GABA-induced neurite outgrowth of cerebellar granule cells is mediated by GABA A receptor...

GABA-induced neurite outgrowth of cerebellar granule cells

is mediated by GABAA receptor activation, calcium influx

and CaMKII and erk1/2 pathways

Laura N. Borodinsky,*,1 Deirdre O’Leary,� Joseph H. Neale,§ Stefano Vicini,� Omar A. Coso�and Monica L. Fiszman*

*Instituto de Investigaciones Farmacologicas-CONICET, Buenos Aires, Argentina

�Laboratorio de Fisiologıa y Biologıa Molecular, Buenos Aires, Argentina

�Department of Physiology and Biophysics, School of Medicine, Georgetown University, Washington DC, USA

§Biology Department, Georgetown University, Washington DC, USA

Abstract

During neuronal development, GABAA-mediated responses

are depolarizing and induce an increase in the intracellular

calcium concentration. Since calcium oscillations can modu-

late neurite outgrowth, we explored the capability of GABA to

induce changes in cerebellar granule cell morphology. We find

that treatment with GABA (1–1000 lM) induces an increase in

the intracellular calcium concentration through the activation

of GABAA receptors and voltage-gated calcium channels of

the L-subtype. Perforated patch-clamp recordings reveal that

this depolarizing response is due to a chloride reversal

potential close to )35 mV. When cells are grown in depolar-

izing potassium chloride concentrations, a shift in reversal

potential (Erev) for GABA is observed, and only 20% of the

cells are depolarized by the neurotransmitter at day 5 in vitro.

On the contrary, cells grown under resting conditions are

depolarized after GABA application even at day 8. GABA

increases the complexity of the dendritic arbors of cerebellar

granule neurons via a calcium-dependent mechanism trig-

gered by voltage-gated calcium channel activation. Specific

blockers of calcium-calmodulin kinase II and mitogen-activa-

ted protein kinase kinase (KN93 and PD098059) implicate

these kinases in the intracellular pathways involved in the

neuritogenic effect of GABA. These data demonstrate that

GABA exerts a stimulatory role on cerebellar granule cell

neuritogenesis through calcium influx and activation of

calcium-dependent kinases.

Keywords: calcium influx, CaMKII, depolarization, erk1/2,

GABAA receptors, neuritogenesis.

J. Neurochem. (2003) 84, 1411–1420.

GABA is the main inhibitory neurotransmitter in the adult

central nervous system. During nervous system development,

this neurotransmitter exerts depolarizing responses through

the activation of GABAA receptors in many structures,

including the hippocampus, hypothalamus, striatum and spinal

cord (Ben-Ari et al. 1989; Fiszman et al. 1990, 1993; Mandler

et al. 1990; Obrietan and van den Pol 1995; Rohrbough and

Spitzer 1996). A higher intracellular concentration of chloride

yielding a depolarized chloride equilibrium potential explains

these GABA-induced depolarizing responses during develop-

ment in a number of cases (Reichling et al. 1994; Ben-Ari

et al. 1997; Rivera et al. 1999; Ganguly et al. 2001).

GABAA receptor-mediated depolarizing responses activate

voltage-gated calcium channels (VGCC), thus increasing the

intracellular calcium concentration ([Ca2+]i; (Connor et al.

Received October 2, 2002; revised manuscript received December 11,

2002; accepted December 11, 2002.

Address correspondence and reprint requests to Dr Monica L. Fisz-

man, ININFA-CONKET, Junın 956 5�Piso, 1113, Buenos Aires,

Argentina. E-mail: [email protected] present address of Laura N. Borodinsky is Neurobiology Section,

Biology Division, University of California, San Diego, 9500 Gilman Dr.,

La Jolla, CA 92093, USA.

Abbreviations used: [Ca2+]I, intracellular calcium; CaMKII, Ca2+-

calmodulin-dependent protein kinase II; CGC, cerebellar granule cells;

D, neuronal fractal dimension; DIV, days in vitro; Erev, reversal

potential; erk1/2, extracellular signal-regulated kinase 1 and 2; FRC-

HRP, tetanus toxin fragment C–horseradish peroxidase conjugated;

MAPK, mitogen-activated protein kinase; MEK1, MAPK kinase;

VGCC, voltage-gated calcium channels; VGCCL, VGCC of the L

subtype.

