Publications using the RPvdsEx

Memory encoding and retrieval rely on specific interactions across multiple brain areas. Although connections between individual brain areas have been extensively studied, the anatomical and functional specificity of neuronal circuit organization underlying information transfer across multiple brain areas remains unclear. Here, we combine transsynaptic viral tracing, optogenetic manipulations, and calcium dynamics recordings to dissect the multisynaptic functional connectivity of the amygdala. We identify a distinct basolateral amygdala (BLA) subpopulation that connects disynaptically to the periaqueductal gray (PAG) via the central amygdala (CeA). This disynaptic pathway serves as a core circuit element necessary for the learning and expression of conditioned fear and exhibits learning-related plasticity. Together, our findings demonstrate the utility of multisynaptic approaches for functional circuit analysis and indicate that disynaptic specificity may be a general feature of neuronal circuit organization.

Neurons that process sensory information exhibit bursts of electrical activity during development, providing early training to circuits that will later encode similar features of the external world. In the mammalian auditory system, this intrinsically generated activity emerges from the cochlea prior to hearing onset, but its role in maturation of auditory circuitry remains poorly understood. We show that selective suppression of cochlear supporting cell spontaneous activity disrupts patterned burst firing of central auditory neurons without affecting cell survival or acoustic thresholds. However, neurons in the inferior colliculus of these mice exhibit enhanced acoustic sensitivity and broader frequency tuning, resulting in wider isofrequency laminae. Despite this enhanced neural responsiveness, total tone-responsive regions in the auditory cortex are substantially smaller. Thus, disruption of pre-hearing cochlear activity causes profound changes in neural encoding of sound, with important implications for restoration of hearing in individuals who experience reduced activity during this critical developmental period.

Fear and anxiety are brain states that evolved to mediate defensive responses to threat. While it is clear that the defense reaction includes multiple interacting behavioural, autonomic and endocrine adjustments, their integrative nature is poorly understood. In particular, threat has been associated with various cardiac changes, yet a clear consensus on their relevance for the integrated defense reaction is missing. We here define rapid microstates associated with specific behaviours and heart rate dynamics, both affected by long-lasting macrostates and reflecting context-dependent threat levels. In addition, we demonstrate that one of the most commonly used defensive behavioural responses, freezing measured by immobility, is part of an integrated cardio-behavioural microstate mediated by specific midbrain circuit elements. Our work puts forth a framework for systematic integration of cardiac and behavioural readouts that presents the basis for a better understanding of complex neural defensive states and their associated systemic functions.

Among the different autism spectrum disorders, Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. Sensory and especially auditory hypersensitivity is a key symptom in patients, which is well mimicked in the Fmr1 -/- mouse model. However, the physiological mechanisms underlying FXS’s acoustic hypersensitivity in particular remain poorly understood. Here, we categorized spike response patterns to pure tones of different frequencies and intensities from neurons in the inferior colliculus (IC), a central integrator in the ascending auditory pathway. Based on this categorization we analyzed differences in response patterns between IC neurons of wild-type (WT) and Fmr1 -/- mice. Our results report broadening of frequency tuning, an increased firing in response to monaural as well as binaural stimuli, an altered balance of excitation-inhibition, and reduced response latencies, all expected features of acoustic hypersensitivity. Furthermore, we noticed that all neuronal response types in Fmr1 -/- mice displayed enhanced offset-rebound activity outside their excitatory frequency response area. These results provide evidence that the loss of Fmr1 not only increases spike responses in IC neurons similar to auditory brainstem neurons, but also changes response patterns such as offset spiking. One can speculate this to be an underlying aspect of the receptive language problems associated with Fragile X syndrome.

