In vivo imaging of cortical organization and plasticity

 The cortex is a laminated structure that is thought to underlie sequential information processing. Sensory input enters layer 4 (L4) from which activity quickly spreads to superficial layers 2/3 (L2/3) and deep layers 5/6 (L5/6). Sensory responses themselves depend on ongoing, i.e. spontaneous cortical activity, as well as the state and behavioral context of the animal. Receptive field properties of neurons can rapidly and adaptively be reshaped when an animal is engaged in a behavioral task, indicating that even in primary auditory cortex encoding of stimuli is dependent on task- or context-dependent state (Francis et al 2018, Francis et al. 2022). Responses also depend on ongoing cortical dynamics in a lamina-dependent fashion. However, we do not know how neuronal circuits shape these emergent dynamics within and between laminae, and we do not know which neurons encode which aspect of a sensory stimulus. In a collaborative project (together with J. Maunsell, D. Rinberg, D. Plenz, M. Histed, S. Shoham, B. Babadi, T. Fellin, S. Panzeri, W. Losert, D. Chialvo) funded by the NIH BRAIN initiative, we use in vivo 2-photon imaging and stimulation technology that allows rapid imaging and stimulation in multiple focal planes and by developing new computational and analysis techniques based on dynamic systems and graph theoretic measures to extract network dynamics at the single neuron and population level. We use these new techniques to investigate the 3D single cell and population activity patterns in the auditory cortex in mice and identify the influence of single neurons relative to the synergistic influence of specific groups of neurons (the crowd) on network dynamics and ultimately behavior of the animal. We already found that populations of neurons in A1 show neuronal avalanches (Bowen et al. 2019) and that they show a frctured columnar small world functional organization (Bowen et al. 2024). Using single holographic stimulation we showed that co-tuned networks in A1 rapidly rebalance their activity (Kang et al. 2025)

Our work already identified the functional micro-architecture of the auditory cortex, and how this architecture can change and how change is influenced by top-down projections from the frontal cortex (see Bandyopadhyay et al Nature Neuroscience 2010, Winkowski & Kanold 2013, Winkowski et al. 2013, Meng et al. 2019, Liu et al. 2019, Mittelstadt & Kanold 2023).

What happens then to the cortex when you learn? We started to investigate this by tracking auditory cortical responses when animals learned an auditory tone discrimination task. We used a fully automated system combining automatic head-fixation and wide field imaging to minimize experimenter interaction with the animal. This was we could track daily performance and neural activity changes. We find that learning recruits higher-order auditory fields and enhances the representation of behavioral variables in these areas (Wang et al. 2024). Moreover, learning increases the functional connectivity between primary and higher-order areas (Wang et al. 2024). Thus learning and auditory task leads to a functional reorganization of the auditory cortex.

In vitro studies of cortical micro-circuits

To understand how neurons are wired up we need to reveal their connections with each other. To do this we use in vitro single and 2-photon photostimulation, coupled with patch clamp recordings and 2-photon imaging. Photostimulation allows us to selectively stimulate neurons and we can then observe which other neurons are responding. Thus we essentially can create input and output connection maps of targeted neurons. By performing these studies across layers and across ages we aim to assemble a wiring diagram of the adult and developing brain. Our work already identified the micro-circuitry of L2/3 of auditory cortex (Watkins et al. 2014, Meng et al. 2017) and how it can change (Meng et al. 2015, 2017, 2019).

Effects of Developmental insults and Aging on sound processing

We are very interested in understanding how changes in cortical processing leads to changes in perception. Some changes in cortical processing and perception seem to happen quite “naturally”, e.g. when we age, and we are engaged in multiple studies unraveling what happens to sound encoding in old age.

While aging happens to all of us, developmental insults can also create life-long changes to the way the brain processes information. We thus also investigate the consequences of developmental manipulations or insults (e.g. perinatal drug exposure) in adult.

Clinical relevance

This work is aimed at revealing how cortical circuits process sound information in the adult. This information is obviously important in understanding how the brain works, but also serves as a crucial reference data for evaluating the effects  of injury or aging. Moreover, understanding how neural circuits in the auditory cortex process sounds and amplify important sound sources and suppress distractors can also lead to the design of better hearing aids, cochlear implants, and AI systems.

