Electrophysiology of Synaptic Transmission: Membrane Potentials and Ion Channels, Lecture notes of Physiology

Download Electrophysiology of Synaptic Transmission: Membrane Potentials and Ion Channels and more Lecture notes Physiology in PDF only on Docsity! 12 Synaptic Electrophysiology of the Drosophila Neuromuscular Junction Bing Zhang1 and Bryan Stewart2 IDepartment of Zoology, University of Oklahoma, Norman, Oklahoma 73019; 'Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario LSL 1C6, Canada ABSTRACT Chemical synaptic transmission is an importa11t means of neuronal communication in the nerv­ ous system. On the arrival of an action potential, the nerve terminal experiences an influx of cal­ cium ions, which in turn trigger the exocytosis of synaptic vesicles (SVs) and the release of neuro­ transmitters into the synaptic cleft. Transmitters elicit synaptic responses in the postsynaptic cell by binding to and activating specific receptors. This is followed by the recycling of SV s at pre­ synaptic terminals. The Drosophila larval neuro­ muscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synap­ tic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mecha­ nisms of synaptic transmission. In this chapter, we describe the theory and practice of electro­ physiology as applied to the Drosophila larval Int roduct ion, 172 Box 1. Electrical Terminology, 172 Box 2. Basic Equations and RC Circu its, 173 The Resting Membrane Potential, 175 Synaptic Transmission, 181 Using Drosophila Larval NMj to Study Synaptic Transmission, 183 Protocol 1: Electrophys iological Recording from a "Model" Cell, 188 Protocol 2: Recording from Drosophila Larval Body Wall Muscles: Passive M embrane Properties and Basic Features of Synaptic Transmission, 193 Protocol 3: Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval NMj , 200 Box 3. A Few Words of Caution Concerni ng Voltage-Clamp Experiments, 201 Protocol 4: Focal Recording of Synaptic Currents from Single Boutons at the Larval NMj, 204 Protocol 5: Fabrication of Microelectrodes, Suct ion Electrodes, and Focal Electrodes, 206 Recipe, 210 Acknowledgments, 210 References, 211 WWW Resources: Suppliers of M ajor Electrophysiology Equipment, 213 NMJ preparation. First, the basics of membrane potentials are introduced, with an emphasis on the rest­ ing potential and synaptic potential. Second, the equipment and methods required to set up an electro­ physiology rig are presented. Third, protocols are provided that explain how to use the rig to record from muscles, determine the passive membrane properties of the muscle (i.e., input resistance and time con­ stant), record synaptic potentials both intracellularly and extracellularly, detect synaptic currents by two­ electrode voltage clamp, perform quantal analysis, and study short-term synaptic plasticity. 171 1 72 / Chapter 12 INTRODUCTION Neurons use electrical and chemical signals to receive, process, and communicate information. The basic currency of signaling by excitable cells (neurons and muscles ) is electrical current. Accordingly, electrophysiology is the study of the electrical properties of cells, and we draw on the theoretical background of electrical phenomenon to help understand the behavior of the nervous system. By analogy, the cell m embrane is similar to a simple resistor-capacitor (RC) circuit (see Boxes 1 and 2) . In an electronic circuit, a battery (voltage source) is connected via conductors (wires) to a resistor and a capacitor arranged in parallel, with a return wire to the battery that completes the circuit. Relatively simple physical relationships describe the flow of electrons through the wires. The cellu­ lar equivalents to an electronic circuit are the resting membrane potential (see below), which acts as the battery; the extracellular and intracellular fluids, which act as the low-resistance conductors; ion channels within cell m embranes, which act as the resistors; and the phospholipid bilayer, which acts as a capacitor. An important difference between electronic and cellular circuits is that electrons are the charged particles that flow through an electronic circuit, whereas in a biological circuit a variety of cations and anions (e.g., Na+, K+, Ca2+, Cl-) generate membrane currents. Drosophila has been used to great advantage as a model system for elucidating the molecular and genetic mechanisms that underlie bioelectric and biochemical signaling. The introduction of the lar­ val NMJ as an experimental preparation established a system that permits relatively easy access to large, electrically excitable cells (Jan and Jan 1976a,b). Other electrical techniques applied to Drosophila include electroretinograms (Hotta and Ben zer 1969; see Chapter 14), extracellular recording from the Box 1. ELECTRICAL TERMINOLOGY Charge (Q): Electri c charge is a fund amenta l prope lty of subatomic particles. By