REVIEW ARTICLE

GABA Effects During Neuronal Differentiation of Stem Cells

Patricia Salazar Æ Marco A. Velasco-Velazquez ÆIvan Velasco

Accepted: 21 February 2008 / Published online: 21 March 2008

� Springer Science+Business Media, LLC 2008

Abstract Gamma-amino butyrate (GABA) is the most

prevalent inhibitory neurotransmitter in the adult brain. In this

review, we summarize the pharmacology and regulation of

GABAergic transmission components (biosynthetic enzymes,

receptors and transporters) in adult non-neurogenic brain

regions. The effects of targeted mutations in genes relevant for

GABAergic functions and how they influence specific neu-

ronal circuits and pathological states are presented. We then

review GABA actions on neuronal differentiation. During

brain development, GABA has depolarizing activity in cere-

brocortical neural precursors, controlling cell division and

contributing to neuronal migration and maturation. In the

adult forebrain there are two neurogenic regions exposed to

synaptic and non-synaptic GABA release. Neural stem cells

and neuronal progenitors express GABA receptors in sub-

ventricular and subgranular zones. GABA effects in these

cells are very similar to those found in embryonic cortical

precursor cells, and therefore it is possible that this amino acid

has important roles during adult brain plasticity.

Keywords GAD � GABA receptors � GAT � GABAergic

depolarization � Neuronal migration � Adult neurogenesis

GABA Synthesis

GABA is synthesized preferentially in central nervous

system (CNS) from glutamate by glutamate decarboxylase

(GAD) enzymes. Early kinetic and inhibition studies

showed two GAD isoforms with different affinity for its

coenzyme, pyridoxal-50-phosphate, GAD65 and GAD67 [1–

4]. These proteins are encoded by different, independently

regulated genes. GAD67, located on cell bodies and den-

drites, synthesizes a cytoplasmic pool of GABA, and

GAD65 synthesizes synaptic GABA in nerve endings [5].

Once produced, GABA is stored into secretory vesicles by

an active transporter called vesicular GABA transporter

(VGAT), that has been cloned and characterized [6].

Vesicular GABA will be released by exocytosis.

Glutamate decarboxylase knockout mice were generated

a decade ago. Since then, it is known that GAD67-deficient

mice present cleft palate and die after birth [7], and that

GAD65 knockout mice show susceptibility to seizures,

indicating a less severe weakness of GABAergic transmis-

sion [8, 9]. Currently, GAD-mediated GABA synthesis has

been implicated in the regulation of activity in specific

GABAergic neurons, and seems to cooperate in the estab-

lishment of proper neuronal networks in definite regions of

the Nervous System. GABA synthesized by GADs is

required for development of the neural network that controls

the respiratory system in rodents. Mouse fetuses lacking

GAD67 die from respiratory failure, even when their CNS is

not affected macroscopically [10]. Given that in these mice a

small amount of GABA is still synthesized by GAD65, Fujii

et al. [11] studied the bursting activity of respiratory motor

nerves and the activity of single respiratory neurons in

GAD67/ GAD65 double knockout mice. These mice die after

birth similarly to GAD67 knockouts, but their forebrain

is totally depleted of GABA [11, 12]. In these

Special issue article in honor of Dr. Ricardo Tapia.

P. Salazar � I. Velasco (&)

Departamento de Neurociencias, Instituto de Fisiologıa Celular,

Universidad Nacional Autonoma de Mexico, AP 70-253,

Mexico, DF 04510, Mexico

e-mail: ivelasco@ifc.unam.mx

M. A. Velasco-Velazquez

Departamento de Farmacologıa, Facultad de Medicina,

Universidad Nacional Autonoma de Mexico, Mexico,

DF 04510, Mexico

123

Neurochem Res (2008) 33:1546–1557

DOI 10.1007/s11064-008-9642-8

GAD67-/-:GAD65

-/- mice, respiratory discharges recorded

from ventral roots are decreased in embryonic day (E)15

fetuses, and totally absent in E18 fetuses [11]. Accordingly,

neurons from the rostroventrolateral medulla show no

respiratory discharges. Application of substance P, which

produces hyperpolarization, induces respiratory discharges

in those neurons. In contrast, substance P has no effect in

neurons from VGAT-deficient mice, suggesting that also

glycine (affected because it is also introduced to synaptic

vesicles by VGAT) is essential for the function of respiratory

neurons [11]. Another cerebral region in which GABAergic

transmission is important is the visual cortex. Maturation of

perisomatic synapses in the adolescent visuocortex of

GAD67 knockdown mice is significantly impaired. Basket

interneurons from GAD67+/- and GAD67

-/- but not GAD65-/-

mice show reduced axonal branching and synapse formation

in organotypic cultures [13]. These abnormalities can be

partially rescued by inhibiting the GABA transport system

with 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]

oxy]ethyl]-3-pyridinecarboxylic acid (NO-711), or by the

administration of modulators of the GABAA (diazepam), or

GABAB (baclofen) receptors [13]. In contrast, perisomatic

innervation in visual cortex is normal in GAD65-knockdown

mice [13]. These results show that GAD67-mediated GABA

synthesis is specifically involved in GABAergic axonal and

synaptic morphogenesis.

Some of the genetic modifications to alter GAD expres-

sion make organisms unviable, and even before development

of knockout technology, other approaches have been tested

to chemically inhibit GAD activity in the postnatal period.

The consistent result of these experiments is that low GAD

activity results in epileptic convulsions [14–17].

GABA Receptors

Once GABA is formed, it is released, synaptically or non-

synaptically, to act on specific receptors. There are many

different compounds that modify responses of the GABAergic

system by acting on three types of widely described GABA

receptors: the ionotropic GABAA and GABAC receptors [18,

19], and the metabotropic GABAB receptor [20].

GABAA Receptors

GABAA receptors are ligand-gated ion channels permeable

to chloride, distributed throughout the mammalian CNS.

These receptors have shown high diversity by the different

combination of subunits: a1-6, b1-4, c1-4, d, e, p, and h,

that confer distinct characteristics, including differences in

channel kinetics, affinity for GABA, and rate of desensi-

tization, that will ultimately affect GABA function as

anxiolitic, anesthetic or anticonvulsant [19, 21].

The first GABA receptor described, GABAA, was iden-

tified by its sensitivity to inhibition by the specific

antagonists bicuculline and picrotoxin [22]. Even when

different agonists (i.e. muscimol) of GABAA receptor have

been discovered, modulators are the most important GABAA

receptor-interacting drugs. GABAA receptors have different

high-affinity drug-binding sites that confer depressant and

antiepileptic properties [23–25]. Benzodiazepines, neuros-

teroids and barbiturates bind to different sites of GABAA

receptors with the same consequence: an increase in the

period of pore opening and an enhancement in Cl- current

through the receptors [21]. The pharmacology of these

receptors depends on subunit composition [19].

Benzodiazepines are positive allosteric modulators of

GABAA receptors. Diazepam potentiates GABA inhibitory

actions, and therefore has CNS depressant properties [23,

24]. Benzodiazepines have been used for treatment of

epilepsy and chronic anxiety. Binding studies in transfected

cells revealed that benzodiazepine pharmacology was

dependent on subunit composition, particularly of the asubunit [26]. Compounds that show preferential affinity for

a1 subunit, such as zolpidem, have mainly hypnotic

activity. In contrast, a2 containing receptors, but not those

that contain a3, mediate the anxiolytic activity and myo-

relaxant effects of diazepam [18, 27, 28]. Another subunit

involved in the potentiating effect of benzodiazepines is the

c subunit, while b2 subunit is responsible for the receptor

desensitization [19].

Neurosteroids, such as allopregnanolone, enhance Cl-

passing through GABAA receptors, and protect against

different models of epilepsy like those induced by picro-

toxin or 4-aminopyridine in rat hippocampal slices [29].

Chronic administration of allopregnanolone increases

expression of the a4 and d subunits of the GABAA

receptors in several areas of the CNS [30]. Barbiturates

actions depend largely on the b subunit, although their

GABA-potentiating activity is influenced by a subunit

type. Pentobarbital is more effective than GABA only

when receptors contain an a6 subunit [31].

