Zebrafish Get Connected: Investigating Neurotransmission Targets and Alterations in Chemical Toxicity

Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning, and memory. Exposure to drugs and chemicals can alter neurotransmission, often through unknown pathways and mechanisms. The zebrafish (Danio rerio) model system is increasingly being used to study the brain and chemical neurotoxicity. In this review, the major neurotransmitter systems, including glutamate, GABA, dopamine, norepinephrine, serotonin, acetylcholine, histamine, and glutamate are surveyed and pathways of synthesis, transport, metabolism, and action are examined. Differences between human and zebrafish neurochemical pathways are highlighted. We also review techniques for evaluating neurological function, including the measurement of neurotransmitter levels, assessment of gene expression through transcriptomic analysis, and the recording of neurobehavior. Finally examples of chemical toxicity studies evaluating alterations in neurotransmitter systems in the zebrafish model are reviewed.


Introduction
Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning and memory, and even maintenance of life. The nervous system is exceedingly complex, and many enzymes, transporters, and receptors all work in concert to maintain these functions. Neurotransmission can be altered by exposure to drugs, pharmaceuticals, chemotherapeutic agents, radiation, food additives, and environmental toxicants including pesticides and heavy metals ( [1,2] and as reviewed by [3][4][5][6]). Alterations in neurotransmission have been linked to a number of diseases including movement disorders, neuropsychiatric disorders, and depression (reviewed by [7][8][9]).
The zebrafish (Danio rerio) research model is increasingly being used in neurotoxicity studies (as reviewed by [3,[10][11][12][13]). Zebrafish share the common neurotransmitter pathways with mammals and have similar neuroanatomy in many areas such as the spinal cord, hindbrain and retina, but as the brain develops by eversion rather than inversion, some classical regions of the mammalian brain, such as the hippocampus, amygdala, and substantia nigra, are not present as such in zebrafish. The function of these areas appears to be maintained elsewhere in the brain, allowing functional comparisons between zebrafish and mammals [14]. As an additional resource, Mueller and Wullimann have recently published a second edition of their "Atlas of Early Zebrafish Brain Development", which characterizes neuronal development and provides excellent figure panels for neuroanatomy [15].
Although the neurochemistry of zebrafish has been reviewed previously [11,16], this review will specifically focus on reviewing the application of the zebrafish in chemical toxicology studies    * Zebrafish grm2b has previously been known as grm7.
During embryonic and post embryonic neurogenesis, proneural and neuronal cells express genes coding for basic helix-loop-helix (Bhlh) transcription factors and the patterns of expression can help identify neuronal populations [68]. The expression of neurogenin 1 (Neurog1)/Neurogenic differentiation 1 (Neurod; NeuroD1) has been linked to the development of glutamatergic neurons [69].
Identification of glutamatergic neurons in adult brains is often made based on the presence of transporter proteins. The SLC17A (VGLUT) genes SLC17A6 and SLC17A7 are often used as markers of glutamatergic neurons; however, glial cells can also express these transporters [33,70,71]. Glutamate itself is a poor marker due to its role in many metabolic pathways. Although the various receptor subunits are differentially expressed throughout the brain, glutamate receptors are also expressed on glia [72]. For a further account on distribution of these receptors in zebrafish please see Haug et al. [64] and Huang et al. [65].

