Contributions of Hearing Loss and Traumatic Brain Injury to Blast-Induced Cortical Parvalbumin Neuron Loss and Auditory Processing Deficits.

Auditory processing disorder is the most common problem affecting veterans after blast exposure, but the distinct impacts of blast-related traumatic brain injury and blast-related hearing loss are unknown. Independently, both hearing loss and blast exposure affect the entire auditory processing pathway at the molecular and physiological levels. Here, we identified distinct changes to the primary auditory cortex (AI) and temporal processing in mice following blast exposure both with and without protected hearing. Our results show that blast-exposure alone activated microglia in AI, but hearing loss was required for reductions in the density of parvalbumin-expressing interneurons. Although blast exposure impaired the temporal following response, these impairments were more severe with concurrent unilateral hearing loss, further resulting in impairments in behavioral gap detection. Taken together, these results indicate that protecting hearing during blast exposure can prevent most impairments to auditory processing but does not fully protect temporal processing.

Altered synaptic plasticity of the longitudinal dentate gyrus network in noise-induced anxiety.

Anxiety is characteristic comorbidity of noise-induced hearing loss (NIHL), which causes physiological changes within the dentate gyrus (DG), a subfield of the hippocampus that modulates anxiety. However, which DG circuit underlies hearing loss-induced anxiety remains unknown. We utilize an NIHL mouse model to investigate short- and long-term synaptic plasticity in DG networks. The recently discovered longitudinal DG-DG network is a collateral of DG neurons synaptically connected with neighboring DG neurons and displays robust synaptic efficacy and plasticity. Furthermore, animals with NIHL demonstrate increased anxiety-like behaviors similar to a response to chronic restraint stress. These behaviors are concurrent with enhanced synaptic responsiveness and suppressed short- and long-term synaptic plasticity in the longitudinal DG-DG network but not in the transverse DG-CA3 connection. These findings suggest that DG-related anxiety is typified by synaptic alteration in the longitudinal DG-DG network.

Correlation of Electrophysiological and Gene Transcriptional Dysfunctions in Single Cortical Parvalbumin Neurons After Noise Trauma.

Parvalbumin-expressing (PV+) interneurons in the sensory cortex form powerful inhibitory synapses on the perisomatic compartments and axon initial segments of excitatory principal neurons (PNs), and perform diverse computational functions. Impaired PV+ interneuron functions have been reported in neural developmental and degenerative disorders. Expression of the unique marker parvalbumin (PV) is often used as a proxy of PV+ interneuron functions. However, it is not entirely clear how PV expression is correlated with PV+ interneuron properties such as spike firing and synaptic transmission. To address this question, we characterized electrophysiological properties of PV+ interneurons in the primary auditory cortex (AI) using whole-cell patch clamp recording, and analyzed the expression of several genes in samples collected from single neurons using the patch pipettes. We found that, after noise induced hearing loss (NIHL), the spike frequency adaptation increased, and the expression of PV, glutamate decarboxylase 67 (GAD67) and Shaw-like potassium channel (KV3.1) decreased in PV+ neurons. In samples prepared from the auditory cortical tissue, the mRNA levels of the target genes were all pairwise correlated. At the single neuron level, however, the expression of PV was significantly correlated with the expression of GAD67, but not KV3.1, maximal spike frequency, or spike frequency adaptation. The expression of KV3.1 was correlated with spike frequency adaptation, but not with the expression of GAD67. These results suggest separate transcriptional regulations of PV/GAD67 vs. KV3.1, both of which are modulated by NIHL.

Chemogenetic Activation of Cortical Parvalbumin-Positive Interneurons Reverses Noise-Induced Impairments in Gap Detection.

