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. 2018 May 1:12:282.
doi: 10.3389/fnins.2018.00282. eCollection 2018.

Src Kinase Dependent Rapid Non-genomic Modulation of Hippocampal Spinogenesis Induced by Androgen and Estrogen

Mika Soma  1 , Jonghyuk Kim  1 , Asami Kato  1 , Suguru Kawato  1   2   3
Affiliations

Src Kinase Dependent Rapid Non-genomic Modulation of Hippocampal Spinogenesis Induced by Androgen and Estrogen

Mika Soma et al. Front Neurosci. .

Abstract

Dendritic spine is a small membranous protrusion from a neuron's dendrite that typically receives input from an axon terminal at the synapse. Memories are stored in synapses which consist of spines and presynapses. Rapid modulations of dendritic spines induced by hippocampal sex steroids, including dihydrotestosterone (DHT), testosterone (T), and estradiol (E2), are essential for synaptic plasticity. Molecular mechanisms underlying the rapid non-genomic modulation through synaptic receptors of androgen (AR) and estrogen (ER) as well as its downstream kinase signaling, however, have not been well understood. We investigated the possible involvement of Src tyrosine kinase in rapid changes of dendritic spines in response to androgen and estrogen, including DHT, T, and E2, using hippocampal slices from adult male rats. We found that the treatments with DHT (10 nM), T (10 nM), and E2 (1 nM) increased the total density of spines by ~1.22 to 1.26-fold within 2 h using super resolution confocal imaging of Lucifer Yellow-injected CA1 pyramidal neurons. We examined also morphological changes of spines in order to clarify differences between three sex steroids. From spine head diameter analysis, DHT increased middle- and large-head spines, whereas T increased small- and middle-head spines, and E2 increased small-head spines. Upon application of Src tyrosine kinase inhibitor, the spine increases induced through DHT, T, and E2 treatments were completely blocked. These results imply that Src kinase is essentially involved in sex steroid-induced non-genomic modulation of the spine density and morphology. These results also suggest that rapid effects of exogenously applied androgen and estrogen can occur in steroid-depleted conditions, including "acute" hippocampal slices and the hippocampus of gonadectomized animals.

Keywords: Src kinase; androgen; estrogen; hippocampus; non-genomic; spine; synapse.

