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Institut de Neurobiologie de la Méditerranée, Institut National de la Santé et de la Recherche Médicale U. 29, Marseille, France
ABSTRACT I. INTRODUCTION AND HISTORICAL PERSPECTIVES II. BASIC PROPERTIES OF GABA SIGNALING A. GABAA Receptors B. GABAB Receptors III. DEPOLARIZING/EXCITATORY ACTIONS OF GABA DURING DEVELOPMENT A. Early Studies on Actions of GABA on Immature Neurons B. Multiple Facets of Depolarizing and Excitatory GABA During Development 1. GABA depolarizes immature neurons A) INTRACELLULAR RECORDINGS. B) GRAMICIDIN PERFORATED PATCH RECORDINGS. C) CELL-ATTACHED RECORDINGS OF GABAA CHANNELS. D) CELL-ATTACHED RECORDINGS OF K+ AND NMDA CHANNELS. 2. GABA triggers sodium action potentials 3. GABA increases [Ca2+]i 4. GABA reduces the voltage-dependent magnesium block of NMDA channels 5. GABA interferes with ionotropic glutamatergic transmission C. Developmental Changes in Chloride Homeostasis D. Bicarbonate-Mediated GABAA Excitation E. Voltage-Gated Chloride Channels F. Time Course of the Excitatory to Inhibitory Developmental Switch G. Intrinsic and Extrinsic Factors Modulate the Developmental Switch 1. GABA itself regulates the developmental switch in the action of GABA 2. Brain-derived neurotrophic factor and tyrosine kinase receptors regulate the developmental switch H. A Dramatic Shift of EGABA During Delivery I. Depolarizing Actions of GABA in Other Brain Structures and Animal Species IV. EARLY OPERATION OF GABAA SIGNALING PRIOR TO SYNAPSE FORMATION A. GABA Signaling Is Present at a Very Early Stage B. A Nonvesicular Release of GABA in Developing Cortical Networks C. GABA Influences DNA Synthesis in Precursor Neocortical Cells D. GABA Modulates Neuronal Migration E. Early Trophic Actions of GABA F. Conclusion: GABA Is an Ancillary Communicating Signal With Multiple Actions at Early Developmental Stages V. GABAERGIC SYNAPSES ARE ESTABLISHED BEFORE GLUTAMATE SYNAPSES A. GABA Receptor Antagonists Block Early Ongoing Activity in the Hippocampus B. GABAergic Synapses Are the First Functional Synapses on Principal Cells of the Hippocampal Formation C. GABAergic Interneurons Mature Before Principal Neurons and Follow the Same Sequence D. GABAergic Interneurons Innervate First the Dendrites of Principal Neurons E. Use of Knockout Strategies to Determine the Role of GABA in Development F. GABA Transporters are Functional After Glutamate Transporters G. The GABA-Glutamate Sequence in Primate Hippocampal Neurons in Utero H. Glutamatergic Mossy Fiber Synapses Have an Early Mixed GABA/Glutamate Phenotype in the Developing Hippocampus I. The GABA-Glutamate Sequence in Proliferating Neurons in Adult Networks J. The GABA-Glutamate Sequence in Other Brain Regions 1. The GABA-Glutamate sequence in the neocortex 2. The GABA-glutamate sequence in the spinal cord 3. The GABA-glutamate sequence in the auditory system 4. Other structures K. A Developmental Switch From GABA to Glycine L. Conclusion: A Model That Integrates These Findings VI. PRIMITIVE PATTERNS IN THE DEVELOPING BRAIN ARE LARGELY BASED ON EXCITATORY GABA A. GDPs in the Hippocampus: A Prototype of Early Network Patterns B. A Problem of Terminology C. How Are GDPs Generated? How do They Propagate? 1. The GABA D-H population model 2. The gap junction pacemaker hilar interneurons model 3. A combined intrinsic current and recurrent glutamatergic excitation model D. GDPs in Subhuman Primates E. Other Early Patterns in the Developing Hippocampus 1. Periodic inward currents 2. Gap junction-mediated oscillations F. Oscillations in Other Developing Structures 1. The neocortex A) IN VITRO PATTERNS. B) IN VIVO PATTERNS. 2. The spinal cord 3. Retinal waves G. Conclusions VII. PLASTICITY OF DEVELOPING GABAergic AND GLYCINERGIC SYNAPTIC TRANSMISSION A. Induction of Long-Term Alterations of Synaptic Efficacy in Developing GABAergic and Glycinergic Synapses B. Long-Term Changes in the Efficacy of GABAergic and Glycinergic Synapses are Mediated by Presynaptic Mechanisms C. Long-Term Plasticity: Contribution to the Establishment of GABAergic and Glycinergic Synapses in the Developing Brain D. Conclusion VIII. PATHOGENIC ASPECTS OF DEPOLARIZING GABA A. GABA and the High Incidence of Seizures of the Immature Brain B. Seizures Beget Seizures: GABA and High-Frequency Oscillations C. Excitatory GABA in Migration Disorders IX. GENERAL CONCLUSIONS: A SEQUENCE THAT EQUILIBRATES GABA AND GLUTAMATE DURING DEVELOPMENT GRANTS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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-aminobuytric acid (GABA), which operates primarily via chloride-permeable GABAA receptor channels. 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. This triggers sodium spikes, activates voltage-gated calcium channels, and acts in synergy with NMDA channels by removing the voltage-dependent magnesium block. GABA signaling is also established before glutamatergic transmission, suggesting that GABA is the principal excitatory transmitter during early development. In fact, even before synapse formation, GABA signaling can modulate the cell cycle and migration. The consequence of these rules is that developing networks generate primitive patterns of network activity, notably the giant depolarizing potentials (GDPs), largely through the excitatory actions of GABA and its synergistic interactions with glutamate signaling. These early types of network activity are likely required for neurons to fire together and thus to "wire together" so that functional units within cortical networks are formed. In addition, depolarizing GABA has a strong impact on synaptic plasticity and pathological insults, notably seizures of the immature brain. In conclusion, it is suggested that an evolutionary preserved role for excitatory GABA in immature cells provides an important mechanism in the formation of synapses and activity in neuronal networks. |
I. INTRODUCTION AND HISTORICAL PERSPECTIVES |