The cochlea decomposes sounds into separate frequency channels, from which the auditory brain must reconstruct the auditory scene. To do this the auditory system must make decisions about which frequency information should be grouped together, and which should remain distinct. Two key cues for grouping are temporal coherence, resulting from coherent changes in power across frequency, and temporal predictability, resulting from regular or predictable changes over time. To test how these cues contribute to the construction of a sound scene we present listeners with a range of precursor sounds, which act to prime the auditory system by providing information about each sounds structure, followed by a fixed masker in which participants were required to detect the presence of an embedded tone. By manipulating temporal coherence and/or temporal predictability in the precursor we assess how prior sound exposure influences subsequent auditory grouping. In Experiment 1, we measure the contribution of temporal predictability by presenting temporally regular or jittered precursors, and temporal coherence by using either narrow or broadband sounds, demonstrating that both independently contribute to masking/unmasking. In Experiment 2, we measure the relative impact of temporal coherence and temporal predictability and ask whether the influence of each in the precursor signifies an enhancement or interference of unmasking. We observed that interfering precursors produced the largest changes to thresholds.

Background: Sensory impairments commonly occur in patients with autism or intellectual disability. Fragile X syndrome (FXS) is one form of intellectual disability that is often comorbid with autism. In electroencephalographic (EEG) recordings obtained from humans with FXS, the ability of cortical regions to consistently synchronize, or "phase-lock", to modulated auditory stimuli is reduced compared to that of typically developing individuals. At the same time, less time-locked, "non-phase-locked" power induced by sounds is higher. The same changes occur in the Fmr1 knockout (KO) mouse - an animal model of FXS. We determined if Fmr1 deletion in a subset of brainstem auditory neurons plays any role in these EEG changes in the mouse. Methods: We reinstated FMRP expression in a subpopulation of brainstem auditory neurons in an otherwise Fmr1 KO control (conditional on; cON Fmr1) mouse and used EEG recordings to determine if reinstatement normalized, or "rescued", the phase-locking phenotype observed in the cON Fmr1 mouse. In determining rescue, this also meant that Fmr1 deletion in the same neuron population was necessary for the phenotype to occur. Results: We find that Fmr1 reinstatement in a subset of brainstem neurons rescues certain aspects of the phase-locking phenotype but does not rescue the increase in non-phase-locked power. Unexpectedly, not all electrophysiological phenotypes observed in the Fmr1 KO were observed in the cON Fmr1 mouse used for the reinstatement experiments, and this was likely due to residual expression of FMRP in these Fmr1 KO controls. Conclusions: Fmr1 deletion in brainstem neurons is necessary for certain aspects of the decreased phase-locking phenotype in the Fmr1 KO, but not necessary for the increase in non-phase-locked power induced by a sound. The most likely brainstem structure underlying these results is the inferior colliculus. We also demonstrate that low levels of FMRP can rescue some EEG phenotypes but not others. This latter finding provides a foundation for how symptoms in FXS individuals may vary due to FMRP levels and that reinstatement of low FMRP levels may be sufficient to alleviate particular symptoms.

Urethane has little effect on nervous system and is often used in neuroscience studies. However, the effect of urethane in neurons is not thoroughly clear. In this study, we investigated changes in neuron responses to tones in inferior colliculus during urethane anesthesia. As urethane was metabolized, the best and characteristic frequencies did not obviously change, but the minimal threshold (MT) remained relatively stable or was elevated. The frequency tuning bandwidth at 60 dB SPL (BW 60dBSPL ) remained unchanged or decreased, and the average evoked spike of effective frequencies at 60 dB SPL (ES 60dBSPL ) gradually decreased. Although the average evoked spike of effective frequencies at a tone intensity of 20 dB SPL above MT (ES 20dBSPLaboveMT ) decreased, the frequency tuning bandwidth at a tone intensity of 20 dB SPL above MT (BW 20dBSPLaboveMT ) did not change. In addition, the changes in MT, ES 60dBSPL , BW 60dBSPL , and ES 20dBSPLaboveMT increased with the MT in pre-anesthesia awake state (MT pre−anesthesiaawake ). In some neurons, the MT was lower, BW 60dBSPL was broader, and ES 60dBSPL and ES 20dBSPLaboveMT were higher in urethane anesthesia state than in pre-anesthesia awake state. During anesthesia, the inhibitory effect of urethane reduced the ES 20dBSPLaboveMT , but did not change the MT, characteristic frequency, or BW 20dBSPLaboveMT . In the recording session with the strongest neuron response, the first spike latency did not decrease, and the spontaneous spike did not increase. Therefore, we conclude that urethane can reduce/not change the MT, increase the evoked spike, or broaden/not change the frequency tuning range, and eventually improve the response of auditory neurons to tone with or without “pushing down” the tonal receptive field in thresholding model. The improved effect increases with the MT pre−anesthesiaawake of neurons. The changes induced by the inhibitory and improved effects of urethane abide by similar regularities, but the change directions are contrary. The improvement mechanism may be likely due to the increase in the ratio of excitatory/inhibitory postsynaptic inputs to neurons.

