Yu-Chia Chen

The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases.

Modulatory neurotransmitters which signal through G protein-coupled receptors control brain functions which deteriorate in degenerative brain diseases. During the past decade many of these systems have been mapped in the zebrafish brain. The main architecture of the systems in zebrafish brain resembles that of the mammals, despite differences in the development of the telencephalon and mesodiencephalon. Modulatory neurotransmitters systems which degenerate in human diseases include dopamine, noradrenaline, serotonin, histamine, acetylcholine and orexin/hypocretin. Although the number of G protein-coupled receptors in zebrafish is clearly larger than in mammals, many receptors have similar expression patterns, binding and signaling properties as in mammals. Distinct differences between mammals and zebrafish include duplication of the tyrosine hydroxylase gene in zebrafish, and presence of one instead of two monoamine oxidase genes. Zebrafish are sensitive to neurotoxins including MPTP, and exposure to this neurotoxin induces a decline in dopamine content and number of detectable tyrosine hydroxylase immunoreactive neurons in distinct nuclei. Sensitivity to important neurotoxins, many available genetic methods, rapid development and large-scale quantitative behavioral methods in addition to advanced quantitative anatomical methods render zebrafish an optimal organism for studies on disease mechanisms.

Adaptive changes in zebrafish brain in dominant–subordinate behavioral context

Male zebrafish were held in dyadic social stress situation for a period of 5 days, to characterize stress coping styles and to investigate the role of the underlying neuroendocrine mechanisms in establishing dominant-subordinate relationships. A strong consistent dominant-subordinate relationship was formed in ten out of the sixteen pairs of fish (62.5%). Both dominant (DOM) and subordinate (SUB) individuals showed statistically significant higher trunk cortisol concentration than controls. Expression of genes encoding proteins involved in the functioning of the hypothalamus-hypophysis-interrenal axis (corticotropin releasing factor, CRF; glucocorticoid receptor, GR; mineralocorticoid receptor, MR); arginine vasotocin, AVT), in the biosynthesis and catabolism of catecholamines (tyrosine hydroxylase, TH1 and TH2; DOPA decarboxylase, DDC), dopamine β-hydroxylase, DBH; catechol-O-methyl transferase, COMT), in the biosynthesis of histamine (histidine decarboxylase, HDC) and in the general stress response (galanin, GAL; hypocretin/orexin, Hcrt) was examined. The MR/GR ratio was higher in dominant and subordinate fish than in controls (P=0.016). The mRNA levels of TH2 and HDC were up-regulated in DOM, of AVT in SUB, while COMT mRNA levels were down-regulated in both DOM and SUB compared to control fish. In addition, mRNA levels of hypocretin/orexin (Hcrt) were up-regulated in dominant compared to subordinate and control males. There was a statistically significant correlation between mRNA expression levels of TH2, HDC, Hcrt, GR, MR and CRF genes. The obtained results provide new evidences for the use of zebrafish as an animal model to study social stress and allostasis in vertebrates.

Dopaminergic cell damage and vulnerability to MPTP in Pink1 knockdown zebrafish

The functions of PTEN (phosphatase/tensin homolog)-induced putative kinase (PINK1), which is mutated in early-onset Parkinsons disease, are poorly understood. We characterized a PINK1 antibody and found colocalization of PINK1-like immunoreactivity with aminergic markers. We inactivated translation of Pink1 using morpholino-oligonucleotides (MOs) in larval zebrafish. Dopaminergic neurons consisted of two sets of neuron populations, marked by complementary expression of two tyrosine hydroxylase genes th1 and th2. Translation inhibition of pink1 resulted in reduction of both th mRNA forms until day 5 or 7, respectively. The affected dopaminergic neurons were in one group expressing th1 and three groups expressing th2. Lack of Pink1 sensitized the fish to subeffective doses of MPTP, which caused a locomotor deficit and facilitated loss of th1 in one diencephalic dopaminergic cell group. Control experiments with pink1 mRNA and control MO suggested that effects with the splice site targeting MO were specific. Distinct groups of dopaminergic neurons are thus sensitive to loss of Pink1. Sensitization of the pink1 morphant fish to MPTP toxicity suggests that genetic factors play a role in toxin-induced Parkinsons disease.

Dopamine from the Brain Promotes Spinal Motor Neuron Generation during Development and Adult Regeneration

Coordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.

