Astrocytes close a motor circuit critical period.

Critical periods-brief intervals during which neural circuits can be modified by activity-are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood1,2. Here we define a critical period in a developing Drosophila motor circuit and identify astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure3, and astrocyte ablation prolonged the critical period. Finally, we used a genetic screen to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses4; we show here that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour.

A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity.

Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system (CNS), where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single-cell RNA sequencing data of Drosophila glia in 2 well-characterised tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila, and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.

Peripheral nerve development in zebrafish requires muscle patterning by tcf15/paraxis.

The vertebrate peripheral nervous system (PNS) is an intricate network that conveys sensory and motor information throughout the body. During development, extracellular cues direct the migration of axons and glia through peripheral tissues. Currently, the suite of molecules that govern PNS axon-glial patterning is incompletely understood. To elucidate factors that are critical for peripheral nerve development, we characterized the novel zebrafish mutant, stl159, that exhibits abnormalities in PNS patterning. In these mutants, motor and sensory nerves that develop adjacent to axial muscle fail to extend normally, and neuromasts in the posterior lateral line system, as well as neural crest-derived melanocytes, are incorrectly positioned. The stl159 genetic lesion lies in the basic helix-loop-helix (bHLH) transcription factor tcf15, which has been previously implicated in proper development of axial muscles. We find that targeted loss of tcf15 via CRISPR-Cas9 genome editing results in the PNS patterning abnormalities observed in stl159 mutants. Because tcf15 is expressed in developing muscle prior to nerve extension, rather than in neurons or glia, we predict that tcf15 non-cell-autonomously promotes peripheral nerve patterning in zebrafish through regulation of extracellular patterning cues. Our work underscores the importance of muscle-derived factors in PNS development.

Postembryonic screen for mutations affecting spine development in zebrafish.

The spine gives structural support for the adult body, protects the spinal cord, and provides muscle attachment for moving through the environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant zebrafish mutations, leading to overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1229 F1 animals, and subsequently bred to homozygosity in F3 families; from these, 314 haploid genomes were screened for adult-viable recessive phenotypes affecting general body shape. We cumulatively found 40 adult-viable (3 dominant and 37 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing of 16 mutant lines within this phenotypic group revealed at least 9 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report multiple recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Our results provide evidence of monogenic traits that are essential for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo.

Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mbp mRNA transport in zebrafish, it is not entirely clear how mbp transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in actr10, which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the actr10 mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mbp mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mbp mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged Mbp mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests Mbp mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates Mbp mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mbp mRNA transport and myelination in vivo.

A Longitudinal Examination of Decisions to Ride and Decline Rides with Drinking Drivers.

BACKGROUND: Riding with a drinking driver (RWDD) is a serious concern that leads to numerous preventable deaths every year. There is a significant gap in research on empirically tested predictors of RWDD that could be implemented in prevention efforts. College students are in need of such prevention efforts, as they have some of the highest rates of alcohol-related crash fatalities and may engage in RWDD more than their noncollege peers. This study utilized behavioral decision-making approach to examine predictors of RWDD and declining a ride from a drinking driver (Decline) in older college students. METHODS: Students (n = 791) in their third year of college were enrolled from 3 large and diverse universities. Psychosocial (e.g., expectancies, norms) and decision-making variables (willingness to RWDD and intentions to use alternatives) were assessed in the fall of their third year. One year later, RWDD and Decline behaviors were assessed. Zero-inflated Poisson analyses were used to assess how decision-making variables predicted RWDD and Decline behavior. Associations between psychosocial and decision-making variables were also assessed. RESULTS: Thirteen percent of students reported RWDD and ~28% reported Decline behavior. Willingness to RWDD and typical weekly drinking were both associated with increases in RWDD (odds ratio [OR] = 1.58 and 1.40, respectively), whereas intentions to use alternatives, sex, and ethnicity were not associated with RWDD. Only weekly drinking was associated with Decline, with an increase in drinking associated with increased Decline (OR = 1.48). All psychosocial variables were significantly associated with the decision-making variables except positive expectancies. CONCLUSIONS: Results provide evidence that willingness to RWDD is a predictor of future RWDD, even if students intend to use safe alternatives. Future research is needed to better understand decision-making factors that influence Decline. Results also suggest prevention and interventions efforts, such as brief motivational intervention, Parent-Based Interventions, and normative feedback interventions could be adapted to reduce RWDD.

Who Will Experience the Most Alcohol Problems in College? The Roles of Middle and High School Drinking Tendencies.

