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Originally published In Press as doi:10.1074/jbc.M305872200 on August 26, 2003

J. Biol. Chem., Vol. 278, Issue 45, 44097-44102, November 7, 2003
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A Rapid Increase in the Total Number of Cell Surface Functional GABAA Receptors Induced by Brain-derived Neurotrophic Factor in Rat Visual Cortex*

Yoshito Mizoguchi{ddagger}, Takashi Kanematsu§, Masato Hirata§, and Junichi Nabekura{ddagger}

From the {ddagger}Cellular and Systems Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 and the §Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan

Received for publication, June 4, 2003 , and in revised form, August 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The number of postsynaptic {gamma}-aminobutyric acid type A (GABAA) receptors is a fundamental determinant of the variability of inhibitory synaptic responses in the central nervous system. In rat visual cortex, [3H]SR-95531 binding assays revealed that brain-derived neurotrophic factor (BDNF), one of the neurotrophins, induced a rapid increase in the total number of cell surface GABAA receptors, through the activation of Trk B receptor tyrosine kinases. We also demonstrated that BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents, using nystatin-perforated patch clamp recordings, in visual cortical layer 5 pyramidal neurons freshly isolated from P14 rats. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and phospholipase C-{gamma}. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses induced by BDNF. The selective increase in mean miniature inhibitory postsynaptic (mIPSC) current amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABAA receptors in visual cortical pyramidal neurons. These results suggest that BDNF could alter the number of cell surface GABAA receptors in a region-specific manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The {gamma}-aminobutyric acid type A (GABAA)1 receptors are ligand-gated ion channels that mediate fast synaptic inhibition in the central nervous system (1). GABAergic transmission is characterized by high variability of synaptic responses attributable to many factors at both pre- and postsynaptic sites (2).

The elevation of intracellular Ca2+ levels, leading to the activation of protein kinases or phosphatases, either potentiates (3–5) or suppresses (6–8) the postsynaptic GABAA receptor responses. These complex results suggest that region-specific phosphorylation or dephosphorylation processes regulate GABAergic function in a cell-specific manner.

The number of postsynaptic GABAA receptors is a fundamental determinant of the variability of postsynaptic responses (2). The endocytosis of GABAA receptors occurs both in hippocampal (9) and in cortical (10) neurons. In contrast, the ubiquitin-like protein Plic-1 increases the number of GABAA receptors available for the recruitment to the plasma membrane (11). In hippocampus, insulin induces a rapid recruitment of functional GABAA receptors to the postsynaptic membranes through the activation of insulin receptor tyrosine kinase, thereby increasing the amplitude of GABAA receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) without effects on their time courses (12).

Brain-derived neurotrophic factor (BDNF), one of the neurotrophins, rapidly modulates postsynaptic GABAA receptor function. In hippocampus of postnatal day (P) 14 rat, BDNF acutely inhibits postsynaptic GABAA responses by elevating intracellular Ca2+ levels via the activation of Trk B receptor tyrosine kinase and subsequent phospholipase C (PLC) {gamma} phosphorylation (13, 14), but at P6, BDNF reversibly potentiates postsynaptic GABAA responses (14).

BDNF also alters the number of cell surface GABAA receptors. In cultured hippocampal (15) and cerebellar granule (16) cells, BDNF rapidly induces an internalization of postsynaptic cell surface GABAA receptors, through the Trk B receptor tyrosine kinase activation, thereby decreasing the amplitudes of mIPSCs.

In visual cortex, the timing of the critical period for ocular dominance columns formation (which is normally between P23 and P33) is determined, not simply by visual stimuli but also by the maturation of the inhibitory circuits themselves (17). Mice overexpressing BDNF exhibit a precocious maturation of GABAergic inhibition in visual cortex and accelerated decline of the critical period for ocular dominance plasticity (18).

In developing rat visual cortex, the expression of Trk B receptor is most prominent in layer 5 pyramidal neurons (19), and the levels of both BDNF mRNA (20) and BDNF protein (21) are highest in layers 2, 3, and 5. In layer 5 of rat visual cortex, the level of BDNF mRNA rapidly increases around P14 (when the eyes have just opened) (20) and thereafter decreases to reach a plateau level at P18 (22). Thus, increasing evidence suggests that BDNF has important roles on GABAergic transmission in developing visual cortex, but the potent mechanisms underlying these effects are unclear.

We tested the rapid effect of BDNF on the cell surface expression of GABAA receptors in visual cortex of P14 rats, using [3H]SR-95531 binding assays. [3H]SR-95531 binding assays revealed that BDNF induced a rapid increase in the total number of cell surface GABAA receptors in rat visual cortex, through the activation of Trk B receptor tyrosine kinases.

We also demonstrated that BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents, using nystatin-perforated patch clamp recordings, in visual cortical layer 5 pyramidal neurons isolated from P14 rats. The selective increase in mean mIPSC amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABAA receptors in visual cortical pyramidal neurons.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
All experiments conformed to the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan, and all efforts were made to minimize the number of animals used and their suffering.

[3H]SR-95531 Binding Assays—Visual cortical cells were freshly isolated from rats using papain dissociation system (Worthington Biochemical Corporation, Lakewood, NJ) intended for the cell dissociation and culture procedures developed by Huettner and Baughman (23), by which high yields of viable and morphologically intact neurons were dissociated from visual cortex of P1–15 rats. In brief, 14-day-old Wistar rats were decapitated under pentobarbital anesthesia (50 mg kg–1, intraperitoneally). Brains were quickly removed and bathed in cold incubation medium bubbled with 95% O2, 5% CO2. Blocks of tissue comprising visual cortex were removed from the occipital cortex of both hemispheres and were gently minced or cut into small pieces. The tissue was placed in the Earle's balanced salt solution, equilibrated with 95% O2, 5% CO2, including papain (at a final concentration of 20 units ml–1) and DNase (0.005%). The mixed solution was incubated for 15 min at 37 °C. After albumin-ovomucoid inhibitor was added to the mixed solution, the mixture was gently triturated with 10-ml pipette three times and centrifuged at 1,100 r.p.m. for 5 min. The supernatant was discarded, and the obtained visual cortical cells were immediately resuspended with external standard solution. Cell viability of the obtained visual cortical cells, determined by the trypan blue exclusion method (24), was 87.8 ± 2.5% (n = 4 rats). The trypan blue exclusion method was as follows. The cell suspension was incubated for 5 min in 0.4% trypan blue solution (at a final concentration of 0.2%; Sigma) and washed twice in external standard solution. In each experiment, five randomly chosen optical fields (at least 250 cells in each field) were analyzed by a phase contrast, bright field microscopy at low magnification. Viable cells are determined as dye-negative, whereas nonviable cells with damaged cell membrane are stained blue. Cell viability was expressed as a percent of the number of total viable (unstained) cells relative to the total (unstained and stained) cells. The final value was obtained as mean ± S.E. of four separate experiments.

Visual cortical cells were incubated in standard solution with vehicle (standard solution), BDNF (20 ng ml–1) alone, or BDNF plus K252a (200 nM) for 10 min at room temperature. The cells were washed with standard solution by centrifugation at 1,000 r.p.m. for 5 min and resuspended in ice-cold standard solution. The protein concentration of the cell suspension was measured using a Lowry method (25) with bovine serum albumin as the standard protein. Twenty µl of cell suspension was added to the assay buffer (50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% bovine serum albumin) containing 20 nM [3H]SR-95531, a competitive GABAA receptor antagonist (specific radioactivity 2,186.7 GBq mmol–1) (PerkinElmer Life Sciences) with or without 10 µM SR-95531, and incubated for 20 min at 4 °C (26). To calculate the specific binding, nonspecific binding values (determined in the presence of 10 µM SR-95531) were subtracted from the total binding values (in the absence of 10 µM SR-95531). For Scatchard analysis (27), assay buffers containing 5–30 nM [3H]SR-95531 were used. Samples were filtered under negative pressure over Whatman GF/C filters, which were then rapidly rinsed two times with 5 ml of ice-cold assay buffer. Radioactivity on filters was measured by liquid scintillation counting.

