Activity‐dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live‐cell imaging

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning‐related synaptic plasticity and potentiating N‐methyl‐D ‐aspartate (NMDA) receptor‐dependent signaling. These findings suggest that tPA may localize to, and undergo activity‐dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization‐induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense‐core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca⁺² concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse‐pinch‐linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA‐type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Experience-dependent pruning of dendritic spines in visual cortex by tissue plasminogen activator

Sensory experience physically rewires the brain in early postnatal life through unknown processes. Here, we identify a robust anatomical consequence of monocular deprivation (MD) in layer II/III of visual cortex that corresponds to the rapid, functional loss of responsiveness preceding any changes in axonal input. Protrusions on pyramidal cell apical dendrites increased steadily after eye opening, but were transiently lost through competitive mechanisms after brief MD only during the physiological critical period. Proteolysis by tissue-type plasminogen activator (tPA) conversely declined with age and increased with MD only in young mice. Targeted disruption of tPA release or its upstream regulation by glutamic acid decarboxylase (GAD65) prevented MD-induced spine loss that was pharmacologically rescued concomitant with critical period plasticity. An extracellular mechanism for structural remodeling that is limited to the binocular zone upon proper detection of competing inputs thus links early sensory experience to visual function.     

Calcium signaling in dendritic spines of hippocampal neurons

Intracellular Ca²⁺ dynamics have been measured using imaging techniques in dendrites and spines of CA3 hippocampal neurons in brain slice under both acute and tissue culture conditions. In response to presynatic stimulation, micromolar levels of Ca²⁺ are rapidly reached in spines of distal dendrites. If stimulus parameters are chosen judiciously so as to minimize postsynaptic firing, then the dendrite shaft increases are far less. Spine Ca²⁺ increases are largely dependent upon activation of NMDA receptors. At the large mossy fiber synapses, presynaptic stimuli also produce large Ca²⁺ increases but the differences in shaft-spine Ca²⁺ levels are much less; often they are insignificant. Also at these locations, postsynaptic firing, without presynaptic stimulation is sufficient to produce large increase in spine Ca²⁺ levels. 1994 John Wiley & Sons, Inc.     

Activity-dependent growth of new dendritic spines is regulated by the proteasome

Growth of new dendritic spines contributes to experience-dependent circuit plasticity in the cerebral cortex. Yet the signaling mechanisms leading to?new spine outgrowth remain poorly defined. Increasing evidence supports that the proteasome is an important mediator of activity-dependent neuronal signaling. We therefore tested the role of the proteasome in activity-dependent spinogenesis. Using pharmacological manipulations, glutamate uncaging, and two-photon imaging of GFP-transfected hippocampal pyramidal neurons, we demonstrate that acute inhibition of the proteasome blocks activity-induced spine outgrowth. Remarkably, mutation of serine 120 to alanine of the Rpt6 proteasomal subunit in individual neurons was sufficient to block activity-induced spine outgrowth. Signaling through NMDA receptors and CaMKII, but not PKA, is required to facilitate spine outgrowth. Moreover, abrogating CaMKII binding to the NMDA receptor abolished activity-induced spinogenesis. Our data support a model in which neural activity facilitates spine outgrowth via an NMDA receptor- and CaMKII-dependent increase in local proteasomal degradation.     

Spike-associated fast contraction of dendritic spines in cultured hippocampal neurons.

Dendritic spines have long been known to contain contractile elements and have recently been shown to express apparent spontaneous motility. Using high-resolution imaging of dendritic spines of green-fluorescent protein (GFP)-expressing, patch-clamped hippocampal neurons in dissociated culture, we find that bursts of action potentials, evoked by depolarizing current pulses, cause momentary contractions of dendritic spines. Blocking calcium currents with cobalt prevented these twitches. In additional experiments with neurons loaded via a micropipette with calcium-sensitive and insensitive dyes, spontaneous calcium transients were associated with a rapid contraction of the spine head. The spine twitch was prolonged by tetraethylammonium or bicuculline, which enhance calcium transients, and was blocked by the actin polymerization antagonist latrunculin-B. The spine twitch may be instrumental in modulating reactivity of the NMDA receptor to afferent stimulation, following back-propagating action potentials.     

