Spine Morphology, Memory and Stress
During the last few years I have been able to extend the use of a new Golgi staining technique. This technique allows for a 3-dimensional reconstruction of dendritic spines that can be combined with immunohistochemical staining allowing for co-localization of selected synaptic markers within various spine shapes. Spine shape and number are important characteristics in delineating effects on synaptic plasticity and memory. Up to now, the only other assessment available to understand how changes in structure affect synaptic connectivity and function requires nanoscale 3-dimensional reconstruction from serial section electron microscopy (EM). In lieu of using EM, we apply this new 3-dimensional reconstruction technique. Our recent publication using this technique identifies rapid changes within particular spine shapes after an acute stress and changes in synaptic protein markers within these spines (Sebastian et al., PloS One, 2013). This paper is the first demonstration characterizing the functional aspects of changing spine morphology with this technique. Our quantification shows that a short-term stress can induce rapid and significant changes in spine morphology across various subregions within the hippocampus.
Workflow utilized for Sebastian et al., 2013. Rats were trained and testes for long-term memory on the 8-radial arm maze, were perfused and brains were processed for golgi-impregnation. Sections of 100 micron thickness were processed for dual processing of golgi-spine morphology and IHC localization using confocal microscopy and IMARIS 3-d reconstruction. This allowed us to link the structural data to the behavior of rats in-vivo, lending a novel understanding of the underlying mechanisms that allow for neural processing of long-term memories.
I have subsequently applied this same technique on rats housed under environmental enrichment. Recently, we found that 1-month of environmental enrichment (EE) from adolescence through young adulthood improves spatial learning and also results in significant increases in mushroom spines as a consequence of EE (Alliger 2016, submitted). This dynamic switch in specific spine shapes includes an increase in the AMPA receptor subunit GluA2, providing a mechanism for memory enhancement in these EE housed animals. Conversely, social defeat stress in adolescent animals reduced GluA2 containing mature spines illustrating how various environmental manipulations can alter hippocampal spine formation (Iñiguez 2016, submitted). In time this new confocal imaging technique will add a new understanding to how we analyze and understand the role of changing spine dynamics in the brain. As this technique will now allow for the quantification of particular spine shapes rather than quantifying overall changes in spine densities (currently the standard with Golgi imaging).
Amber Alliger Ph.D.
Hunter College Psychology
Golgi-spine and immunohistochemistry analysis in CA1 pyramidal dendrite after EE
In my lab, this new technique is also being utilized in the context of traumatic brain injury (TBI) and in developmental studies. Indeed, young organisms do not maintain memories as long as adults, but the mechanisms for this ontogenetic difference are unknown. This work is significant because, even though young animals forget more rapidly than adults, early learning and especially traumatic experiences can have consequences that last a lifetime. I am currently the PI on an NIH R21 grant on this topic, in collaboration with Dr. Shair (Columbia University). In this proposal we focus on a molecular cascade that has a significant impact on spine density, as well as trafficking of synaptic markers within these spines. Recent literature and our own preliminary data have led us to formulate the following hypothesis: Juvenile fear memory is weaker than in adults due to decreased activity of PI3 and MAP kinase and a prevalence of immature spines. Our proposed experiments will delineate the molecular mechanisms of fear memory capacity across specific stages of development.
Harry Shair Ph.D.
Columbia U. Psychiatry
Molecular mechanisms active in juvenile rats that control spinogenesis on neuronal dendrites
Molecular mechanisms active in adult rats that control spinogenesis on neuronal dendrites
Methamphetamine-induced Memory Deficits
Another thriving area of research in my lab investigates the effects of drugs of abuse on memory and learning and the role that inflammation plays in these deficits. I currently have a NIH-DIDARP (Diversity-Promoting Institution Drug Abuse Research Program) training grant. What we have found is that methamphetamine (MA) produces an insult on the brain that triggers a cascade of neuroinflammation involving the upregualtion of cycloxygenase-2 (COX-2) and the prostaglandin J2 (PGJ2). Additionally, we have found that MA impairs memory in a PKMζ dependent mechanism, which we believe may be linked to the upregulation of COX-2. From my collaboration with Dr. Pereira and Rockwell (both a Hunter College, Department of Biology) we know that MA-induced increases in COX-2 is also associated with the ubiquitin and proteosome impairment. Our current research now involves how the trafficking of PKMζ is impaired during these drug insults.
