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Early depolarizing GABA controls critical-period plasticity in the rat visual cortex

Nature Neuroscience volume 18, pages 87–96 (2015)Cite this article

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Abstract

Hyperpolarizing and inhibitory GABA regulates critical periods for plasticity in sensory cortices. Here we examine the role of early, depolarizing GABA in the control of plasticity mechanisms. We report that brief interference with depolarizing GABA during early development prolonged critical-period plasticity in visual cortical circuits without affecting the overall development of the visual system. The effects on plasticity were accompanied by dampened inhibitory neurotransmission, downregulation of brain-derived neurotrophic factor (BDNF) expression and reduced density of extracellular matrix perineuronal nets. Early interference with depolarizing GABA decreased perinatal BDNF signaling, and a pharmacological increase of BDNF signaling during GABA interference rescued the effects on plasticity and its regulators later in life. We conclude that depolarizing GABA exerts a long-lasting, selective modulation of plasticity of cortical circuits by a strong crosstalk with BDNF.

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Figure 1: Early GABAergic interference prolongs critical-period plasticity in the visual cortex in vivo.
Figure 2: Early GABAergic interference prolongs critical-period plasticity in the visual cortex in vitro.
Figure 3: Early GABAergic interference does not alter the overall structural development of the visual system.
Figure 4: Early GABAergic interference does not alter the overall functional development of the visual cortex.
Figure 5: The effect of early bumetanide treatment on visual cortical plasticity is due to regulation of cation-Cl− co-transporters rather than the regulation of osmolarity.
Figure 6: Early GABAergic interference decreases the cortical inhibitory tone, reduces BDNF expression and impairs the maturation of PNNs at P35.
Figure 7: DHF treatment during early GABAergic interference rescues the bumetanide-induced effect on plasticity.
Figure 8: DHF treatment during early GABAergic interference rescues the bumetanide-induced effect on regulators of plasticity.

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Acknowledgements

We thank K. Kaila (University of Helsinki) for providing brain tissue from NKCC1 knockout brains for control experiments. We thank M. Cristina Cenni (CNR, Pisa) for providing the protocol for retinogeniculate axon labeling. We thank M.V. Chao (New York University School of Medicine) for providing anti-pTrkB. We thank F. Benfenati (Istituto Italiano di Tecnologia) for reading the manuscript. The work was supported by Compagnia di San Paolo (grant 2008.1267 to L.C. and M.C.), Telethon (grants GGP10135 to L.C., GGP11116 to M.C. and GGP13187 to L.C.), the Italian Ministry of Research (PRIN 2010-2011 grant 2010N8PBAA_002 to Y.B.) and the University of Trento (Centre for Integrative Biology (CIBIO) start-up grant to Y.B.).

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Author notes

  1. Gabriele Deidda and Manuela Allegra: These authors contributed equally to this work.

  2. Matteo Caleo and Laura Cancedda: These authors jointly directed this work.

Authors and Affiliations

  1. Neuroscience and Brain Technologies Department, Istituto Italiano di Tecnologia, Genova, Italy

    Gabriele Deidda, Shovan Naskar, Guillaume Bony & Laura Cancedda

  2. Scuola Normale Superiore, Pisa, Italy

    Manuela Allegra

  3. CNR Neuroscience Institute, Pisa, Italy

    Manuela Allegra, Chiara Cerri, Yuri Bozzi & Matteo Caleo

  4. Laboratory of Molecular Neuropathology, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy

    Giulia Zunino & Yuri Bozzi

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  1. Gabriele Deidda
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Contributions

G.D. performed in vitro electrophysiological recordings, western blot experiments, in utero electroporation and immunohistochemistry and wrote the manuscript. M.A. performed in vivo electrophysiological recordings, western blot experiments, immunohistochemistry, behavioral testing and retinogeniculate axon labeling and wrote the manuscript. C.C. contributed to in vivo electrophysiological recordings. G.B. and S.N. performed patch-clamp experiments. G.Z. and Y.B. performed RT-PCR and immunohistochemistry. M.C. and L.C. designed the experiments and wrote the manuscript. M.C., Y.B. and L.C. provided financial support. All authors read and revised the manuscript.

Corresponding author

Correspondence to Laura Cancedda.

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Competing interests

L.C. is an inventor on the US Provisional Application US 61/919,195 filed on December 20, 2013.

Integrated supplementary information

Supplementary Figure 1 NKCC1 and KCC2 are developmentally regulated in the rat primary visual cortex.

