Publications using TDT

Central serotonin (5-HT) orchestrates myriad cognitive processes and lies at the core of many stress-related psychiatric illnesses. However, the basic relationship between its brain-wide axonal projections and functional dynamics is not known. Here we combine optogenetics and fMRI to produce a brain-wide 5-HT evoked functional map. We find that DRN photostimulation leads to an increase in the hemodynamic response in the DRN itself, while projection areas predominately exhibit a reduction of cerebral blood volume mirrored by suppression of cortical delta oscillations. We find that the regional distribution of post-synaptically expressed 5-HT receptors better correlates with DRN 5-HT functional connectivity than anatomical projections. Our work suggests that neuroarchitecture is not the primary determinant of function for the DRN 5-HT. With respect to two 5-HT elevating stimuli, we find that acute stress leads to circuit-wide blunting of the DRN output, while the SSRI fluoxetine noticeably enhances DRN functional connectivity. These data provide fundamental insight into the brain-wide functional dynamics of the 5-HT projection system.

Cisplatin-induced ototoxicity is one of the major adverse effects in cisplatin chemotherapy, and hearing protective approaches are unavailable in clinical practice. Recent work unveiled a critical role of autophagy in cell survival in various types of hearing loss. Since the excessive activation of autophagy can contribute to apoptotic cell death, whether the activation of autophagy increases or decreases the rate of cell death in CDDP ototoxicity is still being debated. In this study, we showed that CDDP induced activation of autophagy in the auditory cell HEI-OC1 at the early stage. We then used rapamycin, an autophagy activator, to increase the autophagy activity, and found that the cell death significantly decreased after CDDP injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly increased cell death. In accordance with in vitro results, rapamycin alleviated CDDP-induced death of hair cells in zebrafish lateral line and cochlear hair cells in mice. Notably, we found that CDDP-induced increase of Sirtuin 1 (SIRT1) in the HEI-OC1 cells modulated the autophagy function. The specific SIRT1 activator SRT1720 could successfully protect against CDDP-induced cell loss in HEI-OC1 cells, zebrafish lateral line, and mice cochlea. These findings suggest that SIRT1 and autophagy activation can be suggested as potential therapeutic strategies for the treatment of CDDP-induced ototoxicity.

Microelectrode arrays designed to map and modulate neuronal circuitry have enabled greater understanding and treatment of neurological injury and disease. Reliable detection of neuronal activity over time is critical for the successful application of chronic recording devices. Here, we assess device-related plasticity by exploring local changes in ion channel expression and their relationship to device performance over time. We investigated four voltage-gated ion channels (Kv1.1, Kv4.3, Kv7.2, and Nav1.6) based on their roles in regulating action potential generation, firing patterns, and synaptic efficacy. We found that a progressive increase in potassium channel expression and reduction in sodium channel expression accompanies signal loss over 6 weeks (both LFP amplitude and number of units). This motivated further investigation into a mechanistic role of ion channel expression in recorded signal instability. We employed siRNA in neuronal culture to find that Kv7.2 knockdown (as a model for the transient downregulation observed at 1 day in vivo) mimics excitatory synaptic remodeling around devices. This work provides new insight into the mechanisms underlying signal loss over time.

Processing incentive and drive is essential for control of goal-directed behavior. The limbic part of the basal ganglia has been emphasized in these processes, yet the exact neuronal mechanism has remained elusive. In this study, we examined the neuronal activity of the ventral pallidum (VP) and its upstream area, the rostromedial caudate (rmCD), while two male macaque monkeys performed an instrumental lever release task in which a visual cue indicated the forthcoming reward size. We found that the activity of some neurons in VP and rmCD reflected the expected reward size transiently following the cue. Reward size coding appeared earlier and stronger in VP than in rmCD. We also found that the activity in these areas was modulated by the satiation level of monkeys, which also occurred more frequently in VP than in rmCD. The information regarding reward size and satiation level was independently signaled in the neuronal populations of these areas. The data thus highlighted the neuronal coding of key variables for goal-directed behavior in VP. Furthermore, pharmacological inactivation of VP induced more severe deficit of goal-directed behavior than inactivation of rmCD, which was indicated by abnormal error repetition and diminished satiation effect on the performance. These results suggest that VP encodes incentive value and internal drive and plays a pivotal role in the control of motivation to promote goal-directed behavior. SIGNIFICANCE STATEMENT The limbic part of the basal ganglia has been implicated in the motivational control of goal-directed action. Here, we investigated how the ventral pallidum (VP) and the rostromedial caudate (rmCD) encode incentive value and internal drive and control goal-directed behavior. Neuronal recording and subsequent pharmacological inactivation revealed that VP had stronger coding of reward size and satiation level than rmCD. Reward size and satiation level were independently encoded in the neuronal population of these areas. Furthermore, VP inactivation impaired goal-directed behavior more severely than rmCD inactivation. These results highlight the central role of VP in the motivational control of goal-directed action.

