Glycinergic Synapse Development, Plasticity, and Homeostasis in Zebrafish

The zebrafish glial glycine transporter 1 (GlyT1) mutant provides an animal model in which homeostatic plasticity at glycinergic synapses restores rhythmic motor behaviors. GlyT1 mutants, initially paralyzed by the build-up of the inhibitory neurotransmitter glycine, stage a gradual recovery that is associated with reductions in the strength of evoked glycinergic responses. Gradual motor recovery suggests sequential compensatory mechanisms that culminate in the down-regulation of the neuronal glycine receptor. However, how motor recovery is initiated and how other forms of plasticity contribute to behavioral recovery are still outstanding questions that we discuss in the context of (1) glycinergic synapses as they function in spinal circuits that produce rhythmic motor behaviors, (2) the proteins involved in regulating glycinergic synaptic strength, (3) current models of glycinergic synaptogenesis, and (4) plasticity mechanisms that modulate the strength of glycinergic synapses. Concluding remarks (5) explore the potential for distinct plasticity mechanisms to act in concert at different spatial and temporal scales to achieve a dynamic stability that results in balanced motor behaviors

Drugs prescribed for Phelan-McDermid syndrome differentially impact sensory behaviors in shank3 zebrafish models. [version 1; peer review: 1 approved with reservations]

Background: Altered sensory processing is a pervasive symptom in individuals with Autism Spectrum Disorders (ASD); people with Phelan McDermid syndrome (PMS), in particular, show reduced responses to sensory stimuli. PMS is caused by deletions of the terminal end of chromosome 22 or point mutations in Shank3. People with PMS can present with an array of symptoms including ASD, epilepsy, gastrointestinal distress, and reduced responses to sensory stimuli. People with PMS are often medicated to manage behaviors like aggression and/or self-harm and/or epilepsy, and it remains unclear how these medications might impact perception/sensory processing. Here we test this using zebrafish mutant shank3ab PMS models that likewise show reduced sensory responses in a visual motor response (VMR) assay, in which increased locomotion is triggered by light to dark transitions. Methods: We screened three medications, risperidone, lithium chloride (LiCl), and carbamazepine (CBZ), prescribed to people with PMS and one drug, 2-methyl-6-(phenylethynyl) pyridine (MPEP) tested in rodent models of PMS, for their effects on a sensory-induced behavior in two zebrafish PMS models with frameshift mutations in either the N- or C- termini. To test how pharmacological treatments affect the VMR, we exposed larvae to selected drugs for 24 hours and then quantified their locomotion during four ten-minute cycles of lights on-to-off stimuli. Results: We found that risperidone normalized the VMR in shank3 models. LiCl and CBZ had no effect on the VMR in any of the three genotypes. MPEP reduced the VMR in wildtype (WT) to levels seen in shank3 models but caused no changes in either shank3 model. Finally, shank3 mutants showed resistance to the seizure-inducing drug pentylenetetrazol (PTZ), at a dosage that results in hyperactive swimming in WT zebrafish. Conclusions: Our work shows that the effects of drugs on sensory processing are varied in ways that can be highly genotype- and drug-dependent

Action potential waveform voltage clamp shows significance of different Ca2+ channel types in developing ascidian muscle

Early in development, ascidian muscle cells generate spontaneous, long-duration action potentials that are mediated by a high-threshold, inactivating Ca2+ current. This spontaneous activity is required for appropriate physiological development.Mature muscle cells generate brief action potentials only in response to motor neuron input. The mature action potential is mediated by a high-threshold sustained Ca2+ current.Action potentials recorded from these two stages were imposed as voltage-clamp commands on cells of the same and different stages from which they were recorded. This strategy allowed us to study how immature and mature Ca2+ currents are optimized to their particular functions.Total Ca2+ entry during an action potential did not change during development. The developmental increase in Ca2+ current density exactly compensated for decreased spike duration. This compensation was a function purely of Ca2+ current density, not of the transition from immature to mature Ca2+ current types.In immature cells, Ca2+ entry was spread out over the entire waveform of spontaneous activity, including the interspike voltage trajectory. This almost continuous Ca2+ entry may be important in triggering Ca2+-dependent developmental programmes, and is a function of the slightly more negative voltage dependence of the immature Ca2+ current.In contrast, Ca2+ entry in mature cells was confined to the action potential itself, because of the slightly more positive voltage dependence of the mature Ca2+ current. This may be important in permitting rapid contraction-relaxation cycles during larval swimming.The inactivation of the immature Ca2+ current serves to limit the frequency and burst duration of spontaneous activity. The sustained kinetics of the mature Ca2+ current permit high-frequency firing during larval swimming

Spontaneous activity regulates calcium-dependent K+ current expression in developing ascidian muscle

In embryonic ascidian muscle, outward K + currents develop in two stages: the initial expression of a slowly activating, voltage-gated K + current ( I Kv ) near the time of neurulation is followed about 6 h later by a rapidly activating calcium-activated K + current ( I K(Ca) ). During this 6 h interval, inward Ca 2+ currents ( I Ca ) appear and the inward rectifier ( I K(IR) ), the sole resting conductance, is transiently downregulated. These events predict a period of spontaneous activity. The following experiments were designed to test this prediction and to examine the relevance of spontaneous activity for muscle cell development. By recording activity in cell-attached patches, we have found that muscle cells generate spontaneous action potentials during this 6 h window of time when I K(IR) is downregulated and outward K + currents are slow. Action potentials occur at a mean frequency of 13.9 min −1 . When activity is blocked by the transient application of the Ca 2+ channel blocker Cd 2+ , I K(Ca) fails to develop. This disruption is specific for I K(Ca) : I K(IR) and I Ca develop normally in activity-blocked cells. Application of Cd 2+ either before or after the window of activity has no effect. The reappearance of I K(IR) and the development of I K(Ca) and the mature form of I Ca are all prevented by transcription blockers, with a sensitive period corresponding to the period of activity. These data show that, although the expression of three channel types depends on transcription during the period of spontaneous activity, only the development of I K(Ca) depends on activity

