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The Journal of Neuroscience, July 2, 2003, 23(13):5662-5673
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Brain-Derived Neurotrophic Factor Mediates Activity-Dependent Dendritic Growth in Nonpyramidal Neocortical Interneurons in Developing Organotypic Cultures
Xiaoming Jin,1 Hang Hu,1 Peter H. Mathers,2,3,4,5 andAriel Agmon1,5 Departments of 1Neurobiology and Anatomy, 2Otolaryngology, 3Biochemistry and Molecular Pharmacology, and 4Ophthalmology, and 5The Sensory Neuroscience Research Center, West Virginia University, Morgantown, West Virginia 26506-9128
Abstract
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Abstract
Introduction
Brain-derived neurotrophic factor (BDNF) promotes postnatal maturation of GABAergic inhibition in the cerebral and cerebellar cortices, and its expression and release are enhanced by neuronal activity, suggesting that it acts in a feedback manner to maintain a balance between excitation and inhibition during development. BDNF promotes differentiation of cerebellar, hippocampal, and neostriatal inhibitory neurons, but its effects on the dendritic development of neocortical inhibitory interneurons remain unknown. Here, we show that BDNF mediates depolarization-induced dendritic growth and branching in neocortical interneurons. To visualize inhibitory interneurons, we biolistically transfected organotypic cortical slice cultures from neonatal mice with green fluorescent protein (GFP) driven by the glutamic acid decarboxylase (GAD)67 promoter. Nearly all GAD67–GFP-expressing neurons were nonpyramidal, many contained GABA, and some expressed markers of neurochemically defined GABAergic subtypes, indicating that GAD67–GFP-expressing neurons were GABAergic. We traced dendritic trees from confocal images of the same GAD67–GFP-expressing neurons before and after a 5 d growth period, and quantified the change in total dendritic length (TDL) and total dendritic branch points (TDBPs) for each neuron. GAD67–GFP-expressing neurons growing in control medium exhibited a 20% increase in TDL, but in 200 ng/ml BDNF or 10 mM KCl, this increase nearly doubled and was accompanied by a significant increase in TDBPs. Blocking action potentials with TTX did not prevent the BDNF-induced growth, but antibodies against BDNF blocked the growth-promoting effect of KCl. We conclude that BDNF, released by neocortical pyramidal neurons in response to depolarization, enhances dendritic growth and branching in nearby inhibitory interneurons.
Key words: biolistic; BDNF; GABA; GAD67; gene gun; inhibitory interneuron; neocortical development; neurotrophins; organotypic slice
Introduction
GABAergic inhibition in the rodent cerebral cortex, although already functional at birth (Agmon et al., 1996; Wells et al., 2000
An attractive hypothesis that accounts for the delayed maturation of GABAergic inhibition suggests that inhibition is up-regulated by neuronal excitation, thus establishing a negative feedback loop that counteracts the developmental increase in glutamatergic excitation, albeit with a lag (Marty et al., 1997
BDNF promotes neurochemical and dendritic differentiation of inhibitory neurons of the cerebellum, neostriatum, and hippocampus (Nawa et al., 1994
Materials and Methods
Organotypic culture preparation. Organotypic cortical slice cultures were prepared as described previously (Stoppini et al., 1991Gene gun-mediated transfection. GAD67–GFP plasmid DNA was cloned from a 10.3 kb segment of the mouse GAD67 promoter region (Szabo et al., 1996
Confocal microscopy. Confocal images were first acquired at least 2 d after gene gun transfection to allow complete GFP filling of distal dendrites. Cultured brain slices were removed by cutting out the underlying Millicell membrane (leaving it adherent to the culture) and placed under sterile conditions in an imaging chamber filled with freshly oxygenated ACSF at room temperature. The imaging chamber was made of two round glass coverslips separated by 4 mm and held by a stainless-steel frame with a 17 mm-wide circular opening to allow imaging. Well separated neurons with bright GFP expression were selected for imaging. Neurons were imaged with a 20x, 0.7 numerical aperture (NA) dry objective on an inverted Zeiss (Thornwood, NY) LSM 510 laser-scanning confocal microscope, using the 488 nm argon laser line and a 505–550 nm bandpass emission filter. Image stacks (typically 10–20 optical sections per stack) were collected at 1.5–2.5 µm z-axis steps through the full extent of the dendritic tree and saved for off-line tracing and analysis. Images were taken with the lowest practical laser intensity and the shortest practical illumination time to limit photodynamic damage, and slices were kept outside the incubator for <1 hr per session. This imaging protocol was found not to cause any apparent ill effects to the imaged neurons or the slice culture, because most neurons remained viable and exhibited the same general dendritic morphology when imaged again 5 or 10 d later (see Fig. 3), and some remained viable even after repeated imaging over several weeks (data not shown). At the end of each imaging session, low-power (10x) images were taken in fluorescence and transmission modes to record the locations of the GFP-expressing neurons in the cortex relative to the pia and white matter. The slices with their adherent membranes were then placed back into multiwell plates under sterile conditions and returned to the incubator. The same neurons were imaged again 5 d later.
