Regulation of an inactivating potassium current (IA) by the extracellular matrix protein vitronectin in embryonic mouse hippocampal neurones

doi:10.1113/jphysiol.2002.036889

Regulation of an inactivating potassium current (I_A) by the extracellular matrix protein vitronectin in embryonic mouse hippocampal neurones

Dmitry V. Vasilyev and Michael E. Barish

Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Integrins are a class of intrinsic membrane receptors for extracellular matrix ligands. In the central nervous system, integrins and their ligands influence neuronal growth and synaptic function, but relatively little is known about their potential to regulate intrinsic excitability. To explore this area, we examined the effects of matrix components on potassium currents in developing mouse hippocampal neurones, using electrophysiological and immunochemical approaches. We tested the effects of three integrin ligands present in the hippocampus, fibronectin, laminin and vitronectin, on electrogenesis in late embryonic hippocampal pyramidal neurones. Explants cultured in serum-free medium were exposed to ligands (fibronectin at 3 µg ml^-1, laminin at 5 µg ml^-1, vitronectin at 10 µg ml^-1) for 3-4 days, and voltage-gated potassium currents were recorded from presumptive CA3 pyramidal neurones. Of the three matrix components, only vitronectin affected potassium currents, selectively increasing the amplitude of the inactivating potassium current (I_A, or A-current) by about 75 % over control levels, and its density (current per unit area) by about 40 % (measured after 3 day exposures from embryonic day 15.5). Other potassium currents were spared, except to the extent that membrane area was increased. The actions of vitronectin were sensitive to RGD (Arg-Gly-Asp)-sequence-containing peptide, indicating the involvement of integrins as vitronectin receptors. The kinetic properties of I_A, including the voltage-dependence of activation and inactivation, inactivation rate and the rate of recovery from inactivation, were minimally affected by vitronectin and were consistent with enhanced functional expression of Kv4-family subunits. Analyses of Kv4.2 and Kv1.4 immunoreactivity also suggested a preferential increase in Kv4.2 levels, with lesser effects on Kv1.4 levels. These results indicate that vitronectin can selectively regulate I_A, and together with other observations suggest that modulation of neuronal excitability by integrins and their ligands occurs commonly.

(Resubmitted 1 December 2002; accepted 23 December 2002; first published online 24 January 2003)
Corresponding author M. E. Barish: Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. Email: mbarish{at}coh.org

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

The intrinsic excitability of neurones is controlled by multiple influences, some of which are cell autonomous, and others of which depend on electrical activity or the extracellular environment. We are particularly interested in the activities of signals resident in the extracellular matrix. While the brain does not have a basal lamina, growth factors, proteoglycans and other signalling molecules are immobilized in the extracellular space, and some of these molecules serve as ligands for integrins. These are integral membrane proteins that mediate numerous cell-matrix and cell-cell interactions (Hynes, 1992).

Integrins expressed in the nervous system (see Pinkstaff et al. 1999) have been investigated primarily as regulators of neural migration and morphology, disease and recovery, and, more physiologically, synaptic function (Sanes, 1983, 1989; Staubli et al. 1990; Chen & Grinnell, 1995; Jones, 1996; Grotewiel et al. 1998; Archelos et al. 1999; Ellison et al. 1999; Murase & Schuman, 1999; Benson et al. 2000; Rohrbough et al. 2000; Chavis & Westbrook, 2001). Recently there has been increased interest in integrins and other matrix proteins as acute and developmental modulators of electrogenesis (see Discussion), but this area remains largely unexplored.

The matrix components fibronectin, laminin and vitronectin are all present in the developing brain (Pearlman & Sheppard, 1996; Sobeih & Corfas, 2002). We hypothesized that as part of their influence on nervous system development, these integrin ligands might also modulate neuronal excitability. To address this issue, we examined their effects on the potassium currents of hippocampal neurones, applying these ligands to developing organotypic explant cultures of late embryonic hippocampus for periods of 3-4 days. In the hippocampus, pyramidal neurones undergo terminal division between embryonic day (E)10 and E18 (Angevine, 1975). Soon thereafter, they express multiple voltage-gated potassium currents (Storm, 1990) including the transient potassium current I_A (or A-current) and the sustained currents I_D and I_K (Ficker & Heinemann, 1992; Wu & Barish, 1992). Of the three matrix components tested, only vitronectin affected potassium currents, selectively enhancing I_A, most probably by preferentially upregulating functional expression of one or more Kv4-family subunits, including Kv4.2. Other voltage-gated potassium currents were not altered, except as expected from the increase in membrane area. Enhancement of I_A was sensitive to RGD (Arg-Gly-Asp)-sequence-containing peptide, suggesting that vitronectin signalling passed through one or more integrins. These observations thus indicate that vitronectin- and integrin-mediated signalling can regulate I_A, and suggest the possibility of other similarly specific linkages.

A report of some of these results has been published in abstract form (Vasilyev & Barish, 2000).

