Neuroscience: Hippocampal Slice Preparation

Pharmacological Dissection of Field Responses

• This study focuses on pharmacological analysis of responses within the hippocampus.
• Methods used include hippocampal slice preparation.
• The study analyses excitatory field potentials within the hippocampus, focusing on precise measurements and quantification of PPF, STP, and LTP.
• The learning outcomes involve introducing hippocampal slice preparation, discussing field EPSPs (Excitatory Postsynaptic Potentials) in the CA1 area of the Schaffer collaterals and their analysis, pharmacological isolation and characterization of excitatory field potentials in the hippocampus
• Quantitative pharmacological and physiological studies using field potentials are further analyzed.

Hippocampal Slice Preparation

• Transverse hippocampal slices from rodent hippocampi (dorsal or ventral poles) are used.
• Slices are prepared in artificial cerebrospinal fluid (ACSF) at ~4°C.
• The hippocampi are dissected and transverse slices are created using a McIlwain Tissue Chopper.
• Recovery of the slices occurs at room temperature for 2 hrs before recordings begin.

The Tri-Synaptic Circuit of the Hippocampus

• The hippocampus has a well-organized tri-synaptic circuit preserved in transverse hippocampal slices.
• Granule cells receive synaptic input from the entorhinal cortex.
• They send mossy fibers to CA3 pyramidal cells which then activate the CA3 pyramidal cells.
• The axon of CA3 pyramidal cells (Schaffer collaterals) innervate CA1 pyramidal cells.

Pyramidal Cells of the Hippocampus

• Granule cell bodies, CA3 and CA1 pyramidal neurons, and their dendrites and axons are arranged in a laminar fashion.
• When stimulating Schaffer collaterals near the CA3/CA1 border, predictable biological responses are recorded in the stratum pyramidale (st.p.) and stratum radiatum (st.r.) of the CA1 area.

Current Source Density (CSD) Analysis

• Field potentials evoked by weak activation (single electric stimuli) of fibers projecting to the stratum oriens in area CA1 are recorded.
• The CSD analysis illustrates a sink in the stratum oriens and source in the cell body layer and proximal dendrites.
• Extracellular field EPSPs (Excitatory Postsynaptic Potentials) reflect intracellular events, showing a phase advance relative to the intracellular EPSPs.

Extrascellular Recording in the Hippocampal Slice

• Stratum radiatum (St.r.) responses consist of a negative presynaptic fiber volley (afferent volley), reflecting synchronized action potentials in the Schaffer collateral fibers.
• This is followed by the f-EPSP (field excitatory postsynaptic potential), whose slope is primarily mediated by AMPA receptors within the CA1 area of the St.r.).
• A positive f-EPSP is followed by a negative-going synchronized action potential discharge in CA1 pyramidal neuron bodies (population spike).

Basic Pharmacology of Evoked f-EPSPs

• f-EPSPs depend on neurotransmitter release but can be abolished by removing extracellular Ca2+.
• f-EPSPs are also dependent on ionotropic receptors; this is demonstrated using kynurenic acid (a broad-spectrum excitatory amino acid inhibitor) which abolishes f-EPSPs but preserves the afferent volley.
• Using AMPA and kainate receptor antagonist (NBQX) and sodium channel blocker (TTX) isolates the afferent volley from the fEPSP.

Paired Pulse Facilitation of f-EPSPs

• When two stimuli are applied at a short inter-pulse interval (e.g.,20-50ms), the second response (2nd f-EPSP) shows an increased slope compared to the first f-EPSP.
• This is due to increased neurotransmitter release probability.
• The ratio of slope of the 2nd EPSP to 1st EPSP (x100) can estimate paired pulse facilitation.
• An application of GABAA receptor antagonist (picrotoxin) has no effect on the early slope of f-EPSPs after termination of the fiber volley.

Short- and Long-Term Potentiation of f-EPSPs

• High-frequency stimulation (e.g., theta-burst stimulation) increases the f-EPSP slope recorded in the stratum radiatum (St.r.).
• The latency of population spikes is decreased, and their amplitude is increased with high frequency stimulation.
• Population spikes reduce the amplitude of f-EPSPs recorded in the stratum radiatum, indicating the early slope of f-EPSPs is appropriate measure of synaptic transmission.
• Short-term potentiation (STP) and long-term potentiation (LTP) measurements are less affected by measurement distortions.

Pharmacological Isolation of Field Responses

• Various experiments on pharmacological isolation of field responses demonstrate the effects of different drugs.
• These investigations examine the time course of potentiation and inhibition (e.g., by UBP145, a ketamine analogue; Ket) and the corresponding IC50 values for various substances (e.g., ketamine).

Case Story - Perampanel

• Perampanel is an AMPA receptor antagonist with an independent effect on AMPA-mediated f-EPSPs that doesn't require stimulation.
• The effects of perampanel, on AMPA receptors are unrelated to use-dependence.
• Perampanel has no effect on NMDA receptors or kainate receptor-mediated responses.

Perampanel is More Potent than GYKI 52466

• Perampanel exhibits concentration-dependent inhibition of AMPA receptor-mediated f-EPSPs.
• Perampanel shows significantly stronger inhibition compared to GYKI 52466 (IC50 values).

Perampanel has No Effect on NMDA Receptor-Mediated f-EPSPs

• Perampanel has no effect on NMDA receptor-mediated f-EPSPs, in contrast to D-AP5, an NMDA receptor antagonist.

Perampanel Has No Effect on Kainate Receptor-Mediated Responses

• Perampanel does not affect kainate receptor-mediated responses as demonstrated using NBQX and TTX blocking.

Example Questions L6

• The molecular target of AP5 is the NMDA receptor.

This quiz focuses on the key aspects of preparing transverse hippocampal slices for research purposes. It covers the significance of slice preparation techniques, circuit components involved, and the physiological processes that are measured. Test your knowledge on this essential methodology in neuroscience.