Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines
- SUNY Downstate Medical Center, United States
- Tianjin Medical University, China
Figures
Figure 1 with 2 supplements
Synaptic pruning of CA1 hippocampus of adolescent female mice is prevented in the α4 knock-out.
Pub, pubertal; post-Pub, post-pubertal. (a) CA1 hippocampal pyramidal cells, Pub and post-Pub (8-week old) WT and α4 KO female mouse hippocampus. Upper panel, neurolucida images, scale, 50 µm; lower …
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Figure 1—source data 1
Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells for Figure 1b for wild-type (WT) and α4 knock-out (KO) female mice assessed at puberty (Pub, PND 35, identified by vaginal opening) and post-puberty (Post-pub, PND 56).
- https://doi.org/10.7554/eLife.15106.004
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Figure 1—source data 2
Spine counts/10 μm for different spine-types on dendrites of CA1 hippocampal pyramidal cells for Figure 1c for Pub and Post-pub WT and Post-pub α4 KO.
Spines were identified as: mushroom, stubby, thin or bifurcated (Bif).
- https://doi.org/10.7554/eLife.15106.005
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Figure 1—source data 3
Figure 1f. mEPSC frequency, # mEPSCs/s recorded from CA1 hippocampal pyramidal cells using whole cell patch clamp techniques for post-pubertal WT (left) and α4 KO mice.
- https://doi.org/10.7554/eLife.15106.006
Figure 1—figure supplement 1
Neurolucida images of spine density across pubertal stage and α4 genotype.
Representative Neurolucida drawings. Scale, 10 μm.
Figure 1—figure supplement 2
Synaptic pruning of CA1 hippocampus of adolescent male mice is prevented in the α4 knock-out.
Pub, pubertal; post-Pub, post-pubertal. (a) CA1 hippocampal pyramidal cells, Pub and post-Pub (8-week old) WT and α4 KO male mouse hippocampus. (a) Representative z-stack (100x) images; scale, 5 µm. …
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Figure 1—figure supplement 2—source data 1
Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells for Figure 1b for wild-type (WT) and α4 knock-out (KO) male mice assessed at puberty (Pub, PND 35) and post-puberty (Post-pub, PND 56).
- https://doi.org/10.7554/eLife.15106.009
Figure 2
NMDA receptors maintain spines during puberty.
(a) Representative EPSCs (black) and NMDA EPSCs (red) recorded during puberty in WT or α4 KO hippocampus, in some cases during α5 (50 nM L655) or total (120 μM SR95531) GABAR blockade. In all other …
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Figure 2— source data 1
Figure 2b: NMDA EPSC/ AMPA EPSC ratios recorded from CA1 hippocampal pyramidal cells using whole cell patch clamp techniques for post-pubertal WT (a), α4 KO mice (b), WT hippocampus with SR95531 (c) and WT hippocampus with L-655,708 (L655) (d).
- https://doi.org/10.7554/eLife.15106.013
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Figure 2—source data 2
Figure 2d: Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells – proximal (left) and distal (right) for pubertal (Pub), Post-pubertal (Post-pub) – vehicle (VEH), and Post-pub MK-801 (treated with MK-801 during the pubertal period).
- https://doi.org/10.7554/eLife.15106.014
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Figure 2—source data 3
Figure 2e: Spine counts/10 μm for different spine-types on dendrites of CA1 hippocampal pyramidal cells for Figure 1c for Pub, Post-pub vehicle (VEH) and Post-pub MK-801 (treated with MK-801during the pubertal period).
Spines were identified as: mushroom, stubby, or thin.
- https://doi.org/10.7554/eLife.15106.015
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Figure 2—source data 4
Figure 2g: Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells – proximal (left) and distal (right) for α4 KO: pubertal (Pub), Post-pubertal (Post-pub) – vehicle (VEH), and Post-pub memantine (treated with memantine during the pubertal period).
- https://doi.org/10.7554/eLife.15106.016
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Figure 2—source data 5
Figure 2h: Spine counts/10 μm for different spine-types on dendrites of CA1 hippocampal pyramidal cells for Figure 1c for α4 KO: Pub, Post-pub vehicle (VEH) and Post-pub memantine (treated with memantine during the pubertal period).
Spines were identified as: mushroom, stubby, thin or bifurcated (Bif).
