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Effect of tryptophan administration on serotonin and 5-hydroxyindoleacetic acid content in the brainstem of Lilly 110140-pretreated rats
Life Sciences Vol. 17, pp . 523-530 Priate8 in the II.S .A.
Pergemon Press
EFFECT OF TRYPTOPHAN ADMINLSTRATION ON SEROTONIN AND 5-HYDROXYINDOLEACETIC ACID CONTENT ]N THE BRALNSTEM OF LILLY 110140-PRETREATED RATS* William W. Morgen, P. Kenia Rudeen and Karla A. Pfeil Department of Anatomy, The University of Tezae Health Science Center at San Antonio, San Antonio, Texas 78284 (Received in final form July 7, 1975) Summary Adult male Sprague-Dawley rate were divided into 3 groups . One group was pretreated with Lilly 110140 (10 mg/kg) 27 hours and again 3 hours before sacrifice while a second group received T "illy 110140 only 3 hours before sacrifice . The third or control group received only equivalent volumes of saline . Animals from each group were administered 25 mg/kg L tryptophan intraperitoaeally (i. p. ) 0, 30, 60 or 90 minutes before sacrifice. Equivalent elevations in serum and also brainetem tryptophan content were observed in all three groups with the peak observed at 30 minutes. Brainetem serotonin content was significantly elevated in bow groups of Lilly 110140-pretreated rate but not in the control group. Brainstem 5-hydrozyindoleacetic acid was significantly elevated after tryptophan administration in the control and the 3 hour and 27 hour, Lilly 110140-pretreated groups but not in the 3 hour Lilly 110140 pretreated group. The results indicate that neither 3 or 3 hours and 27 hours of Lilly 110140 pretreatment appreciably affects the increase in brainetem serotonin synthesis induced by the i. p. administration of 25 mg/kg of L tryptophan . Drugs which inhibit the reuptake of eerotoain (5-HT) have been shown to decrease the turnover of 5-HT la the brain (1, 2) and further to decrease the firing rate of raphe neurone (3). This decrease in 5-HT turnover is believed to result from an increase in 5-HT content outside the neuron with a subse quent increased activation of 5-HT receptors . The physiological route by which the level of activation of 5-HT receptors affects 5-HT turnover has not been clearly identified although the effect could result from a decrease in the uptake of tryptophaa by eerotonergic neurons (1). The intraperitoneal administration of tryptophan has been shown to increase the synthesis and turnover of 5-HT (4). Originally, the elevation of 5-HT synthesis and turnover was related to a lack of saturation of brain trypbophan hydroxylaee with its substrate tryptophan (4, 5) . However, recent data obtained with the natural * Supported by NASA Grant NSG 9012
523
524
Brainstem
5-HT and 5-HIAA
Vol . 17, No .
4
cofactor for tryptophaa hydroxylase indicates that this enzyme is normally saturated with substrate. Although the underlying mechanism is unclear, the brain level of tryptophan has been shown to affect the synthesis and turnover of 5-HT . If the inhibition of 5-HT reuptake produces a decreaee in 5-HT turnover by decreasing the uptake of tryptophan into serotonergic neurons, then the inhibition of 5-HT reuptake might also decreaee the response of brain 5-HT synthesis to the intraperitoaeal administration of tryptophan, Ia this study Lilly 110140 was administered once 3 hours before sacrifice or twice at 27 and at 3 hours before sacrifice to decrease 5-HT turnover in the brains of rate . This compound rather selectively inhibits 5-HT reup take and subsequently decreases 5-HT turnover (2, 7) . The ability of 25 mg/kg tryptophan, administered intraperitoaeally, to affect 5-HT content in the brainetem of these animals was then determined. Methode Male Sprague-Dawley rate (150 grams) were housed for 1 week in an animal room and exposed to a 14 :10 light-dark lighting regimen. The lights were on from 0600-2000 daily. The animals were provided with food and water _ad libitum. The rate were randomly divided into 3 groups . One group was pretreated with Lilly 110140 (3-(p-Trifluoromethylpheaozy)-N-methyl-3phenylpropylamine) (Lilly Laboratories, IIi Lilly and Company), (LO mg/kg) iatraperitoneally (i . p. ) 27 hours before sacrifice while the remaining 2 groups received equivalent volumes of saline . Three hours before sacrifice the Lilly 110140-pretreated group received a second administration of drug, a second group received their first dosage of Lilly 110140 while the third or control group again received as equivalent volume of saline . Ani*r,a1 8 is each of dress 3 groups were given 25 mg/kg L tryptophaa i. p. is saline 0, 30, 60 or 90 minutes before sacrifice. The Lilly 110140 pretreatments, the tryptophan administration and the sacrifices were carried out at 3 minute intervals according to a completely randomized schedule . The brain of each animal was removed and the right half of the teleacephaloa (cerebral cortex, striatum, hippocampal formation) and the brainetem were dissected. The braiaatem included that area of the brain from the superior colliculus aateriorly to the gracile nuclei poeteriorly. These brain regions were quick frozen on dry ice, weighed and stored at -55 ° C for 24-48 hours before analysis . Blood was collected from the cervical vessels and allowed to clot for a few minutes at 37 ° C . The blood samples were centrifuged at 1500 g and serum was collected. Brain 5-HT, tryptophaa, 5-hydroxyindoleacetic acid (5-HIAA) were extracted by a column chromatographic procedure (8). The content of brain 5-HT was determined by the method of Maickel et al . (9 ), Brain and also serum tryptophaa content were determined by the method of Lia et al . (10) . Brain 5-HIAA was quaatitated by the method of Curzon and Green (11) . The statistical significance of differences is the parameters after different Lilly 110140 pretreatmente or at different periods following tryptophaa administration was determined by a two way analysis of variance (ANOVA) for unequal samples sizes (12 ). Subsequent statistical analysis were performed
