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As the field of stem cell therapy advances, it is important to develop reliable methods to overcome host immune responses in animal models. This ensures survival of transplanted human stem cell grafts and enables predictive efficacy testing. Immunosuppressive drugs derived from clinical protocols are frequently used but are often inconsistent and associated with toxic side effects. Here, using a molecular imaging approach, we show that immunosuppression targeting costimulatory molecules CD4 and CD40L enables robust survival of human xenografts in mouse brain, as compared to conventional tacrolimus and mycophenolate mofetil.Human neural stem cells were modified to express green fluorescent protein and firefly luciferase. Cells were implanted in the fimbria fornix of the hippocampus and viability assessed by non-invasive bioluminescent imaging. Cell survival was assessed using traditional pharmacologic immunosuppression as compared to monoclonal antibodies directed against CD4 and CD40L. This paradigm was also implemented in a transgenic Alzheimer's disease mouse model.Graft rejection occurs within 7 days in non-immunosuppressed mice and within 14 days in mice on a traditional regimen. The addition of dual monoclonal antibody immunosuppression extends graft survival past 7 weeks (p.001) on initial studies. We confirm dual monoclonal antibody treatment is superior to either antibody alone (p.001). Finally, we demonstrate robust xenograft survival at multiple cell doses up to 6 months in both C57BL/6J mice and a transgenic Alzheimer's disease model (p.001). The dual monoclonal antibody protocol demonstrated no significant adverse effects, as determined by complete blood counts and toxicity screen.This study demonstrates an effective immunosuppression protocol for preclinical testing of stem cell therapies. A transition towards antibody-based strategies may be advantageous by enabling stem cell survival in preclinical studies that could inform future clinical trials.

Emotions are characterized not only by their valence but also by whether they are stable or labile. Yet, we do not understand the molecular or circuit mechanisms that control the dynamic nature of emotionality. We have shown that glucocorticoid receptor overexpression in the forebrain (GRov) leads to a highly reactive mouse with increased anxiety behavior coupled with greater swings in emotional responses. This phenotype is established early in development and persists into adulthood. However, the neural circuitry mediating this lifelong emotional lability remains unknown. In the present study, optogenetic stimulation in ventral dentate gyrus (vDG) of GRov mice led to a greater range and a prolonged duration of anxiety behavior. cFos expression showed that the amplified behavioral response to vDG activation in GRov mice is coupled to increased neuronal activity in specific brain regions. Relative to wild type mice, GRov mice displayed glutamatergic/GABAergic activation imbalance in ventral CA1 (vCA1) and selectively increased glutamatergic activation in the basal posterior amygdaloid complex. Fluorescence in situ hybridization chain reaction studies showed that forebrain GR overexpression led to increased activation of molecularly distinct subpopulations of neurons within the hippocampus and the posterior basolateral amygdala (pBLA). Increased cFos labeling was observed in the Calbindin 1 (Calbn1+) glutamatergic neurons in vCA1 and in the DARPP-32/Ppp1r1b+ glutamatergic neurons in pBLA. We propose that a molecularly distinct hippocampal-amygdala circuit is shaped by stress early in life and tunes the dynamics of emotional responses.

Background: In animal models and tissue preparations, calcium dyshomeostasis is a biomarker of aging and Alzheimer’s disease that is associated with synaptic dysfunction, neuritic pruning, and dysregulated cellular processes. It is unclear, however, whether the onset of calcium dysregulation precedes, is concurrent with, or is the product of pathological cellular events (e.g., oxidation, amyloid-β production, and neuroinflammation). Further, neuronal calcium dysregulation is not always present in animal models of amyloidogenesis, questioning its reliability as a disease biomarker. Objective: Here, we directly tested for the presence of calcium dysregulation in dorsal hippocampal neurons in male and female 5×FAD mice on a C57BL/6 genetic background using sharp electrodes coupled with Oregon-green Bapta-1 imaging. We focused on three ages that coincide with the course of amyloid deposition: 1.5, 4, and 10 months old. Methods: Outcome variables included measures of the afterhyperpolarization, short-term synaptic plasticity, and calcium kinetics during synaptic activation. Quantitative analyses of spatial learning and memory were also conducted using the Morris water maze. Main effects of sex, age, and genotype were identified on measures of electrophysiology and calcium imaging. Results: Measures of resting Oregon-green Bapta-1 fluorescence showed significant reductions in the 5×FAD group compared to controls. Deficits in spatial memory, along with increases in Aβ load, were detectable at older ages, allowing us to test for temporal associations with the onset of calcium dysregulation. Conclusion: Our results provide evidence that reduced, rather than elevated, neuronal calcium is identified in this 5×FAD model and suggests that this surprising result may be a novel biomarker of AD.

