Amphetamine-induced inhibition of central noradrenergic neurons: A pharmacological analysis

Life Sciences, Vol . 24, pp . 2245-2254 Printed in the U .S .A .

Pergamon Press

AMPHETAMINE-INDUCED INHIBITION OF CENTRAL NORADRENERGIC NEURONS: A PHARMACOLOGICAL ANALYSIS Gäran Ergberg and Tagny H . Svensson* Department of Pharmacology University of Göteborg, Fack, 5-400 33 Göteborg, Sweden (Received in final form April 17, 1979)

Summary The amphetamine-induced inhibition of brain noradrenaline (NA) containing neurons in the rat locus coeruleus (LC) was pharmacologically analyzed utilizing single unit recording techniques . The presynaptic cY-receptor blocking agent yohimbine (10 mg~kg i .p ., 30 min before) largely prevented the amphetamine-induced depression of LC units in contrast to prazoain (0 .6 mg~kg i .p ., 30 min) or phenoxybenzamine (20 mg/kg, 30 min) which both show preference for postsynaptic a-receptors . The ß-receptor blocking agent, propranolol (10 mg/kg, 30 min), as well as the peripherally but not centrally active a-receptor blocking drug phentolamine (10 mg/kg, i .p ., 30 min), also did not block the amphetamine effect . The LC inhibition by amphetamine was blocked by pretreatment with reserpine (10 mg/kg, i.p ., 5 h), which caused almost total depletion of brain catecholamines . However, unlike the amphetamine-induced inhibition of central dopamine (DA) neurons the NA cell inhibition was not blocked by pretreatment with a tyraaine hydroxylase inhibitor (a-MT, 50 or 250 mg~kg i .p ., 30 min) . These results suggest that the amphetamine-induced inhibition of NA neurons in the LC is an indirect effect, mediated via activation of central a-receptors of presynaptic character . The lack of antogonism by a-MT indicate that the NA release by amphetamine, unlike its effect on brain DA, is not critically dependent on the rate of tyrosine hydroxylation . Thua the euphoriant action of amphetamine, which is blocked by a-MT, may be associated with release of DA rather than NA in brain . A large body of behavioural (1-5) and biochemical (6-9) evidence indicates that the central stimulation and euphoria induced by amphetamine is mediated by increased release of brain catecholanines (CA), thereby indirectly stimulating paatsynaptic CA-re ceptors . Based on biochemical evidence Corrodi et al . (10) suggested this poatsynaptic activation to cause a reduced impulse flow in the CA-neurons via neuronal feed-back pathways (c .f . 11) . Indeed, subsequent electrophysiological experiments have shown that amphetamine in low doses cause depression of firing of central dopamine (DA) neurons (12,13) as well as noradrenaline (NA) containing cells of the nucleus locus coeruleus (LC,14) . With respect to the inhibition of the DA neurons of the zone compacts, * To whom reprint requests should be addressed . 0024-3205/79/242245-09$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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substantia nigra, there exists also evidence in favor of the proposed neuronal, striatonigral feed-back pathway (15), The mechanism involved in the amphetamine induced inhibition of central NA neuron is, however, not known . Since this effect was attenuated by treatment with either chlorpromazine or piperoxane it may be an a-receptor mediated phenomenon (14,19) . Recent studies indicate the existence of different types of central (and priphral) a-adrenoreceptas (16,17), Thus, the LC neurons are equipped with adrenergic receptors, which pharmacologically behave like presynaptic aYeceptars (18-21) in con trast to the classical postsynaptic central a-receptors (17,21) . Receptors located paatsynaptically in identified projection areas to the LC are of ß-adrenergic type (22-25) although conceivably other LC synapses may yet be of a-character . Consequently, in the present study utilizing single unit recording techniques various adrenergic receptor blocking agents were used to characterize pharmacologically the receptor mediating the amphetamine-induced inhibition of LC neurons . Since there are documented peripheral inputs to the LC (26-28) phentolomine, which effectively blocks peripheral but poorly central a-odrenergic receptors (29), was also included in the study. Theoretically, the amphetamine induced inhibition of LC neurons could be either a direct effect on ackenergic receptors on the neurons (c .f . above) or, more likely, an indirect action (e,g . secondary to increased synaptic availability of NA similar to the LC inhibition after tricyclic antidepressants (30,31) . The indirect nature of the amphetamine-induced inhibition of DA neurorn in the SN was previously shown by its rorol prevention by pretreatment with D, L a-methyl-p-tyrosine methylester ( a-MT), a tyrosine hydroxylase inhibitor (13) . Thus, in the present study the putative antagonistic action of a-MT was explored on the amphetamine-induced LC inhibition . Since but little antagonism was seen, the possible protective action of previous depletion of endogenous neurotransmitter stores by resrpine was investigated, as our previous studies suggest that resrpine prevents the amphetamine induced release of NA (but not DA) in the central nervous system (5, 32) . Methods The electrophysiological exprimerMs were prformed essentially as has been described previously (18,31) on male Sprague Dawley rats weighing 250-300 g. Briefly, the animals were anesthetized with chloral hydrate (400mg/kg i .p .) and mounted in a streotaxic apparatus. Additional injections were given as needed . A burr hole was drilled with its centre at 1 .1 mm lateral and 1 .1 mm posterior to lambda . For recording a micropipette with a tip diameter of approximately 1 Eon filled with 2 M NaCI saturated with fast-green (impedance in vitro 3 - 6 Mil, measured at 135 Hz) was lowered into the brain by a hydraulic mlcrodrive . The electrode potentials were passed through a high input-impedance amplifier and filters . Each spike was discriminated and fed into an integrator being reset every 10 or 1 sec and finally displayed on an osclllascope, an audiomonita and an oscillographic recorder . The body temperature of the animals was kept at 36~i7°C by mearo of a heating pad . The recording sites were marked at tfie end of each expriment by iontopharetlc ejection of fast-green . The rats wre then prfused through the heart with 1096 formaldehyde and srial 50 ft frozen sectioro of the brain wre cut, mounted and stained with cresylviolet and counterstained with neutral red . The neurophysiological charactriatics of the cells wre identical to those previously described far NA neurons of the rat LC (14, 33) . Identified neuroro wre located In the LC .