- https://doi.org/10.7554/eLife.15106.017
Figure 3 with 1 supplement
Effect of GABAR blockade on spine density in the post-pubertal hippocampus.
Inset, Drug treatment during puberty (PND 35–44) was tested for its effect on post-pubertal spine density (PND 56). Drugs: PTX, picrotoxin, a GABAR antagonist; L655, L-655,708, an inverse agonist at …
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Figure 3—source data 1
Figure 3c: Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells – proximal (left) and distal (right) for Post-pubertal (Post-pub) mice treated with L-655,708 (L655, left), vehicle (VEH, middle) or picrotoxin (Picro, right), during the pubertal period.
- https://doi.org/10.7554/eLife.15106.019
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Figure 3—source data 2
Figure 3d: Spine counts/10 μm for different spine-types on dendrites of CA1 hippocampal pyramidal cells for Post-pubertal (Post-pub) mice treated with L-655,708 (L655, left), vehicle (VEH, middle) or picrotoxin (Picro, right), during the pubertal period. Spines were identified as: mushroom, stubby, or thin.
- https://doi.org/10.7554/eLife.15106.020
Figure 3—figure supplement 1
Pubertal lorazepam treatment does not alter spine density in post-pubertal mice.
(a) Representative z-stack images; (b) Averaged spine densities for proximal (left) and distal (right) dendrites of post-pubertal female mice treated during the pubertal period with the positive …
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Figure 3—figure supplement 1—source data 1
Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells – proximal (left) and distal (right) for post-pubertal mice treated with MK-801 during the pubertal period.
- https://doi.org/10.7554/eLife.15106.022
Figure 4 with 1 supplement
NMDA receptor-dependent Kalirin-7 expression decreases at puberty.
(a,c,e,g) Representative images, scale, 2.5 μm. Arrows, spines. (a) Phalloidin (Phal), Kalirin-7 (Kal7) and merged images from pre-pub and pub CA1 hippocampus. (b) Mean pixel intensity, *t-test, …
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Figure 4—source data 1
Figure 4b,d,f,h: Measurements of Kalirin-7 (Kal7) luminescence taken from CA1 hippocampal pyramidal cells for Pre-pub and Pub WT (4b), Pub, WT and α4 KO (4d), Pub WT-treated with MK-801 or vehicle (VEH) (4f) and Post-pub WT-treated with memantine or VEH (4h).
- https://doi.org/10.7554/eLife.15106.024
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Figure 4—source data 2
Figure 4j: Spine counts/20 μm on dendrites of CA1 hippocampal pyramidal cells – proximal (left) and distal (right) for pubertal (Pub) and post-pubertal (Post-pub) Kal7 KO mice.
- https://doi.org/10.7554/eLife.15106.025
Figure 4—figure supplement 1
Kalirin-7 expression varies across pubertal stage, α4 genotype and level of pubertal NMDAR expression.
Representative images of Kalirin-7 (Kal7) expression in CA1 hippocampus across pubertal stage (a), in pubertal WT and α4 KO hippocampus (b), following pubertal treatment with vehicle or MK-801 to …
Figure 5 with 2 supplements
Induction of long-term depression and re-learning are impaired under conditions of high spine density in the α4 KO mouse.
(a) Induction of long-term depression (LTD) using low frequency stimulation (arrow). WT, black, α4 KO, red. *t-test, t(6)=3.56, p=0.01, power=0.84; n=4/group. (Figure 5—source data 1) Inset, …
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Figure 5—source data 1
Figure 5a: Percent baseline slope of field EPSPs recorded after low frequency (1 Hz) stimulation to induce LTD for post-pubertal WT and α4 KO CA1 hippocampus (120 min, 30 s intervals).
Each column is a separate slice recording.
- https://doi.org/10.7554/eLife.15106.028
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Figure 5—source data 2
Figure 5b: Left, Percent baseline slope of field EPSPs recorded after theta burst stimulation to induce LTP for post-pubertal WT and α4 KO CA1 hippocampus (final 20 min 100 min after LTP induction, 30 s intervals).
Each column is a separate slice recording. Right, Averaged values for the final 20 min. segment.
- https://doi.org/10.7554/eLife.15106.029
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Figure 5—source data 3
Figure 5c: Learning acquisition (left) and retention (right) for zone 1 of the active place avoidance task (APA).
