Engineering a Memory with Long-Term Depression (LTD) and Long-Term Potentiation (LTP)

Engineering a Memory with Long-Term Depression (LTD) and Long-Term Potentiation (LTP)

Authors

• Nabavi et al.: Mackenzie Lynch, Reva Joshi, Sawyer Sullivan, Astrid Bell, Serena Yañez, Amanda Feng, Devani Solberg, Rae Rodriguez, Mihir Shah.

Introduction

Research Question

• Is there a causal relationship between LTP/LTD and associative memory formation/reduction?

Background

• Long-Term Potentiation (LTP):

  • A process thought to be a catalyst for memory formation via synapse strengthening.

• Long-Term Depression (LTD):

  • Associated with memory reduction through synapse weakening.

• Previous Work:

  • Indicated a correlational relationship between LTP and LTD but lacked evidence of causation.

Basic Methods

• Animal Model: Adult male rats.

• Experimental Design:

  • Utilized fear conditioning combining a tone with a foot shock to create associative memory.

  • Replaced the tone with optogenetic stimulation of auditory inputs in the lateral amygdala to analyze specific synapses.

  • Conducted LTP and LTD protocols using stimulation paired with shocks to "activate" and "deactivate" associative memory.

  • Measured the AMPAR:NMDAR ratio in brain slices to assess LTP/LTD occurrence.

  • Note: Optogenetic stimulation provided direct control over synaptic plasticity for memory engineering.

Methods Overview

General Set-up

• Subject: Male Sprague-Dawley rats.

  • Younger (6-8 weeks): Used for virus injection and surgery.

  • Older (10-12 weeks): Used for behavioral and electrophysiological studies.

• Housing: Standard cages with a 12-hour light/dark cycle.

Optogenetics Set-up

• Viral Injection:

  • AAV expressing oChIEF, a variant of the light-activated channel ChR2, was injected into the auditory nuclei (MGN + auditory cortex) of younger rats.

• Surgical Implantation:

  • After 3-4 weeks, an optic fiber was implanted above the dorsal tip of the lateral amygdala.

  • This setup permitted stimulation of auditory inputs to the amygdala with blue light (473 nm) instead of actual sound, ensuring control over specific neurons.

Methods: Behavioral Assays

Response Measures

• Lever Press Training:

  • Associated lever pressing with a reward of 40 μl of 10% sucrose water to establish baseline behavior.

• Fear Conditioning:

  • Normal Conditioning:

  • Tone Conditioning:

    • A tone (Conditioned Stimulus - CS) paired with a foot shock (Unconditioned Stimulus - US) leads to learned fear response.

    • Behavioral measure reflects reduced lever pressing or freezing during the tone, indicating fear memory encoded by the amygdala.

  • Optogenetics Conditioning:

  • Blue light (473 nm) was used to activate auditory neurons projecting to the amygdala during foot shock, creating "engineered fear memory".

  • Behavioral measure: observed reduced lever pressing during light activation signifies learned fear.

Figure Representations

Figure 1: Fear Conditioning with Tone or Optogenetics

• Goals of Study:

  • Validate that optogenetic stimulation produces responses equivalent to those generated by a tone.

  • Assess the importance of temporal stimulus pairing.

  • Investigate the relationship between synaptic plasticity and behavior.

• Preliminary Findings:

  • Confirmed effective CS-US pairings elicit strong conditioned response (CR).

  • Verified that optogenetic inputs reach axon terminals in the lateral amygdala.

  • Established that optical CS alone does not yield CR and that blocking NMDA receptors interrupts CR to CS, indicating NMDA receptor necessity for associative learning.

Time Plots and Bar Graphs

• Normalized Lever Presses:

  • Comparison of lever presses during previously learned cued lever-press task analyzed over time.

• Bar Graph Findings:

  • Panel A: Comparisons of tone paired with foot shock vs. unpaired conditioning indicated significant differences in conditioned responses.

  • Panel B: Results of optogenetically driven input stimulation paired with foot shock to assess associative fear memory formation, showcasing timing importance.

