Hypoplasia of dopaminergic neurons by hypoxia-induced neurotoxicity is associated with disrupted swimming development of larval zebrafish.

Hypoxic injury to the developing brain increases the risk of permanent behavioral deficits, but the precise mechanisms of hypoxic injury to the developing nervous system are poorly understood. In this study, we characterized the effects of developmental hypoxia (1% pO2 from 24 to 48 h post-fertilization, hpf) on diencephalic dopaminergic (DA) neurons in larval zebrafish and the consequences on the development of swimming behavior. Hypoxia reduced the number of diencephalic DA neurons at 48 hpf. Returning zebrafish larvae to normoxia after the hypoxia (i.e., hypoxia-recovery, HR) induced reactive oxygen species (ROS) accumulation. Real-time qPCR results showed that HR caused upregulation of proapoptotic genes, including p53 and caspase3, suggesting the potential for ROS-induced cell death. With HR, we also found an increase in TUNEL-positive DA neurons, a persistent reduction in the number of diencephalic DA neurons, and disrupted swimming development and behavior. Interestingly, post-hypoxia (HR) with the antioxidant N-acetylcysteine partially restored the number of DA neurons and spontaneous swimming behavior, demonstrating potential recovery from hypoxic injury. The present study provides new insights for understanding the mechanisms responsible for motor disability due to developmental hypoxic injury.

Fingerpad-Inspired Multimodal Electronic Skin for Material Discrimination and Texture Recognition.

Human skin plays a critical role in a person communicating with his or her environment through diverse activities such as touching or deforming an object. Various electronic skin (E-skin) devices have been developed that show functional or geometrical superiority to human skin. However, research into stretchable E-skin that can simultaneously distinguish materials and textures has not been established yet. Here, the first approach to achieving a stretchable multimodal device is reported, that operates on the basis of various electrical properties of piezoelectricity, triboelectricity, and piezoresistivity and that exceeds the capabilities of human tactile perception. The prepared E-skin is composed of a wrinkle-patterned silicon elastomer, hybrid nanomaterials of silver nanowires and zinc oxide nanowires, and a thin elastomeric dielectric layer covering the hybrid nanomaterials, where the dielectric layer exhibits high surface roughness mimicking human fingerprints. This versatile device can identify and distinguish not only mechanical stress from a single stimulus such as pressure, tensile strain, or vibration but also that from a combination of multiple stimuli. With simultaneous sensing and analysis of the integrated stimuli, the approach enables material discrimination and texture recognition for a biomimetic prosthesis when the multifunctional E-skin is applied to a robotic hand.

User-Interactive Thermotherapeutic Electronic Skin Based on Stretchable Thermochromic Strain Sensor.

User-interactive electronic skin (e-skin) with a distinguishable output has enormous potential for human-machine interfaces and healthcare applications. Despite advances in user-interactive e-skins, advances in visual user-interactive therapeutic e-skins remain rare. Here, a user-interactive thermotherapeutic device is reported that is fabricated by combining thermochromic composites and stretchable strain sensors consisting of strain-responsive silver nanowire networks on surface energy-patterned microwrinkles. Both the color and heat of the device are easily controlled through electrical resistance variation induced by applied mechanical strain. The resulting monolithic device exhibits substantial changes in optical reflectance and temperature with durability, rapid response, high stretchability, and linear sensitivity. The approach enables a low-expertise route to fabricating dynamic interactive thermotherapeutic e-skins that can be used to effectively rehabilitate injured connective tissues as well as to prevent skin burns by simultaneously accommodating stretching, providing heat, and exhibiting a color change.

Dopaminergic Co-Regulation of Locomotor Development and Motor Neuron Synaptogenesis is Uncoupled by Hypoxia in Zebrafish.

Hypoxic injury to the developing human brain is a complication of premature birth and is associated with long-term impairments of motor function. Disruptions of axon and synaptic connectivity have been linked to developmental hypoxia, but the fundamental mechanisms impacting motor function from altered connectivity are poorly understood. We investigated the effects of hypoxia on locomotor development in zebrafish. We found that developmental hypoxia resulted in decreased spontaneous swimming behavior in larva, and that this motor impairment persisted into adulthood. In evaluation of the diencephalic dopaminergic neurons, which regulate early development of locomotion and constitute an evolutionarily conserved component of the vertebrate dopaminergic system, hypoxia caused a decrease in the number of synapses from the descending dopaminergic diencephalospinal tract (DDT) to spinal cord motor neurons. Moreover, dopamine signaling from the DDT was coupled jointly to motor neuron synaptogenesis and to locomotor development. Together, these results demonstrate the developmental processes regulating early locomotor development and a requirement for dopaminergic projections and motor neuron synaptogenesis. Our findings suggest new insights for understanding the mechanisms leading to motor disability from hypoxic injury of prematurity.

