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GABA and glycine in the developing brain

Abstract

GABA and glycine are major inhibitory neurotransmitters in the CNS and act on receptors coupled to chloride channels. During early developmental periods, both GABA and glycine depolarize membrane potentials due to the relatively high intracellular Cl concentration. Therefore, they can act as excitatory neurotransmitters. GABA and glycine are involved in spontaneous neural network activities in the immature CNS such as giant depolarizing potentials (GDPs) in neonatal hippocampal neurons, which are generated by the synchronous activity of GABAergic interneurons and glutamatergic principal neurons. GDPs and GDP-like activities in the developing brains are thought to be important for the activity-dependent functiogenesis through Ca2+ influx and/or other intracellular signaling pathways activated by depolarization or stimulation of metabotropic receptors. However, if GABA and glycine do not shift from excitatory to inhibitory neurotransmitters at the birth and in maturation, it may result in neural disorders including autism spectrum disorders.

The subject of this review is the depolarizing and excitatory effects of GABA and glycine in the developing hippocampus and other CNSs.

GABAA receptors and strychnine-sensitive glycine receptors are ligand-gated chloride ion channels that belong to the Cys-loop pentameric ligand-gated ion channels (LGIC) superfamily, which also includes nicotinic acetylcholine receptors and 5-HT3 receptors [1]. Since the equilibrium potential of chloride ions is usually more negative than the threshold of action potentials, GABA and glycine are regarded as major inhibitory neurotransmitters in the adult CNS. However, in the 1960s, Obata and colleagues [2, 3] reported that GABA and glycine were excitatory in embryonic chick spinal neurons.

The first studies that gathered attention regarding the importance of GABA as an excitatory neurotransmitter in the developing CNS included a paper by Ben-Ari et al. in 1989 [4] and a subsequent short but comprehensive review in 1991 [5]. They [4] studied immature rat hippocampal CA3 neurons and found giant depolarizing potentials (GDPs), which were concluded to be primarily driven by synchronous pulsatile activity of GABAergic neurons. GDPs consist of low frequency pulsatile activity (between 0.005 and 0.2 Hz) when measured using intracellular recording in CA3 neurons (Fig. 1a). GDP begins with a rather steep depolarization lasting several hundred milliseconds and several concomitant action potentials, and is usually followed by hyperpolarization (Fig. 1b). Interestingly, stimulation of the hilus can evoke a large depolarization (evoked GDP) that is nearly identical to spontaneous GDPs (Fig. 1c). Even though GDPs have been shown to be primarily driven by GABA and completely blocked by specific GABAA receptor blocker bicuculline, antagonists for NMDA receptors also block spontaneous and evoked GDPs, suggesting that glutamatergic activity contributes to GDPs through the NMDA receptor. AMPA-type glutamate receptors have also been shown to act in GDP generation [6], and GDPs are thought to be generated by the synergistic action of GABA and glutamate. GDPs disappeared early in the second postnatal week, though spontaneous hyperpolarizing potentials, similar to GDPs in duration and frequency, were sometimes observed at the beginning of the second postnatal week.

Fig. 1
figure1

Schematic representation of a recording of GDPs in an immature rat hippocampal CA3 neuron. a GDPs are seen as large depolarizing events among GABAergic spontaneous synaptic potentials. GDPs are GABA-mediated spontaneous pulsatile depolarizing events that occur at a frequency of 0.005–0.2 Hz. b A typical GDP rises abruptly concomitant with several action potentials, decays within 200–300 ms, and is followed by transient hyperpolarization. c Electrical stimulation at the hilus evokes a depolarizing event similar to a GDP after a stimulus intensity dependent short latency

NMDA receptors are thought to be important in developing CNS. They are often present at silent synapses, in which other types of glutamatergic ionotropic receptors are absent [7] or presynaptic glutamate release is insufficient to activate them [8]. NMDA receptors have three important characteristics [9]. First, they can pass Ca2+ as well as Na+ and K+. Second, they cannot be opened by glutamate unless the Mg2+ block is removed by sufficient depolarization. Thus, NMDA receptors work as “AND-gates” for GABAergic depolarization and glutamatergic stimulation causing Ca2+ influx as outputs (Fig. 2). Synchronized activation of GABAergic and glutamatergic input in GDPs and GDP-like network activity have been shown to increase intracellular Ca2+ and potentiate connectivity in neural circuits in several regions of CNS [10, 11]. Third, they require glycine as co-agonist of glutamate, though a relatively low concentration of glycine is sufficient. Gaïarsa and colleagues showed that glycine can modulate GDP network activity by binding to the glycine binding site on the NMDA receptor in a strychnine insensitive manner [12, 13].

