CHOLINERGIC ACTION IN THE NUCLEUS ACCUMBENS: MODULATION OF DOPAMINE AND ACETYLCHOLINE RELEASE

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1 Br. J. Pharma. (1982), 75, CHOLINERGIC ACTION IN THE NUCLEUS ACCUMBENS: MODULATION OF DOPAMINE AND ACETYLCHOLINE RELEASE J.S. de BELLEROCHE & I.M. GARDINER Departments of Neurology and Biohemistry, Charing Cross Hospital and Medial Shool, Fulham Palae Road, London, W6 8RF 1 The ation of oxotremorine and aetylholine on the release of dopamine and aetylholine from tissue slies of the rat nuleus aumbens was studied. 2 Oxotremorine signifiantly enhaned the release of [14C]-dopamine evoked by 34 mmk+ and the EC5 for this ation was 1.5 x 1 7 M. A maximal enhanement (3%) for this effet was reahed at 2 x 1 7 M oxotremorine. A further enhanement of dopamine release ourred at onentrations of oxotremorine greater than 1-4 M. 3 The ation of oxotremorine on [14C]-dopamine release was alium-dependent and bloked by atropine (1-4M) but not meamylamine (up to 1-4 M). 4 Oxotremorine affeted [3HI-aetylholine release differentially, inhibiting the K+-evoked release of [3H]-aetylholine at onentrations greater than 1- M. The IC5 for this proess was 4.3x1 M. 5 Aetylholine (8 x 1 4 M) showed a similar pattern of ation to oxotremorine: it enhaned the K+-evoked release of [14C]-dopamine (5%) and inhibited the K+-evoked release of [3H]- aetylholine (3%). 6 The mehanism of ation of oxotremorine on dopamine release is disussed in terms of a presynapti reeptor-mediated proess. Introdution High onentrations of aetylholine, holine aetyltransferase and dopamine are found in the nuleus aumbens (Dahlstrom & Fuxe, 1965; Shute & Lewis, 1967; Cheney, Le Fevre & Raagni, 1975). An interation appears to our between these transmitters in this region sine the holinergi agonist, oxotremorine, and physostigmine, elevate levels of homovanilli aid in vivo (Anden & Bedard, 1971; Bartholini, Keller & Pletsher, 1975). This relationship is likely to our diretly (in the nuleus aumbens) sine intra-aumbens injetions of arbahol and oxotremorine initially enhane the loomotor hyperativity indued by bilateral injetions of dopamine into the nuleus aumbens (Jones, Morgenson & Wu, 1981; de Bellerohe, Herberg, Winn, Murzi & Williams, 198 la). A diret ation of aetylholine on dopaminergi transmission ould be mediated via presynapti holinoeptors on dopamine terminals. Studies of the speifi binding of musarini agonists and antagonists do indeed indiate that this ould be the ase, sine the onentration of musarini reeptors in the nuleus aumbens is depleted following 6-hydroxydopamine inje /82/ $1. tion in the ventral tegmental area (de Bellerohe, Kilpatrik, Birdsall & Hulme, 1981). The aim of this projet was to eluidate the ation of holinergi agents on dopamine release in the nuleus aumbens. Methods Female Sprague-Dawley rats (2-25 g body wt.) were stunned and killed by ervial disloation. Two oronal uts were made at right angles to the axis of the brain. The first ut was made 1 mm anterior to the opti hiasma and the seond ut was 1.5 mm anterior to the first ut. The nuleus aumbens was disseted out from this setion, a rat brain atlas (Konig & Klippel, 1963) being used for referene. Four tissue slies of the disseted nuleus aumbens were ut in a plane parallel to the setion (.35 mm thikness, approximately). The tissue slies were immediately immersed in Krebs-biarbonate medium of the following omposition (mm): NaCI 118, KCI 4.7, MgSO4.7H2 1.2, NaHCO3 25, KH2PO4 1.2, Mamillan Publishers Ltd 1982

