PHYSCI 147 Lecture 8 Audio Notes

Role of Long-Term Potentiation (LTP) in Learning and Memory

• Discussion initiated on the role of LTP and its relation to learning and memory.

  • Previous lectures focused on experiments involving the Morris Water Maze, pharmacological antagonists to NMDA receptors, and genetic techniques using knockout mice.

  • Concluded previous studies were insufficient due to too many alternative interpretations and gaps in the evidence. This insufficiency arose because the experimental designs often lacked the specificity to isolate NMDA receptor-dependent LTP definitively from other forms of plasticity or confounding behavioral changes, leading to multiple plausible explanations for the observed effects.

New Experiments on NMDA-Dependent LTP

• New evidence aims to strengthen the association between LTP and learning/memory by employing more refined techniques, such as targeted pharmacological manipulations or advanced genetic tools, to specifically isolate the role of NMDA receptors in different learning paradigms.

  • First focus: role of NMDA-dependent LTP in sea slug Aplysia.

  • Second focus: role of NMDA-dependent LTP in fear conditioning in mice and rats.

    • The evidence generated from these studies is suggestive of the importance of NMDA receptor-dependent LTP for associative learning.

Classical Conditioning in Aplysia

• Introduction to Hebb's Postulate:

  • Definition: "When the axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency as one of the cells firing increases."

  • Significance: highlights the relationship between presynaptic and postsynaptic activity, leading to Hebbian potentiation. Hebbian potentiation refers to the strengthening of a synaptic connection that occurs when the presynaptic neuron's activity consistently contributes to the firing of the postsynaptic neuron. These "growth processes or metabolic changes" can include structural alterations like increased synapse size, changes in the number or type of neurotransmitter receptors, or altered protein synthesis that enhances synaptic transmission.

  • Mechanism: The simultaneous firing of presynaptic and postsynaptic neurons activates postsynaptic NMDA receptors. This co-activation is crucial because NMDA receptors require both the binding of glutamate (released from the presynaptic neuron) and sufficient postsynaptic depolarization (to remove the $Mg^{2+}$ block from the receptor pore) to open and allow $Ca^{2+}$ influx.

• Overview of the Karru et al. Experiment (1984):

  • Objective: Test whether Hebb’s hypothesis holds in classical conditioning in Aplysia (siphon withdrawal).

  • Experimental Setup:

    • Two conditioned stimuli (CS): a weak siphon tap (CS, initially neutral) and a mild tail shock (US, unconditioned stimulus, inherently noxious).

    • Animals undergo differential conditioning with paired (CS+US) and unpaired (CS-US) stimuli. In paired training (CS+US), the siphon tap is consistently followed by the tail shock, while in unpaired conditions (CS-US), the stimuli are presented separately or explicitly decorrelated. This allows differentiation of associative learning from non-associative effects like sensitization.

    • The neural circuit for siphon withdrawal involves sensory neurons from the siphon projecting to siphon motor neurons, which were the focus of investigating synaptic change after conditioning.

    • Measurement was based on the extent of siphon withdrawal, a behavioral output.

• Findings from Karru et al.:

  • Animals conditioned with the siphon tap and tail shock showed greater response rates to siphon taps compared to mantle taps. Specifically, the siphon tap (CS+) when paired with tail shock (US) led to a significantly enhanced siphon withdrawal reflex when the siphon was subsequently tapped alone, compared to control groups or taps on the mantle (a different body part, possibly serving as a CS- or an untrained stimulus).

  • Enhancement in siphon withdrawal response was observed in paired training groups, indicating a direct consequence of associative learning where the CS alone now elicits a stronger response due to its association with the US.

    • Comparison of paired versus unpaired results strengthened claims of associative learning by ruling out non-associative effects like sensitization (a general increase in responsiveness due to US exposure), demonstrating that the enhancement was specific to the associative pairing of CS+ and US.