Exposure to loud noises not only leads to trauma and loss of output from the ear but also alters downstream central auditory circuits. A perceptual consequence of noise-induced central auditory disruption is impairment in gap-induced prepulse inhibition, also known as gap detection. Recent studies have implicated cortical parvalbumin (PV)-positive inhibitory interneurons in gap detection and prepulse inhibition. Here, we show that exposure to loud noises specifically reduces the density of cortical PV but not somatostatin (SOM)-positive interneurons in the primary auditory cortex in mice (C57BL/6) of both sexes. Optogenetic activation of PV neurons produced less cortical inhibition in noise-exposed than sham-exposed animals, indicative of reduced PV neuron function. Activation of SOM neurons resulted in similar levels of cortical inhibition in noise- and sham-exposed groups. Furthermore, chemogenetic activation of PV neurons with the hM3-based designer receptor exclusively activated by designer drugs completely reversed the impairments in gap detection for noise-exposed animals. These results support the notions that cortical PV neurons encode gap in sound and that PV neuron dysfunction contributes to noise-induced impairment in gap detection.SIGNIFICANCE STATEMENT Noise-induced hearing loss contributes to a range of central auditory processing deficits (CAPDs). The mechanisms underlying noise-induced CAPDs are still poorly understood. Here we show that exposure to loud noises results in dysfunction of PV-positive but not somatostatin-positive inhibitory interneurons in the primary auditory cortex. In addition, cortical PV inhibitory neurons in noise-exposed animals had reduced expression of glutamic acid decarboxylases and weakened inhibition on cortical activity. Noise exposure resulted in impaired gap detection, indicative of disrupted temporal sound processing and possibly tinnitus. We found that chemogenetic activation of cortical PV inhibitory interneurons alleviated the deficits in gap detection. These results implicate PV neuron dysfunction as a mechanism for noise-induced CAPDs.

Neuroinflammation and Tinnitus.

Neuroinflammation is the central nervous system's response to: injury, infection, and abnormal neural activity. Inflammatory processes are known to mediate many diseases, and recently evidence indicates that neuroinflammation underlies hearing disorders such as presbyacusis, middle-ear disease, ototoxicity, noise-induced hearing loss, and tinnitus. This chapter provides a review of the role of neuroinflammation in the etiology and treatment of tinnitus. Specifically, our research team has demonstrated that both tumor necrosis factor alpha (TNF-α) and calpain signaling pathways are involved in noise-induced tinnitus and that blocking them yielded therapeutic effects on tinnitus. Other efforts such as controlling acute inflammatory response via specialized pro-resolving mediators may help provide insight into preventing and treating tinnitus-related inflammatory processes.

Long-Term Enhancement of NMDA Receptor Function in Inhibitory Neurons Preferentially Modulates Potassium Channels and Cell Adhesion Molecules.

Effectively enhancing the activity of inhibitory neurons has great therapeutic potentials since their reduced function/activity has significant contributions to pathology in various brain diseases. We showed previously that NMDAR positive allosteric modulator GNE-8324 and M-8324 selectively increase NMDAR activity on the inhibitory neurons and elevates their activity in vitro and in vivo. Here we examined the impact of long-term administering M-8324 on the functions and transcriptional profiling of parvalbumin-containing neurons in two representative brain regions, primary auditory cortex (Au1) and prelimbic prefrontal cortex (PrL-PFC). We found small changes in key electrophysiological parameters and RNA levels of neurotransmitter receptors, Na+ and Ca2+ channels. In contrast, large differences in cell adhesion molecules and K+ channels were found between Au1 and PrL-PFC in drug-naïve mice, and differences in cell adhesion molecules became much smaller after M-8324 treatment. There was also minor impact of M-8324 on cell cycle and apoptosis, suggesting a fine safety profile.

Diffusible Tumor Necrosis Factor-Alpha (TNF-α) Promotes Noise-Induced Parvalbumin-Positive (PV+) Neuron Loss and Auditory Processing Impairments.

Neuroinflammation has been implicated in noise-induced auditory processing disorder and tinnitus. Certain non-auditory neurological disorders can also increase the levels of proinflammatory cytokines in the brain. To investigate the impact of increased brain proinflammatory cytokine levels on the central auditory pathway, we infused recombinant TNF-α into the right lateral cerebral ventricle, and examined auditory processing and cytoarchitecture of the auditory cortex. Microglial deramification was observed in the auditory cortex of mice that had received both TNF-α infusion and exposure to an 86-dB noise, but not in mice that had received either TNF-α infusion or noise exposure alone. In addition, we observed reduced cortical PV+ neuron density and impaired performances in gap detection and prepulse inhibition (PPI) only in mice that received both TNF-α infusion and the noise exposure. These results suggest that disease-related increase in brain proinflammatory cytokine release could be a risk factor for noise-induced auditory processing disorder and tinnitus.