Intracortical microstimulation (ICMS) of the somatosensory cortex (S1) can restore sensory function in patients with paralysis. Studies assessing the stability of ICMS have reported heterogeneous responses across electrodes and over time, potentially hindering the implementation and translatability of these technologies. The foreign body response (FBR) and the encapsulating glial scar have been associated with a decay in chronic performance of implanted electrodes. Moreover, the morphology, intrinsic properties, and function of cells vary across cortical layers, each potentially affecting the sensitivity to ICMS as well as the degree of the FBR across cortical depth. However, layer-by-layer comparisons of the long-term stability of ICMS as well as the extent of the astrocytic glial scar change across cortical layers have not been well explored. Here, we implanted silicon microelectrodes with electrode sites spanning all the layers of S1 in rats. Using a behavioral paradigm, we obtained ICMS detection thresholds from all cortical layers for up to 40 weeks. Our results showed that the sensitivity and long-term performance of ICMS is indeed layer dependent. Overall, detection thresholds decreased during the first 7 weeks post-implantation (WPI). This was followed by a period in which thresholds remained stable or increased depending on the interfacing layer: thresholds in L1 and L6 exhibited the most consistent increases over time, while those in L4 and L5 remained the most stable. Furthermore, histological investigation of the tissue surrounding the electrode showed a biological response of microglia and macrophages which peaked at L1, while the area of the astrocytic glial scar peaked at L2/3. Interestingly, the biological response of these FBR markers is less exacerbated at L4 and L5, suggesting a potential link between the FBR and the long-term stability of ICMS. These findings suggest that interfacing depth can play an important role in the design of chronically stable implantable microelectrodes.

Aging leads to degeneration of the peripheral and central auditory systems, hearing loss, and difficulty understanding sounds in noise. Aging is also associated with changes in susceptibility to or recovery from damaging noise exposures, although the effects of the interaction between acute noise exposure and age on the perception of sounds are not well studied. We tested these effects in the CBA/CaJ mouse model of age-related hearing loss using operant conditioning procedures before and after noise exposure and longitudinally measured changes in their sensitivity for detecting tones in quiet or noise backgrounds. Cochleae from a subset of the behaviorally tested mice were immunolabeled to examine organ of Corti damage relative to what is expected based on aging alone. Mice tested in both quiet and noise background conditions experienced worse behavioral sensitivity immediately after noise exposure, but mice exposed at older ages generally showed greater threshold shifts and reduced recovery over time. Surprisingly, day-to-day stability in thresholds was markedly higher for mice detecting signals in the presence of a noise masker compared with detection in quiet conditions. Cochlear analysis revealed decreases in the total number of outer hair cells (OHCs) and the number of ribbons per inner cell in high-frequency regions in aged, noise-exposed mice relative to aging alone. Our findings build on previous work showing interactions between age and noise exposure and add that background noise can increase the stability of behavioral hearing sensitivity after noise damage.

