Behavioral Neuroscience publications

The temporal structure of sensory inputs contains essential information for their interpretation. Sensory cortex represents these temporal cues through two codes: the temporal sequences of neuronal activity and the spatial patterns of neuronal firing rate. However, it is unknown which of these coexisting codes causally drives sensory decisions. To separate their contributions, we generated in the mouse auditory cortex optogenetically driven activity patterns differing exclusively along their temporal or spatial dimensions. Mice could rapidly learn to behaviorally discriminate spatial but not temporal patterns. Moreover, large-scale neuronal recordings across the auditory system revealed that the auditory cortex is the first region in which spatial patterns efficiently represent temporal cues on the timescale of several hundred milliseconds. This feature is shared by the deep layers of neural networks categorizing time-varying sounds. Therefore, the emergence of a spatial code for temporal sensory cues is a necessary condition to efficiently associate temporally structured stimuli with decisions.

Speech intelligibility declines with age and sensorineural hearing damage (SNHL). However, it remains unclear whether cochlear synaptopathy (CS), a recently discovered form of SNHL, significantly contributes to this issue. CS refers to damaged auditory-nerve synapses that innervate the inner hair cells and there is currently no go-to diagnostic test available. Furthermore, age-related hearing damage can comprise various aspects (e.g., hair cell damage, CS) that each can play a role in impaired sound perception. To explore the link between cochlear damage and speech intelligibility deficits, this study examines the role of CS for word recognition among older listeners. We first validated an envelope-following response (EFR) marker for CS using a Budgerigar model. We then applied this marker in human experiments, while restricting the speech material’s frequency content to ensure that both the EFR and the behavioral tasks engaged similar cochlear frequency regions. Following this approach, we identified the relative contribution of hearing sensitivity and CS to speech intelligibility in two age-matched (65-year-old) groups with clinically normal ( n = 15, 8 females) or impaired audiograms ( n = 13, 8 females). Compared to a young normal-hearing control group ( n = 13, 7 females), the older groups demonstrated lower EFR responses and impaired speech reception thresholds. We conclude that age-related CS reduces supra-threshold temporal envelope coding with subsequent speech coding deficits in noise that cannot be explained based on hearing sensitivity alone.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here, we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Hearing is an active process in which listeners must detect and identify sounds, segregate and discriminate stimulus features, and extract their behavioral relevance. Adaptive changes in sound detection can emerge rapidly, during sudden shifts in acoustic or environmental context, or more slowly as a result of practice. Although we know that context- and learning-dependent changes in the sensitivity of auditory cortical (ACX) neurons support many aspects of perceptual plasticity, the contribution of subcortical auditory regions to this process is less understood. Here, we recorded single- and multiunit activity from the central nucleus of the inferior colliculus (ICC) and the ventral subdivision of the medial geniculate nucleus (MGV) of male and female Mongolian gerbils under two different behavioral contexts: as animals performed an amplitude modulation (AM) detection task and as they were passively exposed to AM sounds. Using a signal detection framework to estimate neurometric sensitivity, we found that neural thresholds in both regions improve during task performance, and this improvement is largely driven by changes in the firing rate rather than phase locking. We also found that ICC and MGV neurometric thresholds improve as animals learn to detect small AM depths during a multiday perceptual training paradigm. Finally, we revealed that in the MGV, but not the ICC, context-dependent enhancements in AM sensitivity grow stronger during perceptual training, mirroring prior observations in the ACX. Together, our results suggest that the auditory midbrain and thalamus contribute to changes in sound processing and perception over rapid and slow timescales.

