Cortical circuits modulate mouse social vocalizations.

Vocalizations provide a means of communication with high fidelity and information rate for many species. Diencephalon and brainstem neural circuits have been shown to control mouse vocal production; however, the role of cortical circuits in this process is debatable. Using electrical and optogenetic stimulation, we identified a cortical region in the anterior cingulate cortex in which stimulation elicits ultrasonic vocalizations. Moreover, fiber photometry showed an increase in Ca2+ dynamics preceding vocal initiation, whereas optogenetic suppression in this cortical area caused mice to emit fewer vocalizations. Last, electrophysiological recordings indicated a differential increase in neural activity in response to female social exposure dependent on vocal output. Together, these results indicate that the cortex is a key node in the neuronal circuits controlling vocal behavior in mice.

High-throughput morphometric and transcriptomic profiling uncovers composition of naïve and sensory-deprived cortical cholinergic VIP/CHAT neurons.

Cortical neuronal networks control cognitive output, but their composition and modulation remain elusive. Here, we studied the morphological and transcriptional diversity of cortical cholinergic VIP/ChAT interneurons (VChIs), a sparse population with a largely unknown function. We focused on VChIs from the whole barrel cortex and developed a high-throughput automated reconstruction framework, termed PopRec, to characterize hundreds of VChIs from each mouse in an unbiased manner, while preserving 3D cortical coordinates in multiple cleared mouse brains, accumulating thousands of cells. We identified two fundamentally distinct morphological types of VChIs, bipolar and multipolar that differ in their cortical distribution and general morphological features. Following mild unilateral whisker deprivation on postnatal day seven, we found after three weeks both ipsi- and contralateral dendritic arborization differences and modified cortical depth and distribution patterns in the barrel fields alone. To seek the transcriptomic drivers, we developed NuNeX, a method for isolating nuclei from fixed tissues, to explore sorted VChIs. This highlighted differentially expressed neuronal structural transcripts, altered exitatory innervation pathways and established Elmo1 as a key regulator of morphology following deprivation.

Synaptic Input and ACh Modulation Regulate Dendritic Ca2+ Spike Duration in Pyramidal Neurons, Directly Affecting Their Somatic Output.

Nonlinear synaptic integration in dendrites is a fundamental aspect of neural computation. One such key mechanism is the Ca2+ spike at the apical tuft of pyramidal neurons. Characterized by a plateau potential sustained for tens of milliseconds, the Ca2+ spike amplifies excitatory input, facilitates somatic action potentials (APs), and promotes synaptic plasticity. Despite its essential role, the mechanisms regulating it are largely unknown. Using a compartmental model of a layer 5 pyramidal cell (L5PC), we explored the plateau and termination phases of the Ca2+ spike under input current perturbations, long-step current-injections, and variations in the dendritic high-voltage-activated Ca2+ conductance (that occur during cholinergic modulation). We found that, surprisingly, timed excitatory input can shorten the Ca2+ spike duration while inhibitory input can either elongate or terminate it. A significant elongation also occurs when the high-voltage-activated Ca2+ channels (CaHVA) conductance is increased. To mechanistically understand these phenomena, we analyzed the currents involved in the spike. The plateau and termination phases are almost exclusively controlled by the CaHVA inward current and the Im outward K+ current. We reduced the full model to a single-compartment model that faithfully preserved the responses of the Ca2+ spike to interventions and consisted of two dynamic variables: the membrane potential and the K+-channel activation level. A phase-plane analysis of the reduced model provides testable predictions for modulating the Ca2+ spike and reveals various dynamical regimes that explain the robust nature of the spike. Regulating the duration of the Ca2+ spike significantly impacts the cell synaptic-plasticity window and, as we show, its input-output relationship.SIGNIFICANCE STATEMENT Pyramidal neurons are the cortex's principal projection neurons. In their apical tuft, dendritic Ca2+ spikes significantly impact information processing, synaptic plasticity, and the cell's input-output relationship. Therefore, it is essential to understand the mechanisms regulating them. Using a compartmental model of a layer 5 pyramidal cell (L5PC), we explored the Ca2+ spike responses to synaptic perturbations and cholinergic modulation. We showed a counterintuitive phenomenon: early excitatory input shortens the spike, whereas weak inhibition elongates it. Also, we demonstrated that acetylcholine (ACh) extends the spike. Through a reduced model containing only the membrane potential and the K+-channel activation level, we explained these phenomena using a phase-plane analysis. Our work provides new information about the robustness of the Ca2+ spike and its controlling mechanisms.

