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REPORT

BDNF Induces Calcium Elevations Associated With I_BDNF, a Nonselective Cationic Current Mediated by TRPC Channels

Department of Neurobiology, Civitan International Research Center and McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 16 July 2007; accepted in final form 9 August 2007

	ABSTRACT

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Brain-derived neurotrophic factor (BDNF) has potent actions on hippocampal neurons, but the mechanisms that initiate its effects are poorly understood. We report here that localized BDNF application to apical dendrites of CA1 pyramidal neurons evoked transient elevations in intracellular Ca²⁺ concentration, which are independent of membrane depolarization and activation of N-methyl-D-aspartate receptors (NMDAR). These Ca²⁺ signals were always associated with I_BDNF, a slow and sustained nonselective cationic current mediated by transient receptor potential canonical (TRPC3) channels. BDNF-induced Ca²⁺ elevations required functional Trk and inositol-tris-phosphate (IP₃) receptors, full intracellular Ca²⁺ stores as well as extracellular Ca²⁺, suggesting the involvement of TRPC channels. Indeed, the TRPC channel inhibitor SKF-96365 prevented BDNF-induced Ca²⁺ elevations and the associated I_BDNF. Thus TRPC channels emerge as novel mediators of BDNF-induced intracellular Ca²⁺ elevations associated with sustained cationic membrane currents in hippocampal pyramidal neurons.

	INTRODUCTION

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Brain-derived neurotrophic factor (BDNF) is a potent modulator of neuronal structure and function (Amaral et al. 2007; Bramham and Messaoudi 2005; Lu 2003; Poo 2001; Tyler et al. 2002). Because Ca²⁺ plays a critical role in these fundamental processes, it is significant that BDNF modulates intracellular Ca²⁺ levels. One of the signaling cascades activated by neurotrophin Trk receptors, the hydrolysis of phosphatidylinositol bisphosphate (PIP₂) by phospholipase C gamma (PLC) leading to IP₃ formation, causes intracellular Ca²⁺ mobilization (Segal and Greenberg 1996). However, direct evidence of such neurotrophin-initiated Ca²⁺ signals is sparse, controversial, and mostly limited to embryonic cultured neurons. BDNF increased Ca²⁺ levels in cultured hippocampal (Berninger et al. 1993; Canossa et al. 1997; Finkbeiner et al. 1997; Li et al. 1998; Marsh and Palfrey 1996) and cortical neurons (Behar et al. 1997; Matsumoto et al. 2001; Mizoguchi and Nabekura 2003; Mizoguchi et al. 2002; Yang and Gu 2005; Zirrgiebel et al. 1995). In contrast, BDNF failed to affect Ca²⁺ levels in cultured cerebellar granule cells (Gaiddon et al. 1996; but see Jia et al. 2007; Numakawa et al. 2001) and in acute slices from visual cortex (Pizzorusso et al. 2000). BDNF also potentiated spontaneous Ca²⁺ oscillations in cultured hippocampal neurons (Numakawa et al. 2002; Sakai et al. 1997); however, this effect was due to enhanced network activity leading to voltage-dependent Ca²⁺ influx (Sakai et al. 1997). Also, BDNF increased Ca²⁺ levels within presynaptic terminals of cultured Xenopus neuromuscular junctions (Boulanger and Poo 1999; Stoop and Poo 1996). Unfortunately, nearly all published Ca²⁺ imaging studies of BDNF actions on intracellular Ca²⁺ levels were done without simultaneous membrane voltage control, making it difficult to differentiate the contribution of voltage-gated and receptor-operated Ca²⁺ influx to the observed Ca²⁺ signals. In fact, most studies to date conclude that a significant fraction of the BDNF-induced Ca²⁺ elevations is sensitive to glutamate receptor antagonists (e.g., Yang and Gu 2005). It should be noted that dendritic and spine Ca²⁺ elevations induced by BDNF in hippocampal dentate granule cells were sensitive to voltage-gated Ca²⁺ channel blockers (Kovalchuk et al. 2002) and always associated with the membrane depolarization proposed to be mediated by Na_v1.9 channels (Blum et al. 2002; Kafitz et al. 1999).

The controversial state of our understanding of BDNF actions on intracellular Ca²⁺ levels prompted us to perform simultaneous whole cell recording and microfluorometric imaging in voltage-clamped neurons. We present evidence that localized BDNF application to apical dendrites of CA1 pyramidal neurons in hippocampal slice cultures evoked transient elevations in intracellular Ca²⁺ concentration, which are independent of voltage-gated Ca²⁺ channels and N-methyl-D-aspartate (NMDA) receptors. These Ca²⁺ signals were always associated with I_BDNF, a slow and sustained nonselective cationic current mediated by TRPC3 channels (Amaral and Pozzo-Miller 2007; Li et al. 1999). BDNF-induced Ca²⁺ elevations required functional Trk and IP₃ receptors, full intracellular Ca²⁺ stores, as well as extracellular Ca²⁺, suggesting the involvement of TRPC channels. Indeed, the TRPC channel inhibitor SKF-96365 prevented BDNF-induced Ca²⁺ elevations and the associated I_BDNF. Thus TRPC channels emerge as novel mediators of BDNF-induced intracellular Ca²⁺ elevations in hippocampal pyramidal neurons.

	METHODS

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Organotypic slice culture

All procedures performed on experimental animals adhered to national and international guidelines for the ethical use of research animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham. Briefly, hippocampi were dissected from anesthetized postnatal day 7–11 Sprague Dawley rats (Harlan, Indianapolis, IN, or Charles River, Wilmington, MA) and cut transversely into 400-µm-thick slices using a custom-made wire-slicer fitted with 20-µm-thick gold-coated platinum wire (Pozzo-Miller et al. 1995). Hippocampal slices were individually plated on Millicell-CM filter inserts (Millipore; Billerica, MA) and cultured in 36°C, 5% CO₂, 98% relative humidity incubators (Thermo-Forma; Waltham, MA). Slices were maintained in culture media (Neurobasal-A plus B27, InVitrogen; Carlsbad, CA) containing 20% equine serum for the first 4 days in vitro (div). To avoid the confounding effects of hormones and growth factors in the serum, its concentration was gradually reduced over a period of 48 h starting at 4 div (24 h each in 10% and 5% serum), as described (Tyler and Pozzo-Miller 2001). After a period of 24 h in serum-free media (Neurobasal-A plus B27), 7–10 div slices were used for simultaneous electrophysiology and Ca²⁺ imaging.

Simultaneous electrophysiology and Ca²⁺ imaging

Individual 7–10 div slices were transferred to a recording chamber mounted on a fixed-stage upright microscope (Zeiss Axioskop FS; Oberkochen, Germany) and continuously perfused (2 ml/min) with artificial cerebrospinal fluid (ACSF) at room temperature (24°C), containing (in mM) 124 NaCl, 2 KCl, 1.24 KH₂PO₄, 1.3 MgSO₄, 17.6 NaHCO₃, 2.5 CaCl₂, 10 glucose, and 29.2 sucrose (310–320 mosM); ACSF was bubbled with 95% O₂-5% CO₂ (pH 7.4). Superficial CA1 pyramidal neurons were visualized with a water-immersion 40x objective (0.9 NA) using IR-DIC microscopy. Simultaneous whole cell recording and microfluorometric Ca²⁺ imaging was performed as described (McCutchen et al. 2002; Petrozzino et al. 1995; Pozzo-Miller 2006; Pozzo-Miller et al. 1996, 1999). Briefly, unpolished patch pipettes contained (in mM) 120 Cs-gluconate, 17.5 CsCl, 10 Na-HEPES, 4 Mg-ATP, 0.4 Na-GTP, 10 Na₂ creatine phosphate, and 0.2 mM fura-2 (or 0.5 mM bis-fura-2); 280–290 mosM; pH 7.2 (resistance 3–4 M). Nominally calcium-free extracellular ACSF was prepared by replacing CaCl₂ with an equimolar concentration of MgCl₂. Some drugs were dissolved in DMSO (0.01%) and others directly into the ACSF or intracellular solution; vehicle controls using 0.01% DMSO were routinely performed yielding no effects on membrane currents or BDNF-induced responses. Membrane currents were recorded in the voltage-clamp mode at a holding potential of –65 mV using an Axoclamp 200B amplifier (Molecular Devices; Sunnyvale, CA), filtered at 2 kHz, and digitized at 10 kHz. Recordings were accepted only if access (series) resistance was 30 M. CA1 neurons had whole cell capacitances of 100 pF. Input resistance (R_i) was measured with hyperpolarizing voltage pulses (50 ms, –20 mV), and cells were discarded if any of those cell parameters (C_m, R_i, R_s) changed by 20% during the course of an experiment. All experiments were performed in the presence of TTX (0.5 µM) to block voltage-gated Na⁺ channels. As noted, some experiments included Cd²⁺ (200 µM) and D,L-2-amino-5-phosphonovaleric acid (D,L-APV, 50 µM) to block voltage-gated Ca²⁺ channels and NMDA receptors, respectively.