BACKGROUND: Previous work examining college drinking tendencies has identified a disproportionately small (20%), but uniquely high-risk group of students who experience nearly 50% of the reported alcohol-related consequences (i.e., the multiple repeated consequences, or MRC, group). With the goal of reducing drinking-related consequences later in college, this study sought to identify potential MRC group members in their first semester by examining: (i) early-risk subgroups based on analysis of early-risk screening constructs (e.g., age of drinking onset, middle school alcohol exposure, high school drinking, and consequences); and (ii) their association with MRC criteria early in the first semester of college. METHODS: A random sample of 2,021 first-year college student drinkers (56% female) completed a web-based drinking survey in their first semester on campus. RESULTS: Latent class analysis revealed 4 early-risk subgroups: (i) an early-onset risk group who endorsed early age of drinking onset and engaged in heavy middle and high school drinking (10%); (ii) a late-onset risk group who engaged in weekend drinking and drunkenness and experienced 6 or more unique consequences as seniors in high school (32%); (iii) an early-onset limited risk group who only endorsed early age of onset and middle school drinking (3%); and (iv) a minimal risk group who did not engage in any early-risk behaviors (55%). Members of both the early- and late-onset risk groups had significantly higher odds of MRC membership in their first semester of college (9.85 and 6.79 greater, respectively). CONCLUSIONS: Results suggest age of onset, middle and high school drinking and drunkenness, and frequency of unique consequences could be particularly useful in brief screening tools. Further, findings support early screening and prevention efforts for MRC membership prior to college matriculation.

OptoChamber: A Low-cost, Easy-to-Make, Customizable, and Multi-Chambered Electronic Device for Applying Optogenetic Stimulation to Larval Drosophila melanogaster.

Optogenetics is a powerful tool used to manipulate physiological processes in animals through cell-specific expression of genetically modified channelrhodopsins. In Drosophila melanogaster, optogenetics is frequently used for temporal control of neuronal activation or silencing through light-dependent actuation of cation and anion channelrhodopsins, respectively. The high setup costs and complexity associated with commercially available optogenetic systems prevents many investigators from exploring the use of this technology. We developed a low-cost, customizable, and easy-to-make optogenetics chamber (OptoChamber) and verified its functionality in a robust cellular assay: activity-dependent remodeling of larval motor neurons in Drosophila embryos.

Glial Regulation of Circuit Wiring, Firing, and Expiring in the Drosophila Central Nervous System.

Molecular genetic approaches in small model organisms like Drosophila have helped to elucidate fundamental principles of neuronal cell biology. Much less is understood about glial cells, although interest in using invertebrate preparations to define their in vivo functions has increased significantly in recent years. This review focuses on our current understanding of the three major neuron-associated glial cell types found in the Drosophila central nervous system (CNS)-astrocytes, cortex glia, and ensheathing glia. Together, these cells act like mammalian astrocytes and microglia; they associate closely with neurons including surrounding neuronal cell bodies and proximal neurites, regulate synapses, and engulf neuronal debris. Exciting recent work has shown critical roles for these CNS glial cells in neural circuit formation, function, plasticity, and pathology. As we gain a more firm molecular and cellular understanding of how Drosophila CNS glial cells interact with neurons, it is clear that they share significant molecular and functional attributes with mammalian glia and will serve as an excellent platform for mechanistic studies of glial function.

Transdermal alcohol concentration features predict alcohol-induced blackouts in college students.

BACKGROUND: Alcohol-induced blackouts (AIBs) are common in college students. Individuals with AIBs also experience acute and chronic alcohol-related consequences. Research suggests that how students drink is an important predictor of AIBs. We used transdermal alcohol concentration (TAC) sensors to measure biomarkers of increasing alcohol intoxication (rise rate, peak, and rise duration) in a sample of college students. We hypothesized that the TAC biomarkers would be positively associated with AIBs. METHODS: Students were eligible to participate if they were aged 18-22 years, in their second or third year of college, reported drinking 4+ drinks on a typical Friday or Saturday, experienced ≥1 AIB in the past semester, owned an iPhone, and were willing to wear a sensor for 3 days each weekend. Students (N = 79, 55.7% female, 86.1% White, Mage = 20.1) wore TAC sensors and completed daily diaries over four consecutive weekends (89.9% completion rate). AIBs were assessed using the Alcohol-Induced Blackout Measure-2. Logistic multilevel models were conducted to test for main effects. RESULTS: Days with faster TAC rise rates (OR = 2.69, 95% CI: 1.56, 5.90), higher peak TACs (OR = 2.93, 95% CI: 1.64, 7.11), and longer rise TAC durations (OR = 4.16, 95% CI: 2.08, 10.62) were associated with greater odds of experiencing an AIB. CONCLUSIONS: In a sample of "risky" drinking college students, three TAC drinking features identified as being related to rising intoxication independently predicted the risk for daily AIBs. Our findings suggest that considering how an individual drinks (assessed using TAC biomarkers), rather than quantity alone, is important for assessing risk and has implications for efforts to reduce risk. Not only is speed of intoxication important for predicting AIBs, but the height of the peak intoxication and the time spent reaching the peak are important predictors, each with different implications for prevention.

Freud's Red Thread: Explorations of the Unconscious Sense of Guilt.

The concept of an "unconscious sense of guilt" bedevils Freud throughout his life, rearing its head in at least twenty-four of his major works and working behind the scenes in many others. In a sense, we can see Freud's oeuvre, and psychoanalysis more generally, as a discourse of unconscious guilt. While Freud frames the oedipus complex as the central defining dynamic of human experience, the unconscious sense of guilt is arguably the underbelly that both precedes and exceeds that complex. By unraveling a range of complexities within Freud's conceptualization of unconscious guilt, we will come to see that guilt is an unavoidable by-product of the human condition, intrinsically interwoven with libidinal desire.

Neuronal cadherins: The keys that unlock layer-specific astrocyte identity?

An astrocyte's intricate morphology is essential for proper brain function, but the intrinsic and extrinsic cues that set astrocyte morphology are largely unknown. In this issue, Tan et al. (https://doi.org/10.1083/jcb.202303138) show that layer-specific expression of neuronal cadherins locally regulates astrocyte morphogenesis and heterogeneity.

Glial regulation of critical period plasticity.

Animal behavior, from simple to complex, is dependent on the faithful wiring of neurons into functional neural circuits. Neural circuits undergo dramatic experience-dependent remodeling during brief developmental windows called critical periods. Environmental experience during critical periods of plasticity produces sustained changes to circuit function and behavior. Precocious critical period closure is linked to autism spectrum disorders, whereas extended synaptic remodeling is thought to underlie circuit dysfunction in schizophrenia. Thus, resolving the mechanisms that instruct critical period timing is important to our understanding of neurodevelopmental disorders. Control of critical period timing is modulated by neuron-intrinsic cues, yet recent data suggest that some determinants are derived from neighboring glial cells (astrocytes, microglia, and oligodendrocytes). As glia make up 50% of the human brain, understanding how these diverse cells communicate with neurons and with each other to sculpt neural plasticity, especially during specialized critical periods, is essential to our fundamental understanding of circuit development and maintenance.

Dose-Rate Effects of Protons and Light Ions for DNA Damage Induction, Survival and Transformation in Apparently Normal Primary Human Fibroblasts.

We report on effects of low-dose exposures of accelerated protons delivered at high-dose rate (HDR) or a simulated solar-particle event (SPE) like low-dose rate (LDR) on immediate DNA damage induction and processing, survival and in vitro transformation of low passage NFF28 apparently normal primary human fibroblasts. Cultures were exposed to 50, 100 and 1,000 MeV monoenergetic protons in the Bragg entrance/plateau region and cesium-137 γ rays at 20 Gy/h (HDR) or 1 Gy/h (LDR). DNA double-strand breaks (DSB) and clustered DNA damages (containing oxypurines and abasic sites) were measured using transverse alternating gel electrophoresis (TAFE) and immunocytochemical detection/scoring of colocalized γ-H2AX pS139/53BP1 foci, with their induction being linear energy transfer (LET) dependent and dose-rate sparing observed for the different damage classes. Relative biological effectiveness (RBE) values for cell survival after proton irradiation at both dose-rates ranged from 0.61-0.73. Transformation RBE values were dose-rate dependent, ranging from ∼1.8-3.1 and ∼0.6-1.0 at low doses (≤30 cGy) for HDR and LDR irradiations, respectively. However peak transformation frequencies were significantly higher (1.3-7.3-fold) for higher doses of 0.5-1 Gy delivered at SPE-like LDR. Cell survival and transformation frequencies measured after low-dose 500 MeV/n He-4, 290 MeV/n C-12 and 600 MeV/n Si-28 ion irradiations also showed an inverse dose-rate effect for transformation at SPE-like LDR. This work demonstrates the existence of inverse dose-rate effects for proton and light-ion-induced postirradiation cell survival and in vitro transformation for space mission-relevant doses and dose rates.

What Predicts Willingness to Experience Negative Consequences in College Student Drinkers?

