The Journal of , December 1992, 72(12): 461 l-4623

The Expression of Nicotinic Receptors by PC1 2 Cells Treated with NGF

Scott W. Rogers, L* Allan Mandelzys,3 Evan S. Deneris,4 Ellis Cooper,3 and Stephen Heinemann* ‘Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, 2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, 3Department of Physiology, McGill University, Montreal, Quebec HSGlYG, Canada, and 4Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106

The expression of neuronal nicotinic ACh receptors (nAChRs) tion. PC12 cells have the advantage of not requiring NGF for and the subunits that compose these receptors by PC12 survival in culture, but upon addition of NGF they extend neu- cells exposed to NGF has been studied. The analysis of total rite-like processesand acquire many biochemical and physio- RNA reveals that the neuronal nAChFl subunits a,, a,, &, &, logical properties that are typical of autonomic neurons. No- and B,, but not a2 and a,, are expressed in our PC12 cells. tably, the addition of NGF to PC12 cells correlates with an Within 48 hr of adding NGF to cultures, the RNA correspond- increasein the electrical excitability of thesecells to ACh (Di- ing to at, as, &, and /3* is decreased, but the & RNA increases chter et al., 1977; Patrick and Stallcup, 1977; Ifune and Stein- for up to 8 d after NGF treatment. To determine the influence bath, 1990), a processthat is mediated through the altered ex- of NGF treatment on subunit protein expression, subunit- pressionor function of nAChRs. specific antisera were prepared. lmmunocytochemistry de- The application of methodsof molecularcloning has revealed tected antigen for 03, a,, &, &, and fi, (but not a2 and aJ in an extended family of neuronal-specificnAChR subunits(Boul- both NGF-treated and nontreated PC12 cells. The expres- ter et al., 1986, 1990; Deneris et al., 1988) that are expressed sion of nAChR subunit proteins, as measured by direct bind- throughout the PNS and CNS (for review, see Deneris et al., ing of antibodies to PC1 2 cells, does not change subsequent 1991) and in PC12 cells (Boulter et al., 1986, 1990; Deneris et to 6 d of treatment with NGF. Whole-cell recording of PC12 al., 1988).The mammalian neuronal nAChR family consistsof cells shows that both the individual cell current density and at least four a-like subunits, LY*(Wada et al., 1988), (Ye(Boulter response to the agonist cytisine were not altered after 5-7 et al., 1986), LY~(Goldman et al., l987), and (Ye(Boulter et al., d in NGF. However, the number of cells exhibiting detectable 1990), and three p-like subunits, & (Deneris et al., 1988) & ACh-induced currents doubled. These results indicate that (Deneris et al., 1989), and p4 (Duvoisin et al., 1989). The pair- NGF increases the number of PC12 cells expressing ACh- wiseexpression ofan (Y-and a B-subunitin various combinations sensitive nAChR currents but the activation is not the result in Xenopus oocytes has shownthat numerousreceptor subtypes of altering the amounts of individual nAChR subunit proteins. arecreated that can bedistinguished by both their pharmacology These data, taken together with the decrease in most nAChR and single-channelproperties (Boulter et al., 1987; Duvoisin et subunit RNAs (except &), suggest that NGF regulation of al., 1989; Papke et al., 1989, 1991; Luetje et al., 1990; Luetje nAChRs may be through a posttranscriptional mechanism. and Patrick, 1991). Since the expressionof RNA for each sub- unit in the mammalian CNS has been shown to be unique but receptors play a central role in regulating the overlapping among the respective subunits(Wada et al., 1989), capacity of neuronsto communicate, and they have the intrigu- there exists the potential for substantialcomplexity in the reg- ing ability to be modified commensuratewith their use. Our ulation, structure, and function of neuronal nAChRs. interest is to investigate the mechanismsthat regulate the ex- PC12 cells have been reported to expressRNA for multiple pressionof neuronal nicotinic ACh receptors (nAChRs) in the nAChR subunitsincluding (Ye,as, &, andp, (Boulter et al., 1990). mammalian nervous system. As a model system,we have em- Lindstrom and colleagues(Whiting et al., 1987)have observed ployed PC 12 cells, a rat pheochromocytoma cell line (Greene that upon addition of NGF to PC12 cells, the surface binding and Tischler, 1982) that has been used extensively to study sitesfor the monoclonal antibody (mAb) 270, which is thought NGF-induced neuronaldifferentiation and altered nAChR func- to bind to the & subunit (Whiting et al., 1991), increasedby six- to sevenfold concomitant with a sixfold induction in car- bachol-stimulated 86Rbflux. However, the simultaneousmea- Received Feb. 26, 1992; revised May 28, 1992; accepted June 5, 1992. surement of the regulation of nAChR subunit RNA, subunit We thank Drs. Jim Boulter, Robert Duvoisin, Lorise Gahring, and David Johnson for their efforts that contributed to the completion of these studies. This protein, and receptor function in PC I2 cells subsequentto ex- work was supported by an NIH postdoctoral fellowship and NIH grant posureto NGF hasnot beenreported. In this study, we examine NS30990R29-01 to S.W.R.; grants from the NIH, Muscular Dystrophy Associ- by Northern blot and RNase protection assaysthe effect NGF ation, and University of California Tobacco-Related Research Program to S.H.; Grant 47 15 from the Northeast Ohio affiliate of the American Heart Association has on the expressionof the respective nAChR subunit RNAs to E.S.D.: and fundine from the MRC of Canada to E.C. and the effect of NGF on the expression of nAChR subunit Correspondence sh&ld be addressed to Scott W. Rogers, Department of Phar- protein as measuredusing subunit-specific antisera developed macology, C-236, University ofColorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. for this study, and the functional expressionof nAChRs is quan- Copyright 0 1992 Society for Neuroscience 0270-647419211246 l l-13%05.00/O tified using electrophysiological techniques. 4612 Rogers et al. - Nicotinic Receptor Expression in PC1 2 Cells

