Hippocampal Slice Preparation and Field Responses
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Questions and Answers

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

  • Increase in synaptic transmission
  • Complete inhibition of synaptic transmission (correct)
  • Partial inhibition of synaptic transmission
  • No effect on synaptic transmission

In the experiments, what dosage of GYKI 52466 was used?

  • 10 𝜇M (correct)
  • 20 𝜇M
  • 15 𝜇M
  • 5 𝜇M

Which parameter was measured to assess the effect of perampanel?

  • Synaptic transmission frequency
  • Calcium ion flux
  • EPSP slope (correct)
  • Receptor density

Which outcome was not associated with the application of 0.3 𝜇M perampanel according to the provided information?

<p>Increased f-EPSP slope (B)</p> Signup and view all the answers

What does f-EPSP stand for in the context of this study?

<p>Field excitatory postsynaptic potential (D)</p> Signup and view all the answers

How does high-frequency stimulation affect the population spikes recorded in St.p?

<p>It decreases the latency and increases the amplitude. (D)</p> Signup and view all the answers

What does a decrease in amplitude of f-EPSPs recorded in St.r indicate?

<p>A reduction in synaptic transmission efficacy. (A)</p> Signup and view all the answers

Which measure is considered appropriate for assessing the efficacy of synaptic transmission after the termination of AV?

<p>Early slope of f-EPSPs. (A)</p> Signup and view all the answers

What happens to distortions of measurements over time concerning f-EPSPs and PSs?

<p>They become less significant. (D)</p> Signup and view all the answers

What does an increase in amplitude of population spikes suggest about neural activity?

<p>Increased synaptic efficacy. (C)</p> Signup and view all the answers

How is the slope of f-EPSP measured?

<p>After the termination of AV. (A)</p> Signup and view all the answers

What is the consequence of high-frequency stimulation on the efficacy of synaptic transmission?

<p>It enhances the efficacy of synaptic transmission. (C)</p> Signup and view all the answers

What is primarily observed in St.r in response to population spikes?

<p>Reduced amplitude in f-EPSPs. (B)</p> Signup and view all the answers

What concentration of perampanel was applied to reduce AMPA receptor-mediated f-EPSPs?

<p>0.3 µM (A)</p> Signup and view all the answers

Which compound does not affect the slope of AMPA receptor-mediated f-EPSPs when present with perampanel?

<p>100 µM cyclothiazide (CTZ) (B)</p> Signup and view all the answers

What was observed regarding the effectiveness of perampanel during the wash-in period?

<p>It did not require stimulation. (C)</p> Signup and view all the answers

During the wash-out of perampanel, what condition was noted regarding recovery?

<p>Recovery was independent of stimulation. (D)</p> Signup and view all the answers

Which statement is true regarding perampanel’s effect on AMPA receptor-mediated responses?

<p>It reduces AMAP receptor-mediated f-EPSPs. (C)</p> Signup and view all the answers

What does 'n = 2 and 3' signify in the context of the study findings?

<p>The number of experimental trials conducted. (C)</p> Signup and view all the answers

Which effect did application of 0.3 µM perampanel NOT produce?

<p>Dependency on prior stimulation. (B)</p> Signup and view all the answers

What does the term 'IC50' represent in the context of drug inhibition?

<p>The concentration required to achieve half-maximal inhibition (C)</p> Signup and view all the answers

What was the IC50 value reported for the AMPA receptor inhibition?

<p>0.23 µM (A)</p> Signup and view all the answers

Which experimental condition displayed the highest concentration of drug used?

<p>100 µM (A)</p> Signup and view all the answers

What does the notation 'n=4' signify in the experimental groups?

<p>Four samples were used in each condition (A)</p> Signup and view all the answers

How many different drug concentrations were tested for the control group in the experiments?

<p>3 (A)</p> Signup and view all the answers

What type of receptor is being studied through the experiments described?

<p>AMPA receptor (B)</p> Signup and view all the answers

What is the significance of the green traces in the provided data?

<p>They show the results of complete inhibition tests (A)</p> Signup and view all the answers

What is the implication of the range given with the IC50 value (0.18-0.3 µM)?

<p>The value indicates variability among different trials (A)</p> Signup and view all the answers

What type of cells make up the primary structure of the hippocampus?

<p>CA1 and CA3 pyramidal neurons (B)</p> Signup and view all the answers

When stimulating the Schaffer collaterals, which layers of the CA1 area show predictable responses?

<p>Stratum radiatum and stratum pyramidale (C)</p> Signup and view all the answers

What characterizes the field potentials recorded from the stratum oriens in area CA1 during weak fiber activation?

<p>Evoking a pure synaptic response (A)</p> Signup and view all the answers

What is the significance of the sink and source identified in current source density (CSD) analysis?

