Maturation of Heterogeneity in Afferent Synapse Ultrastructure in the Mouse Cochlea

Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 μm2) and less round as their short axes shortened predominantly on the modiolar side, from 770 to 360 nm. Membrane-associated synaptic vesicles decreased in number from 53 to 30 per synapse from p17 to p34. Anatomical coupling, measured as PSD to ribbon distance, tightened predominantly on the pillar side. Ribbons became less spherical as long-axes lengthened only on the modiolar side of the IHC, from 372 to 541 nm. A decreasing gradient of synaptic ribbon size along the modiolar-pillar axis was detected only at p34 after aligning synapses of adjacent IHCs to a common reference frame (median volumes in nm3 × 106: modiolar 4.87; pillar 2.38). The number of ribbon-associated synaptic vesicles scaled with ribbon size (range 67 to 346 per synapse at p34), thus acquiring a modiolar-pillar gradient at p34, but overall medians were similar at p17 (120) and p34 (127), like ribbon surface area (0.36 vs. 0.34 μm2). PD and PSD morphologies were tightly correlated to each other at individual synapses, more so at p34 than p17, but not to ribbon morphology. These observations suggest that PDs and PSDs mature according to different cues than ribbons, and that ribbon size may be more influenced by cues from the IHC than the surrounding tissue.

C1ql1 is expressed in adult outer hair cells of the cochlea in a tonotopic gradient

Hearing depends on the transduction of sounds into neural signals by the inner hair cells of the cochlea. Cochleae also have outer hair cells with unique electromotile properties that increase auditory sensitivity, but they are particularly susceptible to damage by intense noise exposure, ototoxic drugs, and aging. Although the outer hair cells have synapses on afferent neurons that project to the brain, the function of this neuronal circuit is unclear. Here, we created a novel mouse allele that inserts a fluorescent reporter at the C1ql1 locus which revealed gene expression in the outer hair cells and allowed creation of outer hair cell-specific C1ql1 knockout mice. We found that C1ql1 expression in outer hair cells corresponds to areas with the most sensitive frequencies of the mouse audiogram, and that it has an unexpected adolescence-onset developmental timing. No expression was observed in the inner hair cells. Since C1QL1 in the brain is made by neurons, transported anterogradely in axons, and functions in the synaptic cleft, C1QL1 may serve a similar function at the outer hair cell afferent synapse. Histological analyses revealed that C1ql1 conditional knockout cochleae may have reduced outer hair cell afferent synapse maintenance. However, auditory behavioral and physiological assays did not reveal a compelling phenotype. Nonetheless, this study identifies a potentially useful gene expressed in the cochlea and opens the door for future studies aimed at elucidating the function of C1QL1 and the function of the outer hair cell and its afferent neurons.

Soluble Guanylate Cyclase As the Key Enzyme in the Modulating Effect of NO on Metabotropic Glutamate Receptors

The synaptic plasticity of the afferent synapse of the vestibular apparatus is defined by the dynamic interaction of ionotropic and metabotropic glutamate receptors and the modulators of synaptic transmission. It was shown that nitric oxide modulates iGluR responses. In this paper, the effect of NO on the function of the afferent synapse mGluR was investigated. Inhibitor of nitric oxide synthase lowered the level of background activity but increased the amplitude of the responses of groups I and II mGluR agonist ACPD. Donor NO SNAP increased the level of background activity. Short-term perfusion of the synaptic region with low concentrations of SNAP led to a decrease in the amplitude of the answers of mGluR agonists ACPD and DHPG. The inhibitory effect of the NO donor was eliminated under blockade of soluble guanylate cyclase with a specific inhibitor ODQ. A prolonged application of NO did not cause a statistically significant change in the amplitude of the ACPD response. However, SNAP at concentrations of 10 and 100 M increased the amplitude of the mGluR agonist responses 30 and 15 minutes, respectively, after termination of the NO donor exposure. The obtained data show the multidirectional effect of NO on the function of mGluR and testify to the existence of a complex modulating mechanism of the afferent flow from vestibular organs to the central nervous system.

Dendritic HCN Channels Shape Excitatory Postsynaptic Potentials at the Inner Hair Cell Afferent Synapse in the Mammalian Cochlea

Synaptic transmission at the inner hair cell (IHC) afferent synapse, the first synapse in the auditory pathway, is specialized for rapid and reliable signaling. Here we investigated the properties of a hyperpolarization-activated current ( Ih), expressed in the afferent dendrite of auditory nerve fibers, and its role in shaping postsynaptic activity. We used whole cell patch-clamp recordings from afferent dendrites directly where they contact the IHC in excised postnatal rat cochlear turns. Excitatory postsynaptic potentials (EPSPs) of variable amplitude (1–35 mV) were found with 10–90% rise times of about 1 ms and time constants of decay of about 5 ms at room temperature. Current–voltage relations recorded in afferent dendrites revealed Ih. The pharmacological profile and reversal potential (−45 mV) indicated that Ih is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The HCN channel subunits HCN1, HCN2, and HCN4 were found to be expressed in afferent dendrites using immunolabeling. Raising intracellular cAMP levels sped up the activation kinetics, increased the magnitude of Ih and shifted the half activation voltage ( Vhalf) to more positive values (−104 ± 3 to −91 ± 2 mV). Blocking Ih with 50 μM ZD7288 resulted in hyperpolarization of the resting membrane potential (∼4 mV) and slowing the decay of the EPSP by 47%, suggesting that Ih is active at rest and shortens EPSPs, thereby potentially improving rapid and reliable signaling at this first synapse in the auditory pathway.

Group III Metabotropic Glutamate Receptors (mGluRs) Modulate Transmission of Gustatory Inputs in the Brain Stem

Glutamate is the principal neurotransmitter at the primary sensory afferent synapse in the medulla for the taste system. At this synapse, glutamate activates N-methyl-d-aspartate (NMDA) and non-NMDA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] and kainate) ionotropic receptors to effect a response in the second-order neurons. The current experiment is the first to examine the role of metabotropic glutamate receptors (mGluRs) in the transmission of taste information. In an in vitro slice preparation of the primary vagal gustatory nucleus in goldfish, primary gustatory afferent fibers were stimulated electrically, whereas evoked dendritic field potentials were recorded in the sensory layers. Recordings were made before, during, and after bath application of mGluR agonists for various mGluR groups and subtypes. Whereas l-AP4, a group III agonist, reduced the field potential, group I and group II agonists had no effect. Furthermore, the selective mGluR4 agonist ACPT-III and mGluR8 agonist PPG were effective at reducing the field potential, whereas agonists selective for mGluR6 and 7 were not. MAP4, a group III mGluR antagonist, attenuated frequency-dependent depression, indicating that endogenous glutamate binds to presynaptic mGluRs under normal conditions. Furthermore, polymerase chain reaction showed that mRNA for mGluR4 and 8 is expressed in the vagal ganglia, a prerequisite if those receptors are expressed presynaptically in the vagal lobe. Collectively, these experiments indicate that mGluR4 and 8 are presynaptic at the primary gustatory afferent synapse and that their activation inhibits glutamatergic release.