The Role of Calcium Entry Via Synaptically Activated NMDA Receptors in the Induction of Long-Term Potentiation 20post-synaptic.jpg

The Role of Calcium Entry Via Synaptically Activated NMDA Receptors in the Induction of

Long-Term Potentiation

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The Role of Calcium Entry Via Synaptically Activated NMDA Receptors in the Induction of Long-Term Potentiation

Background:Background: The paper was written in The paper was written in

1993 by David J. Perkel, 1993 by David J. Perkel, Jeffrey J. Petrozzino, Jeffrey J. Petrozzino, Roger A. Nicoll, and John Roger A. Nicoll, and John A. ConnorA. Connor

The influx of Calcium The influx of Calcium through the NMDA-Rthrough the NMDA-Rwas widely accepted as a was widely accepted as a trigger for manytrigger for manyforms of neural plasticityforms of neural plasticity

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Approach

Prior to this paper, there was no experimental evidence that Prior to this paper, there was no experimental evidence that calcium enters post-synaptic cells exclusively through calcium enters post-synaptic cells exclusively through NMDA-R. This is because there are also voltage-sensitive NMDA-R. This is because there are also voltage-sensitive calcium channels on the post-synaptic cell that may calcium channels on the post-synaptic cell that may contribute to calcium influx.contribute to calcium influx.

A steady post-synaptic depolarization eliminated activation A steady post-synaptic depolarization eliminated activation of voltage-sensitive calcium channels and allowed of voltage-sensitive calcium channels and allowed experimenters to see the effects of NMDA-R alone.experimenters to see the effects of NMDA-R alone.

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Approach

While holding the post-synaptic cell at 0mV While holding the post-synaptic cell at 0mV (reversal potential), the experimenters observed (reversal potential), the experimenters observed synaptically induced calcium transients.synaptically induced calcium transients.

The calcium transients were blocked by APV, which The calcium transients were blocked by APV, which is an NMDA-R antagonist.is an NMDA-R antagonist.

To demonstrate further the role of calcium influx To demonstrate further the role of calcium influx through NMDA-R in synaptic plasticity, through NMDA-R in synaptic plasticity, experimenters observed the magnitude of Long experimenters observed the magnitude of Long Term Potentiation (LTP) at different post-synaptic Term Potentiation (LTP) at different post-synaptic membrane potentials.membrane potentials.

Purpose of The Experiment

This study focused on twoThis study focused on tworelated questions concerningrelated questions concerningthe induction of LTP:the induction of LTP:

(1)(1) Can one directly measure the postulated Can one directly measure the postulated entry of Caentry of Ca2+ through the synaptic NMDA through the synaptic NMDA Receptors?Receptors?

(2) Is this Ca(2) Is this Ca2+ entry necessary for LTP? entry necessary for LTP?

METHODSEffect of Steady Depolarization on Intracellular Free Calcium Concentration in a CA3 Pyramidal Cell.

o Hippocampal sections were used from 3-5 week old guinea pigsHippocampal sections were used from 3-5 week old guinea pigso Recordings were carried out with sections submerged beneath a Recordings were carried out with sections submerged beneath a

continuously superfusing bathing mediumcontinuously superfusing bathing mediumo Free cytoplasmic CaFree cytoplasmic Ca2+ concentration was measured by dye-loading concentration was measured by dye-loading

cells with the Cacells with the Ca2+ indicator fura-2 dissolved CsCl using a indicator fura-2 dissolved CsCl using a hyperpolarizing current. (blocks K+ channels & facilitates hyperpolarizing current. (blocks K+ channels & facilitates depolarization)depolarization)

o Intracellular recordings were made using microelectrodes containing Intracellular recordings were made using microelectrodes containing fura-2 and CsClfura-2 and CsCl

o Intracellular recordings were obtained from CA1 and CA3 pyramidal Intracellular recordings were obtained from CA1 and CA3 pyramidal cells. cells.

o CaCa2+ levels were determined radiometrically from acquired levels were determined radiometrically from acquired fluorescence images using excitation illumination at wavelengths of fluorescence images using excitation illumination at wavelengths of 360nm and 880nm360nm and 880nm

o Cells were depolarized to 0mV and observedCells were depolarized to 0mV and observed

RESULTSEffect of Steady Depolarization on Intracellular Free Calcium Concentration in a CA3 Pyramidal Cell.

Figure A-C Shows the Fluorescent Figure A-C Shows the Fluorescent Images of the depolarization of the Images of the depolarization of the cell to 0mV.cell to 0mV.