ml 5-bromo-4-chloro-3-indolyl phosphate. The reaction was stopped Materials and Methods with PBS containing 1 mM EDTA. PC12 cell culture. PC12 cells (obtained from I. Verma, Salk Institute) ELISA assays were conducted to determine the serum titer and further were cultured in Dulbecco’s modified Eagle’s medium [low glucose for- determine the amount of cross-reactivity between the antisera produced mulation, GIBCO/Bethesda Research Labs (BRL)] containing 10% fetal in this study. Overproduced proteins were dissolved in freshly prepared bovine serum (HYCLONE) and 5% heat-inactivated horse serum (HY- 8 M urea (10 ml per gram of protein) at room temperature for 1 hr. The CLONE). The heat inactivation of the horse serum was conducted for solution was clarified by centrifugation and the supematant diluted 1: 1 hr at 56°C. Cells were grown in a humidified 7.5% CO, atmosphere 10 bv the slow addition of a solution of 50 mM KC1 and 50 mM NaCl at 37°C. At confluencv. the cells were dislodged from the culture dish (pH 10.5). After an additional hour of stirring at room temperature, the by shaking or triturati& with medium from a pipette, and a portion of solution ‘was brought to pH 8 with 1 N HCl and repeatedly dialyzed these cells were replated into new culture dishes. This process selects against 10 mM NaCl in 10 mM sodium phosphate buffer (pH 7.2) at against flat cells that adhere tightly to the dish and are not responsive 4°C. The concentration of soluble protein was then measured by stan- to NGF (see Results). Trypsin and EDTA were avoided. For experi- dard Biuret assay (Bio-Rad). Immulon microtiter plates were then pre- ments, PC 12 cells that were used in immunocytochemistry and radio- pared. Solubilized antigen (5 pdwell) in 100 ~1 of coupling buffer (50 label binding assays were cultured on gelatin-treated culture surfaces. mM Tris, pH 9.5) was added to each well for 14 hr at 4°C. Wells were Analysis of RNA. RNA was isolated from l-5 x lo6 PC12 cells by thoroughly washed with PBS and blocked with blocking PBS (PBS con- the method of Chomczynski and Sacchi (1987). The transfer of frac- taining 5% heat-inactivated calf serum and 0.05% Tween) for 45 min tionated RNA to Nytran (Schleicher and Schuell) and hybridization of at room temperature. Rabbit serum at various dilutions (e.g., 1:50, 1: Northern blots were conducted as described by Boulter et al. (1990) 150, 1:450, etc.) in blocking PBS was then added, and dishes were except that saline-sodium citrate was substituted for saline-sodium incubated for 4 hr at room temperature or overnight at 4°C. Plates were phosphate-EDTA. Random primed probes were prepared from the full- washed five times with blocking PBS before adding goat anti-rabbit length cDNA of each neuronal nAChR subunit using the Amersham alkaline phosphatase-coupled second antibody (1:2000) for 45 min at random-prime kit. Probes typically had a specific activity of 0.5-l x room temperature. Plates were again washed with PBS and developed 1O9 cpmlpg. with p-nitrophenyl phosphate (1 mg/ml) in 50 mM potassium carbonate RNase protection assays were performed as described by Krieg and and 1 mM magnesium chloride (pH 9.8) for approximately 15 min at Melton (1987). Antisense probes were synthesized using SP6 or T7 room temperature. The reaction was stopped with 50 rl/well of 0.3 N polymerase from the pGEM or pSP64 vector (Promega) containing NaOH and plates were scanned on a Teritek multiscan ELISA reader subcloned portions of the cDNA encoding each neuronal nAChR sub- at 410 nm. unit. The following regions for each cDNA were used: (Y*[HYP16(9); Measurement of nAChR subunit protein. The detection of neuronal Wada et al., 19881, nucleotides 1748-l 932, protected length 184 bases; nAChR subunits was measured by direct binding of antibodies to PC 12 (Ye[pPCAlpha 48E(4); Boulter et al., 19861, nucleotides 1557-1815, cells. PC 12 cells were distributed to gelatin-coated six-well culture dishes protected length 258; (Ye[pHYAlpha 231E(l); Goldman et al., 19871, (1 O5 cells/well). PC1 2 cells were washed gently and fixed with freshly nucleotides O-553, protected length 553 bases; (Ye(Alpha 5; Boulter et prepared 2% paraformaldehyde (Electron Microscopy Sciences) in 0.1 al., 1990), nucleotides 1160-1639, protected length 479 bases; & M cacodylate buffer (pH 7.4) for 30 min at room temperature. We found (pGPR49; Deneris et al., 1988) nucleotides 16 17-2 195, protected length the purity and concentration of paraformaldehyde to be crucial to ob- 578 bases; & (pESD7; Deneris et al., 1989) nucleotides 568-1021, taining reproducible results. Concentrations of less than 1.5% or greater protected length 453 bases; p, (pZPC13; Duvoison et al., 1989), nucle- than 2% resulted in diminished immunoreactivity (not shown). The otides 2049-2460, protected length 4 11 bases. fixative was removed, and the cells were washed gently with PBS, and Constructs and bacterial overexpression. Complementary DNA clones then permeabilized using blocking PBS (PBS with either 1% heat-in- encoding subunits of neuronal nicotinic subunits activated calf serum or normal goat serum; GIBCO) containing 0.3% were used for constructs. Portions of the cDNA encoding the amino Triton X- 100 (Pharmacia) for 30 min at room temperature. Cells were acids (numbered from the initiation methionine) of subunits 01~(37 l- washed in blocking PBS, and rabbit antiserum diluted in blocking PBS 5 11; Wada et al., 1988) 01~(284-470; Boulter et al., 1986), 01~.,(461- was then added to the cells for 4 hr at room temperature or overnight 594; Goldman et al., 1987), 01~,(345452; Boulter et al., 1990) & (394- at 4°C. To minimize background, antisera were first adsorbed against 503; Deneris et al., 1988a), p, (330-464; Deneris et al., 1989) and p, Rat 2 fibroblasts. Rat 2 fibroblasts were grown to confluency in 150 (328-426; Duvoisin et al., 1989) were removed by restriction digest mm culture dishes as described previously (Rogers et al., 199 1a), fixed, and subcloned in the appropriate PATH vector (trpE bacterial over- permeabilized, and placed in blocking PBS as described above for PC1 2 production system; Dieckmann and Tzagolott, 1985). The host strain cells. The preimmune or primary antiserum was then diluted to the was Escherichia coli strain HB 10 1. Overexpression of fusion proteins working concentration in blocking PBS (final volume of approximately was done as described previously (Rogers et al., 1991a,b). 5 ml) and then adsorbed over the monolayer of Rat 2 cells for 4 hr at Gel electrophoresis and immunization. Overproduced proteins were room temperature. This preparation was then used immediately for fractionated using sodium dodecyl sulfate polyacrylamide gel electro- direct binding or immunocytochemistry (see below). The cells were phoresis (SDS-PAGE), and protein bands were visualized by soaking washed three times in blocking PBS and then rocked in a fourth change the gel in ice-cold 0.25 M KC1 for 1 hr, and then were cut from the gel of blocking PBS for 30-60 min at room temperature. Blocking PBS (see Rogers et al., 1991a,b). The gel fragments were macerated, emul- containing radioiodinated secondary antibody was then added to the sified in complete Freund’s adjuvant (GIBCO/BRL), and injected sub- cells for 1 hr at room temperature. Cells were gently washed in four cutaneouslv into vouna New Zealand White rabbits. Each rabbit re- changes of PBS, the well was removed from the culture dish using a hot ceived 20&00 pi of irotein. Three weeks later, rabbits were boosted wire, and the total remaining radioiodine was quantitated by gamma with 200-400 pg of antigen as above except emulsification was in in- counting using a Beckman Gamma 300 counter at 30% efficiency. comnlete Freund’s adiuvant (GIBCO/BRL). Serum was collected 12 d Radioiodination of goat anti-rabbit second antibody (Cappel) was later. Subsequent boosts and sera collections were done at 1 month done by the lactoperoxidase method as described by Harlow and Lane intervals. (1988). Typical specific activities were lo6 cpm/pg, which corresponds Antibody testing. Specificity of the antisera was tested using Western to a substitution ratio of approximately 0.5 mol of 12SI/mol of protein. blot and ELISA analysis. For Western blot analysis, overproduced pro- The specific binding was calculated as the binding of either (r2 (control), teins were separated by SDS-PAGE and transferred to nitrocellulose (Ye,(Ye, &, or 0, antiserum minus the respective preimmune serum per filters (Kyhse-Andersen, 1984). Filters were then blocked in PBS con- cell. The value for PC12 cells not treated with NGF was set to 100, and taining 5% Carnation nonfat dry milk (Blotto) for 1 hr at room tem- the relative proportion of binding to this value was calculated for PC 12 perature. To each blot was added rabbit serum (either complete or cells treated with NGF. Measurements were made in duplicate for each absorbed with proteins from bacteria containing the PATH 1 vector) experiment. in Blotto ( 1:3000). After 14 hr at 4°C. blots were washed in three changes Immunocytochemistry. PC 12 cells were fixed, permeabilized, and re- of Blotto.‘Goat anti-rabbit alkaline phosphatase-conjugated second an- acted with the designated antisera as described above. The secondary tibody was added in Blotto (1: 1000; Cappel) for 2 hr at room temper- antibody (alkaline phosphatase-conjugated goat anti-rabbit, Cappel) was ature. Blots were subsequently rinsed in developing buffer [50 mM so- then added to the cells for 1 hr at room temperature. The cells were dium carbonate (pH 9.5) 2 mM MgCl,] and then visualized in developing washed with three changes of PBS and twice with 50 mM sodium car- buffer containing 0.1 mg/ml p-nitro blue tetrazolium chloride, 0.05 mg/ bonate buffer containing 2 mM magnesium chloride (pH 9.5), and the The Journal of Neuroscience, De-eembr 1992, 72(12) 4613