Resistance to noise-induced gap detection impairment in FVB mice is correlated with reduced neuroinflammatory response and parvalbumin-positive neuron loss.

Exposure to loud noises results in neuroinflammatory responses in the central auditory pathway. Noise-induced neuroinflammation is implicated in auditory processing deficits such as impairment in gap detection. In this study, we examined whether strain differences between the FVB and C57BL/6 mice in noise-induced impairment in gap detection are correlated with strain differences in neuroinflammatory responses. We found that noise induced more robust TNF-α expression in C57BL/6 than in FVB mice. Noise-induced microglial deramification was observed in C57BL/6 mice, but not in FVB mice. Furthermore, noise exposure resulted in a reduction in parvalbumin-positive (PV+) neuron density in the C57BL/6 mice, but not in FVB mice. These results suggest that neuroinflammatory responses and loss of PV+ neurons may contribute to strain differences in noise-induced impairment in gap detection.

Increasing endogenous activity of NMDARs on GABAergic neurons increases inhibition, alters sensory processing and prevents noise-induced tinnitus.

Selective enhancement of GABAergic inhibition is thought to impact many vital brain functions and interferes with the genesis and/or progression of numerous brain disorders. Here, we show that selectively increasing NMDA receptor activity in inhibitory neurons using an NMDAR positive allosteric modulator (PAM) elevates spiking activity of inhibitory neurons in vitro and in vivo. In vivo infusion of PAM increases spontaneous and sound-evoked spiking in inhibitory and decreases spiking in excitatory neurons, and increases signal-to-noise ratio in the primary auditory cortex. In addition, PAM infusion prior to noise trauma prevents the occurrence of tinnitus and reduction in GABAergic inhibition. These results reveal that selectively enhancing endogenous NMDAR activity on the GABAergic neurons can effectively enhance inhibitory activity and alter excitatory-inhibitory balance, and may be useful for preventing diseases that involve reduced inhibition as the major cause.

Pharmacological DNA Demethylation Weakens Inhibitory Synapses in the Auditory Cortex and Re-opens the Critical Period for Frequency Map Plasticity.

The critical period is a time of maximal plasticity within the cortex. The progression of the critical period is marked by experience-dependent transcriptional alterations in cortical neurons, which in turn shifts the excitatory-inhibitory balance in the brain, and accordingly reduces plasticity. Epigenetic mechanisms, such as DNA methylation, control the transcriptional state of neurons, and have been shown to be dynamically regulated during the critical period. Here we show that adult animals have a significantly higher concentration of DNA methylation than critical period animals. Pharmacological reduction of DNA methylation in adult animals re-establishes critical period auditory map plasticity. Furthermore, the reduction of DNA methylation in adult animals, reverted intrinsic characteristics of inhibitory synapses to an immature state. Our data suggest that accumulation of DNA methylation during the critical period confers a mature phenotype to cortical neurons, which in turn, facilitates the reduction in plasticity seen after the critical period.

Tinnitus Correlates with Downregulation of Cortical Glutamate Decarboxylase 65 Expression But Not Auditory Cortical Map Reorganization.