There is a paucity in the development of new mechanistic insights and therapeutic approaches for treating psychiatric disease. One of the major challenges is reflected in the growing consensus that risk for these diseases is not determined by a single gene, but rather is polygenic, arising from the action and interaction of multiple genes. Canonically, experimental models in mice have been designed to ascertain the relative contribution of a single gene to a disease by systematic manipulation (e.g., mutation or deletion) of a known candidate gene. Because these studies have been largely carried out using inbred isogenic mouse strains, in which there is no (or very little) genetic diversity among subjects, it is difficult to identify unique allelic variants, gene modifiers, and epigenetic factors that strongly affect the nature and severity of these diseases. Here, we review various methods that take advantage of existing genetic diversity or that increase genetic variance in mouse models to (1) strengthen conclusions of single-gene function; (2) model diversity among human populations; and (3) dissect complex phenotypes that arise from the actions of multiple genes.

The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies.SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.

Alzheimer’s disease (AD) is a neurodegenerative disease that affects cognition and memory. Mouse models such as 5xFAD, incorporate mutations found in familial or early-onset (EOAD) AD; these mutations increase the production of the toxic AB species that contributes to plaque formation in the brain. However, not all brain regions in the 5xAD animals accumulate plaques to the same degree. For example, the cerebellum appears to be resistant to AB plaque formation, while other regions such as the cortex and subiculum develop copious plaques. The mechanism(s) underlying this regional specificity remains unclear. Thus, we used a fluorescent antibody to APP/AB to quantify the amount of these proteins in several brain regions of interest, including subiculum, cortex, and cerebellum. We found that the cerebellum had less APP/AB than the other regions quantified, which demonstrates a correlation between the levels of APP/AB and plaque formation. Taken together, this suggests that the regional specificity of AD pathology may be a result of different levels of protein expression.

International audience; Background Voltage-gated calcium (Ca(v)1) channels contribute to T-lymphocyte activation. Ca(v)1.2 and Ca(v)1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. Methods We generated mice deleted for Ca(v)1.2 in T cells or Ca(v)1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Ca(v)1.2 or Ca(v)1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Ca(v)1.2 and Ca(v)1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. Results We demonstrated non-redundant and synergistic functions of Ca(v)1.2 and Ca(v)1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Ca(v)1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Ca(v)1.2 and Ca(v)1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Ca(v)1.2 and Ca(v)1.3 must be co-expressed within the same CD4(+) T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Ca(v)1.2 and Ca(v)1.3, the expression of both channels by activated CD4(+) T cells from asthmatic children was associated with increased Th2-cytokine transcription. Conclusions Thus, Ca(v)1.2 and Ca(v)1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.

Voltage-gated calcium (CaWe generated mice deleted for CaWe demonstrated non-redundant and synergistic functions of CaThus, Ca

Optimizing antiepileptic drug therapy is very challenging due to the absence of a reliable method to assess how brain activity changes between seizures. This work uses the Taxonomy of Seizure Dynamics (Saggio et al., 2020) to investigate how anticonvulsants influence seizure onset dynamotypes. The no Mg2+ /high K+ mouse brain-slice seizure model (N = 92) was used to generate consistent epileptiform onsets. We compared the onset bifurcations of controls with slices treated with either GABA or carbamazepine. Each anticonvulsant uniquely changed the types of bifurcations in the slices. This experiment provides proof-of-concept evidence that brain states exist on a “map” of seizure dynamics, and that antiepileptic drugs with different mechanisms can change the positioning of the brain states on the map.Impact statementAntiepileptic drugs modify underlying brain states and influence the pathway into seizure onset in brain slices.

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aβ fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aβ42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aβ antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aβ aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.

Acute physical or psychological stress can elicit adaptive behaviors that allow an organism maintain homeostasis. However, intense and/or prolonged stressors often have the opposite effect, resulting in maladaptive behaviors and curbing goal-directed action; in the extreme, this may contribute to the development of psychiatric conditions like generalized anxiety disorder, major depressive disorder, or post-traumatic stress disorder. While treatment of these disorders generally focuses on reducing reactivity to potentially threatening stimuli, there are in fact impairments across multiple domains including valence, arousal, and cognition. Here, we use the genetically stress-susceptible 129S1 mouse strain to explore the effects of stress across multiple domains. We find that 129S1 mice exhibit a potentiated neuroendocrine response across many environments and paradigms, and that this is associated with reduced exploration, neophobia, decreased novelty- and reward-seeking, and spatial learning and memory impairments. Taken together, our results suggest that the 129S1 strain may provide a useful model for elucidating mechanisms underlying myriad aspects of stress-linked psychiatric disorders as well as potential treatments that may ameliorate symptoms.

mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9-13) in the pseudokinase STE20-related kinase adaptor alpha (STRAD-α; STRADA), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited Strada mouse N2a cells, a germline mouse Strada knockout (KO-/-) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the STRADA founder mutation. Strada KO in vitro leads to enhanced mTOR signaling and iPSC-derived neurons from PS individuals exhibit enhanced cell size and mTOR signaling activation, as well as subtle alterations in electrical firing properties e.g., increased input resistance, a more depolarized resting membrane potential, and decreased threshold for action potential (AP) generation. Strada-/- mice exhibit high rates of perinatal mortality and out of more than 100 litters yielding both WT and heterozygous pups, only eight Strada-/- animals survived past P5. Strada-/- mice are hypotonic and tremulous. Histopathological examination (n = 5 mice) revealed normal gross brain organization and lamination but all had ventriculomegaly. Ectopic neurons were seen in all five Strada-/- brains within the subcortical white matter mirroring what is observed in human PS brain tissue. These distinct experimental platforms demonstrate that STRADA modulates mTOR signaling and is a key regulator of cell size, neuronal excitability, and cortical lamination.

