The role of neuronal nitric oxide and its pathways in the protection and recovery from neurotoxin-induced de novo hypokinetic motor behaviors in the embryonic zebrafish (Danio rerio).

Neuronal nitric oxide (nNO) has been shown to affect motor function in the brain. Specifically, nNO acts in part through regulation of dopamine (DA) release, transporter function, and the elicitation of neuroprotection/neurodegeneration of neurons in conditions such as Parkinson's disease (PD). Recently, the zebrafish has been proposed to be a new model for the study of PD since neurotoxin damage to their nigrostriatal-like neurons exhibit PD-like motor dysfunctions similar to those of mammalian models and human patients. Results from this study demonstrate that treatment of 5 days post fertilization (dpf) fish with a nNO synthase inhibitor as a co-treatment with 6-OHDA facilitates long-term survival and accelerates the recovery from 6-OHDA-induced hypokinesia-like symptoms. These findings are unique in that under conditions of neurotoxin-induced stress, the inhibition of the NO-related S-nitrosylation indirect pathway dramatically facilitates recovery from 6-OHDA treatment but inhibition of the NO-sGC-cGMP direct pathway is essential for survival in 5 dpf treated fish. In conclusion, these results indicate that nNOS and the inhibition of the NO-linked S-nitrosylation pathway plays an important role in antagonizing the protection and recovery of fish from neurotoxin treatment. These data begin to help in the understanding of the role of NO as a neuroprotectant in dopaminergic pathways, particularly those that influence motor dysfunctions.

The Relationship between Estrogen and Nitric Oxide in the Prevention of Cardiac and Vascular Anomalies in the Developing Zebrafish (Danio Rerio).

It has been known that both estrogen (E2) and nitric oxide (NO) are critical for proper cardiovascular system (CVS) function. It has also been demonstrated that E2 acts as an upstream effector in the nitric oxide (NO) pathway. Results from this study indicate that the use of a nitric oxide synthase (NOS) inhibitor (NOSI) which targets specifically neuronal NOS (nNOS or NOS1), proadifen hydrochloride, caused a significant depression of fish heart rates (HR) accompanied by increased arrhythmic behavior. However, none of these phenotypes were evident with either the inhibition of endothelial NOS (eNOS) or inducible NOS (iNOS) isoforms. These cardiac arrhythmias could also be mimicked by inhibition of E2 synthesis with the aromatase inhibitor (AI), 4-OH-A, in a manner similar to that of nNOSI. In both scenarios, by using an NO donor (DETA-NO) in either NO + nNOSI or E2 + AI co-treatments, fish could be significantly rescued from decreased HR and increased arrhythmias. However, the addition of an NOS inhibitor (L-NAME) to the E2 + AI co-treatment fish prevented the rescue of low heart rates and arrhythmias, which strongly implicates the NO pathway as a downstream E2 targeted molecule for the maintenance of healthy cardiomyocyte contractile conditions in the developing zebrafish. Cardiac arrhythmias could be mimicked by the S-nitrosylation pathway inhibitor DTT (1,4-dithiothreitol) but not by ODQ (1H-[1-3]oxadiazolo[4,3-a]quinoxalin-1-one), the inhibitor of the NO receptor molecule sGC in the cGMP-dependent pathway. In both the nNOSI and AI-induced arrhythmic conditions, 100% of the fish expressed the phenotype, but could be rapidly rescued with maximum survival by a washout with dantrolene, a ryanodine Ca2+ channel receptor blocker, compared to the time it took for rescue using a control salt solution. In addition, of the three NOS isoforms, eNOS was the one most implicated in the maintenance of an intact developing fish vascular system. In conclusion, results from this study have shown that nNOS is the prominent isoform that is responsible, in part, for maintaining normal heart rates and prevention of arrhythmias in the developing zebrafish heart failure model. These phenomena are related to the upstream stimulatory regulation by E2. On the other hand, eNOS has a minimal effect and iNOS has little to no influence on this phenomenon. Data also suggests that nNOS acts on the zebrafish cardiomyocytes through the S-nitrosylation pathway to influence the SR ryanidine Ca2+ channels in the excitation-coupling phenomena. In contrast, eNOS is the prominent isoform that influences blood vessel development in this model.

Effects of Estrogen, Nitric Oxide, and Dopamine on Behavioral Locomotor Activities in the Embryonic Zebrafish: A Pharmacological Study.

