D-Amphetamine Exposure Differentially Disrupts Signaling Across Ontogeny in the Zebrafish.

Background: Prescriptive and illicit amphetamine (AMPH) use continues to increase along with the likelihood that during an individual's lifetime, the drug deleteriously influences the growth and connectivity of behavior circuits necessary for survival. Throughout ontogeny, neural circuits underlying these behaviors grow in complexity, gradually integrating many sensory inputs that trigger higher order coordinated motor responses. In the present study, we examine how AMPH disrupts the establishment of these circuits at critical neurodevelopmental periods, as well as the communication among established survival circuits. Materials and Methods: Zebrafish embryos (from 1 hpf) were raised in AMPH solutions, growth parameters and escape behavior were assessed at 24 and 48 hpf, and spinal cord tissues analyzed for differences in excitatory-inhibitory signaling balance among treatments. Adult fish were fed an acute dosage of AMPH over an 11-day conditioned place preference (PP) paradigm during which behaviors were recorded and brain tissues analyzed for alterations in dopaminergic signaling. Results: AMPH negatively affects embryonic growth and slows the execution of escape behavior, suggesting an imbalance in locomotor signaling. Although local spinal circuits provide primary escape modulation, no differences in inhibitory glycinergic, and excitatory glutamatergic signaling were measured among spinal neurons. AMPH also influenced place preference in adult zebrafish and resulted in the increased expression of dopamine signaling proteins (DRD1) in brain areas governing survival behaviors.

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.