Increased excitability of hippocampal neurons in mature synaptopodin-knockout mice.

Synaptopodin (SP) is localized within the spine apparatus, an enigmatic structure located in the neck of spines of central excitatory neurons. It serves as a link between the spine head, where the synapse is located, and the endoplasmic reticulum (ER) in the parent dendrite. SP is also located in the axon initial segment, in association with the cisternal organelle, another structure related to the endoplasmic reticulum. Extensive research using SP knockout (SPKO) mice suggest that SP has a pivotal role in structural and functional plasticity. Consequently, young adult SPKO mice were shown to be deficient in cognitive functions, and in ability to undergo long-term potentiation of reactivity to afferent stimulation. However, although SP expresses differently during maturation, its role in synaptic and intrinsic neuronal mechanisms in adult SPKO mice is still unclear. To address this knowledge gap we analyzed hippocampus bulk mRNA in SPKO mice, and we recorded the activity of CA1 neurons in the mouse hippocampus slice, with both extracellular and patch recording methods. Electrophysiologically, SPKO cells in CA1 region of the dorsal hippocampus were more excitable than wild type (wt) ones. In addition, exposure of mice to a complex environment caused a higher proportion of arc-expressing cells in SPKO than in wt mice hippocampus. These experiments indicate that higher excitability and higher expression of arc staining may reflect SP deficiency in the hippocampus of adult SPKO mice.

Diffusion in a dendritic spine: the role of geometry.

Dendritic spines, the sites where excitatory synapses are made in most neurons, can dynamically regulate diffusing molecules by changing their shape. We present here a combination of theory, simulations, and experiments to quantify the diffusion time course in dendritic spines. We derive analytical formulas and compared them to Brownian simulations for the mean sojourn time a diffusing molecule stays inside a dendritic spine when either the molecule can reenter the spine head or not, once it is located in the spine neck. We show that the spine length is the fundamental regulatory geometrical parameter for the diffusion decay rate in the neck only. By changing the spine length, dendritic spines can be dynamically coupled or uncoupled to their parent dendrites, which regulates diffusion, and this property makes them unique structures, different from static dendrites.

Calcium dynamics in dendritic spines, modeling and experiments.

Dendritic spines are microstructures, about one femtoliter in volume, where excitatory synapses are made with incoming afferents, in most neurons of the vertebrate brain. The spine contains all the molecular constituents of the postsynaptic side of the synapse, as well as a contractile element that can cause its movement in space. It also contains calcium handling machineries to allow fast buffering of excess calcium that influx through voltage and NMDA gated channels. The spine is connected to the dendrite through a thin neck that serves as a variable barrier between the spine head and the parent dendrite. We review a novel modeling approach that is more suitable for the accurate description of the stochastic behavior of individual molecules in microstructures. Using this approach, we predict the calcium handling ability of the spine in complex situations associated with synaptic activity, spine motility and plasticity.

Calcium dynamics in dendritic spines and spine motility.

A dendritic spine is an intracellular compartment in synapses of central neurons. The role of the fast twitching of spines, brought about by a transient rise of internal calcium concentration above that of the parent dendrite, has been hitherto unclear. We propose an explanation of the cause and effect of the twitching and its role in the functioning of the spine as a fast calcium compartment. Our molecular model postulates that rapid spine motility is due to the concerted contraction of calcium-binding proteins. The contraction induces a stream of cytoplasmic fluid in the direction of the dendritic shaft, thus speeding up the time course of spine calcium dynamics, relative to pure diffusion. Simulations indicate that chemical reaction rate theory at the molecular level can explain spine motility. They reveal two time periods in calcium dynamics, as measured recently by other researchers. It appears that rapid motility in dendritic spines increases the efficiency of calcium conduction to the dendrite and speeds up the emptying of the spine. This could play a major role in the induction of synaptic plasticity. A prediction of the model is that alteration of spine motility will modify the time course of calcium in the dendritic spine and could be tested experimentally.

Regulation of dendritic spine motility in cultured hippocampal neurons.

