One of the major technological breakthroughs in human
neuroscience research has been the development of functional magnetic resonance
imaging (fMRI). This technique, for the first time allowed us to measure
changes in blood oxygen levels (and by extension brain activity) while subjects
were performing a specific task. Over the years, this technique has been
improved and refined especially in spatial, which can now be as small as 1 mm.
While structural MRI has also been developed for rats and
mice, fMRI is technically very challenging, mainly because of the risk of
movement artefact. fMRI requires subjects to be completely still, and thus
animals have to be either sedated or anesthetised, which obviously precludes
any behavioural studies. One potential alternative to fMRI is Near Infrared
Spectroscopy (NIRS). NIRS is based on the principle that while most biological
tissues are virtually transparent to infrared light, haemoglobin and oxygenated
haemoglobin both absorb infrared light. Thus like fMRI, NIRS can be used to
measure blood oxygen levels in the brain. Moreover, compared to fMRI, this can
be done at a fraction of the co st.
In the project, we aim to develop a wireless NIRS system to
measure brain blood oxygenation in freely moving animals. This project is a collaboration with Professor
Bor Shyh Lin from the National Chiao Tung University in Taiwan. Prof Lin is
working a miniaturizing a prototype NIRS system that sends data via Bluetooth
to a computer. Within our group, we will test the system in freely moving rats,
which will help us to further refine the system. This project is supported by a
Catalyst: Seeding grant.
One of the main challenges in behavioural neuroscience is to
improve the translational validity of the animal models for the human condition.
The lack of success in the development of new drugs for psychiatric disorders
has led many pharmaceutical companies to abandon research in the area
completely, despite the substantial need for better drugs for mental disorders.
To improve the chances of identifying more successful
psychoactive drugs, we are evaluating the usefulness of heart rate variability
(HRV). HRV refers to the beat-to-beat variation in individual heartbeats. Studies
in healthy volunteers have found that low HRV is typically associated with low
emotional stability and with low cognitive flexibility. As a result of this,
reductions in HRV are often reported in clinical populations, such as patients
suffering from schizophrenia, major depressive disorder or autism spectrum
One of the major benefits of HRV is that it can be assessed
in humans and rat with virtually identical methods. In our rat studies, we use
an implantable probe system which allows continuous recording in freely moving
animals. However, there are many different components to HRV. For instance, HRV
can be assessed using linear methods in either the time or the frequency domain.
While this represents the most often used methodology, they actually are a
simplification of the real process of HRV, which iss more accurately described
as a non-linear system.
In this project, we investigate the neurobiological
mechanisms underlying HRV in order to assess its potential applicability as a biomarker
for specific mental disorders. One of the aspects of the project to investigate
how HRV in rats is affected by specific drugs, or genetic alterations. In
addition to assessing the standard linear parameters, we also investigate
several non-linear methods. This will allow us to investigate the exact
contribution of different neurotransmitter system and brain regions in the
regulation of HRV.
Although rats do make audible sounds, most communication,
particularly between rats takes places at a frequency beyond our human hearing.
These so-called ultrasonic vocalizations (USVs) in rats range from roughly 22
to 90 kHz. Traditionally, USV research has shown that rat calls in the 20 – 25
kHz range are typically associated with negative affect, while calls in the 40
– 90 kHz range are typically associated with positive affect. Rat pups, when
separated from their mothers usually make calls in the 30 – 45 kHz range.
While this subdivision in three bands is still largely
correct, recent research focusses more on the classification of individual calls
within each of these ranges. We aim to investigate the specific language rats
use to communicate their emotional state. For that purpose, we expose rats to
different conditions (play behaviour, anticipatory pleasure, social
interaction, tickling) and record USV. Using Deepsqueak we categorize these
calls into different types to see whether rats use different types of calls
under different circumstances. Additionally, we will investigate whether
pharmacological and/or genetic manipulations alter the USV characteristics. In
the long run, we expect that this will give us a better understanding of the
emotional state of rats.
The brain is undoubtedly the most complex organ in our body
and analysis of its function requires a large variety of specialisms, such as
behavioural analysis, electrophysiological analysis, cellular, molecular and
biochemical techniques. While our research group traditionally focussed on behaviour,
we have also often included immunohistochemical techniques to assess brain
activity and cellular functioning.
In recent years, mainly through the collaboration with Dr
Darren Day from the School of Biological Sciences, we have expanded our portfolio
to include a variety of additional techniques, such as quantitative PCR,
Western blot and primary cell cultures. Recently we also implemented the RNAscope
technique. This technique allows us to assess mRNA and proteins within the same
brain section with high sensitivity and accuracy.
Several of our projects focus around further developing and
assessing the value of specific methodologies.
LPS, lipopolysaccharide, or better lipopolysaccharides are a
group of large molecules consisting of a lipid attached to a polysaccharide.
They are found on the outer cell membrane of Gram-negative bacteria and act as
an endotoxin. It binds to the CD14/TLR4/MD2 receptor complex that can be found
on the membrane of several immune cells (monocytes, dendritic cells,
macrophages and B cells). Like PolyI:C, prenatal LPS, while not crossing the
blood placental barrier, activates the maternal immune system which ultimately
affect the foetus.
We currently have two projects aimed at investigating the long-term consequences of prenatal LPS treatment. In the first project, we specifically investigate how the timing of the maternal immune activation affects behaviour. Specifically, we have exposed pregnant rats to two consecutive injections of LPS on gestational days 10 and 11, 15 and 16 or 18 and 19. We have found that exposure on gestational days 10 and 11 (but not 15/16 or 18/19) leads to cognitive deficits in, among others, working memory capacity and selective attention. Additionally, we found that exposure of gestational days 15 and 16 (but not 10/11 and 18/19) leads to emotional deficits in, among others, reward sensitivity anticipatory pleasure and social interaction.
In the second project, we investigate the epigenetic changes
induced by LPS. Specifically, we aim to perform both a targeted and a genome wide
methylation approach. DNA methylation involves the addition of a methyl group
on specific DNA nucleotides, typically (though not exclusively) cytosines. In
most situations, DNA methylation leads to transcriptional repression, and thus
to a reduction in protein production. By investigating different brain regions
after LPS administration on gestational days 10 and 11 or 15 and 16, we hope to
gain more insight into the neurobiological basis of the cognitive and emotional
deficits induced by prenatal LPS.
This research is in part sponsored by a grant from the
While we know that
most psychiatric disorders have a strong genetic component, we equally know
that none are purely determined by genetic factors alone. Studies in
monozygotic (identical twins) show that if one half of the twin has a mental
disorder, the other has a chance of about 40 to 60% of also developing the
disorder. While this is much higher than the risk in the general population it
is also much less than 100% even though both twins share all of their genes. This
clearly indicates that non-genetic, environmental factors must also contribute
to the development of mental disorders, such as major depression, schizophrenia
and autism spectrum disorders. Unfortunately, environmental factors are much
more difficult to identify than genetic factors. This is in part due to the
fact that environmental factors often affect individuals long before the actual
disorder develops and therefore are often only identified in retrospect.
Genetic factors, on the other hand are in general constant and can be
identified at any point in time.
One of the
environmental factors that has often been implicated in mental disorders is a
viral or bacterial infection during pregnancy. Over the last two decades, multiple
epidemiological studies have linked such infections to the development of autism
spectrum disorder and schizophrenia. However, recent studies have also provided
evidence that such infections can increase the risk of substance abuse
disorders and affective disorders such as anxiety disorder, major depression
and bipolar disorders).
To investigate the long-term
consequences of such infections, we use two different animal models using
prenatal injections of either polyI:C or LPS leading to maternal immune
activation (MIA). The reasons for using these drugs rather than actual
infections, is partly because it is difficult to limit an infection to a single
mother. Additionally, by using drugs we have more control regarding the
duration of the maternal immune response, allowing us to investigate whether
MIA at different points during development leads to different long-term
acid (PolyI:C) is an immunostimulant that simulates viral infections. Like
viruses, it does not see to cross the blood placental barrier but activates the
maternal immune system by stimulating the TLR3 receptor (which is found on
several immune cells, such as B-cells, macrophages and dendritic cells). Components
of the maternal immune system, such as interleukin-6 and others are known to cross
the blood placental barrier to reach the foetal brain. Interleukins are known
to activate microglia (the brain’s immune system) and during development can
cause, what is known as a cytokine storm. This is then thought to subsequently
alter the connectivity between different brain regions as well as neurotransmitter
functioning, such as serotonin.
We currently have
two different projects investigating the long-term consequences of polyI:C, one
focussing on behaviour, one on the neurobiological changes in the brain.
In the behavioural
project, we are investigating whether polyI:C exposure on gestational day 15 leads
to changes in social behaviour throughout the lifetime of the rat. Specifically,
we study changes in maternal separation induced ultrasonic vocalizations, social
approach avoidance and empathy, using a helping behaviour paradigm in which
rats can help a trapped rat escape. In addition, we will investigate whether
environmental enrichment from very early on reverses some of these behavioural
behavioural experiments, the brains of the animals exposed to polyi:C and/or
environmental enrichment will be investigated. In this project, we will be
using a variety of standard immunohistochemical and molecular techniques and
will focus predominantly on oligodendrocytes and synaptic connectivity.
Oligodendrocytes are the glial cells that are principally involved in forming
the myelin sheet around axons of nerve cells and there is some clinical
evidence suggesting that the myelination is altered in for instance
schizophrenia and autism spectrum disorder. Likewise, both disorders have been
associated with altered synaptic connectivity, although interestingly in
opposite directions. Thus, while research has found an increased spine density
in autism spectrum disorder, a decrease has been reported in schizophrenia.
