Preface: Special issue: 14th International Conference on Brain Energy Metabolism: Energy substrates and microbiome govern brain bioenergetics and cognitive function with aging.

This Preface introduces the Special Issue entitled, "Energy Substrates and Microbiome Govern Brain Bioenergetics and Cognitive Function with Aging", which is comprised of manuscripts contributed by invited speakers and program/organizing committee members who participated in the 14th International Conference on Brain Energy Metabolism (ICBEM) held on October 24-27, 2022 in Santa Fe, New Mexico, USA. The conference covered the latest developments in research related to neuronal energetics, emerging roles for glycogen in higher brain functions, the impact of dietary intervention on aging, memory, and Alzheimer's disease, roles of the microbiome in gut-brain signaling, astrocyte-neuron interactions related to cognition and memory, novel roles for mitochondria and their metabolites, and metabolic neuroimaging in aging and neurodegeneration. The special issue contains 25 manuscripts on these topics plus three tributes to outstanding scientists who have made important contributions to brain energy metabolism and participated in numerous ICBEM conferences. In addition, two of the manuscripts describe important directions and the rationale for future research in many thematic areas covered by the conference.

A tribute to Arne Schousboe's contributions to neurochemistry and his innovative and enduring research in GABA, glutamate, and brain energy metabolism.

This is a tribute to Arne Schousboe, Professor Emeritus at the University of Copenhagen, an eminent neurochemist and neuroscientist who was a leader in the fields of GABA, glutamate, and brain energy metabolism. Arne was known for his keen intellect, his wide-ranging expertise in neurochemistry and neuropharmacology of GABA and glutamate and brain energy metabolism. Arne was also known for his strong leadership, his warm and engaging personality and his enjoyment of fine wine and great food shared with friends, family, and colleagues. Sadly, Arne passed away on February 27, 2024, after a short illness. He is survived by his wife Inger Schousboe, his two children, and three wonderful grandchildren. His death is a tremendous loss to the neuroscience community. He will be greatly missed by his friends, family, and colleagues. Some of the highlights of Arne's career are described in this tribute.

Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

A tribute to John Edmond: His leadership role in founding the International Conference on Brain Energy Metabolism (ICBEM) meetings and key contributions to substrate metabolism in brain cells.

This is a tribute to John Edmond, professor emeritus of biological chemistry in the David Geffen School of Medicine at UCLA, a renowned neurochemist who had a leadership role in founding the ICBEM meeting series in 1993. John was known for his very warm and engaging personality and his innovative approaches to studying the developing brain and auditory system. He was a brilliant scientist and a fun and delightful person. Without John Edmond's enthusiasm and contributions, we would not have the biennial ICBEM meetings which as noted by Dienel et al. "have had a high impact on conceptual and experimental advances" … "in the energetics and metabolism underlying neural functions"… and "on promoting collaborative interactions among neuroscientists." Sadly, John Edmond passed away on February 18, 2022, following a cerebral hemorrhage. He will be greatly missed by his colleagues and friends.

A tribute to Leif Hertz: The historical context of his pioneering studies of the roles of astrocytes in brain energy metabolism, neurotransmission, cognitive functions, and pharmacology identifies important, unresolved topics for future studies.