Journal of Neurochemistry, 2003, 84, 1411–1420 doi:10.1046/j.1471-4159.2003.01638.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1411–1420 1411

1987; Yuste and Katz 1991; Obrietan and van den Pol 1996;

Owens et al. 1996). Calciumentry throughVGCCcan activate

several kinases, including calcium2+-calmodulin-dependent

protein kinases (CaMKs), extracellular signal-regulated kinase

1 and 2 (erk1/2) and p38 (Ghosh and Greenberg 1995; Mao

et al. 1999). Activation of these different pathways in turn can

modulate various aspects of neuronal development such as

proliferation (LoTurco et al. 1995; Borodinsky and Fiszman

1998; Fiszman et al. 1999; Haydar et al. 2000), migration

(Behar et al. 1996), arborization (Wu and Cline 1998),

synaptic plasticity (Wang and Kelly 1995; Otmakhov et al.

1997) and survival (Gallo et al. 1987; Mao et al. 1999;

Borodinsky et al. 2002).

In the present report we have characterized the depolarizing

responses induced by GABA in cerebellar granule cells

(CGC), grown in vitro under resting and depolarizing condi-

tions, using Fura-2measurements and patch-clamp recordings.

Using this culture system, it has been possible to demonstrate

that exposure of the granule cells to GABA induces ultra-

structural changes (Hansen et al. 1984). The modulation of the

complexity of neuronal morphology induced by GABA was

assessed by determining changes in the fractal dimension (D)

of the cells. The results show thatGABA induces an increase in

[Ca2+]i through the activation of GABAA receptors and VGCC

of the L-subtype (VGCCL) in immature CGC. These depolar-

izing responses are due to a depolarized chloride equilibrium

potential. The exposure of immature CGC to GABA induces

an increase in the complexity of neuronal morphology through

calcium influx and the activation of CaMKII and erk1/2

pathways. These results suggest that GABA can modulate the

differentiation of cerebellar granule cells during development

through a calcium-dependent mechanism.

Materials and methods

Cell cultures

Cerebellar granule cells (CGC) were prepared from 6- to 8-day old

(P6-8) Sprague Dawley rats. Cerebella were excised, cut into 1 mm

pieces and placed in a Krebs solution containing 0.035% MgSO4,

0.6% glucose and 0.3% bovine serum albumin (BSA). The tissue

was incubated for 12 min at 37�C in the same buffer containing

0.02% trypsin, dissociated with fire-polished Pasteur pipettes in a

Krebs solution containing 0.01% DNAse (Boehringer Mannheim,

Mannheim, Germany) and 0.05% trypsin inhibitor, and then

centrifuged for 4 min at 150 g. Cells were inoculated in serum-

free medium (Neurobasal), supplemented with B27 (Gibco,

Rockville, MD, USA) containing 5 mM or 25 mM KCl, in plates

pre-coated with laminin, at a density of 7 · 103 cells/cm2 for

morphometrical studies, and at a density of 2.6 · 105 cells/cm2 for

calcium imaging and electrophysiological recordings.

Intracellular calcium measurements

Cells were grown for 1–8 days in serum-free medium, which was

then removed and replaced by Locke’s medium containing 2 lM

Fura-2 and 0.01% pluronic acid for 30 min. The cells were washed

twice with Locke’s medium and calcium imaging was performed

using an inverted Zeiss microscope (Thornwood, NY, USA) with a

40· fluorescence objective under continuous superfusion at a rate of

1 mL/min. Cells were illuminated at 340 and 380 nm using a xenon

lamp with quartz collector lenses. A shutter and a filter wheel were

controlled by computer. Emitted light was passed through a 400 nm

dichroic mirror, filtered at 490 nm and collected by a CCD camera.

Images were digitized in an image processor connected to a

computer equipped with Attofluor software (Atto Bioscience,

Rockville, MD, USA) which enables data acquisition from 99 cells

simultaneously. An internal calibration curve was constructed. Rmax

and Rmin are ratios at saturating and zero [Ca2+]i, respectively; they

were obtained by perfusing cells with a salt solution containing

10 mM CaCl2 and 10 lM ionomycin and, subsequently, with a Ca2+-

free salt solution containing 10 mM EGTA.