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In a few rats the brain CA depletion induced by reserpine (10 mg/kg i .p . 5 h before sacrifice) was studied. The animals were killed by decapitation and the brairn were quickly taken out. Following homogenization in ice cold perchloric acid NA and DA were determined after isolation on a single Dowex 50 resin ~ previously described (34-36) . For statistical calculations either one-way analysis of variance followed by t-test (Table 1), a p x q factorial design (37, Figure 1) or Student's t-test (biochemical data) were used . Generally, p-values less than 0.05 were regarded as significant . Results d,Amphetamine alone produced in low doses coruistently inhibition of the IdA-neurons in the LC (see Fig . 1 and also Table 1) . The effect was clearly dose-dependent 0 0 a

mu

100 80 60 40

m

20 O

s/

O.is

b O.s

O.E

b bb _________ b 1 .2 L5

Dose amphetamine, mq/kp

3.0

FIG . 1 Log dose-response curve far the amphetamine-induced inhibition of NA-cells in the LC and this effect after pretreatment with prazaain (0 .6 mg/kg i .p ., 30 min, n = 5) yohimbine (10 mg/kg i .p ., 30 min, n=8) p--~p or reserpine (lOmg/kg i.p ., 5h, n=4) ß-~ . Lines have been plotted using lines regression analysis . Each value represents mean t S. E .M . A p x q factorial test (37) revealed that yohimbine~ pretreatment reduced ( p< 0.001 ) the amphetamine inhibition, whereas prazoain had no significant effect in this regard .