Stem cell transplantation therapies are currently under investigation for central nervous system disorders. Although preclinical models show benefit, clinical translation is somewhat limited by the absence of reliable noninvasive methods to confirm targeting and monitor transplanted cells in vivo. Here, we assess a novel magnetic resonance imaging (MRI) contrast agent derived from magnetotactic bacteria, magneto‐endosymbionts (MEs), as a translatable methodology for in vivo tracking of stem cells after intracranial transplantation. We show that ME labeling provides robust MRI contrast without impairment of cell viability or other important therapeutic features. Labeled cells were visualized immediately post‐transplantation and over time by serial MRI in nonhuman primate and mouse brain. Postmortem tissue analysis confirmed on‐target grft location, and linear correlations were observed between MRI signal, cell engraftment, and tissue ME levels, suggesting that MEs may be useful for determining graft survival or rejection. Overall, these findings indicate that MEs are an effective tool for in vivo tracking and monitoring of cell transplantation therapies with potential relevance to many cellular therapy applications., Translation of cell therapies for central nervous system disorders is often limited by an inability to noninvasively identify and track transplanted cells in vivo. McGinley et al report a novel MRI contrast agent based on magnetotactic bacteria that enables visualization and long‐term tracking of cell grafts in nonhuman primate and murine brain.

Significance The interplay between the anorexigenic and orexigenic neurons in the arcuate nucleus that contributes to the control of feeding remains elusive. Using optogenetic stimulation, we show that activation of POMC neurons rapidly inhibits feeding behavior in fasted animals. However, simultaneous stimulation of both POMC neurons and a subset of the orexigenic neurons that express AgRP is sufficient to reverse that inhibition and trigger intense feeding behavior. We used 3D imaging and functional studies to illuminate the anatomical underpinning of both the inhibitory and excitatory events. Our work suggests that translational applications that aim to control appetite need to target the activation rather than the inhibition mechanisms., Two classes of peptide-producing neurons in the arcuate nucleus (Arc) of the hypothalamus are known to exert opposing actions on feeding: the anorexigenic neurons that express proopiomelanocortin (POMC) and the orexigenic neurons that express agouti-related protein (AgRP) and neuropeptide Y (NPY). These neurons are thought to arise from a common embryonic progenitor, but our anatomical and functional understanding of the interplay of these two peptidergic systems that contribute to the control of feeding remains incomplete. The present study uses a combination of optogenetic stimulation with viral and transgenic approaches, coupled with neural activity mapping and brain transparency visualization to demonstrate the following: (i) selective activation of Arc POMC neurons inhibits food consumption rapidly in unsated animals; (ii) activation of Arc neurons arising from POMC-expressing progenitors, including POMC and a subset of AgRP neurons, triggers robust feeding behavior, even in the face of satiety signals from POMC neurons; (iii) the opposing effects on food intake are associated with distinct neuronal projection and activation patterns of adult hypothalamic POMC neurons versus Arc neurons derived from POMC-expressing lineages; and (iv) the increased food intake following the activation of orexigenic neurons derived from POMC-expressing progenitors engages an extensive neural network that involves the endogenous opioid system. Together, these findings shed further light on the dynamic balance between two peptidergic systems in the moment-to-moment regulation of feeding behavior.

Epileptogenic mechanisms in focal cortical dysplasia (FCD) remain elusive, as no animal models faithfully recapitulate FCD seizures, which have distinct electrographic features and a wide range of semiologies. Given that DEPDC5 plays significant roles in focal epilepsies with FCD, we used in utero electroporation with clustered regularly interspaced short palindromic repeats gene deletion to create focal somatic Depdc5 deletion in the rat embryonic brain. Animals developed spontaneous seizures with focal pathological and electroclinical features highly clinically relevant to FCD IIA, paving the way toward understanding its pathogenesis and developing mechanistic-based therapies. Ann Neurol 2018;83:140-146.

The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.

SUMMARY Dentate gyrus (DG) development requires specification of granule cell (GC) progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP) domain and Rap1 small GTPases. In adult Plxna2−/− but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2−/− mice revealed deficits in associative learning, sociability, and sensorimotor gating—traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health., In Brief Zhao et al. find that Sema5A-PlexinA2 forward signaling through Rap1 GTPases is required for progenitor distribution in the developing mouse dentate gyrus. Adult Plxna2−/−, but not Plxna2-GAP-deficient, mice show defects in dentate morphology, neurogenesis, and mossy fiber connectivity. Plxna2−/− and Plxna2-GAP mice exhibit behavioral defects suggestive of neuropsychiatric illness.

