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Effects of dopamine agonists on excitatory inputs to nucleus accumbens neurons from the amygdala: modulatory actions of cholecystokinin
85
Brain Research, 554 (1991) 85-94 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116781P BRES 16781
Effects of dopamine agonists on excitatory inputs to nucleus accumbens neurons from the amygdala: modulatory actions of cholecystokinin R.Z. Liang, M. Wu, C.C.Y. Yim and G.J. Mogenson Department of Physiology, University of Western Ontario, London, Ontario (Canada) (Accepted 12 February 1991)
Key words: CCK-8; Dopamine; SKF 38393; LY 171555; Nucleus accumbens; Amygdala; Neuromodulation
The interaction of the cholecystokinin octapeptide (CCK-8) with dopamine (DA) and dopamine agonists on neurons in the nucleus accumbens was investigated using single unit recording and iontophoretic techniques in urethane-anaesthetized rats. Neurons in the nucleus accumbens were activated by single pulse stimulation of amygdala. Using seven-barrel microelectrodes, the effects of iontophoretic application of CCK-8, DA, dopamine D 1 and/or D 2 receptor agonists (SKF 38393 and LY 171555 respectively) were compared. The iontophoretic application of DA, LY 171555 and LY 171555 + SKF 38393 attenuated by 50-60% the excitatory responses of accumbens neurons to electrical stimulation of basolaterai amygdala whereas SKF 38393 attenuated the response by less than 30%. The iontophoretic application of CCK reduced these attenuating effects of DA, LY 171555 and SKF 38393 + LY 171555. With CCK there was a rather small reduction of the attenuating effect of SKF 38393. These observations provide additional electrophysiologicalevidence of the interaction of CCK and dopamine and suggest that the interaction is associated mainly with dopamine D2 mechanisms. INTRODUCTION Following the d e m o n s t r a t i o n that cholecystokinin ( C C K ) co-exists with d o p a m i n e in neurons that project from the m i d b r a i n to the ventral striatum there has been a k e e n interest in their possible functional interaction 6,12. E x p e r i m e n t s utilizing microdialysis and in vivo voltamm e t r y have shown that C C K influences the release of d o p a m i n e into the nucleus accumbens 1a'19'26. Results of electrophysiological experiments suggest that C C K modulates the release and possibly the postsynaptic effects of d o p a m i n e 29'33. Behavioural experiments have also provided evidence that there are interactions of C C K and d o p a m i n e in the striatum 6'7,s,21'3°,31. Recently the interaction of C C K and d o p a m i n e in the nucleus accumbens has been investigated using a different a p p r o a c h 4°-43. It is based on earlier demonstrations that d o p a m i n e , either applied by iontophoresis or released endogenously by electrical stimulation of the ventral t e g m e n t a l area, reduces the excitatory responses of neurons of the nucleus accumbens to single-pulse stimulation of the basolateral amygdala 38. W h e n proglumide, a C C K antagonist, was applied iontophoretically, this reduction of the excitatory responses of accumbens neurons to a m y g d a l a stimulation due to V T A stimulation
was enhanced 42 whereas when C C K was applied, there was a reversal of the reduction from iontophoretically applied d o p a m i n e . These results suggest that C C K m a y be an e n d o g e n o u s functional antagonist of d o p a m i n e . Since d o p a m i n e is known to bind to separate r e c e p t o r subtypes (D1, D2) in the accumbens, and the 2 r e c e p t o r subtypes p r e s u m a b l y m e d i a t e different functions 4, it is of interest to explore w h e t h e r or not C C K has preferential effects on the r e c e p t o r subtypes. The objective of the present experiments is to investigate the possible interaction of C C K and d o p a m i n e D 1 and D 2 agonists on the interaction b e t w e e n C C K and d o p a m i n e itself following the same e x p e r i m e n t a l p a r a d i g m used in a previous study.
MATERIALS AND METHODS Eighty-three male Wistar rats weighing 250--350 g were used in this study. They were anaesthetized with urethane (1.15 g/kg) and mounted in a stereotaxic apparatus, with the incisor bar fixed at 5 mm above the interaural plane. Body temperature of the animal was maintained at 37 °C with a regulated DC heating pad (Harvard). Stimulating and recording electrodes were lowered through burr holes in the skull to the appropriate sites according to the stereotaxic coordinates obtained from the standard atlas of Pellegrino and Cushman TM. Coordinates used for the basolateral amygdala were 0.5-0.8 mm posterior to bregma, 4.6-4.8 mm lateral to the sagittal
Correspondence: R.Z. Liang, Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
86 sinus and 8.0-8.4 mm below the surface of the cortex. Those for the nucleus accumbens were 3.0-3.4 mm anterior to the bregma, 1.2-1.4 mm lateral to the sagittal sinus and 5.6-7.3 mm below the surface of the cortex. Stainless steel concentric bipolar electrodes (NE-100, Rhodes Medical, 0.5 mm diameter, 0.5 mm tip separation) were used for electrical stimulation of the amygdala. Electrical stimulation was generated by a Grass S-44 stimulator coupled to a Grass stimulation isolation unit (SIU-6). Monophasic square pulses of 0.15 ms duration, 150-800 p A were used. Seven-barrel glass micropipettes were used for recording. Each side barrel was filled separately with dopamine hydrochloride (1 M, pH = 3), SKF 38393 (2,3,4, 5-tetrahydro-7,8-dihydroyl-phenyl-lH-3-benzazepine, 0.05 M, pH = 3), LY 171555 (quinpirole hydrochloride, 0.05 M), CCK (20 pg/ml, pH = 5), glutamate (0.15 M, pH = 8) and saline (0.9%, pH = 3) for iontophoretic injection. The centre barrel was filled with 0.5 M sodium acetate containing 2% Pontamine sky blue for recording and dye deposit. Dopamine, SKF 38393, LY 171555, and saline were ejected as cations at 10-30 nA, while the glutamate and CCK-8 were ejected as anions at 10-30 nA. Currents were driven by a Dagan 6400 iontophoretic unit. The iontophoretic currents were matched for the various compounds (20-30 nA) so that comparisons could be made of their effects on the excitatory response of accumbens neurons to single pulse stimulation of the amygdala. In one series of rats, conditioning stimulation (10 pulses, 10 Hz) was delivered to the ventral tegmental area (VTA), the site of mesolimbic dopamine neurons, ending 100 ms before stimulation to the amygdala (for details see Yim and Mogenson3S). The D 1 antagonist, SCH 23390 (R[ + ]-7-Chloro-8-hydroxy-3-methyl- 1-phenyl-2,3,4,5-tetrahydro-lH-3-benzazepine HCI, 0.05 M), and D 2 antagonist, sulpiride (5-[Aminosulfonyl]-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide, 0.10 M, pH = 5.5), were administered by iontophoresis to investigate the effects on the attenuation by VTA conditioning stimulation of the amygdala-enhanced excitatory responses of accumbens neurons. Signal recorded from the microelectrode was fed into a DC
preamplifier (Axoprobe, Axon Instruments) and then to a Tektronix oscilloscope. Extracellular action potentials were isolated from background noise with a window discriminator which were then sampled in real time by an IBM-AT microcomputer. Sampling and data analysis were done with custom programs developed in our laboratory. As described elsewhere changes of 10% or more in the discharge rate of accumbens neurons were considered statistically significant*~. At the end of each track of recording, recording sites were marked by injecting Pontamine sky blue as an anion with a cathodal current of 10 ~A for 10 min. At the end of the experiment, stimulation sites were marked by an iron deposit by passing 10 p A anodal current through the stimulating electrode for 1 rain. The animal was then sacrificed by an overdose of urethane, and perfused with 50 ml of saline followed by 50 ml of 10% buffered formalin. The brain was removed and fixed in formalin for 24 h. Frozen coronal section of 80 pM were cut with a microtome from the appropriate regions. The sections were mounted on glass slides and stained with thionin for examination.