1A:1A: Normal, resting conditions; Added Normal, resting conditions; Added Fura 2.Fura 2.

1B:1B: The cell was held at -70mV. The cell was held at -70mV. There was a low resting calcium There was a low resting calcium concentration (50-100nM).concentration (50-100nM).

1C:1C: The cell was depolarized to 0mV. The cell was depolarized to 0mV. The Calcium Concentration rose The Calcium Concentration rose due to the constant firing of due to the constant firing of calcium action potentials in the calcium action potentials in the membrane. This figure shows membrane. This figure shows peak calcium concentration levels.peak calcium concentration levels.

David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

METHODSNMDA Receptor-Dependent Calcium Changes in the Apical Dendrites of a CA3 Pyramidal Neuron in Response to Stimulation of Afferent Fibers during Steady Depolarization to 0mV

Same cells as before are usedSame cells as before are used

During stable CaDuring stable Ca2+ concentrations a train of concentrations a train of stimuli of about 50Hz for a duration of 1s was stimuli of about 50Hz for a duration of 1s was given to the afferent fibers in the stratum given to the afferent fibers in the stratum radiatumradiatum

Starting 1-2s before the stimulus and until 5 -10s Starting 1-2s before the stimulus and until 5 -10s afterward, image pairs were collected at afterward, image pairs were collected at approximately 3.5Hzapproximately 3.5Hz

RESULTS NMDA Receptor-Dependent Calcium

Changes in the Apical Dendrites of a CA3 Pyramidal Neuron in Response to Stimulation of Associational/Commissural Fibers during Steady Depolarization to 0mV

Figure BFigure B Shows calcium Shows calcium response to a train of response to a train of synaptic stimuli to synaptic stimuli to associational afferent associational afferent fibers. fibers.

Calcium concentrations Calcium concentrations rose briefly (note yellow rose briefly (note yellow and red regions) and and red regions) and recovered within several recovered within several seconds.seconds.

Figure CFigure C Shows that the Shows that the Calcium concentrations Calcium concentrations returned to resting returned to resting calcium concentrations calcium concentrations (compare FIG A and C).(compare FIG A and C).


METHODSNMDA Receptor-Dependent Calcium Changes in the Apical Dendrites of a CA3 Pyramidal Neuron in Response to Stimulation of Associational/Commissural Fibers during Steady Depolarization to 0mV

Test to see if the rise in CaTest to see if the rise in Ca2+ concentration concentration depended of the activation of NMDA-Rdepended of the activation of NMDA-R

The depolarization and stimuli was The depolarization and stimuli was repeated as before except there was an repeated as before except there was an addition of the NMDA-R antagonist APV to addition of the NMDA-R antagonist APV to the bathing solutionthe bathing solution

RESULTSNMDA Receptor-Dependent Calcium Changes in the Apical Dendrites of a CA3 Pyramidal Neuron in Response to Stimulation of Associational/Commissural Fibers during Steady Depolarization to 0mV

FIGURES D-F Show FIGURES D-F Show Stimulus strains in the Stimulus strains in the presence of APV lead presence of APV lead to little detectable to little detectable change in calcium change in calcium concentrations.concentrations.


METHODSQuantitative Analysis of Regions of the Apical Dendrite of a Different CA3 Neuron Showing Calcium Transients

Selection of another pyramidal cell with a small apical Selection of another pyramidal cell with a small apical dendrite field that showed clear Cadendrite field that showed clear Ca2+ transient transient

Measured the CaMeasured the Ca2+ concentration in that region as a concentration in that region as a function of timefunction of time

Gave tetanic stimulus while the post synaptic cell was Gave tetanic stimulus while the post synaptic cell was held at +10mVheld at +10mV

Quantitatively examined the time course change of CaQuantitatively examined the time course change of Ca2+ concentration in dendritesconcentration in dendrites

RESULTSQuantitative Analysis of Regions of the Apical Dendrite of a Different CA3 Neuron Showing Calcium Transients

The The OPEN SQUARESOPEN SQUARES Show a Show a change in calcium concentration in change in calcium concentration in response to a tetanic stimulation response to a tetanic stimulation (1s, 100Hz) while the postsynaptic (1s, 100Hz) while the postsynaptic cell was held at +10mV.cell was held at +10mV.

TRACE aTRACE a shows the synaptic shows the synaptic response was reversed, indicating response was reversed, indicating that voltage-gated calcium that voltage-gated calcium channels could not have channels could not have contributed substantially to the contributed substantially to the observed rise in intracellular observed rise in intracellular calcium.calcium.

