Context-dependent hyperactivity in syngap1a and syngap1b zebrafish autism models.

Background and Aims: SYNGAP1 disorder is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. Methods: We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. Results: We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab larvae are hyperactive compared to wild type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, with overall movement increasing with the number of mutant syngap1 alleles. Conclusions: Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.

Drugs prescribed for Phelan-McDermid syndrome differentially impact sensory behaviors in shank3 zebrafish models.

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.

Gastrointestinal Dysfunction in Genetically Defined Neurodevelopmental Disorders.

Gastrointestinal 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.

Restoring Shank3 in the rostral brainstem of shank3ab-/- zebrafish autism models rescues sensory deficits.

People with Phelan-McDermid Syndrome, caused by mutations in the SHANK3 gene, commonly exhibit reduced responses to sensory stimuli; yet the changes in brain-wide activity that link these symptoms to mutations in the shank3 gene remain unknown. Here we quantify movement in response to sudden darkness in larvae of two shank3 zebrafish mutant models and show that both models exhibit dampened responses to this stimulus. Using brain-wide activity mapping, we find that shank3-/- light-sensing brain regions show normal levels of activity while sensorimotor integration and motor regions are less active. Specifically restoring Shank3 function in a sensorimotor nucleus of the rostral brainstem enables the shank3-/- model to respond like wild-type. In sum, we find that reduced sensory responsiveness in shank3-/- models is associated with reduced activity in sensory processing brain regions and can be rescued by restoring Shank3 function in the rostral brainstem. These studies highlight the importance of Shank3 function in the rostral brainstem for integrating sensory inputs to generate behavioral adaptations to changing sensory stimuli.

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.

Elevated preoptic brain activity in zebrafish glial glycine transporter mutants is linked to lethargy-like behaviors and delayed emergence from anesthesia.

Delayed emergence from anesthesia was previously reported in a case study of a child with Glycine Encephalopathy. To investigate the neural basis of this delayed emergence, we developed a zebrafish glial glycine transporter (glyt1 - / -) mutant model. We compared locomotor behaviors; dose-response curves for tricaine, ketamine, and 2,6-diisopropylphenol (propofol); time to emergence from these anesthetics; and time to emergence from propofol after craniotomy in glyt1-/- mutants and their siblings. To identify differentially active brain regions in glyt1-/- mutants, we used pERK immunohistochemistry as a proxy for brain-wide neuronal activity. We show that glyt1-/- mutants initiated normal bouts of movement less frequently indicating lethargy-like behaviors. Despite similar anesthesia dose-response curves, glyt1-/- mutants took over twice as long as their siblings to emerge from ketamine or propofol, mimicking findings from the human case study. Reducing glycine levels rescued timely emergence in glyt1-/- mutants, pointing to a causal role for elevated glycine. Brain-wide pERK staining showed elevated activity in hypnotic brain regions in glyt1-/- mutants under baseline conditions and a delay in sensorimotor integration during emergence from anesthesia. Our study links elevated activity in preoptic brain regions and reduced sensorimotor integration to lethargy-like behaviors and delayed emergence from propofol in glyt1-/- mutants.

Genetic compensation in a stable slc25a46 mutant zebrafish: A case for using F0 CRISPR mutagenesis to study phenotypes caused by inherited disease.

A phenomenon of genetic compensation is commonly observed when an organism with a disease-bearing mutation shows incomplete penetrance of the disease phenotype. Such incomplete phenotypic penetrance, or genetic compensation, is more commonly found in stable knockout models, rather than transient knockdown models. As such, these incidents present a challenge for the disease modeling field, although a deeper understanding of genetic compensation may also hold the key for novel therapeutic interventions. In our study we created a knockout model of slc25a46 gene, which is a recently discovered important player in mitochondrial dynamics, and deleterious mutations in which are known to cause peripheral neuropathy, optic atrophy and cerebellar ataxia. We report a case of genetic compensation in a stable slc25a46 homozygous zebrafish mutant (hereafter referred as "mutant"), in contrast to a penetrant disease phenotype in the first generation (F0) slc25a46 mosaic mutant (hereafter referred as "crispant"), generated with CRISPR/Cas-9 technology. We show that the crispant phenotype is specific and rescuable. By performing mRNA sequencing, we define significant changes in slc25a46 mutant's gene expression profile, which are largely absent in crispants. We find that among the most significantly altered mRNAs, anxa6 gene stands out as a functionally relevant player in mitochondrial dynamics. We also find that our genetic compensation case does not arise from mechanisms driven by mutant mRNA decay. Our study contributes to the growing evidence of the genetic compensation phenomenon and presents novel insights about Slc25a46 function. Furthermore, our study provides the evidence for the efficiency of F0 CRISPR screens for disease candidate genes, which may be used to advance the field of functional genetics.

Intestinal dysmotility in a zebrafish (Danio rerio) shank3a;shank3b mutant model of autism.

Background and aims: Autism spectrum disorder (ASD) is currently estimated to affect more than 1% of the world population. For people with ASD, gastrointestinal (GI) distress is a commonly reported but a poorly understood co-occurring symptom. Here, we investigate the physiological basis for GI distress in ASD by studying gut function in a zebrafish model of Phelan-McDermid syndrome (PMS), a condition caused by mutations in the SHANK3 gene. Methods: To generate a zebrafish model of PMS, we used CRISPR/Cas9 to introduce clinically related C-terminal frameshift mutations in shank3a and shank3b zebrafish paralogues (shank3abΔC). Because PMS is caused by SHANK3 haploinsufficiency, we assessed the digestive tract (DT) structure and function in zebrafish shank3abΔC+/- heterozygotes. Human SHANK3 mRNA was then used to rescue DT phenotypes in larval zebrafish. Results: Significantly slower rates of DT peristaltic contractions (p < 0.001) with correspondingly prolonged passage time (p < 0.004) occurred in shank3abΔC+/- mutants. Rescue injections of mRNA encoding the longest human SHANK3 isoform into shank3abΔC+/- mutants produced larvae with intestinal bulb emptying similar to wild type (WT), but still deficits in posterior intestinal motility. Serotonin-positive enteroendocrine cells (EECs) were significantly reduced in both shank3abΔC+/- and shank3abΔC-/- mutants (p < 0.05) while enteric neuron counts and overall structure of the DT epithelium, including goblet cell number, were unaffected in shank3abΔC+/- larvae. Conclusions: Our data and rescue experiments support mutations in SHANK3 as causal for GI transit and motility abnormalities. Reductions in serotonin-positive EECs and serotonin-filled ENS boutons suggest an endocrine/neural component to this dysmotility. This is the first study to date demonstrating DT dysmotility in a zebrafish single gene mutant model of ASD.

Zebrafish: A Pharmacogenetic Model for Anesthesia.

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery.

A defect in the inner kinetochore protein CENPT causes a new syndrome of severe growth failure.

Primordial growth failure has been linked to defects in the biology of cell division and replication. The complex processes involved in microtubule spindle formation, organization and function have emerged as a dominant patho-mechanism in these conditions. The majority of reported disease genes encode for centrosome and centriole proteins, leaving kinetochore proteins by which the spindle apparatus interacts with the chromosomes largely unaccounted for. We report a novel disease gene encoding the constitutive inner kinetochore member CENPT, which is involved in kinetochore targeting and assembly, resulting in severe growth failure in two siblings of a consanguineous family. We herein present studies on the molecular and cellular mechanisms that explain how genetic mutations in this gene lead to primordial growth failure. In both, affected human cell lines and a zebrafish knock-down model of Cenpt, we observed aberrations in cell division with abnormal accumulation of micronuclei and of nuclei with increased DNA content arising from incomplete and/or irregular chromosomal segregation. Our studies underscore the critical importance of kinetochore function for overall body growth and provide new insight into the cellular mechanisms implicated in the spectrum of these severe growth disorders.

Spatial patterning of excitatory and inhibitory neuropil territories during spinal circuit development.

To generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitatory inputs that trigger action potentials and inhibitory inputs that promote a stable resting potential (E/I balance). Previous studies have focused on individual neurons and have shown that, over a short spatial scale, excitatory and inhibitory (E/I) synapses tend to form structured territories with inhibitory inputs enriched on cell bodies and proximal dendrites and excitatory inputs on distal dendrites. However, systems-level E/I patterns, at spatial scales larger than single neurons, are largely uncharted. We used immunostaining for PSD-95 and gephyrin postsynaptic scaffolding proteins as proxies for excitatory and inhibitory synapses, respectively, to quantify the numbers and map the distributions of E/I synapses in zebrafish spinal cord at both an embryonic stage and a larval stage. At the embryonic stage, we found that PSD-95 puncta outnumber gephyrin puncta, with the number of gephyrin puncta increasing to match that of PSD-95 puncta at the larval stage. At both stages, PSD-95 puncta are enriched in the most lateral neuropil corresponding to distal dendrites while gephyrin puncta are enriched on neuronal somata and in the medial neuropil. Significantly, similar to synaptic puncta, neuronal processes also exhibit medial-lateral territories at both developmental stages with enrichment of glutamatergic (excitatory) processes laterally and glycinergic (inhibitory) processes medially. This establishment of neuropil excitatory-inhibitory structure largely precedes dendritic arborization of primary motor neurons, suggesting that the structured neuropil could provide a framework for the development of E/I balance at the cellular level. J. Comp. Neurol. 525:1649-1667, 2017. © 2016 Wiley Periodicals, Inc.

