WWW.JNEUROSCI.ORG
-
The Journal of Neuroscience
QUICK SEARCH:   [advanced]


     
-


HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (130)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Balkowiec, A.
Right arrow Articles by Katz, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Balkowiec, A.
Right arrow Articles by Katz, D. M.

 Previous Article  |  Next Article 

The Journal of Neuroscience, October 1, 2000, 20(19):7417-7423

Activity-Dependent Release of Endogenous Brain-Derived Neurotrophic Factor from Primary Sensory Neurons Detected by ELISA In Situ

Agnieszka Balkowiec and David M. Katz

Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To define activity-dependent release of endogenous brain-derived neurotrophic factor (BDNF), we developed an in vitro model using primary sensory neurons and a modified ELISA, termed ELISA in situ. Dissociate cultures of nodose-petrosal ganglion cells from newborn rats were grown in wells precoated with anti-BDNF antibody to capture released BDNF, which was subsequently detected using conventional ELISA. Conventional ELISA alone was unable to detect any increase in BDNF concentration above control values following chronic depolarization with 40 mM KCl for 72 hr. However, ELISA in situ demonstrated a highly significant increase in BDNF release, from 65 pg/ml in control to 228 pg/ml in KCl-treated cultures. The efficacy of the in situ assay appears to be related primarily to rapid capture of released BDNF that prevents BDNF binding to the cultured cells. We therefore used this approach to compare BDNF release from cultures exposed for 30 min to either continuous depolarization with elevated KCl or patterned electrical field stimulation (50 biphasic rectangular pulses of 25 msec, at 20 Hz, every 5 sec). Short-term KCl depolarization was completely ineffective at evoking any detectable release of BDNF, whereas patterned electrical stimulation increased extracellular BDNF levels by 20-fold. In addition, the magnitude of BDNF release was dependent on stimulus pattern, with high-frequency bursts being most effective. These data indicate that the optimal stimulus profile for BDNF release resembles that of other neuroactive peptides. Moreover, our findings demonstrate that BDNF release can encode temporal features of presynaptic neuronal activity.

Key words: BDNF release; chronic depolarization; electrical field stimulation; ELISA; ELISA in situ; frequency; patterned stimulation; P-CREB; primary sensory neurons


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There is increasing evidence that brain-derived neurotrophic factor (BDNF) plays a trans-synaptic role in regulating transmission between primary sensory neurons and second-order sensory relay cells. BDNF is expressed by subsets of sensory ganglion cells (Schecterson and Bothwell, 1992; Wetmore and Olson, 1995; Apfel et al., 1996; Zhou et al., 1998; Brady et al., 1999), can be transported in the central projections of dorsal root ganglion (DRG) neurons (Zhou and Rush, 1996; Tonra, 1999), and is localized to dense-core vesicles within DRG central axon terminals (Michael et al., 1997). Our studies demonstrated that survival of sensory neurons that both express and depend on BDNF can be supported by long-term exposure to elevated potassium, indicating that BDNF can be released under depolarizing conditions in culture (Brady et al., 1999). More recently we found that BDNF acutely inhibits AMPA-mediated currents in second-order sensory relay neurons, indicating that BDNF may modulate glutamatergic primary afferent transmission (Balkowiec et al., 2000). In addition, Kerr et al. (1999) demonstrated that BDNF can potentiate nociceptive spinal reflexes by enhancing NMDA receptor-mediated responses. Despite these findings, little is known about activity-dependent release of endogenous BDNF, either from primary sensory neurons or other neuronal cell types.

Analysis of regulated secretion of endogenous neurotrophins from identified neurons has been hampered by the limited ability of conventional assays to detect the relatively small quantities of these factors released during physiological stimulation. Studies to date have used ELISA to detect neurotrophin release either from tissue slices or following neurotrophin overexpression in transfected cells (Blöchl and Thoenen, 1995, 1996; Goodman et al., 1996; Heymach et al., 1996; Canossa et al., 1997; Krüttgen et al., 1998; Griesbeck et al., 1999). It is unknown, however, whether overexpression to very high concentrations alters normal routes of BDNF trafficking and release. Moreover, most studies of regulated neurotrophin release have stimulated cells using continuous membrane-depolarizing agents, including elevated extracellular potassium, veratridine, or glutamate receptor agonists (Ghosh et al., 1994; Blöchl and Thoenen, 1995; Androutsellis-Theotokis et al., 1996; Goodman et al., 1996; Heymach et al., 1996; Griesbeck et al., 1999). It is well established, however, that release of classical as well as peptide transmitters depends on nerve impulse pattern (Lundberg et al., 1989; Whim and Lloyd, 1994).

To address these issues, the present study compared the effects of continuous chemical depolarization and patterned electrical stimulation on BDNF release from primary sensory neurons, using a modification of conventional ELISA methodology, termed ELISA in situ. This technique, described by Beech et al. (1997) for measuring cytokine release from T-cells, incorporates a substrate-bound monoclonal antibody against the peptide of interest into the cell culture system, so that the released peptide is immediately captured for subsequent detection by colorimetric methods. We found that, using this technique, we can readily detect release of endogenous BDNF from newborn primary sensory neurons following short-term patterned electrical stimulation. Moreover, we found that short-term stimulation with high-frequency bursts is strikingly more effective at releasing BDNF than KCl-induced depolarization over the same time period.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation and culture. Newborn rats (Sprague Dawley strain; Zivic-Miller, Zelienople, PA) were deeply anesthetized by hypothermia and decapitated. Nodose and petrosal ganglia (NPG) were (1) aseptically removed from the animals, (2) digested in 0.1% trypsin (Worthington Biochemical, Lakewood, NJ) with 0.01% deoxyribonuclease I (Sigma, St. Louis, MO) dissolved in Ca2+- and Mg2+-free HBSS (Mediatech, Herndon, VA) for 30 min at 37°C in a humidified atmosphere of 5% CO2 and 95% air, (3) rinsed in 0.1% soybean trypsin inhibitor (Worthington) dissolved in Ca2+- and Mg2+-containing Dulbecco's phosphate-buffered salt solution (Mediatech), (4) transferred to culture medium, and (5) triturated through siliconized, fire-polished Pasteur pipettes. Dissociated NPG neurons were plated in UV-sterilized, 96-well flat-bottom ELISA plates (MaxiSorp; Nalge Nunc International, Naperville, IL) at a density of one NPG per well. Cultures of NPG neurons were grown for 3 d in Neurobasal-A medium supplemented with B-27 serum-free supplement, 0.5 mM L-glutamine, 0.025 mM glutamic acid, and 1% penicillin-streptomycin-neomycin antibiotic mixture (Life Technologies, Gaithersburg, MD).

BDNF immunoassays. BDNF protein was measured with both a conventional and a modified sandwich ELISA using the BDNF Emax immunoassay system (Promega, Madison, WI) according to the protocol of the manufacturer, except that the concentrations of the anti-BDNF monoclonal antibody and anti-human BDNF polyclonal antibody were 5 and 2 µg/ml, respectively, and the dilution of the anti-IgY-HRP antibody was 1:1000. All reagents used prior to cell plating were sterilized with 0.2 µm Acrodisc syringe filters (Pall, Ann Arbor, MI).

Conventional BDNF ELISA. NPG cells were grown in uncoated 96-well ELISA plates. In some control experiments, wells were precoated with an irrelevant monoclonal antibody (anti-NGF; Promega) to rule out any potential influence of antibody presence on BDNF release. These wells were treated prior to cell plating as described below for anti-BDNF monoclonal antibody. On the day of the assay, a standard curve was generated for each plate using BDNF diluted in the same medium used for cell culture. Standards (in duplicate) and undiluted fresh samples of cell-conditioned culture medium (in duplicate or triplicate) were incubated in ELISA plates precoated with anti-BDNF monoclonal antibody, according to the manufacturer's protocol. Following the incubation and washing steps, anti-human BDNF polyclonal antibody was applied (see below).