OBJECTIVE: Research has shown that students who were more willing to experience consequences reported higher rates of alcohol consumption and negative consequences. The present study used a longitudinal design to examine intra- and interpersonal consequence-specific predictors of willingness to experience negative consequences. METHOD: Students (N = 2,024) were assessed in the fall (Time [T] 1) and spring (T2) semesters of their first year in college. Intrapersonal constructs (i.e., expectancies, subjective evaluations, self-efficacy), interpersonal constructs (i.e., peer descriptive, injunctive norms), and personality constructs (i.e., self-regulation, impulsivity, sensation seeking) were assessed at T1 and willingness to experience negative alcohol-related consequences was assessed 6 months later. A structural path model examined the relationship between T1 predictors and T2 willingness. T1 drinking and sex were included as covariates. RESULTS: These results demonstrated significant positive relationships between T1 participants' subjective evaluations of consequences, expectancies of experiencing consequences, and T2 willingness to experience negative consequences. Further, impulsivity, sensation seeking, and T1 drinking showed significant, positive associations with willingness, whereas higher self-regulation was significantly associated with lower willingness. Men were significantly more willing to experience negative consequences than women. No significant associations were observed between normative perceptions and willingness. CONCLUSIONS: Intrapersonal and personality constructs, as well as previous drinking, were significantly associated with willingness to experience consequences whereas interpersonal constructs were not. Men were significantly more willing to experience negative consequences. College student interventions may benefit from focusing on significant constructs identified in the current study (e.g., enhancing self-regulation) and focusing on students with higher willingness to experience negative consequences.

The role of astrocyte-mediated plasticity in neural circuit development and function.

Neuronal networks are capable of undergoing rapid structural and functional changes called plasticity, which are essential for shaping circuit function during nervous system development. These changes range from short-term modifications on the order of milliseconds, to long-term rearrangement of neural architecture that could last for the lifetime of the organism. Neural plasticity is most prominent during development, yet also plays a critical role during memory formation, behavior, and disease. Therefore, it is essential to define and characterize the mechanisms underlying the onset, duration, and form of plasticity. Astrocytes, the most numerous glial cell type in the human nervous system, are integral elements of synapses and are components of a glial network that can coordinate neural activity at a circuit-wide level. Moreover, their arrival to the CNS during late embryogenesis correlates to the onset of sensory-evoked activity, making them an interesting target for circuit plasticity studies. Technological advancements in the last decade have uncovered astrocytes as prominent regulators of circuit assembly and function. Here, we provide a brief historical perspective on our understanding of astrocytes in the nervous system, and review the latest advances on the role of astroglia in regulating circuit plasticity and function during nervous system development and homeostasis.

Creatine-mediated crosstalk between adipocytes and cancer cells regulates obesity-driven breast cancer.

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.

Impossible Ethics.

The proper practice of psychoanalysis repudiates a rule-based code of ethical conduct. A conflict exists, however, between Freud's rejection of the Biblical commandment to love one's neighbor as oneself and his development of psychoanalytic techniques that demand something very much of this ilk. Other essential conflicts in analytic practice include the impossibility of removing the analyst's desire from the analytic relationship, the unruly nature of unconscious processes in both analyst and analysand, and the après-coup nature of ethical recognition. A discourse of ethics is recommended in which analysts are called on to consider the ethical demands of each clinical moment. Ethical demands on the analysand, as well as the analyst, bring to light the way in which analysis rests on the foundational ethical situation into which humankind is born.

A Diagnosis for Psychoanalysis in the 21st Century: Freud as Medicine.

This paper assesses the history of psychoanalysis in the United States in order to inform a "professional memoir" of the author's experience of analytic training in the 21st Century. The mix of historical and personal landscapes supports a contention that there is something missing or lost in American psychoanalysis, that psychoanalysis has lost sight of the radical and subversive nature of unconscious processes. I argue that only by returning to a study of rigorous and comprehensive theory, seated in Freud's work, can this absence be addressed.

More Than Mortar: Glia as Architects of Nervous System Development and Disease.

Glial cells are an essential component of the nervous system of vertebrates and invertebrates. In the human brain, glia are as numerous as neurons, yet the importance of glia to nearly every aspect of nervous system development has only been expounded over the last several decades. Glia are now known to regulate neural specification, synaptogenesis, synapse function, and even broad circuit function. Given their ubiquity, it is not surprising that the contribution of glia to neuronal disease pathogenesis is a growing area of research. In this review, we will summarize the accumulated evidence of glial participation in several distinct phases of nervous system development and organization-neural specification, circuit wiring, and circuit function. Finally, we will highlight how these early developmental roles of glia contribute to nervous system dysfunction in neurodevelopmental and neurodegenerative disorders.