Relevant publications:

L. Xiang, M Wang, P.O. Kanold*, A. Charles*, “Precise Extraction of Neural Motifs Reveals Multiscale, Parallel Encoding Schemes in Auditory Cortex”, bioRxiv 2025

Y Chen,  CT Chen, Y Gui, P.O. Kanold, “Robust representation and non-linear spectral integration of simple and complex harmonic sounds in layers 4 and 2/3 of primary auditory cortex”, bioRxiv 2025

P. Jendrichovsky, S. Khosravi, A. Rupasinghe, K. Maximov, P. Guo, B. Babadi, P.O. Kanold, “Patchy harmonic functional connectivity of the mouse auditory cortex”, Proc. Natl. Acad. Sci. U.S.A. 122 (27)

H. Kang, T. Babola, P. O. Kanold,“Rapid rebalancing of co-tuned ensemble activity in the auditory cortex” eLife 2025, preprint bioRxiv 2024

T. A. Babola, N. Donovan, S.S. Darcy, C. D. Spjut, P. O. Kanold, “Limiting hearing loss in transgenic mouse models”, eNeuro 2025, preprint in bioRxiv 2024

D. Luo, J. Liu, R. Auksztulewicz, T. Ka-Wing Yip, P.O. Kanold, J. W. H. Schnupp, “Hierarchical Deviant Processing in Auditory Cortex of Awake Mice”, Hearing Research 2025, bioRxiv 2023

H. Kang, P.O.Kanold,”Sparse representation of neurons for encoding complex sounds in the auditory cortex”, Progress in Neurobiology 2024, preprint in bioRxiv 2023

Z. Bowen, D. De Zoysa, K. Shilling-Scrivo, S. Aghayee, G. Di Salvo, A. Smirnov, P. Kanold, and W. Losert, “NeuroART: Real-time analysis and targeting of neuronal population activity during calcium imaging for informed closed loop experiments” eNeuro 2024

M. Wang, P. Jendrichovsky, P. O. Kanold, “Auditory discrimination learning differentially modulates neural representation in auditory cortex subregions and interareal connectivity” Cell Reports 2024

S. Mukherjee, P. Jendrichovsky, P. O. Kanold, B. Babadi, “Reinforcement Learning-Guided Optogenetic Stimulation Policies for Robust Functional Network Discovery,” IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP) 2024.

Z. Bowen, K. Shilling-Scrivo, W. Losert P. O. Kanold , “Fractured columnar small-world functional network organization in volumes of L2/3 of mouse auditory cortex’ PNAS Nexus 2024

T. Ribeiro, S. Yu. D. Martin, P. Jendrichovsky, P.O. Kanold, D. Chialvo, D. Plenz, “Trial-by-trial variability in cortical responses exhibits scaling in spatial correlations predicted from critical dynamics”, Cell Reports 2024, preprint in bioRxiv 2020

 J. Mittelstadt, P. O. Kanold , “Orbitofrontal cortex conveys stimulus and task information to the auditory cortex” Current Biology 2023

G. Calhoun, C.-T. Chen, P. O. Kanold,“Bilateral widefield calcium imaging reveals circuit asymmetries and lateralized functional activation of the mouse auditory cortex” PNAS 2023

L. Koçillari, M. Celotto, N. A. Francis, S. Mukherjee, B. Babadi, P. O. Kanold, S. Panzeri, “Measuring stimulus-related redundant and synergistic functional connectivity with single cell resolution in auditory cortex”, Brain Informatics 2023 ,preprint in  bioRxiv 2023

Bowen, Z., Magnusson, G., Diep, M., Ayyangar, U., Smirnov, A., Kanold, P. O.,  Losert, W. .NeuroWRAP: Integrating, validating, and sharing neurodata analysis workflows. Frontiers in Neuroinformatics, 17. 2023

J.Liu, P. O. Kanold , “Interactive auditory task reveals complex sensory-action integration in mouse primary auditory cortex”, bioRxiv 2022

J.Liu, K. Maximov, P. O. Kanold , “Automated head-fixation training system with high levels of animal participation in psychoacoustic tasks”, PLOS One 2025, Preprint in bioRxiv 2022

N. Francis , S. Mukherjee, L. Koçillari , S. Panzeri, B. Babadi, P. O. Kanold , “Sequential Transmission of Task-Relevant Information in Cortical Neuronal Networks”, Cell Reports 2022, preprint in bioRxiv 2021

W. Shao, M. Chang, K. Emmerich, P. O. Kanold, J. S Mumm, Ji, Yi “Mesoscopic oblique plane microscopy (Meso-OPM) – enabling large-scale 4D isotropic cellular resolution imaging with a diffractive light sheet”, Optica 2022, preprint in bioRxiv 2022 

