BDNF-Mediated Modulation of GABA and GlycineRelease in Dorsal Horn Lamina II from Postnatal Rats

Rita Bardoni,1 Alessia Ghirri,1 Chiara Salio,2 Massimiliano Prandini,1

Adalberto Merighi2,3

1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

2 Department of Veterinary Morphophysiology, University of Turin, Turin, Italy

3 Istituto Nazionale di Neuroscienze, Turin, Italy

Received 19 September 2006; revised 6 December 2006; accepted 13 January 2007

ABSTRACT: Recent studies show that excitatory

glutamatergic transmission is potentiated by BDNF in

superficial dorsal horn, both at the pre- and the postsy-

naptic site. The role of BDNF in modulating GABA and

glycine-mediated inhibitory transmission has not been

fully investigated. To determine whether the neurotrophin

is effective in regulating the spontaneous release of the

two neurotransmitters, we have recorded miniature inhib-

itory postsynaptic currents (mIPSCs) in lamina II of post-

natal rats. We show that application of BDNF enhanced

the spontaneous release of GABA and glycine, in presence

of tetrodotoxin. The effect was blocked by the trk-recep-

tor inhibitor k-252a. Amplitude and kinetics of mIPSCs

were not altered. Evoked GABA and glycine IPSCs

(eIPSCs) were depressed by BDNF and the coefficient of

variation of eIPSC amplitude was significantly increased.

By recording glycine eIPSCs with the paired-pulse proto-

col, an increase of paired-pulse ratio during BDNF appli-

cation was observed. We performed parallel ultrastruc-

tural studies to unveil the circuitry involved in the effects

of BDNF. These studies show that synaptic interactions

between full length functional trkB receptors and GABA-

containing profiles only occur at non peptidergic synaptic

glomeruli of types I and II. Expression of trkB in pre-

synaptic vesicle-containing dendrites originating from

GABAergic islet cells, indicates these profiles as key struc-

tures in the modulation of inhibitory neurotransmission

by the neurotrophin. Our results thus describe a yet

uncharacterized effect of BDNF in lamina II, giving fur-

ther strength to the notion that the neurotrophin plays an

important role in pain neurotransmission. ' 2007 Wiley

Periodicals, Inc. Develop Neurobiol 67: 960–975, 2007

Keywords: neurotrophin; inhibitory transmission; spinal

cord; pain; presynaptic modulation

INTRODUCTION

The neurotrophin BDNF is involved in neuronal dif-

ferentiation and survival during development and in

synaptic transmission and plasticity, in immature and

mature stages. BDNF has been reported to regulate

both excitatory and inhibitory synapses in several

areas of central nervous system (CNS), by acting on

presynaptic terminals (Frerking et al., 1998; McLean

Bolton et al., 2000; Xu et al., 2000) or by modulating

postsynaptic receptor expression and activity (Levine

et al., 1995, 1998; Tanaka et al., 1997; Brunig et al.,

2001; Pezet and McMahon, 2006).

In spinal cord dorsal horn, BDNF has been impli-

cated in the control of nociceptive neurotransmission

on behavioral, physiological, and pharmacological

grounds. Behavioral studies have shown complex

effects of both endogenous and exogenous BDNF.

Endogenous BDNF, derived from a sub-population of

Correspondence to: R. Bardoni; (bardoni@unimo.it)Contract grant sponsor: Italian MIURContract grant sponsor: Compagnia di San Paolo, Turin, ItalyContract grant sponsor: Fondazione CRT, Turin, Italy

' 2007 Wiley Periodicals, Inc.Published online 28 February 2007 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dneu.20401

960

nociceptors, can be released following high frequencystimulation of nociceptive primary afferent fibers(Lever et al., 2001), and plays an important role inregulating inflammatory pain thresholds and second-ary hyperalgesia (Thompson et al., 1999; Zhao et al.,2006). On the other hand exogenously applied BDNFcauses thermal analgesia and attenuates mechanicalallodynia (Siuciak et al., 1994; Cejas et al., 2000).

At the cellular level, the neurotrophin is able topotentiate glutamatergic synaptic transmission be-tween primary afferent fibers and dorsal horn neu-rons, by modulating postsynaptic NMDA receptors(Slack et al., 2004; Garraway et al., 2003). Recentevidence has been provided that BDNF, acting ontrkB receptors, causes an increase of spontaneous glu-tamate release in dorsal horn lamina II, both in con-trol animals and during inflammation (Bardoni et al.,2004; Matayoshi et al., 2005).

Nonetheless, a modulatory action of BDNF on in-

hibitory synaptic transmission in dorsal horn has also

been reported: BDNF released from microglia cells

causes a modulation of Cl-transporter expression in

lamina I neurons, and a shift of chloride equilibrium

potential to positive values (Coull et al., 2005). More-

over, the application of BDNF to the isolated adult

rat spinal cord increases KCl-evoked GABA content

in the collected superfusate (Pezet et al., 2002).

The role of BDNF in regulating GABAergic andglycinergic transmission in dorsal horn, however, hasnot been fully investigated. Furthermore, although thedistribution of full length (fl) functional trkB receptorshas recently been investigated in rat and mouse laminaII (Salio et al., 2005), the presence of these receptorson inhibitory interneurons in dorsal horn has not beenreported yet. In this study, we tested the hypothesisthat BDNF can control the release of GABA and gly-cine in superficial dorsal horn. Since local GABAergicand glycinergic inhibition is critical for gating of noci-ceptive transmission (Malcangio and Bowery, 1996),any effect of BDNF on inhibitory synapses could havea great impact on pain signaling, especially in patho-logical conditions of persistent pain. By using thepatch-clamp technique, we have studied the modula-tory effects of BDNF on spontaneous and evokedrelease of GABA and glycine in lamina II neuronsfrom neonatal rats. Furthermore, by performing ultra-structural immunocytochemical studies, we havedetermined the pattern of expression of fl-trkB recep-tors at GABAergic synapses in this lamina.