Leif Hertz, M.D., D.Sc. (honoris causa) (1930-2018), was one of the original and noteworthy participants in the International Conference on Brain Energy Metabolism (ICBEM) series since its inception in 1993. The biennial ICBEM conferences are organized by neuroscientists interested in energetics and metabolism underlying neural functions; they have had a high impact on conceptual and experimental advances in these fields and on promoting collaborative interactions among neuroscientists. Leif made major contributions to ICBEM discussions and understanding of metabolic and signaling characteristics of astrocytes and their roles in brain function. His studies ranged from uptake of K+ from extracellular fluid and its stimulation of astrocytic respiration, identification, and regulation of enzymes specifically or preferentially expressed in astrocytes in the glutamate-glutamine cycle of excitatory neurotransmission, a requirement for astrocytic glycogenolysis for fueling K+ uptake, involvement of glycogen in memory consolidation in the chick, and pharmacology of astrocytes. This tribute to Leif Hertz highlights his major discoveries, the high impact of his work on astrocyte-neuron interactions, and his unparalleled influence on understanding the cellular basis of brain energy metabolism. His work over six decades has helped integrate the roles of astrocytes into neurotransmission where oxidative and glycogenolytic metabolism during neurotransmitter glutamate turnover are key aspects of astrocytic energetics. Leif recognized that brain astrocytic metabolism is greatly underestimated unless the volume fraction of astrocytes is taken into account. Adjustment for pathway rates expressed per gram tissue for volume fraction indicates that astrocytes have much higher oxidative rates than neurons and astrocytic glycogen concentrations and glycogenolytic rates during sensory stimulation in vivo are similar to those in resting and exercising muscle, respectively. These novel insights are typical of Leif's astute contributions to the energy metabolism field, and his publications have identified unresolved topics that provide the neuroscience community with challenges and opportunities for future research.

A tribute to Sebastián Cerdán and his key contributions to brain metabolism.

This is a tribute to Sebastián Cerdán, a brilliant and innovative NMR spectroscopist whose studies contributed greatly to the fundamental information to the understanding of brain metabolism, particularly in regard to multinuclear magnetic resonance spectroscopy (MRS) techniques. Sebastián Cerdán sadly passed away in May 2022. He was a wonderful mentor and colleague who will be greatly missed.

Targeting glycans for CAR therapy: The advent of sweet CARs.

Chimeric antigen receptor (CAR) T cell therapy has created a paradigm shift in the treatment of hematologic malignancies but has not been as effective toward solid tumors. For such tumors, the primary obstacles facing CAR T cells are scarcity of tumor-specific antigens and the hostile and complex tumor microenvironment. Glycosylation, the process by which sugars are post-translationally added to proteins or lipids, is profoundly dysregulated in cancer. Abnormally glycosylated glycoproteins expressed on cancer cells offer unique targets for CAR T therapy as they are specific to tumor cells. Tumor stromal cells also express abnormal glycoproteins and thus also have the potential to be targeted by glycan-binding CAR T cells. This review will discuss the state of CAR T cells in the therapy of solid tumors, the cancer glycoproteome and its potential for use as a therapeutic target, and the landscape and future of glycan-binding CAR T cell therapy.

Combinatorial antigen targeting strategies for acute leukemia: application in myeloid malignancy.

BACKGROUND AIMS: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse. METHODS: For co-transduction with the authors' previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens. RESULTS: The in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors. CONCLUSIONS: Overall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.

Clinical CAR-T Cell and Oncolytic Virotherapy for Cancer Treatment.

Immunotherapy has recently garnered success with the induction of clinical responses in tumors, which are traditionally associated with poor outcomes. Chimeric antigen receptor T (CAR-T) cells and oncolytic viruses (OVs) have emerged as promising cancer immunotherapy agents. Herein, we provide an overview of the current clinical status of CAR-T cell and OV therapies. While preclinical studies have demonstrated curative potential, the benefit of CAR-T cells and OVs as single-agent treatments remains limited to a subset of patients. Combinations of different targeted therapies may be required to achieve efficient, durable responses against heterogeneous tumors, as well as the microenvironment. Using a combinatorial approach to take advantage of the unique features of CAR-T cells and OVs with other treatments can produce additive therapeutic effects. This review also discusses ongoing clinical evaluations of these combination strategies for improved outcomes in treatment of resistant malignancies.

Mesenchymal stromal cell delivery of oncolytic immunotherapy improves CAR-T cell antitumor activity.

The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids in vitro and suppresses tumor growth in two orthotopic lung cancer models in vivo. Treatment with CAd MSCs increases the overall numbers of human T cells in vivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.

Choline Improves Neonatal Hypoxia-Ischemia Induced Changes in Male but Not Female Rats.

