Effects of iron-deficient diet on sleep onset and spinal reflexes in a rodent model of Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is a common sensorimotor and a sleep disorder that affects 2.5-10% of the European and North American populations. RLS is also often associated with periodic leg movements during sleep (PLMS). Despite ample evidence of genetic contributions, the underlying mechanisms that elicit the sensory and motor symptoms remain unidentified. Clinically, RLS has been correlated with an altered central iron metabolism, particularly in the brain. While several animal models have been developed to determine the outcome of an altered iron homeostasis on brain function, the potential role of an altered iron homeostasis on sleep and sensorimotor circuits has not yet been investigated. Here, we utilize a mouse model to assess the effects of an iron-deficient (ID) but non-anemic state on sleep time and episodes, and sensorimotor reflexes in male and female mice. We found that animals on the ID diet displayed an increased expression of the transferrin receptor in the spinal cord, confirming the results of previous studies that focused only on the impact of ID in the brain. We also demonstrate that the ID diet reduced hematocrit levels compared to controls but not into the anemic range, and that animals on the ID diet exhibited RLS-like symptoms with regard to sleep onset and spinal cord reflex excitability. Interestingly, the effects on the spinal cord were stronger in females than in males, and the ID diet-induced behaviors were rescued by the return of the animals to the control diet. Taken together, these results demonstrate that diet-induced ID changes to CNS function are both inducible and reversible, and that they mimic the sleep and sensorimotor RLS symptoms experienced in the clinic. We therefore propose replacing the commonly used phrase "brain iron deficiency" (BID) hypothesis in the RLS research field with the term "iron deficiency in the central nervous system" (ID-CNS), to include possible effects of altered iron levels on spinal cord function.

Putative Animal Models of Restless Legs Syndrome: A Systematic Review and Evaluation of Their Face and Construct Validity.

Restless legs syndrome (RLS) is a sensorimotor disorder that severely affects sleep. It is characterized by an urge to move the legs, which is often accompanied by periodic limb movements during sleep. RLS has a high prevalence in the population and is usually a life-long condition. While its origins remain unclear, RLS is initially highly responsive to treatment with dopaminergic agonists that target D2-like receptors, in particular D2 and D3, but the long-term response is often unsatisfactory. Over the years, several putative animal models for RLS have been developed, mainly based on the epidemiological and neurochemical link with iron deficiency, treatment efficacy of D2-like dopaminergic agonists, or genome-wide association studies that identified risk factors in the patient population. Here, we present the first systematic review of putative animal models of RLS, provide information about their face and construct validity, and report their role in deciphering the underlying pathophysiological mechanisms that may cause or contribute to RLS. We propose that identifying the causal links between genetic risk factors, altered organ functions, and changes to molecular pathways in neural circuitry will eventually lead to more effective new treatment options that bypass the side effects of the currently used therapeutics in RLS, especially for long-term therapy.

D3 Receptors and Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is a sensorimotor disorder that severely affects sleep. It is characterized by an urge to move the legs that is often accompanied by periodic limb movements during sleep (PLMS). RLS has a high prevalence in the population and is usually a life-long condition. While its origins remain unclear, RLS is initially highly responsive to treatment with dopaminergics that target the D3 receptor. However, over time patients often develop a gradual tolerance that can lead to the emergence of adverse effects and the augmentation of the symptoms. While the basal ganglia and the striatum control leg movements, the lumbar spinal cord is the gateway for the sensory processing of the symptoms and critical for the associated leg movements. D3 receptors are highly expressed in nucleus accumbens (NAc) of the striatum and the sensory-processing areas of the spinal dorsal horn. In contrast, D1 receptors are strongly expressed throughout the entire striatum and in the ventral horn of the spinal cord. Long-term treatment with D3 receptor full agonists is associated with an upregulation of the D1 receptor subtype, and D3 and D1 receptors can form functional heteromers, in which the D3R controls the D1R function. It is conceivable that the switch from beneficial treatment to augmentation observed in RLS patients after prolonged D3R agonist exposure may be the result of unmasked D1-like receptor actions.

Consensus guidelines on the construct validity of rodent models of restless legs syndrome.

