D1 Receptor Activation Research Articles

PHARMACOLOGICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF DOPAMINE D4 RECEPTOR IN HUMAN UMBILICAL ARTERY AND VEIN.

Aims - To investigate the effects of the dopamine D4 receptor agonist A-412997 and the D4 antagonist sonepiprazole in human isolated umbilical artery (HUA) and vein (HUV) and the expression of the D4 receptor by immunohistochemistry in these vessels. Main methods - A segment of the umbilical cord (10-20 cm) from the insertion point in the placenta and 5 cm from the umbilicus was removed by the obstetrician and placed in a container with Krebs-Henseleit's solution (KHS). The Wharton's jelly was removed and HUA and HUV and rings (3-mm) of HUA and HUV were suspended in 10-mL organ baths containing oxygenated (95%O2:5%CO2) and heated (37°C) KHS. For immunohistochemistry, the vessels were fixed in 10% formalin, embedded in paraffin wax and sectioned (4µm). Key findings - A-412997 did not induce contractions in the HUA rings. In HUA pre-contracted rings, A-412997 induced concentration-dependent relaxations, which were reduced when the HUA rings were pre-incubated with L-NAME. A-412997 caused concentration-dependent contractions of HUV rings, which were potentiated by pre-treatment with L-NAME, and reduced by pre-incubation with 6-nitrodopamine. In HUV pre-contracted rings, A-412997 failed to induce relaxations. Sonepiprazole antagonized A-412997 induced contractions in HUV rings and provoked concentration-dependent relaxations in pre-contracted HUA and HUV rings. Dopamine receptor D4 was positive in both HUA and HUV, especially in the endothelium, and detected only in HUV smooth muscle cells. Significance - Activation of HUA D4 receptor is associated with relaxation, whereas in HUV, it leads to contraction. Differential expression of D4 receptors may modulate umbilical-placental blood flow.

Overcoming boundary conditions for object location memory destabilization in male rats involves dopamine D1 receptor activation.

Consolidated long-term memories can undergo strength or content modification via protein synthesis-dependent reconsolidation. This is the process by which a reminder cue initiates reactivation of the memory trace, triggering destabilization. Older and more strongly encoded spatial memories can resist destabilization due to biological boundary conditions. The present study investigated the role of dopamine (DA) at D1 receptors (D1Rs) in object location memory destabilization and overcoming boundary conditions for older ("remote"; tested with a 48-h rather than a 24-h delay between sample and reactivation) memory destabilization. Using male rats in a modified object location task, we found that administering the D1R antagonist SCH23390 (0.1mg/kg, i.p.) prior to reactivation blocked destabilization of recently encoded memories, as well as novelty-induced destabilization of remote memories. Using remote parameters, systemically administered D1R agonist SKF38393 (5mg/kg, i.p.) induced destabilization of remote object location memories in the absence of salient novelty. Intra-dorsal hippocampus administration of SCH23390 (2μg/μL) also blocked destabilization of remote object location memories when a salient novel cue was present. These results are consistent with previous findings implicating DA in memory destabilization as well as demonstrate a role for D1-receptor activation in the destabilization of boundary condition protected-object location memories.

Dopamine D1 receptor activation in the striatum is sufficient to drive reinforcement of anteceding cortical patterns.

Timed dopamine signals underlie reinforcement learning, favoring neural activity patterns that drive behaviors with positive outcomes. In the striatum, dopamine activates five dopamine receptors (D1R-D5R), which are differentially expressed in striatal neurons. However, the role of specific dopamine receptors in reinforcement is poorly understood. Using our cell-specific D1R photo-agonist, we find that D1R activation in D1-expressing neurons in the dorsomedial striatum is sufficient to reinforce preceding neural firing patterns in defined ensembles of layer 5 cortico-striatal neurons of the mouse motor cortex. The reinforcement is cumulative and time dependent, with an optimal effect when D1R activation follows the selected neural pattern after a short interval. Our results show that D1R activation in striatal neurons can selectively reinforce cortical activity patterns, independent of a behavioral outcome or a reward, crucially contributing to the fundamental mechanisms that support cognitive functions like learning, memory, and decision-making.

