Midbrain muscarinic receptor mechanisms underlying regulation of mesoaccumbens and nigrostriatal dopaminergic transmission in the rat

Laterodorsal (LDT) and pedunculopontine (PPT) tegmental nuclei in the mesopontine project cholinergic inputs to the midbrain ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), respectively, to directly and indirectly influence the activity of dopamine neuronal cells via actions on muscarinic and nicotinic receptors. The present study investigated the role of midbrain muscarinic receptors in the functional modulation of VTA and SNc dopamine cell activity as reflected by alterations in, respectively, nucleus accumbens (NAc) and striataldopamine efflux. In vivo chronoamperometry was used to measure changes in basal dopamine efflux via stearate-graphite paste electrodes implanted unilaterally in the NAc or striatum of urethane-anaesthetized rats, following blockade or activation of, respectively, VTA or SNc muscarinic receptors. Intra-VTA or -SNc infusion of the muscarinic antagonist scopolamine (200 microg/microL) reduced, respectively, NAc and striatal dopamine efflux while infusion of the muscarinic and nicotinic agonist carbachol (0.5 microg/microL) or the prototypical muscarinic agonist muscarine (0.5 microg/microL) increased NAc and striatal dopamine efflux. Transient decreases in dopamine efflux preceded these increases selectively in the striatum, suggesting a reduction in excitatory or increase in inhibitory drive to the SNc by preferential activation of M3 muscarinic receptors on GABA interneurons and glutamatergic inputs. This was confirmed by showing that selective blockade of M3 receptors with p-F-HHSiD (0.5 microg/microL) increased striatal, but not NAc, dopamine efflux. Together, these findings suggest that midbrain muscarinic receptors, probably M5 subtypes on VTA and SNc dopamine neurons, contribute to the tonic excitatory regulation of forebrain basal dopamine transmission whereas presynaptic M3 receptors serve to counter excessive excitation of nigral dopamine cell activity.     

Laterodorsal tegmental stimulation elicits dopamine efflux in the rat nucleus accumbens by activation of acetylcholine and glutamate receptors in the ventral tegmental area

Cholinergic and glutamatergic neurons in the laterodorsal tegmentum (LDT) and neighbouring mesopontine nuclei are thought to influence mesolimbic dopaminergic neuronal activity involved in goal-directed behaviours. We measured the changes in dopamine oxidation current (corresponding with dopamine efflux) in the nucleus accumbens (NAc) in response to electrical stimulation of the LDT using in vivo chronoamperometry in urethane-anaesthetized rats. LDT stimulation (35 Hz pulse trains for 60 s, 1 s intertrain interval) evoked a three-component change in dopamine efflux in the NAc: (i) an initial stimulation time-locked increase in the dopamine signal above baseline, followed by (ii) an immediate decrease below baseline, and thereafter by (iii) a prolonged increase in the dopamine signal above baseline. Intra-VTA infusion of the nicotinic receptor antagonist mecamylamine (5 microg/0.5 microL) or the ionotropic glutamate receptor antagonist kynurenate (10 microg/microL) attenuated the first LDT-elicited component. The second suppressive component was abolished by intra-LDT infusions of either the nonselective or the M2-selective muscarinic receptor antagonists scopolamine (100 microg/microL) and methoctramine (50 microg/microL), respectively. In contrast, intra-VTA infusions of scopolamine (200 microg/microL) resulted in a selective attenuation of the third facilitatory component, whereas both second and third components were abolished by systemic injections of scopolamine (5 mg/kg). These results suggest that the initial increase, subsequent decrease, and final prolonged increase in extracellular dopamine levels in the NAc are selectively mediated by LDT-elicited activation of (i) nicotinic and glutamatergic receptors in the VTA, (ii) muscarinic M2 autoreceptors on LDT cell bodies, and (iii) muscarinic receptors in the VTA, respectively.     

