Neuronal growth patterns and synapse formation are mediated by distinct activity-dependent mechanisms

Abstract

All brain functions in animals rely upon neuronal connectivity that is established during early development. Although the activity-dependent mechanisms are deemed important for brain development and adult synaptic plasticity, the precise cellular and molecular mechanisms remain however, largely unknown. This lack of fundamental knowledge regarding developmental neuronal assembly owes its existence to the complexity of the mammalian brain as cell-cell interactions between individual neurons cannot be investigated directly. Here, we used individually identified synaptic partners from Lymnaea stagnalis to interrogate the role of neuronal activity patterns over an extended time period during various growth time points and synaptogenesis. Using intracellular recordings, microelectrode arrays, and time-lapse imaging, we identified unique patterns of activity throughout neurite outgrowth and synapse formation. Perturbation of voltage-gated Ca²⁺ channels compromised neuronal growth patterns which also invoked a protein kinase A mediated pathway. Our findings underscore the importance of unique activity patterns in regulating neuronal growth, neurite branching, and synapse formation, and identify the underlying cellular and molecular mechanisms.

Fig. 1

Endogenous neuronal activity patterns are correlated with distinct growth patterns in paired and unpaired LPeD1 neurons. (A) An unpaired LPeD1 neuron exhibiting bursts consisting of greater number of spikes concomitant with extensive growth (Group A, n = 8) (recorded at 48–50 h). (B) Spontaneous bursting consisting of fewer spikes in unpaired LPeD1 neurons, which exhibited a growth pattern most commonly observed in LPeD1 neurons (Group B, n = 8) (recorded at 48–50 h). (C) Unpaired LPeD1 neurons were hyperpolarized with 0.2-0.3nA current prior to growth initiation (at 4–6 h) (Group C, n = 10). (D) Unpaired LPeD1 neurons that were hyperpolarized at 4–6 h post-plating (prior to exhibiting growth) and examined at 48–50 h post-plating, exhibited thinner neurites which often remained unbranched. (E) Unpaired LPeD1 neurons were impaled with an electrode (at 4–6 h and recordings maintained for 48–50 h) prior to growth initiation (Group D, n = 12) but not hyperpolarized. (F) Ratio of growth endings to primary neurites from unpaired LPeD1 neurons following 48-hours of growth. (G) Mean spikes per burst and (H) IBI for endogenous activity from paired and unpaired LPeD1 neurons. (I) Paired LPeD1 neuronal activity patterns before (12-hours) and after synapse formation (18–24 h) with VD4 (n = 6). The blue box indicates where the synapse likely formed, and recordings from VD4 (pre-synaptic) and LPeD1 (post-synaptic) verified synapse formation with 1:1 excitatory post-synaptic potentials (EPSPs). Statistics: (F-H) used a one-way ANOVA (ratio of growth endings to primary neurites after 48-hours: F[3, 34] = 29.43 (P < 0.0001), Spikes per Burst: F[2, 19] = 22.17 (P < 0.0001), IBI: F[2, 19] = 1.535 (P = 0.2411) followed by Tukey’s multiple comparisons test. A paired t-test was used to compare LPeD1 neurons cultured with VD4 before and after synapse formation for spikes per burst [t = 1.732, df = 5, P = 0.2876] and IBI [t = 13.07, df = 5, P = 0.0004]. Holm-Bonferroni correction was applied to account for family-wise error rate. Error-bars represent SEM, all comparison tests were two-tailed. n.s p > 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig. 2

