Tinnitus mechanisms and the need for an objective electrophysiological tinnitus test.

Tinnitus, the perception of sound with no external auditory stimulus, is a complex, multifaceted, and potentially devastating disorder. Despite recent advances in our understanding of tinnitus, there are limited options for effective treatment. Tinnitus treatments are made more complicated by the lack of a test for tinnitus based on objectively measured physiological characteristics. Such an objective test would enable a greater understanding of tinnitus mechanisms and may lead to faster treatment development in both animal and human research. This review makes the argument that an objective tinnitus test, such as a non-invasive electrophysiological measure, is desperately needed. We review the current tinnitus assessment methods, the underlying neural correlates of tinnitus, the multiple tinnitus generation theories, and the previously investigated electrophysiological measurements of tinnitus. Finally, we propose an alternate objective test for tinnitus that may be valid in both animal and human subjects.

Comparison of two behavioral tests for tinnitus assessment in mice.

Animal research focused on chronic tinnitus associated with noise-induced hearing loss can be expensive and time-consuming as a result of the behavioral training required. Although there exist a number of behavioral tests for tinnitus; there have been few formal direct comparisons of these tests. Here, we evaluated animals in two different tinnitus assessment methods. CBA/CaJ mice were trained in an operant conditioning, active avoidance (AA) test, and a reflexive, gap-induced pre-pulse inhibition of acoustic startle (GPIAS) test, or both. Tinnitus was induced in awake mice by unilateral continuous sound exposure using a 2-kHz- or 1 2 octave-wide noise centered at 16 kHz and presented at 113- or 116-dB SPL. Tinnitus was assessed 8 weeks after sound overexposure. Most mice had evidence of tinnitus behavior in at least one of the two behaviors. Of the mice evaluated in AA, over half (55%) had tinnitus positive behavior. In GPIAS, fewer animals (13%) were positive than were identified using the AA test. Few mice were positive in both tests (10%), and only one was positive for tinnitus behavior at the same spectral frequency in both tests. When the association between tinnitus behavior and spontaneous activity recorded in the inferior colliculus was compared, animals with tinnitus behavior in AA exhibited increased spontaneous activity, while those positive in GPIAS did not. Thus, it appears that operant conditioning tests, like AA, maybe more reliable and accurate tests for tinnitus than reflexive tests.

Long-Duration Sound-Induced Facilitation Changes Population Activity in the Inferior Colliculus.

The inferior colliculus (IC) is at the midpoint of the auditory system and integrates virtually all information ascending from the auditory brainstem, organizes it, and transmits the results to the auditory forebrain. Its abundant, excitatory local connections are crucial for this task. This study describes a long duration sound (LDS)-induced potentiation in the IC that changes both subsequent tone-evoked responses and spontaneous activity. Afterdischarges, changes of spontaneous spiking following an LDS, were seen previously in single neurons. Here, we used multi-channel probes to record activity before and after a single, tetanic sound and describe the changes in a population of IC neurons. Following a 60 s narrowband-noise stimulation, a subset of recording channels (∼16%) showed afterdischarges. A facilitated response spike rate to tone pips following an LDS was also observed in ∼16% of channels. Both channels with an afterdischarge and channels with facilitated tone responses had higher firing rates in response to LDS, and the magnitude of the afterdischarges increased with increased responses to the LDS. This is the first study examining the effect of LDS stimulation on tone-evoked responses. This observed facilitation in vivo has similarities to post-tetanic potentiation in vitro as both manner of induction (strong stimulation for several seconds) as well as time-course of the facilitation (second to minute range) are comparable. Channels with and without facilitation appear to be intermixed and distributed widely in the central nucleus of IC, and this suggests a heretofore unknown property of some IC neurons or their circuits. Consequently, this sound-evoked facilitation may enhance the sound-evoked output of these neurons, while, simultaneously, most other IC neurons have reduced or unchanged output in response to the same stimulus.

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.

Neuronal sensitivity to the interaural time difference of the sound envelope in the mouse inferior colliculus.

We examined the sensitivity of the neurons in the mouse inferior colliculus (IC) to the interaural time differences (ITD) conveyed in the sound envelope. Utilizing optogenetic methods, we compared the responses to the ITD in the envelope of identified glutamatergic and GABAergic neurons. More than half of both cell types were sensitive to the envelope ITD, and the ITD curves were aligned at their troughs. Within the physiological ITD range of mice (±50 µs), the ITD curves of both cell types had a higher firing rate when the contralateral envelope preceded the ipsilateral envelope. These results show that the circuitry to process ITD persists in the mouse despite its lack of low-frequency hearing. The sensitivity of IC neurons to ITD is most likely to be shaped by the binaural interaction of excitation and inhibition in the lateral superior olive.

