Molecular mechanisms of dendritic spine morphogenesis

Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.     

Regulation of dendritic spine morphology and synaptic function by Shank and Homer

The Shank family of proteins interacts with NMDA receptor and metabotropic glutamate receptor complexes in the postsynaptic density (PSD). Targeted to the PSD by a PDZ-dependent mechanism, Shank promotes the maturation of dendritic spines and the enlargement of spine heads via its ability to recruit Homer to postsynaptic sites. Shank and Homer cooperate to induce accumulation of IP3 receptors in dendritic spines and formation of putative multisynapse spines. In addition, postsynaptic expression of Shank enhances presynaptic function, as measured by increased minifrequency and FM4-64 uptake. These data suggest a central role for the Shank scaffold in the structural and functional organization of the dendritic spine and synaptic junction.     

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.     

Cerebellar Golgi cell models predict dendritic processing and mechanisms of synaptic plasticity

The Golgi cells are the main inhibitory interneurons of the cerebellar granular layer. Although recent works have highlighted the complexity of their dendritic organization and synaptic inputs, the mechanisms through which these neurons integrate complex input patterns remained unknown. Here we have used 8 detailed morphological reconstructions to develop multicompartmental models of Golgi cells, in which Na, Ca, and K channels were distributed along dendrites, soma, axonal initial segment and axon. The models faithfully reproduced a rich pattern of electrophysiological and pharmacological properties and predicted the operating mechanisms of these neurons. Basal dendrites turned out to be more tightly electrically coupled to the axon initial segment than apical dendrites. During synaptic transmission, parallel fibers caused slow Ca-dependent depolarizations in apical dendrites that boosted the axon initial segment encoder and Na-spike backpropagation into basal dendrites, while inhibitory synapses effectively shunted backpropagating currents. This oriented dendritic processing set up a coincidence detector controlling voltage-dependent NMDA receptor unblock in basal dendrites, which, by regulating local calcium influx, may provide the basis for spike-timing dependent plasticity anticipated by theory.     

A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi

A family of three structurally related proteins were cloned from human cDNA libraries by their ability to interact preferentially with the activated form of human ADP-ribosylation factor 3 (ARF3) in two-hybrid assays. The specific and GTP-dependent binding was later confirmed through direct protein binding of recombinant proteins. The three proteins share large ( approximately 300 residues) domains at their N termini that are 60-70% identical to each other and a shorter (73 residues) domain at their C termini with 70% homology to the C-terminal "ear" domain of gamma-adaptin. Although GGA1 is found predominantly as a soluble protein by cell fractionation, all three proteins were found to localize to the trans-Golgi network (TGN) by indirect immunofluorescence. The binding of GGAs to TGN was sensitive to brefeldin A, consistent with this being an ARF-dependent event. Thus, these proteins have been named Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins, or GGAs. The finding that overexpression of GGAs was sufficient to alter the distribution of markers of the TGN (TGN38 and mannose 6-phosphate receptors) led us to propose that GGAs are effectors for ARFs that function in the regulation of membrane traffic through the TGN.     

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.     

Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin-induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.     

Running as fast as it can: How spiking dynamics form object groupings in the laminar circuits of visual cortex

How spiking neurons cooperate to control behavioral processes is a fundamental problem in computational neuroscience. Such cooperative dynamics are required during visual perception when spatially distributed image fragments are grouped into emergent boundary contours. Perceptual grouping is a challenge for spiking cells because its properties of collinear facilitation and analog sensitivity occur in response to binary spikes with irregular timing across many interacting cells. Some models have demonstrated spiking dynamics in recurrent laminar neocortical circuits, but not how perceptual grouping occurs. Other models have analyzed the fast speed of certain percepts in terms of a single feedforward sweep of activity, but cannot explain other percepts, such as illusory contours, wherein perceptual ambiguity can take hundreds of milliseconds to resolve by integrating multiple spikes over time. The current model reconciles fast feedforward with slower feedback processing, and binary spikes with analog network-level properties, in a laminar cortical network of spiking cells whose emergent properties quantitatively simulate parametric data from neurophysiological experiments, including the formation of illusory contours; the structure of non-classical visual receptive fields; and self-synchronizing gamma oscillations. These laminar dynamics shed new light on how the brain resolves local informational ambiguities through the use of properly designed nonlinear feedback spiking networks which run as fast as they can, given the amount of uncertainty in the data that they process.     

Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer''s disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.     