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Cultures

To prepare organotypic cultures, developing hippocampal structures (Fig. 7) were dissected free from E15.5 (and occasionally E16.5) mouse brains. Embryos were derived from timed-pregnant Swiss-Webster mice, which were anaesthetized with halothane before being killed by cervical dislocation. Explants were placed ventricle-side up on Millicell CM (PICM03050; Millipore, Bedford, MA, USA) tissue plate inserts, as originally described by Stoppini et al. (1991), and grown at 35.5 °C in a humidified 5 % CO₂/air atmosphere in (serum-free) Neurobasal/B27 medium (GibcoBRL, Rockville, MD, USA). When appropriate, explants were exposed to experimental reagents from 2 h after the time of culture. Reagents were both added to the culture medium and applied in a drop to the surface of the explant, after which the excess fluid slowly passed through the membrane into the underlying reservoir. Neurones were allowed to develop in vitro for between 1 and 4 days, as appropriate. Integrin ligands were purchased: mouse vitronectin from GibcoBRL (no. 12172-011), fibronectin and laminin from BD Biosciences (Franklin Lakes, NJ, USA). All procedures involving animals were performed in accordance with NIH guidelines and were approved by the City of Hope Research Animal Care Committee.

Electrophysiology

I_A was recorded from visually identified (using differential interference contrast optics and an Olympus BX50WI microscope; Olympus Optical, Tokyo, Japan) neurones in cultured explants using standard whole-cell techniques. Electrodes were pulled from borosilicate glass capillaries (TW150F; World Precision Instruments, Sarasota, FL, USA), to a resistance of 2-3 M $Omega$ when filled with intracellular solution. The intracellular solution consisted of (mM): 100 potassium gluconate, 50 KCl, 5 MgCl₂, 1 CaCl₂, 5 EGTA, 20 Hepes, pH adjusted to 7.4 with Tris-Cl. The extracellular solution, artificial cerebrospinal fluid, contained (mM): 140 NaCl, 3 KCl, 26.5 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 2 MgCl₂, 20 glucose, bubbled with carbogen (5 % CO₂/95 % O₂). Tetrodotoxin (0.5 µM) was added to suppress sodium currents and action potentials. Data were collected using an Axopatch 200B amplifier, and digitized using a DigiData 1200 interface and pClamp 8 software (all from Axon Instruments, Union City, CA, USA). Current traces were filtered at 1-2 kHz and digitized at 10 kHz. Series resistance was compensated by 50-70 %. Membrane capacitance was calculated from the charging current elicited by a 5 mV hyperpolarizing voltage step from -50 mV. Capacitance transients and leakage currents linear with voltage were subtracted using a P/-4 voltage protocol. Recordings were made at room temperature (22-24 °C).

Data were analysed using pClamp 8 and Origin 6 (OriginLab, Northampton, MA, USA). The statistical significance of differences between means was evaluated by t test (two-tailed) or ANOVA using Instat (GraphPad, San Diego, CA, USA) or Excel (Microsoft, Bellevue, WA, USA).

Immunochemistry

Purified rabbit antiserum against Kv1.4 (Barry et al. 1995) was a gift from Dr Jeanne Nerbonne (Washington University School of Medicine, St Louis, MO, USA). Purified rabbit antiserum against Kv4.2 was purchased from Chemicon (AB5928; Temecula, CA, USA). Purified rabbit antiserum against mouse vitronectin (Seiffert et al. 1994) was a gift from Dr David Loskutoff (The Scripps Research Institute, La Jolla, CA, USA).

For immunostaining of acute embryonic hippocampal sections, the entire brain was removed from the embryo and fixed in 4 % paraformaldehyde in PBS, pH 7.4, for 2 h at 4 °C, and then rinsed (3 times 15 min each) in PBS. For immunostaining of the cultured hippocampal formation, the tissue and adjacent culture membrane were excised from the Millicell insert and fixed as above. In both cases, tissues were then permeabilized with 0.1 % Triton-X (Sigma) in PBS containing 3 % BSA (bovine serum albumin) and 5 % normal goat serum for 1 h at room temperature, and then incubated for 12-14 h at 4 °C in primary antibody (anti-Kv1.4 or Kv4.2 at 0.5-1.0 µg ml^-1; anti-vitronectin at 1:25 dilution or 30 µg ml^-1) in PBS with 3 % BSA. After rinsing in PBS (3 times 20 min each), sections were incubated in fluorescein-conjugated goat anti-rabbit IgG (Zymed, South San Francisco, CA, USA) diluted 1:100 in PBS containing 5 % normal goat serum for 1 h at room temperature. Finally, sections were rinsed in PBS (3 times 20 min each) and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA).

Images were collected on a laser-scanning confocal microscope (Zeiss 310; Jena, Germany) using times 10 air and times 40 oil-immersion objectives. Montage acquisition software (written locally) was used to scan large brain areas.