Cellular Analogs of Classical Conditioning

• The study utilized electrophysiological approaches:

  • The central nervous system (CNS) in Aplysia was meticulously dissected in vitro to isolate the specific siphon-withdrawal reflex circuit, ensuring the synaptic connections between sensory and motor neurons remained intact for electrophysiological recording and analysis of synaptic changes.

  • Experiment tested the sufficiency and necessity of postsynaptic activity to induce Hebbian potentiation, seeking definitive insights into NMDA receptor roles in learning. This involved manipulating the activity of the postsynaptic neuron while controlling presynaptic input.

• Limitations and Alternative Approaches:

  • Tail shock vs. direct motor neuron stimulation was pivotal to understanding postsynaptic effects. Tail shock is a natural, unconditioned stimulus that activates presynaptic inputs and causes postsynaptic firing. Direct motor neuron stimulation, conversely, bypasses presynaptic inputs, forcing postsynaptic activity intentionally to see if only postsynaptic depolarization (when presynaptic activity is not associatively enhanced) is sufficient for potentiation.

  • The inability of direct postsynaptic motor neuron stimulation to induce the expected Hebbian potentiation strongly indicated that only postsynaptic activity is insufficient for classical conditioning in this context. This finding highlighted the critical role of presynaptic activity (e.g., from the tail shock) in driving the associative changes and suggested that the complex interplay between presynaptic release and postsynaptic depolarization is necessary for effective NMDA receptor engagement and the induction of robust synaptic plasticity underlying learning.

Conclusions on Mechanisms of Classical Conditioning

• Resulting insights revealed ambiguity regarding Hebbian potentiation's role in classical conditioning in Aplysia. The initial findings were ambiguous because simply firing the postsynaptic neuron wasn't enough, suggesting that a purely Hebbian, postsynaptic-driven LTP wasn't the sole mechanism. Findings suggested dual mechanisms:

  1. Presynaptic associative facilitation (due to tail shock).

  2. Postsynaptic Hebbian LTP via NMDA receptor influence demonstrating a necessity for calcium-mediated processes in synaptic modifications.

  • 1. Presynaptic associative facilitation: This mechanism involves the tail shock (US) activating facilitatory interneurons that release neuromodulators (like serotonin) onto the presynaptic terminals of the siphon sensory neurons. This leads to an enhancement of neurotransmitter release from the sensory neurons, strengthening the sensory-motor synapse presynaptically when paired with CS.

  • 2. Postsynaptic Hebbian LTP: This component involves the NMDA receptor. When the presynaptic sensory neuron (CS) fires simultaneously with a postsynaptic motor neuron (driven by the US or robust CS+), the NMDA receptors become active. Their activation leads to a significant influx of $Ca^{2+}$ ions into the postsynaptic neuron, which triggers intracellular signaling cascades crucial for long-term synaptic modifications (e.g., insertion of AMPA receptors, changes in dendritic spine morphology).

• Emphasis on the significance of serotonin in the facilitation of associative changes:

  • Specifically, serotonin release from facilitatory interneurons acts on presynaptic terminals to enhance cAMP-PKA pathways, which contribute to increased neurotransmitter release. This presynaptic change, combined with the postsynaptic NMDA receptor activation and $Ca^{2+}$ influx, provides a comprehensive view of how simultaneous activity drives both immediate functional and long-term structural changes at the synapse, reinforcing the associative link. Potentiation and its correlation with structural synaptic modification thus depend upon simultaneous activations of presynaptic and postsynaptic elements.

Experimental Progression: Antonoff et al. Study

• Detailed the advanced experiments performed by Antonoff et al., establishing stronger links of the NMDA receptor in Aplysia's classical conditioning. Antonoff et al.'s experiments represented a significant progression. While retaining the behavioral paradigm of siphon tap (CS) with tail shock (US), they incorporated refined pharmacological interventions and more precise electrophysiological recordings in vivo and in vitro. This allowed for targeted manipulation of specific molecular pathways and direct observation of cellular responses, providing enhanced precision and control over previous studies.

