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Lia Calcines Rodriguez - PhD Candidate, Neuroscience Degree Program

In Alzheimer’s disease (AD), the most common form of dementia, women seem to be more susceptible as they show greater rates of cognitive decline, brain atrophy, and increased global pathology compared to men. AD is characterized by amyloid-β (Aβ) plaques, neurofibrillary tangles and neuroinflammation. In multiple rodent models of AD, sex differences have been identified in which pathology and neuroinflammation are more severe in females. Microglia, the resident innate immune cells of the central nervous system and key drivers of neuroinflammation, have been implicated in AD pathogenesis. Interestingly, recent studies have demonstrated that female AD mice exhibit an accelerated progression into the disease-associated microglia (DAM) state relative to male mice. Moreover, females exhibit impaired phagocytosis and higher Aβ plaque burden compared to male mice. In human post-mortem AD brains, sex differences in microglia morphology and transcriptome have also been reported where females exhibit a more inflammatory profile. Despite these observations, the contributions of microglia to the establishment of sex differences in AD is largely unexplored. We hypothesize that microglia possess cell-intrinsic sex-specific properties that drive differences in their functional responses to Aβ pathology and that these contribute to the sex differences in Aβ pathology. To address this question, we will isolate microglia of each sex from adult C57BL/6 (WT) mice and transplant them into adult microglia-deficient 5xFAD (5xFAD-FIRE) host brains of the same or opposite sex prior to the development of Aβ pathology.

Specific Aim 1: Characterize sex differences in the 5xFAD mouse model of amyloidosis. We will first characterize changes in Aβ burden, microglial reactivity, and microglia-plaque interaction in male and female 5.5-month-old 5xFAD mice using immunohistochemistry (IHC). We will also isolate microglia using fluorescent-activated cell sorting (FACS) and assess expression changes in DAM genes using real-time quantitative polymerase chain reaction (RT-qPCR).

Specific Aim 2: Determine whether microglia possess sex-specific cell-intrinsic differential responses to Aβ pathology and whether this drives the sex differences in Aβ pathology. First, we will isolate WT microglia via magnetic-activated cell sorting and confirm their purity and homeostatic state. We will then intracranially transplant female or male WT microglia into the male or female hippocampi of 2-month-old 5xFAD-FIRE mice, a model that exhibits aggressive amyloidosis and congenitally lacks microglia. Following transplantation, we will analyze the brains at 5.5 months of age as described in Aim 1. Moreover, using RT-qPCR, we will confirm that microglia retain their sex identity with a panel of sexually dimorphic genes that have previously been shown to remain unchanged upon transplantation. To our knowledge, no other study has sought to disentangle the intrinsic or extrinsic factors that give rise to the sex differences in AD. We believe that unraveling sex-specific cell-intrinsic properties of microglia in AD is the gateway to precision medicine.

 Dec 15, 2023 @ 12:00 p.m.

 Medical Center | Lower Adolph Aud (1-7619)

Host: Kerry O’Banion, MD, PhD - Advisor

Lia Calcines Rodriguez - PhD Candidate, Neuroscience PhD Program

Thesis Proposal

In Alzheimer’s disease (AD), the most common form of dementia, women seem to be more susceptible as they show greater rates of cognitive decline, brain atrophy, and increased global pathology compared to men. AD is characterized by amyloid-β (Aβ) plaques, neurofibrillary tangles and neuroinflammation. In multiple rodent models of AD, sex differences have been identified in which pathology and neuroinflammation are more severe in females. Microglia, the resident innate immune cells of the central nervous system and key drivers of neuroinflammation, have been implicated in AD pathogenesis. Interestingly, recent studies have demonstrated that female AD mice exhibit an accelerated progression into the disease-associated microglia (DAM) state relative to male mice. Moreover, females exhibit impaired phagocytosis and higher Aβ plaque burden compared to male mice. In human post-mortem AD brains, sex differences in microglia morphology and transcriptome have also been reported where females exhibit a more inflammatory profile. Despite these observations, the contributions of microglia to the establishment of sex differences in AD is largely unexplored.

We hypothesize that microglia possess cell-intrinsic sex-specific properties that drive differences in their functional responses to Aβ pathology and that these contribute to the sex differences in Aβ pathology. To address this question, we will isolate microglia of each sex from adult C57BL/6 (WT) mice and transplant them into adult microglia-deficient 5xFAD (5xFAD-FIRE) host brains of the same or opposite sex prior to the development of Aβ pathology.

Specific Aim 1: Characterize sex differences in the 5xFAD mouse model of amyloidosis. We will first characterize changes in Aβ burden, microglial reactivity, and microglia-plaque interaction in male and female 5.5-month-old 5xFAD mice using immunohistochemistry (IHC). We will also isolate microglia using fluorescent-activated cell sorting (FACS) and assess expression changes in DAM genes using real-time quantitative polymerase chain reaction (RT-qPCR).

Specific Aim 2: Determine whether microglia possess sex-specific cell-intrinsic differential responses to Aβ pathology and whether this drives the sex differences in Aβ pathology. First, we will isolate WT microglia via magnetic-activated cell sorting and confirm their purity and homeostatic state. We will then intracranially transplant female or male WT microglia into the male or female hippocampi of 2-month-old 5xFAD-FIRE mice, a model that exhibits aggressive amyloidosis and congenitally lacks microglia. Following transplantation, we will analyze the brains at 5.5 months of age as described in Aim 1. Moreover, using RT-qPCR, we will confirm that microglia retain their sex identity with a panel of sexually dimorphic genes that have previously been shown to remain unchanged upon transplantation. To our knowledge, no other study has sought to disentangle the intrinsic or extrinsic factors that give rise to the sex differences in AD. We believe that unraveling sex-specific cell-intrinsic properties of microglia in AD is the gateway to precision medicine.

 Dec 15, 2023 @ 12:00 p.m.

 Medical Center | Lower Adolph Aud (1-7619)

Host: Kerry O’Banion, MD, PhD - Advisor

Linh Le - PhD Candidate, Neuroscience Graduate Program

Thesis Defense

Alzheimer’s disease (AD) is the most common cause of age-related dementia, characterized by well-known pathological hallmarks including extracellular amyloid β (Aβ) plaque deposition and neurofibrillary tangle accumulation. In the past decade, neuroinflammation has emerged as a crucial contributor to disease pathogenesis thanks to GWAS studies revealing various genetic variants of immune receptors as AD risk factors. These receptors are largely expressed by microglia, the resident innate immune cells of the central nervous system (CNS), making them a promising translational target for disease-modifying therapies. Here, we sought to elucidate the effects of two different approaches to modulating microglia functions in AD-like mouse models.

