Dopamine D1-D2 receptor heteromer expression in key brain regions of rat and higher species: Upregulation in rat striatum after cocaine administration.

BACKGROUND: Dopamine receptors interact with other receptors to form heterooligomers. One such complex, the D1-D2 heteromer, demonstrated in cultured striatal neurons and rat striatum has been linked to drug addiction, Parkinson's disease, schizophrenia, depression and anhedonia. METHODS: D1-D2 heteromer expression was evaluated using in situ proximity ligation assay, in parallel with cellular colocalization of D1 and D2 mRNA using in situ hybridization in 19 different key rat brain regions. Expression in higher species and changes in rat striatum after repeated cocaine administration were evaluated. RESULTS: Differences in D1-D2 heteromer expression in striatal subregions are documented in higher species with nonhuman primate and human demonstrating higher density of heteromer-expressing neurons compared to rodents. All species had higher density of D1-D2 neurons in nucleus accumbens compared to dorsal striatum. Multiple other brain regions are identified where D1-D2 heteromer is expressed, prominently in cerebral cortical subregions including piriform, medial prefrontal, orbitofrontal and others; subcortical regions such as claustrum, amygdala and lateral habenula. Three categories of regions are identified: D1-D2 heteromer expressed despite little to no observed D1/D2 mRNA colocalization, likely representing heteromer on neuronal projections from other brain regions; D1-D2 heteromer originating locally with the density of neurons expressing heteromer matching neurons with colocalized D1/D2 mRNA; regions with both a local origin and targeted inputs projecting from other regions. Repeated cocaine administration significantly increased density of neurons expressing D1-D2 heteromer and D1/D2 mRNA colocalization in rat striatum, with changes in both direct and indirect pathway neurons. CONCLUSION: The dopamine D1-D2 heteromer is expressed in key brain cortical and subcortical regions of all species examined. Species differences in striatum revealed greater abundance in human>nonhuman-primate>rat>mouse, suggesting an evolutionary biologic role for the D1-D2 heteromer in higher CNS function. Its upregulation in rat striatum following cocaine points to regulatory significance with possible relevance for clinical disorders such as drug addiction. The dopamine D1-D2 receptor heteromer may represent a potential target for neuropsychiatric and neurodegenerative disorders, given its distribution in highly relevant brain regions.

Maternal Separation Model of Postpartum Depression: Potential Role for Nucleus Accumbens Dopamine D1-D2 Receptor Heteromer.

Postpartum depression is a mood disorder with a distinct neurobiological and behavioural profile occurring during and after the postpartum period. Dopamine pathways in the limbic regions of the brain such as the nucleus accumbens (NAc) have been shown to be involved in the etiology of depressive disorders. Selective activation of the dopamine D1-D2 receptor heteromer has been demonsrated to cause depressive- and anxiogenic-like behaviours in rats. The maternal separation model involving three hour daily maternal separation (MS) from pups on PPD 2-15 on anxiety-, depression- and anhedonia-like behaviors in the dams was investigated, together with plasma corticosterone, oxytocin and D1-D2 heteromer expression in the NAc core and shell in non-MS and MS dams. Depression, anxiety and anhedonia-like behaviours were measured using the forced swim test, elevated plus maze and sucrose preference test, respectively. In comparison to non-MS controls, MS dams displayed slightly higher depressive and anxiety-like behaviours with no difference in anhedonia-like behaviours. The MS dams displayed significantly increased care of pups after their retrieval with higher bouts of nursing, lower latency to nurse, lower bouts of out nest behaviour and decreased self-care. There was no significant alteration in D1-D2 heteromer expression in NAc core and shell between mothers of either group (MS, non-MS) or between postpartum rats and nonpregnant female rats, remaining higher than in male rats. This data provides evidence for the maternal separation model in producing a postpartum depression-like profile, but with maternal resilience able to modify behaviours to counteract any potential deleterious consequences to the pups.

