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The striatum collects and integrates information from many different areas of the brain and propels this forward to the basal ganglia (BG) output structures. In this way, the striatum is playing a pivotal role in control of voluntary movements and is implicated in debilitating movement disorders such as Parkinson's disease. The functional backbone of the striatum is represented by direct pathway (dSPN) Drd1-expressing and indirect pathway (iSPN) Drd2-expressing spiny projection neurons (SPN), exerting opposite effects on movement. In rodent models of striatal function, unilateral dopamine deprivation is known to induce ipsilateral rotational behavior. To further study imbalance of the BG circuit and striatal domain influence on behavioral outcome, we employed a viral approach based on tetanus toxin light chain (TeLC) activity for permanent inhibition of dSPN activity in dorsomedial striatum (DMS). Cre-dependent TeLC injected unilaterally into the DMS of Drd1-Cre mice resulted in robust expression of TeLC in the dSPN cell populations as shown by immunohistochemistry. In the TeLC expressing mice, but not in control mice, we observed ipsilateral rotations that were enhanced upon administration of amphetamine to augment striatal dopamine levels. We argue that the observed single turns of ipsilateral rotations occur because of TeLC-mediated silencing of dSPN activity in one hemisphere, causing unresponsiveness to dopamine transients during movement initiation. This evokes a temporal BG circuit imbalance manifested as short bursts of rotations, particular evident during extrinsic dopaminergic modulation.

Dopamine serves an important role in supporting both locomotor control and higher brain functions such as motivation and learning. Dopaminergic dysfunction is implicated in an equally multidimensional spectrum of neurological and neuropsychiatric diseases. Extracellular dopamine levels are known to be tightly controlled by presynaptic dopamine transporters (DAT), which is also a main target of psychostimulants. Still, detailed data on dopamine dynamics in space and time is needed to fully understand how dopamine signals are encoded and translated into cellular and behavioral responses, and to uncover the pathological effects of dopamine-related diseases. The recently developed genetically encoded fluorescent dopamine sensors enable unprecedented monitoring of dopamine dynamics and have changed the field of in vivo dopamine recording. However, the potential of these sensors to be used for in vitro and ex vivo assays remains unexplored. Here, we demonstrate a generalizable blueprint for making “sniffer” dopamine cells for multimodal detection of dopamine in vitro and ex vivo. We generated sniffer cell lines with inducible expression of six different dopamine sensors and performed a head-to-head comparison of sensor properties to guide users in sensor selection. In proof-of-principle experiments, we show how the sniffer cells can be applied to measure release of endogenous dopamine from cultured neurons and striatal slices, and for determining total dopamine content in striatal tissue. Furthermore, we use the sniffer cells to quantify DAT-mediated dopamine uptake, and AMPH-induced and constitutive dopamine efflux as a radiotracer free, high-throughput alternative to electrochemical- and radiotracer-based assays. Importantly, the sniffer cells framework can readily be applied to other transmitter systems for which the list of genetically encoded fluorescent sensors is rapidly growing.

Chemical neurotransmission in the brain typically occurs through synapses, which are structurally and functionally defined as sites of close apposition between an axon terminal and a postsynaptic domain. Ultrastructural examinations of axon terminals established by monoamine neurons in the brain often failed to identify a similar tight pre- and postsynaptic coupling, giving rise to the concept of “diffuse” or “volume” transmission. Whether this results from intrinsic properties of such modulatory neurons remains undefined. Using an efficient co-culture model, we find that dopaminergic neurons establish an axonal arbor that is distinctive compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. Furthermore, while most dopaminergic varicosities express key proteins involved in exocytosis such as synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in a subset of dopaminergic terminals that are in proximity to a target cell. Irrespective of their structure, a majority of dopaminergic terminals were found to be active. Finally, we found that the presynaptic protein Nrxn-1αSS4- and the postsynaptic protein NL-1AB, two major components involved in excitatory synapse formation, play a critical role in the formation of synapses by dopamine neurons. Taken together, our findings support the idea that dopamine neurons in the brain are endowed with a distinctive developmental program that leads them to adopt a fundamentally different mode of connectivity, compared to glutamatergic and GABAergic neurons involved in fast point-to-point signaling.SIGNIFICANCE STATEMENTMidbrain dopamine (DA) neurons regulate circuits controlling movement, motivation, and learning. The axonal connectivity of DA neurons is intriguing due to its hyperdense nature, with a particularly large number of release sites, most of which not adopting a classical synaptic structure. In this study, we provide new evidence highlighting the unique ability of DA neurons to establish a large and heterogeneous axonal arbor with terminals that, in striking contrast with glutamate and GABA neurons, actively avoid contact with the target cells. The majority of synaptic and non-synaptic terminals express proteins for exocytosis and are active. Finally, our finding suggests that, NL-1A+B and Nrxn-1αSS4-, play a critical role in the formation of synapses by DA neurons.