O-O

0-0 ,

and 100 per cent inhibition was obtained at 0 .6 - 0.8 mg/kg. The various adrenergic receptor blocking agents differed markedly in their effect on the amphetamine-induced LC inhibition, also when the different a~eceptar blocking agents ore compared . Thus, pretreatment with yohimbine, a drug which probably preferentially blocks a-receptors of presynaptic character (c .f . discussion), lagely blocked the LC inhibition by amphetanine (Fig . 1 and 2) and, in fact, it was impassible to obtain more than about 50 per cent inhibition of the NA cell firing rate regardless of the dose of d~nphetamine used . In contrast prazoain, which in the dose used preferentially blocks the classical pastsynaptic

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a-receptors (38), did not cause any significant antagonism of the amphetamine effect (Fig . 1 and 3) . Pherwxybenzamine appeared to have same antagonistic action, although the ED 50 value fa the amphetamine effect after this drug was not significantly different TABLE I Effect of carious pretreatments on the amphetamine-induced inhibition of NA neurons in the LC Treatment

(n)

d-amphetamine (A) prazosin + A phertoxyberrzam ine + A propranolol + A phentolamine + A a-MT ( 50 mg/kg ) + A a-MT ( 250 mg/kg ) + A

4 5 4 4 2 5 5

ED50 0.24 ± 0.26 ± 0.42 ± 0.26 ± 0.30 ± 0.39 ± 0.36 ±

0.04 0.04 0. 05 0.05 0.02 0.09 0.05

The different adrenergic blocking agents (prazoain HCI, 0.6 mg/kg ; phenoxybenzamine HCI, 10 mg/kg ; D, L-propranolol, 10 mg/kg ; phentolamine HCI, 10 mg/kg ) as well as D, Lax-methyltyrosine methylester HCI (a-MT ) were generally administered intraperitoneally 30 min prior to d-amphetamine, which was given intravenously in small incremental doses until 50 per cent inhibition of firing was attained ( ED50 ) . Shown are means ± S .E .M .s of (n) experiments . None of the combinations proved statistically significant from d-amphetamine alone. A ~lllllllllll~l~ll~

5 MIN FIG . 2 Antagonism by pretreatment with yohimbine ( lOmg/kg i .p . 30 min ) of the inhibitory effect of d-amphetamine ( A, 0.2 mg/kg at each arrow ) on the firing rate of a neuron in the LC .

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A 40

2249

YOH YOH

l~ l

l

U W N

N W Y N

OJ 5 MIN FIG . 3 Lack of antagonism by prazosin (0 .6 mg/kg i .p ., 30 min before ) of the d-amphetamine-induced ( A, 0.2 mg/kg at each arrow )depression of firing of a NA cell in the LC . The inhibition was, in contrast, antagonized by intravenously administered yohimbine ( YOH, 1 .0 + 1 .0 mg/kg ) .

from that obtained by amphetamine alone . Thus, the result with phenoxybsnzamine resembled that obtained with prazosin . The ß-blocking agent propranolol did not antagonize the amphetamine-induced inhibition of the LC neurons and also phentolamine, the peripherally but not centrally active a-adrenergic receptor blocking agent, was without significant effect in this regard . Pretreatment with the tyrosine hydroxylase inhibitor a-MT in two different doses (Table 1, Fig. 4) did not produce any significant antagonism of the amphetamine induced inhibition of the LC neurons. Some antagonism might, however, still exist although this effect was negligible when compared with the effect of reserpine pretreatment (Fig . 5), which almost totally blocked the amphetamine induced inhibition of LC neurons: The reduction in firing rate was less than 10 percent even when d-amphetamine in doses up to 55 mg/kg i .v . was administered . In contrast, the directly acting a-receptor agonist clonidine (18) produced its usual inhibition of the NA neurons in spite of the reserpine pretreatment and this effect was easily reversed by yohimbine (Fig . 5) . The reserpine regimen used caused an almost total depletion of brain CA (Table 2) .

LC Inhibition by Amphetamine

2250

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25 -

V W H O N W

Y d

N

0

FIG. 4 Lack of antagonistic action of pretreatment with a-MT ( 50 mg/kg i. p ., 30 min before ) on the inhibition of a cell in the LC by d-arlphetomine ( A, 0.15 mg/kg i .v . at arrows ), which was subsequently reversed by yohimbine ( YOH, 2 mg/kg i .v . ) .