Mice heterozygous for the Cdh23/Ahl1 mutation show age-related deficits in auditory temporal processing.

A mutation in the Cdh23 gene is implicated in both syndromic and nonsyndromic hearing loss in humans and age-related hearing loss in C57BL/6 mice. It is generally assumed that human patients (as well as mouse models) only have a hearing loss phenotype if the mutation is homozygous. However, a major complaint for patients with a hearing disability is a reduced speech intelligibility that may be related to temporal processing deficits rather than just elevated thresholds. In this study, we used the amplitude modulation following response (AMFR) to test whether mice heterozygous for Cdh23735A > G have an auditory phenotype that includes temporal processing deficits. The hearing of mice heterozygous for the Cdh23735A > G mutation was compared with age-matched mice homozygous for either the mutation or the wild type in 3 cohorts of mice of both sexes at 2-3, 6, and 12 months of age. The AMFR technique was used to generate objective hearing thresholds for all mice across their range of hearing and to test their temporal processing. We found a genotype-dependent hearing loss in mice homozygous for the mutation starting at 5-11 weeks of age, an age when mice on the C57BL/6 background are often presumed to have normal hearing. The heterozygous animals retained normal hearing thresholds up to one year of age. Nevertheless, the heterozygous animals showed a decline in temporal processing abilities at one year of age that was independent of their hearing thresholds. These results suggest that mice heterozygous for the Cdh23 mutation do not have truly normal hearing.

Intravenously-injected gold nanoparticles (AuNPs) access intracerebral F98 rat gliomas better than AuNPs infused directly into the tumor site by convection enhanced delivery.

BACKGROUND: Intravenously (IV)-injected gold nanoparticles (AuNPs) powerfully enhance the efficacy of X-ray therapy of tumors including advanced gliomas. However, pharmacokinetic issues, such as slow tissue clearance and skin discoloration, may impede clinical translation. The direct infusion of AuNPs into the tumor might be an alternative mode of delivery. MATERIALS AND METHODS: Using the advanced, invasive, and difficult-to-treat F98 rat glioma model, we have studied the biodistribution of the AuNPs in the tumor and surrounding brain after either IV injection or direct intratumoral infusion by convection-enhanced delivery using light microscopy immunofluorescence and direct gold visualization. RESULTS: IV-injected AuNPs localize more specifically to intracerebral tumor cells, both in the main tumor mass and in the migrated tumor cells as well as the tumor edema, than do the directly infused AuNPs. Although some of the directly infused AuNPs do access the main tumor region, such access is largely restricted. CONCLUSION: These data suggest that IV-injected AuNPs are likely to have a greater therapeutic benefit when combined with radiation therapy than after the direct infusion of AuNPs.

Identified GABAergic and Glutamatergic Neurons in the Mouse Inferior Colliculus Share Similar Response Properties.

GABAergic neurons in the inferior colliculus (IC) play a critical role in auditory information processing, yet their responses to sound are unknown. Here, we used optogenetic methods to characterize the response properties of GABAergic and presumed glutamatergic neurons to sound in the IC. We found that responses to pure tones of both inhibitory and excitatory classes of neurons were similar in their thresholds, response latencies, rate-level functions, and frequency tuning, but GABAergic neurons may have higher spontaneous firing rates. In contrast to their responses to pure tones, the inhibitory and excitatory neurons differed in their ability to follow amplitude modulations. The responses of both cell classes were affected by their location regardless of the cell type, especially in terms of their frequency tuning. These results show that the synaptic domain, a unique organization of local neural circuits in the IC, may interact with all types of neurons to produce their ultimate response to sound.SIGNIFICANCE STATEMENT Although the inferior colliculus (IC) in the auditory midbrain is composed of different types of neurons, little is known about how these specific types of neurons respond to sound. Here, for the first time, we characterized the response properties of GABAergic and glutamatergic neurons in the IC. Both classes of neurons had diverse response properties to tones but were overall similar, except for the spontaneous activity and their ability to follow amplitude-modulated sound. Both classes of neurons may compose a basic local circuit that is replicated throughout the IC. Within each local circuit, the inputs to the local circuit may have a greater influence in determining the response properties to sound than the specific neuron types.

