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doi: 10.1242/10.1242/dev.00351

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BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K⁺/Cl^- co-transporter KCC2

Fernando Aguado^1,*, Maria A. Carmona^1,*, Esther Pozas¹, Agustín Aguiló¹, Francisco J. Martínez-Guijarro², Soledad Alcantara¹, Victor Borrell¹, Rafael Yuste³, Carlos F. Ibañez⁴ and Eduardo Soriano^1,

¹ Department of Cell Biology Faculty of Biology, and Barcelona Science Park, University of Barcelona, Barcelona 08028, Spain
² Department of Cell Biology, Faculty of Biological Sciences, University of Valencia, 46100 Burjassot, Spain
³ Department of Biological Sciences, Columbia University, New York, New York 10027, USA
⁴ Department of Neurosciences, Karolinska Institute, Stockholm 17177, Sweden

View larger version (71K): [in a new window]   Fig. 1. Developmental regulation of spontaneous [Ca2+]i oscillations in the embryonic and postnatal hippocampus. (A) Horizontal hippocampal slice from an E18 embryo loaded with fura-2-AM. The principal cells (pyramidal and granular neurons) and some interneurons show intense fura-2 fluorescence. Scale bar: 400 µm. (B,C) Representative plots of F/F over time showing spontaneous [Ca2+]i transients in CA1 hippocampal neurons at E17 (B) and P2 (C). The onset of each [Ca2+]i oscillation is indicated by a bar on the x axis. Note the greater number of spontaneous Ca2+ events at postnatal ages than at embryonic ages. (D,E) Histograms illustrating the developmental regulation of the number of spontaneously active cells/2,000 µm2 (D) and their activation rate ([Ca2+]i oscillations/cell/104 seconds) (E) from E16 to P5. Both parameters increase around birth and peak at P2-P5. Values are shown as mean ± s.e.m. CA1 and CA3, hippocampal regions; DG, dentate gyrus; h, hilus; gcl, granule cell layer; ml, molecular layer; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum.

View larger version (68K): [in a new window]   Fig. 2. Synchronous patterns of spontaneous network activity in the hippocampal CA1 region emerge after birth. (A-E) Correlation network analysis of the CA1 hippocampal region at E16. (A,B) Paired photomicrographs showing the same field viewed under fura-2 fluorescence (A) and DIC optics (B) illustrating dozens of cells loaded with the Ca2+ indicator. Black squares indicate cells with spontaneous changes of fura-2 fluorescence signal over time. (C) Raster plot representing the activity profile of each of the 12 spontaneously active cells shown in A and B over 800 seconds. The activity profiles of individual cells are represented by horizontal lines and each tick mark labels the onset of each Ca2+ transient. Dotted lines indicate simultaneous onset of [Ca2+]i oscillations of at least two cells of the plot. Note the small number of co-activations at E16 (e.g. cells no. 6 and no. 11). (D) Spatiotemporal correlation map illustrating all the active cells shown in A-C, in which pairs of cells with statistically significant correlation coefficients are linked by lines. Only 5 out of 12 active cells, located in the stratum oriens and in the pyramidal layer, show significant synchrony. The P value reflects the probability that the overall degree of synchronous correlation present in this network is caused by chance (see E). (E) Distribution of pair-wise correlations found in the real data (arrow) and in 1,000 simulations obtained by the Monte Carlo test (bell-shaped curve) of the active cells shown in A-D (see Materials and Methods). In this case, the number of correlated events in the real data set (arrow) does not exceed that expected by chance in simulated data, P=0.25. (F-I) Correlation network analysis of the CA1 hippocampal region at P1-P2. (F) CA1 region of a P2 hippocampal slice viewed with fura-2 fluorescence. Note the greater number of active cells (squares) than at E16 (A). (G) Raster plot illustrating the activity profile of each of the 70 cells shown in F. Highly synchronous activity patterns, in which network oscillations involve virtually the entire population of active neurons, are easily recognized. (H) Spatiotemporal correlation map of the CA1 region of a P1 slice illustrating complex synchronous networks, which recruit vast populations of active cells. Note the greater overall complexity than at E16. Furthermore, the lines connecting co-active cells are thicker than at E16, indicating higher degrees of correlation. The P value reflecting the probability that the overall degree of synchronous correlation present in this network is caused by chance is 0 (see I). (I) The number of simultaneous co-activations present in the network represented in H (arrow) exceeds the frequency distribution of random experiments created by Monte Carlo simulation. (J) Percentages of active cells involved in synchronous correlated networks at different ages (E16-P5). (K) Average of P values representing network correlation during development. The number of cases with significant synchronous correlated networks (P<0.05) in relation to the total number of cases analyzed is shown at the top of each bar. (L) Graph illustrating the correlation between the rise in network synchrony (P values, left, solid line) and the increase in the number of active cells (dotted line; see Fig. 1D) in the hippocampal CA1 region during development. Values in J and K are given as mean ± s.e.m. Scale bars, 40 µm. Abbreviations as in Fig. 1.

