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i iiii i v i e w p o i n t
GABA:an exdtatory transmitterin earlypostnatal life Enrico Cherubini, Jean L. Gaiarsa and Yehezkel Ben-Aft In the adultmammalianCNS, GABAis the maininhibitory transmitter. It inhibits neuronal firing by increasinga Cl- conductance. Bicuculline blocksthis effectandinducesinterictal discharges.A different picture is present in neonatal hippocampal neurones, wheresynaptically released or exogenously applied GABA depolarizes and excites neuronal membranes - an effect that is due to a different CIgradient. In fact, dunngthe earlyneonatalperiod, GABA acting on GABAA receptorsprovides most of the excitatory dr/ve, whereas excitatory glutamatergic synapsesare quiescent. It is suggestedthat during development GABAexerts mainly atrophic action through membranedepolarization and a risein intracellularCa2+.
postnatal life are reviewed in this article. These studies show that during the first postnatal week GABA depolarizes hippocampal neurones and provides most of the excitatory drive to pyramidal cells. During this period, GABA depolarization subserves a trophic role. Because of the important interactions between GABA, glycine and excitatory amino acids, developmental changes in excitatory amino acids and glycine-mediated events are also reviewed, and their possible functional relevance is discussed.
EnricoCherubiniisat the Biophysics
Laboratory, InternationalSchool forAdvancedStudies, StradaCostiera11, 34014 Trieste,Italy, andJeanLuc 6aiarsa and YehezkdBen-Ari areat INSERMU. 29, 123Bdde PortRoyal, 75014Paris,France.
GABAA mediates excitation in neonatal hippocampal neurones Perhaps the most striking demonstration of GABA is the major inhibitory transmitter in the adult mammalian CNS. It activates GABAA receptors changes taking place in the role of GABAA receptors and inhibits neuronal firing by increasing a CI- con- in early postnatal life comes from the experiments in ductance, Blockade of GABAA receptors by bicu- which the effects of bicuculline were tested on CA3 culline generates epileptic activity. The receptor- hippocampal neurones recorded in slices5'6. Beyond channel complex, which has been sequenced, can postnatal day 8 (PS), as in adults, bicuculline be allosterically modulated by drugs such as benzo- generates interictal activity due to the block of diazepines and barbiturates, known to be thera- the GABAergic inhibitory drive. By contrast, bepeutically active in several CNS disorders (e.g. tween P0 and P7, bicuculline induces a membrane epilepsy and anxiety) ~. In addition to its classical hyperpolarization and fully blocks spontaneous and effect on CI- channels, GABA inhibits neuronal evoked excitatory synaptic activity (Fig. 1), sugactivity by activating GABAB receptors coupled to gesting that at this early stage of development, K + channels 1. In the adult mammalian CNS, the excitatory A B drive is provided mainly by glutamate acting on --40 mV "L//""~"'~ N-methyI-D-aspartate (NMDA) and non-NMDA receptors. Stimulation of afferent pathways elicits a mixed EPSP in cortical and non-cortical structures, composed primarily of a fast non-NMDA compon--64 mV --J ent. A slow NMDA component can be observed "6 in certain conditions that remove the voltagedependent Mg 2+ block from the NMDA channel. I I l I • ! I I' The EPSP is followed by a fast and slow IPSP Q. mediated by GABAA and GABAB receptors, respect\ ] -20 E --85 mV ~J ' ~ 20 mV Membrane potential < ively. However, the rigorous distinction between excitatory and inhibitory transmitters has been 400 ms recently hampered by the observations that excitatory amino acids such as glutamate may modulate the response to GABA (Ref. 2), and that inhibitory amino acids such as glycine serve as allosteric regulators of the excitatory neurotrans- (3 Bic (10 p~4) mission mediated by NMDA receptors 3. +30 min Glycine is also an inhibitory transmitter, which like GABA - inhibits neuronal firing by increasing a CI- conductance. It can be viewed as the phylogenetically older inhibitory transmitter, which is --65 mV ~ . present mainly in the lower parts of the neuraxis .~'-"~'IT-; . ' " '~20 mV such as the brain stem and the spinal cord 4. I rain However, glycine clearly does not play a major role as an inhibitory transmitter in the cortex, limbic Fig. 1. Giant depolarizing potentials (GDPs) are~*nediated by GABA acting on system and most of the diencephalic structures of GABAA receptors. (A) GDPs recorded at different potentials using a microelectrode containing potassium methylsulphate inserted into a CA3 hippoadult neurones. Several lines of evidence indicate that major campal neurone in a slice obtained from a four-day-old rat. (B) The ampfitude the potentials shown in (A) is plotted against the membrane potential The changes in receptor distribution and function occur of responses reversed at - 5 2 mV. (C) In another neurone, bath application early in development. Recent data on GABA- of bicuculline (bar) reversibly blocked GDPs and induced a membrane hypermediated synaptic responses that occur in early polarization. (Taken, with permission, from Ref. 6.) TINS, Vol. 14, No. 12, 1991