Information processing by nonspiking interneurons: passive and active properties of dendritic membrane determine synaptic integration

Nonspiking interneurons control activities of postsynaptic cells without generating action potentials in the central nervous system of many invertebrates. Physiological characteristics of their dendritic membrane have been analyzed in previous studies using single electrode current- and voltage-clamp techniques. We constructed a single compartment model of an identified nonspiking interneuron of crayfish. Experimental results allowed us to simulate how the passive and active properties of the dendritic membrane influence the integrative processing of synaptic inputs. The results showed that not only the peak amplitude but also the time course of synaptic potentials were dependent on the membrane potential level at which the synaptic activity was evoked. When the synaptic input came sequentially, each individual input was still discernible at depolarized levels at which the membrane time constant was short due to depolarization-dependent membrane conductances. In contrast, synaptic potentials merged with each other to develop a sustained potential at hyperpolarized levels where the membrane behaved passively. Thus, synaptic integration in a single nonspiking interneuron depends on the value of membrane potential at which it occurs. This probably reflects the temporal resolution required for specific types of information processing.     

miR-132 enhances dendritic morphogenesis, spine density, synaptic integration, and survival of newborn olfactory bulb neurons

An array of signals regulating the early stages of postnatal subventricular zone (SVZ) neurogenesis has been identified, but much less is known regarding the molecules controlling late stages. Here, we investigated the function of the activity-dependent and morphogenic microRNA miR-132 on the synaptic integration and survival of olfactory bulb (OB) neurons born in the neonatal SVZ. In situ hybridization revealed that miR-132 expression occurs at the onset of synaptic integration in the OB. Using in vivo electroporation we found that sequestration of miR-132 using a sponge-based strategy led to a reduced dendritic complexity and spine density while overexpression had the opposite effects. These effects were mirrored with respective changes in the frequency of GABAergic and glutamatergic synaptic inputs reflecting altered synaptic integration. In addition, timely directed overexpression of miR-132 at the onset of synaptic integration using an inducible approach led to a significant increase in the survival of newborn neurons. These data suggest that miR-132 forms the basis of a structural plasticity program seen in SVZ-OB postnatal neurogenesis. miR-132 overexpression in transplanted neurons may thus hold promise for enhancing neuronal survival and improving the outcome of transplant therapies.     

Conditions that affect sleep alter the expression of molecules associated with synaptic plasticity

Many theories propose that sleep serves a purpose in synaptic plasticity. We tested the hypothesis, therefore, that manipulation of sleep would affect the expression of molecules known to be involved in synaptic plasticity. mRNA expression of four molecules [brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (Arc), matrix metalloproteinase-9 (MMP-9), and tissue plasminogen activator (tPA)] was determined after 8 h of sleep deprivation and after 6 h of a mild increase in ambient temperature, a condition that enhances sleep in rats. After sleep deprivation, BDNF, Arc, and tPA mRNAs in the cerebral cortex increased while MMP-9 mRNA levels decreased. Conversely, after enhanced ambient temperature, BDNF, Arc, and tPA mRNAs decreased while MMP-9 mRNA increased. In the hippocampus, sleep deprivation did not significantly affect BDNF and tPA expression, although Arc mRNA increased and MMP-9 mRNA decreased. Brain temperature enhancement decreased Arc mRNA levels in the hippocampus but did not affect BDNF, MMP-9, or tPA in this area. Results are consistent with the notion that sleep plays a role in synaptic plasticity.     

Loss of the actin regulator cyclase-associated protein 1 (CAP1) modestly affects dendritic spine remodeling during synaptic plasticity

Dendritic spines form the postsynaptic compartment of most excitatory synapses in the vertebrate brain. Morphological changes of dendritic spines contribute to major forms of synaptic plasticity such as long-term potentiation (LTP) or depression (LTD). Synaptic plasticity underlies learning and memory, and defects in synaptic plasticity contribute to the pathogeneses of human brain disorders. Hence, deciphering the molecules that drive spine remodeling during synaptic plasticity is critical for understanding the neuronal basis of physiological and pathological brain function. Since actin filaments (F-actin) define dendritic spine morphology, actin-binding proteins (ABP) that accelerate dis-/assembly of F-actin moved into the focus as critical regulators of synaptic plasticity. We recently identified cyclase-associated protein 1 (CAP1) as a novel actin regulator in neurons that cooperates with cofilin1, an ABP relevant for synaptic plasticity. We therefore hypothesized a crucial role for CAP1 in structural synaptic plasticity. By exploiting mouse hippocampal neurons, we tested this hypothesis in the present study. We found that induction of both forms of synaptic plasticity oppositely altered concentration of exogenous, myc-tagged CAP1 in dendritic spines, with chemical LTP (cLTP) decreasing and chemical LTD (cLTD) increasing it. cLTP induced spine enlargement in CAP1-deficient neurons. However, it did not increase the density of large spines, different from control neurons. cLTD induced spine retraction and spine size reduction in control neurons, but not in CAP1-KO neurons. Together, we report that postsynaptic myc-CAP1 concentration oppositely changed during cLTP and cTLD and that CAP1 inactivation modestly affected structural plasticity.     

