Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein

While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.     

Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser133 residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser133 phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters revealed bands at approximately 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-D-aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 microM], or native glutamate (1 microM) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser133. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 microM) or forskolin (1 microM). Simultaneous treatment with melatonin (1 microM) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir was high, whereas expression of pCREB-ir was low. Treatment with glutamate (1 microM) or dopamine (1 microM) had no effect on total CREB-ir, but increased pCREB-ir in approximately 50 and 30% of cells, respectively, whereas forskolin (1 microM) increased pCREB-ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) revealed that glutamate (1 microM) increased nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in gamma-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 microM) was more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increased pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei.     

Plasticity of synapses in the rat neostriatum after unilateral lesion of the nigrostriatal dopaminergic pathway

In the 6-hydroxydopamine model of Parkinson's disease in the rat, there is a significant reduction in the number of dendritic spines on the principal projection neurons in the neostriatum, presumably attributable to loss of the nigrostriatal dopamine input. These spines invariably receive input from terminals forming asymmetric synapses that originate mainly from the cortex. The object of the present study was to determine the fate of those terminals after the loss of dendritic spines. Unbiased estimates of synaptic density and absolute numbers of synapses in a defined volume of the neostriatum were made using the "disector" and Cavalieri techniques. Numerical synaptic density of asymmetric synaptic contacts was 17% lower in the neostriatum deprived of dopamine innervation and, in absolute terms, there were 3 billion (19%) fewer contacts. The numerical density of a subpopulation of asymmetric contacts on dendritic spines that have complex or perforated synaptic specializations and normally make up 9% of the asymmetric population was 44% higher on the experimental side. Asymmetric synapses were found to be enriched in glutamate using postembedding immunogold labeling. The present observations demonstrate that the loss of spines previously reported after 6-hydroxydopamine lesions is accompanied by a loss of asymmetric synapses rather than by the movement of synapses from spines to other postsynaptic targets. The study also demonstrates that there is an increase in complex synaptic interactions that have been implicated in synaptic plasticity in other regions of the CNS after experimental manipulations.     

Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.     

Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons

The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated by phosphorylation and dephosphorylation. We have previously determined that threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, we now report the decreased phosphorylation state of high molecular weight microtubule-associated protein 2 during the development of cultured cerebellar granule neurons. Phosphorylation of high molecular weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, it is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site which can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate.     

Unique regulation of immediate early gene and tyrosine hydroxylase expression in the odor-deprived mouse olfactory bulb

Tyrosine hydroxylase (TH), expressed in a population of periglomerular neurons intrinsic to the olfactory bulb, displays dramatic down-regulation in response to odor deprivation. To begin to elucidate the importance of immediate early genes (IEG) in TH gene regulation, the present study examined expression of IEGs in the olfactory bulb in response to odor deprivation. In addition, the composition of TH AP-1 and CRE binding complexes was investigated in control and odor-deprived mice. Immunocytochemical studies showed that c-Fos, Fos-B, Jun-D, CRE-binding protein (CREB), and phosphorylated CREB (pCREB) are colocalized with TH in the dopaminergic periglomerular neurons. Unilateral naris closure resulted in down-regulation of c-Fos and Fos-B, but not Jun-D, CREB, or pCREB, in the glomerular layer of the ipsilateral olfactory bulb. Gel shift assays demonstrated a significant decrease (32%) in TH AP-1, but not CRE, binding activity in the odor-deprived bulb. Fos-B was found to be the exclusive member of the Fos family present in the TH AP-1 complex. CREB, CRE modulator protein (CREM), Fos-B, and Jun-D, but not c-Fos, all contributed to the CRE DNA-protein complex. These results indicated that Fos-B, acting through both AP-1 and CRE motifs, may be implicated in the regulation of TH expression in the olfactory bulb dopaminergic neurons.     

Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines

Neurons contain distinct compartments including dendrites, dendritic spines, axons and synaptic terminals. The molecular mechanisms that generate and distinguish these compartments, although largely unknown, may involve the small GTPases Rac and Cdc42, which appear to regulate actin polymerization. Having shown that perturbations of Rac1 activity block the growth of axons but not dendrites of Drosophila neurons, we investigated whether this also applies to mammals by examining transgenic mice expressing constitutively active human Rac1 in Purkinje cells. We found that these mice were ataxic and had a reduction of Purkinje-cell axon terminals in the deep cerebellar nuclei, whereas the dendritic trees grew to normal height and branched extensively. Unexpectedly, the dendritic spines of Purkinje cells in developing and mature cerebella were much reduced in size but increased in number. These 'mini' spines often form supernumerary synapses. These differential effects of perturbing Rac1 activity indicate that there may be distinct mechanisms for the elaboration of axons, dendrites and dendritic spines.     