Subunit-specific GABAA receptor knockouts have been

generated. These studies have provided evidence that some

subunits are more important for a given nervous system

disorder or pharmacological response. Genetic deletion of

the a1-subunit produces a variable phenotype that can

include strong body tremor, spontaneous seizures, lower

body weight [32, 33], and increased susceptibility to the

locomotor stimulants effects of ethanol [34, 35]. A pro-

nounced up-regulation of other GABAA receptor subunits,

without functional compensation, ocurrs in regions where

a1-GABA receptors are absent [36]. In vitro studies have

shown that inhibitory postsynaptic currents (IPSC) are

especially altered in visual cortex [37] and hippocampus

[36]. Moreover, cerebral Purkinje cells show a complete

Neurochem Res (2008) 33:1546–1557 1547

123

loss of GABAA mediated transmission [38]. The fact that

neurons lacking a1 subunit have different functional

properties suggest that the plasticity of GABAergic circuits

probably includes formation of novel synaptic connections

in order to ensure stable function of neuronal networks [36,

39]. b3 subunit of the GABAA receptor is important for

normal CNS function, including the response to anesthetics

[40]. This subunit also participates in the pathogenesis of

neurodevelopmental disorders such as Angelman Syn-

drome [41–43] and autism spectrum disorder [44–46].

GABAA receptor b3-subunit knockout mice show cleft

palate abnormalities [47] and high neonatal mortality [48].

In addition, these mice present compensatory adaptations

[49, 50] and numerous behavioral abnormalities. Recently,

a system that allows conditional inactivation of the b3 gene

in a tissue and/or developmentally specific manner was

engineered [51]. When this subunit is deleted only from

CNS, palate development occurs normally, but survival

ratio is low. In contrast, mice with a selective forebrain

knockout of GABAA b3 survived the neonatal period, but

show reduced reproductive fitness, decreased sensitivity to

the anesthetic etomidate, were hyperactive, and some

became obese [51]. These results confirm that the b3

subunit of the GABAA receptor is involved in develop-

mental processes, normal physiology of behavior and the

onset of pharmacological responses, and suggest that it

may be involved in body weight control. Knockout of the dsubunit produces altered behavioral responses to alcohol

[52] or neurosteroids [53].

GABAA receptors have also shown to be located

extrasynaptically. These receptors could modulate GABA

release to control neuronal excitability in cerebellum and

hippocampus, where GABAA receptors away from the

GABA release sites could interfere with the propagation of

the action potentials by inducing tonic inhibition [54, 55].

Such extrasynaptic GABA receptors have also been found

in retina, where drugs and neurohormones that act on

GABAA receptors could affect the firing patterns critical

for the establishment of adult neural circuits [56].

As we will discuss later, activation of GABAA

receptors in embryonic or adult neural precursors causes

depolarization. This phenomenon, possibly related to a

locally reversed Cl- gradient, can also occur in some

non-neurogenic adult regions. However, in the majority

of adult CNS, the principal action for GABA is to

produce inhibition.

GABAB Receptors

The metabotropic GABAB receptor is coupled to G pro-

teins and its activation causes a presynaptic inhibition by

(i) suppressing the activation of N- and P/Q-type voltage-

gated Ca2+ channels that allow Ca2+ entrance important to

trigger neurotransmitter release, and (ii) by increasing the

opening of voltage-gated K+ channels [57]. GABAB

receptor is not blocked by bicuculline and is activated by

the specific agonist baclofen [58]. Even when other specific

agonists have been discovered, baclofen is still the most

studied GABAB receptor activator. Baclofen induces a

centrally mediated muscle relaxant effect and thus is

effective treating spasticity. This GABAB agonist presents

antinociceptive effects, reduces the craving for cocaine,

show anti-bronchoconstriction and antitussive activities

[59], as well as anxiolytic activity in some clinical assays

[60, 61]. Two allosteric positive modulators of GABAB

receptors have been reported: CGP7930 and GS39783 [62,

63]. Both compounds accentuate the effects of GABA and

baclofen, even when they do not have a direct agonist

activity.

GABAB receptors consist of two subunits: GABAB1

and GABAB2. Deletion of either of the subunits results

in a complete loss of GABAB functions and induces a

highly anxious phenotype [64, 65]. For GABAB1 subunit,

one gene generates two isoforms: GABAB1a is expressed

as a presynaptic heteroreceptor in the hippocampus and

lateral amigdala, and GABAB1b is predominantly located

postsynaptically in these structures [66]. Mice with

genetic mutation of GABAB1a show impaired object

recognition [66, 67] and fail to acquire a conditioned

taste aversion [68], indicating that this isoform is

essential for object recognition and discrimination. In

contrast, GABAB1b-/- mice acquired conditional taste

aversion normally, but failed to extinguish the learned

avoidance up to 30 days later [68], suggesting that this

isoform participates in the retrieval or long-term storage

of memory. Both isoform-deficient mice show similar

innate anxiety and impairments in a test indicative of

spatial working memory [67]. Together, these studies

indicate that both isoforms of the GABAB1 subunit

contribute to cognitive capability, but each conveys

specific components of cognitive processes.

GABAC Receptors

GABAC ionotropic receptors are formed by q1-3 subunits

[69]. This receptor does not respond to bicuculline or

baclofen [70] and can be specifically inhibited by TPMPA

[(1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid]

[71] and activated by CACA (cis-4-amino-crotonic acid) or

CAMP (cis-2-aminomethyl-cyclopropane carboxilic acid)

[72]. Being both ionotropic receptors, GABAC receptor

shares with GABAA receptor: (i) the susceptibility to pic-

rotoxin, and (ii) the capability of being activated by TACA

(trans-4-aminocrotonic acid) or muscimol [73].

The physiological roles of GABAC q1 subunit are

poorly understood compared to GABAA, given that this

1548 Neurochem Res (2008) 33:1546–1557

123

subunit is relatively new discovered. GABAC receptor q1

subunit is highly expressed in the retina [74]. Mice lacking

q1 subunit present retinal alterations with deficient visual

information processing [75]. Recently, q1 subunit was

found to be highly expressed in mitral cells of the olfactory

bulb. When this GABAC subunit is eliminated by genetic

deletion, olfactory sensitivity is enhanced, which may be

caused by an increased sensitivity and over-excitation of

the primary olfactory neurons, secondary to an attenuated

inhibition in the mitral cell layer of the olfactory bulb [76].

Thus, GABAC q1 subunit has also a specific role in the

physiological function of the olfactory sensory system.

GABA Uptake

After GABA has acted on its neuronal receptors, it is

removed from extracellular space by specific transporters

expressed on neuronal and glial cells. Inside glial cells,

GABA is catabolized by GABA transaminase to succinic

semialdehyde, with concomitant transamination of a-keto-

glutarate to form glutamate. Then, glutamate is transformed

to glutamine, via glutamine synthetase, to leave the cell and

reach a GABAergic neuron, where a glutaminase enzyme

transforms glutamine to glutamate, the GABA precursor

[5].

GABA uptake is carried out by high-affinity sodium/

chloride-dependent transporters. Four GABA transporters

(GATs) have been identified. The classical GABA uptake

inhibitors nipecotic acid, guvacine, and THPO (4,5,6,7-

tetrahydroisoxazolo [4,5-c]pyridin-3-ol) were essential for

the structural elucidation and classification of GATs [77].

Guvacine and nipecotic acid show high affinity for rat

(r)GAT-1 and rGAT-2 [mouse (m)GAT3 homologue], and

low affinity for rGAT-3 (mGAT4 homologue) or human

BGT-1 (mGAT2 homologue) [78]. Tiagabine and NO-711

are lipophilic derivatives of nipecotic acid. Interestingly,

these compounds are typically at least four orders of

magnitude more potent at GAT-1 than at any of the other

GATs [79]. Classical GABA uptake inhibitors show a low

permeability at the blood–brain barrier; therefore, these

compounds are inefficient in systemic therapy [77]. On the

other hand, their lipophilic derivatives have been subjected

to extensive animal behavioral studies primarily as anti-

convulsant agents. Tiagabine as well as other derivatives of

nipecotic acid are used clinically as a therapeutic agents for

the treatment of epilepsy [80] as well as for anxiety dis-

orders [81].

The major plasma membrane transporter responsible for

GABA uptake in adult CNS is GAT-1, which is abundant

in axonal terminals and present in some astroglia processes

[82–84]. In GAT-1-/- mice, extracellular levels of GABA

are increased, resulting in a largely enhanced GABAA

receptor-mediated tonic conductance in several brain

regions [85, 86]. These animals displayed altered behav-

ioral response to ethanol [87], tremor, ataxia and

nervousness [86], reduced aggression and lower level of

depression and anxiety-like behaviors, suggesting that

GAT1 is involved in the pathophysiology of depression

[88].

GAT-2 is located in ependymal cells, meninges and

choroid plexus, with rare expression in neurons or astro-

cytes, and therefore is thought that it participates in the

regulation of GABA concentration in cerebrospinal fluid.

