Association between vascular endothelial growth factor-mediated blood-brain barrier dysfunction and stress-induced depression.

Several lines of evidence suggest that stress induces the neurovascular dysfunction associated with increased blood-brain barrier (BBB) permeability, which could be an important pathology linking stress and psychiatric disorders, including major depressive disorder (MDD). However, the detailed mechanism resulting in BBB dysfunction associated in the pathophysiology of MDD still remains unclear. Herein, we demonstrate the role of vascular endothelial growth factor (VEGF), a key mediator of vascular angiogenesis and BBB permeability, in stress-induced BBB dysfunction and depressive-like behavior development. We implemented an animal model of depression, chronic restraint stress (RS) in BALB/c mice, and found that the BBB permeability was significantly increased in chronically stressed mice. Immunohistochemical and electron microscopic observations revealed that increased BBB permeability was associated with both paracellular and transcellular barrier alterations in the brain endothelial cells. Pharmacological inhibition of VEGF receptor 2 (VEGFR2) using a specific monoclonal antibody (DC101) prevented chronic RS-induced BBB permeability and anhedonic behavior. Considered together, these results indicate that VEGF/VEGFR2 plays a crucial role in the pathogenesis of depression by increasing the BBB permeability, and suggest that VEGFR2 inhibition could be a potential therapeutic strategy for the MDD subtype associated with BBB dysfunction.

Comparison of physiological and behavioral responses to chronic restraint stress between C57BL/6J and BALB/c mice.

Rodent models of chronic restraint stress (CRS) are often used as simple models of depressive disorder. However, these models of stress have been mainly developed in rats, and the behavioral phenotypes of CRS models are still controversial. In this study, we compared the physiological and behavioral responses of C57BL/6J (B6) and BALB/c mice, which are commonly used in genetic and behavioral studies, to CRS. In addition to measuring physiological parameters and the levels of corticosterone (a stress hormone) in response to stress, we also examined changes in the levels of testosterone (an anti-stress hormone), which have rarely been studied in stressed mice. The mice were exposed to CRS for 6 h a day for 21 days. In both B6 and BALB/c mice, CRS elicited several physiological stress responses, including decreased body weight gain and changes in the tissue weights of stress-related organs. Accumulated corticosterone in the hair was measured, and BALB/c mice had significantly greater levels than control mice and B6 mice after CRS. On the other hand, in the case of accumulated testosterone in the hair, both B6 mice and BALB/c mice showed significantly higher concentrations than control mice, but the degree of change was not different between the two strains. In the sucrose preference test, BALB/c mice, but not B6 mice, showed anhedonia-like behavior after CRS. However, neither strain showed depressive-like behavior in the forced swim or tail suspension test. Our results show that the physiological and behavioral stress responses of BALB/c mice are greater than those of B6 mice, although anti-stress responses to CRS are similar in both strains. This suggests that BALB/c mice are likely to be advantageous for use as a CRS-induced depression model.

Construction of the common cortical space by spontaneous activity and its application in the mouse cortex.

Wide-field optical imaging of the animal brain is a useful technique for measuring brain dynamics, including spatial structure. However, quantitative inter-animal comparison is difficult due to lack of the common cortical space that can normalize individually imaged brains as done in human functional MRI studies. Here, by using wide-field functional Ca2+ imaging on anesthetized transgenic mice expressing G-CaMP7 in astrocytes and excitatory neutrons, we attempted to establish the common cortical space in mice, which can be useful as a standard of functional brain map. We initially reconstructed cortical areas embedded within spontaneous activity as the functional connectivity maps for the individual mice, then matched them in size, shape, and location across mice by geometric transformation. Finally, we assigned all the recorded signals into the transformed space, to make spatially normalized signals in the common cortical space. Using this method, we managed to extract activity patterns commonly observed across mice. These results suggest that the presented method is available to facilitate inter-animal comparison of brain dynamics, and has the potential to identify common brain activity across animals.

Interhemispheric Synchrony of Spontaneous Cortical States at the Cortical Column Level.