Brainstem
Vol. 17, No . 4
and
5-HT
525
5-HIAA
using a one way analysis of variance (13) or the Student's t test (14) . In order to simplify the presentation of the results only the most pertine~ statistical findings are presented. Results Although the administration of 25 mg/kg of L tryptophan significantly elevated tryptophan content in the telencephalon, the elevation was not sufficient to significantly elevate either 5-HT or 5-HIAA content in the teleacephalon of control or Lilly 110140-pretreated rate . Thus the presentation of results will be restricted to the brainetem and serum only. The intraperitoaeal administration of 25 mg/kg L tryptophan significantly elevated serum tryptophan content (p< 0. 001) (Table 1) . Lilly 110140 pretreatment had no effect on the elevation of serum tryptophan following i. p. tryptophan administration . In all three groups the peak is serum tryptophan was observed 30 minutes after tryptophan administration and by 90 minutes serum tryptophan had returned to 0 time levels . TABLE 1 The Effect ad Tryptophaa Administration oa Serum Tryptophan Content (Wg/ml) in Rats Pretreated with Lilly 110140 Treatment
Results of One Way ANOYA
Time 0
30
60
90
Saline
17 . 7 t0. 8 (11)
44 . 8 t 3. 4 (5)
31 . 2 f 2. 1 (7)
19 . 7 t 1. 3 (7)
p < 0. 001
Lilly 110140 1
18 . 9 f 1. 1 (8)
39 . 911 . 5 (81
30. 711 . 9 (8)
21 . O t 1 . 1 (9)
p < 0. 001
Lilly110140 2
18 .3t2 .4 (9)
41 .St2 .4 (6)
30 .Ot2 .1 (8)
19 .Ot1 .2 (8)
p<0 .001
The mean t standard error is given for each treatment group. The values read across the table are the mean values for the parameter observed in rats treated with saline or Lilly 110140 but sacrificed 0, 30, 60 or 90 minâtes after tryptophan administration . The number in parenthesis indicates the number of animals is the group. The statistical significance of changes in the parameter within the line is shown at the end of the line. Only those statistical differemes which are relevant to the discussion are presented. Similar elevations in brainetem tryptophan content (p< 0. 001) were observed following tryptophan administration (Table 2) . Lilly 110140 pretreat ment had ao effect on the elevation of tryptophan content in the brainstem. As with serum tryptophan, the highest levels of brainstem tryptophan content was observed 30 minutes following tryptophan administration, but brainstem
Brainstem 5-&T and 5-Hrnn
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Vol . 17, No . 4
tryptophaa content was still significantly elevated (p< 0.001) over zero time levels 90 minutes after tryptophaa administration. TAB LE 2 The Effect of Tryptophaa Administration on Tryptophan Content (Wg/gm) in the Brainstem of Rate Pretreated with Lilly 110140 Treatment
Results of Oae Way AN OVA
Time 0
30
60
90
Saline
3. 26 t 0. 14 (11)
10 . 56 f 0 . 63 (5)
7 . 45 f 1 . 17 (7)
4. 35 t 0. 49 P< 0. 001 (7)
Lilly 110140 1
3. 45 f 0. 14 (8)
11 . 34 ~ 0. 39 (8)
6 . 66 f 0 . 27 (8)
4 . 72 f 0. 48 p < 0. 001 (9)
Lilly 110140 2
3. 65 f 0. 40 (9)
11 . 15 f 0. 55 (6)
8. 54 f 0. 45 (8)
5 . 06 f 0. 67 p < 0. 001 (8)
The mean t standard error is given for each treatment group. The values read across the table are the mean values for the parameter observed is rats treated with saline or Lilly 110140 but sacrificed 0, 30, 60 or 90 minutes after tryptophaa administration . The number is parenthesis indicates the number of animals in the group. The statistical significance of changes in the parameter within the line is shown at the end of the line . Only those statistical differences which are relevant to the discussion are presented. The administration of 25 mg/lcg tryptophaa significantly elevated braiastem 5-HT in both groups of Lilly 110140-pretreated rate (p<0 .005) but not in the saline controls (Table 3) . The Student's t teat showed that the level of 5-HT in the brainetem of the 3 hour Lilly 110140 pretreated rate was eigaificaatly elevated at both 60 and 90 minutes but only at 60 minutes in the 27 hour Lilly 110140-pretreated rats . Significant elevations in brainetem 5-HIA.A content were observed in the saline treated (p< 0.005) and the 3 hour and 27 hour Lilly 110140-pretreated rate (p< 0. 025) following the administration of 25 mg/kg L tryptophan (Table 4) . Brainstem 5-HIAA content in 3 hour Lilly 110140-pretreated rate was not eignlficantly elevated during the first 90 minutes following tryptophaa admiaistratioa. Comparable results were observed is 2 separate studies (experiment 1 and experiment 2, Table 4) . Diecuesion Bruimels (1) showed that imipramine, a tricyclic antidepressant compound which inhibits the reuptake of 5-HT and suppresses 5-HT turnover, also decreases the increase in 5-HT content Induced by tryptophaa administration.