The bed nucleus of the stria terminalis (BNST) is involved in feeding, reward, aversion, and anxiety-like behavior. We identify BNST neurons defined by the expression of vesicular glutamate transporter 3, VGluT3. VGluT3 neurons were localized to anteromedial BNST, were molecularly distinct from accumbal VGluT3 neurons, and co-express vesicular GABA transporter (VGaT). Cell-type specific presynaptic processes were identified in arcuate nucleus (ARC) and the paraventricular nucleus of the hypothalamus (PVN), regions critical for feeding and homeostatic regulation. Whole-cell patch-clamp electrophysiology revealed that, while these neurons co-express VGluT3 and VGaT, they functionally transmit GABA to both ARC and PVN, with rare glutamate co-transmission to ARC. Neuronal recordings of VGluT3 BNST neurons showed greater calcium-dependent signaling in response to sucrose consumption while sated compared with fasted. When fasted, optogenetic stimulation of BNST VGluT3 neurons decreased sucrose consumption using several stimulation conditions but not when stimulation occurred prior to sucrose access, suggesting that BNST VGluT3 activation concurrent with consumption in the fasted state reduces feeding. BNST VGluT3 activation during anxiety-like paradigms (novelty-suppressed feeding, open field, and elevated zero maze) and real-time place conditioning resulted in no changes in anxiety-like or reward/aversion behavior. We interpret these data such that VGluT3 BNST neurons represent a unique cellular population within the BNST that provides inhibitory input to hypothalamic regions to decrease feeding without affecting anxiety-like or reward/aversion behavior.

Noise exposure is one of the most common causes of hearing loss and hyperacusis. Studies have shown that noise exposure can induce a cortical gain to compensate for reduced input of the cochlea, which may contribute to the increased sound sensitivity. However, many people with hyperacusis have no measurable cochlear lesion after being exposed to loud sound. In this experiment, we studied the neurological alterations in the cortical and subcortical areas following a prolonged moderate level of noise exposure (84 dB SPL, 8 h/day for 4 weeks) in the laboratory mice. The cochlear function was monitored by auditory brainstem responses (ABRs). The behavioral auditory sensitivity and temporal processing were evaluated using the acoustic startle response (ASR) and gap‐induced prepulse inhibition (gap‐PPI). The central auditory functions were determined by electrophysiological recordings of the inferior colliculus (IC) and the auditory cortex (AC). Our results showed that although there was no significant difference in the ABR thresholds, the noise group showed enhanced ASR and gap‐PPI compared to the control group. Increased neural activity in both the IC and the AC was recorded in the noise‐exposed mice compared to the control group, suggesting a central gain in both the subcortical and cortical regions. The current source density (CSD) analysis of the AC response revealed an increased columnar excitation and reduced corticocortical projection in the noise group, different from the central gain model of noise‐induced hearing loss. Our results suggest that chronic “nondestructive” noise can increase the gain of the central auditory system by altering the balance of auditory thalamocortical and intracortical inputs, which may contribute to the increased sound sensitivity in people with normal hearing.

Objective: Dietary protein restriction induces adaptive changes in food preference, increasing protein consumption over carbohydrates or fat. We investigated whether motivation and reward signaling underpin these preferences. Methods and results: In an operant task, protein-restricted male mice responded more for liquid protein rewards, but not carbohydrate, fat, or sweet rewards compared to non-restricted mice. When the number of responses required to access protein reward varied, protein-restricted mice exhibited higher operant responses at moderate to high response requirements. The protein restriction-induced increase in operant responding for protein was absent in Fgf21-KO mice and mice with neuron-specific deletion of the FGF21 co-receptor beta-Klotho (KlbCam2ka). Fiber photometry recording of VTA dopamine neurons revealed that oral delivery of maltodextrin triggered a larger dopamine neuron activation than casein in control diet-fed mice, while casein triggered a larger activation in low-protein diet-fed mice. This restriction-induced shift in nutrient-specific VTA dopamine signaling was lost in Fgf21-KO mice. Conclusion: These data suggest that the increased FGF21 during protein restriction acts in the brain to induce a protein-specific appetite by specifically enhancing the reward value of protein-containing foods and the motivation to consume them.

Objectives: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na+, K+, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. Methods: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. Results: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. Conclusions: We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.

Associative learning links sensory signals to their behavioral meaning by combining bottom-up inputs with top-down contextual information, enabling decision-making based on expected outcomes. It triggers plastic changes of neuronal responses in both excitatory and inhibitory cell populations. However, the role of inhibition in shaping this plasticity remains debated. Here we used chronic extracellular electrophysiology and optogenetic manipulation of inhibitory neurons and top-down inputs in mice learning an auditory discrimination task. We found that learning enhances stimulus selectivity in a subset of neurons in the primary auditory cortex. Interestingly, somatostatin-expressing (SST) interneurons decrease their response to the rewarded cue and bidirectionally regulate associative learning. More specifically, an increased activity of SST neurons impairs learning by altering bottom-up signaling, whereas the reduction of their activity facilitates learning by gating top-down inputs from the orbitofrontal cortex. These findings demonstrate that inhibition plays a critical role in gating top-down inputs to primary sensory cortices involved in associative learning.

