Spatio-temporal dynamics of lateral Na+ diffusion in apical dendrites of mouse CA1 pyramidal neurons

Abstract

Sodium ions $\left({\text{Na}}^{+}\right)$ are major charge carriers mediating neuronal excitation and play a fundamental role in brain physiology. Glutamatergic synaptic activity is accompanied by large transient ${\text{Na}}^{+}$ increases, but the spatio-temporal dynamics of ${\text{Na}}^{+}$ signals and properties of ${\text{Na}}^{+}$ diffusion within dendrites are largely unknown. To address these questions, we employed multi-photon ${\text{Na}}^{+}$ imaging combined with whole-cell patch-clamp in dendrites of CA1 pyramidal neurons in tissue slices from mice of both sexes. Fluorescence lifetime microscopy revealed a dendritic baseline ${\text{Na}}^{+}$ concentration of ~10 mM. Using intensity-based line-scan imaging we found that local, glutamate-evoked ${\text{Na}}^{+}$ signals spread rapidly within dendrites, with peak amplitudes decreasing and latencies increasing with increasing distance from the site of stimulation. Spread of ${\text{Na}}^{+}$ along dendrites was independent of dendrite diameter, order or overall spine density in the ranges measured. Our experiments also show that dendritic ${\text{Na}}^{+}$ readily invades spines and suggest that spine necks may represent a partial diffusion barrier. Experimental data were well reproduced by mathematical simulations assuming normal diffusion with a diffusion coefficient of ${\text{D}}_{{\text{Na}}^{+}}=600\mu {\text{m}}^2/\text{s}$ . Modeling moreover revealed that lateral diffusion is key for the clearance of local ${\text{Na}}^{+}$ increases at early time points, whereas when diffusional gradients are diminished, ${\text{Na}}^{+}/{\text{K}}^{+}$ -ATPase becomes more relevant. Taken together, our study thus demonstrates that ${\text{Na}}^{+}$ influx causes rapid lateral diffusion of ${\text{Na}}^{+}$ within spiny dendrites. This results in an efficient redistribution and fast recovery from local ${\text{Na}}^{+}$ transients which is mainly governed by concentration differences.

Figure 1:. FLIM-based analysis of Na+i and line scan recording of Na+i changes.

A: Color-coded image of ING2 fast Fluorescence Lifetime (FL) of a CA1 neuron. Color-code on the right illustrates ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ as determined by in situ calibration. The dotted box: region enlarged on the right; PP: patch pipette. Upper Right: dotted lines indicate $10\mu \text{m}$ long ROIs from which FL was analyzed. Note that pipette and soma (encircled by magenta) are depicted with the same color code as dendrites, calibration properties, however, differ in these compartments. B: Box plots illustrating baseline ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ along primary dendrites at different distances from the soma (n=8, N=6). C: Color-coded image of ING2 FL of a cell exposed to TTX. Again, pipette and soma are depicted with the same color code as dendrites, but calibration properties differ. Upper Right: current-clamp recording in control and with TTX. D: Box plots showing ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ in control (16 recordings in 8 cells) and with TTX (24 recordings in 12 cells; p=0.006, Mann-Whitney test). E: Box plots showing ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ in primary dendrites in control conditions from cells patched with a pipette saline containing either 5, 11 or 17 mM ${\text{Na}}^{+}$. B, D and E: Shown are single data points (diamonds), mean (squares), median (horizontal line), quantiles (box), and SD (whiskers). D: Grey diamonds: primary dendrites, red diamonds: secondary dendrites; statistical significance in indicated by asterisks with 0.01<**p<0.001. F: Left: Projection of an SBFI-filled primary dendrite. Red line: position of the scan line; SOI 0: segment exhibiting the largest relative change in fluorescence. IP: iontophoresis pipette; arrowhead indicates flow of bath perfusion. Top Right: False color-coded (x,t) image of the baseline-corrected line scan, depicting ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ changes induced by glutamate iontophoresis (100 ms). Vertical box: position of SOI 0. Center: Glutamate-induced somatic inward current. Bottom Right: Glutamate-induced change in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ in SOI 0. Black: baseline corrected trace (subjected to 50 Hz low pass FFT), red: filtered trace as given by the Python-based program. G: Somatic inward current, its calculated first deviation and the accompanying change in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ in SOI 0. H: Glutamate-induced dendritic ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ transients and somatic currents taken under control conditions (left), after wash in of glutamate receptor blockers APV and NBQX (middle), and after washout of the blockers (right). I: Peak-normalized, averaged traces from experiments conducted at 22 °C (n=18, N=18; red) and at 32 °C (n=15, N=11; green). J: Boxplot comparing the decay time constants $\left(\tau \right)$ of ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ recovery at 22 °C and 32 °C. Shown are individual data points (diamonds), means (squares), medians (horizontal lines), quantiles (boxes), and standard deviations (whiskers). Data was statistically analyzed using a Mann-Whitney test (p=0.704). K: Scatterplot showing correlation between $\Delta {\left[{\text{Na}}^{+}\right]}_{\text{i}}$ and $\tau$ of the signal measured at SOI 0 in experiments taken from DIV 10–25 primary dendrites. The dotted line shows a linear fit (R² < 0.001; slope=0.02).

