake (Fernstrom and Wurt man, 1971). Thus, either the dietary intake of TRP, or any disturbance of TRF ut111zation are likely to have important implications in the regulation behavior (Gibbons et oZ., 1979), and i- psychla~ric diseases
(Wurtman
of and
Partridge, 1979). Several studies have dealt with TRP t~ansport into syna~tosomes
~e]~
and Pujol, 1973; Grahame-Smith and Parfitt, 1970; Knapp and ~qndell,
1973);
and TRP accumulation by glial cells (Bauman e~ ~Z., 1974) and
slices
5rain
(K{ely and Sourkes, 1972; Vahveiainen and oja~ 1972); however, so information + Supported No.
by C.N.R.
79,O1815,O4,
(Consigl~o
Nazionale
Ricerche,
Roma)
grant
1979.
O0,31-6989~o~go877-O6/~p02.00~
© 1980ThehallanPftarm~otog~calSoclety
Pharmacological Research Commun~ations, Vol. 12, No. 9, 1980
878
has focused on possible differences in regional ugtake of TRP. This point was taken into consideration more recently ( D e n l z ~ and Sourkes, 1977;
Martinet
et oZ., 1979) in studies of TRP uptake in brain slices and synaptosomes prepared from brain regions
known to vary in the number of serotonergic
cell
bodies and nerve endings. However, TRP is delivered to the interior of brain cells depending on the transport of the amlnoacid through, first,
the brain
capillary walls (blood-braln barrier), and, second, the
membrane.
neuronal
Since TRP transport through the membrane of neuronal cells and nerve endings is much faster than through the blood-brain barrier (Partridge, 1977),
the
rate-limiting step in brain uptake of plasma TRP is its transport through the capillary walls (Wurtman and Partridge, 1979). This consideration prompted us to study the regional distribution of an orally administered tryptophan load into discrete areas of the rat brain.
MATERIALS AND METHODS Adult CD-COBS male rats (Charles River, Italy), weighing 200+15 g
(54
days old), housed 5 per cage (transparent Makrolon R cages, 42x26x15 cm)
were
used. The animals were maintained at a constant room temperature (21°C~I) and relative humidity (60%), with controlled light-dark cycles (light on from 8:00 a.m. to 8:00 p.m.) and were given laboratory chow (Altromln D~)and
water
ad
Zibitum. L-tryptophan, 25 mg/kg, was administered by oral route, dissolved inluk~ warm (35°C) distilled water, and the rats, ten for each time,
were killed by
decapitation O, 30, 60, and 90 mins after TRP administration. The brains were quickly removed, dissected into the different areas as
described
elsewhere
(Valzelli and Garattlnl, 1968), and stored on frozen CO 2 at -22°C until
bio-
chemical assay. Brain tryptophan was extracted according to a slightly modifi m
Pharmaco/ogicat Research Communications, VoL 12, No. 9, 1980 ed version Dewey
of the revised
(1967).
method
The resulting
sed using an A m i n c o - B o w m a n
(Bloxam and Warren,
samples were
879 1974)
of Denckla and
then s p e c t r o f l u o r i m e t r i e a l l y
analy~
apparatus.
RESULTS AND D I S C U S S I O N The results are shown
in Table
I.
Table Tryptophan
uptake by discrete
areas
after oral a d m i n i s t r a t i o n
Brain areas
1 of rat brain at v a r i o u s
of the aminoaeid
Brain
tryptophan
times
(25 mg/kg).
ug/giS.E,
after
Omln
Ch %
30 min
Ch %
60 min
Ch %
90 min
Ch %
Cerebellum
4.56 ±O.19
-
6.49 ±0.36 °
+ 42
7.22 ±O.29 °
+ 58
5.36 -+0.45
+ 18
C. Q u a d r i g e m i n a
6.80 ±0.33
-
13.49 ±0.54 °
+ 92
13.58 ±O.48 °
+I00
9.03 -+0.48 °
+ 33
Diencephalon
4.96 ±0.26
-
9.02 ±O.25 °
+ 82
9.58 ±O.23 °
+ 93
7.67 +-0.34 °
+ 55
Mesencephalon
4.04 ±0.23
-
7.60 ±O.30 °
+ 88
7.88 ±O.38 °
+ 95
5.91 /O.23 °
+ 46
A m y g d a l a + Hyppocampus
4.95 ±0.25
-
10.20 ±O.57 °
+106
9.84 ±O.23 °
+ 99
7.26 ±0.22 °
+ 47
Corpora
6.87 ±O.17
-
15.36 ±0.53 °
+123
14.29 ±O.47 °
+108
10.33 ±O.49 °
+ 51
Hemispheres
4.65 ±0.15
-
9.07 ±0.37 °
+ 95
8.78 iO.25 °
+ 89
6.64 iO.3b °
+ 43
Whole b r a i n (without c e r e b e l l u m
4.26 ±0.08
-
9.71 ±0.43 °
+127
0.42 +0.29 °
+ 98
5.79 ±0.36 °
+ 36
striata
Ch % - Change %. o .
p < 0.01
Statistical
compared
evaluation
to
basal
value.
by D u n n e t t t s
test.
