Female GluA3-KO mice show early onset hearing loss and afferent swellings in ambient sound levels.

AMPA-type glutamate receptors (AMPAR) mediate excitatory cochlear transmission. However, the unique roles of AMPAR subunits are unresolved. Lack of subunit GluA3 (Gria3KO) in male mice reduced cochlear output by 8-weeks of age. Since Gria3 is X-linked and considering sex differences in hearing vulnerability, we hypothesized accelerated presbycusis in Gria3KO females. Here, auditory brainstem responses (ABR) were similar in 3-week-old female Gria3WT and Gria3KO mice. However, when raised in ambient sound, ABR thresholds were elevated and wave-1 amplitudes were diminished at 5-weeks and older in Gria3KO. In contrast, these metrics were similar between genotypes when raised in quiet. Paired synapses were similar in number, but lone ribbons and ribbonless synapses were increased in female Gria3KO mice in ambient sound compared to Gria3WT or to either genotype raised in quiet. Synaptic GluA4:GluA2 ratios increased relative to Gria3WT, particularly in ambient sound, suggesting an activity-dependent increase in calcium-permeable AMPARs in Gria3KO. Swollen afferent terminals were observed by 5-weeks only in Gria3KO females reared in ambient sound. We propose that lack of GluA3 induces sex-dependent vulnerability to AMPAR-mediated excitotoxicity.

Sex differences in glutamate AMPA receptor subunits mRNA with fast gating kinetics in the mouse cochlea.

Evidence shows that females have increased supra-threshold peripheral auditory processing compared to males. This is indicated by larger auditory brainstem responses (ABR) wave I amplitude, which measures afferent spiral ganglion neuron (SGN)-auditory nerve synchrony. However, the underlying molecular mechanisms of this sex difference are mostly unknown. We sought to elucidate sex differences in ABR wave I amplitude by examining molecular markers known to affect synaptic transmission kinetics. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory transmission in mature SGN afferent synapses. Each AMPAR channel is a tetramer composed of GluA2, 3, and 4 subunits (Gria2, 3, and 4 genes), and those lacking GluA2 subunits have larger currents, are calcium-permeable, and have faster gating kinetics. Moreover, alternatively spliced flip and flop isoforms of each AMPAR subunit affect channel kinetics, having faster kinetics those AMPARs containing Gria3 and Gria4 flop isoforms. We hypothesized that SGNs of females have more fast-gating AMPAR subunit mRNA than males, which could contribute to more temporally precise synaptic transmission and increased SGN synchrony. Our data show that the index of Gria3 relative to Gria2 transcripts on SGN was higher in females than males (females: 48%; males: 43%), suggesting that females have more SGNs with higher Gria3 mRNA relative to Gria2. Analysis of the relative abundance of the flip and flop alternatively spliced isoforms showed that females have a 2-fold increase in fast-gating Gria3 flop mRNA, while males have more slow-gating (2.5-fold) of the flip. We propose that Gria3 may in part mediate greater SGN synchrony in females. Significance Statement: Females of multiple vertebrate species, including fish and mammals, have been reported to have enhanced sound-evoked synchrony of afferents in the auditory nerve. However, the underlying molecular mediators of this physiologic sex difference are unknown. Elucidating potential molecular mechanisms related to sex differences in auditory processing is important for maintaining healthy ears and developing potential treatments for hearing loss in both sexes. This study found that females have a 2-fold increase in Gria3 flop mRNA, a fast-gating AMPA-type glutamate receptor subunit. This difference may contribute to greater neural synchrony in the auditory nerve of female mice compared to males, and this sex difference may be conserved in all vertebrates.

GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphology.

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3-knockout mice (i.e., subunit GluA3KO) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3KO and wild-type (GluA3WT) mice reared in ambient sound pressure level (SPL) of 55-75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3WT, but not GluA3KO, demonstrating GluA3 is required for modiolar-pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3KO and GluA2-lacking synapses were observed only in GluA3KO. GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3KO, despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3KO than GluA3WT. However, GluA3KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca2+-permeable AMPARs.

