Pedunculopontine tegmental stimulation evokes striatal dopamine efflux by activation of acetylcholine and glutamate receptors in the midbrain and pons of the rat

The pedunculopontine tegmental nucleus appears to influence striatal dopamine activity via cholinergic and glutamatergic afferents to dopaminergic cells of the substantia nigra pars compacta. We measured changes in striatal dopamine oxidation current (dopamine efflux) in response to electrical stimulation of the pedunculopontine tegmental nucleus using in vivo electrochemistry in urethane-anaesthetized rats. Pedunculopontine tegmental nucleus stimulation evoked a three-component change in striatal dopamine efflux, consisting of: (i) an initial rapid increase of 2 min duration; followed by (ii) a decrease below prestimulation levels of 9 min duration; then by (iii) a prolonged increase lasting 35 min. Intra-nigral infusions of the ionotropic glutamate receptor antagonist kynurenate (10 microg/ microL) or the nicotinic cholinergic receptor antagonist mecamylamine (5 microg/0.5 microL) selectively attenuated the rapid first component, while systemic injections of the muscarinic cholinergic antagonist scopolamine (5 mg/kg, i.p.) diminished the second and third components. In addition, intra-pedunculopontine tegmental nucleus infusions of the M2 muscarinic antagonist methoctramine (50 microg/ microL) selectively abolished the inhibitory second component, while intranigral infusions of scopolamine (200 microg/ microL) selectively abolished the prolonged third component. Intra-nigral infusions of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (2 microg/ microL) had no effect on pedunculopontine tegmental nucleus-elicited striatal dopamine efflux. These results suggest that the pedunculopontine tegmental nucleus utilizes nicotinic and ionotropic glutamate receptors in the substantia nigra to mediate rapid activation, M2-like muscarinic autoreceptors in the pedunculopontine tegmental nucleus to mediate decreased activation, and muscarinic receptors in the substantia nigra (probably of the M5 subtype) to mediate prolonged activation, of the nigrostriatal dopaminergic system.     

Association Basolateral amygdala stimulation evokes glutamate receptor-dependent dopamine efflux in the nucleus accumbens of the anaesthetized rat

Afferents from the basolateral amygdala and dopamine projections from the ventral tegmental area to the nucleus accumbens have both been implicated in reward-related processes. The present study used in vivo chronoamperometry with stearate-graphite paste electrodes in urethane-anaesthetized rats to determine how basolateral amygdala efferents to the nucleus accumbens synaptically regulate dopamine efflux. Repetitive-pulse (20 Hz for 10 s) electrical stimulation of the basolateral amygdala evoked a complex pattern of changes in monitored dopamine oxidation currents in the nucleus accumbens related to dopamine efflux. These changes were characterized by an initial increase that was time-locked to stimulation, a secondary decrease below baseline, followed by a prolonged increase in the dopamine signal above baseline. The effects of burst-patterned stimulation (100 Hz, 5 pulses/burst, 1-s interburst interval, 40 s) of the basolateral amygdala on the basal accumbens dopamine signal were similar to those evoked by 20 Hz stimulation, with the lack of a secondary suppressive component. Infusions of the ionotropic glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (APV) or 6,7-dinitroquinoxaline-2,3-dione (DNQX) into the nucleus accumbens dose-dependently blocked or attenuated the initial and prolonged increases in the dopamine signal following 20 Hz or burst-patterned basolateral amygdala stimulation. Infusions of the metabotropic glutamate receptor antagonist (+)-α-methyl-4-carboxyphenylglycine selectively blocked the intermediate suppressive effect of 20 Hz basolateral amygdala stimulation on dopamine oxidation currents. Blockade of glutamate receptors or inhibition of dopamine neuronal activity via infusions of either APV + DNQX, lidocaine or γ-hydroxybutyric acid, respectively, into the ventral tegmental area did not effect the pattern of changes in the accumbens dopamine signal evoked by basolateral amygdala stimulation. These data suggest that the glutamatergic basolateral amygdala inputs to nucleus accumbens dopamine terminals synaptically facilitate or depress dopamine efflux, and these effects are independent of dopamine neuronal firing activity. Moreover, these results imply that changes in nucleus accumbens dopamine levels following presentation of reward-related stimuli may be mediated, in part, by the basolateral amygdala.     