For comparisons of potassium channel subunit expression, pairs of cultured explants (control and experimental) were processed in parallel, and images were acquired using the same microscope parameters. A semi-quantitative analysis of luminance in multiple pairs of images (control and experimental) was performed by establishing a threshold for each pair and extracting all pixel luminance values above the threshold (Optimas 6.2; Media Cybernetics, Silver Spring, MD, USA). The appropriate threshold luminance was subtracted from each pixel luminance value and, for each image pair, values of pixel luminance were then normalized relative to the mean (threshold-subtracted) luminance of the control image. Statistical comparisons were made of mean relative pixel luminance (t test), and of cumulative probability of relative pixel luminance (log rank test) using Prism 3 (GraphPad). Software limitations required the luminance values for the millions of pixels in each image to be reduced to about 900 points (Fig. 6c and f), a number that was adequate for this analysis.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

We prepared organotypic cultures by severing the developing hippocampus where it joins with extrahippocampal cortex (dotted line in Fig. 7, which shows a cryostat section of an E14.5 hippocampus; see also Grove et al. 1998; Tole & Grove, 2001) and placing it on the tissue culture insert ventricle-side up. At this stage, cells in the embryonic hippocampus, but not those of adjacent extrahippocampal cortex, express the neuronal marker class III $beta$ -tubulin (Grove et al. 1998) and the CA3 regional marker KA1 (Tole et al. 1997; Grove & Tole, 1999), making them most likely to be developing CA3 pyramidal neurones.

Over the next 3 days the explant thinned, and a layer of cells emerged in an arc along the medial side inside the cortical hem (region enclosed by the dotted lines in Fig. 1Aa) opposite the edge where the hippocampus was cut free of extrahippocampal cortex. Cells within this medial region express the CA3 marker KA1, while those more laterally positioned (i.e. outside the indicated region) express the CA1 marker SCIP (suppressed cyclic AMP-inducible POU domain protein) (Grove & Tole, 1999; Tole & Grove, 2001). Recordings were made from cells within the medial band, most likely nascent CA3 neurones, as indicated by the fluorescent neurobiotin-filled neurones in Fig. 1Ab (from the same explant as panel Aa). Figure 1Ac shows a more detailed view of a neurone from a different explant, with its dendritic field and its single axon emerging to the left. We did not observe noticeable variations in recordings made elsewhere in the explants (not shown), suggesting that the differences in I_A between CA3 and CA1 neurones noted in recordings from more mature cells (not shown) emerge later in development.

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Figure 1. Explant culture of embryonic day E15.5 hippocampus
Aa, image (dark field illumination) of an explant after 3 days in culture; the explant was grown ventricle-side up after separation from the extrahippocampal cortex (see Fig. 7). The region of the cortical hem is down, and the cut edge abutting the extrahippocampal cortex is up. The dotted line encloses the medial band region from which recordings were made; cells in this region express the CA3 marker KA1 (Grove & Tole, 1999; Tole & Grove, 2001). Ab, fluorescence image of two neurobiotin-filled neurones in the explant shown in Aa, illustrating their placement within the medial band. Ac, more detailed fluorescence view of a neurobiotin-filled neurone in a different explant with its dendritic field and leftwards-emerging axon. B, separation of I_A and 'delayed currents' from the total potassium current. Currents were recorded during step depolarizations from -120 mV to voltages between -60 and +60 mV, with and without a 100 ms prepulse to -40 mV. I_A was isolated by point-by-point subtraction as the current sensitive to the inactivating prepulse that spared delayed currents, and was similar in form and kinetics to I_A recorded from other embryonic and neonatal neurones.

Recordings were made from cell bodies, and thus reflect channel activity restricted to somatic and proximal dendritic membrane (Brown & Johnston, 1983). I_A was isolated by subtraction of traces recorded with and without an inactivating prepulse (Fig. 1B). The outward currents remaining after the prepulse, which include I_D and I_K (Storm, 1990), were considered together as 'delayed currents' and were not further separated.

Three extracellular matrix components of the developing hippocampus

In our initial experiments we screened fibronectin, laminin and vitronectin on cultures of E15.5-E16.5 hippocampal explants for any effects they might have on potassium currents. As illustrated in the summary shown in Fig. 2, neither fibronectin nor laminin affected either I_A or delayed currents, except for a small depression of delayed current density induced by laminin. Vitronectin, however, significantly enhanced I_A (Fig. 3), and we therefore pursued this aspect further.

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Figure 2. Potassium currents are not altered by exposure to fibronectin (3 µg ml^-1) or to laminin (5 µg ml^-1)
Explant cultures of the E16.5 hippocampus were exposed to reagents for 4 days. The only significant change noted was a laminin-induced decrease in delayed current density, probably as a consequence of the increase in membrane area (reflected in measures of capacitance). Number of cells: 9 for control, 9 for +fibronectin, 8 for +laminin. In this and subsequent figures, data are means ± S.D. with significance levels: *P < 0.05, **P < 0.01 and ***P < 0.001.

