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The effect of acute raphe lesion on serotonin synthesis and metabolism in the rat forebrain and hippocampus
Brain Research, 110 (1976) 189-193
189
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The effect of acute raphe lesion on serotonin synthesis and metabolism in the rat forebrain and hippocampus
BETTY E. H E R R AND ROBERT H. ROTH
Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.) (Accepted March 16th, 1976)
Previous studies provide indirect evidence which ~uggcsts that the decrease in brain serotonin turnover observed after administration of some hallucinogenic drugs may be secondary to an inhibition of neuronal impulse flow within ascending central serotonergic neurons. For example, using extracellular recording techniques, it has been shown that D-lysergic acid diethylamide (LSD) and N,N-dimethyltryptamine (DMT) inhibit the spontaneous firing of serotonergic neurons located in the midbrain raphe nuclei z. These drugs have also been shown to decrease brain serotonin turnover 3,13,16.
In addition, there is a growing body of direct evidence demonstrating that changes in neuronal impulse flow within serotonergic neurons can alter serotonin synthesis. Direct electrical stimulation of central serotonergic neurons at or near their origin in the midbrain raphe nuclei results in increased synthesis of serotonin in the forebrain 17. Conversely, acute transection of the spinal cord which interrupts impulse flow within descending serotonergic and other monoamine fibers decreases serotonin synthesis in the distal portion of that tissue 6. Within the brain, however, a direct relationship between interruption of impulse flow and serotonin synthesis has not been demonstrated convincingly4, 6. Previous investigators have used a brain hemisection as a model for interruption of impulse flow. However brain hemisection as well as spinal cord transection are not specific techniques since both non-serotonergic as well as serotonin-containing neurons are severed. Biochemical studies have shown that serotonergic terminals in the hippocampus are projections of neurons whose perikarya are located almost exclusively within the median midbrain raphe nuclei s. Cell bodies of serotonergic neurons which project to other parts of the forebrain are located primarily, but not exclusively, within the dorsal and median midbrain rapheS, 1°. Thus, after total midbrain raphe lesion one would expect a complete and specific cessation of firing within the serotonergic neurons with projections to the hippocampus, while a few serotonergic neurons with terminals in other parts of the forebrain would continue to carry impulses. It is assumed, furthermore, that within the first few hours after raphe lesion the biochemical apparatus of the neurons remains intact, since the half life for the disappearance
190 of tryptophan hydroxylase after raphe lesion is about two days 1~ and it takes at~oul the same time for anatomical signs of neuronal degeneration to be observed ~. In these experiments, parameters of serotonin synthesis and metabolism were measured in the hippocampus and remaining forebrain 1-3 h after placement of lesions in the midbrain dorsal and median raphe nuclei. These acute raphe lesions provide a specific model for interruption of impulse flow within otherwise intact serotonergic neurons, especially those which project to the hippocampus. Male Sprague-Dawley rats, 230 280 g, were anesthetized with halothane and cathodal lesions were placed in the midbrain raphe nuclei by means of the technique described by Kuhar et al.l~. 1n one group of experiments radiofrequency lesions were made with a Grass LM4 lesion maker (10-15 mA, 20 sec at each electrode position). Sham-operated rats were prepared in the same manner as lesioned animals but ilo current was passed. Animals were decapitated 1-3 h after the lesion and the forebrain was separated from the brain stem by a cut passing from the rostral border of the superior colliculi to the caudal border of the hypothalamus. In some cases the two hippocampi, including the subiculum and dentate gyrus, were dissected from the rest of the forebrain. The brain stem, with attached cerebellum, was fixed in 5 "/,ogluteraldehyde-0.9',,'~il NaCI solution, then sectioned and stained with cresyl violet for precise histological localization of" the lesion. Serotonin in the hippocampi and remaining forebrain was extracted by the method of Bogdanski el al. a and measured using ninhydrin reagent 18. Recoveries of added serotonin were 79 ~lilin the forebrain and 68 o/in the hippocampus. 