Fig. 2
figure2

Ca2+ influx by GABAergic neural inputs in an immature neuron in immature neurons, NKCC1 is more active than KCC2 and intracellular Cl concentration is maintained at a relatively high level. GABA released from GABAergic interneurons opens GABAA receptor Cl channels and Cl efflux results in the depolarization of the neuron. Depolarization opens voltage dependent calcium channels (VDCC) and removes bound Mg2+ from NMDA receptors, resulting in Ca2+ influx through VDCCs and glutamate-activated NMDA receptors. (The glycine binding site on NMDA receptors has been omitted)

GDP network activity is, in a sense, quite robust. It can be seen, as originally observed, in hippocampal slice preparation and it has been observed in vivo in hippocampus using extracellular recordings as “sharp waves” [14]. In addition, it can be seen in cultured neuron clusters [15] and organotypic slice cultures [16]. Furthermore, GDPs can be seen in small portions of sectioned slices of the hippocampus [17]. Using rat neonatal slice preparations containing both hippocampi and medial septa, Leinekugel et al. [17] showed that the GDPs from the temporal portion of the left and the right hippocampus and the septa were synchronized to the GDPs at the septal portion of hippocampi with a slight delay. The separated septum alone cannot generate GDPs whereas a small section of a hippocampus can still generate GDPs. The frequency of GDPs in temporal portion of a hippocampal slice greatly decreases, when separated from the septal portion of hippocampus, whereas the frequency in the septal portion remains unchanged. From these observations and other studies [14, 18], GDPs are thought to be initiated in the hippocampal CA3 region near the septum by relatively small clusters of neurons. Afterwards, the GDPs synchronously propagate to both hippocampi, spread to the septum, and then spread farther in the limbic system.

Although the role of glycine as a neurotransmitter in forebrain seems to be quite limited, in the adult CNS, the expression of functional glycine receptors has been found almost everywhere in the developing brain [19, 20]. In 1991, Ito and Cherubini found strychnine-sensitive glycine receptors in rat hippocampal CA3 neurons in the first postnatal week [21]. The postsynaptic cell response to glycine was quite similar to its response to GABA. It is excitatory during the first neonatal week and the reversal potential is identical to that of GABA. The polarity changes from depolarization to hyperpolarization early in the second postnatal week. However, the response to glycine disappears at the end of the second postnatal week, whereas the response to GABA persists in adult neurons (Fig. 3).

Fig. 3
figure3

Schematic membrane potential responses of rat neonatal CA3 neurones to GABA and glycine bath application of GABA and glycine lead to quite identical responses on neonatal CA3 neurone in the presence of TTX, except that GABA and glycine were specifically antagonized by bicuculline and strychnine, respectively, and glycine response disappeared in the 3rd neonatal week. Responses changed polarity from depolarization to hyperpolarization between the 1st and the 2nd postnatal weeks, despite the resting membrane potential remaining unchanged at approximately −65 mV

In addition to GDPs, spontaneous postsynaptic potentials (SPSPs) are also seen in neonatal CA3 neurons (Fig. 1a). Hosokawa et al. [22] studied these SPSPs and showed that SPSPs during the first postnatal week are mainly GABAergic despite being excitatory and that glutamatergic SPSPs are scarce. GABAergic SPSPs shift from depolarizing to hyperpolarizing at the end of the first neonatal week concomitant with the disappearance of GDPs and the gradual increase of glutamatergic excitatory SPSPs. Interestingly, Safiulina et al. observed [23] that the frequency of SPSPs becomes significantly higher prior to the onset of each GDP.

The depolarizing and excitatory effects of GABA and glycine are seen throughout the developing CNSs in various vertebrates, as summarized in the review by Ben-Ari et al. [24]. However, Bregestovski and Bernard raised several questions about the excitatory effects of GABA and glycine in the developing brain [25]. They discussed the possibility that pathologically high intracellular Cl concentration in in vitro artificial conditions result in misleading excitatory activity including GDPs. Their arguments were immediately refuted by Ben-Ari et al. [26], and it is improbable that all GDPs and GDP-like activities including in vivo observations are mere artefacts. We must still be aware that GDPs in the slice preparation may not accurately represent occurrences in the hippocampus of intact neonates.

The role of extra-synaptic receptors and the tonic effects of their agonists have been studied during the developmental period [2731] and have been shown to modulate neural network activities including GDP. Tonic effects of agonists on extra-synaptic receptors can be revealed by a shift in the resting membrane potential, or tonic current in a voltage clamp, by specific antagonists and, especially, by specific uptake blockers of putative agonists. Naturally, GABA and glycine have been shown to be tonic agonists on GABAA and glycine receptors [2628]. In addition, taurine and β-alanine are proposed to be tonic glycine receptor agonists because they are abundant in the extracellular environment and the uptake blocker of taurine potentiates glycine receptor-mediated tonic effects [30, 31].