  • Experiment setup included direct taps to the siphon, with paired training using electrodes for tail shock, similar to previous methodologies but with enhanced precision and control.

• Key discoveries:

  • PKA and NMDA receptor activity: Their work confirmed that both PKA, primarily involved in presynaptic facilitation, and NMDA receptor activity, crucial for postsynaptic plasticity, are indispensable for the induction and maintenance of classical conditioning. Inhibition of either pathway significantly impaired associative learning.

  • Observed changes in sensory neuron excitability leading to enhanced responsiveness during paired training versus unpaired circumstances, affirmed through controlled measurements of action potentials upon stimulation. They demonstrated that paired training led to an increase in the excitability of siphon sensory neurons themselves, meaning these neurons fired more action potentials for a given stimulus strength. This enhanced intrinsic excitability, measured by increased action potential firing upon direct current injection, contributed to the stronger withdrawal response during conditioned stimuli. This change was specific to paired training.

  • Confirmed interactions between presynaptic facilitation and postsynaptic LTP dynamics, bolstered suspicions of retrograde signaling processes guiding synaptic adjustments during conditioning. The studies provided strong evidence for interactions between presynaptic facilitation (serotonin acting on PKA) and postsynaptic LTP (NMDA receptor activated $Ca^{2+}$ influx). This interaction is hypothesized to involve retrograde messengers (e.g., nitric oxide, endocannabinoids) released by the postsynaptic neuron which then diffuse back to the presynaptic terminal, influencing neurotransmitter release and further enhancing long-term synaptic changes. This 'cross-talk' between pre- and postsynaptic elements ensures a robust and sustained associative memory trace.

Establishing Linkages Between Mechanisms

• Integration of results across studies led to a consensus on the necessity of both presynaptic and postsynaptic mechanisms in classical conditioning: The integrated findings from Karru et al. and Antonoff et al. converged on a unified model for Aplysia classical conditioning, emphasizing that neither presynaptic facilitation nor postsynaptic LTP alone is sufficient. Instead, their synergistic action, particularly mediated by crucial retrograde signaling molecules (such as nitric oxide or carbon monoxide in Aplysia) which convey information from the postsynaptic to the presynaptic terminal, ensures robust and persistent synaptic strengthening.

  • Key retrograde signaling mechanisms strengthen associative processes hypothesized to function similarly across species.

  • Confirmed NMDA receptor engagement is crucial in long-term synaptic changes that mediate associative learning and memory storage. This dual mechanism is now theorized to represent a conserved principle for associative learning across diverse species, including mammals, where NMDA receptor-dependent LTP is widely recognized as a critical cellular substrate for long-term synaptic plasticity and memory formation.

Summary and Implications

• Current theories emphasize an intricate balance between excitatory synaptic changes and cellular excitability dictated by both postsynaptic and presynaptic activities facilitated predominantly by NMDA receptor dynamics and regulatory processes involving retrograde signaling mechanisms. In essence, effective associative learning involves a finely tuned interplay: excitatory synaptic changes (strengthening of the synapse due to increased postsynaptic receptor efficacy and/or presynaptic release probability) are combined with alterations in intrinsic cellular excitability (the neuron's readiness to fire). These changes are tightly regulated by NMDA receptor dynamics (which act as coincidence detectors) and further modulated by sophisticated retrograde signaling processes that ensure coordinated modifications across the synaptic cleft, forming an enduring memory trace.

• Future directions include upcoming discussions on fear conditioning in mammals, establishing parallels among associative memory mechanisms across species. The foundational understanding gained from Aplysia studies provides a crucial comparative framework for investigating more complex forms of associative learning, such as fear conditioning in mammals. By identifying common underlying molecular and cellular mechanisms (e.g., NMDA receptor involvement, presynaptic/postsynaptic dialogue, retrograde signaling), researchers can establish powerful parallels across evolutionarily distant species, revealing universal principles of memory formation.

• End of Lecture - Questions Facilitated for Further Understanding.