First, building on a multitude of evidence on the anti-inflammatory effects of the neurotransmitter norepinephrine (NE) and our previous work revealing that NE inhibits microglia surveillance activity via the β2 adrenergic receptor (AR), we explored the contribution of microglial β2 adrenergic signaling to AD pathology in 5xFAD mice, a commonly used model of amyloidosis. We observed an early degeneration of NE projections followed by locus coeruleus (LC) neuronal loss in more advanced disease stages, accompanied by a mild decrease in the levels of NE and its metabolite normetanephrine. Interestingly, we found that microglia in 5xFAD mice lost their sensitivity to β2AR signaling early and this was particularly evident in microglia that were in close proximity to Aβ plaques. We also described the important role of microglial β2AR signaling on AD, revealing opposing effects on amyloid pathology, whereby activation of microglial β2AR attenuated plaque deposition whereas inhibition worsened plaque pathology.

We next asked whether global pharmacologically-induced renewal of microglia, which is suggested to have “rejuvenating” and beneficial effects, could be a potential pathology-modifying therapy for AD. We induced microglial repopulation by depleting microglia with PLX5622 (a colony stimulating factor 1 receptor (CSF1R) inhibitor) and allowing them to replenish upon PLX5622withdrawal in two common models of AD: APP/PS1 and 3xTg mice. However, we observed no changes in amyloid pathology after forced repopulation of microglia, accompanied by a lack of cognitive improvement in a battery of behavioral tests.

We then set out to address sexual dimorphism in microglia, which potentially underlies the well-known differences in amyloid pathology in male versus female mice, with pathogenesis in females progressing much faster. To first understand how male and female microglia might differ in their survival and proliferation mechanisms, we examined the sex-specific effects of CSF1R inhibition using PLX3397. We confirmed that CSF1R inhibition resulted in significantly less depletion in female mice compared to male mice. Transcriptomic analysis of microglia revealed differential upregulation of autophagy, mitochondrial dynamics, and surveillance in PLX3397-treated female microglia compared to male microglia. Further studies are warranted to establish mechanistic links between these observations.

Taken together, our results suggested that specific, rather than global, manipulation of microglia might be effective in treating AD. Specifically, we highlighted the potential of leveraging microglial β2AR signaling for disease-modifying therapy.

 Dec 11, 2023 @ 12:00 p.m.

 Medical Center | Ryan Case Method Rm (1-9576)

Host: Ania Majewska, PhD & Kerry O’Banion, MD, PhD - Advisors

Mark Osabutey - PhD Candidate, Neuroscience PhD Program

Thesis Proposal

Radiation-induced neuroinflammation has emerged as a pivotal clinical challenge, particularly for cancer patients undergoing radiotherapy. These adverse neurological effects are attributed to the activation of various immune cell lines, leading to cognitive impairments. Microglia, the primary immune cells of the central nervous system (CNS), have been identified as key players in these processes. Their essential functions, such as synaptic spine clearance and inflammatory response, undergo significant dysregulation post-radiation. Following genotoxic stress from ionizing radiation exposure, self-DNA is released from nuclear, mitochondrial, and extracellular sources into the cytosol. The cGAS-STING-Interferon pathway, a vital signaling cascade activated by binding to double-stranded DNA, is posited to play a central role in the dysregulated activation of microglia following cranial radiation. This pathway, integral to cellular innate immunity, offers a promising avenue for exploration, especially given the current limited understanding of its role in microglial activation following radiation. Therefore, deciphering the complex interplay between ionizing radiation exposure, microglial activation, and the cGAS-STING-Interferon pathway may contribute to targeted therapeutic strategies that prevent adverse effects. The primary hypothesis is that the cGAS-STING pathway is a key determinant of microglial behavior post-radiation and that its modulation can counteract radiation-induced cognitive impairments.

Aim 1 will employ in vitro techniques to investigate alterations in microglial states post-radiation exposure, emphasizing the role of the cGAS-STING pathway in mediating these changes. Furthermore, Aim 1 will explore the molecular mechanisms by which this pathway influences microglial activation and response.

Aim 2 is centered on elucidating the broader implications of microglial dysregulation on cognitive function post-radiation, utilizing a combination of behavioral tests and histological analyses. Based on preliminary findings, the hypothesis is that modulation of the cGAS-STING pathway can mitigate radiation-induced microglial dysregulation and its associated cognitive deficits.

Aim 3 will focus on potential therapeutic strategies targeting this pathway by using cGAS-STING knockout mice to test efficacy in ameliorating radiation-induced neurological consequences. Collectively, the outcomes from these aims will offer insights into radiation-induced neuroinflammation and pave the way for novel therapeutic interventions.

 Dec 08, 2023 @ 1:30 p.m.

 Medical Center | Lower Adolph (1-7619)

Host: M. Kerry O’Banion, MD, PhD & John Olschowka, PhD - Advisors

Emily Przysinda - PhD Candidate, Neuroscience Graduate Program

Thesis Defense

One often overlooked symptom of schizophrenia (SCZ) is marked and persistent difficulties with social information processing. Since social interactions hinge upon understanding our external environment, it’s possible that known language deficits in SCZ could contribute to social difficulties. Here, we aim to understand the neural basis for social deficits and potential underlying language processing deficits in SCZ while participants view naturalistic video stimuli. We examine differences between SCZ patients and neurotypical controls (NTC) using complementary neuroimaging methods, fMRI and EEG, in separate studies with mostly overlapping participants.

For study 1, we chose to focus on a network of brain regions supporting a subset of social processing, theory of mind (ToM), as previous research has shown ToM deficits in SCZ. The fMRI analysis revealed between group differences in the extent to which ToM brain regions were engaged during socially awkward events. Specifically, the SCZ group showed reduced recruitment of the dorsal medial prefrontal cortex (mPFC) compared to controls. Using graph theory methods, we also found a prominent decrease in global efficiency in the dorsal and middle mPFC for SCZ compared to NTC. Our findings converge with previous literature showing decreased functional connectivity during explicit ToM tasks in SCZ, and here we show here that these findings can be replicated in a more naturalistic paradigm. Analysis of these same ToM regions using EEG in study 2 allows us to discern fluctuations in connectivity between ToM regions that may be sensitive to different aspects of the neural signal compared to fMRI.

