The distribution of β-phenylethylamine in discrete regions of the rat brain and its effect on brain noradrenaline, dopamine and 5-hydroxytryptamine levels

Neurophormacology.

1973,

12, 663-668

Pergamon Press.Printedin Gt. Britain.

THE DISTRIBUTION OF /3-PHENYLETHYLAMINE IN DISCRETE REGIONS OF THE RAT BRAIN AND ITS EFFECT ON BRAIN NORADRENALINE, DOPAMINE AND 5-HYDROXYTRYPTAMINE LEVELS D. M. JACKSONand D. B. SMYTHE Department

of Pharmacology,

The University of Sydney, Sydney, N.S.W. 2006, Australia (Accepted

30 November

1972)

Summary-/?-Phenylethylamine (100 mg/kg) injected interperitoneally into rats 1 hr before death, caused a significant depletion of whole brain levels of noradrenaline and dopamine, but not of 5-hydroxytryptamine. An investigation of discrete regions of the brain showed that the same dose of phenylethylamine caused significant depletion of dopamine in the cerebellum, corpus striatum, midbrain, and cortex, but not in the hypothalamus or medulla oblongata-pons region. All regions were depleted of noradrenaline, while only the midbrain and cortex were significantly depleted of 5-hydroxytryptamine. Administration of [4-T]-/%phenylethylamine, 100 mg/kg, showed that the distribution of labelled phenylethylamine in various regions of the brain was significantly correlated with the distribution of dopamine (both in controls and after 100 mg &phenylethylamine/kg). These results and their signiticance are discussed.

/?-Phenylethylamine (PE) is a naturally occurring sympathomimetic amine (NAKAJIMA, KAKIMOTO and SANO,1964; JACKSON and TEMPLE,1970). High concentrations of PE have been found in the urine of phenylketonuric patients (OATES,NIRENBERG, JEPSON,SJOERDSMA and UDENFRIEND,1963; LEVINE,NIRENBERG,UDENFRIENDand SJOERDSMA,1964), while the concentrations were lower than normal in the urine of subjects suffering from endogenous depression (FISCHER,HELLERand MIRO, 1968; BOULTONand MILWARD,1971; FISCHER, HELLER,SPATZ and REGGIANO,1972). /?-Phenylethylamine also occurs in the brains of rats (FISCHERet al., 1972), and rabbits (NAKAJIMA et al., 1964). The highest concentration of PE in rabbit brain after phenylalanine pretreatment and monoamineoxidase inhibition was found in the caudate nucleus (NAKAJIMAet al., 1964; EDWARDSand BLAU, 1972). Injection of PE into animals produced a depletion of brain noradrenaline (NA) in guinea pigs (JACKSON, 1971), both NA and dopamine (DA) in rats (JONSSON,GROBECKER and HOLTZ, 1966; FUXE, GROBECKER and JONSSON,1967), and NA, DA and 5-hydroxytryptamine (5-HT) in mice (JACKSONand SMYTHE,unpublished observations). Because of the presence of PE in brain, and the suggestion by FISCHER,SAAVEDRA and HELLER(1968) and SAAVEDRA and FISCHER(1970) that it may play a physiological role as the ergotrophic hormone postulated by HESS(1959), and because of its possible importance in both phenylketonuria, endogenous depression (see above references), and in the cardiovascular system (JACKSON,1970), it was considered important to determine the effect of injected PE on NA, DA and 5-HT levels in both whole brain and in discrete regions of the brain. An attempt was also made to relate by tracer techniques any effect observed on the distribution of brain amines with the distribution of PE. 663

664

D. M. JACKSON and D. B. SMYTHE METHODS

Male Sprague-Dawley rats (200-300 g) were used in all experiments and allowed food and water ad lib. up to the time of experimentation. For assays of DA and NA in whole brain, the homogenate from one rat brain was used, while 5-HT was determined on a separate brain homogenate. Where brains were dissected into discrete areas, five brains were pooled and used.for each determination. Noradrenaline and DA were extracted by the method of ANTON and SAYRE ( 1962) and the former estimated according to HAGGENDAL (1963) and the latter according to ANTON and SAYRE (1964). 5-Hydroxytryptamine levels were determined by the method of BOGDANSKI, PLETSCHER, BRODIE and UDENFRIEND (1956), because PE was found to significantly interfere with the more sensitive ninhydrin method of ANSELL and BEESON(1968). All amine values reported have been corrected for percentage recoveries (NA = 64.3 rtr 4.5%, DA = 73.0 + 4*9x, 5-HT = 86.7 f 2.2%. (Each recovery was determined from seven experiments.) For distribution studies (4-T)-/I-phenylethylamine hydrochloride (specific activity 1800 mCi/mmol, Aktiebolaget Atomenergi, Studsvik, Sweden) was used. This material was mixed with a carrier solution of PE to produce a concentration of 0.1 mg or 10 mg PE/ml solution containing 4 ,uCi of (4-T)-PE/ml. This was injected into rats 1 hr before death, with or without previous medication with iproniazid phosphate, 100 mg/kg, 24 hr previously. After light ether anaesthesia and decapitation, the brains were rapidly removed, cooled, and dissected into discrete regions. Five brains were combined for each determination. The tissues were homogenised in 10 ml ice cold 0.4~ perchloric acid, centrifuged at 18,000 g for 20 min, and the supernatant decanted. The precipitate was rinsed with 5 ml 0.4~ perchloric acid, centrifuged, and both supernatants combined. An aliquot was then mixed with toluene-Triton X mixture, and the radioactivity determined in a Philips scintillation counter. The results obtained were corrected and expressed as percentage disintegrations per min (dis/min) per g of wet tissue. For this purpose, the sum of the dis/min per g of the six brain regions examined was considered to be 100x, and the dis/min per g of each region then expressed as a percentage. All injections were made via the intraperitoneal route, and all drugs were dissolved in distilled water in a dose volume of 1 ml/l00 g body weight. All doses of drugs are expressed as the salts. RESULTS Table 1 shows that a dose of 100 mg PE/kg administered 1 hr prior to death, produced significant depletion in whole brain NA and DA levels with a small, but insignificant fall in 5-HT. When discrete regions of the brain were examined for amine levels after a dose of 100 mg PE/kg, a different picture emerged (Table 2). All regions of the brain examined were significantly depleted of NA, maximum depletion occurring in the cerebellum. Although maximum depletion of DA by PE also occurred in the cerebellum, the depletion was less marked than the depletion of NA (Table 2). No change in DA concentration was observed in the medulla oblongata-pons or hypothalamus regions. Changes in 5-HT levels were not marked although small but significant depletions occurred in the midbrain and cortex regions. When the respective per cent depletions of NA and DA after a dose of 100 mg PE/kg were compared for each brain region a correlation coefficient of 0.6034 (P > 0.05 < 0.1) was obtained.

/%Pheneylthylamine

and brain amines

665

Table 1. The effect of an intraperitoneal injection of p-phenylethylamine (100 mg/kg) on noradrenaline, dopamine and 5-hydroxytryptamine levels in rat whole brain Control

100 mg PE/kg

0.834 * 0.072 (8) 1.150 & 0.098 (8) 0.716 j, 0.042 (6)

0.211 & 0.024* (10) 0.854 * 0.043* (10) 0.619 & 0.061 (6)

Amine Noradrenaline Dopamine 5-Hydroxytryptamine

zdepletion 74.7 25.7 13.5

* P < 0.05. PE, or a water control, was injected 1 hr before death. DA and NA were estimated in the one series of brains, while 5-HT was estimated in a separate series of brains. The data is expressed as pg amine/g of brain (wet weight) * S.E.M. The results have been corrected for per cent recovery. Significance levels were determined by Student’s t-test. The number of estimations (n) is in brackets. Table 2. Noradrenaline,

dopamine and 5-hydroxytryptamine brain

Dopamine Brain region HYPOthalamus CIWZbellum oblongataMedulla

Control @410 * 0.069 *l&4

PE 100 mg/kg

concentrations

Noradrenaline % ratiot

0,541 $080

132

0.051

@OlO *($IO3*

20

0166 :$025

0.167 :$038

I01

in discrete regions of the rat

5-Hydroxytryptamine % ratiot

PE 100 mg/kg

2.776 f 0,337 #@44

I.057 f 0.082t 0~051:(~~ol2t

I.055 :70094

0.450 $(80;038t

43

0.895 1(5q.O41 0.862 ‘(2”040

96 88

0.309

38 17

Control

PE 100 mgjkg

0,817 f 0.042 (5)

0.883 * 0.033 (5)

z$ rattot.

