Staggered development of GABAergic and glycinergic transmission in the MNTB

Maturation of some brain stem and spinal inhibitory systems is characterized by a shift from GABAergic to glycinergic transmission. Little is known about how this transition is expressed in terms of individual axonal inputs and synaptic sites. We have explored this issue in the rat medial nucleus of the trapezoid body (MNTB). Synaptic responses at postnatal days 5-7 (P5-P7) were small, slow, and primarily mediated by GABA(A) receptors. By P8-P12, an additional, faster glycinergic component emerged. At these ages, GABA(A), glycine, or both types of receptors mediated transmission, even at single synaptic sites. Thereafter, glycinergic development greatly accelerated. By P25, evoked inhibitory postsynaptic currents (IPSCs) were 10 times briefer and 100 times larger than those measured in the youngest group, suggesting a proliferation of synaptic inputs activating fast-kinetic receptors. Glycinergic miniature IPSCs (mIPSCs) increased markedly in size and decay rate with age. GABAergic mIPSCs also accelerated, but declined slightly in amplitude. Overall, the efficacy of GABAergic inputs showed little maturation between P5 and P20. Although gramicidin perforated-patch recordings revealed that GABA or glycine depolarized P5-P7 cells but hyperpolarized P14-P15 cells, the young depolarizing inputs were not suprathreshold. In addition, vesicle-release properties of inhibitory axons also matured: GABAergic responses in immature rats were highly asynchronous, while in older rats, precise, phasic glycinergic IPSCs could transmit even with 500-Hz stimuli. Thus development of inhibition is characterized by coordinated modifications to transmitter systems, vesicle release kinetics, Cl- gradients, receptor properties, and numbers of synaptic inputs. The apparent switch in GABA/glycine transmission was predominantly due to enhanced glycinergic function.     

Cotransmission of GABA and glycine to brain stem motoneurons.

Using whole cell patch-clamp recording in a rat brain stem slice preparation, we found that gamma-aminobutyric acid (GABA) and glycine act as cotransmitters to hypoglossal motoneurons (HMs). Focal application of GABA and glycine onto a single HM revealed that GABAA and glycine receptors are present on the same neuron. To demonstrate that HMs receive both GABAergic and glycinergic synaptic inputs, we simultaneously recorded GABAA- and glycine-receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in single HMs. GABAergic and glycinergic mIPSCs were differentiated based on their kinetics and modulation by pentobarbital. Specifically, GABAA-receptor-mediated events decayed more slowly than glycine-receptor-mediated events. GABAergic response decay kinetics were prolonged by pentobarbital, whereas glycinergic response decay kinetics remained unchanged. The distinct kinetics of the glycine- and GABAA-receptor-mediated synaptic events allowed us to record dual component mIPSCs, mIPSCs that are mediated by both receptor types. These data suggest that GABA and glycine are colocalized in the same presynaptic vesicle and are coreleased from presynaptic terminals opposed to motoneurons.     