5-Hydroxyindoleacetic acid (5-HIAA) in the hippocampi and remaining forebrain was determined after ether extraction and reaction with o-phthaldialdehyde (OPT) it as modified by Korf and Valkenburgh-Sikkema 9. After reaction with OPT, tubes were washed with chloroform to remove precipitated OPT 19. Recovery of added 5-HIAA was 77,6 i 1.4~,, (in -=: 6). The accumulation of [aH]tryptophan and newly synthesized [aH]serotonin was determined in the whole forebrain including hippocampus after a 20 rain tail vein infusion with tracer amounts of [aH]tryptophan (5/~Ci/min). It has been shown previously that this technique gives a good approximation of relative rates of serotonin synthesis lz,16. [aH]Serotonin was separated from [aH]tryptophan by a modification of the column method of Minard and Grant 15. Extracts of whole forebrain were placed on 25 mm ~ 6 mm (i.d.) Amberlite CG-50 (100-200 mesh) columns previously equilibrated with 0.4 M sodium phosphate buffer pH 6.0. Nearly 100 °/~i of the tryptophan was recovered in the effluent plus 1.5 ml of sodium phosphate buffer wash. [aH]Tryptophan was measured by liquid scintillation counting techniques and total tryptophan was measured after conversion to norharman by the method of DenckIa and Dewey 7. Columns were washed with 20.0 ml of water and 7,0 ml of 2'),,~ boric acid. Serotonin was eluted from the columns in 2.5 ml of 0.5 N H C l containing cysteine (1 mg/ml). A 1.5 ml aliquot was used for determination of [aH]serotonin and a 0.5 ml aliquot was used for fluorometric determination of total serotonin alter reaction with OPT and chloroform wash. Recovery of serotonin was approximately 98 °il. Results in Fig. 1 show that there was no significant change in levels of endoge-
FOREBRAIN
HIPPOCAMPUS
•36 F g / g , v / / / /
100
I
2
/ / / /
e¢:
/ / / /
I / I /
/ / / /
/ / / /
Y/½
o o
I
/ / i / / / / /
I / / /
SHAM RAPHE'
/ / / / / / / / / / / /
/ / / / / / / / / / / /
/
LESION
/ / / / / / / /
/ / 1 1
o
v
50
./J/ / / / / I / . / I l l .
¢//.
/ / / /
p. "rIO
. / . I 1 / 1 1
/ / / /
iJ
"///
¢//,
~/// ~/// ¢ / / /
/ / / / / / / /
.%'.'2 N
®N.
®N
I Hour
1 Hour
2 Hours
Fig. 1. Effect of acute raphe lesions on endogenous serotonin in the forebrain and hippocampus. Electrolytic lesions were placed in the raphe nuclei 1 or 2 h before sacrifice. The forebrain and hippocampus were analyzed for content of serotonin as described in Methods. The vertical bars represent the standard error of the mean. The number of experiments is indicated at the bottom of each column. FOREBRAIN
H IPPOCAMPUS 152%
CONTROL
0.60 ,//J ,//,
+113% 0.50
17721 PROBENECID
t//, ,//,
,/// ,11,
0.40 ::L +75% "I"
0.30
r.-// ,/// r/z1 ¢//t
¢//J
¢///
;///,
/// //i f//
I0
+59%
r/¢~, //is r//., ///s 1//1 r/i.~
/// ///
0.20
r//., t/// ~-///
0. I 0
,'/// ,-//l r#/~ ~'///
®N Sham Lesion
Raphe' Lesion
Sham Lesion
Rophe' Lesion
Fig. 2. Probenecid-induced accumulation of 5-H1AA in the forebrain and hippocampus after acute lesion. Rats were sacrificed 3 h after placement of electrolytic lesions in the raphe nuclei. The hippocampi and remaining forebrain were analyzed separately for 5-HIAA as described in Methods. Some animals were injected with probenecid (200 mg/kg, i.p.) 1 h prior to decapitation. Vertical bars represent the standard error of the mean. The number of experiments is indicated at the bottom of each column. When hippocampal 5-HIAA is compared in control and probenecid-pretreated raphe lesioned rats, P < 0.02. In all other cases, values for probenecid-treated animals are significantly different from corresponding controls, P -< 0.001. Control forebrain 5-H IAA is significantly reduced in raphe-lesioned compared to sham-lesioned rats, P < 0.005.
