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

Abstract

The mechanisms underlying the development and maturation of inhibitory synapses in the central nervous system (CNS) are still poorly understood. This is mainly due to the fact that inhibitory neurones are mostly interneurones which are diffusely distributed within a brain region. In the adult CNS, fast synaptic inhibition is mediated by the release of GABA or glycine and subsequent activation of GABAA receptors (GABAARs) or glycine receptors (GlyRs). These receptors are ion channels permeable to Cl¦ and, to a much lesser extent, to other anions such as bicarbonate (HCOצ) (Bormann et al. 1987). A flux of Cl¦ into neurones causes a hyperpolarization of the membrane potential and, hence, inhibition of neuronal activity. However, GABA and glycine depolarize neurones in many CNS areas during embryonic and neonatal development, e.g. in the hippocampus (Ben-Ari et al. 1989;Cherubini et al. 1990), cerebral cortex (Luhmann & Prince, 1991;Lo Turco et al. 1995), hypothalamus (Chen et al. 1996), brainstem (Kandler & Friauf, 1995b;Singer et al. 1998) 1. The inhibitory neurotransmitter glycine can elicit depolarizing responses in immature neurones. We investigated the changes in glycine responses and their ionic mechanism in developing neurones of the rat lateral superior olive (LSO), an auditory brainstem nucleus involved in sound localization. 2. Whole-cell and gramicidin perforated-patch recordings were performed from visually identified LSO neurones in brain slices and glycine was pressure applied for 3-100 ms to the soma. Glycine-evoked currents were reversibly blocked by strychnine. They were mostly monophasic, but biphasic responses occurred in •30% of P8-11 neurones in perforatedpatch recordings. 3. In whole-cell recordings from P2-11 neurones, the reversal potential of glycine-evoked currents (EGly) was determined by the transmembranous Cl¦ gradient and corresponded closely to the Nernst potential for Cl¦, regardless of age. This indicates that Cl¦ is the principle ion permeating glycine receptors, but is also consistent with a low relative (10-20%) permeability for HCOצ. The Cl¦ gradient also determined the polarity and amplitude of glycine-evoked membrane potential changes. 4. Leaving the native intracellular [Cl¦] undisturbed with gramicidin perforated-patch recordings, we found a highly significant, age-dependent change of EGly from −46·8 ± 1·8 mV (P1-4, n = 28) to −67·6 ± 3·3 mV (P5-8, n = 10) to −82·2 ± 4·1 mV (P9-11, n = 18). The majority of P1-4 neurones were depolarized by glycine (•80%) and spikes were evoked in •30%. In contrast, P9-11 neurones were hyperpolarized. 5. In perforated-patch recordings, EGly was influenced by the voltage protocol and the glycine application interval; it could be shifted in the positive and negative direction. For a given application interval, these shifts were always larger in P1-4 than in P8-11 neurones, pointing to less effective Cl¦ regulation mechanisms in younger neurones. 6. Furosemide (frusemide), a blocker of cation-Cl¦ cotransporters, reversibly shifted EGly in the...

Glycinergic/GABAergic synapses in the lateral superior olive are excitatory in neonatal C57Bl/6J mice

“…These data indicate that MNTB-evoked PSPs are primarily depolarizing up to P3 and hyperpolarizing thereafter. Thus, compared to rats [11] or gerbils [12] MNTB-evoked PSPs in mice become hyperpolarizing 5-7 days earlier, consistent with the somewhat faster maturation of the mouse auditory system [5].Recently, it has been demonstrated that glycine can elicit action potentials (APs) in immature rat LSO neurons [7] suggesting that glycine can act as an excitatory neurotransmitter in the LSO. However, in these studies glycine was applied iontophoretically to the cell body, a somewhat artificial situation because it activates both synaptic, as well as extrasynaptic, glycine receptors.…”

“…Recently, it has been demonstrated that glycine can elicit action potentials (APs) in immature rat LSO neurons [7] suggesting that glycine can act as an excitatory neurotransmitter in the LSO. However, in these studies glycine was applied iontophoretically to the cell body, a somewhat artificial situation because it activates both synaptic, as well as extrasynaptic, glycine receptors.…”

“…During early postnatal development, the spatial connectivity pattern of this inhibitory MNTB-LSO pathway is sharpened by activity-dependent mechanisms resulting in the precise tonotopic organization seen in adult animals (for review see [15,16]). During the first postnatal week, glycinergic/GABAergic MNTB-LSO synapses are not hyperpolarizing as in adult animals, but are depolarizing due to a high internal chloride concentration of LSO neurons [7,11]. It has been suggested that this depolarization renders the MNTB pathway excitatory and that the excitatory nature is part of the mechanism by which activity in the MNTB-LSO pathways exerts its effect on neuronal growth, survival, and synaptic In the present study, we examined whether and at what age the MNTB-LSO pathway is depolarizing and whether it can elicit action potentials in LSO neurons of mice.…”

Oligomerization of KCC2 Correlates with Development of Inhibitory Neurotransmission

“…In the vast majority of mature neurons, a low intracellular chloride concentration ([Cl Ϫ ] i ) causes Cl Ϫ influx during channel opening, resulting in hyperpolarization (Eccles, 1966;Kaila, 1994). In contrast, a high [Cl Ϫ ] i in immature neurons results in Cl Ϫ efflux at the resting membrane potential (Zhang et al, 1991;Wang et al, 1994;Obrietan and van den Pol, 1995;Ehrlich et al, 1999;Kakazu et al, 1999). This efflux depolarizes the neurons and can even elicit action potentials.…”

Inhibitory circuits in sensory maps develop through excitation

“…The time course of refinement follows the development of inhibition in the MNTB -LSO system. As is seen during the development of other inhibitory neurons [14], the Cl 2 reversal potential in neurons of the LSO is relatively positive (2 48 mV) at birth but becomes more negative (2 82 mV) by the time of hearing onset [15]. During the refinement period, therefore, glycinergic and GABAergic inputs depolarize the postsynaptic cell [16], which can produce increases in postsynaptic Ca 2þ concentration and can result in action potentials [17].…”