УДК 577.25


© 2012 P. Bregestovski, Yu. Zilberter

INSERM-U751, Université de la Méditerranée, 13285Marseille, France; e-mail: piotr. bregestovski@univmed.fr Received 27.08.2011

A man with his head is something much more than a man's body plus his separate head.

J. Miller

During the last two decades, it has become widely accepted that GABA, the main inhibitory neurotransmitter in mammalian nervous system, exhibits excitatory action at the early stages of postnatal development. This results from a high intracellular Cl- concentration at these stages of the development and is associated with spontaneous synchronized network discharges known as Giant Depolarizing Potentials (GDPs). It has been hypothesized that the excitatory action of GABAergic system stimulates synaptogenesis and the development of neuronal networks. However, accumulating recent observations challenge this view. Here we present a brief review of the current concepts and problems they face in the light of new data.

Keywords: ion homeostasis, energy substrates, network activity, GABAergic transmission, developing brain.

This review as well as the next one (Yu. Zilberter, P. Bregestovski, Fueling Brain Neuronal Activity, this issue) are dedicated to B.I. Khodorov on the occasion of his 90th Jubilee. One of us (Yu. Z.) was fortunate to work on his PhD project under supervision of Boris Izrailevich, as were several generations of researchers. Dr. Khodorov's prolific works in the fields of neurobiology and biophysics can hardly be overestimated. Every meeting with him is an inspiring lesson and a brainstorm. One of the directions of Khodorov's research is an analysis of energy homeostasis and its relation to neuronal functions [1—3]. This theme is closely linked to the subjects of our reviews. We wish Boris Izrailevich good health, fruitful work, new original ideas and many happy days to come.



In the nervous system of vertebrates, activity of neuronal networks is determined primarily by excitatory glutamatergic and inhibitory GABAergic synapses. In mammals, glutamate activates several subtypes of cation-selective channels, and this leads to the membrane depolarization. GABA activates the anion-selective channels triggering the transmembrane fluxes of chloride (Cl-), one of the main types of anions present in biological organisms. Normally, concentration of Cl- in mammalian neurons is low (<10 mM, see rev. [4]) with the reversal potential (ECl) more neg-

ative than the cell resting potential. Therefore, the activation of GABA receptor channels results in hyper-polarization, i.e., the generation of inhibitory postsynaptic potentials (IPSPs), and consequently, inhibition of neuronal activity. This viewpoint is widely accepted in relation to adult brain.

However, at the early stages of postnatal development, the situation is different. Initial observations with the use of intracellular recordings on kitten hippocampus in vivo suggested that inhibition is a predominant form of synaptic activity at the early postnatal ages [5]. On the other hand, in vitro experiments on hippocampal slice preparations from young kittens suggested that the excitatory synaptic events are more common, while the inhibitory synaptic activity becomes predominant on the later developing stages [6]. These observations were confirmed on the hippocam-pal slices from rabbits [7] and rats [8, 9]. For instance, Muller et al. wrote in 1984: "Microapplication of GABA (via pressure ejection) in stratum pyramidale in slices from mature rabbits (age 1 month) evoked a hy-perpolarization of CA1 pyramidal neurons. The reversal potential (Erev) for this response was approximately -70 mV. In contrast, local application of GABA into stratum pyramidale in slices from immature rabbits (age 7-10 days) produced a depolarizing response with an Erev of approximately -54 mV" [10]. As the subtypes and ionic permeability of ionitropic GABA receptors were not yet established at that time, the authors suggested that during the development, two types



Fig. 1. Chloride homeostasis and signaling by the inhibitory neurotransmitter GABA. Conventional view. A, left panel: Levels of intracellular Cl- are high, and the equilibrium potential for Cl-, Ea, is positive relative to the membrane potential, Vm. Opening of Cl- channels by activation of the GABAa receptor depolarizes the cell. A, right panel: Expression of the KCC2 transporter maintains a low level of Cl-. Eq is negative relative to Vm and activation of the GABAa receptor inhibits the cell. Immature hip-pocampal and spinal neurons start life as cells depolarized by GABA (left panel), and in mature cells GABA has a hyperpolarizing action (rightpanel) (from [34]). B, The age dependence of the effect of isoguvacine on neuronal firing. Note that the switch in GABAa signaling from excitation to inhibition occurs at P13.5 (from [13]).


lnhibition ^


15 20 Age, days






of GABA receptor channels coexist: a hyperpolarizing type permeable to Cl- and a depolarizing type with weakly selective channels permeable to sodium and/or calcium as well as to Cl-. Only several years later, the compelling evidence on Cl--selective nature of iono-tropic GABA receptor channels was provided [11].

