Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists.

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with ßarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max values and ßarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

Neurotensin triggers dopamine D2 receptor desensitization through a protein kinase C and beta-arrestin1-dependent mechanism.

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of ß-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of ß-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.

Contribution of Kv1.2 voltage-gated potassium channel to D2 autoreceptor regulation of axonal dopamine overflow.

Impairments in axonal dopamine release are associated with neurological disorders such as schizophrenia and attention deficit hyperactivity disorder and pathophysiological conditions promoting drug abuse and obesity. The D2 dopamine autoreceptor (D2-AR) exerts tight regulatory control of axonal dopamine (DA) release through a mechanism suggested to involve K(+) channels. To evaluate the contribution of Kv1 voltage-gated potassium channels of the Shaker gene family to the regulation of axonal DA release by the D2-AR, the present study employed expression analyses, real time measurements of striatal DA overflow, K(+) current measurements and immunoprecipitation assays. Kv1.1, -1.2, -1.3, and -1.6 mRNA and protein were detected in midbrain DA neurons purified by fluorescence-activated cell sorting and in primary DA neuron cultures. In addition, Kv1.1, -1.2, and -1.6 were localized to DA axonal processes in the dorsal striatum. By means of fast scan cyclic voltammetry in striatal slice preparations, we found that the inhibition of stimulation-evoked DA overflow by a D2 agonist was attenuated by Kv1.1, -1.2, and -1.6 toxin blockers. A particular role for the Kv1.2 subunit in the process whereby axonal D2-AR inhibits DA overflow was established with the use of a selective Kv1.2 blocker and Kv1.2 knock-out mice. Moreover, we demonstrate the ability of D2-AR activation to increase Kv1.2 currents in co-transfected cells and its reliance on Gßγ subunit signaling along with the physical coupling of D2-AR and Kv1.2-containing channels in striatal tissue. These findings underline the contribution of Kv1.2 in the regulation of nigrostriatal DA release by the D2-AR and thereby offer a novel mechanism by which DA release is regulated.

The endocannabinoid 2-arachidonoylglycerol inhibits long-term potentiation of glutamatergic synapses onto ventral tegmental area dopamine neurons in mice.

Drugs of abuse cause changes in the mesocorticolimbic dopamine (DA) system, such as a long-term potentiation (LTP)-like phenomenon at glutamatergic synapses onto ventral tegmental area (VTA) DA neurons. Abolishing this LTP interferes with drug-seeking behavior. Endocannabinoids (ECs) can be released by DA neurons in response to repetitive activation, which can inhibit glutamate release. Therefore, we hypothesized that ECs may act as negative regulators of LTP. Here we tested the induction of LTP in DA neurons of the VTA in mice expressing enhanced green fluorescent protein under the control of the tyrosine hydroxylase promoter. Immunohistochemistry showed colocalization of CB1 receptors with vesicular glutamate transporter (VGLUT)1 in terminals near DA neuron dendrites, with less extensive colocalization with VGLUT2. In addition, a CB1 receptor agonist, as well as trains of stimulation leading to EC production, decreased glutamate release onto DA neurons. We found that blocking CB1 receptors or synthesis of the EC 2-arachidonoylglycerol (2-AG) was without effect on basal excitatory postsynaptic potential amplitude; however, it facilitated the induction of LTP. As previously reported, antagonizing γ-aminobutyric acid (GABA)(A) transmission also facilitated LTP induction. Combining GABA(A) and CB1 receptor antagonists did not lead to larger LTP. LTP induced in the presence of CB1 receptor blockade was prevented by an N-methyl-d-aspartate receptor antagonist. Our observations argue in favor of the hypothesis that 2-AG acts as a negative regulator of LTP in the VTA. Understanding the factors that regulate long-term synaptic plasticity in this circuit is critical to aid our comprehension of drug addiction in humans.

[Recent discoveries on the function and plasticity of central dopamine pathways]. / Découvertes récentes sur la fonction et la plasticité des voies dopaminergiques du cerveau.