Choline is an essential nutrient with many roles in brain development and function. Supplementation of choline in early development can have long-lasting benefits. Our experiments aimed to determine the efficacy of choline supplementation in a postnatal day (PND) 10 rat model of neonatal hypoxia ischemia (HI) at term using both male and female rat pups. Choline (100 mg/kg) or saline administration was initiated the day after birth and given daily for 10 or 14 consecutive days. We determined choline's effects on neurite outgrowth of sex-specific cultured cerebellar granule cells after HI with and without choline. The magnitude of tissue loss in the cerebrum was determined at 72 h after HI and in adult rats. The efficacy of choline supplementation in improving motor ability and learning, tested using eyeblink conditioning, were assessed in young adult male and female rats. Overall, we find that choline improves neurite outgrowth, short-term histological measures and learning ability in males. Surprisingly, choline did not benefit females, and appears to exacerbate HI-induced changes.

Metabolism of [1,6-13 C]glucose in the cerebellum of 18-day-old rats: Comparison with cerebral metabolism.

There is little information on metabolism in developing cerebellum despite the known importance of this region in cognition and motor tasks. Ex vivo 1 H- and 13 C-NMR spectroscopy were used to determine metabolism during late postnatal development in cerebellum and cerebrum from 18-day-old rat pups after intraperitoneal (i.p.) injection of [1,6-13 C]glucose. The concentration of several metabolites in cerebellum was distinctly different than cerebrum; alanine, glutamine, creatine and myo-inositol were higher in cerebellum than cerebrum, the concentrations of lactate, GABA, aspartate and N-acetylaspartate (NAA) were lower in cerebellum than in cerebrum, and levels of glutamate, succinate, choline and taurine were similar in both brain regions. The incorporation of label from the metabolism of [1,6-13 C]glucose into most isotopomers of glutamate (GLU), glutamine (GLN), GABA and aspartate was lower in cerebellum than in cerebrum. Incorporation of label into the C2 position of lactate via the pyruvate recycling pathway was found in both brain regions. The ratio of newly synthesized GLN/GLU was significantly higher in cerebellum than in cerebrum indicating relatively active metabolism via glutamine synthetase in cerebellar astrocytes at postnatal day 18. This is the first study to determine metabolism in the cerebellum and cerebrum of male and female rat brain.

Regulation of erythrocyte Na+/K+/2Cl- cotransport by an oxygen-switched kinase cascade.

Many erythrocyte processes and pathways, including glycolysis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na+/K+-ATPase activity, ankyrin-band 3 interactions, and nitric oxide (NO) release, are regulated by changes in O2 pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O2 dependence of glycolysis, PPP, and ankyrin-band 3 interactions (affecting RBC rheology) are controlled by O2-dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O2 dependence of Na+/K+/2Cl- cotransport (catalyzed by Na+/K+/2Cl- cotransporter 1 [NKCC1]) might similarly originate from competition between deoxyHb and a protein involved in NKCC1 regulation for a common binding site on band 3. Using three transgenic mouse strains having mutated deoxyhemoglobin-binding sites on band 3, we found that docking of deoxyhemoglobin at the N terminus of band 3 displaces the protein with no lysine kinase 1 (WNK1) from its overlapping binding site on band 3. This displacement enabled WNK1 to phosphorylate oxidative stress-responsive kinase 1 (OSR1), which, in turn, phosphorylated and activated NKCC1. Under normal solution conditions, the NKCC1 activation increased RBC volume and thereby induced changes in RBC rheology. Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O2 regulation pathway may also occur in the regulation of other O2-regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O2 modulation of ion transporters in erythrocytes.

Focal Cerebral Arteriopathy in Young Adult Patients With Stroke.

Background and Purpose- Focal cerebral arteriopathy is monophasic inflammatory stenosis of the distal internal carotid artery or the proximal segment of the middle cerebral artery. It is one of the most common causes of acute arterial ischemic stroke in young children but is a less familiar entity for adult neurologists. Methods- We retrospectively reviewed stroke service radiology records at a tertiary referral center from January 2013 to December 2014. Focal cerebral arteriopathy was defined as nonprogressive unifocal and unilateral stenosis/irregularity of the distal internal carotid artery or its proximal branches. Only patients aged 16 to 55 years with stroke were included. Results- There were 5 cases of focal cerebral arteriopathy: 2 males and 3 females. Three cases were from the cohort of 123 acute presentations of young stroke, and 2 cases were outpatient referrals. The mean age (range) was 43 (32-55) years. The majority presented with recurrent transient ischemic attacks/minor strokes within a single vascular territory over days to weeks. All cases had characteristic radiological features. Interval imaging demonstrated resolution in 1 case and improvement in 3 cases. Functional outcome was excellent with discharge modified Rankin Scale score ranging from 0 to 1. Recurrence occurred in 1 case. Conclusions- Focal cerebral arteriopathy is a rare cause of arterial ischemic stroke in young adults. Follow-up intracranial imaging is essential to differentiate from progressive arteriopathies. Evidence-based treatment warrants further investigation. Prognosis is favorable.

Novel role of prostate apoptosis response-4 tumor suppressor in B-cell chronic lymphocytic leukemia.

Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is downregulated in many cancers including renal cell carcinoma, glioblastoma, endometrial, and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL-derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1-to-S cell-cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eµ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with US Food and Drug Administration (FDA)-approved drugs caused a decrease in Par-4 messenger RNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase, and Bruton tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel progrowth rather than proapoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR-signaling inhibitors.

Chronic Lymphocytic Leukemia-Derived IL-10 Suppresses Antitumor Immunity.

Chronic lymphocytic leukemia (CLL) patients progressively develop an immunosuppressive state. CLL patients have more plasma IL-10, an anti-inflammatory cytokine, than healthy controls. In vitro human CLL cells produce IL-10 in response to BCR cross-linking. We used the transgenic Eµ-T cell leukemia oncogene-1 (TCL1) mouse CLL model to study the role of IL-10 in CLL associated immunosuppression. Eµ-TCL mice spontaneously develop CLL because of a B cell-specific expression of the oncogene, TCL1. Eµ-TCL1 mouse CLL cells constitutively produce IL-10, which is further enhanced by BCR cross-linking, CLL-derived IL-10 did not directly affect survival of murine or human CLL cells in vitro. We tested the hypothesis that the CLL-derived IL-10 has a critical role in CLL disease in part by suppressing the host immune response to the CLL cells. In IL-10R-/- mice, wherein the host immune cells are unresponsive to IL-10-mediated suppressive effects, there was a significant reduction in CLL cell growth compared with wild type mice. IL-10 reduced the generation of effector CD4 and CD8 T cells. We also found that activation of BCR signaling regulated the production of IL-10 by both murine and human CLL cells. We identified the transcription factor, Sp1, as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway. Our results suggest that incorporation of IL-10 blocking agents may enhance current therapeutic regimens for CLL by potentiating host antitumor immune response.

Patients' Experiences of Remote Neurology Consultations during the COVID-19 Pandemic.

Telemedicine has been widely implemented during the COVID-19 global pandemic to enable continuity of care of chronic illnesses. We modified our general neurology clinic to be conducted using remote audio-only telephone consultations. We included all patients over a 10-week period who agreed to both a telephone consultation and a questionnaire afterwards in order to ascertain the patient's perspective of the experience. There were 212 participants consisting of men (43.8%) and women (56.2%). The mean ± standard deviation of age was 47.8 ± 17.0 (range 17-93) years. For the most part, patients found remote consultations either "just as good" (67.1%) or "better" (9.0%) than face-to-face consultations. Those who deemed it to be "not as good" were significantly older (52.3 ± 17.9 years vs. 46.6 ± 16.6 years, p =0.045) or were more likely to have a neurological disorder that required clinical examination, namely, a neuromuscular condition (66.7%, p = 0.002) or an undiagnosed condition (46.7%, p = 0.031). At the height of the COVID-19 global pandemic, most patients were satisfied with remote consultations. The positive feedback for remote consultations needs to be verified outside of this unique scenario because the results were likely influenced by the patients' apprehension to attend the hospital amongst other factors.

Fundamentals of CNS energy metabolism and alterations in lysosomal storage diseases.

