Actin-binding Rho activating protein is expressed in the central nervous system of normal adult rats

Previous studies show that actin-binding Rho activating protein (Abra) is expressed in cardiomyocytes and vascular smooth muscle cells.In this study,we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence.Results showed that Abra immunostaining was located in neuronal nuclei,cytoplasm and processes in the central nervous system,with the strongest staining in the nuclei;in the cerebral cortex,Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer,external granular layer,external pyramidal layer,internal granular layer,internal pyramidal layer and polymorphic layer;in the hippocampus,the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer,with positive processes in molecular layer and orien layer;in the cerebellar cortex,Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer;in the spinal cord,Abra-immunopositive products covered the whole gray matter and white matter;co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes,but weakly in the nuclei.In addition,in the hippocampus,Abra was co-stained with F-actin only in neuronal processes,but not in the cell body.This study for the first time presents a comprehensive overview of Abra expression in the central nervous system,providing insights for further investigating the role of Abra in the mature central nervous system.     

Contributions of dendritic spines and perforated synapses to synaptic plasticity

The dynamic nature of synaptic connections has presented morphologists with considerable problems which, from a structural perspective, have frustrated the development of ideas on synaptic plasticity. Gradually, however, progress has been made on concepts such as the structural remodelling and turnover of synapses. This has been considerably helped by the recent elaboration of unbiased stereological procedures. The major emphasis of this review is on naturally occurring synaptic plasticity, which is regarded as an ongoing process in the postdevelopmental CNS. The focus of attention are PSs, with their characteristically discontinuous synaptic active zone, since there is mounting evidence that this synaptic type is indicative of synaptic remodelling and turnover in the mature CNS. Since the majority of CNS synapses can only be considered in terms of their relationship to dendritic spines, the contribution of these spines to synaptic plasticity is discussed initially. Changes in the configuration of these spines appears to be crucial for the plasticity, and these can be viewed in terms of the significance of the cytoskeleton, of various dendritic organelles, and also of the biophysical properties of spines. Of the synaptic characteristics that may play a role in synaptic plasticity, the PSD, synaptic curvature, the spinule, coated vesicles, polyribosomes, and the spine apparatus have all been implicated. Each of these is assessed. Especial emphasis is placed on PSs because of their ever-increasing significance in discussions of synaptic plasticity. The possibility of their being artefacts is dismissed on a number of grounds, including consideration of the results of serial section studies. Various roles, other than one in synaptic plasticity have been put forward in discussing PSs. Although relevant to synaptic plasticity, these include a role in increasing synaptic efficacy, as a more permanent type of synaptic connection, or as a route for the intercellular exchange of metabolites or membrane components. The consideration of many estimates of synaptic density, and of PS frequency, have proved misleading, since studies have reported diverse and sometimes low figures. A recent reassessment of PS frequency, using unbiased stereological procedures, has provided evidence that in some brain regions PSs may account for up to 40% of all synapses. All ideas that have been put forward to date regarding the role of PSs are examined, with particular attention being devoted to the major models of Nieto-Sampedro and co-workers, Carlin and Siekevitz, and Dyson and Jones. New ideas based on recent analysis of quantitative data and of 3-dimensional reconstructions of PSs are discussed. According to these, PSs constitute a separate, and more enduring, component of the synaptic population than NPSs, and have a major role in the maintenance of synaptic efficacy in the mature CNS.     

The calcium sensor synaptotagmin 1 is expressed and regulated in hippocampal postsynaptic spines

Synaptotagmin 1 is a presynaptic calcium sensor, regulating SNARE‐mediated vesicle exocytosis of transmitter. Increasing evidence indicate roles of SNARE proteins in postsynaptic glutamate receptor trafficking. However, a possible postsynaptic expression of synaptotagmin 1 has not been demonstrated previously. Here, we used postembedding immunogold electron microscopy to determine the subsynaptic localization of synaptotagmin 1 in rat hippocampal CA1 Schaffer collateral synapses. We report for the first time that synaptotagmin 1 is present in rat hippocampal postsynaptic spines, both on cytoplasmic vesicles and at the postsynaptic density. We further investigated whether postsynaptic synaptotagmin 1 is regulated during synaptic plasticity. In a rat model of chronic temporal lobe epilepsy, we found that presynaptic and postsynaptic concentrations of the protein are reduced compared to control animals. This downregulation may possibly be an adaptive measure to decrease both presynaptic and postsynaptic calcium sensitivity in excitotoxic conditions.     