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-Aminobutyric acid (GABA) is an inhibitory transmitter, acting on a receptor channel complex permeable mainly to chloride anions that act to reduce neuronal excitability. As such, GABAergic signaling plays a major role in brain physiology, and dysfunction of GABAergic signaling can result in pathological conditions such as epilepsies that are generated when the balance between excitation and inhibition is impaired (16, 178, 179, 181, 284, 525–527). Recent studies suggest a more complex scope of functions for GABAergic signaling than just global inhibition. For example, the heterogeneity in types of GABAergic synapses and interneurons unraveled in the last decade suggests that an array of GABAergic signaling functions may exist (139, 165, 201, 497, 497). GABAergic neurons also control the generation of behaviorally relevant patterns and oscillations that may turn out to be far more important than inhibition per se. In addition, GABA depolarizes neurons because of a "reversed" chloride gradient in a wide range of neuron types and animal species, notably invertebrates (82, 163, 177, 178, 182, 229, 285, 355, 356, 674). Even in adult mammalian cortical neurons, dendritic GABAergic action is depolarizing because of a locally reversed Cl– gradient and not a different ionic mechanism, as was thought for some years (14; also see Refs. 111, 244, 418, 587; for reviews, see Refs. 420, 589). However, the vast majority of central actions of GABA are inhibitory. In this review, we discuss these issues from the standpoint of brain maturation. Studies on brain development have greatly increased our understanding of how the brain operates and how cortical networks integrate neuronal activity. The observation that has renewed interest in studying GABA in development is the discovery of a higher [Cl–]i in immature neurons that leads to excitatory actions of GABA in immature neurons (50). The progressive reduction of [Cl–]i has now been confirmed in every animal species and brain structure investigated, suggesting that the depolarizing to hyperpolarizing (D-H) switch associated with an excitatory to inhibitory (E-I) shift has been preserved during evolution and provides a solution to a major developmental problem. Hence, the central issues are as follows: Why is the chloride gradient reduced during brain maturation? What are the underlying mechanisms and functional significance? What are the implications of these rules in the construction of cortical networks? It is has been suggested (49) that this sequence enables developing neurons and networks to equilibrate glutamatergic and GABAergic drives and avoid transient overexcitation or overinhibition if the former or the latter predominate.
Here, we first review the main features of GABA receptors and GABAergic synapses. Bearing in mind that GABA exerts a multitude of actions on developmental processes well before synapses are functional, we shall then review the early actions of GABA on migration, cell growth, and synapse formation. The earlier formation of GABAergic synapses, the initial excitatory actions of GABA, and the generation of primitive activity patterns are then analyzed. The GABAB metabotropic receptor G protein-activated channels during development will be briefly reviewed. We then review the mechanisms of GABAergic synapse plasticity. Finally, we discuss the role of depolarizing GABA in relation to the high prevalence of seizures during early development and the pathological plasticity of GABA signaling in epileptogenesis. Since studies using the hippocampus have provided many of the initial observations and the concepts derived from them, we shall review these first before discussing other brain structures. We shall review only in brief the organization of GABA receptor subunits as this has been extensively reviewed recently (490, 492).
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II. BASIC PROPERTIES OF GABA SIGNALING |
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GABAA receptors consist of pentameric assembly of distinct subunits that forms a central ion channel permeable to chloride, and to a lesser extent, bicarbonate anions. To date, 19 GABAA receptors subunits have been cloned in the mammalian CNS. This diversity offers a great potential heterogeneity of GABAA receptor subunit composition, which is further increased by alternative splicing. The molecular composition of the GABAA receptors has important functional consequences as it determines the properties, pharmacological modulation, and targeting of the native receptors.
GABAA receptors are ligand-gated ion channels permeable to chloride and bicarbonate with a net effect that depends on the electrochemical gradient of these anions (297). Under physiological conditions, GABAA receptor activation generates a membrane hyperpolarization and a reduction of action potential firing. However, this classical view has been challenged by recent studies showing that GABAA receptor-mediated responses reversal potential (EGABA) is close to, or even at a more depolarized potential than, the resting membrane potential (Em), thus leading to a membrane depolarization (244, 418). Shunting inhibition is an alternative mechanism of inhibition, in which hyperpolarizing and depolarizing GABAA receptor-mediated responses reduce dendritic excitatory glutamatergic responses via a local increase in conductance across the plasma membrane. GABAA receptor-mediated shunting occurs in a narrow window near the peak of GABAA receptor-induced synaptic responses and requires a close temporal overlap between glutamatergic and GABAergic synaptic responses (244, 586). Hyperpolarizing and depolarizing GABAA receptor-mediated synaptic responses can enhance cell excitability; thus hyperpolarizing responses trigger rebound spikes that can pace population activity (202). Dendritic GABAergic depolarizing responses combined with subthreshold membrane depolarization can elicit action potentials in adult cortical pyramidal neurons (244). In some cerebellar interneurons, GABAA receptor-mediated responses reversed at –58 mV, and activation of presynaptic GABAergic afferents leads to postsynaptic firing (111). The polarity of GABAA receptor-mediated responses can also change during physiological cycles or pathological conditions. In the suprachiasmatic nucleus, GABA triggers excitation during the day and inhibition during the night (645). Following repeated activation, GABAA receptor-mediated responses can switch from a hyperpolarizing to depolarizing direction and can enhance cell firing (499). This activity-dependent switch also occurs during epileptiform activity where it may contribute to generate epileptiform activity (206, 325, 339).