GAT-3, on the other hand, is present in neurons and glia in

adult CNS, but at lower levels than GAT-1 [89]. However,

during development, GAT-3 is the most prevalent GAT in

the cerebral cortex: it appears in neuropil, around blood

vessels, and in numerous cells in late embryonic life

[90, 91].

The uptake of GABA is a very efficient process. For

example, in cortical cultures containing neurons and glial

cells, it has been shown that addition of 100 lM GABA is

decreased to 20 lM after 6 h, and only the addition of

100 lM GABA together with uptake inhibitors caused an

increase in GABA concentration around 50 lM, which was

sufficient to inhibit glutamate neurotoxicity [92].

Although a physiologic role for GABA outside the

nervous system is unclear, this amino acid has been found

in peripheral tissues, including liver, kidney, pancreas,

testis, oviduct, adrenal, sympathetic ganglia, gastrointesti-

nal tract and circulating erythrocytes [93]. It is reported

that insulinoma pancreatic cells possess GAD activity,

synthesize GABA and present low GABA uptake [94].

However, different from that reported for brain, pancreatic

GAD activity was not enhanced by addition of pyridoxal-

50-phosphate. Immunofluorescence and electron micros-

copy studies in pancreatic islets of Langerhans, show that

a- and b-cells contain GABA in their microvesicles and

secretory granules, and these cells also contain VGAT and

a plasma membrane transporter (GAT-3) for GABA uptake

[95]. These findings suggest that pancreatic cells possess

the required components to synthesize and release GABA,

which could participate in paracrine signaling.

After reviewing some aspects of GABAergic transmis-

sion in adult CNS, the next sections are devoted to

descriptions of GABA actions during cerebral cortex

development, and the effects of GABA on adult neural stem

cells from the subventricular zone (SVZ) and subgranular

zone (SGZ) of the hippocampus. Neural stem cells are

undifferentiated cells that self-renew and can differentiate

to neurons and glial (astrocytes and oligodendrocytes) cells

[96, 97]. Only neuronal differentiation will be considered

here. There is compelling evidence that GABA regulates

different aspects of neurogenesis in these three cerebral

regions.

Neurochem Res (2008) 33:1546–1557 1549

123

Neurogenesis in the Developing Cerebral Cortex

During early CNS development, the region close to the

ventricle is a neurogenic epithelium constituted by asyn-

chronously dividing cells presenting interkinetic nuclear

movement. Cerebrum cortices are thus formed from multi-

potent neural stem cells that proliferate initially in the

ventricular zone (VZ), and later on the SVZ. These NSC

produce neuroblasts that migrate away from VZ to pass the

intermediate zone (IZ) and stopping just before the marginal

zone (MZ). Neuroblasts reaching the cortical plate (CP)

produce neurons that accommodate themselves in one of the

six layers present in the mature cerebral cortex. One of the

defining features of cortical development is the presence of

glial fibrillary acidic protein (GFAP)-positive cells called

radial glia (RG), that span the thickness of this structure from

the VZ to the CP, and serve as scaffolds for radial migration

of neurons to the CP [98]. Although there is also tangential

migration, especially relevant for GABAergic interneurons

generation, radial migration has been studied extensively

[99]. Early-born neurons can be found in deeper layers and

late-born cells are closer to the pial surface. During this well-

described developmental process, dividing progenitors can

be found in VZ and SVZ. This latter region prevails into

adulthood, producing neural precursors that generate neu-

rons that migrate to the olfactory bulb, which will be

described later. No cell divisions can be observed in the CP

until the gliogenic phase of cortical development, which is

delayed compared with neurogenesis. Glial production will

not be discussed here. Recently, a portion of RG cells have

been identified as the neural stem cells in cerebrocortical

region [100, 101]. Once asymmetrically generated, neurons

migrate to the CP (Fig. 1a). This migration through RG

shafts has been associated with a leading process directed to

the pial end. Migration is dependent on a high number of

membrane proteins, quimioactive molecules and transcrip-

tion factors. One converging mechanism for regulating

radial neuronal migration is the synaptic-independent regu-

lation of intracellular calcium. Cells stop migrating when

they pass previously generated neurons and reach the CP/MZ

interphase, as they adopt their final position in the CP. Cor-

tical neurons are not generated exclusively in the cortex, but

are also born in the ganglionic eminence of the ventral tel-

encephalon and follow migratory routes through the IZ to

reach the cortex and contribute with GABAergic interneu-

rons [102, 103].

The appearance of GABA in cerebral cortex is very

early in development, much before that synaptic contacts

are apparent. At E10 in mice, GABA immunoreactivity

appears with strong staining in the neuroepithelial pial

surface, although diffuse labeling can be found in the entire

neural tissue. At E12 and E14, GABA labeling is still

stronger in the outer layers above the germinal area. By

E16, essentially there is no GABA mark in the proliferative

zone but the labeling remains in the CP and IZ [104].

Knock-in mice that express Green Fluorescent Protein

(GFP) under GAD67 promoter have shown that this enzyme

is present from E15 in IZ and MZ of cerebral cortices

[105]. It is very unlikely that synaptic release is occurring

at these stages, since no structural evidence of synapses are

in place at VZ nor IZ. GATs are also present in early stages

of forebrain development. At E18, GAT-1 and GAT-3 are

expressed in brain cortex, and after birth extended to the

entire cortex [106]. Ambient GABA in developing cerebral

cortex could be released by growth cones [107] or by

GATs from GABAergic neurons [108]. In addition to the

biosynthetic machinery for GABA, during brain develop-

ment, GABAA receptor subunits are expressed and show

different regional and temporal expression. The subunits

a2, a3, and a5 are expressed during the embryonic stages,

while the others are expressed after birth [109, 110].

The inhibitory hyperpolarizing action of GABA in mature

CNS is not present in developing cerebral cortex. In fact,

activation of GABAA receptors in proliferating neuroblast of

the VZ causes a depolarization that lasts longer and required

lower GABA concentrations than GABA responses in

mature neurons. The first evidence that GABA regulates

cortical neurogenesis came from explants where it was

demonstrated that GABA depolarizes neural cells at the VZ,

causing decreased DNA synthesis. This cell cycle arrest was

correlated with depolarization and calcium entry, since high

potassium and glutamate had the same effects. GABA

depolarization was not present with furosemide, indicating

that chloride currents were responsible of this excitatory

action. Endogenous tonic GABA release is present in the

cortical slices, since adding bicuculline caused a shift in

current [111]. A more detailed analysis of GABA effects on

proliferation evidenced that in fact 30 lM GABA caused a

shortening in cell cycle duration in VZ cells, but a decrease in

proliferation of SVZ cells. When analyzed as a general

proliferating population, the overall effect of GABA or

muscimol was to prevent proliferation, which are essentially

the results reported earlier. Regarding neuronal differentia-

tion, GABA prevents neurogenesis by maintaining

precursors of the VZ proliferating, because GABA-treated

slices had less neuroblasts migrating above the SVZ and

therefore decreased number of postmitotic neurons in the CP

[104]. Cortical precursor cells divide in culture when

exposed to basic fibroblast growth factor (bFGF). This

cytokine caused an increase in the proportion of cells

expressing a1 GABAA receptor subunit. GABA blocked

bFGF-induced proliferation (Fig. 1a) and caused neuronal

differentiation of cortical precursors [112].

Later on, it was shown that neuroblasts contained a

higher content (around 30 mM) of Cl- than mature neu-

rons (close to 10 mM), and since GABAA receptors are

1550 Neurochem Res (2008) 33:1546–1557

123

chloride channels, the net effect in the first case is excita-

tion, and hyperpolarization in the second [113]. This high

Cl- concentration is due to the expression of NKCC1, a

Na+/K+/2Cl- inward transporter that causes intracellular

accumulation of chloride in precursor cells. Mature neu-

rons, in addition to NKCC1, also express KCC2, a K+/Cl-

transporter that extrudes chloride from the cells and shift

GABA action from excitation to inhibition in cortical [114]

and hippocampal cells [115]. Recently, in utero electro-

poration of KCC2 switched GABA action from excitation

to inhibition in VZ cells. Postnatal analysis of KCC2

overexpressor cells showed correct migration and cortical

layering. However, a maturation defect was observed

in vivo, consisting of decreased length and number of

branches in neuronal processes. To correlate GABA-

induced depolarization with these alterations, prenatal

transfection was performed with Kir2.1 potassium channel,

which resulted in cells with lower membrane potential, and

caused similar structural changes in postnatal cerebral

cortex [116]. These findings can be related to experiments

showing that GABA regulates neurite outgrowth in cul-

tured neurons in a chloride-dependent fashion [117].