Long-Lasting Sound-Evoked Afterdischarge in the Auditory Midbrain.

Different forms of plasticity are known to play a critical role in the processing of information about sound. Here, we report a novel neural plastic response in the inferior colliculus, an auditory center in the midbrain of the auditory pathway. A vigorous, long-lasting sound-evoked afterdischarge (LSA) is seen in a subpopulation of both glutamatergic and GABAergic neurons in the central nucleus of the inferior colliculus of normal hearing mice. These neurons were identified with single unit recordings and optogenetics in vivo. The LSA can continue for up to several minutes after the offset of the sound. LSA is induced by long-lasting, or repetitive short-duration, innocuous sounds. Neurons with LSA showed less adaptation than the neurons without LSA. The mechanisms that cause this neural behavior are unknown but may be a function of intrinsic mechanisms or the microcircuitry of the inferior colliculus. Since LSA produces long-lasting firing in the absence of sound, it may be relevant to temporary or chronic tinnitus or to some other aftereffect of long-duration sound.

Functional organization of the local circuit in the inferior colliculus.

The inferior colliculus (IC) is the first integration center of the auditory system. After the transformation of sound to neural signals in the cochlea, the signals are analyzed by brainstem auditory nuclei that, in turn, transmit this information to the IC. However, the neural circuitry that underlies this integration is unclear. This review consists of two parts: one is about the cell type which is likely to integrate sound information, and the other is about a technique which is useful for studying local circuitry. Large GABAergic (LG) neurons receive dense excitatory axosomatic terminals that originate from the lower brainstem auditory nuclei as well as local IC neurons. Dozens of axons coming from both local and lower brainstem neurons converge on a single LG soma. Excitatory neurons in IC can innervate many nearby LG somata in the same fibrodendritic lamina. The combination of local and ascending inputs is well suited for auditory integration. LG neurons are one of the main sources of inhibition in the medial geniculate body (MGB). LG neurons and the tectothalamic inhibitory system are present in a wide variety of mammalian species. This suggests that the circuitry of excitatory and inhibitory tectothalamic projections may have evolved earlier than GABAergic interneurons in the MGB, which are found in fewer species. Cellular-level functional imaging provides both morphological and functional information about local circuitry. In the last part of this review, we describe an in vivo calcium imaging study that sheds light on the functional organization of the IC.

Differences in the strength of cortical and brainstem inputs to SSA and non-SSA neurons in the inferior colliculus.

In an ever changing auditory scene, change detection is an ongoing task performed by the auditory brain. Neurons in the midbrain and auditory cortex that exhibit stimulus-specific adaptation (SSA) may contribute to this process. Those neurons adapt to frequent sounds while retaining their excitability to rare sounds. Here, we test whether neurons exhibiting SSA and those without are part of the same networks in the inferior colliculus (IC). We recorded the responses to frequent and rare sounds and then marked the sites of these neurons with a retrograde tracer to correlate the source of projections with the physiological response. SSA neurons were confined to the non-lemniscal subdivisions and exhibited broad receptive fields, while the non-SSA were confined to the central nucleus and displayed narrow receptive fields. SSA neurons receive strong inputs from auditory cortical areas and very poor or even absent projections from the brainstem nuclei. On the contrary, the major sources of inputs to the neurons that lacked SSA were from the brainstem nuclei. These findings demonstrate that auditory cortical inputs are biased in favor of IC synaptic domains that are populated by SSA neurons enabling them to compare top-down signals with incoming sensory information from lower areas.

Differential distribution of GABA and glycine terminals in the inferior colliculus of rat and mouse.

The inferior colliculus (IC), the midbrain component of the auditory pathway, integrates virtually all inputs from the auditory brainstem. These are a mixture of excitatory and inhibitory ascending inputs, and the inhibitory transmitters include both gamma-aminobutyric acid (GABA) and glycine (GLY). Although the presence of these inhibitory inputs is well established, their relative location in the IC is not, and there is little information on the mouse. Here, we study the distribution of glutamic acid decarboxylase (GAD)67 and GLY transporter 2 (T2) in axonal terminals to better understand the relative contributions of these inputs. Large-scale mosaic composite images of immunohistochemistry sections of rat and mice were used to isolate the signals related to the concentrations of these axonal terminals in the tissue, and the ratio of GLYT2/GAD67 in each pixel was calculated. GLYT2 was seen only in the central nucleus of the IC (ICC), whereas GAD67 was seen throughout the IC. The map of the GAD67 and GLYT2 axonal distribution revealed a gradient that runs from ventrolateral to dorsomedial along the axis of the laminae of the ICC and perpendicular to the tonotopic axis. Although anatomically different, both the mouse and the rat had relatively more GAD67 dorsomedially in the ICC and relatively more GLYT2 ventrolaterally. This organization of GABA and GLY inputs may be related to functional zones with different properties in ICC that are based, in part, on different sets of inhibitory inputs to each zone.