BDNF ELISA in situ. Ninety-six-well ELISA plates were UV-sterilized for 30 min and coated with anti-BDNF monoclonal antibody at 4°C for 16.5 hr. Next, plates were washed and blocked, followed by two 1 hr incubations with culture medium to remove any residue of the ELISA washing solution. Then the NPG neurons were prepared as described above, plated in anti-BDNF-coated wells, and grown for 3 d under various experimental conditions (see Results). BDNF samples used to generate the standard curves were incubated in the same plate as the cells. At the end of the culture period, plates were extensively washed to remove all cells and cell debris, and the anti-human BDNF polyclonal antibody was applied, followed by subsequent steps according to the manufacturer's protocol. In experiments designed to compare the conventional BDNF ELISA with BDNF ELISA in situ, all steps of the protocol, beginning with the application of the anti-human BDNF antibody, were performed simultaneously for both assays. Absorbance values were read at 450 nm in a plate reader (Vmax; Molecular Devices, Sunnyvale, CA). For control wells in which anti-BDNF monoclonal antibody was omitted, absorbance values were not significantly different from the absorbance of blank wells.

Electrical field stimulation of NPG neurons. NPG cultures were prepared as described above for BDNF ELISA in situ. After an initial 3-d incubation, three adjacent culture wells were connected to each other in series through thin strips of 1% agarose gel permeated with culture medium, and to the stimulator (MultiStim System; Digitimer) through Ag:AgCl stimulating electrodes (modified from the methods of Brevet et al., 1976; McDonough et al., 1994). Three additional wells were also connected to each other by agarose bridges but were not connected to the stimulator and served as controls. The plate was put back to the incubator, and the neurons were stimulated for 30 or 60 min with biphasic rectangular pulses delivered at various patterns (see Results). In experiments comparing the effects of patterned electrical stimulation with potassium-induced continuous depolarization, KCl was added to three additional wells, to a final concentration of 40 mM, at the beginning of the stimulation period. In addition, BDNF standards were prepared in the same plate, also at the beginning of the stimulation period. After stimulation, all wells were vigorously washed prior to the ELISA steps described above.

Calculations and statistical analysis. BDNF levels were calculated from the standard curve prepared for each plate, using SOFTmax PRO version 3.0 software (Molecular Devices). The standard curves were linear within the range used (0-500 pg/ml), and the quantities of BDNF in experimental samples were always within the linear range of the standard curve. Data are expressed as mean ± SE. Samples were compared using ANOVA followed by Duncan's multiple-comparison procedure, and p < 0.05 was considered significant.

Immunocytochemistry. Cultures for immunocytochemical staining were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 30 min at room temperature. Protein gene product (PGP) 9.5 immunostaining was performed as previously described (Brady et al., 1999). The number of neurons in each culture was evaluated by counting all PGP9.5-immunoreactive cells per well. Experiments were performed three times with three cultures per experimental group. Values were compared using ANOVA followed by Duncan's multiple-comparison procedure, and p < 0.05 was considered significant. TrkB and phospho-cAMP response element-binding protein (P-CREB) immunostaining were performed as previously described (Brady et al., 1999), using rabbit polyclonal anti-TrkB (Chemicon, Temecula, CA) or rabbit anti-phospho-CREB IgG (Upstate Biotechnology, Lake Placid, NY) and goat anti-rabbit biotinylated IgG (Vector Laboratories, Burlingame, CA). Control cultures, in which primary antibody was omitted, were completely devoid of staining.

Anti-TrkB IgG1 (clone 47; Transduction Laboratories, Lexington, KY; catalog #T16020, special order, without additives) was used to inhibit binding of BDNF to TrkB receptors (see Results). We established that 5 µg/ml anti-TrkB was sufficient to inhibit survival of BDNF-dependent embryonic day 16.5 NPG neurons (Erickson et al., 1996) grown in the presence of 10 ng/ml BDNF. Survival of newborn NPG neurons, which are not BDNF-dependent (Brady et al., 1999), was not affected by treatment with 10 µg/ml antibody (1197.56 ± 102.78 neurons per well in control and 1025.5 ± 80.49 neurons per well with anti-TrkB; n = 9; p = 0.37578).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Initial studies sought to compare extracellular levels of BDNF in cultures of newborn NPG neurons grown for 72 hr in the absence (control) or presence of depolarizing concentrations of KCl (40 mM; Brosenitsch et al., 1998; Brady et al., 1999) using a conventional BDNF ELISA protocol. Using this approach, we were able to detect only very low levels of BDNF in control cultures and saw no significant change in BDNF concentration in KCl-treated groups compared with controls (Fig. 1; control, 2.88 ± 0.84 pg/ml; n = 23; KCl-treated, 4.92 ± 0.81 pg/ml; n = 21; p = 0.88256). This result was at odds with previous findings from our laboratory that endogenous BDNF can support survival of NPG neurons in culture following chronic exposure to elevated KCl (Brady et al., 1999). We therefore sought to improve detectability of BDNF in our culture system using a modification of the conventional ELISA, termed ELISA in situ, in which cells are grown in wells precoated with a monoclonal antibody against the peptide of interest, which is thus immobilized and subsequently detected using standard colorimetric methods (Beech et al., 1997). Indeed, using BDNF ELISA in situ, we were able not only to measure higher levels of BDNF in control cultures but also to detect significant release of BDNF following chronic depolarization. Specifically, the concentration of BDNF averaged 65.34 ± 5.29 pg/ml (n = 23) in 72 hr control NPG cultures and 228.16 ± 19.47 pg/ml (n = 21) following 72 hr treatment with elevated KCl (p = 0.00011; Fig. 1). The increase in BDNF levels detected in KCl-treated cultures was not attributable to increased neuronal survival (1234.3 ± 71.05 neurons per well in control cultures vs 1429.6 ± 91.22 neurons per well in KCl-treated cultures; n = 9; p = 0.53294). Similarly, survival was not significantly increased by the presence of the BDNF antibody during the cell culture period (1234.3 ± 71.05 neurons per well in the presence of anti-BDNF vs 1197.56 ± 102.78 neurons per well in the absence of anti-BDNF; n = 9; p = 0.70989). To determine whether the presence of the capture antibody by itself stimulated BDNF release, sister cultures were grown in wells precoated with an irrelevant anti-NGF monoclonal antibody, in the presence or absence of elevated KCl, and extracellular BDNF levels were measured using conventional BDNF ELISA. The presence of the monoclonal antibody had no effect on BDNF levels, either in control cultures (0.67 ± 0.45 pg/ml in the presence of anti-NGF vs 0.73 ± 0.26 pg/ml in the absence of anti-NGF; n = 8; p = 0.97377) or KCl-treated cultures (5.02 ± 2.28 pg/ml in the presence of anti-NGF vs 4.76 ± 1.46 pg/ml in the absence of anti-NGF; n = 8; p = 0.89129). However, we cannot exclude the possibility that the presence of the antibody stimulated release of low levels of BDNF, below the limits of detectability by conventional ELISA. To rule out the possibility that the observed increase in BDNF release was due simply to the increased osmolarity of the KCl-supplemented medium, we compared cultures grown in control medium with cultures supplemented with 40 mM NaCl and found that BDNF levels, measured using ELISA in situ, were unchanged in the presence of elevated NaCl (92.38 ± 13.24 vs 76.76 ± 7.38 pg/ml in control groups; n = 9; p = 0.46864).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1.   Long-term exposure to elevated extracellular potassium induces BDNF release from newborn NPG neurons in culture. Mean BDNF levels were measured with standard ELISA and ELISA in situ in sister cultures grown for 72 hr in the absence (Control, gray bars) or presence of 40 mM potassium (KCl, black bars). n = 21; ***p < 0.001; n.s., not significant.

