Background Long Term Potentiation. EGTA. NMDA Receptors.

Intracellular Injection of EGTA Blocks Induction of Hippocampal Long Term Potentiation

Question Is LTP from Pre or Postsynaptic changes? Paper suggests that LTP is a result of modification of postynaptic neurons and its induction depends on intracellular Ca++ concentration

Experimental Setup Placed electrode into the Stratum radiatum to stimulate Schaffer Collaterals. Second recording electrode was placed in the apical dendritic zone of a CA1 cell. A third electrode was placed in the soma of the CA1 cell.  Some had 4M Potassium acetate (control)  Others had EGTA (experimental)

EGTA Ethylene-glycol-tetra acetic acid. A calcium buffer which acts by chelating free calcium.

Methods Injected EGTA into the Postsynaptic Cell (CA1). Normal baseline readings to test for an EPSP. Series of tetanus to ensure the stability of the neuronal connection between the Schaffer Collaterals and the CA1 cell. Slow firing testing proceeded for minutes to test for LTP.

Results Control – Higher EPSP after the series of tetanus With EGTA – No Change in the EPSP after the series of tetanus

Results Control – Percent change in the average of EPSP amplitudes With EGTA – Percent change in the average of EPSP amplitudes

Effects of EGTA Does EGTA affect resting membrane potential or membrane conductance? If the resting membrane potential or the membrane conductance was affected, then the lack of LTP could be the result of EGTA.

Effects of EGTA on Membrane Potential A baseline was achieved even after the injection of EGTA and was similar to the control. This shows that EGTA has a negligible effect on the resting membrane potential. The remaining 3 columns show the effect of EGTA on the EPSP amplitudes after the train of tetanus.

Paired Pulse Stimulation Used to artificially create an LTP without a tetanus. First they would depolarize the neuron. Then they would stimulate the neuron.

After-hyperpolarization (AHP) The overshooting (hyperpolarization) of the membrane when returning to resting potential after an action potential. The AHP induced by a paired-pulse is theorized to be Ca++ mediated. If a paired-pulse stimulated EGTA neuron is the only neuron that blocks AHP then more evidence that EGTA only effects calcium mediated processes

Results of AHP Control – stimulated by paired-pulse, there is an AHP. With EGTA – stimulated by paired-pulse there is no detectable AHP. Control – stimulated by tetanus, there is an AHP. With EGTA – stimulated by tetanus, there is an AHP.

Post Tetanic Potentiation Stimulation A temporary increase in release of neurotransmiter after a period of high frequency stimulation and lasts a few minutes. This process is not mediated by calcium ions and is not blocked in neurons containing EGTA.

Conclusions Adding EGTA to the postsynaptic neuron blocks LTP. This paper supports the hypothesis that LTP is due to Calcium stimulated modification of the postsynaptic neuron. This paper proposes that EGTA sequesters Calcium that activates enyzmatic machinery required for LTP.

“Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites.” Wade G. Regehr and David W. Tank

Purpose Current models suggest that calcium conductance is the basis of LTP What accounts for the calcium basis of LTP? The study found a transient component at the dendritic areas

Methods Stimulate fibers in the distal region of stratum radiatum Amplitude and reliability of LTP induction increases with increasing stimulus frequency

Microfluorometric Measurements Fura-2  a fluorescent Ca 2+ indicator By using microfluorimetric techniques calcium movement can be visualized The microfluorimetric technique was used to visualize the calcium accumulation in tetanized regions

Resting calcium distribution (no stimulus) Calcium distribution at moderate stimulation (20Hz for 5s) without AP5 Calcium distribution at moderate stimulation (20Hz for 5s) with AP5 Difference of calcium accumulation of trial with AP5 from trial without AP5

Resting calcium distribution (no stimulus) Calcium distribution at high stimulation (100Hz for 1s) without AP5 Calcium distribution at high stimulation (100Hz for 1s) with AP5 Difference of calcium accumulation of trial with AP5 from trial without AP5

After a series of stimulations on the distal apical dendrites, calcium accumulations were measured  Distal apical dendrites Calcium inside the cell with AP5 = nM Calcium inside the cell without AP5 = nM  Basal dendrites Calcium inside the cell with AP5 = nM Calcium inside the cell without AP5 = nM Basically, two types of calcium influx on the postsynaptic dendrites:  AP5 sensitive  AP5 insensitive

Is the calcium accumulation due to proximity of stimulated afferents? Experimental Test… Stimulate afferent fibers intersecting stratum radiatum OR stratum oriens, with and without AP5.

In both instances calcium influx was seen at the points of stimulation. In areas further away from the point of stimulation calcium influx was mainly due to AP5-insensitive current

Stimulate the afferent fibers that intersect the distal apical dendrites. How is AP5 sensitivity related to distance from point of stimulation?

What does this mean? AP5 sensitive NMDA receptor activity is localized to the region where stimulation occurs. Stimulation occurred at PD1 showing a strong AP5 sensitive region. The sensitivity of AP5 decreased with increasing distance from the point of stimulation.

Conclusions Calcium accumulations are divided into two components; AP5 insensitive and AP5 sensitive accumulations. AP5-insensitive - widely distributed over apical and basal regions, and produced in moderate and high frequency tetanus. AP5-sensitive - accumulations of calcium are found at stimulated postsynaptic areas (specificity of synapses) and verifies the NMDA receptor theory of a calcium pool basis for LTP induction at high frequency tetanus.

Credits Invitrogen. Easton, Dextor M. July July 2000 Wade et al “Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites” (June 1990). Lynch et al “Intracellular injections of EGTA block induction of hippocampal long-term potentiation” (1983). “Wikipedia.org – the free encylopedia” Squire et al. Fundamental Neuroscience. New York: Elsevier Science and Technology, Kullmann DM, Perkel DJ, Manabe T, Nicoll RA. Ca2+ entry via postsynaptic voltage-sensitive Ca2+ channels can transiently potentiate excitatory synaptic transmission in the hippocampus. Neuron Dec;9(6): W.G. Regher et al. Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells. Journal of Neuroscience Vol 12: BIPN 148 Lecture Notes. Journal of General Physiology.

Thank you. Any questions?