Acute modulation of synaptic plasticity of pyramidal neurons by activin in adult hippocampus

Abstract

Activin A is known as a neuroprotective factor produced upon acute excitotoxic injury of the hippocampus (in pathological states). We attempt to reveal the role of activin as a neuromodulator in the adult male hippocampus under physiological conditions (in healthy states), which remains largely unknown. We showed endogenous/basal expression of activin in the hippocampal neurons. Localization of activin receptors in dendritic spines (=postsynapses) was demonstrated by immunoelectron microscopy. The incubation of hippocampal acute slices with activin A (10 ng/mL, 0.4 nM) for 2 h altered the density and morphology of spines in CA1 pyramidal neurons. The total spine density increased by 1.2-fold upon activin treatments. Activin selectively increased the density of large-head spines, without affecting middle-head and small-head spines. Blocking Erk/MAPK, PKA, or PKC prevented the activin-induced spinogenesis by reducing the density of large-head spines, independent of Smad-induced gene transcription which usually takes more than several hours. Incubation of acute slices with activin for 2 h induced the moderate early long-term potentiation (moderate LTP) upon weak theta burst stimuli. This moderate LTP induction was blocked by follistatin, MAPK inhibitor (PD98059) and inhibitor of NR2B subunit of NMDA receptors (Ro25-6981). It should be noted that the weak theta burst stimuli alone cannot induce moderate LTP. These results suggest that MAPK-induced phosphorylation of NMDA receptors (including NR2B) may play an important role for activin-induced moderate LTP. Taken together, the current results reveal interesting physiological roles of endogenous activin as a rapid synaptic modulator in the adult hippocampus.

Keywords: LTP; activin; hippocampus; kinase; rapid effect; spine; synapse.

Figure 1

Expression and localization of activin in the adult male rat hippocampus under physiological conditions. (A) Immunohistochemical staining of activin with anti-activin IgG. (A1) Coronal section of CA1 region. (A2) Coronal section of CA3 region. (A3) Coronal section of DG. so, stratum oriens; pcl, pyramidal cell layer; sr, stratum radiatum. Scale bar, 200 μm. Representative images are shown from approx. 18 photographs from 6 independent slices from 3 animals. (Supplementary Figure 1A4) No staining after preadsorption treatments with activin. (B) RT-PCR analysis of mRNA for inhibin β_A, α, β_B, β_C and β_E subunits in the hippocampus (31 cycles). (B1) The expression of inhibin β_A subunit transcripts. From left to right, size marker (Marker), hippocampus (Hi), ovary (Ov). (B2) Inhibin α and β_B subunit. (B3) Inhibin β_C and β_E subunits. (+): with reverse transcriptase added; (−): without reverse transcriptase, a negative control. P: Ethidium bromide staining of PCR products. GAPDH (21 cycles) was used as an internal control for PCR amplification. Ovary (Ov) or Liver (Li) was used for positive control. The image is a representative one from duplicate determinations for each rat of total 4 rats.

Figure 2

Localization of activin type IB receptor in the rat hippocampus. (A) Immunohistochemical staining of the hippocampal slice (coronal section) of adult male rat probed with anti-activin receptor IgG. (A1) CA1, (A2) CA3, (A3) DG. Scale bar, 200 μm. so, stratum oriens; pcl, pyramidal cell layer; sr, stratum radiatum. Representative images are shown from approx. 18 photographs from 6 independent slices from 3 animals. (Supplementary Figure 2A4) No staining after preadsorption treatments with activin receptor. (B) Immunoelectron microscopic analysis, using anti-activin receptor IgG, of the distribution of activin receptor within the axospinous synapses of the hippocampal principal neurons in the stratum radiatum of CA1 (B1), stratum radiatum and lucidum of CA3 (B2), and hilus of DG (B3). Representative images are shown from approx. 100 photographs from 27 independent slices from 4 animals. Gold particles (arrowheads) were localized in the pre- and postsynaptic regions (B1–3). In spines (postsynapses), gold particles were associated with PSD regions as well as within the spine head (B1). In the presynaptic terminus, gold particles were often associated with small synaptic vesicles. In dendrites of neurons (B4, CA1 region), gold particles were often found in cytoplasmic space. A 1:20000 dilution anti-activin receptor IgG was used to prevent non-specific labeling. pre, presynaptic region; post, postsynaptic region; den, dendrite. Scale bar, 200 nm for (B1,B3), 300 nm for (B2,B4).