Ovarian-derived estrogen can signal non-canonically at membrane-associated receptors in the brain to rapidly regulate neuronal function. Early alcohol drinking confers greater risk for alcohol use disorder in women than men, and binge alcohol drinking is correlated with high estrogen levels, but a causal role for estrogen in driving alcohol drinking has not been established. We found that female mice displayed greater binge alcohol drinking and reduced avoidance when estrogen was high during the estrous cycle than when it was low. The pro-drinking, but not anxiolytic, effect of high endogenous estrogen occurred via rapid signaling at membrane-associated estrogen receptor alpha in the bed nucleus of the stria terminalis, which promoted synaptic excitation of corticotropin-releasing factor neurons and facilitated their activity during alcohol drinking. Thus, this study demonstrates a rapid, nongenomic signaling mechanism for ovarian-derived estrogen in the brain controlling behavior in gonadally intact females.

Alcohol use disorder (AUD) is a chronic relapsing brain disorder characterized by an impaired ability to stop or control alcohol consumption despite adverse social, occupational, or health consequences. AUD affects nearly one-third of adults at some point during their lives, with an associated cost of approximately $249 billion annually in the U.S. alone. The effects of alcohol consumption are expected to increase significantly during the COVID-19 pandemic, with alcohol sales increasing by approximately 54%, potentially exacerbating health concerns and risk-taking behaviors. Unfortunately, existing pharmacological and behavioral therapies for AUD are associated with poor success rates, with approximately 40% of individuals relapsing within three years of treatment.Pre-clinical studies have shown that chronic alcohol consumption leads to significant changes in synaptic function within the dorsal medial striatum (DMS), one of the brain regions associated with AUD and responsible for mediating goal-directed behavior. Specifically, chronic alcohol consumption has been associated with hyperactivity of dopamine receptor 1 (D1) medium spiny neurons (MSN) and hypoactivity of dopamine receptor 2 (D1) MSNs within the DMS. Optogenetic, chemogenetic, and transgenic approaches have demonstrated that reducing the D1/D2 MSN signaling imbalance decreases alcohol self-administration in rodent models of AUD.Here, we present an electrical stimulation approach that uses ultra-low (≤ 1 Hz) frequency (ULF) spike-timing-dependent plasticity (STDP) in mouse models of AUD to reduce DMS D1/D2 MSN signaling imbalances by stimulating D1-MSN afferents into the GPi and ACC glutamatergic projections to the DMS in a time-locked stimulation sequence. Our data suggest that GPi/ACC ULF-STDP selectively decreases DMS D1-MSN hyperactivity leading to reduced alcohol consumption without evoking undesired affective behaviors using electrical stimulation rather than approaches requiring genetic modification. This work represents a step towards fulfilling the unmet need for a reliable method of treating severe AUD through cell-type-specific control with clinically available neuromodulation tools.

Background Recent studies have identified hearing loss (HL) as a primary risk factor for Alzheimer’s disease (AD) onset. However, the mechanisms linking HL to AD are not fully understood. This study explored the effects of drug-induced hearing loss (DIHL) on the expression of proteins associated with AD progression in mouse models. Methods DIHL was induced in 5xFAD and Tg2576 mice aged 3 to 3.5 weeks using kanamycin (700 mg/kg, subcutaneous) and furosemide (600 mg/kg, intraperitoneal). The accumulation and expression of beta-amyloid (Aβ), ionized calcium-binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP) were measured through immunohistochemistry and immunoblotting. Additionally, the expression of proteins involved in the mammalian target of rapamycin (mTOR) pathway, including downstream effectors p70 ribosomal S6 kinase (p70S6K) and S6, as well as proinflammatory cytokines, was analyzed. Results Compared to control conditions, HL led to a significant increase in the accumulation of Aβ in the hippocampus and cortex. Elevated levels of neuroinflammatory markers, including Iba1 and GFAP, as well as proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), were observed. Moreover, DIHL enhanced phosphorylation of mTOR, p70S6K, and S6, indicating activation of the mTOR pathway. Conclusions HL significantly increases Aβ accumulation in the brain. Furthermore, HL activates astrocytes and microglia, leading to increased neuroinflammation and thereby accelerating AD progression. These findings strongly suggest that HL contributes autonomously to neuroinflammation, highlighting the potential for early intervention in HL to reduce AD risk.

Bilateral cochlear implant (CI) patients exhibit significant limitations in spatial hearing. Their ability to process interaural time differences (ITDs) is often impaired, while their ability to process interaural level differences (ILDs) remains comparatively good. Clinical studies aiming to identify the causes of these limitations are often plagued by confounds and ethical limitations. Recent behavioral work suggests that rats may be a good animal model for studying binaural hearing under neuroprosthetic stimulation, as rats develop excellent ITD sensitivity when provided with suitable CI stimulation. However, their ability to use ILDs has not yet been characterized. Objective of this study is to address this knowledge gap. Neontally deafened rats were bilaterally fitted with CIs, and trained to lateralize binaural stimuli according to ILD. Their behavioral ILD thresholds were measured at pulse rates from 50 to 2400 pps. CI rats exhibited high sensitivity to ILDs with thresholds of a few dB at all tested pulse rates. We conclude that early deafened rats develop good sensitivity, not only to ITDs but also to ILDs, if provided with appropriate CI stimulation. Their generally good performance, in line with expectations from other mammalian species, validates rats as an excellent model for research on binaural auditory prostheses.

Animacy perception, the ability to discern living from non-living entities, is crucial for survival and social interaction, as it includes recognizing abstract concepts such as movement, purpose, and intentions. This process involves interpreting cues that may suggest the intentions or actions of others. It engages the temporal cortex (TC), particularly the superior temporal sulcus (STS) and the adjacent region of the inferior temporal cortex (ITC), as well as the dorsomedial prefrontal cortex (dmPFC). However, it remains unclear how animacy is dynamically encoded over time in these brain areas and whether its processing is distributed or localized. In this study, we addressed these questions by employing a symbolic categorization task involving animate and inanimate objects using natural movie stimuli. Simultaneously, electrocorticography were conducted in both the TC and dmPFC. Time-frequency analysis revealed region-specific frequency representations throughout the observation of the movies. Spatial searchlight decoding analysis demonstrated that animacy processing is represented in a distributed manner. Regions encoding animacy information were found to be dispersed across the fundus and lip of the STS, as well as in the ITC. Next, we examined whether these dispersed regions form functional networks. Independent component analysis revealed that the spatial distribution of the component with the most significant animacy information corresponded with the dispersed regions identified by the spatial decoding analysis. Furthermore, Granger causality analysis indicated that these regions exhibit frequency-specific directional functional connectivity, with a general trend of causal influence from the ITC to STS across multiple frequency bands. Notably, a prominent feedback flow in the alpha band from the ITC to both the ventral bank and fundus of the STS was identified. These findings suggest a distributed and functionally interconnected neural substrate for animacy processing across the STS and ITC.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD). Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine consolidate sleep. Furthermore, inhibiting LC-NE neurons restores memory. Finally, rabies-mediated screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory-regulatory regions in 16p11.2 deletion mice. Our findings identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Some individuals are susceptible to chronic stress, and others are more resilient. While many brain regions implicated in learning are dysregulated after stress, little is known about whether and how neural teaching signals during stress differ between susceptible and resilient individuals. Here, we seek to determine if activity in the lateral habenula (LHb), which encodes a negative teaching signal, differs between susceptible and resilient mice during stress to produce different outcomes. After (but not before) chronic social defeat stress, the LHb is active when susceptible mice are in proximity of the aggressor strain. During stress, activity is higher in susceptible mice during aggressor interactions, and activation biases mice toward susceptibility. This manipulation generates a persistent and widespread increase in the balance of subcortical vs. cortical activity in susceptible mice. Taken together, our results indicate that heightened activity in the LHb during stress produces lasting brainwide and behavioral substrates of susceptibility.

The neuromodulators dopamine (DA) and serotonin (5-hydroxytryptamine; 5HT) powerfully regulate associative learning1-8. Similarities in the activity and connectivity of these neuromodulatory systems have inspired competing models of how DA and 5HT interact to drive the formation of new associations9-14. However, these hypotheses have not been tested directly because it has not been possible to interrogate and manipulate multiple neuromodulatory systems in a single subject. Here we establish a mouse model that enables simultaneous genetic access to the brain's DA and 5HT neurons. Anterograde tracing revealed the nucleus accumbens (NAc) to be a putative hotspot for the integration of convergent DA and 5HT signals. Simultaneous recording of DA and 5HT axon activity, together with genetically encoded DA and 5HT sensor recordings, revealed that rewards increase DA signalling and decrease 5HT signalling in the NAc. Optogenetically dampening DA or 5HT reward responses individually produced modest behavioural deficits in an appetitive conditioning task, while blunting both signals together profoundly disrupted learning and reinforcement. Optogenetically reproducing DA and 5HT reward responses together was sufficient to drive the acquisition of new associations and supported reinforcement more potently than either manipulation did alone. Together, these results demonstrate that striatal DA and 5HT signals shape learning by exerting opponent control of reinforcement.

Inhibitory neuronal circuits within the lateral septum (LS) play a key role in regulating mood and stress responses. Even though glial cells can modulate these circuits, the impact of astrocytes on LS neural circuits and their functional interactions remains largely unexplored. Here, we demonstrate that astrocytes exhibit increased intracellular Ca²⁺ levels in response to aversive sensory and social stimuli in both male and female mice. This astrocytic Ca²⁺ elevation inhibits neighboring LS neurons by reducing excitatory synaptic transmissions through A1R-mediated signaling in both the dorsal (LSd) and intermediate LS (LSi) and enhancing inhibitory synaptic transmission via A2AR-mediated signaling in the LSi. At the same time, astrocytes reduce inhibitory tone on distant LS neurons. In the LSd, astrocytes promote social avoidance and anxiety, as well as increased heart rate in socially stressed male mice. In contrast, astrocytes in the LSi contribute to elevated heart rate and heightened blood corticosterone levels in unstressed male mice. These results suggest that the dynamic interactions between astrocytes and neurons within the LS modulate physiological and behavioral responses to stressful experiences.

Background/Objectives: Tinnitus is a debilitating auditory disorder commonly described as a ringing in the ears in the absence of an external sound source. Sound trauma is considered a primary cause. Neuronal hyperactivity is one potential mechanism for the genesis of tinnitus and has been identified in the cochlear nucleus (CN) and the auditory cortex (AC), where there may be an imbalance of excitatory and inhibitory neurotransmissions. However, no study has directly correlated tinnitus with the extracellular levels of amino acids in the CN and the AC using microdialysis, which reflects the functions of these neurochemicals. In the present study, rats were exposed to acoustic trauma and then subjected to behavioural confirmation of tinnitus after one month, followed by microdialysis. Methods: Rats were divided into sham (aged, n = 6; young, n = 6); tinnitus-positive (aged, n = 7; young, n = 7); and tinnitus-negative (aged, n = 3; young, n = 3) groups. In vivo microdialysis was utilized to collect samples from the CN and the AC, simultaneously, in the same rat. Extracellular levels of amino acids were quantified using high-performance liquid chromatography (HPLC) coupled with an electrochemical detector (ECD). The effects of sound stimulation and age on neurochemical changes associated with tinnitus were also examined. Results: There were no significant differences in either the basal levels or the sound stimulation-evoked changes of any of the amino acids examined in the CN and the AC between the sham and tinnitus animals. However, the basal levels of serine and threonine exhibited age-related alterations in the AC, and significant differences in threonine and glycine levels were observed in the responses to 4 kHz and 16 kHz stimuli in the CN. Conclusions: These results demonstrate the lack of a direct link between extracellular levels of amino acids in the CN and the AC and tinnitus perception in a rat model of tinnitus.