John B. Pierce Laboratory

Alzheimer's disease (AD) poses a significant global health challenge, being the most prominent cause of dementia with prevalence increasing as the population ages. While the majority of AD cases are late-onset (LOAD), current animal models predominantly represent the more aggressive, faster progressing early-onset AD (EOAD), limiting their ability in assessing early biomarkers and gaining deeper understanding of LOAD progression. This study explores a promising translatable model, the APOE4.TREM2 mouse, which combines the APOE4 allele and the Trem2 p.R47H mutation, both linked to increased AD risk in the human population. We performed behavioral phenotyping and measured hemodynamics and neurovascular coupling in dorsal olfactory bulbs (dOB) during odor stimulation of the APOE4.TREM2 mouse line. Experimental evidence of olfactory dysfunction prior to clinical symptoms suggests the opportunity of utilizing smell testing and fMRI as tools for screening of AD, both for preclinical and clinical studies. Here we assess and confirm the translatability of the APOE4.TREM2 mouse LOAD model, reporting exacerbated anxiety, deficits in odor-based foraging and spatial memory, and exacerbated odor-evoked dOB neural and intrinsic responses, but stable neurovascular coupling, in an age-dependent manner.

Creating vascular structures in vitro via bioprinting to replace damaged or missing vasculature has significant advantages over current surgical methods of vessel replacement. Using rat fibroblasts and smooth muscle cells, we have bioprinted a rat aorta using a rotating mandrel method to create the tubular replacement structure. Then, the 3D-bioprinted aortas were implanted into rats to determine their functionality long-term in vivo. The implanted vascular conduits were well-tolerated, well-incorporated into native vasculature, and showed the physiological behavior of a native vessel. The development and deployment of 3D-bioprinted vessels for repair of large vessels in an animal model paves the way for advancements in the treatment of vascular disease in humans.

Voltage imaging is a powerful tool for studying the dynamics of neuronal activities in the brain. However, voltage imaging data are fundamentally corrupted by severe Poisson noise in the low-photon regime, which hinders the accurate extraction of neuronal activities. Self-supervised deep learning denoising methods have shown great potential in addressing the challenges in low-photon voltage imaging without the need for ground-truth but usually suffer from the trade-off between spatial and temporal performances. We present DeepVID v2, a self-supervised denoising framework with decoupled spatial and temporal enhancement capability to significantly augment low-photon voltage imaging. DeepVID v2 is built on our original DeepVID framework, which performs frame-based denoising by utilizing a sequence of frames around the central frame targeted for denoising to leverage temporal information and ensure consistency. Similar to DeepVID, the network further integrates multiple blind pixels in the central frame to enrich the learning of local spatial information. In addition, DeepVID v2 introduces a new spatial prior extraction branch to capture fine structural details to learn high spatial resolution information. Two variants of DeepVID v2 are introduced to meet specific denoising needs: an online version tailored for real-time inference with a limited number of frames and an offline version designed to leverage the full dataset, achieving optimal temporal and spatial performances. We demonstrate that DeepVID v2 is able to overcome the trade-off between spatial and temporal performances and achieve superior denoising capability in resolving both high-resolution spatial structures and rapid temporal neuronal activities. We further show that DeepVID v2 can generalize to different imaging conditions, including time-series measurements with various signal-to-noise ratios and extreme low-photon conditions. Our results underscore DeepVID v2 as a promising tool for enhancing voltage imaging. This framework has the potential to generalize to other low-photon imaging modalities and greatly facilitate the study of neuronal activities in the brain.

We tested the hypothesis that hyperandrogenemia in androgen excess polycystic ovary syndrome (AE-PCOS) is a primary driver in blood pressure (BP) dysregulation via altered sympathetic nervous system activity (SNSA), reduced integrated baroreflex gain and increased renin-angiotensin system (RAS) activation. We measured resting SNSA (microneurography), integrated baroreflex gain, and RAS with lower body negative pressure in obese insulin-resistant (IR) women with AE-PCOS [n = 8, 23 ± 4 yr; body mass index (BMI) = 36.3 ± 6.4 kg/m2] and obese IR controls (n = 7, control, 29 ± 7 yr; BMI = 34.9 ± 6.8 kg/m2), at baseline (BSL), after 4 days of gonadotropin-releasing hormone antagonist (ANT, 250 μg/day) and 4 days of ANT + testosterone (ANT + T, 5 mg/day) administration. Resting BP was similar between groups for systolic blood pressure (SBP; 137 ± 14 vs. 135 ± 14 mmHg, AE-PCOS, control) and diastolic BP (89 ± 21 vs. 76 ± 10 mmHg, AE-PCOS, control). BSL integrated baroreflex gain was similar between groups [1.4 ± 0.9 vs. 1.0 ± 1.3 forearm vascular resistance (FVR) U/mmHg], but AE-PCOS had lower SNSA (10.3 ± 2.0 vs. 14.4 ± 4.4 burst/100 heartbeats, P = 0.04). In AE-PCOS, T suppression increased integrated baroreflex gain, which was restored to BSL with ANT + T (4.3 ± 6.5 vs. 1.5 ± 0.8 FVR U/mmHg, ANT, and ANT + T, P = 0.04), with no effect in control. ANT increased SNSA in AE-PCOS (11.2 ± 2.4, P = 0.04). Serum aldosterone was greater in AE-PCOS versus control (136.5 ± 60.2 vs. 75.7 ± 41.4 pg/mL, AE-PCOS, control, P = 0.04) at BSL but was unaffected by intervention. Serum angiotensin-converting enzyme was greater in AE-PCOS versus control (101.9 ± 93.4 vs. 38.2 ± 14.7 pg/mL, P = 0.04) and reduced by ANT in AE-PCOS (77.7 ± 76.5 vs. 43.4 ± 27.3 µg/L, ANT, and ANT + T, P = 0.04) with no impact on control. Obese, IR women with AE-PCOS showed decreased integrated baroreflex gain and increased RAS activation compared with control.NEW & NOTEWORTHY Here we present evidence for an important role of testosterone in baroreflex control of blood pressure and renal responses to baroreceptor unloading in women with a common, high-risk androgen excess polycystic ovary syndrome (AE-PCOS) phenotype. These data indicate a direct effect of testosterone on the vascular system of women with AE-PCOS independent of body mass index (BMI) and insulin-resistant (IR). Our study indicates that hyperandrogenemia is a central underlining mechanism of heightened cardiovascular risk in women with PCOS.

