J. Neurochem.(2012) 122, 900-910. ABSTRACT: GABAergic transmission in the neostriatum plays a central role in motor coordination, in which a plethora of GABA-A receptor subunits combine to modulate neural inhibition. GABAρ receptors were originally described in the mammalian retina. These receptors possess special electrophysiological and pharmacological properties, forming a characteristic class of ionotropic receptors. In previous studies, we suggested that GABAρ receptors are expressed in the neostriatum, and in this report we show that they are indeed present in all the calretinin-positive interneurons of the neostriatum. In addition, they are located in calbindin-positive interneurons and projection neurons that express the dopamine D(2) receptor. GABAρ receptors were also located in 30% of the glial fibrillary acidic protein-positive cells, and may therefore also contribute to gliotransmission. Quantitative reverse transcription-PCR suggested that the mRNAs of this receptor do not express as much as in the retina, and that GABAρ2 is more abundant than GABAρ1. Electrophysiological recordings in brain slices provided evidence of neurons expressing a cis-4-aminocrotonic acid-activated, 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid-sensitive ionotropic GABA receptor, indicating the presence of functional GABAρ receptors in the neostriatum. Finally, electron-microscopy and immunogold located the receptors mainly in perisynaptic as well as in extrasynaptic sites. All these observations reinforce the importance of GABAρ receptors in the neostriatum and contribute to the diversity of inhibitory regulation in this area.

A bicuculline-resistant and TPMPA-sensitive GABAergic component was identified in hippocampal neurons in culture and in acute isolated brain slices. In both preparations, total GABAergic activity showed two inactivation kinetics: fast and slow. RT-PCR, in situ hybridization (ISH) and immunohistochemistry detected expression of GABAρ subunits. Immunogold and electron microscopy indicated that the receptors are mostly extrasynaptic. In addition, by RT-PCR and immunofluorescence we found GABAρ present in amygdala and visual cortex.

Gamma-aminobutyric acid (GABA)ρ receptors are selectively targeted to the axon terminals of the retinal bipolar neurons. The traffic of a green fluorescent protein-tagged GABAρ2 was examined in retinal bipolar neurons and cerebellar astrocytes. In bipolar neurons, time-lapse laser confocal microscopy revealed that the fluorescence emitted by GABAρ2-green fluorescent protein accumulates first, in clusters, in the soma and is then distributed along the axon in at least two populations: one that remains relatively immobile and a second population of smaller clusters that moved constantly to and from the axon end. In astrocytes, the fluorescent clusters were relatively immobile and located mainly in the soma.

In the present study, we provide evidence for the expression of all three GABA(C) receptor rho subunits through development of the rat cerebellum. Injection of cerebellum mRNA into frog oocytes gave rise to the expression of both GABA(A) and GABA(C) receptors. qRT-PCR of RNA isolated from postnatal developing cerebella showed that the expression of each rho subunit is relatively low, with a relative comparative expression of rho3>rho1>rho2. In situ hybridization and immunohistochemistry revealed a limited distribution of GABA(C) receptors in the Purkinje and Golgi neurons whereas electron microscopy detected the rho1 and rho2 subunits in the soma and dendritic tree of the Purkinje cells. The expression of GABA(C) receptors in the cerebellum adds a new dimension to the regulation of GABAergic neurotransmission and suggests further experiments to determine their functional consequences.

GABA(C) receptors were originally found in the mammalian retina and recent evidence shows that they are also expressed in several areas of the brain, including caudate nucleus, brain stem, pons and corpus callosum. In this study, plasma membranes from the caudate nucleus were microinjected into X. laevis oocytes. This led the oocyte plasma membrane to incorporate functional bicuculline-resistant, Cl(-) conducting bovine GABA receptors, similar to those of the retina. Immunolocalization of the GABA rho1 subunit revealed its expression in bovine neurons in the head of the caudate as well as in the olive, cuneiform and reticular nuclei of the brain stem. The same antibodies failed to show expression in the callosum and pons, where the GABA rho1 mRNA was previously detected. The cloned GABA rho1 sequence predicts a protein with 473 amino acids and 74-93% similarity to other GABA rho1 subunits. Oocytes injected with the cDNA express a non-desensitizing, homomeric receptor with a GABA EC(50)=6.0muM and a Hill coefficient of 1.8. The results confirm the presence of GABA(C) receptor mRNAs in several areas of the mammalian brain and show that some of these areas express functional GABA rho1 receptors that have the classic GABA(C) receptor characteristics.

GABAρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after postnatal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABAρ1 gene, includes putative transcription factor binding sites, two Inr elements, and it lacks a TATA-box. A RACE assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in STC-1 cells and retina. Finally, the 0.232-kb fragment drives GFP expression to the inner nuclear layer, where bipolar cells are present. © 2012 International Society for Neurochemistry, J. Neurochem.(2012) 10.1111/jnc.12067.

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

GABAρ1 receptors are highly expressed in bipolar neurons of the retina and to a lesser extent in several areas of the central nervous system (CNS), and dopamine and serotonin are also involved in the modulation of retinal neural transmission. Whether these biogenic amines have a direct effect on ionotropic GABA receptors was not known. Here, we report that GABAρ1 receptors, expressed in X. laevis oocytes, were negatively modulated by dopamine and serotonin and less so by octopamine and tyramine. Interestingly, these molecules did not have effects on GABA(A) receptors. 5-Carboxamido-tryptamine and apomorphine did not exert evident effects on any of the receptors. Schild plot analyses of the inhibitory actions of dopamine and serotonin on currents elicited by GABA showed slopes of 2.7 ± 0.3 and 6.1 ± 1.8, respectively, indicating a noncompetitive mechanism of inhibition. The inhibition of GABAρ1 currents was independent of the membrane potential and was insensitive to picrotoxin, a GABA receptor channel blocker and to the GABAρ-specific antagonist (1,2,5,6-tetrahydropyridine-4-yl)methyl phosphinic acid (TPMPA). Dopamine and serotonin changed the sensitivity of GABAρ1 receptors to the inhibitory actions of Zn(2+). In contrast, La(3+) potentiated the amplitude of the GABA currents generated during negative modulation by dopamine (EC(50) 146 μM) and serotonin (EC(50) 196 μM). The functional role of the direct modulation of GABAρ receptors by dopamine and serotonin remains to be elucidated; however, it may represent an important modulatory pathway in the retina, where GABAρ receptors are highly expressed and where these biogenic amines are abundant.

Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at -60 mV revealed that ATP (20-250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ~60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ~5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at -60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known F(in) and S(in)-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using whole-cell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ~16% of the FC held at -60 mV. The net Ang II response, mediated by the activation of the AT(1) receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes.

The Casiopeínas® are mixed chelate copper (II) complexes and promising antineoplastics agents against cancer cells and tumors in vitro and in vivo. However, the action mode of these compounds is poorly characterized. In this work the effect of the antineoplastic Casiopeína IIIEa on the metabolism and ultrastructure of the yeast Saccharomyces cerevisiae was investigated. Exposure of cells growing in rich or in low-iron medium to 5 μM of the compound decreased duplication time and reduced oxygen consumption. Those cells formed smaller colonies when growing in a non-fermentable carbon source and low-iron medium, and under the light microscope, multiple folds were observed along the plasma membrane accompanied with a reduction in the diameter of the yeast. These observations were confirmed under the electron microscope, which also revealed a slight reduction of the mitochondrial size. A correlation was found with smaller colonies exhibiting lower rates of oxygen consumption, and yeast labelled with fluorescent MitoTracker(TM) consistently exhibited reduced mitochondrial activity. It appears that Casiopeína IIIEa gives rise to smaller yeast and petite-like colonies by reducing the mitochondrial respiratory activity without significantly affecting the mitochondrial structure.

