Eric Erbs

Receptor subtypes involved in the presynaptic and postsynaptic actions of dopamine on striatal interneurons

By stimulating distinct receptor subtypes, dopamine (DA) exerts presynaptic and postsynaptic actions on both large aspiny (LA) cholinergic and fast-spiking (FS) parvalbumin-positive interneurons of the striatum. Lack of receptor- and isoform-specific pharmacological agents, however, has hampered the progress toward a detailed identification of the specific DA receptors involved in these actions. To overcome this issue, in the present study we used four different mutant mice in which the expression of specific DA receptors was ablated. In D1 receptor null mice, D1R-/-, DA dose-dependently depolarized both LA and FS interneurons. Interestingly, SCH 233390 (10 μm), a D1-like (D1 and D5) receptor antagonist, but not l-sulpiride (3–10 μm), a D2-like (D2, D3, D4) receptor blocker, prevented this effect, implying D5 receptors in this action. Accordingly, immunohistochemical analyses in both wild-type and D1R-/- mice confirmed the expression of D5 receptors in both cholinergic and parvalbumin-positive interneurons of the striatum. In mice lacking D2 receptors, D2R-/-, the DA-dependent inhibition of GABA transmission was lost in both interneuron populations. Both isoforms of D2 receptor, D2L and D2S, were very likely involved in this inhibitory action, as revealed by the electrophysiological analysis of the effect of the DA D2-like receptor agonist quinpirole in two distinct mutants lacking D2L receptors and expressing variable contents of D2S receptors. The identification of the receptor subtypes involved in the actions of DA on different populations of striatal cells is essential to understand the circuitry of the basal ganglia and to develop pharmacological strategies able to interfere selectively with specific neuronal functions.

Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites.

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [35S]GTPγS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.

Ligand-Directed Trafficking of the δ-Opioid Receptor In Vivo: Two Paths Toward Analgesic Tolerance

δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80—but not ARM390—administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca2+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.

Chronic Haloperidol Promotes Corticostriatal Long-Term Potentiation by Targeting Dopamine D2L Receptors

Reduced glutamate-mediated synaptic transmission has been implicated in the pathophysiology of schizophrenia. Because antipsychotic agents might exert their beneficial effects against schizophrenic symptoms by strengthening excitatory transmission in critical dopaminoceptive brain areas, in the present study we have studied the effects of acute and chronic haloperidol treatment on striatal synaptic plasticity. Repetitive stimulation of corticostriatal terminals in slices induced either long-term depression or long-term potentiation (LTP) of excitatory transmission in control rats, whereas it invariably induced NMDA receptor-dependent LTP in animals treated chronically with haloperidol. Haloperidol effects were mimicked and occluded in mice lacking both D2L and D2S isoforms of dopamine D2 receptors (D2R-/-), in mice lacking D2L receptors and expressing normal levels of D2S receptors (D2R-/-;D2L-/-), and in mice lacking D2L receptors and overexpressing D2S receptors (D2L-/-). These data indicate that the blockade of D2L receptors was responsible for the LTP-favoring action of haloperidol in the striatum. In contrast, overexpression of D2S receptors uncovered a facilitatory role of this receptor isoform in LTP formation because LTP recorded from D2L-/- mice, but not those recorded from wild-type, D2R-/-, and D2R-/-;D2L-/- mice, was insensitive to the pharmacological blockade of D1 receptors. The identification of the cellular, molecular, and receptor mechanisms involved in the action of haloperidol in the brain is essential to understand how antipsychotic agents exert their beneficial and side effects.

A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks.

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.

Differential contribution of dopamine D2S and D2L receptors in the modulation of glutamate and GABA transmission in the striatum.

