Effects of gonadectomy and dihydrotestosterone on neuronal plasticity in motivation and reward related brain regions in the male rat

Gonadal hormones affect neuronal morphology to ultimately regulate behaviour. In female rats, oestradiol mediates spine plasticity in hypothalamic and limbic brain structures, contributing to long‐lasting effects on motivated behaviour. Parallel effects of androgens in male rats have not been extensively studied. Here, we investigated the effect of both castration and androgen replacement on spine plasticity in the nucleus accumbens shell and core (NAcSh and NAcC), caudate putamen (CPu), medial amygdala (MeA) and medial preoptic nucleus (MPN). Intact and castrated (gonadectomy [GDX]) male rats were treated with dihydrotestosterone (DHT, 1.5 mg) or vehicle (oil) in three experimental groups: intact‐oil, GDX‐oil and GDX‐DHT. Spine density and morphology, measured 24 hours after injection, were determined through three‐dimensional reconstruction of confocal z‐stacks of DiI‐labelled dendritic segments. We found that GDX decreased spine density in the MPN, which was rescued by DHT treatment. DHT also increased spine density in the MeA in GDX animals compared to intact oil‐treated animals. By contrast, DHT decreased spine density in the NAcSh compared to GDX males. No effect on spine density was observed in the NAcC or CPu. Spine length and spine head diameter were unaffected by GDX and DHT in the investigated brain regions. In addition, immunohistochemistry revealed that DHT treatment of GDX animals rapidly increased the number of cell bodies in the NAcSh positive for phosphorylated cAMP response‐element binding protein, a downstream messenger of the androgen receptor. These findings indicate that androgen signalling plays a role in the regulation of spine plasticity within neurocircuits involved in motivated behaviours.


| INTRODUC TI ON
Gonadal hormones are key regulators of rewarding behaviour. 1,2 Oestrogen, progesterone and androgen signalling in the brain is involved in the display of motivated behaviours such as copulation, aggression and physical activity. 3 Moreover, gonadal hormones have been shown to influence the susceptibility to addiction-like behaviour. 4 To understand how hormones affect behaviour, it is important to investigate the underlying neurobiological mechanisms.
One mechanism through which gonadal hormones exert their influence on motivated behaviours is by affecting the structural plasticity of neurones. Previous research has shown that spine density,spinemorphologyanddendritelengthcanbeimpactedbygonadal hormones in multiple brain regions involved in motivation. [5][6][7][8] These hormone-induced structural reorganisations are both sexually dimorphic and strikingly different between brain regions, and have been linked to motivated behaviour, learning, memory and addiction. 4,[9][10][11] Copulation is a naturally occurring motivated behaviour reliant on gonadal hormones. Earlier research has shown that structural neuronal plasticity could be at the basis of hormonal effects on sexual behaviour. 12 For example, within the hypothalamus, oestradiol appearstoenhanceneuronalconnectivity,essentialforlordosis. [13][14][15][16] Oestrogens impact additional structures in the female limbic system.

Forexample,spinedensityinthehippocampusfluctuatesduringthe
oestrous cycle and oestradiol increases spine density in ovariectomised animals. 17,18 Bycontrast,oestradioladministrationtoovariectomised hamsters or rats produces a decrease in spine density within thenucleusaccumbenscore(NAcC). 8,19 Castration gradually ceases all sexual behaviour in male rats and hormonal replacement fully restores copulation. 20 Yet, in males, it remains grossly unknown what neurobiological mechanisms underliethelossofsexualbehaviourfollowinglossofgonadalhormones, and whether hormone effects on structural plasticity could be involved. Although some studies have shown spine plasticity in responsetotestosteroneinmales,itremainsuncleartowhatextent this is mediated by oestrogen formed through aromatisation of testosterone. [21][22][23] Itis,however,evidentthatoestrogensdonotsimply have the same effects on spine plasticity in males as in females. For example, as mentioned, the hippocampal CA1 region exhibits increasedspinedensityuponoestrogentreatmentinfemales,butis unresponsive to oestrogens in males. 24,25 Instead,CA1spinedensity in males is induced by dihydrotestosterone (DHT), a high-affinity ligand of the androgen receptor that is not aromatised into oestradiol. 24 Our laboratory also recently reported similar effects in the nucleusaccumbens,wherespineplasticityisaffectedbyoestrogens infemalesandbyDHTinmales,againindicatingthattheseeffectsin males are caused by androgens rather than oestrogens. 8,26 Although the effects of gonadal hormones on spine plasticity are sexually dimorphic, there are indications that the underlying mechanisms through which these effects arise are homologous.
Specifically, hormone-induced spine plasticity in the nucleus accumbens is mediated by activation of metabotropic glutamate receptor (mGluR) signalling, via oestradiol in females and DHT in males. 8,26 In females, the oestrogen-induced spine plasticity is reliantonmembrane-boundoestrogenreceptorsthatarecoupledto mGluRs,whichareactivateduponoestrogenbindingtotheoestrogen receptor. The activation of mGluRs can induce a downstream phosphorylation pathway leading to increased phosphorylation of cAMP response-element binding protein (CREB). 27,28 CREB is a transcription factor involved in numerous behavioural outputs and implicated in spine plasticity. 29,30 Becauseandrogen-inducedspine plasticity in the nucleus accumbens in males is also mediated by mGluRs,itcouldbeexpectedthatandrogensignallinginmaleshas similar effects on CREB phosphorylation as oestradiol in females, perhapsmediatedbymembrane-associatedandrogenreceptors. [31][32][33] Inthepresentstudy,weinvestigatedtheeffectsofgonadectomy (GDX)andandrogenreplacementonneuronalplasticityinputatively important brain regions for sexual motivation in male rats. We hy-
The used DHT dose in the present study is based on a recent study from our laboratory showing significant effect on spine density in theNAcShofcastratedmales. 26 The experiment was run in batches oftwoanimals(cagemates)atatime.Thetwoanimalsineachbatch were in the same group, and treatment groups were randomised accordingtoalatinsquaredesign,sothataveragecastrationduration did not differ between castrated groups. Animals were killed 24hoursafterhormoneorvehicletreatment.
The tissue was prepared and DiOlistically labelled as described previously. 34 DiOlistic labelling involves the ballistic delivery of tungsten microparticles coated with a lipophilic fluorescent dye (here:DiI)totissuesections.DiIlabelsmembranesofallneurones in which a tungsten bead is embedded, providing a Golgi-like labelling of neurones, with higher throughput and without bias.

