Inhibiting the endocannabinoid degrading enzymes FAAH and MAGL during zebrafish embryogenesis alters sensorimotor function.

The endocannabinoid system (eCS) plays a critical role in a variety of homeostatic and developmental processes. Although the eCS is known to be involved in motor and sensory function, the role of endocannabinoid (eCB) signaling in sensorimotor development remains to be fully understood. In this study, the catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) were inhibited either simultaneously or individually during the first ∼24 h of zebrafish embryogenesis, and the properties of contractile events and escape responses were studied in animals ranging in age from 1 day post-fertilization (dpf) to 10 weeks. This perturbation of the eCS resulted in alterations to contractile activity at 1 dpf. Inhibition of MAGL using JZL 184 and dual inhibition of FAAH/MAGL using JZL 195 decreased escape swimming activity at 2 dpf. Treatment with JZL 195 also produced alterations in the properties of the 2 dpf short latency C-start escape response. Animals treated with JZL 195 exhibited deficits in escape responses elicited by auditory/vibrational stimuli at 5 and 6 dpf. These deficits were also present during the juvenile developmental stage (8- to 10-week-old fish), demonstrating a prolonged impact to sensory systems. These findings demonstrate that eCS perturbation affects sensorimotor function, and underscores the importance of eCB signaling in the development of motor and sensory processes.

Early suppression of the endocannabinoid degrading enzymes FAAH and MAGL alters locomotor development in zebrafish.

The fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes are the predominant catabolic regulators of the major endocannabinoids (eCBs) anadamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively. The expression and roles of eCBs during early embryogenesis remain to be fully investigated. Here, we inhibited FAAH and MAGL in zebrafish embryos during the first 24 h of life and examined motor neuron and locomotor development at 2 and 5 days post fertilization (dpf). Application of the dual FAAH/MAGL inhibitor, JZL195 (2 µmol l-1), resulted in a reduction in primary and secondary motor neuron axonal branching. JZL195 also reduced nicotinic acetylcholine receptor (nAChR) expression at neuromuscular junctions. Application of URB597 (5 µmol l-1), a specific inhibitor of the FAAH enzyme, also decreased primary motor neuron branching but did not affect secondary motor neuron branching and nAChR expression. Interestingly, JZL184 (5 µmol l-1), a specific inhibitor of MAGL, showed no effects on motor neuron branching or nAChR expression. Co-treatment of the enzyme inhibitors with the CB1R inhibitor AM251 confirmed the involvement of CB1R in motor neuron branching. Disruption of FAAH or MAGL reduced larval swimming activity, and AM251 attenuated the JZL195- and URB597-induced locomotor changes, but not the effects of JZL184. Together, these findings indicate that inhibition of FAAH, or augmentation of AEA acting through CB1R during early development, may be responsible for locomotor deficiencies.

CB1 and CB2 receptors play differential roles in early zebrafish locomotor development.

Endocannabinoids (eCBs) mediate their effects through actions on several receptors, including the cannabinoid receptors CB1R and CB2R. The role played by eCBs in the development of locomotor systems is not fully understood. In this study, we investigated the roles of the eCB system in zebrafish development by pharmacologically inhibiting CB1R and CB2R (with AM251 and AM630, respectively) in either the first or second day of development. We examined the morphology of motor neurons and we determined neuromuscular outputs by quantifying the amount of swimming in 5 days post-fertilization larvae. Blocking CB2R during the first day of development resulted in gross morphological deficits and reductions in heart rate that were greater than those following treatment with the CB1R blocker AM251. Blocking CB1Rs from 0 to 24 h post-fertilization resulted in an increase in the number of secondary and tertiary branches of primary motor neurons, whereas blocking CB2Rs had the opposite effect. Both treatments manifested in reduced levels of swimming. Additionally, blocking CB1Rs resulted in greater instances of non-inflated and partially inflated swim bladders compared with AM630 treatment, suggesting that at least some of the deficits in locomotion may result from an inability to adjust buoyancy. Together, these findings indicate that the eCB system is pivotal to the development of the locomotor system in zebrafish, and that perturbations of the eCB system early in life may have detrimental effects.