The properties of glycine receptors (GlyRs) depend upon their subunit composition. While the prevalent adult forms of GlyRs are heteromers, previous reports suggested functional α homomeric receptors in mature nervous tissues. Here we show two functionally different GlyRs populations in the rat medial nucleus of trapezoid body (MNTB). Postsynaptic receptors formed α1/β-containing clusters on somatodendritic domains of MNTB principal neurons, colocalizing with glycinergic nerve endings to mediate fast, phasic IPSCs. In contrast, presynaptic receptors on glutamatergic calyx of Held terminals were composed of dispersed, homomeric α1 receptors. Interestingly, the parent cell bodies of the calyces of Held, the globular bushy cells of the cochlear nucleus, expressed somatodendritic receptors (α1/β heteromers) and showed similar clustering and pharmacological profile as GlyRs on MNTB principal cells. These results suggest that specific targeting of GlyR β-subunit produces segregation of GlyR subtypes involved in two different mechanisms of modulation of synaptic strength.

Early onset long term depression (LTD) during the first postnatal week has rarely been demonstrated at the medial nucleus of trapezoid body (MNTB)-lateral superior olive (LSO) synapses in spite of many favorable conditions, such as depolarizing synapses and glutamate co-release from MNTB terminals. Thus, we tested the early expression of LTD at MNTB-LSO synapses during the first postnatal week using circling mice, whose main transmitter is glutamate at MNTB-LSO synapses. Tetanic stimulation on MNTB elicited LTD of postsynaptic currents recorded at LSO neurons in P0∼P3 homozygous (cir/cir) mice (45.8±0.3% of the control, n=7) and heterozygous (+/cir) mice (43.3±0.4% of the control, n=7). The magnitude of LTD decreased in P8 ∼ P12 heterozygous (+/cir) mice (84.5±0.3% of the control, n=7), but was maintained in P8 ∼P12 homozygous (cir/cir) mice (38.2±0.3% of the control, n=9). Glutamatergic LTD observed in homozygous (cir/cir) mice and glycinergic LTD observed heterozygous (+/cir) mice showed similar pattern of change. As currents induced by the pressure application of glycine on LSO neurons were reduced by tetanic stimulation in P0∼P3 heterozygous (+/cir) mice, LTD was thought to occur at postsynaptic sites. Our results suggest that LTD might occur in vivo and participate in the synaptic silencing and strengthening of MNTB-LSO synapses, which is most active during the first postnatal week.

The organization of developing auditory circuits depends on the elimination of aberrant connections and strengthening of appropriate ones. Endocannabinoid mediated plasticity is a proposed mechanism for this refinement. Here we investigated for the anatomical presence of cannabinoid receptors (CB1R) in the lateral superior olive (LSO) and medial nucleus of the trapezoid body (MNTB) of developing rats. We found that CB1R is present within the LSO and that it colocalized with vesicular glutamate transporter (VGLUT3), a presynaptic marker for MTNB terminals. Both before (P5) and around hearing onset (P12), the expression levels of CB1R were higher in the lateral limb of the LSO than in the medial limb. These results suggest that endocannabinoid signaling can modulate the strength of the developing MNTB-LSO synapse.

The calyx of Held is an axosomatic terminal in the auditory brainstem that has attracted anatomists because of its giant size and physiologists because of its accessibility to patch-clamp recordings. The calyx allows the principal neurons in the medial nucleus of the trapezoid body (MNTB) to provide inhibition that is both well timed and sustained to many other auditory nuclei. The special adaptations that allow the calyx to drive its principal neuron even when frequencies are high include a large number of release sites with low release probability, a large readily releasable pool, fast presynaptic calcium clearance and little delayed release, a large quantal size, and fast AMPA-type glutamate receptors. The transformation from a synapse that is unremarkable except for its giant size into a fast and reliable auditory relay happens in just a few days. In rodents this transformation is essentially ready when hearing starts. Expected final online publication date for the Annual Review of Physiology Volume 74 is February 11, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