The activation of GABAA receptors by the release of GABA leads to both phasic inhibitory postsynaptic currents (IPSCs) and tonic currents as revealed by the outward holding current and decrease in background noise induced by GABAA receptors antagonists (303, 477, 554). Tonic GABAA receptor-mediated currents were observed early in pre- and postnatal life (158) but not in adult pyramidal cells, unless the concentration of GABA was increased (96, 554, 665). The tonic current results from GABA spillover acting on extrasynaptic receptors with different subunit composition and pharmacological profile compared with the synaptic receptors (250, 477, 554, 590). The functional role of the tonic current remains to be determined. The net effect of the tonic current is an increase in input conductance, thus decreasing the input-output relationship of the neurons (107). Moreover, the total charge carried by the tonic current in granule cells and interneurons is larger than the averaged charge carried by the spontaneous phasic current, thus pointing to an important role in regulating the network excitability.
GABA also acts on GABAB receptors that operate through Gi and Go proteins (68, 140, 449) localized on both pre- and postsynaptic membranes. Activation of postsynaptic receptors generally causes activation of inwardly rectifying potassium channels (GIRK or Kir3) that underlie the late phase of inhibitory postsynaptic potentials (170, 407). Activation of presynaptic GABAB receptors decreases neurotransmitter release by inhibiting voltage-activated Ca2+ channels of the N or P/Q types (13, 435, 448, 508, 545), although mechanisms independent of changes in membrane conductance have also been proposed (449). Activation of GABAB receptors also modulates cAMP production (256, 564), leading to a wide range of actions on ion channels and proteins that are targets of the cAMP-dependent kinase (protein kinase A or PKA), and thus modulate neuronal and synaptic functions (228, 538).
To date, genes encoding two different subunits, GABAB1 and GABAB2, have been identified (81, 283, 310, 503, 551). Fully functional GABAB receptors require the coassembly of the two different subunits, since neither the GABAB1 nor the GABAB2 is active when expressed independently (194, 294, 311, 361, 524, 662). However, when coexpressed, recombinant GABAB1,2 receptors mediate all predominant effects of native receptors, i.e., modulation of cAMP production, activation of GIRK channels, and inhibition of P/Q- and N-type Ca2+ channels (175, 194, 416). Moreover, in GABAB1 or GABAB2 knockout mice, all GABAB receptor-mediated functions were absent (226, 511, 547). However, the general assumption that heterodimerization of GABAB1 and GABAB2 subunits is required has been recently challenged by the observation of responses with receptor subunit expressed in isolation (226, 417).
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III. DEPOLARIZING/EXCITATORY ACTIONS OF GABA DURING DEVELOPMENT |
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Contradictory observations were made in early studies concerning the maturation of GABAergic inhibition (174, 254, 512, 549). Thus in vivo studies of kitten hippocampus suggested that inhibition is the predominant form of early synaptic activity (512). In contrast, studies in hippocampal slices suggested that excitatory synaptic events are more common in young animals and that inhibitory synaptic activity appears fairly late in the kitten (549), rabbit (548), and rat (166, 254) hippocampus. Probably the first suggestion of a developmentally regulated shift of GABA actions was made by Obata et al. (481) in spinal cord neurons. Applications of GABA or glycine depolarized 6-day-old chick spinal neurons in culture and hyperpolarized 10-day-old embryos (481). Using intracellular recordings, Schwartzkroin and colleagues found depolarizing responses to somatic GABA application and a depolarizing GABAergic component of synaptic responses in neonatal (P6–10) rabbit hippocampal CA1 pyramidal neurons. The Em was of –53 mV, and the reversal potentials of the somatic responses to GABA and GABAergic postsynaptic potentials were of –36 and –46 mV, respectively (467), although a more negative value of –54 mV of the somatic EGABA was reported in a previous study (466). The authors suggested that depolarizing GABA inhibits via shunting mechanisms. In mature pyramidal cells, the Em was of –59 mV, and the reversal potentials of the somatic responses to GABA and GABAergic IPSPs were of –71 and –67 mV, respectively. The authors suggested that these developmental changes are due to two types of GABA receptors/channels: a hyperpolarizing type permeable to chloride and a depolarizing type permeable to sodium and/or calcium in addition to chloride. Although subsequent studies suggested different actions of GABA in dendrites and somata of adult neurons (9, 10, 14, 612), the developmental changes in GABAergic signaling are clearly due to alterations of [Cl–]i.
In a study performed in 1989, the developmental changes of GABAergic signaling in neonatal hippocampal slices were investigated using intracellular recordings from CA3 pyramidal cells (56). The principal findings of this study can be summarized as follows: 1) GABA acting via GABAA receptors depolarizes and excites the immature neurons, due to an elevated concentration of [Cl–]i in immature cells that is reduced progressively with development; 2) neuronal activity at an early developmental stage is provided by a network primarily driven by synchronized GABAA-mediated giant depolarizing potentials (GDPs); 3) GABAergic activity is expressed first and precedes glutamatergic (AMPA receptor-mediated) synaptic transmission during development; and 4) early glutamatergic synapses are predominantly based on postsynaptic NMDA receptors. The developmental excitatory to inhibitory (E-I) switch in the action of GABA and reversal potential of GDPs and GABAergic responses occurred at postnatal day P5–P7. Although various details of these observations have been recently revised (see below), the principal conclusions of this study have been confirmed in a wide range of preparations suggesting that the progressive reduction of [Cl–]i is a general developmental rule that has been conserved throughout evolution.
B. Multiple Facets of Depolarizing and Excitatory GABA During Development
1. GABA depolarizes immature neurons
A) INTRACELLULAR RECORDINGS.