METHODS

Animals

Experiments were carried out in Sprague-Dawley postnatal

rats, ranging in age from 8 (P8) to 30 days (P30). Patch-

clamp experiments were carried out in P8–P16 animals,

whereas histology was performed in young-adult rats (P25–

P30). All experimental procedures were in strict accordance

with the Italian and EU regulation on animal welfare, and

previously authorized by the Italian Ministry of Health.

Electrophysiological Recordings

Slice Preparation. Postnatal Sprague-Dawley rats were

anesthetized with halothane and decapitated, the spinal cord

and vertebrae were rapidly removed and placed in ice-cold

Krebs’ solution (composition in mM: 125 NaCl, 2.5 KCl, 1.25

NaH2PO4, 26 NaHCO3, 25 glucose, 1 MgCl2, and 2 CaCl2, pH

7.4, 320 mOsm), bubbled with carboxygen (95% O2, 5%

CO2). The lumbar part of the spinal cord was isolated, laid

down on an agarose block, and transverse slices (500 lm thick)

were obtained using a vibrating microtome (Campden Instru-

ments, UK). Slices were incubated in oxygenated Krebs’ solu-

tion at 358C for 1 h and used for recording within 6–7 h.

Patch-Clamp Recording and Focal Synaptic Stimula-tion. Patch-clamp recording in whole-cell configuration was

performed on visually identified lamina II neurons at room

temperature. Neurons were visualized by using a Zeiss Axio-

skop microscope, fitted with Nomarski optics and connected

to a camera (Dage-MTI, USA). Slices were perfused at 1–

2 mL/min. Thick-walled borosilicate pipettes, having a resist-

ance of 3–5 mO, were filled with a solution having the fol-

lowing composition (in mM): 130 Cs-gluconate, 10 CsCl, 11

EGTA, 1 CaCl2, 10 HEPES, and 2 Mg2þ -ATP, pH adjusted

to 7.2 with NaOH, osmolarity adjusted to 305 with sucrose.

Junction potential was corrected after recording.

Data were recorded and acquired using a Multiclamp

700A amplifier and pClamp 9 software (Molecular Devices,

USA). Sampling rate was 10 kHz, and data were filtered at

2 kHz. Both miniature inhibitory postsynaptic currents

(mIPSCs) and evoked inhibitory postsynaptic currents (eIPSCs)

were recorded in Krebs’ solution, in constant presence of

10 lM NBQX and 50 lM D-APV; only for mIPSCs recording,

0.5 lM tetrodotoxin (TTX) was added to the bath solution.

Evoked IPSCs were obtained by focally stimulating the

tissue around the recorded cell with a patch-clamp pipette,

filled with Krebs’ solution. The second pole was repre-

sented by a silver wire placed close to the slice. Stimuli had

intensities of 0.1–1 mA and duration of 0.05–0.1 ms.

Evoked IPSCs with constant latency and no failures during

1 Hz stimulation were considered monosynaptic and tested

for BDNF.

Data Analysis and Statistics. Miniature IPSC amplitudes,

frequencies, and kinetics were determined using Minianaly-

sis software (Synaptosoft, Decatur, USA). Detection thresh-

old for mIPSCs was set at 8–10 pA. Selection of mIPSCs

was performed manually and events with rise-time higher

than 3 ms were usually discarded (the exclusion of slower

events did not affect the results of the analysis). Analysis of

mIPSCs was performed on the same time window (10 min)

during control and when the BDNF effect was stabilized.

BDNF Modulates GABA and Glycine in Lamina II 961

Developmental Neurobiology. DOI 10.1002/dneu

Effects of BDNF on mIPSC inter-event intervals and ampli-

tudes were assessed for each cell by comparing control and

test plots in cumulative probability histograms, using the

Kolmogorov–Smirnov 2-sample test. In neurons were an

insufficient number of events was detected (<100), the

unpaired t-test was applied. Decays of isolated GABA or

glycine mIPSCs were interpolated with a single exponential

function, by using Minianalysis software. Events that did

not return to the baseline or composed by multiple peaks

were not included in the kinetic analysis. Kinetic parame-

ters (10–90% rise-time and decay time constants) were

compared by using the unpaired t-test. Values of p < 0.05

were considered significant.

Analysis of peak amplitudes and kinetics of evoked

IPSCs was performed by using Clampfit (pClamp9, Molec-

ular Devices, USA). Graphs and statistical analysis were

obtained by using Sigmaplot 8 (SPSS, USA). Evoked IPSC

peaks were manually detected for each trace, in the same

2 ms window. Decay phase fitting was obtained by interpo-

lating the 10–90% portion of current decay with a two ex-

ponential function. Statistical analysis on amplitudes and

kinetic parameters was performed by using the unpaired t-test. Coefficient of variation (CV) of eIPSC amplitudes was

determined as standard deviation/mean, considering at least

30 traces in control and in BDNF, at the time when the

effect had fully developed. For paired pulse experiments,

the analysis of the second peak was determined by extrapo-

lating the fitting of the first response decay phase to the

time of the second peak and subtracting this value from the

peak amplitude of the second response. Mean paired pulse

ratio (PPR) was determined by averaging PPRs deriving

from 20–30 traces in each condition. Comparison of CV

and PPR in control and BDNF were obtained by applying

paired t-tests. p values <0.05 were considered significant.

Data were expressed as mean value 6 S.E.

Drugs. All chemicals were obtained from Sigma Aldrich

(St. Louis, MO), except the following: bicuculline, NBQX,

and TTX were obtained from Tocris Cookson (UK),

D-APV from Ascent scientific (UK), BDNF from Peprotech

(UK), and k-252a from Calbiochem (USA).

