Serotonin and catecholamine concentrations in brain of rats injected intracerebrally with 5,6-dihydroxytryptamine

304

SHORT COM MU NI CATIONS

Serotonin and catecholamine concentrations in brain of rats injected intracerebrally with S,6-dihydroxytryptamine Baumgarten et aL 1 reported a selective and long lasting depletion1 of brain serotonin (5-hydroxytryptamine, 5-HT) in rats receiving 5,6-dihydroxytryptamlne (5,6-HT) creatinine sulfate salt intracerebrally. It was proposed that 5,6-HT is concentrated selectively in serotonergic neurons, where it then causes biochemical lesions similar to those caused by 6-hydroxydopamine m adrenergic neurons. A sample of 5.6-HT creatinine sulfate (hereafter referred to as 5.6-HT-I) provided by Baumgarten et al. 1 was analyzed mass spectrometrically and found to be impure since it gave r~se to significant ions (m/e 217~ of higher mass than that of the molecular ion of the parent compound (m/e 192). Another sample of 5.6-HT creatinine sulfate (hereafter referred to as 5,6-HT-II) prepared from 5.6-dibenzyloxytryptamine was pure by mass spectral analysis (see below). This report concerns a comparison of the effects of 5.6HT-I and -II on brain monoamine concentrations. Both samples were injected through a permanent polyethylene cannuta s (Clay Adams. P.E. No. 10: inner diameter. 0.011 m.. outer diameter. 0.025 in.I placed in the right cerebral ventricle of rats t0 48 h prior to the injection. In another group of experiments the compounds were injected intracerebrally as suggested by Noble et al. 7. Sprague-Dawley male rats of 180-220 g were housed at 20 °C. 8 per cage for at least 5 days after their arrival from Zivic Miller Laboratories (Pittsburgh. Pa, t. Samples o f 5.6-HT were dissolved in equimolar concentrations of ascorbic acid : 20 lzl of the solution contained 0.39 ,umoles of either 5.6-HT-I or 5.6-HT-II. This volume was injected 14 days before sacrifice. The central nervous system of rats injected with 5.6H T and that of rats injected with ascorbic acid solution was dissected in 5 parts: spinal cord, medulla ports, hypothalamus, cerebellum and telencephalon. Two transversal sections, one at the upper limit of the ports, and the other at the apex o f the fourth ventricle delimited the medulla pons from mesencephalon and spinal cord. respectively. The other parts were dissected as described by Meyerson'L Immediately after dissection, these brain parts were frozen and kept at 20 °C until assay at a later time. We measured concentrations of 5-HT. norepinephrine (NE) and dopamine (DA) with a method previously reported 6. This method separates the amines from their amino acid precursor and their acid metabolites by ion exchange chromatography using Dowex 50 X 4W, 200-400 mesh buffered at pH 6.5 with N a ~. Each of the 3 amines is eluted separately from the column and is further purified either by A1203 absorption (catecholamines~ or by butanol extraction {5-HT). Catechotamine concentrations in the final formic acid f0.4 NI eluate are measured by the trihydroxyindole method, the 5-HT concentrations were measured by forming a fluorophore with ninhydrin according to Snyder et al. 9 5.6-HT-II was synthesized as follows. 5,6-Dibenzyloxytryptamine formate* * Dibenzyloxytryptamine formate salt was provided by Dr. A. A. Manian. Psychopharmacotogy Service Center. National Institute of Mental Health. Synthesis proceeded from 5.6-dibenzyloxyindole by: (1) reaction with oxalylchloride. (2) conversion to the 3-glyoxylamide with ammonia, and (3) reduction to 5.6-dibenzyloxytryptamine with,llithium aluminum hydride, and was carried out by Regis Chemical Co.. Chicago, 111.. under Research Contract No. SA 43-pH 3021. Brain Research. 44 ~1972) 304-308

