49 Molecular Mechanisms of Memory: Hippocampus

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Zooming in beyond the level of anatomy, the substrates of learning can be found at the level of synapses. Synapses change in a phenomenon called plasticity. The word “plasticity” refers to a change in synaptic strength, which may be an increase or a decrease. This change may persist for minutes, hours, days, or in some cases, even a whole lifetime.

When synaptic strength is increased and remains elevated, we call this long-term potentiation (LTP). A prolonged weakness of a synapse is called long-term depression (LTD). In our current limited understanding of plasticity, both phenomena are important for a healthy brain, and neither one is always good or always bad. It is also important to clarify that both excitatory synapses and inhibitory synapses can be subject to either LTP or LTD.

The Hippocampus

The hippocampus, meaning “seahorse” in Greek, was named based on its morphology. It is located along the ventral and medial surface of the brain. The hippocampus serves as one of the critical structures of the limbic system, a series of subcortical brain structures that are involved in several different complex behaviors, such as emotions and memory. The synaptic connectivity of the hippocampus is very well characterized. Hippocampal synaptic connectivity was first described by Ramon y Cajal, and is made up of three main synaptic connections; sometimes called the trisynaptic circuit.

First, the entorhinal cortex serves as the major input to the hippocampus. This white matter signaling tract is called the perforant pathway, and the neurons synapse onto the granule cells within an area of the hippocampus called the dentate gyrus. The dentate gyrus neurons send their axons, called mossy fibers, to the pyramidal cells of the CA3 region of the hippocampus. The CA3 neurons have axonal projections called Schaffer collaterals that project out of the hippocampus via the fornix and also to neurons within an area of the hippocampus called CA1, which are also neurons that serve as an output of the hippocampus.

Image showing the different cells and connections within the hippocampus. Details in caption and text.
Figure 49.1. Rodent hippocampal trisynaptic circuit. Neurons from the entorhinal cortex serve as the input to the hippocampus. The axons of these cells form the perforant path (orange). The entorhinal cortex neurons synapse onto granule cells in the dentate gyrus (red). The dentate gyrus cells extend their axons, called mossy fibers, to cells in a region of the hippocampus called CA3. The CA3 neurons (purple) extend axons, called Shaffer collaterals, to another area of the hippocampus called the CA1 region (blue). ‘Rodent hippocampal trisynaptic circuit’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Long-Term Potentiation (LTP)

Long-term potentiation (LTP) is a long-lasting increase in synaptic strength, measured by the amplitude of the post-synaptic potential. Plasticity can be measured throughout these connections, but for our purposes we will examine how to create LTP within the Schaffer collateral.

To create LTP within the Schaffer collateral, a brief electrical stimulus must be provided to the presynaptic axons coming from the CA3 cells via a stimulating electrode. The EPSP generated in the postsynaptic CA1 neurons is then measured with a recording electrode to establish a baseline.

Image of the hippocampus and the placement of stimulating and recording electrodes to measure LTP in the Schaffer collateral. Details in caption and text.
Figure 49.2. Inducing Schaffer collateral LTP. To induce LTP through the Schaffer collateral, a stimulating electrode is positioned to electrically excite the Schaffer collaterals, which are also the axon terminal of the CA3 neurons. A recording electrode is placed postsynaptically in the CA1 neurons. First, baseline measurements are taken by providing brief stimulation to the CA3 neurons and recording the amplitude of the EPSP postsynaptic response. After establishing a baseline, a tetanus is delivered the the CA3 neuron, consisting of short burst of high frequency stimulation. Following the delivery of the tetanus, a single brief stimulation is provided to the CA3 neuron and the postsynaptic response in the CA1 neuron is measured. An increased amplitude of the EPSP in the CA1 neuron indicates LTP. ‘Inducing Schaffer collateral LTP’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Using Hebb’s theory about plasticity, if “cells that fire together, wire together,” then it stands that repeated stimulation of that synapse would induce a rewiring of the connection, resulting in LTP. To induce LTP within these cells, a tetanus, or a very intense electrical stimulation consisting of 100 stimulations a second (100 Hz) is delivered to the presynaptic CA3 cells for 3 seconds. Following the delivery of tetanus, a single stimulus is provided again and the EPSP is then measured in the postsynaptic CA1 neuron. The delivery of the high frequency stimulation results in an increased amplitude of the postsynaptic EPSP in response to a single stimulus, demonstrating that LTP is a measurable phenomenon.