192 nous serotonin in the hippocampus 1 and 2 h after raphe lesion or in the serotonin content of the remaining forebrain 1 h after lesion. Three hours after raphe lesion baseline levels of 5-HIAA were reduced in both the forebrain and hippocampus; however, the decrease in 5-HIAA was statistically significant only in the forebrain (Fig. 2). In sham-operated rats probenecid (200 mg/kg, i.p.) injected 1 h before sacrifice caused a 132 °,o increase in hippocampal 5-HIAA levels, while in rats with raphe lesion pFobenecid caused only a 59 '}i, increase in 5-HIAA. In the remaining forebrain probenecid caused a 113 °/ooincrease in 5-HIAA in sham-operated rats, while in rats with raphe lesions the increase was only 75"~,i. Table I shows that after a 20 min tail vein infusion with tracer amounts of [3H]tryptophan (5/,Ci/min) the accumulation of newly synthesized [aH]serotonin in the whole forebrain was reduced by 33 % in rats with acute raphe lesions as compared to sham-operated rats. Accumulation of [aH]tryptophan was the same in both groups of rats. In addition, after an acute raphe lesion, endogenous levels of tryptophan and serotonin were unchanged. In summary, these results show that acute lesioning of the midbrain raphe nuclei had no effect on steady state levels of endogenous serotonin in either the hippocampus or forebrain (Fig. 1, Table I). The dynamics of serotonin synthesis and metabolism was altered significantly by this procedure however. Acute raphe lesion caused a 33 °,'o reduction in forebrain serotonin synthesis as determined by measuring the accumulation of newly synthesized serotonin after a 20 rain tail vein infusion with tracer amounts of [aH]tryptophan. This reduction in serotonin synthesis was not associated with any alteration in endogenous tryptophan or accumulation of [aH]tryptophan precursor (Table I). There was a similar inhibition in the metabolism of serotonin in the forebrain while in the hippocampus the inhibition was
TABLE I EFFECT OF ACUTE RAPHE LESION ON ACCUMULATION OF [3H]TRYPTOPHAN AND NEWLY SYNTHESIZED [3H]SEROTONIN IN THE RAT FOREBRAIN IN VIVO AFTER INFUSION OF [3H]TRYPTOPHAN
Rats with radiofrequency or sham lesions placed in the midbrain raphe nuclei 1-3 h earlier were anesthetized with chloral hydrate (400 mg/kg, i,p.) and infused through a tail vein for 20 min with a total of 1.5 ml of 0.9~ NaCI containing [aH]tryptophan (100/~Ci). Forebrains were analyzed for endogenous and labeled tryptophan and serotonin as described in methods. Sham lesion
Tryptophan TryptophanLug/g) [3H]Tryptophan (disint./min/g/20min x 10-3) Serotonin Serotonin Lug/g) [3HlSerotonin (disint./min/g/20 rain >." 10 a)
6.0 ~ 0.4 389.0 kF 14.0
Acute raphe lesion
6.6 i
% change
0.5
418.0 ± 19.0
0.36 34z 0.01
0.39 :k 0.01
3.13 ± 0.21
2.10 ± 0.13"
* Significantly different from sham-lesioned controls, P < 0.001.