Estradiol induces formation of dendritic spines in hippocampal neurons: functional correlates

Dissociated cultured rat hippocampal pyramidal neurons respond to estradiol with a time-dependent, twofold increase in density of their dendritic spines. This effect is mediated by an estrogen receptor, probably of the alpha nuclear receptor type. In searching for the molecular mechanisms leading from the initial activation of the estrogen receptor to the final formation of new dendritic spines, we found that estradiol acts on GABAergic interneurons expressing the estrogen receptor by decreasing their inhibitory tone. In culture, this is assumed to cause a shift in the balance between excitation and inhibition toward enhanced excitation, overactivation of the pyramidal neurons, and subsequent formation of novel dendritic spines. The action of estradiol on spine formation is mediated by phosphorylation of cyclic AMP response element binding protein in the pyramidal neurons and is blocked when inhibition is enhanced by diazepam and when excitation is blocked by tetrodotoxin. Progesterone blocks the effect of estradiol on dendritic spines through its conversion to tetrahydroprogesterone, which enhances GABAergic inhibition. Subsequent to formation of novel dendritic spines, there is an increase in the density of glutamatergic receptors in the affected cells, an increase in the cellular calcium response to glutamate, and an increase in network synaptic activity among the cultured neurons.     

Rapid WAVE dynamics in dendritic spines of cultured hippocampal neurons is mediated by actin polymerization

The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE) complex has been proposed to link Rho GTPase activity with actin polymerization but its role in neuronal plasticity has never been documented. We now examined the presence, distribution and dynamics of WAVE3 in cultured hippocampal neurons. WAVE3 was localized to dendritic spines via its N-terminal domain. Green fluorescent protein (GFP)-tagged WAVE3 clusters demonstrate an F-actin-dependent high rate of local motility. Constitutive Rac activation translocates WAVE3 (via the N-terminus), to the leading edge of lamellipodia. Also, spinogenesis is associated with actin-based motility of the WAVE3 protein. Brain specific WAVE1 showed similar localization and effects on spine density. Cytoplasmic fragile X mental retardation protein interacting protein (CYFIP) and non-catalytic region of tyrosine kinase adaptor protein 1 (NCK-1), proteins that are assumed to complex with WAVE, have a somewhat similar cellular distribution and motility. We propose that the WAVE complex is a downstream effector of the Rac signaling cascade, localized to sites of novel synaptic contacts by means of its N-terminal domain, to guide local actin polymerization needed for morphological plasticity of neurons.     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.     

Matrix metalloproteinase-7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro-environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)-7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP-7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom-shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F-actin from spine heads into thick, rope-like structures in the dendritic shaft. Strikingly, MMP-7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel-specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.     

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).     

A factor from endothelium facilitates activation of plasminogen by tissue plasminogen activator

A component extracted from endothelium and partially purified has been found to have a capacity to enhance the rate of plasminogen activation by tissue-type plasminogen activator. The mechanism of action of this cofactor differs from that of others, such as fibrin.     

Conductance transients onto dendritic spines in a segmental cable model of hippocampal neurons.

Dendritic shaft (Zd) and spine (Zsp) input impedances were computed numerically for sites on hippocampal neurons, using a segmental format of cable calculations. The Zsp values for a typical spine appended onto a dendritic shaft averaged less than 2% higher than the Zd values for the adjacent dendritic shaft. Spine synaptic inputs were simulated by a brief conductance transient, which possessed a time integral of 12 X 10(-10)S X ms. This input resulted in an average peak spine response of 20 mV for both dentate granule neurons and CA1 pyramidal cells. The average spine transient was attenuated less than 2% in conduction across the spine neck, considering peak voltage, waveform parameters, and charge transfer. The spine conductance transient resulted in an average somatic response of 100 microV in the dentate granule neurons, because of passive electrotonic propagation. The same input transient was also applied to proximal and distal sites on CA1 pyramidal cells. The predicted responses at the soma demonstrated a clear difference between the proximal and distal inputs, in terms of both peak voltage and waveform parameters. Thus, the main determinant of the passive propagation of transient electrical signals in these neurons appears to be dendritic branching rather than signal attenuation through the spine neck.     

