Postmortem neuroimaging: Temporal and spatial sensitivity of manganese-enhanced magnetic resonance imaging (MEMRI) and impact of Mn2+ uptake.

Magnetic resonance imaging data collection and analysis have been challenges in the field of auditory neuroscience. Recent studies have addressed these concerns by using manganese-enhanced magnetic resonance imaging (MEMRI). Basic challenges for in vivo application of MEMRI in rodents includes how to set inclusion criteria for adequate Mn2+ uptake and whether valid data can be collected from brains postmortem. Since brain Mn2+ uptake is complete within 2-4 h and clearance can take 2-4 weeks, one assumption has been that Mn2+-enhanced R1 values continue to reliably reflect the degree of Mn2+-uptake for some indeterminate time after death. To address these issues, the impact of death on R1 values was determined in rats administered Mn2+ and rats that were not. Images of auditory nuclei were collected at fixed intervals from rats before and after death for up to 10 h postmortem. By taking a ratio of pituitary and muscle T1-W intensities (P/M), a reliable quantitative method for assessing adequate brain Mn2+ uptake was created and suggest that P/M ratios should be adopted to objectively measure the quality of the Mn2+ injection. Postmortem R1 values decreased in all brain regions in both the After Mn2+ and No Mn2+ groups. However, the time-course of postmortem changes in R1 was dependent on brain region and degree of Mn2+ uptake. Thus, postmortem R1 values not only differ after death, but vary with time and across brain regions. Postmortem R1 values in unfixed brain tissue, including the auditory nuclei, should be interpreted with caution.

Dopamine in Auditory Nuclei and Lemniscal Projections is Poised to Influence Acoustic Integration in the Inferior Colliculus.

Dopamine (DA) modulates the activity of nuclei within the ascending and descending auditory pathway. Previous studies have identified neurons and fibers in the inferior colliculus (IC) which are positively labeled for tyrosine hydroxylase (TH), a key enzyme in the synthesis of dopamine. However, the origins of the tyrosine hydroxylase positive projections to the inferior colliculus have not been fully explored. The lateral lemniscus (LL) provides a robust inhibitory projection to the inferior colliculus and plays a role in the temporal processing of sound. In the present study, immunoreactivity for tyrosine hydroxylase was examined in animals with and without 6-hydroxydopamine (6-OHDA) lesions. Lesioning, with 6-OHDA placed in the inferior colliculus, led to a significant reduction in tyrosine hydroxylase immuno-positive labeling in the lateral lemniscus and inferior colliculus. Immunolabeling for dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), enzymes responsible for the synthesis of norepinephrine (NE) and epinephrine (E), respectively, were evaluated. Very little immunoreactivity for DBH and no immunoreactivity for PNMT was found within the cell bodies of the dorsal, intermediate, or ventral nucleus of the lateral lemniscus. The results indicate that catecholaminergic neurons of the lateral lemniscus are likely dopaminergic and not noradrenergic or adrenergic. Next, high-pressure liquid chromatography (HPLC) analysis was used to confirm that dopamine is present in the inferior colliculus and nuclei that send projections to the inferior colliculus, including the cochlear nucleus (CN), superior olivary complex (SOC), lateral lemniscus, and auditory cortex (AC). Finally, fluorogold, a retrograde tracer, was injected into the inferior colliculus of adult rats. Each subdivision of the lateral lemniscus contained fluorogold within the somata, with the dorsal nucleus of the lateral lemniscus showing the most robust projections to the inferior colliculus. Fluorogold-tyrosine hydroxylase colocalization within the lateral lemniscus was assessed. The dorsal and intermediate nuclei neurons exhibiting similar degrees of colocalization, while neurons of the ventral nucleus had significantly fewer colocalized fluorogold-tyrosine hydroxylase labeled neurons. These results suggest that several auditory nuclei that project to the inferior colliculus contain dopamine, dopaminergic neurons in the lateral lemniscus project to the inferior colliculus and that dopaminergic neurotransmission is poised to play a pivotal role in the function of the inferior colliculus.

