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Synaptosomal uptake and levels of serotonin in rat brain areas after p-chloroamphetamine or B-9 lesions
European Journal o f Pharmacology, 33 (1975) 419--422 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication S Y N A P T O S O M A L U P T A K E A N D L E V E L S O F S E R O T O N I N IN R A T B R A I N A R E A S A F T E R p - C H L O R O A M P H E T A M I N E O R B-9 LESIONS V. J O H N M A S S A R I and E L A I N E S A N D E R S - B U S H
Department o f Pharmacology, Vanderbilt University School o f Medicine, and Tennessee Neuropsychiatric Institute, Nashville, Tennessee, U.S.A. Received 1 August 1975, accepted 6 August 1975
V.J. MASSARI and E. SANDERS-BUSH, Synaptosomal uptake and levels o f serotonin in rat brain areas after p-chloroamphetamine or B-9 lesions, European J. Pharmacol. 33 (1975) 419--422. In contrast to the pronounced fall in 5HT levels and synaptosomal uptake caused by p-chloroamphetamine, bilateral lesions of the B-9 cell group caused minimal regional changes, except for 35% decreases in the metathalamus--thalamus. We conclude therefore that the prolonged biochemical effects of p-chloroamphetamine are not due to a selective cytotoxic action on B-9 cells; and that a lateral 5HT pathway, possibly from the B-9 cell group, projects to the metathalamus--thalamus. p-Chloroamphetamine
B-9 lesions
Serotonin
1. Introduction After the administration of a single i.p. dose of p-chloroamphetamine (PCA) to rats, a prompt and prolonged reduction in brain levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid occurs. Reductions in the activity of tryptophan hydroxylase and in the high affinity uptake of 5HT were also found (Sanders-Bush et al., 1972, 1975). This spectrum of effects is analogous to those caused by the intraventricular administration of the neurotoxic agents, 5,6- and 5,7-dihydroxytryptamine (Bj6rklund et al., 1974). Recently, Harvey et al. (1975)presented histological evidence of cytopathology in the brains of rats pretreated with PCA. The reactive cells were predominantly restricted to the B-9 cell group of DahlstrSm and Fuxe (1964), and the authors suggested that the biochemical effects of PCA are due to a selective neurotoxic action of the drug on this cell group. If this is the case,
Synaptosomal uptake
electrolytic lesions of these cells should cause changes in various parameters of serotonergic cell function similar to those found after injection of PCA. In the present paper such a comparison is presented.
2. Materials and methods 2.1. Experimental animals and drugs Male albino rats (280--330 g) were purchased from Sprague--Dawley Company (Madison, Wisconsin, U.S.A.). Individually housed animals were fed Purina Rat Chow and water ad libitum, and maintained on a 12 hr light--dark cycle. A single dose of 15 mg/kg (as free base) of d,l-p-chloroamphetamine hydrochloride (Regis Chemical Co.) was administered i.p. to one: group of 6 animals. Another group was injected with saline. The ambient temperature was maintained at about 15°C for
420 12 hr following injection. Subsequently, room temperature was kept at 24 + 3°C. 2. 2. B-9 lesions
Animals were anesthetized with chloral hydrate (300 mg/kg) and placed in a Kopf stereotaxic instrument with the incisor bar oriented 6.1 mm below the ear bars. Stereotaxic zero was 0.4 mm dorsal to the intersection of the interaural line and the midsagittal plane. After exposing the skull, bilateral burr holes were drilled. A stainless steel guide cannula (0.6 mm, o.d.) was placed 1.1 m m anterior to stereotaxic zero, and then lowered along a line forming an angle of 30 ° with respect to the interaural line. A pointed stainless steel stylet was inserted in the cannula and a small hole was drilled through the tough connective tissue surrounding the trigeminal nerve by spinning the styler in the chuck of a dental drill. The stylet was withdrawn and a wire electrode (0.3 mm, o.d.), insulated except for I mm at the tip, was inserted to points 0.8 and 1.8 mm lateral to the midsagittal plane. At each of these sites 1.0 m A m p of anodal current was passed for 9 sec. An ear bar served as the cathode. Control animals were treated identically except that the electrode was not lowered into the brain. All lesions were verified microscopically on frozen, 60 p serial sections stained with cresyl violet. Improperly lesioned brains were rejected from further analysis. 2. 3. Dissection