Compelling evidence indicates that the long (D2L) and the short (D2S) isoform of dopamine (DA) D2 receptors serve distinct physiological functions in vivo. To address the involvement of these isoforms in the control of synaptic transmission in the striatum, we measured the sensitivity to D2 receptor stimulation of glutamate- and GABA-mediated currents recorded from striatal neurons of three mutant mice, in which the expression of D2L and D2S receptors was either ablated or variably altered. Our data indicate that both isoforms participate in the presynaptic inhibition of GABA transmission in the striatum, while the D2-receptor-dependent modulation of glutamate release preferentially involves the D2S receptor. Accordingly, the inhibitory effects of the DA D2 receptor agonist quinpirole (10 microM) on GABA(A)-mediated spontaneous inhibitory postsynaptic currents (IPSCs)correlate with the total number of D2 receptor sites in the striatum, irrespective of the specific receptor isoform expressed. In contrast, glutamate-mediated spontaneous excitatory postsynaptic currents (EPSCs) were significantly inhibited by quinpirole only when the total number of D2 receptor sites, normally composed by both D2L and D2S receptors in a ratio favoring the D2L isoform, was modified to express only the D2S isoform at higher than normal levels. Understanding the physiological roles of DA D2 receptors in the striatum is essential for the treatment of several neuropsychiatric conditions, such as Parkinsons disease, Tourettes syndrome, schizophrenia, and drug addiction.

The BAR Domain Protein Arfaptin-1 Controls Secretory Granule Biogenesis at the trans-Golgi Network

BAR domains can prevent membrane fission through their ability to shield necks of budding vesicles from fission-inducing factors. However, the physiological role of this inhibitory function and its regulation is unknown. Here we identify a checkpoint involving the BAR-domain-containing protein Arfaptin-1 that controls biogenesis of secretory granules at the trans-Golgi network (TGN). We demonstrate that protein kinase D (PKD) phosphorylates Arfaptin-1 at serine 132, which disrupts the ability of Arfaptin-1 to inhibit the activity of ADP ribosylation factor, an important component of the vesicle scission machinery. The physiological significance of this regulatory mechanism is evidenced by loss of glucose-stimulated insulin secretion due to granule scission defects in pancreatic β cells expressing nonphosphorylatable Arfaptin-1. Accordingly, depletion of Arfaptin-1 leads to the generation of small nonfunctional secretory granules. Hence, PKD-mediated Arfaptin-1 phosphorylation is necessary to ensure biogenesis of functional transport carriers at the TGN in regulated secretion.

Insulin secretory granules control autophagy in pancreatic β cells

Too hungry to eat, too hungry not to eat Pancreatic beta cells, the source of insulin in response to food, employ an unusual mechanism to adapt to nutrient depletion. Goginashvili et al. found that starvation of beta cells induced selective degradation of newly formed insulin granules through their fusion with lysosomes, the cells garbage disposal units (see the Perspective by Rutter). The nutrient sensor mTOR is recruited to these lysosomes, leading to its local activation and the suppression of autophagy—a process by which cells “eat” their own constituents. Protein kinase D, a major regulator of insulin granule biogenesis, controls this granule degradation in response to nutrient availability. Thus, unlike most other cells, autophagy is not the strategy of choice in beta cells to adapt to starvation. Science, this issue p. 878; see also p. 826 Newly formed insulin granules are degraded by lysosomes to prevent insulin release upon fasting. [Also see Perspective by Rutter] Pancreatic β cells lower insulin release in response to nutrient depletion. The question of whether starved β cells induce macroautophagy, a predominant mechanism maintaining energy homeostasis, remains poorly explored. We found that, in contrast to many mammalian cells, macroautophagy in pancreatic β cells was suppressed upon starvation. Instead, starved β cells induced lysosomal degradation of nascent secretory insulin granules, which was controlled by protein kinase D (PKD), a key player in secretory granule biogenesis. Starvation-induced nascent granule degradation triggered lysosomal recruitment and activation of mechanistic target of rapamycin that suppressed macroautophagy. Switching from macroautophagy to insulin granule degradation was important to keep insulin secretion low upon fasting. Thus, β cells use a PKD-dependent mechanism to adapt to nutrient availability and couple autophagy flux to secretory function.

Distribution of delta opioid receptor-expressing neurons in the mouse hippocampus

Delta opioid receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. We examined the distribution of delta receptor-expressing cells in the hippocampus using fluorescent knock-in mice that express a functional delta receptor fused at its carboxyterminus with the green fluorescent protein in place of the native receptor. Colocalization with markers for different neuronal populations was performed by immunohistochemical detection. Fine mapping in the dorsal hippocampus confirmed that delta opioid receptors are mainly present in GABAergic neurons. Indeed, they are mostly expressed in parvalbumin-immunopositive neurons both in the Ammons horn and dentate gyrus. These receptors, therefore, most likely participate in the dynamic regulation of hippocampal activity.

In Vivo Visualization of Delta Opioid Receptors upon Physiological Activation Uncovers a Distinct Internalization Profile

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.