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

The role of glycinergic and GABAergic systems in mediating spontaneous synaptic transmission in newly formed neural networks was examined in motoneurons in the developing rat spinal cord. Properties of action potential-independent miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine and GABA(A) receptors (GlyR and GABA(A)R) were studied in spinal cord slices of 17- to 18-day-old embryos (E17-18) and 1- to 3-day-old postnatal rats (P1-3). mIPSC frequency and amplitude significantly increased after birth, while their decay time decreased. To determine the contribution of glycinergic and GABAergic synapses to those changes, GlyR- and GABA(A)R-mediated mIPSCs were isolated based on their pharmacological properties. Two populations of pharmacologically distinct mIPSCs were recorded in the presence of glycine or GABA(A) receptors antagonists: bicuculline-resistant, fast-decaying GlyR-mediated mIPSCs, and strychnine-resistant, slow-decaying GABA(A)R-mediated mIPSCs. The frequency of GABA(A)R-mediated mIPSCs was fourfold higher than that of GlyR-mediated mIPSCs at E17-18, indicating that GABAergic synaptic sites were functionally dominant at early stages of neural network formation. Properties of GABA(A)R-mediated mIPSC amplitude fluctuations changed from primarily unimodal skewed distribution at E17-18 to Gaussian mixtures with two to three discrete components at P1-3. A developmental shift from primarily long-duration GABAergic mIPSCs to short-duration glycinergic mIPSCs was evident after birth, when the frequency of GlyR-mediated mIPSCs increased 10-fold. This finding suggested that either the number of glycinergic synapses or the probability of vesicular glycine release increased during the period studied. The increased frequency of GlyR-mediated mIPSCs was associated with more than a twofold increase in their mean amplitude, and in the number of motoneurons in which mIPSC amplitude fluctuations were best fitted by multi-component Gaussian curves. A third subpopulation of mIPSCs was apparent in the absence of glycine and GABA(A) receptor antagonists: mIPSCs with both fast and slow decaying components. Based on their dual-component decay time and their suppression by either strychnine or bicuculline, we assumed that these were generated by the activation of co-localized postsynaptic glycine and GABA(A) receptors. The contribution of mixed glycine-GABA synaptic sites to the generation of mIPSCs did not change after birth. The developmental switch from predominantly long-duration GABAergic inhibitory synaptic currents to short-duration glycinergic currents might serve as a mechanism regulating neuronal excitation in the developing spinal networks.     

Inhibitory synapses in the developing auditory system are glutamatergic

Activity-dependent synapse refinement is crucial for the formation of precise excitatory and inhibitory neuronal circuits. Whereas the mechanisms that guide refinement of excitatory circuits are becoming increasingly clear, the mechanisms guiding inhibitory circuits have remained obscure. In the lateral superior olive (LSO), a nucleus in the mammalian sound localization system that receives inhibitory input from the medial nucleus of the trapezoid body (MNTB), specific elimination and strengthening of synapses that are both GABAergic and glycinergic (GABA/glycinergic synapses) is essential for the formation of a precise tonotopic map. We provide evidence that immature GABA/glycinergic synapses in the rat LSO also release the excitatory neurotransmitter glutamate, which activates postsynaptic NMDA receptors (NMDARs). Immunohistochemical studies demonstrate synaptic colocalization of the vesicular glutamate transporter 3 with the vesicular GABA transporter, indicating that GABA, glycine and glutamate are released from single MNTB terminals. Glutamatergic transmission at MNTB-LSO synapses is most prominent during the period of synapse elimination. Synapse-specific activation of NMDARs by glutamate release at GABAergic and glycinergic synapses could be important in activity-dependent refinement of inhibitory circuits.     

Distinct profiles of refilling of inhibitory neurotransmitters into presynaptic terminals projecting to spinal neurones in immature rats

Corelease of glycine and GABA from the single synaptic terminal (synaptic bouton) is well accepted in immature rat spinal cord and brainstem. However, it raises the question of how glycine and GABA are accumulated in the same synaptic vesicles and coreleased. To address this issue, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and focally evoked IPSCs (eIPSCs) mediated via a single synapse were recorded from synaptic bouton preparations of the rat immature sacral dorsal commissural nucleus (SDCN) neurones by whole-cell patch recording. Focal stimulation of a single synaptic bouton revealed that three different quantal releases occur from a single synaptic bouton: i.e. pure glycine, pure GABA, and mixed. Prolonged treatment with bafilomycin A1, a vacuolar-type H⁺/ATPase inhibitor, to the SDCN neurone greatly suppressed frequency and amplitude of the mIPSCs. During washing out of bafilomycin A1, complete recovery in the amplitude of glycinergic mIPSCs was observed, while that of GABAergic and mixed mIPSCs was incomplete. These observations indicate that three types of vesicles coexist in single synaptic terminals, and that refilling of glycine into the synaptic vesicle predominantes over GABA after pretreatment with bafilomycin A1 in immature rats. This could be explained by the decrease in the cytosolic concentration of GABA, or by the presence of subtypes of vesicular inhibitory amino acid transporter in the synaptic vesicle membrane.     

GABA(B) modulation of GABA(A) and glycine receptor-mediated synaptic currents in hypoglossal motoneurons

We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.     