convention an elec­ tron has a charge of - 1 and a proton has a charge of + 1. In general , parti cles with the same charge repe l each other, and particles with opposite charges attract each othe r. The uni t of charge is known as the coulomb (C) and 1 C is equal to 6.25 x 1018 charges. In biological cells, the charge is carried by ions such as Na +, K+, Ca2+, and CI-. Current (I): The d irectional movement of charged parti cles is electric current, such as electrons mov­ ing throu gh a metal conductor. Current is measured in amperes (A); 1 A is the movement of 1 C of charge pe r second . Voltage (V): Also known as electrical pote nti al or potential difference, voltage refe rs to the difference in electric pote nti al between two points within an e lectric field , such as a circuit. By definit~on , potential diffe rence is the amount of work required to move a unit of cha rge. A 1-V potential differ­ ence requires 1 J of work to separate 1 C of chal"ge. Conductance (g! or Resistance (R or Q) : The fl ow of current between two points is called conduc­ tance; the greate r the conductance, the easier it is for current to fl ow. Resistance is the inverse of conductance; the greate r the resistance, the harder it is fo r current to flow. Conductance is measured in siemens (S) and resistance is measured in ohms (Q ). The biological equ ivalent of conductance is the pe rmeability of ion channels. The higher the conductance of a membrane, the greate r the num­ ber of open ion channe ls. Capacitance (C): If two conducting mate ri als are separated by aninsu lating layer, a capacitor is formed. This could be two metal plates separated by a nonconducting material; in the ce ll , the lipid bilayer of the membrane separates the conducting intrace llular and extrace llular fluids. If there is an excess of pos itive charge on one of the conductors and an excess of negative charge on the other, a potential difference develops between the two conductors. Capacitance is measured in farads (F): A 1-F capacitor will have an electri cal potential of 1 V when + 1 C of charge is on one conductor and - 1 C is on the othe r conductor. An electric current will change the electric potential of a capacitor by moving charge from one conductor to the othe r. This is called a capacitive current (lc) . Lipid bilay­ ers se rve as biological capacito rs" Synaptic Electrophysiology of the Drosophila NMJ / 175 al. 2004; Lima and Miesenbock 2005; Rasse et al. 2005; see Chapters 19-21), have expanded the reper­ toire of techniques available to challenge and measure physiological processes used by neurons. THE RESTING MEMBRANE POTENTIAL The physical basis of electrical signaling in cells is the resting membrane potential. Simply stated, there is a charge difference across cell membranes because the inside of a cell is electrically negative when compared with the outside. Understanding the origins of this electrical potential is the foun­ dation for learning how changes in the membrane potential constitute signaling. How do we know that cells have a resting membrane potential? Julius Bernstein in 1902 was among the first to predict that a cell would have a negative resting potential. Direct evidence of cell resting potentials, however, was not available until electrophysiologists measured it in squid giant axons in the 1940s (Hodgkin and Huxley 1945; Hodgkin and Katz 1949). Today, using well-honed techniques, it is relatively straightforward to gain electrical access to the inside of most cells. Glass micropipettes, having a tip diameter on the order of several hundred nanometers (for typical intra­ cellular recording pipettes) are carefully inserted through the plasma membrane and into the cell lumen. Using the appropriate electronic hqrdware connected to the micropipette, cell resting poten­ tials can be accurately measured and are typically -50-80 mY less than the outside. How Is the Membrane Potential Generated? Two basic factors determine a cell's resting potential: unequal distribution of ions between the inside and outside of a cell and the selective permeability of cell membranes. Unequal Distribution of Ions between the Inside and Outside of a Cell The ionic composition of the extracellular fluid is different from that in the intracellular fluid. The extracellular fluid usually contains Na+ as the major cation, which is electrically balanced by a com­ bination of CI- and HC03- ions to maintain electrical neutrality. Smaller contributions to the extra­ cellular charge come from K+, Ca2+, and Mg++, which again are balanced by Cl- and HC03- ions. The major cation of the intracellular fluid is K+, with a smaller contribution from Na+. Intracellular free [Ca2+] is usually near 0, because Ca2+ are bound to buffers, pumped into intracel­ lular stores, or pumped out of the cell. Intracellular fluid has a greater abundance (compared with the