Electrophysiological recordings

Cultured granule cells grown for 1–8 days in vitro (DIV)were voltage-

clamped at various holding potentials in the whole-cell configuration

using the perforated patch-clamp technique on the stage of an inverted

microscope (Nikon) at room temperature. The recording pipette

contained (inmM) 145K-gluconate, 2MgCl2, 11EGTA, 2NaATP and

10 mM HEPES, brought to pH 7.2 with NaOH. Amphotericin B was

added at 100 lg/mL to allow access to the cell without altering the

endogenous chloride concentration (Reichling et al. 1994).Cellswere

bathed in a solution containing (in mM) 145 NaCl, 5 KCl, 2 CaCl2 and

5 HEPES, brought to pH 7.2 with NaOH. Osmolarity was adjusted to

325 mOsm/L with sucrose. The culture dish in the recording

chamber (< 500 lL total volume) was continuously superperfused

(5 mL/min). GABA was dissolved in bath solution and applied

directly by a gravity-fed Y-tubing delivery system (Murase et al.

1989) placed within 100 lm of the recorded cell. Currents were

monitored with an Axoclamp 2B patch amplifier (Axon Instrument,

Foster City, CA, USA), filtered at 1.5 kHz (8-pole low-pass Bessel)

and digitized in an IBM-PC computer with Pclamp8 software

(Axopatch 1D; Axon Instrument) for off-line analysis. Results are

expressed as mean ± SEM. Origin (MicroCal Software, Northampton,

MA, USA) was used for figure preparation. Statistical analysis was

performed using ANOVA or a paired t-test, and values were considered

significantly different for a p < 0.05 or p < 0.01, respectively.

Determination of neuronal complexity

Tetanus toxin fragment C cell staining

Tetanus toxin fragment C–horseradish peroxidase conjugate (FRC-

HRP; List Biological Laboratories, Inc., Campbell, CA, USA) was

used as previously described (Neale et al. 1993) with slight

modifications (Borodinsky and Fiszman 1998). FRC-HRP was

diluted to a concentration of 5 lg/mL in HEPES/BSA (in mM):

NaCl 145, KCl 5, CaCl2 2, MgCl2 1, HEPES 10 and glucose 10,

adjusted to pH 7.4 with NaOH; BSA was added to a final

concentration of 0.1%. Cultures were rinsed three times with

HEPES/BSA, incubated for 30 min at 22�C in FRC-HRP, rinsed

again three times in HEPES/BSA, and fixed for 30 min in 4%

paraformaldehyde. Samples were then washed three times in 0.01 M

phosphate-buffered saline (PBS) and incubated in 3-3¢diaminobenzidine (0.75 mg/mL) in Tris buffer (50 mM) at pH 7.4,

1412 L. N. Borodinsky et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1411–1420

containing 0.01% H2O2 (substrate), for 30 min. The reaction was

stopped with four washes in 50 mM Tris-HCl.

Measurement of fractal dimension

Neuronal complexity was determined by measurement of the fractal

dimension of neurons inoculated at low density for 48 h and then

fixed and stained with FRC-HRP as described previously (Borodin-

sky and Fiszman 2001). GABA was added 24 h before fixing and

staining the cells. Nifedipine, MgCl2 (Sigma, St. Louis, MO, USA),

PD98059 (New England Biolabs Inc., Beverly, MA, USA) and

KN93 (Seikagaku Corporation, Falmouth, MA, USA), were added

1 h before the addition of GABA. Photomicrographs from stained

cells grown in low-density cultures were taken and scanned. Binary

silhouettes were obtained using ScionImage software (Scion Corp.,

Frederick, MD, USA) and the fractal dimension (D) was calculated

using the dilation method (Smith et al. 1996). Briefly, the cell

perimeter was determined by replacing each border pixel with a disc,

which varied in size from 4 to 128 pixels. The equivalent perimeter

length after each dilation was plotted against the diameter of the

dilating disc on a log-log scale. The points were fitted by least

squares regression and D was calculated from the linear slope (S) by

the formula D ¼ 1–S. Neuronal fractal dimensions range between 1

and 2, with higher D indicating greater complexity. The fractal

dimension of neurons increases with the ruggedness of the cellular

border, the degree of branching and with space-filling capacity

(Neale et al. 1993; Smith et al. 1996).

