LeukoScan protocol.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
Biomedical subjects
S Schroeter
Publications and source records attributed to S Schroeter.
Ultrastructural localization of the serotonin transporter in superficial and deep layers of the rat prelimbic prefrontal cortex and its spatial relationship to dopamine terminals.
Dopamine levels within the prefrontal cortex (PFC) can be manipulated by selective inhibitors of the serotonin transporter (SERT). However, the cellular mechanisms underlying these effects are not clear. The present study sought to examine the distribution of immunogold-silver labeling for SERT (SERT-ir) in the rat prelimbic PFC and to describe its ultrastructural spatial relationship to dopamine axons labeled by immunoperoxidase staining for tyrosine hydroxylase (TH-ir). SERT was localized to axonal profiles that ranged in size from fine caliber fibers containing dense SERT-ir, primarily along the membrane, and rarely forming synapses to large, spherical varicosities exhibiting less dense staining, mainly within the cytoplasm, and more commonly forming synapses. Synaptic contacts of SERT profiles were typically asymmetric, axospinous, and more frequent in superficial (38%) than deep (19%) layers. For TH-ir profiles, only 24% were within the same 13.8 microm(2) microenvironment as SERT-ir profiles. Furthermore, TH-ir and SERT-ir profiles were rarely directly apposed to each other or convergent onto common dendritic structures. Instead, these two profiles were typically separated by an average distance of 1.30 microm in the coronal plane, a value that did not vary with the size of SERT-ir axons, the amount of SERT labeling, or the cortical layer examined. These results are consistent with two populations of SERT profiles within the rat prelimbic PFC that may arise from different raphe nuclei or that represent varicose and intervaricose portions of the same axons. Moreover, the functional interactions between cortical serotonin and dopamine systems that may contribute to the therapeutic efficacy of antidepressant drugs are likely to occur over distances greater than 1 microm.
Animals↗
Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter.
Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585-602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine- and dopamine-containing neurons. Intracerebroventricular anti-dopamine beta-hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP-25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states.
Animals↗
Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins.
Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) mediate antidepressant-sensitive clearance of 5-HT following release. Although we have been aware for decades that SERT-mediated 5-HT clearance can be modulated by exogenous agents including serotonin-selective reuptake inhibitors, amphetamines, and cocaine, we have had little reason to speculate that SERT activity was actively controlled through endogenous pathways. Recent studies indicate that SERTs are likely to be trafficked to specific plasma membrane subdomains to achieve localized clearance of 5-HT, and that the number of SERTs resident in the plasma membrane is controlled through kinase- and phosphatase-linked pathways. In particular, roles for protein kinase C and phosphatase 2A become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter redistribution and loss of functional uptake capacity. Based on our findings, and the studies of others in native tissues and transfected cells, we propose a model whereby SERTs are organized in a macromolecular complex in the plasma membrane that may serve to locate reuptake activity near release sites. Although many elements of this model remain hypothetical, our findings suggest a much more dynamic picture of transporter-mediated 5-HT reuptake than typically described and suggest opportunities both for the development of new SERT regulatory agents and for the identification of regulatory pathways that may be compromised in mental illness.
Animals↗
Acute regulation of norepinephrine transport: II. PKC-modulated surface expression of human norepinephrine transporter proteins.
Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, beta-PMA or beta-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km. Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, beta-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetine Kd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.
Binding Sites↗
Polarized expression of the antidepressant-sensitive serotonin transporter in epinephrine-synthesizing chromaffin cells of the rat adrenal gland.
Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localization in vivo are lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2 termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intra-cellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.
Adrenal Glands↗
Cloning and expression of the mouse serotonin transporter.
A mouse brain cDNA encoding the high-affinity serotonin transporter (SERT) has been identified and characterized. The mouse transporter sequence (mSERT) encodes a protein of 630 amino acids which contains twelve potential transmembrane domains (TMDs), N-linked glycosylation and kinase-mediated phosphorylation sites, and high levels of homology with rat and human SERTs. Heterologous expression of mSERT in COS-I cells resulted in a [3H]serotonin transport activity characterized by kinetic saturability (Km = 403 +/- 42 nM. Vmax = 1.02 +/- 0.10 pmol/mg/min), Na1 and Cl- dependences (5HT:Na+:Cl- coupling ratio of 1:1:1), and sensitivity to known inhibitors of serotonin transport (including antidepressant and psychostimulant agents). Northern analysis using mSERT cDNA as probe revealed a single 3.4 kb mRNA species expressed in mouse lung, midbrain and brainstem regions, and absent from heart and liver. In situ hybridization studies further established the specific localization of mSERT gene expression to the raphe nuclei of the mouse midbrain. The identified mSERT cDNA sequence provides a new tool for the evaluation of serotonin transport pharmacology in heterologous expression systems and provides an opportunity for the evaluation of mSERT gene expression in a well-characterized model of mammalian development.
Animals↗
Drug targets in the embryo. Studies on the cocaine- and antidepressant-sensitive serotonin transporter.
Explore the source record for details and available documents.
Amino Acid Sequence↗
On-call nuclear medicine service.
Explore the source record for details and available documents.
Holidays↗
Spatial and temporal patterns of muscle cleavage in the chick thigh and their value as criteria for homology.