Actin-binding proteins and signalling pathways associated with the formation and maintenance of dendritic spines

Introduction

Dendritic spines are the main sites of excitatory synaptic contacts. Moreover, they present plastic responses to different stimuli present in synaptic activity or damage, ranging from an increase or decrease in their total number, to redistribution of progenitor dendritic spines, to variations in their size or shape. However, the spines can remain stable for a long time.

Background

The use of experimental models has shown that different molecules of the F-actin binding and signalling pathways are closely related to the development, maintenance and plasticity of excitatory synapses, which could affect the number, size and shape of the dendritic spines; these mechanisms affect and depend on the reorganisation of the actin cytoskeleton.

Development

It is proposed that the filopodia are precursors of dendritic spines. Drebrin is an F-actin binding protein, and it is responsible for concentrating F-actin and PSD-95 in filopodia that will guide the formation of the new spines.

Conclusion

The specific mechanisms of actin regulation are an integral part in the formation, maturing process and plasticity of dendritic spines in association with the various actin cytoskeleton-binding proteins The signalling pathways mediated by small GTPases and the equilibrium between G-actin and F-actin are also involved.     

Clustering of delta glutamate receptors is regulated by the actin cytoskeleton in the dendritic spines of cultured rat Purkinje cells

The interaction of neurotransmitter receptors with the cytoskeleton is an important mechanism for the targeting of receptors to the postsynaptic membrane. Using cytoskeleton-perturbing agents, it was demonstrated that delta glutamate receptors, predominantly expressed on the dendritic spines of cerebellar Purkinje cells, are anchored to the actin cytoskeleton. The number of delta glutamate receptor-immunoreactive clusters was dramatically decreased following treatment of the Purkinje cells with the actin-disrupting agents, cytochalasin D or latrunculin A, without any significant effect on the number of presynaptic contacts of the granule cell axons. The clusters disrupted by latrunculin A were re-established 24 h after removal of the drug. These results suggest that morphological changes in the actin cytoskeleton regulate the delta glutamate receptor clustering on the dendritic spines, and may affect synaptic efficacy and plasticity.     

Organization of postsynaptic density proteins and glutamate receptors in axodendritic and dendrodendritic synapses of the rat olfactory bulb

Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.     

RIM3gamma is a postsynaptic protein in the rat central nervous system

RIMs (Rab3-interacting molecules) are synaptic proteins essential for neural transmission and plasticity. RIM1alpha has been implicated in membrane trafficking and regulation of secretory vesicle exocytosis in eukaryotic cells. Little information is as yet available on RIM3gamma. In the present study, we investigated the cellular expression, subcellular distribution, and possible functions of RIM3gamma in the rat CNS. Rim3gamma cDNA was subcloned and the protein expressed in vitro for the generation and purification of a rabbit anti-RIM3gamma polyclonal antibody. In situ hybridization histochemistry, immunohistochemistry, and immunoelectron microscopy were performed to map expression of the mRNA and protein in the rat CNS. Our results indicated widespread distribution of RIM3gamma in diverse CNS neuronal cell types. The mRNA was found mainly in the cell bodies, whereas the protein immunoreactivity was localized chiefly to neuronal dendrites and to the postsynaptic densities as visualized under the light and electron microscope. This postsynaptic placement of RIM3gamma is distinct from the presynaptic localization of RIM1alpha but may contribute to regulating synaptic transmission and plasticity. The identification of RIM3gamma as a postsynaptic protein has functional implications for CNS synapse functions.     