Author(s): Bushart, David D; Chopra, Ravi; Singh, Vikrant; Murphy, Geoffrey G; Wulff, Heike; Shakkottai, Vikram G | Abstract: ObjectivePurkinje neuron dysfunction is associated with cerebellar ataxia. In a mouse model of spinocerebellar ataxia type 1 (SCA1), reduced potassium channel function contributes to altered membrane excitability resulting in impaired Purkinje neuron spiking. We sought to determine the relationship between altered membrane excitability and motor dysfunction in SCA1 mice.MethodsPatch-clamp recordings in acute cerebellar slices and motor phenotype testing were used to identify pharmacologic agents which improve Purkinje neuron physiology and motor performance in SCA1 mice. Additionally, we retrospectively reviewed records of patients with SCA1 and other autosomal-dominant SCAs with prominent Purkinje neuron involvement to determine whether currently approved potassium channel activators were tolerated.ResultsActivating calcium-activated and subthreshold-activated potassium channels improved Purkinje neuron spiking impairment in SCA1 mice (P l 0.05). Additionally, dendritic hyperexcitability was improved by activating subthreshold-activated potassium channels but not calcium-activated potassium channels (P l 0.01). Improving spiking and dendritic hyperexcitability through a combination of chlorzoxazone and baclofen produced sustained improvements in motor dysfunction in SCA1 mice (P l 0.01). Retrospective review of SCA patient records suggests that co-treatment with chlorzoxazone and baclofen is tolerated.InterpretationTargeting both altered spiking and dendritic membrane excitability is associated with sustained improvements in motor performance in SCA1 mice, while targeting altered spiking alone produces only short-term improvements in motor dysfunction. Potassium channel activators currently in clinical use are well tolerated and may provide benefit in SCA patients. Future clinical trials with potassium channel activators are warranted in cerebellar ataxia.

Exploring the molecular mechanisms that drive the maturation of oligodendrocyte progenitor cells (OPCs) during the remyelination process is essential to developing new therapeutic tools to intervene in demyelinating diseases such as multiple sclerosis. To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for OPC development during remyelination, we generated an inducible conditional knock-out mouse in which the L-VGCC isoform Cav1.2 was deleted in NG2-positive OPCs (Cav1.2KO). Using the cuprizone (CPZ) model of demyelination and mice of either sex, we establish that Cav1.2 deletion in OPCs leads to less efficient remyelination of the adult brain. Specifically, Cav1.2KOOPCs mature slower and produce less myelin than control oligodendrocytes during the recovery period after CPZ intoxication. This reduced remyelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, during the remyelination phase of the CPZ model, the corpus callosum of Cav1.2KOanimals presented a significant decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons compared with controls. In addition, in a mouse line in which the Cav1.2KOOPCs were identified by aCrereporter, we establish that Cav1.2KOOPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca2+influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca2+channel for OPC maturation during the remyelination of the adult brain.SIGNIFICANCE STATEMENTIon channels implicated in oligodendrocyte differentiation and maturation may induce positive signals for myelin recovery. Voltage-gated Ca2+channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To determine whether voltage Ca2+entry is involved in oligodendrocyte differentiation and remyelination, we used a conditional knockout mouse for VGCCs in OPCs. Our results indicate that VGCCs can modulate oligodendrocyte maturation in the demyelinated brain and suggest that voltage-gated Ca2+influx in OPCs is critical for remyelination. These findings could lead to novel approaches for obtaining a better understanding of the factors that control OPC maturation in order to stimulate this pool of progenitors to replace myelin in demyelinating diseases.

Hippocampus oxidative stress is considered pathogenic in neurodegenerative diseases, such as Alzheimer disease (AD), and in neurodevelopmental disorders, such as Angelman syndrome (AS). Yet clinical benefits of antioxidant treatment for these diseases remain unclear because conventional imaging methods are unable to guide management of therapies in specific hippocampus subfields in vivo that underlie abnormal behavior. Excessive production of paramagnetic free radicals in nonhippocampus brain tissue can be measured in vivo as a greater-than-normal 1/T1 that is quenchable with antioxidant as measured by quench-assisted (Quest) MRI. Here, we further test this approach in phantoms, and we present proof-of-concept data in models of AD-like and AS hippocampus oxidative stress that also exhibit impaired spatial learning and memory. AD-like models showed an abnormal gradient along the CA1 dorsal–ventral axis of excessive free radical production as measured by Quest MRI, and redox-sensitive calcium dysregulation as measured by manganese-enhanced MRI and electrophysiology. In the AS model, abnormally high free radical levels were observed in dorsal and ventral CA1. Quest MRI is a promising in vivo paradigm for bridging brain subfield oxidative stress and behavior in animal models and in human patients to better manage antioxidant therapy in devastating neurodegenerative and neurodevelopmental diseases.—Berkowitz, B. A., Lenning, J., Khetarpal, N., Tran, C., Wu, J. Y., Berri, A. M., Dernay, K., Haacke, E. M., Shafie-Khorassani, F., Podolsky, R. H., Gant, J. C., Maimaiti, S., Thibault, O., Murphy, G. G., Bennett, B. M., Roberts, R. In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome.

