1. The Role of Calcium Entry Via Synaptically Activated NMDA Receptors in the Induction of Long-Term Potentiation http://www.georgiapainphysicians.com/downloads/m1_slides/9.%20post-synaptic.jpg

2. The Role of Calcium Entry Via Synaptically Activated NMDA Receptors in the Induction of Long-Term Potentiation Background: The paper was written in 1993 by David J. Perkel, Jeffrey J. Petrozzino, Roger A. Nicoll, and John A. Connor The paper was written in 1993 by David J. Perkel, Jeffrey J. Petrozzino, Roger A. Nicoll, and John A. Connor The influx of Calcium through the NMDA-R The influx of Calcium through the NMDA-R was widely accepted as a trigger for many forms of neural plasticity http://www.gcarlson.com/images/synapse.jpg

3. Approach Prior to this paper, there was no experimental evidence that calcium enters post-synaptic cells exclusively through NMDA-R. This is because there are also voltage-sensitive calcium channels on the post-synaptic cell that may contribute to calcium influx. Prior to this paper, there was no experimental evidence that calcium enters post-synaptic cells exclusively through NMDA-R. This is because there are also voltage-sensitive calcium channels on the post-synaptic cell that may contribute to calcium influx. A steady post-synaptic depolarization eliminated activation of voltage-sensitive calcium channels and allowed experimenters to see the effects of NMDA-R alone. A steady post-synaptic depolarization eliminated activation of voltage-sensitive calcium channels and allowed experimenters to see the effects of NMDA-R alone. http://www.alz.org/brain/images/00a.jpg

4. Approach While holding the post-synaptic cell at 0mV (reversal potential), the experimenters observed synaptically induced calcium transients. While holding the post-synaptic cell at 0mV (reversal potential), the experimenters observed synaptically induced calcium transients. The calcium transients were blocked by APV, which is an NMDA-R antagonist. The calcium transients were blocked by APV, which is an NMDA-R antagonist. To demonstrate further the role of calcium influx through NMDA-R in synaptic plasticity, experimenters observed the magnitude of Long Term Potentiation (LTP) at different post-synaptic membrane potentials. To demonstrate further the role of calcium influx through NMDA-R in synaptic plasticity, experimenters observed the magnitude of Long Term Potentiation (LTP) at different post-synaptic membrane potentials.

5. Purpose of The Experiment  This study focused on two related questions concerning the induction of LTP: (1) Can one directly measure the postulated entry of Ca through the synaptic NMDA Receptors? (1) Can one directly measure the postulated entry of Ca 2+ through the synaptic NMDA Receptors? (2) Is this Ca entry necessary for LTP? (2) Is this Ca 2+ entry necessary for LTP?

6. METHODS Effect of Steady Depolarization on Intracellular Free Calcium Concentration in a CA3 Pyramidal Cell. o Hippocampal sections were used from 3-5 week old guinea pigs o Recordings were carried out with sections submerged beneath a continuously superfusing bathing medium o Free cytoplasmic Ca concentration was measured by dye-loading cells with the Ca indicator fura-2 dissolved CsCl using a hyperpolarizing current. (blocks K+ channels & facilitates depolarization) o Free cytoplasmic Ca 2+ concentration was measured by dye-loading cells with the Ca 2+ indicator fura-2 dissolved CsCl using a hyperpolarizing current. (blocks K+ channels & facilitates depolarization) o Intracellular recordings were made using microelectrodes containing fura-2 and CsCl o Intracellular recordings were obtained from CA1 and CA3 pyramidal cells. o Ca levels were determined radiometrically from acquired fluorescence images using excitation illumination at wavelengths of 360nm and 880nm o Ca 2+ levels were determined radiometrically from acquired fluorescence images using excitation illumination at wavelengths of 360nm and 880nm o Cells were depolarized to 0mV and observed

7. RESULTS Effect of Steady Depolarization on Intracellular Free Calcium Concentration in a CA3 Pyramidal Cell. Figure A-C Shows the Fluorescent Images of the depolarization of the cell to 0mV. Figure A-C Shows the Fluorescent Images of the depolarization of the cell to 0mV. 1A: Normal, resting conditions; Added Fura 2. 1B: The cell was held at -70mV. There was a low resting calcium concentration (50-100nM). 1C: The cell was depolarized to 0mV. The Calcium Concentration rose due to the constant firing of calcium action potentials in the membrane. This figure shows peak calcium concentration levels. David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

8. METHODS NMDA 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 used Same cells as before are used During stable Ca concentrations a train of stimuli of about 50Hz for a duration of 1s was given to the afferent fibers in the stratum radiatum During stable Ca 2+ concentrations a train of stimuli of about 50Hz for a duration of 1s was given to the afferent fibers in the stratum radiatum Starting 1-2s before the stimulus and until 5 -10s afterward, image pairs were collected at approximately 3.5Hz Starting 1-2s before the stimulus and until 5 -10s afterward, image pairs were collected at approximately 3.5Hz

