Interactions between d -Ala-Met-enkephalin, A10 dopaminergic neurones, and spontaneous behaviour in the rat

Behavioural Brain Research, I (1980) 3-24 © Elsevier/North-Holland Biomedical Press

3

Research Papers INTERACTIONS BETWEEN I>ALA-MET-ENKEPHALIN, A10 DOPAMINERGIC NEURONES, AND SPONTANEOUS BEHAVIOUR IN THE RAT**

ANN E. KELLEY*, LOUIS STINUS and SUSAN D. IVERSEN

Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge CB2 3EB ( U.K.) and ( L.S.) Laboratoire de Neurobiologie des Comportements, Universit6 de Bordeaux 1, Avenue des Facult~s, 33405 Talence (France) (Received July 24th. 1979) (Revised version accepted October 4th. 1979)

SUMMARY

We have investigated the interaction between opioid peptides and dopaminergic AI0 (DA-AI0) neurones in the ventral tegmental area (VTA). The behavioural consequences of VTA infusion of D-Ah-Met-enkephalinamide (DALA) were analyzed. DALA elicited a dose-dependent increase in locomotor activity measured in photocell cages and the circular corridor. Observations in the open field and in a hole box revealed thet DAI.A-induced behavioural stimulation was characterized by enhancement of locomotion, rearing, and number of hole visits, while grooming time and duration of hdle visits were decreased. DALA-induced stimulation was reserved by naloxone, and was completely blocked by 6-OHDA destruction of DA-AI0 terminals. D-Amphetamineinduced behavioural activation was potentiated by simultaneous VTA infusion

* To whom correspondence should be sent at present address: Mailman Research Laboratories. McLean Hospital, Belmont, Mass., U.S.A. ** This work was presented in pan at the IIl European Neurosciences Meeting, Rome, September 11-14, 1979,

4

of DALA which indicates that the behavioural response to DALA is dependent on DA-AI0 neuronal activity. It is postulated that stimulation of opiate receptors exerts a pres)/naptic inhibition of an inhibitory input to DA-AI0 neurones (e.g. GABA or dendritic D,~.), thus releasing dopaminergic activity. In contrast to the acute effect, the D-amphetamine response was strongly attenuated 4 h, l and 6 days after VTA infusion of DALA, and returned to normal only at 14 days. This long-lasting modification may reflect decreased activity of opioid neurones, releasing the inhibition of DA-AI0 neurones. Our findings suggest that endogenous opioid peptides may exert a modulatory influence on the dopaminergic mesocorticolimbic system.

INTRODUCTION

The discovery of the existence of endogenous opioid peptides in the brain has opened a new field of investigation of neurotransmitter function. In parallel, extensive studies have been carried out on the distribution and binding properties of the receptor for morphine [48, 60]. It is believed that the peptides Leu- and Met-enkephalin and the endorphins are the endogenous ligands for morphine receptors, although at present the exact relationship is not clearly understood. Many studies have focussed both on the role of these opioid peptides in the CNS and on mechanisms of action of narcotic drugs. Neuronal localization of opioid peptides [13, 16, 25, 47, 58] has provided the basis for electrophysiological, biochemical and behavioural investigations. Presently there is little information concerning the precise distribution of enkephalin and endorphin containing neurones; however, there is evidence that the highest concentration of enkephalin and opiate receptors are found in the limbic and extrapyramidal system [2, 3, 24, 42], areas implicated in motivational mechanisms and sensory-motor integration. In addition there is a distinct overlap between the distribution of enkephalin immanofluorescence, opiate receptors and dopamine (DA) cell bodies and terrc,iaals in areas such as substantia nigra, ventral tegrnental area, striatum and nucleus accumbens [2, 3, 24, 30, 69]. There are a number of complex pharmacological interactions between opiates and other transmitters, lontophoretic application of Met-enkephalin, fl-end0rphin and morphine has revealed that opiate substances have an inhibitory action in most brain sites tested [7, 20-22, 45, 75]. Moreover, opiates have been shown to be potent inhibitors of noradrenaline [671, acetylcholine [29] and DA release [39, 64], effects all of which are blocked by opiate receptor antagonists such as naloxone. There is substantial evidence which favours an opiate-DA interaction: (i) the parallel CNS distribution, (ii) DA synthesis and turnover is