Effects of Noise Exposure on the Vestibular System: A Systematic Review.

Despite our understanding of the impact of noise-induced damage to the auditory system, much less is known about the impact of noise exposure on the vestibular system. In this article, we review the anatomical, physiological, and functional evidence for noise-induced damage to peripheral and central vestibular structures. Morphological studies in several animal models have demonstrated cellular damage throughout the peripheral vestibular system and particularly in the otolith organs; however, there is a paucity of data on the effect of noise exposure on human vestibular end organs. Physiological studies have corroborated morphological studies by demonstrating disruption across vestibular pathways with otolith-mediated pathways impacted more than semicircular canal-mediated pathways. Similar to the temporary threshold shifts observed in the auditory system, physiological studies in animals have suggested a capacity for recovery following noise-induced vestibular damage. Human studies have demonstrated that diminished sacculo-collic responses are related to the severity of noise-induced hearing loss, and dose-dependent vestibular deficits following noise exposure have been corroborated in animal models. Further work is needed to better understand the physiological and functional consequences of noise-induced vestibular impairment in animals and humans.

Publisher Correction: Novel QUEST MRI In Vivo Measurement of Noise-induced Oxidative Stress in the Cochlea.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The rat as a model for studying noise injury and otoprotection.

A major challenge for those studying noise-induced injury pre-clinically is the selection of an animal model. Noise injury models are particularly relevant in an age when people are constantly bombarded by loud noise due to occupation and/or recreation. The rat has been widely used for noise-related morphological, physiological, biochemical, and molecular assessment. Noise exposure resulting in a temporary (TTS) or permanent threshold shift (PTS) yields trauma in peripheral and central auditory related pathways. While the precise nature of noise-related injuries continues to be delineated, both PTS and TTS (with or without hidden hearing loss) result in homeostatic changes implicated in conditions such as tinnitus and hyperacusis. Compared to mice, rats generally tolerate exposure to loud sounds reasonably well, often without exhibiting other physical non-inner ear related symptoms such as death, loss of consciousness, or seizures [Skradski, Clark, Jiang, White, Fu, and Ptacek (2001). Neuron 31, 537-544; Faingold (2002). Hear. Res. 168, 223-237; Firstova, Abaimov, Surina, Poletaeva, Fedotova, and Kovalev (2012). Bull Exp. Biol. Med. 154, 196-198; De Sarro, Russo, Citraro, and Meldrum (2017). Epilepsy Behav. 71, 165-173]. This ability of the rat to thrive following noise exposure permits study of long-term effects. Like the mouse, the rat also offers a well-characterized genome allowing genetic manipulations (i.e., knock-out, viral-based gene expression modulation, and optogenetics). Rat models of noise-related injury also provide valuable information for understanding mechanistic changes to identify therapeutic targets for treatment. This article provides a framework for selection of the rat as a model for noise injury studies.

Novel QUEST MRI In Vivo Measurement of Noise-induced Oxidative Stress in the Cochlea.

Effective personalized therapeutic treatment for hearing loss is currently not available. Cochlear oxidative stress is commonly identified in the pathogenesis of hearing loss based upon findings from excised tissue, thus suggesting a promising druggable etiology. However, the timing and site(s) to target for anti-oxidant treatment in vivo are not clear. Here, we address this long-standing problem with QUEnch-assiSTed Magnetic Resonance Imaging (QUEST MRI), which non-invasively measures excessive production of free radicals without an exogenous contrast agent. QUEST MRI is hypothesized to be sensitive to noise-evoked cochlear oxidative stress in vivo. Rats exposed to a loud noise event that resulted in hair cell loss and reduced hearing capability had a supra-normal MRI R1 value in their cochleae that could be corrected with anti-oxidants, thus non-invasively indicating cochlear oxidative stress. A gold-standard oxidative damage biomarker [heme oxidase 1 (HO-1)] supported the QUEST MRI result. The results from this study highlight QUEST MRI as a potentially transformative measurement of cochlear oxidative stress in vivo that can be used as a biomarker for improving individual evaluation of anti-oxidant treatment efficacy in currently incurable oxidative stress-based forms of hearing loss.

