Lecture 18: Long Term Potentiation

Types of Memory

• 2 basic classes of memory

  • Declarative (explicit) memory

  • Non-declarative (implicit) memory

• These different types of memory are supported by and stored in different brains regions

Declarative (Explicit) Memory

• Conscious/aware

  • Knowing what/where/when e.g. facts and events

• Associated with the hippocampus

Non-Declarative (Implicit) Memory

• Not conscious/aware

• Knowing how to (procedural)

  • e.g. skills, associative (priming classical conditions - subconscious) non-associative (sensitisation/ habituation)

Test For Declarative Memory

• Learning a list of words; delay then recall e.g. for an exam=

• Identify how objects move in space – object recognition task – visual memory and ability to state what is going on

Test of Spatial Memory

• It is a form of declarative memory,

• Tested by the identification of a pattern and then seeing how it has changed

  • Uses the hippocampus to process the information in the object recognition and placement tasks

Henry Molaison

• Suffered an accident that resulted in damage to the hippocampus resulting in seizures

• Removing the hippocampus resulted in declarative memory loss, but not procedural memory loss

  • Had anterior grade amnesia – couldn’t make new memories, but could remember things before his accident

• If doing the object recognition or location task – sit in a scanner and the MRI lights up when conducting these tasks

Recordings of Long Term Potentiation in the Hippocampus

• Studied by Terje Lomø in Norwegian rabbit hippocampus (in vivo, under anaesthesia).

• Perforant path input to dentate gyrus was activated and responsiveness of the DG was recorded for a period of time at a low frequency stimulation then increased, before returning back to the LFS.

  • Low-frequency stimulation (LFS): 1 stimulus every 10s.

  • Then high-frequency stimulation (100Hz, brief bursts) for 3-4s.

Findings from Long Term Potentiation in the Hippocampus

• Increased responsiveness in the dentate gyrus, lasting several hours.

• Steeper rise time (slope) of extracellular field EPSP - generation of upward field potentials

  • can be measured to estimate its size/ measure size of the populatin spike

• Larger population spike (more cells firing action potentials) - has a large downward deflection due to the shift in responsiveness

• Enduring change in synaptic transmission was observed even in awake rabbit (not anesthesia artifact).

Recording of LTPs in Hippocampal Slices

• In-vitro techniques developed to target drugs to these processes and manipulate the underlying cellular processes supporting the changes

Experimental Set-Up For In Vitro LTP in the CA1 Region

• A 300-500μm hippocampal slice, where the CA3 region can be visually identified and its output pathway activated and recorded via visual identification of the CA1 region is used

• Extra- and intracellular recordings were taken from CA1 pyramidal neurons.

• Low-frequency stimulation (<0.1Hz) to establish baseline EPSPs (glutamatergic neurotransmission).

• High-frequency stimulation/tetanus (100Hz for 1s) induces LTP.

• Post-HFS, return to low-frequency stimulation to assess changes in synaptic responsiveness.

Methods of Recording In-Vitro LTP Experiments at CA3→CA1

• EPSC (Excitatory Post-Synaptic Current): Measured using a voltage clamp to assess synaptic responsiveness.

• EPSP (Excitatory Post-Synaptic Potential): Can convert sub-threshold EPSPs into action potentials.

• Field EPSP:

  • Recorded from dendrites in CA1 (not from the cell body layer).

  • Fields are negative, but effective slope measurement estimates the size of the field EPSP.

  • Inflexions from neuronal firing can interfere with true field EPSP size.

• The protocol increases synaptic strength or "weight."

Factors Affecting the Duration and Magnitude of LTP at CA3→CA1 In-Vitro

• The timing of the tetanus delivery affects the outcome.

• Transmission monitored at <0.1Hz before and after 100Hz HFS.

• Post-Tetanic Potentiation (PTP)/Short-Term Potentiation (STP): Initial potentiation lasts ~10 min, then decays to a sustained level.

• Single 100Hz Stimulus: Results in decay to baseline after two hours, with an upregulation lasting ~1 hour (early LTP).

  • Fades after 90 minutes, and returns to baseline.

• Four 100Hz Stimuli (1 every 5 min): Can last up to 24 hours, generating robust early LTP + late LTP.

• LTP is dependent on the amount of activation from high-frequency stimuli and determines both its size and endurance.

Three Component Phase of LTP

• Induction phase - high-frequency stimulation (100Hz) initates the LTP by increasing the responsivenes of synaptic transmission

• Transient or Early Phase - seen after single 100Hz stimulation;

  • the reversible early LTP following induction - fades over time

• Consolidation or Late Phase - permanent change occurs leading to the maintenance of late-LTP

• Likely that specific mechanisms are present, supporting each

What is The Induction of LTP Dependent on

• NDMA receptor

• Postsynaptic rise in intracellular Ca2+

• Postsynaptic depolarisation

AP5

• An NDMA receptor antagonist that blocks LTP is applied during HSF, but not after induction

• Demonstrated that the induction of LTP was dependent on the NDMA receptor

EGTA

• A (Ca²⁺ chelator): Blocks LTP induction when injected intracellularly, preventing rise in intracellular [Ca²⁺]

Injection of Negative Current/ Freqency Stimulation

• Results in the hyperpolarisation of the membrane potential and prevents postsynaptic depolarisation during HFS,

Inital Post-Tetanic Potentiation

• Occurs in response to short-term mechanism

• Induction of LTP begins with short-term potentiation due to high-frequency stimulation (tetanus).

• Elevated presynaptic Ca²⁺ due to activation of presynaptic terminals enhances release probability, but effects fade after 10 minutes.

Role of NMDA receptors during LTP induction at the CA3-CA1 synapse

• Baseline neurotransmission at low-frequency stimulation (<0.1 Hz) is primarily AMPA-mediated EPSPs.

• NMDA receptors are blocked by Mg²⁺ at hyperpolarised membrane potentials of -80mV (due to internal negativity), so they contribute little to baseline transmission.

• AMPA receptors are responsible for baseline neurotransmission but do not allow Ca²⁺ entry.

• D-AP5 (an NMDA receptor antagonist) has little effect on EPSPs evoked by <0.1Hz stimulation.

Effect of Frequency Stimultation on NMDA receptors during LTP induction at the CA3-CA1 synapse?

• Postsynaptic responses are influenced by the frequency of stimulation and the time course of the EPSP, leading to temporal summation and depolarisation.

• Higher-frequency stimulation or stronger synaptic activation causes greater depolarization.

• NMDA receptors contribute more to neurotransmission when the postsynaptic membrane is depolarised enough to remove the Mg²⁺ block.

• Coincidental activation of AMPA and NMDA receptors results in an influx of Ca²⁺ into the postsynaptic cell, which is essential for LTP.

Morris Water Maze

• Rodent trained rodents to swim in an opaque to a hidden platform – identifiable with a flag      

• Mice use spatial cues to identify where the platform is      

• Once they have effectively learnt where it is, the flag can be removed and the mice swim directly to the platform due to spatial cues e.g. checkerboard and yellow triangle

Morris Water Maze - Rodent Treated With AP5/ KO NMDA-Rs

• Mice don’t learn where the platform is - poor spatial memory

  • Observations shows LTP’s sensitivity to NMDA receptor block and that they may contribute to learning

Application of AP5

• No LTP block if applied after induction

  • Independent of NMDA receptor activation

• Specific to the induction

Protein Kinases

• Activated in response to increased Ca2+ involved in the induction of LTP

Application of PKI

• LTP induction blocked by PKC_19-31 or CaMKII_273-302

• Dependent upon activation of protein kinase(s) protein kinase C (PKC) and/or calmodulin-dependent Protein Kinase II (CaMKII) – likely both are involved

Role of PKC and CaMKII in Early LTP at CA3→CA1 synapses

• These kinases regulate AMPA receptor function through phosphorylation, which increases AMPA receptor conductance.

• Both kinases target the same phosphorylation site, leading to

  • enhanced ion channel conductance,

  • increased current flow through the channels,

  • larger EPSP (due to higher macroscopic EPSC amplitude).

    • EPSP is proportional to channel current flow

Protein Kinase A

• An enzyme involved in the late phase LTP by increasing cAMP which activates the genomic process to synthesise new proteins to maintain synaptic changes of increased synaptic responsiveness

• The synthesis of new proteins stabilises these synaptic modifications

• It can explain the distinction between single high-frequency stimulation and the delivery of 4 successive stimuli

H89

• A PKA selective inhibitor

• It transforms the long-lasting late EPSP into a transient initial response that fades over time, demonstrating the necessity of PKA for sustained synaptic modifications.

Early Phase LTP

• It involves local biochemical processes like Ca2+ activation of the Ca2+-dependent kinases like PKC and CaMKII for phosphorylation of AMPA receptors

Late Phase LTP

• It relies on PKA for genomic activation and protein synthesis, resulting in more permanent synaptic modification - maintains the level of responsiveness overtime

Anisomycin and Actinomycin D

• A translational inhibitor and transcriptional inhibitor prevent new protein and mRNA synthesis, respectively, by blocking the late phase of LTP that relies on protein synthesis.

Effects of Protein Synthesis Inhibition in Late Phase LTP

• It prevents the maintenance of long-lasting synaptic modifications, as new proteins are required to support sustained changes in synaptic strength.

Problems with Long-Term Potentiation (LTP) as the sole mechanism for memory and learning

• If it is the only mechanism for long-term synaptic changes, all synapses would eventually become fully potentiated.

• Synaptic weights would reach ‘the ceiling’ where no further increases in efficacy are possible.

• This limits the potential for learning and memory.

• To maintain flexibility, there must be a way to reset or disassociate synaptic connections through disassociation (resting) to allow subsequent re-association of synaptic activity.

• Solution: Long-Term Depression (LTD) allows for the reversal of LTP, enabling further learning and memory encoding.