Fura-2 or bis-fura-2 (Molecular Probes; Carlsbad, CA) were alternatively excited at 360 and 380 nm using a monochromator (Polychrome-II, TILL Photonics; Munich, Germany), and its emission (>510 nm) filtered and detected with a frame-transfer cooled CCD camera (PXL-37, Roper Scientific; Duluth, GA); digital image pairs were acquired every 4 s (50-ms exposures for 100 x 200 pixel sub-arrays, 1 x 1 binning). Background-subtracted fluorescence intensity measurements were obtained within regions of interest (ROIs) defined over apical primary and secondary dendrites, as well as over neuronal cell bodies. The average ratio of 360 and 380 nm fluorescence within each ROI was used as an estimate of intracellular Ca²⁺ concentration as these two parameters are directly proportional to each other (Grynkiewicz et al. 1985). Electrical and optical data were simultaneously acquired on a single G4 Macintosh computer (Apple; Cupertino, CA) running custom-written software (TIWorkBench, kindly provided Dr. T. Inoue, Waseda University, Japan). All of the chemicals used for these experiments were obtained from Sigma (St. Louis, MO), Calbiochem (San Diego, CA), or Tocris (Ellisville, MO).

Recombinant human mature BDNF (supplied by Amgen; Thousand Oaks, CA) was pressure-applied from glass pipettes (5 M) using a Picospritzer-III (Parker Hannifin; Cleveland, OH). An application pipette was positioned 100 µm above the slice and 200 µm away from the soma of the CA1 neuron under recording, aimed at its apical dendrites within stratum radiatum (150 µm from the soma) and against the direction of ACSF perfusion flow. This arrangement produced a stream of BDNF solution that overshoots the cell under recording and flows back over the slice, already diluted in the ACSF. Application of glutamate (100 µM, 8 psi, 9 s) from similar pipettes was used to optimize this arrangement, yielding highly reproducible and stable transient membrane currents. In addition, food coloring was used to assess the spatial spreading of the applied solution over the slice. Pressure pulses of 30 psi lasting 25–30 s delivered a total volume of 2 µl of solution from 5 M glass pipettes. In most experiments, the pipette contained 100 µg/ml BDNF in 0.0001–0.1% bovine serum albumin (BSA). BDNF denatured by boiling (10 min; 100 µg/ml), BSA alone (0.0001 or 0.1%), and ACSF were used as pressure application controls, which yielded neither changes in membrane currents (Amaral and Pozzo-Miller 2007) nor intracellular Ca²⁺ levels.

Statistical analyses

All data are presented as means ± SE; the SD of the mean was used to calculate the coefficient of variance (CV = mean/SD), which is provided as a measure of consistency. All data were statistically analyzed using unpaired Student's t-test or ANOVA tests with the Prism software package (GraphPad Software; San Diego, CA). Probability values lower than 0.05 were considered statistically significant (i.e., P < 0.05, <5% probability that the observations are due to chance). When lower than this cut-off value, the actual P values 4 decimal points are given in RESULTS (rather than just the statement "greater than" or "less than") to provide readers with more detailed information regarding the outcome of the statistical analyses. Compromise power analyses were performed to determine the statistical power given the number of observations, sample means and SDs, using G*Power (Erdfelder et al. 1996). These power analyses yielded values of statistical Power (1-; where is the Type-II error) larger than 0.95 (i.e., 95% confidence of accepting the null hypothesis when is true).