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Figure 3. Functional expression of I_A is enhanced by exposure to vitronectin
A, representative I_A traces recorded at +40 mV (sufficient for near-maximal activation; see Fig. 5) from the somata of control and vitronectin-exposed cells. B, summary data for recordings from control (ctl), vitronectin (vn, 10 µg ml^-1)-exposed, and vitronectin-GRGDSP peptide (vn + rgd, 200 µg ml^-1)-exposed cells. Upper, current amplitudes showing significantly larger I_A and delayed currents in only the vitronectin-exposed neurones, with the actions of vitronectin blocked by co-incubation with RGD-sequence-containing peptide. Lower left, significantly increased whole-cell capacitance in vitronectin-exposed cells. Lower right, only I_A density (expressed as pA pF^-1 to normalize for changes in membrane area) was increased in vitronectin-exposed cells, indicating a selective effect on I_A beyond what would be expected from the increase in membrane area. Number of cells: 45 for control, 57 for +vitronectin, 18 for +vitronectin and RGD peptide.

Vitronectin and I_A

Vitronectin (Hayman et al. 1983) is a widely expressed (Seiffert et al. 1991, 1994; Seiffert, 1997) and multifunctional component of serum and extracellular matrix. In the mouse nervous system, vitronectin mRNA is found in the floor plate as early as E10 (Seiffert et al. 1995), and vitronectin influences development of the retina, spinal cord and cerebellum (Neugebauer et al. 1991; Martinez-Morales et al. 1995, 1997; Pons & Marti, 2000). In the more mature brain, vitronectin is detected under both normal and pathological conditions (Akiyama et al. 1991; Neugebauer et al. 1991; McGeer et al. 1992; Yasuhara et al. 1994; Gladson et al. 1995; Niquet et al. 1996; Murase & Hayashi, 1998; Walker & McGeer, 1998).

I_A is particularly interesting in the context of an investigation of integrins and membrane currents because in young neurones it appears to be particularly susceptible to regulation by extracellular influences (McFarlane & Cooper, 1993; Dourado & Dryer, 1994; Raucher & Dryer, 1994; Wu & Barish, 1994; see Barish, 1995). In pyramidal neurones, I_A is mediated primarily by spatially segregated populations of potassium channels incorporating Shal (Kv4) family subunits (e.g. Kv4.2 and Kv4.3), or Shaker (Kv1) family subunits (e.g. Kv1.4; see Discussion). Physiologically, I_A influences the approach of the membrane potential to threshold in response to excitatory input (Connor & Stevens, 1971; Rogawski, 1985).

To obtain consistent recordings we followed a standard protocol involving culture of hippocampal explants at E15.5 and 3 day exposure to vitronectin (10 µg ml^-1) added 2 h after the cultures were established. Under these conditions, vitronectin selectively enhanced I_A amplitude, as illustrated by the representative recordings from control and vitronectin-exposed neurones shown in Fig. 3A. The amplitudes of both I_A and the delayed current (Fig. 3B, upper) were larger in vitronectin-exposed neurones; peak I_A amplitude was increased by ~75 % (from 0.8 ± 0.5 to 1.4 ± 0.6 nA; mean ± S.D., P < 0.001, n = 45 for control and 57 for +vitronectin). At the same time, whole-cell capacitance (lower left), an indicator of membrane area, was also about 31 % larger in these neurones. When current amplitudes were normalized to capacitance and considered as current densities (pA pF^-1; Fig. 3B, lower centre and right), only I_A density was significantly affected by vitronectin (increase of ~40 %; from 48.9 ± 24.5 to 67.6 ± 27.6 pA pF^-1; P < 0.01). Vitronectin thus selectively enhanced the amplitude of I_A to a greater extent than was expected from the increase in cell size alone.

The amino acid sequence RGD (Arg-Gly-Asp) defines an integrin binding region that was originally described for fibronectin, and which is present on numerous other integrin ligands (Ruoslahti & Pierschbacher, 1987). Peptides incorporating this sequence can disrupt integrin-mediated signalling that uses this motif. Including an RGD sequence-containing peptide (GRGDSP; 200 µg ml^-1) with vitronectin blocked increases in whole-cell capacitance and potassium current amplitudes (Fig. 3B). This observation suggests that vitronectin signalling involves a combination of its RGD-sequence-containing domain (Schvartz et al. 1999) with one or more of several vitronectin-responsive integrins potentially expressed in the developing brain (see Discussion).

The time course of changes in potassium currents is illustrated in Fig. 4. After culture at E15.5, vitronectin-induced differences in I_A amplitude and density were evident as early as 1 day in culture (right upper and lower). Between days 1 and 3, amplitudes of control I_A and delayed current increased 16- and 8-fold, respectively (above), and both were affected by vitronectin, However, whole-cell capacitance also changed (lower left), and when currents were evaluated as density, only I_A was affected by vitronectin (lower right). Vitronectin thus appeared to selectively amplify the normal course of I_A development in cultured explants. The decrease in both I_A and delayed current densities by the 4th day in culture was also seen in pyramidal neurones of the same age in dissociated cell culture (Wu & Barish, 1992).

