Effect of intraventricular infusion of dopamine and norepinephrine on motor activity

PhyMology and Behavior, Vol. 8, pp. 653-658, Brain Research Publications Inc., 1972. Printed in Great Britain.

Effect of Intraventricular Infusion of Dopamine and Norepinephrine on Motor Activity' M A R K A. G E Y E R , D A V I D S. S E G A L A N D A R N O L D J. M A N D E L L

Department of Psychiatry, School of Medicine, University of California, San Diego, La Jolla, California 92037 U.S.A. (Received 16 December 1971)

GEYER,M. A., D. S. SEo,~ ANDA. J. MANDELL.Effectofintraventricularinfusionofdopamineand norepinephrineon motor activity. PHYSIOL.Bra-~v. 8 (4) 653-658, 1972.--Dose-dependent increases in free-field activity resulted from intraventricular infusions of dopamine and norepinephrine in unrestrained rats. Norepinephrine was more potent than dopamine in producing this hyperactivity. Pretreatment with imipramine, which blocks neuronal uptake of cateeholamines, prevented the activation induced by infused dopamine without affecting the response to norepinephrine. However, the effects of dopamine and norepinephrine infusions were not differentially altered by haloperidol, an alleged dopamine receptor blocker. These results suggested that the activity produced by dopamine was due to its conversion to or displacement of norepinephrine and consequent excitation of noradrenergic receptors. Dopamine Norepinephrine Intraventricular infusion Rat

Cateeholamines

Locomotor activity

A GREATDEALof converging evidence has accumulated which is consistent with the alleged role of brain catecholamines (CA) in mediating states of behavioral excitation. It appears that increased functional levels of CAs are associated with arousal while deficits in these amines result in sedation or depression [24, 26]. Since neither dopamine (DA) nor norepinephrine (NE) readily passes the blood brain barrier, most of the support for this hypothesis stems from indirect manipulations of brain D A and NE with the use of various drugs or precursors. As a consequence of these indirect techniques, it has proven difficult to determine the relative contributions of noradrenergic and dopaminergic systems to the observed drug effects on behavior. This distinction is particularly important since recent studies indicate that in addition to its role as a precursor to NE, D A is also a neurotransmitter substance in several brain pathways [2]. Both NE and D A have been implicated in behavioral arousal. In the case of NE, although sedation or stupor has been observed following its direct injection into the brain [3, 19], more recent findings with relatively low doses are consistent with a behavioral activating role for NE [5, 15, 28]. For example, with intraventfieular infusions of low doses of N E in unrestrained rats, Segal and Mandell [28] found a dose-dependent increase in activity both in a free-field situation and in a Sidman avoidance task. In the case of DA, other investigators have suggested that the hyperactive states induced by such drugs as amphetamine, cocaine and apomorphine are primarily due to effects on brain D A [13, 20, 36]. Everett and Wiegand [7] and others [5, 26] have reported that motor excitation and the reversal of reserpineinduced behavioral sedation produced by the administration

Imipramine

Haloperidol

of dihydroxyphenylalanine (DOPA) (a precursor of D A and NE) paralleled increases in brain D A more closely than the alterations produced in NE levels. These findings, as well as the observed increase in behavioral activity following intracerebral injection of D A [3], have led some to suggest that D A rather than NE mediates behavioral arousal [8, 35]. However, more recent studies have suggested that the primary role of brain dopaminergic systems involves the regulation of stereotyped behavior patterns [10, 11, 18]. Fuxe and Ungerstedt [12] and Taylor and Snyder [33] have suggested that amphetamine induced increases in locomotor activity are due to the effects of amphetamine on brain noradrenergic systems while stereotyped behaviors produced by amphetamine are more closely associated with alterations in D A mediated neural pathways. The present study was designed to provide a direct comparison of the effects of D A and NE on various patterns of behavior. Using a modification of the technique devised by Segal [27], unanesthetized.'rats were chronically infused, intraventcicularly, with graded doses of D A or NE. It was concluded that the stimulation produced by D A was due to an indirect effect on noradrenergic neurons since imipramine pretreatment selectively prevented the increased behavioral activity following D A administration. No D A induced increase in stereotyped behavior patterns was observed. EXPERIMENT 1

Method Animals. Sixty male Sprague-Dawley rats (Simonsen) weighing 200-300 g were housed individually in constant