These findings demonstrated that markedly higher levels of BDNF were detected in control and KCl-treated NPG cultures using ELISA in situ compared with conventional ELISA, and that this difference could not be attributed to increased neuronal survival or other nonspecific effects. We hypothesized, therefore, that the in situ assay protocol increased BDNF detectability by rapidly capturing and immobilizing released BDNF and thereby protecting the peptide from binding to cells and/or degradation. To test these possibilities, 500 pg/ml exogenous human recombinant BDNF (Promega) was added to culture wells containing either medium alone or NPG dissociate cultures. Following 72 hr of incubation, BDNF levels were compared in both groups using conventional ELISA and ELISA in situ. No significant differences were found between the levels of BDNF detected by standard ELISA (310.83 ± 39.31 pg/ml; n = 17) and ELISA in situ (304.64 ± 27.13 pg/ml; n = 10; p = 0.88643; Fig. 2) in wells containing culture medium alone plus BDNF. This experiment demonstrated that there are no intrinsic differences in the sensitivity of the two assays. However, when compared among wells containing NPG neurons plus BDNF, there was a highly significant (p = 0.00047) difference between the levels of BDNF detected with the two assays. Specifically, using standard ELISA, the BDNF concentration was only 32.12 ± 5.42 pg/ml (n = 19) after 72 hr, a value that was not significantly different from BDNF levels in medium from control NPG cultures grown without added BDNF (p = 0.30941; Fig. 2). This result indicates that, in the presence of NPG cells, BDNF is lost over time from the culture medium, perhaps through degradation or binding to the high-affinity BDNF receptor TrkB, which is expressed by newborn NPG neurons (Zhuo and Helke, 1996; present study, Fig. 3A). In fact, treatment of cultures with a function blocking anti-TrkB antibody (Transduction Laboratories; for details, see Materials and Methods) significantly increased the ability of conventional ELISA to detect BDNF added to standard NPG cultures. Specifically, addition of the anti-TrkB antibody increased detection of BDNF by conventional ELISA by nearly threefold compared with control cultures grown without anti-TrkB (with anti-TrkB, 211.58 ± 62.71 pg/ml; n = 5; vs controls, 68.36 ± 11.44 pg/ml; n = 13; p = 0.00056; Fig. 3B). These data suggest that the relative inability of conventional ELISA to detect BDNF release in NPG cultures is attributable, in large part, to binding of BDNF to TrkB on the cultured cells.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 2.   Detectability of exogenous BDNF by standard ELISA versus ELISA in situ. BDNF (500 pg/ml) was added at plating to newborn NPG cultures (hatched gray bars) and to culture medium (Med.) alone (hatched white bars) and incubated for 72 hr in the absence (Standard ELISA) or presence of anti-BDNF monoclonal capture antibody (ELISA in situ). BDNF levels were also measured in control cultures (solid gray bars) to which exogenous BDNF was not added. ***p < 0.001; n.s., not significant.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 3.   Inhibition of BDNF binding to cells increases BDNF detectability by standard ELISA. A, Immunostaining with an antibody against the extracellular domain of TrkB (Chemicon) in newborn NPG cultures grown for 3 d in the absence (Control) or presence of exogenous BDNF (+BDNF, 500 pg/ml). B, Mean levels of BDNF, detected with standard ELISA, in the absence (Control) or presence of anti-TrkB antibody (Anti-TrkB Ab, 10 µg/ml) in NPG cultures (hatched gray bars) and culture medium alone (hatched white bars) 48 hr after addition of 500 pg/ml BDNF. BDNF levels were also measured in control cultures (solid gray bars) to which exogenous BDNF was not added. ***p < 0.001;*p < 0.05; n.s., not significant.

In contrast to the results obtained with standard ELISA, ELISA in situ detected 337.33 ± 25.93 pg/ml BDNF (n = 18) after 72 hr, a level not significantly different from that in wells to which BDNF was added in the absence of cells (p = 0.92444; Fig. 2). These data demonstrate that the substrate-bound anti-BDNF, which is present throughout the culture period in the in situ paradigm, successfully competes with BDNF binding to TrkB on cells, thereby enhancing detectability of BDNF in the culture medium.

Previous studies of activity-dependent neurotransmitter release demonstrated that chronic depolarization is markedly less effective than high-frequency electrical stimulation at releasing both classical transmitters and peptide co-transmitters (Belai et al., 1987; Agoston et al., 1988). To examine whether BDNF release is similarly regulated, we compared the effects of patterned electrical field stimulation for 30 min (50 biphasic rectangular pulses of 25 msec, at 20 Hz, every 5 sec) with 30 min of continuous depolarization by 40 mM KCl, on BDNF release from newborn NPG neurons, using ELISA in situ. To determine whether these stimulation protocols were effective at activating NPG neurons, we performed immunostaining with an antibody against the phosphorylated form of CREB, a marker of neuronal depolarization (Ghosh et al., 1994; Moore et al., 1996). Both KCl treatment and patterned electrical field stimulation led to marked increases in P-CREB staining in the vast majority of cells (Fig. 4A), indicating that both protocols were effective at activating neurons in these cultures.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4.   Patterned electrical stimulation is markedly more effective at releasing BDNF from newborn NPG neurons than KCl-induced continuous depolarization. A, P-CREB immunostaining of newborn NPG cultures after 30 min electrical field stimulation (20 Hz; Electrical stimulation) or 30 min continuous depolarization (40mM KCl) compared with unstimulated controls. B, Mean levels of BDNF released in sister cultures of newborn NPG neurons during 30 min of control conditions (no stimulation), electrical field stimulation (50 biphasic rectangular pulses of 25 msec, at 20 Hz, every 5 sec), or continuous depolarization with 40 mM KCl. Each value represents the difference between the BDNF level measured after stimulation and the level measured in sister cultures at the beginning of the stimulus period. ***p < 0.001; n.s., not significant.

BDNF levels were compared following 30 min of either control conditions, patterned electrical stimulation, or KCl-induced chronic depolarization. Patterned electrical stimulation at 20 Hz resulted in a highly significant increase in BDNF release from NPG neurons (62.95 ± 4.19 pg/ml vs 2.87 ± 1.11 pg/ml in control; n = 16; p = 0.00011; Fig. 4B). In contrast, KCl-induced chronic depolarization over the same time period was completely ineffective at increasing detectable BDNF release (-1.35 ± 3.58 pg/ml; n = 20; p = 0.87134; Fig. 4B). The release of BDNF induced by electrical stimulation was abolished by treatment of cultures with 1.5 µM tetrodotoxin (TTX), an inhibitor of voltage-dependent Na+ channels, before stimulation (1.50 ± 0.70 pg/ml with TTX vs 35.12 ± 0.94 pg/ml without TTX; n = 4; p = 0.0000001), indicating that activation of voltage-gated sodium channels is required for this release. To rule out the possibility that the enhanced BDNF release was due to damage of cells by electrical activation, we compared cell survival between control cultures and cultures stimulated for 30 min with bursts of 50 biphasic rectangular pulses of 25 msec, at 20 Hz, delivered every 5 sec. Twenty-four hours after stimulation, there was no significant difference in the number of cells in control and stimulated cultures (per well: control, 1575 ± 60.35; stimulated, 1480 ± 57.15; n = 9; p = 0.28485).