Electrophysiological Recordings—Visual cortical pyramidal neurons from 14-day-old Wistar rats were freshly dissociated using procedures similar to those described previously (14, 28). Briefly, rats were decapitated under pentobarbital anesthesia. Brains were quickly removed and transversely sliced at a thickness of 370 µm (VT-1000; Leica; Nussloch; Germany). The slices were kept in the incubation medium saturated with 95% O2 and 5% CO2 at room temperature for at least 1 h. After 30 min of enzyme digestion, the slices were transferred into a 35-mm culture dish, and layer 5 of the visual cortex was identified under a binocular microscope (x20, SMZ-1; Nikon, Tokyo, Japan). A fire-polished glass pipette was touched lightly onto the surface of the region and was vibrated horizontally at 30–50 Hz for 3 min using an apparatus developed in our laboratory (29, 30). Slices were removed from the dish, and dissociated pyramidal neurons with a large pyramidal-shaped soma, a prominent apical dendrite, and a skirt of basal dendrites (31, 32) adhered to the bottom of the dish within 20 min (Fig. 3A).



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  		FIG. 3.
BDNF-induced sustained potentiation of GABAA responses in visual cortical pyramidal neurons isolated from rats. A, photomicrograph of a freshly isolated visual cortical pyramidal neuron. B, representative inward currents in response to 10 µM GABA application for 3 s. Drug solutions were applied, using the Y-tube perfusion system, allowing rapid exchange of the solution surrounding a cell. The three traces shown in the left panel show a control response (left), a response in the presence of 5 ng ml–1 BDNF (middle), and the response obtained following washout of BDNF (right). In C, the graph on the right shows the time course of the effects of BDNF on the amplitude of the GABAA receptor response elicited by the application of GABA each 2.5 min. The period of the application of BDNF is indicated by a filled bar. In C, filled squares represent the response to BDNF (5 ng ml–1), whereas the filled diamonds represent the response to 20 ng ml–1 BDNF. Data are presented as mean ± S.E., normalized to the amplitude of the response just before BDNF application.

 

Electrical measurements were performed using the nystatin-perforated patch recording method (14, 28, 33). All recordings were performed using voltage clamp at a holding potential of –50 mV, using patch clamp amplifier (EPC-7; List Biologic, Campbell, CA). Patch pipettes were made from borosilicate capillary glass tubes (G-1.5; Narishige, Tokyo, Japan) in two stages on a vertical pipette puller (PB-7; Narishige). The resistance between the patch pipette filled with the internal solution and the reference electrode in the normal external solution was 4–6 megaohms. Neurons were visualized with phase-contrast equipment on an inverted microscope (Diaphoto; Nikon). Current and voltage were continuously monitored on an oscilloscope (VC-6725; Hitachi, Tokyo, Japan) and a pen recorder (Recti-Horiz-8K; Sanei, Tokyo, Japan) and recorded on a digital-audio tape recorder (RD-120TE; TEAC). Membrane currents were filtered at 1 kHz (E-3201A Decade Filter; NF Electronic Instruments, Tokyo, Japan), and data were digitized at 4 kHz. For mIPSCs recording, the extracellular solution was supplemented with 6-cyano-nitroquinoxaline-2,3-dione (10 µM) and 2-amino-5-phosphonopentanoic acid (20 µM). The mIPSCs were completely blocked by the competitive GABAA receptor antagonist, bicuculline (10 µM; data not shown). mIPSCs were detected and analyzed using MiniAnalysis program (Synaptosoft, NJ). All experiments were performed at room temperature (27 ± 1 °C). All data are expressed as mean ± S.E., and statistical analysis was performed using Student's t test, with p < 0.05 being considered significantly different.

Solutions and Drugs—The ionic composition of the internal (patch pipette) solution was 40 mM methanesulphonic acid potassium salt, 110 mM KCl, 10 mM HEPES. The pH of internal solution was adjusted to 7.2 with Tris-OH. Nystatin (Sigma) was dissolved in acidified methanol at 10 mg ml–1. This stock solution was diluted with internal pipette solution just before use to a final concentration of 100–200 µgml–1. The ionic composition of the incubation medium was 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 24 mM NaHCO3, 2.4 mM CaCl2, 1.3 mM MgSO4, and 10 mM glucose. Following equilibration with 95% O2, 5% CO2, the pH was 7.4. The ionic composition of the external standard solution was 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES. The pH of the external standard solution was adjusted to 7.4 with Tris-OH. Drug solutions were applied, using the Y-tube perfusion system, allowing rapid exchange of the solution surrounding a cell within 20 ms (34–36).