Pharmacology of Medical Cannabis.

The Cannabis plant has been used for many of years as a medicinal agent in the relief of pain and seizures. It contains approximately 540 natural compounds including more than 100 that have been identified as phytocannabinoids due to their shared chemical structure. The predominant psychotropic component is Δ9-tetrahydrocannabinol (Δ9-THC), while the major non-psychoactive ingredient is cannabidiol (CBD). These compounds have been shown to be partial agonists or antagonists at the prototypical cannabinoid receptors, CB1 and CB2. The therapeutic actions of Δ9-THC and CBD include an ability to act as analgesics, anti-emetics, anti-inflammatory agents, anti-seizure compounds and as protective agents in neurodegeneration. However, there is a lack of well-controlled, double blind, randomized clinical trials to provide clarity on the efficacy of either Δ9-THC or CBD as therapeutics. Moreover, the safety concerns regarding the unwanted side effects of Δ9-THC as a psychoactive agent preclude its widespread use in the clinic. The legalization of cannabis for medicinal purposes and for recreational use in some regions will allow for much needed research on the pharmacokinetics and pharmocology of medical cannabis. This brief review focuses on the use of cannabis as a medicinal agent in the treatment of pain, epilepsy and neurodegenerative diseases. Despite the paucity of information, attention is paid to the mechanisms by which medical cannabis may act to relieve pain and seizures.

Targeted mutation of the gene encoding prion protein in zebrafish reveals a conserved role in neuron excitability.

The function of the cellular prion protein (PrP(C)) in healthy brains remains poorly understood, in part because Prnp knockout mice are viable. On the other hand, transient knockdown of Prnp homologs in zebrafish (including two paralogs, prp1 and prp2) has suggested that PrP(C) is required for CNS development, cell adhesion, and neuroprotection. It has been argued that zebrafish Prp2 is most similar to mammalian PrP(C), yet it has remained intransigent to the most thorough confirmations of reagent specificity during knockdown. Thus we investigated the role of prp2 using targeted gene disruption via zinc finger nucleases. Prp2(-/-) zebrafish were viable and did not display overt developmental phenotypes. Back-crossing female prp2(-/-) fish ruled out a role for maternal mRNA contributions. Prp2(-/-) larvae were found to have increased seizure-like behavior following exposure to the convulsant pentylenetetrazol (PTZ), as compared to wild type fish. In situ recordings from intact hindbrains demonstrated that prp2 regulates closing of N-Methyl-d-aspartate (NMDA) receptors, concomitant with neuroprotection during glutamate excitotoxicity. Overall, the knockout of Prp2 function in zebrafish independently confirmed hypothesized roles for PrP, identifying deeply conserved functions in post-developmental regulation of neuron excitability that are consequential to the etiology of prion and Alzheimer diseases.

Conversion of short-term potentiation to long-term potentiation in mouse CA1 by coactivation of ß-adrenergic and muscarinic receptors.

Encoding new information requires dynamic changes in synaptic strength. The brain can boost synaptic plasticity through the secretion of neuromodulatory substances, including acetylcholine and noradrenaline. Considerable effort has focused on elucidating how neuromodulatory substances alter synaptic properties. However, determination of the potential synergistic interactions between different neuromodulatory systems remains incomplete. Previous results indicate that coactivation of ß-adrenergic and cholinergic receptors facilitated the conversion of STP to LTP through an extracellular signal-regulated kinase (ERK)-dependent mechanism. ERK signaling has been linked to synaptically localized translation regulation. Thus, we hypothesized that costimulation of noradrenergic and cholinergic receptors could initiate the transformation of STP to LTP through up-regulation of protein synthesis. Our results indicate that a protocol which yields STP (5 Hz, 5 sec) when paired with coapplication of the ß-adrenergic agonist, isoproterenol (ISO), and the cholinergic agonist, carbachol (CCh), induces translation-dependent LTP in mouse CA1. This form of LTP requires both ß1-adrenergic and M1 muscarinic receptor activation, as blocking either receptor subtype prevented LTP induction. Blocking ERK, mTOR, or translation reduced the expression of LTP induced with ISO + CCh. Taken together, our data demonstrate that coactivation of ß-adrenergic and muscarinic receptors facilitates the conversion of STP to LTP through a mechanism requiring translation initiation.