outside of the cell) of large impermeable anions, chiefly proteins, DNA, and RNA. These nega­ tive charges are balanced by intracellular K+, and it is for this reason that there is an imbalance of K+ across the cell membrane. Selective Membrane Permeability The cell membrane is a phospholipid bilayer with associated peripheral and transmembrane pro­ teins. The lipid bilayer portion is impermeable to most things, noteworthy exceptions being °2, CO2, and steroid compounds. Charged ions such as K+ and Na+ do not readily cross the bilayer. They move in and out of the cell with the aid of transmembrane protein channels and transporters. Among these proteins is a family of ion channels, known as leakage channels, that primarily trans­ port K+ (Goldstein et al. 1996), making the cell membrane relatively permeable to potassium. The membrane, however, remains relatively impermeable to other ions when it is in the resting state. The Physical Basis for Membrane Potential We see now that the resting membrane potential is a function of the ionic composition of the intra­ cellular and extracellular milieus, the charge difference that results, and the selective movement of ions across the membrane with the assistance of ion-selective protein channels and transporters. To 1 76 / Chapter 12 A B 0.01 M KCI 0.1 M KCI o volts I I lon-selective membrane (only K+. not Cr) I 0.01 M KCI I No net movement of K+ • 1 I 0.01 M KCI I 6-@ @ 1 6 @JI6 G:) --r- @ -@ Dl i @'0t~6 Initial concentration - 6 -~ @@P @ (0 -16 00@1 @ r?-@.@ V _ + New Equilibrium concentration - I( electricai FIGURE 1. Physical basis of the membrane potential. (A ) In a tank two KCI solutions are separated by a membrane that is on ly permeable to K+. A voltmeter measures the electrical difference between the two solutions. If the solu­ t ions on each side of the membrane are equal, there is no net movement of ions, so the voltmeter reads 0 V. (8) If the solution on the left side is increased to 0.1 M KCI, then there is an initial concentration grad ient drivi ng K+ and CI­ from left to right. Because the membrane is permeable on ly to K+, some K+ ions wi ll be separated from counterbal­ ancing CI- ions at the membrane. This charge separation instantly creates an electrical force, which is equal to but in the opposite direction from the concentration gradient, establishing a new equilibrium. The voltmeter now indicates there is an electrical potential d ifference between the two solutions. help us visualize how these factors generate a potential, consider a tank divided into two compart­ ments by a membrane that is only permeable to K+ (Fig. O. Each compartment is filled with 0.01 M KCl, and a voltmeter is used to measure the electrical difference across the membrane (Fig. IA). The meter indicates that there is no potential difference- that is, there is zero voltage across the mem­ brane_ This illustrates that selective permeability without a chemical gradient is insufficient to gen­ erate a voltage difference across the membrane. Next replace the solution in one of the compartments with a 0.1 M KCI solution . Now there is an unequal distribution of ions across the membrane and a concentration gradient exists that could drive K+ and Cl- from high concentration to low. If the membrane was equally permeable to K+ and Cl- (not shown in Fig. 1), then all of the ions in the two compartments would redistribute and even­ tually reach equal concentrations on both sides of the membrane. At this point, the voltage differ­ ence across the membrane would again be O. Thus, having a concentration difference and unrestricted permeability will not generate a potential difference. Let us now examine the situation when the membrane is only permeable to K+ and when there is a concentration gradient for KCI across the membrane (Fig. IB). Following the second law of thermo­ dynamics, K+ will start to diffuse across the membrane, but the counterbalancing negatively charged Cl- ions cannot follow these positive charges. A separation of charge therefore occurs at the membrane interface. There are now two forces at play: the K+ concentration gradient moving ions in one direc­ tion and a newly developed electrical gradient, which opposes the concentration gradient, because the negatively charged ions attract the positively charged ions. A new equilibrium condition is quickly established in which the force of the concentration gradient is exactly balanced by an opposing elec­ trical force. This electrical force is the membrane potential of this artificial cell. Therefore, both unequal distribution of ions and selective permeability are required to generate a membrane potential. Synaptic Electrophysiology of the Drosophil a NMJ / 177 Biological systems develop a similar separation of charge across the plasma membrane and this is what we measure as the membrane potential. The K+ leakage channels