Research report
Glycine facilitates transmitter release at developing synapses: a patch clamp study from Purkinje neurons of the newborn rat

https://doi.org/10.1016/S0165-3806(03)00159-7Get rights and content

Abstract

Synaptic currents in immature Purkinje cells from rats on postnatal days 0–14 (P0–P14) were studied using whole-cell patch-electrodes applied to cerebellar slices (200 μm in thickness). Purkinje cells (held at −40 mV) showed excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) spontaneously. From P2 to P12 the frequencies of miniature EPSCs and miniature IPSCs in the Purkinje cells increased by 10-fold or more, suggesting progressive formation of functional synapses during this period. Application of glycine (100 μM) to an immature Purkinje cell at P3–10 immediately increased the frequencies of both EPSCs and IPSCs. The effects of glycine showed maximum at P5–6 for EPSCs and at P9–10 for IPSCs and decreased thereafter. Facilitatory effects of glycine were suppressed by strychnine (1 μM), a specific blocker of the ionotropic glycine receptor, while the effects were also induced by other glycinergic agonists, including α-l-alanine (1 mM), l-serine (1 mM) and taurine (500 μM). The site of glycinergic effects was studied by removing the action potential generation in cerebellar slices. Following the addition of tetrodotoxin (TTX, 1 μM), the glycine-induced facilitation of EPSC almost disappeared, while that of IPSC remained (i.e. miniature IPSCs) and reached more than half of the value without TTX. These findings suggest that the ionotropic glycinergic receptors are expressed transiently but profoundly in the developing cerebellum, and that the distributions of these receptors causing excitation are different at excitatory and inhibitory presynaptic neurons. The glycine receptors may play distinct roles in the maturation and organization of cerebellar neural circuits.

Introduction

Glycine is a major inhibitory transmitter in the central nervous system, principally in the spinal cord and brain stem [27], [28]. Recent molecular studies have shown that glycine receptors are distributed more extensively than previously thought, particularly in the higher brain of mammals, including the hippocampus, cerebral cortex, hypothalamus and cerebellum [5], [13], [24], [33], [34], [41]. Expressions of glycine receptors in these areas are regulated distinctly in terms of subunit compositions and amounts.

During the early stage of neural development, glycine causes excitation rather than inhibition of neurons because the intracellular concentration of Cl is higher than the equilibrium concentration as estimated from the resting membrane potential [11], [24], [27], [38]. Such excitation is presumably essential for synaptic maturation or neural development, since depolarization causes activation of voltage-dependent Ca2+ channels, enabling Ca2+ to permeate into neurons and thus increasing the intracellular concentration of Ca2+ and evoking specific functions of the cell [12], [21], [28]. Glycine receptors have recently been found on presynaptic terminals of auditory brain stem neurons, and activation of these receptors has been demonstrated to cause membrane depolarization and enhanced transmitter release [45], [46]. Thus, ionotropic glycine receptors might play diverse roles, being regulated more intricately than previously thought [1], [15], [27].

In the cerebellum, glycinergic synaptic currents have been identified in Golgi cells, a subset of local inhibitory neurons in the cerebellar cortex, and it has been suggested that a portion of local neurons, identified as Lugaro cells, release glycine [8], [9]. Also on the granule cells of the cerebellar cortex, functional glycinergic receptors are found. Most of the receptors seem to operate only during a specific period of development and may exert a pivotal influence on the establishment of synaptic connections [13], [22], [47]. Based on the findings of neurological abnormalities caused by mutations of glycine receptors or the high concentration of glycine in the immature brain, it has also been suggested that glycine receptors play a critical role in the development of balanced neural circuits [28], [32], [39].

The cerebellar cortex of the rat has been focused on developmental studies because it shows remarkable maturational changes after birth [2], [7]. In the present study by focusing on functional maturation of synapses on Purkinje cells, the expression and functional properties of glycine receptors were studied using a slice-patch technique and a ‘Y tube’ method modified for rapid drug application to a brain slice preparation [23]. Immediately after birth, spontaneous synaptic activities on Purkinje cells were weak but the levels of activity began to increase after 2–3 days. Application of glycine to Purkinje cells at postnatal days 3–10 markedly increased the frequencies of both excitatory and inhibitory postsynaptic currents. The author characterized the properties of this facilitation and has found that strychnine-sensitive glycine receptors are expressed transiently and distinctively on presynaptic neurons of developing cerebellar Purkinje cells, inducing remarkable presynaptic excitation.

Section snippets

Slice preparation

All experiments were carried out in accordance with the Guiding Principles of the Physiological Society of Japan. The procedures for preparing and preserving thin slices from a mammalian brain and for cleaning cells in the slices for patch-clamp recordings have been described in detail elsewhere [23]. Briefly, newborn rats of Wistar strain at postnatal days 0–14 (P0–P14; both males and females) were killed by decapitation after ether anesthesia, and then the cerebellum was quickly dissected out

Postnatal development of synapses on Purkinje cells

During the early postnatal period, Purkinje cells rigorously extend their dendrites in the molecular layers and receive inhibitory and excitatory synaptic inputs from local and afferent neurons [2], [23]. Fig. 1A and B shows miniature synaptic currents in Purkinje cells on postnatal days 2 to 12 (P2–P12) measured in the presence of tetrodotoxin (TTX, 1 μM), a blocker of Na+-dependent action potential. The membrane potential of Purkinje cells was held at −40 mV, at which excitatory and

