Amphetamine-induced increase in rat cerebral blood flow: Apparent lack of catecholamine involvement

Brain Research, 149 (1978) 165-174 © Elsevier/North-HollandBiomedicalPress

165

AMPHETAMINE-INDUCED I N C R E A S E I N R A T C E R E B R A L B L O O D FLOW: APPARENT LACK OF CATECHOLAMINE INVOLVEMENT

KARIN NORBERG, BERNARD SCATTON and JAKOB KORF Synthdlabo, Department of Biology, Cerebral Circulation and Metabolism Unit, Neurochemistry Unit, 92220 Bagneux (France)

(Accepted October 20th, 1977)

SUMMARY The influence of O,L-amphetamine (5 mg/kg i.p.) on regional cerebral blood flow (CBF) in rats has been studied after surgically or pharmacologically induced depletion of brain catecholamines. (1) Bilateral removal of the superior cervical ganglion (one week before the experiment) did not prevent the amphetamine-induced augmentation of CBF present in intact animals to 2-4 times above the control value. Maximal changes occurred in the frontal and parietal cortex. (2) Destruction of the ascending noradrenergic pathways by uni- or bilateral injections of 6-hydroxydopamine, which decreased the noradrenaline (NA) level in the frontal cortex by 89 ~, was ineffective in abolishing the increase in CBF caused by the drug in the frontal cortex. (3) The involvement of other catecholaminergic systems was excluded by pretreatment of the rats with reserpine plus ct-methyl-p-tyrosine which reduced the levels of NA, dopamine and adrenaline in the frontal cortex with 92, 97 and 99 ~ respectively. Such treatment did not alter the effect of amphetamine on CBF in the frontal cortex. The results support the hypothesis that the action of amphetamine on CBF is not mainly mediated by catecholamines.

INTRODUCTION Amphetamine increases cerebral blood flow (CBF) in baboons and ratsa, 12. This effect has been claimed to occur via release of catecholamines12 which may be involved in the physiological regulation of CBF. In fact the intracerebral vessels are innervated by noradrenergic fibers from the superior cervical ganglion15,16 and by adrenergic projections of central origin (locus coeruleus), although the latter is less clearly established 6,s. Furthermore, changes in CBF may occur via catecholamine-induced altera-

166 tions of cerebral metabolism 11. However, no direct evidence exists, as yet, for the mediation by catecholamines of the amphetamine action on CBF. We have therefore investigated the effect of this drug on blood flow in rat brain virtually deprived of noradrenergic innervation of central and peripheral origin, as compared to the intact animal. MATERIALS AND METHODS

Experimental protocol The experiments were performed on male rats weighing 240-360 g (C.O.B.X., C.D.-strain, Charles River, France) housed at 22 °C and maintained on a 12 h light/dark cycle, and with free access to food and water. In some animals the superior cervical ganglion was removed bilaterally one week before measuring CBF. In others the ascending noradrenergic pathways (dorsal and ventral) were destroyed by uni- or bilateral injections of 6-hydroxydopamine (2/~g/#l) laterally to the pedunculus cerebellaris superior 21. The coordinates were anterior +0.25 mm, lateral ± 1.4 mm and vertical --1.8 mm 1°. These animals were allowed to recover for 3-5 weeks before further experimentation. Another group of rats were injected i.p. with reserpine (5 mg/kg Serpasil, Ciba-Geigy) and a-methyl-p-tyrosine methyl ester (250 mg/kg, Sigma) 27 and 3 h before CBF measurements, respectively. Anaesthesia was induced by 2-3 ~ halothane and the animals were maintained on artificial respiration with 70 ~ N20 and 30 ~ 02. The animals were ventilated to a pCO2 of 35-40 mm Hg. The body temperature was adjusted to 37 °C. Femoral arteries and femoral veins were cannulated to allow continuous blood pressure recording, anaerobic sampling of arterial blood and CBF measurements. After the operation the animals were allowed a stabilizing period of 20-30 min. O,L-amphetamine (5 mg/kg) was then given i.p. and CBF were measured 30 min later.

