Pharmacological dissection of field responses Flashcards
Hippocampal slice preparation
- Transverse hippocampal slices are prepared from either dorsal or ventral poles of rodent hippocampi
- The procedure involves the brain being cooled down to ~4 oC in artificial cerebrospinal fluid (ACSF).
- The hippocampi are dissected free and transverse slices are prepared using McIlwain Tissue Chopper.
- After the preparation the slices are allowed to recover at room temperature for ~ 2 hours, before recordings can be commenced.
How is the tri-synaptic circuitry well preserved in transverse hippocampal slices?
The hippocampus is arranged in a very orderly fashion therefore the tri-synaptic circuitry of the hippocampus is well preserved in transverse hippocampal slices.
The tri-synaptic circuit of the hippocampus
The granule cells (gc) receive synaptic input from the entorhinal cortex (pp) and send their axons (mossy fibres) to activate the CA3 pyramidal cells. The CA3 cell axons (the Schaffer collaterals, Sch) innervate the CA1 pyramidal cells.
Pyramidal cells of the hippocampus
The cell bodies of the granule cells and the CA3 and CA1 pyramidal neurons, and their dendrites and axons, are arranged in a “laminar” fashion
Thus, when stimulating the Schaffer collaterals at the boarder between CA3 and CA1 areas, predictable biological responses to single electric stimuli can be recorded the stratum pyramidale (st.p.) and in the stratum radiatum (st.r.) of the CA1 area.
Current source density (CSD) analysis:
A. 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.
B. CSD analysis reveals a sink (positive) in stratum oriens and a source (negative) in the cell body layer and proximal dendrites.
C. 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 fibre volley (afferent volley, AV) that reflects synchronised action potential discharges in the Schaffer collateral fibre bundle traveling past the recording electrode. The AV is followed by the field excitatory postsynaptic potential (f-EPSP) the slope of which, in the CA1 area of the St.r., is primarily mediated by AMPA receptors.
The stratum pyramidale (St.p.) waveform is composed of a positive f-EPSP that is followed by a negative going synchronised action potential discharge in the CA1 pyramidal neuron bodies (the population spike, PS). PS is positive in St.r. and affects the measurement of the peak negative amplitude of the f-EPSP.
Pharmacology of evoked f-EPSPs:
f-EPSPs, evoked by stimulation of the Schaffer collaterals, are dependent on release of neurotransmitter and can be abolished by removal of extracellular Ca2+. Note that the axonal fibre volley (AV) is not dependent on Ca2+ and can be seen returning back to baseline, after inhibition of the f-EPSP. This demonstrates that there is a component of the f-EPSP that is contaminated by the FV and also vice versa.
f-EPSPs are also dependent on the opening of ionotropic receptors, as demonstrated by the application of a broad spectrum excitatory amino acid inhibitor, kynurenic acid. Kynurenate abolishes f-EPSP whilst preserving AV.
AV is also seen in isolation from f-EPSP in experiments in which AMPA and kainate receptor antagonist NBQX is applied to the preparation whereas inhibition of sodium channels, whilst using TTX, abolishes the fibre volley.
Paired pulse facilitation of f-EPSPs:
When two stimuli are given at a short inter- pulse interval, IPI (e.g. 20 – 500 ms) the second response in the St.r. rises with a steeper slope than the first due to increased probability of neurotransmitter release. A ratio of the two slope measurements (slope of f- EPSP2 / slope of EPSP1 * 100) can be used to estimate the amount of paired pulse facilitation (PPF).
Application of GABAA receptor antagonist picrotoxin has no effect on the measurement of the early slope of the f-EPSPs, after the termination of the fibre volley. This demonstrates that inhibitory neurotransmission does not affect the measurement of f-EPSP and that the strength of excitatory neurotransmission can be reliably quantified.
Note: the epilptiform activity produced by application of picrotoxin. It manifests itself in the St.r. as positive going deflections of the population spikes, which also reduce the maximal negative amplitudes of the f-EPSPs
Short- and long-term potentiation of f-EPSPs:
The f-EPSP slope, recorded in the St.r., increases after high frequency stimulation is applied to the Schaffer collaterals (e.g. theta- burst stimulation, TBS [4 pulses @ 100Hz repeated 10 times @ 5 Hz frequency]).
High frequency stimulation also decreases the latency and increases the amplitude of population spikes, recorded in St.p..
Population spikes reduce the amplitude of f- EPSPs recorded in the St.r., indicating that the early slope of f-EPSPs (after the termination of the AV) is the appropriate measure of the efficacy of synaptic transmission.
Distortions of the measurements become less significant as the magnitudes of f-EPSPs and the latencies of PSs decrease with time. Notably, measurements of the peak amplitude of f-EPSP result in under-estimation of the magnitude and duration of short-term potentiation (STP). Measurements of long- term potentiation (LTP) are somewhat less affected by the method of measurement.
Pharmacological isolation of field responses:
Important for quantification of drug effects on plasticity
Ketamine gets rid of STP but preserves LTP in the slice —> we can inhibit STP selectively without affecting LTP (component of plasticity associated with working memory —> inhibition can lead to secondary effects in the brain)
Perampanol
Epilepsy treatment
Effects of perampanel on AMPAR-mediated transmission are not use dependent
Does perampanol have an effect on NMDA receptor-mediated f-EPSPSs?
No, had no effects in NMDA receptor0mediated f-ESPSPs
Whereas 1 𝜇M D-AP5 provides a significant inhibition of NMDA receptor-mediated f-EPSP and 30 𝜇M D-AP5 provides a full block
Does perampanol have an effect on Kianate receptor-mediated responses?
No, had no effect on facilitation of mossy fibres
Whereas NBQX abolished the fifth population spike evoked through activation of kainate receptors
What is the molecular target of AP5?
NMDA receptors