The application of BDNF to CA1 pyramidal neurons evoked transient fura-2 ratio elevations that preceded I_BDNF with a mean amplitude of 1.26 ± 0.14 (n = 3; CV = 0.19); this value was five to six times the SD of the pre-BDNF baseline fura-2 ratio (0.89 ± 0.03, n = 7, P = 0.0043 vs. peak amplitude; CV = 0.08). The peak of the sustained fura-2 ratio elevations that were simultaneous with I_BDNF reached a mean amplitude of 1.71 ± 0.09 (n = 7 of 7 cells; CV = 0.14), which was 17 times the SD of the pre-BDNF baseline fura-2 ratio (0.89 ± 0.03, n = 7, P < 0.0001 vs. peak amplitude; CV = 0.08). Therefore the fura-2 responses to BDNF applications were always above "noise" fluctuations in fura-2 ratio values.

	RESULTS

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In the present experiments, we chose to locally apply BDNF from pipettes placed 100 µm above hippocampal slice cultures to avoid pressure and mechanical artifacts, and over s. radiatum dendrites 200 µm away from CA1 neuron cell bodies to reproduce the release profile of a paracrine neuropeptide (Lessmann et al. 2003). Under these conditions, a single 25- to 30-s application of BDNF activated I_BDNF, a delayed and slowly developing nonselective cationic conductance described originally in acutely dissociated pontine neurons (Li et al. 1999). I_BDNF was observed in every hippocampal CA1 pyramidal neuron from which we recorded (Amaral and Pozzo-Miller 2007), having a mean amplitude of 577.23 ± 41.07 pA (n = 24). Considering that I_BDNF was sensitive to the Ca²⁺ chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (Amaral and Pozzo-Miller 2007), TRPC channel activity is enhanced by intracellular Ca²⁺ elevations (Clapham 2003), and plasma membrane TRPC channels mediating I_BDNF are permeable to Ca²⁺ ions (Li et al. 1999), we set out to perform simultaneous whole cell recording and microfluorometric imaging in voltage-clamped CA1 pyramidal neurons filled with Ca²⁺ indicators (200 µM fura-2 or 500 µM bis-fura-2 in the patch pipette).

Within 100 s of its application, BDNF evoked transient elevations in the 360/380 nm ratio in the targeted apical dendrites that preceded the onset of I_BDNF in three of seven cells tested in the presence of TTX (0.5 µM), despite the fact that every cell expressed I_BDNF (Amaral and Pozzo-Miller 2007). These initial fura-2 ratio elevations were restricted to apical dendrites 100 µm from the cell body and had peak amplitudes of 1.26 ± 0.14 (n = 3; CV = 0.19). To rule out the potential contribution of voltage-gated Ca²⁺ channels and NMDA receptors (NMDAR) to BDNF-induced Ca²⁺ elevations, Cd²⁺ (200 µM) and D,L-APV (50 µM) were included in an additional set of experiments. In two of four cells, BDNF evoked transient fura-2 ratio increases that preceded I_BDNF in the absence of Ca²⁺ channel and NMDAR activity (peak amplitudes 1.05–1.6; Fig. 1 A, expanded in B). The effects of BDNF were confirmed to be specific, as vehicle alone (0.1% BSA) or BDNF denatured by boiling were entirely ineffective (Amaral and Pozzo-Miller 2007).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Brain-derived neurotrophic factor (BDNF) induces intracellular Ca2+ elevations that precede and are simultaneous with IBDNF activation. A: representative example of BDNF-evoked Ca2+ signals in the presence of TTX, Cd2+ and D,L-2-amino-5-phosphonovaleric acid (D,L-APV). These Ca2+ elevations included a transient and localized dendritic signal during BDNF application () and a secondary widespread elevation associated with IBDNF. The initial Ca2+ transient (*) is shown in B at an expanded time scale. B: expanded view of the initial Ca2+ elevation that preceded IBDNF. Here and in the other figures, the fluorescence images (360 nm excitation) show the color-coded regions of interest (ROIs) plotted with the simultaneously recorded membrane currents.

