APP Regulation of Male Sexual Behavior in Rodents

AMYLOID PRECURSOR PROTEIN IN MALE SEXUAL BEHAVIOR
1
Amyloid precursor protein regulation of male sexual behavior
Fundamental Neuroscience: Vic Shao-Chih Chiang, 2019

2
Background & Aims
Sexual behavior is traditionally viewed as motivated by human reproduction, however, the impetus
is in fact largely diverse. People have reported engaging in sexual behavior due to attraction,
pleasure, expression of love, fun, adventure, curiosity, reciprocation, practice, spirituality etc.
(Meston & Buss, 2007). There are also less frequent reasons like getting a job, being promoted,
for punishment, humiliation, breaking up another relationship, to get gifts, peer pressure, fame etc
(Meston & Buss, 2007). The diversity of these motivations indicate sexual behavior to not only be
a biological imperative, but a paramount preoccupation for humans.
Rodents have been used for a long time to study male sexual behavior (MSB) since the first
publication in 1931 when Bacq investigated rat sexual behavior with sympathetic denervation
experiments(Bacq, 1931). Male rodents generally begin sexual behavior from examining the face
and anogenital region of the female(Angoa-Pérez & Kuhn, 2015). This is followed by mounting
the female’s rear and thrusting his pelvis. With these thrusts, he attempts to insert his penis into
her vagina(Angoa-Pérez & Kuhn, 2015). When he succeeds, this process of intromission occurs
several times until he ejaculates(Angoa-Pérez & Kuhn, 2015). One can observe the ejaculation
from the longer and deeper thrusts followed by the male dismounting slowly (Angoa-Pérez &
Kuhn, 2015). Rodent sexual behavior is divided into either appetitive or consummatory (Le Moëne
& Ågmo, 2019). Appetitive behavior is those that are courtship-based rituals to bring sexual
partners to contact, for instance, anogenital scent marking in male rodents (Le Moëne & Ågmo,
2019). In terms of consummatory behavior, these are those involved in copulation, notably, the
mounting, intromission and ejaculation and these can be measured scientifically by the latencies,
duration and frequencies of these events (Le Moëne & Ågmo, 2019). Sex steroid hormones are
well-established to be involved in sexual behavior, and in male rodents, these are driven by
testosterone which derives from Leydig cell secretion in the testes followed by aromatization into
estrogen or 5-alpha reduction to dihydrotestosterone (Hull & Dominguez, 2007).
The brain orchestrates male sexual behavior and several neuroanatomies have been identified to
be cardinal. Firstly, the medial preoptic area (mPOA) has been demonstrated in numerous studies
to be important for mounting, intromission and ejaculation and some of the key studies are

3
addressed as follows (Sakamoto, 2012). Implanting an electrode to mPOA with stimulation at
300uA and 50ms pulse induced MSB by changes in number and latencies of mounting and
intromission, as well as post ejaculation interval (Merari & Ginton, 1975). Tailing that in the 1990s,
a study lesioned the mPOA through radiofrequency at 55dC for 1 minute, which beget decreases
in mounting, intromission and ejaculation in Wistar male rats (Kondo, Shinoda, Yamanouchi, &
Arai, 1990). A much more recent study carried out fibre photometry and optogenetics on the
mPOA in mice and found calcium activity to precede MSB, the optogenetic stimulation to activate
MSB as well as the involvement of estrogen receptors (Wei et al., 2018).
Other regions that have shown to influence MSB includes the parvocellular subparafasicular
thalamic nucleus (SPFp), paraventricular nucleus, caudodorsal part of the posteromedial part of
the bed nucleus of the stria terminalis (BNSTpm), posterodorsal part of the medial amygdala
(MeApd) and the brain stem (Sakamoto, 2012). For the SPFp, ibotenic acid lesioning of the
posterior thalamus (which contains the SPFp) eliminates MSB in Long-Evans rats (Maillard &
Edwards, 1991). Vis-à-vis the MeApd, after MSB, the rats given one hour rest followed by
perfusion for immunohistochemistry, revealed activated neurons in the MeApd through Fos
immunoreactivity (Veening & Coolen, 1998). With respect to the BNSTpm, in male Wistar rats,
perfusion after one hour of rest followed by 75um sections by vibratome, also uncovered activated
neurons in the region through Fos immunoreactivity (Coolen, Peters, & Veening, 1997). Given the
importance of the mPOA, tracing studies have targeted this region to find circuitries with other
regions, which ferreted out mPOA connections with the medial forebrain bundle, SPFp and brain
stem (Sakamoto, 2012). As I mentioned the distinct grouping of MSB into appetitive and
consummatory behavior, discrete brain regions have similarly been divided accordingly. Those
involved in the appetitive aspect are amongst others, the BNSTpm, MeApd, and medial preoptic
nucleus (a part of the mPOA) (Snoeren, Veening, Olivier, & Oosting, 2014). For the
consummatory aspect, the mPOA, MeApd and BNSTpm were found to participate (Snoeren et al.,
2014).
At the cellular level, several molecules have been attributed to espouse MSB. For example, the
oxytocin-expressing neurons in paraventricular nucleus engage MSB neurocircuitry and the
abolishment of MSB by oxytocin antagonist infers the cardinal role of oxytocin(Sakamoto, 2012).

4
Another molecule, serotonin has been attributed with a modulatory and facilitatory role in MSB
(Snoeren et al., 2014). This was exhibited by studies with 5HTA1A receptor agonist in the mPOA
to decrease latency to first ejaculation, as well as further support of this postulation from studies
using selective serotonin reuptake inhibitors (Snoeren et al., 2014). It should, however, be noted
that these effects were dependent on the extracellular environment of how prevalent serotonin was
(Snoeren et al., 2014). Dopamine has likewise been implicated in MSB, as microinjections of a
D1/D2 receptor agonist to mPOA reduced time to reach ejaculation and decrease the time between
subsequent ejaculations, while dopamine antagonist exerted an opposite effect (Will, Hull, &
Dominguez, 2014). The endocannabinoid system ma be engaged by the same token, on the
grounds that CB1/CB2 receptor agonist increased latency to mount, intromit and ejaculate
(Androvicova, Horacek, Stark, Drago, & Micale, 2017). In addition to all of these above, nitric
oxide commensurately exert influential roles on MSB. This relationship was first discovered in
1995, where mice of C57B6J and SvEv 129 strains with gene deficiency in neuronal nitric oxide
synthase displayed an increase in mounting to reach ejaculation (Nelson et al., 1995). Following
that study, in 2004, injection of L-NAME, a NO synthesis inhibitor to the mPOA blocks copulation
in Long-Evans rats (Lagoda, Muschamp, Vigdorchik, & Hull, 2004). In the same year, the same
lab found sexual experience for two hours for a consecutive of three days augmented nNOS levels
in the mPOA that is not attributable to acute mating (J. M. Dominguez, Muschamp, Schmich, &
Hull, 2004).
Inasmuch as the well-established roles of sex steroid hormones in MSB as mentioned above, it is
tantalizing to ascribe these neurological mechanisms to them. Alternatively, steroid independent
mechanisms may be analogously important based on evidence from a few human epidemiological
studies and rodent models. This stance is buttressed with how sexual desire, erectile function, and
sexual function to be only minimally correlated with testosterone, estradiol and sex hormone-
binding globulin levels in the serum (Cunningham et al., 2015). Another example is a substantial
proportion of men remaining sexually active post-castration with 37% having sex several times
per week, and only 8% reporting to becoming non-sexual post-castration (Handy, Jackowich,
Wibowo, Johnson, & Wassersug, 2016).

5
Apropos to rodent models, Park and colleagues found the persistence of MSB in 40% of Siberian
hamsters 19 weeks post-castration (Park et al., 2004). Later work by Park and colleagues
discovered similar events in B6D2F1 mice where 30% of them continued to exhibit MSB at least
20 weeks post-castration, a phenomenon termed as SI-MSB (Park, Bonthuis, Ding, Rais, &
Rissman, 2009). Seeing how abstruse this phenomenon is, the same team gained further molecular
insight by comparing those that continue to mate post-castration (maters) vs those that did not
(non-maters). The maters displayed up-regulated mRNA of amyloid precursor protein and
microtubule-associated protein tau in the medial preoptic area (mPOA) (Bharadwaj et al., 2013;
Park, Bonthius, Tsai, Bekiranov, & Rissman, 2010). Congruently in the mPOA, it was identified
in maters an increased immunoexpression of synaptic proteins, synaptophysin and spinophilin, as
well as increased dendritic spine density (Bharadwaj et al., 2013).
APP is a transmembrane protein with a large extracellular domain and short cytoplasmic tail
(Müller, Deller, & Korte, 2017). It undergoes proteolytic processing either through the
amyloidogenic pathway to form amyloid beta or the non-amyloidogenic one (Müller et al., 2017).
There are also other non-canonical pathways discovered that generates Jcasp, C31 and other N-
terminal APP fragments (Müller et al., 2017). They impart interactions with a wide range of
molecules including NMDAR, RELN, NOGOR, SHC, NUMB etc and acts on axon growth &
guidance, neuromuscular junctions, synaptic function, plasticity, learning & memory, CNS injury
and protection (Müller et al., 2017). In addition to the APP up-regulation in maters, Park and
colleagues also found APP transgenic mice similarly exhibit SI-MSB (Park et al., 2010). The
transgenic mice had a 650kb YAC transgene with an entire human APP gene (transgene insert 8.9)
transfected into 129S2/SvPas mice D3 embryonic stem cells and crossed with C57BL/6J using the
method developed by Bruce T Lamb (Park et al., 2010). All of the mRNA and protein of the human
APP isoform were increased in the brain by >70% and they retained SI-MSB 12 weeks after
castration (Park et al., 2010). These results provide substantiation of APP to possibly partake in
SI-MSB, but the underlying mechanism is unknown.
I hypothesize a potential mechanism through nitric oxide (NO), on the basis of two frameworks.
Firstly as I have mentioned previously NO facilitate MSB and secondly, there are a few studies
showing APP to regulate NO. What follows is an account of these studies. This begins with the

6
study that infused amyloid beta through microdialysis to the Sprague-Dawley rat striatum, to
increase reactive oxygen species production (Parks, Smith, Trimmer, Bennett, & Parker, 2001).
NO was found to endow this process, on the grounds that the application of a nitric oxide synthase
(NOS) inhibitor (nitro-L-arginine) to block this effect (Parks et al., 2001). Next, it was observed
PC12 and human embryonic kidney cells with Swedish double mutation in the APP burgeoned in
NO levels (Keil et al., 2004). Following that study, the injection of amyloid beta to neonate
hippocampus CA1 impaired spatial memory after 90 days through NO (Díaz et al., 2010). They
unbosom this NO dependence as the administration with a NOS inhibitor ( L-NAME) to abolish
this effect (Díaz et al., 2010). Another vindication of APP and NO relationship was the pro-
inflammatory and toxic effect of amyloid beta in neurons co-cultured with glia was reduced
through NOS inhibitor (Yuste, Tarragon, Campuzano, & Ros-Bernal, 2015). Finally, in human
lens epithelial cells, amyloid beta accumulation from interferon and lipopolysaccharide treatment
bestowed nitric oxide production and this process was delineated to mediate through inducible
NOS (iNOS) from iNOS inhibitor (AG) experiments (Nagai, Ito, Shibata, Kubo, & Sasaki, 2017).
In light of these studies, many questions remain esoteric surrounding APP and SI-MSB including
the cell heterogeneity of APP in the mPOA, effects of APP knockdown, and the molecular
mechanisms governing APP regulation of SI-MSB. Ergo, this leads to three aims proposing to
answer these questions.
1. To characterize the differences in APP-expressing cell types in the mPOA of maters
compared to non-maters using intelligent-activated cell sorting followed by deep single-
cell RNA sequencing.
2. To determine if knockdown of APP in excitatory neurons through dCas9-based CRISPR
interference can impair SI-MSB.
3. To elucidate if amyloid beta treatment to primary mPOA culture neurons result in nitric
oxide production through the involvement of inducible NOS using live cell imaging by
fluorogenic RNA aptamers.

7
Methodology
Animals (Park et al., 2010)
Male B6D2F1 hybrid mice are bred by crossing C57BL/6J females with DBA/2J males. All
males are raised in the animal facility at the University of Massachusetts, Boston, and will be
weaned at 21 days. After weaning, they will be housed with littermates in cages individually
ventilated by negative airflow to prevent inter-cage transmission of olfactory cues. All mice
will be allowed ad libitum access to water and chow containing minimal phytoestrogen content
(Teklad Global Rodent Diet 2016, Harlan Laboratories, Inc.). The housing room is maintained
on a 12:12 light: dark cycle, with lights off at 1200 h EST. All procedures will be authorized
and carried out in accordance with the University of Massachusetts, Boston IACUC
(#IACUC2010) & AALAC guidelines.
Male sexual behaviour (Park et al., 2009)
The optimal sample size will be calculated using G power to carry out power analysis, with
effect size from the literature, accepted margin of error in the literature, 5% type I error, 80%
power, two-tailed test, and 10% attrition.
From 55 days of age, male B6D2F1 mice will be given four weekly MSB testing until
orchidectomy. For this, the males will be placed into Plexiglas arenas (17.8 cm w×17.8 cm
h×25.4 cm l) with home cage bedding which had not been changed for at least 1 week, and
then habituated to the arena for at least 30 min. All behavioural testing will be conducted under
dim red illumination in our behaviour testing room, during the dark phase of the light cycle, at
similar times of the day.
Stimulus females for sexual experience will be female C57BL/6J mice that are ovariectomized
in adulthood (70 – 74 days of age) and group-housed. Behavioural estrus will be induced with
subcutaneous injections of estradiol benzoate (10 μg in 0.1 mL sesame oil) 48 h prior to mating
followed by progesterone administration (400 μg in 0.05 mL sesame oil) 3–6 h prior to mating.
The stimulus female will be placed into the Plexiglas arenas for 120 min to allow mating. This
will be digital video recorded and manually noted by a blinded observer for the presence of
MSB components (mounting, intromission, ejaculation). Males must have ejaculated on at least
3 of the 4 tests to proceed to the next stage of the experiment.

8
Orchidectomy (Bharadwaj et al., 2013)
Orchidectomies will be performed under light isoflurane for a duration of 10 – 20 minutes. The
abdominal area of the mouse is shaved and cleaned with betadine (Providone-Iodine 7.5%).
Thereafter, a midline abdominal incision is made first through the skin, and then through the
abdominal muscle. After the testes are removed via cauterization, the muscle and skin will be
sutured. Animals will be individually housed for the remainder of the experiment. For
postoperative pain, buprenorphine (0.6 mg/kg) is administered intraperitoneally immediately
after surgery and 24 hours postoperatively.
Male sexual behaviour post-orchidectomy (Templin et al., 2019)
B6D2F1 hybrid males will be tested for steroid-independent male sexual behavior (SI-MSB)
every two weeks for 10 weeks after orchidectomy. Males are identified as “maters” if they
ejaculate on at least three out of the last four behavioral tests. They are identified as “non-
maters” if they did not display any of the MSB components (mounting, intromission and
ejaculation) during the last 4 tests.
Single Cell Dissociation (Moffitt et al., 2018)
Mice will be deeply anaesthetized with isofluorane and then decapitated. After that, brains are
removed from the skull and transferred to an adult mouse brain matrix to rapidly dissect the
mPOA in ice-cold 1x phosphate-buffered saline. The dissection will result in a ~2.5 mm × 2.5
mm × 1.1 mm (Bregma +0.5 to -0.6) tissue block spanning the preoptic region, and then placed
into papain dissociation buffer: 8 U/ml Papain, 100 U/ml DNase1, 50 U/ml chondroitinase
ABC, 0.07% hyaluronidase, 0.8 mM kynurenic acid, 1X Glutamax, 0.05 mM (2R)-amino-5-
phosphonovaleric acid (APV), 0.01 mM Y27632 dihydrochloride, 0.2X B27 supplement, and
1% w/v D(+)trehalose in hibernate A media. Following from that, the tissue will be washed
thrice in Hibernate A buffer: 0.8 mM kynurenic acid; 1X Glutamax; 0.05 mM APV; 1% w/v
D(+)trehalose; 0.2X B27 supplement; 0.01 mM Y27632 dihydrochloride in 1X Hibernate A
media, containing 0.1mg/mL trypsin inhibitor. Next, the sample is triturated in Hibernate A
buffer and then filtered with 20 μm nylon mesh. To sort cells that are viable and neuronal, the
neuron L1CAM antibody (neuron cell-surface marker) and propidium iodide will be applied in
Hibernate A buffer.

9
Intelligent image –activated cell sorting (ilACS) (Isozaki et al., 2019; Nitta et al., 2018)
This method uses machine-intelligent technology to sort single cells beyond one-dimensional
signals to unique spatial, biochemical and morphological traits. The ilACS machine consists of
a liquid pump, a cell focuser, a microscope, a speed meter, an image processor and a cell sorter.
To characterize, count the sort, and execute sort-decision functions, ilACS is equipped with
ImageJ, R, NIS-Elements AR and C++. Cells are first gated based on propidium iodide negative
cells to obtain viable cells. Subsequent gating will be made based on area, perimeter and shape
of bright field images that resembled mPOA neurons in previous studies (Takagi & Kawashima,
1993). Final gating is made from the fluorescence signal intensity of the neuron cell-surface
marker L1CAM.
Deep single-cell RNA sequencing (scRNA-seq) (Li et al., 2019)
Single cells are sorted into RNA extraction buffer followed by capturing on poly(T)
oligonucleotides that contain unique molecular identifier sequences, single-cell specific
barcodes and adaptor for subsequent amplification. After reverse transcription and second-
strand synthesis with hairpin primer, the transcriptome is amplified by polymerase chain
reaction. The amplicons are then fragmented by tagmentation and the quality assessed through
the fragment analyzer and concentration by Qubit. A final amplification step allows the
sequencing adaptors to be attached, and the resulting library is assessed with Bioanalyzer for
size distribution and Qubit for concentration. Illumina sequencing is next carried out to a depth
of at least 1 million raw reads per cell.
Bioinformatics (Lafzi, Moutinho, Picelli, & Heyn, 2018)
The raw data is presented in FastQ and pre-processed to trim the adaptor, normalized to the
housekeeping gene, and analyzed for different quality metrics. Alignment is then made to
detect alternative splicing and obtain quantities of the transcripts. Quality control measures are
in place to filter out transcripts with low quality. Normalization of batch effects and spike-in
controls are then made. Tailing from that, subpopulations of single cells are clustered with t-
distributed stochastic neighbor embedding. Differential gene expression between single cells
is analyzed with Find All Markers with Wilcoxon rank-sum tests. The data is subsequently
reduced of its dimensions for simplicity of visualization in two dimensional formats. Next,
gene ontology analysis on the function and relation is carried out to determine neuron
populations for comparison between maters and non-maters.

10
dCas9-based CRISPR interference (CRISPRi) (Zheng et al., 2018)
Cas9 nuclease can be navigated by single-guide RNA (sgRNA) to induce double-strand breaks
in a specific gene of interest. dCas9 is the nuclease-null form of Cas9 with H840A & D10A
mutations to the catalytic residues NUC & HNH. It is incorporated into the CRISPRi system,
and illustrated superior target specificity with minimal off-target effects when compared to
RNAi methods (Zheng et al., 2018). The replacement of Cas9 by dCas9 prevents random
cleavage repairs and instead functions through its conjugation with KRAB to suppress
transcription at the transcription start site (TSS).
I will use CRISPRi to knockdown APP expression in excitatory neurons. Firstly, the CRISPRi
construct will be made using the lentiCRISPR v2 vector as a template. The hU6 promoter will
be retained whilst the puromycin cassette will be replaced by enhanced green fluorescent
protein (EGFP), that is expressed with dCas9 through the flanking of self-cleaving 2A peptide.
KRAB will be conjugated to the N-terminus of dCas9 and sgRNA to APP will be designed to
target the DNA region from -50 to 300bp relative to the TSS of APP. Since I want to target
excitatory neurons, the elongation factor-1a short promoter will be replaced by the mouse
pCaMKIIa promoter. To package these plasmids into viruses, HEK293FT cells will be co-
transfected with the plasmids and virus packaging vectors using polyethyleneimine, followed
by harvesting and titer quantification.
To inject the virus into the mPOA, mice are anaesthetized and craniotomy is carried out to
access the mPOA. The lentivirus will be injected bilaterally to the mPOA using a
microinjection pump based on coordinates to the bregma, AP, −0.16 mm; ML, ±0.4 mm; DV,
−5.150 mm (Moffitt et al., 2018). After 2 weeks of injection, SI-MSB is tested and brains are
immediately harvested to confirm correct target site and gene knockdown by EGFP
immunofluorescence and APP expression, respectively.
For comparison, B6D2F1 mice will be tested for SI-MSB one week after-castration, tailgating
by virus injection with either the APP CRISPRi or scrambled CRISPRi control, and then re-
testing of SI-MSB after 2 weeks. The comparison of the percentage of those that ejaculated
will be made with two-tailed student’s t-test. P values less than 0.05 (p<0.05) is considered as
statistically significant.
Primary mPOA neurons (Parent et al., 2017)
The mPOA region is dissected at embryonic day 16.5 with brain matrix as described above.
The tissue is then trypsinized, homogenized and seeded onto six-well plates pre-filled with

11
Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 20mM KCl,
0.25% glucose, 15mM HEPES, 0.1% Penicillin-Streptomycin. The seed density will be
approximately 1 x 106
cells per mL in a volume of 1.5mL per well and then cultured at 5% CO2
and 37o
C. To prevent glial cell growth, 20μM 5’-fluorodeoxyuridine is also added.
Fluorogenic RNA aptamer live cell imaging (Wu et al., 2019)
Fluorogenic RNA aptamers bind to non-fluorescent molecules and convert them into
fluorescent forms, which allows visualization of RNA under fluorescence microscopy. To
facilitate this, a t-Deg tag is created, which is a bifunctional peptide that contains a degron
sequence and an RNA-binding peptide, Tat. When Tat binds to trans-activation response
element (TAR), it would be shielded from degradation. T-Deg can be tagged to fluorescent
proteins such as mNeonGreen, to engender the same effect, where fluorescence only occurs
when TAR is bound.
To create an RNA aptamer, TAR is expressed as a circular RNA, here on which would be
referred to as the “Pepper” tag. In order to enhance the recruitment of multiple fluorescent
proteins to the aptamer to increase fluorescence, 20 concatenated Pepper sequences will be
inserted into one construct. Each of these Pepper tags will be conjugated to an RNA three-way
junction sequence (F30) to improve folding.
For the mNeonGreen-tDeg plasmid, I will use the pcDNA3.1+ vector as a template followed
by digestion with restriction enzymes to enable insertion and ligation of a min-CMV promoter,
a restriction site for downstream digestion, a Kozak sequence for translation initiation, and the
gene encoding mNeonGreen, fused with tDeg. For CMV-iNOS--(F30-2xPepper)10, the
pcDNA3.1+ vector will similarly be used as a template for subsequent digestion and ligation
of the relevant components.
These plasmids will be co-transfected into mPOA neurons using FuGENE HD and then
incubated with 1μM DAF-FM after 40 hours of transfection to measure NO production. Cells
will then be treated with an optimized level of amyloid beta for 10 minutes, and then imaged
under an epifluorescence-inverted microscope with Hoechst33342 to stain for the nucleus. The
dynamics of iNOS mRNA and NO production to amyloid beta treatment will be visualized and
recorded for 48 hours. The median fluorescence intensity of NO production at 12, 24, 36 and
48 hours across six independent experiments will be analyzed. These will be used to compare
to vehicle control using one-way ANOVA and p-values less than 0.05 (p<0.05) will be
considered as statistically significant.

12
Expected Outcome & Implications
Although Park and colleagues previously found mPOA APP to be altered in SI-MSB, it is
unknown which cells contribute to this, as well as how the cell heterogeneity landscape changes
in SI-MSB (Park et al., 2010). Therefore, I propose to perform scRNA-seq on sorted neurons
from the mPOA to a depth of 1 million raw reads to reveal subtle differences between neuron
subpopulations (Li et al., 2019).
This method generates major cell classes of inhibitory and excitatory neurons based on Vgat
and Vglut2 expression, respectively. Further analysis of these populations would identify 20 –
40 neuronal subpopulations as observed in the previous scRNA-Seq study on the mPOA
(Moffitt et al., 2018). Given that APP is up-regulated in maters (Park et al., 2010), I expect
maters would possess larger clusters of APP expressing neurons.
A weakness to this experiment is that only a modicum of studies conducts flow cytometer
sorting of neurons due to their low viability, and therefore the markers used to isolate neurons
has been equivocal across the field. To address this weakness, validation experiments will need
to be conducted to compare my use of L1CAM with other known neuronal markers for FACS
such as NeuN and Thy1 (Bedrosian, Quayle, Novaresi, & Gage, 2018; McCullough et al.,
2016). On the plus side, the use of ilACS allows better discrimination of mPOA from not only
their biochemical traits but also morphological ones, that are informed from previous studies
(Takagi & Kawashima, 1993).
If I do not find differences in the size of APP-expressing neuron populations between maters
and non-maters, the APP differences previously observed may instead be attributable to non-
neuronal cells. In this case, I will shift my focus to another molecule such as tau, as it has
likewise illustrated to be up-regulated in the mPOA of maters (Bharadwaj et al., 2013). The
reason why APP is prioritized is on the grounds of existing studies that unearthed interactions
with MSB-related molecules including NO, serotonin, glutamate, endocannabinoids, and
dopamine.
Next, I proposed the use of CRISPRi to knockdown APP in mPOA excitatory neurons. Despite
APP over-expression using APP transgenic mice imparted MSB (Park et al., 2010), this effect
does not limit to specific parts of the body, and may, in a similar fashion, be argued to attribute
to developmental changes. It is expected that injection with APP CRISPRi will result in
decrease the percentage of males ejaculating when the APP is successfully inactivated after

13
two weeks of injection (Zheng et al., 2018), when compared to castrated males injected with
the scrambled CRISPRi control.
However, I acknowledge my approach is limited by my assumption that the mPOA excitatory
neurons are the drivers of the process. This is based on a priori studies that demonstrate
glutamate signalling to be important for MSB. Firstly, it was shown after ejaculation, that a
300% surge of glutamate was evident in the mPOA as measured by microdialysis, and reverse-
dialyzing glutamate to this region increased the number of ejaculation (Juan M. Dominguez,
Gil, & Hull, 2006). Another study found MSB-activated neurons in the mPOA expressed
higher amount of NR1, which is a subunit of NMDA receptor, and the infusion of its inhibitor
inhibited MSB (Juan M. Dominguez et al., 2007). Further supporting evince found antagonist
to NMDA infused to mPOA of Long-Evans rats to impair MSB (Vigdorchik, Parrish, Lagoda,
McHenry, & Hull, 2012). In spite of these, it is possible that inhibitory neurons are the bona
fide drivers of this process, and therefore alternative experiments will be conducted by
introducing inhibitory neuron markers, amongst others, Dlx1/2 into the APP CRISPRi
construct.
Strengths of this method include the CRISPRi being a system that is less laborious and time-
consuming compared to creating conventional conditional knockout mice, Cre-lox mice and so
forth (Zheng et al., 2018). CRISPRi enables the spatiotemporal targeting of APP as controlled
by the injection time and site of the virus, and thus prevents developmental aberrations and
confounding effects from other brain regions. Furthermore, the use of the nuclease-null mutant
avoids the random repair of the cleavage site and instead commits knockdown through KRAB.
In addition to this, CRISPRi knockdown was demonstrated to be better than RNA interference
strategies (Zheng et al., 2018).
Finally, the last experiment I propose is to use mPOA primary neurons as a model system to
study the direct effects of amyloid beta, which is a conventional product derived from APP
cleavage. There are no studies that investigate the molecular machinery of how APP may
regulate MSB, thus much work remains to be done to decipher this. I propose to examine their
effects on NO production through iNOS inasmuch as their role in MSB (Will et al., 2014) and
previous studies showing amyloid beta to affect this system.
Quasi to the study looking at the relationship between NO and amyloid beta in human lens
epithelial cells, I expect NO to steadily accrue across 12, 24, 36 and 48 hours after amyloid
beta treatment (Nagai et al., 2017). Apropos the dynamics of iNOS, these enzyme has been

14
shown to localize in a variety of subcellular compartments namely in Golgi, caveolae,
sarcoplasmic reticulum, mitochondria, nucleus and cytoskeleton (Villanueva & Giulivi, 2010).
Their translocation has also been shown to affect downstream signalling, gene transcription,
and their own activity (Villanueva & Giulivi, 2010). Although very sparse information on how
amyloid beta interacts with iNOS, I expect iNOS would have preferential compartmentation
upon initial transfection, and translocation occurs after amyloid beta is applied. This will be
evinced by mNeonGreen fluorescent signals present at different locations across time and
coincides with signals from the fluorescent marker of NO.
In comparison to conventional methods to measure mRNA or protein which locks them at a
cross-section in time, this method enables continuous imaging of their dynamics in live cells.
Researchers have also conducted several experiments that corroborated the superiority of this
method. This method was revealed to exhibit reduced fluorescent background, high signal-to-
noise ratio, and that the concatenate Pepper tag does not affect the stability of the mRNA nor
does it affect total cellular proteasome activity (Wu et al., 2019). To do this, they transfected
HEK293FT cells and treated them with actin D to block new transcription followed by qPCR
that found no differences to mCherry transcript stability with or without the tag (Wu et al.,
2019). Notwithstanding, they found the tag to give rise to relatively lesser levels of mRNA and
protein, but this was also evident in an irrelevant tag, 24xMS2, leading to the speculation that
this is possibly due to the longer transcript (Wu et al., 2019). Upon normalizing the protein
levels to the mRNA, the translation efficiency appears the same with non-tagged constructs
(Wu et al., 2019). Next, since the degron sequence leads to ubiquitination of fluorescent protein
leading to recruitment of the proteasome system, they wondered if this would increase the
overall level of proteasome degradation (Wu et al., 2019). With the use of western blot of
ubiquitinated proteins, there were no differences unless proteasome inhibitor was applied (Wu
et al., 2019).
Despite the above benefits, a weakness could be the unknown contribution from endogenous
NOS activity, and this aspect must be fully disentangled before proceeding to the use
fluorogenic RNA aptamers. Under the circumstance no changes in NO production and iNOS
dynamics is observed, I would focus on other molecular targets that have been implicated in
MSB, glutamate and endocannabinoid systems, to mention a few.
In the scenario that these hypothesized outcomes are fulfilled, several future studies will be
warranted. Firstly, after understanding the cell heterogeneity of mPOA from maters, we can

15
further discern underlying epigenetic mechanisms such as through single-cell DNA adenine
methyltransferase identification (DamID) with messenger RNA sequencing of the same cell
(scDam&T-seq) to enable simultaneous quantification of protein-DNA contacts and the
transcriptome. With regards to the CRISPRi results, optogenetic or chemogenetic stimulation
of these APP-expressing excitatory neurons can be conducted to determine their effects on SI-
MSB. These tools have begun to be used to study sexual behavior. One of the studies
optogenetically silenced the accessory olfactory bulb mitral cells and found lordosis to be
abated, which is a critical female sexual behavior (McCarthy et al., 2017). Another study
chemogenetically silenced medial amygdala neurons, which also disrupted lordosis (Ishii et al.,
2017). Finally, optogenetic stimulation of mPOA produced mounting behavior in both male
and female mice in addition to the normal range of mounting behavior (Wei et al., 2018). Vis-
à-vis follow-up studies from the neuron culture work, other signalling pathways important for
NO production should be investigated, for instance, NFκB, STAT and JNK signaling pathways
(Villanueva & Giulivi, 2010).
No work has been conducted to determine if APP is involved in human sexual behavior, and
only two tangentially related studies are found. In a cohort of over 3000 adults with dementia
that are 62-91, including some that are theorized to be driven by amyloid, a prevalence of 77%
sexual dysfunction was uncovered (Lindau et al., 2018). In another study, those with familial
amyloid polyneuropathy, patients often exhibited symptoms of erectile dysfunction
(Kavousanaki et al., 2019). These hardly touches the question of whether APP takes part in
human sexual behavior, and thus much more mechanistic understanding in animal models are
required before warranting further studies in humans.
At a more macroscopic level, through the understanding of rodent sexual behavior, we can
begin unravelling the complexities in human sexual behavior. This has practical significance
in male sexual dysfunction, that spans feature of desire and interest, erectile dysfunction,
ejaculation dysfunction, orgasm and dyspareunia (McCabe et al., 2016). The prevalence of
erectile dysfunction escalates with ageing, and is estimated to be 20 – 40% between 60 – 69
years of age, and rises above to 50 – 100% for men in their 70 and 80s (McCabe et al., 2016;
Quilter, Hodges, von Hurst, Borman, & Coad, 2017). Male sexual dysfunction is instigated by
multiple factors, the co-morbidity with diseases like prostate cancer (Albaugh, Sufrin, Lapin,
Petkewicz, & Tenfelde, 2017), and type 2 diabetes (Algeffari et al., 2018), or the use of drugs

16
such as finasteride (Ali, Heran, & Etminan, 2015) as an illustration. It is unfortunate the neglect
of this area due to the fact that sexual dysfunction is neither a direct cause of death nor appears
ostensibly, a great expense to the society. With that being said, it is no doubt sexual behavior
is fundamental to human well-being (Blanchflower & Oswald, 2004; Cheng & Smyth, 2015;
Todd B. Kashdan, Fallon R. Goodman, Melissa Stiksma, Cayla R. Milius, 2017) and sexual
dysfunction has dramatic impact on the quality of life (Rosen et al., 2009).
Furthermore, there is increasing recognition and discussion around the globe surrounding
sexual diversity in terms of characteristics (eg effeminate men), gender identities (eg
transgender), relationship paradigms (eg polyamory), fetishes (eg BDSM) that can vary over
time and context (Gupta, 2012). Figuring out the mechanisms of MSB is a critical step forward
to begin understanding the intricacy of sexual diversity. It is important to highlight current
definitions of sexual diversity will also continue to evolve and is evident in recent debates
challenging the assumption of homogeneity in sexual orientation, as stimulated by studies that
suggest a continuum paradigm (Roselli, 2018). Albeit, an important step forward is for future
neuroscience studies to consider the sexuality demographics, as this is an area that has long
been neglected in neuroscience, and helps address challenges due to possible political
misinterpretation. Science predominantly conceptualize human existence from a heterosexual
perspective, resulting in a parochial view of the world. The current proposal hopes to offer
baby steps towards understanding this labyrinth of human sexuality.

17
References
Albaugh, J. A., Sufrin, N., Lapin, B. R., Petkewicz, J., & Tenfelde, S. (2017). Life after
prostate cancer treatment: A mixed methods study of the experiences of men with sexual
dysfunction and their partners. BMC Urology, 17(1). https://doi.org/10.1186/s12894-
017-0231-5
Algeffari, M., Jayasena, C. N., MacKeith, P., Thapar, A., Dhillo, W. S., & Oliver, N. (2018,
February 1). Testosterone therapy for sexual dysfunction in men with Type 2 diabetes: a
systematic review and meta-analysis of randomized controlled trials. Diabetic Medicine,
Vol. 35, pp. 195–202. https://doi.org/10.1111/dme.13553
Ali, A. K., Heran, B. S., & Etminan, M. (2015). Persistent Sexual Dysfunction and Suicidal
Ideation in Young Men Treated with Low-Dose Finasteride: A Pharmacovigilance
Study. Pharmacotherapy, 35(7), 687–695. https://doi.org/10.1002/phar.1612
Androvicova, R., Horacek, J., Stark, T., Drago, F., & Micale, V. (2017, January 1).
Endocannabinoid system in sexual motivational processes: Is it a novel therapeutic
horizon? Pharmacological Research, Vol. 115, pp. 200–208.
https://doi.org/10.1016/j.phrs.2016.11.021
Angoa-Pérez, M., & Kuhn, D. M. (2015). Neuroanatomical dichotomy of sexual behaviors in
rodents: A special emphasis on brain serotonin. Behavioural Pharmacology, 26, 595–
606. https://doi.org/10.1097/FBP.0000000000000157
Bacq, Z. M. (1931). IMPOTENCE OF THE MALE RODENT AFTER SYMPATHETIC
DENERVATION OF THE GENITAL ORGANS. American Journal of Physiology-
Legacy Content, 96(2), 321–330. https://doi.org/10.1152/ajplegacy.1931.96.2.321
Bedrosian, T. A., Quayle, C., Novaresi, N., & Gage, F. H. (2018). Early life experience
drives structural variation of neural genomes in mice. Science, 359(6382), 1395–1399.
https://doi.org/10.1126/science.aah3378
Bharadwaj, P., McInnis, C., Madden, A. M. K., Bonthuis, P. J., Zup, S., Rissman, E. F., &
Park, J. H. (2013). Increased Dendritic Spine Density and Tau Expression Are
Associated with Individual Differences in Steroidal Regulation of Male Sexual
Behavior. PLoS ONE, 8(7). https://doi.org/10.1371/journal.pone.0069672
Blanchflower, D. G., & Oswald, A. J. (2004). Money, Sex and Happiness: An Empirical
Study. Scandinavian Journal of Economics, 106(3), 393–415.

18
https://doi.org/10.1111/j.0347-0520.2004.00369.x
Cheng, Z., & Smyth, R. (2015). Sex and happiness. Journal of Economic Behavior and
Organization, 112, 26–32. https://doi.org/10.1016/j.jebo.2014.12.030
Coolen, L. M., Peters, H. J. P. W., & Veening, J. G. (1997). Distribution of Fos
immunoreactivity following mating versus anogenital investigation in the male rat brain.
Neuroscience, 77(4), 1151–1161. https://doi.org/10.1016/S0306-4522(96)00542-8
Cunningham, G. R., Stephens-Shields, A. J., Rosen, R. C., Wang, C., Ellenberg, S. S.,
Matsumoto, A. M., … Snyder, P. J. (2015). Association of sex hormones with sexual
function, vitality, and physical function of symptomatic older men with low testosterone
levels at baseline in the testosterone trials. Journal of Clinical Endocrinology and
Metabolism, 100(3), 1146–1155. https://doi.org/10.1210/jc.2014-3818
Díaz, A., De Jesús, L., Mendieta, L., Calvillo, M., Espinosa, B., Zenteno, E., … Limón, I. D.
(2010). The amyloid-β25-35 injection into the CA1 region of the neonatal rat
hippocampus impairs the long-term memory because of an increase of nitric oxide.
Neuroscience Letters, 468(2), 151–155. https://doi.org/10.1016/j.neulet.2009.10.087
Dominguez, J. M., Muschamp, J. W., Schmich, J. M., & Hull, E. M. (2004). Nitric oxide
mediates glutamate-evoked dopamine release in the medial preoptic area. Neuroscience,
125(1), 203–210. https://doi.org/10.1016/j.neuroscience.2004.01.022
Dominguez, Juan M., Balfour, M. E., Lee, H. S., Brown, J. L., Davis, B. A., & Coolen, L. M.
(2007). Mating Activates NMDA Receptors in the Medial Preoptic Area of Male Rats.
Behavioral Neuroscience, 121(5), 1023–1031. https://doi.org/10.1037/0735-
7044.121.5.1023
Dominguez, Juan M., Gil, M., & Hull, E. M. (2006). Preoptic glutamate facilitates male
sexual behavior. Journal of Neuroscience, 26(6), 1699–1703.
https://doi.org/10.1523/JNEUROSCI.4176-05.2006
Gupta, K. (2012). Protecting Sexual Diversity: Rethinking the Use of Neurotechnological
Interventions to Alter Sexuality. AJOB Neuroscience, 3(3), 24–28.
https://doi.org/10.1080/21507740.2012.694391
Handy, A. B., Jackowich, R. A., Wibowo, E., Johnson, T. W., & Wassersug, R. J. (2016).
Gender Preference in the Sexual Attractions, Fantasies, and Relationships of Voluntarily
Castrated Men. Sexual Medicine, 4(1), e51–e59.

19
https://doi.org/10.1016/j.esxm.2015.11.001
Hull, E. M., & Dominguez, J. M. (2007). Sexual behavior in male rodents. Hormones and
Behavior, 52(1), 45–55. https://doi.org/10.1016/j.yhbeh.2007.03.030
Ishii, K. K., Osakada, T., Mori, H., Miyasaka, N., Yoshihara, Y., Miyamichi, K., & Touhara,
K. (2017). A Labeled-Line Neural Circuit for Pheromone-Mediated Sexual Behaviors in
Mice. Neuron, 95(1), 123-137.e8. https://doi.org/10.1016/j.neuron.2017.05.038
Isozaki, A., Mikami, H., Hiramatsu, K., Sakuma, S., Kasai, Y., Iino, T., … Goda, K. (2019).
A practical guide to intelligent image-activated cell sorting. Nature Protocols, 14(8),
2370–2415. https://doi.org/10.1038/s41596-019-0183-1
Kavousanaki, M., Tzagournissakis, Zaganas, I., Stylianou, K. G., Patrianakos, A. P.,
Tsilimbaris, M. K., … Samonakis, D. N. (2019). Liver Transplantation for Familial
Amyloid Polyneuropathy (Val30Met): Long-Term Follow-up Prospective Study in a
Nontransplant Center. Transplantation Proceedings, 51(2), 429–432.
https://doi.org/10.1016/j.transproceed.2019.01.071
Keil, U., Bonert, A., Marques, C. A., Scherping, I., Weyermann, J., Strosznajder, J. B., …
Eckert, A. (2004). Amyloid β-induced changes in nitric oxide production and
mitochondrial activity lead to apoptosis. Journal of Biological Chemistry, 279(48),
50310–50320. https://doi.org/10.1074/jbc.M405600200
Kondo, Y., Shinoda, A., Yamanouchi, K., & Arai, Y. (1990). Role of septum and preoptic
area in regulating masculine and feminine sexual behavior in male rats. Hormones and
Behavior, 24(3), 421–434. https://doi.org/10.1016/0018-506X(90)90019-T
Lafzi, A., Moutinho, C., Picelli, S., & Heyn, H. (2018, December 1). Tutorial: guidelines for
the experimental design of single-cell RNA sequencing studies. Nature Protocols, Vol.
13, pp. 2742–2757. https://doi.org/10.1038/s41596-018-0073-y
Lagoda, G., Muschamp, J. W., Vigdorchik, A., & Hull, E. M. (2004). A nitric oxide synthesis
inhibitor in the medial preoptic area inhibits copulation and stimulus sensitization in
male rats. Behavioral Neuroscience, 118(6), 1317–1323. https://doi.org/10.1037/0735-
7044.118.6.1317
Le Moëne, O., & Ågmo, A. (2019). Modeling Human Sexual Motivation in Rodents: Some
Caveats. Frontiers in Behavioral Neuroscience, 13.
https://doi.org/10.3389/fnbeh.2019.00187

20
Li, Q., Cheng, Z., Zhou, L., Darmanis, S., Neff, N. F., Okamoto, J., … Barres, B. A. (2019).
Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep
Single-Cell RNA Sequencing. Neuron, 101(2), 207-223.e10.
https://doi.org/10.1016/j.neuron.2018.12.006
Lindau, S. T., Dale, W., Feldmeth, G., Gavrilova, N., Langa, K. M., Makelarski, J. A., &
Wroblewski, K. (2018). Sexuality and Cognitive Status: A U.S. Nationally
Representative Study of Home-Dwelling Older Adults. Journal of the American
Geriatrics Society, 66(10), 1902–1910. https://doi.org/10.1111/jgs.15511
Maillard, C. A., & Edwards, D. A. (1991). Excitotoxin lesions of the zona incerta/lateral
tegmentum continuum: effects on male sexual behavior in rats. Behavioural Brain
Research, 46(2), 143–149. https://doi.org/10.1016/S0166-4328(05)80107-X
McCabe, M. P., Sharlip, I. D., Lewis, R., Atalla, E., Balon, R., Fisher, A. D., … Segraves, R.
T. (2016). Incidence and Prevalence of Sexual Dysfunction in Women and Men: A
Consensus Statement from the Fourth International Consultation on Sexual Medicine
2015. Journal of Sexual Medicine, 13(2), 144–152.
https://doi.org/10.1016/j.jsxm.2015.12.034
McCarthy, E. A., Kunkhyen, T., Korzan, W. J., Naik, A., Maqsudlu, A., Cherry, J. A., &
Baum, M. J. (2017). A comparison of the effects of male pheromone priming and
optogenetic inhibition of accessory olfactory bulb forebrain inputs on the sexual
behavior of estrous female mice. Hormones and Behavior, 89, 104–112.
https://doi.org/10.1016/j.yhbeh.2016.12.011
McCullough, K. M., Choi, D., Guo, J., Zimmerman, K., Walton, J., Rainnie, D. G., &
Ressler, K. J. (2016). Molecular characterization of Thy1 expressing fear-inhibiting
neurons within the basolateral amygdala. Nature Communications, 7.
https://doi.org/10.1038/ncomms13149
Merari, A., & Ginton, A. (1975). Characteristics of exaggerated sexual behavior induced by
electrical stimulation of the medial preoptic area in male rats. Brain Research, 86(1),
97–108. https://doi.org/10.1016/0006-8993(75)90641-1
Meston, C. M., & Buss, D. M. (2007). Why humans have sex. Archives of Sexual Behavior,
36(4), 477–507. https://doi.org/10.1007/s10508-007-9175-2
Moffitt, J. R., Bambah-Mukku, D., Eichhorn, S. W., Vaughn, E., Shekhar, K., Perez, J. D., …

21
Zhuang, X. (2018). Molecular, spatial, and functional single-cell profiling of the
hypothalamic preoptic region. Science, 362(6416).
https://doi.org/10.1126/science.aau5324
Müller, U. C., Deller, T., & Korte, M. (2017, May 1). Not just amyloid: Physiological
functions of the amyloid precursor protein family. Nature Reviews Neuroscience, Vol.
18, pp. 281–298. https://doi.org/10.1038/nrn.2017.29
Nagai, N., Ito, Y., Shibata, T., Kubo, E., & Sasaki, H. (2017). A positive feedback loop
between nitric oxide and amyloid β (1-42) accelerates mitochondrial damage in human
lens epithelial cells. Toxicology, 381, 19–30. https://doi.org/10.1016/j.tox.2017.02.014
Nelson, R. J., Demas, G. E., Huang, P. L., Fishman, M. C., Dawson, V. L., Dawson, T. M., &
Snyder, S. H. (1995). Behavioural abnormalities in male mice lacking neuronal nitric
oxide synthase. Nature, 378(6555), 383–386. https://doi.org/10.1038/378383a0
Nitta, N., Sugimura, T., Isozaki, A., Mikami, H., Hiraki, K., Sakuma, S., … Goda, K. (2018).
Intelligent Image-Activated Cell Sorting. Cell, 175(1), 266-276.e13.
https://doi.org/10.1016/j.cell.2018.08.028
Parent, C., Wen, X., Dhir, S. K., Ryan, R., Diorio, J., & Zhang, T. Y. (2017). Maternal care
associates with differences in morphological complexity in the medial preoptic area.
Behavioural Brain Research, 326, 22–32. https://doi.org/10.1016/j.bbr.2017.02.047
Park, J. H., Bonthius, P. J., Tsai, H. W., Bekiranov, S., & Rissman, E. F. (2010). Amyloid β
precursor protein regulates male sexual behavior. Journal of Neuroscience, 30(30),
9967–9972. https://doi.org/10.1523/JNEUROSCI.1988-10.2010
Park, J. H., Bonthuis, P., Ding, A., Rais, S., & Rissman, E. F. (2009). Androgen- and
estrogen-independent regulation of copulatory behavior following castration in male
B6D2F1 mice. Hormones and Behavior, 56(2), 254–263.
Park, J. H., Takasu, N., Alvarez, M. I., Clark, K., Aimaq, R., & Zucker, I. (2004). Long-term
persistence of male copulatory behavior in castrated and photo-inhibited Siberian
hamsters. Hormones and Behavior, 45(3), 214–221.
Parks, J. K., Smith, T. S., Trimmer, P. A., Bennett, J. P., & Parker, W. D. (2001). Neurotoxic
Aβ peptides increase oxidative stress in vivo through NMDA-receptor and nitric-

22
oxide-synthase mechanisms, and inhibit complex IV activity and induce a
mitochondrial permeability transition in vitro. Journal of Neurochemistry, 76(4),
1050–1056. https://doi.org/10.1046/j.1471-4159.2001.00112.x
Quilter, M., Hodges, L., von Hurst, P., Borman, B., & Coad, J. (2017). Male Sexual Function
in New Zealand: A Population-Based Cross-Sectional Survey of the Prevalence of
Erectile Dysfunction in Men Aged 40–70 Years. Journal of Sexual Medicine, 14(7),
928–936. https://doi.org/10.1016/j.jsxm.2017.05.011
Roselli, C. E. (2018). Neurobiology of gender identity and sexual orientation. Journal of
Neuroendocrinology, 30(7), e12562. https://doi.org/10.1111/jne.12562
Rosen, R. C., Shifren, J. L., Monz, B. U., Odom, D. M., Russo, P. A., & Johannes, C. B.
(2009). ORIGINAL RESEARCH—EPIDEMIOLOGY: Correlates of Sexually Related
Personal Distress in Women with Low Sexual Desire. The Journal of Sexual Medicine,
6(6), 1549–1560. https://doi.org/10.1111/j.1743-6109.2009.01252.x
Sakamoto, H. (2012, February). Brain-spinal cord neural circuits controlling male sexual
function and behavior. Neuroscience Research, Vol. 72, pp. 103–116.
https://doi.org/10.1016/j.neures.2011.11.002
Snoeren, E. M. S., Veening, J. G., Olivier, B., & Oosting, R. S. (2014). Serotonin 1A
receptors and sexual behavior in male rats: A review. Pharmacology Biochemistry and
Behavior, Vol. 121, pp. 102–114. https://doi.org/10.1016/j.pbb.2013.11.007
Takagi, K., & Kawashima, S. (1993). Culture of rat brain preoptic area neurons: Effects of
sex steroids. International Journal of Developmental Neuroscience, 11(1), 63–70.
https://doi.org/10.1016/0736-5748(93)90035-C
Templin, J. S., Wyrosdic, J. C., David, C. D., Wyrosdic, B. N., Lapp, H. E., Bala, A., …
Park, J. H. (2019). Peripubertal gonadal steroids are necessary for steroid-independent
male sexual behavior in castrated B6D2F1 male mice. Hormones and Behavior, 113,
38–46. https://doi.org/10.1016/j.yhbeh.2019.04.013
Todd B. Kashdan, Fallon R. Goodman, Melissa Stiksma, Cayla R. Milius, P. E. M. (2017).
Sexuality Leads to Boosts in Mood and Meaning in Life With No Evidence for the
Reverse Direction: A Daily Diary Investigation. Emotion, 18(4), 563–576.
Veening, J. G., & Coolen, L. M. (1998). Neural activation following sexual behavior in the
male and female rat brain. Behavioural Brain Research, 92(2), 181–193.

23
https://doi.org/10.1016/S0166-4328(97)00190-3
Vigdorchik, A. V., Parrish, B. P., Lagoda, G. A., McHenry, J. A., & Hull, E. M. (2012). An
NMDA antagonist in the MPOA impairs copulation and stimulus sensitization in male
rats. Behavioral Neuroscience, 126(1), 186–195. https://doi.org/10.1037/a0026460
Villanueva, C., & Giulivi, C. (2010, August). Subcellular and cellular locations of nitric
oxide synthase isoforms as determinants of health and disease. Free Radical Biology
and Medicine, Vol. 49, pp. 307–316.
https://doi.org/10.1016/j.freeradbiomed.2010.04.004
Wei, Y. C., Wang, S. R., Jiao, Z. L., Zhang, W., Lin, J. K., Li, X. Y., … Xu, X. H. (2018).
Medial preoptic area in mice is capable of mediating sexually dimorphic behaviors
regardless of gender. Nature Communications, 9(1). https://doi.org/10.1038/s41467-017-
02648-0
Will, R. G., Hull, E. M., & Dominguez, J. M. (2014). Influences of dopamine and glutamate
in the medial preoptic area on male sexual behavior. Pharmacology Biochemistry and
Behavior, 121, 115–123. https://doi.org/10.1016/j.pbb.2014.02.005
Wu, J., Zaccara, S., Khuperkar, D., Kim, H., Tanenbaum, M. E., & Jaffrey, S. R. (2019). Live
imaging of mRNA using RNA-stabilized fluorogenic proteins. Nature Methods, 16(9),
862–865. https://doi.org/10.1038/s41592-019-0531-7
Yuste, J. E., Tarragon, E., Campuzano, C. M., & Ros-Bernal, F. (2015, August 17).
Implications of glial nitric oxide in neurodegenerative diseases. Frontiers in Cellular
Neuroscience, Vol. 9. https://doi.org/10.3389/fncel.2015.00322
Zheng, Y., Shen, W., Zhang, J., Yang, B., Liu, Y. N., Qi, H., … Yao, J. (2018). CRISPR
interference-based specific and efficient gene inactivation in the brain. Nature
Neuroscience, 21(3), 447–454. https://doi.org/10.1038/s41593-018-0077-5

APP Regulation of Male Sexual Behavior in Rodents

Recomendados

Recomendados

Mais conteúdo relacionado

Mais procurados

Mais procurados (20)

Semelhante a APP Regulation of Male Sexual Behavior in Rodents

Semelhante a APP Regulation of Male Sexual Behavior in Rodents (20)

Mais de Vic Shao-Chih Chiang

Mais de Vic Shao-Chih Chiang (14)

Último

Último (20)

APP Regulation of Male Sexual Behavior in Rodents