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Figure 3. Dendritic growth of GAD67–GFP-expressing neurons in control medium occurred only in younger cultures. A, Confocal projection of a representative GAD67–GFP-expressing neuron first imaged at EP7 (left) and again 5 d later (right). B, Another neuron first imaged at EP22 (left) and again 10 d later (right). Scale bar: (in B) A, B, 100 µm. C, TDL growth ratio of all of the neurons in our sample during a 5 d period in normal medium, plotted against the equivalent age at first imaging. A TDL ratio of 1 (dashed line) indicates no change. Note that dendritic growth was observed in neurons first imaged at EP7–EP9 (left of the vertical dotted line), but there was no change on average in neurons first imaged at later ages (right of the vertical dotted line).
Pharmacological treatments. In some slices, one or more of the following were added to the culture medium: 200 ng/ml recombinant human BDNF (Alomone Labs, Jerusalem, Israel), 250 ng/ml recombinant human neurotrophin (NT)4/5 (courtesy of Genentech, South San Francisco, CA), 50 µg/ml rabbit anti-BDNF polyclonal antibody (Chemicon, Temecula, CA), 200 nM K252a, and 1 µM tetrodotoxin (TTX) (both from Alomone Labs). Treatments were refreshed on medium change. Pharmacological treatments started the day after the first imaging session; therefore, treatments lasted 4 of the 5 d between imaging sessions. Control experiments (slices cultured in normal medium) were interspersed throughout the treatment experiments.
Biocytin filling and histochemistry. To verify that GFP fluorescence revealed the full dendritic morphology, a subset of GFP-expressing neurons were filled with biocytin after confocal imaging. For biocytin filling, slices with the underlying Millicell membranes were transferred to a submersion recording chamber and continuously superfused with ACSF saturated with 95% O2–5% CO2 at room temperature. GFP-expressing neurons were visualized with a 40x, 0.8 NA water immersion objective under an Olympus Optical (Melville, NY) BX50 upright microscope equipped with fluorescence and differential interference contrast optics and a Hamamatsu (Bridgewater, NJ) CCD camera. Patch pipettes were pulled from 1.5 mm outer diameter glass capillary tubes (World Precision Instruments) and filled with a solution of 136 mM potassium gluconate, 2 mM MgCl2, 0.6 mM EGTA, 10 mM HEPES, 2 mg/ml biocytin, with pH adjusted to 7.3 and osmolarity to 275–285 mOsm. A whole-cell clamp configuration was achieved using the Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA) and maintained for
1hr to allow for complete diffusion of biocytin. Slices were then fixed overnight in 0.1 M PBS with 4% paraformaldehyde at 4°C. After three rinses in PBS, fixed slices were incubated for 2 hr with ABC solution (Vector Laboratories, Burlingame, CA), followed by additional rinses in PBS and staining with diaminobenzidine (DAB) (0.7 mg/ml) and H2O2 (0.3%) in PBS. The reaction was stopped by transferring the slices to cold PBS.
Immunocytochemistry. For immunocytochemistry, GAD67–GFP-transfected slices were fixed for
Neuronal tracing. Neurons with strong GFP fluorescence throughout their dendritic tree were reconstructed digitally from the confocal image stacks using Neurolucida software (MicroBrightField, Colchester, VT). GFP-expressing neurons that were filled with biocytin were traced a second time with Neurolucida after processing the DAB reaction, using a 20x, 0.7 NA objective and a CCD video camera attached to an Olympus Optical AX70 microscope.
Data analysis. Quantitative analysis on the traced data were done using Neuroexplorer software (MicroBrightField). Numerical data (total dendritic length and branch points) generated from the second imaging session (day 5) were divided by the values derived from the first imaging session (day 0) to yield a ratio; geometrical means and SEs of the ratios were then calculated for each treatment group and for the control group.
