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.