The brain has a very high requirement for energy. Adult brain relies on glucose as an energy substrate, whereas developing brain can utilize alternative substrates as well as glucose for energy and for the biosynthesis of lipids and proteins required for brain development. Metabolism provides the energy required to support all cellular functions and brain development and building blocks for macromolecules. Lysosomes are organelles involved in breakdown of biological compounds including proteins and complex lipids in the body and brain. Recent studies suggest that lysosomal dysfunction can damage neurons and/or alter neurotransmitter homeostasis. Several studies also implicate mitochondrial dysfunction in the pathophysiology of brain damage in lysosomal storage diseases. This manuscript provides a brief review of energy metabolism and the key pathways involved in metabolism in brain. Roles of lysosomes related to metabolism and neurotransmission are discussed, and evidence for mitochondrial dysfunction in several lysosomal storage diseases is presented. This article is part of the Special Issue "Lysosomal Storage Disorders".

A Practical Guide to Gynecologic and Reproductive Health in Women Undergoing Hematopoietic Stem Cell Transplant.

Optimum care of female transplant recipients requires gynecologic care at several stages through the allogeneic hematopoietic stem cell transplantation (HCT) process. Sex-based considerations in women post-HCT span gynecologic sequelae of transplant along with assessment and maintenance of optimal sexual and gynecologic health. Pre-HCT, managing menstruation and abnormal uterine or genital bleeding, considering fertility preservation, and assessing for sexually transmitted infections, including human papillomavirus (HPV)-related disease and cervical cancer, enhance women's health. While inpatient during transplant when women are thrombocytopenic, menstrual bleeding requires suppression. Whenever graft-versus-host disease (GVHD) is assessed, screening for genital GVHD merits consideration. After the first 100 days, periodic assessments include obtaining a menstrual history, assessing ovarian function, and reviewing current hormonal use and contraindications to hormonal methods. Regular assessment for primary ovarian insufficiency, dyspareunia, and intimacy guides provision of contraception and hormone replacement options. As part of ongoing screening for genital GVHD and HPV-related disease, including sexually transmitted infections, periodic pelvic examinations are performed. Once successful long-term survival is achieved, planning for fertility may be considered. This article offers a comprehensive approach to these aspects of gynecologic care of patients throughout the trajectory of HCT and beyond into survivorship. We review the effects of HCT treatment on sexual health, ovarian function, and resulting menstrual changes and fertility challenges. Identification, treatment, and prevention of subsequent malignancies, including breast cancer, are discussed, with a focus on regular assessment of genital HPV disease and GVHD in long-term follow-up.

White Matter Alterations in Fmr1 Knockout Mice during Early Postnatal Brain Development.

Fragile X syndrome (FXS) is the most commonly inherited form of intellectual disability ascribed to the autism spectrum disorder. Studies with FXS patients have reported altered white matter volume compared to controls. The Fmr1 knockout (KO) mouse, a model for FXS, showed evidence of delayed myelination during postnatal brain development. In this study, we examined several white matter regions in the male Fmr1 KO mouse brain compared to male wild-type (WT) mice at postnatal days (PND) 18, 21, 30, and 60, which coincide with critical stages of myelination and postnatal brain development. White matter volume, T2 relaxation time, and magnetization transfer ratio (MTR) were measured using magnetic resonance imaging and myelin content was determined with histological staining of myelin. Differences in the developmental accumulation of white matter and myelin between Fmr1 KO and WT mice were observed in the corpus callosum, external and internal capsules, cerebral peduncle, and fimbria. Alterations were more predominant in the external and internal capsules and fimbria of Fmr1 KO mice, where the MTR was lower at PND 18, then elevated at PND 30, and again lower at PND 60 compared to the corresponding regions in WT mice. The pattern of changes in MTR were similar to those observed in myelin staining and could be related to the altered protein synthesis that is a hallmark of FXS. While no significant changes in white matter volumes and T2 relaxation time between the Fmr1 KO and WT mice were observed, the altered pattern of myelin staining and MTR, particularly in the external capsule, reflecting the abnormalities associated with myelin content is suggestive of a developmental delay in the white matter of Fmr1 KO mouse brain. These early differences in white matter during critical developmental stages may contribute to altered brain networks in the Fmr1 KO mice.