1This work was supported in part by research grants ( # MH-18065 and ~ MH-14360) from National Institute of Mental Health. We thank P. Josephine Branson for her assistance with surgery and Ronald Browne for running Sidman animals. 653

654 light and had ad lib access to food and water. Each animal was permanently implanted with a stainless steel cannula extending to a point 1 m m above the fight lateral ventricle (De G r e e t : A 5.4, M L 2.0, H+4.0), using sodium nembutal anesthesia (40 mg/kg). When in place, the infusion needle protruded 1 mm below the cannula into the ventricle. Apparatus. Activity was measured in 18 × 18 × 11 in. soundproofed Plexiglas chambers, the floors of which were electronically divided into quadrants. Cross-overs from one quadrant to another and reafings (touching the wall 5.5 in. above the floor) were recorded automatically. The infusion apparatus consisted of a counterbalanced arm with a polyethylene tube leading through a swivel to the infusion needle. This needle was attached to the cannula, leaving the animal unrestrained. All infusions were done with a constant speed mechanical infusion pump at a rate of 20 ~1 per hour. Procedure. The design of the present infusion apparatus obviated the need for any restraining saddle or prolonged habituation sessions as used previously [27]. Instead, 4 or 5 days after cannulation, each animal was placed in the experimental chamber for one hour of adaptation. The animals were tested 1, 3, and in some cases 5 days later. A test session consisted of a 1 hr warm-up period after which either isotonic saline, 1.0, 3.0, 6.0, 12.0 ~tg/~l D A (3-hydroxytyramine HCI, Calbiochem), or 1.0, 2.0, 3.0, 6.0 tzg/l~l NE (d 1-arterenol HC1, Calbioehem) was infused for one hour. Thus the hour of infusion was relatively unaffected by handling or the variable activity typically exhibited during the first hr in the chamber. The infusion was begun by simply switching on the infusion pump, without disturbing the animals. All drugs were weighed and dissolved in isotonic saline immediately before use. Each animal received the same treatment on each of the 2 or 3 test days. Since there were no significant differences between the results obtained for the first, second, and third infusions, these data were pooled. Subsequent infusions of Methylene Blue dye and gross dissections of the brain were used to verify the cannula placements. These tests revealed that the dye had effectively perfused much of the ventricular system, typically reaching the third and fourth ventricles. Statistical significance was determined with the use of the Mann-Whitney U test [30].

Results Both D A (Fig. 1, open bars) and NE (Fig. 2, open bars) produced significant dose-dependent increases in locomotor activity during the one hr period of infusion (p<0.05). Similar increases were observed in the number of rearings by D A or NE infused animals when compared to the saline infused controls (p <0.025). Several differences between the effects of NE and D A infusions were apparent. While D A infusions resulted in progressive increases in ambulation and rearing up to a dose of 12.0 ~tg/~l, the maximally effective dose of NE was 2.0 I~g/Id. Furthermore N E was considerably more effective in producing hyperactivity than was DA. In fact, the infusion of 2.0 i~g/~tlN E produced a significantly greater increase in both cross-overs and rearing than did the 12.0 tzg/Izl dose of D A (p < 0.01). These effects appeared within 15 min after initiation of infusion for all the test animals with the exception of those receiving the lowest dose of D A (1.0 ~g/~zl). This group did not show a marked increase in activity until the final 15 min of infusion. Periodic observation of the animals during infusion revealed no obvious occurrence of bizarre behavior patterns. However, consistent differences between the behavioral

GEYER, SEGAL AND MANDELL

100

THE EFFECT OF IMIPRAMINE PRETREATMENT ON OOPAMINE INFUSIONS

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1"

80

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=1=

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> o

I

o r~

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10

0 SALINE

DA 1.0

4 DA 3.0

I15~ DA 6.0

9 DA 12.0

FIG. 1. Mean number of cross-overs ( i S E M ) during one hour infusion of graded doses of dopamine (DA) or saline following pretreatment with saline or 5.0 mg/kg i.p. imipramine. Imipramine pretreatment prevented the DA-induced increase in cross-overs at all doses of DA tested. Numbers within blocks represent number of infusion sessions included in mean. * =significantly greater than saline controls p<0.05.

effects of D A and NE infusions were obselved. Even at the lowest dose, animals infused with NE exhibited more sniffing and chewing, and were more reactive to handling than either normals or D A infused animals. In addition, NE infused rats showed a marked flaccidity when held gently. Dopamine infusions, on the other hand, did not produce the continuous motor activity characteristic of NE. Rather, these animals often lay flat and occasionally moved in a crawling posture.