Neurovascular coupling, cerebrovascular remodeling and hemodynamic changes are critical to brain function, and dysregulated in neuropathologies such as brain tumors. Interrogating these phenomena in freely behaving animals requires a portable microscope with multiple optical contrast mechanisms. Therefore, we developed a miniaturized microscope with: a fluorescence (FL) channel for imaging neural activity (e.g., GCaMP) or fluorescent cancer cells (e.g., 9L-GFP); an intrinsic optical signal (IOS) channel for imaging hemoglobin absorption (i.e., cerebral blood volume); and a laser speckle contrast (LSC) channel for imaging perfusion (i.e., cerebral blood flow). Following extensive validation, we demonstrate the microscope’s capabilities via experiments in unanesthetized murine brains that include: (i) multi-contrast imaging of neurovascular changes following auditory stimulation; (ii) wide-area tonotopic mapping; (iii) EEG-synchronized imaging during anesthesia recovery; and (iv) microvascular connectivity mapping over the life-cycle of a brain tumor. This affordable, flexible, plug-and-play microscope heralds a new era in functional imaging of freely behaving animals.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique that is widely used to stimulate the sensorimotor cortex, and yet the mechanism by which it influences the natural activity of cortical networks is still under debate. Here, we characterize the effects of anodal and cathodal tDCS on underlying neurons in active macaque sensorimotor cortex across a range of doses. We find changes in spike rates that are sensitive to both current intensity and polarity, behavioral state, and that are cell-type specific. At high currents, effects persist after the offset of stimulation, and the spatiotemporal activity associated with motor activity of the contralateral limb, measured by dynamics of neural ensembles, are altered. These data suggest that tDCS induces reproducible and noticeable changes in cortical neuron activity and support the theory that it affects brain activity through a combination of single neuron polarization and network interactions.

How does the human brain rapidly process incoming information in working memory? In growing divergence from a single-region focus on the prefrontal cortex (PFC), recent work argues for emphasis on how distributed neural networks are rapidly coordinated in support of this central neurocognitive function. Previously, we showed that working memory for everyday "what," "where," and "when" associations depends on multiplexed oscillatory systems, in which signals of different frequencies simultaneously link the PFC to parieto-occipital and medial temporal regions, pointing to a complex web of sub-second, bidirectional interactions. Here, we used direct brain recordings to delineate the frontoparietal oscillatory correlates of working memory with high spatiotemporal precision. Seven intracranial patients with electrodes simultaneously localized to prefrontal and parietal cortices performed a visuospatial working memory task that operationalizes the types of identity and spatiotemporal information we encounter every day. First, task-induced oscillations in the same delta-theta (2-7 Hz) and alpha-beta (9-24 Hz) frequency ranges previously identified using scalp electroencephalography (EEG) carried information about the contents of working memory. Second, maintenance was linked to directional connectivity from the parietal cortex to the PFC. However, presentation of the test prompt to cue identity, spatial, or temporal information changed delta-theta coordination from a unidirectional, parietal-led system to a bidirectional, frontoparietal system. Third, the processing of spatiotemporal information was more bidirectional in the delta-theta range than was the processing of identity information, where alpha-beta connectivity did not exhibit sensitivity to the contents of working memory. These findings implicate a bidirectional delta-theta mechanism for frontoparietal control over the contents of working memory.

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor-stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

Evidence shows that selective attention to visual stimuli modulates the gain of cochlear responses, probably through auditory-cortex descending pathways. At the cerebral cortex level, amplitude and phase changes of neural oscillations have been proposed as a correlate of selective attention. However, whether sensory receptors are also influenced by the oscillatory network during attention tasks remains unknown. Here, we searched for oscillatory attention-related activity at the cochlear receptor level in humans. We used an alternating visual/auditory selective attention task and measured electroencephalographic activity simultaneously to distortion product otoacoustic emissions (a measure of cochlear receptor-cell activity). In order to search for cochlear oscillatory activity, the otoacoustic emission signal, was included as an additional channel in the electroencephalogram analyses. This method allowed us to evaluate dynamic changes in cochlear oscillations within the same range of frequencies (1-35 Hz) in which cognitive effects are commonly observed in electroencephalogram works. We found the presence of low frequency (<10 Hz) brain and cochlear amplifier oscillations during selective attention to visual and auditory stimuli. Notably, switching between auditory and visual attention modulates the amplitude and the temporal order of brain and inner ear oscillations. These results extend the role of the oscillatory activity network during cognition in neural systems to the receptor level.

