HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/11/1267 03-1368fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by ROMANOVA, E. V. Articles by SWEEDLER, J. V. Search for Related Content PubMed PubMed Citation Articles by ROMANOVA, E. V. Articles by SWEEDLER, J. V.

Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning

Department of Chemistry, Beckman Institute and Frederick Seitz Materials Research Laboratory, University of Illinois, Urbana, Illinois, USA

¹ Correspondence: Department of Chemistry, University of Illinois, 71 Roger Adams Lab, 600 S. Mathews Ave., Urbana, IL 61801, USA. E-mail: sweedler{at}scs.uiuc.edu

SPECIFIC AIMS

We wanted to know how localized extracellular stimuli control the shape and physiological properties of neurons. To answer these questions, we examined the morphological, biochemical and electrophysiological parameters of individual Aplysia neurons cultured in the chemically and spatially defined environment of micropatterned surfaces.

PRINCIPAL FINDINGS

1. The spatial features presented by a micropatterned substrate of growth permissive molecules determine the nerve cell morphology
We find that patterns of covalently immobilized adhesion molecules, polylysine (PLL) and collagen IV, alternated with nonadhesive regions of BSA guide the neurite extension in cultured Aplysia neurons and modulate neuron structural characteristics. PLL and collagen IV have profoundly different effects on cell morphology in cultures on conventional uniform substrates. When presented as surface patterns, however, these growth permissive molecules induce changes in cellular morphology dictated by the shape of the pattern. Computer graphical analysis of branching patterns of newly formed neurites demonstrates that neurons plated on micropatterned substrates exhibit highly anisotropic outgrowth strongly biased in the direction of the growth permissive surface pattern (Fig. 1 ). Neurons on regular surfaces, in comparison, exhibit a relatively uniform distribution of outgrowth in all directions. The total neurite outgrowth per cell is significantly lower for neurons cultured on patterned surfaces (P<0.01). This difference is from a decreased number of primary neurites that originate from the cell soma (P<0.01) and a lower number of branch points per primary neurite (P<0.01). The significantly shorter mean length of a neurite (P<0.05) on the patterned substrates may indicate an overall slower rate of growth. The observed preference in the orientation of neurite outgrowth and simplification of the cellular morphology indicates that spatial constraints exerted by the limited adhesion area of growth-guiding pattern serve as a primary factor in determining the morphology of neurons. The outgrowth from cells growing on patterns never achieved the same extent as neurons plated on the analogous uniform substrates, even if kept in culture for considerably longer.

View larger version (61K): [in this window] [in a new window]   Figure 1. BCNs conform to the growth guiding patterns of vertebrate collagen IV (A–C) and PLL (D–F) and exhibit pattern-defined morphology. Fan-In projection diagrams display the geometry, orientation and length of every neurite on representative coverslips of corresponding substrates (B, E) and demonstrate that newly formed neurites have a preferred orientation. Polar histograms demonstrate that the amount of outgrowth for neurons on representative patterned surfaces (C, F) is greater in the direction aligned with the pattern. For collagen IV surface, n = 12 cells; PLL surface, n = 21 cells.

2. The physicochemical properties of the substrate influence the electrophysiological parameters of cultured nerve cells
Aplysia bag cell neurons (BCNs) grown on patterned and uniform substrates exhibit substrate-dependent changes in dynamic parameters of the action potential (AP), indicating differences in the excitability of the neuron plasma membrane. The changes observed in the dynamic parameters of the AP develop gradually in a series of repetitive electric stimulations of the cell. All neurons studied (n=23) maintain a negative membrane potential, generate APs upon repetitive positive current injections, and exhibit a characteristic progressive increase in duration of individual spikes in a train of APs (broadening). Neurons cultured on uniform PLL and collagen IV substrates show similar AP parameters. Because of a faster AP repolarization phase, neurons cultured on PLL micropatterns exhibit a significantly smaller broadening of the AP than neurons grown on regular PLL substrate (Fig. 2 ). This change in the repolarization phase of the AP can be attributed to modulations of the activity and/or abundance of K⁺ and Ca²⁺ voltage-gated ion channels. Gradual broadening of individual spikes in a train of APs, a characteristic feature of peptidergic neurons, has been associated with enhancement of neurotransmitter release. Our observation of a decrease in AP broadening in neurons on PLL patterns suggests that the ability of cultured BCNs to release neuropeptides can be modulated by the support surface. The decreased AP broadening in neurons grown on patterned PLL substrates may result in weaker excitatory postsynaptic potentials that may lead to a reduction in signal transmission between cells.