increased by morphine and enkephalin [I, 5, 36, 65, 71], (iii) a number of be-havioural and physiological responses to opiates can be blocked by specific DA antagonists [31, 37, 70], although DA and opiate receptors can be differentiated [28], (iv) opiate receptors are known to be present on striatal and mesolimbic DA neurones [51, 52]. However, there are numerous contradictions in the opiate literature. For example, Gauchy et al. IOund that morphine increases DA synthesis and also increases DA release [19], whereas B-endorphin and enkephalin have been found to inhibit K~-evoked DA release [39, 64]. Behavioural studies are also conflicting. Uhilateral injection of morphine into the substantia nigra (SN) elicits contralateral turning which is blocked by unilateral 6-OHDA lesion of DA cell bodies in the SN [2~. A further study implicating stimulation of DA systems demonstrated morphine potentiation of apomorphine stereotypy [55]. In contrast, morphine has also been shown to block stereotypy induced by amphetamine or apomorphine [53] and to attenuate amphetamine-induced rotation after unilateral 6-OHDA lesion of the caudate nucleus [6]. The discrepancies noted on both biochemical and behavioural experiments are partly due to the fact that much work is based on systemic or intracerebral ventricular administration of opiates, which involves stimulating opiate receptors at many different levels of the CNS. Considering that there are multiple opiate receptors [40, 41], opiate substances may have differential effects depending on the site of action. It would appear that local infusion of opiate agonists and antagonists into discrete CNS areas can more clearly assess the functional role of endogenous opiate peptides. A number of recent investigations found that infusion of morphine or enkephalin into the area of DA cell bodies elicits a behavioural activation [8, 31]. There are high concentrations of enkephalin in the ventral tegmental area (VTA), near the origin of the mesocorticolimbic DA AI0 neurones (DA-AI0) [69]. We have therefore conducted a systematic investigation of the behavioural responses to enkephalin infusion into the VTA and have evaluated the involvement of DA-A10 neurones in the observed effects. The experiments have been carried out using D-Ala-Met-enkephalinamide, a stable synthetic analog of Met-enkephalin [49]. METHODS

Animals and surgery Male Sprague-Dawley rats weighing 300-330 g at the time of surgery were used. All rats were stereotaxicaUy implanted with bilateral stainless steel guide cannulae (23-gauge) aimed at the VTA (coordinates in mm: - 3.3 from bregma, + 0.5 lateral, - 5.6 from skull, incisor Bar 5.0 above interaural line). At the time of surgery the inner infusion cannulae (30-gauge) to be used in experiments were lowered through the guide to the VTA ( - 8.9 from skull), in order to minimize

non-specific effects of tissue damage during experiment infusions. The cannulae guides were kept clear with wire stylets. AH animals were housed singly or in twos, and were allowed at least one week recovery period before the start of behavioural testing. In a lesion experiment (IV), in addition to the above procedures, rats were infused bilaterally with 6-hydroxydopamine (6-OHDA) (8/~g in 2/~1 over 14 min) in the nucleus accumbens (n. accumbens). Control rats were infused with the vehicle solution alone (0.9% saline containing 0.2 mg/ml ascorbic acid). Thirty minutes before infusion of 6-OHDA the rats were pretreated with pargyline (50 mg/kg i.p.).

Microinfusions and materials D-Ala-Met-enkephalinamide (DALA) (Bachem, Torrance, Calif.) was stored at - 20 °C in freeze-dried aliquots of 200 #g. For each experiment the appropriate concentrations of DALA were prepared by the addition of sterile 0.9% saline (151 mM). All microinfusions were bilateral. The inner infusion cannulae were connected to a microdrive pump by means of polyethylene tubing. Before infusion the guide cannulae were cleared with dental square broaches. During the infusion procedure the infusion cannulae were lowered to the VTA, and DALA or saline was infused in a volume of i #1 over 2 min, followed by I rain diffusion time, removal ofthe infusion cannulae, and replacement of stylets. The rats were allowed to move freely in a small open cage during actual infusion. Before any testing, all rats were given a preliminary saline infusion in order to adapt them to the procedure and also to minimize mechanical effects during experimental infusions. Peripheral injections were administered either intraperitoneaily (i.p.) or subcutaneously (s.c.). Drugs administered in this manner were D-amphetamine sulphate (D-amph) (Smith, Kline and French), naloxone hydrochloride (Endo Laboratories) and apomorphine sulphate (APO) (MacFarlan Ltd.). Histology and statistics At the completion of each experiment, rats were deeply anaesthetized, perfused intracardially with 109/o formalin and the brains removed. The brains were subsequently cut and stained with cresyl violet for location of cannulae tracks. Statistical analysis of all data was conducted by multifactor analysis of variance [73], with repeated measures on factors where appropriate (e.g. when animals were used as their own control).