Braiastem
Vol. 17, No . 4
$-$T
an8 5-HIAA
52 7
TABLE 3 The Effect of Tryptophan Administration on Serotonin Content (Wg/gm) in üie Brainetem of Rats Pretreated with Lilly 110140 Results of One Way ANOVA
Time
Treatment 0
30
Saline
0. 97
f 0.
Lilly 110140 1
0. 95
f 0. 02 (8)
Lilly 110140 2
0. 94
(10)
06
t 0. 05 (9)
1 . 20 t 0. 07 (5)
1 . 32 1 . 12
t 0. 11
(8)
f0. 06 (7)
60
90
1. 02 t 0. 05
0. 96
1 . 24 f0. 06 (8)P< 0.01
1 . 22 t 0. 06 (9) P< 0.01
(8)
t 0. 05 (7)
1 . 25 t 0. 06 1 . 05 f 0. 06 (8)p< 0.01 " (8)
N. 5 . p < 0. 005 p< 0. 005
The mean f standard error is given for each treatment group. The values read across the table are the mean values for the parameter observed in rate treated wilü saline or Lllly 110140 but sacrificed 0, 30, 60 or 90 minutes after tryptophan adminietr~tion . The number in parenthesis indicates the number of animals is the group. The statistical significance of changes is the parameter within the line is shown at the end of the line . Only those statistical differences which are relevant to ~e discussion are presented. The statistical significance (determined by the t teat) (14) of any mean within a column from the saline mean is the same column is designated by a p value under the appropriate mesa. N. S. = not significant . He concluded that imipramine inhibited the biosynthesis of 5-HT by inhibiting the uptake of tryptophan. A similar mechanism could ezplain the decrease in 5-HT synthesis and turnover observed after the administration of other inhibitors of 5-HT reuptake, i. e . Lilly 110140 . However, the results of the present study provide no support for the hypothesis that the Inhibition of 5-HT reuptake by Lilly 110140 produces a subsequent decrease in 5-HT synthesis and turnover by appreciably decreasing the uptake of tryptophan. The elevations of 5-HT content In the brainstem following the administration of 25 mg/kg tryptophsa appeared to be greater in the Lilly 110140 pretreated rats and were in fact only statistically significant in these animals . The increase is 5-HT synthesis in the saline treated controls following tryptophan administration was better reflected by tire significant elevations of 5-Hrea content in these animals . Since 5-HT reuptake was inhibited in the Lilly 110140 pretreated animals, the increased 5-HT in these groups were comparatively protected from iatraaeuronal degradation and 5-HLAA levels were initially lower and less elevated following tryptophaa administration .