Cerebral bloodflow CBF was measured by the tissue uptake method 18 as modified for [14C]ethanolT. The tracer was infused i.v. for 30 sec. Arterial blood was sampled every 3 sec, during the infusion. Immediately after the last sample, saturated KCI was injected i.v., the rat was decapitated and the head was frozen in liquid nitrogen. The frontal, parietal and occipital cortex, hippocampus, thalamus, hypothalamus, pons and cerebellum were dissected at --12 °C for blood flow calculations 7.

Biochemistry Brain levels of dopamine (DA), noradrenaline (NA), and in some cases adrenaline (A) were determined using a radioenzymatic methodL The presence of [14C]ethanol did not interfere with this assay.

Statistics Statistical analyses were performed using two-tailed Student's t-test.

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169 RESULTS Effects o f amphetamine on rat CB F after bilateral removal o f the superior cervical ganglion In the rats sympathectomized one week prior to the experiment no changes of physiological parameters occurred apart from the arterial blood pressure which decreased by 20-30 mm Hg (Table I, lower part). Removal of the superior cervical ganglion did not prevent the amphetamine-induced increase in CBF seen in intact animals (Table I, upper part), but rather tended to enhance the response to the drug, especially in the frontal cortex. Effect o f amphetamine on rat C B F after destruction of the ascending noradrenergic pathways by 6-hydroxydopamine (1) Unilateral destruction. The experiments included three groups of rats: (a) Sham operated animals (intracerebral injection of vehicle); (b) Rats treated with 6-hydroxydopamine, and (c) Rats injected with both the neurotoxin and amphetamine. The lesioned rats showed a bilaterally increased CBF, although not significant, as compared to the sham operated animals (Table II, upper part). When amphetamine was administered to these animals, an increase in CBF of the same order of magnitude as that observed in unoperated rats (compare Tables I and II) was observed bilaterally in most of the brain regions studied. No alterations in the mean arterial blood pressure, body temperature, pO2, pCO2 and blood pH were observed (Table II, lower part). (2) Bilateral destruction. Table III shows that the bilateral lesion of the ascending noradrenergic pathwayby 6-hydroxydopamine did not prevent the effect of amphetamine on CBF in the frontal cortex and the pons (other areas were not investigated). The physiological parameters remained within the normal range as in the previous experiments. The lesion of the noradrenergic pathways by 6-hydroxydopamine resulted in a decrease of 89 ~ of NA levels in the frontal cortex, ipsilateral to the injection side. The

TABLE III Effect of amphetamine on rat regional CBFand on bloodpressureafter bilateraldestruction of the ascending noradrenergic pathways

All values are expressed as means with S.E.M. Treatment

Number Regional CBF in ml/ ( lO0 g • min) Mean arterial blood of rats pressure (mm Hg) Frontal Pons cortex

Controls 10 Sham operated 6 6-hydroxydopamine (H) bilateral 10 H + D,L-amphetamine 10

192 & 14 197 d: 20

107 -4- 5 110 d: 7

252 d: 30 128 :~ 8 394 ~: 36** 157 i 9*

125 4- 3 134 ± 4 127 -4- 2 135 ± 2

Significantlydifferent from controls *0.01 < P < 0.05; **0.001 < P < 0.01.

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171 TABLE V Effect of amphetamine on regional CBF of the rat after pretreatment with reserpine and a-methyl-ptyrosine Treatment

Number CBF in ml/( lO0 g.min) of rats Frontal cortex Pons

Reserpine + amethyl-p-tyrosine 10 Reserpine + amethyl-p-tyrosine + D,L-amphetamine 10

Mean arterial blood pressure (mmHg)

PaCOz (mmHg)

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178 :i: 15"

96 ± 7 106 i 7

37.9 5z 0.4

The values are expressed as means with S.E.M.; * P < 0.001.

levels of NA (means 4- S.E.M., 4 animals) in the frontal cortex of the control and the lesioned side were 429 dz 52 and 47 i 14 ng/g wet tissue, respectively. Effect o f amphetamine on rat C B F after treatment with reserpine plus a-methyl-p-tyrosine

