J Mol Neurosci (2010) 41:329339 DOI 10.

1007/s12031-010-9369-2

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells
Arthur A. Nery & Rodrigo R. Resende & Antonio H. Martins & Cleber A. Trujillo & Vesna A. Eterovic & Henning Ulrich

Received: 3 February 2010 / Accepted: 7 April 2010 / Published online: 12 May 2010 # Springer Science+Business Media, LLC 2010

Abstract Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for 3, 5, 7, 2 and 4 subunits was present during the course of differentiation, while mRNAs coding for 2, 4 and 3 subunits were not expressed in PC12 cells. 7 subunit expression was highest following 1 day of induction to differentiation. Activity of 7 nAChRs, however, was most
A. A. Nery : C. A. Trujillo : H. Ulrich (*) Departamento de Bioqumica, Instituto de Qumica, Universidade de So Paulo, So Paulo, Brazil e-mail: henning@iq.usp.br R. R. Resende Department of Physics, Institute of Exact Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil R. R. Resende Federal University of So Joo Del-Rei-Campus Centro-Oeste, Divinpolis-MG, Brazil A. H. Martins : V. A. Eterovic Department of Biochemistry, University Central del Caribe, Bayamon, Puerto Rico

elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the 7 agonist choline. Increased 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation. Keywords Nicotinic acetylcholine receptors . PC12 pheochromocytoma cells . Alpha7 subtypes . Whole-cell recording . Nicotinic receptor expression during differentiation Abbreviations nAChR nicotinic acetylcholine receptor MLA Methyllycaconitine citrate CCh Carbamoylcholine b-FGF basic fibroblast growth factor dbcAMP dibutyril cAMP

Introduction Nicotinic acetylcholine receptors (nAChRs) as part of the superfamily of ligand-gated ion channels are formed by assembly of five subunits. At least nine (210) and three (24) subunits are known to assemble functional neuronal nAChRs from heteromeric subunits, while 7, 8 and 9 subunits form homomeric receptor channels (Kalamida et al. 2007). Activated nicotinic receptor channels allow fluxes of Na+, K+ and Ca2+ ions across the plasma membrane, with the 7 subtype being the most

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permeable for Ca2+ fluxes. The Ca2+/Na+ permeability ratio of subtypes obtained using different / combinations is in the range of 1:1.5 compared to approximately 20:1 when only homomeric 7 receptors were studied (McGehee and Role 1995; Role and Berg 1996; Gotti et al. 1997). Moreover, 7 homomeric receptor activity can be distinguished from other nAChRs by its fast desensitization rate (Hatton and Yang 2002; Matsubayashi et al. 2004). This receptor subtype is regularly expressed in the adult brain and is one of the first neurotransmitter receptors present in brain development (reviewed by Wonnacott et al. 2005). Initial expression of nAChR subunits does not depend on functional synapses, suggesting functions for these ionotropic receptors in directing neurogenesis (Zoli et al. 1995). Furthermore, nAChRs are diffusely distributed throughout the nerve fibre surface prior to formation of synaptic contacts, while after innervation, nAChRs cluster at high concentrations at the neuronal junctions (Brehm and Henderson 1988; Brenner et al. 1990). Using the P19 embryonal carcinoma cell line as in vitro stem cell model for early neurogenesis, we have shown that nAChRs and most prominently the 7 subtype are already expressed and functional in embryonic cells (Resende et al. 2008a). Additionally, nicotinic receptor activation contributed to induction of proliferation and further neuronal differentiation of the progenitor stage of this cell line (Resende et al. 2008b). Functionality of 7 nAChRs during early in vitro and in vivo neurogenesis has been suggested based on their ability to promote calcium fluxes, expression levels of this receptor and binding studies with radiolabelled bungarotoxin (Resende et al. 2008a; Liu et al. 2007; Falk et al. 2003; Adams et al. 2002), In principle, recombinant heterologous expression of nAChR subtypes in cell lines, which do not express any endogenous nicotinic receptors, would be most appropriate for characterization of receptor kinetics. However, such studies have turned out to be difficult in case of nAChRs as functional receptors are expressed at low concentrations at the cell surface and, therefore, do not produce considerable agonist-induced whole-cell currents. For instance, wholecell currents evoked by 300 M nicotine in SH-EP1 cells expressing recombinant human 7 receptors did not exceed 100 pA (Peng et al. 2005), although some authors claimed to obtain larger currents as much as 1 nA with the same cells (Zhao et al. 2003). Therefore, most pharmacological studies involving 7 receptors have been frequently using two-electrode oocyte-voltage clamping (Grnlien et al. 2007; Whiteaker et al. 2007; Papke et al. 2007), which as a result of its low time resolution, this technique does not provide the same information about ion flow and receptor desensitization as the whole-cell recording technique does. PC12 pheochromocytoma cells (Tischler and Greene 1975) have been extensively used as an in vitro model for