um
was then used for statistical analysis. Measurements of spine length and head diameter were pooled for each treatment condition and thenplottedinviolinplots,aswellasbinnedcumulativeprobability distributions(binsizes:spinelength,0.5μm;headdiameter,0.1μm).

| Dendritic spine plasticity
To investigate the effects of both GDX and androgen replacement on dendritic spine plasticity,we compared intact males treatedwith oil (vehicle) to GDX males treatedwith oil, aswell as to GDX males

| pCREB expression
To investigate the potential rapid effects of DHT we focused on the striatum,abrainregioninwhichrapidphosphorylationofCREBhas been documented. 27 We determined the number of cells expressing

| D ISCUSS I ON
Gonadal hormones are known to regulate synaptic plasticity. 42,43 Althoughtheliteraturehassofarmostlycharacterisedtheeffectsof oestrogensinfemales,someevidenceexistsforeffectsofgonadal steroids in males as well. 24,31,44 In the present study, we aimed to examine the effects of loss and subsequent replacement of gonadal hormones on spine plasticity in males. We focused on brain regions thatareinvolvedinneuralcircuitsof(sexual)motivation:theNAcC, NAcShandCPu,whicharepartofdopaminergicrewardprocessing, Treatment with testosterone given systemically or locally into the MPN facilitates copulation in GDX males. [51][52][53][54] In addition, testosterone rescues GDX-induced spine loss in the MPN of male hamsters. 23 Furthermore, functional aromatisation of testosterone into oestrogen is necessary for the display of the full range of sex- Oestrogens induce rapid membrane-mediated signalling cascades, which are followed by longer lasting transcriptional activation via nuclear receptors. 67 We envision a similar set of actions for male sexualbehaviourinwhichandrogensprovidebothrapidandlongterm plasticity.
The small numbers of dendritic spines measured in these studies sometimes raise questions about the functional significance of these spine changes. For striatal medium spiny neurones, Golgi studies suggest that the cumulative dendritic length may be on the order of 2100 µm, 68 whereas cell fills put the number closer to about 3000 µm. 69 With an increase of three spines per 10 µm,aswesee inthenucleusaccumbensshell,thistranslatestoupwardsof1000 excitatory synapses per medium spiny neurone, producing a substantial impact on the electrotonic potential of these neurones. 69 AlimitationoftheDiOlisticlabellingapproachtakeninthisexperiment is that it is not possible to differentiate between specific neuronalcell-typessuchasD1vsD2mediumspinystriatalneurones.In theMeAandMPN,neuroneswereselectedbasedonsimilargross morphology,whichonlypartiallyaddressesneuronalheterogeneity.
Future research will aim to refine and combine methods in order to distinguish different neuronal populations.
Androgens can exert their action on neurones through multiple signalling pathways. 70  This leads to activation of a downstream signalling cascade culminating into phosphorylation of CREB, thereby enhancing its gene transcription properties. There is a large body of literature on the functionofCREB,which,amongstothers,isinvolvedinlearningand memory and synaptic plasticity. 29 Whether this proposed mecha-nismofDHT-inducedplasticity,throughmGluR5orothermGluRs, canalsobeappliedtotheeffectswefoundintheMPNandMeAwill be part of future research.

| CON CLUS IONS
We conclude that both GDX and androgen differentially affect spine plasticity in the MPN, MeA and NAcSh, whereas NAcC and CPu remain unaffected. In the NAcSh, DHT may exert its effects throughpCREBinductionmediatedbyandrogenreceptoractivation ofmGluR5.

ACK N OWLED G EM ENTS
Financial support was received from NIH DA041808 to PGM and from Norwegian Research Council grant #251320 to EMS. PTH was supported by a Personal Overseas Research Grant from the NorwegianResearchCouncil.WethankAnnaPeylafortechnicalassistance with immunohistochemistry.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are available from the corresponding author upon reasonable request.