Nerve fibre morphometry with transmission electron microscopy: Application of the nucleator probe in ImageJ.

Stereology and semiautomated binary image histomorphometry are two common methods used for morphometry of nerve fibres. Nucleator probe can be used for the estimation of morphometric parameters like diameter, perimeter, area and volume of a structure that is approximately either a circle or a sphere. In this study, we estimated these parameters with the help of ImageJ software on calibrated transmission electron micrographs. We procured samples of the cochlear nerve (CN) during winter months, within 6-12 hours of death, to reduce post-mortem autolytic changes. The temporal bones containing the CN were fixed by immersion in chilled paraformaldehyde. After dissecting out from the petrous part of the temporal bone, the CN were osmicated and processed for embedding in resin. From the resin blocks, silver coloured (70 nm) ultrathin sections were cut and picked on 300-mesh copper grids, stained with uranyl acetate and lead citrate and viewed under Tecnai G2-20 transmission electron microscope. The transmission electron micrographs had scale bars embedded into them by the software at the time of imaging, and the morphometric parameters of randomly selected nerve fibres were measured using the ImageJ software. The ImageJ software could become a low-cost and dependable tool for nerve fibre morphometry.•Nucleator probe is used for the estimation of morphometric parameters like diameter, perimeter, area or volume•Morphometric parameters were estimated by the ImageJ software on calibrated transmission electron micrographs•The ImageJ software could become a low-cost and dependable tool for nerve fibre morphometry.

The ultrastructural study of human cochlear nerve at different ages.

Ultrastructural and molecular changes in the myelin of the cochlear nerve (CN) have been associated with decreased hearing-acuity with increasing age. But most of these are animal studies or with very few human samples. Hence, we studied the ultrastructure of the human CN at different ages. We obtained samples of CN from persons, who at the time of death belonged to young, middle or old age-groups; defined as ≤ 30, 31 to 50, and ≥ 51 years of age, respectively. These were processed for viewing under a transmission electron microscope (TEM). Morphology and morphometry were assessed after blinding the observer. Measurements of diameter (whole nerve fibre, axon), myelin thickness and calculation of G-ratio were made on calibrated images using ImageJ software. K-Means cluster analysis was performed based on total and inner nerve fibre area. Middle and old age CN showed degenerating axons, splitting of myelin sheath and myelin balloons. Between the middle and old age groups there was significant decrease in axon diameter (p<0.001), inner nerve fibre area (p<0.001), myelin thickness (p<0.001), nerve fibre diameter (p<0.001), and G-ratio (p<0.001). By clustering, we identified three distinct populations of myelinated nerve fibres: large, medium and small. The large fibres (by size), seen in the young, disappeared in the old age-group. We were unable to find any unmyelinated nerve fibres in this study. The morphological deterioration CN fibres may be a visible sign of molecular degeneration and contribute to decreased hearing-acuity.

Trinucleotide Cap Analogue Bearing a Locked Nucleic Acid Moiety: Synthesis, mRNA Modification, and Translation for Therapeutic Applications.

The synthesis of a new trinucleotide cap analogue containing a locked nucleic acid (LNA) moiety such as m7(LNA)G(5')ppp(5')AmpG and its molecular biology applications are described. The most appealing feature is that this new cap analogue outperforms the standard trinucleotide cap m7G(5')ppp(5')AmpG and the anti-reverse cap analogue m27,3'-OG(5')ppp(5')G by a factor of 5 in terms of translational efficiency.

Age-related changes in the number of cresyl-violet-stained, parvalbumin and NMDAR 2B expressing neurons in the human spiral ganglion.