RESULTS Extracellular recordings were made from more than 200
neurons
in
the
medial
nucleus
accumbens
not
r e p o r t e d p r e v i o u s l y . T h e s a m p l e i n c l u d e s q u i e s c e n t as well as s p o n t a n e o u s l y a c t i v e n e u r o n s t h a t w e r e a c t i v a t e d b y single p u l s e s t i m u l a t i o n o f t h e b a s o l a t e r a l n u c l e u s o f amygdala.
Examples
of
the
excitatory
responses
to
a m y g d a l a s t i m u l a t i o n a r e s h o w n in r e c o r d i n g s o f a c t i o n p o t e n t i a l s in Fig. 1 A a n d in v e r t i c a l r a s t e r p l o t s in Fig. lB. These excitatory responses were attenuated by the iontophoretic
application of dopamine
(Fig.
1), In a
s e r i e s of 126 a c c u m b e n s n e u r o n s t h e e x c i t a t o r y r e s p o n s e
TABLE I The effects o f CCK, DA and its agonists on the excitatory inputs to nucleus accumbens from the arnygdala (A) The effects o f DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation No. o f accumbens neurons Attenuation
Facilitation No change
CON
DA
CON
SKF
CON
LY
CON
SKF + L Y
1.80
0.66
1.65
1.30
1.60
0.89
1.37
0.66
126 (~ = -63 + 1.6%) 0 0
38 (~ = -28 _+2.3%) 4 8
89 (~ = -51 _+2.9%) 6 4
53 (~ = -53 _+2.7%) 0 2
(B) The effects o f CCK on the attenuating effects o f DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation No. o f accumbens neurons Attenuation
Facilitation No change
CON
CCK + DA
CON
CCK + SKF
CON
CCK + LY
CON
CCK + SKF + LY
1.54
1.34
1.56
1.20
1.50
1.37
1.24
0.74
122 (~ = -13 _+2.5%) 0 4
15 (~ = -4 _+6.5%) 12 11
77 (~ = -2 _+3.3%) 4 8
28 (~ = -31 _+4.5%) 10 15
87
.a. CCK 3O DA 30 SAL 30
DA 30
28 Z e~
r. t~
8
300 s
B SAL 30
CCK 30
DA 30
DA 30
mo
~oo
~oo
9oo
~oo
'
Fig. 1. A: a time frequency histogram showing excitatory responses of a neuron of the nucleus accumbens to stimulation of the basolateral amygdala and the effects of the iontophoretic application of saline (SAL, 30 hA), dopamine (DA, 30 nA) and CCK (30 hA). B: a vertical raster plot for this neuron to show the effect of iontophoretically applied SAL, DA and CCK on the excitatory responses elicited by the electrical stimulation of basolateral amygdala. Activities recorded from the neuron are plotted along the y axis and successive sweeps are displayed along the x axis. The line of dots along the 40 ms time mark represents stimulus artifacts from the amygdala stimulation delivered at that time. The cluster of dots are the responses of the neuron to the stimulations. The horizontal bars mark iontophoretic application of drugs. Iontophoretic application of DA (30 hA) reduced the excitatory inputs from amygdala by 72%. This DA effect on accumbens neurons was reversed by iontophoretic application of CCK (from -72% to -4.5%). As a control 30 nA of current was applied to a barrel of the micropipette containing isotonic saline.
to a m y g d a l a stimulation was r e d u c e d by 10% o r m o r e (arbitrarily considered to be significant 44) in all 126 neurons (100%). This attenuating effect of d o p a m i n e was in turn reversed or suppressed when C C K was applied iontophoretically before and during the application of d o p a m i n e to accumbens neurons (see Fig. 1). This action of C C K was o b s e r v e d in 122 of the 126 accumbens neurons and, as shown in Table I, C C K reduced the attenuating effects of d o p a m i n e on the excitatory response to amygdala stimulation from - 6 3 % to - 1 3 % . T h e excitatory response of accumbens neurons to a m y g d a l a stimulation was r e d u c e d by the iontophoretic application of the D 2 agonist, LY 171555, as shown in Figs. 2 and 3. This effect of LY 171555 was o b s e r v e d in 89 of a series of 99 accumbens neurons (90%) in which the average reduction of the excitatory response was 51% (see Table I). The i o n t o p h o r e t i c application of the D 1 agonist, S K F 38393, also r e d u c e d the excitatory response
of accumbens neurons to a m y g d a l a stimulation (see Figs. 4 and 5). This reduction occurred in 38 of 50 accumbens neurons (76%) and as shown in Table I the reduction was 28%. The iontophoretic application of C C K suppressed or reversed the attenuating effects of the 2 D A agonists on the excitatory response of accumbens neurons to amygdala stimulation (see Figs. 2, 3, 4 and 5), the suppression being greater for LY 171555 than for S K F 38393. This effect was observed in 77 of the 89 neurons in which attenuation occurred to LY 171555 and the reversal from C C K was from - 5 1 % to - 2 % (see Table I). F o r S K F 38393 the reversal occurred in 15 of 38 accumbens neurons and was from - 2 8 % to - 4 % . The effects of the c o m b i n e d i o n t o p h o r e t i c applications of the O 1 and D 2 agonists on the excitatory responses of accumbens neurons to a m y g d a l a stimulation were also investigated as illustrated in Fig. 6. In a series of 55
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accumbens neurons the excitatory responses were reduced in 53 neurons by an average of 53% (see Table I). In 28 of these neurons the application of CCK suppressed the effects of the co-administration of SKF 38393 and LY 171555 to a 31% reduction of the excitatory responses of
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Fig. 3. B a r h i s t o g r a m s to s h o w the c h a n g e s in the r e s p o n s e s for a c c u m b e n s n e u r o n to a m y g d a l a s t i m u l a t i o n a s s o c i a t e d with the i o n t o p h o r e t i c t r e a t m e n t s p r e s e n t e d in Fig. 2.
accumbens neurons (see Fig. 7 and Table I). In a series of 9 accumbens neurons it was possible to compare in the same neuron the effects of CCK on the responses to dopamine, SKF 38393 and LY 171555. In all 9 neurons the excitatory response to amygdala stimulation was reduced (average reduction shown in brackets) by the iontophoretic application of dopamine (-74%), of LY 171555 (-45%), and of SKF + LY (-70%). The iontophoretic application of SKF 38393 reduced the excitatory response to amygdala stimulation in 5 of the 9 neurons with the average reduction of 28%. As shown in Table II these effects were suppressed by CCK, for dopamine to - 1 5 % (n = 9), for LY 171555 to - 2 0 % (n = 7) and for LY + SKF to - 3 4 % (n = 6). In another series of 32 accumbens neurons, the excitatory response to stimulation of the basolateral amygdala was reduced by a train of conditioning pulses to the ventral tegmental area, as reported previously 38. A comparison was then made of the effects of the iontophoretic application of the D 1 antagonist, SCH 23390, and of the D2 antagonist, sulpiride. Suipiride attenuated the effects of ventral tegmental area stimulation in 30 of 32 neurons whereas SCH 23390 attenuated the effects of ventral tegmental area stimulation in 10 of 32 neurons.