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Figure 4. Developmental progression of cell growth and membrane currents over 4 days in culture
Upper, I_A and delayed current amplitudes; both increased dramatically over the first 3 days in culture from E15.5 and then stabilized. Lower left, whole-cell capacitance also increased over this same period, and in control cells stabilized. Lower right, I_A density was consistently and significantly larger in vitronectin-exposed cells, while showing the same pattern of increase and decline; delayed current density was not altered. Number of cells: at 1 day, 7 for control and 9 for +vitronectin; at 3 days, 23 for control and 25 for +vitronectin; at 4 days, 20 for control and 20 for +vitronectin. Note that the data shown in Figs 3 and 4 were taken from different groups of experiments within which populations of neurones were grown and assayed in parallel. Neither control nor +vitronectin data differed significantly between the two data sets (P > 0.05).

Intrinsic I_A properties

The effects of I_A on excitability will depend on its amplitude, its activation and inactivation rates, and its time course of recovery from inactivation. As shown in Fig. 5, vitronectin exposure resulted in only small changes in the intrinsic kinetic properties of I_A that may reflect changes in phosphorylation or other states of underlying potassium channels.

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Figure 5. The intrinsic kinetic properties of I_A did not differ between control and vitronectin-exposed neurones
A1, I_A from representative control and experimental neurones, scaled to peak amplitude. The inactivation time course was fitted with the sum of two exponential functions:
Y = A_fastexp(-t/ $tau$ _fast) + A_slowexp(-t/ $tau$ _slow) + C;
the fitted curves are superimposed in red. A2, summary of parameters derived from these fits. Exposure to vitronectin increased the absolute amplitudes of fast and slow components, A_fast and A_slow, with A_slow showing a greater percentage increase (196 % vs. 102 % for A_fast). The intrinsic time constants of these components, $tau$ _fast and $tau$ _slow, were not affected. Number of cells: 5 for control (ctl) and 12 for +vitronectin (vn). B, voltage dependence of I_A activation and inactivation, fitted with Boltzmann relationships of the general form G/G_max = 1/(1 + exp[(V - V_1/2)/k]), where V is the activation voltage, V_1/2 is the half-activation voltage and k is the slope factor. Changes in activation and inactivation were minimal, with a trend towards negative shifts in the activation curves of vitronectin-exposed neurones. Number of cells: 6 for control and 9 for +vitronectin. C, the time course of recovery from inactivation was not altered by vitronectin. Neurones were held at -40 mV to inactivate I_A, hyperpolarized to -110 mV for varying periods to remove inactivation, and then stepped to -30 mV to assess I_A availability. Number of cells: 3 for control and 3 for +vitronectin.

First, the increase in I_A amplitude was accompanied by a small but consistent slowing of inactivation. Illustrated in Fig. 5A1 are representative I_A records, scaled to peak amplitudes, with the inactivation time course fitted with the sum of two exponential functions (curves superimposed in red). The current from the vitronectin-exposed neurone relaxes slightly more slowly. As indicated in Fig. 5A2, the time constants characterizing the fast and slow components, $tau$ _fast (~26 ms) and $tau$ _slow (~169 ms), respectively, were not affected by vitronectin exposure, while the amplitudes of each component (A_fast and A_slow, respectively) increased, by 102 % and 196 %, respectively. The larger increase in A_slow was reflected in a small and statistically insignificant increase in its contribution to the total I_A: 32.8 % in vitronectin-exposed neurones vs. 25.0 % in control neurones. Thus in control and experimental neurones, three-quarters to two-thirds of the total I_A was contributed by the more rapidly inactivating component.

Second, the voltage dependence of I_A activation and inactivation (Fig. 5B) was also not significantly altered by exposure to vitronectin. Activation was assessed from I_A amplitude at test voltages between -60 and +60 mV; steady-state inactivation was determined from I_A amplitude at -40 mV (at which I_A will be the only time-dependent current active) after a 250 ms test hyperpolarizations to voltages between -160 and -40 mV. Fits of normalized current amplitudes with Boltzmann relationships indicated a trend towards a negative shift in activation voltage (one-half activation voltage, V_1/2, of about -19 mV for control and about -23 mV for vitronectin-exposed neurones; P = 0.55, n = 6 for control and 9 for +vitronectin) that could contribute to the increase in I_A amplitude, particularly at voltages near V_1/2. In contrast, the voltage dependence of inactivation, and thus I_A availability, was minimally altered (V_1/2 of about -86 mV for control and about -83 mV for vitronectin-exposed neurones; P > 0.05).

Finally, there was no apparent change in the rate of recovery from inactivation after vitronectin exposure (Fig. 5C). While we did not examine early time points in detail, clearly the time for one-half recovery was less than 150 ms, and there was no indication of a slow (seconds-long) component. These results are consistent with a dominant contribution of Kv4-family channels to these somatic recordings (see Discussion), and suggest that the axonal compartment containing Kv1.4 channels (detected immunochemically; see below) was not electrically accessible.