GABA
GABA is the major inhibitory neurotransmitter in the central nervous system (CNS) and GABAergic neurons are widely present throughout the brain. As an inhibitory neurotransmitter, GABA mainly acts to modulate neural systems and the activity of postsynaptic cells [73]. GABA has been associated with the regulation of neural transmission and perturbances in the GABAergic system have been associated with epilepsy, depression, schizophrenia, and sleep dysfunction [74][75][76].
As reviewed by Ben-Ari and Reynolds et al. [77,78], in early development neurons have higher intracellular levels of chloride than mature neurons due to the expression of the sodium-potassium-chloride cotransporter 1 (NKCC1; SLC12A2) in the absence of potassium-chloride cotransporter 2 (KCC2; SLC12A5) expression [79]. Therefore, the classic inhibitory neurotransmitters GABA and glycine act to depolarize, and excite the immature neuron. Concurrent with neuronal maturation, SLC12A5 is expressed and this transporter reverses the chloride gradient, establishing the adult chloride gradient and causing GABA and glycine to act as inhibitory neurotransmitters [80].
GABA is synthesized in neurons through the GABA shunt. In the first step, α-ketoglutarate from the TCA cycle is transaminated by the 4-aminobutyrate transaminase (ABAT; GABA α-ketoglutarate transaminase; GABA-T) enzyme into L-glutamic acid. In the second step, glutamate decarboxylase (GAD) removes the carboxyl group and produces GABA. In mammals, two genes code for GAD, GAD1 and GAD2 (also known as GAD 67 and GAD 65 , respectively) [81].
GABA is packaged into synaptic vesicles by the SLC32A1 solute carrier, also known as Vesicular GABA transporter (VGAT). Upon presynaptic depolarization, the vesicles are released into the synaptic space. SLC6A family members, SLC6A1, SLC6A11, and SLC6A12, also known as GABA transporters (GAT1, GAT3, and BGT1) are responsible for transporting GABA out of the synapse [82]. SLC6A13 (GAT2) does not appear to have a significant role in the brain [73]. Neurons may recycle the collected GABA back into synaptic vesicles or GABA can be metabolized to succinic semialdehyde by ABAT [83]. In glia, which lack GAD, the succinic semialdehyde is oxidized by succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5 family member A1; ALDH5A) into succinic acid, which enters the TCA cycle and can be cycled through to α-ketoglutarate to again produce glutamine [73,84].
GABA has two classes of receptors: GABA A and GABA B . As mentioned previously, GABA A receptors are ligand-gated ion channel receptors and mediate postsynaptic membrane hyperpolarization through the influx of chloride through their integral channel [85]. Like all ligand-gated ion channels, GABA A receptors are pentamers and 19 subunits in 7 classes provide basis of the regional variations and differential actions [86].
GABA B receptors are metabotropic and therefore can mediate a variety of effects through their coupling with G proteins [87][88][89]. GABA B receptors can activate certain potassium channels, regulate IP3, or inhibit cAMP production [89]. Presynaptic GABA receptors may inhibit presynaptic neurotransmitter release [90]. Two GABA B receptor subunits have been identified in mammals, GABABR1 and GABABR2 (reviewed in [87,91]). Table 2 list genes important for the synthesis, metabolism, and action of GABAergic neurons in humans and the known zebrafish paralogs. Zebrafish have a single copy of some GABAergic genes, such as the transaminase abat, the VGAT transporter slc32a1, and aldh5a1 (SSADH). Other genes such as gad1 (glutamate decarboxylase), slc6a1 (GAT1), and slc6a11 (GAT3) have two zebrafish paralogs [92]. Corresponding zebrafish orthologs or paralogs have not been identified for all human GABA receptor subunits (e.g., GABRA2 and GABRA4), but some human genes have more than one paralog in zebrafish (e.g., GABBR1: gabbr1a and gabbr1b).  GAD has only been identified in GABAergic neurons, therefore providing a specific marker for GABA producing neurons. Additionally, zebrafish achaete-scute homolog 1a (Zash1a) expression has been linked to the development of GABA producing, inhibitory neurons, and has been used as a marker for GABAergic neurons during development [93,94].
In zebrafish, GABA is widely produced in the brain and spinal cord by interneurons [95]. The postembryonic (3 days post fertilization; dpf) subpallium, preoptic region, ventral and sections of the dorsal thalamus, and hypothalamus have been shown to produce GABA [93]. In the adult, GABA has been identified in the olfactory bulb, subpallium, preoptic, pretectal, ventral thalamic, hypothalamic, and posterior tubercular nuclei with scattered Gad1 positive cells in the pallial zones and the bed nucleus of the stria medullaris [96,97]. GABA, Gad2, Gabra1, and Gabbr1 are expressed in the zebrafish cerebellum and have a similar distribution to mammals [98].

