Association cortical areas in the mouse contain a large population of fast-spiking GABAergic neurons that do not express parvalbumin.

GABAergic neurons represent 10-15% of the neuronal population of the cortex but exert a powerful control over information flow in cortical circuits. The largest GABAergic class in the neocortex is represented by the parvalbumin-expressing fast-spiking neurons, which provide powerful somatic inhibition to their postsynaptic targets. Recently, the density of parvalbumin interneurons has been shown to be lower in associative areas of the mouse cortex as compared with sensory and motor areas. Modelling work based on these quantifications linked the low-density of parvalbumin interneurons with specific computations of associative cortices. However, it is still unknown whether the total GABAergic population of association cortices is smaller or whether another GABAergic type can compensate for the low density of parvalbumin interneurons. In the present study, we investigated these hypotheses using a combination of neuroanatomy, mouse genetics and neurophysiology. We found that the GABAergic population of association areas is comparable with that of primary sensory areas, and it is enriched of fast-spiking neurons that do not express parvalbumin and were not accounted for by previous quantifications. We developed an intersectional viral strategy to demonstrate that the population of fast-spiking neurons is comparable across cortical regions. Our results provide quantifications of the density of fast-spiking GABAergic neurons and offers new biological constrains to refine current models of cortical computations.

Exercised blood plasma promotes hippocampal neurogenesis in the Alzheimer's disease rat brain.

BACKGROUND: Exercise training promotes brain plasticity and is associated with protection against cognitive impairment and Alzheimer's disease (AD). These beneficial effects may be partly mediated by blood-borne factors. Here we used an in vitro model of AD to investigate effects of blood plasma from exercise-trained donors on neuronal viability, and an in vivo rat model of AD to test whether such plasma impacts cognitive function, amyloid pathology, and neurogenesis. METHODS: Mouse hippocampal neuronal cells were exposed to AD-like stress using amyloid-ß and treated with plasma collected from human male donors 3 h after a single bout of high-intensity exercise. For in vivo studies, blood was collected from exercise-trained young male Wistar rats (high-intensity intervals 5 days/week for 6 weeks). Transgenic AD rats (McGill-R-Thy1-APP) were injected 5 times/fortnight for 6 weeks at 2 months or 5 months of age with either (a) plasma from the exercise-trained rats, (b) plasma from sedentary rats, or (c) saline. Cognitive function, amyloid plaque pathology, and neurogenesis were assessed. The plasma used for the treatment was analyzed for 23 cytokines. RESULTS: Plasma from exercised donors enhanced cell viability by 44.1% (p = 0.032) and reduced atrophy by 50.0% (p < 0.001) in amyloid-ß-treated cells. In vivo exercised plasma treatment did not alter cognitive function or amyloid plaque pathology but did increase hippocampal neurogenesis by ∼3 fold, regardless of pathological stage, when compared to saline-treated rats. Concentrations of 7 cytokines were significantly reduced in exercised plasma compared to sedentary plasma. CONCLUSION: Our proof-of-concept study demonstrates that plasma from exercise-trained donors can protect neuronal cells in culture and promote adult hippocampal neurogenesis in the AD rat brain. This effect may be partly due to reduced pro-inflammatory signaling molecules in exercised plasma.

Amidst an amygdala renaissance in Alzheimer's disease.

The amygdala was highlighted as an early site for neurofibrillary tau tangle pathology in Alzheimer's disease in the seminal 1991 article by Braak and Braak. This knowledge has, however, only received traction recently with advances in imaging and image analysis techniques. Here, we provide a cross-disciplinary overview of pathology and neuroimaging studies on the amygdala. These studies provide strong support for an early role of the amygdala in Alzheimer's disease and the utility of imaging biomarkers of the amygdala in detecting early changes and predicting decline in cognitive functions and neuropsychiatric symptoms in early stages. We summarize the animal literature on connectivity of the amygdala, demonstrating that amygdala nuclei that show the earliest and strongest accumulation of neurofibrillary tangle pathology are those that are connected to brain regions that also show early neurofibrillary tangle accumulation. Additionally, we propose an alternative pathway of neurofibrillary tangle spreading within the medial temporal lobe between the amygdala and the anterior hippocampus. The proposed existence of this pathway is strengthened by novel experimental data on human functional connectivity. Finally, we summarize the functional roles of the amygdala, highlighting the correspondence between neurofibrillary tangle accumulation and symptomatic profiles in Alzheimer's disease. In summary, these findings provide a new impetus for studying the amygdala in Alzheimer's disease and a unique perspective to guide further study on neurofibrillary tangle spreading and the occurrence of neuropsychiatric symptoms in Alzheimer's disease.