Analysis of an excitable dendritic spine with an activity-dependent stem conductance

 Dendritic spines are the major target for excitatory synaptic inputs in the vertebrate brain. They are tiny evaginations of the dendritic surface consisting of a bulbous head and a tenuous stem. Spines are considered to be an important locus for plastic changes underlying memory and learning processes. The findings that synaptic morphology may be activity-dependent and that spine head membrane may be endowed with voltage-dependent (excitable) channels is the motivation for this study. We first explore the dynamics, when an excitable, yet morphologically fixed spine receives a constant current input. Two parameter Andronov–Hopf bifurcation diagrams are constructed showing stability boundaries between oscillations and steady-states. We show how these boundaries can change as a function of both the spine stem conductance and the conductance load of the attached dendrite. Building on this reference case an idealized model for an activity-dependent spine is formulated and analyzed. Specifically we examine the possibility that the spine stem resistance, the tunable “synaptic weight” parameter identified by Rall and Rinzel, is activity-dependent. In the model the spine stem conductance depends (slowly) on the local electrical interactions between the spine head and the dendritic cable; parameter regimes are found for bursting, steady states, continuous spiking, and more complex oscillatory behavior. We find that conductance load of the dendrite strongly influences the burst pattern as well as other dynamics. When the spine head membrane potential exhibits relaxation oscillations a simple model is formulated that captures the dynamical features of the full model. Received: 10 January 1997/Revised version: 25 March 1997     

Dietary low linolenic acid compared with docosahexaenoic acid alter synaptic plasma membrane phospholipid fatty acid composition and sodium-potassium ATPase kinetics in developing rats

The objective of this study was to investigate if maternal dietary 20:4n-6 arachidonic acid (AA) and 22:6n-3 compared with adequate or low levels of 18:3n-3 linolenic acid (LNA) increases synaptic plasma membrane (SPM) cholesterol and phospholipid content, phospholipid 20:4n-6 and 22:6n-3 content, and Na,K-ATPase kinetics in rat pups at two and five weeks of age. At parturition, Sprague-Dawley rats were fed semi-purified diets containing either AA + docosahexaenoic acid (DHA), adequate LNA (control; 18:2n-6 : 18:3n-3 ratio of 7.1 : 1) or low LNA (18:2n-6 : 18:3n-39 ratio of 835 : 1). During the first two weeks of life, the rat pups received only their dams' milk. After weaning, pups received the same diet as their respective dams to five weeks of age. No significant difference was observed among rat pups fed the diet treatments for SPM cholesterol or total and individual phospholipid content at two and five weeks of age. Fatty acid analysis revealed that maternal dietary AA + DHA, compared with feeding the dams the control diet or the low LNA diet, increased 20:4n-6 in phosphatidylserine and 22:6n-3 content of SPM phospholipids. Rats fed dietary AA + DHA or the control diet exhibited a significantly increased Vmax for SPM Na,K-ATPase. Diet treatment did not alter the Km (affinity) of SPM Na,K-ATPase in rat pups at two and five weeks of age. It is concluded that dietary AA + DHA does not alter SPM cholesterol and phospholipid content but increases the 22:6n-3 content of SPM phospholipids modulating activity of Na,K-ATPase.     

Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex

In this study we characterized the responses of neocortical neurons to iontophoretically applied gamma-aminobutyric acid (GABA) and examined how these GABA responses as well as the inhibitory postsynaptic potentials (IPSPs) were affected by the presence of penicillin or pentobarbital. Intracellular recordings were obtained from slices of rat neocortex maintained in vitro; injection of the dye Lucifer yellow indicated that recordings were primarily from pyramidal neurons. Orthodromically evoked responses were always depolarizing at the cell's resting membrane potential. Such depolarizing responses could easily be reversed in polarity by depolarizing the cell 10-15 mV, suggesting that the response consisted partly of an IPSP. In some cases, depolarization unmasked a small, short-latency excitatory postsynaptic potential (EPSP). Responses to iontophoretically applied GABA were also depolarizing at rest. Biphasic hyperpolarizing-depolarizing responses were occasionally observed upon depolarization of the neuron. Bath application of penicillin (1.7-3.4 mM) decreased the amplitude of the IPSPs and increased their time to peak, an effect associated with the development of epileptiform activity. Penicillin also reduced the maximum response to iontophoretically applied GABA without affecting the dose required to obtain a half-maximal response, suggesting a noncompetitive antagonism. Pentobarbital (100-200 microM) prolonged the time course and increased the amplitude of the IPSPs while producing a leftward shift in the GABA charge-response relation. These results suggest that the convulsant penicillin and the anticonvulsant pentobarbital have opposing actions on GABAergic inhibition in the neocortex.     

Lipid hapten containing membrane targets can trigger specific immunoglobulin E-dependent degranulation of rat basophil leukemia cells

We have studied the binding of liposomes containing dinitrophenylated lipid to rat basophil leukemia cells armed with monoclonal anti-dinitrophenyl IgE. The liposomes were either "fluid" at 37 degrees C (dimyristoylphosphatidylcholine or an equimolar binary mixture dipalmitoylphosphatidylcholine and cholesterol) or "solid" (dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, or dibehanoylphosphatidylcholine). We have also studied the immune mediated degranulation of these cells induced by the above lipid membrane targets. In some cases both studies were carried out with liposomes containing various surface densities of lipid haptens. From these studies we conclude that freely mobile nonaggregated lipid haptens in bilayer membrane targets can trigger efficient serotonin release from rat basophil leukemia cells in the presence of specific antihapten IgE. Solid target membranes are also effective as stimulators of serotonin release. The release of serotonin depends strongly on the surface density of lipid haptens over a narrow range of surface densities. These studies with lipid membrane targets having well defined physical properties indicate the need for generalized molecular models of receptor-mediated cell triggering.