Role of Glial Cells in Neuronal Function, Mood Disorders, and Drug Addiction.

Mood disorders and substance use disorder (SUD) are of immense medical and social concern. Although significant progress on neuronal involvement in mood and reward circuitries has been achieved, it is only relatively recently that the role of glia in these disorders has attracted attention. Detailed understanding of the glial functions in these devastating diseases could offer novel interventions. Here, following a brief review of circuitries involved in mood regulation and reward perception, the specific contributions of neurotrophic factors, neuroinflammation, and gut microbiota to these diseases are highlighted. In this context, the role of specific glial cells (e.g., microglia, astroglia, oligodendrocytes, and synantocytes) on phenotypic manifestation of mood disorders or SUD are emphasized. In addition, use of this knowledge in the potential development of novel therapeutics is touched upon.

Beyond life: Exploring hemodynamic patterns in postmortem mice brains.

We utilize Laser Speckle Contrast Imaging (LSCI) for visualizing cerebral blood flow in mice during and post-cardiac arrest. Analyzing LSCI images, we noted temporal blood flow variations across the brain surface for hours postmortem. Fast Fourier Transform (FFT) analysis depicted blood flow and microcirculation decay post-death. Continuous Wavelet Transform (CWT) identified potential cerebral hemodynamic synchronization patterns. Additionally, non-negative matrix factorization (NMF) with four components segmented LSCI images, revealing structural subcomponent alterations over time. This integrated approach of LSCI, FFT, CWT, and NMF offers a comprehensive tool for studying cerebral blood flow dynamics, metaphorically capturing the 'end of the tunnel' experience. Results showed primary postmortem hemodynamic activity in the olfactory bulbs, followed by blood microflow relocations between somatosensory and visual cortical regions via the superior sagittal sinus. This method opens new avenues for exploring these phenomena, potentially linking neuroscientific insights with mysteries surrounding consciousness and perception at life's end.

Bilateral Whisker Representations in the Primary Somatosensory Cortex in Robo3cKO Mice Are Reflected in the Primary Motor Cortex.

In Robo3cKO mice, midline crossing defects of the trigeminothalamic projections from the trigeminal principal sensory nucleus result in bilateral whisker maps in the somatosensory thalamus and consequently in the face representation area of the primary somatosensory (S1) cortex (Renier et al., 2017; Tsytsarev et al., 2017). We investigated whether this bilateral sensory representation in the whisker-barrel cortex is also reflected in the downstream projections from the S1 to the primary motor (M1) cortex. To label these projections, we injected anterograde viral axonal tracer in S1 cortex. Corticocortical projections from the S1 distribute to similar areas across the ipsilateral hemisphere in control and Robo3cKO mice. Namely, in both genotypes they extend to the M1, premotor/prefrontal cortex (PMPF), secondary somatosensory (S2) cortex. Next, we performed voltage-sensitive dye imaging (VSDi) in the left hemisphere following ipsilateral and contralateral single whisker stimulation. While controls showed only activation in the contralateral whisker barrel cortex and M1 cortex, the Robo3cKO mouse left hemisphere was activated bilaterally in both the barrel cortex and the M1 cortex. We conclude that the midline crossing defect of the trigeminothalamic projections leads to bilateral whisker representations not only in the thalamus and the S1 cortex but also downstream from the S1, in the M1 cortex.

Neurophotonic methods in approach to in vivo animal epileptic models: Advantages and limitations.

Neurophotonic technology is a rapidly growing group of techniques that are based on the interactions of light with natural or genetically modified cells of the neural system. New optical technologies make it possible to considerably extend the tools of neurophysiological research, from the visualization of functional activity changes to control of brain tissue excitability. This opens new perspectives for studying the mechanisms underlying the development of human neurological diseases. Epilepsy is one of the most common brain disorders; it is characterized by recurrent seizures and affects >1% of the world's population. However, how seizures occur, spread, and terminate in a healthy brain is still unclear. Therefore, it is extremely important to develop appropriate models to accurately explore the causal relationship of epileptic activity. The use of neurophotonic technologies in epilepsy research falls into two broad categories: the visualization of neural epileptic activity, and the direct optical influence on neurons to induce or suppress epileptic activity. An optogenetic variant of the classical kindling model of epileptic seizures, in which activatable cells are genetically defined, is called optokindling. Research is also underway concerning the application of neurophotonic techniques for suppressing epileptic activity, aiming to bring these methods into clinical practice. This review aims to systematize and describe new approaches that use combinations of different neurophotonic methods to work with in vivo models of epilepsy. These approaches overcome many of the shortcomings associated with classical animal models of epilepsy and thus increase the effectiveness of developing new diagnostic methods and antiepileptic therapy.

