Fukuda et al. 2008Fukuda et al. 2008

Working MemoryWorking 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 Protocadherin17 was highly enriched in focal regions of the human prefrontal cortexof the human prefrontal cortex

Cadherins – adhesion moleculesCadherins – adhesion molecules

Cadherin superfamily: Cadherin superfamily: Classical Cadherins and ProtocadherinsClassical Cadherins and Protocadherins

Protocadherins were found in vertebrate and invertebrate speciesThis 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 moleculesMutations in protocadherin genes and their expression may play a role in schizophrenia and Usher Syndrome

Animal and behavioral experiments

Animals used:

Experimental proceduresExperimental proceduresAnimal and behavioral experimentsMotor function testsWire hang test: mouse was placed on lid of a wire cage and then inverted so that mouse gripped the wireThe latency to fall was recorded with 60 s cutoff time

Open field testMice placed in center of the field and total time spent in the center was recorded for2hr

Light/dark transition testOne 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 activityMice were observed for 3 days and total distance traveled during night & day wasrecorded

Elevated plus mazeMice placed in central square facing the closed arm and time spent in open arm was recorded for 10mins

Porsolt forced swim testMice placed in a filled cylinder & their behavior was recorded for 10min, time of immobility was recorded

Pain testMice were placed on hot plate at 550C and latency to paw lick or foot shake was recorded

Contextual and cued fear conditioningTraining day: Tone for 30s as conditional stimulus followed by 2 sec foot shock as unconditional stimulus1-2 more tone-shock at 2min interval and then returned to home cage24hr after: Freezing behavior was measured1hr after: Mice placed into white chamber and tone was turned on and freezing was recorded at 3min interval

Morris water maze4 trials per day for 9 days: Latency and distance traveled to platform, avg. swim speed & time spent at the perimeter of the pool were recorded10th day: platform was removed and probe test was conducted

Eight-arm radial maze testMice were maintained at 80-85% body weight After pretraining, maze acquisition trials for 15 daysFollowing were scored: Choice of arms

latency to obtain all the pelletsdistance travellednumber of arms chosen within first 8 choicesnumber of working memory errors

Morris Water MazeLearning and Memory

•Morris water maze test is the most popular spatial learning test for rodents.

8-arm Radial MazeLearning 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 MazeFear/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 TestPain/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

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

memorymemory

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 typemore time spent in the center of open field and open arms of elevated maze

Mutant showed different behavior than wild typeIn 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 testHowever, mutant was similar to wild type in cued tests

Contextual fear conditioning requires normal functioning of both hippocampus and amygdalaConditioning 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 inneo/ neo

Fig3

No difference was seen between neo/ neo and WT during probe test

Similar cognitive impairmentSimilar escape latencySimilar swimming speedSimilar 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 testThus, suggesting that lower levels of Pcdh- A isoform results in disparities in spatial working memory

Fig. 5

Fig. 5

Fig. 6

neo/ neo showed more freezing in the first 3 mins of contextual tests and no change in training and cued testsThus, confirming the role of A isoform of Pcdhin enhanced freezing during contextual fear conditioning

Fig. 7 Eight arm maze test

neo/ neo showed lower working memory errors than WT in the first five trialsAll results were similar to neo/ neo miceHence, confirming the role of A isoform of Pcdhin 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 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 formationPcdh-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 integrinsIntegrins play a role in regulating synaptic plasticity in CA1 synapse of hippocampusThus, Pcdh- and integrins may regulate synaptic plasticity in hippocampus

Thus may play a role in structural and scaffolding role in neuronsNeurofilament 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