Catecholamines
Dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline) are the major catecholamine neurotransmitters. Structurally, this group is characterized by a catechol group (benzene group with two adjacent hydroxyl groups), with an ethylamine side chain and an amine group. Catecholamines are considered modulatory neurotransmitters and have been linked to reward, movement, memory, and neuropsychiatric disorders [99][100][101].
Catecholamines are formed from the amino acid tyrosine and oxygen. Tyrosine hydroxylase (tyrosine 3-monooxygenase; TH) is the first enzyme in the synthesis pathway and is the rate limiting step. TH produces 3,4-dihydroxyl-l-phenylalanine (L-DOPA) with (6R)-L-erythro-tetrahydrobiopterin (BH4) and Fe 2+ acting as cofactors in the hydroxylase step [102]. L-DOPA is converted to dopamine (DA) by the enzyme aromatic amino acid decarboxylase (AAAD), also known as DOPA decarboxylase [103]. AAAD is the preferred enzyme name as AAAD is also important in the monoamine serotonin synthesis pathway [82]. Although TH immunoreactivity is considered specific for dopaminergic neurons, AAAD can be found in non-monoamine producing neurons and glial cells. AAAD can also alternatively produce trace amines such as tryptamine, tyramine, and 2-phenylethylamine [104].
Catecholamines are transported into vesicles through members of the SLC18 family, namely SLC18A2 (also known as VMAT2) in the brain (as reviewed by [105,106]). SLC18A2 can transport serotonin and histamine as well as catecholamines. Dopamine and norepinephrine transporters belong to the SLC6 family of carriers, with, at least in mammals, SLC6A2 functioning as the norepinephrine transporter (NET), and SLC6A3 as the dopamine transporter (DAT) [107].
Catecholamines are metabolized by a monoamine oxidase (MAO) enzyme. In humans, there are two isoforms of MAO in mammals, MAO-A and MAO-B, with different substrate specificity, pharmacology, and anatomic localization [108]. The catecholamine substrates are degraded into aldehydes, and aldehyde dehydrogenase and aldehyde reductase further degrade the products into alcohols or glycols respectively. Catechol-O-methyltransferase (COMT) adds a methyl group to catecholamines and their metabolites, which assists in elimination of the neurotransmitters and their metabolites. A major DA metabolite is 3,4-dihydroxyphenylacetic acid (DOPAC), which is the product of MAO and aldehyde dehydrogenase. DOPAC can be further metabolized by COMT to form homovanillic acid (HVA). Norepinephrine is mostly metabolized by MAO and aldehyde reductase, forming 3,4-dihydroxyphenylglycol (DHPG). Likewise, DHPG can be further metabolized to 3-methoxy-4-hydroxyphenylgylcol (MHPG) [104].
Catecholamines bind to G-protein-coupled receptors to modulate neurotransmission. As reviewed by Callier et al., there are five dopaminergic receptors in mammals: D1-D5, with D1 and D5 (D1-like) and D2-D4 (D2-like) sharing similar mechanisms [109]. D1-like receptors classically activate G s G-proteins causing an increase in cAMP while D2-like activate Gα i /G o G-proteins which inhibits adenylyl cyclase activity [110]. Beaulieu et al. extensively reviewed the mechanisms of dopamine receptor signaling [111]. Norepinephrine has nine receptors organized into three families, α 1 , α 2 , and β, each containing three receptors. α 1 receptors activate G q /G 11 proteins and activate phospholipase C and increase intra cellular calcium and protein kinase C activation. The α 2 adrenergic receptors are coupled to G i /G o proteins and inhibit adenylyl cyclase and stimulate phospholipase A2. The β adrenergic receptors are coupled to G s and activate adenylyl cyclase [104].
Originally two Th encoding paralogs were identified in teleosts, th1 and th2 [112]. th1-negative, th2-positive neurons were identified in zebrafish brain [113][114][115] and although these neurons appeared to be immunoreactive for slc18a2 (vmat2), aaad, and slc6a3 (dat), consistent with a dopaminergic phenotype, it was discovered that the gene encoded by th2 appears to function as a tryptophan hydroxylase when isolated in vitro [115,116]. Therefore, th1 is referred to as th by the Zebrafish Information Network (ZFIN) and is considered the only tyrosine hydroxylase in zebrafish. Currently, only one ortholog of AAAD (aaad), DBH (dbh), and of the transporters SLC18A2 (slc18a2), SLC6A2 (slc6a2), and SLC6A3 (slc6a3) have been identified. Although mammals have two isozymes, MAO-A and MAO-B, zebrafish have only one paralogous enzyme, Mao (also known as Zmao) [117]. Zebrafish Mao may have a structure and function more similar to MAO-A, but is inhibited by deprenyl, a MAO-B specific inhibitor [118,119]. Two putative COMT genes have been identified, comta and comtb. However, the gene products have not been fully characterized, and some protein products may not be functional [16,120]. Table 3 outlines known paralogs of dopamine and adrenergic receptors [121,122]. The receptor genes drd1a and drd1b likely have D1-like activity and the other receptor genes likely have D2-like functions [122]. Table 3. Zebrafish genes involved in catecholamine neurotransmission.
In order to help compare neuroanatomy between species, dopaminergic populations have been labeled numerically based on rostral to caudal location in the brain. As reviewed by Schweitzer et al. [123], Rink and Wullimann [124] labeled the dopaminergic populations 1-8 in larvae and 0-8 in adults, with 0 representing the ventral thalamic area. Sallinen [119] used a 17 population classification scheme. Both classification systems localize dopaminergic neurons to the olfactory bulb, subpallium, posterior tuberculum, hypothalamus, and pretectum [123]. Zebrafish brain lack a substantia nigra and ventral tegmental area, however, the posterior tuberculum has populations of dopaminergic cells with projections that extend to the subpallium and spinal cord [125,126]. Figure 1 outlines the locations of the modulatory neurotransmitters in humans and zebrafish. and ventral tegmental area, however, the posterior tuberculum has populations of dopaminergic cells with projections that extend to the subpallium and spinal cord [125,126]. Figure 1 outlines the locations of the modulatory neurotransmitters in humans and zebrafish. Neurons producing dopamine or norepinephrine also produce either glutamate or GABA and therefore have two transmitter profiles. Dopaminergic neurons in the dopaminergic posterior tubercular groups 2, 4, and 6 and hypothalamic group 5 and some norepinephrine producing cells in the area postrema produce glutamate and all other dopamine or norepinephrine producing neurons produce GABA [134]. Neurons producing dopamine or norepinephrine also produce either glutamate or GABA and therefore have two transmitter profiles. Dopaminergic neurons in the dopaminergic posterior tubercular groups 2, 4, and 6 and hypothalamic group 5 and some norepinephrine producing cells in the area postrema produce glutamate and all other dopamine or norepinephrine producing neurons produce GABA [134].
DBH, the enzyme that converts dopamine to epinephrine, is also present and is used as a marker of adrenergic neurons. Dbh is only found in zebrafish hindbrain, specifically at the locus coeruleus [135,136]. The locus coeruleus projects to the pallium, the subpallium and the thalamus [137,138].