9. 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 B Shows calcium response to a train of synaptic stimuli to associational afferent fibers. Figure B Shows calcium response to a train of synaptic stimuli to associational afferent fibers. Calcium concentrations rose briefly (note yellow and red regions) and recovered within several seconds. Calcium concentrations rose briefly (note yellow and red regions) and recovered within several seconds. Figure C Shows that the Calcium concentrations returned to resting calcium concentrations (compare FIG A and C). Figure C Shows that the Calcium concentrations returned to resting calcium concentrations (compare FIG A and C). David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

10. METHODS 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 Test to see if the rise in Ca concentration depended of the activation of NMDA-R Test to see if the rise in Ca 2+ concentration depended of the activation of NMDA-R The depolarization and stimuli was repeated as before except there was an addition of the NMDA-R antagonist APV to the bathing solution The depolarization and stimuli was repeated as before except there was an addition of the NMDA-R antagonist APV to the bathing solution

11. 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 FIGURES D-F Show Stimulus strains in the presence of APV lead to little detectable change in calcium concentrations. FIGURES D-F Show Stimulus strains in the presence of APV lead to little detectable change in calcium concentrations. David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

12. METHODS Quantitative Analysis of Regions of the Apical Dendrite of a Different CA3 Neuron Showing Calcium Transients Selection of another pyramidal cell with a small apical dendrite field that showed clear Ca transient Selection of another pyramidal cell with a small apical dendrite field that showed clear Ca 2+ transient Measured the Ca concentration in that region as a function of time Measured the Ca 2+ concentration in that region as a function of time Gave tetanic stimulus while the post synaptic cell was held at +10mV Gave tetanic stimulus while the post synaptic cell was held at +10mV Quantitatively examined the time course change of Ca concentration in dendrites Quantitatively examined the time course change of Ca 2+ concentration in dendrites

13. RESULTS Quantitative Analysis of Regions of the Apical Dendrite of a Different CA3 Neuron Showing Calcium Transients The OPEN SQUARES Show a change in calcium concentration in response to a tetanic stimulation (1s, 100Hz) while the postsynaptic cell was held at +10mV. The OPEN SQUARES Show a change in calcium concentration in response to a tetanic stimulation (1s, 100Hz) while the postsynaptic cell was held at +10mV. TRACE a shows the synaptic response was reversed, indicating that voltage-gated calcium channels could not have contributed substantially to the observed rise in intracellular calcium. TRACE a shows the synaptic response was reversed, indicating that voltage-gated calcium channels could not have contributed substantially to the observed rise in intracellular calcium. RESULTS: There was a rapid rise in calcium concentration which decayed back to baseline value over several seconds. David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

14. RESULTS Quantitative Analysis of Regions of the Apical Dendrite of a Different CA3 Neuron Showing Calcium Transients The CLOSED CIRCLES show the effect of APV (an NMDA-R blocker) on the calcium response to the same stimulus. The CLOSED CIRCLES show the effect of APV (an NMDA-R blocker) on the calcium response to the same stimulus. TRACE b shows the membrane potential did not depolarize during the synaptic stimulation. TRACE b shows the membrane potential did not depolarize during the synaptic stimulation. RESULTS: In the presence of APV, the synaptic stimulation led to only a small increase in the calcium concentration, which measured 5% of the control response. David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

15. RESULTS Quantitative Analysis of Regions of the Apical Dendrite of a Different CA3 Neuron Showing Calcium Transients The CLOSED SQUARES show that the effect of stimulation following a wash of the APV. The CLOSED SQUARES show that the effect of stimulation following a wash of the APV. TRACE c shows the membrane potential did not depolarize during the synaptic stimulation. TRACE c shows the membrane potential did not depolarize during the synaptic stimulation. RESULTS: The effect of APV was partially reversible upon removal from the perfusion medium, recovering to 37% of the control response. David J. Perkel,*+ Jeffrey J. Petrozzino,* Roger A. Nicoll,* and John A. Connor*

16. METHODS Blockade of LTP When Low Frequency Synaptic Stimuli Were Paired with Extreme Depolarization to +70mV Took advantage of the increased control of the membrane potential available with whole cell recording Took advantage of the increased control of the membrane potential available with whole cell recording During baseline recording period (holding potential at - 80mV) EPSCs were elicited alternately in two independent afferent pathways at.1Hz During baseline recording period (holding potential at - 80mV) EPSCs were elicited alternately in two independent afferent pathways at.1Hz Stimulation was ceased and the potential was shifted to a test value between -30mV and +100mV Stimulation was ceased and the potential was shifted to a test value between -30mV and +100mV After holding current stabilized, 50 stimuli were given to one of the pathways at 0.2-0.7 Hz After holding current stabilized, 50 stimuli were given to one of the pathways at 0.2-0.7 Hz Shifted the membrane potential to the synaptic reversal potential (approx. +10mV) Shifted the membrane potential to the synaptic reversal potential (approx. +10mV) Second pathway given 50 stimuli with the membrane potential held at the reversal potential Second pathway given 50 stimuli with the membrane potential held at the reversal potential