Details of the dissection procedure will be published in a subsequent paper. 2 weeks after surgery or the injection of PCA, animals were decapitated. Brains were rapidly removed and placed in ice cold saline. Coronal sections of the chilled brains of about 1 mm thickness were made using a simple slicing apparatus and razor blade while viewing the ventral surface of the brain through a dissecting microscope. Selected nuclear areas were punched out of these slices using a trephine with internal diameter of 2 mm. Area wet weights (mean + S.E.M., n = 36)
v.J. MASSARI, E. SANDERS-BUSH are listed below in parentheses. The 'metathalamus--thalamus' (MTH, 4.6 -+ 0.1 mg) mainly included the dorsal and ventral nuclei of the lateral geniculate body and portions of the nuclei ventralis and lateralis thalami. The 'cortex-hippocampus' sample (CC-HC, 106.2 +- 2.6 mg) consisted of the entire telencephalic component of the slice containing MTH. The 'basal hypothalamus' (HTH, 2.1 -+ 0.1 mg) included the suprachiasmatic nuclei and parts of the nuclei hypothalamicus anterior and hypothalamicus periventricularis. The 'striatum' (ST, 42.8 + 0.7 mg) contained predominantly the nuclei caudatus--putamen and globus pallidus. All other areas beginning with the slice containing MTH and continuing to the anterior margin of the olfactory tubercle were pooled for the 'forebrain' (FB, 680 + 6 mg). 2. 4. B i o c h e m i c a l assays
For synaptosomal uptake, samples were homogenized in 200 volumes of 0.25 M sucrose in a Potter-Elvehjem tissue homogenizer. After centrifugation at 1000 × g for 10 min at 5°C, duplicate 0.3 ml aliquots of the supernatants were placed in ice cold test tubes containing buffered media as described by Sanders-Bush et al. (1975). After 5 min at 37°C, 3H-5HT (11 Ci/mmole) was added to give a final concentration of 0.01 pM. The samples were incubated for an additional 2.5 min, during which uptake was linear, and then immediately filtered through Millipore filters (AAWPO2500) by suction. The filters were washed with 50 ml of saline, placed in 15 ml of Aquasol (New England Nuclear, Boston, Mass.) and counted in a Nuclear Chicago Isocap/300 Liquid Scintillation System. Since preliminary studies showed no significant variation in the 'ice blank' obtained from different brain areas, aliquots of the FB samples maintained at 2°C in the presence of I pM chlorimipramine were used to correct for nonspecific binding of 3H-5HT. 5HT was assayed by fluorescence after derivatization with o-phthalaldehyde (Maickel and Miller, 1966). The m e t h o d was similar to that of Curzon and Green (1970), except that
BRAIN 5HT: p-CHLOROAMPHETAMINE VS. B-9 LESIONS
the supernatant from butanol-extracted tissue was washed with pH 10.0 borate buffer before extraction into acid. 2. 5. Statistics
The data was analyzed statistically using a Student's two-tailed t-test. 3. Results The B-9 lesions destroyed major portions of the medial lemniscus and some of the surround-
421
ing reticular formation and pons from the level of the posterior margin of the inferior coUiculus to the posterior third of the interpeduncular nucleus (fig. 1). The dorsal and median raphe nuclei were completely spared. Damage to the lateral parts of the superior cerebellar peduncle was minor and was usually restricted to the most anterior portion of the lesions. After B-9 lesions, the uptake of 5HT was reduced 35% in the MTH sample (fig. 2). Although uptake in all of the other areas was apparently reduced by 10--20%, these values were not significantly different from control. 5HT levels in the MTH were also reduced by 37% (control, n = 5, 0.72 + 0.08 ng/mg; lesion, n = 5, 0.45 +- 0.01 ng/mg, p < 0.01). In the other areas, levels were reduced slightly; absolute values in ng/rng of tissue + S.E.M. in control vs. lesioned rats were: HTH, 0.60 + 0.13 (n = 4) vs. 0.57 + 0.11 (n = 5), p > 0.05; ST, 0.32 + 0.01 (n = 10) vs. 0.28_+ 0.02 (n = l l ) , p > 0.05; CC-HC, 0.32 _+ 0.01 (n = 9) vs. 0.29 _+0.01 (n = 11), p < 0.05; FB, 0.37 _+0.01 (n = 11) vs. 0.33 _+0.01 (n = l l ) , p < 0.01. After the injection of PCA, the uptake of 5HT was reduced by 75% or more in all areas studied (fig. 2).