In rodents, the progression of extrastriate areas located laterally to primary visual cortex (V1) has been assigned to a putative object-processing pathway (homologous to the primate ventral stream), based on anatomical considerations. Recently, we found functional support for such attribution (Tafazoli et al., 2017), by showing that this cortical progression is specialized for coding object identity despite view changes, the hallmark property of a ventral-like pathway. Here, we sought to clarify what computations are at the base of such specialization. To this aim, we performed multielectrode recordings from V1 and laterolateral area LL (at the apex of the putative ventral-like hierarchy) of male adult rats, during the presentation of drifting gratings and noise movies. We found that the extent to which neuronal responses were entrained to the phase of the gratings sharply dropped from V1 to LL, along with the quality of the receptive fields inferred through reverse correlation. Concomitantly, the tendency of neurons to respond to different oriented gratings increased, whereas the sharpness of orientation tuning declined. Critically, these trends are consistent with the nonlinear summation of visual inputs that is expected to take place along the ventral stream, according to the predictions of hierarchical models of ventral computations and a meta-analysis of the monkey literature. This suggests an intriguing homology between the mechanisms responsible for building up shape selectivity and transformation tolerance in the visual cortex of primates and rodents, reasserting the potential of the latter as models to investigate ventral stream functions at the circuitry level.SIGNIFICANCE STATEMENT Despite the growing popularity of rodents as models of visual functions, it remains unclear whether their visual cortex contains specialized modules for processing shape information. To addresses this question, we compared how neuronal tuning evolves from rat primary visual cortex (V1) to a downstream visual cortical region (area LL) that previous work has implicated in shape processing. In our experiments, LL neurons displayed a stronger tendency to respond to drifting gratings with different orientations while maintaining a sustained response across the whole duration of the drift cycle. These trends match the increased complexity of pattern selectivity and the augmented tolerance to stimulus translation found in monkey visual temporal cortex, thus revealing a homology between shape processing in rodents and primates.

Interaural time differences (ITD) are one of several principle cues for localizing sounds. However, ITD are in the sub-millisecond range for most animals. Because the neural processing of such small ITDs pushes the limit of temporal resolution, the precise ITD-range for a given species and its usefulness - relative to other localization cues - was a powerful selective force in the evolution of the neural circuits involved. Birds and other non-mammals have internally coupled middle ears working as pressure-difference receivers that may significantly enhance ITD, depending on the precise properties of the interaural connection. Here, the extent of this internal coupling was investigated in chickens, specifically under the same experimental conditions as typically used in neurophysiology of ITD-coding circuits, i.e. with headphone stimulation. Cochlear microphonics (CM) were recorded simultaneously from both ears of anesthetized chickens under monaural and binaural stimulation, using pure tones from 0.1 to 3 kHz. Interaural transmission peaked at 1.5 kHz at a loss of only −5.5 dB; the mean interaural delay was 264 μs. CM amplitude strongly modulated as a function of ITD, confirming significant interaural coupling. The “ITD heard” derived from the CM phases in both ears showed enhancement, compared to the acoustic stimuli, by a factor of up to 1.8. However, the closed sound delivery systems impaired interaural transmission at low frequencies (< 1 kHz). We identify factors that need to be considered when interpreting neurophysiological data obtained under these conditions, and relating them to the natural free-field condition. Summary statement The interaural time differences that chickens can use for sound localization are significantly greater than their small head size suggests. Closed-system sound stimulation can, however, produce complex artefacts.