Despite the fact that the neurotransmitter dopamine was discovered more than 50 years ago, we still have limited knowledge of its physiological and pathological roles. Recent work has unveiled novel and surprising properties of dopamine neurons and of other key players involved in regulating the dopamine system. For example, the integration of the dopamine signal by its receptors depends on many proteins of diverse signaling pathways and also of other types of receptors that interact with and regulate dopamine receptors: many new promising interactions have been reported during the past few years. Also, we are beginning to discover that chronic treatment with dopamine receptor ligands or other molecules affecting dopaminergic pathways induce long-term molecular, structural and functional rearrangements that could ultimately force us to revisit the mechanism of action of established therapeutic agents. Finally, the discovery of glutamate co-release by dopamine neurons is leading us to reconsider some keys aspects of dopamine neuron physiology.

Long-acting antipsychotic medication as first-line treatment of first-episode psychosis with comorbid substance use disorder.

AIM: Substance use disorder (SUD) is highly prevalent among patients with first-episode psychosis (FEP) and associated with poor adherence and worst treatment outcomes. Although relapses are frequent in FEP, current literature on long-acting injectable antipsychotics (LAI-AP) use in FEP is scarce and studies often exclude patients with SUD. OBJECTIVES: To determine the impact of LAI-AP as first-line treatment on psychotic relapses or rehospitalizations in FEP patients with comorbid SUD (FEP-SUD). METHODS: This is a naturalistic, longitudinal, 3-year prospective and retrospective study on 237 FEP-SUD admitted in two EIS in Montreal, between 2005 and 2012. The patients were divided on the basis of first-line medication introduced, either oral antipsychotics (OAP, n = 206) or LAI-AP (n = 31). Baseline characteristics were compared using χ² test and analysis of variance, and Kaplan-Meier survival analysis was performed on relapse and rehospitalization. RESULTS: Compared to the OAP group, patients in the LAI-AP group presented worse prognostic factors (eg, history of homelessness). Despite this, the LAI-AP group presented a lower relapse rate (67.7% vs 76.7%), higher relapse-free survival time (694 vs 447 days, P = 0.008 in Kaplan-Meier analysis), and trends for reduced rehospitalization rates (48.4% vs 57.3%) and hospitalization-free survival time (813 vs 619 days, P = 0.065 Kaplan-Meier analysis). Of those receiving OAP as first-line, 41.3% were eventually switched to LAI-AP and displayed worst outcome in relapse and rehospitalization. CONCLUSION: LAI-AP should be strongly considered as first-line treatment of FEP-SUD patients since this pharmacological option reduces the risk of relapse and rehospitalization even in the individuals with poor prognostic factors.

Homeostatic regulation of excitatory synapses on striatal medium spiny neurons expressing the D2 dopamine receptor.

Striatal medium spiny neurons (MSNs) are contacted by glutamatergic axon terminals originating from cortex, thalamus and other regions. The striatum is also innervated by dopaminergic (DAergic) terminals, some of which release glutamate as a co-transmitter. Despite evidence for functional DA release at birth in the striatum, the role of DA in the establishment of striatal circuitry is unclear. In light of recent work suggesting activity-dependent homeostatic regulation of glutamatergic terminals on MSNs expressing the D2 DA receptor (D2-MSNs), we used primary co-cultures to test the hypothesis that stimulation of DA and glutamate receptors regulates the homeostasis of glutamatergic synapses on MSNs. Co-culture of D2-MSNs with mesencephalic DA neurons or with cortical neurons produced an increase in spines and functional glutamate synapses expressing VGLUT2 or VGLUT1, respectively. The density of VGLUT2-positive terminals was reduced by the conditional knockout of this gene from DA neurons. In the presence of both mesencephalic and cortical neurons, the density of synapses reached the same total, compatible with the possibility of a homeostatic mechanism capping excitatory synaptic density. Blockade of D2 receptors increased the density of cortical and mesencephalic glutamatergic terminals, without changing MSN spine density or mEPSC frequency. Combined blockade of AMPA and NMDA glutamate receptors increased the density of cortical terminals and decreased that of mesencephalic VGLUT2-positive terminals, with no net change in total excitatory terminal density or in mEPSC frequency. These results suggest that DA and glutamate signaling regulate excitatory inputs to striatal D2-MSNs at both the pre- and postsynaptic level, under the influence of a homeostatic mechanism controlling functional output of the circuit.

