Retrosplenial inputs drive visual representations in the medial entorhinal cortex.

The importance of visual cues for navigation and goal-directed behavior is well established, although the neural mechanisms supporting sensory representations in navigational circuits are largely unknown. Navigation is fundamentally dependent on the medial entorhinal cortex (MEC), which receives direct projections from neocortical visual areas, including the retrosplenial cortex (RSC). Here, we perform high-density recordings of MEC neurons in awake, head-fixed mice presented with simple visual stimuli and assess the dynamics of sensory-evoked activity. We find that a large fraction of neurons exhibit robust responses to visual input. Visually responsive cells are located primarily in layer 3 of the dorsal MEC and can be separated into subgroups based on functional and molecular properties. Furthermore, optogenetic suppression of RSC afferents within the MEC strongly reduces visual responses. Overall, our results demonstrate that the MEC can encode simple visual cues in the environment that may contribute to neural representations of location necessary for accurate navigation.

Developmental trajectory of cortical somatostatin interneuron function.

GABAergic inhibition is critical to the proper development of neocortical circuits. However, GABAergic interneurons are highly diverse and the developmental roles of distinct inhibitory subpopulations remain largely unclear. Dendrite-targeting, somatostatin-expressing interneurons (SST-INs) in the mature cortex regulate synaptic integration and plasticity in excitatory pyramidal neurons (PNs) and exhibit unique feature selectivity. Relatively little is known about early postnatal SST-IN activity or impact on surrounding local circuits. We examined juvenile SST-INs and PNs in mouse primary visual cortex. PNs exhibited stable visual responses and feature selectivity from eye opening onwards. In contrast, SST-INs developed visual responses and feature selectivity during the third postnatal week in parallel with a rapid increase in excitatory synaptic innervation. SST-INs largely exerted a multiplicative effect on nearby PN visual responses at all ages, but this impact increased over time. Our results identify a developmental window for the emergence of an inhibitory circuit mechanism for normalization.

Traumatic brain injury disrupts state-dependent functional cortical connectivity in a mouse model.

Traumatic brain injury (TBI) is the leading cause of death in young people and can cause cognitive and motor dysfunction and disruptions in functional connectivity between brain regions. In human TBI patients and rodent models of TBI, functional connectivity is decreased after injury. Recovery of connectivity after TBI is associated with improved cognition and memory, suggesting an important link between connectivity and functional outcome. We examined widespread alterations in functional connectivity following TBI using simultaneous widefield mesoscale GCaMP7c calcium imaging and electrocorticography (ECoG) in mice injured using the controlled cortical impact (CCI) model of TBI. Combining CCI with widefield cortical imaging provides us with unprecedented access to characterize network connectivity changes throughout the entire injured cortex over time. Our data demonstrate that CCI profoundly disrupts functional connectivity immediately after injury, followed by partial recovery over 3 weeks. Examining discrete periods of locomotion and stillness reveals that CCI alters functional connectivity and reduces theta power only during periods of behavioral stillness. Together, these findings demonstrate that TBI causes dynamic, behavioral state-dependent changes in functional connectivity and ECoG activity across the cortex.

Rapid fluctuations in functional connectivity of cortical networks encode spontaneous behavior.

Experimental work across species has demonstrated that spontaneously generated behaviors are robustly coupled to variations in neural activity within the cerebral cortex. Functional magnetic resonance imaging data suggest that temporal correlations in cortical networks vary across distinct behavioral states, providing for the dynamic reorganization of patterned activity. However, these data generally lack the temporal resolution to establish links between cortical signals and the continuously varying fluctuations in spontaneous behavior observed in awake animals. Here, we used wide-field mesoscopic calcium imaging to monitor cortical dynamics in awake mice and developed an approach to quantify rapidly time-varying functional connectivity. We show that spontaneous behaviors are represented by fast changes in both the magnitude and correlational structure of cortical network activity. Combining mesoscopic imaging with simultaneous cellular-resolution two-photon microscopy demonstrated that correlations among neighboring neurons and between local and large-scale networks also encode behavior. Finally, the dynamic functional connectivity of mesoscale signals revealed subnetworks not predicted by traditional anatomical atlas-based parcellation of the cortex. These results provide new insights into how behavioral information is represented across the neocortex and demonstrate an analytical framework for investigating time-varying functional connectivity in neural networks.

The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

Retrosplenial inputs drive diverse visual representations in the medial entorhinal cortex.

