Declan W. Ali

NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity

Regulation of postsynaptic glutamate receptors is one of the main mechanisms for altering synaptic efficacy in the central nervous system. Recent studies have given insight into the upregulation of the NMDA receptor by Src family tyrosine kinases, which bind to scaffolding proteins in the NMDA receptor complex. Src acts as a common step in signalling cascades that link G-protein-coupled receptors with protein kinase C via the intermediary cell-adhesion kinase beta. This signalling to NMDA receptors is required for long-term potentiation in the CA1 region of the hippocampus.

Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.

Children exposed to alcohol in utero have significantly delayed gross and fine motor skills, as well as deficiencies in reflex development. The reasons that underlie the motor deficits caused by ethanol (EtOH) exposure remain to be fully elucidated. The present study was undertaken to investigate the effects of embryonic alcohol exposure (1.5%, 2% and 2.5% EtOH) on motor neuron and muscle fiber morphology in 3 days post fertilization (dpf) larval zebrafish. EtOH treated fish exhibited morphological deformities and fewer bouts of swimming in response to touch, compared with untreated fish. Immunolabelling with anti-acetylated tubulin indicated that fish exposed to 2.5% EtOH had significantly higher rates of motor neuron axon defects. Immunolabelling of primary and secondary motor neurons, using znp-1 and zn-8, revealed that fish exposed to 2% and 2.5% EtOH exhibited significantly higher rates of primary and secondary motor neuron axon defects compared to controls. Examination of red and white muscle fibers revealed that fish exposed to EtOH had significantly smaller fibers compared with controls. These findings indicate that motor neuron and muscle fiber morphology is affected by early alcohol exposure in zebrafish embryos, and that this may be related to deficits in locomotion.

Mechanisms underlying metabolic and neural defects in zebrafish and human multiple acyl-CoA dehydrogenase deficiency (MADD).

In humans, mutations in electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETFDH) lead to MADD/glutaric aciduria type II, an autosomal recessively inherited disorder characterized by a broad spectrum of devastating neurological, systemic and metabolic symptoms. We show that a zebrafish mutant in ETFDH, xavier, and fibroblast cells from MADD patients demonstrate similar mitochondrial and metabolic abnormalities, including reduced oxidative phosphorylation, increased aerobic glycolysis, and upregulation of the PPARG-ERK pathway. This metabolic dysfunction is associated with aberrant neural proliferation in xav, in addition to other neural phenotypes and paralysis. Strikingly, a PPARG antagonist attenuates aberrant neural proliferation and alleviates paralysis in xav, while PPARG agonists increase neural proliferation in wild type embryos. These results show that mitochondrial dysfunction, leading to an increase in aerobic glycolysis, affects neurogenesis through the PPARG-ERK pathway, a potential target for therapeutic intervention.

Impaired hippocampal LTP in inbred mouse strains can be rescued by β-adrenergic receptor activation

Long‐term potentiation (LTP), an activity‐dependent enhancement of synaptic strength, and memory can be influenced by neuromodulatory transmitters such as norepinephrine (NE) and also by genetic background. β‐Adrenergic receptor activation can facilitate the expression of hippocampal CA1 LTP induced by weak stimulus patterns, but its influence on LTP induced by stronger stimulus patterns is unclear. We examined neural NE and dopamine (DA) levels, β‐adrenergic receptor expression and hippocampal LTP in genetically diverse inbred mouse strains. Brain tissue levels of NE were significantly lower in strains 129S1/SvImJ (129), BALB/cByJ (BALB) and C3H/HeJ (C3H) than in C57BL/6NCrlBR (B6). Western blot analysis showed that hippocampal β1‐adrenergic receptor expression was similar in strains B6, 129 and C3H, but was increased in BALB. LTP was induced in area CA1 of hippocampal slices by four trains of high‐frequency stimulation (HFS) of the Schaeffer collaterals in the four inbred strains. Two hours after induction, LTP was significantly reduced in strains 129, BALB and C3H compared to B6, correlating with neural NE levels. We rescued hippocampal LTP in strains 129, BALB and C3H to levels seen in B6 by bath application of 1 µm isoproterenol, a β‐adrenergic receptor agonist, during HFS. Propranolol, a β‐adrenergic receptor antagonist, blocked this rescue in 129, BALB and C3H but did not affect LTP in strain B6. Thus, although this form of multitrain LTP does not rely on β‐adrenergic receptor activation, our data show that pharmacological activation of β‐adrenergic receptors during multiple trains of HFS can rescue CA1 LTP in genetically diverse strains with impaired LTP.