View larger version (24K): [in this window] [in a new window]   Figure 2. Substrate-dependent modulation of the AP decay time in neurons cultured on growth guiding patterned surfaces. APs in neurons cultured on patterned PLL decayed significantly faster starting with 7th action potential (A). No significant change in the AP decay time was observed in neurons cultured on patterned and uniform collagen IV surface (B). Inserts show representative 30th APs acquired from neurons cultured on patterned (lower traces) and uniform (upper traces) surfaces. All data points represent value mean ± SE (n=6 cells for all traces except for the patterned collagen, where n=5).

In comparison to uniform collagen surfaces, substrates with patterned collagen induce changes in the depolarization phase of the APs that can be related to the alterations in activity and/or expression of Na⁺ and/or Ca²⁺ voltage-gated ion channels involved in formation of this phase. The neurons on collagen patterns exhibit a significantly less negative membrane potential and smaller amplitude of the AP. The time point of the maximum of the left slope of the AP depolarization phase significantly increases in neurons cultured on collagen patterns.

It appears that physical and chemical features of the surface contribute to the electrophysiological changes observed. Nonspecific protein adsorption used in conventional substrate preparation results in random orientation of molecules on the surface. In protein immobilization procedures using covalent interactions, protein molecules are linked to the surface at particular sites, which likely results in a more ordered orientation of molecules on the surface. Cell binding domains may be hindered or exposed differently in the later case, ultimately leading to differential cellular response. This is especially relevant to the collagen IV patterns investigated in this work, as a control antibody binding study clearly demonstrated preservation of the biospecificity of collagen IV on these patterns. Changes in the conformational state of covalently immobilized PLL may also modulate the cellular response of cultured neurons via changes in surface nanotopography or by redistribution of charged PLL sites on the substrate surface.

3. The neuropeptide content of nerve cells grown on micropatterned and uniform surfaces is similar
We determine if the complement of signaling molecules changes under different culturing conditions. BCNs are known to express and release bioactive peptides that have been confirmed in freshly isolated and cultured BCNs by mass spectrometry. We use single-cell MALDI-MS to compare the neuropeptides in BCNs cultured on uniform and protein-bearing patterned substrates. In all sample types, the expected bioactive peptides are observed: egg-laying hormone, acidic peptide, -, ß-, -, and -bag cell peptides. The similarity in the neuropeptide profiles between BCNs from uniform and patterned culture substrates suggests that the neuropeptide prohormone processing pathways in BCNs are not affected and apparently are regulated independent of intracellular mechanisms sensitive to the surface interaction factors.

CONCLUSIONS AND SIGNIFICANCE

Our results suggest that the morphological and electrophysiological parameters of neurons can be predictably altered/engineered by modulation of the chemical, physical, and topographical features of culture substrates. Neurons grown on patterns exhibit a preference in the orientation of neurite outgrowth and simplification of neurite morphology, which suggests that the shape and size of the growth permissive region on a micropatterned surface influence the production of primary neurites and determines their branching pattern and their direction of extension. We hypothesize that there is an inverse correlation between the degree of alignment and extent of growth.

Properties of the support layer influence the electrical activity of the neuron. While the formation of similar cellular morphologies is observed with different immobilized proteins, the protein identity causes a change in the cell’s electrophysiological parameters. The observed variations of membrane excitability in neurons on uniform and patterned substrates with the same adhesion molecule may be attributed to the differences in molecular conformation of the extracellular matrix altering the interaction of the substrate with cell receptors or to differences in neuronal morphology causing different distributions of ion channels around the cell. Differences in electrophysiological properties between neurons grown on different protein patterns, PLL and collagen IV, may reflect the influence of the chemical nature of the extracellular substrate. The decrease in the broadening of individual APs in neurons on PLL patterns suggests that the ability of cultured bag cell neurons to release neuropeptides can be modulated by the chemistry of the support layer. It will be important to investigate the mechanisms responsible for the reduction of described spike broadening.

We conclude that by controlling both shape and chemistry of the surface, the electrophysiological, morphological, and biochemical properties of individual neurons can be independently studied and manipulated in culture. We suggest a novel idea that the physical and chemical details of microfabricated support layer influence the operation or type of cell ion channels present in the neuron membrane and thus can be used to manipulate the excitability of neurons and their assembles in culture. Modeling of the neuronal circuits where just a few identified neurons are involved in generation of a particular behavior of an animal can be beneficial for an investigation of mechanisms of learning and memory.

View larger version (43K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrates that the surface used as a culture substrate influence the morphological and physiological features of cultured neurons. Understanding of the neuronal response to different surface cues enables the rational design of model systems for control of the cellular function via cell-substrate interactions.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1368fje; doi: 10.1096/fj.03-1368fje

This Article Full Text (PDF) All Versions of this Article: 18/11/1267 03-1368fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by ROMANOVA, E. V. Articles by SWEEDLER, J. V. Search for Related Content PubMed PubMed Citation Articles by ROMANOVA, E. V. Articles by SWEEDLER, J. V.