305

SHORT COMMUNICATIONS

(209 rag, 0.5 mmole) was dissolved in 25 ml H20 with 100 mg 10%opalladium on charcoal catalyst. Reduction with hydrogen gas at atmospheric pressure was complete in 4-5 h. The sample was filtered under a stream of nitrogen gas into 20 ml of H..,O containing 0.5 mmoles H2SO4 and 0.5 mmoles of creatinine. The solution was lyophilized and the residue triturated with 5 ml ice-cold water-acetone (1:1) under nitrogen and filtered to yield 270 mg of 5,6-dihydroxytryptamine creatinine sulfate salt (5,6-HT-1I). The 5,6-HT salts were analyzed with a Finnigan 3000 Quadrupole Mass Spectrometer. As expected in the mass spectrometric analysis of a creatinine sulfate organic salt, the sulfate moiety is undetected, leaving only the creatinine and in this case the 5,6-HT moieties to be detected. Therefore, the normalized spectra of 5,6-HT-I and -1I (Fig. 1) can be viewed as composite spectra of 5,6-HT and creatinine. The spectrum for 5,6-HT-I may be analyzed as follows: m/e 192 is the molecular ion of 5,6-HT, m/e 113 is the molecular ion of creatinine, m/e 162 is the base peak of 5,6-HT corresponding to the a-/~ carbon cleavage in the side chain with loss of CH2NH.) and m/e 42 is the base peak of creatinine. The spectrum of 5,6-HT-I also contains two unidentified peaks (m/e 149, m/e 217), the latter of which is of equal intensity to the molecular ion (m/e 192) of 5,6-HT. The peak with m/e 217 cannot result from 5,6-HT creatinine sulfate. The spectrum of 5,6-HT-II does not contain the two mass peaks (m/e 149, m/e 217). In addition, the ratio of the molecular creatinine peak to the molecular 5,6-HT peak is smaller by a factor of approximately 180. This suggests that 5,6-HT-I contains I00- 42

5,6-HT-I /--XIO0 I /

45

1162

l/

I I I I I

50-

l ~lF t

I jf

M+ I r°9~ 84 l

_

fbo

40

-~ I00-

42

2bo

15o O~

N.H

"~N jL'N H

43

50-

250

CH3 CREATININE M W 113

H••'CH2CH2N H2

5,6 DIHYDROXYTRYPTAMI NE M W 192

162

0 4O

217

5,6-HT- Tr

uJ

~-

M+ 192

69~ 84L

M* 192

,bo

,go

26o

2~o

m/e

Fig. 1. Mass spectra of the two preparations (5,6-HT-I and 5,6-HT-II) of 5,6-dihydroxytryptamine creatinine sulfate dihydrate.

Brain Research, 44 (1972) 304-308

taa

4~

1.8±0.06 9.0 _~ 0.73 1.8 + 0.07 5.6 5- 0.33 4.6 i 0.11

(NE) AND DOPAM1NE

1.5±0.05 9.8 ± 0.69 1.6 2_- 0.095 3.6 ~ 0.11 2.0 =~-0.08

( D A ) IN

1,6 8.6 1.5 2.8 2.0

~z 0,06 5- 0.37 5- 0.07 5- 0.22* ~ 0.04

3.8_4-0.13 -----

Control

3.8±0.13 -----

5,6-HT-1

3.6 ~ 0.1 -----

5,6-HT-I1

BRAIN PARTS OF RATS INJECTED W I T H 5 , 6 - D I H Y D R O X Y T R Y P T A M I N E

1.65-0.05 8.1 5- 0.38* 1.6 5- 0.083 2.8 5- 0.21" 2.1 5- 0.13

5,6-HT-I1

DA (nmoles/g + S.E.M.)

Teiencephalon Cerebellum Hypothalamus Medulla-ports

Brain area

i.9 1.8 9.5 4.9

-~ 0.12 .I-.:0.14 ± 1.5 5- 0~57

Control 1.3 _+ 0.12" 1 . 6 ~ 0,04 9.5 5- 1!4 5.2 5-0.51

5,6-HT-1

5-HT (nmoles/g _6=S.E,M.)

I 3,51.6± 7.7 ± 5.7 5-

0.1 * 0115 0.59 0.21

5,6,HT-11

1.5 1.6 11.0 3.5

~ 0.l ± 0.19 :L 0.76 + 0~23

Control

115 1.5 l0 3.0

± 0.04 ± 0.13 :~ 0:84 5- 0~26

5,6-HT-I

NE (nmoles/g ~ S.E:M.)