528
Hraiast :e~ 5-HT and 5-BIAA
Vol . 17, No . 4
TABLE 4 The Effect of Tryptophan Administration on 5-Hydrozyiadoleactic Acid (Flg/gm) Content in the Brainstem of Rate Pretreated with Lilly 110140 Ezperiment 1 Treatment
Results of Oae Way ANOVA
Time 0
30
60
90
0. 71 t 0 . 04 (6)
0. 90 t 0. 02 (3)
1 . 12 t 0. 09 (7)
0. 88 f 0. 05 (6)
Lilly 110140
0. 58 f 0. O6 (8)
0. 82 f 0. 10 (8)
0. 72 t 0. 07 (5)p< 0.01
0. 72 t 0. 04 (8)p< 0 .025
Lilly 110140 2
0. 52 t 0. 03 0. 72 f 0. 07 (8)p< 0.005 (7)p< 0 .05
0 . 88 f 0. 07 (7)
0. ?6 f 0 . 12 (7)
Saline
1
p < 0. 00 5 N. S. p < 0. 025
Ezperimeat 2 Treatment
Results of One Way ANOVA
Time 0
30
60
Saline
0. 69 t 0, 03 (8)
0. 95 f 0. 11 (7)
1 . 04 f 0. 11
Lilly 110140 2
0 .50 t0 . 04 0.7110. 07 (7)p< 0.005 (7)
(8)
90
0. 93 f 0. 07 (8)
0. 6810 . 03 0, 6010 . 05 (8)p< 0, 005 (8)p< 0.005
p < 0. 05 p< 0. 025
The meaatstandard error ie given for each treatment group. The valuee read across the table are the mesa valuee for the parameter observed is rate treated with saline or Lilly 110140 but sacrificed 0, 30, 60 or 90 minutes after tryptophan administration. The number in parenthesis indicates the number of animals is ~e group. The statistical significance of changes in the parameter within the line ie shown at the end of the line . Only those statistical differences which are relevant to the discussion are presented . The statistical significance (determined by ~e t test) (14) of any mean within a column from the saline mesa in the same column is designated by a p value under the appropriate mean. N. S. = not significant. Experiment 2 was a repeat ezperiment. The elevation of braiastem 5-HT following tryptophan administration was more prolonged in the 3 hour than is the 3 hours sad 27 hour Lilly 110140 pretreated rats . These data suggest that the increased 5-HT was more rapidly removed from the brainsteme of the rate pretreated with 2 injections of Lilly 110140, one at 27 hours sad a second at 3 hours before sacrifice . During the 27 hour period the brain may have adapted to remove the elevated eatraneuronal
Hrainstem
Vol. 17, No . 4
5-AT and 5-HiAA
529
5-HT . It is unlikely that 5-HT is the 3 hour and 27 hour Lilly 110140-pretreated rats ie being more rapidly metabolized to 5-HTAA since 5-HTAA levels in the 2 groups of Lilly pretreated rats are not eignülcantly different. It is unlikely that the level of Lilly 110140 was any lower in the animals which received 2 injections of die drug since they also received a second injection of Lilly 110140 3 hours before eacrüice . However, an increase in Lilly 110140 clearance from these animals ca~ot be ezcluded. These data ehaav that the inhibition of 5-HT reuptake by Lilly 110140 pretreatment does not affect the ability of intraperitoneally administered tryptophan to increase 5-HT synthesis. Ia a related study Jacoby et al . (15) observed that chloroimipramiae, another drug which inhibits 5-HTreuptake and also decreases 5-HT synthesis, did not inhibit an increase in brain tryptophan and 5-HIAA induced by fasting. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
J. BRUINVELS, Eur. J. Pharmacol. 20 231-237 (1972) . R. W. FULLER, W. PERRY, and B . B. MOLLOY, Lüe Sçi. _15 1161-1171 (1974). G. K. AGHAJANIAN, B . S. BUNNEY, and M. J. KUHAR, New Concepts is Neurotransmitter Regulation , A. J. Mande]1 editor, pp " 115-134 (1973) . J. D. FERNSTROM and R. J. WURTMAN, Science 173 149-152 (1971) . E. JEQUIER, W. LOVENBERG, and A. SJOERDSMA, Mol. Pharmacol. 3 275-278 (1967) . P. A. FRIEDMAN, A. H. KAPPELMAN, and S. KAUFMAN, J. Biol . Chem . 242 4165-4173 (1972) . D. T. WONG, J. S. HORNG, F. P. BYMASTER, K. L. HAUSER, and B. B. MOLLOY, Life Séi. 15 471-479 (1974) . F. HERY, E. ROUER, and J. GLOWiNSKI, Brain Res. _43 445-465 (1972) . R. P. MAICKEL, R. H. COX, JR . , J. SAILLANT, and F. P. MILLER, Neuropharmacology _7 275-281 (1968) . R. C. LIN, E. COSTA, N. H. NEFF, C. T. WANG, and S. A. NGAI, J. Pharmacol. ~. Ther. _170 232-238 (1969) . G. CURZON and A. R. GREEN, Br . J. Pharmacol. 39 653-655 (1970) . D. J. VELDMAN, Fortran Progra**,* _~r,o _for the Behavioral Sciences, page 257-268, Holt, Rinehart and Wineton, New York (1967) . R. l~. SOKAL and F. J. ROHLF, Biometry , pp . 208-209 . W . H. Freema n and Compa~r, San Francisco (1969) . R. R. SOKAL and F. J. ROHLF, Biometry, pp . 222-223, W. H. Freeman and Company, San Francisco (1969) . J. H. JACOBY, J. Z. COLMENARES, and R. J. WURTMAN, _Fed . Proc . 33 562 (1974) .