50-, V W N O

W W

Y N

OJ 5 MIN FIG . 5 Prevention by resxpine pretreatment ( 10 mg/kg i .p ., 5 h before ) of the d-amphetanine-induced ( A, 0.5 mg/kg i .v . at arrows ) inhibition of a LC neuron . The directly acting a~eceptor ogonist clonidine ( CLON, 20~g i .v . ) rapidly silenced the cell, however, and this action was subsequently reversed by yohimbine ( YOH, 1 .0 mg/kg i .v . ) .

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TABLE 2 Depletion by reserpine of the endogenous stores of DA and NA in rat brain Treatmen t Vehicle Reserpine

DA 747 ± 26 (5) 11 . 3 ± 0.3 (3)

NA 315 ± 15 16 . 3 ± 1 .9

(5) (3)

Shown are means ± S . E. M. s of (n) determinations of the amines, expressed as na/g tissue . The reduction ore highly significant ( p < 0.001, both ). Discussion Previous data indicate that in the doses used yohimbine is a powerful antagonist at presynaptic a-receptors with much less effect on the postsynaptic a-receptors, whereas phenoxybenzamine preferentially blocks the postsynoptic a-receptors, with little effect on the presynaptic a-receptors (16,17,21) . Prazosin should effectively block the postsynaptic a-receptors almost exclusively (38,39) . Consequently, the results of this study suggest that the amphetamine-induced inhibition of noradrenergic neurons in the LC of the rat is mediated via a-receptors of presynaptic character. Since pretreatment with propranolol, which has a high lipid solubility and easily penetrates into the brain (40, 41) did not antagonize the LC inhibition by amphetamine, this effect is probably not a postsynaptically mediated response via a feed-back loop from e. g. cerebellum or hippocampus (22-25). A peripheral a-receptor mediated effect by amphetamine, secondarily inhibiting the LC, seems not likely in view of the virtual lack of antagonistic action of phentolamine (c .f . introduction), which blocks both pre- and postsynaptic peripheral a-receptors (42) . Thus, the previously found a-receptor in the LC of presynaptic character (c . f. introduction) may well mediate the inhibitory effect of amphetamine on these neurons. If so, amphetamine could cause activation of a collateral, inhibitory system (43) or, alternatively, release of adrenaline from topically active, inhibitory afferents originating in medulla oblongata and terminating in the LC (44) . Since previous depletion of endogenous monoamines by reserpine (45) almost totally blocked the amphetamine-induced inhibition of LC-neurons, one can conclude that in all probability it is an indirect effect mediated by NA (or epinephrine) and not a direct effect . The electrophysiological results thus are in excellent agreement with our previous behavioral and biochemical data (c . f. introduction) . Interestingly, pretreatment with a-MT did not prevent the amphetamine inhibition of the NA-cells of the LC in contrast to the previous experiments on DA-neurons (13) in which a-MT ( 50 mg/kg, 15-30 min before) completely blocked the amphetamine-induced inhibition of firing . Since earlier data (46,47) indicate maximal synthesis inhibition to occur within 30 min after administration of a-MT, the effect of amphetamine on brain NA neurons seem not to be dependent on intact tyrosine hydroxylation. Thus, whereas the DA effect is blocked by previous synthesis inhibition but largely unaffected by depletion of the bulk store of transmitter by reserpine (5,32), the effect on NA neurons is largely unaffected by inhibition of tyrosine hydroxylass (present data, 49) but blocked by previous ressrpine-induced depletion of NA (32) . This difference, which could reflect fundamental differences in the mode of operation of central DA and NA neurons (48), may also have clinical implications . Since pretreatment with a-MT largely blocked the euphoriant action of amphetamine in man (50), this effect may be linked to release of DA rather than NA .

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In conclusion, the amphetamine-induced inhibition of central NA neurons in the LC is an indirect effect mediated via activation of a-receptors of pharmacologically characterized presynaptic type . Acknowledgements Supported by the Swedish Medical Research Council (grant No . 4747), "Torsten och Ragnar Säderbergs Stiftelse", "Magnus Bergvalls Stiftelse" . Vilhelm och Martins Lundgrens Vetenskapsfond", "Fonden fär Neurobiologisk Forskning", and the Medical Faculty, University of Göteborg . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .

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