For study 3, we used linear methods to relate the EEG signal to the auditory and language features of the episode, which yields quantitative values that index how well the brain is tracking these features. We found that EEG tracking of acoustic envelope and lexical surprisal was reduced in SCZ versus NTC. This suggests that patients with SCZ may have difficulty in processing basic acoustic and language features of naturalistic video. The overlap of subjects between these studies allows us to make within-subject comparisons of both language and social processing measures, while also capitalizing on the strengths of both neuroimaging methods.

Zoom Information:  Meeting ID: 945 3098 2212; Passcode: 052687

 Nov 29, 2023 @ 12:00 p.m.

 Medical Center | Adolph (Lower) Auditorium (1-7619)

Host: Ed Lalor & David Dodell-Feder - Advisors

Catalina Guzman - PhD Candidate, Neuroscience PhD Program

Thesis Proposal

Adolescence is a critical period marked by protracted dopamine (DA) innervation to the frontal cortex – a process crucial in the development of cognitive functions. Delta-9-tetrahydrocannabinol (THC) is the main psychotropic phytocannabinoid in cannabis, and its chronic use in adolescence has been linked to long term deficits in neurocognitive capabilities in both preclinical models and clinical populations. In contrast, cannabidiol (CBD), a non-psychotropic cannabis component, shows therapeutic and pro-cognitive potential. The prefrontal cortex's rich abundance of CB1 cannabinoid receptors raises the possibility that the endocannabinoid (eCB) system may modulate prefrontal DA release and cognitive outcomes.

The overall objective of this proposal is to investigate the in vivo mechanisms underpinning the impact of chronic cannabis consumption during adolescence on the mesoprefrontal DA circuit. Several electrophysiological studies have indicated that THC administration can elevate DA transmission in certain brain regions, while CBD may potentially reduce DA release. Additionally, chronic THC exposure can downregulate CB1 receptors and the level of an eCB degradation enzyme FAAH. However, the effects of eCB signaling are brain region and circuit dependent. The impact of chronic THC or THC:CBD administration during adolescence on mesoprefrontal circuit development and function remains unknow.

We hypothesize that adolescent THC exposure disrupts the development of prefrontal DA innervation and release via its impact on eCB signaling, which may be ameliorated by the antagonist action of CBD. We will employ a combination of histochemical and pharmacological techniques, alongside in vivo fiber photometry to monitor DA and eCB signaling in the medial prefrontal cortex (mPFC). We outline the following specific aims: 1) Evaluate the influence of chronic oral administration of THC and THC:CBD (1:1 ratio) during adolescence on the density of mPFC DA innervations and CB1 expression levels in adulthood; 2) Assess changes in mPFC DA release during cognitive tasks in adulthood after chronic oral administration of THC and THC:CBD in adolescence; and 3) Examine changes in mPFC eCB release during cognitive tasks in adulthood after chronic oral administration of THC and THC:CBD in adolescence. This research will provide valuable insights into the enduring effects of chronic cannabis exposure on cognitive development and function, shedding light on the potential therapeutic role of CBD in mitigating cannabis-induced cognitive deficits.

 Nov 03, 2023 @ 11:00 a.m.

 Medical Center | Ryan Case Methods Rm (1-9576)

Host: Kuan Hong Wang, PhD - Advisor

Estephanie Balbuena - PhD Candidate, Neuroscience Graduate Program

Thesis Proposal

Cerebrospinal fluid (CSF) movement into and throughout the brain is supported via a network of perivascular spaces known as the glymphatic system. The glymphatic system is comprised of blood vessels enclosed by astrocytic endfeet expressing the aquaporin 4 (AQP4) water channel, and clears waste such as lactate and β-amyloid from the brain. Recent studies have established that circadian rhythms, endogenous 24-hour cycles of physiological changes, increase glymphatic function during the rest phase. What drives this circadian rhythm of glymphatic function remains unknown. Two well studied circadian neuropeptides arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) found in the suprachiasmatic nucleus (SCN), the central pacemaker of the brain, also are found in the CSF. AVP is known for its role in regulating circadian behavior, is important for tissue-water homeostasis, and is a potent vasoconstrictor. VIP synchronizes circadian rhythms, can stimulate water secretion in the periphery, and is a potent vasodilator. Both AVP and VIP receptors are expressed in astrocytes, the glial cells important for maintaining glymphatic function. Based on this information, these neuropeptides are great candidates to regulate glymphatic function.

Aim 1 will establish whether AVP and VIP can mediate glymphatic function in a circadian manner. Most studies on circadian properties of AVP and VIP have been on ex vivo slices of the SCN, with little to no information on how these peptides work in vivo.

Aim 2 will test whether in vivo SCN-driven changes in AVP and VIP regulate glymphatic function. AVP and VIP have been implicated in ischemic stroke pathology and recovery. Stroke onset and severity are regulated by time-of-day. The glymphatic system is also implicated in ischemic strokes pathology, suggesting that regulators of glymphatic function may contribute to stroke.

Aim 3 will determine whether AVP and VIP concentration varies before and after ischemia across the brain, potentially driving circadian-related pathology. Together, these studies may provide novel insight into how neuropeptides contribute to maintaining circadian regulation of the glymphatic system, and following ischemic stroke.

 Oct 30, 2023 @ 2:00 p.m.