Figure 2:. Spread of Na+ along primary dendrites.

A: Projection of an SBFI-filled primary dendrite. Red line: position of the scan line; with SOIs spanning $5.1\mu \text{m}$ each. IP: iontophoresis pipette; arrowhead indicates flow of bath perfusion. Scale bar: $10\mu \text{m}$. Right: False color-coded (x,t) image of the baseline-corrected line scan with SOI 0 outlined. Vertical boxes indicate SOIs, dotted box indicates glutamate application (100 ms). B: Glutamate-induced ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ transients in SOIs 0–8. Black: baseline corrected traces (subjected to 50 Hz low pass FFT), red: filtered traces as given by the Python-based program. Grey area indicates glutamate application. Grey triangles indicate the peak time points. Trace on the top right shows the somatic current. C: Overlaid traces of ${\text{Na}}^{+}$ transients from SOI 0, 2, 4, 6 and 8 at higher time resolution. D: Projection of an SBFI-filled primary dendrite subjected to imaging where SOI 0 is outlined. IP: iontophoresis pipette; arrowhead indicates flow of bath perfusion. Scale bar: $10\mu \text{m}$. Right: False color-coded (x,t) image of the baseline-corrected line scan with SOI 0 outlined; dotted rectangular box indicates glutamate application (100 ms). E: Data of 18 experiments showing normalized peak changes in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}\left(\text{norm}.\left.\Delta {\left[{\text{Na}}^{+}\right]}_{\text{i}}\right)\right.$ versus distance from SOI 0 for a SOI length of $5.1\mu \text{m}$ (Top) and of $1.02\mu \text{m}$ (Bottom). Negative distances indicate dendritic sections within the perfusion direction relative to SOI 0. Shown are individual data points (grey symbols), means (black symbols) and standard deviations (whiskers). Black dotted line shows exponential fit for SOIs within perfusion direction (R²=0.99), black line for SOIs against the perfusion flow (R²=1.00). F: Peak time points after stimulation versus distance from SOI 0; same data set and illustration as shown in E.

Figure 3:. Morphological characteristics of dendrites and diffusion of Na+ from dendrites into spines.

A: Maximal projections of primary (pri) and secondary (sec) dendrites filled with SBFI in slices cultured for 3–6, 10–25 or 35–50 days. B, C: Boxplots showing dendrite diameter (left) and spine density (right) of primary dendrites (DIV 10–25; n=18, N=18) and of secondary dendrites at DIV 3–6 (n=17, N=17), DIV 10–25 (n=18, N=18) and DIV 35–50 (n=13, N=13). Shown are individual data points (diamonds), means (squares), medians (horizontal lines), quantiles (boxes), and standard deviations (whiskers). For statistical analysis, normal distribution of the data was determined by the Shapiro Wilk test and one-way ANOVA with Bonferroni post hoc analysis was used for normal distributed data. Statistical significance is indicated by asterisks: ***p<0.001. C: Maximal projection of an SBFI filled spiny dendrite at DIV 10–25. IP: iontophoresis pipette; arrowhead indicates flow of bath perfusion. Inset: Section shown at higher magnification, indicating the placement of the line scan (red line) over the dendrite and two adjacent spines (s1, s2). D: False color image of a line scan, taken from the dendrite shown in A with a binning of 2 pixels. Black line below indicates the glutamate application (100 ms). Scale bars represent the following: Horizontal scale bar 1 s, vertical scale bar $1\mu \text{m}.$ E: ${\text{Na}}^{+}$ transients taken from the line scan shown in D, filtered as given by the Python-based program. The recovery to baseline was fitted monoexponentially (colored lines). F: Top. Averaged traces taken from dendrites (n=8, N=8; black trace) and spines (n=9, N=8; grey trace), after binning individual measurements to 50 Hz. Colored traces represent monoexponential fits of the decay. Center: Peak-normalized traces. Bottom: traces at higher temporal resolution. Linear fits depict the slopes of the rising phase. Arrowheads point at the peak time points. G, H: Histograms showing peak changes in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}\left(\Delta {\left[{\text{Na}}^{+}\right]}_{\text{i}}\right)$ and decay time constants $\left(\tau \right)$ of the recovery from ${\text{Na}}^{+}$ transients within dendrites (n=8, N=8) and directly adjacent spines (n=9, N=8). Shown are individual data points (black diamonds) and means (short horizontal lines). Data points derived from a given dendrite and adjacent spine are connected by grey lines. Normal distribution of the data was determined by the Shapiro Wilk test; paired sample t-tests were used to determine the statistical significance between data sets and is indicated as: 0.05<*p<0.01.

Figure 4:. Spread of Na+ along secondary dendrites.

