Electrophysiology and RNA-Mediated Memory Transfer in Aplysia

Overview of Dr. Glanzman’s Research and the Provocative Hypothesis

• Research Context: These notes cover the second part of a presentation regarding Dr. Glanzman's research. Previous discussions established that long-term sensitization (a form of learning) in Aplysia (sea slugs) can be transferred from a trained animal to a fresh one via RNA injection.
• Pharmacological Evidence: Yesterday’s final experiment demonstrated at a pharmacological level (using drug testing) that this behavior transfer is dependent on epigenetics, specifically DNA methylation.
• The Running Hypothesis: RNA from a trained sea slug, when injected into a fresh slug, induces a specific DNA methylation pattern in the recipient. This new methylation pattern alters gene expression to recreate the memory of training (long-term sensitization). This process can be blocked by specific chemical inhibitors targeting DNA methylation.
• Provocation in Science: This research is considered provocative because it contradicts current mainstream theories of the neurobiology of memory, which typically focus on synaptic changes rather than RNA-mediated epigenetic inheritance. The speaker emphasizes that science involves presenting contradictory evidence and "battling it out collegially" through peer critique and replication.
• The Neuroscientific Question: Dr. Glanzman transitioned from purely behavioral and pharmacological studies to a neuroscientific approach. He sought to detect physical or electrical changes in the neurons involved in the learning circuit to provide a cellular correlation for the observed behavioral changes. Without neural correlates, the behavioral data would be harder to validate.

Biological Fundamentals of the Aplysia Nervous System

• Organism Selection: The first researchers to use Aplysia for memory studies were led by Eric Kandel (Columbia University) in the 1970s. Kandel chose this organism because of its simple behaviors and accessible nervous system, for which he eventually won the Nobel Prize.
• Neuroanatomy (Ganglia vs. Brain):
  • Invertebrates like Aplysia do not have a centralized brain like vertebrates. Instead, they possess a collection of interconnected clusters of neurons called ganglia.
  • Collectively, these ganglia function as the brain, though they are structurally decentralized.
  • Specific Ganglia Names and Locations:
    • Buccal Ganglia: Located near the mouth.
    • Cerebral Ganglia: Located toward the head.
    • Pleural Ganglia: Located near the gills; contains neurons for the gill-withdrawal reflex.
    • Pedal Ganglia: Located near the foot.
    • Abdominal Ganglion: Involved in processing tail shocks.
• Simplified Circuitry: A major advantage of Aplysia is that its circuitry is easily understood and consistent across animals, making it an ideal model for testing hypotheses about simple learning circuits.

Mechanisms of Learning: Long-Term Potentiation (LTP)

• The Monosynaptic Reflex: At its most basic, the gill-withdrawal reflex is a single synapse connection between a sensory neuron (detecting touch on the siphon) and a motor neuron (retracting the gill muscles).
• The Circuitry of Sensitization:
  • Sensory Neuron: Detects stimuli on the siphon.
  • Motor Neuron: Controls the muscles for gill withdrawal.
  • Facilitatory Interneuron (Yellow): Activated by a tail shock. These interneurons release the neurotransmitter Serotonin ($\text{5-HT}$) onto the axon terminal of the sensory neuron.
• Long-Term Potentiation (LTP):
  • When a tail shock and siphon touch occur simultaneously, two excitatory inputs hit the synapse. This causes a massive release of neurotransmitters, increasing the firing of the motor neuron.
  • Repeated training (tail shocks) causes a change in the synapse called synaptic plasticity. The synapse remains "stronger" for days or weeks, even without further tail shocks.
  • As a result, a simple touch to the siphon now causes a much stronger gill withdrawal than before because the synapse releases more synaptic vesicles.

Electrophysiological Measurement and "The RIG"

• Intracellular Recording: Since the 1950s, technology has allowed scientists to place an electrode inside a single neuron. This allows for the measurement and quantifying of electrical activity.
• Two-Electrode Setup: Voltages are relative; therefore, a recording requires:
  1. Recording Electrode: Placed inside the cell body (soma).
  2. Reference Electrode: Placed in the surrounding medium (water, salts, buffers).
• "The RIG": This is the colloquial term for the collection of equipment used for recordings, including:
  • Microscope: Used to guide the tiny electrodes to the cell body.
  • Amplifiers: Used to record and boost small electrical signals and to inject electrical current into the cell.
  • Computer: Receives data and allows for software-based control of the experiment.
• Protection from Noise:
  • Faraday Cage: A metal shield around the microscope to block electromagnetic radiation (e.g., cell phone signals, radio waves).
  • Anti-vibration Table: Prevents physical vibrations from moving the electrode or destroying the cell.
• Audio Conversion: Some neuroscientists connect the equipment to speakers so they can "hear" the action potentials as beep sounds.