Dopamine receptors D1, D2, and D4 modulate electrical synapses and excitability in the thalamic reticular nucleus.

The thalamic reticular nucleus (TRN) is a thin shell of gap junction coupled GABAergic inhibitory neurons that regulate afferent sensory relay of the thalamus. The TRN receives dopaminergic innervation from the midbrain, and it is known to express high concentrations of D1 and D4 receptors. Although dopaminergic modulation of presynaptic inputs to TRN has been described, the direct effect of dopamine on TRN neurons and its electrical synapses is largely unknown. Here, we confirmed D1 and D4 expression and show that D2 receptors are also expressed in TRN. To characterize how dopamine affects both neuronal excitability and electrical synapse coupling strength in the TRN, we performed dual whole-cell patch-clamp recordings of TRN neurons and injected them with 500-ms current pulses to measure input resistance, rheobase, spiking frequency, and coupling conductance. Measurements were taken before and after bath application of dopamine or agonists for either D1, D2, or D4 receptors. Our results show that bath application of dopamine did not consistently modulate excitability or electrical synapse strength. However, application of specific dopamine receptor agonists revealed that activation of D1 and D4 receptors increase input resistance, and activation of D2-like receptors lower maximum tonic spike rate. Notably, D2 and D4 receptors depressed electrical synapses. Together, our results suggest that coactivation of D1, D2, and D4 receptors may result in crosstalk due to opposing signaling cascades. Further, we show that selective dopamine receptor engagement has substantial potential to modulate TRN circuitry.

Dopamine D4 receptors in the lateral habenula regulate anxiety-related behaviors in a rat model of Parkinson's disease

Although the output of the lateral habenula (LHb) controls the activity of midbrain dopamine (DA) and 5-hydroxytryptamine (5-HT) containing systems, which are implicated in the pathophysiology of anxiety, it is not clear how activation and blockade of LHb D4 receptors affects anxiety-like behaviors, particularly in Parkinson’s disease related anxiety. In this study, unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) in rats induced anxiety-like behaviors, which attribute to hyperactivity of LHb neurons and decrease in the level of DA in the medial prefrontal cortex (mPFC), amygdala and ventral hippocampus (vHip) compared to sham-operated rats. Intra-LHb injection of D4 receptor agonist A412997 induced or increased the expression of anxiety-like behaviors, while injection of D4 receptor antagonist L741742 showed anxiolytic effects in sham-operated and the SNc-lesioned rats. However, the doses producing behavioral effects in the lesioned rats were higher than those of sham-operated rats. Intra-LHb injection of A412997 increased firing rate of LHb neurons, and decreased levels of DA and 5-HT in the mPFC, amygdala and vHip; conversely, L741742 decreased firing rate of LHb neurons, and increased levels of DA and 5-HT in two groups of rats. Compared to sham-operated rats, the duration of A412997 and L741742 action on the firing rate of neurons was markedly shortened in the lesioned rats. Collectively, these findings suggest that D4 receptors in the LHb are involved in the regulation of anxiety-like behaviors, and degeneration of the nigrostriatal pathway down-regulates function and/or expression of these receptors.

Decrease of KATP channel expression through D3 receptor-mediated GSK3β signaling alleviates levodopa-induced dyskinesia (LID) in Parkinson's disease mouse model

AimsThe standard Parkinson's disease (PD) treatment is L-3,4-dihydroxyphenylalanine (L-DOPA); however, its long-term use may cause L-DOPA-induced dyskinesia (LID). Aberrant activation of medium spiny neurons (MSNs) contributes to LID, and MSN excitability is regulated by dopamine D3 receptor (D3R) and ATP-sensitive potassium (KATP) channel activity. Nevertheless, it remains unclear if D3R and KATP channels may be linked in the context of LID. MethodsWild-type and tyrosine hydroxylase (TH)-specific Kir6.2 knockout mice were injected with 6-hydroxydopamine (6-OHDA) to generate a PD mouse model, then chronically treated with L-DOPA to induce LID. Analyses included immunohistochemical staining, biochemical endpoints, and behavior tests. The mechanisms by which D3R/KATP channels regulate LID in the PD/LID mouse model were probed by treatment with a D3R antagonist, KATP channel opener and glycogen synthase kinase 3β (GSK3β) inhibitor, followed by evaluation of abnormal involuntary movements (AIMs). Key findingsThe D3R antagonist FAUC365 alleviated LID, reducing AIMs and protecting against degeneration of the nigrostriatal pathway, which occurred through a direct interaction between D3Rs and KATP channels. In line with this mechanism, activation of D3R/GSK3β signaling increased KATP channel expression in the striatum of PD/LID mice. Additionally, the KATP channel opener Diz slowed LID progression and preserved nigrostriatal projections. Consistently, mice with TH-specific knockout of Kir6.2 exhibited reduced PD-like symptoms and less severe LID. SignificanceD3Rs act through GSK3β signaling to regulate expression of KATP channels, which may subsequently modulate LID. Inhibition of KATP channels in TH-positive cells is sufficient to reduce AIMs in a mouse model of PD/LID.

Striatal cholinergic transmission in an inducible transgenic mouse model of paroxysmal non-kinesiogenic dyskinesia

Altered interaction between striatonigral dopaminergic (DA) inputs and local acetylcholine (ACh) in striatum has long been hypothesized to play a central role in the pathophysiology of dystonia and dyskinesia. Indeed, previous research using several genetic mouse models of human isolated dystonia identified a shared endophenotype with paradoxical excitation of striatal cholinergic interneuron (ChIs) activity in response to activation of dopamine D2 receptors (D2R). These mouse models lack a dystonic motor phenotype, which leaves a critical gap in comprehending the role of DA and ACh transmission in the manifestations of dystonia. To tackle this question, we used a combination of ex vivo slice physiology and in vivo monitoring of striatal ACh dynamics in the inducible, phenotypically penetrant, transgenic mouse model of paroxysmal non-kinesiogenic dyskinesia (PNKD), an animal with both dystonic and dyskinetic features. We found that, similarly to genetic models of isolated dystonia, the PNKD mouse displays D2R-induced paradoxical excitation of ChI firing in ex vivo striatal brain slices. In vivo, caffeine triggers dystonic symptoms while reversing the D2R-mediated excitation of ChIs and desynchronizing ACh release in PNKD mice. In WT littermate controls, caffeine stimulates spontaneous locomotion through a similar but reversed mechanism involving an excitatory switch of the D2R control of ChI activity, associated with enhanced synchronization of ACh release. These observations suggest that the “paradoxical excitation” of cholinergic interneurons described in isolated dystonia models could represent a compensatory or protective mechanism that prevents manifestation of movement abnormalities and that phenotypic dystonia is possible only when this is absent. These findings also suggest that D2Rs may play an important role in synchronizing the ChI network leading to rhythmic ACh release during heightened movement states. Dysfunction of this interaction and corresponding desynchrony of ACh release may contribute to aberrant movements.

Dopamine D1 receptors activation rescues hippocampal synaptic plasticity and cognitive impairments in the MK-801 neonatal schizophrenia model

Schizophrenia is a disorder with a higher cognitive decline in early adulthood, causing impaired retention of episodic memories. However, the physiological and behavioral functions that underlie cognitive deficits with a potential mechanism to ameliorate and improve cognitive performance are unknown. In this study, we used the MK-801 neurodevelopmental schizophrenia-like model. Rats were divided into two groups: one received MK-801, and the other received saline for five consecutive days (7–11 postnatal days, PND). We evaluated synaptic plasticity late-LTP and spatial memory consolidation in early adolescence and young adulthood using extracellular field recordings in acute hippocampal slices and the Barnes maze task. Next, we examined D1 receptor (D1R) activation as a mechanism to ameliorate cognitive impairments. Our results suggest that MK-801 neonatal treatment induces impairment in late-LTP expression and deficits in spatial memory retrieval in early adolescence that is maintained until young adulthood. Furthermore, we found that activation of dopamine D1R ameliorates the impairments and promotes a robust expression of late-LTP and an improved performance in the Barnes maze task, suggesting a novel and potential therapeutic role in treating cognitive impairments in schizophrenia.

The activation of D2-like dopamine receptors increases NMDA currents in the dorsal raphe serotonergic neurons

The dorsal raphe nucleus (DRN) receives dopaminergic inputs from the ventral tegmental area (VTA). Also, the DRN contains a small population of cells that express dopamine (DRNDA neurons). However, the physiological role of dopamine (DA) in the DRN and its interaction with serotonergic (5-HT) neurons is poorly understood. Several works have reported moderate levels of D1, D2, and D3 DA receptors in the DRN. Furthermore, it was found that the activation of D2 receptors increased the firing of putative 5-HT neurons. Other studies have reported that D1 and D2 dopamine receptors can interact with glutamate NMDA receptors, modulating the excitability of different cell types. In the present work, we used immunocytochemical techniques to determine the kind of DA receptors in the DRN. Additionally, we performed electrophysiological experiments in brainstem slices to study the effect of DA agonists on NMDA-elicited currents recorded from identified 5-HT DRN neurons. We found that D2 and D3 but not D1 receptors are present in this nucleus. Also, we demonstrated that the activation of D2-like receptors increases NMDA-elicited currents in 5-HT neurons through a mechanism involving phospholipase C (PLC) and protein kinase C (PKC) enzymes. Possible physiological implications related to the sleep-wake cycle are discussed.

Astrocyte D1/D5 Dopamine Receptors Govern Non-Hebbian Long-Term Potentiation at Sensory Synapses onto Lamina I Spinoparabrachial Neurons.

Recent work demonstrated that activation of spinal D1 and D5 dopamine receptors (D1/D5Rs) facilitates non-Hebbian long-term potentiation (LTP) at primary afferent synapses onto spinal projection neurons. However, the cellular localization of the D1/D5Rs driving non-Hebbian LTP in spinal nociceptive circuits remains unknown, and it is also unclear whether D1/D5R signaling must occur concurrently with sensory input in order to promote non-Hebbian LTP at these synapses. Here we investigate these issues using cell-type-selective knockdown of D1Rs or D5Rs from lamina I spinoparabrachial neurons, dorsal root ganglion (DRG) neurons, or astrocytes in adult mice of either sex using Cre recombinase-based genetic strategies. The LTP evoked by low-frequency stimulation of primary afferents in the presence of the selective D1/D5R agonist SKF82958 persisted following the knockdown of D1R or D5R in spinoparabrachial neurons, suggesting that postsynaptic D1/D5R signaling was dispensable for non-Hebbian plasticity at sensory synapses onto these key output neurons of the superficial dorsal horn (SDH). Similarly, the knockdown of D1Rs or D5Rs in DRG neurons failed to influence SKF82958-enabled LTP in lamina I projection neurons. In contrast, SKF82958-induced LTP was suppressed by the knockdown of D1R or D5R in spinal astrocytes. Furthermore, the data indicate that the activation of D1R/D5Rs in spinal astrocytes can either retroactively or proactively drive non-Hebbian LTP in spinoparabrachial neurons. Collectively, these results suggest that dopaminergic signaling in astrocytes can strongly promote activity-dependent LTP in the SDH, which is predicted to significantly enhance the amplification of ascending nociceptive transmission from the spinal cord to the brain.

Spatiomolecular Characterization of Dopamine D2 Receptors Cells in the Mouse External Globus Pallidus.

The external globus pallidus (GPe) is part of the basal ganglia circuit and plays a key role in controlling the actions. Although, many evidence indicate that dopamine through its activation of dopamine D2 receptors (D2Rs) modulates the GPe neuronal activity, the precise spatiomolecular characterization of cell populations expressing D2Rs in the mouse GPe is still lacking. By combining single molecule in situ hybridization, cell type-specific imaging analyses, and electrophysiology slice recordings, we found that GPe D2R cells are neurons preferentially localized in the caudal portion of GPe. These neurons comprising pallido-striatal, pallido-nigral, and pallido-cortical neurons segregate into two distinct populations displaying molecular and electrophysiological features of GPe GABAergic PV/NKX2.1 and cholinergic neurons respectively. By clarifying the spatial molecular identity of GPe D2R neurons in the mouse, this work provides the basis for future studies aiming at disentangling the action of dopamine within the GPe.

Revealing a role of brainstem monoaminergic nuclei on the pronociceptive effect of sleep restriction

Sleep disturbances and persistent pain conditions are public health challenges worldwide. Although it is well-known that sleep deficit increases pain sensitivity, the underlying mechanisms remain elusive. We have recently demonstrated the involvement of nucleus accumbens (NAc) and anterior cingulate cortex (ACC) in the pronociceptive effect of sleep restriction. In this study, we found that sleep restriction increases c-Fos expression in NAc and ACC, suggesting hyperactivation of these regions during prolonged wakefulness in male Wistar rats. Blocking adenosine A2A receptors in the NAc or GABAA receptors in the ventral tegmental area (VTA), dorsal raphe nucleus (DRN), or locus coeruleus (LC) effectively mitigated the pronociceptive effect of sleep restriction. In contrast, the blockade of GABAA receptors in each of these nuclei only transiently reduced carrageenan-induced hyperalgesia. Pharmacological activation of dopamine D2, serotonin 5-HT1A and noradrenaline alpha-2 receptors within the ACC also prevented the pronociceptive effect of sleep restriction. While pharmacological inhibition of these same monoaminergic receptors in the ACC restored the pronociceptive effect which had been prevented by the GABAergic disinhibition of the of the VTA, DRN or LC. Overall, these findings suggest that the pronociceptive effect of sleep restriction relies on increased adenosinergic activity on NAc, heightened GABAergic activity in VTA, DRN, and LC, and reduced inhibitory monoaminergic activity on ACC. These findings advance our understanding of the interplay between sleep and pain, shedding light on potential NAc-brainstem-ACC mechanisms that could mediate increased pain sensitivity under conditions of sleep impairment.

Dopamine D2 Receptor Activation Blocks GluA2/ROS Positive Feedback Loop to Alienate Chronic-Migraine-Associated Pain Sensitization.

Chronic migraine is a disabling disorder without effective therapeutic medicine. AMPA receptors have been proven to be essential to pathological pain and headaches, but the related regulatory mechanisms in chronic migraine have not yet been explored. In this study, we found that the level of surface GluA2 was reduced in chronic migraine rats. Tat-GluR23Y (a GluA2 endocytosis inhibitor) reduced calcium inward flow and weakened synaptic structures, thus alleviating migraine-like pain sensitization. In addition, the inhibition of GluA2 endocytosis reduced the calcium influx and alleviated mitochondrial calcium overload and ROS generation in primary neurons. Furthermore, our results showed that ROS can induce allodynia and GluA2 endocytosis in rats, thus promoting migraine-like pain sensitization. In our previous study, the dopamine D2 receptor was identified as a potential target in the treatment of chronic migraine, and here we found that dopamine D2 receptor activation suppressed chronic-migraine-related pain sensitization through blocking the GluA2/ROS positive feedback loop in vivo and in vitro. Additionally, ligustrazine, a core component of ligusticum chuanxiong, was shown to target the dopamine D2 receptor, thereby alleviating ROS production and abnormal nociception in CM rats. This study provides valuable insight into the treatment of chronic migraine.