Inhibitory synaptogenesis in the rat anteroventral cochlear nucleus

Spherical cells in the anteroventral division of the cochlear nucleus, which relay excitatory inputs from the auditory nerve, also receive both GABAergic and glycinergic inhibitory synapses. Inhibition mediated by GABA and glycine fulfils essential roles in the processing abilities of these and other auditory neurons. However, the developmental program leading to a mature complement of GABAergic and glycinergic synapses and microcircuits is largely unknown. Because of their relatively simple geometry, spherical cells provide an excellent model for unraveling basic developmental patterns of inhibitory synaptogenesis. Using a combination of high resolution immunocytochemical methods, we report that, in the rat, synapses containing GABA or glycine are deployed on spherical cell bodies over a time period extending well beyond hearing onset. Such postnatal developmental recruitment of inhibitory endings is progressive, although there are two distinct leaps in their numbers. The first occurs by the end of the first postnatal week, prior to hearing onset, and the second, during the third postnatal week, after hearing onset. This pattern suggests that adjustments in inhibition could be driven by acoustic experience. While GABAergic and glycinergic endings are maturing and growing in number and size, their neurotransmitter content also appears to be developmentally regulated. Quantitative ultrastructural immunocytochemistry with colloidal gold suggests that GABA and glycine accumulation in synaptic endings follows a staggered pattern, with labeling stabilizing at adult levels by postnatal day 21. This may account for adjustments in synaptic efficacy and strength.     

Differential effects of ethanol on GABA(A) and glycine receptor-mediated synaptic currents in brain stem motoneurons

Ethanol potentiates glycinergic synaptic transmission to hypoglossal motoneurons (HMs). This effect on glycinergic transmission changes with postnatal development in that juvenile HMs (P9-13) are more sensitive to ethanol than neonate HMs (P1-3). We have now extended our previous study to investigate ethanol modulation of synaptic GABA(A) receptors (GABA(A)Rs), because both GABA and glycine mediate inhibitory synaptic transmission to brain stem motoneurons. We tested the effects of ethanol on GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from neonate and juvenile rat HMs in an in vitro slice preparation. Bath application of 30 mM ethanol had no significant effect on the GABAergic mIPSC amplitude or frequency recorded at either age. At 100 mM, ethanol significantly decreased the GABAergic mIPSC amplitude recorded from neonate (6 +/- 3%, P < 0.05) and juvenile (16 +/- 3%, P < 0.01) HMs. The same concentration of ethanol increased the GABAergic mIPSC frequency recorded from neonate (64 +/- 17%, P < 0.05) and juvenile (40 +/- 15%, n.s.) HMs. In contrast, 100 mM ethanol robustly potentiated glycinergic mIPSC amplitude in neonate (31 +/- 3%, P < 0.0001) and juvenile (41 +/- 7%, P < 0.001) HMs. These results suggest that glycine receptors are more sensitive to modulation by ethanol than GABA(A) receptors and that 100 mM ethanol has the opposite effect on GABA(A)R-mediated currents in juvenile HMs, that is, inhibition rather than enhancement. Further, comparing ethanol's effects on GABAergic mIPSC amplitude and frequency, ethanol modulates GABAergic synaptic transmission to HMs differentially. Presynaptically, ethanol enhances mIPSC frequency while postsynaptically it decreases mIPSC amplitude.     

Contribution of single-channel properties to the time course and amplitude variance of quantal glycine currents recorded in rat motoneurons

