Edited by: Takayoshi Ubuka, Monash University Malaysia, Malaysia
Reviewed by: Barney A. Schlinger, University of California, Los Angeles, United States; Arturo Ortega, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico
This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience
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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.
Accumulating evidence over the past two decades supports the conclusion that some sex steroid responses in the brain involve rapid non-genomic mechanisms (Mukai et al.,
On the other hand, androgen (T and DHT)-induced rapid effects on synaptic modulation in the hippocampus or other brain regions have not been extensively investigated (Foradori et al.,
Rapid synaptic action may require new arrangement of sex steroid receptor systems. In the hippocampus, androgen receptor (AR), and estrogen receptor (ER), classic nuclear steroid receptors, appear to be primarily located in the glutamatergic neurons (Simerly et al.,
Since adult hippocampus locally synthesizes androgen and estrogen (Hojo et al.,
We here investigated the possible involvement of Src tyrosine kinase in rapid spine modulation of DHT, T, and E2, with considering similarity and difference in signal pathways between DHT, T, and E2 in male rats. Although Src kinase was known to be activated by androgen and E2 in prostate and breast cancer cells (Migliaccio et al.,
Young adult male Wistar rats (12 week old, 320–360 g) were purchased from Tokyo Experimental Animals Supply (Japan). All animals were maintained under a 12 h light/12 h dark cycle and free access to food and water. The experimental procedure of this research was approved by the Committee for Animal Research of Teikyo University.
DHT, T and PP2 were purchased from Sigma-Aldrich (USA). Estradiol was from Wako Pure Chemicals (Japan). Lucifer Yellow was obtained from Molecular Probes (USA).
Adult male rats were deeply anesthetized by isoflurane and decapitated. Immediately after decapitation, the brain was removed from the skull and placed in ice-cold oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 22 NaHCO3, and 10 D-glucose (all from Wako); pH was set at 7.4. The hippocampus was then dissected and 400 μm thick transverse slices to the long axis, from the middle third of the hippocampus, were prepared with a vibratome (Dosaka, Japan). These slices were “fresh” slices without ACSF incubation. Slices were then incubated in oxygenated ACSF for 2 h (slice recovery processes) in order to obtain widely used “acute slices.”
The “acute” slices (used worldwide) were incubated for 2 h with 10 nM DHT, 10 nM T, or 1 nM E2, together with 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2), a Src kinase inhibitor. Slices were then fixed with 4% paraformaldehyde in PBS at 4°C overnight. Neurons within slices were visualized by an injection of Lucifer Yellow (Molecular Probes, USA) under Nikon E600FN microscope (Japan) equipped with a C2400-79H infrared camera (Hamamatsu Photonics, Japan) and with a 40× water immersion lens (Nikon, Japan).
Current injection was performed with glass electrode filled with 4% Lucifer Yellow for 2 min, using Axopatch 200B (Axon Instruments, USA). Approximately two neurons within a depth of 100–200 μm from the surface of a slice were injected with Lucifer Yellow (Duan et al.,
The imaging was performed from sequential z-series scans with super-resolution confocal microscope (Zeiss LSM880; Carl Zeiss, Germany) using Airy Scan Mode, at high zoom (× 3.0) with a 63 × oil immersion lens, NA 1.4. For Lucifer Yellow, the excitation and emission wavelengths were 458 and 515 nm, respectively. For analysis of spines, three-dimensional image was reconstructed from ~30 sequential z-series sections of every 0.45 μm. The applied zoom factor (×3.0) yielded 23 pixels per 1 μm. The confocal lateral resolution was ~0.14 μm. The z-axis resolution was ~0.40 μm. Our resolution limits were regarded to be sufficient to allow the determination of the head diameter of spines in addition to the density of spines. Confocal images were deconvoluted with the measured point spread function using Processing Mode of LSM880.
The density of spines as well as the head diameter were analyzed with Spiso-3D (automated software calculating mathematically geometrical parameters of spines) developed by Bioinformatics Project of Kawato's group (Mukai et al.,
Drug-treated dendrite images were used for spine analysis, and typical images were shown in Figures
Effects of Src kinase blocker (PP2) on DHT-induced spine increase and change in morphology in hippocampal slices.
We investigated the involvement of Src protein kinase in the modulation effects of DHT, T, and E2 on spinogenesis. Dendritic spine imaging was performed for Lucifer Yellow-injected glutamatergic neurons in acute hippocampal slices of male rats. We analyzed secondary branches of the apical dendrites located 100–200 μm distant from the pyramidal cell body around the middle of the stratum radiatum of CA1 region.
The morphological changes in spine head diameter induced after 2 h treatments of drugs were analyzed. Since observing the total spine density cannot describe well the complicated different kinase effects, the changes in spine head diameter distribution were also analyzed. Because the majority of spines (>93–95%) had distinct heads and necks, and stubby spines and filopodia did not contribute much to overall changes (<5–7%), we analyzed spines having distinct heads. We classified these spines with clear heads into three categories based 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.