Immunocytochemistry

Tissue Preparation. Under deep pentobarbital anesthesia

(60 mg/100 g), animals were injected intracardially with

1 mL heparin (5000 U/mL) and perfused through the de-

scending aorta with Ringer solution followed by cold fixa-

tive solution (4% paraformaldehyde þ 0.5% glutaraldehyde

in sodium cacodylate buffer 0.05 M, pH 7.4). Segments

from lumbar spinal cord were carefully dissected out, and

postfixed for 2 additional hours in the same aldehyde mix-

ture. Coronal sections were cut on a vibratome at a thick-

ness of 50 lm.

Immunolabeling. Dual labeling experiments were carried

out simultaneously to localize high affinity functional

BDNF receptor (fl-trkB receptor) and GABA immunoreac-

tivities at the ultrastructural level.

The immunolocalization of fl-trkB receptors was carried

out by a pre-embedding protocol with ultra small gold

(Salio et al., 2005). Briefly, free-floating spinal cord vibra-

tome sections were incubated overnight with a chicken

anti-fl-trkB primary antibody (Promega Corporation, USA),

followed by anti-chicken IgY biotinylated secondary anti-

body (Vector, USA), and finally by AlexaFluor 488-Fluoro-

nanogoldTM-Streptavidin (Nanoprobes, USA). Sections

were postfixed in osmium ferrocyanide, dehydrated in

increasing concentrations of ethanol, and embedded in

Araldite. FluoronanogoldTM particles were intensified

directly on grid by a Gold Enhancement Kit (Nanoprobes).

Fl-trkB-labeled ultrathin sections were subsequently

stained for GABA following a post-embedding immuno-

staining protocol on grid (Merighi and Polak, 1993).

Briefly, after sodium metaperiodate etching sections were

placed on drops of an anti-GABA antiserum (Merighi et al.,

1989) and incubated overnight. After extensive rinsing in

buffer, they were transferred in goat anti-rabbit IgGs conju-

gated with 10-nm gold (BioCell, UK). Section were then

fixed in 2.5% glutaraldehyde in cacodylate buffer, 0.05 M,

pH 7.4, and finally washed in distilled water.

After routine counterstaining, grids were observed with

a Philips CM10 electron microscope.

RESULTS

BDNF Causes the Increase of MiniatureGABA and Glycine IPSC Frequency

Patch-clamp recordings were obtained from 72 neu-

rons in rat dorsal horn lamina II, by using the volt-

age-clamp modality and holding the recorded cell at

0/þ10 mV. To test whether BDNF can regulate

GABA and glycine release in lamina II, we first

recorded miniature GABA and glycine IPSCs

(mIPSCs), in continuous presence of TTX (0.5 lM)

and the glutamatergic receptor antagonists NBQX

(10 lM) and D-APV (50 lM). In most cells both

GABA and glycine mIPSCs were present, as revealed

by blocking mIPSCs with the GABAA receptor antag-

onist bicuculline (10 lM) and the glycine receptor an-

tagonist strychnine (3 lM) at the end of the experi-

ment. In one neuron mIPSCs were mediated by

GABA receptors only, since they were completely

blocked by bicuculline. This cell was excluded from

this first set of experiments and added to the group of

neurons where GABA mIPSCs were pharmacologi-

cally isolated (see later).

Application of 100 ng/mL BDNF caused a signifi-

cant increase of GABA and glycine mIPSC frequency

in 9 out of 13 lamina II neurons tested [Kolmogorov–

Smirnov test, p < 0.01; Fig. 1(A,B,D)]. The effect

962 Bardoni et al.

Developmental Neurobiology. DOI 10.1002/dneu

Figure 1 BDNF application increases GABA and glycine mIPSC frequency. (A) Patch-clamp

recordings of GABA and glycine mIPSCs from a lamina II neuron from a P9 rat, in control and in

presence of 100 ng/mL BDNF. (B, C) Cumulative probability curves obtained from the analysis of

the neuron shown in (A), representing the distribution of mIPSC inter-event intervals (B) and

amplitudes (C), in control (solid lines) and in BDNF (dashed lines). Kolmogorov–Smirnov test per-

formed on the two distributions showed an effect of BDNF on mIPSC frequency (p < 0.01), but

not on amplitude (p > 0.05). (D) Averaged data showing the BDNF-induced significant change of

mIPSC frequency in 9 out of 13 lamina II neurons tested. Co-application of BDNF with k-252a

(400 ng/mL) did not cause any significant frequency variation (5 cells out of 5). Frequency values

in BDNF and k-252a are expressed as percentage of control. (E) BDNF did not significantly alter

mIPSC mean amplitude in the 9 neurons where the frequency was significantly increased.

BDNF Modulates GABA and Glycine in Lamina II 963

Developmental Neurobiology. DOI 10.1002/dneu

started after 15 min of BDNF application and usually

persisted during removal of the neurotrophin. In three

cells a partial recovery was observed after about

20 min wash out. The mean frequency of mIPSCs in

control was 0.19 6 0.04 Hz, in BDNF was 0.42 6

0.1. Co-application of BDNF with the trk receptor an-

tagonist k-252a (400 ng/mL) did not cause any signif-

icant change of mIPSC frequency in all 5 neurons

tested [Kolmogorov–Smirnov test, p > 0.05; Fig.

1(D)]. Miniature IPSC amplitude was not signifi-

cantly altered during BDNF application in 8 of the 9

cells responsive to the neurotrophin [Kolmogorov–

Smirnov test, p > 0.05; Fig. 1(C,E)]. One cell exhib-

ited a bimodal distribution of mIPSC amplitudes in

control; in BDNF, the mIPSC population of larger

amplitude was preferentially potentiated, causing a

significant change of mean mIPSC amplitude.

