Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code

Data collection
IntraCell software (custom-made, LIN Magdeburg, Germany) was used for data collection by LTP measurements Data analysis For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
-OriginPro 2018 and pClamp10 software (Molecular Devices) were used for analysis and recordings of electrophysiological data in organ otypic preparations. -Custom written Matlab scripts were used for preprocessing and analysis of FRET biosensors data and F-and G-acti n results.
-ImageJ was used for analysis of fluorescence intensity of images obtained by confocal microscopy -Spine Magick (patent no. WO/2013/021001) was used for spine analysis in cultured neurons. -3DSpAn and Imaris were used for 3D visualition of dendritic spines from organotypic culture. -SigmaPlot 12.0 and GraphPad Prizm8 were used for statistical data analysis.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request 2 nature research | reporting summary

October 2018
Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No predetermine sample-size calculation was performed; all sample sizes were based on statistically significant difference between experimental groups. Sample size was selected according to previous experience and publications.
Data exclusions In general, no data were excluded from the analyses.
During the LTP measurements, several slices were excluded independently of LTP values, only if a technical problem appeared during recordin gs (dust of recording electrode, insufficient solution oxygenation), which manifested as sudden changes in fEPSP amplitude. All such cases are properly documented in the lab book. In addition, Western blots with air bubbles affecting quantification were excluded from analysis.

Replication
All experimental findings showed the reproducibility and were replicated through the repeated experiments.
In case of LTP, no extra-replication was done as this is quite reliable assay used for many years in the lab of Dr. Dityatev Randomization In general, all samples were allocated in a random way. Tissue samples were also randomly allocated into experimental groups for both organotypic and acute slice preparations in electrophysiological and 2P florescence imaging experiments.
In case of LTP measurement, slices from the same mouse were randomly attributed to control or treatment groups.
Blinding -The analysis of the spines morphology, N1E-115 cell morphology, F/G-actin ratio were done blindly. The analysis of biosensors were done by Matlab script automatically and ROI selection was done blindly.
-Blinding was not relevant to the electrophysiological data of neuronal excitability/synaptic transmission where agonists were acutely applied to tissue preparations or neuronal cultures (i.e. before-after treatment).
-In case of LTP measurements, no blinding was performed as this methodological approach is robust and there are no subjective decisions du ring data acquisition and analysis.
-In case of Western blot, blinding was not relevant because the method used is robust and there are no subjective decisions during data acquisition and analysis Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Wild animals
The study did not involve wild animals

Field-collected samples
The study did not involve samples collected from the field

Ethics oversight
All procedures performed on animals were according to the guidelines of the European Commission (European Communities Council Directive 2010/63/EU) and the United Kingdom Home Office (Scientific Procedures) Act (1986). Experiments were approved by the local animal care committee (Landesverwaltungsamt Sachsen-Anhalt).
Note that full information on the approval of the study protocol must also be provided in the manuscript.