Early demonstrations of depolarizing actions of GABA on immature neurons were obtained mainly using intracellular recordings (56, 405, 467, 481). However, intracellular recordings introduce several sources of errors including alterations in the intracellular ionic composition which affects EGABA and neuronal depolarization, both errors being particularly important in immature neurons. Indeed, sharp electrodes used for intracellular recordings are filled with electrolyte in the molar range that exceeds severalfold the ionic composition in intact cells. Dialysis of the cell during recordings with such electrodes will alter intracellular ionic composition. For example, using intracellular recordings, EGABA in the neonatal CA3 pyramidal cells at P2–5 was estimated at –25 mV with KCl-filled electrodes and at –51 mV with potassium methylsulfate-filled electrodes (56). In addition to direct dialysis of anions, [Cl–]i may change as a result of the alteration in the activity of the cation-chloride cotransporters. For example, potassium-filled sharp electrodes could elevate [K+]i and increase KCC2 driving force, whereas cesium-filled electrodes could block KCC2. Besides the dialysis problem, intracellular recordings using sharp electrodes introduce leak conductance in the range of 500 M
that could also affect [Cl–]i because of exchange of Cl– between the cell and external solution via leak conductance. Leak conductance also introduces an important error in the estimation of Em causing neuronal depolarization, the artifact being maximal in small neurons with high membrane resistance (37, 626). Nevertheless, the depolarizing effects of GABA have now been confirmed with less invasive recording techniques.
B) GRAMICIDIN PERFORATED PATCH RECORDINGS. To overcome the problem of intracellular dialysis, Marty and colleagues have developed a technique of perforated patch recordings. This is based on ionophores (polyene antibiotics) that are inserted in plasma membrane in cell-attached configuration to obtain electrical access to cell (278, 419, 514). Polypeptide antibiotic gramicidin forms channels in membranes that are selectively permeable to small cations but not anions (469) and therefore are suitable for noninvasive recordings of GABAA and glycine responses (1, 176, 364, 517).
With the use of gramicidin perforated patch, GABA- and glycine-evoked depolarization were found in cultured rat dorsal horn neurons (515, 653), cortical neurons (244, 399, 418, 488, 632), hippocampal pyramidal neurons (31, 215, 367, 625) and interneurons (31, 205), cerebellar interneurons and Purkinje cells (111), hypothalamic neurons (113), and chick cochlear neurons (401) (Table 1). Examples of the depolarizing and excitatory responses evoked by the GABAA agonist isoguvacine and synaptic activation of the GABAA receptors in a P2 CA3 pyramidal cell recorded using gramicidin perforated patch are shown in Figure 1. In CA3 pyramidal cells, EGABA measured using gramicidin perforated patch was of –55 mV during the early neonatal period and progressively shifted to –74 mV by the end of the second postnatal week (625) (see also Ref. 31). However, while gramicidin perforated patch recordings eliminate the problem of intracellular dialysis and thus provide accurate estimate of EGABA, it does not solve the problem of leak conductance and associated neuronal depolarization that particularly affects immature cells with a gigaohms range membrane resistance. For example, gramicidin perforated patch measurements gave an estimate of Em in P0–2 CA3 pyramidal cells at around –40 to –50 mV, whereas noninvasive measurements of Em using cell-attached recordings of NMDA channels gave value of –77 mV (626).
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D) CELL-ATTACHED RECORDINGS OF K+ AND NMDA CHANNELS. Cell-attached recordings of K+ (204, 401, 573, 638, 638, 679) and NMDA channels (377, 624, 626) used to monitor membrane potential have also revealed depolarizing actions of GABA in immature neurons. This technique was first used in adult neurons by Zhang and Jackson (679), who used changes in the amplitude of currents through single K+ channels to monitor changes in the membrane potential in response to GABA in the membranes of peptidergic nerve terminals of the posterior pituitary (679). With K+ concentration in the recording pipette equal to intracellular K+ concentration, K+ channels reversed at pipette potential equivalent to the membrane potential. A similar approach was used to determine the depolarizing actions of GABA on adult granular cells and hilar neurons (573). Miles and colleagues (638) studied the developmental profile of GABA actions using K+ channels to monitor membrane potential in hippocampal interneurons. Application of GABAA agonists depolarized CA1 interneurons but seldom excited neurons from P1-P21 rats. However, Chavas and Marty (111) raised some concern on this technique and suggested that K+ channel reversal method gives a value that may be too hyperpolarized.
NMDA channels recorded in cell-attached configuration were also used to monitor membrane potential and to study the depolarizing effects of GABA (377, 626). Since currents through NMDA channels reverse near 0 mV (475), NMDA currents should reverse their polarity at a holding potential on the pipette Vp = Em. With the use of this approach, it was shown that bath application of the GABAA agonist isoguvacine causes depolarization of P2–5 CA3 pyramidal cells from –82 to –59 mV (in the presence of tetrodotoxin to block network-driven activity) (377).
GABA-mediated depolarization is due to the efflux of chloride ions via GABAA channels, and because the chloride ions are negatively charged, efflux of negative ions results in inward electric current producing depolarization ·V = IGABA x Rm. Efflux of chloride ions results in a reduction of intracellular negative charge (·V =·Q/C, where Q is the charge and C is the cell capacitance). Therefore, a similar current will produce a larger depolarization in small neurons having high Rm and small C. Theoretical maximal level of GABA-mediated depolarization is equal to EGABA. However, in reality, GABA-mediated depolarization affects the activity of voltage-dependent membrane conductance, and the integral response to GABA is a result of complex interactions between the GABAA and voltage-dependent sodium, calcium, and potassium channels. In physiological conditions, GABA responses also interact with other conductance (including glutamate-activated AMPA/kainate and NMDA receptors), and modeling of such interactions in simplified systems reveals that GABA receptors can produce different types of responses depending on the temporal and spatial context of their activation. Prolonged activation of GABAA receptors can also result in a redistribution of chloride ions ("collapse of chloride gradient") and increase the relative contribution of bicarbonate permeability of GABAA channels producing a delayed depolarization (587). This mechanism does not, however, operate in immature cells because of delayed expression of intracellular carbonic anhydrase (520, 533).
