Mikiya Asako

Deafness-related plasticity in the inferior colliculus: gene expression profiling following removal of peripheral activity

The inferior colliculus (IC) is a major center of integration in the ascending as well as descending auditory pathways, where both excitatory and inhibitory amino acid neurotransmitters play a key role. When normal input to the auditory system is decreased, the balance between excitation and inhibition in the IC is disturbed. We examined global changes in gene expression in the rat IC 3 and 21 days following bilateral deafening, using Affymetrix GeneChip arrays and focused our analysis on changes in expression of neurotransmission‐related genes. Over 1400 probe sets in the Affymetrix Rat Genome U34A Array were identified as genes that were differentially expressed. These genes encoded proteins previously reported to change as a consequence of deafness, such as calbindin, as well as proteins not previously reported to be modulated by deafness, such as clathrin. A subset of 19 differentially expressed genes was further examined using quantitative RT–PCR at 3, 21 and 90 days following deafness. These included several GABA, glycine, glutamate receptor and neuropeptide‐related genes. Expression of genes for GABA‐A receptor subunits β2, β3, and γ2, plus ionotropic glutamate receptor subunits AMPA 2, AMPA 3, and kainate 2, increased at all three times. Expression of glycine receptor α1 initially declined and then later increased, while α2 increased sharply at 21 days. Glycine receptor α3 increased between 3 and 21 days, but decreased at 90 days. Of the neuropeptide‐related genes tested with qRT–PCR, tyrosine hydroxylase decreased approximately 50% at all times tested. Serotonin receptor 2C increased at 3, 21, and 90 days. The 5B serotonin receptor decreased at 3 and 21 days and returned to normal by 90 days. Of the genes tested with qRT–PCR, only glycine receptor α2 and serotonin receptor 5B returned to normal levels of expression at 90 days. Changes in GABA receptor β3, GABA receptor γ2, glutamate receptor 2/3, enkephalin, and tyrosine hydroxylase were further confirmed using immunocytochemistry.

Deafness‐related decreases in glycine‐immunoreactive labeling in the rat cochlear nucleus

There is increasing evidence of activity‐related plasticity in auditory pathways. The present study examined the effects of decreased activity on immunolocalization of the inhibitory neurotransmitter glycine in the cochlear nucleus of the rat after bilateral cochlear ablation. Specifically, glycine‐immunoreactive puncta adjacent to somatic profiles were compared in normal hearing animals and animals deafened for 14 days. The number of glycine‐immunoreactive puncta surrounding somatic profiles of spherical and globular bushy cells, glycine‐immunoreactive type I stellate multipolar cells, radiate neurons (type II stellate multipolar cells), and fusiform cells decreased significantly. In addition, the number of glycine immunopositive tuberculoventral (vertical or corn) cells in the deep layer of the dorsal cochlear nucleus also decreased significantly. These results suggest that decreased inhibition reported in cochlear nucleus after deafness may be due to decreases in glycine.

Deafness associated changes in expression of two-pore domain potassium channels in the rat cochlear nucleus

Two-pore domain potassium channels (K(2PD)+) play an important role in setting resting membrane potential by regulating background leakage of potassium ions, which in turn controls neuronal excitability. To determine whether these channels contribute to activity-dependent plasticity following deafness, we used quantitative real-time PCR to examine the expression of 10 K(2PD)+ subunits in the rat cochlear nucleus at 3 days, 3 weeks and 3 months after bilateral cochlear ablation. There was a large sustained decrease in the expression of TASK-5, a subunit that is predominantly expressed in auditory brain stem neurons, and in the TASK-1 subunit which is highly expressed in several types of cochlear nucleus neurons. TWIK-1 and THIK-2 also showed significant decreases in expression that were maintained across all time points. TWIK-2, TREK-1 and TREK-2 showed no significant change in expression at 3 days but showed large decreases at 3 weeks and 3 months following deafness. TRAAK and TASK-3 subunits showed significant decreases at 3 days and 3 weeks following deafness, but these differences were no longer significant at 3 months. Dramatic changes in expression of K(2PD)+ subunits suggest these channels may play a role in deafness-associated changes in the excitability of cochlear nucleus neurons.

Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling

The physiology and circuitry associated with dorsal cochlear nucleus neurons (DCN) have been well described. The ability to remotely manipulate neuronal activity in these neurons would represent a step forward in the ability to understand the specific function of DCN neurons in hearing. Although, optogenetics has been used to study the function of pathways in other systems for several years, in the auditory system only neurons in the auditory cortex have been studied using this technique. Adeno-associated viral vectors with either channelrhodopsin-2 fused with GFP (ChR2-GFP) or halorhodopsin fused with mCherry (HaloR-mCherry), capable of expressing light sensitive cation channels or chloride pumps, respectively, were delivered into the dorsal cochlear nucleus (DCN). One to 18 months later, expression of ChR2 and HaloR was observed throughout the DCN. Rhodopsin distribution within the DCN was determined to be within several cell types identified based on morphology and location within the DCN. Expression of ChR2-GFP and HaloR-mCherry was found at both the injection site as well as in regions receiving projections from the site. Wavelength appropriate optical stimulation in vivo resulted in neuronal activity that was significantly increased over pre-stimulation levels with no return to baseline levels during the time of the light exposure. We also examined the effects of optically driven neuronal activity on subsequent tone driven responses in the DCN. In the DCN 75% of the 16 electrode sites showed decreased neuronal activity in response to a tone immediately following light stimulation while six percent were decreased following tone stimulation and 19% of the electrode sites showed no change. This is in contrast to tone driven neuronal activity prior to the light exposure in which the majority of electrode sites showed increased neuronal activity. Our results indicate that expression and activation of rhodopsin within neurons involved in auditory processing does not appear to have deleterious effects on hearing even 18 months following expression. In addition, virally targeted rhodopsins may be useful as tract tracers to delineate as well as modulate the activity of pathways and specific neurons. In the future rhodopsins can be targeted to specific subpopulations of auditory neurons. Ultimately, photostimulation may provide a physiologically relevant method for modulating the function of auditory neurons and affecting hearing outcomes. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Changes in glycine immunoreactivity in the rat superior olivary complex following deafness.

The balance between inhibitory and excitatory amino acid neurotransmitters contributes to the control of normal functioning of the auditory brainstem. Changes in the level of neuronal activity within the auditory brainstem pathways influence the balance between inhibition and excitation. Activity‐dependent plasticity in the auditory pathways can be studied by creating a large decrease in activity through peripheral deafening. Deafness‐related decreases in GABA have previously been shown in the inferior colliculus. However, glycine is a more prevalent inhibitory transmitter in the mature superior olivary complex (SOC). The present study therefore examined if there were deafness‐related changes in glycine in the SOC using postembedding immunocytochemistry. Animals were bilaterally deafened by an intrascalar injection of neomycin. Five nuclei in the SOC, the lateral superior olive (LSO), superior paraolivary nucleus (SPoN), and the medial, lateral, and ventral nuclei of the trapezoid body (MNTB, LNTB, and VNTB) were examined 14 days following the deafening and compared to normal hearing age‐matched controls. The LSO and SPoN were divided into high and low frequency regions. The number of glycine immunoreactive puncta on the somata of principal cells showed significant decreases in all regions assessed, with changes ranging from 50% in the VNTB to 23% in the LSO. J. Comp. Neurol. 494:179–189, 2006.

Thyroid-like low-grade nasopharyngeal papillary adenocarcinoma: Report of two cases

Thyroid‐like low‐grade nasopharyngeal papillary adenocarcinoma (LGNPPA) is extremely rare; only four cases have been reported. Herein are presented the case reports of two Japanese male patients with thyroid‐like LGNPPA. Macroscopically, these tumors were pedunculated polypoid masses on the roof of the nasopharynx. Microscopically, they were characterized by papillary and glandular epithelial proliferation. The papillae were complex and tightly packed with hyalinized fibrovascular cores and lined by columnar and pseudostratified cells with intervening spindle‐shaped cells. Both cell types had round to oval vesicular nuclei with tiny nucleoli and mildly eosinophilic cytoplasm. Mitotic figures were not evident and necrosis was not observed. Psammoma bodies were seen focally in one of the patients. Transition from normal surface epithelium to tumor cells was identified in both cases. On immunohistochemistry the tumor cells were positive for cytokeratin (CK)7, CK19, thyroid transcription factor‐1 (TTF‐1) and vimentin. They were negative for CK5/6, CK20, thyroglobulin, S‐100 protein and CD15. In situ hybridization for EBV was negative. Nasopharyngeal tumors with similar morphological appearance should be examined for TTF‐1 immunoreactivity, and patients should be clinically followed to determine the course of this unusual disease and the significance of TTF‐1 expression.

Spatial and temporal patterns of evoked neural activity from auditory nuclei in chick brainstem detected by optical recording.

In order to detect the spatial patterning of the auditory projection of the embryonic chick brainstem, anatomical methods such as orthograde transport of horseradish peroxidase have been used. However, these methods do not provide the continuous information required about the absolute value and time-course of varying neural excitement. Furthermore, the use of conventional electrophysiological methods makes it difficult or impossible to detect the transmembrane voltage change because of the small size and fragility of the cells of the young chick brainstem. We thus believe that optical measurement of membrane potential might be beneficial in circumstances where electrodes are difficult to use for reasons of cell size, complexity, or membrane topology. In the present work, we therefore examined the feasibility of an optical method for delineating the synaptic transmission of afferent input in the auditory nuclei in the chick brainstem. We used embryonic chick brainstem slice preparations featuring an intact eighth nerve, and loaded depolarizing square current pulses from tungsten microelectrodes into the auditory nerve for stimulation of these preparations. In this approach, we used a multiple-site optical recording system comprising a 16 x 16-element photodiode array and a voltage sensitive dye (NK-2761). Neural excitation evoked by stimulation to the left auditory nerve was propagated to the dorsal side of the brainstem. This area in which the optical signal was detected is located on the auditory nuclei. Since the physiological spatial patterning of the auditory nerve projection could be roughly estimated by the optical technique, the technique is considered useful for examining the electrical activity generated from auditory nuclei in the brainstem. This is the first report of spatial patterning of auditory neurons in the embryonic chick brainstem generated through optical recording.

The Presence of Platelet-Activating Factor-Acetylhydrolase in Human Middle Ear Effusions

Factors related to inflammation, including platelet-activating factor (PAF), apparently have a role in chronic otitis media with effusion. PAF is metabolized to the biologically inactive lyso-PAF by the enzyme PAF-acetylhydrolase (PAF-AH). We have obtained evidence that PAF-AH activity is present in human middle ear effusions in patients with chronic otitis media with effusion. The present study revealed the enzyme in human middle ear effusions to be the plasma type PAF-AH. We suggest that PAF-AH may be involved in regulating inflammation in the middle ear by inactivating PAF, the potent proinflammatory autacoid.

Residual exhaled nitric oxide elevation in asthmatics is associated with eosinophilic chronic rhinosinusitis.

Abstract Objective: Eosinophilic chronic rhinosinusitis (ECRS) is as a subgroup of chronic rhinosinusitis (CRS) with nasal polyps. ECRS is a refractory disease closely related to bronchial asthma. Fractionated exhaled nitric oxide (FeNO) levels were reportedly elevated in some asthmatics with CRS after adequate treatment, suggesting that residual eosinophilic airway inflammation or ECRS might affect FeNO levels. Methods: To investigate the association between asthma with ECRS and FeNO levels, we examined FeNO levels in 133 asthmatics (99 with ECRS and 34 without ECRS) and 13 patients with ECRS without asthma. The severity of asthma was defined by the Global Initiative for Asthma guidelines and that of sinusitis was evaluated by the sinus CT score based on the Lund–Mackay scale. Results and conclusions: FeNO levels were elevated even in well-controlled asthmatics with ECRS, whereas asthmatics without ECRS and ECRS patients without asthma did not have high FeNO levels (>50 ppb). Although FeNO levels were not correlated with asthma severity, they were positively correlated with the sinus CT score. In asthmatics with ECRS, patients with higher FeNO levels had more severe ECRS and asthma. There is a possibility of having comorbid ECRS, particularly in asthmatics with high FeNO levels even after adequate treatment, including ICS, suggesting that asthma and ECRS may be closely associated as one airway disease with eosinophilic inflammation. Continual awareness of the coexistent ECRS is ideally recommended for asthmatics with high FeNO levels.

Downregulation of N-methyl-D-aspartate receptor ζ1 subunit (GluN1) gene in inferior colliculus with aging.

Presbycusis is the impairment of auditory function associated with aging, which stems from peripheral cochlear lesions and degeneration of the central auditory process. The effect of age-induced peripheral hearing loss on the central auditory process is not fully understood. C57Bl/6 (C57) mice present accelerated peripheral hearing loss, which is well developed by middle-age and mimics the human presbycusis pattern. The aim of this study was to elucidate the molecular effects of peripheral hearing loss in the inferior colliculus (IC) with age between young and middle-aged C57 mice using cDNA microarray. Glutamate receptor ionotropic NMDA ζ1 (GluN1) exhibited the greatest decrease in the middle-aged group as determined using cDNA microarray and by further assessment using real-time PCR (qPCR). Histological assessment with in situ hybridization of GluN1 showed significantly decreased expression in all IC subdivisions of the middle-aged group. GluN1 is a receptor for excitatory neurotransmission, and significant downregulation of this gene may be subsequent to the decline of afferent input from the cochlea in aging C57 mice. Consequently, using the combination of microarray, qPCR, and in situ hybridization, we showed that the decline of GluN1 in the IC of aging animals might have a key role in the pathogenesis of presbycusis.