K.
Pergemon Press
EFFECT OF TRYPTOPHAN ADMINLSTRATION ON SEROTONIN AND 5-HYDROXYINDOLEACETIC ACID CONTENT ]N THE BRALNSTEM OF LILLY 110140-PRETREATED RATS* William W. Morgen, P. Kenia Rudeen and Karla A. Pfeil Department of Anatomy, The University of Tezae Health Science Center at San Antonio, San Antonio, Texas 78284 (Received in final form July 7, 1975) Summary Adult male Sprague-Dawley rate were divided into 3 groups . One group was pretreated with Lilly 110140 (10 mg/kg) 27 hours and again 3 hours before sacrifice while a second group received T "illy 110140 only 3 hours before sacrifice . The third or control group received only equivalent volumes of saline . Animals from each group were administered 25 mg/kg L tryptophan intraperitoaeally (i. p. ) 0, 30, 60 or 90 minutes before sacrifice. Equivalent elevations in serum and also brainetem tryptophan content were observed in all three groups with the peak observed at 30 minutes. Brainetem serotonin content was significantly elevated in bow groups of Lilly 110140-pretreated rate but not in the control group. Brainstem 5-hydrozyindoleacetic acid was significantly elevated after tryptophan administration in the control and the 3 hour and 27 hour, Lilly 110140-pretreated groups but not in the 3 hour Lilly 110140 pretreated group. The results indicate that neither 3 or 3 hours and 27 hours of Lilly 110140 pretreatment appreciably affects the increase in brainetem serotonin synthesis induced by the i. p. administration of 25 mg/kg of L tryptophan . Drugs which inhibit the reuptake of eerotoain (5-HT) have been shown to decrease the turnover of 5-HT la the brain (1, 2) and further to decrease the firing rate of raphe neurone (3). This decrease in 5-HT turnover is believed to result from an increase in 5-HT content outside the neuron with a subse quent increased activation of 5-HT receptors . The physiological route by which the level of activation of 5-HT receptors affects 5-HT turnover has not been clearly identified although the effect could result from a decrease in the uptake of tryptophaa by eerotonergic neurons (1). The intraperitoneal administration of tryptophan has been shown to increase the synthesis and turnover of 5-HT (4). Originally, the elevation of 5-HT synthesis and turnover was related to a lack of saturation of brain trypbophan hydroxylaee with its substrate tryptophan (4, 5) . However, recent data obtained with the natural * Supported by NASA Grant NSG 9012
523
524
Brainstem
5-HT and 5-HIAA
Vol . 17, No .
4
cofactor for tryptophaa hydroxylase indicates that this enzyme is normally saturated with substrate. Although the underlying mechanism is unclear, the brain level of tryptophan has been shown to affect the synthesis and turnover of 5-HT . If the inhibition of 5-HT reuptake produces a decreaee in 5-HT turnover by decreasing the uptake of tryptophan into serotonergic neurons, then the inhibition of 5-HT reuptake might also decreaee the response of brain 5-HT synthesis to the intraperitoaeal administration of tryptophan, Ia this study Lilly 110140 was administered once 3 hours before sacrifice or twice at 27 and at 3 hours before sacrifice to decrease 5-HT turnover in the brains of rate . This compound rather selectively inhibits 5-HT reup take and subsequently decreases 5-HT turnover (2, 7) . The ability of 25 mg/kg tryptophan, administered intraperitoaeally, to affect 5-HT content in the brainetem of these animals was then determined. Methode Male Sprague-Dawley rate (150 grams) were housed for 1 week in an animal room and exposed to a 14 :10 light-dark lighting regimen. The lights were on from 0600-2000 daily. The animals were provided with food and water _ad libitum. The rate were randomly divided into 3 groups . One group was pretreated with Lilly 110140 (3-(p-Trifluoromethylpheaozy)-N-methyl-3phenylpropylamine) (Lilly Laboratories, IIi Lilly and Company), (LO mg/kg) iatraperitoneally (i . p. ) 27 hours before sacrifice while the remaining 2 groups received equivalent volumes of saline . Three hours before sacrifice the Lilly 110140-pretreated group received a second administration of drug, a second group received their first dosage of Lilly 110140 while the third or control group again received as equivalent volume of saline . Ani*r,a1 8 is each of dress 3 groups were given 25 mg/kg L tryptophaa i. p. is saline 0, 30, 60 or 90 minutes before sacrifice. The Lilly 110140 pretreatments, the tryptophan administration and the sacrifices were carried out at 3 minute intervals according to a completely randomized schedule . The brain of each animal was removed and the right half of the teleacephaloa (cerebral cortex, striatum, hippocampal formation) and the brainetem were dissected. The braiaatem included that area of the brain from the superior colliculus aateriorly to the gracile nuclei poeteriorly. These brain regions were quick frozen on dry ice, weighed and stored at -55 ° C for 24-48 hours before analysis . Blood was collected from the cervical vessels and allowed to clot for a few minutes at 37 ° C . The blood samples were centrifuged at 1500 g and serum was collected. Brain 5-HT, tryptophaa, 5-hydroxyindoleacetic acid (5-HIAA) were extracted by a column chromatographic procedure (8). The content of brain 5-HT was determined by the method of Maickel et al . (9 ), Brain and also serum tryptophaa content were determined by the method of Lia et al . (10) . Brain 5-HIAA was quaatitated by the method of Curzon and Green (11) . The statistical significance of differences is the parameters after different Lilly 110140 pretreatmente or at different periods following tryptophaa administration was determined by a two way analysis of variance (ANOVA) for unequal samples sizes (12 ). Subsequent statistical analysis were performed