It is well established that peptide neurotransmitter release can be differentially regulated by distinct patterns of neuronal activity (Lundberg et al., 1986, 1989; Whim and Lloyd, 1994; Vilim et al., 1996). To examine the effect of stimulus pattern on the release of BDNF from NPG neurons, we used a paradigm in which the overall number of pulses, and consequently, average frequency, as well as the number of pulses in individual bursts, remained constant, whereas intraburst frequency and interburst interval were varied. Specifically, BDNF levels were compared following 60 min of either control conditions or electrical field stimulation with 50 biphasic rectangular pulses of 10 msec, delivered at 5, 10, 20, and 50 Hz, with interburst intervals, respectively, of 0 (tonic stimulation), 10, 15, and 18 sec (Fig. 5A). BDNF release was significantly higher during stimulation with high-frequency bursts (20 Hz, 34.95 ± 4.98 pg/ml; p = 0.0144; 50 Hz, 48.07 ± 7.18 pg/ml; p = 0.0005) compared with tonic stimulation at 5 Hz (18.09 ± 2.79 pg/ml; n = 10; Fig. 5B). When compared among different bursting patterns, stimulation with 2 sec 50 Hz bursts delivered every 20 sec was most effective, despite the short burst duration and long interburst interval characteristic of this pattern (Fig. 5).



View larger version (51K):
[in this window]
[in a new window]
  		Figure 5.   Activity-dependent release of BDNF is regulated by the pattern of stimulation. A, Schematic representation of the stimulation patterns applied to each group of cultures. B, Mean levels of BDNF released from newborn NPG neurons during 60 min of electrical field stimulation with 50 biphasic rectangular pulses of 10 msec, delivered at 5, 10, 20, and 50 Hz, with interburst intervals, respectively, of 0, 10, 15, and 18 sec as shown in A. ***p < 0.001; *p < 0.05; n.s., not significant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that primary sensory neurons can release BDNF in an activity-dependent manner. The amount of released BDNF is regulated by both stimulus frequency and pattern, and high-frequency bursts are markedly more effective at evoking release than either continuous depolarization with elevated extracellular KCl or tonic electrical stimulation. Moreover, our results demonstrate that the detectability of released BDNF by ELISA in situ is greatly enhanced compared with conventional ELISA. Thus, we are able to quantify release of endogenous BDNF from dissociated neurons, without the need to enhance peptide levels by genetic overexpression, as in other studies (Blöchl and Thoenen, 1995, 1996; Goodman et al., 1996; Heymach et al., 1996; Canossa et al., 1997; Krüttgen et al., 1998; Griesbeck et al., 1999).

Previous analyses of neurotrophin release have used continuous depolarization, induced by elevated potassium, veratridine, or glutamate agonists to activate cells (Ghosh et al., 1994; Blöchl and Thoenen, 1995, 1996; Androutsellis-Theotokis et al., 1996; Goodman et al., 1996; Heymach et al., 1996; Griesbeck et al., 1999). Indeed, continuous depolarization is highly effective at inducing calcium influx and activation of intracellular signaling pathways required for both genomic and nongenomic responses (Sheng et al., 1990; Ginty et al., 1991; Bito, 1998; Brosenitsch et al., 1998; Tao et al., 1998). In the present study, for example, short-term exposure to 40 mM KCl was sufficient to increase CREB phosphorylation, a marker of neuronal depolarization and activation of multiple intracellular signaling cascades (Sheng et al., 1990; Davis et al., 1996; Deisseroth et al., 1996; Moore et al., 1996). Despite this, 30 min of KCl depolarization was completely ineffective at evoking detectable release of BDNF. Similarly, Griesbeck et al. (1999) reported that short-term KCl-induced depolarization was ineffective at releasing BDNF from primary cultures of hippocampal neurons. In contrast, we found that 30 min of patterned electrical stimulation led to a marked 20-fold rise in the concentration of extracellular BDNF. These data suggest that, rather than depolarization per se, activation of specific signaling pathways by patterned stimulation is required to evoke detectable BDNF release (see also Buonanno and Fields, 1999).

We did find that exposure to KCl for 3 d evokes detectable release of BDNF from NPG neurons, albeit much less than only 30 min of high-frequency electrical stimulation. However, such release likely reflects multiple sequelae of long-term continuous depolarization, including increased BDNF expression (Shieh et al., 1998; Shieh and Ghosh, 1999), and is therefore probably not a useful model for elucidating mechanisms that govern release of preexisting BDNF pools.

Our results demonstrate for the first time that the amount of BDNF release depends on stimulus pattern, indicating that BDNF can encode temporal features of presynaptic neuronal activity. This finding may be of particular significance in light of the proposed role of BDNF in activity-dependent mechanisms of neuronal development and function (Cabelli et al., 1995; Thoenen, 1995; Acheson and Lindsay, 1996; Bonhoeffer, 1996; Galuske et al., 1996; Katz and Shatz, 1996; McAllister et al., 1996; Snider and Lichtman, 1996; Stoop and Poo, 1996; Cabelli et al., 1997; Marty et al., 1997; Black, 1999; Lu and Chow, 1999; McAllister et al., 1999), including homeostatic regulation of synaptic strength (Rutherford et al., 1998; Turrigiano, 1999). For example, by encoding afferent firing patterns, BDNF could provide a mechanism for distinguishing among competing inputs during activity-dependent refinement of synaptic connections. Once released from presynaptic terminals, BDNF could act directly or, alternatively, by modulating responses to classical neurotransmitters. We recently found, for example, that BDNF, acting through TrkB, strongly inhibits AMPA responses of developing sensory relay neurons (Balkowiec et al., 2000).

Studies of other peptide transmitter systems, such as neuropeptide Y, vasoactive intestinal polypeptide, or the small cardioactive peptide, all indicate that the pattern of nerve impulses is critical for coding peptide release (Lundberg et al., 1986; Agoston et al., 1988; Lundberg et al., 1989; Pernow et al., 1989; Whim and Lloyd, 1994). Specifically, high-frequency stimulation releases larger amounts of neuropeptides compared with low-frequency stimulation. Moreover, KCl depolarization is significantly less effective, or even completely ineffective, at stimulating the release of vasoactive intestinal polypeptide compared with high-frequency electrical impulses (Belai et al., 1987; Agoston et al., 1988). Therefore, in this regard, activity-dependent BDNF release resembles that of other peptide neurotransmitters. In addition, studies of the intracellular distribution of BDNF have shown that the peptide is localized to dense-core vesicles in sensory axon terminals (Michael et al., 1997), as is typical of other sensory neuropeptides (Zupanc, 1996), and to vesicles of the regulated secretory pathway in cortical neurons (Fawcett et al., 1997; Haubensak et al., 1998). Thus, our current findings provide functional data, consistent with the subcellular distribution of BDNF, that support its role as a peptide neuromodulator at sensory synapses (Kerr et al., 1999; Balkowiec et al., 2000) as well as other synapses (Lohof et al., 1993; Lessmann et al., 1994; Kang and Schuman, 1995; Lessmann and Heumann, 1998; Levine et al., 1998). Moreover, the stimulus frequencies applied in the current study to evoke BDNF release from NPG neurons are within the physiological range for these cells (Jaffe and Sampson, 1976; Thoren, 1976; Coleridge et al., 1987).

In conclusion, the present study shows that primary sensory neurons can release endogenous BDNF in an activity-dependent manner, and that the magnitude of release depends on the pattern and frequency of stimulation. Given that transient, repetitive electrical stimulation resembles patterns of nerve activity in vivo more closely than continuous depolarization, we believe that this model provides new opportunities for defining physiological mechanisms of BDNF release and, consequently, BDNF roles in synaptic development and function.


