Dopamine Binding Sites Research Articles

Highly sensitive and selective detection of dopamine using atomic layer deposited HfO2 ultra-thin films

The development of non-enzymatic and binder-free biosensors plays a vital role, and it possesses main benefits for effective biomolecule recognition with high sensitivity and preferred selectivity. Herein, an atomic layer deposition (ALD) was employed to develop the hafnium oxide (HfO2) nanofilm on silicon (Si) for fabricating binder-free selective and sensitive determination of the non-enzymatic electrochemical dopamine sensor. The prepared HfO2 nanofilm selectively acted as a local dopamine binding site because of the hydrophobic-hydrophobic interaction between fabricated film and dopamine, electrostatic attraction between negatively charged hydroxyl group on film surfaces, and positively charged amino group of dopamine. The developed insufficient oxygen vacancies on the HfO2 could have behaved as a charge-trapping site for better sensing properties. The developed sensor demonstrated excellent realization in sensing dopamine with a very low detection limit (0.4 pM) and swift response time of 3s which is quite precise compared with various reported literature. The fabricated HfO2/Si electrode sensor has exhibited high sensitivity, stability, and repeatability. The selectivity of dopamine sensors was at least three orders greater than other interfering biomolecules. This work proposes the essential track for the practical realization of dopamine detection in human serum and biomedical applications.

Overview of the structure and function of the dopamine transporter and its protein interactions.

The dopamine transporter (DAT) plays an integral role in dopamine neurotransmission through the clearance of dopamine from the extracellular space. Dysregulation of DAT is central to the pathophysiology of numerous neuropsychiatric disorders and as such is an attractive therapeutic target. DAT belongs to the solute carrier family 6 (SLC6) class of Na+/Cl- dependent transporters that move various cargo into neurons against their concentration gradient. This review focuses on DAT (SCL6A3 protein) while extending the narrative to the closely related transporters for serotonin and norepinephrine where needed for comparison or functional relevance. Cloning and site-directed mutagenesis experiments provided early structural knowledge of DAT but our contemporary understanding was achieved through a combination of crystallization of the related bacterial transporter LeuT, homology modeling, and subsequently the crystallization of drosophila DAT. These seminal findings enabled a better understanding of the conformational states involved in the transport of substrate, subsequently aiding state-specific drug design. Post-translational modifications to DAT such as phosphorylation, palmitoylation, ubiquitination also influence the plasma membrane localization and kinetics. Substrates and drugs can interact with multiple sites within DAT including the primary S1 and S2 sites involved in dopamine binding and novel allosteric sites. Major research has centered around the question what determines the substrate and inhibitor selectivity of DAT in comparison to serotonin and norepinephrine transporters. DAT has been implicated in many neurological disorders and may play a role in the pathology of HIV and Parkinson's disease via direct physical interaction with HIV-1 Tat and α-synuclein proteins respectively.

T Allele of the DRD2 Taq1 A Gene Polymorphism Increases the Predisposition to Drug Addiction in Indonesian Population

Background: Dopamine plays an important role in mediating the rewarding properties in the abuse of drugs. The Taq1A polymorphism is a commonly studied DRD2 gene variant whereby carriers of the low-function T allele (T/T or T/C genotypes) show reduced brain dopamine function. Therefore, individuals who have the DRD2 Taq1A polymorphism will experience higher levels of drug addiction because the T allele is associated with a reduced number of dopamine binding sites in the brain. A study of this gene has been conducted in some areas, but there is no research for the population of Indonesia. Objective: This study will focus on the frequency of the DRD2 Taq1A gene polymorphism in the population of Indonesia and define its association with drug addiction. Methods: This is a cross sectional study in which 182 subjects were divided into 91 drugaddicted patients and 91 non-drug-addicted control subjects. The genotype analysis was carried out by a modified allele-specific Polymerase Chain Reaction (PCR) method. Results: The frequency of the T/T and C/T was significantly higher in the addicted than control subjects. They are 6.6% and 63.7% compared to 0% and 3.3%. Likewise, the T allele is more frequent in the addicted equal to 38%, compared to only 2% in the control subjects. The frequency of the T allele between the addicted and control subjects shows a significant difference (p-value < 0.0001; 95% CI), with the addicted being at a higher risk of having the T allele (OR = 37.3; 95% CI [11.46-121.29]). Conclusion: There is a significant difference in the frequency of the DRD2 Taq1A gene polymorphism between addicted patients and control subjects. Thus, there is an association between this gene polymorphism and the development of drug addiction with T allele increases the predisposition to addiction.