Top, Cropped images of a western blot experiment from fresh visual cortical samples collected at P3, P8 and P35 from naïve rats. Full-length blots are presented in Supplementary Figure 11c. Bottom, quantification of the relative amount of protein showed an opposite pattern of NKCC1 and KCC2 expression during development. A total of 4 animals were used for each experimental age.

Supplementary Figure 2 Bumetanide-treated animals display a depression of VEPs elicited by the closed eye at P35, a typical sign of juvenile-like plasticity.

Quantification of absolute VEP amplitudes revealed a significant decrease of contralateral, deprived eye responses and a potentiation of ipsilateral eye inputs (data not shown) upon MD in bumetanide- (blue; Mann Whitney Rank Sum Test, P = 0.002), but not vehicle-treated animals (orange; Mann Whitney Rank Sum Test, P = 0.647). The data are summarized by a box chart, where the horizontal lines denote the 25th, 50th, and 75th percentile values, whereas the square indicates the mean. Statistical significance: **P < 0.01.

Supplementary Figure 3 Early GABAergic interference does not alter morphological maturation of pyramidal neurons in the visual cortex.

(a) Schematic representation of the experimental protocol with tripolar in utero electroporation of the visual cortex. (b) Neuromorphological reconstruction of isolated EGFP-positive pyramidal neurons located in layer II/III in animals perinatally treated with vehicle (orange) or bumetanide (blue). Scale bar, 100 μm. (c, d) Quantification of the total branch number (c) and length (d) of EGFP neurons revealed no significant differences between bumetanide and vehicle groups (Student’s t-test; c: P = 0.502, d: P = 0.646). The histograms depict average ± SEM. Numbers in parentheses: processed cells, animals.

Supplementary Figure 4 Early GABAergic interference with bumetanide does not affect body-weight gain.

The graph shows average weight gain (± SEM) of vehicle- (orange) and bumetanide-treated pups (blue) during treatment (P3 to P8). Data are expressed as the percentage of body-weight gain from P3. Numbers in parentheses: processed animals.

Supplementary Figure 5 Basic membrane properties of layer II/III visual cortical neurons in whole-cell patch-clamp recordings at P35.

(a) Membrane resistance, (b) membrane capacitance (c) and membrane resting potential did not show any significant difference between neurons from bumetanide- (blue) and vehicle-treated animals (orange; Student’s t-test; a: P = 0.426, b: P = 0.068, c: P = 0.779). Histograms depict average ± SEM. Numbers in parentheses: recorded cells. (d, e) The kinetics of GABAergic mIPSCs assessed in terms of tau (d) and rise time (e) were similar in layer II/III visual cortical neurons from bumetanide- (blue) and vehicle-treated animals (orange) at P35 (Student’s t-test/Mann-Whitney rank sum test, d: P = 1.000, e: P = 0.090). Histograms depict average ± SEM. Numbers in parentheses: recorded cells.

Supplementary Figure 6 Treatment with diazepam during MD rescues the bumetanide-induced effect on plasticity in adulthood.

(a) Schematic cartoon of the experimental protocol. A group of rats was treated perinatally with bumetanide (from P3 to P8) and then monocularly deprived for 3 days at P35 followed by VEP recordings. During MD, rats received daily i.p. injections of either diazepam (2 mg/Kg) or vehicle. (b) C/I VEP ratios measured in bumetanide animals treated with either diazepam (orange) or vehicle (blue) during 3 days of MD at P35. Diazepam significantly reduced the magnitude of the OD shift in bumetanide-treated animals (Student’s t-test, P = 0.025). The histogram depicts average ± SEM, whereas circles indicate data from single animals. The dotted blue line represents the mean C/I of bumetanide-treated nonMD rats for comparison.

Supplementary Figure 7 Early GABAergic interference does not affect the density of presynaptic PV-positive boutons around pyramidal neurons at P35.

(a) Representative images from coronal sections of the visual cortex from vehicle- and bumetanide-treated animals at P35, labeled for parvalbumin (PV, white). Arrows point to presynaptic PV-boutons around cell somas. Scale bar, 10 μm. (b) Quantification of the number of boutons around each cell revealed no difference between bumetanide- (blue) and vehicle-treated animals (orange; Mann-Whitney rank sum test, P = 0.931). At least 15 cells for each animal were analyzed. The histograms depict average values ± SEM. Numbers in parentheses: processed animals.

Supplementary Figure 8 Early GABAergic interference has no effect on cortical inhibitory tone or BNDF expression at P26.

