What are Neurotransmitters?
Neurotransmitters are the biochemical messengers of the central nervous system that play a crucial role in physiological and physical health, specifically for proper brain function. They are responsible for human motor control, perception, and a wide range of cognitive functions such as learning, memory, emotions, sleep, to name just a few. Aside from being involved in physiological function, neurotransmitter research is important because the dysregulation of normal levels of NTs has also been associated with a number of neurodegenerative diseases and psychological conditions. Alzheimer’s Disease, Parkinson’s, Epilepsy, Schizophrenia, Addiction, Depression, and many other conditions affecting the nervous system can be better understood and potentially treated from what is learned using neurotransmitter detection technologies.
For researchers, the difficulty with neurotransmitter detection is that they are present at very low concentrations and some are only present in certain regions of the nervous system. As a result, robust and selective methods have had to be developed to detect these messenger molecules during both in vitro and in vivo experiments. The first neurotransmitter detected was Acetylcholine, discovered in 1921 by a pharmacologist named Otto Loewi. Since this discovery, more than 200 molecules classified as neurotransmitters (NTs) have been identified, with many of their functions still unknown.
Main Neurotransmitters and Classification
Although all NTs contribute to the normal functioning of the nervous system, only a small number are involved in the most common neuropathologies. As a result, the existing detection techniques for neurotransmitter detection are usually optimized for the following NTs:
Classifying Neurotransmitters for Detection
Neurotransmitters are in most cases classified into categories by their function, action, and structure. However, some neurotransmitters can act as both inhibitory and exhibitory, or have multiple actions in different regions of the nervous system. Therefore, they are mostly classified by their biochemical structure (amino acids, biogenic amines, or soluble gases).
Another layer to understanding and studying NTs lies in understanding their electroactive properties – i.e., do they naturally respond to a voltage or to an enzyme?
Some NTs are activated from the action potential generated in a neuron when it is firing, while other neurotransmitters are activated by enzymes. This means that different techniques are needed to detect different types of neurotransmitters depending on whether they have electroactive or non-electroactive properties.
When broken down and classified into these categories of amino acids, amines, or gases with different electrochemical properties, researchers can use a number of different analytical techniques to detect and monitor neurotransmitter levels in both in vivo and in vitro conditions.
This is the basis of how neurotransmitter detection systems are supplied by Green Leaf Scientific work: electrochemical detection of neurotransmitters (FSCV, microdialysis, and HPLC), or by enzyme-based biosensors. These systems work by detecting different properties of the neurotransmitters and making use of the technology to classify the molecule based on its properties.
Different Detection Techniques
Although the detection and monitoring of NTs in the brain has been a significant challenge for scientific researchers, several scientific research companies have developed innovative ways of quantifying neurotransmitters in vivo and in vitro, including Pinnacle Technologies and EICOM.
Compared to non-invasive methods such as PET and SPECT imaging, which are mostly used in humans to identify where NTs are concentrated in the brain, invasive in vivo and vitro techniques can be applied to animal lab research studies to better understand NTs form and functions.
FSCV is a powerful electrochemical technique that is suitable for both in vivo and in vitro applications but is more commonly used during in vivo studies due to its high temporal resolution capabilities. It can detect electroactive NTs such as dopamine, adrenaline, noradrenaline, and serotonin. It uses carbon-based biosensors, which are electroactive by nature.
It involves measuring changes in neurotransmitter concentration as a rapidly cycled voltage flows between two implanted electrodes. Neurotransmitters around or near one of the electrodes are oxidized and the change of state is observed on the acquisition system monitoring the currents. Depending on the neurotransmitter, a different shape is observed over time, multiple scans are combined together to analyze the changes in NT kinetics.
The advantages of FSCV are its low cost, small size, ease of use, fast response time and it is possible to detect two or more analytes simultaneously. Pinnacle’s FSCV system can measure spontaneous sub-second neurotransmitter release while conducting detailed behavioral studies. Both the wireless and tethered systems scan up to 1,000 V/s in a user-selectable range spanning -0.6 to +1.5 V. All systems have built-in support for controlling an external stimulus.