NO NGF Remove NGF NGF (Days) (Days) - 12 3 412 3

28s - a3 4.. 1

18s -

Figure I. Northern blot hybridization analysis of total RNA from PC1 2 cells. Total RNA was isolated from l-5 x P2 28s - -’ . ’ lo6 PC1 2 cells that were either not treated with NGF, treated for l-4 d with NGF, or treated with NGF for 4 d and 18s - then returned to NGF-free culture me- dium for l-3 d. Twenty micrograms of total RNA for each sample were elec- trophoresed through 1% agarose gels containing formaldehyde, transferred to Nytran blotting membrane, and then probed with full-length cDNA random- 28s - primed probes for each subunit as de- p4 scribed in Materials and Methods. The exposure was for 14 hr at -7O”c, and 18s- the locations of the 28s and 18s ribo- somal RNA are indicated. alkaline phosphatase was visualized by developing the cells at room pettes (10-20 am), positioned at an appropriate distance (30-40 pm) temperature in the same buffer with freshly added nitro blue tetrazolium from the PC12 cell, so that upon application of light pressure (usually (1 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.5 mg/ml). De- 20-30 kPa) we could perfuse a large area (see Mandelzys et al., 1990). velopment was usually for 30-60 mitt, with fresh developing solution The cultures were continuously perfused at the rate of 1 mumin with added every 15 min. To stop the reaction, PBS supplemented to 2 mM extracellular solution (see below) to ensure that the drug did not accu- EDTA was added. Successful permeabilization of PC 12 cells was mon- mulate during the experiment. The ACh pipettes were filled with 50 PM itored in cells in adjacent wells with fluoresceinated anti-tubulin anti- ACh dissolved in the extracellular solution. This concentration of ACh body (Boehringer Mannheim; not shown). was chosen to ensure that we could detect cells with ACh-gated currents DNA synthesis by PC12 cells was visualized using immunocyto- but avoid agonist-induced desensitization. The extracellular solution chemistry for the incorporation of 5-bromo-2-deoxyuridine (BUdR). was composed of 140 mM NaCl, 5.4 mM KCl, 2.8 mM C&l,, 0.18 mM PC 12 cells were grown without NGF and in the presence of NGF for MgCl,, 10 mM HEPES, 5.6 mM glucose., 2 mM glutamine, 30 U/ml 2-5 d. For the final 24 hr of culture, BUdR (40 PM final) was added, penicillin, and 30 &ml streptomycin. The intracellular solution con- and cells were rinsed with PBS, and fixed with 95% ethanol, 5% acetic sisted of 80 mM KF, 60 mM K-acetate, 5 mM NaCl, 1 mM MgCl,, 10 acid for 30 min at room temperature. The cells were rinsed with distilled mM EGTA, and 10 mM HEPES. All solutions were adjusted to pH 7.2- water and the DNA was denatured with 2 N HCl for 30 min. The cells 7.4 with NaOH for the extracellular solution and KOH for the intra- were rinsed with PBS, placed in blocking PBS containing mouse anti- cellular solution. The limit of resolution in this study was determined BUdR (1: 100; Becton-Dickinson) for 30 mitt, rinsed with PBS, placed to be 5-10 pA, which is equivalent to three to five receptors open in blocking PBS containing goat anti-mouse alkaline phosphatase-con- simultaneously. Since the average cell capacitance is approximately 30 jugated secondary antibody (Cappel), and visualized as described above. pF, our limit of detection is 0.3 pA/pF. PC12 cells with ACh-gated Electrophysiology.Sensitivity of PC12 cells to ACh was measured currents below this value were grouped as insensitive. electrophysiologically using whole-cell patch-clamp techniques (Hamill To determine the relationship between currents evoked by ACh and et al., 198 1). All recordings were done with a List EPC-7 amplifier at those evoked by cytisine (Aldrich Chemical Co.), we constructed double- 22-24°C. The recording electrodes typically had resistances of 2-6 MQ, barreled pipettes so that the tip of each barrel was fused side by side; and the current signal balanced to zero. During the experiments, mem- each tip was 15-20 pm. One barrel contained 50 PM ACh, and the other brane currents and voltages were filtered at 3 kHz, digitized at 44 kHz barrel contained 50 PM cytisine. All ACh currents are normalized to by a pulse code modulation unit (PCM 701, Sony Corp.), and stored cell capacitance by integrating the capacity current evoked by a 5 mV on a Beta videocassette recorder. Round PC12 cells with clear nuclei hyperpolarizing voltage step from a holding potential of - 50 mV. Un- were selected at random. In pilot experiments, we found that flat cells treated PC1 2 cells had capacitances that ranged from 13 pF to 18 pF, that adhered tightly to the culture surface were insensitive to ACh, and whereas NGF-treated PC 12 cells had capacitances ranging from 22 pF these were not examined further (not shown). For most PC 12 cells, the to 30 pF. The distributions of ACh current densities represent values membrane potential was held at two potentials, -50 mV and - 100 obtained in response to 50 PM ACh at a holding potential of -50 mV mV, while ACh (ACh iodide, Sigma Chemical Co.) was applied to the and were plotted on a log scale to cover the wide range of values (from cell body, as well as the proximal processes of the NGF-treated cells. 0.3 pA/pF to 28.1 pA/pF) that exist on different PC 12 cells. The zero To measure the overall ACh current density of each PC12 cell, we column in each distribution reflects the proportion of neurons in which applied the drug by pressure ejection from relatively large-tipped pi- there was no detectable response. We used the Mann-Whitney U test 4614 Rogers et al. l Nicotinic Receptw Expression in PC12 Cells z Days with NGF 2 $ nAChR ‘0 2 5 6’ 5 5: subunit