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Fig. 4. A vertical raster plot shows that iontophoretic application of DA D~ agonist SKF 38393 reduced the amygdala excitatory input to accumbens neuron (about 34% reduction)• Iontophoretic application of CCK had little or no effect on this SKF 38393 elicited reduction (from -34% to-17%)•
Also, as shown in Fig. 8, the degree of attenuation was less for SCH 23390 than for sulpiride (from - 5 9 % to - 5 4 % as compared to - 5 9 % to -7.9%). The effects of the combined iontophoretic application of SCH 23390 and
sulpiride were similar to those of SCH 23390 alone. The iontophoretic application of CCK reduced the attenuating effects of ventral tegrnental stimulation from - 5 9 % to -18.9% (see Fig. 8) but had little effect when administered along with SCH 23390 and sulpiride. A representative recording site in the accumbens and the stimulation site in the amygdala are shown in Fig. 9.
O
DISCUSSION
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Fig. 5. Bar histograms to show the changes in the responses of an accumbens neuron to amygdala stimulation associated with the iontophoretic treatments presented in Fig. 4.
Neurons of the medial region of the nucleus accumbens were strongly activated by single pulse electrical stimulation of the basolateral amygdala. This observation is consistent with the results of previous studies a8,4°. These excitatory responses appear to be mediated by glutamatergic axons projecting to the medial accumbens from the basolateral amygdala 3,15,23. The excitatory responses of accumbens neurons to amygdala stimulation were reduced substantially by the iontophoretic application of dopamine, confirming previous observations 38. It is now well-established that inputs to accumbens neurons from amygdala and hippocampus are modulated by dopamine, applied exoge-
90 CCK
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Fig. 6. Iontophoretic application of the combination of DA D 1 and D 2 receptor agonists (SKF 38393 and LY 171555, respectively) reduced the excitatory input from amygdala by 67%. This inhibition elicited by the combination of D~ and D 2 agonists was attenuated by iontophoretic application of CCK (from -67% to -38%). nously or r e l e a s e d endogenously 16'36'38-4°. The iontophoretic application of D~ and D 2 agonists also reduced the excitatory responses of accumbens neurons to amygdala stimulation although, as shown in Figs. 3 and 5, LY
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171555 (D 2 agonist) had a more potent effect than SKF 38393 (D1 agonist). When LY 171555 and SKF 38393 were applied concurrently the reduction in the amygdalaevoked excitatory responses of accumbens neurons was similar to that for the application of LY 171555 alone. It appears that the dominant action of the D 2 agonist might have masked the weak effect of the D 1 agonist in modulating the amygdala-evoked excitatory response of the accumbens neurons. In addition, several iontophoretic studies have demonstrated that D 1 agonist 'enable' the expression of the inhibitory action of D= agonist recorded from spontaneous firing accumbens neurons when endogenous dopamine has been depleted acutely 27"
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Fig. 7. Bar histograms to show the changes in the responses of an accumbens neuron to amygdala stimulation with the iontophoretic treatments presented in Fig. 6.
A presynaptic action of dopamine on excitatory afferent inputs to the accumbens was suggested from in vitro intracellular recording experiments in an accumbens slice preparation as bath-applied dopamine was found to suppress EPSPs evoked from periaccumbens-stimulation, while postsynaptic glutamate-evoked depolarization in these neurons was not altered by dopamine 9. The De antagonist (-)-sulpiride only partially suppressed the action of dopamine on the evoked EPSPs, whereas
91 TABLE II Direct comparison of the modulatory effects of CCK on the excitatory inputs to accumbens from amygdala after iontophoretically applied DA and its agonists (A) The effects of DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation
CON
DA
CON
SKF
CON
LY
CON
SKF + L Y
1.57
0.32
1.60
1.31
1.53
0.78
1,67
0.51
No. of accumbens neurons
Attenuation Facilitation No change
9 (~ = -74 +9.0%) 0 0
5 (~ = -28 +1.1%) 0 4
9 (~ = -45 +7.5%) 0 0
9 (£ = -70 +4.4%) 0 0
(B) The effects of CCK on the attenuated effects of DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation
CON
CCK + DA
CON
CCK + SKF
CON
CCK + LY
CON
CCK + SKF + LY
1.60
1.36
2.01
1.48
1.58
1.27
1.67
0.95
No. of accumbens neurons
Attenuation Facilitation No change
9 (~ ----15 +14.4%) 0 0
forskolin or dibutyryl cyclic AMP (which presumably mediate the effect of D1 receptor activation) mimicked the suppressive action of dopamine on the evoked EPSP 9. It is likely that the dopaminergic modulation of the amygdala-evoked excitatory accumbens response involves dopamine receptors on the amygdala-accumbens axonal terminals (pre-synaptic), as well as dopamine receptors on the accumbens neurons (post-synaptic). This suggestion requires further investigation since the interaction of D 1 and D 2 receptors is very complex 4'9'2z. The excitatory responses of accumbens neurons to amygdala stimulation were reduced by conditioning stimulation of the ventral tegmental area which results in the release of dopamine into the nucleus accumbens. This response was substantially reduced by the iontophoretic application of sulpiride (D 2 antagonist) to accumbens neurons, consistent with previous observations which implicated D 2 receptors in the neuromodulatory effects of dopamine on inputs to striatal and ventral striatal neurons 25'37. A small reduction in one-third of the accumbens neurons was observed with the iontophoretic application of SCH 23390 (D1 antagonist). The combined iontophoretic application of sulpiride and SCH 23390 had an effect similar to SCH 23390 alone. The reason for this observation is unclear. As experimental evidence was accumulating for the neuromodulatory effects of dopamine it was reported
1 l~I 3 1
7 (~ = -20 +7.7%) 1 1
6 (£ = -34 +4.4%) 0 3
that CCK co-exists with dopamine in some mesolimbic neurons 1°'ll. In the present study the iontophoretic application of CCK was shown to reduce the attenuating effects of iontophoretically applied dopamine on the excitatory responses of accumbens neurons to amygdala stimulation. Most recordings were from neurons in the posteromedial region of the nucleus accumbens, which has the densest CCK innervation 2°,45. This electrophysiological finding is consistent with the results of experiments by Yim and Mogenson 41,44 showing that the attenuating effects of dopamine released endogenously from electrical stimulation of the ventral tegmental area, on the excitatory responses of accumbens neurons to amygdala stimulation, was partially reversed by the iontophoretic application of CCK to the accumbens neurons. In doses of 20-30 nA, CCK had no effect on the excitatory responses of accumbens neurons to amygdala neurons (see Figs. 1 and 2) although higher doses of CCK (>60 nA) enhanced the excitatory responses to amygdala stimulation and, as reported in earlier studies 29,3°,33, increased the spontaneous activity of accumbens neurons. Additional evidence of the interaction of dopamine and CCK has come from electrophysiological experiments in which the CCK antagonist, proglumide, was administered. Yim and Mogenson 42 reported that proglumide increased the attenuating effects of conditioning stimulation of the ventral tegmental area on amygdala-
92
A SCH
SUL
SUL
o
,IL -TO
B 0
-II0 -,110
410
-70
Fig. 8. The excitatory response of neurons of the nucleus accumbens to single pulse stimulation of the basolateral amygala (0.15 ms, 400-500 ~A) are reduced by conditioning stimulation (600-800/~A, 10 pulses, 10 Hz) to the ventral tegmental area (VTA); A: the reduction of the amygdala-evoked activation of accumbens neurons by VTA conditioning stimulation was reduced by iontophoretic application of sulpiride (SUL), a D 2 antagonist (30-40/IA), but not by the iontophoretic application of SCH 23390 (SCH), a D 1 antagonist (30-40/~A), or by the combined application of the D1 and D 2 antagonists (SCH + SUL). B: prior and concurrent iontophoretie application of CCK (30-40/zA) reduced the effects of VTA conditioning stimulation shown in A but had no effect when combined with sulpiride or with SCH 23390.