The endocannabinoid system's involvement in motor development relies on cannabinoid receptors, TRP channels, and Sonic Hedgehog signaling.

The endocannabinoid system (eCS) plays critical roles in locomotor function and motor development; however, the roles of non-canonical cannabinoid receptor systems such as transient receptor potential (TRP) channels and the Sonic Hedgehog (SHH) signaling pathway in conjunction with the eCS in sensorimotor development remains enigmatic. To investigate the involvement of canonical and non-canonical cannabinoid receptors, TRP channels, and the SHH pathway in the development of sensorimotor function in zebrafish, we treated developing animals with pharmacological inhibitors of the CB1R, CB2R, TRPA1/TRPV1/TRPM8, and a smoothened (SMO) agonist, along with inhibitors of the eCS catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) during the first ~24 h of zebrafish embryogenesis. Locomotor function was examined by assessing touch-evoked escape swimming at 2 days post-fertilization. We report that FAAH inhibition had no effect on swimming while MAGL inhibition using JZL 184 reduced swimming distance and the dual FAAH/MAGL inhibitor JZL 195 impaired swimming distance and mean swimming velocity. The CB1R antagonist AM 251 prevented locomotor deficits caused by eCS perturbation but the CB2R antagonist AM 630 did not. Inhibition of TRPA1/TRPV1/TRPM8 using AMG 9090 rescued the locomotor reductions caused by FAAH/MAGL inhibition, but not by MAGL inhibition alone. The SMO agonist purmorphamine attenuated the effects of JZL 184 and JZL 195 on swimming distance, but not mean velocity. Together, these findings provide one of the first investigations examining the interactions between the eCS and its non-canonical receptor systems in vertebrate motor development.

PKCgamma-induced trafficking of AMPA receptors in embryonic zebrafish depends on NSF and PICK1.

The trafficking of AMPA receptors (Rs) to and from synaptic membranes is a key component underlying synaptic plasticity mechanisms such as long-term potentiation (LTP) and long-term depression (LTD), and is likely important for synaptic development in embryonic organisms. However, some of the key biochemical components required for receptor trafficking in embryos are still unknown. Here, we report that in embryonic zebrafish, the activation of PKCgamma by phorbol 12-myristate 13-acetate, strongly potentiates the amplitude of AMPAR-mediated miniature excitatory postsynaptic currents (AMPA-mEPSCs) via a N-ethylmaleimide-sensitive fusion (NSF) and protein interacting with C-kinase-1 (PICK1)-dependent process. We found that the mEPSC potentiation is DAG- and Ca(2+)-dependent, and occurs on application of active PKCgamma. Peptides that prevent the association of NSF and PICK1 with the GluR2 subunit, and the actin-polymerization blocker, latrunculin B, prevented the increase in mEPSC amplitude. Also, application of tetanus toxin (TeTx), which cleaves SNARE proteins, also blocked the increase in mEPSC amplitude. Last, application of a 5 mM K(+) medium led to an enhancement in mEPSC amplitude that was prevented by addition of the PKCgamma and NSF-blocking peptides, and the NMDA receptor blocker, 2-amino-5-phosphonovaleric acid (APV). Thus, activation of PKCgamma is necessary for the activity-dependent trafficking of AMPARs in embryonic zebrafish. This process is NMDA and SNARE-dependent and requires AMPARs to associate with both NSF and PICK1. The present data further our understanding of AMPAR trafficking, and have important implications for synaptic development and synaptic plasticity.

Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ9-tetrahydrocannabinol.

In light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) until the end of gastrulation (1-10 h post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented Calcium-Modulated Photoactivatable Ratiometric Integrator (CaMPARI) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and their receptors to understand the implications of cannabis consumption on fetal development.

Protein kinase Cgamma is a signaling molecule required for the developmental speeding of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor kinetics.