Hypoxia and connectivity in the developing vertebrate nervous system.

The developing nervous system depends upon precise regulation of oxygen levels. Hypoxia, the condition of low oxygen concentration, can interrupt developmental sequences and cause a range of molecular, cellular and neuronal changes and injuries. The roles and effects of hypoxia on the central nervous system (CNS) are poorly characterized, even though hypoxia is simultaneously a normal component of development, a potentially abnormal environmental stressor in some settings, and a clinically important complication, for example of prematurity. Work over the past decade has revealed that hypoxia causes specific disruptions in the development of CNS connectivity, altering axon pathfinding and synapse development. The goals of this article are to review hypoxia's effects on the development of CNS connectivity, including its genetic and molecular mediators, and the changes it causes in CNS circuitry and function due to regulated as well as unintended mechanisms. The transcription factor HIF1α is the central mediator of the CNS response to hypoxia (as it is elsewhere in the body), but hypoxia also causes a dysregulation of gene expression. Animals appear to have evolved genetic and molecular responses to hypoxia that result in functional behavioral alterations to adapt to the changes in oxygen concentration during CNS development. Understanding the molecular pathways underlying both the normal and abnormal effects of hypoxia on CNS connectivity may reveal novel insights into common neurodevelopmental disorders. In addition, this Review explores the current gaps in knowledge, and suggests important areas for future studies.

Temporal Dysynchrony in brain connectivity gene expression following hypoxia.

BACKGROUND: Despite the fundamental biological importance and clinical relevance of characterizing the effects of chronic hypoxia exposure on central nervous system (CNS) development, the changes in gene expression from hypoxia are unknown. It is not known if there are unifying principles, properties, or logic in the response of the developing CNS to hypoxic exposure. Here, we use the small vertebrate zebrafish (Danio rerio) to study the effects of hypoxia on connectivity gene expression across development. We perform transcriptional profiling at high temporal resolution to systematically determine and then experimentally validate the response of CNS connectivity genes to hypoxia exposure. RESULTS: We characterized mRNA changes during development, comparing the effects of chronic hypoxia exposure at different time-points. We focused on changes in expression levels of a subset of 1270 genes selected for their roles in development of CNS connectivity, including axon pathfinding and synapse formation. We found that the majority of CNS connectivity genes were unaffected by hypoxia. However, for a small subset of genes hypoxia significantly affected their gene expression profiles. In particular, hypoxia appeared to affect both the timing and levels of expression, including altering expression of interacting gene pairs in a fashion that would potentially disrupt normal function. CONCLUSIONS: Overall, our study identifies the response of CNS connectivity genes to hypoxia exposure during development. While for most genes hypoxia did not significantly affect expression, for a subset of genes hypoxia changed both levels and timing of expression. Importantly, we identified that some genes with interacting proteins, for example receptor/ligand pairs, had dissimilar responses to hypoxia that would be expected to interfere with their function. The observed dysynchrony of gene expression could impair the development of normal CNS connectivity maps.

Methamphetamine-, d-Amphetamine-, and p-Chloroamphetamine-Induced Neurotoxicity Differentially Effect Impulsive Responding on the Stop-Signal Task in Rats.

Abused amphetamines, such as d-amphetamine (AMPH) and methamphetamine (METH), are highly addictive and destructive to health and productive lifestyles. The abuse of these drugs is associated with impulsive behavior, which is likely to contribute to addiction. The amphetamines also differentially damage dopamine (DA) and serotonin (5-HT) systems, which regulate impulsive behavior; therefore, exposure to these drugs may differentially alter impulsive behavior to effect the progression of addiction. We examined the impact of neurotoxicity induced by three amphetamines on impulsive action using a stop-signal task in rats. Animals were rewarded with a food pellet after lever pressing (i.e., a go trial), unless an auditory cue was presented and withholding lever press gained reward (i.e., a stop trial). Animals were trained on the task and then exposed to a neurotoxic regimen of either AMPH, p-chloroamphetamine (PCA), or METH. These regimens preferentially reduced DA transporter levels in striatum, 5-HT transporter levels in prefrontal cortex, or both, respectively. Assessment of performance on the stop-signal task beginning 1 week after the treatment revealed that AMPH produced a deficit in go-trial performance, whereas PCA did not alter performance on either trial type. In contrast, METH produced a deficit in stop-trial performance (i.e., impulsive action) but not go-trial performance. These findings suggest that the different neurotoxic consequences of substituted amphetamines are associated with different effects on inhibitory control over behavior. Thus, the course of addiction and maladaptive behavior resulting from exposure to these substances is likely to differ.

Transgenic FingRs for Live Mapping of Synaptic Dynamics in Genetically-Defined Neurons.

