Ultramicroscopy 111 (2011) 1417–1422

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Ultramicroscopy

0304-39

doi:10.1

n Corr

300052nn Cor

E-m

yanjun_

journal homepage: www.elsevier.com/locate/ultramic

Investigation of morphological and functional changes during neuronaldifferentiation of PC12 cells by combined Hopping Probe Ion ConductanceMicroscopy and patch-clamp technique

Xi Yang a, Xiao Liu a, Xiaofan Zhang a, Hujie Lu a, Jianning Zhang b,c,d,n, Yanjun Zhang a,nn

a Nanomedicine Laboratory, China National Academy of Nanotechnology and Engineering, Tianjin 300457, Chinab Tianjin Medical University General Hospital, Tianjin 300052, Chinac Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin 300052, Chinad Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Tianjin 300052, China

a r t i c l e i n f o

Article history:

Received 1 March 2011

Received in revised form

9 May 2011

Accepted 22 May 2011Available online 1 June 2011

Keywords:

SICM

SPM

Patch-clamp

Ion channel

Neuron

91/$ - see front matter & 2011 Elsevier B.V. A

016/j.ultramic.2011.05.008

esponding author at: Tianjin Medical Univers

, China. Tel.: þ86 22 60362026; fax: 86 22 27

responding author. Tel: þ86 22 62002900 11

ail addresses: jianningzhang@hotmail.com (J

zhang@hotmail.com (Y. Zhang).

a b s t r a c t

PC12 cells derived from rat pheochromocytoma can differentiate into sympathetic-neuron-like cells in

response to nerve growth factor (NGF). These cells have been proved to be a useful cell model to study

neuronal differentiation. NGF induces rapid changes in membrane morphology, neurite outgrowth,

and electrical excitability. However, the relationship between the 3D morphological changes of

NGF-differentiated PC12 cells and their electrophysiological functions remains poorly understood.

In this study, we combined a recently developed Hopping Probe Ion Conductance Microscopy

(HPICM) with patch-clamp technique to investigate the high-resolution morphological changes and

functional ion-channel development during the NGF-induced neuronal differentiation of PC12 cells.

NGF enlarged TTX-sensitive sodium currents of PC12 cells, which associated with cell volume,

membrane surface area, surface roughness of the membrane, and neurite outgrowth. These results

demonstrate that the combination of HPICM and patch-clamp technique can provide detailed

information of membrane microstructures and ion-channel functions during the differentiation of

PC12 cells, and has the potential to become a powerful tool for neuronal research.

& 2011 Elsevier B.V. All rights reserved.

1. Introduction

Neurites outgrowth and ion-transporter development arecharacteristics of neuronal differentiation [1–3]. Rat pheochro-mocytoma cells (PC12 cells) can exhibit neuron-like neuriteoutgrowth in response to nerve growth factor (NGF) and havelong been used as a cell model to study neuronal differentiation[4,5]. The NGF-induced PC12 differentiation includes three majorcellular processes: morphology changes, neurite outgrowth, anddeveloping electrical excitability [6]. However, the actual rela-tionship between the morphological changes and associatedelectrophysiological functions remains to be determined.

The development of Scanning Probe Microscopy (SPM) family,such as feedback-controlled Atomic Force Microscopy (AFM) [7]and Scanning Ion Conductance Microscopy (SICM) [8–10] allowsus to study of 3D cell membrane structures on nanometer scale

ll rights reserved.

ity General Hospital, Tianjin

813550.

39, fax: þ86 22 62002984.

. Zhang),

and in real time. The latter also provide insights into functionalion-channel development during neuronal differentiation.A recent study, comparing SICM to AFM of the same cells hasdemonstrated that non-contact SICM imaging provides truetopographical information of soft samples at a comparable reso-lution [11]. In contrast, continuous feedback-controlled SICM islimited to imaging relatively flat surfaces, making the high-resolution observation of living neuron membrane challenging[12–15]. Backstep [12], floating backstep [13,14,16], and standingoperating procedures [15] have been developed to overcomethese disadvantages in neuron imaging. Novak et al. [13] hasrecently developed Hopping Probe Ion Conductance Microscopy(HPICM) in order to achieve the non-contact imaging complexsurfaces of living neurons at lateral nanoscale resolutions.Because of probing a membrane surface with minimal contact,HPICM can be coupled with the widely used electrophysiologicalpatch-clamp recording [17–20], which makes itself an idealtool to investigate the relationship between physiological func-tions and morphology changes of PC12 cells during neuronaldifferentiation.