Serotonin
The neurotransmitter serotonin (5-hydroxytrptamine; 5-HT) is a biologic amine. 5-HT has an indole nucleus with a hydroxyl group and an amine group. L-tryptophan is the base of the molecule, with dietary protein being the major source. 5-HT is a modulatory neurotransmitter and has been associated with brain development, appetite, motor function, arousal and mood, neuroendocrine function, circadian rhythms, and depression [139][140][141][142].
The first enzyme in the 5-HT synthesis pathway is tryptophan hydroxylase (L-tryptophan-5monooxygenase; TPH), which converts tryptophan to 5-hydroxytryptophan (5-HTP). The conversion of tryptophan to 5-HTP is considered the rate limiting step in the synthesis pathway and is unique to serotonergic neurons. In mammals there are two genes that code for TPH. TPH1 is expressed in the periphery and TPH2 is expressed exclusively in the brain and can be used as a marker for serotonergic neurons [143]. Once formed, 5-HTP is quickly converted to 5-HT by AAAD.
5-HT is transported into synaptic vesicles by vesicular transporter SLC18A2 (VMAT2) and released from the vesicle via exocytosis [144]. The serotonin transporter SLCl6A4 (also known as SERT) is responsible for the uptake/reuptake of 5-HT, although glia and non-serotonergic neurons can take up serotonin through organic cation transporter (OCT), plasma membrane monoamine transporter (PMAT), or through SLC6A2 or SLC6A3 (NET and DAT, respectively) [145].
In mammals there are three families of 5-HT receptors (HTR) that act through G-proteins, the 5-HT 1 family, the 5-HT 2 family, and a family that includes the 5-HT 4 , 5-HT 6 , and 5-HT 7 receptors [142]. 5-HT 3 is a cation specific ligand-gated ion channel and is considered its own family [145]. The 5-HTR 1 family generally acts to inhibit adenylyl cyclase through the G i/o family of G proteins while the 5-HTR 2 family acts through G q/11 family G proteins by stimulating phospholipase C. The 5-HTR 4 , 5-HTR 6 , and 5-HTR 7 family mainly act through the G s family G proteins to stimulate adenylyl cyclase [146].
As a result of the teleost gene duplication event, zebrafish have four paralogous genes encoding TPH, although the nomenclature has not been standardized. Bellipanni et al. first identified two paralogs of TPH in the developing zebrafish brain, tphD1, expressed in the preoptic nuclei and the posterior tubercular in the diencephalon, and tphD2, expressed in the pineal gland and transiently in the preoptic nuclei [147]. Teraoka et al. identified a third paralog of Tph, tphR, expressed in the raphe nuclei and pineal gland [148]. In later reviews of the serotonin neurotransmitter system, these genes are referred to as tph1a, tph1b, and tph2, respectively [149][150][151]. Further research found that zebrafish th2 encodes for a fourth tph gene in the ventral diencephalon and caudal hypothalamus [116]. This gene has also been called tph3 to better reflect its function [150]. As mentioned previously, zebrafish have only one ortholog of AAAD and MAO [117]. There are two paralogs of the serotonin transporter gene SLC6A4, slc6a4a and slc6a4b [152]. In mammals, over 15 HTR genes have been identified. Table 4 outlines the critical genes in serotonin synthesis and metabolism as well as listing known paralogous genes for the serotonin HTR receptor families [121].   As reviewed by Lillesaar [149], in larval zebrafish 5-HT positive cells are located in the pineal gland, the pretectum, the posterior tuberculum, the hypothalamus, and the superior and inferior raphe. Panula et al. outlined a labeling scheme for the adult zebrafish with serotoninergic nuclei identified in the pretectal complex, the anterior, intermediate, and posterior paraventricular organ nuclei, the dorsal, median, and ventrolateral raphe, the inferior raphe, and the caudal raphe [16,150]. Adult zebrafish also have scattered serotonergic neurons within the medulla oblongata [137]. The distribution of 5-HT in the zebrafish brain compared to humans is shown in Figure 1C.
When identifying serotonergic populations, the Ets-domain transcription factor pet1 (pheochromocytoma 12 ETS [E26 transformation-specific]) is a specific developmental marker of the raphe serotonergic nuclei [151]. TPH is typically used as the serotonergic specific marker in imaging studies [149].

