Can LDOPA Rescue Zebrafish Danio rerio from n

Can L-DOPA Rescue Zebrafish (Danio rerio) from n. NOSI-induced Parkinson -like Symptoms? John Mc. Kelvey BI ‘ 16 Mentor: Dr. Turner ‘ 65

Basal Ganglia Disorders • • Parkinson’s disease (PD) Huntington’s disease Tourette syndrome Hemiballismus Dystonia OCD Psychostimulant addiction

What is the basal ganglia? • Cluster of neurons in the base of forebrain • Strongly interconnected with the cerebral cortex, thalamus, and brainstem, as well as several other brain areas • Involved with voluntary movements and procedural learning

Why Focus On Parkinson’s Disease? • Second most common neurodegenerative disorder after Alzheimer’s disease – 7 million affected globally (1 million in US) – 103 K deaths annually (2013 figures, up from 44 K in 1990 ) – US total burden = $23 billion yearly • Symptoms – Motor: shaking, rigidity, slowness of movement, difficulty walking, balance problems (gait) – Cognition: dementia, depression – Other: sensory, sleep, and emotional problems http: //www. nejm. org/doi/full/10. 1056/NEJMic m 0810287

Parkinson’s Disease • Cause – undetermined • Pathology – – Dopaminergic neuron degeneration in the substantia nigra compacta (SNc) – Accumulation of Lewey bodies in affected neurons • Treatment – no cure; strategy is to increase dopamine content in brain by: – L-Dopa administration – dopamine intermediate that crosses the B-B barrier – DAT antagonists – MOA inhibitors

Nitric Oxide (NO) – How Does It Fit Into Basal Ganglia Function and PD? • What is NO? – A gaseous neurotransmitter with second messenger signaling abilities • Role of NO in integration of BG motor function – NO donors and NOSIs affect motor behaviors – Dopamine (DA) and glutamate regulate NO synthesis – NO regulates DA release • Does NO paly a role in PD patients? – 50% loss of nitrites in CSF – < in NOS expression and n. NOS protein levels

Why Use Zebrafish? • • • Vertebrates Easy to manage Short generation time Lots of offspring produced Translucent

Why Use Zebrafish? • Can we link, for the first time, the E 2 -NO-DA interactions in the zebrafish ‘listless’ model as a cause and effect in generating these Parkinson-like symptoms? • Can the zebrafish be used as a model for the study of motor disorders? • Human Parkinson Symptoms 1. Postural instability 2. Slowness of movement 3. Rigidity 4. Resting tremor • Zebrafish ‘Listless’ Phenotype 1. Vestibular loss 2. Decreased swimming speed 3. Immobile, hyprflexed bodies 4. Unusual swimming movements

What have I done so far? Question #1: When is the best time to treat embryonic zebrafish for NO studies? Is there is a developmental regulation of the NO response system in embryonic zebrafish? What is the best treatment time for NO studies?

The effect of developmental age on embryonic zebrafish response to 50 µM n. NOSI in developing the ‘listless’ phenotype • Have determined that treating 6 days post fertilization (dpf) fish with n. NOSI is ideal for my experiments in order to induce listlessness

The effect of developmental age on the ability of embryonic zebrafish to recover from the ‘listless’ phenotype after 24 hours of treatment with 50 µM n. NOSI Have determined that after n. NOSI treatment at 6 days post fertilization (dpf) fish can completely recover their locomotor skills after a 24 hr washout with ERS

So now what? Zebrafish as a PD-like model? Question #2: Can zebrafish be rescued from the ‘listless’ phenotype caused by NO deprivation through dopamine treatment?

Materials and Methods • 4 groups – ERS control, L-DOPA, n. NOSI (50 µM), L-DOPA + n. NOSI • Warm water bath – L-DOPA (10 m. M) preparation • Change solutions every 24 hours using transfer pipettes • Check for results at 48 hours – Spontaneous swimming movements – Vestibular/balance – Sensory-motor response – probe • Data analysis - Z test calculator for multiple population proportions

The effect of treatment on percentage of zebrafish that respond to probing Have shown that L-DOPA co-treatment can re-establish the normal sensorymotor response in NO deprived zebrafish.

The effect of treatment on percentage of zebrafish that swim spontaneously Have shown that L-DOPA co-treatment can partly re-establish normal sontaneous swimming in NO deprived zebrafish.

Future work • Repeat L-DOPA co-treatment rescue experiment • Use other pharmacological tools to augment the presence of dopamine in the NO deprived zebrafish – Dopamine transporter (DAT) inhibitor – Monoamine oxidase (MAO) inhibitor • Immunohistochemical studies to identify effects of treatments on: : – TH/DA neurons – DAT/DA neurons