Neurotransmitters Analysis Service

What are Neurotransmitters?

Neurotransmitters, also named as chemical transmitter, are a great variety of chemical agents released by neurons to stimulate neighbouring neurons. Through this way, impulses to are transferred from one cell to the next throughout the nervous system. The synapse is the site where neurons meet, consisting of the axon terminal of one cell and the dendrite of the next. A synaptic cleft is a microscopic gap existing between the neurons. When arriving at the axon terminal of one neuron, the nerve impulse would elicit the presynaptic membrane to release a chemical substance. The release of the chemical substance is stimulated by the electrical activity of the neuron. The chemical substance can be transported at extremely high speed to the postsynaptic membrane of the adjoining neuron. It will take only milliseconds for the chemical substance to transfer across the synaptic cleft. The chemical substances like this are called neurotransmitters. The identified neurotransmitters include dopamine, norepinephrine, acetylcholine, and serotonin.

The precursors of neurotransmitters are plentiful and simple such as amino acids and biogenic amines. These precursors are readily available from the diet and only simple biosynthetic steps are needed to convert the precursors to neurotransmitters. Though only one kind of neurotransmitter are produced and released by some neurons, most neurons make and release more than one neurotransmitter at any given time. Because of the coexistence of more than one neurotransmitter in the synapse, it is possible for the cell to exert several influences at the same time. In the axon terminal on the presynaptic side of a synapse, there are some synaptic vesicles clustered beneath the membrane. Neurotransmitters are packaged into these synaptic vesicles. Though low-level baseline release of neurotransmitters occurs without electrical stimulation, peak-level release of neurotransmitters usually is stimulated by an action potential at the synapse or a graded electrical potential.

In recent years, neurotransmitters have been the subject of intensive investigations. For example, because of its effect on alertness, memory and learning, an essential neurotransmitter in the central nervous system, acetylcholine has been widely studied. Several issues must be considered, when select a method for separation and quantification. Since the concentration and the volume of most neurotransmitters in the extracellular space is rather low, the developed analytical method should be able to work with the smallest sample volume and provide detection limits lower than the lowest concentration in the dialysate.

Creative Proteomics for Neurotransmitters Analysis

Creative Proteomics providing an extensive range of specialized services designed to meet the unique needs of our clients. Our neurotransmitter analysis services encompass a variety of detailed and precise approaches, ensuring comprehensive insights into neurotransmitter dynamics. Below are the specific services we offer:

Quantitative Analysis

We provide accurate quantification of neurotransmitters in various biological matrices, which is crucial for understanding their physiological and pathological roles. Our quantitative analysis services include:

• Absolute Quantification: Utilizing standards to provide precise concentration values of neurotransmitters.
• Relative Quantification: Comparing neurotransmitter levels across different samples or conditions.

Profiling Studies

Our profiling studies offer a holistic view of neurotransmitter levels in different biological contexts. This service includes:

• Baseline Profiling: Establishing baseline neurotransmitter levels in normal physiological conditions.
• Disease Profiling: Identifying changes in neurotransmitter levels associated with specific diseases or disorders.
• Temporal Profiling: Monitoring neurotransmitter levels over time to study dynamic changes.

Metabolic Pathway Analysis

Understanding the metabolic pathways of neurotransmitters is vital for elucidating their roles in the body. Our metabolic pathway analysis services include:

• Pathway Mapping: Identifying and mapping metabolic pathways involving neurotransmitters.
• Flux Analysis: Studying the flow of neurotransmitters through metabolic pathways.
• Enzyme Activity Analysis: Measuring the activity of enzymes involved in neurotransmitter metabolism.

Comparative Analysis

We offer comparative analysis services to investigate variations in neurotransmitter levels between different sample sets. This service is essential for:

• Cross-Species Comparisons: Comparing neurotransmitter levels between different species.
• Tissue Comparisons: Analyzing neurotransmitter levels in different tissues or regions of the brain.
• Condition Comparisons: Comparing neurotransmitter levels under different experimental conditions.

Pharmacokinetic Studies

Pharmacokinetic studies are essential for understanding how drugs affect neurotransmitter levels. Our services in this area include:

• Drug Response Analysis: Measuring changes in neurotransmitter levels in response to drug administration.
• Dose-Response Studies: Analyzing how different doses of a drug affect neurotransmitter levels.
• Time-Course Studies: Monitoring neurotransmitter levels over time following drug administration.

Biomarker Discovery

Identifying biomarkers for neurological diseases can facilitate early diagnosis and treatment. Our biomarker discovery services include:

• Biomarker Identification: Detecting potential neurotransmitter biomarkers for various diseases.
• Biomarker Validation: Confirming the reliability and specificity of identified biomarkers.
• Clinical Correlation: Correlating biomarker levels with clinical data to validate their relevance.