4. Discussion
...... I l l Fig. 1. Schematic illustration on 3 coronal planes of the extent of cavitation and gliosis following B-9 lesions. Abbreviations: CI, inferior colliculus; CS, superior coUiculus; DR, dorsal raphe nucleus; IP, interpeduncular nucleus; LM, medial lemniscus; MLF, medial longitudinal fasciculus; MR, median raphe nucleus; PCS, superior cerebeUar peduncle; TP, nucleus tegmenti pontis; VT, nucleus tegrnenti ventralis; VS, sensory component of trigeminal nerve.
The administration of PCA causes large widespread decreases in the high affinity uptake of 5HT. In contrast, large lesions o f the B-9 cell group cause only small or insignificant reductions in the level and uptake o f 5HT. We conclude, therefore, that a selective neurotoxic action on the B-9 cell group cannot entirely explain the long lasting and pronounced biochemical effects of PCA. Apparently, only a minor portion of the 5HT nerve terminals in the forebrain arise from cells in the B-9 group. This is consistent with the results of Kuhar et al. (1972) who found a 70--90% decrement in 5HT levels, uptake, and t r y p t o p h a n hydroxylase activity in most forebrain areas after extensive lesions o f the dorsal and median raphe nuclei, which spared the B-9 cell group. Specific information on the projec-
422
z
z
~ o
13_
V.J. MASSARI, E. SANDERS-BUSH
16-
E Z CONTROL ~ B-9 LESION
~ ~
CONTROL PCA
L 16
z
14 -
14
12-
12
z
io
s
io-
c
~-
~ ~
g
4 ~
6
MTH
HTH
ST
CC-HC
FB
MTH
HTH
6
ST
CC-HC
FB
Fig. 2. Synaptosomal uptake of 5HT 2 weeks after B-9 lesions or PCA (15 mg/kg, i.p.). Abbreviations: MTH, metathalamus--thalamus; HTH, basal hypothalamus; ST, striatum; CC-HC, cerebral cortex--hippocampus; FB, forebrain. Asterisks indicate significant differences between lesion and control groups. *p < 0.05, **p < 0.01, ***p < 0.001. Vertical bars represent S.E.M.
tions of the B-9 cell group is sparse. Histochemical fluorescence data (Fuxe and Jonsson, 1974) suggest a lateral projection to the caudate--putamen and 'extrapyramidal m o t o r system'. The present results indicate that a lateral serotonergic pathway, possibly from the B-9 cell group, projects to the metathalamus-thalamus.
Acknowledgements This study was supported in part by USPHS Grants MH-08107 and MH-11468 and a Grant-in-Aid from the Vanderbilt University Research Council. Dr. E. Sanders-Bush is a recipient of NIMH Research Scientist Development Award MH-70898. The authors wish to thank Ms. Jan A. Bushing for skillful technical assistance.
References Bj6rklund, A., H.G. Baumgarten and A. Nobin, 1974, Chemical lesioning of central monoamine axons by means of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine, Advan. Biochem. Psychopharmacol. 10, 13.
Curzon, G. and A.R. Green, 1970, Rapid method for the determination of 5-hydroxytryptamine and 5hydroxyindoleacetic acid in small regions of rat brain, Brit. J. Pharmacol. 3 9 , 6 5 3 . Dahlstr6m, A. and K. Fuxe, 1964, Evidence for the existence of monamine neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand. 64, Suppl. 232. Fuxe, K. and G. Jonsson, 1974, Further mapping of central 5-hydroxytryptamine neurons: studies with the neurotoxic dihydroxytryptamines, Advan. Biochem. Psychopharmacol. 10, 1. Harvey, J., S. McMaster and L. Yunger, 1975, p-Chloroamphetamine: selective neurotoxic action in brain, Science 187, 841. Kuhar, M., G. Aghajanian and R. Roth, 1972, Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesions: correlations with serotonin levels and histochemical fluorescence, Brain Res. 44, 165. Maickel, R.P. and F.P. Miller, 1966, Fluorescent products formed by reaction of indole derivatives and o-phthalaldehyde, Anal. Chem. 38, 1937. Sanders-Bush, E., J. Bushing and F. Sulser, 1972, Long-term effects of p-chloroamphetamine on tryptophan hydroxylase activity and on the levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in brain, European J. Pharmacol. 20,385. Sanders-Bush, E., J. Bushing and F. Sulser, 1975, Long-term effects of p-chloroamphetamine and related drugs on central serotonergic mechanisms, J. Pharmacol. Exptl. Therap. 192, 33.
