Increased excitability in hippocampal neurons of synaptopodin-knockout mouse

Synaptopodin (SP) is localized within the spine apparatus, an enigmatic structure located in the neck of 35 spines of central excitatory neurons. It serves as a link between the spine head, where the synapse is 36 located, and the endoplasmic reticulum (ER) in the parent dendrite (Vlachos et al. 2009, Korkotian and 37 Segal, 2011, Zhang et al. 2013). SP is also located in the axon initial segment, in association with the 38 cisternal organelle, another structure related to endoplasmic reticulum. Extensive research using SP 39 knockout (SPKO) mice suggests that SP has a pivotal role in structural and functional plasticity (Deller et al. 2003, Deller et al. 2007). Consequently, SPKO mice were shown to be deficient in cognitive functions, 41 and in ability to undergo long term potentiation of reactivity to afferent stimulation (Deller et al. 2003). In 42 contrast, neurons of SPKO mice appear to be more excitable than their wild type (wt) counterparts(Bas Orth 43 et al, 2007). To address this discrepancy, we have now recorded activity of CA1 neurons in the mouse 44 hippocampus slice, with both extracellular and patch recording methods. Electrophysiologically, SPKO 45 cells in CA1 region of the dorsal hippocampus were more excitable than wt ones. In addition, exposure of 46 mice to a complex environment caused a higher proportion of arc-expressing cells in SPKO than in wt mice 47 hippocampus. These experiments indicate that higher excitability and higher expression of arc staining may 48 reflect SP deficiency in the hippocampus of adult SPKO mice.

field, mice were sacrificed. All mice explored the space and ran in the rotating disk, but their behavior was 86 apparently not different between the two groups, and was not quantified.

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Detector and amplifier gain were initially set to obtain pixel densities within a linear range. Eight image 92 stacks were recorded for each hippocampus. Arc-positive and с-Fos -positive cells were counted from each 93 field size of 135x135 μm (63x/1.40 oil DIC objectives). Cell count and fluorescence levels were measure 94 using Image-J software. Measurements were made in a double-blind procedure by an independent observer 95 to assure unbiased analysis. Statistical comparisons were made using Origin software.

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Mice were rapidly decapitated with a guillotine, their brain removed and the hippocampus was sliced into  Nickel-Chromium electrode. Two stimulating electrodes were located on both sides of the recording 106 electrode, with both stimulating the schaffer collateral pathway. Data acquisition and off-line analysis were 107 performed using pCLAMP 9.2 (Axon Instruments) in a blind procedure.

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Whole cell patch recordings:

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Mice were rapidly decapitated with a guillotine, their brain removed and sliced using vibratome into

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Action potential kinetics analysis: 124 Current-clamp recordings were imported in Matlab where the first ten action potentials (AP) that did not 125 arrive in bursts or too close to the end of the current pulse where collected with 5ms pre-peak and 65ms 126 post-peak; these spikes were aligned at peak, averaged and this average was used to calculate a phase plot.

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The average and standard error were calculated for these phase plots within every group. Numeric voltage 128 derivative was calculated as difference between the voltages recorded at neighboring sampling points; 129 multiplied by sampling rate (per ms) when appropriate. AP onset was calculated as a time-point where

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The higher elevation of arc in active SPKO mice compared to wt suggests a higher excitability of 144 hippocampal neurons. To test this directly, experiments with hippocampal slices were conducted. First, 145 population EPSPs in field recording were measured at three different stimulation intensities. CA1 cells 146 produced significantly larger population EPSPs in SPKO compared to wt (Fig 1A). Furthermore, SPKO 147 slices produced population spikes at lower stimulation intensities than wt slices. In addition, Paired pulse 148 facilitation was measured at three different inter-pulse intervals (IPI). We found a trend of higher paired 149 pulses facilitation in the SPKO group compared to wt mice, in all three IPI's tested. However, there was no 150 statistically significant difference between the two groups ( Fig 1B).

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Asterisks denote AP threshold (see methods), AP half-amplitude, AP peak and AP after-hyperpolarization.

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The present study addressed a discrepancy among published results on the role of SP in synaptic plasticity.

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Earlier studies were able to localize SP to the spine apparatus, an enigmatic structure found at a strategic 216 location in the neck of mature dendritic spines. In addition, SP was recently found to be expressed in the

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In the present study we found that SPKO mice at 6 months of age are hyper-excitable compared to control.

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This was found in both spike properties and spontaneous PSC's. In an earlier study Bas Orth et al (2007) 243 showed that SPKO animals are not different in several properties of action potentials. Interestingly, while 244 there were no significant differences, the SPKO animals expressed higher firing rates than wt mice. These 245 experiments were conducted with 3 month old mice, while our experiments showing significant differences 246 were conducted with 6 month old mice. This indicates the enhanced excitability matures between 3 and 6 247 months of age. At the present time it is apparent that SP regulates excitability in both the dendritic spine 248 and the axon initial segment, which is enriched with calcium stores (Segal 2018).

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The enhanced excitability in SPKO mice hippocampus brings up an interesting possibility, namely, that SP 250 actually functions to reduce excitability, via activation of some calcium gated K currents. This is hinted in 251 the significantly reduced AHP in the SPKO cells (Fig 2D). Assuming that native SP reduces excitability, 252 how then is this related to its documented function in synaptic plasticity? One possible interpretation is that

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Consequently, the electrophysiological changes we observed might result from other homeostatic 264 mechanisms that SPKO cell employ to balance the enhanced excitability and sensitivity of the arc gene.

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Further investigations are needed to analyze the types of molecular regulators of synaptic and potential 266 properties in these neurons.