Glycine and GABA, the dominant inhibitory neurotransmitters in the CNS, assume a depolarizing role in early development, leading to increased cytoplasmic Ca 2ϩ levels and action potentials. The effect is thought to be of some significance for maturation. The depolarization is caused by Cl Ϫ efflux, and chloride transporters contribute to the phenomenon by raising the intracellular Cl

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...

Synaptic refinement via the elimination of inappropriate synapses and strengthening of appropriate ones is crucially important for the establishment of specific, topographic neural circuits. The mechanisms driving these processes are poorly understood, particularly concerning inhibitory projections. Here, we address the refinement of an inhibitory topographic projection in the auditory brainstem in functional and anatomical mapping studies involving patch-clamp recordings in combination with minimal and maximal stimulation, caged glutamate photolysis, and single axon tracing. We demonstrate a crucial dependency of the refinement on Ca V 1.3 calcium channels: Ca V 1.3 Ϫ/Ϫ mice displayed virtually no elimination of projections up to hearing onset. Furthermore, strengthening was strongly impaired, in line with a reduced number of axonal boutons. The mediolateral topography was less precise and the shift from a mixed GABA/ glycinergic to a purely glycinergic transmission before hearing onset did not occur. Together, our findings provide evidence for a Ca V 1.3-dependent mechanism through which both inhibitory circuit formation and determination of the neurotransmitter phenotype are achieved.

The inhibitory transmitters glycine and GABA undergo a developmental shift from depolarizing to hyperpolarizing action (D/H-shift). To analyse this shift in functionally related nuclei of the rat superior olivary complex (SOC), we employed voltage-sensitive dye recordings in auditory brainstem slices. Complementarily, we analysed single neurons in gramicidin perforated-patch recordings. Our results show a differential timing of the D/H-shift in the four SOC nuclei analysed. In the medial superior olive (MSO), the shift occurred at postnatal day (P) 5-9. In the superior paraolivary nucleus (SPN), it occurred between embryonic day (E) 18 and P1. No D/H-shift was observed in the medial nucleus of the trapezoid body (MNTB) until P10. This is in line with the finding that most of the patched MNTB neurons displayed glycine-induced depolarizations between P0-9. While no regional differences regarding the D/H-shift were found within the MSO, SPN, and MNTB, we observed such differences in the lateral superior olive (LSO). All LSO regions showed a D/H-shift at P4-5. However, in the high-frequency regions, hyperpolarizations were large already at P6, yet amplitudes of this size were not present until P8 in the low-frequency regions, suggesting a delayed development in the latter regions. Our physiological results demonstrate that D/H-shifts in SOC nuclei are staggered in time and occur over a period of almost two weeks. Membrane-associated immunoreactivity of the Cl- outward transporter KCC2 was found in every SOC nucleus already at times when glycine was still depolarizing. This implies that the mere presence of KCC2 does not correlate with functional Cl- outward transport.

Within the Ca v 1 family of voltage-gated calcium channels, Ca v 1.2 and Ca v 1.3 channels are the predominant subtypes in the brain. Whereas specific functions for each subtype were described in the adult brain, their role in brain development is poorly understood. Here we assess the role of Ca v 1.3 subunits in the activity-dependent development of the auditory brainstem. We used Ca v 1.3-deficient (Ca v 1.3 Ϫ/Ϫ ) mice because these mice lack cochlea-driven activity that deprives the auditory centers from peripheral input. We found a drastically reduced volume in all auditory brainstem centers (range 25-59%, total 35%), which was manifest before hearing onset. A reduction was not obvious outside the auditory system. The lateral superior olive (LSO) was strikingly malformed in Ca v 1.3 Ϫ/Ϫ mice and had fewer neurons (1/3 less). The remaining LSO neurons displayed normal dendritic trees and received functional glutamatergic input, yet they fired action potentials predominantly with a multiple pattern upon depolarization, in contrast to the single firing pattern prevalent in controls. The latter finding appears to be due to a reduction of dendrototoxin-sensitive potassium conductances, presumably mediated through the K v 1.2 subtype. Fura2 imaging provided evidence for functional Ca v 1.3 channels in the LSO of wild-type mice. Our results imply that Ca v 1.3 channels are indispensable for the development of the central auditory system. We propose that the unique LSO phenotype in Ca v 1.3 Ϫ/Ϫ mice, which hitherto was not described in other hereditary deafness models, is caused by the synergistic contribution of two factors: on-site loss of Ca v 1.3 channels in the neurons plus lack of peripheral input.