Hearing loss is the biggest risk factor for tinnitus, and hearing-loss-related pathological changes in the auditory pathway have been hypothesized as the mechanism underlying tinnitus. However, due to the comorbidity of tinnitus and hearing loss, it has been difficult to differentiate between neural correlates of tinnitus and consequences of hearing loss. In this study, we dissociated tinnitus and hearing loss in FVB mice, which exhibit robust resistance to tinnitus following monaural noise-induced hearing loss. Furthermore, knock-down of glutamate decarboxylase 65 (GAD65) expression in auditory cortex (AI) by RNA interference gave rise to tinnitus in normal-hearing FVB mice. We found that tinnitus was significantly correlated with downregulation of GAD65 in the AI. By contrast, cortical map distortions, which have been hypothesized as a mechanism underlying tinnitus, were correlated with hearing loss but not tinnitus. Our findings suggest new strategies for the rehabilitation of tinnitus and other phantom sensation, such as phantom pain.SIGNIFICANCE STATEMENT Hearing loss is the biggest risk factor for tinnitus in humans. Most animal models of tinnitus also exhibit comorbid hearing loss, making it difficult to dissociate the mechanisms underlying tinnitus from mere consequences of hearing loss. Here we show that, although both C57BL/6 and FVB mice exhibited similar noise-induced hearing threshold increase, only C57BL/6, but not FVB, mice developed tinnitus following noise exposure. Although both strains showed frequency map reorganization following noise-induced hearing loss, only C57BL/6 mice had reduced glutamate decarboxylase 65 (GAD65) expression in the auditory cortex (AI). Knocking down GAD65 expression in the AI resulted in tinnitus in normal-hearing FVB mice. Our results suggest that reduced inhibitory neuronal function, but not sensory map reorganization, underlies noise-induced tinnitus.

Neuroinflammation mediates noise-induced synaptic imbalance and tinnitus in rodent models.

Hearing loss is a major risk factor for tinnitus, hyperacusis, and central auditory processing disorder. Although recent studies indicate that hearing loss causes neuroinflammation in the auditory pathway, the mechanisms underlying hearing loss-related pathologies are still poorly understood. We examined neuroinflammation in the auditory cortex following noise-induced hearing loss (NIHL) and its role in tinnitus in rodent models. Our results indicate that NIHL is associated with elevated expression of proinflammatory cytokines and microglial activation-two defining features of neuroinflammatory responses-in the primary auditory cortex (AI). Genetic knockout of tumor necrosis factor alpha (TNF-α) or pharmacologically blocking TNF-α expression prevented neuroinflammation and ameliorated the behavioral phenotype associated with tinnitus in mice with NIHL. Conversely, infusion of TNF-α into AI resulted in behavioral signs of tinnitus in both wild-type and TNF-α knockout mice with normal hearing. Pharmacological depletion of microglia also prevented tinnitus in mice with NIHL. At the synaptic level, the frequency of miniature excitatory synaptic currents (mEPSCs) increased and that of miniature inhibitory synaptic currents (mIPSCs) decreased in AI pyramidal neurons in animals with NIHL. This excitatory-to-inhibitory synaptic imbalance was completely prevented by pharmacological blockade of TNF-α expression. These results implicate neuroinflammation as a therapeutic target for treating tinnitus and other hearing loss-related disorders.

A circuit mechanism of time-to-space conversion for perception.

Sensory information in a temporal sequence is processed as a collective unit by the nervous system. The cellular mechanisms underlying how sequential inputs are incorporated into the brain has emerged as an important subject in neuroscience. Here, we hypothesize that information-bearing (IB) signals can be entrained and amplified by a clock signal, allowing them to efficiently propagate along in a feedforward circuit. IB signals can remain latent on individual dendrites of the receiving neurons until they are read out by an oscillatory clock signal. In such a way, the IB signals pass through the next neurons along a linear chain. This hypothesis identifies a cellular process of time-to-space and sound-to-map conversion in primary auditory cortex, providing insight into a mechanistic principle underlying the representation and memory of temporal sequences of information.

Blocking Tumor Necrosis Factor-Alpha Expression Prevents Blast-Induced Excitatory/Inhibitory Synaptic Imbalance and Parvalbumin-Positive Interneuron Loss in the Hippocampus.

