Publications using TDT

Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.

Studying the in-vivo mechanical and electrophysiological cochlear responses in several species helps us to have a comprehensive view of the sensitivity and frequency selectivity of the cochlea. Different species might use different mechanisms to achieve the sharp frequency-place map. The outer hair cells (OHC) play an important role in mediating frequency tuning. In the present work, we measured the OHC-generated local cochlear microphonic (LCM) and the motion of different layers in the organ of Corti using optical coherence tomography (OCT) in the first turn of the cochlea in guinea pig. In the best frequency (BF) band, our observations were similar to our previous measurements in gerbil: a nonlinear peak in LCM responses and in the basilar membrane (BM) and OHC-region displacements, and higher motion in the OHC region than the BM. Sub-BF the responses in the two species were different. In both species the sub-BF displacement of the BM was linear and LCM was nonlinear. Sub-BF in the OHC-region, nonlinearity was only observed in a subset of healthy guinea pig cochleae while in gerbil, robust nonlinearity was observed in all healthy cochleae. The differences suggest that gerbils and guinea pigs employ different mechanisms for filtering sub-BF OHC activity from BM responses. However, it cannot be ruled out that the differences are due to technical measurement differences across the species.

The middle ear is a high-fidelity, broadband impedance transformer that transmits acoustic stimuli at the eardrum to the inner ear. It is home to the two smallest muscles in mammalian species, which modulate middle ear transmission. Of this pair, the function of the tensor tympani muscle (TTM) has remained obscure. We investigated the acoustic effects of this muscle in young adult gerbils. We measured changes in middle ear vibration produced by pulse-train-elicited TTM contraction - in the time-domain with a click stimulus and in the frequency-domain with multitone zwuis stimuli. In our click experiments, there was generally a small reduction in the primary peak of the response and a slight increase in the subsequent ringing, but there was essentially no change in the delay of the click response at the umbo (less than 1 µs change). In our multitone experiments, there were consistent patterns of attenuation and enhancement in the velocity responses at the umbo and ossicles. TTM contraction produced a narrow band of enhancement around 6 kHz (maximally ~5 dB) that can be modeled with an increased stiffness of an overdamped spring-mass resonance. At frequencies below 2 kHz and above 35 kHz, TTM contraction attenuated middle ear vibrations by as much as fivefold.

Three experiments explored the effects of abrupt changes in stimulus properties on streaming dynamics. Listeners monitored 20-s-long low- and high-frequency (LHL–) tone sequences and reported the number of streams heard throughout. Experiments 1 and 2 used pure tones and examined the effects of changing triplet base frequency and level, respectively. Abrupt changes in base frequency (±3–12 semitones) caused significant magnitude-related falls in segregation (resetting), regardless of transition direction, but an asymmetry occurred for changes in level (±12 dB). Rising-level transitions usually decreased segregation significantly, whereas falling-level transitions had little or no effect. Experiment 3 used pure tones (unmodulated) and narrowly spaced (±25 Hz) tone pairs (dyads); the two evoke similar excitation patterns, but dyads are strongly modulated with a distinctive timbre. Dyad-only sequences induced a strongly segregated percept, limiting scope for further build-up. Alternation between groups of pure tones and dyads produced large, asymmetric changes in streaming. Dyad-to-pure transitions caused substantial resetting, but pure-to-dyad transitions sometimes elicited even greater segregation than for the corresponding interval in dyad-only sequences (overshoot). The results indicate that abrupt changes in timbre can strongly affect the likelihood of stream segregation without introducing significant peripheral-channeling cues. These asymmetric effects of transition direction are reminiscent of subtractive adaptation in vision.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