Tools for genetically-determined visualization of synaptic circuits and interactions are necessary to build connectomics of the vertebrate brain and to screen synaptic properties in neurological disease models. Here we develop a transgenic FingR (fibronectin intrabodies generated by mRNA display) technology for monitoring synapses in live zebrafish. We demonstrate FingR labeling of defined excitatory and inhibitory synapses, and show FingR applicability for dissecting synapse dynamics in normal and disease states. Using our system we show that chronic hypoxia, associated with neurological defects in preterm birth, affects dopaminergic neuron synapse number depending on the developmental timing of hypoxia.

A Serotonin Circuit Acts as an Environmental Sensor to Mediate Midline Axon Crossing through EphrinB2.

Modulation of connectivity formation in the developing brain in response to external stimuli is poorly understood. Here, we show that the raphe nucleus and its serotonergic projections regulate pathfinding of commissural axons in zebrafish. We found that the raphe neurons extend projections toward midline-crossing axons and that when serotonergic signaling is blocked by pharmacological inhibition or by raphe neuron ablation, commissural pathfinding is disrupted. We demonstrate that the serotonin receptor htr2a is expressed on these commissural axons and that genetic knock-down of htr2a disrupts crossing. We further show that knock-down of htr2a or ablation of the raphe neurons increases ephrinB2a protein levels in commissural axons. An ephrinB2a mutant can rescue midline crossing when serotonergic signaling is blocked. Furthermore, we found that regulation of serotonin expression in the raphe neurons is modulated in response to the developmental environment. Hypoxia causes the raphe to decrease serotonin levels, leading to a reduction in midline crossing. Increasing serotonin in the setting of hypoxia restored midline crossing. Our findings demonstrate an instructive role for serotonin in axon guidance acting through ephrinB2a and reveal a novel mechanism for developmental interpretation of the environmental milieu in the generation of mature neural circuitry. SIGNIFICANCE STATEMENT: We show here that serotonin has a novel role in regulating connectivity in response to the developmental environment. We demonstrate that serotonergic projections from raphe neurons regulate pathfinding of crossing axons. The neurons modulate their serotonin levels, and thus alter crossing, in response to the developmental environment including hypoxia. The findings suggest that modification of the serotonergic system by early exposures may contribute to permanent CNS connectivity alterations. This has important ramifications because of the association between premature birth and accompanying hypoxia, and increased risk of autism and evidence associating in utero exposure to some antidepressants and neurodevelopmental disorders. Finally, this work demonstrates that the vertebrate CNS can modulate its connectivity in response to the external environment.

Perseverative behavior in rats with methamphetamine-induced neurotoxicity.

Methamphetamine induces monoamine depletions thought to contribute to cognitive and behavioral dysfunctions. Previously, we reported that methamphetamine-induced neurotoxicity is associated with impaired formation of stimulus-response associations. Additionally, subjective observations suggested that behavioral flexibility might be affected. Thus, the present study examined whether methamphetamine neurotoxicity induces perseverative behavior. Rats were pretreated with (±)-methamphetamine (4 × 10 mg/kg, 2-hr intervals) or saline. Three weeks later, rats were trained to press a lever on one side of an operant chamber and then retrieve the reinforcer from a magazine on the opposite side until they reached criterion (>50 reinforcers/30-min). After four consecutive sessions performing the task at criterion, rats were sacrificed and brains removed for monoamine determinations. Methamphetamine-pretreated rats had ∼50% loss of striatal dopamine and prefrontal serotonin. Methamphetamine- and saline-pretreated rats were not different in the number of sessions required to reach criterion or in the total numbers of lever presses and/or head entries made across the four consecutive sessions at criterion-level performance. However, methamphetamine-pretreated rats earned fewer reinforcers, because they made extra lever-presses and head entries when they should have been retrieving the reinforcer or returning to the lever. Latencies for methamphetamine-pretreated rats to switch between the two behaviors also were significantly slower than latencies for controls. Interestingly, the degree of additional lever-presses negatively correlated with serotonin-transporter binding in the prefrontal cortex, even in saline-pretreated controls. These data suggest that methamphetamine-induced partial monoamine toxicity is associated with perseveration and that the degree of perseveration may depend on serotonin innervation of the frontal cortex.

Impaired formation of stimulus-response, but not action-outcome, associations in rats with methamphetamine-induced neurotoxicity.