2 36 J.S. DE BELLEROCHE & I.M. GARDINER CaCl2.6H2 2.5, gluose 11.1, asorbate 2.3 and pargyline 1, ph 7.4 gassed with 95% 2: 5% Co2. Tissue slies were inubated in Krebs-biarbonate medium (2 ml) ontaining.65 Mm [methyl-3h]- holine hloride (77 Ci/mmol) and 4.4 pm [7-14C]- dopamine (56mCi/mmol) at 37 C for 3 min. The tissue slies were then transferred to a perspex blok ontaining 8 tissue hambers and were superfused with Krebs-biarbonate medium, maintained at 37 C, at a rate of 1 ml/min for a further 3 min. The initial perfusion period was arried out in order to reah a steady and onstant base line rate of release. At the end of this period, tissue was treated differently for studying the effet of either oxotremorine or aetylholine. Effet of oxotremorine Tissue slies were transferred with fine foreps to Krebs-biarbonate medium (1 ml) and inubated for a 1 min period at 37 C under ontrol onditions or in the presene of drug (oxotremorine, atropine, meamylamine) as indiated. The slies obtained from one nuleus aumbens were divided equally between ontrol and test inubations. The tissue slies were then transferred to fresh medium ontaining 34 mm K+ for a 5 min inubation period at 37 C under ontrol or test onditions (drug present) as before. The tissue slies were removed and aliquots (.8 ml) from the two inubation periods were taken for analysis of the 14C and tritium ontent using double-label liquid sintillation ounting. Aquasol 2 (New England Nulear Chemials, Massahusetts) was used as the sintillant. The 14C and tritium ontent of the tissue slies were also determined following solubilization in Soluene 35 (Pakard Instrument Company In., Illinois). Purifiation of the [3H]-aetylholine and [14C]-dopamine was not arried out as these onditions have previously been found to yield [14C]-dopamine as the main '4C_ labelled onstituent (9%) of the samples and [3H]- aetylholine, the main tritium labelled onstituent when physostigmine is ontained in the medium (de Bellerohe, unpublished observations; de Bellerohe & Neal, 1981). Efflux of [3H]-aetylholine or [14C]- dopamine is expressed as the amount released in eah inubation period as a perentage of the total remaining (released plus tissue ontent) at the end of that inubation period divided by the time of the inubation in minutes (e.g. % tissue [3H]-aetylholine released/min). Colletion of samples by superfusion was not onsidered neessary for the oxotremorine experiments as a marked and signifiant response was obtained at low onentrations of oxotremorine. However, it was onsidered important in the experiments on the effet of aetylholine (next paragraph), where rapid inativation of aetylholine by the ation of aetylholinesterase ours, to attempt to maintain the external aetylholine onentration by the ontinuous superfusion method. Effet of aetylholine After the initial superfusion period, olletion of superfusate samples (2.4 min) was started. A K+ pulse was applied by superfusion with oxygenated medium ontaining 34 mm KCI for 4.8 min. A seond K+ pulse ontaining aetylholine (8.2 x 1 4 M) and physostigmine (1 4M) was applied after 12 min of superfusion with ontrol medium, and a third pulse of K+ (aetylholine and physostigmine absent) was applied after a similar period (12 min) of superfusion with ontrol medium. The 14C and tritium ontent of eah superfusate sample and the tissue slies after superfusion were determined as desribed above. Transmitter release in serial samples is expressed 16r *151 14O 13- h. -W S t , 'a, 1O (n) ~~. I, Oxotremorine (M) lo Figure 1 The effet of oxotremorine on the K - evoked release of [14C]-dopamine (frational release x 1/min) from tissue slies of nuleus aumbens is related (%) to parallel ontrol inubations arried out in 34 mm K+ in the absene of oxotremorine. Tissue slies derived from the same nuleus aumbens were preinubated together and divided into two and used for ontrol and test onditions respetively. Values are means for the number of experiments indiated (n); vertial lines show s.e.means. The values of frational release/min derived from the same nuleus aumbens for ontrol and test onditions were ompared using a paired Student's t test. *Indiates that the presene of oxotremorine signifiantly enhaned release, P<.2. The mean ontrol value (frational release x 1/min) of K+-evoked release of [ C]-dopamine (no drug) was 3.41 ±.5/min for 14 experiments.