The effect of non-informational speech spectrum noise as a distractor on cognitive and listening comprehension ability was examined in fifty-three young, normal hearing adults. Time-controlled tasks were used to measure auditory working memory (WM) capacity and attention switching (AS) ability. Listening comprehension was measured using a lecture, interview, and spoken narratives test. Noise level was individually set to achieve at least 90% or higher speech intelligibility. Participants’ listening comprehension in the presence of distracting noise was better on inference questions compared to listening in quiet. Their speed of information processing was also significantly faster in WM and AS tasks in noise. These results were consistent with the view that noise may enhance arousal levels leading to faster information processing during cognitive tasks. Whereas the speed of AS was faster in noise, this rapid switching of attention resulted in more errors in updating items. Participants who processed information faster in noise and did so accurately, more effectively switched their attention to refresh/rehearse recall items within WM. More efficient processing deployed in the presence of noise appeared to have led to improvements in WM performance and making inferences in a listening comprehension task. Additional research is required to examine these findings using background noise that can cause informational masking.

The corticostriatal circuit has been identified as a vital pathway for associative learning. However, how learning is implemented when the sensory striatum is permanently impaired remains unclear. Using chemogenetic techniques to suppress layer five auditory cortex (AC) input to the auditory striatum, learning of a sound discrimination task was significantly impacted in freely moving Mongolian gerbils, in particular when this suppression occurs early on during learning. Whole-cell recordings sampled throughout learning revealed a transient reduction in postsynaptic (GABAA) inhibition in both striatal D1 and D2 cells in normal-hearing gerbils during task acquisition. In contrast, when the baseline striatal inhibitory strengths and firing rates were permanently reduced by a transient period of developmental sensory deprivation, learning was accompanied by augmented inhibition and increased firing rates. Direct manipulation of striatal inhibition in vivo and in vitro revealed a key role of the transient inhibitory changes in task acquisition. Together, these results reveal a flexible corticostriatal inhibitory synaptic plasticity mechanism that accompanies associative auditory learning.

A dopaminergic neural circuit projecting from the VTA to the hindbrain regulates feeding by transducing meal satiation signals.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.

Background Cerebellar electrical stimulation has shown promise in improving motor recovery post-stroke in both rodent and human studies. Past studies have used motor evoked potentials (MEPs) to evaluate how cerebellar stimulation modulates ongoing activity in the cortex, but the underlying mechanisms are incompletely understood. Here we used invasive electrophysiological recordings from the intact and stroke-injured rodent primary motor cortex (M1) to assess how epidural cerebellar stimulation modulates neural dynamics at the level of single neurons as well as at the level of mesoscale dynamics. Methods We recorded single unit spiking and local field potentials (LFPs) in both the intact and acutely stroke-injured M1 contralateral to the stimulated cerebellum in adult Long-Evans rats under anesthesia. We analyzed changes in the firing rates of single units, the extent of synchronous spiking and power spectral density (PSD) changes in LFPs during and post-stimulation. Results Our results show that post-stimulation, the firing rates of a majority of M1 neurons changed significantly with respect to their baseline rates. These firing rate changes were diverse in character, as the firing rate of some neurons increased while others decreased. Additionally, these changes started to set in during stimulation. Furthermore, cross-correlation analysis showed a significant increase in coincident firing amongst neuronal pairs. Interestingly, this increase in synchrony was unrelated to the direction of firing rate change. We also found that neuronal ensembles derived through principal component analysis were more active post-stimulation. Lastly, these changes occurred without a significant change in the overall spectral power of LFPs post-stimulation. Conclusions Our results show that cerebellar stimulation caused significant, long-lasting changes in the activity patterns of M1 neurons by altering firing rates, boosting neural synchrony and increasing neuronal assemblies’ activation strength. Our study provides evidence that cerebellar stimulation can directly modulate cortical dynamics. Since these results are present in the perilesional cortex, our data might also help explain the facilitatory effects of cerebellar stimulation post-stroke.

