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. Increasing evidence indicates that interactions between different types of neurotransmitter receptors represent a major mechanism of neuronal plasticity. A variety of physiological and pharmacological stimuli alter the efficacy of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic transmission (1, 2). These changes in signaling have been accounted for in large part by changes in the distribution of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors between spines and the interior of the cell (3, 4). Although signaling in the dopaminergic pathway also shows a great deal of plasticity, little information is available about its cellular basis. In the present study, we examined the possibility that an alteration in the distribution of dopamine receptors might contribute to the ability of N-methyl-D-aspartate (NMDA) to alter the balance between D1 and D2 receptor signaling pathways. Materials and MethodsCell Culture. Cultures of striatal neurons were prepared from 18-to 19-day rat embryos. The cells were plated on poly-D-lysinecoated glass coverslips and cultured in Eagle's MEM plus F-12 (1:1). The cultures were grown in the presence of 5% FBS, penicillin, or streptomycin (100 mg͞ml). To suppress the growth of glial cells, FBS was equilibrated with N2 (1%) 2 days after plating, and cytosine arabinoside (5 M) was added for 48 h to the culture medium on day 5. Cells were maintained in culture for 2-3 weeks before experiments; at that time, virtually all cells from embryonic rat striatum seemed to be mature neurons. They resembled typical medium spiny neurons and stained positively for neuronal markers (5).Immunostaining. Cells were fixed for 20 min in ice-cold 2% paraformaldehyde, permeabilized with 0....

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.

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 ϩ -K ϩ -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 ϩ -K ϩ -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.AT 1 receptors; Na ϩ -K ϩ -ATPase; calcium signaling RENAL SODIUM EXCRETION IS bidirectionally regulated by angiotensin II (ANG II) and dopamine (13). Long-term dopamine exposure is known to decrease AT 1 receptors (AT1R) in renal proximal tubular cells (7). Furthermore, studies by Zeng et al. (21) have shown that long-term stimulation of AT1R results in an upregulation of D1-like receptors (D1R). This effect was not observed in spontaneously hypertensive rats, indicating that the interaction between AT1R and D1R has an impact on blood pressure regulation. Since sodium excretion must be regulated with great precision over a short period of time, it is important that control mechanisms are able to exert their effects within a short time frame. The aim of the current study has been to explore the short-term effects of ANG II exposure on D1R and the short-term effects of a D1-agonist on AT1R. Our approach has been to test the hypothesis that AT1R and D1R form a heteromeric signaling complex, where activation of either receptor may cause internalization and/or interruption of the signaling capacity of the other.The studies were performed using rat proximal tubule cells, since these cells express both AT1R and D1R in both the apical and the basolateral membrane (12,18). Previous studies from our laboratory have shown that in these cells Na ϩ -K ϩ -ATPase, the enzyme responsible for active sodium transport, is bidirectionally regulated by ANG II and dopamine (2). MATERIALS AND METHODS Cells and tissue.All studies were performed using outer cortical tissue from young (3-5 wk) male Sprague-Dawley rats. Immediately after the animals were killed, 250-m slices were taken from the outer renal cortex using a microtome. The outer 250-m region of the rat renal cortex contains Ͼ90% proxi...

The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusiontrap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy.organotypic cultures ͉ fluorescence recovery after photo-bleaching ͉ lateral diffusion ͉ receptor movement A variety of pharmacological, biochemical, and molecular genetic evidence indicates that the schizophrenic state is associated with hypoglutamatergia and a high ratio of D2 dopaminergic to D1 dopaminergic signaling (1, 2). We recently reported a cellular interaction between NMDA receptors and D1 receptors (D1Rs) that can explain how such abnormalities in glutamate and dopamine signaling might be causally related. Specifically, activation of NMDA receptors was shown to increase recruitment of D1Rs but not of D2Rs in the plasma membrane of primary cultures of striatal neurons (3). In view of the potential clinical relevance of NMDA-induced selective recruitment of dopamine D1Rs, we have, in the present study, explored the underlying mechanism. Here, we provide evidence that dopamine D1Rs, laterally diffusing in the dendritic plasma membrane, can be trapped in spines by ligand-occupied NMDA receptors. ResultsTo monitor its mobility in live neurons, the D1R was tagged with the fluorescent protein Venus (D1R-Venus) (4) and expressed in organotypic cultures from rat striatum (Fig. 1a). The fluorescent signal was evenly distributed in the cell body and plasma membrane. A strong signal was also recorded from the dendritic tree (Fig. 1b). Approximately five D1R-positive spines were detected per 10 m of dendrite. D1R-positive spines were mushroom shaped, thin, or stubby. Filopodia-like D1R-positive structures were rare. The subcellular distribution of D1R-Venus was similar to that described in electron microscope studies of endogenous D1R (5, 6). No distinct D1R signal was observed in glial cells. This lack of signal from glial cells minimized the background signal and made this study feasible (Fig. 1c). The selective distribution of t...