Weight‐loss response to naltrexone/bupropion is modulated by the Taq1A genetic variant near DRD2 (rs1800497): A pilot study

Naltrexone/bupropion (NB) is a US Food and Drug Administration-approved antiobesity medication. Clinical trials have shown variable weight loss, with responders and non-responders. NB is believed to act on central dopaminergic pathways to suppress appetite. The Taq1A polymorphism near DRD2 (rs1800497) is associated with the density of striatal dopamine D2 receptors, with individuals carrying the A allele (AA or AG; termed A1+) having 30%-40% fewer dopamine binding sites than those who do not carry the A allele (GG; termed A1-). We performed a pilot study to assess the association of the rs1800497 ANKK1 c.2137G > A (p.Glu713Lys) variant with weight loss with NB treatment in 33 subjects. Mean (SD) weight loss was 5.9% (3.2%) for the A1+ genotype group (n = 15) and 4.2% (4.2%) for the A1- genotype group (n = 18). The mean weight loss for the A1+ genotype group was significantly greater than the predefined clinically significant 4% weight-loss target (one-sample t-test, P = .035), whereas the mean weight loss for the A1- genotype group was not (P = .85). Individuals with the A1+ genotype appear to respond better to NB than A1- individuals.

Enzyme-Free Plasmonic Biosensor for Direct Detection of Neurotransmitter Dopamine from Whole Blood

Complex biological fluids without pretreatment, separation, or purification impose stringent limitations on the practical deployment of label-free plasmonic biosensors for advanced assays needed in point of care applications. In this work, we present an enzyme-free plasmonic neurotransmitter dopamine biosensor integrated with a microfluidic plasma separator. This integrated device allows the in-line separation of plasma directly from the bloodstream and channels it to the active detection area, where inorganic cerium oxide nanoparticles function as local selective dopamine binding sites through strong surface redox reaction. A thorough understanding and engineering of the nanoparticles is carried out to maximize its dopamine sensitivity and selectivity. We obtain detection of dopamine at 100 fM concentration in simulated body fluid and 1 nM directly from blood without any prior sample preparation. The detection selectivity is found to be at least five-times higher compared to the common interfering species. This demonstration shows the feasibility of the practical implementation of the proposed plasmonic system in detection of variety of biomarkers directly from the complex biological fluids.

Elucidation of self‐mediated enhancement of dopamine transport by the dopamine transporter which can be modulated by extracellular gate and N‐terminal residues

The dopamine (DA) and serotonin (5‐HT) transporters (DAT and SERT respectively) are members of the SLC6 family of neurotransmitter:sodium symporters (NSSs). DAT and SERT clear substrate from perisynaptic spaces by the generally accepted alternating access mechanism in which the transporters transition between outward‐facing and inward‐facing conformational states. This mechanism involves a sequential closing and opening of extracellular (EC) and intracellular (IC) gates along the permeation pathway. The EC and IC gates are almost exclusively composed of acidic and basic residues capable of interacting through salt bridge formation, closing the primary substrate binding site to the intracellular and extracellular milieus. Previously, we identified that the human SERT acidic/basic residue pair is critical for the recognition of 3,4‐methylenedioxymethamphetamine (MDMA) as a substrate. In contrast, mutations in the EC gate had no effect on 5‐HT recognition. Our current efforts focus on determining if the EC gate of the human DAT has similar mechanistic characteristics for substrate selectivity. While investigating that possibility, we identified a previously unreported phenomenon of self‐mediated enhancement of DA transport in hDAT expressing cells. In homologous competitive uptake experiments utilizing [3H]DA, a 200% to 300% increase in [3H]DA uptake is observed upon addition of micromolar concentrations of unlabeled DA. This enhancement is further modulated by the identity of the acidic EC gate residue. Substituting the Asp with the polar amino acid Asn (D476N) produces a 600% increase in [3H]DA uptake. Experiments utilizing heterologous competition with [3H]DA and the known hDAT substrates 1‐methyl‐4‐phenylpyridinium (MPP+), 4‐(4‐dimethylamino)phenyl‐1‐methylpyridinium(APP+) and tyramine indicate that the enhancement effect is not exclusive to DA nor is it observed with all DAT substrates. Notably, elimination of phosphorylation at N‐terminal residue Thr53 virtually eliminates the DA self‐enhancement phenotype. The apparent ability of DA to enhance its own uptake is consistent with reports proposing a second substrate binding site (S2) that can act as a trigger for release of substrate from the primary DA binding site (S1). However, the presence of this S2 site remains controversial. Continued investigation of this phenomenon may provide insight into molecular determinants on both the substrate and transporter that are important for substrate identification and translocation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dopamine, Noradrenaline and Serotonin Receptor Densities in the Striatum of Hemiparkinsonian Rats following Botulinum Neurotoxin-A Injection

