Master/PhD in Neurosciences Faculdade de Medicina Universidade de Lisboa 2009/ /10/2009 Diogo Rombo Credit: Graham Johnson, Graham Johnson Medical Media, Boulder, Colorado

The human body is an integrated group of systems

e.g. Gap junctions between adjacent cells of the cardiac muscle Contact dependent signaling e.g.. Notch signalling, that controls cell differentiation during embryonic development

Targets only cells in the vicinity of the emitting cell e.g. Neurotransmitters or Growth factors Long distance signalling in both plants & animals e.g. Hormones

Purves, 2004 Bidirectional transmission Extremely fast Direct cell-to-cell contact (gap junctions) Unidirectional transmission Paracrine signalling

Neurotransmitter Chemical Synapse Presynaptic cell Postsynaptic cell Signal transmission between neurons is mediated by Ion Channels Produce transient electrical signals that are essential for carrying time-sensitive information rapidly over long distances

Extracell Intracell V R = 8,31 J/K. Mol T = 310 K (~37ºC) F = C/mol

Extracell Intracell V R = 8,31 J/K. Mol T = 310 K (~37ºC) F = C/mol

= -57 mV V Na = 56 mV V Cl = -76 mV V K = -102 mV

Threshold Threshold Excitatory cellInhibitory cell GABA R Target cell Glu R [Na + ] [Cl - ]

Threshold Resting potential Action Potential Resting membrane potential – Selective permeability to K + (ion channels) and and large K + concentration gradient (active transport). 2 Action potential – Membrane becomes very permeable to Na + (Activation of voltage-dependent Na + channels). 3 Repolarization – The rise in Na + permeability is transient and the membrane becomes again primarily permeable to K + (Na + channels inactivate). 3 Hyperpolarization – The K + conductances hyperpolarize the membrane (by gradually opening the voltage-gated K + channels).

20 mV 200 ms Study of the electrical properties of biological cells and tissues, through measurements of voltage changes or electrical current flows on a wide variety of scales (from single ion channel proteins to whole tissues like the heart)

The voltage clamp technique is used to measure ion currents across a neuronal membrane while holding (clamping) the membrane potential at a set level

Axon of giant squid First preparation used to voltage clamp a transmembrane current Axon of giant squid First preparation used to voltage clamp a transmembrane current

Biological preparation: Living organismExcised tissue (acute or cultured) Dissociated cells from excised tissue

- Recording from single cell - Recording from cell population

Associative memory and learning Very easily removed from the brain Well defined circuitry Ramón y Cajal, 1911

Wistar rat Lopes, LV, 2003 Accutely prepared hippocampal slices Extracellular recordings Patch-clamp recordings Tissue Chopper Vibratome

Electrical stimulation of the Schaffer collateral commissural fibbers Results from delivery of the electrical stimulus Complex field potential that results from the sum of the action potentials fired by the afferent fibers stimulated Negative wave resulting from the sum of inhibitory and excitatory postsynaptic potentials recorded from that group of postsynaptic neurons

Changes in the initial slope of fEPSPs are taken as a measure of synaptic strength, as they are less prone to contamination

Neuron Gigaseal ( GΩ) Ohm’s Law V = I x R -5mV Low voltage square wave applied to the pipette to monitor seal resistance When the pipette comes into close contact with the cell surface the current decreases → increase in seal resistance Gentle suction is applied to the pipette to obtain a giga-seal Any current now recorded will have to pass across the cell membrane, through the opening of membrane ion pores or channels

1) Allows the study of ion channel behavior on the membrane section attached to the recording electrode; 2) One can manipulate the environment affecting the intracellular portion of ion channels e.g. Range of [cAMP] to study channels that are activated by intracellular cAMP

1) Allows perfusing the same patch with different solutions 2) With ligand-gated channels, one can obtain dose-response curves 3) If the membrane patch is small enough, it is possible to perform single- channel recordings of neuronal activity

1) Allows the control of both the bath solution and internal milieu of the recorded cell 2) Permits the study of the electrical behavior of the entire cell as a whole Disadvantage → Dialysis of cytoplasmic contents

Recording electrode is filled with a solution containing an antibiotic (Nystatin, Amphotericin-B, Gramicidin...) Small perforations are made on the patch of membrane attached to the electrode - Reduces the dialysis of cell contents that occurs in whole cell recordings 1) Higher access resistances, which decreases current resolution and increases recording noise. 2) A min period can occur before the antibiotic perforates the membrane. 3) The patch of membrane under the electrode tip is weakened by the perforations and tends to rupture → whole-cell mode (and with antibiotic inside the cell).

