Biomedical subjects
G Banker
Publications and source records attributed to G Banker.
Development of subcellular mRNA compartmentation in hippocampal neurons in culture.
Neurons possess an RNA transport system that is present in dendrites (but not axons) and sort mRNAs so that some mRNAs are restricted to cell bodies while a few others (like the mRNA for MAP2) are present in dendrites. The present study evaluates when dendrite-specific RNA transport and mRNA sorting into cell body and somatodendritic compartments first appear in developing hippocampal neurons maintained in culture. A 3H-uridine pulse-chase paradigm was used to evaluate transport of newly synthesized RNA from the site of synthesis in the nucleus into the developing neurites. The intracellular distribution of mRNAs encoding actin, tubulin, GAP-43, and MAP2 as well as polyA RNA and rRNA was evaluated by in situ hybridization at different stages of development. Newly synthesized RNA was translocated into both developing axons and dendrites early in development, but only into dendrites as the neurons matured. Tubulin, GAP-43, and actin mRNAs, which are restricted to cell bodies in mature neurons, were found exclusively in neuronal cell bodies at all developmental stages. MAP2 mRNA, which is present in the dendrites of mature neurons, was present at very low levels in neurons at 2 or 3 d in culture and was not detectable within dendrites. The overall levels of MAP2 mRNA increased over time, and by 5-7 d in culture, MAP2 mRNA was detectable in some dendrites. PolyA RNA and rRNA were detectable in developing neurites including axons. Levels of polyA and rRNA increased in dendrites as neurons matured while labeling of axons diminished. By 10 d in culture, axonal labeling for polyA and rRNA had virtually disappeared. The increase in the levels of polyA, rRNA, and MAP2 mRNA in dendrites between 5 and 7 d in culture corresponds roughly with the appearance of other dendritic characteristics and the beginning of dendritic outgrowth.
Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development.
Hippocampal neurons in culture develop extensive axonal and dendritic arbors and form numerous synapses. Presynaptic specializations occur at sites of contact between axons and somata or dendrites but they do not appear until day 3 in culture, even though numerous contacts between cells develop within the first 24 hr (Fletcher et al., 1991). To determine whether this delay in the appearance of presynaptic specializations could be related to maturational events in the presynaptic axon or in the postsynaptic target, "heterochronic" cocultures were prepared by adding newly dissociated neurons to cultures containing mature neurons. The competence of axons to form presynaptic vesicle clusters in response to contact with the somata or dendrites of mature or immature neurons was determined by immunofluorescent staining for synapsin I or synaptophysin. After only 1 d of coculture, there was a fivefold increase in the number of synapses along the somata and dendrites of the mature neurons, compared to mature neurons cultured alone. If newly dissociated neurons were labeled with a fluorescent dye before coculture, dye-labeled axons frequently were colocalized with presynaptic specializations on mature cells. In contrast, when the axons of mature neurons contacted immature neurons, synapses were first observed only after coculture for 3 d. These results suggest that the axons of hippocampal neurons have the capacity to form presynaptic specializations soon after they emerge, provided they encounter appropriate targets, but that the cell bodies and dendrites of hippocampal neurons are not capable of inducing the formation of presynaptic specializations until they reach a critical stage of maturation.
Use of HSV-1 amplicon vectors to study RNA and protein targeting in cultured hippocampal neurons.
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Inhibition of protein synthesis alters the subcellular distribution of mRNA in neurons but does not prevent dendritic transport of RNA.
This study evaluates whether protein synthesis plays a role in targeting RNA molecules to different subcellular domains within neurons. Transport of newly synthesized RNA (labeled with [3H]uridine) was examined in the presence of the protein synthesis inhibitors puromycin and cycloheximide. In situ hybridization was used to determine whether inhibition of protein synthesis altered the subcellular distribution of mRNAs. Transport of recently synthesized RNA was not disrupted after prolonged exposure to either inhibitor. However, inhibition of protein synthesis caused several mRNAs that are normally confined to the cell body to appear in dendrites. The distribution of mRNAs that are normally present in dendrites was unaffected. These findings suggest that protein synthesis is not required to translocate RNA into the dendrites but may play a role in restricting particular mRNAs to the neuronal cell body.
Subcellular distribution of rRNA and poly(A) RNA in hippocampal neurons in culture.