Experimental procedures (I) Time course and dose-dependence of DALA-induced behavioural activation Locomotor activity was measured in two kinds of testing apparatus, in order to ensure that any effects observed were not dependent on the actual method of testing. Activity was measured in wire cages (35 cm × 25 cm × 25 cm) equipped with 2 photocells, whose interruption registered counts on a recorder outside the testing room. Locomotor activity was also measured in a circular corridor, 12 cm wide and 170 cm long, equipped with 4 photocells. In this and other locomotor activity experiments the rats were well habituated to the apparatus prior to behavioural testing. On a test day they were habituated for 1 h before infusion, infused with DALA or saline, and returned to the apparatus. The behavioural response to DALA was observed in two separate experiments: (i) the effect of VTA infusion of 2.5 or 5.0 ttg (3.9 or 7.8 nmol) DALA on activity in photocell cages and (ii) the effect of VTA infusion of a wider range of doses of DALA (0.02, 0.1, 0.5, 2.5, 12.5/~g) (0.03, 0.15, 0.78, 3.9, 7.8 and 12.5 nmol) on activity in the circular corridor. These amounts of the drug did not modify the total molar concentration of the isotonic saline solution (151 mM).

(il) Bet;avioural analysis of DA LA-induced activation (i) Openfield. Qualitative analysis of spontaneous activity was conducted in an open field after infusion of DALA (1 ~ug, 1.56 nmol) or saline. The white. square open field (100 cm x 100 cm x 70 cm) was brightly illuminated (0.14 ft.lamberts) and had the floor divided into squares. Masking white noise was present (approx. 70 dB). Behaviours recorded over a 10 min period were locomotion and rearing, along the walls and in the centre, and grooming. Behaviour was observed via video and recorded with an event recorder. Prior to testing the rats were habituated to the open field, for two 20 min habituation sessions. On a test day they were infused and placed in the open field ! 5 rain later. (ii) 8-Hole box. Behaviour was recorded in an 8-hole box after infusion of DALA (2.5/Jg, 3.9 nmol) or saline. The 8-hole box was a circular open field (70 cm diameter, 50 cm high) with 8 holes (2.5 cm diameter) spaced equally around the wall. A photocell fixed outside each hole measured the number and duration of hole visits. A radar detector was located !.5 m above the box which measured global motor activity (reference, type MRXI 9AC, Phillips RTC). The rats were previously habituated to the box for 1 h prior to testing. On a test day they were infused and then immediately placed in the hole box for 30 min.

(III) Opiate specificity of DALA-induced activation The opiate specificity of the DALA response was tested in photocell cages with naloxone. On a control day one group of rats received a VTA infusion of

8

saline, and saline i.p., and another received saline-VTA and naloxone i.p. (1 mg/kg) (in order to determine effects of naloxone alone). On a test day the two groups were infused with DALA (2.5 pg, 3.9 nmol); 40 rain later one group was injected i.p. with naloxone, the other with saline.