Western blot analysis

To assess the levels of phosphorylated erk1/2 and phosphorylated

CaMKII, CGC at 2 DIV were treated with 25 mM KCl or 100 lM

GABA for 5, 15 or 30 min at 37�C. Following treatment, the neurons

were washed with ice-cold PBS and harvested in a lysis buffer

containing 25 mM HEPES, pH 7.5, 300 mM NaCl, 1.5 mM MgCl2,

0.2 mM EDTA, 0.1% Triton X100, 0.5 mM glycerolphosphate,

0.1 mM sodium vanadate, 2 lg/mL leupeptin, 0.6 mM phenyl-

methanesulfonyl fluoride (PMSF) and 0.5 mM 1,4-dithiothreitol

(DTT). Samples were centrifuged and the pellet was discarded. The

amount of protein present in the supernatant fluids was measured in

each sample as described by Lowry et al. (1951). Aliquots of 200 lg

were subjected to sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) on 10% polyacrylamide gels. After elec-

trophoretic separation, proteins were transferred to nitrocellulose

membranes (0.45 lm; Bio-Rad Laboratories, Hercules, CA, USA),

blocked with 5% non-fat dry milk in Tris-buffered saline containing

0.05% Tween-20, and probed with specific antibodies (anti-phos-

phorylated erk, 1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA,

USA; anti-phosphorylated CaMKII, 1 : 5000, Promega, Madison,

WI, USA). Immunoreactive bands were visualized using a chemilu-

minescence method and quantified by NIH IMAGE software (NIH

Software, Bethesda, MD, USA).

Results

GABA stimulates calcium elevation in cultured

postnatal cerebellar granule cells

GABA has been shown to exert depolarizing responses in

many developing central nervous system structures (Ben-Ari

et al. 1989; Fiszman et al. 1990, 1993; Mandler et al. 1990;

Obrietan and van den Pol 1995; Rohrbough and Spitzer

1996). In particular, we have previously reported that GABA

increases proliferation of immature cerebellar granule cells

through the activation of GABAA receptors and voltage-

dependent calcium channels (Fiszman et al. 1999). In the

present report, we further characterized the responses

triggered by GABA in immature CGC by carrying out

calcium imaging experiments using Fura-2 in CGC grown

for 2 DIV. The average basal [Ca2+]i was 55 ± 5 nM (mean ±

SEM, n ¼ 20 experiments). Addition of GABA induced an

increase in [Ca2+]i in a concentration-dependent manner with

a threshold concentration of 1 lM (Fig. 1a). The addition of a

maximal effective concentration of GABA (100 lM) induced

a 3.5-fold increase in [Ca2+]i over resting values, reaching a

peak of 150 ± 14 nM (mean ± SEM, n ¼ 16; Fig. 1a). The

rise in [Ca2+]i was completely blocked by 10 lM picrotoxin

(Fig. 1b), a chloride channel blocker, or by 10 lM bicucul-

line, a specific GABAA receptor antagonist (Fig. 1c). The

effect induced by GABA was also blocked by 1 lM

nifedipine, a VGCC blocker of the L-subtype (Fig. 1d).

These results show that GABA increases the intracellular

calcium concentration in young CGC through the activation

of GABAA receptors, and VGCCL are involved.