Our understanding of the causes and natural course of restless legs syndrome (RLS) is incomplete. The lack of objective diagnostic biomarkers remains a challenge for clinical research and for the development of valid animal models. As a task force of preclinical and clinical scientists, we have previously defined face validity parameters for rodent models of RLS. In this article, we establish new guidelines for the construct validity of RLS rodent models. To do so, we first determined and agreed on the risk, and triggering factors and pathophysiological mechanisms that influence RLS expressivity. We then selected 20 items considered to have sufficient support in the literature, which we grouped by sex and genetic factors, iron-related mechanisms, electrophysiological mechanisms, dopaminergic mechanisms, exposure to medications active in the central nervous system, and others. These factors and biological mechanisms were then translated into rodent bioequivalents deemed to be most appropriate for a rodent model of RLS. We also identified parameters by which to assess and quantify these bioequivalents. Investigating these factors, both individually and in combination, will help to identify their specific roles in the expression of rodent RLS-like phenotypes, which should provide significant translational implications for the diagnosis and treatment of RLS.

Morphine Resistance in Spinal Cord Injury-Related Neuropathic Pain in Rats is Associated With Alterations in Dopamine and Dopamine-Related Metabolomics.

Opioids are not universally effective for treating neuropathic pain following spinal cord injury (SCI), a finding that we previously demonstrated in a rat model of SCI. The aim of this study was to determine analgesic response of morphine-responsive and nonresponsive SCI rats to adjunct treatment with dopamine modulators and to establish if the animal groups expressed distinct metabolomic profiles. Thermal thresholds were tested in female Long Evans rats (N = 45) prior to contusion SCI, after SCI and following injection of morphine, morphine combined with dopamine modulators, or dopamine modulators alone. Spinal cord and striatum samples were processed for metabolomics and targeted mass spectrometry. Morphine provided analgesia in 1 of 3 of SCI animals. All animals showed improved analgesia with morphine + pramipexole (D3 receptor agonist). Only morphine nonresponsive animals showed improved analgesia with the addition of SCH 39166 (D1 receptor antagonist). Metabolomic analysis identified 3 distinct clusters related to the tyrosine pathway that corresponded to uninjured, SCI morphine-responsive and SCI morphine-nonresponsive groups. Mass spectrometry showed matching differences in dopamine levels in striatum and spinal cord between these groups. The data suggest an overall benefit of the D3 receptor system in improving analgesia, and an association between morphine responsiveness and metabolomic changes in the tyrosine/dopamine pathways in striatum and spinal cord. PERSPECTIVE: Spinal cord injury (SCI) leads to opioid-resistant neuropathic pain that is associated with changes in dopamine metabolomics in the spinal cord and striatum of rats. We present evidence that adjuvant targeting of the dopamine system may be a novel pain treatment approach to overcome opioid desensitization and tolerance after SCI.

Loss of Function in Dopamine D3 Receptor Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation in vitro.

Evidence suggests the existence of an intracardiac dopaminergic system that plays a pivotal role in regulating cardiac function and fibrosis through G-protein coupled receptors, particularly mediated by dopamine receptor 3 (D3R). However, the expression of dopamine receptors in cardiac tissue and their role in cardiac fibroblast function is unclear. In this brief report, first we determined expression of D1R and D3R both in left ventricle (LV) tissue and fibroblasts. Then, we explored the role of D3R in the proliferation and migration of fibroblast cell cultures using both genetic and pharmaceutical approaches; specifically, we compared cardiac fibroblasts isolated from LV of wild type (WT) and D3R knockout (D3KO) mice in response to D3R-specific pharmacological agents. Finally, we determined if loss of D3R function could significantly alter LV fibroblast expression of collagen types I (Col1a1) and III (Col3a1). Cardiac fibroblast proliferation was attenuated in D3KO cells, mimicking the behavior of WT cardiac fibroblasts treated with D3R antagonist. In response to scratch injury, WT cardiac fibroblasts treated with the D3R agonist, pramipexole, displayed enhanced migration compared to control WT and D3KO cells. Loss of function in D3R resulted in attenuation of both proliferation and migration in response to scratch injury, and significantly increased the expression of Col3a1 in LV fibroblasts. These findings suggest that D3R may mediate cardiac fibroblast function during the wound healing response. To our knowledge this is the first report of D3R's expression and functional significance directly in mouse cardiac fibroblasts.