induction of neuronal differentiation in the presence of nerve growth factor (NGF) or basic fibroblast growth factor (b-FGF) and dibutyryl cAMP (dbcAMP) (Greene and Tischler 1976; Michel et al. 1995; Ho and Raw 1992; Huang et al. 1996; Richter-Landsberg and Jastorff 1986). Several published studies have already shown the expression of nAChR subunits in PC12 cells. The results of these studies were mostly based on radioligand-binding studies and reverse transcriptase polymerase chain reaction (RT-PCR) assays (Avila et al. 2003), but to our knowledge, no expression and activity profile of nAChRs, including the 7 subtype, along differentiation of PC12 cells into neurons has been determined so far (Table 1). Using real-time PCR analysis and fast kinetic whole-cell recording, we have shown differential nAChR subunit gene expression and receptor activity along differentiation of PC12 cells into neurons. Analysis of current decay rates in whole-cell recording experiments as a measure of receptor desensitization in the presence of the nAChR agonist carbamoylcholine (CCh) indicated a peak in 7 receptor activity on day 2 of differentiation. Measurement of CChinduced whole-cell currents in the presence of methyllycaconitine citrate (MLA), which at 10 nM concentration specifically inhibits the 7 subtype (Alkondon and Albuquerque 1994) and the nAChR responsiveness to the specific 7 subtype agonist choline (Alkondon et al. 1997), confirmed these data. Therefore, PC12 cells induced to differentiation with b-FGF and dbcAMP represent a suitable model for studying 7 receptor function and activity.

Materials and Methods Cell Culture and Neuronal Differentiation Rat pheochromocytoma cells (PC12) (Greene and Tischler 1976) were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose; Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (Cultilab, Campinas, Brazil) and 5% of horse serum (Invitrogen), 10 mM L-glutamine, 2 mM sodium pyruvate, 30 mM sodium bicarbonate and 10 mM HEPES, pH 7.4, in the presence of 100 IU/ml of penicillin and 100 g/ml streptomycin at 37C in a water-saturated atmosphere containing 5% CO2. Cell cultures were weekly split and seeded at a density of 5105 cells/culture flask (175 cm2), and culture media were replaced every 2 days. For neuronal differentiation, cells were plated in the above-mentioned culture medium at densities of 2.5 104 cells/ml in 75 cm2 culture flasks. Following 24 h of culture, differentiation was induced by addition of 30 ng/ml b-FGF and 250 M dbcAMP. These experimental conditions were used for induction of PC12 cells to neuronal

J Mol Neurosci (2010) 41:329339 Table 1 Primer for RT-PCR and real-time PCR amplification Subunit (GenBank access no.) Alpha 2 (NM_133420) Alpha 3 (NM_052805) Alpha 4 (NM_024354) Alpha 5 (NM_017078) Alpha 7 (NM_012832) Beta 2 (NM_019297) Beta 3 (NM_133597) Beta 4 (NM_052806) -Actin (NM_007393) Alpha 3 real-time (NM_052805) Alpha 5 real-time (NM_017078) Alpha 7 real-time (NM_012832) Beta 2 real-time (NM_019297) Beta 4 real-time (NM_052806) -Actin real-time (NM_007393) Forward primer 5-TTTGGAGGCTACAATCGCTG-3 5-CCTGTGGCTGAAGCAAATCTG-3 5-GCCATCTATAAGAGCTCCTGC-3 5-CAGGTACAACGGCACTGTCAC-3 5-CTGTACAAGGAGCTGGTCAAG-3 5-CTCTGAGCTGGTGACTGTACA-3 5-GTCTCTCTGAAGCAGACGTCA-3 5-CAGGAATGGACTGACTACCGC-3 5-AGGAAGAGGATGCGGCAGTGG-3 5-CGTGACCTACTTCCCATTCGA-3 5-ATTAAGCGGCTGCCTCTCTTC-3 5-TCATGCCAGCAACATCTGATTC-3 5-CATGCAAGATTGAGGTGAAGCA-3 5-CGCTCATTGGCAAGTACCT-3 5-CTGGCCTCACTGTCCACCTT-3 Reverse primer

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5-GTCCAGGAGCCAAACTTCATC-3 5-GGTGATCACCAGGAGAAAGAC-3 5-GAAGACGGTGAGAGAAAGCAG-3 5-CCAATCTTCAACAACCTCGCG-3 5-GAGCTCTTGAATATGCCTGGAG-3 5-CGTACATGCCGTCAGCATTGT-3 5-GTTGCCCTTCATCCCCTTTGC-3 5-CACACACAGTGGTGACGATGG-3 5-CGAGGCCCAGAGCAAGAGAG-3 5-CCAGGTCGATCTTTGCCTTGT-3 5-TCAGAAATGAGAGCCCAATGC-3 5-AGAGAGGCCCACGATGATCAT-3 5-AGCGAAACTTCATGGTGCAAA-3 5-CACAGTGGTGACGATGGAA-3 5-CGGACTCATCGTACTCCTGCTT-3