To determine whether BDNF acts on both GABA

and glycine mediated mIPSCs, we performed experi-

ments where a single population was isolated by

application of 10 lM bicuculline or 3 lM strychnine

(Fig. 2). A significant increase of mIPSC frequency

in BDNF was observed for both GABA and glycine

mIPSCs, respectively in 5 out of 9 and 5 out of 8

tested neurons [Kolmogorov–Smirnov test, p < 0.01;

Fig. 2(A,B)]. Mean GABA mIPSC frequency was

0.22 6 0.09 Hz in control and 0.45 6 0.14 in BDNF;

mean glycine mIPSC frequency increased from 0.20

6 0.09 in control to 0.65 6 0.3 Hz in BDNF. The

effects of BDNF on GABA and glycine mIPSC fre-

quency are summarized in Figure 2(C).

BDNF did not affect the peak amplitude of pure

GABA and glycine mIPSCs [Kolmogorov–Smirnov

test, p > 0.05; Fig. 2(B,D)]. GABA and glycine

mIPSC 10–90% rise time values and decay time con-

stants (obtained by fitting the mIPSC decay with a

single exponential function) were also not altered by

BDNF [t-test, p > 0.05; Fig. 2(E)]. The increase of

mIPSC frequency produced by BDNF, without any

significant effect on mIPSC amplitude or kinetics,

would suggest a presynaptic action of the neurotro-

phin on GABAergic and glycinergic synapses in su-

perficial dorsal horn.

Evoked IPSCs Are Depressed inPresence of BDNF

To further investigate the role of BDNF in regulating

GABAergic and glycinergic transmission in lamina

II, we have studied the effects of the neurotrophin on

evoked GABA and glycine IPSCs (eIPSCs), obtained

by focally stimulating the superficial dorsal horn.

Mixed GABA and glycine IPSCs were recorded in

presence of 10 lM NBQX and 50 lM D-APV, at the

holding potential of 0 mV; stimulation intensity was

set right above the threshold and the stimulus was

delivered at the frequency of 0.03 Hz. Experiments

performed in control conditions showed that stimula-

tion at this frequency did not produce by itself any

significant depression of eIPSC during long time

recordings (45–60 min) (data not shown).

BDNF (100 ng/mL) caused a significant depres-

sion of eIPSC peak amplitude in 9 out of 16 lamina II

neurons tested [t-test, p < 0.05; Fig. 3(A,B)]. The

time course of BDNF effect was similar to that

observed during mIPSC recordings, the onset of

effect occurring after about 15 min from the begin-

ning of application and the maximum of depression

developing after 20–30 min. Only in three cells a par-

tial recovery was obtained during BDNF wash out.

At the end of the experiments, application of 10 lMbicuculline blocked a substantial part of eIPSCs (67.4

6 8.1% of peak amplitude). Addition of 3 lM strych-

nine to bicuculline caused the complete block of

eIPSCs, showing that both GABAA and glycine

receptors contributed to the generation of inhibitory

currents.

Incubation of slices in the trk receptor antagonist

k-252a (400 ng/mL) for at least 20 min before BDNF

application and during BDNF prevented any depress-

ing action of the neurotrophin on eIPSCs in all lamina

II neurons tested, suggesting a BDNF-induced activa-

tion of trkB receptors [t-test, p > 0.05, n ¼ 5; Fig.

3(B)]. Incubation of slices in k-252a before BDNF

application did not cause any significant alteration of

eIPSC amplitude, indicating that endogenous BDNF

was not able to modulate inhibitory synaptic trans-

mission in our experimental conditions.

Since recordings were performed in whole cell

configuration, a shift of chloride equilibrium potential

(ECl) was unlikely. Nevertheless, an incomplete diffu-

sion of the intracellular solution into the cytoplasm

could have partially preserved the intracellular chlo-

ride concentration. In this case, a shift of ECl towards

more positive values could determine a depression of

eIPSCs at 0 mV and an increase of IPSC amplitude at

potentials more negative than ECl. In our experimen-

tal conditions ECl was �60.7 mV. Evoked IPSCs

recorded at �90 mV in three neurons showed a sig-

nificant depression in presence of BDNF, excluding

an alteration of intracellular chloride concentration

due to BDNF (data not shown).

The depressing action of BDNF on eIPSCs could

be due to a presynaptic inhibition of GABA and gly-

cine release or rather to a direct modulation of post-

synaptic GABA and glycine receptors. To assess

whether the neurotrophin modulates inhibitory synap-

964 Bardoni et al.

Developmental Neurobiology. DOI 10.1002/dneu

Figure 2 (See legend on following page)

BDNF Modulates GABA and Glycine in Lamina II 965

Developmental Neurobiology. DOI 10.1002/dneu

Figure 2 BDNF increases the frequency of pharmacologically isolated GABA and glycine mIPSCs.

(A) Recordings of mIPSCs mediated by GABA (left) and by glycine (right) receptors, obtained from

two different lamina II neurons, in control and in 100 ng/mL BDNF. The mIPSCs were recorded in

presence of 3 lM strychnine or 10 lM bicuculline, respectively. (B) Cumulative probability histo-

grams obtained from the analysis of the two neurons in (A), representing the effect of BDNF on inter-

event interval and amplitude distributions (control: solid lines, BDNF: dashed lines). BDNF caused a

significant decrease of inter-event intervals (Kolmogorov–Smirnov test, p < 0.01), leaving mIPSC am-

plitude unaffected (Kolmogorov–Smirnov test, p > 0.05). (C) Averaged data demonstrating BDNF

effect on isolated GABA and glycine mIPSC frequency. BDNF caused a significant increase of fre-

quency of both GABA (5 positive cells out of 9) and glycine mIPSCs (5 positive neurons out of 8).

(D) Mean amplitudes of GABA and glycine mIPSCs were not significantly affected by BDNF; (E)

BDNF did not significantly change 10–90% rise time and decay time of GABA and glycine mIPSCs.

Decay time was determined as the time constant of the single exponential function fitting the mIPSC

decay phase. Analysis was performed on the cells responsive to BDNF.