17. What is Whole-Cell Recording? Advantages: Larger opening at the tip of the patch clamp electrode provides lower resistance and access to the inside of the cell. Larger opening at the tip of the patch clamp electrode provides lower resistance and access to the inside of the cell.Disadvantages: The volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode The volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode

18. RESULTS Blockade of LTP When Low Frequency Synaptic Stimuli Were Paired with Extreme Depolarization to +70mV This is one graph the authors showed, but many experiments were done This is one graph the authors showed, but many experiments were done Cell was depolarized to an extreme potential (+70mV) while the postsynaptic cell was held at -80mV Cell was depolarized to an extreme potential (+70mV) while the postsynaptic cell was held at -80mV The “test” pathway was stimulated 50 times at 0.5Hz The “test” pathway was stimulated 50 times at 0.5Hz The membrane potential was then changed to the reversal potential (~10mV) The membrane potential was then changed to the reversal potential (~10mV) The “reference” pathway was given the same stimulation The “reference” pathway was given the same stimulation Membrane potential returned to -80mV and alternate stimuli was repeated in order to assess the presence of LTP Membrane potential returned to -80mV and alternate stimuli was repeated in order to assess the presence of LTP   Is Ca 2+ influx through NMDAR necessary for LTP induction?

19. RESULTS Voltage Dependence of Induction of LTP by Pairing Low Frequency Stimulation with Depolarization The Experiment Logic: The Experiment Logic: “…if Ca entry were necessary, then a “…if Ca 2+ entry were necessary, then a reduced driving force on Ca2+ during activation of NMDA receptors should result in little or no LTP.”

20. RESULTS Voltage Dependence of Induction of LTP by Pairing Low Frequency Stimulation with Depolarization There is a direct correlation between amount of Ca influx and relative amount of potentiation. There is a direct correlation between amount of Ca 2+ influx and relative amount of potentiation.

21. Let’s Discuss!!

22. Two Key Questions: 1) “Can one directly measure the postulated entry of Ca through the synaptic NMDA receptor channel?” 1) “Can one directly measure the postulated entry of Ca 2+ through the synaptic NMDA receptor channel?” 2) “Is this Ca entry a trigger for LTP?” 2) “Is this Ca 2+ entry a trigger for LTP?”

23. Measurement of Ca2+ Transients During Synaptic Activation of NMDA Receptors Observations: Transient increases in the free [Ca 2+ ] of the dendrites in response to presynaptic stimulation. Done at 0mV in order to block Ca 2+ influx from Voltage sensitive Ca 2+ channels. The changes in [Ca 2+ ] were greatly reduced in the presence of APV The changes were partially restored after a washout of APV

24. Measurement of Ca2+ Transients During Synaptic Activation of NMDA Receptors This data indicates that synaptic stimulation can lead to rises in intracellular [Ca 2+ ] through NMDA channels without a contribution from Voltage sensitive Ca 2+ channels.

25. Various factors allowed the observation of a synaptically mediated Ca 2+ signal:   The use of depolarization to unmask NMDA currents. Virtually complete removal of Mg 2+ blockade of the NMDA receptor when the cell is held at +10mV.   The use of dihydropyridine Ca 2+ channel antagonist nimodipine to reduce the steady-state Ca 2+ influx through noninactivating Ca 2+ channels.   Maintains background Ca 2+ levels low.

26. What Does This Mean? Synaptic stimulation gives rise to increased intracellular Ca 2+ This rise in Ca 2+ is directly dependent on Ca 2+ influx through NMDA receptors.

27. Key Question #2: “Is Ca 2+ entry a trigger for LTP?”

28. The Voltage Dependence of LTP Induction What we know: The presence of postsynaptic Ca 2+ buffers blocks LTP. What we want to know: Whether there is simply a minimum level of Ca 2+ necessary to enable the biochemical process for LTP, OR whether a specific rise in intracellular Ca 2+ is necessary.

29. Voltage Dependence of Induction of LTP by Pairing Low Frequency Stimulation With Depolarization Results provide evidence for a direct instructive role for Ca 2+ in LTP. LTP was blocked at extreme depolarization (no Ca 2+ influx) Direct correlation between amount of Ca 2+ influx to relative amount of potentiation.

30. CONCLUSION THE ORIGINAL QUESTION: Is it possible to measure the entry of Ca 2+ through the synaptic NMDA receptor channel, and if so is this Ca 2+ entry a trigger for LTP? NMDA Receptor http://homepage.psy.utexas.edu/homepage/class/Psy332/Salinas/Neurotransmitters/Slide13.GIF

31. CONCLUSION ANSWER: YES! It is possible and…   NMDA mediated Calcium influx is necessary for LTP induction!

32. 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.

33. EXCEPTIONS/ THINGS TO THINK ABOUT  What about contributions from the activation of metabotropic glutamate receptors?  QUESTION: What about contributions from the 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.

34. 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.

35. 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.

36. 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. 1. Possible slowing effect due to buffering of Calcium by fura-2 2. 2. Prolonged Calcium entry through altered NMDA receptor kinetics 3. 3. Alteration in activity of sodium/calcium exchanger by depolarization

37. Questions…