Acetylcholine
Acetylcholine (ACh) is the major neurotransmitter in the parasympathetic nervous system and is the neurotransmitter at neuromuscular junctions [153]. Additionally, ACh neurotransmission is widespread in the CNS and can help modulate the release of other neurotransmitters such as GABA, and has been implicated in arousal, reward, and learning and memory [153][154][155].
ACh is formed from acetyl-CoA and choline via choline acetyltransferase (ChAT). Two SLC family transport systems, a high affinity (SLC5A7; HAChU) and a low affinity (SLC44 family; LAChU), concentrate choline in terminals to provide a reserve for ACh synthesis. SLC44 is present ubiquitously throughout the body, but SLC5A7 is only found in cholinergic nerve termini [156]. The rate of SLC5A7 transport is regulated by the rate of ACh release and SLC5A7 is the rate limiting step in ACh production. Once ACh has been synthesized by ChAT it is packaged into vesicles by vesicular ACh transporter (VAChT) which is coded by SLC18A3 [106]. ACh is then released in quanta into the synaptic cleft in a calcium dependent manner. Unlike other neurotransmitters which have transporter mediated uptake/reuptake to clear the synapse, ACh is metabolized by acetylcholinesterase (AChE) within the synaptic space and broken into acetate and choline [156].
There are two major types of cholinergic receptors, nicotinic (nAChR) and muscarinic receptors (mAChR). The receptors were respectively named after nicotine and muscarine, their drug agonists. nAChRs are a member of the cys-loop family of ligand-gated ion channel receptors and are formed by combinations of receptor subunits [156,157]. In mammals, there are 17 nicotinic receptor subunits including muscle and neural specific subunits. nAChRs are located throughout the brain, though many subtypes of nAChR are located on presynaptic termini or cell bodies and function to modulate neurotransmitter release [154]. mAChRs are G-protein-coupled receptors and are located throughout the CNS and PNS. There are five subtypes of mAChR receptors in mammals, M1-M5. M1, M3, and M5 couple to G q/11 family proteins to increase phospholipase C. M2 and M4 receptors act to decrease adenylyl cyclase through G i /G o receptor activation. mAChR are expressed throughout the brain, but are not uniformly distributed (reviewed by Brown [158]).
The important genes in the cholinergic system are outlined in Table 5. Zebrafish have two paralogs of the HAChU SLC5A7 (slc5a7a and slc5a7b), although slc5a7b has not been well described. The gene that produces ChAT has two paralogs in zebrafish, chata and chatb. slc18a3a and slc18a3b are two paralogs of SLC18A3 (VAChT). There is only one paralog of ACHE (ache). Currently, there are 12 putative genes encoding nAChR subunits in zebrafish and 10 putative paralogs of mAChR [159][160][161]. Table 5. Zebrafish genes involved in cholinergic neurotransmission. Cholinergic neurons have been identified through immunohistochemical staining against choline-acetyltransferase (ChAT) [94,162]. In zebrafish, cholinergic neurons are found in both the brain and spinal cord, specifically in the octavolateralis cells and modulatory or sensory neurons, the ventral telencephalic area, the central, dorsal, and subcommissural nuclei of the ventral telencephalic areas, the preoptic area, dorsal thalamus, pretectal nuclei, hypothalamus, optic tectum, and tegmentum [137,[163][164][165]. The distribution of ACh in zebrafish and human brains is shown in Figure 1D.

Histamine
Histamine is a signaling molecule present in many tissues, serving functions in the stomach, skin, and immune systems. Histamine also has a role in neurotransmission [166]. Within the CNS, histamine is associated with wakefulness, feeding and drinking, and learning and memory [130,167,168].
The structure of histamine, 2-(4-imidazolyl)ethylamine, is similar to 5-HT, NE, and EP, but histamine has an imidazole nucleus and therefore has tautomeric properties that may be associated with receptor affinity. In mammals, mast cells of bone marrow origin reside in perivascular spaces, choroid plexus, and meninges and can produce significant amounts of histamine within the brain although the only neurons that produce histamine are located within hypothalamic tuberomamilary neurons within the posterior hypothalamus [169]. Zebrafish, on the other hand, do not have stores of histamine outside of the brain, suggesting any histamine is of importance to neurotransmission [130,170]. The periventricular cells of the caudal hypothalamus are the only cell group that contains histaminergic neurons in zebrafish brain, similar to mammals, although the axons project throughout the CNS [137,170]. These histamine producing neurons also contain other signaling molecules including GABA, neuropeptides, and thyrotropin-releasing hormone [171].
Histamine is synthesized by L-histidine decarboxylase (HDC). The rate of biosynthesis is controlled by the availability of L-histidine and the rate limiting enzyme, HDC. Once formed, histamine is transported into vesicles by SLC18A2 (VMAT2) [172]. Most histamine in the brain is released via non-synaptic mechanisms and often acts on both presynaptic and postsynaptic receptors. There is no evidence of a neuronal histamine transporter [169].
The metabolism of histamine can occur either by diamine oxidase (DAO; amine oxidase AOC1), which oxidizes histamine to imidazole acetic acid (IAA), or by histamine N-methyltransferase (HNMT), which methylates histamine and forms tele-methylhistamine (t-MH) and is then further metabolized by MAO (MAO-B in mammals) to tele-methylimidazole acetic acid (t-MIAA). The methylation metabolism pathway is more common in vertebrate brains and HMT is widely distributed throughout the brain [168,169].
Mammals have four histamine receptors that are found in the brain, H1, H2, H3, and H4. H1 and H2 are considered excitatory while H3 is inhibitory and often acts as an autoreceptor. H4 is the most recently discovered but does appear to localize to the brain [173]. Histamine receptors are linked to G proteins in both neurons and glia. H1 receptors are linked to G q and stimulate phospholipase C. H2 receptors may couple with G q or G s but act through the stimulation of adenylyl cyclase. H3 receptors are linked to G i/o and inhibit adenylyl cyclase. H3 receptors may also activate MAPK, Akt/GSK-3β, and phospholipase A 2 pathways [174][175][176]. The H4 receptor is similar to the H3 receptor and acts through G i/o proteins to inhibit adenylyl cyclase [173].
In zebrafish there is one ortholog each of HDC (L-histidine decarboxylase), AOC1 (diamine oxidase), and HNMT (histamine N-methyltransferase) ( Table 6). To date, there are four known genes in zebrafish that code for histamine receptors, hrh1, hrh2a, hrh2b, and hrh3 [177]. In zebrafish, histamine containing neurons have been localized only to the ventrocaudal hypothalamus, though the projections are widespread [137,178]. The distribution of histamine in the zebrafish brain compared to humans is shown in Figure 1E. Table 6. Zebrafish genes involved in histamine neurotransmission.