Figure 3

Changes in the density and morphology of spines by activin and drugs in hippocampal slices. Spines were analyzed along the secondary dendrites of pyramidal neurons in the stratum radiatum of CA1 neurons. (A) Representative images of confocal micrographs; spines along dendrite without drug-treatments (Cont) and spines along dendrite after activin treatment for 2 h (Act). Maximal intensity projection onto XY plane from z-series confocal micrographs (MAX-XY), image analyzed by Spiso-3D (S) and 3 dimensional model (Model) are shown together. Bar, 3 μm. (B) Effect of treatments by activin and glutamate receptor blockers on the total spine density in CA1 neurons. Vertical axis is the average number of spines per 1 μm. A 2-h treatment in ACSF without drugs (Control, total spine numbers = 552, 8 neurons), with 10 ng/mL activin A (Act), with 10 ng/mL activin and 100 ng/mL follistatin (Act + Fol), with 10 ng/mL activin and 20 μM CNQX (Act + CNQX, total spine numbers = 977, 11 neurons, P = 0.005), with 10 ng/mL activin and 50 μM MK-801 (Act + MK). Statistical significance is calculated against activin treated group and indicated by stars. ^*P < 0.05, ^**P < 0.01. (C) Histogram of spine head diameters after a 2 h treatment in ACSF without drugs (Control, closed black diamond), with 10 ng/mL activin (closed red square), and with 10 ng/mL activin A and 100 ng/mL follistatin (closed blue diamond), with 10 ng/mL activin and 20 μM CNQX (closed green triangle), with 10 ng/mL activin A and 50 μM MK-801 (closed purple triangle). (D) Density of three subtypes of spines. Abbreviations are same as in (A). Vertical axis is the number of spines per 1 μm of dendrite. From left to right, small-head spines (Small), middle-head spines (Middle), and large-head spines (Large) type. ACSF without drugs (open column), Act (orange column), Act + Fol (blue column), Act + CNQX (green column), Act + MK801 (purple column) are shown. Statistical significance is calculated against activin treated group in each spine subtypes and comparisons reached significance are indicated by stars. The significance yielded P < 0.05. ^*P < 0.05, ^**P < 0.01. In (B,D), results are reported as mean ± s.e.m. For each drug treatment, we investigated 3 rats, 7 slices, 14 neurons, 28 dendrites and 1400–2000 spines. For control, we used 5 rats, 8 slices, 16 neurons, 31 dendrites and approx. 1700 spines.

Figure 4

Effects by inhibition of kinases on changes of the density and morphology of spines in the presence of activin A. Spines were analyzed along the secondary dendrites of CA1 pyramidal neurons. (A) Total spine density. Effect of kinase inhibitors in the presence of activin in CA1 neurons. Vertical axis is the average number of spines per 1 μm. A 2-h treatment in ACSF without drugs (Control), with 10 ng/mL activin (Act), with 10 ng/mL activin and 20 μM PD98059 (Erk MAPK inhibitor) (Act + PD), with 10 ng/mL activin and 10 μM SB203580 (p38 MAPK inhibitor) (Act + SB), with 10 ng/mL activin and 10 μM H-89 (PKA inhibitor) (Act + H89), with 10 ng/mL activin and 10 μM chelerythrine (PKC inhibitor) (Act + CHEL), with 10 ng/mL activin A and 10 μM LY294002 (PI3K inhibitor) (Act + LY), with 10 ng/mL activin and 1 μM cyclosporin A (calcineurin inhibitor) (Act + CsA), and with 10 ng/mL activin and 1 μM KN-93 (CaMKII inhibitor) (Act + KN93). Statistical significance is calculated against activin treated group and indicated by stars. ^*P < 0.05, ^**P < 0.01. (B) Histogram of spine head diameters. Abbreviations are the same as in (A). Vertical axis is the number of spines per 1 μm of dendrite. After a 2-h treatment in ACSF without drugs (Control, closed black diamond), Act (closed red square), Act + PD (closed brown triangle), Act + SB (closed orange diamond), with Act + H89 (closed blue triangle), with Act + CHEL (open blue triangle), with Act + LY (open green diamond), and Act + CsA (closed purple circle), and Act + KN93 (open orange square). (C) Density of three subtypes of spines. Abbreviations are the same as in (A). Vertical axis is the average number of spines per 1 μm of dendrite. From left to right, small-head spines (Small), middle-head spines (Middle), and large-head spines (Large). In each group, control (open column), Act (closed orange column), Act + PD (closed blue column), Act + SB (closed green column), Act + H89 (hatched orange column), Act + CHEL (hatched blue column), Act + LY (hatched green column), and Act + CsA (hatched purple column), and Act + KN93 (closed purple column). Statistical significance is calculated against activin treated group in each spine subtypes and comparisons reached significance are indicated by stars. The significance yielded P < 0.05. ^*P < 0.05, ^**P < 0.01. (D) No effect of kinase inhibitors alone on the total spine density in CA1 neurons. Abbreviations are same as in (A). (E) Representative spine images of confocal micrographs used for (A–C): activin plus KN-93 treatment (Act+KN93) and only KN-93 treatment (KN93); activin plus H-89 treatment (Act+H89) and only H-89 treatment (H89); activin plus PD98059 treatment (Act+PD); activin plus chelerythrine treatment (Act+CHEL). Maximal intensity projection onto XY plane from z-series (MAX-XY), image analyzed by Spiso-3D (S) and 3 dimensional model (Model) are shown together. Bar, 3 μm. In (A,C,D), results are reported as mean ± s.e.m. For each drug treatment, we investigated 3 rats, 7 slices, 14 neurons, 28 dendrites and 1400–2000 spines. For control, we used 5 rats, 8 slices, 16 neurons, 31 dendrites and approx. 1700 spines.