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity-producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1- to 2-h-old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.     

Simultaneous transport of different localized mRNA species revealed by live-cell imaging

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.     

Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells

We have examined the effect of phorbol esters and cAMP elevating compounds on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) secretion. Phorbol esters induce a time- and dose-dependent increase in tPA release from endothelial cells, while forskolin, isobutylmethylxanthine, dibutyryl cAMP, and 8-bromo-cAMP had no significant stimulatory effect on tPA secretion. However, elevation of cAMP simultaneously with phorbol ester treatment potentiated the phorbol ester-induced release of tPA 6 times from 22.2 ng/ml with phorbol myristate acetate (PMA) alone to 122.1 ng/ml (PMA and forskolin). Potentiation was dose-dependent (half-maximal potentiation = 4 microM forskolin), and tPA release was enhanced at all stimulatory concentrations of PMA with no change in the PMA concentrations causing half-maximal or maximum tPA release. The kinetics of release was also similar in PMA versus PMA-forskolin-treated cells. A 4-h delay was observed, enhanced release was transient, and was followed by the onset of a refractory period. In contrast, elevation of cAMP reduced constitutive secretion of PAI-1 by 30-40% and prevented the increase in PAI-1 secretion stimulated by PMA. Elevated cAMP also decreased the rate of PAI-1 deposition into the endothelial substratum. These studies indicate that activation of a cAMP-dependent pathway(s) in coordination with phorbol ester-induced responses plays a central role in modifying the tPA and PAI-1 secretion from endothelial cells, leading to a profibrinolytic state in the endothelial environment.     

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.     

Mechanisms of thrombin-induced plasminogen activator release

The protein kinase C inhibitor H7 (10(-5) mol/l) is able to inhibit the thrombin-induced t-PA release in the isolated perfused pig ear. The thrombin-induced t-PA release can be blocked by increasing the intracellular c-AMP via either the activation of adenylate cyclase by means of forskolin, or the inhibition of the phosphodiesterase by means of motapizone or milrinone. Protein kinase C is assumed to be involved in the process of thrombin-induced t-PA release.     

Non-proteolytic neurotrophic effects of tissue plasminogen activator on cultured mouse cerebrocortical neurons

Most biological effects of tissue plasminogen activator (tPA), such as fibrinolysis, are mediated by its protease activity. Recent studies, however, have demonstrated that tPA also has several protease-independent effects such as: neuroprotection, microglial activation, and promoting LTP formation. In order to gain a better understanding of how tPA affects neurons, we examined neurite outgrowth and cell survival in low density cerebrocortical neuronal culture in the presence of tPA. tPA enhanced neurite elongation and neuronal survival. tPA protease inhibitors, PAI-1 or PMSF, did not alter either effect. Consistent with neurotrophic effects, tPA activated Raf-K/ERK, PKC and PI3-K/Akt, 5-60 min after treatment. In addition, specific inhibitors of these kinases reduced tPA-induced neurite outgrowth. Interestingly, survival-promoting effect of tPA was attenuated only by PI3-K inhibitors. Activation of signaling kinases suggests that tPA activates an upstream membrane receptor. Thus far, three membrane proteins, low density lipoprotein receptor-related protein (LRP), mannose receptor (MR), and annexin-II (AII), have been identified to bind tPA. While inhibiting LRP or MR did not change tPA-induced neurite outgrowth and cell survival, inhibiting AII blocked neurotrophic effects of tPA. Taken together, our results indicate that tPA has novel, non-proteolytic neurotrophic effects on cultured cortical neurons, which are likely mediated by AII.