differentiation in the absence or presence of 1.5 mM of the 7 nAChR agonist choline. Culture medium was replaced every 24 h for fresh medium with or without choline. For whole-cell recording, cells were induced for differentiation in 35-mm cell culture dishes (TPP Techno Plastic Products, Trasadingen, Switzerland) and then used for experiments within 0 to 7 days. Immunofluorescence Studies Cells were fixed with 4% para-formaldehyde in phosphatebuffered saline (PBS) for 20 min, washed with PBS and permeabilized in the presence of 0.05% Triton X-100 in PBS for 10 min at room temperature. Cells were washed with PBS, and unspecific binding sites were blocked by incubation for 1 h with PBS containing 2% bovine serum albumin (BSA). Following three additional washing steps, expression of heavy neurofilament subunit (NF-200) was detected using NF 200-specific rabbit antibodies (Sigma, St. Luis, MO), at 1:7,000 dilution followed by addition of a 1:100 dilution of Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Groove, PA). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for visualization of entire cell populations. RNA Extraction, PCR and Real-Time PCR Total RNA extraction was performed using the TRIzol reagent (Invitrogen) as recommended by the manufacturer. The extracted RNA was reverse transcribed to cDNA in a total volume of 20 l, containing 2 l of reverse transcription buffer (100 mM TrisHCl, 500 mM KCl, 0.8% Nonidet P40, pH 8.8), 1 l of each MgCl2 (50 mM), random Primers

(50 ng/l), oligo-dT-18 (1 g/l), dNTPs (10 mM), M-MLVreverse transcriptase (200 U) and RNAse inhibitor (40 U/l) (Applied Biosystems, Foster City, CA). Following treatment with DNAse 1 (Applied Biosystems), 4 l of the reverse transcription reaction was amplified by PCR in 50-l reactions, with 1 PCR buffer in the presence of 3 l of MgCl2 (50 mM), 1 l of dNTPs (10 mM), 1 l of nicotinic receptor-subtype specific reverse and forward primers (7.5 pmol/l) and 2.5 U of Taq DNA polymerase (Fermentas, Glen Burnie, MD). For quantification of relative gene expression by real-time PCR, 3 l of the reverse transcription reaction were amplified in the presence of 1 l of primers (18 M) and 6 l of SYBR Green MIX (Applied Biosystems) in an Applied Biosystems 7300 Real-time cycler. Relative gene expression of nicotinic receptor subunits was calculated through the delta-delta-CT method (Al-Robaiy et al. 2001) using for normatization -actin mRNA transcription levels. Whole-Cell Current Recording and Rapid Application of Ligand Solutions For whole-cell recording experiments, PC12 cells were plated and induced to differentiation at a density of 20 to 100 cells/mm2 in 35-mm cell culture dishes. Whole-cell currents from day 0 to day 7 of differentiation were recorded at room temperature and a transmembrane voltage of -70 mV and pH 7.4, using an Axopatch 200B amplifier (MDS Inc., Toronto Canada), and low-pass Bessel filtered at 2 kHz. Glass pipettes were pulled on a Sutter P-30 pipette puller (Sutter Instrument, Novato, CA) and fire-polished on a Microforge MF-830 (Narishige, Tokyo, Japan). Pipette resistance ranged from 2 to 6 M. The filtered signals were digitized using a

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Figure 1 Differentiation of PC12 pheochromocytoma cells into neurons. Cells were induced to neuronal differentiation by addition of 30 ng/ml of b-FGF and 250 M of dbcAMP. a. Undifferentiated PC12 cells, b. day 01, c. day 02, d. day 03, e. day 04, f. day 05, g. day 06 and h. day 07. Progress of differentiation was confirmed by neurite

outgrowth and immunofluorescence (IF) staining against neurofilament NF-200, a protein expressed by mature neurons. NF-200 immunostaining was observed on days 6 and 7 of differentiation: i. day 06 phasecontrast, j day 06 IF, k. day 07 phase-contrast, l. day 07 IF image. Scale bar indicates a size of 100 m

Digidata 1322A interface and recorded using the pCLAMP software package (MDS Inc.). The solution in the recording pipette contained 145 mM KCl, 10 mM NaCl, 2 mM MgCl2, 1 mM EGTA and 25 mM HEPES, pH 7.4. The extracellular bath solution contained 145 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose and 25 mM HEPES, pH 7.4. CCh, an agonist for all nAChR subtypes and choline as specific agonist of 7 nAChRs (Alkondon et al. 1997) were used to determine nicotinic receptor activity during PC12 cell differentiation into neurons. MLA at 10 nM concentration acting as a specific antagonist of 7 nAChR activity (Bray et al. 2005) was used to determine the participation of this nAChR subtype in the overall receptor response induced by 1.5 mM CCh. Carbamoylcholine-induced currents were recorded by whole-cell recording in combination with a rapid ligand delivery system (cell-flow

technique) with a time resolution of 10 ms (Udgaonkar and Hess 1987). For ligand application, a U-shaped stainless steel capillary tube (250 nm inner diameter) with a circular porthole of 150 nM diameter at the base of the U was connected to pumps on both ends. The ligand solution flew into the tube at one end and was removed through the other end at twice the entry flow rate. The porthole was placed 100 nm away from a cell attached to the recording pipette. Upon closing a solenoid valve between the U-tube and the suction pump by an electric trigger, agonist or agonist and inhibitor solutions were applied to the cell in a laminar flow. Correction for Receptor Desensitization in Cell-Flow Measurements The maximum amplitude of the current is a measure of the concentration of open channels. As receptor desensitization