Expression of potassium channel subunits Kv4.2 and Kv1.4

Kv4.2 and Kv1.4 are part of a larger group of subunits potentially contributing to I_A in developing hippocampal neurones that includes, in addition, Kv3.4, 4.1, Kv4.3 as well as cognate accessory $beta$ subunits. We focussed on Kv4.2 and Kv1.4 because the results of other studies have suggested that channels incorporating Kv4.2 are major carriers of I_A in the somatic-dendritic compartment (see Discussion). We excluded Kv3.4 because it appears to make its most significant contribution to the transient potassium current in the granule cells of the dentate gyrus (Rudy et al. 1999; Riazanski et al. 2001), and Kv4.1 because it is not highly expressed in pyramidal neurones (Martina et al. 1998; Serôdio & Rudy, 1998).

Subunit-selective antibodies were used to examine the magnitude and distribution Kv4.2 and Kv1.4 immunoreactivity (Fig. 6). Even at this relatively immature state (E15.5 + 3 or 4 days in culture), Kv4.2 immunoreactivity appeared to be restricted to rounded cell bodies (Fig. 6a and a') while Kv1.4 immunoreactivity was found in long thin structures, presumably axons, especially prominent in the outgrowth zone emerging from the explant edge (Fig. 6d and d').

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Figure 6. Distribution of Kv4.2 and Kv1.4 potassium channel subunits and changes induced by exposure to vitronectin
Above, Kv4.2 immunoreactivity in control (a) and vitronectin-exposed (b) explants. Immunofluorescence appeared restricted to cell bodies (a', b'). Cumulative probability analyses of relative luminance (c; luminance in each pixel normalized to the mean luminance of the control explant; see Methods) illustrates the generalized vitronectin-induced increase in luminance evident in comparison of a and b; P < 0.0001 (log rank test). Number of experiments: 3 explant pairs (control/experimental). Below, Kv1.4 immunoreactivity in control (d) and vitronectin-exposed (e) explants. Immunofluorescence appeared limited to thin processes, particularly at the edges of the explants (d'and e'). A cumulative probability analysis of relative luminance (f) illustrates the relatively smaller but significant increase in luminance observed; P < 0.01. Number of experiments: 3 explant pairs (control/experimental).

We assessed whether exposure to vitronectin was affecting Kv4.2 and/or Kv1.4 subunit expression using immunofluorescence images. Visual comparisons of Kv4.2 and Kv1.4 immunoreactivity between control and vitronectin-exposed explants (Fig. 6a', b', d' and e') did not indicate major subcellular redistributions of subunit proteins. At the same time, an increase in Kv4.2 immunoreactivity was evident to the eye (Fig. 6a and b), while Kv1.4 immunoreactivity appeared relatively unchanged (Fig. 6d and e). We performed a semi-quantitative analysis by normalizing luminance values of individual pixels in each control/experimental explant pair to the mean luminance of the control explant (see Methods). Evaluated in this way, increases in mean relative luminance were 31 ± 20 % (mean ± S.D.; P = 0.051; n = 3) for Kv4.2 and 26 ± 23 % for Kv1.4 (P > 0.05; n = 3), results suggesting a likely preferential action of vitronectin on Kv4.2 expression. Visualized more graphically (Fig. 6c and f), cumulative probability histograms of relative luminance show a clear and highly significant (P > 0.001) shift towards greater luminance for Kv4.2 immunoreactivity, while Kv1.4 immunoreactivity shows a smaller but consistent trend (also statistically significant; P > 0.01).

Vitronectin immunoreactivity in the late embryonic hippocampus

Figure 7 illustrates vitronectin immunoreactivity in a cryostat section of an acutely isolated E14.5 hippocampus. Vitronectin appears to surround the cells of the ventricular and subventricular zone (upper inset) and the nascent hippocampus (lower inset), and is thus positioned to potentially influence hippocampal differentiation, as it does elsewhere in the CNS (see Discussion).

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Figure 7. Presence of vitronectin in the E14.5 hippocampus
Confocal immunofluorescence image (see Methods) illustrating vitronectin immunoreactivity surrounding cells in the ventricular and subventricular zones (detail in upper left) and developing cell body layer of the hippocampus (detail in lower right). The dotted line (right) marks the point where the hippocampal region was separated from the extrahippocampal cortex to be placed into explant culture. In control images, only a very faint signal was detected when the primary antibody (1 ° ab) was omitted (lower left). Furthermore, a minimal signal was detected when the primary antibody was preabsorbed with normal mouse serum (which contains vitronectin at a titre of hundreds of micrograms per millilitre: Hayman et al. 1983; data not shown).

Note the location of the cortical hem (Grove et al. 1998) close to the eventual position of the fimbria and opposite the extrahippocampal cortex, landmarks used for isolation of the hippocampus and orientation of the cultured explants.

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

Vitronectin is a component of the extracellular matrix that can be detected by immunofluorescence in the embryonic hippocampus. Our data indicate that vitronectin can enhance I_A amplitude in developing hippocampal neurones, probably through combination with appropriate integrins. This regulation appears to be selective for I_A, in that other outward potassium currents were affected only to the extent that somatic and proximal dendritic membrane area also increased.