View larger version (29K): [in this window] [in a new window]   FIG. 2. BDNF-induced Ca2+ signals require functional Trk and IP3 receptors. A: BDNF-evoked Ca2+ signals and IBDNF are sensitive to the Trk inhibitor k-252a (200 nM). B: The IP3R inhibitor xestospongin-C (1 µM intracellular) blocked BDNF-evoked Ca2+ signals and IBDNF.

View larger version (34K): [in this window] [in a new window]   FIG. 3. BDNF-induced Ca2+ signals require full intracellular Ca2+ stores, Ca2+ influx from the extracellular space, and activation of TRPC channels. A: depletion of intracellular Ca2+ stores with thapsigargin (1 µM) abolished BDNF Ca2+ responses and IBDNF. B: applying BDNF in the absence of extracellular Ca2+ failed to evoke Ca2+ elevations and significantly reduced IBDNF. C: The TRPC inhibitor SKF-96365 (30 µM) prevented Ca2+ signals and IBDNF.

	DISCUSSION

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Here we present novel insights into the immediate actions of BDNF on hippocampal neurons. First, BDNF caused intracellular Ca²⁺ elevations in CA1 pyramidal neurons under voltage-clamp and in the absence of voltage-gated Na⁺ and Ca²⁺ channels as well as NMDA receptor activation. Second, these Ca²⁺ signals were always associated with I_BDNF, a sustained nonselective cationic current mediated by TRPC3 channels (Amaral and Pozzo-Miller 2007; Li et al. 1999). Third, BDNF-induced Ca²⁺ elevations required IP₃ receptors, full intracellular Ca²⁺ stores and extracellular Ca²⁺. Consistent with this last observation and the role of TRPC channels in BDNF-induced membrane currents, Ca²⁺ signals evoked by BDNF were sensitive to a TRPC/SOC inhibitor. We propose that BDNF binding to the TrkB receptor activates the PLC pathway. PLC then hydrolyzes PIP₂ to IP₃; IP₃ binds to its receptor (IP₃R) on the smooth endoplasmic reticulum (SER) and causes Ca²⁺ to be released. TRPC3 channels are then activated and mediate Ca²⁺ entry into the neuron.

It has been known for a while that BDNF elicits somatic Ca²⁺ elevations in cultured hippocampal neurons (Berninger et al. 1993), but the mechanism(s) underlying these responses has remained elusive (Amaral and Pozzo-Miller 2005; Amaral et al. 2007; McCutchen et al. 2002). BDNF-induced somatic Ca²⁺ elevations in cultured neurons were reduced—but not completely blocked—in the absence of extracellular Ca²⁺ (Finkbeiner et al. 1997; Li et al. 1998), suggesting that both Ca²⁺ influx and mobilization from intracellular stores contribute to the responses. Some features of these Ca²⁺ signals resemble capacitative Ca²⁺ entry (Putney 2003), a mechanism postulated to be mediated by some members of the TRPC channel subfamily (Birnbaumer et al. 1996; Mikoshiba 1997; Montell et al. 2002; but see Clapham 2003). Indeed TRPC3/6 channels mediate BDNF-evoked Ca²⁺ signals in growth cones (Li et al. 2005) and somata (Jia et al. 2007) of cultured cerebellar granule cells, whereas xTRPC1, a Xenopus homologue of TRPC1, plays a similar role in BDNF-induced growth cone turning in vitro (Wang and Poo 2005). Consistently, the TRPC/SOC inhibitor SKF-96365 completely prevented BDNF-induced Ca²⁺ responses and I_BDNF. It was originally reported that SKF-96365 also inhibited voltage-gated Ca²⁺ channels in GH3 pituitary cells and rabbit ear-artery smooth muscle cells (Merritt et al. 1990); however, a broad-spectrum Ca²⁺ channel blocker (i.e., 200 µM Cd²⁺) did not affect I_BDNF or BDNF-induced Ca²⁺ signals in CA1 pyramidal neurons in our experiments. Furthermore, siRNA-mediated TRPC3 knockdown, or intracellular application of anti-TRPC3 antibodies—but not anti-TRPC5—prevented the activation of I_BDNF in CA1 neurons (Amaral and Pozzo-Miller 2007). Thus our results suggest that ion channels containing at least TRPC3 subunits mediate I_BDNF and its associated Ca²⁺ elevations.

