The Journal of Neuroscience, November 1989, 9(11): 3978-3987

Regulation of Ionic Currents in Cells by and

Sarah S. Garber, Toshinori Hoshi, and Richard W. Aldrich Department of Neurobiology, Stanford University, Stanford, California 94305

Growth factors and hormones induce differentiation of clonal panied by changesin electrical properties(see Spitzer, 1985, for pheochromocytoma (PC1 2) cells, which are derived from rat review). Many lines of evidence suggestthat the presenceof chromaffin cells. On application of nerve nerve growth factor (NGF) and glucocorticoids are important growth factor (NGF), PC1 2 cells extend neurites and express to the differentiation of postnataladrenal medullary cells.Clonal properties characteristic of autonomic ganglion cells. In con- PC12 cells, derived from rat adrenal chromaffin cells, have been trast, incubation of PC12 cells with a corticosteroid, dexa- usedas a model systemfor the study of neuronaldifferentiation. methasone (DEX), does not induce neurite formation but Rat adrenal chromaffin cellsand rat pheochromocytomasshow causes an increase in tyrosine hydroxylase activity, sug- similar changesin processoutgrowth, catecholaminecontent, gesting that the cells become -like. The ability and tyrosine hydroxylase activity when treated with NGF or of NGF and DEX to regulate ionic currents has been less glucocorticoids (Tishler et al., 1982b, 1983). well studied. Therefore, we examined how long-term NGF PC 12cells arenormally sphericalin shapeand do not generate and DEX treatments affected voltage-dependent Na, Ca, and action potentials. In the presenceof NGF, however, the cells K currents in PC12 cells. Voltage-dependent Na currents develop long, branching processes.This morphological differ- were observed only in a small fraction of the PC12 cells in entiation is accompaniedby increasedsynthesis and storageof the absence of NGF or DEX. Virtually all NGF-treated cells ACh (Greene and Rein, 1977) and development of electrical expressed Na currents within 7 d. DEX increased the number excitability (Dichter et al., 1977), which is, in part, a result of of cells expressing voltage-dependent Na current slowly over the enhancedexpression of Na+ channels(Rudy et al., 1982; 3 weeks, but, unlike NGF, DEX did not change Na current Mandel et al., 1988).PC1 2 cellshave also beenshown to respond density. Both NGF and DEX also affected the expression of to cortical steroidssuch as dexamethasone(DEX) by increasing voltage-dependent Ca currents. Most of the untreated cells the activity of tyrosine hydroxylase, an enzyme involved in the had only sustained, high-threshold voltage-dependent Ca synthesisof .Glucocorticoids, suchas DEX, also currents. Chronic application of NGF or DEX increased the inhibit neurite outgrowth (Unsicker et al., 1978; Tishler et al., fractions of the cells that showed transient, low-threshold 1982b). It has been suggestedthat NGF causesPC12 cells to T-type Ca currents in addition to the high-threshold currents. develop toward autonomic neurons, whereas glucocorticoids The T-type Ca current density, however, increased signifi- causethem to develop toward a chromaffin cell-like phenotype cantly only in NGF-treated cells. Neither DEX nor NGF af- (Tishler et al., 1982b, 1983). Little is known, however, about fected the voltage-dependent K currents. These results sug- the effect of DEX on the electrophysiologicalproperties of PC12 gest that the expression of voltage-dependent Na and Ca cells. currents are differentially regulated by NGF and DEX. The Several different types of CaZ+channels, such as L-, N-, and distinction between treated and untreated cells decreased T-type Ca2+channels, have beencharacterized (seeTsien et al., after 3 weeks in culture as older untreated cells showed 1987 for review). High-threshold (L and N) Ca2+channels ac- increases in the fraction of cells expressing both Na and tivate at relatively depolarized voltages (> - 10 mV) and can low-threshold Ca currents. A PC1 2 subline selected for ad- be distinguishedby their slow inactivation and pharmacological herence to uncoated plastic also showed increased fraction properties. Low-threshold, T-type Ca*+ channelsbegin to ac- of cells expressing these currents, suggesting that inter- tivate at more hyperpolarized voltages (< - 70 mV) and inac- actions with substrate may also influence ionic current tivate rapidly with maintaineddepolarization. Eachtype ofCa2+ expression. channel may serve a different physiological function. The dis- tribution of the types of voltage-dependent Ca2+ channelsis Peptide growth factors and steroid hormones play important different among different cell types. Adrenal chromaffin cells, rolesin the differentiation of excitable cells suchas neuronsand for example, do not expressT-type Ca*+ channels,while cells endocrinecells. Differentiation of excitable cells is often accom- from many other tissuesexpress both T-type and L-type CaZ+ channels(Tsien et al., 1987). Both L- and N-type channelshave Received Mar. 22, 1989; revised June 1, 1989; accepted June 8, 1989. beenimplicated in the control of neurotransmitter release(Rane We thank Drs. C. Bitler, R. Maue, M. P. Nusbaum, and D. Shelton for critical et al., 1987; Hirning et al., 1988). reading of this manuscript, Dr. R. Tsien for helpful discussion, and Drs. M. Morganfield and C. Burnett for their constant support and encouragement. We Pharmacological evidence has suggestedthat undifferentia- are indebted to Drs. D. L. Shelton and E. Shooter for making clone B and PC12 ted PC12 cells, like chromaffin cells, expressprimarily a dihy- cell lines available to us. Supported by NS07 158 to S.S.G., NIH and American Heart (CA affilate) postdoctoral fellowships to T.H., and NS23294 to R.W.A. dropyridine-sensitive, high-threshold voltage-dependent Ca Correspondence should be addressed to Sarah S. Garber at the above address. current (Kongsamut and Miller, 1986). A minor dihydropyri- Copyright 0 1989 Society for Neuroscience 0270-6474/89/l 13976-12$02.00/O dine-insensitive Ca2+component which increasesafter the ap- The Journal of Neuroscience, November 1989, 9(11) 3977

