Figure 1.
Timing of the stages of neuronal differentiation.
A) Schematic overview of different stages of the development of hippocampal neurons in culture. Developing hippocampal neurons were harvested at seven indicated time points at basal state and following the induction of neuronal activity using different chemical stimulation protocols. Synaptic activity was blocked with the voltage-gated sodium channel blocker tetrodotoxin (TTX, 2 μM, 48 h) or enhanced with the GABAA receptor antagonist bicuculline (Bicuc, 40 μM, 48 h). Bath application of 50 μM NMDA for 5 min induces “chemical” long-term depression (LTD) and activation of synaptic NMDARs with 200 μM glycine for 5 min triggering “chemical” long-term potentiation (LTP) in cultured neurons. B) Representative images of hippocampal neurons in culture, fixed at the indicated times and stained for the nucleus (DAPI), neuron specific tubulin (tubulin-ßIII), axon marker Tau, dendrite specific protein MAP2 and postsynaptic protein PSD-95. Scale bar, 20 μm C) Western blot analysis of extract from hippocampal neurons in culture for 6 hours (stage 2), 20 hours (stage 3), 4 days (stage 4) and 3 weeks (stage 5) using indicated antibodies. D) Quantification of the percentage of MAP2 and GFAP positive cells in hippocampal neurons culture for 6 hours (stage 2), 20 hours (stage 3), 4 days (stage 4) and 3 weeks (stage 5). Note that in hippocampal culture of 3 weeks, ∼50% of cells are GFAP positive. E) Representative images of hippocampal neurons in culture, fixed at 3 weeks and stained for dendrite specific protein MAP2 and glial fibrillary acidic protein (GFAP), a marker for astrocytes.
Figure 2.
miRNA expression profiles in developing and mature hippocampal neurons.
A–C) Heat maps showing average expression levels of miRNAs in hippocampal neurons during differentiation (6 h, 20 h, 48 h, 3 day and 5 days) compared to mature hippocampal neurons (21 days) in a blue (low relative expression) to yellow (high relative expression) scale. The selection criterion was an average expression value of >50. Using the following threshold signal values >400, 100–400 and 50–100, we subdivided the neuronal miRNAs in mature hippocampal neurons in respectively high (A), intermediate (B) and low expressing levels (C). The mean values were calculated from 4 independent experiments. One of the duplicate probes is shown in the figure D) Expression analysis of miRNAs by qPCR. Higher Ct value means low expression level of the miRNA. The expression of let-7c was changed with time, 6 hours versus 8 days. Single factor ANOVA was performed for statistical significance. Error bars represent standard deviation. ***p<0.0001.
Figure 3.
miRNA expression is regulated during early and late neuronal development.
A–B) Heat maps showing relative expression levels of miRNAs in hippocampal neurons at 6 h, 20 h, 48 h, 3 day and 5 days compared to mature neurons (21 days) after plating in a green (negative fold-change) to red (positive fold-change) scale. Selection criteria; in all samples an absolute fold change value greater than or equal to 5 for negative fold changes P<0.01 and 1.5 for positive fold changes (P<0.05). Expression levels of miRNAs that are changed during early development (6 hours –48 hours) are shown in (A). Expression levels of miRNAs that are altered during late development (3 days –8 days) are shown in (B). (C–H) Graphs of relative signals (normalized fold changes) of miRNA expression during early neuronal development (C–E) and late neuronal differentiation (F–H). The blue and red lines indicate the two probes used to detect the expression of indicated miRNAs, which were spotted in duplicate on the LNA array.
Figure 4.
Sustained expression of subset of miRNAs modulates axonal outgrowth.
A) Hippocampal dissociated neurons were co-transfected with GFP vector and miRIDIAN mimics for let-7c, let-7e, let-7i, miR-7b, miR-26a, miR-28, miR-30c, miR-30d, miR-30e, miR-135b, miR-200a, miR-221, miR-292-5p, miR-378 and miRNA. Three representative images of miRNA mimic transfected hippocampal neurons are shown. Scale bar, 50 μm. B) Graph represents measurement of total axon length. The average axon length of miRNA mimic expressing neurons is longer compared to control neurons (175.5 ± 10.2 μm). * p<0.01, ** p<0.001, *** p<0.0001, **** p<0.00001 in one way ANOVA with bonferroni correction for multiple testing.
Figure 5.
miRNA expression profiling following NMDA receptor-dependent synaptic plasticity.
A) Heat map showing relative expression levels of miRNAs in mature hippocampal neurons (21 days) that are significantly changed following 5 min of 200 μM glycine treatment and recovered for 30 min (chemical LTP), 5 min of 50 μM NMDA treatment recovered for 30 min and 2 hours (chemical LTD) compared to non-treated neurons in a green (negative fold-change) to red (positive fold-change) scale. To control for the specificity of the treatments, all glycine en NMDA experiments were also performed in the presence of NMDA receptor inhibitor APV. Selection criteria; absolute fold change value greater than or equal to 1.4 with P<0.01 for NMDA P<0.05 for glycine and expression changes are blocked by APV. B–E) Graphs of relative signal (normalized fold changes) of miRNA expression following glycine and NMDA treatment compared to non-treated mature hippocampal neurons. The blue and red lines indicate the two probes used to detect the expression of indicated miRNAs, which were spotted in duplicate on the LNA array.
Figure 6.
miRNA expression profiling following prolonged changes in global network activity.
A) Heat map showing relative expression levels of miRNAs in mature hippocampal neurons (21 days) that are significantly changed following 4 hours and 48 hours 2 μM TTX and 4 hours and 48 hours 40 μM Bicuculine treatment compared to non-treated neurons in a green (negative fold-change) to red (positive fold-change) scale. To control for the specificity of the Bicuculine treatments, these experiments were also performed in the presence of APV and CNQX. Selection criteria were: absolute fold change value greater than or equal to 1.5 for 4 hour treatments and 1.4 for 48 hour treatments (P<0.05) and changes in Bicuculine expression are blocked by APV/CNQX. B–G) Graphs of relative signal (normalized fold changes) of miRNA expression following TTX and Bicuculine treatments compared to non-treated mature hippocampal neurons. The blue and red lines indicate the two probes used to detect the expression of indicated miRNAs, which were spotted in duplicate on the LNA array.