The claustrum (CLA) is a thin and elongated brain structure that is located between the insula and lateral striatum and is implicated in a wide range of behaviors. It is characterized by its extensive synaptic connectivity with multiple cortical regions. While CLA projection neurons are glutamatergic, several studies have shown an inhibitory impact of CLA on its cortical targets, suggesting the involvement of inhibitory cortical interneurons. We employed both in vivo and ex vivo electrophysiology and optogenetics in mice to dissect the synaptic organization of projections from the CLA to the anterior cingulate cortex (ACC), a robust claustro-cortical pathway implicated in cognitive functions. Optogenetic stimulation of CLA neurons expressing calcium/calmodulin-dependent protein kinase II alpha (CaMKIIa) or vesicular glutamate transporter-2 (VGLUT2) evoked distinct multiphasic excitatory and inhibitory responses in the ACC that depended on the stimulated CLA neuron population and the recipient layer in the ACC. Stimulation of CLA-ACC axons evoked monosynaptic responses in pyramidal neurons and four types of molecularly defined interneurons. Synaptic responses were uniquely determined by the presynaptic CLA projection type, the recipient cortical layer, and the type of postsynaptic neuron, with interneurons displaying the highest specificity. This intricate organization of the CLA-ACC pathway explains the diverse and complex influence of CLA on cortical activity.

OBJECTIVE:Ultrasound can noninvasively penetrate deep into the brain for neuromodulation, demonstrating good potential for clinical application. However, the underlying mechanisms are unclear. So far most in vitro studies have focused on the activation of individual neurons by ultrasound with calcium imaging. As the focal region of ultrasound is typically millimeter or submillimeter size, it is important to investigate yet so far unclear how the mechanical effects of ultrasound would influence the synaptic circuit activity of neurons. METHODS:Low-intensity pulse ultrasound was used to stimulate cultured hippocampal neurons. Postsynaptic currents were recorded in individual cells with the whole-cell patch-clamp technique. We also simultaneously imaged intracellular calcium, along with neuronal electrical signals, to resolve neuronal network dynamics during ultrasound stimulation. RESULTS:Excitatory postsynaptic currents (EPSCs) were evoked by ultrasound in high-density neuronal cultures with increased frequency and amplitude, indicating enhanced glutamatergic synaptic transmission. The probability of evoking responses and the total charge of EPSCs increased with ultrasound intensity. Mechanistic analysis reveals that extracellular calcium influx, action potential firing and synaptic transmission are necessary for the responses to ultrasound in high-density culture. In contrast, EPSCs were not enhanced in low-density culture. Simultaneous calcium imaging of neuronal network activity indicates that recurrent excitatory network activity is recruited during ultrasound stimulation in high-density cultures, which lasts over tens to hundreds of seconds. CONCLUSION:Our study provides insights into the mechanisms involved in the response of the brain to ultrasound and illuminates the potential to use ultrasound to regulate synaptic function in neurological disorders.

Because of the important roles of both serotonin (5-HT) and the cerebellum in regulating anxiety, we asked whether 5-HT signaling within the cerebellum is involved in anxiety behavior. Physiological 5-HT levels were measured in vivo by expressing a fluorescent sensor for 5-HT in lobule VII of the cerebellum, while using fiber photometry to measure sensor fluorescence during anxiety behavior on the elevated zero maze. Serotonin increased in lobule VII when male mice were less anxious and decreased when mice were more anxious. To establish a causal role for this serotonergic input in anxiety behavior, we photostimulated or photoinhibited serotonergic terminals in lobule VII while mice were in an elevated zero maze. Photostimulating these terminals reduced anxiety behavior in mice, while photoinhibiting them enhanced anxiety behavior. Our findings add to evidence that cerebellar lobule VII is a topographical locus for anxiety behavior and establish that 5-HT input into this lobule is necessary and sufficient to bidirectionally influence anxiety behavior. These results represent progress toward understanding how the cerebellum regulates anxiety behavior and provide new evidence for a functional connection between the cerebellum and the serotonin system within the anxiety circuit.

