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extracellular field potentials (Andersen 2007). In addition, the hippocampus
is the site in the brain where AD pathology usually first appears, making it
highly relevant to study considering the aims of this thesis.
The acute hippocampal slice preparation was chosen for most of the
electrophysiological studies because it leaves much of the circuitry intact
while still allowing for pharmacological manipulation.
Figure 1.
A typical fEPSP. The first transient deflection seen in this picture (1.) is the
shock artefact generated by the electrical stimulation. The second, smaller deflection
(2.) is the fibre volley which corresponds to the action potentials evoked by the
stimulation. The action potentials will subsequently promote release of transmitter in
the presynaptic terminals, leading to the third larger deflection (3.) which is the
postsynaptic potential.
3.3.2
Extracellular field recordings
The field EPSP (fEPSP) recorded in a hippocampal slice is generated by the
actions of hundreds of neurones, and subsequently tens of thousands of
synapses. The pyramidal neurons in CA1 are organised in one layer with their
apical dendrites projecting perpendicular from that layer. The currents
generated by synaptic activity in these dendrites will flow intracellularly in
the same general direction towards the soma where they will exit and flow
back along the apical dendrites in the extracellular space, thereby generating
an electrical field. Experimentally, such field potentials can easily be evoked
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20
by electrical stimulation of the axons of the pyramidal cells in CA3, called
the Shaffer collaterals. The recorded fEPSP will reflect the events of its
making (fig. 1) and constitutes a great sample of synapses.
The strength in this approach is also its weakness; while many synapses are
sampled and thus provide a robust measurement, it is impossible to
differentiate between different types of synapses. The relevance of such a
differentiation in the present thesis is, however, very limited.
Input/output measurements
Input/output measurements are an excellent and very straight forward way to
measure synaptic efficacy. The size of the fibre volley corresponds to the
number of axons stimulated (Andersen et al. 1978), whereas the fEPSP is the
response from the activated population of synapses. By testing different
stimulation intensities and plotting the magnitude of the fEPSP against that of
the volley a measure of synaptic efficacy per axon can be obtained.
The strength of this method is its simplicity; however, this is also its
weakness, since we cannot know which quantal parameters (i.e. n, p or q) are
changing.
Paired pulse recordings
This type of stimulation paradigm, where two synaptic responses are elicited
in close succession, is commonly used to detect changes in the quantal
parameter p (Branco and Staras 2009). The size of the second pulse in
relation to the first pulse is described by the paired pulse ratio (PPR), and
calculated as the second pulse divided by the first pulse. When the PPR is
larger than 1, the plasticity is called facilitation and taken as a sign of a low
initial p (Zucker and Regehr 2002).
Interpretation of PPR data should be made with caution, however; changes in
release probability as a consequence of changes in vesicle pool size, results in
no or very modest changes in PPR (Hanse and Gustafsson 2001;
Abrahamsson et al. 2005). Moreover, if postsynaptic changes (i.e. changes in
q) occur in a subpopulation of either high- or low-release probability
synapses, the PPR would change, thus falsely hint on a change in p.
Nevertheless, paired pulse recordings are simple, and an excellent way to
probe for changes in release probability.
MK- 801 experiments
MK-801 is an irreversible open channel blocker, specific for the NMDAR.
This can be utilised to estimate release probability in an almost direct manner
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since the rate of decay of the NMDAR EPSP magnitude is dependent on the
presynaptic release probability (Hessler et al. 1993; Wasling et al. 2004).
When a synaptic vesicle is released, the opposing NMDARs open; the
resulting EPSP is recorded, but since MK-801 is present, these NMDARs are
irreversibly blocked, which means that the synaptic NMDARs are turned off
in a release-dependent way. A limitation of this method is that the decay also
depends on the open probability of the individual NMDARs. In this study we
have no information on this, and cannot exclude that there is a difference
between the studied groups. There is, however, no reason to believe that
knockout of exon 24 in C3 should affect the open probability of the
NMDARs.
Burst recordings
Single pulses are very common in electrophysiological experiments;
however, in the hippocampus in vivo, neurons often fire bursts of action
potentials. This is why it can be of importance to investigate how some
physiological phenomena respond to burst stimulation. A problem is that the
EPSPs become distorted by back propagating action potentials, to a large
extent this problem can be overcome by measuring the initial slope of the
EPSP, instead of measuring the amplitude or perhaps the area under the
whole burst response.
3.3.3
Whole cell patch clamp recordings
The patch-clamp technique was invented by Sakmann and Neher in 1976
(Neher and Sakmann 1976). Briefly, the technique is based on establishing a
high resistance seal between a glass pipette containing an electrode, and a
cell, thus enabling control of the cell membrane voltage or current. By setting
the voltage over the membrane, or clamping the membrane potential as it is
normally put, currents flowing over the cell membrane can be measured. In
the present work, this has been utilised in the simplest possible way by
measuring miniature synaptic currents. The major advantage of whole cell
recordings is that they enable a dissection of the quantal parameters.
In the present investigations, field recordings were done when possible, but
to estimate quantal size, patch clamp whole cell recordings were necessary.
Aside from being more technically demanding, whole cell experiments also
carry the disadvantage of being further invasive by disrupting the
intracellular milieu; this have been shown to greatly affect the possibility of
inducing LTP (Malinow and Tsien 1990), but it has most likely not affected
the results presented here.