 Medical Center | K-207 (2-6408)

Host: Lauren Hablitz, PhD - Advisor

Kathryn Toffolo - PhD Candidate, Neuroscience Graduate Program

Thesis Defense

The ongoing context of a sentence is often used to comprehend meaning (semantics) within spoken language. One can measure this semantic processing using electroencephalography (EEG) and functional magnetic resonance imaging (fMRI). Using EEG, it was found that context-dependent predictions result in amplitude modulations of the N400 event-related potential (ERP) component and the late positivity component (LPC/P600). Additionally, fMRI research of neurotypical (NT) adults has shown that semantic information is processed in the left medial/posterior Superior Temporal Gyrus (m/pSTG) and/or the Inferior Frontal Gyrus (IFG). However, there is no consensus about the neurophysiology of semantic processing in individuals with autism spectrum disorder (ASD), who often have atypical semantic comprehension, and these differences have not been characterized throughout development. Because this research is crucial to understanding whether the language differences in children with ASD are due to a language delay, an alternative language trajectory, or both, this project aimed to investigate the development of auditory semantic processing in individuals with ASD using fMRI and EEG. To accomplish this, a standardized semantic stimulus set was created for children and verified to evoke semantic processes in adults (i.e. the N400 and LPC/P600). This stimulus set was also tested in passive vs. active contexts to see if semantic processing could be investigated in minimally verbal children with ASD. Lastly, in response to the same semantic stimuli, semantic processing was localized to the left IFG and bilateral pSTG in 20 NT adults. With an outline of the temporal properties and localization of mature semantic processing, this project investigated language development in children with and without autism in distinct age groups (6-7 years, 8-9 years, and 10-12 years) using both EEG and fMRI. Preliminary data show an effect of age on the amplitude and latency of these components and that there are similarities between the ERPs of ASD and NT children. Conversely, older autistic children have different functional activation in response to semantic information than their NT peers, specifically bilateral activation in the IFG and additional activity increases in the fusiform and STG. These data suggest that autistic children may process semantic information differently than NT children.

 Oct 27, 2023 @ 1:00 p.m.

 Medical Center | K-207 (2-6408)

Host: John Foxe, PhD & Edward Freedman, PhD - Advisors

Alexis Feidler - PhD Candidate

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and the most common monogenic cause of autism spectrum disorder (ASD). Phenotypically and behaviorally, FXS is characterized by specific facial features, anxiety, hyperactivity, social deficits, learning disabilities, sensory hypersensitivity, seizures and macroorchidism. FXS is a repeat expansion disorder where >200 repeats at the CpG island in the 5’UTR of the Fragile X messenger ribonucleoprotein 1 (Fmr1) gene leads to methylation, silencing and subsequent loss of Fragile X messenger ribonucleoprotein (FMRP) expression. FMRP is an RNA binding protein expressed throughout the brain, ovaries and testes. It interacts with 4% of mRNAs in the brain. While the function of FMRP is not well characterized, it has been implicated as a negative regulator of translation. In FXS patients and animal models, loss of FMRP leads to changes in dendritic spine architecture in brain regions associated with modulating and responding to sensory experiences as well as learning and memory, which mirrors the behavioral phenotype in FXS patients. While mixed results exist, studies have primarily found a reduction in mature, mushroom shaped spines, an increase in immature, filipodia-like spines and an increase in spine density. These likely contribute to changes in long-term potentiation, long-term depression and hyperexcitability in FXS that underlie learning disabilities, sensory sensitivities and seizures. Microglia, the resident immune cells of the central nervous system, have many roles in the brain including synapse monitoring and pruning. Deficits in microglia have been shown to contribute to spine phenotypes, in particular, aberrant excitatory synapse maturation. In addition, it has been shown that mutations in microglia can induce ASD-like behaviors and changes in cortical excitability, and microglia and general immune dysfunctions are implicated in other neurodevelopmental disorders. While this suggests that microglia could play a significant role in FXS, to date, few studies have examined this cell type in this disorder. Considering the roles microglia play in synapse remodeling and their roles in other neurodevelopmental disorders,

I propose to test the hypothesis that microglia dysfunction contributes to the aberrant spine phenotype affecting circuit level functions of synaptic gain and plasticity and excitability as well as behavior in FXS.

 Sep 19, 2023 @ 12:00 p.m.

 Medical Center | CEL Rm. 2-7520

Host: Advisor: Ania Majewska, PhD

Julia Granato - PhD Candidate, Advisor: Jennetta Hammond

 Aug 21, 2023 @ 12:00 p.m.

 Medical Center | K-307 (3-6408)

Anjali Sinha - PhD Candidate in Neuroscience

The peripheral vestibular system detects head position and movement through activation of vestibular hair cells (HCs) in semicircular canal cristae and otolithic organs. HCs transmit this information to the CNS by way of primary vestibular afferent neurons, which are critical for maintaining balance, gaze stability and spatial navigation. The CNS, in turn, modulates HCs and afferents via the efferent vestibular system (EVS) and activation of cholinergic signaling mechanisms. In mice, we previously demonstrated that activation of muscarinic acetylcholine receptors (mAChRs), during EVS stimulation, gives rise to a slow excitation that takes seconds to peak and tens of seconds to decay back to baseline. This slow excitation is mimicked by muscarine and ablated by the non-selective mAChR blockers scopolamine, atropine, and glycopyrrolate. While there are five distinct mAChRs (M1-M5), the distinct subtypes driving EVS-mediated slow excitation remain unidentified and downstream details on how these mAChRs alter vestibular function are not well understood. Using a panel of mAChR-subtype selective drugs, we determined that M3mAChRs were the main regulator of EVS-mediated slow excitation, with a possible contribution from M1mAChRs. In M3mAChR-KO mice, a loss of EVS-mediated slow excitation was observed in irregular, but not regular firing afferents. Consistent with these observations, the combined enhancement of P1N1 amplitudes and shortening of P1 latencies in vestibular sensory-evoked potentials, attributed to activation of mAChRs on irregular vestibular afferents, was significantly reduced in M3mAChR-KO mice. Finally, M3mAChR KO mice also had a number of deficits in various vestibular-related behaviors including thermal regulation, open-field activity, balance beam performance, body sway measurements, and forced swim test. M3mAChRs are Gq/11-coupled GPCRs which engage phospholipase-C (PLC) to deplete PIP2 in the plasma membrane, which is normally required to keep voltage-gated potassium channels, belonging to the KCNQ (Kv7.X) family, open. Closure of KCNQ channels in vestibular afferents, following EVS-mediated mAChR activation, gives rise to a depolarization that enhances the resting discharge rate of vestibular afferent neurons. To this end, multiple members of the KCNQ channel family, including KCNQ2 and KCNQ3, are localized to several microdomains within vestibular afferent endings, where they influence afferent excitability and could be targeted by EVS neurons. Our pharmacological analyses, using KCNQ subunit-selective openers and blockers, revealed that the closure of KCNQ2/3 channels likely gives rise to EVS-mediated slow excitation, while KCNQ3/4 channels appear to play a role in maintaining afferent excitability. Finally, we found that mice lacking the KCNQ3 subunit had an altered distribution of afferent firing rates and EVS-mediated slow responses. KCNQ3 KO mice also had fewer high-firing (>80sp/s) regular vestibular afferents again suggesting that KCNQ3 likely contributes to discharge properties in these afferents. Collectively, our pharmacological data in conjunction with M3mAChR and KCNQ3 KO mice reveal that EVS-mediated slow response is mediated primarily by activation of M3mAChRs which leads to the closure of KCNQ2/3 channels on vestibular afferents. Our studies suggest possible roles for EVS-mediated slow excitation in vestibular behaviors.