In a functional neuron, tetrodotoxin (TTX) sensitive Naþ

channels serve to initiate and conduct action potentials, while

X. Yang et al. / Ultramicroscopy 111 (2011) 1417–14221418

Kþ channels play a key role in the limitation of its propagation[21]. In neuron differentiation, these ion channel activities play acrucial role in membrane morphology and outgrowth of neurites[22,23]. For example, Kþ is important for neurite outgrowthduring neuronal differentiation [24], but the key to develope theneuronal electrical excitability is the TTX-sensitive sodium chan-nels [6]. In this study, we used the recently developed HPICMtechnology combined the patch-clamping to investigate morpho-logical changes in high-resolution and how the morphologicalchanges associate with functional ion-channel development ofdifferentiated PC12 cells. We also quantitatively evaluated the 3Dmorphological changes of neuronal PC12 in cell volume, height,membrane surface area, and roughness of membrane surface(RMS). These experimental results could provide a detailedinformation about the association between cellular morphologicalchanging and electrophysiological functions of PC12 cells duringNGF induction.

2. Materials and methods

2.1. PC12 cells culture and NGF treatment

PC12 cells were obtained from Cell Bank of Type CultureCollection of Chinese Academy of Sciences (Shanghai, China).They were grown in 85% DMEM (Hyclone, Logan, Utah, USA)supplemented with 10% horse serum (Gibco, Langley, OK,USA), 5% heat-inactivated fetal bovine serum (Gibco), 100 U/mlpenicillin (Gibco) and 100 mg/ml streptomycin (Gibco) at 37 1C ina humidified atmosphere containing 5% CO2 in air. Cells werepassaged twice per week with medium changed every 2–3 days.For electrophysiological studies and morphological analysis, cellswere plated onto 35 mm plates (Corning, USA) at a density of2�104 cells/plate and differentiated with 50 ng/ml NGF 2.5 S(Invitrogen, San Diego, CA, USA) for 3–9 days. The cell culturemedium was changed to CO2-independent Leibovitz’s L15medium (Gibco 21083) and equilibrated for 10 min before morpho-logical imaging. When a patch-clamp recording was required, theL15 medium was changed to the standard extracellular solutionand equilibrated for 10 min before patch-clamping. All subse-quent experiments were completed within 2 h at room tempera-ture (2572 1C).

2.2. Preparation of scanning nanopipette and patch-clamping

micropipette

The pipettes were pulled from borosilicate glass (O.D.1.00 mm, I.D. 0.59 mm, length 90 mm, Vitalsense Scientific Instru-ments, China) using a P-2000 laser-based puller (Sutter Instru-ments Co., USA). For HPICM scanning, the resistances ofnanopipettes ranged from 130 to 150 MO when filled with L15medium. For whole-cell patch-clamp recordings, glass micropip-ettes had a resistance of 2–6 MO when filled with electrodeinternal solution.

2.3. Hopping Probe Ion Conductance Microscope (HPICM) setup

Hopping Probe Ion Conductance Microscope (HPICM) setupwas upgraded from a commercial ICnano Scanning ion conduc-tance microscope (SICM) scanner controller (Ionscope Ltd, UK)and a sample scan head SH01 (Ionscope Ltd, UK) as describedpreviously [13]. Briefly, the SH01 scan head (Ionscope Ltd, UK)was placed on the platform of inverted TiU microscope (NikonCorporation, Japan). The ICnano SICM Controller (Ionscope Ltd,UK) controlled the vertical Z direction LISA Piezo Stage (25 mm,P-753.21C, Physik Instrumente, Germany) to perform positioning

and hopping of the nanopipette probe, and also the scanningmovement of PC12 cell in the XY plane by two PIHera PiezoNanopositioning Stage (100 mm, P-621.2C, Physik Instrumente,Germany).

An external Axon MultiClamp 700B amplifier (MolecularDevices, USA) was used to monitor the ion current flowing intothe nanopipette tip and supply a DC voltage of þ200 mV betweennanopipette electrode and bath electrode. When the hoppingprobe was approaching to PC12 cell surface, a sample–pipettedistance with 0.4% reduction of the reference DC current flowinginto the nanopipette was set to maintain the separation betweennanopipette and cell surface, the Z-position of nanopipette wasrecorded as the height of the cell membrane at this XY scanningpoint, which was then used to generate a topographical imageof the cell. During HPICM adaptive imaging of PC12 cells, aspreviously reported by Novak et al. [13], the raw topography datawere obtained with a pre-scan hop amplitude from 4 to 6 mm attwo resolution levels of 256�256 and 64�64 pixels, and theroughness threshold value was set as 100 nm. In our experiments,the time required to scan a big area of 60�60 or 90�90 mm2 wasabout 30–40 min, while a higher resolution 10�10 mm2 scanningwas about 15 min.

2.4. Electrophysiology

Ion channel currents were recorded with the whole-cellconfiguration of patch-clamp technique [25], using Axopatch700B amplifier connected to a Digidata 1440 A analog-to-digitalconverter (Molecular Devices, USA). After forming a gigaseal,the pipette resistance and capacitance were compensated. Thenthe membrane was ruptured with a gentle suction to obtain thewhole-cell voltage clamp configuration, and series resistanceswere compensated for 70–80%. Evoked currents were sampled at10 kHz and filtered by a low-pass Bessel filter set at 2 kHz.Standard extracellular solution contained (in mM): NaCl 140,KCl 3, MgCl2 1, CaCl2 2, HEPES 10, Glucose 10, pH 7.4. Electrodeinternal solution contained (in mM): KCl 140, MgCl2 2, HEPES 10,EGTA 10, Mg–ATP 2, pH 7.3. Tetrodotoxin (TTX) was purchasedfrom Hebei Fisheries Research Institute (Hebei, China). Tetraethy-lammonium chloride (TEA-Cl) and 4-aminopyridine (4-AP) werepurchased from Sigma-Aldrich (St. Louis, MO, USA).

2.5. Image processing and data analysis

The raw topography data were processed and analyzed bySICM Image Viewer software (Ionscope Ltd, UK). All patch-clamprecording data were analyzed using Clampfit 10.2 (Axon Instru-ments, USA) and Microcal Origin software version 8.0 (MicrocalSoftware Inc., USA). Values were presented as mean7SEM.Statistical significance was determined by the Student’s t-test.A difference between means at level of po0.05 was consideredstatistically significant.

3. Results

3.1. Neurites outgrowth and electrophysiological functions of NGF-

treated PC12 cells

When compared with untreated PC12 cells (Fig. 1A), phase-contrast images of PC12 cells treated with NGF for nine daysshowed neurites outgrowth (Fig. 1B). HPICM was then used toscan the cells (in black-dotted square from Fig. 1B), showing thatNGF-treated PC12 cells formed neurites that were interconnectedto form networks (Fig. 1C).

Fig. 1. Images from optical phase-contrast microscopy for untreated PC12 cells (A) and those treated with NGF for nine days (B). Panel C shows HPICM topographical

image of those NGF-treated PC12 cells (black-dotted square marked area in B).

Fig. 2. Whole-cell ion channel recordings from PC12 cells that were either untreated or treated with NGF for nine days. (A) Typical outward ion channel currents of an

untreated PC12 cell (upper) that was blocked by 4-AP and TEA-Cl (lower). (B) Typical ion channel currents of a 9-day NGF-treated PC12 cell (upper) that could be blocked

by TTX (lower). (C) Ion currents recordings are obtained with 250 ms depolarizing pulses from holding potential from �70 to þ60 mV at 10 mV steps.

X. Yang et al. / Ultramicroscopy 111 (2011) 1417–1422 1419

Patch-clamp experiments on untreated PC12 cells recordedoutward ion currents that had a peak amplitude of 1835 pA andwere completely inhibited by Kþ channel blockers 4-AP andTEA-Cl (Fig. 2A) [26]. The outward Kþ currents were measuredat a holding potential of �70 mV, which were increased toþ60 mV in 10 mV steps with the test pulses lasting for 250 ms(Fig. 2C). A few of untreated PC12 cells had less than 50 pA inwardion currents (Fig. 2A red-dotted circle marked part), a finding thatis consistent with previous reports on PC12 cells [6].

Key to the neuronal electrical excitability is the developmentof TTX-sensitive Naþ channels [6]. We measured inward Naþ

currents on PC12 cells that had been treated with NGF for ninedays (Fig. 2B). In order to block the outward Kþ currents, patch-clamping was performed in the presence of 3 mM 4-AP and25 mM TEA-Cl in bath solution. The inward Naþ currents werealso measured at a holding potential of �70 mV, which wereincreased to þ60 mV in 10 mV steps with the test pulses lastingfor 250 ms (Fig. 2C). The recorded inward currents had a peakamplitude of about 1200 pA (Fig. 1B NGF nine days, the red-dotted ellipse marked area) that was blocked by 1 mM TTX (Fig. 2BNGF 9dþTTX). We observed the development of TTX-sensitiveNaþ channels in differentiated PC12 cells, which is also consistentwith a previous report [6].

3.2. NGF-induced changes in morphology and ion channel activity of

PC12 cells

To assess morphological microstructures and functions ofNGF-differentiated PC12 cells, we combined HPICM with patch-clamping to investigate the responses of PC12 cells to NGF on third,sixth, and ninth day after treatment. The morphology changes ofthe treated PC12 cells were firstly examined with large scale HPICMscanning (Fig. 3A column). As shown in Fig. 3A top panel, controlPC12 cells were spherically shaped with a diameter of approxi-mately 22.5 mm, and sprouted no neurites. After treated with NGF,these cells increased their sizes and underwent neurite outgrowthon third day of NGF treatment and progressed over time (Fig. 3A).The high resolution zoom-in HPICM scannings was used to revealthe roughnesses of membrane surface (RMS, Fig. 3B) [27]. Com-pared with untreated cells, the surface of PC12 cells treated withNGF for three days had less microvilli-like structures, resulting in asignificant reduction in RMS values (0.5470.03 mm for untreatedand 0.4070.02 mm for treated cells, n¼12–15, po0.05).

To examine the development of voltage-gated ion channels inNGF-treated PC12 cells, the whole-cell voltage clamp was usedto record Kþ and Naþ currents (Fig. 3C). These voltage-gated ioncurrents were consistently evoked by applying 10 mV steps of

Fig. 3. Effects of NGF on PC12 cell morphology and ionic currents. (A) HPICM images of PC12 cells that were either untreated or treated with NGF for three, six, and nine

days. (B) High-resolution HPICM zoom-in images of the central areas of cells presented in column A. (C) Whole-cell recordings from control and NGF-treated PC12 cells.

The red-dotted circles represent the TTX-sensitive inward sodium currents.

X. Yang et al. / Ultramicroscopy 111 (2011) 1417–14221420

240 ms duration from a holding potential of �60 to þ30 mV inPC12 cells. We found that a longer treatment with NGF resulted in agreater TTX-sensitive Naþ current (the red-dotted circle marked partof the inward currents in Fig. 3C). These data suggest that differ-entiated PC12 cells developed voltage-gated Kþ and Naþ channels.

3.3. Association between morphological changes and the

development of functional ion channels in NGF-treated PC12 cells

Analyses on ion-channel currents and cell morphology demon-strate that NGF treatment induces the development of TTX-sensitiveNaþ currents in a time-dependent manner (Fig. 4A). We also founda correlation between Naþ current amplitude and cell size. Onepossible explanation is that a greater ion current is resulted from anenlarged membrane surface with more ion channels. This possibilityis evaluated by normalizing whole-cell currents with the cellcapacitance and calculating the current density of the recordedcurrents (Fig. 4B). The increasing ion current density of PC12 cellstreated with NGF for 3–6 days reflected an increase in the number ofion channels produced by the cells and trafficked to cell membrane,whereas the similar ion current density between ninth-day andsixth-day NGF-treated cells indicated that a growing cell bodycontained more ion channels at an identical channel density.

The morphological characteristics of cells treated with NGF forthree, six, or nine days such as heights, volumes, RMS, and surfacearea were measured using a SICM Image Viewer program in order

to conduct quantitative analyses (Fig. 4). We found that cellvolume, RMS, and surface area increased with the duration ofNGF treatment, reaching the maximal on day six. In contrast, cellheight remained constant during these periods.

4. Discussion

The pattern of electrical excitability in developing neurons hasa profound influence on neuronal differentiation. It depends onthe expression of functional voltage-sensitive Kþ and Naþ chan-nels [28]. The neuronal differentiation also results in neuritesoutgrowth and morphological changes [29,30]. We investigatedmembrane microstructural and functional changes in NGF-trea-ted PC12 cells with high-resolution morphological scanning andwhole-cell patch-clamping to gain new insight into the structuresand functions correlation during cell differentiation.

It is well documented that NGF up-regulates the expression ofNaþ [31,32] and Kþ channels [33] in PC12 cells. In this study, weshow that PC12 cells developed functional voltage-gated Kþ andTTX-sensitive Naþ channels upon NGF stimulation in a time-dependent manner. The increase of inward ion current density incells treated with NGF for 3–6 days indicates an increase in thenumber of functional ion channels in the membrane, primarily dueto cell morphological changes upon NGF treatment as demonstratedby the time-dependent increase in the cell volume, RMS, and surface

Fig. 4. (A) The peak of the recorded inward TTX-senstive Naþ currents in three-, six-, and nine-day NGF-treated PC12 cells. Values are the average of three experiments.

(B) Normalized current density of ion-channel recordings of three-, six-, and nine-day NGF-treated PC12 cells. The measurement results and statistical analysis of cell

height (C), cell volume (D), roughness of membrane surface (RMS, E), and surface area (F) for three-, six-, and nine-day NGF-treated PC12 cells are represented,

respectively. (*po0.05 vs. NGF 3d, NS¼no significant difference).

X. Yang et al. / Ultramicroscopy 111 (2011) 1417–1422 1421

area. Our study shows that the combination of HPICM and patch-clamp technique was approved suitable to image complex 3Dmorphological microstructures and monitor the development ofion-channel functions of NGF-differentiated PC12 cells, which hadthe potential to become a powerful tool for neuronal research.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China [no. 30971184].

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