Statistics. Exact, distribution-independent permutation tests were used to compare experimental treatments with control (Manly, 1997
Results
Transfecting GABAergic interneurons with GAD67–GFP
To visualize nonpyramidal neocortical interneurons, we transfected cortical organotypic slices with a DNA construct in which the GAD67 promoter drives the expression of GFP (Jin et al., 2001
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Figure 1. Neurons expressing GAD67–GFP were nonpyramidal, but neurons expressing CMV–GFP had mixed morphologies. A, C, Neurons labeled by transfection with GAD67–GFP, imaged at low (10x) and high (20x) power, respectively. B, D, Neurons labeled by transfection with CMV–GFP, imaged at low and high power, respectively. The images in A–D are from four different slices. Arrowhead in A points to a pair of apparently fused neurons; arrowheads in B indicate apical dendrites of pyramidal neurons. Arrows in C and D indicate the initial segment of the axon, which exits toward the pial surface in the interneuron (C) and toward the white matter in the pyramidal neuron (D). Solid lines in this and subsequent figures indicate the pial surface, and dashed lines indicate the border between layer 6 and the white matter. Scale bars: (in B) A, B, 200 µm; (in D) C, D, 100 µm.
In many slices, a small number of GFP-expressing neurons were observed to form clusters of two to three closely situated cells that appeared to be fused together at a single contact point on their somata or their proximal dendrites (Fig. 1A, arrowhead). Because such fused neurons were found in both GAD67–GFP- and CMV–GFP-transfected slices, we assume that the fusion was induced by the mechanical effect of the biolistic transfection; however, the precise mechanism remains to be determined. Fused neurons were excluded from additional study.
As an additional test of the neurotransmitter content of GAD67–GFP-expressing neurons, we stained them immunocytochemically with antibodies to GABA (Fig. 2A,B) and to GAD67 (Fig. 2C,D). Forty-two percent of GAD67–GFP-expressing neurons examined were immunopositive for GABA (n = 24), but only 26% were immunopositive for GAD67 (n = 34). Moreover, GAD67–GFP-expressing neurons staining immunopositive for either GABA or GAD67 were among the least intensely stained neurons in their microscopic field (Fig. 2B,D). This could reflect a low level of GAD67 (and therefore GABA) expression in GFP-expressing neurons, possibly because of competition for limited transcription factors between the endogenous and exogenous GAD67 promoters.
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Figure 2. Some, but not all, of the GAD67–GFP-expressing neurons were immunopositive for GABA and GABAergic subtype markers. A, C, E, G, Dual-channel confocal images of immunofluorescence (pseudocolored red) and GAD67–GFP fluorescence (pseudocolored green). B, D, F, H, The same fields with immunofluorescence only. Immunostaining is for antibodies against GABA (A, B), GAD67 (C, D), and somatostatin (E–H). Arrows in B, D, F, and H indicate the location of the GFP-expressing neuron shown in A, C, E, and G, respectively. Note that the GFP-expressing neurons in A, C, and E were immunopositive, but the neuron in G was immunonegative. Light arrows in D indicate non-GFP-expressing immunopositive neurons. Scale bar: (in H) A, B, E–H, 100 µm; C, D, 40 µm. NCtx, Neocortex; S Oriens, stratum oriens; L. VI, layer 6. Solid and dashed lines are as described in Figure 1.