Statistical analyses based on classification of the spines into three categories were performed. In control slices (without sex steroids supplementation), the spine density was 0.52 spines/μm for small-head spines, 0.30 spines/μm for middle-head spines, and 0.20 spines/μm for large-head spines (Figure
The treatments with DHT and PP2 induced significant changes in the total spine density (
From spine head diameter analysis, after 2 h treatments with DHT, the density of middle- and large-head spines significantly increased (
Note that, only PP2 did not significantly affect the total spine density, implying that the observed inhibitory effects are not due to simple non-specific effects by blockers (Figure
T and PP2 treatments induced significant changes in the total spine density (
Effects of Src kinase blocker on T-induced spine increase and change in morphology in hippocampal slices.
From spine head diameter analysis, upon treatments with T, the density of small- and middle-head spines significantly increased (
E2 and PP2 treatments induced significant changes in the total spine density (
Effects of Src kinase blocker on E2-induced spine increase and change in morphology.
From spine head diameter analysis, treatments with E2 significantly increased the density of small-head spines (
Airy scan mode of LSM880 super resolution confocal microscopy is equipped with 32 channel area detectors with honeycomb arrangement in the back of confocal pinhole. With these detectors we can directly measure the main part of the point spread function with which we perform deconvolution of confocal images. Therefore, the resultant final spine images were much clearer particularly for neck images with super resolution confocal microscopic imaging (Figure
Comparison of spine images obtained by super resolution confocal microscopy and conventional confocal microscopy.
We quantitatively compared control dendritic spines (without steroid supplementation) obtained from super resolution confocal microscopic analysis (LSM880) with those obtained from conventional confocal microscopic analysis (with Zeiss PASCAL confocal microscopy) (Hasegawa et al.,
We consider and discuss about Src kinase dependent signaling mechanisms in non-genomic modulation of sex steroid-induced dendritic spinogenesis.
Involvement of Src kinase (nonreceptor tyrosine kinase) in non-genomic rapid signaling upon androgen and estrogen stimulation has been extensively investigated in non-neuronal cells, including prostate cancer cells (e.g., LNCaP cells) (Migliaccio et al.,
Both T and E2 induced complex formation of AR, ERβ and Src kinase in LNCaP (Migliaccio et al.,
Taken together, upon stimulation of T or E2, AR or ER may form complex with Src kinase, leading to activation of MAPK in prostate cancer cells, breast cancer cells, or other gonadal tissues (Foradori et al.,
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.
We observed association of Src kinase, Erk MAPK, and ERα with post synaptic density (PSD), using Western blotting of PSD fractions prepared from male rat hippocampi, implying the spine membrane binding of Src kinase, MAPK, and ER (Mukai et al.,
Until recently, however, inadequate amounts of investigations had been accumulated for Src kinase signaling upon androgen and estrogen stimulation in central nervous systems. In one of a few reports, Src tyrosine kinase involvement was suggested from PP2-induced suppression of E2-enhanced LTP in acute hippocampal slices (Bi et al.,
On the other hand, downstream signaling of Src kinase, from MAPK to spine increase, has been extensively studied in neurons (see section MAPK-Mediated Signaling, Downstream of Src Kinase in Spinogenesis).
Erk MAPK (serine/threonine kinase) was involved in the rapid non-genomic effects on CA1 hippocampal spinogenesis through DHT, T, and E2 treatments (Hasegawa et al.,
Since both steroid receptors (AR and ER), Src kinase and MAPK are present in dendritic spines, an efficient coupling between these proteins could occur in spines, resulting in activation of Src kinase, followed by activation of Erk MAPK (Figure
In spinogenesis, the target of Erk MAPK may be cortactin. Erk MAPK phosphorylates cortactin which is associated with actin (MacQueen et al.,
The involvement of classic AR in androgen-induced rapid action was indicated from suppressing effects of hydroxyflutamide, a specific inhibitor of AR, on androgen-induced spinogenesis in acute slices of male rat hippocampus (Hatanaka et al.,
The involvement of classic ER in rapid E2 action was indicated from suppressing effects of ICI182,780 (ICI), a specific antagonist of both ER, on E2-induced spinogenesis in male hippocampal slices (Mukai et al.,
Expression of ERα in neurons of CA1 in rat and mouse hippocampus was demonstrated by immunostaining with purified antibody RC-19 (Mukai et al.,
Although treatments with DHT, T, and E2 increased the total spine density to almost the same level, three subclass analysis showed clear differences between DHT, T, and E2 in modulation of the spine morphology of hippocampal CA1 neurons (Figures
The T-effect is not dependent on the conversion from T to E2, and neither from T to DHT. This is concluded from the fact that T-effect was not blocked by inhibition of P450arom (E2 synthetase) and 5α-reductase (DHT synthetase) (Hatanaka et al.,
Sex steroid levels in the hippocampus play a key role for rapid action through synaptic AR and ER as modulators of synaptic plasticity. The average concentrations of male rat hippocampal DHT, T, and E2
The currently obtained knowledge encourages analysis of
SK: conceived and designed the study; MS, JK, and AK: conducted the experiments and analysis of the data; SK: wrote the manuscript. All authors provided feedback on the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Mr. J. Murakami, Mr. W. Tokuyama for spine data analysis. Profs. K. Inoue, J. Kurihara, and A. Kittaka (Teikyo University) are acknowledged for helpful discussions.