plication of NGF has also been observed (Takahashi et al., 1985; currents primarily from the cell body. There were no marked qualitative Kongsamut and Miller, 1986; Streit and Lux, 1987). These ob- differences in the capacitance measurements or the current recordings obtained from the cells using either of these 2 methods or those made servations are consistent with the development of a neuronal from intact cells with neurites. phenotype in the presence of NGF. The cell capacitance was determined from the capacitative transient The amount and temporal pattern of Ca influx can be regu- current in response to a 20 mV voltage step. The whole-cell results lated by voltage-dependent Na, Ca, and K channels. Because presented are based on recordings from >900 cells. Single-channel re- cordings were made from 25 cells. the regulation ofintracellular levels ofCa2+ is important to many Solutions. Whole-cell Ca currents were recorded with external solu- cellular processes, including the differentiation of excitable cells tions containing (in mM) 10 CaCl,; 130 tetraethylammonium (TEA); 2 (see Kater et al., 1988, and Miller, 1988), we hypothesized that MgCl,; 10 HEPES; 5 4-aminopyridine; internal solutions contained (in DEX or NGF would differentially affect the expression of these mM) 120 CsCl; 20 TEA, 2 MgCl,; 11 EGTA, 1 CaCl,; 10 HEPES; 10 ion channels. Although the effects of NGF on voltage-dependent nM free Ca2+. Whole-cell Na and K currents were recorded with external solutions with 140 NaCl; 2.8 KCl; 2 MgCl,; 1 CaCl,; 10 HEPES (so- Na currents of PC12 cells have been studied (e.g., Mandel et lutions contained up to 40 mM sucrose); internal solutions normally al., 1988) neither the effects of NGF on other ion currents nor contained (in mM) 140 KCl; 2 MgCl,; 11 EGTA, 1 CaCl,; 10 HEPES; the effects of DEX on the electrical properties of PC 12 cells have 10 nM free Ca2+. All solutions were brought to pH 7.2 with N-meth- been systematically examined. We show here that chronic in- ylglucamine (NMG). Single-channel recordings were made in the cell- attached configuration. The pipette was filled with (in mM) 105 BaCl,; cubation of PC 12 cells with DEX and/or NGF induces distinct 10 HEPES: pH 7.2 with TEAOH. The bath solution contained (in mM) changes in cellular morphologies and changes in voltage-depen- 140 K-g&&rate; 3 MgCl,; 1 EGTA, 10 HEPES; pH 7.2 with NMG. dent Na and Ca currents, but not in voltage-dependent K cur- Other solutions used are described in the figure legends. Salts and DEX rents ( grade) were obtained from Sigma Chemical Co. (St. Louis,

-‘--I.MO\ Statistical analysis. Recordings from cells were scored on the absence Materials and Methods or presence of transient T-type Ca current, Na current, or transient K current. Recordings made from cells within each treatment group were Cell culture. PC12 cells were obtained from the laboratory of Dr. E. M. distributed over 30 d after plating. Data from each group, at each time Shooter (Department of Neurobiology, Stanford University, CA). The point, were pooled for statistical comparison. We found that changes cells were maintained in plastic Lux tissue culture dishes (Miles Lab- in the numbers of cells expressing the ionic current in question and oratories, Naperville, IL). with Dulbecco’s modified Eagle’s medium current densities occurred gradually. These changes could be summa- with or without 25 mM HEPES (GIBCO. Grand Island, NY). supnle- rized by pooling the data into groups which were treated up to (and mented with 5% horse- and 5%-supplemented calf sera (Hyclone-Lab- inclusive of) 2 1 d, over 2 1 d, or up to 30 d. Percentages of cells expressing oratories, Logan, UT). Two different lots of each sera type produced eitherT-type Ca current or Na currentwere determined as a simple indistinguishable results. Levels of glucocorticoids in the media were average of cells within a treatment group over an indicated time period. ~30 nM. For most of the electrophysiological experiments, the cells Differences in the fractions of the cells expressing the ion current of were plated on borosilicate glass coverslips (Bellco Biological Glassware, interest between different treatment groups were evaluated using the Vineland, NJ). The coverslips were seeded such that cells became con- 2-tailed x2 test. A level of p = 0.05 was considered to be significant. fluent after 3-3.5 weeks. The length of time a culture was maintained Current densities were determined as an average of [peak current am- was measured from the day a new plating was made. P-NGF (200 ng/ plitude/cell capacitance]. ml) and DEX (1 PM) were added at the time of the new plating and were present thereafter in the medium. The medium was changed twice per week. In some experiments, coverslips were coated with Cell-Tak Results (a preparation of barnacle glue shown to-increase attachment of tissue Morphological changes induced by NGF and DEX culture cells: BioPolvmers. Inc.. Farmington, CT) or with nitrocellulose Treatment of PC12 cells with NGF, DEX, or both NGF and (Lagenaur and Lemmon, ‘1987). P-NGF was a’gift from Prof. E. M. Shooter. DEX together (DEX/NGF) resulted in distinct morphological An adherent PC12 subclone, clone B, was obtained from Dr. D. L. changes(Fig. 1). Untreated PC12 cells remained mostly round, Shelton (Department of Neurobiology, Stanford University, Stanford with occasionaloval and rare flat cells, over the course of 4 CA) and selected from the parent PC12 stock as follows: PC12 cells weeksin culture (Fig. 1A). DEX-treated cells becamelarger and were plated on Costar (Cambridge, MA) dishes. After 24 hr, medium was removed and fresh medium was vigorously and repeatedly squirted more irregular in shapeover 2-3 weeks (Fig. 1B). These DEX- into the dish in an attempt to remove all the cells. Cells which remained treated cultures did not becomeconfluent, suggestingthat cell adherent to the dish were then fed with fresh medium. This procedure division had been drastically slowed (Goodman et al., 1979; was repeated every other day. Discrete colonies of very adherent cells Schubert et al., 1980). Cultures treated with DEX for 4 weeks were apparent after 4 weeks. These adherent colonies were then grown showed a large proportion of cells in clumps with occasional for 2 weeks in the presence of NGF. Colonies which were well separated and exhibited vigorous neurite outgrowth were picked individually. cytoplasmic degeneration (Harris et al., 1986). Isolated cells These clones were placed in NGF-free medium and allowed to prolif- were chosen for recording in order to increasefidelity of the erate. voltage clamp. Cells treated with NGF ceaseddividing and Electrophysiology. PC12 cells were voltage-clamped using the tight- extended long processeswithin 2-3 d from the beginning of the seal whole-cell patch-clamp method (Hamill et al., 198 1). Electrophys- iological data were collected and analyzed essentially as described pre- treatment (Fig. lc). The presenceof both DEX and NGF to- viously (Hoshi and Aldrich, 1988a, b). Capacitative and leak currents gether prevented the extensive neurite outgrowth observed in have been subtracted from the data shown. All experiments were per- the presenceof NGF alone. Only short, stubby processeswere formed at room temperature (20 ? 2°C). Unless otherwise noted, the observed on DEX/NGF-treated cells during the first weeks of data were obtained from the cells grown on uncoated coverslips. culture (Fig. 10). Longer, thick processeswere observed on The following 2 methods were used to improve voltage-clamp of NGF-treated cells. (1) NGF-treated cells were grown in suspension. The DEX/NGF-treated cells after 4 weeksin culture. Theseresults cells in suspension do not extend neurites. The cells were then plated of DEX and NGF treatment are similar to those observed in on glass coverslips 10-20 min before recording. (2) NGF-treated cells adrenal chromaffin cells (Unsicker et al., 1978; Tishler et al., with extensive neurites were collected with a 25 gauge needle and tritu- 1982b; Lillien and Claude, 1985). rated. The cells were replated on a new coverslip 10-20 min before recording. This procedure either removed or retracted the cellular pro- Changesin membranearea, as measuredby cell capacitance, cesses, making the cells suitable for voltage-clamp experiments. Re- were also observed with NGF and DEX. The capacitanceof cordings made from cells treated in this manner represent the ionic untreated cells, ranging from 4 to 12 pF, remained stable with 3978 Garber et al. l Regulation of Ionic Currents in PC12 Cells

Figure 1. PC1 2 cells grown (A) untreated (12 d) or in the presence of(B) DEX (20 d), (C) NGF (12 d), and both (0) DEX and NGF (12 d) have distinct morphologies. Untreated cells remain round and 1 S-20 pm in diameter. DEX-treated cells become larger with increased time in culture, and often appear to lose distinct cellular boundaries. The extensive net&e-like processes observed within days of the application of NGF alone appeared as short, stubby processes in the presence of DEX and NGF. Scale bar, 50 pm. Photographs were taken using DIC (Nomarski) optics. time in culture (Fig. 2A). DEX, or NGF alone, however, in- Cells were scoredas expressingvoltage-dependent Na current creasedthe capacitancein some cells by 2- to 3-fold (Fig. 2B, if transient inward currents greater than 25 pA in amplitude C). The measuredcapacitance of NGF-treated cells reflectspri- were recorded at - 10 mV following a 1 set hyperpolarizing marily that of the cell bodiesbecause most of the neurites were prepulseto a voltage 5 - 100 mV, usingNa+-containing external sheared off for more accurate electrophysiological measure- solutions. In cells expressingNa current, little or no inward ments (seeMaterials and Methods). The capacitance of cells current was observed following a prepulse to -50 mV. In a treated with both DEX and NGF increased4- to 5-fold over majority of the experiments, Na currents were recorded with an that of the untreated population within the first week of treat- internal solution containing K+, resulting in the presenceof ment (Fig. 20). It was not possible to reliably determine the voltage-dependent outward currents at voltages I - 10 mV. capacitanceof NGF- and DEX/NGF-treated cells after 2 weeks However, the outward currents activated much more slowly in culture becauseof the largesize ofthe cells. It wasalso difficult than the voltage-dependentNa currents at voltages ~0 mV (see to obtain high resistanceseals with older NGF- and DEX/NGF- Fig. 3). Thus, the measurementsof the peak inward currents treated cells, resulting in smaller test populations for thesecat- observed at - 10 mV were only slightly contaminated by the egories.These cultures, however, remained viable for several outward K currents. Becausethe replacement of external Na+ weeks.The capacitancemeasurements made on DEX-, NGF-, with NMG+ almost completely abolished any inward currents and DEX/NGF-treated cells representa lower estimate of the observed, Ca currents contributed very little to the inward cur- membranearea as our bias wasto record currents from smaller, rent measured with these solutions. The relative proportion of isolated cells in order to increase the fidelity of the voltage- cells expressing Na current changed over time in response to clamp. different treatments. The properties of the voltage-dependent Na currentsrecorded from cells under different treatments,how- DEX antagonizesNGF-induced increasein voltage-dependent ever, did not differ noticeably. The presenceof the Na current Na current density wasnot correlated with any specificcellular morphology or with PC12 cells without (Fig. 3A) and with (Fig. 3B) voltage-depen- membrane area. dent Na currents were found in all treatment groups (untreated, Voltage-dependent Na current was observed only in a mi- DEX, NGF, and DEX/NGF). The peak current-voltage [1(I)] nority of the untreated cells (24%, n = 39) over a 21 d period relationshipsof representativecurrents are shownin Figure 3C. after plating (Fig. 4A). The number of untreated cells expressing The Journal of Neuroscience, November 1989, 9(11) 3979