The delayed entry of thoracic neural crest cells into the dorsolateral path is a consequence of the late emigration of melanogenic neural crest cells from the neural tube

Recent studies have shown that changes in dendritic architecture are an important component of functional plasticity in the adult central nervous system. In the present study, we determined whether gonadectomy induces changes in dendritic architecture in the arcuate nucleus, a target tissue for gonadal hormones. A combination of retrograde labeling with systemically injected Fluoro-Gold and intracellular injection of neurons in a fixed-slice preparation was used to examine the morphology of neuroendocrine neurons in the rat arcuate nucleus. Intracellullary filled arcuate neuroendocrine neurons (8-21 neurons per brain) from intact (n = 5) and orchidectomized (n = 5) animals were reconstructed with the aid of a computer microscope. A quantitative analysis revealed that orchidectomy had no effect on the number and distribution of Fluoro-Gold-labeled neuroendocrine neurons in the rat arcuate nucleus. The morphology of arcuate neuroendocrine neurons in intact animals was relatively simple, with the majority of neurons (79%) having only two primary dendrites and few dendritic spines. Compared with intact controls, arcuate neuroendocrine neurons in the orchidectomized group had significantly larger somatic profile areas and exhibited significant increases in dendrite length, dendrite volume, terminal branch number, and spines per unit length of dendrite. The increase in terminal branch number in orchidectomized animals was due primarily to the appearance of short branches that gave a striking, claw-like appearance to many of the distal dendrites. These results provide evidence for hormonal regulation of dendritic morphology of arcuate neuroendocrine neurons in adult mammals.     

Neurons and synaptic patterns in the deep layers of the superior colliculus of the cat. A Golgi and electron microscopic study

Golgi and electron microscopic observations were made on the neurons in the deep layers (below the stratum opticum) of the cat superior colliculus. Large neurons, 35-60 micrometers in somal diameter, occur mainly in the lateral two-thirds of the colliculus. They have numerous somatic and dendritic spines and receive a large number of axon terminals (bouton covering ratio: more than 70%). The medium-sized neurons (20-30 micrometer), with a moderate number of dendritic spines, show a lower bouton covering ratio (25-30%). The ratio for small neurons (8-15 micrometers), with very few dendritic spines, is less than 10%. The medium-sized and small neurons are distributed throughout the colliculus and show marked variability in the dendritic arrangement. Seven different types of axon terminals were distinguished: types I, II, V, and VII form asymmetrical and types III, IV, and VI symmetrical synapses. Type I terminals represent small boutons containing predominantly spherical vesicles, and are in contact mainly with small dendritic profiles. Type II terminals are medium-sized and slender, contain a mixture of spherical and slightly oval vesicles, and make synaptic contacts with small to medium-sized dendrites and somatic spines. This type of terminal is occasionally presynaptic to vesicle-containing dendrites (type VIII). Type III terminals are small, contain flattened vesicles predominantly, and are presynaptic to a wide variety of neuronal elements in the deep layers of the superior colliculus. Type IV terminals are represented by medium to large-sized boutons that contain pleomorphic vesicles and make synaptic contacts chiefly with the large neurons. Type V and VI terminals exhibit a quite dense axoplasmic matrix and mainly contact the large neurons. Type VII terminals are often in the form of boutons en passant and contain numerous large granular vesicles. Pleomorphic vesicle-containing dendrites (type VIII terminals) are also observed to participate in the axodendrodendritic serial synapses.     

Calcium-evoked dendritic exocytosis in cultured hippocampal neurons. Part I: trans-Golgi network-derived organelles undergo regulated exocytosis

Exocytosis is a widely observed cellular mechanism for delivering transmembrane proteins to the cell surface and releasing signaling molecules into the extracellular space. Calcium-evoked exocytosis, traditionally thought to be restricted to presynaptic specializations in neurons, has been described recently in many cells. Here, calcium-evoked dendritic exocytosis (CEDE) is visualized in living cultured hippocampal neurons. Organelles that undergo CEDE are in somata, dendrites, and perisynaptic regions, identified by using immunocytochemistry and correlative light and electron microscopy. CEDE is regulated developmentally: neurons <9 d in vitro do not show CEDE. In addition, CEDE is blocked by tetanus toxin, an inhibitor of regulated exocytosis, and nocodazole, an inhibitor of microtubule polymerization. Organelles that undergo CEDE often are found on the base of spines, putative sites of synaptic plasticity. CEDE therefore could be involved in structural and functional modification of spines and could play a role in synaptic plasticity, where it might involve changes in receptor/channel density, release of active compounds having effect on pre- and postsynaptic function, and/or growth of synaptic structures.     