    FOOTNOTES

Received May 22, 2000; revised July 5, 2000; accepted July 13, 2000.

This work was supported by US Public Health Service National Heart, Lung, and Blood Institute grants to D.M.K.

Correspondence should be addressed to Dr. David M. Katz, Department of Neurosciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail: dmk4{at}po.cwru.edu.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

  • Acheson A, Lindsay RM (1996) Non target-derived roles of the neurotrophins. Philos Trans R Soc Lond B Biol Sci 351:417-422[Web of Science][Medline].
  • Agoston DV, Conlon JM, Whittaker VP (1988) Selective depletion of the acetylcholine and vasoactive intestinal polypeptide of the guinea-pig myenteric plexus by differential mobilization of distinct transmitter pools. Exp Brain Res 72:535-542[Web of Science][Medline].
  • Androutsellis-Theotokis A, McCormack WJ, Bradford HF, Stern GM, Pliego-Rivero FB (1996) The depolarisation-induced release of [125I]BDNF from brain tissue. Brain Res 743:40-48[Web of Science][Medline].
  • Apfel SC, Wright DE, Wiideman AM, Dormia C, Snider WD, Kessler JA (1996) Nerve growth factor regulates the expression of brain-derived neurotrophic factor mRNA in the peripheral nervous system. Mol Cell Neurosci 7:134-142[Web of Science][Medline].
  • Balkowiec A, Kunze DL, Katz DM (2000) Brain-derived neurotrophic factor acutely inhibits AMPA-mediated currents in developing sensory relay neurons. J Neurosci 20:1904-1911[Abstract/Free Full Text].
  • Beech JT, Bainbridge T, Thompson SJ (1997) Incorporation of cells into an ELISA system enhances antigen-driven lymphokine detection. J Immunol Methods 205:163-168[Web of Science][Medline].
  • Belai A, Ralevic V, Burnstock G (1987) VIP release from enteric nerves is independent of extracellular calcium. Regul Pept 19:79-89[Medline].
  • Bito H (1998) The role of calcium in activity-dependent neuronal gene regulation. Cell Calcium 23:143-150[Web of Science][Medline].
  • Black IB (1999) Trophic regulation of synaptic plasticity. J Neurobiol 41:108-118[Web of Science][Medline].
  • Blöchl A, Thoenen H (1995) Characterization of nerve growth factor (NGF) release from hippocampal neurons: evidence for a constitutive and an unconventional sodium-dependent regulated pathway. Eur J Neurosci 7:1220-1228[Web of Science][Medline].
  • Blöchl A, Thoenen H (1996) Localization of cellular storage compartments and sites of constitutive and activity-dependent release of nerve growth factor (NGF) in primary cultures of hippocampal neurons. Mol Cell Neurosci 7:173-190[Web of Science][Medline].
  • Bonhoeffer T (1996) Neurotrophins and activity-dependent development of the neocortex. Curr Opin Neurobiol 6:119-126[Web of Science][Medline].
  • Brady R, Zaidi SIA, Mayer C, Katz DM (1999) BDNF is a target-derived survival factor for arterial baroreceptor and chemoafferent primary sensory neurons. J Neurosci 19:2131-2142[Abstract/Free Full Text].
  • Brevet A, Pinto E, Peacock J, Stockdale FE (1976) Myosin synthesis increased by electrical stimulation of skeletal muscle cell cultures. Science 193:1152-1154[Abstract/Free Full Text].
  • Brosenitsch TA, Salgado-Commissariat D, Kunze DL, Katz DM (1998) A role for L-type calcium channels in developmental regulation of transmitter phenotype in primary sensory neurons. J Neurosci 18:1047-1055[Abstract/Free Full Text].
  • Buonanno A, Fields RD (1999) Gene regulation by patterned electrical activity during neural and skeletal muscle development. Curr Opin Neurobiol 9:110-120[Web of Science][Medline].
  • Cabelli RJ, Hohn A, Shatz CJ (1995) Inhibition of ocular dominance column formation by infusion of NT-4/5 or BDNF. Science 267:1662-1666[Abstract/Free Full Text].
  • Cabelli RJ, Shelton DL, Segal RA, Shatz CJ (1997) Blockade of endogenous ligands of trkB inhibits formation of ocular dominance columns. Neuron 19:63-76[Web of Science][Medline].
  • Canossa M, Griesbeck O, Berninger B, Campana G, Kolbeck R, Thoenen H (1997) Neurotrophin release by neurotrophins: implications for activity-dependent neuronal plasticity. Proc Natl Acad Sci USA 94:13279-13286[Abstract/Free Full Text].
  • Coleridge HM, Coleridge JC, Schultz HD (1987) Characteristics of C fibre baroreceptors in the carotid sinus of dogs. J Physiol (Lond) 394:291-313[Abstract/Free Full Text].
  • Davis GW, Schuster CM, Goodman CS (1996) Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity. Neuron 17:669-679[Web of Science][Medline].
  • Deisseroth K, Bito H, Tsien RW (1996) Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity. Neuron 16:89-101[Web of Science][Medline].
  • Erickson JT, Conover JC, Borday V, Champagnat J, Barbacid M, Yancopoulos G, Katz DM (1996) Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing. J Neurosci 16:5361-5371[Abstract/Free Full Text].
  • Fawcett JP, Aloyz R, McLean JH, Pareek S, Miller FD, McPherson PS, Murphy RA (1997) Detection of brain-derived neurotrophic factor in a vesicular fraction of brain synaptosomes. J Biol Chem 272:8837-8840[Abstract/Free Full Text].
  • Galuske RA, Kim DS, Castrén E, Thoenen H, Singer W (1996) Brain-derived neurotrophic factor reverses experience-dependent synaptic modifications in kitten visual cortex. Eur J Neurosci 8:1554-1559[Web of Science][Medline].
  • Ghosh A, Carnahan J, Greenberg ME (1994) Requirement for BDNF in activity-dependent survival of cortical neurons. Science 263:1618-1623[Abstract/Free Full Text].
  • Ginty DD, Glowacka D, Bader DS, Hidaka H, Wagner JA (1991) Induction of immediate early genes by Ca2+ influx requires cAMP-dependent protein kinase in PC12 cells. J Biol Chem 266:17454-17458[Abstract/Free Full Text].
  • Goodman LJ, Valverde J, Lim F, Geschwind MD, Federoff HJ, Geller AI, Hefti F (1996) Regulated release and polarized localization of brain-derived neurotrophic factor in hippocampal neurons. Mol Cell Neurosci 7:222-238[Web of Science][Medline].
  • Griesbeck O, Canossa M, Campana G, Gärtner A, Hoener MC, Nawa H, Kolbeck R, Thoenen H (1999) Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity. Microsc Res Tech 45:262-275[Web of Science][Medline].
  • Haubensak W, Narz F, Heumann R, Lessmann V (1998) BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons. J Cell Sci 111:1483-1493[Abstract].
  • Heymach JV, Krüttgen A, Suter U, Shooter EM (1996) The regulated secretion and vectorial targeting of neurotrophins in neuroendocrine and epithelial cells. J Biol Chem 271:25430-25437[Abstract/Free Full Text].
  • Jaffe RA, Sampson SR (1976) Analysis of passive and active electrophysiologic properties of neurons in mammalian nodose ganglia maintained in vitro. J Neurophysiol 39:802-815[Abstract/Free Full Text].
  • Kang H, Schuman EM (1995) Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science 267:1658-1662[Abstract/Free Full Text].
  • Katz LC, Shatz CJ (1996) Synaptic activity and the construction of cortical circuits. Science 274:1133-1138[Abstract/Free Full Text].
  • Kerr BJ, Bradbury EJ, Bennett DL, Trivedi PM, Dassan P, French J, Shelton DB, McMahon SB, Thompson SW (1999) Brain-derived neurotrophic factor modulates nociceptive sensory inputs and NMDA-evoked responses in the rat spinal cord. J Neurosci 19:5138-5148[Abstract/Free Full Text].
  • Krüttgen A, Möller JC, Heymach JV, Shooter EM (1998) Neurotrophins induce release of neurotrophins by the regulated secretory pathway. Proc Natl Acad Sci USA 95:9614-9619[Abstract/Free Full Text].
  • Lessmann V, Gottmann K, Heumann R (1994) BDNF and NT-4/5 enhance glutamatergic synaptic transmission in cultured hippocampal neurones. NeuroReport 6:21-25[Web of Science][Medline].
  • Lessmann V, Heumann R (1998) Modulation of unitary glutamatergic synapses by neurotrophin-4/5 or brain-derived neurotrophic factor in hippocampal microcultures: presynaptic enhancement depends on pre-established paired-pulse facilitation. Neuroscience 86:399-413[Web of Science][Medline].
  • Levine ES, Crozier RA, Black IB, Plummer MR (1998) Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing N-methyl-D-aspartic acid receptor activity. Proc Natl Acad Sci USA 95:10235-10239[Abstract/Free Full Text].
  • Lohof AM, Ip NY, Poo M-M (1993) Potentiation of developing neuromuscular synapses by the neurotrophins NT-3 and BDNF. Nature 363:350-353[Medline].
  • Lu B, Chow A (1999) Neurotrophins and hippocampal synaptic transmission and plasticity. J Neurosci Res 58:76-87[Web of Science][Medline].
  • Lundberg JM, Rudehill A, Sollevi A, Fried G, Wallin G (1989) Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport. Neuroscience 28:475-486[Web of Science][Medline].
  • Lundberg JM, Rudehill A, Sollevi A, Theodorsson-Norheim E, Hamberger B (1986) Frequency- and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen. Neurosci Lett 63:96-100[Web of Science][Medline].
  • Marty S, Berzaghi MP, Berninger B (1997) Neurotrophins and activity-dependent plasticity of cortical interneurons. Trends Neurosci 20:198-202[Web of Science][Medline].
  • McAllister AK, Katz LC, Lo DC (1996) Neurotrophin regulation of cortical dendritic growth requires activity. Neuron 17:1057-1064[Web of Science][Medline].
  • McAllister AK, Katz LC, Lo DC (1999) Neurotrophins and synaptic plasticity. Annu Rev Neurosci 22:295-318[Web of Science][Medline].
  • McDonough PM, Stella SL, Glembotski CC (1994) Involvement of cytoplasmic calcium and protein kinases in the regulation of atrial natriuretic factor secretion by contraction rate and endothelin. J Biol Chem 269:9466-9472[Abstract/Free Full Text].
  • Michael GJ, Averill S, Nitkunan A, Rattray M, Bennett DL, Yan Q, Priestley JV (1997) Nerve growth factor treatment increases brain-derived neurotrophic factor selectively in TrkA-expressing dorsal root ganglion cells and in their central terminations within the spinal cord. J Neurosci 17:8476-8490[Abstract/Free Full Text].
  • Moore AN, Waxham MN, Dash PK (1996) Neuronal activity increases the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in rat hippocampus and cortex. J Biol Chem 271:14214-14220[Abstract/Free Full Text].
  • Pernow J, Schwieler J, Kahan T, Hjemdahl P, Oberle J, Wallin BG, Lundberg JM (1989) Influence of sympathetic discharge pattern on norepinephrine and neuropeptide Y release. Am J Physiol 257:H866-H872[Abstract/Free Full Text].
  • Rutherford LC, Nelson SB, Turrigiano GG (1998) BDNF has opposite effects on the quantal amplitude of pyramidal neuron and interneuron excitatory synapses. Neuron 21:521-530[Web of Science][Medline].
  • Schecterson LC, Bothwell M (1992) Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons. Neuron 9:449-463[Web of Science][Medline].
  • Sheng M, McFadden G, Greenberg ME (1990) Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB. Neuron 4:571-582[Web of Science][Medline].
  • Shieh PB, Ghosh A (1999) Molecular mechanisms underlying activity-dependent regulation of BDNF expression. J Neurobiol 41:127-134[Web of Science][Medline].
  • Shieh PB, Hu S-C, Bobb K, Timmusk T, Ghosh A (1998) Identification of a signaling pathway involved in calcium regulation of BDNF expression. Neuron 20:727-740[Web of Science][Medline].
  • Snider WD, Lichtman JW (1996) Are neurotrophins synaptotrophins? Mol Cell Neurosci 7:433-442[Web of Science][Medline].
  • Stoop R, Poo M-M (1996) Synaptic modulation by neurotrophic factors. Prog Brain Res 109:359-364[Web of Science][Medline].
  • Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME (1998) Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 20:709-726[Web of Science][Medline].
  • Thoenen H (1995) Neurotrophins and neuronal plasticity. Science 270:593-598[Abstract/Free Full Text].
  • Thoren PN (1976) Atrial receptors with nonmedullated vagal afferents in the cat. Discharge frequency and pattern in relation to atrial pressure. Circ Res 38:357-362[Abstract/Free Full Text].
  • Tonra JR (1999) Classical and novel directions in neurotrophin transport and research: anterograde transport of brain-derived neurotrophic factor by sensory neurons. Microsc Res Tech 45:225-232[Web of Science][Medline].
  • Turrigiano GG (1999) Homeostatic plasticity in neuronal networks: the more things change, the more they stay the same. Trends Neurosci 22:221-227[Web of Science][Medline].
  • Vilim FS, Cropper EC, Price DA, Kupfermann I, Weiss KR (1996) Release of peptide cotransmitters in Aplysia: regulation and functional implications. J Neurosci 16:8105-8114[Abstract/Free Full Text].
  • Wetmore C, Olson L (1995) Neuronal and nonneuronal expression of neurotrophins and their receptors in sensory and sympathetic ganglia suggest new intercellular trophic interactions. J Comp Neurol 353:143-159[Web of Science][Medline].
  • Whim MD, Lloyd PE (1994) Differential regulation of the release of the same peptide transmitters from individual identified motor neurons in culture. J Neurosci 14:4244-4251[Abstract].
  • Zhou X-F, Chie ET, Rush RA (1998) Distribution of brain-derived neurotrophic factor in cranial and spinal ganglia. Exp Neurol 149:237-242[Web of Science][Medline].
  • Zhou X-F, Rush RA (1996) Endogenous brain-derived neurotrophic factor is anterogradely transported in primary sensory neurons. Neuroscience 74:945-951[Web of Science][Medline].
  • Zhuo H, Helke CJ (1996) Presence and localization of neurotrophin receptor tyrosine kinase (TrkA, TrkB, TrkC) mRNAs in visceral afferent neurons of the nodose and petrosal ganglia. Brain Res Mol Brain Res 38:63-70[Medline].
  • Zupanc GK (1996) Peptidergic transmission: from morphological correlates to functional implications. Micron 27:35-91.