© 1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236/91/$02.00
51 5
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most of the excitatory drive is mediated by GABAA receptors and that glutamatergic synapses are rather quiescent 5'6. In fact, until P5-P7, the spontaneous activity of CA3 neurones is characterized by the presence of network-driven giant depolarizing potentials (GDPs), which are mediated by GABA acting on GABAA receptors 5'6 (Fig. 1); GDPs have the same reversal potential as the responses to exogenously applied GABA (Refs 5,6). Electrical stimulation of neonatal slices also evokes GDPs that are mediated by GABAA receptors 5. Recently, it has been proposed that GDPs are generated by the release of GABA, which is controlled by endogenous Zn 2+ via an inhibitory action on pre- and postsynaptic GABAB receptors7. Although this hypothesis (which is based on the observation that specific Zn2+-chelating agents block GDPs) is attractive, it cannot be reconciled with the delayed development of the mossy fibres 8 that constitute the principal source of endogenous Zn 2+ in the hippocampus of neonatal animals. Previous electrophysiological data of the synaptic responses in the CA1 area of the hippocampus in vivo 9 or in vitro ~°'1~ have emphasized the relatively slow maturation of the GABAergic inhibitory system in early postnatal life. Thus, the paired-pulse inhibition that probably reflects recurrent inhibition by basket cells on CA1 pyramidal neurones has never been observed before P6 (Ref. 10). Intracellular recordings have confirmed that the predominant form of synaptic activity in immature animals is excitation. It has been shown that immature rabbit hippocampal neurones 12 ' 13 and rat neocortical neurones TM exhibit prolonged depolarizing postsynaptic potentials in response to stimulation of the stratum radiatum or the white matter. IPSPs cannot be detected in neurones of animals less than one week old TM, Several differences between adult and neonatal GABA responses have been described. (1) In neonatal neurones, GABA depolarizes neuronal membranes s'6'13'15'16 (Fig. 2). This has been confirmed using flow cytometric techniques combined with voltage-sensitive dyes in embryonic and early postnatal hippocampal cells in suspension 17. As in the slice preparation, bicuculline prevents muscimol-elicited membrane depolarization in acutely dissociated neurones, and induces a hyperpolarization that reflects the block of depolarizing ambient GABA (Ref. 17). GABA depolarization in neonatal neurones, which is Cl- dependent, may be due to a modified CI- gradient that results from the reversed operation of the Cl- membrane pump TM. An alternative view is that GABA-activated channels are also permeable to bicarbonate ions (HCO3-). In this case, the reversal potential for GABA responses will deviate significantly from the CI- equilibrium potential towards the bicarbonate equilibrium potential 19. Since intracellular HCO3- is determined by the pH regulatory mechanisms of the cell2°, developmental changes in the cell metabolism might shift the HCO3- equilibrium potential and therefore the GABA equilibrium potential. GABAA receptormediated depolarizing responses can be obtained from adult hippocampal neurones as well as from 516
neonatal neurones. Application of GABA onto the dendrites of adult pyramidal cells elicits a membrane depolarization due to a reversed CI- gradient 2~. A GABAA-mediated depolarizing synaptic response can be elicited in CA1 neurones by high-intensity afferent stimulation or application of 4-aminopyridine 22. It would be interesting to see whether the adult (dendritic) and neonatal GABAA receptors have a similar structure and pharmacological profile. During the second postnatal week, in concomitance with the shift of the GABA response from the depolarizing to the hyperpolarizing direction, spontaneous hyperpolarizing GABA-mediated potentials appear. Bicuculline blocks these events and induces the appearance (as in the adult) of interictal discharges5. (2) In neonatal neurones, GABA currents show little desensitization6; this will considerably enhance the effects of GABA. (3) GABA currents in neonatal neurones are almost linearly related to the membrane potential in the range - 8 0 to - 4 0 m V . They show little outward rectification at more positive potentials 23. (4) GABA currents are potentiated by barbiturates but are insensitive to benzodiazepines in neonatal neurones 24.
