Autophosphorylation at Thr 286 of the  Calcium- Calmodulin Kinase II in LTP and Learning Giese KP, Fedorov NB, Filipkowski RK, Silva AJ., Science Vol. 279, (1998)

Mechanisms for generating and maintaining LTP

LTP Induction results in transient increases in intracellular [Ca ++ ]

Structure of CaMKII

Regulation States of CaMKII Regulation States of CaMKII

Previous Experiments Pharmacological and genetic lesions of CaMKII impair LTP and learning (2-4) Pharmacological and genetic lesions of CaMKII impair LTP and learning (2-4) Increased active [CaMKII] affects LTP and learning (5-6) Increased active [CaMKII] affects LTP and learning (5-6) Autophosphorylation at Thr 286 promotes switch from CaM-dependent to CaM-independent kinase activity in CaMKII Autophosphorylation at Thr 286 promotes switch from CaM-dependent to CaM-independent kinase activity in CaMKII LTP induction triggers a persistent increase in autophosphorylated form of CaMKII (9,10) LTP induction triggers a persistent increase in autophosphorylated form of CaMKII (9,10)

Investigator’s Question To determine whether autophosphorylation of CaMKII is required for LTP and spatial learning To determine whether autophosphorylation of CaMKII is required for LTP and spatial learning

 CaMKII T B6F2 mutants PCR introduced genetic point mutations T286A and HincII (targeting construct) PCR introduced genetic point mutations T286A and HincII (targeting construct) Mutant Identification using electrophosphoresis Mutant Identification using electrophosphoresis Confirmed normal expression of  CaMKII in somata and dendrites of WT and mutant mice using Immunoblot and Immunocytochemistry Confirmed normal expression of  CaMKII in somata and dendrites of WT and mutant mice using Immunoblot and Immunocytochemistry

Long-term potentiation impairments in the  CaMKII mutant mice Extracellular field recordings in CA1 stratum radiatum region Extracellular field recordings in CA1 stratum radiatum region Tetanus (100Hz/1s) protocol Tetanus (100Hz/1s) protocol Additional protocols: Additional protocols: –10Hz protocol –1Hz protocol –AP5/Tetanus protocol NMDA dependency NMDA dependency Normalized measure of synaptic transmission Normalized measure of synaptic transmission

Pairing-induced LTP deficits in the  CaMKII mutant mice Whole cell recordings in CA1 neurons Whole cell recordings in CA1 neurons Pairing protocol Pairing protocol –Postsynaptic depolarization (+10mV) by injection –Stimulation (2Hz/50s) GABA receptor inhibition blocked with PTX in A GABA receptor inhibition blocked with PTX in A –Removal of GABA inhibition influence in experiment GABA and AMPA blocked in C and D via PTX and CNQX antagonists GABA and AMPA blocked in C and D via PTX and CNQX antagonists –Similar NMDA-R currents and I/V curves

Spatial learning deficits of the  CaMKII mutant mice Morris water maze Morris water maze –Hidden platform version Hippocampus-dependent Hippocampus-dependent Mutants showed impaired learning Mutants showed impaired learning –Visible platform version Hippocampus-independent Hippocampus-independent Mutants demonstrated their ability to “learn” to escape the maze Mutants demonstrated their ability to “learn” to escape the maze Possessed necessary vision, motor skills, and motivation Possessed necessary vision, motor skills, and motivation Transfer Tests Transfer Tests –Mutants didn’t search selectively target quadrant or platform location

Summary of Results Extracellular Field Recordings (Stratum Radiatum Region) Extracellular Field Recordings (Stratum Radiatum Region) –Observed reduced LTP in mutant mice with theta burst and tetanus protocols –Observed no depression of potentiation with low frequency stimulation –Observed loss of potentiation in WT mice in tetanus protocol with addition of AP5—NMDA antagonist –Observed similar synaptic transmission in WT and mutant mice (figure D— normalized fEPSP/fiber volley) Whole Cell Recordings (CA1 Neurons) Whole Cell Recordings (CA1 Neurons) –Observed reduced LTP in mutant mice with pairing protocol that included blocking of GABA inhibition –Observed similar NMDA-R current response with GABA and AMPA antagonist present –Observed similar NMDA-R I/V curves in WT and mutant CA1 neurons Morris Water Mazes Morris Water Mazes –Mutants did not selectively search for the hidden platform –Mutants did not show any somatosensory, motor, or motivational deficits –Mutants were able to “learn” to escape the maze in the visible platform version

Conclusion Autophosphorylation of R286 is required for NMDAR- dependent hippocampal CA1 LTP and hippocampus- dependent spatial learning Autophosphorylation of R286 is required for NMDAR- dependent hippocampal CA1 LTP and hippocampus- dependent spatial learning Observed LTP deficits were not due to: Observed LTP deficits were not due to: –An influence due to GABA inhibition –Differences in synaptic transmission before or during tetanus –Variation in NMDA receptor currents or voltage dependency The deficits observed were probably due to disturbances downstream of the calcium influx through NMDA receptors, such as phosphorylation of glutamate receptor subunits The deficits observed were probably due to disturbances downstream of the calcium influx through NMDA receptors, such as phosphorylation of glutamate receptor subunits