--33
193 somewhat greater. In the forebrain acute raphe lesion caused a 30 ~ reduction in the probenecid-induced accumulation of 5-HIAA compared to 55 ~o in the hippocampus (Fig. 2). This finding is consistent with the fact that almost all of the serotonin in the hippocampus is derived from neurons with cell bodies located within the midbrain raphe, while some of the serotonin in other parts of the forebrain is derived from neurons located outside the midbrain raphe s,l°. These experiments thus establish a direct cause and effect relationship between interruption of impulse flow in central serotonergic neurons and a decrease in serotonin turnover. Since similar changes in serotonin metabolism are observed after acute raphe lesioning and treatment with drugs which inhibit raphe cell firinga,la, 16, the data suggest that these changes in serotonin turnover may be due to a common mechanism. The molecular mechanism responsible for this decrease in serotonin turnover following acute interruption of impulse flow is currently under investigation 1 AGHAJANIAN, G. K., BLOOM, E. E., AND SHEARD, M. H., Electron microscopy of degeneration within the serotonin pathway of rat brain, Brain Research, 13 (1969) 266-273. 2 AGHAJANIAN,G. K., FOOTE, W. E., AND SHEARD, M. H., Action of psychotogenic drugs on single midbrain raphe neurons, J. Pharmacol. exp. Ther., 171 (1970) 178-187. 3 ANDEN, N. E., CORRODI, H., AND FUXE, K., Hallucinogenic drugs of the indolealkylamine type and central monoamine neurons, J. Pharmacol. exp. Ther., 179 (1971) 236-249. 4 BEDARD, P., CARESSON, A., AND LINDQVIST, M., Effect of a hemisection on 5-hydroxytryptamine metabolism in the rat brain, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 272 (1972) 1 15. 5 BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B., AND UDENFRIEND,S., Identification and assay of serotonin in brain, J. Pharmacol. exp. Ther., 117 0956) 82-88. 6 CARLSSON, A., BEDARD, P., LINDQVIST, M., AND MAGNUSSON, T., The influence of nerve-impulse flow in the synthesis and metabolism of 5-hydroxytryptamine in the central nervous system, Bioehem. Soc. Syrup., 36 (1972) 17-32. 7 DENCKLA, W. D., AND DEWEY, H. K., The determination of tryptophan in plasma, liver, and urine, J. Lab. clin. Med., 69 (1967) 160-169. 8 JACOBS,B. L., WISE, W. D., AND TAYLOR, K. M., Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats, Brain Research, 79 (1974) 353 361. 9 KORF, J., AND VALKENBURGH-SIKKEMA,T., Fluorometric determination of 5-hydroxyindoleacetic acid in h u m a n urine and cerebrospinal fluid, Clin. chim. Acta, 26 (1969) 301 306. 10 KUHAR, M. J., AGHAJANIAN, G. K., AND ROTH, R. H., Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesions : correlations with serotonin levels and histochemical fluorescence, Brain Research, 44 (1972) 165-176. II KUHAR, M. J., ROTH, R. H., AND AGHAJANIAN, G. K., Selective reduction of tryptophan hydroxylase activity in rat forebrain after midbrain raphe lesions, Brain Research, 35 (1971) 167-176. 12 LIN, R. C., COSTA, E., NEFF, N. H., WANG, C. T., AND NGAI, S. H., In vivo measurement of 5hydroxytryptamine turnover rate in the rat brain from the conversion of C 14 tryptophan to C TM5hydroxytryptamine, J. Pharmacol. exp. Ther., 170 (1969) 232-238. 13 LIN, R. C., NGA1, S. H., AND COSTA, E., Lysergic acid diethylamine: role in conversion of plasma tryptophan to brain serotonin (5-hydroxytryptamine), Science, 166 (1969) 237-239. 14 MAICKEL,R. P., AND MILLER, F. P., Fluorescent products formed by reaction of indole derivatives and o-phthalaldehyde, Analyt. Chem., 38 (1966) 1937-1938. 15 MINARD, F. N., AND GRANT, D. S., A convenient method for the chromatographic analysis of norepinephrine, dopamine and serotonin, Biochem. Med., 6 (1972) 46-52. 16 SCHUBERT,J., NYB.~CK, H., AND SEDVALL, G., Accumulation and disappearance of [aH]5-hydroxytryptamine formed from [aH]tryptophan in mouse brain - - effect of LSD-25, Europ. J. Pharmacol., l0 (1970) 215 224. 17 SHIELDS, P. J., AND ECCEESTON, D., Effect of electrical stimulation of rat midbrain on 5-hydroxytryptamine synthesis as determined by a sensitive radioisotope method, J. Neurochem., 19 (1972) 265-272. [ 8 SNYDER, S. H., AXELROD, J., AND ZWEIG, M., A sensitive and specific fluorescence assay for tissue serotonin, Biochem, Pharmacok, 14 (1965) 831-835. 19 THOMPSON, J., SPEZIA, C. A., AND ANGULO, M., FIuorometric detection of serotonin using o-phthaldialdehyde: an improvement, Experientia (Basel), 26 (1967) 327 329.