In vivo laser speckle contrast imaging of 4-aminopyridine- or pentylenetetrazole-induced seizures.

Clinical and preclinical studies on epileptic seizures are closely linked to the study of neurovascular coupling. Obtaining reliable information about cerebral blood flow (CBF) in the area of epileptic activity through minimally invasive techniques is crucial for research in this field. In our studies, we used laser speckle contrast imaging (LSCI) to gather information about the local blood circulation in the area of epileptic activity. We used two models of epileptic seizures: one based on 4-aminopyridine (4-AP) and another based on pentylenetetrazole (PTZ). We verified the duration of an epileptic seizure using electrocorticography (ECoG). We applied the antiepileptic drug topiramate (TPM) to both models, but its effect was different in each case. However, in both models, TPM had an effect on neurovascular coupling in the area of epileptic activity, as shown by both LSCI and ECoG data. We demonstrated that TPM significantly reduced the amplitude of 4-AP-induced epileptic seizures (4-AP+TPM: 0.61 ± 0.13 mV vs 4-AP: 1.08 ± 0.19 mV; p < 0.05), and it also reduced gamma power in ECoG in PTZ-induced epileptic seizures (PTZ+TPM: 38.5% ± 11.9% of the peak value vs PTZ: 59.2% ± 3.0% of peak value; p < 0.05). We also captured the pattern of CBF changes during focal epileptic seizures induced by 4-AP. Our data confirm that the system of simultaneous cortical LSCI and registration of ECoG makes it possible to evaluate the effectiveness of pharmacological agents in various types of epileptic seizures in in vivo models and provides spatial and temporal information on the process of ictogenesis.

Arc protein, a remnant of ancient retrovirus, forms virus-like particles, which are abundantly generated by neurons during epileptic seizures, and affects epileptic susceptibility in rodent models.

A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or Arc protein) of retroviral ancestry resides in the genome of all tetrapods for millions of years and is expressed endogenously in neurons. It is a well-known protein, very important for synaptic plasticity and memory consolidation. Activity-dependent Arc expression concentrated in glutamatergic synapses affects the long-time synaptic strength of those excitatory synapses. Because it modulates excitatory-inhibitory balance in a neuronal network, the Arc gene itself was found to be related to the pathogenesis of epilepsy. General Arc knockout rodent models develop a susceptibility to epileptic seizures. Because of activity dependence, synaptic Arc protein synthesis also is affected by seizures. Interestingly, it was found that Arc protein in synapses of active neurons self-assemble in capsids of retrovirus-like particles, which can transfer genetic information between neurons, at least across neuronal synaptic boutons. Released Arc particles can be accumulated in astrocytes after seizures. It is still not known how capsid assembling and transmission timescale is affected by seizures. This scientific field is relatively novel and is experiencing swift transformation as it grapples with difficult concepts in light of evolving experimental findings. We summarize the emergent literature on the subject and also discuss the specific rodent models for studying Arc effects in epilepsy. We summarized both to clarify the possible role of Arc-related pseudo-viral particles in epileptic disorders, which may be helpful to researchers interested in this growing area of investigation.

Adjuvant effect of dendritic cells activator Imiquimod in genetic immunization with HIV-1 p55 Gag.

Dendritic cells (DC) are important antigen-presenting cells that have abilities to induce and maintain T-cell immunity, or attenuate it during hyperimmunization. Additional activation of DCs may be useful for vaccination purposes. Imiquimod is known to be a specific agonist of the Toll-like receptors (TLR7), which are located mainly on DCs. To study the effect of DC stimulation on the effectiveness of an HIV-1 p55 gag DNA vaccine in a mice model, we employed 25, 50, and 100 nM of Imiquimod as an adjuvant. Subsequently, Western blot analysis was used to quantify p55 protein production after the immunization. To characterize T-cells immune response, both the frequency of IFN-γ -secreting cells and IFN-γ and IL-4 production were measured, via an ELIspot assay and ELISA, respectively. Low concentrations of Imiquimod were found to effectively stimulate Gag production and the magnitude of the T-cell immune response, whereas higher concentrations reduced vaccination effects. Our results show that the adjuvant effects of Imiquimod depend on concentration. The use of Imiquimod may be helpful to study DC to T cell communication, including possible induction of immunotolerance.