Cellular and subcellular distribution of spinophilin, a PP1 regulatory protein that bundles F-actin in dendritic spines

Spinophilin is an actin binding protein that positions protein phosphatase 1 next to its substrates in dendritic spines. It contains a single PDZ domain and has the biochemical characteristics of a cytoskeletal scaffolding protein. Previous studies suggest that spinophilin is present in most spines, but the concentration of spinophilin varies from brain region to region in a manner that does not simply reflect differences in spine density. Here, we show that spinophilin is enriched in the great majority of dendritic spines in cerebral cortex, caudatoputamen, hippocampal formation, and cerebellum, irrespective of regional differences in spinophilin concentration. In addition, spinophilin is present postsynaptic to asymmetrical contacts on interneuronal dendritic shafts. We further show that, in hippocampus and ventral pallidum, spinophilin is occasionally present in dendritic shafts adjacent to gamma-aminobutyric acid-containing contacts. Thus, the functional role of spinophilin may not be exclusively restricted to excitatory synapses and may be significant at a small fraction of inhibitory contacts. These data also suggest that the concentration of spinophilin per spine is variable and is likely regulated by local physiological factors and/or regional influences.     

Change in the shape and density of dendritic spines caused by overexpression of acidic calponin in cultured hippocampal neurons

Dendritic spines are morphing structures believed to provide a cellular substrate for synaptic plasticity. It has been suggested that the actin cytoskeleton is the target of molecular mechanisms regulating spine morphology. Here we hypothesized that acidic calponin, an actin-binding protein, is one of the key regulators of actin filaments during spine plasticity. Our data showed that the overexpression of acidic calponin-GFP (green fluorescent protein) in primary cultures of rat hippocampal neurons causes an elongation of spines and an increase of their density as compared with those of GFP-expressing neurons. These effects required the actin-binding domains of acidic calponin. The close apposition of the presynatic marker synaptophysin to these long spines and the presence of specific postsynaptic markers actin, PSD-95, NR1, and GluR1 suggested the existence of functional excitatory synaptic contacts. Indeed, electrophysiological data showed that the postsynaptic overexpression of acidic calponin enhanced the frequency of miniature excitatory postsynaptic currents as compared with that of GFP-expressing neurons, but did not affect their properties such as amplitude, rise time, and half width. Studies in heterologous cells revealed that acidic calponin reorganized the actin filaments and stabilized them. Taken together, these findings show that acidic calponin regulates dendritic spine morphology and density, likely via regulation of the actin cytoskeleton reorganization and dynamic. Furthermore, the acidic calponin-induced spines are able to establish functional glutamatergic synapses. Such data suggest that acidic calponin is a key factor in the regulation of spine plasticity and synaptic activity.     

Microanatomy of dendritic spines: emerging principles of synaptic pathology in psychiatric and neurological disease.

Psychiatric and neurologic disorders ranging from mental retardation to addiction are accompanied by structural and functional alterations of synaptic connections in the brain. Such alterations include abnormal density and morphology of dendritic spines, synapse loss, and aberrant synaptic signaling and plasticity. Recent work is revealing an unexpectedly complex biochemical and subcellular organization of dendritic spines. In this review, we highlight the molecular interplay between functional domains of the spine, including the postsynaptic density, the actin cytoskeleton, and membrane trafficking domains. This research points to an emerging level of analysis--a microanatomical understanding of synaptic physiology--that will be critical for discerning how synapses operate in normal physiologic states and for identifying and reversing microscopic changes in psychiatric and neurologic disease.     

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.     

Selective localization of high concentrations of F-actin in subpopulations of dendritic spines in rat central nervous system: a three-dimensional electron microscopic study

Dendritic spines differ considerably in their size, shape, and internal organization between brain regions. We examined the actin cytoskeleton in dendritic spines in hippocampus (areas CA1, CA3, and dentate gyrus), neostriatum, and cerebellum at both light and electron microscopic levels by using a novel high-resolution photoconversion method based in the high affinity of phalloidin for filamentous (F)-actin. In all brain regions, labeling was strongest in the heads of dendritic spines, diminishing in the spine neck. The number of labeled spines varied by region. Compared with the cerebellar molecular layer and area CA3, where nearly every dendritic spine was labeled, less than half the spines were labeled in CA1, dentate gyrus, and neostriatum. Serial section reconstructions of spines in these areas indicated that phalloidin labeling was restricted to the largest and most morphologically diverse dendritic spines. The resolution of the photoconversion technique allowed us to examine the localization and organization of actin filaments in the spine. The most intense staining for actin was found in the postsynaptic density and associated with the spines internal membrane system. In mushroom-shaped spines, F-actin staining was particularly strong between the lamellae of the spine apparatus. Three-dimensional reconstruction of labeled spines by using electron tomography showed that the labeled dense material was in continuity with the postsynaptic density. These results highlight differences in the actin cytoskeleton between different spine populations and provide novel information on the organization of the actin cytoskeleton in vivo.     

