Search

G-Protein coupled D2 autoreceptors expressed on dopamine neurons (D2Rs) inhibit transmitter release and cell firing at axonal endings and somatodendritic compartments. Mechanistic details of somatodendritic dopamine release remain unresolved, partly due to insufficient information on the subcellular distribution of D2Rs. Previous studies localizing D2Rs have been hindered by a dearth of antibodies validated for specificity in D2R knockout animals and have been limited by the small sampling areas imaged by electron microscopy. This study utilized sub-diffraction fluorescence microscopy and electron microscopy to examine D2 receptors in a superecliptic pHlourin GFP (SEP) epitope tagged D2 receptor knockin mouse. Incubating live slices with an anti-SEP antibody achieved the selective labeling of plasma membrane associated receptors for immunofluorescent imaging over a large area of the substantia nigra pars compacta (SNc). SEP-D2Rs appeared as puncta-like structures along the surface of dendrites and soma of dopamine neurons visualized by antibodies to tyrosine hydroxylase (TH). TH-associated SEP-D2Rs displayed a cell surface density of 0.66 puncta per μm(2), which corresponds to an average frequency of 1 punctum every 1.50 μm. Separate ultrastructural experiments using silver-enhanced immunogold revealed that membrane-bound particles represented 28% of total D2Rs in putative dopamine cells within the SNc. Structures immediately adjacent to dendritic membrane gold particles were unmyelinated axons or axon varicosities (40%), astrocytes (19%), other dendrites (7%), or profiles unidentified (34%) in single sections. Some apposed profiles also expressed D2Rs. Fluorescent and ultrastructural analyses also provided the first visualization of membrane-D2Rs at the axon initial segment, a compartment critical for action potential generation. The punctate appearance of anti-SEP staining indicates there is a population of D2Rs organized in discrete signaling sites along the plasma membrane, and for the first time a quantitative estimate of spatial frequency is provided.

Background We describe a 4-generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant. Objectives The objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant. Methods After detailed clinical and neurological examination, whole-exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington-like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein-mediated inhibition of adenylyl cyclase determined in a cell model, and G protein-regulated inward-rectifying potassium channels measured in midbrain slices of mice. Result We identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease-causing variants. We demonstrated that the D2(S/L)-(IF)-F-212 receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2(S)-(IF)-F-212 receptor exhibited aberrant receptor function in mouse midbrain slices. Conclusions Our results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4-generation pedigree. (c) 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

SUMMARY Neuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition., Graphical Abstract, In brief Somatodendritic release of dopamine via D2 autoreceptors activates an inhibitory postsynaptic current. The synaptic current is dependent on a transiently high concentration of dopamine. Condon et al. use a combination of optical and electrical approaches to show the spatial and temporal substrates of dendrodendritic transmission in the substantia nigra.

The dopamine D2 receptor has two splice variants, D2S (Short) and D2L (Long). In dopamine neurons, both variants can act as autoreceptors to regulate neuronal excitability and dopamine release, but the roles of each variant are incompletely characterized. In a previous study we used viral receptor expression in D2 receptor knockout mice to show distinct effects of calcium signaling on D2S and D2L autoreceptor function (Gantz et al., 2015). However, the cocaine-induced plasticity of D2 receptor desensitization observed in wild type mice was not recapitulated with this method of receptor expression. Here we use mice with genetic knockouts of either the D2S or D2L variant to investigate cocaine-induced plasticity in D2 receptor signaling. Following a single in vivo cocaine exposure, the desensitization of D2 receptors from neurons expressing only the D2S variant was reduced. This did not occur in D2L-expressing neurons, indicating differential drug-induced plasticity between the variants.

Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged µ-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

Summary GABA release from interneurons in VTA, projections from the nucleus accumbens (NAc), and rostromedial tegmental nucleus (RMTg) was selectively activated in rat brain slices. The inhibition induced by μ-opioid agonists was pathway dependent. Morphine induced a 46% inhibition of IPSCs evoked from the RMTg, 18% from NAc, and IPSCs evoked from VTA interneurons were almost insensitive (11% inhibition). In vivo morphine treatment resulted in tolerance to the inhibition of RMTg, but not local interneurons or NAc, inputs. One common sign of opioid withdrawal is an increase in adenosine-dependent inhibition. IPSCs evoked from the NAc were potently inhibited by activation of presynaptic adenosine receptors, whereas IPSCs evoked from RMTg were not changed. Blockade of adenosine receptors selectively increased IPSCs evoked from the NAc during morphine withdrawal. Thus, the acute action of opioids, the development of tolerance, and the expression of withdrawal are mediated by separate GABA afferents to dopamine neurons.

Alcohol addiction is a complex, uniquely human disease. Breaking addiction down into contributing endophenotypes enables its study in a variety of model systems. The Drosophila model system has been most often used to study alcohol sensitivity, tolerance, and physiological dependence. However, none of these endophenotypes can account for the near-permanent quality of the addicted state. It has been recently discussed that addictive drugs may hijack the learning-and-memory machinery to produce persistent behavioral changes. Learning and memory is amenable to experimental study, and provides us with a window into how alcohol affects higher-order mental functions that are likely to contribute compulsive drug use. Here, we review the Drosophila literature that links alcohol-related behaviors to learning and memory.