Parkinson’s disease (PD) is characterized by a degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) that causes a dopamine (DA) deficit in the caudate-putamen (CPu) accompanied by compensatory changes in other neurotransmitter systems. These changes result in severe motor and non-motor symptoms. To disclose the role of various receptor binding sites for DA, noradrenaline, and serotonin in the hemiparkinsonian (hemi-PD) rat model induced by unilateral 6-hydroxydopamine (6-OHDA) injection, the densities of D1, D2/D3, α1, α2, and 5HT2A receptors were longitudinally visualized and measured in the CPu of hemi-PD rats by quantitative in vitro receptor autoradiography. We found a moderate increase in D1 receptor density 3 weeks post lesion that decreased during longer survival times, a significant increase of D2/D3 receptor density, and 50% reduction in 5HT2A receptor density. α1 receptor density remained unaltered in hemi-PD and α2 receptors demonstrated a slight right-left difference increasing with post lesion survival.In a second step, the possible role of receptors on the known reduction of apomorphine-induced rotations in hemi-PD rats by intrastriatally injected Botulinum neurotoxin-A (BoNT-A) was analyzed by measuring the receptor densities after BoNT-A injection. The application of this neurotoxin reduced D2/D3 receptor density, whereas the other receptors mainly remained unaltered.Our results provide novel data for an understanding of the postlesional plasticity of dopaminergic, noradrenergic and serotonergic receptors in the hemi-PD rat model. The results further suggest a therapeutic effect of BoNT-A on the impaired motor behavior of hemi-PD rats by reducing the interhemispheric imbalance in D2/D3 receptor density.

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist.

Family A G protein-coupled receptors (GPCRs) control diverse biological processes and are of great clinical relevance. Their archetype rhodopsin becomes naturally light sensitive by binding covalently to the photoswitchable tethered ligand (PTL) retinal. Other GPCRs, however, neither bind covalently to ligands nor are light sensitive. We sought to impart the logic of rhodopsin to light-insensitive Family A GPCRs in order to enable their remote control in a receptor-specific, cell-type-specific, and spatiotemporally precise manner. Dopamine receptors (DARs) are of particular interest for their roles in motor coordination, appetitive, and aversive behavior, as well as neuropsychiatric disorders such as Parkinson's disease, schizophrenia, mood disorders, and addiction. Using an azobenzene derivative of the well-known DAR ligand 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), we were able to rapidly, reversibly, and selectively block dopamine D1 and D2 receptors (D1R and D2R) when the PTL was conjugated to an engineered cysteine near the dopamine binding site. Depending on the site of tethering, the ligand behaved as either a photoswitchable tethered neutral antagonist or inverse agonist. Our results indicate that DARs can be chemically engineered for selective remote control by light and provide a template for precision control of Family A GPCRs.

Rational design of supramolecular hemin/G-quadruplex-dopamine aptamer nucleoapzyme systems with superior catalytic performance.