0 5

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Figure 2. Capacitance of (A) untreat- ed PC 12 cells remained 8 f 4 pF for 4 weeks. The capacitance of some cells increased up to 2- to 3-fold in the pres- ence of (B) DEX and (C) NGF and 0 ) , , , , , , . , I 4- to 5-fold in the presence of (0) DEX/ 0 5 10 15 20 25 30 lc; 1; 2b 25 NGF. Each data point shown is an av- #Days in culture #Days in culture eragecapacitance of at least 3 cells.

voltage-dependent Na current also increased with time in cul- this protocol, L-type Ca2+ channels would be activated but a ture (Fig. 4A), while the current density decreased (Fig. 4B). proportion of N-type Ca z+ channel were probably inactivated. Voltage-dependent Na currents were expressed in 88% of NGF- We do not make a distinction between L and N Ca currents treated cells (p = 0.0001, n = 33: Fig. 4A). The Na current and have denoted nontransient, sustained Ca currents as high- density in NGF-treated cells increased 3-fold with respect to threshold Ca currents. The L and N components of high-thresh- untreated cultures (Fig. 4B). Significantly more cells exhibited old Ca currents are difficult to separate in PC12 cells without Na currents when incubated with DEX than untreated cells determining dihydropyridine sensitivity (Plummer et al., 1989). (72%, p = 0.0004, 12 = 46: Fig. 4C). This increase was not In cells expressing the T-type Ca current, very little or no tran- accompanied by an increase in Na current density (Fig. 4D), sient Ca current was observed following a 1 set prepulse to -40 suggesting that, along with the increase in membrane area (Fig. mV. 2B), there is an increase in the total number of Na+ channels. Untreated, DEX-, NGF-, and DEX/NGF-treated cells each Treatment with both DEX and NGF also increased the per- fell into 2 populations: those that expressed only high-threshold centage of cells expressing Na current (p = 0.005, it = 5: Fig. Ca currents (Fig. 54) and those that expressed both high-thresh- 4c). This increase was significant despite the small sample size. old and T-type currents (Fig. 5B). High-threshold currents were The presence of DEX, however, antagonized the increase in the observed in virtually all cells examined. Peak Z(v) relationships Na current density induced by the presence of NGF alone. When (Fig. 5C) showed that high-threshold Ca current had a maximal compared with the large increase in Na current density in NGF amplitude at +20 mV, whereas T-type Ca current had a max- alone, the increase in DEX/NGF-treated cells was only to 1.2- imal amplitude at - 20 to - 10 mV following a prepulse to - 90 fold over untreated cells (Fig. 40). This antagonistic effect of mV. Representative openings of the single channels which un- DEX on current density is at least partly due to the larger in- derlie the T-type and high-threshold Ca currents are shown in crease in membrane area of these cells (Fig. 20). No significant Figure 6. In agreement with the properties of the whole-cell Ca changes in Na current density over time in culture were observed currents, the probability of the T-type channel being open rap- in DEX- or DEX/NGF-treated cells. idly increased after a depolarization to -20 mV and then de- clined rapidly (Fig. 6A). In contrast, the probability of a single Both DEX and NGF alter voltage-dependent Ca current high-threshold-type channel being open did not decline notice- Two distinct types of voltage-dependent Ca current were ob- ably during a pulse to 0 mV (Fig. 6B). The amplitude and gating served in PC 12 cells; sustained high-threshold Ca current and properties suggest that these records are from an L-type channel. low-threshold, transient T-type Ca current. All whole-cell Ca We did not, however, confirm this by testing dihydropyridine current experiments were done under conditions in which Ca*+ sensitivity. The unitary current amplitude of the T-type channel was the only charge carrier. The criteria for the presence of was 2-3 times smaller than that of the high-threshold channel T-type Ca current was the appearance of transient inward cur- in the range of -20 to 0 mV. The mean open duration of the rent, greater than 5 pA in amplitude in response to a depolarizing T-type channel was, however, markedly greater than that of the pulse to - 10 mV from a holding potential of I - 80 mV. Under high-threshold single channel. Both T-type and high-threshold 3990 Garber et al. l Regulation of Ionic Currents in PC1 2 Cells

A. -50 to 0 mV -50 to 0 mV -120 t Bm -120 F . Without Na+ current 0 With Na+ current Untreated mV

100 pA L1.3 ms

100 pA 100 pA 1.3 ms L L1.3 ms

mV NGF

V

400 pA L1.3 ms Figure 3. Representative families of the voltage-dependent Na currents from untreated, DEX-, NGF-, and DEX/NGF-treated cells. In each treatment group, some cells (A) expressed no or little Na current and others (B) expressed appreciable amounts of Na current. The currents were elicited at the voltages shown following 1 set prepulses to voltages 5 - 100 mV. C, Peak Z(v) curves were obtained from cells which did not express the Na current (black squares) and those which expressed the Na current (circles). The voltage-dependent Na currents were often recorded using an internal solution containing K+, thus resulting in outward voltage-dependent K currents at positive voltages. However, the voltage-dependent K currents activated much more slowly than the voltage-dependent Na current at the voltages more negative than + 10 mV (see DEX records in A). In other experiments (untreated and NGF records in A and B), outward K currents were reduced substantially by using an internal solution with Cs+ (see Materials and Methods). Some fidelity of the voltage-clamp in DEX/NGF cells was lost because of the very large cell size (see Fig. 20). The apparent inward currents observed in DEX/NGF cells in A were artifacts of the imperfect leak current subtraction [see the corresponding Z(v) curve in C].

currents observedin this study closely resemblethose described n = 44: Fig. 7A) and resulted in a 3-fold increasein the density in other preparations(Tsien et al., 1987). Plummer et al. (1989) of T-type current with respectto untreated cells(Fig. 8c). NGF have also describedsingle, high-threshold L and N Ca2+chan- treatment did not markedly changethe high-threshold Ca cur- nels in PC 12 cells. ren: density. High-threshold currents were assayedas previously The relative proportion of cells expressingT-type current described. Under theseconditions, not all the N channelsmay changedin responseto the length of time cells were treated with be available for opening. These experiments would not detect DEX, NGF, or DEX/NGF. Only 20% of untreated cells (n = changesin current density ofhigh-threshold channelswhich may lOl), grown in culture up to 3 weeks, expressedthe T-type require longer times to recover from inactivation. The presence current in addition to the high-threshold current (Fig. 7A). In of the T-type voltage-dependentCa current in any of the treat- the presenceof DEX, the fraction of cells expressingT-type Ca ment groupswas not correlated with any specificcellular mor- currents increasedslowly over the courseof 3 weeksto 40% (p phology or with cell capacitance.Voltage-dependence of T-type = 0.0008, n = 142; Fig. 7A). There was a small increase in and high-threshold Ca currents did not differ markedly among T-type current densitieswith the DEX treatment (Fig. 8B). Ca treatment groups. current densitiesdid not changewith time in DEX-treated cells. Despite the ability of either DEX or NGF to increase the Treatment with NGF alone increasedthe fraction of cells ex- fraction of cells expressingT-type Ca current, the percentageof pressingthe T-type current within 7-10 d to - 60% (p = 0.0 17, DEX/NGF-treated cells with T-type Ca current was not signif- The Journal of Neuroscience. November 1989, 9(11) 3981

P Y. 0

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Figure 4. A, The fraction of untreatedcontrol cellsexpressing Na currentgrown in cultureless than 21 d comparedto NGF-treatedcells and untreatedcultures grown for over 21 d. B, Averagedensities of the voltage-dependentNa currentin untreatedcells grown for lessthan 21 d in culturecompared to NGF-treatedcells and untreatedcells grown for morethan 2 1 d in culture.C, DEX- and DEX/NGF-treatedcells compared to untreatedpopulations grown over 30 d in culture. No significantdifferences were observed between DEX- and DEX/NGF-treatedcells grown up to and over 21 d in culture;thus, these populations were pooled for eachtreatment. D, Na currentdensity of DEX- and DEX/NGF-treated cellscompared to that of untreatedcells grown in culture over a 30 d period.The total numberof cellsrecorded in eachgroup is given as n. Significantdifferences between untreated groups and treatment groups are given asp andwere determined using x2 analysis.Average current density at - 10mV is unlikely to be contaminatedby the voltage-dependentoutward currents. SEM are shownas error bars.

icantly different from the control level of untreated cells over recorded from up to 3 weeks in culture, while that of high- the sametime period (p = 0.35, n = 19: Fig. 7B). The presence threshold current increased(Fig. 8E). of DEX prevented the increase in T-type current density ob- served in the presenceof NGF alone (Fig. 80). Effect of substrate on T-type Ca current density Recent reports have suggestedthat cellular functions such as T-type Ca current density increases in confluent untreated neurite outgrowth in cultured cells are substrate-dependent(Lin cultures and Levitan, 1988; Tomaselli et al., 1986). Bethea and Kozak As untreated cells becameconfluent after 3 weeks in culture, (1984, 1985) have also shown that PC12 cells grown in the the fraction of the cells expressingT-type Ca current increased presenceof extracellular matrix releasemore and store lessdo- to 60% 0, = 0.001, n = 34: Fig. 7A), compared with 20% found pamine then cells grown on plastic. Although coverslips in the in the O-2 1 d untreated cells. This increaseis accompaniedby present experiments were uncoated borosilicateglass, confluent an increasein the T-type current density in untreated cells cul- PC12 cells are in contact with neighboring cells. This contact tured over 21 d (Fig. 8A). The percentageof untreated cells may provide a substratequite different from that encountered grown in culture for more than 2 1 d expressingT-type Ca current by a PC 12 cell, freshly seededat low density. Cell-cell and cell/ surpassedthe level achieved by DEX treatment after 3 weeks extracellular matrix interactions, for example, have resultedin and approachedthat inducedby NGF over a shorter time period modulation of the NGF responsein PC12 cells (Doherty et al., (Fig. 7C). Becauseof this increasein T-type current in >21 d 1987). We therefore consideredthe possibility that interaction untreated cells, there was no significant differencein the fraction with a more adherent, nonspecificsubstrate was responsiblefor of cells with T-type Ca current between the >21 d untreated the changein the fraction of cells expressingT-type Ca current cells and DEX-treated cells over 21 d (p = 0.75, n = 45: Fig. between untreated cells grown lessthan 2 1 d and over 2 1 d in 70. The density of T-type Ca current of the over 2 1 d untreated culture. control cells decreasedslightly with respectto DEX-treated cells Cells grown on glasscoverslips coated with Cell-Tak to pro- 3992 Garber et al. l Regulation of Ionic Currents in PC12 Cells