Amyloid Deposition and Dendritic Complexity of Corticocortical Projection Cells in Five Familial Alzheimer's Disease Mouse.

In Alzheimer's disease and related dementias, amyloid beta (Aß) and amyloid plaques can disrupt long-term synaptic plasticity, learning and memory and cognitive function. Plaque accumulation can disrupt corticocortical circuitry leading to abnormalities in sensory, motor, and cognitive processing. In this study, using 5xFAD (five Familial Alzheimer's Disease - FAD - mutations) mice, we evaluated amyloid plaque formation in different cortical areas, and whether differential amyloid accumulation across cortical fields correlates with changes in dendritic complexity of layer 3 corticocortical projection neurons and functional responses in the primary somatosensory cortex following whisker stimulation. We focused on three cortical areas: the primary somatosensory cortex (S1), the primary motor cortex (M1), and the prefrontal cortex (PFC including the anterior cingulate, prelimbic, and infralimbic subdivisions). We found that Aß and amyloid plaque accumulation is not uniform across 5xFAD cortical areas, while there is no expression in littermate controls. We also found that there are differential layer 3 pyramidal cell dendritic complexity changes across the three areas in 5xFAD mice, compared to same age controls, with no apparent relation to differential amyloid accumulation. We used voltage-sensitive dye imaging (VSDi) to visualize neural activity in S1, M1 and PFC following whisker activation. Control mice show normal physiological responses in all three cortical areas, whereas 5xFAD mice only display physiological responses in S1. Taken together our results show that 5xFAD mutation affects the overall dendritic morphology of layer 3 pyramidal cells across sensory-motor and association cortex irrespective of the density and distribution of the Aß amyloid proteins. Corticocortical circuitry between the sensory and motor/association areas is most likely disrupted in 5xFAD mice as cortical responses to whisker stimulation are altered.

Amount of Melanin Granules in Human Hair Defines the Absorption and Conversion to Heat of Light Energy in the Visible Spectrum.

One of the known important functions of hair is protection from extensive sunlight. This protection is accomplished in large part due to natural hair pigmentation which is known to reflect the number of melanin granules (melanosomes) in the hair shaft, and melanin variants. Melanin takes in excessive light energy and converts it to heat in a process called absorption; heat is then dissipated into the environment as infrared radiation, thereby protecting the underlying skin. We used transmission electron microscopy (TEM) to visualize the melanosome counts in samples of human hair, and used thermal microscopy to measure the temperature changes of the samples when exposed to green and blue light lasers. In our experiments green light conversion to heat was highly correlated to the number of melanosomes, whereas blue light conversion to heat was less correlated, which may be because the reddish melanosomes it contains are less effective in absorbing energy from the blue spectrum of light. Anyway, we have shown the metals accumulation in the melanin can be easily visualized with TEM. We confirmed that the amount of melanin granules in human hair defines the conversion to heat of light energy in the visible spectrum.

Layers 3 and 4 Neurons of the Bilateral Whisker-Barrel Cortex.

In Robo3R3-5cKO mouse brain, rhombomere 3-derived trigeminal principal nucleus (PrV) neurons project bilaterally to the somatosensory thalamus. As a consequence, whisker-specific neural modules (barreloids and barrels) representing whiskers on both sides of the face develop in the sensory thalamus and the primary somatosensory cortex. We examined the morphological complexity of layer 4 barrel cells, their postsynaptic partners in layer 3, and functional specificity of layer 3 pyramidal cells. Layer 4 spiny stellate cells form much smaller barrels and their dendritic fields are more focalized and less complex compared to controls, while layer 3 pyramidal cells did not show notable differences. Using in vivo 2-photon imaging of a genetically encoded fluorescent [Ca2+] sensor, we visualized neural activity in the normal and Robo3R3-5cKO barrel cortex in response to ipsi- and contralateral single whisker stimulation. Layer 3 neurons in control animals responded only to their contralateral whiskers, while in the mutant cortex layer 3 pyramidal neurons showed both ipsi- and contralateral whisker responses. These results indicate that bilateral whisker map inputs stimulate different but neighboring groups of layer 3 neurons which normally relay contralateral whisker-specific information to other cortical areas.