The serotonin transporter (SERT), a member of the neurotransmitter:sodium symporter family, is responsible for termination of serotonergic signaling by re-uptake of serotonin (5-HT) into the presynaptic neuron. Its key role in synaptic transmission makes it a major drug target, e.g. for the treatment of depression, anxiety and post-traumatic stress. Here, we apply hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs. Our results reveal significant changes in dynamics in regions TM1, EL3, EL4, and TM12 upon binding co-transported ions (Na+/K+) and ligand-mediated changes in TM1, EL3 and EL4 upon binding 5-HT, the drugs S-citalopram, cocaine and ibogaine. Our results provide a comprehensive direct view of the conformational response of SERT upon binding both biologically relevant substrate/ions and ligands of pharmaceutical interest, thus advancing our understanding of the structure-function relationship in SERT., The serotonin transporter (SERT) is responsible for re-uptake of serotonin into the presynaptic neuron and plays a key role in synaptic transmission. Here, the authors use hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs.

Attention-deficit/hyperactivity disorder (ADHD) is a psychiatric disorder characterized by inattention, aberrant impulsivity, and hyperactivity. Although the underlying pathophysiology of ADHD remains unclear, dopamine and norepinephrine signaling originating from the ventral tegmental area (VTA) and locus coeruleus (LC) is thought to be critically involved. In this study, we employ Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) together with the mouse 5-Choice Serial Reaction Time Task (5-CSRTT) to investigate the necessary roles of these catecholamines in ADHD-related behaviors, including attention, impulsivity, and motivation. By selective inhibition of tyrosine hydroxylase (TH)-positive VTA dopamine neurons expressing the Gi-coupled DREADD (hM4Di), we observed a marked impairment of effort-based motivation and subsequently speed and overall vigor of responding. At the highest clozapine N-oxide (CNO) dose tested (i.e. 2 mg/kg) to activate hM4Di, we detected a reduction in locomotor activity. DREADD-mediated inhibition of LC norepinephrine neurons reduced attentional performance in a variable stimulus duration test designed to increase task difficulty, specifically by increasing trials omissions, reducing mean score, and visual processing speed. These findings show that VTA dopamine and LC norepinephrine neurons differentially affect attention, impulsive and motivational control. In addition, this study highlights how molecular genetic probing of selective catecholamine circuits can provide valuable insights into the mechanisms underlying ADHD-relevant behaviors.

For more than 60 years, dopamine (DA) has been known as a critical modulatory neurotransmitter regulating locomotion, reward-based motivation, and endocrine functions. Disturbances in DA signaling have been linked to an array of different neurologic and psychiatric disorders, including Parkinson’s disease, schizophrenia, and addiction, but the underlying pathologic mechanisms have never been fully elucidated. One major obstacle limiting interpretation of standard pharmacological and transgenic interventions is the complexity of the DA system, which only appears to widen as research progresses. Nonetheless, development of new genetic tools, such as chemogenetics, has led to an entirely new era for functional studies of neuronal signaling. By exploiting receptors that are engineered to respond selectively to an otherwise inert ligand, so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), chemogenetics enables pharmacological remote control of neuronal activity. Here we review the recent, extensive application of this technique to the DA field and how its use has advanced the study of the DA system and contributed to our general understanding of DA signaling and related behaviors. Moreover, we discuss the challenges and pitfalls associated with the chemogenetic technology, such as the metabolism of the DREADD ligand clozapine N -oxide (CNO) to the D2 receptor antagonist clozapine. We conclude that despite the recent concerns regarding CNO, the chemogenetic toolbox provides an exceptional approach to study neuronal function. The huge potential should promote continued investigations and additional refinements to further expound key mechanisms of DA signaling and circuitries in normal as well as maladaptive behaviors.

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDATWT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

The dopamine transporter utilizes the transmembrane sodium gradient to mediate reuptake of dopamine from the extracellular space. The dopamine transporter can form dimers and possibly also higher order structures in the plasma membrane, and this oligomerization has been implicated in both trafficking and transport. However, we still do not fully understand its biological importance. A study by Sorkina et al. now describes a series of small molecules that link transporter conformation to oligomerization and endocytosis, providing an interesting step forward in an intricate dance.

The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. In a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of the transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine (DA) adaptations and changes in DA-related behaviors different from those seen in other genetic DAT mouse models. DA levels in striatum are reduced to ∼45% of wild type (WT), accompanied by elevated DA turnover. Nonetheless, Fast-Scan Cyclic Voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels while immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. Summarized, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics.