A: Projection of an SBFI-filled secondary dendrite. The red line indicates the scan line, SOIs are $5.1\mu \text{m}$ each. IP: iontophoresis pipette; arrowhead indicates flow of bath perfusion. Right: False color-coded (x,t) image of the baseline-corrected line scan. Vertical boxes indicate SOIs with SOI 0 outlined, the dotted rectangular box indicates the glutamate application (100 ms). B: Glutamate-induced ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ transients in SOIs 0–9. Somatic current is shown on the top right. Black: baseline corrected traces (subjected to 50 Hz low pass FFT), red: filtered trace as given by the Python-based program. Grey area indicates glutamate application. Grey triangles indicate the peak time points. C: Overlaid traces from SOI 0, 2, 4, 6 and 8 at higher time resolution. D, E, F: Normalized peak changes in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ (norm. $\Delta {\left[{\text{Na}}^{+}\right]}_{\text{i}}$; diamonds) and peak time points after stimulation (circles) versus distance from SOI 0 in DIV 10–25 secondary dendrites (n=18, N=18), DIV 3–6 secondary dendrites (n=17, N=17) and DIV 35–50 secondary dendrites (n=13, N=13). Shown are individual data points (grey symbols), means (black symbols) and standard deviations (whiskers). Colored lines represent monoexponential fits of the data, with corresponding R² values indicated (full line: $\Delta {\left[{\text{Na}}^{+}\right]}_{\text{i}}$; dotted line: peak time points).

Figure 5:. Impact of dendrite morphology on the diffusional spread of Na+.

A: Spatial profiles of normalized changes in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ for all investigated DIV 10–25 secondary dendrites. Shown are concentration profiles taken every 100 ms (10 Hz). Color code depicts the point in time at which spatial ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ distributions were determined. B: Plots showing changes in variance over time in DIV 10–25 primary dendrites (black), in secondary dendrites at DIV 3–6 (green), DIV 10–25 (dark blue) and DIV 35–50 (light blue), and for all secondary dendrites (pooled data, red), calculated from profiles such as shown in A. Dotted lines illustrate the mathematical analysis of the diffusional spread using the boundary effects and initial conditions provided by the respective conditions employing the $eff{D}_{{\mathit{\text{Na}}}^{+}}$ for each condition. C: Apparent ${\text{Na}}^{+}$ diffusion coefficients $\left({\text{D}}_{\text{app}}\right)$ resulting from variances depicted in B. Dotted lines illustrate the analysis using the boundary effects and initial conditions employing the $eff{D}_{{\mathit{\text{Na}}}^{+}}$ for each condition.

Figure 6:. Modeling of Na+ dynamics in dendrites.

A: 2-Dimensional projection of the measured CA1 pyramidal neuron (NeuroMorpho.org; (Buchin et al., 2022) NMO_276156; (Tecuatl et al., 2024). The soma is indicated as large red oval, the dendrite used for the simulation is highlighted in red (also see Insert). The arrow points to the dendrite segment subjected to an increase in ${\text{Na}}^{+}$. B: Color-coded images, showing the ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ in the dendrite at t=0 s and t=0.5 s after reaching the maximum peak at the segment associated with the highest ${\text{Na}}^{+}$ amplitude (at $0\mu \text{m}$; indicated by an arrow). C: Image of the color-coded line scan, showing the ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ spread through the dendrite over time. D: Spatial concentration profiles of normalized changes in ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ taken every 20 ms. Color-code depicts the point in time at which ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ distributions were determined. E: Left: ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ transients in the stimulated segment $\left(0\mu \text{m}\right)$, as well as segments at 5.8, 9.2, 12.1, 15.5 and $22.2\mu \text{m}$ distance. Right: Corresponding experimental data from primary dendrites (n=18, N=18; see Fig. 2). Individual traces were averaged and normalized to the peak at $0\mu \text{m}$ distance. The grey column indicates the duration of the glutamate application. F: ${\text{Na}}^{+}$ fluxes at the before mentioned distances from the stimulated site, including diffusion fluxes $\left({\mathit{\text{Na}}}^{+}\text{-}{\mathit{\text{Flux}}}_{\mathit{\text{Diff}}}\right)$ and NKA-mediated extrusion fluxes $\left({\mathit{\text{Na}}}^{+}\text{-}{\mathit{\text{Flux}}}_{NKA}\right)$. Inserted boxes show blown up images of the traces on the left. G: Correlation coefficient of ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ transients with ${\mathit{\text{Na}}}^{+}\text{-}{\mathit{\text{Flux}}}_{\mathit{\text{Diff}}}$ and ${\mathit{\text{Na}}}^{+}\text{-}{\mathit{\text{Flux}}}_{NKA}$ over time. Note that correlation coefficient of 0 and −1, respectively, indicate no correlation and strong correlation between ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ and respective flux. H, I: ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ normalized with respect to the peak value at the stimulated segment as a function of distance from the stimulated segment at different times (color-coded) using normal NKA-mediated clearance (H) and enhanced (10 times that of normal) NKA-mediated clearance (I). J: Peak ${\left[{\text{Na}}^{+}\right]}_{\text{i}}$ (black) and time of the peak (red) as functions of distance from the stimulated segment at normal NKA-mediated clearance (solid lines) and enhanced (10 times that of normal) NKA-mediated clearance (dashed lines).