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Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 39 untranslated region (39UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 39UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pulldown assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 39UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD.

Efforts to phenotype pancreatic islets have contributed tremendously to our present understanding of endocrine function and diabetes. A continued evolution in approaches to study islet physiology is important given the need to establish reference points for mature islet functionality, understanding biological variation amongst individuals and cells, and the ongoing appreciation of the role for islets in diabetes susceptibility. Recent efforts in islet biology have focused on technological improvements in imaging, molecular profiling and data analysis, along with a push for enhanced transparency and reporting. The integration of these approaches within a classical islet physiology framework, and approaches to link these data with in vivo human phenotypes, will be critical as we move towards a better understanding of islet function in health and disease. Here we discuss what we feel are important issues and useful approaches to consider as we move forward as a field in islet and beta cell phenotyping.

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.

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 badrenergic 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 b-adrenergic agonist, isoproterenol (ISO), and the cholinergic agonist, carbachol (CCh), induces translationdependent LTP in mouse CA1. This form of LTP requires both b1-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 b-adrenergic and muscarinic receptors facilitates the conversion of STP to LTP through a mechanism requiring translation initiation.Memory formation is believed to rely on an activity-dependent, enduring modification of synaptic strength known as long-term potentiation (LTP) (Bliss and Lomo 1973;Bliss and Collingridge 1993;Kandel 2001). LTP has been demonstrated in the hippocampus following events which induce behavioral changes indicative of learning and memory (Whitlock et al. 2006). The hippocampus plays a time-limited role in the establishment of new memories (Scoville and Milner 1957;Zola-Morgan et al. 1986;Neves et al. 2008), a process subject to modification through the release of neuromodulatory transmitters.Cholinergic and noradrenergic neuromodulatory systems can enhance synaptic plasticity (Hu et al. 2007;Dringenberg et al. 2008;Fernández de Sevilla et al. 2008) and long-term memory formation (Cahill et al. 1994 Although the individual contributions of b-and muscarinic receptors to synaptic function have been investigated, the effects of simultaneous activation of these receptors have yet to be fully elucidated. Previous research has demonstrated that coactivation of a-adrenergic and muscarinic receptors facilitates the induction of long-term depression (LTD) (Scheiderer et al. 2008). LTD induced by a-adrenergic and M1 receptors is facilitated through a synergistic elevation of extracellular signal-regulated kinase (ERK) phosphorylation. A similar effect on ERK stimulation was observed when the b-adrenergic receptor agonist, isoproterenol (ISO), was paired with carbachol (CCh), a broad-spectrum mu...

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals 1 . We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact 2,3,4 . The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated) 5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

γ-Aminobutyric acid (GABA) binds to ionotropic GABAA receptors to mediate fast inhibitory synaptic transmission in the central nervous system (CNS). GABAA receptors are pentameric structures composed of receptor subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1-3) with various stoichiometries. They play important roles in the control of neural networks and are the pharmacological targets for the treatment of diseases such as epilepsy, autism, and schizophrenia. Thus far, there has been no report on GABA synaptic transmission in developing zebrafish. Here we used whole-cell patch-clamp electrophysiology to record GABAA-mediated miniature postsynaptic currents from the Mauthner cells of embryonic zebrafish. Spontaneous GABAA currents occurred infrequently and were low in amplitude (27.2 ± 0.9 pA). Analysis of their kinetics suggested the existence of three main types of events: the first (group I) is mediated by a single type of receptor with decay kinetics of 54 ± 1.6 ms; the second (group II) is also mediated by a single receptor type, but exhibits significantly longer decay kinetics (151 ± 7.2 ms); and the third type of synapse (group III) contains multiple receptor types with fast (τ1=28.7 ± 2.5 ms) and slow (τ2=153 ± 11 ms) kinetics. Thus, for the first time, we report the properties of GABA synaptic currents associated with the Mauthner cells of zebrafish.