Developmental changes in the structure of GABAA receptors During the past few years, a considerable amount of work has been devoted to the characterization of the molecular structure of the GABAA receptor 25'26. This receptor is a hetero-oligomeric protein composed of several distinct polypeptide types (o~, 13, "h 6). When expressed in heterologous cells these polypeptides produce GABA-activated CI- channels. The functional characteristic of the receptor is determined by the structure and assembly of different subunits. The minimal requirement for the functional vertebrate GABAA receptor channel is obtained by the combination of 0~, 13and Y2 subunits (Ref. 27); the Y2 subunit being responsible for the potentiating effect of benzodiazepines 28'29, while the 132subunit is responsible for the desensitization and outward rectification of the GABA current 3°. It has recently been shown that the oq subunit of the GABA receptor, which is also a part of the benzodiazepine type-1 receptor 31, appears later in development 32. It is possible, therefore, that in analogy with the nicotinic ACh receptor 33'34, changes in structure or assembly of different subunits may determine developmental modifications in function. Functional GABAB receptors are already present at birth From birth, activation of GABAB receptors by baclofen elicits a membrane hyperpolarization in the hippocampal subfield of CA3 pyramidal cells15'35. This effect is due to an increase in K+ conductance15. However, in neonatal neurones, synaptically released GABA does not induce a membrane hyperpolarization. Hyperpolarizing responses can be seen after blockade of the depolarizing GABAA-mediated response by bicuculline 5,6. These responses reverse at the equilibrium potential for K+, suggesting that TINS, Vo/. 14, No. 12, 1991
vlewpo
III GABA8 receptors coupled to K÷ channels may be involved in their generation. We suggest that, in physiological conditions, synaptic GABAB-mediated hyperpolarizing responses are 'masked' by GDPs.
nl:;
C
A
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GABA (0.5 mM, P4)
- 12
[] [] --37, mV
--61, mV
Z~ J l0 mV
-70
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-50 -30i i i i
-8
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i iiii i v i e w p o i n t
GABA:an exdtatory transmitterin earlypostnatal life Enrico Cherubini, Jean L. Gaiarsa and Yehezkel Ben-Aft In the adultmammalianCNS, GABAis the maininhibitory transmitter. It inhibits neuronal firing by increasinga Cl- conductance. Bicuculline blocksthis effectandinducesinterictal discharges.A different picture is present in neonatal hippocampal neurones, wheresynaptically released or exogenously applied GABA depolarizes and excites neuronal membranes - an effect that is due to a different CIgradient. In fact, dunngthe earlyneonatalperiod, GABA acting on GABAA receptorsprovides most of the excitatory dr/ve, whereas excitatory glutamatergic synapsesare quiescent. It is suggestedthat during development GABAexerts mainly atrophic action through membranedepolarization and a risein intracellularCa2+.
postnatal life are reviewed in this article. These studies show that during the first postnatal week GABA depolarizes hippocampal neurones and provides most of the excitatory drive to pyramidal cells. During this period, GABA depolarization subserves a trophic role. Because of the important interactions between GABA, glycine and excitatory amino acids, developmental changes in excitatory amino acids and glycine-mediated events are also reviewed, and their possible functional relevance is discussed.
EnricoCherubiniisat the Biophysics
Laboratory, InternationalSchool forAdvancedStudies, StradaCostiera11, 34014 Trieste,Italy, andJeanLuc 6aiarsa and YehezkdBen-Ari areat INSERMU. 29, 123Bdde PortRoyal, 75014Paris,France.