It has been reported that GABA exerts a depolarizing

response only in immature neurons (Rivera et al. 1999), and

that depolarizing culture conditions induce a GABA switch

to hyperpolarizing responses (Ganguly et al. 2001). There-

fore it was interesting to explore the influence of depolarizing

KCl concentration, a condition that promotes survival and

maturation of these cells (Gallo et al. 1987; Borodinsky

et al. 2002), on GABA-induced responses during different

times in vitro. Accordingly, we carried out calcium imaging

to explore GABA responses in cells plated in 5 or 25 mM

KCl. We found in 1–8 DIV cultures grown in 5 mM KCl that

the exogenous application of 100 lM GABA induced an

increase in [Ca2+]i (Fig. 2a, white bars), with 70% of the

cells being responsive (Fig. 2b). Interestingly, in 25 mM

KCl-containing cultures, the increase in [Ca2+]i induced by

GABAwas observed in cells from 1 to 4 DIV. However, after

5 or more days, GABA failed to increase [Ca2+]i (Fig. 2a,

black bars), and only 20% of the cells responded to GABA

with an increase in [Ca2+]i at day 5 (Fig. 2b, black bars). To

elucidate the ionic mechanism that underlies the change in

the response to GABA we performed recordings from CGC

as early as 24 h after plating. The resting membrane potential

of cells at 1–3 DIV measured in current-clamp perforated

patch recordings was )66 ± 2 mV (n ¼ 6) and ) 65 ± 3 mV

(n ¼ 3) in 5 and 25 mM KCl containing cultures, respect-

ively. GABA applied to these neurons grown under 5 or

25 mM KCl produced a depolarization (Fig. 3a). We then

compared the response induced by 100 lM GABA in

voltage-clamped CGC at various holding potentials to

GABAA receptor-mediated neurite outgrowth 1413


determine the Erev. When neurons were grown in 25 mM

KCl, currents evoked by GABA application to cells voltage-

clamped at )45 mV were inward at 1 DIV and outward at

8 DIV. At 1–3 DIV the Erev of GABA currents was

)36 ± 1 mV (n ¼ 3). In contrast, at 8–9 DIV the Erev was

)47 ± 2 mV (n ¼ 3, Fig. 3b). On the other hand, when

neurons were grown in 5 mM KCl for 6–8 DIV, currents

evoked by GABA application were still inward and the Erev

was )34 ± 4 mV (n ¼ 5), not significantly different from

the Erev measured at 1–3 DIV ()36 ± 2 mV, n ¼ 3).

CGC neuritogenesis induced by GABA

Because GABA exerts depolarizing responses in immature

CGC grown in vitro and calcium can regulate neurite

outgrowth (Connor 1986; Fields et al. 1993; Gomez and

Spitzer 1999; Borodinsky et al. 2002), it was of interest to

determine whether sustained application of GABA modu-

lates neuritogenesis.

Experiments were carried out with CGC grown under low-

density conditions to avoid formation of synaptic contacts,

and the fractal dimension (D) of the neurons was measured as

a parameter that reflects the complexity of the dendritic arbor

as previously described (Neale et al. 1993; Borodinsky and

Fiszman 2001).

We found that cells grown in isolation under resting

conditions have a bipolar morphology (Figs 4a and b) and a

D close to one (Fig. 4c) that corresponds to a low order of

complexity of neuronal morphology. The addition of 100 lM

GABA induces changes in neuronal morphology, when

compared with control cultures, that consists, in this

particular example, of an increase in the number of primary

neurites (Figs 4d, e and f). We quantified the neuronal fractal

dimension and found a significant increase in D induced

by GABA (Dcontrol ¼ 1.08 ± 0.01; DGABA ¼ 1.26 ± 0.02;

n ¼ 18, p < 0.0001, Fig. 5b).

Since GABA stimulates calcium elevations that could

activate Ca2+-dependent kinases, we tested the action of

blockers of calcium influx and inhibitors of kinases on the

neuritogenic effect induced by GABA to elucidate the

mechanisms involved. Binary silhouettes of three examples

for each treatment are shown in Fig. 5(a). We found that the

changes in neuronal morphology induced by GABA were

Fig. 1 GABA increases [Ca2+]i. Representative traces of Fura-2

measurements recorded from 99 CGC grown in vitro for 2 days

registered in a single experiment. Periods of drug application are

indicated by bars above the traces. Antagonists were added before

GABA as indicated in the figure. (a) Dose–response to GABA. (b) In

the presence of picrotoxin (PTX). (c) In the presence of bicuculline

(BIC). (d) In the presence of nifedipine (NIF).



prevented by calcium influx blockers such as 10 mM MgCl2or 1 lM nifedipine, or with MEK1 and CaMKII kinase

inhibitors, PD98059 (75 lM) and KN93 (10 lM), respect-

ively (Fig. 5b).

Western blot assays revealed that GABA induced a two to

fourfold increase in the levels of the phosphorylated forms of

erk1/2 and CaMKII. As shown in Fig. 6, 100 lM GABA was

as effective as 25 mM KCl in increasing the levels of the

active forms of CaMKII and erk1/2 as early as 5 min after

treatment. These changes were not due to an increase in the

total levels of the kinases, since levels of the total forms of

erk and CaMKII assessed using antibodies against these two

kinases did not change (data not shown). After 30 min of

stimulation (100 lM GABA or 25 mM KCl), the levels of

erk1/2 remained as high as in 5 min-treated samples, but

levels of pCaMKII were lower than in 5 min-stimulated

cultures (Fig. 6). This decrease in pCaMKII after longer

stimulation might be explained by an autoinhibitory mech-

anism and/or by the activation of specific phosphatase that

modulates the kinetics of CaMKII activation (Kubota and

Bower 2001; Hudmon and Schulman 2002).

