Measurements of Calcium Transients in the Soma, Neurite, and Growth Cone of Single Cultured Neurons

The Journal of Neuroscience July 1966, 6(7): 1934-1940

Stephen R. BoIsover* and llan Spector-f *Department of Physiology, University College, London, London WClE 6BT, England, and TDepartment of Anatomical Sciences, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794

Voltage-gated changes in cytosolic free calcium ion concentra- interpret because they cannot give precise information about tion were measured in single, differentiated cells of mouse neu- the behavior of a local region of the cell or detect the presence roblastoma clone NlE-115 using the calcium-sensitive dye ar- of active calcium channels that do not contribute significantly senazo III (AIII). In cells bathed in normal medium containing to the action potential. To examine the spatial distribution of 10 mM calcium, the changes in AI11 absorbance during a single voltage-gated calcium entry in the neuronal cell membrane, we action potential indicated an increase of 1.4 nM in cytosolic used the calcium indicator dye arsenazo III (AIII) (Brown et al., calcium. When 10 mrw tetraethylammonium (TEA) was added 1975). This dye presents two important advantages. First, it can to the bath, the action potential became prolonged and the change diffuse rapidly inside the cell (R. F. Rakowski, personal com- in cytosolic calcium increased to 3.9 nM. Under these conditions, munication), thereby allowing measurements of changes in cy- repetitive stimulation at 0.5 Hz or faster caused a gradual de- tosolic calcium concentration ([Ca”],) in all regions of a single cline in the amplitude and duration of the action potential and neuron. Second, the response is directly proportional to the a gradual decline of the change in cytosolic calcium associated calcium concentration change. Therefore, the changes in optical with each action potential. The amplitude of the prolonged af- absorbance on adding calcium to a volume of dye (e.g., the cell ter-hyperpolarization (AHP) that follows the action potential soma) are independent of the spatial distribution of the added was found to reflect the magnitude of the change in cytosolic calcium (Smith and Zucker, 1980). To date, this technique has calcium. An action potential elicited in the cell soma caused an been used to examine changes in [Ca2+], in the cell soma region increase in cytosolic calcium in the soma, neurite, and growth of a variety of neuronal preparations (Ahmed and Connor, 1979; cone regions of a single cell, indicating that the membrane of Smith and Zucker, 1980; Smith et al., 1983) and in the presyn- all three regions possesses voltage-gated calcium channels. Es- aptic terminal region of invertebrate cells (Miledi and Parker, timation of calcium flux per unit area of membrane suggests a 198 1; Stockbridge and Ross, 1984) but no studies have ex- distinct topographical organization of calcium channels. Cal- amined changes in [Ca?+], in all regions of a single neuron. cium channel densities in the growth cone and cell soma regions Clonal lines of the C 1300 mouse neuroblastoma provide a are similar and significantly higher than that in the neurite. highly suitable mammalian preparation to study localization of calcium entry in various regions of the cell, to examine the The plasma membrane of nerve cells contains calcium channels establishment of this localization during development, and to that subserve different functions (for reviews, see Campbell, relate this entry to different neuronal functions. In these cells 1983; Hille, 1984) but the spatial distribution of these channels calcium has been shown to play an important role as a charge over the neuronal membrane is not known. Several studies of carrier across the membrane (Fishman and Spector, 1981; invertebrate cells have suggested that calcium currents contrib- Moolenaar and Spector, 1979a, b) and as an intracellular mes- ute significantly to the total membrane current in the soma and senger that regulates a variety of neuronal functions, such as the in the presynaptic terminal, but are negligible in the axon &ado, permeability of the cell membrane to various ions (Moolenaar 1973; Llinds et al., 1981; Meves and Vogel, 1973; Miledi and and Spector, 1979a, b, Yellen, 1982), neurotransmitter secretion Parker, 1981; Stockbridge and Ross, 1984). These studies and (Nirenberg et al., 1983) and growth cone movement (Anglister the fundamental role of calcium in transmitter release (Katz, et al., 1982). In the present study we used AI11 to measure [Ca2+], 1969) have led to the widely accepted view that calcium chan- transients resulting from a single action potential in the cell nels are concentrated in the soma and presynaptic terminals of soma, neurite, and growth cone of dimethylsulfoxide (DMSO)- all neurons (for a review, see Hagiwara and Byerly, 1981). At- differentiated cells of clone N 1E- 115. tempts to localize calcium currents in regions other than the cell soma of mammalian neurons have been limited to intracellular recordings of calcium action potentials in dendrites (Llinas and Materials and Methods Sugimori, 1980) and to indirect recordings of calcium action N 1E- 115 cells were grown and induced to differentiate as described potentials in neurites and growth cones of cultured nerve cells previously (Moolenaar and Spector, 1978). Experiments were performed using extracellular electrodes or optical methods (Anglister et on cells grown on polylysine-coated glass coverslips for 7-16 d after al., 1982; Dichter and Fischbach, 1977; Grinvald and Farber, replating in growth medium containing 2% DMSO. The coverslip was 198 1; Willard, 1980). However, these studies are difficult to placed in a chamber mounted on the stage of a compound microscope and superfused with a solution containing 130 mM NaCl, 5.5 mM KCl, Received July 26, 1985; revised Nov. 18, 1985; accepted Dec. 18, 1985. 10 mM CaCl,, 1 mM MgCl,, 25 mM glucose, and 20 mM HEPES, pH We thank Drs. J. E. Brown, P. DeWeer, and G. D. Fischbach for helpful crit- 7.4. Where noted, 10 mM tetraethylammonium chloride (TEA) was icism. This work was supported by National Institutes of Health Grants NS 20857 added without other adjustments. The cell soma was impaled with a to S.R.B. and NS 22028 to IS. single micropipette containing 30 mM AIII, 5 mM 3-(N-morpholino) Correspondence should be addressed to Dr. Spector at the above address. propane sulfonic acid (MOPS), and 100 mM KCl, pH 7.8. This micro- Copyright 0 1986 Society for Neuroscience 0270-6474/86/071934-07$02.00/O pipette was used to pass current and to record voltage. AI11 was injected