Glycine
Glycine is an amino acid that serves as a signaling molecule and neurotransmitter in the brainstem and spinal cord. Glycine is the simplest amino acid, with only a hydrogen for its side chain. Glycine, typically considered an inhibitory neurotransmitter, is involved with interneuron differentiation in neurodevelopment, mediation of spinal reflexes, and reflex behaviors such as breathing [179,180].
Glycine can be formed through the conversion of serine to glycine with either glycine dehydrogenase (GLDC), also known as glycine decarboxylase (GDC) or glycine-cleavage system, or serine hydroxylmethyltransferase (SHMT). Glycine is transported into synaptic vesicles via the vesicular inhibitory amino acid transporter, SLC32A1 (VIAAT, also known as vesicular GABA transporter (VGAT)) [180].
The glycine receptors are in the ligand gated ion channel superfamily. In mammals, glycine receptor subunits arise from two separate gene families. The α gene family has four subunit genes (α 1 -α 4 ) and the β gene group has only one member (reviewed by Bowery and Smart [182]). Glycine also acts on NMDA receptors and modulates the amplitude and time course of the glutamate-elicited response [183]. Interestingly, although no metabotropic counterparts have been identified, the glycine receptor subunit α can interact with G protein βγ subunits, which potentiates the response to glycine [184].
As outlined in Table 7, zebrafish tend to have a single known ortholog for most of the genes important for glycine metabolism [185,186]. The exception is the presence of two paralogs for glycine receptor α subunit 4 gene and the glycine receptor β gene. Glycinergic neurons are identified via positive immunoreactivity for glycine transporter (slc6A9) immunohistochemistry. In the developing zebrafish, glycine producing cells are limited to the hindbrain and spinal cord, and appear as early as 20 h post fertilization (hpf) [187,188]. In adult zebrafish, the vast majority of glycinergic neurons are within the medulla oblongata, though a few positive neurons are within a ventral tegmental equivalent nucleus [129,187]. Table 7. Zebrafish genes involved in glycine neurotransmission.

Other Neurotransmitters
Other substances can also act as neurotransmitters in the brain, including purines, peptides, nitric oxide, and endocannabinoids [189,190]. Please see Rico et al. [11] for a review of the purine nucleotides and nucleosides in zebrafish and Panula et al. [16] for a review of neuropeptides.

Evaluation of Neurotoxicity
The neurotransmitter systems can serve as targets of chemical toxicity. The individual enzymes, transporters, and receptors may be altered by chemical toxicants through changes in gene expression or changes in activity of the enzyme or receptor. Multiple methods can be used to evaluate neurotransmitter systems in zebrafish, from the measuring of neurotransmitter levels in the brain, to evaluation of gene expression, to the functional testing of behavioral assays. These methods are reviewed below.

Evaluation of Neurotransmitters
In the evaluation of chemical toxicants, measuring neurotransmitter levels can provide information on the functional alterations in the brain resulting from chemical treatment. The quantity of neurotransmitters can be measured in the zebrafish brain. Sallinen et al. [119] and Chattererjee et al. [191] have both described methods for measuring neurotransmitter levels via high performance liquid chromatography (HPLC) that have been modified and used in other laboratories [192,193].
Experimental neurotransmitter results may be comparable within one laboratory, but not comparable across multiple laboratories due to differences in experimental methods and equipment used. Furthermore, one difficulty in comparing neurotransmitter levels across studies and laboratories is the lack of reference intervals and the tendency to normalize neurotransmitter values to controls rather than reporting absolute values, which limits comparison. For example, Pan et al. [194] reported the difference in neurotransmitter levels between the AB and short-fin wildtype strains of zebrafish as a ratio between zebrafish strains, rather than average concentration. Table 8 lists reported neurotransmitter levels from control zebrafish. Comparison of values in the table is difficult between studies in part to different methodologies used for normalization. Neurotransmitter levels can be normalized to protein content or number of fish pooled for the sample, and it is difficult to equate between the two methods.
Other methods for the measurement of neurotransmitters include an analytical method described by Tufi et al. [195] of hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) that has been used to measure neurotransmitter levels in zebrafish larvae. The levels of neurotransmitters and major metabolites were measured in 0-6 dpf zebrafish larvae. Additionally, Jones et al. [196] describe a technique to detect neurotransmitter release and reuptake in brain tissue slices through fast scan cyclic voltammetry (FSCV).