The rational design of a set of hemin/G-quadruplex (hGQ)-dopamine binding aptamer (DBA) conjugates, acting as nucleoapzymes, is described. The nucleoapzyme constructs consist of a hGQ DNAzyme as a catalytic unit and DBA as a substrate binding unit that are brought into spatial proximity by a duplex scaffold composed of complementary oligonucleotide strands. When the hGQ unit is linked to the duplex scaffold via a single-strand DNA tether of variable length, the resulting nucleoapzymes reveal a moderate catalytic enhancement toward the H2O2-mediated oxidation of dopamine to aminochrome as compared to the process stimulated by the separated hGQ and DBA units (5-7 fold enhancement). This limited enhancement is attributed to inappropriate spatial positioning of the hGQ in respect to the dopamine binding site, and/or to the flexibility of the tether that links the hGQ catalytic site to the double-stranded scaffold. To solve this, rigidification of the hGQ/DBA conjugates by triplex oligonucleotide structures that anchor the hGQ to a duplex domain associated with the DBA units was achieved. By the sequential, programmed, triplex-controlled rigidification of the hGQ/DBA structure, a nucleoapzyme with superior catalytic activity toward the oxidation of dopamine to aminochrome is identified (30-fold catalytic enhancement). Molecular dynamics simulations reveal that in the resulting highly active rigidified nucleoapzyme structure, the hGQ catalytic site is positioned in spatial proximity to the opening of the DBA substrate binding site, thus rationalizing and supporting the enhanced catalytic functions of the system. Finally, the most active nucleoapzyme system was subjected to fuel- and anti-fuel strands that separate and re-assemble the nucleoapzyme structure, allowing "ON" and "OFF" switching of the nucleoapzyme catalytic functions.

Cocaine Photo‐affinity Analogs Bind in the S1 Binding Pocket of the Dopamine Transporter Providing a Mechanism for Competitive Inhibition of Dopamine Uptake

Background Cocaine inhibition of dopamine (DA) uptake through the dopamine transporter (DAT) has been proposed to occur by both competitive and non-competitive mechanisms. Current pharmacological and computational data support the existence of two independent cocaine binding sites on DAT, a centrally located site (S1) and a more extracellularly positioned site (S2), which may underlie the complexity of cocaine inhibition of DAT. Objective: Identify the binding site(s) of the high-affinity cocaine photo-affinity analogs RTI 82, MFZ 2 24, and JHC 2 48. Results: Computational docking of the analogs to DAT as well as biochemical peptide mapping analyses of crosslinked DAT complexes revealed independent sites of adduction of the 4ʹ-azido-3ʹ-iodophenylethyl ester moiety on each analog. Our computational and crosslinking data place the core tropane pharmacophore in the S1 binding pocket, which was verified biochemically using the substituted cysteine accessibility method. Cocaine and the analogs could protect S1, but not S2 residues from reacting with methanethiosulfonate reagents. Conclusion The tropane pharmacophore binds to the S1 site, which overlaps the putative DA binding site, supporting a competitive mechanism for cocaine inhibition of DA uptake. Grant Support: INBRE Program of the NCRR and NIDA-IRP (AHN), DA027845 (LKH & RAV), P20 RR017699 from the COBRE Program of the NCRR, and P20 RR016741 from ND EPSCoR IIG (RAV & JDF).

Mechanism of action of salsolinol on tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in dopamine synthesis. Dopamine regulates TH as an end-product inhibitor through its binding to a high and low affinity site, the former being abolished by Ser40 phosphorylation only, and the latter able to bind and dissociate according to intracellular dopamine levels. Here, we have investigated TH inhibition by a dopamine metabolite found in dopaminergic brain regions, salsolinol (SAL). SAL is known to decrease dopamine in the nigrostriatal pathway and mediobasal hypothalamus, and to also decrease plasma catecholamines in rat stress models, however a target and mechanism for the effects of SAL have not been found. We found that SAL inhibits TH activity in the nanomolar range in vitro, by binding to both the high and low affinity dopamine binding sites. SAL produces the same level of inhibition as dopamine when TH is non-phosphorylated. However, it produces 3.7-fold greater inhibition of Ser40-phosphorylated TH compared to dopamine by competing more strongly with tetrahydrobiopterin, the cofactor of this enzymatic reaction. SAL’s potent inhibition of phosphorylated TH would prevent TH from being fully activated to synthesise dopamine.