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current rise time; the peak amplitude of this corrected current is called Imax (that is, the maximum current that would be obtained in the absence of desensitization), and this current was the one used to analyze the inhibition and activation rates during this work (Udgaonkar and Hess 1987; Ulrich et al. 2008). Origin 7.0 software (MicroCal, Northampton, MA) was used to estimate the rate of current decay in the presence of agonist. Equation 1 was fitted to the decreasing part of the recording, and the observed maximum current amplitude was corrected for receptor desensitization.

Results Neuronal differentiation of PC12 cells induced by b-FGF and dbcAMP was visualized by morphological changes towards a neuron-like phenotype (Fig. 1ah). Expression of NF-200, an indicator protein for mature neurons, was detected on day 6 and 7 of differentiation (Fig. 1il) being in accordance with previous works (Rydel and Greene 1987). Gene expression of 3, 5, 7, 2 and 4 nAChR subunits was detected in undifferentiated cells and throughout differentiation as already previously reported (Magdesian et al. 2005). Transcription of mRNAs coding for 4, 6 and 3 subunits was below the detection limit of the RTPCR method (Fig. 2). Real-time PCR experiments revealed that transcription levels of mRNAs coding for nAChR subunits were regulated during the differentiation. Gene expression of 7 nAChRs, with the highest permeability for flow of Ca2+ ions among nicotinic receptors, was highest at the onset of differentiation on day 1, whereas gene expression of other nAChR subunits such 3 and 5 increased on days 3 and 4 of differentiation, respectively. Expression of other subunits (2 and 4) decreased following onset of differentiation and augmented again on day 4 of differentiation (Fig. 3). Following induction to differentiation by b-FGF and dbcAMP, PC12 cells were responsive to CCh application as determined by the whole cell recording technique. Maximal receptor activity was obtained in the presence of 1.5 mM CCh with EC50 values of 24894 M for receptor-ligand binding on day 2 of differentiation being in agreement with a previous study (Magdesian et al. 2005) (Fig. 4a) Whole-cell current recording in the presence of 1.5 mM CCh as nicotinic receptor agonist revealed highest receptor responses on day 7 of differentiation with measured currents in the range of 7.20.6 nA. Lowest currents (1.60.6 nA) were observed on day 1 of differentiation. No significant CCh-induced current amplitude could be measured in undifferentiated PC12 cells (1128 pA), suggesting that nicotinic receptor activity is being developed during differentiation of PC12 cells into neurons (Fig. 4b,c).

Figure 2 Gene expression of nAChR subunits along differentiation of PC12 cells. Total RNAs were collected from nondifferentiated cells (ND) and days 1 to 7 of differentiation and reverse-transcribed to cDNAs followed by PCR amplification in the presence of subunitspecific forward and reverse primers. Gene expression of 2, 4 and 3 subunits were below the detection limit of the RT-PCR assay. Control RT-PCR assays were performed in the presence of primers specific for -actin cDNAs in order to verify the integrity of the isolated RNAs. An 1-kbp ladder (MBI-Fermentas) was used for determination of the size of PCR-amplified DNA fragments

may occur while the ligand solution is flushed over the cell surface, the observed current amplitudes were corrected for receptor desensitization, using the following equation: It I1 et=T1 I2 et=T2 Ie 1

where I(t) is the observed whole-cell current at time t; I1, I2 and Ie are the amplitudes of the two exponential and the equilibrium terms and T 1 and T 2 are the apparent desensitization time constants of the first and second exponential terms, respectively. In our experimental system, current rise time is approximately 10 ms, whereas T1 is on average 75 ms (at 1.5 mM CCh) (Ulrich et al. 2008); therefore, measurable receptor desensitization occurs during current rise time. To determine the true response to a certain agonist (and inhibitor) concentration, we corrected the observed current I(t) for receptor desensitization during

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Figure 3 Differential gene expression of nicotinic receptor subunits along neuronal differentiation of PC12 cells. Relative mRNA transcription levels were determined by real-time PCR using the SYBR green method as detailed in Materials and Methods. -Actin mRNA transcription was determined as an internal control for

normalization of nAChR gene expression (a. 3, b. 5, c. 7, d. 2 and e. 4). f. Comparisson of gene expression of all subunits (days of differentiation 1-7). The shown data are representative for at least three independent experiments carried out in triplicate