Multilayer subwavelength gratings or sandwiches with periodic structure shape light reflection in the tapetum lucidum of taxonomically diverse vertebrate animals.

Eye shine in the dark has attracted many researchers to the field of eye optics, but the initial studies of subwavelength arrangements in tapetum began only with the development of electronic microscopy at the end of the 20th century. As a result of a number of studies, it was shown that the reflective properties of the tapetum are due to their specialized cellular subwavelength microstructure (photonic crystals). These properties, together with the mutual orientation of the crystals, lead to a significant increase in reflection, which, in turn, enhances the sensitivity of the eye. In addition, research confirmed that optical mechanisms of reflection in the tapetum are very similar even for widely separated species. Due to progress in the field of nano-optics, researchers now have a better understanding of the main principles of this phenomenon. In this review, we summarize electron microscopic and functional studies of tapetal structures in the main vertebrate classes. This allows data on the microstructure of the tapetum to be used to improve our understanding of the visual system.

The Anti-Epileptic Effects of Carbenoxolone In Vitro and In Vivo.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

Methodological aspects of studying the mechanisms of consciousness.

There are at least two approaches to the definition of consciousness. In the first case, certain aspects of consciousness, called qualia, are considered inaccessible for research from a third person and can only be described through subjective experience. This approach is inextricably linked with the so-called "hard problem of consciousness", that is, the question of why consciousness has qualia or how any physical changes in the environment can generate subjective experience. With this approach, some aspects of consciousness, by definition, cannot be explained on the basis of external observations and, therefore, are outside the scope of scientific research. In the second case, a priori constraints do not constrain the field of scientific investigation, and the best explanation of the experience in the first person is included as a possible subject of empirical research. Historically, in the study of cause-and-effect relationships in biology, it was customary to distinguish between proximate causation and ultimate causation existing in biological systems. Immediate causes are based on the immediate influencing factors [1]. Proximate causation has evolutionary explanations. When studying biological systems themselves, such an approach is undoubtedly justified, but it often seems insufficient when studying the interaction of consciousness and the brain [2,3]. Current scientific communities proceed from the assumption that the physical substrate for the generation of consciousness is a neural network that unites various types of neurons located in various brain structures. Many neuroscientists attach a key role in this process to the cortical and thalamocortical neural networks. This question is directly related to experimental and clinical research in the field of disorder of consciousness. Progress in this area of medicine depends on advances in neuroscience in this area and is also a powerful source of empirical information. In this area of consciousness research, a large amount of experimental data has been accumulated, and in this review an attempt was made to generalize and systematize.

Wireless Optogenetic Modulation of Cortical Neurons Enabled by Radioluminescent Nanoparticles.

While offering high-precision control of neural circuits, optogenetics is hampered by the necessity to implant fiber-optic waveguides in order to deliver photons to genetically engineered light-gated neurons in the brain. Unlike laser light, X-rays freely pass biological barriers. Here we show that radioluminescent Gd2(WO4)3:Eu nanoparticles, which absorb external X-rays energy and then downconvert it into optical photons with wavelengths of ∼610 nm, can be used for the transcranial stimulation of cortical neurons expressing red-shifted, ∼590-630 nm, channelrhodopsin ReaChR, thereby promoting optogenetic neural control to the practical implementation of minimally invasive wireless deep brain stimulation.

In Vitro and In Vivo Study of the Short-Term Vasomotor Response during Epileptic Seizures.

Epilepsy remains one of the most common brain disorders, and the different types of epilepsy encompass a wide variety of physiological manifestations. Clinical and preclinical findings indicate that cerebral blood flow is usually focally increased at seizure onset, shortly after the beginning of ictal events. Nevertheless, many questions remain about the relationship between vasomotor changes in the epileptic foci and the epileptic behavior of neurons and astrocytes. To study this relationship, we performed a series of in vitro and in vivo experiments using the 4-aminopyridine model of epileptic seizures. It was found that in vitro pathological synchronization of neurons and the depolarization of astrocytes is accompanied by rapid short-term vasoconstriction, while in vivo vasodilation during the seizure prevails. We suggest that vasomotor activity during epileptic seizures is a correlate of the complex, self-sustained response that includes neuronal and astrocytic oscillations, and that underlies the clinical presentation of epilepsy.

In vivo voltage-sensitive dye imaging of mouse cortical activity with mesoscopic optical tomography.