Itch is an unpleasant sensation that elicits robust scratching and aversive experience. However, the identity of the cells and neural circuits that organize this information remains elusive. Here, we show the necessity and sufficiency of chloroquine-activated neurons in the central amygdala (CeA) for both itch sensation and associated aversion. Further, we show that chloroquine-activated CeA neurons play important roles in itch-related comorbidities, including anxiety-like behaviors, but not in some aversive and appetitive behaviors previously ascribed to CeA neurons. RNA-sequencing of chloroquine-activated CeA neurons identified several differentially expressed genes as well as potential key signaling pathways in regulating pruritis. Finally, viral tracing experiments demonstrate that these neurons send projections to the ventral periaqueductal gray that are critical in modulation of itch. These findings reveal a cellular and circuit signature of CeA neurons orchestrating behavioral and affective responses to pruritus in mice.

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared with caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ∼30% inhibition. Dopamine transporter blockade with either cocaine or GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL/6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared with saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm.SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is a region highly implicated in mediating stress responses; however, the dBNST also receives dopaminergic inputs from classically defined drug reward pathways. Here we used various techniques to demonstrate that dopamine signaling within the dBNST region has inhibitory effects on population activity. We show that cocaine, an abused psychostimulant, augments both catecholamine release and dopamine-mediated cellular inhibition in this region. We also demonstrate that cocaine administration reduces population activity in the dBNST, in vivo Together, these data support a mechanism of dopamine-mediated inhibition within the dBNST, providing a means by which drug-induced elevations in dopamine signaling may inhibit dBNST activity to promote drug reward.

Acoustic communication signals are inevitably challenged by ambient noise. In response to noise, many animals adjust their calls to maintain signal detectability. However, the mechanisms by which the auditory system adapts to the adjusted pulses are unclear. Our previous study revealed that the echolocating bat, Hipposideros pratti , increased its pulse intensity in the presence of background white noise. In vivo single-neuron recording demonstrated that the auditory midbrain neurons tuned to the second harmonic (H2 neurons) increased their minimal threshold (MT) to a similar degree as the increment of pulse intensity in the presence of the background noise. Furthermore, the H2 neurons exhibited consistent spike rates at their best amplitudes and sharper intensity tuning with background white noise compared with silent conditions. The previous data indicated that sound intensity analysis by auditory midbrain neurons was adapted to the increased pulse intensity in the same noise condition. This study further examined the echolocation pulse frequency and frequency analysis of auditory midbrain neurons with noise conditions. The data revealed that H. pratti did not shift the resting frequency in the presence of background noise. The auditory midbrain neuronal frequency analysis highly linked to processing the resting frequency with the presence of noise by presenting the constant best frequency (BF), frequency sensitivity, and frequency selectivity. Thus, our results suggested that auditory midbrain neuronal responses in background white noise are adapted to process echolocation pulses in the noise conditions.

Understanding how neural populations encode natural stimuli with complex spatiotemporal structure to give rise to perception remains a central problem in neuroscience. Here we investigated population coding of natural communication stimuli by hindbrain neurons within the electrosensory system of weakly electric fish Apteronotus leptorhynchus . Overall, we found that simultaneously recorded neural activities were correlated: signal but not noise correlations were variable depending on the stimulus waveform as well as the distance between neurons. Combining the neural activities using an equal-weight sum gave rise to discrimination performance between different stimulus waveforms that was limited by redundancy introduced by noise correlations. However, using an evolutionary algorithm to assign different weights to individual neurons before combining their activities (i.e., a weighted sum) gave rise to increased discrimination performance by revealing synergistic interactions between neural activities. Our results thus demonstrate that correlations between the neural activities of hindbrain electrosensory neurons can enhance information about the structure of natural communication stimuli that allow for reliable discrimination between different waveforms by downstream brain areas.