Interestingly, knocking out Dlx1 caused a reduced number

in a subset of GABAergic interneurons in the cortex, and

the surviving cells had smaller dendrite length and reduced

branching; these mutant animals developed epilepsy [118].

Cortical layers

A GABAARGABA

A cellsB cell

C cell

GABAnaptic)

GABABR

GABACRRadialglia

bFGF

Developingcerebralcortex

GABA

B Olfactory bulb(non-sy RMS

SVZ

Ependymal cells

Type-1cell

CGABA Dentate gyrus

SGZ

Type-2 (D)cell GABA

Fig. 1 Schemes showing neurogenesis and GABA actions in devel-

oping cerebral cortex, subventricular zone (SVZ) and subgranular

zone (SGZ). In the left side of each panel, multipotent neural stem

cells (NSC) are identified by a self-pointing arrow that indicates self-

renewal. Neurogenic events continue to the right side in the grey cells.

GABAA receptor activation in precursor cells cause depolarization

and calcium entry. ? indicates positive modulation and ‘ symbol

means a negative effect on proliferation, migration or maturation. (a)

Radial glia cells are NSC in the cerebral cortex. These cells divide

asymmetrically to generate basic fibroblast growth factor (bFGF)-

responsive neuroblasts that migrate radially to the cortical plate.

GABA stops proliferation in neuroblasts. Radial migration takes place

for neurons to accommodate in cortical layers, being layer 1 the

deepest and earliest-generated, and the sixth layer the last to be

formed, and the most external one. Radial migration is regulated by

different GABA receptors. GABAC receptor (GABACR) activation

allow exit from ventricular and subventricular zones. GABA binding

to GABABR promotes the migration from the intermediate zone to

cortical plate, and GABAAR stimulation at the marginal zone

indicates that cell must stop and integrate to the corresponding

cortical layer. GABA depolarization is important for neuronal

processes branching and growth. One important difference with

adult-born neurons is that during development, neurons that project to

other regions of the CNS are generated, whereas in the adult brain

only interneurons are produced. (b) NSC in the SVZ are GFAP+/

Nestin+ and are called B cells. These cells generate transit amplyfing

C cells that produce migrating neuroblasts (A cells). The rostral

migratory stream (RMS) brings A cells to the Olfactory bulb, where

neurons fully differentiate. A cells non-synaptically release GABA

(dotted arrow), which in turn decreases proliferation of B cells trough

GABAAR activation and also slows rostral migration. A cells express

GAT-3/4 and not GAT-1. (c) Type-1 cells are NSC in the SGZ. Few

of these cells respond to GABA. Type-2 cells, which are PSA-

NCAM+/Doublecortin+/Nestin+ and divide more actively are depo-

larized by GABAAR activation. The resulting effect is a decrease in

proliferation and enhanced neuronal differentiation. GABA depolar-

ization also promotes the growth of processes in granule neurons

when they reach the dentate gyrus

Neurochem Res (2008) 33:1546–1557 1551

123

GABA produces chemotaxis (directed migration to

GABA source) at submicromolar concentrations, and che-

mokinesis (random motility) when applied in micromolar

ranges in dissociated E15 cortical cells [119]. GABA has

also been shown to affect neuroblast radial migration: E18

cerebrocortical slices were incubated with the mitotic

marker BrdU and analyzed 48 h later. Controls show

BrdU-positive cells that migrated to CP. Addition of

saclofen (GABAB antagonist) caused accumulation in IZ,

whereas picrotoxin (GABAA+C receptor blocker) caused

labeled cells to remain in VZ/SVZ. On the other hand,

bicuculline did not block, but enhanced migration to the CP

and caused increased thickness. A model was proposed in

which transit from VZ/SVZ was dependent on GABAC

receptors, the pass from IZ to CP was promoted by GABAB

activation and the stop signal at the MZ required GABAA

receptors [120]. In vivo application of GABAergic drugs

also alters migration. Implanting slabs that acutely release

muscimol or bicuculline in the surface of cerebral cortex of

neonatal rats caused heterotopia (an increased number of

neurons in the surface of the cortex), just underneath the

application points, after 7 or 14 days. In brain slices,

bicuculline, and to a lesser extent muscimol, caused an

increase in migration of cells to the cortical surface. No

mechanism is provided to explain the fact that GABAA

agonist and antagonist had the same effect [121].

Thus, GABA has depolarizing effects on cerebrocortical

cells, causes decreased proliferation of neuroblasts, par-

ticipate in neuronal migration and promotes neurite

extension during development. Collectively, the evidence

points to very important roles of GABA during neuronal

circuit formation in rodents.

Neurogenesis in Adult Brain

Subventricular Zone

The region in the adult brain that contains the largest

germinal zone for neurons is the SVZ. This area contains

multipotent neural stem cells [122–125] that are thought to

originate from the embryonic VZ [126], and produce

neuroblasts that migrate to their final differentiation site,

the olfactory bulb, through the rostral migratory stream

(RMS, Fig. 1b). This migration is independent of RG or

other glial elements [127]. Structural and functional anal-

yses have provided a detailed architecture of the SVZ (see

Fig. 1b). Neural stem cells (called B cells) express GFAP

and are in close apposition with ependymal cells of the

ventricle. These cells divide and generate transit amplify-

ing (C) cells that differentiate into migratory neuroblast

(A cells) that generates GABAergic and dopaminergic

interneurons in the olfactory bulb. Migrating neuroblast,

similar to what it is observed during CNS development,

express doublecourtin [128, 129].

Similar to the effects reported in cortical precursors dur-

ing development, GABA elicited a depolarizing (+14 to

+19 mV) chloride current resulting from GABAA receptor

activation in precursor cells from postnatal and adult SVZ

[130, 131]. Precursors in culture express GAD67 and GAD65,

and are immunopositive for GABA. Their subunit combi-

nation of GABAA receptor include a2-5, b1-3 and c1-3, since

these subunits were detected by RT-PCR [131, 132]. A

proportion of PSA-NCAM+ precursor cells, cultured as

neurospheres, responded with an intracellular calcium rise

upon GABA application, which caused decreased prolifer-

ation via mitogen-activated protein kinases MEK 1 and 2.

Release of endogenous GABA to the culture medium is

likely, since GABAA antagonists prevented these responses,

and the positive GABAergic modulators clonazepam and

pentobarbital decreased proliferation. Interestingly, EGF

application lowered GABA concentration in the cultures,

suggesting a feedback loop where EGF directly stimulates

proliferation and indirectly decreased GABA extrusion to

prevent GABA anti-proliferative effects [132].

In saggital cerebral slices that encompass SVZ and RMS,

application of 10 lM GABA or pentobarbital decreased A

cells’ rostral migratory rate, which was in fact activated by

bicuculline, pointing to endogenous GABA modulation of

migration in the RMS. This was further supported by the fact

that elevating GABA levels, by causing depolarization-

induced release, or by inhibition of GATs, decreased

migration speed [133]. These ex-vivo studies were extended

by the same group using coronal slices of transgenic animals

that express GFP in GFAP-positive B cells. When 100 lM

GABA was applied, all GFP+ cells presented a GABAA

receptor-mediated inward current close to chloride equilib-

rium potential that resulted in cell depolarization. Addition

of exogenous GABA was potentiated by an uptake blocker

selective for GAT 3/4, and unaffected by NO-711 (GAT1-

specific). Slices were electrically stimulated and a GABAA

response was observed in GFP-expressing cells. GABA

release by neuroblasts (A cells) was taking effect, although

amino acid extrusion was not synaptic in nature, since it was

tetrodotoxin-, vesicular release- and extracellular calcium-

independent. This group nicely showed that GABA release

had a non-proliferating effect on SVZ B cells (NSC) and

conversely, bicuculline augmented NSC division [134]. In

this scheme, GABA secreted by A cells would act as a par-

acrine factor for B cells regulating cell number, and also as an

autocrine molecule to influence migration in the RMS.

Subgranular Zone in the Hippocampus

Hippocampal multipotent neural stem cells reside in the

SGZ of the dentate gyrus, between the hilus and the

1552 Neurochem Res (2008) 33:1546–1557

123

granule cell layer, and express GFAP [135] and Nestin.