e v o k e d excitatory responses of accumbens neurons. T h e effects of C C K on the attenuating effects of D] and D 2 agonists on the excitatory responses of accumbens neurons to amygdala stimulation were investigated for the first time. A s shown in Tables I and II, C C K r e d u c e d substantially the attenuating effects of LY 171555, the D 2 agonist, in 85% of the neurons, but p r o d u c e d a reduction of the attenuating effects of S K F 38393, the D 1 agonist, in less than 40% of the neurons. The attenuating effect of LY 171555 + S K F 38393 on the excitatory responses of accumbens neurons to amygdala stimulation was reduced by C C K in only half of the neurons and the magnitude of the attenuation was relatively small. This contrasts with the substantial interaction of C C K and D A referred to above. T h e presence of C C K 2 as well as d o p a m i n e D 1 and D2 receptors on accumbens n e u r o n s 1A4'34 suggests that this
Fig. 9. Representative photomicrographs showing a recording site in nucleus accumbens (A) and an electrical stimulation site in basolateral nucleus (B) of amygdala. Calibration bar = 1 mm. may be the site of the interaction of C C K and dopamine. H o w e v e r , as indicated above, the d o p a m i n e m o d u l a t i o n of the a m y g d a l a - e v o k e d excitatory response of accumbens neurons by a presynaptic D 2 mechanism raises the possibility that this is also a site of C C K - D A interaction. F r o m the functional perspective it is of considerable interest to investigate the effects of C C K on d o p a m i n e m e d i a t e d behaviors. H o w e v e r , behavioral studies have not yielded consistent findings. Some investigators rep o r t e d that C C K enhances D A - m e d i a t e d behaviors 7"a7'35 and o t h e r investigators have r e p o r t e d antagonistic e f f e c t s 8'21'3]. Crawley 5 has suggested that the nature of the interaction between C C K and D A may d e p e n d on the dose of C C K administered. Vaccarino and R a n k i n 24 r e p o r t e d that differential effects on a m p h e t a m i n e - i n duced l o c o m o t o r activity resulted from microinjections of C C K into the rostral and caudal nucleus accumbens. M o r e recently Yim and M o g e n s o n 43 p r e s e n t e d preliminary evidence suggesting that the C C K - D A interaction d e p e n d s on the a m o u n t of d o p a m i n e released into the nucleus accumbens. Injecting p r o g l u m i d e , a C C K antagonist, into the accumbens e n h a n c e d l o c o m o t o r activity when a relatively small dose of picrotoxin was injected into the ventral t e g m e n t a l area but r e d u c e d l o c o m o t o r activity when a larger dose of picrotoxin was injected into the ventral tegmental area. Injecting picrotoxin into the ventral tegmental area blocks G A B A synapses which
93 disinhibits mesolimbic dopamine n e u r o n s and the con-
n e u r o m o d u l a t o r y action of dopamine. In addition, it is
comitant e n d o g e n o u s release of dopamine into the
shown that the d o p a m i n e D2 agonist LY 171555 produced
nucleus accumbens increases locomotor activity tS. The observations are consistent with a conclusion of Wang 28
a similar (though less potent) action as dopamine. The D1 agonist SKF 38393 had a small effect on its own.
based on electrophysiological experiments that "...the
Co-administration of the 2 agonists produced an effect that was similar to that of dopamine. C C K antagonized the action of LY 171555 as well as the combined action
particular alteration in the activity of a given nucleus accumbens n e u r o n during co-administration of C C K and D A depends on the relative amounts of the two substances being administered". (page 364). In summary, the results of the present experiments confirm those from previous studies that dopamine modulates excitatory inputs from the amygdala to the accumbens and that CCK is a functional antagonist of the
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of LY 171555 and SKF 38393 and had a smaller effect on SKF 38393.
Acknowledgements. Funding for this research was provided by grants from the Medical Research Council of Canada to G.J.M. and C.C.Y. We thank Bixia Shen for technical assistance at the beginning of the study.
