Biomedical subjects

Bernardo L Sabatini

Publications and source records attributed to Bernardo L Sabatini.

14 recordsLinked to original sources

PubMed2007

Nonlinear regulation of unitary synaptic signals by CaV(2.3) voltage-sensitive calcium channels located in dendritic spines.

The roles of voltage-sensitive sodium (Na) and calcium (Ca) channels located on dendrites and spines in regulating synaptic signals are largely unknown. Here we use 2-photon glutamate uncaging to stimulate individual spines while monitoring uncaging-evoked excitatory postsynaptic potentials (uEPSPs) and Ca transients. We find that, in CA1 pyramidal neurons in acute mouse hippocampal slices, CaV(2.3) voltage-sensitive Ca channels (VSCCs) are found selectively on spines and act locally to dampen uncaging-evoked Ca transients and somatic potentials. These effects are mediated by a regulatory loop that requires opening of CaV(2.3) channels, voltage-gated Na channels, small conductance Ca-activated potassium (SK) channels, and NMDA receptors. Ca influx through CaV(2.3) VSCCs selectively activates SK channels, revealing the presence of functional Ca microdomains within the spine. Our results suggest that synaptic strength can be modulated by mechanisms that regulate voltage-gated conductances within the spine but do not alter the properties or numbers of synaptic glutamate receptors.

Animals↗

PubMed2006

Retraction of synapses and dendritic spines induced by off-target effects of RNA interference.

RNA interference (RNAi), which allows selective gene silencing, has been proposed for functional genomic analysis and for the treatment of human disease. However, induction of RNAi in mammalian cells by expression of double-stranded RNA can activate innate antiviral response pathways that perturb off-target gene expression. The activation and functional consequences of these effects in neurons are unknown. We find that expression of subsets of short hairpin RNAs (shRNAs) in rat hippocampal pyramidal neurons can have off-target effects that reduce the complexity of dendritic arbors and trigger the loss of dendritic spines. Morphological changes are accompanied by electrophysiological perturbations in passive membrane properties and a decrease in the number and strength of excitatory and inhibitory synapses. These perturbations depend on the shRNA sequence and are independent of the identity of the targeted protein. Our results indicate that off-target effects of RNAi severely perturb neuronal structure and function and may lead to the functional withdrawal of affected cells from the brain circuitry.

Adaptation, Physiological↗

PubMed2006

Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells.

Here we demonstrate that cerebellar stellate cells diffusionally isolate synaptically evoked signals in dendrites and are capable of input-specific synaptic plasticity. Sustained activity of parallel fibers induces a form of long-term depression that requires opening of calcium (Ca(2+))-permeable AMPA-type glutamate receptors (CP-AMPARs) and signaling through class 1 metabotropic glutamate receptors (mGluR1) and CB1 receptors. This depression is induced by postsynaptic increases in Ca(2+) concentration ([Ca(2+)]) and is limited to activated synapses. To understand how synapse-specific plasticity is induced by diffusible second messengers in aspiny dendrites, we examined diffusion of Ca(2+) and small molecules within stellate cell dendrites. Activation of a single parallel fiber opened CP-AMPARs, generating long-lived Ca(2+) transients that were confined to submicron dendritic stretches. The diffusion of Ca(2+) was severely retarded due to interactions with parvalbumin and a general restriction of small molecule mobility. Thus stellate cell dendrites spatially restrict signaling cascades that lead from CP-AMPAR activation to endocannabinoid production and trigger the selective regulation of active synapses.

Animals↗

PubMed2006

Mutual information-based feature selection in studying perturbation of dendritic structure caused by TSC2 inactivation.

In this study, the effect of protein Tuberous sclerosis 2 (TSC2) on the dendritic spine density and length was demonstrated by using TSC2-RNAinactivation. In addition, the role of rapamycin, an antagonist of the molecular target of rapamycin, in the morphological changes of spine caused by TSC2 silencing was investigated. The features were extracted from highresolution three-dimensional image stacks collected by two-photon laser scanning microscopy of green fluorescing pyramidal cells expressing TSC2-RNA interference (RNAi), or TSC2-RNAi and rapamycin treatment in rat hippocampal slice cultures. We proposed to apply the lognormal distribution method for feature extraction. The extracted features of three cases under investigation, namely, (1) green-fluorescent protein GFP vs TSC2-RNAi, (2) GFP vs TSC2-RNAi and rapamycin, and (3) TSC2-RNAi vs TSC2-RNAi and rapamycin, were analyzed by mutual information-based feature selection and evaluated by three classifiers, K-nearest neighbor, Perceptron, and two-layer neural networks. The results showed that both the spine density and length have significant morphological changes after TSC2-RNAi treatment. However, rapamycin treatment could reverse the effect of TSC2-RNAi on spine length but not on spine density. These results are consistent with the results reported in the scientific literature. Finally, we explored the application of pattern recognition method in a small sample with richer feature properties, namely bootstrap mutual information estimation and a mutual information- based feature selection method.