Background:APOE4 and APP are two of the main genetic risk factors for Alzheimer's disease (AD). Although there have been suggestions that these two factors interact, most of the in vivo evidence for such interactions comes from transgenic mouse models that suffer from complications associated with protein overexpression. Our goal was to examine the consequences of interactions between APOE4 and APP on brain function while avoiding the use of transgenic mice. Methods:We generated and characterized double-mutant knock-in mice incorporating familial APP mutations and humanized APOE4. Results:In the brains of 3-month-old double-mutant mice there were significant alterations in vascular remodeling genes, vascular structure and blood-brain barrier permeability. These changes were not observed in either APOE4 or APP single-mutant mice and, thus, were caused by interactions between the two genes. These interaction effects were transient, because they were absent in 8-month-old double-mutant mice. Conclusion:These findings indicate that early vascular changes, driven by the interaction of APP and APOE4, may influence the progression of AD. Our work highlights the need to focus on the synergistic vascular actions of APOE4 and APP, particularly at younger ages.

The social transmission of food preference, a rudimentary form of social learning, has primarily been studied in pairs of adult rodents. Here, we present a protocol to explore the parent-offspring context in social learning using an adaptation of this classic paradigm for rodent dam-pup dyads. We describe steps for studying weanling mice from the same mother and present a worked example using weight-based (food consumption) and time-based (exploration) indices of social learning.

Lower urinary tract (LUT) function is controlled by the central nervous system, including higher-order cognitive brain regions. The anterior cingulate cortex (ACC) is one of these regions, but the role of its activity in LUT function remains poorly understood. In the present study, we conducted optogenetic experiments to manipulate neural activity in mouse ACC while monitoring bladder pressure to elucidate how the activity of ACC regulates LUT function. Selective optogenetic stimulation of excitatory neurons in ACC induced a sharp increase in bladder pressure, whereas activation of inhibitory neurons in ACC prolonged the interval between bladder contractions. Pharmacological manipulation of ACC also altered bladder contractions, consistent with those observed in optogenetic experiments. Optogenetic mapping of the cortical area responsible for eliciting the increase in bladder pressure revealed that stimulation to ACC showed more potent effects than the neighboring motor cortical areas. These results suggest that ACC plays a crucial role in initiating the bladder pressure change and the micturition reflex. Thus, the balance between excitation and inhibition in ACC may regulate the reflex bidirectionally.

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2a WT ), but not the tetramerization-deficient mutant K337Q (Syn2a K337Q ), fully rescued these effects. Single-particle tracking revealed that Syn2a K337Q mEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool.

Although the cerebellum is traditionally known for its role in motor functions, recent evidence points toward the additional involvement of the cerebellum in an array of non-motor functions. One such non-motor function is anxiety behavior: a series of recent studies now implicate the cerebellum in anxiety. Here, we review evidence regarding the possible role of the cerebellum in anxiety-ranging from clinical studies to experimental manipulation of neural activity-that collectively points toward a role for the cerebellum, and possibly a specific topographical locus within the cerebellum, as one of the orchestrators of anxiety responses.

The concept of spatial confinement is the basis of cell positioning and guidance in in vitro studies. In vivo, it reflects many situations faced during embryonic development. In vitro, spatial confinement of neurons is achieved using different technological approaches: adhesive patterning, topographical structuring, microfluidics and the use of hydrogels. The notion of chemical or physical frontiers is particularly central to the behaviors of growth cones and neuronal processes under confinement. They encompass phenomena of cell spreading, boundary crossing, and path finding on surfaces with different adhesive properties. However, the most universal phenomenon related to confinement, regardless of how it is implemented, is the acceleration of neuronal growth. Overall, a bi-directional causal link emerges between the shape of the growth cone and neuronal elongation dynamics, both in vivo and in vitro. The sensing of adhesion discontinuities by filopodia and the subsequent spatial redistribution and size adaptation of these actin-rich filaments seem critical for the growth rate in conditions in which adhesive contacts and actin-associated clutching forces dominate. On the other hand, the involvement of microtubules, specifically demonstrated in 3D hydrogel environments and leading to ameboid-like locomotion, could be relevant in a wider range of growth situations. This review brings together a literature collected in distinct scientific fields such as development, mechanobiology and bioengineering that highlight the consequences of confinement and raise new questions at different cellular scales. Its ambition is to stimulate new research that could lead to a better understanding of what gives neurons their ability to establish and regulate their exceptional size.