A. Rupasinghe , N. Francis , J. Liu , Z. Bowen , P. O. Kanold , B. Babadi, “Direct Extraction of Signal and Noise Correlations from Two-Photon Calcium Imaging of Ensemble Neuronal Activity”, eLife 2021

J. Liu, P.O. Kanold, “Diversity of receptive fields and sideband inhibition with complex thalamocortical and intracortical origin in L2/3 of mouse primary auditory cortex”, in press  J Neuroscience 2021

Z. Bowen, D. E. Winkowski, P. O. Kanold, “Functional Organization of Mouse Primary Auditory Cortex in adult C57BL/6 and F1 (CBAxC57) mice”, Scientific Reports 2020, Preprint in bioRxiv 2019

N. Francis, K. Bohlke, P. O. Kanold, “Automated Behavioral Experiments in Mice Reveal Periodic Cycles of Task Engagement within Circadian Rhythms. “, eNeuro 2019

Z. Bowen, D. E Winkowski, S. Seshadri, D. Plenz, P. O. Kanold, “Neuronal avalanches in input and associative layers of auditory cortex” Frontiers in Systems Neuroscience 2019, preprint in bioRxiv 2019

 J. Liu, M.R. Whiteway, A. Sheikhattar, D.A. Butts, B. Babadi, P. O. Kanold, “Parallel Processing of Sound Dynamics across Mouse Auditory Cortex via Spatially Patterened Thalamic Inputs and Distinct Areal Intracortical Circuits”, Cell Reports 2019

A. Sheikhattar, S. Miran, J. Liu, J.B. Fritz, S.A. Shamma, P. O. Kanold, B. Babadi, “Extracting neuronal functional network dynamics via adaptive Granger causality analysis” Proceedings of the National Academy of Sciences 2018

N. Francis, D. E Winkowski, A. Sheikhattar, K. Armengol, B. Babadi, P. O. Kanold “Small networks Encode Decision-Making in Auditory Cortex”, Neuron 2018

S. Seshradi, A. Klaus, D. E Winkowski, P. O. Kanold, D. Plenz, “Altered avalanche dynamics in a developmental NMDR hypofunction model of cognitive impairment”, Translational Psychiatry 2018

S. Aghayee, D. E Winkowski, Z. Bowen, E. E Marshall, M. J Harrington, P. O. Kanold, W. Losert, “Particle Tracking Facilitates Real Time Capable Motion Correction in 2D or 3D Two-Photon Imaging of Neuronal Activity” Frontiers in Neural Circuits 2017

S. Aghayee, D. E Winkowski, Z. Bowen, E. E Marshall, M. J Harrington, P. O. Kanold, W. Losert, “Particle Tracking Facilitates Real Time Capable Motion Correction in 2D or 3D Two-Photon Imaging of Neuronal Activity” Frontiers in Neural Circuits 2017

A. Kazemipour, J. Liu, K. Solarana, D. Nagode, P. Kanold, M. Wu, B. Babadi, “Fast and Stable Signal Deconvolution via Compressible State-Space Models” IEEE Trans. on Biomedical Engineering 2017

N. Francis N., P.O. Kanold ,”Automated Operant Conditioning in the Mouse Home Cage”, Frontiers Neural Circuits 2017

D. Winkowski, D. Nagode, K. Donaldson, P. Yin, S.A. Shamma, J.B. Fritz, P.O. Kanold, “Orbitofrontal cortex neurons respond to sound and activate primary auditory cortex neurons”, Cerebral Cortex 2017

P.O. Kanold*, I. Nelken*, D.B. Polley*,”Local versus global scales of organization in auditory cortex”, Trends in Neuroscience 2014

PV. Watkins, JPY Kao, P.O. Kanold,”Spatial pattern of intra-laminar connectivity in supragranular mouse auditory cortex “, Frontiers in Neural Circuits 2014

D. Winkowski*, S. Bandyopadhyay*, S. Shamma, P.O. Kanold,”Frontal cortex activation causes rapid plasticity of auditory cortical processing”, Journal of Neuroscience 2013

D. Winkowski, P.O. Kanold,”Laminar transformation of frequency organization in auditory cortex”, Journal of Neuroscience 2013

S. Bandyopadhyay, S. Shamma, P.O. Kanold,”Dichotomy of functional organization in the mouse auditory cortex”,  Nature Neuroscience, 2010.  see News & Views by Castro and Kandler