189
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The effect of acute raphe lesion on serotonin synthesis and metabolism in the rat forebrain and hippocampus
BETTY E. H E R R AND ROBERT H. ROTH
Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.) (Accepted March 16th, 1976)
Previous studies provide indirect evidence which ~uggcsts that the decrease in brain serotonin turnover observed after administration of some hallucinogenic drugs may be secondary to an inhibition of neuronal impulse flow within ascending central serotonergic neurons. For example, using extracellular recording techniques, it has been shown that D-lysergic acid diethylamide (LSD) and N,N-dimethyltryptamine (DMT) inhibit the spontaneous firing of serotonergic neurons located in the midbrain raphe nuclei z. These drugs have also been shown to decrease brain serotonin turnover 3,13,16.
In addition, there is a growing body of direct evidence demonstrating that changes in neuronal impulse flow within serotonergic neurons can alter serotonin synthesis. Direct electrical stimulation of central serotonergic neurons at or near their origin in the midbrain raphe nuclei results in increased synthesis of serotonin in the forebrain 17. Conversely, acute transection of the spinal cord which interrupts impulse flow within descending serotonergic and other monoamine fibers decreases serotonin synthesis in the distal portion of that tissue 6. Within the brain, however, a direct relationship between interruption of impulse flow and serotonin synthesis has not been demonstrated convincingly4, 6. Previous investigators have used a brain hemisection as a model for interruption of impulse flow. However brain hemisection as well as spinal cord transection are not specific techniques since both non-serotonergic as well as serotonin-containing neurons are severed. Biochemical studies have shown that serotonergic terminals in the hippocampus are projections of neurons whose perikarya are located almost exclusively within the median midbrain raphe nuclei s. Cell bodies of serotonergic neurons which project to other parts of the forebrain are located primarily, but not exclusively, within the dorsal and median midbrain rapheS, 1°. Thus, after total midbrain raphe lesion one would expect a complete and specific cessation of firing within the serotonergic neurons with projections to the hippocampus, while a few serotonergic neurons with terminals in other parts of the forebrain would continue to carry impulses. It is assumed, furthermore, that within the first few hours after raphe lesion the biochemical apparatus of the neurons remains intact, since the half life for the disappearance
190 of tryptophan hydroxylase after raphe lesion is about two days 1~ and it takes at~oul the same time for anatomical signs of neuronal degeneration to be observed ~. In these experiments, parameters of serotonin synthesis and metabolism were measured in the hippocampus and remaining forebrain 1-3 h after placement of lesions in the midbrain dorsal and median raphe nuclei. These acute raphe lesions provide a specific model for interruption of impulse flow within otherwise intact serotonergic neurons, especially those which project to the hippocampus. Male Sprague-Dawley rats, 230 280 g, were anesthetized with halothane and cathodal lesions were placed in the midbrain raphe nuclei by means of the technique described by Kuhar et al.l~. 1n one group of experiments radiofrequency lesions were made with a Grass LM4 lesion maker (10-15 mA, 20 sec at each electrode position). Sham-operated rats were prepared in the same manner as lesioned animals but ilo current was passed. Animals were decapitated 1-3 h after the lesion and the forebrain was separated from the brain stem by a cut passing from the rostral border of the superior colliculi to the caudal border of the hypothalamus. In some cases the two hippocampi, including the subiculum and dentate gyrus, were dissected from the rest of the forebrain. The brain stem, with attached cerebellum, was fixed in 5 "/,ogluteraldehyde-0.9',,'~il NaCI solution, then sectioned and stained with cresyl violet for precise histological localization of" the lesion. Serotonin in the hippocampi and remaining forebrain was extracted by the method of Bogdanski el al. a and measured using ninhydrin reagent 18. Recoveries of added serotonin were 79 ~lilin the forebrain and 68 o/in the hippocampus. 