Phenotyping Protocols for Pipeline: EUMODIC Pipeline 2 ESLIM_002

Week 9

Open-field ESLIM_007_001 (v.1.0)

The Open Field test is used to assess anxiety and exploratory drive. It is based on the natural tendency of an animal to explore and avoidance reaction to protect itself, which make normal animals to spend more time in the corners and the periphery than in the centre (the most anxiogenic area).

Grip-Strength ESLIM_009_001 (v.1.0)

The grip test is used to measure the maximal muscle strength of forelimbs and combined forelimbs and hind limbs as a primary phenotype screen. In the EMPReSSslim pipeline it is performed directly following the modified SHIRPA. Three trials are carried out consecutively measuring forelimb-strength only, followed by three trials testing consecutively the combined forelimb/hindlimb grip strength.

Modified SHIRPA ESLIM_008_001 (v.1.0)

Use modified SHIRPA for a simple first-line phenotyping screen.

Week 10

Rotarod ESLIM_010_001 (v.1.0)

The rotarod test is used to assess motor coordination and balance in rodents. Mice have to keep their balance on a rotating rod. It is measured the time (latency) ittakes the mouse to fall off the rod rotating at different speeds or under continuous acceleration (e.g. from 4 to 40rpm).

Week 11

Acoustic Startle&PPI ESLIM_011_001 (v.1.0)

An acoustic startle model of sensorimotor gating, in which a weak acoustic stimulus (the pre-pulse) is used to decrease the reflex response (startle) producedby a second, more intense, stimulus (the pulse) in mice. Pre-pulse inhibition (PPI) provides an operational measure of sensorimotor gating which reflects the abilityof an animal to inhibit sensory information properly. Several clinical studies have shown that schizophrenic patients have a reduced PPI. The lack of sufficient sensory gating mechanism is thought to lead to an overflow of the sensory stimulation and disintegration of the cognitive functions. The startle reflex paradigm is therefore largely used to assess the effects of putative anti-psychotics and to explore genetic and neurobiological mechanisms underlying behaviours of relevance to psychosis (Geyer, 1999; Ouagazzal et al., 2001).

Week 12

Hot Plate ESLIM_012_001 (v.1.0)

The hot plate test is used to assess acute pain sensitivity to a thermal stimulus.

Week 13

Clinical Chemistry ESLIM_015_001 (v.1.0)

For the determination of biochemical parameters in plasma including enzymatic activities, specific substrates and electrolytes using an Olympus AU400 analyser (Olympus Diagnostics).

Indirect ophthalmoscopy ESLIM_013_001 (v.1.0)

This test can be used to determine whether a mouse presents with abnormalities of the fundus, such as retinal degeneration, optic disc coloboma, or vascular problems.

Slit Lamp ESLIM_014_001 (v.1.0)

For viewing the eye structures in detail

Haematology ESLIM_016_001 (v.1.0)

For the determination of blood cell counts (white blood cells, red blood cells, platelets), haemoglobin measurement and the calculation of haematological indexes (mean cell volume, mean corpuscular haemoglobin and mean cell haemoglobin concentration). Haematology measurements are obtained using either the Beckman Coulter AcT Diff or Siemens Advia 2120.

Immunoglobulin ESLIM_019_001 (v.1.0)

For the determination of antibodies found in blood.

FACs Analysis ESLIM_018_001 (v.1.0)

The characterisation of of main leukocyte subsets in peripheral blood cells from mice by flow cytometry.Reliable values of frequencies of leukocyte clusters are very much dependent on the appropriate preparation, acquisition and gating of leukocytes. This methodinvolves ammonium chloride erythrocyte lyses, thus preventing interference by larger amounts of erythrocytes. For all stainings, a monoclonal antibody to thecell surface glycoprotein CD45 is used to be able to create a CD45+ gate, allowing discrimination of leukocytes from debris, erythrocytes and thrombocytes. Furthermore, staining with propidium iodide (PI) gates out dead cells. Samples are acquired until a number of 30000 living CD45+ cells is reached for each sample (ref Adler and Busch 2007, submitted). Data are compensated analysed and using FlowJo software.This protocol is part of the EMPReSSslim pipeline and is carried out on a cohort of 10 males and 10 females (age matched) at a time.