Energy metabolism, leptin, and biochemical parameters are altered in rats subjected to the chronic administration of olanzapine

Olanzapine, an atypical antipsychotic drug with affinities for dopamine, serotonin, and histamine binding sites appears to be associated with substantial weight gain and metabolic alterations. The aim of this study was to evaluate weight gain and metabolic alterations in rats treated with olanzapine on a hypercaloric diet. We used 40 rats divided into 4 groups: Group 1, standard food and water conditions (control); Group 2, standard diet plus olanzapine; Group 3, cafeteria diet (hypercaloric); and Group 4, olanzapine plus cafeteria diet. Olanzapine was administered by gavage at a dose of 3 mg/kg for 9 weeks. RESULTS There were no significant changes in the cholesterol levels in any group. Glucose levels increased in Group 3 by the fourth week. Triglyceride levels were altered in group 2 toward the end of the experiment. Leptin levels decreased in Groups 2 and 4. Complex II activity in the muscles and liver was altered in Group 2 (muscle), and Groups 2, 3, and 4 (liver). Complex IV activity was altered only in the liver in Group 2, without significant alterations within the muscles. These results suggest that olanzapine is correlated with weight gain and the risks associated with obesity.

Investigation of Dopamine Receptor Structure and Function by Structure Prediction and Unnatural Amino Acid Mutagenesis

D2 and D4 dopamine receptors were investigated by structure prediction and unnatural amino acid mutagenesis. Active and inactive structures of the D2 dopamine receptor were predicted and the dopamine binding site was modeled. Aromatic residues at and near the binding site of D2 and D4 receptors were interrogated by unnatural amino acid mutagenesis, revealing a network of aromatic-aromatic interactions. Conserved proline residues within five of the D2 receptor's transmembrane helices were also interrogated by unnatural amino acid mutagenesis, revealing a key role for lack of a backbone hydrogen bond donor and importance of a substituent on the backbone N at the Pro5.50 site.

Interaction of tyrosine 151 in norepinephrine transporter with the 2β group of cocaine analog RTI-113

Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100× more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2β position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2β position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies.

A Possible Biological Mechanism for the B Vitamins Altering Behaviour in Attention-Deficit/Hyperactivity Disorder

There is a growing body of recent evidence showing that micronutrients (combinations of minerals, vitamins and amino acids) improve the symptoms of attention-deficit/hyperactivity disorder (ADHD). Dopamine agonists, such as methylphenidate, have long been identified as effective in treating ADHD symptoms, by inhibiting dopamine transporter (DAT) function. This article explores the role that B vitamins might have in the treatment of ADHD symptoms by investigating the structural similarities between B vitamins and methylphenidate. We suggest that the presence of B vitamins and their postulated structure activity relationships (SARs) with dopamine may be responsible for the observed pharmacological effect. This pharmacological activity is likely to be via their competitive binding to the DAT dopamine binding site with a concomitant increase in synaptic dopamine concentration, which in turn might activate the postsynaptic dopamine D2 receptor and thus ameliorate the symptoms ofADHD.Further research is required to assess the validity of the intriguing possibility that B vitamins and methylphenidate share a common neurochemical mechanism of action.

The binding sites for benztropines and dopamine in the dopamine transporter overlap

Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational modeling together with site-directed mutagenesis to characterize the binding site for BZTs in DAT. Docking into molecular models based on the structure of the bacterial homolog LeuT supported a BZT binding site that overlaps with the substrate-binding pocket. In agreement, mutations of residues within the pocket, including 2 2 Number in superscript describes residue number according to generic numbering scheme (see Materials and Methods). Val152 3.46 to Ala or Ile, Ser422 8.60 to Ala and Asn157 3.51 to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [ 3H]dopamine uptake inhibition assays and/or [ 3H]CFT competition binding assay. A putative polar interaction of one of the phenyl ring fluorine substituents in JHW007 with Asn157 3.51 was used as a criterion for determining likely binding poses and establish a structural context for the mutagenesis findings. The analysis positioned the other fluorine-substituted phenyl ring of JHW007 in close proximity to Ala479 10.51/Ala480 10.52 in transmembrane segment (TM) 10. The lack of such an interaction for BZT led to a more tilted orientation, as compared to JHW007, bringing one of the phenyl rings even closer to Ala479 10.51/Ala480 10.52. Mutation of Ala479 10.51 and Ala480 10.52 to valines supported these predictions with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine.