1934 The Journal of Neuroscience Ca Transients in Different Regions of a Neuron 1935

500 600 700 nm free AI11 and a Mg:AIII complex, and that if a Ca:AIII complex is present, its concentration is below detection. This result further indicates I I I I I I I that the cytosolic free calcium concentration is much lower than 2 PM, which is the dissociation constant (K,) for Ca:AIII under these condi- Aroma tions (Brown and Rydqvist, 198 1). i The fraction of the total intracellular AI11 present as free dye R = [AIII]/([AIII] + [Mg:AIII]) can be calculated from the optical absorbance at any two wavelengths at which AI11 absorbs light. Using 540 and 660 nm

R= (1)

x where e denotes the specific absorptivity measured in a defined solution in vitro, and a the measured intracellular absorbance at 540 nm divided Figure 1. In vitro and in vivo absorbance spectra of arsenazo III. Con- by the measured intracellular absorbance at 660 nm. In nine cells, R tinuous lines, In vitro absorbance spectra of AI11 in different media: was found to be 0.75 & 0.03. Using this value and the dissociation AIII, free; Ca:AIII, bound to calcium; Mg:AZZZ, bound to magnesium. constant (K,). -, for Mn:AIII. it is oossible to calculate the cvtosolic free Filled circles, Absorbance of AI11 in a soma of a NlE-115 cell bathed magnesium concentration’in N I-E- 115 cells provided the resting intra- in normal Na+ medium containing 10 mM Ca2+ and 10 mM TEA. The cellular pH is known, since the Kd for Mg:AIII is a function of pH absorbance increase produced by injecting AI11 solution, to an estimated (Baylor et al., 1982). Moolenaar et al. (1984) have found the resting intracellular pH of N 1E- 115 cells to be 7.24, which yields a K,, for Mg: total concentration of 0.8 mM, into the cell soma was measured at five different wavelengths. The soma pathlength was 56.5 pm. Abscissa, AI11 of 2.0 mM. Using our measurements of R = 0.75, we calculated Wavelength in nm; left ordinate, absorptivity values of the in vitro the cytosolic free magnesium concentration in N 1E- 115 cells to be 0.67 spectra; right ordinate, absorbance of the injected cell soma (see text for rnM. details). The optical absorbance of intracellular AI11 is linearly proportional to both the intracellular concentration of AI11 and the optical pathlength. The total intracellular concentration of AI11 can therefore be calculated by applying pressure (l-5 psi) to the back of the pipette to a final as absorbance of 0.02-0.04 at 660 nm. To allow for the dye to diffuse [AIII] + [Mg:AIII] = A&& . pathlength) throughout the cell, measurements were made at least 10 min after dye (2) injection. Comparison of signals from the soma, neurite,and growth where Ae6,, = measured absorbance at 660 nm, and &; = R . &t + cone was not made until at least 30 min after dye injection. Light from (1 - R) . e$&A1ll. The optical pathlength through different regions of a a quartz-halogen source was limited to a single wavelength band by single cell was measured by focusing first on the upper surface and then interference filters (10 nm full-width, half-maximum) and focused onto on the lower surface of the cell using Hoffman modulation contrast the cell by a microscope condenser. Light passing through the cell was optics. From these measurements, the mean total intracellular concen- collected by a long-working-distance objective and measured with a tration of AI11 was calculated to be 1.5 * 0.5 mM. photodiode. Light reaching the photodiode was limited by two pairs of Considering that the intracellular AI11 is present as a mixture of free razor blades that could be adjusted to define a specific rectangular