L6 Pharmacological Dissection of Field Responses PDF

Pharmacological dissection of field responses @Arturas Volianskis Pharmacological dissection of field responses Learning outcomes: 1. Introduce hippocampal slice preparation 2. Discuss field EPSPs in the CA1 area of the Schaffer collaterals...

Pharmacological dissection of field responses @Arturas Volianskis Pharmacological dissection of field responses Learning outcomes: 1. Introduce hippocampal slice preparation 2. Discuss field EPSPs in the CA1 area of the Schaffer collaterals and their analyses 3. Discuss pharmacological isolation and characterisation of excitatory field potentials in the hippocampus 4. Discuss the measurements and quantification of PPF, STP and LTP 5. Discuss quantitative pharmacological and physiological studies using field potentials. BI2432: Fundamental neuropharmacology Pharmacological dissection of field responses Villers & Ris, JOVE 2013, http://www.jove.com/video/50483, doi:10.3791/50483 BI2432: Fundamental neuropharmacology 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. Adapted from: Tidball, P. et al. (2017) Differential ability. Brain Neurosci Adv 1, 239821281668979. BI2432: Fundamental neuropharmacology The tri-synaptic circuit of the hippocampus 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 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. Skrede, K. K. & Westgaard, R. H. The transverse hippocampal slice: a well-defined cortical structure maintained in vitro. Brain Res 35, 589–593 (1971). BI2432: Fundamental neuropharmacology Pyramidal cells of the hippocampus The cell bodies of the granule cells (not shown here) 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. The Hippocampus Book BI2432: Fundamental neuropharmacology Current source density (CSD) analysis C 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. The Hippocampus Book BI2432: Fundamental neuropharmacology Extracellular recording in the hippocampal slice f-EPSP St.p. responses Stim. PS 1 mV 1 ms PS St.r. responses AV f-EPSP 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. Volianskis, A. & Jensen, M. S. Transient and J Physiol (Lond) 550, 459–492 (2003) BI2432: Fundamental neuropharmacology Basic pharmacology of evoked f-EPSPs f-EPSPs, evoked by stimulation of the Schaffer collaterals, are dependent on 0 mM Ca2+ 4 mM Mg2+ Kynurenate, 5 mM release of neurotransmitter and can be abolished by removal of extracellular Ca2+. 1 mV 1 mV Note that the axonal fibre volley (AV) is not dependent on Ca2+ and can be seen 1 ms 1 ms returning back to baseline, after inhibition of the f-EPSP. This demonstrates that there is a component of the f-EPSP that is AV amplitude 10 µM NBQX contaminated by the FV and also vice versa. 125 f-EPSP slope 1 µM TTX f-EPSP and AV (% of baseline) a b c f-EPSPs are also dependent on the opening 100 of ionotropic receptors, as demonstrated by the application of a broad spectrum 75 excitatory amino acid inhibitor, kynurenic acid. Kynurenate abolishes f-EPSP whilst 50 preserving AV. 25.25 mV AV is also seen in isolation from f-EPSP in 0 1 ms experiments in which AMPA and kainate 0 10 20 30 40 receptor antagonist NBQX is applied to the Time (min) preparation whereas inhibition of sodium channels, whilst using TTX, abolishes the fibre volley. Volianskis, A. & Jensen, M. S. Transient and J Physiol (Lond) 550, 459–492 (2003) & unpublished. BI2432: Fundamental neuropharmacology Paired pulse facilitation of f-EPSPs When two stimuli are given at a short inter- pulse interval, IPI (e.g. 20 – 500 ms) the f-EPSP 1 (a, b) 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- 1 mV EPSP2 / slope of EPSP1 * 100) can be used f-EPSP 2 (c, d) 5 ms to estimate the amount of paired pulse facilitation (PPF). 300 80 ms IPI Application of GABAA receptor antagonist 250 picrotoxin has no effect on the measurement f-EPSP (%) & PPF (%) of the early slope of the f-EPSPs, after the 200 t e r m i n a t i o n o f t h e f i b r e v o l l e y. T h i s 150 demonstrates that inhibitory neurotransmission ac bd does not affect the measurement of f-EPSP 100 and that the strength of excitatory neurotransmission can be reliably quantified. 