It is worth noting that dendritic and spine Ca²⁺ elevations induced by BDNF in hippocampal dentate granule cells were sensitive to voltage-gated Ca²⁺ channel blockers (Kovalchuk et al. 2002) and always associated with fast and brief membrane depolarizations proposed to be mediated by Na_v1.9 channels (Blum et al. 2002; Kafitz et al. 1999). In addition, I_BDNF in pontine (Li et al. 1999) and CA1 pyramidal neurons (Amaral and Pozzo-Miller 2007) is markedly different from those faster and transient TTX-insensitive Na⁺ current activated by TrkB ligands in several regions of the brain (Kafitz et al. 1999). Furthermore, fast BDNF-activated Na⁺ currents were blocked by the Na⁺ channel blocker saxitoxin (Blum et al. 2002), whereas I_BDNF in CA1 pyramidal neurons is not (Amaral and Pozzo-Miller 2007). It has been recently reported that brief and focal BDNF applications elicited fast and local Ca²⁺ elevations near synaptic sites on apical dendrites of immature CA3 pyramidal neurons (slice cultures prepared from P0 to P2 rats and cultured for 3 div), which required the activation of voltage-gated Ca²⁺ and Na⁺ channels (Lang et al. 2007). In contrast with the observations presented here regarding more developed CA1 neurons (slice cultures prepared from P7 to P11 rats and cultured for 7–11 div), fast and local Ca²⁺ elevations evoked by BDNF in immature CA3 neurons were unaffected by store depletion with CPA or inhibition of SOC/TRPC channels with SKF-96365 (Lang et al. 2007). It remains to be tested whether BDNF-induced Ca²⁺ elevations in immature CA3 neurons are a secondary response to the membrane depolarization caused by activation of Na_v1.9 channels (Blum et al. 2002; Kafitz et al. 1999) as shown in dentate granule neurons (Kovalchuk et al. 2002).

Is there a role for these BDNF-induced membrane currents and Ca²⁺ elevations? Because BDNF increases spine density in CA1 neurons (Alonso et al. 2004; Tyler and Pozzo-Miller 2001, 2003), it is tempting to speculate that sustained intracellular Ca²⁺ elevations such as those induced by brief BDNF applications and mediated by TRPC channels trigger the cytoskeletal rearrangements necessary for dendritic spine remodeling and/or formation. Indeed, siRNA-mediated TRPC3 knockdown as well as TRPC inhibitors prevented the increase in spine density by BDNF (Amaral and Pozzo-Miller 2007). Together with the observations that Ca²⁺ mobilization from intracellular stores (Korkotian and Segal 1999) and activation of group-I mGluRs (Vanderklish and Edelman 2002) also induce changes in spine form and promote spine formation, our results suggest a convergence of mGluR and TrkB signaling pathways on TRPC channels to engage a program of spine formation and/or maturation via Ca²⁺-dependent, actin-based structural remodeling. In this view, TRPC channels emerge as novel effectors of BDNF-mediated dendritic remodeling through the activation of a sustained depolarization associated with intracellular Ca²⁺ elevations.

	GRANTS

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This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1-NS-40593 to L. Pozzo-Miller and by the Civitan International Foundation. L. Pozzo-Miller is a McNulty Civitan Scientist.

	ACKNOWLEDGMENTS

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We are indebted to Dr. Takafumi Inoue (Waseda University) for continuous support of the acquisition and analysis software (TIWorkBench). We thank AMGEN for the supply of BDNF. We also thank the technical assistance of the UAB Neuroscience Cores (P30-NS-47466, P30-HD-38985, P30-NS-57098).

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Pozzo-Miller, Dept. of Neurobiology, SHEL-1002, University of Alabama at Birmingham, 1825 University Blvd., Birmingham, AL 35294-2182 (E-mail: lucaspm{at}uab.edu)

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