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Fukuda et al. 2008
Working Memory
Both the medial prefrontal cortex (mPFC) and
hippocampus are implicated in working memory
Using microarray analysis identified various genes that are
differentially expressed between superior temporal gyrus (STG)
and the remaining cerebral cortex.
-Transcription factors enriched in STG
-Cell adhesion molecules and ECM in cortical regions
Used QPCR and In situ hybridization to confirm these genes
Protocadherin17 was highly enriched in focal regions
of the human prefrontal cortex
Cadherins – adhesion molecules
Cadherin superfamily:
Classical Cadherins and Protocadherins
Protocadherins were found in vertebrate and invertebrate
species
This prevalence in a wide range of species suggested that these
cadherins eveolved from an ancient cadherin and were thus
termed "Protocadherins" as the "first cadherins"
Protocadherins are the largest subfamily of cadherins present in
mammals.
They are involved in homophilic adhesion, and also act as
signaling or receptor molecules
Mutations in protocadherin genes and their expression may play
a role in schizophrenia and Usher Syndrome
Animal and behavioral experiments
Animals used:
All tests were performed with male mice that were 10 weeks old
at the start of testing.
4 mice per cage in a room with a 12 h light ⁄ dark cycle
Experimental procedures
Animal and behavioral experiments
Motor function tests
Wire hang test: mouse was placed on lid of a wire cage and then inverted so that
mouse gripped the wire
The latency to fall was recorded with 60 s cutoff time
Open field test
Mice placed in center of the field and total time spent in the center was recorded for2hr
Light/dark transition test
One dark and one bright chamber, mice were placed into the light chamber and
allowed to move freely for 10min between two chambers.
Total number of transitions and total time in light chamber were recorded.
Home-cage activity
Mice were observed for 3 days and total distance traveled during night & day was
recorded
Elevated plus maze
Mice placed in central square facing the closed arm and time spent in open arm was
recorded for 10mins
Porsolt forced swim test
Mice placed in a filled cylinder & their behavior was recorded for 10min, time of immobility
was recorded
Pain test
Mice were placed on hot plate at 550C and latency to paw lick or foot shake was recorded
Contextual and cued fear conditioning
Training day:
Tone for 30s as conditional stimulus followed by 2 sec foot shock as unconditional stimulus
1-2 more tone-shock at 2min interval and then returned to home cage
24hr after: Freezing behavior was measured
1hr after: Mice placed into white chamber and tone was turned on and freezing was recorded
at 3min interval
Morris water maze
4 trials per day for 9 days: Latency and distance traveled to platform, avg. swim speed & time
spent at the perimeter of the pool were recorded
10th day: platform was removed and probe test was conducted
Eight-arm radial maze test
Mice were maintained at 80-85% body weight
After pretraining, maze acquisition trials for 15 days
Following were scored:
Choice of arms
latency to obtain all the pellets
distance travelled
number of arms chosen within first 8 choices
number of working memory errors
Morris Water Maze
Learning and Memory
8-arm Radial Maze
Learning and Memory
•Morris water maze test is the most
popular spatial learning test for
rodents.
•8-arm radial maze is used to a test
for spatial working memory.
•Animals must remember the arms
which they previously visited.
Hot Plate Test
Pain/Analgesia
Elevated Plus Maze
Fear/Anxiety
•The elevated plus maze is a test
for anxiety-like behavior.
•A mouse is placed in the center
of the maze and the number of
entries and amount of time spent
in the open and closed arms are
recorded during a brief trial.
•Hot plate test evaluates the reaction
time of mice dropped on to a heated
surface. Mice are removed from the
apparatus immediately after they
reacted or 15 seconds have elapsed,
so that they dont get hurt.
•A metal hot-plate is heated to a
temperature of 55 degrees C.
Behavioral tests in mice
Role of Pcdh-a A isoform in contextual
fear conditioning and spatial working
memory
Fig1
Fig1
No differences were seen between mutant and wild type in:
Motor tests, wire hanging test, responses in light/dark transitions tests and home cage activity
Mutant displayed anxious or fearful phenotype
as compared to wild type
more time spent in the center of open field and
open arms of elevated maze
Mutant showed different behavior than wild
type
In Porsolt forced swim test [Depression]
Hot plate test [Pain]
Fig2
Mutant showed significant increase in freezing
as compared to wild type during contextual fear
conditioning test
However, mutant was similar to wild type in
cued tests
Contextual fear conditioning requires normal
functioning of both hippocampus and amygdala
Conditioning to tones requires amygdala and not
hippocampus.
DB/ DB did not show any difference from WT in any
of these tests
Enhanced freezing may be due to downregulation of A isoform of
Pcdh a in DBneo/ DBneo
Fig3
No difference was seen between DBneo/ DBneo
and WT during probe test
Similar cognitive impairment
Similar escape latency
Similar swimming speed
Similar time spent at the perimeter
Hence, no abnormalities were seen in the mutant
mice in Morris water maze learning test
Spatial working memory (Eight-arm radial maze test)
Fig4
DBneo/ DBneo mutant mice showed lower
working memory errors than WT for first six
trials
DB/DB showed no difference from WT in eight
arm radial maze test
Thus, suggesting that lower levels of Pcdh-a A
isoform results in disparities in spatial working
memory
Fig. 5
Fig. 5
Fig. 6
DAneo/ DAneo showed more freezing in the first
3 mins of contextual tests and no change in
training and cued tests
Thus, confirming the role of A isoform of Pcdha
in enhanced freezing during contextual fear
conditioning
Fig. 7
Eight arm maze test
DAneo/ DAneo showed lower working memory
errors than WT in the first five trials
All results were similar to DBneo/ DBneo mice
Hence, confirming the role of A isoform of
Pcdha in regulating learning and memory
abilites
Fig. 8
Fig. 8
Looked at levels of noradrenalin, 5-HIAA, 5-HT,
dopamine, 3,4-dihydroxyphenylacetic acid (dopamine
metabolites) or homovanillic acid
(dopaminemetabolites)
No difference was seen in levels of any of the monoamines
in frontal cortex between WT and DBneo/ DBneo , DB/DB
and DAneo/ DAneo
However, there was increase in level of 5-HT in DBneo/ DBneo
and DAneo/ DAneo mice in hippocampus as compared to WT
Thus, suggesting that 5-HT levels in hippocampus are
associated with Pcdh-a A isoform
Pcdh-a A isoform may regulate learning,
working memory and hippocampal 5-HT levels
Pcdh-g null mice die due to increased neuronal
death and decreased synapse formation
Pcdh-g and Pcdh-a A can form a protein
complex on membrane surface and also
colocalize in hippocampus in areas where
synapses are enriched
Protocadherins can bind to integrins
Integrins play a role in regulating synaptic plasticity
in CA1 synapse of hippocampus
Thus, Pcdh-a and integrins may regulate synaptic
plasticity in hippocampus
Thus may play a role in structural and
scaffolding role in neurons
Neurofilament M also forms a protein complex
with NMDA receptor
Plays a role in synaptic plasticity, learning and
memory in hippocampus
Bipolar disorder pathogenesis
One of the alleles of Pcdh-a cluster have been
identified in patients with this disease