Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression.

gma σ1 and σ2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ1R interacting with dopamine D1 and D2 receptors, the potential regulation of dopaminergic transmission by σ2R is also unknown. We here demonstrate that σ2R may form heteroreceptor complexes with D1 but not with D2 receptors. Remarkably σ1, σ2, and D1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ2R induces bias in signal transduction as σ2R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ1R, suggest that the D1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ1, σ2, and D1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit. KEYWORDS: ERK1/2 phosphorylation; acute; addiction; cAMP; chronic; dopamine D1 and D2 receptors; label-free; signalin

Inhibitory Control of Basolateral Amygdalar Transmission to the Prefrontal Cortex by Local Corticotrophin Type 2 Receptor

Background: Basolateral amygdalar projections to the prefrontal cortex play a key role in modulating behavioral responses to stress stimuli. Among the different neuromodulators known to impact basolateral amygdalar-prefrontal cortex transmission, the corticotrophin releasing factor (CRF) is of particular interest because of its role in modulating anxiety and stress-associated behaviors. While CRF type 1 receptor (CRFR1) has been involved in prefrontal cortex functioning, the participation of CRF type 2 receptor (CRFR2) in basolateral amygdalar-prefrontal cortex synaptic transmission remains unclear. Methods: Immunofluorescence anatomical studies using rat prefrontal cortex synaptosomes devoid of postsynaptic elements were performed in rats with intra basolateral amygdalar injection of biotinylated dextran amine. In vivo microdialysis and local field potential recordings were used to measure glutamate extracellular levels and changes in long-term potentiation in prefrontal cortex induced by basolateral amygdalar stimulation in the absence or presence of CRF receptor antagonists. Results: We found evidence for the presynaptic expression of CRFR2 protein and mRNA in prefrontal cortex synaptic terminals originated from basolateral amygdalar. By means of microdialysis and electrophysiological recordings in combination with an intra-prefrontal cortex infusion of the CRFR2 antagonist antisauvagine-30, we were able to determine that CRFR2 is functionally positioned to limit the strength of basolateral amygdalar transmission to the prefrontal cortex through presynaptic inhibition of glutamate release. Conclusions: Our study shows for the first time to our knowledge that CRFR2 is expressed in basolateral amygdalar afferents projecting to the prefrontal cortex and exerts an inhibitory control of prefrontal cortex responses to basolateral amygdalar inputs. Thus, changes in CRFR2 signaling are likely to disrupt the functional connectivity of the basolateral amygdalar-prefrontal cortex pathway and associated behavioral responses

Differential effect of amphetamine over the corticotropin-releasing factor CRF2 receptor, the orexin OX1 receptor and the CRF2-OX1 heteroreceptor complex

Stress is one of the factors underlying drug seeking behavior that often goes in parallel with loss of appetite. We here demonstrate that orexin 1 receptors (OX1R) may form complexes with the corticotropin releasing factor CRF2 receptor. Two specific features of the heteromer were a cross-antagonism and a blockade by CRF2 of OX1R signaling. In cells expressing one of the receptors, agonist-mediated signal transduction mechanisms were potentiated by amphetamine. Sigma 1 (σ1) and 2 (σ2) receptors are targets of drugs of abuse and, despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is not known. We here show that σ1 receptors interact with CRF2 receptors and that σ2 receptors interact with OX1R. Moreover, we show that amphetamine effect on CRF2 receptors was mediated by σ1R whereas the effect on OX1 receptors was mediated by σ2R. Amphetamine did potentiate the negative cross-talk occurring within the CRF2-OX1 receptor heteromer context, likely by a macromolecular complex involving the two sigma receptors and the two GPCRs. Finally, in vivo microdialysis experiments showed that amphetamine potentiated orexin A-induced dopamine and glutamate release in the ventral tegmental area (VTA). Remarkably, the in vivo orexin A effects were blocked by a selective CRF2R antagonist. These results show that amphetamine impacts on the OX1R-, CRF2R- and OX1R/CRF2R-mediated signaling and that cross-antagonism is instrumental for in vivo detection of GPCR heteromers

Bases biológicas de la adicción

Charla de Katia Gysling, neurofarmacóloga, académica e investigadora de la Pontificia Universidad Católica de Chile. V Conferencia Internacional de Cultura Científica UNAB. Santiago de Chile, 29 de noviembre de 2017.https://www.facebook.com/CienciaUNAB/videos/1767830856581865

Regulation of transmitter synthesis and release in mesolimbic dopaminergic nerve terminals: effect of ethanol