Discussion

The properties of developing synapses on Purkinje cells and the glycinergic facilitation of transmitter release from these synapses were studied using slices of cerebella obtained from immature rats (at P0–P14). When Purkinje cells were whole-cell-clamped at −40 mV, spontaneous synaptic currents with inward and outward directions, which have been identified as glutamatergic EPSCs and GABAergic IPSCs, respectively, were observed (Fig. 1; also Ref. [23]). In the presence of TTX, frequencies of

Acknowledgements

I thank Mr S. Sai and Mr S. Ohsawa for their technical support and Mr S. Chisholm for reading the manuscript. This work was supported by a grant from Toyota Physical and Chemical Research Institute. This study was also supported by a CREST grant from the Japan Science and Technology Corporation (JST).

References (50)

  • K. Sato et al.

    Regional distribution of cells expressing glycine receptor α2 subunit mRNA in the rat brain

    Brain Res.

    (1992)
  • O.A. Sergeeva et al.

    Glycine receptor mediated responses in rat histaminergic neurons

    Neurosci. Lett.

    (2001)
  • C. Virginio et al.

    Glycine-activated whole cell and single channel currents in rat cerebellar granule cells in culture

    Brain Res. Dev. Brain Res.

    (1997)
  • S. Ahmadi et al.

    PGE2 selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons

    Nat. Neurosci.

    (2002)
  • J. Altman et al.

    Development of the Cerebellar System: Evolution, structure, and functions

    (1997)
  • A.M. Batchelor et al.

    GABAB receptors in the parallel fibre pathway of rat cerebellum

    Eur. J. Neurosci.

    (1992)
  • J.E. Chatterton et al.

    Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

    Nature

    (2002)
  • S.C. Chattipakorn et al.

    Pharmacological characterization of glycine-gated chloride currents recorded in rat hippocampal slices

    J. Neurophysiol.

    (2002)
  • F. Crepel

    Excitatory and inhibitory processes acting upon cerebellar Purkinje cells during maturation in the rat; influence of hypothyroidism

    Exp. Brain Res.

    (1974)
  • S. Dieudonne

    Glycinergic synaptic currents in Golgi cells of the rat cerebellum

    Proc. Natl. Acad. Sci. USA

    (1995)
  • A. Dumoulin et al.

    IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells

    J. Neurosci.

    (2001)
  • E.D. Eggers et al.

    Developmental changes in the modulation of synaptic glycine receptors by ethanol

    J. Neurophysiol.

    (2000)
  • I. Ehrlich et al.

    Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl regulation

    J. Physiol.

    (1999)
  • J. Eilers et al.

    GABA-mediated Ca2+ signalling in developing rat cerebellar Purkinje neurones

    J. Physiol.

    (2001)
  • B.-X. Gao et al.

    Transition from GABAergic to glycinergic synaptic transmission in newly formed spinal networks

    J. Neurophysiol.

    (2001)
  • Cited by (24)

    • Electrophysiological evidence of increased glycine receptor-mediated phasic and tonic inhibition by blockade of glycine transporters in spinal superficial dorsal horn neurons of adult mice

      2017, Journal of Pharmacological Sciences
      Citation Excerpt :

      Since the amplitude of mIPSCs was unaltered, this effect appears to be mediated by presynaptic mechanisms (29,30). Recently, presynaptic strychnine-sensitive glycine receptors have been shown to facilitate release of glutamate (31) and GABA (32) in the presence of TTX. A mechanism of enhanced release due to activation of Ca2+ channels coupled with a weakly depolarizing Cl− current via glycine receptors in the nerve terminal has been proposed (31).

    • Propofol depresses cerebellar Purkinje cell activity via activation of GABA<inf>A</inf> and glycine receptors in vivo in mice

      2015, European Journal of Pharmacology
      Citation Excerpt :

      In the cerebellum, glycinergic synaptic currents have been identified in granule cells and Golgi cells in the cerebellar cortex (Dieudonne, 1995; Dumoulin et al., 2001; Elster et al., 1998). In a previous study (Kawa, 2003), the glycine-induced slow outward currents were observed in cerebellar PCs at postnatal 0–10 days, which were blocked by glycine receptor antagonist but were resistant to GABAA receptor antagonist. However, the glycine-induced slow outward currents were not exhibited after postnatal 13–15 days, indicated that the ionotropic glycinergic receptors were expressed transiently but profoundly in the developing cerebellar PCs (Kawa, 2003).

    • Pregnenolone sulfate increases glutamate release at neonatal climbing fiber-to-Purkinje cell synapses

      2011, Neuroscience
      Citation Excerpt :

      Nonetheless, in the presence of strychnine, the PregS-induced increase in AMPA-mEPSC frequency was not affected. These results are not surprising given that the glycine-induced increase in sEPSC frequency was almost completely abolished by TTX (Kawa, 2003), and that PregS has been shown to inhibit recombinant glycine receptors (reviewed in Gibbs et al., 2006). Another plausible candidate for the effect of PregS on PCs was the Ca2+ permeable α7nACh receptor.

    • Developmental expression of glycine receptor subunits in rat cerebellum

      2008, International Journal of Developmental Neuroscience
    View all citing articles on Scopus
    View full text