 Aug 08, 2023 @ 11:00 a.m.

 Medical Center | K-207 Auditorium (2-6408)

Host: Dr. J. Christopher Holt, Neuroscience, University of Rochester School of Medicine & Dentistry

Leslie Gonzales - PhD Candidate, Advisor: Ken Henry, PhD

As humans age it is common to lose afferent auditory-nerve fibers (ANFs), and this loss of synapses between inner hair cells and ANFs is termed cochlear synaptopathy (CS). While some studies have found that CS produces an associated impairment in the perception of complex sounds in noise (i.e., hidden hearing loss), many others have shown no effect. Greater clarity into hidden hearing loss requires a detailed knowledge of CS effects on neural encoding in surviving ANFs, but these remain controversial due to very few studies. Therefore, the first aim of the current study is to characterize a new animal model (the budgerigar, parakeet), in order to determine how ANF physiology is affected by CS. Budgerigars are the chosen animal model due to their hearing range, which includes lower frequencies humans rely on for speech comprehension. Furthermore, budgerigars are widely used in behavioral auditory research, including ongoing behavioral studies of CS in our lab, and show performance comparable to humans for various complex auditory discrimination tasks. Intracochlear infusions of 1-2 mM kainic acid, the glutamate analog, are performed to produce substantial ANF loss while sparing hair cells. We hypothesize that ANF responses in normal-hearing budgerigars will be fundamentally similar to those in other birds and mammals, including the dynamic range of rate-level functions, threshold distribution pattern, and similar tuning curves. We hypothesize that induced CS will cause an increase in onset synchrony of single-fiber responses, based on recent discoveries in mouse and gerbil pointing to altered temporal dynamics of hair-cell ribbon synapses. On the other hand, we expect no major changes with CS in other ANF response properties (e.g. dynamic range), nor preferential degradation of any specific spontaneous-rate group of fiber. Second, the study seeks to optimize tools for detecting CS. Despite its prevalence in humans, there are no sufficient non-invasive tools to detect CS. The envelope-following response (EFR) is a scalp potential evoked with amplitude-modulated stimuli, that may be more sensitive to CS in humans due to its relatively high amplitude. EFRs are typically measured using sinusoidally amplitude-modulated (SAM) tones, but a recent modeling study suggests that square wave modulated (SWM) tones might provide more synchronous ANF responses and therefore provide a more sensitive diagnostic tool to detect CS. Hence, the current study will probe EFRs to SAM and SWM tones in control animals and in animals with experimentally induced CS. Preliminary data in the budgerigar demonstrate EFRs evoked by SWM stimuli provide a better indication of histological ANF damage in our animal model of CS than SAM EFRs. Finally, ANF responses to SAM and SWM tones will be recorded in control and CS ears to test for the hypothesized increase in synchrony to the modulation frequency for SWM tones (expected in both groups). Ultimately, the proposed experiments will characterize changes in ANF response properties following CS and optimize non-invasive methods that can be used to detect this all-too-common cochlear pathology.

 Jul 12, 2023 @ 9:00 a.m.

 Medical Center | Lower Auditorium (1-7619)

Eleni Patelaki - PhD Candidate, Advisors: Ed Freedman, PhD and John Foxe, PhD

Aging is associated with decline in locomotion, with this decline being aggravated in Parkinson’s disease (PD), the most common and fast-growing motoric neurodegenerative disease. Dysfunctional ambulation can have a severe negative impact on the quality of life, leading to increased incidence of falls and fall-related injuries, as well as loss of independence. Converging evidence indicates that, in healthy aging and during the early stages of PD, compensatory mechanisms work to mask incipient deficits in the gait-motoric circuitry, leading to the manifestation of seemingly normal gait patterns under less demanding and unperturbed walking conditions. However, when a cognitive task, such as texting or engaging in a conversation, is added to walking, decrements in walking and/or cognitive performance become amplified to a degree that they can no longer be masked. As such, combining walking and a concurrent cognitive task (dual-task walking) may be an effective methodology for revealing and diagnosing motor and cognitive pathologies at the early stages, through taxing the underlying neural resources. Starting with young healthy adults and then proceeding to older healthy adults and PD patients with and without treatment, this dissertation used the MoBI modality to investigate the neural substrates of the interactions between walking and a cognitive task requiring inhibitory control, a core executive function known to weaken in aging and PD. Overall, our findings showed that, surprisingly, most young adults improve cognitive task performance during dual-task walking, and this improvement is subserved by more flexible recruitment of fronto-cortical neural circuits. Conversely, performance in most older adults declines under the same conditions, and this decline is associated with reduced ability to efficiently recruit multiple neural circuits during walking. However, a small—potentially ‘super-aging’—group of older adults exhibited improvement during dual-task walking, underpinned by preserved flexibility in using the same frontal circuits linked to improvement in young adults. PD patients manifested pronounced decrements in cognitive and gait performance compared to healthy older adults, as well as pathological neural processing over multiple cortical regions, during dual-task walking. At the behavioral level, the combination of deep brain stimulation (DBS) with dopaminergic medication was the treatment approach that benefited gait and cognition the most. At the neural level, we found right-hemisphere prefrontal and parietal signatures of DBS, presumably reflecting DBS-related facilitation in neural processing.

Flyer

Zoom Link:  https://rochester.zoom.us/j/95293342672  Passcode: 834456

 May 12, 2023 @ 1:00 p.m.