Abstract-Slices from rat olfactory tubercle were incubated in freshly oxygenated Krebs-Ringer phosphate (KRP) and in the presence of L-tyrosine[“C-U] as dopamine (DA) precursor. Thereafter, the newly synthesized [‘“C]DA and the [“C]DA released into the incubation media were isolated by Alumina column, and ion-exchange, chromatography. The presence of K” depolarizing concentrations (25-70mM) in the incubation media markedly increased the formation of [“C]DA from [“Cltyrosine. following a rather complex and biphasic pattern. Dibutyryl cyclic AMP (dB-CAMP) and theophylline also increased the formation of newly synthesized (“CIDA. Ethanol (0.2 to 0.4%. w/v) significantly blocked the stimulation of [l’C]DA biosynthesis that was induced by low K’ depolarizing concentrations (25 mM) and by dB-CAMP (5 x lo-’ M) or theophylline (I x IO ’ M). In contrast, only higher ethanol concentrations (0.8 to 1.10/c, w/v) blocked the increase in DA formation induced by high K- depolarizing concentrations (40 and 55 mM). Potassium depolarization (JO mM) markedly evoked the release of newly synthesized [‘HIDA or [‘HIDA previously taken up by the slices. The release was shown to be dependent upon the presence of Ca” and inhibited by an excess of Mg” (I2 mM). Ethanol (0.8 to 1.10/c, w/v) produced no effect on K’-induced release of [‘HIDA. The model described in this paper can be used as a simple experimental tool to study neurotransmitter synthesis and release from nerve terminals belonging to the mesolimbic dopaminergic system. The results reported suggest the existence of at least two mechanisms by which neuronal depolarization increases transmitter lormation in mesolimbic dopaminergic terminals. Ethanol. at relatively low concentrations. seems to produce a specific inhibitory effect upon the mechanism that predominates under low depolarizing conditions. The possibility is raised that the effects described for ethanol may play a role in the neuropharmacological responses induced by this agent in vivo

Molecular Modeling of Structures and Interaction of Human Corticotropin-Releasing Factor (CRF) Binding Protein and CRF Type-2 Receptor

The corticotropin-releasing factor (CRF) system is a key mediator of the stress response and addictive behavior. The CRF system includes four peptides: The CRF system includes four peptides: CRF, urocortins I–III, CRF binding protein (CRF-BP) that binds CRF with high affinity, and two class B G-protein coupled receptors CRF1R and CRF2R. CRF-BP is a secreted protein without significant sequence homology to CRF receptors or to any other known class of protein. Recently, it has been described a potentiation role of CRF-BP over CRF signaling through CRF2R in addictive-related neuronal plasticity and behavior. In addition, it has been described that CRF-BP is capable to physically interact specifically with the α isoform of CRF2R and acts like an escort protein increasing the amount of the receptor in the plasma membrane. At present, there are no available structures for CRF-BP or for full-length CRFR. Knowing and studying the structure of these proteins could be beneficial in order to characterize the CRF-BP/CRF2αR interaction. In this work, we report the modeling of CRF-BP and of full-length CRF2αR and CRF2βR based on the recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human CRF1R, in addition with the resolved N-terminal extracellular domain of CRFRs. These models were further studied using molecular dynamics simulations and protein–protein docking. The results predicted a higher possibility of interaction of CRF-BP with CRF2αR than CRF2βR and yielded the possible residues conforming the interacting interface. Thus, the present study provides a framework for further investigation of the CRF-BP/CRF2αR interaction

An Amphipathic Alpha-Helix in the Prodomain of Cocaine and Amphetamine Regulated Transcript Peptide Precursor Serves as Its Sorting Signal to the Regulated Secretory Pathway

<div><p>Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1–41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. <i>In silico</i> modeling predicted an alpha-helix structure located between residues 24–37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCART_L34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCART_L34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting.</p> </div

proCART-EGFPm and proCART_L34F-EGFPm are released upon barium stimulation, and proCART_MISS-EGFPm is secreted constitutively.

<p>(A) Basal secretion. The 48 h incubation media of 3 independent samples for proCART-EGFPm, proCART_L34F-EGFPm and proCART_MISS-EGFPm were immunoblotted with an anti-GFP antibody to determine the secretion in the absence of stimulus. Twenty μg of each cell lysate were immunoblotted with anti-GFP and anti-GAPDH antibodies. Secreted levels were plot as percentage of fusion protein released/total fusion protein expressed. (*P<0.0001; One-way ANOVA followed by Dunnet post-hoc test). (B) Stimulated secretion. PC12 cells transfected with either proCART-EGFPm, proCART_L34F-EGFPm or proCART_MISS-EGFPm and 48 h after, cells were incubated for 30 min in basal medium (− lanes) and thereafter for additional 30 min in 2 mM Ba²⁺-medium (+ lanes). proCART-EGFPm, proCART_L34F-EGFPm and proCART_MISS-EGFPm secreted in the respective media were analyzed by western blotting with anti-GFP antibody. In all cases immunoblots were also revealed with anti-SgII antibody to control for the presence of the normal response to barium-stimulation.</p

Subcellular distribution of CART-EGFPm fusion proteins.

<p>Confocal images of PC12 cells transfected with SigP-EGFPm (A), proCART_1–9-EGFPm (B), proCART_1–26-EGFPm (C), proCART_1–41-EGFPm (D) or proCART_MISS-EGFPm (E) expression vectors. Aldehyde-fixed cells were subjected to immunofluorescence protocols using SgII antibody. (A–E) Left panel: EGFPm signal. Middle: SgII-ir signal. Right panel: Merging of both signals. (F) Pearson's coefficient values for the colocalization of each of the fusion protein with SgII-ir (* P<0.001, One-way ANOVA followed by Dunnet's post-hoc test).</p