GABA is a modulator, rather than a classical transmitter, in the medial nucleus of the trapezoid body-lateral superior olive sound localization circuit.

KEY POINTS: The lateral superior olive (LSO), a brainstem hub involved in sound localization, integrates excitatory and inhibitory inputs from the ipsilateral and the contralateral ear, respectively. In gerbils and rats, inhibition to the LSO reportedly shifts from GABAergic to glycinergic within the first three postnatal weeks. Surprisingly, we found no evidence for synaptic GABA signalling during this time window in mouse LSO principal neurons. However, we found that presynaptic GABAB Rs modulate Ca2+ influx into medial nucleus of the trapezoid body axon terminals, resulting in reduced synaptic strength. Moreover, GABA elicited strong responses in LSO neurons that were mediated by extrasynaptic GABAA Rs. RNA sequencing revealed highly abundant δ subunits, which are characteristic of extrasynaptic receptors. Whereas GABA increased the excitability of neonatal LSO neurons, it reduced the excitability around hearing onset. Collectively, GABA appears to control the excitability of mouse LSO neurons via extrasynaptic and presynaptic signalling. Thus, GABA acts as a modulator, rather than as a classical transmitter. ABSTRACT: GABA and glycine mediate fast inhibitory neurotransmission and are coreleased at several synapse types. Here we assessed the contribution of GABA and glycine in synaptic transmission between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO), two nuclei involved in sound localization. Whole-cell patch-clamp experiments in acute mouse brainstem slices at postnatal days (P) 4 and 11 during pharmacological blockade of GABAA receptors (GABAA Rs) and/or glycine receptors demonstrated no GABAergic synaptic component on LSO principal neurons. A GABAergic component was absent in evoked inhibitory postsynaptic currents and miniature events. Coimmunofluorescence experiments revealed no codistribution of the presynaptic GABAergic marker GAD65/67 with gephyrin, a postsynaptic marker for GABAA Rs, corroborating the conclusion that GABA does not act synaptically in the mouse LSO. Imaging experiments revealed reduced Ca2+ influx into MNTB axon terminals following activation of presynaptic GABAB Rs. GABAB R activation reduced the synaptic strength at P4 and P11. GABA appears to act on extrasynaptic GABAA Rs as demonstrated by application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, a δ-subunit-specific GABAA R agonist. RNA sequencing showed high mRNA levels for the δ-subunit in the LSO. Moreover, GABA transporters GAT-1 and GAT-3 appear to control extracellular GABA. Finally, we show an age-dependent effect of GABA on the excitability of LSO neurons. Whereas tonic GABA increased the excitability at P4, leading to spike facilitation, it decreased the excitability at P11 via shunting inhibition through extrasynaptic GABAA Rs. Taken together, we demonstrate a modulatory role of GABA in the murine LSO, rather than a function as a classical synaptic transmitter.

Impaired Glycine Receptor Trafficking in Neurological Diseases.

Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycine-gated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level.

PKA and PKC Modulators Affect Ion Channel Function and Internalization of Recombinant Alpha1 and Alpha1-Beta Glycine Receptors.

Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-ß GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 µM and 0.5 µM. EC50 of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-ß GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3-6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.