Graph of EPSP magnitude over time showing LTP. Details in caption and text.
Figure 49.3. Graphing LTP. In this graph the magnitude of the postsynaptic EPSP is being measured on the y-axis over time. After establishing a baseline, a high frequency stimulation (tetanus) is delivered. After the high frequency stimulation, the magnitude of the postsynaptic response increases and remains elevated for a long-period of time, indicating LTP. ‘Graphing LTP’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

In many cases, LTP is shown graphically by measuring the EPSP amplitude as a percent of the control EPSP amplitude. Prior to tetanus delivery, the amplitude will be measured as 100%, or the same as control. Following tetanus, the amplitude of the EPSP will be larger than it was baseline, for instance 130% indicating a 30% increase in amplitude. Increasing the amplitude of the postsynaptic EPSP increases the likelihood of a neuron firing an action potential by increasing the neuron membrane potential such that it is closer to the threshold potential for the cell.

It has been demonstrated in rodents that the elevated postsynaptic EPSP response is long-lasting and can remain elevated for upwards of one year. In humans, we theorize that some synaptic connections may remain potentiated for our entire lifetime, however investigating this is in humans is ethically constrained.

Graph of postsynaptic EPSP before and after high frequency stimulation, graphed as a percent of control. Details in caption and text.
Figure 49.4. Graphing postsynaptic response as a percent of control. Here, the postsynaptic EPSP amplitude is being measured as a percent of the control measurements. Prior to the high frequency stimulation, the EPSP amplitude is 100%. Following the high frequency stimulation, the EPSP amplitude is increased above the control, indicating that there is a larger postsynaptic response. In this example, the EPSP amplitude remains elevated for upwards of one year of time, indicating that LTP can be long-lasting. ‘Graphing postsynaptic response as a percent of control’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Glutamate Receptors and LTP

Long-lasting changes in synaptic strength, such as the LTP, are made possible through a series of molecular and cellular level changes. One form of LTP results from a change in the types of glutamatergic receptors. Of the three classes of ionotropic glutamate receptors, two are important for this form of LTP: the AMPA and the NMDA receptors (Chapter 16).

When a molecule of glutamate binds to the active site of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor, the ligand-gated ion channel changes to the open conformation and allows the cations, sodium and potassium, to cross the cell membrane. Sodium moves into the cells more than potassium leaves the cell, leading to depolarization. The NMDA (N-methyl-D-aspartate) receptor requires the binding of glutamate to open, but it is also dependent on voltage. The pore of the NMDA ligand-gated ion channel is blocked by a molecule of magnesium when the membrane potential is below or near resting membrane potential, preventing ions from moving through the channel. Once the cell depolarizes, the magnesium block is expelled from the receptor, which allows sodium, potassium, and calcium to cross the membrane.

The voltage change needed to open the NMDA receptor is usually a result of AMPA receptor activation. Released glutamate binds to both AMPA and NMDA receptors, and sodium influx occurs through open AMPA channels, which depolarizes the cell enough to expel the magnesium ion and allow ion flow through the NMDA receptors.

Images showing the movement of ions through AMPA and NMDA receptors. Details in caption and text.
Figure 49.5. AMPA and NMDA. (A) When below the voltage threshold, glutamate binds to AMPA receptors, causing the ionotropic receptor to open and allow passage of both sodium and potassium. The movement of sodium into the cell dominates, indicated by the bold arrow. However, the binding of glutamate to the NMDA receptor does not allow the movement of ions across the membrane due to a magnesium ion blocking the pore of the channel. (B) When the membrane is depolarized above the threshold voltage of the NMDA receptor, the magnesium ion is expelled from the pore of the channel, allowing movement of calcium, sodium, and potassium through the NMDA channel. The movement of calcium and sodium into the cell dominates, indicated by the bold arrows.

LTP and Intracellular Calcium

The calcium that enters the cell through the open NMDA receptors activates various kinases including, protein kinase A (PKA), protein kinase C (PKC), and calcium-calmodulin-dependent kinase II (CAMKII). Kinases phosphorylate target proteins within the cell, including AMPA receptors. Phosphorylation of AMPA receptors increases their conductance, allowing more ions to pass through the receptors and leading to a greater degree of depolarization. Further, increased intracellular calcium can lead to the insertion of additional AMPA receptors into the postsynaptic cell membrane, which will also allow more sodium to move into the cell and lead to increased depolarization.

Glutamatergic synapse showing calcium influx through NMDA receptors and the effects of calcium within the cell. Details in caption and text.
Figure 49.6. Calcium and LTP. Calcium enters the cells through NMDA receptors. When intracellular calcium levels are high, the calcium activates protein kinases within the cell. The protein kinases phosphorylate AMPA receptors expressed on the cell surface, increasing their conductance and allowing more sodium to enter the cell. Kinases also cause additional AMPA receptors to be inserted into the membrane of the cell, thereby increasing the available receptors that glutamate can bind. ‘Calcium and LTP’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Long-Term Depression (LTD)

Long-term potentiation increases the strength of synapses. Changes in activity can also cause synapses to be weakened through the process of long-term depression (LTD). Again, using the Schaffer collateral as an example, long-term depression can be induced at the synapses between the CA3 and CA1 neurons by replacing the tetanus stimulation with low-frequency stimulation for longer periods of time. For instance, a 15 min exposure to 1 Hz stimulation will lead to a decreased EPSP amplitude.