Regions of lower cell density, called cleavage zones, emerge within the dorsal and ventral muscle masses in the vertebrate limb to separate distinct muscles. In the chick thigh, the stereotyped patterns of separation have been broadly outlined, but differences in interpretation exist because no criteria for separation have been defined, and the tissues of the limb are indistinct early in development. We have examined the cleavage process using modern applications of light microscopy and immunocytochemistry to completely detail the spatial and temporal progression of cleavage in stage 27-32 embryos. We find that each muscle has a complex but characteristic pattern of separation along the proximodistal axis. The complex pattern of separation is not related to the positions of muscles within the thigh, locations of blood vessels, activity patterns of muscles, or innervation patterns. The initial separation patterns are more straightforward than later separations and may be of value in determining the phylogenetic history of limb muscles since the same patterns are common to many tetrapods. Our detailed documentation clarifies the ontogeny of the thigh musculature and reveals more complex separation patterns between muscles than previously described.
Animals↗
Ultrastructural and morphometric analysis of the separation of two thigh muscles in the chick.
Limb muscles separate from one another in a complex but highly stereotyped sequence and spatial pattern. The process of separation is characterized by the progression of a region of increased extracellular space, the cleavage zone, along the proximodistal axis between the individual muscle anlagen. We analyzed ultrastructurally the muscles and cleavage zone during the separation of two representative muscles, the developing sartorius and iliotibialis in the chick thigh, to establish an accurate baseline for an analysis of the mechanisms of separation. Comparisons of the morphology and distribution of cells before and after separation show no evidence that muscles became separated by the massive influx of an exterior cell population; if populations invade the cleavage zone, they are small. We do find characteristic transitions within the cell population of the cleavage zone in situ that could accomplish cleavage without invoking massive cell movements. These progressive transitions within the cleavage zone include a loss of close cell-cell interactions, an increase in extracellular space, the assumption of a more stellate morphology by mesenchyme cells, and a gradual alteration in the composition of the extracellular matrix from one typical of early muscle to one typical of loose connective tissue. Myotubes do differentiate between the incipient muscles, ruling out the possibility that the location where muscles will separate is defined by sites where myotubes fail to differentiate. Instead, the myotubes in the cleavage zone gradually diminish in number and appear to be specifically recognized and removed from the cleavage zone by phagocytes. We suggest that the transitions within the cleavage zone, including the loss of muscle cells, are a result of the progressive differentiation of loose connective tissue. If so, then the spatial pattern and process of cleavage is a consequence of spatially programmed cell differentiation.
Animals↗
Distribution and projection pattern of motoneurons that innervate hindlimb muscles in the quail.
We characterized the motoneuron pool positions and projection patterns in the embryonic quail hindlimb and compared them to those in the chick to determine the degree of similarity and to form a baseline for future chimeric experiments. We find that the most similar parameters of pool position correlate with the major axonal pathway choices. First, the medial-lateral pool position, which is highly conserved among birds and mammals, is identical in the quail and chick and correlates with the dorsal-ventral pathway choice, the first and least plastic of the choices within the limb. Second, although quail pools were known to be compressed into seven rather than eight segments, we show that the map of pools is compressed about a central point (segment three) that preserves the spatial relationships between anterior pools and the crural plexus, and between posterior pools and the sciatic plexus. Access to guidance cues that are restricted to each plexus region is thus maintained between species. Third, pool position along the anterior-posterior axis is the least similar parameter between species. In fact, the entire lumbosacral motor complex may shift by +/- half a segment in individual quail. Despite the consequent differences in segmental projections, the specific projection pattern within each quail hindlimb is identical to that in the chick. There is no need to preserve the exact segmental pattern either phylogenetically or during development, because motoneurons accommodate to modest variations in their position along the anterior-posterior axis by sorting out at the limb base. The contrast between variable segmental and constant limb projections also demonstrates that neither the specification nor the precise projection of motoneurons is dependent upon the specification or development of somites.
Animals↗
Cell death delineates axon pathways in the hindlimb and does so independently of neurite outgrowth.
We wished to know whether the cell death and phagocytosis seen near the outgrowing nerve front in the hindlimb delineate axon pathways and, if so, whether the cells died only in the presence of growth cones. We unilaterally deleted the lumbosacral neural tube and reconstructed the patterns of neurite outgrowth and phagocytes during the stage when neurites first begin to colonize the thigh. In the control limbs, sensory and motor nerve pathways coincided with sites of phagocytosis, including those pathways that had yet to be colonized by growth cones. For instance, phagocytes were clustered at foci within the muscle masses where muscle nerves form a day later. However, they were not seen in adjacent, nonpathway regions such as posterior sclerotome or dorsal and ventral to the region of the plexus in which axons extend only posteriorly. Phagocytes were also seen in defined regions that are probably inaccessible to growth cones because they are too distant from pathways (i.e., subjacent to the apical ectodermal ridge) or express substances that are typical of precartilagenous tissues which may prohibit axon advance. In the experimental limbs, we conservatively estimated that neurite outgrowth was reduced to less than one-tenth (neurites were visible only with electron microscopy) or less than one-third of normal. Outgrowth extended less far distally and, in half the cases, motor innervation was completely abolished. Despite the extensive reduction in neurite outgrowth, the distribution of phagocytes was indistinguishable from that of the control side. Furthermore, the number of phagocytes did not differ significantly. We conclude that cell death delineates axon pathways remarkably well and does so without an interaction with growth cones; it is an independent characteristic of the axonal pathways and may be directly or indirectly important to axonal pathfinding. This is the first identification of a feature that characterizes prospective nerve pathways in the hindlimb.
Animals↗