Excitatory responses to GABA and glycine in immature neurons are explained by the relatively high intracellular Cl concentration. As shown in Fig. 2, the intracellular Cl concentration is regulated mainly by two cation-chloride cotransporters, NKCC1 and KCC2 [32]. NKCC1 imports Cl whereas KCC2 extrudes intracellular Cl. In addition, a relatively lower expression of KCC2 is thought to cause a high intracellular Cl concentration in developing neurons [33, 34]. Thus, the reversal potential of Cl is kept higher than the resting membrane potential and an efflux of Cl through receptor chloride channels activated by GABA or glycine causes a depolarizing response. It must be emphasised that the intracellular concentration of Cl is actively controlled with considerable energy cost when the reversal potential differs from the membrane potential.

The intracellular Cl concentration gradually decreases during the early developmental period due to changes in the balance of activities of NKCC1 and KCC2 [35], whereas the resting potential remains relatively constant (Fig. 4a). In the case of rat CA3 neurons, the shift from depolarization to hyperpolarization (D/H shift) occurs at the end of the first postnatal week [4, 21]. Tyzio and colleagues found an amazing phenomenon in mouse hippocampal and cortical neurons that occurs at birth (Fig. 4a) [36]. Although GABAergic response is excitatory both in the embryonic and early postnatal periods, it transiently shifts to inhibitory at birth. This shift was found to be caused by maternal oxytocin which crosses the placenta, blocks NKCC1 and results in a lower intracellular Cl concentration. They hypothesized that this transient D/H shift works by calming down neural activity, and that it protects the immature brain from harmful stress such as excessive catecholamine release during delivery.

Fig. 4
figure4

The time course of the change in neural intracellular Cl concentration during early developmental periods. a During the embryonic and early neonatal periods, NKCC1 is more active than KCC2 and the reversal potential for Cl is maintained above the resting potential. However, at the birth, maternal oxytocin acts on the fetal neurons and suppresses NKCC1 activity resulting in a transient hyperpolarizing shift of Cl reversal potential. After birth, KCC2 becomes gradually more active than NKCC1 and the Cl reversal potential finally becomes more negative than the resting potential. Failure of sufficient oxytocin action at birth has been suggested to be a cause of pathological conditions such as autism spectrum disorders. b Responses to GABA and glycine change from excitatory to inhibitory between the 1st and the 2nd postnatal weeks due to the increase of intracellular Cl concentration

A transient D/H shift during delivery and a gradual decrease of intracellular Cl after birth were found to be impaired in autism models in rodents [37]. Pre-treatment with bumetanide, a specific NKCC1 blocker, can improve symptoms of autism by lowering intracellular Cl. While oxytocin itself has been recently tested as a treatment for autism spectrum disorders (ASDs) [3840], trials of bumetanide for ASD patients are now ongoing with some significantly positive results [41, 42].

In conclusion, spontaneous synaptic events including SPSPs and GDP-like activity work to refine neural networks, and the tonic effects of agonists on extra-synaptic receptors in the microenvironment control the excitability of neurons. Both are the most important mechanisms for activity-dependent morphogenesis and functiogenesis in which GABA and glycine play major roles. On the other hand, failure of a shift to inhibitory at the appropriate timing causes pathological problems of the brain function.

Abbreviations

AMPA:

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ASD:

Autism spectrum disorder

CA3:

Cornu ammonis area 3

CNS:

Central nervous system

GABA:

γ-Aminobutyric acid

GDP:

Giant depolarizing potential

KCC2:

Potassium chloride cotransporter 2

NKCC1:

Sodium potassium chloride cotransporter 1

NMDA:

N-methyl-d-aspartate

SPSP:

Spontaneous postsynaptic potential

References

  1. 1.

    Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, Peters JA, Harmar AJ (2013) The concise guide to pharmacology 2013/14: ligand-gated ion channels. Br J Pharmacol 170:1582–1606

  2. 2.

    Obata K (1974) Transmitter sensitivities of some nerve and muscle cells in culture. Brain Res 73:71–88

  3. 3.

    Obata K, Oide M, Tanaka H (1978) Excitatory and inhibitory actions of GABA and glycine on embryonic chick spinal neurons in culture. Brain Res 144:179–184

  4. 4.

    Ben-Ari Y, Cherubini E, Corradetti R, Gaïarsa JL (1989) Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol 416:303–325

  5. 5.

