The suction pipette method for internal perfusion and voltage clamp of small excitable cells

Journal of Neuroscience Methods, 2 (1980) 51--78 51 © Elsevier/North-Holland Biomedical Press

THE SUCTION PIPETTE METHOD FOR INTERNAL PERFUSION AND VOLTAGE CLAMP OF SMALL EXCITABLE CELLS

K.S. LEE, N. AKAIKE and A.M. BROWN *

Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550 (U.S.A.)

(Received October 12th, 1979) (Accepted October 22nd, 1979)

A full description of a suction pipette method for voltage clamp and internal perfusion of isolated nerve cell bodies is presented. Particular emphasis is placed upon manufacture and assembly of the pipette and the limitations of the method.

INTRODUCTION

Functional investigation of excitable membranes is aided considerably when both the current flow through the membrane and the voltage drop across the membrane are under experimental control . Two methods which are of ten combined allow such control ; these are voltage clamp and internal perfusion. The voltage clamp method introduced by Cole (1949) represented a substantial conceptual achievement and revolutionized the experimental investigation of excitable membranes. The method requires an accurate measurement of cellular membrane potential. This measurement is then compared with a command signal and if the signals differ current is passed through the membrane by an amplifier until they are equal. The current measurement which is the dependent variable should include only current coming from the region of membrane under uniform potential control . The me thod was capitalized on quickly by Hodgkin and Huxley (1952) and led to their famous analysis of the action potential of squid giant axon. By holding the voltage steady over a range of potentials, it was possible to demon- strate that the action potent ial was produced by a sequence in which a Na conductance was activated and then inactivated. Short ly after Na activation, a K conductance was also activated. The inward flow of Na ions and the outward flow of K ions were responsible for the rise and fall of the action potential . Fur ther investigations were directed towards the underlying molecular mechanisms and were aided considerably by the development of

* To whom correspondence should be addressed.

52

a method for internal perfusion of squid axon (Oikawa et al., 1961; Baker et al., 1961). Internal perfusion allowed control of the ionic composition on both sides of the plasma membrane and by combining this method with voltage clamp, separation of individual ionic conductances was more readily accomplished. The details of permeation and excitation were now accessible to experimental investigation and much of our present knowledge on ionic interactions with channels, channel gating, the chemical composition of channel constituents and anesthetic actions, has resulted from experiments using the combined approach.

In smaller excitable cells the axial wire voltage clamp method is impossible and different approaches have been taken to achieve voltage control. Huxley and St~impfli (1951) and Frankenhaeuser (1957) used potentiometric methods for measuring membrane potential of node of Ranvier which was isolated by two Vaseline seals and an air gap. With this as a basis, Dodge and Frankenhaeuser (1958) were able to voltage clamp a single node. Limited access to the node's interior was attained by diffusion from adjacent nodal pools and the combined method has proven quite successful (Hille, 1971). Sucrose isolation was introduced by St~impfli (1954) and applied to lobster axon by Julian et al. (1962) who made an artificial node using two sucrose streams. Subsequently, sucrose isolation methods were extended particularly to the syncytia of cardiac and smooth muscle. Hybrid techniques which combine micropipette recording of voltage with current injections via a compartment isolated by sucrose have also been used in these situations. Spatial control of voltage and temporal resolution are limited, mainly because of the syncytial nature of the tissues in the case of cardiac and smooth muscle, the RC characteristics of high impedance micropipettes, and inhomogeneities arising from 'point ' clamping of spherical cells or cell aggregates. The use of sucrose also introduces complications, in particular the hyperpolarizing effect noted by Julian et al. (1962). Furthermore the methods do not provide for perfusion of the interior of cells.

Hagiwara and Saito (1959) introduced a two micropipette technique for voltage clamp of nerve cell bodies and similar approaches have also been used in skeletal, cardiac and smooth muscle. The method usually cannot resolve frequencies faster than 1 kHz and spatial control is inadequate when in the case of nerve cell bodies, the axon is attached. To avoid some of the difficulties Neher and Lux (1969) measured local or patch currents with a thin-walled micropipette pressed against a neuron's surface to achieve isolation. Kado (1973) used a somewhat different approach to make a similar measurement. Subsequently, Neher and Sakmann (1976) used their patch method to record from single channels at denervated end-plate. In these patch measurements of current, voltage clamp was applied through a separate circuit. Methods for voltage clamp of a small patch of membrane itself were first introduced for skeletal muscle by Strickholm (1961) and later extended by Hencek et al. (1969). The first true patch clamp of neural membrane was achieved by Fishman in 1975 for squid giant axon. Sucrose

53

was used in obtain patch isolation and a high shunt resistance. This method was applied to neurons but we found it difficult to sustain the necessary isolation for reasonable experimental times (Brown and Lee, unpublished observations).