<p>The sink represents positive events, while the source indicates negative ones. (B)</p> Signup and view all the answers

Which receptor type is primarily responsible for mediating the slope of the field excitatory postsynaptic potential (f-EPSP) in the CA1 area?

<p>AMPA receptors (B)</p> Signup and view all the answers

What does the presynaptic fiber volley (affernet volley) from the stratum radiatum indicate?

<p>Synchronous action potential discharges in Schaffer collateral fibers (D)</p> Signup and view all the answers

What structure is mainly involved when recording responses in the CA1 hippocampal slice?

<p>Hippocampal fimbria (A)</p> Signup and view all the answers

What is the primary response observed when the Schaffer collaterals are stimulated?

<p>Predictable biological responses recorded in specific layers (B)</p> Signup and view all the answers

What was the effect of 10 𝜇M perampanel on NMDA receptor-mediated f-EPSP?

<p>It had no effect on the response. (C)</p> Signup and view all the answers

What was the percentage of baseline response for f-EPSP at 1 𝜇M D-AP5?

<p>58.5 ± 3.4% (A)</p> Signup and view all the answers

How did the effects of 10 𝜇M perampanel and 1 𝜇M D-AP5 compare statistically?

<p>They differed significantly with P &lt; 0.01. (D)</p> Signup and view all the answers

What does the data suggest about perampanel's effect on kainate receptor-mediated responses?

<p>It has no effect on kainate receptor-mediated responses. (C)</p> Signup and view all the answers

At what concentration does D-AP5 provide a full block of NMDA receptor-mediated f-EPSP?

<p>30 𝜇M (A)</p> Signup and view all the answers

Which of the following drugs was used in conjunction with 10 𝜇M perampanel in the experiments?

<p>NBQX (A)</p> Signup and view all the answers

What was the purpose of using a paired t-test in the experiment?

<p>To determine if there were significant differences in responses. (B)</p> Signup and view all the answers

What does the abbreviation f-EPSP stand for?

<p>Fast excitatory postsynaptic potential (A)</p> Signup and view all the answers

Flashcards

Perampanel's effect on AMPA receptors

A drug that completely blocks the transmission of signals through AMPA receptors at a concentration of 3 𝜇M.

f-EPSP slope

A measure of the rate of change of the f-EPSP, which reflects the strength of synaptic transmission.

AMPA receptor

A type of glutamate receptor critical for excitatory synaptic transmission in the brain.

GYKI 52466

A drug known to inhibit AMPA receptors, but with a different mechanism than perampanel.

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f-EPSP (field excitatory postsynaptic potential)

A type of electrical signal generated in a neuron that reflects the strength of the synaptic connection.

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Long-term potentiation (LTP)

A type of synaptic plasticity where the strength of a synapse is increased following repeated stimulation.

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Synaptic depression (STP)

A type of synaptic plasticity where the strength of a synapse is decreased following repeated stimulation.

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Slope of the f-EPSP

A measure of the efficacy of synaptic transmission. It is the rate of rise of the f-EPSP after the termination of the action potential.

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f-EPSP (fiber-evoked EPSP)

A brief, rapid depolarization of the postsynaptic membrane caused by the arrival of an action potential at the presynaptic terminal.

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Population spikes (PS)

A synchronous firing of a large number of neurons, which can be measured by the electrical activity of the brain.

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Amplitude of the f-EPSP

A measure of the size of the f-EPSP. It reflects the strength of the synaptic connection.

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Effect of HFS on population spikes

High-frequency stimulation (HFS) of a synapse can lead to a reduction in the latency and an increase in the amplitude of population spikes.

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Effect of HFS on f-EPSP amplitude

High-frequency stimulation (HFS) can reduce the amplitude of f-EPSPs, indicating a weakening of the synaptic connection.

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IC50

A measurement of the concentration of a drug that is required to inhibit a biological response by 50%.

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Concentration-response curve

A type of experiment where the effect of different concentrations of a drug is measured on a biological response.

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Antagonist

A chemical that binds to a receptor and blocks its activation.

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Complete inhibition

To completely prevent or stop a biological response from occurring.

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Perampanel

A drug that selectively blocks the AMPA receptor, preventing glutamate from activating it.

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Laminar organization of the hippocampus

The arrangement of neurons and their projections in the hippocampus, creating distinct layers with predictable responses to stimulation.

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Stratum pyramidale (st.p.)

The area of the hippocampus that receives input from the CA3 region, located just below the stratum radiatum.

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Stratum radiatum (st.r.)

The area of the hippocampus that receives input from the CA3 region, located above the stratum pyramidale.