It has been demonstrated that kanamycin treatment during early developmental period induces partial cochlear destruction and enhanced glutamatergic transmission at the medial nucleus of the trapezoid body (MNTB)- the lateral superior olive (LSO) synapses in the superior olivary complex (SOC). As c-fos was expected to be expressed in the SOC by kanamycin-induced cochlear damage, the expression of c-fos protein (Fos) was investigated using immunohistochemistry in kanamycin-treated rat pups. In the control rat pups less than postnatal (P) day 9 in age, Fos-like immunoreactivity (Fos-IR) was transiently observed in the MNTB and LSO on P6, but disappeared on P9, which reflects a physiologic process. In contrast, in kanamycin-treated rats, Fos-IR was consistently observed through P9. Because a significant increase in terminal uridine deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) and glial fibrillary acidic protein (GFAP) IR was not demonstrated in the MNTB and LSO of kanamycin-treated rats, the increased Fos-IR does not appear to indicate an ongoing pathologic process, but may be related to the increased activity caused by the disturbance in excitatory and inhibitory balance between brainstem auditory circuits.

The calyx of Held synapse is a giant axosomatic synapse that has a fast relay function within the sound localization circuit of the brainstem. In the adult, each principal neuron of the medial nucleus of the trapezoid body (MNTB) is contacted by a single calyx terminal. In rodents, the calyx of Held synapse forms around the third postnatal day (P3). Here, we studied the developmental changes in the intrinsic excitability of the principal neurons during the first postnatal week by making whole-cell recordings from brainstem slices. In slices from P0-1 rats, about 20% of the principal neurons were spontaneously active, whereas after P3, no spontaneously active cells were observed. Already at P0, principal neurons received both glutamatergic and GABAergic/glycinergic inputs. The occurrence of spontaneous action potentials depended upon the presence of spontaneous glutamatergic inputs; summation of only a few quanta was enough to reach action potential threshold. The main cause for this high excitability was a high resting membrane resistance, which decreased at least four-fold during the first postnatal week. A relatively slow decay of synaptic currents and a relatively depolarized membrane potential may have contributed as well. We conclude that the decrease in the excitability of principal neurons in the MNTB matches the increase of the strength of the synaptic inputs resulting from the formation and maturation of the calyx of Held synapse during the first postnatal week. This decrease in excitability will make it progressively more difficult for non-calyceal inputs to trigger action potentials.

There is a well-established link between hyperbilirubinaemia and hearing loss in paediatrics, but the cellular mechanisms have not been elucidated. Here we used the Gunn rat model of hyperbilirubinaemia to investigate bilirubin-induced hearing loss. In vivo auditory brainstem responses revealed that Gunn rats have severe auditory deficits within 18 h of exposure to high bilirubin levels. Using an in vitro preparation of the auditory brainstem from these rats, extracellular multi-electrode array recording from the medial nucleus of the trapezoid body (MNTB) showed longer latency and decreased amplitude of evoked field potentials following bilirubin exposure, suggestive of transmission failure at this synaptic relay. Whole-cell patch-clamp recordings confirmed that the electrophysiological properties of the postsynaptic MNTB neurons were unaffected by bilirubin, with no change in action potential waveforms or current-voltage relationships. However, stimulation of the trapezoid body was unable to elicit large calyceal EPSCs in MNTB neurons of hyperbilirubinaemic rats, indicative of damage at a presynaptic site. Multi-photon imaging of anterograde-labelled calyceal projections revealed axonal staining and presynaptic profiles around MNTB principal neuron somata. Following induction of hyperbilirubinaemia the giant synapses were largely destroyed. Electron microscopy confirmed loss of presynaptic calyceal terminals and supported the electrophysiological evidence for healthy postsynaptic neurons. MNTB neurons express high levels of neuronal nitric oxide synthase (nNOS). Nitric oxide has been implicated in mechanisms of bilirubin toxicity elsewhere in the brain, and antagonism of nNOS by 7-nitroindazole protected hearing during bilirubin exposure. We conclude that bilirubin-induced deafness is caused by degeneration of excitatory synaptic terminals in the auditory brainstem.