In situ hybridization was used to assess the subcellular distribution of rRNA and poly(A) RNA in hippocampal neurons maintained in culture. Labeling produced with 35S-labeled probes to either rRNA or poly(A) was heaviest over the cell body with lighter, patchy labeling of proximal dendrites. In contrast, 3H-labeled probes labeled dendrites throughout their length, and the ratio of dendritic to cell body labeling was higher with 3H-labeled probes. There was no detectable labeling of axons of mature neurons with either probe. The pattern of hybridization produced by 35S-labeled oligonucleotide probes to rRNA varied depending on the concentration of the oligonucleotide. These studies provide the first detailed study of the subcellular distribution of rRNA and poly(A) RNA in neurons, and highlight technical issues to consider when evaluating results of hybridizations carried out with 35S- and 3H-labeled probes on cells in culture.
The distribution of glutamate receptors in cultured rat hippocampal neurons: postsynaptic clustering of AMPA-selective subunits.
The distribution of several glutamate receptor subunits was investigated in cultured rat hippocampal neurons by in situ hybridization and immunocytochemistry. The AMPA/kainate-selective receptors GluR1-6 exhibited two patterns of mRNA expression: most neurons expressed GluR1, R2, and R6, whereas only about 20% expressed significant levels of GluR3, R4, and R5. By immunocytochemistry, the metabotropic glutamate receptor mGluR1 alpha was detectable only in a subpopulation of GABAergic interneurons. GluR1 and GluR2/3 segregated to the somatodendritic domain within the first week in culture, even in the absence of synaptogenesis. Glutamate receptor-enriched spines developed later and were present only on presumptive pyramidal cells, not on GABAergic interneurons. Clusters of GluR1 and GluR2/3 completely colocalized and were restricted to a subset of postsynaptic sites. Thus, glutamate receptor subunits exhibit both a cell type-specific expression and a selective subcellular localization.
Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.
The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.
Temperature-dependent blockade of nucleocytoplasmic transport of newly synthesized RNA in neurons.
This study evaluates the temperature sensitivity of transport of recently synthesized RNA from the nucleus to the cytoplasm (nucleocytoplasmic transport) in CNS neurons. Rat hippocampal slices were incubated with [3H]uridine for 1 h to label recently synthesized RNA. Slices were then fixed immediately or maintained at 27 degrees C or 37 degrees C for chase intervals of 3, 4.5, and 6 h to allow for nucleocytoplasmic transport of recently synthesized RNA. The time-dependent translocation of recently synthesized RNA was evaluated autoradiographically. At the end of the 1 h pulse at either 27 degrees C or 37 degrees C, the label was localized exclusively over nuclei. In slices maintained at 37 degrees C, labeling expanded to cover the cell body and proximal dendrites. However, in slices that were labeled and maintained at room temperature, labeling remained confined to the nucleus. In slices that were pulse-labeled at room temperature, and then transferred to 37 degrees C medium, cytoplasmic labeling increased as a function of time. Nucleocytoplasmic transport of RNA in cultured rat hippocampal neurons showed a comparable temperature sensitivity. The inhibition of nucleocytoplasmic transport of RNA at room temperature provides an opportunity to evaluate neuronal function when no new RNA molecules can reach the cytoplasm.
Neuronal polarity.
The axonal and somatodendritic domains of neurons differ in their cytoskeletal and membrane composition, complement of organelles, and capacity for macromolecular synthesis. Recently there has been progress in elucidating the cellular mechanisms that underlie the establishment and maintenance of neuronal polarity, including microtubule organization and the sorting, transport, and anchoring of membrane proteins.
Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides.
Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.
Intracellular organization of hippocampal neurons during the development of neuronal polarity.
In culture, hippocampal neurons initially establish several short, apparently identical processes; of these, only one acquires axonal characteristics, the remainder becoming dendrites. We examined the organization of cytoplasmic constituents that might influence which of the initial processes becomes the axon. The Golgi complex was visualized using either fluorescent wheat germ agglutinin or a specific antibody. Presumptive microtubule-organizing centers were identified by depolymerizing microtubules with nocodazole, then allowing them to repolymerize for brief periods. As judged by light microscopy, hippocampal neurons contained a single Golgi region and a single microtubule-organizing center, which were frequently localized together adjacent to a shallow indentation in the nucleus. In cells fixed shortly after the axons had emerged, there was no correlation between the position of the Golgi complex or the microtubule organizing center and the site of origin of the axon. Based on nuclear shape, the position of the Golgi complex and microtubule-organizing center could also be inferred in living cells. When axonal outgrowth was followed in individual cells by time-lapse microscopy, so that the location of the Golgi complex and microtubule-organizing center could be determined at the exact moment when the axon emerged, no correlation was apparent. Antibodies that recognize specific posttranslational modifications of alpha-tubulin--acetylation and de-tyrosination--were used to assess the distribution of arrays of stable microtubules. Stable microtubules were present in all processes, both before and after the emergence of the axon. They were not confined to the axon. Thus the localization of these cellular constituents does not play a major role in determining which of the processes initially extended by hippocampal neurons becomes the definitive axon.