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94 28 Wang, R.Y., Cholecystokinin, dopamine, and schizophrenia: recent progress and current problems, Ann. N.Y.. Acad. Sci., 537 (1988) 362-379. 29 Wang, R.Y. and Hu, X.T., Does cholecystokinin potentiate dopamine action in the nucleus accumbens?, Brain Research, 380 (1986) 363-367. 30 Wang, R.Y., White, F.J. and Voigt, M.M., Cholecystokinin, dopamine and schizophrenia, Trends Pharmacol. Sci., 5 (1984) 436-438. 31 Weiss, F., Tanzer, D.J. and Ettenberg, A., Opposite actions of CCK-8 on amphetamine-induced hyperlocomotion and stereotype following intracerebroventricular and intra-accumbens injections in rat, Pharmacol. Biochem. Behav., 30 (1988) 309-317. 32 White, EJ., D 1 dopamine receptor stimulation enables the inhibition of nucleus accumbens neurons by a D 2 receptor agonist, Eur. J. Pharmacol., 135 (1987) 101-105. 33 White, F.J. and Wang, R.Y., Pharmacological characterization of dopamine autoreceptors in the rat VTA: microiontophoretic studies, J. Pharmacol. Exp. Therap., 231 (1984) 275-280. 34 White, EJ. and Wang, R.Y., Electrophysiological evidence for the existence of both D 1 and D 2 dopamine receptors in the rat nucleus accumbens, J. Neurosci., 6 (1986) 274-280. 35 Worms, P., Martinez, J., Briet, C., Castro, B. and Biziere, K., Evidence for dopaminomimetic effect of intrastriatally injected cholecystokinin octapeptide in mice, Eur. J. PharmacoL, 121 (1986) 395-401. 36 Yang, C.R. and Mogenson, G.J., Electrophysiological responses of neurons in the nucleus accumbens to hippocampal stimulation and attenuation of the excitatory responses by the mesolimbic dopaminergic system, Brain Research, 324 (1984) 69-84. 37 Yang, C.R. and Mogenson, G.J., Hippocampal signal transmis-
38
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sion to the pedunculopontine nucleus and its regulation by dopamine D 2 receptors in the nucleus accumbens: an electrophysioiogical and behavioral study, Neuroscience, 23 (1987) 10411055. Yim, C.Y. and Mogenson, G.J., Response of nucleus accumbens neurons to amygdala stimulation and its modification by dopamine, Brain Research, 239 (1982) 401-415. Yim, C.Y. and Mogenson, G.J., Mesolimbic dopamine projection modulates amygdala-evoked EPSP in nucleus accumbens: an in vivo study, Brain Research, 369 (1986) 347-352. Yim, C.Y. and Mogenson, G.J., Neuromodulatory action of dopamine in the nucleus accumbens: an in vivo intracellular study, Neuroscience, 26 (1988) 403-415. Yim, C.Y. and Mogenson, G.J., Cholecystokinin modifies the neuromodulatory action of dopamine in nucleus accumbens: electrophysiological evidence, Proc. Soc. Neurosci., 14 (1988) 114, Abstr. 46.15. Yim, C.Y. and Mogenson, G.J., Effect of cholecystokinin blockade on neuromodulatory action of dopamine in the accumbens, Proc. Can. Fed. Biol. Soc., 31 (1988)72, Abstr. 123. Yim, C.Y. and Mogenson, G.J., Behavioral evidence of interaction between endogenous dopamine and cholecystokinin in nucleus accumbens, Proc. Can. Fed. Biol. Soc., 32 (1989) 55, Abstr. 028. Yim, C.Y. and Mogenson, G.J., Electrophysiological evidence of modulatory interaction between dopamine and cholecystokinin in the nucleus accumbens, Brain Research, 541 (1991) 12-20. Zaborszky, L., Alheid, G.F., Beinfield, M.C., Eidens, L.E., Heimer, L. and Palkovits, M., Cholecystokinin innervation of the ventral striatum: a morphological and radioimmunological study, Neuroscience, 14 (1985) 427-353.
Brain Research, 554 (1991) 85-94 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116781P BRES 16781
Effects of dopamine agonists on excitatory inputs to nucleus accumbens neurons from the amygdala: modulatory actions of cholecystokinin R.Z. Liang, M. Wu, C.C.Y. Yim and G.J. Mogenson Department of Physiology, University of Western Ontario, London, Ontario (Canada) (Accepted 12 February 1991)
Key words: CCK-8; Dopamine; SKF 38393; LY 171555; Nucleus accumbens; Amygdala; Neuromodulation
The interaction of the cholecystokinin octapeptide (CCK-8) with dopamine (DA) and dopamine agonists on neurons in the nucleus accumbens was investigated using single unit recording and iontophoretic techniques in urethane-anaesthetized rats. Neurons in the nucleus accumbens were activated by single pulse stimulation of amygdala. Using seven-barrel microelectrodes, the effects of iontophoretic application of CCK-8, DA, dopamine D 1 and/or D 2 receptor agonists (SKF 38393 and LY 171555 respectively) were compared. The iontophoretic application of DA, LY 171555 and LY 171555 + SKF 38393 attenuated by 50-60% the excitatory responses of accumbens neurons to electrical stimulation of basolaterai amygdala whereas SKF 38393 attenuated the response by less than 30%. The iontophoretic application of CCK reduced these attenuating effects of DA, LY 171555 and SKF 38393 + LY 171555. With CCK there was a rather small reduction of the attenuating effect of SKF 38393. These observations provide additional electrophysiologicalevidence of the interaction of CCK and dopamine and suggest that the interaction is associated mainly with dopamine D2 mechanisms. INTRODUCTION Following the d e m o n s t r a t i o n that cholecystokinin ( C C K ) co-exists with d o p a m i n e in neurons that project from the m i d b r a i n to the ventral striatum there has been a k e e n interest in their possible functional interaction 6,12. E x p e r i m e n t s utilizing microdialysis and in vivo voltamm e t r y have shown that C C K influences the release of d o p a m i n e into the nucleus accumbens 1a'19'26. Results of electrophysiological experiments suggest that C C K modulates the release and possibly the postsynaptic effects of d o p a m i n e 29'33. Behavioural experiments have also provided evidence that there are interactions of C C K and d o p a m i n e in the striatum 6'7,s,21'3°,31. Recently the interaction of C C K and d o p a m i n e in the nucleus accumbens has been investigated using a different a p p r o a c h 4°-43. It is based on earlier demonstrations that d o p a m i n e , either applied by iontophoresis or released endogenously by electrical stimulation of the ventral t e g m e n t a l area, reduces the excitatory responses of neurons of the nucleus accumbens to single-pulse stimulation of the basolateral amygdala 38. W h e n proglumide, a C C K antagonist, was applied iontophoretically, this reduction of the excitatory responses of accumbens neurons to a m y g d a l a stimulation due to V T A stimulation
was enhanced 42 whereas when C C K was applied, there was a reversal of the reduction from iontophoretically applied d o p a m i n e . These results suggest that C C K m a y be an e n d o g e n o u s functional antagonist of d o p a m i n e . Since d o p a m i n e is known to bind to separate r e c e p t o r subtypes (D1, D2) in the accumbens, and the 2 r e c e p t o r subtypes p r e s u m a b l y m e d i a t e different functions 4, it is of interest to explore w h e t h e r or not C C K has preferential effects on the r e c e p t o r subtypes. The objective of the present experiments is to investigate the possible interaction of C C K and d o p a m i n e D 1 and D 2 agonists on the interaction b e t w e e n C C K and d o p a m i n e itself following the same e x p e r i m e n t a l p a r a d i g m used in a previous study.