A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation

Identification of conserved proteins that act to establish the neuronal phenotype has relied predominantly on structural homologies of the underlying genes. In the case of the repressor element 1 silencing transcription factor (REST), a central player in blocking the neuronal phenotype in vertebrate non-neural tissue, the invertebrate homolog is absent, raising the possibility that distinct strategies are used to establish the CNS of invertebrates. Using a yeast two-hybrid screen designed specifically to identify functional analogs of REST, we show that Drosophila melanogaster uses a strategy that is functionally similar to, but appears to have evolved independently of, REST. The gene at the center of the strategy in flies encodes the repressor Tramtrack88 (Ttk88), a protein with no discernable homology to REST but that nonetheless is able to interact with the same transcriptional partners. Ttk88 uses the REST corepressor Drosophila CoREST to coordinately regulate a set of genes encoding the same neuronal hallmarks that are regulated by REST in vertebrates. Our findings indicate that repression is an important mechanism for regulating neuronal phenotype across phyla and suggest that co-option of a similar corepressor complex occurred to restrict expression of genes critical for neuronal function to a compartmentalized nervous system

Gastrointestinal Dysfunction in Genetically Defined Neurodevelopmental Disorders

AbstractGastrointestinal symptoms are common in most forms of neurodevelopment disorders (NDDs) such as in autism spectrum disorders (ASD). The current patient-reported outcome measures with validated questionnaires used in the general population of children without NDDS cannot be used in the autistic individuals. We explore here the multifactorial pathophysiology of ASD and the role of genetics and the environment in this disease spectrum and focus instead on possible diagnostics that could provide future objective insight into the connection of the gut-brain-microbiome in this disease entity. We provide our own data from both humans and a zebrafish model of ASD called Phelan-McDermid Syndrome. We hope that this review highlights the gaps in our current knowledge on many of these profound NDDs and that it provides a future framework upon which clinicians and researchers can build and network with other interested multidisciplinary specialties

The Gut-Brain-Microbiome Axis and Its Link to Autism: Emerging Insights and the Potential of Zebrafish Models

Research involving autism spectrum disorder (ASD) most frequently focuses on its key diagnostic criteria: restricted interests and repetitive behaviors, altered sensory perception, and communication impairments. These core criteria, however, are often accompanied by numerous comorbidities, many of which result in severe negative impacts on quality of life, including seizures, epilepsy, sleep disturbance, hypotonia, and GI distress. While ASD is a clinically heterogeneous disorder, gastrointestinal (GI) distress is among the most prevalent co-occurring symptom complex, manifesting in upward of 70% of all individuals with ASD. Consistent with this high prevalence, over a dozen family foundations that represent genetically distinct, molecularly defined forms of ASD have identified GI symptoms as an understudied area with significant negative impacts on quality of life for both individuals and their caregivers. Moreover, GI symptoms are also correlated with more pronounced irritability, social withdrawal, stereotypy, hyperactivity, and sleep disturbances, suggesting that they may exacerbate the defining behavioral symptoms of ASD. Despite these facts (and to the detriment of the community), GI distress remains largely unaddressed by ASD research and is frequently regarded as a symptomatic outcome rather than a potential contributory factor to the behavioral symptoms. Allowing for examination of both ASD’s impact on the central nervous system (CNS) as well as its impact on the GI tract and the associated microbiome, the zebrafish has recently emerged as a powerful tool to study ASD. This is in no small part due to the advantages zebrafish present as a model system: their precocious development, their small transparent larval form, and their parallels with humans in genetics and physiology. While ASD research centered on the CNS has leveraged these advantages, there has been a critical lack of GI-centric ASD research in zebrafish models, making a holistic view of the gut-brain-microbiome axis incomplete. Similarly, high-throughput ASD drug screens have recently been developed but primarily focus on CNS and behavioral impacts while potential GI impacts have not been investigated. In this review, we aim to explore the great promise of the zebrafish model for elucidating the roles of the gut-brain-microbiome axis in ASD

Synaptic Homeostasis in a Zebrafish Glial Glycine Transporter Mutant

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects. These experiments are consistent with previous studies that demonstrate regulation of global glycine levels in the CNS as a primary function of GlyT1. As GlyT1 mutants mature, their ability to mount an escape response naturally recovers. To understand the basis of this recovery, we assay synaptic transmission in primary spinal motor neurons by measuring stimulus-evoked postsynaptic potentials. At the peak of the motility defect, inhibitory synaptic potentials are both significantly larger and more prolonged indicating a prominent role for GlyT1 in shaping fast synaptic transmission. However, as GlyT1 mutants naturally regain their ability to swim, the amplitude of inhibitory potentials decreases to below wild-type levels. In parallel with diminishing synaptic potentials, the glycine concentration required to evoke the mutant motility defect increases 61-fold during behavioral recovery. Behavioral recovery is also mirrored by a reduction in the levels of both glycine receptor protein and transcript. These results suggest that increased CNS glycine tolerance and reduced glycine receptor expression in GlyT1 mutants reflect compensatory mechanisms for functional recovery from excess nervous system inhibition

CoREST: A functional corepressor required for regulation of neural-specific gene expression

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity