Electrophysiological and Morphological Characterization of RatEmbryonic Motoneurons in a Defined System

Mainak Das, Peter Molnar, Halagowder Devaraj, Matthew Poeta, andJames J. Hickman*

Department of Bioengineering, Clemson University, 420 Rhodes Hall, Clemson, South Carolina 29634-0905

In an attempt to integrate biological components with silicon-based devices andsystems, artificial silane surfaces have been successfully used to grow motoneuronsin a defined environment. In this study we characterized the morphology andelectrophysiology of purified rat embryonic (E14) motoneurons grown on a self-assembled monolayer (SAM) of N-1[3-(trimethoxysilyl)propyl]diethylenetriamine (DETA)versus that on ornithine/laminin surfaces in serum-free media. On DETA motoneuronswere flat and grew more processes, whereas on ornithine/laminin they tended toaggregate. The membrane time constant, a characteristic associated with electrotoniccompactness, was significantly longer for motoneurons grown on DETA. Otherelectrophysiological parameters were similar for the motoneurons on the differentsurfaces. This is the first study where purified ventral horn motoneurons were culturedin a completely defined (nonbiological surface, serum-free) environment.

Introduction

The integration of living cells with silicon-based sys-tems is essential for the frontier areas of bioengineering,biosensor development, prosthetics, high throughputdrug/toxin screening, neurocomputing, and biorobotics(1-3). An enabling factor for research into these topicsis to create in vitro systems where a majority of thevariables are known or defined. A representative systemof the type of in vitro model that would have a tremen-dous impact if developed is the reflex arc, one of the mostfundamental circuits for motor control. The recreationof this system in a defined in vitro environment will allowresearchers to address a number of the above topics andalso to gain a more fundamental understanding of thebasic neuroscience of this complex system.

The first step to create an in vitro system composed ofall of the elements of the reflex arc integrated withsilicon-based systems (i.e., microelectromechanical sys-tems, or MEMS, devices) involves the modification of thesurface of the silicon to enable the development and thelong-term survival of cells in a defined hybrid environ-ment. In addition, for many cases biological variabilityin the components of the culture media are not desirablefor the above applications and the neurons need to becultured in a completely defined media that approximatescerebral spinal fluid (CSF) (4). We have experimentallyestablished a system where all of the major neuronalculture parameters are known and quantifiable (5), whichconsists of a reproducible dissociated culture methodol-ogy, serum-free media, and a surface composed of self-assembled monolayers (SAMs) (4).

Motoneurons are one of the most extensively charac-terized neuronal cell types (6-8). Recently, much atten-tion has been focused on motoneuron physiology driven

by the partial success in the repair of spinal cord injuries(9). With the development of tissue engineered nerveconstructs (3), the study of the interaction of motoneuronswith engineered surfaces has become crucial for theirsuccessful application. However, there is little reportedeffort to create a defined in vitro system for studyingisolated motoneurons or motoneurons in conjunction withother components of the reflex arc, except for rudimen-tary systems such as Campenot chambers (10). It isimportant to coculture the motoneurons with theirtargets because, without the appropriate intracellularand extracellular signals, motoneurons do not survive forsignificant periods of time (9). Motoneurons can bemaintained in serum-free culture media with the additionof several growth factors (8, 11), but in most of thepublished methods motoneurons are initially plated inserum-containing media, where serum is an undefinedcomponent and not reproducible. Thus, this research isimportant because, although the electrophysiologicalcharacteristics of developing motoneurons have beenstudied extensively (6, 7, 12), no reports of their mor-phological or electrophysiological properties in a defined,reproducible environment have been reported.

The use of SAMs for chemical surface modificationenables that surface to be equipped with a variety offunctional groups that possess required specific electrical,optical, or chemical properties (13). SAMs have alreadybeen used for studying cell-surface interactions (4), thecontrol of protein adsorption (14), prosthetic devicebiocompatibility (15), and most importantly for recreatinga Campenot-like system, cell patterning (16). In additionto the above reasons, serum-containing culture mediashould not be used in chemical patterning of neuronalnetworks because proteins deposited from serum cancover the original chemical pattern on the substrate (17).Previously, a self-assembled monolayer of N-1[3-(tri-methoxysilyl)propyl]diethylenetriamine (DETA) was suc-

* To whom correspondence should be addressed. Phone: (864)656-7168. Fax: (864) 656-4466. E-mail: hickman@clemson.edu.

1756 Biotechnol. Prog. 2003, 19, 1756−1761

10.1021/bp034076l CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical EngineersPublished on Web 09/09/2003

cessfully used to pattern (5, 18) and determine polarity(16) of hippocampal pyramidal cells.

In this study, which is the first step in creating an invitro reflex arc, we demonstrate that purified rat embry-onic motoneurons could be cultured in a defined environ-ment (serum-free) on a synthetic surface (DETA), andwe report their electrophysiological characterizationcompared to a more biologically based system (ornithine/laminin). The passive membrane properties of the mo-toneurons were determined because they are the majordeterminants of the summation of synaptic inputs (6, 19).The development of the specific ionic conductances onthe soma are also necessary for sustained repetitivefiring of action potentials and ultimately muscle action(20).

Materials and MethodsSurface Modification. DETA. Glass coverslips were

cleaned using HCl/methanol (1:1), soaked in concentratedH2SO4 for 30 min, and then rinsed in double distilledH2O. Coverslips were next boiled in deionized water,rinsed with acetone, and then oven dried. The DETA filmwas formed by reaction of cleaned surfaces with a 0.1%(v/v) mixture of the organosilane in toluene. The DETAcover glasses were heated to just below the boiling pointof toluene, rinsed with toluene, reheated to just belowthe boiling temperature, and then oven dried.

Surface Characterization. Surfaces were character-ized by static contact angle measurements using aCam 200 contact angle goniometer (KSV) utilizing stan-dard protocols and X-ray photoelectron spectroscopy(XPS). XPS is a technique for the characterization of thetop 50-100 Å of a surface that identifies elementspresent in the surface layer, their oxidation states, andtheir relative amounts. For XPS analysis, the Kratos 165XPS instrument was used according to a publishedprotocol (21).

Ornithine/Laminin. Stock solution of polyornithine(Sigma, 1.5 mg/mL /1000×/) was prepared in water,whereas the stock solution of laminin (natural mouse,Invitrogen, CA, 1.5 mg/mL /500×/) was prepared inphosphate-buffered saline (PBS) (Invitrogen). Coverslips(Thomas Scientific, Swedesboro, NJ) cleaned by acidtreatment were incubated with polyornithine (1:1000dilution from stock in water) overnight at room temper-ature. Coverslips were dried in the laminar flow hood.Laminin was dissolved in the culture medium at adilution of 1:500. Ornithine-coated coverslips were placedin 35 mm cell culture dishes, and then 3 mL of thelaminin solution was added and incubated in a CO2incubator overnight at 37 °C (8).

Rat Embryonic Motoneuron Culture. Rat spinalmotoneurons were purified from ventral cords of embry-onic day 14 (E14) embryos as described by Henderson etal. (8). Briefly, rats were anaesthetized and killed byinhalation of an excess of CO2. This procedure was inagreement with the Animal Research Council of ClemsonUniversity, which adheres to IACUC policies. Ventralspinal cells from the embryo were dissociated aftertrypsin (Invitrogen, 0.05%) treatment and centrifuged for15 min at 500g over a 6.5% metrizamide cushion (22).The large cells remaining above the cushion were furtherselected using the immune interaction between moto-neurons and the 192 antibody (1:2 dilution, ICN Bio-medicals, Akron, OH) coated on the dishes (8). Theantibody recognizes the low affinity NGF receptor ex-pressed only by ventral motoneurons at this age (23).

Purified motoneurons were plated on 22 × 22 mm2

ornithine/laminin-coated coverslips and DETA-coated

coverslips at a density of 1.5-2.0 × 103 cells/mm2. Theculture medium was Neurobasal (Gibco-BRL) supple-mented with B27 (2% v/v; Invitrogen), L-glutamine (0.5mM), and 2-mercaptoethanol (25 µM). Three growth-promoting factors were added: glial cell line-derivedneurotrophic factor (100 pg mL-1 GDNF; Invitrogen),brain-derived neurotrophic factor (100 pg mL-1 BDNF;Invitrogen), and ciliary neurotrophic factor (1 ng mL-1

CNTF; Cell Sciences) (24). The culture medium waschanged every 5 days and L-glutamate (25 µM) was addedto the culture medium during the first 5 days of growth.Motoneurons were maintained in vitro for 14 days.

Identification of Motoneurons by Islet-1 Im-munostaining and Live-Dead Assay. The purity of themotoneurons was verified using Islet-1 immunostaining.Briefly, purified motoneurons on coverslips were fixedusing 4% paraformaldehyde in PBS plus 0.1% glutaral-dehyde at 4 °C for 15 min. Quenching of excess aldehydegroups and permeabilization were performed with 50 mMlysine plus 0.1% Triton X-100 for 15 min at roomtemperature. Nonspecific staining was blocked using 2%BSA and 2% goat serum in PBS. Anti-Islet antibody 4D5(Developmental Studies Hybridoma Bank, University ofIowa) was used at a dilution of 1:200. The secondary anti-mouse antibody was coupled to Cy3 (Jackson labs).Molecular Probe’s L-3224 Live/Dead Assay kit was usedfor the live-dead assays.

Morphological Analysis. Phase-contrast pictureswere taken with a commercial Nikon Coolpix 990 camerausing the 40× objective of a Zeiss Axiovert S100 micro-scope. Pictures were enhanced in Photoshop (Adobe) andanalyzed using Scion Image Software (Scion Corp.,Maryland). The long and the short axes of the soma aswell as the number of processes originating in the somawere measured.

Electrophysiology. Whole-cell patch clamp record-ings were performed in a recording chamber on the stageof a Zeiss Axioscope 2 FS Plus upright microscope. Thechamber was continuously perfused (2 mL/min) with theextracellular solution (Neurobasal culture medium; pHwas adjusted to 7.3 with HEPES, 35 °C). Patch pipetswere prepared from borosilicate glass (BF150-86-10;Sutter, Novato, CA) with a Sutter P97 pipet puller andfilled with intracellular solution (in mM: K-gluconate140, EGTA 1, MgCl2 2, Na2ATP 2, phosphokreatine 5,phosphokreatine kinase 2.4 mg, Hepes 10; pH ) 7.2). Theresistance of the electrodes was 6-8 MΩ. Voltage clampand current clamp experiments were performed with aMulticlamp 700A amplifier (Axon, Union City, CA).Signals were filtered at 2 kHz and digitized at 20 kHzwith an Axon Digidata 1322A interface. Data recordingand analysis were performed with pClamp 8 software(Axon). Membrane potential was corrected by subtractionof a 15 mV tip potential, which was calculated usingAxon’s pClamp 8 program. The membrane resistance andcapacitance were calculated using a 50 ms voltage stepfrom -85 to -95 mV without any whole-cell or seriesresistance compensation. Sodium and potassium currentswere measured in voltage clamp mode using voltage stepsfrom a -85 mV holding potential. Whole cell capacitanceand series resistance was compensated using a p/6protocol. Only cells with access resistance less than 22MΩ were analyzed. The membrane time constant wasdetermined using a 1 ms -0.8 nA current impulse incurrent clamp mode. Six traces were averaged and 3exponentials were fitted to the averaged trace using thepClamp program. The electrotonic length of neurons wascalculated using the ratio of the 0th and 1st order time

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constants (L ) π[(τ0/τ1) - 1]-1/2) (25). Repetitive firing,firing thresholds, and after-hyperpolarization were mea-sured with 1 s depolarizing current injections from a -85mV holding potential.

Statistical Analysis. A two-sample t test was per-formed for the statistical analysis of morphological andelectrophysiological data. Parameters obtained fromDETA-plated neurons were compared with the ornithine/laminin controls.

Results

Surface Modification. The surface modification con-trols were tested by contact angle and X-ray photoelec-tron spectroscopy (XPS). The contact angle for DETAcovered coverslips was 40.62 ( 2.92° (mean ( SD), whichhad been shown previously to be acceptable (4). XPSindicated that the glass surface was modified by acomplete layer of DETA (characterized by the 399 eV Npeak) (4).

Survival of Motoneurons in Defined Environ-ment. Purified rat embryonic motoneurons, plated eitheron DETA or ornithine/laminin surfaces (Figure 1), sur-vived at least 12 days in serum-free Neurobasal culturemedia. The initial number of attached cells was signifi-cantly higher on DETA than on ornithine/laminin, butthe number of surviving cells decreased more rapidly onDETA during the first 4 days (Figure 2). Islet-1 immu-nostaining was performed after day 6 in culture. Morethan 90% of the purified motoneurons were positive toIslet-1.

Effect of DETA Surface on the Morphology ofMotoneurons. The morphology of motoneurons on DETAand on ornithine/laminin was different (Figures 1 and2, Table 1). On DETA the motoneurons were flat, basedon phase contrast microscopy observation, and sent outmore processes, whereas on ornithine/laminin they tendedto aggregate after day 10 in culture. The number ofprocesses originating from the soma was significantlyhigher with neurons plated on DETA than with neuronsplated on ornithine/laminin (Figure 2, Table 1). Therewas no significant difference in the size of the cells onthe two surfaces. The diameter of the cells (long axis inTable 1) increased as the cells matured in the culturesystem (Table 1).

Effect of Surface on Electrophysiological Char-acteristics of the Motoneurons. The resting mem-brane potential of the motoneurons became more nega-tive with time and stabilized by day 6 (Table 2). Theamplitudes of the voltage-dependent sodium and potas-sium currents also increased with time, and the neuronsbecame capable of sustained repetitive firing after ap-

Figure 1. Purified rat embryonic motoneurons plated on DETA(left column) and ornithine/laminin (right column) surfaces. (A,B) Day 3 cultures, phase contrast. Scalebar: 25 µm. (C, D) Day6 cultures, phase contrast. Scalebar: 25 µm. (E, F) Day 6cultures, epifluorescent picture. Scalebar: 100 µm.

Figure 2. Survival and morphological characteristics of purified rat embryonic motoneurons on DETA and on ornithine/lamininsurfaces. (A) Neurons survived at least 12 days in serum-free conditions. (B) The number of processes originating from the soma wassignificantly higher (two-sample Student’s t test) on neurons plated on DETA than on neurons plated on ornithine/laminin. (Mean( SEM; ***P < 0.005)

Table 1. Summary of Morphological Parameters of Motoneurons Grown on DETA and Ornithine/Laminin Surfaces

day 3 day 6 day 9 day 12

DETA SD O/L SD DETA SD O/L SD DETA SD O/L SD DETA SD O/L SD

long axis (µm) 17.13 2.90 18.40 3.83 19.25 3.53 20.91 4.38 21.40 4.76 22.72 4.54 24.69 4.37 25.60 5.84short axis (µm) 9.98 1.87 10.68 2.95 12.47 2.31 13.57 3.57 12.28 1.76 14.30 2.48 14.27 2.94 12.36 2.76num. proc. 9.10a 2.66 4.50 2.04 8.48a 3.19 4.44 1.54 10.13 3.54 7.20 2.59 10.25a 2.79 5.77 2.20n 21 20 21 18 16 6 16 13

a P < 0.005, two-sample Student’s t test.

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proximately 6 days (Figure 3). There was no significantdifference between the passive or active membraneproperties of motoneurons plated on DETA or ornithine/laminin surfaces except for the membrane time constant,which was longer for neurons grown on DETA (Figure3). This difference was observable for each time periodchecked. The calculated electrotonic length of motoneu-rons was shorter for the DETA surface than for ornithine/laminin. On the basis of firing properties of the neuronsthey were classified into 4 “firing type” categories:category 0, no action potential; category 1, 1 or 2 actionpotentials; category 3, repetitive firing but duration ofthe firing is less than 1 s; category 4, continuousrepetitive firing. The maximum firing frequency of thematurated motoneurons was 18 Hz, observed during thefirst 1 s measurement period.

Discussion

In this study purified rat embryonic motoneurons werecultured on a nonbiological surface, DETA, and on astandard biological surface, ornithine/laminin, in a com-pletely serum-free environment. Immunostaining withIslet-1 was over 90% positive for motoneurons in bothcases. The motoneurons survived and matured during a12-day study period on both surfaces and were compa-rable to previous experiments with the control surface(11). The morphology and electrophysiology of motoneu-rons on DETA and ornithine/laminin were different;however, the only electrophysiological characteristic thatdiffered significantly between the two surfaces was themembrane time constant.

The development of purified embryonic motoneuronson the biological surface (ornithine/laminin) was verysimilar to that described previously (7). Despite thegeneral similarity of the results on both surfaces, how-ever, there were some differences noted. The differencesobserved include the total number of neurons adheringto the surface after initial plating, the growth dynamicsduring the first 4 days following plating, the neuronalgeometry, and the membrane time constant. Possibleexplanations for the higher initial cell number on the

DETA surface is that cell attachment to DETA is muchstronger than to ornithine/laminin or this combinationof factors provides a more favorable initial environmentfor the neurons (4, 5, 16, 21, 26-28). On DETA, moto-neurons were flat and grew more processes versus thebiological surface, and in the older cultures (9-12 days)the motoneurons tended to aggregate on ornithine/laminin but not on DETA, as noted by qualitative visualobservations. As noted above, a possible explanation forthis is that the attachment of neurons to DETA is muchstronger than to ornithine/laminin. Also, on ornithine/laminin, it is possible that the migration of the cells isfacilitated. This is an area of continuing research.Another possible explanation might be that the lamininpresent in the substrate activated cell surface receptors(i.e., integrins), which then influenced the morphologyof the motoneurons. This specific signaling could bemissing or there could be different receptors activatedin the case of DETA.

It is important to determine if embryonic motoneuronsmaintained in cell culture go through the proper devel-opmental stages and become functional. During thematuration process for neurons, the membrane potentialbecomes more negative until it reaches a stable value ofabout -45 mV. In addition, during normal development,the amplitude of voltage-sensitive sodium and potassiumcurrents increases, thus enabling the repetitive firingproperties of the motoneurons. Our data are in goodagreement with the published results for the emergenceof the normal electrophysiology of the embryonic moto-neurons (7, 25). The only observed difference, in themembrane time constant, could be related to the alteredmorphology of the motoneurons on the DETA surface.The electrotonic length is a major parameter used incompartmental models of motoneurons, and it is highlydependent on the morphology of the neuron (29). Thecalculated electrotonic length was longer on ornithine/laminin than on DETA, indicating a possible differencein the physiology of the motoneurons, and this couldbe important because the electrotonic length is a param-eter involved in the description of the summation of

Table 2. Passive and Active Membrane Properties of Embryonic Motoneurons on DETA and on Ornithine/LamininSurfacesa

day 3 day 6 day 9 day 12

DETA STD OL STD DETA STD OL STD DETA STD OL STD DETA STD OL STD

VM (mV) -39.5 5.2 -37.5 7.0 -41.8 6.8 -45.7 6.2 -48.8 6.0 -46.1 5.1 -49.7 4.4 -44.6 7.5RN (MΩ) 1233 810 947 414 583 275 475 108 396 216 390 190 419 200 458 213CM (pF) 17.7 10.7 17.4 9.0 18.4 11.7 12.8 5.0 18.4 8.0 22.0 10.7 18.5 4.4 17.6 20.1L τ0/τ1 1.97 0.47 2.29 0.37 1.41b 0.42 1.96 0.39 1.34b 0.28 2.19 0.68 1.71 0.56 1.83 0.72τ0 (ms) 56.37b 31.0 20.7 9.0 54.02b 23.7 22.3 5.4 45.7b 16.9 20.0 6.3 51.9b 30.1 28.0 16.0TVCi1 (ms) 0.46 0.22 0.54 0.30 0.83 0.30 1.18 0.33 1.04 0.47 0.94 0.38 1.11 0.76 1.32 0.46Na curent (pA) -1520 930 -1243 651 -2640 1172 -2929 1588 -4917 3411 -2429 1110 -3541 2408 -3501 1718K current(pA) 1229 714 1416 749 2596 1030 3833 2340 3165 1213 4018 1928 4536 2556 4672 2572Vthr (mV) -53.7 4.7 -51.9 5.6 -61.0 3.5 -57.7 2.0 -60.2 4.4 -54.8 4.8 -59.8 3.6 -57.3 5.0Ithr (pA) 31.5 26.0 38.6 19.9 36.5 23.8 41.8 13.8 52.0 32.2 92.9 52.0 52.8 31.0 55.3 57.5firing type 0.80 0.42 0.89 0.60 2.40 1.35 3.22 0.67 3.13 1.13 3.00 1.15 3.67 0.52 3.57 1.13max firing (Hz) 0.80 0.42 0.89 0.60 5.10 4.72 10.56 6.71 9.63 6.93 8.57 7.98 12.67 8.78 10.00 4.20AP ampl (mV) 54.7 9.8 53.5 6.9 66.7 12.1 69.5 8.0 80.6 8.9 60.4 13.6 84.6 9.0 72.6 11.7AP duration (ms) 7.99 2.86 6.69 2.87 3.67 1.79 2.85 1.51 2.68 1.09 2.44 1.06 2.43 0.53 2.72 0.66AHP Ampl. (mV) -5.16 3.69 -4.67 1.18 -4.01 3.05 -4.29 2.22 -4.89 1.97 -4.99 2.61 -5.62 2.21 -4.94 2.14AHP duration (ms) 89.4 53.8 69.1 34.3 70.4 35.3 82.7 41.2 93.3 29.8 68.6 40.5 101.0 31.9 65.6 32.1n 10 9 10 9 8 7 6 7

a Electrophysiological parameters were measured using conventional voltage clamp and current clamp protocols during whole cellpatch-clamp recordings from motoneurons growing on DETA and ornithine/laminin surfaces. VM: resting membrane potential. RN:input resistance. CM: membrane capacitance. L: electrotonic length. τ0: zero-order membrane time constant. TVCi1: longest membranetime constant in voltage clamp mode. Vthr: action potential threshold voltage. Ithr: action potential threshold current. Firing type: empiricalclassification of neurons (0 from 4) based on their repetitive firing properties. b Significant difference (two-sample Student’s t test) comparedto ornithine/laminin surface.

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incoming synaptic inputs. This difference could be relatedto different receptors being activated on the differentsurfaces and will be one focus of the continuation of theseinvestigations. However, these minor variations do notappear to be a hindrance in using this defined system tostudy the properties of developing motoneurons or thecreation of a new in vitro system to study the segmentsof the reflex arc.

Conclusion

This is the first study where purified motoneuronswere cultured and morphologically and electophysiologi-cally characterized on an artificial surface in a serum-free environment. In future experiments these artificialsurfaces will be used to pattern immunopurified embry-onic motoneurons, as seen with hippocampal neurons (5,30), and to integrate them with silicon-based hybridconstructs to create the first segment of the reflexarc: motoneuron to muscle. Moreover, in this definedsystem the substrate composition can be systematicallyvaried to study the contact signaling pathways influenc-ing motoneuron survival, differentiation, and regenera-tion.

Acknowledgment

We thank Dr. Estevez, Department of Physiology andBiophysics, University of Alabama at Birmingham for hiskind help in streamlining purification, immunopanning,and immnostaining protocols; Developmental StudiesHybridoma Bank at the University of Iowa for providingthe Islet-1 antibody; and Mark Shalinsky for his helpfulcomments. This work was supported by DARPA grantDARPA-ITO N65236-01-1-7400.

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Figure 3. Electrophysiological characterization of rat embryonic motoneurons grown on DETA and on ornithine/laminin surfaces.(A) Representative recording of sodium and potassium currents obtained from a day 6 neuron on the DETA surface (-85 mV holdingpotential, 10 mV steps). (B) Representative recording of membrane time constants obtained from day 6 neurons on DETA and onornithine/laminin surfaces. (C, D) Maximum action potential firing rate of a day 3 (C) and a day 9 (D) motoneuron plated on a DETAsurface. (E) Maximum firing rate of motoneurons on DETA and ornithine/laminin surfaces. (F) Membrane time constants. (Mean (SEM; *P < 0.05; **P < 0.01; ***P < 0.005, two-sample Student’s t test.)

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Accepted for publication July 2, 2003.

BP034076L

Biotechnol. Prog., 2003, Vol. 19, No. 6 1761