The ability of rodents to use visual cues for successful navigation and goal-directed behavior has been long appreciated, although the neural mechanisms supporting sensory representations in navigational circuits are largely unknown. Navigation is fundamentally dependent on the hippocampus and closely connected entorhinal cortex, whose neurons exhibit characteristic firing patterns corresponding to the animal's location. The medial entorhinal cortex (MEC) receives direct projections from sensory areas in the neocortex, suggesting the ability to encode sensory information. To examine this possibility, we performed high-density recordings of MEC neurons in awake, head-fixed mice presented with simple visual stimuli and assessed the dynamics of sensory-evoked activity. We found a large fraction of neurons exhibited robust responses to visual input that shaped activity relative to ongoing network dynamics. Visually responsive cells could be separated into subgroups based on functional and molecular properties within deep layers of the dorsal MEC, suggesting diverse populations within the MEC contribute to sensory encoding. We then showed that optogenetic suppression of retrosplenial cortex afferents within the MEC strongly reduced visual responses. Overall, our results demonstrate the the MEC can encode simple visual cues in the environment that can contribute to neural representations of location necessary for accurate navigation.

Spatiotemporally heterogeneous coordination of cholinergic and neocortical activity.

Variation in an animal's behavioral state is linked to fluctuations in brain activity and cognitive ability. In the neocortex, state-dependent circuit dynamics may reflect neuromodulatory influences such as that of acetylcholine (ACh). Although early literature suggested that ACh exerts broad, homogeneous control over cortical function, recent evidence indicates potential anatomical and functional segregation of cholinergic signaling. In addition, it is unclear whether states as defined by different behavioral markers reflect heterogeneous cholinergic and cortical network activity. Here, we perform simultaneous, dual-color mesoscopic imaging of both ACh and calcium across the neocortex of awake mice to investigate their relationships with behavioral variables. We find that higher arousal, categorized by different motor behaviors, is associated with spatiotemporally dynamic patterns of cholinergic modulation and enhanced large-scale network correlations. Overall, our findings demonstrate that ACh provides a highly dynamic and spatially heterogeneous signal that links fluctuations in behavior to functional reorganization of cortical networks.

Spatiotemporal dynamics in large-scale cortical networks.

Investigating links between nervous system function and behavior requires monitoring neuronal activity at a range of spatial and temporal scales. Here, we summarize recent progress in applying two distinct but complementary approaches to the study of network dynamics in the neocortex. Mesoscopic calcium imaging allows simultaneous monitoring of activity across most of the cortex at moderate spatiotemporal resolution. Electrophysiological recordings provide extremely high temporal resolution of neural signals at multiple targeted locations. A number of recent studies have used these tools to reveal novel patterns of activity across distributed cortical subnetworks. This growing body of work strongly supports the hypothesis that the dynamic coordination of spatially distinct regions is a fundamental aspect of cortical function that supports cognition and behavior.

Building bridges: simultaneous multimodal neuroimaging approaches for exploring the organization of brain networks.

Brain organization is evident across spatiotemporal scales as well as from structural and functional data. Yet, translating from micro- to macroscale (vice versa) as well as between different measures is difficult. Reconciling disparate observations from different modes is challenging because each specializes within a restricted spatiotemporal milieu, usually has bounded organ coverage, and has access to different contrasts. True intersubject biological heterogeneity, variation in experiment implementation (e.g., use of anesthesia), and true moment-to-moment variations in brain activity (maybe attributable to different brain states) also contribute to variability between studies. Ultimately, for a deeper and more actionable understanding of brain organization, an ability to translate across scales, measures, and species is needed. Simultaneous multimodal methods can contribute to bettering this understanding. We consider four modes, three optically based: multiphoton imaging, single-photon (wide-field) imaging, and fiber photometry, as well as magnetic resonance imaging. We discuss each mode as well as their pairwise combinations with regard to the definition and study of brain networks.

Simultaneous cortex-wide fluorescence Ca2+ imaging and whole-brain fMRI.

Achieving a comprehensive understanding of brain function requires multiple imaging modalities with complementary strengths. We present an approach for concurrent widefield optical and functional magnetic resonance imaging. By merging these modalities, we can simultaneously acquire whole-brain blood-oxygen-level-dependent (BOLD) and whole-cortex calcium-sensitive fluorescent measures of brain activity. In a transgenic murine model, we show that calcium predicts the BOLD signal, using a model that optimizes a gamma-variant transfer function. We find consistent predictions across the cortex, which are best at low frequency (0.009-0.08 Hz). Furthermore, we show that the relationship between modality connectivity strengths varies by region. Our approach links cell-type-specific optical measurements of activity to the most widely used method for assessing human brain function.