A key step in the maturation of glutamate synapses is the developmental speeding of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPA-R) kinetics, which occurs via a switch in receptor subtypes. However, the molecular components required for the switch in receptors are unknown. Here, we used the zebrafish preparation to show that activation of protein kinase C (PKC)gamma is necessary for the developmental speeding of AMPA-R kinetics. Targeted knockdown of PKCgamma with an antisense morpholino oligonucleotide [PKCgamma-morpholino (PKCgamma-MO)], prevents the normal speeding up of AMPA-R kinetics in Mauthner cells. PKCgamma-MO-injected embryos are incapable of trafficking AMPA-Rs following application of phorbol 12-myristate 13-acetate or PKCgamma. PKCgamma-MO-injected embryos do not hatch or exhibit the C-start escape response. Increasing synaptic activity (33 h post-fertilization embryos) by application of an elevated K(+) medium or by application of N-methyl-D-aspartate induces rapid PKCgamma-dependent trafficking of fast AMPA-Rs to synapses. Our findings reveal that PKCgamma is a molecular link underlying the developmental speeding of AMPA-Rs in zebrafish Mauthner cells.

Characterization of ionic currents and electrophysiological properties of goldfish somatotropes in primary culture.

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca(2+) channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca(2+) concentration ([Ca(2+)]i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of -78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of -90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and -30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K(+), tetrodotoxin (TTX)-sensitive Na(+), and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca(2+) channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca(2+)]i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca(2+)]i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K(+) channels regulate excitability while TTX-sensitive Na(+) channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca(2+) channels during AP firing leads to increases in [Ca(2+)]i.

Early Exposure to THC Alters M-Cell Development in Zebrafish Embryos.

Cannabis is one of the most commonly used illicit recreational drugs that is often taken for medicinal purposes. The psychoactive ingredient in cannabis is Δ9-Tetrahydrocannabinol (Δ9-THC, hereafter referred to as THC), which is an agonist at the endocannabinoid receptors CB1R and CB2R. Here, we exposed zebrafish embryos to THC during the gastrulation phase to determine the long-term effects during development. We specifically focused on reticulospinal neurons known as the Mauthner cells (M-cell) that are involved in escape response movements. The M- cells are born during gastrulation, thus allowing us to examine neuronal morphology of neurons born during the time of exposure. After the exposure, embryos were allowed to develop normally and were examined at two days post-fertilization for M-cell morphology and escape responses. THC treated embryos exhibited subtle alterations in M-cell axon diameter and small changes in escape response dynamics to touch. Because escape responses were altered, we also examined muscle fiber development. The fluorescent labelling of red and white muscle fibers showed that while muscles were largely intact, the fibers were slightly disorganized with subtle but significant changes in the pattern of expression of nicotinic acetylcholine receptors. However, there were no overt changes in the expression of nicotinic receptor subunit mRNA ascertained by qPCR. Embryos were allowed to further develop until 5 dpf, when they were examined for overall levels of movement. Animals exposed to THC during gastrulation exhibited reduced activity compared with vehicle controls. Together, these findings indicate that zebrafish exposed to THC during the gastrula phase exhibit small changes in neuronal and muscle morphology that may impact behavior and locomotion.

Autoinhibition of TRPV6 Channel and Regulation by PIP2.

Transient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N and C termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis, and cancer. The TRPV6 function and regulation remain poorly understood. Here we show that the TRPV6 intramolecular S4-S5 linker to C-terminal TRP helix (L/C) and N-terminal pre-S1 helix to TRP helix (N/C) interactions, mediated by Arg470:Trp593 and Trp321:Ile597 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on the L/C interaction but not reversely. Three cationic residues in S5 or C terminus are involved in binding PIP2 to suppress both interactions thereby activating TRPV6. This study reveals "PIP2 - intramolecular interactions" regulatory mechanism of TRPV6 activation-autoinhibition, which will help elucidating the corresponding mechanisms in other TRP channels.

Analysis of leukemia inhibitory factor and leukemia inhibitory factor receptor in embryonic and adult zebrafish (Danio rerio).

Leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family that functions in the survival, repair and formation of neurons as well as in the maintenance of neural and embryonic stem cells. The functions of LIF have been well documented in mammals, however until recently, the presence of IL-6 family cytokines in ectothermic vertebrates has only been speculated. We report on the identification of lif and lifr transcripts in the zebrafish and document the expression of these molecules in the developing embryos and tissues of adult zebrafish. We also examined the phylogenetic relationship between these molecules and other IL-6 cytokine family members known in mammals. In adult zebrafish, lif is expressed in the kidney and brain while lifr is expressed in the kidney, gill, brain, spleen and liver. During zebrafish embryogenesis, lif and lifr are both expressed as early as 12 hours postfertilization (hpf). In developing zebrafish, lif is expressed in the otic vesicle, retina and cranial sensory ganglia, and lifr is strongly expressed in the notochord, forebrain, otic vesicle, cranial ganglia and the retina. Morpholino knockdown of Lif and Lifr in developing embryos suggests that Lifr, but not Lif is required for proper neural development. lifr morpholino-injected embryos exhibit defects in the trigeminal, facial and vagal branchiomotor neurons, and improper axonal development as measured by acetylated tubulin staining. These embryos also display severe hydrocephaly by 48 hpf. This suggests that Lifrs are involved in proper neural development in zebrafish. This is the first evidence of the expression and role of an LIFR-like molecule in developing fish.

Development of skeletal muscle sodium and potassium currents in zebrafish sofa potato mutants.

In some cells, the development of voltage-gated channels requires synaptic input, while in others it does not. Here we investigate whether the sodium and potassium currents in the skeletal muscle of zebrafish sofa potato (sop(-/-)) mutants develop normally. Zebrafish sop(-/-) mutants do not express nicotinic acetylcholine receptors at neuromuscular junctions, and therefore do not exhibit synaptic activity in muscle. We find that in both red and white muscle fibers, sop(-/-) mutants are able to support normal potassium current development during early stages of development [1-3 days post fertilization (dpf)], but at 6 dpf the potassium current density is significantly smaller than that in their phenotypically wild-type siblings (sop(+/?)). In contrast, sodium current density is unaffected. The steady-state properties of potassium currents are unaltered in the sop(-/-) mutants, but there is a significant difference in the V(50) of inactivation of sodium currents. This is the first study in zebrafish to investigate activity-dependent mechanisms of ion channel development and our results indicate that some aspects of ion current development in skeletal muscle require synaptic activity, whereas others do not.

Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish.

Zebrafish embryos exhibit spontaneous contractions of the musculature as early as 18-19 h post fertilization (hpf) when removed from their protective chorion. These movements are likely initiated by early embryonic central nervous system activity. We have made the observation that narrowminded mutant embryos (hereafter, nrd(-/-)) lack normal embryonic motor output upon dechorionation. However, these mutants can swim and respond to tactile stimulation by larval stages of development. nrd(-/-) embryos exhibit defects in neural crest development, slow muscle development and also lack spinal mechanosensory neurons known as Rohon-Beard (RB) neurons. At early developmental stages (i.e. 21-22 hpf) and while still in their chorions, nrd siblings (nrd(+/?)) exhibited contractions of the musculature at a rate similar to wild-type embryos. Anatomical analysis indicated that RB neurons were present in the motile embryos, but absent in the non-motile embryos, indicating that the non-motile embryos were nrd(-/-) embryos. Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development. These errors were reversed when nrd(-/-) embryos were raised in high [K(+)] beginning at 21 hpf, indicating that the abnormal axonal phenotypes may be related to a lack of depolarizing activity early in development. When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos. These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

Motor neuron development in zebrafish is altered by brief (5-hr) exposures to THC (∆9-tetrahydrocannabinol) or CBD (cannabidiol) during gastrulation.