a3 -

ii Days with NGF 2s nAChR Figure 2 RNaseprotection of n4ChR '0 2 4 5 6’55 subunit subunitsfrom total RNA preparedfrom PC12 cells treated with NGF. Total RNA from PC12 cellstreated with NGF P2+ +P2 for the indicatedtime periodwas pre- paredand analyzedwith subunit-spe- p3+ -3 P cific probesas describedin Materials r, andMethods. Protected transcripts were P4 734 observedfor nAChR subunitsa,, cz5,&, &, and@,, but not (Y*or LX,. andthe x2test to assessthe sigpificanceof differencesbetween the current The more sensitive method of RNaseprotection was applied densitydistributions. to measure(Ye and to test for the presenceof 1y2,(Ye, and & which are not observed using Northern blot analysis (Boulter et al., Results 1990;not shown). This method offers the additional advantage NGF alters nAChR subunit RNA expression of allowing the simultaneousanalysis of more than one nAChR RNA sampleswere prepared from PC 12 cellsthat were exposed transcript in an RNA preparation sinceprobes of different lengths to 50 rig/ml of NGF for l-6 d as described in Materials and can be used. The results of these assaysfor (Ye,(Y,, a4, and (Ye Methods. At day 4, one group of cells was returned to culture and for &, /I,, and @.,are shown in Figure 2. As expected from media not supplementedwith NGF. Northern blot analysis of Northern blot analysis, the expression of 1~~and & decreased 20 Mg of total RNA per lane is shown in Figure 1. CX,and & rapidly following exposure to NGF and the expression of & RNA decreasedwithin 48 hr of exposure to NGF and decreased increasedslowly as seenabove. The expressionof cx5decreased only slightly thereafter, but & RNA was observed to increase approximately 50% in responseto NGF treatment. No protected slowly over the 6 d period. The removal of NGF from the culture speciescorresponding to (r2 or LY.,was detected in PC12 cells. media subsequentto 4 d of NGF treatment resultedin the rapid However, a protected speciescorresponding to & was observed, return (within 24 hr) of all and j!& RNA to approximately 80% and the expressionof this RNA was decreasedsubsequent to of the level seenin PC 12 cells never exposedto NGF. RNA for NGF treatment. These results demonstrate that PC12 cells ex- & also returned to pre-NGF treatment levels, but in contrast pressRNA correspondingto the nAChR subunits of (Y,, 01~,&, to aJ and & this return wasgradual over the 3 d tested.Northern &, and & but not a2and LY.,,and that the relative concentrations blot analysis of total RNA detects at least two forms of RNA of these RNAs are altered by the presenceNGF in the culture for each subunit asreported previously (seeBoulter et al., 1986, medium. 1990). We found that the various transcript forms were altered similarly in responseto NGF (seeFig. 1; quantitative data not Antisera to neuronal nAChR subunits shown), which suggeststhat NGF treatment and the regulation To determine the influence of NGF on the expressionof nAChR of nAChR subunit RNA do not occur through selectively al- subunit protein, we prepared rabbit polyclonal antiseraagainst tering the level of one RNA form relative to the other. the putative cytoplasmic domains of the subunitsa*, (Ye,(Y.,, as, The Journal of Neuroscience, December 1992, f2(12) 4615