5-Hydroxyindoleacetic acid (5-HIAA) in the hippocampi and remaining forebrain was determined after ether extraction and reaction with o-phthaldialdehyde (OPT) it as modified by Korf and Valkenburgh-Sikkema 9. After reaction with OPT, tubes were washed with chloroform to remove precipitated OPT 19. Recovery of added 5-HIAA was 77,6 i 1.4~,, (in -=: 6). The accumulation of [aH]tryptophan and newly synthesized [aH]serotonin was determined in the whole forebrain including hippocampus after a 20 rain tail vein infusion with tracer amounts of [aH]tryptophan (5/~Ci/min). It has been shown previously that this technique gives a good approximation of relative rates of serotonin synthesis lz,16. [aH]Serotonin was separated from [aH]tryptophan by a modification of the column method of Minard and Grant 15. Extracts of whole forebrain were placed on 25 mm ~ 6 mm (i.d.) Amberlite CG-50 (100-200 mesh) columns previously equilibrated with 0.4 M sodium phosphate buffer pH 6.0. Nearly 100 °/~i of the tryptophan was recovered in the effluent plus 1.5 ml of sodium phosphate buffer wash. [aH]Tryptophan was measured by liquid scintillation counting techniques and total tryptophan was measured after conversion to norharman by the method of DenckIa and Dewey 7. Columns were washed with 20.0 ml of water and 7,0 ml of 2'),,~ boric acid. Serotonin was eluted from the columns in 2.5 ml of 0.5 N H C l containing cysteine (1 mg/ml). A 1.5 ml aliquot was used for determination of [aH]serotonin and a 0.5 ml aliquot was used for fluorometric determination of total serotonin alter reaction with OPT and chloroform wash. Recovery of serotonin was approximately 98 °il. Results in Fig. 1 show that there was no significant change in levels of endoge-
FOREBRAIN
HIPPOCAMPUS
•36 F g / g , v / / / /
100
I
2
/ / / /
e¢:
/ / / /
I / I /
/ / / /
/ / / /
Y/½
o o
I
/ / i / / / / /
I / / /
SHAM RAPHE'
/ / / / / / / / / / / /
/ / / / / / / / / / / /
/
LESION
/ / / / / / / /
/ / 1 1
o
v
50
./J/ / / / / I / . / I l l .
¢//.
/ / / /
p. "rIO
. / . I 1 / 1 1
/ / / /
iJ
"///
¢//,
~/// ~/// ¢ / / /
/ / / / / / / /
.%'.'2 N
®N.
®N
I Hour
1 Hour
2 Hours
Fig. 1. Effect of acute raphe lesions on endogenous serotonin in the forebrain and hippocampus. Electrolytic lesions were placed in the raphe nuclei 1 or 2 h before sacrifice. The forebrain and hippocampus were analyzed for content of serotonin as described in Methods. The vertical bars represent the standard error of the mean. The number of experiments is indicated at the bottom of each column. FOREBRAIN
H IPPOCAMPUS 152%
CONTROL
0.60 ,//J ,//,
+113% 0.50
17721 PROBENECID
t//, ,//,
,/// ,11,
0.40 ::L +75% "I"
0.30
r.-// ,/// r/z1 ¢//t
¢//J
¢///
;///,
/// //i f//
I0
+59%
r/¢~, //is r//., ///s 1//1 r/i.~
/// ///
0.20
r//., t/// ~-///
0. I 0
,'/// ,-//l r#/~ ~'///
®N Sham Lesion
Raphe' Lesion
Sham Lesion
Rophe' Lesion
Fig. 2. Probenecid-induced accumulation of 5-H1AA in the forebrain and hippocampus after acute lesion. Rats were sacrificed 3 h after placement of electrolytic lesions in the raphe nuclei. The hippocampi and remaining forebrain were analyzed separately for 5-HIAA as described in Methods. Some animals were injected with probenecid (200 mg/kg, i.p.) 1 h prior to decapitation. Vertical bars represent the standard error of the mean. The number of experiments is indicated at the bottom of each column. When hippocampal 5-HIAA is compared in control and probenecid-pretreated raphe lesioned rats, P < 0.02. In all other cases, values for probenecid-treated animals are significantly different from corresponding controls, P -< 0.001. Control forebrain 5-H IAA is significantly reduced in raphe-lesioned compared to sham-lesioned rats, P < 0.005.