International audience; The aged population is growing rapidly, which has sparked tremendous interest in elucidating mechanisms of aging in both the body and the brain. Animal models have become an indispensable tool in biomedical science, but because of the cost and extended timeframe associated with aging animals to appropriate time points, studies that rely on using aged animals are often not feasible. Somewhat surprisingly, there are relatively few animal models that have been specifically engineered to mimic physiological changes known to occur during "normal" aging. Developing transgenic animal models that faithfully mimic key aspects of aging would likely be of great utility in studying both age-related deficits in the absence of overt pathology as well as an adjunct for transgenic models of diseases where aging is a primary risk factor. In particular, there are several alterations in the aged brain that are amenable to being modeled genetically. We have focused on one key aspect that has been repeatedly demonstrated in aged animals - an increase in the L-type voltage-gated calcium channel CaV1.3. Here we present a novel transgenic mouse line in which expression of CaV1.3 is increased by approximately 50% in the forebrain of young mice. These mice do not display any overt physical or non-cognitive deficits, exhibiting normal exploratory behavior, motor function, and affective-like responses, suggesting that these mice can be successfully deployed to assess the impact of an "aged brain" in a variety of conditions

The mammalian amygdala represents one of the central testing grounds for key questions in neuroscience. In particular, it has been the focus of many questions regarding the relationship between neural plasticity (i.e., long-term potentiation) and learning. Early foundational studies established the role of the amygdala in associative learning, particularly in fear learning. These studies identified the neuroanatomy, neurocircuitry, and basic neurophysiology of cells in the amygdala and correlated their structure and function to learning and behavior. More recently, with the advancements in neurotechnology that facilitate in vivo monitoring and control over the activation of specified cell populations in the brain, the direct links between neural plasticity and learning have been drawn. These studies indicate that Hebbian forms of plasticity are necessary for associative fear learning; however, Hebbian plasticity alone is not sufficient. In this chapter, we review some of the significant studies supporting this conclusion and consider additional neural mechanisms that support the induction of synaptic plasticity during learning. We conclude with the future directions of research for the cellular transduction of associative forms of learning beyond Hebbian plasticity.

Dysregulated adult hippocampal neurogenesis occurs in many temporal lobe epilepsy (TLE) models. Most dentate granule cells (DGCs) generated in response to an epileptic insult develop features that promote increased excitability, including ectopic location, persistent hilar basal dendrites (HBDs), and mossy fiber sprouting. However, some appear to integrate normally and even exhibit reduced excitability compared to other DGCs. To examine the relationship between DGC birthdate, morphology, and network integration in a model of TLE, we retrovirally birthdated either early-born [EB; postnatal day (P)7] or adult-born (AB; P60) DGCs. Male rats underwent pilocarpine-induced status epilepticus (SE) or sham treatment at P56. Three to six months after SE or sham treatment, we used whole-cell patch-clamp and fluorescence microscopy to record spontaneous excitatory and inhibitory currents from birthdated DGCs. We found that both AB and EB populations of DGCs recorded from epileptic rats received increased excitatory input compared with age-matched controls. Interestingly, when AB populations were separated into normally integrated (normotopic) and aberrant (ectopic or HBD-containing) subpopulations, only the aberrant populations exhibited a relative increase in excitatory input (amplitude, frequency, and charge transfer). The ratio of excitatory-to-inhibitory input was most dramatically upregulated for ectopically localized DGCs. These data provide definitive physiological evidence that aberrant integration of post-SE, AB DGCs contributes to increased synaptic drive and support the idea that ectopic DGCs serve as putative hub cells to promote seizures. SIGNIFICANCE STATEMENT Adult dentate granule cell (DGC) neurogenesis is altered in rodent models of temporal lobe epilepsy (TLE). Some of the new neurons show abnormal morphology and integration, but whether adult-generated DGCs contribute to the development of epilepsy is controversial. We examined the synaptic inputs of age-defined populations of DGCs using electrophysiological recordings and fluorescent retroviral reporter birthdating. DGCs generated neonatally were compared with those generated in adulthood, and adult-born (AB) neurons with normal versus aberrant morphology or integration were examined. We found that AB, ectopically located DGCs exhibit the most pro-excitatory physiological changes, implicating this population in seizure generation or progression.

Fear conditioning is an associative learning process by which organisms learn to avoid environmental stimuli that are predictive of aversive outcomes. Fear extinction learning is a process by which avoidance of fear-conditioned stimuli is attenuated when the environmental stimuli is no longer predictive of the aversive outcome. Aberrant fear conditioning and extinction learning are key elements in the development of several anxiety disorders. The 129S1 inbred strain of mice is used as an animal model for maladaptive fear learning because this strain has been shown to generalize fear to other nonaversive stimuli and is less capable of extinguishing fear responses relative to other mouse strains, such as the C57BL/6. Here we report new environmental manipulations that enhance fear and extinction learning, including the ability to discriminate between an aversively paired tone and a neutral tone, in both the 129S1 and C57BL/6 strains of mice. Specifically, we show that discontinuous (“pipped”) tone stimuli significantly enhance within-session extinction learning and the discrimination between neutral and aversively paired stimuli in both strains. Furthermore, we find that extinction training in novel contexts significantly enhances the consolidation and recall of extinction learning for both strains. Cumulatively, these results underscore how environmental changes can be leveraged to ameliorate maladaptive learning in animal models and may advance cognitive and behavioral therapeutic strategies.

We have found a significant upregulation of L-type voltage-operated Ca(++) channels (VOCCs) in reactive astrocytes. To test if VOCCs are centrally involved in triggering astrocyte reactivity, we used in vitro models of astrocyte activation in combination with pharmacological inhibitors, siRNAs and the Cre/lox system to reduce the activity of L-type VOCCs in primary cortical astrocytes. The endotoxin lipopolysaccharide (LPS) as well as high extracellular K(+) , glutamate, and ATP promote astrogliosis in vitro. L-type VOCC inhibitors drastically reduce the number of reactive cells, astrocyte hypertrophy, and cell proliferation after these treatments. Astrocytes transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents as well as Cav1.2 knockout astrocytes showed reduce Ca(++) influx by ∼80% after plasma membrane depolarization. Importantly, Cav1.2 knock-down/out prevents astrocyte activation and proliferation induced by LPS. Similar results were found using the scratch wound assay. After injuring the astrocyte monolayer, cells extend processes toward the cell-free scratch region and subsequently migrate and populate the scratch. We found a significant increase in the activity of L-type VOCCs in reactive astrocytes located in the growing line in comparison to quiescent astrocytes situated away from the scratch. Moreover, the migration of astrocytes from the scratching line as well as the number of proliferating astrocytes was reduced in Cav1.2 knock-down/out cultures. In summary, our results suggest that Cav1.2 L-type VOCCs play a fundamental role in the induction and/or proliferation of reactive astrocytes, and indicate that the inhibition of these Ca(++) channels may be an effective way to prevent astrocyte activation. GLIA 2016. GLIA 2016;64:1396-1415.

Agonist‐triggered downregulation of β‐adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of β2 AR signaling highly specific for Cav1.2. We find that β2‐AR binding to Cav1.2 residues 1923–1942 is required for β‐adrenergic regulation of Cav1.2. Despite the prominence of PKA‐mediated phosphorylation of Cav1.2 S1928 within the newly identified β2 AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the β2 AR from Cav1.2 upon β‐adrenergic stimulation rendering Cav1.2 refractory for several minutes from further β‐adrenergic stimulation. This effect is lost in S1928A knock‐in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, β2 AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the β2 AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized β2 AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT‐LTP) depends on Cav1.2 and its regulation by channel‐associated β2 AR.

Over the last two decades there has been significant progress towards understanding the neural substrates that underlie age-related cognitive decline. Although many of the exact molecular and cellular mechanisms have yet to be fully understood, there is consensus that alterations in neuronal calcium homeostasis contribute to age-related deficits in learning and memory. Furthermore, it is thought that the age-related changes in calcium homeostasis are driven, at least in part, by changes in calcium channel expression. In this review, we focus on the role of a specific class of calcium channels: L-type voltage-gated calcium channels (LVGCCs). We provide the reader with a general introduction to voltage-gated calcium channels, followed by a more detailed description of LVGCCs and how they serve to regulate neuronal excitability via the post burst afterhyperpolarization (AHP). We conclude by reviewing studies that link the slow component of the AHP to learning and memory, and discuss how age-related increases in LVGCC expression may underlie cognitive decline by mediating a decrease in neuronal excitability.

The ubiquitin-binding proteasomal shuttle protein UBQLN2 is implicated in common neurodegenerative disorders due to its accumulation in disease-specific aggregates and, when mutated, directly causes familial frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). Like other proteins linked to FTD/ALS, UBQLN2 undergoes phase separation to form condensates. The relationship of UBQLN2 phase separation and accumulation to neurodegeneration, however, remains uncertain. Employing biochemical, neuropathological and behavioral assays, we studied the impact of overexpressing WT or mutant UBQLN2 in the CNS of transgenic mice. Expression of UBQLN2 harboring a pathogenic mutation (P506T) elicited profound and widespread intraneuronal inclusion formation and aggregation without prominent neurodegenerative or behavioral changes. Both WT and mutant UBQLN2 formed ubiquitin- and P62-positive inclusions in neurons, supporting the view that UBQLN2 is intrinsically prone to phase separate, with the size, shape and frequency of inclusions depending on expression level and the presence or absence of a pathogenic mutation. Overexpression of WT or mutant UBQLN2 resulted in a dose-dependent decrease in levels of a key interacting chaperone, HSP70, as well as dose-dependent profound degeneration of the retina. We conclude that, at least in mice, robust aggregation of a pathogenic form of UBQLN2 is insufficient to cause neuronal loss recapitulating that of human FTD/ALS. Our results nevertheless support the view that altering the normal cellular balance of UBQLN2, whether wild type or mutant protein, has deleterious effects on cells of the CNS and retina that likely reflect perturbations in ubiquitin-dependent protein homeostasis.

Stem cell transplantation offers a potentially transformative approach to treating neurodegenerative disorders. The safety of cellular therapies is established in multiple clinical trials, including our own in amyotrophic lateral sclerosis. To initiate similar trials in Alzheimer’s disease, efficacious cell lines must be identified. Here, we completed a preclinical proof-of-concept study in the APP/PS1 murine model of Alzheimer’s disease. Human neural stem cell transplantation targeted to the fimbria fornix significantly improved cognition in two hippocampal-dependent memory tasks at 4 and 16 weeks post-transplantation. While levels of synapse-related proteins and cholinergic neurons were unaffected, amyloid plaque load was significantly reduced in stem cell transplanted mice and associated with increased recruitment of activated microglia. In vitro, these same neural stem cells induced microglial activation and amyloid phagocytosis, suggesting an immunomodulatory capacity. Although long-term transplantation resulted in significant functional and pathological improvements in APP/PS1 mice, stem cells were not identified by immunohistochemistry or PCR at the study endpoint. These data suggest integration into native tissue or the idea that transient engraftment may be adequate for therapeutic efficacy, reducing the need for continued immunosuppression. Overall, our results support further preclinical development of human neural stem cells as a safe and effective therapy for Alzheimer’s disease.

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (MR)of the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

Epileptogenic mechanisms in focal cortical dysplasia (FCD) remain elusive, as no animal models faithfully recapitulate FCD seizures, which have distinct electrographic features and a wide range of semiologies. Given that DEPDC5 plays significant roles in focal epilepsies with FCD, we used in utero electroporation with clustered regularly interspaced short palindromic repeats gene deletion to create focal somatic Depdc5 deletion in the rat embryonic brain. Animals developed spontaneous seizures with focal pathological and electroclinical features highly clinically relevant to FCD IIA, paving the way toward understanding its pathogenesis and developing mechanistic-based therapies. Ann Neurol 2018;83:140-146.

Objective A growing body of evidence suggests that increased blood–brain barrier (BBB) permeability can contribute to the development of seizures. The protease tissue plasminogen activator (tPA) has been shown to promote BBB permeability and susceptibility to seizures. In this study, we examined the pathway regulated by tPA in seizures. Methods An experimental model of kainate-induced seizures was used in genetically modified mice, including mice deficient in tPA (tPA−/−), its inhibitor neuroserpin (Nsp−/−), or both (Nsp:tPA−/−), and in mice conditionally deficient in the platelet-derived growth factor receptor alpha (PDGFRα). Results Compared to wild-type (WT) mice, Nsp−/− mice have significantly reduced latency to seizure onset and generalization; whereas tPA−/− mice have the opposite phenotype, as do Nsp:tPA−/− mice. Furthermore, interventions that maintain BBB integrity delay seizure propagation, whereas osmotic disruption of the BBB in seizure-resistant tPA−/− mice dramatically reduces the time to seizure onset and accelerates seizure progression. The phenotypic differences in seizure progression between WT, tPA−/−, and Nsp−/− mice are also observed in electroencephalogram recordings in vivo, but absent in ex vivo electrophysiological recordings where regulation of the BBB is no longer necessary to maintain the extracellular environment. Finally, we demonstrate that these effects on seizure progression are mediated through signaling by PDGFRα on perivascular astrocytes. Interpretation Together, these data identify a specific molecular pathway involving tPA-mediated PDGFRα signaling in perivascular astrocytes that regulates seizure progression through control of the BBB. Inhibition of PDGFRα signaling and maintenance of BBB integrity might therefore offer a novel clinical approach for managing seizures.

