Integrative Analysis of Long Isoform Sequencing and Functional Data Identifies Distinct Cortical Layer Neuronal Subtypes Derived from Human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated non-neuronal cells in the culture as compared to those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, Beta tubulin, and Synapsin I), and whole-cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.

Implications of altered pyramidal cell morphology on clinical symptoms of neurodevelopmental disorders.

The prevalence of pyramidal cells (PCs) in the mammalian cerebral cortex underscore their value as they play a crucial role in various brain functions, ranging from cognition, sensory processing, to motor output. PC morphology significantly influences brain connectivity and plays a critical role in maintaining normal brain function. Pathological alterations to PC morphology are thought to contribute to the aetiology of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. This review explores the relationship between abnormalities in PC morphology in key cortical areas and the clinical manifestations in schizophrenia and ASD. We focus largely on human postmortem studies and provide evidence that dendritic segment length, complexity and spine density are differentially affected in these disorders. These morphological alterations can lead to disruptions in cortical connectivity, potentially contributing to the cognitive and behavioural deficits observed in these disorders. Furthermore, we highlight the importance of investigating the functional and structural characteristics of PCs in these disorders to illuminate the underlying pathogenesis and stimulate further research in this area.

The effects of abnormal visual experience on neurodevelopmental disorders.

Normal visual development is supported by intrinsic neurobiological mechanisms and by appropriate stimulation from the environment, both of which facilitate the maturation of visual functions. However, an offset of this balance can give rise to visual disorders. Therefore, understanding the factors that support normal vision during development and in the mature brain is important, as vision guides movement, enables social interaction, and allows children to recognize and understand their environment. In this paper, we review fundamental mechanisms that support the maturation of visual functions and discuss and draw links between the perceptual and neurobiological impairments in autism spectrum disorder (ASD) and schizophrenia. We aim to explore how this is evident in the case of ASD, and how perceptual and neurobiological deficits further degrade social ability. Furthermore, we describe the altered perceptual experience of those with schizophrenia and evaluate theories of the underlying neural deficits that alter perception.

Topological Sholl descriptors for neuronal clustering and classification.

Neuronal morphology is a fundamental factor influencing information processing within neurons and networks. Dendritic morphology in particular can widely vary among cell classes, brain regions, and animal species. Thus, accurate quantitative descriptions allowing classification of large sets of neurons is essential for their structural and functional characterization. Current robust and unbiased computational methods that characterize groups of neurons are scarce. In this work, we introduce a novel technique to study dendritic morphology, complementing and advancing many of the existing techniques. Our approach is to conceptualize the notion of a Sholl descriptor and associate, for each morphological feature, and to each neuron, a function of the radial distance from the soma, taking values in a metric space. Functional distances give rise to pseudo-metrics on sets of neurons which are then used to perform the two distinct tasks of clustering and classification. To illustrate the use of Sholl descriptors, four datasets were retrieved from the large public repository https://neuromorpho.org/ comprising neuronal reconstructions from different species and brain regions. Sholl descriptors were subsequently computed, and standard clustering methods enhanced with detection and metric learning algorithms were then used to objectively cluster and classify each dataset. Importantly, our descriptors outperformed conventional morphometric techniques (L-Measure metrics) in several of the tested datasets. Therefore, we offer a novel and effective approach to the analysis of diverse neuronal cell types, and provide a toolkit for researchers to cluster and classify neurons.

Surgical Strategies Used to Successfully Re-implant an Implant that Failed Four Times at the Same Site: A Case Report.

Although implants have been shown to have high success rates, complications such as implant failure can occur. This presents a challenging dilemma for clinicians when attempting another implant placement in the failed site. The patient in this clinical case report presented with implant failure four times at the same site. This case report describes implant placement in a site where four failed implants were previously removed and evaluates the approach used to achieve a successful outcome.

Developmental refinement of visual callosal inputs to ferret area 17.

Corticocortical connections link visual cortical areas in both the ipsilateral and contralateral hemispheres. We studied the postnatal refinement of callosal connections linking multiple cortical areas with ferret area 17 during the period from just before eye opening (4 weeks) to 10 weeks of age. We aimed to determine (1) whether callosal projections from multiple visual cortical areas to area 17 refine with a similar rate and (2) whether the refinement of callosal projections parallels that of intrahemispheric cortical circuits. We injected the bidirectional tracer CTb into area 17, and mapped the areal and laminar distribution of labeled cells in visual areas of the contralateral hemisphere. Like intrahemispheric projections, callosal inputs to area 17 before eye opening are dominated by Suprasylvian area Ssy (with lesser and comparable input from areas 17, 18, 19, and 21), but within 2 weeks of eye opening are jointly dominated by area 18 and Ssy inputs; however, there are fewer labeled cells in the contralateral hemisphere. Unlike intrahemispheric projections, there is no laminar reorganization of callosal inputs; in all visual areas and at all ages studied, the greatest proportion of callosal projections arises from the infragranular layers. Also, unlike intrahemispheric projections, the peak density of callosal cells in each area projecting to area 17 declines more modestly. These results reveal important similarities and differences in the postnatal reorganization of inter- and intrahemispheric projections to area 17.

Developmental Changes in Pyramidal Cell Morphology in Multiple Visual Cortical Areas Using Cluster Analysis.

Neuronal morphology is characterized by salient features such as complex axonal and dendritic arbors. In the mammalian brain, variations in dendritic morphology among cell classes, brain regions, and animal species are thought to underlie known differences in neuronal function. In this work, we obtained a large dataset from http://neuromorpho.org/ comprising layer III pyramidal cells in different cortical areas of the ventral visual pathway (V1, V2, V4, TEO, and TE) of the macaque monkey at different developmental stages. We performed an in depth quantitative analysis of pyramidal cell morphology throughout development in an effort to determine which aspects mature early in development and which features require a protracted period of maturation. We were also interested in establishing if developmental changes in morphological features occur simultaneously or hierarchically in multiple visual cortical areas. We addressed these questions by performing principal component analysis (PCA) and hierarchical clustering analysis on relevant morphological features. Our analysis indicates that the maturation of pyramidal cell morphology is largely based on early development of topological features in most visual cortical areas. Moreover, the maturation of pyramidal cell morphology in V1, V2, V4, TEO, and TE is characterized by unique developmental trajectories.

Visual Corticocortical Inputs to Ferret Area 18.

Visual cortical areas in the adult mammalian brain are linked by a network of interareal feedforward and feedback circuits. We investigated the topography of feedback projections to ferret (Mustela putorius furo) area 18 from extrastriate areas 19, 21, and Ssy. Our objective was to characterize the anatomical organization of the extrastriate feedback pool to area 18. We also wished to determine if feedback projections to area 18 share similar features as feedback projections to area 17. We injected the tracer cholera toxin B subunit (CTb) into area 18 of adult ferrets to visualize the distribution and pattern of retrogradely labeled cells in extrastriate cortex. We find several similarities to the feedback projection to area 17: (i) Multiple visual cortical areas provide feedback to area 18: areas 19, 21, Ssy, and weaker inputs from posterior parietal and lateral temporal visual areas. Within each area a greater proportion of feedback projections arises from the infragranular than from the supragranular layers. (ii) The cortical area immediately rostral to area 18 provides the greatest proportion of total cortical feedback, and has the greatest peak density of cells providing feedback to area 18. (iii) The spacing (peak cell density and nearest neighbor distances) of cells in extrastriate cortex providing feedback to areas 17 and 18 are similar. However, peak density of feedback cells to area 18 is comparable in the supra- and infragranular layers, whereas peak density of feedback cells to area 17 is higher in the infragranular layers. Another prominent difference is that dorsal area 18 receives a cortical input that area 17 does not: from ventral cortex representing the upper visual field; this appears to be roughly 25% of the feedback input to area 18. Lastly, area 17 receives a greater proportion of cortical feedback from area 21 than from Ssy, whereas area 18 receives more feedback from Ssy than from area 21. While the organization of feedback projections from extrastriate cortex to areas 17 and 18 is broadly similar, the main difference in input topography might arise due to differences in visual field representations of the two areas.

Postnatal Development of Visual Cortical Function in the Mammalian Brain.

This review aims to discuss (1) the refinement of mammalian visual cortical circuits and the maturation of visual functions they subserve in primary visual cortex (V1) and other visual cortical areas, and (2) existing evidence supporting the notion of differential rates of maturation of visual functions in different species. It is well known that different visual functions and their underlying circuitry mature and attain adultlike characteristics at different stages in postnatal development with varying growth rates. The developmental timecourse and duration of refinement varies significantly both in V1 of various species and among different visual cortical areas; while basic visual functions like spatial acuity mature earlier requiring less time, higher form perception such as contour integration is more complex and requires longer postnatal time to refine. This review will highlight the importance of systematic comparative analysis of the differential rates of refinement of visual circuitry and function as that may help reveal underlying key mechanisms necessary for healthy visual development during infancy and adulthood. This type of approach will help future studies to establish direct links between various developmental aspects of different visual cortical areas in both human and animal models; thus enhancing our understanding of vision related neurological disorders and their potential therapeutic remedies.

A mechanized device for mounting histological tissue sections.

