Neuroscience: Hippocampal Slice Preparation

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary purpose of preparing transverse hippocampal slices in this context?

To allow for analysis of field responses and excitatory field potentials (correct)

To facilitate the recovery of brain tissue at lower temperatures

To enable the manipulation of mossy fiber synapses

To enhance synaptic input from CA3 pyramidal cells

Which component of the brain does NOT receive direct input from the entorhinal cortex in the tri-synaptic circuit?

Granule cells

CA1 pyramidal cells (correct)

CA3 pyramidal cells

Mossy fibers

What is the significance of cooling the brain to ~4°C during slice preparation?

It enhances the excitability of the mossy fibers

It allows for increased synaptic transmission in the CA1 area

It improves blood flow to the hippocampal region

It prevents enzymatic degradation during dissection (correct)

After preparation, how long are the hippocampal slices allowed to recover before recording begins?

2 hours Signup and view all the answers

Field potentials are analyzed to measure which of the following phenomena?

Long-term potentiation (LTP), short-term potentiation (STP), and paired-pulse facilitation (PPF) Signup and view all the answers

What is the function of the Schaffer collaterals in the hippocampal circuitry?

They connect CA3 pyramidal cells to CA1 pyramidal cells. Signup and view all the answers

What role does artificial cerebrospinal fluid (ACSF) play in slice preparation?

It maintains ionic balance and provides nutrients to the brain tissue. Signup and view all the answers

What effect did 0.3 µM perampanel have on AMPA receptor-mediated synaptic transmission?

It resulted in complete inhibition. Signup and view all the answers

Which compound was used as a comparative drug to perampanel in the experiments?

GYKI 52466 Signup and view all the answers

At what concentration did GYKI 52466 exhibit its effects in the experiments?

10 µM Signup and view all the answers

What was the primary measured outcome in the experiments involving perampanel and GYKI 52466?

Inhibition of f-EPSP slope Signup and view all the answers

What does the term f-EPSP refer to in the context of the experimental results?

Fast excitatory postsynaptic potential Signup and view all the answers

What effect does high-frequency stimulation have on the latency of population spikes?

Decreases latency Signup and view all the answers

How does the amplitude of population spikes change with high-frequency stimulation?

Increases in amplitude Signup and view all the answers

What is indicated by the early slope of f-EPSPs following the termination of AV?

Serves as a measure of synaptic transmission efficacy Signup and view all the answers

What happens to the amplitude of f-EPSPs as population spikes are recorded?

It decreases due to population spikes Signup and view all the answers

What trend is observed regarding the magnitudes of f-EPSPs over time?

Become less significant in measurements Signup and view all the answers

Which of the following best describes the relationship between high-frequency stimulation and synaptic distortion?

Reduces significance of synaptic distortion Signup and view all the answers

What is suggested by the reduction in latency and increase in amplitude with high-frequency stimulation?

Enhanced synaptic efficacy Signup and view all the answers

With a high frequency of stimulation, what characteristic of f-EPSPs is seen regarding their early slope?

They represent synaptic transmission efficacy Signup and view all the answers

What can be inferred about the relationship between amplitude and latency in population spikes and f-EPSPs over time?

Lower amplitudes lead to shorter latencies Signup and view all the answers

What does the term f-EPSP slope likely refer to in this context?

The rate of change in the postsynaptic potential Signup and view all the answers

Based on the data, what effect does Perampanel at 10 µM have on the f-EPSP slope compared to baseline?

It decreases the f-EPSP slope significantly Signup and view all the answers

Which drug concentration is indicated as potentially effective in impacting f-EPSP slope?

10 µM Perampanel Signup and view all the answers

What might you infer about the 'No stim' condition in the experiment?

It indicates a baseline response without stimuli Signup and view all the answers

How long was the observed response measured in this experiment?

60 minutes Signup and view all the answers

What annotation follows the peak response in the provided data?

Stim. Signup and view all the answers

What does a higher f-EPSP slope percentage relative to baseline suggest?

Increased postsynaptic activity Signup and view all the answers

What concentration of Perampanel corresponds to the most significant observed decrease in f-EPSP slope?

10 µM Signup and view all the answers

What does the amount '0.3 µM' represent in the context of the data?

A concentration threshold of CTZ Signup and view all the answers

Which time point would logically show maximum f-EPSP activity after stimulus application?

Immediately after stimulation Signup and view all the answers

What is the primary method used to isolate and study field responses in neuronal studies, as alluded to in the content?

Pharmacological Isolation Signup and view all the answers

According to the content, what is the primary difference between short-term potentiation (STP) and long-term potentiation (LTP)?

LTP is more susceptible to underestimation when measured using peak amplitude. Signup and view all the answers

Based on the data provided on perampanel, what is the effect of 0.3 µM perampanel on the f-EPSP slope percentage relative to baseline?

A statistically significant decrease in f-EPSP slope Signup and view all the answers

What is the likely role of the compound NBQX in the experiment shown in the data on perampanel?

To block NMDA receptors and isolate AMPA receptor signaling Signup and view all the answers

What is the most likely interpretation of the data point marked as CTZ (100 µM) with respect to the study of perampanel?

CTZ acts as a reference point to quantify the effects of perampanel in isolation. Signup and view all the answers

Which of the following best describes the principle of 'pharmacological isolation' in relation to the study of perampanel?

Isolating perampanel's effect on synaptic transmission from other neurotransmitters. Signup and view all the answers

What is the most likely reason for the underestimation of STP magnitude and duration when using peak amplitude measurements, as mentioned in the content?

Peak amplitude measurements rely on a single data point, failing to account for the integrated change in amplitude over time. Signup and view all the answers

Given the information about underestimation of STP when using peak amplitude, which measurement technique would likely yield a more accurate representation of STP?

Calculating the area under the f-EPSP curve. Signup and view all the answers

If the researchers wanted to investigate the potential involvement of GABA receptors in the effects of perampanel on f-EPSPs, which additional experimental approach would be most appropriate?

Using a GABA receptor antagonist in conjunction with perampanel. Signup and view all the answers

Based on the content, what is the most likely relationship between the methods of measurement and the accuracy of assessing both STP and LTP?

The accuracy of measurements depends on the specific experimental conditions and the duration of the studied phenomena. Signup and view all the answers

Study Notes

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.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

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.