50 100 µM Picrotoxin 0 Please note the epilptiform activity produced 0 15 30 45 by application of picrotoxin. It manifests itself Time (min) in the St.r. as positive going deflections of the population spikes, which also reduce the maximal negative amplitudes of the f-EPSPs. Volianskis, A. & Jensen, M. S. Transient and J Physiol (Lond) 550, 459–492 (2003) BI2432: Fundamental neuropharmacology Short- and long-term potentiation of f-EPSPs The f-EPSP slope, recorded in the St.r., f-EPSP increases after high frequency stimulation is a St.p. responses applied to the Schaffer collaterals (e.g. theta- burst stimulation, TBS [4 pulses @ 100Hz Stim. PS repeated 10 times @ 5 Hz frequency]). b 1 mV High frequency stimulation also decreases the latency and increases the amplitude of 1 ms population spikes, recorded in St.p.. PS St.r. responses a AV Population spikes reduce the amplitude of f- f-EPSP b EPSPs recorded in the St.r., indicating that the early slope of f-EPSPs (after the termination of 250 the AV) is the appropriate measure of the Slope (Rate of rise) of fEPSP efficacy of synaptic transmission. Amplitude of fEPSP STP f-EPSP (% of baseline) 200 LTP Distortions of the measurements become less 150 significant as the magnitudes of f-EPSPs and the latencies of PSs decrease with time. 100 Notably, measurements of the peak amplitude 0.5 mV of f-EPSP result in under-estimation of the TBS 5 ms magnitude and duration of short-term 50 a b c d potentiation (STP). Measurements of long- 0 0.5 1 1.5 2 term potentiation (LTP) are somewhat less Time (h) affected by the method of measurement. Volianskis et al. J Physiol (Lond) 550, 459–492 (2003) & Brain Res 1621, 5–16 (2015). BI2432: Fundamental neuropharmacology Pharmacological isolation of field responses Ingram, R. et al. Some distorted thoughts Neuropharmacology 142, 30–40 (2018). BI2432: Fundamental neuropharmacology Case story - perampanel A Perampanel (0.3 µM) 150 f-EPSP slope (% of baseline) CTZ (100 µM) 10 µM NBQX Baseline NBQX (10 µM) 0.3 µM 100 Peramp. 0.2 mV Baseline 5 ms 50 0.3 µM Peramp. 0 CTZ (100 µM) AMPAR f-EPSP 0 30 60 90 120 Time (min) B Perampanel (1 µM) C Perampanel (10 µM) 150 150 f-EPSP slope (% of baseline) f-EPSP slope (% of baseline) No stim ( ) No stim ( ) Baseline 100 100 50 50 Stim. throughout Stim. throughout Stim. off for 30 min Stim. off for 30 min during washin during washout 0 0 0 30 60 90 0 60 120 180 240 300 Time (min) Time (min) Effects of perampanel on AMPAR-mediated transmission are not use dependent. (A) Application of 0.3 𝜇M perampanel resulted in a reduction of AMPA receptor-mediated f-EPSPs (filled circles, application of compounds in this and subsequent figures are indicated by thick lines above the trace). The waveforms from this experiment (inset to the right), visualise the effects of 0.3 𝜇M perampanel (red) and 10 𝜇M NBQX (green) on AMPA receptor-mediated control responses (black). Note that application of 100 𝜇M cyclothiazide (CTZ, blue), in the presence of perampanel, had no effect on the slope of AMPA receptor mediated f-EPSPs, but prolonged their decay time. (B) Inhibition by perampanel of AMPA receptor-mediated f-EPSPs did not require stimulation during the wash-in (open vs. closed circles, as indicated) of the compound (n = 2 and 3, with and without the delay respectively). (C) Recovery during wash-out of perampanel was also independent of the stimulation (n = 2 and 5, with and without the delay respectively). Ceolin, L. et al. A novel anti-epileptic agent, perampanel. Neurochemistry International 61, 517–522 (2012). BI2432: Fundamental neuropharmacology Perampanel is more potent than GYKI 52466 A (A) A series of experiments showing concentration- 150 Perampanel 3 µM Perampanel dependent inhibition of AMPA receptor-mediated f- f-EPSP slope (% of baseline) 0.3 µM EPSPs by perampanel (4–7 slices per group, mean ± S.E.M.). 0.01 𝜇M perampanel (open circles) had no Baseline 100 0.2 mV Control (n=4) Baseline 5 ms significant effect on AMPA receptor-mediated f- 50 0.01µM (n=5) 0.1 µM (n=4) 100 µM GYKI EPSPs when compared to the control (filled circles, P > 0.05, NKMCT), whereas application 0.1 𝜇M (or any 0.3 µM (n=4) 1 µM (n=5) 10 µM 3 µM (n=6) 0 10 µM (n=7) 0.2 mV higher concentration) reduced f-EPSPs significantly Baseline 5 ms (filled squares, P < 0.001, NKMCT). Application of 3 0 30 60 90 𝜇M perampanel resulted in a complete inhibition of Time (min) AMPA receptor-mediated synaptic transmission. Inset B GYKI 52466 C shows representative waveforms from two individual 150 f-EPSP slope (% of baseline) Inhibition of f-EPSP (%) 100 Perampanel experiments using perampanel (0.3 𝜇M; red) and Baseline GYKI 52466 100 75 GYKI52466 (GYKI; 10 𝜇M; red). The green traces for Control (n=4) 1µM (n=4) 50 3 𝜇M perampanel and 100 𝜇M GYKI are from separate experiments and are shown to illustrate their 3 µM (n=4) 50 10 µM (n=4) 25 complete inhibition of the AMPA receptor mediated 30 µM (n=4) 100 µM (n=4) 0 0 IC50 = 0.23 µM (0.18-0.3) responses. IC = 7.82 µM (5.9-10.4) 50 0 30 60 90 Ctrl 0.001 0.1 10 1000 (B) Effects of GYKI52466 on AMPA receptor- Time (min) Antagonists (µM) mediated f-EPSPs (4 slices per group). (C) Perampanel (IC50 = 0.23 𝜇M) is 34-times more potent than GYKI52466 (IC50 = 7.82 𝜇M, P < 0.001, confidence intervals for the IC50 values are given in parentheses). Ceolin, L. et al. A novel anti-epileptic agent, perampanel. Neurochemistry International 61, 517–522 (2012). BI2432: Fundamental neuropharmacology Perampanel has no effect on NMDA receptor-mediated f-EPSPs A Perampanel (10 µM, b) B 150 Inhibition of NMDA f-EPSP (%) f-EPSP amplitude (% of baseline) AP5 (1 µM, c) ** Baseline (a) 100 AP5 (30 µM) 100 75 50 50 0.2 mV 25 20 ms 0 0 NMDAR f-EPSP NBQX (10 µM ) 0 30 60 90 10 µM 1 µM Time (min) Peramp. AP5 (A) a group of experiments showing that 10 𝜇M perampanel has no effect on NMDA receptor-mediated f-EPSP (98.9 ± 1.9% of baseline response) whereas 1 𝜇M D-AP5 provides a significant inhibition of NMDA receptor-mediated f-EPSP (58.5 ± 3.4%) and 30 𝜇M D-AP5 provides a full block (n = 4). (B) Effects of 10 𝜇M perampanel and 1 𝜇M D-AP5 differed significantly (P < 0.01, paired t- test). Ceolin, L. et al. A novel anti-epileptic agent, perampanel. Neurochemistry International 61, 517–522 (2012). BI2432: Fundamental neuropharmacology Perampanel has no effect on kainate receptor-mediated responses A B n.s. s1 s2 s3 s4 s5 ** Popspike amplitude (%) f-EPSP 100 0.5 mV 75 100 µM GYKI, 50 µM AP5, 50 µM PTX 100 ms 50 + 10 µM NBQX 5th popspike + 1 µM TTX 25 0 0.5 mV GYKI, AP5, PTX Ctrl 10 µM 1 µM 10 µM + 10 µM Perampanel 100 ms NBQX TTX Peramp. (A) Top panel shows representative waveforms from an experiment in which activation of kainate receptors induced facilitation of the population spike (black), which was blocked by 10 𝜇M NBQX (red). The residual, non-synaptic component of the response was blocked by 1 𝜇M TTX (green). Bottom panel depicts that perampanel (10 𝜇M, blue) had no effect on facilitation of mossy fibre responses. (B) For presentation purposes the control data from the two sets of experiments that are depicted in panel A were pooled (n = 8, Ctrl). Application of perampanel did not significantly affect the size of the fifth population spike when compared to the control (88.0 ± 13.4%, n = 4, P = 0.4, paired t test) whereas NBQX abolished the fifth population spike evoked through activation of kainate receptors (30.9 ± 7.6%, n = 4, P = 0.03, paired t test). The residual component of the response in NBQX was blocked by TTX (4.4 ± 2.0%). Ceolin, L. et al. A novel anti-epileptic agent, perampanel. Neurochemistry International 61, 517–522 (2012). BI2432: Fundamental neuropharmacology Example questions L6: Q1: What is the molecular target of AP5? (A) NMDA receptor (B) Voltage gated Na+ channel (C) AMPA receptor (D) Voltage gated K+ channel (E) Kainate receptor BI2432: Fundamental neuropharmacology Study materials: BI2432: Fundamental neuropharmacology Weekly schedule of the fundamental neuropharmacology Friday 29.11.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L1. Introduction to fundamental neuropharmacology Rang & Dale’s Pharmacology, L2. Basic principles of neuropharmacology I & lecture materials Friday 06.12.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L3. Basic principles of neuropharmacology II Rang & Dale’s Pharmacology L4. Techniques in neuropharmacology & lecture materials Friday 10.12.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L5. Acetylcholine and Glutamate (and a bit of Glycine) Rang & Dale’s Pharmacology L6. Pharmacological dissection of field responses The Hippocampus Book pages 27-30 & lecture materials Tuesday 07.01.2025 (13:10-14:00);C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L7. GABA and Glycine Rang & Dale’s Pharmacology & lecture materials Friday 10.01.2025 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L8. Catecholamines Rang & Dale’s Pharmacology L9. Serotonin & lecture materials Friday 27.01.2025 (13:00-13:45 & 14:00-14:45); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L10. Neuropharmacology of drug dependence and addiction I Rang & Dale’s Pharmacology L11. Neuropharmacology of drug dependence and addiction II & lecture materials Tuesday 21.01.2025 (13:10-14:00); C/-1.04 Tuesday 23.01.2025 Neuroanatomy L12. Exam preparation 2 and Neuropharmacology ICA BI2432: Fundamental neuropharmacology