Regulation of dendritic spine motility was studied in dissociated cultures of the rat and mouse hippocampus, using green fluorescent protein-labeled neurons or neurons loaded with the calcium-sensitive dye Oregon Green-1. Cells were time-lapse-photographed on a confocal laser-scanning microscope at high resolution to detect movements as well as spontaneous fluctuations of intracellular calcium concentrations in their dendritic spines. Active presynaptic terminals attached to the spines were labeled with FM4-64, which marks a subset of synaptophysin-labeled terminals. Dendritic spines were highly motile in young, 4- to 7-d-old cells. At this age, neurons had little spontaneous calcium fluctuation or FM4-64 labeling. Within 2-3 weeks in culture, dendritic spines were much less motile, they were associated with active presynaptic terminals, and they expressed high rates of spontaneous calcium fluctuations. Irrespective of age, and even on the same dendrite, there was an inverse relationship between spine motility and presence of FM4-64-labeled terminals in contact with the imaged spines. Spine motility was blocked by latrunculin, which prevents actin polymerization, and was disinhibited by blockade of action potential discharges with tetrodotoxin. It is proposed that an active presynaptic terminal restricts motility of dendritic spines.

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.

Structure-function relations in dendritic spines: is size important?

The recent use of novel high-resolution imaging methods of living neurons in vitro has led to a change in the view of the dendritic spine, from a stable, long-term memory storage device to that of a dynamic structure, which can undergo fast morphological changes over periods of hours and even minutes. While the functional significance of these changes in spine dimensions is still obscure, we have obtained evidence to indicate that the length of the spine has a critical role in determining the degree of interaction between the spine head and the parent dendrite, such that longer spines are more independent of the parent dendrite than the short ones. We have now studied the role of intracellular calcium stores in determining the magnitude and time course of spine responses to a calcium surge evoked in response to glutamate, which causes an influx of calcium, and the results indicate that spine morphology has an important role in determining the involvement of the stores in calcium responses. Since spines can change their length over a rather short time, these results indicate that changes in spine length serve to fine-tune the interaction between the spine head and the parent dendrite on a continuous basis.

Bidirectional regulation of dendritic spine dimensions by glutamate receptors.

The recent conflicting observations on the effects of excitatory afferent activity on dimensions of dendritic spines of central neurons led us to examine the possibility that the same spine can either increase or decrease its length in response to different stimuli. Cultured hippocampal neurons labeled with calcein, were 3D reconstructed in a confocal laser scanning microscope. Their responses to pulse application of glutamate were examined. Short pulses of glutamate caused elongation of dendritic spines, while long pulses caused fast shrinkage of the same set of spines. Thus, the same spine can undergo two opposite responses to application of glutamate, depending on the stimulation intensity/duration. These observations have important implications for understanding the roles of dendritic spines in information processing in central neurons.

Release of calcium from stores alters the morphology of dendritic spines in cultured hippocampal neurons.

The ability to monitor ongoing changes in the shape of dendritic spines has important implications for the understanding of the functional correlates of the great variety of shapes and sizes of dendritic spines in central neurons. We have monitored and three-dimensionally reconstructed dendritic spines in cultured hippocampal neurons over several hours of observation in a confocal laser scanning microscope. In the absence of extrinsic stimulation, the dimensions of dendritic spines of 3-week-old cultured neurons did not change to any significant degree over 3-4 hr in the culture dish, unlike the case with younger cultures. Releasing calcium from stores with pulse application of caffeine causes a transient rise of [Ca(2+)](i) in dendrites and spines, monitored with the calcium dye Oregon-green. Application of caffeine to a dendrite imaged with calcein caused a fast and significant increase in the size of existing dendritic spines and could lead to formation of new ones. This effect is mediated by calcium released from the ryanodine-sensitive stores, as application of caffeine in the presence of ryanodine blocked this effect on the morphology of dendritic spines. Thus, release of calcium from stores is sufficient to produce significant changes in the shape of dendritic spines of cultured hippocampal neurons.

Geometry of dendritic spines affects calcium dynamics in hippocampal neurons: theory and experiments.