Figure 3 Evoked IPSCs are depressed by BDNF (A) Individual traces of GABA and glycine

mediated evoked IPSCs (eIPSCs), recorded from a lamina II neuron (from a P9 rat) and evoked by

focal stimulation of lamina II. BDNF effect started after about 15 min. (B) Summary of BDNF

effects on eIPSC amplitude: the neurotrophin induced a significant depression in 9 out of 16 lamina

II neurons tested. Peak amplitudes are expressed as percentage of control. Co-application of BDNF

and k-252 did not cause any significant depression of evoked IPSC amplitude (n ¼ 5). (C) BDNF

altered coefficient of variation (CV) of eIPSC amplitudes, causing a significant increase (n ¼ 8).

CV is represented as percentage of control. No significant increase of CV was observed in cells

negative to BDNF (n ¼ 7).

966 Bardoni et al.

Developmental Neurobiology. DOI 10.1002/dneu

ses at the presynaptic site, we determined the effect

of BDNF on the eIPSC variability (expressed as CV

of eIPSC amplitudes, CV). In a subpopulation of

lamina II cells positive to BDNF, the CV of peak am-

plitude was calculated on 30–40 traces in control and

during the test. BDNF caused a significant increase of

CV in all neurons [paired t-test, p < 0.05, n ¼ 8; Fig.

3(C)]: the mean value of CV was 0.16 6 0.03 in con-

trol and 0.26 6 0.04 in BDNF. In the three neurons

where a recovery of peak amplitude was observed in

wash, CV returned close to the values in control. No

significant changes in CV were observed in the cells

where BDNF did not cause a significant depression of

eIPSC amplitude (paired t-test, p > 0.05, n ¼ 7).

The depression of eIPSC amplitude was not

accompanied by a change of decay kinetics (Fig. 4).

Evoked eIPSCs were usually fitted by a double expo-

nential function. Neither the fast nor the slow decay

time constants (tau1 and tau2) were significantly

affected in cells responsive to BDNF [t-test, p >0.05, n ¼ 9; Fig. 4(B)]. The relative amplitudes of the

fast (A1) and slow components (A2) were not altered

in presence of the neurotrophin [t-test, p > 0.05, n ¼9; Fig. 4(C)].

To further confirm the presynaptic nature of

BDNF inhibition of eIPSCs, paired pulse protocol

was applied to glycinergic eIPSCs, recorded in pres-

ence of 10 lM bicuculline to block the GABA medi-

ated component [Fig. 5(A)]. Glycinergic eIPSCs were

used because of their fast decay kinetics: in most

cells, at the inter-pulse interval of 50 ms, the first

response produced only a negligible contamination of

the second peak. Paired pulse protocol caused the

facilitation of the second peak in the majority of cells

tested; in some traces, where the first peak had large

amplitude, the second peak was depressed. Applica-

tion of BDNF caused a significant depression of the

first peak in 9 cells out of 16 tested [t-test, p < 0.05;

Fig. 5(B)], whereas the second peak was either

depressed or unchanged. In all positive cells BDNF

increased the amount of facilitation produced by the

paired pulse protocol, expressed as a significant

Figure 4 BDNF does not alter evoked IPSC decay kinetics. (A) (left and center) Averaged traces

of GABA/glycine eIPSCs recorded from a lamina II neuron (P12 rat). BDNF caused a strong

depression of eIPSC amplitude. Right: The graph, obtained by normalizing the trace in BDNF to

that in control and by superimposing the two traces, shows that BDNF does not alter the eIPSC

kinetics. (B, C) Fast and slow decay time constants (tau1 and tau2, B) and the relative contribution

of the fast and slow components (C) are not significantly altered by BDNF. The analysis was per-

formed on the 9 neurons where eIPSCs were depressed by BDNF.

BDNF Modulates GABA and Glycine in Lamina II 967

Developmental Neurobiology. DOI 10.1002/dneu

increase of the PPR in presence of the neurotrophin

[paired t-test, p < 0.05, n ¼ 9; Fig. 5(C)]. These

results provide additional evidence of BDNF-medi-

ated modulation of glycine release in dorsal horn

lamina II.

Localization of trkB Receptors on DorsalHorn Inhibitory Interneurons

The ultrastructural distribution of fl-trkB receptors in

rat and mouse lamina II has been recently described

in details on both qualitative and quantitative bases

(Salio et al., 2005). Here we have performed a series

of dual labeling experiments to combine together

fl-trkB and GABA immunolocalization to assess

whether the two labels were expressed at the same

synapses, and, in the affirmative, if they coexisted

within individual profiles. According to the parame-

ters employed in the gold intensification procedure

for rendering the ultra-small 1-nm gold particles of

the FluoroNanogoldTM easily visible and recogniz-

able under the electronic beam, the positive signal

appeared in the form of very electrondense particles

of relatively large sizes (25–30 nm) and irregular

shapes, that were very easily distinguishable from the

unintensified 10-nm colloidal gold particles used to

visualize GABA immunoreactive profiles (Figs. 6

and 7).

In our double labeled preparations, synaptic inter-

actions between fl-trkB and GABA immunoreactive

Figure 5 BDNF application changes the paired-pulse ratio of evoked glycinergic IPSCs. (A)

Averaged traces of evoked glycinergic IPSCs, recorded using the paired-pulse protocol (lamina II

neuron from a P10 rat). The IPSCs are represented in control, in presence of 100 ng/mL BDNF and

superimposed (the trace in BDNF is normalized to that in control and represented as dashed line).

BDNF causes a depression of both peaks, but the amount of depression was larger for the first

peak. (B) Average amplitude of the first and the second peak in BDNF, expressed as percentage of

control, for 9 cells responsive to BDNF (out of 16 neurons tested). BDNF causes a large and signif-

icant depression of the first peak in all 9 cells, while the second peak is less affected. (C) PPR (2nd

peak/1st peak) is significantly increased in BDNF (n ¼ 9).

968 Bardoni et al.

Developmental Neurobiology. DOI 10.1002/dneu

profiles were consistently observed only at the level

of synaptic glomeruli, although they represent a mi-

nority (about 5%) of the synapses in lamina II. Syn-

aptic glomeruli are multi-synaptic complexes whose

core, commonly referred to as C bouton, is formed by

an axon terminal derived from a primary afferent

fiber. Two main types of glomeruli, named type I and

II, have been described in rat (see Ribeiro-da-Silva,

2004). Type I glomeruli may have a particularly

electron-dense C bouton filled by small agranular

Figure 6 Dual labeling immunocytochemistry of fl-trkB receptors and GABA in rat spinal cord

lamina II. (A, B) Synaptic glomeruli of the Ia (nonpeptidergic) type displaying peripheral labeling

for fl-trkB (gold-intensified FluoronanogoldTM) and GABA (10 nm gold). Note that fl-trkB has a

postsynaptic localization in one of the ‘‘plain’’ dendrites engaged in the glomerulus (D), as well as

a presynaptic localization in one vesicle-containing or presynaptic dendrite (V1). All V1 dendrites

are immunoreactive for GABA, but not for fl-trkB (A). The area indicated by the rectangle in B is

shown at higher magnification in the bottom insert at down right corner. A symmetric synaptic spe-

cialization is clearly visible between the V1 profile and the central bouton of the glomerulus. (C) A

synaptic glomerulus of the IIa type displaying a peripheral V1 profile immunolabeled for trkB

(gold-intensified FluoronanogoldTM) and GABA (10 nm gold). The area indicated by the rectangle

is shown at higher magnification in the top insert at down right corner. Scale bars ¼ 500 nm; inserts

¼ 100 nm.

BDNF Modulates GABA and Glycine in Lamina II 969

Developmental Neurobiology. DOI 10.1002/dneu

synaptic vesicles and usually display one V1 (presyn-

aptic dendrite) and one V2 (axon) peripheral profiles

(type Ia or type I nonpeptidergic), whereas others

show a C bouton enriched with a number of peptide-

containing large granular vesicles (type Ib or type I

peptidergic). Type II glomeruli have a larger, lighter,

and less irregular C bouton, particularly rich in

mitochondria. Type II glomeruli also exist in two

subcategories: type IIa, with a C bouton devoid of

neurofilaments; and type IIb, with a C bouton rich of

neurofilaments.

Concurrent presence of trkB and GABA immunor-

eactivities was observed at glomeruli of the Ia [Fig.

6(A,B)], IIa [Fig. 6(C)] and IIb (Fig. 7) types. Irre-

spectively of the glomerular type, fl-trkB receptors

were identified in plain dendrites (D), and vesicle-

containing presynaptic dendrites (V1). Fl-trkB immu-

nolabeled plain dendrites were never positive for

GABA, whereas certain V1 profiles were dual labeled

for fl-trkB and GABA, the remaining ones being

only singularly immunostained by the anti-GABA

antiserum.

It seems noteworthy to remark here that, although

axons constituted the 33.2% of fl-trkB immunoreac-

tive profiles in rat lamina II (Salio et al., 2005), we

never observed fl-trkB immunopositive axons in con-

tact with dendritic or axonal profiles that stained posi-

tively for GABA.

DISCUSSION

In this article we show, for the first time, that exoge-

nous application of BDNF modulates the release of

both the inhibitory neurotransmitters GABA and gly-

cine onto lamina II neurons. In a subpopulation of

lamina II neurons the neurotrophin caused the

increase of GABA and glycine spontaneous mIPSC

frequency, leaving mIPSC amplitude and kinetics

unchanged. Action potential-dependent release of the

two neurotransmitters was also affected by BDNF:

the depression of eIPSCs was accompanied by

changes of CV and PPR, usually considered as indi-

cators of presynaptic modulation. Ultrastructural

studies, showing the co-localization of fl-trkB recep-

tors and GABA at presynaptic V1 profiles in synaptic

glomeruli, give further support to the role of BDNF

as a modulator of GABA and glycine release in spinal

cord dorsal horn.

Modulation of GABAergic andGlycinergic Miniature IPSCs

In our recordings most neurons exhibited both ‘‘pure

mIPSCs’’, mediated by a single type of receptor,

exhibiting monophasic decay and small amplitude,

and ‘‘mixed mIPSCs’’, mediated by both receptors,

larger and biphasic, with partial time constants simi-

lar to those of GABAergic and glycinergic mIPSCs.

This was not surprising, since GABA and glycine are

often co-released in dorsal horn, whereas a minority

of inhibitory synapses is mediated by GABA only

(Todd and Sullivan, 1990). Moreover, our recordings

were obtained from rats in the second postnatal week:

at this developmental stage mIPSCs can be mediated

by GABA or glycine or by both neurotransmitters, as

shown by previous studies (Keller et al., 2001). Fre-

quency of pure and mixed mIPSCs was increased by

BDNF, while the amplitude was not affected, sug-

gesting a presynaptic effect of the neurotrophin. The

frequency of pharmacologically isolated GABA and

glycine mIPSCs was also significantly increased, con-

firming a modulatory action of BDNF on both neuro-

transmitters. Moreover, the lack of effect on both

GABA and glycine mIPSC amplitude and kinetics

would likely exclude a modulation of the postsynap-

tic receptor function.