MATERIALS AND METHODS Eighty-three male Wistar rats weighing 250--350 g were used in this study. They were anaesthetized with urethane (1.15 g/kg) and mounted in a stereotaxic apparatus, with the incisor bar fixed at 5 mm above the interaural plane. Body temperature of the animal was maintained at 37 °C with a regulated DC heating pad (Harvard). Stimulating and recording electrodes were lowered through burr holes in the skull to the appropriate sites according to the stereotaxic coordinates obtained from the standard atlas of Pellegrino and Cushman TM. Coordinates used for the basolateral amygdala were 0.5-0.8 mm posterior to bregma, 4.6-4.8 mm lateral to the sagittal
Correspondence: R.Z. Liang, Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
86 sinus and 8.0-8.4 mm below the surface of the cortex. Those for the nucleus accumbens were 3.0-3.4 mm anterior to the bregma, 1.2-1.4 mm lateral to the sagittal sinus and 5.6-7.3 mm below the surface of the cortex. Stainless steel concentric bipolar electrodes (NE-100, Rhodes Medical, 0.5 mm diameter, 0.5 mm tip separation) were used for electrical stimulation of the amygdala. Electrical stimulation was generated by a Grass S-44 stimulator coupled to a Grass stimulation isolation unit (SIU-6). Monophasic square pulses of 0.15 ms duration, 150-800 p A were used. Seven-barrel glass micropipettes were used for recording. Each side barrel was filled separately with dopamine hydrochloride (1 M, pH = 3), SKF 38393 (2,3,4, 5-tetrahydro-7,8-dihydroyl-phenyl-lH-3-benzazepine, 0.05 M, pH = 3), LY 171555 (quinpirole hydrochloride, 0.05 M), CCK (20 pg/ml, pH = 5), glutamate (0.15 M, pH = 8) and saline (0.9%, pH = 3) for iontophoretic injection. The centre barrel was filled with 0.5 M sodium acetate containing 2% Pontamine sky blue for recording and dye deposit. Dopamine, SKF 38393, LY 171555, and saline were ejected as cations at 10-30 nA, while the glutamate and CCK-8 were ejected as anions at 10-30 nA. Currents were driven by a Dagan 6400 iontophoretic unit. The iontophoretic currents were matched for the various compounds (20-30 nA) so that comparisons could be made of their effects on the excitatory response of accumbens neurons to single pulse stimulation of the amygdala. In one series of rats, conditioning stimulation (10 pulses, 10 Hz) was delivered to the ventral tegmental area (VTA), the site of mesolimbic dopamine neurons, ending 100 ms before stimulation to the amygdala (for details see Yim and Mogenson3S). The D 1 antagonist, SCH 23390 (R[ + ]-7-Chloro-8-hydroxy-3-methyl- 1-phenyl-2,3,4,5-tetrahydro-lH-3-benzazepine HCI, 0.05 M), and D 2 antagonist, sulpiride (5-[Aminosulfonyl]-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide, 0.10 M, pH = 5.5), were administered by iontophoresis to investigate the effects on the attenuation by VTA conditioning stimulation of the amygdala-enhanced excitatory responses of accumbens neurons. Signal recorded from the microelectrode was fed into a DC
preamplifier (Axoprobe, Axon Instruments) and then to a Tektronix oscilloscope. Extracellular action potentials were isolated from background noise with a window discriminator which were then sampled in real time by an IBM-AT microcomputer. Sampling and data analysis were done with custom programs developed in our laboratory. As described elsewhere changes of 10% or more in the discharge rate of accumbens neurons were considered statistically significant*~. At the end of each track of recording, recording sites were marked by injecting Pontamine sky blue as an anion with a cathodal current of 10 ~A for 10 min. At the end of the experiment, stimulation sites were marked by an iron deposit by passing 10 p A anodal current through the stimulating electrode for 1 rain. The animal was then sacrificed by an overdose of urethane, and perfused with 50 ml of saline followed by 50 ml of 10% buffered formalin. The brain was removed and fixed in formalin for 24 h. Frozen coronal section of 80 pM were cut with a microtome from the appropriate regions. The sections were mounted on glass slides and stained with thionin for examination.
RESULTS Extracellular recordings were made from more than 200
neurons
in
the
medial
nucleus
accumbens
not
r e p o r t e d p r e v i o u s l y . T h e s a m p l e i n c l u d e s q u i e s c e n t as well as s p o n t a n e o u s l y a c t i v e n e u r o n s t h a t w e r e a c t i v a t e d b y single p u l s e s t i m u l a t i o n o f t h e b a s o l a t e r a l n u c l e u s o f amygdala.
Examples
of
the
excitatory
responses
to
a m y g d a l a s t i m u l a t i o n a r e s h o w n in r e c o r d i n g s o f a c t i o n p o t e n t i a l s in Fig. 1 A a n d in v e r t i c a l r a s t e r p l o t s in Fig. lB. These excitatory responses were attenuated by the iontophoretic
application of dopamine
(Fig.
1), In a
s e r i e s of 126 a c c u m b e n s n e u r o n s t h e e x c i t a t o r y r e s p o n s e
TABLE I The effects o f CCK, DA and its agonists on the excitatory inputs to nucleus accumbens from the arnygdala (A) The effects o f DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation No. o f accumbens neurons Attenuation
Facilitation No change
CON
DA
CON
SKF
CON
LY
CON
SKF + L Y
1.80
0.66
1.65
1.30
1.60
0.89
1.37
0.66
126 (~ = -63 + 1.6%) 0 0
38 (~ = -28 _+2.3%) 4 8
89 (~ = -51 _+2.9%) 6 4
53 (~ = -53 _+2.7%) 0 2
(B) The effects o f CCK on the attenuating effects o f DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation No. o f accumbens neurons Attenuation
Facilitation No change
CON
CCK + DA
CON
CCK + SKF
CON
CCK + LY
CON
CCK + SKF + LY
1.54
1.34
1.56
1.20
1.50
1.37
1.24
0.74
122 (~ = -13 _+2.5%) 0 4
15 (~ = -4 _+6.5%) 12 11
77 (~ = -2 _+3.3%) 4 8
28 (~ = -31 _+4.5%) 10 15
87
.a. CCK 3O DA 30 SAL 30
DA 30
28 Z e~
r. t~
8
300 s
B SAL 30
CCK 30
DA 30
DA 30
mo
~oo
~oo
9oo
~oo
'
Fig. 1. A: a time frequency histogram showing excitatory responses of a neuron of the nucleus accumbens to stimulation of the basolateral amygdala and the effects of the iontophoretic application of saline (SAL, 30 hA), dopamine (DA, 30 nA) and CCK (30 hA). B: a vertical raster plot for this neuron to show the effect of iontophoretically applied SAL, DA and CCK on the excitatory responses elicited by the electrical stimulation of basolateral amygdala. Activities recorded from the neuron are plotted along the y axis and successive sweeps are displayed along the x axis. The line of dots along the 40 ms time mark represents stimulus artifacts from the amygdala stimulation delivered at that time. The cluster of dots are the responses of the neuron to the stimulations. The horizontal bars mark iontophoretic application of drugs. Iontophoretic application of DA (30 hA) reduced the excitatory inputs from amygdala by 72%. This DA effect on accumbens neurons was reversed by iontophoretic application of CCK (from -72% to -4.5%). As a control 30 nA of current was applied to a barrel of the micropipette containing isotonic saline.