Cortical GABAergic interneurons can be classified into several different subtypes with distinct neurochemical identities (Kubota et al., 1994
The incidence of GABAergic subtype markers in GAD67–GFP-expressing neurons was well below the incidence of the same markers in the GABAergic population of the mature cortex in vivo; for example, approximately one-half of all of the GABAergic neurons are estimated to express PV in the adult rat neocortex (Ren et al., 1992
Unlike parvalbumin, globally reduced expression could not underlie the low incidence of somatostatin in GAD67–GFP-expressing cells, as demonstrated by two contrasting examples in Figure 2E–H. The large GFP-expressing neuron in Figure 2E, with a cell body at the oriens–alveus border of CA1, was by its laminar location and dendritic morphology a typical oriens–lacunosum moleculare interneuron, a class of hippocampal interneurons known to express SOM (Freund and Buzsaki, 1996
Dendritic development of nonpyramidal cortical interneurons in organotypic slice cultures
Because GFP expression in our cultures revealed the detailed morphology of living neurons, we were able to follow the morphological development of individual neurons over time by imaging the same neuron two or more times at 5 d intervals, digitally reconstructing the three-dimensional (3-D) dendritic morphology of the neuron from stacks of confocal images, and quantifying the TDL and TDBPs at each imaging time point.We first followed the growth of GFP-expressing neurons in normal medium (Fig. 3). When first imaged, the age of our cultures varied between equivalent postnatal day 7 (EP7) (equivalent postnatal day = postnatal day at culturing + days in vitro) and EP24. We quantified dendritic growth over the next5dby calculating the ratio between TDL at second imaging (day 5) and TDL at first imaging (day 0). This ratio is plotted in Figure 3C against equivalent age at first imaging. The mean TDL ratio was 1.22 ± 0.07 (n = 19 neurons) for neurons first imaged at EP10 or earlier (left of vertical dotted line), but was 1.01 ± 0.03 (n = 26) for neurons first imaged at older ages (right of dotted line). The difference between the means of the two groups was highly significant (p = 0.0008). Notably, the SD of the TDL ratios in the younger group was twice as large as in the older group, indicating a higher variability in growth rate in younger slices. As an additional measure of growth, we also compared the TDBP ratio of the second to the first imaging. The mean TDBP ratio (0.93 ± 0.09 and 1.03 ± 0.06, respectively, for the younger and older groups) was not significantly different from 1 for either group (p = 0.5 and 0.18, respectively), indicating that, in our control conditions, there was a balance between the addition and elimination of new branches, even when (as in the younger age group) there was a net elongation of dendrites. These data suggest that dendritic growth in our nonpyramidal cortical interneurons occurred mostly during the first 2 weeks in organotypic culture, and that dendritic branching patterns may have been already fully established by the age of our earliest imaging (EP7). Because our goal was to study the role of BDNF during normal development, all of the subsequent experiments were done on the younger age group (EP7–EP10 at first imaging).
Because organotypic slices grow thinner and more translucent with time in culture, and because the level of GFP expression often appeared to increase with time in culture, we were concerned that any apparent increase in dendritic length between the two imaging sessions could be the result of incomplete visualization or incomplete GFP filling of dendrites at the earliest imaging time point. To test this possibility, we compared dendritic filling and visualization between GFP and biocytin, a much smaller molecule commonly used for reconstruction of neuronal morphology after intracellular recordings. Seven GFP-expressing neurons were imaged with a confocal microscope and then injected with biocytin through a patch pipette, processed for the histochemical DAB reaction, and traced with a computerized system. The 3-D dendritic morphology was then compared between reconstructed confocal images and biocytin tracings of the same neuron (Fig. 4). We found that, in older neurons, dendritic reconstructions from GFP and biocytin closely overlapped, after adjusting for shrinkage (Fig. 4A). In some of the younger neurons, however, GFP tracings from confocal stacks missed a terminal portion of one or two dendritic branches, as evident from comparison with the biocytin image (Fig. 4B, top tracing, arrow). This was not caused by insufficient GFP filling, but rather by loss of optical signal from dendritic branches growing deep into the tissue, as illustrated by the x–z projection in Figure 4B, bottom tracing, arrow. This situation was easy to identify, because such branches gradually disappeared from view in deeper optical sections; these branches were thereby excluded from analysis in all of the images taken from that particular neuron.
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Figure 4. GAD67–GFP fluorescence revealed the full dendritic morphology. GAD67–GFP-expressing neurons were imaged 3 weeks (A) or 3 d (B) after transfection and then injected with biocytin through a patch pipette. The 3-D reconstructions of the dendritic trees from the confocal images (green) are superimposed on the 3-D reconstructions from the biocytin tracings of the same neurons (red). Note that the GFP fluorescence was at least as extensive as the biocytin labeling, but one secondary dendrite in the younger neuron in B appeared shorter in the confocal image compared with the corresponding biocytin tracing (B, top tracing, arrow). The terminal part of the dendrite was missing from the confocal reconstruction, because it was located deep in the slice, as shown by the x–z image of the same neuron (B, bottom tracing, arrow). Scale bar, 100 µm (for both images).
