CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND 4. SYNAPTIC TRANSMISSION II: GLUTAMATERGIC TRANSMISSION Video 4-1: Observations and glutamate receptor channels
Synaptic transmission II 1 Constance Hammond
Observation 2 whole-cell configuration current-clamp mode stimulation glutamatergic presynaptic neuron recording postsynaptic response
Excitatory postsynaptic potential (EPSP) 3 whole-cell configuration current-clamp mode stimulation action potential recording excitatory postsynaptic potential 2 ms EPSP = transient depolarization of the postsynaptic membrane
Excitatory postsynaptic potential (EPSP) 4 control + APV 2 mv 2 ms EPSP APV APV (40 µm) modifies the EPSP time course
Excitatory postsynaptic potential (EPSP) 5 + APV 2 mv 2 ms EPSP APV + NBQX + APV + NBQX 1 µm APV + NBQX (1 µm) abolish the EPSP
Excitatory postsynaptic potential (EPSP) 6 + APV 2 mv 2 ms EPSP + APV + NBQX 1 µm APV + NBQX Sometimes : residual response
Excitatory postsynaptic potential (EPSP) 7 + APV control + APV 2 mv + APV + NBQX 1 µm + APV + APV + NBQX 1 µm
Glutamate receptors 8 ionotropic receptors (= receptor channels) metabotropic receptors
Structure of a glutamate receptor-channel 9 NH 2 glutamate N-terminal domain agonist ligand-binding domain out out transmembrane domains 4 subunits in in COOH C-terminal domain
Structure of a glutamate receptor-channel 10 3 types of glutamate receptor-channels: AMPA (GluA1-4 subunits) Kainate (GluK1-5 subunits) NMDA (GluN1-2A-D subunits) agonist out NH 2 N-terminal domain ligand-binding domain transmembrane domains in COOH C-terminal domain
Take home message 11 3 types of glutamate receptorchannels: AMPA, NMDA & Kainate AMPA receptors are made up of four GluA subunits (GluA1 to 4) NMDA receptors are made up of 4 GluN subunits (GluN1, GluN2A-D) Kainate receptors are made up of 4 GluK subunits (GluK1-5)
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND 4. SYNAPTIC TRANSMISSION II: GLUTAMATERGIC TRANSMISSION Video 4-2: Unitary AMPA receptor-mediated current
AMPA glutamate receptor-channel 12 glutamate O O HO OH AMPA HO NH 2 O NH 2 OH N O
Unitary AMPA receptor-mediated current 13 outside-out configuration voltage-clamp mode AMPA i o AMPA
Unitary AMPA receptor-mediated current 14 AMPA Voltage-independent AMPA receptor channel Cation current
Unitary AMPA receptor-mediated current 15 glutamate out in C O
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND 4. SYNAPTIC TRANSMISSION II: GLUTAMATERGIC TRANSMISSION Video 4-3: Total AMPA receptor-mediated current
Total AMPA receptor-mediated current 16 whole-cell configuration voltage-clamp mode spontaneous synaptic glutamatergic currents recording Glutamate afferent GABA afferent
Total AMPA receptor-mediated current 17 whole-cell configuration voltage-clamp mode spontaneous synaptic glutamatergic currents recording To isolate AMPA currents Blockers: gabazine (10 µm), GABA channel antagonist APV (40 µm) vs NMDA channel antagonist specific blocker of kainate channels, if necessary Glutamate afferent GABA afferent
Total AMPA receptor-mediated current and AMPA EPSP 18 The total spontaneous AMPA current is inward. decay
Summary on AMPA receptor-channels 19 Agonists: - glutamate - AMPA Antagonist: NBQX (1 µm) Voltage-independent Permeable to Na + and K + ions Permeability to Ca 2+ ions depends on the presence of the GluA2 subunit (see appendix)
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND 3. SYNAPTIC TRANSMISSION II: GLUTAMATERGIC TRANSMISSION Video 4-4: Unitary NMDA receptor-mediated current
NMDA receptor channel 20 glutamate O O HO OH NH 2 NMDA O HO HN OH
Unitary NMDA receptor-mediated current 21 outside-out configuration voltage-clamp mode
Unitary NMDA receptor-mediated current 22 outside-out configuration voltage-clamp mode
NMDA receptor blockade by Mg 2+ ions 23 outside-out configuration voltage-clamp mode Extracellular Mg 2+ ions block NMDA receptors at hyperpolarized potentials.
NMDA receptor blockade by Mg 2+ ions 24 glutamate Ca 2+ Mg 2+ Na + K + Voltage-sensitive Mg 2+ blockade
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND 4. SYNAPTIC TRANSMISSION II: GLUTAMATERGIC TRANSMISSION Video 4-5: Total NMDA receptor-mediated current
Total NMDA receptor-mediated current 25 whole-cell configuration voltage-clamp mode recording spontaneous synaptic currents Glutamate afferent GABA afferent
Total NMDA receptor-mediated current 26 whole-cell configuration voltage-clamp mode recording spontaneous synaptic currents To isolate NMDA currents: Blockers: gabazine (10 µm) vs GABA channels CNQX (10 µm) vs AMPA/KA channels V H = +40 mv (no Mg 2+ blockade) Glutamate afferent GABA afferent
Total NMDA receptor-mediated current 28 NMDA receptors are permeable to Ca 2+ ions and blocked by Mg 2+ ions.
Total NMDA receptor-mediated current 29 I N
NMDA channel opening in physiological conditions 30
Summary on NMDA receptor-channels 31 Agonists: - glutamate - NMDA Antagonist: APV Voltage-dependent (due to Mg 2+ blockade - see appendix) Permeable to cations: Na +, K + and Ca 2+
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND 4. SYNAPTIC TRANSMISSION II: GLUTAMATERGIC TRANSMISSION Video 4-6: Conclusion
Overview of glutamatergic transmission 32 Presynaptic membrane : - Ca 2+ channels - anchoring proteins - synaptic vesicle release sites - glutamate transporters (uptake) Postsynaptic membrane: - AMPA and/or KA and/or NMDA receptor-channels - mglur metabotropic receptors postsynaptic element
Overview of glutamatergic transmission 33 3 types of glutamate receptor-channels: AMPA, KA and NMDA. All composed of 4 subunits (tetramers) AMPA receptors: GluA1 to GluA4 subunits NMDA receptors: 2 GluN1 subunits + 2 GluN2A to D subunits
Overview of glutamatergic transmission 34 2 types of AMPA receptors: With the GluA2 subunit: permeable to Na + and K + cations impermeable to Ca 2+ ions (see appendix) the most common AMPA receptors Without the GluA2 subunit: permeable to Na +, K + and Ca 2+ cations (see appendix)
Overview of glutamatergic transmission 35 NMDA receptors Combined binding of two ligands: glutamate on GluN2 (A-D) subunits glycine on the GluN1 subunit (see appendix) Permeable to Na +, K + and Ca 2+ cations Voltage-sensitive (blockade by Mg 2+ ions at hyperpolarized potentials - see appendix) Slow kinetics
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND Professor Constance Hammond Project manager Isabelle Virard Graphic designer Géraldine Fohr Movie director Marine Chabrolin Music Tetoma - Hicham Chahidi Translation Isabelle Virard et Atenao 2016