Rapid increase of spines by dihydrotestosterone and ...

1 Online at ReportRapid increase of spines by dihydrotestosteroneand testosterone in hippocampal neurons:Dependence on synaptic androgen receptorand kinase networksYusuke Hatanakaa,1, Yasushi Hojoa,b,1, Hideo Mukaia,b, Gen Murakamia,b,Yoshimasa Komatsuzakia, Jonghyuk Kima, Muneki Ikedaa, Ayako Hiragushia,Tetsuya Kimotoa, Suguru Kawatoa,b,nQ1aDepartment of Biophysics and Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, JapanbBioinformatics Project of Japan Science and Technology Agency, The University of Tokyo, Tokyo, Japanarticle infoArticle history.

2 Accepted 3 December 2014 Keywords:AndrogenAndrogen receptorDihydrotestosteroneHippocampusSp ineSynapseTestosteroneabstractRapid modulation of hippocampal synaptic plasticity by locally synthesized androgen isimportant in addition to circulating androgen. Here, we investigated the Rapid changes ofdendritic spines in response to the elevation of dihydrotestosterone (DHT) and testoster-one (T), by using hippocampal slices from adult male rats, in order to clarify whether thesesignaling processes include synaptic/extranuclear androgen receptor (AR) and activation ofkinases.

3 We found that the application of 10 nM DHT and 10 nM T increased the totaldensity of spines by approximately within 2 h, by imaging Lucifer Yellow-injectedCA1 pyramidal neurons. Interestingly, DHT and T increased different head-sized DHT increased middle- and large-head spines , T increased small-head spinogenesis was suppressed by individually blocking Erk MAPK, PKA,PKC, p38 MAPK, LIMK or calcineurin. On the other hand, blocking CaMKII did not inhibitspinogenesis. Blocking PI3K altered the spine head diameter distribution, but did notchange the total spine density.

4 Blocking mRNA and protein synthesis did not suppress theenhancing effects induced by DHT or T. The enhanced spinogenesis by androgens wasblocked by AR antagonist, which AR was localized postsynaptically. Taken together, theseresults imply that enhanced spinogenesis by DHT and T is mediated by synaptic/extranuclear AR which rapidly drives the kinase article is part of a Special Issue entitled SI: Brain and Published by Elsevier : 43978 Model7pp: 1212 col:fig:: NIL 1211221231241251261271281291301311321331 3413513613713813914014114214314414514614 7148149150151152153154155156157158159160 1611621631641651661671681691701711721731 7417517617717817918018118218318418518618 7 Published by Elsevier author at.

5 Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo,3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan. Fax: 81 3 5454 Kawato).1 Contributed equally to the present research](]]]])]]] ]]]Please cite this article as: Hatanaka, Y., et al., Rapid increase of spines by dihydrotestosterone and testosterone inhippocampal neurons:.. Brain Research (2014), synthesis of dihydrotestosterone (DHT) and testos-terone (T) has been demonstrated in male rat hippocampalneurons (Hojo et al., 2004). Mass-spectrometric analysis has rev-ealed that the level of adult male hippocampal androgen ishigher than that of plasma androgen (Hojo et al.

6 , 2009). Theseresults suggest that hippocampal T ( 17 nM) may be the sum ofhippocampus-synthesized T ( 3 nM) and circulating T ( 14 nM),which penetrates into the hippocampus. The level of hippocam-pal DHT is approximately 7 nM which is much higher than thatof circulating DHT ( ).Therefore,itisimportanttoinve-stigate the modulating effects on synaptic plasticity by hippo-campal over a decade, slow modulating effects on synapticplasticity by circulating androgen have been investigated. Gona-dectomy decreases spine synapses in male rat hippocampus,and the replacement of DHT or T through subcutaneous injec-tion rescues the level of spine synapsedensityofCA1neuronsin gonadectomized rats, after three days (Leranth et al.

7 , 2003).Electrophysiological investigations in rat hippocampal sliceshave shown that T application rescues excitatory postsynapticpotentiation (EPSP), which is decreased by castration (Smithet al., 2002). For rats castrated during puberty, T decr-eases hippocampal long-term potentiation (LTP) in vivo (Harleyet al., 2000), and an antagonist of androgen receptor (AR),flutamide, suppresses this reduction (Hebbard et al., 2003).Gonadectomized male rats show impaired cognitive perfor-mance, and androgen replacement rescues this impairment(Edinger and Frye, 2004;Frye and Seliga, 2001).

8 A direct applica-tion of androgen to the rat hippocampus in vivo has anti-anxietyeffects mediated via AR (Edinger and Frye, 2005,2006).Candidates of receptors for androgen action have beeninvestigated in the hippocampus. The expression of AR in thehippocampal neurons (Brown et al., 1995;Simerly et al., 1990)implies that the hippocampal neurons are the target of DHTand T. In the CA1 area of the hippocampus, in which ARappears to be primarily located in the pyramidal neurons(Clancy et al., 1992;Kerr et al., 1995). AR is located not only inthe cytoplasm and in the nuclei but also within dendriticspines and axon terminals (Tabori et al.)

9 , 2005). These resultssuggest that androgen may have Rapid effects on synapticfunction via synaptic/extranuclear AR, in addition to slowgenomic mechanisms of Rapid effects of androgen in thehippocampus are, however,still largely unknown (Foradoriet al., 2008;Hajszan et al., 2008), in contrast to deep under-standings of estrogen-induced Rapid synaptic effects. Leranth,MacLusky and their coworkers conclude that remodeling ofspine synapses by androgen is not driven via AR (Hajszan et al.,2008). Our earlier study has shown androgen-induced rapidincrease of dendritic thorns in CA3 stratum lucidum via MAPKand PKC, but not via PKA (Hatanaka et al.

10 , 2009). The rapideffect of androgen on the spinogenesis may play an importantrole in memory processes via producing new spines for creatingnew neuronal contacts. Here, we performed experiments to testthe hypothesis that androgen rapidly modulates dendriticspines via nongenomic signaling, including activation of synap-tic/extranulear AR and several kinases (including MAPK, PKA,PKC, LIMK). We also investigated different effects of DHT and Ton spine head diameter Rapid effect of androgen on CA1 spinogenesisWe investigated the effect of DHT and T on the modulation ofthe dendritic spine density and morphology in the hippo-campal CA1 stratum radiatum.