Localization of dopamine D2 receptors on cholinergic interneurons of the dorsal striatum and nucleus accumbens of the rat

Striatal cholinergic interneurons located in the dorsal striatum and nucleus accumbens are amenable to influences of the dopaminergic mesolimbic pathway, which is a pathway involved in reward and reinforcement and targeted by several drugs of abuse. Dopamine and acetylcholine neurotransmission and their interactions are essential to striatal function, and disruptions to these systems lead to a variety of clinical disorders. Dopamine regulates acetylcholine release through dopamine receptors that are localized directly on striatal cholinergic interneurons. The dopamine D2 receptor, which attenuates acetylcholine release, has been implicated in drug relapse and is targeted by therapeutic drugs that are used to treat a variety of neurological disorders including Tourette Syndrome, Parkinson's disease and schizophrenia. The present study provides the first direct evidence for the localization of dopamine D2 receptors on striatal cholinergic interneurons of the rat brain using dual labeling immunocytochemistry procedures. Using light microscopy, dopamine D2 receptors were localized on the cell somata and dendritic and axonal processes of striatal cholinergic interneurons in the dorsal striatum and nucleus accumbens of the rat brain. These findings provide a foundation for understanding the specific roles that cholinergic neuronal network systems and interacting dopaminergic signaling pathways play in striatal function and in a variety of clinical disorders including drug abuse and addiction.     

Dopamine D-5 receptor modulates hippocampal acetylcholine release

Dopamine is intimately involved in cognitive processes in the brain. Of the several subtypes of dopamine receptors, the possible role of dopamine D1-like receptors in brain functions, especially in learning and memory, has recently generated much interest. However, molecularly the D1-like receptors are comprised of at least two subtypes, namely D-1 and D-5, and it has not been possible to ascertain which of these two receptor classes is responsible for these functions due to the lack of selective ligands. In the present study, utilizing a combined antisense-in vivo dialysis approach, we show that the D-5 subtype is the dopamine D1-like receptor involved in modulating hippocampal acetylcholine (ACh) release, a transmitter implicated in a variety of cognitive processes. This is one of the first evidence for a functional role for the D-5 receptor.     

Molecular phenotype of rat striatal neurons expressing the dopamine D5 receptor subtype

Dopamine is one of the principal neurotransmitters in the basal ganglia, where it plays a critical role in motor control and cognitive function through its interactions with the specific dopamine receptors D1 to D5. Although the activities mediated by most dopamine receptor subtypes have already been determined, the role of the D5 receptor subtype in the basal ganglia has still not been established. Furthermore, it is often difficult to distinguish between dopamine D5 and D1 receptors as they are stimulated by the same ligands, and they have a similar molecular structure and pharmacology. In an effort to understand the differences between these two receptor subtypes, we have studied the distribution of neurons containing D5 receptors in the striatum, and their molecular phenotype. As a result, we show that the D5 receptor subtype is present in two different populations of striatal neurons, projection neurons and interneurons. Overall, the abundance of this receptor subtype in the striatum is low, particularly in striatal projection neurons of both the direct and indirect projection pathways. In contrast, the expression of D5 receptors in striatal interneurons (cholinergic, somatostatin- or parvalbumin-positive neurons) is high, while low to moderate expression was observed in calretinin-positive neurons. Our results demonstrate the presence of D5 receptors in all the striatal cell populations so far described, although at different intensities in each. The fact that a large number of striatal neurons express the D5 receptor subtype suggests that this receptor fulfils an important function in the process of integrating information in the striatum.     

Differential expression of D1 and D2 dopamine and m4 muscarinic acetylcholine receptor proteins in identified striatonigral neurons

Large families of genetically distinct G-protein coupled receptor subtypes mediate dopamine's (D1–D5) and acetylcholine's effects (m1–m5). A functional balance of dopamine and acetylcholine may be based in part on the differential expression of receptor subtypes by distinct neuron subpopulations. The localization of the D1 and D2 receptors, the predominant dopamine receptors in neostriatum, to distinct subpopulations of striatal projection neurons has been controversial. In addition, m4 receptor localization to specific striatal projection neuron subpopulations is also at question. To determine whether rat striatonigral neurons differentially express D1, D2, and m4 receptor proteins, we combined immunocytochemistry by using receptor subtype specific antibodies and retrograde tracing with cholera toxin-colloidal gold. D1 and m4 receptor immunoreactivity was visualized in 95% and 92% of identified striatonigral neurons, respectively. By contrast, D2 receptor immunoreactivity was visualized in only 1% of these neurons. These findings support models of basal ganglia in which D1 and D2 receptors are segregated, as well as indicate that D1 and m4 are colocalized. These cellular distributions may be important substrates for the putative DA/ACh balance that is implicated in certain movement disorders. Synapse 27:357–366, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Dopamine D1-like receptor subtypes in human peripheral blood lymphocytes.

