The COBRE Neuroimaging and Electrophysiology Facility (NIEF) has developed into a state-of the- art resource for the Center for Neuroplasticity investigators and the larger UPR biomedical research community. During COBRE Phase II we propose to further expand these efforts by transforming the NIEF into a self-sustained imaging and electrophysiology instrumentation service facility. The first step (Specific Aim 1) will be to establish a web-based cloud data analysis computing network available to UPR scientists located at all major sites/campuses. The NIEF will invest in new computers and software tools necessary to provide onsite and remote data analysis services to all users. The second step (Specific Aim 2) will be to continue upgrading and integrating imaging and electrophysiological applications within our existing NIEF facilities. The NIEF will provide existing and new users the ability to combine electrophysiological recordings, optical modulation of action potentials and genetic methods through LED-based optogenetic techniques. This aim will be reinforced by a $20M investment by the UPR in to create a Neuroplasticity floor and a state-of-the-art animal facility at the Molecular Sciences Research Center. The third step (Specific Aim 3) will be to further increase the equipment flexibility and detection sensitivity for light and fluorescence-based applications. This objective will include purchase of a super-resolution/TIRF (total internal fluorescent microscopy) microscopy system. Fulfillment of these aims will enable the COBRE NIEF to become a completely self-sustained neuroimaging research facility and a role model for Puerto Rico. A detailed description of our plans to accomplish these goals appears in the NIEF Core section of this proposal.

Pilot Program

Pilot Project Director. Dr. Walter I. Silva Ortiz.
The COBRE Center for Neuroplasticity Pilot Project Program (PPP) will identify and stimulate development of promising early career UPR researchers. Funds ($75k/year/project) are allocated to support two cohorts of three 2-year projects. During the initial six months of COBRE Phase II, the availability of funds will be disseminated and eligible faculty will be encouraged to apply via a web-based system. We will target non-tenured faculty across the UPR system. Proposals will utilize the NIH format and reviewers (internal and external) will be instructed to score them using the NIH scoring system and criteria (Significance, Investigator(s), Innovation, Approach, Environment). Reviewers and the COBRE EAC will also consider each proposal’s relevance to the goals of the Center for Neuroplasticity and long-range promise for patents or other means of stimulating the island’s economy. Guillermo Yudowski, a competitively funded UPR neuroscientist with considerable mentoring experience, will direct the Pilot Project mentoring program using the strategies outlined in the Administrative Core section of this proposal. Pilot Project investigators will be required to comply with all applicable federal policies, rules, and guidelines for research involving human subjects, vertebrate animals, and/or biohazards. As part of their mentoring, they will receive guidance from the UPR IACUC and the Biosafety Committee at their respective campuses. The COBRE evaluation team will be charged with monitoring scientific progress of the Pilot Projects and measuring whether they are leading to increased publications, presentations and grant submissions. During YR03 of the COBRE Phase II, Pilot Project investigators will have the opportunity to compete for subproject funding, and a second call for Pilot Project proposals will be announced. All COBRE investigators (Subprojects and Pilot Projects) will be strongly encouraged to participate in the Early Career Reviewer (ECR) Program of the NIH Center for Scientific Review.

(ECR) Program
The ECR Program was developed to recruit and educate qualified scientists who do not have prior CSR experience. It exposes them directly as panelists to the peer review process. Detailed descriptions of our COBRE Phase II early career mentoring program, academic and industry partnerships, and faculty development activities appear in the Administrative Core section of this proposal.

Mentored Projects

1-Contributions of Traumatic Brain Injury to Fear Extinction and Avoidance.

  • This project explores common mechanisms that contribute to the neurobiology of traumatic brain injury (TBI) and post-traumatic stress disorder (PTSD). Traumatic brain injury affects approximately 4 million civilians and soldiers each year, many of which are diagnosed with mental health conditions like post-traumatic stress disorder. Epidemiological studies of combat veterans show a strong correlation between sustaining TBI and developing PTSD. However, animal studies show conflicting results. To help evaluate the extent to which there is a relationship between TBI and PTSD, a biological link must be examined using reliable brain injury models and appropriate behavioral tests. This project will therefore utilize the Controlled Cortical Impact model of brain injury, and will test its effect on extinction of conditioned fear. Results from this work could lead to the development of novel approaches for treatment of patients with TBI and PTSD.

2-Alpha-7 nAChR: Potential Therapeutic Target for Obesity Induced Cognitive Decline

  • This project takes a cellular and molecular approach toward understanding obesity, a demonstrated risk factor for many conditions including diabetes, hypertension, dyslipidemia, stroke, heart disease, several cancers, and arthritis. Obesity has also been shown to be associated with certain mental health conditions, including depression and cognitive impairments that occur in Alzheimer’s disease. The proposed study will utilize α7 nAChR conditional knockout mice developed as part of Colón’s postdoctoral research at NIEHS. Crossing this line with inducible microglia selective Cre recombinase mice (CX3CR1CreER) will result in the production of microglia selective, tamoxifen-inducible α7 nAChR knock-out mice (α7 nAChRmicKO). This mouse model will present Colón with the unique opportunity to dissect the role of α7 nAChR in microglia activation and subsequent neuroinflammation caused by a high fat diet (HFD), without affecting the α7 nAChR found on other cell types such as neurons, astrocytes or peripheral myeloid cells.