2. GABA triggers sodium action potentials
Depolarizing GABA is also often excitatory in immature neurons, i.e., neurons generate action potentials in response to GABA. This occurs when GABAA-mediated depolarization reaches directly or via voltage-gated conductance the threshold for the generation of action potentials. For example, synaptic activation of GABAA receptors by electrical stimulation in the presence of the glutamate receptors antagonists (CNQX and APV) evoked one or two action potentials in P2–5 CA3 pyramidal cells and interneurons recorded in the cell-attached configuration, and the response was blocked by the GABAA antagonist bicuculline (Fig. 2) (325, 326, 377). Similar excitatory responses can be recorded with gramicidin perforated patch recordings (625). Excitatory responses are also evoked by application of GABA or GABAA agonists (325, 625). Multiple unit activity recorded with extracellular electrodes is also enhanced by application of the GABAA agonists (325, 625). Synaptic activation of GABAA receptors augmented and reduced multiple unit activity (MUA), respectively, in neonatal and adult hippocampal neurons; both effects were sensitive to the GABAA antagonist bicuculline (325, 625). The positive allosteric modulator of GABAA receptors diazepam, which prolongs openings of GABAA channels and slows down the decay of the GABAergic responses, also increased GDPs frequency, synaptic activity, and MUA in neonatal rats (316). Blockade of GABAA receptors reduces and increases MUA in neonatal and adult rat CA3 hippocampus neurons, respectively (126, 172), probably via suppression of background GABAergic tone (567). Thus GABA clearly excites immature pyramidal cells and interneurons (see Table 1 for excitatory actions of GABA in other brain structures).
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Activation of GABAA receptors increases [Ca2+]i in virtually all immature types of neurons. This effect is mainly due to the activation of voltage-gated calcium channels as it persists when sodium channels are blocked and is suppressed by calcium channel blockers. Interestingly, blockade of GABAA receptors induces a significant decrease in [Ca2+]i, indicating that tonic release of GABA is sufficient to increase Ca2+ in immature neurons (484).
Connor et al. (132) were among the first to use digital imaging of the Ca2+ to study the depolarizing responses of developing granule cells in culture to GABA. Application of GABA induced a transient increase in membrane conductance and caused [Ca2+]i increase that outlasted the exposure to GABA by several minutes. Glutamate or kainate also elevated [Ca2+]i, but unlike GABA, this [Ca2+]i response reversed rapidly upon removal of the transmitter. In keeping with these results, GABA and glycine increased [Ca2+]i in a number of embryonic and neonatal neurons including neocortex (223, 385, 399, 675), hippocampus (222, 377, 378), spinal cord (359), dorsal horn neurons (515, 653), hypothalamus, olfactory bulb, cortex, medulla, striatum, thalamus, hippocampus, and colliculus (484) (see Table 1).
Synaptically released GABA also increases intracellular calcium in immature neurons. Obrietan and van den Pol (484) have shown that addition of bicuculline to monosynaptically connected hypothalamic neurons decreased [Ca2+]i, indicating that hypothalamic neurons were secreting GABA at an early age of development, and that sufficient GABA was released to elicit an increase in [Ca2+]i. This effect was seen even after blocking all glutamatergic activity with glutamate receptor antagonists (484). Leinekugel et al. (378) have demonstrated that electrical stimulation of afferent fibers induces a transient increase in [Ca2+]i in neonatal pyramidal cells and interneurons (P5). This elevation of [Ca2+]i was reversibly blocked by bicuculline but not by glutamate receptor antagonists. During simultaneous electrophysiological recording in current-clamp mode and [Ca2+]i monitoring from P5 pyramidal cells, electrical stimulation of afferent fibers, in the presence the glutamate receptors antagonists, caused synaptic depolarization accompanied by a few action potentials and a transient increase in [Ca2+]i. In voltage-clamp mode, however, there was no increase in [Ca2+]i following synaptic stimulation, showing that it is depolarization dependent (378).
An additional factor to consider is that voltage dependence of calcium conductance is developmentally regulated. Ganguly et al. (215) have shown that mild depolarization produced by application of 6 mM extracellular potassium evokes robust increase in [Ca2+]i in P7 neurons but no response at P13 in organotypic slices (215). Only 8–10 mM [K+]o produced calcium signals in P13 neurons. Whole cell study of the voltage dependence of calcium currents revealed a developmental shift in the activation profile of calcium currents toward more hyperpolarized potentials (215). Calcium fluorescence measurements can be also affected by the developmental changes in the Ca2+-buffering properties of neurons. Several developmental studies indicate that calcium-binding proteins are progressively expressed during development in various types of neurons including calretinin (543), parvalbumin (130), and calbindin (79). Recently, Chavas and Marty (111) raised concern on using [Ca2+]i to monitor depolarizing actions of GABA showing that, in cerebellar interneurons, GABAA agonists induce a somatodendritic [Ca2+]i rise that persists at least until postnatal day 20 and is not mediated by depolarization-induced Ca2+ entry. A local [Ca2+]i elevation could likewise be elicited by repetitive stimulation of presynaptic GABAergic afferent fibers. Following GABAA receptor activation, bicarbonate-induced Cl– entry led to cell depolarization, Cl– accumulation, and osmotic tension. The authors proposed that this tension induces the [Ca2+]i rise as part of a regulatory volume decrease reaction (110).