The role of dendritic spine morphology in the regulation of the spatiotemporal distribution of free intracellular calcium concentration ([Ca2+]i) was examined in a unique axial-symmetrical model that focuses on spine-dendrite interactions, and the simulations of the model were compared with the behavior of real dendritic spines in cultured hippocampal neurons. A set of nonlinear differential equations describes the behavior of a spherical dendritic spine head, linked to a dendrite via a cylindrical spine neck. Mechanisms for handling of calcium (including internal stores, buffers, and efflux pathways) are placed in both the dendrites and spines. In response to a calcium surge, the magnitude and time course of the response in both the spine and the parent dendrite vary as a function of the length of the spine neck such that a short neck increases the magnitude of the response in the dendrite and speeds up the recovery in the spine head. The generality of the model, originally constructed for a case of release of calcium from stores, was tested in simulations of fast calcium influx through membrane channels and verified the impact of spine neck on calcium dynamics. Spatiotemporal distributions of [Ca2+]i, measured in individual dendritic spines of cultured hippocampal neurons injected with Calcium Green-1, were monitored with a confocal laser scanning microscope. Line scans of spines and dendrites at a <1-ms time resolution reveal simultaneous transient rises in [Ca2+]i in spines and their parent dendrites after application of caffeine or during spontaneous calcium transients associated with synaptic or action potential discharges. The magnitude of responses in the individual compartments, spine-dendrite disparity, and the temporal distribution of [Ca2+]i were different for spines with short and long necks, with the latter being more independent of the dendrite, in agreement with prediction of the model.

Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons.

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca(2+)](i) response to glutamate, and a large increase in [Ca(2+)](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co-incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B(1), completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca(2+)](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C(8)-glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, suggesting that [Ca(2+)](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.

Fast confocal imaging of calcium released from stores in dendritic spines.

The emerging significance of calcium stores in neuronal plasticity and the assumed involvement of dendritic spines in long-term plastic properties of neurons have led us to examine the presence and possible regulation of calcium stores in dendritic spines. Immunohistochemical staining for ryanodine receptors was found in dendritic spines of cultured hippocampal neurons. Confocal microscopic imaging of calcium transients in dendritic spines of these neurons in response to caffeine allowed us to demonstrate an independent and unique calcium store in spines. The response to caffeine was blocked by thapsigargin and ryanodine, and maintained in calcium-free medium. The calcium stores were depleted faster in the spines than the dendrites. Furthermore, when calcium was released from stores under calcium-free conditions, and diffused passively between the spine and the dendrite, the length of the spine neck determined the degree of spine independence. Finally, the caffeine-sensitive ryanodine receptor-linked calcium store was instrumental in regulating the response of neurons to glutamate. These results have important implications for understanding the roles of dendritic spines in neuronal integration and plasticity.

Calcium-containing organelles display unique reactivity to chemical stimulation in cultured hippocampal neurons.

Cultured rat hippocampal neurons grown on glass coverslips for 1-3 weeks were loaded with the calcium-sensitive fluorescent dye Fluo-3 and viewed with a confocal laser scanning microscope. Large pyramidal-shaped neurons were found to contain dye-accumulating organelles in their somata, primarily around nuclei and near the base of their primary dendrites. These organelles varied in size and increased in density over weeks in culture, and were not colocalized with the endoplasmic reticulum or with mitochondria. The Fluo-3 fluorescence in these calcium-containing organelles (CCOs) was transiently quenched by exposure to Mn2+, indicating that the dye is a genuine [Ca2+] reporter and is not just a site of accumulating Fluo-3 dye. Recovery of fluorescence in the CCOs after washout of Mn2+ involved activation of a thapsigargin-sensitive process. CCOs responded to stimuli that evoke a rise of cytosolic [Ca2+] ([Ca]i) in a unique manner; perfusion of caffeine caused a prolonged rise of [Ca] in the CCOs ([Ca]C), whereas it caused only a transient rise of [Ca]i. Pulse application of caffeine also caused a faster effect on [Ca]C than on [Ca]i. Glutamate caused a transient rise of both [Ca]i and [Ca]C, followed by a prolonged fall of only [Ca]C to below rest level. This fall was blocked by preincubation with thapsigargin. Ryanodine blocked the cytosolic effects of caffeine but not its effect on [C]C. A clear distinction between CCOs and the known calcium stores was seen in digitonin-permeabilized cells; in these, remaining Fluo-3 reported changes in store calcium, i.e., caffeine caused a reduction in Fluo-3 fluorescence in permeabilized cells, whereas it still caused an increase in [Ca]C. A possible role of CCOs in regulation of release of calcium from ryanodine-sensitive stores was indicated by the observation that CCO-containing cells exhibited a larger and faster response to caffeine than cells that did not have them. We propose that CCOs constitute a unique functional compartment involved in release of calcium from calcium-sensitive stores.

