Lena Scott

Selective up-regulation of dopamine D1 receptors in dendritic spines by NMDA receptor activation

Glutamate, by activating N-methyl-d-aspartate (NMDA) receptors, alters the balance between dopamine D1 and D2 receptor signaling, but the mechanism responsible for this effect has not been known. We report here, using immunocytochemistry of primary cultures of rat neostriatal neurons, that activation of NMDA receptors recruits D1 receptors from the interior of the cell to the plasma membrane while having no effect on the distribution of D2 receptors. The D1 receptors were concentrated in spines as shown by colocalization with phalloidin-labeled actin filaments. The effect of NMDA on D1 receptors was abolished by incubation of cells in calcium-free medium and was mimicked by the calcium ionophore ionomycin. Recruitment of D1 receptors from the interior of the cell to the membrane was confirmed by subcellular fractionation. The recruited D1 receptors were functional as demonstrated by an increase in dopamine-sensitive adenylyl cyclase activity in membranes derived from cells that had been pretreated with NMDA. These results provide evidence for regulated recruitment of a G protein-coupled receptor in neurons, provide a cell biological basis for the effect of NMDA on dopamine signaling, and reconcile the conflicting hyperdopaminergic and hypoglutamatergic hypotheses of schizophrenia.

Spatial distribution of Na + -K + -ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy

BackgroundThe Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons.ResultsWith help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.ConclusionsA compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

Negative Reciprocity between Angiotensin II type 1 and Dopamine D1 receptors in rat renal proximal tubule cells

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling. Similarly, exposure of rat proximal tubule cells and renal tissue to angiotensin II resulted in a rapid partial internalization of D1R and abolishment of D1R signaling. D1R and AT1R were, by use of coimmunoprecipitation studies and glutathione-S-transferase pull-down assays, shown to be partners in a multiprotein complex. Na+-K+-ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of D1R (residues 397-416) was found to interact with both AT1R and Na+-K+-ATPase. Results indicate that AT1R and D1R function as a unit of opposites, which should provide a highly versatile and sensitive system for short-term regulation of sodium excretion.

Interaction between N-methyl-d-aspartic acid receptors and D1 dopamine receptors: An important mechanism for brain plasticity

Dopamine and glutamate may be the most extensively studied neurotransmitters in the brain, and single components of their signaling pathways have been well characterized. In recent years integration of the dopamine and glutamate signaling pathways has received increasing attention. This research has been fueled by the fact that many psychiatric conditions, including schizophrenia, seem to be due to imbalances in both the glutamatergic and the dopaminergic system, and that many addictive drugs seem to affect both systems. Thus more knowledge about the interaction between the glutamatergic and dopaminergic systems will have important implications for the generation of new treatment for psychiatric disorders. This review will focus on the intraneuronal interaction between the glutamate and dopamine systems.

Developmental changes in frequency of the ciliary somatostatin receptor 3 protein

Primary cilia extend from the surface of most vertebrate cells and display several signaling molecules, including the somatostatin receptor 3 (SSTR3), enabling cilia to play essential roles as chemical, osmotic and mechanical sensors. The SSTR3 is widely distributed in the adult rat brain, and also influences cell proliferation and apoptosis. To establish whether the SSTR3 is positioned to influence these developmental processes, we examined, using immunohistochemistry, the embryonic and postnatal development of SSTR3 expression in the rat hippocampal formation, and its association with newly born and mature neurons in adult rats. Elongated SSTR3-immunoreactive (-ir) cilia first appeared in the hippocampal formation CA3 region of postnatal day (P) 0 animals, and their density increased to high levels by P2, remained at high levels through to P30, but were at low levels in 5-month old rats. A similar developmental pattern was observed in the CA1 region, where SSTR3-ir ciliated structures were first detected on P2. In contrast, density levels in the granular cell layer of the dentate gyrus were very high by P30, and remained elevated in adult rats. SSTR3-ir cilia did not colocalize with neuroblasts in the hippocampal formation or olfactory bulb, but appeared to be localized to more mature cells in these regions. A few SSTR3-ir neurons were also observed in the hippocampal formation. These findings support the hypothesis that the ciliary SSTR3 is well positioned to influence the cell cycle and apoptotic processes during postnatal development, and in neurogenic regions of the adult rat brain.

Nearest neighbor analysis of dopamine D1 receptors and Na+-K+-ATPases in dendritic spines dissected by STED microscopy

Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super‐resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual‐color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+‐ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+‐ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+‐ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.

