Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, Bip

To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.     

The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment

The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.     

Emerging aspects of membrane traffic in neuronal dendrite growth

Polarized growth of the neuron would logically require some form of membrane traffic to the tip of the growth cone, regulated in conjunction with other trafficking processes that are common to both neuronal and non-neuronal cells. Unlike axons, dendrites are endowed with membranous organelles of the exocytic pathway extending from the cell soma, including both rough and smooth endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Dendrites also have satellite Golgi-like cisternal stacks known as Golgi outposts that have no membranous connections with the somatic Golgi. Golgi outposts presumably serve both general and specific local trafficking needs, and could mediate membrane traffic required for polarized dendritic growth during neuronal differentiation. Recent findings suggest that dendritic growth, but apparently not axonal growth, relies very much on classical exocytic traffic, and is affected by defects in components of both the early and late secretory pathways. Within dendrites, localized processes of recycling endosome-based exocytosis regulate the growth of dendritic spines and postsynaptic compartments. Emerging membrane traffic processes and components that contribute specifically to dendritic growth are discussed.     

The dynamics of engineered resident proteins in the mammalian Golgi complex relies on cisternal maturation

After leaving the endoplasmic reticulum, secretory proteins traverse several membranous transport compartments before reaching their destinations. How they move through the Golgi complex, a major secretory station composed of stacks of membranous cisternae, is a central yet unsettled issue in membrane biology. Two classes of mechanisms have been proposed. One is based on cargo-laden carriers hopping across stable cisternae and the other on “maturing” cisternae that carry cargo forward while progressing through the stack. A key difference between the two concerns the behavior of Golgi-resident proteins. Under stable cisternae models, Golgi residents remain in the same cisterna, whereas, according to cisternal maturation, Golgi residents recycle from distal to proximal cisternae via retrograde carriers in synchrony with cisternal progression. Here, we have engineered Golgi-resident constructs that can be polymerized at will to prevent their recycling via Golgi carriers. Maturation models predict the progress of such polymerized residents through the stack along with cargo, but stable cisternae models do not. The results support the cisternal maturation mechanism.     

Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation

Objectives: Our previous study has shown that slow or “controlled” reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO⁻) formation induced by hypoxia/reoxygenation (H/R). Methods: Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation. Results: H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO⁻ formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO⁻ scavenger uric acid (UA) at reoxygenation significantly decreased ONOO⁻ production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO⁻ formation (P < 0.01). Conclusion: These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO⁻ formation following reoxygenation. H.-C. Wang and H.-F. Zhang contributed equally to this study.     

Hypoxia, reactive oxygen, and cell injury

Hypoxia usually decreases the formation of reactive oxygen species by oxidases and by autoxidation of components of cellular electron transfer pathways and of quinoid compounds such as menadione. In the case of menadione reactive oxygen species are liberated to a significant extent only at non-physiologically high oxygen partial pressures (PO2). At physiological and hypoxic PO2 values electron shuttling of menadione in the mitochondrial respiratory chain predominates. In contrast, lipid peroxidation induced by halogenated alkanes, such as carbon tetrachloride, in liver leads to an increase in the formation of reactive oxygen and thus in cell injury under hypoxic conditions. Reactive oxygen species may also be generated during reoxygenation of a previously hypoxic tissue. Based on experiments with isolated hepatocytes a three-zone-model of liver injury due to hypoxia and reoxygenation is presented; 1) a zone where the cells die by hypoxia; 2) a zone where the cells are destroyed upon reoxygenation, presumably mediated by an increase in the cellular ATP content; and 3) a zone where cell injury occurs upon reoxygenation, mediated by reactive oxygen species possibly liberated by xanthine oxidase.     