Copyright © 2000 Society for Neuroscience  0270-6474/00/20197417-07$05.00/0


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
G. Nagappan, E. Zaitsev, V. V. Senatorov Jr., J. Yang, B. L. Hempstead, and B. Lu
Control of extracellular cleavage of ProBDNF by high frequency neuronal activity
PNAS, January 27, 2009; 106(4): 1267 - 1272.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. de Wit, R. F. Toonen, and M. Verhage
Matrix-Dependent Local Retention of Secretory Vesicle Cargo in Cortical Neurons
J. Neurosci., January 7, 2009; 29(1): 23 - 37.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. H. Kao, B. D. Wright, and A. J. Doupe
Neurons in a Forebrain Nucleus Required for Vocal Plasticity Rapidly Switch between Precise Firing and Variable Bursting Depending on Social Context
J. Neurosci., December 3, 2008; 28(49): 13232 - 13247.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
R. A. Crozier, C. Bi, Y. R. Han, and M. R. Plummer
BDNF Modulation of NMDA Receptors Is Activity Dependent
J Neurophysiol, December 1, 2008; 100(6): 3264 - 3274.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
M. Bergami, S. Santi, E. Formaggio, C. Cagnoli, C. Verderio, R. Blum, B. Berninger, M. Matteoli, and M. Canossa
Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes
J. Cell Biol., October 20, 2008; 183(2): 213 - 221.
[Abstract] [Full Text] [PDF]

Home page
 Cereb CortexHome page
N. Heck, A. Golbs, T. Riedemann, J.-J. Sun, V. Lessmann, and H. J. Luhmann
Activity-Dependent Regulation of Neuronal Apoptosis in Neonatal Mouse Cerebral Cortex
Cereb Cortex, June 1, 2008; 18(6): 1335 - 1349.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
R. W. Bavis and G. S. Mitchell
Long-term effects of the perinatal environment on respiratory control
J Appl Physiol, April 1, 2008; 104(4): 1220 - 1229.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
H. Sha, J. Xu, J. Tang, J. Ding, J. Gong, X. Ge, D. Kong, and X. Gao
Disruption of a novel regulatory locus results in decreased Bdnf expression, obesity, and type 2 diabetes in mice
Physiol Genomics, October 19, 2007; 31(2): 252 - 263.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. Ogier, H. Wang, E. Hong, Q. Wang, M. E. Greenberg, and D. M. Katz
Brain-Derived Neurotrophic Factor Expression and Respiratory Function Improve after Ampakine Treatment in a Mouse Model of Rett Syndrome
J. Neurosci., October 3, 2007; 27(40): 10912 - 10917.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Kolarow, T. Brigadski, and V. Lessmann
Postsynaptic Secretion of BDNF and NT-3 from Hippocampal Neurons Depends on Calcium Calmodulin Kinase II Signaling and Proceeds via Delayed Fusion Pore Opening
J. Neurosci., September 26, 2007; 27(39): 10350 - 10364.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
K. Kohara, H. Yasuda, Y. Huang, N. Adachi, K. Sohya, and T. Tsumoto
A Local Reduction in Cortical GABAergic Synapses after a Loss of Endogenous Brain-Derived Neurotrophic Factor, as Revealed by Single-Cell Gene Knock-Out Method
J. Neurosci., July 4, 2007; 27(27): 7234 - 7244.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. P. Magby, C. Bi, Z.-Y. Chen, F. S. Lee, and M. R. Plummer
Single-Cell Characterization of Retrograde Signaling by Brain-Derived Neurotrophic Factor
J. Neurosci., December 27, 2006; 26(52): 13531 - 13536.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
H. Wang, S.-a. Chan, M. Ogier, D. Hellard, Q. Wang, C. Smith, and D. M. Katz
Dysregulation of brain-derived neurotrophic factor expression and neurosecretory function in mecp2 null mice.
J. Neurosci., October 18, 2006; 26(42): 10911 - 10915.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
J. D. King Jr. and S. D. Meriney
Proportion of N-Type Calcium Current Activated by Action Potential Stimuli
J Neurophysiol, December 1, 2005; 94(6): 3762 - 3770.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
T. Brigadski, M. Hartmann, and V. Lessmann
Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins
J. Neurosci., August 17, 2005; 25(33): 7601 - 7614.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
Q. Yao, M. A. Haxhiu, S. I. Zaidi, S. Liu, A. Jafri, and R. J. Martin
Hyperoxia enhances brain-derived neurotrophic factor and tyrosine kinase B receptor expression in peribronchial smooth muscle of neonatal rats
Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L307 - L314.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
W. J. Moody and M. M. Bosma
Ion Channel Development, Spontaneous Activity, and Activity-Dependent Development in Nerve and Muscle Cells
Physiol Rev, July 1, 2005; 85(3): 883 - 941.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. Villadiego, S. Mendez-Ferrer, T. Valdes-Sanchez, I. Silos-Santiago, I. Farinas, J. Lopez-Barneo, and J. J. Toledo-Aral
Selective Glial Cell Line-Derived Neurotrophic Factor Production in Adult Dopaminergic Carotid Body Cells In Situ and after Intrastriatal Transplantation
J. Neurosci., April 20, 2005; 25(16): 4091 - 4098.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
V. L. Brooks and A. F. Sved
Pressure to change? Re-evaluating the role of baroreceptors in the long-term control of arterial pressure
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2005; 288(4): R815 - R818.
[Full Text] [PDF]

Home page
 J. Neurosci.Home page
P. Baldelli, J.-M. Hernandez-Guijo, V. Carabelli, and E. Carbone
Brain-Derived Neurotrophic Factor Enhances GABA Release Probability and Nonuniform Distribution of N- and P/Q-Type Channels on Release Sites of Hippocampal Inhibitory Synapses
J. Neurosci., March 30, 2005; 25(13): 3358 - 3368.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
D.-Y. He, N. N. H. McGough, A. Ravindranathan, J. Jeanblanc, M. L. Logrip, K. Phamluong, P. H. Janak, and D. Ron
Glial Cell Line-Derived Neurotrophic Factor Mediates the Desirable Actions of the Anti-Addiction Drug Ibogaine against Alcohol Consumption
J. Neurosci., January 19, 2005; 25(3): 619 - 628.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
N. N. H. McGough, D.-Y. He, M. L. Logrip, J. Jeanblanc, K. Phamluong, K. Luong, V. Kharazia, P. H. Janak, and D. Ron
RACK1 and Brain-Derived Neurotrophic Factor: A Homeostatic Pathway That Regulates Alcohol Addiction
J. Neurosci., November 17, 2004; 24(46): 10542 - 10552.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Koyama, M. K. Yamada, S. Fujisawa, R. Katoh-Semba, N. Matsuki, and Y. Ikegaya
Brain-Derived Neurotrophic Factor Induces Hyperexcitable Reentrant Circuits in the Dentate Gyrus
J. Neurosci., August 18, 2004; 24(33): 7215 - 7224.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. Y. Yang, T. Verhovshek, and D. R. Sengelaub
Brain-Derived Neurotrophic Factor and Androgen Interact in the Maintenance of Dendritic Morphology in a Sexually Dimorphic Rat Spinal Nucleus
Endocrinology, January 1, 2004; 145(1): 161 - 168.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. P. Hibbert, S. J. Morris, N. G. Seidah, and R. A. Murphy
Neurotrophin-4, Alone or Heterodimerized with Brain-derived Neurotrophic Factor, Is Sorted to the Constitutive Secretory Pathway
J. Biol. Chem., November 28, 2003; 278(48): 48129 - 48136.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
C. Chabbert, I. Mechaly, V. Sieso, P. Giraud, A. Brugeaud, J. Lehouelleur, F. Couraud, J. Valmier, and A. Sans
Voltage-gated Na+ channel activation induces both action potentials in utricular hair cells and brain-derived neurotrophic factor release in the rat utricle during a restricted period of development
J. Physiol., November 15, 2003; 553(1): 113 - 123.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
K. Kohara, A. Kitamura, N. Adachi, M. Nishida, C. Itami, S. Nakamura, and T. Tsumoto
Inhibitory But Not Excitatory Cortical Neurons Require Presynaptic Brain-Derived Neurotrophic Factor for Dendritic Development, as Revealed by Chimera Cell Culture
J. Neurosci., July 9, 2003; 23(14): 6123 - 6131.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
B. Jiang, Y. Akaneya, Y. Hata, and T. Tsumoto
Long-Term Depression Is Not Induced by Low-Frequency Stimulation in Rat Visual Cortex In Vivo: A Possible Preventing Role of Endogenous Brain-Derived Neurotrophic Factor
J. Neurosci., May 1, 2003; 23(9): 3761 - 3770.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
J. L. Goldberg
How does an axon grow?
Genes & Dev., April 15, 2003; 17(8): 941 - 958.
[Full Text] [PDF]

Home page
 Learn. Mem.Home page
B. Lu
BDNF and Activity-Dependent Synaptic Modulation
Learn. Mem., March 1, 2003; 10(2): 86 - 98.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
G. S. Mitchell and S. M. Johnson
Plasticity in Respiratory Motor Control: Invited Review: Neuroplasticity in respiratory motor control
J Appl Physiol, January 1, 2003; 94(1): 358 - 374.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. L. Carroll
Plasticity in Respiratory Motor Control: Invited Review: Developmental plasticity in respiratory control
J Appl Physiol, January 1, 2003; 94(1): 375 - 389.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
T. Saarelainen, P. Hendolin, G. Lucas, E. Koponen, M. Sairanen, E. MacDonald, K. Agerman, A. Haapasalo, H. Nawa, R. Aloyz, et al.
Activation of the TrkB Neurotrophin Receptor Is Induced by Antidepressant Drugs and Is Required for Antidepressant-Induced Behavioral Effects
J. Neurosci., January 1, 2003; 23(1): 349 - 357.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
A. Balkowiec and D. M. Katz
Cellular Mechanisms Regulating Activity-Dependent Release of Native Brain-Derived Neurotrophic Factor from Hippocampal Neurons
J. Neurosci., December 1, 2002; 22(23): 10399 - 10407.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Haapasalo, I. Sipola, K. Larsson, K. E. O. Akerman, P. Stoilov, S. Stamm, G. Wong, and E. Castren
Regulation of TRKB Surface Expression by Brain-derived Neurotrophic Factor and Truncated TRKB Isoforms
J. Biol. Chem., November 1, 2002; 277(45): 43160 - 43167.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
T. M. Brushart, P. N. Hoffman, R. M. Royall, B. B. Murinson, C. Witzel, and T. Gordon
Electrical Stimulation Promotes Motoneuron Regeneration without Increasing Its Speed or Conditioning the Neuron
J. Neurosci., August 1, 2002; 22(15): 6631 - 6638.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
K Tucker and D. Fadool
Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent
J. Physiol., July 15, 2002; 542(2): 413 - 429.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Gartner and V. Staiger
Neurotrophin secretion from hippocampal neurons evoked by long-term-potentiation-inducing electrical stimulation patterns
PNAS, April 30, 2002; 99(9): 6386 - 6391.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. K. Cook and D. A. Fadool
Two Adaptor Proteins Differentially Modulate the Phosphorylation and Biophysics of Kv1.3 Ion Channel by Src Kinase
J. Biol. Chem., April 5, 2002; 277(15): 13268 - 13280.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Canossa, E. Giordano, S. Cappello, C. Guarnieri, and S. Ferri
Nitric oxide down-regulates brain-derived neurotrophic factor secretion in cultured hippocampal neurons
PNAS, February 20, 2002; (2002) 42504299.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
K. L. Eagleson, L. D. Fairfull, S. R. J. Salton, and P. Levitt
Regional Differences in Neurotrophin Availability Regulate Selective Expression of VGF in the Developing Limbic Cortex
J. Neurosci., December 1, 2001; 21(23): 9315 - 9324.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
H. Boukhaddaoui, V. Sieso, F. Scamps, and J. Valmier
An Activity-Dependent Neurotrophin-3 Autocrine Loop Regulates the Phenotype of Developing Hippocampal Pyramidal Neurons before Target Contact
J. Neurosci., November 15, 2001; 21(22): 8789 - 8797.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
P. A. Heppenstall and G. R. Lewin
BDNF but not NT-4 is required for normal flexion reflex plasticity and function
PNAS, July 3, 2001; 98(14): 8107 - 8112.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
I. J. Lever, E. J. Bradbury, J. R. Cunningham, D. W. Adelson, M. G. Jones, S. B. McMahon, J. C. G. Marvizon, and M. Malcangio
Brain-Derived Neurotrophic Factor Is Released in the Dorsal Horn by Distinctive Patterns of Afferent Fiber Stimulation
J. Neurosci., June 15, 2001; 21(12): 4469 - 4477.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
G. S. Mitchell, T. L. Baker, S. A. Nanda, D. D. Fuller, A. G. Zabka, B. A. Hodgeman, R. W. Bavis, K. J. Mack, and E. B. Olson Jr.
Physiological and Genomic Consequences of Intermittent Hypoxia: Invited Review: Intermittent hypoxia and respiratory plasticity
J Appl Physiol, June 1, 2001; 90(6): 2466 - 2475.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
G. S. Pollock, E. Vernon, M. E. Forbes, Q. Yan, Y.-T. Ma, T. Hsieh, R. Robichon, D. O. Frost, and J. E. Johnson
Effects of Early Visual Experience and Diurnal Rhythms on BDNF mRNA and Protein Levels in the Visual System, Hippocampus, and Cerebellum
J. Neurosci., June 1, 2001; 21(11): 3923 - 3931.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
T. A. Brosenitsch and D. M. Katz
Physiological Patterns of Electrical Stimulation Can Induce Neuronal Gene Expression by Activating N-Type Calcium Channels
J. Neurosci., April 15, 2001; 21(8): 2571 - 2579.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
K. Kohara, A. Kitamura, M. Morishima, and T. Tsumoto
Activity-Dependent Transfer of Brain-Derived Neurotrophic Factor to Postsynaptic Neurons
Science, March 23, 2001; 291(5512): 2419 - 2423.
[Abstract] [Full Text]

Home page
 J. Neurosci.Home page
J. T. Erickson, T. A. Brosenitsch, and D. M. Katz
Brain-Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor Are Required Simultaneously for Survival of Dopaminergic Primary Sensory Neurons In Vivo
J. Neurosci., January 15, 2001; 21(2): 581 - 589.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Canossa, E. Giordano, S. Cappello, C. Guarnieri, and S. Ferri
Nitric oxide down-regulates brain-derived neurotrophic factor secretion in cultured hippocampal neurons
PNAS, March 5, 2002; 99(5): 3282 - 3287.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Yin, G. M. Edelman, and P. W. Vanderklish
The brain-derived neurotrophic factor enhances synthesis of Arc in synaptoneurosomes
PNAS, February 19, 2002; 99(4): 2368 - 2373.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
K. R. Leslie, S. B. Nelson, and G. G. Turrigiano
Postsynaptic Depolarization Scales Quantal Amplitude in Cortical Pyramidal Neurons
J. Neurosci., October 1, 2001; 21(19): RC170 - RC170.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (130)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Balkowiec, A.
Right arrow Articles by Katz, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Balkowiec, A.
Right arrow Articles by Katz, D. M.

-

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help
-
Copyright 2009 by Society for Neuroscience ONLINE ISSN: 1529-2401
-