It can also be paired with other in vivo technology such as electrophysiology equipment (EEG or microelectrode arrays) to better study NT relationships with LFPs.
Enzymatic based biosensors, which are non-electroactive in nature, are used to detect enzyme-activated molecules such as Glutamate, as well as other molecules involved in neurotransmitter synthesis such as Glucose, Lactate, and Ethanol. These biosensors can be used for in vitro detection, but their ability to be used in vivo, implanted into specific regions of the brain, is where their strengths lie. When used during in vivo experiments they can be coupled with EEG/EMG systems and optogenetics to correlate the data from the systems.
They have a wide range of applications, including drug screening, identification of biomarkers, and investigating a wide range of cognition functions such as behaviour, learning, memory, sleep, seizures, and stress.
Pinnacle’s biosensors function by the enzyme-mediated processing of the molecule of interest. This enzyme is layered on the surface of the electrode being used, which reacts with the analyte and produces hydrogen peroxide which is detected by the electrode.
The advantage of these biosensors for analyte detection is real-time data with low analyte consumption, high specificity for the analytes of interest, and a fast sample rate and response time. The biggest factor to consider when conducting experiments using these biosensors is that due to the biological nature of the enzymes, the in vivo half-life is a lot shorter than carbon-based biosensors.
EICOM supplies systems for both microdialysis and HPLC for neurotransmitter detection. Combining both methods allows for the detection of a wide number of molecules involved in normal nervous system functioning as well as disease states. Microdialysis was first developed in the 1960s and is one of the most well-established neurotransmitter detection techniques in the field of neuroscience.
The basis of Microdialysis is the continuous measurement of free molecule concentrations in the extracellular tissue of the brain. It involves the minimally invasive insertion of a microdialysis probe into the brain region of interest of a living animal. The probe is semi-permeable, meaning it mimics the composition of the surrounding tissues of the brain which allows the molecules of interest in the extracellular fluid to diffuse according to their gradient. Used alongside HPLC and mass spectrometry, analyzing this diffusion gradient is what allows researchers to detect the molecules.
One major advantage of using microdialysis is the ability to detect inflammatory markers and larger molecules such as Amyloid-beta and Tau proteins, both hallmarks of Alzheimer’s disease. Apart from animal research, it has been used in the human brain to detect acetylcholine, neuropeptides, amino acids, and biogenic amines in the human brain.
HPLC is an analytic chemistry technique widely used in many different branches of scientific research. It involves separating molecules in a sample based on their binding properties to the medium being used. While it can be combined with Microdialysis to detect several molecules from the brain, it is also used in many different fields of scientific research to detect many different molecules based on their chemical structure.
In neuroscience, when paired with mass spectrometry, it can be used to detect different transmitters, including GABA, glutamate, dopamine, serotonin, and acetylcholine. It has also been used for amino acid detection and biogenic amine detection of other neurotransmitters.
EICOM has developed a range of protocols to make the detection of specific molecules in different samples straightforward. A list of their protocols can be found on their website.
Selecting a System – In Vitro or in Vitro?
When choosing the right neurotransmitter detection solution for your lab, there are multiple elements to consider. In vitro studies are suitable for clinical applications, whereas in vivo is more applicable for continuous monitoring to detect the state of disease in a timely manner. However, in vivo often gives rise to more complex electrodes with lower sensitivity and higher cost.
The major advantage of in vivo experiments is understanding how the NTs relate to behaviour and physiological function, which is extremely useful for studying behavioural paradigms, for example, learning and memory. In vitro studies aid researchers in understanding the intrinsic workings of neurotransmitters in their local neuron connections on a microscopic cellular level. However, when using biological samples in vitro, neurotransmitter concentrations are relatively low meaning they require highly selective and sensitive technologies to detect them.
Not sure what solution is best for your lab? Contact us today and we will let you know what product best suits your needs.