Automated high-throughput mouse transsynaptic viral tracing using iDISCO+ tissue clearing, light-sheet microscopy, and BrainPipe.

Transsynaptic viral tracing requires tissue sectioning, manual cell counting, and anatomical assignment, all of which are time intensive. We describe a protocol for BrainPipe, a scalable software for automated anatomical alignment and object counting in light-sheet microscopy volumes. BrainPipe can be generalized to new counting tasks by using a new atlas and training a neural network for object detection. Combining viral tracing, iDISCO+ tissue clearing, and BrainPipe facilitates mapping of cerebellar connectivity to the rest of the murine brain. For complete details on the use and execution of this protocol, please refer to Pisano et al. (2021).

A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia.

In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

Early-onset cerebellar ataxia in a patient with CMT2A2.

A 9-yr 8-mo-old right-handed female presented with a history of gait difficulties, which first became apparent at age 9 mo of age, along with slurred speech and hand tremors while holding a tray. Her past medical history was significant for global developmental delay, and she was attending fourth grade special education classes. On examination, she had an ataxic gait, dysarthria, absent deep tendon reflexes, and flexor plantar responses. There were no signs of optic atrophy or hearing loss. Nerve conduction studies were consistent with an axonal neuropathy. A fascicular sural nerve biopsy showed a marked decrease of myelinated fibers larger than 6 µm in diameter as compared with an age-matched control. By electron microscopy, clusters of degenerating axonal mitochondria in both myelinated and unmyelinated fibers were frequently found. Whole-exome sequencing revealed a heterozygous c.314C > T (p.Thr105Met) missense variant in MFN2 in the patient but not in her mother. The father was unavailable for testing. The phenotypes with MFN2 variants can be quite variable, including intellectual disability, optic atrophy, auditory impairment, spinal atrophy with or without hydromyelia, and hydrocephalus. We report here that early onset ataxia with intellectual disability can also be associated with MFN2-related Charcot-Marie-Tooth, Type 2A2A diagnosis, the most common type of autosomal dominant axonal neuropathy.

Ultrasonic vocalization sex differences in 5-HT1A-R deficient mouse pups: Predictive phenotypes associated with later-life anxiety-like behaviors.

Anxiety disorders affect nearly twice as many women as men. However, little is known regarding sex-dependent developmental behavioral differences and whether there is an association with later life anxiety disorders. The present study assessed the developmental-behavioral milestones (DBMs) and their relationship with later life anxiety-like behaviors by comparing postnatal ultrasonic vocalizations (USVs) with open field (OF), elevated plus maze (EPM), and light/dark (LD) anxiety test outcomes using the serotonin 1A receptor knockout (KO) mouse model of anxiety. The USVs and DBMs (i.e., grasping, righting, and startle reflexes) were examined on postnatal day 6 (P6), P8, and P10. Adult anxiety-like behaviors were examined on P60 to compare the genotype and sex-dependent differences in anxiety-like behaviors and to correlate them with the USVs. The total number of USVs observed on P8 correlated with later life anxiety-like behaviors in a genotype-, age-, and sex-dependent manner. Interestingly, female KO (KOF) mice exhibited elevated levels of anxiety-like behavior within the OF, EPM, and LD tests. Additionally, an investigation of the USV subtypes, as well as USV sequence structure and repertoire variation, revealed that the KOF mice produced less complex USVs and complex USV-containing sequences on P10. The present study provides an intriguing, predictive "P8/10-USV-to-P60" anxiety-like behavioral model that may prove useful in future characterization, psychopharmacology, and drug rescue studies directed towards sex-specific anxiety treatment.

Aberrant hippocampal Atp8a1 levels are associated with altered synaptic strength, electrical activity, and autistic-like behavior.