Isoform specificity in the relationship of actin to dendritic spines

Dendritic spines contain high concentrations of actin, but neither the isoforms involved nor the mechanism of accumulation is known. In situ hybridization with specific probes established that beta- and gamma-cytoplasmic actins are selectively expressed at high levels by spine-bearing neurons. Transfecting cultured hippocampal neurons with epitope-tagged actin isoforms showed that cytoplasmic beta- and gamma-cytoplasmic actins are correctly targeted to spines, whereas alpha-cardiac muscle actin, which is normally absent from neurons, formed aggregates in dendrites. The transfected actin cDNAs contained only coding domains, suggesting that spine targeting involves amino acid sequences in the proteins, an interpretation supported by experiments with chimeric cDNAs in which C-terminal actin sequences were found to be determinative in spine targeting. By contrast to actin, microtubule components, including tubulin and MAP2, were restricted to the dendritic shaft domain. The close association of cytoplasmic actins with spines together with their general involvement in cell surface motility further supports the idea that actin motility-based changes in spine shape may contribute to synaptic plasticity.     

Tap withdrawal circuit interneurons require CREB for long-term habituation in Caenorhabditis elegans.

We investigated the role of the Caenorhabditis elegans CREB (cAMP response element binding protein) homologue, crh-1, in response to tap (nonlocalized mechanosensory stimulation) and tap habituation. Worms with a loss-of-function mutation in crh-1 performed smaller reversals in response to tap than did wild-type worms and did not show long-term memory for spaced training 24-hr posttraining; however, they did show short-term habituation to tap stimuli when stimuli were presented at both 10-s and 60-s interstimulus intervals, and showed 12-hr intermediate memory for spaced habituation training (intermediate-term memory). Expressing CRH-1 broadly throughout the nervous system and in a subset of interneurons of the tap withdrawal circuit, but not in the mechanosensory neurons, rescued the long-term memory defects observed in crh-1 mutants. Here we show for the first time that CREB is required for long-term habituation and show that the interneurons of the tap withdrawal response circuit are the locus of plasticity for long-term mechanosensory habituation in C. elegans. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.     

Dendritic remodeling of dentate granule cells following a combined entorhinal cortex/fimbria fornix lesion

This study examined the time course of dendritic reorganization of dentate granule neurons of the hippocampus following the loss of input from both the fimbria fornix (FF) and the entorhinal cortex (EC). We used the Golgi-Cox stain to assess the morphology of dentate granule neurons at six postlesion time points (4, 8, 14, 30, 45, and 60 days) and dendritic measures included total dendritic length, number of segments, number of branch points, and spine density. We found that as early as 4 days postlesion, total dendritic length and number of segments were significantly decreased with the greatest change occurring in the distal parts of the dendritic arbor located in the outer molecular layer of the dentate gyrus. Dendritic measures related to segment number and dendritic length returned to 70% of intact values by 30 days postlesion and were not significantly different from unlesioned rats at 45 and 60 days postlesion. In contrast, the recovery of spine density was transient. Spine density in the outer molecular layer of the dentate gyrus decreased by 60% at 4 days postlesion and returned to 87% of intact values by 30 days postlesion. However, there was a second loss of dendritic spines along the distal portion of the dendrite between 30 and 60 days postlesion. These data provide evidence that the ability of granule neurons to recover a dendritic morphology similar to that of unlesioned rats is impaired following the combined EC/FF lesion and that the "secondary loss" of dendritic spine density on granule neurons may significantly limit the chances of the hippocampus reforming a synaptic circuitry that could lead to functional recovery after the EC/FF lesion.     

Long-lasting morphological changes in dendritic spines of dentate granular cells following stimulation of the entorhinal area

Stimulation of the perforant path induces a long-lasting increase in the area of dendritic spines, which are sites of termination of the stimulated pathway in the distal third of the dentate molecular layer. No enlarged spines were found in the proximal third of the dentate molecular layer, where the commissural afferents terminate. Following a single tetanic stimulus of 30 sec duration at 30/sec, spines became significantly larger by 15%, 38%, 35% and 23% within poststimulation intervals of 2-6 min, 10-60 min, 4-8 h, and 23 h, respectively. Axon terminals decreased their area by 15% within the 2-6 min interval and the vesicle density was decreased by 19% within the 10-60 min interval. Both changes were reversible and terminals resumed their prestimulation condition at longer intervals (greater than 4 h). The initial enlargement of spines was interpreted as being due to a glutamate-induced increase in the sodium permeability of the spine membrane, whereas for the long-lasting enlargement an increase in protein synthesis was postulated. The long-lasting enlargement of dendritic spines in the dentate molecular layer following a short train of stimuli delivered to the perforant path, supports the postulate which links such a change to the mechanism of long-lasting postactivation potentiation observed in this pathway.