GABAA mediates excitation in neonatal hippocampal neurones Perhaps the most striking demonstration of GABA is the major inhibitory transmitter in the adult mammalian CNS. It activates GABAA receptors changes taking place in the role of GABAA receptors and inhibits neuronal firing by increasing a CI- con- in early postnatal life comes from the experiments in ductance, Blockade of GABAA receptors by bicu- which the effects of bicuculline were tested on CA3 culline generates epileptic activity. The receptor- hippocampal neurones recorded in slices5'6. Beyond channel complex, which has been sequenced, can postnatal day 8 (PS), as in adults, bicuculline be allosterically modulated by drugs such as benzo- generates interictal activity due to the block of diazepines and barbiturates, known to be thera- the GABAergic inhibitory drive. By contrast, bepeutically active in several CNS disorders (e.g. tween P0 and P7, bicuculline induces a membrane epilepsy and anxiety) ~. In addition to its classical hyperpolarization and fully blocks spontaneous and effect on CI- channels, GABA inhibits neuronal evoked excitatory synaptic activity (Fig. 1), sugactivity by activating GABAB receptors coupled to gesting that at this early stage of development, K + channels 1. In the adult mammalian CNS, the excitatory A B drive is provided mainly by glutamate acting on --40 mV "L//""~"'~ N-methyI-D-aspartate (NMDA) and non-NMDA receptors. Stimulation of afferent pathways elicits a mixed EPSP in cortical and non-cortical structures, composed primarily of a fast non-NMDA compon--64 mV --J ent. A slow NMDA component can be observed "6 in certain conditions that remove the voltagedependent Mg 2+ block from the NMDA channel. I I l I • ! I I' The EPSP is followed by a fast and slow IPSP Q. mediated by GABAA and GABAB receptors, respect\ ] -20 E --85 mV ~J ' ~ 20 mV Membrane potential < ively. However, the rigorous distinction between excitatory and inhibitory transmitters has been 400 ms recently hampered by the observations that excitatory amino acids such as glutamate may modulate the response to GABA (Ref. 2), and that inhibitory amino acids such as glycine serve as allosteric regulators of the excitatory neurotrans- (3 Bic (10 p~4) mission mediated by NMDA receptors 3. +30 min Glycine is also an inhibitory transmitter, which like GABA - inhibits neuronal firing by increasing a CI- conductance. It can be viewed as the phylogenetically older inhibitory transmitter, which is --65 mV ~ . present mainly in the lower parts of the neuraxis .~'-"~'IT-; . ' " '~20 mV such as the brain stem and the spinal cord 4. I rain However, glycine clearly does not play a major role as an inhibitory transmitter in the cortex, limbic Fig. 1. Giant depolarizing potentials (GDPs) are~*nediated by GABA acting on system and most of the diencephalic structures of GABAA receptors. (A) GDPs recorded at different potentials using a microelectrode containing potassium methylsulphate inserted into a CA3 hippoadult neurones. Several lines of evidence indicate that major campal neurone in a slice obtained from a four-day-old rat. (B) The ampfitude the potentials shown in (A) is plotted against the membrane potential The changes in receptor distribution and function occur of responses reversed at - 5 2 mV. (C) In another neurone, bath application early in development. Recent data on GABA- of bicuculline (bar) reversibly blocked GDPs and induced a membrane hypermediated synaptic responses that occur in early polarization. (Taken, with permission, from Ref. 6.) TINS, Vol. 14, No. 12, 1991
© 1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236/91/$02.00
51 5
w e w p o
.........
most of the excitatory drive is mediated by GABAA receptors and that glutamatergic synapses are rather quiescent 5'6. In fact, until P5-P7, the spontaneous activity of CA3 neurones is characterized by the presence of network-driven giant depolarizing potentials (GDPs), which are mediated by GABA acting on GABAA receptors 5'6 (Fig. 1); GDPs have the same reversal potential as the responses to exogenously applied GABA (Refs 5,6). Electrical stimulation of neonatal slices also evokes GDPs that are mediated by GABAA receptors 5. Recently, it has been proposed that GDPs are generated by the release of GABA, which is controlled by endogenous Zn 2+ via an inhibitory action on pre- and postsynaptic GABAB receptors7. Although this hypothesis (which is based on the observation that specific Zn2+-chelating agents block GDPs) is attractive, it cannot be reconciled with the delayed development of the mossy fibres 8 that constitute the principal source of endogenous Zn 2+ in the hippocampus of neonatal animals. Previous electrophysiological data of the synaptic responses in the CA1 area of the hippocampus in vivo 9 or in vitro ~°'1~ have emphasized the relatively slow maturation of the GABAergic inhibitory system in early postnatal life. Thus, the paired-pulse inhibition that probably reflects recurrent inhibition by basket cells on CA1 pyramidal neurones has never been observed before