CCh-induced nAChR activity in PC12 cells on day 2 rapidly desensitized such as 7 nAChR response typically do (Hatton and Yang 2002; Matsubayashi et al. 2004), whereas slower receptor desensitization was observed during the remaining days of differentiation due to participation of other nicotinic receptor subunits in the formation of functional nAChRs (Fig. 4b). The participation of 7 homomeric receptors in CCh-induced whole-cell currents was confirmed by experiments in the presence of MLA, which at 10 nM concentration specif-

ically inhibits this nAChR subtype (Alkondon et al. 2000) (Fig. 5a). Strong inhibition of CCh-induced receptor activity by MLA was obtained on day 2 of differentiation with 4911% reduction of receptor activity being due to 7 subtype inhibition. At remaining days of differentiation, the presence of 10 nM MLA resulted in percentages of inhibition between 12% and 28%, suggesting less contribution of 7 nAChRs to nicotinic receptor responses (Fig. 5b,c). The observation that there is residual 7 receptor activity along

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Figure 4 Nicotinic-receptor induced whole-cell currents in PC12 cells during neuronal differentiation. CCh-provoked whole-cell currents were recorded and analyzed as stated in Materials and Methods. a. Agonist (CCh) doseresponse curve performed with PC12 cells on the second day of the differentiation. Measured currents were normalized to responses obtained in the presence of 1.5 mM, which were considered as 100%. b. Representative whole-cell currents of PC12 cells of days 0 to 7 of differentiation were obtained following application of 1.5 mM CCh by using the cell-flow technique (Udgaonkar and Hess 1987). Results were confirmed in at least three independent experiments. Measured whole-cell currents were corrected for receptor desensitization as detailed in Eq. 1. c Averages (mean values SD) of currents obtained from at least five cells along the differentiation process

on days 5, 6 and 7, suggesting decreasing contributions of 7 receptors to nAChR-induced whole cell current during maturation of PC12 cells to neurons (Fig. 6a). T1 values as measures for receptor desensitization observed following activation of nicotinic receptor currents in PC12 from different days of differentiation were in good agreement with percentages of inhibition of CCh (1.5 mM)-induced whole-cell currents in the presence of MLA (Fig. 6a). Receptor activities recorded at the second day of differentiation with highest sensitivity to MLA inhibition of CCh-induced whole cell currents revealed the lowest T1 constants (Fig. 6a). Rapid receptor desensitization, major characteristic of the 7 homomeric channels (Hatton and Yang 2002), measured as a decrease in T1 on day 2 compared to T1 values measured on remaining days of differentiation (Fig. 6b,c) indicates again 7 nAChR activity. T2 constants as measures for the second and slow phase of desensitization revealed no significant differences during days 1 to 7 of differentiation (Fig. 6b,c). Confirming the results shown in Fig. 6b, the 7 receptor agonist choline at 1.5 mM concentrations induced highest current amplitudes on day 2 of differentiation (Fig. 7a,b). Moreover, choline-induced receptor responses were potentiated when neuronal differentiation was induced in the presence of a 1.5 mM concentration of this agonist. On day 1, the cholineinduced responses increased from 125% to 3915% of the whole-cell current obtained with 1.5 mM CCh when cells were cultured in the presence of 1.5 mM choline. On day 2 of differentiation, the response reached 5217% of the response obtained by 1.5 mM CCh when cells had been cultured with choline (Fig. 7a,b). The presence of the 7-subtype specific agonist during neuronal differentiation did not result in any morphological changes of the obtained neuron-like phenotypes (data not shown). However, the presence of choline during induction of differentiation resulted in an up-regulation of 7 nAChR activity by 400% on day 1 of differentiation when compared to PC12 cells differentiated in the absence of choline (Fig. 7a, b). A slight increase in 7 nAChR activity was also visible on day 2, whereas on days 3 and 4 of differentiation no such effect was observed.

the course of differentiation of PC12 cells is in agreement with gene expression data obtained by RT-PCR and realtime PCR (Figs. 2 and 3). Results indicating that 7 nAChR expression and activity are high on day 2 but not on any other day of differentiation are supported by analysis of the time constants of current decay as measures for kinetics of receptor desensitization of CCh-induced whole-cell currents. Time constants for the fast phase of receptor desensitization following receptor activation by 1.5 mM CCh were in the range of 10 to 50 ms on day 2 of differentiation, and T1 constants, as rate constants for fast current decay, were in the range of 20 to 380 ms on days 1, 3 and 4 of differentiation and increased even further

Discussion Gene expression of nAChR subunits and the formation of functional homomeric and heteromeric receptors are regulated following induction of PC12 cells to neurons by b-FGF and dbcAMP. Nicotinic receptor activity gradually increased when cells started neurite outgrowth and differentiated into neurons. The 7 subtype with the highest permeability for calcium flow among nAChRs, ranging around PCa/PNa>10 (Sgula et al. 1993), was highly expressed and functional during the initial phase of differentiation.

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Figure 5 Contribution of 7 nAChRs to nicotinic receptor-induced whole-cell currents. a. Representative whole-cell currents of PC12 cells of days 0 to 7 of differentiation were obtained following application of 1.5 mM CCh and 10 nM of MLA using the cell-flow technique. b. For desensitization corrected whole-cell currents induced by 1.5 mM CCh in PC12 during differentiation into neurons were determined in the absence and presence of 10 nM MLA as a specific inhibitor of 7 nAChR activity. The plotted results represent mean

values SD of at least three independent experiments (A and Ai are CCh-induced whole-cell currents in the absence or presence of 10 nM MLA, respectively). c. Data from b. were used for calculation of percentages of inhibition of nAChR responses in PC12 cells induced to differentiation. Percentage of inhibition of agonist-induced receptor inhibition by MLA was calculated for each cell, followed by mean value determination. Statistical analysis was done by ANOVA, *p< 0.05 compared with control data

Gene expression of 7 subunits peaked on day 1 of differentiation, whereas following protein expression of functional homomeric 7 receptors was delayed to day 2. On day 2, around 50% of CCh-induced receptor activity was due to 7 subtype activation as determined by inhibition experiments in the presence of MLA, while residual CChinduced whole-cell current resulted from heteromeric nAChR activity. Nicotinic receptor desensitization in the presence of 1.5 mM CCh was fastest on the second day when compared to any other day of neuronal differentiation. Moreover, cells revealing fast nAChR desensitization on day 2 of differentiation were most susceptible to inhibition by 10 nM MLA. Time constants for fast desensitization (T1) of 37.614.6 ms, indicating major participation of 7 subtype activity in PC12 cells, were increased to values of 12394 to 464326 ms in cells from days of differentiation with less 7 subunit expression. The suggestion that increased T1 constants indicate 7 nAChR activity in agreement with results obtained with SH-SY5Y cells expressing a mixture of 3- and 7-subunit containing nAChRs (Sokolova et al.

2005). Moreover, the high responsiveness to choline application on day 2 of differentiation supports such hypothesis. The function of nicotinic receptors in neuronal transmission has been widely studied (Kalamida et al. 2007). However, recent studies have revealed that functional nAChRs are also expressed by cells that do not form synaptic contacts, including embryonic cells and neural progenitor cells (reviewed by Trujillo et al. 2009). In these cells, nAChRs and, most importantly, 7 receptors with highest Ca2+ conductance participate in differentiation into neural phenotypes. In addition to their suggested importance in contributing to transient increases of free intracellular calcium concentration [Ca2+]i during early development, resulting in Ca2+-dependent activation of neuron-specific gene expression, 7 receptors function is implicated in various physiological and pathological processes including long-term plasticity (Taly et al. 2005). Moreover, our group has shown in a recent work that nicotinic receptor-induced transients are already present in

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characteristics for neural progenitor cells (Angelastro et al. 2003). Differentiate cells express NF-200, a marker protein of mature neurons (Fig. 1). In this regard, the stage of differentiation of undifferentiated PC12 cells are similar to P19

Figure 6 Correlation between nAChR desensitization and potency of MLA (10 nM)-induced inhibition of nAChR activity on days 1 to 7 of differentiation. a. Percentage of inhibition of CCh (1.5 mM) induced whole-cell currents in the presence of 10 nM MLA observed in single cells. b. The same cells were analyzed for time constants of current decay (T1) as measure for receptor desensitization following application of 1.5 mM CCh. It can be noted that cells on day 2 of differentiation, which were most sensitive to inhibition by MLA, also revealed fastest receptor desensitization (T1<100 ms). Bars within the figures indicate mean values of T1 constants. c Averages of T1 and T2 constants along the neural differentiation process. T1 and T2 values were calculated by using Eq. 1 as stated in Materials and Methods

pluripotent P19 embryonal carcinoma cells used as in vitro model for early neurogenesis (Resende et al. 2008a). Nicotinic receptor activity, presumably with major participation of 7 subtypes, participated in acetylcholine-induced proliferation and differentiation of the neural progenitor cell stage of P19 cells (Resende et al. 2008b). Undifferentiated PC12 pheochromocytoma cells already express proteins which are

Figure 7 Increase of 7 nAChR receptor activity during neuronal differentiation in the presence of choline. PC12 cells were induced to differentiation by addition of b-FGF and dbcAMP in the absence or presence of 1.5 mM of the 7 nAChR agonist choline, and agonistinduced whole-cell currents were measured as detailed in the Materials and Methods. Obtained current amplitudes were corrected for receptor desensitization by using Eq. 1. a. Agonist (1.5 mM of CCh or choline)-induced receptor responses obtained from PC12 cells (day 1 and 2) following induction to neuronal differentiation in the absence or presence of 1.5 mM choline. b. Mean values SD of three independent experiments of choline-induced whole currents of cells on day 1 or 2 of differentiation (without choline and with choline: neuronal differentiation was carried out in the absence or presence of 1.5 mM choline, respectively). Shown are mean values S.E. representative for at least three independent experiments with three measurements from each cell. Whole-cell responses induced by choline were plotted as percentages of those obtained in the presence of 1.5 mM CCh. *** p<0.001 compared with control data