RESULTS:RESULTS: There was a rapid rise in There was a rapid rise incalcium concentration which decayed calcium concentration which decayed back to baseline value over several back to baseline value over several seconds.seconds.



The CLOSED CIRCLES show the The CLOSED CIRCLES show the effect of APV (an NMDA-R effect of APV (an NMDA-R blocker) on the calcium response blocker) on the calcium response to the same stimulus.to the same stimulus.

TRACE b shows the membrane TRACE b shows the membrane

potential did not depolarize during potential did not depolarize during the synaptic stimulation.the synaptic stimulation.

RESULTS: In the presence of RESULTS: In the presence of APV, the synaptic stimulation led APV, the synaptic stimulation led to only a small increase in the to only a small increase in the calcium concentration, which calcium concentration, which measured 5% of the control measured 5% of the control response.response.



The The CLOSED SQUARESCLOSED SQUARES show that the effect of show that the effect of stimulation following a wash of stimulation following a wash of the APV. the APV.

TRACE cTRACE c shows the shows the membrane potential did not membrane potential did not depolarize during the synaptic depolarize during the synaptic stimulation.stimulation.

RESULTS:RESULTS: The effect of APV The effect of APV was partially reversible upon was partially reversible upon removal from the perfusion removal from the perfusion medium, recovering to 37% of the medium, recovering to 37% of the control response.control response.


METHODS Blockade of LTP When Low Frequency Synaptic Stimuli Were Paired with Extreme Depolarization to +70mV

Took advantage of the increased control of the Took advantage of the increased control of the membrane potential available with whole cell recordingmembrane potential available with whole cell recording

During baseline recording period (holding potential at -During baseline recording period (holding potential at -80mV) EPSCs were elicited alternately in two 80mV) EPSCs were elicited alternately in two independent afferent pathways at .1Hzindependent afferent pathways at .1Hz

Stimulation was ceased and the potential was shifted to Stimulation was ceased and the potential was shifted to a test value between -30mV and +100mVa test value between -30mV and +100mV

After holding current stabilized, 50 stimuli were given to After holding current stabilized, 50 stimuli were given to one of the pathways at 0.2-0.7 Hzone of the pathways at 0.2-0.7 Hz

Shifted the membrane potential to the synaptic reversal Shifted the membrane potential to the synaptic reversal potential (approx. +10mV)potential (approx. +10mV)

Second pathway given 50 stimuli with the membrane Second pathway given 50 stimuli with the membrane potential held at the reversal potentialpotential held at the reversal potential

What is Whole-Cell Recording?

Advantages:Advantages: Larger opening at the tip of Larger opening at the tip of

the patch clamp electrode the patch clamp electrode provides lower resistance provides lower resistance and access to the inside of and access to the inside of the cell. the cell.

Disadvantages:Disadvantages: The volume of the The volume of the

electrode is larger than the electrode is larger than the cell, so the soluble contents cell, so the soluble contents of the cell's interior will of the cell's interior will slowly be replaced by the slowly be replaced by the contents of the electrodecontents of the electrode

RESULTS Blockade of LTP When Low Frequency Synaptic Stimuli Were Paired with Extreme Depolarization to +70mV

This is one graph the authors showed, This is one graph the authors showed, but many experiments were donebut many experiments were done

Cell was depolarized to an extreme Cell was depolarized to an extreme potential (+70mV) while the potential (+70mV) while the postsynaptic cell was held at -80mVpostsynaptic cell was held at -80mV

The “test” pathway was stimulated 50 The “test” pathway was stimulated 50 times at 0.5Hztimes at 0.5Hz

The membrane potential was then The membrane potential was then changed to the reversal potential changed to the reversal potential (~10mV)(~10mV)

The “reference” pathway was given the The “reference” pathway was given the same stimulationsame stimulation

Membrane potential returned to -80mV Membrane potential returned to -80mV and alternate stimuli was repeated in and alternate stimuli was repeated in order to assess the presence of LTPorder to assess the presence of LTP

Is Ca2+ influx through NMDAR necessary for LTP induction?

RESULTS Voltage Dependence of Induction of LTP by Pairing Low Frequency Stimulation with Depolarization

The Experiment Logic:The Experiment Logic:

“…“…if Caif Ca2+ entry were necessary, then a entry were necessary, then a

reduced driving force on Ca2+ during reduced driving force on Ca2+ during

activation of NMDA receptors should result activation of NMDA receptors should result

in little or no LTP.”in little or no LTP.”

RESULTS Voltage Dependence of Induction of LTP by Pairing Low Frequency Stimulation with Depolarization

There is a direct There is a direct correlation between correlation between amount of Caamount of Ca2+ influx influx and relative amount and relative amount of potentiation.of potentiation.