A multifaceted architectural framework of the mouse claustrum complex.

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CLCX), this has yet to be fully accomplished. The CLCX is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are debated. To address this, we conducted a multifaceted analysis of fiber- and cytoarchitecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to CLCX, including an online reference atlas. Our data indicated four distinct subregions within CLCX, subdividing both CL and DEn into two. Additionally, we conducted brain-wide tracing of inputs to CLCX using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of CLCX and its subregions. Myelinated fibers were abundant dorsally in CL but absent ventrally, whereas PV expressing fibers occupied the entire CL. CB staining revealed a central gap within CL, also visible anterior to the striatum. The Nr2f2, Npsr1, and Cplx3 genes expressed specifically within different subregions of the CLCX, and Rprm helped delineate the CL-insular border. Furthermore, cells in CL projecting to the retrosplenial cortex were located within the myelin sparse area. By combining own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework.

Mice are a highly tractable model for studying the claustrum complex (CLCX). However, without a consensus on how to delineate the CLCX in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CLCX, to match the level of detail needed to study its functional properties. To improve and expand upon preexisting delineation schemes, we used the combinatorial expression of several markers to create a comprehensive guide to delineate the CLCX and its subregions, including an online reference atlas. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CLCX.

The efferent connections of the orbitofrontal, posterior parietal, and insular cortex of the rat brain.

The orbitofrontal, posterior parietal, and insular cortices are sites of higher-order cognitive processing implicated in a wide range of behaviours, including working memory, attention guiding, decision making, and spatial navigation. To better understand how these regions contribute to such functions, we need detailed knowledge about the underlying structural connectivity. Several tract-tracing studies have investigated specific aspects of orbitofrontal, posterior parietal and insular connectivity, but a digital resource for studying the cortical and subcortical projections from these areas in detail is not available. We here present a comprehensive collection of brightfield and fluorescence microscopic images of serial coronal sections from 49 rat brain tract-tracing experiments, in which discrete injections of the anterograde tracers biotinylated dextran amine and/or Phaseolus vulgaris leucoagglutinin were placed in the orbitofrontal, parietal, or insular cortex. The images are spatially registered to the Waxholm Space Rat brain atlas. The image collection, with corresponding reference atlas maps, is suitable as a reference framework for investigating the brain-wide efferent connectivity of these cortical association areas.

Transcriptional development of the hippocampus and the dorsal-intermediate-ventral axis in rats.

Risk and resilience for neuropsychiatric illnesses are established during brain development, and transcriptional markers of risk may be identifiable in early development. The dorsal-ventral axis of the hippocampus has behavioral, electrophysiological, anatomical, and transcriptional gradients and abnormal hippocampus development is associated with autism, schizophrenia, epilepsy, and mood disorders. We previously showed that differential gene expression along the dorsoventral hippocampus in rats was present at birth (postnatal day 0, P0), and that a subset of differentially expressed genes (DEGs) was present at all postnatal ages examined (P0, P9, P18, and P60). Here, we extend the analysis of that gene expression data to understand the development of the hippocampus as a whole by examining DEGs that change with age. We additionally examine development of the dorsoventral axis by looking at DEGs along the axis at each age. Using both unsupervised and supervised analyses, we find that the majority of DEGs are present from P0 to P18, with many expression profiles presenting peaks or dips at P9/18. During development of the hippocampus, enriched pathways associated with learning, memory, and cognition increase with age, as do pathways associated with neurotransmission and synaptic function. Development of the dorsoventral axis is greatest at P9 and P18 and is marked by DEGs associated with metabolic functions. Our data indicate that neurodevelopmental disorders like epilepsy, schizophrenia and affective disorders are enriched with developmental DEGs in the hippocampus, regardless of dorsoventral location, with the greatest enrichment of these clinical disorders seen in genes whose expression changes from P0-9. When comparing DEGs from the ventral and dorsal poles, the greatest number of neurodevelopmental disorders is enriched with DEGs found at P18. Taken together, the developing hippocampus undergoes substantial transcriptional maturation during early postnatal development, with expression of genes involved in neurodevelopmental disorders also showing maximal expression changes within this developmental period.