These type-1 cells divide slowly and generate highly

dividing progenitors (type-2 cells, D cells) that migrate a

short distance to integrate as neurons into the granule cell

layer of the dentate gyrus (Fig. 1c). Migrating neuroblasts

express doublecourtin and PSA-NCAM, and terminally

differentiated neurons are positive for NeuN and Calbindin

[136]. There is also evidence for hippocampal neurogenesis

in humans [137]. Since hippocampal formation has been

related to several memory and learning functions, the

possibility that adult hippocampal neurogenesis could play

a physiological role in such tasks has been suggested [138,

139]. Significant numbers of adult-born neurons are pro-

duced in the hippocampus [140], although a proportion

undergoes cell death. There are several factors that modify

the amount of neurons produced in this structure such as

hormones, enriched environment, exercise, stroke and

small modulatory RNAs [141–145].

Using mice that express GFP under Nestin regulatory

elements, two groups described fluorescent type-1 neural

stem cells that have one thick process and enter cell cycle

sporadically, whereas GFP+ type-2 cells are PSA-

NCAM+, have a more round morphology and actively

divide. Type-2 cells showed spontaneous and evoked

depolarizing currents sensitive to bicuculline [146, 147].

Intracellular chloride concentration in these cells was 30

vs. 5 mM in mature granule cells. This was correlated with

NKCC1 expression in type-2 cells, and not in granule

neurons. GABA application caused increased intracellular

calcium, and this was followed by expression of the neu-

rogenic transcription factor Neuro D [146]. Other works

have established that early-born neurons do not respond to

zolpidem, a GABAA receptor a1 subunit potentiator, while

mature neurons show increased GABA hyperpolarizing

responses in its presence [148, 149]; a1 is expressed by

full-grown neurons but absent in newborn neurons [149].

Recently, a double retroviral labeling technique, with

two different fluorescent proteins injected at E15 and in

adulthood, allowed to study the functional integration of

embryonic-born hippocampal neurons and adult-differen-

tiated granule cells in the same slice. After analyzing

evoked responses in both neuronal populations, they con-

clude that recorded synaptic responses are very similar, and

just subtle differences are present [150]. In these studies, it

was established that newborn neurons receive GABA

afferents first and then glutamatergic synaptic contacts,

similar to what is observed in developing cerebral cortex.

Using GFP retrovirus, it has been shown that there is

tonic GABAA receptor-mediated depolarization in new-

born neurons. The depolarizing action of GABA is due to a

high chloride content in immature cells that express

NKCC1, and as cells mature they start expressing KCC2,

reducing chloride concentration and responding to GABA

by hyperpolarization. Injecting retrovirus that express short

hairpin RNA targeting Nkcc1, the authors knock down

expression of NKCC1 and reversed the depolarizing action

of GABA. This was accompanied by a delayed and reduced

integration of the newborn neurons to GABA and gluta-

mate synaptic contacts. Remarkably similar to what was

observed in developing cerebral cortex, manipulation of

cells to lower intracellular chloride concentration, and

therefore inhibition of GABA-induced depolarization,

caused a marked decrease in the length and arborization of

dendrites [151].

In vivo application of GABAA receptor-interacting

drugs had no effect on type-1 cells, but increased prolif-

eration (antagonists) or arrested (agonists) type-2 cells.

Positive modulators of GABAA receptors caused a long-

term increase in the number BrdU+ Calbindin+ neurons 28

days after treatment [146]. These results are hard to

interpret because both mature hippocampal circuitry and

progenitor cells would be exposed to these pharmacologi-

cal agents.

In conclusion, the actions of GABA in developing CNS

and germinal niches in adult brains are different from

GABAergic inhibitory hyperpolarization in non-neuro-

genic regions of adult CNS. In neural precursors, GABA

causes depolarization and affects proliferation, migration,

and neuronal maturation. Therefore, GABAergic players

are important during developmental and adult neurogene-

sis. The results summarized here show that genetic or

pharmacological modifications of the GABAergic system

cause alterations in neuronal circuit formation, emphasiz-

ing the role of GABA in CNS plasticity.

Acknowledgements Our laboratories are supported by grants from

PAPIIT of Universidad Nacional Autonoma de Mexico, Conacyt

(M.A.V-V. and I.V.), National Institute for Neurological Disorders

and Stroke, Fundacion Aleman and TWAS (I.V.).

References

1. Bayon A, Possani LD, Tapia M et al (1977) Kinetics of brain

glutamate decarboxylase. Interactions with glutamate, pyridoxal

50-phosphate and glutamate-pyridoxal 50-phosphate Schiff base.

J Neurochem 29:519–525

2. Covarrubias M, Tapia R (1980) Brain glutamate decarboxylase:

properties of its calcium-dependent binding to liposomes and

kinetics of the bound and the free enzyme. J Neurochem

34:1682–1688

3. Erlander MG, Tobin AJ (1991) The structural and functional

heterogeneity of glutamic acid decarboxylase: a review. Neu-

rochem Res 16:215–226

4. Kaufman DL, Houser CR, Tobin AJ (1991) Two forms of the

gamma-aminobutyric acid synthetic enzyme glutamate decar-

boxylase have distinct intraneuronal distributions and cofactor

interactions. J Neurochem 56:720–723

5. Soghomonian JJ, Martin DL (1998) Two isoforms of glutamate

decarboxylase: why? Trends Pharmacol Sci 19:500–505

Neurochem Res (2008) 33:1546–1557 1553

123

6. McIntire SL, Reimer RJ, Schuske K et al (1997) Identification

and characterization of the vesicular GABA transporter. Nature

389:870–876

7. Asada H, Kawamura Y, Maruyama K et al (1997) Cleft palate

and decreased brain gamma-aminobutyric acid in mice lacking

the 67-kDa isoform of glutamic acid decarboxylase. Proc Natl

Acad Sci USA 94:6496–6499

8. Kash SF, Johnson RS, Tecott LH et al (1997) Epilepsy in mice

deficient in the 65-kDa isoform of glutamic acid decarboxylase.

Proc Natl Acad Sci USA 94:14060–14065

9. Asada H, Kawamura Y, Maruyama K et al (1996) Mice lacking

the 65 kDa isoform of glutamic acid decarboxylase (GAD65)

maintain normal levels of GAD67 and GABA in their brains but

are susceptible to seizures. Biochem Biophys Res Commun

229:891–895

10. Kuwana S, Okada Y, Sugawara Y et al (2003) Disturbance of

neural respiratory control in neonatal mice lacking GABA

synthesizing enzyme 67-kDa isoform of glutamic acid decar-

boxylase. Neuroscience 120:861–870

11. Fujii M, Arata A, Kanbara-Kume N et al (2007) Respiratory

activity in brainstem of fetal mice lacking glutamate decar-

boxylase 65/67 and vesicular GABA transporter. Neuroscience

146:1044–1052

12. Ji F, Kanbara N, Obata K (1999) GABA and histogenesis in fetal

and neonatal mouse brain lacking both the isoforms of glutamic

acid decarboxylase. Neurosci Res 33:187–194

13. Chattopadhyaya B, Di Cristo G, Wu CZ et al (2007) GAD67-

mediated GABA synthesis and signaling regulate inhibitory

synaptic innervation in the visual cortex. Neuron 54:889–903

14. Tapia R, Pasantes-Morales H, Taborda E et al (1975) Seizure

susceptibility in the developing mouse and its relationship to

glutamate decarboxylase and pyridoxal phosphate in brain.

J Neurobiol 6:159–170

15. Salazar P, Montiel T, Brailowsky S et al (1994) Decrease of

glutamate decarboxylase activity after in vivo cortical infusion

of gamma-aminobutyric acid. Neurochem Int 24:363–368

16. Massieu L, Rivera A, Tapia R (1994) Convulsions and inhibition

of glutamate decarboxylase by pyridoxal phosphate-gamma-

glutamyl hydrazone in the developing rat. Neurochem Res

19:183–187

17. Arias C, Valero H, Tapia R (1992) Inhibition of brain glutamate

decarboxylase activity is related to febrile seizures in rat pups.

J Neurochem 58:369–373

18. Mohler H (2006) GABA(A) receptor diversity and pharmacol-

ogy. Cell Tissue Res 326:505–516

19. Benarroch EE (2007) GABAA receptor heterogeneity, function,

and implications for epilepsy. Neurology 68:612–614

20. Lujan R (2007) Subcellular regulation of metabotropic GABA

receptors in the developing cerebellum. Cerebellum 6:123–129

21. Macdonald RL, Olsen RW (1994) GABAA receptor channels.

Annu Rev Neurosci 17:569–602

22. Bormann J (1988) Electrophysiology of GABAA and GABAB

receptor subtypes. Trends Neurosci 11:112–116

23. Atack JR (2005) The benzodiazepine binding site of GABA(A)

receptors as a target for the development of novel anxiolytics.