Mesoscopic Imaging: Shining a Wide Light on Large-Scale Neural Dynamics.

Optical imaging has revolutionized our ability to monitor brain activity, spanning spatial scales from synapses to cells to circuits. Here, we summarize the rapid development and application of mesoscopic imaging, a widefield fluorescence-based approach that balances high spatiotemporal resolution with extraordinarily large fields of view. By leveraging the continued expansion of fluorescent reporters for neuronal activity and novel strategies for indicator expression, mesoscopic analysis enables measurement and correlation of network dynamics with behavioral state and task performance. Moreover, the combination of widefield imaging with cellular resolution methods such as two-photon microscopy and electrophysiology is bridging boundaries between cellular and network analyses. Overall, mesoscopic imaging provides a powerful option in the optical toolbox for investigation of brain function.

NMDAR-Dependent Emergence of Behavioral Representation in Primary Visual Cortex.

Although neocortical sensory areas are generally thought to faithfully represent external stimuli, cortical networks exhibit considerable functional plasticity, allowing them to modify their output to reflect ongoing behavioral demands. We apply longitudinal 2-photon imaging of activity in the primary visual cortex (V1) of mice learning a conditioned eyeblink task to investigate the dynamic representations of task-relevant information. We find that, although all V1 neurons robustly and stably encode visual input, pyramidal cells and parvalbumin-expressing interneurons exhibit experience-dependent emergence of accurate behavioral representations during learning. The functional plasticity driving performance-predictive activity requires cell-autonomous expression of NMDA-type glutamate receptors. Our findings demonstrate that accurate encoding of behavioral output is not inherent to V1 but develops during learning to support visual task performance.

Diverse Spatiotemporal Scales of Cholinergic Signaling in the Neocortex.

ACh is a signaling molecule in the mammalian CNS, with well-documented influence over cognition and behavior. However, the nature of cholinergic signaling in the brain remains controversial, with ongoing debates focused on the spatial and temporal resolution of ACh activity. Generally, opposing views have embraced a dichotomy between transmission as slow and volume-mediated versus fast and synaptic. Here, we provide the perspective that ACh, like most other neurotransmitters, exhibits both fast and slow modes that are strongly determined by the anatomy of cholinergic fibers, the distribution and the signaling mechanisms of receptor subtypes, and the dynamics of ACh hydrolysis. Current methodological approaches remain limited in their ability to provide detailed analyses of these underlying factors. However, we believe that the continued development of novel technologies in combination with a more nuanced view of cholinergic activity will open critical new avenues to a better understanding of ACh in the brain.Dual Perspectives Companion Paper: Forebrain Cholinergic Signaling: Wired and Phasic, Not Tonic, and Causing Behavior, by Martin Sarter and Cindy Lustig.

Layer 5 Circuits in V1 Differentially Control Visuomotor Behavior.

Neocortical sensory areas are thought to act as distribution hubs, transmitting information about the external environment to downstream areas. Within primary visual cortex, various populations of pyramidal neurons (PNs) send axonal projections to distinct targets, suggesting multiple cellular networks may be independently engaged during behavior. We investigated whether PN subpopulations differentially support visual detection by training mice on a novel eyeblink conditioning task. Applying 2-photon calcium imaging and optogenetic manipulation of anatomically defined PNs, we show that layer 5 corticopontine neurons strongly encode sensory and motor task information and are selectively necessary for performance. Our findings support a model in which target-specific cortical subnetworks form the basis for adaptive behavior by directing relevant information to distinct brain areas. Overall, this work highlights the potential for neurons to form physically interspersed but functionally segregated networks capable of parallel, independent control of perception and behavior.

Simultaneous mesoscopic and two-photon imaging of neuronal activity in cortical circuits.

Spontaneous and sensory-evoked activity propagates across varying spatial scales in the mammalian cortex, but technical challenges have limited conceptual links between the function of local neuronal circuits and brain-wide network dynamics. We present a method for simultaneous cellular-resolution two-photon calcium imaging of a local microcircuit and mesoscopic widefield calcium imaging of the entire cortical mantle in awake mice. Our multi-scale approach involves a microscope with an orthogonal axis design where the mesoscopic objective is oriented above the brain and the two-photon objective is oriented horizontally, with imaging performed through a microprism. We also introduce a viral transduction method for robust and widespread gene delivery in the mouse brain. These approaches allow us to identify the behavioral state-dependent functional connectivity of pyramidal neurons and vasoactive intestinal peptide-expressing interneurons with long-range cortical networks. Our imaging system provides a powerful strategy for investigating cortical architecture across a wide range of spatial scales.