The drugs used in the present study included GABA, 2-amino-5-phosphonopentanoic acid, 6-cyano-nitroquinoxaline-2,3-dione, bicuculline, diazepam, muscimol, and BAPTA-AM (Sigma), TTX (Wako, Tokyo, Japan), K252a, U73122 [GenBank] , and KN-62 (Calbiochem). Drugs that are insoluble in water were first dissolved in dimethyl sulfoxide (Me2SO), and then diluted in the external solution. The final concentration of Me2SO was always less than 0.1% and did not affect neuronal responses observed in the present study. Human recombinant BDNF (Sigma) was dissolved (100 µg ml–1) in phosphate buffer solution containing 0.1% bovine serum albumin and stored below –20 °C. Before the experiment, this stock solution was diluted with external solution to obtain the final concentration (5 or 20 ng ml–1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
A Rapid Increase in the Total Number of Cell Surface GABAA Receptors Induced by BDNF in Rat Visual Cortex—We examined the expression of cell surface GABAA receptor {alpha}/{beta} subunits of BDNF-(20 ng ml–1) treated and -untreated cells in visual cortex of P14 rats, by measuring the binding of the hydrophilic GABAA receptor competitive antagonist [3H]SR-95531. The hydrophilic nature of [3H]SR-95531 restricts its binding to GABAA receptors on the cell surface (37, 38).

To examine whether BDNF modulates the cell surface GABAA receptor number or its affinity or both, total specific [3H]SR-95531 radioligand binding activity expressed by visual cortical cells was determined by Scatchard analysis. In 20 ng/ml BDNF-treated cells, total binding (maximal binding capacity, Bmax) was increased by 2.25-fold as compared with the BDNF-untreated control cells (n = 4 rats), whereas the dissociation constant (Kd) remained unchanged (n = 4; p > 0.05, Fig. 1). The values are summarized in Fig. 1B, and a representative Scatchard plot is shown in Fig. 1A. This result suggests that BDNF induced a rapid increase in the total number of cell surface GABAA receptors in rat visual cortex, without effects on the receptor affinity.



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  		FIG. 1.
BDNF-induced rapid increase in the total binding of cell surface GABAA receptors in rat visual cortex, without effects on GABAA receptors affinity. In A, a representative Scatchard plot is shown for BDNF-untreated (open squares) and -treated (filled circles) cells. For Scatchard analysis, assay buffers containing 5–30 nM [3H]SR-95531, a competitive GABAA receptor antagonist, were used. Each point is the mean of triplicate. B, a summary of the [3H]SR-95531 binding parameters (Kd and Bmax) in BDNF-untreated (Control; n = 4) and -treated cells (BDNF; n = 4).

 

BDNF specifically binds to Trk B, a neurotrophin receptor, which contains a catalytic domain of tyrosine kinase (39). The specific binding of 20 nM [3H]SR-95531 to BDNF-treated cells was significantly increased as compared with the control cells, by 72.6 ± 5.6% (n = 4 rats; p < 0.005). On the other hand, the inclusion of 200 nM K252a, a membrane-permeant inhibitor of Trk receptor tyrosine kinases (40, 41) to the BDNF-treated cells, blocked the elevation of [3H]SR-95531 binding (by 6.2 ± 2.4%, n = 4, Fig. 2). All together, these results suggest that BDNF induced a rapid increase in the total number of cell surface GABAA receptors in rat visual cortex, through the activation of Trk B receptor tyrosine kinases.



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  		FIG. 2.
Involvement of the activation of Trk B receptor tyrosine kinases in the rapid increase in the binding of cell surface GABAA receptors induced by BDNF. Binding of 20 nM [3H]SR-95531 to BDNF-treated cells was measured in the presence or absence of 200 nM K252a, a membrane-permeant Trk receptor tyrosine kinase inhibitor, using 20 µl of cell suspension (protein concentration was 15–30 µg). Nonspecific binding (80–90 dpm) was also assayed in the presence of cold 25 µM SR-95531 and was subtracted to provide the specific binding in the y axis. The symbols (open circles, open triangles, open squares, and filled diamonds) represent specific binding of four independent assays done in duplicate. Each value was normalized as dpm/20 µg of protein.