Expert Opin Investig Drugs 14:601–618

24. Banfi S, Cornelli U, Fonio W et al (1982) A screening method

for substances potentially active on learning and memory.

J Pharmacol Method 8:255–263

25. Macdonald RL, Kelly KM (1994) Mechanisms of action of

currently prescribed and newly developed antiepileptic drugs.

Epilepsia 35(Suppl 4):S41–50

26. Pritchett DB, Luddens H, Seeburg PH (1989) Type I and type II

GABAA-benzodiazepine receptors produced in transfected

cells. Science 245:1389–1392

27. Crestani F, Low K, Keist R et al (2001) Molecular targets for

the myorelaxant action of diazepam. Mol Pharmacol 59:

442–445

28. Crestani F, Martin JR, Mohler H et al (2000) Mechanism of

action of the hypnotic zolpidem in vivo. Br J Pharmacol

131:1251–1254

29. Salazar P, Tapia R, Rogawski MA (2003) Effects of neurosteroids

on epileptiform activity induced by picrotoxin and 4-aminopyri-

dine in the rat hippocampal slice. Epilepsy Res 55:71–82

30. Smith SS, Shen H, Gong QH et al (2007) Neurosteroid regula-

tion of GABA(A) receptors: Focus on the alpha4 and delta

subunits. Pharmacol Ther 116:58–76

31. Drafts BC, Fisher JL (2006) Identification of structures within

GABAA receptor alpha subunits that regulate the agonist action

of pentobarbital. J Pharmacol Exp Ther 318:1094–1101

32. Sur C, Wafford KA, Reynolds DS et al (2001) Loss of the major

GABA(A) receptor subtype in the brain is not lethal in mice.

J Neurosci 21:3409–3418

33. Kralic JE, Korpi ER, O’Buckley TK et al (2002) Molecular and

pharmacological characterization of GABA(A) receptor alpha1

subunit knockout mice. J Pharmacol Exp Ther 302:1037–1045

34. Kralic JE, Wheeler M, Renzi K et al (2003) Deletion of

GABAA receptor alpha 1 subunit-containing receptors alters

responses to ethanol and other anesthetics. J Pharmacol Exp

Ther 305:600–607

35. Blednov YA, Walker D, Alva H et al (2003) GABAA receptor

alpha 1 and beta 2 subunit null mutant mice: behavioral

responses to ethanol. J Pharmacol Exp Ther 305:854–863

36. Schneider Gasser EM, Duveau V, Prenosil GA et al (2007)

Reorganization of GABAergic circuits maintains GABAA

receptor-mediated transmission onto CA1 interneurons in

alpha1-subunit-null mice. Eur J Neurosci 25:3287–3304

37. Bosman LW, Rosahl TW, Brussaard AB (2002) Neonatal

development of the rat visual cortex: synaptic function of

GABAA receptor alpha subunits. J Physiol 545:169–181

38. Fritschy JM, Panzanelli P (2006) Molecular and synaptic orga-

nization of GABAA receptors in the cerebellum: effects of

targeted subunit gene deletions. Cerebellum 5:275–285

39. Marder E, Goaillard JM (2006) Variability, compensation and

homeostasis in neuron and network function. Nat Rev Neurosci

7:563–574

40. Boehm SL II, Ponomarev I, Jennings AW et al (2004) gamma-

Aminobutyric acid A receptor subunit mutant mice: new per-

spectives on alcohol actions. Biochem Pharmacol 68:1581–1602

41. DeLorey TM, Handforth A, Anagnostaras SG et al (1998) Mice

lacking the beta3 subunit of the GABAA receptor have the

epilepsy phenotype and many of the behavioral characteristics

of Angelman syndrome. J Neurosci 18:8505–8514

42. Saitoh S, Kubota T, Ohta T et al (1992) Familial Angelman

syndrome caused by imprinted submicroscopic deletion

encompassing GABAA receptor beta 3-subunit gene. Lancet

339:366–367

43. Wagstaff J, Knoll JH, Fleming J et al (1991) Localization of the

gene encoding the GABAA receptor beta 3 subunit to the

Angelman/Prader-Willi region of human chromosome 15. Am J

Hum Genet 49:330–337

44. Shao Y, Cuccaro ML, Hauser ER et al (2003) Fine mapping of

autistic disorder to chromosome 15q11-q13 by use of pheno-

typic subtypes. Am J Hum Genet 72:539–548

45. DeLorey TM (2005) GABRB3 gene deficient mice: a potential

model of autism spectrum disorder. Int Rev Neurobiol 71:

359–382

46. Buxbaum JD, Silverman JM, Smith CJ et al (2002) Association

between a GABRB3 polymorphism and autism. Mol Psychiatry

7:311–316

1554 Neurochem Res (2008) 33:1546–1557

123

47. Culiat CT, Stubbs LJ, Montgomery CS et al (1994) Phenotypic

consequences of deletion of the gamma 3, alpha 5, or beta 3

subunit of the type A gamma-aminobutyric acid receptor in

mice. Proc Natl Acad Sci USA 91:2815–2818

48. Homanics GE, DeLorey TM, Firestone LL et al (1997) Mice

devoid of gamma-aminobutyrate type A receptor beta3 subunit

have epilepsy, cleft palate, and hypersensitive behavior. Proc

Natl Acad Sci USA 94:4143–4148

49. Ramadan E, Fu Z, Losi G et al (2003) GABA(A) receptor beta3

subunit deletion decreases alpha2/3 subunits and IPSC duration.

J Neurophysiol 89:128–134

50. Wong SM, Cheng G, Homanics GE et al (2001) Enflurane

actions on spinal cords from mice that lack the beta3 subunit of

the GABA(A) receptor. Anesthesiology 95:154–164

51. Ferguson C, Hardy SL, Werner DF et al (2007) New insight into

the role of the beta3 subunit of the GABAA-R in development,

behavior, body weight regulation, and anesthesia revealed by

conditional gene knockout. BMC Neurosci 8:85

52. Mihalek RM, Bowers BJ, Wehner JM et al (2001) GABA(A)-

receptor delta subunit knockout mice have multiple defects in

behavioral responses to ethanol. Alcohol Clin Exp Res 25:

1708–1718

53. Zhu WJ, Wang JF, Krueger KE et al (1996) Delta subunit

inhibits neurosteroid modulation of GABAA receptors. J Neu-

rosci 16:6648–6656

54. Glykys J, Mody I (2007) The main source of ambient GABA

responsible for tonic inhibition in the mouse hippocampus.

J Physiol 582:1163–1178

55. Kullmann DM, Ruiz A, Rusakov DM et al (2005) Presynaptic,

extrasynaptic and axonal GABAA receptors in the CNS: where

and why? Prog Biophys Mol Biol 87:33–46

56. Wang CT, Blankenship AG, Anishchenko A et al (2007)

GABA(A) receptor-mediated signaling alters the structure of

spontaneous activity in the developing retina. J Neurosci

27:9130–9140

57. MacDermott AB, Role LW, Siegelbaum SA (1999) Presynaptic

ionotropic receptors and the control of transmitter release. Annu

Rev Neurosci 22:443–485

58. Bormann J (2000) The ‘ABC’ of GABA receptors. Trends

Pharmacol Sci 21:16–19

59. Bowery NG, Bettler B, Froestl W et al (2002) International

Union of Pharmacology. XXXIII. Mammalian gamma-amino-

butyric acid(B) receptors: structure and function. Pharmacol Rev

54:247–264

60. Drake RG, Davis LL, Cates ME et al (2003) Baclofen treatment

for chronic posttraumatic stress disorder. Ann Pharmacother

37:1177–1181

61. Breslow MF, Fankhauser MP, Potter RL et al (1989) Role of

gamma-aminobutyric acid in antipanic drug efficacy. Am J

Psychiatry 146:353–356

62. Urwyler S, Pozza MF, Lingenhoehl K et al (2003) N,N0-di-

cyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine

(GS39783) and structurally related compounds: novel allosteric

enhancers of gamma-aminobutyric acidB receptor function.

J Pharmacol Exp Ther 307:322–330

63. Urwyler S, Mosbacher J, Lingenhoehl K et al (2001) Positive

allosteric modulation of native and recombinant gamma-ami-

nobutyric acid(B) receptors by 2,6-Di-tert-butyl-4-(3-hydroxy-

2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog

CGP13501. Mol Pharmacol 60:963–971

64. Bowery NG (2006) GABAB receptor: a site of therapeutic

benefit. Curr Opin Pharmacol 6:37–43

65. Mombereau C, Kaupmann K, Froestl W et al (2004) Genetic

and pharmacological evidence of a role for GABA(B) receptors

in the modulation of anxiety- and antidepressant-like behavior.