P,, p,, and p4, respectively (see Materials and Methods). As present, we are uncertain if this punctate staining reflects clus- shown in Figure 3, Western blot analysis of these bacterially tering of the receptors. overproduced proteins indicates that each antiserum reacts only PC12 cells treated with NGF generally cease cell division with the subunit protein to which it was made. Similarly, ELISA (Greene and Tischler, 1982; Rudkin et al., 1989), although DNA analysis showed that cross-reactivity between sera was not sig- synthesis and cell division may persist within some cells of the nificant unless only low dilutions (less than 1:300) were used population (Rudkin et al., 1989). To determine if PC12 cells (data not shown). There remains the possibility that these sera that express nAChRs and respond to NGF by altering their could react with other subunits, although additional studies in- morphology also stop cell division, DNA synthesis in PC 12 cells dicate that this is not the case. First, overlapping portions of that were exposed to NGF for varying periods was measured. each subunit were used for antigen, and there is essentially no PC12 cells were distributed to culture dishes and cultured for sequence identity between these regions ofthe receptor subunits. 24 hr before adding 50 rig/ml of NGF for 2, 3, 4, and 5 d, Second, each antibody has been used to study Rat 2 cells that respectively. For the final 24 hr of culture, the cells were grown were transfected with either the subunit combination C& (Rog- in the presence of 40 PM BUdR (see Materials and Methods) ers et al., 199 1a) or cu& (not shown). For the cell line expressing and the nuclei of cells undergoing DNA syntheses were visu- (YJ&, immunoprecipitation and immunocytochemistry were alized using an antibody to BUdR as described in Materials and successful only with antisera to (Yeor & (Rogers et al., 1991a; Methods. As shown in Figure 5 for PC12 cells cultured without not shown). Third, antisera to LYEor &, respectively, immuno- NGF or in the presence of NGF for 2 or 5 d, the nuclei of cells precipitate high-affinity 3H-nicotine binding sites from the rat that responded to NGF by extending processes failed to stain brain. Immunoprecipitation is not altered by preclearing the for BUdR immunoreactivity. In contrast, the nuclei of PC12 membrane preparations with a3, (Ye,&, or p4 receptor subunit cells that did not extend processes in the presence of NGF stained antisera, but it can be inhibited by preclearing with either an- for BUdR immunoreactivity. This result supports the conclu- tisera to (Yeor &, respectively (Flores et al., 1992). These results sion that cells in our PC 12 cell population that fail to respond taken together with the results in the next section suggest that to NGF and fail to exhibit staining with nAChR subunit antisera the antisera used in this study exhibit subunit specificity. correspond to those that also continue DNA synthesis (see Dis- cussion). Immunocytochemistry reveals PC12 cell heterogeneity Immunocytochemical analysis of PC12 cells not treated with NGF or treated for 5 d is shown in Figure 4A for each antiserum. NGF does not alter neuronal nAChR subunit protein Immunostaining was observed in PC1 2 cells for antisera raised expression to a3, cr,, &, and p,. No staining was observed with antisera to Immunocytochemistry reveals reactivity toward nAChR sub- either (Yeor LY+Although immunoreactivity toward & is seen, units CY,,(Ye, &, and 0, in both NGF-treated and untreated PC 12 the intensity of this staining varied between experiments. The cells. Since there is a dramatic change in the relative mRNA data in Figure 4A illustrate the point that the treatment of PC1 2 levels of these subunits in response to NGF, we determined if cells with NGF does not obviously change either the number corresponding changes in the protein concentrations of these of cells expressing antigen or the amount of antigen present per subunits could be detected through measuring the direct binding cell (see below). As is seen in Figure 4B, immunoreactivity of antibody to fixed and permeabilized PC 12 cells (see Materials toward neuronal nAChR subunits (Yeand & reveals PC12 cell and Methods). In three experiments, when binding was calcu- heterogeneity. At least two cell types can be distinguished based lated by subtracting (Y*immune binding and the results presented upon their relative adherence to the culture surface and their as a ratio to PC 12 cells not treated with NGF, no change between response to NGF. The first group (Fig. 4B) consists of flat cells the ratio of radioactivity bound by PC 12 cells for up to 6 d was that attach firmly to the culture dish, they fail to extend neurites detected (Table 1). Similar results were obtained,when the bind- in response to NGF, they rarely exhibit immunocytochemical ing by preimmune serum was substituted for the binding to staining, and they do not contain ACh-sensitive currents (see antisera to (Ye(not shown). The amount of binding or antibody below). The relative abundance of these cells increases with to PC12 cells that were not permeabilized with Triton X-100 prolonged culture and changes if culture conditions are altered did not exceed 20% of the total counts (not shown). This binding (e.g., reduced serum concentrations or the use of trypsinization probably reflects specific binding to the cytoplasmic domain of for subculture; not shown). In our experiments, these cells rep- nAChR subunits since the immunoreactivity toward tubulin resent less than 10% of the total cell population. The second could be visualized in some PC12 cells fixed with 2% but not group of cells are rounded and attach loosely to the dish; they 4% paraformaldehyde (not shown). This result suggests that extend neurites in response to NGF, they exhibit immunoreac- some membrane permeabilization occurs at the relatively low tivity toward neuronal nAChR-directed antisera, and they ex- fixative concentration of 2% required to preserve immunoreac- hibit ACh-sensitive ion channels (Fig. 4B). Within this group tivity toward the nAChR subunits. of NGF-responsive PC 12 cells, there is consistently observed a The sensitivity of this assay was determined using the (Ye subpopulation of cells that appear to be smaller and oval in subunit. Crude PC 12 membranes were prepared (see Rogers et shape, and to extend shorter neurites (Fig. 4B). They often stain al., 1991a,b), adsorbed against prediluted immune serum (in less intensely for nAChR subunits than do PC12 cells that ex- blocking PBS) overnight at 4°C removed by centrifugation, and tend extensive net&es. These cell types are also observed in the supematant was used for binding assays by radioiodinated Figure 4A. antibody to fixed and permeabilized PC 12 cells as described in Antigen appears to concentrate in the cell body (Fig. 4B). Materials and Methods. Membranes prepared from Rat 2 cul- Although staining of neurite-like processes is observed occa- tured fibroblasts were used to control adsorption to membranes sionally, the majority remain unstained except for a punctate not containing nAChRs, and a2 immune serum was used to staining that is observed on some well-developed processes. At measure background binding (not shown). These assays indi- 4616 Rogers et al. l Nicotinic Receptor Expression in PC1 2 Cells

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50 ---

39 ---

Figure 3. Specificity of antisera raised to neuronal nAChR subunit fusion proteins. At the upper left is shown a Coomassie-stained gel of insoluble protein from E. cofi strain HBlOl containing either the parent PATH 1 plasmid (trpE) or a fusion construct containing a portion of the cDNA encoding the putative cytoplasmic portion of each nAChR subunit (see Materials and Methods). The upper right panel shows a Western blot of an identical gel visualized using alkaline phosphatase staining after incubation with rabbit anti-a, immune serum diluted 1:3000. In the remaining Western blots, immunoreactivity toward trpE and other bacterial proteins was removed from each immune serum. Immunoreactivity to one bacterial protein was not removed in the & antiserum (arrow). Lower molecular weight bands are degradation products of the fusion protein and vary between protein preparations. Prestained molecular weight markers (Bio-Rad), which do not migrate true to their indicated molecular weight in our system, are indicated for the relative comparison of gels. The Journal of Neuroscience, December 1992, 72(12) 4617 cated that the detection sensitivity of our assay is approximately 3 f 1 ng per lo6 cells (not shown). Discussion In this study, we have analyzed the effect of NGF on the ex- ACh-activated currents are not altered by NGF treatment pression of neuronal nAChR subunit RNA, subunit protein, The above measurements indicate that the relative nAChR sub- and receptor function. We observed a rapid change in the amount unit protein concentration is not altered in response to NGF of each nAChR subunit RNA expressed by PC 12 cells subse- treatment despite significant changes in the respective subunit quent to NGF exposure. RNA for & increased slowly over the RNA levels. Nevertheless, it is well documented that NGF treat- 6 d NGF treatment period, but RNA for (Ye, +, /Is, and p4 ment of PC 12 cells increases ACh-activated currents. To ensure decreased. This decrease appears to follow three courses: the this was true of our PC1 2 cells and to study further the influence rapid loss of approximately 75% of the RNA followed by a slow of NGF on expression of functional nAChRs, we used whole- but continuous loss over the next 5 d of NGF exposure as cell patch-clamp techniques to compare the current densities observed for &,, a relatively rapid drop of approximately 50% and agonist sensitivities of nAChRs on untreated and NGF- followed by a period of essentially no change in the RNA level treated PC12 cells. As noted in Materials and Methods, PC12 as observed for the (Yeand ,f3,subunits, and finally the loss of cells with a flat morphology were found to contain no detectable approximately 75% of the original RNA level over 3 d of NGF ACh-activated currents, consistent with immunocytochemical followed by a subsequent period of slow loss as observed for results, and they were not included in the following analysis. a3. These observations suggest that the RNA for each subunit Figure 6 shows the ACh current density distribution for 66 PC 12 is regulated independently subsequent to NGF exposure, but it cells that were not treated with NGF. Thirty percent of the cells is not known if this regulation is transcriptional or posttran- show ACh sensitivity, and their current densities range from scriptional. These differences are also of interest since the genes 0.6 pA/pF to 28.9 pA/pF with a mean of 4.4 f 1.0. Figure 6 encoding the nAChR subunits (Y,, (Ye,and p, that are tightly also shows the current distribution for 72 PC12 cells treated linked (Boulter et al., 1990). with NGF for 5-7 d. After treatment with NGF, approximately Immunocytochemical staining of PC1 2 cells was observed for 60% of the cells respond to ACh and their current densities CQ,(Ye, &, p,, and p4, but the intensity of staining was particularly range from 0.3 pA/pF to 28.1 pA/pF with a mean of 2.9 + 0.6. strong for (1~~and &. Whether this staining reflects the amount These results demonstrate that exposure to NGF results in a of antigen present or if it is the result of the particular antiserum twofold increase in the proportion (from 30% to 58%) of PC12 remains to be determined. Nevertheless, this suggests that the cells expressing functional nAChRs, but there is little effect on nAChR of PC 12 cells could be composed of multiple subunits, the current density of nAChRs on individual cells. and the potential for substantial complexity exists. Further, re- Recently, Luetje and Patrick (1991) observed that a,/& re- cent reports of additional nAChR subunits have been made. ceptors expressed in Xenopus oocytes were loo-fold less sen- These subunits include the preliminary reports of (Ye(Lamar et sitive to the agonist cytisine relative to ACh. In contrast, cytisine al., 1990) and the discovery of the a-bungarotoxin binding sub- was two- to threefold more potent than ACh on the LY,/& func- units (Y, and (Y*from chick (Couturier et al., 1990; Schoepfer et tional receptor. This observation suggests that cytisine may be al., 1990). At present, only the chick cY,-subunit has been re- used to infer the presence of & or & in functional receptors. An ported to have function when introduced into Xenopus oocytes example of a typical response from an ACh-sensitive PC 12 cell and this receptor is blocked by cY-bungarotoxin (Couturier et al., is shown in Figure 7 for ACh and cytisine, respectively. The 1990). PC1 2 cells also express an a-bungarotoxin binding sub- application of agonist causes a rapid inward current that peaks unit (Patrick and Stallcup, 1977), but the relationship of this in 100-200 msec after the drug is applied, and the response subunit to the toxin-binding protein of PC 12 remains unclear shows a small degree of desensitization that occurs with a rel- since cu-bungarotoxin does not block ACh or nicotine-elicited atively slow time course. We found that cytisine produced a 2.4 current in these cells (Patrick and Stallcup, 1977; Rogers et al., * 0.3-fold increase in the evoked current from PC12 cells not 1991a). We have observed RNA for the rat homolog of (Y, in exposed to NGF relative to ACh at equal concentrations (see our PC12 cells, and the RNA amount is decreased for these Fig. 7). This result suggests that functional nAChRs on PC12 species when NGF is added to the culture (D. Johnson, J. Boul- cells contain the p4 subunit or that it dominates this pharma- ter, and S. Rogers, unpublished observation). cology. Similarly, the cytisine:ACh ratio of receptors on PC12 In this study, immunocytochemistry and electrophysiology cells treated with NGF is 2.6, which indicates that treatment revealed that nAChR expression in PC 12 cells was limited to with NGF does not alter the relative receptor sensitivity to the those cells that responded to NGF by ceasing DNA synthesis two agonists. and altering their morphology, particularly by extending neurite-

Figure 4. Immunocytochemistry of PC12 cells. A, PC12 cells that were either not treated with NGF or treated for 4 d were prepared for immunocytochemistry with either preimmune or immune rabbit anti-a,, -c+, -(Ye,-(Y>, -&, -&, or -j3, diluted 1:2000 as described in the Materials and Methods. Immunoreactivity of the preimmune sera toward PC12 cells exposed to NGF was similar to that presented for non-NGF-treated cells and is not shown. Immunoreactivity toward (r), (rs, &, &, and & is observed, but not toward (Y*or a0 B, Higher magnification of PC12 cells treated with NGF for 4 d that were stained for immunoreactivity toward nAChR subunit (Yeor &. PC12 cells that extend neurites in response to NGF (double arrows) or are smaller but are poorly attached to the dish surface and become elongated (single solid arrow) exhibit a3 and fi2 (double arrows) as well as (Yeand 8, (see above) immunoreactivity. Cells that adhere tightly to the culture dish but do not extend neurites in response to NGF fail to show immunoreactivity toward nAChR antisera (arrowheuds). A flat cell that is extending neurites and also shows (Yeimmunoreactivity is indicated by the open arrow. Immunoreactivity is seen predominantly in the soma, and it is rarely observed in the extended processes. However, a fine “punctate” pattern of staining can be seen on the processes extended from the cells marked by double arrows. Scale:A, 10 mm = 50 pm; B, 10 mm = 12 pm.

4626 Rogers et al. * Nicotinic Receptor Expression in PC12 Cells

NO NGF _ ‘ UNffEA-l-ED

r ,i’ / I/

‘, .

IL .3 1 3 10 30 100 NGF, 2 DAYS

+ NGF \- ,‘j>”

0 .3 1 3 10 30 100

CURRENT DENSITY (pA@FJ Figure 6. The distribution of ACh current densities for untreated PC12 cells. In the upper panel is shown the results from 66 PC 12 cells. In the lower panel are the results from 72 PC1 2 cells treated with NGF for 5- 7 d. In both cases, 50 PM ACh was applied to PC12 cells that were voltage clamped at -50 mV.