NMDA receptors in nucleus accumbens modulate stress-induced dopamine release in nucleus accumbens and ventral tegmental area

Converging evidence suggests that dopamine (DA) transmission in nucleus accumbens (NAcc) is modulated locally by an excitatory amino acid (EAA)-containing input possibly originating in medial prefrontal cortex (PFC). In the present study, we examined the effects of intra-NAcc administration of EAA receptor antagonists on stress-induced increases of NAcc DA levels and of dendritically released DA in the ventral tegmental area (VTA). Local injection of the NMDA receptor antagonist—AP-5 (0.05, 0.5, and 5.0 nmoles)—dose-dependently potentiated increases in NAcc DA levels elicited by 15 min of restraint stress. In contrast, local application of equivalent doses of the kainate/AMPA receptor antagonist—DNQX—failed to alter the NAcc DA stress response reliably. In a separate experiment, we found that intra-NAcc injection of AP-5 also potentiated stress-induced increases in VTA DA levels. These results indicate that EAAs acting at NMDA receptors in NAcc can modulate stress-induced DA release in this region. Our data indicate, however, that this action exerts an inhibitory influence on the NAcc DA stress response, suggesting that the relevant population of NMDA receptors are not located on NAcc DA terminals. The fact that intra-NAcc AP-5 injections also potentiated the DA stress response in VTA suggests instead an action mediated by NMDA receptors located on NAcc neurons that feedback, directly or indirectly, to cell bodies of the mesocorticolimbic DA system. Synapse 26:225–234, 1997. © 1997 Wiley-Liss Inc.     

Extracellular dopamine in the rat ventral tegmental area and nucleus accumbens following ventral tegmental infusion of cocaine

Rats were implanted with dual dialysis probes, one in the ventral tegmental area, and another one ipsilateral in the nucleus accumbens. Infusion of cocaine (10, 100, 1000 mM) into the ventral tegmental area gradually increased extracellular dopamine to 164, 329 and 991% of baseline in the ventral tegmental area, but reduced dopamine to 76, 47 and 38% of baseline in the nucleus acumbens. These results are consistent with cocaine-induced feedback regulation of dopamine cell activity involving somatodendritec impulse-regulating dopamine D₂ autoreceptors.     

Systemic nicotine-induced dopamine release in the rat nucleus accumbens is regulated by nicotinic receptors in the ventral tegmental area

Stimulation of the mesolimbic dopamine (DA) system is considered of major importance for the rewarding and dependence producing properties of nicotine (NIC). To identify the site of this stimulatory action, simultaneous microdialysis was performed in the ventral tegmental area (VTA) and the ipsilateral nucleus accumbens (NAC) of awake rats. Extracellular concentrations of DA and its metabolites were measured in the NAC. NIC (0.5 mg/kg, s.c.) increased DA and its metabolites by ～50%.Concomitant infusion of the nicotinic receptor antagonist mecamylamine (MEC, 100 μ) through the VTA probe, starting 40 min before NIC injection, antagonized the NIC induced increases of DA and its metabolites. In contrast, similar MEC pretreatment (40 or 140 min) in the NAC did not affect DA or metabolite responses to systemic NIC. Infusion of NIC (1,000 μ) in the NAC or the VTA increased DA release by 49% and 48%, respectively, whereas only the VTA infusion increased metabolite concentrations by -25%. MEC infusion (1–1,000 μ) in the VTA did not affect DA or its metabolites, whereas the 1,000 μ concentration infused in the NAC increased DA by 77%. These results suggest that nicotinic receptors in the somatodendritic region may be of greater importance than those located in the terminal area for the stimulatory action of systemic NIC on the mesolimbic DA system. Furthermore, our findings support the notion that the mesolimbic dopaminergic system is phasically rather than tonically regulated by nicotinic receptor activation within the VTA. © 1994 Wiley-Liss, Inc.     

Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat

The mesopontine rostromedial tegmental nucleus (RMTg) is a mostly γ-aminobutyric acid (GABA)ergic structure believed to be a node for signaling aversive events to dopamine (DA) neurons in the ventral tegmental area (VTA). The RMTg receives glutamatergic inputs from the lateral habenula (LHb) and sends substantial GABAergic projections to the VTA, which also receives direct projections from the LHb. To further specify the topography of LHb projections to the RMTg and VTA, small focal injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin were aimed at different subdivisions of the LHb. The subnuclear origin of LHb inputs to the VTA and RMTg was then confirmed by injections of the retrograde tracer cholera toxin subunit b into the VTA or RMTg. Furthermore, we compared the topographic position of retrogradely labeled neurons in the RMTg resulting from VTA injections with that of anterogradely labeled axons emerging from the LHb. As revealed by anterograde and retrograde tracing, LHb projections were organized in a strikingly topographic manner, with inputs to the RMTg mostly arising from the lateral division of the LHb (LHbL), whereas inputs to the VTA mainly emerged from the medial division of the LHb (LHbM). In the RMTg, profusely branched LHb axons were found in close register with VTA projecting neurons and were frequently apposed to the latter. Overall, our findings demonstrate that LHb inputs to the RMTg and VTA arise from different divisions of the LHb and provide direct evidence for a disynaptic pathway that links the LHbL to the VTA via the RMTg.     

Chemical stimulation of the ventral hippocampus elevates nucleus accumbens dopamine by activating dopaminergic neurons of the ventral tegmental area.

Dual-probe microdialysis (with HPLC and electrochemical detection) in freely moving rats and single-unit recording in anesthetized rats were used to study the extent to which impulse flow through the ventral tegmental area (VTA) contributes to elevations in nucleus accumbens (NAS) dopamine (DA) evoked by stimulation of the ventral subiculum (VS). During perfusion of artificial extracellular fluid into the VTA, injections of 0.74 microgram of the excitatory amino acid NMDA into the VS elevated accumbens DA to >150% of basal values. During intra-VTA perfusion of either 1 microM tetrodotoxin (which blocks impulse flow) or 1 mM kynurenic acid (which blocks excitatory glutamate receptors), injections of NMDA into the VS failed to elevate accumbens DA. Thus, increased impulse flow through VTA DA neurons, mediated by excitatory glutamate inputs to this region, appears critical for VS stimulation to elevate NAS DA. Increased impulse flow through VTA DA neurons was confirmed using single-unit recording in anesthetized rats. Intra-VS NMDA injections increased the firing rates of 45% (14 of 31), decreased the firing rates of 13% (4 of 31), and had no effect on 42% (13 of 31) of VTA DA neurons. Increases in firing rates were evident within 15 min of NMDA injections, a time at which VS NMDA injections elevate accumbens DA in awake animals. The results of the present experiments identify the VTA as a critical site through which outputs from the VS modulate NAS dopaminergic neurotransmission.     

Inhibition from ventral tegmental area of nucleus accumbens neurons in the rat

The role of the ventral tegmental area (VTA), which is rich in dopamine-containing cell bodies, on nucleus accumbens (Acc) neurons was examined. In Acc neurons receiving input from parafascicular nucleus (Pf) of thalamus, VTA conditioning stimulation produced an inhibition of spike generation with Pf stimulation. In contrast, VTA conditioning stimulation did not affect Acc neurons receiving input from limbic structures such as the amygdala nucleus and hippocampus.     

Electrical stimulation of the ventral tegmental area and catecholamine metabolism in discrete regions of the rat brain

Data are presented which show a different pattern of dopamine and noradrenaline utilization in terminal regions of the A9 and A10 dopaminergic systems and in terminal regions of the dorsal noradrenergic bundle after electrical stimulation of the ventral tegmental area and an adjacent area. At the sites of the electrodes an enhanced turnover of noradrenaline was found. These results are discussed relating the location of the electrode sites and the location of the dopaminergic cell bodies A9 and A10 and catecholaminergic fibers passing through the MFB innervating forebrain and limbic structures. It is concluded that activation of parts of the A10 dopaminergic system and the A6 noradrenergic system is correlated with intracranial selfstimulation.     