Brainstem
Vol. 17, No . 4
and
5-HT
525
5-HIAA
using a one way analysis of variance (13) or the Student's t test (14) . In order to simplify the presentation of the results only the most pertine~ statistical findings are presented. Results Although the administration of 25 mg/kg of L tryptophan significantly elevated tryptophan content in the telencephalon, the elevation was not sufficient to significantly elevate either 5-HT or 5-HIAA content in the teleacephalon of control or Lilly 110140-pretreated rate . Thus the presentation of results will be restricted to the brainetem and serum only. The intraperitoaeal administration of 25 mg/kg L tryptophan significantly elevated serum tryptophan content (p< 0. 001) (Table 1) . Lilly 110140 pretreatment had no effect on the elevation of serum tryptophan following i. p. tryptophan administration . In all three groups the peak is serum tryptophan was observed 30 minutes after tryptophan administration and by 90 minutes serum tryptophan had returned to 0 time levels . TABLE 1 The Effect ad Tryptophaa Administration oa Serum Tryptophan Content (Wg/ml) in Rats Pretreated with Lilly 110140 Treatment
Results of One Way ANOYA
Time 0
30
60
90
Saline
17 . 7 t0. 8 (11)
44 . 8 t 3. 4 (5)
31 . 2 f 2. 1 (7)
19 . 7 t 1. 3 (7)
p < 0. 001
Lilly 110140 1
18 . 9 f 1. 1 (8)
39 . 911 . 5 (81
30. 711 . 9 (8)
21 . O t 1 . 1 (9)
p < 0. 001
Lilly110140 2
18 .3t2 .4 (9)
41 .St2 .4 (6)
30 .Ot2 .1 (8)
19 .Ot1 .2 (8)
p<0 .001
The mean t standard error is given for each treatment group. The values read across the table are the mean values for the parameter observed in rats treated with saline or Lilly 110140 but sacrificed 0, 30, 60 or 90 minâtes after tryptophan administration . The number in parenthesis indicates the number of animals is the group. The statistical significance of changes in the parameter within the line is shown at the end of the line. Only those statistical differemes which are relevant to the discussion are presented. Similar elevations in brainetem tryptophan content (p< 0. 001) were observed following tryptophan administration (Table 2) . Lilly 110140 pretreat ment had ao effect on the elevation of tryptophan content in the brainstem. As with serum tryptophan, the highest levels of brainstem tryptophan content was observed 30 minutes following tryptophan administration, but brainstem
Brainstem 5-&T and 5-Hrnn
526
Vol . 17, No . 4
tryptophaa content was still significantly elevated (p< 0.001) over zero time levels 90 minutes after tryptophaa administration. TAB LE 2 The Effect of Tryptophaa Administration on Tryptophan Content (Wg/gm) in the Brainstem of Rate Pretreated with Lilly 110140 Treatment
Results of Oae Way AN OVA
Time 0
30
60
90
Saline
3. 26 t 0. 14 (11)
10 . 56 f 0 . 63 (5)
7 . 45 f 1 . 17 (7)
4. 35 t 0. 49 P< 0. 001 (7)
Lilly 110140 1
3. 45 f 0. 14 (8)
11 . 34 ~ 0. 39 (8)
6 . 66 f 0 . 27 (8)
4 . 72 f 0. 48 p < 0. 001 (9)
Lilly 110140 2
3. 65 f 0. 40 (9)
11 . 15 f 0. 55 (6)
8. 54 f 0. 45 (8)
5 . 06 f 0. 67 p < 0. 001 (8)
The mean t standard error is given for each treatment group. The values read across the table are the mean values for the parameter observed is rats treated with saline or Lilly 110140 but sacrificed 0, 30, 60 or 90 minutes after tryptophaa administration . The number is parenthesis indicates the number of animals in the group. The statistical significance of changes in the parameter within the line is shown at the end of the line . Only those statistical differences which are relevant to the discussion are presented. The administration of 25 mg/lcg tryptophaa significantly elevated braiastem 5-HT in both groups of Lilly 110140-pretreated rate (p<0 .005) but not in the saline controls (Table 3) . The Student's t teat showed that the level of 5-HT in the brainetem of the 3 hour Lilly 110140 pretreated rate was eigaificaatly elevated at both 60 and 90 minutes but only at 60 minutes in the 27 hour Lilly 110140-pretreated rats . Significant elevations in brainetem 5-HIA.A content were observed in the saline treated (p< 0.005) and the 3 hour and 27 hour Lilly 110140-pretreated rate (p< 0. 025) following the administration of 25 mg/kg L tryptophan (Table 4) . Brainstem 5-HIAA content in 3 hour Lilly 110140-pretreated rate was not eignlficantly elevated during the first 90 minutes following tryptophaa admiaistratioa. Comparable results were observed is 2 separate studies (experiment 1 and experiment 2, Table 4) . Diecuesion Bruimels (1) showed that imipramine, a tricyclic antidepressant compound which inhibits the reuptake of 5-HT and suppresses 5-HT turnover, also decreases the increase in 5-HT content Induced by tryptophaa administration.