Antinociceptive role of the thalamic dopamine D3 receptor in descending modulation of intramuscular formalin-induced muscle nociception in a rat model of Parkinson's disease

Pain in Parkinson's disease (PD) has been validated as one of the major non-motor dysfunctions affecting the quality of life and subsequent rehabilitation. In the present study, we investigated the role of the dopamine D3 receptor in the thalamic mediodorsal (MD) and ventromedial (VM) nuclei mediated descending control of nociception and intramuscular (i.m.) 2.5% formalin-induced persistent muscle nociception. Paw withdrawal reflexes were measured in naive rats and rats subjected to PD induced by unilateral microinjection of 6 μg 6-OHDA into the rat striatum. Formalin-induced muscle nociception in phase 1, inter-phase, and phase 2 was significantly greater in PD rats compared to naive and vehicle-treated rats (P < 0.001). PD rats exhibited bilaterally mechanical hyperalgesia and heat hypoalgesia in formalin-induced muscle nociception. Microinjection of SK609, a dopamine D3 receptor agonist, at various doses (2.5–7.5 nmol/0.5 μl) into the thalamic VM nucleus dose-dependently prolonged heat-evoked paw withdrawal latencies in both naive and PD rats. Administration of SK609 to either the MD or VM nuclei had no effect on noxious mechanically evoked paw withdrawal reflexes. Pre-treatment of the thalamic MD nucleus with SK609 significantly attenuated formalin-induced nociception, and reversed mechanical hyperalgesia, but not heat hypoalgesia. Pre-treatment of the thalamic VM nucleus with SK609 inhibited formalin-induced nociception in the late phase of phase 2 (30–75 min) and heat hypoalgesia, but not mechanical hyperalgesia (P < 0.05). It is suggested that the dopamine D3 receptors in the thalamus play an antinociceptive role in the descending modulation of nociception. Activation of D3 receptors within the thalamic MD and VM nuclei attenuates descending facilitation and enhances descending inhibition in rats during PD.

Integrating UPLC-MS/MS with in Silico and in Vitro Screening Accelerates the Discovery of Active Compounds in Stephania epigaea

Traditional Chinese medicines (TCMs) are popular in clinic because of their safety and efficacy. They contain abundant natural active compounds, which are important sources of new drug discovery. However, how to efficiently identify active compounds from complex ingredients remains a challenge. In this study, a method combining UHPLC-MS/MS characterization and in silico screening was developed to discover compounds with dopamine D2 receptor (D2R) activity in Stephania epigaea (S. epigaea). By combining the compounds identified in S. epigaea by UHPLC-MS/MS with reported compounds, a virtual library of 80 compounds was constructed for in silico screening. Potentially active compounds were chosen based on screening scores and subsequently tested for in vitro activity on a transfected cell line CHO-K1-D2 model using label-free cellular phenotypic assay. Three D2R agonists and five D2R antagonists were identified. (-)-Asimilobine, N-nornuciferine and (-)-roemerine were reported for the first time as D2R agonists, with EC50 values of 0.35 ± 0.04 μM, 1.37 ± 0.10 μM and 0.82 ± 0.22 μM, respectively. Their target specificity was validated by desensitization and antagonism assay. (-)-Isocorypalmine, (-)-tetrahydropalmatine, (-)-discretine, (+)-corydaline and (-)-roemeroline showed strong antagonistic activity on D2R with IC50 values of 92 ± 9.9 nM, 1.73 ± 0.13 μM, 0.34 ± 0.02 μM, 2.09 ± 0.22 μM and 0.85 ± 0.08 μM, respectively. Their kinetic binding profiles were characterized using co-stimulation assay and they were both D2R competitive antagonists. We docked these ligands with human D2R crystal structure and analyzed the structure-activity relationship of aporphine-type D2R agonists and protoberberine-type D2R antagonists. These results would help to elucidate the mechanism of action of S. epigaea for its analgesic and sedative efficacy and benefit for D2R drug design. This study demonstrated the potential of integrating UHPLC-MS/MS with in silico and in vitro screening for accelerating the discovery of active compounds from TCMs.

Effects of the Synthetic Cathinone α-Pyrrolidinobutiothiophenone (α-PBT) on Discriminative Stimulus Effects and Intracranial Self-Stimulation Thresholds in Male Rats.