 

Rapid Potentiation of GABAA Responses Induced by BDNF in Rat Visual Cortical Pyramidal Neurons—We next tested whether BDNF rapidly potentiates GABAA responses in pyramidal neurons. Fresh pyramidal neurons were dissociated from layer 5 of the visual cortex isolated from P14 rats (Fig. 3A). Voltage clamp recordings were obtained at a holding potential of –50 mV using nystatin-perforated patch clamp recordings, which preserves Ca2+ and other soluble intracellular constituents intact. The application of 10 µM GABA or muscimol, using the Y-tube perfusion system, to these neurons induced a GABAA receptor-mediated inward current (Fig. 3B, control), which was completely blocked by 10 µM bicuculline (n = 3, data not shown). In the presence of BDNF (5 ng ml–1), this GABAA response was potentiated in every neuron tested (by 71.6 ± 20.0%, n = 12; Fig. 3B), within 5 min of BDNF application, and in the longest recordings obtained, persisted more than 60 min after BDNF washout (Fig. 3C). A higher BDNF concentration (20 ng ml–1) caused an even more marked potentiation (by 154.3 ± 26.6%, n = 12). These results show that BDNF rapidly induces a sustained potentiation of GABAA receptor-mediated currents in rat visual cortical pyramidal neurons.

Intracellular Regulatory Pathway Involved in the Potentiating Effect of BDNF on GABAA Responses—In the presence of K252a (200 nM), BDNF (20 ng ml–1) failed to potentiate the GABAA responses in visual cortical pyramidal neurons (n = 5; Fig. 4, A and B). K252a (200 nM), by itself, had no effect on the control GABAA responses (n = 5). Thus, BDNF potentiated GABAA responses through the activation of Trk B receptor tyrosine kinases.



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  		FIG. 4.
Intracellular regulatory pathway involved in the potentiating effect of BDNF on GABAA responses in visual cortical pyramidal neurons. A, representative traces showing that, in the presence of K252a (200 nM), BDNF (20 ng ml–1) failed to potentiate GABAA responses. B, time course of the effects of BDNF on GABAA receptor responses in the presence of K252a. C, time course of the effects of BDNF (20 ng ml–1) on the GABAA responses, in the presence of BAPTA-AM (open squares) or 5 µM U73122 [GenBank] (filled squares). D, time course of the effects of BDNF on GABAA receptor responses in the presence of 5 µM KN-62 (open circles). In the inset, representative inward currents are shown as a response in the presence of KN-62 (a) and a response following the application of 20 ng ml–1 BDNF (b).

 

In addition to causing tyrosine kinase activity, the binding of BDNF to the Trk B receptor activates many intracellular signaling pathways, including the PLC-{gamma} pathway, leading to generation of inositol trisphosphate and the mobilization of intracellular Ca2+ from endoplasmic reticulum (42, 43). BDNF stimulates PLC-{gamma}1 phosphorylation within 20 s (42) and elevates intracellular Ca2+ levels within 1–2 min (44, 45).

In the presence of 150 µM BAPTA-AM, a membrane-permeant Ca2+ chelator, BDNF (20 ng ml–1) failed to potentiate the GABAA responses (n = 5; Fig. 4C). In the presence of U73122 [GenBank] (5 µM), a membrane-permeant PLC-{gamma} inhibitor (46), again BDNF (20 ng ml–1), failed to potentiate the GABAA responses (n = 5; Fig. 4C).

In mouse cortical (5), rat cerebellar Purkinje (4), and immature rat hippocampal neurons (14), an increase in intracellular Ca2+ enhances postsynaptic GABAA responses via an activation of Ca2+/calmodulin-dependent protein kinase 2 (CaMK-2). In the presence of KN-62 (5 µM), a membrane-permeant specific CaMK-2 inhibitor (48), BDNF (20 ng ml–1), potentiated the GABAA responses.

These results suggest that BDNF potentiated GABAA responses through the activation of Trk B receptor tyrosine kinase and PLC-{gamma}. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses induced by BDNF. In contrast, CaMK-2 was not involved in the sustained potentiation of GABAA responses induced by BDNF in rat visual cortical pyramidal neurons.