192 nous serotonin in the hippocampus 1 and 2 h after raphe lesion or in the serotonin content of the remaining forebrain 1 h after lesion. Three hours after raphe lesion baseline levels of 5-HIAA were reduced in both the forebrain and hippocampus; however, the decrease in 5-HIAA was statistically significant only in the forebrain (Fig. 2). In sham-operated rats probenecid (200 mg/kg, i.p.) injected 1 h before sacrifice caused a 132 °,o increase in hippocampal 5-HIAA levels, while in rats with raphe lesion pFobenecid caused only a 59 '}i, increase in 5-HIAA. In the remaining forebrain probenecid caused a 113 °/ooincrease in 5-HIAA in sham-operated rats, while in rats with raphe lesions the increase was only 75"~,i. Table I shows that after a 20 min tail vein infusion with tracer amounts of [3H]tryptophan (5/,Ci/min) the accumulation of newly synthesized [aH]serotonin in the whole forebrain was reduced by 33 % in rats with acute raphe lesions as compared to sham-operated rats. Accumulation of [aH]tryptophan was the same in both groups of rats. In addition, after an acute raphe lesion, endogenous levels of tryptophan and serotonin were unchanged. In summary, these results show that acute lesioning of the midbrain raphe nuclei had no effect on steady state levels of endogenous serotonin in either the hippocampus or forebrain (Fig. 1, Table I). The dynamics of serotonin synthesis and metabolism was altered significantly by this procedure however. Acute raphe lesion caused a 33 °,'o reduction in forebrain serotonin synthesis as determined by measuring the accumulation of newly synthesized serotonin after a 20 rain tail vein infusion with tracer amounts of [aH]tryptophan. This reduction in serotonin synthesis was not associated with any alteration in endogenous tryptophan or accumulation of [aH]tryptophan precursor (Table I). There was a similar inhibition in the metabolism of serotonin in the forebrain while in the hippocampus the inhibition was
TABLE I EFFECT OF ACUTE RAPHE LESION ON ACCUMULATION OF [3H]TRYPTOPHAN AND NEWLY SYNTHESIZED [3H]SEROTONIN IN THE RAT FOREBRAIN IN VIVO AFTER INFUSION OF [3H]TRYPTOPHAN
Rats with radiofrequency or sham lesions placed in the midbrain raphe nuclei 1-3 h earlier were anesthetized with chloral hydrate (400 mg/kg, i,p.) and infused through a tail vein for 20 min with a total of 1.5 ml of 0.9~ NaCI containing [aH]tryptophan (100/~Ci). Forebrains were analyzed for endogenous and labeled tryptophan and serotonin as described in methods. Sham lesion
Tryptophan TryptophanLug/g) [3H]Tryptophan (disint./min/g/20min x 10-3) Serotonin Serotonin Lug/g) [3HlSerotonin (disint./min/g/20 rain >." 10 a)
6.0 ~ 0.4 389.0 kF 14.0
Acute raphe lesion
6.6 i
% change
0.5
418.0 ± 19.0
0.36 34z 0.01
0.39 :k 0.01
3.13 ± 0.21
2.10 ± 0.13"
* Significantly different from sham-lesioned controls, P < 0.001.