Developmental dyslexia is a common neurodevelopmental disorder characterized by difficulties in reading and writing. Although underlying biological and genetic mechanisms remain unclear, anomalies in phonological processing and auditory processing have been associated with dyslexia. Several candidate risk genes have also been identified, with KIAA0319 as a main candidate. Animal models targeting the rodent homolog ( Kiaa0319 ) have been used to explore putative behavioral and anatomic anomalies, with mixed results. For example after downregulation of Kiaa0319 expression in rats via shRNA, significant adult rapid auditory processing impairments were reported, along with cortical anomalies reflecting atypical neuronal migration. Conversely, Kiaa0319 knockout (KO) mice were reported to have typical adult auditory processing, and no visible cortical anomalies. To address these inconsistencies, we tested Kiaa0319 KO mice on auditory processing tasks similar to those used previously in rat shRNA knockdown studies. Subsequent neuroanatomic analyses on these same mice targeted medial geniculate nucleus (MGN), a receptive communication‐related brain structure. Results confirm that Kiaa0319 KO mice exhibit significant auditory processing impairments specific to rapid/brief stimuli, and also show significant volumetric reductions and a shift toward fewer large and smaller neurons in the MGN. The latter finding is consistent with post mortem MGN data from human dyslexic brains. Combined evidence supports a role for KIAA0319 in the development of auditory CNS pathways subserving rapid auditory processing functions critical to the development of speech processing, language, and ultimately reading. Results affirm KIAA0319 variation as a possible risk factor for dyslexia specifically via anomalies in central acoustic processing pathways.

Failure in the structural maintenance of the hair cell stereocilia bundle and ribbon synapse causes hearing loss. Here, we have studied how ER stress elicits hair cell pathology, using mouse models with inactivation of Manf (mesencephalic astrocyte-derived neurotrophic factor), encoding an ER-homeostasis-promoting protein. From hearing onset, Manf deficiency caused disarray of the outer hair cell stereocilia bundle and reduced cochlear sound amplification capability throughout the tonotopic axis. In high-frequency outer hair cells, the pathology ended in molecular changes in the stereocilia taper region and in strong stereocilia fusion. In high-frequency inner hair cells, Manf deficiency degraded ribbon synapses. The altered phenotype strongly depended on the mouse genetic background. Altogether, the failure in the ER homeostasis maintenance induced early-onset stereociliopathy and synaptopathy and accelerated the effect of genetic causes driving age-related hearing loss. Correspondingly, MANF mutation in a human patient induced severe sensorineural hearing loss from a young age onward. Thus, we present MANF as a novel protein and ER stress as a mechanism that regulate auditory hair cell maintenance in both mice and humans.

Several studies have shown that type IV fibrocytes, located in the spiral ligament, degenerate first after noise exposure. Interestingly, this is the region where Coch expression is most abundant. As it is suggested that cochlin plays a role in our innate immune system, our goal is to investigate hearing thresholds and inner ear inflammation after noise exposure in Coch knockout (Coch−/−) mice compared to Coch wildtype (Coch+/+) mice. Animals were randomly allocated to a noise exposure group and a control group. Vestibular and auditory testing was performed at 48 h and one week after noise exposure. Whole mount staining and cryosectioning of the cochlea was performed in order to investigate hair cells, spiral ganglion neurons, inner ear inflammation, Coch expression and fibrocyte degeneration. Hearing assessment revealed that Coch+/+ mice had significantly larger threshold shifts than Coch−/− mice after noise exposure. We were unable to identify any differences in hair cells, neurons, fibrocytes and influx of macrophages in the inner ear between both groups. Interestingly, Coch expression was significantly lower in the group exposed to noise. Our results indicate that the absence of Coch has a protective influence on hearing thresholds after noise exposure, but this is not related to reduced inner ear inflammation in the knockout.

Background Individuals with Fragile X syndrome (FXS) and autism spectrum disorder (ASD) exhibit an array of symptoms, including sociability deficits, increased anxiety, hyperactivity, and sensory hyperexcitability. It is unclear how endocannabinoid (eCB) modulation can be targeted to alleviate neurophysiological abnormalities in FXS as behavioral research reveals benefits to inhibiting cannabinoid (CB) receptor activation and increasing endocannabinoid ligand levels. Here, we hypothesize that enhancement of 2-arachidonoyl-sn-glycerol (2-AG) in Fragile X mental retardation 1 gene knock-out ( Fmr1 KO) mice may reduce cortical hyperexcitability and behavioral abnormalities observed in FXS. Methods To test whether an increase in 2-AG levels normalized cortical responses in a mouse model of FXS, animals were subjected to electroencephalography (EEG) recording and behavioral assessment following treatment with JZL-184, an irreversible inhibitor of monoacylglycerol lipase (MAGL). Assessment of 2-AG was performed using lipidomic analysis in conjunction with various doses and time points post-administration of JZL-184. Baseline electrocortical activity and evoked responses to sound stimuli were measured using a 30-channel multielectrode array (MEA) in adult male mice before, 4 h, and 1 day post-intraperitoneal injection of JZL-184 or vehicle. Behavior assessment was done using the open field and elevated plus maze 4 h post-treatment. Results Lipidomic analysis showed that 8 mg/kg JZL-184 significantly increased the levels of 2-AG in the auditory cortex of both Fmr1 KO and WT mice 4 h post-treatment compared to vehicle controls. EEG recordings revealed a reduction in the abnormally enhanced baseline gamma-band power in Fmr1 KO mice and significantly improved evoked synchronization to auditory stimuli in the gamma-band range post-JZL-184 treatment. JZL-184 treatment also ameliorated anxiety-like and hyperactivity phenotypes in Fmr1 KO mice. Conclusions Overall, these results indicate that increasing 2-AG levels may serve as a potential therapeutic approach to normalize cortical responses and improve behavioral outcomes in FXS and possibly other ASDs.

Serotonin transporter (SERT) modulates the level of 5-HT and significantly affects the activity of serotonergic neurons in the central nervous system. The manipulation of SERT has lasting neurobiological and behavioral consequences, including developmental dysfunction, depression, and anxiety. Auditory disorders have been widely reported as the adverse events of these mental diseases. It is unclear how SERT impacts neuronal connections/interactions and what mechanism(s) may elicit the disruption of normal neural network functions in auditory cortex. In the present study, we report on the neuronal morphology and function of auditory cortex in SERT knockout (KO) mice. We show that the dendritic length of the fourth layer (L-IV) pyramidal neurons and the second-to-third layer (L-II/III) interneurons were reduced in the auditory cortex of the SERT KO mice. The number and density of dendritic spines of these neurons were significantly less than those of wild-type neurons. Also, the frequency-tonotopic organization of primary auditory cortex was disrupted in SERT KO mice. The auditory neurons of SERT KO mice exhibited border frequency tuning with high-intensity thresholds. These findings indicate that SERT plays a key role in development and functional maintenance of auditory cortical neurons. Auditory function should be examined when SERT is selected as a target in the treatment for psychiatric disorders.

Slow waves and cognitive output have been modulated in humans by phase-targeted auditory stimulation. However, to advance its technical development and further our understanding, implementation of the method in animal models is indispensable. Here, we report the successful employment of slow waves’ phase-targeted closed-loop auditory stimulation (CLAS) in rats. To validate this new tool both conceptually and functionally, we tested the effects of up- and down-phase CLAS on proportions and spectral characteristics of sleep, and on learning performance in the single-pellet reaching task, respectively. Without affecting 24 hr sleep-wake behavior, CLAS specifically altered delta (slow waves) and sigma (sleep spindles) power persistently over chronic periods of stimulation. While up-phase CLAS does not elicit a significant change in behavioral performance, down-phase CLAS exerted a detrimental effect on overall engagement and success rate in the behavioral test. Overall CLAS-dependent spectral changes were positively correlated with learning performance. Altogether, our results provide proof-of-principle evidence that phase-targeted CLAS of slow waves in rodents is efficient, safe, and stable over chronic experimental periods, enabling the use of this high-specificity tool for basic and preclinical translational sleep research.