Convergence of Lemniscal and Local Excitatory Inputs on Large GABAergic Tectothalamic Neurons.

Large GABAergic (LG) neurons form a distinct cell type in the inferior colliculus (IC), identified by the presence of dense VGLUT2-containing axosomatic terminals. Although some of the axosomatic terminals originate from local and commissural IC neurons, it has been unclear whether LG neurons also receive axosomatic inputs from the lower auditory brainstem nuclei, i.e., cochlear nuclei (CN), superior olivary complex (SOC), and nuclei of the lateral lemniscus (NLL). In this study we injected recombinant viral tracers that force infected cells to express GFP in a Golgi-like manner into the lower auditory brainstem nuclei to determine whether these nuclei directly innervate LG cell somata. Labeled axons from CN, SOC, and NLL terminated as excitatory axosomatic endings, identified by colabeling of GFP and VGLUT2, on single LG neurons in the IC. Each excitatory axon made only a few axosomatic contacts on each LG neuron. Inputs to a single LG cell are unlikely to be from a single brainstem nucleus, since lesions of individual nuclei failed to eliminate most VGLUT2-positive terminals on the LG neurons. The estimated number of inputs on a single LG cell body was almost proportional to the surface area of the cell body. Double injections of different viruses into IC and a brainstem nucleus showed that LG neurons received inputs from both. These results demonstrated that both ascending and intrinsic sources converge on the LG somata to control inhibitory tectothalamic projections.

Asymmetric temporal interactions of sound-evoked excitatory and inhibitory inputs in the mouse auditory midbrain.

In the auditory midbrain, synaptic mechanisms responsible for the precise temporal coding of inputs in the brainstem are absent. Instead, in the inferior colliculus (IC), the diverse temporal firing patterns must be coded by other synaptic mechanisms, about which little is known. Here, we demonstrate the temporal characteristics of sound-evoked excitatory and inhibitory postsynaptic currents (seEPSCs and seIPSCs, respectively) in vivo in response to long-duration tones. The seEPSCs and seIPSCs differ in the variability of their temporal properties. The seEPSCs have either early or late current peaks, and the early-peaked currents may be either transient or sustained varieties. The seIPSCs have only early-peaked sustained responses but often have offset responses. When measured in a single neuron, the seIPSC peaks usually follow early, transient seEPSCs, but the seIPSCs precede latest-peaking seEPSCs. A model of the firing produced by the integration of asymmetric seEPSCs and seIPSCs showed that the temporal pattern of the early-peaked sustained neurons was easily modified by changing the parameters of the seIPSC. These results suggest that the considerable variability in the peak time and duration of the seEPSCs shapes the overall time course of firing and often precedes or follows the less variable seIPSC. Despite this, the inhibitory currents are potent in modifying the firing patterns, and the inhibitory response to sound offset appears to be one area where the integration of excitatory and inhibitory synaptic currents is lacking. Thus, the integration of sound-evoked activity in the IC often employs the asymmetric temporal interaction of excitatory and inhibitory synaptic currents to shape the firing pattern of the neuron.

Local and commissural IC neurons make axosomatic inputs on large GABAergic tectothalamic neurons.

Large GABAergic (LG) neurons are a distinct type of neuron in the inferior colliculus (IC) identified by their dense vesicular glutamate transporter 2 (VGLUT2)-containing axosomatic synaptic terminals. Yet the sources of these terminals are unknown. Since IC glutamatergic neurons express VGLUT2, and IC neurons are known to have local collaterals, we tested the hypothesis that these excitatory, glutamatergic axosomatic inputs on LG neurons come from local axonal collaterals and commissural IC neurons. We injected a recombinant viral tracer into the IC which enabled Golgi-like green fluorescent protein (GFP) labeling in both dendrites and axons. In all cases, we found terminals positive for both GFP and VGLUT2 (GFP+/VGLUT2+) that made axosomatic contacts on LG neurons. One to six axosomatic contacts were made on a single LG cell body by a single axonal branch. The GFP-labeled neurons giving rise to the VGLUT2+ terminals on LG neurons were close by. The density of GFP+/VGLUT2+ terminals on the LG neurons was related to the number of nearby GFP-labeled cells. On the contralateral side, a smaller number of LG neurons received axosomatic contacts from GFP+/VGLUT2+ terminals. In cases with a single GFP-labeled glutamatergic neuron, the labeled axonal plexus was flat, oriented in parallel to the fibrodendritic laminae, and contacted 9-30 LG cell bodies within the plexus. Our data demonstrated that within the IC microcircuitry there is a convergence of inputs from local IC excitatory neurons on LG cell bodies. This suggests that LG neurons are heavily influenced by the activity of the nearby laminar glutamatergic neurons in the IC.