Differential dopamine modulation of spinal reflex amplitudes is associated with the presence or absence of the autonomic nervous system.

The spinal cord contains a highly collateralized network of descending dopamine (DA) fibers that stem from the dorso-posterior hypothalamic A11 region in the brain, however, the modulatory actions of DA have generally only been assessed in lumbar segments L2-L5. In contrast to these exclusively sensorimotor segments, spinal cords segments T1-L2 and, in mouse, L6-S2, additionally contain the intermediolateral (IML) nucleus, the origin of autonomic nervous system (ANS). Here, we tested if the different spinal circuits in sensorimotor and IML-containing segments react differently to the modulation of the monosynaptic reflex (MSR) by DA. Bath-application of DA (1 µM) led to a decrease of MSR amplitude in L3-L5 segments; however, in IML-containing segments (T10-L2, and S1/2) the MSR response was facilitated. We did not observe any difference in the response between thoracic (sympathetic) and lumbosacral (parasympathetic) segments. Application of the D2-receptor agonists bromocriptine or quinpirole mimicked the effects of DA, while blocking D2 receptor pathways with raclopride or application with the D1-receptor agonist SKF 38393 led to an increase of the MSR in L3-L5 segments and a decrease of the MSR in IML-containing segments. In contrast, in the presence of the gap-junction blockers, carbenoloxone and quinine, DA modulatory actions in IML-containing segments were similar to those of sensorimotor L3-L5 segments. We suggest that DA modulates MSR amplitudes in the spinal cord in a segment-specific manner, and that the differential outcome observed in ANS segments may be a result of gap junctions in the IML.

Consensus Guidelines on Rodent Models of Restless Legs Syndrome.

Restless legs syndrome (RLS) is a chronic sensorimotor disorder diagnosed by clinical symptoms. It is challenging to translate the diagnostic self-reported features of RLS to animals. To help researchers design their experiments, a task force was convened to develop consensus guidelines for experimental readouts in RLS animal models. The RLS clinical diagnostic criteria were used as a starting point. After soliciting additional important clinical features of RLS, a consensus set of methods and outcome measures intent on capturing these features-in the absence of a face-to-face interview-was generated and subsequently prioritized by the task force. These were, in turn, translated into corresponding methods and outcome measures for research on laboratory rats and mice and used to generate the final recommendations. The task force recommended activity monitoring and polysomnography as principal tools in assessing RLS-like behavior in rodents. Data derived from these methods were determined to be the preferred surrogate measures for the urge to move, the principal defining feature of RLS. The same tools may be used to objectively demonstrate sleep-state features highly associated with RLS, such as sleep disturbance and number and periodicity of limb movements. Pharmacological challenges and dietary or other manipulations that affect iron availability are desirable to aggravate or improve RLS-like behavior and lend greater confidence that the animal model being proffered replicates key clinical features of RLS. These guidelines provide the first consensus experimental framework for researchers to use when developing new rodent models of RLS. © 2020 International Parkinson and Movement Disorder Society.

Dopamine receptor D3 agonist (Pramipexole) reduces morphine-induced cardiac fibrosis.