Background Work in humans has shown that impulsivity can be advantageous in certain settings. However, evidence for so-called functional impulsivity is lacking in experimental animals. Aims This study investigated the contexts in which high impulsive (HI) rats show an advantage in performance compared with mid- (MI) and low impulsive (LI) rats. We also assessed the effects of dopaminergic and noradrenergic agents to investigate underlying neurotransmitter mechanisms. Methods We tested rats on a variable inter-trial interval (ITI) version of the 5-choice serial reaction time task (5CSRTT). Rats received systemic injections of methylphenidate (MPH, 1 mg/kg and 3 mg/kg), atomoxetine (ATO, 0.3 mg/kg and 1 mg/kg), amphetamine (AMPH, 0.2 mg/kg), the alpha-2a adrenoceptor antagonist atipamezole (ATI, 0.3 mg/kg) and the alpha-1 adrenoceptor agonist phenylephrine (PHEN, 1 mg/kg) prior to behavioural testing. Results Unlike LI rats, HI rats exhibited superior performance, earning more reinforcers, on short ITI trials, when the task required rapid responding. MPH, AMPH and ATI improved performance on short ITI trials and increased impulsivity in long ITI trials, recapitulating the behavioural profile of HI. In contrast, ATO and PHEN impaired performance on short ITI trials and decreased impulsivity, thus mimicking the behavioural profile of LI rats. The effects of ATO were greater on MI rats and LI rats. Conclusions These findings indicate that impulsivity can be advantageous when rapid focusing and actions are required, an effect that may depend on increased dopamine neurotransmission. Conversely, activation of the noradrenergic system, with ATO and PHEN, led to a general inhibition of responding.

Dopaminergic dysfunction is central to movement disorders and mental diseases. The dopamine transporter (DAT) is essential for the regulation of extracellular dopamine but the genetic and mechanistic link between DAT function and dopamine-related pathologies remains elusive. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here we identify a novel coding DAT variant, DAT-K619N, in a patient with early-onset parkinsonism and comorbid neuropsychiatric disease and in 22 individuals from exome-sequenced samples of neuropsychiatric patients. The variant localizes to the critical C-terminal PDZ-binding motif of DAT and causes reduced uptake capacity, decreased surface expression, and accelerated turnover of DAT in vitro. In vivo, we demonstrate that expression of DAT-K619N in mice and dropsophila imposes impairments in dopamine transmission with accompanying changes in dopamine-directed behaviors. Importantly, both cellular studies and viral overexpression of DAT-K619N in mice show that DAT-K619N has a dominant-negative effect which collectively implies that a single dominant-negative genetic DAT variant can confer risk for neuropsychiatric disease and neurodegenerative early-onset parkinsonism.

SummaryBin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that shape negatively charged curved lipid membranes during membrane remodeling processes. The BAR domain proteins ICA69, PICK1 and arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at thetrans-Golgi network. Here, we identify four coding variants in the PICK1 gene from a Danish whole-exome screening of diabetic patients, that all involve change of positively charged residues in the PICK1 BAR domain. All four coding variants failed to rescue the insulin content in INS-1E cells upon KD of endogenous PICK1. Moreover, two variants showed dominant negative properties. Interestingly,in vitroassays addressing the BAR domain function suggest that the coding variants compromised membrane binding capacity but increased capacity to cause fission of liposomes.Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin from ISGs during the maturation process and that the coding variants may cause premature budding possibly explaining their dominant negative function.

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2–12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF(549) and JF(646)). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [(3)H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF(549)-conjugated DG3–80 (3) and JF(646)-conjugated DG4–91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

EMBO molecular medicine 12(6), e11248 (2020). doi:10.15252/emmm.201911248, Maladaptive plasticity involving increased expression of AMPA‐type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell‐permeable, high‐affinity (~2 nM) peptide inhibitor, Tat‐P$_4$‐(C5)$_2$, of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat‐P$_4$‐(C5)$_2$ disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA‐receptor surface expression in vivo. Moreover, Tat‐P$_4$‐(C5)$_2$ administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat‐P$_4$‐(C5)$_2$ as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non‐tandem protein–protein interaction domains., Published by EMBO Press, Heidelberg

The serotonin transporter (SERT) terminates serotonin signaling by rapid presynaptic reuptake. SERT activity is modulated by antidepressants, e.g., S-citalopram and imipramine, to alleviate symptoms of depression and anxiety. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). In this study, our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. The SERT inhibition by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we reveal a S1-S2 coupling mechanism that will facilitate rational design of high-affinity SERT allosteric inhibitors., The serotonin transporter (SERT) terminates serotonin signaling and its activity is modulated by antidepressants. Here authors reveal the mechanistic details underlying the coupling between the two binding sites in SERT and a high-affinity ligand for the allosteric site.

In rodents, only a single dose of cocaine or amphetamine is required to cause a marked increase in extracellular dopamine, induce hyperlocomotion and cause persistent plasticity changes within dopaminergic neurons of the ventral tegmental area (VTA). The initial drug experience is suggested to predict vulnerability of developing addiction, but only few studies have assessed the perception of reward accompanying this initial exposure. We recently presented an approach to assess the initial rewarding effects of cocaine in mice with a single-exposure place preference (sePP) protocol, avoiding repeated drug injections. Here, we demonstrate a condensed version of the sePP, allowing assessment of initial subjective reward-perception within a day. By using this protocol, we demonstrate that a single exposure to both cocaine and amphetamine is sufficient to induce place preference. Furthermore, we use chemogenetics ( Designer Receptors Exclusively Activated by Designer Drugs [DREADD]) to show that both inhibitory and stimulatory modulation of VTA DA signalling disrupts cocaine-induced place preference in the condensed sePP. Our findings support the presence of initial reward-perception of both cocaine and amphetamine, and the formation of drug-context association. In addition, our data support that VTA DA signalling prior to drug exposure affects either reward-perception or the time during which associations are formed, thereby preventing induction of cocaine-induced place preference in the sePP. The easy and timesaving sePP protocol should form a critical basis for further deciphering the complex mechanisms underlying the progression from the initial drug experience to escalating drug intake and addiction.

Genetic factors are known to significantly contribute to the etiology of psychiatric diseases such as attention deficit hyperactivity disorder (ADHD) and autism spectrum and bipolar disorders, but the underlying molecular processes remain largely elusive. The dopamine transporter (DAT) has received continuous attention as a potential risk factor for psychiatric disease, as it is critical for dopamine homeostasis and serves as principal target for ADHD medications. Constrain metrics for the DAT-encoding gene, solute carrier family 6 member 3 (SLC6A3), indicate that missense mutations are under strong negative selection, pointing to pathophysiological outcomes when DAT function is compromised. Here, we systematically characterized six rare genetic variants of DAT (I312F, T356M, D421N, A559V, E602G, and R615C) identified in patients with neuropsychiatric disorders. We evaluated dopamine uptake and ligand interactions, along with ion coordination and electrophysiological properties, to elucidate functional phenotypes, and applied Zn(2+) exposure and a substituted cysteine–accessibility approach to identify shared structural changes. Three variants (I312F, T356M, and D421N) exhibited impaired dopamine uptake associated with changes in ligand binding, ion coordination, and distinct conformational disturbances. Remarkably, we found that all three variants displayed gain-of-function electrophysiological phenotypes. I312F mediated an increased uncoupled anion conductance previously suggested to modulate neuronal excitability. T356M and D421N both mediated a cocaine-sensitive leakage of cations, which for T356M was potentiated by Zn(2+), concurrent with partial functional rescue. Collectively, our findings support that gain of disruptive functions due to missense mutations in SLC6A3 may be key to understanding how dopaminergic dyshomeostasis arises in heterozygous carriers.

Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine's actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.

The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. Using a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine (DA) adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of wild type (WT), accompanied by elevated DA turnover. Nonetheless, Fast-Scan Cyclic Voltammetry (FSCV) recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, while immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

The precise regulation of synaptic dopamine (DA) content by the dopamine transporter (DAT) ensures the phasic nature of the DA signal, which underlies the ability of DA to encode reward prediction error, thereby driving motivation, attention, and behavioral learning. Disruptions to the DA system are implicated in a number of neuropsychiatric disorders, including attention deficit hyperactivity disorder (ADHD) and, more recently, Autism Spectrum Disorder (ASD). An ASD-associated de novo mutation in the SLC6A3 gene resulting in a threonine to methionine substitution at site 356 (DAT T356M) was recently identified and has been shown to drive persistent reverse transport of DA (i.e. anomalous DA efflux) in transfected cells and to drive hyperlocomotion in Drosophila melanogaster. A corresponding mutation in the leucine transporter, a DAT-homologous transporter, promotes an outward-facing transporter conformation upon substrate binding, a conformation possibly underlying anomalous dopamine efflux. Here we investigated in vivo the impact of this ASD-associated mutation on DA signaling and ASD-associated behaviors. We found that mice homozygous for this mutation display impaired striatal DA neurotransmission and altered DA-dependent behaviors that correspond with some of the behavioral phenotypes observed in ASD.

PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. Our data demonstrate how multivalent protein-protein and protein-lipid interactions provide critical avidity for the strong binding between the PDZ domain scaffold proteins, PICK1 and PSD-95, and their cognate transmembrane binding partners. The kinetics of the binding were remarkably slow and binding strength two-three orders of magnitude higher than the intrinsic affinity for the isolated PDZ interaction. Interestingly, discrete changes in the intrinsic PICK1 PDZ affinity did not affect overall binding strength but instead revealed dual scaffold modes for PICK1. Our data supported by simulations suggest that intrinsic PDZ domain affinities are finely tuned and encode specific cellular responses, enabling multiplexed cellular functions of PDZ scaffolds., eLife digest Inside a cell, many different signals carry information that is essential for the cell to remain healthy and perform its role in the body. It is, therefore, very important that the signals are sent to the right places at the right times. Scaffold proteins play an essential role in organizing these signals by bringing specific proteins and other molecules into close contact at particular times and locations within the cell. Defects in scaffolding proteins can lead to cancer, psychiatric disorders and other diseases, so these proteins represent potential new targets for medicinal drugs. Many scaffolding proteins assemble groups of proteins on the surface of the membrane that surrounds the cell. Previous studies have shown that scaffolding proteins are able to bind to several other proteins as well as the membrane itself at the same time. However, the precise way in which scaffolding proteins assemble such groups is not clear because it is technically challenging to study this process in living cells. To overcome this challenge, Erlendsson, Thorsen et al. used a new experimental setup known as supported cell membrane sheets – which provides direct access to the side of the cell membrane that usually faces into the cell – to study two scaffolding proteins known as PICK1 and PSD-95. The experiments show that PICK1 and PSD-95 bind to their partner proteins up to 100 times more strongly than previously observed using other approaches. This is due to the scaffolding proteins binding more strongly to both their partners and the membrane. Unexpectedly, the experiments show that the shape and physical characteristics of the partner protein have no effect on the increase in the strength of the binding. Further experiments suggest that altering the ability of the PDZ domain of PICK1 to bind to partner proteins changes the mode of action of the PICK1 protein so that it can activate different responses in the cell. Together these findings imply that the ability of scaffolding proteins to bind to their partner proteins is finely tuned to encode specific responses in cells in different situations – a hypothesis that Erlendsson, Thorsen et al. are planning to test in intact cells.

Background and purpose The 5-HT transporter (SERT) is a target for antidepressant drugs. SERT possesses two binding sites: the orthosteric (S1) binding site, which is the presumed target for current SERT inhibitors, and an allosteric (S2) site for which potential therapeutic effects are unknown. The antidepressant drug citalopram displays high-affinity S1 binding and low-affinity S2 binding. To elucidate a possible therapeutic role of allosteric inhibition of SERT, a drug that specifically targets the allosteric site is required. The purpose of this study was to find a compound having higher selectivity towards the S2 site. Experimental approach We performed a systematic structure-activity relationship study based on the scaffold of citalopram and the structurally closely related congener, talopram, which shows low-affinity S1 binding in SERT. The role of the four chemical substituents, which distinguish citalopram from talopram in conferring selectivity towards the S1 and S2 site, respectively, was assessed by determining the binding of 14 citalopram/talopram analogous to the S1 and S2 binding sites in SERT using membranes of COS7 cells transiently expressing SERT. Key results The structure-activity relationship study revealed that dimethyl citalopram possesses the highest affinity for the allosteric site relative to the S1 site in SERT and has approximately twofold selectivity for the allosteric site relative to the S1 site in SERT. Conclusions and implications The compound could be a useful lead for future synthesis of drugs with high affinity and high selectivity towards the allosteric binding site.

Introduction A solid body of preclinical evidence shows that glucagon-like peptide-1 receptor (GLP-1R) agonists attenuate the effects of substance use disorder related behaviors. The mechanisms underlying these effects remain elusive. In the present study, we hypothesized that GLP-1R activation modulates dopaminetransporter (DAT) and thus dopamine (DA) homeostasis in striatum. This was evaluated in three different experiments: two preclinical and one clinical. Methods Rat striatal DA uptake, DA clearance and DAT cell surface expression was assessed following GLP-1 (7-36)-amide exposure in vitro. DA uptake in mice was assesed ex vivo following systemic treatment with the GLP-1R agonist exenatide. In addition, DA uptake was measured in GLP-1R knockout mice and compared with DA-uptake in wild type mice. In healthy humans, changes in DAT availability was assessed during infusion of exenatide measured by single-photon emission computed tomography imaging. Results In rats, GLP-1 (7-36)-amide increased DA uptake, DA clearance and DAT cell surface expression in striatum. In mice, exenatide did not change striatal DA uptake. In GLP-1R knockout mice, DA uptake was similar to what was measured in wildtype mice. In humans, systemic infusion of exenatide did not result in acute changes in striatal DAT availability. Conclusions The GLP-1R agonist-induced modulation of striatal DAT activity in vitro in rats could not be replicated ex vivo in mice and in vivo in humans. Therefore, the underlying mechanisms of action for the GLP-1R agonists-induced efficacy in varios addiction-like behavioural models still remain.

