Eduard Korkotian

Dendritic spine formation and pruning: common cellular mechanisms?

The recent advent of novel high-resolution imaging methods has created a flurry of exciting observations that address a century-old question: what are biological signals that regulate formation and elimination of dendritic spines? Contrary to the traditional belief that the spine is a stable storage site of long-term neuronal memory, the emerging picture is of a dynamic structure that can undergo fast morphological variations. Recent conflicting reports on the regulation of spine morphology lead to the proposal of a unifying hypothesis for a common mechanism involving changes in postsynaptic intracellular Ca2+ concentration, [Ca2+]i: a moderate rise in [Ca2+]i causes elongation of dendritic spines, while a very large increase in [Ca2+]i causes fast shrinkage and eventual collapse of spines. This hypothesis provides a parsimonious explanation for conflicting reports on activity-dependent changes in dendritic spine morphology, and might link these changes to functional plasticity in central neurons.

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 [Ca2+] i response to glutamate, and a large increase in [Ca2+] 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 B1, completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca2+] i stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C8-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 [Ca2+] 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.

Synaptopodin Regulates Plasticity of Dendritic Spines in Hippocampal Neurons

The spine apparatus is an essential component of dendritic spines of cortical and hippocampal neurons, yet its functions are still enigmatic. Synaptopodin (SP), an actin-binding protein, is tightly associated with the spine apparatus and it may play a role in synaptic plasticity, but it has not yet been linked mechanistically to synaptic functions. We studied endogenous and transfected SP in dendritic spines of cultured hippocampal neurons and found that spines containing SP generate larger responses to flash photolysis of caged glutamate than SP-negative ones. An NMDA-receptor-mediated chemical long-term potentiation caused the accumulation of GFP-GluR1 in spine heads of control but not of shRNA-transfected, SP-deficient neurons. SP is linked to calcium stores, because their pharmacological blockade eliminated SP-related enhancement of glutamate responses, and release of calcium from stores produced an SP-dependent increase of GluR1 in spines. Thus, SP plays a crucial role in the calcium store-associated ability of neurons to undergo long-term plasticity.

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.

Formation of dendritic spines in cultured striatal neurons depends on excitatory afferent activity.

The role of afferent innervation in the formation of dendritic spines was studied in cultured rat striatum. The striatum is a unique structure in that it contains highly spiny GABAergic projection neurons, with no known local excitation. Grown alone in culture, striatal neurons did not express spontaneous network activity and were virtually devoid of dendritic spines. Adding GFP‐expressing mouse cortical neurons to the striatal culture caused the appearance of spontaneous and evoked excitatory synaptic currents in the striatal neurons and a 10‐fold increase in the density of spines on their dendrites. This effect was blocked by a continuous presence of TTX in the growth medium, while removal of the drug caused a rapid appearance of spines. Exposure to glutamate, or the presence of cortex‐conditioned medium did not mimic the effect of cortical neurons on formation of spines in the striatal neurons. Also, the cortical innervation did not cause a selective enhancement of survival of specific subtypes of spiny striatal neurons. These experiments demonstrate that excitatory afferents are necessary for the formation of dendritic spines in striatal neurons.

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.

Dynamic regulation of spine-dendrite coupling in cultured hippocampal neurons.

We investigated the role of dendritic spine morphology in spine–dendrite calcium communication using novel experimental and theoretical approaches. A transient rise in [Ca2+]i was produced in individual spine heads of Fluo‐4‐loaded cultured hippocampal neurons by flash photolysis of caged calcium. Following flash photolysis in the spine head, a delayed [Ca2+]i transient was detected in the parent dendrites of only short, but not long, spines. Delayed elevated fluorescence in the dendrite of the short spines was also seen with a membrane‐bound fluorophore and fluorescence recovery from bleaching of a calcium‐bound fluorophore had a much slower kinetics, indicating that the dendritic fluorescence change reflects a genuine diffusion of free [Ca2+]i from the spine head to the parent dendrite. Calcium diffusion between spine head and the parent dendrite was regulated by calcium stores as well as by a Na–Ca exchanger. Spine length varied with the recent history of the [Ca2+]i variations in the spine, such that small numbers of calcium transients resulted in elongation of spines whereas large numbers of calcium transients caused shrinkage of the spines. Consequently, spine elongation resulted in a complete isolation of the spine from the dendrite, while shrinkage caused an enhanced coupling with the parent dendrite. These studies highlight a dynamically regulated coupling between a dendritic spine head and its parent dendrite.

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.

The Spine Apparatus, Synaptopodin, and Dendritic Spine Plasticity

The spine apparatus (SA) is an essential component of mature dendritic spines of cortical and hippocampal neurons, yet its functions are still enigmatic. Synaptopodin (SP), an actin-binding protein, colocalizes with the SA. Hippocampal neurons in SP-knockout mice lack SA, and they express lower LTP. SP probably plays a role in synaptic plasticity, but only recently it is being linked mechanistically to synaptic functions. These authors and others have studied endogenous and transfected SP in dendritic spines of cultured hippocampal neurons. They found that spines containing SP generate twice as large responses to flash photolysis of caged glutamate than SP-negative ones. An N-methyl-d-aspartate receptor—mediated chemical LTP caused accumulation of GFP-GluR1 in spine heads of control but not of shRNA transfected, SP-deficient neurons. SP is linked to calcium stores, because their pharmacological blockade eliminated SP-related enhancement of glutamate responses. Furthermore, release of calcium from stores produces an SP-dependent delivery of GluR1 into spines. Thus, SP plays a crucial role in the calcium store-associated ability of neurons to undergo long-term plasticity.

Synaptopodin Regulates Spine Plasticity: Mediation by Calcium Stores

The role of synaptopodin (SP), an actin-binding protein residing in dendritic spines, in synaptic plasticity was studied in dissociated cultures of hippocampus taken from control and SP knock-out (SPKO) mice. Unlike controls, SPKO cultures were unable to express changes in network activity or morphological plasticity after intense activation of their NMDA receptors. SPKO neurons were transfected with SP-GFP, such that the only SP resident in these neurons is the fluorescent species. The localization and intensity of the transfected SP were similar to that of the native one. Because less than half of the spines in the transfected neurons contained SP, comparisons were made between SP-containing (SP(+)) and SP lacking (SP(−)) spines in the same dendritic segments. Synaptic plasticity was induced either in the entire network by facilitation of the activation of the NMDA receptor, or specifically by local flash photolysis of caged glutamate. After activation, spines that were endowed with SP puncta were much more likely to expand than SP(−) spines. The spine expansion was suppressed by thapsigargin, which disables calcium stores. The mechanism through which SP may promote plasticity is indicated by the observations that STIM-1, the sensor of calcium concentration in stores, and Orai-1, the calcium-induced calcium entry channel, are colocalized with SP, in the same dendritic spines. The structural basis of SP is likely to be the spine apparatus, found in control but not in SPKO cells. These results indicate that SP has an essential, calcium store-related role in regulating synaptic plasticity in cultured hippocampal neurons.