Perforated synapses on double-headed dendritic spines: a possible structural substrate of synaptic plasticity

Examination of axospinous synapses in serial sections obtained from the middle molecular layer of the rat dentate gyrus has revealed that some of them involve double-healed dendritic spines. Each spine head is apposed by a separate axon terminal with which it always forms a perforated synaptic contact distinguished by a discontinuous postsynaptic density. The number of perforated synapses on double-headed spines was estimated as a synapse-to-neuron ratio with the aid of the disector technique and found to be significantly increased in rats kindled via medial perforant path stimulation. These results support the notion that perforated synapses involving double-headed dendritic spines represent a structural modification related to enhanced synaptic efficacy.     

Plasticity of dendritic spines: Molecular function and dysfunction in neurodevelopmental disorders

Dendritic spines are tiny postsynaptic protrusions from a dendrite that receive most of the excitatory synaptic input in the brain. Functional and structural changes in dendritic spines are critical for synaptic plasticity, a cellular model of learning and memory. Conversely, altered spine morphology and plasticity are common hallmarks of human neurodevelopmental disorders, such as intellectual disability and autism. The advances in molecular and optical techniques have allowed for exploration of dynamic changes in structure and signal transduction at single‐spine resolution, providing significant insights into the molecular regulation underlying spine structural plasticity. Here, I review recent findings on: how synaptic stimulation leads to diverse forms of spine structural plasticity; how the associated biochemical signals are initiated and transmitted into neuronal compartments; and how disruption of single genes associated with neurodevelopmental disorders can lead to abnormal spine structure in human and mouse brains. In particular, I discuss the functions of the Ras superfamily of small GTPases in spatiotemporal regulation of the actin cytoskeleton and protein synthesis in dendritic spines. Multiple lines of evidence implicate disrupted Ras signaling pathways in the spine structural abnormalities observed in neurodevelopmental disorders. Both deficient and excessive Ras activities lead to disrupted spine structure and deficits in learning and memory. Dysregulation of spine Ras signaling, therefore, may play a key role in the pathogenesis of multiple neurodevelopmental disorders with distinct etiologies.     

Tissue-type plasminogen activator is a homeostatic regulator of synaptic function in the central nervous system

Membrane depolarization induces the release of the serine proteinase tissue-type plasminogen activator(t PA) from the presynaptic terminal of cerebral cortical neurons.Once in the synaptic cleft this t PA promotes the exocytosis and subsequent endocytic retrieval of glutamate-containing synaptic vesicles,and regulates the postsynaptic response to the presynaptic release of glutamate.Indeed,t PA has a bidirectional effect on the composition of the postsynaptic density(PSD) that does not require plasmin generation or the presynaptic release of glutamate,but varies according to the baseline level of neuronal activity.Hence,in inactive neurons t PA induces phosphorylation and accumulation in the PSD of the Ca~(2+)/calmodulin-dependent protein kinase IIα(pCa MKIIα),followed by pCa MKIIα-induced phosphorylation and synaptic recruitment of Glu R1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA) receptors.In contrast,in active neurons with increased levels of pCa MKIIα in the PSD t PA induces pCa MKIIα and p Glu R1 dephosphorylation and their subsequent removal from the PSD.These effects require active synaptic N-methyl-D-aspartate(NMDA) receptors and cyclin-dependent kinase 5(Cdk5)-induced phosphorylation of the protein phosphatase 1(PP1) at T320.These data indicate that t PA is a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate via bidirectional regulation of the pCa MKIIα/PP1 switch in the PSD.     