(IV) Involvement of dopamine in DALA-induced activation Amphetamine-DALA interaction, it was postulated that DALA-induced stimulation might be related to a change in dopaminergic activity; this could be either an inhibition of DA neurones, since enkephalila is generally inhibitory, or an excitation since increases in locomotor activity are often related to stimulation of the DA-AI0 system [12, 50]. The behavioural response to D-amph is known to be dependent on DA-AI0 neurones [34]. Therefore an analysis of the interaction between the locomotor response to D-:'~'oh and the response to DALA was conducted in two situations. (i) This experiment tested the locomotor response in the photocell cages to VTA infusion of DALA (0.5/~g, 0.78 nmol) with simultaneous i.p. injection of 0.75 mg/kg D-amph. On previous control days the rats were infused with saline in the VTA and injected with D-~mph i.p., or 0.5/~g DALA in the VTA and saline i.p. (ii) In this experiment rats were infused with 0.02/~g (0.03 nmol) DALA~or saline 30 min after i.p. injection of 0.75 mg/kg D-amph and tested in the circular corridor. The DALA group was previously infused with 0.02/~g DALA tO determine activity scores after this dose. 6-OHDA lesion. An additional experiment investigated the possibility that DALA-induced stimulation might be blocked by DA terminal destruction. Two groups of rats (lesioned and sham) were tested in the circular corridor in 4 situations: spontaneous activity, D-amph 1.5 mg/kg i.p., APO 0.1 mg/kg s.c., and 2.5/~g (3.9 nmol) DALA infused into the VTA. The D-amph and APO probes were utilized to ascertain the extent of the lesion, since it is known that 6-OHDA lesions of the area of the n.accumbens block the D-amph locomotor" response and induce behaviourai supersensitivity to APO [34].

(II) Long-term effects of DALA infusion into the VTA The three groups of rats in the time course experiment (I) were injected with D-amph (1.5 mg/kg i.p.) 4 h after the V I A infusion, and the response tested in the photocell cages. Four days later, 5 rats which had received DALA were infused with 2.5/~g (3.9. nmoi) DALA, and 5 control animals were similarly infused with saline. Subsequently the locomotor response to 0.75 mg/kg D-amph was tested in the two groups l, 2, 6, and 14 days after this second infusion. All vertical bars represent mean score + standard error of the mean. Further experimental details for all testing are given in the figures and legends.

9 RESULTS

(I) Time-course and dose-dependence of DALA-induced activation (i) Fig. 1 shows that infusion of DALA into the VTA elicited a significant increase in locomotor activity (overall group effect F = 12.55, d f = 2,12, P < 0.001). Infusion of 2.5/~g DALA induced a pronounced activation, and levels of activity after this dose remained elevated for approximately 3 h. Infusion of 5.0 #g DALA also stimulated activity, but this response was a complex combination ofactivation and other competing behavioural patterns. As it is apparent in Fig. 1, after infusion of the high dose, the level of stimulation (measured by activity counts) never reached that of the 2.5 gg group. Upon observation, the rats in the 5.0/~g group appeared to be engaged in stereotypy such as lateral swaying of the head and intense head-down sniffing. (ii) Fig. l also shows that locomotor activity in the circular corridor was enhanced by VTA infusion of DALA in a dose-dependent fashion. Infusion of 2.5 #g appeared to be optimal for eliciting the most pronounced response, although much lower doses also induced activation. The threshold for elicitation of the DALA response was between 0.02 and 0.1 #g (between 0.03 and 0.15 nmol). The highest dose, 12.5 #g, also induced an overall increase in activity; however,

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Fig, !. T i m e course and dose-dependence o f D A L A - i n d u c e d activation. Left: rats were habituated for ! h in the photocell cage~ a n d then infused with D A L A (2.5/~g, n = 4; 5.0/Jg, n = 6) o r saline (n = 5). Right: experiment conducted in the circular corridor. Rats were habituated for l h, then infused (saline, n = 3; 0.02, n --- 4; 0.1, n -- 3; 0.5, n = 4; 2.5, n = 4; 12.5, n = 3).

10 this increase was characterized by initial suppression followed by development of very high levels of activity over the course of the hour.

(II) Behaviouralanalysis of DALA inducedactivation (i) Open field. Open field activity after VTA infusion of DALA (1/~g) or saline is displayed in Fig. 2. DALA evoked a marked increase in locomotion in the periphery (F---- 11.6, d f = 1,10, P < 0.01), but no concomitant increase in centre locomotor activity. The increase in peripheral locomotion was accompanied by a significant increase inrearing in the periphery (F= 99.6, d f = 1,10, P < 0.01). Rearing in the centre was not affected. Grooming was significantly attenuated by DALA ( F = 13.9, d f = 1,10, P < 0.01); in addition there was a significant group x time interaction ( F = 6.88, d f = 1,10, P < 0.05), indicating that while the control group increased their grooming score in the second 5 min, grooming in the DALA group remained low. In general, the DALA-infused animalsappeared very activated, confined all their activity along the walls, moved in short, .jerky bursts, and showed very little grooming or resting as did the controls.