Pham~Tacolog~al ResearchCommun~ations, VoL 12, No. 9, 1980
880
In basal conditions (0 time), significant1~ higher (p < 0.01) concentr~ tions of TRP were found in the corpora striata and corpora quadrigemina than in the other brain areas considered. In general, peak concentrations
were
reached between 30 and 60 mln from l-tryptophan administration, with
lower
values at 90 m i ~ and in the cerebellum TRP had already \ returned to normal by '\
this time. The most intense uptake of administered TRP was seen in the corp~ ra striata, followed by the llmbic areas pf amygdala and hippocampus, and by the corpora quad.rigemina, even if the apparent differences do not raise significant value.
Of the rat brain areas considered the cerebellum
any
showed
the lowest and most short-lasting TRP incorporation. This may agree with pr~ vious observations that synaptosomes fromlthis area are scarcely effective in accumulating TRP (Denizeau and Sourkes, 1977), and that serotonergic
nerve
endings are present only in small numbers in the cerebellum. In previous studies dealing with brain TRP distribution in rats
(Knott
and Curzon, 1974), the changes induced by an intraperitoneal load of TRP do~ ble that here employed (50 mg/kg) were measured at only one time, 2hrs, after administration. Others (Smith et aZ., 1976) examined the changes of TRP in 4 brain areas of pigeons engaged in multiple operant behavior schedules an intramuscular TRP load of 300 mg/kg. These studies are thus not
after
directly
comparable with the present experiment. Findings ~n vit~,o show that an acute injection or a constant infusion of TRP to rats leads to greater serotonin release in the corpus striatum
and
cortex/hippocampus than in~ehypothalamusand brainstem (Ternaux et aZ.,1976). In addition, a 5-HT receptor blocker such as methiothepln facilitates TRP ul take by brain slices, suggesting that changes of TRP uptake could depend the functional
state
been proposed that ters
represented Thus,
of serotonergic regional
(Hamon e t aZ,,
u p t a k e o f TRP may c o r r e l a t e
by s e r o t o n i n
the functional
neurons
u p t a k e and r e l e a s e
level
of the brain
and J t h a s
w i t h t h e two p a r a m e -
(Martinet
serotonerglc
1976),
on
et aZ.,
1979).
s y s t e m and t h e b e -
Pharmacological Research Communicat~n~ VoL 1~ No. ~ 1980
881
havloral activity of animals probably regulate the passage of TRP
into
the
brain through the blood-brain barrier and penetration of the amlnoacid into nerve cells. Further experiments are obviously required to clarify thispoin~
REFERENCES Bauman, A., Bourgoin, S., Panda, P., Glowlnski, J., and Hamon, H. 3rein Re~. 66, 253.
(1974).
Belin, M.-F., and Pujol, J.-F. (1973). Experientia 29,~411. Bloxam, D.L.,and
Warren, W.H. (1974). Anal.Biochem. 60, 621.
Denckla, W.D., and Dewey, H.K. (1967). J.Lab.clin.~d.
69, 160.
Denizeau, F., and Sourkes, T.L. (1977). J.Neurochem. 28, 951. Fernstr~m, J.D., b~dras, B.K., ~nro, H.N., and Wurtman, R.J. I n: Aromatic Amino Acid8 in the Brain, Ciba Foundatlon Symposium 22 (new series), Amsterdam, Elsevier, p. 153, 1974. Fernstr~m, J.D. and Wurtman, R.J. (1971). Science 174, 1023. Gessa, G.L., Bigglo, G., Fadda, F., Corsini, G.U., and Tagliamonte, A. (1974). J..Neurochrm. 22, 869. Gibbons, J.L., Barr, G.A., Bridger, W.H., and Lelbowitz, S.F. (1979). Brain Res. 169, 139. Grahame-Smith, D.G., and Parfitt, A.G. (1970). J.Neurochem. I?, 1339. Hamon, M., Bourgoln, S., Hery, F., Ternaux, J.P. and Glowlnski, J. (1976). Nature 2~0, 61. Kiely, M., and Sourkes, T.L. (1972). J.Neurochem. 19, 2863. Knapp, S., and b~ndell, A.J. (1973). Science 180, 645. Knott, P.J., and Curzon, O. (1974). J.Neurochem. 22, I065. Martinet,
hi., F o n l u p t ,
P . , and Pachco, H. (1979). PsyohopharmacoZouH ~6, 63.
P a r d r i d g e , W.M. In: N u t r i t i o n and the Brain, Vol. 1, R . J . Wurtman and J . J . Wurtman ( E d s - ) . New York, Raven P r e s s , p. 141, 1977. Smith, J . E . , Hingten, J . N . , 26, 537.
Lane, J . D . ,
Tagliamonte, A., B i g g i o , a . , Vargiu, L . , 20, 909.
and Aprison, N.H. (1976). J.Neurochem. and Gessa, G.L. (1973). J. Neurochem.
882
Pharmacological Research Communications, Vol. 12, No. 9, 1980
Ternaux, J.P., Boireau, A., Bourgoin, S., Hamon, ~., Hery, F., and Glowinski, J. (1976). Brain Res. I01, 533. Vahvelainen, M.-L., and Oja, S.S. (1972). Brain Res. 40, 477. Valze111, L., and Garattini, S. (1968). J. Neuroche, n. 15, 259. Wurtman, R.J., and Pardridge, W.M. (1979). 227.
J.Neural Transmission suppZ. 15,