Dissociating distinct cortical networks associated with subregions of the human medial temporal lobe using precision neuroimaging.

Tract-tracing studies in primates indicate that different subregions of the medial temporal lobe (MTL) are connected with multiple brain regions. However, no clear framework defining the distributed anatomy associated with the human MTL exists. This gap in knowledge originates in notoriously low MRI data quality in the anterior human MTL and in group-level blurring of idiosyncratic anatomy between adjacent brain regions, such as entorhinal and perirhinal cortices, and parahippocampal areas TH/TF. Using MRI, we intensively scanned four human individuals and collected whole-brain data with unprecedented MTL signal quality. Following detailed exploration of cortical networks associated with MTL subregions within each individual, we discovered three biologically meaningful networks associated with the entorhinal cortex, perirhinal cortex, and parahippocampal area TH, respectively. Our findings define the anatomical constraints within which human mnemonic functions must operate and are insightful for examining the evolutionary trajectory of the MTL connectivity across species.

Projections of the insular cortex to orbitofrontal and medial prefrontal cortex: A tracing study in the rat.

The dense fiber pathways that connect the insular cortex with frontal cortices are thought to provide these frontal areas with interoceptive information, crucial for their involvement in executive functions. Using anterograde neuroanatomical tracing, we mapped the detailed organization of the projections from the rat insular cortex to its targets in orbitofrontal (OFC) and medial prefrontal (mPFC) cortex. In OFC, main insular projections distribute to lateral and medial parts, avoiding ventral parts. Whereas projections from the primary gustatory cortex densely innervate dorsolateral OFC, likely corresponding to what in primates is known as the secondary gustatory cortex, these projections avoid mPFC. Instead, mPFC is targeted almost exclusively by projections from agranular fields of the insular cortex. Finally, "parietal" domains of the insular cortex project specifically to the dorsolateral OFC, and strongly innervate ventral portions of mPFC, i.e., the dorsal peduncular cortex.

Visuomotor interactions in the mouse forebrain mediated by extrastriate cortico-cortical pathways.

Introduction: The mammalian visual system can be broadly divided into two functional processing pathways: a dorsal stream supporting visually and spatially guided actions, and a ventral stream enabling object recognition. In rodents, the majority of visual signaling in the dorsal stream is transmitted to frontal motor cortices via extrastriate visual areas surrounding V1, but exactly where and to what extent V1 feeds into motor-projecting visual regions is not well known. Methods: We employed a dual labeling strategy in male and female mice in which efferent projections from V1 were labeled anterogradely, and motor-projecting neurons in higher visual areas were labeled with retrogradely traveling adeno-associated virus (rAAV-retro) injected in M2. We characterized the labeling in both flattened and coronal sections of dorsal cortex and made high-resolution 3D reconstructions to count putative synaptic contacts in different extrastriate areas. Results: The most pronounced colocalization V1 output and M2 input occurred in extrastriate areas AM, PM, RL and AL. Neurons in both superficial and deep layers in each project to M2, but high resolution volumetric reconstructions indicated that the majority of putative synaptic contacts from V1 onto M2-projecting neurons occurred in layer 2/3. Discussion: These findings support the existence of a dorsal processing stream in the mouse visual system, where visual signals reach motor cortex largely via feedforward projections in anteriorly and medially located extrastriate areas.

Lowering levels of reelin in entorhinal cortex layer II-neurons results in lowered levels of intracellular amyloid-ß.

Projection neurons in the anteriolateral part of entorhinal cortex layer II are the predominant cortical site for hyper-phosphorylation of tau and formation of neurofibrillary tangles in prodromal Alzheimer's disease. A majority of layer II projection neurons in anteriolateral entorhinal cortex are unique among cortical excitatory neurons by expressing the protein reelin. In prodromal Alzheimer's disease, these reelin-expressing neurons are prone to accumulate intracellular amyloid-ß, which is mimicked in a rat model that replicates the spatio-temporal cascade of the disease. Two important findings in relation to this are that reelin-signalling downregulates tau phosphorylation, and that oligomeric amyloid-ß interferes with reelin-signalling. Taking advantage of this rat model, we used proximity ligation assay to assess whether reelin and intracellular amyloid-ß directly interact during early, pre-plaque stages in anteriolateral entorhinal cortex layer II reelin-expressing neurons. We next made a viral vector delivering micro-RNA against reelin, along with a control vector, and infected reelin-expressing anteriolateral entorhinal cortex layer II-neurons to test whether reelin levels affect levels of intracellular amyloid-ß and/or amyloid precursor protein. We analysed 25.548 neurons from 24 animals, which results in three important findings. First, in reelin-expressing anteriolateral entorhinal cortex layer II-neurons, reelin and intracellular amyloid-ß engage in a direct protein-protein interaction. Second, injecting micro-RNA against reelin lowers reelin levels in these neurons, amounting to an effect size of 1.3-4.5 (Bayesian estimation of Cohen's d effect size, 95% credible interval). This causes a concomitant reduction of intracellular amyloid-ß ranging across three levels of aggregation, including a reduction of Aß42 monomers/dimers amounting to an effect size of 0.5-3.1, a reduction of Aß prefibrils amounting to an effect size of 1.1-3.5 and a reduction of protofibrils amounting to an effect size of 0.05-2.1. Analysing these data using Bayesian estimation of mutual information furthermore reveals that levels of amyloid-ß are dependent on levels of reelin. Third, the reduction of intracellular amyloid-ß occurs without any substantial associated changes in levels of amyloid precursor protein. We conclude that reelin and amyloid-ß directly interact at the intracellular level in the uniquely reelin-expressing projection neurons in anteriolateral entorhinal cortex layer II, where levels of amyloid-ß are dependent on levels of reelin. Since amyloid-ß is known to impair reelin-signalling causing upregulated phosphorylation of tau, our findings are likely relevant to the vulnerability for neurofibrillary tangle-formation of this entorhinal neuronal population.

Organization of projections from the entorhinal cortex to the hippocampal formation of the Egyptian fruit bat Rousettus aegyptiacus.

The hippocampal formation and entorhinal cortex are crucially involved in learning and memory as well as in spatial navigation. The conservation of these structures across the entire mammalian lineage demonstrates their importance. Information on a diverse set of spatially tuned neurons has become available, but we only have a rudimentary understanding of how anatomical network structure affects functional tuning. Bats are the only order of mammals that have evolved true flight, and with this specialization comes the need to navigate and behave in a three dimensional (3D) environment. Spatial tuning of cells in the entorhinal-hippocampal network of bats has been studied for some time, but whether the reported tuning in 3D is associated with changes in the entorhinal-hippocampal network is not known. Here we investigated the entorhinal-hippocampal projections in the Egyptian fruit bat (Rousettus aegyptiacus), by injecting chemical anterograde tracers in the entorhinal cortex. Detailed analyses of the terminations of these projections in the hippocampus showed that both the medial and lateral entorhinal cortex sent projections to the molecular layer of all subfields of the hippocampal formation. Our analyses showed that the terminal distributions of entorhinal fibers in the hippocampal formation of Egyptian fruit bats-including the proximo-distal and longitudinal topography and the layer-specificity-are similar to what has been described in other mammalian species such as rodents and primates. The major difference in entorhinal-hippocampal projections that was described to date between rodents and primates is in the terminal distribution of the DG projection. We found that bats have entorhinal-DG projections that seem more like those in primates than in rodents. It is likely that the latter projection in bats is specialized to the behavioral needs of this species, including 3D flight and long-distance navigation.

Early amygdala and ERC atrophy linked to 3D reconstruction of rostral neurofibrillary tau tangle pathology in Alzheimer's disease.

Previous research has emphasized the unique impact of Alzheimer's Disease (AD) pathology on the medial temporal lobe (MTL), a reflection that tau pathology is particularly striking in the entorhinal and transentorhinal cortex (ERC, TEC) early in the course of disease. However, other brain regions are affected by AD pathology during its early phases. Here, we use longitudinal diffeomorphometry to measure the atrophy rate from MRI of the amygdala compared with that in the ERC and TEC in cognitively unimpaired (CU) controls, CU individuals who progressed to mild cognitive impairment (MCI), and individuals with MCI who progressed to dementia of the AD type (DAT), using a dataset from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Our results show significantly higher atrophy rates of the amygdala in both groups of 'converters' (CU→MCI, MCI→DAT) compared to controls, with rates of volume loss comparable to rates of thickness loss in the ERC and TEC. We localize atrophy within the amygdala within each of these groups using fixed effects modeling. Controlling for the familywise error rate highlights the medial regions of the amygdala as those with significantly higher atrophy in both groups of converters than in controls. Using our recently developed method, referred to as Projective LDDMM, we map measures of neurofibrillary tau tangles (NFTs) from digital pathology to MRI atlases and reconstruct dense 3D spatial distributions of NFT density within regions of the MTL. The distribution of NFTs is consistent with the spatial distribution of MR measured atrophy rates, revealing high densities (and atrophy) in the amygdala (particularly medial), ERC, and rostral third of the MTL. The similarity of the location of NFTs in AD and shape changes in a well-defined clinical population suggests that amygdalar atrophy rate, as measured through MRI may be a viable biomarker for AD.

Dissection and culturing of adult lateral entorhinal cortex layer II neurons from APP/PS1 Alzheimer model mice.

BACKGROUND: Primary neuronal cultures enable cell-biological studies of Alzheimer's disease (AD), albeit typically non-neuron-specific. The first cortical neurons affected in AD reside in layer II of the lateralmost part of the entorhinal cortex, and they undergo early accumulation of intracellular amyloid-ß, form subsequent tau pathology, and start degenerating pre-symptomatically. These vulnerable entorhinal neurons uniquely express the glycoprotein reelin and provide selective inputs to the hippocampal memory system. Gaining a more direct access to study these neurons is therefore highly relevant. NEW METHOD: We demonstrate a methodological approach for dissection and long-term culturing of adult lateral entorhinal layer II-neurons from AD-model mice. RESULTS: We maintain adult dissected lateralmost entorhinal layer II-neurons beyond two months in culture. We show that they express neuronal markers, and that they are electrophysiologically active by 15 days in vitro and continuing beyond 2 months. COMPARISON WITH EXISTING METHODS: Primary neurons are typically harvested from embryonic or early postnatal brains because such neurons are easier to culture compared to adult neurons. Methods to culture adult primary neurons have been reported, however, to our knowledge, culturing of adult entorhinal neuron-type specific primary neurons from AD-model animals have not been reported. CONCLUSIONS: Our methodological approach offers a window to study initial pathological changes in the AD disease-cascade. This includes the study of proteinopathy, single-neuron changes, and network-level dysfunction.

Hippocampal-medial entorhinal circuit is differently organized along the dorsoventral axis in rodents.

The general understanding of hippocampal circuits is that the hippocampus and the entorhinal cortex (EC) are topographically connected through parallel identical circuits along the dorsoventral axis. Our anterograde tracing and in vitro electrophysiology data, however, show a markedly different dorsoventral organization of the hippocampal projection to the medial EC (MEC). While dorsal hippocampal projections are confined to the dorsal MEC, ventral hippocampal projections innervate both dorsal and ventral MEC. Further, whereas the dorsal hippocampus preferentially targets layer Vb (LVb) neurons, the ventral hippocampus mainly targets cells in layer Va (LVa). This connectivity scheme differs from hippocampal projections to the lateral EC, which are topographically organized along the dorsoventral axis. As LVa neurons project to telencephalic structures, our findings indicate that the ventral hippocampus regulates LVa-mediated entorhinal-neocortical output from both dorsal and ventral MEC. Overall, the marked dorsoventral differences in hippocampal-entorhinal connectivity impose important constraints on signal flow in hippocampal-neocortical circuits.

Projections from the five divisions of the orbital cortex to the thalamus in the rat.