In cat early visual cortex, neural activity patterns resembling evoked orientation maps emerge spontaneously under anesthesia. To test if such patterns are synchronized between hemispheres, we performed bilateral imaging in anesthetized cats using a new improved voltage-sensitive dye. We observed map-like activity patterns spanning early visual cortex in both hemispheres simultaneously. Patterns virtually identical to maps associated with the cardinal and oblique orientations emerged as leading principal components of the spontaneous fluctuations, and the strength of transient orientation states was correlated with their duration, providing evidence that these maps are transiently attracting states. A neural mass model we developed reproduced the dynamics of both smooth and abrupt orientation state transitions observed experimentally. The model suggests that map-like activity arises from slow modulations in spontaneous firing in conjunction with interplay between excitation and inhibition. Our results highlight the efficiency and functional precision of interhemispheric connectivity.

Critical dynamics, anesthesia and information integration: Lessons from multi-scale criticality analysis of voltage imaging data.

Critical dynamics are thought to play an important role in neuronal information-processing: near critical networks exhibit neuronal avalanches, cascades of spatiotemporal activity that are scale-free, and are considered to enhance information capacity and transfer. However, the exact relationship between criticality, awareness, and information integration remains unclear. To characterize this relationship, we applied multi-scale avalanche analysis to voltage-sensitive dye imaging data collected from animals of various species under different anesthetics. We found that anesthesia systematically varied the scaling behavior of neural dynamics, a change that was mirrored in reduced neural complexity. These findings were corroborated by applying the same analyses to a biophysically realistic cortical network model, in which multi-scale criticality measures were associated with network properties and the capacity for information integration. Our results imply that multi-scale criticality measures are potential biomarkers for assessing the level of consciousness.

Mechanisms for shaping receptive field in monkey area TE.

Visual object information is conveyed from V1 to area TE along the ventral visual pathway with increasing receptive field (RF) sizes. The RFs of TE neurons are known to be large, but it is largely unknown how large RFs are shaped along the ventral visual pathway. In this study, we addressed this question in two aspects, static and dynamic mechanisms, by recording neural responses from macaque area TE and V4 to object stimuli presented at various locations in the visual field. As a component related to static mechanisms, we found that in area TE, but not in V4, response latency to objects presented at fovea were different from objects in periphery. As a component of the dynamic mechanisms, we examined effects of spatial attention on the RFs of TE neurons. Spatial attention did not affect response latency but modulated response magnitudes depending on attended location, shifting of the longitudinal axis of RFs toward the attended locations. In standard models of large RF formation, downstream neurons pool information from nearby RFs, and this process is repeated across the visual field and at each step along the ventral visual pathway. The present study revealed that this mechanism is not that simple: 1) different circuit mechanisms for foveal and peripheral visual fields may be situated between V4 and area TE, and 2) spatial attention dynamically changes the shape of RFs.NEW & NOTEWORTHY Receptive fields (RFs) of neurons are progressively increased along the ventral visual pathway so that an RF at the final stage, area TE, covers a large area of the visual field. We explored the mechanism and suggested involvement of parallel circuit mechanisms between V4 and TE for foveal and peripheral parts of visual field. We also found a dynamic component of RF shape formation through attentional modulation of responses in a location-dependent manner.

Reduction in calcium responses to whisker stimulation in the primary somatosensory and motor cortices of the model mouse with trigeminal neuropathic pain.

OBJECTIVE: Chronic constriction injury (CCI) of the infraorbital nerve induces neuropathic pain, such as allodynia and hyperalgesia, in the orofacial area. However, the changes in the local circuits of the central nervous system following CCI remain unclear. This study aimed to identify the changes following CCI in Thy1-GCaMP6s transgenic mice. METHODS: Neural activity in the primary somatosensory cortex (S1) and motor cortex (M1) following whisker stimulation was assessed using in vivo Ca2+ imaging. CCI-induced changes in responses were analyzed. RESULTS: Before CCI, whisker stimulation induced a greater Ca2+ response in the contralateral S1 than in the ipsilateral S1 and contralateral M1. The peak Ca2+ response amplitude in the bilateral S1 and contralateral M1 decreased two days after CCI compared to before CCI. Decreased Ca2+ response amplitude in these regions was observed until four days after CCI. Seven days after CCI, the Ca2+ response amplitude in the contralateral S1 decreased, whereas that in the ipsilateral S1 and contralateral M1 recovered to control levels. CONCLUSION: These results suggest that neural activity in regions receiving excitatory inputs via corticocortical pathways recovers earlier than in regions receiving thalamocortical inputs. (185/250 words).