A radical departure from these methods was taken by Kostyuk et al. (1975) who succeeded in inserting a nerve cell body, enzymatically dispersed from its neighbors, into a part i t ion between two chambers. One hemisphere of the spherical soma was made leaky by exposure to zero Ca solutions leaving a single intact hemisphere of membrane between the two chambers. The chambers were connected via low resistance macroelectrodes to a voltage clamp circuit. The membrane could now be clamped rapidly and perfused on both sides. Our own efforts were along similar lines but we found the sequence of cell dispersion and subsequent insertion into a parti- t ion too tedious. We therefore adopted a different approach based on a suction pipette method. In our approach (Lee et al., 1977, 1978) strong suction is the me thod by which a high shunt resistance is achieved. A large pipet te is used to aspirate a single neuron, rupture the aspirated membrane, measure intracellular potential , voltage clamp the cell and perfuse the cell's interior using the negative pressure of the suction. The axon may also be severed in the process, thus isolating the neuron soma completely. If currents larger than 10--20 X 10 - gA are anticipated, a separate micropipet te or a second suction pipette is used to moni tor intracellular potential. Following in t roduct ion of the suction pipette method it was employed in an inverted form by Takahashi and Yoshii (1978) to study the electrical properties of egg cells. Kostyuk et al. (1978) have also used suction pipette techniques in studies on neuroblastoma cells. Recently single cardiac muscle cells have been examined with the suction pipette method after dispersion of the cardiac syncyt ium into single cells (Lee et al., 1979). Hence it appears possible that the suction pipet te method may have wide application and for this reason a detailed description of the manufacture of the pipettes, its use and its limitations seems desirable. The present report a t tempts to do this while recognizing that many of our procedures can undoubted ly be improved upon or modified to suit a particular user or situation.

METHODS

The general method including isolation of nerve cell bodies will be described first and then details on manufacture and assembly of the pipette will be presented.

General approach In the case of neurons, cells located in the subesophageal ganglion of

Helix aspersa are used. The ganglion is removed and the connective tissue stripped off with jeweller's forceps revealing clusters of neurons which float in the snail Ringer solution. Individual neurons 50--150 pm in diameter are then partially aspirated under negative pressure of about --30 mm Hg so as

54

b c_

d •

Fig. 1. Aspiration and separation of a nerve cell body from the subesophageal ganglion of Helix aspersa, a: aspiration with the suction pipette (Sp) using negative pressure, b: stretch axon by pulling manipulator on which the suction pipette is mounted, c: removal of other small neurons and stripping residual connective tissue with a micro-tool such as a glass micropipette, d, e: sever the axon. f: isolated nerve cell body recovers and is ready to provide new experimental data.

,° . ~ •

:... '" :."

: 'iiii:, '. A ~ B

i T

I Rs-2

~ Rm Rsh

Fig. 2. A punctured aspirated neuron. A: scheme of the aspirated neuron on the suction pipette. The inlet tubing (filled with dots) and the Pt-Ir wire (solid instrument) used to puncture the neuron are also shown. B: equivalent electrical circuit of A. Rs.z, series resistance inside the tip of the pipette; Rs-2, series resistance outside the pipette; Cm, Rm, membrane capacitance and resistance; Rsh , shunt resistance.

55

to occlude the 35--50 pm diameter pipette tip (Fig. 1). The suction pipette is mounted on a micromanipulator and by moving the manipulator it is possible to stretch the axon and then sever it with a micro-tool. The result is a nerve cell body attached to the suction pipette and isolated completely from other cells. The next step is to rupture the aspirated membrane using a sharpened Pt-Ir wire which may be either left inside the neuron or may be withdrawn (Fig. 2). Following this step internal perfusion with the desired solution is begun. Where a separate measurement of membrane potential, EM, is required, a glass micropipette of about 1 pm tip diameter and 106 ~ tip resistance filled with 3 M KC1 may be inserted into the same cell. The micropipette is coated with a conductive paint {Silver Print, Wholesale Electronic Supply, Dallas) then insulated using polystyrene Q dope (G.C. Electronics, Ill.) and is connected by a driven shield to a unity gain operational amplifier. Intracellular potentials are measured single-ended by either the micropipette or suction pipette connected to a buffer amplifier (Fig. 3) or differentially using a macro- or micropipette of 30--100 k~2 resistance placed near the cell as the reference electrode. In the latter case the intra- and extracellular pipettes are each connected to a buffer amplifier and the two outputs connected to a differential amplifier. For current clamp, CC, a current pump indicated by Vp in Fig. 3 is used. When voltage clamp, VC, is performed using E M measured by the suction pipette, a differential operational amplifier (Analog Devices, 48K) with a bandwidth of 15 × 106 Hz is used to supply current. When the micropipette is used a differential amplifier such as the Tektronix 502A operational amplifier with an extended bandwidth and a range of frequency and gain controls is employed, because of the frequency with which instabilities occur. The feedback resistors (10 M~2 and 100 M ~ in Fig. 3) are used with the 48K operational amplifier for measurement of microscopic current fluctuations to obtain the best signal/ noise performance from the clamp system (Poussart, 1971). Current is measured by a current to voltage converter one side of which is held at virtual ground. Series resistance compensation is provided by adding a portion of the membrane current to the command voltage to nullify the voltage drop across the series resistance. In current clamp the compensation is set to nullify the instantaneous changes in E M that occur when current steps are turned on and off. Leakage and shunt currents can also be offset electroni- cally using a summing amplifier. However in most experiments leakage and shunt currents and the linear portion of the capacitative current transient are subtracted by adding the current responses to equal and opposite voltage steps.

For experiments of isolated, dispersed single ventricular myocytes, a double suction pipette arrangement such as that shown in Fig. 4 is used. The tip diameter of the pipettes used is about 15 pm. With this arrangement, the voltage control and time resolution are greatly enhanced. Also changes in intracellular ionic composition can be accelerated by a pressure gradient between the two pipettes.

56

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fl

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vc

Fig. 3. Experimental arrangement. CC, current clamp circuit; Vp, voltage applied to current pump; VC, voltage clamp circuit; IN, current measuring point for registering microscopic current fluctuations (noise); Vc, command pulse; VM, membrane potential; IM, macroscopic membrane current measured by the current--voltage converter (operational amplifier, labeled b); Sp, membrane potential recorded by suction pipette via a calomel half cell. In this si tuation the reference electrode (not shown) is also a calomel half-cell connected to the bath by a bridge of 1% Agar in 3 M KC1; Mp, membrane potential recorded by a glass micropipet te about t pm in tip diameter and filled with 3 M KC1. In this case the reference electrode is a broken glass micropipet te placed close to the suc- t ion pipette. SR, series resistance compensation circuit. This is the usual type of positive feedback summed with the command potential at the differential clamp amplifier, a, to nullify the IR drop across the series resistance sources shown in Fig. 2; Lc, leak and shunt current compensation provided by supplying sufficient current via a potent iometer to a summing amplifier, c, to nullify the leakage and shunt currents produced by hyperpolarizing voltage steps. Another method for removing these components is to add the currents produced by equal voltage steps of opposite sign.

Manufacture of the suction pipette A macropipette is pulled from Pyrex tubing 3.0 mm o.d. and tapered over

a length of 1.0--2.5 cm from its tip (Fig. 5). Shorter tapers are better for internal perfusion (Fig. 5Aa.1). The shank is scored by hand with a diamond knife at a distance which varies depending upon the internal tip diameter ult imately desired. For a diameter of 30--50 pm the score can be made

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8ram Fig. 5. Manufacture of the suction pipette. A: two types of suction pipette, a-l, short- shank type used for applying several internal solutions successively; a-2, long-shank type used to perfuse with a single solution; a-3, orifice after fracture of the shank at the dotted lines in a-l, a-2. B: fire-polishing of fractured tip. b-l, fractured tip is fire-polished in gas-flame; b-2, enlarged scheme of the tip orifice before and after (shown by heavy lines) fire-polishing. C: Vaseline coating of the tip. After dipping it into small amount of Vaseline, the electrode tip is inserted into a larger heated glass tube resulting in a hydro- phobic coating. D: fitting of vinyl tube (denoted with i in Fig. 6) to hold pipette gasket (h in Fig. 6) connecting pipette to antechamber (A).

a b o u t 1.0 cm back f r o m the t ip and then b r o k e n a t this p o i n t (Fig. 5Aa-e,a-z). We use pressure against a f inger to do this b u t o the r m e t h o d s m a y be m o r e rel iable. The crucial p o i n t is t h a t the f r ac tu re m u s t be p e r f e c t with- o u t a n y jagged edges {Fig. 5Aa.3). T h e f r ac tu red t ip is t h e n f i re-pol ished as s h o w n in Fig. 5Bb.1 resu l t ing in t he shape s h o w n in Fig. 5Bb.2 whe re the wall th i ckness a t t he t ip is a b o u t 20 p m . T h e t ip is t hen m a d e h y d r o p h o b i c b y d ipp ing it in a 50 : 50 m i x t u r e o f Vasel ine and minera l oil and inser t ing the t ip in to a h e a t e d glass (Fig. 5C). The p i pe t t e is t h e n r eady fo r inser t ion in to a p o l y e t h y l e n e gaske t as s h o w n in Fig. 5D which c o n n e c t s it to the ante-

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I--4.9 Fig. 6. Overall scheme o'f the suction pipette. A, antechamber; B, a t tachment for recording membrane potential; C, suction pipette at tachment, a, outlet tube for suction and internal perfusate; b, Ag-wire to apply constant current during current clamp and feedback current during voltage clamp; c, port ion insulated by polyethylene tube to allow puncture wire to be hand driven; d, guide filled with silicone grease or Vaseline for the Ag-wire; e, inlet tube for internal perfusate; f, rubber plug; g, tapered polyethylene tube used for inflow; h, polyethylene gasket connecting the suction pipette to the antechamber; i, vinyl fitting; j, glass suction pipette; k, puncture wire (90% Pt platinized-10% Ir) soldered to Ag-wire and sharpened electrolytically to have 1--2 pm tip diameter; 1, single cell; m, connection between calomel half-cell and antechamber. Details shown in Fig. 10; n, Ringer 1% Agar bridge; o, 0.15 M KCl; p, calomel half-cell.

c h a m b e r as s h o w n in F ig . 6 A . T h e c o n n e c t i o n t o t h e a n t e c h a m b e r w h i c h is m a d e f r o m a p l a s t i c s y r i n g e is s h o w n in m o r e d e t a i l in F ig . 7. T h e c o m p o - n e n t s f o r p e r f u s i o n a n d m e m b r a n e r u p t u r e a r e a s s e m b l e d in t h e a n t e c h a m b e r a n d t h e p r o c e d u r e f o r i n t e r n a l p e r f u s i o n is d e s c r i b e d n e x t .

60

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Intracellular per fusion Internal perfusion is preceded by disrupting part of the neuronal mem-

brane aspirated into the tip of the suction pipette. This is accomplished by inserting a platinized platinum--iridium wire having a diameter of 2 pm at its tip through the membrane and into the cytoplasm. Insertion is done by manually advancing the wire down the pipette shaft. The tip was sharpened electrolytically using wire 20 ~m in diameter and a solution of 95 ml H3PO4, 5 ml H2SO4 and 1 g gelatin. The sharpened wire was soldered onto a silver wire as shown in Fig. 8D. The wire was inserted into the bung at the top of the suction pipette through a guide filled with silicone grease {Fig. 8C).

The polyethylene tubing used for inflow is drawn to a taper in a flame and positioned down near t h e tip of the suction pipette as shown in Fig. 6C. The outlet tube is situated in the antechamber as shown in Fig. 6A.

Inflow was delivered from a group of parallel reservoirs located about 30 cm above the neuron. Negative pressure was applied via two trap bottles {Fig. 9) and was adjusted so as to provide maximum sealing of the soma from the extracellular fluid; an overzealous approach results in complete aspiration of the neuron. The negative pressure together with the hydro- static pressure resulted in a flow rate through the suction pipette of about 0.5 ml/min.

61

1 L --t- do1

A • • B 2 •

3 t. " - - ~ d - 2

. . . . . j 4 L . . . . . .

I - - t - - b -1

s L . . . . c l - 3

..d-4

Fig. 8. Manufacture and assembly of suction pipette components for insertion into antechamber. A: outlet to suction. B: inlet for internal perfusate. C: wire for applying current and puncture in aspirated membrane, c-1, Ag-wire lead to apply current in CC and VC; c-2, insulated portion for manual insertion and retraction of puncture wire; c-3, Vaseline or silicone grease for insulation and for smooth movement of the wire inside polyethylene sleeve; c-4, solder joint of platinized platinum--iridium wire to Ag-wire; c-5, terminal part of puncture wire with 1--2/~m tip. Silicone grease fills the space between the two parts of the rubber plug. D: scheme for soldering Pt--Ir wire to Ag-wire (d-1 to d-4). The terminal portion of the silver wire tip is ground to a flat stage (d-l) then an appropriate length (about one half the total length) of the etched Pt--Ir wire is folded around and soldered to the stage (d-2 to d-4). A sleeve of PE tubing adds mechanical support to the soldered region.

The sequence involved in aspirat ion and perfus ion begins by filling the p ipe t te wi th the internal per fus ion solut ion by gentle suct ion. Then the suc- t ion is t u rned of f while leaving the valve to the internal so lut ion open. The residual negative pressure inside the a n t e c h a m b e r will soon equil ibrate with a tmospher ic pressure in the bath solut ion. Next , the p ipet te is b rough t very close to the cell o f interest and the suct ion is tu rned on. The cell jumps into the tip o f the p ipet te and the aspirated m e m b r a n e surface is then rup tu red by manual inser t ion of the Pt-Ir wire. I t is also possible to aspirate the cell with the sharpened wire set at the p ipe t te tip. The Pt-Ir wire will au tomat i - cally advance a few pm into the cell thus rup tur ing the m e m b r a n e at the

62

6

, \ Fig. 9. Inflow and outflow of internal perfusion system, a, internal perfusate reservoir; b, bubble trap; c, valve; d, suction pipette. Position of outlet and inlet tubing and puncture wire (inside aspirated cell) are shown; e, valve; f, waste trap bott le; g, trap bott le connected to earth through 3 M KC1 solution and Ag--AgCl plate; h, negative pressure generator (vacuum pump or water aspirator). The pressure at this point is more negative that --30 mm Hg; i, j, sequence for filling the pipette with internal perfusate. First, the tip is filled with Ringer solution by opening the suction valve e, while valve c is closed (i). Then, valve c is opened and the internal perfusion solution runs through the system quickly. When the antechamber is filled with the internal solution (black area in j), suction is turned off. Drops continue to run into the bubble trap for a brief period. Valve c is closed when drops cease to flow. A Schlieren effect is observed through the dissecting microscope as fluid emerges from the tip of the suction pipette (shown with an arrow in j); k, appearance of the tip (1--2 pm) of the puncture wire through dissecting microscope. To the left of the wire is the polyethylene inflow tube.

t i m e o f a s p i r a t i o n . T h e w i r e m a y b e l e f t in p l a c e o r w i t h d r a w n a n d i n t e r n a l p e r f u s i o n is b e g u n .

Measurement of membrane potential B e c a u s e o f f l u c t u a t i o n s in p r e s s u r e a n d o c c u r r e n c e o f b u b b l e s in t h e suc-

t i o n p i p e t t e i t was n e c e s s a r y t o i n t r o d u c e o n e b a f f l e a n d a d a m p i n g c h a m b e r b e t w e e n t h e p i p e t t e a n t e c h a m b e r a n d t h e c a l o m e l ha l f - ce l l u s e d t o m e a s u r e i n t r a c e l l u l a r p o t e n t i a l . T h e a r r a n g e m e n t is s h o w n in F ig . 10 a n d i n v o l v e d t h e use o f t w o 1% A g a r p l u g s a t a -3 a n d b -3 as we l l as a c o m b i n a t i o n g a u z e a n d w o o d p l u g a t a-1 a n d a-2 . T h e s o l u t i o n s u s e d t o m a k e u p t h e A g a r w e r e t h e s a m e as t h e p a r t i c u l a r i n t e r n a l p e r f u s a t e a n d c o n n e c t i o n t o t h e h a l f - c e l l ( c - l ) w a s m a d e t h r o u g h t h e b r i d g e a t b-3 w h i c h was 1% A g a r m a d e u p in 0 . 1 5 o r 3 .0 M KC1. A s e c o n d c a l o m e l h a l f - c e l l w a s u s e d t o m e a s u r e t h e b a t h p o t e n t i a l

63

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Fig. 10. Connection of the calomel half-cell to pipette antechamber. A: adaptor connection to the antechamber. B: connection between adaptor and chamber C which holds the calomel half-cell, a-l , gauze impregnated with perfusate surrounding a wooden wedge (a-2) making a tight fit to the antechamber. This prevents aspiration of contents of A and B into the pipette antechamber; a-2, wooden wedge; a-3, Ringer 1% Agar bridge; a-4, rubber O-ring; a-5, 0.15 M KC1; b-l , air purge valve; b-2, connection to 0.15 M KC1 reservoir (see Fig. 6); b-3, Ringer--Agar bridge; b-4, 0.15 M KCI; c-l , 0.15 M KCl.

and was also connected to the bath via a 0.15 M or 3.0 M KC1 1% Agar bridge. The resistance of either half-cell arrangement in snail Ringer is 1--2 k~2.

RESULTS

Evaluation o f electrical measurements

The most important electrical parameters to be evaluated were the shunt and series resistances. The shunt resistance, Rsh (Figs. 2 and 11), isolates the neuron from the bathing fluid and was estimated in several ways. During isolation, voltage pulses were passed between the bath ground and the voltage electrode in the suction pipette. Isolation was usually successful when the current responses were reduced between 20- and 80-fold, and further increases in negative pressure had no additional effects (Fig. 11). Another simple test of satisfactory isolation was the amplitude of the action potential which should be similar to values obtained with intracellular micropipettes (Fig. 12). A more quantitative estimate was made by inserting two micropipettes, one for injecting transmembrane current, the other for measuring transmembrane voltage.The voltage deflections of the micropipette and of the voltage-sensing electrode in the suction pipette were compared before and after puncture of the aspirated portion of neuronal membrane (Fig. 13). The equivalent electrical circuits are shown in Fig. 13.

64

A B

Rs

! : Rs h nA

o L ~ l i

30see

Fig. 11. A s p i r a t i o n o f a nerve cell body . A: l oca t ion of series res is tance at p ipe t t e t ip (Rs) and s h u n t res is tance (Rsh) . B: ef fec ts of a sp i ra t ion on the cu r r en t s el ic i ted by c o n s t a n t vol tage pulses appl ied b e t w e e n the suc t ion p ipe t t e ' s in te r io r and b a t h g round . The m a r k e d r e d u c t i o n in c u r r e n t ind ica tes good i so la t ion in this case of a Helix aspersa n e u r o n 120 p m in d iamete r .

A _.F'I_

\ S

\ S

h i i l i l l l i

25 rnV

0.5 sec 50 m$'c Fig. 12. Comparison of successive voltage responses to constant current steps in an isolated neuron using the arrangement shown in A recorded by an intracellular micropipette (B and E) and the suction pipette (C and D). Constant current pulses of 2 nA were used in B and C (top traces). The larger deflections in the suction pipette records are due to t he IR d r o p across the p i p e t t e t ip. Resul ts f rom a n o t h e r cell are s h o w n in D and E. Depola r iz ing cu r r en t s ( n o t s h o w n ) are used in t o p t races of D and E and hyperpo la r i z ing cu r r en t s in b o t t o m t races D and E: T he gaps in the records i m m e d i a t e l y fo l lowing tu rn- o n and t u r n - o f f o f t he c u r r e n t s teps in D are due to t he vol tage d rop across the t ip o f the suc t ion p ipe t te .

65

A B SE V| V 2

C

o

I Rm Rsh Va

0

Fig. 13. Evaluation of Rsh, membrane resistance R m and patch resistance, Rp. A: experimental procedures; see text for description. B: equivalent circuit prior to puncture. C: equivalent circuit following puncture.

Before puncture the relevant equations are:

I = V1/Rm + V2/Rsh (1)

and

Ip = (V1 -- V2)/Rp = V2/Rsh = I~h (2)

where I is the current injected from the micropipette, V1 is the transmem- brahe voltage change, V2 is the voltage change in the suction pipette, Ip and Ish are the patch and shunt currents, and Rm, Rsh, and Rp are the membrane, shunt, and patch resistances, respectively. After puncture we have:

V3 _ Rm • Rsh

I R m + Rsh (3)

where V3 is the voltage change in the suction pipette elicited by the same transmembrane current. In using this method to evaluate the system's electrical properties we have found that R m in Helix nerve cell bodies ranges from 10 to 20 M ~ and in satisfactory experiments Rsh has values of 10 X Rm or greater.

The same arrangement allowed us to compare the current clamp responses of the suction pipette and micropipette systems using an artificial cell made of passive components which simulated the equivalent circuit of Fig. 13, but also included a parallel capacitor of 1--5 X 10 -l° F. The cell was grounded, and current and voltage connections between it and either of the two pipette systems were made through the bath. The voltage responses to hyperpolarizing current steps were similar in amplitude and time-course. The current responses to voltage clamp steps were not. The rise times of the voltage steps for an unshielded micropipette system and for the suction pipette were 2 msec and 10 psec, respectively. The time constants for the respective current transients were 3--7 msec and 100--500 psec for snail neurons, respectively. As we shall see next, the values for the suction pipette are influenced by its tip diameter.

66

The voltage responses to constant current pulses (Fig. 12) passed via the suction pipette and were measured by both the suction pipette and a separate intracellular micropipette. The voltage responses to hyperpolarizing current steps were similar in time-course, as were the action potentials elicited by depolarizing current pulses (Fig. 12), but the deflections in the suction pipette were larger due to the IR drop across the tip of the pipette. The value can be estimated from the purely resistive voltage response at the onset and offset of the current step. As expected, the IR drop depended upon the tip diameter of the suction pipette and the resultant resistance to current flow at this site; the relationship between pipette resistance and tip diameter is shown in Fig. 14. When a separate micropipette was used to record voltage the series resistance estimated from the instantaneous voltage response to a step input of current was 30--50 k~ .

In experiments using suction pipettes with larger tip diameters the voltage deflections elicited by steps of current at T = 0 usually gave values for the series resistance of l0 s ~ . The voltage clamp capacitative current transient was fitted by a single exponential (Fig. 7 of Lee et al., 1978) and had time constants ranging from 100 to 500 psec. Hence the series resistance was largely due to the pipettes' tip resistance. The membrane capacitance was calculated from the integrated clamp current transient and ranged from 300 to 600 pF. The lower values correlate reasonably with calculated values assuming a spherical cell body without significant infoldings of ~100 M in diameter corrected for ~20% membrane loss with the suction pipette and a specific capacitance of 10 -6 Fcm -2. The higher values may have been asso- ciated with larger cells but this was not examined systematically.

When tip diameters of 50 pm or more were used and the currents were 20 X 10 -9 A or less, series resistance compensation provided a suitable adjust- ment of EM to the command potential, V2. This is shown in Fig. 15 where

1.0

0.1

0 . 0 5

I I I 0 50 100 150

DIAMETER (~m) 200

Fig. 14. Re l a t i onsh ip b e t w e e n d i a m e t e r of the tip of the suction pipette and res is tance of p ipe t t e to c u r r e n t flow.

67

300

E /

/

e=o

o

r r

200 //~ / / ~ R s c o m p e n s a t i o n

! ! I ! ~, -200 -- I00 ' . v ~ O I00 200 500

VH ~ ] Expected membrane potential (mV)

/ t-,oo -200

Fig. 15. Evaluat ion of effect iveness of series resistance compensa t ion . Membrane potentials are measured by a separate intracellular glass mic rop ipe t te and are plot ted vs command voltage c lamp pulses (labeled expec ted membrane potent ia l ) delivered through the suct ion pipet te . Broken line indicates the ideal relat ion wi thou t series resistance.

recordings of E M measured with a separate intracellular micropipette are compared with the command voltages with and wi thout Rs compensation. Between --150 and +100 to +150 mV, Rs compensation provides adequate EMS. In Fig. 16 the inward and outward currents recorded with equal voltage clamp steps, using only the suction pipette to record E M and deliver current wi thout Rs compensat ion (A), with Rs compensation (B) and using a separate intracellular micropipette for recording EM, to be employed by the differential clamp amplifier (C), are compared. B and C compare favorably for the smaller inward currents but B underestimates the larger outward current and the outward tail current. For larger currents or pipettes with smaller diameters it is clearly preferable to use a separate pipette for measurement of E M. The isopotentiati ty of the cell can also be checked with the two intracellular pipettes provided current is passed through the pipette not recording E M.

A

I

C

[

68

+15mV

B

. . . . . f 0 nA

VH : - 6 0 m V 5 mse¢

Fig. 16. Ef fec ts of Rs c o m p e n s a t i o n on isola ted Helix n e u r o n cur ren ts . Cur ren t s are e l ic i ted by vol tage c l amp pulses to +15 m V t h r o u g h the suc t ion p ipe t te . Na cu r r en t is suppressed by Tris s u b s t i t u t i o n for Na. Cur ren t s are no t co r rec t ed for leakage. A: E M is measu red by t h e suc t i on p i pe t t e and R s - c o m p e n s a t i o n is n o t used. B: same a r r a n g e m e n t as A, b u t R s c o m p e n s a t i o n is used. C: E M is measu red by a separa te m i c r o p i p e t t e and R s c o m p e n s a t i o n is used. The c u r r e n t t race is noisier , t he o u t w a r d cu r r en t , and the tail cu r r en t s are m o r e subs tan t i a l t h a n cu r r en t s in B.

Adequacy of internal perfusion

This was evaluated directly by following the changes in concentration of intracellular K ÷ or C1- when the internal perfusion fluid was switched to K ÷- free or C1--rich solutions (Fig. 17). Intracellular K ÷ and C1- liquid ion exchanger microelectrodes were fabricated and used as described by Russell and Brown (1972a, b). In the case of CI-, the neuron was perfused with IC aspartate which was then changed to KC1. Within 2--3 rain the intracellular C1- had risen from a few mM to 105 mM. When the aspartate perfusate was reinstituted, a quick fall in Cl-i occurred. The few mM Cl-i present in K aspartate reflects the selectivity of the C1- electrode (C1- : aspartate- selec- tivity ratio is 20--50}.

The measured changes of K+i were similar. Replacing K aspartate with

140

120

100

3E

so

E

.u~

o

40

20

Znse

r t;

on

135

Cl~

OC

I

\

105

C I

I I

oc,

I 0

With

drow

ol

135C

I

B

100

80 ~ ~

K +

or

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Mp

Inse

rtio

n

[ ,o

5 K

.

2 I:

60

40

20

| I

I 0

A I

I0

20

30

40

0 70

TIM

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(ra

in)

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wal

105

K

OK

[

5K

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i 10

20

30

40

50

60

TIM

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(rai

n)

Fig

. 17

. A

: ch

ang

es i

n i

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on

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ere

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.77

. A

t th

e fi

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arro

w t

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lec

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de

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in

to t

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ron

wh

ich

was

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fuse

d w

ith

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10

5 m

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l- w

as t

hen

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bst

itu

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fo

r as

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-. (

e),

extr

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+ c

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70

Tris C1 resulted in a fall of K÷i from 105 mM to 10 mM within periods ranging from 2.5 to 5 min. The 10 mM residual levels of K ÷ in Tris perfusate reflect the selectivity of the K ÷ electrode {about 10--30 to 1 for K ÷ over Tris+).

The exchange times are affected by the shape of the suction pipette and the position of the perfusion tubing. The most rapid exchanges occur when the tip of the pipette has a very shallow taper and the perfusion tubing is located very close to the neuron (Fig. 17).

Another method of evaluating internal exchange was to follow the block- age of the outward clamp currents after switching the internal perfusion fluid from K aspartate to Cs aspartate. The outward currents were usually completely blocked within 5--10 min of such a solution change (Fig. 18). Notice that as the outward current disappears the inward current becomes

A + 25mY

-t ~ J 20nA

5msec

B vH = -60mY

30 < c

~, 20

U ~ lO

0 c

- 1 0

? u -20

4" 0

z I I I

o 5 lO i..5 INTRACELLULAR PERFUSION TIME (MIN)

Fig. 18. Time-course of changes in membrane currents after intracellular perfusion with Cs aspartate. Currents are corrected for leakage. A: currents evoked by steps from --60 to +25 inV. B: corresponding values for peak inward and outward currents. Zero K ÷ current occurred 13 rain after Cs perfusion.

A

B

C

,t15

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5

-- +

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Fig

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= -

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. In

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as w

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Rin

ger

. a,

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efo

re s

ep

ara

tio

n;

b,

in T

TX

5

× 1

0 -

~ M

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he

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d

cu

rre

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mV

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b-

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s "

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, P

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the

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n (

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% f

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ain

) p

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ut

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; c,

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X

and

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for

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2+ i

n

the

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on

. T

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cu

rre

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÷ c

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its

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pli

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mal

ler.

72

larger. The inward current does not inactivate completely (Akaike et al,, 1978).

The ease with which ionic currents can be separated is shown by the sequence in Fig. 19. Fig. 19A shows the voltage clamp currents in an isolated neuron soma. The records show an early inward current and a later outward current. The outward current is then suppressed by substituting Cs + for K * intracellularly leaving a transient inward current that inactivates slowly (Fig. 19B). The inward current can be separated into its two components shown in Fig. 19Cb ana c. The larger, slower componen t is present when Tris is substi tuted for Na ÷ in the external solution or when TTX is added in the presence of Na ÷ and the small faster componen t is revealed when Co 2÷ is added to the external solution which contains Na ÷.

Isola t ion o f the neuron soma f rom the neuron axon

Satisfactory spatial control can only be achieved when the nerve cell body has been separated from its axon. This is accomplished as shown in Fig. 1. Separation is usually accompanied by a fall in input resistance but there is a subsequent recovery as shown in Fig. 20. The effects of severing the axon on the voltage responses to current steps are shown in Fig. 21. The voltage responses before rupture of the aspirated neuronal membrane are shown in Fig. 21A. It is clear that only limited electrical access to the cell's interior has been attained wi thout rupture of the membrane. Between A and B the membrane was ruptured and depolarizing current produces 4 action potentials. After severing the axon, a single action potential is produced along with a maintained depolarization. A comparison of current records obtained from a nerve cell body under voltage clamp with intact axon (A) and severed axon (B) is shown in Fig. 22. It is clear tha t the axon is not under clamp control as inverted action potentials are recorded in the soma.

30

.~ 2;

Z

~ 2c

z 15

S j

AFTER SEPARATION (MIN)

Fig. 20. Time course of recovery of input resistance af ter m e m b r a n e punc ture .

A B

73

With A x o n

i i ! L

J 4 o m V

200msec

W i t h o u t ~

1 s e c

Fig. 21. M e m b r a n e po t en t i a l responses to c o n s t a n t cu r r en t s teps before (A) and af te r (B) r u p t u r e of the asp i ra ted m e m b r a n e . Before r u p t u r e the ef fec ts of charging the mem- b rane capac i t ance are no t ev iden t and the to ta l res is tance to cu r r en t f low is greater. Depolar iz ing s teps do n o t p r oduce ac t ion poten t ia l s . Af te r rup tu re , depo l a r i za t i on of cell w i th a x o n p roduces a bur s t of ac t ion po ten t i a l s whereas a single po t en t i a l and sus ta ined depo la r i za t ion is observed fo l lowing removal of the axon.

A

J 8 0 n A

20inlet

+ 9 6

B

/

~'~'~ -37

J 2 0 n A

2msec

Fig. 22. A: vol tage c l amp cu r r en t s f rom a n e u r o n wi th a t t a c h e d axon . The vol tage s teps were to - -45 , - -40 , - -39 , - -33 , - -23 , and --4 m V and were appl ied f rom ho ld ing po ten t i a l , VH of - -60 inV. The inward cu r r en t s arise a f te r some l a t ency and increase very l i t t le wi th increasing vol tage c l amp steps. Repe t i t ive responses occur at larger depo la r iza t ions . B: m e m b r a n e cu r r en t s a f te r severing the n e u r o n soma f rom its axon. VH = - -60 mV. The vol tages o f the s teps are given wi th each c u r r e n t t race.

7~

Microscopic membrane currents (current noise)

Another advantage of using the suction pipette method is the low noise of the recording system which makes it possible to record small signal fluctuations. Since microscopic current fluctuations are the desired measurements, voltage clamp should be used and the proper precautions taken regarding tip size and magnitude of currents. The single suction pipette method is quite suitable for steady state recording of Ca current which is only a few nA but unsuitable for steady K currents where a separate measurement of E M should be made. Measurements of K and Ca current noise in isolated nerve cell bodies of Helix have been reported by Akaike et al. (1978b) using the suc-

a

K + and Ca 2 + Conduction Noise and Current

\ - 20 mV

I l O n A

Ca2"j f ° ,

in Ni J 20 nA

15 sec 15 $ e c

VH= - 6 0 m V

Fig. 23. a: membrane currents before, during and after prolonged depolar iza t ion of an isolated, internal ly-perfused, vol tage-clamped snail neurone. Holding potent ia l was - -60 mV and the voltage was s tepped to - -20 inV. The left-hand panel shows the current noise traces and the large transients due to the high-pass fi l ter; r ight-hand panels show the corresponding low gain recordings of membrane currents I m af ter leakage correct ion. Current noise was analyzed during the 15 sec periods indicated by the hor izontal bars. Note the d i f ferent current cal ibrat ions for the noise and I m panels, b: af ter suppression of I K with Cs +. Addi t ion of t e t r odo tox in or subs t i tu t ion of Tris for Na + had no fur ther effect , and the current noise in b is a t t r ibu ted to f luctuat ions in Ica and leakage current. I m w a s c o r r e c t e d for leakage and has transient and steady inward components , c: af ter addi t ion o f Ni 2÷ to the external solut ion. The difference in noise be tween b and c is a t t r ibu ted to suppression o f Ica by Ni 2+. The residual ou tward current is leakage current.

75

A

b r

B

a 5 x lO-12A

b _ _ / . ~ ~ ~ ~ " 5 x 10"9A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

t l0-4M ACh 5 s e c

Fig. 24. Current noise from two isolated nerve cell bodies of Helix recorded with the suction pipette, Cell A shows a depolarizing response (inward current, Ab) to ACh 10 .4 M added to the bathing solution. This is accompanied by a large increase in current noise (Aa). Cell B shows a large outward current when ACh is added and an increase in noise as well.

t ion p i p e t t e m e t h o d and fo r Ca cu r r en t noise b y Krishtal and P idop l i chko {1977) using the m e t h o d o f K o s t y u k et al. (1975) . The p o w e r dens i ty spec t r a had the L o r e n t z i a n shape assoc ia ted wi th channe l noise (Kolb and Lauger , 1977) and similar values fo r the un i t c o n d u c t a n c e o f the Ca channe l (10 -13 S) were o b t a i n e d b y b o t h groups . E x a m p l e s of K and Ca cu r r en t noise are s h o w n in Fig. 23. Na cu r r en t was suppressed using Tris subs t i t u t i on fo r Na. T h e K noise inc luded a small c o n t r i b u t i o n o f Ca noise which b e c a m e m o r e a p p a r e n t a f t e r b lockage of K + c o n d u c t i o n wi th Cs ÷. T ransmi t t e r - ac t iva ted noise is also readi ly a p p a r e n t using the suc t ion p ipe t t e m e t h o d . Fig. 24 shows the large cu r ren t noise p r o d u c e d in t w o d i f f e ren t neu rons b y add ing 1 0 - 6 M ACh to the ex te rna l pe r fusa te . In one cell ACh p roduces inward cu r r en t at a ho ld ing p o t e n t i a l o f - -50 m V and in t he o the r cell out- wa rd cu r r en t results.

DISCUSSION

The suc t ion p ipe t t e m e t h o d has p roved to be useful p rov ided its l imita- t ions are recognized . The l imi ta t ions are the shun t res is tance re la ted to the

76

seal between the aspirated cell and the pipette and the series resistance at the pipette tip, when a single pipette is being used for voltage recording and current supply. The simple solution to the latter problem is to record membrane potential separately with another pipette. In snail neurons we use an intracellular micropipette. In dispersed single heart muscle cells we use a second suction pipette. The filtering action of the second suction pipette on voltage recording is 1--2 orders less than that of a 10 6 ~2 micropipette so that the rise time of the voltage clamp step is less than 5 psec. Furthermore perfusion of the particular cell is enhanced by applying a gradient in suction between the two pipettes. Shunt resistance is made large by the suction and by the seal made between the partially aspirated cell membrane and the treated glass wall of the suction pipette. In some cells shunt resistance cannot be made large enough because of a poor seal. This appears to be the case for Aplysia neurons whose cell bodies have numerous infoldings and a complete glial covering. It is not the case in Helix neurons where the cell body surface is relatively smooth and the glial covering is incomplete. Nor is it the case for dispersed single cardiac myocytes where our experiments indicate that the action potentials recorded with suction pipettes and micropipettes are equivalent.

The main virtue o f the suction pipette technique is that it is a relatively simple method of producing voltage clamp and internal perfusion in smaller cells. With proper precautions spatial control is adequate and temporal resolution at the 10 -4 sec level is possible. Moreover in the case of neurons the method itself is a useful way of severing the axon from the cell body manually, thereby avoiding enzymatic dispersion techniques which may alter membrane properties (Lee et al., 1977). Finally, the method is useful for measurement of microscopic current fluctuations because of its low inherent noise.

ACKNOWLEDGEMENTS

We would like to thank Dr. Y. Tsuda for his help with this paper. This work was supported by N.I.H. Grants NS-11453, NS-12655 and NS-

13778.

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77

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The suction pipette method for internal perfusion and voltage clamp of small excitable cells