3-Epigenetic control of transcription dynamics in long-term alcohol neuroadaptation

  • This project explores the neuroadaptations that lead to alcoholism. A progressive increase in alcohol tolerance, coupled with the emergence of physiological dependence, are thought to contribute to the uncontrolled urge to consume alcohol by influencing the brain reward system. Ghezzi’s project seeks to uncover the molecular mechanisms by which alcohol initiates and perpetuates changes in gene expression, neural physiology, and behavior that drive the alcoholic state. He will test the hypothesis that temporally distinct epigenetic modifications contribute to the dynamics of alcohol neuroadaptations by orchestrating multi-gene transcriptional reprogramming. Specifically, these experiments will explore the role of epigenetic histone modifications in the dynamics of alcohol tolerance in the Drosophila model system. Identification of such mechanisms is crucial for the development of more effective treatments for alcoholism.

4-Role of microglial α7nAChR in diet induced brain inflammation

  • Research in my laboratory focuses on dissecting the mechanisms underlying Brain Development. Using rodent models, we study the influence of genetic and environmental factors on dynamic neurodevelopmental processes, such as neurogenesis, cell differentiation & migration, early activity and connectivity. We also assess development in human infants using high-density electroencephalography (hd-EEG) and correlate brain activity with physiological and cognitive tools to help map the developmental time-course and possible deviations in at-risk groups for neurodevelopmental conditions. With this translational approach, we aim to connect basic and clinical components of Developmental Neuroscience and contribute to the early identification and treatment of neurodevelopmental disorders.

Pilots Projects

1-Establishing an experimental model for probing the neural and molecular basis of abnormally repetitive behavior

  • Mutations in different genes can give rise to behaviors that are abnormally repetitive, a hallmark of many behavioral disorders such as autism spectrum disorder (ASD) and obsessive-compulsive disorder (OCD). Yet, little is known concerning the range of different neural circuit functions that become defective as a consequence of such mutations to cause repetitive behavior. The objective of this pilot project is to establish an experimental framework that will allow us to bridge the gap between the mutations that cause repetitive behavior, and the neural circuits that become defective as a consequence of these mutations.

2-Polymorphism in neuroplasticity-related genes and its association with anxiety and depressive symptoms severity in Caribbean Hispanic patients

  • Our current research aims to identify genetic variants that can be used as predictors for treatment response in psychiatric conditions. Depressive and anxiety symptoms are more frequent psychiatric health conditions that contribute to lower quality of life. Also, depression and anxiety are the principal comorbidities in many chronic conditions. It is known their prevalence is different across ethnicity, in the case of Caribbean Hispanic, Puerto Rican have higher prevalence compared to other Hispanic. With a better understanding of the effects of these genetic variants, we will plan to develop risk scores using genetic markers as well as clinical factors that are relevant to our Puerto Rican population.

3-Role of Nicotinic Alpha 7 Autoreceptors in Striatal Cholinergic Interneurons

4-Chromatin remodeling during butterfly brain development

  • I use a well-studied butterfly native to Puerto Rico, Heliconius charithonia or zebra longwing butterfly, to study how gene regulation affects tissue development and brain functioning. Heliconius butterflies display complex behaviors and have a large brain with several unique facets. They thus represent a well-suited model to study the molecular mechanisms that regulate tissue development and brain functioning. I do this by investigating changes in the folding of DNA (~chromatin structure), which give hints about DNA fragments that regulate gene expression and, potentially, behaviors.

Collaborative seeds project

1-Ex vivo and in vivo models of Gulf War Illness Allow for Mechanistic Studies and the Search for Antidotes

  • Operation Desert Storm (1990-1991) achieved a swift and decisive victory liberating Kuwait. The US casualties were low, 269 killed and 458 wounded. However, the unexpected “casualties” of this war were enormous; by the end of the war, more than 200,000 US military personnel were afflicted with the Gulf War Illness (GWI). It has been established that the main cause was an unfortunate combination of products usually regarded as safe issued to the soldiers. Pyridostigmine bromide (PB), administered as prophylactic against a sarin attack; permethrin (PER), and the insect repellent Off (DEET) for insect control. To this drug mix, a low concentration of sarin in-theater was added.The goal of this study is to develop a pharmacological therapy for GWI and identify hippocampal morphological alterations caused by the disease. To achieve that we will test the effectiveness of 4R-cembratrienediol (4R) to stop the persistence of the GWI in an ex vivo hippocampal slices and an in vivo mouse models of GWI. The outcome measurement will be evaluating the persistence of neuronal inflammation and synaptic integrity. The 4R neuroprotective efficacy has been already demonstrated against other brain injuries like Parkinson’s disease and ischemic stroke, thus suggesting that it will be beneficial for neurodegenerative GWI.