3 CHOLINERGIC ACTION IN THE NUCLEUS ACCUMBENS 361 as a frational rate oeffiient, i.e. the efflux at eah time period as a perentage of the total radioativity remaining at that time (e.g. % tissue [3H]- aetylholine released/min). This is defined as the amount of radioativity olleted during a 2.4min period divided by the arithmeti mean of the total radioativity present in the tissue at the beginning and end of the same olletion period and divided by the time of olletion. The K+-evoked release is expressed as a perentage of the resting release in the sample immediately preeding the K+ pulse (e.g. % inrease in release of [3H]-aetylholine due to K+). Effet of oxotremorine on dopamine uptake Dopamine uptake into tissue slies and homogenates of nuleus aumbens was measured in the same inubation medium as used for the release experiments. Tissue slies or unfrationated homogenates were preinubated (.5 ml) for 3 min at 37 C in Krebs-biarbonate medium ontaining gluose (1mM), asorbate (2.3 mm) and pargyline (1 mm). [14C]-dopamine (56 mci/mmol) was then added to give a final onentration of.7 x 1-7M dopamine. Inubation was terminated after 5 min with unlabelled medium (3. ml) and the samples were immediately entrifuged. The pellet was washed 3 times with isotope-free inubation medium. The final pellet was digested with NaOH, mixed with Aquasol 2 and the radioativity in the samples measured in a liquid sintillation spetrometer. The uptake into tissue slies was related to the wet weight of eah tissue slie and uptake into homogenates was related to the protein ontent determined by the method of Lowry, Rosebrough, Farr & Randall (195 1). Materials All materials used were of Analar quality. Radioisotopes were obtained from The Radiohemial Centre, Amersham, Buks. Oxotremorine and aetylholine perhlorate were obtained from Sigma. Results Effet ofoxotremorine on the release of[14c]- dopamine from tissue slies of nuleus aumbens Oxotremorine had little effet on the resting release of [14C]-dopamine. However, the K+-evoked release of [14C]-dopamine was signifiantly enhaned by oxotremorine at onentrations of 1.6 x 1 M and above (Figure 1). The inrease in release due to oxotremorine was 3% at 2 x 1-7M oxotremorine and this inrease remained onstant up to a onentration of 1-4 M. The EC5 value for this stimulation v 14 ; 1 I-,,- 12 I ~ 8C._e 4 4). Co *6 2C 2C I-. 3 I Ca2+ absent Figure 2 The effet of oxotremorine, in the presene and absene of alium, on the K -evoked release of [ C]-dopamine from tissue slies of nuleus aumbens is related to parallel ontrol inubations arried out in 34mM K+ in the absene of oxotremorine (%). The medium ontaining no aliuon ontained MgCl2 (2.5 mm) to maintain isotoniitv. Values are means for 8 experiments; vertial lines show s.e.mean, *Lndiates that oxotremorine signifiantly enhaned the K - evoked release of [ C]-dopamine, P< t). 1. The mea.- ontrol value (frational release x 1t)(/min) of K evoked release of [ C]-dopamine was /min (8). Control inubation (stippled olumns) superimposed on 34 mm K+ inubation (open olumns). Oxotremorine 3 x 1 M inubation (heavily hathed olumns) superimposed on oxotremorine plus 34 mm K+ inubation (lightly hathed olumns. was estimated to be 1.5 x 1 7M. At onentrations of oxotremorine above 1 4M, a further inrease in the K+-evoked release of ['4C]-dopamine was also seen. The effet of oxotremorine was not studied above 1 3M. The enhanement of the K+-evoked release of [14Cj-dopamine inreased up to 5% at 3 1- M oxotremorine (Figure 1). When CaCl2 was replaed in the inubation medium with MgCl2, the K+-evoked release of dopamine was redued by 66% (Figure 2). Oxotremorine up to a onentration of 3 x 1-4 M did not signifiantly inrease the release of [14C]-dopamine in the medium laking Ca2+. The K+-evoked release of [14C]-dopamine in the presene of oxotremorine was redued by 72% in the alium-free medium. These results indiated that alium was neessary for both the K+-evoked release and the enhanement of this effet by oxotremorine. Atropine (3 x 1 4M) signifiantly antagonized the ation of oxotremorine (Figure 3) but meamylamine (1 4M) was without effet.