--33
193 somewhat greater. In the forebrain acute raphe lesion caused a 30 ~ reduction in the probenecid-induced accumulation of 5-HIAA compared to 55 ~o in the hippocampus (Fig. 2). This finding is consistent with the fact that almost all of the serotonin in the hippocampus is derived from neurons with cell bodies located within the midbrain raphe, while some of the serotonin in other parts of the forebrain is derived from neurons located outside the midbrain raphe s,l°. These experiments thus establish a direct cause and effect relationship between interruption of impulse flow in central serotonergic neurons and a decrease in serotonin turnover. Since similar changes in serotonin metabolism are observed after acute raphe lesioning and treatment with drugs which inhibit raphe cell firinga,la, 16, the data suggest that these changes in serotonin turnover may be due to a common mechanism. The molecular mechanism responsible for this decrease in serotonin turnover following acute interruption of impulse flow is currently under investigation 1 AGHAJANIAN, G. K., BLOOM, E. E., AND SHEARD, M. H., Electron microscopy of degeneration within the serotonin pathway of rat brain, Brain Research, 13 (1969) 266-273. 2 AGHAJANIAN,G. K., FOOTE, W. E., AND SHEARD, M. H., Action of psychotogenic drugs on single midbrain raphe neurons, J. Pharmacol. exp. Ther., 171 (1970) 178-187. 3 ANDEN, N. E., CORRODI, H., AND FUXE, K., Hallucinogenic drugs of the indolealkylamine type and central monoamine neurons, J. Pharmacol. exp. Ther., 179 (1971) 236-249. 4 BEDARD, P., CARESSON, A., AND LINDQVIST, M., Effect of a hemisection on 5-hydroxytryptamine metabolism in the rat brain, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 272 (1972) 1 15. 5 BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B., AND UDENFRIEND,S., Identification and assay of serotonin in brain, J. Pharmacol. exp. Ther., 117 0956) 82-88. 6 CARLSSON, A., BEDARD, P., LINDQVIST, M., AND MAGNUSSON, T., The influence of nerve-impulse flow in the synthesis and metabolism of 5-hydroxytryptamine in the central nervous system, Bioehem. Soc. Syrup., 36 (1972) 17-32. 7 DENCKLA, W. D., AND DEWEY, H. K., The determination of tryptophan in plasma, liver, and urine, J. Lab. clin. Med., 69 (1967) 160-169. 8 JACOBS,B. L., WISE, W. D., AND TAYLOR, K. M., Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats, Brain Research, 79 (1974) 353 361. 9 KORF, J., AND VALKENBURGH-SIKKEMA,T., Fluorometric determination of 5-hydroxyindoleacetic acid in h u m a n urine and cerebrospinal fluid, Clin. chim. Acta, 26 (1969) 301 306. 10 KUHAR, M. J., AGHAJANIAN, G. K., AND ROTH, R. H., Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesions : correlations with serotonin levels and histochemical fluorescence, Brain Research, 44 (1972) 165-176. II KUHAR, M. J., ROTH, R. H., AND AGHAJANIAN, G. K., Selective reduction of tryptophan hydroxylase activity in rat forebrain after midbrain raphe lesions, Brain Research, 35 (1971) 167-176. 12 LIN, R. C., COSTA, E., NEFF, N. H., WANG, C. T., AND NGAI, S. H., In vivo measurement of 5hydroxytryptamine turnover rate in the rat brain from the conversion of C 14 tryptophan to C TM5hydroxytryptamine, J. Pharmacol. exp. Ther., 170 (1969) 232-238. 13 LIN, R. C., NGA1, S. H., AND COSTA, E., Lysergic acid diethylamine: role in conversion of plasma tryptophan to brain serotonin (5-hydroxytryptamine), Science, 166 (1969) 237-239. 14 MAICKEL,R. P., AND MILLER, F. P., Fluorescent products formed by reaction of indole derivatives and o-phthalaldehyde, Analyt. Chem., 38 (1966) 1937-1938. 15 MINARD, F. N., AND GRANT, D. S., A convenient method for the chromatographic analysis of norepinephrine, dopamine and serotonin, Biochem. Med., 6 (1972) 46-52. 16 SCHUBERT,J., NYB.~CK, H., AND SEDVALL, G., Accumulation and disappearance of [aH]5-hydroxytryptamine formed from [aH]tryptophan in mouse brain - - effect of LSD-25, Europ. J. Pharmacol., l0 (1970) 215 224. 17 SHIELDS, P. J., AND ECCEESTON, D., Effect of electrical stimulation of rat midbrain on 5-hydroxytryptamine synthesis as determined by a sensitive radioisotope method, J. Neurochem., 19 (1972) 265-272. [ 8 SNYDER, S. H., AXELROD, J., AND ZWEIG, M., A sensitive and specific fluorescence assay for tissue serotonin, Biochem, Pharmacok, 14 (1965) 831-835. 19 THOMPSON, J., SPEZIA, C. A., AND ANGULO, M., FIuorometric detection of serotonin using o-phthaldialdehyde: an improvement, Experientia (Basel), 26 (1967) 327 329.