High-speed low-light two-photon voltage imaging is an emerging tool to simultaneously monitor neuronal activity from a large number of neurons. However, shot noise dominates pixel-wise measurements and the neuronal signals are difficult to be identified in the single-frame raw measurement. We developed a self-supervised deep learning framework for voltage imaging denoising, DeepVID, without the need for any high-SNR measurements. DeepVID infers the underlying fluorescence signal based on independent temporal and spatial statistics of the measurement that is attributable to shot noise. DeepVID achieved a 15-fold improvement in SNR when comparing denoised and raw image data.

Polycystic ovary syndrome (PCOS) is associated with endothelial dysfunction; whether this is attributable to comorbid hyperandrogenism and/or obesity remains to be established. Therefore, we 1) compared endothelial function between lean and overweight/obese (OW/OB) women with and without androgen excess (AE)-PCOS and 2) examined androgens as potential modulators of endothelial function in these women. The flow-mediated dilation (FMD) test was applied in 14 women with AE-PCOS (lean: n = 7; OW/OB: n = 7) and 14 controls (CTRL; lean: n = 7, OW/OB: n = 7) at baseline (BSL) and following 7 days of ethinyl estradiol supplementation (EE; 30 µg/day) to assess the effect of a vasodilatory therapeutic on endothelial function; at each time point we assessed peak increases in diameter during reactive hyperemia (%FMD), shear rate, and low flow-mediated constriction (%LFMC). BSL %FMD was attenuated in lean AE-PCOS versus both lean CTRL (5.2 ± 1.5 vs. 10.3 ± 2.6%, P < 0.01) and OW/OB AE-PCOS (5.2 ± 1.5 vs. 6.6 ± 0.9%, P = 0.048). A negative correlation between BSL %FMD and free testosterone was observed in lean AE-PCOS only (R2 = 0.68, P = 0.02). EE increased %FMD in both OW/OB groups (CTRL: 7.6 ± 0.6 vs. 10.4 ± 2.5%, AE-PCOS: 6.6 ± 0.9 vs. 9.6 ± 1.7%, P < 0.01), had no impact on %FMD in lean AE-PCOS (5.17 ± 1.5 vs. 5.17 ± 1.1%, P = 0.99), and reduced %FMD in lean CTRL (10.3 ± 2.6 vs. 7.6 ± 1.2%, P = 0.03). Collectively, these data indicate that lean women with AE-PCOS exhibit more severe endothelial dysfunction than their OW/OB counterparts. Furthermore, endothelial dysfunction appears to be mediated by circulating androgens in lean but not in OW/OB AE-PCOS, suggesting a difference in the endothelial pathophysiology of AE-PCOS between these phenotypes.NEW & NOTEWORTHY We present evidence for marked endothelial dysfunction in lean women with androgen excess polycystic ovary syndrome (AE-PCOS) that is 1) associated with free testosterone levels, 2) impaired relative to overweight/obese women with AE-PCOS, and 3) unchanged following short-term ethinyl estradiol supplementation. These data indicate an important direct effect of androgens on the vascular system in women with AE-PCOS. Our data also suggest that the relationship between androgens and vascular health differs between phenotypes of AE-PCOS.

Women who have had preeclamptic (PE) pregnancies are at increased risk for developing cardiovascular diseases later in life. One potential mechanism that may mediate this increased risk is sympathetic dysregulation. Indeed, there is evidence for elevated muscle sympathetic nerve activity (MSNA) both during and following PE pregnancies. Moreover, previously PE (PPE) women may demonstrate impaired MSNA responses during stress, although this has only been examined to a limited extent. Therefore, we tested the hypothesis that MSNA responses to isometric hand grip exercise (HG) would be greater in PPE women (n=6, age 31±6 years, BMI 29±5 kg/m2, 17±4 months postpartum) compared with women who had a healthy pregnancy (HP; n=8, age 29±2, BMI 25±4 kg/m2, 14±4 months postpartum). MSNA (peroneal nerve microneurography), mean arterial pressure (MAP; finger photoplethysmography), and total peripheral resistance (TPR; MAP/cardiac output), were assessed at baseline, during a 2‐min HG protocol, and during 2‐min post‐exercise circulatory occlusion (PECO). As previously reported, baseline MSNA was higher in PPE than HP (22±7 vs 13±5 bursts/min, P=0.01). Baseline MAP was not different between PPE and HP (94±11 vs 89±9 mmHg, P=0.23), nor was TPR (13±3 vs 13±2 mmHg/L/min, P=0.63). During HG, MSNA was greater in PPE than HP (24±8 vs 16±6 bursts/min, P=0.03), whereas MAP was not different between PPE and HP (95±12 vs 95±11 mmHg, P=0.99), nor was TPR (13±3 vs 14±2 mmHg/L/min, P=0.39). During PECO, no differences were observed between PPE and HP women in MSNA (26±8 vs 18±6 bursts/min, P=0.08), MAP (93±12 vs 96±12 mmHg, P=0.70), or TPR (12±3 vs 15±2 mmHg/L/min, P=0.18). These findings indicate that PPE women demonstrate exaggerated MSNA during isometric HG exercise relative to HP women, although the role of the metaboreflex in mediating this effect, as assessed using PECO, remains unclear. Importantly, these data also demonstrate that young PPE women were able to buffer the deleterious cardiovascular outcomes of elevated MSNA such that MAP and TPR outcomes were similar to age‐matched HP women during all conditions.

Chronic pain is associated with anhedonia and decreased motivation. These behavioral alterations have been linked to alterations in the limbic brain and could explain the increased risk for obesity in pain patients. The mechanism of these behavioral changes and how they set in in relation to the development of chronic pain remain however poorly understood. Here we asked how eating behavior was affected in low-back pain patients before and after they transitioned to chronic pain, compared to patients whose pain subsided. Additionally, we assessed how the hedonic perception of fat-rich food, which is altered in chronic pain patients, related to the properties of the nucleus accumbens in this patients' population. We hypothesized that the accumbens would be directly implicated in the hedonic processing of fat-rich food in pain patients because of its well-established role in hedonic feeding and fat ingestion, and its emerging role in chronic pain. Accordingly, we used behavioral assays and structural brain imaging to test sub-acute back pain patients (SBP) and healthy control subjects at baseline and at approximately one-year follow-up. We also studied a sample of chronic low-back pain patients (CLBP) at one time point only. We found that SBP patients who recovered at follow-up (SBPr) and CLBP patients showed disrupted eating behaviors. In contrast, SBP patients who persisted in having pain at follow-up (SBPp) showed intact eating behavior. From a neurological standpoint, only SBPp and CLBP patients showed a strong and direct relationship between hedonic perception of fat-rich food and nucleus accumbens volume. This suggests that accumbens alterations observed in SBPp patients in previous works might protect them from hedonic eating disruptions during the early course of the illness. We conclude that disrupted eating behavior specifically sets in after pain chronification and is accompanied by structural changes in the nucleus accumbens.

Polycystic ovary syndrome (PCOS) is characterized by reproductive and metabolic dysfunction, and elevated blood pressure (BP). The cardiometabolic consequences of maternal hyperandrogenemia on offspring, either as adults or with aging, have not been well studied. We previously found that male offspring of hyperandrogenemic female (HAF) rats, a model of PCOS, are normotensive but have an exaggerated pressor response to angiotensin (Ang) II. In this study, the hypothesis was tested that adult and aging female offspring of HAF rats develop a metabolic and hypertensive phenotype. Control and HAF rats were implanted prepubertally with placebo or dihydrotestosterone pellets, which continued throughout pregnancy and lactation. Female offspring of HAF dams had lower birth weight than female control offspring. Although female HAF offspring (aged 16-24 weeks) had no differences in intrarenal Ang II, plasma lipids or proteinuria, they did have lower intrarenal Ang (1-7) and lower nitrate/nitrite excretion than controls. Adult HAF offspring had similar baseline BP as controls, but had an attenuated pressor response to Ang II. With aging (16-20 months), female HAF offspring remained normotensive with an attenuated pressor response to Ang II and high salt diet but more proteinuria and higher intrarenal Ang(1-7) than controls. Taken together, these data suggest that female HAF offspring are protected from developing hypertension, but may be at risk for renal injury with aging. Future studies are necessary to determine whether adult and postmenopausal offspring of PCOS women are at increased risk for cardiovascular dysfunction.Graphical abstract:http://links.lww.com/HJH/B820.

SUMMARYMonitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4×0.4mm2 field of view, and; a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise limited signals. Through these combined advances, we achieved simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.