Potassium channel subunits underlying I_A

Vitronectin exhibited striking specificity in its effects on particular potassium channel subunits. Immunochemically, significant vitronectin-induced increases in mean relative pixel luminance, and in overall luminance across entire explants, were observed for Kv4.2. At the same time, increases in Kv1.4 immunoreactivity were consistent but smaller; these could reflect a growth response induced by vitronectin (evident in the whole-cell capacitance increases noted). Electrophysiological evidence also indicated that virtually all I_A observed was carried by Kv4-family channels, and that this was not altered by vitronectin exposure. Homomeric Kv4-family, but not Kv1-family, channels are characterized by rapid recovery from inactivation (Ruppersberg et al. 1990; Petersen & Nerbonne, 1999), and under both control and experimental conditions, transient current exhibited almost 80 % recovery from inactivation after 150 ms. Changes in I_A occurred without alteration of the normal subcellular segregation of Kv4-family (i.e. Kv4.2 and Kv4.3) subunits into somatodendritic and Kv1-family (i.e. Kv1.4) subunits into axonal compartments (Sheng et al. 1992; Maletic-Savatic et al. 1995; Serôdio et al. 1996; Debanne et al. 1997; Johns et al. 1997; Keros & McBain, 1997; Martina et al. 1998; Serôdio & Rudy, 1998; R.-L. Wu et al. 1998; Geiger & Jonas, 2000). Note that within the context of Kv4-family subunits, an additional contribution of Kv4.3 cannot be excluded since these subunits are expressed in hippocampal pyramidal neurones (Martina et al. 1998; Serôdio & Rudy, 1998) and their currents cannot easily be separated electrophysiologically (Coetzee et al. 1999).

Vitronectin did not appear to effect functional expression of the currents considered collectively as delayed currents. These, I_D and I_K, most likely reflect the activity of Kv1.2 (Coetzee et al. 1999; Bekkers & Delaney, 2001) and Kv2.1/Kv2.2 (Martina et al. 1998; Murakoshi & Trimmer, 1999; Du et al. 2000) subunits, respectively.

Vitronectin-linked signalling pathways

Sensitivity to RGD-sequence-containing peptide suggests that vitronectin affects I_A by combining with one or more integrins via its RGD domain (Schvartz et al. 1999), but precisely which integrins is difficult to infer because vitronectin combines with multiple integrins. The most common vitronectin-responsive subunits include $alpha$ v $beta$ 1, $alpha$ v $beta$ 3 and $alpha$ v $beta$ 5 (Felding-Habermann & Cheresh, 1993), subunits for which mRNA is found in the mature brain (Pinkstaff, 1999). However, because fibronectin shares with vitronectin activity on integrins $alpha$ v $beta$ 1 and $alpha$ v $beta$ 3 (Felding-Habermann & Cheresh, 1993) but fails to influence potassium currents, it is possible that $alpha$ v $beta$ 5 or other less common vitronectin-responsive integrins (Nishimura et al. 1998; Gladson et al. 2000) could link vitronectin with I_A.

Under what circumstances might regulation of I_A by vitronectin occur?

Vitronectin might regulate I_A during development, since we detected vitronectin in the embryonic hippocampus and, as mentioned above, vitronectin is an active regulator of neuronal differentiation in other CNS contexts. Curiously, however, RGD peptide alone did not affect I_A development during the 3 day test interval (not shown), despite its efficacy against exogenous vitronectin during this same period, an observation reminiscent of the apparent absence of overt phenotype in vitronectin-deficient mice (Zheng et al. 1995). This paradox may reflect the persistence of previously activated promoters of I_A expression not necessarily involving integrins. Other possibilities lie in not well understood aspects of vitronectin -integrin signalling, such as compartmentalization, immobilization or conformational specificity that limits exposure of vitronectin's integrin (RGD-sequence-containing) binding domain (Seiffert & Smith, 1997; Minor & Peterson, 2002).

In other contexts, because vitronectin expression is generally upregulated at sites of tissue inflammation and injury (Seiffert, 1997), modulation of apparently developmental processes may foreshadow future physiological reactions (Aihara & Barish, 2001, 2002). Thus exposure of susceptible naïve tissues to vitronectin could elicit regulatory responses having deleterious consequences. A dramatic example, found outside the CNS, is control of embryonic smooth muscle phenotype (Dahm & Bowers, 1998), in which exposure of contractile muscle cells to vitronectin elicits their transformation into proliferating fibroblast-like cells through an $alpha$ v $beta$ 1-mediated mechanism.

Regulation of I_A by vitronectin is one example of an increasingly recognized association of matrix- and integrin-based signalling with excitability (see Mistry et al. 2002) and/or intracellular Ca²⁺ levels (Gomez et al. 2001; Wildering et al. 2002). Significantly, many of these links are between particular integrins and/or ligands, and specific ion channels and aspects of cell physiology. These include membrane hyperpolarization and activation of inward-rectifier potassium current in neuroblastoma cells (Arcangeli et al. 1993, 1996), vasoconstriction or dilation in vascular smooth muscle cells through modulation of voltage-gated calcium (X. Wu et al. 1998; Waitkus-Edwards et al. 2002) or potassium (Platts et al. 1998) channels (see Hool, 2002), enhancement of the delayed-rectifier potassium current in murine erythroleukaemia cells (Becchetti et al. 1992), and physical and functional associations with heterologously expressed inward-rectifier potassium channels (McPhee et al. 1998) or with delayed-rectifier potassium channels of T cells and melanoma cells (Levite et al. 2000; Artym & Petty, 2002).

Complexity of I_A regulation by integrins

Additional observations suggest that there are probably other presently unidentified integrin ligands in serum that regulate I_A. We have noted that when these organotypic hippocampal cultures are grown in serum-containing media, exposure to RGD-sequence-containing peptide, or substitution of serum-free medium, results in an increase in I_A amplitude (D. Vasilyev & M. E. Barish, unpublished observations), contrary to what might be expected if vitronectin uniquely upregulated I_A. Thus while vitronectin enhances I_A, other integrin ligands in serum appear to limit the functional expression of I_A.

REFERENCES

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Abstract
Introduction
Methods
Results
Discussion
References

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Acknowledgements

We thank Drs David Loskutoff and Jeanne Nerbonne for generous gifts of antibodies, and Ms Jill Brantley for assistance with the manuscript. This work was supported by grants from the March of Dimes (1FY00328) and the National Institutes of Health (R01NS23857).

Author's present address

D. V. Vasilyev: Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA.

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Aihara Y , & Barish ME (2001) Increase in vitronectin (an integrin ligand). during organotypic slice culture of postnatal mouse hippocampus: a model CNS injury response? Soc Neurosci Abs 27, program 213
Akiyama H, Kawamata T, Dedhar S & McGeer PL (1991). Immunohistochemical localization of vitronectin, its receptor and $beta$ 3 integrin in Alzheimer brain tissue. J Neuroimmunol 32, 19-28	[Medline]
Angevine JB Jr, (1975). Development of the hippocampal region. In The Hippocampus, vol. 1, Structure and Development, ed. Isaacson RL & Pribram KH, pp. 61-94. Plenum, New York
Arcangeli A, Becchetti A, Mannini A, Mugnai G, De Filippi P, Tarone G, Del Bene MR, Barletta E, Wanke E & Olivotto M (1993). Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels. J Cell Biol 122, 1131-1143	[Abstract]
Arcangeli A, Faravelli L, Bianchi L, Rosati B, Gritti A, Vescovi A, Wanke E & Olivotto M (1996). Soluble or bound laminin elicit in human neuroblastoma cells short- or long-term potentiation of a K⁺ inwardly rectifying current: relevance to neuritogenesis. Cell Adhes Commun 4, 369-385	[Medline]
Archelos JJ, Previtali SC & Hartung HP (1999). The role of integrins in immune-mediated diseases of the nervous system. Trends Neurosci 22, 30-38	[Medline]
Artym VV , & Petty HR (2002). Molecular proximity of Kv1. 3 voltage-gated potassium channels and $beta$ ₁-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers. J Gen Physiol 120, 29-38	[Abstract/Full Text]
Barish ME, (1995). Modulation of the electrical differentiation of neurons by interactions with glia and other non-neuronal cells. Perspect Dev Neurobiol 2, 357-370	[Medline]
Barry DM, Trimmer JS, Merlie JP & Nerbonne JM (1995). Differential expression of voltage-gated K⁺ channel subunits in adult rat heart. Circ Res 77, 361-369	[Abstract/Full Text]
Becchetti A, Arcangeli A, Del Bene MR, Olivotto M & Wanke E (1992). Response to fibronectin-integrin interaction in leukaemia cells: delayed enhancing of a K⁺ current. Proc R Soc Lond B Bio Sci 248, 235-240
Bekkers JM , & Delaney AJ (2001). Modulation of excitability by $alpha$ -dendrotoxin-sensitive potassium channels in neocortical pyramidal neurons. J Neurosci 21, 6553-6560	[Abstract/Full Text]
Benson DL, Schnapp LM, Shapiro L & Huntley GW (2000). Making memories stick: cell-adhesion molecules in synaptic plasticity. Trends Cell Biol 10, 473-482	[Medline]
Brown TH , & Johnston D (1983). Voltage-clamp analysis of mossy fiber synaptic input to hippocampal neurons. J Neurophysiol 50, 487-507	[Medline]
Chavis P , & Westbrook G (2001). Integrins mediate functional pre- and postsynaptic maturation at a hippocampal synapse. Nature 411, 317-321	[Medline]
Chen BM , & Grinnell AD (1995). Integrins and modulation of transmitter release from motor nerve terminals by stretch. Science 269, 1578-1580
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