Nigrostriatal dopamine release modulated by mesopontine muscarinic receptors

The present study investigated the regulation of substantia nigra pars compacta (SNc) dopamine neuronal activity by pedunculopontine (PPT) cholinergic neurons. Changes in dopamine efflux following chemical activation or blockade of muscarinic acetylcholine receptors in the PPT were measured at stearate-carbon paste electrodes in the striatum of urethane (1.5 g/kg) anaesthetized male rats using in vivo chronoamperometry (30 s sampling rate). Intra-PPT infusions of a mixed muscarinic/nicotinic (carbachol 8 microg/microl) or M2/4-selective muscarinic (oxotremorine 0.5 microg/microl) receptor agonist attenuated striatal dopamine efflux, whereas a non-selective (scopolamine 100 microg/microl) or M2/4-selective (methoctramine 50 microg/microl) muscarinic receptor antagonist enhanced striatal dopamine efflux. These results suggest that M2/4 muscarinic receptors in the mesopontine tonically influence SNc basal dopamine cell activity and striatal dopamine release.     

Midbrain acetylcholine and glutamate receptors modulate accumbal dopamine release

This study determined the role of ventral tegmental area acetylcholine and glutamate receptors in modulating laterodorsal tegmentum stimulation-evoked dopamine efflux in the nucleus accumbens. Rapid changes in dopamine oxidation current were measured at carbon fiber microelectrodes using fixed potential amperometry in urethane anesthetized male mice. Intraventral tegmental area infusions of the muscarinic acetylcholine receptor antagonist scopolamine, the nicotinic acetylcholine receptor antagonist mecamylamine, or the ionotropic glutamate receptor antagonist kynurenate significantly diminished dopamine efflux in the nucleus accumbens evoked by brief electrical stimulation of the laterodorsal tegmentum. These findings suggest that acetylcholine and ionotropic glutamate receptors influence rapid dopaminergic activity and thus the communication of behaviorally relevant information from ventral tegmental area dopamine cells to forebrain areas.     

Motor activity-induced dopamine release in the substantia nigra is regulated by muscarinic receptors

Nigro-striatal neurons release dopamine not only from their axon terminals in the striatum, but also from somata and dendrites in the substantia nigra. Somatodendritic dopamine release in the substantia nigra can facilitate motor function by mechanisms that may act independently of axon terminal dopamine release in the striatum. The dopamine neurons in the substantia nigra receive a cholinergic input from the pedunculopontine nucleus. Despite recent efforts to introduce this nucleus as a potential target for deep brain stimulation to treat motor symptoms in Parkinson's disease; and the well-known antiparkinsonian effects of anticholinergic drugs; the cholinergic influence on somatodendritic dopamine release is not well understood. The aim of this study was to investigate the possible regulation of locomotor-induced dopamine release in the substantia nigra by endogenous acetylcholine release. In intact and 6-OHDA hemi-lesioned animals alike, the muscarinic antagonist scopolamine, when perfused in the substantia nigra, amplified the locomotor-induced somatodendritic dopamine release to approximately 200% of baseline, compared to 120-130% of baseline in vehicle-treated animals. A functional importance of nigral muscarinic receptor activation was demonstrated in hemi-lesioned animals, where motor performance was significantly improved by scopolamine to 82% of pre-lesion performance, as compared to 56% in vehicle-treated controls. The results indicate that muscarinic activity in the substantia nigra is of functional importance in an animal Parkinson's disease model, and strengthen the notion that nigral dopaminergic regulation of motor activity/performance is independent of striatal dopamine release.     

A cholinergic input to the substantia nigra pars compacta increases striatal dopamine metabolism measured by in vivo voltammetry

3,4-Dihydroxyphenylacetic acid (DOPAC) and ascorbic acid (AA) were measured by differential pulse voltammetry in the neostriatum of anesthetized rats. Physostigmine (2.3 nmol) applied into the substantia nigra pars compacta (SNc), increased DOPAC concentration in the ipsilateral neostriatum, but did not modify AA levels. The largest increase of striatal DOPAC (37 +/- 8% above basal) was observed when physostigmine was applied at less than 0.5 mm from SNc, and decreased with increasing distance of the injection site from the pars compacta region. Chemical stimulation of the pedunculopontine tegmental nucleus (PPN) with kainic acid (2.3 nmol) increased both DOPAC and AA concentration in the ipsilateral neostriatum. Pretreatment with the muscarinic antagonist scopolamine (5 mg/kg, i.p.) inhibited the increase of striatal DOPAC from 20 to 70 min after kainic acid injection into the PPN, whereas the increase of AA was reduced from 90 to 160 min. By contrast, the nicotinic antagonist mecamylamine (4 mg/kg, i.p.) did not inhibit neither DOPAC nor AA increase elicited by the chemical stimulation of PPN. These results support the existence of cholinergic neurotransmission within the SNc that increases the firing rate of nigrostriatal dopaminergic neurons, enhancing dopamine turnover in neostriatum without changes in AA release. They also suggest that the PPN could be the origin of cholinergic afferents to the SNc that modulate the activity of dopaminergic neurons, through activation of muscarinic cholinergic receptors. Finally, the activation of a multisynaptic loop involving a cholinergic pathway which modulates the activity of the glutamatergic corticostriatal neurons is postulated to explain the increase of AA in neostriatum observed after PPN stimulation.     