Figure 5

Effects of protein and mRNA synthesis on changes in the density and morphology of spines by activin. Spines were analyzed along the secondary dendrites of CA1 pyramidal neurons as in Figure 3. (A) Total spine density. Effect of inhibitors for protein or mRNA synthesis in the presence of activin on CA1 neurons. Vertical axis is the average number of spines per 1 μm. A 2-h treatment in ACSF without drugs (Control), with 10 ng/mL activin (Act), with 10 ng/mL activin and 20 μM cycloheximide (Act + CHX), and with 10 ng/mL activin and 4 μM actinomycin D (Act + actD). Statistical significance is calculated against activin treated group. ^*P < 0.05, ^**P < 0.01. (B) Histogram of spine head diameters. Abbreviations are same as in (A). Vertical axis is the number of spines per 1 μm of dendrite. After a 2-h treatment in ACSF without drugs (Control, closed black diamond), Act (closed orange square), Act + CHX (closed blue triangle), and Act + ActD (closed green triangle). (C) Density of three subtypes of spines. Abbreviations are same as in (A). Vertical axis is the average number of spines per 1 μm of dendrite. From left to right, small-head spines (Small), middle-head spines (Middle), and large-head spines (Large). ACSF without drugs (open column), Act (orange column), Act + CHX (blue column), Act + actD (green column). Statistical significance is calculated against activin treated group in each spine subtypes and comparisons reached significance are indicated by stars. The significance yielded P < 0.05. ^*P < 0.05, ^**P < 0.01. In (A,C) results are reported as mean ± s.e.m. For each drug treatment, we investigated 3 rats, 6 slices, 12 neurons, 24 dendrites and 1100–1800 spines. For control, we used 5 rats, 8 slices, 16 neurons, 31 dendrites and approx. 1700 spines.

Figure 6

(A) Induction of moderate LTP by weak-TBS stimulation after short incubation (~2 h) with activin in the CA1 of hippocampal slices. Slices with 0 ng/ml activin (control, open square, n = 10 slices, 10 rats), with 10 ng/ml activin (closed circle, n = 10 slices, 10 rats), with 10 ng/ml activin plus 100 ng/ml follistatin (closed triangle, n = 7 slices, 7 rats), with respectively. The number of independent experiments is indicated as n. Vertical axis indicates EPSP slope. Here, 100% refers to the EPSP slope value of the average of t = −10 to −1 min prior to weak-TBS stimulation. LTP was induced at time t = 0. Illustrated data points and error bars represent the mean ± s.e.m. from n of independent slices. (B) Co-incubation of activin with MAPK inhibitor PD98059 (20 μ M) prevented the induction of LTP (open circle, n = 7 slices, 7 rats). Co-incubation of activin with NR2B inhibitor Ro25-6981 (1 μ M) prevented the induction of LTP (open square, n = 7 slices, 7 rats). Activin-treated slices (closed circle, n = 10 slices, 10 rats). Maximal LTP by full-TBS is also shown (closed circle, n = 7, 7 rats). (C) Comparison of modulation effects by activin upon weak-TBS as shown in (A) and (B). From left to right; slices without drugs (Cont), with 10 ng/ml activin (Act), with activin plus follistatin (+Fol), activin plus PD98059 (+PD), activin plus Ro25-6981 (+Ro) and full-TBS (full-TBS). The significance yielded p < 0.05. ^*P < 0.05, ^**P < 0.01. (D) Representative raw traces of EPSP, showing sample recordings prior to (black line) or after (gray line) weak-TBS stimulation. Control (0 ng/ml activin), Act (10 ng/ml activin), Act+ PD (activin plus PD98059), Act + Ro (activin plus Ro25-6981). EPSP trace for full-TBS is also shown.

Figure 7

Schematic illustration of activin-induced spinogenesis via multiple kinase pathways. (A) Upon activation by activin receptor complex (ActR), early LTP is suppressed by anti-inflammatory action of activin. (B) After 2 h of ActR activation, PKA, PKC and MAPK may promote actin polymerization process, leading to the formation of new spines. (C) After 2 h of ActR activation, MAPK may phosphorylate NR2B of NMDA receptors, thereafter moderate LTP is induced upon weak-TBS.