Morphine is routinely used for pain management in heart failure patients. However, extended morphine exposure associates with major adverse cardiovascular events. Reports link the dopamine receptor D2-family with morphine-induced nociception modulation. This study first assessed whether morphine induces cardiac remodeling in healthy mice, then whether DRD3 agonist (DRD3ag, D2-family member) adjunct therapy prevents morphine-induced cardiac remodeling. Mice received morphine (2 mg/kg/day i. p.) for 7 days (D7) and were either euthanized at D7 or kept 7 more days without morphine (i.e. withdrawal period, D8-D14): G1, morphine; G2, morphine/DRD3ag; G3, morphine + withdrawal; G4, morphine/DRD3ag + withdrawal; G5, morphine + withdrawal/DRD3ag. A separate cohort of animals were used as naïve tissues. We evaluated functional and molecular parameters of cardiac remodeling. Although we did not observe significant differences in systolic function, morphine induced both interstitial fibrosis and cardiomyocyte hypertrophy. Interestingly, DRD3ag abolished these effects. Compared to naïve tissues, collagen 1 increased after withdrawal in G3 and G4 and collagen 3 increased in G1-G4 but at higher levels in G1 and G2. Only G5 did not show collagen differences compared to naïve, suggesting DRD3ag treatment during withdrawal may be beneficial and prevent morphine-induced fibrosis. Smad2/3 phosphorylation increased during withdrawal, indicating a likely upstream pathway for the observed morphine-induced fibrosis. Overall, our data suggest that DRD3ag adjunct therapy decreases morphine-induced adverse cardiac remodeling.

BTBD9 and dopaminergic dysfunction in the pathogenesis of restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an urge to move legs, usually accompanied by uncomfortable sensations. RLS symptoms generally happen at night and can be relieved by movements. Genetic studies have linked polymorphisms in BTBD9 to a higher risk of RLS. Knockout of BTBD9 homolog in mice (Btbd9) and fly results in RLS-like phenotypes. A dysfunctional dopaminergic system is associated with RLS. However, the function of BTBD9 in the dopaminergic system and RLS is not clear. Here, we made use of the simple Caenorhabditis elegans nervous system. Loss of hpo-9, the worm homolog of BTBD9, resulted in hyperactive egg-laying behavior. Analysis of genetic interactions between hpo-9 and genes for dopamine receptors (dop-1, dop-3) indicated that hpo-9 and dop-1 worked similarly. Reporter assays of dop-1 and dop-3 revealed that hpo-9 knockout led to a significant increase of DOP-3 expression. This appears to be evolutionarily conserved in mice with an increased D2 receptor (D2R) mRNA in the striatum of the Btbd9 knockout mice. Furthermore, the striatal D2R protein was significantly decreased and Dynamin I was increased. Overall, activities of DA neurons in the substantia nigra were not altered, but the peripheral D1R pathway was potentiated in the Btbd9 knockout mice. Finally, we generated and characterized the dopamine neuron-specific Btbd9 knockout mice and detected an active-phase sleepiness, suggesting that dopamine neuron-specific loss of Btbd9 is sufficient to disturb the sleep. Our results suggest that increased activities in the D1R pathway, decreased activities in the D2R pathway, or both may contribute to RLS.

Dopamine D1 or D3 receptor modulators prevent morphine tolerance and reduce opioid withdrawal symptoms.

The long-term treatment of chronic pain by opioids is limited by tolerance and risk of addiction/dependence. Previously, we have shown that combination treatment of morphine with a dopamine D1 or D3 receptor modulator restored morphine analgesia in morphine-resistant neuropathic pain and decreased morphine's reward potential in an acute setting. Here, we investigated whether such adjunct therapy with a dopamine D1 receptor preferring antagonist (SCH 39166) or a dopamine D3 receptor preferring agonist (pramipexole) could prevent morphine tolerance and reduce withdrawal symptoms. Initially, tolerance to the combination of morphine + pramipexole was assessed in mice. Mice receiving intraperitoneal injections of morphine showed reduced thermal thresholds on Day 7 whereas those receiving morphine + pramipexole maintained analgesia at Day 7. Next, tolerance and withdrawal to both combinations were tested over 14 days in rats. Rats were assigned one of four drug conditions, (1) saline, 2) morphine, 3) morphine + SCH 39166, 4) morphine + pramipexole), for chronic administration via osmotic pumps. Chronic administration of morphine over 14 days resulted in a significant reduction of morphine analgesia. However, analgesia was maintained when morphine was administered with either the dopamine D1 receptor preferring antagonist or the D3 receptor preferring agonist. Withdrawal symptoms peaked at 48 h and were decreased in rats receiving either combination compared to morphine alone. The data suggests that adjunct therapy with dopamine D1 or D3 receptor preferring modulators prevents morphine tolerance and reduces the duration of morphine withdrawal symptoms, and thus this combination has potential for long-term pain management therapy.