Obesity is a serious global health problem because of its increasing prevalence and comorbidities, but its treatments are limited. The serotonin 2C receptor (5-HT2CR), a G-protein-coupled receptor, activates proopiomelanocortin (POMC) neurons in the arcuate nucleus of hypothalamus (ARH) to reduce appetite and weight gain. However, several 5-HT analogs targeting this receptor, e.g., lorcaserin (Lor), suffer from diminished efficacy to reduce weight after prolonged administration. Here, we show that barbadin (Bar), a novel β-arrestin/β2-adaptin inhibitor, can prevent 5-HT2CR internalization in cells and potentiate long-term effects of Lor to reduce appetite and body weight in male mice. Mechanistically, we demonstrate that Bar co-treatment can effectively maintain the sensitivity of the 5-HT2CR in POMCARH neurons, despite prolonged Lor exposure, thereby allowing these neurons to be activated through opening the transient receptor potential cation (TRPC) channels. Thus, our results prove the concept that inhibition of 5-HT2CR desensitization can be a valid strategy to improve the long-term weight loss effects of Lor or other 5-HT2CR agonists, and also provide an intellectual framework to develop effective long-term management of weight by targeting 5-HT2CR desensitization.SIGNIFICANCE STATEMENT By demonstrating that the combination of barbadin (Bar) with a G-protein-coupled receptor (GPCR) agonist can provide prolonged weight-lowering benefits in a preclinical setting, our work should call for additional efforts to validate Bar as a safe and effective medicine or to use Bar as a lead compound to develop more suitable compounds for obesity treatment. These results prove the concept that inhibition of serotonin 2C receptor (5-HT2CR) desensitization can be a valid strategy to improve the long-term weight loss effects of lorcaserin (Lor) or other 5-HT2CR agonists. Since GPCRs represent a major category as therapeutic targets for various human diseases and desensitization of GPCRs is a common issue, our work may provide a conceptual framework to enhance effects of a broad range of GPCR medicines.

The mechanisms that govern thalamocortical transmission are poorly understood. Recent data have shown that sensory stimuli elicit activity in ensembles of cortical neurons that recapitulate stereotyped spontaneous activity patterns. Here, we elucidate a possible mechanism by which gating of patterned population cortical activity occurs. In this study, sensory-evoked all-or-none cortical population responses were observed in the mouse auditory cortex in vivo and similar stochastic cortical responses were observed in a colliculo-thalamocortical brain slice preparation. Cortical responses were associated with decreases in auditory thalamic synaptic inhibition and increases in thalamic synchrony. Silencing of corticothalamic neurons in layer 6 (but not layer 5) or the thalamic reticular nucleus linearized the cortical responses, suggesting that layer 6 corticothalamic feedback via the thalamic reticular nucleus was responsible for gating stochastic cortical population responses. These data implicate a corticothalamic-thalamic reticular nucleus circuit that modifies thalamic neuronal synchronization to recruit populations of cortical neurons for sensory representations.

To examine the oxidative stress and the antioxidant response of p62-Keap1-Nrf2 pathway in cochleae during age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL), and then elucidate the function of full-length and variant p62/Sqstm1 (referred to here as p62) in the regulation of Nrf2 activation. Cochlear damage was assessed by analyzing auditory brainstem response (ABR) and by counting hair cells (HCs). For oxidative stress detection, the malondialdehyde (MDA) levels and 7, 8-dihydro-8-oxoguanine (8-oxoG) expression in the cochleae was measured by assay kits and immunohistochemistry, respectively. The expression of full-length and variant p62 in cochleae, hippocampus (HIP) and auditory cortex (AC) was found by western blotting; For the Keap1-Nrf2 pathway activation, the Nrf2 nuclear translocation and the Nrf2 target genes HO-1/NQO-1 expression was detected by western blotting and qRT-PCR, respectively. The oxidative function of full-length and variant p62 was examined in HEI-OC-1 cells by flow cytometry. The results showed hearing loss and cochlear hair cell loss was associated with MDA accumulation and 8-oxoG expression during ARHL and NIHL. Nrf2 showed no obvious changes in nuclear protein. Expression levels mRNA for HO-1 and NQO1 was lower in old mice and mildly greater in AT Mice. The expression of p62 splicing variant lacking the Keap1-interacting region was greater than full-length p62 in cochleae. However, the expression of p62 splicing variant was lesser than full-length p62 in HIP and AC. For HEI-OC-1 cells, high expression of full-length p62 decreased ROS levels induced by H 2 O 2 . Oxidative stress is closely related to ARHL and NIHL. Changing the ratio of full-length to variant p62 protein expression may be a new target to reduce the level of oxidative stress in cochleae.