The application of K252a (200 nM) did not affect the GABAA responses potentiated by BDNF (20 ng ml–1) (n = 3; Fig. 5A). In addition, either U73122 [GenBank] (5 µM) or BAPTA-AM (150 µM) failed to suppress the GABAA responses potentiated by BDNF (Fig. 5B).



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  		FIG. 5.
The activation of Trk B-PLC {gamma} was not important for the maintenance of the BDNF-induced sustained potentiation of GABAA responses. A, time course of the effect of 200 nM K252a on the GABAA responses potentiated by BDNF. B, effects of K252a, U73122 [GenBank] , and BAPTA-AM on the maintenance of the BDNF-induced sustained potentiation of GABAA responses. Each column represents the normalized GABAA responses obtained at 10 min after a 10-min BDNF treatment.

 

Potentiation of mIPSCs by BDNF in Rat Visual Cortical Pyramidal Neurons—We next recorded mIPSCs in rat visual cortical pyramidal neurons at P14 to examine whether BDNF could also potentiate the response of GABAA receptors to released GABA in the synaptic cleft. We used an enzyme-free dissociation procedure so as to preserve functional presynaptic boutons adherent to the isolated neurons (49). BDNF (20 ng ml–1) caused a significant increase, of approximately 78%, in the mean amplitude of mIPSCs (from –19.3 ± 2.2 pA to –34.3 ± 2.7 pA, p < 0.001, n = 5). BDNF increased mIPSC amplitude without affecting the mIPSC frequency (67.3 ± 2.3 events/3 min in control conditions and 68.0 ± 2.1 events/3 min during BDNF application, p > 0.1, n = 5). Fig. 6, A and B, show representative currents and corresponding amplitude histograms from one of these neurons. Thus, BDNF potentiates the GABAA receptor responses to both exogenously applied and released GABA in the synaptic cleft in rat visual cortical pyramidal neurons.



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  		FIG. 6.
Potentiation of the amplitudes of mIPSCs in rat visual cortical pyramidal neurons by BDNF. A, representative traces of GABAA receptor-mediated mIPSCs, at a holding potential of –50 mV, before (control; left panel) and 5 min after (20 ng ml–1; right) addition of BDNF. B, histogram showing the mIPSC amplitude distribution of all mIPSCs observed during a 3-min recording period in the absence (control; left panel) and presence of BDNF (right panel). C, representative traces of individual mIPSCs control, in the presence of diazepam (1 µM) and BDNF.

 

We compared the actions of BDNF on mIPSCs with a well established GABAA receptor modulator, the benzodiazepine, diazepam. Diazepam (1 µM), which increased the affinity of GABAA receptors (50), prolonged the mIPSCs decay without markedly affecting peak amplitude (n = 5; Table I and Fig. 6C). In contrast, BDNF caused a significant increase in mean mIPSC amplitude and no change in either the mIPSC rise time or the decay time constant (n = 5). If GABA released from a single vesicle is sufficient to nearly saturate the cluster of postsynaptic GABAA receptors in our experimental condition, then one possible reason for the increase in mIPSC amplitude is that BDNF induces a rapid increase in the total number of cell surface GABAA receptors in rat visual cortical pyramidal neurons.


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  		TABLE I
Effect of BDNF or diazepam on mIPSCs

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The present experiments demonstrated that BDNF induced a rapid increase in the total number of cell surface GABAA receptors in visual cortex of P14 rats, through the activation of Trk B receptor tyrosine kinases. In visual cortical pyramidal neurons isolated from P14 rats, BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and PLC-{gamma}. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses by BDNF. The selective increase in mean mIPSC amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABAA receptors in visual cortical pyramidal neurons.

In the binding assays, we used visual cortical cells as preparations, which include not only pyramidal neurons but also interneurons or glial cells. A rapid increase in the total number of cell surface GABAA receptors induced by BDNF that we observed might not simply reflect the BDNF effect on pyramidal neurons.