D3 and D1 receptors: The Yin and Yang in the treatment of restless legs syndrome with dopaminergics.

Dopaminergic treatments targeting the D3 receptor subtype to reduce the symptoms of RLS show substantial initial clinical benefits but fail to maintain their efficacy over time. Sensorimotor circuits in the spinal cord are the gateway for the sensory processing of the symptoms and critical for the associated leg movements that relieve the symptoms and the periodic limb movements that often develop during sleep. There is a high preponderance of the inhibitory D3 receptor in the sensory-processing areas of the spinal cord (dorsal horn), whereas the motor areas in the ventral horn more strongly express the excitatory D1 receptor subtype. D3 and D1 receptors can form functional heteromeric ensembles that influence each other. In the spinal cord, long-term treatment with D3 receptor agonists is associated with the upregulation of the D1 receptor subtype and block of D1 receptor function at this stage can restore the D3 receptor effect. Alternate scenarios for a role of dopamine involve a role for the D5 receptor in regulating motor excitability and for the D4 receptor subtype in controlling D3-like effects. A model emerges that proposes that the behavioral changes in RLS, while responsive to D3 receptor agonists, may be ultimately be the result of unmasked increased D1-like receptor activities.

Adenosine A1-Dopamine D1 Receptor Heteromers Control the Excitability of the Spinal Motoneuron.

While the role of the ascending dopaminergic system in brain function and dysfunction has been a subject of extensive research, the role of the descending dopaminergic system in spinal cord function and dysfunction is just beginning to be understood. Adenosine plays a key role in the inhibitory control of the ascending dopaminergic system, largely dependent on functional complexes of specific subtypes of adenosine and dopamine receptors. Combining a selective destabilizing peptide strategy with a proximity ligation assay and patch-clamp electrophysiology in slices from male mouse lumbar spinal cord, the present study demonstrates the existence of adenosine A1-dopamine D1 receptor heteromers in the spinal motoneuron by which adenosine tonically inhibits D1 receptor-mediated signaling. A1-D1 receptor heteromers play a significant control of the motoneuron excitability, represent main targets for the excitatory effects of caffeine in the spinal cord and can constitute new targets for the pharmacological therapy after spinal cord injury, motor aging-associated disorders and restless legs syndrome.

Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is often and successfully treated with dopamine receptor agonists that target the inhibitory D3 receptor subtype, however there is no clinical evidence of a D3 receptor dysfunction in RLS patients. In contrast, genome-wide association studies in RLS patients have established that a mutation of the MEIS1 gene is associated with an increased risk in developing RLS, but the effect of MEIS1 dysfunction on sensorimotor function remain unknown. Mouse models for a dysfunctional D3 receptor (D3KO) and Meis1 (Meis1KO) were developed independently, and each animal expresses some features associated with RLS in the clinic, but they have not been compared in their responsiveness to treatment options used in the clinic. We here confirm that D3KO and Meis1KO animals show increased locomotor activities, but that only D3KO show an increased sensory excitability to thermal stimuli. Next we compared the effects of dopaminergics and opioids in both animal models, and we assessed D1 and D3 dopamine receptor expression in the spinal cord, the gateway for sensorimotor processing. We found that Meis1KO share most of the tested behavioral properties with their wild type (WT) controls, including the modulation of the thermal pain withdrawal reflex by morphine, L-DOPA and D3 receptor (D3R) agonists and antagonists. However, Meis1KO and D3KO were behaviorally more similar to each other than to WT when tested with D1 receptor (D1R) agonists and antagonists. Subsequent Western blot analyses of D1R and D3R protein expression in the spinal cord revealed a significant increase in D1R but not D3R expression in Meis1KO and D3KO over WT controls. As the D3R is mostly present in the dorsal spinal cord where it has been shown to modulate sensory pathways, while activation of the D1Rs can activate motoneurons in the ventral spinal cord, we speculate that D3KO and Meis1KO represent two complementary animal models for RLS, in which the mechanisms of sensory (D3R-mediated) and motor (D1R-mediated) dysfunctions can be differentially explored.