 Medical Center | Upper Auditorium (3-7619)

Host: University of Rochester Department of Biomedical Engineering

Katherine Andersh - PhD Candidate, Advisor: Rick Libby, PhD

Glaucoma is the second leading cause of blindness worldwide, with vision loss occurring due to retinal ganglion cell (RGC) death. The major risk factors for developing glaucoma are age and elevated intraocular pressure (IOP). Currently, the only treatment for glaucoma patients is reducing IOP, but this does not halt the disease or restore lost vision. Therefore, identifying the molecular mechanisms underlying how glaucomatous injury leads to RGC death is crucial to identify therapeutic targets. Growing literature suggests a role for extrinsic signaling driving RGC death in glaucoma. After a glaucomatous insult, microglial cells are thought to produce three potentially neurotoxic cytokines: complement component 1 subcomponent q (C1q), interleukin 1 alpha (IL1A) and tumor necrosis factor (TNF). In an age-related model of ocular hypertension, C1q, Il1a, and Tnf were upregulated at early stages of glaucomatous neurodegeneration. In addition, others have shown that deficiency of C1q, Il1a, and Tnf resulted in near complete RGC protection after optic nerve injury.

C1Q and TNF have been extensively studied in the context of glaucoma, however, IL1A remains understudied for its direct effects on RGCs. Here, I test whether IL1A is sufficient for RGC death and define the localization of downstream IL1A signaling following cytokine insult. IL1A was intravitreally injected alone or in combination with TNF. Injection of IL1A alone did not result in RGC loss, however, when IL1A and TNF were injected together, RGC death occurred. Loss was present at 14 days, much earlier than the 12 weeks typically seen in TNF alone conditions, indicating that IL1A acts in a rapid, synergistic manner with TNF to facilitate RGC degeneration. We found that IL1A+TNF mediated RGC death required the expression of the sole IL1 receptor: IL1R1. Loss of Il1r1 globally or in RGCs and macroglia resulted in RGC survival following IL1A+TNF. However, loss of Il1r1 in primarily RGCs failed to confer RGC protection, indicating a requirement for IL1R1 signaling in multiple cell types and/or macroglia following IL1A+TNF. Overall, these studies support the direct, detrimental role that neurotoxic cytokines play in neurodegeneration and highlight the importance of assessing the molecular mechanisms by which these cytokines act.

 May 01, 2023 @ 11:00 a.m.

 Medical Center | Ryan Case Method Room (1-9576)

Allison Murphy - PhD Candidate, Advisor: Farran Briggs, PhD

In the visual system, information enters as light hitting the retina of the eye. This signal is transmitted to the dorsal lateral geniculate nucleus (LGN) in the thalamus, then to primary visual cortex (V1). In highly visual animals, this feedforward visual pathway is organized into parallel informational streams, known as the magnocellular, parvocellular, and koniocellular streams in primates and the somewhat homologous X, Y, and W streams in carnivores, including ferrets. While these feedforward stream have been extensively studied, relatively little is known about the corticogeniculate (CG) feedback pathway that projects from V1 to the LGN. Although corticogeniculate feedback synapses in the LGN greatly outnumber feedforward retinal inputs, their modulatory nature has made their functional role difficult to study. The purpose of this thesis is to examine the structure of this pathway and its functional role in vision by answering the following questions: 1) Is a parallel stream structure maintained in the feedback pathway? 2) What is the functional impact of feedback in the LGN? To answer these questions, we performed neurophysiological recordings simultaneously in LGN and V1 of anesthetized animals. For the first question, we found functionally connected pairs of corticogeniculate and LGN neurons using cross-correlation of their spike trains. We analyzed the physiological response properties of these pairs of neurons to determine whether distinct populations of CG neurons maintain stream-specific projections to the LGN. Additional anatomical analyses provide support for distinct morphological subtypes of CG neurons. For the second question, we used a modified rabies virus to selectively infect corticogeniculate neurons with a light sensitive ion channel and performed optogenetic manipulation during presentation of visual stimuli. We then examined how manipulation of corticogeniculate feedback affected visual response properties of LGN neurons, including the variability and information content of their spike trains. Together, these experiments demonstrate that corticogeniculate feedback is a pathway with a complex organizational structure that plays a subtle role in shaping LGN visual responses.

 Apr 11, 2023 @ 11:00 a.m.

 Medical Center | K-307 (3-6408)

Thomas Delgado - PhD Candidate, Advisor: Gail Johnson, PhD

Astrocytes are essential for maintaining neuronal function in resting and disease states. Following injury, astrocytes take on reactive phenotypes that grade from neurotoxic to neuroprotective, which impact subsequent neuronal recovery processes, such as axonal regeneration. Only recently has the heterogeneity of reactive astrocyte populations begun to be described, and little is known about the contextual requirements and molecular inflection points that underlie these graded responses. Accumulating data from my lab suggests that one of these inflection points is transglutaminase 2 (TG2). TG2 is complex in that it regulates signaling of numerous molecular pathways at the cell membrane, in the cytosol, and in the nucleus; thus, it can provide multiple levels of context-dependent input into gene regulation. Importantly, when TG2 is depleted from astrocytes, they better protect neurons in culture from oxygen-glucose deprivation (OGD), improve motor function recovery in a mouse spinal cord injury model, and better facilitate neurite outgrowth in vitro on an injury-relevant, growth-inhibitory matrix. Additionally, TG2 depletion upregulates lipid handling (lipid uptake and formation of lipid droplets) and lipid metabolism pathways in an injury-dependent manner. As stressed neurons accumulate reactive oxygen species and peroxidated lipids, and cannot metabolize these toxic lipids on their own, they export them to astrocytes. Astrocytic lipid uptake and metabolism eliminates these oxidized lipids and recycles them to produce energy substrates for neurons. This mechanism may partly underlie the protective effect of TG2-/- astrocytes after injury.

In aim 1, I will test the hypothesis that depletion of astrocytic TG2 improves axonal regeneration through an inhibitory extracellular matrix (ECM) after CNS injury using an optic nerve crush model in mice. These studies are built on previous spinal cord injury data from my lab, with the unique addition that, in the optic nerve, axonal regeneration can only occur through the inhibitory ECM deposited at the crush site, while in the spinal cord, regrowth may occur via collaterals around the injury site, through permissive matrices.