The striatum of the basal ganglia is pivotal for voluntary movements and is implicated in debilitating movement disorders such as Parkinsonism and dystonia. Striatum projects to downstream nuclei through direct (dSPN) and indirect (iSPN) pathway projection neurons thought to exert opposite effects on movement. In rodent models of striatal function, unilateral dopamine deprivation induces ipsiversive rotational behavior. The dSPNs of the dorsal striatum are believed to engage distinct motor programs but underlying mechanisms remain unclear. Here, we show by employing chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] that unilateral inhibition of dorsomedial dSPNs is sufficient to selectively impair contraversive movement and elicit ipsiversive rotational behavior in mice. Adeno-associated virus (AAV) encoding Cre-dependent Gi-coupled DREADD was injected unilaterally into the dorsomedial striatum of Drd1-Cre mice, resulting in expression of the modified human M4 muscarinic receptor (hM4Di) in ∼20% of dorsostriatal dSPNs. Upon hM4Di activation, a striking positive linear correlation was found between turn ratio and viral expression, which corroborates a relationship between unilateral inhibition of dorsomedial dSPNs and rotational behavior. Bursts of ipsiversive rotations were interspersed with normal ambulation. However, partial unilateral inhibition of ∼20% of dorsostriatal dSPNs did not affect horizontal and vertical locomotion or forelimb use preference. Overall, our results substantiate a unique role of dSPNs in promoting response bias in rotational behavior and show this to be a highly sensitive measure of dSPN performance.

Amphetamine (AMPH) acts as a substrate of the dopamine transporter (DAT) and causes a dramatic increase in extracellular dopamine (DA). Upon entering DA neurons, AMPH promotes DA efflux via DAT through a mechanism implicating depletion of DA from vesicular stores, activation of kinase pathways and transporter phosphorylation. Despite the role of intracellular signaling for AMPH action, it remains elusive how the response to AMPH is affected in vivo by metabotropic regulation via G protein coupled receptor signaling pathways. Here, we show by employment of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that the acute hyperlocomotor response to AMPH is bidirectionally regulated by metabotropic input to VTA DA neurons with a markedly enhanced response upon activation of a Gs-coupled pathway and a markedly decreased locomotor response upon activation of a Gi-coupled pathway. The unique mechanism of action for AMPH was underlined by the absence of an effect of Gs activation on the locomotor response to the DAT inhibitor cocaine. Regardless of the profound effect on the acute AMPH response, repeated Gs activation or Gi activation did not affect development of AMPH sensitization. Furthermore, activation of a Gs-pathway or activation of a Gi-pathway in DA neurons did not have any effect on the AMPH-induced locomotor response in the AMPH sensitized mice. This suggests induction of alterations in DA neuronal functions that overrule the stimulatory or inhibitory effect of metabotropic input seen in drug-naïve mice. The data thereby underline the remarkable strength of maladaptive changes that occur upon intake of strong psychostimulants. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Dopamine plays a key role in the cellular and behavioral responses to drugs of abuse, but the implication of metabotropic regulatory input to dopaminergic neurons on acute drug effects and subsequent drug-related behavior remains unclear. Here, we used chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] to modulate dopamine signaling and activity before cocaine administration in mice. We show that chemogenetic inhibition of dopaminergic ventral tegmental area (VTA) neurons differentially affects locomotor and reward-related behavioral responses to cocaine. Stimulation of Gi-coupled DREADD (hM4Di) expressed in dopaminergic VTA neurons persistently reduced the locomotor response to repeated cocaine injections. An attenuated locomotor response was seen even when a dual-viral vector approach was used to restrict hM4Di expression to dopaminergic VTA neurons projecting to the nucleus accumbens. Surprisingly, despite the attenuated locomotor response, hM4Di-mediated inhibition of dopaminergic VTA neurons did not prevent cocaine sensitization, and the inhibitory effect of hM4Di-mediated inhibition was eliminated after withdrawal. In the conditioned place-preference paradigm, hM4Di-mediated inhibition did not affect cocaine-induced place preference; however, the extinction period was extended. Also, hM4Di-mediated inhibition had no effect on preference for a sugar-based reward over water but impaired motivation to work for the same reward in a touchscreen-based motivational assay. In addition, to support that VTA dopaminergic neurons operate as regulators of reward motivation toward both sugar and cocaine, our data suggest that repeated cocaine exposure leads to adaptations in the VTA that surmount the ability of Gi-signaling to suppress and regulate VTA dopaminergic neuronal activity.