View larger version (120K): [in a new window]   Fig. 3. Histological organization of the hippocampus in transgenic BDNF-overexpressing embryos. (A) Transgenic (BDNF) and control (C) E18 embryos are identified by PCR analysis of the nestin-BDNF construct. As revealed by northern blot analysis, nestin-BDNF transgenic forebrains express about 30-fold higher levels of BDNF mRNA than controls (including endogenous and transgenic mRNAs). Cyclophilin mRNA levels were used as an internal standard. (B,C) Nissl staining of BDNF-overexpressing and control hippocampal sections. The overall hippocampal cytoarchitectonics is preserved in transgenic hippocampi, although transgenic embryos that integrated many construct copies show a double pyramidal layer (arrows in C). (D,E) Calretinin immunostaining shows the correct location of Cajal-Retzius cells in the stratum lacunosum-moleculare (arrows) in both transgenic and wild-type hippocampi. (F) Section from the hippocampus of a BDNF transgenic embryo immunolabeled by the axonal marker L1, showing correct axonal patterns with L1-positive fascicles in the white matter (wm), fimbria (fi) and in the stratum lacunosum-moleculare (slm), mainly corresponding to commissural and entorhino-hippocampal axons. (G) Patterns of entorhino-hippocampal innervation in a BDNF transgenic hippocampus following a DiI injection in the entorhinal cortex. Entorhinal fibers (red) are seen in the fimbria (fi) and in the stratum lacunosum-moleculare (slm). Section counterstained with bisbenzimide (blue). Scale bars, 400 µm. Abbreviations as in Fig. 1.

View larger version (101K): [in a new window]   Fig. 7. GAD65/67 expression and GABAergic synaptic contacts are greater in the E18 BDNF-overexpressing hippocampus. (A,B) Non-radioactive in situ hybridization showing the expression patterns of GAD65/67 in the embryonic wild-type (A) and transgenic (B) hippocampus at E18. Note the increased hybridization signals in the transgenic hippocampus. (C) Northern blot analysis of E18 forebrain showing a 3-fold increase in GAD67 mRNA expression in transgenic embryos (BDNF) compared to control embryos (C). Cyclophylin mRNA levels were used as an internal standard. (D,E) High magnification photomicrographs illustrating calbindin-immunopositive interneurons (arrows) in the stratum oriens of control (D) and transgenic (E) E18 hippocampi. Note the larger calbindin-positive neurons in the BDNF transgenic embryos compared to controls (arrows). (F-H) Electron microphotographs showing GABA immunogold labeling (arrows) in the stratum radiatum of E18 wild-type (F,G) and transgenic (H) hippocampus exhibiting GABA-positive (F,H) and GABA-negative (G) axon terminals (AT) with dendrites (D). (I,J) Histograms representing the greater number of GABA-positive axon terminals (I) and GABA-negative axon terminals (J) in BDNF-overexpressing hippocampus (black bar) than in wild-type hippocampus (white bar). *P<0.05; **P<0.001. Scale bars: 300 µm (A,B); 20 µm (D,E); 0.4 µm (F,G). Abbreviations as in Fig. 1.

View larger version (62K): [in a new window]   Fig. 4. BDNF-overexpressing hippocampi show increased spontaneous correlated network activity at E18. (A-H) Representative examples of network activity in the hippocampal CA1 region in control (A-D) and transgenic (E-H) E18 embryos. (A) Fura-2-loaded control hippocampal slice showing few active neurons (squares). (B) Plot of F/F over time illustrating spontaneous [Ca2+]i changes in a control neuron. The initiation of [Ca2+]i transients is indicated by bars on the x axis. (C) Raster plots of all active cells shown in A. Horizontal lines represent the activity profile over 800 seconds of each active neuron and vertical thick lines mark the onset of [Ca2+]i oscillations. (D) Correlation map illustrating a significant (P<0.01) spatio-temporal co-activation among all cell pairs shown in A and C assayed with 2 contingency tables. The thickness of the lines is proportional to the degree of significance. The P value indicated at the bottom (P=0.15) reflects the overall level of co-activation obtained by Monte Carlo simulation, and represents the probability that the co-activations present in this field are caused by chance. (E) Fura-2-loaded transgenic hippocampal slice illustrating more active neurons (squares) than in controls. (F) Representative plot of F/F over time showing spontaneous [Ca2+]i oscillations in a hippocampal neuron from a transgenic embryo. (G) Raster plot of all active neurons shown in E, illustrating the activity profile of each of the 49 cells over 800 seconds. Note the greater number of active neurons, [Ca2+]i transients and co-activations than in C. (H) Correlation map showing the spatio-temporal co-activations present in the transgenic CA1 region shown in E. BDNF-overexpression increases the complexity of spontaneous correlated networks in the hippocampus. Furthermore, the overall synchrony of this network also increases in BDNF transgenic embryos, as indicated by the P value through the Monte Carlo simulation test (P=0.001). (I-K) Histograms showing significant increases in the percentage of active neurons out of the total of fura-2-loaded cells (I), the activation rates (Ca2+ transients/cell/104 seconds) (J) and in number of active cells involved in correlated networks (K), in BDNF-overexpressing hippocampi (black bars) compared to controls (white bars). (L) The average of P values obtained by Monte Carlo simulation in co-active networks of transgenic slices (black bars) is much lower than in controls (white bars). *P<0.01, **P<0.001 Scale bars: 50 µm.

View larger version (65K): [in a new window]   Fig. 5. Expression and contribution of neurotransmitter receptors in the BDNF-overexpressing hippocampus. (A) Western blot analysis of the GABAA receptor 1 subunit and the glutamate receptor NR1, NR2A, GluR1 and GluR2/3 subunits in membrane fractions isolated from wild-type (C) and transgenic (BDNF) forebrains at E18. There are no differences between groups. (B-G) Immunostaining for the GABAA 2 (B,E), NR1 (C,F) and GluR2/3 subunits (D,G) in control (B-D) and BDNF-overexpressing (E-G) E18 hippocampal sections. These subunits are distributed in a similar way throughout the hippocampal formation of control and transgenic embryos. In addition, no major changes in the density of immunostaining were observed. (H,I) Representative paired correlation maps showing spontaneous correlated network activity in the CA1 region of a transgenic hippocampal slice before (H) and after (I) blocking GABAA receptors by administration of 30 µM BMI. Note that GABAA receptor blockade decreases correlated network complexity. (J-L) Histograms showing the percentage of active neurons (J), the activation rate (K) and the number of active cells in spontaneous correlated networks (L) in the E18 CA1 region after incubations with the GABAA, NMDA and AMPA/kainate antagonists BMI, APV and CNQX, respectively. Data are stardardized to baseline values. White bars represent wild-type and black bars, transgenic embryos. The number of slices used ranged from 3 to 10 for each experimental condition. BMI incubation blocks spontaneous network activity in both wild-type and transgenic slices. Data are mean ± s.e.m. Significance levels: *P<0.05, **P<0.01, Student t-test. Scale bars, 200 µm. Abbreviations as in Fig. 1.

View larger version (120K): [in a new window]   Fig. 6. Transgenic BDNF-overexpressing embryos show increased hippocampal synaptogenesis. (A) Immunoblot analysis of the synaptic vesicle proteins synaptophysin and synapsin I, and of the t-SNARE syntaxin1 in forebrain membrane fractions from control (C) and transgenic (BDNF) E18 embryos reveals no major differences between groups. (B,C) Photomicrographs illustrating similar patterns of immunolabeling were observed for synaptophysin in hippocampal sections from wild-type (B) and BDNF-overexpressing (C) E18 embryos. (D,E) Electron micrographs illustrating synaptic contacts (black arrows) in the stratum radiatum of control (D) and transgenic (E) E18 embryos. Note the distinct patterns of distribution of synaptic vesicles in the axon terminal and the increased area of postsynaptic dendrite of transgenic synapses. Open arrow in E points to a putative contact. (F-J) Quantitative analysis illustrating structural alterations in synaptic profiles of E18 transgenic hippocampi. BDNF-overexpressing embryos show a significant increase in the number of synaptic contacts per area (F) and in the number of docked synaptic vesicles per contact (H), whereas the total number of synaptic vesicles per contact is significantly decreased in these embryos compared to wild-type embryos (G). The area of postsynaptic dendrites was significantly increased in transgenic embryos (I) whereas no differences were found for axon terminal area between both groups of animals (J). Values are mean ± s.e.m. White bars, wild type; black bars, transgenic embryos. *P<0.01; **P<0.0001. Scale bars, 400 µm (B,C); 0.5 µm (D,E). AT, axon terminal; D, dendrite; so, sp, sr, slm, CA1, CA3 and DG as in Fig. 1.

View larger version (51K): [in a new window]   Fig. 8. BDNF-overexpression alters KCC2 mRNA expression and GABAA receptor-evoked responses in E18 transgenic embryos. (A) Semiquantitative RT-PCR analysis showing the expression levels of KCC2 mRNA in two wild-type (control) and two transgenic (BDNF) forebrains. Note the marked increase in KCC2 mRNA levels in BDNF-overexpressing embryos compared to controls. GAPDH RT-PCR analysis was used as reference. (B,C) Non-radioactive in situ hybridizations illustrating neuronal somata expressing consistent levels of KCC2 mRNA through the hippocampus of transgenic BDNF-overexpressing embryos (C), whereas KCC2 mRNA expression levels in the control hippocampus are close to background (B). (D,E) Plot of F/F over time illustrating representative examples of GABAA-evoked responses in wild-type (control) and transgenic (BDNF) neurons from E18 hippocampal slices. Administration of 10 µM muscimol (arrows) caused a [Ca2+]i increase in both control and transgenic neurons, although the response was impaired in BDNF-overexpressing embryos (E). (F) Histograms representing the percentage of neurons responding to muscimol by [Ca2+]i increase and the F/F amplitude in control (white bars) and BDNF-overexpressing hippocampi (black bars). Values are mean ± s.e.m. Scale bar: 200µm. *P<0.0001. Abbreviations as in Fig. 1.