The Role of Hub Neurons in Modulating Cortical Dynamics.

Many neurodegenerative diseases are associated with the death of specific neuron types in particular brain regions. What makes the death of specific neuron types particularly harmful for the integrity and dynamics of the respective network is not well understood. To start addressing this question we used the most up-to-date biologically realistic dense neocortical microcircuit (NMC) of the rodent, which has reconstructed a volume of 0.3 mm3 and containing 31,000 neurons, ∼37 million synapses, and 55 morphological cell types arranged in six cortical layers. Using modern network science tools, we identified hub neurons in the NMC, that are connected synaptically to a large number of their neighbors and systematically examined the impact of abolishing these cells. In general, the structural integrity of the network is robust to cells' attack; yet, attacking hub neurons strongly impacted the small-world topology of the network, whereas similar attacks on random neurons have a negligible effect. Such hub-specific attacks are also impactful on the network dynamics, both when the network is at its spontaneous synchronous state and when it was presented with synchronized thalamo-cortical visual-like input. We found that attacking layer 5 hub neurons is most harmful to the structural and functional integrity of the NMC. The significance of our results for understanding the role of specific neuron types and cortical layers for disease manifestation is discussed.

Single cortical neurons as deep artificial neural networks.

Utilizing recent advances in machine learning, we introduce a systematic approach to characterize neurons' input/output (I/O) mapping complexity. Deep neural networks (DNNs) were trained to faithfully replicate the I/O function of various biophysical models of cortical neurons at millisecond (spiking) resolution. A temporally convolutional DNN with five to eight layers was required to capture the I/O mapping of a realistic model of a layer 5 cortical pyramidal cell (L5PC). This DNN generalized well when presented with inputs widely outside the training distribution. When NMDA receptors were removed, a much simpler network (fully connected neural network with one hidden layer) was sufficient to fit the model. Analysis of the DNNs' weight matrices revealed that synaptic integration in dendritic branches could be conceptualized as pattern matching from a set of spatiotemporal templates. This study provides a unified characterization of the computational complexity of single neurons and suggests that cortical networks therefore have a unique architecture, potentially supporting their computational power.

Cortical VIP+ /ChAT+ interneurons: From genetics to function.

One of the urgent tasks of neuroscience is to understand how neuronal circuits operate, what makes them fail, and how to repair them when needed. Achieving this goal requires identifying the principal circuitry elements and their interactions with one another. However, what constitutes 'an atom' of a neuronal circuit, a neuronal type, is a complex question. In this review we focus on a class of cortical neurons that are exclusively identified by the expression of vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The genetic profile of these VIP+ /ChAT+ interneurons suggests that they can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). This hints to a specific potential role in the cortical circuitry. Yet the VIP+ /ChAT+ interneurons are sparse (a mere 0.5% of the cortical neurons), which raises questions about their potential to significantly affect the circuit function. In view of recent developments in genetic techniques that allow for direct manipulation of these neurons, we provide a thorough and updated picture of the properties of the VIP+ /ChAT+ interneurons. We discuss their genetic profile, their physiological and structural properties, and their input-output mapping in sensory cortices and the medial prefrontal cortex (mPFC). Then, we examine possible amplification mechanisms for mediating their function in the cortical microcircuit. Finally, we discuss directions for further exploration of the VIP+ /ChAT+ population, focusing on its function during behavioral tasks as compared to the VIP+ /ChAT- population.

Astrocytes contribute to remote memory formation by modulating hippocampal-cortical communication during learning.