P6 (Ref. 10). Intracellular recordings have confirmed that the predominant form of synaptic activity in immature animals is excitation. It has been shown that immature rabbit hippocampal neurones 12 ' 13 and rat neocortical neurones TM exhibit prolonged depolarizing postsynaptic potentials in response to stimulation of the stratum radiatum or the white matter. IPSPs cannot be detected in neurones of animals less than one week old TM, Several differences between adult and neonatal GABA responses have been described. (1) In neonatal neurones, GABA depolarizes neuronal membranes s'6'13'15'16 (Fig. 2). This has been confirmed using flow cytometric techniques combined with voltage-sensitive dyes in embryonic and early postnatal hippocampal cells in suspension 17. As in the slice preparation, bicuculline prevents muscimol-elicited membrane depolarization in acutely dissociated neurones, and induces a hyperpolarization that reflects the block of depolarizing ambient GABA (Ref. 17). GABA depolarization in neonatal neurones, which is Cl- dependent, may be due to a modified CI- gradient that results from the reversed operation of the Cl- membrane pump TM. An alternative view is that GABA-activated channels are also permeable to bicarbonate ions (HCO3-). In this case, the reversal potential for GABA responses will deviate significantly from the CI- equilibrium potential towards the bicarbonate equilibrium potential 19. Since intracellular HCO3- is determined by the pH regulatory mechanisms of the cell2°, developmental changes in the cell metabolism might shift the HCO3- equilibrium potential and therefore the GABA equilibrium potential. GABAA receptormediated depolarizing responses can be obtained from adult hippocampal neurones as well as from 516
neonatal neurones. Application of GABA onto the dendrites of adult pyramidal cells elicits a membrane depolarization due to a reversed CI- gradient 2~. A GABAA-mediated depolarizing synaptic response can be elicited in CA1 neurones by high-intensity afferent stimulation or application of 4-aminopyridine 22. It would be interesting to see whether the adult (dendritic) and neonatal GABAA receptors have a similar structure and pharmacological profile. During the second postnatal week, in concomitance with the shift of the GABA response from the depolarizing to the hyperpolarizing direction, spontaneous hyperpolarizing GABA-mediated potentials appear. Bicuculline blocks these events and induces the appearance (as in the adult) of interictal discharges5. (2) In neonatal neurones, GABA currents show little desensitization6; this will considerably enhance the effects of GABA. (3) GABA currents in neonatal neurones are almost linearly related to the membrane potential in the range - 8 0 to - 4 0 m V . They show little outward rectification at more positive potentials 23. (4) GABA currents are potentiated by barbiturates but are insensitive to benzodiazepines in neonatal neurones 24.
Developmental changes in the structure of GABAA receptors During the past few years, a considerable amount of work has been devoted to the characterization of the molecular structure of the GABAA receptor 25'26. This receptor is a hetero-oligomeric protein composed of several distinct polypeptide types (o~, 13, "h 6). When expressed in heterologous cells these polypeptides produce GABA-activated CI- channels. The functional characteristic of the receptor is determined by the structure and assembly of different subunits. The minimal requirement for the functional vertebrate GABAA receptor channel is obtained by the combination of 0~, 13and Y2 subunits (Ref. 27); the Y2 subunit being responsible for the potentiating effect of benzodiazepines 28'29, while the 132subunit is responsible for the desensitization and outward rectification of the GABA current 3°. It has recently been shown that the oq subunit of the GABA receptor, which is also a part of the benzodiazepine type-1 receptor 31, appears later in development 32. It is possible, therefore, that in analogy with the nicotinic ACh receptor 33'34, changes in structure or assembly of different subunits may determine developmental modifications in function. Functional GABAB receptors are already present at birth From birth, activation of GABAB receptors by baclofen elicits a membrane hyperpolarization in the hippocampal subfield of CA3 pyramidal cells15'35. This effect is due to an increase in K+ conductance15. However, in neonatal neurones, synaptically released GABA does not induce a membrane hyperpolarization. Hyperpolarizing responses can be seen after blockade of the depolarizing GABAA-mediated response by bicuculline 5,6. These responses reverse at the equilibrium potential for K+, suggesting that TINS, Vo/. 14, No. 12, 1991
vlewpo
III GABA8 receptors coupled to K÷ channels may be involved in their generation. We suggest that, in physiological conditions, synaptic GABAB-mediated hyperpolarizing responses are 'masked' by GDPs.
nl:;
C
A
[]
GABA (0.5 mM, P4)
- 12
[] [] --37, mV
--61, mV
Z~ J l0 mV
-70
i i
-50 -30i i i i
-8