Algorithms↗

PubMed2005

Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2.

Mutations in the TSC1 or TSC2 tumor suppressor genes lead to tuberous sclerosis complex (TSC), a dominant hamartomatous disorder that often presents with mental retardation, epilepsy and autism. The etiology of these neurological symptoms is unclear and the function of the TSC pathway in neurons is unknown. We found that in post-mitotic, hippocampal pyramidal neurons of mice and rats, loss of Tsc1 or Tsc2 triggered enlargement of somas and dendritic spines and altered the properties of glutamatergic synapses. Furthermore, loss of a single copy of the Tsc1 gene was sufficient to perturb dendritic spine structure. Morphological changes required regulation of the actin-depolymerization factor cofilin at a conserved LIM-kinase phosphorylation site, the phosphorylation of which was increased by loss of Tsc2. Thus, the TSC pathway regulates growth and synapse function in neurons, and perturbations of neuronal structure and function are likely to contribute to the pathogenesis of the neurological symptoms of TSC.

Animals↗

PubMed2005

Neuronal activity regulates diffusion across the neck of dendritic spines.

In mammalian excitatory neurons, dendritic spines are separated from dendrites by thin necks. Diffusion across the neck limits the chemical and electrical isolation of each spine. We found that spine/dendrite diffusional coupling is heterogeneous and uncovered a class of diffusionally isolated spines. The barrier to diffusion posed by the neck and the number of diffusionally isolated spines is bidirectionally regulated by neuronal activity. Furthermore, coincident synaptic activation and postsynaptic action potentials rapidly restrict diffusion across the neck. The regulation of diffusional coupling provides a possible mechanism for determining the amplitude of postsynaptic potentials and the accumulation of plasticity-inducing molecules within the spine head.

Action Potentials↗

PubMed2005

SK channels and NMDA receptors form a Ca2+-mediated feedback loop in dendritic spines.

Small-conductance Ca(2+)-activated K(+) channels (SK channels) influence the induction of synaptic plasticity at hippocampal CA3-CA1 synapses. We find that in mice, SK channels are localized to dendritic spines, and their activity reduces the amplitude of evoked synaptic potentials in an NMDA receptor (NMDAR)-dependent manner. Using combined two-photon laser scanning microscopy and two-photon laser uncaging of glutamate, we show that SK channels regulate NMDAR-dependent Ca(2+) influx within individual spines. SK channels are tightly coupled to synaptically activated Ca(2+) sources, and their activity reduces the amplitude of NMDAR-dependent Ca(2+) transients. These effects are mediated by a feedback loop within the spine head; during an excitatory postsynaptic potential (EPSP), Ca(2+) influx opens SK channels that provide a local shunting current to reduce the EPSP and promote rapid Mg(2+) block of the NMDAR. Thus, blocking SK channels facilitates the induction of long-term potentiation by enhancing NMDAR-dependent Ca(2+) signals within dendritic spines.

Animals↗

PubMed2004

State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons.

Striatal medium spiny neurons (MSNs) in vivo undergo large membrane depolarizations known as state transitions. Calcium (Ca) entry into MSNs triggers diverse downstream cellular processes. However, little is known about Ca signals in MSN dendrites and spines and how state transitions influence these signals. Here, we develop a novel approach, combining 2-photon Ca imaging and 2-photon glutamate uncaging, to examine how voltage-sensitive Ca channels (VSCCs) and ionotropic glutamate receptors contribute to Ca signals in MSNs. We find that upstate transitions switch the VSCCs available in dendrites and spines, decreasing T-type while enhancing L-type channels. Moreover, these transitions change the dominant synaptic Ca source from Ca-permeable AMPA receptors to NMDA receptors. Finally, pairing bAPs with synaptic inputs generates additional synaptic Ca signals due to enhanced Ca influx through NMDA receptors. By altering the sources, amplitude, and kinetics of spine Ca signals, state transitions may gate synaptic plasticity and gene expression in MSNs.