Short communication S Y N A P T O S O M A L U P T A K E A N D L E V E L S O F S E R O T O N I N IN R A T B R A I N A R E A S A F T E R p - C H L O R O A M P H E T A M I N E O R B-9 LESIONS V. J O H N M A S S A R I and E L A I N E S A N D E R S - B U S H
Department o f Pharmacology, Vanderbilt University School o f Medicine, and Tennessee Neuropsychiatric Institute, Nashville, Tennessee, U.S.A. Received 1 August 1975, accepted 6 August 1975
V.J. MASSARI and E. SANDERS-BUSH, Synaptosomal uptake and levels o f serotonin in rat brain areas after p-chloroamphetamine or B-9 lesions, European J. Pharmacol. 33 (1975) 419--422. In contrast to the pronounced fall in 5HT levels and synaptosomal uptake caused by p-chloroamphetamine, bilateral lesions of the B-9 cell group caused minimal regional changes, except for 35% decreases in the metathalamus--thalamus. We conclude therefore that the prolonged biochemical effects of p-chloroamphetamine are not due to a selective cytotoxic action on B-9 cells; and that a lateral 5HT pathway, possibly from the B-9 cell group, projects to the metathalamus--thalamus. p-Chloroamphetamine
B-9 lesions
Serotonin
1. Introduction After the administration of a single i.p. dose of p-chloroamphetamine (PCA) to rats, a prompt and prolonged reduction in brain levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid occurs. Reductions in the activity of tryptophan hydroxylase and in the high affinity uptake of 5HT were also found (Sanders-Bush et al., 1972, 1975). This spectrum of effects is analogous to those caused by the intraventricular administration of the neurotoxic agents, 5,6- and 5,7-dihydroxytryptamine (Bj6rklund et al., 1974). Recently, Harvey et al. (1975)presented histological evidence of cytopathology in the brains of rats pretreated with PCA. The reactive cells were predominantly restricted to the B-9 cell group of DahlstrSm and Fuxe (1964), and the authors suggested that the biochemical effects of PCA are due to a selective neurotoxic action of the drug on this cell group. If this is the case,
Synaptosomal uptake
electrolytic lesions of these cells should cause changes in various parameters of serotonergic cell function similar to those found after injection of PCA. In the present paper such a comparison is presented.
2. Materials and methods 2.1. Experimental animals and drugs Male albino rats (280--330 g) were purchased from Sprague--Dawley Company (Madison, Wisconsin, U.S.A.). Individually housed animals were fed Purina Rat Chow and water ad libitum, and maintained on a 12 hr light--dark cycle. A single dose of 15 mg/kg (as free base) of d,l-p-chloroamphetamine hydrochloride (Regis Chemical Co.) was administered i.p. to one: group of 6 animals. Another group was injected with saline. The ambient temperature was maintained at about 15°C for
420 12 hr following injection. Subsequently, room temperature was kept at 24 + 3°C. 2. 2. B-9 lesions
Animals were anesthetized with chloral hydrate (300 mg/kg) and placed in a Kopf stereotaxic instrument with the incisor bar oriented 6.1 mm below the ear bars. Stereotaxic zero was 0.4 mm dorsal to the intersection of the interaural line and the midsagittal plane. After exposing the skull, bilateral burr holes were drilled. A stainless steel guide cannula (0.6 mm, o.d.) was placed 1.1 m m anterior to stereotaxic zero, and then lowered along a line forming an angle of 30 ° with respect to the interaural line. A pointed stainless steel stylet was inserted in the cannula and a small hole was drilled through the tough connective tissue surrounding the trigeminal nerve by spinning the styler in the chuck of a dental drill. The stylet was withdrawn and a wire electrode (0.3 mm, o.d.), insulated except for I mm at the tip, was inserted to points 0.8 and 1.8 mm lateral to the midsagittal plane. At each of these sites 1.0 m A m p of anodal current was passed for 9 sec. An ear bar served as the cathode. Control animals were treated identically except that the electrode was not lowered into the brain. All lesions were verified microscopically on frozen, 60 p serial sections stained with cresyl violet. Improperly lesioned brains were rejected from further analysis. 2. 3. Dissection
