Long-term potentiation in a hippocampal slice

The hippocampus is a curled structure deep in the temporal lobe that is needed to form new memories of places and events. Its main circuit runs in one direction: axons from the entorhinal cortex (the perforant path) excite the granule cells of the dentate gyrus; these excite the pyramidal cells of area CA3; and the axons of CA3 cells, called Schaffer collaterals, excite the pyramidal cells of area CA1. Because this circuit lies flat in a transverse slice, a slice about 0.4 mm thick keeps it intact and alive for many hours in oxygenated artificial cerebrospinal fluid (ACSF).

When the Schaffer collaterals are stimulated, thousands of synapses onto CA1 dendrites are activated together. An electrode placed among those dendrites, in the layer called stratum radiatum, records a small negative voltage called the field excitatory postsynaptic potential (fEPSP). Its initial slope is a measure of synaptic strength. Just before it comes a brief fibre volley, the signal of the stimulated axons themselves, which shows how many axons were activated.

In anaesthetised rabbits, brief trains of high-frequency stimulation of the perforant path potentiated the responses of the dentate gyrus for between 30 minutes and 10 hours (Bliss and Lømo, 1973): this lasting increase is long-term potentiation (LTP), a leading cellular model of memory. In rat hippocampal slices, the antagonist APV (also called AP5) left the Schaffer collateral fEPSP unchanged but prevented its potentiation by high-frequency stimulation (Collingridge et al., 1983), showing that induction of this LTP needs NMDA receptors; the same drug infused into the brain blocked LTP and impaired place learning in rats (Morris et al., 1986).

The experiment follows the classic design: control slices are compared with slices from animals allocated to AP5, with the animal, not the slice, as the unit of analysis. All recordings are simulated from a model.

Learning objectives

• Name the parts of the hippocampal slice (dentate gyrus, CA3, CA1, Schaffer collaterals and the layers of CA1) and explain why stimulating and recording electrodes both go in stratum radiatum
• Prepare healthy slices and explain what oxygenation, temperature, thickness and recovery do to a slice
• Read a field recording: stimulus artefact, fibre volley, fEPSP and its 10–90% slope, and recognise a population spike
• Build an input–output curve, choose a test intensity, and interpret the paired-pulse ratio as a presynaptic measure
• Record a stable baseline, induce LTP with high-frequency or theta-burst stimulation, and predict the effect of the NMDA-receptor antagonist AP5
• Analyse potentiation with animals as the unit, with exclusion criteria set in advance, and explain why counting slices as independent is pseudoreplication

Ethics and safety

Slices are taken from animals killed humanely by an approved method; the work needs approval by an Institutional Animal Ethics Committee and trained, authorised staff; in India such work is governed by the CCSEA under the Prevention of Cruelty to Animals Act 1960, with equivalent frameworks in the European Union (Directive 2010/63/EU), the United States (Animal Welfare Act and PHS Policy) and the United Kingdom (Animals (Scientific Procedures) Act 1986). The 3Rs apply: several slices from each animal reduce the number of animals, experiments are planned with enough animals to give a clear answer and no more, and simulations such as this one can replace some practice experiments. Studies are reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020), giving both the number of slices and the number of animals. Laboratory safety: the vibratome blade is very sharp, carbogen cylinders must be secured, and drugs such as AP5 are handled as the local risk assessment requires.

Key terms

• Hippocampal slice: A transverse slice of the hippocampus, about 0.3–0.4 mm thick, kept alive in oxygenated ACSF so that its circuit can be studied for several hours.
• ACSF: Artificial cerebrospinal fluid: a salt solution with glucose that matches the fluid around the brain, bubbled with carbogen (95% oxygen, 5% carbon dioxide) to supply oxygen and keep the pH near 7.4.
• Schaffer collaterals: The axons of CA3 pyramidal cells that run to area CA1 and make excitatory synapses on the dendrites of CA1 pyramidal cells in stratum radiatum.
• Stratum radiatum: The layer of CA1 just beneath the cell bodies, containing the apical dendrites of the pyramidal cells and the Schaffer collateral synapses onto them.
• Stratum pyramidale: The thin layer of CA1 and CA3 that contains the cell bodies of the pyramidal neurons.
• Field EPSP (fEPSP): The summed excitatory synaptic response of many neurons, recorded outside the cells; in stratum radiatum it is a negative wave whose initial slope measures synaptic strength.
• Fibre volley: A small, brief wave just before the fEPSP made by the action potentials of the stimulated axons; it shows how many axons were activated.
• Population spike: A sharp negative wave recorded near the cell bodies when many neurons fire action potentials together; it is not a measure of synaptic strength.
• Input–output curve: The response plotted against stimulus intensity, used to find the maximal response and to choose a test intensity.
• Paired-pulse ratio: The second of two responses divided by the first. A ratio above 1 (facilitation) generally suggests synapses with a low probability of transmitter release.
• LTP and LTD: Long-term potentiation and long-term depression: lasting increases or decreases in synaptic strength after particular patterns of activity.
• HFS, TBS and LFS: Conditioning protocols: high-frequency stimulation (100 pulses at 100 Hz), theta-burst stimulation (short bursts at 100 Hz repeated five times a second) and low-frequency stimulation (pulses at 1 Hz for 15 minutes).
• AP5 (APV): A selective antagonist of NMDA-type glutamate receptors, the receptors whose calcium entry triggers this form of LTP.
• Pseudoreplication: Treating measurements that are not independent, such as several slices from one animal, as if they were independent samples.