Results: Lever Press and AMPA/NMDA Ratios

• AMPA/NMDA Ratio Findings:

  • Naive Ratio: $( rac{2.4 ext{ ± } 0.2}{n=11 ext{ from } 6 ext{ rats}})$

  • Unpaired Ratio: $( rac{2.1 ext{ ± } 0.2}{n=10 ext{ from } 6 ext{ rats}})$

  • Paired Ratio: $( rac{4.4 ext{ ± } 0.6}{n=8 ext{ from } 4 ext{ rats}})$

Synaptic Modification Model

• LTP was determined after pairing optical CS with foot shock, confirming potentiation of the CS-induced CR.

• AMPA and NMDA receptor components measured via amygdala slices confirmed LTP occurrence.

Protocols for Learning and Memory Manipulation

LTD and LTP Induction

• LTD Induction:

  • Applied 1 Hz stimulation for 15 minutes.

• LTP Induction:

  • Applied 100 Hz stimulation in 5 bursts with 3-minute intervals.

Conditioning Protocol Summary

• Paired Conditioning: Conditioned response evidenced by reduced lever pressing when light condition was on denoting active memory.

• Observations under LTD: No conditioned response; lever pressing persisted indicating inactive memory.

• Under LTP: Conditioned response returned, memory was active again.

• Observations on repeated manipulations: Confirmation of bidirectional plasticity across trials.

Cellular Model of Synaptic Modification

Analysis of Synaptic Effects of LTP and LTD

• LTD Protocol Effects:

  • Weakening of synapses in the lateral amygdala, denoted by decreased AMPA receptors leading to inactivation of CS-triggered fear memory.

• LTP Protocol Effects:

  • Strengthening of synapses in the lateral amygdala, indicated by increased AMPA receptors resulting in reactivation of CS-triggered fear memory.

Results Summary & Implications

Figures Related to Memory Manipulation

• Figure 3 Summary:

  • Aims to explore whether LTP or LTD alone can create, erase, or restore fear memory, and whether this depends on prior conditioning.

• Key Methodologies outlaid:

  • Utilization of paired conditioning, optical LTD, and optical LTP protocols to determine effects on naïve rats.

Main Conclusions

• Functional Insights:

  • LTP alone post-conditioning was able to restore fear memory, whereas LTD could inhibit it, while LTP alone could not create memory.

  • LTD lacks the capacity to erase fear memory entirely.

Electrophysiological Responses

• Goals of Electrophysiological Assessments:

  • Confirm optogenetic stimulation protocols produced expected synaptic changes.

• Findings:

  • 10 Hz stimulation had no effect on synaptic strength, while 1 Hz and 100 Hz stimulation confirmed LTD and LTP effects, respectively, indicating these protocols alter synaptic transmission strength.

Strengths of the Study

• Operational Control:

  • Precise optogenetic manipulation verified in identified auditory-amygdala pathways.

• Data Correlation:

  • Results combined behavioral, electrophysiological, and in vitro data linking synaptic changes to behavior.

• Reversibility:

  • Manipulation display of reversible conditions provides causative evidence for memory control.

• Novelty:

  • Demonstration of toggling specific memories on and off is unprecedented.

Limitations of the Study

• Scope:

  • Focused primarily on a singular conditioned fear memory and one neural pathway.

• Reproducibility of Conditions:

  • Optogenetic excitation may not adequately replicate intricate natural neural activity.

• Molecular Mechanisms:

  • The exploration did not delve into the molecular mechanisms or considerations of long-term systems-level consolidation.

• Complexity:

  • It's uncertain if similar memory control applies to more complex or sophisticated distributed memories.

Main Conclusions

Synopsis on LTP and LTD's Role in Memory

• Thesis Point:

  • Demonstrated LTP and LTD as foundational mechanisms for memory storage.

• Causal Evidence:

  • Clear evidence that synaptic changes in specific pathways directly control memory expression.

• Neural Encoding Insight:

  • Memory storage proposed to be based on synaptic connectivity rather than mere activity patterns.

Key Findings and Clinical Significance

Toggle Mechanism of Memory

• LTD Effects:

  • Low-frequency stimulation weakens synaptic connections, inhibiting memory expression.

• LTP Effects:

  • High-frequency stimulation regenerates synaptic connections, reactivating memory.

• Reversibility:

  • Memories modifiable without retraining animals; indicative of specificity within neural pathways.

Importance Reiterations

• Theoretical Impact:

  • Strong backing for Synaptic Plasticity Hypothesis concerning learning paradigms.

• Translational Opportunity:

  • Potential clinical applications for targeted manipulation of memory.

  • Opening avenues for future research into reversible modifications in memory.