Type IV ATPases are putative aminophospholipid translocases (APLTs), more commonly known as flippases. A pronounced induction of the flippase Atp8a1 was observed in post-mortem tissue homogenates from the hippocampus and temporal lobe of juvenile autistic subjects compared to age-matched controls. In order to simulate the human data, C57BL/6 mice were allowed to develop after intra-hippocampal injection of recombinant lentivirus expressing Atp8a1 at the early developmental stage of postnatal day 6 (P6). Transmission electron microscopy (TEM) analysis of the lentivirus-Atp8a1 treated (Atp8a1+) mice in adulthood revealed fewer and weaker excitatory synapses in the hippocampal CA1 region compared to mice injected with empty virus. Significant inhibition of the Schaffer collateral pathway was observed in the Atp8a1+ mice in paired-pulse recording (PPR) at 20-ms inter-stimulus interval. In the three-chambered sociability test, the Atp8a1+ mice displayed no preference for an encaged stranger mouse over a novel object, which is a characteristic autistic-like behavior. In sharp contrast, Atp8a1 (-/-) mice displayed a preference for a stranger mouse over the novel object, which is characteristic of neurotypical mouse behavior. However, similar to the Atp8a1+ mice, the Atp8a1 (-/-) mice harbored fewer and weaker excitatory synapses in CA1 compared to wild-type controls, and displayed inhibition at 20-ms inter-stimulus interval in PPR. These findings suggest that both elevated and diminished levels of Atp8a1 during early development are detrimental to brain connectivity, but only elevated Atp8a1 is associated with aberrant social behavior. Mice with augmented levels of Atp8a1 may therefore serve as a potential model in autism research.

TSPO Finds NOX2 in Microglia for Redox Homeostasis.

During the past decade, translocator protein 18 kDa (TSPO), previously named peripheral benzodiazepine receptor, has gained a great deal of attention based on its use as a clinical biomarker of neuroinflammation with therapeutic potential. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. Here, we identify a novel function of TSPO in microglia that is not associated with steroidogenesis. We propose that a TSPO interaction with NADPH oxidase (NOX2) links the generation of reactive oxygen species (ROS) to the induction of an antioxidant response to maintain redox homeostasis. This line of investigation may provide a greater understanding of TSPO glial cell biology, and the knowledge gained may prove beneficial in devising therapeutic strategies.

Presynaptic mechanisms of lead neurotoxicity: effects on vesicular release, vesicle clustering and mitochondria number.

Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development.

Increasing maternal or post-weaning folic acid alters gene expression and moderately changes behavior in the offspring.

BACKGROUND: Studies have indicated that altered maternal micronutrients and vitamins influence the development of newborns and altered nutrient exposure throughout the lifetime may have potential health effects and increased susceptibility to chronic diseases. In recent years, folic acid (FA) exposure has significantly increased as a result of mandatory FA fortification and supplementation during pregnancy. Since FA modulates DNA methylation and affects gene expression, we investigated whether the amount of FA ingested during gestation alters gene expression in the newborn cerebral hemisphere, and if the increased exposure to FA during gestation and throughout the lifetime alters behavior in C57BL/6J mice. METHODS: Dams were fed FA either at 0.4 mg or 4 mg/kg diet throughout the pregnancy and the resulting pups were maintained on the diet throughout experimentation. Newborn pups brain cerebral hemispheres were used for microarray analysis. To confirm alteration of several genes, quantitative RT-PCR (qRT-PCR) and Western blot analyses were performed. In addition, various behavior assessments were conducted on neonatal and adult offspring. RESULTS: Results from microarray analysis suggest that the higher dose of FA supplementation during gestation alters the expression of a number of genes in the newborns' cerebral hemispheres, including many involved in development. QRT-PCR confirmed alterations of nine genes including down-regulation of Cpn2, Htr4, Zfp353, Vgll2 and up-regulation of Xist, Nkx6-3, Leprel1, Nfix, Slc17a7. The alterations in the expression of Slc17a7 and Vgll2 were confirmed at the protein level. Pups exposed to the higher dose of FA exhibited increased ultrasonic vocalizations, greater anxiety-like behavior and hyperactivity. These findings suggest that although FA plays a significant role in mammalian cellular machinery, there may be a loss of benefit from higher amounts of FA. Unregulated high FA supplementation during pregnancy and throughout the life course may have lasting effects, with alterations in brain development resulting in changes in behavior.

Cued and contextual fear conditioning in BTBR mice is improved with training or atomoxetine.