Lasting effects of glutamate on nuclear calcium concentration in cultured rat hippocampal neurons: regulation by calcium stores.

1. Changes in free intracellular calcium concentrations ([Ca2+]i) were measured in the nucleus and perinuclear regions of cultured rat hippocampal neurons using either fura-2 or fluo-3 calcium indicators. 2. Brief application of glutamate caused a transient rise of [Ca2+]i in all cell compartments, which recovered to pre-drug levels in all but the nuclear region. The new, higher level of nuclear calcium ([Ca2+]n) was sustained for as long as the cell was monitored. 3. The new level of [Ca2+]n was dependent on the magnitude of the calcium transient, and was higher in older cells in culture, but it did not affect responses to subsequent applications of glutamate. 4. The sustained elevation of [Ca2+]n was prevented by drugs which affect calcium stores (caffeine, ryanodine and Ruthenium Red), indicating that an extranuclear calcium store interacts with [Ca2+]n.

Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons.

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.

[The neuronal background impulse activity of the cerebellar dentate nucleus in the rat]. / Fonovaia impul'snaia aktivnost' neironov zubchatogo iadra mozzhechka krysy.

The analysis of serial correlograms of spontaneous neuronal activity of the rat cerebellum dentate nucleus revealed six main dynamic versions of the impulse flows. Independent haphazard distribution of interspike intervals, irregular sequences of short and long intervals, periodic changes of the impulse flow rate, were predominant.

[The background impulse activity of the neurons of the nucleus fastigii in the rat cerebellum]. / Fonovaia impul'snaia aktivnost' neironov fastigial'nogo iadra mozzhechka krysy.

Background impulse activity (BIA) of fastigial nucleus (NF) of the rat cerebellum has been analyzed. Prevalence of stationary impulse flows (IFs) and their irregularity of various types are found out, regular components of IF occurring frequently. Nonstationary IFs are detected as well, but they are studied only in 15% of a total number of units. A serial correlation analysis of duration of interspike intervals (ISIs) in case of stationary IFs and nonstationary ones permits stating six main varieties of ISI dynamics: 1) independent accidental distribution of impulse sequences; 2) irregular changes (acceleration or deceleration) of IF rate with positive correlation coefficients (CCs) in a range of initial five ISI orders; 3) irregular appearance of combined short and long intervals with negative CC in neighbouring ISIs; 4) periodic changes of IF rate, in particular, as grouped discharges varying in total duration and frequencies (4 and 5 IF varieties); 5) in case of negative values of CCs of the first order the recorded IFs in the primary complex serial correlograms have been determined as the 5th dynamic variant; and, finally, 6) prolonged cycles of BIA acceleration or deceleration with positive CCs up to 10-20 and higher orders. One third of NF units recorded have other BIA variations with the characteristics similar to types 2-3 and 6 mentioned above. They are considered to be induced by various correlations existing also at high-order ISIs. Formation of IFs registered and their pattern changes can serve as an adequate index of current state of cellular activity recorded extracellularly from small size cells of the CNS.

[The background impulse activity of the fastigial neurons of the rat cerebellum]. / Fonovaia impul'snaia aktivnost' fastigial'nykh neironov mozzhechka krysy.

Background impulse activity (BIA) of rat fastigial neurons was studied. Mean frequencies of BIA were 6-130 imp/s (45+5.6 imp/s, n = 105). Monomodality and positive asymmetry of interval histograms were typical of a majority of neurons analyzed. About 1/3 of neurons studied have got Gaussian type of interspike interval distribution.