Neuropsychopharmacology 29:1050–1062

66. Vigot R, Barbieri S, Brauner-Osborne H et al (2006) Differen-

tial compartmentalization and distinct functions of GABAB

receptor variants. Neuron 50:589–601

67. Jacobson LH, Kelly PH, Bettler B et al (2007) Specific roles of

GABA(B(1)) receptor isoforms in cognition. Behav Brain Res

181:158–162

68. Jacobson LH, Kelly PH, Bettler B et al (2006) GABA(B(1))

receptor isoforms differentially mediate the acquisition and

extinction of aversive taste memories. J Neurosci 26:8800–8803

69. Zhang D, Pan ZH, Zhang X et al (1995) Cloning of a gamma-

aminobutyric acid type C receptor subunit in rat retina with a

methionine residue critical for picrotoxinin channel block. Proc

Natl Acad Sci USA 92:11756–11760

70. Feigenspan A, Bormann J (1998) GABA-gated Cl- channels in

the rat retina. Prog Retin Eye Res 17:99–126

71. Chebib M, Mewett KN, Johnston GA (1998) GABA(C) receptor

antagonists differentiate between human rho1 and rho2 receptors

expressed in Xenopus oocytes. Eur J Pharmacol 357:227–234

72. Kusama T, Spivak CE, Whiting P et al (1993) Pharmacology of

GABA rho 1 and GABA alpha/beta receptors expressed in

Xenopus oocytes and COS cells. Br J Pharmacol 109:200–206

73. Bormann J, Feigenspan A (1995) GABAC receptors. Trends

Neurosci 18:515–519

74. Cutting GR, Lu L, O’Hara BF et al (1991) Cloning of the

gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA

receptor subunit highly expressed in the retina. Proc Natl Acad

Sci USA 88:2673–2677

75. McCall MA, Lukasiewicz PD, Gregg RG et al (2002) Elimi-

nation of the rho1 subunit abolishes GABA(C) receptor

expression and alters visual processing in the mouse retina.

J Neurosci 22:4163–4174

76. Chen Y, Zhou D, Zhou K et al (2007) Study on olfactory

function in GABA(C) receptor/channel rho(1) subunit knockout

mice. Neurosci Lett 427:10–15

77. Krogsgaard-Larsen P, Frolund B, Frydenvang K (2000) GABA

uptake inhibitors. Design, molecular pharmacology and thera-

peutic aspects. Curr Pharm Des 6:1193–1209

78. Borden LA (1996) GABA transporter heterogeneity: pharma-

cology and cellular localization. Neurochem Int 29:335–356

79. Dhar TG, Borden LA, Tyagarajan S et al (1994) Design, syn-

thesis and evaluation of substituted triarylnipecotic acid

derivatives as GABA uptake inhibitors: identification of a ligand

with moderate affinity and selectivity for the cloned human

GABA transporter GAT-3. J Med Chem 37:2334–2342

80. LaRoche SM (2007) A new look at the second-generation

antiepileptic drugs: a decade of experience. Neurologist 13:

133–139

81. Crane D (2003) Tiagabine for the treatment of anxiety. Depress

Anxiety 18:51–52

82. Gadea A, Lopez-Colome AM (2001) Glial transporters for

glutamate, glycine, and GABA: II. GABA transporters. J Neu-

rosci Res 63:461–468

83. Guastella J, Nelson N, Nelson H et al (1990) Cloning and

expression of a rat brain GABA transporter. Science 249:

1303–1306

84. Schousboe A, Sarup A, Larsson OM et al (2004) GABA trans-

porters as drug targets for modulation of GABAergic activity.

Biochem Pharmacol 68:1557–1563

85. Jensen K, Chiu CS, Sokolova I et al (2003) GABA transporter-1

(GAT1)-deficient mice: differential tonic activation of GABAA

versus GABAB receptors in the hippocampus. J Neurophysiol

90:2690–2701

86. Chiu CS, Brickley S, Jensen K et al (2005) GABA transporter

deficiency causes tremor, ataxia, nervousness, and increased

GABA-induced tonic conductance in cerebellum. J Neurosci

25:3234–3245

Neurochem Res (2008) 33:1546–1557 1555

123

87. Cai YQ, Cai GQ, Liu GX et al (2006) Mice with genetically altered

GABA transporter subtype I (GAT1) expression show altered

behavioral responses to ethanol. J Neurosci Res 84:255–267

88. Liu GX, Cai GQ, Cai YQ et al (2007) Reduced anxiety and

depression-like behaviors in mice lacking GABA transporter

subtype 1. Neuropsychopharmacology 32:1531–1539

89. Conti F, Minelli A, Melone M (2004) GABA transporters in the

mammalian cerebral cortex: localization, development and

pathological implications. Brain Res Brain Res Rev 45:196–212

90. Evans JE, Frostholm A, Rotter A (1996) Embryonic and post-

natal expression of four gamma-aminobutyric acid transporter

mRNAs in the mouse brain and leptomeninges. J Comp Neurol

376:431–446

91. Minelli A, Barbaresi P, Conti F (2003) Postnatal development of

high-affinity plasma membrane GABA transporters GAT-2 and

GAT-3 in the rat cerebral cortex. Brain Res Dev Brain Res

142:7–18

92. Velasco I, Tapia R (2002) High extracellular gamma-aminobu-

tyric acid protects cultured neurons against damage induced

by the accumulation of endogenous extracellular glutamate.

J Neurosci Res 67:406–410

93. Tillakaratne NJ, Medina-Kauwe L, Gibson KM (1995) gamma-

Aminobutyric acid (GABA) metabolism in mammalian neural

and nonneural tissues. Comp Biochem Physiol A Physiol

112:247–263

94. Salazar P, del Carmen Sanchez-Soto M, Hiriart M et al (2001)

Biochemical characteristics of the gamma-aminobutyric acid

system in the insulinoma cell lines HIT-T15, RIN-m5F, betaTC3,

and comparison with rat brain. Arch Med Res 32:419–428

95. Gammelsaeter R, Froyland M, Aragon C et al (2004) Glycine,

GABA and their transporters in pancreatic islets of Langerhans:

evidence for a paracrine transmitter interplay. J Cell Sci

117:3749–3758

96. Gage FH (2000) Mammalian neural stem cells. Science

287:1433–1438

97. McKay R (1997) Stem cells in the central nervous system.

Science 276:66–71

98. Rakic P (2006) A century of progress in corticoneurogenesis:

from silver impregnation to genetic engineering. Cereb Cortex

16(Suppl 1):13–17

99. Metin C, Baudoin JP, Rakic S et al (2006) Cell and molecular

mechanisms involved in the migration of cortical interneurons.

Eur J Neurosci 23:894–900

100. Noctor SC, Flint AC, Weissman TA et al (2001) Neurons

derived from radial glial cells establish radial units in neocortex.

Nature 409:714–720

101. Noctor SC, Flint AC, Weissman TA et al (2002) Dividing pre-

cursor cells of the embryonic cortical ventricular zone have

morphological and molecular characteristics of radial glia.

J Neurosci 22:3161–3173

102. Ang ES Jr, Haydar TF, Gluncic V et al (2003) Four-dimensional

migratory coordinates of GABAergic interneurons in the

developing mouse cortex. J Neurosci 23:5805–5815

103. Lopez-Bendito G, Sturgess K, Erdelyi F et al (2004) Preferential

origin and layer destination of GAD65-GFP cortical interneu-

rons. Cereb Cortex 14:1122–1133

104. Haydar TF, Wang F, Schwartz ML et al (2000) Differential

modulation of proliferation in the neocortical ventricular and

subventricular zones. J Neurosci 20:5764–5774

105. Tamamaki N, Yanagawa Y, Tomioka R et al (2003) Green

fluorescent protein expression and colocalization with calretinin,

parvalbumin, and somatostatin in the GAD67-GFP knock-in

mouse. J Comp Neurol 467:60–79

106. Jursky F, Nelson N (1996) Developmental expression of GABA

transporters GAT1 and GAT4 suggests involvement in brain

maturation. J Neurochem 67:857–867

107. Taylor J, Docherty M, Gordon-Weeks PR (1990) GABAergic

growth cones: release of endogenous gamma-aminobutyric acid

precedes the expression of synaptic vesicle antigens. J Neuro-

chem 54:1689–1699

108. Taylor J, Gordon-Weeks PR (1991) Calcium-independent

gamma-aminobutyric acid release from growth cones: role of

gamma-aminobutyric acid transport. J Neurochem 56:273–

280

109. Fritschy JM, Paysan J, Enna A et al (1994) Switch in the

expression of rat GABAA-receptor subtypes during postnatal

development: an immunohistochemical study. J Neurosci

14:5302–5324

110. Laurie DJ, Wisden W, Seeburg PH (1992) The distribution of

thirteen GABAA receptor subunit mRNAs in the rat brain. III.

