Presentation on theme: "Biophysics 702 Patch Clamp Techniques Stuart Mangel, Ph.D."— Presentation transcript:
1Biophysics 702 Patch Clamp Techniques Stuart Mangel, Ph.D. LEARNING OBJECTIVESPrinciples that underlie different electrical recording techniquesPhysiological and biophysical information the techniques provide1. Extracellular recording and multi-electrode arrays- spiking (all-or-none) information, neural codes conveyed by individual neurons and by groups of neurons2. Intracellular recording- measurements of input resistance, synaptic input, and synaptic integration3. Patch-clamp recording (cell-attached; whole-cell; inside-out patch; outside-out patch)- measurements of input resistance, synaptic input, synaptic integration; characteristics of voltage-gated ion channels and single ion channel events
2EXTRACELLULAR VS. INTRACELLULAR RECORDING Extracellularly and intracellularly recorded voltages arein the microvolt and millivolt ranges, respectively.
3Maintaining the resting membrane potential The Goldman-Hodgkin-Katz (GHK) Equation:The steady state membrane potential for a given set of ionic concentrations inside and outside the cell and the relative permeability of the membrane to each ionVm = lnRTFpK[K+]o + pNa[Na+]o + pCl[Cl-]ipK[K+]i + pNa[Na+]i + pCl[Cl-]oextracellularintracellularENa = +56Na+ (150)EK = -102K+ (3)ECl = -76Cl- (120)ECa = +125Ca2+ (1.2)Na+ (18)K+ (135)Cl- (7)Ca2+ (0.1 µM)Na+,K+-ATPase-60 to -75 mVNSCC
4INTRACELLULAR RECORDING Measuring EMMeasure the potential difference between two electrodes using a D.C. amplifierExpected value of the membrane potential is in millivolts (not microvolts), so the gain does not need to be as high
5Intracellular Recording When a fine-tipped electrode penetrates the membrane of a cell, one observes a sudden change in the measured potential to a more negative value.Typical problemsHigh impedance μEDamage when cell penetrated
6MEASURING THE INPUT RESISTANCE Wheatstone BridgeUsed to measure an unknown resistanceDiscovered by Hunter Christie, 1833Popularized by Charles Wheatstone
7BALANCING THE BRIDGE R1 = Fixed R R2 = Variable R R3 = Fixed R R4 = Unknown R?To get R2/R1 = R4/R3,adjust R2, so that there isno current across B, CR4 = (R2/R1)·R3
8CALCULATING THE INPUT RESISTANCE OF A CELL “Balanced”“Out of Balance”Balance the bridge before entering the cellAfter impaling the cell,the bridge is “out of balance” by the R value of the cellI is known, measure V, and calculate R using Ohm’s Law (V = IR)R = V/IDid R increase or decrease?Did channels open or close?
9PATCH-CLAMP RECORDING Neher and Sakmann, Nobel Prize, 1991Tremendous technical breakthrough that improved the signal to noise ratio of the recordingRecord from whole cells or from a small patch of cell membrane, so only a few ion channels (or one) can be studiedHigh resistance (in giga-ohms) and high mechanical strength of the seal between the glass electrode and the cell membrane enable one to observe very small currents.The diameter of the tip of patch electrodes can be larger than that of fine-tipped intracellular microelectrodes (1.0 micron vs microns), so that the resistance of patch electrodes is lower (e.g. 5 MΩ vs 200 MΩ). The lower resistance of patch electrodes makes voltage clamping easier.
18Inhibitory actions of GABA synapses result from the opening of ion channels selective for Cl-
19ROLE OF ION TRANSPORTERS IN NEURAL NETWORK FUNCTION GABA-evoked depolarizationGABA-evoked hyperpolarizationFig. 1Fig. 3Fig. 3. The GABA reversal potential at the starburst amacrine cell (SAC) distal dendrite is more hyperpolarized than at the proximal dendrite due to KCC2 activity. (A, B) GABA was applied onto the proximal dendrite (A) and onto the distal dendrite (B) ~ 100 m from the cell body of a SAC in the presence of cobalt (2 mM) to block synaptic transmission. (C) Average EGABA of the proximal and distal dendrites of SACs were significantly different (p < 0.01). (D) Average EGABA of distal dendrites before and during bath application of FUR (25 M), a selective inhibitor of KCC2 activity, were significantly different (p < 0.01).Fig. 1. The chloride cotransporters, Na-K-2Cl (NKCC) and K-Cl (KCC2), determine whether the neurotransmitter GABA, which opens Cl- channels, depolarizes or hyperpolarizes neurons, respectively.Fig. 2Fig. 2. The dendrites of starburst amacrine cells (green), a type of interneuron in the retina, hyperpolarize to light stimuli that move from the periphery to the cell body (bottom left) and depolarize to light stimuli that move from the cell body to the periphery (bottom right). These directionally-selective responses are generated in part by the differential distribution of the Na-K-2Cl (NKCC) cotransporter (pink) on the cell body and proximal dendrites and the K-Cl (KCC2) cotransporter (blue) on the distal dendrites. The expression patterns of Na-K-2Cl and K-Cl are represented as pink to purple and purple to blue gradients, respectively, on the dendrites and cell body of this starburst cell.- modified from Gavrikov et al., 2006, PNAS
20SODIUM CHANNEL CURRENTS RECORDED FROM CELL-ATTACHED PATCH