The atypical dopamine receptor agonist SKF 83959 enhances hippocampal and prefrontal cortical neuronal network activity in a rat model of cognitive dysfunction.

Deficits in neuronal network synchrony in hippocampus and prefrontal cortex have been widely demonstrated in disorders of cognitive dysfunction, including schizophrenia and Alzheimer's disease. The atypical dopamine agonist SKF 83959 has been shown to increase brain-derived neurotrophic factor signalling and suppress activity of glycogen synthase kinase-3 in PFC, two processes important to learning and memory. The purpose of this study was to therefore evaluate the impact of SKF 83959 on oscillatory deficits in methylazoxymethanol acetate (MAM) rat model of schizophrenia. To achieve this, local field potentials were recorded simultaneously from the hippocampus and prefrontal cortex of anesthetized rats at 15 and 90 min following both acute and repeated administration of SKF 83959 (0.4 mg/kg). In MAM rats, but not controls, repeated SKF 83959 treatment increased signal amplitude in hippocampus and enhanced the spectral power of low frequency delta and theta oscillations in this region. In PFC, SKF 83959 increased delta, theta and gamma spectral power. Increased HIP-PFC theta coherence was also evident following acute and repeated SKF 83959. In apparent contradiction to these oscillatory effects, in MAM rats, SKF 83959 inhibited spatial learning and induced a significant increase in thigmotactic behaviour. These findings have uncovered a previously unknown role for SKF 83959 in the positive regulation of hippocampal-prefrontal cortical oscillatory network activity. As SKF 83959 is known to have affinity for a number of receptors, delineating the receptor mechanisms that mediate the positive drug effects on neuronal oscillations could have significant future implications in disorders associated with cognitive dysfunction.

A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism.

Although the dopamine D1-D2 receptor heteromer has emerging physiological relevance and a postulated role in different neuropsychiatric disorders, such as drug addiction, depression, and schizophrenia, there is a need for pharmacological tools that selectively target such receptor complexes in order to analyze their biological and pathophysiological functions. Since no selective antagonists for the D1-D2 heteromer are available, serial deletions and point mutations were used to precisely identify the amino acids involved in an interaction interface between the receptors, residing within the carboxyl tail of the D1 receptor that interacted with the D2 receptor to form the D1-D2 receptor heteromer. It was determined that D1 receptor carboxyl tail residues (404)Glu and (405)Glu were critical in mediating the interaction with the D2 receptor. Isolated mutation of these residues in the D1 receptor resulted in the loss of agonist activation of the calcium signaling pathway mediated through the D1-D2 receptor heteromer. The physical interaction between the D1 and D2 receptor could be disrupted, as shown by coimmunoprecipitation and BRET analysis, by a small peptide generated from the D1 receptor sequence that contained these amino acids, leading to a switch in G-protein affinities and loss of calcium signaling, resulting in the inhibition of D1-D2 heteromer function. The use of the D1-D2 heteromer-disrupting peptide in vivo revealed a pathophysiological role for the D1-D2 heteromer in the modulation of behavioral despair. This peptide may represent a novel pharmacological tool with potential therapeutic benefits in depression treatment.

Phasic D1 and tonic D2 dopamine receptor signaling double dissociate the motivational effects of acute nicotine and chronic nicotine withdrawal.

Nicotine, the main psychoactive ingredient of tobacco smoke, induces negative motivational symptoms during withdrawal that contribute to relapse in dependent individuals. The neurobiological mechanisms underlying how the brain signals nicotine withdrawal remain poorly understood. Using electrophysiological, genetic, pharmacological, and behavioral methods, we demonstrate that tonic but not phasic activity is reduced during nicotine withdrawal in ventral tegmental area dopamine (DA) neurons, and that this pattern of signaling acts through DA D2 and adenosine A2A, but not DA D1, receptors. Selective blockade of phasic DA activity prevents the expression of conditioned place aversions to a single injection of nicotine in nondependent mice, but not to withdrawal from chronic nicotine in dependent mice, suggesting a shift from phasic to tonic dopaminergic mediation of the conditioned motivational response in nicotine dependent and withdrawn animals. Either increasing or decreasing activity at D2 or A2A receptors prevents the aversive motivational response to withdrawal from chronic nicotine, but not to acute nicotine. Modification of D1 receptor activity prevents the aversive response to acute nicotine, but not to nicotine withdrawal. This double dissociation demonstrates that the specific pattern of tonic DA activity at D2 receptors is a key mechanism in signaling the motivational effects experienced during nicotine withdrawal, and may represent a unique target for therapeutic treatments for nicotine addiction.