4. GABA reduces the voltage-dependent magnesium block of NMDA channels
The depolarization produced by GABA also attenuates the voltage-dependent magnesium block of NMDA channels (Fig. 2). Using cell-attached recordings of single NMDA channels from P2–5 CA3 pyramidal cells, Leinekugel et al. (377) have shown that activation of GABAA receptors strongly reduces the magnesium block of NMDA channels by reducing the affinity of magnesium ions to NMDA channels from 16 to 118 µM. This effect was entirely due to neuronal depolarization from –82 to –59 mV. Confocal microscopy with the permanent dye fluo 3-AM revealed that in the presence of the calcium channels blocker D600, applications of isoguvacine and NMDA increase [Ca2+]i when applied together but not separately. In the presence of an AMPA receptor antagonist (CNQX), electrical stimulation evoked on average 3.6 action potentials in immature pyramidal cells and interneurons (326); adding an NMDA receptor antagonist (APV) further reduced the response to 1.4 action potentials, and the remaining spikes were fully blocked by bicuculline (Fig. 2). Therefore, synaptic activation of GABAA receptors attenuates the magnesium voltage-dependent block of NMDA receptors. This "synergistic" interaction between GABAA and NMDA receptors contributes to the generation of the physiological pattern of GDPs (59, 326, 377) (and see below).
5. GABA interferes with ionotropic glutamatergic transmission
In developing hypothalamic neurons in culture, GABA acting via GABAA receptors exerts depolarizing actions that will exert different effects on AMPA-mediated responses depending on the delay between the activation of GABAA and AMPA receptors (219). GABAergic depolarization reduced and augmented glutamatergic postsynaptic responses at short and longer latencies, respectively. The reduction is due to the shunting effect of GABAA-mediated conductance. In contrast, subthreshold glutamatergic responses summated with GABAA-mediated depolarization generated spikes if they occurred at the end of GABAA depolarization, when the shunting GABAA conductance ceased. These observations suggest that under certain temporal conditions GABAA and AMPA/kainate receptors may work in synergy to excite the immature neurons. Similar synergistic excitatory actions of GABA-mediated depolarization and glutamatergic EPSPs may also occur in mature cortical neurons (244).
C. Developmental Changes in Chloride Homeostasis
Developmental changes in GABA signaling are determined by the progressive negative shift in EGABA that in turn reflects the developmental reduction of [Cl–]i. In addition to electrophysiological and calcium-imaging experiments (see above), elevated [Cl–]i in the immature neurons has been demonstrated directly with Cl– indicator dyes. With the use of membrane-permeable MQAE and two-photon microscopy, activation of GABAA receptors induced a chloride influx and efflux in second and first postnatal week, respectively, in CA1 pyramidal neurons (412). Immature dissociated neurons also possess a somatodendritic chloride gradient (253). Kuner and Augustine (362) constructed an optical indicator for chloride ions by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein (clomeleon) and showed that [Cl–]i decreases during development of hippocampal neurons in culture (362).
Neuronal chloride homeostasis is controlled by the activity of several chloride cotransporters, exchangers, and channels (reviewed in Refs. 157, 436, 499, 506). Developmental changes in two cation-chloride cotransporters, accumulating chloride NKCC1 and chloride extruder KCC2, play a pivotal role in the developmental changes in [Cl–]i (Fig. 3).
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KCC2 is the principal transporter for Cl– extrusion from neurons. KCC2 extrudes K+ and Cl– using the electrochemical gradient for K+. Cl– extrusion is weak in immature neurons and increases with neuronal maturation (330, 405, 678). The KCC2 isoform of KCC cotransporters is expressed in mature neurons, thus underlying the developmental changes in Cl– extrusion (400, 521, 562, 651, 671). K+-Cl– cotransport also contributes to the low [Cl–]i in mature neurons (156, 286, 450, 613–615). Additionally, the KCC1, KCC3, and KCC4 isoforms have been also found in the central nervous system but with a limited expression in neurons (499).
Using ribonuclease protection analysis and in situ hybridization, Clayton et al. (121) determined the developmental expression of the members of the cation-Cl– cotransporter gene family in rat brain (121). Of the inwardly directed cotransporters, NKCC-1, NKCC-2, and NCC-1, only NKCC-1 was detected at significant levels in brain. NKCC-1 was expressed in neurons, appearing first in the cortical plate but not in the ventricular or subventricular zone. Expression levels peaked by the third postnatal week and were maintained in adults. Outwardly directed cotransporters demonstrated a different time course of expression: KCC-1 was expressed prenatally at low levels that increased slightly over the course of development; KCC-2 expression appeared around birth and increased dramatically after the first week of postnatal life.
Using single-cell PCR, Rivera et al. (521) showed that at birth, KCC2 mRNA was barely detectable; a steep increase in the expression was evident at P5, reaching adult level by P15. Interestingly, mature dorsal root ganglion neurons that have depolarizing GABA (160) did not express KCC2. In contrast, KCC2 mRNA was present in abundant amounts at embryonic day E42 in the hippocampus of guinea pig, a species with early maturation. Spatiotemporal expression pattern of KCC2 mRNA expression follows a caudorostral gradient reflecting functional maturation of various brain areas. Electrophysiological recordings using Cl–-free sharp electrodes revealed strong correlation between the hyperpolarizing GABAA-mediated responses and KCC2 mRNA expression in the pyramidal hippocampal neurons of the rat, guinea pig, and dorsal root ganglion neurons. Antisense oligodeoxynucleotides against KCC2 mRNA produced a fivefold reduction in KCC2 protein in P11–13 rat hippocampal slices associated with a strong reduction of DFGABA (from –10.9 to –2.8 mV). Similarly, early overexpression of KCC2 in immature cortical neurons, before the upregulation of KCC2, produced a negative shift in GABA reversal potential and reduced GABA-elicited calcium responses in cultured neurons (118, 374, 588). Taken together, these results indicated that developmental expression of KCC2 is pivotal for development of hyperpolarizing GABAA-mediated inhibition.
