Biology Reference
In-Depth Information
4. Technical Notes
1. Cutting healthy brain slices:
The health and viability of slices are critical in the success of the
above experiments. Factors that affect the health of slices and
are controllable include the age of animal, slicing and ACSF
solutions, researcher's skill and speed in dissecting and slicing
the brains, and the condition of instruments. Brains need to be
dissected out quickly and gently to avoid ischemia and any
physical damage. If one is not skillful and quick in dissecting
out the brain, it is helpful to submerge the brain in a Petri dish
fi lled with ice-cold ACSF, and fi nish dissecting in ACSF. The
vibration amplitude and cutting speed of a Vibratome should
be adjusted according to the age of rats and region of brain.
Slower cutting speed and larger amplitude should be used for
old brains.
2. When to perform FPR after injuries:
Theoretically, FPR can be applied any time after ischemia or
TBI. The specifi c time for postinjury FPR should be carefully
selected based on the type and severity of injury and, more
importantly, the specifi c questions to be addressed. For example,
TBI suppresses LTP between 2-3 and 48 h after injury (
10, 11
)
while maintenance of LTP can remain disrupted for up to
8 weeks post injury (
12
). With regard to short-term synaptic
plasticity, TBI is found to cause an elevation of PPF during
1-7 days post injury (
13
) but a signifi cant reduction in PPF
and epileptic seizure 7-9 weeks after injury (
6, 14
).
3. Interpretation of paired pulse ratio:
As mentioned earlier, short-term synaptic plasticity is believed
to be due to changes in the probability for neurotransmitter
release from presynaptic terminals. The mechanism of paired
pulse facilitation (PPF) is generally explained by the “residual
Ca
2+
hypothesis”: facilitation of neurotransmitter release is
caused by accumulation of “residual Ca
2+
” in the presynaptic
terminals after stimulation with short interval. In the paired-
pulse protocol, the fi rst AP causes extracellular Ca
2+
to enter
synaptic terminals through voltage-gated Ca
2+
channels. At
short interstimulus intervals, the second AP arrives while residual
Ca
2+
from the fi rst AP still remains in the synaptic terminals,
which causes “residual Ca
2+
” to accumulate, and leads to higher
release probability and facilitation of the second synaptic
response (
5, 15
). Paired pulse ratio is, therefore, a measure of
the probability of neurotransmitter release. Paired pulse depres-
sion (PPD) suggests higher release probability of neurotrans-
mitter because the fi rst AP signifi cantly depletes the readily
releasable pool of neurotransmitter vesicles, whereas PPF
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