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
Pathway Signaling in the Consolidation of Long-Term Memory


Background
Background Signaling in the Consolidation of Long-Term Memory

  • 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
Definitions Signaling in the Consolidation of Long-Term Memory

  • 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
*Abstract: Biochemical Aspect Signaling in the Consolidation of Long-Term Memory

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
Dominant Negative CaMKIV Signaling in the Consolidation of Long-Term Memory

  • 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
Effects of dnCaMKIV Signaling in the Consolidation of Long-Term Memory

  • 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
Localization of dnCaMKIV to Forebrain Signaling in the Consolidation of Long-Term Memory

  • 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
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
Method for Testing the Effects of Stimulation on Mutants proteins

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

    • 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
    Long-term LTP (L-LTP) is reduced in dnCaMKIV mutants 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
    Points of Concern mutants

    • 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
    Conclusion mutants

    • 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
    Abstract: Behavioral mutants

    • 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 mutants

    • 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
    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
    Morris Water Maze: Fixed visible platform location)

    • 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
    Contextual Fear Conditioning: Context location)

    • 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
    Cued Fear Conditioning location)

    • 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.



    Limitation of Experiment location)

    • 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.


    Conclusion1
    Conclusion location)

    • The acquisition phase appeared unaffected

    • Short-term retention also unaffected

    • Specifically, the consolidation/retention was affected



    Input output curves synaptic transmission is normal in c34
    Input-Output Curves: Synaptic Transmission is Normal in C34 location)

    • Fiber Volley Amplitude: Input (presynaptic)

    • Field EPSP Slope: Output (postsynaptic)


    Open field and plus maze test
    *Open-field and Plus Maze Test location)

    • 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
    CaMKIV mutants location)

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