Evaluation of D1 and D2 dopamine receptor segregation in the developing striatum using BAC transgenic mice.

The striatum is predominantly composed of medium spiny neurons (MSNs) that send their axons along two parallel pathways known as the direct and indirect pathways. MSNs from the direct pathway express high levels of D1 dopamine receptors, while MSNs from the indirect pathway express high levels of D2 dopamine receptors. There has been much debate over the extent of colocalization of these two major dopamine receptors in MSNs of adult animals. In addition, the ontogeny of the segregation process has never been investigated. In this paper, we crossed bacterial artificial chromosome drd1a-tdTomato and drd2-GFP reporter transgenic mice to characterize these models and estimate D1-D2 co-expression in the developing striatum as well as in striatal primary cultures. We show that segregation is already extensive at E18 and that the degree of co-expression further decreases at P0 and P14. Finally, we also demonstrate that cultured MSNs maintain their very high degree of D1-D2 reporter protein segregation, thus validating them as a relevant in vitro model.

Dopamine facilitates dendritic spine formation by cultured striatal medium spiny neurons through both D1 and D2 dopamine receptors.

Variations of dopamine (DA) levels induced by drugs of abuse or in the context of Parkinson's disease modulate the number of dendritic spines in medium spiny neurons (MSNs) of the striatum, showing that DA plays a major role in the structural plasticity of MSNs. However, little is presently known regarding early spine development in MSNs occurring before the arrival of cortical inputs and in particular about the role of DA and D1 (D1R) and D2 (D2R) DA receptors. A cell culture model reconstituting early cellular interactions between MSNs, intrinsic cholinergic interneurons and DA neurons was used to study the role of DA in spine formation. After 5 or 10 days in vitro, the presence of DA neurons increased the number of immature spine-like protrusions. In MSN monocultures, chronic activation of D1R or D2R also increased the number of spines and spinophilin expression in MSNs, suggesting a direct role for these receptors. In DA-MSN cocultures, chronic blockade of D1R or D2R reduced the number of dendritic spines. Interestingly, the combined activation or blockade of both D1R and D2R failed to elicit more extensive spine formation, suggesting that both receptors act through a mechanism that is not additive. Finally, we found increased ionotropic glutamate receptor responsiveness and miniature excitatory postsynaptic current (EPSC) frequency in DA-MSN co-cultures, in parallel with a higher number of spines containing PSD-95, suggesting that the newly formed spines present functional post-synaptic machinery preparing the MSNs to receive additional glutamatergic contacts. These results represent a first step in the understanding of how dopamine neurons promote the structural plasticity of MSNs during the development of basal ganglia circuits.

Culture of postnatal mesencephalic dopamine neurons on an astrocyte monolayer.

This unit presents a protocol for primary culture of postnatal mesencephalic dopamine neurons grown on an astrocyte monolayer, which can be used to investigate cellular and molecular mechanisms regulating dopamine neuron function. Using this in vitro approach, dopamine neurons survive for an extended period of time and establish functional axon terminals and dendrites that display properties similar to those observed in vivo and in brain slices. An alternate protocol is provided for a microculture system in which astrocytes are grown on a spatially limited surface where single or small groups of dopamine neurons develop. Under such conditions, isolated neurons establish synaptic contacts, or autapses, onto their own somatodendritic compartment, thus facilitating morphological and physiological experiments.