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J Mol Neurosci (2010) 41:329339 Alkondon M, Albuquerque EX (1994) Presence of alpha-bungarotoxinsensitive nicotinic acetylcholine receptors in rat olfactory bulb neurons. Neurosci Lett 176:152156 Alkondon M, Pereira EF, Cortes WS, Maelicke A, Albuquerque EX (1997) Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur J Neurosci 9:27342742 Alkondon M, Braga MF, Pereira EF, Maelicke A, Albuquerque EX (2000) Alpha7 nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus. Eur J Pharmacol 393:5967 Al-Robaiy S, Rupf S, Eschrich K (2001) Rapid competitive PCR using melting curve analysis for DNA quantification. Biotechniques 31 (13821386):1388 Angelastro JM, Ignatova TN, Kukekov VG, Steindler DA, Stengren GB, Mendelsohn C et al (2003) Regulated expression of ATF5 is required for the progression of neural progenitor cells to neurons. J Neurosci 23:45904600 Avila AM, Dvila-Garca MI, Ascarrunz VS, Xiao Y, Kellar KJ (2003) Differential regulation of nicotinic acetylcholine receptors in PC12 cells by nicotine and nerve growth factor. Mol Pharmacol 64:974986 Bray C, Son JH, Meizel S (2005) Acetylcholine causes an increase of intracellular calcium in human sperm. Mol Hum Reprod 11:881889 Brehm P, Henderson L (1988) Regulation of acetylcholine receptor channel function during development of skeletal muscle. Dev Biol 129:111 Brenner HR, Witzemann V, Sakmann B (1990) Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature 344:544547 Buisson B, Bertrand D (2002) Nicotine addiction: the possible role of functional upregulation Trends Pharmacol Sci 23:130136 Cho CH, Song W, Leitzell K, Teo E, Meleth AD, Quick MW et al (2005) (2005) Rapid upregulation of alpha7 nicotinic acetylcholine receptors by tyrosine dephosphorylation. J Neurosci 25:37123723 Corringer PJ, Sallette J, Changeux JP (2006) Nicotine enhances intracellular nicotinic receptor maturation: a novel mechanism of neural plasticity? J Physiol Paris 99:162171 El Kouhen R, Hu M, Anderson DJ, Li J, Gopalakrishnan M (2009) Pharmacology of alpha7 nicotinic acetylcholine receptor mediated extracellular signalregulated kinase signalling in PC12 cells. Br J Pharmacol 156:638648 Falk L, Nordberg A, Seiger A, Kjaeldgaard A, Hellstrm-Lindahl E (2003) Higher expression of alpha7 nicotinic acetylcholine receptors in human fetal compared to adult brain. Brain Res Dev Brain Res 142:151160 Gotti C, Fornasari D, Clementi F (1997) Human neuronal nicotinic receptors. Prog Neurobiol 53:199237 Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73:24242428 Grnlien JH, Hkerud M, Ween H, Thorin-Hagene K, Briggs CA, Gopalakrishnan M, Malysz J (2007) Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol Pharmacol 72:715724 Hatton GI, Yang QZ (2002) Synaptic potentials mediated by alpha 7 nicotinic acetylcholine receptors in supraoptic nucleus. J Neurosci 22:2937 Ho PL, Raw I (1992) Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells. J Cell Physiol 150:647656 Huang CM, Tsay KE, Kao LS (1996) Role of Ca2+ in differentiation mediated by nerve growth factor and dibutyryl cyclic AMP in PC12 cells. J Neurochem 67:530539 Kalamida D, Poulas K, Avramopoulou V, Fostieri E, Lagoumintzis G, Lazaridis K et al (2007) Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J 274:3799 3845

progenitor cells regarding their differentiation stage, and 7type nAChRs participate in neuronal differentiation of PC12 cells, like it has been suggested for P19 cell differentiation. In agreement with neuroprotective functions of 7-nAChRs, treatment of PC12, cell with positive modulators of this receptor subtype augmented ERK phosphorylation, which is involved in neuroprotective processes (El Kouhen et al. 2009). Positive modulation of receptor activity was also observed in the presence of the 7-receptor agonist choline. 7-nAChRmediated whole-cell currents were increased when cells were induced to neuronal differentiation in the presence of 1.5 mM choline. Up-regulation of nAChR activity has been observed before in SH-SY5Y neuroblastoma cells following chronic treatment with nicotine (Sokolova et al. 2005). This phenomenon was studied in detail using recombinant receptor expression. As possible mechanisms, receptors could be stabilized in a form with high affinity for their agonist. High- and low-affinity receptor states for nicotine have been described for the 42 subtype, with high-affinity types mediating larger responses (Buisson and Bertrand 2002; Vallejo et al. 2005). On the other hand, it has been postulated that the presence of the agonist could convert preexisting pools of immature nAChR subunits into high-affinity receptors by favoring specific intersubunit interactions (Corringer et al. 2006; Sallette et al. 2004) or simply could induce upregulation of nAChRs by dephosphorylation, as already suggested for 7-type receptors (Cho et al. 2005). In summary, we have shown that PC12 cells induced to neuronal differentiation are an adequate model for studying 7 nAChR expression and activity in an environment in which various nAChR subunits are expressed. 7 nAChR expression is up-regulated at the onset of differentiation. and its activity can even be further potentiated following chronic exposure to choline. This is the first time that expression and functionality of a single nicotinic subtype has been studied in a context of various endogenous active nAChRs. In view of that, PC12 cells are a suitable model for screening of drug effects on 7 nAChR activity and expression.
Acknowledgments The work was supported by research grants from Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP), project no.: 2006/61285-9, and Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) Brazil, awarded to H.U.; A.A. N.'s and C.A.T.'s Ph.D. theses are supported by fellowships from FAPESP, Brazil. A.H.M and V.A.E. acknowledge the NIH grant support (UPRPRAABREP20RR016470 and G12RR03035-24), R.R.R is grateful for grants from CNPq and FAPEMIG.

References
Adams CE, Broide RS, Chen Y, Winzer-Serhan UH, Henderson TA, Leslie FM et al (2002) Development of the alpha7 nicotinic cholinergic receptor in rat hippocampal formation. Brain Res Dev Brain Res 139:175187

J Mol Neurosci (2010) 41:329339 Liu Z, Zhang J, Berg DK (2007) Role of endogenous nicotinic signaling in guiding neuronal development. Biochem Pharmacol 74:11121119 Magdesian MH, Nery AA, Martins AH, Juliano MA, Juliano L, Ulrich H et al (2005) Peptide blockers of the inhibition of neuronal nicotinic acetylcholine receptors by amyloid beta. J Biol Chem 280:3108531090 Matsubayashi H, Inoue A, Amano T, Seki T, Nakata Y, Sasa M et al (2004) Involvement of alpha7- and alpha4beta2-type postsynaptic nicotinic acetylcholine receptors in nicotine-induced excitation of dopaminergic neurons in the substantia nigra: a patch clamp and single-cell PCR study using acutely dissociated nigral neurons. Brain Res Mol Brain Res 129:17 McGehee DS, Role LW (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 57:521546 Michel PP, Vyas S, Agid Y (1995) Synergistic differentiation by chronic exposure to cyclic AMP and nerve growth factor renders rat phaeochromocytoma PC12 cells totally dependent upon trophic support for survival. Eur J Neurosci 7:251260 Papke RL, Dwoskin LP, Crooks PA (2007) The pharmacological activity of nicotine and nornicotine on nAChRs subtypes: relevance to nicotine dependence and drug discovery. J Neurochem 101:160167 Peng JH, Fryer JD, Hurst RS, Schroeder KM, George AA, Morrissy S et al (2005) High-affinity epibatidine binding of functional, human alpha7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells. J Pharmacol Exp Ther 313:2435 Resende RR, Gomes KN, Adhikari A, Britto LR, Ulrich H (2008a) Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro. Cell Calcium 43:107121 Resende RR, Alves AS, Britto LR, Ulrich H (2008b) Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells. Exp Cell Res 314:14291443 Richter-Landsberg C, Jastorff B (1986) The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells. J Cell Biol 102:821829 Role LW, Berg DK (1996) Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16:10771085 Rydel RE, Greene LA (1987) Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci 7:36393653

339 Sallette J, Bohler S, Benoit P, Soudant M, Pons S, Le Novre N et al (2004) An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine. J Biol Chem 279:1876718775 Sgula P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW (1993) Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J Neurosci 13:596604 Sokolova E, Matteoni C, Nistri A (2005) Desensitization of neuronal nicotinic receptors of human neuroblastoma SH-SY5Y cells during short or long exposure to nicotine. Br J Pharmacol 146:10871095 Taly A, Delarue M, Grutter T, Nilges M, Le Novre N, Corringer PJ et al (2005) Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism. Biophys J 88:39543965 Tischler AS, Greene LA (1975) Nerve growth factor-induced process formation by cultured rat pheochromocytoma cells. Nature 258:341342 Trujillo CA, Schwindt TT, Martins AH, Alves JM, Mello LE, Ulrich H (2009) Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics. Cytometry A 75:3853 Udgaonkar JB, Hess GP (1987) Acetylcholine receptor: channel-opening kinetics evaluated by rapid chemical kinetic and single-channel current measurements. Biophys J 52:873883 Ulrich H, Akk G, Nery AA, Trujillo CA, Rodriguez AD, Eterovi VA (2008) Mode of cembranoid action on embryonic muscle acetylcholine receptor. J Neurosci Res 86:93107 Vallejo YF, Buisson B, Bertrand D, Green WN (2005) Chronic nicotine exposure upregulates nicotinic receptors by a novel mechanism. J Neurosci 25:55635572 Whiteaker P, Christensen S, Yoshikami D, Dowell C, Watkins M, Gulyas J et al (2007) Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist. Biochemistry 46:66286636 Wonnacott S, Sidhpura N, Balfour DJ (2005) Nicotine: from molecular mechanisms to behaviour. Curr Opin Pharmacol 5:5359 Zhao L, Kuo YP, George AA, Peng JH, Purandare MS, Schroeder KM et al (2003) Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharmacol Exp Ther 305:11321141 Zoli M, Le Novre N, Hill JA Jr, Changeux JP (1995) Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems. J Neurosci 15:19121939