Research reportGlycine facilitates transmitter release at developing synapses: a patch clamp study from Purkinje neurons of the newborn rat
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).
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