to a m y g d a l a stimulation was r e d u c e d by 10% o r m o r e (arbitrarily considered to be significant 44) in all 126 neurons (100%). This attenuating effect of d o p a m i n e was in turn reversed or suppressed when C C K was applied iontophoretically before and during the application of d o p a m i n e to accumbens neurons (see Fig. 1). This action of C C K was o b s e r v e d in 122 of the 126 accumbens neurons and, as shown in Table I, C C K reduced the attenuating effects of d o p a m i n e on the excitatory response to amygdala stimulation from - 6 3 % to - 1 3 % . T h e excitatory response of accumbens neurons to a m y g d a l a stimulation was r e d u c e d by the iontophoretic application of the D 2 agonist, LY 171555, as shown in Figs. 2 and 3. This effect of LY 171555 was o b s e r v e d in 89 of a series of 99 accumbens neurons (90%) in which the average reduction of the excitatory response was 51% (see Table I). The i o n t o p h o r e t i c application of the D 1 agonist, S K F 38393, also r e d u c e d the excitatory response
of accumbens neurons to a m y g d a l a stimulation (see Figs. 4 and 5). This reduction occurred in 38 of 50 accumbens neurons (76%) and as shown in Table I the reduction was 28%. The iontophoretic application of C C K suppressed or reversed the attenuating effects of the 2 D A agonists on the excitatory response of accumbens neurons to amygdala stimulation (see Figs. 2, 3, 4 and 5), the suppression being greater for LY 171555 than for S K F 38393. This effect was observed in 77 of the 89 neurons in which attenuation occurred to LY 171555 and the reversal from C C K was from - 5 1 % to - 2 % (see Table I). F o r S K F 38393 the reversal occurred in 15 of 38 accumbens neurons and was from - 2 8 % to - 4 % . The effects of the c o m b i n e d i o n t o p h o r e t i c applications of the O 1 and D 2 agonists on the excitatory responses of accumbens neurons to a m y g d a l a stimulation were also investigated as illustrated in Fig. 6. In a series of 55
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accumbens neurons the excitatory responses were reduced in 53 neurons by an average of 53% (see Table I). In 28 of these neurons the application of CCK suppressed the effects of the co-administration of SKF 38393 and LY 171555 to a 31% reduction of the excitatory responses of
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accumbens neurons (see Fig. 7 and Table I). In a series of 9 accumbens neurons it was possible to compare in the same neuron the effects of CCK on the responses to dopamine, SKF 38393 and LY 171555. In all 9 neurons the excitatory response to amygdala stimulation was reduced (average reduction shown in brackets) by the iontophoretic application of dopamine (-74%), of LY 171555 (-45%), and of SKF + LY (-70%). The iontophoretic application of SKF 38393 reduced the excitatory response to amygdala stimulation in 5 of the 9 neurons with the average reduction of 28%. As shown in Table II these effects were suppressed by CCK, for dopamine to - 1 5 % (n = 9), for LY 171555 to - 2 0 % (n = 7) and for LY + SKF to - 3 4 % (n = 6). In another series of 32 accumbens neurons, the excitatory response to stimulation of the basolateral amygdala was reduced by a train of conditioning pulses to the ventral tegmental area, as reported previously 38. A comparison was then made of the effects of the iontophoretic application of the D 1 antagonist, SCH 23390, and of the D2 antagonist, sulpiride. Suipiride attenuated the effects of ventral tegmental area stimulation in 30 of 32 neurons whereas SCH 23390 attenuated the effects of ventral tegmental area stimulation in 10 of 32 neurons.
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Also, as shown in Fig. 8, the degree of attenuation was less for SCH 23390 than for sulpiride (from - 5 9 % to - 5 4 % as compared to - 5 9 % to -7.9%). The effects of the combined iontophoretic application of SCH 23390 and
sulpiride were similar to those of SCH 23390 alone. The iontophoretic application of CCK reduced the attenuating effects of ventral tegrnental stimulation from - 5 9 % to -18.9% (see Fig. 8) but had little effect when administered along with SCH 23390 and sulpiride. A representative recording site in the accumbens and the stimulation site in the amygdala are shown in Fig. 9.
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Neurons of the medial region of the nucleus accumbens were strongly activated by single pulse electrical stimulation of the basolateral amygdala. This observation is consistent with the results of previous studies a8,4°. These excitatory responses appear to be mediated by glutamatergic axons projecting to the medial accumbens from the basolateral amygdala 3,15,23. The excitatory responses of accumbens neurons to amygdala stimulation were reduced substantially by the iontophoretic application of dopamine, confirming previous observations 38. It is now well-established that inputs to accumbens neurons from amygdala and hippocampus are modulated by dopamine, applied exoge-
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Fig. 6. Iontophoretic application of the combination of DA D 1 and D 2 receptor agonists (SKF 38393 and LY 171555, respectively) reduced the excitatory input from amygdala by 67%. This inhibition elicited by the combination of D~ and D 2 agonists was attenuated by iontophoretic application of CCK (from -67% to -38%). nously or r e l e a s e d endogenously 16'36'38-4°. The iontophoretic application of D~ and D 2 agonists also reduced the excitatory responses of accumbens neurons to amygdala stimulation although, as shown in Figs. 3 and 5, LY
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171555 (D 2 agonist) had a more potent effect than SKF 38393 (D1 agonist). When LY 171555 and SKF 38393 were applied concurrently the reduction in the amygdalaevoked excitatory responses of accumbens neurons was similar to that for the application of LY 171555 alone. It appears that the dominant action of the D 2 agonist might have masked the weak effect of the D 1 agonist in modulating the amygdala-evoked excitatory response of the accumbens neurons. In addition, several iontophoretic studies have demonstrated that D 1 agonist 'enable' the expression of the inhibitory action of D= agonist recorded from spontaneous firing accumbens neurons when endogenous dopamine has been depleted acutely 27"
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Fig. 7. Bar histograms to show the changes in the responses of an accumbens neuron to amygdala stimulation with the iontophoretic treatments presented in Fig. 6.