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Current source density (CSD) analysis

A technique used to measure the electrical activity of different layers in the hippocampus by analyzing the changes in voltage across these layers.

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Afferent volley (AV)

The electrical signal generated by the synchronous firing of presynaptic neurons traveling towards the recording electrode.

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Field excitatory postsynaptic potential (f-EPSP)

The electrical signal generated by the synaptic activation of postsynaptic neurons, resulting in a change in their membrane potential.

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What is perampanel?

Perampanel is a drug that reduces the activity of AMPA receptors, which are involved in the transmission of excitatory signals in the brain.

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What are AMPA receptors?

AMPA receptors are a type of glutamate receptor that plays a crucial role in excitatory neurotransmission.

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What was the effect of perampanel in the experiment?

The experiment demonstrated that perampanel inhibits AMPA receptor-mediated f-EPSPs, leading to a decrease in excitatory synaptic transmission.

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What is f-EPSP?

f-EPSP refers to the excitatory postsynaptic potential, a change in the electrical potential of a postsynaptic neuron caused by excitatory neurotransmitters.

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Is the effect of perampanel dependent on stimulation?

The experiment showed that the inhibitory effect of perampanel on AMPA receptor-mediated f-EPSPs was not dependent on the presence of stimulation during the drug's application.

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What was observed during the washout period?

The study also found that the recovery of AMPA receptor-mediated f-EPSPs after perampanel washout was independent of stimulation.

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What is Cyclothiazide (CTZ)?

Cyclothiazide (CTZ) is a drug that prolongs the decay time of AMPA receptor-mediated f-EPSPs by blocking their deactivation.

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What is NBQX?

NBQX, an AMPA receptor antagonist, completely blocked the AMPA receptor-mediated f-EPSPs, confirming the specific action of perampanel on these receptors.

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Perampanel's Lack of Effect on f-EPSPs

The study found that 10 μM Perampanel had no impact on NMDA receptor-mediated f-EPSPs, essentially leaving them at their baseline level. This suggests that Perampanel doesn't interact with NMDA receptors.

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D-AP5 Effect on NMDA Receptors

D-AP5 is a drug that acts as an antagonist for NMDA receptors. At a low concentration (1 μM), D-AP5 significantly inhibits NMDA receptor activity, reducing f-EPSPs by almost half. A higher concentration (30 μM) completely blocks the activity of NMDA receptors.

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Statistical Test Comparison

A paired t-test was used to compare the effect of Perampanel and D-AP5 on f-EPSPs. The results of the t-test showed a significant difference between the two drugs, indicating that their effects were not the same.

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Perampanel's Lack of Effect on Kainate Receptors

Perampanel does not affect the response of neurons driven by kainate receptors. This is shown by the lack of change in the amplitude of popspikes (a type of neuronal firing) in the presence of Perampanel.

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What are Popspikes?

Popspikes are brief, highly synchronized bursts of neuronal firing often observed in brain slices. They are measured by their amplitude, which reflects their intensity.

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Drugs Used in the Experiment

The experiment used several drugs to block different types of receptors: GYKI to block AMPA receptors, AP5 to block NMDA receptors, PTX to block GABA receptors, NBQX to block AMPA and kainate receptors, and TTX to block sodium channels.

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Measuring Neuronal Activity

The experiment measured the amplitude of popspikes in brain slices, which is a measure of the intensity of neuronal firing. Changes in popspike amplitude indicate changes in neuronal activity.

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Study Notes

Hippocampal Slice Preparation

  • Transverse hippocampal slices are prepared from either dorsal or ventral poles of rodent hippocampi.
  • The brain is cooled to approximately 4°C in artificial cerebrospinal fluid (ACSF).
  • The hippocampi are dissected free.
  • Transverse slices are prepared using a McIlwain Tissue Chopper.
  • The slices are allowed to recover at room temperature for 2 hours before recordings.

Pharmacological Dissection of Field Responses

  • Learning Outcomes:
    • Introduce hippocampal slice preparation.
    • Discuss field EPSPs in the CA1 area of the Schaffer collaterals and their analyses.
    • Discuss pharmacological isolation and characterization of excitatory field potentials in the hippocampus.
    • Discuss measurements and quantification of PPF, STP, and LTP.
    • Discuss quantitative pharmacological and physiological studies using field potentials.

The Tri-synaptic Circuit of the Hippocampus

  • The hippocampus is arranged in a very orderly fashion.
  • The tri-synaptic circuitry of the hippocampus is well preserved in transverse hippocampal slices.
  • Granule cells (GCs) receive synaptic input from the entorhinal cortex (PP) and send axons (mossy fibers) to activate CA3 pyramidal cells.
  • CA3 pyramidal cells axons (Schaffer collaterals, Sch) innervate the CA1 pyramidal cells.

Pyramidal Cells of the Hippocampus

  • Cell bodies of granule cells (not shown), CA3, and CA1 pyramidal neurons, and their dendrites and axons are arranged in a laminar fashion.
  • When stimulating the Schaffer collaterals at the border between CA3 and CA1 areas, predictable biological responses to single electric stimuli can be recorded in the stratum pyramidale (st.p.) and the stratum radiatum (st.r.) of the CA1 area.

Current Source Density (CSD) Analysis

  • Field potentials evoked by weak activation of fibers projecting to stratum oriens in area CA1, evoking a pure synaptic response without a superimposed population spike.
  • CSD analysis reveals a sink (positive) in stratum oriens and a source (negative) in the cell body layer and proximal dendrites.
  • The extracellular field EPSP reflects intracellular events, but is phase-advanced with respect to the intracellular EPSP.

Extracellular Recording in the Hippocampal Slice

  • The stratum radiatum (St.r.) responses are comprised of a negative going presynaptic fiber volley (afferent volley, AV) that reflects synchronized action potentials.
  • The AV is followed by the field excitatory postsynaptic potential (f-EPSP), the slope of which is primarily mediated by AMPA receptors.
  • The St.p. waveform is composed of a positive f-EPSP that is followed by a negative going synchronized action potential discharge in CA1 pyramidal neuron bodies (population spike, PS).
  • PS is positive in the St.r. and affects the measurement of the peak negative amplitude of the f-EPSP.

Basic Pharmacology of Evoked f-EPSPs

  • f-EPSPs evoked by stimulation of the Schaffer collaterals are dependent on neurotransmitter release and can be abolished by removal of extracellular Ca2+.
  • f-EPSPs also depend on the opening of ionotropic receptors, demonstrated by application of a broad spectrum excitatory amino acid inhibitor (kynurenate).
  • Kynurenate abolishes f-EPSPs while preserving the afferent volley (AV).

Paired Pulse Facilitation of f-EPSPs

  • When two stimuli are given at a short interval (IPI), the second response has a steeper slope than the first due to increased probability of neurotransmitter release.
  • A ratio of the slopes of the two f-EPSPs can estimate the amount of paired pulse facilitation (PPF).
  • The application of GABAA receptor antagonist picrotoxin has no effect on early f-EPSP slope measurements, indicating that inhibitory neurotransmission does not affect f-EPSP measurements.
  • Picrotoxin application produces epileptiform activity.

Short- and Long-Term Potentiation of f-EPSPs

  • f-EPSP slope increases after high-frequency stimulation (e.g., theta-burst stimulation).
  • High-frequency stimulation decreases latency and increases amplitude of population spikes.
  • Population spikes reduce the amplitude of early f-EPSPs, indicating that the early slope of f-EPSPs is the appropriate measure of synaptic transmission efficiency.
  • Distortions of measurements become less significant over time, as magnitudes of f-EPSPs and latencies of population spikes decrease.

Pharmacological Isolation of Field Responses

  • Details of experiments and results related to pharmacological isolation of field responses and the effects on different types of responses are given.

Case Study - Perampanel

  • Perampanel's effect on AMPA receptor-mediated transmission is not use-dependent, meaning the action occurs independently of prior stimulation.
  • Application of perampanel decreases AMPA receptor-mediated f-EPSPs but does not affect their decay time.
  • Perampanel's action on AMPA receptor-mediated f-EPSPs is independent of stimulation during wash-in and wash-out.

Perampanel is More Potent than GYKI 52466

  • Perampanel (IC50 = 0.23 µM) is significantly more potent than GYKI 52466 (IC50 = 7.82 µM) in inhibiting AMPA receptor-mediated f-EPSPs.

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

  • Perampanel doesn't affect NMDA receptor-mediated f-EPSPs.
  • D-AP5 (NMDA receptor antagonist) significantly inhibits NMDA receptor-mediated f-EPSPs.

Perampanel Has No Effect on Kainate Receptor-Mediated Responses

  • Perampanel doesn't affect kainate receptor-mediated responses.
  • NBQX (kainate receptor antagonist) blocks the facilitation of population spikes evoked by activation of kainate receptors.

Example Questions L6

  • Q1: What is the molecular target of AP5?
    • (A) NMDA receptor

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L6 Pharmacological Dissection of Field Responses PDF

Description

This quiz covers the process of preparing transverse hippocampal slices from rodent brains and analyzing field responses in the CA1 area. You will explore pharmacological dissection, including the characterization of excitatory field potentials and the quantitative study of phenomena such as paired-pulse facilitation and long-term potentiation. Test your understanding of these crucial processes in neurophysiology.

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