Muscarinic control of rostromedial tegmental nucleus GABA neurons and morphine‐induced locomotion

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express μ‐opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid‐induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co‐localized with μ‐opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno‐associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D‐expressing mice, clozapine‐N‐oxide increased morphine‐induced, but not vehicle‐induced, locomotion. In M3D‐expressing mice, clozapine‐N‐oxide blocked morphine‐induced, but not vehicle‐induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine‐dependent and dopamine‐independent opioid‐induced rewards and locomotion.     

Effects of scopolamine on dopamine neurons in the substantia nigra: Role of the pedunculopontine tegmental nucleus

Previous neurochemical and behavioral studies suggest that muscarinic receptor antagonism has an excitatory effect on the nigrostriatal dopamine (DA) system. Using in vivo extracellular single unit recording, this study examined whether blockade of the muscarinic receptor by scopolamine alters the firing properties of DA neurons in the substantia nigra (SN). Scopolamine was administered either systemically or locally to DA neurons using microiontophoresis. Surprisingly, scopolamine did not cause any significant change in either the firing rate or pattern of the spontaneously active DA neurons. However, systemic injection of scopolamine significantly increased the number of active DA neurons in the SN. Local infusion of scopolamine into the pedunculopontine tegmental nucleus (PPT) mimicked the effect induced by systemically administered scopolamine, significantly increasing the number of active DA neurons without altering the firing rate and pattern. These results suggest that the reported increase in striatal DA release induced by scopolamine is in part mediated by activation of silent nigral DA neurons. The experiments with PPT local infusion further suggest that part of the effect of scopolamine may be due to its blockade of the inhibitory muscarinic autoreceptors on PPT cholinergic cells. The latter effect may lead to activation of quiescent DA neurons by increasing acetylcholine (ACh) release in the SN or in other brain areas providing inputs to DA neurons. Further understanding of the mechanism of action of scopolamine may help us further understand the role of ACh in both the pathophysiology and treatment of DA‐related disorders including schizophrenia and Parkinson's disease. Synapse 63:673–680, 2009. © 2009 Wiley‐Liss, Inc.     

Acetylcholine–Dopamine Interactions in the Pathophysiology and Treatment of CNS Disorders

Dopaminergic neurons in the substantia nigra pars compacta and ventral tegmental area of the midbrain form the nigrostriatal and mesocorticolimbic dopaminergic pathways that, respectively, project to dorsal and ventral striatum (including prefrontal cortex). These midbrain dopaminergic nuclei and their respective forebrain and cortical target areas are well established as serving a critical role in mediating voluntary motor control, as evidenced in Parkinson's disease, and incentive‐motivated behaviors and cognitive functions, as exhibited in drug addiction and schizophrenia, respectively. Although it cannot be disputed that excitatory and inhibitory amino acid‐based neurotransmitters, such as glutamate and GABA, play a vital role in modulating activity of midbrain dopaminergic neurons, recent evidence suggests that acetylcholine may be as important in regulating dopaminergic transmission. Midbrain dopaminergic cell tonic and phasic activity is closely dependent upon projections from hindbrain pedunculopontine and the laterodorsal tegmental nuclei, which comprises the only known cholinergic inputs to these neurons. In close coordination with glutamatergic and GABAergic activity, these excitatory cholinergic projections activate nicotinic and muscarinic acetylcholine receptors within the substantia nigra and ventral tegmental area to modulate dopamine transmission in the dorsal/ventral striatum and prefrontal cortex. Additionally, acetylcholine‐containing interneurons in the striatum also constitute an important neural substrate to provide further cholinergic modulation of forebrain striatal dopaminergic transmission. In this review, we examine neurological and psychopathological conditions associated with dysfunctions in the interaction of acetylcholine and dopamine and conventional and new pharmacological approaches to treat these disorders.     