A presynaptic action of dopamine on excitatory afferent inputs to the accumbens was suggested from in vitro intracellular recording experiments in an accumbens slice preparation as bath-applied dopamine was found to suppress EPSPs evoked from periaccumbens-stimulation, while postsynaptic glutamate-evoked depolarization in these neurons was not altered by dopamine 9. The De antagonist (-)-sulpiride only partially suppressed the action of dopamine on the evoked EPSPs, whereas
91 TABLE II Direct comparison of the modulatory effects of CCK on the excitatory inputs to accumbens from amygdala after iontophoretically applied DA and its agonists (A) The effects of DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation
CON
DA
CON
SKF
CON
LY
CON
SKF + L Y
1.57
0.32
1.60
1.31
1.53
0.78
1,67
0.51
No. of accumbens neurons
Attenuation Facilitation No change
9 (~ = -74 +9.0%) 0 0
5 (~ = -28 +1.1%) 0 4
9 (~ = -45 +7.5%) 0 0
9 (£ = -70 +4.4%) 0 0
(B) The effects of CCK on the attenuated effects of DA and its agonists on the excitatory inputs to nucleus accumbens from the amygdala
Evoked responses per stimulation
CON
CCK + DA
CON
CCK + SKF
CON
CCK + LY
CON
CCK + SKF + LY
1.60
1.36
2.01
1.48
1.58
1.27
1.67
0.95
No. of accumbens neurons
Attenuation Facilitation No change
9 (~ ----15 +14.4%) 0 0
forskolin or dibutyryl cyclic AMP (which presumably mediate the effect of D1 receptor activation) mimicked the suppressive action of dopamine on the evoked EPSP 9. It is likely that the dopaminergic modulation of the amygdala-evoked excitatory accumbens response involves dopamine receptors on the amygdala-accumbens axonal terminals (pre-synaptic), as well as dopamine receptors on the accumbens neurons (post-synaptic). This suggestion requires further investigation since the interaction of D 1 and D 2 receptors is very complex 4'9'2z. The excitatory responses of accumbens neurons to amygdala stimulation were reduced by conditioning stimulation of the ventral tegmental area which results in the release of dopamine into the nucleus accumbens. This response was substantially reduced by the iontophoretic application of sulpiride (D 2 antagonist) to accumbens neurons, consistent with previous observations which implicated D 2 receptors in the neuromodulatory effects of dopamine on inputs to striatal and ventral striatal neurons 25'37. A small reduction in one-third of the accumbens neurons was observed with the iontophoretic application of SCH 23390 (D1 antagonist). The combined iontophoretic application of sulpiride and SCH 23390 had an effect similar to SCH 23390 alone. The reason for this observation is unclear. As experimental evidence was accumulating for the neuromodulatory effects of dopamine it was reported
1 l~I 3 1
7 (~ = -20 +7.7%) 1 1
6 (£ = -34 +4.4%) 0 3
that CCK co-exists with dopamine in some mesolimbic neurons 1°'ll. In the present study the iontophoretic application of CCK was shown to reduce the attenuating effects of iontophoretically applied dopamine on the excitatory responses of accumbens neurons to amygdala stimulation. Most recordings were from neurons in the posteromedial region of the nucleus accumbens, which has the densest CCK innervation 2°,45. This electrophysiological finding is consistent with the results of experiments by Yim and Mogenson 41,44 showing that the attenuating effects of dopamine released endogenously from electrical stimulation of the ventral tegmental area, on the excitatory responses of accumbens neurons to amygdala stimulation, was partially reversed by the iontophoretic application of CCK to the accumbens neurons. In doses of 20-30 nA, CCK had no effect on the excitatory responses of accumbens neurons to amygdala neurons (see Figs. 1 and 2) although higher doses of CCK (>60 nA) enhanced the excitatory responses to amygdala stimulation and, as reported in earlier studies 29,3°,33, increased the spontaneous activity of accumbens neurons. Additional evidence of the interaction of dopamine and CCK has come from electrophysiological experiments in which the CCK antagonist, proglumide, was administered. Yim and Mogenson 42 reported that proglumide increased the attenuating effects of conditioning stimulation of the ventral tegmental area on amygdala-
92
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SUL
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B 0
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410
-70
Fig. 8. The excitatory response of neurons of the nucleus accumbens to single pulse stimulation of the basolateral amygala (0.15 ms, 400-500 ~A) are reduced by conditioning stimulation (600-800/~A, 10 pulses, 10 Hz) to the ventral tegmental area (VTA); A: the reduction of the amygdala-evoked activation of accumbens neurons by VTA conditioning stimulation was reduced by iontophoretic application of sulpiride (SUL), a D 2 antagonist (30-40/IA), but not by the iontophoretic application of SCH 23390 (SCH), a D 1 antagonist (30-40/~A), or by the combined application of the D1 and D 2 antagonists (SCH + SUL). B: prior and concurrent iontophoretie application of CCK (30-40/zA) reduced the effects of VTA conditioning stimulation shown in A but had no effect when combined with sulpiride or with SCH 23390.
e v o k e d excitatory responses of accumbens neurons. T h e effects of C C K on the attenuating effects of D] and D 2 agonists on the excitatory responses of accumbens neurons to amygdala stimulation were investigated for the first time. A s shown in Tables I and II, C C K r e d u c e d substantially the attenuating effects of LY 171555, the D 2 agonist, in 85% of the neurons, but p r o d u c e d a reduction of the attenuating effects of S K F 38393, the D 1 agonist, in less than 40% of the neurons. The attenuating effect of LY 171555 + S K F 38393 on the excitatory responses of accumbens neurons to amygdala stimulation was reduced by C C K in only half of the neurons and the magnitude of the attenuation was relatively small. This contrasts with the substantial interaction of C C K and D A referred to above. T h e presence of C C K 2 as well as d o p a m i n e D 1 and D2 receptors on accumbens n e u r o n s 1A4'34 suggests that this
Fig. 9. Representative photomicrographs showing a recording site in nucleus accumbens (A) and an electrical stimulation site in basolateral nucleus (B) of amygdala. Calibration bar = 1 mm. may be the site of the interaction of C C K and dopamine. H o w e v e r , as indicated above, the d o p a m i n e m o d u l a t i o n of the a m y g d a l a - e v o k e d excitatory response of accumbens neurons by a presynaptic D 2 mechanism raises the possibility that this is also a site of C C K - D A interaction. F r o m the functional perspective it is of considerable interest to investigate the effects of C C K on d o p a m i n e m e d i a t e d behaviors. H o w e v e r , behavioral studies have not yielded consistent findings. Some investigators rep o r t e d that C C K enhances D A - m e d i a t e d behaviors 7"a7'35 and o t h e r investigators have r e p o r t e d antagonistic e f f e c t s 8'21'3]. Crawley 5 has suggested that the nature of the interaction between C C K and D A may d e p e n d on the dose of C C K administered. Vaccarino and R a n k i n 24 r e p o r t e d that differential effects on a m p h e t a m i n e - i n duced l o c o m o t o r activity resulted from microinjections of C C K into the rostral and caudal nucleus accumbens. M o r e recently Yim and M o g e n s o n 43 p r e s e n t e d preliminary evidence suggesting that the C C K - D A interaction d e p e n d s on the a m o u n t of d o p a m i n e released into the nucleus accumbens. Injecting p r o g l u m i d e , a C C K antagonist, into the accumbens e n h a n c e d l o c o m o t o r activity when a relatively small dose of picrotoxin was injected into the ventral t e g m e n t a l area but r e d u c e d l o c o m o t o r activity when a larger dose of picrotoxin was injected into the ventral tegmental area. Injecting picrotoxin into the ventral tegmental area blocks G A B A synapses which
93 disinhibits mesolimbic dopamine n e u r o n s and the con-
n e u r o m o d u l a t o r y action of dopamine. In addition, it is
comitant e n d o g e n o u s release of dopamine into the
shown that the d o p a m i n e D2 agonist LY 171555 produced
nucleus accumbens increases locomotor activity tS. The observations are consistent with a conclusion of Wang 28
a similar (though less potent) action as dopamine. The D1 agonist SKF 38393 had a small effect on its own.
based on electrophysiological experiments that "...the
Co-administration of the 2 agonists produced an effect that was similar to that of dopamine. C C K antagonized the action of LY 171555 as well as the combined action
particular alteration in the activity of a given nucleus accumbens n e u r o n during co-administration of C C K and D A depends on the relative amounts of the two substances being administered". (page 364). In summary, the results of the present experiments confirm those from previous studies that dopamine modulates excitatory inputs from the amygdala to the accumbens and that CCK is a functional antagonist of the
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of LY 171555 and SKF 38393 and had a smaller effect on SKF 38393.
Acknowledgements. Funding for this research was provided by grants from the Medical Research Council of Canada to G.J.M. and C.C.Y. We thank Bixia Shen for technical assistance at the beginning of the study.
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