BDNF, but not NT4/5, enhanced dendritic growth in nonpyramidal interneurons
To investigate the role of BDNF and activity in regulating dendritic growth of nonpyramidal interneurons, we followed the morphological development of individual GAD67–GFP-expressing neurons over a 5 d period and compared TDL and TDBP ratios between neurons from cultures treated with 200 ng/ml BDNF, or one of several other treatments, to neurons from untreated control cultures. Representative neurons are illustrated in Figure 5, and a summary plot of all of the experiments is presented in Figure 6. As illustrated in Figure 5A, growth in control conditions was modest (note the small rightward shift in the cumulative histogram). As reported above, the mean control TDL ratio (i.e., TDL at day 5 divided by TDL at day 0) was 1.22 ± 0.07 (n = 19); this change was significantly different from 1 (p = 0.01), but there was no significant change in TDBPs. In contrast, GAD67–GFP-expressing neurons developing in the presence of BDNF (Fig. 5B) exhibited considerably more growth. The mean TDL ratio in the presence of BDNF was 1.42 ± 0.05 (n = 27), representing a fractional increase (42%) nearly double that in control medium (22%). BDNF also promoted branching of existing dendrites (but not addition of primary dendrites): the mean TDBP ratio in BDNF was 1.25 ± 0.09. Both TDL and TDBP ratios in the BDNF group were significantly different from those of the control group (p = 0.03 and 0.01, respectively) (Fig. 6A,B). These results indicate that BDNF promotes dendritic growth and complexity in nonpyramidal neocortical interneurons.
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Figure 5. BDNF and KCl enhanced dendritic growth of GAD67–GFP-expressing neurons. Left column, Morphological reconstructions of representative dendritic trees of neurons before (green) and after (red) a 5 d period in culture medium supplemented by one of the following: no supplement (A), 200 ng/ml BDNF (B), 200 ng/ml BDNF and 1 µM TTX (C), 10 mM KCl (D), or 10 mM KCl and 50 µg/ml anti-BDNF (E). Right column, Cumulative histogram of TDL in each treatment group; each data point represents the fraction of neurons in the treatment group with TDL values equal to or smaller than the corresponding x value. , TDL at day 0 (before treatment).
, TDL at day 5 (after treatment). Note that the rightward shift of the curves (indicating an overall increase in total dendritic length) was considerably larger in B, C, and D, compared with control. Scale bar, 100 µm (for all images).
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Figure 6. Summary and statistical analysis of the effects of all of the tested treatments on dendritic growth and branching. Ratio change in TDL (left) and TDBPs (right) after 5 d of the treatment indicated. Each box spans the 25th to 75th percentile of the data points, with the median represented by a vertical line inside the box; the whiskers span the 5th to 95th percentiles. The number of neurons tested is indicated in parentheses after the treatment label. The dashed vertical lines at x = 1 indicate no change. (*)p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.005; p values are from pairwise comparisons with the control group.
To test whether the growth-promoting effect of BDNF on nonpyramidal, GABAergic interneurons depended on electrical activity, we allowed cultures to develop in the presence of both 200 ng/ml BDNF and 1 µM TTX (Fig. 5C). Under these conditions, there was still enhanced dendritic growth in GAD67–GFP-expressing neurons, and the mean TDL ratio was 1.62 ± 0.11 (n = 17), significantly larger than control (p = 0.001) (Fig. 6A) (the increase in TDL ratio compared with BDNF alone was only marginally significant at the p = 0.06 level). TTX did seem to prevent branching (Fig. 6B)(p = 0.6). These data suggest that, unlike the effect of BDNF on pyramidal neurons (McAllister et al., 1996
To study the role of endogenous BDNF in dendritic development in culture, we compared dendritic growth in control slices with growth in cultures in which endogenous BDNF was neutralized with a high titer (50 µg/ml) of anti-BDNF antibodies, shown to block the effect of endogenous BDNF (Ghosh et al., 1994
Effect of neuronal activity on GABAergic neuron dendritic growth
Electrical activity can increase several parameters of GABAergic function (Rutherford et al., 1997Because electrical activity enhances cortical BDNF levels (Castren et al., 1992
To test whether growth in normal culture medium is dependent on electrical activity, we cultured slices in the presence of 1 µM TTX. Mean TDL ratio in TTX was 0.96 ± 0.03 (n = 12), very significantly different from control (p = 0.0002) (Fig. 6A), indicating that TTX counteracted the increase in TDL that occurred in normal medium and suggesting that sodium-dependent electrical activity is required for normal dendritic growth in culture.
Finally, to test whether BDNF and depolarization affect dendritic growth in a similar manner, we used Sholl analysis to quantify dendritic growth within consecutive concentric rings around the cell body, and compared Sholl plots showing the increase in dendritic length overa5d growth period among control, BDNF, and high K+ conditions (Fig. 7). As illustrated, dendritic growth was distributed within a radius of
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Figure 7. Sholl analysis reveals a similar pattern of dendritic growth in response to BDNF and KCl. Absolute increase in TDL in the control ( ), high K+ (
), and BDNF (
) groups was calculated separately for each consecutive, 20 µm-wide concentric ring, and averaged between neurons. Note that the high K+ and BDNF groups exhibited a greater average increase in TDL, compared with control, but the radial extent of the increase was similar among all three groups. Error bars represent SEMs.
