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HANDBOOK OF PHYSIOLOGY 



NEUROPHYSIOLOGY 



ins; potentials. But when stimulating or polarizing 

 currents are to be delivered to the penetrated cell (see 

 page 274) or when ions are to be deli\ered through 

 the tip bv iontophoresis then pipettes must be selected 

 for their current-carrying ability. 



Cajiacilnnce. The ability of a micropipettc to follow 

 rapid changes in the electrical potential at its tip 

 varies inversely with the electrical capacitance across 

 its walls (see below). The capacitance across the 

 glass wall of a micropipettc between inside and out- 

 side electrolytes depends upon the thickness of the 

 wall and on the length of that portion of the pipette 

 which is immersed in the external \olume conductor. 

 Since in a drawn glass pipette the ratio of wall thick- 

 ness to diameter remains approximately constant the 

 capacity is proportional to the immersed length. Frey- 

 gang (28) calculates the capacity across a pipette 

 drawn from Pyrex tubing No. 7740 having a ratio 

 OD/ID of 2 as 0.4 /i/if per mm. This is close to the 

 measured value for an actual pipette of 0.37 fifif per 

 mm of immersion. The total capacity is linear with 

 immersion depth except for a minor change occurring 

 as the shoulder of the pipette enters the external con- 

 ductor. Nastuk's figure of i /x^f per mm would indi- 

 cate a smaller ratio of OD, ID (47). 



Tip potential. Nastuk & Hodgkin (47), who used 

 micropipettes filled with 3 m KCl, assumed pro- 

 visionally that measurements of potential made with 

 .such pipettes are not altered by a junction potential 

 at the electrode tip. However, Nastuk (46), del Cas- 

 tillo & Katz (17) and Adrian (3) showed that with 

 many such fine tipped pipettes there is a potential 

 diflference across the tip which may be as large as 70 

 mv, inside negative, and that this tip potential may 

 change suddenly with movement of the pipette in 

 muscle or nervous tissue. Thus measurements of 

 steady potential differences, e.g. the membrane po- 

 tential of a cell, will be in error if the tip potential of 

 the micropipettc differs at two points. Such a differ- 

 ence can arise in two ways. The tip potential of the 

 pipette may be changed by clogging or unclogging the 

 tip as it moves through the nervous tissue. In this case 

 alternate measurements at two points would not 

 generally be expected to repeat the error. A more con- 

 sistent error will be encountered if the two points 

 whose potential difference is to be measured are in 

 regions of different ionic composition, and if the tip 

 potential of the micropipettc is different in the two 

 regions. Adrian (3), attempting to clarify this point, 

 measured the tip potential of a series of pipettes in 

 both 100 mmole KCl and 100 mmole NaCl. He found 

 that the difference in tip potential in the two solutions 



was proportional to the tip potential in 100 mmole 

 KCl. Adrian considers the mechanism of the tip po- 

 tential to be a selective reduction in mobility of some 

 of the ions, particularly that of the anion and probably 

 due to some form of blocking. He argues that if a 

 pipette has a small tip potential it is less likely to 

 show a change or introduce an error due to tip po- 

 tential. On this basis he measures the resting potential 

 of muscle membrane by selecting only those pipettes 

 whose tip potentials are less than 5 mv, inside nega- 

 ti\e. \Vhile it is true that such a pipette would be ex- 

 pected to introduce only a small error of i or 2 mv in 

 the measurement of potential difference between two 

 test solutions (100 mmole KC'.l and 100 mmole NaCl), 

 there seems to be less certainty that a pipette having 

 an initially small tip potential will not increase its tip 

 potential during its movement through tissue. In the 

 particular case of the resting potential across a cell 

 memijrane, errors introduced by variation of tip 

 potential cannot be eliminated by repeated compari- 

 sons since the cell memijrane is generally damaged by 

 repeated penetration. For this reason, micropipettc 

 measurements of steady potential difference between 

 different points in the nervous system are subject to 

 considerable uncertainty at the present time. 



Frequency response. Metal microelectrodes are gen- 

 erallv poor for measurement of d.c. or of very slowly 

 varying potentials as described above, but at higher 

 frequencies their impedance drops to relatively low 

 values. The resistance of a glass micropipettc on the 

 other hand is quite independent of frequency at low 

 frequencies. This does not mean that an ordinary 

 amplifier connected to a micropipettc necessarily re- 

 cords biological potentials with a good frequency 

 response. Indeed, this is one of the failings of these 

 electrodes. 



A micropipettc must generally pass through a 

 region of grounded volume conductor before reaching 

 the highlv localized region whose potential is to be 

 recorded. The capacity across the glass wall between 

 the inside electrolyte and this external volume con- 

 ductor (.see above) tends to reduce the high-fre- 

 quency response of the electrode. Since most of the 

 resistance of the pipette is located very near its tip, it 

 can be well represented in such an application by 

 the equivalent circuit of figure 4. If the input im- 

 pedance of the amplifier is high enough to be neglected 

 in comparison to Re and Ce, a sudden change in 

 \ oltage E will be recorded as an exponential rise hav- 

 ing a time constant r = Re Ce; that is, the recorded 

 N'oltage \' will rise to about 63 per cent of E in the 

 time T. For anv form of voltage signal E fed in, the 