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.     

The limbic circuitry underlying cocaine seeking encompasses the PPTg/LDT

The direct glutamatergic projection from the medial prefrontal cortex (mPFC) to the nucleus accumbens plays a critical role in mediating the reinstatement of cocaine seeking behavior. The mPFC also sends glutamatergic projections to the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDT), which in turn send glutamatergic and cholinergic efferents to the ventral tegmental area (VTA) where they synapse on dopaminergic cells that innervate limbic structures including the nucleus accumbens. The goal of these experiments was to examine a potential role for the PPTg/LDT in the reinstatement of cocaine seeking. All rats were trained to self-administer cocaine (0.25 mg, i.v.) on a fixed-ratio 5 schedule of reinforcement. Cocaine self-administration behavior was extinguished and a series of subsequent pharmacological experiments were performed to assess the potential role of the mPFC, PPTg/LDT and VTA in the reinstatement of cocaine seeking. Administration of the D1-like dopamine receptor agonist SKF-81297 (1.0 μg) directly into the mPFC produced a small, but statistically significant, increase in cocaine seeking behavior. Furthermore, microinjection of the ionotropic glutamate receptor antagonist CNQX (0.3 μg) into the PPTg/LDT attenuated the reinstatement of drug seeking induced by a priming injection of cocaine (10 mg/kg, i.p.). Intra-VTA administration of CNQX, the nicotinic receptor antagonist mecamylamine (10.0 μg) or the muscarinic receptor antagonist scopolamine (24.0 μg) also blocked cocaine seeking. Taken together, these results suggest that cocaine priming-induced reinstatement of drug seeking is mediated in part by a serial polysynaptic limbic subcircuit encompassing the mPFC, PPTg/LDT and VTA.     

Muscarinic receptor antagonism causes a functional alteration in nucleus accumbens mu-opiate-mediated feeding behavior

Intra-nucleus accumbens (Acb) infusion of cholinergic muscarinic antagonist, scopolamine (10 microg/0.5 microl), markedly reduced fat intake elicited by intra-Acb treatment of the mu-opioid receptor agonist, DAMGO, with 30 min and 4h pretreatment intervals. Intra-Acb scopolamine infusions also reduced food intake in food-deprived rats, but not water intake in water-deprived rats. Hence, Acb muscarinic manipulations exhibit some specificity for feeding, perhaps via interactions with the striatal opioid system.     

How best to consider the structure and function of the pedunculopontine tegmental nucleus: evidence from animal studies

This review presents the hypothesis that the best way to consider the pedunculopontine tegmental nucleus is by analogy with the substantia nigra. The substantia nigra contains two main compartments: the pars compacta and the pars reticulata. The former contains dopamine neurons that project widely within the basal ganglia while the latter is in receipt of corticostriatal output. Similarly, the PPTg contains the Ch5 acetylcholine containing neurons that project to the thalamus and corticostriatal systems (notably the pars compacta of substantia nigra and the subthalamic nucleus) while the non-cholinergic neurons of the pedunculopontine are in receipt of corticostriatal output. Assessment of the location, composition and connections of the pedunculopontine tegmental nucleus is made to support the hypothesis that it has structural similarities with substantia nigra. Assessment of the motor, sensory and cognitive functions of the pedunculopontine is also made, suggesting functional similarities exist also. Having a clear model of pedunculopontine structure and function is a matter of some importance. It is clearly involved in Parkinson's disease and could potentially be a target for therapeutic intervention. If this is to be realized it will be best to have as clear an understanding as possible of pedunculopontine structure and function in order to maximize positive benefits.     

Selective blockade by scopolamine of synaptic responses in cat's caudate nucleus and its modification by lesions of the substantia nigra.