While evidence of a postsynaptic modulation of

glycine receptors by BDNF in CNS has not been pro-

vided to our knowledge, several studies performed in

different areas of the brain have shown that BDNF

can rapidly alter the functionality of postsynaptic

GABAA receptors, causing inhibition and receptor

Figure 7 Dual labeling immunocytochemistry for fl-trkB

receptors and GABA in rat spinal cord lamina II. Synaptic

glomerulus of the IIb type displaying two peripheral vesi-

cle-containing presynaptic dendrites (V1) immunoreactive

for GABA (10 nm gold). The V1 profile at right is also pos-

itively stained for fl-trkB (gold-intensified Fluoronano-

goldTM). The area indicated by the rectangle is shown at

higher magnification in the insert. Scale bars ¼ 500 nm;

insert ¼ 100 nm.

970 Bardoni et al.

Developmental Neurobiology. DOI 10.1002/dneu

internalization (Tanaka et al., 1997; Brunig et al.,

2001; Hennebeger et al., 2002; Cheng and Yeh, 2003;

Jovanovic et al., 2004). Differently from our data, in

these studies a significant depression of mIPSC

amplitude was usually observed, which was rarely

accompanied by a change of mIPSC frequency.

However, studies in several CNS areas have dem-

onstrated that BDNF can modulate GABAergic syn-

apses by regulating transmitter release. Thus, in

agreement with our results, facilitatory effects on

spontaneous GABA release have usually been re-

ported after acute (10–20 min) and chronic (h or

days) application of BDNF (McLean Bolton et al.,

2000; Wardle and Poo, 2003; Henneberger et al.,

2005; Baldelli et al., 2005).

In spinal cord dorsal horn, recent studies have pre-

viously provided evidence for BDNF-mediated con-

trol of neurotransmitter release. We have shown that

the acute application (10 min) of BDNF increases

AMPA mediated mEPSC frequency in neonatal rats,

without altering their amplitude or decay time (Bar-

doni et al., 2004). Matayoshi et al. (2005) report that

a very short application of BDNF (2 min) is sufficient

to increase mEPSC frequency in lamina II neurons

from adult rats, but only after induction of peripheral

inflammation. A facilitatory action of BDNF on GABA

release has been previously reported by Pezet et al.

(2002), showing that acute BDNF application induces

the increase of KCl-elicited GABA release in the

superfusate collected from the isolated spinal cord in

adult rats.

Effect of BDNF on GABAergic andGlycinergic Evoked IPSCs

While BDNF caused a facilitation of spontaneous

release of GABA and glycine, evoked IPSCs

recorded at 0 mV were depressed by the neurotro-

phin. Time course of BDNF action was similar to that

observed for mIPSCs (about 15–20 min) and wash

out was difficult to obtain. Depression of eIPSCs was

likely not simply due to the stimulation protocol,

since pre-incubation of the slices in the trk receptor

antagonist k-252a prevented the amplitude decrease

over the same time period. BDNF caused an increase

of synaptic variability of GABA/Glycine eIPSC peak

amplitude, expressed by the increase of CV, indicat-

ing a possible presynaptic modulatory site. Further-

more, the experiments performed on glycinergic

IPSCs applying the paired pulse protocol showed that

the PPR increases, suggesting a decrease of probabil-

ity of glycine release due to BDNF. Similar results

have been reported by Frerking et al. (1998) in CA1

pyramidal neurons, where BDNF causes the depres-

sion of GABAergic eIPSCs, inducing changes in CV

and PPR. Although the depressing action of BDNF

on eIPSCs is seemingly in contrast with the facilita-

tion of mIPSCs, a differential regulation of spontane-

ous and evoked release by BDNF has been already

reported. In cultured hippocampal neurons, at specific

synaptic types, BDNF causes an increase in mEPSC

frequency, which is not accompanied by a change of

evoked synaptic responses (Schinder et al., 2000).

Furthermore and in keeping with our results, Wardle

and Poo (2003) have observed in the same neuronal

type that BDNF causes a depression of eIPSCs, while

mIPSC frequency is increased. Moreover, examples

of opposite modulation of spontaneous and evoked

GABA and glycine release have also been observed

in dorsal horn neurons, involving ionotropic and

metabotropic presynaptic receptors (Jang et al., 2001;

Kerchner et al., 2001; Engelman et al., 2006). Cellu-

lar mechanisms regulating spontaneous, TTX resist-

ant, and action potential-dependent evoked release

can be different. The first can be regulated by pre-

synaptic receptors expressed close to the terminal,

controlling the intracellular calcium concentration.

Evoked release can also be affected by mechanisms

modulating action potential generation and propaga-

tion in the presynaptic neuron. Moreover, presynaptic

proteins can also contribute to the independent regu-

lation of spontaneous and evoked neurotransmitter

vesicular release: lack of functional synaptotagmin,

for instance, reduces evoked transmitter release but

increases mEPSC frequency (Broadie et al., 1994;

Geppert et al., 1994). These considerations indicate

that the final outcome of the BDNF modulatory

action on GABA and glycine release in dorsal horn

results by a complex interaction of multiple factors,

such as the cellular type(s) involved in synaptic activ-

ity, the subcellular localization of trkB receptors, and

the specific intracellular pathway(s) that are activated

by the neurotrophin.

Circuitry and Functional Significance

The ultrastructural studies presented in this article

show that co-localization of fl-trkB receptors and

GABA is present only at the level of V1 profiles in

any type of glomeruli, with the exception of type Ib

(peptidergic). In addition fl-trkB and GABA immuno-

reactive profiles were never engaged together in any

type of synaptic arrangement outside of glomeruli,

i.e. axo-dendritic or axo-somatic synapses. These

synaptic contacts, which represent the majority of

profiles in lamina II (Ribeiro-da-Silva, 2004), did not

BDNF Modulates GABA and Glycine in Lamina II 971

Developmental Neurobiology. DOI 10.1002/dneu

display the two labels together as pre and postsynap-

tic partners in any possible configuration. Although

we have performed these studies only on GABA

expression, it has been suggested that virtually all

glycinergic neurons in laminae I-III are also

GABAergic (Todd, 1990, 1996; Todd and Sullivan,

1990). This would suggest that co-localization of gly-

cine with trkB receptors follows a similar pattern.

Figure 8 summarizes in a schematic fashion one

possible circuital arrangement that is compatible with

the results of the present work.

V1 profiles in glomeruli (in red) are presynaptic

vesicular dendrites that are known to belong to

GABAergic islet cells, which often also contain gly-

cine (Spike and Todd, 1992). Fl-trkB receptor expres-

sion was detected not only in this type of profiles at

glomeruli, but also on plain dendrites, lacking of

GABA vesicles (D–in yellow): since it appears that

most GABAergic interneurons possess GABA-con-

taining vesicles in their dendrites (Spike and Todd,

1992), we can safely assume that these D profiles

belong to neurons other than the islet cells.

Our electron microscopy analysis shows that fl-

trkB receptors can be localized at V1 presynaptic ve-

sicular dendrites that contain, and likely release

GABA/glycine (Carton and Hayes, 1991; Spike and

Todd, 1992) and provide support for a presynaptic

action of BDNF on spontaneous release of these neu-

rotransmitters. Involvement of these presynaptic den-

drites in the regulation of evoked release is more dif-

ficult to explain, since they do not seem to be capable

to antidromically conduct action potentials (Safronov,

1999). In olfactory bulb mitral cells, release of

GABA from presynaptic dendrites has been reported

to be caused by calcium entry through NMDA recep-

tors and by membrane depolarization and subsequent

activation of voltage-dependent calcium channels

(Schoppa et al., 1998; Isaacson, 2001). The mecha-

nisms responsible for dendritic transmitter release in

dorsal horn glomeruli are still unknown: in our exper-

imental conditions, membrane depolarization of pre-

synaptic dendrites could be generated by focal stimu-

lation, evoking the release of the two inhibitory neu-

rotransmitters. Further studies are needed to confirm

this hypothesis.

The lack of expression of fl-trkB receptors on

GABAergic extraglomerular axo-dendritic and axo-

somatic synapses would exclude a direct and local

modulation of BDNF at these presynaptic terminals.

However, control of GABA and glycine release

(spontaneous and evoked) could result from the inter-

action between BDNF and fl-trkB on the dendrites,

triggering intracellular signals that rapidly propagate

up to the axon terminals or the modulation of ionic

Figure 8 Circuitry involved in the response of lamina II

neurons to BDNF. Schematic drawing representing a sim-

plified neuronal circuitry that may be responsible for the in-

hibitory response to BDNF of lamina II neurons. The cir-

cuitry is derived from the electrophysiological and ultrastruc-

tural experiments described in the result section. A

GABAergic islet cell (red) expressing fl-trkB receptors (blue

dots) at its V1 dendrites in glomeruli releases GABA at ter-

minal making axo-dendritic and/or axo-somatic synapses

onto the recorded neuron (orange). V1 profiles at glomeruli

are known to be bi-directionally connected (blue/red arrows)

with the central bouton (light blue). Dendro-dendritic synap-

ses between V1 and D profiles at glomeruli are also

described (see Ribeiro-da-Silva, 2004). The red arrows indi-

cate the putative sites of GABA release from V1 profiles.

Modulation at this level may be also linked to activation of

metabotropic glutamate receptors (not represented) that have

been described to be specifically localized at this type of glo-

merular profiles in non-peptidergic glomeruli. D profiles

expressing fl-trkB receptors (yellow–blue dots) are unlikely

to participate to modulation of inhibitory neurotransmission.

They may belong to excitatory neurons, whose influence

could be minimal in our experimental conditions of this

study, since ionotropic glutamate receptors are blocked (see

text).

972 Bardoni et al.

Developmental Neurobiology. DOI 10.1002/dneu

channels affecting action potential conduction (see

Blum and Konnerth, 2005).

Finally and strictly related to our present findings,

ultrastructural studies provided evidence for expres-

sion of metabotropic glutamate receptors specifically

at V1 profiles in non-peptidergic glomeruli (Jia et al.,

1999): this suggests that BDNF-mediated modulation

of inhibitory synaptic transmission could also involve

more indirect mechanisms, such as glutamate release.

BDNF is an important modulator of pain transmis-

sion in dorsal horn, exerting, in most cases, a pronoci-

ceptive action (see Merighi et al., 2004; Pezet and

McMahon, 2006). Our data are the first functional

demonstration that the neurotrophin can also exert

complex effects on inhibitory synaptic transmission

in lamina II. It is generally hold that peptidergic noci-

ceptors express low basal levels of BDNF, which are

upregulated following inflammation and peripheral

NGF stimulation. However, we have recently pro-

vided evidence that a subpopulation of these nocicep-

tors possesses substantial levels of the neurotrophin

also under basal conditions (Salio et al., 2005), and

that the neurotrophin is anterogradely transported to

terminals that form the C bouton in peptidergic type

Ib glomeruli (Salio et al., 2007). Interestingly, this

glomerular type does not have V1 profiles colocaliz-

ing fl-trkB receptors and GABA. One is therefore

tempted to speculate that in normal conditions of pain

transmission, low levels of BDNF are locally released

at type Ib glomeruli and do not significantly affect

inhibitory synaptic transmission. Upregulation of

BDNF in inflammatory and neuropathic pain condi-

tions would lead to a more abundant synaptic and/or

extrasynaptic release of the neurotrophin that could

diffuse to nearby glomeruli with fl-trkB/GABA con-

taining V1 profiles, and regulate, on a rapid time

scale (min), GABAergic and glycinergic transmission

in this population of islet cells. In particular, the con-

trol of evoked release could determine a general

depression of inhibition, compatible with the previ-

ously reported pronociceptive effects of BDNF (see

Pezet and McMahon, 2006), whereas the regulation

of spontaneous release could exert a local modulation

of the inhibitory tone and the control of primary

afferent terminal function at the glomerular level.

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