Fast extraction of neuron morphologies from large-scale SBFSEM image stacks

Neuron morphology is frequently used to classify cell-types in the mammalian cortex. Apart from the shape of the soma and the axonal projections, morphological classification is largely defined by the dendrites of a neuron and their subcellular compartments, referred to as dendritic spines. The dimensions of a neuron’s dendritic compartment, including its spines, is also a major determinant of the passive and active electrical excitability of dendrites. Furthermore, the dimensions of dendritic branches and spines change during postnatal development and, possibly, following some types of neuronal activity patterns, changes depending on the activity of a neuron. Due to their small size, accurate quantitation of spine number and structure is difficult to achieve (Larkman, J Comp Neurol 306:332, 1991). Here we follow an analysis approach using high-resolution EM techniques. Serial block-face scanning electron microscopy (SBFSEM) enables automated imaging of large specimen volumes at high resolution. The large data sets generated by this technique make manual reconstruction of neuronal structure laborious. Here we present NeuroStruct, a reconstruction environment developed for fast and automated analysis of large SBFSEM data sets containing individual stained neurons using optimized algorithms for CPU and GPU hardware. NeuroStruct is based on 3D operators and integrates image information from image stacks of individual neurons filled with biocytin and stained with osmium tetroxide. The focus of the presented work is the reconstruction of dendritic branches with detailed representation of spines. NeuroStruct delivers both a 3D surface model of the reconstructed structures and a 1D geometrical model corresponding to the skeleton of the reconstructed structures. Both representations are a prerequisite for analysis of morphological characteristics and simulation signalling within a neuron that capture the influence of spines.     

Axonal and dendritic endocytic pathways in cultured neurons

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.     

Epigenetic regulation of neuronal dendrite and dendritic spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity, which is correlated with learning and memory. The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time. It has been shown that specific molecules are needed not only at the spine’s point of contact, but also at a distance, providing signals that initiate a cascade of events leading to synapse formation. The specific molecules that act to signal neuronal differentiation, dendritic morphology, and synaptogenesis are tightly regulated by genetic and epigenetic programs. It has been shown that the dendritic spine structure and distribution are altered in many diseases, including many forms of mental retardation (MR), and can also be potentiated by neuronal activities and an enriched environment. Because dendritic spine pathologies are found in many types of MR, it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR. Epigenetic mechanisms, including DNA methylation, chromatin remodeling, and the noncoding RNA-mediated process, have profound regulatory roles in mammalian gene expression. The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding. Despite extensive efforts to understand the molecular regulation of dendrite and spine development, epigenetic mechanisms have only recently been considered. In this review, we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines. We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR, such as is seen in fragile X and Rett syndromes. We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Actin filaments and microtubules in dendritic spines

Dendritic spines are small protrusions emerging from their parent dendrites, and their morphological changes are involved in synaptic plasticity. These tiny structures are composed of thousands of different proteins belonging to several subfamilies such as membrane receptors, scaffold proteins, signal transduction proteins, and cytoskeletal proteins. Actin filaments in dendritic spines consist of double helix of actin protomers decorated with drebrin and ADF/cofilin, and the balance of the two is closely related to the actin dynamics, which may govern morphological and functional synaptic plasticity. During development, the accumulation of drebrin‐binding type actin filaments is one of the initial events occurring at the nascent excitatory postsynaptic site, and plays a pivotal role in spine formation as well as small GTPases. It has been recently reported that microtubules transiently appear in dendritic spines in correlation with synaptic activity. Interestingly, it is suggested that microtubule dynamics might couple with actin dynamics. In this review, we will summarize the contribution of both actin filaments and microtubules to the formation and regulation of dendritic spines, and further discuss the role of cytoskeletal deregulation in neurological disorders.     

