PKD regulates the stabilization of the F-actin network within dendritic spines upon chemically induced plasticity changes and is needed for proper hippocampal LTP and spatial memory formation.
CASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.
Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons. During the search for future presynaptic partners, their shape and size change dynamically, with a direct impact on the formation, stabilization and maintenance of synaptic connections both in vivo and in vitro. In order to reveal molecular players regulating synapse formation, quantitative analysis of dendritic filopodia motility is needed. Defining the length or the tips of these protrusions manually, however, is time consuming, limiting the extent of studies as well as their statistical power. Additionally, area detection based on defining a single intensity threshold can lead to significant errors throughout the image series, as these small structures often have low contrast in fluorescent images. To overcome these problems, the open access Dendritic Filopodia Motility Analyzer, a semi-automated ImageJ/Fiji plugin was created. Our method calculates the displacement of the centre of mass (CoM) within a selected region based on the weighted intensity values of structure forming pixels, selected by upper and lower intensity thresholds. Using synthetic and real biological samples, we prove that the displacement of the weighted CoM reliably characterizes the motility of dendritic protrusions. Additionally, guidelines to define optimal parameters of live cell recordings from dendritic protrusions are provided. V C 2014 International Society for Advancement of Cytometry Key terms neuron; dendritic filopodia; motility analysis; time-lapse MOST excitatory synapses in the mammalian brain terminate on dendritic spines.During neuronal development, transient actin-rich protrusions named dendritic filopodia appear first along the dendrites. Filopodia are highly motile structures, which actively search for future presynaptic contact sites. In the absence of a suitable axonal contact, filopodia retract. On the other hand, initial contact triggers the formation of synaptic machinery and the appearance of dendritic spines (1). Filopodia can change their shape by a nodding motion when the angle between the neck and the head or dendritic shaft is changing. In addition, their length or the width of their head can also alter in time (2-4). Developmental regulation of filopodial motility is required for synapse formation: initial movements and elongations are needed to get into contact with the future presynaptic partner, while motility is attenuated for the successful maintenance of the synaptic connections (5).In contrast to filopodia, dendritic spines have established synaptic connections, possessing postsynaptic components within the head region of dendritic spines. Stubby spines are shorter, while thin and mushroom spines are usually longer than 1 mm with a well-extended head region in case of mushroom spines. The size and shape of established dendritic spines can also alter during development, in correlation with the stability and the strength of the synapse (1).To characterize dendritic filopodia and spine motility, som...
Alzheimer's disease is associated with a significant decrease in the cholinergic input to the neocortex. In a rat model of this depletion, we analyzed the subsequent long-term changes in cholinergic fiber density in two well-defined areas of the frontal and parietal cortices: Fr1, the primary motor cortex, and HL, the hindlimb area of the somatosensory (parietal) cortex, two cortical cholinergic fields that receive inputs from the nucleus basalis magnocellularis (nBM). A specific cholinergic lesion was induced by the intraparenchymal injection of 192 IgG-saporin into the nBM. Choline acetyltransferase (ChAT) immunohistochemistry was applied to identify the loss of cholinergic neurons in the nBM, while acetylcholinesterase (AChE) enzyme histochemistry was used to analyze the decreases in the number of cholinoceptive neurons in the nBM and the cholinergic fiber density in the Fr1 and HL cortical areas in response to the nBM lesion. The immunotoxin differentially affected the number of ChAT- and AChE-positive neurons in the nBM. 192 IgG-saporin induced a massive, irreversible depletion of the ChAT-positive (cholinergic) neurons (to 11.7% of the control level), accompanied by a less dramatic, but similarly persistent loss of the AChE-positive (cholinoceptive) neurons (to 59.2% of the control value) in the nBM within 2 weeks after the lesion. The difference seen in the depletion of ChAT- and AChE-positive neurons is due to the specificity of the immunotoxin to cholinergic neurons. The cholinergic fiber densities in cortical areas Fr1 and HL remained similarly decreased (to 62% and 68% of the control values, respectively) up to 20 weeks. No significant rebound in AChE activity occurred either in the nBM or in the cortices during the period investigated. This study therefore demonstrated that, similarly to the very extensive reduction in the number of ChAT-positive neurons in the nBM, cortical areas Fr1 and HL underwent long-lasting reductions in the number of AChE-positive fibers in response to specific cholinergic lesioning of the nBM.
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