The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish

The histaminergic and hypocretin/orexin (hcrt) neurotransmitter systems play crucial roles in alertness/wakefulness in rodents. We elucidated the role of histamine in wakefulness and the interaction of the histamine and hcrt systems in larval zebrafish. Translation inhibition of histidine decarboxylase (hdc) with morpholino oligonucleotides (MOs) led to a behaviorally measurable decline in light‐associated activity, which was partially rescued by hdc mRNA injections and mimicked by histamine receptor H1 (Hrh1) antagonist pyrilamine treatment. Histamine‐immunoreactive fibers targeted the dorsal telencephalon, an area that expresses histamine receptors hrh1 and hrh3 and contains predominantly glutamatergic neurons. Tract tracing with DiI revealed that projections from dorsal telencephalon innervate the hcrt and histaminergic neurons. Translation inhibition of hdc decreased the number of hcrt neurons in a Hrh1‐dependent manner. The reduction was rescued by overexpression of hdc mRNA. hdc mRNA injection alone led to an up‐regulation of hcrt neuron numbers. These results suggest that histamine is essential for the development of a functional and intact hcrt system and that histamine has a bidirectional effect on the development of the hcrt neurons. In summary, our findings provide evidence that these two systems are linked both functionally and developmentally, which may have important implications in sleep disorders and narcolepsy.—Sundvik, M., Kudo, H., Toivonen, P., Rozov, S., Chen, Y.‐C., Panula, P. The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish. FASEB J. 25, 4338–4347 (2011). www.fasebj.org

Glucocerebrosidase 1 deficient Danio rerio mirror key pathological aspects of human Gaucher disease and provide evidence of early microglial activation preceding alpha-synuclein-independent neuronal cell death

Autosomal recessively inherited glucocerebrosidase 1 (GBA1) mutations cause the lysosomal storage disorder Gauchers disease (GD). Heterozygous GBA1 mutations (GBA1+/−) are the most common risk factor for Parkinsons disease (PD). Previous studies typically focused on the interaction between the reduction of glucocerebrosidase (enzymatic) activity in GBA1+/− carriers and alpha-synuclein-mediated neurotoxicity. However, it is unclear whether other mechanisms also contribute to the increased risk of PD in GBA1+/− carriers. The zebrafish genome does not contain alpha-synuclein (SNCA), thus providing a unique opportunity to study pathogenic mechanisms unrelated to alpha-synuclein toxicity. Here we describe a mutant zebrafish line created by TALEN genome editing carrying a 23 bp deletion in gba1 (gba1c.1276_1298del), the zebrafish orthologue of human GBA1. Marked sphingolipid accumulation was already detected at 5 days post-fertilization with accompanying microglial activation and early, sustained up-regulation of miR-155, a master regulator of inflammation. gba1c.1276_1298del mutant zebrafish developed a rapidly worsening phenotype from 8 weeks onwards with striking reduction in motor activity by 12 weeks. Histopathologically, we observed marked Gaucher cell invasion of the brain and other organs. Dopaminergic neuronal cell count was normal through development but reduced by >30% at 12 weeks in the presence of ubiquitin-positive, intra-neuronal inclusions. This gba1c.1276_1298del zebrafish line is the first viable vertebrate model sharing key pathological features of GD in both neuronal and non-neuronal tissue. Our study also provides evidence for early microglial activation prior to alpha-synuclein-independent neuronal cell death in GBA1 deficiency and suggests upregulation of miR-155 as a common denominator across different neurodegenerative disorders.

High Mobility Group Box-1 (HMGB1; Amphoterin) Is Required for Zebrafish Brain Development

Hmgb1 (high mobility group box-1; amphoterin) is highly expressed in brain during early development of vertebrate and nonvertebrate species. However, its role in brain development remains elusive. Here we have cloned the zebrafish Hmgb1 and specifically manipulated Hmgb1 expression using injection of morpholino antisense oligonucleotides or Hmgb1 cRNA. The HMGB1 knockdown morphants produced by injection of three different morpholino oligonucleotides display a characteristic phenotype with smaller size, smaller brain width, and shorter distance between the eyes. Closer examination of the phenotype reveals severe defects in the development of the forebrain that largely lacks catecholaminergic neural networks. The HMGB1 morphant is deficient in survival and proliferation of neural progenitors and displays fewer cell groups expressing the transcription factor Pax6a in the forebrain and aberrant Wnt8 signaling. The mechanism of HMGB1-dependent progenitor survival involves the neuronal transmembrane protein AMIGO (amphoterin-induced gene and orf), the expression of which is regulated by HMGB1 in vivo. Our data demonstrate that HMGB1 is a critical factor for brain development, enabling survival and proliferation of neural progenitors that will form the forebrain structures.

Acute ethanol treatment upregulates th1, th2, and hdc in larval zebrafish in stable networks

Earlier studies in zebrafish have revealed that acutely given ethanol has a stimulatory effect on locomotion in fish larvae but the mechanism of this effect has not been revealed. We studied the effects of ethanol concentrations between 0.75 and 3.00% on 7-day-old larval zebrafish (Danio rerio) of the Turku strain. At 0.75-3% concentrations ethanol increased swimming speed during the first minute. At 3% the swimming speed decreased rapidly after the first minute, whereas at 0.75 and 1.5% a prolonged increase in swimming speed was seen. At the highest ethanol concentration dopamine levels decreased significantly after a 10-min treatment. We found that ethanol upregulates key genes involved in the biosynthesis of histamine (hdc) and dopamine (th1 and th2) following a short 10-min ethanol treatment, measured by qPCR. Using in situ hybridization and immunohistochemistry, we further discovered that the morphology of the histaminergic and dopaminergic neurons and networks in the larval zebrafish brain was unaffected by both the 10-min and a longer 30-min treatment. The results suggest that acute ethanol rapidly decreases dopamine levels, and activates both forms or th to replenish the dopamine stores within 30 min. The dynamic changes in histaminergic and dopaminergic system enzymes occurred in the same cells which normally express the transcripts. As both dopamine and histamine are known to be involved in the behavioral effects of ethanol and locomotor stimulation, these results suggest that rapid adaptations of these networks are associated with altered locomotor activity.

Presenilin1 Regulates Histamine Neuron Development and Behavior in Zebrafish, Danio rerio

Modulatory neurotransmitters, including the histaminergic system, are essential in mediating cognitive functions affected in Alzheimers disease (AD). The roles of disease genes associated with AD, most importantly the presenilin1 gene (psen1), are poorly understood. We studied the role of psen1 in plasticity of the brain histaminergic system using a novel psen1 mutant zebrafish, Danio rerio. We found that in psen1−/− zebrafish, the histaminergic system is altered throughout life. At 7 d postfertilization (dpf) the histamine neuron number was reduced in psen1−/− compared with wild-type (WT) fish; at 2 months of age the histamine neuron number was at the same level as that in WT fish. In 1-year-old zebrafish, the histamine neuron number was significantly increased in psen1−/− fish compared with WT fish. These changes in histamine neuron number were accompanied by changes in histamine-driven behaviors. Treatment with DAPT, a γ-secretase inhibitor, similarly interfered with the development of the histaminergic neurons. We also assessed the expression of γ-secretase-regulated Notch1a mRNA and β-catenin at different time points. Notch1a mRNA level was reduced in psen1−/− compared with WT fish, whereas β-catenin was slightly upregulated in the hypothalamus of psen1−/− compared with WT fish at 7 dpf. The results reveal a life-long brain plasticity in both the structure of the histaminergic system and its functions induced by altered Notch1a activity as a consequence of psen1 mutation. The new histaminergic neurons in aging zebrafish brain may arise as a result of phenotypic plasticity or represent newly differentiated stem cells.

Galanin gene expression and effects of its knock-down on the development of the nervous system in larval zebrafish

Despite the known importance of galanin in the nervous system of vertebrates, the galanin gene structure and expression and the consequences of galanin deficiency in developing zebrafish are unknown. We cloned the galanin gene and analyzed its expression by using in situ hybridization, PCR, and immunocytochemistry throughout the early development of zebrafish until the end of the first week of life. The single zebrafish galanin gene encoded for a single amidated galanin peptide and a galanin message‐associated peptide. Two forms resulting from alternative processing were identified. Galanin mRNA was maternally expressed and found in developing fish throughout early development. In situ hybridization showed the first positive neurons in three groups in the brain at 28 hours postfertilization. At 2 days postfertilization, three prosencephalic neuron groups were seen in the preoptic area and in rostral and caudal periventricular hypothalamus. In addition, two other groups of weakly stained neurons were visible, one in the midbrain and another in the hindbrain. Translation inhibition of galanin mRNA with morpholino oligonucleotides caused complete disappearance of galanin immunoreactivity in the brain until 7 dpf and did not induce known cascades of nonspecific pathways or morphological abnormalities. A minor disturbance of sensory ganglia was found. Galanin knockdown did not alter the expression of tyrosine hydroxylases 1 and 2, choline acetyltransferase, histidine decarboxylase, or orexin mRNA. The results suggest that galanin does not regulate the development of these key markers of specific neurons, although galanin‐expressing fibers were in a close spatial proximity to several neurons of these neuronal populations. J. Comp. Neurol. 520:3846–3862, 2012.