Molecular biology studies have shown that human peripheral blood lymphocytes express a dopamine D5 receptor, whereas no information is available on dopamine D receptor, the other dopamine D1-like receptor subtype. Radioligand binding assay investigations with the nonsubtype selective dopamine D1-like receptor antagonist [3H]SCH 23390 as radioligand have suggested the presence of a dopamine D5 receptor in human peripheral blood lymphocytes. However, so far no evidence was provided as whether or not human peripheral blood lymphocytes express a dopamine D1 receptor. In this study, we have investigated dopamine D1 and D5 receptor mRNA and the influence of antibodies against dopamine D1 and D5 receptors on [3H]SCH 23390 binding to intact human peripheral blood lymphocytes. The two receptors were also analyzed by immunocytochemistry. Dopamine D5 receptor, but not D1 mRNA, was detected in human peripheral blood lymphocytes. Anti-dopamine D5 receptor antibodies, but not anti-dopamine D1 receptor antibodies, significantly decreased [3H]SCH 23390 binding to human peripheral blood lymphocytes. A dark-brown immunoreactivity was visualized in cytospin centrifuged human peripheral blood lymphocytes exposed to anti-dopamine D5, but not to anti-dopamine D1 receptor antibodies. These data collectively indicate that dopamine D5 receptor is the only dopamine D1-like receptor subtype expressed by human peripheral blood lymphocytes.     

Dopamine in neurotoxicity and neuroprotection: what do D2 receptors have to do with it?

Accurate control of dopamine levels and/or the resulting dopamine-receptor interaction is essential for brain function. Indeed, several human neurological and psychiatric disorders are characterized by dysfunctions of the dopaminergic system. Dopamine has been reported to exert either protective or toxic effects on neurons, yet it is unclear whether these effects are receptor-dependent and, if so, which dopamine receptor could be involved. The D(2) dopamine receptor occupies a privileged position because its signalling might be neuroprotective in human diseases, such as Parkinson's disease, ischaemia and epilepsy. Unravelling the role of D(2) receptors in neuronal death and survival might be central to understanding the mechanisms that underlie several neuropathologies.     

Expression and localization of muscarinic receptors in P19-derived neurons

The muscarinic acetylcholine receptors are important in a variety of physiological processes such as induction of secretion from various glands and regulation of pacemaker activity, muscle tone, and neurotransmission. To date, the muscarinic receptor family includes five members (designated m1–m5), of which m1–m4 are abundant in brain and in peripheral tissues, and m5 is found exclusively in brain, and even there at very low levels. The expression of m1–m5 receptor subtypes was studied in neurons derived from the murine embryonal carcinoma cell line P19. These cells serve as a model system for differentiation and maturation of neurons resembling CNS neurons. Our results show that P19 neurons express mainly the m2, m3, and m5 subtypes. Low levels of m1 receptors are also detected and m4 subtype is practically absent. Furthermore, muscarinic receptors in P19 neurons are functional in activating second messenger signaling pathways. The localization of m2 receptors is predominantly presynaptic, whereas the m5 subtype is mainly postsynaptic. Consequently, P19 cells provide a model system for the study of pre- and postsynaptic muscarinic acetylcholine-receptor subtypes in a proper neuronal context. This is particularly valid for the rare m5 receptors.     