The amplitude of spontaneous, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in hypoglossal motoneurons (HMs) in an in vitro brain stem slice preparation increased over the first 3 postnatal weeks, from 42 +/- 6 pA in neonate (P0-3) to 77 +/- 11 pA in juvenile (P11-18) HMs. Additionally, mIPSC amplitude distributions were highly variable: CV 0.68 +/- 0.05 (means +/- SE) for neonates and 0.83 +/- 0.06 for juveniles. We wished to ascertain the contribution of glycine receptor (GlyR)-channel properties to this change in quantal amplitude and to the amplitude variability and time course of mIPSCs. To determine whether a postnatal increase in GlyR-channel conductance accounted for the postnatal change in quantal amplitude, the conductance of synaptic GlyR channels was determined by nonstationary variance analysis of mIPSCs. It was 48 +/- 8 pS in neonate and 46 +/- 10 pS in juvenile HMs, suggesting that developmental changes in mIPSC amplitude do not result from a postnatal alteration of GlyR-channel conductance. Next we determined the open probability (Popen) of GlyR channels in outside-out patches excised from HMs to estimate the contribution of stochastic channel behavior to quantal amplitude variability. Brief (1 ms) pulses of glycine (1 mM) elicited patch currents that closely resembled mIPSCs. The GlyR channels' Popen, calculated by nonstationary variance analysis of these currents, was approximately 0.70 (0.66 +/- 0.09 in neonates and 0.72 +/- 0.05 in juveniles). The decay rate of patch currents elicited by brief application of saturating concentrations of glycine (10 mM) increased postnatally, mimicking previously documented changes in mIPSC time course. Paired pulses of glycine (10 mM) were used to determine if rapid GlyR-channel desensitization contributed to either patch current time course or quantal amplitude variability. Because we did not observe any fast desensitization of patch currents, we believe that fast desensitization of GlyRs underlies neither phenomenon. From our analysis of glycinergic patch currents and mIPSCs, we draw three conclusions. First, channel deactivation is the primary determinant of glycinergic mIPSC time course, and postnatal changes in channel deactivation rate account for observed developmental changes in mIPSC decay rate. Second, because GlyR-channel Popen is high, differences in receptor number between synapses rather than stochastic channel behavior are likely to underlie the majority of quantal variability seen at glycinergic synapses throughout postnatal development. We estimate the number of GlyRs available at a synapse to be on average 27 in neonate neurons and 39 in juvenile neurons. Third, this change in the calculated number of GlyRs at each synapse may account for the postnatal increase in mIPSC amplitude.     

Pre- and postsynaptic properties of glutamatergic transmission in the immature inhibitory MNTB-LSO pathway

The lateral superior olive (LSO) integrates excitatory inputs driven by sound arriving at the ipsilateral ear with inhibitory inputs driven by sound arriving at the contralateral ear in order to compute interaural intensity differences needed for localizing high-frequency sound sources. Specific mechanisms necessary for developmental refinement of the inhibitory projection, which arises from the medial nucleus of the trapezoid body (MNTB), have only been partially deciphered. The demonstration that immature MNTB-LSO synapses release glutamate has led to a model in which early glutamate neurotransmission plays a major role in inhibitory plasticity. We used whole cell electrophysiology in acute auditory brain stem slices of neonatal rats to examine glutamatergic transmission in the developing MNTB-LSO pathway. Unexpectedly, AMPA receptor (AMPAR)-mediated responses were prevalent at the earliest ages. We found a salient developmental profile for NMDA receptor (NMDAR) activation, described both by the proportion of total glutamate current and by current durations, and we found evidence for distinct release probabilities for GABA/glycine and glutamate in the MNTB-LSO pathway. The developmental profile of NMDAR is consistent with the possibility that the inhibitory MNTB-LSO pathway experiences a sensitive period, driven by cochlear activity and mediated by GluN2B-containing NMDARs, between postnatal days 3 and 9. Differing neurotransmitter release probabilities could allow the synapse to switch between GABA/glycinergic transmission and mixed glutamate/GABA/glycinergic transmission in response to changing patterns of spiking activity.     

Excitatory action of an immature glycinergic/GABAergic sound localization pathway

Most mammals determine the azimuthal direction of incoming sound using auditory cues arising from differences in interaural sound intensity. The first station in the ascending auditory pathway, which processes interaural intensity differences, is the lateral superior olive (LSO), a binaural nucleus in the auditory brainstem. LSO neurons encode interaural intensity differences by integrating excitatory input from the ipsilateral cochlea and inhibitory input from the contralateral cochlea. Both inputs converge on single neurons in a highly organized, frequency-specific manner. The correct development of the precise arrangement of these inputs and their physiological properties depends on neuronal activity. Previous studies have shown that inhibitory, glycinergic/GABAergic inputs to the LSO are transiently depolarizing, and it has been hypothesized that this depolarizing action enables developing inhibitory inputs to act as excitatory inputs. In support of this hypothesis, we recently demonstrated that depolarizing glycinergic/GABAergic inputs can increase the intracellular calcium concentration in immature LSO neurons and elicit action potentials. These results provide support for the notion that the influence of glycinergic/GABAergic synaptic activity on development of the LSO involves calcium-dependent signaling mechanisms.     

Inhibitory synaptic transmission differs in mouse type A and B medial vestibular nucleus neurons in vitro

Fast inhibitory synaptic transmission in the medial vestibular nucleus (MVN) is mediated by GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs). To assess their relative contribution to inhibition in the MVN, we recorded miniature inhibitory postsynaptic currents (mIPSCs) in physiologically characterized type A and type B MVN neurons. Transverse brain stem slices were prepared from mice (3-8 wk old), and whole cell patch-clamp recordings were obtained from visualized MVN neurons (CsCl internal; Vm = -70 mV; 23 degrees C). In 81 MVN neurons, 69% received exclusively GABA(A)ergic inputs, 6% exclusively glycinergic inputs, and 25% received both types of mIPSCs. The mean amplitude of GABA(A)R-mediated mIPSCs was smaller than those mediated by GlyRs (22.6 +/- 1.8 vs. 35.3 +/- 5.3 pA). The rise time and decay time constants of GABA(A)R- versus GlyR-mediated mIPSCs were slower (1.3 +/- 0.1 vs. 0.9 +/- 0.1 ms and 10.5 +/- 0.3 vs. 4.7 +/- 0.3 ms, respectively). Comparison of type A (n = 20) and type B (n = 32) neurons showed that type A neurons received almost exclusively GABA(A)ergic inhibitory inputs, whereas type B neurons received GABA(A)ergic inputs, glycinergic inputs, or both. Intracellular labeling in a subset of MVN neurons showed that morphology was not related to a MVN neuron's inhibitory profile (n = 15), or whether it was classified as type A or B (n = 29). Together, these findings indicate that both GABA and glycine contribute to inhibitory synaptic processing in MVN neurons, although GABA dominates and there is a difference in the distribution of GABA(A) and Gly receptors between type A and type B MVN neurons.     

GABAergic and glycinergic inhibition modulate monaural auditory response properties in the avian superior olivary nucleus

The superior olivary nucleus (SON) is the primary source of inhibition in the avian auditory brainstem. While much is known about the role of inhibition at the SON's target nuclei, little is known about how the SON itself processes auditory information or how inhibition modulates these properties. Additionally, the synaptic physiology of inhibitory inputs within the SON has not been described. We investigated these questions using in vivo and in vitro electrophysiological techniques in combination with immunohistochemistry in the chicken, an organism for which the auditory brainstem has otherwise been well characterized. We provide a thorough characterization of monaural response properties in the SON and the influence of inhibitory input in shaping these features. We found that the SON contains a heterogeneous mixture of response patterns to acoustic stimulation and that in most neurons these responses are modulated by both GABAergic and glycinergic inhibitory inputs. Interestingly, many SON neurons tuned to low frequencies have robust phase-locking capability and the precision of this phase locking is enhanced by inhibitory inputs. On the synaptic level, we found that evoked and spontaneous inhibitory postsynaptic currents (IPSCs) within the SON are also mediated by both GABAergic and glycinergic inhibition in all neurons tested. Analysis of spontaneous IPSCs suggests that most SON cells receive a mixture of both purely GABAergic terminals, as well as terminals from which GABA and glycine are coreleased. Evidence for glycinergic signaling within the SON is a novel result that has important implications for understanding inhibitory function in the auditory brainstem.     

Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons

Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.     

Development of synaptic transmission to respiratory motoneurons

Respiratory motoneurons provide the exclusive drive to respiratory muscles and therefore are a key relay between brainstem neural circuits that generate respiratory rhythm and respiratory muscles that control moment of gases into and out of the airways and lungs. This review is focused on postnatal development of fast ionotropic synaptic transmission to respiratory motoneurons, with a focus on hypoglossal motoneurons (HMs). Glutamatergic synaptic transmission to HMs involves activation of both non-NMDA and NMDA receptors and during the postnatal period co-activation of these receptors located at the same synapse may occur. Further, the relative role of each receptor type in inspiratory-phase motoneuron depolarization is dependent on the type of preparation used (in vitro versus in vivo; neonatal versus adult). Respiratory motoneurons receive both glycinergic and GABAergic inhibitory synaptic inputs. During inspiration phrenic and HMs receive concurrent excitatory and inhibitory synaptic inputs. During postnatal development in HMs GABAergic and glycinergic synaptic inputs have slow kinetics and are depolarizing and with postnatal development they become faster and hyperpolarizing. Additionally shunting inhibition may play an important role in synaptic processing by respiratory motoneurons.     

Development of chloride-mediated inhibition in neurons of the anteroventral cochlear nucleus of gerbil (Meriones unguiculatus)

At the initial stages in neuronal development, GABAergic and glycinergic neurotransmission exert depolarizing responses, assumed to be of importance for maturation, which in turn shift to hyperpolarizing in early postnatal life due to development of the chloride homeostasis system. Spherical bushy cells (SBC) of the mammalian cochlear nucleus integrate excitatory glutamatergic inputs with inhibitory (GABAergic and glycinergic) inputs to compute signals that contribute to sound localization based on interaural time differences. To provide a fundamental understanding of the properties of GABAergic neurotransmission in mammalian cochlear nucleus, we investigated the reversal potential of the GABA-evoked currents (E GABA) by means of gramicidin-perforated-patch recordings in developing SBC. The action of GABA switches from depolarizing to hyperpolarizing by the postnatal day 7 due to the negative shift in E GABA. Furthermore, we studied the expression pattern of the K+-Cl(-)-extruding cotransporter KCC2, previously shown to induce a switch from neonatal Cl(-) efflux to the mature Cl(-) influx in various neuron types, thereby causing a shift from depolarizing to hyperpolarizing GABA action. The KCC2 protein is expressed in SBC already at birth, yet its activity is attained toward the end of the first postnatal week as indicated by pharmacological inhibition. Interruption of the Cl(-) extrusion by [(dihydroindenyl)oxy] alkanoic acid or furosemide gradually shifted E(GABA) in positive direction with increasing maturity, suggesting that KCC2 could be involved in maintaining low [Cl(-)]i after the postnatal day 7 thereby providing the hyperpolarizing Cl(-)-mediated inhibition in SBC.     

Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex

Previous work demonstrates that the cerebellum uses glycine as a fast inhibitory neurotransmitter [Ottersen OP, Davanger S, Storm-Mathisen J (1987) Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon, Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake. Exp Brain Res 66(1):211-221; Ottersen OP, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450(1-2):342-353; Dieudonne S (1995) Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci U S A 92:1441-1445; Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057; Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498; Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123-141]. In the rat cerebellum glycine is not released by itself but is released together with GABA by Lugaro cells onto Golgi cells [Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057] and by Golgi cells onto unipolar brush and granule cells [Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498]. Here we report, from immunolabeling evidence in Macaca cerebellum, that interneurons in the granular cell layer are glycine+ at a density of 120 cells/linear mm. Their morphology indicates that they include Golgi and Lugaro cell types with the majority containing both glycine and GABA or glutamic acid decarboxylase. These data are consistent with the proposal that, as in the rat cerebellum, these granular cell layer interneurons corelease glycine and GABA in the primate cerebellum. The patterns of labeling for glycine and GABA within Golgi and Lugaro cells also indicate that there are biochemical sub-types which are morphologically similar. Further, we find that glycine, GABA and glutamic acid decarboxylase identified candelabrum cells adjacent to the Purkinje cells which is the first time that this interneuron has been reported in primate cerebellar cortex. We propose that candelabrum cells, like the majority of Golgi and Lugaro cells, release both glycine and GABA.     