Preserving the balance: diverse forms of long-term GABAergic synaptic plasticity.

Cellular mechanisms that regulate the interplay of synaptic excitation and inhibition are thought to be central to the functional stability of healthy neuronal circuits. A growing body of literature demonstrates the capacity for inhibitory GABAergic synapses to exhibit long-term plasticity in response to changes in neuronal activity. Here, we review this expanding field of research, focusing on the diversity of mechanisms that link glutamatergic signalling, postsynaptic action potentials and inhibitory synaptic strength. Several lines of evidence indicate that multiple, parallel forms of plasticity serve to regulate activity at both the input and output domains of individual neurons. Overall, these varied phenomena serve to promote both stability and flexibility over the life of the organism.

Ketamine disrupts neuromodulatory control of glutamatergic synaptic transmission.

A growing body of literature has demonstrated the potential for ketamine in the treatment of major depression. Sub-anesthetic doses produce rapid and sustained changes in depressive behavior, both in patients and rodent models, associated with reorganization of glutamatergic synapses in the prefrontal cortex (PFC). While ketamine is known to regulate N-methyl-D-aspartate (NMDA) -type glutamate receptors (NMDARs), the full complement of downstream cellular consequences for ketamine administration are not well understood. Here, we combine electrophysiology with 2-photon imaging and glutamate uncaging in acute slices of mouse PFC to further examine how ketamine alters glutamatergic synaptic transmission. We find that four hours after ketamine treatment, glutamatergic synapses themselves are not significantly affected. However, levels of the neuromodulatory Regulator of G-protein Signaling (RGS4) are dramatically reduced. This loss of RGS4 activity is associated with disruption of the normal compartmentalization of synaptic neuromodulation. Thus, under control conditions, α2 adrenergic receptors and type B γ-aminobutyric acid (GABAB) receptors selectively inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type glutamate receptors (AMPARs) and NMDARs, respectively. After ketamine administration and reduction in RGS4 activity, this selectivity is lost, with both modulatory systems broadly inhibiting glutamatergic transmission. These results suggest a novel mechanism by which ketamine may influence synaptic signaling and provide new avenues for the exploration of therapeutics directed at treating neuropsychiatric disorders, such as depression.

Potassium channels contribute to activity-dependent regulation of dendritic inhibition.

GABAergic inhibition plays a critical role in the regulation of neuronal activity. In the neocortex, inhibitory interneurons that target the dendrites of pyramidal cells influence both electrical and biochemical postsynaptic signaling. Voltage-gated ion channels strongly shape dendritic excitability and the integration of excitatory inputs, but their contribution to GABAergic signaling is less well understood. By combining 2-photon calcium imaging and focal GABA uncaging, we show that voltage-gated potassium channels normally suppress the GABAergic inhibition of calcium signals evoked by back-propagating action potentials in dendritic spines and shafts of cortical pyramidal neurons. Moreover, the voltage-dependent inactivation of these channels leads to enhancement of dendritic calcium inhibition following somatic spiking. Computational modeling reveals that the enhancement of calcium inhibition involves an increase in action potential depolarization coupled with the nonlinear relationship between membrane voltage and calcium channel activation. Overall, our findings highlight the interaction between intrinsic and synaptic properties and reveal a novel mechanism for the activity-dependent regulation of GABAergic inhibition.

Input-Specific NMDAR-Dependent Potentiation of Dendritic GABAergic Inhibition.

Preservation of a balance between synaptic excitation and inhibition is critical for normal brain function. A number of homeostatic cellular mechanisms have been suggested to play a role in maintaining this balance, including long-term plasticity of GABAergic inhibitory synapses. Many previous studies have demonstrated a coupling of postsynaptic spiking with modification of perisomatic inhibition. Here, we demonstrate that activation of NMDA-type glutamate receptors leads to input-specific long-term potentiation of dendritic inhibition mediated by somatostatin-expressing interneurons. This form of plasticity is expressed postsynaptically and requires both CaMKIIα and the ß2 subunit of the GABA-A receptor. Importantly, this process may function to preserve dendritic inhibition, as genetic deletion of NMDAR signaling results in a selective weakening of dendritic inhibition. Overall, our results reveal a new mechanism for linking excitatory and inhibitory input in neuronal dendrites and provide novel insight into the homeostatic regulation of synaptic transmission in cortical circuits.