Neuronal sub‐compartmentalization: a strategy to optimize neuronal function

Neurons are highly polarized cells that consist of three main structural and functional domains: a cell body or soma, an axon, and dendrites. These domains contain smaller compartments with essential roles for proper neuronal function, such as the axonal presynaptic boutons and the dendritic postsynaptic spines. The structure and function of these compartments have now been characterized in great detail. Intriguingly, however, in the last decade additional levels of compartmentalization within the axon and the dendrites have been identified, revealing that these structures are much more complex than previously thought. Herein we examine several types of structural and functional sub‐compartmentalization found in neurons of both vertebrates and invertebrates. For example, in mammalian neurons the axonal initial segment functions as a sub‐compartment to initiate the action potential, to select molecules passing into the axon, and to maintain neuronal polarization. Moreover, work in Drosophila melanogaster has shown that two distinct axonal guidance receptors are precisely clustered in adjacent segments of the commissural axons both in vivo and in vitro, suggesting a cell‐intrinsic mechanism underlying the compartmentalized receptor localization. In Caenorhabditis elegans, a subset of interneurons exhibits calcium dynamics that are localized to specific sections of the axon and control the gait of navigation, demonstrating a regulatory role of compartmentalized neuronal activity in behaviour. These findings have led to a number of new questions, which are important for our understanding of neuronal development and function. How are these sub‐compartments established and maintained? What molecular machinery and cellular events are involved? What is their functional significance for the neuron? Here, we reflect on these and other key questions that remain to be addressed in this expanding field of biology.     

Epigenetic regulation of neuronal dendrite and dendrite spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity,which is correlated with learning and memory.The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time.It has been shown that specific molecules are needed not only at the spine's point of contact,but also at a distance,providing signals that initiate a cascade of events leading to synapse formation.The specific molecules that act to signal neuronal differentiation,dendritic morphology,and synaptogenesis are tightly regulated by genetic and epigenetic programs.It has been shown that the dendritic spine structure and distribution are altered in many diseases,including many forms of mental retardation(MR),and can also be potentiated by neuronal activities and an enriched environment.Because dendritic spine pathologies are found in many types of MR,it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR.Epigenetic mechanisms,including DNA methylation,chromatin remodeling,and the noncoding RNA-mediated process,have profound regulatory roles in mammalian gene expression.The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding.Despite extensive efforts to understand the molecular regulation of dendrite and spine development,epigenetic mechanisms have only recently been considered.In this review,we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines.We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR,such as is seen in fragile X and Rett syndromes.We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Multiple subcellular mRNA distribution patterns in neurons: A nonisotopic in situ hybridization analysis

Previous studies have established that most of the mRNAs that neurons express are localized in the cell body and very proximal dendrites, whereas a small subset of mRNAs is present at relatively high levels in dendrites. It is not clear, however, whether particular mRNAs have the same subcellular distribution in different types of neurons or whether different types of neurons sort mRNAs in different ways. The present study was undertaken to address these questions. Nonisotopic in situ hybridization techniques were used to define the subcellular localization of representative mRNAs including β-tubulin, low-molecular-weight neurofilament protein (NF-68), high-molecular-weight microtubule-associated protein (MAP2), growth-associated protein 43 (F1/GAP43), the alpha subunit of calcium/calmodulin-dependent protein kinase II (αCaMII kinase), and poly(A⁺) mRNA. The mRNAs for β-tubulin, neurofilament 68, and F1/GAP43 were restricted to the region of the cell body and very proximal dendrites in most neurons. In some neuron types, however, labeling for NF-68 extended for considerable distances into dendrites. In some neurons that express MAP2, the mRNA was present at the highest levels in the proximal third to half of the dendritic arbor, whereas in other neurons the highest levels of labeling were in the cell body. In most neurons that express αCaMII kinase, the highest levels of the mRNA were in the cell body, but labeling was also present throughout dendrites. However, in a few types of neurons, αCaMII kinase mRNA was largely restricted to the cell body. The fact that there are no general rules for mRNA localization that apply to all neuron types implies the existence of neuron type-specific mechanisms that regulate mRNA distribution. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 473–493, 1997     

Eph receptors are involved in the activity-dependent synaptic wiring in the mouse cerebellar cortex