The orbital cortex (ORB) of the rat consists of five divisions: the medial (MO), ventral (VO), ventrolateral (VLO), lateral (LO), and dorsolateral (DLO) orbital cortices. No previous report has comprehensively examined and compared projections from each division of the ORB to the thalamus. Using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin, we describe the efferent projections from the five divisions of the ORB to the thalamus in the rat. We demonstrated that, with some overlap, each division of the ORB distributed in a distinct (and unique) manner to nuclei of the thalamus. Overall, ORB projected to a relatively restricted number of sites in the thalamus, and strikingly distributed entirely to structures of the medial/midline thalamus, while completely avoiding lateral regions or principal nuclei of the thalamus. The main termination sites in the thalamus were the paratenial nucleus (PT) and nucleus reuniens (RE) of the midline thalamus, the medial (MDm) and central (MDc) divisions of the mediodorsal nucleus, the intermediodorsal nucleus, the central lateral, paracentral, and central medial nuclei of the rostral intralaminar complex and the submedial nucleus (SM). With some exceptions, medial divisions of the ORB (MO, VO) mainly targeted "limbic-associated" nuclei such as PT, RE, and MDm, whereas lateral division (VLO, LO, DLO) primarily distributed to "sensorimotor-associated" nuclei including MDc, SM, and the rostral intralaminar complex. As discussed herein, the medial/midline thalamus may represent an important link (or bridge) between the orbital cortex and the hippocampus and between the ORB and medial prefrontal cortex. In summary, the present results demonstrate that each division of the orbital cortex projects in a distinct manner to nuclei of the thalamus which suggests unique functions for each division of the orbital cortex.

Retrosplenial and subicular inputs converge on superficially projecting layer V neurons of medial entorhinal cortex.

The medial entorhinal cortex (MEC) plays a pivotal role in spatial processing together with hippocampal formation. The retrosplenial cortex (RSC) is also implicated in this process, and it is thus relevant to understand how these structures interact. This requires precise knowledge of their connectivity. Projections from neurons in RSC synapse onto principal neurons in layer V of MEC and some of these neurons send axons into superficial layers of MEC. Layer V of MEC is also the main target for hippocampal efferents from the subiculum and CA1 field. The aim of this study was to assess whether the population of cells targeted by RSC projections also receives input from the hippocampal formation and to compare the distribution of synaptic contacts on target dendrites. We labeled the cells in layer V of MEC by injecting a retrograde tracer into superficial layers. At the same time, we labeled RSC and subicular projections with different anterograde tracers. 3D-reconstruction of the labeled cells and axons revealed likely synaptic contacts between presynaptic boutons of both origins and postsynaptic MEC layer V basal dendrites. Moreover, these contacts overlapped on the same dendritic segments without targeting specific domains. Our results support the notion that MEC layer V neurons that project to the superficial layers receive convergent input from both RSC and subiculum. These data thus suggest that convergent subicular and RSC information contributes to the signal that neurons in superficial layers of EC send to the hippocampal formation.

Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

Stellate cells are principal neurons in the entorhinal cortex that contribute to spatial processing. They also play a role in the context of Alzheimer's disease as they accumulate Amyloid beta early in the disease. Producing human stellate cells from pluripotent stem cells would allow researchers to study early mechanisms of Alzheimer's disease, however, no protocols currently exist for producing such cells. In order to develop novel stem cell protocols, we characterize at high resolution the development of the porcine medial entorhinal cortex by tracing neuronal and glial subtypes from mid-gestation to the adult brain to identify the transcriptomic profile of progenitor and adult stellate cells. Importantly, we could confirm the robustness of our data by extracting developmental factors from the identified intermediate stellate cell cluster and implemented these factors to generate putative intermediate stellate cells from human induced pluripotent stem cells. Six transcription factors identified from the stellate cell cluster including RUNX1T1, SOX5, FOXP1, MEF2C, TCF4, EYA2 were overexpressed using a forward programming approach to produce neurons expressing a unique combination of RELN, SATB2, LEF1 and BCL11B observed in stellate cells. Further analyses of the individual transcription factors led to the discovery that FOXP1 is critical in the reprogramming process and omission of RUNX1T1 and EYA2 enhances neuron conversion. Our findings contribute not only to the profiling of cell types within the developing and adult brain's medial entorhinal cortex but also provides proof-of-concept for using scRNAseq data to produce entorhinal intermediate stellate cells from human pluripotent stem cells in-vitro.

