Dietary cadmium and benzo(a)pyrene increased intestinal metallothionein expression in the fish Fundulus heteroclitus.

Fish were individually fed food pellets containing cadmium, benzo(a)pyrene, or a combination of the two, then analyzed for metallothionein mRNA expression in the intestine, liver, and gill using real-time RT-qPCR. An initial experiment using only cadmium showed that ingestion of pellets varied in individual fish, and estimates of cadmium dose from the numbers of ingested pellets indicated considerable individual variability in cadmium dose. Induction of intestinal metallothionein mRNA was apparent, however, and a linear dose-response relationship was observed for metallothionein expression and cadmium dose in the intestine, but not the other organs, which showed no induction. In a second experiment, the entire daily cadmium dose was provided in a single contaminated pellet that was consumed by all treated fish, effectively eliminating the effect of variable ingestion rates on dose, and the interaction between cadmium and benzo(a)pyrene was also investigated. The intestine was again the primary organ for metallothionein induction by cadmium. When benzo(a)pyrene was administered together with cadmium, induction of metallothionein was potentiated by the presence of benzo(a)pyrene, with the main effect seen in the intestine, where already high levels of induction by cadmium alone increased by 1.74-fold when benzo(a)pyrene was present.

Focal motility determines the geometry of dendritic spines.

The geometry of dendritic spines has a major impact on signal transmission at excitatory synapses. To study it in detail we raised transgenic mice expressing an intrinsic green fluorescent protein-based plasma membrane marker that directly visualizes the cell surface of living neurons throughout the brain. Confocal imaging of developing hippocampal slices showed that as dendrites mature they switch from producing labile filopodia and polymorphic spine precursors to dendritic spines with morphologies similar to those reported from studies of adult brain. In images of live dendrites these mature spines are fundamentally stable structures, but retain morphological plasticity in the form of actin-rich lamellipodia at the tips of spine heads. In live mature dendrites up to 50% of spines had cup-shaped heads with prominent terminal lamellipodia whose motility produced constant alterations in the detailed geometry of the synaptic contact zone. The partial enveloping of presynaptic terminals by these cup-shaped spines coupled with rapid actin-driven changes in their shape may operate to fine-tune receptor distribution and neurotransmitter cross-talk at excitatory synapses.

Cytoskeletal microdifferentiation: a mechanism for organizing morphological plasticity in dendrites.

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubule-associated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Actin dynamics in dendritic spines: a form of regulated plasticity at excitatory synapses.

Dendritic spines form the postsynaptic element at most excitatory synapses in the brain. The spine cytoskeleton consists of actin filaments which, in time-lapse recordings of living neurons expressing actin labeled with green fluorescent protein, can be seen to undergo rapid, dynamic changes. Because actin dynamics are associated with changes in cell shape, these cytoskeletal rearrangements may form a molecular basis for the morphological plasticity at brain synapses. The rapidity of these dynamic events in dendritic spines raises new questions. First, do the changes in actin cytoskeleton that are visible by light microscopy really correspond to changes in spine morphology, or do they represent changes in the relationship between actin and its many binding partners at postsynaptic sites? Second, how are these changes regulated by synaptic transmission? Third, to what extent do these changes occur in organized brain tissue? Answers to these questions are now beginning to emerge.

Actin-based plasticity in dendritic spines.

The central nervous system functions primarily to convert patterns of activity in sensory receptors into patterns of muscle activity that constitute appropriate behavior. At the anatomical level this requires two complementary processes: a set of genetically encoded rules for building the basic network of connections, and a mechanism for subsequently fine tuning these connections on the basis of experience. Identifying the locus and mechanism of these structural changes has long been among neurobiology's major objectives. Evidence has accumulated implicating a particular class of contacts, excitatory synapses made onto dendritic spines, as the sites where connective plasticity occurs. New developments in light microscopy allow changes in spine morphology to be directly visualized in living neurons and suggest that a common mechanism, based on dynamic actin filaments, is involved in both the formation of dendritic spines during development and their structural plasticity at mature synapses.

Glutamate receptors regulate actin-based plasticity in dendritic spines.

Dendritic spines at excitatory synapses undergo rapid, actin-dependent shape changes which may contribute to plasticity in brain circuits. Here we show that actin dynamics in spines are potently inhibited by activation of either AMPA or NMDA subtype glutamate receptors. Activation of either receptor type inhibited actin-based protrusive activity from the spine head. This blockade of motility caused spines to round up so that spine morphology became both more stable and more regular. Inhibition of spine motility by AMPA receptors was dependent on postsynaptic membrane depolarization and influx of Ca 2+ through voltage-activated channels. In combination with previous studies, our results suggest a two-step process in which spines initially formed in response to NMDA receptor activation are subsequently stabilized by AMPA receptors.

