Relating a calcium indicator signal to the unperturbed calcium concentration time-course

BACKGROUND: Optical indicators of cytosolic calcium levels have become important experimental tools in systems and cellular neuroscience. Indicators are known to interfere with intracellular calcium levels by acting as additional buffers, and this may strongly alter the time-course of various dynamical variables to be measured. RESULTS: By investigating the underlying reaction kinetics, we show that in some ranges of kinetic parameters one can explicitly link the time dependent indicator signal to the time-course of the calcium influx, and thus, to the unperturbed calcium level had there been no indicator in the cell

Spine neck plasticity regulates compartmentalization of synapses

Dendritic spines have been proposed to transform synaptic signals through chemical and electrical compartmentalization. However, the quantitative contribution of spine morphology to synapse compartmentalization and its dynamic regulation are still poorly understood. We used time-lapse super-resolution stimulated emission depletion (STED) imaging in combination with fluorescence recovery after photobleaching (FRAP) measurements, two-photon glutamate uncaging, electrophysiology and simulations to investigate the dynamic link between nanoscale anatomy and compartmentalization in live spines of CA1 neurons in mouse brain slices. We report a diversity of spine morphologies that argues against common categorization schemes and establish a close link between compartmentalization and spine morphology, wherein spine neck width is the most critical morphological parameter. We demonstrate that spine necks are plastic structures that become wider and shorter after long-term potentiation. These morphological changes are predicted to lead to a substantial drop in spine head excitatory postsynaptic potential (EPSP) while preserving overall biochemical compartmentalization

Lobe Specific Ca2+-Calmodulin Nano-Domain in Neuronal Spines: A Single Molecule Level Analysis

Calmodulin (CaM) is a ubiquitous Ca2+ buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca2+-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca2+-CaM-dependent enzymes: Ca2+/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca2+ and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca2+ ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca2+ and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca2+ signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca2+-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca2+ channels, and to the microscopic injection rate of Ca2+ ions. We also demonstrate that Ca2+ saturation takes place via two different pathways depending on the Ca2+ injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca2+ sensors that can differentially transduce Ca2+ influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca2+-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity

Calmodulin Activation by Calcium Transients in the Postsynaptic Density of Dendritic Spines

The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM) and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways

PAK1 Protein Expression in the Auditory Cortex of Schizophrenia Subjects

Deficits in auditory processing are among the best documented endophenotypes in schizophrenia, possibly due to loss of excitatory synaptic connections. Dendritic spines, the principal post-synaptic target of excitatory projections, are reduced in schizophrenia. p21-activated kinase 1 (PAK1) regulates both the actin cytoskeleton and dendritic spine density, and is a downstream effector of both kalirin and CDC42, both of which have altered expression in schizophrenia. This study sought to determine if there is decreased auditory cortex PAK1 protein expression in schizophrenia through the use of quantitative western blots of 25 schizophrenia subjects and matched controls. There was no significant change in PAK1 level detected in the schizophrenia subjects in our cohort. PAK1 protein levels within subject pairs correlated positively with prior measures of total kalirin protein in the same pairs. PAK1 level also correlated with levels of a marker of dendritic spines, spinophilin. These latter two findings suggest that the lack of change in PAK1 level in schizophrenia is not due to limited sensitivity of our assay to detect meaningful differences in PAK1 protein expression. Future studies are needed to evaluate whether alterations in PAK1 phosphorylation states, or alterations in protein expression of other members of the PAK family, are present in schizophrenia

Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images

A fundamental challenge in understanding how dendritic spine morphology controls learning and memory has been quantifying three-dimensional (3D) spine shapes with sufficient precision to distinguish morphologic types, and sufficient throughput for robust statistical analysis. The necessity to analyze large volumetric data sets accurately, efficiently, and in true 3D has been a major bottleneck in deriving reliable relationships between altered neuronal function and changes in spine morphology. We introduce a novel system for automated detection, shape analysis and classification of dendritic spines from laser scanning microscopy (LSM) images that directly addresses these limitations. The system is more accurate, and at least an order of magnitude faster, than existing technologies. By operating fully in 3D the algorithm resolves spines that are undetectable with standard two-dimensional (2D) tools. Adaptive local thresholding, voxel clustering and Rayburst Sampling generate a profile of diameter estimates used to classify spines into morphologic types, while minimizing optical smear and quantization artifacts. The technique opens new horizons on the objective evaluation of spine changes with synaptic plasticity, normal development and aging, and with neurodegenerative disorders that impair cognitive function

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8–5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall

GABA Receptors and the Pharmacology of Sleep

Current GABAergic sleep-promoting medications were developed pragmatically, without making use of the immense diversity of GABAA receptors. Pharmacogenetic experiments are leading to an understanding of the circuit mechanisms in the hypothalamus by which zolpidem and similar compounds induce sleep at α2βγ2-type GABAA receptors. Drugs acting at more selective receptor types, for example, at receptors containing the α2 and/or α3 subunits expressed in hypothalamic and brain stem areas, could in principle be useful as hypnotics/anxiolytics. A highly promising sleep-promoting drug, gaboxadol, which activates αβδ-type receptors failed in clinical trials. Thus, for the time being, drugs such as zolpidem, which work as positive allosteric modulators at GABAA receptors, continue to be some of the most effective compounds to treat primary insomnia