BACKGROUND: Traditional methods for mounting tissue sections onto slides are suboptimal as the amount of labor required quickly multiplies with increasing number of samples. Methods to accelerate the tissue mounting process while reducing the associated risk of tissue damage are needed. NEW METHOD: We designed and 3D printed a mechanized device with an inclined platform used to mount tissue sections onto slides in buffer solution. The main advantage of this design is to reduce the time required for mounting sections as well as minimize the possibility of damaging delicate or thin tissue sections. RESULTS: Using our device, we illustrate and describe in detail the steps required to mount smaller coronally cut mouse brain sections, as well as bigger tangentially cut ferret brain sections. This method's efficiency was assessed by comparing the time required to mount an entire slide of ferret brain sections using our method and the conventional method. Using our device reduced the tissue mounting time by 60%. COMPARISON WITH EXISTING METHOD(S): Compared to existing conventional tissue mounting methods, our device is a simple and user friendly alternative that substantially reduces the time required to mount tissue sections while preserving tissue section quality. CONCLUSIONS: Using our device can streamline histological processing and prove to be especially useful for a variety of tissue types as the platform was designed to accommodate different size microscope slides, and thus use for varying tissue section sizes.

Postnatal refinement of interareal feedforward projections in ferret visual cortex.

We studied the postnatal refinement of feedforward (FF) projections from ferret V1 to multiple cortical targets during the period around eye opening. Our goal was to establish (a) whether the developmental refinement of FF projections parallels that of feedback (FB) cortical circuits, and (b) whether FF pathways from V1 to different target areas refine with a similar rate. We injected the tracer CTb into V1 of juvenile ferrets, and visualized the pattern of labeled axon terminals in extrastriate cortex. Bouton density of FF projections to target areas 18, 19, and 21 declined steadily from 4 to 8 weeks postnatal. However, in area Ssy this decline was delayed somewhat, not occurring until after 6 weeks. During this postnatal period, mean interbouton intervals along individual FF axons to all visual areas increased, and we observed a concomitant moderate decrease in axon density in areas 18, 21, and Ssy. These data suggest that FF circuits linking V1 to its main extrastriate targets remodel largely synchronously in the weeks following eye opening, that FF and FB cortical circuits share a broadly similar developmental timecourse, and that postnatal visual experience is critical for the refinement of both FF and FB cortical circuits.

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain.

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

Developmental remodeling of corticocortical feedback circuits in ferret visual cortex.

Visual cortical areas in the mammalian brain are linked through a system of interareal feedforward and feedback connections, which presumably underlie different visual functions. We characterized the refinement of feedback projections to primary visual cortex (V1) from multiple sources in juvenile ferrets ranging in age from 4-10 weeks postnatal. We studied whether the refinement of different aspects of feedback circuitry from multiple visual cortical areas proceeds at a similar rate in all areas. We injected the neuronal tracer cholera toxin B (CTb) into V1 and mapped the areal and laminar distribution of retrogradely labeled cells in extrastriate cortex. Around the time of eye opening at 4 weeks postnatal, the retinotopic arrangement of feedback appears essentially adult-like; however, suprasylvian cortex supplies the greatest proportion of feedback, whereas area 18 supplies the greatest proportion in the adult. The density of feedback cells and the ratio of supragranular/infragranular feedback contribution declined in this period at a similar rate in all cortical areas. We also found significant feedback to V1 from layer IV of all extrastriate areas. The regularity of cell spacing, the proportion of feedback arising from layer IV, and the tangential extent of feedback in each area all remained essentially unchanged during this period, except for the infragranular feedback source in area 18, which expanded. Thus, while much of the basic pattern of cortical feedback to V1 is present before eye opening, there is major synchronous reorganization after eye opening, suggesting a crucial role for visual experience in this remodeling process.

Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex.

A critical question in brain development is whether different brain circuits mature concurrently or with different timescales. To characterize the anatomical and functional development of different visual cortical areas, one must be able to distinguish these areas. Here, we show that zinc histochemistry, which reveals a subset of glutamatergic processes, can be used to reliably distinguish visual areas in juvenile and adult ferret cerebral cortex, and that the postnatal decline in levels of synaptic zinc follows a broadly similar developmental trajectory in multiple areas of ferret visual cortex. Zinc staining in all areas examined (17, 18, 19, 21, and Suprasylvian) is greater in the 5-week-old than in the adult. Furthermore, there is less laminar variation in zinc staining in the 5-week-old visual cortex than in the adult. Despite differences in staining intensity, areal boundaries can be discerned in the juvenile as in the adult. By 6 weeks of age, we observe a significant decline in visual cortical synaptic zinc; this decline was most pronounced in layer IV of areas 17 and 18, with much less change in higher-order extrastriate areas during the important period in visual cortical development following eye opening. By 10 weeks of age, the laminar pattern of zinc staining in all visual areas is essentially adultlike. The decline in synaptic zinc in the supra- and infragranular layers in all areas proceeds at the same rate, though the decline in layer IV does not. These results suggest that the timecourse of synaptic zinc decline is lamina specific, and further confirm and extend the notion that at least some aspects of cortical maturation follow a similar developmental timecourse in multiple areas. The postnatal decline in synaptic zinc we observe during the second postnatal month begins after eye opening, consistent with evidence that synaptic zinc is regulated by sensory experience.