Methamphetamine (METH) induces neurotoxic changes, including partial striatal dopamine depletions, which are thought to contribute to cognitive dysfunction in rodents and humans. The dorsal striatum is implicated in action-outcome (A-O) and stimulus-response (S-R) associations underlying instrumental learning. Thus, the present study examined the long-term consequences of METH-induced neurotoxicity on A-O and S-R associations underlying appetitive instrumental behavior. Rats were pretreated with saline or a neurotoxic regimen of METH (4 × 7.5-10 mg/kg). Rats trained on random ratio (RR) or random interval (RI) schedules of reinforcement were then subjected to outcome devaluation or contingency degradation, followed by an extinction test. All rats then were killed, and brains removed for determination of striatal dopamine loss. The results show that: (1) METH pretreatment induced a partial 45-50% decrease in striatal dopamine tissue content in dorsomedial and dorsolateral striatum; (2) METH-induced neurotoxicity did not alter acquisition of instrumental behavior on either RR or RI schedules; (3) outcome devaluation and contingency degradation similarly decreased responding in saline- and METH-pretreated rats trained on the RR schedule, suggesting intact A-O associations guiding behavior; (4) outcome devaluation after training on the RI schedule decreased extinction responding only in METH-pretreated rats, suggesting impaired S-R associations. Overall, these data suggest that METH-induced neurotoxicity, possibly due to impairment of the function of dorsolateral striatal circuitry, may decrease cognitive flexibility by impairing the ability to automatize behavioral patterns.

Signal intensities of radiolabeled cRNA probes used alone or in combination with non-isotopic in situ hybridization histochemistry.

This study addressed the question of whether radioactive hybridization signal intensities are reduced in combined isotopic and non-isotopic double in situ hybridization (DISH) compared with those in single in situ hybridization (ISH). Non-isotopic digoxigenin (Dig)-labeled hybrids were detected using an alkaline phosphatase (AP) enzymatic reaction which results in nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)-salt precipitation that could shield S35-radiation from penetrating to the surface. Sections were plastic coated of with 2% parlodion to prevent a chemical reaction between AP and developer during processing of the photosensitive emulsion, which could further reduce radioactive hybridization signal detection by autoradiography. We used DISH with a hybridization cocktail of radioactive S35- and Dig-labeled GAD67 cRNA probes. In order to avoid competition for the same complementary sequence, the probes were directed towards different sequences of the glutamic acid decarboxylase (GAD67) mRNA, resulting in co-detection of isotopic and non-isotopic hybrids in close to 100% of GAD67 positive cells. Quantitation of autoradiograms showed that there was no reduction of autoradiographic signal intensity from S35-labeled hybrids in the presence of Dig-labeled hybrids. Plastic coating of single or dual hybridized sections did not reduce the radioactive signal intensity. When mRNAs for nicotinic acetylcholine receptor (nAChR) subunits were detected with subunit specific S35-labeled cRNA probes in GAD67 hippocampal interneurons the total numbers of nAChR subunit expressing cells remained the same in single or double hybridized sections even for low abundant mRNAs. Together, these results indicate that combined radioactive and non-radioactive DISH does not interfere with the detection of the radiation signal from the S35-labeled hybrids, and neither specificity nor sensitivity is compromised.

Chronic neonatal nicotine exposure increases mRNA expression of neurotrophic factors in the postnatal rat hippocampus.

Nicotine, the psychoactive ingredient in tobacco, can be neuroprotective but the mechanism is unknown. In the adult hippocampus, chronic nicotine can increase expression of growth factors which could contribute to nicotine's neuroprotective effects. During development, nicotine could also increase expression of neurotrophic factors. Therefore, we determined whether chronic neonatal nicotine (CNN) exposure increased mRNA expression levels of brain-derived neurotrophic factor (BDNF), nerve-growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). Nicotine (6 mg/kg/day in milk formula) or milk formula (controls) were delivered in three daily doses via oral gastric intubation to rat pups from postnatal day (P)1 to P8, and then sacrificed. Brains were processed for in situ hybridization using specific (35)S-labeled cRNA probes. At P8, CNN had a significant stimulant treatment effect on the expression of BDNF, FGF-2, NT-3 and IGF-1 [p<0.01], but not NGF. Specifically, BDNF mRNA expression, detected in CA1, CA3 stratum (s.) pyramidal and granule cell layer of the dentate gyrus (DG), was increased by 27.4%, 23.26% and 27.3%, respectively. FGF-2 mRNA expression, detected in neurons and astrocytes in CA1 s. radiatum, CA2 and CA3 s. pyramidale, and molecular layer of the DG, was increased by 34.0%, 8.9%, 31.0% and 23.1%, respectively. NT-3 mRNA expression in CA2 s. pyramidale was increased by 80.0%, and CNN increased the number of IGF-1-expressing cells in CA1 (18.0%), CA3 (20.9%) and DG (17.7%). Thus, nicotine exposure during early postnatal development differentially up-regulated expression of neurotrophic factor mRNAs in the hippocampus, which could increase neurotrophic tone and alter developmental processes.

Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons.

Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.