    Cherubini E, Gaïarsa JL, Ben-Ari Y (1991) GABA: an excitatory transmitter in early postnatal life. Trends Neurosci 14:515–519

  6. 6.

    Bolea S, Avignone E, Berretta N, Sanchez-Andres JV, Cherubini E (1999) Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. J Neurophysiol 81:2095–2102

  7. 7.

    Durand GM, Kovalchuk Y, Konnerth A (1996) Long-term potentiation and functional synapse induction in developing hippocampus. Nature 381:71–75

  8. 8.

    Gasparini S, Saviane C, Voronin LL, Cherubini E (2000) Silent synapses in the developing hippocampus: lack of functional AMPA receptors or low probability of glutamate release? Proc Natl Acad Sci USA 97:9741–9746

  9. 9.

    Blanke ML, Van Dongen AMJ (2009) Activation mechanisms of the NMDA receptor. In: Van Dongen AMJ (ed) Biology of the NMDA receptor. CRC, Boca Raton

  10. 10.

    Kasyanov AM, Safiulina VF, Voronin LL, Cherubini E (2004) GABA-mediated giant depolarizing potentials as coincidence detectors for enhancing synaptic efficacy in the developing hippocampus. Proc Natl Acad Sci USA 101:3967–3972

  11. 11.

    Mohajerani MH, Cherubini E (2006) Role of giant depolarizing potentials in shaping synaptic currents in the developing hippocampus. Crit Rev Neurobiol 18:13–23

  12. 12.

    Gaïarsa JL, Corradetti R, Cherubini E, Ben-Ari Y (1990) The allosteric glycine site of the N-methyl-d-aspartate receptor modulates GABAergic-mediated synaptic events in neonatal rat CA3 hippocampal neurons. Proc Natl Acad Sci USA 87:343–346

  13. 13.

    Gaïarsa JL, Corradetti R, Cherubini E, Ben-Ari Y (1991) Modulation of GABA-mediated synaptic potentials by glutamatergic agonists in neonatal CA3 rat hippocampal neurons. Eur J Neurosc 3:301–309

  14. 14.

    Leinekugel X, Khazipov R, Cannon R, Hirase H, Ben Ari Y, Buzsaki G (2002) Correlated bursts of activity in the neonatal hippocampus in vivo. Science 296:2049–2052

  15. 15.

    Voigt T, Opitz T, De Lima AD (2001) Synchronous oscillatory activity in immature cortical network is driven by GABAergic preplate neurons. J Neurosci 21:8895–8905

  16. 16.

    Mohajerani MH, Cherubini E (2005) Spontaneous recurrent network activity in organotypic rat hippocampal slices. Eur J Neurosci 22:107–118

  17. 17.

    Leinekugel X, Khalilov I, Ben-Ari Y, Khazipov R (1998) Giant depolarizing potentials: the septal pole of the hippocampus paces the activity of the developing intact septohippocampal complex in vitro. J Neurosci 18:6349–6357

  18. 18.

    Menendez de la Prida L, Sanchez-Andres JV (2000) Heterogenous populations of cells mediate spontaneous synchronous bursting in the developing hippocampus through a frequency-dependent mechanism. Neuroscience 97:227–241

  19. 19.

    Bets H (1991) Glycine receptors: heterogeneous and widespread in the mammalian brain. Trends Neurosci 14:458–461

  20. 20.

    Avila A, Nguyen L, Rigo JM (2013) Glycine receptors and brain development. Front Cell Neurosci 7:184

  21. 21.

    Ito S, Cherubini E (1991) Strychnine sensitive glycine responses of neonatal rat hippocampal neurones. J Physiol 440:67–83

  22. 22.

    Hosokawa Y, Sciancalepore M, Stratta F, Martina M, Cherubini E (1994) Developmental changes in spontaneous GABAA-mediated synaptic events in rat hippocampal CA3 neurons. Eur J Neurosci 6:805–813

  23. 23.

    Cherubini E, Griguoli M, Safiulina V, Lagostena L (2011) The depolarizing action of GABA controls early network activity in the developing hippocampus. Mol Neurobiol 43:97–106

  24. 24.

    Ben-Ari Y, Gaïarsa JL, Tyzio R, Khazipov R (2007) GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev 87:1215–1284

  25. 25.

    Bregestovski P, Bernard C (2012) Excitatory GABA: how a correct observation may turn out to be an experimental artifact. Front Pharmacol 3:65

  26. 26.