In hippocampus at P6, Ca2+/calmodulin-dependent protein kinase 2 (CaMK-2) plays important roles in the reversible potentiation of postsynaptic GABAA responses by BDNF (14). In visual cortical pyramidal neurons isolated from P14 rats, CaMK-2 was not involved in the sustained potentiation of GABAA responses induced by BDNF (Fig. 4D). Thus, different intracellular regulatory pathways seem to underlie the potentiating effect of BDNF on GABAA responses in hippocampus of P6 rat and visual cortex at P14.

In rat hippocampus at P14, BDNF acutely inhibits postsynaptic GABAA responses by elevating postsynaptic Ca2+ levels via the activation of Trk B receptor tyrosine kinase and PLC-{gamma} (13, 14). A similar transduction mechanism seems to underlie the potentiating effect of BDNF on GABAA responses observed in isolated visual cortical pyramidal neurons. However, the activation of the Trk B-PLC {gamma} pathway was not important for the maintenance of the sustained potentiation of GABAA responses induced by BDNF (Fig. 5). This also supports the idea that BDNF increases the total number of functional GABAA receptors in visual cortical pyramidal neurons.

In cultured hippocampal (15) and cerebellar granule (16) cells, BDNF induces a rapid down-regulation of cell surface GABAA receptors. We proposed an opposite effect of BDNF in visual cortex, i.e. a rapid increase in the total number of cell surface GABAA receptors. Thus, in the CNS, BDNF may rapidly increase or decrease the cell surface expression of GABAA receptors through the activation of Trk B receptor tyrosine kinases in a region-specific manner.

In our experimental conditions, GABA released from a single vesicle is likely to be sufficient to saturate the cluster of postsynaptic GABAA receptors, as reported at other central nervous system synapses (51, 52). However, the degree of receptor occupancy and the use of benzodiazepines for determining postsynaptic GABAA receptor occupancy remain controversial. In layer 5 pyramidal neurons of visual cortex, GABAA receptors were not shown to be saturated by the synaptically released GABA (53, 54), but in layers 2 and 3, GABAA receptors were fully occupied (54). Zolpidem, a benzodiazepine, enhances the amplitudes of mIPSCs if applied at room temperature (22–25 °C) but not at physiological temperature (35 °C), suggesting that zolpidem cannot be used as a tool to determine the GABAA receptor occupancy at physiological temperature (53). The variation in the GABA concentration in the synaptic cleft underlies the amplitude variability of postsynaptic responses (55). There might be differences in the GABA concentration in the synaptic cleft between our experimental conditions and those in previous reports.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
BDNF induced a rapid increase in the total number of cell surface GABAA receptors in visual cortex of P14 rats, through the activation of Trk B receptor tyrosine kinases. In visual cortical pyramidal neurons isolated from P14 rats, BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and PLC-{gamma}. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses induced by BDNF. The selective increase in mean mIPSC amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABAA receptors in visual cortical pyramidal neurons.

In visual cortex, BDNF plays important roles in the formation of ocular dominance columns (56, 57). Mice overexpressing BDNF exhibit a precocious maturation of GABAergic inhibition in the visual cortex and an accelerated decline of the critical period for ocular dominance plasticity (which is normally between P23 and P33) (18). Both the onset and the close of this critical period are determined, not simply by visual stimuli, but also by the maturation of the inhibitory circuits themselves (17, 47, 58, 59). A rapid increase in the total number of cell surface GABAA receptors induced by BDNF in visual cortex of P14 rats may be important for the maturation of the inhibitory circuits and the development of visual cortex.


   FOOTNOTES
 
* This work was supported by Grants-in-aid for Scientific Research on Priority Areas (C)-Advanced Brain Project (15016082) and Research Grants (15390065, 15650076) from the Ministry of Education, Culture, Sports, and Science and Technology, Japan to J. Nabekura. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 81-92-642-6090; Fax: 81-92-642-6094; E-mail: nabekura{at}mailserver.med.kyushu-u.ac.jp.

1 The abbreviations used are: GABAA, {gamma}-aminobutyric acid type A; BDNF, brain-derived neurotrophic factor; mIPSC, miniature inhibitory postsynaptic current; PLC, phospholipase C; BAPTA-AM, 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy-methyl) ester; P, postnatal day. Back


   ACKNOWLEDGMENTS
 
We thank A. Moorhouse for critical comments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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