ObjectiveBrain-derived neurotrophic factor (BDNF) seems to play an important role in the course of depression including the response to antidepressants in patients with depression. We aimed to study the effect of an antidepressant intervention on peripheral BDNF in healthy individuals with a family history of depression.MethodsWe measured changes in BDNF messenger RNA (mRNA) expression and whole-blood BDNF levels in 80 healthy first-degree relatives of patients with depression randomly allocated to receive daily tablets of escitalopram 10 mg versus placebo for 4 weeks.ResultsWe found no statistically significant difference between the escitalopram and the placebo group in the change in BDNF mRNA expression and whole-blood BDNF levels. Post hoc analyses showed a statistically significant negative correlation between plasma escitalopram concentration and change in whole-blood BDNF levels in the escitalopram-treated group.ConclusionThe results of this randomised trial suggest that escitalopram 10 mg has no effect on peripheral BDNF levels in healthy individuals.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson's disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases. Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas. DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay. Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Background The potential of abused drugs to induce addiction and compulsive drug-related behavior is associated with their ability to alter dopamine signaling. Dopamine plays a key role in reward signaling and it has been of great interest to investigate how various drugs of abuse alter reward-related behavior. Comparison with existing methods In rodents, the rewarding effects of drugs have often been assessed in self-administration or place preference paradigms; both involving repeated drug exposure and weeks of training and testing. New method Our investigation describes a valid approach to assess the initial rewarding effects of cocaine in mice with a single exposure place preference (sePP) paradigm, avoiding repeated drug injections. Results We present the sePP paradigm with a 3-day protocol to assess the initial rewarding effects of cocaine. Interestingly, only male mice exhibit sePP to cocaine. To assess subsequent drug-related behavior, the protocol was extended by 3 days of extinction followed by reinstatement on day 10. Conclusion The sePP paradigm provides a reliable and convenient approach to assess the initial rewarding effects of cocaine, circumventing the need for repeated drug injections. The sePP protocol allows further dissection of the mechanism and influence of initial cocaine exposure on subsequent drug-related behaviors by including extinction and reinstatement. The lack of sePP in female mice may reflect a biologically relevant sex difference in the initial subjective perception of cocaine-induced reward. This could relate to and explain why males and females have been reported to respond differently to cocaine and cocaine-associated cues.

Attention is a fundamental cognitive process involved in nearly all aspects of life. Abnormal attentional control is a symptom of many neurological disorders, most notably recognized in ADHD (attention deficit hyperactivity disorder). Although attentional performance and its malfunction has been a major area of investigation, it has proven difficult to accurately associate specific neuronal projections, cell types, neurotransmitter systems and receptors with distinct phenotypes owing to its complexity. In this MiniReview, we present a recently invented technology known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD technology is an emerging and transformative method that allows selective manipulation of G protein-coupled receptor (GPCR) signalling, and its broad-ranging usefulness in attention research is now beginning to emerge. We first describe the different DREADDs available and explain how unprecedented specificity of neuronal signalling can be achieved using DREADDs. We next discuss various studies performed in animal models of visual attention, where different brain regions and neuronal populations have been probed by DREADDs. We highlight the interplay between the dopamine (DA) and noradrenaline (NA) catecholamine systems in visual attention and explain why DREADD technology can untangle and help us better understand such complex systems in normal and malfunctioning conditions.

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input., The dopamine transporter (DAT) has a crucial role in the regulation of neurotransmission. Here, the authors use super-resolution imaging to show that DAT clusters into cholesterol-dependent membrane regions that are reversibly regulated by ionotropic glutamate receptors activation.

The serotonin transporter (SERT) terminates serotonergic signaling and enables refilling of synaptic vesicles by mediating reuptake of serotonin (5-HT) released into the synaptic cleft. The molecular and cellular mechanisms controlling SERT activity and surface expression are not fully understood. Here we demonstrate that the substrate 5-HT itself causes acute down-regulation of SERT cell surface expression. To assess surface SERT expression by ELISA, we used a SERT variant (TacSERT) where the N-terminus of SERT was fused to the intracellular tail of the extracellularly FLAG-tagged single-membrane spanning protein Tac. In stably transfected HEK293 cells, 5-HT caused a dose-dependent reduction in TacSERT surface signal with an EC50 value equivalent to the Km value observed for 5-HT uptake. The 5-HT-induced reduction in surface signal reached maximum within 40-60 min and was blocked by the selective SERT inhibitor S-citalopram. 5-HT-induced reduction in SERT expression was further supported by surface biotinylation experiments showing 5-HT-induced reduction in wild type SERT plasma membrane levels. Moreover, preincubation with 5-HT lowered the Vmax for 5-HT uptake in cultured raphe serotonergic neurons, indicting that endogenous cell-surface resident SERT likewise is down-regulated in the presence of substrate.

Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Rare genetic mutations in genes such as Parkin, Pink1, DJ-1, α-synuclein, LRRK2 and GBA are found to be responsible for the disease in about 15% of the cases. A key unanswered question in PD pathophysiology is why would these mutations, impacting basic cellular processes such as mitochondrial function and neurotransmission, lead to selective degeneration of SNc DA neurons? We previously showed in vitro that SNc DA neurons have an extremely high rate of mitochondrial oxidative phosphorylation and ATP production, characteristics that appear to be the result of their highly complex axonal arborization. To test the hypothesis in vivo that axon arborization size is a key determinant of vulnerability, we selectively labeled SNc or VTA DA neurons using floxed YFP viral injections in DAT-cre mice and showed that SNc DA neurons have a much more arborized axon than those of the VTA. To further enhance this difference, which may represent a limiting factor in the basal vulnerability of these neurons, we selectively deleted in mice the DA D2 receptor (D2-cKO), a key negative regulator of the axonal arbour of DA neurons. In these mice, SNc DA neurons have a 2-fold larger axonal arborization, release less DA and are more vulnerable to a 6-OHDA lesion, but not to α-synuclein overexpression when compared to control SNc DA neurons. This work adds to the accumulating evidence that the axonal arborization size of SNc DA neurons plays a key role in their vulnerability in the context of PD., Author summary Parkinson’s disease motor symptoms have been linked to age-dependent degeneration of a class of neurons in the brain that release the chemical messenger dopamine. The reason for the selective loss of these neurons represents a key unsolved mystery. One hypothesis is that the neurons most at risk in this disease are those with the most extensive and complex connectivity in the brain, which would make these cells most dependent on high rates of mitochondrial energy production and expose them to higher rates of oxidative stress. Here we selectively deleted in dopamine neurons a key gene providing negative feedback control of the axonal arbor size of these neurons, in the objective of producing mice in which dopamine neurons have more extensive connectivity. We found that deletion of the dopamine D2 receptor gene in dopamine neurons leads to dopamine neurons with a longer and more complex axonal domain. We also found that in these mice, dopamine neurons in a region of the brain called the substantia nigra show increased vulnerability to a neurotoxin often used to model Parkinson’s disease in rodents. Our findings provide support for the hypothesis that the scale of a neuron’s connectivity directly influences its vulnerability to cellular stressors that trigger Parkinson’s disease.

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca2+-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects. Background: The significance of dopamine transporter (DAT) C-terminal protein-protein interactions for amphetamine-induced dopamine efflux is unsettled. Results: Cell-permeable C-terminal DAT peptides attenuate amphetamine-induced dopamine efflux and locomotor activity in mice. Conclusion: DAT C-terminal protein-protein interactions are critical for the effects of amphetamine in vivo. Significance: Targeting protein-protein interactions might be a way of inhibiting the effects of psychostimulants.

The effect of selective serotonin reuptake inhibitors (SSRI) on healthy individuals remains unclear. We tested the hypothesis that escitalopram decreases perceived stress and salivary cortisol. The trial has a randomised, blinded, placebo-controlled, parallel-group design. After informed consent 80 healthy first-degree relatives to patients with depression were randomly allocated to receive daily tablets of escitalopram 10mg or placebo for 4 weeks. The area under the curve (AUC) for awakening and all day salivary cortisol was analysed in samples taken immediately after awakening and at 15-min intervals for the next hour, and at 12:00, 18:00 and 23:00. The salivary cortisol awakening response, all day salivary cortisol, and scale scores on sleep, pain, aggression, quality of life, and perceived stress assessed at entry were compared to values following 4 weeks of intervention. Statistically significant decreases were found in awakening salivary cortisol ( P =0.04) and in all day salivary cortisol ( P =0.02) in the escitalopram group compared with the placebo group. There were no statistically significant differences in perceived stress between the intervention groups. These findings from a randomised clinical trial suggest that a long-term escitalopram administration to healthy participants results in a decrease in the hypothalamic–pituitary–adrenal (HPA) activity measured by salivary cortisol compared with inert placebo. However, change in salivary cortisol was one out of multiple outcome measures. The results of the present trial do not refute salivary cortisol as a potential endophenotype for depression.

The 5-choice serial reaction time task (5-CSRTT) is widely used to measure rodent attentional functions. In humans, many attention studies in healthy and clinical populations have used testing based on Bundesen’s Theory of Visual Attention (TVA) to estimate visual processing speeds and other parameters of attentional capacity. We aimed to bridge these research fields by modifying the 5-CSRTT’s design and by mathematically modelling data to derive attentional parameters analogous to human TVA-based measures. C57BL/6 mice were tested in two 1-h sessions on consecutive days with a version of the 5-CSRTT where stimulus duration (SD) probe length was varied based on information from previous TVA studies. Thereafter, a scopolamine hydrobromide (HBr; 0.125 or 0.25 mg/kg) pharmacological challenge was undertaken, using a Latin square design. Mean score values were modelled using a new three-parameter version of TVA to obtain estimates of visual processing speeds, visual thresholds and motor response baselines in each mouse. The parameter estimates for each animal were reliable across sessions, showing that the data were stable enough to support analysis on an individual level. Scopolamine HBr dose-dependently reduced 5-CSRTT attentional performance while also increasing reward collection latency at the highest dose. Upon TVA modelling, scopolamine HBr significantly reduced visual processing speed at both doses, while having less pronounced effects on visual thresholds and motor response baselines. This study shows for the first time how 5-CSRTT performance in mice can be mathematically modelled to yield estimates of attentional capacity that are directly comparable to estimates from human studies.

Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signalling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways.

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.