    Ben-Ari Y, WoodinM A, Sernagor E, Cancedda L, Vinay L, Rivera C, Legendre P, Luhmann HJ, Bordey A, Wenner P, Fukuda A, vad den Pol AN, Gaïarsa JL, Cherubini E (2012) Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever! Front Cell Neurosci 6:1–18

  27. 27.

    Marchionni I, Omrani A, Cherubini E (2007) In the developing rat hippocampus a tonic GABAA-mediated conductance selectively enhances the glutamatergic drive of principal cells. J Physiol 581:515–528

  28. 28.

    Gomeza J, Hülsmann S, Koji Ohno, Eulenburg V, Szöke K, Richter D, Betz H (2003) Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40:785–796

  29. 29.

    Sipila ST, Spoljaric A, Virtanen MA, Hiironniemi I, Kaila K (2014) Glycine transporter-1 controls nonsynaptic inhibitory actions of glycine receptors in the neonatal rat hippocampus. J Neurosci 34:10003–10009

  30. 30.

    Mori M, Gähwiler BH, Gerber U (2002) β-alanine and taurine as endogenous agonists at glycine receptors in rat hippocampus in vitro. J Physiol 539:191–200

  31. 31.

    Chen R, Okabe A, Sun H, Sharopov S, Hanganu-Opatz IL, Kolbaev SN, Fukuda A, Luhmann HJ, Kilb W (2014) Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus. J Physiol 592:2153–2168

  32. 32.

    Kaila K, Price TJ, Payne JA, Puskarjov M, Voipio J (2014) Cation-chloride cotransporters in neuronal development, plasticity and disease. Nat Rev Neurosci 15:637–654

  33. 33.

    Wang C, Shimizu-Okabe C, Watanabe K, Okabe A, Matsuzaki H, Ogawa T, Mori N, Fukuda A, Sato K (2002) Developmental changes in KCC1, KCC2, and NKCC1 mRNA expressions in the rat brain. Brain Res Dev Brain Res 139:59–66

  34. 34.

    Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A (2004) Cl uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1. J Physiol 557:829–841

  35. 35.

    Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K (1999) The K/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251–255

  36. 36.

    Tyzio R, Cossart R, Khalilov I, Minlebaev M, Hubner CA, Represa A, Ben-Ari Y, Khazipov R (2006) Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery. Science 314:1788–1792

  37. 37.

    Tyzio R, Nardou R, Ferrari DC, Tsintsadze T, Shahrokhi A, Eftekhari S, Khalilov I, Tsintsadze V, Brouchoud C, Chazal G, Lemonnier E, Lozovaya N, Burnashev N, Ben-Ari Y (2014) Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring. Science 343:675–679

  38. 38.

    Hollander E, Bartz J, Chaplin W, Phillips A, Sumner J, Soorya L, Anagnostou E, Wasserman S (2007) Oxytocin increases retention of social cognition in autism. Biol Psychiatry 61:498–503

  39. 39.

    Gordon I, Wyk BCV, Bennett RH, Cordeaux C, Lucas MV, Eilbott JA, Zagoory-Sharon O, Leckman JF, Feldman R, Pelphrey KA (2013) Oxytocin enhances brain function in children with autism. Proc Natl Acad Sci USA 110(52):20953–20958

  40. 40.

    Watanabe T, Mi Kuroda, Kuwabara H, Aoki Y, Iwashiro N, Tatsunobu N, Takao H, Nippashi Y, Kawakubo Y, Kunimatsu A, Kasai K, Yamasue H (2015) Clinical and neural effects of six-week administration of oxytocin on core symptoms of autism. Brain 138:3400–3412

  41. 41.

    Lemonnier E, Degrez C, Phelep M, Tyzio R, Josse F, Grandgeorge M, Hadjikhani N, Ben-Ari Y (2012) A randomised controlled trial of bumetanide in the treatment of autism in children. Transl Psychiatry 2:e202

  42. 42.

    Curatolo P, Ben-Ari Y, Bozzi Y, Catana MV, D’Angelo E, Mapelli L, Oberman LM, Rosenmund C, Cherubini E (2014) Synapses as therapeutic targets for autism spectrum disorders: an international symposium held in Pavia on July 4th. Front Cell Neurosci 8:309. doi:10.3389/fncel.2014.00309

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Acknowledgments

I am grateful to Prof. Enrico Cherubini for his valuable remarks and suggestions. I am also grateful to Miss Keiko Narazaki for help in preparation of figures.

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Correspondence to Susumu Ito.

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Ito, S. GABA and glycine in the developing brain. J Physiol Sci 66, 375–379 (2016). https://doi.org/10.1007/s12576-016-0442-7

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Keywords

  • GABA
  • Glycine
  • Giant depolarizing potentials
  • Activity dependent functiogenesis