In aim 2, I will identify the major molecular pathways and mechanisms of gene and protein regulation underlying the unique function of TG2-/- astrocytes. As TG2 is a known interactor of transcription machinery and chromatin regulators in the nucleus, I will use ATAC-seq to analyze differences in chromatin accessibility between TG2-/- and wild type (WT) astrocytes and integrate these data with differentially regulated transcripts identified through RNA sequencing. I will compare differential enrichment in upstream signaling pathways with differential protein expression identified by tandem mass spectrometry to better approximate the functional impact of these molecular changes.

In aim 3, following data that lipid handling and metabolism is differentially regulated in TG2-/- astrocytes, I hypothesize that TG2-/- astrocytes more efficiently uptake and metabolize lipids that are released by stressed neurons (as peroxidated lipids), ultimately providing better control of oxidative stress and increased energy supply to neurons in injury conditions. Therefore, I will measure the ability of WT and TG2-/- astrocytes to take up and metabolize lipids released by neurons, in control and stressed conditions, as indicated by lipid droplet accumulation. Further, I will measure metabolic flux through ketone and cholesterol synthesis pathways, downstream of fatty acid oxidation, in WT and TG2-/- astrocytes by labeling them with C13-palmitate and measuring C13-labeled metabolites by LC-MS. I will then pair neurons with either astrocyte group to track the export of C13-labeled energy substrates to neurons.

Overall, this project will explore the role of TG2 as a key regulator of astrocyte reactivity after CNS injury and, within this scope, better characterize the astrocytic metabolic pathways that are integral for neuronal and functional recovery.

 Apr 07, 2023 @ 2:00 p.m.

 Medical Center | Lower Adolph Auditorium (1-7619)

Garrick Salois - PhD Candidate, Advisor: Margot Mayer-Proschel, PhD

Iron deficiency is the most common micro-nutrient deficiency worldwide and is especially common amongst pregnant women. Gestational iron deficiency (gID) has been correlated with a wide range of timing, dose, and duration-dependent behavioral and neurophysiological consequences that are often not corrected by iron supplementation later in life. Despite its prevalence and consequences, little is known about how gID affects the developing human nervous system.

Data from mouse models suggests that gID may affect the balance of excitation and inhibition in adult neural circuits and that the timing of iron deficiency may alter both the resulting phenotype as well as the potential for treatment via iron supplementation. Using immunofluorescence in our mouse model of gID, in which overt brain iron deficiency begins by embryonic day 12, we observed increased embryonic expression of Nkx2.1, a transcription factor critical for fate determination of inhibitory neural precursors in the ganglionic eminence. Furthermore, despite iron supplementation at birth, the offspring show a disruption in cortical inhibitory interneuron subtypes that persists at least until day 100.

To examine whether these observations in mice extend to human neurodevelopment, we established a human ventral forebrain organoid model of gID that recapitulates relevant iron levels and allows for the identification of changes in the transcription factor Nkx2.1 as well as subsequent impairments in the proliferation, differentiation, and maturation of developing interneurons. We then used a variety of techniques including immunofluorescence, confocal microscopy, ICPMS, qRT-PCR, flow cytometry, and sc-RNAseq to measure the effect of iron deficiency on organoid development. Using this novel organoid system, we observed that three major hallmarks identified in our animal model are preserved: (i) disrupted divalent metal homeostasis, (ii) increased Nkx2.1 expression, (ii) and sustained changes in cell type specification. Our novel human model of gID allows us to now decipher the mechanisms that leads to these iron deficiency-associated disruptions and offers a unique opportunity to gain insight into the impact of this prevalent condition on fetal brain development.

 Mar 29, 2023 @ 2:00 p.m.

 Medical Center | 1-7619 Adolph Lower Aud.

Host: University of Rochester School of Medicine and Dentistry
The Neuroscience Graduate Program

Dennisha P. King - PhD Candidate, Advisor: Julie Fudge, MD

In primates, the amygdala’s basal nucleus is evolutionarily expanded, and matures postnatally. The ventral (“parvicellular”) basal nucleus (Bpc) is the last region to develop in the fetus; postnatally, it is bordered by the paralaminar nucleus (PL) which contains immature neurons. Between infancy and adolescence, the PL gains mature neurons (concomitantly reducing immature ones) implying that synapse formation is also occurring. Microglia form, remodel and maintain neural circuitry by pruning synapses that are non-functional, weak, or redundant. Our preliminary data have shown that the density, area, and perimeter of the microglia differ between the PL and adjacent Bpc, and increase in both regions from infancy to adolescence. Aim 1 will first characterize microglial morphology and their role in synaptic pruning in the PL and Bpc in control infant and adolescent monkeys during normal development. Aim 1A will extend preliminary morphologic analyses of Iba1-immunoreactive (IR) cells (microglial marker), using larger cell numbers, and more sophisticated analyses (Imaris software) in control infant and adolescent PL and Bpc. I expect that the microglia will increase their process length and complexity between infancy and adolescence in the PL and Bpc. Aim 1B will examine connectivity between pre-synaptic (synapsin-1) contacts and excitatory spines (PSD-95) in the PL and Bpc between infancy and adolescence. I hypothesize that synaptic contacts onto excitatory spines are greater in the Bpc which is more cellularly mature than the PL, and that contacts in both regions increase between infancy and adolescence. Aim 1C will focus on microglia’s role in pruning excitatory spines by examining PSD-95 engulfment by microglial lysosomes (PSD95 colocalization with CD68/Iba1IR). I hypothesize that PSD-95 engulfment, representing spine pruning, occurs in both the PL and Bpc more frequently in adolescence correlating with increased phagocytic activity in the microglia (CD68/Iba1).

During development, the PL’s postmitotic neurons continue differentiating and thus may be susceptible to being influenced by early life events. Our preliminary data shows that early life stress (ELS) in the form of maternal deprivation, results in a downregulation of PL transcripts that promote neural growth and migration, and an upregulation in complement pathways, a key mediator of synaptic pruning, during infancy. However, we still do not understand the dynamic relationship between the neurons and microglia in the PL and Bpc, which are both impacted by ELS. Aim 2A will determine whether ELS is associated with changes in infant monkey PL microglia morphology, suggesting increased synaptic pruning. Aim 2B will determine whether there is a net increase or decrease in synapses in the infant PL after the ELS conditions. In Aim 2C, I will interrogate PL transcription changes in ELS versus control infants to test the idea that complement mediated mechanisms are increased in ELS. Using a curated list of microglia transcripts, I hypothesize that many upregulated transcripts will be complement and immune-related molecules. I will also perform a pathway analysis (whole genome) to investigate the most changed pathways following ELS.