Because it is commonly believed that acetylcholine is a synaptic transmitter in the caudate nucleus and that the reduction of striatal biogenic amines in Parkinson's disease leads to acetylcholine supersensitivity in the caudate nucleus, we investigated the effects of the muscarinic blocking agent scopolamine on synaptic responses of neurons in the intact feline caudate nucleus and in the caudate nucleus depleted of dopamine by long-standing nigrostriatal lesions. In the intact caudate nucleus, micro-iontophoretic application of scopolamine selectively blocked the neuronal responses to stimulation of the caudate nucleus near the recording site without affecting the responses to stimulation of the sensorimotor cortex or the substantia nigra in the same fashion. This suggests that acetylcholine is a synaptic transmitter of caudate interneurons. Responses to thalamic stimuli were also blocked by scopolamine, suggesting that acetylcholine may be a transmitter of thalamic afferents although the course of these afferents is unclear. In the dopamine-depleted caudate nucleus scopolamine was more effective than in the intact caudate nucleus blocking the neuronal responses to stimulation of the caudate nucleus. This greater blocking effect by scopolamine suggests an increased effect of endogenous acetylcholine in this response and supports previous observations of an increased excitatory effect of iontophoretic acetylcholine in the dopamine-depleted caudate nucleus. These results suggest that the acetylcholine supersensitivity which follows nigrostriatal degeneration may be due to increased effectiveness of synaptic transmission by cholinergic interneurons in the caudate nucleus.     

Cholinergic systems in the rat brain: III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia, and basal forebrain

The ascending cholinergic projections of the pedunculopontine and dorsolateral tegmental nuclei, referred to collectively as the pontomesencephalotegmental (PMT) cholinergic complex, were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Propidium iodide, true blue, or Evans blue was infused into the anterior, reticular, mediodorsal, central medial, and posterior nuclear areas of the thalamus; the habenula; lateral geniculate; superior colliculus; pretectal/parafascicular area; subthalamic nucleus; caudate-putamen complex; globus pallidus; entopeduncular nucleus; substantia nigra; medial septal nucleus/vertical limb of the diagonal band area; magnocellular preoptic/ventral pallidal area; and lateral hypothalamus. In some animals, separate injections of propidium iodide and true blue were made into two different regions in the same rat brain, usually a dorsal and a ventral target, in order to assess collateralization patterns. Retrogradely transported fluorescent labels and ChAT and/or AChE were analyzed microscopically on the same brain section. All of the above-delimited targets were found to receive cholinergic input from the PMT cholinergic complex, but some regions were preferentially innervated by either the pedunculopontine or dorsolateral tegmental nucleus. The former subdivision of the PMT cholinergic complex projected selectively to extrapyramidal structures and the superior colliculus, whereas the dorsolateral tegmental nucleus was observed to provide cholinergic input preferentially to anterior thalamic regions and rostral portions of the basal forebrain. The PMT cholinergic neurons showed a tendency to collateralize extensively.     

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.     

Post-training intra-striatal scopolamine or flupenthixol impairs radial maze learning in rats

Systemic treatments with acetylcholine (ACh) or dopamine (DA) receptor antagonists during hours 0-4 but not during hours 5-8 following training on a radial arm maze (RAM) or lesions of the dorsal striata impair learning. This suggested that intra-striatal infusions of ACh or DA receptor antagonists during hours 0-4 following training may impair learning. Rats were randomly assigned to groups (ns=5-11) receiving dorsal striatal infusions of the ACh receptor antagonist scopolamine (0-18 microg/microL at 0 and 2h or at 4 and 6h after training), the DA receptor antagonist cis-flupenthixol (0-25 microg/microL at 0, 4 or 12h after training) or the inactive isomer trans-flupenthixol (6 microg/microL at 0 h after training). Scopolamine and cis-flupenthixol impaired the habit-learning version of the task. Given after hours 0-4 following training, the effects of scopolamine were diminished but those of cis-flupenthixol were not. Trans-flupenthixol produced less impairment than cis-flupenthixol. Results suggest that ACh and DA receptors in the dorsal striatum during hours 0-4 following training play a role in habit learning.     