The balance of excitatory and inhibitory synaptic inputs for coding sound location.

The localization of high-frequency sounds in the horizontal plane uses an interaural-level difference (ILD) cue, yet little is known about the synaptic mechanisms that underlie processing this cue in the inferior colliculus (IC) of mouse. Here, we study the synaptic currents that process ILD in vivo and use stimuli in which ILD varies around a constant average binaural level (ABL) to approximate sounds on the horizontal plane. Monaural stimulation in either ear produced EPSCs and IPSCs in most neurons. The temporal properties of monaural responses were well matched, suggesting connected functional zones with matched inputs. The EPSCs had three patterns in response to ABL stimuli, preference for the sound field with the highest level stimulus: (1) contralateral; (2) bilateral highly lateralized; or (3) at the center near 0 ILD. EPSCs and IPSCs were well correlated except in center-preferred neurons. Summation of the monaural EPSCs predicted the binaural excitatory response but less well than the summation of monaural IPSCs. Binaural EPSCs often showed a nonlinearity that strengthened the response to specific ILDs. Extracellular spike and intracellular current recordings from the same neuron showed that the ILD tuning of the spikes was sharper than that of the EPSCs. Thus, in the IC, balanced excitatory and inhibitory inputs may be a general feature of synaptic coding for many types of sound processing.

Gene expression identifies distinct ascending glutamatergic pathways to frequency-organized auditory cortex in the rat brain.

A conserved feature of sound processing across species is the presence of multiple auditory cortical fields with topographically organized responses to sound frequency. Current organizational schemes propose that the ventral division of the medial geniculate body (MGBv) is a single functionally homogenous structure that provides the primary source of input to all neighboring frequency-organized cortical fields. These schemes fail to account for the contribution of MGBv to functional diversity between frequency-organized cortical fields. Here, we report response property differences for two auditory fields in the rat, and find they have nonoverlapping sources of thalamic input from the MGBv that are distinguished by the gene expression for type 1 vesicular glutamate transporter. These data challenge widely accepted organizational schemes and demonstrate a genetic plurality in the ascending glutamatergic pathways to frequency-organized auditory cortex.

The basic circuit of the IC: tectothalamic neurons with different patterns of synaptic organization send different messages to the thalamus.

The inferior colliculus (IC) in the midbrain of the auditory system uses a unique basic circuit to organize the inputs from virtually all of the lower auditory brainstem and transmit this information to the medial geniculate body (MGB) in the thalamus. Here, we review the basic circuit of the IC, the neuronal types, the organization of their inputs and outputs. We specifically discuss the large GABAergic (LG) neurons and how they differ from the small GABAergic (SG) and the more numerous glutamatergic neurons. The somata and dendrites of LG neurons are identified by axosomatic glutamatergic synapses that are lacking in the other cell types and exclusively contain the glutamate transporter VGLUT2. Although LG neurons are most numerous in the central nucleus of the IC (ICC), an analysis of their distribution suggests that they are not specifically associated with one set of ascending inputs. The inputs to ICC may be organized into functional zones with different subsets of brainstem inputs, but each zone may contain the same three neuron types. However, the sources of VGLUT2 axosomatic terminals on the LG neuron are not known. Neurons in the dorsal cochlear nucleus, superior olivary complex, intermediate nucleus of the lateral lemniscus, and IC itself that express the gene for VGLUT2 only are the likely origin of the dense VGLUT2 axosomatic terminals on LG tectothalamic neurons. The IC is unique since LG neurons are GABAergic tectothalamic neurons in addition to the numerous glutamatergic tectothalamic neurons. SG neurons evidently target other auditory structures. The basic circuit of the IC and the LG neurons in particular, has implications for the transmission of information about sound through the midbrain to the MGB.