Activity-regulated, cytoskeleton-associated protein (Arc) is essential for visceral endoderm organization during early embryogenesis.

Activity-regulated, cytoskeleton-associated protein (Arc) was first identified as an immediate-early gene regulated by synaptic activity. We have studied its functional role in vivo using a gene-targeting approach. We found that Arc is encoded by a single exon, and Arc mRNA is ubiquitously expressed in early mouse embryos. Homozygous Arc mutants are severely growth-retarded, fail to gastrulate and subsequently die before day 8.5 of embryogenesis. Further analysis revealed severe disorganization of visceral endoderm formation, and total separation and ectopic location of embryonic and extraembryonic structure. These findings demonstrate that Arc function is essential for early embryo development and patterning in mice, and support the hypothesis that signaling from visceral endoderm is essential for normal patterning of the extraembryonic and embryonic structure.

Protein isoaspartyl methyltransferase protects from Bax-induced apoptosis.

Protein L-isoaspartyl methyltransferase (Pimt) is a highly conserved enzyme utilising S-adenosylmethionine (AdoMet) to methylate aspartate residues of proteins damaged by age-related isomerisation and deamidation. We have been particularly interested in this enzyme since addition of the compound CGP3466 to primary rat astroglia cell cultures resulted in an upregulation of Pimt at the mRNA level, as shown here by semi-quantitative RT-PCR. CGP3466 is a compound related to the anti-Parkinson's drug R-(-)-deprenyl, which has been shown to protect from neural apoptosis induced by trophic factor withdrawal [Tatton et al., 1994. J. Neurochem. 63, 1572]. The pro-apoptotic gene Bax is required in the cascade of events following withdrawal [Deckwerth et al., 1996. Neuron 17, 401]. We therefore investigated whether Pimt overexpression was able to affect Bax-induced apoptosis in primary mouse cortical neurons. Our results show that Pimt is indeed able to protect from Bax-induced apoptosis. Furthermore, this activity is not restricted to brain-specific cell types, since the same effect is also demonstrated in COS1 cells. In addition, mutational analysis suggests that the protective effect is dependent on the adenosine methionine-binding motif, which is well conserved in protein methyltransferases, and that a mutation destroying this motif crucially affects cytoskeletal structures of the cell.

Postsynaptic actin and neuronal plasticity.

In the adult brain, actin is concentrated in dendritic spines where it can produce rapid changes in their shape. Through various synaptic junction proteins, this postsynaptic actin is linked to neurotransmitter receptors, influencing their function and, in turn, being influenced by them. Thus, the actin cytoskeleton is emerging as a key mediator between signal transmission and anatomical plasticity at excitatory synapses.

Volatile anesthetics block actin-based motility in dendritic spines.

Dendritic spines form the postsynaptic contact sites for most excitatory synapses in the brain. Spines occur in a wide range of different shapes that can vary depending on an animal's experience or behavioral status. Recently we showed that spines on living neurons can change shape within seconds in a process that depends on actin polymerization. We have now found that this morphological plasticity is blocked by inhalational anesthetics at concentrations at which they are clinically effective. These volatile compounds also block actin-based motility in fibroblasts, indicating that their action is independent of neuron-specific components and thus identifying the actin cytoskeleton as a general cellular target of anesthetic action. These observations imply that inhibition of actin dynamics at brain synapses occurs during general anesthesia and that inhalational anesthetics are capable of influencing the morphological plasticity of excitatory synapses in the brain.

Lithium reduces tau phosphorylation: effects in living cells and in neurons at therapeutic concentrations.

BACKGROUND: The mechanism of action of lithium remains to be determined satisfactorily. Recent studies suggested a possible role in inhibiting glycogen synthase kinase-3 (GSK-3), previously shown to phosphorylate the protein tau. Tau is expressed mainly in neurons, where it functions to stabilize microtubules in a phosphorylation-dependent manner. METHODS: Neurons and transfected non-neuronal cells were treated with lithium and the phosphorylation of tau at multiple epitopes examined by western blotting and by immunocytochemistry. Using green fluorescent protein as a tag we examined the effects of lithium on phosphorylated tau in living cells. RESULTS: Lithium reversibly reduced tau phosphorylation at therapeutic concentrations, and even at high concentrations did not alter neuronal morphology. Green fluorescent protein tagged-tau when phosphorylated by GSK-3 was diffusely distributed; treatment with lithium resulted in association with microtubules and then bundle formation. Removing lithium allowed observation of the dissolution of bundles and gradual dissociation of tau from microtubules in living cells. CONCLUSIONS: Lithium may have multiple effects in brain, but at least one action is demonstrated to be a relative inhibition of GSK-3-induced tau phosphorylation. These results carry implications for future studies of the actions of mood-stabilizing drugs and indeed of the molecular mechanisms of affective disorders.

Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.

Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruitment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminin-treated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.

The low density lipoprotein receptor gene family. Differential expression of two alpha2-macroglobulin receptors in the brain.

LR7/8B is a member of the low density lipoprotein receptor gene family that is specifically synthesized in the brain. Here we have functionally expressed in 293 cells the splice variant harboring eight ligand binding repeats (LR8B). As assessed by confocal microscopy, the expressed receptor is localized to the plasma membrane. Importantly, in cell binding experiments, we demonstrate that this protein is a receptor for activated alpha2-macroglobulin. Because to date low density lipoprotein receptor-related protein (LRP) has been shown to be the only alpha2-macroglobulin receptor in brain, we became interested in the expression pattern of both proteins at the cellular level in the brain. LR7/8B is expressed in large neurons and Purkinje cells of the cerebellum and in cells constituting brain barrier systems such as the epithelial cells of the choroid plexus, the arachnoidea, and the endothelium of penetrating blood vessels. Anti-LR7/8B antibody stains the plasma membrane, dendrites, and vesicular structures close to the cell membrane of neurons, especially of Purkinje cells. In contrast, LRP is present in patchy regions around large neurons and most prominently in the glomeruli of the stratum granulare of the cerebellum. This suggests that, contrary to LR7/8B, LRP is expressed in synaptic regions of the neurons; furthermore, there is a striking difference in the expression patterns of LR7/8B and LRP in the choroid plexus. Whereas LRP shows baso-lateral and apical localization in the epithelial cells, LR7/8B is restricted to the apical cell aspect facing the cerebrospinal fluid. Finally, these studies were extended to cultured primary rat neurons, where double immunofluorescence labeling with anti-LR7/8B and anti-microtubuli-associated protein 2 (MAP2) confirmed the somatodendritic expression of the receptor. Based upon these data, we propose that LR7/8B is involved in the clearance of alpha2-macroglobulin.proteinase complexes and/or of other substrates bound to alpha2-macroglobulin from the cerebrospinal fluid and from the surface of neurons.

GFP illuminates the cytoskeleton.

Until recently, cytoskeleton research has relied primarily on immunofluorescence microscopy techniques, requiring fixation and hence killing of the specimen before the analysis. The sole method for visualizing cytoskeletal dynamics in living cells has been the microinjection of purified and fluorescently labelled protein, but technical difficulties have precluded its widespread use. The recent introduction of green fluorescent protein (GFP) has enabled visualization of proteins and cytoskeletal dynamics with only minimal perturbations of the living cell and has opened new horizons for studying the cytoskeleton.

Rapid actin-based plasticity in dendritic spines.

Dendritic spines have been proposed as primary sites of synaptic plasticity in the brain. Consistent with this hypothesis, spines contain high concentrations of actin, suggesting that they might be motile. To investigate this possibility, we made video recordings from hippocampal neurons expressing actin tagged with green fluorescent protein (GFP-actin). This reagent incorporates into actin-containing structures and allows the visualization of actin dynamics in living neurons. In mature neurons, recordings of GFP fluorescence revealed large actin-dependent changes in dendritic spine shape, similar to those inferred from previous studies using fixed tissues. Visible changes occurred within seconds, suggesting that anatomical plasticity at synapses can be extremely rapid. As well as providing a molecular basis for structural plasticity, the presence of motile actin in dendritic spines implicates the postsynaptic element as a primary site of this phenomenon.

Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules.

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at approximately 20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.

Cytoskeletal maturation in cultured hippocampal slices.

We have studied the expression of genes for neuronal microtubule-associated proteins in organotypic hippocampal slice cultures using immunoblotting and polymerase chain reaction combined with reverse transcription on a single-slice basis. We found that for microtubule-associated protein 2 and tau the same developmental transition from embryonic to adult splice variants occurs in the cultures as has been described previously in intact brain. This finding indicates that the maturation profile of these proteins is not determined by extrinsic inputs but by a cell-autonomous programme or local factors within the hippocampus. Our study corroborates previous data for the maturation of hippocampal slice cultures and is also the first biochemical analysis on the level of the neuronal cytoskeleton of this widely used model system for the hippocampus.