Parallel development of orientation maps and spatial frequency selectivity in cat visual cortex.

In an early stage of the postnatal development of cats, orientation maps mature and spatial frequency selectivity is consolidated. To investigate the time course of orientation map maturation associated with the consolidation of spatial frequency selectivity, we performed optical imaging of intrinsic signals in areas 17 and 18 of cats under the stimulation of drifting square-wave gratings with different orientations and spatial frequencies. First, orientation maps for lower spatial frequencies emerged in the entire part of the lateral gyrus, which includes areas 17 and 18, and then these orientation maps in the posterior part of the lateral gyrus disappeared as orientation maps for higher spatial frequencies matured. Independent of age, an anteroposterior gradient of response strengths from lower to higher spatial frequencies was observed. This indicates that the regional distribution of spatial frequencies is innately determined. The size of iso-orientation domains tended to decrease as the stimulus spatial frequency increased at every age examined. In contrast, orientation representation bias changed with age. In cats younger than 3 months, the cardinal (vertical and horizontal) orientations were represented predominantly over the oblique orientations. However, in young adult cats from 3 to 9 months old, the representation bias switched to predominantly oblique orientations. These age-dependent changes in the orientation representation bias imply that orientation maps continue to elaborate within postnatal 1 year with the consolidation of spatial frequency selectivity. We conclude that both intrinsic and mutual factors lead to the development of orientation maps and spatial frequency selectivity.

Repetitive nociceptive stimulation increases spontaneous neural activation similar to nociception-induced activity in mouse insular cortex.

Recent noninvasive neuroimaging technology has revealed that spatiotemporal patterns of cortical spontaneous activity observed in chronic pain patients are different from those in healthy subjects, suggesting that the spontaneous cortical activity plays a key role in the induction and/or maintenance of chronic pain. However, the mechanisms of the spontaneously emerging activities supposed to be induced by nociceptive inputs remain to be established. In the present study, we investigated spontaneous cortical activities in sessions before and after electrical stimulation of the periodontal ligament (PDL) by applying wide-field and two-photon calcium imaging to anesthetized GCaMP6s transgenic mice. First, we identified the sequential cortical activation patterns from the primary somatosensory and secondary somatosensory cortices to the insular cortex (IC) by PDL stimulation. We, then found that spontaneous IC activities that exhibited a similar spatiotemporal cortical pattern to evoked activities by PDL stimulation increased in the session after repetitive PDL stimulation. At the single-cell level, repetitive PDL stimulation augmented the synchronous neuronal activity. These results suggest that cortical plasticity induced by the repetitive stimulation leads to the frequent PDL stimulation-evoked-like spontaneous IC activation. This nociception-induced spontaneous activity in IC may be a part of mechanisms that induces chronic pain.

A new phenotype identification method with the fluorescent expression in cross-sectioned tails in Thy1-GCaMP6s transgenic mice.

PURPOSE: Unless the phenotype of the transgenic mice is distinguishable, genotyping in each mouse is required prior to experiments. This study aimed to establish a new identification method for the phenotype in Thy1-GCaMP6s transgenic mice to reduce the cost and time. METHODS: Tail biopsies (2 mm) were performed under general anesthesia with isoflurane in 3 to 4-week-old mice. Then, the resected tail was cut again with a sharp razor, and the cross-sections were observed with two-photon microscopy (excitation wavelength = 940 nm). The emitted light was split into green and red light by a dichroic mirror (570 nm) with bandpass filters (495-540 nm for green, 575-645 nm for red). RESULTS: Two types of expressed fluorescent pattern were found in the tail tissue: the presence of green fluorescent structures (type 1) and the absence of the structures (type 2). Cortical imaging confirmed that type 1 expressed the cortical GCaMP6s, while type 2 did not. CONCLUSION: These results suggest that observation of the cross-sectioned tail in Thy1-GCaMP6s mice enabled to identify the phenotype within approximately 10 min/mouse, which reduces the cost and time for genotyping.

Sirtuin 6 is a regulator of dendrite morphogenesis in rat hippocampal neurons.

Sirtuin 6 (SIRT6), a member of the Sirtuin family, acts as nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase, mono-adenosine diphosphate (ADP)-ribosyltransferase, and fatty acid deacylase, and plays critical roles in inflammation, aging, glycolysis, and DNA repair. Accumulating evidence has suggested that SIRT6 is involved in brain functions such as neuronal differentiation, neurogenesis, and learning and memory. However, the precise molecular roles of SIRT6 during neuronal circuit formation are not yet well understood. In this study, we tried to elucidate molecular roles of SIRT6 on neurite development by using primary-cultured hippocampal neurons. We observed that SIRT6 was abundantly localized in the nucleus, and its expression was markedly increased during neurite outgrowth and synaptogenesis. By using shRNA-mediated SIRT6-knockdown, we show that both dendritic length and the number of dendrite branches were significantly reduced in the SIRT6-knockdown neurons. Microarray and subsequent gene ontology analysis revealed that reducing SIRT6 caused the downregulation of immediate early genes (IEGs) and alteration of several biological processes including MAPK (ERK1/2) signaling. We found that nuclear accumulation of phosphorylated ERK1/2 was significantly reduced in SIRT6-knockdown neurons. Overexpression of SIRT6 promoted dendritic length and branching, but the mutants lacking deacetylase activity had no significant effect on the dendritic morphology. Collectively, the presented findings reveal a role of SIRT6 in dendrite morphogenesis, and suggest that SIRT6 may act as an important regulator of ERK1/2 signaling pathway that mediates IEG expression, which leads to dendritic development.

Presynaptic NK1 Receptor Activation by Substance P Suppresses EPSCs via Nitric Oxide Synthesis in the Rat Insular Cortex.

Substance P (SP) regulates inhibitory synaptic transmission mediated by GABAA receptors in the cerebral cortex; however, SP-mediated regulation of excitatory synaptic transmission remains poorly understood. We performed whole-cell patch-clamp recordings from pyramidal neurons to examine the effects of SP on excitatory postsynaptic currents (EPSCs) mediated via AMPA receptors in the insular cortex (IC), which is involved in nociceptive information processing. First, EPSCs evoked by minimal electrical stimulation (eEPSCs) including stepwise EPSCs and failure events, were examined. SP dose-dependently suppressed mean eEPSC amplitude, partially due to an increase in the failure rate of eEPSCs. The SP-induced suppression of eEPSCs was accompanied by an increase in the paired-pulse ratio and was inhibited by the preapplication of SR140333, an NK1 receptor antagonist. [Sar9,Met(O2)11]-substance P, an NK1 receptor-selective agonist, mimicked the effects of SP on eEPSCs and decreased the frequency of miniature EPSCs (mEPSCs) without changing the average mEPSC amplitude. Considering that most NK1 receptors in the cerebral cortex are expressed in nitric oxide synthase (NOS)-positive GABAergic neurons, the SP-induced suppressive effect on EPSCs may be mediated by nitric oxide (NO) in this subtype of GABAergic neurons. NO imaging using the fluorescent probe DAX-J2 Red supports this hypothesis: SP increased the fluorescence intensity of DAX-J2 Red in some GABAergic neurons. Furthermore, both L-NAME, an NOS inhibitor, and PTIO, an NO scavenger, diminished the SP-induced suppression of eEPSCs. These results suggest that the activation of presynaptic NK1 receptors contributes to SP-induced eEPSC suppression by activating the NO synthesis pathway in GABAergic neurons. (246 words).

Development and Reorganization of Orientation Representation in the Cat Visual Cortex: Experience-Dependent Synaptic Rewiring in Early Life.