Function Over Form: Modeling Groups of Inherited Neurological Conditions in Zebrafish.

Zebrafish are a unique cell to behavior model for studying the basic biology of human inherited neurological conditions. Conserved vertebrate genetics and optical transparency provide in vivo access to the developing nervous system as well as high-throughput approaches for drug screens. Here we review zebrafish modeling for two broad groups of inherited conditions that each share genetic and molecular pathways and overlap phenotypically: neurodevelopmental disorders such as Autism Spectrum Disorders (ASD), Intellectual Disability (ID) and Schizophrenia (SCZ), and neurodegenerative diseases, such as Cerebellar Ataxia (CATX), Hereditary Spastic Paraplegia (HSP) and Charcot-Marie Tooth Disease (CMT). We also conduct a small meta-analysis of zebrafish orthologs of high confidence neurodevelopmental disorder and neurodegenerative disease genes by looking at duplication rates and relative protein sizes. In the past zebrafish genetic models of these neurodevelopmental disorders and neurodegenerative diseases have provided insight into cellular, circuit and behavioral level mechanisms contributing to these conditions. Moving forward, advances in genetic manipulation, live imaging of neuronal activity and automated high-throughput molecular screening promise to help delineate the mechanistic relationships between different types of neurological conditions and accelerate discovery of therapeutic strategies.

Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy.

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder.

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis.

Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1-2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes.

Knock-down DHDDS expression induces photoreceptor degeneration in zebrafish.

A mutation in the dehydrodolichol diphosphate synthase (DHDDS) was recently identified as the cause of a subtype of recessive retinitis pigmentosa (RP). Molecular modeling indicates that this mutation could result in low enzymatic efficiency of DHDDS. To investigate the possible link between insufficient DHDDS activity and photoreceptor degeneration, the expression of DHDDS was knocked down by morpholino oligonucleotides (MO) injected into zebrafish one cell embryos. The general appearance and behavior of 4-day-old MO-injected fish were normal, but they failed to respond to light-off, suggesting loss of visual function. Morphological analysis showed that photoreceptor outer segments in retinas of MO-injected fish are very short and in many cases completely missing. Peanut agglutinin (PNA) staining confirmed the absence of cone outer segments. These results demonstrate that suppression of DHDDS expression in zebrafish leads to the loss of photoreceptor outer segments and visual function. These results support the hypothesis that insufficient DHDDS function leads to retinal degeneration.

Distinct phenotypes in zebrafish models of human startle disease.

Startle disease is an inherited neurological disorder that causes affected individuals to suffer noise- or touch-induced non-epileptic seizures, excessive muscle stiffness and neonatal apnea episodes. Mutations known to cause startle disease have been identified in glycine receptor subunit (GLRA1 and GLRB) and glycine transporter (SLC6A5) genes, which serve essential functions at glycinergic synapses. Despite the significant successes in identifying startle disease mutations, many idiopathic cases remain unresolved. Exome sequencing in these individuals will identify new candidate genes. To validate these candidate disease genes, zebrafish is an ideal choice due to rapid knockdown strategies, accessible embryonic stages, and stereotyped behaviors. The only existing zebrafish model of startle disease, bandoneon (beo), harbors point mutations in glrbb (one of two zebrafish orthologs of human GLRB) that cause compromised glycinergic transmission and touch-induced bilateral muscle contractions. In order to further develop zebrafish as a model for startle disease, we sought to identify common phenotypic outcomes of knocking down zebrafish orthologs of two known startle disease genes, GLRA1 and GLRB, using splice site-targeted morpholinos. Although both morphants were expected to result in phenotypes similar to the zebrafish beo mutant, our direct comparison demonstrated that while both glra1 and glrbb morphants exhibited embryonic spasticity, only glrbb morphants exhibited bilateral contractions characteristic of beo mutants. Likewise, zebrafish over-expressing a dominant startle disease mutation (GlyR α1(R271Q)) exhibited spasticity but not bilateral contractions. Since GlyR ßb can interact with GlyR α subunits 2-4 in addition to GlyR α1, loss of the GlyR ßb subunit may produce more severe phenotypes by affecting multiple GlyR subtypes. Indeed, immunohistochemistry of glra1 morphants suggests that in zebrafish, alternate GlyR α subunits can compensate for the loss of the GlyR α1 subunit. To address the potential for interplay among GlyR subunits during development, we quantified the expression time-course for genes known to be critical to glycinergic synapse function. We found that GlyR α2, α3 and α4a are expressed in the correct temporal pattern and could compensate for the loss of the GlyR α1 subunit. Based on our findings, future studies that aim to model candidate startle disease genes in zebrafish should include measures of spasticity and synaptic development.

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.

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.

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.