Recently, the abuse of synthetic cathinones is increasing among young people. α-Pyrrolidinobutiothiophenone (α-PBT), a synthetic cathinone, is a designer drug that is freely traded online with no legal restrictions. Moreover, there is currently no scientific basis for legal regulation. Here, we examined the addictive properties of α-PBT using a drug discrimination (DD) task. We also investigated the role of α-PBT in brain stimulation reward (BSR) using an intracranial self-stimulation (ICSS) paradigm in rats. Initially, the rats were trained to discriminate between cocaine and saline. After the discrimination training criteria were met, we determined the dose-effect curves of cocaine and conducted generalization tests with α-PBT and α-pyrrolidinopentiothiophenone (α-PVT) using a cumulative dosing protocol. In a separate set of studies, we examined the dopaminergic mechanisms underlying the function of α-PBT as an interoceptive stimulus (17.8 mg/kg) by intraperitoneally injecting either the dopamine (DA) D1 antagonist SCH23390 (0.06 and 0.12 mg/kg) or the D2 antagonist eticlopride (0.05 and 0.1 mg/kg) 15 min before DD testing. Brain reward function was measured using an ICSS procedure to examine the effects of α-PBT on ICSS threshold under the frequency-rate procedure. Our results showed that α-PBT functioned as a discriminative cue similar to cocaine in rats. More importantly, SCH23390 abolished the effects of α-PBT as an interoceptive stimulus in a dose-dependent manner in rats trained to press a lever to receive cocaine. Similarly, eticlopride dose-dependently attenuated the effect of α-PBT used as a discriminative cue. Additionally, cumulative α-PBT administration dose-dependently lowered ICSS thresholds compared with those in saline-treated rats. Furthermore, α-PBT-induced potentiation of BSR was abolished by pretreatment with both SCH23390 and eticlopride. Taken together, our results suggest that α-PBT can function as a cocaine-like discriminative cue via the activation of D1 and D2 receptors. α-PBT also appears to influence BSR by reducing the brain reward threshold via changes in D1 and D2 receptors. The present study suggests that α-PBT could have addictive properties through DA D1 and D2 receptors and thus poses a threat to humans.

Dopamine-mediated plasticity preserves excitatory connections to direct pathway striatal projection neurons and motor function in a mouse model of Parkinson's disease.

The cardinal symptoms of Parkinson's disease (PD) such as bradykinesia and akinesia are debilitating, and treatment options remain inadequate. The loss of nigrostriatal dopamine neurons in PD produces motor symptoms by shifting the balance of striatal output from the direct (go) to indirect (no-go) pathway in large part through changes in the excitatory connections and intrinsic excitabilities of the striatal projection neurons (SPNs). Here, we report using two different experimental models that a transient increase in striatal dopamine and enhanced D1 receptor activation, during 6-OHDA dopamine depletion, prevent the loss of mature spines and dendritic arbors on direct pathway projection neurons (dSPNs) and normal motor behavior for up to 5 months. The primary motor cortex and midline thalamic nuclei provide the major excitatory connections to SPNs. Using ChR2-assisted circuit mapping to measure inputs from motor cortex M1 to dorsolateral dSPNs, we observed a dramatic reduction in both experimental model mice and controls following dopamine depletion. Changes in the intrinsic excitabilities of SPNs were also similar to controls following dopamine depletion. Future work will examine thalamic connections to dSPNs. The findings reported here reveal previously unappreciated plasticity mechanisms within the basal ganglia that can be leveraged to treat the motor symptoms of PD.

Enhancing Osteogenic Potential: Controlled Release of Dopamine D1 Receptor Agonist SKF38393 Compared to Free Administration.