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11 (ii) 8-hole box. VTA infusion of 2.5/~g DALA significantly modified the behaviour of animals in the hole box, as shown in Table I. The radar detector indicated that total motor activity was enhanced (F= 23.3, d r = 1,4, P < 0.01). The total number of holes visited by DALA infused rats was increased (F = 11.96, df = 1,4, P < 0.05); however, this was accompanied by a clear reduction in the mean duration of a hole visit (F = 13.0, df = 1,4, P < 0.05).

(1II) Opiate specificity of DALA-induced activation Fig. 3 shows that the behavioural response to infusion of 2.5/~g DALA into the VTA was rapidly and completely blocked by i.p. injection of naloxone (1 mg/kg). (F= 5.61, df =- i,8, P < 0.05").This dose of naloxone had no effect on activity when the rats were infused with saline (mean + S.E./180 min: saline, 160 + 25; naloxone 181 + 70). Therefore it appeared that the behavioural stimulation was a specific opiate-receptor mediated effect.

(IV) Involvement of dopamine in DALA-induced activation Amphetamine-DALA interaction. Results from this experiment are shown in Fig. 4. It is clear that low doses of DALA did not inhibit the locomotor response to D-amph but iiJ fact enhanced it (Fig. 4, left). The simultaneous administration

TABLE !

Effect of DALA injiision into VTA on behaviour in an 8-hole box Two groups of rats were tested in an 8-hole box. The radar measured global motor activity. The last row presents statistical analysis on different behaviours.

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EFFECTOF NALOXONEON cl-olo-met.enkephalin(DALA)INDUCEDACTIVATION Fig. 3. Naloxone blockade of DALA-induced activation measured in photocell cages, Rats were given an i.p. injection of saline or naloxone 40 rain after VTA local infusion of DALA. Naloxone per se did not have any effect on the activity of VTA-saline-infused rats (see tex0.

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•POTENTIATIONOF AMPHETAMINERESPONSEFOLLOWINGd-ala-met-enkepholin (DALA) INTO VTA (d-omph o.75mg/kg) Fig. 4. Interaction between response to V I A infusion of DALA and i.p. injection of D-amph, Shaded area in both graphs represents difference between activity scores after DALA + D-amph administered together, and scores obtained by addition of the effect of DALA and D-amph vere

inei.p. sted sion of saline or 0.02/Ag DALA. The DALA group was previously infused with DALA 30 min after saline i.p. in order to determine the potentiation effect.

13 of 0.5/tg DALA and 0.75 D-amph induced a greater response than DALA or D-amph alone, which was also greater than the simple addition of the effects of DALA alone and D-amph alone. (This dose of DALA elicited a small response as determined by infusion of 0.5/~g DALA alone, and as is apparent from the dose-response curve in Fig. 1.) The lower demarcation of the shaded area represents the predicted additive effect of DALA alone plus D-amph alone, while the upper demarcation represents the actual scores obtained when the drugs were administered together. A comparison between the actual curve of DALA + D-amph with the hypothetical additive one reveals that they are significantly different (F= 8.16, d f = 1,5, P < 0.05) (Fig. 4, right). In the second experiment rats were infused with a dose of DALA (0.02/zg) which per se does not elicit a significant response. In this condition the potentiation effect is more apparent. During the 30 min following VTA infusion the response to D-amph in the DALA group was significantly greater than the saline infused group (F= 5.7, df = 1,12, P < 0.03). 6-OHDA lesion. The results from this study, carried out in the circular corridor, are shown in Fig. 5. N.accumbens terminal destruction by 6-OHDA resulted in spontaneous hypoactivity, confirming previous findings from this laboratory. The lesion blocked the locomotor response after administration of !.5 mg/kg D-amph (F= 32,8. dr= 1,11, P < 0.001), and induced supersensitivity to apomorphine (F= 125.5, df = 1,9,P < 0.001). Both results are typical of the

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Fig. 5. Drug responses in 6-OHDA-lesioned and control rats. Activity in the sham and lesioned rats was tested in the circular corridor under 4 conditions, spaced at least 4 days apart: (1) spontaneous activity, (2) systemic D-amph, (3) systemic APO and (4) VTA local infusion of DALA. The experiment was conducted between the 16th to the 32nd day post-lesion.

14 profde after this lesion. Furthermore, the lesion completely blocked the locomotor response to VTA infusion of 2.5 #g DALA (F= 110.6, dr= i,9, P < 0,001).

(V) Long-term effects of DALA infusion into the VTA Four hours after infusion of DALA into the VTA, when the acute effect had subsided, the locomotor response to 1.5 mg/kg i.p. D-amph was attenuated in a dose-dependent manner (Fig. 6). After prior infusion of 5.0/tg DALA, D-amph activation was significantly reduced in comparison with saline infused controls (F--- 5.73, df= 1,9, P < 0.05). Prior infusion of 2.5/tg DALA also reduced the response, although this effect was not significant. Observation of the D-amph locomotor response at much longer time intervals after DALA pretreatment (2.5 #g) revealed a long-term suppression of the D-amph response (overall group effect over 4time points: F - 6.14, dr= 1,8, P < 0.05) (Fig. 7). The activity of the DALA group expressed as percentage of the saline group was: 1 day post DALA, 47%, 2 days 60%, 6 days 68%, 14 days 92%, indicating that the response returns to normal only after 14 days at the most.

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Histology One example of brain slices stained with cresyl violet depicting cannulae tracks, is shown in Fig. 8. Although some cellular destruction in this area does occur, it is clear from the histology that the extent of the damage is minimal, even with repeated injections. Cannulae placements from all experiments were well located in the VTA. This section depicts the most active site for DALA behavioural stimulation but active sites ranged from just anterior to the interpeduncular nucleus m the middle of this nucleus.

16

Fig. 8. A representative section (30 pm) stained with cresyl violet depicting cannulae placements in VTA. Active sites for DALA-induced bebavioural activation ranged from just anterior to the interpeduncular nucleus to the middle of this nucleus.

DISCUSSION

The present results provide evidence that VTA infusion of the stable analog of Met-enkephalin, DALA, into the Al0 dopaminergic area elicits a potent, long-lasting, dose-dependent and opiate-specific behavioural activation. We have found further that this activation is likely to be,due to increased release of DA in the mesocortical-limbicareas. This hypothesis is supported by three factors: first, destruction of DA-A 10 terminals abolishes the response to DALA; second. we have recently found that the dopamine antagonist ~-flupenthixoi specifically blocks the DALA response while the inactive isomer p-flupenthixol which has no antagonistic activity does not [32]; third, VTA infusion of DALA potentiates the D-amph-induced locomotor stimulation. There are a number of questions which we have not yet been able to answer; such factors as dose-response relationships and possible sequential effects of drug testing (particularly in the DALA-D-amph potentiation experiment) must be taken into account in pursuing this work. Nevertheless, the findings taken together indicate that DA-AI0 neurones are n ~ s m r y for the expression of the DALA response and that this response is dependent on the activation of these neurones. The implications of the results can be discussed within frameworks o f both behavioural and neurochemical mechanisms. While the results from the photocell cages and circular corridor indicate

17 an enhancement of general motor activity, the data from the open field and 8-hole board provide a further analysis of the type of activation. The open field data is especially interesting in comparison with the profile observed in the same apparatus after VTA infusion of another endogenous peptide, substance P (SP). VTA infusion of approximately equimolar doses of SP elicits a behavioural activation quite different from that observed after DALA, but also dependent on DA-AI0 neuronal activation [33, 62]. This includes overall increased locomotion and rearing but particularly in the centre area, and no modification of grooming. DALA increases locomotion and rearing but only along the walls; the spatial distribution of activity is not changed as it is after SP. In view of the fact that rats tend to distinctly avoid the centre of the open field, presumably because of the aversive properties of the open field space, it is possible that the SP response reflects changes in exploratory patterns and emotionality, whereas the DALA response does not. Amphetamine, which preferentially releases DA and inhibits its re-uptake at the terminals [72], produces a profile markedly similar to SP [32]. Generally speaking, it is interesting to compare different patterns of behaviour which result from different manipulations of the DA-A10 system; however, actual interpretation must be made with caution, as open field behaviour in the rat is factorially very complex [14]. The hole board perhaps provides a better measure of exploration. VTA infusion of DALA increased the actual number of holes visited but concurrently decreased the duration of each visit; this again contrasts with results of a similar experiment with SP, which increased both the number and duration of hole visits (in preparation). It may be that activation of the DA-AI0 system in one manner such as with SP (an endogenous peptide) enhances certain natural tendencies such as exploration, while activation with DALA (a long lasting analogue of the natural peptide met-enkephalin) is characterized by a disruptive, overaroused state. In other words, two substances could induce release of DA at the terminals but via different VTA mechanisms and thus produce different behavioural consequences. A word can be mentioned about the rewarding properties of DALA and the motivational significance of DALA-induced activation. Broekkamp et al. [9] found that infusion of 2.5 gg DALA into the VTA enhances self-stimulation rates from electrodes placed in the lateral hypothalamus, which suggest that the endogenous peptide may be involved in modulation of reward. Strong evidence for rewarding effects of enkephalin is that it is self-administered by animals [4] as is morphine [23, 68]. It should be emphasized that it is clearly premature to claim that enkephalin is the endogenous neuropeptide of primary importance in the VTA. One cannot disregard the possible role of the endorphins, and indeed Stinus et al. [63] have recently found that endorphins also have a potent stimulatory effect at this s!te. However, in considering general neurochemical mechanisms, two major ques-

18

tions arise: what are the synaptic events responsible for the acute response, and what is the neural basis of the long-term attenuation of the amphetamine response ? Opiates are inhibitory in most neurophysiological studies [45] and have been proposed to inhibit DA at the level of DA-terminals [I 1, 57]. It is difficult to imagine that enkephalin muses DA release by direct inhibition of DA cell bodies. it is more likely that opiate receptors in the V,TA are not located directly on DA neurones, but influence DA as part of the integrative circuity in the VTA. In this case DALA induced behavioural activation could be explained by the presynaptic inhibition of an inhibitory input onto DA-A 10 neurones [56]. There are several possibilities of opiate-DA interaction. A schematic model of possible connections is presented in Fig. 9. The model emphasizes presynaptic enkephalinergic control of GABA neurones, which modulate the activity of the DA-AI0 system. Although the existence of GABA neurones in the VTA is documented [17, 43] it is still unclear whether GABA is localized in short interneurones, in forebrain neurones projecting to VTA, or in both. ~ccording to the model, GABA FOREBRAIN

VENTRAL TEGMENTAL AREA

: : OPiATe (-)

Fig. 9. Schematic diagram of hypotheses for opiate-DA interaction at the level of the DA-AI0 soma. Activation of DA neurones could be mediated by opiate inhibition of an inhibitory input to DA soma such as (!) GABA neurones originating in the forebrain (e.g.n. accumbens), (2) small GABA interneurones located in VTA, (3) dendritic release of DA, (4) undetermined transmitter pathways. Origin of enkephalin neurones is not presently known. Also represented is a possible direct excitatory input to DA soma by substance P (SP) neurones. Part" of this SP innervation originates in the medial habenula, which receives afferents from some DA terminal structures (septum, n.accubens, diagonal band).