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Figures

Figure 1
Figure 1
Effects of Src kinase blocker (PP2) on DHT-induced spine increase and change in morphology in hippocampal slices. (A) Spines were analyzed along the secondary dendrites of pyramidal neurons in the stratum radiatum of CA1 neurons. Dendrite after DHT-treatment for 2 h (DHT) and dendrite after DHT plus PP2 treatment for 2 h (PP2+DHT). (Spiso) shows the image of dendrite and spines analyzed with Spiso-3D software. Maximal intensity projections onto XY plane is shown. Traced dendrite is shown in red color and spines are indicated in yellow color. (Model) shows 3 dimensional model illustration of (Spiso) image. Bar, 5 μm. (B) Effect of treatments by DHT or PP2 on the total spine density in CA1 neurons. Vertical axis is the average number of spines per 1 μm of dendrite. A 2 h treatment in ACSF without drugs (Control), with 10 nM DHT (DHT), with 10 nM DHT and 10 μM PP2 (PP2 + DHT), and with PP2 only (PP2). (C) Histogram of spine head diameters after a 2 h treatment in ACSF without drugs (Control, black dashed line), with 10 nM DHT (black line), with 10 nM DHT and 10 μM PP2 (red line). Spines were classified into three categories depending on their head diameter, e.g., 0.2–0.4 μm as small-head spines, 0.4–0.5 μm as middle-head spines, and larger than 0.5 μm as large-head spines. Vertical axis is the number of spines per 1 μm of dendrite. (D) Density of three subtypes of spines. Abbreviations are same as in (B). 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 (Control, open column), DHT (black column), PP2 + DHT (red column). Vertical axis is the number of spines per 1 μm of dendrite. Results are represented as mean ± SEM. Statistical significance yielded *P < 0.05, **P < 0.01 vs. DHT sample. For DHT, PP2+DHT, and PP2 only treatments, we investigated, 50 dendrites with 2,300–2,700 spines from 3 rats, 12 slices, and 30 neurons. For control, we used 80 dendrites with ~4,000 spines from 6 rats, 24 slices and 50 neurons.
Figure 2
Figure 2
Effects of Src kinase blocker on T-induced spine increase and change in morphology in hippocampal slices. (A) Spines were analyzed along the secondary dendrites of pyramidal neurons in the stratum radiatum of CA1 neurons as in Figure 1. Dendrite after T-treatment for 2 h (T) and dendrite after T plus PP2 treatment for 2 h (PP2+T). (Spiso) shows the image of dendrite and spines analyzed with Spiso-3D software. Maximal intensity projections onto XY plane is shown. Traced dendrite is shown in red color and spines are indicated in yellow color. (Model) shows 3 dimensional model illustration of (Spiso) image. Bar, 5 μm. (B) Effect of treatments by T or PP2 on the total spine density in CA1 neurons. Vertical axis is the average number of spines per 1 μm of dendrite. A 2 h treatment in ACSF without drugs (Control), with 10 nM T (T), with 10 nM T and 10 μM PP2 (PP2 + T). (C) Histogram of spine head diameters after a 2 h treatment in ACSF without drugs (Control, black dashed line), with 10 nM T (black line), with 10 nM T and 10 μM PP2 (PP2 + T, red line). (D) Density of three subtypes of spines. Abbreviations are same as in (B). From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. ACSF without drugs (Control, open column), T (black column), PP2 + T (red column). Results are represented as mean ± SEM. Statistical significance yielded *P < 0.05, **P < 0.01 vs. T sample. For T and PP2 + T treatments, we investigated 50 dendrites with 2300–2700 spines from 3 rats, 12 slices, 30 neurons. For control, we used 80 dendrites with ~4,000 spines from 6 rats, 24 slices, and 50 neurons.
Figure 3
Figure 3
Effects of Src kinase blocker on E2-induced spine increase and change in morphology. (A) Spines were analyzed along the secondary dendrites of pyramidal neurons in the stratum radiatum of CA1 neurons as in Figure 1. Dendrite after E2-treatment for 2 h (E2) and dendrite after E2 plus PP2 treatment for 2 h (PP2+E2). (Spiso) shows the image of dendrite and spines analyzed with Spiso-3D software. Maximal intensity projections onto XY plane is shown. Traced dendrite is shown in red color and spines are indicated in yellow color. (Model) shows 3 dimensional model illustration of (Spiso) image. Bar, 5 μm. (B) Effect of treatments by E2 and PP2 on the total spine density. Vertical axis is the average number of spines per 1 μm of dendrite. A 2 h treatment in ACSF without drugs (Control), with 1 nM E2 (E2), and with 1 nM E2 and 10 μM PP2 (PP2 + E2). (C) Histogram of spine head diameters. A 2 h treatment in ACSF without drugs (Control, dashed line), with E2 (black line), with E2 + PP2 (red line). (D) Density of three subtypes of spines. From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. In each group, control (open column), E2 (black column), and PP2+E2 (red column). Results are represented as mean ± SEM. Statistical significance was defined as *p < 0.05, **p < 0.01 vs. E2 sample. For E2 and PP2+E2 treatments, we investigated 50 dendrites with 2300–2700 spines from 3 rats, 12 slices, and 30 neurons. For control, we used 80 dendrites with ~4,000 spines from 6 rats, 24 slices, and 50 neurons.
Figure 4
Figure 4
Comparison of spine images obtained by super resolution confocal microscopy and conventional confocal microscopy. (A,B) Typical dendrites with spines of CA1 neurons are shown after analysis with Spiso-3D software. Maximal intensity projections onto XY plane are shown. Traced dendrites are shown in red color and spines are indicated in yellow color. Images are obtained with (A) Super resolution confocal microscopy and (B) Conventional confocal microscopy. Bar, 5 μm. (C) Comparison of the total spine density of control dendrites (without steroid supplementation) obtained with super resolution confocal microscopy (Super resolution) and Conventional confocal microscopy (Conventional). Vertical axis is the average number of spines per 1 μm of dendrite. (D) Histogram of spine head diameters of control. Super resolution (red line), and Conventional (black line). (E) Density of three subtypes of spines of control dendrites. From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large). In each group, Super resolution (red column), and Conventional (black column). Abbreviations are same as in (C). Results are represented as mean ± SEM. No statistical significance was observed. For Super resolution, we investigated 80 dendrites with ~4,000 spines, 6 rats, 24 slices, and 50 neurons. For Conventional, we used 40 dendrites with ~2,000 spines from 4 rats, 10 slices, and 21 neurons.
Figure 5
Figure 5
Model illustration of Src kinase-mediated signaling in non-genomic effects of sex steroids on spinogenesis. DHT, T and E2 bind to synaptic receptors (AR and ER) within spines. Then, Src kinase may form complex with AR and ER, resulting in activation of Src kinase. Erk MAPK is then activated, leading to phosphorylation of cortactin, resulting in actin polymerization and new spine formation. AR and ER are anchored to the membrane via palmitoylation. Src kinase is localized to the membrane via myristoylation.
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