Morphological diversification and functional maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain.

Astrocytes, the most abundant glial cell type in the brain, are underrepresented in traditional cortical organoid models due to the delayed onset of cortical gliogenesis. Here we introduce a new glia-enriched cortical organoid model that exhibits accelerated astrogliogenesis. We demonstrated that induction of a gliogenic switch in a subset of progenitors enabled the rapid derivation of astroglial cells, which account for 25-31% of the cell population within 8-10 weeks of differentiation. Intracerebral transplantation of these organoids reliably generated a diverse repertoire of cortical neurons and anatomical subclasses of human astrocytes. Spatial transcriptome profiling identified layer-specific expression patterns among distinct subclasses of astrocytes within organoid transplants. Using an in vivo acute neuroinflammation model, we identified a subpopulation of astrocytes that rapidly activates pro-inflammatory pathways upon cytokine stimulation. Additionally, we demonstrated that CD38 signaling has a crucial role in mediating metabolic and mitochondrial stress in reactive astrocytes. This model provides a robust platform for investigating human astrocyte function.

Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation.

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

Neuronal activity-related transcription is blunted in immature compared to mature dentate granule cells.

Immature dentate granule cells (DGCs) generated in the hippocampus during adulthood are believed to play a unique role in dentate gyrus (DG) function. Although immature DGCs have hyperexcitable membrane properties in vitro, the consequences of this hyperexcitability in vivo remain unclear. In particular, the relationship between experiences that activate the DG, such as exploration of a novel environment (NE), and downstream molecular processes that modify DG circuitry in response to cellular activation is unknown in this cell population. We first performed quantification of immediate early gene (IEG) proteins in immature (5-week-old) and mature (13-week-old) DGCs from mice exposed to a NE. Paradoxically, we observed lower IEG protein expression in hyperexcitable immature DGCs. We then isolated nuclei from active and inactive immature DGCs and performed single-nuclei RNA-Sequencing. Compared to mature nuclei collected from the same animal, immature DGC nuclei showed less activity-induced transcriptional change, even though they were classified as active based on expression of ARC protein. These results demonstrate that the coupling of spatial exploration, cellular activation, and transcriptional change differs between immature and mature DGCs, with blunted activity-induced changes in immature cells.

AAV ablates neurogenesis in the adult murine hippocampus.

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hr post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentategyrus (DG)- without ablating adult neurogenesis- can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo two-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system should be carefully evaluated.

Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis.

Neurogenesis in the adult hippocampus declines with age, a process that has been implicated in cognitive and emotional impairments. However, the mechanisms underlying this decline have remained elusive. Here, we show that the age-dependent downregulation of lamin B1, one of the nuclear lamins in adult neural stem/progenitor cells (ANSPCs), underlies age-related alterations in adult hippocampal neurogenesis. Our results indicate that higher levels of lamin B1 in ANSPCs safeguard against premature differentiation and regulate the maintenance of ANSPCs. However, the level of lamin B1 in ANSPCs declines during aging. Precocious loss of lamin B1 in ANSPCs transiently promotes neurogenesis but eventually depletes it. Furthermore, the reduction of lamin B1 in ANSPCs recapitulates age-related anxiety-like behavior in mice. Our results indicate that the decline in lamin B1 underlies stem cell aging and impacts the homeostasis of adult neurogenesis and mood regulation.

The role of adult hippocampal neurogenesis in brain health and disease.

Adult neurogenesis in the dentate gyrus of the hippocampus is highly regulated by a number of environmental and cell-intrinsic factors to adapt to environmental changes. Accumulating evidence suggests that adult-born neurons may play distinct physiological roles in hippocampus-dependent functions, such as memory encoding and mood regulation. In addition, several brain diseases, such as neurological diseases and mood disorders, have deleterious effects on adult hippocampal neurogenesis, and some symptoms of those diseases can be partially explained by the dysregulation of adult hippocampal neurogenesis. Here we review a possible link between the physiological functions of adult-born neurons and their roles in pathological conditions.

A novel environment-evoked transcriptional signature predicts reactivity in single dentate granule neurons.

Activity-induced remodeling of neuronal circuits is critical for memory formation. This process relies in part on transcription, but neither the rate of activity nor baseline transcription is equal across neuronal cell types. In this study, we isolated mouse hippocampal populations with different activity levels and used single nucleus RNA-seq to compare their transcriptional responses to activation. One hour after novel environment exposure, sparsely active dentate granule (DG) neurons had a much stronger transcriptional response compared to more highly active CA1 pyramidal cells and vasoactive intestinal polypeptide (VIP) interneurons. Activity continued to impact transcription in DG neurons up to 5 h, with increased heterogeneity. By re-exposing the mice to the same environment, we identified a unique transcriptional signature that selects DG neurons for reactivation upon re-exposure to the same environment. These results link transcriptional heterogeneity to functional heterogeneity and identify a transcriptional correlate of memory encoding in individual DG neurons.

An in vivo model of functional and vascularized human brain organoids.

Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.

Mechanisms of dietary flavonoid action in neuronal function and neuroinflammation.

Flavonoids are a class of plant-derived dietary polyphenols that have attracted attention for their pro-cognitive and anti-inflammatory effects. The diversity of flavonoids and their extensive in vivo metabolism suggest that a variety of cellular targets in the brain are likely to be impacted by flavonoid consumption. Initially characterized as antioxidants, flavonoids are now believed to act directly on neurons and glia via the interaction with major signal transduction cascades, as well as indirectly via interaction with the blood-brain barrier and cerebral vasculature. This review discusses potential mechanisms of flavonoid action in the brain, with a focus on two critical transcription factors: cAMP response element-binding protein (CREB) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). To advance beyond current understanding of cellular targets, critical bioavailability studies need to be performed to verify the identity and concentration of flavonoid metabolites reaching the brain after ingestion and to validate that these metabolites are produced not just in rodent models but also in humans. Recent advances in human induced pluripotent stem cell (iPSC) differentiation protocols to generate human neuronal and glial cell types could also provide a unique tool for clinically relevant in vitro investigation of the mechanisms of action of bioavailable flavonoid metabolites in humans.

In vivo imaging of dendritic pruning in dentate granule cells.

We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs.

Nuclear RNA-seq of single neurons reveals molecular signatures of activation.

Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.

Increasing the resolution of the adult neurogenesis picture.

The birth of new neurons in the adult mammalian brain-once thought impossible-is now a well-accepted phenomenon that takes place in the subventricular zone of the lateral ventricles and the hippocampus. This review focuses on the recent work that has sharpened our views of how hippocampal newborn neurons are regulated and function. Areas of study include (a) how neurogenesis contributes to behavioral pattern separation, (b) how pattern separation may be influenced by the properties and circuitry of newborn neurons, (c) differences along the dorsal-ventral axis of how neurogenesis is regulated and functions, and (d) adult neurogenesis in primates, including new human data. These current avenues of research reveal new details of adult neurogenesis and foreshadow what we may learn about this exciting phenomenon in the near future.

Effects of CB1 and CRF1 receptor antagonists on binge-like eating in rats with limited access to a sweet fat diet: lack of withdrawal-like responses.

Positive reinforcement (e.g., appetitive, rewarding properties) has often been hypothesized to maintain excessive intake of palatable foods. Recently, rats receiving intermittent access to high sucrose diets showed binge-like intake with withdrawal-like signs upon cessation of access, suggesting negative reinforcement mechanisms contribute as well. Whether intermittent access to high fat diets also produces withdrawal-like syndromes is controversial. The present study therefore tested the hypothesis that binge-like eating and withdrawal-like anxiety would arise in a novel model of binge eating based on daily 10-min access to a sweet fat diet (35% fat kcal, 31% sucrose kcal). Within 2-3 weeks, female Wistar rats developed binge-like intake comparable to levels seen previously for high sucrose diets (~40% of daily caloric intake within 10 min) plus excess weight gain and adiposity, but absent increased anxiety-like behavior during elevated plus-maze or defensive withdrawal tests after diet withdrawal. Binge-like intake was unaffected by pretreatment with the corticotropin-releasing factor type 1 (CRF(1)) receptor antagonist R121919, and corticosterone responses to restraint stress did not differ between sweet-fat binge rats and chow-fed controls. In contrast, pretreatment with the cannabinoid type 1 (CB(1)) receptor antagonist SR147778 dose-dependently reduced binge-like intake, albeit less effectively than in ad lib chow or sweet fat controls. A priming dose of the sweet fat diet did not precipitate increased anxiety-like behavior, but rather increased plus-maze locomotor activity. The results suggest that CB(1)-dependent positive reinforcement rather than CRF(1)-dependent negative reinforcement mechanisms predominantly maintain excessive intake in this limited access model of sweet-fat diet binges.

