Intracellular measurement of ionic activity

280 requires a certain threshold level for activation, or is especially sensitive to certain pharmacological agents, inferences can be made by demonstrating that specific components of surface recorded potentials also have these characteristics. Clearly, these tests require tenuous assumptions concerning the neural events. Data are often not available to confirm these assumptions. For these reasons, the origins of most longlatency components of surface recorded evoked potentials are controversial. One unspoken assumption in the preceding discussion is that evoked potentials are generated by neuronal activity. This assumption is not necessarily applicable. There are many possible sources of electrical potentials in nervous tissue other than electrical current across neuronal membranesL For instance, glial cells may generate field potentials. Ionic gradients in the extracellular space may produce diffusion potentials. In fact, the potentials need not be exclusively current-related; changes in static charge distribution may alter surface potentials. Such possibilities have not been adequately studied or ruled out. tn conclusion, the value of surface recorded evoked potentials should not be judged solely in comparison with more classical neurophysiological approaches. The virtue of surface potential techniques stems from the unprecedented opportunity they provide for non-invasive study of human and animal nervous systems. In many situations, this virtue outweighs the limitations of the technique. Effective strategies have been devised to overcome some of the limitations. In this article, a guarded but optimistic view of the field is offered.

TINS - N o v e m b e r 1981 7 Rebert, C. S. (1978) in Muhidisciplinary Perspectives in Event-Related Brain Potential Research, Document EPA-600/9-77-043, U.S. Environmental Protection Agency 8 Regan, D. (1972) Evoked Potentials in PsychoL ogy, Sensory Physiology and Clinical Medicine, Chapman and Hall, London 9 Start. A., Sohmer, H. and Celesia, G. G. (1978) in Event-Related Brain Potentials in Man (Callaway,

Intr

Press. New York 4 Goff, W. R., Allison. T. and Vaughan, H. G. (19781 in Event.Related Brain Potential.~"in Man (( allaway,E., Tueting, P. and Kostow,S. H.. eds), pp. 1-92, AcademicPress, New York 5 Henderson. C. J.. Butler, S. R. and Glass, A. (1975) Electroencephalogr. Clin. Neurophysiol. 39, 117-1311 6 John, E. R. 119771Neurometrics:ClinicalApplications of Quantitative Eleetrophysiology. Functional Neuroscience Series, Vol, 2, J. Wiley & Stms,New York ~3 EIsevieffNorth-HollandBiomedicalPress 1981 11378 5912/81/0000- ~11100/$0275

Wize Young i~ at the New York Universi~" Medical (.'entre, Sehool o f Medicine, 550 Fimt Avenue, New York, N Y 10016, U.S.A.

ltutar measurement of ionic activity M. B. A. Djamgoz and P. J. Laming

The measurement o f intracellular ionic activity is crucial to a quantitative understanding o f neuronal function. Intracelhdar ion-sensitive microelectrodes (1SMs) can now be routinely made which are capable o f determining the ionic activities o f Na +, K +, Cl , H +and Ca 2÷and o f monitoring their changes even in small neurones. This article outlines some o f the theoretical and practical aspects underlying the use o f lSMs, and provides details o f some applications. Interested readers should also consult the recent article by Charles Nieholson on extracellular measurement (TINS 1980, pp. 218-218).

The ionic basis of electrical activity in the nervous system was first formalized by Bernstein using the electrochemical theories of Nernst. He suggested that in the resting state the potential difference across the nerve membrane could be accounted for by the m e m b r a n e ' s selective permeability to potassium ions, and that excitation inw)lved a general increase in membrane permeability to Na +, K ~ and CI- which effectively short circuited this potential. Later, however, Hodgkin, Huxley and Katz showed that during excitation the interior of the nerve becomes transiently positive, due to a selective increase in the m e m b r a n e ' s permeability to Na ~. It is now generally accepted that the sigAcknowledgements nalling ability of excitable cells depends on Supported by NIH NINCDS grants R01- the transmembranc concentration graNS15590 and 2P50-NS10164. dients o f N a +, K +, CI . Ca 2+ ions. Each ion has its characteristic equilibrium potential Reading list which is expressed by the Nernst cquation: I Dawson, G. D. (1951)J. Physiol. (London) 115. 2P-3P 2 Donchin, E. andHeffiey, E.(1978)inMultidisciplinary Perspectives in Event-Related Brain Potential Research. Document EPA-60019-77-1143. U.S. Environmental Protection Agency 3 (;laser. E. M. and Ruchkin, D. S. (1976) Principles of Neurobiological Signal Analysis, Academic

t:.., Tueting, P. and Koslow. S. t|. cds). pp. 155-221, Academic Pres~, New York 10 Thatcher, R. W. and John, E. R. (1977) ~?~undatiott~ o f Cognitive Processes, Functional Neuroscience Serie.s, Vol. 1, J. Wiley & Sons. New York

R T In a'i

membrane permeability to a particular ion displaces the potential towards the equilibrium value for that species. In order to be able to determine the contribution of a particular ion to the active and passive properties of a particular cell both the intracellular and extracellular concentrations must be determined. Clearly, the most difficult of these measurements is the intracellular one. In the case of the squid giant axon sufficient cytoplasm can be extruded to carry out ion analysis by conventional chemical means (e.g. flame photometry). However, this technique is obviously impractical for small cells in the ncrw)us system. The ISM however allows these measurements to be cartied out in vivo. The theory and application of this technique have been described in greater detail by several authors LT,~r'~-2x what flfllows is a brief summary of the aspccts most pertinent to the general reader,

F~Y = . . . .

Z,F

a ',

where E~ is the equilibrium potential for the ion 'x'; R, the universal gas constant; T, the absolute temperature; F, the Faraday constant, a'~ and a', the activities* of the ion x on the outside and the inside of the membrane, respectively, and Z~ its valency. The membrane potcntial is a function of the Nernstian potential h)r these four ions, but usually it is dominated by thc contribution of a single species. Increasing the *The activity of an itm. a~, is related Ic~ its concentration Cv, by the relationship ax - ~.('~. where ",/~ is known as the activity coefficient.

Classification and construction 1SMs consist of a conventional glass micropipette electrode filled with the appropriate reference solution (Fig. 1)whose tip is plugged with a material which is ionsensitive/selective. Since the tip can be a micron or less in diameter, it can be used for intracellular recording. ISMs can be classified according to the physical nature of the ion-selective sensor (liquid-state and solid-state ISMs), and the ionic species to which it is primarily sensitive (K+-sensitivc ISM, C1--sensitive ISM, etc.). An overall classification is given in Table I.

T I N S - N o v e m b e r 1981

281

TABLE I. A classification of intracellular is available for a given ion, the latter type of only for recording from relatively large ion-sensitivemicroeleetrodes ISM is almost always preferred. For exam- cells since the exposed ion-sensitive comple, solid-state K+-sensitive ISMs are now ponent is usually several microns in length. hardly ever used, and there is increasing Metal based ISMs are manufactured in Type of Primary Reference demand for a suitable Na t liquid ion an analogous way. For example, a 1SMsensor sensitivity exchanger in preference to using the Na ÷- chlorided silver wire (Ag:AgCI) behaves as sensitive glass. At present, however, glass a C1--sensitive electrode, so a recessed-tip Liquid state: membrane ISMs for Na t and H+(pH) or a protruding-tip type CI- ISM can be Liquid ion K+, CI , 1,3, 7, remain the best sensors available for these constructed by sealing a sharpened exchanger Na+, Ca2÷ 17.20 Neutral ion K+, Ca2+,Na+ 1,7, 8, 13, ions. The first step in the manufacture of AG:AgCI wire into the end of a glass micarrier 17, 19 this type of ISM is to pull a microelectrode cropipette (Fig. le) ~7. This type of CI- ISM Solidstate: from the ion-sensitive glass and seal its tip has been used to check results obtained Glass Na+, H+, K+ 6,7,15, using a heated microforge element. The with liquid ion exchanger type ISMs TM.The membrane 16. 17 sealed glass is lowered into an insulating reliability of the antimony-based pH ISM, Metal CI . H+ 7, 10, 14. 17 glass micropipette (aluminosilicate rather however, has been doubtful TM. than borosilicate glass for improved duraOperational characteristics The liquid sensors for ISMs are water- bility) of similar tip shape and size until the immisciblelT'tL They would normally be two tips are separated by some 10/zm. The The voltage response Vx of an ISM sensidisplaced from the tip of a glass microelec- ion-sensitive conical glass membrane is seal- tive primarily to an ion x of valency Zx, in trode by the aqueous reference solution ed locally onto the inside of the outer glass the presence of a number of interfering that fills the back of the electrode, since a by applying heat from the outside to a reions y of valencies Zy, is Nernstian and glass surface is normally hydrophilic. Hav- gion 50-100/~m above the tip of the inner given by the Nikolskii equation: ing pulled a glass micropipette of the pipette (Fig. lb). The electrodes are then desired tip size and shape, the next crucial filled with the appropriate reference soluRT (Z.,'~, V ~ : V . - - ~--~ I n [ a , + ~ kav, ., e x p k - ~ + ) j . step is to make its inside wall hydrophobic tion (e.g. 0.1 M NaCI + 1 M E D T A for Na t (water-repellant) by treating it with an ISM; 0.1 M NaCI buffered to pH 6 with organic silicon compoundlL Not surpris- 0.1 M citrate buffer for pH ISM) ~7. Finally, (+ cationic ISM; - anionic ISM) where Vo ingly, a variety of different silanes and a Ag - AgCI wire is sealed into the back of is a constant reference potential, ax and ay silanizing procedures have been used for the ISM. This recessed-tip type solid-state are the activities of the primary and interthis reason, as the subsequent performance ISM was introduced by Thomas ~,~, and feting ions, respectively, and k+r is the of a liquid-state ISM to a great extent will has the advantage of ensuring that the ion- selectivity 'constant' of the ISM for the depend on how well it was silanized in- sensitive glass membrane comes into con- primary ion over each of the interfering itially. In general, under-silanization will tact with the intracellular medium only. ions. In the simple case of a K t ISM for lead to the filling solution gradually creep- This is achieved at the expense of a tip size which the main interfering ion is Na t , the ing to the tip of the ISM and forming a thin (1 t~m to a few micrometres) and response response potential is given by: layer of relatively high conductivity time (several seconds are required to VK = Vo + S loglo(aK + kKN~aN,) thereby shunting some of the ISM elec- achieve diffusional equilibrium at the tip of trochemical potential. On the other hand, the ISM). An alternative to this is the where S is known as the slope constant of over-silanization will partially (if not com- Hinke-type design in which the ion- the ISM and has a value of approximately pletely) block ISM tips and induce undesir- sensitive glass membrane cone protrudes 58 mV at 20°C. The selectivity 'constant' ably high electrode resistances. Typically from the tip of the insulating glass u. The k~y of an ISM does not necessarily have a 3% tri-N-butylchlorosilane in chloronaph- latter type of ISM is faster, but suitable fixed value. It has been shown to depend thalene is used to silanize single-barrel ISMs aT and the vapour from dimethyl dia b c chlorosilane to silani~e the active side of a double-barrel ISM3.L Following the introduction of the silane solution into the microelectrode shafts, the latter are baked at 100°C for 1 h to complete the silanization procedure. A small quantity of the liquid sensor is then injected as far down the shank of the electrode as possible and made to fill 200/zm to a few millimetres W..,~_tiquid from the tip by applying pressure or using the capillary action of a cat's whisker. The rest of the microelectrode is filled with a reference solution (e.g. 0.5 M KCI for K +and CI--sensitive ISMs) into which the |~/ ion-sensitive rl chlorided end of a silver wire is sealed to |~glass membrane II complete the ISM assembly. Solid-state ISMs are, in general, more Fig. 1. The tip assembly of three different types of lS M (not drawn to scale). (a) Liquid-state IS M. The length of the difficult to manufacture than the liquid- liqm'dsensor column can be 200ta~nto several millimetres; the inside wall of the micropipette nmtsrbesilanized. (b) Solid-state, glass membrane, recessed-tiptype ISM; the recess volume is criticaland determines the response time of state variety, and if a suitable liquid sensor the electrode. (c) Solid-state, metallic sur]~ce type ISM. (Modifiedand redrawnfromThomas'7.)

z++

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282

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i

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Fig. 2. ('alihration traees and graphs fbr a double barrel K'ISM. (a) The voltage responses o f both the potassium acetate filled reference electrode (Vr) and that ]or the ISM barrel (VIe) v;,ere monitored simultaneously. The calibrating solutions contained 1, 5, 10. 50, IO0 mM o f K + concentration; the ionic strength wa~ kept constant at 100 mM with eorresponding amount~ of NaCI. VK for 100 mM K* was arbitrarily fixed as 0 inV. (b) The voltage readings, VK (mM) from (a) plotted against the logarithms o f the K +activities aK (m st 3,1~"= O. 770. Open circles, calibration values obtained prior to the experiment; full circles, calibration value~' obtained fi)r the same ISM following the completion o f the recording session. The calibration points can be fitted by the Nikolskii equation to within ± I m V assuming kKN. equaL~ 1.4 × I0 2.

on the ionicstrengthof the solution4andtip diameterL A number of different methods have been described for the assessment of selectivity constantsL The ISM selectivity problem is worst when ionic activity measurements arc desired in cells in which the activity of the primary ion is low compared with the activity of the main interfering ions e.g. Na* v. K +, Ca ~+ v. K + and Na ~. ISMs have electrical resistances in the range 10~ - 10nl~, due mainly to their ionsensitive tip components. To record an electrochemical potential correctly one must match the 1SM resistance with a high input impedence (1015D,) electrometer which will draw negligible current from the ISM. Such high resistance ISMs suffer from external electrical interference pick-ups and require careful electrical shielding of the preparation with a metallic enclosure and of the ISM itself by a signal-driven guard. Furthermore, capacity compensation is often required to improve the response time of the electrodes.

When an ISM impales a cell, the voltage it records is the sum of the transmembrane (resting) potential (Era) plus the electrochemical potential associated with the ionic activity. To obtain the latter measurement alone, E,, must be recorded independently and subtracted from the ISM potential. With large cells (e.g. snail neurones, barnacle photoreceptors) it is possible to impale the unit under visual control with a second (reference electrode) for the measurement of Em alone. If this is impossible, double-barrel microelectrodes must be used; one barrel is an ISM whilst the other one is the reference (ioninsensitive) electrode. The ionic activity levels recorded by an ISM will depend to some extent on the nature of the electrolyte filling the reference barrel because of leakage, and because the reference electrode itself can be slightly ion-sensitive depending on the liquid junction potentials. It is therefore, crucial to ensure that the reference electrodes are as ion-insensitive as possible and that any leakage from them are not sensed by the ISMs. Prior to using an ISM in an experiment, its w~ltage response is calibrated in a series of standard solutions containing varying amounts of the primary ion detected. The relative merits of various calibratkm pro-

0

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4631-

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1981

cedures have been discuss0d clsewherc'L The general rule is that {he calibration media should resemble, as (:losely as possible, the intraccllular pool of ions from which activity measurements ,*ill he made. Fig. 2 illustrates the calibration responses of a dot, bit-barrel K* ISM in a series of KCI ~ NaCI solutions (o~ constant ionic strength 100 mM; activity coefficient 0.770). The reference barrel is filled with 0.5 ~alithium acetate and does not respond to the changes in the K + activity (Fig. 2a). The potassium signal, VK, is given by the differential measuremen I (ISM potential minus reference electrode potential) and this is plotted as a function of the potassium activity in Fig. 2b. At high ionic activity the response of this clectrgde incrcases by 56 mV for each t0-fold increase in K ~ activity but it deviates from the straight line predicted by the Ncrnstian relatkmship at low K + levels - here interference from Na ~ ions becomes significant. Fig. 2h illustrates another important procedure in ISM experiments, namely the necessity to calibrate the ISM before and after a recording session - the two calibrations mnst not vary by more than a few milliw)lts. The following sections provide examples of the vast range of applications for which ISMs have been used in neurobiology.

b

20~

a&l {mM) 17

I

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Em(mY)

Em(mV)

0-7

o

-20

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Fig. 3. lntraeellular recordings ]tom L~-type horizontal ('ells with double-barrel K + ISM (a) and (7 ISM (b). Membrane potentials (Era), and K* ISM and CI- ISM response voltages were recorded simultaneott~ly. "Thelarge negative deflection in each case marks the impalement o f the cells upon which light-evoked hyperpolarizing respon.~es were obtained (presentation o f the test light stimuli are marked by the dots and short bars below the Em trace). During the light-evoked changes in Vm, apparent transient changes in VK and V o are seen due to the relatively slow response time o f the ISMs. No capacity compensation was possible in these experiments. In all cases, it was found that all voltage readings Stabilised within some seconds" o f cell impalement. The horizontal 'time bars' denote 5 s' in (a) and 20 s in (h).

T I N S - N o v e m b e r 1981

Vertebrate retinal horizontal cells Horizontal cells are a type of interneurone in the vertebrate retina, They are driven by the photoreceptor ceils (rods and three spectral classes of cone) through chemical synapses and, in response to light generate slow, graded hyperpolarizing and depolarizing electrical responses (Spotentials) 9. We have started an analysis of horizontal cell ionic mechanisms in the fish (roach, Rutilus rutilus) retina using ISMs. Fig. 3a is a chart recording showing the impalement of a red-sensitive L~-type unit with a double barrel K + liquid ion exchanger ISM. With this type of ISM, the m e m b r a n e potential (Era, in this case, - 3 9 m Y ) and the K + signal (upper trace) are recorded simultaneously from the same neurone. In these units, ak had an average value of about 60 mM ( a ~ 3.5 mM), and E~ was consistently more negative than Em by at least 20 mV. From these measurements, it is deduced that in the dark the L~-type horizontal cell membrane is permeable to other ion(s) 'x' with E~ more positive than E~. One candidate is CI , since in these units E o is more positive than Em by at least 15 m V (Fig. 3b). During synaptic blockage by light stimulation or application of 1-2 mM CoCk, the membrane potential tends towards EK, and at saturation E m = EK thereby showing that the non-synaptic conductance mechanisms of the horizontal cell m e m b r a n e is K + driven.

Invertebrate photoreceptors The intracellular K +, Cl-, Na +, Ca ~+ and H + levels have been measured in barnacle photoreceptors by Brown and coworkersS-~'~4'=L The dark-adapted photoreceptor was found to have a significant chloride permeability such that Cl- was distributed passively (Ecr = E~). The potassium equilibrium potential was in the range 70-80 m V negative whilst that for sodium was greater than + 6 0 inV. The lightevoked depolarization of the photoreceptor was accompanied by a decrease in the internal p H (a ~) and an increase in a ~ ~.~. It was suggested that changes in intracellular p H of the photoreceptor induced by light stimulation might modulate its sensitivity.

Molluscan neurones Several of the neurones in molluscs are unusually large, and, as a result, have been subject to extensive electrophysiological investigation with ISMs. Perhaps the best known is the correlation between a~, and the electrogenic sodium pump made by Thomas in 1969. (He later found, with a sharper microelectrode, that a~, was rather

283 lower than previously thought.) The intracellular CI- activity in both H and D cells of Helix aspersa was found to be about 8.3 raM, giving an E a of about - 5 8 m V (Ref. 12). The action of acetylcholine on these cells was to increase both the Na + and the CI- permeabilities. The intracellular p H of snail neurones was found by T h o m a s to be regulated by a mechanism that involves the simultaneous influx of Na + and H C O L and the effiux of C1- and H +. It was suggested that such a regulation might be mediated by a single membrane carrier with separate transport sites for each of these ions TM.

Acknowledgements We wish to thank R, C. Thomas and his students for teaching one of us (M.D.) the ISM technique. We also thank him, H. M. Brown, T. Gillett, R. W. Meech and W. R. Schlue for discussions and advice, the SRC for financial support and Guliz Onkal for technical assistance. Reading list 1 Armstrong, W. McD. and Garcia-Diaz, J.F. (1980) Fed, Proc. Fed. Am. Soe. Exp. Biol. 39, 2851-2859 2 Bailey, P. L. (1980) Analysis with Ion-selective Electrodes, 2nd edn, Heyden, London 3 Berridge, M. J. and Schlue. W. R. (1978) J. Exp. BioL 72, 203-216 4 BoRon, T. B. and Vaughan-Jones,R. D. (1977) J. Physiol. (London) 270, 801-833 5 Brown. H, M. (1976) J. Gen. PhysioL 68,

281-296 6 Brown, H. M. and Meech, R, W. (1979) J. Physiol. (London) 297, 73--93 7 Brown, H. M. and Owen, J.D. (1979) Ionselective Electrode Rev. t, 145-186 8 Coles, J. A. and Tsacopoulos, H. (1979) J. Physiol. (London) 290, 525-549 9 Djamgoz, M. B. A. and Ruddock. K. H. (1979) Vision Res. 19, 413~$18 1(I Green, R. and Giebisch,G, ( 1974) in Ion-selective Microelectrodes (Berman. H.J. and Herbert, N. C., eds), pp. 43-53, Plenum, New York ll Hinke. J. A. M. (1959) Nature (London) 184, 1257-1258 12 Neild, T. O. and Thomas, R. C. (1974) J, Physiol. (London) 242,453-470 13 O'Doherty, L, Garcia-Diaz,J. F. and Armstr
A brain stem generator for saccadic eye movements Albert F. Fuchs and Chris R. S. Kaneko The extremely rapid conjugate shiJ~s o f gaze called saccades are accomplished by a pulsestep change o f excitation in the extraocular muscles, Within the last several years, the premotoneuronal circuitry responsible for this innervation pattern has been studied intensively, and this paper summarizes our current understanding o f the brain stem mechanisms involved in generating the saccadic trajectory.

We scan our visual environment with saceadic eye movements, conjugate shifts of both eyes that aim each fovea at the object of interest. Saccades can be elicited by asking a subject to fixate a spot of light as it jumps from point to point. After the target jumps, a delay of 200-300 ms elapses before the eyes move. During this time, the CNS must identify the target, decide whether to move the eyes toward it, and initiate the saceade. Once the saccade is triggered, the eyes accelerate rapidly to m a x i m u m velocities exceeding 500 deg s -1 at mid-trajectory, and decelerate to bring the foveae accurately on to the target, usually with no overshoot or oscillations. Thus,

saccades are not only the fastest but also the best controlled movements of which the body musculature is capable. The central structures implicated in the control of eye m o v e m e n t s include parts of the thalamus, large areas of the cortex, the basal ganglia, the cerebellum and the superior colliculus. Unfortunately, it has been difficult to determine how these structures are involved in saccade generation, possibly because most of them seem to lie at the difficult, if exciting, interface between visual and motor events. On the other hand, neurons in the brain stem surrounding the various oculomotor nuclei have discharge patterns related primarily, O Elsevier/Norlh-Holland Biomedical Press 198 l 0378 - 5912/81/0000 - (K900/$02.75