Discussion The concomitant dose-dependent increases in cross-overs and rearings during the infusion of D A confirm and extend previous findings of hyperactivity resulting from direct administration of D A into the brains of cats [3]. Corroborative evidence with respect to the generality of this hyperactivity has been obtained in this laboratory using a continuous Sidman avoidance task (unpublished observations). While a dose of 3.0 ~.g/t.d D A d i d not significantlyaffect the rate of avoidance responding, infusions of 6.0 ~g/~zl D A o r 3.0 ~tg/~tl NE resulted in significant increases over the baseline response rates. The results presented here with NE infusions further confirm the work of Segal and Mandell [28] and provide for a comparison of the potencies of D A and N E in the experimental situation.

CATECHOLAMINES AND BEHAVIOR

200-

655

THE EFFECT OF IMIPRAMINE PRETREATMENT ON NOREPINEPHRINE INFUSIONS

to NE intraneuronally by dopamine-13-hydroxylase (DBH) [21, 32,] IMI pretreatment should preclude hyperactivity in D A infused animals if D A is increasing activity via its conversion to or displacement of NE.

180 .

Saline

Method Thirty-six male rats were tested according to the procedures described for Experiment 1 with the exception that these animals were pretreated with 5.0 mg/kg IP IMI (Tofranil, Geigy Pharmaceuticals) immediately before being placed in the activity chambers. In rats, this dose o f l M I has been shown to potentiate the behavioral effects of amphetamine [31] and NE infusions [27], while having no significant effect by itself. The D A or the N E infusions were begun one hour after the IMI injections.

160 •

B Irnipramine 140

° el-

10080. °

80° 40-

ii 2 o

SAUNE

NE 1.0

NE 2.0

NE 3.0

NE 6.0

FIG. 2. Mean number of cross-overs (+SEM) during one hour infusion of graded doses of norepinephrine (NE) or saline following pretreatment with saline or 5.0 mg/kg i.p. imipramine. Imipramine pretreatment did not significantly alter the NE induced increase in cross-overs. Numbers within blocks represent number of infusion sessions included in mean. *=significantly greater than saline controls p < 0.05.

Since the racemic mixture of NE has been used in these studies even though the dextro isomer is relatively inactive (unpublished observations), the effective dose of 12.0 ~g/~l D A is approximately 12 times that of 2.0 I~g/IzlNE on a molar equivalent basis. That this dose of N E is significantly more potent than the higher dose of D A argues against the possibility that infused NE might be having its primary effect on dopaminergic systems. On the assumption that D A stimulates D A receptors as effectively as does N-E, D A should be at least as potent as equimolar doses of NE. This assumption is supported by studies showing intracerebral D A to be more potent than NE in producing postural asymmetries mediated by dopaminergic neurons [34]. rxr~rcr

Results Figure 1 shows the comparison between the mean ( + S E M ) number of cross-overs produced during one hour of infusion with various doses of D A or saline following saline (open bars) or IMI (shaded bars) pretreatment. There was no significant effect of IMI in the saline infused group; however IMI pretreatment did prevent the D A induced increase in locomotor activity at all doses of D A tested Co <0.05). Imipramine pretreatment also prevented the increase in rearing produced by DA infusions. There was no significant difference in either index of behavioral activity between D A and saline infused animals pretreated with IMI. In contrast, the N E induced increase in cross-overs and rearings was unaltered by IMI pretreatment (Fig. 2). Discussion Imipramine pretreatment did not significantly alter the activity of either control of NE infused animals, although a significant increase was reported by Segal [27] using the same doses of NE and IMI, but with no intervening warm-up period as necessitated in these earlier studies. On the other hand, such pretreatment did prevent the behavioral stimulation produced by exogenously administered DA. Since responses to both the low (1.0 ~g/l~l) and high (6.0 ~tg/~tl) doses of D A wele blocked by IMI, this effect is probably not attributable to an overstimulaUon by an excess of functionally available DA. Indeed, the IMI groups receiving D A were virtually indistinguishable behaviorally from the IMI controls (Fig. 1). Since IMI blocks the uptake of CAs into central adrenergic neurons [13, 25], it may have prevented the D A induced behavioral activity by reducing the amount of NE converted from or displaced by the infused DA. The present findings with IMI pretreatment indicate that infused D A may be stimulating activity indirectly via excitation of central noradrenergic systems, rather than by a direct effect on dopaminergic or noradrenergic receptors.

