Neurochemistry and Behaviour

A promising approach to increase the diagnostic accuracy for dementia diagnosis is the use of biochemical markers (biomarkers) that are present in easily accessible body fluids like serum, plasma or cerebrospinal fluid (CSF).Within the Reference Center for Biological Markers of Memory Disorders, we contribute to the development and characterization of biomarkers, meanwhile developing and validating biomarkers-based diagnostic models that can easily be applied in routine clinical diagnosis.Indeed, several studies have confirmed the relevance of total tau-protein (T-tau) and β-amyloid peptide (Aβ1-42) in the pathogenic processes associated with dementias of neurodegenerative origin.The combined assessment of CSF A β42, T-tau, and tau phosphorylated at threonine 181 (P-tau181P) levels is routinely performed by means of commercially available ELISA kits and was demonstrated to have added diagnostic value for AD diagnosis and to increase specificity for discriminating AD from other (degenerative) dementias. Should you wish to send future CSF samples for biomarker analyses, please fill out the request form. It is moreover important to pay attention to the following guidelines in order to ensure reliable biomarker results (see as well Technical directions and forms):

• Please sample CSF in polypropylene vials
• Minimal sample volume: 2 ml
• CSF samples that are macroscopically hemorrhagic, should be excluded
• Avoid freeze-thaw cycles:
• In case the samples are shipped at room temperature, it should arrive at the lab within 24 hours
• In case the samples are frozen, they should be shipped on dry ice

Please inform your patients that the cost price for these analyses is €165, which is not covered by the regular Belgian insurance companies (refunding is however provided by some supplementary hospitalization insurances).

Behavioural phenotyping is crucially based upon test batteries of behavioural paradigms assessing different brain and behaviour functions. This type of research is often both laborious and time consuming and demands a specific know-how and sophisticated equipment. The Rodent Behavioural Research Unit at the Laboratory of Neurochemistry & Behaviour holds a considerable expertise in the field of behavioural phenotyping of (transgenic) rodent models for human neurological conditions. The Research Unit is fully equipped for behavioural observations in rodents (mice and rats), including the evaluation of pharmacological interventions in specific models. The behavioural observation ranges from evaluation of simple reflexes to the systematic registration of complex learned responses as studied in the Morris water maze.Motor performance, equilibrium and coordination are tested with an accelerating rotarod (Ugo Basile), the wire suspension test, the stationary beam test, and employing a semi-automated analysis of gait patterns. General activity and exploration levels are assessed in an open field arena complemented with a computerized video-tracking system (Ethovision system, Noldus). Latter tracking system is as well employed for recording of social interactive behaviour. Cage activity recording patterns of individually housed animals can be recorded over a period of several days by means of a computerized system.Learning and memory performance can be evaluated in a variety of behavioural paradigms. The Morris water maze combined with the Ethovision video-tracking system assesses hippocampal-dependent visual-spatial memory. In addition, a plus-shaped water maze is available. Passive avoidance learning is screened in a step-through box linked with a small animal shocker. Protocols for evaluation of short and long-term memory can be applied in the various mazes. Conditioning protocols are analysed in Skinner or operant conditioning boxes (Coulbourn Instruments). These paradigms are also employed to study eating and drinking patterns over periods of several days. Fear responses are measured with a contextual fear protocol, as well as with an automated startle reflex system (Med Associates Inc). Anxiety can be quantified employing an elevated plus maze linked to the Ethovision video-tracking system, as well as a dark-light transition box and a holeboard.The research unit has moreover ample experience in behavioural observations related to isolation-induced aggression, male sexual behaviour, depression-related symptoms, vision and olfaction, and epilepsy.

Expertise

The Reversed-Phase Ultra-High-Performance Liquid Chromatography (RP-UHPLC) Unit of the Laboratory of Neurochemistry and Behavior is an analytical task force that holds considerable expertise in the field of neurochemical analysis of different biogenic amines and metabolites in biofluids and brain tissue samples.

More specifically, we quantitatively measure the levels of (nor)adrenaline, dopamine, serotonin and their main metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and, 5-hydroxyindoleacetic acid (5-HIAA)) in serum/plasma/urine/cerebrospinal fluid (CSF) or frozen brain tissue samples of (a) (transgenic) rodent models (preclinical level), or, (b) patients (clinical level).

We primarily focus on miscellaneous neurodegenerative conditions, such as:

• Alzheimer’s disease (AD);
• frontotemporal dementia;
• Parkinson’s disease;
• dementia with Lewy bodies;
• amyotrophic lateral sclerosis;
• multiple sclerosis;

and related conditions, such as Down syndrome.

By applying this technique, for instance, neurotransmitter fluctuations in behaviorally perturbed dementia patients (aggression, depression, other neuropsychiatric symptoms) can be monitored, pharmacological interventions in specific mice models can be evaluated, or, the additional discriminative value of biogenic amines combined with traditional AD biomarkers might be investigated.

Equipment

This research unit is fully equipped with one optimized AlexysTM Dual Monoamines Analyzer with electrochemical detection (Antec Leyden BV, Zoeterwoude, The Netherlands). This device comprises two LC110 pumps, operating at an isocratic flow rate of 40µL/min. Samples (5 µL) are loaded with an AlexysTM AS 100 Autosampler on microbore ALF-125 columns (250 mm × 1 mm, C18, 3  µm particle size) maintained at a constant temperature of 36°C.