Postnatal development of projections of the postrhinal cortex to the entorhinal cortex in the rat.

The ability to encode and retrieve contextual information is an inherent feature of episodic memory that starts to develop during childhood. The postrhinal cortex, an area of the parahippocampal region, has a crucial role in encoding object-space information and translating egocentric to allocentric representation of local space. The strong connectivity of POR with the adjacent entorhinal cortex, and consequently the hippocampus, suggests that the development of these connections could support the postnatal development of contextual memory. Here, we report that postrhinal cortex projections of the rat develop progressively from the first to the third postnatal week starting in the medial entorhinal cortex before spreading to the lateral entorhinal cortex. The increased spread and complexity of postrhinal axonal distributions is accompanied by an increased complexity of entorhinal dendritic trees and an increase of postrhinal - entorhinal synapses, which supports a gradual maturation in functional activity.SIGNIFICANCE STATEMENTPostrhinal-entorhinal cortical interplay mediates important aspects of encoding and retrieval of contextual information that is important for episodic memory. To better understand the function of the postrhinal interactions with the entorhinal cortex we studied the postnatal development of the connection between the two cortical areas. Our study describes the postnatal development of the postrhinal-to-entorhinal projections as established with neuroanatomical and electrophysiological methods. The projections gradually reach functionally different areas of the entorhinal cortex, reaching the area involved in spatial functions first, followed by the part involved in representing information about objects and sequences of events.

Prenatal development of the human entorhinal cortex.

Little is known about the development of the human entorhinal cortex (EC), a major hub in a widespread network for learning and memory, spatial navigation, high-order processing of object information, multimodal integration, attention and awareness, emotion, motivation, and perception of time. We analyzed a series of 20 fetal and two adult human brains using Nissl stain, acetylcholinesterase (AChE) histochemistry, and immunocytochemistry for myelin basic protein (MBP), neuronal nuclei antigen (NeuN), a pan-axonal neurofilament marker, and synaptophysin, as well as postmortem 3T MRI. In comparison with other parts of the cerebral cortex, the cytoarchitectural differentiation of the EC begins remarkably early, in the 10th week of gestation (w.g.). The differentiation occurs in a superficial magnocellular layer in the deep part of the marginal zone, accompanied by cortical plate (CP) condensation and multilayering of the deep part of CP. These processes last until the 13-14th w.g. At 14 w.g., the superficial lamina dissecans (LD) is visible, which divides the CP into the lamina principalis externa (LPE) and interna (LPI). Simultaneously, the rostral LPE separates into vertical cell-dense islands, whereas in the LPI, the deep LD emerges as a clear acellular layer. In the 16th w.g., the LPE remodels into vertical cell-dense and cell-sparse zones with a caudorostral gradient. At 20 w.g., NeuN immunoreactivity is most pronounced in the islands of layer II cells, whereas migration and differentiation inside-out gradients are seen simultaneously in both the upper (LPE) and the lower (LPI) pyramidal layers. At this stage, the EC adopts for the first time an adult-like cytoarchitectural organization, the superficial LD becomes discernible by 3T MRI, MBP-expressing oligodendrocytes first appear in the fimbria and the perforant path (PP) penetrates the subiculum to reach its molecular layer and travels along through the Cornu Ammonis fields to reach the suprapyramidal blade of the dentate gyrus, whereas the entorhinal-dentate branch perforates the hippocampal sulcus about 2-3 weeks later. The first AChE reactivity appears as longitudinal stripes at 23 w.g. in layers I and II of the rostrolateral EC and then also as AChE-positive in-growing fibers in islands of superficial layer III and layer II neurons. At 40 w.g., myelination of the PP starts as patchy MBP-immunoreactive oligodendrocytes and their processes. Our results refute the possibility of an inside-out pattern of the EC development and support the key role of layer II prospective stellate cells in the EC lamination. As the early cytoarchitectural differentiation of the EC is paralleled by the neurochemical development, these developmental milestones in EC structure and connectivity have implications for understanding its normal function, including its puzzling modular organization and potential contribution to consciousness content (awareness), as well as for its insufficiently explored deficits in developmental, psychiatric, and degenerative brain disorders.