2

Since D A is the biological precursor to NE, the increased locomotor activity produced by D A infusions may be due to the conversion of D A to NE, rather than to a direct effect of D A on dopaminergic systems. Alternatively, D A may displace intraneuronal NE, causing an increase in its release; or D A may have a direct effect on N E receptors. To test the hypothesis that D A is acting indirectly, animals were pretreated with imipramine (IMI), which prevents the uptake of CAs into adrenergic neurons [13, 25]. Since D A is converted

EXVra~MENT 3 If D A infusions increase locomotor activity indirectly via noradrenergic systems, then blockade of central D A receptors should either have no affect on the response to infused D A or similarly affect the response to D A and NE infusions. This hypothesis was tested by pretreating animals with the presumed potent D A receptor blocker haloperidol (HAL) [1, 17, 20].

656

GEYER, SEGAL AND MANDELL

Method Forty male Sprague-Dawley rats weighing 200-300 g were housed and cannulated as in the previous experiments. Activity was measured as cross-overs in 15 × 15 × 12 in. sound-proofed chambers divided into quadrants by photo-beams. All animals were given a 30 min adaptation session on Day 1. Activity was monitored for one hr with or without infusions immediately after an hour warm-up on Days 2, 4, and in some cases 6. Immediately before being placed in the chambers, the animals were injected IP with isotonic saline or 0.1, 0.3 or 0.5 mg/kg H A L (Haldol Concentrate, McNeil). Experimental animals were infused with either 1.0 ~tg/~l NE or 3.0 0tg/tzl DA. Each individual animal received the same treatment on successive test days. Results and Discussion As in Experiment 1, both 3.0 ~tg/~l D A and 1.0 ~g/~l NE infusions produced significant increases in activity when compared to the saline pretreated controls (p<0.001). Haloperidol pretreatment resulted in a dose-dependent reduction in the activity of both control and experimental animals (see Fig. 3). This dose range of H A L has been reported to selectively block D A receptors in a variety of experimental situations [1, 17, 20], yet the response to infused D A in this study was not totally antagonized by HAL. While the reductions produced by the 0.3 and 0.5 mg/kg

THE EFFECTS OF HALOPERIDOL ON DOPAMINE A N D NOREPINEPHRINE

INFUSIONS

200-

~ DA

160

D NE cn ft. 120

uJ > O I

O n.0

80

40

SALINE

HAL 0.1 ~mg

HAL 0.3 m.~Kg HAL 0.5 ~ g

FIG. 3. Comparison of the effects of graded doses of haloperidol (i.p.) on cross-overs induced by intraventricular infusion of NE (1.0 ~tg/~.l)and DA (3.0 ~zg/~l). Haloperidol proportionally reduced the effects of both amines. Numbers within blocks indicate number of infusion sessions included in mean. * =significantly greater than appropriate controls p < 0.05.

doses were significant for both control and experimental animals (p<0.05), D A and NE infusions still resulted in increased activity at each dose when compared to the appropriate controls (p <0.05). In addition, the decrease in NE and D A induced activity was approximately proportional for all doses of H A L tested. Thus the infusion of D A and NE was not differentially affected by H A L pretreatment.

DISCUSSION

Experiment 1 demonstrated that increasing brain D A or NE by the relatively direct method of gradual intraventricular infusion leads to behavioral activation in the rat. This hyperactivity has been measured as cross-overs and rearings in an open field and as the rate of response in a Sidman avoidance task. Norepinephrine proved to be more potent than D A in increasing locomotor activity. These findings lend further support to the general hypothesis of a direct relationship between central CA activity and behavioral excitation. The stimulation produced by infused D A reported hele may not be due to a direct effect of D A on D A receptors, as indicated by the results of Experiment 2. In order to be converted to NE, D A must be taken up into the neurons in which DBH is located [21, 32]. Similarly, to cause the release of NE by displacement, D A must be taken up by NE containing cells. Since I M I blocks this uptake process at the cell membrane [13], it prevents both the conversion of infused D A to NE and the displacement of NE by D A but may in fact increase the exposure of infused N E or D A to postsynaptic receptors. The results showed that while I M I did not alter the effects of NE infusion it blocked the excitatory effects of infused DA. These results are consistent with the view that D A infusions do not increase spontaneous motor activity by a direct action of D A on dopaminergic receptors. Rather the behavioral activation by exogenously administered D A may be attributable to excitation of noradrenergic systems following the displacement of NE or the conversion of D A to NE. The rate and extent of this conversion or displacement may also explain the relative potencies of the two amines in increasing ambulation, and the delayed onset of effect produced by low doses of DA. In a recent study, Sanghvi, Urquiaga, and Gershon [22] reported an analogous effect with IMI in dogs. They found that pretreatments with I M I prevented the behavioral effects of L-DOPA. They attributed this result to a lack of conversion of L-DOPA to D A and/or N E following blockade of the C A membrane pump by IMI. In contrast, IMI given after L-DOPA potentiated the behavioral effects of L-DOPA Presumably the effect of the D A and/or NE synthesized from the L - D O P A was augmented by the IMI inhibition of uptaka inactivation. Imipramine has also been reported to block amine uptake by neurons containing 5-hydroxytryptamine (5HT) [4]. If 5HT can produce behavioral activation as some investigators have suggested [7, 14], then the hyperactivity produced by infused D A may be a result of the displacement of 5HT by D A and consequent release of 5HT. As with NE, I M I pretreatment would prevent this displacement by inhibiting the uptake process at the 5HT cell membrane. While intraventricular infusions of 5HT in the free-field situation have not resulted in large idecreases, 5HT (6.0 oi 12.0 ~tg/izl) infusions significantly lowered the response rate in the Sidman avoidance

CATECHOLAMINES AND BEHAVIOR

657

task (unpublished observations) in contrast to the increased rate found with infused DA. However, further studies with other uptake inhibitors related to I M I are in progress to examine the possible contribution of 5HT displacement to the D A induced hyperactivity. Another alternative explanation for the results of Experiments 1 and 2 must also be considered. Dopamine is thought to be extremely important in the mediation of some patterns of stereotyped behavior [11, 12, 16, 18, 23]. It may well be that some of these behavioral vatterns are incompatible with gross locomotor hyperactivity. Thus, the relative ineffectiveness of infused D A in increasing ambulation might be due to the fact that it more effectively stimulates D A receptors than does infused N E [34]. Therefore, D A would result in more competing behavior than is produced by N E infusions. Pretreatment with IMI may simply increase the relative amount of stereotyped behavior by preventing the inactivation of D A by re-uptake, thus increasing the relative effective contribution of D A receptor stimulation. However, this explanation is questionable since little if any stereotypical behavior was exhibited by the D A infused animals, especially those pretreated with IMI. The results of Experiment 3 also conflict with this competing response explanation. This explanation would suggest that blockade of central D A receptors by H A L might have the opposite effect of 1MI on the response to D A infusions.

Since H A L reportedly blocks D A receptors in the brain [1, 17, 20], pretreatment with H A L should prevent the direct effect of D A on dopaminergic receptors and the consequent stereotyped behavior known to be associated with stimulation of these receptors. Thus the effect of D A following its conversion to N E should predominate, resulting in more ambulation than without H A L pretreatment. The low dose of H A L (0.1 mg/kg) did not significantly increase the activation produced by infused DA, and in fact a mean decrease was obtained (see Fig. 3). The failure of H A L to augment the response to D A is inconsistent with the explanation that N E is more potent than D A because of the elicitation, by DA, of competing stereotyped responses. Conversely, the results with H A L are compatible with the hypothesis that D A affects locomotor activity primarily via displacement of or conversion to NE. Haloperidol has been shown to be a far more potent blocker of central D A receptors than of NE receptors [1, 17, 20]. Therefore the proportional reduction of both D A and NE induced activity by H A L pretreatment suggests that the action of infused N E and D A occurred at the same site, presumably either at dopaminergic or noradrenergic synapses. In view of the results with I M I pretreatment, it seems reasonable to attribute the reduction in activity by H A L to its effect on N E receptors and the hyperactivity produced by D A to its conversion to or displacement of NE.

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