Comorbidities, treatment, and pathophysiology in restless legs syndrome.

Restless legs syndrome, also known as Willis-Ekbom disease, is a common neurological condition whose manifestation is affected by complex environmental and genetic interactions. Restless legs syndrome can occur on its own, mostly at a young age, or with comorbidities such as cardiovascular disease, diabetes, and arterial hypertension, making it a difficult condition to properly diagnose. However, the concept of restless legs syndrome as being two entities, primary or secondary to another condition, has been challenged with genetic data providing further insight into the pathophysiology of the condition. Although dopaminergic treatment was formerly the first-line therapy, prolonged use can result in a serious worsening of symptoms known as augmentation. Clinical studies on pregabalin, gabapentin enacarbil, oxycodone-naloxone, and iron preparations have provided new treatment options, but most patients still report inadequate long-term management of symptoms. Studies of the hypoxic pathway activation and iron deficiency have provided valuable information about the pathophysiology of restless legs syndrome that should now be translated into new, more effective treatments for restless legs syndrome.

The value of endoscope assistance during transcranial surgery for tuberculum sellae meningiomas.

OBJECTIVE In tuberculum sellae meningioma (TSM) surgery, endonasal approaches are claimed to have a superior visual outcome compared with transcranial approaches. The authors question whether this is always true and analyze their series of cases of endoscope-assisted transcranial TSM surgery with special regard to the postoperative visual outcome in order to explore this issue. METHODS All surgical procedures for TSM performed between 2003 and 2015 in the Department of Neurosurgery, University Medicine Greifswald, were retrospectively analyzed. Special attention was paid to the postoperative visual outcome. RESULTS During the study period, 15 patients (12 female and 3 male) underwent surgery for TSM. Gross-total resection was achieved in 14 cases (93.3%) and near-total resection in 1. One patient suffered from a major stroke during surgery and had to be excluded from further analyses. No other complications occurred. Preoperatively, visual acuity was disturbed in 12 patients (80%) and visual field deficits were present in 11 patients (73.3%). In 3 patients (20%), the TSM was an incidental finding. Postoperatively, ophthalmological examination revealed an improvement of visual acuity in 10 (90.9%) of 11 patients and improvement of visual field deficits in 9 (90%) of 10 patients; no deterioration of visual acuity or visual field was seen in any patient. Visual acuity and visual field improvement was observed in all patients who had surgery within 3 years after the onset of visual disturbances. No tumor recurrence was observed during follow-up (mean 32 months, range 3-134 months). TSMs were approached via a frontolateral craniotomy in 7 patients and via a supraorbital craniotomy in 8. The use of the endoscope as an assistive device led to improved tumor visualization and consequent removal in areas that were hidden in the microscopic view in 6 patients (40%). CONCLUSIONS The present series confirms a favorable visual outcome after TSM surgery via supraorbital or frontolateral endoscope-assisted approaches. With endoscopic visualization, major manipulation of the optic apparatus could be avoided, perhaps affecting the favorable visual outcome.

Long-term treatment with dopamine D3 receptor agonists induces a behavioral switch that can be rescued by blocking the dopamine D1 receptor.

Restless legs syndrome (RLS) is commonly treated with the dopamine agonists pramipexole, and rotigotine, which target the inhibitory dopamine receptor subtype D3R. While initially highly effective, these compounds lose their efficacy in treating RLS over time, and long-term therapy regularly leads to a worsening of the symptoms (augmentation). This dopamine agonist-induced augmentation has become a prime concern in the treatment of RLS, and while alternate therapies are being proposed, the mechanisms leading to augmentation remain opaque. Evidence suggests that the prolonged D3R treatment may lead to a hyper-dopaminergic state and involve the excitatory dopamine D1 receptor (D1R) subtype. We here present an animal model in which we can test acute and long-term effects of dopamine D3R agonists in a behavior setting relevant to RLS and in which we can induce a switch of the drug effect similar to the one observed in RLS patients under chronic therapy. We also present evidence that we can reverse this long-term effect by blocking the D1R. Together, data from our new animal model indicate that the mechanisms leading to augmentation in RLS patients after long-term use of the currently used dopamine receptor agonists may be related to a D3R-induced upregulation of the D1R system. As such, our model can be used to assess the interactions between D3R and D1R and unravel the mechanisms that lead to augmentation, and it has the potential to serve as a Launchpad for the development of new pharmacological strategies for the treatment of both RLS and augmentation.