Synapsins cluster synaptic vesicles (SVs) to provide a reserve pool (RP) of SVs that maintains synaptic transmission during sustained activity. However, it is unclear how synapsins cluster SVs. Here we show that either liquid-liquid phase separation (LLPS) or tetramerization-dependent cross-linking can cluster SVs, depending on whether a synapse is excitatory or inhibitory. Cell-free reconstitution reveals that both mechanisms can cluster SVs, with tetramerization being more effective. At inhibitory synapses, perturbing synapsin-dependent LLPS impairs SV clustering and synchronization of gamma-aminobutyric acid (GABA) release, while preventing synapsin tetramerization does not. At glutamatergic synapses, the opposite is true: synapsin tetramerization enhances clustering of glutamatergic SVs and mobilization of these SVs from the RP, while synapsin LLPS does not. Comparison of inhibitory and excitatory transmission during prolonged synaptic activity reveals that synapsin LLPS serves as a brake to limit GABA release, while synapsin tetramerization enables rapid mobilization of SVs from the RP to sustain glutamate release.

The cholinergic system of the basal forebrain plays an integral part in behaviors ranging from attention to learning, partly by altering the impact of noise in neural populations. The circuit computations underlying cholinergic actions are confounded by recent findings that forebrain cholinergic neurons corelease both acetylcholine (ACh) and GABA. We have identified that corelease of ACh and GABA by cholinergic inputs to the claustrum, a structure implicated in the control of attention, has opposing effects on the electrical activity of claustrum neurons that project to cortical vs. subcortical targets. These actions differentially alter neuronal gain and dynamic range in the two types of neurons. In model networks, the differential effects of ACh and GABA toggle network efficiency and the impact of noise on population dynamics between two different projection subcircuits. Such cholinergic switching between subcircuits provides a potential logic for neurotransmitter corelease in implementing behaviorally relevant computations.

Social recognition memory (SRM) is a key determinant of social interactions. While the cerebellum emerges as an important region for social behavior, how cerebellar activity affects social functions remains unclear. We selectively increased the excitability of molecular layer interneurons (MLIs) to suppress Purkinje cell firing in the mouse cerebellar vermis. Chemogenetic perturbation of MLIs impaired SRM without affecting sociability, anxiety levels, motor coordination or object recognition. Optogenetic interference of MLIs during distinct phases of a social recognition test revealed the cerebellar engagement in the retrieval, but not encoding, of social information. c-Fos mapping after the social recognition test showed that cerebellar manipulation decreased brain-wide interregional correlations and altered network structure from medial prefrontal cortex and hippocampus-centered to amygdala-centered modules. Anatomical tracing demonstrated hierarchical projections from the central cerebellum to the social brain network integrating amygdalar connections. Our findings suggest that the cerebellum organizes the neural matrix necessary for SRM.

The distinct disease progression patterns of severe acute respiratory syndrome coronavirus clade 2 (SARS‐CoV‐2) indicate diverse host immune responses. SARS‐CoV‐2 severely impairs type I interferon (IFN) cell signaling, resulting in uncontrolled late‐phase lung damage in patients. For better pharmacological properties, cytokine modifications may sometimes result in a loss of biological activity against the virus. Here, we employed the genetic code expansion and engineered IFN‐β, a phase II clinical cytokine with 3‐amino tyrosine (IFN‐β‐A) that reactivates STAT2 expression in virus‐infected human cells through JAK/STAT cell signaling without affecting signal activation and serum half‐life. This study identified that genetically encoded IFN‐β‐A might stabilize the protein‐receptor complex and trigger JAK‐STAT cell signaling, which is a promising modality for controlling SARS‐CoV‐2 infection.

The claustrum (CLA) is a brain nucleus located between the insula and lateral striatum, implicated in a wide range of behaviors. Underpinning the different behavioral phenotypes is the connectivity between the claustrum and various cortical regions, including the anterior cingulate cortex (ACC). CLA projection neurons are glutamatergic neurons, however, the impact of CLA on its cortical targets has been shown in some studies to be inhibitory. Such inhibition is likely to arise from claustral activation of cortical interneurons, however, the intricate synaptic connectivity between different CLA and cortical cell types is not known. Here, we combine in vivo and ex vivo electrophysiology and optogenetics to reveal the functional organization of the CLA-ACC pathway according to the identity of its pre- and postsynaptic populations. Optogenetic stimulation of CLA neurons in awake mice resulted in multiphasic excitatory and inhibitory responses in ACC cells, which depended on the layer, cell type, and stimulated CLA population. Using ex vivo paired recordings in ACC, monosynaptic responses were recorded in pyramidal cells and different types of interneurons following photostimulation of CLA-ACC synaptic terminals. CLA axons formed monosynaptic connections in all ACC cortical layers, but the probability and strength of synaptic responses depended on the type of CLA projection, target layer in ACC, and the type of postsynaptic neuron. This intricate organization of the CLA-ACC pathway may explain the complex impact of CLA on ACC and other cortical regions, thus resolving some of the discrepancies in the field and shedding light on the functional role CLA plays in cortical function.

