A Review of the Association of Blue Food Coloring With Attention Deficit Hyperactivity Disorder Symptoms in Children.

Attention Deficit Hyperactivity Disorder, also known as ADHD, is a neurodevelopmental disorder diagnosed in children. The exact cause of ADHD is not known, but, along with genetic factors, it is possible that environmental factors including toxins and diet may affect symptom severity. Of these dietary components, artificial food coloring (AFC), while approved by the Food and Drug Administration (FDA), has been suspected to be associated with ADHD symptoms. Of the nine FDA-certified food colors, two are used for artificial blue coloring: Blue No. 1 and Blue No. 2. There is limited literature describing the possible role of blue AFC in causing symptoms of ADHD in children. This paper provides a review of the literature surrounding artificial food coloring and its ability to affect the neurodevelopment of children in a way that could increase the behavioral indicators of ADHD. To do this, search criteria were established using a combination of MeSH terms about blue AFCs and ADHD and were entered into PubMed, along with limits on article types and publication dates from January 2000 to June 2022. There was a total of 20 articles that met this search criterion. These articles were reviewed by authors, and the ones not relevant to the topic were excluded. In total, four studies were chosen to be included in this article. After reviewing the literature, it was found that restriction diets, specifically those excluding AFCs, may affect symptom severity. The source of these changes is not known, but possible mechanisms include AFCs causing nutritional deficiencies and allergic reactions or altering neurotransmitter levels. More research is necessary to describe the neurotoxicity of artificial blue dyes in humans.

Genomic strategies for the identification of dopamine receptor genes in zebrafish.

In this chapter, we describe the identification and cloning of D2-like dopamine receptor (DR) genes in zebrafish, a vertebrate model genetic organism. To identify DR genes, we performed searches of the zebrafish genomic sequence database that yielded contig segments of several D2-like DR genes. From these sequences, we amplified full-length cDNAs encoding three D2, one D3, and three D4 DR receptor subtypes via RT-PCR. The predicted proteins displayed 57-72% amino acid identity when compared to their human DR counterparts. To validate the identity of zebrafish DR genes, each of the genes was mapped by using the T51 radiation hybrid panel. With the exception of drd2b and drd4b, each of the zebrafish DR genes mapped to chromosomal positions that were syntenic with regions of human chromosomes containing orthologs of the zebrafish DR genes. To further validate the identity of the D2-like DR genes in zebrafish, we conducted phylogenetic analysis which supported the predicted identities of the cloned DR receptor cDNAs.

Identification of zebrafish A2 adenosine receptors and expression in developing embryos.

The A2A adenosine receptor (AdR) subtype has emerged as an attractive target in the pursuit of improved therapy for Parkinson's disease (PD). This report focuses on characterization of zebrafish a2 AdRs. By mining the zebrafish EST and genomic sequence databases, we identified two zebrafish a2a (adora2a.1 and adora2a.2) genes and one a2b (adora2b) AdR gene. Sequence comparisons indicate that the predicted zebrafish A2 AdR polypeptides share 62-74% amino acid identity to mammalian A2 AdRs. We mapped the adora2a.1 gene to chromosome 8, the adora2a.2 gene to chromosome 21, and the adora2b gene to chromosome 5. Whole mount in situ hybridization analysis indicates zebrafish a2 AdR genes are expressed primarily within the central nervous system (CNS). Zebrafish are known to be sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes selective loss of dopaminergic neurons and PD-like symptoms in humans as well as in animal models. Here we show that caffeine, an A2A AdR antagonist, is neuroprotective against the adverse effects of MPTP in zebrafish embryos. These results suggest that zebrafish AdRs may serve as useful targets for testing novel therapeutic strategies for the treatment of PD.

Neuronal calcium sensor-1 gene ncs-1a is essential for semicircular canal formation in zebrafish inner ear.

We have analyzed the functional role of neuronal calcium sensor-1 (Ncs-1) in zebrafish development. We identified two orthologs of the mammalian NCS-1 gene. Full-length cDNAs encoding zebrafish Ncs-1a and Ncs-1b polypeptides were cloned and characterized. Whole-mount in situ hybridization revealed that ncs-1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs-1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs-1a expression was detected primarily in the brain. Expression of ncs-1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs-1b was expressed at low levels throughout the brain. Knockdown of ncs-1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs-1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function.

Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish.

We mined the zebrafish genomic sequence database and identified contigs containing segments of several dopamine receptor genes. By using a polymerase chain reaction amplification strategy, we generated full-length cDNAs encoding a single dopamine D3 receptor and three distinct D2 receptor subtypes. Zebrafish dopamine receptor genes were mapped by using the T51 radiation hybrid panel. The D3 receptor gene (drd3) mapped to linkage group (LG) 24. The three D2 receptor genes were localized to LG 15 (drd2a), LG 16, (drd2b), and LG 5 (drd2c). With the exception of the drd2b gene, each of these map positions was syntenic with regions of human chromosomes containing orthologs of the zebrafish dopamine receptor genes. Whole-mount in situ hybridization was used to investigate expression of the D2 and D3 receptor genes. Expression of the drd3 gene was first detected at mid-somitogenesis and was particularly prominent in somites. Thereafter, the drd3 gene was expressed diffusely throughout the brain and spinal cord. The three D2 receptor genes were expressed throughout the central nervous system (CNS) in distinct but overlapping patterns. In early embryos, the drd2a gene was expressed exclusively in the epiphysis, whereas the drd2c gene was localized to the notochord. After 24 hpf, the drd2a, drd2b, and drd2c genes were differentially expressed throughout the CNS. The identification of dopamine receptor genes in zebrafish should allow us to use the power of zebrafish genetics to analyze the functional properties of this important class of neurotransmitter receptors.

Differential expression of Na,K-ATPase alpha and beta subunit genes in the developing zebrafish inner ear.

We have used whole-mount in situ hybridization to analyze Na,K-ATPase alpha and beta subunit gene expression in the developing zebrafish ear. Four alpha1-like (alpha1a.1, alpha1a.2, alpha1a.4, and alpha1a.5) and two beta (beta1a and beta2b) subunit genes are expressed in ear beginning at mid-somitogenesis. Each gene exhibits a distinct spatial and temporal expression pattern. The alpha1a.1 gene was ubiquitously expressed in the otic epithelium from mid-somitogenesis to 24 hr postfertilization (hpf). Expression of this gene was gradually reduced and by 48 hpf, alpha1a.1 transcripts were no longer detectable in the ear. The alpha1a.2 and alpha1a.5 genes were expressed in regions that correspond to the anterior macula, lateral crista, and semicircular canal projections up to 48 hpf. At later stages, expression of these genes was limited to cells in the dorsolateral septum and semicircular canal projections. alpha1a.4 and beta1a transcripts were ubiquitously expressed during ear development and were present in most otic tissues at 5 days postfertilization (dpf). Expression of the beta2b gene, on the other hand, was restricted to subsets of cells that form sensory epithelia. These results strongly suggest different functional roles for individual Na,K-ATPase genes in zebrafish ear development. Na,K-ATPase genes are likely to represent useful markers for the analysis of zebrafish otogenesis.