Mechanism for Cocaine Blocking the Transport of Dopamine: Insights from Molecular Modeling and Dynamics Simulations

Molecular modeling and dynamics simulations have been performed to study how cocaine inhibits dopamine transporter (DAT) for the transport of dopamine. The computationally determined DAT-ligand binding mode is totally different from the previously proposed overlap binding mode in which cocaine- and dopamine-binding sites are the same (Beuming, T.; et al. Nat. Neurosci. 2008, 11, 780-789). The new cocaine-binding site does not overlap with, but is close to, the dopamine-binding site. Analysis of all results reveals that when cocaine binds to DAT, the initial binding site is likely the one modeled in this study because this binding site can naturally accommodate cocaine. Then cocaine may move to the dopamine-binding site after DAT makes some necessary conformational change and expands the binding site cavity. It has been demonstrated that cocaine may inhibit the transport of dopamine through both blocking the initial DAT-dopamine binding and reducing the kinetic turnover of the transporter following the DAT-dopamine binding. The relative contributions to the phenomenological inhibition of the transport of dopamine from blocking the initial binding and reducing the kinetic turnover can be different in different types of assays. The obtained general structural and mechanistic insights are consistent with available experimental data and could be valuable for guiding future studies toward understanding cocaine's inhibiting of other transporters.

The Low Affinity Dopamine Binding Site on Tyrosine Hydroxylase: The Role of the N-Terminus and In Situ Regulation of Enzyme Activity

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.

Restricted Cortical and Amygdaloid Removal of Vesicular Glutamate Transporter 2 in Preadolescent Mice Impacts Dopaminergic Activity and Neuronal Circuitry of Higher Brain Function

A major challenge in neuroscience is to resolve the connection between gene functionality, neuronal circuits, and behavior. Most, if not all, neuronal circuits of the adult brain contain a glutamatergic component, the nature of which has been difficult to assess because of the vast cellular abundance of glutamate. In this study, we wanted to determine the role of a restricted subpopulation of glutamatergic neurons within the forebrain, the Vglut2-expressing neurons, in neuronal circuitry of higher brain function. Vglut2 expression was selectively deleted in the cortex, hippocampus, and amygdala of preadolescent mice, which resulted in increased locomotor activity, altered social dominance and risk assessment, decreased sensorimotor gating, and impaired long-term spatial memory. Presynaptic VGLUT2-positive terminals were lost in the cortex, striatum, nucleus accumbens, and hippocampus, and a downstream effect on dopamine binding site availability in the striatum was evident. A connection between the induced late-onset, chronic reduction of glutamatergic neurotransmission and dopamine signaling within the circuitry was further substantiated by a partial attenuation of the deficits in sensorimotor gating by the dopamine-stabilizing antipsychotic drug aripiprazole and an increased sensitivity to amphetamine. Somewhat surprisingly, given the restricted expression of Vglut2 in regions responsible for higher brain function, our analyses show that VGLUT2-mediated neurotransmission is required for certain aspects of cognitive, emotional, and social behavior. The present study provides support for the existence of a neurocircuitry that connects changes in VGLUT2-mediated neurotransmission to alterations in the dopaminergic system with schizophrenia-like behavioral deficits as a major outcome.

Neurotransmitter Receptor Imbalances in Motor Cortex and Basal Ganglia in Hepatic Encephalopathy

Hepatic encephalopathy (HE) in chronic liver disease is characterized by neuropsychiatric and motor disturbances and associated with a net increase of inhibitory neurotransmission. Though many studies, mostly carried out in animal models, have linked dysfunctions of single neurotransmitter systems with the pathogenesis of HE, reports concerning neurotransmitter receptor alterations are controversial. Little is known about the situation in humans. We carried out a multireceptor assessment of HE-associated changes in neurotransmitter receptor densities and affinities in human post-mortem brain samples. Dissociation constants and densities of different binding sites for glutamate, GABA, acetylcholine, norepinephrine, serotonin, dopamine and adenosine were determined by in vitro binding assays and quantitative receptor autoradiography in the motor cortex and putamen of HE and control brains. HE cases do not build a homogeneous group, but differ concerning direction and intensity of binding site density divergences from control values. The acetylcholine M2 binding site dissociation constant was significantly higher in HE brains. Nicotinic acetylcholine and adenosine type 1 and 2A densities were significantly down-regulated in the putamen of HE brains. Our data suggest that neurotransmitter alterations are probably not the primary key factor responsible for the neuropsychiatric and motor disturbances associated with HE.