µ-δ opioid receptor heteromer-specific signaling in the striatum and hippocampus.

The µ-δ opioid receptor heteromer activates the pertussis toxin-resistant Gαz GTP-binding protein following stimulation by the δ-agonist deltorphin-II whereas µ- and δ-receptors activate the pertussis toxin-sensitive Gαi3 protein following stimulation by µ- and δ-agonists, respectively. Although the regulation of the µ-δ heteromer is being investigated extensively in vitro, its physiological relevance remains elusive owing to a lack of available molecular tools. We investigated µ-δ heteromer signaling under basal conditions and following prolonged morphine treatment in rodent brain regions highly co-expressing µ- and δ-receptors and Gαz. Deltorphin-II induced Gαz activation in the striatum and hippocampus, demonstrating the presence of µ-δ heteromer signaling in these brain regions. Prolonged morphine treatment, which desensitizes µ- and δ-receptor function, had no effect on µ-δ heteromer signaling in the brain. Our data demonstrate that µ-δ heteromer signaling does not desensitize and is regulated differently from µ- and δ-receptor signaling following prolonged morphine treatment.

Dopamine D1-D2 receptor heteromer regulates signaling cascades involved in addiction: potential relevance to adolescent drug susceptibility.

Adolescence is a developmental period that has been associated with heightened sensitivity to psychostimulant-induced reward, thus placing adolescents at increased risk to develop drug addiction. Although alterations in dopamine-induced synaptic plasticity are perhaps the most critical factor in mediating addiction processes, developmental differences in the cell signaling mechanisms that contribute to synaptic plasticity, and their contribution to adolescent reward sensitivity, has been grossly understudied. The most abundant dopamine receptors, the D1 and D2 receptors, as well as the dopamine D1-D2 receptor heteromer, exhibit age-dependent and brain region-specific changes in their expression and function and are responsible for regulating cell signaling pathways known to significantly contribute to the neurobiological mechanisms underlying addiction. The D1-D2 receptor heteromer, for instance, has been associated with calcium calmodulin kinase IIα, brain-derived neurotrophic factor and glycogen synthase kinase 3 (GSK-3) signaling, three proteins highly implicated in the regulation of glutamate transmission and synaptic plasticity and which regulate addiction to amphetamine, opioids and cocaine. Therefore, in this review the importance of these signaling proteins as potential mediators of addiction susceptibility in adolescence will be highlighted, and the therapeutic potential of the D1-D2 receptor heteromer in addiction will be discussed. It is the overall goal of this review to draw attention to the research gap in dopamine-induced cell signaling in the adolescent brain--knowledge that would provide much-needed insights into adolescent addiction vulnerability.

Neonatal hypocalcemia, neonatal seizures, and intellectual disability in 22q11.2 deletion syndrome.