Control

108

pans striatum Corpus

8.1 I4 ;7y508

5,575 ;$22lt

69

0.322 T7y2

@153 :8?015t

48

@522 :5W6

0,457 tS”,.O46

Midbrain

0786 57q.086

58

0.630 :;;026

0,251 ‘($342’

40

0.883 $5y026

0.767 $5;021*

87

Cortex

0673 ;;040

a452 i 0.1 l7* (8) 0.349 f$OOlt

52

0.321 f;O48

0,112 ;gOOl4*

35

Yzz”

:6zg

0.4669 >O,I

0.4759 >O.l

-

0.435 *0.014* (5) -0.0667 20.1

81

;

0.535 iO~O22 (5) -0.2798 >O,l

-

-

* P < 0.05 when compared to its water control. t P < 0401. : Noradrenaline, dopamine and 5-hydroxytryptamine concentrations after a dose of 100 mg PE/kg compared to control values expressed as a percentage of the control level of each amine. Animals were killed I hr after either water or 100 mg PE/kg. DA and NA results were obtained from the pooling of the discrete brain regions of five rats. 5-HT was determined in a separate series of brains. The number of experiments performed is in brackets and the data are expressed as erg amine/g of wet brain i S.E.M. All values have been corrected forpercentrecovery. Thecorrelation coefficient r and P have been calculated using the data in this table and column 4 of Table 3 for distribution of (CT)-PE after 100 mg PEjkg (see text).

When a dose of 1 mg (4-T)-PE/kg was injected i.p. 1 hr before death a fairly uniform distribution of (CT)-PE was observed in the various regions of the brain examined (Table 3). This 1 mg/kg dose of PE was a subeffective dose in that it had no effect on brain amine levels or any observable behavioural effects with or without iproniazid pretreatment. Since 100 mg PE/kg had a marked effect on the brain levels and distribution of the amines examined, 100 mg PE/kg was administered to a number of animals to determine whether such a dose of PE might alter the distribution of the labelled PE. Table 3 shows that this dose of PE changed the distribution of labelled PE so that highest concentrations were found primarily in the striatal region, and secondarily, in the hypothalamic region. When the distribution of labelled PE after a carrier dose of 100 mg/kg (Table 3) was correlated with the distribution of DA, NA and 5-HT in various regions of the brain both in control animals and after 100 mg PE/kg (Table 2). a significantly positive correlation was seen (Table 2) between DA levels and (CT)-PE levels. This finding suggests that PE tends

666

D.M. Table 3. Distribution

JACKSON

andD.B.

SMYTHE

of (4-T)-@phenylethylamine in rat brain after intraperitoneal injection 1 hr prior to death Treatment

Hypothalamus Cerebellum Medulla oblongata-pons Corpus striatum Midbrain Cortex

1 mg PE/kg

Iproniazid plus 1 mg PE/kg

1664 + 0.42 (5) 17.51 f 0.25 (5)

17.75 + 0.78 (5) 16.18 * 0.22 (5)

19.30 * 0.73 (7) 1540 & 0.24 (7)

17.13 f 16.20 k 16.08 * 1644 f

15.73 * 17.01 i 17.70 + 15.63 Jr

14.65 i 1944 * 16.05 + 15.15 +

0.61 0.49 0.28 0.31

(5) (5) (5) (5)

0.15 0.43 0.47 0.43

(5) (5) (5) (5)

100 mg PE/kg

0.32 0.96 0.14 0.25

(7) (7) (7) (7)

The carrier was either 1 or 100 mg PE/kg including 40 @i/kg (CT)-PE. When iproniazid phosphate 100 mg/kg was administered, the pretreatment occurred 24 hr prior to the 1 mg PE/kg dose. The results are expressed as per cent dis/min per g weight i S.E.M.(seeMethods).Thenumberofexperimentsperformedareinbrackets.

to concentrate in regions of high DA concentration. A similar, though trend was observed between (4-T)-PE levels and NA levels (both before mg PE/kg) but no trend was seen with 5-HT.

nonsignificant and after 100

DISCUSSION

The results show that PE depletes both NA and DA in rat whole brain confirming earlier work of JONSSON ef al. (1966) and FUXE et al. (1967). Although JACKSON and SMYTHE (unpublished observations) showed a significant fall in 5-HT levels in mouse brain after PE injection, no significant depletion of 5-HT was noted in rat whole brain in this study, although a trend suggesting there was a fall was supported by the significant depletion of 5-HT in midbrain and cortex regions. An examination of the amine concentrations in discrete regions of the brain after PE shows clearly that PE exerts a selective effect on various regions of the brain, with an indication that the depleting effect of PE in a discrete region of the brain on DA levels is positively correlated (although not significantly) with the effect on NA levels. These results confirm once again the necessity of examining discrete areas of the brain, rather than endogenous amine levels in whole brain, since in this study, for example, the fall in whole brain DA concentration was compounded by a rise in the hypothalamic region, no change in the medulla oblongata-pons region and a fall in cerebellar, striatal, midbrain and cortex regions. In spite of the expectation that (CT)-PE levels in various regions of the brain might mirror the effect on amine levels in the corresponding regions, no such relationship was in fact found using a subeffective dose of carrier PE (I mg/kg) with or without prior iproniazid pretreatment. However, when the high dose of 100 mg/kg of PE was used, the dose used for the amine depletion studies, a positive correlation was found between DA levels (both in controls and after 100 mg PE/kg) and the distribution of (4-T)-PE, indicating that this dose of PE showed altered distribution to an extent where the (4-T)-PE concentrated in DA rich areas. A similar trend was observed between NA and (CT)-PE distribution, but not with 5-HT. With the high dose of PE, the (4-T)-PE tended to concentrate primarily in the corpus striatum and the hypothalamus, agreeing with previously published results of NAKAJIMA

p-Phenylethylamine and brain amines

667

et al. (1964) and EDWARDS and BLAU (1972). This finding also shows that the dose of a stimulant such as PE is critical for its brain regional distribution, and ipso facto for pharmacological activity. No traces of administered PE can be detected in the brains of mice more than 20-30 min after injection (NAKAJIMA et al., 1964; JACKSONand SMYTHE,unpublished observation). Since peak effects after administration of PE on brain amine levels in guinea pigs (JACKSON, 1971), locomotor activity in mice (JACKSON,1972), and hyper- and hypothermic effects in mice (JACKSONand SMYTHE,1972) occur more than 20-30 min after injection, this would suggest firstly, that the radioactivity estimated in the present series of experiments (at least where iproniazid was not used) was not due wholly to PE, but at least partially to some metabolite of PE, and secondly, that many of the observed pharmacological effects of PE are due to metabolites and not to PE itself (JACKSON,197la). Two possible metabolites are phenylacetate, produced rapidly from PE by monoamineoxidase (BLASCHKO,1952), and phenylethanolamine, produced by the action of dopamine-fl-oxidase (CREVELING,DALY, WITKOPand UDENFRIEND, 1962). Phenylethylamine could compete with DA for dopamineP-oxidase, an event which would make more DA available at active receptor sites. This hypothesis is compatible with the positive correlation found between DA levels and (CT)-PE in the various regions of the brain, and is supported by preliminary experiments in this laboratory which indicate that agents which make more DA available in the CNS potentiate the behavioural effects of PE on locomotor activity in mice. Acknowledgeme~nts-We

wish to acknowledge the excellent assistance given us in the preparation manuscript by Dr. D. M. TEMPLEand Miss F. PENGLIS.

of the

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NP Vol. I2 No. 7-E

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D. M. JACKSON and D.B.

SMYTHE

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