Details of the dissection procedure will be published in a subsequent paper. 2 weeks after surgery or the injection of PCA, animals were decapitated. Brains were rapidly removed and placed in ice cold saline. Coronal sections of the chilled brains of about 1 mm thickness were made using a simple slicing apparatus and razor blade while viewing the ventral surface of the brain through a dissecting microscope. Selected nuclear areas were punched out of these slices using a trephine with internal diameter of 2 mm. Area wet weights (mean + S.E.M., n = 36)
v.J. MASSARI, E. SANDERS-BUSH are listed below in parentheses. The 'metathalamus--thalamus' (MTH, 4.6 -+ 0.1 mg) mainly included the dorsal and ventral nuclei of the lateral geniculate body and portions of the nuclei ventralis and lateralis thalami. The 'cortex-hippocampus' sample (CC-HC, 106.2 +- 2.6 mg) consisted of the entire telencephalic component of the slice containing MTH. The 'basal hypothalamus' (HTH, 2.1 -+ 0.1 mg) included the suprachiasmatic nuclei and parts of the nuclei hypothalamicus anterior and hypothalamicus periventricularis. The 'striatum' (ST, 42.8 + 0.7 mg) contained predominantly the nuclei caudatus--putamen and globus pallidus. All other areas beginning with the slice containing MTH and continuing to the anterior margin of the olfactory tubercle were pooled for the 'forebrain' (FB, 680 + 6 mg). 2. 4. B i o c h e m i c a l assays
For synaptosomal uptake, samples were homogenized in 200 volumes of 0.25 M sucrose in a Potter-Elvehjem tissue homogenizer. After centrifugation at 1000 × g for 10 min at 5°C, duplicate 0.3 ml aliquots of the supernatants were placed in ice cold test tubes containing buffered media as described by Sanders-Bush et al. (1975). After 5 min at 37°C, 3H-5HT (11 Ci/mmole) was added to give a final concentration of 0.01 pM. The samples were incubated for an additional 2.5 min, during which uptake was linear, and then immediately filtered through Millipore filters (AAWPO2500) by suction. The filters were washed with 50 ml of saline, placed in 15 ml of Aquasol (New England Nuclear, Boston, Mass.) and counted in a Nuclear Chicago Isocap/300 Liquid Scintillation System. Since preliminary studies showed no significant variation in the 'ice blank' obtained from different brain areas, aliquots of the FB samples maintained at 2°C in the presence of I pM chlorimipramine were used to correct for nonspecific binding of 3H-5HT. 5HT was assayed by fluorescence after derivatization with o-phthalaldehyde (Maickel and Miller, 1966). The m e t h o d was similar to that of Curzon and Green (1970), except that
BRAIN 5HT: p-CHLOROAMPHETAMINE VS. B-9 LESIONS
the supernatant from butanol-extracted tissue was washed with pH 10.0 borate buffer before extraction into acid. 2. 5. Statistics
The data was analyzed statistically using a Student's two-tailed t-test. 3. Results The B-9 lesions destroyed major portions of the medial lemniscus and some of the surround-
421
ing reticular formation and pons from the level of the posterior margin of the inferior coUiculus to the posterior third of the interpeduncular nucleus (fig. 1). The dorsal and median raphe nuclei were completely spared. Damage to the lateral parts of the superior cerebellar peduncle was minor and was usually restricted to the most anterior portion of the lesions. After B-9 lesions, the uptake of 5HT was reduced 35% in the MTH sample (fig. 2). Although uptake in all of the other areas was apparently reduced by 10--20%, these values were not significantly different from control. 5HT levels in the MTH were also reduced by 37% (control, n = 5, 0.72 + 0.08 ng/mg; lesion, n = 5, 0.45 +- 0.01 ng/mg, p < 0.01). In the other areas, levels were reduced slightly; absolute values in ng/rng of tissue + S.E.M. in control vs. lesioned rats were: HTH, 0.60 + 0.13 (n = 4) vs. 0.57 + 0.11 (n = 5), p > 0.05; ST, 0.32 + 0.01 (n = 10) vs. 0.28_+ 0.02 (n = l l ) , p > 0.05; CC-HC, 0.32 _+ 0.01 (n = 9) vs. 0.29 _+0.01 (n = 11), p < 0.05; FB, 0.37 _+0.01 (n = 11) vs. 0.33 _+0.01 (n = l l ) , p < 0.01. After the injection of PCA, the uptake of 5HT was reduced by 75% or more in all areas studied (fig. 2).