Elimination of the Kv1.3 voltage-dependent potassium channel gene produces striking changes in the function of the olfactory bulb, raising the possibility that this channel also influences other sensory systems. We have examined the cellular and subcellular localization of Kv1.3 in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, a nucleus in which neurons fire at high rates with high temporal precision. A clear gradient of Kv1.3 immunostaining along the lateral to medial tonotopic axis of the MNTB was detected. Highest levels were found in the lateral region of the MNTB, which corresponds to neurons that respond selectively to low-frequency auditory stimuli. Previous studies have demonstrated that MNTB neurons and their afferent inputs from the cochlear nucleus express three other members of the Kv1 family, Kv1.1, Kv1.2, and Kv1.6. Nevertheless, confocal microscopy of MNTB sections coimmunostained for Kv1.3 with these subunits revealed that the distribution of Kv1.3 differed significantly from other Kv1 family subunits. In particular, no axonal staining of Kv1.3 was detected, and most prominent labeling was in structures surrounding the somata of the principal neurons, suggesting specific localization to the large calyx of Held presynaptic endings that envelop the principal cells. The presence of Kv1.3 in presynaptic terminals was confirmed by coimmunolocalization with the synaptic markers synaptophysin, syntaxin, and synaptotagmin and by immunogold electron microscopy. Kv1.3 immunogold particles in the terminals were arrayed along the plasma membrane and on internal vesicular structures. To confirm these patterns of staining, we carried out immunolabeling on sections from Kv1.3(-/-) mice. No immunoreactivity could be detected in Kv1.3(-/-) mice either at the light level or in immunogold experiments. The finding of a tonotopic gradient in presynaptic terminals suggests that Kv1.3 may regulate neurotransmitter release differentially in neurons that respond to different frequencies of sound. J. Comp. Neurol. 518:3205-3220, 2010.(c) 2010 Wiley-Liss, Inc.

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca(2+) responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca(2+) activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca(2+) responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.

Kv7.5/KCNQ5, a voltage-dependent potassium channel that generates a subthreshold K+ current (also called M-current), is localized in excitatory endings of auditory brainstem nuclei in the adult rat. Here, we focus on how specific targeting develops from birth to adulthood in the rat. We first analyzed by immunocytochemistry the distribution of KCNQ5 during postnatal development of neurons in the anteroventral cochlear nucleus (AVCN) and their targets in the medial nucleus of the trapezoid body (MNTB). From postnatal days (P) 0 to 12, KCNQ5 immunoreactivity was restricted to cell bodies, whereas from P13 onward a shift in labeling pattern was seen, with KCNQ5 immunoreactivity becoming confined to synaptic endings in both the AVCN and MNTB. The developmental synaptic targeting was also accompanied by a downregulation of KCNQ5 transcripts in the cochlear nucleus from P13 onward, as seen with quantitative reverse transcriptase polymerase chain reaction. We further tested whether auditory nerve activity at hearing onset (approximately P12) regulates synaptic targeting of the channel. Cochleae were removed at P10, before hearing onset. In the MNTB, 3 days after cochlear ablation, at P13, KCNQ5 immunoreactivity was seen in calyces of Held, as in normal age-matched controls. However, immunolabeling virtually disappeared from MNTB calyces 40 days after cochlear ablation but reappeared in the somata of neurons in AVCN. These findings suggest that synaptic targeting of KCNQ5 in brainstem auditory neurons occurs around the time of hearing onset, regardless of auditory nerve activity. However, long-term synaptic localization after hearing onset depends on peripheral input. J. Comp. Neurol. 518:1301-1314, 2010.(c) 2009 Wiley-Liss, Inc.