Suppression of attack behavior in cats by stimulation of ventral tegmental area and nucleus accumbens

Low frequency (6 pps) stimulation of ventral tegmental area (VTA) and nucleus accumbens (NA) produced EEG synchronization and suppressed attack behavior elicited by hypothalamic stimulation. Both quiet biting and affective attack with rage were suppressed. Autonomic and non-directed somatic motor components of the attack reaction were unaffected.High frequency (60 pps) stimulation of VTA failed to suppress any components of the attack reaction; high frequency stimulation of NA, however, did produce suppression of attack.Low frequency (6 pps) sensory stimulation, delivered by photic or lateral geniculate stimulation, produced EEG synchronization but failed to cause suppression of attack. These results indicate that low frequency stimulation per se does not cause suppression of ongoing behavior.This study demonstrates that VTA and NA, components of the mesolimbic dopamine system, are involved in the inhibition of emotional-type behaviors.     

Injections of N-methyl-D-aspartate into the ventral hippocampus increase extracellular dopamine in the ventral tegmental area and nucleus accumbens

The nucleus accumbens septi receives inputs from dopaminergic neurons of the ventral tegmental area (VTA) and glutamatergic neurons of the ventral subiculum (VS). The convergence of these inputs in the NAS is important for the normal expression of exploratory locomotion; stimulation of the VS by injection of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) causes dopamine-dependent increases in locomotion. In the present study, in vivo microdialysis in conjunction with high-performance liquid chromatography and electrochemical detection (HPLC-EC) was used to estimate changes in extracellular dopamine in the VTA and NAS in response to intra-VS injections of NMDA (0.074, 0.28, 0.74 microg). NMDA injections caused dose-dependent elevations in extracellular dopamine in each region. Each dose of NMDA clearly increased extracellular dopamine in the NAS, whereas only the two higher doses increased dopamine significantly in the VTA. The highest dose of NMDA elevated extracellular dopamine to approximately 180% of baseline in each region. Whereas elevations in NAS dopamine might be induced by impulse-independent local mechanisms, elevations of dopamine in the VTA are presumed to reflect increased somatodendritic release associated with increased impulse flow through dopamine neurons. Thus, the present study suggests that the modulation of dopaminergic neurotransmission by the ventral subiculum results from a trans-synaptic activation of dopamine cell bodies in the VTA.     

Muscarinic receptor blockade in the ventral tegmental area attenuates cocaine enhancement of laterodorsal tegmentum stimulation‐evoked accumbens dopamine efflux in the mouse

The reinforcing properties of cocaine have been related to increased extracellular concentrations of dopamine in the nucleus accumbens (NAc). M5 muscarinic acetylcholine receptors (mAChRs) on dopamine cells in the ventral tegmental area (VTA) facilitate mesoaccumbens dopamine transmission and are critically involved in mediating natural and drug reinforcement. We investigated the effects of pharmacological blockade of mAChRs in the VTA on cocaine's ability to enhance electrically evoked NAc dopamine efflux. Using fixed potential amperometry together with carbon fiber recording microelectrodes positioned in the NAc core, we quantified dopamine oxidation currents (dopamine efflux) evoked by brief stimulation (15 monophasic pulses at 50 Hz every 30 s) of the laterodorsal tegmentum (LDT) in urethane (1.5 g/kg, i.p.) anesthetized mice. Compared to predrug baseline responses, cocaine (5 or 10 mg/kg, i.p.) dose‐dependently enhanced LDT stimulation‐evoked NAc dopamine efflux, whereas the nonsubtype selective mAChR antagonist scopolamine (10 μg/0.5 μl) microinfused into the VTA diminished LDT‐evoked NAc dopamine efflux. Preinfusion of scopolamine into the VTA diminished the facilitatory actions of cocaine on LDT stimulation‐evoked NAc dopamine efflux, and when infused at the peak effect of cocaine attenuated LDT‐evoked dopamine efflux to below predrug baseline levels. These findings suggest that LDT cholinergic inputs to dopamine neurons in the VTA, via activation of mAChRs (probably of the M5 subtype), are involved in modulating the facilitatory effects of cocaine on NAc dopamine neurotransmission. They also suggest that the development of antagonists aimed at selectively disrupting M5 receptor function may be valuable in reducing abuse liability of psychostimulants. Synapse 64:216–223, 2010. © 2009 Wiley‐Liss, Inc.     