PURPOSE: Hypocalcemia is a common endocrinological condition in 22q11.2 deletion syndrome. Neonatal hypocalcemia may affect neurodevelopment. We hypothesized that neonatal hypocalcemia would be associated with rare, more severe forms of intellectual disability in 22q11.2 deletion syndrome. METHODS: We used a logistic regression model to investigate potential predictors of intellectual disability severity, including neonatal hypocalcemia, neonatal seizures, and complex congenital heart disease, e.g., interrupted aortic arch, in 149 adults with 22q11.2 deletion syndrome. Ten subjects had moderate-to-severe intellectual disability. RESULTS: The model was highly significant (P < 0.0001), showing neonatal seizures (P = 0.0018) and neonatal hypocalcemia (P = 0.047) to be significant predictors of a more severe level of intellectual disability. Neonatal seizures were significantly associated with neonatal hypocalcemia in the entire sample (P < 0.0001), regardless of intellectual level. There was no evidence for the association of moderate-to-severe intellectual disability with other factors such as major structural brain malformations in this sample. CONCLUSION: The results suggest that neonatal seizures may increase the risk for more severe intellectual deficits in 22q11.2 deletion syndrome, likely mediated by neonatal hypocalcemia. Neonatal hypocalcemia often remains unrecognized until the postseizure period, when damage to neurons may already have occurred. These findings support the importance of early recognition and treatment of neonatal hypocalcemia and potentially neonatal screening for 22q11.2 deletions.

Prevalence of hypocalcaemia and its associated features in 22q11·2 deletion syndrome.

BACKGROUND: 22q11.2 deletion syndrome (22q11.2DS) is a relatively common yet under-recognized genetic syndrome that may present with endocrine features. We aimed to address the factors that contribute to the high prevalence of hypocalcaemia. METHODS: We investigated hypocalcaemia in a well-characterized sample of 138 adults with 22q11.2DS (65 m, 73 F; mean age 34.2, SD 11.8, years) using laboratory studies and lifelong medical records. Logistic regression modelling was used to identify features associated with lifetime prevalence of hypocalcaemia. RESULTS: Of the total sample, 111 (80.4%) had a lifetime history of hypocalcaemia. Eleven (84.6%) of 13 subjects with neonatal hypocalcaemia had documented recurrence of hypocalcaemia. Lifetime history of hypocalcaemia was associated with lifetime prevalence of hypoparathyroidism (P < 0.0001) and hypothyroidism (P = 0.04), as statistically independent factors. Hypomagnesaemia was associated with concurrent hypocalcaemic measurements, especially in the presence of concurrent hypoparathyroidism (P = 0.02). CONCLUSIONS: The results suggest that, in addition to the major effect of hypoparathyroidism, hypothyroidism may play a role in hypocalcaemia in 22q11.2DS and that there is a high recurrence rate of neonatal hypocalcaemia. Hypomagnesaemia may contribute to hypocalcaemia by further suppressing parathyroid hormone (PTH). Although further studies are needed, the findings support regular lifelong follow-up of calcium, magnesium, PTH and TSH levels in patients with 22q11.2DS. At any age, hypocalcaemia with hypoparathyroidism and/or hypothyroidism may suggest a diagnosis of 22q11.2DS.

D5 dopamine receptor carboxyl tail involved in D5-D2 heteromer formation.

We have demonstrated that D(5) and D(2) dopamine receptors exist as heteromers in cells, and determined these receptor interact through amino acids in the cytoplasmic regions of each receptor. Specifically involved in heteromer formation we identified in the carboxyl tail of the D(5) receptor three adjacent glutamic acid residues, and in intracellular loop 3 of the D(2) receptor two adjacent arginine residues. Any pairing of these three D(5) receptor glutamic acids were sufficient for heteromer formation. These identified residues in D(5) and D(2) receptors are oppositely charged and likely interact by electrostatic interactions.

Enhanced brain-derived neurotrophic factor signaling in the nucleus accumbens of juvenile rats.