are the major contributor to the resting permeability of the plasma membrane, but the ionic and cellular environment within and around a cell is obviously more complicated than the above illustration. Many more species of ions are involved, and, importantly, the plasma membrane is permeable to K+ and at smaller per­ meability values to other ions as well. Equilibrium Potentials of Individual Ions The simplified experiment in the previous section illustrates that having unequal concentrations of ions across a semipermeable membrane gives rise to an electrical force. If the concentrations of ions are known, then the electrical force that is required to balance the force of the concentration gradi­ ent can be predicted using the Nernst equation: E. = RT In [ion outside cell] IOn zF [ion inside cell] , in which Eion is the equilibrium potential for the ion, R is the universal gas constant, T is absolute temperature, z is the valence of the ion, and F is Faraday's constant. This equation is often simplified for monovalent cations when the cell is at 200 e: E ion (mV) = 58 log [i~n ~ut~ide cell]. [IOn mSlde cell] Note the change from natural logarithm to logarithm base-lO. In our example above, if the 0.1 M KCl solution is considered to be inside the cell and the 0.01 M KCl solution to be outside the cell, then EK+ = -58 m V. This relationship can be used to calculate the equilibrium potential for any ion. The reader will see that this value is independent of permeability. Cations that are at a higher con­ centration inside the cell will have a negative equilibrium potential, whereas cations that are at a higher concentration outside the cell will have a positive equilibrium potential. For example, typi­ cally [Na+]outside = 100 mM, whereas [Na+Lnside = 5 mM, so the equilibrium potential for sodium is E Na+ = +75 mV. The Resting Potential for an Entire Cell Cells are in the presence of many different ions, both inside and outside the cell. To understand the resting potential for an entire cell, we need to consider the concentrations of those ions and their rel­ ative permeability. The major contributing cations are Na+ and K+, and Cl- is the major anion. The Goldman-Hodgkin-Katz (GHK) equation describes the relationship between the voltage of the cell membrane (Vn,), the permeability (P), and the concentrations of ions as V = RT In P K [K + t t + P Na [N a + lout + PCI [Cl - t . m zF PK[K +ln+PNa [Na+ln+PCI[Cl-Lt Note the reversal of terms for Cl-, which reflects the negative valence of the chloride ion. Once again converting to logarithm base-lO and assuming a temperature of 20°C, the relationship becomes V-I PK [K + lout + PNa [Na+ lout + PCI [Cl - lin m - 58 og + [+ _ . PK [K lin + PNa Na In + PCI [Cl lout Much of the work to derive this relationship was performed on the squid axon, in which it was found that at rest the permeabilities of K+:Na+:Cl- are 1:0.03:0.1. That is, in the resting squid axon, the permeability of K+ is 10 times greater than that of Cl- and 33.3 times greater than that of Na+. If [K+]out = 10 mM, [K+Ln = 400 mM, [Na+]out = 460 mM, [Na+Ln = 50 mM, [Cl- lout = 540 mM, [Cl-Ln = 40 mM, then 180 / Chapter 12 is a negative value (e.g., -70 mV), whereas E Na+ is usually a positive value (e.g., +50 mY). Therefore, in this example the driving force for Na+ is 120 mv' IfNa+ channels are fully open under these con­ ditions, then there is a large force that will push Na+ through the channel. In contrast, Na+ influx at the peak of an action potential (e.g., +48 mY) will be minimal even if all of the Na+ channels are open. This is because the driving force (2 mY) is very small when the peak of the action potentials closely approaches ENa+. Hence, ion permeability, as well as the driving force, determines the impact of an ion on the membrane potential. The concepts of relative permeability and driving force and their dynamics can be illustrated by a "spring" model, as shown in Figure 2. At rest, the thick and thin springs represent a high permeabil­ ity for K+ and a low permeability for Na+, respectively. Because the resting potential is close to EK+, the driving force (the length of the spring) for K+ is much smaller (shorter) than the driving force for Na+. Although the equilibrium potentials for Na+ and K+ do not usually change for a given cell, both the permeability and driving force can be highly dynamic. For example, the Na+ spring becomes much stronger relative to the K+ spring at the peak of an action potential. At this point, the driving force for Na+ reaches a minimal level, whereas it is at its largest for K+. Readers should keep in mind that after a brief delay the K+ spring strengthens dramatically, whereas the Na+ spring weakens during repolar­ ization of the action potential (not shown). This spring model also applies to synaptic potentials. The example shown here is an excitatory postsynaptic