Nerve terminal glutamate immunoreactivity in the rat nucleus accumbens and ventral tegmental area after a short withdrawal from cocaine

Cocaine administration has been shown to alter glutamate transmission in numerous studies. Using quantitative electron microscopic immunogold labeling, our laboratory has previously reported that nerve terminal glutamate immunoreactivity is transiently altered following cocaine administration. The present study was undertaken to examine presynaptic nerve terminal glutamate immunoreactivity at shorter time points after withdrawal from cocaine. Animals received saline or cocaine for 7 days followed 3 days later by a cocaine or saline challenge. Most (>75%) cocaine-challenged animals had a heightened locomotor response to cocaine compared to the first day of cocaine and were considered behaviorally sensitized. One day after the challenge, glutamate immunogold-labeling was quantified in nerve terminals making asymmetrical synaptic contacts within the core and shell of the nucleus accumbens and ventral tegmental area. A single dose of cocaine did not alter the density of presynaptic nerve terminal glutamate immunoreactivity in the nucleus accumbens (NAc) or ventral tegmental area (VTA). The density of nerve terminal glutamate immunoreactivity in the shell, but not the core, was significantly increased in the animals receiving repeated cocaine. In the VTA the density of nerve terminal glutamate immunoreactivity did not change in the cocaine-sensitized group, but was significantly increased in the nonsensitized group. The finding that repeated cocaine treatment increased glutamate nerve terminal immunolabeling within the nucleus accumbens shell, but not the core, supports the hypothesis that glutamate synapses in the core and shell are differentially sensitive to repeated cocaine administration. Overall, our study does not support a role for changes in presynaptic glutamate in the development of behavioral sensitization.     

Levels of CCK immunoreactivity and dopamine in the nucleus accumbens after 6-hydroxydopamine lesions of the ventral tegmental area and the nucleus accumbens

The effects of 6-hydroxydopamine (6-OHDA) injected into either the ventral tegmental area (VTA) or the nucleus accumbens on dopamine and its metabolite DOPAC and CCK-immunoreactivity in the nucleus accumbens were studied. Measurements were made 1, 3 and 6 weeks after the lesion in both the anterior and posterior part of the nucleus accumbens. 6-OHDA applied in the VTA reduced the level of DA and DOPAC, but did not affect the CCK content in the nucleus accumbens. 6-OHDA lesions in the anterior part of the nucleus accumbens produced depletion of DA and DOPAC from one week after the injection, and decreased the CCK content 6 weeks after the injection. The results suggest that in the nucleus accumbens DA and CCK are localized, at least for the major part, in different nerve terminals.     

Repeated ventral tegmental area amphetamine administration alters dopamine D1 receptor signaling in the nucleus accumbens

Neuroadaptations of the mesoaccumbens dopamine (DA) system likely underlie the emergence of locomotor sensitization following the repeated intermittent systemic administration of amphetamine (AMPH). In the nucleus accumbens (NAc), such neuroadaptations include enhanced DA overflow in response to a subsequent AMPH challenge as well as increased sensitivity to the inhibitory effects of D1 DA receptor (D1R) activation and an altered profile of D1R-dependent induction of immediate early genes (IEGs). Previous results indicate that AMPH acts in the ventral tegmental area (VTA) to initiate those changes leading to sensitization of the locomotor activity and NAc DA overflow produced by systemic administration of this drug. These observations are intriguing, given that acute infusion of AMPH into the VTA does not stimulate locomotor activity or, as we report presently, increase extracellular NAc DA concentrations. Two experiments, therefore, assessed the ability of repeated VTA AMPH to produce adaptations in D1R signaling in the NAc. Rats were administered three bilateral VTA infusions of saline or AMPH (2.5 microg/0.5 microl/side, one every third day). In the first experiment, in vivo extracellular electrophysiological recordings revealed that previous exposure to VTA AMPH enhanced the sensitivity of NAc neurons to the inhibitory effects of iontophoretic application of the D1R agonist SKF 38393. This effect was observed early (2-3 days) and at 1 month of withdrawal, but not after 2 months. Similarly, in the second experiment it was found that the D1R-dependent induction by AMPH of Fos, FosB, and JunB, but not NGFI-A, in the NAc was enhanced in rats exposed 1 week earlier to repeated VTA AMPH. These findings indicate that repeated VTA AMPH administration initiates relatively long-lasting adaptations in D1R signaling in the NAc that may, together with presynaptic adaptations affecting DA overflow, contribute to the expression of locomotor sensitization by this drug.     