Brain-derived neurotrophic factor (BDNF) signaling through its receptor, tropomyosin receptor kinase B (TrkB), plays a critical role in neural plasticity and its dysregulation in striatum and prefrontal cortex (PFC) has been implicated in the etiology of mental health disorders such schizophrenia and drug addiction. In the present study, we characterized age-dependent differences in BDNF signaling and TrkB expression within the nucleus accumbens (NAc), caudate putamen (CP) and PFC in rats and determined the effects of administration of the dopamine agonist, SKF 83959, which activates the Gq-coupled dopamine receptors, the dopamine D5 receptor and the D1-D2 receptor heteromer. As proBDNF binds with high affinity to the p75 neurotrophin receptor (p75NTR), expression levels of these proteins were also assessed. The present findings showed that juvenile rats (aged 26-28 days) exhibited significantly elevated basal BDNF expression and activation of full-length TrkB (TrkBfull) in NAc compared to their adult counterparts, as evidenced by increased TrkBfull phosphorylation. These changes were concomitant with an increase in the relative expression of TrkBfull compared to the truncated isoform, TrkB.T1, in NAc and CP. Conversely, in PFC the basal expression of BDNF in juvenile rats was significantly lower than in adult rats with an elevated relative expression of TrkBfull. Acute administration of SKF 83959 to juvenile rats abolished the age-dependent differences in BDNF expression in NAc and PFC, and in the relative expression of TrkBfull in NAc and CP. Together these findings indicate that the expression and/or signaling of BDNF and TrkB in striatum and PFC of juvenile rats is fundamentally different from that of adult rats, a finding that may have implications in neuropsychiatric disorders that exhibit age-dependent susceptibility such as schizophrenia and drug addiction.

A physiological role for the dopamine D5 receptor as a regulator of BDNF and Akt signalling in rodent prefrontal cortex.

The dopamine D5 receptor (D5R) exhibits a wide distribution in prefrontal cortex (PFC) but its role in this region has not yet been elucidated. In the present study, we identified a novel physiological function for the D(5)R as a regulator of brain-derived neurotrophic factor (BDNF) and Akt signalling in PFC. Specifically, acute activation of the D(5)R by the dopamine agonist SKF 83959 enhanced BDNF expression and signalling through its receptor, tropomyosin receptor kinase B (TrkB), in rats and in mice gene-deleted for the D1 receptor but not the D(5)R. These changes were concomitant with increased expression of GAD67, a protein whose down-regulation has been implicated in the aetiology of schizophrenia. Furthermore, D(5)R activation increased phosphorylation of Akt at the Ser(473) site, consequently decreasing the activity of its substrate GSK-3ß. These findings could have wide-reaching implications given evidence showing activation of these pathways in PFC has therapeutic effects in neuropsychiatric disorders such as drug addiction, schizophrenia and depression.

Striatal development involves a switch in gene expression networks, followed by a myelination event: implications for neuropsychiatric disease.

Because abnormal development of striatal neurons is thought to be the part of pathology underlying major psychiatric illnesses, we studied the expression pattern of genes involved in striatal development and of genes comprising key striatal-specific pathways, during an active striatal maturation period, the first two postnatal weeks in rat. This period parallels human striatal development during the second trimester, when prenatal stress is though to lead to increased risk for neuropsychiatric disorders. To identify genes involved in this developmental process, we used subtractive hybridization, followed by quantitative real-time PCR, which allowed us to characterize the developmental expression of over 60 genes, many not previously known to play a role in neuromaturation. Of these 12 were novel transcripts, which did not match known genes, but which showed strict developmental expression and may play a role in striatal neurodevelopment. An additional 89 genes were identified as strong candidates for involvement in this neurodevelopmental process. We show that during the first two postnatal weeks in rat, an early gene expression network, still lacking key striatal-specific signaling pathways, is downregulated and replaced by a mature gene expression network, containing key striatal-specific genes including the dopamine D1 and D2 receptors, conferring to these neurons their functional identity. Therefore, before this developmental switch, striatal neurons lack many of their key phenotypic characteristics. This maturation process is followed by a striking rise in expression of myelination genes, indicating a striatal-specific myelination event. Such strictly controlled developmental program has the potential to be a point of susceptibility to disruption by external factors. Indeed, this period is known to be a susceptibility period in both humans and rats.

Endocannabinoid System and Exogenous Cannabinoids in Depression and Anxiety: A Review.

