Zebrafish Models for Investigating the Effects of Chronic Benzodiazepine Exposure on Cognitive Dysfunction

Benzodiazepines (BZDs) is one of the most prescribed medication in outpatient clinic for patients with anxiety, insomnia, chronic pain, spasticity, panic disorder, agitation, tremor and seizure disorder. Long-term use due to addiction and increasing dose due to tolerance are commonly seen. Although acute intoxication of BZDs has been well-known to cause amnesia and confusion, long-term effects of using BZDs is poorly understood. Recently, many clinical studies pointed out that long-term use of BZDs may increase the risk of dementia. Given the high prevalence of dementia of all causes, the social economic burden caused by drug side-effect should not be overlooked. However, there are still debates whether there is association between BZDs and dementia and a causal effect remains to be proved. Surprisingly, animal models of chronic exposure of BZDs have been lacking and therefore no experimental evidence to support this hypothesis. Zebrafish (Danio rerio) is a vertebrate and one of the model organisms that is especially suitable for neuropharmacology testing and neuroscience research. In this proposal, we aim to create zebrafish models for testing if chronic use of BZDs may cause cognitive decline. Our specific aims are:Aim 1: Test the hypothesis that chronic exposure of benzodiazepine in young and aged zebrafish may impact differently on their cognitive function.We will use adult zebrafish of different age groups (6, 12, 24 months) and expose them in anxiolytic dosage of BZDs (e.g. Diazepam) for 3-6 months. Behavioral tests will be performed to determine if there are adverse effects of cognitive decline following long term exposure of BZDs. Immunostaining of downstream targets of BZDs, immunostaining of post-synaptic marker protein and morphological studies using Golgi staining on zebrafish lateral pallium will be performed. Aim 2: Test the hypothesis that chronic benzodiazepine exposure may not alleviate the cognitive dysfunction caused by chronic stress in zebrafish models.Adult zebrafish will undergo a series of physical and emotional stress using the unexpected chronic stress (UCS) protocol for one month and combined UCS and anxiolytic dosage of BZDs for another 3 months. Both anxiolytic effects and cognitive function will be evaluated through a battery of cognitive behavioral tests. Transcriptome analysis with RNA-seq and proteomic analysis using mass spectrometry will be performed for the evaluation of gene expression after long-term UCS and BZDs treatments. Aim 3: Test the hypothesis that chronic exposure of benzodiazepine in the amyloid-β42 neurotoxic zebrafish model may aggravate the learning deficits.Middle-aged zebrafish will be subject to cerebroventricular microinjection (CVMI) for planting amyloid-β42 into zebrafish brain. The amyloid-β42 will be dispersed through cerebrovascular fluid (CVF) to target periventricular cells. Control experiments will be done to ensure amyloid-β42 effects to cause apoptosis, neurogenesis, microglia activation and synaptic alteration. Amyloid-β42 treated zebrafish will be subdivided into two groups: one treated with BZDs for 3 month and another with placebo. Behavior test, Golgi staining, immunoblotting, immunostaining, as well as transcriptomic and proteomic studies will be performed 4 months after microinjection.

Project ID:PC10708-0889
External Project ID:MOST107-2314-B182-019