4. Discussion
...... I l l Fig. 1. Schematic illustration on 3 coronal planes of the extent of cavitation and gliosis following B-9 lesions. Abbreviations: CI, inferior colliculus; CS, superior coUiculus; DR, dorsal raphe nucleus; IP, interpeduncular nucleus; LM, medial lemniscus; MLF, medial longitudinal fasciculus; MR, median raphe nucleus; PCS, superior cerebeUar peduncle; TP, nucleus tegmenti pontis; VT, nucleus tegrnenti ventralis; VS, sensory component of trigeminal nerve.
The administration of PCA causes large widespread decreases in the high affinity uptake of 5HT. In contrast, large lesions o f the B-9 cell group cause only small or insignificant reductions in the level and uptake o f 5HT. We conclude, therefore, that a selective neurotoxic action on the B-9 cell group cannot entirely explain the long lasting and pronounced biochemical effects of PCA. Apparently, only a minor portion of the 5HT nerve terminals in the forebrain arise from cells in the B-9 group. This is consistent with the results of Kuhar et al. (1972) who found a 70--90% decrement in 5HT levels, uptake, and t r y p t o p h a n hydroxylase activity in most forebrain areas after extensive lesions o f the dorsal and median raphe nuclei, which spared the B-9 cell group. Specific information on the projec-
422
z
z
~ o
13_
V.J. MASSARI, E. SANDERS-BUSH
16-
E Z CONTROL ~ B-9 LESION
~ ~
CONTROL PCA
L 16
z
14 -
14
12-
12
z
io
s
io-
c
~-
~ ~
g
4 ~
6
MTH
HTH
ST
CC-HC
FB
MTH
HTH
6
ST
CC-HC
FB
Fig. 2. Synaptosomal uptake of 5HT 2 weeks after B-9 lesions or PCA (15 mg/kg, i.p.). Abbreviations: MTH, metathalamus--thalamus; HTH, basal hypothalamus; ST, striatum; CC-HC, cerebral cortex--hippocampus; FB, forebrain. Asterisks indicate significant differences between lesion and control groups. *p < 0.05, **p < 0.01, ***p < 0.001. Vertical bars represent S.E.M.
tions of the B-9 cell group is sparse. Histochemical fluorescence data (Fuxe and Jonsson, 1974) suggest a lateral projection to the caudate--putamen and 'extrapyramidal m o t o r system'. The present results indicate that a lateral serotonergic pathway, possibly from the B-9 cell group, projects to the metathalamus-thalamus.
Acknowledgements This study was supported in part by USPHS Grants MH-08107 and MH-11468 and a Grant-in-Aid from the Vanderbilt University Research Council. Dr. E. Sanders-Bush is a recipient of NIMH Research Scientist Development Award MH-70898. The authors wish to thank Ms. Jan A. Bushing for skillful technical assistance.
References Bj6rklund, A., H.G. Baumgarten and A. Nobin, 1974, Chemical lesioning of central monoamine axons by means of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine, Advan. Biochem. Psychopharmacol. 10, 13.
Curzon, G. and A.R. Green, 1970, Rapid method for the determination of 5-hydroxytryptamine and 5hydroxyindoleacetic acid in small regions of rat brain, Brit. J. Pharmacol. 3 9 , 6 5 3 . Dahlstr6m, A. and K. Fuxe, 1964, Evidence for the existence of monamine neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand. 64, Suppl. 232. Fuxe, K. and G. Jonsson, 1974, Further mapping of central 5-hydroxytryptamine neurons: studies with the neurotoxic dihydroxytryptamines, Advan. Biochem. Psychopharmacol. 10, 1. Harvey, J., S. McMaster and L. Yunger, 1975, p-Chloroamphetamine: selective neurotoxic action in brain, Science 187, 841. Kuhar, M., G. Aghajanian and R. Roth, 1972, Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesions: correlations with serotonin levels and histochemical fluorescence, Brain Res. 44, 165. Maickel, R.P. and F.P. Miller, 1966, Fluorescent products formed by reaction of indole derivatives and o-phthalaldehyde, Anal. Chem. 38, 1937. Sanders-Bush, E., J. Bushing and F. Sulser, 1972, Long-term effects of p-chloroamphetamine on tryptophan hydroxylase activity and on the levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in brain, European J. Pharmacol. 20,385. Sanders-Bush, E., J. Bushing and F. Sulser, 1975, Long-term effects of p-chloroamphetamine and related drugs on central serotonergic mechanisms, J. Pharmacol. Exptl. Therap. 192, 33.