Braiastem
Vol. 17, No . 4
$-$T
an8 5-HIAA
52 7
TABLE 3 The Effect of Tryptophan Administration on Serotonin Content (Wg/gm) in üie Brainetem of Rats Pretreated with Lilly 110140 Results of One Way ANOVA
Time
Treatment 0
30
Saline
0. 97
f 0.
Lilly 110140 1
0. 95
f 0. 02 (8)
Lilly 110140 2
0. 94
(10)
06
t 0. 05 (9)
1 . 20 t 0. 07 (5)
1 . 32 1 . 12
t 0. 11
(8)
f0. 06 (7)
60
90
1. 02 t 0. 05
0. 96
1 . 24 f0. 06 (8)P< 0.01
1 . 22 t 0. 06 (9) P< 0.01
(8)
t 0. 05 (7)
1 . 25 t 0. 06 1 . 05 f 0. 06 (8)p< 0.01 " (8)
N. 5 . p < 0. 005 p< 0. 005
The mean f standard error is given for each treatment group. The values read across the table are the mean values for the parameter observed in rate treated wilü saline or Lllly 110140 but sacrificed 0, 30, 60 or 90 minutes after tryptophan adminietr~tion . The number in parenthesis indicates the number of animals is the group. The statistical significance of changes is the parameter within the line is shown at the end of the line . Only those statistical differences which are relevant to ~e discussion are presented. The statistical significance (determined by the t teat) (14) of any mean within a column from the saline mean is the same column is designated by a p value under the appropriate mesa. N. S. = not significant . He concluded that imipramine inhibited the biosynthesis of 5-HT by inhibiting the uptake of tryptophan. A similar mechanism could ezplain the decrease in 5-HT synthesis and turnover observed after the administration of other inhibitors of 5-HT reuptake, i. e . Lilly 110140 . However, the results of the present study provide no support for the hypothesis that the Inhibition of 5-HT reuptake by Lilly 110140 produces a subsequent decrease in 5-HT synthesis and turnover by appreciably decreasing the uptake of tryptophan. The elevations of 5-HT content In the brainstem following the administration of 25 mg/kg tryptophsa appeared to be greater in the Lilly 110140 pretreated rats and were in fact only statistically significant in these animals . The increase is 5-HT synthesis in the saline treated controls following tryptophan administration was better reflected by tire significant elevations of 5-Hrea content in these animals . Since 5-HT reuptake was inhibited in the Lilly 110140 pretreated animals, the increased 5-HT in these groups were comparatively protected from iatraaeuronal degradation and 5-HLAA levels were initially lower and less elevated following tryptophaa administration .
