The function o f CaMKK(3 in learning and memory

Probe trial on day 10: One animal o f each gender and genotype was removed to analyse phosphorylation o f CREB. The remaining animals were over-trained for a further 4 days in order to test if the deficit in spatial learning can be overcome by prolonged training.

When a probe trial was given at the end o f training day 10, male mutants and WT controls spent equal amounts o f time searching in the target quadrant (fig. 3 -14a; mutants: 44.3 ± 6.5 %; WT: 48.6 ± 4.6 %; F ijg = 0.31, p = 0.58). Male mutants searched selectively according to search time = 10.23, p<0.001; p<0.001 for all post-hoc com parisons). M ale m utants also searched selectively according to the proxim ity m easure (fig. 3-14d; F] ,3 6 = 9.62, p<0.001) and according to platform

crossings (fig. 3-14c; p<0.05, ANOVA on ranks). M ale W T controls searched selectively according to search time (F3,4o= 26.71, p<0.001; p<0.001 for all post-hoc

comparisons), proxim ity (F3,4o= 24.32, p<0.001) and platform crossings (p<0.001,

ANOVA on ranks). Thus, male C am kkl null mutant mice can overcome the deficit in

spatial memory form ation i f over-trained. This further supports the earlier notion that

the impairments in spatial learning on day 6 are not due to a performance deficit.

Female mutants and WT controls used a spatial strategy and they spent equal amounts o f time searching in the target quadrant as WT controls (fig. 3 -14b; % time in the target quadrant: mutants 51.0 ± 4.5 %, WT 52.5 ± 5.4 %, Fi,2o= 0.05, p = 0.83; searching

mutants: F3,4o= 28.82, p<0.001, searching WT: F3,4o = 22.77, p<0.001). As with male

mutants, female CamkkP null mutant mice searched selectively according to proximity (mutants: F3,4o = 31.84, p<0.001; WT: F3,4o = 22.78, p<0.001). They also searched

selectively according to platform crossings (mutants: p<0.001; WT: p<0.001, ANOVA on ranks). There was no difference in the time spent searching in the target quadrant between male and female W T controls (Fi,2o= 0.31, p = 0.59), indicating that male and

Chapter 3 : The function o f CaMKKp in learning and memory 120

Retention o f spatial memory: To test whether spatial memory is stable in Camkk2 null mutant mice an additional probe trial was given 7 days after the end o f training at day 10.

Analysis o f searching revealed that male mutants and control littermates spent equal amounts o f time searching in the target quadrant (fig. 3-14e; mutants 41.1 ± 7.0 %, WT 41.4 ± 5.7 %, F]j9<0.01, p = 0.98). Male mutants used a spatial strategy to locate the hidden platform (F3,36 = 6.85, p<0.001, Tukey post-hoc comparison: p<0.001 for TQ vs

AL, p<0.01 for TQ vs OP, p = 0.73 for TQ vs AR). WT control males also searched selectively (F3,4o= 8.48, p<0.001, Tukey post-hoc comparison: p<0.001 for TQ vs AL, p

< 0.01 for TQ vs AR, p<0.05 for TQ vs OP). This was confirmed by analysis o f proxim ity (mutants: p3 ,3 6 = 6.76, p<0.001; WT: p3,4o = 4.89, p<0.01) and platform

crossings (mutants: p<0.01, WT: p<0.01, ANOVA on ranks). Hence, spatial memory acquired by over-training was preserved in male mutants.

Similarly, the time spent searching in the target quadrant did not differ between female mutants and WT controls (fig. 3-14f; mutants 45.3 ± 4.0 %; WT 46.5 ± 4.9 %, Fi,2o =

0.04, p = 0.85). Female mutants and WT mice used a spatial strategy to locate the hidden platform according to the time spent searching in the different quadrants o f the MWM (mutants: F3,4o = 20.36, p<0.001, WT: F3,4o = 13.10, p<0.001; all post-hoc

comparisons p<0.001). They also searched selectively according to proximity (mutants: F3,4o= 13.30, p<0.001; WT: F3,4o= 12.76, p<0.001) and platform crossings (mutants:

p<0.001; WT: p<0.001; ANOVA on ranks). Thus, spatial memory was also preserved in female Camkk2 null mutant mice.

Chapter 3: The function o f CaMKJCP in learning and memory 121

probe trial day 10 male B probe trial day 10 female

«= 30

40

J-uJL

T Q A L O P A R T Q A L O P A R T Q A L O P A R T Q A L O P A R

platform crossings day 10 _D proximity day 10

T O AL O P A R male WT

T Q AL O P A R T Q A L O P A R

male mut female WT

T Q AL O P A R female mut

T Q O P T Q O P T Q O P T Q O P

male WT male mut fem. WT fem, mut

memory probe trial male memory probe trial female

50 40 •I 30 10 50 40 I 30 10 T Q A L O P A R T O A L O P A R T Q A L O P A R T Q A L O P A R

Fig. 3-14 Normal spatial LTM after over-training o f Camkk2 null mutant mice.

A and B) Time spent searching in the quadrants o f the pool during the probe trial on day 10. (C) Platform crossings in the probe trial on day 10.

(D) Proximity to the platform position in TQ and to the equivalent position in the opposite quadrant on day 10. ***: p<0.001 in post-hoc analysis.

E and F) Time spent searching in the pool quadrants in the mem ory probe trial given 7 days after the end o f training.

C h a p te r 3: T h e function o f C a M K K P in learning and m e m o ry 122

3 . 1 4 Im p aired activation of C R E B after spatial learning, a prelim inary study

H ippocam pi w ere rem oved from one male m utant and W T control 30 min after the probe trial on day 6. Phosphorylation o f C R EB was studied in hippocam pal protein extracts and c o m p a re d to levels o f pC R EB in naive anim als (baseline pCREB). This was done as a first step to characterize activation o f C R E B in C am kk2 null mutant mice.

There was no obvious difference in pCR EB in the naive m utant male as com pared to the W T control. A fter training, levels o f pCR EB were increased approximately 2 fold in the trained W T anim al, whereas levels o f pC R E B stayed at baseline in the trained m utant (fig. 3-15). T here was no gross difference in the levels o f total CR EB in the m utant a nd W T h ip p o ca m p u s . A lth o u g h prelim in ary (see discussion), the results suggest that activation o f CREB is impaired in male mutants.

WT mut WT mut

10 5 10 5 10 5 1 0 5 total protein

s. MMtfi -4— anti pCREB blot MM mmm mmüm mww . ^— anti CREB blot male naive male trained

F ig . 3 - 1 5 P h o sph orylation o f CREB in n aive a n d tra in ed m ale C am kkI null m utant mice. left: L e v e ls o f C R E B and p C R E B in on e n aive m ale m utant and W T control.