Morphine responsiveness to thermal pain stimuli is aging-associated and mediated by dopamine D1 and D3 receptor interactions.

Morphine actions involve the dopamine (DA) D1 and D3 receptor systems (D1R and D3R), and the responses to morphine change with age. We here explored in differently aged wild-type (WT) and D3R knockout mice (D3KO) the interactions of the D1R/D3R systems with morphine in vivo at three different times of the animals' lifespan (2months, 1year, and 2years). We found that: (1) thermal pain withdrawal reflexes follow an aging-associated phenotype, with relatively longer latencies at 2months and shorter latencies at 1year, (2) over the same age range, a dysfunction of the D3R subtype decreases reflex latencies more than aging alone, (3) morphine altered reflex responses in a dose-dependent manner in WT animals and changed at its higher dose the phenotype of the D3KO animals from a morphine-resistant state to a morphine-responsive state, (4) block of D1R function had an aging-dependent effect on thermal withdrawal latencies in control animals that, in old animals, was stronger than that of low-dose morphine. Lastly, (5) block of D1R function in young D3KO animals mimicked the behavioral phenotype observed in the aged WT. Our proof-of-concept data from the rodent animal model suggest that, with age, block of D1R function may be considered as an alternative to the use of morphine, to modulate the response to painful stimuli.

Connectome and molecular pharmacological differences in the dopaminergic system in restless legs syndrome (RLS): plastic changes and neuroadaptations that may contribute to augmentation.

Restless legs syndrome (RLS) is primarily treated with levodopa and dopaminergics that target the inhibitory dopamine receptor subtypes D3 and D2. The initial success of this therapy led to the idea of a hypodopaminergic state as the mechanism underlying RLS. However, multiple lines of evidence suggest that this simplified concept of a reduced dopamine function as the basis of RLS is incomplete. Moreover, long-term medication with the D2/D3 agonists leads to a reversal of the initial benefits of dopamine agonists and augmentation, which is a worsening of symptoms under therapy. The recent findings on the state of the dopamine system in RLS that support the notion that a dysfunction in the dopamine system may in fact induce a hyperdopaminergic state are summarized. On the basis of these data, the concept of a dynamic nature of the dopamine effects in a circadian context is presented. The possible interactions of cell adhesion molecules expressed by the dopaminergic systems and their possible effects on RLS and augmentation are discussed. Genome-wide association studies (GWAS) indicate a significantly increased risk for RLS in populations with genomic variants of the cell adhesion molecule receptor type protein tyrosine phosphatase D (PTPRD), and PTPRD is abundantly expressed by dopamine neurons. PTPRD may play a role in the reconfiguration of neural circuits, including shaping the interplay of G protein-coupled receptor (GPCR) homomers and heteromers that mediate dopaminergic modulation. Recent animal model data support the concept that interactions between functionally distinct dopamine receptor subtypes can reshape behavioral outcomes and change with normal aging. Additionally, long-term activation of one dopamine receptor subtype can increase the receptor expression of a different receptor subtype with opposite modulatory actions. Such dopamine receptor interactions at both spinal and supraspinal levels appear to play important roles in RLS. In addition, these interactions can extend to the adenosine A1 and A2A receptors, which are also prominently expressed in the striatum. Interactions between adenosine and dopamine receptors and dopaminergic cell adhesion molecules, including PTPRD, may provide new pharmacological targets for treating RLS. In summary, new treatment options for RLS that include recovery from augmentation will have to consider dynamic changes in the dopamine system that occur during the circadian cycle, plastic changes that can develop as a function of treatment or with aging, changes in the connectome based on alterations in cell adhesion molecules, and receptor interactions that may extend beyond the dopamine system itself.