Using perforated patch-clamp method and single-cell multiplex RT-PCR to measure cation-Cl– cotransporter mRNAs in postnatal rat neocortical neurons, Yamada et al. (671) reported that the mRNA expression levels of NKCC1 and KCC2 were positively and negatively correlated, respectively, with EGABA (671). [Cl–]i and NKCC1 mRNA were higher in cortical plate (CP) neurons than in the presumably older layer V/VI pyramidal neurons in a given slice. The pharmacological effects of selective NKCC1 blocker bumetanide on EGABA were consistent with the different expression levels of NKCC1 mRNA. The expression of NKCC1 and KCC2 was also analyzed by Western blot and immunofluorescence and double labeling in the rat and human cortex (173). In the rat cortex, NKCC1 expression reached 14-fold higher levels between P3–14 than at P21 and adulthood. In contrast, KCC2 levels were significantly lower during the first two postnatal weeks than at P21 and adulthood. These ontogenic findings were confirmed by immunofluorescence double labeling using the neuronal marker NeuN and either NKCC1 or KCC2 specific antibodies. In human cortex, NKCC1 expression was significantly higher at 31–41 postconceptional weeks than at 1 year and older. During the first year of life, NKCC1 expression rapidly decreased to levels of the adult. Between 31 and 41 postconceptional weeks, when NKCC1 levels were peaking, KCC2 expression was 2–25% of adult levels, rising over the first year of life. Immunofluorescence double labeling with neuronal marker NeuN and antibodies against NKCC1 and KCC2 was consistent with Western blot results. Whole cell recordings from P4–6 rat CA1 pyramidal cells demonstrated that blockade of NKCC1 activity with bumetanide shifted EGABA from –37 ± 2.7 to –40.4 ± 2.7 mV. Modest effect of bumetanide on EGABA in these experiments could be due to the error of EGABA measurements introduced by whole cell recordings. Taken together, these observations suggest that neocortical neurons, like hippocampal neurons, express a D-H change mediated by a developmental shift from NKCC1 to KCC2.
D. Bicarbonate-Mediated GABAA Excitation
Strong activation of GABA receptors produces depolarizing shift in EGABA and excitatory action of GABA in mature neurons (9, 10, 14, 298, 533, 570, 587, 612). This effect is maximal in small cell compartments with high receptor-to-volume ratio such as dendrites (587). The activity-dependent GABAA-mediated depolarization/excitation is contingent on HCO3–, which is permeable via GABAA receptor channels (74, 297). HCO3– equilibrium potential is set at approximately –10 mV, and therefore, bicarbonate currents via GABAA receptors are depolarizing. Efflux of HCO3– is compensated by rapid synthesis of HCO3– from CO2 by carbonic anhydrase (498). Depolarization caused by the HCO3– current leads to accumulation of [Cl–]i, a positive shift in EGABA (299, 300, 644), and an increase of extracellular K+ that depolarizes neurons to more positive levels than EGABA (300, 570). Furthermore, inhibitors of carbonic anhydrase suppress GABAergic excitation (533).
However, HCO3–-dependent excitation is expressed relatively late in development and does not contribute to depolarizing and excitatory actions of GABA in immature neurons. Also, GABA-mediated excitation of immature neurons persists after removal of bicarbonate/CO2 from the external medium and its substitution for HEPES-based buffer (378). Also, carbonic anhydrase VII, a key molecule in the generation of HCO3–-dependent GABAergic excitation, is not expressed at an early stage, and high-frequency stimulation of GABAergic inputs generates excitatory actions of GABA at P10–12 but not before (520, 533). HCO3– efflux however contributes to the depolarizations mediated by GABA and glycine in fetal spinal cord motoneurons (359).
E. Voltage-Gated Chloride Channels
Several members of the voltage-gated chloride channel family are expressed in the CNS including ClC-2, ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7 (121, 122). ClC-2 channels are inwardly rectifying, with significant conductance only at membrane potentials more negative than Cl– equilibrium potential. They play a role in regulation of cell volume regulation and the maintenance of a balance between electrolytes and intracellular ions (241; see also Ref. 534). Acute hyposmotic challenges increase cell volume and activate ion fluxes (K+ and Cl–), and conversely, hyperosmotic challenge causes shrinkage consequently to the activation of K+ and Cl– conductances. These channels are intracellular, membrane bound, or both (119). In mature hippocampal neurons, hyperpolarization activated Cl– conductance mediated by ClC-2 channels is of sufficient magnitude and duration to stabilize the relationship between ECl and resting membrane potential independently of electroneutral Cl– transport (that is KCC2) (571, 584). Transgenic expression of ClC-2 in neurons that accumulate Cl– is sufficient to reduce intracellular Cl– levels to concentrations that approach the passive Cl– equilibrium potential (585). ClC-2 is expressed early in development and may play a role in the developmental decline in [Cl–]i (122). The relatively low expression of ClC-2 at an early stage may contribute to the depolarizing actions of GABA and glycine in the hippocampus (451). More recent studies suggest that CLC-3 may play an important role in maturation. Thus Strobawa et al. (592) have reported an almost complete degeneration of the hippocampus and retina in CLC-3 knockouts and have shown that this effect is mediated by an intracellular action of the protein since the volume changes evoked identical responses in controls and CLC-3 knockouts. More recently however, Nelson and co-workers (655) showed a chloride conductance activated by calmodulin kinase II that in hippocampal cultures increases the duration and amplitude of exogenous and synaptic currents generated by NMDA receptors in immature neurons at a time when [Cl–]i is elevated. This effect is reduced with maturation, when [Cl–]i is lower, and thus acts to augment excitability and NMDA receptor-mediated events in immature neurons. The channels are postsynaptically located close to NMDA receptors and may play an important role in parallel to the synergistic actions of GABA with glutamate to augment the contribution of NMDA receptor-mediated events. This important observation points to an alternative mechanism for increasing excitability when [Cl–]i is elevated.