To date, numerous mathematical models have been proposed on the basis of some types of Hebbian synaptic learning to account for the activity-dependent development of orientation maps as well as neuronal orientation selectivity. These models successfully reproduced orientation map-like spatial patterns. Nevertheless, we still have questions: (1) How does synaptic rewiring occur in the visual cortex during the formation of orderly orientation maps in early life? (2) How does visual experience contribute to the maturation of orientation selectivity of visual cortical neurons and reorganize orientation maps? (3) How does the sensitive period for orientation plasticity end? In this study, we performed animal experiments and mathematical modeling to understand the mechanisms underlying synaptic rewiring for experience-dependent formation and reorganization of orientation maps. At first, we visualized orientation maps from the intrinsic signal optical imaging in area 17 of kittens reared under single-orientation exposure through cylindrical-lens-fitted goggles. The experiments revealed that the degree of expansion of cortical domains representing the experienced orientation depends on the age at which the single-orientation exposure starts. As a result, we obtained the sensitive period profile for orientation plasticity. Next, we refined our previously proposed mathematical model for the activity-dependent self-organization of thalamo-cortical inputs on the assumption that rewiring is caused by the competitive interactions among transient synaptic contacts on the same dendritic spine. Although various kinds of molecules have been reported to be involved in such interactions, we attempt to build a mathematical model to describe synaptic rewiring focusing on brain-derived neurotrophic factor (BDNF) and its related molecules. Performing computer simulations based on the refined model, we successfully reproduced orientation maps reorganized in kittens reared under single-orientation exposure as well as normal visual experience. We also reproduced the experimentally obtained sensitive period profile for orientation plasticity. The excellent agreement between experimental observations and theoretical reproductions suggests that the BDNF-induced competitive interaction among synaptic contacts from different axons on the same spine is an important factor for the experience-dependent formation and reorganization of orientation selectivity and orientation maps.

Anisotropy in the representation of direction preferences in cat area 18.

Higher visual cortical areas are involved in the perception of complex stimuli, such as the optic flow created by self-motion. On the other hand, area 18 is thought to extract primitive visual features, feeding higher cortical areas for further processing. In this study, we applied optical imaging of intrinsic signals in the central, lower visual field of cat area 18, and reconstructed direction preference and direction selectivity maps in each hemisphere. We observed a significant overrepresentation of downward and temporal directions, in accordance with previous electrophysiological results. Cardinal orientations were not overrepresented, however. Downward directions were overrepresented at the highest direction selectivity domains. Temporal direction representation, on the other hand, decreased with direction selectivity. Our findings therefore suggest the existence of a neural substrate for the processing of optic flow in cat area 18.

Experience-dependent orientation plasticity in the visual cortex of rats chronically exposed to a single orientation.

We used the intrinsic signal optical imaging technique to assess the effect of orientation-restricted visual experience on response properties of the rat visual cortex. We placed young animals wearing goggles fitted with plano-convex cylindrical lenses in a stimulus-enriched environment for 3 weeks. Experienced orientation was over-represented in the visual cortex, which was associated with the under-representation of orthogonal orientation. These findings suggest that chronic exposure to a single orientation can modify orientation preferences even in rats lacking in orderly arrangement of preferred orientations.

A postnatal critical period for orientation plasticity in the cat visual cortex.

Orientation selectivity of primary visual cortical neurons is an important requisite for shape perception. Although numerous studies have been previously devoted to a question of how orientation selectivity is established and elaborated in early life, how the susceptibility of orientation plasticity to visual experience changes in time remains unclear. In the present study, we showed a postnatal sensitive period profile for the modifiability of orientation selectivity in the visual cortex of kittens reared with head-mounted goggles for stable single-orientation exposure. When goggle rearing (GR) started at P16-P30, 2 weeks of GR induced a marked over-representation of the exposed orientation, and 2 more weeks of GR consolidated the altered orientation maps. GR that started later than P50, in turn, induced the under-representation of the exposed orientation. Orientation plasticity in the most sensitive period was markedly suppressed by cortical infusion of NMDAR antagonist. The present study reveals that the plasticity and consolidation of orientation selectivity in an early life are dynamically regulated in an experience-dependent manner.