Methods

1
Activation of SK (KCa) ion channels in dorsal CA1
hippocampus impairs object
recognition and delayed fear conditioning Kyle A.
Vick, Michael Guidi and Robert W. Stackman Jr.
Department of Psychology, Florida Atlantic
University, Boca Raton, Florida
428.25
Object Recognition
Fear Conditioning
Introduction Small-conductance Ca2-activated K
(SKCa1-3 KCa2.1-2.3) channels are voltage
insensitive, but open in response to micromolar
increases in intracellular calcium (Pedarzani et
al, 2000). SK channels modulate the encoding of
hippocampal-dependent spatial, contextual and
object memory in mice. Blocking SK channels with
apamin facilitates object memory encoding,
delayed fear conditioning and the induction of
long-term changes in synaptic strength in the
hippocampal slice (Stackman et al, 2002 Hammond
et al, 2006). The chemical compound
1-ethyl-2-benzimidazolinone (1-EBIO) selectively
stimulates the activation of SK channels by
increasing the channel sensitivity to
intracellular calcium.
Day 12 Arena Habituation, mock infusion Day 3
Sample, infusion with 1-EBIO or vehicle Day 4
Test, infusion with vehicle
Day 1 Pre-exposure, infusion with vehicle Day 2
Conditioning, infusion with 1-EBIO or vehicle Day
3 Context Tone, infusion with vehicle
Methods Animals All subjects were naïve 8- to
12-week-old C57BL/6N mice. Control animals, may
not have been naïve, and were not subject to any
prior pharmacological manipulations. Animals were
housed in a temperature and humidity controlled
environment with ad libitum access to food and
water and kept on a 12 hour light/dark cycle.
Male mice were used in all experiments. Surgery
and Procedure Twenty-four animals were
introduced into a stereotaxic device.
Intracranial guide holes were drilled bilaterally
through the skull above the CA1 region of the
dorsal hippocampus. All coordinates measured
2.000 mm posterior to bregma and 1.500 mm
lateral to the midline. Bilateral guide cannulae
inserted 0.5 mm ventral to the cortical surface.
All animals were given 7-10 days to recover
before any experimentation took place. Prior to
any behavioral tasks, all mice were handled for
at least two days. In addition animals were
habituated to the infusion protocol using dummy
injectors which were shorter than the normal
injectors to prevent mechanical damage to the
tissue. Object Recognition Animals were
habituated to the object recognition arena for 10
minutes a day for two consecutive days. During
the sample day two identical objects were placed
in opposing corners of the arena and all animals
received infusions of either 1-EBIO or vehicle
(dimethyl sulfoxide, DMSO). Time spent exploring
the objects was recorded and mice failing to
amass 38 seconds of exploration of either
familiar object were excluded from the study. A
five minute test session was held after a 24 hour
delay, with a novel object in place of one
familiar object. Delay Fear Conditioning During
the first day animals were infused with vehicle,
placed in the chamber for five minutes and
allowed free exploration. During the second day
mice were infused with either 1-EBIO or vehicle
and placed in the same chamber. After a one
minute delay a 90 dB tone was emitted for 30
seconds which coterminated with a one second 0.5
mA footshock. The tone footshock pairings
occurred three times each separated with a 120
second delay. Freezing was measured with
computational software and defined as the lack of
activity, other than respiration, which lasted
for longer than 0.6 seconds. Twenty-four hours
later all mice were given 1-EBIO and contextual
freezing measured by returning animals to the
same chamber for five minutes. Tone familiarity
was tested by placing the animals in a different
and disguised chamber, emitting the same tone
twice and measuring freezing.
Results
Results
Discussion
Acknowledgements
National Science Foundation Grant Number 0630522