Through LTP and LTD, synapses demonstrate bidirectional plasticity that is dependent on the type of stimulation that the synapse receives.

Graph of EPSP magnitude over time showing LTD. Details in caption and text.
Figure 49.7. Graphing LTD.  In this graph the magnitude of the postsynaptic EPSP is being measured on the y-axis over time. After establishing a baseline, a low frequency stimulation is delivered. After the low frequency stimulation, the magnitude of the postsynaptic response decreases and remains lowered for a long-period of time, indicating LTD. ‘Graphing LTD’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

LTD and Intracellular Calcium

Even with low-frequency stimulation of the synapse, glutamate will bind to post-synaptic NMDA receptors and allow for the influx of calcium into the cell. Weak depolarization of the membrane leads to a low amount of calcium entering the cell, which activates a separate class of enzymes call protein phosphatases, specifically protein phosphatase 1 and protein phosphatase 2, that remove phosphate groups from target proteins. Further, LTD causes AMPA receptors to be internalized, or removed from the postsynaptic membrane. Removal of AMPA receptors will decrease excitability.

Image of a glutamatergic synapse showing influx of calcium through NMDA receptors and the effects of low levels of calcium on protein phosphatases. Details in caption and text.
Figure 49.8. Calcium and LTD. Calcium enters the cells through NMDA receptors. When intracellular calcium levels are low, the calcium activates protein phosphatases within the cell. The protein phosphatases de-phosphorylate AMPA receptors expressed on the cell surface, decreasing their conductance of sodium. Further, phosphatases cause AMPA receptors to be internalized and removed from the cell surface, thereby decreasing the available receptors that glutamate can bind. ‘Calcium and LTD’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Relating LTP to Learning

To test how LTP And LTD are directly related to memory, a robust model of learning is used called Inhibitory Avoidance. In an inhibitory avoidance task, a rat learns to associate an environment with an aversive experience. Typically, a two-chamber compartment is used with one light side and one dark side. The animal is permitted to move from the light chamber to the dark chamber, where it receives an electric foot shock. After just one trial with this protocol, the animal will learn to avoid the dark chamber where it received the foot shock.

One trial of inhibitory avoidance led to measurable LTP within the Schaffer collaterals, demonstrating that the behavioral learning corresponded to LTP during this task. Administration of an NMDA receptor blocker to the hippocampus prior to inhibitory avoidance training results in the animals being unable to learn that the dark chamber leads to a foot shock and inhibition of the corresponding LTP.

Image of the inhibitory avoidance protocol and a graph of LTP following one trial of inhibitory avoidance. Details in caption and text.
Figure 49.9. Inhibitory avoidance. In this task, rodents learn to associate an environment with an aversive experience, in this case a foot shock. In a two-chamber compartment, one side is light and one side is dark to distinguish the two compartments.  When the animal is placed in the dark compartment it receives an electric foot shock. After just one trial with this protocol, the animal learns to avoid the dark chamber where it received the foot shock. One trial of inhibitory avoidance led to measurable LTP within the Schaffer collaterals, demonstrating that the behavioral learning corresponded to LTP during this task. ‘Inhibitory avoidance’ by Valerie Hedges is licensed under a Creative Commons Attribution Non-Commercial Share-Alike (CC-BY-NC-SA) 4.0 International License.

Key Takeaways

  • Plasticity in the form of LTP and LTD occurs at both inhibitory and excitatory synapses.
  • The hippocampus has a trisynaptic circuit of connections.
  • Long-term potentiation (LTP) is a long-term strengthening of a synapse, whereas long-term depression (LTD) is a long-term weakening of a synapse.
  • LTP is demonstrated by observing an increased EPSP amplitude following a tetanus.
  • LTD is demonstrated by observing a decreased EPSP amplitude following low frequency stimulation.
  • AMPA and NMDA glutamate receptors are important in LTP.
  • High levels of intracellular calcium from high frequency stimulation activates protein kinases, which leads to increased conductance of AMPA receptors and insertion of AMPA receptors in the cell membrane.
  • Low levels of intracellular calcium from low frequency stimulation activates protein phosphatases, which leads to decreased conductance of AMPA receptors and removal of AMPA receptors in the cell membrane.
  • Inhibitory avoidance is a robust learning model in which animals only need one trial before learning occurs. It has been show to also induce LTP.

Attributions

This chapter is adapted from “Molecular Mechanisms of Memory: Hippocampus” in Introduction to Neuroscience by Valerie Hedges which is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

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Introduction to Neurobiology Copyright © 2024 by Avinash Singh is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted.

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