4 362 J.S. DE BELLEROCHE & I.M. GARDINER 4- ) - Oxotremorine 3 X 1-5M o 1o Control - ae o n m a) +~ Atropine Atropine Meamy- 3 X 1 4M 3 X 1-4M lamine 1-4M Figure 3 The effet of oxotremorine, in the presene and absene of atropine (3 x 1 M) and meamylamine (1 M), on the K -evoked release of [14C]-dopamine from tissue slies of nuleus aumbens is related so parallel ontrol inubations arried out in 34mM K+ in the absene of drugs (%). Values are vieans for 5 experiments, vertial lines show s.e.mean. Indiates that atropine signifiantly redued the effet of oxotremorine, P <.5. Further details as for Figure 1. Effet ofoxotremorine on the release of[3h]- aetylholine Oxotremorine did not affet the release of [3H]- aetylholine up to 1-5 M, whih overed most of the range of onentrations ausing the initial enhanement of the K+-evoked release of [14C]-dopamine (Figure 4). However, at 1-4 M oxotremorine, a signifiant inhibition (3%) of the K+-evoked release of [3H]-aetylholine was seen. The seond phase of stimulation of [14C]-dopamine release was not evident at this onentration of oxotremorine. The IC5 for this inhibitory ation of oxotremorine was estimated to be 4.3 x 1i5 M. Effet of aetylholine on [14C1-dopamine and [3H]- aetylholine release from nuleus aumbens Aetylholine, 8.2 x 1 M in the presene of physostigmine (1 4M) produed similar effets to the higher onentrations of oxotremorine, e.g. 1-4M. Thus, aetylholine signifiantly enhaned the K+-evoked release of [14C]-dopamine (5%) and also dereased the release of [3HI-aetylholine (3%) from tissue slies of nuleus aumbens (Fig [ 6-53 * (n) Oxotremorine (M) Figure 4 The effet of oxotremorine on the K+ - evoked release of [3H]-aetylholine (frational release x 1/min) from tissue slies of nuleus aumbens is related to the release measured in parallel inubations (%) arried out in 34mM K+ in the absene of oxotremorine. Tissue slies derived from the same nuleus aumbens were preinubated together, divided and used for both ontrol and test onditions. Values are means for the number of experiments indiated (n), vertial lines show s.e.mean. *Indiates that the values are signifiantly dereased by the presene of oxotremorine, P<.2. The mean ontrol value of K - evoked release (frational release x 1/min) of [ 3Haetylholine (no drug) was /min for 78 experiments. Further details as for Figure 1. ure 5). The magnitude of these effets on the K+evoked release of [14CJ-dopamine was similar and in the same diretion as those obtained with oxotremorine, although a superfusion method was used for measuring the effet of aetylholine ompared to a S min inubation period for the latter. Using the superfusion method, it was possible to determine whether the effet of aetylholine was reversible by applying a third pulse of K+ in the absene of aetylholine. The K+-evoked release of [14CJ-dopamine in the third K+ pulse was similar to the original response (first pulse), whereas the release of [ H]- aetylholine had only partially returned to the original response. Effet of oxotremorine on dopamine uptake In order to test whether the stimulatory effet of oxotremorine on dopamine release was partially due to an inhibition of dopamine uptake, the effet of oxotremorine on dopamine uptake into tissue slies and homogenates of nuleus aumbens was measured in the same inubation medium as used for the release experiments. Oxotremorine did not affet the uptake of [14C]-dopamine (.7 x 1 7M) into tissue

5 CHOLINERGIC ACTION IN THE NUCLEUS ACCUMBENS C,,o 2 L a) ) o C - e m.= 1) W ) ) ) CU ) lo'or [ a, 8 a) 6 2) 4 a) C.) X 2 n) I. I. I- ACh 8.2 x 1-4M H * P<O.2 * P<A.21 I~~~~~~~~~~~~~~~~~~~~ m I 1st P<O.1 2nd I 3rd n = 6 Pulse Figue 5 The effet of aetylholine on the 34 mm K -evoked release of [ 14C]-dopamine and 3H]- aetylholine from ontinuously superfused tissue slies of nuleus aumbens. Three pulses of K+ were applied (4.8 min); aetylholine was only present in the seond pulse. The K +-evoked release (frational release/min) is expressed as a perentage of the restin release in the sample immediately preeeding the K -pulse (% inrease in release due to K ). Values are means for n experiments as indiated; vertial lines show s.e.mean. Further details desribed in the Methods.