Background The receptor for advanced glycation end-products (RAGE) is involved in neuroinflammation. This study investigated the changes in RAGE expression following noise-induced hearing loss. Methods Three-week-old female Sprague–Dawley rats were exposed to 115 dB SPL white noise for 4 h daily for 3 d (noise group, n = 16). In parallel, age and sex-matched control rats were raised under standard conditions without noise exposure (control group, n = 16). After 2 h (noise immediate, n = 8) and 4 wk (noise 4-week, n = 8) of noise exposure, the auditory cortex was harvested and cytoplasmic and nuclear fractions were isolated. The gene expression levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6), interleukin 1 beta (IL1β), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and RAGE were evaluated using real-time reverse transcription polymerase chain reaction. The protein expression levels of nuclear RAGE and cytosolic RAGE were evaluated using western blotting. Additionally, matrix metalloproteinase 9 (MMP9) was pharmacologically inhibited in the noise immediate group, and then nuclear and cytosolic RAGE expression levels were evaluated. Results The noise immediate and noise 4-week groups exhibited increased auditory thresholds at 4, 8, 16, and 32 kHz frequencies. The genes encoding the pro-inflammatory cytokines TNF-α, IL6, IL1β, and NF- κB were increased 3.74, 1.63, 6.42, and 6.23-fold in the noise immediate group, respectively (P = 0.047, 0.043, 0.044, and 0.041). RAGE mRNA expression was elevated 1.42-fold in the noise 4-week group (P = 0.032). Cytosolic RAGE expression was increased 1.76 and 6.99-fold in the noise immediate and noise 4-week groups, respectively (P = 0.04 and 0.03). Nuclear RAGE expression was comparable between the noise and control groups. matrix metalloproteinase 9 (MMP9) inhibition reduced cytosolic RAGE expression in the noise immediate group (P = 0.004). Conclusions Noise exposure increased the expression of cytosolic RAGE in the auditory cortex and upregulated pro-inflammatory genes, but this response could be alleviated by MMP9 inhibition.

Obesity remains prevalent in the US. One potential treatment is vagus nerve stimulation (VNS), which activates the sensory afferents innervating the stomach that convey stomach volume and establish satiety. However, current VNS approaches and stimulus optimization could benefit from additional understanding of the underlying neural response to stomach distension. In this study, obesity-prone Sprague Dawley rats consumed a standard, high-carbohydrate, or high-fat diet for several months, leading to diet-induced obesity in the latter two groups. Under anesthesia, the neural activity in the vagus nerve was recorded with a penetrating microelectrode array while the stomach was distended with an implanted balloon. Vagal tone during distension was compared to baseline tone prior to distension. Responses were strongly correlated with stomach distension, but the sensitivity to distension was significantly lower in animals that had been fed the nonstandard diets. The results indicate that both high fat and high carbohydrate diets impair vagus activity.

Aggregation and self-sorting of cells in three dimensional cultures have been described for non-neuronal cells. Despite increased interest in engineered neural tissues for treating brain injury or for modeling neurological disorders in vitro, little data is available on collective cell movements in neuronal aggregates. Migration and sorting of cells may alter these constructs’ morphology and, therefore, the function of their neural circuitry. In this work, linear, adhered rat and human 3D neuronal-astrocyte cultures were developed to enable the study of aggregation and sorting of these cells. An in silico model of the contraction, clustering, and cell sorting in the 3D cultures was also developed. Experiments and computational modeling showed that aggregation was mainly a neuron mediated process, and formation of astrocyte-rich sheaths in 3D cultures depended on differential attraction between neurons and astrocytes. In silico model predicted formation of self-assembled neuronal layers in disk-shaped 3D cultures. Neuronal activity patterns were found to correlate with local morphological differences. This model of neuronal and astrocyte aggregation and sorting may benefit future design of neuronal constructs.