X-ray phase contrast for CO2 microangiography

We demonstrate a laboratory method for imaging small blood vessels using x-ray propagation-based phase-contrast imaging and carbon dioxide (CO(2)) gas as a contrast agent. The limited radiation dose in combination with CO(2) being clinically acceptable makes the method promising for small-diameter vascular visualization. We investigate the possibilities and limitations of the method for small-animal angiography and compare it with conventional absorption-based x-ray angiography. Photon noise in absorption-contrast imaging prevents visualization of blood vessels narrower than 50 µm at the highest radiation doses compatible with living animals, whereas our simulations and experiments indicate the possibility of visualizing 20 µm vessels at radiation doses as low as 100 mGy. Experimental computed tomography of excised rat kidney shows blood vessels of diameters down to 60 µm with improved image quality compared to absorption-based methods. With our present prototype x-ray source, the acquisition time for a tomographic dataset is approximately 1 h, which is long compared to the 1-20 min common for absorption-contrast micro-CT systems. Further development of the liquid-metal-jet microfocus x-ray sources used here and high-resolution x-ray detectors shows promise to reduce exposure times and make this high-resolution method practical for imaging of living animals.

Alteration of dopamine D1 receptor-mediated motor inhibition and stimulation during development in rats is associated with distinct patterns of c-fos mRNA expression in the frontal-striatal circuitry

Dopamine D1 receptors have been implicated in various neurodevelopmental disorders, including attention‐deficit/hyperactivity disorder. However, little is known about potential late maturational changes of the motor inhibitory and stimulatory role of these receptors. Here, we investigated the effects of a full and selective D1 receptor agonist, SKF‐81297, on motor activity and expression of the plasticity‐associated gene, c‐fos, in the prefrontal cortex and striatum of juvenile and adolescent male rats. In general, SKF‐81297 produced a biphasic effect on motor activity (locomotor and rearing activity), which consisted of an initial short inhibition followed by a long‐lasting stimulation. These effects were dose‐ and age‐ dependent. The inhibitory phase was more pronounced in adolescent than in juvenile rats whereas the opposite was true for the stimulatory phase. During the initial inhibitory phase of the drug, c‐fos mRNA expression was increased in the prefrontal cortex of juvenile rats but reduced in adolescent rats. There was also an increase in c‐fos mRNA expression in the medial‐dorsal striatum and olfactory tubercle, which was more evident in juvenile rats. In contrast, during the stimulatory phase, c‐fos mRNA expression was increased in both the dorsal and ventral striatum, especially in the nucleus accumbens, as well as in the prefrontal cortex, in both age groups. The increase of c‐fos mRNA in the dorsal striatum, however, was more pronounced in juvenile rats. These results indicate the presence of two distinct D1 receptor populations within the frontal‐striatal circuitry, which have opposite effects on motor activity, and which have different maturational profiles.

Recruitment of renal dopamine 1 receptors requires an intact microtubulin network

Abstract. Renal dopamine1 receptor (D1R) can be recruited from intracellular compartments to the plasma membrane by D1R agonists and endogenous dopamine. This study examines the role of the cytoskeleton for renal D1R recruitment. The studies were performed in LLCPK-1 cells that have the capacity to form dopamine from L-dopa. In approximately 50% of the cells treated with L-dopa the D1R was found to be translocated from intracellular compartments towards the plasma membrane. Disruption of the microtubulin network by nocodazole significantly prevented translocation. In contrast, depolymerization of actin had no effect. In control cells D1R colocalized with NBD-C6-ceramide, a trans-Golgi fluorescent marker. This colocalization was disrupted in L-dopa-treated cells. Tetanus toxin, an inhibitor of exocytosis, prevented L-dopa-induced receptor recruitment. L-Dopa treatment resulted in activation of protein kinase C (PKC). To test the functional effect of D1R recruitment, the capacity of D1R agonists to activate PKC was studied. Activation of D1R significantly translocated PKC-α from intracellular compartments to the plasma membrane. Disruption of microtubules abolished D1R-mediated – but not phorbol-ester-mediated – translocation of PKC. We conclude that renal D1R recruitment requires an intact microtubulin network and occurs via Golgi-derived vesicles. These newly recruited receptors couple to the PKC signaling pathway.

Binding of Losartan to Angiotensin AT1 Receptors Increases Dopamine D1 Receptor Activation

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling.