Obesity impairs sex: what is the electrophysiology at the single neuron level?

1. 1
TITLE PAGE
Title of the study project
Obesity impairs sex: what is the electrophysiology at the single neuron level?
Name
CHIANG, Vic Shao-Chih
Date of the entire proposed project period
5th
January 2023 – 5th
January 2024
Abstract
Sexual behaviour behoves our well-being and in sexual dysfunction, our quality of life
cripples detrimentally. Unhealthy dietary patterns lead to obesity, which poses a major risk
factor for sexual dysfunction. Globally, one in ten people is obese, which burgeons in the US
with over 40% obese. Only a paucity of studies exist that investigated the neurological
mechanism between sexual dysfunction and obesity, and the electrophysiological mechanisms
remain unknown.
With the research group led by Dr Ole Paulsen in the University of Cambridge, we will
identify the aberrant neuron electrophysiological activity in obese mice in their nucleus
accumbens, which is a brain region imperative for male sexual behaviour.
To study this, we will adopt a novel approach that has never been used in the field of
sexual behaviour. Using the transgenic mice with a +/+ CaMKII-α-Cre on a C57BL/6
background, this enables electrophysiological responses to be traced specifically to excitatory
neurons using optogenetics. To induce obesity, we will raise our transgenic mice with a high-
fat diet for 24 weeks and confirm their sexual dysfunction by their latency to ejaculate.
We will then surgically implant an optic fibre with a recording electrode assembly into
their nucleus accumbens. Following that, the infection with AAV2-EF1a-DIO-ChR2-mCherry
virus warrants optogenetic tagging precisely in CaMKII-α-expressing cells. Ensuing in vivo
electrophysiology are recorded during sociosexual interactions with a hormone-primed
ovariectomized conspecific female. Plexon software and statistical analysis will determine
neural correlates to sexual behaviour.
Our goal facilitates the understanding of how obesity changes neuronal activity in
sexual dysfunction. This buttresses the development of advanced therapeutics for sexual
dysfunction, unravel the complexity of sexual diversity and promote sexual diversity in fields
of science, technology, engineering, and mathematics.

2. 2
SPECIFIC AIMS
Sexual behaviour constitutes a human preoccupation 1
, and rodent studies attribute the nucleus
accumbens as one of the paramount brain regions for sexual behaviour 2
. The dysfunction of
sexual behaviour affects 40% of men above the age of 60 3
and obesity posits as a substantial
risk factor 4
. The relationship was deemed causal in rodent studies, but questions remain on the
electrophysiological mechanisms 5
. Through in vivo electrophysiology and optogenetic tagging
in transgenic male mice during sociosexual interactions with female conspecifics, this grant
unveils the mechanisms of obesity-induced sexual dysfunction to test the following hypothesis:
1. Excitatory neurons in the nucleus accumbens have perturbed electrophysiology in
obese male mice during sociosexual interactions.
2. Inhibitory interneurons in the nucleus accumbens have perturbed electrophysiology in
obese male mice during sociosexual interactions.
3. Neurons in the nucleus accumbens have perturbed oscillation patterns in obese male
mice during sociosexual interactions.
BACKGROUND
Sexual Behaviour
The traditional view on sexual behaviour rests on its motivations in human reproduction,
however, the impetus of sexual behaviour is myriad. People have reported engaging in sexual
behaviour due to attraction, pleasure, expression of love, fun, adventure, curiosity,
reciprocation, practice, spirituality etc. 1
. Less frequent reasons included getting a job, being
promoted, for punishment, humiliation, breaking up another relationship, to get gifts, peer
pressure, fame etc 1
. The diversity of these motivations indicates sexual behaviour to not only
be a biological imperative but a paramount preoccupation for humans.
Sexual Dysfunction
The dysfunction of sexual behaviour expressed in males spans features of desire and interest,
erectile dysfunction, ejaculation dysfunction, orgasm and dyspareunia 3
. Notably, the
prevalence of erectile dysfunction escalates with ageing, with estimations to inflict 20 – 40%
males between 60 – 69 years of age, and rises above to 50 – 100% for men in their 70 and 80s
3,6
. Male sexual dysfunction instigates from multiple factors. As an illustration, these comprise
of the co-morbidity with diseases like prostate cancer 7
and type 2 diabetes 8
, or the use of drugs
such as finasteride 9
.
Rodents Sexual Behavior
In 1931, investigations of sexual behaviour began from sympathetic denervation experiments
10
. Since then, rodents became an indispensable model to study male sexual behaviour. Male
rodents begin their sexual repertoire from examining the face and anogenital region of the
female 11
. This follows with mounting the female’s rear and thrusting his pelvis in attempts to
insert his penis into her vagina 11
. When he succeeds, this process of intromission occurs several
times until he ejaculates 11
. We can observe the ejaculation from the longer and deeper thrusts
followed by the male dismounting slowly 11
.
Rodent sexual behaviour can be considered either as appetitive or consummatory 12
. Appetitive
behaviour refers to courtship-based rituals to bring sexual partners to contact, for instance,
anogenital scent marking in male rodents 12
. In terms of consummatory behaviour, these allude
to mounting, intromission and ejaculation, which quantifies with measurements on their
latencies, durations and 12
.

3. 3
Neurological Basis
The brain orchestrates male sexual behaviour and several neuroanatomies identify as cardinal.
To name a few, these encompass the medial preoptic area, parvocellular sub parafascicular
thalamic nucleus, paraventricular nucleus, caudodorsal part of the posteromedial part of the
bed nucleus of the stria terminalis, posterodorsal part of the medial amygdala, and the brain
stem 13
.
The nucleus accumbens likewise coordinates male sexual behaviour, beginning with support
in 1986 from drug injections to the region 14
. More compelling evidence emerged from lesion
studies, where males did not intromit with nucleus accumbens lesions 15
. Further countenance
derives from examining male mice chemosensory attraction to females whereby the nucleus
accumbens dopaminergic system was perforce 16
.
Electrophysiology and Sexual Behaviour
Aside from the neuroanatomical basis of sexual behaviour, the neuron firing patterns in these
regions are similarly important. Such endeavours began in 1973, with the first article publishing
on the electrophysiological correlates in male rat sexual behaviour 17
. They implanted
electrodes onto regions of the frontal and parietal cortex over the skull and determined
correlating rhythms from the hippocampus electroencephalograph 17
. Another study repeated
this a decade later in addition to correlating the ultrasonic responses that were involved 18
. This
extended to the use of electrocorticogram, which features higher spatial resolution and higher
signal to noise ratio to electroencephalography, and discovered distinct bands of frequencies to
mounting, intromission and ejaculation 19
. Further electroencephalography studies ran a decade
later in deeper brain structures with the implant of electrodes into the nucleus accumbens and
ventral tegmental area 20
. Male sexual behaviour in rats responded with changes in the relative
power of different frequency bands to these regions 20
.
Moving the field forward was the first single-unit neuron electrophysiology recording for local
field potential in 2012 during male rat sexual behaviour, with a recording electrode assembly
implanted into the nucleus accumbens shell 21
. This enabled different types of neurons to be
assessed during sexual behaviour and found electrophysiological responses specifically to fast-
spiking interneurons and medium spiny neurons including their frequency oscillation 21
.
Obesity and Sexual Dysfunction
Obesity has proliferated worldwide in the past 50 years, accumulating to more than 10% of
adults 22
. This alarms western countries in particular, on the grounds of obesity affecting nearly
40% of the US population 22
. Globally, this currently attributes to a financial burden of USD 2
trillion each year for direct healthcare costs and economic productivity 23
.
In addition to that, obesity portrays as a risk factor for developing sexual dysfunction. For
example, in 500 participants, an association imparted between higher BMI with greater
impaired sexual quality of life in terms of enjoyment, desire, and performance 24
. A review
came out the next year that summarized results from prospective and cross-sectional studies
and further espoused this correlation 25
. A decade later, another review ferreted this relationship
to stem from direct effects of adipose tissue, comorbidities, and psychological factors 26
. Most
recently in 2019, an additional review continues to buttress the correlation between sexual
dysfunction with obesity 4
.
Animal models have drawn causality in this relationship. For example, the Zucker obese
diabetic rat exhibited erectile dysfunction, with the obesity-induced through a missense
mutation in the leptin receptor 27
. Furthermore, male rats fed with a high-fat diet demonstrated
several features of sexual dysfunction from erectile responses and decreased sex-related
hormone concentrations 28
. The mechanisms of these changes ascribe in part to leptin inhibition
of testicular steroidogenesis via several genes such as SF-1, StAR, P450scc 29
. Newer studies

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evinced the melanocortin system to implicate in this mechanism through Sim1 and oxytocin
expressing neurons5
. How the neuron electrophysiology engages in this is an unaddressed
question.
INNOVATION
Sufficient molecular understanding of the relationship between obesity and sexual dysfunction
in rodent models endorses further electrophysiology work. This domain limited to
electroencephalography studies except in 2012, with the only study that looks at the single
neuron level 21
. With this pioneering study, it informs the current study to investigate the
unknown electrophysiological mechanisms of how obesity at the single neuron level,
contributes to sexual dysfunction. Furthermore, the recent advances in optogenetic tagging in
electrophysiology studies enables our study to identify with precision, the activity from specific
neurons at the single neuron level 30,31
.
APPROACH
The in vivo electrophysiology and optogenetics follows the method from the host institution,
Dr Ole Paulsen’s lab in the University of Cambridge and using their CaMKIIα-Cre transgenic
mice 32–35
. Overall, we will study obese mice during their sociosexual interactions with
ovariectomized, hormonally-primed conspecific females. In order to examine the
electrophysiology of excitatory neurons in the nucleus accumbens, we adopt optogenetic
tagging of these neurons through injecting viruses expressing channelrhodopsin into the
nucleus accumbens in the CaMKIIα-Cre mice. In vivo electrophysiology will then be carried
out with an implanted optetrode to examine spiking and oscillation patterns, as well as allowing
additional classification of inhibitory interneurons signals.
Mice
We will use Cre mice with a CaMKIIα promoter on a 129S6 genetic background. The new-
borns wean at 21 days of age and then housed individually with ventilation by negative airflow
to prevent inter-cage transmission of olfactory cues. The circadian rhythm keeps at a 12:12
light, dark cycle with lights off at 1200. Using G power, based on power analysis to calculate
the optimal sample size with effect size and accepted margin of error from the literature, 5%
type I error, 80% power, two-tailed t-test, and 10% attrition, we will use a sample size of 20
mice in each group.
For diet-induced obesity, we will feed mice with a 60% high-fat diet after weaning for 24 weeks,
while the control mice consume a normal chow diet. We will also record their body weights
and food intake on a weekly basis.
Viral constructs & adeno-associated virus packaging
To express the light-sensitive channelrhodopsin gene in CaMKIIα-expressing cells, we will
use the AAV2-EF1a-DIO-ChR2-mCherry adeno-associated virus. For this, we will package
the adeno-associated virus vectors carrying DIO-ChR2-mCherry into AAV2/9 serotype, and
reach titres of 1-5 × 1012
viral particles / mL.
Precisely, we co-transfect the viral construct and the adeno-associated virus vector into
HEK293FT cells using polyethyleneimine. The virus release through freeze-thaw cycles and
sonication, then purified with a calcium chloride density gradient ultra-centrifugation. Finally,
we dialyze the virus into HEPES buffer and the titre measured using quantitative real-time
polymerase chain reaction.

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Ovariectomy
For sexual behaviour, male mice pair with C57BL/6 stimulus ovariectomized females. We
perform this surgery under light isoflurane of 10 – 20 minutes. First, we shave their abdominal
area and clean with betadine (Providone-iodine 7.5%). Thereafter, we incise their midline
abdomen region first through the skin then the abdominal muscle. After the ovaries
cauterization, we will suture the muscle and skin. For postoperative pain, we administer
intraperitoneal buprenorphine (0.6mg/kg) immediately after surgery and 24 hours
postoperatively.
After the surgery, the mice house singly for one week then two or three additional
ovariectomized females group together. Prior to sexual pairing with males, we inject estradiol
benzoate (10 μg in 0.1 mL sesame oil) 48 h before mating to induce behavioural oestrus in
stimulus females, followed by progesterone administration (400 μg in 0.05 mL sesame oil) 3–
6 h before mating.
Male sexual behaviour
Once males reach adulthood at 55 days of age, we will give them sexual experience every two
weeks until one month before electrophysiology recording. Each mouse interacts with a
random stimulus female in their home cage overnight.
One month before electrophysiology recording, they undergo weekly sexual behaviour testing.
For this, we place males into Plexiglas arenas (17.8 cm width ×17.8 cm height ×25.4 cm length)
with home cage bedding unchanged for at least 48 hours, and then habituated to the arena for
at least 30 minutes. We conduct all behavioural testing under dim red illumination during the
dark phase of the light cycle. Tailgating that, we place the stimulus females into the arena for
120 min to allow mating. We record this with a video camera and a blinded observer will assess
male sexual behaviour: mounting, intromission and ejaculation. The latency to ejaculate,
defined as the time from the first intromission to ejaculation, measures the extent of sexual
dysfunction.
Neurosurgery and virus injection
We anaesthetize the mice with pentobarbital (80mg/kg) and then place them onto a mouse
stereotaxic instrument. Subsequently, we disinfect the region of the incision with 0.3%
hydrogen peroxide and then make the scalp incision. After, the skull is cleaned with hydrogen
peroxide, followed by a craniotomy based on the coordinates for the upper part of the left
nucleus accumbens shell: 1.5–1.9 mm rostral from the bregma, 1.3mm lateral from the midline,
and 7.3mm below the brain surface.
To inject 500nl virus solution, we use a microsyringe pump to the target region at a rate of 40nl
/ min. Ensuing that, we leave the microcapillary pipette for an additional five minutes before
slowly withdrawn. Above the craniotomy, we will situate a Microdrive with a protruding
optetrode, consisting of four tetrodes and one optical ferrule. The ceramic fibre optic ferrule
houses the optic fibre and then glued together with the rest of the assembly. Next, we lower the
optetrode to 0.7mm above the nucleus accumbens shell and secure this with screws and dental
acrylic. Thereafter, we allow the mice to recover for two weeks and for the adeno-associated
virus to be expressed.
In vivo electrophysiology
For in vivo electrophysiology, the male sexual behaviour testing component mirrors what we
mentioned above, except for the testing time and the additional electrophysiology component.
First, we check the neuron activity for stable signals over 10 minutes. If no signal is found, we
lower the electrode assembly by ~22 – 88 μm and then return the mice to their home cage for
later testing. We are including two phases of five-minute recordings: phase 1 when we place

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the mice into the arena and phase 2 when we place the stimulus female into the arena to interact
sociosexually with the male. Given that the obese mice are sexually dysfunctional, they may
not exhibit the full repertoire of male sexual behaviour. Therefore, we will only investigate the
electrophysiological differences when they interact with the female.
After recording, we will lower the electrode assembly by ~22 – 88 μm through turning the
Microdrive screw so we can record new neurons in the next session. Afterwards, we place the
mice back to their home cages and allow at least four days before the next testing.
For electrophysiology recording, we will use a 16-channel amplifier with built-in bandpass
filters (0.5 – 3.6 kHz) to amplify extracellular spiking signals. To minimize moving artefact,
one of the channels will act as a virtual reference. The analogue signals digitalize at 25 kHz
and collected using Power 1401 digitizer and Spike2 software. We will sort the single units
offline using Spike2 software. In order to remove the interference of torque during movements,
a torque-controlled servomotor will pull a 25-channel commutator.
Optogenetic tagging
To confirm the cell type of the recorded single units, we deploy optogenetic tagging through
the AAV2-EF1a-DIO-ChR2-mCherry virus injected into the nucleus accumbens shell of our
CaMKIIα-Cre mice. We deliver light pulses at 5ms through the optic fibre to evoke spike firing
of ChR2-expressing neurons. We will also lower the intensity to 0.1 or 10 Hz to reduce spike
jittery.
Down the line of data collection, we determine the cell type by calculating different parameters
from the recording. First is the reliability of light-evoked spiking within 10ms from the light
onset. Then we have the correlation coefficient of spike waveform for spontaneous spikes and
evoked spikes (above 0.85). Next, we obtain the latency of triggering spikes after light onset.
We will compare the distribution latencies of light-evoked spikes and the bootstrapped
distribution of latencies of spontaneous spikes to ascertain if light stimulation truly evoked the
spikes. The statistical P-value for this test should be below 0.001 to consider as bona fide.
We must furthermore verify the fibre placement and recording site, plus the viral targeting to
the nucleus accumbens shell. To do this, we anaesthetize the mice to extract the brain and fix
it in 4% paraformaldehyde. After procuring 50 μm cryosections, we will examine them under
a fluorescent microscope for the location of fibre and recording site, in tandem with mCherry
signals as an indicator of the target virus infection.
Spike sorting and classification of neurons
From the Plexon software, we isolate neuron activity into single units based on the waveform
components. These include positive, negative, entire spike amplitudes, spike duration,
amplitude windows immediately prior to and after the initial negative-going peak, and time
until the maximum of positive and negative peaks. In order to check for consistency, we will
compare the superimposed waveforms of the isolated units to the recordings.
By the same token, we will use Plexon software to perform spike sorting by plotting spikes
into two or three-dimensional feature spaces of different waveform features. To ensure
boundaries separate distinctly and the waveform shapes align with action potentials, we will
check each cluster in the feature space. For each isolated cluster, we will construct an inter-
spike interval histogram. With the intention to exclude confounding multiple units, we will
account for a legitimate absolute refractory period of at least 1.0ms. For the purpose of
checking for consistency, we will again compare the superimposed waveforms of the isolated
units to the recordings.
So as to classify neurons, we will use the following parameters: post-spike suppression (the
period before neuron activity return to average firing rate after each action potential), the initial
slope of valley decay of the waveform, valley half decay time of the waveform, and mean firing

7. 7
rate. On top of optogenetic tagging excitatory neurons, this method jointly allows identification
of cholinergic interneurons (putative tonically active neurons) based on their post-spike
suppression >50 ms, and mean firing rate > 2Hz. It commensurately permits identification of
putative fast-spiking interneurons based on their mean firing rate of >2 Hz, the initial slope of
valley decay of the waveform >22, valley half decay time of the waveform > 250us, and post-
spike suppression <50ms. Putative medial spiny projection neurons identify as initial slope of
valley decay of the waveform <22, valley half decay time of the waveform >240us, and post-
spike suppression <50ms. Other neurons categorize as “unclassified”.
Correlation between neuron activity & behaviour
We include several behaviours in this analysis from the onset of chasing, anogenital sniffing,
mounting, and intromission. The subsequent analysis will depend on the prevalence of each
behaviour across the mice as obese mice may not exhibit mounting or intromission during the
recording timeframe. On the grounds of this, we will conduct two types of comparative analysis.
First is to directly compare the spike firing of the recording timeframe regardless of the
behaviour. In the circumstance where identical behaviours are present for direct comparison,
we compare their spike firing specifically to that behaviour. From this data, we will create peri-
event histograms that are aligned to the behaviour or the timeframe. Given that chasing always
precedes another behaviour beforehand, we analyse the chasing in tandem with the behaviour.
In the following diagram, we define the behavioural periods for statistical analysis
(Modified from 21
)
We compare the average firing rates of these periods with the baseline activity through repeated
measures one-way ANOVA followed by Bonferroni post-hoc tests. For significance, we define
this as >1.0 Hz average difference and p<0.05. In terms of neuron response, we classify them
as follows: excitatory if the firing rate increases in at least 1 period, inhibitory if the firing rate
decreased in at least 1 period, both excitatory and inhibitory, or no response. For the purpose
of assessing statistical significance for the ratio of different neuron responses across and within
all neurons, coupled with those between each neuron type, we will conduct Chi-squared tests
(df = 1, p< 0.05).
Periodicity of firing
We will analyse the periodic firing patterns in the 1 – 20 Hz range during different behaviours.
So as to visualize the serial correlation over time of 2s, we will generate an autocorrelation plot.
Furthermore, we will apply a Gaussian filter that focuses on the full width at half maximum,
with the aim to reduce noise. Using the following equation, we can calculate the primary
oscillation frequency:
𝑓(𝑡) = 𝐴 ∙ exp⁡((−
𝑡
𝜎1
)2⁡
∙ cos(2𝜋𝑣(𝑡)) + 𝑂 + 𝐵⁡ ∙ exp((−
𝑡
𝜎2
)2
• 𝐴 ∙ exp⁡((−
𝑡
𝜎1
)2⁡
∙ cos(2𝜋𝑣(𝑡)) : Gabor function

8. 8
• 𝐵⁡ ∙ exp((−
𝑡
𝜎2
)2
: Gaussian function to consider a central modulation of the auto-
correlogram
• O: offset
• t: time
• A, σ1, v: amplitude, decay constant, wave frequency of the Gabor function
• V: frequency of oscillation of a given neuron i.e. wave frequency of the Gabor function
We impose three criteria to elucidate if the neuron oscillates significantly at a given frequency
(v). First, the function regresses with the effective coefficient of A and v (p<0.05). Second, the
σ1 is larger than 1/v*0.8, meaning that the function has at least one satellite peak. Finally, the
number of spikes within the autocorrelogram exceeds 150. We define the frequency ranges of
oscillation as follows: delta (1- 4 Hz), low theta (4 – 7 Hz), high theta (7-12 Hz), low gamma
(40 – 60Hz), high gamma (60 – 80 Hz), and alpha (12 – 20 Hz).
For each frequency range, we analyse the number of total neurons and the number of each
neuron type. This will then inform the patterns of oscillation by using Chi-squared test to assess
the ratio of the neurons for each frequency range, compared to the total number of neurons with
>150 spikes (p<0.05, df = 3). To validate the regression model, we will over and above conduct
residual analysis with a significance level set at > 2.0.
EXPECTED OUTCOME
On the question of how obesity impairs sexual behaviour, we use diet-induced obese mice and
study their electrophysiological responses during sociosexual interaction with hormonally
primed ovariectomized female conspecifics. With the optetrode implantation into the nucleus
accumbens shell, we obtain spiking and oscillation patterns from the inhibitory interneurons:
putative fast-spiking neurons and medium spiny neurons. This couples with optogenetic
tagging to further identify signals from excitatory neurons. We expect aberrations in the
electrophysiological patterns of these neurons. In particular, there would be fewer inhibitory
interneurons to exhibit inhibitory responses and fewer neurons in general that exhibit delta and
high gamma oscillation.
BROADER IMPACT
Treatment for Sexual Dysfunction
Our society does not prioritize sexual dysfunction research over other illnesses such as heart
disease, cancer and respiratory diseases, because it neither causes direct death nor appears
ostensibly, as a tremendous expense to society. With that being said, sexual behaviour
consummates human well-being 36–38
and sexual dysfunction has a dramatic impact on the
quality of life 39
.
A variety of recourses can improve male sexual dysfunction, namely through prostaglandin E1
creams, penis pumps, surgery, and prosthesis 40
. These however only act as adjuvants and
require more radical changes 40
. Apropos to the currently Food and Drug Administration
approved pharmaceuticals for male sexual dysfunction, hitherto, we limit to only
phosphodiesterase 5 inhibitors, whilst others targeting nitric oxide synthase, aromatase and
cytochrome P450 17 displays insufficient efficacy 40
. In response to these, understanding
electrophysiological mechanisms may beget novel therapeutics. Supplementing that, this may
engender grasping the underlying mechanisms of alternative therapies used for sexual
dysfunction such as meditation 41
, diet 42
, and traditional Chinese medicine 43
. Our research
espouses the current efforts in developing cutting edge male sexual dysfunction treatments
including stem cell therapy, platelet-rich plasma, vascular stent, penile transplant and
shockwave therapy 44,45
.

9. 9
Understanding Sexual Diversity
By the same token, the world burgeons with recognition and discussion surrounding sexual
diversity in terms of characteristics (eg effeminate men), gender identities (eg transgender),
relationship paradigms (eg polyamory) and fetishes (eg BDSM) that can vary across time and
context (Gupta, 2012). Figuring out the mechanisms of sexual behaviour manifests as a critical
step forward to begin understanding the intricacy of sexual diversity.
This relates to the current grant on obese-induced sexual dysfunction for several reasons. First,
a sexual orientation factor exists in obesity46,47
, and sexual dysfunction 48–50
. Second, we still
only have a rudimentary knowledge of sexual behaviour, and this grant bestows
electrophysiological understanding that paves the way to extend beyond the heterosexual
binary paradigm. For instance, this can be further investigated in animal models with partner
preference testing, in addition to interaction periods and genital arousal with same-sex
conspecifics51
. These have been elaborated with changes in perinatal and pubertal sex steroid
hormones 51
, and a recent study that used optogenetics to induce male sexual behaviour in
females 52
. Finally, just as gender, religion, spirituality, and ethnicity partake in therapeutic
processes, as evinced with the field of precision medicine 53–55
, a similar inference can be made
with sexual diversity that begets the precision necessary for treating illnesses.
A myriad of efforts exists in this domain, for instance, the search for the genes relating to sexual
diversity using next-generation sequencing from samples of homosexual men 56
and
transgender people 57
. A slightly earlier study has attempted to investigate this from the
perspective of the mother which found the increase in antibodies against neuroligin 4 Y-linked
protein in mothers with subsequent pregnancies, and this correlated with the likelihood of
attaining a homosexual son 58
. On a similar note, neuroimaging studies strive to understand
sexual diversity 59
. Amongst others, these embodied studies on cerebral responses to human
voices, size of interstitial nuclei of the anterior hypothalamus and cortex parameters 60 61
.
Additionally, we accentuate that the current definitions of sexual diversity will continue to
evolve in tandem with recent debates challenging the assumption of homogeneity in sexual
orientation, as supported by studies that suggest instead a paradigm of sexuality continuum 51
.
Albeit, an important step forward necessitates future neuroscience studies to consider the
sexuality demographics, which has long been neglected in the field and hold duties to address
challenges of possible political misinterpretation. Science predominantly conceptualizes
human existence from a heterosexual perspective, resulting in a parochial view of the world.
The current research hopes to offer baby steps towards understanding this labyrinth of human
sexuality.
Sexual Diversity in Society
Coupled with the actual understanding of sexual diversity, by the same token, this research
project raises awareness of the importance of sexual diversity in fields of science, technology,
engineering, and mathematics. We observe emanating forces in this domain such as the 500
Queer Scientists visibility campaign for LGBTQ+ people to act as role models for inspiring
other and next-generation LGBTQ+ individuals to pursue a career in fields of science,
technology, engineering, and mathematics. Unfortunately, this remains largely a lacuna and the
current project espouses this cause. Our research strives to understand the neurological
underpinnings of sexual behaviour and reveals the intricacies of the neurological connectome
involved. This paves ground to inspire other scientists to partake in quasi research, LGBTQ+
to feel recognized, as well as provides a scientific basis for conversations and activisms that
addresses issues of sexual diversity.

10. 10
TIMELINE

11. 11
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BUDGET

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PERSONAL STATEMENT
My research began with my Bachelors in Food Sciences at the University of
Auckland, New Zealand conducting nutrition clinical trials and exploring how nutrition affects
our microRNAs. I also hosted a nationwide weekly live radio show on nutrition and during my
preparation, I stumbled across nutrition professor, Richard Bazinet’s review in November 2014
on how dietary lipids affect the brain. This sparked my fascination to understand how foods
interact with the brain, such as through the gut microbiome, and appetite neurocircuitry
amongst others. With this predilection, I pursued a Masters in University College London, UK,
a top 10 university in the world, for neuroscience training, using transgenic mice to unravel
brain cancer mechanisms. In June 2016, during reading the Nature interviews featuring science
stars of Asia, I came across the neuroscientist, Nancy Ip. Since whether to pursue a PhD
bedevilled me for several years, I sought an opportunity to consult her on this through skype.
She tipped my balance in favour of pursuing a PhD, by affirming its concordance with my
visions of intellectual challenge, elite networking, and societal contributions. However, I
hesitated to commit 6+ years to a PhD before I can foresee a suitable lab. Therefore, in the next
few years, in accordance with my career goals, I took on work positions using transgenic mice
to tackle Alzheimer’s disease at HKUST, Hong Kong and food reward-coding neurons at
Tsinghua, Beijing.
During this period, I started hosting Open Mind events to debate controversial
topics and two major themes anchored deeply within me: climate change and minorities. As I
prepared for these events, reading widely across Aeon, Lapham’s quarterly, the Point etc., I
saw a 2016 interview with the bioethicist, Peter Singer in the Conversation, who accentuated
that over 750 million refugees will emerge as climate change render their countries inhospitable.
This incubus fomented my volunteering efforts to establish green lab practices at HKUST and
help Health in Action increase healthcare access to refugees. Continuing this endeavour in
Toronto, I co-founded SAVYN, an award-winning social start-up that uses neuroscience to
help refugees. Another neglected minority population in neuroscience are LGBTQ+. My
intersectional minority identity as a Taiwanese LGBTQ+ introvert provides a germane lens on
this. In recent years, I have annexed more sexual diversity into my life, for instance by attending
the gender justice movements unconference in Hong Kong in May 2017, and diversity & equity
conference in Toronto in November 2018. In tandem with my own existential search, I came
to UMass Boston for a PhD to study the neuroscience of sexual behaviour. This decision
propelled with the lab being small, collaborative, non-hierarchal and welcomes my sexuality,
in addition to the university awards as a minority-serving institution. Furthermore, I appreciate
the lab to accept my professional commitments and ambitions outside of academia.
This sinuous journey informs my aspiration to use nutrition neuroscience as a
puissant tool to start social ventures that address the intersections between sexual diversity,
sexual dysfunction and climate change. This begins with the neurological understanding of
sexual diversity, and its unique junctions with sexual dysfunctions. Climate relates to this as it
impacts much more detrimentally to sexual minorities. My social ventures will address these
problems through precision neuro-medicine to help us in this climate change inequity. Climate
change also increases the minorities’ sexual abuse (violence, disease transmission etc), and
exacerbates our healthcare inequity (access to contraceptives, hormone treatments etc).
Therefore, my start-ups will additionally devote to increasing sexual minority representation
in climate change policymaking, as well as prioritize our career opportunities to bestow
autonomy and healthcare access. My PhD will provide the techniques, networks, critical
thinking and adaptability that are perforce for realizing this vision. In conjunction with this
grant, I will pursue additional activities to catalyse this process, namely through leveraging
deep learning to study behaviomics by automating sexual behavioural analyses, collaborations
in deploying chemomagnetics as an innovative neuromodulation to manipulate sexual

17. 17
behaviour, and increasing efforts in my video channel to interview minority scientists and
communicate their research to the public.
CONTRIBUTION TO SCIENCE
Red meat has been associated with cancer in epidemiological studies, but these
conditions on the cooking methods. We conducted a clinical trial to determine the postprandial
response to red meat cooked differently. This unravelled differences in plasma levels of
hormones, cytokines, metabolites and trace elements, findings imperative to inform dietary
guidelines. I contributed to the conceptualization, data curation, formal analysis, investigation,
methodology, project administration, visualization, writing original draft and editing.
• U. K. Prodhan, S. Pundir, V. S.-C. Chiang, A. M. Milan, M. P. G. Barnett, G. C. Smith, J.
F. Markworth, S. O. Knowles, D. Cameron-Smith. (2020) Comparable Postprandial Amino
Acid and Gastrointestinal Hormone Responses to Beef Steak Cooked Using Different
Methods: A Randomised Crossover Trial, Nutrients, doi: 10.3390/nu12020380
• M. P. G. Barnett, V. S. C. Chiang, A. M. Milan, S. Pundir, T. A. Walmsley, S. Grant, J. F.
Markworth, S. Y. Quek, P. M. George, D. Cameron-Smith. (2018) Plasma elemental
responses to red meat ingestion in healthy young males and the effect of cooking method,
Eur J Nutr, doi:10.1007/s00394-018-1620-6
• V. S. C. Chiang, S. Y. Quek (2017) The relationship of red meat with cancer: Effects of
thermal processing and related physiological mechanisms, Crit Rev Food Sci Nutr, doi:
10.1080/10408398.2014.967833
• A. Nuora, V. S. C. Chiang, A. M. Milan, M. Tarvainen, S. Pundir, S. Y. Quek, G. C. Smith,
J. F. Markworth, M. Ahotupa, D. Cameron-Smith, K. M. Linderborg (2015) The impact of
beef steak thermal processing on lipid oxidation and postprandial inflammation-related
responses, Food Chem, doi: 10.1016/j.foodchem.2015.03.059
MicroRNAs regulates genes at the post-transcription level and we discovered their roles
in the benefits of nutrition and exercise. By uncovering these molecular mechanisms, we can
develop better health interventions for ageing-related diseases. Given that the lab had no prior
experience with microRNAs, I contributed to establishing all practices and protocols
surrounding microRNAs i.e. the data curation and methodology of the lab’s projects.
• F. Ramzan, C. J. Mitchell, A. M. Milan, W. Schierding, N. Zeng, P. Sharma, S. M. Mitchell,
R. F. D’Souza, S. O. Knowles, N. C. Roy, A. Sjödin, K. Wagner, D. Cameron‐Smith (2019)
Comprehensive Profiling of the Circulatory miRNAome Response to a High Protein Diet
in Elderly Men: A Potential Role in Inflammatory Response Modulation, Mol Nutr Food
Res, doi: 10.1002/mnfr.201800811
• R. F. D’Souza, N. Zeng, V. C. Figueiredo, J. F. Markworth, B. R. Durainayagam, S. M.
Mitchell, A. C. Fanning, S. D. Poppitt, D. Cameron-Smith, C. J. Mitchell (2018) Dairy
Protein Supplementation Modulates the Human Skeletal Muscle microRNA Response to
Lower Limb Immobilization, Mol Nutr Food Res, doi: 10.1002/mnfr.201701028
• R. F. D’Souza, J. S. T. Woodhead, N. Zeng, C. Blenkiron, T. L. Merry, D. Cameron-Smith,
C. J. Mitchell (2018) Circulatory exosomal miRNA following intense exercise is unrelated
to muscle and plasma miRNA abundances, Am J Physiol Endocrinol Metab, doi:
10.1152/ajpendo.00138.2018
• V. S.-C. Chiang (2014) Post-harvest consideration factors for microRNA research in
cellular, tissue, serum and plasma samples, Cell Bio Int, doi: 10.1002/cbin.10346
Alzheimer’s disease affects 10% of elderlies with a financial burden of over $600 billion
USD. We unearthed the gene variants for increased Alzheimer’s risk in the Chinese population.
At the same time, we used mice models to garner mechanistic insights of Alzheimer’s in
relation to microglial phagocytosis and adult neurogenesis. I focused on the fluorescence-

18. 18
activated cell sorting, and blood genomic sample preparation parts of the project, thereby
contributing to the methodology and data curation.
• S. F. Lau, C. Chen, W. Y. Fu, J. Y. Qu, T. H. Cheung, A. K. Y. Fu, N. Y. Ip (2020) IL-33-
PU.1 Transcriptome Reprogramming Drives Functional State Transition and Clearance
Activity of Microglia in Alzheimer’s Disease, Cell Rep, doi:
/10.1016/j.celrep.2020.107530
• Y. T. Su, S. F. Lau, J. P. K. Ip, K. Cheung, T. H. T. Cheung, A. K. Y. Fu, N. Y. Ip (2019)
α2-Chimaerin is essential for neural stem cell homeostasis in mouse adult neurogenesis,
PNAS, doi: 10.1073/pnas.1903891116
• X. Zhou, Y. Chen, K. Y. Mok, T. C. Y. Kwok, V. C. T. Mok, Q. Guo, F. C. Ip, Y. Chen,
N. Mullapudi, P. Giusti-Rodríguez, P. F. Sullivan, J. Hardy, A. K. Y. Fu, Y. Li, N. Y. Ip
(2019) Non-coding variability at the APOE locus contributes to the Alzheimer’s risk, Nat
Commun, doi: 10.1038/s41467-019-10945-z
• X. Zhou, Y. Chen, K. Y. Mok, Q. Zhao, K. Chen, Y. Chen, J. Hardy, Y. Li, A. K. Y. Fu,
Q. Guo, N. Y. Ip (2018) Identification of genetic risk factors in the Chinese population
implicates a role of the immune system in Alzheimer’s disease pathogenesis, PNAS, doi:
10.1073/pnas.1715554115
Memories are essential for our survival including remembering rewards, aversive
experiences, or spatial navigation. In this regard, we vindicated the mechanism of memory to
engage the dorsal raphe nucleus dopamine neurons, and nucleus incertus neuromedin B
neurons. Understanding memory facilitates therapeutic development for dementia, addiction
amongst others. Although my involvement lent towards the lab’s food reward-coding neurons
project, the techniques I deployed similarly benefited memory projects. These encompass virus
construction and packaging, neurosurgery in addition to implants for optogenetics and fibre
photometry. Thereby, I contributed to the methodology and data curation of these parts of the
project.
• L. Lu, Y. Ren, T. Yu, Z. Liu, S. Wang, L. Tan, J. Zeng, Q. Feng, R. Lin, Y. Liu, Q. Guo,
M. Luo (2020) Control of locomotor speed, arousal, and hippocampal theta rhythms by the
nucleus incertus, Nat Commun, doi: 10.1038/s41467-019-14116-y
• R. Lin, J. Liang, R. Wang, T. Yan, Y. Zhou, Y. Liu, Q. Feng, F. Sun, Y. Li, A. Li, H. Gong,
M. Luo (2020) The Raphe Dopamine System Controls the Expression of Incentive Memory,
Neuron, doi: 10.1016/j.neuron.2020.02.009