The Decade II electrochemical detector is equipped with thin layered electrochemical VT03 flow cells each fitted with a glassy carbon 0.7 mm working electrode and an in situ Ag/AgCl (ISAAC) reference electrode. Integration of chromatograms (41 minutes runtime) is performed with channel integration M018/EN25B Clarity software (DataApex Ltd., Prague,The Czech Republic). We use a fast and simple sample preparation protocol for both serum/plasma/CSF and brain tissue samples.

Our second Alexys Neurotransmitter Analysis device has recently been adapted to a RP-UHPLC setting, using one LC110S pump delivering backpressures up to 700 bar. Separation is achieved by using a short 15cm Waters Acquity Column (BEH C18, 1mm diameter, particle size 1.7µm), delivering optimal performances for monoamine analysis. Total runtime for each sample is under 20 minutes after which all 8 monoamines and metabolites are detected.

Interestingly, both devices can be (relatively easy) adapted or customized for analyses of other neurochemical compounds, such as GABA, glutamate, acetylcholine/choline, glutathione and nitrotyrosine.

Samples

Cerebrospinal fluid (CSF); serum; plasma; brain tissue; urine (of rodent or human origin)

Project examples

“Neurochemical characterization of behavioral disturbances in dementia”

“Unraveling the monoaminergic pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis”

“The noradrenergic system in Down syndrome with/without Alzheimer’s disease: the discriminative role of serum MHPG”

“Defining the prefrontal monoaminergic neurotransmission in frontotemporal dementia compared to Alzheimer’s disease”

“Monoaminergic brain topochemistry in Alzheimer’s disease versus healthy elderly”

“The serotonergic system and ageing”

Selected publications

• Nguyen, A.T., Aerts, T., Van Dam, D., De Deyn, P.P. (2010) Biogenic amines and their metabolites in mouse brain tissue: development, optimization and validation of an analytical HPLC method. J. Chromatogr. B. 878, 3003-3014. 
• Van Dam, D., Vermeiren, Y., Aerts, T., De Deyn, P.P. (2014) Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical detection for the simultaneous and fast determination of eight biogenic amines and metabolites in human brain tissue. J. Chromatogr. A. 1353, 28-39. 
• Vermeiren, Y., Van Dam, D., Aerts, T., Engelborghs, S., De Deyn, P.P. (2014) Monoaminergic neurotransmitter alterations in postmortem brain regions of depressed and aggressive patients with Alzheimer’s disease. Neurobiol. Aging. 35(12): 2691-2700. 
• Vermeiren, Y., Van Dam, D., Aerts, T., Engelborghs, S., Martin, JJ., De Deyn, P.P. (2015) The monoaminergic footprint of depression and psychosis in dementia with Lewy bodies compared to Alzheimer’s disease. Alzheimers Res. Ther. 7:7, DOI 10.1186/s13195-014-0090-1. 
• Dekker, A.D., Coppus, A.M.W., Vermeiren, Y., Aerts, T., van Duijn, C.M., Kremer, B.P., Naudé, P.J.W., Van Dam, D., De Deyn, P.P. (2015) Serum MHPG strongly predicts conversion to Alzheimer’s disease in behaviorally characterized subjects with Down syndrome. J. Alzheimers Dis. 43(3), 871-891.

• Vermeiren, Y., De Deyn, P.P. (2017) Targeting the norepinephrinergic system in Parkinson’s disease and related disorders: the locus coeruleus story. Neurochem. Int. 102: 22-32.

• Dekker, A.D., Vermeiren, Y., Carmona-Iragui, M., Benejam, B., Videla, L., Gelpi, E., Aerts, T., Van Dam, D., Fernández, S., Lleó, A., Videla, S., Sieben, A., Martin, J.-J., Netherlands Brain Bank, Blesa, R., Fortea, J., De Deyn, P.P. (2018) Monoaminergic impairment in Down syndrome with Alzheimer’s disease compared to early-onset Alzheimer’s disease. Alzheimers Dement. (Amst). 10: 99-111.

• van der Zee, S., Vermeiren, Y., Fransen, E., Van Dam, D., Aerts, T., Gerritsen, M.J., Spikman, J.M., van Laar, T., De Deyn, P.P. (2018) Monoaminergic markers across the cognitive spectrum of Lewy body disease. J. Parkinsons Dis. 8(1): 71-84.

• Janssens, J., Vermeiren, Y., Fransen, E., Aerts, T., Van Dam, D., Engelborghs, S., De Deyn, P.P. (2018) Cerebrospinal fluid and serum MHPG improve Alzheimer’s disease versus dementia with Lewy bodies differential diagnosis. Alzheimers Dement. (Amst). 10: 172-181.

• Janssens, J., Atmosoerodjo, S.D., Vermeiren, Y., Absalom, A.R., den Daas, I., De Deyn, P.P. (2019) Sampling issues of cerebrospinal fluid and plasma monoamines: investigation of the circadian rhythm and rostrocaudal concentration gradient. Neurochem. Int., In Press, doi: 10.1016/j.neuint.2019.04.015.