Intracortical microelectrodes are neuroprosthetic devices used in brain-machine interfaces to both record and stimulate neural activity in the brain. These technologies have been improved by advances in microfabrication, which have led to the creation of subcellular and high-density microelectrodes. The greater number of independent stimulation channels in these devices allows for improved neuromodulation selectivity, compared to single-site microelectrodes. Elements of electrode design such as electrode-site placement can influence the long-term performance of neuroprostheses. Previous studies have shown that electrode-sites placed on the edge of a planar microelectrode have greater chronic recording functionality than sites placed in the center. However, the effect of electrode-site placement on long-term intracortical microstimulation (ICMS) is still unknown. Here, we show that, in rats chronically implanted with custom-made planar silicon microelectrodes, electrode-sites on the tip of the device outperformed those on both the edge and center in terms of the effect per charge delivered, though there is still a slight advantage to using edge sites over center sites for ICMS. Longitudinal analysis of ICMS detection thresholds over a 16-week period revealed that while all sites followed a similar trend over time, the tip and edge sites consistently elicited the behavioral response with less charge compared to center sites. Furthermore, we quantified channel activity over time and found that edge sites remained more active than center sites over time, though the rate of decay of active sites for center and edge sites was comparable. Our results demonstrate that electrode-site placement plays an important role in the long-term stability of intracortical microstimulation and could be a potential factor to consider in the design of future intracortical electrodes.

The prevalence of noise-induced hearing loss (NIHL) continues to increase, with limited therapies available for individuals with cochlear damage. We have previously established that the transcription factor FOXO3 is necessary to preserve outer hair cells (OHCs) and hearing thresholds up to two weeks following mild noise exposure in mice. The mechanisms by which FOXO3 preserves cochlear cells and function are unknown. In this study, we analyzed the immediate effects of mild noise exposure on wild-type, Foxo3 heterozygous ( Foxo3 +/− ), and Foxo3 knock-out ( Foxo3 −/− ) mice to better understand FOXO3’s role(s) in the mammalian cochlea. We used confocal and multiphoton microscopy to examine well-characterized components of noise-induced damage including calcium regulators, oxidative stress, necrosis, and caspase-dependent and caspase-independent apoptosis. Lower immunoreactivity of the calcium buffer Oncomodulin in Foxo3 −/− OHCs correlated with cell loss beginning 4 h post-noise exposure. Using immunohistochemistry, we identified parthanatos as the cell death pathway for OHCs. Oxidative stress response pathways were not significantly altered in FOXO3’s absence. We used RNA sequencing to identify and RT-qPCR to confirm differentially expressed genes. We further investigated a gene downregulated in the unexposed Foxo3 −/− mice that may contribute to OHC noise susceptibility. Glycerophosphodiester phosphodiesterase domain containing 3 (GDPD3), a possible endogenous source of lysophosphatidic acid (LPA), has not previously been described in the cochlea. As LPA reduces OHC loss after severe noise exposure, we treated noise-exposed Foxo3 −/− mice with exogenous LPA. LPA treatment delayed immediate damage to OHCs but was insufficient to ultimately prevent their death or prevent hearing loss. These results suggest that FOXO3 acts prior to acoustic insult to maintain cochlear resilience, possibly through sustaining endogenous LPA levels.