I have also extended this line of investigation to include the role of HIV in methamphetamine induced memory deficits. MA is a highly toxic and addictive drug of abuse and enhances high-risk sexual behaviors, thereby increasing the likelihood of HIV infection. MA use also is associated with higher HIV viral loads, immune dysfunction, and antiretroviral resistance. Moreover, animal studies demonstrate that MA enhances HIV infection/replication. HIV positive (+) patients are also at risk of developing HIV associated neurocognitive disorders (HAND), which progress over time. I focus on MA and HIV not only because MA abuse is common among HIV+ individuals but also because neurocognitive outcomes tend to be worse in MA users with HIV. Our latest studies with mice show that MA induces spatial learning deficits six weeks after an acute neurotoxic dose. These long-term cognitive deficits are associated with a decrease in protein kinase M zeta (PKMζ), which is important for memory. Moreover, we established that an acute neurotoxic MA dose induced up-regulation of the proinflammatory enzyme COX-2 in mice exhibiting spatial learning deficits. It is unknown how prostaglandins (PGs) affect synaptic plasticity linked to learning and memory deficits induced by MA and HIV. Currently, I am revising a grant proposal on this topic that will be reviewed Nov 2016. This proposal utilizes the HIV/gp120 transgenic mouse model for investigating the combined effects of MA/HIV on cognition, as the short and long-term effects of MA abuse, in combination with chronic HIV infection. These transgenic mice express the envelope protein gp120 of HIV-1 in their brain under the control of the promoter for glial fibrillary acidic protein (HIV-1 gp120tg). Importantly, this mouse model shows similarities to HIV+ positive human brains by exhibiting reactive astrocytosis as a marker of brain injury and damage to cortical pyramidal neurons. Capitalizing the use of the HIV/gp120 transgenic mouse model, my goal is to address the role of sustained or escalating levels of inflammation on the cognitive impairment observed as a function of MA and/or HIV. Together, with an evaluation of synaptic markers important for memory and their role in changing spine morphology, these studies will have a significant impact on understanding the effects of acute MA addiction by itself and in combination with HIV on cognition.
Inflammation and Neurodegenerative Disorders
My interest in protein kinase activity during memory and synaptic plasticity has grown to include how learning and memory are compromised during brain insults and neurodegenerative disorders that produce products of inflammation. This interest fostered collaborations with Professors Pereira and Rockwell. I am particularly interested in how the inflammatory response produces increased levels of prostaglandins J2 (PGJ2) that develop over time lending to increased cognitive deficits affecting hippocampal function. High levels of PGJ2 are found in the brains of individuals after stroke. Thus, we use the highly toxic and endogenous PGJ2 to model neurodamage in both neuronal cultures and in vivo. We focus on the downstream mechanisms that are linked to the PGJ2 pathology, such as mitochondrial dysfunction, tau and APP dysregulation, and memory deficits associated with impaired trafficking of synaptic proteins important for memory. We hypothesize that inflammatory markers such as microglia and (cyclooxygenase-2) COX-2 are triggered by the PGJ2 insult and have a long-term negative impact on cognitive function. We currently have an NIH R01 application (as multiple PIs with Drs. Pereira and Rockwell) on this topic. In this proposal, we investigate the effects of overexpressing ATF5, a transcription factor that regulates neuroprotective mictochrondrial genes in response to mitochondrial dysfunction. If awarded this proposal could have a significant impact on the treatment of neurodamage induced by PGJ2 and potentially reduce cognitive deficits associated with stroke and Alzheimer’s disease. In this collaboration, we bring together a significant amount of experience across a broad spectrum of fields necessary to execute this multidisciplinary project.
Synthesis and transport pathways for PGJ2, a neurotoxic product of inflammation relevant to neurodegenrative disorders like Alzheimer's and the deterioration of cognitive functions
Mechanisms and sites of action for PGJ2, a neurotoxic product of inflammation relevant to neurodegenrative disorders like Alzheimer's and the deterioration of cognitive functions
Maria E. Figueiredo-Pereira Ph.D. & Patricia Rockwell Ph.D., Biology Hunter College
Ultimately, my research remains focused on the signal transduction mechanisms involved when stress significantly alters the morphology of dendritic spines affecting synaptic plasticity, learning and memory. It is understood that depending on the stress, the effect on memory and learning can be enhanced or impaired. My lab is specifically interested in how stress can regulate the expression of various synaptically localized proteins that are important for memory, such as PKMζ. I incorporate various model systems involving inflammation, neurodegenerative disorders, drugs abuse, traumatic brain injury, HIV, and stress. These models collectively provide brain insults to better understand neural mechanisms associated with memory deficits and remediation by environmental enrichment. We use animal models to assess behavioral parameters on memory and learning. We also use several different molecular and imaging techniques to asses changes in protein expression and spine shape morphology and evaluate synaptic strength using electrophysiology involving long-term potentiation. My lab has several different projects, which provide a unique perspective on the interaction between several brain regions including the hippocampus, amygdala and frontal cortex as they relate to stress, traumatic brain injury and Alzheimer’s disease.
Ongoing collaborators include: