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 speciesvertebrateinvertebrate 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. mammals They are involved in homophilic adhesion, and also act as signaling or receptor moleculesadhesionreceptormolecules Mutations in protocadherin genes and their expression may play a role in schizophrenia and Usher SyndromeschizophreniaUsher Syndrome

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 Animal and behavioral experiments Animals used:

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 55 0 C 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 10 th 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 Morris water maze test is the most popular spatial learning test for rodents. 8-arm Radial Maze Learning and Memory 8-arm radial maze is used to a test for spatial working memory. Animals must remember the arms which they previously visited.

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 Pain/Analgesia 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 Behavioral tests in mice Behavioral tests in mice Behavioral tests in mice

Role of Pcdh-  A isoform in contextual fear conditioning and spatial working memory

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.  /  did not show any difference from WT in any of these tests Enhanced freezing may be due to downregulation of A isoform of Pcdh  in  neo /  neo

Fig3

No difference was seen between  neo /  neo 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

Fig4 Spatial working memory (Eight-arm radial maze test)

 neo /  neo mutant mice showed lower working memory errors than WT for first six trials  /  showed no difference from WT in eight arm radial maze test Thus, suggesting that lower levels of Pcdh-  A isoform results in disparities in spatial working memory

Fig. 5

Fig. 6

 neo /  neo 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 Pcdh  in enhanced freezing during contextual fear conditioning

Fig. 7Eight arm maze test

 neo /  neo showed lower working memory errors than WT in the first five trials All results were similar to  neo /  neo mice Hence, confirming the role of A isoform of Pcdh  in regulating learning and memory abilites

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  neo /  neo,  /  and  neo /  neo However, there was increase in level of 5-HT in  neo /  neo and  neo /  neo mice in hippocampus as compared to WT Thus, suggesting that 5-HT levels in hippocampus are associated with Pcdh-  A isoform

Pcdh-  isoform may regulate learning, working memory and hippocampal 5-HT levels

Pcdh-  null mice die due to increased neuronal death and decreased synapse formation Pcdh-  and Pcdh-  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-  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-  cluster have been identified in patients with this disease