Remote memories depend on coordinated activity in the hippocampus and frontal cortices, but the timeline of these interactions is debated. Astrocytes sense and modify neuronal activity, but their role in remote memory is scarcely explored. We expressed the Gi-coupled designer receptor hM4Di in CA1 astrocytes and discovered that astrocytic manipulation during learning specifically impaired remote, but not recent, memory recall and decreased activity in the anterior cingulate cortex (ACC) during retrieval. We revealed massive recruitment of ACC-projecting CA1 neurons during memory acquisition, which was accompanied by the activation of ACC neurons. Astrocytic Gi activation disrupted CA3 to CA1 communication in vivo and reduced the downstream response in the ACC. In behaving mice, it induced a projection-specific inhibition of CA1-to-ACC neurons during learning, which consequently prevented ACC recruitment. Finally, direct inhibition of CA1-to-ACC-projecting neurons spared recent and impaired remote memory. Our findings suggest that remote memory acquisition involves projection-specific functions of astrocytes in regulating CA1-to-ACC neuronal communication.

Temporal structure of mouse courtship vocalizations facilitates syllable labeling.

Mice emit sequences of ultrasonic vocalizations (USVs) but little is known about the rules governing their temporal order and no consensus exists on the classification of USVs into syllables. To address these questions, we recorded USVs during male-female courtship and found a significant temporal structure. We labeled USVs using three popular algorithms and found that there was no one-to-one relationships between their labels. As label assignment affects the high order temporal structure, we developed the Syntax Information Score (based on information theory) to rank labeling algorithms based on how well they predict the next syllable in a sequence. Finally, we derived a novel algorithm (Syntax Information Maximization) that utilizes sequence statistics to improve the clustering of individual USVs with respect to the underlying sequence structure. Improvement in USV classification is crucial for understanding neural control of vocalization. We demonstrate that USV syntax holds valuable information towards achieving this goal.

Barrel cortex VIP/ChAT interneurons suppress sensory responses in vivo.

Cortical interneurons expressing vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT) are sparsely distributed throughout the neocortex, constituting only 0.5% of its neuronal population. The co-expression of VIP and ChAT suggests that these VIP/ChAT interneurons (VChIs) can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). In vitro physiological studies quantified the response properties and local connectivity patterns of the VChIs; however, the function of VChIs has not been explored in vivo. To study the role of VChIs in cortical network dynamics and their long-range connectivity pattern, we used in vivo electrophysiology and rabies virus tracing in the barrel cortex of mice. We found that VChIs have a low spontaneous spiking rate (approximately 1 spike/s) in the barrel cortex of anesthetized mice; nevertheless, they responded with higher fidelity to whisker stimulation than the neighboring layer 2/3 pyramidal neurons (Pyrs). Analysis of long-range inputs to VChIs with monosynaptic rabies virus tracing revealed that direct thalamic projections are a significant input source to these cells. Optogenetic activation of VChIs in the barrel cortex of awake mice suppresses the sensory responses of excitatory neurons in intermediate amplitudes of whisker deflections while increasing the evoked spike latency. The effect of VChI activation on the response was similar for both high-whisking (HW) and low-whisking (LW) conditions. Our findings demonstrate that, despite their sparsity, VChIs can effectively modulate sensory processing in the cortical microcircuit.

Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. They have been steadily increasing in children over the past several years, with no effective treatment. BTBR T+tf/J (BTBR) mice are an accepted model of evaluating autistic-like behaviors as they present all core symptoms of ASD. We have previously shown that transplantation of human bone marrow mesenchymal stem cells (MSC) to the lateral ventricles of BTBR mice results in long lasting improvement in their autistic behavioral phenotypes. Recent studies point exosomes as the main mediators of the therapeutic effect of MSC. Here, we tested whether treatment with the exosomes secreted from MSC (MSC-exo) will show similar beneficial effects. We found that intranasal administration of MSC-exo increased male to male social interaction and reduced repetitive behaviors. Moreover, the treatment led to increases of male to female ultrasonic vocalizations and significant improvement in maternal behaviors of pup retrieval. No negative symptoms were detected following MSC-exo intranasal treatments in BTBR or healthy C57BL mice. The marked beneficial effects of the exosomes in BTBR mice may translate to a novel, non-invasive, and therapeutic strategy to reduce the symptoms of ASD.