Perturbations of electrical activity in LPeD1 neurons via direct intracellular current injection influence growth and axonal branching. (A) Shows an unpaired LPeD1 neuron being impaled with an intracellular electrode at (30–36 h) when the neuron had exhibited growth and branching. LPeD1 was hyperpolarized (0.2-0.3nA) for 1 h, the starting point indicated at the red line and was subsequently released from hyperpolarization at the purple line. (B) Shows an image of another LPeD1 neuron that was released from hyperpolarization (purple line). Within 30 min to 1–2 h of resumption of endogenous bursting, this triggered subsequent branching (n = 12). (C) Shows another LPeD1 neuron where following the release of hyperpolarization, bursting activity was triggered by injecting 0.1-0.2nA depolarizing current (purple line) which was followed by the resumption of neuritic/growth cone branching within 1 (n = 6). (D) LPeD1 growth cone prior to triggering prolonged bursts for action potentials (3–5 s) over a period of 5–10 min. (E) Same LPeD1 growth cone collapsed followed by neurite retraction within 10–15 min (n = 7). (F) Spikes per burst of unpaired LPeD1 neurons during endogenous (Groups A and B) and triggered bursting activity. (G) IBI of unpaired LPeD1 neurons during endogenous (Groups A and B) and triggered bursts. Statistics: (F) and (G) used one-way ANOVA (for spikes per burst: F[3, 25] = 12.49 (P < 0.0001), for IBI: F[3, 25] = 10.37 (P = 0.0001) followed by Tukey’s multiple comparisons test. A Holm-Bonferroni correction was applied to all p-values to account for family-wise error rate. Error bars indicate SEM, all comparison tests were two-tailed. n.s p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001. A scale bar is provided for all images.

Fig. 3

Activity evoked calcium influx regulates the extent of growth in LPeD1 neurons. (A) Representative non-ratiometric calcium imaging of a LPeD1 neuron during endogenous activity using Fluo-4 dye (at 48–50 h) (n = 7). (B) Non-ratiometric calcium imaging using Fluo-4 dye for LPeD1 growth cones at 24 h during exogenous bursting stimulation (n = 9). (C) LPeD1 neuronal growth (after 40–48 h) when the neurons were cultured in the presence of 5 μm (n = 16 added after 2–4 h of plating) and (D) 10 μm nifedipine (n = 11). (E) LPeD1 neuronal growth (photographed at 48–50 h) when cultured in the presence of 5 μm (n = 20), and (F) 10 μm cadmium (n = 25 added after 2–4 h of cell plating). (G) Blocking protein kinase A with 5 μm staurosporine (cells photographed after 48–50 h of cell plating) inhibited neuronal branching in LPeD1 neurons (n = 18, the drug was added after 2–4 h of cell plating). For all inhibition experiments, exogenous stimulations (as above) were elicited (0.1-0.2nA) in attempts to induce branching, while also monitoring the health of neurons. (H) Ratio of the number of growth endings to number of neurites present after 48-hours for unperturbed LPeD1 neurons in Groups A and B, as compared to instances of inhibiting Ca²⁺ or PKA. Statistics: (H) used a one-way ANOVA (F[6, 99] = 368.1, P < 0.0001) followed by Tukey’s multiple comparisons test to compare all Ca²⁺ inhibitors alongside staurosporine to Group A and B neurons, alongside staurosporine to all Ca²⁺ inhibitors. A Holm-Bonferroni correction was applied to all p-values to account for family wise error rate. Error bars indicated SEM. A scale bar is provided for all images.

Fig. 4

Characteristic activity patterns of LPeD1 neurons plated with VD4 on MEAs. (A) Example of individual LPeD1 and VD4 neurons plated on MEA exhibiting growth. 1 refers to LPeD1, 4 refers to VD4, a relative scale bar is provided for the image. (B) LPeD1 neurons predominantly exhibited either single spikes that were interspaced with prolonged periods of quiescence or prolonged tonic activity during the first 5–8 h of following plating (n = 8). (C) Spontaneous bursting activity began occurring consistently after 8–11 h following plating (n = 8). (E) These bursts began to consist of more spikes nearing the 16–18 h of recording (n = 8). (F) Tetanic activity was often seen during or following physical contact between the synapsing pairs (generally when growth cones were within 35–50 μm of target, n = 6 clear instances of contact between partners). (G) Nearing synapse formation which usually occurred around 19–24 h, long tonic trains would also occur (n = 6).