Inhibition of synaptically evoked cortical acetylcholine release by intracortical glutamate: involvement of GABAergic neurons

Cortical acetylcholine (ACh) has been shown to regulate diverse cognitive processes and its release can be regulated by neuromodulators that act presynaptically at cholinergic terminals. The neocortex receives dense glutamatergic input from thalamocortical and other fibres. The present study used in vivo microdialysis to examine, and pharmacologically characterize, the effect of glutamate on cortical ACh release evoked by electrical stimulation of the pedunculopontine tegmental nucleus in urethane-anaesthetized rats. All drugs were administered locally within the cortex by reverse dialysis. Application of glutamate had no detectable effect on spontaneous ACh release but reduced evoked cortical ACh efflux in a concentration-dependent manner. This effect was mimicked by the glutamate transporter blocker L-trans-pyrrolidine-2,4-dicarboxylic acid, as well as by the ionotropic glutamate receptor agonists N-methyl-D-aspartic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and was blocked by the ionotropic glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and (+/-)-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid. Glutamate application also increased extracellular adenosine levels but the simultaneous delivery of the broad-spectrum adenosine receptor antagonist caffeine failed to affect the inhibitory action of glutamate on evoked ACh release. However, the effect of glutamate was fully blocked by simultaneous delivery of the GABAA receptor antagonist bicuculline and partially blocked by the GABAB receptor antagonist phaclofen. These results suggest that ionotropic glutamate receptor activation by glutamate inhibits evoked cortical ACh release via an indirect pathway involving GABAergic neurons in the cortex.     

Cholinergic neuronal modulation alters dopamine D2 receptor availability in vivo by regulating receptor affinity induced by facilitated synaptic dopamine turnover: positron emission tomography studies with microdialysis in the conscious monkey brain.

To evaluate the cholinergic and dopaminergic neuronal interaction in the striatum, the effects of scopolamine, a muscarinic cholinergic antagonist, on the striatal dopaminergic system were evaluated multi-parametrically in the conscious monkey brain using high-resolution positron emission tomography in combination with microdialysis. l-3,4-Dihydroxyphenylalanine (l-[beta-(11)C]DOPA) and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([beta-(11)C]CFT) were used to measure dopamine synthesis rate and dopamine transporter (DAT) availability, respectively. For assessment of dopamine D(2) receptor binding in vivo, [(11)C]raclopride was applied because this labeled compound, which has relatively low affinity to dopamine D(2) receptors, was hypothesized to be sensitive to the striatal synaptic dopamine concentration. Systemic administration of scopolamine at doses of 10 and 100 microg/kg dose-dependently increased both dopamine synthesis and DAT availability as measured by l-[beta-(11)C]DOPA and [beta-(11)C]CFT, respectively. Scopolamine decreased the binding of [(11)C]raclopride in a dose-dependent manner. Scopolamine induced no significant changes in dopamine concentration in the striatal extracellular fluid (ECF) as determined by microdialysis. However, scopolamine dose-dependently facilitated the striatal ECF dopamine induced by the DAT inhibitor GBR12909 at a dose of 0.5 mg/kg. Scatchard plot analysis in vivo of [(11)C]raclopride revealed that scopolamine reduced the apparent affinity of dopamine D(2) receptors. These results suggested that the inhibition of muscarinic cholinergic neuronal activity modulates dopamine turnover in the striatum by simultaneous enhancement of the dynamics of dopamine synthesis and DAT availability, resulting in no significant changes in apparent "static" ECF dopamine level but showing a decrease in [(11)C]raclopride binding in vivo attributable to the reduction of affinity of dopamine D(2) receptors.     

Striatal muscarinic receptor antagonism reduces 24-h food intake in association with decreased preproenkephalin gene expression

Cholinergic interneurons of the striatum respond to motivationally relevant stimuli and are involved in appetitive learning. However, there has been relatively little inquiry into the role of striatal acetylcholine in food motivation. Here we show in rats that a single infusion of the muscarinic receptor antagonist scopolamine (0, 5.0 or 10.0 microg/0.5 microL bilaterally) potently reduced 24-h food intake following injections into either the ventral or dorsal striatum, without affecting water intake. Furthermore, muscarinic receptor blockade induced reliable and widespread reductions in striatal preproenkephalin, but not preprodynorphin, mRNA expression. These data suggest a novel role for striatal acetylcholine in modulating feeding behavior via its effects on enkephalin gene expression. As prior research indicates a critical role for striatal enkephalin in consummatory behaviors and palatability, we hypothesize that cholinergic interneurons assist in translating hypothalamic energy state signals into food-directed behaviors via their regulation of striatal opioid peptides.