Osteoporosis is the most common metabolic bone disorder and is characterized by decreased bone density, which has a relationship with the quality of life among the aging population. Previous research has found that activation of the dopamine D1 receptor can improve bone mass formation. SKF38393 is an agonist of dopamine D1 receptors. However, as a small-molecule drug, SKF38393 is unstable and releases quickly. The aim of this study was to prototype polylactic-co-glycolic acid (PLGA)/SKF38393 microspheres and assess their potential osteogenic effects compared to those under the free administration of SKF38393. The cytocompatibility of PLGA/SKF38393 was determined via CCK-8 and live/dead cell staining; the osteogenic effects in vitro were determined with ALP and alizarin red staining, qRT-PCR, and Western blotting; and the in vivo effects were assessed using 25 Balb/c mice. We also used a PCR array to explore the possible signaling pathway changes after employing PLGA/SKF38393. Our experiments demonstrated that the osteogenic effect of D1Rs activated by the PLGA/SKF38393 microsphere was better than that under free administration, both in vitro and in vivo. According to the PCR array, this result might be associated with six signaling pathways (graphical abstract). Ultimately, in this study, we prototyped PLGA/SKF38393, demonstrated its effectiveness, and preliminarily analyzed its mechanism of action.

Dopamine D2 Receptor Modulates Exercise Related Effect on Cortical Excitation/Inhibition and Motor Skill Acquisition.

Exercise is known to benefit motor skill learning in health and neurological disease. Evidence from brain stimulation, genotyping, and Parkinson's disease studies converge to suggest that the dopamine D2 receptor, and shifts in the cortical excitation and inhibition (E:I) balance, are prime candidates for the drivers of exercise-enhanced motor learning. However, causal evidence using experimental pharmacological challenge is lacking. We hypothesized that the modulatory effect of the dopamine D2 receptor on exercise-induced changes in the E:I balance would determine the magnitude of motor skill acquisition. To test this, we measured exercise-induced changes in excitation and inhibition using paired-pulse transcranial magnetic stimulation (TMS) in 22 healthy female and male humans, and then had participants learn a novel motor skill-the sequential visual isometric pinch task (SVIPT). We examined the effect of D2 receptor blockade (800 mg sulpiride) on these measures within a randomized, double-blind, placebo-controlled design. Our key result was that motor skill acquisition was driven by an interaction between the D2 receptor and E:I balance. Specifically, poorer skill learning was related to an attenuated shift in the E:I balance in the sulpiride condition, whereas this interaction was not evident in placebo. Our results demonstrate that exercise-primed motor skill acquisition is causally influenced by D2 receptor activity on motor cortical circuits.

Abstract 533: Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models

Abstract Triple Negative breast cancer (TNBC) accounts for 10-15% of all breast cancers where patients encounter poor clinical outcomes. Treatment modalities for TNBC are conventional cytotoxic chemotherapy and radiation leads to 35%-40% relapse within 5 years of diagnosis. There is a growing need for more effective targeted therapeutics to inhibit TNBC growth. The role of EZH2 in maintaining tumor growth and metastasis in TNBC is well documented. EZH2 promotes breast tumor-initiating cell expansion (BITC) and cancer progression by impairing the DNA damage repair process favoring activation of oncogenes. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. Here we hypothesize that the presence of dopamine D1 receptor agonist (A77636) enhances the efficacy of EZH2 inhibitors (GSK126) to inhibit in vitro TNBC tumor growth and metastasis. To test the efficacy of the combination we employed a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. We also employed a 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor) to study the effect of the drug combination on metastasis in vitro. The SynTumor MPS devices replicate the pathophysiological architecture of native vascularized breast tumors. The combination treatment enhanced a significant reduction of tumor spheroid diameter compared to the vehicle and indicated spheroid regression over treatment. Furthermore, the combination therapy induced necrosis and caused complete inhibition of EZH2 expression. The knockdown of EZH2 inhibited tumor spheroid formation and displayed cytoskeleton de-arrangement. In the microfluidic SynTumor model, circulating tumor cell numbers were reduced by half at 96 hrs after EZH2 combination treatment. Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated in vitro tumor growth and metastasis (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: xilal Rima, Gautam Sarathy, Chunyu Hu, Divya S. Patel, Deborah Ramsey, Lara Rizotto, Dario Palmeri, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegu, Eswar Shankar. Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 533.