Astrocytic Activation Generates De Novo Neuronal Potentiation and Memory Enhancement.

Astrocytes respond to neuronal activity and were shown to be necessary for plasticity and memory. To test whether astrocytic activity is also sufficient to generate synaptic potentiation and enhance memory, we expressed the Gq-coupled receptor hM3Dq in CA1 astrocytes, allowing their activation by a designer drug. We discovered that astrocytic activation is not only necessary for synaptic plasticity, but also sufficient to induce NMDA-dependent de novo long-term potentiation in the hippocampus that persisted after astrocytic activation ceased. In vivo, astrocytic activation enhanced memory allocation; i.e., it increased neuronal activity in a task-specific way only when coupled with learning, but not in home-caged mice. Furthermore, astrocytic activation using either a chemogenetic or an optogenetic tool during acquisition resulted in memory recall enhancement on the following day. Conversely, directly increasing neuronal activity resulted in dramatic memory impairment. Our findings that astrocytes induce plasticity and enhance memory may have important clinical implications for cognitive augmentation treatments.

Adrenergic Modulation Regulates the Dendritic Excitability of Layer 5 Pyramidal Neurons In Vivo.

The excitability of the apical tuft of layer 5 pyramidal neurons is thought to play a crucial role in behavioral performance and synaptic plasticity. We show that the excitability of the apical tuft is sensitive to adrenergic neuromodulation. Using two-photon dendritic Ca2+ imaging and in vivo whole-cell and extracellular recordings in awake mice, we show that application of the α2A-adrenoceptor agonist guanfacine increases the probability of dendritic Ca2+ events in the tuft and lowers the threshold for dendritic Ca2+ spikes. We further show that these effects are likely to be mediated by the dendritic current Ih. Modulation of Ih in a realistic compartmental model controlled both the generation and magnitude of dendritic calcium spikes in the apical tuft. These findings suggest that adrenergic neuromodulation may affect cognitive processes such as sensory integration, attention, and working memory by regulating the sensitivity of layer 5 pyramidal neurons to top-down inputs.

Intensify3D: Normalizing signal intensity in large heterogenic image stacks.

Three-dimensional structures in biological systems are routinely evaluated using large image stacks acquired from fluorescence microscopy; however, analysis of such data is muddled by variability in the signal across and between samples. Here, we present Intensify3D: a user-guided normalization algorithm tailored for overcoming common heterogeneities in large image stacks. We demonstrate the use of Intensify3D for analyzing cholinergic interneurons of adult murine brains in 2-Photon and Light-Sheet fluorescence microscopy, as well as of mammary gland and heart tissues. Beyond enhancement in 3D visualization in all samples tested, in 2-Photon in vivo images, this tool corrected errors in feature extraction of cortical interneurons; and in Light-Sheet microscopy, it enabled identification of individual cortical barrel fields and quantification of somata in cleared adult brains. Furthermore, Intensify3D enhanced the ability to separate signal from noise. Overall, the universal applicability of our method can facilitate detection and quantification of 3D structures and may add value to a wide range of imaging experiments.

Rich cell-type-specific network topology in neocortical microcircuitry.

Uncovering structural regularities and architectural topologies of cortical circuitry is vital for understanding neural computations. Recently, an experimentally constrained algorithm generated a dense network reconstruction of a ∼0.3-mm3 volume from juvenile rat somatosensory neocortex, comprising ∼31,000 cells and ∼36 million synapses. Using this reconstruction, we found a small-world topology with an average of 2.5 synapses separating any two cells and multiple cell-type-specific wiring features. Amounts of excitatory and inhibitory innervations varied across cells, yet pyramidal neurons maintained relatively constant excitation/inhibition ratios. The circuit contained highly connected hub neurons belonging to a small subset of cell types and forming an interconnected cell-type-specific rich club. Certain three-neuron motifs were overrepresented, matching recent experimental results. Cell-type-specific network properties were even more striking when synaptic strength and sign were considered in generating a functional topology. Our systematic approach enables interpretation of microconnectomics 'big data' and provides several experimentally testable predictions.