Let’s Discuss!!Let’s Discuss!!

Two Key Questions:

1)1) ““Can one directly measure the postulated Can one directly measure the postulated entry of Caentry of Ca2+ through the synaptic NMDA through the synaptic NMDA receptor channel?”receptor channel?”

2)2) ““Is this CaIs this Ca2+ entry a trigger for LTP?” entry a trigger for LTP?”

Measurement of Ca2+ Transients During Synaptic Activation of NMDA Receptors

Observations:Transient increases in the free [Ca2+] of the

dendrites in response to presynaptic stimulation.

Done at 0mV in order to block Ca2+ influx from Voltage sensitive Ca2+ channels.

The changes in [Ca2+] were greatly reduced in the presence of APV

The changes were partially restored after a washout of APV

Measurement of Ca2+ Transients During Synaptic Activation of NMDA Receptors

This data indicates that synaptic stimulation can lead to rises in intracellular [Ca2+] through NMDA channels without a contribution from Voltage sensitive Ca2+ channels.

Various factors allowed the observation

of a synaptically mediated Ca2+ signal:

The use of depolarization to unmask NMDA currents.

Virtually complete removal of Mg2+ blockade of the NMDA receptor when the cell is held at +10mV.

The use of dihydropyridine Ca2+ channel antagonist nimodipine to reduce the steady-state Ca2+ influx through noninactivating Ca2+ channels.

Maintains background Ca2+ levels low.

What Does This Mean?

Synaptic stimulation gives rise to increased intracellular Ca2+

This rise in Ca2+ is directly dependent on Ca2+ influx through NMDA receptors.

Key Question #2:

“Is Ca2+ entry a

trigger for LTP?”

The Voltage Dependence of LTP Induction

What we know: The presence of postsynaptic

Ca2+ buffers blocks LTP.

What we want to know: Whether there is simply a

minimum level of Ca2+ necessary to enable the biochemical process for LTP, OR whether a specific rise in intracellular Ca2+ is necessary.

Voltage Dependence of Induction of LTP by Pairing Low Frequency Stimulation With Depolarization

Results provide evidence for a direct instructive role for Ca2+ in LTP. LTP was blocked at

extreme depolarization (no Ca2+ influx)

Direct correlation between amount of Ca2+ influx to relative amount of potentiation.

CONCLUSION

THE ORIGINAL

QUESTION:Is it possible to measure the entry of Ca2+ through the synaptic NMDA receptor channel, and if so is this Ca2+ entry a trigger for LTP?

NMDA Receptor

http://homepage.psy.utexas.edu/homepage/class/Psy332/Salinas/Neurotransmitters/Slide13.GIF

CONCLUSION

ANSWER: YES! It is possible and…

NMDA mediated Calcium influx is necessary for LTP induction!

EXCEPTIONS/THINGS TO THINK ABOUT

While the almost complete blockade of calcium transients by APV indicates a strong dependence on activation of NMDA-Rs it’s possible that the increase in calcium came from other sources as well.

Example: We do not know to what degree calcium induced calcium release from intracellular stores may have amplified the calcium influx.

EXCEPTIONS/ THINGS TO THINK ABOUT

QUESTION: What about contributions from the What about contributions from the activation of metabotropic glutamate receptors?activation of metabotropic glutamate receptors?

ANSWER: Since APV, which does not affect other receptors, blocked calcium transients by 85%, they concluded that the non-NMDA Ionotropic and metabotropic receptors can only account for a small fraction of the response.

LIMITATIONS

In the imaging study, we have our first limitation: the dendritic spines were difficult to resolve.

In addition, it wasn’t clear if the Calcium levels observed were due to the averages of signals from numerous spines or spillover from spines to dendritic shafts.

LIMITATIONS

Our 2nd limitation arose as a direct result of using the current-clamp method. No precise value for the membrane potential could be

established.

Thus, we couldn’t compare quantitatively the dependence on membrane potential of Calcium entry and LTP induction.

LIMITATIONS

The 3rd limitation involved the validity of time course of the Calcium transients in relation to how long it would take under normal conditions.

Three things must be taken into account:

1. Possible slowing effect due to buffering of Calcium by fura-2

2. Prolonged Calcium entry through altered NMDA receptor kinetics

3. Alteration in activity of sodium/calcium exchanger by depolarization

Questions…

Documents

The Role of Calcium Entry Via Synaptically Activated NMDA Receptors in the Induction of Long-Term Potentiation 20post-synaptic.jpg