The dark side of food addiction.

In drug addiction, the transition from casual drug use to dependence has been linked to a shift away from positive reinforcement and toward negative reinforcement. That is, drugs ultimately are relied on to prevent or relieve negative states that otherwise result from abstinence (e.g., withdrawal) or from adverse environmental circumstances (e.g., stress). Recent work has suggested that this "dark side" shift also is a key in the development of food addiction. Initially, palatable food consumption has both positively reinforcing, pleasurable effects and negatively reinforcing, "comforting" effects that can acutely normalize organism responses to stress. Repeated, intermittent intake of palatable food may instead amplify brain stress circuitry and downregulate brain reward pathways such that continued intake becomes obligatory to prevent negative emotional states via negative reinforcement. Stress, anxiety and depressed mood have shown high comorbidity with and the potential to trigger bouts of addiction-like eating behavior in humans. Animal models indicate that repeated, intermittent access to palatable foods can lead to emotional and somatic signs of withdrawal when the food is no longer available, tolerance and dampening of brain reward circuitry, compulsive seeking of palatable food despite potentially aversive consequences, and relapse to palatable food-seeking in response to anxiogenic-like stimuli. The neurocircuitry identified to date in the "dark" side of food addiction qualitatively resembles that associated with drug and alcohol dependence. The present review summarizes Bart Hoebel's groundbreaking conceptual and empirical contributions to understanding the role of the "dark side" in food addiction along with related work of those that have followed him.

Sigma-1 receptor knockout mice display a depressive-like phenotype.

Activation of sigma-1 receptors (Sig-1R) reportedly has antidepressant-like action. Limited data suggest that Sig-1Rs also modulate anxiety-related behaviors. The present experiments measured depressive-like, anxiety-like and motor behavior in Sig-1R knockout mice and their wildtype littermates. Sig-1R knockout mutants showed increased immobility in the forced swimming test, a depressive-like phenotype, but normal anxiety-like behavior in the elevated plus-maze and light/dark box tests and normal locomotor activity. The results further suggest that Sig-1Rs inversely modulate depressive-like behavior.

Gonadal steroids mediate the opposite changes in cocaine-induced locomotion across adolescence in male and female rats.

Evidence from both human studies and animal models indicates that cocaine elicits more behavioral stimulation in females than males. The present study sought to determine whether sex-specific responses to cocaine emerge during adolescence and to determine if gonadal steroid action during puberty affects adult responsiveness to cocaine. We administered cocaine using an escalating dose model in male and female rats at ages postnatal (PN) 28, 42, and 65 days. To assess the effects of pubertal gonadal steroid action, we compared the effects of binge cocaine administration on intact and prepubertally gonadectomized male and female rats in adulthood. Cocaine responses changed in opposite directions in males and females as they progressed through adolescence. At most doses, adolescent males were more responsive than adult males whereas adult females were more responsive than adolescent females. Ambulatory activity was age-dependent in males whereas non-ambulatory activity was age-dependent in females. Prepubertal gonadectomy increased behavioral responsiveness to the highest dose of cocaine in males whereas it decreased behavioral responsiveness to lower doses of cocaine in females. We conclude that sex differences in behavioral responses to cocaine arise during adolescence from a concurrent decrease in male responsiveness and increase in female responsiveness. Our results suggest that gonadal steroids exert lasting and opposing effects on the sensitivity of males and females to psychostimulants during development.