Adult Drosophila melanogaster has long been a popular model for learning and memory studies. Now the larval stage of the fruit fly is also being used in an increasing number of classical conditioning studies. In this study, we employed heat shock as a novel negative reinforcement for larvae and obtained high learning scores following just one training trial. We demonstrated heat-shock conditioning in both reciprocal and non-reciprocal paradigms and observed that the time window of association for the odor and heat shock reinforcement is on the order of a few minutes. This is slightly wider than the time window for electroshock conditioning reported in previous studies, possibly due to lingering effects of the high temperature. To test the utility of this simplified assay for the identification of new mutations that disrupt learning, we examined flies carrying mutations in the dnc gene. While the sensitivity to heat shock, as tested by writhing, was similar for wild type and dnc homozygotes, dnc mutations strongly diminished learning. We confirmed that the learning defect in dnc flies was indeed due to mutation in the dnc gene using non-complementation analysis. Given that heat shock has not been employed as a reinforcement for larvae in the past, we explored learning as a function of heat shock intensity and found that optimal learning occurred around 41 °C, with higher and lower temperatures both resulting in lower learning scores. In summary, we have developed a very simple, robust paradigm of learning in fruit fly larvae using heat shock reinforcement.

D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors. DOI: http://dx.doi.org/10.7554/eLife.09358.001, eLife digest Dopamine is an important component of the brain's reward system and is commonly referred to as a ‘feel-good’ chemical. It is mainly released from neurons in the brain in response to natural rewards, such as food or sex, and following exposure to, or in anticipation of, certain drugs of abuse (including cocaine). Dopamine-releasing neurons also sense dopamine, and just like someone can change the volume of their voice by hearing themselves speak, dopamine neurons regulate how much dopamine is released based on how much dopamine they sense. This feedback system is known as autoinhibition. These neurons sense dopamine when it binds to, and activates, so-called ‘dopamine D2 receptors’ on their cell surface. But not all D2 receptors are alike. Instead there are two variants called D2S and D2L. Previous studies have shown that D2 receptor signaling in dopamine neurons is altered by the concentration of calcium ions inside these cells. Furthermore, exposure to cocaine and other drugs is known to change how these calcium ions regulate D2 receptor signaling. Now, Gantz et al. have used mice that produce only a single variant of the D2 receptor (either D2S or D2L) in their dopamine neurons to uncover similarities and differences between the two variants. The experiments show that localized increases in calcium ion concentration make D2S less capable of autoinhibition, like D2 receptors in neurons from wild type mice, without affecting autoinhibition by D2L. In further experiments, some of these mice were given cocaine before D2 receptor signaling was assessed. In dopamine neurons from wild type mice, a single exposure to cocaine eliminates the calcium-dependent regulation; thus, cocaine treatment causes a D2L-like response. In contrast, cocaine treatment did not affect the calcium-dependent regulation when only one variant of the D2 receptor was present. This implies that dopamine neurons must have both D2S and D2L receptors before the drug can induce changes in D2 receptor signaling. These findings also challenge the long-held view that the D2S receptor is the predominant form involved in autoinhibition. The next challenge is to determine how cocaine induces an apparent switch from D2S to D2L and the implications of this switch for the development of cocaine addiction. DOI: http://dx.doi.org/10.7554/eLife.09358.002

Alcohol addiction is a disease that includes a diverse set of phenotypes. Functional alcohol tolerance is an adaptation to the effects of alcohol that restores neuronal homeostatic balance while the drug is present. When the drug is suddenly withheld, these adaptations unbalance the nervous system and are thought to be the origin of some withdrawal symptoms. Withdrawal symptoms, which can be a motivating factor for alcoholics to relapse, are taken as evidence of physiological ethanol dependence. Both tolerance and withdrawal symptoms are diagnostic criteria for alcoholism. Recent studies have demonstrated that the larvae of Drosophila show conserved alcohol tolerance and withdrawal phenotypes indicating that Drosophila genetics can now be used in studying this endophenotype of alcohol addiction.

Summary Physiological alcohol dependence is a key adaptation to chronic ethanol consumption that underlies withdrawal symptoms, is thought to directly contribute to alcohol addiction behaviors, and is associated with cognitive problems such as deficits in learning and memory [1–3]. Based on the idea that an ethanol-adapted (dependent) animal will perform better in a learning assay than an animal experiencing ethanol withdrawal will, we have used a learning paradigm to detect physiological ethanol dependence in Drosophila . Moderate ethanol consumption initially degrades the capacity of larvae to learn, but they eventually adapt and are able to learn as well as ethanol-naive animals. However, withholding ethanol from ethanol-adapted larvae impairs learning. Ethanol reinstatement restores the capacity to learn, thus demonstrating cognitive dependence on ethanol. The larval nervous system also shows ethanol-withdrawal hyperexcitability. Larvae reach ethanol concentrations equivalent to 0.05 to 0.08 blood-alcohol concentration—levels that would be mildly intoxicating in humans. These ethanol-induced changes in learning are not the product of sensory deficits or state-dependent learning. This is the first demonstration of cognitive ethanol dependence in an invertebrate genetic model system.

Although the primate insular cortex has been studied extensively, a comprehensive investigation of its neuronal morphology has yet to be completed. To that end, neurons from 20 human subjects (10 males and 10 females; N = 600) were selected from the secondary gyrus brevis, precentral gyrus, and postcentral gyrus of the left insula. The secondary gyrus brevis was generally more complex in terms of dendritic/spine extent than either the precentral or postcentral insular gyri, which is consistent with the posterior-anterior gradient of dendritic complexity observed in other cortical regions. The male insula had longer, spinier dendrites than the female insula, potentially reflecting sex differences in interoception. In comparing the current insular data with regional dendritic data quantified from other Brodmann's areas (BAs), insular total dendritic length (TDL) was less than the TDL of high integration cortices (BA6beta, 10, 11, 39), but greater than the TDL of low integration cortices (BA3-1-2, 4, 22, 44). Insular dendritic spine number was significantly greater than both low and high integration regions. Overall, the insula had spinier, but shorter neurons than did high integration cortices, and thus may represent a specialized type of heteromodal cortex, one that integrates crude multisensory information crucial to interoceptive processes.

Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (∼7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects.