Maldi and Metabolomics

Genetic alterations or early environmental challenges typically lead to many neurobiological changes. While it is certainly possible to predict some of these, the complexity of the brain and the neuronal connectivity make it necessary to use special techniques that go beyond the standard “hypothesis-driven” approach. Over the last decades the research field has developed numerous so-called “hypothesis-free” techniques, such as genome wide association studies, RNA sequencing and proteomics. Together with our collaborators Drs Rob Keijzers and Bill Jordan, we are using two of these techniques: Maldi and metabolomics.

Maldi (Matrix Assisted Laser Desorption/Ionization) is a technique that allows the detection and distribution of many different compounds in biological tissues. The idea is that a brain section is coated with a specific matrix (a chemical coating) and subsequently exposed to a laser beam. This laser created heat which vaporises the top layer of both the matrix and the brain section. The resulting ions can then be detected with mass spectroscopy. The great benefit of Maldi for neuroscience is that the laser can be targeted to a very small region (in the order to 50 micrometers). By moving the laser across a brain slice, we can get a detailed map of the distribution of different neurobiological compounds. By adjusting the matrix, we can assess many different components of the brain (neurotransmitters, lipids etc).

Metabolomics refers to the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”. As such it can be considered the final consequence of the genomics -> transcriptomics -> proteomics -> metabolomics process. There are several different methods that allow us to identify the metabolome of a biological sample (such as brain region or blood plasma), including mass spectroscopy, gas chromatography and nuclear magnetic resonance. Each of these methods have their own advantages and disadvantages in terms of ease of sample preparation, sensitivity and data interpretation. We therefore aim to use and compare all three detection methods while assessing the metabolome changes seen in SERT knock-out rats and in rats expose to maternal immune activation.

This project is in part supported by a grant from “Research for Life” (formerly the Wellington Medical Research Foundation).

Near Infrared Spectroscopy

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.

Heart Rate Variability

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 disorders.

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.

The analysis of ultrasonic vocalizations

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 long-term consequences of maternal LPS exposure

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 Neurological Foundation.

The long-term consequences of maternal polyI:C exposure

Polyinosinic:polycytidylic 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 deficits.

After the 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.

SERT & Drug Addiction

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 serotonin.

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.

Long term biochemical changes in SERT compromised rats

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 metabolites.

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.

The SERT & synaptic plasticity

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.

The SERT & Heart Disease

People with psychiatric disorders such as major depression and anxiety disorders are much more likely to also suffer from with heart problems than the general population. Likewise, individuals suffering from heart problems are more likely to develop major depression or anxiety disorders. In other words, there seems to a causal link between depression, anxiety and heart disease.

In this project we investigate the hypothesis that high levels of serotonin (5-HT) early in life may be this causal link. The reasoning behind this is that genetic reductions in the SERT are a vulnerability factor for major depression, anxiety disorders and heart disease and leads to high levels of 5-HT already very early on in life. From studies in rats and mice, we have learned that 5-HT, during development, plays an important role in shaping the structure and function of the brain as well as the heart.

For this project we will change the extracellular levels of 5-HT early in life through pharmacological means and subsequently investigate whether this leads to changes in the body and behaviour. Behaviourally, we will investigate depressive and anxiety-like symptoms. We will also assess changes in heart rate and especially heart rate variability. In addition, we will investigate changes in the structure and functioning of the brain and heart using immunohistochemistry.

This project is supported by a grant from the New Zealand Heart Foundation