Marijuana is one of the most commonly used illicit recreational drugs and is widely used for medicinal purposes. The psychoactive ingredient in marijuana is ∆9-tetrahydrocannabinol (∆9-THC), whereas the major non-psychoactive ingredient is cannabidiol (CBD). Here, we exposed zebrafish embryos to ∆9-THC or CBD for 5 hours during the critical stage of development known as gastrulation. Embryos were allowed to develop normally and were examined at 2 and 5 days post fertilization. THC and CBD treated embryos exhibited reduced heart rates, axial malformations and shorter trunks. Cannabinoid treatment altered synaptic activity at neuromuscular junctions (NMJs), and fluorescent labelling of primary and secondary motor neurons indicated a change in branching patterns and a reduction in the number of axonal branches in the trunk musculature. Furthermore, there were alterations in the α-bungarotoxin labelling of nicotinic acetylcholine receptors at NMJs. Locomotion studies show that larvae exposed to THC or CBD during gastrulation exhibited drastic reductions in the number of C-start escape responses to sound stimuli, but not to touch stimuli. Together these findings indicate that zebrafish embryos exposed to ∆9-THC or CBD during the brief but critical period of gastrulation exhibited alterations in heart rate, motor neuronal morphology, synaptic activity at the NMJ and locomotor responses to sound.

Differential expression of PKC isoforms in developing zebrafish.

Protein kinase C isozymes are a biologically diverse group of enzymes known to be involved in a wide variety of cellular processes. They fall into three families (conventional, novel and atypical) depending upon their mode of activation. Several classes of zebrafish neurons have been shown to express PKCalpha during development, but the expression of other isoforms remains unknown. In this study we performed immunohistochemistry to determine if zebrafish express various isoforms of PKC. We used antibodies to test for the presence of enzymes that are thought to be preferentially expressed in the nervous system (PKCgamma, betaII, delta, epsilon, theta and zeta). Here, we show that PKCgamma, epsilon, theta and zeta are expressed in the zebrafish CNS. Anti-PKCgamma labels Rohon-Beard sensory neurons and Mauthner cells. PKCepsilon and zeta staining is widespread in the CNS, and PKCtheta and betaII are expressed in skeletal muscle, especially at intersegmental boundaries. Immunoblot experiments confirm the specificity of the antibodies in zebrafish and indicate that the fish isoforms of PKCgamma, betaII, epsilon and zeta are similar to the mammalian isoforms. Interestingly, PKCtheta appears to be similar to PKCthetaII, which, to date, has been found exclusively in mouse testis, but not in the mammalian CNS. Overall, our findings indicate that several different PKC isoforms are expressed in zebrafish, and that Rohon-Beard, Mauthner cells and muscle fibers preferentially express some isoforms over others.

Nicotinic acetylcholine receptors (nAChRs) at zebrafish red and white muscle show different properties during development.

Nicotinic acetylcholine receptors (nAChRs) are highly expressed at the vertebrate neuromuscular junction (NMJ) where they are required for muscle activation. Understanding the factors that underlie NMJ development is critical for a full understanding of muscle function. In this study we performed whole cell and outside-out patch clamp recordings, and single-cell RT-qPCR from zebrafish red and white muscle to examine the properties of nAChRs during the first 5 days of development. In red fibers miniature endplate currents (mEPCs) exhibit single exponential time courses at 1.5 days postfertilization (dpf) and double exponential time courses from 2 dpf onwards. In white fibers, mEPCs decay relatively slowly, with a single exponential component at 1.5 dpf. By 2 and 3 dpf, mEPC kinetics speed up, and decay with a double exponential component, and by 4 dpf the exponential decay reverts back to a single component. Single channel recordings confirm the presence of two main conductance classes of nAChRs (∼45 pS and ∼65 pS) in red fibers with multiple time courses. Two main conductance classes are also present in white fibers (∼55 pS and ∼73 pS), but they exhibit shorter mean open times by 5 dpf compared with red muscle. RT-qPCR of mRNA for nicotinic receptor subunits supports a switch from γ to ε subunits in white fibers but not in red. Our findings provide a developmental profile of mEPC properties from red and white fibers in embryonic and larval zebrafish, and reveal previously unknown differences between the NMJs of these muscle fibers.© 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 916-936, 2016.