Animal-studies associate age-related hearing loss (presbycusis) with decreasing number of spiral ganglion neurons (SGNs) in Rosenthal's canal (RC) of cochlea. The excitatory neurotransmitter for SGNs is glutamate (through its receptor NMDAR 2B), which can be neurotoxic through Ca2+ overload. Neurotoxicity is balanced by calcium-binding proteins (CBPs) like Parvalbumin (PV), which is the predominant CBP of the SGNs. To estimate the volume of the RC and total number of SGNs that are immunoreactive to PV and NMDAR 2B, we used unbiased stereology in 35 human cochleae derived from cadavers of persons from 2nd to 8th decade of life (subsequently statistically divided into two groups) and compared them to the total number of cresyl violet (CV) stained SGNs. We also estimated the volume of individual neurons and their nuclei. Regression analysis was made on estimated parameters against age. Hierarchical-cluster analysis was done on the neuronal against neuronal nuclear volumes.The average volume of the RC did not change with increasing age (p = 0.4115). The total number of SGNs (CV-stained and those separately expressing PV and NMDAR 2B) significantly decreased with age (p < 0.001). We identified three distinct populations of neurons on the basis of their volumes among SGNs. Thus, there is significant age-related decline in the total number of SGNs, which starts early in life. It may be due to ambient noise and inadequate neutralisation of excitotoxicity.

Morphological development of the human cochlear nucleus.

Morphological studies in developing brain determine critical periods of proliferation, neurogenesis, gliogenesis, and apoptosis. During these periods both intrinsic and extrinsic pathological factors can hamper development. These time points are not available for the human cochlear nucleus (CN). We have used design-based stereology and determined that 18-22 weeks of gestation (WG) are critical in the development of the human CN. Twenty-three fetuses and seven postnatal brainstems were processed for cresyl violet (CV) staining and immunoexpression of NeuN (neurons), GFAP (astrocytes), Ki-67 (proliferation) and TUNEL (apoptosis) and 3-D reconstruction. The volume of CN, total number of neurons selected profiles and the volume of neurons and their nuclei were estimated. Data were grouped (G) into: G1:18-20 WG, G2: 21-24 WG, G3: 25-28 WG and G4 >29 WG. The dimensions of morphologically identified neurons were also measured. The CN primordium was first identifiable at 10WG. Definitive DCN (Dorsal cochlear nucleus) and VCN (ventral cochlear nucleus) were identifiable at 16 WG. There was a sudden growth spurt in total volume of CN, number of neurons and astrocytes from 18 WG. We also observed an increase in proliferation and apoptosis after 22 WG. The number of neurons identifiable by CV was significantly lower than that by NeuN-immunostaining till 25 WG (p = 0.020), after which, both methods were equivalent. Eight morphological types of neurons were identifiable by 26 WG and could be resolved into four clusters by volume and diameter. The CN changed orientation from small, flat and horizontal at 10-16 WG to larger and oblique from 18WG onwards. Prevention of exposure to noxious factors at 18-22 WG may be important in preventing congenital deafness.

Morphological and neurochemical changes in GABAergic neurons of the aging human inferior colliculus.

It is well known that quality of hearing decreases with increasing age due to changes in the peripheral or central auditory pathway. Along with the decrease in the number of neurons the neurotransmitter profile is also affected in the various parts of the auditory system. Particularly, changes in the inhibitory neurons in the inferior colliculus (IC) are known to affect quality of hearing with aging. To date, there is no information about the status of the inhibitory neurotransmitter GABA in the human IC during aging. We have collected and processed inferior colliculi of persons aged 11-97 years at the time of death for morphometry and immunohistochemical expression of glutamic acid decarboxylase (GAD67) and parvalbumin. We used unbiased stereology to estimate the number of cresyl-violet and immunostained neurons. Quantitative real-time PCR was used to measure the relative expression of the GAD67 mRNA. We found that the number of total, GABAergic and PV-positive neurons significantly decreased with increasing age (p < 0.05). The proportion of GAD67-ir neurons to total number of neurons was also negatively associated with increasing age (p = 0.004), but there was no change observed in the proportion of PV-ir neurons relative to GABAergic neurons (p = 0.25). Further, the fold change in the levels of GAD67 mRNA was negatively correlated to age (p = 0.024). We conclude that the poorer quality of hearing with increasing age may be due to decreased expression of inhibitory neurotransmitters and the decline in the number of inhibitory neurons in the IC.