Based on these and other findings, it was hypothesized that immaturity of the GABAergic system could underlie the late development of hyperpolarizing inhibitory synaptic activity in hippocampus [10]. Several years later, a hypothesis on the leading role of excitatory GABA in the development was proposed by Ben-Ari and co-authors [12-15] and claimed as a general feature of the nervous system: "In all developing animal species and brain structures investigated, neurons have a higher intracellular chloride concentration at an early stage leading to an efflux of chloride and excitatory actions of GABA in immature neurons" [13]. Excitatory action of GABA in the neonatal brain slices was confirmed in a number of studies (for review, see [13]).

Observations on the rodent brain slices indicated that the switch from the excitatory to inhibitory action of GABA takes place near the second week of postnatal life (Fig. 1). At this stage of development, the neuronal network in neonatal rat hippocampus in vitro exhibits intrinsic population neuronal activities. This phenomenon was first described by Harris and Teyler in 1983 and called "spontaneous unison firing" [9]. It was also observed by Mueller et al. in 1984, who wrote: "Immature neurons often demonstrated spontaneous depolarizations of up to 30 mV amplitude and 30 to 60 sec duration" [10]. Several years later, Ben-Ari and co-authors also described this phenomenon, which they called Giant Depolarizing Potentials (GDPs) [12]. This synchronous neuronal activity is detected on brain slices from animals of P0 to P10 and is infrequent or absent after P12. It was proposed that depolarizing GABA plays a key role in the generation of GDPs, and this spontaneous activity results from syn-ergistic excitatory activities mediated by GABAA and glutamate N-methy-^-aspartate (NMDA) receptors

[14]. As GDPs terminate around the postnatal days 10-11 and coincide with the time of "excitation/inhibition switch" of GABA action, it has been postulated that GDPs represent a primitive pattern activity of the neonatal hippocampus and provide a selective control of hippocampal synaptic formation [16].

Based on these observations, it became widely accepted that the depolarizing GABAergic signalling underlies normal neuronal differentiation. Several points on the crucial role of GABA in the development of neuronal network were stated [13, 15]:

1. In neonatal nervous system of rodents, GABA is excitatory because of high intracellular Cl- concentration.

2. During the early postnatal development, activation of GABAa receptors produces the outflow of Cl-, resulting in membrane depolarization, facilitated bursting, and enhanced network activity.

3. GABA-induced depolarization releases NMDA receptor channels from the block by Mg ions. These channels are highly permeable to calcium (Ca2+), so their activation elevates intracellular Ca2+ concentration, which stimulates a number of intracellular processes underlying synaptogenesis and the development of nervous system. Generation of GDPs plays a key role in this process of neuronal networks development.

4. In rodents, the developmental excitatory to inhibitory switch in the action of GABA and generation of GDPs occurs at the second postnatal week.

Therefore, in this concept, the elevated Cl- concentration, depolarizing GABA and generation of GDPs at the early stages of postnatal development represent a background for nervous system development. This viewpoint is sublimated in the recent review ofvan Welie et al. [17], who wrote: "Depolarizing GABA is required for normal brain development, as it contributes to the morphological maturation of neurons [18], and neuronal circuits [16, 19]. Depolarizing GABA can drive juvenile neurons to fire action potentials [15] and conversely, neuronal activity can regulate Egaba, by either specific patterns of synaptic activation [20, 21], or alterations in postsynaptic activity levels [4] via changes in intracellular Ca2+ [22]."


It should be mentioned that a majority of studies confirming the depolarizing action of GABA have been obtained in the neonatal brain slices or cell cultures. However, several lines of evidence are not in the line with this view.

First, immunocytochemistry and electron microscopy studies seriously question the conclusion that GABAergic system plays a key role in synaptogenesis. Marty et al. [23] demonstrated that in hippocampal

pyramidal cells, there is a developmental shift in the localization of the Cl- transporter (NKCC1)

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