Experimental set-up for patch-clamp recordings

Recording membrane potential variations (e.g., AP activity) while keeping electrical current through the recording electrode very small Physiological tool to distinguish different cell types (e.g., interneurons from pyramidal cells) Physiological tool to distinguish different cell types (e.g., interneurons from pyramidal cells) Stratum radiatum IN Stratum oriens IN CA1 pyramidal cell

Recording electrode axon Spontaneous miniature postsynaptic currents (mEPSCs or mIPSCs) Recording electrode axon Stimulation electrode Excitatory Postsynaptic Currents (EPSCs) Pressure ejection system “Puff” Recording electrode Muscimol evoked postsynaptic currents (PSCs)

Recording electrical signals arising from individual ion channels - Study of drug interactions with ionic channels; - Channel gating mechanisms; - Study of drug interactions with ionic channels; - Channel gating mechanisms;

The patch pipette is used to load the recorded cell with a fluorescent dye (e.g., Lucifer Yellow) The dye spreads by diffusion along the cell, revealing both its morphology and synaptic contacts with other cells

Applied suction Harvested mRNA Reverse transcriptase cDNA construct PCR Amplified cDNA

GiGi GSGS GSGS GiGi Adenylate cyclase ATP cAMP A3A3 A1A1 A 2A A 2B Inhibition Stimulation Adapted from

PresynapticallyPostsynaptically Extra-synaptically

Neuron GABA A Cl - CL - Cl - Pressure ejection system Recording electrode Muscimol Amplifier 100 pA 200 ms Gabazine Control Muscimol

GSGS A 2A GABA A mediated postsynaptic currents are not changed by activation of adenosine A 2A receptors! Pharmacological Tools: CGS – A 2A receptor agonist CPA – A 1 receptor agonist Muscimol – GABA A receptor agonist DPCPX – A 1 receptor antagonist Gabazine – GABA A receptor antagonist

Activation of adenosine A 1 receptors inhibits GABA A mediated postsynaptic currents GiGi A1A1 Pharmacological Tools: CGS – A 2A receptor agonist CPA – A 1 receptor agonist Muscimol – GABA A receptor agonist DPCPX – A 1 receptor antagonist Gabazine – GABA A receptor antagonist

GiGi A1A1 GiGi A1A1 GiGi A1A1 GiGi A1A1 DPCPX facilitates recovery of GABA A mediated currents after inhibition by CPA Pharmacological Tools: CGS – A 2A receptor agonist CPA – A 1 receptor agonist Muscimol – GABA A receptor agonist DPCPX – A 1 receptor antagonist Gabazine – GABA A receptor antagonist

Pharmacological Tools: CGS – A 2A receptor agonist CPA – A 1 receptor agonist Muscimol – GABA A receptor agonist DPCPX – A 1 receptor antagonist Gabazine – GABA A receptor antagonist TTX – Na + channel blocker DL-AP5 – NMDA receptor antagonist CNQX – AMPA/Kainate receptor antagonist axon - TTX (500nM) - DL-AP5 (50uM) - CNQX (10uM)

“The human brain is a network of more than 100 billion individual nerve cells interconnected in systems that construct our perceptions of the external world, fix our attention, and control the machinery of our actions. A first step toward understanding the mind, therefore, is to learn how neurons are organized into signalling pathways and how they communicate by means of synaptic transmission.” In: Principles of Neural Science, by Kandel, Schwartz & Jessel

Curso Livre Experimental Neurociências Básicas Faculdade de Medicina 2009/ /09/2009 Diogo Rombo Credit: Graham Johnson, Graham Johnson Medical Media, Boulder, Colorado