Nitric oxide (NO) has been shown to affect motor function. Specifically, NO has been shown to act through regulation of dopamine (DA) release, transporter function, and the elicitation of neuroprotection/neurodegeneration of neurons. Recently, zebrafish have been proposed to be a new model for the study of various types of motor dysfunctions, since neurotoxin damage to their nigrostriatal-like neurons exhibit motor anomalies similar to those of mammalian models and human patients. Results from this study demonstrate that when NO synthesis is inhibited in zebrafish, using a neuronal NO synthase inhibitor (nNOSI), a condition called 'listless' occurs, where the fish lack swimming abilities, are rigid, and have difficulty maintaining balance. Additionally, co-treatment with either NO or estrogen (E2), an upstream regulator of NO synthase, can rescue fish from the 'listless' phenotype caused by exposure to the neurotoxin 6-hydroxydopamine (6 OHDA). In turn, NO deprived zebrafish were rescued from the 'listless' phenotype when co-treated with L-DOPA, a precursor to DA. Interestingly, the longer fish are exposed to a 6 OHDA + nNOSI co-treatment, the slower the recovery after washout, compared to a single treatment of each. Most significantly, NO involvement in the motor homeostasis of the embryonic zebrafish was shown to be expressed through the NO-cGMP-dependent pathway, and response to nNOSI treatments is developmentally regulated. In conclusion, these results indicate that there is a link between E2, NO, and DA systems that regulate motor functions in the embryonic zebrafish.

Use of a novel cell adhesion method and digital measurement to show stimulus-dependent variation in somatic and oral ciliary beat frequency in Paramecium.

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.

Estrogen prevents cardiac and vascular failure in the 'listless' zebrafish (Danio rerio) developmental model.

The presence of a robust estrogen (E2) response system throughout heart and blood vessel tissues of vertebrates, including humans, has led to the speculation that this ubiquitous hormone may play a prominent role in the health and maintenance of the adult cardiovascular system (CVS). We previously established an embryonic zebrafish model called 'listless', which results from the inhibition of E2 synthesis by treatment with aromatase enzyme inhibitors (AI). These fish have outward symptoms similar to the human condition of congestive heart failure and tamponade. E2 replacement therapy (1) reduced the severity of cardiac sac abnormalities, (2) protected heart function, (3) prevented reduction in heart size, and (4) reduced blood vessel deterioration. Nitric oxide may be a critical downstream mediator of these events. We also demonstrate that removal of fluid around the heart increases survival of AI-treated fish. These results strongly indicate the importance of E2 in the developing CVS of the zebrafish and offer a potential model for the study of its role in CVS development, maintenance, and disease conditions.

Effects of estrogen on the neuromuscular system in the embryonic zebrafish (Danio rerio).

Estrogen (E2) has been shown to play an important role in maintaining central nervous system (CNS) axonal growth, synapse formation, and neurotransmitter release; however, there is less direct evidence for a similar role in the peripheral nervous system (PNS). In a previous study we have shown that when E2 was removed from embryonic zebrafish (Danio rerio) system using the aromatase inhibiter (AI) 4-hydroxyandrostenedione (4-OH-A) fish did not developmentally express normal sensory-motor (S-M) functions such as tactile, vestibular, and swimming behaviors, creating a condition called 'listless.' These findings led to speculation that E2 deprivation, under these conditions, caused a neuromuscular-like "denervation" resulting in the 'listless' condition. Morphometric data analysis reported in this study indicated that there was an absence of vesicular acetylcholine transporter (VAChT) staining in the primary motor neurons as a result of AI treatment compared to controls. In contrast, E2 co-treatment with AI (E2+AI) rescued a significant number of VAChT stained nerve endings and treatment of fish with E2 alone exhibited a significantly higher number of VAChT profiles than in control fish. In addition, in the AI treated group znp-1 antibody staining of the primary motor neurons demonstrated: 1) diminished axon branching; 2) shorter primary axons; and 3) an absence in the posterior trunk regions of fish. In turn, trunk muscles were significantly diminished in size and less organized when treated with AI when compared to controls and E2+AI treatment restored myotome width and height accompanied by some dramatic changes in the α-bungarotoxin-labeled ACh post-synaptic receptor elements of the trunk skeletal muscles. Data from this study suggest that treatment with the AI 4-OH-A essentially denervates the zebrafish trunk skeletal muscles, most likely by compromising the development of the vesicular transport system for ACh preventing it from acting at the synaptic terminals. These findings begin to demonstrate the prominent role that E2 plays in the developing zebrafish PNS, particularly at the neuromuscular level.

Genetic dissection of attractant-induced conductances in Paramecium.

Paramecium tetraurelia is attracted to acetate and biotin by swimming smoothly and fast up gradients of these attractants, and turning immediately and slowing down when leaving these stimuli. We use a group of mutants, each with a different defect in an identified ion conductance, to show that these two stimuli open different ion channels, and the behaviors that occur upon application of stimulus (on-response) and removal of stimulus (off-response) have different roles in attraction to these two stimuli. The most important parameters for successful attraction to acetate are the on-response behaviors of fast swimming with few turns, and the mutants' behavior suggests that I(K(Ca,h)) is the conductance involved that initiates this behavior. I(K(Ca,h or d)) appears to be important to the on-response in biotin; the results with mutants suggest that the biotin off-response depolarization is initiated by an I(Ca), which can be large enough or close enough to channels to open I(K(Ca,d)), I(Na(Ca)) and I(Mg(Ca)).