potential (EPSP) mediated by nonselective cation channels, such as the nicotinic acetylcholine receptor at mammalian NMJs and the glutamate recep­ tor at fly NMJ s. At the reversal potential, when the peak of the EPSP approaches -10m V, the springs for Na+ and K+ are represented as equally strong. Hence, it is the relative strength of the springs-that is, the permeability of permeable ions-that determines the membrane potential. Resting Peak of An AP Peak of An EPSP ___ ,_ENa -..,_ ... - Vm OmV~ ~ OmV _.,._ .... _ Vm ____ EK FIGURE 2. A spring model of relative permeability, driving force, and their dynamics. This cartoon models the rela­ ti ve permeability, the driving force, and membrane potentials of a typical ce ll at three different states. The thickness of the spring correlates positively with the permeability of an ion channel, whereas th e length of the spring represents the driving force . At rest, the dominant strength of the K+ spring (PK+) brings the membrane potential (V m) toward EK+. But the driving force for Na+ is much higher than that for K+ The equilibrium potent ials for Na+ (ENa +) and K+ (EK+) rare ly change for a given ce ll. However, both the permeability and driving force can be highly dynamic. During the peak of an action potential, the spring strength and length reverse for K+ and Na+ such that PNa+ overpowers PK+, thereby bringing the membrane potential near ENa+. In a synaptic potential mediated by nonselective cation channels such as the glutamate receptor at the fly NMj, the spring is usually equal ly strong at the reversal potential. Hence, the peak of the EPSP approaches - 1 0 mV The fundamental take-home message is that it is the strength of the spring (i.e., relative permeability) of permeable ions that determines the membrane potential. Synaptic Electrophysiology of the Drosophil a NMJ / 181 SYNAPTIC TRANSMISSION Synaptic transmission is the process by which neurons secrete neurotransmitter molecules from the nerve terminal onto target cells. Synaptic physiology has been studied for a long time and remains a favorite topic among neuroscientists. Although a complete review of synaptic transmission is beyond the scope of this chapter, highlighting some of the achievements and contemporary advancements is worthwhile. Synaptic physiology began when Bernard Katz and his colleagues recorded the first synaptic response at the frog NMJ in the 1950s using an intracellular microelec­ trode. Their important studies, summarized in an elegant book (Katz 1966), provided the essential elements for our understanding of synaptic transmission. Their major findings were as follows. 1. There appeared to be a spontaneous release of neurotransmitter. 2. Action potentials arriving at the nerve terminal caused the release of neurotransmitter. 3. Transmitter release required that calcium be in the extracellular fluid at the time the action potential arrived. 4. Neurotransmitter is released in defined units, which Katz called quantal units. 5. The units appeared to be the same as the spontaneously released units (Fig. 3). , Indeed in subsequent years much of the effort in synaptic physiology has been to test, retest, extend, and expand on these early ideas. A recent study appears to challenge the basic tenet of Katz's quan­ tal theory (Fredj and Burrone 2009). Nearly simultaneous with the development of Katz's data on synaptic physiology, the first glimpses of the nerve terminal ultrastructure were obtained with an electron microscope. These ini­ tial views of the synapse revealed the presence of small clear vesicular structures in the nerve termi­ nal. This observation naturally gave rise to the notion that Katz's quantal units were in fact the synaptic vesicles. This remained a controversial notion, however, until the important work of Heuser, Reese, and others, who showed a correspondence between quantal release as measured by electrophysiology and vesicular release as observed with the electron microscope (Heuser et al. 1979). In the late 1980s studies began to reveal the molecular nature of synaptic transmission. Synaptophysin, VAMP (vesicle-associated membrane protein)/synaptobrevin, and SNAP-25 (synap­ tosomal-associated protein 25) were among the first synaptic proteins to be identified and charac­ terized (Trimble et al. 1988; Oyler et al. 1989; Sudhof et al. 1989). This was followed by the identification of synaptotagmin (Perin et al. 1990), syntaxin (Bennett et al. 1992), and Rab3 (Fischer et al. 1990) in the early 1990s (also see reviews by Jahn and Sudhof 1994; Hay and Scheller 1997; Chen and Scheller 2001; Jahn et al. 2003). Research into the molecular nature of cellular processes was (of course) not limited to neuroscience, and studies on neural proteins soon became greatly influenced by molecular studies in related vesicle trafficking disciplines within the broader frame­ work of cell biology. Particularly noteworthy was the identification of endocytic protein dynamin (Chen et al. 1991; van der Bliek and Meyerowitz 1991) following earlier discoveries of clathrin (Pearse 1976) and AP2 (Keen 1987), as well as seminal studies emerging from the vesicle trafficking field (see reviews by Cremona and De Camilli 1997; Brodsky et al. 2001; Conner and Schmid 2003). Most noteworthy are the studies that first identified N-ethylmaleimide-sensitive factor (NSF) and the other SNARE (soluble NSF attachment receptor) proteins and the realization that VAMP, syn­ taxin, and SNAP-25 were members of large protein families involved in a host of vesicular transport processes throughout the cell (Sollner and Rothman 1994). It had long been known that tetanus toxin and the botulinum toxins have profound effects on synaptic transmission. The discovery that these toxins were proteases that specifically cleaved VAMP, syntaxin, and SNAP-25 led to studies using them as probes that helped explain synaptic processes at the molecular level (Jahn and Niemann 1994) . In particular, identifying the target molecules of these toxins quickly assigned a function to these recently discovered synaptic molecules. 182 / Chapter 12 B Nerve-evoked c OO[ '4 '~~It. IQ)~ ! __ ~J : : l r; it . ·r .. Spontaneous ; '~ .. I'i l .... J ; ~' Nerve-evoked II FIGURE 3. Quantal units of transmitter release. (A ) A constant feature of synaptic transmission is the sponta­ neous (i.e., in the absence of action potentials) release of neurotransmitter. These miniature synaptic events appear similar to those caused by action potentials at low extracellular [Ca2+]. (8) Nerve-evoked EJPs at medium extracellular [Ca2+ ]. (C) If one collects EJP amplitude data of spontaneous events and nerve-stim­ ulated events (under low-release conditions), two observations can be made. First, the amplitude distri­ bution of the spontaneous events is the same as for the smallest nerve-stimulated events; second, the larger nerve-stimulated EJPs fluctuate in a manner that sug­ gests the larger EJPs are multiples of the smallest EJP. These observations gave rise to the quantal hypothesis, which is that neurotransmitter is released from the nerve terminal in small packets (or quanta). (Reprinted, with permission, from del Castillo and Katz 1954.) Synaptic Elec troph ys iology of the Drosop hil a NMJ / 185 FIGURE 4. Examples of electrophysiology rigs. These pictures illustrate the major components of an electrophysiology setup commonly used in fly neurophysiology laboratories. (1) Vibration isolation (reduction) table; (2) microscopes (com­ pound and stereomicroscope); (3) amplifier; (4) analog-to-digital (AD) and digital-to-analog (DA) interface board; (5) computer, monitor, and acquisition and analysis software (latter two not shown); (6) stimulator and stimulus isolator; (7) micromanipulators (motorized and manual). (A-C) A rig using an upright compound microscope. This configuration can be used for a variety of both basic and sophisticated experiments, including patch clamping of neurons or muscles (in embryos, larvae, and adul ts), two-electrode voltage clamping (TEVC), intracellular recording, single-bouton foca l record­ ing, and optical imaging. (0) A rig using a stereomicroscope. This simple setup is highly functiona l for intracellular record­ ings and TEVC in larvae or adult muscles. It is, however, not suitable for recordings from embryos or single boutons. stages. Make sure that the outer diameter of the capillary glass to be used for manufacturing micro­ electrodes matches the size of the electrode holder. Suggested suppliers are Molecular Devices, HEKA, and Warner Instruments. AD/DA Interface Board: This device changes analog (A) signals to digital (D) form so that a com­ puter can record electrical traces. It also converts digital signals into analog ones, such as when your computer sends command signals to stimulate the nerve. A suggested product is Digidata 1440A (Molecular Devices) . Computer and Software: The software used in the course is called pClamp 10.0, which is compatible with the Digidata 1440A AD/DA interface board. The pClamp package has two separate programs: The Clamp ex program makes it possible to observe and acquire data and the Clamp fit program is used for data analysis. In addition, the Mini Analysis Program (Synaptosoft Inc. ) is used to analyze spontaneous and miniature synaptic potentials. 186 / Chap ter 12 Stimulator: To excite an axon to fire action potentials, a brief electrical shock must be delivered to it. A stimulator and isolator are used to program the stimulation paradigms (such as stimulation fre­ quency, duration, and strength). Suggested products are the S48 square pulse stimulator and SIUS isolator (Grass Technologies/Astro-Med, Inc.) or the Master-8 and ISO-Flex stimulus isolator (A.M.P.I., Israel). If you are only delivering simple pulses (such as a single stimulus or two pulses), a stimulator is not required. Instead, use the built-in software with the Clampex program as a "stim­ ulator" (for safety reasons a stimulus isolator must be used). Micromanipulators: Micromanipulators help position the electrode(s) and assist in impaling the cell membrane with minimal physical damage. Larval NMJ recordings require one "coarse" manual manipulator (e.g., Narishige MM-3; Siskiyou MXllO), which is usually placed on the left side and is used to position the stimulating ("suction") electrode. Two "fine" manipulators are needed to help position the recording electrode and impale muscle fibers. A manipulator with fine movement can be manual (e.g., Narishige NMN-2S), hydraulic (e.g., Narishige MHW-3), or motorized (e.g., Sutter Instruments MP-22S or MP-28S; Burleigh PCS-SOOO). The fine manipulator is a critically important piece of equipment because it is used to direct the electrode to impale muscles and to hold the elec­ trode in a stable position. It is prudent to obtain the best micromanipulator possible. Microelectrode puller: This device is used to pull the high-quality micro electrodes needed for impal­ ing cell membranes. As one of the most important (but often overlooked) pieces of electrophysiol­ ogy equipment, an electrode puller will determine how reliable your electrodes will be over time. Suggested products are the P-97 or P-lOOO Flaming/Brown Micropipette Puller (Sutter Instruments) or the PC-l 0 Puller (Narishige). Other Items: BNC cables Glass capillaries for manufacturing microelectrodes Grounding wires and silver wires Light source MicroFil (World Precision Instruments) Syringe (l-mL), filled with 3 M KCI and to be used with MicroFil to backfill a micro electrode Video Monitor System: This is optional, but for teaching purposes it is extremely useful, because it allows up to five students to simultaneously visualize NMJ preparations and to watch their own actions (such as electrode position/movement) on a large monitor. Oscilloscope: This is optional, although an oscilloscope has more functions than does a computer. No two electrophysiology rigs are identical, because each laboratory configures theirs to suit-their needs. In addition, different experiments require minor modifications to the rig, such as varying the number of micromanipulators needed depending on the number of electrodes being used. Historically, the system for signal observation and data storage has gone through the most dramatic change (and improvement) within the last 20 years. Instead of the traditional oscilloscope, chart recorder, and magnetic tapes, electrophysiological data are now visualized, acquired, and stored in digital formats in a computer using specially designed software. Using Axoclamp 2B and the Digidata data acquisition systems as examples, a diagram of wiring connections for an electrophysiology rig that can be used for both intracellular recording and TEVC is provided in Figure S. Synaptic Electrophysiology of the Drosophila NMJ / 187 Amplifier ~I )r ~ ? I ~1 ~2 + Step Activation INPUTS : Back panel (INPUTS) Front panel (OUTPUTS) OUTPUTS 10Vm 0.1 X 12 V2 ON Stimulus Stimulator : Isolator STIMULATI • : ... ----- ---- ~ --------- ----------------,. - Computer a ADIDA INTERFAC nd E Computer Start & pClamp USB ,..-------- . : [JO[ Digital Analog Analog Outputs Outputs Inputs Digidata FIGURE 5. A wiri ng diagram for an electrophysiology rig. A rig can be divided into four major components based on the flow and processing of electrical signals: inputs, outputs, stimu lation, and interface and data acquisition. Once connected through a BNC cable as illustrated here, these components will become a functional electrophysiology rig. The w iring diagram shown here is based on the AxoCiamp 2B amplifier, Digidata, and pClamp software. However, this diagram can be used as a reference for any other similar products. Membrane potentials recorded by microelec­ trodes are detected by specific probes and then sent into an amplifier through corresponding channels (such as ME1 and ME2J. Once amplified by the amplifier, the electrical signals travel to the AD interface board to be converted into digital forms and then acquired by the computer using specially designed software. The interface board is also capa­ ble of producing signals to command either the stimu lator or the amplifier. A voltage or current delivered through the stimulus isolator and a suction electrode is used to stimu late the motor axons into firing action potentials. A stimula­ tor is used to program different stimu lus parameters so that one can control the stimulus duration and frequency. The software and DA interface board can also be used to control the stimu lus pulse, but they do not have great flexibility for users if one wants to have more complex stimu lus paradigms.