Evaluation of Gene Expression Changes in Neurotransmitter Pathways
Chemical toxicants can affect the expression of genes, and evaluation of these changes can provide information on pathways that may be altered due to chemical exposure. Quantitative PCR (qPCR) can be used to evaluate alterations in gene expression [198]; however, the information is limited to the number of genes investigated.
Microarray studies have been used to evaluate gene expression after chemical exposure in zebrafish [199][200][201][202]. The value of transcriptomic evaluations in zebrafish toxicology studies is well recognized, especially for environmental toxicology [12,203]. Next generation technologies such as RNA-seq are providing precise and powerful options for evaluating the transcriptome [203,204]. Please see the review by Wang et al. [204] and Aanes et al. [205] for information on RNA-seq and its application in zebrafish.

Evaluation of Neurobehavior
The significance of apparent alterations in gene expression and changes in neurotransmitter levels is uncertain without having differences in phenotype. Zebrafish are an accepted model of neurobehavior [206][207][208][209][210][211][212][213][214][215] and have been used to study stress, anxiety-like behavior, and depression, with decreased movement typically associated with anxiety [216][217][218]. Zebrafish are increasingly being used in neurobehavioral research to evaluate learning and neuropsychiatric disorders (reviewed by [207,208,212,219]). Neurobehavioral tests have been developed for both larval and adult zebrafish to assess anxiety-like behavior. Changes in neurobehavior can substantiate changes observed through neurotransmitter analysis or through transcriptomic evaluation; however, changes in neurobehavior cannot be localized to a specific neurotransmitter, pathway, or protein. Please refer to Kalueff et al. [219] and Parker et al. [220] for recent reviews of neurobehavior in zebrafish toxicology research.

Visualization of Neurotransmitters and Neurotransmitter Systems
The visualization of neurotransmitters has been important for evaluating the location, relative quantity, and pathologic alterations of neurotransmitters and neurotransmitter pathway components. A multitude of techniques, including immunohistochemistry, in situ hybridization, immunofluorescence, calcium indicators, selective fluorescent reporters [221][222][223][224][225][226] (and reviewed by [227]) have been used to study neuroanatomy and neurotransmitter systems in zebrafish. The imaging of neurotransmitters, transporters, and receptors in zebrafish brains has classically been used in the study of developmental biology [68] and recently for neural systems mapping [226,228]. A recent review by Arrenberg and Driever [228] highlights the use of optogenetics and calcium indicator activity probes in the development of functional maps of the zebrafish brain. Currently this methodology has been underused in studies of chemical toxicity, but could provide powerful information when combined with the other approaches mentioned.

Pharmacology Screens
The advantages of the zebrafish model system allow for high throughput screening of pharmacologic agents which can help identify neuropathways, mechanisms of toxicity, possible therapeutic drugs, and help classify zebrafish behavior (reviewed by [229][230][231][232][233][234]). The neuropharmacology of the monoamine neurotransmitters was reviewed by Maximino and Herculano [235]. Kalueff et al. [219] have also recently written a highly recommended review that evaluates the zebrafish system in neurobehavior and pharmacology screenings.

Specific Examples of Chemical Toxicity Targeting Neurotransmitter Systems
Zebrafish have been used as a model organism to study the effect of multiple classes of chemicals on neurodevelopment and neural function. Classes of chemicals examined include drugs, especially ethanol, pesticides, and metals. The significant methods and findings from the literature are summarized below and in Table 8.

Drugs
The effects of alcohol have been extensively studied in zebrafish. Rico et al. [236] found that adult zebrafish exposed to ethanol had increased AChE activity in a 1% ethanol treatment group although the mRNA levels of AChE were decreased, suggesting post-transcriptional or post-translational modifications to AChE. Chatterjee and Gerlai [191] found that adult zebrafish exposed to ethanol for 1 h had an increase in brain dopamine levels at all treatment levels, while 5-HT and 5-HIAA increased at the highest (1%) ethanol group. Chatterjee and Shams [237] found a similar increase in dopamine and 5-HT after acute ethanol exposure in AB strain zebrafish, but not in SF strain zebrafish. Puttonen et al. [238], found that larval Turku strain zebrafish with acute exposure to ethanol had increased locomotor activity at lower treatment levels, decreased locomotor activity at the highest treatment level (3%), upregulation of hdc (histidine decarboxylase), th1, and th2 at higher treatment levels, no alterations in the dopaminergic and histaminergic systems according to in situ hybridization and immunohistochemistry, and a decrease in dopamine levels, as measured by HPLC. These results suggest that ethanol has the ability to alter the cholinergic, dopaminergic, and histaminergic neurotransmitter systems and that neurobehavior can be a sensitive measure of altered neurotransmitter systems.
The effects of alcohol on zebrafish behavior appear to have an inverted U shaped dose-response, with increased locomotor activity and shoaling behavior at lower doses and decreased locomotor activity and shoaling behavior at higher doses, as well as either increased or decreased measures of anxiety [237,[239][240][241][242]. Although this finding appears to be dependent on the specific zebrafish strain (AB, SF, WIK, or Turku strain) used and therefore, needs further analysis [237][238][239]243]. Bailey et al. found juvenile AB strain zebrafish with developmental exposure to ethanol had increased locomotor activity overall and after stress or anxiety inducing stimuli [244]. A latent learning neurobehavioral assay performed by Luchiari et al. suggested that AB strain zebrafish have impaired memory recall after ethanol exposure [245]. Echevarria et al. have reviewed additional behavioral outcomes of ethanol exposure in zebrafish [246].
Zenki et al. [247] found that alcohol and its metabolite acetaldehyde decreased the activity of glutamate transporters, based on measuring the in vitro rate of glutamate uptake in tissue sections, in adult zebrafish and that acetaldehyde was more toxic than ethanol when measuring cell viability via 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and extracellular lactate dehydrogenase (LDH) activity.
Nicotine is often used to study nicotinic cholinergic receptors, but nicotine also is a drug known for anxiolytic effects. Levin et al. [248] and Bencan and Levin [249], found that adult zebrafish exposed to nicotine had reduced time spent in the bottom of a novel tank (novel tank test) and found that the anxiolytic effect was mediated through the Chrna7 and Chrna4b receptor subunits [248,249]. Levin and Chen (2004), also found that adult zebrafish exposed to low dose nicotine had improved memory based on a 3-chambered tank test for learning assessment while zebrafish exposed to higher concentrations of nicotine had impaired memory function [250].