Hilar ectopic dentate granule cells (DGCs) are a salient feature of aberrant plasticity in human temporal lobe epilepsy (TLE) and most rodent models of the disease. Recent evidence from rodent TLE models suggests that hilar ectopic DGCs contribute to hyperexcitability within the epileptic hippocampal network. Here we investigate the intrinsic excitability of DGCs from humans with TLE and the rat pilocarpine TLE model with the objective of comparing the neurophysiology of hilar ectopic DGCs to their normotopic counterparts in the granule cell layer (GCL). We recorded from 36 GCL and 7 hilar DGCs from human TLE tissue. Compared with GCL DGCs, hilar DGCs in patient tissue exhibited lower action potential (AP) firing rates, more depolarized AP threshold, and differed in single AP waveform, consistent with an overall decrease in excitability. To evaluate the intrinsic neurophysiology of hilar ectopic DGCs, we made recordings from retrovirus-birthdated, adult-born DGCs 2–4 mo after pilocarpine-induced status epilepticus or sham treatment in rats. Hilar DGCs from epileptic rats exhibited higher AP firing rates than normotopic DGCs from epileptic or control animals. They also displayed more depolarized resting membrane potential and wider AP waveforms, indicating an overall increase in excitability. The contrasting findings between disease and disease model may reflect differences between the late-stage disease tissue available from human surgical specimens and the earlier disease stage examined in the rat TLE model. These data represent the first neurophysiological characterization of ectopic DGCs from human hippocampus and prospectively birthdated ectopic DGCs in a rodent TLE model.

Cardiorespiratory failure is the most common cause of sudden unexplained death in epilepsy (SUDEP). Genetic autopsies have detected "leaky" gain-of-function mutations in the ryanodine receptor-2 (RyR2) gene in both SUDEP and sudden cardiac death cases linked to catecholaminergic polymorphic ventricular tachycardia that feature lethal cardiac arrhythmias without structural abnormality. Here we find that a human leaky RyR2 mutation, R176Q (RQ), alters neurotransmitter release probability in mice and significantly lowers the threshold for spreading depolarization (SD) in dorsal medulla, leading to cardiorespiratory collapse. Rare episodes of sinus bradycardia, spontaneous seizure, and sudden death were detected in RQ/+ mutant mice in vivo; however, when provoked, cortical seizures frequently led to apneas, brainstem SD, cardiorespiratory failure, and death. In vitro studies revealed that the RQ mutation selectively strengthened excitatory, but not inhibitory, synapses and facilitated SD in both the neocortex as well as brainstem dorsal medulla autonomic microcircuits. These data link defects in neuronal intracellular calcium homeostasis to the vulnerability of central autonomic brainstem pathways to hypoxic stress and implicate brainstem SD as a previously unrecognized site and mechanism contributing to premature death in individuals with leaky RYR2 mutations.

L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, CaV1.2 and CaV1.3, using transgenic mice have failed to find a role of either subtype in fear extinction. This discontinuity between the pharmacological studies of LVGCCs and the studies investigating individual subtype contributions could be due to the limited neuronal deletion pattern of the CaV1.2 conditional knockout mice previously studied to excitatory neurons in the forebrain. To investigate the effects of deletion of CaV1.2 in all neuronal populations, we generated CaV1.2 conditional knockout mice using the synapsin1 promoter to drive Cre recombinase expression. Pan-neuronal deletion of CaV1.2 did not alter basal anxiety or fear learning. However, pan-neuronal deletion of CaV1.2 resulted in a significant deficit in extinction of contextual fear, implicating LVGCCs, specifically CaV1.2, in extinction learning. Further exploration on the effects of deletion of CaV1.2 on inhibitory and excitatory input onto the principle neurons of the lateral amygdala revealed a significant shift in inhibitory/excitatory balance. Together these data illustrate an important role of CaV1.2 in fear extinction and the synaptic regulation of activity within the amygdala.

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (MR)of the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

De novo mutations of the voltage-gated sodium channel gene SCN8A have recently been recognized as a cause of epileptic encephalopathy, which is characterized by refractory seizures with developmental delay and cognitive disability. We previously described the heterozygous SCN8A missense mutation p.Asn1768Asp in a child with epileptic encephalopathy that included seizures, ataxia, and sudden unexpected death in epilepsy (SUDEP). The mutation results in increased persistent sodium current and hyperactivity of transfected neurons. We have characterized a knock-in mouse model expressing this dominant gain-of-function mutation to investigate the pathology of the altered channel in vivo. The mutant channel protein is stable in vivo. Heterozygous Scn8a(N1768D/+) mice exhibit seizures and SUDEP, confirming the causality of the de novo mutation in the proband. Using video/EEG analysis, we detect ictal discharges that coincide with convulsive seizures and myoclonic jerks. Prior to seizure onset, heterozygous mutants are not defective in motor learning or fear conditioning, but do exhibit mild impairment of motor coordination and social discrimination. Homozygous mutant mice exhibit earlier seizure onset than heterozygotes and more rapid progression to death. Analysis of the intermediate phenotype of functionally hemizygous Scn8a(N1768D/-) mice indicates that severity is increased by a double dose of mutant protein and reduced by the presence of wild-type protein. Scn8a(N1768D) mutant mice provide a model of epileptic encephalopathy that will be valuable for studying the in vivo effects of hyperactive Nav1.6 and the response to therapeutic interventions.