Presynaptic muscarinic receptors on dopaminergic terminals in the nucleus accumbens

Levels of muscarinic receptors were measured in the nucleus accumbens of rat following 0.8 μg 6-hydroxydopamine or vehicle injections (0.2 μl) into the ventral tegmental area to investigate whether the dopaminergic terminals destroyed by this procedure bear muscarinic receptors. Dopamine levels in the nucleus accumbens ipsilateral to the injection of 6-hydroxydopamine were substantially reduced by 83% as compared to the unlesioned side after 7 days. Significant decreases in the specific binding of [³H]N-methylscopolamine of 9 and 15% were also seen in the nucleus accumbens ipsilateral to the lesion after 7 and 14 days respectively. The class of muscarinic receptor depleted by the lesion was further investigated using [³H]oxotremorine-M to label the ‘super high’ affinity binding sites. Thepercentage occupancy of total muscarinic receptors by [³H]oxotremorine-M was significantly decreased by lesion e.g. 23% after 7 days, indicating a selective loss of ‘super high’ affinity binding sites. The lesion caused no change in the affinity constant for the muscarinic antagonist, propylbenzilylcholine. Studies of the binding of the agonists carbachol and oxotremorine-M by competition with [³H]propylbenzilylcholine showed little change in the concentrations of affinity constants of the ‘high’ and ‘low’ affinity binding sites with the 6-hydroxydopamine lesion.     

Self-stimulation of the nucleus accumbens and ventral tegmental area of tsai attenuated by microinjections of spiroperidol into the nucleus accumbens

The contribution of dopaminergic neurons to self-stimulation of the ventral tegmental area, nucleus accumbens and prefrontal cortex was investigated. The ventral tegmental area is the site of non-striatal dopaminergic neurons and their axons project to the nucleus accumbens and prefrontal cortex. Injections of spiroperidol, a dopamine antagonist, into the nucleus accumbens significantly reduced self-stimulation of the ipsilateral ventral tegmental area but did not influence self-stimulation of the contralateral ventral tegmental area. Injections of spiroperidol into the prefrontal cortex did not reduce self-stimulation of the ipsilateral or contralateral ventral tegmental area. Electrical stimulation of sites in the nucleus accumbens positive for self-stimulation antidromically activated neurons of the ventral tegmental area, and a reduction of discharge of these neurons following administration of apomorphine suggested that they were dopaminergic neurons. These observations provide additional evidence implicating dopaminergic neurons in brain-stimulation reward and suggest that dopaminergic neurons contribute to self-stimulation of the nucleus accumbens but not the prefrontal cortex.     

Synaptic connections of starburst amacrine cells and localization of acetylcholine receptors in primate retinas

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.     

Muscarinic receptors on dopamine terminals in the cat caudate nucleus: Neuromodulation of [H]dopamine release in vitro by endogenous acetylcholine

The directly acting muscarinic receptor agonist oxotremorine (1.8–10 μM) produced an increase in electrically evoked [³H]dopamine release from slices of the cat caudate. The maximal increase caused by oxotremorine was 40%, and was antagonized by the muscarinic receptor blocking agent atropine (0.1 μM). Exposure to the acetylcholinesterase (AChE) inhibitor physostigmine (1 μM) resulted in a 50% increase in electrically evoked [³H]dopamine release. The increase caused by physostigmine was also antagonized by atropine (0.1 μM).Atropine did not, however, alter the modulations in [³H]dopamine release mediated by the dopamine autoreceptor: the increase in electrically evoked [³H]dopamine release caused by the dopamine receptor antagonist S-sulpiride (0.1 μM) and the decrease caused by the dopamine receptor agonist pergolide (30 nM) were unaffected by atropine (0.1 μM). These results indicate that the muscarinic receptor-mediated and dopamine autoreceptor-mediated presynaptic effects on [³H]dopamine release are independent.The present results suggest that in the electrically depolarized caudate slice in vitro, released endogenous acetylcholine may interact with muscarinic receptors faciliting depolarization-evoked [³H]dopamine release,ifAChE is inhibited. These muscarinic receptors may be located on dopamine nerve terminals. In the context of present neuroanatomical knowledge, the action of released endogenous acetylcholine on dopamine terminals may be a non-synaptic neuromodulation.