The role of GABAB receptors in the subcortical pathways of the mammalian auditory system.

GABAB receptors are G-protein coupled receptors for the inhibitory neurotransmitter GABA. Functional GABAB receptors are formed as heteromers of GABAB1 and GABAB2 subunits, which further associate with various regulatory and signaling proteins to provide receptor complexes with distinct pharmacological and physiological properties. GABAB receptors are widely distributed in nervous tissue, where they are involved in a number of processes and in turn are subject to a number of regulatory mechanisms. In this review, we summarize current knowledge of the cellular distribution and function of the receptors in the inner ear and auditory pathway of the mammalian brainstem and midbrain. The findings suggest that in these regions, GABAB receptors are involved in processes essential for proper auditory function, such as cochlear amplifier modulation, regulation of spontaneous activity, binaural and temporal information processing, and predictive coding. Since impaired GABAergic inhibition has been found to be associated with various forms of hearing loss, GABAB dysfunction could also play a role in some pathologies of the auditory system.

Ablation of Gabra5 Influences Corticosterone Levels and Anxiety-like Behavior in Mice.

Stress responses are activated by the hypothalamic-pituitary-adrenal axis (HPA axis), culminating in the release of glucocorticoids. During prolonged periods of secretion of glucocorticoids or inappropriate behavioral responses to a stressor, pathologic conditions may occur. Increased glucocorticoid concentration is linked to generalized anxiety, and there are knowledge gaps regarding its regulation. It is known that the HPA axis is under GABAergic control, but the contribution of the individual subunits of the GABA receptor is largely unknown. In this study, we investigated the relationship between the α5 subunit and corticosterone levels in a new mouse model deficient for Gabra5, which is known to be linked to anxiety disorders in humans and phenologs observed in mice. We observed decreased rearing behavior, suggesting lower anxiety in the Gabra5-/- animals; however, such a phenotype was absent in the open field and elevated plus maze tests. In addition to decreased rearing behavior, we also found decreased levels of fecal corticosterone metabolites in Gabra5-/- mice indicating a lowered stress response. Moreover, based on the electrophysiological recordings where we observed a hyperpolarized state of hippocampal neurons, we hypothesize that the constitutive ablation of the Gabra5 gene leads to functional compensation with other channels or GABA receptor subunits in this model.

Cochlear ablation in neonatal rats disrupts inhibitory transmission in the medial nucleus of the trapezoid body.

Inhibitory circuits in the auditory brainstem undergo multiple postnatal changes that are both activity-dependent and activity-independent. We tested to see if the shift from GABA- to glycinergic transmission, which occurs in the rat medial nucleus of the trapezoid body (MNTB) around the onset of hearing, depends on sound-evoked neuronal activity. We prevented the activity by bilateral cochlear ablations in early postnatal rats and studied ionotropic GABA and glycine receptors in MNTB neurons after hearing onset. The removal of the cochlea decreased responses of GABAA and glycine receptors to exogenous agonists as well as the amplitudes of inhibitory postsynaptic currents. The reduction was accompanied by a decrease in the number of glycine receptor- or vesicular GABA transporter-immunopositive puncta. Furthermore, the ablations markedly affected the switch in presynaptic GABAA to glycine receptors. The increase in the expression of postsynaptic glycine receptors and the shift in inhibitory transmitters were not prevented. The results suggest that inhibitory transmission in the MNTB is subject to multiple developmental signals and support the idea that auditory experience plays a role in the maturation of the brainstem glycinergic circuits.

Modulation of synaptic depression of the calyx of Held synapse by GABA(B) receptors and spontaneous activity.

The calyx of Held synapse of the medial nucleus of the trapezoid body is a giant axosomatic synapse in the auditory brainstem, which acts as a relay synapse showing little dependence of its synaptic strength on firing frequency. The main mechanism that is responsible for its resistance to synaptic depression is its large number of release sites with low release probability. Here, we investigated the contribution of presynaptic GABA(B) receptors and spontaneous activity to release probability both in vivo and in vitro in young-adult mice. Maximal activation of presynaptic GABA(B) receptors by baclofen reduced synaptic output by about 45% in whole-cell voltage clamp slice recordings, which was accompanied by a reduction in short-term depression. A similar reduction in transmission was observed when baclofen was applied in vivo by microiontophoresis during juxtacellular recordings using piggyback electrodes. No significant change in synaptic transmission was observed during application of the GABA(B) receptor antagonist CGP54626 both during in vivo and slice recordings, suggesting a low ambient GABA concentration. Interestingly, we observed that synapses with a high spontaneous frequency showed almost no synaptic depression during auditory stimulation, whereas synapses with a low spontaneous frequency did depress during noise bursts. Our data thus suggest that spontaneous firing can tonically reduce release probability in vivo. In addition, our data show that the ambient GABA concentration in the auditory brainstem is too low to activate the GABA(B) receptor at the calyx of Held significantly, but that activation of GABA(B) receptors can reduce sound-evoked synaptic depression.

Differential distribution of glycine receptor subtypes at the rat calyx of Held synapse.

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/ß-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/ß heteromers) and showed similar clustering and pharmacological profile as GlyRs on MNTB principal cells. These results suggest that specific targeting of GlyR ß-subunit produces segregation of GlyR subtypes involved in two different mechanisms of modulation of synaptic strength.

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells.

Class C G-protein coupled receptors form obligatory dimers. Metabotropic glutamate receptors (mGluRs) are found commonly as homodimers. Alternative splicing of mGluR1 gene results in vivo in the expression of a long variant mGluR1a and at least two short variants mGluR1b and d. The amino acid sequences diverge within their carboxyl-termini six amino acid residues following RRKK motif. This four basic residue sequence was shown to have pronounced impact on function and trafficking of the short variants, while for mGluR1a the long C-terminus reduces the effects caused by presence of the RRKK motif. Here we investigated consequences of interactions between long mGluR1a and short mGluR1b variants. Our results show that mGluR1a interferes with mGluR1b trafficking to the cell surface in HEK293 transfected cells. Expression of a mGlu1a mutant incapable of activating G-proteins with mGluR1b mutated in the glutamate binding site led to the formation of a functional heterodimer. Moreover, we show that swapping long mGluR1a and/or short mGluR1b C-termini with corresponding regions in chimerical GB1 and GB2 gamma-amino butyric acid b (GABAb) receptor subunits do not exclude heterodimerization. These data reveal that the C-terminal ends of mGluR1 do not control subunit association, such that mGluR1 dimers with two distinct C-termini can form and function properly.

The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits.

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.