The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture.
As a first step toward elucidating mechanisms involved in the sorting of synaptic vesicle proteins in neurons, we have used immunofluorescence microscopy to determine the distribution of two synaptic vesicle proteins, synapsin I and synaptophysin, in hippocampal neurons developing in culture. In mature cultures, synapsin I and synaptophysin immunoreactivity was concentrated in puncta that were restricted to sites where axons contacted neuronal cell bodies or dendrites. Electron-microscopic immunocytochemistry demonstrated that these puncta corresponded to vesicle-filled axonal varicosities that were exclusively presynaptic. At early stages of development, before cell-cell contact, both synapsin I and synaptophysin were preferentially localized in axons, where they were particularly concentrated in the distal axon and growth cone. In axons that did not contact other cells, immunostaining for these two proteins had a granular appearance, which persisted for at least 7 d, but focal accumulations of vesicles comparable to those seen at sites of synaptic contact were not observed. When neurons contacted one another, numerous puncta of synapsin I and synaptophysin formed within the first week in culture. Double-label immunofluorescence demonstrated that the two vesicle antigens were closely codistributed throughout these stages of development. These observations demonstrate that synaptic vesicle proteins assume a polarized distribution within nerve cells beginning early in development, as soon as the axon can be identified. In contrast, differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective sorting of some materials in nerve cells, first appear at a subsequent stage of development. The selective distribution of synaptic vesicle proteins to the axon occurs in isolated cells, independent of interactions with other cells. In contrast, the formation of large clusters of vesicles typical of presynaptic specializations requires contact with an appropriate postsynaptic target. Thus, in cultured hippocampal neurons, the localization of synaptic vesicles in presynaptic specializations is the result of sorting mechanisms intrinsic to individual neurons as well as to mechanisms mediated by cell-cell contact.
Differential subcellular localization of particular mRNAs in hippocampal neurons in culture.
In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and beta-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that is selectively distributed in dendrites and cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.
Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions.
Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity.
Changes in the distribution of GAP-43 during the development of neuronal polarity.
GAP-43, a neuron specific growth-associated protein, is selectively distributed to the axonal domain in developing neurons; it is absent from dendrites and their growth cones. Using immunofluorescence microscopy, we have further examined the distribution of GAP-43 during the development of hippocampal neurons in culture, in order to determine when this polarized distribution arises. Cultured hippocampal neurons initially extend several short processes which have the potential to become either axons or dendrites. At this stage, before the morphological expression of polarity, GAP-43 is concentrated in the growth cones of these processes but is distributed more or less equally among them. Polarity becomes established when one of these processes elongates to become the axon. At the earliest stage when the emerging axon can be identified, GAP-43 is preferentially concentrated in its growth cone. During the next few days, as the remaining processes take on dendritic properties, they lose their residual GAP-43 immunoreactivity. Throughout development, GAP-43 remains highly concentrated in the axonal growth cone, but the concentration of GAP-43 in the axon shaft increases, beginning near the growth cone and progressing proximally until GAP-43 is uniformly distributed along the entire axon. At all stages of development, GAP-43 is also concentrated in the region of the Golgi apparatus. These results suggest that the selective sorting of at least one membrane protein into the axon coincides with the morphological expression of polarity. These results also raise the possibility that GAP-43 may play an important role in the early phases of axonal outgrowth, by which the functional polarity of neurons is established.
Experimental observations on the development of polarity by hippocampal neurons in culture.
In culture, hippocampal neurons develop a polarized form, with a single axon and several dendrites. Transecting the axons of hippocampal neurons early in development can cause an alteration of polarity; a process that would have become a dendrite instead becomes the axon (Dotti, C. G., and G. A. Banker. 1987. Nature (Lond.). 330:254-256). To investigate this phenomenon more systematically, we transected axons at varying lengths. The greater the distance of the transection from the soma, the greater the probability for regrowth of the original axon. However, it was not the absolute length of the axonal stump that determined the response to transection, but rather its length relative to the lengths of the cell's other processes. If one process was greater than 10 microns longer than the others, it invariably became the axon regardless of its identity before transection. Conversely, when a cell's processes were nearly equal in length, it was impossible to predict which would become the axon. In these cases, axonal outgrowth began only after a long latency. During this interval, the processes appeared to be in dynamic equilibrium, some growing for short distances while others retracted. When one process exceeded the others by a critical length, it rapidly elongated to become the axon. The establishment of neuronal polarity during normal development may similarly involve an interaction among processes whose identities have not yet been determined. When, by chance, one exceeds the others by a critical length, it becomes specified as the axon.
The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.
We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament-associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to-distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.