Traumatic brain injury (TBI) is a major cause of neurological disorder and death in civilian and military populations. It comprises two components-direct injury from the traumatic impact and secondary injury from ensuing neural inflammatory responses. Blocking tumor necrosis factor-alpha (TNF-α), a central regulator of neural inflammation, has been shown to improve functional recovery after TBI. However, the mechanisms underlying those therapeutic effects are still poorly understood. Here, we examined effects of 3,6'-dithiothalidomide (dTT), a potentially therapeutic TNF-α inhibitor, in mice with blast-induced TBI. We found that blast exposure resulted in elevated expression of TNF-α, activation of microglial cells, enhanced excitatory synaptic transmission, reduced inhibitory synaptic transmission, and a loss of parvalbumin-positive (PV+) inhibitory interneurons. Administration of dTT for 5 days after the blast exposure completely suppressed blast-induced increases in TNF-α transcription, largely reversed blasted-induced synaptic changes, and prevented PV+ neuron loss. However, blocking TNF-α expression by dTT failed to mitigate blast-induced microglial activation in the hippocampus, as evidenced by their non-ramified morphology. These results indicate that TNF-α plays a major role in modulating neuronal functions in blast-induced TBI and that it is a potential target for treatment of TBI-related brain disorders.

Blast Exposure Disrupts the Tonotopic Frequency Map in the Primary Auditory Cortex.

Blast exposure can cause various auditory disorders including tinnitus, hyperacusis, and other central auditory processing disorders. While this is suggestive of pathologies in the central auditory system, the impact of blast exposure on central auditory processing remains poorly understood. Here we examined the effects of blast shockwaves on acoustic response properties and the tonotopic frequency map in the auditory cortex. We found that multiunits recorded from the auditory cortex exhibited higher acoustic thresholds and broader frequency tuning in blast-exposed animals. Furthermore, the frequency map in the primary auditory cortex was distorted. These changes may contribute to central auditory processing disorders.

Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation.

3D silicon neural probe with integrated optical fibers for optogenetic modulation.

Optogenetics is a powerful modality for neural modulation that can be useful for a wide array of biomedical studies. Penetrating microelectrode arrays provide a means of recording neural signals with high spatial resolution. It is highly desirable to integrate optics with neural probes to allow for functional study of neural tissue by optogenetics. In this paper, we report the development of a novel 3D neural probe coupled simply and robustly to optical fibers using a hollow parylene tube structure. The device shanks are hollow tubes with rigid silicon tips, allowing the insertion and encasement of optical fibers within the shanks. The position of the fiber tip can be precisely controlled relative to the electrodes on the shank by inherent design features. Preliminary in vivo rat studies indicate that these devices are capable of optogenetic modulation simultaneously with 3D neural signal recording.

Perceptual learning in the developing auditory cortex.

A hallmark of the developing auditory cortex is the heightened plasticity in the critical period, during which acoustic inputs can indelibly alter cortical function. However, not all sounds in the natural acoustic environment are ethologically relevant. How does the auditory system resolve relevant sounds from the acoustic environment in such an early developmental stage when most associative learning mechanisms are not yet fully functional? What can the auditory system learn from one of the most important classes of sounds, animal vocalizations? How does naturalistic acoustic experience shape cortical sound representation and perception? To answer these questions, we need to consider an unusual strategy, statistical learning, where what the system needs to learn is embedded in the sensory input. Here, I will review recent findings on how certain statistical structures of natural animal vocalizations shape auditory cortical acoustic representations, and how cortical plasticity may underlie learned categorical sound perception. These results will be discussed in the context of human speech perception.

Failed stabilization for long-term potentiation in the auditory cortex of FMR1 knockout mice.

Fragile X syndrome is a developmental disorder that affects sensory systems. A null mutation of the Fragile X Mental Retardation protein 1 (Fmr1) gene in mice has varied effects on developmental plasticity in different sensory systems, including normal barrel cortical plasticity, altered ocular dominance plasticity and grossly impaired auditory frequency map plasticity. The mutation also has different effects on long-term synaptic plasticity in somatosensory and visual cortical neurons, providing insights on how it may differentially affect the sensory systems. Here we present evidence that long-term potentiation (LTP) is impaired in the developing auditory cortex of the Fmr1 knockout (KO) mice. This impairment of synaptic plasticity is consistent with impaired frequency map plasticity in the Fmr1 KO mouse. Together, these results suggest a potential role of LTP in sensory map plasticity during early sensory development.