Latency to enter shock zone (s) for post-pub WT and α4 KO mice. Figure 5d, Re-learning acquisition (left) and retention (right) for zone 2 of the APA. Latency to enter shock zone (s) for post-pub WT and α4 KO mice.
- https://doi.org/10.7554/eLife.15106.030
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Figure 5—source data 4
Figure 5e: #shocks/entry (left) and locomotor activity (right) for post-pub WT and α4 KO mice assessed for the active place avoidance task (APA).
- https://doi.org/10.7554/eLife.15106.031
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Figure 5—source data 5
Figure 5f: Learning acquisition for zones 1–3 for post-pub WT (left) and α4 KO (right) of the multiple placement object relocation task (MPORT).
Duration of time spent, M, moved object; unm, unmoved object; m/unm, ratio of time spent moved versus unmoved object.
- https://doi.org/10.7554/eLife.15106.032
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Figure 5—source data 6
Figure 5g: Locomotor activity (left) and # approached, a measure of interest (right) for for post-pub WT (left) and α4 KO (right) mice using MPORT.
- https://doi.org/10.7554/eLife.15106.033
Figure 5—figure supplement 1
NMDAR antagonist treatment alters behavioral flexibility.
Treatment with MK-801 or memantine during puberty, to increase NR1-NMDAR expression or decrease NMDAR activity (Gao and Tamminga, 1995; Cole et al., 2013), respectively, produces opposite effects on …
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Figure 5—figure supplement 1—source data 1
Learning acquisition for positions (Pos) 2 and 3 for the multiple placement object relocation task (MPORT).
Ratio of time spent for the moved versus unmoved object for post-pubertal mice treated with MK-801 during the pubertal period.
- https://doi.org/10.7554/eLife.15106.035
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Figure 5—figure supplement 1—source data 2
Learning acquisition for positions (Pos) 2 and 3 for the multiple placement object relocation task (MPORT).
Ratio of time spent for the moved versus unmoved object for post-pubertal α4 KO mice treated with memantine during the pubertal period.
- https://doi.org/10.7554/eLife.15106.036
Figure 5—figure supplement 2
Pubertal GABAR antagonist treatment impairs behavioral flexibility post-pubertally.
Mice were treated with picrotoxin (Picro, 3 mg/kg, i.p.), to block GABARs, or vehicle during puberty (PND 35–44) and tested on PND 56 using MPORT. Post-pubertal increases in spine density produced …
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Figure 5—figure supplement 2—source data 1
Learning acquisition for positions (Pos)1–3 for the multiple placement object relocation task (MPORT).
Ratio of time spent for the moved versus unmoved object for post-pubertal mice treated with picrotoxin during the pubertal period.
- https://doi.org/10.7554/eLife.15106.038
Figure 6
Learning and re-learning increase mushroom-type dendritic spines in CA1 hippocampus following adolescent synaptic pruning.
(a) Representative z-stack images from CA1 hippocampal pyramidal cells illustrating changes in spine type and number after hippocampal-dependent learning and re-learning, compared to naïve …
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Figure 6—source data 1
Figure 6b: (proximal), 6c (distal), Spine counts/10 μm for different spine-types on dendrites of CA1 hippocampal pyramidal cells assessed 1–2 hr after learning, re-learning or naïve conditions.
Spines were identified as: mushroom, stubby, or thin.
- https://doi.org/10.7554/eLife.15106.040
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Figure 6—source data 2
Figure 6e: (proximal), 6f (distal), Spine counts/10 μm for different spine-types on dendrites of CA1 hippocampal pyramidal cells assessed 1–2 hr after learning, re-learning or naïve conditions in mice with high spine densities (treated with picrotoxin during the pubertal period).
Spines were identified as: mushroom, stubby, or thin.
- https://doi.org/10.7554/eLife.15106.041
Tables
Table 1
Dendrite length is not altered during adolescence or after α4 knock-out. Mean ± SEM, n=4 neurons (mice)/group. Dendrite length, ANOVA, F(3,15)=0.35, p=0.80.
| Dendrite length (Mean ± SEM) | Pubertal | Post-pubertal |
|---|---|---|
| WT | 190 ± 5.8 | 205 ± 27.5 |
| α4 KO | 183.7 ± 5.5 | 195 ± 9.6 |
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Table 1—source data 1
Dendrite length for pubertal (Pub) and post-pubertal (Post-pub) WT and α4 KO female mice.
- https://doi.org/10.7554/eLife.15106.011