Background: There is a growing liberalization of cannabis-based preparations for medical and recreational use. In multiple instances, anxiety and depression are cited as either a primary or a secondary reason for the use of cannabinoids. Aim: The purpose of this review is to explore the association between depression or anxiety and the dysregulation of the endogenous endocannabinoid system (ECS), as well as the use of phytocannabinoids and synthetic cannabinoids in the remediation of depression/anxiety symptoms. After a brief description of the constituents of cannabis, cannabinoid receptors and the endocannabinoid system, the most important evidence is presented for the involvement of cannabinoids in depression and anxiety both in human and from animal models of depression and anxiety. Finally, evidence is presented for the clinical use of cannabinoids to treat depression and anxiety. Conclusions: Although the common belief that cannabinoids, including cannabis, its main studied components-tetrahydrocannabinol (THC) and cannabidiol (CBD)-or other synthetic derivatives have been suggested to have a therapeutic role for certain mental health conditions, all recent systematic reviews that we report have concluded that the evidence that cannabinoids improve depressive and anxiety disorders is weak, of very-low-quality, and offers no guidance on the use of cannabinoids for mental health conditions within a regulatory framework. There is an urgent need for high-quality studies examining the effects of cannabinoids on mental disorders in general and depression/anxiety in particular, as well as the consequences of long-term use of these preparations due to possible risks such as addiction and even reversal of improvement.

Daily Δ9-Tetrahydrocannabinol and Withdrawal Increase Dopamine D1-D2 Receptor Heteromer to Mediate Anhedonia- and Anxiogenic-like Behavior Through a Dynorphin and Kappa Opioid Receptor Mechanism.

Background: Frequent cannabis use is associated with a higher risk of developing cannabis use disorder and other adverse consequences. However, rodent models studying the underlying mechanisms of the reinforcing and withdrawal effects of the primary constituent of cannabis, Δ9-tetrahydrocannabinol (THC), have been limited. Methods: This study investigated the effects of daily THC (1 mg/kg, intraperitoneal, 9 days) and spontaneous withdrawal (7 days) on hedonic and aversion-like behaviors in male rats. In parallel, underlying neuroadaptive changes in dopaminergic, opioidergic, and cannabinoid signaling in the nucleus accumbens were evaluated, along with a candidate peptide designed to reverse altered signaling. Results: Chronic THC administration induced anhedonic- and anxiogenic-like behaviors not attributable to altered locomotor activity. These effects persisted after drug cessation. In the nucleus accumbens, THC treatment and withdrawal catalyzed increased cannabinoid CB1 receptor activity without modifying receptor expression. Dopamine D1-D2 receptor heteromer expression rose steeply with THC, accompanied by increased calcium-linked signaling, activation of BDNF/TrkB (brain-derived neurotrophic factor/tropomyosin receptor kinase B) pathway, dynorphin expression, and kappa opioid receptor signaling. Disruption of the D1-D2 heteromer by an interfering peptide during withdrawal reversed the anxiogenic-like and anhedonic-like behaviors as well as the neurochemical changes. Conclusions: Chronic THC increases nucleus accumbens dopamine D1-D2 receptor heteromer expression and function, which results in increased dynorphin expression and kappa opioid receptor activation. These changes plausibly reduce dopamine release to trigger anxiogenic- and anhedonic-like behaviors after daily THC administration that persist for at least 7 days after drug cessation. These findings conceivably provide a therapeutic strategy to alleviate negative symptoms associated with cannabis use and withdrawal.

Δ9-Tetrahydrocannabinol does not upregulate an aversive dopamine receptor mechanism in adolescent brain unlike in adults.