Eph receptor tyrosine kinases are involved in many cellular processes. In the developing brain, they act as migratory and cell adhesive cues while in the adult brain they regulate dendritic spine plasticity. Here we show a new role for Eph receptor signalling in the cerebellar cortex. Cerebellar Purkinje cells are innervated by two different excitatory inputs. The climbing fibres contact the proximal dendritic domain of Purkinje cells, where synapse and spine density is low; the parallel fibres contact the distal dendritic domain, where synapse and spine density is high. Interestingly, Purkinje cells have the intrinsic ability to generate a high number of spines over their entire dendritic arborisations, which can be innervated by the parallel fibres. However, the climbing fibre input continuously exerts an activity-dependent repression on parallel fibre synapses, thus confining them to the distal Purkinje cell dendritic domain. Such repression persists after Eph receptor activation, but is overridden by Eph receptor inhibition with EphA4/Fc in neonatal cultured cerebellar slices as well as mature acute cerebellar slices, following in vivo infusion of the EphA4/Fc inhibitor and in EphB receptor-deficient mice. When electrical activity is blocked in vivo by tetrodotoxin leading to a high spine density in Purkinje cell proximal dendrites, stimulation of Eph receptor activation recapitulates the spine repressive effects of climbing fibres. These results suggest that Eph receptor signalling mediates the repression of spine proliferation induced by climbing fibre activity in Purkinje cell proximal dendrites. Such repression is necessary to maintain the correct architecture of the cerebellar cortex.     

Stacks of flattened smooth endoplasmic reticulum highly enriched in inositol 1,4,5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells.

By immunogold electron microscopy we have shown that in mouse cerebellar Purkinje cells fixed by perfusion with formaldehyde-glutaraldehyde solution, the InsP3 receptor are numerously detected on the stacks of flattened cisterns (OTSU et al, (1990) Cell Struct. Funct., 15: 163-173). In the present experiment we investigated distribution, structure and properties of the stacks by conventional electronmicroscopy, lectin cytochemistry and immunoelectron microscopy. The size and number of stacks were variable depending on their intracellular localization; short stacks with 2-4 parallel cisterns predominate in the perikaryon, long stacks with 4-15 cisterns in the proximal dendrite, and long stacks with 3-4 cisterns in the distal dendrites. The flattened cisterns bind with concanavalin A but not with wheat-germ agglutinin and may contain KDEL proteins loaded with Lys-Asp-Glu-Leu at their C-terminin in their lumens, indicating that the cisterns are derived from ER membranes. The electron dense materials sandwiched between the cisternal membranes are composed of small particles, short cylindrical in shape and approximately 20 nm in diameter, and markedly labeled with anti InsP3R antibody. We suggest that they correspond to the tetramer of the InsP3R or their related molecules. It is not clear whether the stacks of flattened cisterns exist per se in the Purkinje cells or smooth ER existing in singlet in vivo in the Purkinje cells forms stacks during fixation. It is strongly suggested, however, that the smooth ER membranes covered by the InsP3R or their related molecules can easily interact and stack each other in the Purkinje cells.     

A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster

Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actin::GFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of >4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actin::GFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment.     

Muscarinic regulation of Kenyon cell dendritic arborizations in adult worker honey bees

The experience of foraging under natural conditions increases the volume of mushroom body neuropil in worker honey bees. A comparable increase in neuropil volume results from treatment of worker honey bees with pilocarpine, an agonist for muscarinic-type cholinergic receptors. A component of the neuropil growth induced by foraging experience is growth of dendrites in the collar region of the calyces. We show here, via analysis of Golgi-impregnated collar Kenyon cells with wedge arborizations, that significant increases in standard measures of dendritic complexity were also found in worker honey bees treated with pilocarpine. This result suggests that signaling via muscarinic-type receptors promotes the increase in Kenyon cell dendritic complexity associated with foraging. Treatment of worker honey bees with scopolamine, a muscarinic inhibitor, inhibited some aspects of dendritic growth. Spine density on the Kenyon cell dendrites varied with sampling location, with the distal portion of the dendritic field having greater total spine density than either the proximal or medial section. This observation may be functionally significant because of the stratified organization of projections from visual centers to the dendritic arborizations of the collar Kenyon cells. Pilocarpine treatment had no effect on the distribution of spines on dendrites of the collar Kenyon cells.     

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.     

Evidence for a satellite secretory pathway in neuronal dendritic spines

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.