19 stimulation of opiate receptors would inhibit the GABA input to DA neurones, thus releasing DA neuronal activity. There is considerable biochemical and behavioural evidence which supports this hypothesis. GABA and GABA agonists have been found to inhibit VTA neurones [46, 74]. Fuxe et al. found that GABA agonists preferentially reduced DA turnover in the nucleus accumbens and olfactory tubercle, as compared with the striatum [! 8] and suggest a strong possibility of GABAergic control ofmesocorticolimbic DA neuronal activity. A number ofbehavioural studies support the GABA-DA link; for example the GABA agonist muscimol antagonizes motility caused by dopaminergic drugs [54]. Furthermore, VTA infusion ofpicrotoxin, a GABA receptor blocker results in motor activation [44~. Opiate-GABA interactions have also been demonstrated. GABA turnover in the nucleus accumbens and striatum is decreased by morphine [l !] and muscimo! antagonizes systemic morphine-induced hypermotility [10]. The GABA-DA-A 10 neuronal relationship has also been studied by Stevens et al. [6 ! ] as an animal model for psychosis, and it is thus possible that enkephalin neurones could be impli.cated in psychopathology. One studywhich contradicts this model found that the VTA infusion of l#l of a molar solution of GABA increases locomotor activity [66]. However, it is unlikely to be a physiological response because of the concentration of GABA. A number of alternative hypotheses could account for the opiate-DA interaction. Inhibitory control of DA activity may be exerted by other neuronal systems, For example opiate receptor stimulation could inhibit serotonergic activity, or dendritic release of DA, both possibly controlling DA-A l0 activity. Some of these possibilities have been studied by Llorens-Cortes et al. [38] who found that opiate receptors in the substantia nigra are located both on DA soma and GABA terminals. In summary, opiates may have an indirect excitatory action on DA cell bodies, although they may directly inhibit DA neurones at the level of the terminals [15, 37, 57]. There remains the question of the surprising long-term modification of the amphetamine response, which we have recently reproducea in the circular corridor test. At various time points after VTA infusions of DALA the D-amph response is diminished, indicating a change in the neuronal substrate for the expression of this response. (This contrasts with the locomotor response to D-amph after a similar VTA infusion of SP, which is sharply potentiated i h and 24 h post-SP) [26, 62]. There are various mechanisms to explain this phenomenon, for example decreased DA synthesis or availability at DA terminals. However, the response to 4 repeated VTA infusions of DALA once a day is not altered [32], indicating that the opiate receptor reactivity is unchanged and that DA is released normally, at least in response to DALA. The long-term change must take place presynaptically to the opiate receptors, possibly in enkephalin neurones which form part of the feedback systems important in the amphetamine

20 response. For example, infusion of enkephalin onto opiate terminals may decrease enkephalin release and thus increase GAB A inhibition of DA neurones. The long-lasting b e h a v i o r ! effects are ~ i c ~ l y interestmg in view of several present hypotheses which p o ~ t e a~ activiW of ~ i o i d neurones as a mechanism u n d e r l i n g tolerance and d ~ d e n c e to narcotics [35, ~ , 59]i However, it should ~ emphasized t ~ t full evaluation o f ~ s phenomenon regarding dose-response relationships a n d pharmacological mechanisms awaits further investigation. Clearly the synaptic organization of VTA is very complex, and further work is needed to test the GABA hypothesis and to elucidate the functional circuitry which controls the DA-AI0 system. In conclusion, it is possible that the acute effects of V I A infusion of DALA are related to stimulant and euphoric properties of drugs such as morphine, and an understanding of the long-term effects may help clarify both mechanisms of addiction and normal modulatory functions of the endogenous opioid peptides. ACKNOWLEDGEMENTS This research was supported by MRC Grant 978/290/N to S.D.I., a Twinning Grant awarded by the European Science Foundation, and INSERM Grant CL79,1,331,6 to L.S, A.E.K. was supported by a Thouron BritishAmerican Exchange Scholarship, REFERENCES i Algcri, S., Calderini, G., Con~loziona, A. and Garattini, S., The effect of methionineenkephalin and D-alaninemethionine enkephalinamide on the concentration of dopamine metabolism in rat striatum. Europ. J. Pharmacol.. 45 (1977)207-209. 2 Atweh,S.F. and Kuhar, M.J., Autoradiographiclocalizationof opiate receptors in rat brain. !!. The brain stem. Brain Res., 129 (!977) !-12. 3 Atweh,S.F. and Kuhar, M.J., Autoradiographiclocalizationof opiate receptors in rat brain. I!!. The telencephalon,Brain Res., 134 (1977) 393-405. 4 Belluzzi,J.P. and Stein, L., Enkephalin may mediate euphoria and drive-reduction reward. Nature (Load.). 266 (1977) 556-558. 5 Biggio,G., Casu, M.. Corda, M.G., Di Bello, C. and Gessa, G.L.. Stimulationof dopamine synthesisin caudate nucleusby intrastriatalenkephalinsand antogonismby naloxone,Science, 200 (1978) 552-554. 6 Blundell,C., Crossman, A.R. and Slater, P., The effect of morphine on turning behavior in i6P. ttophoretically (Lond.), 261 ~halin micro-

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