2-Synthesis of Biological Active Amino Derivatives and Multi-target Drugs for the Treatment of Alzheimer’s Disease

  • The major goal of this project is to design and prepare multi-target benzazepine and nicotine analogues and study their biological activity for the treatment of neuron-degenerative diseases, such as Alzheimer and Parkinson. The development of novel drugs for the treatment of AD that can act as cholinergic agents as well as antioxidants and inhibitors of toxic and soluble aggregates is presently an area of great relevance. The synthesis of new benzazepines with a variety of linkers designed to enhance their bioactivity as nicotinic agonists are presently under study. Their potential as nicotinic receptor agonist and as β-amyloidal inhibitors are of great interest. In addition, novel racemic and nicotinic compounds are being prepared and will be studied as β-amyloidal inhibitors.Novel methods for the preparation and biological activity determination of key organic compounds is an area of great interest due to the concern that new drugs are needed for the treatment of Alzheimer’s disease. In addition, key amino derivatives are important organic compounds used as building blocks for the synthesis of many pharmaceutical products and auxiliaries, particularly for neurodegeneration.

3-Nuclear G-coupled Kinin-B2 Receptors in a Human hCMEC/D3 Blood Brain Barrier Cell Model

  • One of the natural ligands regulating blood-brain barrier (BBB) permeability is the nonapeptide called bradykinin, an agonist of the kinin-B2 receptor (B2BKR). Bradykinin production increases after brain injury, and despite its importance in BBB permeability, neuroprotection and neuroplasticity, it has never been studied in isolated human brain endothelial cells. A prominent human brain endothelial BBB model is the hCMEC/D3 immortalized cell line. Our group has demonstrated the expression of the B2BKR in hCMEC/D3 cell lysates. Interestingly, extracellularly-added bradykinin does not stimulate the expected B2BKR activity, as measured by intracellular calcium mobilization. An examination of B2BKR’s subcellular localization by immunofluorescence and confocal Z-stack analyses showed that this receptor was not localized in the plasma membrane, but in the hCMEC/D3 cell’s nucleus. This novel localization of B2BKR could explain its lack of stimulation. To complete these studies, we will use the NIEF’s electrophysiology facility to test if the nuclear G-coupled B2BKR found in hCMEC/D3 cells is functional. We hypothesize that if this receptor is functional in hCMEC/D3 cells, then measurements of the inward calcium currents by patch clamp made before and after intracellular administration of a B2BKR agonist to the pipette solution, should reveal current changes. These studies will help us test if hCMEC/D3 cells express a functional G coupled B2BKR in the nucleus, providing new mechanistic insights in the regulation of this important BBB model.

4-Age-dependent involvement of inflammation associated receptors in hippocampal plasticity and neuroprotection after an ischemic insult

  • Ischemic stroke is the leading cause for the development of disabilities in the US. Less than 5% of patients can benefit from the current FDA-approved stroke treatment, the anticoagulant tPA, due to its small time-window for eligibility (4.5 h after stroke), and it does not alleviate the chronic neuroinflammation that occurs after the patient survives this neurological event. This collaborative proposal focuses on the study of alternate mechanisms against neuroinflammation to pursue different treatments to tPA, especially for those patients who become ineligible for this therapy. Two of the receptors that we will be focusing on our proposed experiments are the α7 nicotinic acetylcholine receptor (α7nAchR) and the Triggering receptor expressed on myeloid cells-like (TREM)-like transcript-1 (TLT-1). Our general hypothesis is that both receptors play a crucial role in the brain against neuroinflammation, and their down-regulation throughout aging decreases the chances of recovering from insults such as an ischemic stroke. The study of these receptors and their expression throughout age could help us expand our understanding of their role in the modulation of neuroinflammation. This information could lead to the development of neuroprotective treatments targeting these receptors.

5-Lipid Dendrimers as a Convenient Strategy to Construct Nanoparticles for Drug-Delivery Applications

6-Xylazine effects on morphine-induced prefrontal cortex neuroplasticity

  • The goal of the proposed research is to study whether the cutting agent xylazine changes the electrophysiology and behavioral parameters of key brain reward regions altered by opioid addiction. Xylazine, known in Puerto Rico as ‘’anestesia de caballo” has been found as the major adulterant of street heroin. Little has been done to detect the potential addiction profile of xylazine or if it contributes to heroin addiction. Evidence obtained from different addiction models has identified synaptic plasticity as a key molecular mechanism through which drugs of abuse induce the various stages of addiction. A lot of attention has been given to the synaptic plasticity events induced by cocaine and amphetamines at the ventral tegmental area (VTA), and Nucleus Accumbens (NAc), two major areas of the reward circuit. Much less information is available on changes in prefrontal cortex plasticity under the influence of opiates. Investigations regarding the potential effect of xylazine, specifically on opiate-induced plasticity in the reward circuit, has not yet been reported.