Cholinergic modulation of midbrain dopaminergic systems

Dopamine neurons in the midbrain respond to behavioral events and environmental stimuli. Their different patterns of activation in turn modulate the activity of forebrain regions and modulate the expression of selective behavioral responses. However, their activity is closely dependent on the cholinergic systems in the brainstem. Ascending cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei target dopaminergic neurons in the substantia nigra compacta and ventral tegmental area following a topographical gradient. These projections, by means of the activation of acetylcholine receptors, influence the firing of dopamine neurons and therefore their responsiveness, ultimately affecting the release of dopamine in their forebrain targets. Brainstem cholinergic neurons are thus in a position to critically influence the activity of dopaminergic neurons in the midbrain, and thereby have a critical role in the expression of behavior.     

Neurokinins robustly activate the majority of septohippocampal cholinergic neurons

In the brain, tachykinins acting via the three cloned neurokinin (NK) receptors are implicated in stress-related affective disorders. Hemokinin-1 is a novel tachykinin that reportedly prefers NK1 to NK2 or NK3 receptors. Although NK1 and NK3 receptors are abundantly expressed in the brain, NK2-receptor-mediated electrophysiological effects have rarely been described as NK2 receptors are expressed only in a few brain regions such as the nucleus of the medial septum/diagonal band. Medial septal/diagonal band neurons that control hippocampal mnemonic functions also colocalize NK1 and NK3 receptors. Functionally, intraseptal activation of all three NK receptors increases hippocampal acetylcholine release and NK2 receptors have specifically been implicated in stress-induced hippocampal acetylcholine release. Electrophysiological studies on the effects of NKs on septohippocampal cholinergic neurons are lacking and electrophysiological effects of hemokinin-1 have thus far not been reported in brain neurons. In the present study we examined the electrophysiological and pharmacological effects of multiple NKs on fluorescently tagged septohippocampal cholinergic neurons using whole-cell patch-clamp recordings in a rat brain slice preparation. We demonstrate that a vast majority of septohippocampal cholinergic cells are activated by NK1, NK2 and NK3 receptor agonists as well as by hemokinin-1 via direct post-synaptic mechanisms. Pharmacologically, hemokinin-1 recruits not only NK1 but also NK2 and NK3 receptors to activate septohippocampal cholinergic neurons that are the primary source of acetylcholine for the hippocampus.     

Dopamine D2 and D3 receptor agonists limit oligodendrocyte injury caused by glutamate oxidative stress and oxygen/glucose deprivation

Dopamine receptor activation is thought to contribute adversely to several neuropathological disorders, including Parkinson's disease and schizophrenia. In addition, dopamine may have a neuroprotective role: dopamine receptor agonists are reported to protect nerve cells by virtue of their antioxidant properties as well as by receptor-mediated mechanisms. White matter injury can also be a significant factor in neurological disorders. Using real-time RT-PCR, we show that differentiated rat cortical oligodendrocytes express dopamine D2 receptor and D3 receptor mRNA. Oligodendrocytes were vulnerable to oxidative glutamate toxicity and to oxygen/glucose deprivation injury. Agonists for dopamine D2 and D3 receptors provided significant protection of oligodendrocytes against these two forms of injury, and the protective effect was diminished by D2 and D3 antagonists. Levels of oligodendrocyte D2 receptor and D3 receptor protein, as measured by Western blotting, appeared to increase following combined oxygen and glucose deprivation. Our results suggest that dopamine D2 and D3 receptor activation may play an important role in oligodendrocyte protection against oxidative glutamate toxicity and oxygen-glucose deprivation injury.     

Neuronal instability: implications for Rett's syndrome

The maturational changes in the brain and spinal cord do not linearly proceed from immature in infants to mature in adults. Dendrites dynamically extend or retract as neurotrophic factors fluctuate. In certain cases mature neurons can be seen soon after birth, and in other cases immature neurons can be identified in the aged brain. Monoamine ‘neurotransmitter’; such as serotonin (5-HT), dopamine and norepinephrine appear to function as Maintenance Growth Factors since they must be present in order to produce their maturational actions. Serotonin neurons contain TRK-B receptors and are sensitive to availability of the trophic factor, BDNF. 5-HT also functions by promoting the release of the glial extension factor, S-100β. 5-HT and S-100β can provide maturational signals to a variety of neurons, in both cortical and subcortical areas, and appear to be involved in regulating the maturation and release of acetylcholine and dopamine. We have shown that activation of the 5-HT1A receptor is particularly effective in inducing growth of stunted neurons. The mechanism of action of the 5-HT1A receptor involves both a direct inhibition on c-AMP and pCREB formation in postsynaptic neurons and a release of S-100β from glial cells. Both these events are capable of stabilization and elaboration of the cytoskeleton of the neuron and inhibition of apoptosis. 5-HT1A receptors have been shown to effectively reverse stunted neurons and microencephaly produced in animal models of fetal alcohol syndrome and prenatal cocaine administration. I discuss the implications for regressive disorders such as Rett's syndrome and autism, and the feasibility of treatments with 5-HT1A agonists in children with developmental disorders.     

D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons

Dopamine shapes a wide variety of psychomotor functions. This is mainly accomplished by modulating cortical and thalamic glutamatergic signals impinging upon principal medium spiny neurons (MSNs) of the striatum. Several lines of evidence suggest that dopamine D1 receptor signaling enhances dendritic excitability and glutamatergic signaling in striatonigral MSNs, whereas D2 receptor signaling exerts the opposite effect in striatopallidal MSNs. The functional antagonism between these two major striatal dopamine receptors extends to the regulation of synaptic plasticity. Recent studies, using transgenic mice in which cells express D1 and D2 receptors, have uncovered unappreciated differences between MSNs that shape glutamatergic signaling and the influence of DA on synaptic plasticity. These studies have also shown that long-term alterations in dopamine signaling produce profound and cell-type-specific reshaping of corticostriatal connectivity and function.     

Visualization of a dopamine D1 receptor mRNA in human and rat brain.

Using 32P-labeled oligonucleotides derived from the coding region of human dopamine D1 receptor mRNA we have localized in the human and rat brain the cells containing the mRNAs coding for this receptor. Dopamine D1 receptor mRNA in human brain was found to be contained in the neurons of the caudate and putamen nuclei as well as in the nucleus accumbens, some cortical regions and some nuclei of the amygdala. In the rat brain, cells containing D1 receptor mRNA were enriched in caudate-putamen and accumbens nuclei, olfactory tubercle, islands of Calleja, some cortical areas and in several thalamic nuclei. Moreover, in both species, it was absent from the neurons of the substantia nigra both pars compacta and pars reticulata and ventral tegmental area as well as from the globus pallidus pars lateralis and medialis in human and globus pallidus and entopeduncular nucleus in rat. In general, a good agreement was found with the distribution of binding sites labeled with the D1 antagonist SCH 23390. The main exception was the absence of D1 receptor mRNA in globus pallidus and substantia nigra, regions where high densities of receptor sites are found. These data support the notion that sites in these two regions are localized to projections from striatal neurons and that dopaminergic neurons do not express this receptor.     

Dopaminergic regulation of cortical acetylcholine release.

The extent to which the activity of basal forebrain cholinergic neurons is influenced by dopamine (DA) was investigated using in vivo microdialysis of cortical acetylcholine (ACh). Systemic administration of the DA receptor agonist apomorphine significantly increased dialysate concentrations of ACh. Systemic, but not local, administration of d-amphetamine produced similar effects. Both D1 (SCH 23390) and D2 (haloperidol, raclopride) DA receptor antagonists attenuated the amphetamine-induced increase in cortical ACh release; however, only the D1 antagonist significantly reduced basal output of cortical ACh. These findings suggest that the activity of cortically projecting cholinergic neurons in the nucleus basalis is regulated in an excitatory manner by central dopaminergic neurons and that both D1 and D2 receptors are involved.     

Microglia-neuron interaction in inflammatory and degenerative diseases: role of cholinergic and noradrenergic systems

Reciprocal interactions between glia and neurons are essential for many critical functions in brain health and disease. Microglial cells, the brain resident macrophages, and astrocytes, the most prevalent type of cell in brain, are actively involved in the control of neuronal activities both in developing and adult organisms. At the same time, neurons influence glial functions, through direct cell-to-cell interactions as well as the release of soluble mediators. Among signals from neurons that may have an active role in controlling glial activation are two major neurotransmitters: acetylcholine and noradrenaline. Several studies indicate that microglia and astrocytes express adrenergic receptors, whose activation influences the release of pro-inflammatory mediators, controlling the extent of glial reactivity. Acetylcholine receptors are also expressed by glial cells. In particular, microglial cells express the nicotinic receptor alpha7 and its activation attenuates the pro-inflammatory response of microglial cultures, suggesting that acetylcholine may control brain inflammation, in analogy to what demonstrated in peripheral tissues. Deficiencies of noradrenergic and cholinergic systems are linked to important neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD) and it has been suggested that in addition to impairing neuron-to-neuron transmission, noradrenergic and cholinergic hypofunction may contribute to dysregulation of the normal neuron-glia interaction, abnormal glial reaction and, eventually, neurodegeneration. A deeper knowledge of role of cholinergic and noradrenergic systems in controlling neuron-glia interactions may offer new venues for disease treatments.     

De novo expression of dopamine D2 receptors on microglia after stroke

Dopamine is the predominant catecholamine in the brain and functions as a neurotransmitter. Dopamine is also a potent immune modulator. In this study, we have characterized the expression of dopamine receptors on murine microglia. We found that cultured primary microglia express dopamine D1, D2, D3, D4, and D5 receptors. We specifically focused on the D2 receptor (D2R), a major target of antipsychotic drugs. Whereas D2Rs were strongly expressed on striatal neurons in vivo, we did not detect any D2R expression on resident microglia in the healthy brains of wild-type mice or transgenic mice expressing the green fluorescent protein (GFP) under the control of the Drd2 promoter. However, cerebral ischemia induced the expression of D2R on Iba1-immunoreactive inflammatory cells in the infarct core and penumbra. Notably, D2R expression was confined to CD45^hi cells, and GFP BM chimeras revealed that D2R was expressed on activated resident microglia as well as on peripherally derived macrophages in the ischemic brain. Importantly, the D2/3R agonist, pramipexole, enhanced the secretion of nitrite by cultured microglia in response to proinflammatory stimuli. Thus, dopamine may serve as a modulator of microglia function during neuroinflammation.     

Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

We investigated the subcellular distribution of dopamine D(1), D(2) and D(5) receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D(1) receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D(2) receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D(5) receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D(5) receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D(1) receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D(5) and D(2) receptor subtypes were not significantly altered by cocaine treatment. These data imply that D(5) receptors are predominantly cytoplasmic, D(2) receptors are diffusely distributed within the cell, whereas D(1) receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.     

Characterization of mouse striatal precursor cell lines expressing functional dopamine receptors

Dopamine and its receptors appear in the developing brain early in the embryonic period and dopamine receptor activation influences proliferation and differentiation of neuroepithelial precursor cells. Since dopamine D(1) and D(2) receptor activation produces opposing effects on precursor cell activity, dopamine's overall effects may correlate with relative numbers and activity of each receptor subtype on the precursor cells. Dopamine receptor expression and activity in individual precursor cells in the intact brain are difficult to ascertain. Therefore, cell lines with known receptor expression profiles can be useful tools to study dopamine's influence on neuroepithelial cells. We report characterization of dopamine receptor expression and activity profiles in three mouse striatal precursor cell lines and suggest that these cell lines can be valuable tools to study dopamine's effects on striatal precursor cell proliferation and differentiation.     

Brain CCK-B receptors mediate the suppression of dopamine release by cholecystokinin

The sulfated octapeptide of cholecystokinin (CCK-8S) and CCK fragments were administered to mice to determine the subtype and central versus peripheral location of the CCK receptor that modulates dopamine release in the neostriatum. Dopamine release was decreased when unsulfated CCK (CCK-8U) or the butoxycarbonyl tetrapeptide of CCK (t-boc-CCK-4) was infused into the brain ventricles but not when injected subcutaneously. These CCK fragments bind to the brain-type (CCK-B) but not alimentary-type (CCK-A) receptor. Centrally or peripherally administered CCK-8S also lowered dopamine release and this action was not blocked by the selective CCK-A receptor antagonist, L 364,718. The increase in dopamine release following amphetamine administration was attenuated by central injections of t-boc-CCK-4, CCK-8U, or CCK-8S, and this action of CCK-8S was not prevented by L 364,718. These data are the first to demonstrate that CCK-B receptors in brain mediate the suppression of dopamine release by cholecystokinin, especially when release is augmented. CCK-B receptor agonists should be useful for the treatment of psychiatric conditions that result from hyperactive dopamine neurons.