Protracted postnatal development of inhibitory synaptic transmission in rat hippocampal area CA1 neurons

In the CNS, inhibitory synaptic function undergoes profound transformation during early postnatal development. This is due to variations in the subunit composition of subsynaptic GABA(A) receptors (GABA(A)Rs) at differing developmental stages as well as other factors. These include changes in the driving force for chloride-mediated conductances as well as the quantity and/or cleft lifetime of released neurotransmitter. The present study was undertaken to investigate the nature and time course of developmental maturation of GABAergic synaptic function in hippocampal CA1 pyramidal neurons. In neonatal [postnatal day (P) 1-7] and immature (P8-14) CA1 neurons, miniature inhibitory postsynaptic currents (mIPSCs) were significantly larger, were less frequent, and had slower kinetics compared with mIPSCs recorded in more mature neurons. Adult mIPSC kinetics were achieved by the third postnatal week in CA1 neurons. However, despite this apparent maturation of mIPSC kinetics, significant differences in modulation of mIPSCs by allosteric agonists in adolescent (P15-21) neurons were still evident. Diazepam (1-300 nM) and zolpidem (200 nM) increased the amplitude of mIPSCs in adolescent but not adult neurons. Both drugs increased mIPSC decay times equally at both ages. These differential agonist effects on mIPSC amplitude suggest that in adolescent CA1 neurons, inhibitory synapses operate differently than adult synapses and function as if subsynaptic receptors are not fully occupied by quantal release of GABA. Rapid agonist application experiments on perisomatic patches pulled from adolescent neurons provided additional support for this hypothesis. In GABA(A)R currents recorded in these patches, benzodiazepine amplitude augmentation effects were evident only when nonsaturating GABA concentrations were applied. Furthermore nonstationary noise analysis of mIPSCs in P15-21 neurons revealed that zolpidem-induced mIPSC augmentation was not due to an increase in single-channel conductance of subsynaptic GABA(A)Rs but rather to an increase in the number of open channels responding to a single GABA quantum, further supporting the hypothesis that synaptic receptors may not be saturated during synaptic function in adolescent neurons. These data demonstrate that inhibitory synaptic transmission undergoes a markedly protracted postnatal maturation in rat CA1 pyramidal neurons. In the first two postnatal weeks, mIPSCs are large in amplitude, are slow, and occur infrequently. By the third postnatal week, mIPSCs have matured kinetically but retain distinct responses to modulatory drugs, possibly reflecting continued immaturity in synaptic structure and function persisting through adolescence.     

Development of presynaptic inhibition onto retinal bipolar cell axon terminals is subclass-specific.

Synaptic integration is modulated by inhibition onto the dendrites of postsynaptic cells. However, presynaptic inhibition at axonal terminals also plays a critical role in the regulation of neurotransmission. In contrast to the development of inhibitory synapses onto dendrites, GABAergic/glycinergic synaptogenesis onto axon terminals has not been widely studied. Because retinal bipolar cells receive subclass-specific patterns of GABAergic and glycinergic presynaptic inhibition, they are a good model for studying the development of inhibition at axon terminals. Here, using whole cell recording methods and transgenic mice in which subclasses of retinal bipolar cells are labeled, we determined the temporal sequence and patterning of functional GABAergic and glycinergic input onto the major subclasses of bipolar cells. We found that the maturation of GABAergic and glycinergic synapses onto the axons of rod bipolar cells (RBCs), on-cone bipolar cells (ON-CBCs) and off-cone bipolar cells (OFF-CBCs) were temporally distinct: spontaneous chloride-mediated currents are present in RBCs earlier in development compared with ON- and OFF-CBC, and RBCs receive GABAergic and glycinergic input simultaneously, whereas in OFF-CBCs, glycinergic transmission emerges before GABAergic transmission. Because on-CBCs show little inhibitory activity, GABAergic and glycinergic events could not be pharmacologically distinguished for these bipolar cells. The balance of GABAergic and glycinergic input that is unique to RBCs and OFF-CBCs is established shortly after the onset of synapse formation and precedes visual experience. Our data suggest that presynaptic modulation of glutamate transmission from bipolar cells matures rapidly and is differentially coordinated for GABAergic and glycinergic synapses onto distinct bipolar cell subclasses.