For depression project, our study will unravel new therapeutic opportunities, i.e. specifically targeting regulatory elements of glutamatergic signaling, for the treatment of depression. This study will also lay the groundwork for the identification of a predicative biomarker for tailored therapy, which is critically needed for timely and effective health care. For Alzheimer’s project, the potential is to develop a peptide drug to slow the progression of AD and improve the life quality of AD patients. Moreover, the therapeutic pathway uncovered by our peptide may open a new avenue for the development of other small molecules (eg. nanobodies) for AD treatment.

This surgery is essential for the animals' survival, and ensuring their well-being becomes paramount, especially during the postsurgical period. To alleviate any discomfort, we meticulously apply local anesthetic around the incision site and utilize preemptive and postoperative analgesics. While animals generally experience mild chronic stress, certain behavioral tests, such as fear conditioning, can induce more intense stress levels. It's important to note that fear conditioning tests are globally recognized for assessing hippocampal function. Koolhaas (1997) categorized the corticosterone response to various stressors, ranking them from highest to lowest impact: social defeat, social victory, naive swimming, trained swimming, restraint, 2 mA foot shock, 1 mA foot shock, 0.5 mA foot shock (eliciting less than half the corticosterone compared to social defeat), water immersion, shockprod, novel cage, and 100 dBA noise. In our experiment, we will administer a 0.75 mA shock for two seconds, which falls within the range of mild to moderate stress.

The experiments are terminal. Brain tissue is collected as specific time points according to the protocol and then used for biochemical analysis or imaging (immunohistochemistry).

The number of animals used is kept to a minimum by the following measures: 1. Expertise at all steps of the procedure from anesthesia and surgical technique, to implantation of the cannulae, dissection, cutting and storage of brain tissue, and biochemical/molecular analysis. For a technically valid experiment, all steps must work. Effective transfer of these skills to staff and students is essential. As part of this training, we have made videos of all stages of the procedure that must be studied by trainees before they begin. 2. Use of Pilot Studies: Conduct small-scale preliminary studies (n = 5-6) to evaluate feasibility, test procedures, identify potential issues, optimize parameters, validate assumptions, and estimate effect size, thereby refining our hypotheses and avoiding the usage of large numbers of animals in potentially flawed studies. 3. Better Use of Existing Data: A. Meta-Analysis: Combine data from multiple studies to reduce the need for new experiments. B. Data Sharing: Create centralized databases for sharing data to prevent redundancy. 4. Advanced Statistical Methods: A. Refined Statistical Techniques: Use sophisticated statistical models to increase accuracy with fewer animals. B. Sequential Analysis: Analyze data at intervals to potentially conclude studies early if results are clear. 5. Collaborative Studies: We and our collaborators will share resources and data to consolidate animal use. 6. We develop experimental design to address the easiest questions first. This makes it possible to change directions or terminate experiments at an early stage. We believe reduction is best achieved by careful planning of the research strategy and anticipation of methodological challenges.

In our pursuit of refinement, we have implemented a series of measures to optimize animal welfare throughout all stages of our study. Firstly, stringent protocols are followed for anesthesia and analgesia administration during surgical procedures, ensuring that animals experience minimal pain and distress. We utilize carefully selected anesthetic agents and monitor vital signs closely to maintain appropriate levels of sedation while minimizing potential side effects. Post-operative analgesia is provided to alleviate any discomfort and promote rapid recovery. Furthermore, we have established predefined humane endpoints to guide intervention decisions and minimize unnecessary suffering. These endpoints are continually monitored throughout the study, and interventions are promptly implemented if animals exhibit signs of distress or discomfort beyond predefined thresholds. In addition to medical interventions, environmental enrichment strategies are integrated into our housing conditions to promote the psychological well-being of research animals. Enrichment activities, such as social housing with compatible cage mates, access to stimulating toys, and varied feeding methods, are provided to encourage natural behaviors and reduce stress levels. Moreover, housing facilities are maintained at optimal environmental conditions, including appropriate temperature, humidity, and lighting levels, to ensure the overall well-being of the animals. Operating techniques are optimized to minimize discomfort and stress for the animals. Surgical procedures are conducted using refined techniques by skilled personnel to minimize tissue damage and post-operative complications. In our refinement efforts within behavioral testing, we meticulously tailor protocols to ensure the well-being and comfort of research animals. This includes employing non-invasive or minimally invasive techniques wherever feasible to reduce stress and discomfort. By continuously refining our methods and prioritizing animal welfare, we aim to conduct behavioral tests with the utmost ethical consideration and scientific rigor. Our commitment to refinement ensures that the welfare of the animals involved is prioritized at every stage of the study, ultimately contributing to the ethical and responsible use of animals in biomedical research.

For Alzheimer's project: The reason to choose 5XFAD mice is because it generates Aβ-42 almost exclusively and rapidly accumulate massive cerebral levels. In addition, these mice have reduced synaptic marker protein levels, increased p25 levels, neuron loss, and memory impairment in the Y-maze test. 5XFAD transgenic mice rapidly recapitulate major features of Alzheimer's Disease amyloid pathology. Specifically, extracellular amyloid plaques were detected by immunohistochemistry in hippocampus, cortex, and thalamus of 2-month animals (Richard et al., 2015). Electrophysiological evidence of synaptic deficits was obtained from 8- to 12-week mice (Buskila et al., 2013). Impairments of spatial working memory, assessed in a cross-maze test, emerge between 3 and 6 months and worsen with age (Jawhar et al., 2012). Progressive decreases in anxiety, as measured in the elevated plus-maze, also emerge between 3 and 6 months. Abnormal reflexes, specifically hind and forelimb clasping in a tail-suspension test, are seen as early as 5 months (Richard et al., 2015). Reduced anxiety in 5xFAD mice is also seen in the open field, at 9 to 12 months of age. One group reported normal spatial memory function in the Morris water maze at least until 7 months (Richard et al., 2015), while a second group reported memory deficits in the water maze in mice as young as one month (Gu et al., 2018; Wu et al., 2018). For depression project: The reason to choose 2-3 month old C57BL/6 mouse strain is first because it is one of the most widely used strains to generate stress/depression models. Second, because 5XFAD mice is on a congenic C57BL/6 genetic background, this will facilitate breeding and also comparison of the anxiety behavior of 5 X FAD mice and mice exposed to chronic stress.