MRI compatible MS2 nanoparticles designed to cross the blood-brain-barrier: providing a path towards tinnitus treatment.

Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood-brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn2+) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.

Tinnitus and temporary hearing loss result in differential noise-induced spatial reorganization of brain activity.

Loud noise frequently results in hyperacusis or hearing loss (i.e., increased or decreased sensitivity to sound). These conditions are often accompanied by tinnitus (ringing in the ears) and changes in spontaneous neuronal activity (SNA). The ability to differentiate the contributions of hyperacusis and hearing loss to neural correlates of tinnitus has yet to be achieved. Towards this purpose, we used a combination of behavior, electrophysiology, and imaging tools to investigate two models of noise-induced tinnitus (either with temporary hearing loss or with permanent hearing loss). Manganese (Mn2+) uptake was used as a measure of calcium channel function and as an index of SNA. Manganese uptake was examined in vivo with manganese-enhanced magnetic resonance imaging (MEMRI) in key auditory brain regions implicated in tinnitus. Following acoustic trauma, MEMRI, the SNA index, showed evidence of spatially dependent rearrangement of Mn2+ uptake within specific brain nuclei (i.e., reorganization). Reorganization of Mn2+ uptake in the superior olivary complex and cochlear nucleus was dependent upon tinnitus status. However, reorganization of Mn2+ uptake in the inferior colliculus was dependent upon hearing sensitivity. Furthermore, following permanent hearing loss, reduced Mn2+ uptake was observed. Overall, by combining testing for hearing sensitivity, tinnitus, and SNA, our data move forward the possibility of discriminating the contributions of hyperacusis and hearing loss to tinnitus.

Development of manganese-enhanced magnetic resonance imaging of the rostral ventrolateral medulla of conscious rats: Importance of normalization and comparison with other regions of interest.

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) are believed to contribute to pathophysiological alterations in sympathetic nerve activity and the development of cardiovascular disease. The ability to identify changes in the activity of RVLM neurons in conscious animals and humans, especially longitudinally, would represent a clinically important advancement in our understanding of the contribution of the RVLM to cardiovascular disease. To this end, we describe the initial development of manganese-enhanced magnetic resonance imaging (MEMRI) for the rat RVLM. Manganese (Mn2+ ) has been used to estimate in vivo neuronal activity in other brain regions because of both its paramagnetic properties and its entry into and accumulation in active neurons. In this initial study, our three goals were as follows: (1) to validate that Mn2+ enhancement occurs in functionally and anatomically localized images of the rat RVLM; (2) to quantify the dose and time course dependence of Mn2+ enhancement in the RVLM after one systemic injection in conscious rats (66 or 33 mg/kg, intraperitoneally); and (3) to compare Mn2+ enhancement in the RVLM with other regions to determine an appropriate method of normalization of T1 -weighted images. In our proof-of-concept and proof-of-principle studies, Mn2+ was identified by MRI in the rat RVLM after direct microinjection or via retrograde transport following spinal cord injections, respectively. Systemic injections in conscious rats produced significant Mn2+ enhancement at 24 h (p < 0.05). Injections of 66 mg/kg produced greater enhancement than 33 mg/kg in the RVLM and paraventricular nucleus of the hypothalamus (p < 0.05 for both), but only when normalized to baseline scans without Mn2+ injection. Consistent with findings from our previous functional and anatomical studies demonstrating subregional neuroplasticity, Mn2+ enhancement was higher in the rostral regions of the RVLM (p < 0.05). Together with important technical considerations, our studies support the development of MEMRI as a potential method to examine RVLM activity over time in conscious animal subjects.

Dopamine in the auditory brainstem and midbrain: co-localization with amino acid neurotransmitters and gene expression following cochlear trauma.