B. -50 to +30 mV C. n Without “T-Type” current 0 With “T-Type” current Untreated

5 mV I-20 ms v NGF I

100 pA L20 ms

L-20ms Figure 5. Representative families of Ca currents recorded from untreated and DEX-, NGF-, and DEXMGF-treated cells. In each treatment group, some cells (A) expressed only high-threshold Ca current and others (B) expressed both high-threshold and T-type current. The currents shown were elicited at the voltages indicated from a holding potential of - 90 mV (repetition interval = 6 set). C, Peak Z(v) curves obtained from the cells which showed only the high-threshold current (black squares) and those which had both T-type and high-threshold currents (circles). The T-type Ca current shows up as a distinct shoulder at voltages more negative than 0 mV, with peak current occurring at -20 to - 10 mV. In cells expressing T-type Ca current, high-threshold Ca current was also observed. Holding potentials of 5 -80 mV and 1 set prepulses to -40 mV elicited similar high-threshold currents. Some variability of the inactivation kinetics (independent of holding or prepulse potential) of low- and high-threshold currents was observed.

mote cellular adhesionto coverslips did not show a significant es. These cells were generally ovoid and slightly flat in appear- differencein the fraction ofthe cellsexpressing T-type Cacurrent ance.Capacitance of clone B cellswas similarto that of untreated @ = 0.35, n = 16: Fig. 9A), although those grown on nitrocel- cells (data not shown).A significant fraction (77%) of thesecells lulosedid (p = 0.02, n = 12: Fig. 9A). T-type Ca current density expressedT-type current ascompared with untreated cellsgrown also did not changesignificantly in cells grown on Cell-Tak (Fig. up to 2 1 d in culture (p = 0.000 1, IZ= 3 1: Fig. 9A). Other equally 9B), but did increasein cells grown on nitrocellulose (Fig. 9C), adherent subclonedlines also exhibited a larger fraction of cells when compared with untreated cells grown on uncoated cover- with T-type current (data not shown). The T-current density slips. There was also a significant decreasein high-threshold also increased,whereas the high-threshold current density de- current density in cells grown on nitrocellulose (Fig. 9C). Un- creased,with respectto untreated populations (Fig. 9D). treated, NGF-, and DEX-treated populations of cells grown in Plating on Cell-Tak-coated coverslips did not result in a sig- suspensiondid not alter the expressionof the T-type Ca current nificant increasein percentageof cells with Na current (p = 0.28, compared with cultures grown on coverslips under equivalent n = 16: Fig. 1OA). Na current density of cells grown on Cell- conditions (data not shown). Tak did not increase significantly (Fig. 10B). The fraction of To test the possibility that changesin the adherent properties clone B cells expressingNa current, however, was significantly of PC 12 cellsmay also result in a correspondingchange in ionic greater than untreated cells (‘p = 0.002, n = 17: Fig. 1OA). This currents, we made recordings from a subcloned continuous, wasaccompanied by a large increasein Na current density (Fig. adherent PC12 cell line, clone B. Clone B was selectedon the 1OB). Theseobservations suggest that the expressionofadhesion basisof its adherent properties to uncoated Costarculture dish- moleculesand the development of certain ionic currents may The Journal of Neuroscience, November 1989, 9(11) 3983

-20 mV OmV -40 rn3 A. -70my B.

Figure 6. Activity of single voltage- dependent CaZ+ channels recorded from NGF-treated cells using the cell-at- tached recording configuration. A, Rep- resentative openings of the T-type CaZ+ channels elicited at -20 rnv hm a holding voltage of -70 mV. The bot- tom t&e is >n ensemble average of idealized records showing the time course of the probability of the channel being open. The data were filtered at 500 Hz through an 8-pole Bessel filter and digitized at 5 kHz. Pulses were ap- plied every 5 sec. The recording was madefrom the cell body. The neurites of this cell were intact. Open duration histograms of a T-type Ca channel re- corded at -20 mV are shown below 1 pA, 0.5 2 PA, 0.5 traces. E, Representative openings of a 40 msec 40 msec high-threshold Ca channel elicited at 0 mV from a holding voltage of -40 mV. The bottomtrace is an ensemble aver- age of idealized records showing the time Open Duration Histogram 1000 Open Duration Histogram 2 Lo course of the probability of the channel B E being open. The recordings shown are ul probably from an L-type Ca*+ channel. B B Time Constant 0.4 msec b 50 Time Constant 1.8 msec 500 The data were filtered at 1.2 kHz and 2 $ digitized at 10 kHz. The recording was I 2 made from the cell body. The neurites 1 of this cell were intact. Open durations 2 0 z 0 i of the high-threshold Ca channels shown lb 20 0 2 4 6 below current traces were recorded at Duration (msec) Duration (msec) 0 mV.

be linked in PC12 cells. Other processes,such as increasesin to some extent by the time in culture, substrate,and perhaps acetylcholinesteraseactivity in PC12 cells (Lucas et al., 1980; by adhesion.The resultsdescribed in this report are summarized Reigeret al., 1980) and survival of rat chromaffin cells in the below. presenceof DEX (Grothe et al., 1985), have also been shown to be dependent on substrate. Morphology Treatment with NGF alone,DEX alone,or both NGF and DEX Voltage-dependentK currents were not afleetedby NGF or together, increasesthe capacitance, and hence the membrane DEX area, of PC12 cells. However, the extensive neurite outgrowth The whole-cell voltage-dependentK currents in the PC 12 cells observed with NGF is antagonized by DEX. usedin this study activate rapidly and inactivate very slowly, with transient K currents very infrequently observed (Hoshi and Voltage-dependentNa currents Aldrich, 1988a).DEX or NGF treatment up to 4 weeksdid not Both NGF and DEX increasethe fraction of cells which express increasethe appearanceof transient voltage-dependentK cur- voltage-dependentNa current. Only NGF, however, causessig- rents at +lO mV (p = 0.088, n = 66 and p = 0.75, n = 24, nificant increasesin the Na current density. The effect of NGF respectively) or otherwise alter the composition of the whole- on the Na current is attenuated in the presenceof DEX. Both cell K current. Clone B cells or cells grown on Cell-Tak or clone B, a subcloned line selectedfor adherenceto uncoated nitrocellulose did not show any marked differencesin K cur- plastic, and confluent untreated cells showed increasesin the rents. The following treatments also failed to affect these K fraction of cells expressingNa current. currents; dibutryl CAMP and theophylline (2 and 5 mM, re- spectively, for 2 d, p = 0.99, n = 5), P-estradiol (0.4 PM for 3 Voltage-dependentCa currents d, p = 0.29, n = 9), absenceof serain the medium (for 6 d, p Chronic application of DEX or NGF or confluency of untreated = 0.47, n = 2). cells increasesthe occurrenceand density of T-type Ca current. Clone B cells also show increasedoccurrence and density of Discussion T-type Ca current in the absenceof either DEX or NGF. Expres- Both DEX and NGF have distinct effects on the morphology sion of T-type Ca current may be influenced by certain sub- and complement of voltage-dependentionic currents in PC 12 strates.A possibly related result is that fibroblasts, which be- cells. In general, DEX also appearsto antagonize the effectsof come much lessadherent after oncogenictransformation, also NGF. Ionic current composition, however, can also be altered lack T-current after tranformation (Chen et al., 1988). 3984 Garber et al. * Regulation of Ionic Currents in PC12 Cells

Pa, n Untreated up to 21 days I Untreated OVB, 21 days -20 -10 20

-20J - -10 20

E. Go f -,-IO .-.e we, 21 days r m Untreated 8 I 66 DEX -- Figure 7. Percentages of cells with different treatments expressing -lb 26 T-type Ca current. A, The fraction of untreated control cells grown in Voltage (mV) culture less than 21 d compared to DEX- and NGF-treated cells and untreated cultures grown for over 21 d. B, DEX/NGF-treated cells Figure 8. A, Average densities of the voltage-dependentCa current in compared to untreated populations grown over 30 d in culture. C, Un- untreated cells grown for less than 21 d in culture compared to those treated cells grown in culture over 2 1 d compared to DEX-treated cells grown for more than 21 d in culture. Ca current density of(B) DEX- also grown over 2 1 d. The total number of cells recorded in each group and (C’) NGF-treated cells compared to that of untreated cells grown is given as n. Significantdifferences between untreated groups (presented in culture for < 2 1 d. D, DEX/NGF-treated cells compared to untreated on the left side of each graph) and treatment groups are given as p and cells grown over 30 d. E, DEX-treated cells grown in culture over 2 1 d were determined using x2 analysis. ns denotes p > 0.05. compared with untreated cells of the same age. The average current density at - 10 mV primarily represents the T-type Ca current density, while that at +20 mV consists primarily of the high-threshold Ca current density. SEM are shown as error bars. Voltage-dependent K currents Neither NGF or DEX markedly affect voltage-dependent K currents. These results are in contrast to those of Rudy and viously inserted, but nonfunctional, channels are somehow ac- Pollock (1988) who observed that application of NGF to PC1 2 tivated. Alternatively, because a small fraction of nonconfluent, cells cause a decrease in a TEA-sensitive, transient voltage- untreated cells also express T-type Ca currents and Na currents, dependent K current. it is possible that treatment with DEX and/or NGF selectively The straightforward interpretation of the observed increases enhances the division of cells containing these channels. This in the fractions of cells expressing T-type Ca current or Na latter hypothesis is unlikely, however, because PC1 2 cells cease current in response to DEX and/or NGF is that there is an to divide or divide only very slowly in the presence of NGF increase in the number of functional ion channel molecules and DEX (Greene and Tishler, 1976; Goodman et al., 1979; underlying these currents. The increase in capacitance without Schubert et al., 1980). It is possible that with an increase in decreases in current density suggests that new ion channel pro- confluence or age of cells, the numbers of dead or floating cells teins are either inserted into the plasma membrane or that pre- increased, resulting in an unintended selection of more adherent The Journal of Neuroscience, November 1999, 9(11) 3995

p = .OOOl n-31

Figure 9. A, Percentages of clone B, Cell-Tak, and nitrocellulose-treated cultures expressing T-type Ca current compared with untreated cultures grown up to 2 1 d in culture. Average densities of the voltage-dependent Ca currents of untreated cells compared to (I?) Cell- Tak- and (C) nitrocellulose-treated cul- tures and to (0) clone B. The average current density at - 10 mV primarily represents the T-type Ca current den- sity, while that at +20 mV consists pri- marily of the high-threshold Ca current density. All test groups are compared with control cells grown on untreated I Untreated borosilicate glass c&erslips. The total cl w Clone a -20 number of cells recorded in each group -10 20 is given as II. ns denotesp > 0.05. SEM voltage (mvj are shown as error bars. cells with characteristicssimilar to that of clone B. If this were Capacitance measurementsindicate that the average mem- the case,however, continued passagingof PC 12 cells over sev- brane area of the DEX- or NGF-treated PC12 cell bodies in- eral months should result in cell lines with greatly increased creased2- to 3-fold. These observationssuggest that both treat- appearanceof T-type Ca current. This is not observed (unpub- ments result in the addition of membrane to the cell body. In lished results).Another possibility is that channelsare synthe- the caseof NGF, additional membranemay be neededfor neu- sizedat a constant rate, and are maintained at a constant tum- rite extension. The increasedmembrane area in DEX-treated over rate. Thus, channelswould accumulate in slowly dividing cells may be a result of membrane incorporation due to fusion or nondividing cells (such as NGF-, DEX-treated, or confluent of vesiclesassociated with the releaseof neurohormones.The control cells). Mandel et al. (1988) have shown that NGF se- larger increase in the capacitance of DEX/NGF-treated cells lectively inducestype II Na channelmRNA expressionand that suggeststhat while DEX inhibits NGF-mediated neurite out- this induction results in an increasein voltage-dependent Na growth, the additional membraneintended for outgrowth is still current density. This observation suggeststhat, at least in the produced and is insteadadded to the cell body. case of NGF-treated PC12 cells, the increase in Na current Previous studieshave documented differential, often antag- density is in part due to increasedsynthesis of the channel onistic, effects of DEX and NGF on cellular functions. For protein. We suggestthat the DEX- and NGF-induced increases example, the intracellular content of neuropeptide Y (NPY) in in the fractions of the cells with T-type Ca currents and voltage- PC 12 cells increasesin the presenceof NGF but not DEX (Allen dependentNa currents reflect an increasein the number of active et al., 1984). Addition of DEX to NGF-treated cultures of PC- ion channel proteins in the cell membrane. 12 cells prevents any further increasein NPY content (Allen et

A. 100 D z .002 Figure 10. A, Percentages of clone B and Cell-Tak-treated cultures express- ing Na current compared with untreat- ed cultures grown up to 2 1 d in culture. B, Voltage-dependent Na current av- erage density of untreated cells com- pared to clone B and Cell-Tak-treated -10 mV cultures. Average current densities at up to 2ldays - 10 mV are shown. All test groups are I compared with control cells grown on untreated borosilicate glass coverslips. The total number of cells recorded in each group is given as n. ns denotes p > 0.05. SEM are shown as error bars. 3988 Garber et al. * Regulation of Ionic Currents in PC12 Cells

al., 1984). Similar observations were made concerning neuro- on PC 12 cell shape and processing. Mol. Cell Endocrinol. tensin and vasoactive intestinal peptide-like immunoreactivity 37: 319-329. Bethea, C. L., and S. L. Kozak (1985) Further characterization of in primary human pheochromocytoma cultures incubated with substratum influence on PC12 cell shape and dopamine processing. NGF or DEX/NGF (Tishler et al., 1982a, 1984, 1986). In ad- Mol. Cell Endocrinol. 42: 59-72. dition, the specific activity of choline acetyltransferase increases Chen, C., M. J. Corbley, T. M. Roberts, and P. Hess (1988) Voltage- in NGF-treated cells, whereas the specific activity of tyrosine sensitive calcium channels in normal and transformed 3T3 fibro- hydroxylase increases in the presence of glucocorticoids (Greene blasts. Science 239: 1024-1026. Dichter, M. A., A. S. Tishler, and L. A. Greene (1977) Nerve growth and Rein, 1977; Edgar and Thoenen, 1978; Otten and Towbin, factor-induced increase in electrical excitability and acetylcholine sen- 1980; Schubert et al., 1980; Tishler et al., 1983). The activity sitivity of a rat pheochromocytoma cell line. Nature 268: 501-504. of both of these enzymes, however, also increases with increased Doherty, P., D. A. Mann, and F. Walsh (1987) Cell-cell interactions cell density (Greene and Rein, 1977; Edgar and Thoenen, 1978; modulate the responsiveness of PC12 cells to nerve growth factor. Development 101: 605-615. Lucas et al., 1979). It is not clear whether the changes in voltage- Edgar, D. H., and H. Thoenen (1978) Selective enzyme induction in dependent ionic currents reported here and changes in enzy- a nerve growth factor-responsive pheochromocytoma cell line (PC 12). matic activity are associated or simply concurrent. It is likely, Brain Res. 154: 186-190. however, that treatment of PC 12 cells with DEX or NGF results Fenwick, E. M., A. Marty, and E. Neher (1982) Sodium and calcium in a cascade of effects. Two of the many possible end points of channels in bovine chromaffin cells. J. Physiol. (Lond.) 331: 599- 635. these cascades could be alterations of enzymatic or ionic current Goodman, R., C. Chandler, and H. R. Herschman (1979) Pheochro- activity. It would be of interest to determine if the specific mocytoma cell lines as models of neuronal differentiation. In Hor- activity of these enzymes is increased in clone B. manes and Cell Culture, G. H. Sato and R. Ross, eds., pp. 653-669, Our results show that undifferentiated PC 12 cells can express Cold Sminp. Harbor Laboratorv. Cold Snrina Harbor. New York. Greene, L. A:, and G. Rein (1977) Synthesis, storage’ and release of both high- and low-threshold-type Ca currents. This is true for acetylcholine by a noradrenergic pheochromocytoma cell line. Nature both the untreated population of PC 12 cells and of the subclone, 268: 349-35 1. clone B. The existence of more than one type of Ca current in Greene, L. A., and A. S. Tishler (1976) Establishment ofa noradenergic PC12 cells had been suggested in the work of Takahashi et al. clonal line of rat adrenal pheochromocytoma cells which respond to (1985) and Kongsamut and Miller (1986). The presence of T-type nerve growth factor. Proc. Natl. Acad. Science USA 73: 2424-2428. Grothe, C., H.-D. Hofmann, A. A. J. Verhoftad, and K. Unsicker (1985) Ca current differs from the results of Streit and Lux (1987), who Nerve growthfactor and dexamethasone specify the catecholamin- did not observe a T-type Ca current in PC12 cells, although ergic phenotype of cultured rat chromaffin cells:.Dependence on de- high-threshold Ca currents were recorded. Streit and Lux ob- velopmental stages. Dev. Brain Res. 21: 125-132. served, however, that these Ca currents inactivated more rapidly Hamili, 0. 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