Fig. 5

Presence and the pairing configuration of a synaptic partner modulates activity patterns. (A) Burst duration (seconds), (B) number of spikes per burst, and (C) number of total bursts recorded from postsynaptic LPeD1 neurons under different culture configurations during the 10 h post plating (GDSF: n = 8, Unpaired: n = 7, Soma-Soma: n = 8). (D) Representative timelapse and corresponding raster plots for neurons cultured adjacently to each other (n = 8). ‘G’ indicates when growth began, 1 refers to LPeD1, and 4 refers to VD4. Burst heatmap is provided directly underneath timelapse where the proportion of spikes belonging to bursts within an hour bin uses a color gradient (low = white, high = red). Mean hourly spike rate is presented in a heatmap with a color gradient (low = blue, high = red). (E) Change in amplitude (µV) and (F) spike rate (spikes/s) from start of experiment to point of contact for LPeD1 neurons cultured separated from presynaptic VD4 (n = 6 neurons that made clear contact and network maturation). (G) Spike rate in neurons that branched to form synapses (n = 6) as compared to neurons cultured adjacently to each other to form synapses in the absence of growth (n = 8). (H) Comparison of spike waveforms between GDSF (n = 8) and unpaired (n = 7) conditions during hours 0–5, (I) hours 5–10, and (J) hours 10–15 of experiment, and (K) for hours 0–15 following recovery from plating. The average GDSF waveform is indicated with a blue line, the shaded blue region indicates SD. The average unpaired waveform is indicated with a red line, the shaded red region indicates the SD. Statistics: (A), (B) and (C) used a Kruskal Walis Test (Kruskal-Walis Statistic: 7.800 (P = 0.0202), 9.026 (P = 0.0110), 4.005 (P = 0.1350) for (A), (B), and (C) respectively) with Tukey’s Honestly Significant Difference Test. (E) and (F) used a Wilcoxon paired test. (G) used a Mann-U-Whitney Test. (H), (I), (J), and (K) used the Pearson Correlation Coefficient. Error bars indicate SEM. n.s p > 0.05, *p < 0.05, *p < 0.01.

Fig. 6

MEA recordings validate role of bursting activity as observed in intracellular experiments. (A) Distribution of all burst patterns recorded from LPeD1 neurons that were cultured separated from their synaptic partner VD4 (n = 8). (B) Distribution of all bursts recorded from unpaired LPeD1 neurons. Zoom in provides focus on the main part of the distribution (n = 7). (C) Distribution of all bursts recorded from LPeD1 neurons when cultured adjacently with their synaptic partner VD4 in a soma-soma configuration (n = 8). For histograms bursts are indicated in red, non-bursting activity is in blue. (D) Number of hourly bursts recorded from LPeD1 neurons cultured separated from their synaptic partner VD4 throughout 30 h of development following plating (n = 8). (E) Mean hourly bursts recorded from LPeD1 cultured separated from VD4 throughout 10-hour intervals (n = 8). (F) Representative timelapse of postsynaptic LPeD1 establishing physical contact with its presynaptic partner VD4 (n = 6) with mean raster plots of corresponding activity patterns. Burst raster plot is provided directly underneath timelapse where the proportion of spikes belonging to bursts within an hour bin uses a color gradient (low = white, high = red). The overall mean hourly spike rate is presented in a heatmap with a color gradient where blue indicates the lowest spike rate in a one-hour time bin during the experiment, and red represents the highest spike rate in a one-hour time bin. ‘G’ indicates when growth began, S refers to synapse formation, 1 refers to LPeD1, and 4 refers to VD4. A scale bar is provided for all images. Image quality was sharpened to improve visibility of growth when scaling for figure panels. Statistics: (E) used a Friedman test (Friedman statistic: 7.000, P = 0.0303) with Tukey’s Honestly Significant Difference Test. Error bars indicate SEM. n.s p > 0.05, *p < 0.05.