(a) Top: representative mIPSCs recorded at P26 in whole-cell patch-clamp configuration from layer II/III visual cortical neurons in animals treated perinatally with vehicle (orange) and bumetanide (blue). Bottom: quantification revealed no effect on mIPSC frequency (Student’s t-test, P = 0.367). The histogram represents average ± SEM. Numbers in parentheses: recorded cells. Scale bars: vertical, 10 pA; horizontal, 2 s. (b) Top: average traces of 50 mIPSC events recorded from vehicle- (orange) and bumetanide-treated animals (blue) as in panel (a). Bottom: quantification of the amplitude of mIPSCs revealed no difference between bumetanide and vehicle groups (Student’s t-test, P = 0.563). The histogram depicts average ± SEM. Numbers in parentheses: recorded cells. Scale bars: vertical, 2 pA; horizontal, 10 ms. (c) Cropped images of representative immunoblotting for BDNF on protein extracts from visual cortices collected at P26 from vehicle- and bumetanide-treated animals. α-tubulin was used as an internal standard. Full-length blots are presented in Supplementary Figure 11d. Bottom: quantification showed no difference in BDNF expression in bumetanide- (blue) and vehicle-treated animals (orange; Student’s t-test, P = 0.756). The histogram depicts average ± SEM. Numbers in parentheses: processed animals.

Supplementary Figure 9 Early GABAergic interference has no effect on cortical inhibitory tone, BDNF expression or PNNs at P75.

(a) Top: representative whole-cell patch-clamp recordings of mIPSCs from layer II/III visual cortical neurons in P75 animals treated perinatally with vehicle (orange) or bumetanide (blue). Bottom: quantification revealed no effect on mIPSC frequency (Student’s t-test, P = 0.180). The histogram represents average ± SEM. Numbers in parentheses: recorded cells. Scale bars: vertical 10 pA; horizontal, 2 s. (b) Top: average traces of 50 mIPSC events from vehicle- (orange) and bumetanide-treated animals (blue) as in (a). Bottom: quantification of mIPSC amplitudes revealed no difference between bumetanide and vehicle groups (Student’s t-test, P = 0.712). The histogram depicts average ± SEM. Numbers in parentheses: recorded cells. Scale bars: vertical, 2 pA; horizontal, 10 ms. (c) Cropped images of immunoblotting for BDNF on protein extracts from P75 visual cortices of vehicle- and bumetanide-treated animals. α-tubulin was used as internal standard. Full-length blots are presented in Supplementary Figure 11e. Bottom: quantification showed no difference in BDNF expression of bumetanide- (blue) and vehicle-treated animals (orange; Student’s t-test, P = 0.533).The histogram depicts average ± SEM. Numbers in parentheses: processed animals. (d) Representative images from coronal sections of the visual cortex from P75 vehicle- and bumetanide-treated animals, labeled for PNNs (red). Scale bar, 20 μm. Bottom: quantification of the density of WFA-positive (PNN) cells revealed no difference in bumetanide- (blue) and vehicle-treated animals (orange; Student’s t-test, P = 0.766). The data are summarized by a box chart. Horizontal lines denote the 25th, 50th, and 75th percentiles, the square indicates the mean. Numbers in parentheses: processed animals.

Supplementary Figure 10 Early DHF treatment does not interfere per se with plasticity at P26.

(a) Average time course of the increase in the amplitude of layer II/III field synaptic potentials after TBS of the WM in vehicle- (orange) and DHF- treated animals (dark red) at P26. Insets: Average of 10 traces recorded from a slice of a vehicle- (orange) or DHF-treated animal (dark red) before (continuous line) and 30 min after TBS (dashed line). Stimulus artifacts have been deleted from traces for clarity. Scale bars: vertical, 0.2 mV; horizontal, 2 ms. Numbers in parentheses: recorded slices, animals. (b) Average ± SEM of the level of plasticity from slices recorded in panel (a). Treatment with DHF did not interfere with plasticity at P26 (Student’s t-test, P = 0.433). Numbers in parentheses: recorded slices.

Supplementary Figure 11 Full images of cropped blots presented in other figures.

Full images of cropped blots presented in other figures. (a) Full blot for Figure 6c. (b) Full blot for Figure 8b. (c) Full blot for Supplementary Figure S1. (d) Full blot for Supplementary Figure S8c. Over-exposition of the blot (blue spots) was necessary to reveal the low BDNF signal. (e) Full blot for Supplementary Figure S9c. Over-exposition of the blot (blue spots) was necessary to reveal the low BDNF signal.

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Deidda, G., Allegra, M., Cerri, C. et al. Early depolarizing GABA controls critical-period plasticity in the rat visual cortex. Nat Neurosci 18, 87–96 (2015). https://doi.org/10.1038/nn.3890

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