Discussion
In this study, we tested whether exogenous BDNF or depolarization by high KCl enhanced dendritic growth of nonpyramidal cortical interneurons expressing GAD67–GFP in organotypic slice cultures, and whether there was cross-dependency between the effects of BDNF and depolarization. Short-term (minutes to hours) changes in dendritic morphology were tracked previously in living GFP-expressing cortical neurons in vitro (Fischer et al., 1998BDNF effects in pyramidal and nonpyramidal neocortical interneurons are qualitatively different
Exogenous or endogenous BDNF, NT3, and NT4/5 have been shown to promote dendritic elongation and branching in neocortical pyramidal cells developing in organotypic cultures (McAllister et al., 1995In our study, a 4 d treatment with 200 ng/ml BDNF caused a 40% increase in TDL of neocortical interneurons. In contrast, a 36 hr treatment with 200 ng/ml BDNF doubles the TDL and TDBPs of layer 4 pyramidal neurons in the ferret (McAllister et al., 1995
Depolarization promotes dendritic growth in nonpyramidal neocortical interneurons
We found that depolarizing neurons by adding 10 mM KCl to our organotypic cultures enhanced dendritic growth and branching of nonpyramidal interneurons to a level similar to that induced by 200 ng/ml BDNF, whereas blocking action potentials with TTX prevented dendritic growth. Therefore, these data suggest that dendritic growth of cortical interneurons in organotypic cultures requires action potentials and can be enhanced by depolarization. KCl-induced enhancement and/or TTX-induced reduction of dendritic length and complexity have been demonstrated previously in some systems (Reitstetter and Yool, 1998Depolarization effects are mediated by BDNF released by pyramidal neurons
Depolarization can promote dendritic growth in a cell-autonomous manner, e.g., by activating voltage-gated calcium channels, followed by calcium-dependent phosphorylation of the dendritic protein MAP2 (microtubule-associated protein 2) (Quinlan and Halpain, 1996In the cerebral cortex, BDNF is expressed only by pyramidal neurons, but TrkB, the preferred receptor for BDNF, is found in both pyramidal cells and GABAergic interneurons (Cellerino and Maffei, 1996
A larger dendritic tree is likely to receive more axodendritic (mostly excitatory) synapses and thus increase electrical activity in the neuron. Moreover, the larger dendritic trees were most likely accompanied by larger (or denser) axonal arbors, as shown previously in dissociated cultures (Vicario-Abejon et al., 1998
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Figure 8. BDNF mediates a feedback loop that maintains a balance between excitation and inhibition. Left, A pyramidal neuron is assumed to be depolarized (+), causing it to increase its firing rate and thereby release more BDNF into the extracellular space, acting on TrkB receptors in a nearby inhibitory neuron. (Although this figure implies that BDNF is released from dendrites, BDNF may also be released from axon terminals.) Right, The released BDNF caused the inhibitory interneuron to ramify its dendritic and axonal arbors and thereby increase its inhibitory effect on the pyramidal cell (–) counteracting the initial depolarization.
BDNF and activity-dependent maturation of GABAergic function
Electrical and synaptic activity in vitro has been shown previously to promote, and blockade of this activity, to reverse, an increase in the level of expression of GABA and GABAergic markers (Marty et al., 1996b
Footnotes
Received Oct. 1, 2002; revised May. 5, 2003; accepted May. 5, 2003.This work was supported by National Institutes of Health Grants HD33463 (A.A.) and EY12152 (P.H.M.). We thank Albert Berrebi for helpful advice on immunocytochemistry and comments on this manuscript. We also thank Janet Cyr and Jason Wells for helpful comments on this manuscript; Cary Johnson, Jeff Altemus, Colette Ramsburg, and Eric Christenson for excellent technical support; and Dr. Gabor Szabo for providing the GAD67 promoter.
Correspondence should be addressed to Dr. Aric Agmon, Department of Neurobiology and Anatomy, West Virginia University, Health Science Center Drive, Morgantown, WV 26506-9128. E-mail: aagmon{at}wvu.edu.
X. Jin's present address: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305
Copyright © 2003 Society for Neuroscience 0270-6474/03/235662-12$15.00/0
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