F. Time Course of the Excitatory to Inhibitory Developmental Switch
The general conclusion derived from studies on the time course of the developmental E-I switch in GABAergic actions is that it parallels the negative shift in EGABA. The timing of the shift also depends on the species, sex, brain structures, and neuronal type. Studies using viral infections to label proliferating neurons in adult networks strongly suggest that the shift of GABAA signaling reflects the "age" of the recorded neuron (227, 487; and see below). Timing of the E-I switch in the GABA actions has been most thoroughly investigated in the rat hippocampus. Originally it was reported in rat CA3 pyramidal cells that E-I switch occurs at around P5 (56). In agreement with these results, intracellularly recorded inhibitory GABAergic postsynaptic potentials were observed in the majority of CA3 pyramidal cell by P5–6 and by P9 in CA1 pyramidal cells (600). Different estimates were made with whole cell recordings from CA1 pyramidal cells (678). However, these techniques are invasive and will affect EGABA, reflecting the importance of combining noninvasive imaging with electrophysiological techniques.
With measurement of [Ca2+]i using fluorescent calcium-sensitive dyes, the activation of GABA receptors induced an influx of calcium ions via voltage-gated calcium channels at P2–5 CA3 pyramidal neurons and interneurons but not at P12–13, suggesting that the switch occurs during the second postnatal week (378). With the use of a similar approach, the switch in CA1 pyramidal cells was estimated to be around P5–6; at P7–10, 
20% of cells were still excited by GABA, and none from the beginning of the third postnatal week onwards (222). In cultures of hippocampal neurons, the midpoint of disappearance of the GABAA-mediated increase in [Ca2+]i occurred at 11 days in vitro (215).
Noninvasive extracellular and cell-attached recordings revealed a developmental switch in the effect of GABAA agonist isoguvacine on MUA in CA3 Sprague-Dawley rat hippocampus at around P13 (325) (Fig. 4). Similar experiments performed in Wistar rat hippocampus revealed that the effect of isoguvacine switched from an increase to a decrease MUA at around P10 (625), that is 3 days earlier than in Sprague-Dawley rats, suggesting a strain difference in the timing of the E-I switch. The effect of synaptic GABAA-mediated responses evoked by electrical stimulation in the presence of the glutamate ionotropic and GABAB receptors antagonists on MUA in the Wistar rat CA3 pyramidal cell layer switched from an increase to a decrease at around P8 (625). With gramicidin perforated patch recordings, the E-I switch was also centered at P8 (625). The GABAA antagonist bicuculline switched from decreasing to increasing MUA in Wistar CA3 pyramidal cell layer at around P12 (172). The time course of the E-I switch of GABA also depends on the sex of the animal: with the use of the gramicidin perforated patch-clamp recordings from the rat substantia nigra pars reticularis, it has been shown that the switch occurs in males around P17 and in females around P10 (363). Since the postnatal development is associated with dramatic changes in physiological activity patterns and seizure susceptibility, these minor differences may have an important physiological and clinical relevance (see sects. VI and IX).
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What are the genetic and environmental mechanisms underlying the developmental switch? So far, our knowledge on this topic is fragmentary. Information is now available on the developmental expression of molecules that control chloride concentrations (see sect. III), but the control of the expression of these molecules remains to be fully determined.
1. GABA itself regulates the developmental switch in the action of GABA
Ganguly et al. (215) determined the regulation of the developmental switch in rat hippocampal cells culture. Using gramicidin perforated patch recordings and intracellular calcium imaging, the authors showed that GABA increased [Ca2+]i in neurons cultured for 4–9 days but not after day 13. The response was blocked by nimodipine, an antagonist of L-type calcium channels, but not by depleting intracellular calcium stores with thapsigargin, suggesting the GABA-activated rise in [Ca2+]i is mediated by L-type calcium channels. Developmental decrease of the GABA-evoked calcium response was paralleled with a negative shift of EGABA from –44 mV at days 6–7 to –61 mV at days 13–14. Chronic blockade of GABAA, but not glutamate, receptors prevented the loss of calcium responses and the negative shift of EGABA while chronic depolarization with 10 mM KCl accelerated the switch. Blockade of GABAA receptors also decreased by 68% the developmental increase in the expression of mRNA encoding for KCC2. Interestingly, blockade of sodium spikes with tetrodotoxin (TTX) did not affect the switch, and GABAA receptor antagonists prevented the switch in presence of TTX. These observations suggest that the developmental switch is triggered by miniature GABAA postsynaptic potentials generated in the absence of action potentials. These findings suggested that GABAA-activated Ca2+ influx regulates the expression of chloride extruder KCC2, raising the issue of the intracellular cascades underlying this phenomenon. According to this work, GABA may play a self-regulatory role in the kinetics of the developmental switch in the GABA actions. Poo and co-workers (196) have recently also reported that prolonged postsynaptic spiking of hippocampal neurons led to a shift in the reversal potential of GABA-induced Cl– currents toward positive levels in a duration- and frequency-dependent manner. This shift requires an elevation of [Ca2+]i and is occluded by inhibition of KCC2. Interestingly, these changes are larger in mature neurons that express the cotransporter than in immature neurons. These observations further reinforce the link between activity and the actions of GABA (196).
Using a turtle model and calcium imaging, Sernagor and colleagues (379) investigated the effects of bicuculline on the GABAergic polarity switch (from 1 wk before hatching until 4 wk after hatching) and the presence of patterns of spontaneously generated retinal waves in the retinal ganglion cell layer. During that period, spontaneous activity normally switches from propagating waves to stationary patches of coactive cells, until correlated activity completely disappears. The authors reported that in the presence of bicuculline, GABAA responses remain excitatory and spontaneous waves were generated and propagated across the retinal ganglionic layer. This action was associated with a reduction of the developmentally regulated expression of the KCC2 transporter, thus in keeping with the observations of Ganguly et al. (215</