*Indiates that release is signifiantly different from that during previous K pulse. + slies (n = 5), uptake of dopamine was.51±.4 (mean ± s.e.mean) pmol 5 min- l mg- l wet wt in ontrol onditions ompared to.54 ±.4 in the presene of 1, M oxotremorine and.56 ±.5 in the presene of 3 x 1-7M oxotremorine. Similarly, oxotremorine had no effet on an unfrationated homogenate of nuleus aumbens, where the rate of uptake was 34.2±2. pmol 5 mintlmg lprotein (n = 5) under ontrol onditions and 37.3 ± 1.1 pmol 5 min-' mg protein (n = 6) in the presene of oxotremorine (16M). Although optimal rates of dopamine uptake would not be obtained using tissue slies and unfrationated homogenates these results learly showed that under the experimental onditions used, where oxotremorine enhanes dopamine release, it has no ation on dopamine uptake. Disussion Modulation of dopamine release by holinoeptor agonists Both oxotremorine and aetylholine enhaned the K+-evoked release of dopamine. From the studies on oxotremorine, this effet is seen to be bloked by the musarini antagonist, atropine and dependent on the presene of alium. The stimulation of dopamine release was maximal at 2 x 1-7M oxotremorine. At onsiderably higher onentrations, e.g. 1-3 M, a further signifiant inrease in release of dopamine was promoted by oxotremorine. Modulation of aetylholine release Oxotremorine also had an effet on aetylholine release, reduing the K+-evoked release of (3HJaetylholine. The IC5s for this inhibition was approximately 4.3 x 1 M. This ation of oxotremorine ontrasted with those seen on dopamine release, sine it was inhibitory rather than exitatory and also, the half maximal effet ourred at onentrations more than one order of magnitude apart. It seems likely that the inhibition of aetylholine release by a holinoeptor agonist serves as an autoregulatory mehanism to suppress transmitter release from the nerve terminals when it has built up to a ritial level in the synapse. A similar ation of holinoeptor agonists on aetylholine release has also been demonstrated in erebral ortex (Szerb & Somogyi, 1973). Presynapti musarini reeptors on dopamine terminals A diret modulation of dopamine metabolism and release in the nuleus aumbens by a transmitter or drug is likely to be mediated by a presynapti reeptor, for example, the regulation of dopamine synthesis by apomorphine in the striatum (Christiansen & Squires, 1974). The possibility that holinoeptors may be present on dopamine terminals emerges from these release studies in nuleus aumbens presented here and similar studies on other dopaminergially innervated regions suh as orpus striatum (Giorguieff, Le Flo'h, Westfall, Glowinski & Beeson, 1977; de Bellerohe & Bradford, 1978) and is supported by studies on musarini reeptor populations. Thus, 6-hydroxydopamine treatment whih destroys dopamine terminals also depletes the levels of musarini reeptors in orpus striatum (Kato, Carson, Kemel, Glowinski & Giorguieff, 1977; de Bellerohe, Luqmani & Bradford, 1978) and nuleus aumbens (de Bellerohe et al., 198 lb). A detailed examination of the lass of musarini agonist bind-

6 364 J.S. DE BELLEROCHE & I.M. GARDINER ing site depleted in the nuleus aumbens by 6- hydroxydopamine lesion of dopamine ontaining ells of the ventral tegmental area indiated that there is a seletive loss of the highest affinity binding site, known as the 'super high' affinity binding site (de Bellerohe et al., 1981b). Thus, presynapti musarini reeptors apable of being depleted by 6- hydroxydopamine are likely to be mediators of the ation of oxotremorine and aetylholine on dopamine release from nuleus aumbens. Funtional role of interation between holinergi and dopaminergi systems in the nuleus aumbens The dopaminergi innervation of nuleus aumbens plays a key role in modulating loomotor ativity as shown by the ation of dopamine and dopamine agonists applied diretly into the nuleus aumbens of rats to inrease loomotor ativity and exploration (e.g. Pijnenburg & Van Rossum, 1973; Costall & Naylor, 1975). This effet ontrasts with that obtained from injetion of dopamine into the audate nuleus also innervated by a dopamine projetion from the midbrain whih auses gnawing, biting and liking. Other behavioural measures suh as hoarding and maternal behaviour are lost on 6- hydroxydopamine lesion of the ventral tegmental area (Le Moal, Galey & Cardo, 1975; Galey, Simon & Le Moal, 1977) and are therefore dependent on an intat dopaminergi innervation of the nuleus aumbens. The present results indiate that the presynapti ation of holinoeptor agonists modulate dopaminergi ativity. These findings are onsistent with the ation of intra-aumbens injetions of oxotremorine and arbahol to enhane dopamineindued hyperativity (de Bellerohe et al., 198 la; Jones et al., 1981). This stimulatory ation ours within the first hour following injetion of the agonist and ontrasts with the delayed ation of holinergi agents, e.g. areoline and physostigmine, whih after 1-2 h ause a marked inhibition of loomotor ativity (Costall et al., 198). This work was supported finanially by the Mental Health Foundation. We are grateful to Dr Clifford Rose and Professor B.E. Ryman in whose departments this researh was arried out. Referenes ANDEN, N-E. & BEDARD, P. (1981). Influenes of holinergi mehanisms on the funtion and turnover of brain dopamine. J. Pharm. Pharma., 23, BARTHOLINI, G., KELLER, H.H. & PLETSCHER, A. (1975). Drug-indued hanges in dopamine turnover in striatum and limbi system of the rat. J. Pharm. Pharma., 27, DE BELLEROCHE, J.S. & BRADFORD, H.F. (1978). Biohemial evidene for the presene of presynapti reeptors on dopaminergi nerve terminals. Brain Res., 142, DE BELLEROCHE, J., HERBERG, J., WINN, P., MURZI, E. & WILLIAMS, S. (1981a). Modulation of dopaminergi ativity in the nuleus aumbens by oxotremorine: Pro. Int. So. Neurohem., 257A. DE BELLEROCHE, J., KILPATRICK, I.C., BIRDSALL, N.J.M. & HULME, E.C. (1981b). Presynapti musarini reeptors on dopaminergi terminals in the nuleus aumbens. Brain Res. (in press). DE BELLEROCHE, J.S., LUQMANI, Y. & BRADFORD, H.F. (1979). Evidene for presynapti holinergi reeptors on dopaminergi terminals: degeneration studies with 6-hydroxydopamine. Neurosi. Leu., 11, DE BELLEROCHE, J.S. & NEAL, M.J. (1981). Contrasting effets of both aute and hroni neurolepti administration on ACh release from orpus striatum and n. aumbens of rat. J. Physiol., 312, 15-16P. CHENEY, D.L., LE FEVRE, H. & RACAGNI, G. (1975). Choline aetyltransferase ativity and mass fragmentographi measurement of aetylholine in speifi nulei and trats of rat brain. Neuropharmaology, 14, CHRISTIANSEN, J. & SQUIRES, R.F. (1974). Antagonisti effets of apomorphine and haloperidol on rat striatal synaptosomal tyrosine hydroxylase. J. Pharm. Pharma., 26, COSTALL, B. & NAYLOR, R.J. (1975). The behavioural effets of dopamine applied intraerebrally to areas of the mesolimbi system. Eur. J. Pharma., 32, COSTALL, B., HUI, S.C.G. & NAYLOR, R.J. (198). Hyperativity indued by intra-aumbens dopamine: ations and interations of neurolepti, holinomimeti and holinolyti agents. Neuropharmaology, 18, DAHLSTROM, A. & FUXE, K. (1964). Evidene for the existene of monamine ontaining neurones in the entral nervous system. I. Demonstration of monamines in the ell bodies of brain stem neurones. Ata physiol. sand., 62, Suppl. 232, GALEY, D., SIMON, H. & LE MOAL, M. (1977). Behavioural effets of lesions in the A1 dopaminergi area of the rat. Brain Res., 124, GIORGUIEFF, M.F., LE FLOC'H, M.L., WESTFALL, T.C., GLOWINSKI, J. & BEESON, M-J. (1976). Niotini effet of aetylholine on the release of newly synthesised [3H]-dopamine in rat striatal slies and at audate nuleus. Brain Res., 16, JONES, D.L., MORGENSON, G.J. & WU, M. (1981). Injetions of dopaminergi, holinergi, serotoninergi and GABAergi drugs into the nuleus aumbens: effets on loomotor ativity in the rat. Neuropharmaology, 2, KATO, G., CARSON, S., KEMEL, M.L., GLOWINSKI, J. & GIORGUIEFF, M.F. (1978). Changes in striatal speifi [3H]-atropine binding after unilateral 6-

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