Discussion

In the present report we demonstrate that GABA induces

depolarizing responses in immature CGC grown in vitro.

This depolarization is mediated by the activation of GABAA

receptors, which, in turn, induce calcium influx through

VGCCL. The mechanisms that explain GABAA-induced

depolarizing responses have been extensively reviewed, and

evidence has been presented in support of three different

mechanisms. Several investigators have observed an outward

chloride flux at early developmental stages (Reichling et al.

1994; Ben-Ari et al. 1997; Rivera et al. 1999). However,

other evidence supports a link between GABAA receptor

activation and VGCCL opening (Barker and Nicoll 1973;

Andersen et al. 1980; Mueller et al. 1984; Lambert et al.

1991; Segal 1993), and an outward bicarbonate flux through

GABAA receptors has been proposed (Staley et al. 1995).

Since HEPES was used to buffer our electrophysiological

recording solutions, it seems unlikely that a significant

Fig. 3 GABA induces an outward chloride current in immature CGC.

(a) Current clamp recording from a cerebellar granule neuron at 2 DIV.

Resting potential was ) 66 mV. The bar indicates the application of

100 lM GABA. (b) Voltage clamp recordings comparing the GABA-

evoked currents in granule cells grown in 25 mM KCl at different DIV

and at distinct holding voltages. The bars indicate the application of

100 lM GABA.

Fig. 2 Time course of [Ca2+]i increase induced by GABA in 5 and

25 mM KCl-containing cultures. (a) Increase in [Ca2+]i induced by

GABA in 5 and 25 mM KCl-containing cultures. (b) Percentage of cells

in which GABA induced an increase in [Ca2+]i.



contribution of bicarbonate ion permeability contributes to

our results. Our data demonstrate that CGC grown in vitro for

1–3 days have a depolarized chloride equilibrium potential

consistent with an elevated [Cl–]i.

The potassium chloride co-transporter (KCC2) maintains a

low intracellular chloride concentration (Rivera et al. 1999).

As reported by others, neuronal maturation is concomitant

with KCC2 expression (Rivera et al. 1999; Ganguly et al.

2001). We suggest that KCC2 expression may be up-regulated

more rapidly in cultures grown in 25 mM KCl than in 5 mM

KCl-containing cultures, as has been postulated for hippo-

campal neurons (Ganguly et al. 2001). Another possibility is

that depolarization-induced calcium transients are decreased

during development of CGC grown under depolarizing KCl

concentrations, as reported for hippocampal neurons grown

in vitro (Ganguly et al. 2001). However, the addition of

25 mM KCl induced an increase in [Ca2+]i at 1–8 DIV in

these cultures (Borodinsky et al. 2002). Moreover, this

increase was more robust at 5–8 DIV than at 1–3 DIV.

Thus, the possibility of a reduction in the depolarization-

induced elevation of intracellular calcium concentration in

later stages of cultures grown under depolarizing KCl

concentrations seems unlikely. In addition, these results

may reflect the fact that the GABAA system matures

differently in both cell culture conditions. In this regard, it

has been previously reported that GABAA receptor subunit

expression and function depend on the presence of depolar-

izing CGC culture conditions (Harris et al. 1995; Zhu et al.

1995), suggesting that neural activity modulates the matur-

ation of the GABAA receptors in cultured CGC.

In the ontogeny of excitable membranes, GABA induces

depolarizing responses during early development, and then

hyperpolarizing responses in the adult nervous system,

consistent with a role for GABA as a modulator of early

neural development (see review by Waagepetersen et al.

1999). Our results demonstrate that exogenously-applied

GABA increases the complexity of neuronal morphology

measured as the fractal dimension of isolated cultured

neurons. Previous data revealed a role of synaptically-released

GABA on neurite length of cultured hippocampal neurons

(Barbin et al. 1993). The neuritogenic effect induced by

GABA on CGC is mediated by calcium influx through VGCC

of the L-subtype. GABA-induced neurite outgrowth was also

dependent on the activation of CaMKII and erk1/2. The

involvement of calmodulin in glutamate-induced changes in

hippocampal dendrite outgrowth has been previously reported

(Wilson et al. 2000). Also, the MAP kinase signaling pathway

is involved in the neuritogenic effect induced by cell adhesion

molecule L1 on cultured cerebellar granule neurons (Schmid

et al. 2000), in neurite outgrowth of cultured hippocampal

cells (Veeranna et al. 1998), and in NGF-mediated neurite

outgrowth in PC12 cells (Pang et al. 1995). The precise

sequence of the intracellular events responsible for GABA-

induced neuritogenesis remains to be established. Calcium

Fig. 4 Analysis of fractal dimension for

cultured CGC in control and GABA-treated

cultures. CGC were plated at low density

in serum-free medium (a, b, c). After 24 h

in vitro, 100 lM GABA was added (d, e, f).