528
Hraiast :e~ 5-HT and 5-BIAA
Vol . 17, No . 4
TABLE 4 The Effect of Tryptophan Administration on 5-Hydrozyiadoleactic Acid (Flg/gm) Content in the Brainstem of Rate Pretreated with Lilly 110140 Ezperiment 1 Treatment
Results of Oae Way ANOVA
Time 0
30
60
90
0. 71 t 0 . 04 (6)
0. 90 t 0. 02 (3)
1 . 12 t 0. 09 (7)
0. 88 f 0. 05 (6)
Lilly 110140
0. 58 f 0. O6 (8)
0. 82 f 0. 10 (8)
0. 72 t 0. 07 (5)p< 0.01
0. 72 t 0. 04 (8)p< 0 .025
Lilly 110140 2
0. 52 t 0. 03 0. 72 f 0. 07 (8)p< 0.005 (7)p< 0 .05
0 . 88 f 0. 07 (7)
0. ?6 f 0 . 12 (7)
Saline
1
p < 0. 00 5 N. S. p < 0. 025
Ezperimeat 2 Treatment
Results of One Way ANOVA
Time 0
30
60
Saline
0. 69 t 0, 03 (8)
0. 95 f 0. 11 (7)
1 . 04 f 0. 11
Lilly 110140 2
0 .50 t0 . 04 0.7110. 07 (7)p< 0.005 (7)
(8)
90
0. 93 f 0. 07 (8)
0. 6810 . 03 0, 6010 . 05 (8)p< 0, 005 (8)p< 0.005
p < 0. 05 p< 0. 025
The meaatstandard error ie given for each treatment group. The valuee read across the table are the mesa valuee for the parameter observed is rate treated with saline or Lilly 110140 but sacrificed 0, 30, 60 or 90 minutes after tryptophan administration. The number in parenthesis indicates the number of animals is ~e group. The statistical significance of changes in the parameter within the line ie shown at the end of the line . Only those statistical differences which are relevant to the discussion are presented . The statistical significance (determined by ~e t test) (14) of any mean within a column from the saline mesa in the same column is designated by a p value under the appropriate mean. N. S. = not significant. Experiment 2 was a repeat ezperiment. The elevation of braiastem 5-HT following tryptophan administration was more prolonged in the 3 hour than is the 3 hours sad 27 hour Lilly 110140 pretreated rats . These data suggest that the increased 5-HT was more rapidly removed from the brainsteme of the rate pretreated with 2 injections of Lilly 110140, one at 27 hours sad a second at 3 hours before sacrifice . During the 27 hour period the brain may have adapted to remove the elevated eatraneuronal
Hrainstem
Vol. 17, No . 4
5-AT and 5-HiAA
529
5-HT . It is unlikely that 5-HT is the 3 hour and 27 hour Lilly 110140-pretreated rats ie being more rapidly metabolized to 5-HTAA since 5-HTAA levels in the 2 groups of Lilly pretreated rats are not eignülcantly different. It is unlikely that the level of Lilly 110140 was any lower in the animals which received 2 injections of die drug since they also received a second injection of Lilly 110140 3 hours before eacrüice . However, an increase in Lilly 110140 clearance from these animals ca~ot be ezcluded. These data ehaav that the inhibition of 5-HT reuptake by Lilly 110140 pretreatment does not affect the ability of intraperitoneally administered tryptophan to increase 5-HT synthesis. Ia a related study Jacoby et al . (15) observed that chloroimipramiae, another drug which inhibits 5-HTreuptake and also decreases 5-HT synthesis, did not inhibit an increase in brain tryptophan and 5-HIAA induced by fasting. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
J. BRUINVELS, Eur. J. Pharmacol. 20 231-237 (1972) . R. W. FULLER, W. PERRY, and B . B. MOLLOY, Lüe Sçi. _15 1161-1171 (1974). G. K. AGHAJANIAN, B . S. BUNNEY, and M. J. KUHAR, New Concepts is Neurotransmitter Regulation , A. J. Mande]1 editor, pp " 115-134 (1973) . J. D. FERNSTROM and R. J. WURTMAN, Science 173 149-152 (1971) . E. JEQUIER, W. LOVENBERG, and A. SJOERDSMA, Mol. Pharmacol. 3 275-278 (1967) . P. A. FRIEDMAN, A. H. KAPPELMAN, and S. KAUFMAN, J. Biol . Chem . 242 4165-4173 (1972) . D. T. WONG, J. S. HORNG, F. P. BYMASTER, K. L. HAUSER, and B. B. MOLLOY, Life Séi. 15 471-479 (1974) . F. HERY, E. ROUER, and J. GLOWiNSKI, Brain Res. _43 445-465 (1972) . R. P. MAICKEL, R. H. COX, JR . , J. SAILLANT, and F. P. MILLER, Neuropharmacology _7 275-281 (1968) . R. C. LIN, E. COSTA, N. H. NEFF, C. T. WANG, and S. A. NGAI, J. Pharmacol. ~. Ther. _170 232-238 (1969) . G. CURZON and A. R. GREEN, Br . J. Pharmacol. 39 653-655 (1970) . D. J. VELDMAN, Fortran Progra**,* _~r,o _for the Behavioral Sciences, page 257-268, Holt, Rinehart and Wineton, New York (1967) . R. l~. SOKAL and F. J. ROHLF, Biometry , pp . 208-209 . W . H. Freema n and Compa~r, San Francisco (1969) . R. R. SOKAL and F. J. ROHLF, Biometry, pp . 222-223, W. H. Freeman and Company, San Francisco (1969) . J. H. JACOBY, J. Z. COLMENARES, and R. J. WURTMAN, _Fed . Proc . 33 562 (1974) .
K.