Fukuda et al. 2008

1. Down-regulation of protocadherin-a A isoforms in mice changes contextual fear conditioning and spatial workingmemory Fukuda et al. 2008

2. Working Memory Both the medial prefrontal cortex (mPFC) and hippocampus are implicated in working memory

3. 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

4. Protocadherin17 was highly enriched in focal regions of the human prefrontal cortex

5. Cadherins – adhesion molecules

7. Cadherin superfamily: Classical Cadherins and Protocadherins

8. Protocadherins • Protocadherins were found in vertebrate and invertebratespecies • 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 receptormolecules • Mutations in protocadherin genes and their expression may play a role in schizophrenia and Usher Syndrome

9. Animal and behavioral experiments Animals used:

10. 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

11. 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

12. 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

14. Behavioral tests in mice

15. Role of Pcdh-a A isoform in contextual fear conditioning and spatial working memory

16. Fig1

17. Fig1

18. Emotional behaviour of DBneo/DBneo 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

19. Emotional behaviour of DBneo/ DBneo • 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]

20. Contextual fear conditioning test Fig2

21. Contextual fear conditioning test for DBneo/ DBneo • 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 Pcdha in DBneo/ DBneo

22. Spatial learning defects (Morris water Maze) Fig3

23. Spatial learning defects (Morris water Maze) • 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

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

25. Spatial working memory (Eight-arm radial maze test) • 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

26. Lets make a complete Pcdh a A isoform knockout

27. Fig. 5

28. Fig. 5

29. Fig. 6 Learning and memory tests for DAneo/ DAneo

30. Learning and memory tests for DAneo/ DAneo • 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

31. Fig. 7 Eight arm maze test

32. Learning and memory tests for DAneo/ DAneo • 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

33. Levels of monoamines in mutant mice Fig. 8

34. Levels of monoamines in mutant mice Fig. 8

35. Levels of monoamines in mutant mice • 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

36. Discussion • Pcdh-a A isoform may regulate learning, working memory and hippocampal 5-HT levels

37. Pcdh-a A isoform may increase 5-HT levels in hippocampus by generating serotonergic projections from the raphe nucleus to hippocampus

38. Protocadherins and Limbic system • 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

39. Protocadherins may play a role in synapse generation, learning and memory • 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

40. Cytoplasmic tail of Pcdh-a associates with Neurofilament M • 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

41. Protocadherin in diseases • Bipolar disorder pathogenesis • One of the alleles of Pcdh-a cluster have been identified in patients with this disease

42. Thank You !