Pesticides
Pesticides represent a broad group of chemicals that include herbicides, insecticides, fungicides, and rodenticides. Some pesticides are important environmental toxicants while others are more acutely toxic to humans (for example, during application or manufacturing).
The effects of organophosphate pesticides on brain acetylcholinesterase are well known, however, exposure to organophosphates can also affect other brain neurotransmitter systems. Eddins et al. [251] studied the effects of developmental exposure of zebrafish to chlorpyrifos. Adult zebrafish previously exposed to chlorpyrifos had greater startle responses in a startle response and habituation behavioral assay that persisted into the habituation period. Additionally, decreased dopamine and serotonin levels and increased transmitter turnover were measured in larval zebrafish while only the decreased dopamine persisted to adulthood [251].
Atrazine is a commonly used herbicide in the Midwestern United States that often contaminates drinking water supplies. Wirbisky et al. [199] found decreased levels of the serotonin metabolite 5-HIAA and decreased serotonin turnover (5-HIAA/5-HT) in the brain of adult female zebrafish aged 9 months that were exposed to atrazine only during embryogenesis. Transcriptomic analysis via microarray identified multiple molecular pathways related to brain development, function, and behavior that were altered in the treatment groups including several targets associated with the serotoninergic system [199].
Semicarbazide is a contaminant formed from the breakdown of azodicarbonamide, a chemical used to treat flour. Semicarbazide derivatives have also been used as herbicides. Adult zebrafish exposed to semicarbazide for 96 h had increased expression of gad1 while adult zebrafish with 28 day exposure had down regulation of gad1, gabrr1, and gabbr2 [252]. Yu et al. [252] suggest the alterations of the GABAergic pathway genes could indirectly result in alterations in the hypothalamus-pituitary-gonadal axis.

Metals
Some metals, such as lead and mercury are important environmental toxicants [254][255][256]. Developmental lead exposure is linked to a number of CNS effects, including lowered IQ and attention deficit disorders [257][258][259][260]. Wirbisky et al. [193] found that developmental exposure to lead resulted in altered expression of GABAergic pathway genes including gad2, gad1b, slc6a1 (gat1), slc32a1 (vgat), gabbr1, and gabbr1a as well as altering GABA levels during development. Furthermore, Lee and Freeman [261] found that adult zebrafish with a developmental lead exposure had altered gene expression in pathways associated with neurodevelopment and neurotransmission.
In addition, studies have also started to evaluate the impact of mercury exposure on neurotransmitter systems. The toxicity of mercury depends on its chemical form. Methylmercury is associated with Minamata disease and nervous system impairment. Cambier et al. [198] fed adult male fish food contaminated with methylmercury and found changes in gene expression in the GABA synthesis and metabolism pathways. Inorganic mercury is associated with acute toxicity and renal failure; however, Richetti et al. [262] found that adult zebrafish exposed to mercury chloride and lead acetate had decreased activity of acetylcholinesterase, but no alterations in the gene expression of AChE.

Conclusions
The neurotransmitter systems are highly conserved between zebrafish and mammals, making the zebrafish model a powerful tool for the study of mechanisms of chemical neurotoxicity. Although there are some differences in neurochemistry, the pathways of neurotransmitter synthesis, metabolism, and action are highly conserved across species. Although some genes have multiple paralogs in zebrafish, further research into these paralogs may help identify new functions of genes in humans. Furthermore, further research into neurotransmitter receptors may provide more information about basic neurological systems and connectivity.
The nervous system is an important target of chemical toxicants. Neurotransmitter levels, gene expression, and neurobehavior can be evaluated after chemical treatment to discover toxicant based changes in the nervous system. New technologies provide more sensitive and powerful methods; however, the integration of these methods along with other techniques, such as imaging and activity assays, will be necessary to determine the mechanisms and pathogenesis of chemical toxicant related alterations. Overall, relatively few studies have focused on alterations of neurochemical systems in zebrafish, but the completed studies support the utility and application of zebrafish in neurochemical toxicology.