Mesoscale cortical activity can be defined as the organization of activity of large neuron populations into collective action, forming time-dependent patterns such as traveling waves. Although collective action may play an important role in the cross-scale integration of brain activity and in the emergence of cognitive behavior, a comprehensive formulation of the laws governing its dynamics is still lacking. Because collective action processes are macroscopic with respect to neuronal activity, these processes cannot be described directly with methods and models developed for the microscale (individual neurons).To identify the characteristic features of mesoscopic dynamics, and to lay the foundations for a theoretical description of mesoscopic activity in the hippocampus, we conduct a comprehensive examination of observational data of hippocampal local field potential (LFP) recordings. We use the strong correlation between rat running-speed and the LFP power to parameterize the energy input into the hippocampus, and show that both the power and non-linearity of collective action (e.g., theta and gamma rhythms) increase with increased speed. Our results show that collective-action dynamics are stochastic (the precise state of a single neuron is irrelevant), weakly non-linear, and weakly dissipative. These are the principles of the theory of weak turbulence. Therefore, we propose weak turbulence a theoretical framework for the description of mesoscopic activity in the hippocampus. The weak turbulence framework provides a complete description of the cross-scale energy exchange (the energy cascade). It uncovers the mechanism governing major features of LFP spectra and bispectra, such as the physical meaning of the exponent α of power-law LFP spectra (e.g., f -α, where f is the frequency), the strengthening of theta-gamma coupling with energy input into the hippocampus, as well as specific phase lags associated with their interaction. Remarkably, the weak turbulence framework is consistent with the theory of self organized criticality, which provides a simple explanation for the existence of the power-law background spectrum. Together with self-organized criticality, weak turbulence could provide a unifying approach to modeling the dynamics of mesoscopic activity.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

The caudomedial nidopallium (NCM) is a higher level region of auditory cortex in songbirds that has been implicated in encoding learned vocalizations and mediating perception of complex sounds. We made cell-attached recordings in awake adult male zebra finches ( Taeniopygia guttata) to characterize responses of single NCM neurons to playback of tones and songs. Neurons fell into two broad classes: narrow fast-spiking cells and broad sparsely firing cells. Virtually all narrow-spiking cells responded to playback of pure tones, compared with approximately half of broad-spiking cells. In addition, narrow-spiking cells tended to have lower thresholds and faster, less variable spike onset latencies than did broad-spiking cells, as well as higher firing rates. Tonal responses of narrow-spiking cells also showed broader ranges for both frequency and amplitude compared with broad-spiking neurons and were more apt to have V-shaped tuning curves compared with broad-spiking neurons, which tended to have complex (discontinuous), columnar, or O-shaped frequency response areas. In response to playback of conspecific songs, narrow-spiking neurons showed high firing rates and low levels of selectivity whereas broad-spiking neurons responded sparsely and selectively. Broad-spiking neurons in which tones failed to evoke a response showed greater song selectivity compared with those with a clear tuning curve. These results are consistent with the idea that narrow-spiking neurons represent putative fast-spiking interneurons, which may provide a source of intrinsic inhibition that contributes to the more selective tuning in broad-spiking cells. NEW & NOTEWORTHY The response properties of neurons in higher level regions of auditory cortex in songbirds are of fundamental interest because processing in such regions is essential for vocal learning and plasticity and for auditory perception of complex sounds. Within a region of secondary auditory cortex, neurons with narrow spikes exhibited high firing rates to playback of both tones and multiple conspecific songs, whereas broad-spiking neurons responded sparsely and selectively to both tones and songs.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major NADPH-producing enzyme which is essential for maintaining the mitochondrial redox balance in cells. We sought to determine whether IDH2 deficiency induces mitochondrial dysfunction and modulates auditory function, and investigated the protective potential of an antioxidant agent against reactive oxygen species (ROS)-induced cochlear damage in Idh2 knockout (Idh2-/-) mice. Idh2 deficiency leads to damages to hair cells and spiral ganglion neurons (SGNs) in the cochlea and ultimately to apoptotic cell death and progressive sensorineural hearing loss in Idh2-/- mice. Loss of IDH2 activity led to decreased levels of NADPH and glutathione causing abnormal ROS accumulation and oxidative damage, which might trigger apoptosis signal in hair cells and SGNs in Idh2-/- mice. We performed ex vivo experiments to determine whether administration of mitochondria-targeted antioxidants might protect or induce recovery of cells from ROS-induced apoptosis in Idh2-deficient mouse cochlea. MitoQ almost completely neutralized the H2O2-induced ototoxicity, as the survival rate of Idh2-/- hair cells were restored to normal levels. In addition, the lack of IDH2 led to the accumulation of mitochondrial ROS and the depolarization of ΔΨm, resulting in hair cell loss. In the present study, we identified that IDH2 is indispensable for the functional maintenance and survival of hair cells and SGNs. Moreover, the hair cell degeneration caused by IDH2 deficiency can be prevented by MitoQ, which suggests that Idh2-/- mice could be a valuable animal model for evaluating the therapeutic effects of various antioxidant candidates to overcome ROS-induced hearing loss.

