Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells (OPCs). Electrically active neurons may influence OPC function and selectively instruct myelination of an active neural circuit. In this work, we use optogenetic stimulation of the premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural progenitor cells and OPCs, promotes oligodendrogenesis, and increases myelination within the deep layers of the premotor cortex and subcortical white matter. We further show that this neuronal activity–regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal malignancy of the childhood central nervous system, with a median overall survival of 9–11 months. We have previously shown that primary DIPG tissue contains numerous tumor-associated macrophages, and substantial work has demonstrated a significant pathological role for adult glioma-associated macrophages. However, work over the past decade has highlighted many molecular and genomic differences between pediatric and adult high-grade gliomas. Thus, we directly compared inflammatory characteristics of DIPG and adult glioblastoma (GBM). We found that the leukocyte (CD45+) compartment in primary DIPG tissue samples is predominantly composed of CD11b + macrophages, with very few CD3+ T-lymphocytes. In contrast, T-lymphocytes are more abundant in adult GBM tissue samples. RNA sequencing of macrophages isolated from primary tumor samples revealed that DIPG- and adult GBM-associated macrophages both express gene programs related to ECM remodeling and angiogenesis, but DIPG-associated macrophages express substantially fewer inflammatory factors than their adult GBM counterparts. Examining the secretome of glioma cells, we found that patient-derived DIPG cell cultures secrete markedly fewer cytokines and chemokines than patient-derived adult GBM cultures. Concordantly, bulk and single-cell RNA sequencing data indicates low to absent expression of chemokines and cytokines in DIPG. Together, these observations suggest that the inflammatory milieu of the DIPG tumor microenvironment is fundamentally different than adult GBM. The low intrinsic inflammatory signature of DIPG cells may contribute to the lack of lymphocytes and non-inflammatory phenotype of DIPG-associated microglia/macrophages. Understanding the glioma subtype-specific inflammatory milieu may inform the design and application of immunotherapy-based treatments.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0553-x) contains supplementary material, which is available to authorized users.

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi–histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.

Chronic restraint stress produces morphological changes in medial prefrontal cortex and disrupts a prefrontally mediated behavior, retrieval of extinction. To assess potential physiological correlates of these alterations, we compared neural activity in infralimbic and prelimbic cortex of unstressed versus stressed rats during fear conditioning and extinction. After implantation of microwire bundles into infralimbic or prelimbic cortex, rats were either unstressed or stressed via placement in a plastic restrainer (3 h/day for 1 week). Rats then underwent fear conditioning and extinction while activity of neurons in infralimbic or prelimbic cortex was recorded. Percent freezing and neural activity were assessed during all phases of training. Chronic stress enhanced freezing during acquisition of conditioned fear, and altered both prelimbic and infralimbic activity during this phase. Stress did not alter initial extinction or conditioned stimulus (CS)-related activity during this phase. However, stress impaired retrieval of extinction assessed 24 h later, and this was accompanied by alterations in neuronal activity in both prelimbic and infralimbic cortex. In prelimbic cortex, unstressed rats showed decreased activity in response to CS presentation, whereas stressed rats showed no change. In infralimbic cortex, neurons in unstressed rats exhibited increased firing in response to the CS, whereas stressed rats showed no increase in infralimbic firing during the tone. Finally, CS-related firing in infralimbic but not prelimbic cortex was correlated with extinction retrieval. Thus, the stress-induced alteration of neuronal activity in infralimbic cortex may be responsible for the stress-induced deficit in retrieval of extinction.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

The upcoming transition of the United States Medical Licensing Examination Step 1 to pass/fail score reporting has wide-ranging implications for the medical education community. The decision to discontinue 3-digit numeric score reporting comes following advocacy to change the exam because of its disproportionate importance in the residency match process, the negative impact the exam has on student well-being, and the prominent influence the exam has on medical school curricula. Shifting to pass/fail score reporting for Step 1 creates significant uncertainties to be addressed by the stakeholders in the transition from undergraduate medical education to graduate medical education. In this Invited Commentary, the authors reflect on the positive implications of this scoring change, potential negative ramifications that need to be proactively addressed, and future ways that the transition to residency can be optimized. The transition to pass/fail score reporting for Step 1 creates an opportunity to redefine the residency match process, to support applicant decision making, and to encourage a holistic review of applicants. These changes to the review process will require grace and trust among stakeholders, including students, as well as active support for students who might be negatively impacted during a complex implementation phase. By removing the dominance of Step 1 numeric scores from the residency selection process, the change to pass/fail scoring provides a unique opportunity for all stakeholders to work together and redefine the transition to residency while protecting students from unintended negative consequences.

Both stress and dysfunction of prefrontal cortex are linked to psychological disorders, and structure and function of medial prefrontal cortex (mPFC) are altered by stress. Chronic restraint stress causes dendritic retraction in the prelimbic region (PL) of mPFC in rats. Dopamine release in mPFC increases during stress, and chronic administration of dopaminergic agonists results in dendritic remodeling. Thus, stress-induced alterations in dopaminergic transmission in PL may contribute to dendritic remodeling. We examined the effects of dopamine D1 receptor (D1R) blockade in PL during daily restraint stress on dendritic morphology in PL. Rats either underwent daily restraint stress (3 h/day, 10 days) or remained unstressed. In each group, rats received daily infusions of either the D1R antagonist SCH23390 or vehicle into PL prior to restraint; unstressed and stressed rats that had not undergone surgery were also examined. On the final day of restraint, rats were euthanized and brains were processed for Golgi histology. Pyramidal neurons in PL were reconstructed and dendritic morphology was quantified. Vehicle-infused stressed rats demonstrated dendritic retraction compared to unstressed rats, and D1R blockade in PL prevented this effect. Moreover, in unstressed rats, D1R blockade produced dendritic retraction. These effects were not due to attenuation of the HPA axis response to acute stress: plasma corticosterone levels in a separate group of rats that underwent acute restraint stress with or without D1R blockade were not significantly different. These findings indicate that dopaminergic transmission in mPFC during stress contributes directly to the stress-induced retraction of apical dendrites, while dopamine transmission in the absence of stress is important in maintaining normal dendritic morphology.

Previously, we showed that neonatal maternal separation impaired eyeblink conditioning in adult rats. This impairment is correlated with increased glucocorticoid receptor (GR) expression in the cerebellar posterior interpositus nucleus, a critical site of learning-related plasticity. To assess whether increased GR expression is responsible for the separation-induced learning impairment, we infused a GR antagonist (mifepristone) or vehicle into the posterior interpositus during eyeblink conditioning in adult male Long-Evans rats that had undergone control rearing or neonatal maternal separation (1 h/day, postnatal days 2-14). Rats received standard rearing (control) or neonatal maternal separation (separated; 1h/day on postnatal days 2-14). In adulthood, rats underwent surgery for implantation of recording electrodes in the obicularis oculi of the left eyelid, a bipolar stimulating electrode dorsocaudal to the left eye, and an infusion guide cannula positioned over the posterior interpositus. Then, rats underwent 10 daily sessions of eyeblink conditioning. Rats in each group received either 0.2 μl of mifepristone (2ng in 2% EtOH) or vehicle infusion prior to each eyeblink conditioning session. Mifepristone infusions improved conditioning in separated rats, but impaired control rats' performance. Thus, separation-induced increases in GRs may mediate the learning deficit seen in adult neonatally separated rats.