Dopamine (DA) modulates the effects of amino acid neurotransmitters (AANs), including GABA and glutamate, in motor, visual, olfactory, and reward systems (Hnasko et al., 2010; Stuber et al., 2010; Hnasko and Edwards, 2012). The results suggest that DA may play a similar modulatory role in the auditory pathways. Previous studies have shown that deafness results in decreased GABA release, changes in excitatory neurotransmitter levels, and increased spontaneous neuronal activity within brainstem regions related to auditory function. Modulation of the expression and localization of tyrosine hydroxylase (TH; the rate limiting enzyme in the production of DA) in the IC following cochlear trauma has been previously reported (Tong et al., 2005). In the current study the possibility of co-localization of TH with AANs was examined. Changes in the gene expression of TH were compared with changes in the gene expression of markers for AANs in the cochlear nucleus (CN) and inferior colliculus (IC) to determine whether those deafness related changes occur concurrently. The results indicate that bilateral cochlear ablation significantly reduced TH gene expression in the CN after 2 months while in the IC the reduction in TH was observed at both 3 days and 2 months following ablation. Furthermore, in the CN, glycine transporter 2 (GLYT2) and the GABA transporter (GABAtp) were also significantly reduced only after 2 months. However, in the IC, DA receptor 1 (DRDA1), vesicular glutamate transporters 2 and 3 (VGLUT2, VGLUT3), GABAtp and GAD67 were reduced in expression both at the 3 days and 2 months time points. A close relationship between the distribution of TH and several of the AANs was determined in both the CN and the IC. In addition, GLYT2 and VGLUT3 each co-localized with TH within IC somata and dendrites. Therefore, the results of the current study suggest that DA is spatially well positioned to influence the effects of AANs on auditory neurons.

Manganese enhanced magnetic resonance imaging (MEMRI): a powerful new imaging method to study tinnitus.

Manganese enhanced magnetic resonance imaging (MEMRI) is a method used primarily in basic science experiments to advance the understanding of information processing in central nervous system pathways. With this mechanistic approach, manganese (Mn(2+)) acts as a calcium surrogate, whereby voltage-gated calcium channels allow for activity driven entry of Mn(2+) into neurons. The detection and quantification of neuronal activity via Mn(2+) accumulation is facilitated by "hemodynamic-independent contrast" using high resolution MRI scans. This review emphasizes initial efforts to-date in the development and application of MEMRI for evaluating tinnitus (the perception of sound in the absence of overt acoustic stimulation). Perspectives from leaders in the field highlight MEMRI related studies by comparing and contrasting this technique when tinnitus is induced by high-level noise exposure and salicylate administration. Together, these studies underscore the considerable potential of MEMRI for advancing the field of auditory neuroscience in general and tinnitus research in particular. Because of the technical and functional gaps that are filled by this method and the prospect that human studies are on the near horizon, MEMRI should be of considerable interest to the auditory research community. This article is part of a Special Issue entitled .

Evidence of key tinnitus-related brain regions documented by a unique combination of manganese-enhanced MRI and acoustic startle reflex testing.

Animal models continue to improve our understanding of tinnitus pathogenesis and aid in development of new treatments. However, there are no diagnostic biomarkers for tinnitus-related pathophysiology for use in awake, freely moving animals. To address this disparity, two complementary methods were combined to examine reliable tinnitus models (rats repeatedly administered salicylate or exposed to a single noise event): inhibition of acoustic startle and manganese-enhanced MRI. Salicylate-induced tinnitus resulted in wide spread supernormal manganese uptake compared to noise-induced tinnitus. Neither model demonstrated significant differences in the auditory cortex. Only in the dorsal cortex of the inferior colliculus (DCIC) did both models exhibit supernormal uptake. Therefore, abnormal membrane depolarization in the DCIC appears to be important in tinnitus-mediated activity. Our results provide the foundation for future studies correlating the severity and longevity of tinnitus with hearing loss and neuronal activity in specific brain regions and tools for evaluating treatment efficacy across paradigms.