Much of our understanding about how acetylcholine modulates prefrontal cortical (PFC) networks comes from behavioral experiments that examine cortical dynamics during highly attentive states. However, much less is known about how PFC is recruited during passive sensory processing and how acetylcholine may regulate connectivity between cortical areas outside of task performance. To investigate the involvement of PFC and cholinergic neuromodulation in passive auditory processing, we performed simultaneous recordings in the auditory cortex (AC) and PFC in awake head fixed mice presented with a white noise auditory stimulus in the presence or absence of local cholinergic antagonists in AC. We found that a subset of PFC neurons were strongly driven by auditory stimuli even when the stimulus had no associative meaning, suggesting PFC monitors stimuli under passive conditions. We also found that cholinergic signaling in AC shapes the strength of auditory driven responses in PFC, by modulating the intra-cortical sensory response through muscarinic interactions in AC. Taken together, these findings provide novel evidence that cholinergic mechanisms have a continuous role in cortical gating through muscarinic receptors during passive processing and expand traditional views of prefrontal cortical function and the contributions of cholinergic modulation in cortical communication.

Hearing impairment in older people is thought to arise from impaired temporal processing in auditory circuits. We used a systems-level (scalp recordings) and a microcircuit-level (extracellular recordings) approach to investigate how aging affects the sensitivity to temporal envelopes of speech-like sounds in rats. Scalp-recorded potentials suggest an age-related increase in sensitivity to temporal regularity along the ascending auditory pathway. The underlying cellular changes in the midbrain were examined using extracellular recordings from inferior colliculus neurons. We observed an age-related increase in sensitivity to the sound's onset and temporal regularity (i.e., periodicity envelope) in the spiking output of inferior colliculus neurons, relative to their synaptic inputs (local field potentials). This relative enhancement for aged animals was most prominent for multi-unit (relative to single-unit) spiking activity. Spontaneous multi-unit, but not single-unit, activity was also enhanced in aged compared with young animals. Our results suggest that aging is associated with altered sensitivity to a sound's temporal regularities, and that these effects may be due to increased gain of neural network activity in the midbrain.

Contemporary cochlear implants (CI) are generally very effective for remediation of severe to profound sensorineural hearing loss, but outcomes are still highly variable. Auditory nerve survival is likely one of the major factors underlying this variability. Neurotrophin therapy therefore has been proposed for CI recipients, with the goal of improving outcomes by promoting improved survival of cochlear spiral ganglion neurons (SGN) and/or residual hair cells. Previous studies have shown that glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor, and neurotrophin-3 can rescue SGNs following insult. The current study was designed to determine whether adeno-associated virus vector serotype 5 (AAV-5) encoding either green fluorescent protein or GDNF can transduce cells in the mouse cochlea to express useful levels of neurotrophin and to approximate the optimum therapeutic dose(s) for transducing hair cells and SGN. The findings demonstrate that AAV-5 is a potentially useful gene therapy vector for the cochlea, resulting in extremely high levels of transgene expression in the cochlear inner hair cells and SGN. However, overexpression of human GDNF in newborn mice caused severe neurological symptoms and hearing loss, likely due to Purkinje cell loss and cochlear nucleus pathology. Thus, extremely high levels of transgene protein expression should be avoided, particularly for proteins that have neurological function in neonatal subjects.

This paper investigates the safety of a novel 'magnetic injection' method of delivering therapy to the cochlea, in a rodent model. In this method of administration, a magnetic field is employed to actively transport drug-eluting superparamagnetic iron-oxide core nanoparticles into the cochlea, where they then release their drug payload (we delivered the steroid prednisolone). Our study design and selection of control groups was based on published regulatory guidance for safety studies that involve local drug delivery. We tested for both single and multiple delivery doses to the cochlea, and found that magnetic delivery did not harm hearing. There was no statistical difference in hearing between magnetically treated ears versus ears that received intra-tympanic steroid (a mimic of a standard-of-care for sudden sensorineural hearing loss), both 2 and 30 days after treatment. Since our treatment is local to the ear, the levels of steroid and iron circulating systemically after our treatment were low, below mass-spectrometry detection limits for the steroid and no different from normal for iron. No adverse findings were observed in ear tissue histopathology or in animal gross behavior. At 2 and 30 days after treatment, inflammatory changes examined in the ear were limited to the middle ear, were very mild in severity, and by day 90 there was ongoing and almost complete reversibility of these changes. There were no ear tissue scarring or hemorrhage trends associated with magnetic delivery. In summary, after conducting a pre-clinical safety study, no adverse safety issues were observed.