Long term beneficial effect of neurotrophic factors-secreting mesenchymal stem cells transplantation in the BTBR mouse model of autism.

Autism spectrum disorders (ASD) are neurodevelopmental disabilities characterized by severe impairment in social communication skills and restricted, repetitive behaviors. We have previously shown that a single transplantation of mesenchymal stem cells (MSC) into the cerebral lateral ventricles of BTBR autistic-like mice resulted in an improvement across all diagnostic criteria of ASD. We suggested that brain-derived neurotrophic factor (BDNF), a protein which supports the survival and regeneration of neurons secreted by MSC, largely contributed to the beneficial behavioral effect. In this study, we investigated the behavioral effects of transplanted MSC induced to secrete higher amounts of neurotrophic factors (NurOwn®), on various ASD-related behavioral domains using the BTBR mouse model of ASD. We demonstrate that NurOwn® transplantation had significant advantages over MSC transplantation in terms of improving communication skills, one and six months following treatment, as compared to sham-treated BTBR mice. Furthermore, NurOwn® transplantation resulted in reduced stereotypic behavior for as long as six months post treatment, compared to the one month improvement observed in the MSC treated mice. Notably, NurOwn® treatment resulted in improved cognitive flexibility, an improvement that was not observed by MSC treatment. Both MSC and NurOwn® transplantation induced an improvement in social behavior that lasted for six months. In conclusion, the present study demonstrates that a single transplantation of MSC or NurOwn® have long-lasting benefits, while NurOwn® may be superior to MSC treatment.

Neural networks within multi-core optic fibers.

Hardware implementation of artificial neural networks facilitates real-time parallel processing of massive data sets. Optical neural networks offer low-volume 3D connectivity together with large bandwidth and minimal heat production in contrast to electronic implementation. Here, we present a conceptual design for in-fiber optical neural networks. Neurons and synapses are realized as individual silica cores in a multi-core fiber. Optical signals are transferred transversely between cores by means of optical coupling. Pump driven amplification in erbium-doped cores mimics synaptic interactions. We simulated three-layered feed-forward neural networks and explored their capabilities. Simulations suggest that networks can differentiate between given inputs depending on specific configurations of amplification; this implies classification and learning capabilities. Finally, we tested experimentally our basic neuronal elements using fibers, couplers, and amplifiers, and demonstrated that this configuration implements a neuron-like function. Therefore, devices similar to our proposed multi-core fiber could potentially serve as building blocks for future large-scale small-volume optical artificial neural networks.

Social Ultrasonic Vocalization in Awake Head-Restrained Mouse.

Numerous animal species emit vocalizations in response to various social stimuli. The neural basis of vocal communication has been investigated in monkeys, songbirds, rats, bats, and invertebrates resulting in deep insights into motor control, neural coding, and learning. Mice, which recently became very popular as a model system for mammalian neuroscience, also utilize ultrasonic vocalizations (USVs) during mating behavior. However, our knowledge is lacking of both the behavior and its underlying neural mechanism. We developed a novel method for head-restrained male mice (HRMM) to interact with non-restrained female mice (NRFM) and show that mice can emit USVs in this context. We first recorded USVs in a free arena with non-restrained male mice (NRMM) and NRFM. Of the NRMM, which vocalized in the free arena, the majority could be habituated to also vocalize while head-restrained but only when a female mouse was present in proximity. The USVs emitted by HRMM are similar to the USVs of NRMM in the presence of a female mouse in their spectral structure, inter-syllable interval distribution, and USV sequence length, and therefore are interpreted as social USVs. By analyzing the vocalizations of NRMM, we established criteria to predict which individuals are likely to vocalize while head fixed based on the USV rate and average syllable duration. To characterize the USVs emitted by HRMM, we analyzed the syllable composition of HRMM and NRMM and found that USVs emitted by HRMM have a higher proportion of USVs with complex spectral representation, supporting previous studies showing that mice social USVs are context dependent. Our results suggest a way to study the neural mechanisms of production and control of social vocalization in mice using advanced methods requiring head fixation.