Expression of a phosphorylated isoform of MAP1B is maintained in adult central nervous system areas that retain capacity for structural plasticity

Microtubule-associated protein IB (MAP1B) is the first MAP to be detected in the developing nervous system, and it becomes markedly down-regulated postnatally. Its expression, particularly that of its phosphorylated isoform, is associated with axonal growth. To determine whether adult central nervous system (CNS) areas that retain immunoreactivity for MAP1B are associated with morphological plasticity, we compared the distribution of a phosphorylated MAP1B isoform (MAP1B-P) to the distribution of total MAP1B protein and MAP1B-mRNA. Although they were present only at very low levels, both protein and message were found ubiquitously in almost all adult CNS neurons. The intensity of staining, however, varied markedly among different regions, with only a few nuclei retaining relatively high levels. MAP1B-P was restricted to axons, whereas total MAP1B was present in cell bodies and processes. Relatively to total MAP1B protein and its mRNA, MAP1B-P levels decreased more dramatically with maturation, and they were detectable in only a few specific areas that underwent structural modifications. These included primary afferents and motor neurons, olfactory tubercles, habenular and raphe projections to interpeduncular nuclei, septum, and the hypothalamus. The distribution pattern of MAP1B-P was compared to that of the embryonic N-CAM rich in polysialic acid (PSA-NCAM). We found that the PSA-NCAM immunostaining was largely overlapped with that of MAP1B-P in the adult CNS. These results suggest that, like PSA-NCAM, MAP1B may be one of the molecules expressed during brain development that also plays a role in structural remodeling in the adult. © 1996 Wiley-Liss, Inc.     

Palladin is expressed preferentially in excitatory terminals in the rat central nervous system.

Palladin is a recently described intracellular protein associated with the actin cytoskeleton and cell adhesion in fibroblasts. In Western and Northern blot analyses, palladin expression is ubiquitous in embryonic mice, but it is down-regulated dramatically in most adult tissues. Significant amounts of palladin persist in the brain of adult rodents, as assessed by Western blot analysis. With this work, we extend preliminary observations and determine the overall distribution and subcellular location of palladin throughout the rat brain. In sagittal and coronal sections of the central nervous system, immunostain for palladin is present throughout the brain and spinal cord, but not uniformly. The densest regions of immunostain include the olfactory bulb, cerebral and cerebellar cortex, hippocampus, amygdala, superior colliculus, and superficial laminae of the spinal dorsal horn. Because immunostain characteristically is punctate, we performed double staining for palladin and the presynaptic marker synaptophysin. Confocal microscopy showed that palladin-immunopositive puncta are also immunopositive for synaptophysin; the proportion of synaptophysin-immunopositive puncta that also stained for palladin ranged from 100% of mossy fiber terminals in field CA3 of the hippocampus and in the cerebellar cortex to 60--70% of terminals in the cerebral cortex, striatum, and spinal dorsal horn. The presence of palladin in synaptic terminals was confirmed by electron microscopy. Because immunostained terminals commonly establish asymmetric synapses, the selectivity of palladin expression in synaptic terminals was tested by double staining for palladin and gamma-aminobutyric acid. The modest level of colocalization in this material at both the light microscopic and electron microscopic levels suggests a selectivity of palladin for terminals that release excitatory neurotransmitters. As concomitant work in cell cultures has shown that palladin participates in axonal development and migration, the present results suggest that palladin persists at excitatory synapses of the adult nervous system.     

Chol-1 is a cholinergic marker in the human central nervous system.

The cholinergic-specific gangliosides Chol-1 alpha and beta were detected in human brain and spinal cord by immune staining of thin-layer chromatography (TLC) plates on which ganglioside extracts had been separated. The colocalization of choline acetyltransferase (ChAT) and the Chol-1 antigens in ventral horn motoneurons was demonstrated immunocytochemically. In analytical studies, Chol-1 was found to be more concentrated in dorsal cord than ventral but the reverse was true of ChAT. This difference was explained by differences in the subcellular location of the two markers. Within each region of the thoracic cord the levels of ChAT and Chol-1 in different cords showed covariance. The expected fall of ChAT and Chol-1 in amyotrophic lateral sclerosis (ALS) cords was not seen and possible reasons for this are discussed.