Staining with a specific neuronal marker

was performed at 48 h (a, d). Binary sil-

houettes were obtained using Scion Image

software (b, e). Fractal dimension (D) was

calculated using the dilation method (c, f).

The log of the perimeter of the neuron is

plotted against the log of the disc diameter

used to measure the perimeter. The D for

neuron (a) is 1.07; for neuron (d) D ¼ 1.31.



elevations stimulated by GABA may activate CaMKII,

leading to activation of erk1/2, as previously described for

vascular smooth muscle cells (Muthalif et al. 1998). We

previously showed that the addition of 25 mM KCl induced a

CaMKII-dependent increase in neuronal fractal dimension

that was not dependent on erk1/2 activity (Borodinsky et al.

2002). One explanation for this difference could be that

100 lM GABAand not 25 mM KClwas able to activate erk1/2.

However, as shown in the present report, 25 mM KCl and

100 lM GABA were both able to activate erk1/2 to the same

extent, hence excluding this possibility. Alternatively, the

increases in [Ca2+]i induced by 25 mM KCl or GABA are

different in amplitude and duration, and differences in time

course as well as spatial selectivity (e.g. soma or dendrites) of

erk1/2 cascade activation may yield distinct effects. Consis-

tent with this concept, Dudek and Fields (2001) found that the

frequency of Ca2+ entry is a determining factor in the

activation of MAPK in hippocampal slices.

The present report shows a potential interaction between

neural activity mediated by GABA and cytoskeletal dynam-

ics leading to neurite morphological changes. The interplay

between GABA-mediated calcium influx, CaMKII and erk1/2

activation, and molecules that modulate cytoskeletal

dynamics, remains to be established. Actin or microtubule-

associated proteins may be possible target effectors for

GABA-induced neurite outgrowth (van Rossum and Hanisch

1999; Halpain 2000). The Rho family of small GTP-binding

proteins is involved in cytoskeletal organization in non-

neuronal cells (Hall 1994; Chant and Stowers 1995) and in

neural cells (Mackay et al. 1995; Threadgill et al. 1997).

Thus, the activation of erk1/2 and CaMKII may be coupled

with the activation of GTP-binding proteins. Additionally,

Fig. 5 GABA-induced increase in neuronal

complexity is blocked by calcium influx

blockers, MEK1 and CaMKII inhibitors. (a)

Binary silhouettes of typical examples of

neuronal morphologies obtained in control

and treated samples. (b) Pooled data of D

calculated for CGC grown under different

treatments shown in (a).



VGCCL activation leads to cAMP-response element binding

protein (CREB) phosphorylation in cortical neurons

(Dolmetsch et al. 2001), and CREB was found to be required

for neural cell adhesion molecules (N-CAM)-mediated neurite

outgrowth in PC12 cells (Jessen et al. 2001). Moreover, in

developing neurons, GABA increases brain-derived neuro-

trophic factor (BDNF) expression via a MAPK-CREB-

dependent mechanism (Obrietan et al. 2002). These results

suggest that CREB may mediate GABA-induced neuritogen-

esis.

Taken together, these results indicate that GABA exerts

depolarizing responses in immature CGC grown in vitro and

increases the complexity of their morphology. Since CGC are

recipients of GABAergic synapses from Golgi II at day 5,

GABA is detected in Golgi II as early as day 3, and as

GABAA receptors are expressed in the developing CGC at

the time of synaptogenesis (Meinecke and Rakic 1990), this

neurotransmitter may influence the maturation of CGC

morphology and function in vivo.

Acknowledgements

This work was supported by Fundacion Antorchas A-13622/1–122

and Carrillo-Onativia Fellowship (MLF), Buenos Aires, Argentina.

LNB was funded with a Postdoctoral Fellowship of CONICET,

Buenos Aires, Argentina. We thank Dr Nicholas Spitzer for critical

reading of the manuscript and Dr Belen Elghoyen for the loan of

equipment.

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