ap- AI11 and Mg:AIII, an increase in cytosolic free calcium concentration erture in the back focal plane of the objective. The aperture was adjusted (lCa*+l.) will cause the formation of a Ca:AIII comolex that will be under visual control to frame a cell soma, a neurite, or a growth cone. mflecte’d by an increase of optical absorbance at 6 10 and 660 nm, the Action potentials were elicited in the cell soma by depolarizing current maxima of the absorbance spectrum for Ca:AIII (Fig. 1). In this work pulses superimposed on a steady hyperpolarizing current or by anode we have chosen to use the absorbance changes at 660 nm, because this break stimulation from the resting potential. The optical signal was low- wavelength was found to give the best signal-to-noise ratio. pass-filtered using a four-pole Bessel filter with a time constant of 30 Quantitative determination of changes in cytosolic free calcium con- msec. No signal averaging was used. Experiments were performed at centration based on the absorbance changes of AI11 depends on a host room temperature (21-24°C). Results are given as means ? SEM. of factors (Ahmed and Connor, 1979; Smith and Zucker, 1980). None- theless, under the conditions that (1) the fractional changes of [AIII] Calibration of AIII and [Mg:AIII] are small and can be ignored, (2) the fraction of total free intracellular AI11 (R) reequilibrates and remains constant following the Absorbance spectra of AI11 were measured in the following solutions: formation of Ca:AIII; and (3) [Ca2+], is low, so that Ca:AIII does not free AIII, magnesium AI11 (Mg:AIII), and calcium AI11 (Ca:AIII). Mea- contribute significantly to the total absorbance spectrum, the change of surements were carried out in cuvettes of 200 pm pathlength using a intracellular free calcium concentration will be given by DMS 90 spectrophotometer (Varian Corp., Palo Alto, CA). All solutions contained 1 mM sodium AIII, 10 mM HEPES, and 120 mM KC1 and &$aAIII . A“&,, . p* one of the following: 60 mM KC1 for the AI11 solution; 20 mM MgCl, A[Ca2+li = 660 for the Mg:AIII solution; and 20 mM CaCl, for the Ca:AIII solution. R~&o~A&& The DH was adiusted to 7.5 with KOH. Blanks contained the corre- sponding salt and bufferconcentrations. Figure 1 showsthe absorbance where A[Cal+], is the change of intracellular free calcium concentration; spectra of free AIII, Mg:AIII, and Ca:AIII. The spectrum of the dye in eAII1 isthe dissociationconstant for the Ca:AIII complexand is taken a cell, measured at five wavelengths, is depicted by the data points. to be 2 PM, the value appropriate for 1 mM AI11 (Brown and Rydqvist, Specific absorptivity values (e) in absorbance units x M-I x pm-l for 1981); AA,,, is the changeof absorbanceat 660 nm; and Ae& = AIII, Mg:AIII, and Ca:AIII at 540 and 660 nm were &$‘I’ - eg.

e$J = 3.52 1- 0.03; egg = 0.37 Ik 0.01 Results e$F” = 2.55 + 0.03; @&AI” = 1.50 + 0.02 AIII responses in the cell soma during a egp” = 2.22 z!c0.03; e$$“’ = 2.82 xk 0.04 single action potential In normal medium containing 1.8 mM calcium the action po- As shown in Figure 1, the absorbance of AI11 inside the cell was inter- tential of neuroblastomacells consistsof a fast Na+ component mediate between the in vitro absorbance spectrum of free AI11 and that followed by a small Ca2+component (Moolenaar and Spector, of Mg:AIII and was quite distinct from the absorbance spectrum of Ca: 1978). Under theseconditions, the Ca2+current is very small AIII. This indicates that AI11 in the cytosol is present as a mixture of (Moolenaar and Spector, 1979b), and the light signal during a 1936 Bolsover and Spector Vol. 6, No. 7, Jul. 1966