The BTBR T+tf/J (BTBR) strain of mice is a model for autism spectrum disorders (ASDs). These mice display reduced social behavior, altered communication, and high levels of repetitive behavior. BTBR mice have shown a deficit in learning cued and contextual fear conditioning. In this study, experiments were conducted to determine if either changes in training or drug administration would improve learning in BTBR mice when compared to C57BL/6 (B6) mice in contextual and cued fear conditioning. The first experiment examined the effects of three conditioned stimulus-unconditioned stimulus (CS-US) training paradigms; a 1P (1 CS-US pairing), 4P (4 CS-US pairings), and 10P (10 CS-US pairings). Increasing the number of CS-US pairings to 10 caused an increase in freezing behavior by the BTBR mice in contextual and cued conditioning indicating that more training facilitated BTBR learning. B6 mice had a more complex reaction to the increased training; the mice increased freezing behavior in the cued fear conditioning but not contextual fear conditioning. The second experiment determined whether atomoxetine, a noradrenergic reuptake inhibitor that has been shown to improve attention and decrease hyperactivity, impulsivity, and social withdrawal, would enhance learning. There was a significant increase in freezing behavior in contextual fear conditioning following atomoxetine administration in BTBR mice but not in B6 mice. Our data demonstrates that contextual and cued learning in BTBR mice is facilitated by increased training. Furthermore, contextual learning is improved in BTBR mice with use of atomoxetine, which helps to improve attention. Both increased training and pharmacological intervention improved learning in the BTBR mice suggesting a role for the combination of the two.

Taurine's effects on the neuroendocrine functions of pancreatic ß cells.

Taurine plays significant physiological roles, including those involved in neurotransmission. Taurine is a potent γ-aminobutyric acid (GABA) agonist and alters cellular events via GABA(A) receptors. Alternately, taurine is transported into cells via the high affinity taurine transporter (TauT), where it may also play a regulatory role. We have previously demonstrated that treatment of Hit-T15 cells with 1 mM taurine for 24 h significantly decreases insulin and GABA levels. We have also demonstrated that chronic in vivo administration of taurine results in an up-regulation of glutamic acid decarboxylase (GAD), the key enzyme in GABA synthesis. Here, we wished to test if administration of 1 mM taurine for 24 h may increase release of another ß cell neurotransmitter somatostatin (SST) and also directly impact up-regulation of GAD synthesis. Treatment with taurine did not significantly alter levels of SST (p > 0.05) or GAD67 (p > 0.05). This suggests that taurine does not directly affect SST release, nor does it directly affect GAD synthesis. Taken together with our observation that taurine does promote GABA release via large dense-core vesicles, the data suggest that taurine may alter membrane potential, which in turn would affect calcium flux. We show here that 1 mM taurine does not alter intracellular Ca(2+) concentrations from 20 to 80 s post treatment (p > 0.05), but does increase Ca(2+) flux between 80 and 200 s post-treatment (p < 0.005). This suggests that taurine may induce a biphasic response in ß cells. The initial response of taurine via GABA(A) receptors hyperpolarizes ß cell and sequesters Ca(2+). Subsequently, taurine may affect Ca(2+) flux in long term via interaction with K(ATP) channels.

New directions in the treatment of autism spectrum disorders from animal model research.

INTRODUCTION: Currently, there is not an effective pharmacotherapy for the core symptoms of the autism spectrum disorders (ASD), which include aberrant social behavior, delayed communication and repetitive behavior and/or restricted interests. There are several drugs that treat the symptoms associated with autism including irritability, aggressiveness and hyperactivity. Current drug research is based on the ongoing genetic, animal model and neuropathologic research. Two areas in particular, the glutamate and oxytocin systems, provide exciting new avenues for drug discovery. AREAS COVERED: This review examines what approaches have been used for the drugs that are currently being used to treat people with ASD. For the most part, drugs that treat other neuropsychiatric disorders have been examined to treat the people with ASD, unfortunately with little effect on the core symptoms. EXPERT OPINION: Until recently, there was not a plethora of knowledge about the neurobiological substrates of social behavior, pragmatic language usage and repetitive and/or restricted behaviors. Therefore, drug discovery has used the tools available for other neuropsychiatric disorders. Now that more biological information is available, there are many avenues for research for drug targets for ASD.