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.

PKCγ-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 PKCγ 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 Ca2+-dependent, and occurs on application of active PKCγ. 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 PKCγ and NSF-blocking peptides, and the NMDA receptor blocker, 2-amino-5-phosphonovaleric acid (APV). Thus, activation of PKCγ 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.

AMPA receptors associated with zebrafish Mauthner cells switch subunits during development

Glutamate AMPA receptors (AMPARs) are major excitatory receptors in the vertebrate CNS. In many biological systems there is a developmental speeding in AMPAR kinetics, which occurs either because of a switch in AMPAR subunits or a change in synaptic morphology. We studied the development of AMPAR‐mediated miniature excitatory postsynaptic currents (AMPAR‐mEPSCs) in zebrafish Mauthner cells (M‐cells) to determine the reasons underlying the speeding of AMPA mEPSCs in this preparation. We recorded AMPAR‐mEPSCs in zebrafish ranging in age from 33 h postfertilization (hpf) to 72 hpf. We found that the glutamate waveform in the synaptic cleft did not change during development, suggesting that synaptic morphology played little role in shaping the mEPSC. The current–voltage (I–V) relationship was linear at 33 hpf and outwardly rectified in older animals, while AMPAR decay kinetics were slower at positive potentials, compared with negative potentials. The relative change in τ with depolarization was found to be greater at 48 hpf than at 33 hpf. AMPARs in 33 hpf fish had a conductance of ∼9 pS, and in older fish ∼15 pS. Finally, the desensitization blocker, cyclothiazide, increased τ by ∼4‐fold in 48 hpf preparations, but only 1.5‐fold in 33 hpf fish. These results are consistent with the hypothesis that the major mechanism underlying the developmental speeding in AMPAR kinetics in zebrafish CNS is a switch in receptor subunits. To our knowledge this is the first study to suggest that AMPARs change subunits during development in fish.

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 PKCα 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 (PKCγ, βII, δ, ɛ, θ and ζ). Here, we show that PKCγ, ɛ, θ and ζ are expressed in the zebrafish CNS. Anti‐PKCγ labels Rohon‐Beard sensory neurons and Mauthner cells. PKCɛ and ζ staining is widespread in the CNS, and PKCθ and βII 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 PKCγ, βII, ɛ and ζ are similar to the mammalian isoforms. Interestingly, PKCθ appears to be similar to PKCθII, 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.

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.

Embryonic ethanol exposure alters synaptic properties at zebrafish neuromuscular junctions.

Pre-natal alcohol exposure induces delays in fine and gross motor skills, and deficiencies in reflex development via mechanisms that remain to be elucidated. The purpose of the present study was to investigate the effect of embryonic ethanol exposure (16-hour exposure window with 1.5%, 2% or 2.5% EtOH) on synaptic properties at the neuromuscular junction (NMJ) in 3 day post fertilization (dpf) zebrafish larvae. Immunohistochemical studies show that exposure of embryos to 2.5% ethanol for 16 h results in motor neuron axons that display abnormal branching patterns. Co-labelling embryos with pre-synaptic markers such as SV-2 or 3A10, and the post-synaptic marker, α-bungarotoxin, which irreversibly binds to nicotinic acetylcholine receptors (nAChRs), indicates that pre- and post-synaptic sites are properly aligned even when motor neuron axons display abnormal morphology. Miniature endplate currents (mEPCs) recorded from muscle fibers revealed the presence of two types of mEPCs that we dubbed fast and slow. Ethanol treated fish experienced significant changes in the frequencies of fast and slow mEPCs, and an increase in the rise time of slow mEPCs recorded from red muscle fibers. Additionally, embryonic exposure to ethanol resulted in a significant increase in the decay time of fast mEPCs recorded from white fibers. Mean mEPC amplitude was unaffected by ethanol treatment. Together, these results indicate that zebrafish embryos exposed to ethanol may experience altered synaptic properties at the NMJ.