T50mV 50 mV

A “,“,“,“-/L---- 1 OA=~XIO-~

B I9 VI -

Figure 3. Effects of cobalt ions on the action potential and the calcium 2 nA iI I signal in a NlE- 115 cell bathed in normal Na+ medium containing 10 - mM Ca2+ and 10 mM TEA. Recordings ofthe action potential and optical 500 msec absorbance at 660 nM before (A) and after (B) addition of 10 mM Co2+ to the bath. Note, in B, the complete suppression of the increase in Figure 2. Intracellular calcium transients associated with a single ac- cytosolic [Ca2+]. tion potential in the soma of a NlE- 115 cell bathed in normal Na+ medium containing 10 mM Ca2+ before (A) and after (B) addition of 10 mM TEA to the bath. Here and Figure3, trace V representsthe action voltage-gatedcalcium channels,and was not an artifact of cur- potential elicited by a depolarizing current pulse applied at a hyper- rent injection or of membrane depolarization, is indicated by polarized membrane potential of -90 mV, trace Agy represents the the following observations. First, no absorbancechanges were optical absorbance at 660 nm. An upward deflection of the optical signal observed during an action potential before injection of AI11 into indicates an increase in cytosolic [Ca2+]. The lowermost trace in B is the cytosol. Second,after injection of AI11 no absorbancechanges the current pulse. Note, in B, the prolongation of the action potential and the larger rise of the cytosolic [Ca*+]. The absorbance calibration were observed during hyperpolarizing current pulsesor during bar indicates that A&,, = 3 x 10m4and is equivalent to AAA660/A660= depolarizations that did not elicit an action potential. Third, 7 x 10-J. when 10 mM TEA was added to the bath to suppresspotassium currents that normally counteract the inward Ca2+ currents (Moolenaar and Spector, 1979b), both the action potential du- singleaction potential is expected to be too small to be resolved ration and the amplitude of the absorbancechange increased without the use of signal averaging. However, in media con- markedly (Fig. 2B). In TEA-treated cells, L&?/A%y was taining elevated Ca2+,the contribution of Ca2+currents to the (4.8 f 0.4) x 1O-3(nine cells), and the increasein [Ca2+]iwas action potential is enhanced (Moolenaar and Spector, 1979a, calculated to be 3.9 f 0.3 nM. However, the maximum rate of b), and as a result it is possible to examine [CaZ+li transients changeof Argy remained essentiallyunaltered in the presence without signal averaging. Figure 2A showsan action potential of TEA, thus [Ca2+li reached a maximum later, immediately elicited in a cell somainjected with AI11 and bathed in medium before the termination of the TEA-modified action potential containing 10 mM Ca2+ (upper trace), and the simultaneously (Fig. 2B). Finally, as shown in Figure 3, the large absorbance recorded optical signal (lower trace). It can be seen that the changerecorded during the TEA-modified action potential was optical signal exhibits an upward deflection, which represents completely eliminated by application of the calcium antagonist an increasein the absorbanceof light by the cell soma at 660 Co2+ (10 mM), which severely reduced the amplitude of the nm. A smaller downward deflection with a similar time course action potential (Spector et al., 1973). This effect wasreversible was recorded at 540 nm, with a ratio of ti$y/ti&r = on removal of Co2+. -0.68 f 0.08 (three cells).These optical changesare consistent with binding ofcalcium to AI11 and therefore reflect an elevation Relationship between intracellular calcium of [Ca2+], (Brown et al., 1975). They are inconsistent with a transients and AHP nonspecific effect such as a change in light scattering, which In many cell types, an increasein [CaZ+licauses an increasein should also be seenat other wavelengthswhere the absorbance K+ permeability (Meech, 1978). In NlE-115 cells, Moolenaar of the dye is great (Fig. l), but no light signal could be reliably and Spector (1979b) identified a slow, TEA-insensitive, K+ cur- detected at other wavelengths. As shown in Figure 2A, [Ca2+li rent that is dependent on extracellular calcium and is blocked beginsto increaseat the onset of the action potential upstroke, by calcium antagonists.They suggestedthat this slow K+ current reachesa steady level at the termination of the action potential, is activated by a calcium influx that occurs during the action and doesnot return to baselineover a time courseof seconds. potential, and is responsiblefor the prolongedAHP that follows In five cells AAgv/Agy, the fractional changeof absorbanceat an action potential. According to their model, enhancedCa2+ 660 nm d&ng the action potential, was (1.8 * 0.4) x 10-3, entry should increasethe amplitude and duration of the AHP. and the increasein free calcium concentration was calculated To test this hypothesis,action potentials were elicited by anodal to be 1.4 + 0.3 nM using equation 3. This value is somewhat break stimulation from the restingmembrane potential, and the lower than values estimatedfor bullfrog sympathetic neurons, amplitude of the AHP that followed each action potential was 10 nM (Smith et al., 1983), and rat dorsal root ganglion cells, compared with the amplitude of the absorbancechanges. Be- 18 nM (Neering and McBurney, 1984). causethe resting membranepotential is between - 35 and - 55 That the absorbancechange was due to Ca*+ influx through mV and the reversal potential of the AHP is between -75 and The Journal of Neuroscience Ca Transients in Different Regions of a Neuron 1937

V 1100 mV

AA=O.OOl A 660 J 1 I I

Figure 4. Plot of the AHP that follows an action potential versus the 30 set absorbance change at 660 nm (A&,). The action potential was elicited Figure 5. Intracellular calcium transients associated with a train of by anode break stimulation of varying intensity from a resting potential action potentials in the soma of a N 1E- 115 cell bathed in normal Na+ of -53 mV in medium containing 10 mM Ca*+ and 10 mM TEA. The medium containing 10 mM Ca*+ and 10 mM TEA. The action potential amplitude of the AHP was graded with the calcium signal during the train was elicited by repetitive stimulation at 1 Hz. Uppermost truce(i), action potential. Line fitted by linear regression. The injected current; middletrace (I’), the membrane potential; lowest truce (&,J, the absorbance of the cell soma to light of 660 nm. Note the gradual decline in the amplitude and duration ofthe action potential, - 85 mV (Moolenaar and Spector, 1979a),AHP amplitudesof and the progressively smaller increase ofintracellular calcium associated up to about 6 mV will have little effect on the driving force for with each action potential. Note also the persistence of the AHP fol- the AHP. As illustrated in Figure 4, under thesezero external lowing the action potential train. current conditions, the changein [Caz+li and the AHP amplitude were positively correlated over this limited range. A similar to decline. These resultsdemonstrate that, at least in the pres- correlation was found in all cells studied (six cells). Thus, for ence of high extracellular calcium plus TEA, repetitive stimu- thesesmall increasesof cytosolic calcium over the restinglevel, lation at a rate of 0.5 Hz or more causesa gradual decline in the Ca2+-dependentK+ conductance can be regardedas a linear the amplitude and duration of the action potential and a non- function of [Caz+li. This result is consistentwith the behavior linear summation of the [Ca2+li that enters during successive of Ca2+-dependentK+ channelsincorporated into planar lipid action potentials. However, in all casesthe AHP undershoot bilayers (Moczydlowski and Latorre, 1983). reacheda peak value of about - 89 mV, which was maintained throughout the action potential train. This value, which is vir- Intracellular calcium transientsduring tually identical with the equilibrium potential for K+ (Moolenaar posttetanichyperpolarization and Spector, 1979a),began to decline very slowly only after the One of the other roles attributed to the Caz+-dependent K+ end of the train, concomitant with the [CaZ+],signal. This result currents is to underlie the posttetanic hyperpolarization present supportsthe contention that the long duration of the posttetanic in many neurons.It has been proposedthat the persistenceof hyperpolarization directly reflectsa prolongedelevation of [Ca2+li the AHP for secondsfollowing a train of action potentials is (Smith et al., 1983). due to a summation of [Ca2+lithat enters during each action potential and that the kinetics of the posttetanic hyperpolariza- Localization of voltage-gatedcalcium transientsin tion directly reflects the kinetics of the [Ca2+li transient (Smith d$erent regionsof a single cell et al., 1983; Stockbridgeand Ross, 1984). Figure 5 clearly shows In order to directly measurethe voltage-gatedcalcium transients that the progressiveincrease in the [CaZ+],signal during a train in different regionsof a singlecell, we elicited an action potential of 15 action potentials elicited by 1 Hz stimulation is indeed in the cell somaby a depolarizing current pulse, and monitored due to a summation of [Ca2+li that enters during each action separately the optical absorbanceof AI11 at 660 nm inside the potential. However, it can also be seen that there is a gradual soma @ET), the neurite (A;:‘““), and the growth cone (&p) decline in the amplitude and duration of the successiveaction by limiting the light reaching the photodiode to a rectangular potentials and that the summation of the optical signalis non- aperture that framed one of the three regions (seeFig. 6). The linear, with a marked depressionin the responseto the second resultsof suchan experiment are shownin Figure 6 (right panel). impulse. Thus, the increasein cytosolic calcium during the ac- It can be seenthat all three regionsexhibit an increasein [Ca2+li tion potential train reachesa peak level of only 24 nM. In the during the soma-evokedaction potential, but that the magnitude four cells studied, A&; of the second of a pair of action po- of the increaseis different in the three regions.The largestab- tentials 1 set apart was63 + 4% of the control, and the duration sorbancechange was obtained in the cell soma,a slightly smaller of the secondaction potential was 70 f 8% of the control. When signal was obtained from the growth cone, and the smallest action potentials were 2 set apart, ti6:y and the duration of signal from the neurite. In seven cells, the absorbancechange the secondaction potential were 79 f 4% and 84 f 7% of the in the growth cone was not significantly different from the ab- control, respectively. Only when repetitive stimulation was ap- sorbancechange in the cell soma(AA~~IAA~Y = 0.84 + 0.25). plied at a rate of 0.2 Hz or lessdid the [Caz+li changeassociated However, the absorbancechange in the neurite was significantly with each action potential and the action potential duration fail smaller (A&;p*/A Agy = 0.49 + 0.17). These resultsdemon- 1938 Bolsover and Spector Vol. 6, No. 7, Jul. 1986 1 InA 1IOOmV

Figure 6. Right, Recordings of absorbance changes at 660 nm in the soma, neurite, and growth cone of a single NlE-115 cell during an action potential elicited in the cell soma. The cell was bathed in normal Na+ medium containing 10 mM Ca*+ and 10 mM TEA. The uppermost truce (i) represents the current pulse; the second truce (V’), the membrane potential; the three lower truces, absorbance changes to light at 660 nm in the soma (A%?), neurite (A;:$ti” ), and growth cone (&y). Light reaching the photodiode was adjusted to a defined rectangular aperture that framed each of the three regions. The action potential shown was recorded in the soma during absorbance measurements in the soma. Absorbance changes in the neurite and growth cone were measured during a similar action potential elicited by identical stimulation. The sketch, left, shows the horizontal dimensions of the cell, the position of the micropipette used to pass currentand recordvoltage, and the threerectangular apertures in the back focal planeof the objectivethat definedthe light reachingthe photodiode.The light pathlengthsthrough the cytosolwere 48 pm (soma), 13 pm (neurite), and 5 pm (growth cone).

stratethat voltage-gatedcalcium entry and, therefore, a calcium siblefor the increasedcalcium influx during an action potential. current, do occur in all three regions.In several experiments the Our results in NlE-115 cells demonstratea similar effect. The pathlength through the cell was measuredto be 48 f 6 Km (10 initial rate at which [Caz+li rises at the onset of an action po- cells)at the soma, 11 f 1 Mm (three cells) at the neurite, and tential is not changed by TEA, rather, because of the prolon- 5 + 1 pm (four cells)at the growth cone. If we assumea uniform gation of the action potential, the total [Caz+li changeis con- intracellular concentration of AI11 and Mg2+, then the absor- siderably enhanced in the presenceof TEA. Furthermorel we bancechange at 660 nm (A&,,) is proportional to the product have found that the increasein [Ca*+], during the TEA-modified of the changein intracellular free calcium concentration AICaz+li action potential reaches a maximum immediately before the and the pathlength. Thus, although the values of AA,,, in the repolarization phasethat terminates the action potential; thus, somaand growth cone were similar, A[Ca2+li during an action the rate of increaseof cytosolic calcium is abruptly halted by potential, averagedover the total pathlength, may rise to much the repolarization phase. These results are consistent with a higher levels in the flatter growth cone than in the soma. Sim- model according to which the termination of the TEA-modified ilarly, despitethe low value of AA,,, in the neurite, an increase action potential is due to activation of Ca*+-gatedK+ channels of [Ca”], during an action potential may be significant because by raisedcytosolic calcium (Alvarez-Leefmansand Miledi, 1980; of the small pathlength relative to that of the soma. Neering and McBumey, 1984; Rossand Stuart, 1978). The pos- sibility that the primary trigger for repolarization of the TEA- modified action potential is voltage inactivation of Ca*+ chan- Discussion nels is rather unlikely because the rate of increase of cytosolic In addition to demonstratingthe feasibility of combining elec- calcium, and therefore the inward calcium current, hardly de- trophysiological techniquesand direct measurementsof changes clines during the action potential. Another consequenceof the in the cytosolic calcium concentration in a single mammalian activation of Ca2+-gated K+ channelsby raisedcytosolic calcium neuron, our study addressesseveral points pertaining to the in Nl E- 115 cells is the prolonged AHP that follows the action properties and localization of voltage-gatedcalcium channels. potential (Moolenaar and Spector, 1979a).Our resultsshow that Previous work in molluscanneurons using the AI11 technique the amplitude of the AHP is directly proportional to the cyto- has helped to resolve some problemsconcerning the properties solic calcium changeduring the action potential. However, the of calcium currents (Ahmed and Connor, 1979; Gorman and maximum changeof cytosolic calcium that we have measured Thomas, 1980; Smith and Zucker, 1980).For example, although during a Nl E- l 15 action potential is ca. 4 nM, whereasin a it was widely accepted that the primary effect of TEA was to variety of preparations, Ca*+-gated K+ channels are activated block the voltage-gatedK+ current and thereby prolong the ac- by cytosolic calcium in the micromolar range (Barrett et al., tion potential, it has not been clear whether the enhancedCa*+ 1982; Gallin, 1984; Meech, 1978; Moczydlowski and Latorre, current that is observed in the presenceof TEA is due to a direct 1983). Sincethe small changeobtained in NlE- 115 cellsreflects stimulatory effect of TEA on calcium channels. Ahmed and the averagerise in [Caz+li over the whole cell soma, our results Connor (1979) have shown that it is the prolongation of the suggestthat most of the calcium ions that enter the cell soma action potential by external application of TEA that is respon- during the action potential remain within a restricted region The Journal of Neuroscience Ca Transients in Different Regions of a Neuron 1939

close to the membrane (Chad and Eckert, 1984). Within this Thus, the absorbancechange during an action potential in a restricted region the rise in calcium may be several orders of particular cell region is proportional to the density of activated magnitude greater than the average rise in the entire soma. calcium channelsin the membrane of that region. Conditions During a train of action potentials, [Ca2+li increases in a step- (1) and (2) are likely to be valid (Chad and Eckert, 1984; Harary like fashion. However, under conditions that optimize calcium and Brown, 1984; Smith and Zucker, 1980). Conditions (3) and entry during a single action potential, repetitive stimulation at (4) also appear to be valid. The membraneof all three areasis rates of 0.5 Hz or faster causes a decline in the calcium influx free from obvious clefts and infoldings (Anglister et al., 1982; associated with successive action potentials, as well as in the Graham et al., 1974); save at the highly motile ruffled edge of amplitude and duration of these action potentials. Part of the the growth cone, which was excluded from measurementsby depression of the calcium influx may result directly from in- the rectangularaperture. The neuritesand growth conesthat we activation of calcium channels. However, the progressive in- studied were considerably closer to the somathan the 900 pm crease of [Ca2+li during the action potential train will also cause length constant of NlE-115 cells (Grinvald et al., 1981); thus a progressive enhancement of the Ca*+-dependent K+ conduc- the three regions are likely to be equipotential. With regard to tance, thereby decreasing the amplitude and duration of the condition (5) Tillotson and Gorman (1980) have shown that calcium component of the action potential. Therefore, part of cytoplasmic calcium buffering capacity changesalong the radial the reduction of the [Ca*+], change with repetitive stimulation dimension within a molluscan neuron. However, no informa- may be an indirect effect of the decreased amplitude and du- tion is available as to the relative buffering capacity of the soma, ration of the action potential. neurite, and growth cone regions of a single neuron. Even if The present experiments are the first direct measurements of regionaldifferences in buffering capacity do exist, it is extremely voltage-gated calcium transients in the soma, neurite, and growth unlikely that they alone can account for the small absorbance cone regions of a single neuron. The most significant finding of changein the neurite region of N 1E- 115 cells. In fact, although these experiments is that an action potential elicited in the cell calcium action potentials can be elicited by stimulation of either soma causes an increase in cytosolic calcium in all three regions, the soma or the growth cone, the neurite of NlE- 115 cells is the largest increase being in the growth cone. It is unlikely that incapable of initiating calcium action potentials (Grinvald and calcium elevation in the neurite is a consequence of calcium Farber, 198 1). Thus, it seemssafe to concludethat the similarly diffusion from the soma or the growth cone, because diffusion large absorbancechanges recorded from the soma and growth of calcium within the cytoplasm is extremely restricted (Chad cone during an action potential indeed reflect the higher con- and Eckert, 1984; Harary and Brown, 1984; Smith and Zucker, centration of calcium channelsin the membrane of thesetwo 1980). Furthermore, as noted above, calcium entering the soma regions, and the significantly smaller absorbancechange re- of N 1E- 115 cells appears to remain near its site of entry. Hence, corded from the neurite is the consequenceof a significantly our results are clear evidence that voltage-gated calcium chan- lower density of calcium channelsin the neurite membrane. nels are present in the membrane of all specialized regions of a The above conclusions are in accordance with the widely cultured neuron. accepted idea that calcium channels are concentrated in the If certain conditions are met, the absorbance change in a neuronal soma and presynaptic terminal and that these two particular cell region (A&,) during an action potential is directly regions are capableof calcium-dependentelectrical excitation. proportional to the calcium influx per unit area of membrane They also corroborate resultsobtained by indirect recordingsof and depends on neither the total area studied nor the pathlength. calcium action potentials in cultured neurons suggestingthat These conditions are (1) [AIII] and [Mg*+] are identical in the early in neuronal development calcium action potential gener- three regions; (2) the calcium concentration change in one region ation becomesconfined to the soma and growth cone regions is produced only by calcium entering the cell through the mem- (Anglister et al., 1982; Dichter and Fischbach, 1977; Grinvald brane directly below or above this region; (3) the cell membrane and Farber, 1981; Willard, 1980). However, the tentative con- density in the measurement area-that is, the ratio of cell mem- clusion that active calcium channels may therefore be absent brane area to the cross-sectional area of the measurement beam- from the neurites (Grinvald and Farber, 1981) is at odds with is similar in the three regions; (4) the action potential has a the present results, which clearly demonstrate the presenceof similar amplitude and time course in all three regions; and (5) active calcium channelsin the neurite. Although their low den- the ability of the cytosol to buffer calcium is identical in the sity suggeststhat calcium contributes comparatively little to three regions. electrical excitability in this region, localized entry of calcium As noted in the introduction, the linearity of the AI11 response into the neurite might contribute significantly to several im- means that changes in dye absorption are independent of the portant functions. For example, neurites are packed with mi- spatial distribution of the calcium added to a volume of dye. crotubules (Marchisio et al., 1978; Yamada et al., 1970) whose This is in contrast to the response of the photoprotein aequorin assembly and disassemblyappear to be regulated by calcium to the addition of an aliquot of calcium, which is greater when and calmodulin (Schliwa et al., 1981). In addition, recent evi- the calcium remains spatially restricted within the cell volume denceindicates that calcium regulatesa whole range of micro- (Smith and Zucker, 1980). With AIII, we can consider, therefore, filament-basedcell motility phenomena(Jacobs, 1982; Weeds, the simplest case in which calcium entering a region of a cell 1982), including organelletransport in the axon (Brady et al., under study distributes uniformly throughout that region to 1984). Voltage-gatedcalcium channelsin specificlocations along increase the calcium concentration by A[Ca2+]. We can write the neurite might have far-reaching implications for theseand other processesthat involve the nerve cell cytoskeleton. a,,, a A[Ca*+] x d A[Caz+] a 4 x A/V References where d is the path length: 4, the calcium influx per unit area Ahmed, Z., and J. A. Connor (1979) Measurements of calcium influx of membrane; A, the membrane area; and V, cytosol volume. under voltage clamp in molluscan neurones using the metallochromic However, dye arsenazo III. J. Physiol. (Lond.) 286: 6 l-82. Alvarez-Leefmans, F. T.,.and R. Miledi (1980) Voltage sensitive cal- VaAxd cium entry in frog motoneurones. J. Physiol. (Lond.) 308: 241-257. with the constant of proportionality being the samein all three Anglister, L., I. C. Farber, A. Shahar, and A. Grinvald (1982) Local- regionsof the cell if condition (3) is valid. Therefore, ization of voltage-sensitive calcium channels along developing neurites: Their possible role in regulating neurite elongation. Dev. 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