Despite substantialchanges in the expressionof nAChR sub- unit RNA, we did not detect correspondingchanges in subunit protein, ACh current density, or sensitivity to cytisine. As pre- sentedin the results,a threefold changein antigen concentration Figure 5. Mouse anti-BUdR was used to visualize BUdR incorpora- should be detectedby the assaysused in this study (not shown). tion in to PC1 2 cells after 4 d of exposure to NGF. Cells that respond If, for example, the amount of (Yeor & protein parallels the to NGF by extending neurite-like processes do not exhibit BUdR in- subunit RNA, then a decreasein the total protein concentration corporation. for thesesubunits subsequentto NGF treatment should reflect the rate of subunit synthesisand degradation. If the synthetic like processes.One consequenceof continued cell division by rate remains constant and the degradation rate for the PC12 this relatively small subpopulation of cells that do not express receptor is similar to the 20 hr half-life reported for either the nAChRs could be to dilute the relative RNA levels subsequent nAChRs in muscleprior to innervation (Stanley and Drachman, to NGF treatment. This doesnot seemto be the case,however, 1983),in cultured musclecells (Patrick et al., 1977),or in ciliary since& RNA increasesin the sameRNA sampleswhere & and ganglia (Jacob et al., 1986) then as little as 5% of the original p4decrease independently of eachother (seeFig. 2). In addition, receptorsat day 0 could be left after 6 d of NGF treatment. We cell division continues in this subpopulation of PC12 cells found that the whole-cell currents and agonist sensitivity of throughout the RNA analysis period, yet almost the entire de- nAChRs on NGF-treated PC12 cells are the same as on non- creaseof aj and & RNA occurs within 48 hr of initiating NGF treated cells (i.e., & containing; seeResults). However, sincethe treatment, and then slowssubstantially thereafter. Finally, upon RNA for most subunits decreasesrapidly after NGF exposure, removal of NGF, the expressionof nAChR subunit RNA rap- to account for this result either subunit protein synthesisin- idly returns to approximately 80% of the pre-NGF treatment creases,the protein is (or becomes)more stable than assumed, value. or preexisting receptors becomeactive. The Journal of Neuroscience, December 1992, 12(12) 4621

Table 1. Relative binding of neuronal nAChR subunit antisera to PC12 cells treated with NGF

NGF 013 015 P2 A (4 x SE x SE K SE R SE 0 100 - 100 - 100 - 100 - n=7 n=3 n=9 n=3 1 102 7.6 137 41.0 105 5.0 92 11.2 n=7 n=3 n=9 n=3 2 103 7.6 110 24.0 99 6.1 82 10.6 n=7 n=3 n=9 n=3 3 109 14.1 95 25.0 99 6.3 76 19.0 n=4 n=3 n=4 n=3 4 111 24.5 124 - 115 23.0 84 19.1 n=3 n=2 n=3 n&3 5 94 17.9 97 - 121 20.9 75 4.0 n=3 n=2 n=3 n=3

Since our antisera are directed to the nAChR subunit cyto- distribution of an internal receptor pool. Internal pools of plasmic domain and the entire subunit protein pool is measured, nAChRs have been reported for both muscle nAChRs (e.g., our results cannot directly addresswhich of the above possi- Patrick et al., 1977)and nAChRs in chick ciliary neurons(Jacob bilities is most likely. However, Lindstrom and colleagues et al., 1986).Swanson et al. (1987) and Keyser et al. (1988) have (Whiting et al., 1987) have reported that a 3 d exposureto NGF also observed substantial immunoreactivity toward neuronal inducesa sixfold induction of both 86Rbflux and binding mAb nAChRs in the somaof neuronsfrom both the CNS and retina, 270, a monoclonal antibody that binds the & subunit (Whiting respectively, which is consistent with our observations of the et al., 1990) on nonpermeabilized PC12 cells. In our study, we pattern of immunoreactivity in PC12 cells. In any case, since found that NGF treatment increasedthe proportion of PC12 the macroscopiccurrents and agonist sensitivity were the same cells that had ACh currents from 30% to 60%, and the average before and after NGF treatment, the composition of the newly current was two- to threefold greater than on untreated cells inserted or possibly activated receptorsmay be similar to those (although when corrected for cell capacitance, the ACh current that areconstitutively expressedby non-NGF-treated PC 12cells. density was less).Taken together, these results suggesta four- Ifune and Steinbach (1990) observed a five- to sevenfold in- to sixfold increasein functional nAChRs after NGF treatment. creasein the median ACh current density of their PC 12 cells Our observation that nAChR protein doesnot detectably change subsequentto NGF treatment. Although this is not consistent after NGF treatment suggeststhat the increase in functional with our results,the heterogeneity among PC12 cells seemsto receptors on the surface of PC 12 cells could result from a re- be a likely explanation (seeGreene, et al., 1991). The instability