1.6 1.5 10 3.5

-k 0.11 "=0.08 ± 1.5 5- 0.14

5,6-HT-1l

5,6-HT-I 3.8 ± 0.26 4.2 !: 0.28 . . . . . . . . . . . . . . . . . . . . . . .

Control

DA (nmoles/g ± S.E.M.)

--

3.8 ± 0.2

5,6-HT-H

Each value is the average o f 5 experiments. T h e rats were killed 14 days after t h e injection o f 5 , 6 - H T (0.39/~moles/rat) t h r o u g h polyethylene c a n n u l a s implanted into the right lateral ventricle. I a n d lI refer to the source o f 5,6-HT

CONCENTRATIONS OF SEROTONIN

1.45-0.21 8.9 ~ 1.2 1.8 ~ 0.08 4.4 -- 0.13" 2.3 :~ 0.16"

NOREPtNEPHRINE

1.3±0.12" 7.1 ± 0.36* 1.9 5_ 0.06 4.4 :L 0.12" 2.9 ~: 0.21"

5,6-HT-!

Control

5,6-HT-II

Control

5,6-HT-1

NE (nmoles/g 5- S.E.M.)

5-HT (nmoles/g ± S.E.M.)

(5-HT), (5,6-HT) BY A VENTRiCULAR CANNULA

T A B L E II

* P < 0,01.

Telencephalon Hypothalamus Cerebellum Pons a n d m e d u l l a Spinalcord

Brain area

Each value is the average o f 10 experiments. T h e rats were killed 14 days after the injection o f 0 . 3 9 / ~ m o l e s / r a t o f 5,6-HT. 1 a n d 11 refer to t h e source o f 5,6-HT injected.

CEREBRALLY

CONCENTRATIONS OF SEROTONIN (5-HT), NOREPINEPHRINE ( N E ) ANO DOPAMINE ( D A ) JN BATS RECEIVING 5,6-DIHYDROXYTRYPTAMINE (5,6-HT) 1NTBA-

TABLE I

c)

O

SHORT COMMUNICATIONS

307

additional free creatinine sulfate as a contaminant. It is difficult to determine the exact ratios involved since the free creatinine sulfate does seem to be slightly more volatile at the sample temperature chosen (180 °C) for the analysis. Thus, as the analysis proceeds the ratio ofcreatinine to 5,6-HT decreases. The ratios reported in Fig. 1 were obtained using identical procedures thereby giving an estimate of sample purity*. The difference in the mass spectral analysis of 5,6-HT-I and -II prompted us to study the effect of intracerebral injection of both 5,6-HT-I and -II on the monoamine concentrations of various brain parts. Both 5,6-HT-I and -II deplete 5-HT concentrations in telencephalon, medulla pons and spinal cord (Table I). Both compounds fail to deplete cerebellar 5-HT concentrations and 5,6-HT-II does not change the 5-HT content of hypothalamus. The extent of the 5-HT depletion elicited by both samples of 5,6-HT is less than that reported by Baumgarten et al. 1 although the relative proportion of the depletion in different brain regions is similar. In our experiments, the extent of depletion was greatest in the spinal cord. In contrast to Baumgarten's report, we found a decrease of brain NE in hypothalamus and pons medulla with 5,6-HT-I. 5,6-HT-II decreased brain NE only in the medulla pons. Brain DA concentrations were not changed by injection of either sample of 5,6-HT. In order to determine whether the reason for the smaller effect obtained in the present study could have been due to a faulty injection, the experiments were repeated with injection of the drug through cannulas implanted in the lateral ventricle. Usually when drugs are injected through an intraventricular cannula their action tends to be restricted and localized. Various doses of 5,6-HT of up to 3.9 #moles/rat were injected, but a greater depletion of brain 5-HT was not elicited. In the animals receiving the highest doses, recurrent convulsive episodes began 6 h after the injection and lasted for 48-60 h. In some experiments, 0.39/~mole/rat of either 5,6-HT-I or -II was injected through cannulas placed in the lateral ventricle (Table II). The concentration of 5-HT was reduced only in the telencephalon and spinal cord. No change was observed in the DA and NE concentrations. We conclude that the impurity detected mass spectrometrically in 5,6-HT-I is not responsible for the 5-HT depletion elicited by this compound. Both samples of 5,6-HT cause brain 5-HT depletion but this effect is less intense than that reported by Baumgarten et al.l.While the extent of the depletion in spinal cord and telencephalon does not depend on the route of administration of 5,6-HT, it seems that the action of the drug is restricted and localized when administered through an implanted ventricular cannula. We do not have any plausible explanation for the discrepancy between our findings and those of Baumgarten et a L L We assayed the brain tissue after purification with column chromatography and butanol extraction and assayed 5-HT by the ninhydrin method, which at pH 6.5 is specific For 5-HT. Baumgarten et al. 1 used Bertler's method 2 where the tissues are purified only with ionic exchange chromatography