The seminal paper of Crick and Koch (2005) proposed that the claustrum, an enigmatic and thin grey matter structure that lies beside the insular cortex, may be involved in the processing of consciousness. As a result, this otherwise obscure structure has received ever-increasing interest in the search for neural correlates of consciousness. Here we review theories of consciousness and discuss the possible relationship between the claustrum and consciousness. We review relevant experimental evidence collected since the Crick and Koch (2005) paper and consider whether these findings support or contradict their hypothesis. We also explore how future experimental work can be designed to clarify how consciousness emerges from neural activity and to understand the role of the claustrum in consciousness.

Using genetic strategies and viral-based directional tracers, we investigated the topological location and output networks of claustrum (CLA) neuron populations projecting to either the retrosplenial cortex, primary motor cortex, or basolateral amygdala. We found that all three CLA neuron populations clearly reside in distinct topological locations within the CLA complex and project broadly to multiple downstream targets. Each neuron population projects to different targets, suggesting that each CLA subzone coordinates a unique set of brain-wide functions. Our findings establish that the claustrum complex encompasses at least three minimally overlapping networks that are compartmentalized into different topological subzones. Such modularity is likely to be important for CLA function.

The function of a feedback inhibitory circuit between cerebellar Purkinje cells and molecular layer interneurons (MLIs) was defined by combining optogenetics, neuronal activity recordings both in cerebellar slices and in vivo, and computational modeling. Purkinje cells inhibit a subset of MLIs in the inner third of the molecular layer. This inhibition is non-reciprocal, short-range (less than 200 μm) and is based on convergence of one to two Purkinje cells onto MLIs. During learning-related eyelid movements in vivo, the activity of a subset of MLIs progressively increases as Purkinje cell activity decreases, with Purkinje cells usually leading the MLIs. Computer simulations indicate that these relationships are best explained by the feedback circuit from Purkinje cells to MLIs and that this feedback circuit plays a central role in making cerebellar learning efficient.

Despite notable genetic influences, obesity mainly results from the overconsumption of food, which arises from the interplay of physiological, cognitive and environmental factors. In patients with obesity, eating is determined more by external cues than by internal physiological needs. However, how environmental context drives non-homeostatic feeding is elusive. Here, we identify a population of somatostatin (TNSST) neurons in the mouse hypothalamic tuberal nucleus that are preferentially activated by palatable food. Activation of TNSST neurons enabled a context to drive non-homeostatic feeding in sated mice and required inputs from the subiculum. Pairing a context with palatable food greatly potentiated synaptic transmission between the subiculum and TNSST neurons and drove non-homeostatic feeding that could be selectively suppressed by inhibiting TNSST neurons or the subiculum but not other major orexigenic neurons. These results reveal how palatable food, through a specific hypothalamic circuit, empowers environmental context to drive non-homeostatic feeding.

Fluorescent proteins (FP) are an integral part of modern biology due to its diverse biochemical and photophysical properties. The boundaries of FP have been extended through conventional mutagenesis and directed evolution approaches. Engineering of FP based on the standard genetic code consisting of 20 amino acids with limited functional groups restrict its diversification. Degeneracy of genetic code has helped in covering this substantial gap through genetic code engineering, wherein introduction of unnatural amino acid (UAA) analogues resulted in a collection of FP with varying properties. This review features the work carried till date in the area of FP incorporated with UAAs and explores strategies employed for incorporation, impact of UAAs in chromophore and surrounding residues and changes in inherent properties of FP. The long-standing association of FP as a tool for high throughput screening of orthogonal aaRS/tRNA pairs used in site specific incorporation of UAAs is expounded. Insertion of UAAs in FP has enabled their use in contemporary fields such as biophotovoltaics, bioremediation, biosensors, biomaterials and imaging of acidic vesicles. Thus, expansion of genetic code of FP is envisaged to rejig the existing spectra of colors and future research initiative in this direction is expected to glow brighter and brighter.