50 UYhch so UY cyushle A

Figure 7. Cytisine:ACh ratio for un- treated and NGF-treated PC 12 cells. In A are shown two current traces from an untreated PC12 cell. The inward cur- rents represent responses to 50 PM ACh and 50 PM cytisine, respectively. In B is shown the mean cytisine:ACh ratio for untreated (n = 10) and NGF-treated (n = 13) PC12 cells. There is no sig- nificant difference between the ratio of the two groups. The error bars represent UNTREATED . NGF the SEM. 4622 Rogers et al. - Nicotinic Receptor Expression in PC12 Cells of expression by PC 12 cells has been reported elsewhere (Lukas, 1987; Greene, et al., 199 1). We have observed that our PC12 References cells will lose their ability to express nAChRs upon prolonged Amy CM, Bennett EL (1983) Increased sodium ion conductance through culture, particularly if serum is altered or trypsin-EDTA is used nicotine acetylcholine receptor channels in PC12 cells exposed to during cell subculture (not shown). Further, it is likely that our nervegrowth factor. J Neurosci 3: 1547-l 553. Boulter J, Evans K, Goldman D, Martin G, Treco D, Heinemann S, PC 12 cells vary from those distributed by Greene (Greene and Patrick J (1986) Isolation of a cDNA clone coding for a possible Tischler, 1982; Greene et al., 199 l), since our cell division time neuronal nicotinic acetylcholine receptor alpha-subunit. Nature 3 19: is around 17 hr whereas the original PC12 cells divided ap- 368-374. proximately every 3 d. Nevertheless, our PC12 cell line is ad- Boulter J, Connolly J, Deneris E, Goldman D, Heinemann S, Patrick J vantageous for these studies since they grow rapidly, they re- (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc Nat1 Acad spond robustly to NGF, and the nAChR phenotype is very stable Sci USA 8417763-7767. if great care is taken to ensurestandardized culture conditions. Boulter J, O’Shea-Greenfield A, Duvoisin RM, Connolly JG, Wada E, The effect of NGF on nAChR expression is not unique to Jensen A, Gardner PD, Ballivet M, Deneris ES, McKinnon D, Hei- PC12 cells. We have shown previously that NGF can upregulate nemann S, Patrick J (1990) c+, (Ye,and p4: three members of the rat the ACh current density on primary sensoryneurons in culture neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J Biol Chem 265:4472-t482. (Mandelzys et al., 1990; Mandelzys and Cooper, 1992). When Chomczynski P, Sacchi N ( 1987) Single step method of RNA isolation neonatal rat nodose neurons are grown with NGF for 3 weeks by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal and in the absenceof other cell types, there is a six- to sevenfold Biochem 162:256-259. increasein the median ACh current density comparedwith sister Couturier S, Bertrand D, Matter J-M, Hemandez M-C, Bertrand S, Millar N, Valera S, Barkas T, Ballivet M (1990) A neuronal nicotinic cultures grown in the absenceof NGF. Also, the observation acetylcholine receptor subunit (o,) is developmentally regulated and that the regulation of RNA for oj and /I2 occurs in opposite forms a homo-oligomeric channel blocked by a-BTX. Neuron 5:847- directions has been observed in the animal (Senba et al., 1990). 856. This group found that when facial motor neurons are axotom- Deneris E, Connolly J, Rogers SW, Duvoisin R (199 1) Pharmacolog- ical and functional diversity of neuronal nicotinic acetylcholine re- ized, the (YeRNA, as measuredby in situ hybridization, decreases ceptors. Trends Phannacol 12:34-40. whereasthe & signal is enhanced. Deneris ES, Connolly J, Boulter J, Wada E, Wada K, Swanson L, Patrick The lack of a correlation between RNA expression,subunit J, Heinemann S (1988a) Primarv structure and exnression of a,: a protein expression, and the expressionof functional receptors novel subunit of neuronal nicotinic acetylcholine receptors. Neuron is intriguing. Translational or posttranslational regulation of 1:45-54. Deneris ES, Connolly J, Boulter J, Wada E, Wada K, Swanson L, Patrick receptor concentration and function is likely to contribute to J, Heinemann S (198813) &: a new member of nicotinic acetylcholine this process. For example, the regulation of muscle nAChRs receptor gene family is expressed in brain. J Biol Chem 265:4472- occurs both at the level of translation (Horovitz et al., 1989) 4482. and posttranslationally through increasing the receptor stability Dichter MA, Tischler AS, Greene LA (1977) Nerve growth factor- induced increase in electrical excitability and acetylcholine sensitivity by electrical activity and clustering at the neuromuscular junc- of a rat pheochromocytoma cell line. Nature 268:501-506. tion (for review, see Salpeter and Loting, 1985). There is also Dieckmann CL, Tzagoloff A (1985) Assembly of mitochondrial mem- evidence for the role of phosphorylation in altering the number brane system. J Biol Chem 260: 15 13-l 520. of functional receptors of the closely related neuronal nAChRs Downing JEG, Role LW ( 1987) Activators of protein kinase C enhance in chick ciliary neurons, possibly by altering rates of desensi- acetylcholine receptor desensitization in sympathetic ganglion neu- rons. Proc Nat1 Acad Sci USA 84~7739-7743. tization (Downing and Role, 1987; Margiotta et al., 1987; Sim- Duvoisin R, Deneris ES, Patrick J, Heinemann S (1989) The func- mons et al., 1990; Vijayaraghaven et al., 1990), and mammalian tional diversity of neuronal nicotinic acetylcholine receptors is in- receptors (see Huganir and Miles, 1989). Therefore, if a sub- creased by a novel subunit: 6,. Neuron 3~487496. stantial surface pool of nAChRs exist that are susceptible to Plores CM, Rogers SW, Pabreeza LA, Wolfe BB, Kellar KJ (1992) A desensitization, then a change in the phosphorylated state could subtype of nicotinic cholinergic receptor in rat brain is comprised of a-4 and D-2 subunits and is up-regulated by chronic nicotine treat- rapidly alter the ratio of active to nonactive receptors. This ment. Mol Pharmacol 41:31-37. model can describe the activation of nAChRs on receptors pres- Goldman D, Deneris ES, Luyten W, Kochhar A, Patrick J, Heinemann ent in chick ciliary ganglia (Margiotta et al., 1987), but altering S (1987) Members of a nicotinic acetylcholine receptor gene family phosphorylation in PC 12 cells fails to affect ACh-activated cur- are expressed in different regions of the mammalian central nervous rents (Amy and Bennett, 1983). Further, the increase in the system. Cell 48:965-973. Greene LA, Tischler AS (1982) PC1 2 pheochromocytoma cell in neu- number of surface & subunits (Whiting et al., 1987) collectively robiology research. Adv Cell Neurobiol 3:373-414. favors a model where a receptor pool is recruited as suggested Greene LA, Sobeth MM, Teng KK (1991) Methodologies for the by Jacob et al. (1986) or possibly stabilized in response to NGF culture and experimental use of the PC1 2 rat pheochromocytoma cell treatment. In any case, the composition of nAChR subunit pool line. In: Culturing nerve cells (Banker G, Goslin K, eds), pp 207-226. and the function of the receptor would be expected to reflect Cambridge, MA: MIT Press. Hamill OP, Marty A, Neher E, Sakman B, Sigworth FJ (198 1) Im- eventually the change in the RNA observed subsequent to NGF proved patch-clamp techniques for high-resolution current recording treatment. Of course,the nAChR subunit RNA may be present from cells and cell-free membrane patches. Pfluegers Arch 391:85- in excessof what is required to maintain functional nAChR 100. expression. Changesin nAChRs that would reflect these pos- Harlow E, Lane D, eds (1988) Antibodies: a laboratory manual, pp sibilities may require a longer period to occur than was studied 335-336. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. here, or additional signals such as electrical activity, the presence Horovitz 0, Spitsberg V, Salpeter MM (1989) Regulation of acetyl- choline receptor synthesis at the level of translation in rat primary of agonist, or other trophic factors must be present that are muscle cells. J Cell Biol 108:1817-1822. absent in the cultured cell system we employ. Testing these Huganir RL, Miles K (1989) Protein phosphorylation of nicotinic possibilities presentsinteresting avenues for further study. acetylcholine receptors. Crit Rev Biochem Mol Bio124: 182-2 15. The Journal of Neuroscience, December 1992, 12(12) 4923

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