Animals↗

PubMed2004

Imaging calcium concentration dynamics in small neuronal compartments.

Calcium and its regulation play central roles diverse physiologic processes. Quantification of calcium concentrations ([Ca2+]) in small neuronal compartments is crucial to understanding Ca2+-dependent signaling. Here, we describe techniques that are optimized for 2-photon imaging of [Ca2+] dynamics in small compartments such as dendrites and dendritic spines.

Animals↗

PubMed2003

ScanImage: flexible software for operating laser scanning microscopes.

BACKGROUND: Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope. RESULTS: We describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality. CONCLUSIONS: We present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design.

Equipment Design↗

PubMed2003

Plasticity of calcium channels in dendritic spines.

Voltage-sensitive Ca2+ channels (VSCCs) constitute a major source of calcium ions in dendritic spines, but their function is unknown. Here we show that R-type VSCCs in spines of rat CA1 pyramidal neurons are depressed for at least 30 min after brief trains of back-propagating action potentials. Populations of channels in single spines are depressed stochastically and synchronously, independent of channels in the parent dendrite and other spines, implying that depression is the result of signaling restricted to individual spines. Induction of VSCC depression blocks theta-burst-induced long-term potentiation (LTP), indicating that postsynaptic action potentials can modulate synaptic plasticity by tuning VSCCs. Induction of depression requires [Ca2+] elevations and activation of L-type VSCCs, which activate Ca2+/calmodulin-dependent kinase II (CaMKII) and a cyclic adenosine monophosphate (cAMP)-dependent pathway. Given that L-type VSCCs do not contribute measurably to Ca2+ influx in spines, they must activate downstream effectors either directly through voltage-dependent conformational changes or via [Ca2+] microdomains.

Action Potentials↗

PubMed2002

The life cycle of Ca(2+) ions in dendritic spines.

Spine Ca(2+) is critical for the induction of synaptic plasticity, but the factors that control Ca(2+) handling in dendritic spines under physiological conditions are largely unknown. We studied [Ca(2+)] signaling in dendritic spines of CA1 pyramidal neurons and find that spines are specialized structures with low endogenous Ca(2+) buffer capacity that allows large and extremely rapid [Ca(2+)] changes. Under physiological conditions, Ca(2+) diffusion across the spine neck is negligible, and the spine head functions as a separate compartment on long time scales, allowing localized Ca(2+) buildup during trains of synaptic stimuli. Furthermore, the kinetics of Ca(2+) sources governs the time course of [Ca(2+)] signals and may explain the selective activation of long-term synaptic potentiation (LTP) and long-term depression (LTD) by NMDA-R-mediated synaptic Ca(2+).

Action Potentials↗

PubMed2002

Facilitation at single synapses probed with optical quantal analysis.

Many synapses can change their strength rapidly in a use-dependent manner, but the mechanisms of such short-term plasticity remain unknown. To understand these mechanisms, measurements of neurotransmitter release at single synapses are required. We probed transmitter release by imaging transient increases in [Ca(2+)] mediated by synaptic N-methyl-D-aspartate receptors (NMDARs) in individual dendritic spines of CA1 pyramidal neurons in rat brain slices, enabling quantal analysis at single synapses. We found that changes in release probability, produced by paired-pulse facilitation (PPF) or by manipulation of presynaptic adenosine receptors, were associated with changes in glutamate concentration in the synaptic cleft, indicating that single synapses can release a variable amount of glutamate per action potential. The relationship between release probability and response size is consistent with a binomial model of vesicle release with several (>5) independent release sites per active zone, suggesting that multivesicular release contributes to facilitation at these synapses.

2-Chloroadenosine↗

PubMed2002

Structure and function of dendritic spines.

Spines are neuronal protrusions, each of which receives input typically from one excitatory synapse. They contain neurotransmitter receptors, organelles, and signaling systems essential for synaptic function and plasticity. Numerous brain disorders are associated with abnormal dendritic spines. Spine formation, plasticity, and maintenance depend on synaptic activity and can be modulated by sensory experience. Studies of compartmentalization have shown that spines serve primarily as biochemical, rather than electrical, compartments. In particular, recent work has highlighted that spines are highly specialized compartments for rapid large-amplitude Ca(2+) signals underlying the induction of synaptic plasticity.

Animals↗