In rats treated with a combination of reserpine (5 mg/kg i.p.) and a-methyl-ptyrosine (250 mg/kg i.p.) the levels of NA, DA and adrenaline in the frontal cortex were decreased by 92, 97 and 99 %, respectively, whereas the levels of these amines in the pons were no longer reliably detectable. The levels of NA and DA of the adrenal medulla were decreased by more than 80 ~ (Table IV). Administration of amphetamine to these animals resulted in a significant increase of CBF in the frontal cortex (Table V). The small increase in CBF of the pons caused by amphetamine in control animals was no longer significant in rats treated with reserpine and a-methyl-p-tyrosine. There were no significant increases in mean arterial blood pressure and arterial pCO2 (Table V). DISCUSSION There are two major hypotheses concerning the regulation of cerebral blood flow. One emphasizes the neurogenic control whilst the other takes into account metabolic factors. Whatever the sequence or predominance in the influence of these factors may be, there is a tight coupling between the increase in cerebral blood flow and cerebral metabolism 2°. Amphetamine may cause an increase in CBF by influencing one or both of these factors. The possible role of noradrenergic nerves in the control of CBF has been extensively discussed 5,19. In the present study it appeared that removal of the superior cervical ganglia led to slight increase in CBF in some regions of the cerebral cortex. These results as well as those of others 5,19favour the concept of sympathetic influence on the regulation of CBF. Lesions of the cerebral noradrenergic innervation of the forebrain (in particular the cerebral cortex, hypothalamus and the hippocampus) had no major consequences on the regional CBF. Thus, our data do not substantiate the hypothesesS, 17 of the in-

172 volvement of the cerebral adrenergic mechanisms in the regulation of CBF in normal animals. The increase in CBF caused by amphetamine might occur via release of catecholamines lz. Catecholamines appear to affect CBF via two different mechanisms: for instance NA might directly stimulate cerebral vascular receptors and both NA and DA stimulate cerebral energy metabolism, which in term leads to changes in CBF 1~,t3. The present experiments, however, show that the amphetamine-induced increase in CBF is not prevented either by removal of sympathetic innervation from the superior cervical ganglion or by both uni- and bilateral destruction of the ascending cerebral noradrenergic pathways, originating mainly in locus coeruleus. This supports the fact that sympathetic innervation of cerebral vessels as well as noradrenaline-induced metabolic changes in the brain are not involved in the enhancement of CBF following amphetamine administration. However, the action of this drug on CBF might be mediated by other cerebral catecholamines, e.g. DA, and/or by peripheral influence from the adrenal medulla. The experiments with reserpine and a-methyl-p-tyrosine tend to rule out this possibility since this treatment markedly lowered both the cerebral DA levels and the catecholamine content of the adrenal medulla. Urquilla et al. 24 reported that treatment with reserpine alone in doses lower than those used in the present study attenuated the effect of tyramine on CBF of the goat. It is of interest to note here that milder pretreatments that we used abolished any effect of amphetamine on the behaviourt4. These various effects indicate that catecholamines do undoubtedly play a role. Based on our experiments with reserpine and a-methyl-p-tyrosine we conclude that the increase in CBF caused by amphetamine does not need an intact catecholaminergic transmission in brain as well as in the adrenal medulla. The present data do not completely exclude the involvement of catecholamines in the action of amphetamine. In fact, both surgically- and pharmacologically-induced catecholamine depletion were not complete. Moreover, supersensitivity of adrenergic receptors to remaining catecholamines might have developed, possibly explaining the persistence of the amphetamine effect. Alternatively the possibility of a direct action of amphetamine on the smooth muscle cell in the brain vessel walls has to be considered1, 4,23. The action of amphetamine on CBF is possibly mediated by adrenergic fl-receptors of the brain vessels as propanolol was able to inhibit the increased CBF due to the stimulanta. Involvement of amphetamine metabolite(s) is unlikely as the effect of the drug on CBF takes place almost instantaneously after administration and since formation of vasoactive metabolites (e.g. p-hydroxynorephedrine) only occurs in adrenergic nerves 2z. Finally the changes in CBF cannot be attributed to changes in arterial blood pressure, pO2, pCO2 or body temperature. In conclusion, the increase in rat CBF caused by amphetamines does not seem to involve the release of catecholamines in the brain and/or from the adrenal medulla. A possible direct effect of the drug on vascular or neuronal receptors has to be considered for explaining the increase in CBF.

173 ACKNOWLEDGEMENTS The a u t h o r s t h a n k Dr. G. Bartholini for his constructive criticism, Miss Dani61e Pretesacque for C B F measurements, Mrs. A. Bianchetti for catecholamine determina t i o n a n d Mrs. S. Bischoffand B. Leroux for the surgery.

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