Significance: Cellular layering is a hallmark of the mammalian neocortex with layer and cell type-specific connections within the cortical mantle and subcortical connections. A key challenge in studying circuit function within the neocortex is to understand the spatial and temporal patterns of information flow between different columns and layers. Aim: We aimed to investigate the three-dimensional (3D) layer- and area-specific interactions in mouse cortex in vivo. Approach: We applied a new promising neuroimaging method-fluorescence laminar optical tomography in combination with voltage-sensitive dye imaging (VSDi). VSDi is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but it is traditionally used for two-dimensional (2D) imaging. Our mesoscopic technique allows visualization of neuronal activity in a 3D manner with high temporal resolution. Results: We first demonstrated the depth-resolved capability of 3D mesoscopic imaging technology in Thy1-ChR2-YFP transgenic mice. Next, we recorded the long-range functional projections between sensory cortex (S1) and motor cortex (M1) in mice, in vivo, following single whisker deflection. Conclusions: The results show that mesoscopic imaging technique has the potential to investigate the layer-specific neural connectivity in the mouse cortex in vivo. Combination of mesoscopic imaging technique with optogenetic control strategy is a promising platform for determining depth-resolved interactions between cortical circuit elements.

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and in vivo.

The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K+-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability and fluorescence lifetime imaging microscopy (FLIM) we confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. We evaluated FI3 nanoparticles in real-time live cell imaging and found that it is highly useful for monitoring intra- and extracellular K+ dynamics in cultured neurons. Proof-of-concept in vivo brain imaging confirmed applicability of the biosensor for visualization of epileptic seizures. Collectively, this data makes fluoroionophore FI3 a versatile cross-platform fluorescent biosensor, broadly compatible with diverse experimental models and that crown ether-based polymer nanoparticles can provide a new venue for design of efficient fluorescent probes.

Organization of orientation-specific whisker deflection responses in layer 2/3 of mouse somatosensory cortex.

The rodent whisker-barrel system is characterized by its patterned somatotopic mapping between the sensory periphery and multiple regions of the brain. While somatotopy in the whisker system is established, we know far less about how preferences for stimulus orientation or other features are organized. Mouse somatosensation is an increasingly popular model for circuit-based dissection of perceptual decision making and learning, yet our understanding of how stimulus feature representations are organized in the cortex is incomplete. Here, we used in vivo two-photon calcium imaging to monitor activity of populations of layer (L) 2/3 neurons in the mouse primary somatosensory cortex during deflections of a single whisker in two orthogonal orientations (azimuthal or elevational). We split the population response to whisker deflections into an orientation-specific component and a non-specific component that reflected overall excitability in response to deflection of a single whisker. Orientation-specific responses were organized in a locally heterogeneous and spatially distributed manner. Correlations in the stimulus-independent trial-to-trial variability of pairs of neurons were higher among neurons that preferred the same orientation. These correlations depended on similarity in both orientation-specific and non-specific components of responses to single-whisker deflections. Our results shed light on L2/3 organization in mouse somatosensory cortex, and lay a foundation for dissecting circuit mechanisms of perceptual learning and decision-making during orientation discrimination tasks.

Structural and functional differences in the barrel cortex of Mecp2 null mice.

Functional deficits in sensory systems are commonly noted in neurodevelopmental disorders, such as the Rett syndrome (RTT). Defects in methyl CpG binding protein gene (MECP2) largely accounts for RTT. Manipulations of the Mecp2 gene in mice provide useful models to probe into various aspects of brain development associated with the RTT. In this study, we focused on the somatosensory cortical phenotype in the Bird mouse model of RTT. We used voltage-sensitive dye imaging to evaluate whisker sensory evoked activity in the barrel cortex of mice. We coupled this functional assay with morphological analyses in postnatal mice and investigated the dendritic differentiation of barrel neurons and individual thalamocortical axon (TCA) arbors that synapse with them. We show that in Mecp2-deficient male mice, whisker-evoked activity is roughly topographic but weak in the barrel cortex. At the morphological level, we find that TCA arbors fail to develop into discrete, concentrated patches in barrel hollows, and the complexity of the dendritic branches in layer IV spiny stellate neurons is reduced. Collectively, our results indicate significant structural and functional impairments in the barrel cortex of the Bird mouse line, a popular animal model for the RTT. Such structural and functional anomalies in the primary somatosensory cortex may underlie orofacial tactile sensitivity issues and sensorimotor stereotypies characteristic of RTT.