Embryonic and postnatal development. J Neurosci 12:4151–

4172

111. LoTurco JJ, Owens DF, Heath MJ et al (1995) GABA and

glutamate depolarize cortical progenitor cells and inhibit DNA

synthesis. Neuron 15:1287–1298

112. Antonopoulos J, Pappas IS, Parnavelas JG (1997) Activation of

the GABAA receptor inhibits the proliferative effects of bFGF

in cortical progenitor cells. Eur J Neurosci 9:291–298

113. Owens DF, Boyce LH, Davis MB et al (1996) Excitatory GABA

responses in embryonic and neonatal cortical slices demon-

strated by gramicidin perforated-patch recordings and calcium

imaging. J Neurosci 16:6414–6423

114. Owens DF, Kriegstein AR (2002) Is there more to GABA than

synaptic inhibition? Nat Rev Neurosci 3:715–727

115. Rivera C, Voipio J, Payne JA et al (1999) The K+/Cl-

co-transporter KCC2 renders GABA hyperpolarizing during

neuronal maturation. Nature 397:251–255

116. Cancedda L, Fiumelli H, Chen K et al (2007) Excitatory GABA

action is essential for morphological maturation of cortical

neurons in vivo. J Neurosci 27:5224–5235

117. Maric D, Liu QY, Maric I et al (2001) GABA expression

dominates neuronal lineage progression in the embryonic rat

neocortex and facilitates neurite outgrowth via GABA(A)

autoreceptor/Cl- channels. J Neurosci 21:2343–2360

118. Cobos I, Calcagnotto ME, Vilaythong AJ et al (2005) Mice

lacking Dlx1 show subtype-specific loss of interneurons,

reduced inhibition and epilepsy. Nat Neurosci 8:1059–1068

119. Behar TN, Li YX, Tran HT et al (1996) GABA stimulates

chemotaxis and chemokinesis of embryonic cortical neurons via

calcium-dependent mechanisms. J Neurosci 16:1808–1818

120. Behar TN, Schaffner AE, Scott CA et al (2000) GABA receptor

antagonists modulate postmitotic cell migration in slice cultures

of embryonic rat cortex. Cereb Cortex 10:899–909

121. Heck N, Kilb W, Reiprich P et al (2007) GABA-A receptors

regulate neocortical neuronal migration in vitro and in vivo.

Cereb Cortex 17:138–148

122. Reynolds BA, Weiss S (1992) Generation of neurons and

astrocytes from isolated cells of the adult mammalian central

nervous system. Science 255:1707–1710

123. Lois C, Alvarez-Buylla A (1994) Long-distance neuronal

migration in the adult mammalian brain. Science 264:1145–

1148

124. Luskin MB (1993) Restricted proliferation and migration of

postnatally generated neurons derived from the forebrain sub-

ventricular zone. Neuron 11:173–189

125. Doetsch F, Caille I, Lim DA et al (1999) Subventricular zone

astrocytes are neural stem cells in the adult mammalian brain.

Cell 97:703–716

126. Tramontin AD, Garcia-Verdugo JM, Lim DA et al (2003)

Postnatal development of radial glia and the ventricular zone

(VZ): a continuum of the neural stem cell compartment. Cereb

Cortex 13:580–587

1556 Neurochem Res (2008) 33:1546–1557

123

127. Wichterle H, Garcia-Verdugo JM, Alvarez-Buylla A (1997)

Direct evidence for homotypic, glia-independent neuronal

migration. Neuron 18:779–791

128. Gleeson JG, Lin PT, Flanagan LA et al (1999) Doublecortin is a

microtubule-associated protein and is expressed widely by

migrating neurons. Neuron 23:257–271

129. Leker RR, Soldner F, Velasco I et al (2007) Long-lasting

regeneration after ischemia in the cerebral cortex. Stroke

38:153–161

130. Wang DD, Krueger DD, Bordey A (2003) GABA depolarizes

neuronal progenitors of the postnatal subventricular zone via

GABAA receptor activation. J Physiol 550:785–800

131. Stewart RR, Hoge GJ, Zigova T et al (2002) Neural progenitor

cells of the neonatal rat anterior subventricular zone express

functional GABA(A) receptors. J Neurobiol 50:305–322

132. Nguyen L, Malgrange B, Breuskin I et al (2003) Autocrine/

paracrine activation of the GABA(A) receptor inhibits the pro-

liferation of neurogenic polysialylated neural cell adhesion

molecule-positive (PSA-NCAM+) precursor cells from postnatal

striatum. J Neurosci 23:3278–3294

133. Bolteus AJ, Bordey A (2004) GABA release and uptake regulate

neuronal precursor migration in the postnatal subventricular

zone. J Neurosci 24:7623–7631

134. Liu X, Wang Q, Haydar TF et al (2005) Nonsynaptic GABA

signaling in postnatal subventricular zone controls proliferation

of GFAP-expressing progenitors. Nat Neurosci 8:1179–1187

135. Seri B, Garcia-Verdugo JM, McEwen BS et al (2001) Astro-

cytes give rise to new neurons in the adult mammalian

hippocampus. J Neurosci 21:7153–7160

136. Christie BR, Cameron HA (2006) Neurogenesis in the adult

hippocampus. Hippocampus 16:199–207

137. Eriksson PS, Perfilieva E, Bjork-Eriksson T et al (1998) Neu-

rogenesis in the adult human hippocampus. Nat Med 4:1313–

1317

138. Schinder AF, Gage FH (2004) A hypothesis about the role of

adult neurogenesis in hippocampal function. Physiology

(Bethesda) 19:253–261

139. Aimone JB, Wiles J, Gage FH (2006) Potential role for adult

neurogenesis in the encoding of time in new memories. Nat

Neurosci 9:723–727

140. Cameron HA, McKay RD (2001) Adult neurogenesis produces a

large pool of new granule cells in the dentate gyrus. J Comp

Neurol 435:406–417

141. Nakatomi H, Kuriu T, Okabe S et al (2002) Regeneration of

hippocampal pyramidal neurons after ischemic brain injury by

recruitment of endogenous neural progenitors. Cell 110:429–441

142. Cameron HA, McKay RD (1999) Restoring production of hip-

pocampal neurons in old age. Nat Neurosci 2:894–897

143. Kempermann G, Kuhn HG, Gage FH (1997) More hippocampal

neurons in adult mice living in an enriched environment. Nature

386:493–495

144. Kuwabara T, Hsieh J, Nakashima K et al (2004) A small

modulatory dsRNA specifies the fate of adult neural stem cells.

Cell 116:779–793

145. van Praag H, Kempermann G, Gage FH (1999) Running

increases cell proliferation and neurogenesis in the adult mouse

dentate gyrus. Nat Neurosci 2:266–270

146. Tozuka Y, Fukuda S, Namba T et al (2005) GABAergic exci-

tation promotes neuronal differentiation in adult hippocampal

progenitor cells. Neuron 47:803–815

147. Wang LP, Kempermann G, Kettenmann H (2005) A subpopu-

lation of precursor cells in the mouse dentate gyrus receives

synaptic GABAergic input. Mol Cell Neurosci 29:181–189

148. Karten YJ, Jones MA, Jeurling SI et al (2006) GABAergic

signaling in young granule cells in the adult rat and mouse

dentate gyrus. Hippocampus 16:312–320

149. Overstreet Wadiche L, Bromberg DA, Bensen AL et al (2005)

GABAergic signaling to newborn neurons in dentate gyrus.

J Neurophysiol 94:4528–4532

150. Laplagne DA, Kamienkowski JE, Esposito MS et al (2007)

Similar GABAergic inputs in dentate granule cells born during

embryonic and adult neurogenesis. Eur J Neurosci 25:2973–

2981

151. Ge S, Goh EL, Sailor KA et al (2006) GABA regulates synaptic

integration of newly generated neurons in the adult brain. Nature

439:589–593

Neurochem Res (2008) 33:1546–1557 1557

123