Earlier age of cannabis usage poses higher risk of Cannabis Use Disorder and adverse consequences, such as addiction, anxiety, dysphoria, psychosis, largely attributed to its principal psychoactive component, Δ9-tetrahydrocannabinol (THC) and altered dopaminergic function. As dopamine D1-D2 receptor heteromer activation causes anxiety and anhedonia, this signaling complex was postulated to contribute to THC-induced affective symptoms. To investigate this, we administered THC repeatedly to adolescent monkeys and adolescent or adult rats. Drug-naïve adolescent rat had lower striatal densities of D1-D2 heteromer compared to adult rat. Repeated administration of THC to adolescent rat or adolescent monkey did not alter D1-D2 heteromer expression in nucleus accumbens or dorsal striatum but upregulated it in adult rat. Behaviourally, THC-treated adult, but not adolescent rat manifested anxiety and anhedonia-like behaviour, with elevated composite negative emotionality scores that correlated with striatal D1-D2 density. THC modified downstream markers of D1-D2 activation in adult, but not adolescent striatum. THC administered with cannabidiol did not alter D1-D2 expression. In adult rat, co-administration of CB1 receptor (CB1R) inverse agonist with THC attenuated D1-D2 upregulation, implicating cannabinoids in the regulation of striatal D1-D2 heteromer expression. THC exposure revealed an adaptable age-specific, anxiogenic, anti-reward mechanism operant in adult striatum but deficient in adolescent rat and monkey striatum that may confer increased sensitivity to THC reward in adolescence while limiting its negative effects, thus promoting continued use and increasing vulnerability to long-term adverse cannabis effects.

Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1-D2 heteromer formation.

D(1) and D(2) dopamine receptors exist as heteromers in cells and brain tissue and are dynamically regulated and separated by agonist concentrations at the cell surface. We determined that these receptor pairs interact primarily through discrete amino acids in the cytoplasmic regions of each receptor, with no evidence of any D(1)-D(2) receptor transmembrane interaction found. Specifically involved in heteromer formation we identified, in intracellular loop 3 of the D(2) receptor, two adjacent arginine residues. Substitution of one of the arginine pair prevented heteromer formation. Also involved in heteromer formation we identified, in the carboxyl tail of the D(1) receptor, two adjacent glutamic acid residues. Substitution of one of the glutamic acid pair prevented heteromer formation. These amino acid pairs in D(1) and D(2) receptors are oppositely charged, and presumably interact directly by electrostatic interactions.

Disruption of the mu-delta opioid receptor heteromer.

The crystal structure of the mu and kappa opioid receptors has revealed dimeric structural arrangements. Mu-delta receptors heteromers also exist and we have identified discrete cytoplasmic regions in each receptor required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In intracellular loop 3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation.

Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth.

Although the perturbation of either the dopaminergic system or brain-derived neurotrophic factor (BDNF) levels has been linked to important neurological and neuropsychiatric disorders, there is no known signaling pathway linking these two major players. We found that the exclusive stimulation of the dopamine D1-D2 receptor heteromer, which we identified in striatal neurons and adult rat brain by using confocal FRET, led to the activation of a signaling cascade that links dopamine signaling to BDNF production and neuronal growth through a cascade of four steps: (i) mobilization of intracellular calcium through Gq, phospholipase C, and inositol trisphosphate, (ii) rapid activation of cytosolic and nuclear calcium/calmodulin-dependent kinase IIalpha, (iii) increased BDNF expression, and (iv) accelerated morphological maturation and differentiation of striatal neurons, marked by increased microtubule-associated protein 2 production. These effects, although robust in striatal neurons from D5(-/-) mice, were absent in neurons from D1(-/-) mice. We also demonstrated that this signaling cascade was activated in adult rat brain, although with regional specificity, being largely limited to the nucleus accumbens. This dopaminergic pathway regulating neuronal growth and maturation through BDNF may have considerable significance in disorders such as drug addiction, schizophrenia, and depression.

Dopamine D1-D2 receptor Heteromer-mediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2.

We identified that activation of the G(q)-linked dopamine D1-D2 receptor hetero-oligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pretreatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822, which only activates the D1 receptor homo-oligomer, led to rapid desensitization of the D1-D2 receptor heteromer-mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. Bioluminescence resonance energy transfer and co-immunoprecipitation studies indicated an agonist-induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pretreatment with either agonist. Expression of the catalytically inactive and RGS (regulator of G protein signaling)-mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer-mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.