In Aim 3, I will validate the most changed microglial transcripts (Aim 2C) and examine whether ELS leads to increased microglial engulfment of glutamatergic spines in the infant PL, through a complement mechanism, e.g. a net increase of PSD95/C3 engulfment by CD68/Iba1. Using the results of Aim 2C, I will first choose the most changed transcripts involved in microglial pruning for which antibodies are available and ensure that these are changing at the protein level. I will then examine microglia (Iba1/CD68/complement receptor) engulfment of dendritic spines (PSD95, complement protein) in the PL of ELS versus control infants. Depending on the results of the microarray and validation experiments, I may focus on different members of the complement pathway.

Ultimately this project will examine the microglia’s role in supporting synaptic formation and maintenance in the primate PL and Bpc, and investigate whether and how ELS alters this role during infancy.

 Mar 15, 2023 @ 11:30 a.m.

 Medical Center | Ryan Case Method (1-9576)

Michael Giannetto - PhD Candidate, Advisor: Maiken Nedergaard, MD, DMSc

The brain is the most metabolically active organ in the body, yet it lacks a traditional lymphatic system for waste clearance. Instead, the brain utilizes the glymphatic system, a network of fluid filled spaces surrounding blood vessels, termed perivascular spaces (PVSs), which facilitates movement of cerebrospinal fluid (CSF) into the brain along arteries and waste clearance out of the brain along veins. Astrocytic endfeet comprise the outer boundary of the PVS and serve as a point of regulation for CSF flow into the brain. CSF flow in pial artery PVSs is well characterized to follow the same direction as blood flow driven by cardiac pulsations, and CSF flow is increased by large arterial dilations associated with neuronal activity. However, the function, and even existence, of the PVS along capillaries remains unclear. Additionally, the physiological function of pericytes, mural cells that cover capillaries, remains controversial. Some groups claim pericytes are important in blood flow regulation while others have demonstrated they are irrelevant to blood flow, but instead could maintain the extracellular matrix and phagocytose waste. Capillaries make up the bulk of vasculature surface area in the brain, with no brain tissue further than 30 micrometers away from a capillary, and capillaries are continuously covered by pericytes. Thus, capillary PVSs and pericytes are well positioned to clear waste but remain understudied. In this proposal, I will determine if there is directional fluid flow in capillary PVSs, test if pericytes contribute to capillary PVS function, and finally test pericyte and capillary PVS function in aging.

Aim1, I will test the hypothesis that fluid flow in the capillary PVS is directional, following the same direction of blood flow, similar to CSF flow in arterial PVSs. I will utilize in vivo 2-photon imaging of secreted fluorescent protein to label capillary PVSs and measure fluorescent recovery after photobleaching.

Aim 2 will test whether pericytes play a role in clearing waste or maintaining the structure of the capillary PVS. I will use approaches developed in Aim 1 combined with inducible genetic manipulations of pericytes to ablate them or impair their phagocytic and cell matrix maintaining functions, then measure capillary PVSs and glymphatic flow.

Aim3, I will test the hypothesis that capillary PVSs and pericyte dysfunction contribute to glymphatic impairment. I will use the same methods to label capillary PVSs to determine if functional fluid flow decreases, the structure of capillary PVSs changes, or pericyte functions decrease in a cohort of aged mice. I will then attempt to rescue pericyte function in aged mice using PDGF-beta supplementation to improve glymphatic function.

Ultimately this project will answer longstanding questions concerning function of pericapillary PVSs and pericytes, and determine their effect on the glymphatic system in normal aging.

 Feb 08, 2023 @ 2:00 p.m.

 Medical Center | Ryan Case Method (1-9576)

Berke Karaahmet - PhD Candidate in Neuroscience, Advisor: Kerry O’Banion, MD, PhD

Alzheimer’s Disease (AD) is a chronic neurodegenerative disorder that clinically manifests as the most common form of dementia. Due to their surveillance functions and immunocompetence as resident macrophages of the Central Nervous System (CNS), microglia are well-equipped to respond to perturbation of tissue homeostasis. Therefore, they are regarded as promising translational targets in modulating the impact of amyloid and Tau pathologies observed in AD. Here, we sought to elucidate the effect of two peripherally administrated pharmacologic approaches that are hypothesized to modulate microglial activation phenotypes in AD-like models.

In our first approach, we investigated the use of the multiple sclerosis (MS) drug, Glatiramer Acetate (GA), in murine models of aggressive amyloid pathology (5xFAD) or amyloid and Tau pathology combined (3xTg). In response to GA treatment, we observed improvements in cognitive function and molecular pathology in female 3xTg mice. These were associated with minimal transcriptomic changes in microglia, in which Dcstamp was the most upregulated gene. Follow-up analyses of Aβ plaque burden in 5xFAD; DCSTAMP knockout mice showed that the females of this genotype had increased plaque numbers, but this effect did not reach significance. In female 5xFAD mice, we found that GA treatment did not impact plaque burden if started early in life, showed a trend towards decreased plaque burden if started during early disease progression and, unexpectedly, increased plaque burden if started during late disease stages. No changes in plaque burden were observed in 5xFAD males.

In our second approach, we sought to investigate whether microglia that were depleted with a CSF1R inhibitor containing diet and allowed to repopulate could attenuate levels of pathological markers in aged 3xTg mice. We observed no changes in amyloid pathology but found differential effects in several markers of phosphorylated Tau. Single-cell transcriptomic analysis of microglia revealed a cluster that was strongly characterized by Cxcl13 expression. In situ analysis of Cxcl13 showed that it was localized to regions of AD-like pathology in 3xTg mice. This suggests that Cxcl13 upregulation in repopulated microglia responding to AD-like pathology might be one of the driving factors behind the changes observed in phosphorylated Tau levels. Further studies are warranted to establish mechanistic links between these observations.

Altogether, our data indicate that immunomodulatory therapeutics may be beneficial in restricting certain aspects of AD pathology. However, caution must be exercised when designing these therapies since outcomes may depend on the pathological stage of AD.

 Feb 01, 2023 @ 12:00 p.m.

 Medical Center | 1-7619 (Lower Adolph Auditorium)

Host: Dr. Kerry O’Banion, Neuroscience, University of Rochester School of Medicine & Dentistry