Custom Research Projects

We understand that each research project is unique. Therefore, we offer custom research services tailored to your specific needs, including:

• Custom Method Development: Developing bespoke analytical methods to meet your research requirements.
• Specialized Sample Processing: Providing customized sample processing protocols for non-standard samples.
• Collaborative Research: Partnering with you on collaborative research projects to achieve your scientific goals.

List of Detectable Neurotransmitters

Analytical Techniques for Neurotransmitters Analysis

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

LC-MS/MS is a highly sensitive and specific technique for quantifying neurotransmitters in complex biological matrices. It combines the separation capabilities of liquid chromatography with the detection power of mass spectrometry, allowing for:

• High Sensitivity: Detection of neurotransmitters at very low concentrations.
• Specificity: Accurate identification of neurotransmitters and their metabolites.
• Quantification: Absolute and relative quantification of neurotransmitter levels.

Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS is ideal for the analysis of volatile neurotransmitters and metabolic profiling. This technique offers:

• Volatile Compound Analysis: Effective separation and analysis of volatile neurotransmitters.
• High Resolution: Detailed profiling of complex biological samples.
• Quantitative and Qualitative Analysis: Both quantification and identification of neurotransmitter compounds.

High-Performance Liquid Chromatography (HPLC)

HPLC is widely used for separating and quantifying neurotransmitters and their metabolites. Its features include:

• Versatility: Suitable for a wide range of neurotransmitters and metabolites.
• High Precision: Accurate and reproducible quantification.
• Multiple Detection Options: Compatible with various detectors, including UV-Vis and fluorescence.

Capillary Electrophoresis (CE)

CE offers high-resolution separation of neurotransmitter compounds, providing:

• High Efficiency: Rapid and efficient separation of neurotransmitters.
• Small Sample Volumes: Requires minimal sample volumes, preserving precious samples.
• Versatile Detection: Compatible with different detection methods, including laser-induced fluorescence and mass spectrometry.

Fluorescence and UV-Vis Spectroscopy

These techniques are often used as supplementary methods for neurotransmitter analysis, providing:

• Rapid Detection: Quick and straightforward detection of neurotransmitters.
• Non-Destructive Analysis: Minimal sample preparation and preservation of sample integrity.
• Quantitative and Qualitative Data: Both quantification and identification of neurotransmitter compounds.

Sample Requirements for Neurotransmitters Analysis

Report

• A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters.
• Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
• The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

LC–MS/MS analysis of twelve neurotransmitters and amino acids in mouse cerebrospinal fluid

Journal: Journal of Neuroscience Methods

Published: 2020

Background

Neuroactive molecules (NMs) in the cerebrospinal fluid (CSF) play crucial roles in brain function and pathology. While extensive research exists on NM levels in human and rat CSF, data on mouse CSF is sparse. This study aims to fill this gap by introducing a novel method for simultaneous quantification of 12 NMs in mouse CSF using targeted metabolomics services. The method was validated and applied to a diverse group of mice, providing insights into NM levels and their biological relevance.

Technical Methods

Chemicals and Materials: Twelve NMs were obtained from Sigma-Aldrich, including 5-Hydroxytryptamine Hydrochloride, Acetylcholine chloride, l-aspartic acid, and others. Internal standards with isotope labeling were used for accurate quantification. Other chemicals and materials such as formic acid, acetonitrile, and water purification systems were sourced from reputable suppliers.

Preparation of Standard Solutions: Stock solutions (10 mM) of each NM were prepared in water, and a mixed working solution (100 μM) was prepared by dilution with artificial CSF (aCSF). Calibration solutions were further diluted from the working solution and freshly prepared for each analysis.

Animals: Experiments were conducted on C57/Bl6 mice aged 6 to 76 weeks, housed under standard laboratory conditions. Mice were weighed and anesthetized with a ketamine cocktail before CSF collection.

Surgical Procedure and CSF Sampling: CSF was collected from the cisterna magna of euthanized mice using a glass micro-capillary pipette. The surgical site was carefully prepared, and CSF was drawn to avoid blood contamination. Collected samples were centrifuged and inspected for blood contamination before being snap frozen and stored for analysis.

Blood Glucose Measurement: Blood glucose levels were measured using a glucometer after obtaining blood from a tail cut.

Sample Handling and Preparation: Collected CSF samples were diluted with an aqueous solution containing formic acid and internal standards. Samples were vortex mixed, centrifuged, and prepared for LC-MS/MS analysis.

LC-MS/MS Chromatographic Method: The separation was performed using a Waters Acquity UPLC system coupled to a Xevo TQ-MS triple quadrupole mass spectrometer. The eluent system consisted of formic acid and acetonitrile, with a linear gradient used for separation.

MS Parameters: Optimized MS parameters for each NM included cone voltages and collision energies to achieve maximum response. Data was acquired in multiple reaction monitoring (MRM) mode.

Method Validation: The method was validated according to FDA guidelines, testing for linearity, sensitivity, accuracy, precision, carryover, and matrix effect using artificial CSF as the surrogate matrix. Calibration curves were built, and the method's accuracy and precision were confirmed through multiple quality control tests.

Data Analysis: Peak integration and NM quantification were performed using Targetlynx software, with correlation analysis done using MetaboAnalyst software.

Results

NM Detection in Mouse CSF: CSF from 37 mice (14–35 g, 6–76 weeks) was analyzed, and 11 out of 12 NMs were detected in a representative sample from a 9-week-old male mouse. One outlier (sample No. 36) showed unusually high NM levels despite passing visual inspection for blood contamination.

Correlation Analysis: Data for EP, ACh, DA, and MHis were removed for correlation analysis. GABA, Asp, Glu, and Gly levels were highly correlated, and moderately correlated with blood glucose levels, consistent with known metabolic pathways.

Age-related Trends: Age-related trends showed a weak negative correlation of glycine with age (p = 0.03, Spearman r = -0.35). GABA showed a decreasing trend with age, though not statistically significant. The ratio of excitatory to inhibitory NMs remained stable with age.

Comparison with Other Species: Mouse NM data were compared with literature values for rats and humans, showing general consistency, though some differences were noted due to species and methodological variations.

Method Robustness and Sensitivity: The developed method demonstrated high sensitivity and robustness, capable of detecting low concentrations of NMs in mouse CSF. It offers a promising tool for future studies on NM variations across neurological conditions in mouse models.

Overlapped chromatograms of Glycine (left, 76 m/z  → 30 m/z) and Glutamate (right, 148 m/z → 84 m/z) obtained with 0.1% HFBA (in red) and 5 mM NFPA (in black) as ion pairing reagents. The use of HFBA dramatically suppresses the ionization of these analytes.

LC–MS/MS chromatograms of NMs, with the indication of the MRM trace and the absolute area for each peak (A), obtained from a real sample of CSF obtained from a 9-week old male mouse

Reference

1. Blanco, Maria Encarnacion, et al. "LC–MS/MS analysis of twelve neurotransmitters and amino acids in mouse cerebrospinal fluid." Journal of Neuroscience Methods 341 (2020): 108760.

What are the four major types of neurotransmitters?

The four major types of neurotransmitters are:

Acetylcholine (ACh): Responsible for muscle control, learning, and memory.

Biogenic amines (including dopamine, norepinephrine, and serotonin):

• Dopamine: Involved in motivation, reward, and motor control.
• Norepinephrine: Affects alertness and arousal.
• Serotonin: Regulates mood, appetite, and sleep.

Amino acids (including glutamate, GABA, and glycine):

• Glutamate: Acts as an excitatory neurotransmitter, involved in learning and memory.
• Gamma-aminobutyric acid (GABA): Acts as an inhibitory neurotransmitter, regulating anxiety and muscle tone.
• Glycine: Generally inhibitory, involved in motor control and sensory perception.

Neuropeptides: Act as neuromodulators, influencing the activity of other neurotransmitters. Examples include endorphins, which modulate pain perception and mood.

What is each neurotransmitter responsible for?

• Acetylcholine (ACh): Responsible for muscle movement, learning, and memory.
• Dopamine: Regulates motivation, reward processing, and movement.
• Norepinephrine: Affects attention, arousal, and stress response.
• Serotonin: Regulates mood, sleep, appetite, and pain perception.
• Glutamate: Primary excitatory neurotransmitter involved in learning and memory.
• Gamma-aminobutyric acid (GABA): Primary inhibitory neurotransmitter, calming neural activity and reducing anxiety.
• Glycine: Inhibitory neurotransmitter in the spinal cord and brainstem, involved in motor control and sensory processing.
• Neuropeptides (e.g., endorphins): Modulate pain perception, mood, and other physiological processes.

How to balance neurotransmitters?

Balancing neurotransmitters involves several approaches:

• Diet and Nutrition: Consuming a balanced diet rich in essential nutrients like omega-3 fatty acids, vitamins B6 and B12, and magnesium can support neurotransmitter function.
• Exercise: Regular physical activity boosts dopamine and serotonin levels, promoting mood stability and stress reduction.
• Sleep: Adequate sleep supports neurotransmitter replenishment and overall brain health.
• Stress Management: Chronic stress can deplete neurotransmitter levels; practices like meditation, yoga, and deep breathing can help manage stress and balance neurotransmitters.
• Supplements: Under medical supervision, supplements like L-theanine, 5-HTP (precursor to serotonin), and tyrosine (precursor to dopamine) may help rebalance neurotransmitter levels.
• Medication: In cases of neurotransmitter imbalance leading to clinical conditions like depression or anxiety, medications that affect neurotransmitter levels (e.g., SSRIs for serotonin) may be prescribed.