Ohm’s Law and Clamping Techniques

• The Mathematical Relationship: In electrophysiology, voltage and current are interdependent variables governed by Ohm's Law:     $V = I \times R$
  • $V$: Voltage (Potential difference across the membrane).
  • $I$: Current (Flow of charged ions).
  • $R$: Resistance (How many channels are open/present for ions to pass through).
• Resistance Constant: In experiments measured in minutes, resistance is assumed to be constant because changing the number of channels in a membrane usually takes hours or days.
• Clamping: Scientists must "clamp" (fix) one variable to study the other.
  1. Voltage Clamp: The amplifier forces the cell to stay at a specific voltage (e.g., $-70 \text{mV}$). The scientist then measures the current ($I$) required to maintain that voltage.
  2. Current Clamp: The amplifier forces a specific current (e.g., $1 \text{pA}$) into the cell, and the scientist measures the resulting change in voltage ($V$), such as action potentials.

Interpreting Electrophysiological Traces

• Current Convention (Voltage Clamp):
  • Inward Current: Represented as a downward dip in the trace. This signifies the cell becoming more positive (either positive ions entering or negative ions leaving).
  • Outward Current: Represented as an upward deflection in the trace. This signifies the cell becoming less positive (e.g., Potassium $K^+$ leaving).
• Examples of Current Measurement:
  • Sodium ($Na^+$) Flow: When a $\text{5-HT}_3\text{A}$ receptor opens, sodium rushes in, creating a downward dip (inward current).
  • Potassium ($K^+$) Flow: Clamping a cell at a depolarized level (e.g., $+40 \text{mV}$) causes voltage-gated potassium channels to open. Potassium flows out, creating an upward trace (outward current).
• Action Potentials (Current Clamp):
  • Subthreshold Current: A very small current (e.g., $1 \text{pA}$) that changes the membrane potential slightly (e.g., from $-60 \text{mV}$ to $-58 \text{mV}$) but does not trigger a spike.
  • Suprathreshold Current: A higher current (e.g., $2 \text{pA}$ or more) that triggers one or more action potentials.

Experimental Findings: RNA Effects on Neural Excitability

• The "Cheating" Experiment Design: Rather than reconstructing the entire 5-neuron circuit in a dish, Dr. Glanzman simplified the experiment by extracting only sensory and motor neurons from the right ganglia.
• Sensory Neuron Data:
  • Control RNA (Untrained): Showed no increase in the number of action potentials; in some cases, activity slightly decreased.
  • Trained RNA: Showed a qualitative increase in action potentials in the post-test (after RNA application). The cells became more excitable (e.g., gaining an extra spike or two).
• Motor Neuron Data:
  • Result: Across all groups (Vehicle, Control RNA, Trained RNA), there were no significant changes in motor neuron excitability.
  • Interpretation: The RNA-mediated memory transfer appears to skip the motor neurons and specifically target the sensory neurons.

Statistical Analysis and Implications

• Plotting Percentage Change: Data was plotted as the percentage change in the number of action potentials between the pre-test and post-test.
• Statistical Significance Metrics:
  • One Star (*): Typically denotes $p < 0.05$. In this study, comparing the vehicle control to trained RNA yielded one star.
  • Two Stars (): Denotes a more significant p-value. In this study, comparing control RNA to trained RNA yielded $p = 0.004$.
  • NS (Not Significant): No meaningful statistical difference (e.g., comparing Vehicle to Control RNA).
• Conclusions:
  • RNA seemingly mediates long-term sensitization by altering sensory neurons but not motor neurons.
  • This effect is likely achieved through DNA methylation, which modifies the sensory neurons to make them more excitable and stronger in the gill-withdrawal circuit.
  • This provides a potential new model where learning and memory are modulated by RNA and epigenetic strategies.

Questions & Discussion

• Question: Did the RNA transfer the methylation pattern into the DNA of the recipient slug?
• Response: That is the current running hypothesis. The RNA from the trained animal induces a methylation pattern in the fresh animal, which changes the expression pattern of the memory. This was supported by the fact that the behavior could be blocked with a methylation inhibitor.
• Question: Is there a limit to how many axons can interact with a single neuron?
• Response: While there is likely a theoretical limit based on the surface area of the cell, estimates suggest every neuron in our nervous system can receive inputs from between $1,000$ and $10,000$ other neurons.
• Question: Why did they not look at the siphon sensory neurons specifically?
• Response: The rationale is often as simple as ease of dissection. Tail sensory neurons might have been easier to extract, and scientific experiments are often "imperfect" due to funding or technical difficulty.