Background: Auditory hallucinations (which occur when the distinction between thoughts and perceptions is blurred) are common in psychotic disorders. The orbitofrontal cortex (OFC) may be implicated, because it receives multiple inputs, including sound and affective value via the amygdala, orchestrating complex emotional responses. We aimed to elucidate the circuit and neuromodulatory mechanisms that underlie the processing of emotionally salient auditory stimuli in the OFC — mechanisms that may be involved in auditory hallucinations. Methods: We identified the cortico-cortical connectivity conveying auditory information to the mouse OFC; its sensitivity to neuromodulators involved in psychosis and postpartum depression, such as dopamine and neurosteroids; and its sensitivity to sensory gating (defective in dysexecutive syndromes). Results: Retrograde tracers in OFC revealed input cells in all auditory cortices. Acoustic responses were abolished by pharmacological and chemogenetic inactivation of the above-identified pathway. Acoustic responses in the OFC were reduced by local dopaminergic agonists and neurosteroids. Noticeably, apomorphine action lasted longer in the OFC than in auditory areas, and its effect was modality-specific (augmentation for visual responses), whereas neurosteroid action was sex-specific. Finally, acoustic responses in the OFC reverberated to the auditory association cortex via feedback connections and displayed sensory gating, a phenomenon of local origin, given that it was not detectable in input auditory cortices. Limitations: Although our findings were for mice, connectivity and sensitivity to neuromodulation are conserved across mammals. Conclusion: The corticocortical loop from the auditory association cortex to the OFC is dramatically sensitive to dopamine and neurosteroids. This suggests a clinically testable circuit behind auditory hallucinations. The function of OFC input–output circuits can be studied in mice with targeted and clinically relevant mutations related to their response to emotionally salient sounds.

The microRNA miR-96 is important for hearing, as point mutations in humans and mice result in dominant progressive hearing loss. Mir96 is expressed in sensory cells along with Mir182 and Mir183, but the roles of these closely-linked microRNAs are as yet unknown. Here, we analyse mice carrying null alleles of Mir182, and of Mir183 and Mir96 together to investigate their roles in hearing. We found that Mir183/96 heterozygous mice had normal hearing and homozygotes were completely deaf with abnormal hair cell stereocilia bundles and reduced numbers of inner hair cell synapses at 4 weeks of age. Mir182 knockout mice developed normal hearing then exhibited progressive hearing loss. Our transcriptional analyses revealed significant changes in a range of other genes, but surprisingly there were fewer genes with altered expression in the organ of Corti of Mir183/96 null mice compared with our previous findings in Mir96Dmdo mutants, which have a point mutation in the miR-96 seed region. This suggests that the more-severe phenotype of Mir96Dmdo mutants compared with Mir183/96 mutants, including progressive hearing loss in Mir96Dmdo heterozygotes, is likely to be mediated by the gain of novel target genes in addition to the loss of its normal targets. We propose three mechanisms of action of mutant miRNAs: loss of targets that are normally completely repressed, loss of targets for which transcription is normally buffered by the miRNA, and gain of novel targets. Any of these mechanisms could lead to a partial loss of a robust cellular identity and consequent dysfunction.

Network analysis of large-scale neuroimaging data is a particularly challenging computational problem. Here, we adapt a novel analytical tool, the community dynamic inference method (CommDy), for brain imaging data from young and aged mice. CommDy, which was inspired by social network theory, has been successfully used in other domains in biology; this report represents its first use in neuroscience. We used CommDy to investigate aging-related changes in network metrics in the auditory and motor cortices by using flavoprotein autofluorescence imaging in brain slices and in vivo. We observed that auditory cortical networks in slices taken from aged brains were highly fragmented compared to networks observed in young animals. CommDy network metrics were then used to build a random-forests classifier based on NMDA receptor blockade data, which successfully reproduced the aging findings, suggesting that the excitatory cortical connections may be altered during aging. A similar aging-related decline in network connectivity was also observed in spontaneous activity in the awake motor cortex, suggesting that the findings in the auditory cortex reflect general mechanisms during aging. These data suggest that CommDy provides a new dynamic network analytical tool to study the brain and that aging is associated with fragmentation of intracortical networks.