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.

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.

Tyrosine hydroxylase in rat auditory midbrain: distribution and changes following deafness.

Tyrosine hydroxylase (TH), a key enzyme in the catecholaminergic pathway, allows for the differentiation of dopaminergic neurons. We previously showed decreases in TH gene expression in the rat inferior colliculus (IC) 3 and 21 days following deafness. In the present study, we characterized the normal distribution of TH as well as changes following deafness (bilateral cochlear ablation) in the IC and nuclei of the lateral lemniscus. Immunostaining was compared in three groups of rats: normal hearing (n=8), 21 day deaf (n=5) and 90 days following deafening (n=5). Many TH immunoreactive fibers and puncta were identified in the IC and nuclei of the lateral lemniscus of normal hearing animals and labeling was most dense in the external cortex of the IC. We also identified immunolabeling for fibers and puncta for another catecholaminergic enzyme, dopamine beta hydroxylase (DBH), but not phenylethanolamine-N-methyltranferase (PNMT). Neurons immunopositive for TH but not DBH or PNMT were observed in the dorsal cortex and dorsal horn of the central nucleus of the IC and ventral and intermediate lemniscus. In the central nucleus of the IC and dorsal lateral lemniscus many lightly labeled TH neurons were also DBH positive. Although the number of immunopositive cells in the IC and lemniscus declined 3 weeks and 3 months after deafening, the decline was not significant at three weeks in the VNLL nor after three months in the dorsal cortex. Immunolabeling for TH decreased significantly in IC and lemniscus 3 weeks and 3 months following deafening. These results suggest a role for dopaminergic neurons and fibers in deafness-related plasticity.

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 beta2, beta3, and gamma2, plus ionotropic glutamate receptor subunits AMPA 2, AMPA 3, and kainate 2, increased at all three times. Expression of glycine receptor alpha1 initially declined and then later increased, while alpha2 increased sharply at 21 days. Glycine receptor alpha3 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 alpha2 and serotonin receptor 5B returned to normal levels of expression at 90 days. Changes in GABA receptor beta3, GABA receptor gamma2, glutamate receptor 2/3, enkephalin, and tyrosine hydroxylase were further confirmed using immunocytochemistry.

Distribution of methionine and leucine enkephalin neurons within the social behavior circuitry of the male Syrian hamster brain.

Enkephalin plays a role in the social behaviors of many species, but no corresponding role for this peptide has been investigated in the male Syrian hamster, a species in which brain nuclei controlling social behaviors have been identified. Previous studies have shown the distribution of dynorphin and beta-endorphin throughout social behavior circuits within the male hamster brain. To date, the only studies of enkephalin in the hamster brain address the distribution of this peptide in the olfactory bulb and hippocampus. The present study provides a complete map of enkephalinergic neurons within the forebrain and midbrain of the male Syrian hamster and addresses the question of whether enkephalin immunoreactive (Enk-ir) cells are found within brain regions relevant to male hamster social behaviors. Following immunocytochemistry for either methionine enkephalin (met-enkephalin) or leucine enkephalin (leu-enkephalin), we observed enkephalin localization consistent with data that have previously been reported in the rat, with notable exceptions including lateral septum, ventromedial nucleus of the hypothalamus and cingulate gyrus. Additionally, met- and leu-enkephalin localization patterns largely overlap. Consistent with the post-translational processing of preproenkephalin, met-enkephalin was more abundant than leu-enkephalin both within individual cells (darker staining), and within given brain nuclei (more met-enkephalin immunoreactive cells). Two exceptions were the posterointermediate bed nucleus of the stria terminalis, containing more neurons heavily labeled for leu-enkephalin, and the main olfactory bulb, where only met-enkephalin was observed. Of most interest for this study was the observation of Enk-ir cells and terminals in areas implicated in both sexual and agonistic behaviors in this species.