Synaptic elimination and the complement system in Alzheimer’s disease
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3.4
Behavioural testing
To investigate if the C3 KO mice displayed an abnormal learning ability, we
assessed their aptitude for contextual and cued fear conditioning. Two
paradigms of fear conditioning (brief delay and trace), were tested.
Training was carried out in two different settings (differing in visual
appearance and smell), one setting was safe (no shocks delivered) and one
setting was unsafe (shocks delivered). The shocks were paired with tones. In
the brief delay paradigm the tone and shock were temporally close, whereas
they were more separated in the trace paradigm.
The basic concept is that contextual fear conditioning (using the cues that are
present in the conditioning box throughout the entire conditioning session)
tests hippocampal-dependent learning and memory, while cued fear
conditioning (using the cue that is explicitly paired with the shock, the tone in
this case) tests hippocampal-independent/amygdala-dependent learning and
memory. Animals with hippocampal lesions can still learn cued fear
conditioning, but are impaired on contextual fear conditioning (Otto and
Poon 2006). Cued fear conditioning becomes hippocampal-dependent when
there is a longer delay between the end of the tone and the beginning of the
shock. Hence, memory of the tone-shock association using the brief delay
procedure reflects hippocampal-independent memory, while memory of the
contextual cue-shock association in either procedure and memory of the tone-
shock association in the trace delay procedure reflect hippocampal-dependent
memory.
3.5
Antibody- based assays
The biggest issue with antibody-based assays is the specificity of the
antibodies used. In the present work, all antibodies used were commercially
available and specificity was assured by the manufacturers. No additional
validation was performed.
3.5.1
Immunohistochemistry
In
order
to
corroborate
the
electrophysiological
findings,
immunohistochemical detection of synapses was performed. Thin sections of
hippocampal tissue were made. These were incubated with an antibody
specific for the presynaptic protein VGLUT1, the primary antibody. This was
followed by incubation with a fluorescently labelled antibody against the
primary antibody, thus providing amplification of the signal, while
Jonny Daborg
23
maintaining the specificity of it, and enabling visual detection of presynaptic
terminals. Images were then taken by use of confocal microscopy.
Because of the small size and vast amount of synaptic puncta, an automated
high content analysis of the images was performed (Dragunow 2008) by an
investigator blind to the genotype of the sections.
3.5.2
Western blot
Western blot is a commonly used method to detect a specific protein in a
sample. Relative difference in amounts of the protein can also be estimated.
Briefly, proteins were extracted from freshly dissected hippocampal tissue,
and total protein concentrations were measured. Subsequently, equal amounts
of protein were added to a gel, and under the influence of an electric field the
proteins run down the gel, distributed according to size. Then the proteins
were transferred to a membrane that was incubated with a primary antibody,
followed by incubation with an enzyme linked secondary antibody which
catalyses a reaction producing a chemiluminescent signal which is quantified
and taken as a measure of the protein.
The drawback with western blot is that it does not provide any absolute
concentrations, but when the question posed only regards relative differences,
western blot is a sufficient method.
3.5.3
ELISA
ELISA is an abbreviation for enzyme-linked immunosorbent assay, and that
is exactly what it is. An antibody specific for the protein of interest is
attached to a surface (e.g. well, plate, or beads), the sample containing the
protein to be analysed is added to the surface, and as a consequence of the
specific antibodies attached to this, the protein will be adsorbed to the surface
(immunosorbed). This is followed by washing, thus leaving only the protein
of interest on the surface, then an enzyme linked or biotinylated antibody is
added. In the case of a biotin-conjugated antibody, a secondary streptavidin-
conjugated enzyme is added. Thus the protein to be analysed is sandwiched
between two specific antibodies and each such complex is linked to an
enzyme. In the final step, a substrate for the enzyme is added, and the
reaction is quantified using a set of standard samples containing known
concentrations of the analyte.
An ELISA can be more sensitive than a western blot, and allows for
measurement of absolute concentrations. This is necessary for analysis of all
Synaptic elimination and the complement system in Alzheimer’s disease
24
chemical biomarkers where concentrations are indicative as opposed to mere
presence.
3.6
qPCR
Real time quantitative PCR (qPCR) is a PCR-based technique for quantifying
nucleic acids. In the present thesis it was combined with reverse transcription
PCR, where mRNA is transcribed to its corresponding complementary DNA
(cDNA). The cDNA is then amplified by PCR. Instead of quantifying the
PCR product after the reaction, the PCR product is measured during the
reaction in real time. Here this was accomplished by the presence of a
molecule that fluoresce when it binds to double stranded DNA (SYBR
GREEN), thus when the single-stranded DNA recombines in the PCR cycle.
Quantification is based on comparison with a standard curve, and to correct
for variation between different samples, the signal from the gene of interest is
normalised to that of a stably expressed reference gene.
3.7
Statistics
Most science is based on comparisons, e.g. different experimental settings,
study groups or changes over time. The common way of dealing with the
analysis is to evaluate two hypotheses – the null hypothesis, according to
which the difference between the groups is zero, and the alternative
hypothesis, according to which there is a difference in the material. Normal
variation in combination with chance can result in seemingly substantial
differences between different samples; therefore it is important to estimate
the element of chance. This is usually done by calculating a p-value which is
the probability of the observation occurring while the null hypothesis is true.
Differences are denoted as significant if the p-value fall under a stipulated
cut-off level, usually set to 0.05. Thus, if the p-value is lower than 0.05, the
null hypothesis should be refuted, and the alternative hypothesis accepted.
If the null hypothesis is rejected though no true difference actually exists, a
Type I error has been made (the probability for this to occur is the stipulated
significance level). Conversely, if the null hypothesis is accepted while a true
difference actually exists, a Type II error has been made. To avoid Type II
errors it is necessary to have a large enough sample size in respect to the
variability in the variable investigated. The necessary sample size can be
estimated by a power calculation. Statistical power is the probability of
detecting a true difference, if it exists, hence of not making a Type II error.
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