Given that prenatal polyI:C has been used as a model for both disorders, it
will be interesting to see how it affects spine density in rats.
When thinking about addictions, most people tend to think primarily
of dopamine. This is not surprising as dopamine plays a very important role in
reward and all known drugs of abuse increase dopamine release within the
forebrain. However, while dopamine is certainly closely related to the acute
reinforcing effect of addictive substances, other neurotransmitters are
definitely involved as well. In this project we focus primarily on the role of
Genetic studies have indicated that alterations in the SERT transporter
might make individuals more susceptible to the rewarding properties of drugs of
abuse and increase the likelihood of becoming substance abusers. However, from
studies in humans it is difficult to determine the causal relationship between
genetic changes in the SERT and addictive disorders.
Therefore, in this overarching project, we investigate the
effects of addictive substances in rats with a genetic reduction in the SERT. The
project has both a behavioural and a biochemical, molecular component.
Behaviourally we have already shown that rats with a genetic reduction in the
SERT have an increased sensitivity to the rewarding effects of cocaine and MDMA
(the active ingredient of ecstasy). However, they were not more sensitive to
the rewarding effects of heroin. Currently we are looking at the rewarding
properties of alcohol. This will be done both in SERT compromised animals as
well as in their offspring to investigate whether drinking behaviour in the
father affects the sensitivity of the children towards alcohol.
In addition to the behavioural changes induced by addictive
substances we also investigate how such drugs affect the brain. Using an RNA
sequencing technique, we found strong evidence that MDMA changes synaptic
communication between cells, particularly glutamatergic neurotransmission which
takes place on dendritic spines. Using a variety of techniques, such as Western
Blot, quantitative PCR and RNAscope, we are now investigating this further.
Additionally, we are looking at epigenetic changes (such as DNA methylation),
particularly in the offspring of alcohol drinking rats.
These projects have in part been and are funded by grants
from the Neurological Foundation and the Catalyst: Seeding fund and are
performed in close collaboration with Drs Darren Day and Melanie McConnell from
the School of Biological Sciences as well as Prof Tomoaki Shirao from Gunma
University in Japan.
When studying the role of serotonin in mood disorders there
is an obvious paradox. We know that one of the most effective treatment for
depression and anxiety disorders is blocking the SERT through selective
serotonin reuptake inhibitors (SSRIs), leading to an increase in extracellular
5-HT. On the other hand, a genetic reduction in the SERT, which equally leads
to increases in extracellular 5-HT, actually increased the risk of depression
and anxiety disorder.
One possible explanation for this apparent paradox lies in
the timing of the increases in extracellular 5-HT. Thus, in the case of a
genetic reduction in SERT, 5-HT levels are increased already at a very early
age. We know that 5-HT is critically involved in the development of the nervous
system. It is therefore conceivable that the brain (and body) of genetically
compromised SERT animals is fundamentally different from normal (so-called
Wildtype) rats. However, since 5-HT plays such as broad role in development it
is very hard to predict exactly what has changed in the SERT compromised animals.
In this project we therefore take a so-called
“hypothesis-free” approach. So rather than setting a hypothesis a-priori about
what may have changed, we aim to investigate as many changes as we can possible
find. For that we take two different approaches: MALDI-MS and metabolomics. With
MALDI-MS we carefully scan entire brain sections for regional changes in small
molecules such as neurotransmitters and neurotransmitter metabolites. In
metabolomics, we use brain or blood serum samples to investigate many different
This project is in part supported by a grant from the
Wellington Medical Research Foundation and is a collaboration with Drs Robert
Keyzers and Bill Jordan.
While 5-HT is best known for its role in mood, cognition and
reward, it also plays an important role in the development of the central nervous
system. Several studies have found that different serotonin receptors can
affect developmental processes such as axon and dendrite maturation, axon
guidance and spine formation. This latter is very important, as dendritic
spines are essential hubs for neuronal connections, especially excitatory
connections that use glutamate as a neurotransmitter. Moreover, dendritic spine
changes do not only occur during development, they are also a central element
in adult synaptic plasticity.
Serotonin transporter (SERT) knock-out rats have much higher
extracellular levels of 5-HT from very early on. Given its important role in neurodevelopment,
we hypothesize these animals to show changes in neuronal connectivity. In this
project we aim to investigate this with a variety of different techniques, such
as Western Blot, quantitative PCR, immunohistochemistry and RNAscope. We will
also grow neuronal cell cultures as it is easier to visualize dendritic spines
in such culture than in normal brain tissues. To evaluate the changes over time
we will study analyse the brain of young (first two weeks after birth) as well
as adult rat brain tissues.
This project is a collaboration with Dr Darren Day from the
School of Biological Sciences.
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