Tonic inhibition sets the state of excitability in olfactory bulb granule cells.

GABAergic granule cells (GCs) regulate, via mitral cells, the final output from the olfactory bulb to piriform cortex and are central for the speed and accuracy of odour discrimination. However, little is known about the local circuits in which GCs are embedded and how GCs respond during functional network activity. We recorded inhibitory and excitatory currents evoked during a single sniff-like odour presentation in GCs in vivo. We found that synaptic excitation was extensively activated across cells, whereas phasic inhibition was rare. Furthermore, our analysis indicates that GCs are innervated by a persistent firing of deep short axon cells that mediated the inhibitory evoked responses. Blockade of GABAergic synaptic input onto GCs revealed a tonic inhibitory current mediated by furosemide-sensitive GABA(A) receptors. The average current associated with this tonic GABAergic conductance was 3-fold larger than that of phasic inhibitory postsynaptic currents. We show that the pharmacological blockage of tonic inhibition markedly increased the occurrence of supra-threshold responses during an odour-stimulated sniff. Our findings suggest that GCs mediate recurrent or lateral inhibition, depending on the ambient level of extracellular GABA.

Tonic inhibition enhances fidelity of sensory information transmission in the cerebellar cortex.

Tonic inhibition is a key regulator of neuronal excitability and network function in the brain, but its role in sensory information processing remains poorly understood. The cerebellum is a favorable model system for addressing this question as granule cells, which form the input layer of the cerebellar cortex, permit high-resolution patch-clamp recordings in vivo, and are the only neurons in the cerebellar cortex that express the α6δ-containing GABA(A) receptors mediating tonic inhibition. We investigated how tonic inhibition regulates sensory information transmission in the rat cerebellum by using a combination of intracellular recordings from granule cells and molecular layer interneurons in vivo, selective pharmacology, and in vitro dynamic clamp experiments. We show that blocking tonic inhibition significantly increases the spontaneous firing rate of granule cells while only moderately increasing sensory-evoked spike output. In contrast, enhancing tonic inhibition reduces the spike probability in response to sensory stimulation with minimal effect on the spontaneous spike rate. Both manipulations result in a reduction in the signal-to-noise ratio of sensory transmission in granule cells and of parallel fiber synaptic input to downstream molecular layer interneurons. These results suggest that under basal conditions the level of tonic inhibition in vivo enhances the fidelity of sensory information transmission through the input layer of the cerebellar cortex.

Sensitivity to perturbations in vivo implies high noise and suggests rate coding in cortex.

It is well known that neural activity exhibits variability, in the sense that identical sensory stimuli produce different responses, but it has been difficult to determine what this variability means. Is it noise, or does it carry important information-about, for example, the internal state of the organism? Here we address this issue from the bottom up, by asking whether small perturbations to activity in cortical networks are amplified. Based on in vivo whole-cell patch-clamp recordings in rat barrel cortex, we find that a perturbation consisting of a single extra spike in one neuron produces approximately 28 additional spikes in its postsynaptic targets. We also show, using simultaneous intra- and extracellular recordings, that a single spike in a neuron produces a detectable increase in firing rate in the local network. Theoretical analysis indicates that this amplification leads to intrinsic, stimulus-independent variations in membrane potential of the order of +/-2.2-4.5 mV-variations that are pure noise, and so carry no information at all. Therefore, for the brain to perform reliable computations, it must either use a rate code, or generate very large, fast depolarizing events, such as those proposed by the theory of synfire chains. However, in our in vivo recordings, we found that such events were very rare. Our findings are thus consistent with the idea that cortex is likely to use primarily a rate code.