An Important Role of Neural Activity-Dependent CaMKIV Signaling in the Consolidation of Long-Term Me...
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An Important Role of Neural Activity-Dependent CaMKIV Signaling in the Consolidation of Long-Term Memory. Hyejin Kang, Linus D. Sun, Coleen M. Atkins, Thomas R. Soderling, Matthew A. Wilson, and Susumu Tonegawa. Pathway. Background. Goal: Assess CaMKIV’s role in memory

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An Important Role of Neural Activity-Dependent CaMKIV Signaling in the Consolidation of Long-Term Memory

Hyejin Kang, Linus D. Sun, Coleen M. Atkins, Thomas R. Soderling, Matthew A. Wilson,

and Susumu Tonegawa


Pathway


Background

  • Goal: Assess CaMKIV’s role in memory

    • Problems with CREB knockouts

      • compensatory increases in other CREB isoforms?

    • Problems with CaMKIV knockouts:

      • Some knockouts showed significant physiological impairment


Definitions

  • Dominant Negative: mutant protein that blocks function of the WT protein by binding either to the WT protein or a protein upstream or downstream of the WT protein in a pathway.

  • C-Fos: An “immediate early gene” dependent on CREB phosphorylation


*Abstract: Biochemical Aspect

In mice with dnCaMKIV limited to the postnatal forebrain:

  • Basic synaptic function and early LTP (E-LTP) were unaffected.

  • CREB phosphorylation and C-Fos expression were reduced, while other protein levels were unaffected.

  • The dnCaMKIV resulted in a deficit in hippocampal late LTP (L-LTP), analogous to the effects of a protein synthesis inhibitor.


Dominant Negative CaMKIV

  • CP: Regulatory domain of CamKII

  • FLAG: epitope tag

  • SV40: Viral DNA which can be differentiated from endogenous DNA

  • dnCaMKIV

    • HMDT308-311DEDD (autoinhibitory domain mutation)

    • L71A (ATP binding site)

    • T196A (phosphorylation site of CAMKK)


Effects of dnCaMKIV

  • CaMKK enhances CaMKIV activity.

  • dnCaMKIV significantly reduces caCaMKIV activity

  • No statistically significant difference between the CREB mutation and dnCaMKIV…but a trend?


Localization of dnCaMKIV to Forebrain

  • C34: name of animal line expressing dnCaMKIV

  • In situ hybridization to SV40 probe showed dnCAMKIV localized to the cerebral cortex, hippocampus, lower striatum, amygdala, and olfactory bulb


dnCaMKIV: present and not affecting basal levels of synaptic proteins

  • Western Blot:

  • Flag: transgene expression in hippocampal extracts

  • dnCaMKIV does not affect CaMKII or Actin levels

  • Basal CREB, MAPK, and pMAPK levels are also unaffected by the transgene (data not shown).


Method for Testing the Effects of Stimulation on Mutants

  • Hippocampal slices are perfused with saline containing either:

    • 90mM KCl (depolarizes membrane: VGCC allow Calcium influx into soma)

    • 100µM Glutamate

  • 30 minutes later:

    • Ser133-phosphorylated CREB (pCREB) and C-fos expression measured by immunoreactivity analysis


  • After Stimulation, PCREB and C-Fos levels are reduced in C34 mutants

    • Basal levels of pCREB and C-Fos are unaffected by dnCaMKIV

    • After stimulation (Glu and KCl), C34 hippocampal slices show a deficit in pCREB and c-Fos expression compared to WT slices.

    • Fosk (which activates the PKA pathway) is not affected by dnCaMKIV: dnCaMKIV effects seem to be limited to the pathway of interest.


    Early LTP (E-LTP) is Normal in C34 Hippocampal Slices

    • Potentiation through theta-burst stimulation

    • Early synaptic changes (e.g. those mediated by CaMKII) do not seem to be affected.


    Long-term LTP (L-LTP) is reduced in dnCaMKIV mutants

    • Potentiation through 4 x tetanic stimulation

    • WT maintained LTP up to 200 minutes while potentiation in C34 continually decayed

    • Decay in C34 potentiation resembles decay in WT potentiation in the presence of anisomycin (a protein synthesis inhibitor)


    Points of Concern

    • CAMKIV is necessary to produce L(Late phase)-LTP in the hippocampus, but what about other brain regions? (Area of future research!)

    • The loss of L-LTP in C34 cannot be directly linked to the effect of dnCaMKIV on CREB phosphorylation (CaMKIV may regulate other transcription machinery).


    Conclusion

    • dnCaMKIV does not affect basic synaptic function or E-LTP.

    • CaMKIV plays a role in the protein synthesis-dependent component of L-LTP.


    Abstract: Behavioral

    • Morris water maze and Fear conditioning

      • Impairment in long-term memory

        • Specifically, the consolidation/retention was affected

      • Short-term memory was intact

        • The acquisition phase appeared unaffected

        • Short-term retention also unaffected


    Morris Water Maze: Hidden Platform

    • Transgenic mice shows longer escape latencies.

    • No latencies differences during the first two days of training, so it is not from motor impairment.


    Morris Water Maze: Fixed visible platform (in a new location)

    • Transgenic and wild-type shows similar latency curves

      • Therefore, latency from hidden platform is not due to swimming speed and fractional periphery occupancy.

    • Wild type swam to the previous location of the platform instead of the new location

    • Transgenic mice swam directly to the new platform


    Morris Water Maze: Fixed visible platform

    • Wild type mice employ spatial memory strategy even in visible platform of Morris water maze.

    • C34 swim directly to the visible platform

      • Rely on cue-platform association strategy instead of spatial memory strategy


    Contextual Fear Conditioning: Context

    • Since Morris water maze requires training over several days, it is likely that spatial memory can be consolidated.

      • Therefore, Contextual fear conditioning is used to test CaMKIV’s involvement in latter phases.

    • dnCaMKIV transgenic

      • Similar levels of freezing 24 hours after training

      • Reduction in context dependent freezing after 7 days


    Cued Fear Conditioning

    • dnCaMKIV transgenic

      • Similar levels of freezing 24 hours after training and 7 days after training

      • Suggest that transgenic mice have selective deficit in the consolidation/retention phase of context-dependent fear memory.


    CREB and Fear Conditioning


    Limitation of Experiment

    • dnCaMKIV was strongly expressed in the hippocampus as well as the cerebral cortex. The deficits in behavior may reflect decreased CaMKIV activity in either or both areas.


    Conclusion

    • The acquisition phase appeared unaffected

    • Short-term retention also unaffected

    • Specifically, the consolidation/retention was affected


    CREB levels (unphosphorylated) are the same in WT and C34


    Input-Output Curves: Synaptic Transmission is Normal in C34

    • Fiber Volley Amplitude: Input (presynaptic)

    • Field EPSP Slope: Output (postsynaptic)


    *Open-field and Plus Maze Test

    • C34 transgenic and Wild-type mice are indistinguishable in …

      • Percent dwell time in open arm

      • Percent entries in open arm

      • Total number of entries in both open and closed arm

    • Suggest that C34 transgenic mice impairment is due to spatial learning and not general emotional defects (reduce or increase in anxiety).


    CaMKIV mutants

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