* A successivesample of 5,6-HT supplied by Baumgarten shows a 90-foldreduction in free creatinine. Also the m/e 217 peak was decreased by a factor of 2. This reduction was accompanied by a more intense peak at m/e 149. Brain Research, 44 (1972) 304-308

308

SHORT COMMUNICATIONS

and 5 - H T is detected with a m e t h o d specific f o r 5 - O H indoles. Since we d o u b t that 5 - H T is the only i n d o l e a l k y l a m i n e in brain 3A the discrepancy may be related to the different specificities o f the m e t h o d s used by us an d by B a u m g a r t e n et atA to purify and assay brain 5-HT.

Laboratory of Preclinical Pharmacology, National Institute of" Mental Health. Saint Elizabeths Hospital. Washington. D. C. 20032.

Laboratory of Chemistry, National Institute of" Arthritis and Metabolic Diseases. National Institutes of Health, Bethesda. Md. 20014 (U.S.A.)

E. COSTA H. LEFEVRE J. MEEK A, REVUELTA F. SPANO* S. STRADA J. DALY

1 BAUMGARTEN, H . G . , BJORKLUND, A., LACHENMAYER, L.. NOBIN, A., AND STENEVI, U., Long lasting selective depletion of brain serotonin by 5,6-dihydroxytryptamine, Acta physiol, seand., Suppl 373 (1971). 2 BERTLER, A., Effect of reserpine on the storage of catecholamines in brain and other tissues. Aeta physiol, scand., 5I (1961) 75-83. 3 BJ6RKLUND. A., FALCK, B., ~,NDSTENEVI, U.. Classification of monoamine neurons in the rat mesencephalon: distribution of a new monoamine neuron system, Brain Research, 32 (1971) 269-285. 4 GREEN.A. R., KOSLOW.S. H., SPANO,P. F.. ANDCOSTA,E., Identification of melatonin (M) and 5-methoxytryptamine (5-MT) in rat hypothalamus by gas chromatography mass spectrometry (GC-MS), Abstract, 5th int. Meeting Pharmacol., July (1972). 5 MEYERSON,B. J..Centralnervousmonoaminesandhormoneinducedestrusbehaviorinthespayed rat. Acta physiol, stand., 63, Suppl. 241 (1964). 6 NEFF. N. H.. SPANO, P. F., GROPPETrl, A., WANG, C. T., AND COSTA, E., A simple procedure for calculating the synthesis rate of brain norepinephrine, dopamine and serotonin after a pulse injection of radioactive tyrosme and tryptophan, J. Pharmacol. exp. Ther., 176 (1971) 701-710. 7 NOBLE, E. P., WURTMAN,R. J.. AND AXELROO,J., A simple and rapid method for injecting '~Hnorepinephrine into the lateral ventricle of the rat brain, Life Sci., 6 (1967) 281-291. 8 ROBINSON, C. A., HENGEVALD. C. A., AND DEBALBIAN VESTER. F., Improved polyethylene cannulation techniques, PhysioL Behav., 4 (1969) 123-124. 9 SNYDER, S. H.. AXELROD, J., AND ZWEIG, M., A sensitive and specific fluorescence assay for tissue serotonin, Biochem. Pharrnacol., 14 (1965) 831-835. l0 STRADA, S. J., SANDERS-BUSH, E., AND SULSER, F , Temporal relationship between psychomotor stimulation and metabolism of brain norepinephrine, Biochem. Pharrnacol., 19 (1970) 2621-2629.

(Accepted June 9th, 19721

* On leave of absence from the Department of Pharmacology, University of Milan, Milan, Italy.

Brain Research, 44 (1972) 304-308